12th International C. elegans Meeting

12 th International C. elegans Meeting The University of Wisconsin

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Platform Session #1: The Genome: Present & Future Wednesday, June 2, 7:00 p.m. Abstracts 1 - 15 Leilani Miller, Chair

24 May 1999 15:50 1 1 Platform Session #2: Neurons: Fate & Migration Thursday, June 3, 9:00 a.m.

Keynote Address: Reflections on the C. elegans Genome Project Robert H. Waterston, Genome Sequencing Center, Alan Coulson, The Sanger Center

1 Accessing genome project data and the state of ACeDB Members of the Genome Sequencing Consortium and the ACeDB collaboration

2 AcePerl: A New Face for Ace Lincoln Stein, Jean Thierry-Mieg, and Richard Durbin

3 Expressed Genes in C. elegans Jean Thierry-Mieg, Danielle Thierry-Mieg, Tadasu Shin-i and Yuji Kohara

4 Post genomics strategies and resources in C.elegans Yuji Kohara, the Kohara Lab and Collaborators

5 A Gene Knockout Service for C. elegans Gary Moulder, Nathan Cook, Brooke Toland, Jenny Medelberg and Robert Barstead, David Hughes, Chris Johnson, Ngoc-Sung Ly, Richard Durbin and Alan Coulson, Erin Gilchrist, Greg Mullen, Blazej Szczygielski and Ann Rose, Donald Moerman, Steven Jones, Stefan Eimer, Christine Goebel, Bianca Wiesinger and Ralf Baumeister

6 The complete family of G protein genes of Caenorhabditis elegans Gert Jansen, Karen L. Thijssen, Pia Werner, Marieke van der Horst, Esther Hazendonk and Ronald H.A. Plasterk

7 Using DNA microarrays to identify targets of the Ras/MAP kinase signaling pathway Stuart K. Kim, Carrie B. Van Doren and Rebecca R. Begley

8 Identification of the 3’ splice site by both subunits of U2AF Diego A. R. Zorio and Thomas Blumenthal

9 An alternatively spliced U2AF 65 exon capable of keeping a reporter gene mRNA from leaving the nucleus Peg MacMorris, Diego Zorio, and Tom Blumenthal

10 Natural targets of mRNA surveillance in C. elegans Quinn Mitrovich and Phil Anderson

11 Inherited and controllable interference by dsRNA Nektarios Tavernarakis, Shi Liang Wang, Maxim Dorovkov, Alexey Ryazanov, and Monica Driscoll

12 RNA interference depends on genes required for mRNA surveillance Daniel P. Morse, Mary Ellen Domeier, Michael Portereiko, Brenda L. Bass, and Susan E. Mango

13 Genetic analysis of RNA interference in C. elegans Hiroaki Tabara, Madathia Sarkissian, Lisa Timmons, Andrew Fire and Craig C. Mello

14 The Epigenetics of Germline Maintenance Bill Kelly and Andy Fire

15 Transposon silencing in C.elegans. René F. Ketting, Henri G.A.M. van Luenen, Miriam T. Smits and Ronald H.A. Plasterk

Platform Session #2: Neurons: Fate & Migration Thursday, June 3, 9:00 a.m. Abstracts 16 - 31 Adam Antebi, Chair

24 May 1999 15:50 2 2 Platform Session #3: Sex & Reproduction Thursday, June 3, 7:00 p.m.

16 UNC-43 CaMKII regulates the density of central glutamatergic synapses in vivo Christopher Rongo and Joshua Kaplan

17 A C. elegans liprin protein SYD-2 regulates presynaptic active zones Mei Zhen and Yishi Jin

18 Synaptic choice is regulated by UNC-4 and UNC-37-dependent repression of motor neuron-specific genes. Angela Winnier, James Meir, Jennifer Ross, Kim Lickteig, and David Miller

19 A C. elegans JNK signal transduction pathway regulates coordinated movement via type-D GABAergic motor neurons Masato Kawasaki, Naoki Hisamoto, Jun Ninomiya-Tsuji, Kunihiro Matsumoto

20 vab-7 controls the DB neuron fate Behrooz Esmaeili, Jennifer Ross, Cara Neades, David Miller, and Julie Ahringer

21 Genes involved in the formation of ciliated endings of sensory neurons in C. elegans Peter Swoboda and James H. Thomas

22 Cyclic GMP Signaling via ODR-1, DAF-11 and PKG-1 Noelle L’Etoile, Yongmai Zhang and Cori Bargmann

23 The LIM homeobox gene lim-4 distinguishes between two olfactory neuron fates Alvaro Sagasti, Oliver Hobert, Gary Ruvkun, and Cori Bargmann

24 A Forkhead Homolog Involved in Specifying Chemosensory Neuron Cell Fate Trina R. Sarafi-Reinach and Piali Sengupta

25 unc-130 is required to maintain the correct expression pattern of unc-129 in C. elegans. Bruce Nash and Joseph Culotti

26 A Screen for Suppressors of unc-6DC: Interactions between UNC-6 and NPR-1, a Neuropeptide Y Receptor Qun Wang and William G.Wadsworth

27 Mechanisms of axon pathfinding by SAX-3 Tim Yu, Joe Hao, Jennifer Zallen, Do Lee, Ken Prehoda, Marc Tessier-Lavigne, Wendell Lim, and Cori Bargmann

28 UNC-53 is involved in growth cone steering and co-localises with microtubule plus-ends Marc Van de Craen, Luc Maertens, Nina Cromheecke, Marleen Brunain, Peter Verhasselt, Marc De Raeymaeker, Walter Luyten, Christ Platteeuw, Joel Vandekerckhove, Johan Geysen and Thierry Bogaert

29 How the EGL-20/Wnt protein specifies two different migratory behaviors in the Q cell lineage Jennifer Whangbo and Cynthia Kenyon

30 A C. elegans ror receptor tyrosine kinase regulates cell motility and cell polarity Wayne Forrester, Megan Dell, Elliot Perens and Gian Garriga

31 Control of DAF-7 TGF-b expression and neuronal process development by a receptor tyrosine kinase KIN-8 in C. elegans Makoto Koga, Masaya Take-uchi, Tatsuji Tameishi and Yasumi Ohshima

Platform Session #3: Sex & Reproduction Thursday, June 3, 7:00 p.m. Abstracts 32 - 47 Meera Sundaram, Chair

24 May 1999 15:50 3 3 Platform Session #4: Chromosomes & The Cell Cycle Friday, June 4, 9:00 a.m.

32 SDC-2 triggers hermaphrodite sexual development and targets dosage compensation machinery to X chromosomes Heather E. Dawes, Denise M. Lapidus, Jason D. Lieb and Barbara J. Meyer

33 Molecular Similarity of Sex Determining Genes In Worms, Flies, and Perhaps Mammals Woesung Yi, Christopher Raymond, Jae Kettlewell, Emily Parker, and David Zarkower

34 The TRA-1 sex-determination protein regulates sexually dimorphic programmed cell death by transcriptionally repressing the egl-1 cell-death activator gene Barbara Conradt and Bob Horvitz

35 TRA-1 regulates the cellular distribution of the tra-2 mRNA in C. elegans Laura E. Graves, Scott P. Segal and Elizabeth B. Goodwin

36 The Brachyury-related gene mab-9 and cell fate determination in C.elegans: a tale of tails. Alison Woollard and Jonathan Hodgkin

37 Genetic analysis of male mating behavior: What’s lov got to do with it? Maureen M. Barr and Paul W. Sternberg

38 Genes utilized in protracting the male C. elegans spicules L. René García and Paul W. Sternberg

39 The PGL Family of P-granule-associated Proteins Interact and Function Redundantly in C. elegans Germline Development Ichiro Kawasaki, Anahita Amiri, Yuan Fan, and Susan Strome

40 EPS-1 Prevents P-Granule Expression in the Soma Yingdee Unhavaithaya, Tae Ho Shin and Craig C. Mello

41 FBF, NANOS-3 and the regulation of germline fates Sarah Crittenden, Brian Kraemer, Andrei Petcherski, Shanping Wang, Beilin Zhang, Maria Gallegos, Gary Moulder, Robert Barstead, Marvin Wickens and Judith Kimble

42 daz-1, a C. elegans homologue of DAZ (Deleted in Azoospermia), is required for the progression of meiosis in oogenesis Takeshi Karashima, Asako Sugimoto and Masayuki Yamamoto

43 Receptor-Mediated Endocytosis in C. elegans Barth Grant, Yinhua Zhang, Laura Pedraza, David H. Hall and David Hirsh

44 Identification of RNA Targets of GLD-1 Min-Ho Lee, Barth Grant, David Hirsh and Tim Schedl

45 GLD-1 represses tra-2 translation through a poly (A) tail-dependent mechanism Eric Jan and Elizabeth B. Goodwin

46 FOG-1 is a Cytoplasmic Polyadenylation Element Binding protein Suk-Won Jin, Judith Kimble and Ronald E. Ellis

47 CPEBs: a family of related RNA-binding proteins involved in distinct steps of spermatogenesis. C. Luitjens, J. Kimble and M. Wickens

Platform Session #4: Chromosomes & The Cell Cycle Friday, June 4, 9:00 a.m. Abstracts 48 - 63 Ben Williams, Chair

24 May 1999 15:50 4 4 Platform Session #5: Neurons: Synapses & Signals Friday, June 4, 7:00 p.m.

48 Getting Intimate with the Right Partner Dernburg, A.F. and Villeneuve, A.M.

49 Meiosis, Mitosis and Their Relationship to Dosage Compensation Annette Chan, Tammy Wu, Danielle Pasqualone, and Barbara Meyer

50 Synapsis and Chiasma Formation in C. elegans Require HIM-3, a Component of the Axial Element That Functions in Meiotic Chromosome Segregation Monique Zetka, Ichiro Kawasaki, Susan Strome, and Fritz Müller.

51 him-4 encodes an extracellular matrix protein required for cell adhesion and germ-line chromosome segregation Bruce E. Vogel and Edward M. Hedgecock

52 Mutants with post-fertilization meiotic progression defects A. Golden, P. Sadler, G. Holt, M. Wallenfang, G. Seydoux, D. Shakes

53 Genes Involved in Meiotic and Mitotic Spindle Formation Martin Srayko, M. Rhys Dow, Chenggang Lu, and Paul E. Mains

54 Secretion is Required to Complete Cytokinesis in Caenorhabditis elegans Ahna R. Skop, Dominique Bergmann, William A. Mohler, and John G. White

55 Depletion of syntaxins in the early C. elegans embryo reveals a role for membrane fusion events in cytokinesis Verena Jantsch-Plunger and Michael Glotzer

56 HKP-1, a kinetochore-associated protein is assembled onto mitotic chromosomes during prophase and is important for chromosome segregation in Caenorhabditis elegans Landon L. Moore, Mike Morrison, and Mark B. Roth

57 The role of lin-5 in chromosome segregation. Monique Lorson, Marian Walhout, Marc Vidal, Bob Horvitz and Sander van den Heuvel

58 Regulation of post-embryonic G1 cell cycle progression by a cyclin D/cyclin dependent kinase-like complex in C. elegans Morgan Park and Michael Krause

59 Genes, mdf-1 and mdf-2, encoding mitotic checkpoint components are essential in C. elegans Risa Kitagawa and Ann M. Rose

60 Two C. elegans mutants with defects in telomere replication Shawn Ahmed and Jonathan Hodgkin

61 The heterochronic gene lin-42 and circadian rhythms: is there a connection? Mili Jeon, Heather Gardner and Ann Rougvie

62 The heterochronic gene lin-41 encodes a temporally regulated RING finger protein that controls the timing of appearance of LIN-29 protein. Frank Slack and Gary Ruvkun

63 Caenorhabditis Genetics Center Theresa Stiernagle, Sylvia Martinelli, Jonathan Hodgkin, Leon Avery, Robert Herman

Platform Session #5: Neurons: Synapses & Signals Friday, June 4, 7:00 p.m. Abstracts 64 - 78 Asako Sugimoto, Chair

24 May 1999 15:50 5 5 Platform Session #6: Embryos: Patterns & Fates Saturday, June 5, 9:00 a.m.

64 unc-13 mutants accumulate synaptic vesicles and are defective in evoked neurotransmitter release J. E. Richmond, W. S. Davis and E. M. Jorgensen

65 The Rab3 GTP/GDP exchange factor homologue AEX-3 is a regulator of two different pathways for presynaptic activities Kouichi Iwasaki, Erik Jorgensen, and Rika Toyonaga

66 Gqa and Goa Pathways Act Antagonistically to Regulate Synaptic Transmission in C. elegans Ken Miller, Melanie Emerson, and Jim Rand

67 egl-8 and egl-30 define a pathway that modulates cholinergic neurotransmission Mark R. Lackner, Stephen J. Nurrish, and Joshua M. Kaplan

68 Signaling by Go and Gq : suppressors of activated Goa Wen J. Chen, Yvonne M. Hajdu-Cronin and Paul W. Sternberg

69 Serotonin inhibits Acetylcholine release via depletion of Diacylglycerol at the neuromuscular junction. Stephen Nurrish, Laurent Segalat, and Joshua M Kaplan.

70 An Ionotrophic Serotonin Receptor and a Serotonin Reuptake Transporter Are Involved in Experience-Dependent Modulation of Behavior Rajesh Ranganathan, Stephen C. Cannon, and Bob Horvitz

71 Fluoxetine (prozac) resistant mutants identify a novel family of multipass transmembrane proteins Robert K.M. Choy and James H. Thomas

72 Pharyngeal Calcium Transients and Prospects for Excitable Cell Imaging Rex Kerr, Varda Lev-Ram, Roger Y. Tsien, and William R. Schafer

73 EXP-2 is a K + channel that repolarizes the pharyngeal muscle M. Wayne Davis, Richard Fleischhauer, Joe Dent, Rolf Joho, Leon Avery

74 EAT-2 is a beta subunit of a nicotinic acetylcholine receptor involved in neurotransmission from MC to pharyngeal muscle Jim McKay, David Raizen, and Leon Avery

75 Analysis of UNC-49 reveals two structural determinants for steroid modulation of GABA receptors Bruce A. Bamber, Tina Rutar and Erik M. Jorgensen

76 The Genetics of Ivermectin Resistance Joe Dent, Demetrios Vassilatis, McHardy Smith, Leon Avery

77 Mutations in the AMPA-type glutamate receptor, glr-1, block olfactory associative and non-associative learning in C. elegans. Glenn E. Morrison and Derek van der Kooy

78 NMDA Receptors in C. elegans: Their Role in Effective Locomotion Penelope J. Brockie, David M. Madsen and Andres V. Maricq

Platform Session #6: Embryos: Patterns & Fates Saturday, June 5, 9:00 a.m. Abstracts 79 - 93 Patricia Kuwabara, Chair

24 May 1999 15:50 6 6 Platform Session #7: Cues & Morphogenesis Saturday, June 5, 2:00 p.m.

79 PAR-6/PAR-3/PKC-3, a PDZ-mediated protein complex important for establishing early embryonic polarity Tak Hung, Esther Lee, and Ken Kemphues

80 pod-1 links embryonic polarity to cellular architecture Rappleye, C., Smith, C., Paredez, A., McDonald, K, Aroian R.

81 mes-1, a gene required for unequal divisions of the germline in early C. elegans embryos, encodes a membrane protein localized to the boundary between the germline and gut cells. Laura A. Berkowitz and Susan Strome

82 PIE-1 localization to the germ lineage depends on two distinct mechanisms that act on separate domains in the PIE-1 protein K. J. Reese, M. A. Dunn, and G. Seydoux

83 Studies on POS-1 interacting proteins. Ken-ich Ogura and Yuji Kohara

84 Non-destructive, 3-D imaging of GFP-tagged proteins throughout C. elegans embryogenesis James A. Waddle and Robert H. Waterston

85 Regulation of POP-1 asymmetry Rueyling Lin

86 The LIT-1 and MOM-4 Protein Kinases Control Cell-Fate in Response to Anterior/Posterior Polarity Signals Tae Ho Shin, Christian E. Rocheleau, Jun Yasuda and Craig C. Mello

87 MAP Kinase and Wnt signaling Pathways converge to directly down-regulate HMG-domain containing transcription factors in C. elegans and vertebrates Marc D. Meneghini, J. Clayton Carter, Tohru Ishitani, Naoki Hisamoto, Jun Ninomiya-Tsuji, Shin-ichi Nagai, Michiru Nishita, Hiroshi Shibuya, Christopher J. Thorpe, Danielle R. Hamill, Kunihiro Matsumoto, and Bruce Bowerman.

88 src-1 interacts with a Wnt signaling pathway and an APC-related gene to control cell fate decisions in the early C. elegans embryo. Jennifer Hogan, Yanxia Bei, Craig Mello, and John Collins

89 Spatiotemporal control of end-1 expression: positive regulation by the Wnt pathway and lineage-specific repression Jodie J. Kasmir, Eric S. Witze, Jan L. Sumerel, Jiangwen Zhu, and Joel H. Rothman

90 Specification of endomesoderm by the MED-1 and MED-2 GATA factors: similarity of endoderm and mesoderm specification Morris F. Maduro and Joel H. Rothman

91 Does Wnt signaling distribute HAM-1? Nancy Hawkins and Gian Garriga

92 A new Abd-B homolog is required for posterior patterning of the embryo K. Van Auken, D. Weaver, B. Robertson, L. Edgar, and B. Wood

93 The cadherin-catenin complex stabilizes filopodial adhesive contacts during hypodermal morphogenesis William B. Raich, Cristina Agbunag, James R. Priess and Jeff Hardin

Platform Session #7: Cues & Morphogenesis Saturday, June 5, 2:00 p.m.

24 May 1999 15:50 7 7 Platform Session #7: Cues & Morphogenesis Saturday, June 5, 2:00 p.m.

Abstracts 94 - 109 Eric Moss, Chair

94 The C. elegans type XVIII collagen/endostatin homologue is involved in cell migrations and axon guidance Brian D. Ackley and James M. Kramer

95 Nidogen is non-essential for basement membrane assembly but synthetic lethal with a dystroglycan-like gene Seong Hoon Kang, Jeffrey Johnson, Shaoru Wang, and James M. Kramer

96 The gon-1 gene is a metalloprotease required for gonad morphogenesis Robert Blelloch, Sonia Santa Anna-Arriola, Yongjing Li, Jonathan Hodgkin, Judith Kimble

97 MIG-17, an adam family metalloprotease required for dtc migration in C. elegans Kiyoji Nishiwaki, Naoki Hisamoto, Kunihiro Matsumoto, Norio Suzuki

98 The Ephrin VAB-2 interacts with the VAB-1 Eph receptor to regulate neural and epidermal morphogenesis. Ian Chin-Sang, Sean George, Mei Ding and Andrew Chisholm

99 Multiple ephrins control cell organization in C. elegans through kinase-dependent and kinase-independent functions of the VAB-1 Eph receptor Xiangmin Wang, Peter J. Roy, Sacha J. Holland, Lijia W. Zhang, Joseph G. Culotti and Tony Pawson

100 The soc genes in FGF receptor signaling in C. elegans Jennifer L. Schutzman and Michael J. Stern

101 sop-1, -2 and -3: New Components That Regulate C. elegans Hox Gene Expression Hong Zhang and Scott W. Emmons

102 Vulval pattern formation revisited Minqin Wang and Paul W. Sternberg

103 A modular system of docking sites mediates ERK map kinase recognition of substrate proteins such as LIN-1 and KSR-1 Dave Jacobs and Kerry Kornfeld

104 lin-55 DP and an E2F-like gene act in the lin-35 Rb pathway to antagonize let-60 ras signaling during vulval induction Craig Ceol and Bob Horvitz

105 abi-1, a gene required for vulval morphogenesis, encodes a nuclear hormone receptor homolog Zhe Chen and Min Han

106 Patterning and differentiation of the ventral uterine cells that connect to the vulva Anna P. Newman and Hediye Nese Cinar

107 Who said you need two to tango? The development of the real and pseudo vulvae of a let-60 gf mutant Gidi Shemer, Ranjana Sharma-Kishore and Benjamin Podbilewicz

108 The even-skipped homolog Ppa-vab-7 is involved in the phylogenetic restriction of the vulva equivalence group in Pristionchus pacificus Benno Jungblut and Ralf Sommer

109 Genetic analysis of vulva development in Oscheius sp. CEW1 Marie-Anne Félix, Mark Viney, Marie-Laure Dichtel, Sophie Louvet and Paul W. Sternberg

24 May 1999 15:50 8 8 Platform Session #8: Life & Death Sunday, June 6, 9:00 a.m.

Platform Session #8: Life & Death Sunday, June 6, 9:00 a.m. Abstracts 110 - 125 Lesilee Rose, Chair

110 Genetic Dissection of Radiation-Induced Apoptosis and Cell Cycle Arrest in C. elegans Anton Gartner, Stuart Milstein and Michael Hengartner

111 CED-9 and EGL-1 regulate the subcellular localization of CED-4 Brad Hersh, Fangli Chen, Barbara Conradt, Zheng Zhou, and Bob Horvitz

112 Identification and Characterization of Downstream Targets of the Cell-Death Protease CED-3 D. Ledwich, C. Duffy, C. Lau, H. Metters, P. Huynh, and D. Xue

113 A CED-2/Crk-II, CED-5/DOCK180, CED-10/Rac pathway controls cell-corpse engulfment and cell migration Peter Reddien and Bob Horvitz

114 The Cell-corpse Engulfment Gene ced-1 Encodes A Transmembrane Receptor That May Act To Recognize Dying Cells Zheng Zhou and Bob Horvitz

115 A Common Set of Engulfment Genes Mediates the Removal of Both Apoptotic and Necrotic Corpses in C. elegans Sambath Chung, Tina L. Gumienny, Michael Hengartner, and Monica Driscoll

116 Mutations That Suppress Degenerative Cell Death Can Extend Lifespan Keli Xu, Laura A Herndon, Hyuk Wan Ko and Monica Driscoll

117 MEC-6 directly interacts with the mechanosensitive channel subunits, MEC-4 and MEC-10 Dattananda Chelur, Lei Chen and Marty Chalfie

118 ins-14, one of many insulin-related genes in C.elegans, can regulate dauer formation Sarah Pierce, Robert Wisotsky, Leo Liu, and Gary Ruvkun

119 Multiple Layers of Cell Non-autonomy in the Control of C. elegans Lifespan Javier Apfeld and Cynthia Kenyon

120 Cell-Type Specific age-1 Expression Reveals Multiple Outputs for Insulin-like Signaling Catherine A. Wolkow, Mingsum Lee, Kotaro Kimura# and Gary Ruvkun

121 The tkr-1 life-extension gene, which responds to starvation and environmental stress, is regulated by the daf-16 forkhead transcription factor. Shin Murakami and Thomas E Johnson

122 A Cytosolic Catalase, CTL-1, Is Necessary For daf-c- and clk-1-dependent Extension of Adult Life-span in C. elegans James Taub, Joe Lau, Charles Ma, Jang Hee Hahn, Rafaz Hoque, Jonathan Rothblatt, and Martin Chalfie

123 Long-lived C. elegans mutants have reduced metabolic rates. Wayne A. Van Voorhies, Samuel Ward

124 Signals from the Reproductive System that Regulate the Lifespan of C. elegans Honor Hsin and Cynthia Kenyon

125 Illicit Sex in Georgia: inter-species reproductive interactions and speciation in the genus Caenorhabditis Katherine L. Hill and Steven W. L’Hernault

24 May 1999 15:50 9 9 Teaching Posters Thursday, Friday, and Saturday, 2:00 - 5:00 p.m.

Teaching Posters Thursday, Friday, and Saturday, 2:00 - 5:00 p.m. Abstracts 126 - 136

126 C. elegans: A Model Organism for Teaching? Stephanie Aamodt

127 Using C. elegans to Teach Embryonic Development to Undergraduates Raffi Aroian, Diane Johnson, Gabriele Wienhausen

128 C. elegans in Introductory Biology Projects Elizabeth De Stasio

129 Worms in class at NYU David Fitch, E. Jane Albert Hubbard, and Scott Clark

130 Using PCR in an undergraduate lab course to detect deletions in the unc-93 gene James L. Lissemore, Laura L. Lackner, and George D. Fedoriw

131 A project-based laboratory course using C. elegans Leilani M. Miller

132 Lecture/lab combo: P-granule antibody staining Mary K. Montgomery

133 Use of C. elegans in Undergraduate Biology Courses. William R. Morgan

134 Biology Biology 478: Research & Seminar on the Molecular Biology of Model Organisms Joe Pelliccia

135 Development of an Open-ended Muscle Physiology Experiment with C. elegans J. Sulcove and T. Allen

136 Surfing the Genome: Using the C. elegans Genomic Sequence to Teach Molecular Genetics Bruce Wightman

Poster Session #1 Thursday, June 3, 2:00 - 5:00 p.m. Abstracts 137 - 409 Aamodt - Hopper

137 Conservation of sequence and intron/exon structure between the homologous pag-3 genes from C. elegans and C. briggsae Eric Aamodt, Ling Shen, Brandi Rose and Joan McDermott

138 Characterization of the heterochronic gene lin-57, a gene hypostatic to lin-4 Juan E. Abrahante, Eric A. Miller, Ann E. Rougvie

139 Pharmacological analysis on unc-68 suppressor animals of Caenorhabditis elegans Ryota Adachi, Yasuji Sakube and Hiroaki Kagawa

140 adm-2 has an essential role during early embryogenesis in C. elegans Vardit Adir and Benjamin Podbilewicz

141 T-box genes involved in embryonic patterning Julie Ahringer and Michael Mitsch

142 Analysis of a regulator of body length in C. elegans Margareta Aili and Simon Tuck

24 May 1999 15:50 10 10 Poster Session #1 Thursday, June 3, 2:00 - 5:00 p.m.

143 27° in the life of wild and mutant worms Michael Ailion and James H. Thomas

144 Identification and characterization of long-lived mutants in C. elegans J. Alcedo, D. Garigan, N. Arantes-Oliveira, N. Libina, B. Albinder, J. Apfeld, H. Hsin, B. Tsung, and C. Kenyon

145 Structure/ function of C-terminus motor KLPs in C.elegans. M.Yusuf Ali, T. Harada, Shams T. Khan and Shahid S. Siddiqui

146 Multiple kinesins play critical role in chromosomal movement during embryonic cell divisions in C.elegans. M.Yusuf Ali, Shams T. Khan, Azham S. Mohammed and Shahid S. Siddiqui

147 In vivo function of kinesins in C elegans neuromuscular system M. Yusuf Ali, F. Hori, Azham Shah Mohammed, Shahid S. Siddiqui

148 Determining the roles of octopamine and CREB in worm behavior Mark Alkema and Bob Horvitz

149 Biological Roles of Troponin T T. Allen, L. Hong, J. Ward, A. Burkeen, and E.A. Bucher

150 Analysis of C. elegans host defense response to bacterial pathogen Pseudomonas aeruginosa Genevieve Alloing, Rhonda Feinbaum, Shalina Mahajan-Miklos, Man-Wah Tan and Frederick M. Ausubel

151 Regulation of Cell fusion in C. elegans Scott Alper and Cynthia Kenyon

152 Temporal coordination of cell cycle with development in C. elegans larvae. Victor Ambros, Yang Hong, Rosalind Lee, Richard Roy

153 tbx-9 encodes a transcription activator Yoshiki Andachi

154 Molecular genetic analysis of daf-12 and related receptors Adam Antebi, Corrina Kober-Eisermann

155 Three mutants that affect vulva development in C. elegans Igor Antoshechkin, Roda Amaria, and Min Han

156 Identification and tissue expression of CETMIV, the fourth isoform of the tropomyosin gene in Caenorhabditis elegans AKWASI ANYANFUL, YASUJI SAKUBE, HIROAKI KAGAWA.

157 The UNC-45 protein is a component of muscle thick filaments and co-localizes with myosin heavy chain B, but not myosin heavy chain A. Wanyuan Ao and Dave Pilgrim

158 Nucleoside transporters in C. elegans P.J. Appleford, M. Griffiths, S.A. Baldwin, E.G. Chomey, S.Y.M. Yao, D. MacGregor, R.E.Isaac, D.Coates, C.E.Cass and J.D. Young

159 Molecular investigation of smg-4, required for mRNA surveillance in C. elegans Rachel Aronoff, Renee Baran, and Jonathan Hodgkin

160 RNA and Predicted Ionotropic Glutamate Receptors in the C. elegans Genome Rachel Aronoff, Stefan Binnenbose, Carl Petersen, Peter Seeburg, and Rolf Sprengel

161 Homologues of claudin, integral protein of mammalian tight junction, are present and important in C. elegans Akira Asano, Kimiko Asano, Mikio Furuse, Hiroyuki Sasaki, and Shoichiro Tsukita

162 A Cell Cycle Regulator in the Germ Line Neville Ashcroft, Andy Golden

24 May 1999 15:50 11 11 Poster Session #1 Thursday, June 3, 2:00 - 5:00 p.m.

163 Identification of fork head transcription factors expressed during C. elegans embryogenesis. Sobia Aslam, Andrew Mounsey, Petra Bauer, Christine Royall and Ian A. Hope

164 Does the ortholog of the brain development genes ems/EMX have a function in C. elegans? Gudrun Aspöck and Thomas R. Burglin

165 A second look at warthog and groundhog genes Gudrun Aspöck, Hiroshi Kagoshima and Thomas R. Burglin

166 Complete closure of the anterior and posterior hypodermis needs the C. elegans homeobox gene ceh-43. Gudrun Aspöck and Thomas R. Burglin

167 The regulator hypothesis revisited: CAMs, IgSFs, ZIGs and tissue patterning Oscar N. Aurelio, Jun Zhu and Oliver Hobert

168 Genetic Mapping of Multiple Loci Determining Life Span in C. elegans Srinivas Ayyadevara, John J. Thaden and Robert J. Shmookler Reis

169 A quantitative trait locus for body size Ricardo B. R. Azevedo, C. Knight and Armand M. Lero

170 Analysis of a temperature sensitive muation in zen-4 kinesin like protein reveals a requirement during ventral enclosure Baas, T., Raich, W., Hamill, D., and Hardin, J.

171 Cloning and Characterization of the Cytokinesis mutant stu-4. Ananth Badrinath, John White

172 Associatiions of Caenorhabditis species with terrestrial isopods. Scott Everet Baird

173 Genetic and electrophysiological analysis defines the composition of a native GABA receptor in C. elegans Bruce A. Bamber, Janet Richmond and Erik M. Jorgensen

174 Transcriptional regulation of sex specific bi-directional promoter between male tail collagen gene and sperm specific protein gene in Caenorhabditis elegans Tetsuya Bando, Tatsuji Ikeda and Hiroaki Kagawa

175 Characterization of Calcineurin, a Ser/Thr protein phosphatase, in C. elegans Jaya Bandyopadhyay, Jiyeon Lee, and Joohong Ahnn

176 A Screen for Components of the GOA-1 Signaling Pathway I. Amy Bany and Michael R. Koelle

177 Characterization of syd-5, a gene that may be involved in synaptogenesis. Renee Baran and Yishi Jin

178 UCS Domain Functions as a Myosin Assemblase Jose M. Barral and Henry F. Epstein

179 Polarized Localization of PAR-3 and PAR-2 in C. elegansEmbryos Requires ooc-5 and ooc-3 Stephen E. Basham and Lesilee S. Rose

180 Cellular and genetic analysis of Gq alpha mediated signaling pathways in C. elegans Carol A. Bastiani, Shahla Gharib, Paul W. Sternberg, Melvin I. Simon

181 A genetic screen for inhibitors of the vulval induction pathway Gopala Battu and Alex Hajnal

182 Quantification of Transcriptome Distortion After Amplification Using cDNA Micro-Arrays L. Ryan Baugh and Craig P. Hunter

24 May 1999 15:50 12 12 Poster Session #1 Thursday, June 3, 2:00 - 5:00 p.m.

183 A C. elegans homolog of the human gene for X-linked kallmann syndrome Paolo Bazzicalupo, Anna Facciolli, Massimo Hilliard, Elia Di Schiavi, Andrea Ballabio and Elena Rugarli

184 GSK-3 plays a positive role in Wnt signaling in C.elegans embryogenesis Yanxia Bei, Kevin Fitzgerald and Craig C. Mello

185 Cellular, molecular, and genomic analysis of the novel matrix receptor gene mua-3 Mark Bercher, Jim Wahl, Zhaoliang Lu, Bruce Vogel, Edward Hedgecock and John Plenefisch

186 Molecular characterization of the C2H2 zinc finger protein MUA-1 Mark Bercher, Xiao-Feng Zhou, John Plenefisch

187 Establishing the handedness of left/right asymmetry in C. elegans Dominique Bergmann and Bill Wood

188 Olfactory Plasticity in C. elegans: A Concentration-dependent Separation of Habituation and Adaptation. N. Bernhard and D. van der Kooy

189 TEG-1 Functions in the GLD-1 Pathway to Initiate Meiotic Development L. W. Berry, D. Hansen, T. Dang, D. F. Schneider and T. Schedl

190 RNA interference of SMN, the gene causing Spinal Muscular Atrophy in humans, leads to embryonic lethality and to germ cell apoptosis in the nematode Caenorhabditis elegans. Solange Bertrandy, Suzie Lefebvre, Yugi Kohara, Arnold Munnich and Danielle Thierry-Mieg

191 Genetic analysis of neuromuscular junction formation in C. elegans. Jean-Louis Bessereau and Erik Jorgensen

192 Development of a mutagenesis strategy based on heterologous transposition in C. elegans. Jean-Louis Bessereau, Ashley Wright and Erik Jorgensen

193 Behavioral Effects of Exposure to Ethanol Jill C. Bettinger and Steven L. McIntire

194 Investigating the sex-specific expression and function of the putative chemosensor srd-1. Marc Bickle, David Hughes and Jonathan Hodgkin.

195 Analysis of the UNC-44 Ankyrins Brent Bill, Sabrina Chan, Dina Darwis, Marc Domanus, Brian McFaul, Amreen Khan, Kimberly Stergulz, Sarah Windler, Nena Velamparampil, and Anthony Otsuka

196 Preliminary Studies of the ets Gene Family in C. elegans Adam G. Blaszczak, Matthew B. Smith, and Barbara J. Graves

197 The mut-2 mutator of C. elegans Boese, Q and Collins, J

198 Embryonic development of a mononchid nematode G. Borgonie and A. Coomans

199 The Clr Phenotype and FGFR Signaling Christina Z. Borland and Michael J. Stern

200 RNA interference targets the pre-mRNA of the lir-1/lin-26 operon to prevent expression of both genes Julia M. Bosher, Pascale Dufourcq, Satis Sookhareea and Michel Labouesse

201 Investigating apico-basal polarity in the embryonic gut of C. elegans O. Bossinger, A. Klebes, C. Theres, C. Segbert and E. Knust

202 Developmental regulation of the cell cycle in C. elegans Mike Boxem and Sander van den Heuvel

24 May 1999 15:50 13 13 Poster Session #1 Thursday, June 3, 2:00 - 5:00 p.m.

203 Complex alteration of respiration, metabolic potential and ATP stores in long-lived Clk mutants of Caenorhabditis elegans Bart P. Braeckman, Koen Houthoofd, Annemie De Vreese, Jacques R. Vanfleteren

204 Further Analysis of the Gonad-Dependent Mechanism of Sex Myoblast Migration Catherine S. Branda and Michael J.Stern

205 Isolation and Characterization of Suppressors of the C. elegans timing gene clk-1 Robyn Branicky, Jinliu Feng, and Siegfried Hekimi

206 Identification of promoter elements of parasitic nematode genes in transgenic C. elegans Collette Britton, Diane L. Redmond and David P. Knox

207 G protein signaling pathways in C. elegans Lorna A. Brundage, Paul W. Sternberg and Melvin I. Simon

208 ceh-13 is involved in the anterior organization of the C. elegans embryo Karin Brunschwig, Claudia Wittmann, Ralf Schnabel, Thomas R. Buerglin, Heinz Tobler, Fritz Mueller

209 A C. elegans histone H3-like protein localizes to the centromere of both holokinetic and monokinetic chromosomes. Brian J. Buchwitz, Landon L. Moore, Kami Ahmad, Steven Henikoff, Mark B. Roth

210 Inositol Phosphate Signaling in C. elegans Yen K. Bui, Minqin Wang, Paul W. Sternberg

211 Mutations that Affect Synaptic Localization of GLR-1 Michelle Burbea, Christopher G. Rongo and Joshua M. Kaplan

212 Analysis of C. elegans non-coding regions: a bioinformatics approach Mark Burke and Elizabeth F. Ryder

213 In Vivo Analysis of Troponin I Domains in Muscle Function A. Burkeen, J. Sulcove, E.A. Bucher, and T. Allen

214 Molecular characterization of mutations in dig-1, a gene involved in sensory map formation. Christopher Burket, Stacy Hubbard, and Elizabeth F. Ryder.

215 Suppression of the neuropeptide gene flp-1 in Caenorhabditis elegans. E.R. Bush, H. Field and C. Li

216 Studying human disease genes in C. elegans Ned Buttner and Bob Horvitz

217 unc-16 May Play a Role in DD Motorneuron Remodeling in C. elegans D.T.Byrd, M.Walcoff, and Y.Jin

218 mec-14 Encodes a Novel Member of the Oxido-reductase Superfamily That May Modulate Mechanosensory Channels Guy A. Caldwell, Lei Chen, Nora Hom, Sidhu Gangadharan, Yuan Tu, Mingxia Huang, and Martin Chalfie

219 C. elegans Genes Representing a Link Between Nuclear and Neuronal Migration Guy A. Caldwell, Angus L. Dawe, N. Ronald Morris and Martin Chalfie

220 Molecular mechanisms of pop-1 transcriptional regulation Dominica Calvo and Yang Shi

221 pag-3 may specify both neuroblast cell fate and terminal fates during development of the ventral cord Scott Cameron, Joan B. McDermott, Eric Aamodt, and Bob Horvitz

222 A screen for mutations that affect programmed cell death in the ventral cord Scott Cameron, Nancy Tsung, and Bob Horvitz

24 May 1999 15:50 14 14 Poster Session #1 Thursday, June 3, 2:00 - 5:00 p.m.

223 Serotonin Modulation of Locomotion by Regulated Neurotransmission Tija Carey, Stephen J Nurrish, Joshua M. Kaplan

224 tab-1, a Gene Involved in Response to Anterior Touch Lucinda Carnell Brian Harfe Andrew Fire, and Marty Chalfie

225 ts emb mutants: a retrospective (on the g-set) & future uses II: recent news on some neurobiological collaborations Randy CASSADA

226 Excitation-contraction coupling in C. elegans muscle cells as monitored by multiphoton excitiation FRET imaging of calcium indicator proteins. Victoria Centonze, Ed Maryon, David Wokosin Bonnie Saari, Phil Anderson.

227 How are anterior cell migrations guided by mig-13? QueeLim Ch’ng, Lisa Moore, Mary Sym and Cynthia Kenyon

228 Developmental patterning in the C. elegans hindgut Helen M. Chamberlin and James H. Thomas

229 Immunoaffinity purification of the C. elegans dosage compensation complex Raymond C. Chan, Michael Albrecht, Chun Tsai and Barbara J. Meyer

230 SOS-1, a C. elegans homolog of SOS, mediates vulval induction, viability, fertility and spicule development. Chieh Chang, Neil A. Hopper and Paul W. Sternberg

231 Control of Early Germ Line Proliferation Aisha Chaudhary and E. Jane Albert Hubbard

232 ARM-1 is a novel body wall muscle protein that binds UNC-44 Lihsia Chen and Vann Bennett

233 A Study of LAD-1, the C. elegans Homologue of the L1 Family of Neuronal Adhesion Molecules Lihsia Chen, Anthony Otsuka, Vann Bennett

234 Transcriptional control of germ cell fate Pei-Jiun Chen and Ronald Ellis

235 Are Complex N-glycans Essential for the Development of the Nematode C. elegans? Shihao Chen, Sihong Zhou, Mohan Sarkar, Andrew M.Spence and Harry Schachter

236 Dissecting the Meiotic Recombination Pathway Chin, G.M., Kelly, K.A., Hillers, K.J., and Villeneuve, A.M.

237 Functional Characterization of C. elegans calsequestrin, a high capacity calcium-binding protein Jeonghoon Cho, Youngsoo Oh, Kyewon Park, Jaeran Yu, Dohan Kim, and Joohong Ahnn

238 Gene and chromosome specific localization of SDC-1 directs sexual fate and implements dosage compensation. Diana S. Chu, Denise M. Lapidus, Barbara J. Meyer

239 Investigating the function of the SMA-1 SH3 domain in C. elegans morphogenesis Alba I. Cid and Judith Austin

240 Transgenic Strains of the Nematode Caenorhabditis elegans as Biomonitors of Environmental Metal Contamination L. K. Cioci, Ling Qiu and Jonathan H. Freedman

241 GFP transgene expression pattern based mutant screens to identify genes involved in endoderm development in Caenorhabditis elegans. Caroline Clucas and Iain Johnstone

24 May 1999 15:50 15 15 Poster Session #1 Thursday, June 3, 2:00 - 5:00 p.m.

242 Molecular Characterization of the Nuclear Pore Protein, gp210 Merav Cohen and Yosef Gruenbaum

243 Acetylcholinesterase genes in C. elegans. I. ace-1 and ace-2 encoding classes A and B AChEs (ACE-1 and ACE-2) D. Combes, E. Culetto, M. Grauso, Y. Fedon, J.-P. Toutant and M. Arpagaus

244 Acetylcholinesterase genes in C. elegans.II. Tandem organization of the third and fourth genes. D. Combes, E. Culetto, M. Grauso, Y. Fedon, J.-P. Toutant and M. Arpagaus

245 A C. elegans-based study of Huntington Disease-associated pathways John Connolly, Sébastien Holbert, Isabelle Denghien, Coralie Philippe, Stéphanie Morinière, Martin Chalfie, and Christian Néri

246 CeTwist mutants have defective non-striated muscle development A. K. Corsi, S. Kostas, E. Jorgensen, A. Fire, and M. Krause

247 Complex cuticle patterning is revealed with GFP-tagged struts. Jennifer R. Crew and James M. Kramer

248 T04C9 encodes a novel protein related to centaurin and oligophrenin GTPase Activating Proteins Justin Cross, Neda Zahedi, Howard Baylis, Laura Harrington and Trevor Jackson

249 Cholesterol Functions Enantiospecifically in C. elegans C. Michael Crowder, Laura Metz, A. Sampath Kumar, Douglas F. Covey

250 sad-1, sad-2: Cloning Progress, Branching Digress Gage Crump, Cori Bargmann

251 Not all nematodes have constant cell lineages Ana S. Cunha, Ricardo B. R. Azevedo, and Armand M. Leroi

252 Characterization and Cloning of pag-1 Melody Cunningham, Guofeng Xie, Yiwen Jia, Joan B. McDermott and Eric Aamodt

253 Heterotrimeric G proteins of C. elegans: cDNA sequence analysis and interaction studies Edwin Cuppen, Gert Jansen, Ronald H.A. Plasterk

254 The IP 3 Receptor is a Timekeeper for the Defecation Cycle Rhythm Paola Dal Santo, Mary A. Logan, Andrew D. Chisholm, and Erik M. Jorgensen

255 Toward the cloning of vab-6, a gene involved in axon guidance and morphogenesis Gratien Dalpé, Peter Roy and Joseph Culotti

256 Characterization and Mapping of odr-9/egl-4 Susan Daniels and Piali Sengupta

257 Characterisation of sigma class glutathione s-transferases of C.elegans Joanna Daulby, David Coates and R.Elwyn Isaac

258 gut-2 and gut-4 Encode Proteins with Similarity to Sm-like Proteins Andrew Davies, Melinda Moseley and Jocelyn Shaw

259 Functional Overlap between the mec-8 Gene and Five sym Genes in C. elegans Andrew G. Davies, Caroline A. Spike, Jocelyn E. Shaw and Robert K. Herman

260 Mutants with altered sensitivity to the intoxicating effects of ethanol Andrew G. Davies, Catharine L. Eastman, Nicole G. Coufal, HongKyun Kim and Steven L. McIntire

261 Annotation of uncharacterized C. elegans proteins by transferring knowledge from the Yeast Proteome Database (YPD). B. P. Davis, K. J. Roberg-Perez, P. E. Hodges, M. C. Costanzo, A. H. Z. McKee, M. E. Cusick, W. E. Payne, J. E. Brooks and J. I. Garrels

24 May 1999 15:50 16 16 Poster Session #1 Thursday, June 3, 2:00 - 5:00 p.m.

262 dpy-23 encodes a component of a clathrin adaptor complex and is required for the endocytosis of synaptic vesicles Warren S. Davis, Paul Baum, Gian Garriga and Erik M. Jorgensen

263 Cloning and characterization of the class A synthetic multivulva genes Ewa M. Davison and Bob Horvitz

264 The Genetics of C. elegans social behavior Mario de Bono and Cori Bargmann

265 spn-2 and spn-3: two genes involved in spindle orientation Leah R. DeBella and Lesilee S. Rose

266 Evolution of vulval development and P11/12 cell migration in nematodes M.Delattre and M-A. Félix

267 WASP and Disabled work in parallel to UNC-34, a C. elegans homolog of enabled Megan Dell, N. Hawkins, E. Kong, W. Forrester and G. Garriga

268 Expression of LIN-15a, a negative regulator of vulval induction John DeModena, Linda S. Huang and Paul W. Sternberg

269 Mitochondrial DNA mutation rate in Caenorhabditis elegans Dee R. Denver, Krystalynne Morris, Larissa Vassilieva, Michael Lynch and W. Kelley Thomas

270 Analysis of the C. elegans homolog of the FHIT tumor suppressor gene W. Brent Derry, Suzanne van Kreeveld and Joel H. Rothman

271 Characterization of two ras-signaling components in C. elegans Edward DesJardins, Daniel Sherbenou and Min Han

272 The MADS box containing factor CeMef-2 is not required for normal myogenesis and development Daryl Dichoso, Thomas Brodigan, Jin Sook Lee, Kyu Yeong Chwoe, Reymond Llacer, Steve Kostas, Andy Fire, Joo Hong Ahnn, and Michael Krause

273 Nicotine Adaptation is PKC Dependent Kari A. Dickinson

274 Genetic characterization and molecular cloning of the defecation gene aex-1 Motomichi Doi and Kouichi Iwasaki

275 Isolation and characterization of new class A synthetic multivulva genes John Doll, Ewa Davison and Bob Horvitz

276 Three RGS proteins appear to control G oa signaling in C. elegans Meng-Qiu Dong and Michael R. Koelle

277 Structure/function analysis of LIN-31, a winged-helix transcription factor involved in Caenorhabditis elegans vulval development David B. Doroquez, Heather Hess, Noelle Andrews, and Leilani M. Miller

278 Genetic and molecular characterization of sel-8, a suppressor of lin-12 gain-of-function mutants Timothy G. Doyle and Iva Greenwald

279 Function and expression of ceh-32, a sine oculis homologue. Christine Dozier, Giuseppe Cassata, Gisela Niklaus, Hiroshi Kagoshima, and Thomas R. Burglin.

280 Teneurin, a novel morhogenetic factor in C. elegans Krzysztof Drabikowski and Ruth Chiquet-Ehrismann

281 A Novel Suppresor of unc-93(e1500)-induced paralysis in C. elegans Ann Dude, Joseph Donohue, Jason Tennessen, and Elizabeth De Stasio

24 May 1999 15:50 17 17 Poster Session #1 Thursday, June 3, 2:00 - 5:00 p.m.

282 Chromogranin A Immunoreactivity in C. elegans Janet Duerr, Jennifer Gaskin, Lee Eiden, and Jim Rand

283 Development of Neurotransmitter Phenotypes in the VC Motor Neurons Janet Duerr, Jennifer Gaskin, Dennis Frisby, and Jim Rand

284 Analysis of p130, a component of the P/CAF complex, in C. elegans P. Dufourcq, Y. Nakatani, M. Labouesse and Y. Shi.

285 Genome-Wide RNAi-Based Screen to Identify Genes Required for Cell Division Processes in C. elegans. C. Echeverri, P. Gönczy, K. Oegema, S. Pichler, M. Kirkham, M. Brehm, A. Coulson, S. Jones, and A. Hyman

286 Embryonic pal-1 expression in the AB lineage is required for late gastrulation and rectal formation. Lois Edgar and Bill Wood

287 The Chromosome II Genetic Toolkit Mark L. Edgley, Carolyn A. Turner and Donald L. Riddle

288 Genetic Variants and Structural Characterization of the Chromosome II Inversion mIn1 Mark L. Edgley and Donald L. Riddle

289 Cell fusion as an alternative fate for surviving non-vulval Pn.p cells in Pristionchus pacificus. Andreas Eizinger and Ralf Sommer

290 mup-4, a Gene Required for Epidermal Morphogenesis and Muscle Position, is a Member of a Novel Family of Transmembrane Proteins. T. Elbl, L. Hong, J. Ward, C. Franzini-Armstrong, and E. A. Bucher

291 A Nematode Drug Screen For Presenilin Inhibitors In Alzheimer’s Disease BR Ellerbrock, EM Thomas, TG Geary, KA Greenlee, NC Stratman, M-J Miller, JR Brashler, GW Melchior, AE Buhl, DB Carter and ME Gurney

292 Integration of lineal, spatial, temporal and sexual coordinates by a C. elegans hox gene promoter Scott W. Emmons and Henrique B. Ferreira

293 or148 and or209: Two genes involved in cell division timing in early C.elegans embryogenesis Sandra E. Encalada, Danielle, R. Hamill, and Bruce Bowerman

294 UNC-13 and Synaptic Neurotransmission Rebecca Eustance, Janet Duerr, Ichi Maruyama, Hiroko Maruyama, Angie Duke, John McManus and Jim Rand

295 The sequence of the SL2 RNA spliced leader is not required for transcription or trans-splicing Donald Evans and Thomas Blumenthal

296 A biochemical search for factors that localize glp-1 translation in early development. Tom Evans and Simon Bellamy

297 We do it, bees do it, even educated fleas do it, (plants as well), but how do worms fight infections? Jonathan Ewbank, Dominique Ferrandon, Philippe Bulet and Jules Hoffmann

298 Analysis of polyglutamine-mediated cellular dysfunction in C. elegans. Peter Faber, Janet Alter, Jill Spoerke, Beth Westlund, Marcy MacDonald and Anne Hart

299 Molecular Characterization of mes-4 Youyi Fang, Susan Strome

300 Biochemical and Genetic Studies of the ETS-Domain Protein, LIN-1 Douglas A. Fantz and Kerry Kornfeld

24 May 1999 15:50 18 18 Poster Session #1 Thursday, June 3, 2:00 - 5:00 p.m.

301 Genetic and Molecular Analysis of sel-5 Hanna (Johnny) Fares and Iva Greenwald

302 Identification and Analysis of Vulval Morphogenetic Mutants David S. Fay and Min Han

303 vav Function in C. elegans Robert T. Fazzio, Mary C. Beckerle and Andres V. Maricq

304 Genetic and Phenotypic Analysis of spe-32, a Gene Required for Spermatogenesis with an Associated Larval Lethal Phenotype Frederick Fell, Daniel Halperin, Diane Downing, and Samuel Ward

305 cul-2 and the vhl Tumor Suppressor Gene: A genetic study Hui Feng, Gary Moulder, Robert Barstead, and Edward T. Kipreos

306 Functional Analysis of peb-1 during Pharyngeal Development Anthony Fernandez and Peter Okkema

307 Structural requirements for the trigger in RNA-mediated interference Andrew Fire, Jamie Fleenor, SiQun Xu, and Susan Parrish#

308 The Role of C. elegans lin-5 in Mitosis Ridgely Fisk, Marian Walhout, Marc Vidal, and Sander van den Heuvel

309 Evolution of Rhabditidae, a family for C. elegans D. Fitch, N. Eakin, K. Kiontke, C. Nguyen, J. Vanfleteren, A. Vierstraete, P. De Ley, R. De Wachter, M. Sutherlin, W. Sudhaus

310 Evidence for cospeciation between entomopathogenic nematode Heterorhabditis and its bacterial symbiont, Photorhabdus András Fodor, Emília Szállás and Byron J. Adams

311 A screen for mutants with defects in asymmetric cell division C. Andrew Frank, Nancy Hawkins and Gian Garriga

312 Evaluation of Excitation-Contraction Coupling in C. elegans Muscle. C. J. Franks, E. T. Claverol, C. Rogers, J. E. Chad, R. J. Walker and Lindy Holden-Dye.

313 C. elegans Inhibitor of Apoptosis Protein (IAP) homologue BIR-1 plays a conserved role in cytokinesis, not apoptosis Andrew Fraser, Claerwen James, Gerard Evan and Michael Hengartner

314 gon-4 encodes a novel protein required for timing of somatic gonadal cell divisions Lisa C. Friedman, Sonia Santa Anna-Arriola, Jonathan Hodgkin, Judith Kimble

315 Origin and evolution of microsatellites in C. elegans Linda Frisse, Larissa Vassilieva, Michael Lynch and W. Kelley Thomas

316 The APC-related gene apr-1 is required for morphogenesis of the embryo and generation of the vulval equivalence group Erika Froehli and Alex Hajnal

317 An analysis of the 26F4 gene which is expressed in the nuclei of seam cells and a subset of neurons Takashi Fujii, Go Shioi, Yukimasa Shibata, Hajime Fujisawa and Shin Takagi

318 Analysis of an RNA-binding protein ceHuD in C. elegans Masaki Fujita, Taizo Kawano, Hiroshi Sakamoto

319 A novel WD40 protein, CHE-2, acts cell-autonomously in the formation of sensory cilia. Manabi Fujiwara, Takeshi Ishihara, Isao Katsura

320 Watching the elt-2 GATA factor binding to its own promoter inside gut nuclei of developing C. elegans embryos Tetsunari Fukushige, Micheal J. Hendzel, David P. Bazett-Jones and James D. McGhee

24 May 1999 15:50 19 19 Poster Session #1 Thursday, June 3, 2:00 - 5:00 p.m.

321 The elt-2 Gene and The Regulatory Network Controlling C. elegans Gut Development. Tetsunari Fukushige and James D. McGhee

322 Embryonic roles of the cki-1 and cki-2 genes Masamitsu Fukuyama, Steve B. Gendreau, W. Brent Derry, and Joel H. Rothman

323 emb-30 Encodes a Novel Protein Required for Germline Proliferation and Completion of the Meiotic Divisions Tokiko Furuta, Bryan Koch, Roy Auty, Jay Kirchner, and David Greenstein

324 Sensory Neuron Polarity and Interneuron Function Maria E. Gallegos, Jennifer A. Zallen and Cori Bargmann.

325 Genetic and molecular analyses of ced-8, which controls the time of appearance of cell corpses Brendan Galvin and Bob Horvitz

326 vex-2 mutations perturb vulval fate execution at the first vulval-specific cell division David Garbe, Ranjana Kishore and Meera Sundaram

327 lin-42, a molecular link between circadian rhythms and developmental timing Heather Gardner, Mili Jeon, and Ann Rougvie

328 duf-1(zu316cs) - Dorsal UnFused Tamar Gattegno and Benjamin Podbilewicz

329 Cloning and characterization of C. briggsae and C. remanei homologs of fem-1 Jeb Gaudet and Andrew Spence

330 The sex determination protein FEM-3 accumulates in nuclei Jeb Gaudet and Andrew Spence

331 Two Distinct Importin a’s, IMA1 and IMA2, Differentially Regulate Germ Line Cell Fates Kenneth G. Geles and Stephen A. Adam

332 An enhanced gene disruption method is suitable for reverse genetics of Caenorhabditis elegans Keiko Gengyo-Ando and Shohei Mitani

333 Still Searching for components of the VAB-1 pathways Sean E. George and Andrew D. Chisholm

334 Phenotypic and molecular analysis of mig-8 B. Gerisch, C. Weitzel, A. Antebi

335 More Crossed Wires in unc-37 Ventral Cord Eileen A. German1, David M. Miller2, and David H. Hall1

336 The role of the GATA factor elt-3 in hypodermal development J.S. Gilleard and J.D.McGhee

337 The Nuclear Receptor Superfamily: Conservation of Sequence and Function Chris R. Gissendanner, Tim Lindblom, Lisa Hipsher, and Ann E. Sluder

338 nhr-25, the C. elegans ortholog of Ftz-F1, is required for hypodermal and somatic gonad development Chris R. Gissendanner and Ann E. Sluder

339 The netrin UNC-6 can redirect axons in vivo Zemer Gitai, Marc Tessier-Lavigne, and Cori Bargmann

340 let-21, a Homolog of the ect2 Proto-oncogene, is Necessary for Germline Development and Mitotic Cell Cycle Progression A. Godbey, P. Kuwabara, E. Lambie, R. Francis and T. Schedl

24 May 1999 15:50 20 20 Poster Session #1 Thursday, June 3, 2:00 - 5:00 p.m.

341 New Genes Involved in Ras-mediated Induction of Vulval Cell Fates Jessica Goldstein and Kerry Kornfeld

342 FMRFamide-related peptides and egg-laying Steve Golik, Laura Hardaker and William Schafer

343 zyg-8 controls anaphase spindle positioning in the one-cell stage embryo Pierre Gönczy, Titus Kaletta, Heinke Schnabel, Ralf Schnabel and Tony Hyman

344 Autonomy vs. non-autonomy of lin-15 action Aidyl S Gonzalez-Serricchio and Paul W. Sternberg

345 Ionic Currents in C. elegans Mechanosensory Neurons M. B. Goodman, S. R. Lockery, D. Lenzi and M. Chalfie

346 Structure/Function Analysis of the EGL-15 FGF Receptor S.J. Goodman and M.J. Stern

347 odr-7 and the AWA olfactory neurons G. Gopinathrao and P. Sengupta

348 Control of cell division axis choice in C.elegans Monica Gotta and Julie Ahringer

349 Can orthologous homeotic lin-39 genes from Pristionchus and Caenorhabditisfunctionally replace each other? Kaj Grandien and Ralf J. Sommer

350 Axonemal transport proteins in nematodes Grant, W.N., Dubowsky, A., Wisotzkey, R. and Johnson, C.D.

351 SEM-4 regulates vulval cell fate and expression of lin-39 hox Kelly Grant, Wendy Hanna-Rose and Min Han

352 Investigations of the Low Density Lipoprotein Receptor-Related Protein (LRP) in Caenorhabditis elegans Jennifer L. Grimsby and Martha E. Roemer

353 SMG-5 and SMG-7 Interact Directly and are Required to Regulate SMG-2 Phosphorylation Andrew Grimson, Michelle Page, Kirk Anders and Philip Anderson

354 Properties of RNAi inheritance in C. elegans Alla Grishok, Hiroaki Tabara, Tae Ho Shin and Craig C. Mello

355 Localization pathways of the glutamate-gated ion channels NMR-1 and GBR-2 Maria E. Grunwald, Christopher Rongo, and Joshua M. Kaplan

356 Simulation of the Embryogenesis of C. elegans in a Computer Model: Necessity of Active Cell Movements to Establish the Premorphogenetic Regions Markus Gumbel, Ralf Schnabel, and Hans-Peter Meinzer

357 Further studies of programmed germ cell death Tina Gumienny, Eric Lambie, and Michael Hengartner

358 A putative nuclear factor of the inductive signaling pathway specifying vulval cell fates in C. elegans Bhagwati P Gupta, Jing Liu, Paul W Sternberg

359 Quantitative Trait Loci for Chemotaxis in C. elegans : Repulsion Mutants Marjorie C. Gurganus and Patrick C. Phillips

360 Screening Transacting Factors for RNA-duplex Mediated Temporal Down-regulation of LIN-14 Protein. Ilho Ha and Gary Ruvkun

24 May 1999 15:50 21 21 Poster Session #1 Thursday, June 3, 2:00 - 5:00 p.m.

361 tra-2 regulation in Caenorhabditis remanei, a gonochoristic worm Eric S. Haag and Judith Kimble

362 Characterization of an SMC-like protein in C. elegans K. Hagstrom, D. Pasqualone, M. Albrecht, J. Lieb, and B. J. Meyer

363 Regulation of cell-cell associations in the C. elegans male tail hypodermis Andrew C. Hahn and Scott W. Emmons

364 Go May Regulate Behavior by Antagonizing Gq Yvonne M. Hajdu-Cronin, Wen J. Chen and Paul W. Sternberg

365 ric-3, a gene required for acetylcholine receptor function Sarah Halevi, Jim McKay, Millet Treinin

366 Ultrastructural Features of the Adult Hermaphrodite Gonad: Relations Between the Germ Line and Soma David H. Hall, Virginia P. Winfrey, Gareth Blaeuer, Loren H. Hoffman, Tokiko Furuta, Kimberly L. Rose, Oliver Hobert, David Greenstein

367 ram mutations in C. elegans cause hypertrophy of structural and hypodermal cells in the male sensory rays Hall, David H., Nguyen, Can Q., Yu, Raymond Y.L. and Chow, King L.

368 Bridging the GAP: New Insights into SYD-1 Structure and Function Steven Hallam and Yishi Jin

369 Epitope-based functional analysis on the ryanodine receptor of Caenorhabditis elegans Tomoyo Hamada, Yasuji Sakube and Hiroaki Kagawa

370 Characterization of temperature-sensitive embryonic-lethal mutants with early cell cycle defects Danielle R. Hamill, Thimo Kurz, and Bruce A. Bowerman

371 UNC-70 is ß-Spectrin Marc Hammarlund and Erik M. Jorgensen

372 cog-4/egl-26 encodes a novel protein involved in vulva morphogenesis Wendy Hanna-Rose and Min Han

373 Genes involved in axonal branching of an amphid neuron Joe C. Hao, Marc Tessier-Lavigne, and Cornelia I. Bargmann

374 in vitro analysis of C. elegans synaptobrevin in the synaptic vesicles docking to the plasma membrane Shin-ichi Harada, Toshihiro Sassa, and Ryuji Hosono

375 Role of the Caenorhabditis elegans homologs of cdk5 and p35 Thomas Harbaugh and Gian Garriga

376 Genome project Data in the EMBL Nucleotide Sequence Database. Diane Harbison, Peter Sterk and Guenter Stoesser

377 C. elegans Centaurins: Components of a PI 3-Kinase Signalling pathway? Laura S Harrington, Howard A Baylis, Trevor R Jackson

378 Genetic interaction of the VAB-1 receptor tyrosine kinase with the PTP-1 receptor tyrosine phosphatase Robert J. Harrington, Michael Gutch, Michael Hengartner, Nicholas Tonks, Inessa Grinberg, and Andrew Chisholm

379 Mutations in unc-26, the C. elegans synaptojanin homolog, disrupt synaptic vesicle recycling and result in defects in cytoskeletal organization. Todd W.C. Harris, Erika Hartwieg, H.R. Horvitz, and Erik M. Jorgensen

380 Oxygen Mutagenesis in C. elegans Phil Hartman, Becky Ponder, Hiroshi Kasaiand Nao Ishii

24 May 1999 15:50 22 22 Poster Session #1 Thursday, June 3, 2:00 - 5:00 p.m.

381 Developmental switches in the life-cycle of the parasitic nematode Strongyloides ratti. S.C. Harvey and M.E. Viney

382 Role of cysteine protease(s) in C. elegans embryogenesis/development Sarwar Hashmi and Sara Lustigman

383 Does the leucine zipper protein ZIP-1 function as an activator of gene expression in pharyngeal muscle? Christina Haun, Jack D. Thatcher, and Peter G. Okkema

384 exc-7 Encodes a Homologue to the RNA-Processing Protein ELAV Dana Hawkinson, Kevin V. King, and Matthew Buechner

385 DIE-1, an apparent zinc-finger protein, is required for intercalation and actin organization in the dorsal hypodermis Paul J. Heid, Bill Raich, Steven B. Gendreau, Joel H. Rothman, and Jeff Hardin

386 Forkhead genes in C.elegans Marika Hellqvist Greberg and David Baillie

387 Initiation of epithelial polarity during C. elegans intestinal morphogenesis Greg Hermann, Benjamin Leung and James Priess

388 Contributions of degenerative cell death and a WRN-related helicase to C.elegans aging Laura A. Herndon, Keli Xu and Monica Driscoll

389 n3194, identified in a screen for suppressors of ced-9(n1950), may define an ion channel required for cell viability Brad Hersh, Erika Hartwieg, and Bob Horvitz

390 The control of cell polarity and cell migration in C. elegans by WNT signaling and a protein similar to a metastasis-associated factor Susan M. Hettenbach and Michael A. Herman

391 POP-1 expression in mirror symmetric lineages. Russell J. Hill, Rueyling Lin, James R. Priess.

392 Avoidance of water soluble repellents Massimo A. Hilliardand Paolo Bazzicalupo

393 Systematic RNAi experiments with maternal genes. Keiko Hirono, Shuichi Onami and Yuji Kohara

394 Novel functions of the Ras-MAPK signal in neurons of C. elegans Takaaki Hirotsu, Satoshi Saeki and Yuichi Iino

395 Ventral enclosure in C. elegans: getting to the molecules Jeffrey S. Simske, Rebecca D. Hirsch, Deborah T. Locke, Ryan D. Smith and Jeff Hardin

396 Effect of overexpressing mab-21, isolation and identification of mab-21 interacting cellular components in C. elegans by epitope tagging Ho, S. Hong, Wong, Y. Ming and Chow, King L.

397 ceh-10 regulates ttx-3: An initiation-maintenance switch paradigm Oliver Hobert, Zeynep Altun-Gultekin and Gary Ruvkun

398 ttx-3 affects neurite branching and neural connectivity Oliver Hobert, Gary Ruvkun and David H. Hall

399 The neural function of lim-6, a new LIM homeobox genes, suggests a common theme in LIM homeobox gene function Oliver Hobert, Kristin Tessmar and Gary Ruvkun

400 A new bacterial pathogen of C.elegans, and the isolation of resistant mutants Jonathan Hodgkin

24 May 1999 15:50 23 23 Poster Session #2 Friday, June 4, 2:00 - 5:00 p.m.

401 Genetic mapping and genetic nomenclature: the CGC subcontract Jonathan Hodgkin, Richard Durbin, Sylvia Martinelli

402 A CLR example of redundancy: cdh-3 and enc-1 share a redundant function in excretory duct morphogenesis Lisa Hodgson and Jonathan Pettitt

403 Toward Understanding Cell-Type-Specific Cell Death Regulation Daniel J. Hoeppner and Michael O. Hengartner

404 A functional analysis of the role of UNC-11, a nematode AP180 homolog, in synaptic vesicles endocytosis. Andrea Holgado and Aixa Alfonso.

405 The life-extension, oxidative stress resistance and antioxidant enzyme gene expression Yoko Honda and Shuji Honda

406 Isolation and Characterization of Genes Responsible for Ethanol Sensitivity in Caenorhabditis elegans Min Gi Hong and Junho Lee

407 Inositol monophosphatase is required for AWC-mediated chemotaxis to volatile odorants Lee Honigberg, Marie Sutherlin and Carl D. Johnson

408 A region of the myosin rod critical for interaction with paramyosin Pamela E. Hoppe and Robert H. Waterston

409 Negative regulation of egfr signalling in C. elegans. Neil A. Hopper, Chieh Chang, Giovanni M. Lesa and Paul W. Sternberg.

Poster Session #2 Friday, June 4, 2:00 - 5:00 p.m. Abstracts 410 - 681 Hornsten - Portereiko

410 The C. elegans Amyloid Protein Precursor-Related Gene Has An Essential Function Angela Hornsten and Chris Li

411 Central role of UNC-13 and UNC-18 in synaptic vesicle docking Ryuji Hosono, Shin-ichi Harada, James B.Rand, Ichiro N.Maruyama and Toshihiro Sassa

412 him-10 encodes a novel protein involved chromosome segregation Mary Howe, D. G. Albertson, and B. J. Meyer

413 Myotactin, a novel adhesion molecule involved in muscle-hypodermal signaling. Michelle Hresko, Lawrence Schriefer, Paresh Shrimankar and Robert Waterston.

414 Structure-function analysis of LIN-14 Marta Hristova and Victor Ambros

415 Context-dependent gene silencing in the soma of C. elegans Jenny Hsieh, Steve Kostas, and Andrew Fire.

416 Each C. elegans Laminin a Subunit Mediates Distinct Aspects of Morphogenesis C. Huang, P. Yurchenco, and W. Wadsworth

417 A Screen for Mutations that Affect C. elegans Aging Cheng Huang and Kerry Kornfeld

418 Identification of proteins that may interact with MEX-3 to regulate pal-1 translation Nancy Huang, Marian Walhout, Marc Vidal, and Craig Hunter

24 May 1999 15:50 24 24 Poster Session #2 Friday, June 4, 2:00 - 5:00 p.m.

419 SYD-3, a giant protein with a GEF domain, functions in synapse formation Xun Huang, Mei Zhen, and Yishi Jin

420 A Novel Protein, UNC-79, Controls Anesthetic Sensitivity. J. Humphrey, M. Sedensky, P. Morgan

421 Suppressors of syntaxin reduction-of-function regulate general anesthetic sensitivity Stephen Hunt, Bruno van Swinderen, Christine Liu, Ammar Hawasli, and C. Michael Crowder

422 Neuromodulation in C. elegans and unc-34 suppressors Melissa Hunter-Ensor, Bob Horvitz

423 Finding the role of PAR-4 in embryonic asymmetry Daryl Hurd, Diane Morton and Kenneth Kemphues

424 Supramolecular Organization of Coiled-Coil Proteins By Novel Coupling Proteins Alex H. Hutagalung, Feizhou Liu, Christopher C. Bauer, Irving Ortiz and Henry F. Epstein

425 Genes required for axonal fasciculation/target recognition Harald Hutter and Edward M. Hedgecock

426 Inactivation of a VHA-2 gene in Caenorhabditis elegans with the dsRNAi technique Oliver Hüttmann and Einhard Schierenberg

427 Regulation of the C. elegans epidermal growth factor homolog LIN-3 Byung J. Hwang, Nadeem Moghal, Koen Verbrugghe, Jing Liu, and Paul W. Sternberg

428 Nucleotide-sugar biosynthesis and glycosylation are involved in vulval morphogenesis Ho-Yon Hwang and Bob Horvitz

429 Characterization of suppressors of daf-1, daf-8 and daf-14. Takao Inoue and James H. Thomas

430 Analyses of the Interaction of Two Sensory Signals on the Behavior under Well-fed and Starved Conditions Takeshi Ishihara and Isao Katsura

431 An update on the analysis of the ttx-2 gene involved in thermotaxis Kazuyoshi Ishii, Ikue Mori and Yasumi Ohshima

432 Oxidative Stress and Aging in C.elegans: What lessons learned from mev-1? Nao Ishii and Phil Hartman

433 Toward four-dimensional database of gene expression in C. elegans Masahiro Ito, Tomoko Motohashi, Yohei Minakuchi and Yuji Kohara

434 Cloning and characterization of dinhr-6, a putative homolog of the Drosophila ecdysone response gene E75 , in a parasitic nematode. Megan C. Jackson, Claude V. Maina and Kirsten Crossgrove

435 A method for high resolution mapping relative to single nucleotide polymorphisms used to positionally clone cdf-1, a gene involved in vulval development Janelle Jakubowski and Kerry Kornfeld

436 Characterisation of a Neurotransmitter Transporter Gene (T25B6.7) in C.elegans Chela James, David Coates and R.Elwyn Isaac

437 Molecular analysis of daf-9, a gene controlling C. elegans larval development Kailiang Jia, Patrice S. Albert, Ian M. Caldicott and Donald L. Riddle

438 An essential role for aha-1, a PAS-domain-containing regulatory gene in C. elegans Huaqi Jiang, Rong Guo, and Jo Anne Powell-Coffman

439 Mutations in sof-1 and sof-2 can suppress fog-1(q253ts) Suk-Won Jin and Ronald E. Ellis

24 May 1999 15:50 25 25 Poster Session #2 Friday, June 4, 2:00 - 5:00 p.m.

440 A diet of double-stranded RNA allows simple male/female genetics Suk-Won Jin, Pei-Jiun Chen, Daniel Salinsky and Ronald E. Ellis

441 Marker Rescue of Srf-2 Wendy A. Jobling and Samuel M. Politz

442 Genetic Amenability of Chromosome I left Robert C. Johnsen, Steven J. M. Jones and Ann M. Rose

443 Automated sorting and dispensing of C. elegans to wells of microtiter plates Carl D. Johnson, Ralph Clover, Brian Reardon, Petra B. Krauledat, Anthony A. Ferrante and W. Peter Hansen

444 unc-110, egl-23 and a newly recognized unc-93 family Duncan B. Johnstone, Maya Kunkel, Larry Salkoff and James H. Thomas

445 pvl-5 is involved in the early development of the vulva in C. elegans Pradeep Joshi and David M. Eisenmann

446 Regulatory sequence for body wall muscle specific expression of Caenorhabditis elegans Hiroaki Kagawa, Hiroaki Ohta and Tetsuya Bando

447 The LIM homeobox gene ceh-14 is required for sensory neuron differentiation H. Kagoshima, G. Cassata, T.R. Bürglin

448 A screen for new genes involved in osm-9 signaling Amanda H. Kahn, David Tobin, and Cori Bargmann

449 Anteroposterior Axon Guidance Genes Behnam Kahoussi, Catherine Chiu, Timothy Smith and Scott Clark

450 The cytokinesis defective mutant cyk-3 makes a contractile ring, but it fails to ingress Susi Kaitna, Pierre Gönczy, Titus Kaletta, Heinke Schnabel, Ralf Schnabel, Tony Hyman, and Michael Glotzer

451 Different expression patterns and activities of the three forms of PHA-4 John Kalb and Jim McGhee

452 tlp-1 encodes a variant TATA-binding protein that is essential for embryogenesis Linda S. Kaltenbach, Mary Ellen Domeier and Susan E. Mango

453 Expression of Dominant Negative Forms of Laminin b: Evidence for a Functional Role for Polymerized Laminin in Basement Membranes. Gautam Kao, Xingcong Ren and William Wadsworth.

454 Characterization of Laminin beta and gamma Genes. Gautam Kao, Cheng-chen Huang and William Wadsworth

455 fzr-1, a homologue of fizzy-related/HCT1, appears to be required for cell division control in various tissues Takeshi Karashima, Mariko Sawa, Asako Sugimoto and Masayuki Yamamoto

456 PLC210, a conserved Ras/Rap1A-associating phospholipase C is required for fertility in C. elegans Ken-ichi Kariya, Chang-Deng Hu, Mitsushige Shibatohge, Yanhong Liao, Chunhua Song, Xianlong Gao and Tohru Kataoka

457 Investigation into the lin-12 mediated feedback loops regulating the AC/VU decision Xantha Karp and Iva Greenwald

458 The EGL-3 prohormone convertase modulates ASH-interneuron synapses Jamie Kass, Peter Kim, and Joshua Kaplan

459 C. elegans homologue of Sra-1, a target of Rac GTPase, is involved in hypodermal morphogenesis Katsuhisa Kasuya, Hiroshi Qadota, Martha Soto, Craig Mello, Kozo Kaibuchi

24 May 1999 15:50 26 26 Poster Session #2 Friday, June 4, 2:00 - 5:00 p.m.

460 Immune defense of C.elegans: abf operon, ASABF type antimicrobial peptide genes Yusuke Kato

461 Structure of Neural Network of C. elegans Observed by a Random Walker K. Kawamura, S. Morita, K. Oshio, Y. Funabashi, Y. Osana and K. Oka

462 A new mutant which affects longitudinal and circumferential migrations of distal tip cells Takehiro Kawano and Joseph G. Culotti

463 Analysis of SR protein genes in C. elegans Taizo Kawano, Masaki Fujita and Hiroshi Sakamoto

464 CDC-42 is Required for Polarity in the Early Embryo Amanda Kay and Craig P. Hunter

465 Do Impaired Mitochondria of the Nervous System Cause Hypersensitivity to Volatile Anesthetics in C.elegans? E.-B. Kayser, P. Morgan, M. Sedensky

466 Innexins play a role in modulating the pharyngeal pumping rate of dauer larvae. John Keane and Leon Avery

467 Two of the five isoforms of protein synthesis initiation factor eIF4E, which distinguish between mono- and trimethylated mRNA caps, are essential for viability in C. elegans Brett D. Keiper, Barry J. Lamphear, Eric J. Aamodt, Marzena Jankowska, Atul Deshpande, Thomas Blumenthal and Robert E. Rhoads

468 A class VI myosin has a role in fertility Joseph Kelleher, Gary Moulder, Robert Barstead and Margaret Titus

469 A search for genes that interact with unc-31 or act in the same pathway M. Liakot Ali Khan and Kouichi Iwasaki

470 OSM-3 Subfamily of the Kinesin Motors in C.elegans Shams T. Khan, M.Yusuf Ali and Shahid S. Siddiqui

471 Genetic analysis of lj22, lj21 and lj10 genes involved in nicotine adaptation in C. elegans Jinah Kim and William Schafer

472 Function of flp neuropeptide gene family Kyuhyung Kim and Chris Li

473 alt-1, a Gene Required for Patterning Longitudinal Axon Tracts at the Midlines Seonhee Kim and William G. Wadsworth

474 A Telomere Binding Protein in the Nematode C. elegans. SeungHyun Kim and Junho Lee

475 cDNA cloning and functional analysis of the C. elegans dna topoisomerase IIIa gene You-Chan Kim and Hyeon-Sook Koo

476 An approach toward imaging activities of the neural network Kotaro Kimura, Kunihiro Matsumoto and Ikue Mori

477 Ca 2+ /CaM-dependent protein kinases (CaM-K)-mediated signal cascade is conserved in C. elegans Yoshishige Kimura, Ko Eto, Masa-aki Muramatsu and Hiroshi Tokumitsu

478 How are guidance and motility coupled in cells and axons? Rachel Kindt and Cynthia Kenyon

479 Sensory axon guidance defects in C. elegans Susan Kirch, Gage Crump and Cori Bargmann

24 May 1999 15:50 27 27 Poster Session #2 Friday, June 4, 2:00 - 5:00 p.m.

480 Elucidating the roles of GLH-1 and GLH-2, two constitutive P-granule components, in germline development Jay Kirchner, Kathleen Kuznicki, Pliny Smith, Karen Bennett, and Susan Strome

481 Mutant Myosin Expression in Transgenic Lines of Caenorhabditis elegans Antonis Kirmizis, Sara Olson, Anne Peregrine and Elizabeth De Stasio

482 Searching for Regulators of Cell Fate Specification Martha Kirouac and Paul Sternberg

483 KSR-1::GFP expression, and effects of ksr-1 on LIN-12::GFP Ranjana Kishore and Meera Sundaram

484 Difference in habituations induced by mechanical stimuli in Caenorhabditis elegans Kei-ichiro Kitamura, Shigetoyo Amano, Ryuji Hosono

485 Genetic analysis of C. elegans elongation using sma-1 suppressors Sarah Kniss and Judith Austin

486 Growth cone migration in C. elegans Karla M. Knobel, David Warner, Erik M. Jorgensen, Michael J. Bastiani

487 Comparative genomics applications of a comprehensive, non-redundant protein sequence registry Robert G. Knowlton and Jeffrey S. Aaronson

488 Synthesis of glycoprotein is important for molting and male tail sensory ray morphogenesis in Caenorhabditis elegans Ko, Frankie C.F. and Chow, King L.

489 T19E10.1 protein, a putative RhoGEF related to the mouse oncoprotein Ect2, is involved in versatile developmental processes. Yuki Kodama, Asako Sugimoto, Joel Rothman, and Masayuki Yamamoto

490 Site-Specific Recombination Tools in C. elegans Kelly Komachi, Dmitry Blinder, Owen Hughes

491 Toxicity Testing in C. elegans Kelly Komachi, Maria Galenko, Dmitry Blinder, Owen Hughes

492 The role of jam-1 during C. elegans embryogenesis Mathias Köppen, Gary Moulder, Jeff Simske, Anthony Radice, Robert Barstead and Jeff Hardin

493 A genetic screen for genes involved in gut development and differentiation Jay D. Kormish and James D. McGhee

494 Approaches to Identify Mutants of the C. elegans wee-1.3 Gene Mary E. Kosinski and Andy Golden

495 Postembryonic Muscle Patterning Steve Kostas, Andy Fire

496 Genetic dissection of molting pathway Marta Kostrouchova, Michael Krause, Zdenek Kostrouch and Joseph E. Rall

497 Characterization of mutants that mis-traffic synaptic vesicle proteins in touch neurons Sandhya P. Koushika, Anneliese M. Schaefer and Michael L. Nonet

498 Core 2 N-acetylglucosaminyltransferase Homologues in C. elegans: Preliminary Characterization. Aldis Krizus, Charles E Warren, Emily Partridge and James W Dennis

499 Structure-Function Analysis of UNC-73 Terry Kubiseski, Rob Steven, Mike Rosen, Joe Culotti, and Tony Pawson.

24 May 1999 15:50 28 28 Poster Session #2 Friday, June 4, 2:00 - 5:00 p.m.

500 How is ceh-22 expression regulated in pharyngeal muscle? Craig A. Kuchenthal, Wei Chen, Tomas Vilimas, Peter G. Okkema

501 The cloning and characterization of smg-3, a gene required for mrna surveillance in C. elegans Sherry Kuchma, Brian Cali, and Phil Anderson

502 Overexpression of a Novel Gene Causes Meiotic Nondisjunction Lynnette M. Kuervers, Diana L. Janke, and David L. Baillie

503 Mutations in a two-P domain potassium channel cause uncoordinated movement in c. elegans. M. T. Kunkel, D. B. Johnstone, M. L. Nonet, J. H. Thomas, L. Salkoff

504 VAB-17, a novel WD-40 protein involved in morphogenesis Patricia E. Kuwabara, Richard Benton, and Jonathan Hodgkin

505 The C. elegans ptc genes play essential, distinct and apparently Hh-independent developmental roles Patricia E. Kuwabara

506 Unwinding the P granules: evidence from RNAi and glh mutants Kathleen A. Kuznicki, Jay W. Kirchner, Pliny A. Smith, and Karen L. Bennett

507 Transcriptional Mediators in the Nematode Caenorhabditis elegans JaeYoung Kwon, JinMo Park, ByungSoo Gim, YoungJoon Kim and Junho Lee

508 Characterization of a C. elegans homologue of band 4.1 protein Soonjae Kwon, Sunki Jung, Joohong Ahnn

509 Processing of ROP-1, degradation of CeY RNA and formation of dauer larvae. Jean-Claude Labbé, Luis A. Rokeach and Siegfried Hekimi

510 A fate map of organs and tissues at the early gastrula stage Michel Labouesse and Renaud Legouis

511 C. elegans dynamin-related protein drp-1 controls severing of the mitochondrial outer membrane Arnaud M. Labrousse, Mauro D. Zappaterra, Daniel A. Rube and Alexander M. van der Bliek

512 Negative regulation of LET-23 mediated signaling Christopher J. Lacenere and Paul W. Sternberg

513 Luminescent Caenorhabditis elegans: a novel eukaryotic biosensor Cristina Lagido, Jonathan Pettitt and L. Anne Glover

514 Dominant Spermatogenesis-defective mutants in C. elegans Todd Lamitina and S.W. L’Hernault, Ph.D.

515 PKL is targeted to F-actin via a PDZ domain M Land and C.S. Rubin

516 Identification of a gene required for pharyngeal elongation and deleted by mnDf90. Sarah Lange and Susan E. Mango

517 Developmental determination of AWA and AWB chemosensory neurons Anne Lanjuin, Tali Melkman, Piali Sengupta

518 Sodium Azide Induces Thermotolerance In C. elegans By A Mechanism Similar To The Heat Shock Response Michelle R. Massie, Kristy D. Boggs, Karen E. Stine, Glenn E. White

519 Altered Response Of Dauer Mutants To Sodium Azide Exposure And The Acquisition Of Thermotolerance. Elizabeth M. Lapoczka, Karen E. Stine, Glenn E. White

24 May 1999 15:50 29 29 Poster Session #2 Friday, June 4, 2:00 - 5:00 p.m.

520 Evolutionary & ecological aspects of aging in C. elegans Steve Le Comber, Linda Partridge and David Gems

521 Programmed cell death in the ventral epidermis of Pristionchus pacificus Kwang-Zin Lee and Ralf Sommer

522 Integrin signaling in C. elegans Myeongwoo Lee and Jean Schwarzbauer

523 Expressional control of the C. elegans dna topoisomerase i gene Myon Hee Lee and Hyeon-Sook Koo

524 Extension of C. elegans lifespan by a mutation in daf-21/hsp90 Raymond Lee and Gary Ruvkun

525 Genes that are involved in controlling morphogenesis of the embryo. Renaud Legouis, Julia M. Bosher, David L.Baillie, Ann Rose and Michel Labouesse.

526 Organizational Cues for Intestinal Morphogenesis in C. elegans Benjamin Leung, Greg Hermann and James R. Priess

527 mig-5, a dsh Homologue, Controls Cell Migration and Cell Fate Determination in C. elegans Na Li, Chaobo Guo and Edward Hedgecock

528 Involvement of mag-1, a homolog of Drosophila posterior group gene mago nashi, in hermaphrodite germ-line sex determination Weiqing Li, Robert Boswell and Bill Wood

529 Molecular characterization of the novel cadmium-inducible gene, cdr-1 Vivian Hsiu-Chuan Liao and Jonathan H. Freedman

530 Regulation of vesicular proteins by UNC-4 and UNC-37 Kim Lickteig, Janet Duerr, Dennis Frisby, Jim Rand, and David Miller.

531 UNC-3 regulates cholinergic motor neuron differentiation. Kim Lickteig, Dennis Frisby, Janet Duerr, David Miller and Jim Rand

532 Molecular Studies on a Putative Potassium Channel Gene, F08B12.3 Found in Caenorhabditis elegans Genome Hyun-Ho Lim, Chul-Seung Park and Joohong Ahnn

533 Domain C of netrin UNC-6 is Required to Inhibit the Circumferential Migration of Longitudinal Motoneuron Axons in the Ventral Cord Yoo-Shick Lim, Smita Mallapur, Gautam Kao, Xing-Cong Ren, and William G. Wadsworth

534 Specification of the Command Interneurons: A Genetic Approach to Identify Genes Required for Cell Fate Determination Chingju Lin, P. J. Brockie, S. Poulson and Andres V. Maricq

535 isolation OF ADDITIONAL DAF-2 SUPPRESSORS Kui Lin and Cynthia Kenyon

536 Acid Sphingomyelinase Regulates RAS Signaling Functions Xinhua Lin, Michael Hengartner and Richard Kolesnick

537 Isolation of the pat-6and pat-12genes Xinyi Lin and Benjamin D. Williams

538 nhr-8 is Required for Gut Function and May Respond to the Food Signal Tim H. Lindblom and Ann E. Sluder

539 Progress of cloning spe-10 Wesley C. Lindsey and Steven W. L’Hernault

24 May 1999 15:50 30 30 Poster Session #2 Friday, June 4, 2:00 - 5:00 p.m.

540 Genetic analysis of beta amyloid toxicity in transgenic C. elegans Chris Link, Alice Smith, Amy Fluet, and Carolyn J. Johnson

541 C. elegans immunity? A reverse genetic analysis of components of the Toll signaling pathway Elizabeth Malone Link, Laura Nelson, Gary Hardiman, Jonathan Hodgkin, and Leo X. Liu

542 Patterning of dopaminergic identity in C. elegans ray neurons by a DBL-1 signaling pathway and a Hox gene Robyn Lints and Scott W. Emmons

543 Specification of Neuronal Connectivity in the C. elegans Male Tail Jonathan Lipton and Scott W. Emmons

544 Phylogenetic analysis of vertebrate and invertebrate Delta/Serrate/LAG-2 (DSL) James L. Lissemore and William T. Starmer

545 Hox factors and other components in patterning of embryonic and postembryonic myogenic lineages Jun Liu and Andrew Z. Fire

546 CDK-Activating Kinase (CAK): A genomic search Ji Liu and Edward T. Kipreos

547 Molecular, functional and behavioral characterization of unc-11 mutant alleles. Kesheng Liu, Eileen B. Landies, Ami Kurani, Andrea Holgado, Ravit Golan and Aixa Alfonso

548 The C. elegans Engulfment Protein CED-6 Might be Conserved Across Species Qiong A. Liu and Michael O. Hengartner

549 Identification and Analysis of Proximal Proliferation (Pro) Mutants Te-Wen Lo and E. Jane Albert Hubbard

550 Continuing characterization of serotonergic marker genes and serotonin-deficient mutants Curtis M. Loer and G. Patrick Merritt

551 Using RNA interference to screen for new factors involved in the control of glp-1 translation and early embryonic polarity Alex L. Lublin, Scott A. Barbee, Thomas C. Evans

552 TRA-1 is a Phosphoprotein and Interacts with FEM-2, a Protein Type 2C Phosphatase David H. Lum, David Zarkower and Andrew Spence

553 The Cytoplasmic Domain of TRA-2 Localizes to the Nucleus and Interacts with TRA-1 David H. Lum, Patricia Kuwabara, David Zarkower and Andrew Spence

554 Ethanol Tolerance in C. elegans Kirsten Lundin and Patrick C. Phillips

555 UNC-115 and AXM-1 Define a New Axon Guidance Signal Transduction Pathway Erik Lundquist and Cori Bargmann

556 C. elegans homologues of MuSK and rapsyn interact to control motor neuron function Charles Ma, Guy Caldwell, Lucinda Carnell, and Martin Chalfie

557 PAT-4 is a homologue of integrin-linked kinase and is required for assembly of the myofilament lattice in body-wall muscle cells A. Craig Mackinnon and Benjamin D. Williams

558 Mutations Affecting Meiotic Pairing and Synapsis MacQueen, A. and Villeneuve, A.

559 Electrophysiologic analysis of neuromuscular transmission Jon Madison and Joshua Kaplan

24 May 1999 15:50 31 31 Poster Session #2 Friday, June 4, 2:00 - 5:00 p.m.

560 A Zyxin-like Molecule in C. elegans David M. Madsen, R. T. Fazzio, M. C. Beckerle, and A. V. Maricq

561 Genetic hierarchies and GATA factor specificity in ems development Morris F. Maduro and Joel H. Rothman

562 An RNAi screen for embryonic patterning genes Morris F. Maduro, Jan Sumerel and Joel H. Rothman

563 lon-1, a putative downstream target of TGFb signaling Lisa L. Maduzia, Pradnya Shetgiri, Srikant Krishna, Cathy Savage-Dunn, Huang Wang and Richard W. Padgett

564 Isolation and characterization of novel TGFb-like components Lisa L. Maduzia, Huang Wang, Stephen Cohen, Cathy Savage-Dunn and Richard W. Padgett

565 RNAi Screening with a non-redundant cDNA set Ikuma Maeda, Yuji Kohara, Masayuki Yamamoto and Asako Sugimoto

566 A Role for fem-2 During Embryonic Elongation Paul E. Mains

567 Characterization of the role of rho-1 and nuclear migration during P cell migration Christian J. Malone, Satoshi Orita and Min Han

568 Analysis of glutamate transporter knockouts in the worm Itzhak Mano and Monica Driscoll

569 The unc-13 Puzzles in Neurotransmission Ichi Maruyama

570 Revisiting Ryanodine Resistance in Caenorhabditis elegans Ed Maryon, LuAnne Sowinski, and Phil Anderson

571 C. elegans unc-119 mutants define novel neurite development defects Wayne Materi, Morris Maduro and Dave Pilgrim

572 Regulation of the expression of mab-21: a candidate Hox target gene Rie Matsui, Daihachiro Tomotsune and Naoki Takahashi

573 Progress on the cloning of spd-2, a gene involved in centrosome function and the establishment of embryonic polarity Kara N. Maxwell, Kevin F. O’Connell, and John G. White

574 Development of molecular markers that migrate across synapses in the C. elegans nervous system Steven McCarroll and Cori Bargmann

575 Searching for Targets of pag-3 Joan McDermott and Eric Aamodt

576 Constitutively activated integrin during muscle development KEVIN A. McDONALD, JOEL Z. SCHWARZ, and BENJAMIN D. WILLIAMS

577 Whole Genome Analysis of C. elegans Using DNA Microarrays J. McDowall, S. Jones, T. Freeman, A. Coulson, P. Kuwabara

578 LIN-13, a zinc-finger protein that acts in vulval development Alicia Melendez and Iva Greenwald

579 Electrophysiological Analysis of C. elegans Ionotropic Glutamate Receptors Jerry E. Mellem, Y. Zheng, P. J. Brockie, and A. V. Maricq

580 him-5 and him-8 Philip Meneely, Toni Amato, Alex Ensminger, Maria Robinson and Marnie Gelbart

24 May 1999 15:50 32 32 Poster Session #2 Friday, June 4, 2:00 - 5:00 p.m.

581 Interference during Meiosis on the X Chromosome and an Autosome Philip Meneely, Tate Kauffman, Kathryn Strain, Laura Lehnhoff and Anna Farago

582 An Astounding Variety of Neuropeptides Lynn A. Messinger and Antony O. W. Stretton

583 che-14encodes a distant member of the PATCHED receptor family required for differentiation of ectodermal epithelial cells G. Michaux, S. Sookhareea, G. Belliard, C. Hindelang and M. Labouesse

584 Specifying cell fates in the C lineage Katie M. Mickey and James R. Priess

585 Characterization of a C. elegans orthologue of the human SMN gene mutated in spinal muscular atrophy I. Miguel-Aliaga, E. Culetto, D. S. Walker, H.A. Baylis, D.B. Sattelle and K.E. Davies

586 Observing Basement Membrane Structure and Dynamics in C.elegans with epi-1:GFP Nicholas Miliaras and Ed Hedgecock

587 Two-color GFP expression in C. elegans David Miller, Neil Desai, Delia Hardin, David Piston, George Patterson, Jamie Fleenor, SiQun Xu and Andrew Fire

588 GOA-1 (Goa) and RIC-8 interact to Regulate Synaptic Transmission in Adults as well as Centrosome and Nuclear Positioning in Early Embryogenesis Ken Miller and Jim Rand

589 ETR-1, an elav-type RNA-binding protein with homology to human CUG-bp, is essential for muscle development in C. elegans. Catherine A. Milne and Jonathan Hodgkin

590 A screen for genes that control programmed cell death in the germ line Stuart Milstein, Anton Gartner, and Michael Hengartner

591 spf-1 is required for DTC specification and SGP formation during gonadogenesis Jennifer Miskowski, Yongjing Li, Ron Ellis and Judith Kimble

592 Members of a divergent nuclear hormone receptor family are expressed in multiple cell types in C. elegans T. Miyabayashi, M. T. Palfreyman1, F. Slack and P. Sengupta

593 sdf-13/Ce-tbx-2 mutants have abnormality in odorant-specific adaptation. Kohji Miyahara, Norio Suzuki, Takeshi Ishihara, Isao Katsura

594 Conservation of the C. elegans Dpy-20 transcription regulator in Drosophila. Azham S. Mohammed, M. Yusuf Ali, and Shahid S. Siddiqui

595 Hypodermal cell fusions are not required for gross morphogenesis in C. elegans William A. Mohler and John G. White

596 Genetic analysis of cultivation temperature avoidance induced by starvation Akiko Mohri, Mizuho Koike, Ikue Mori

597 Identification of upstream regulatory elements and transcription factors responsible for cell-specific expression of the C. elegans metallothionein genes Lori H. Moilanen, Tetsunari Fukushige, and Jonathan H. Freedman

598 Comparison of gene expression in C. elegans and C. briggsae Laurent Molin and Ian A. Hope

599 Molecular and functional diversity of the nicotinic acetylcholine receptor gene family of C. elegans N.P. Mongan, E. Culetto, N. Gower, V. Raymond and D.B. Sattelle

24 May 1999 15:50 33 33 Poster Session #2 Friday, June 4, 2:00 - 5:00 p.m.

600 Genetic study of G as -coupled signal transduction in C. elegans Celine Moorman, Hendrik C. Korswagen, Alexander M. van der Linden and Ronald H.A. Plasterk

601 The C. elegans Spectrin Based Membrane Skeleton S. Moorthy, L. Chen and V. Bennett

602 Identification of cis- and trans- acting factors controlling the translation of maternal pal-1 mRNA Darcy Mootz and Craig P. Hunter

603 Search of in vivo targets of Hox gene products Toshiharu Morimitsu, Ken Murayama, Rie Matsui and Naoki Takahashi

604 CeBRAM-1A and CeBRAM-2B, new signal mediators of TGF-b signaling pathways in the C. elegans Kiyokazu Morita, Miho Shimizu, Satoru Yoshida, Makoto Mochii, Hiroshi Shibuya and Naoto Ueno

605 The Stressed Worm Project James F Morley, Sanjeev Satyal, Josh Segal, Richard I. Morimoto

606 Quantitative Complementation Testing Reveals Variation in Chemotaxis Caused by the odr-1 Locus in Natural Isolates of C. elegans. Juliet Morphew and Patrick C. Phillips

607 Time-series analysis of pirouettes in C. elegans chemotaxis. T.M. Morse, J.T. Pierce, S.E. Owens, M.L. Moravec, A.B. Bates, T.C. Ferree, and S.R. Lockery.

608 The heterochronic gene lin-46 encodes a non-essential member of the moeA family and may be a target of lin-28 regulation Eric G. Moss, Rosalind C. Lee, Jill E. Johnstone, Denise Au Yeung, and Victor Ambros

609 Systematic analysis of mRNA distribution by whole mount in situ hybridization Tomoko Motohashi, Tokie Ohba, Ikuko Sugiura, Masumi Obara, Sayuri Kitayama, Takami Suzuki Tadasu Shin-i and Yuji Kohara

610 The Unexpected Phenotype of Alpha-actinin Nulls Gary Moulder, Janet Duerr, Steve Fields and Robert Barstead

611 Expression Pattern Data For Genes Predicted In The C. elegans Genome. Andrew Mounsey, Petra Bauer, David McCarroll, and Ian A. Hope

612 Isolating genes asociated with longevity Manuel J. Munoz and Donald L. Riddle

613 Temporal expression of collagen genes in Caenorhabditis elegans during development. Joaquin M. Muriel and Iain L. Johnstone

614 Characterization of the Shank, a novel PSD-family protein, in C. elegans. Yoonseong Na and Joohong Ahnn

615 Isolation of novel body size mutants of C.elegans Yoshiya Nakano, Yasumi Ohshima

616 An analysis of C.elegans plexin genes Fumi Nakao, Takashi Fujii, Fumikazu Suto, Hajime Fujisawa, and Shin Takagi

617 Functional analysis of rapsyn in AchR clustering SeungHee Nam and Junho Lee

618 SPE-12 may function as a plasma membrane signal amplifier inducing spermatids to become spermatozoa Jeremy Nance, Elizabeth Davis, and Samuel Ward

619 Pharmacology and Regulation of the C. elegans Dopamine Transporter Richard Nass, Janet Duerr, Jim Rand, David M. Miller, and Randy D. Blakely

24 May 1999 15:50 34 34 Poster Session #2 Friday, June 4, 2:00 - 5:00 p.m.

620 Analysis of the mode of bar-1 function in vulval cell fate specification Lakshmi Natarajan and David M. Eisenmann

621 Identification and Analysis of Novel C. elegans Neuropeptide Genes Arif Nathoo, Rachael Moeller and Anne Hart

622 Interaction between PIE-1 and RCK/p54, a conserved RNA helicase Rosa E. Navarro, Eun Yong Shim, Amy Walker, Yang Shi and Keith Blackwell

623 Evidence for the involvement of END-2, a member of the steroid hormone receptor family, in endoderm and mesoderm formation. Erin Newman-Smith and Joel H. Rothman

624 The unc-43 Ca 2+ /Calmodulin-dependent kinase II (CaMKII) is a critical neuronal regulator in C. elegans Elizabeth M. Newton and James H. Thomas

625 Diplogasteroides berwigi: A model for the further phylogenetic analysis of nematode vulval evolution Hanh Nguyen, Benno Jungblut and Ralf Sommer

626 New mutants defective in postembryonic morphogenesis of the male tail tip Can Q. Nguyen, Ying Yang, Tania Del Rio and David Fitch

627 unc-61Locus Encodes for C. elegans Septin That Plays a Critical Role in Post-embryonic Cytokinesis Tri Q. Nguyen, Hitoshi Sawa and John G.White

628 Serotonin and Feeding Timothy Niacaris and Leon Avery

629 Electrophysiological Recording from Identified C. elegans Chemosensory Neurons W. T. Nickell, R. Y. K. Pun, C. I. Bargmann and S. J. Kleene

630 An interspecies comparison of lin-25 Lars Nilsson, Teresa Tiensuu and Simon Tuck

631 Expression of carbohydrate antigens in the nematode Caenorhabditis elegans Kazuya Nomura, Kazuko H. Nomura Souhei Mizuguchi, Shinichi Ichikawa Yoshio Hirabayashi

632 Effectors of the RAB-3 synaptic vesicle-associated small GTP protein M. Nonet, J. Staunton, G. Hadwiger, L. Wei and B. Ganetzky

633 RNAi: how low can you go? Jeff Norman, Kelly Galey, Mary K. Montgomery

634 Analysis of alpha spectrin function during muscle development in C. elegans. K.R. Norman and D.G. Moerman

635 Habituation modulates the interaction of multiple response mechanisms mediating olfactory chemotaxis William M. Nuttley, Singh Harbinder and Derek van der Kooy

636 A genetic mosaic screen for mutations affecting the P 1 lineage Josefin Nyström, Helen Stewart, Lars Nilsson, David Baillie and Simon Tuck

637 The zyg-1 Gene Encodes a Serine/Threonine Kinase Required for Bipolar Spindle Formation Kevin O’Connell, Cathy Caron, Kenneth Kemphues, and John White

638 Role of Astral Microtubules in the Establishment of Anterior-Posterior Polarity and Pronuclear Migration Kevin O’Connell, Kara Maxwell, and John White

639 The Representation of C.elegans Genome Sequencing Data in SWISS-PROT Claire O’Donovan

24 May 1999 15:50 35 35 Poster Session #2 Friday, June 4, 2:00 - 5:00 p.m.

640 Analysis of Essential Genes in the sDf125 Region of Chromosome III Nigel J. O’Neil, Douglas Chan, Greg P. Vatcher, Lynnette M. Kuervers, David L. Baillie

641 The PTEN homologue DAF-18 acts in the DAF-2 insulin receptor-like metabolic signaling pathway Scott Ogg and Gary Ruvkun

642 KEL-1, a homologue of Drosophila Kelch, is essential for larval development M. Ohmachi, A. Sugimoto, Y. Iino and M. Yamamoto

643 flr-2 gene, which controls the growth rate of flr-1, flr-3 and flr-4 mutants, encodes a putative TGF-b antagonist. Akane Oishi, Masaya Take-uchi, Takeshi Ishihara, Isao Katsura

644 Isolation of new thermotaxis-defective mutations using the interaction with dafmutations in TGF-b-like signaling pathway Yoshifumi Okochi, Masatoshi Okumura, Ikue Mori

645 Chemosensory Control of Surface Antigen Switching Douglas Olsen, Laura Miceli, and Samuel M. Politz

646 Developmental regulation and mechanism of action of the lin-4 translational repressor RNA. Phil Olsen, Rhonda Feinbaum, Fang Liu, H. Scott Silverman, Eric Moss, Victor Ambros

647 Habituation of C. elegans for the touch sensitivity K. Omata, F. Yonezawa and K. Kawamura

648 Characterization of the neural circuit of C. elegans: model analysis of the touch response K. Omata, F. Yonezawa and K. Kawamura

649 A screen for mutations that affect the localization of CED-4 Daniel Omura, Brad Hersh, Fangli Chen, and Bob Horvitz

650 Protein Expression Pattern Analysis of Maternal mRNAs. Shuichi Onami, Tamami Nagaoka and Yuji Kohara

651 Genetic and biochemical studies on modulators of unc-60 in body wall muscle S. Ono, D. L. Baillie, K. B. Mercer, and G. M. Benian

652 Behavioral, Structural and Genetic Analyses of unc-jd19: A Gene Affecting the Development of the DD Motoneurons. Kavita Oommen and Bill Walthall

653 Neural Network Model for Touch Sensitivity in Caenorhabditis elegans Y. Osana, K. Oshio, S. Morita, Y. Funabashi, K. Oka, M. Hagiwara and K. Kawamura

654 Analysis of neuronal connectivity of C. elegans using random walker K. Oshio, Y. Funabashi, S. Morita, Y. Osana, K. Oka and K. Kawamura

655 lin-18, a gene necessary for vulval cell orientation, encodes a Ryk-like predicted transmembrane tyrosine kinase Helieh S. Oz, Michael L. Goodson, C. Jeffery Goodwin, Shahla Gharib, Paul W. Sternberg and Wendy S. Katz

656 A Muv connection to early embryogenesis Barbara D. Page, Dave Waring and James R. Priess

657 Characterization of bac-1 (big anchor cell) Robert E. Palmer, Keith B. Brown, Bhagwati P. Gupta, Paul W. Sternberg

658 Phylogenetic analysis of entomopathogenic nematode / bacterium symbiotic complexes by using PhastSystem PAGE PCR-RFLP of rDNA spacer sequences Horolma Pamjav, Dimitra Triga, Emília Szállás, András Fodor, Buzás Zsuzsanna

24 May 1999 15:50 36 36 Poster Session #2 Friday, June 4, 2:00 - 5:00 p.m.

659 4D Imaging System to Study C. elegans Development Andrew Papp and Michael Palopoli

660 Characterization of Calreticulin (Crt-1), a calcium binding protein, in Caenorhabditis elegans Byung-Jae Park, Duk-Gyu Lee, and Joohong Ahnn

661 Studies of C. elegans HIP1 Alex Parker and Ann Rose

662 Investigating the fates of trigger RNAs and target transcripts in RNA-mediated interference Susan Parrish and Andrew Fire

663 Egls: a new growth control pathway? Mavji N. Patel and Armand M. Leroi

664 Two conserved domains of the EGL-10 RGS protein cooperate to inhibit G protein signaling in the C. elegans nervous system Georgia A. Patikoglou, Paul B. Sigler and Michael R. Koelle

665 ImmunoEM Localization of YP170::GFP Reveals Yolk Transport Marie-Christine Paupard, Agness Miller, and David H. Hall

666 SUP-9 and SUP-18 may be components of a K+ channel involved in the regulation of muscle contraction Ignacio Perez de la Cruz, Stephen C. Cannon, and Bob Horvitz

667 Assays for interactions between HER-1 and TRA-2 Marc D. Perry, Roxana N. Sultan, and Nimerta Rajwans

668 Identification of cis-acting elements and trans-acting factors responsible for her-1 regulation Marc D. Perry, Michael K. Garroni, and Nimerta Rajwans

669 Isolation of RNAi resistant mutants from C. elegans Andrei Petcherski and Judith Kimble

670 Influence of temperature on reproductive rate in C. elegans: r versus R o Patrick C. Phillips, Barbara L. Armstrong, Christina P. Coucke, and Ray B. Huey

671 RNAi screening for polarity genes Fabio Piano, Aaron Schetter, Ken Kemphues

672 ooc-3, a novel gene required for rotation of the centrosome-nucleus complex in P 1 Silke Pichler, Pierre Gönczy, Heinke Schnabel, Ralf Schnabel and Tony Hyman

673 The role of Rho-dependent kinase (LET-502) and myosin phosphatase (MEL-11) in embryonic elongation A.J. Piekny, A.K. Wissmann and P.E. Mains

674 The chemosensory neuron ASER regulates the initiation but not the execution of pirouettes in C. elegans chemotaxis. J. T. Pierce-Shimomura, M. R. Gaston and S. R. Lockery.

675 C32E8.7, a C.elegans homolog of the diabetes autoantigen ICA69 Marc Pilon, Andrew M. Spence and Hans-Michael Dosch

676 A JAVA Based 4D Lineage Analysis Application Jason N. Pitt, Russell J. Hill, Benjamin Leung, James R. Priess

677 Cold sensitive fusion: kinetic dissection of cell fusion during morphogenesis in N2 and duf-1 Benjamin Podbilewicz, Michael Glickman, Yoed Rabin and Tamar Gattegno

24 May 1999 15:50 37 37 Poster Session #3 Saturday, June 5, 5:00 - 8:00 p.m.

678 In Search of Phenotypes for ptl-1 Paula Polk, Eric Aamodt, Stephanie Aamodt

679 Families of C. elegans tyrosine kinase receptors (RTK): evidence for continuing evolution of metazoan multigene families Cornel Popovici, Régine Roubin, François Coulier, Pierre Pontarotti and Daniel Birnbaum

680 C. elegans genes encoding Tyrosine Kinase Receptors share structural features with mammalian Vascular Endothelial Growth Factor Receptors (VEGFRs) Cornel Popovici, Marie-Josée Santoni, Daniel Birnbaum and Régine Roubin

681 Pharyngeal Morphogenesis Michael F. Portereiko, Mary Ellen Domeier and Susan E. Mango

Poster Session #3 Saturday, June 5, 5:00 - 8:00 p.m. Abstracts 682 - 952 Portman - Zmasek

682 The bHLH proteins LIN-32 and HLH-2 function together in multiple aspects of ray development in C. elegans Douglas S. Portman and Scott W. Emmons

683 Function of the Receptor Tyrosine Kinase cam-1/kin-8. Susan M. Poulson, D. M. Madsen, C. S. Walker and A. V. Maricq

684 X Chromosome Counting Mechanisms in C. elegans Jennifer R. Powell, Kevin Amonlirdviman, and Barbara J. Meyer

685 Identification and characterization of kinesins required for early embryogenesis James Powers, Debra Rose, Olaf Bossinger, Susan Strome, William Saxton

686 SMA-1 is an actin-binding protein required for epithelial cell morphogenesis Vida Praitis and Judith Austin

687 The unc-3 locus encodes an O/E transcription factor, CeO/E, which is required for axonal guidance and chemosensory function. Brinda C. Prasad, Randall R. Reed

688 The C. elegans sex determination gene mog-6 encodes a cyclophilin Paolo Pugnale and Alessandro Puoti

689 unc-53 expression pattern and phenotype Nathalie Pujol and Thierry Bogaert

690 Axonal pathfinding defects in CePhox2/ceh-17 mutants Nathalie Pujol, Jonathan Ewbank, Rémi Terranova, Jean-François Brunet

691 CUL-2 is required for mitotic chromatin condensation George Punkosdy, Hui Feng, Weiwei Zhong, and Edward T. Kipreos

692 DEAH-box proteins and sex determination in C. elegans Alessandro Puoti and Judith Kimble

693 cDNA libraries enriched in clones representing rare mRNAs Anatoliy T. Puzyrev and Donald L. Riddle

694 Functional analysis of C. elegans PKN homologue Hiroshi Qadota, Takayuki Miyauchi, Katsuhisa Kasuya, Kozo Kaibuchi

695 Promoter analysis of the lin-26 gene. Sophie Quintin, Alex Davies, Bart G.W.den Boer and Michel Labouesse

24 May 1999 15:50 38 38 Poster Session #3 Saturday, June 5, 5:00 - 8:00 p.m.

696 Analysis of C05C12.3 and F54D1.5, two genes for gon-2-like putative cation channels Cassandra D Ragnauth and Howard A Baylis

697 Characterization of unc-23 and its suppressors Poupak Rahmani and Donald Moerman

698 UNC-1 and UNC-8 interact to control anesthetic sensitivity in C. elegans S. Rajaram, M. Sedensky and P. Morgan.

699 The RdgC Homolog CePPEF: A Protein Phosphatase Found in Interneurons and the Cilia of Several Sensory Neurons. Pradeep Ramulu and Jeremy Nathans

700 Behavioral Analyses of nmr-1: Does the Mutation Affect Learning in the Worm? C. H. Rankin, E. Phillip, J. Wells, P.J. Brockie, D.M. Madsen, and A.V. Maricq.

701 Characterisation of C.elegans C-terminal kinesin KLP-3 Rao Susmitha G and Manjari Mazumdar

702 Identification of EGL-27 Molecular and Genetic Interactions Thomas M. Ratliff and Michael A. Herman

703 Actions of cholinergic anthelmintics and ivermectin on recombinant homomeric nicotinic acetylcholine receptors chicken a7 and C. elegans ACR-16 V. Raymond, N. P. Mongan and D.B. Sattelle

704 The engulfment of dying cells contributes to the killing process of programmed cell death Peter Reddien, Scott Cameron, and Bob Horvitz

705 Does mab-19 encode an hepatocyte growth factor receptor-related gene? David J. Reiner and Barbara J. Meyer

706 Gene Expression Profiles in the Germline Using Microarrays Valerie Reinke, J. Wang, C.B. Van Doren, R.R. Begley, and Stuart Kim

707 Effects of Heat Shock on Early Embryogenesis and Gut Development K. Resendes and D.C. Shakes

708 Characterization of Ets Transcription Factors in C. elegans Roddie Reventar, Adam Hart, Lijia Zhang, Joseph Culotti and Alan Bernstein

709 Ce-hcr-51 gene, the RAD51/DMC1 homolog in Caenorhabditis elegans: a functional characterization Cinzia Rinaldo, Paolo Bazzicalupo, Sara Ederle, Massimo Hilliard, Marinella Mastrogiacomo, and Adriana La Volpe

710 Isolation and Identification of Suppressors of an Activated Ca ++ /Calmodulin-Dependent Protein Kinase II Homolog in C. elegans Merrilee Robatzek and James H. Thomas

711 The C. ELEGANS and Yeast Proteome Databases: An integrated knowledge system. K. J. Roberg-Perez, B. P. Davis, M. E. Cusick, P. E. Hodges, M. C. Costanzo, A. H. Z. McKee, W. E. Payne, and J. I. Garrels.

712 Genetic analysis of genes involved in hypodermal function in Caenorhabditis elegans Brett Roberts, Caroline Clucas and Iain Johnstone

713 Searching for POP-1 target genes by single-embryo subtractive hybridization Scott Robertson, Melanie Reuben and Rueyling Lin

714 Towards the cloning of two genes involved in sex myoblast migration Matthew K. Robinson and Michael J. Stern

715 UNC-86: The POUer of Interaction S. Roehrig, I. Roeckelein, R. Donhauser, R. Baumeister

24 May 1999 15:50 39 39 Poster Session #3 Saturday, June 5, 5:00 - 8:00 p.m.

716 New mutations involved in growth cone steering of the excretory cell Ingele Roelens, Richard Feichtinger, Isabelle Maillet and Thierry Bogaert

717 The unc-112 and dim-1 genes encode novel proteins required for myofilament lattice assembly and stability Teresa Rogalski, Danelle Devenport, Mary Gilbert, Greg Mullen and Don Moerman

718 Localisation and function of the FMRF-amide-like neuropeptides in C.elegans . C. Rogers, C.J. Franks, R.J. Walker, J.F. Burke and L. Holden-Dye.

719 Feeding For Phenotypes Jim Rogers and E. Jane Albert Hubbard

720 The Caenorhabditis elegans Vps34 Class of PI 3-kinase Lorenz Roggo, Monique Zetka, Vincent Bernard, Ann Rose, Fritz Müller, Matthias P. Wymann

721 Functional Organization of the Nuclear Envelope in C. elegans Tom Rolef Ben-Shahar, Jun Liu, Dieter Reimer, Millet Treinin, Perah Spann, Merav Cohen, Andrew Fire, Klaus Weber and Yosef Gruenbaum

722 UNC-4 and UNC-37 repress VB-specifying genes to define VA traits Jennifer Ross, Eileen German, David Hall, and David Miller

723 Regulation of dauer larva development in Caenorhabditis elegans by daf-18, a homologue of the tumour suppressor PTEN Jean Pierre Rouault, Patricia E. Kuwabara, Olga M. Sinilnikova, Laurent Duret, Danielle Thierry-Mieg, Marc Billaud

724 Cloning and characterization of aex-2 and aex-4, two genes required for defecation Elaine K. Round and James H. Thomas

725 Extra distal tip cells in cki-1(RNAi) animals do not result from duplication of existing distal tip cells. Richard Roy, Eric Lambie and Victor Ambros

726 New Alleles of mec-4 Dewey Royal, Mary Anne Royal, Shubha Narayan, and Monica Driscoll

727 New Investigations of the Phosphatidylinositol Signaling Pathway Dewey Royal, Mary Anne Royal, Nicole Sirotin, and Monica Driscoll

728 Probing the functions of c. elegans dyn-1 binding proteins Daniel A. Rube, Arnaud M. Labrousse, Katie Kong and Alexander M. van der Bliek

729 Evolution of glp-1 and lin-12 genes in Caenorhabditis species David Rudel, Lisa Friedman and Judith Kimble

730 Identification of new loci required for sensitivity to specific odorants R. Rutherford, K. Roayaie and C.I. Bargmann

731 The role of the STAR family in tra-2 3’UTR translational regulation Lisa Saccomanno and Elizabeth B. Goodwin

732 Anucleate C. elegans sperm can crawl, fertilize oocytes, and direct anterior-posterior polarization of the 1-cell embryo. Penny L. Sadler and Diane C. Shakes

733 Getting to anaphase: emb-27 is required for chromosome segregation in the germline of C. elegans P.L. Sadler and D.C. Shakes

734 Another form of associative learning in chemotactic behavior of C. elegans Satoshi Saeki, Masayuki Yamamoto and Yuichi Iino

735 Identifying synaptic regulators by screening for suppressors of a syntaxin hypomorph Owais Saifee and Michael L. Nonet

24 May 1999 15:50 40 40 Poster Session #3 Saturday, June 5, 5:00 - 8:00 p.m.

736 Novel Screens for Regulators of G1 Phase of the Cell-cycle in C. elegans R. Mako Saito, Dayalan Srinivasan and Sander van den Heuvel

737 Progress in cloning mua-6: gene required for post embryonic muscle attachment Kamlai Saiyasisongkhram, Wuraola Omotosho, Samit Patel, Nathan Penning and John Plenefisch

738 Identification and Characterization of Proteins Associated with The Ryanodine Receptor in Caenorhabditis elegans Yasuji Sakube and Hiroaki Kagawa

739 EHS-1, the C.elegans homologue of mammalian Eps15, acts with DYN-1 in synapic vesicle recycling. Anna Elisabetta Salcini, Massimo Antonio Hilliard, Pier Paolo Di Fiore and Paolo Bazzicalupo

740 Investigating A Role For Cholinergic Signaling In The Dauer Formation Pathway Gisela Sandoval and Gary Ruvkun

741 A mut-6 screen for RNAi deficient mutants Madathia Sarkissian, Hiroaki Tabara and Craig C. Mello

742 Differential expression of CeCRMP/DHP-1 and CeCRMP/DHP-2, novel members of CRMP/DRP/DHP/UNC-33 family in C. elegans. Sasaki, Y., Takemoto, T., Kimura, H., Nonaka, M., and Goshima, Y.

743 Cellular and molecular analysis of thermotaxis-defective mutants Hiroyuki Sasakura, Atsushi Kuhara, Hidetoshi Komatsu, Ikue Mori

744 A novel C. elegans gene encodes a nicotinic acetylcholine receptor a subunit is capable of forming a functional hetero-oligomeric receptor in vitro when co-expressed with lev-1 and unc-29 D. B. Sattelle, E. Culetto, N. P. Mongan, K. Matsuda, J. T. Fleming, M. D. Squire, J. Lewis and H. A. Baylis

745 The AFD Thermosensory Neurons: Specification of Cell Fate and Function John S. Satterlee and Piali Sengupta

746 Chaperones to the rescue of misfolded (proteins in) worms? Sanjeev Satyal, Enrico Schmidt, Kazunori Kitagawa, Neal Sondheimer, James M. Kramer, Susan Lindquist and Richard I. Morimoto

747 Protein fold assignment and functional analysis of the C. elegans genome J. Michael Sauder and Roland L. Dunbrack, Jr.

748 Mutations affecting asymmetric T cell division Hitoishi Sawa, Hiroko Kouike and Hideyuki Okano

749 Mutations that Alter Interneuronal Synaptogenesis Anneliese M. Schaefer and Michael L. Nonet

750 Investigating the role of P granules in germ cell development Jennifer Schisa, Jason Pitt, and James Priess

751 unc-120 encodes a SRF transcription factor involved in muscle development Lawrence Schriefer, Ian Caldicott and Robert Waterston

752 mex-5 and ah6.5 act redundantly to establish cell fates in the early embryo Charlotte Schubert, Rueyling Lin, James Priess

753 The Great Divide: Analysis of single-cell cytokinesis-defective mutants in C. elegans Jill Schumacher, Matt Wallenfang, Geraldine Seydoux, and Andy Golden

754 A screen for mutants defective in the specification of programmed cell death in the postdeirid lineage Hillel Schwartz and Bob Horvitz

755 Classical and reverse thermosensory genetics in C. elegans Erich M. Schwarz and Martin Chalfie

24 May 1999 15:50 41 41 Poster Session #3 Saturday, June 5, 5:00 - 8:00 p.m.

756 Studies of PAR-2 localization by ectopic expression in somatic cells Lynn Boyd and P. Schweinsberg

757 Towards a genome-wide collection of transposon insertions L. Ségalat, C. Bessou, F. Schurr, O. Hauser, T. Alvarez, S. Sookhareea, R. Legouis, M. Labouesse

758 Translational regulation of the heterochronic gene lin-28 by the lin-4 RNA and a lin-14-dependent positive feedback loop Kathy Seggerson, Jill E. Johnstone and Eric G. Moss

759 The role of Cyt-1 in oxdative stress-induced mitochondrial dysfunction and apoptosis in C.elegans Nanami Senoo-Matsuda, Shinichi Yoshimura, Ryoko Muto, Akira Akatsuka, Phil Hartman, and Nao Ishii

760 Cytokinesis in C. elegans embryos: analysis of cyk-1 function Aaron F. Severson, J. Clayton Carter, Danielle R. Hamill, Dave L. Baillie and Bruce A. Bowerman

761 Genetic Analysis of Signaling Pathways that Determine the Deaths of Sex-Specific Neurons Manisha Shah, Jo Jo Nyugen, and Ding Xue

762 The Cloning of laf-1: A gene involved in the translational regulation of tra-2 Sejal Shah, Laura E. Graves, Elizabeth B. Goodwin

763 Identification of novel C. elegans caspases, their regulators and targets Shai Shaham, Ira Herskowitz, and Cori Bargmann

764 cip-1, a C. elegans gene that can activate the programmed cell death pathway Shai Shaham and Cori Bargmann

765 Regulation of Body Size by TGF-beta-like Signaling in C. elegans Ayesha N. Shajahan and Scott Everet Baird

766 Progress towards cloning smg-6 Hongyu Shang and Phil Anderson

767 Studies of the mechanisms and possible roles of LIN-12 post-transcriptional downregulation during VPC specification. Daniel Shaye and Iva Greenwald

768 The Nonmuscle Myosin Regulatory Light Chain Gene mlc-4 is Required for Cytokinesis, Anterior-Posterior Polarity, and Body Morphology during C. elegans Embryogenesis Christopher A. Shelton, J. Clayton Carter, and Bruce Bowerman

769 Genetic characterisation of lon-4 gene Zai Zhong Shen, Mavji N. Patel and Armand M. Leroi

770 Global analysis of ABC transporters in C. elegans Jonathan A. Sheps, Ming Yeung Chau, Victor Ling and David L. Baillie

771 Involvement of cog-1 in uterine-vulval attachment David R. Sherwood, Robert A. Palmer and Paul W. Sternberg

772 Possible involvement of octopamine in the hyperactivity caused by feeding defects Yukimasa Shibata, Takashi Fujii, Hajime Fujisawa and Shin Takagi

773 Molecular Interaction of Myotonic Dystrophy Protein Kinase: The Structure and Function of DMPK Family Members in C. elegans. Miho Shimizu and Henry F. Epstein

774 Characterization of Flectin -like protein in C.elegans Jiyeon Shin, Sunja Kim, Jungsu Lee, Joohong Ahnn

775 Zinc-finger and KH domain Proteins in C. elegans Embryogenesis Tae Ho Shin and Craig Mello

24 May 1999 15:50 42 42 Poster Session #3 Saturday, June 5, 5:00 - 8:00 p.m.

776 NEXTDB: The nematode expression pattern map database. Tadasu Shin-i and Yuji Kohara

777 A Screen for Mutants with Abnormal Neural Morphology Go Shioi, Michinari Shoji, Masashi Nakamura, Takeshi Ishihara, Isao Katsura, Hajime Fujisawa and Shin Takagi

778 C.elegans Kinesin Superfamily: Subfamilies and Surprises Shahid S. Siddiqui, M. Y. Ali, S. T. Khan, M. L.A. Khan, F. Hori, T. Harada, K.Nishikawa, K. Fukami-Kobayashi, and Z. K. Siddiqui.

779 A PP2A regulatory subunit positively regulates Ras-mediated signaling during C. elegans vulval induction Derek S. Sieburth, Meera Sundaram and Min Han

780 The gonad as fertile ground for genetic studies of organogenesis Kellee R. Siegfried, Finn-Hugo Markussen, Laura D. Mathies, Lisa C. Friedman, Peggy Kroll-Conner and Judith Kimble

781 Analysis of the ventral enclosure mutant ct254 Kristin Simokat, Jeffrey Simske, Lois Edgar, William Wood, and Jeff Hardin

782 VAB-9 is a junction associated, putative membrane-spanning protein required for coordinated epithelial morphogenesis Jeffrey S. Simske and Jeff Hardin

783 A screen for extragenic suppressors of spe-9 Andrew Singson and Steve L’Hernault

784 EGO-1, required for germline development, is related to plant rna-directed rna polymerases Anne Smardon, Jill Spoerke, Steven Stacey, Nancy Mackin, and Eleanor Maine

785 Genome-wide analysis of C. elegans sperm development Harold Smith, Elizabeth Davis, Jeremy Nance, and Samuel Ward

786 When humans lead the way: uncovering the role of C27H5.1 Jessica Smith, Michelle Chambers, and Dave Pilgrim

787 Identification And Characterization Of Intrinsically Thermotolerant Substrains Of Caenorhabditis elegans Nicole D. Smith, Karen E. Stine, Glenn E. White

788 The GLHs: antibodies, protein biochemistry, and a few mutants. Pliny A. Smith, Kathleen A. Kuznicki, W. M. Alexandra Leung, Annette Estevez, and Karen L. Bennett.

789 Expression of the daf-12 gene Mark I. Snow and Pamela L. Larsen

790 Characterisation of TRA-3, a predicted calpain protease involved in sex determination Sharon B. Sokol, Richard Benton and Patricia E. Kuwabara

791 The C.elegans genes egl-27 and egr-1 are similar to MTA1, a member of a chromatin regulatory complex, and are redundantly required for embryonic patterning. Florence Solari, Alex Bateman, and Julie Ahringer

792 inx-1 is required for body morphogenesis and encodes C. elegans HEM-2, a putative Rac GTPase associated protein Martha Soto, Katsuhisa Kasuya, Hiroshi Qadota, Kozo Kaibuchi, Craig Mello

793 C. elegans Neurexin-1 Jürgen Soutschek, and Jochen Scheel

794 Characterization of smu-2, a Gene Implicated in RNA Splicing Angela Spartz, Robert Herman and Jocelyn Shaw

24 May 1999 15:50 43 43 Poster Session #3 Saturday, June 5, 5:00 - 8:00 p.m.

795 The inhibitor-of-apoptosis (IAP)-like protein BIR-1 is required for cytokinesis E.K. Speliotes, A. Uren, D. Vaux, and H.R. Horvitz

796 smu-1 Affects the Accumulation of Alternatively-Spliced Transcripts from the unc-52 Gene Caroline A. Spike, Robert K. Herman and Jocelyn E. Shaw

797 Creating a C. elegans cell line Dayalan Srinivasan and Sander van den Heuvel

798 Regulation of cell death vs. non-cell death in the ventral epidermis of Pristionchus pacificus. Jagan Srinivasan and Ralf Sommer

799 Characterisation of neprilysin (NEP) and endothelin-converting enzyme (ECE) from C. elegans Patrick Stancombe, Mohammed Sajid, David Coates and R. Elwyn Isaac

800 Analysis of downstream events in the pathway for programmed cell death Gillian Stanfield, Yi-Chun Wu, Erika Hartwieg and Bob Horvitz

801 Cloning and analysis of the cGMP-dependent protein kinase CGK-1 John Stansberry, Eric Baude, Ronald E. Ellis and Michael D. Uhler

802 Localization of Gap Junction Proteins Todd Starich, Agnes Miller, David Hall, and Jocelyn Shaw

803 The roles of unc-83, unc-84, and anc-1 in nuclear migration and anchorage Daniel A. Starr, Christian J. Malone, and Min Han

804 Identification of mutants affecting mitotic chromosome segregation in the embryo Jeffrey H. Stear, Brian J. Buchwitz, Landon L. Moore, and Mark B. Roth

805 Characterization of Muscarinic Acetylcholine Receptors in the Pharynx Kate Steger and Leon Avery

806 Regulation of the actin cytoskeleton: The identification of genes that interact with the Rac activator unc-73 Rob Steven, Joe Culotti and Tony Pawson

807 How does the pharyngeal primordium generate different pharyngeal cell types? Jeff Stevenson, Mary Ellen Domeier, Andrew Chisholm and Susan E. Mango

808 NHR transcription factors and cuticle synthesis in C.elegans Eric J Stewart, Iain Johnstone

809 Cilium Structure Genes osm-1, daf-10, che-11 and che-13 Steven Stone, Andrew Davies, Tracy Ricke and Jocelyn Shaw

810 The Caenorhabditis sex determining protein FEM-2 evolves faster than its paralogs Paul Stothard, Dave Hansen and Dave Pilgrim

811 Homologs of the C. elegans masculinizing gene her-1 in C. briggsae and the filarial parasite B. malayi in C. briggsae and the filarial parasite B. malayi Adrian Streit, Weiqing Li, Barbara Robertson, Jacqueline Schein, Ibrahim H. Kamal, Marco Marra, and Bill Wood

812 Beyond the ENDs: the network of genes regulating gut development Keith Strohmaier, Kyunghee Koh, Ian Hope, and Joel H. Rothman

813 nos-1 and nos-2, two genes related to Drosophila nanos, are required for primordial germ cell development. K. Subramaniam, T. Brodigan, M. Krause and G. Seydoux

24 May 1999 15:50 44 44 Poster Session #3 Saturday, June 5, 5:00 - 8:00 p.m.

814 osm-10, osm-16(rt6) and not(rt32): genes required for signal transduction in the ASH sensory circuit. Hana Sugimoto, Shinya Matsumoto, Rachael Moeller and Anne C. Hart

815 Possible role for a C. elegans homologue of protein phosphatase 4 in the replication and function of centrosomes Eisuke Sumiyoshi, Asako Sugimoto, and Masayuki Yamamoto

816 Towards Cloning of daf-5 Li Sun and Garth Patterson

817 A genetic screen for synthetic Vulvaless/Lethal mutations Meera Sundaram, Alissa Goldman, and Robyn Howard

818 Mediators and Modulators of dbl-1 Functions in Body Size Control Yo Suzuki, Gail Morris, and Bill Wood

819 or191ts and or182ts are Embryonic Lethal, Temperature-Sensitive Mutations that Disrupt Spindle Rotation and Cell Cycle Timing, Respectively, in Two-Cell Stage C. elegans Embryos. Kathryn A. Swan, Danielle R. Hamill and Bruce A. Bowerman

820 A C. elegans model of Niemann-Pick type C disease: two NPC1 homologs act redundantly in dauer formation Mary Sym, Michael Basson and Carl D. Johnson

821 The role of the ER membrane protein calnexin in programmed cell death Mary Sym, Michael Basson and Carl D. Johnson

822 The role of serotonin in C.elegans Ji Ying Sze, Martin Victor, Yang Shi, and Gary Ruvkun

823 Essential role of emb-1 and emb-8 in establishing early embryonic polarity Yo Tabuse and Johji Miwa

824 CeHSF and HSB-1: The heat shock response regulators Li-Jung Tai, Sanjeev Satyal, Sally McFall, Sue Fox, Catherine Airey, Tim Roytman, and Richard Morimoto

825 Pathway analysis of the daf-12 nuclear receptor receptor Danilo Tait, Andreas Ludewig, Adam Antebi Antebi;

826 Cloning, characterization and gene disruption of CeCRMP/DHP-1and -2 T. TAKEMOTO, H. KIMURA, Y. SASAKI, N. HAMAJIMA, Y. GOSHIMA, and M. NONAKA

827 Effects of UNC-2, a a-1 Calcium Channel Subunit, on Neuronal Migration and Development Tobey Tam and William Schafer

828 PGP and MRP homologs in C. elegans protect the nematode from Pseudomonas aeruginosa pathogenicity Man-Wah Tan, Annegien Broeks, Frederick M. Ausubel and Ronald H.A. Plasterk

829 A C. elegans SEK-1 MAP kinase kinase regulates egg-laying and male turning behaviors. Miho Tanaka-Hino, Masato Kawasaki, Kiyoji Nishiwaki, Naoki Hisamoto, and Kunihiro Matsumoto

830 Knockout of Elongation Factor-2 Kinase Extends Lifespan in C. elegans Nektarios Tavernarakis, Charmaine E. Mendola, Karen S. Pavur, Lijia Zhang, Zeynep Altun-Gultekin, William Wadsworth, Monica Driscoll, and Alexey G. Ryazanov.

831 Cytoskeleton dynamics affect the function of membrane channels and interfere with mec-4(d)-induced cell death Nektarios Tavernarakis, Shi Liang Wang, and Monica Driscoll

832 Distinct cytoplasmic and nuclear functions for the germline factor PIE-1? T. Tenenhaus, M. Dunn, M. Wallenfang, K. Subramaniam and G. Seydoux.

24 May 1999 15:50 45 45 Poster Session #3 Saturday, June 5, 5:00 - 8:00 p.m.

833 Functional analysis on the troponin C of the pat-10 mutant of Caenorhabditis elegans Hiromi Terami and Hiroaki Kagawa

834 Abstract withdrawn

835 Comparative genomic analysis: the structure of a complex gene, bli-4 Colin Thacker, Marco A. Marra, Alana Jones, David L. Baillie, and Ann M. Rose

836 Kex2/subtilisin-like Proprotein Convertase activity is required for Cuticle Structure/Formation and Defecation. C. Thacker and A.M. Rose

837 Effects on nitrogen and energy metabolism of a defective DAF-2 insulin receptor-like protein John J. Thaden, kewen Jauss, Heather S. Reis, Henry J. Mroczkowski, Daniel Owen, and Robert J. Shmookler Reis

838 Molecular and genetic analyses of unc-69, a gene required for axon guidance. Suzanne Tharin, Bruce Wightman, Nancy Tsung, Erika Hartwieg, Gian Garriga, Bob Horvitz and Michael Hengartner

839 PEB-1, A Novel DNA Binding Protein Involved In Pharyngeal Gene Expression Jack D. Thatcher, Christina Haun and Peter G. Okkema

840 Analysis of a deletion mutation of del-1, a member of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily Heather A. Thieringer, Nektarios Tavernarakis, and Monica Driscoll

841 Cross-Platform Software Tools for Time-Lapse Imaging of 3D Samples (4D Imaging) Charles Thomas and John G. White

842 Comparison of Cytidine Deaminase in Brugia pahangi and C. elegans Fiona Thompson, Sarah Hunter, Collette Britton and Eileen Devaney.

843 Functional analysis of UNC-51 and UNC-14 involved in axonal formation in C.elegans HuaiZe Tian and Yasumi Ohshima

844 A genetic approach to identify the molecular target of SB-204269 in Caenorhabditis elegans Marcel Tijsterman, Julie Ahringer, Sohaila Rastan, Hugh Herdon, George P. Livi and Ronald H. A. Plasterk

845 On the Systemic Nature of dsRNA-mediated Genetic Interference in C. elegans Lisa Timmons, Jamie Fleenor, and Andrew Fire

846 UNC-96: an intermediate filament protein of muscle? T.L. Tinley, K.B. Mercer, C. Alberico, X. Tang, R. Santoianni, and G.M. Benian

847 UNC-89 participates in Rho-like signaling to organize thick filaments T. Tinley, R. Kindt, E. Baraldi, C. Kenyon and G. Benian

848 Using model organisms to identify genes that affect life span Heidi A. Tissenbaum and Leonard Guarente

849 Roles of osm-9/Capsaicin Receptor Family Members in Chemosensory Behavior David Tobin, David Madsen, Gary Moulder, Robert Barstead, Andres V. Maricq, Cori Bargmann

850 The TGFb Signaling Phenotypes Rafal Tokarz, Ling Yu, Christina Giannikas, and Cathy Savage-Dunn

851 Decreases in Body Size as a Function of Age and Proposal of Adulthood Phases M. K. Tom, R. Lu, E. M. Crimmins, L. H. Smith, and P. L. Larsen

852 Cloning of the C. elegans ADAR Leath A. Tonkin and Brenda L. Bass

853 Characterization of str-2 asymmetric expression Emily Troemel, Alvaro Sagasti, Cori Bargmann

24 May 1999 15:50 46 46 Poster Session #3 Saturday, June 5, 5:00 - 8:00 p.m.

854 Using the C. elegans genome to search for the vesicular glutamate transporter Miles Trupp, Kim Schuske, Marc Hammarlund, Steve McIntire, Erik Jorgensen

855 Searching for new genes involved in dosage compensation Chun Tsai and Barbara Meyer

856 Nematodes with Mitochondrial Diseases William Y. Tsang and Bernard D. Lemire

857 Identification of Genes Required for Spindle Orientation in the Embryo Meng-Fu Tsou, Adam Hayashi, and Lesilee S. Rose

858 Adapting a manual clonal screen to semi-automation Bernadine Tsung, Anthony A. Ferrante W. Peter Hansen, Petra B. Krauledat, Carl Johnson, and Craig P. Hunter

859 A clonal screen to identify genes that control pal-1 expression. Bernadine Tsung, Amanda Kay, Darcy Mootz, Nancy Huang, Amanda J. Wright, Monica Gerber, William Winston, Keuna Cho, and Craig P. Hunter

860 Characterization and cloning of the muscle activation gene unc-58 Monika Tzoneva and James H. Thomas

861 Cloning of che-1 gene involved in chemotaxis of C. elegans Okiko Uchida, Makoto Koga, Yasumi Ohshima

862 Molecular characterization of C. elegans protein phosphatase 1 homologue (CePP1) Hiroko Ueda, Xizhen Xu, Kanako Hirano, Masahiko Nakamura, Shin-ichi Harada, Toshihiro Sassa, Ryuji Hosono

863 The ADM-1 protein: possible roles in development and interacting proteins. Clari Valansi, Gidi Shemer and Benjamin Podbilewicz

864 A genetic screen for recessive mutations that suppress the egg-laying defect of lin-12 gain-of-function mutations Laura G. Vallier, Xantha Karp, Ning Chen, Iskra Katric and Iva Greenwald

865 Genetic analysis of signal transduction components involved in taste perception Claudia van den Berg, Esther Hazendonk, Corry de Vries and Ronald Plasterk

866 Mutations that suppress the lethargic and egg-laying defective phenotype of Gb overexpression Alexander M. van der Linden, Femke Simmer, Hendrik C. Korswagen, Ronald H.A. Plasterk

867 World-wide worm SNPs: Dissecting the molecular evolution of the species using a high density SNP map Henri G.A.M. van Luenen, Romke Koch, Ronald H.A. Plasterk

868 Promoter analysis of mel-32(SHMT) using GFP expression and interspecies DNA comparison Greg P. Vatcher and David L. Baillie

869 Elucidating the function of a novel DNA polymerase Ana Vaz Gomes and Erik Sonnhammer

870 Transgenically improved entomophatogenic nematodes for pest control Tibor Vellai, Attila Molnár, Lóránt Lakatos, Zsófia Bánfalvi, Fritz Müller, and András Fodor

871 ’High density C. elegans screening’: a drug discovery platform applied to the steerin (unc-53) pathway Kris Ver Donck, Isabelle Maillet, Ingele Roelens, Luc Bols, Peter Van Osta, Thierry Bogaert and Johan Geysen

872 Steerins (UNC-53) in health and disease: from C. elegans gene to drug discovery P Verhasselt, C Buesa, M De Raeymaeker, L Maertens, C Platteeuw, H Geerts, K Ver Donck, M Vandecraen, E Stringham, W Luyten, T Bogaert and J Geysen

24 May 1999 15:50 47 47 Poster Session #3 Saturday, June 5, 5:00 - 8:00 p.m.

873 The function of Rap1 and its guanine-nucleotide-exchange factors in C. elegans Mark H.G. Verheijen, Gert Jansen, Ronald H.A Plasterk and Johannes L. Bos

874 CBP-1 in C. elegans embryonic development and differentiation Martin Victor, Craig Mello and Yang Shi

875 The Elusive Rec-1 Vijhee Vijayaratnam and Ann M. Rose

876 Protein-Protein Interactions Involving the Sex Determining Protein, FEM-3 Usha Vivegananthan, Andrew M. Spence

877 Genetic analysis of the entomopathogenic nematode - bacterium symbiosis from bacterial side Antónia Völgyi, András Fodor and Steven Forst

878 A nicotinic acetylcholine receptor involved in regulation of egg-laying behavior. Laura Waggoner, Daniel Poole, and William Schafer

879 The C. elegans protein interaction mapping project: a test-case using proteins involved in vulval development Walhout AJM, Sordella R, Thierry-Mieg N, Brasch M, Temple G, Hartley J, Lorson M, van den Heuvel S, Endoh H, and Vidal M

880 Role of General Transcription Factors in Early C. elegans Gene Regulation Amy Walker, Yang Shi and Keith Blackwell

881 glr-5 and glr-7: Two Glutamate Receptor Subunits That May Have a Role in Thermotaxis. Craig S. Walker, D.M. Madsen, P. J. Brockie and A.V. Maricq

882 Studying the function of inositol 1,4,5-trisphosphate receptors in C. elegans Denise S. Walker and Howard A. Baylis

883 Identification and analysis of temperature sensitive embryonic lethal mutations that disrupt gene expression in early C. elegans embryos M. Wallenfang and G. Seydoux

884 RHA-1: A conserved RNA helicase in C. elegans important for development Katherine M. Walstrom, Kathryn A. Swan

885 Convergent Genetic Programs Create the Synaptic Pattern that Distinguishes the VD and DD Motor Neurons from One Another Bill Walthall and Mimi Zhou

886 Expression of amyloid precursor and related protein in C. elegans Haiyun Wang and Chris Li

887 Identification of Potential mpk-1 Targets by DNA Microarray Analysis Wang J, Reinke V, Van Doren CB, Begley RR, Kim SK

888 cMi-2, a component of histone deacetylase, is essential for C. elegans development Shi-Liang Wang, Yi Zhang, Danny Reinberg, Monica Driscoll

889 Two zinc finger proteins interact with the TRA-2 intracellular domain Shanping Wang and Judith Kimble

890 A molecule containing a Hedgehog-intein (HINT) domain is required for molting in C. elegans. Xiwei Wang, Philip Beachy, Geraldine Seydoux

891 PIE-1 Gets Help from CeUBC9 in Germline Protection Xiaoting Wang, Tae Ho Shin and Craig Mello

892 Molecular Cloning and Characterization of a STAT homologue in C. elegans Yaming Wang and David E. Levy

24 May 1999 15:50 48 48 Poster Session #3 Saturday, June 5, 5:00 - 8:00 p.m.

893 Function, Tissue Distribution, and Mutants of nSLO1 Z. W. Wang, O. Saifee, T. Jegla, M. Nonet, and L. Salkoff

894 gly-2 is an N-acetylglucosaminyltransferase V and Can Rescue the Mammalian Lec4 Mutation. Charles E Warren and James W Dennis

895 Attempts toward larger C. elegans by a sma transgene Naoharu Watanabe, Yasumi Ohshima

896 Examining the role of polyunsaturated fatty acids in growth and development of C. elegans Jennifer Watts and John Browse

897 egl-2 encodes an eag-like potassium channel blocked by imipramine in C. elegans Aguan Wei, David Weinshenker, James Thomas and Lawrence Salkoff

898 The C. elegans KQT-1 potassium channel is essential for normal pharyngeal pumping rhythm and viability: An animal model for long-QT syndrome. Aguan Wei and Lawrence Salkoff

899 ric-6 and ric-7, two genes involved in C. elegans neurotransmission Robby M. Weimer and Erik M. Jorgensen

900 Information Coding in the C. elegans Olfactory System Paul D. Wes and Cori Bargmann

901 A sensitized genetic screen to identify genes that act in basolateral membrane localization of the LET-23 EGF Receptor Charles Whitfield, Mike Povelones, Bob Coffey, Alex Hajnal and Stuart Kim

902 A strategy for the genetic analysis of the perception of taste sub-modalities. Stephen R. Wicks, Marieke A.G. Essers and Ronald H.A. Plasterk

903 Regulative Development in a Nematode Embryo: A Hierarchy of Cell Fate Transformations Oliver Wiegner and Einhard Schierenberg

904 The Roles of fax-1 and unc-20 in Nervous System Development Bruce Wightman, Nicole Carmean, Bryan Ebert, Kelly Klampert, Holbrook Kohrt, Elissa Murphy, Son Nguyen and Galina Radzievsky

905 The Anaphase-Promoting Complex is Required for Meiosis I Anaphase L. Wille, M. Abdolrasulnia, and D. Shakes

906 Identification and analysis of a new mutation affecting T cell polarity Krista L. Williams and Michael A. Herman

907 An Analysis of Cytokinesis in Early C. elegans Embryos by Electron Microscopy Ellen Williams-Masson and John White

908 K-Cl Cotransporters in C. elegans John S. Willis and Edward T. Kipreos

909 Mutations in the L-type calcium channel EGL-19 affect UNC-4 function Angela R. Winnier and David M. Miller, III.

910 A Chemical Genetic Screen for Embryonic Lethality in C. elegans William M. Winston, Stephen J. Haggerty, Stuart L. Schreiber, Craig P. Hunter

911 Candidate catecholamine receptors in C. elegans Richard F. Wintle, Wojciech Kawczynski, Nathaniel R. Santos and Hubert H.M. Van Tol

912 ATP-binding cassette (ABC) transporters form a large and diverse gene family in C. elegans Robert G. Wisotzkey, Warwick Grant, Gary Hardiman and Carl D. Johnson

24 May 1999 15:50 49 49 Poster Session #3 Saturday, June 5, 5:00 - 8:00 p.m.

913 Analysis of Muscle Function in C. elegans collagen IV mutants. Colette M. Witkowski and James M. Kramer

914 Thermal Avoidance: a novel approach to study nociception in C. elegans N. Wittenburg and R. Baumeister

915 Biochemical approaches to the identification of endoderm regulatory factors in the early embryo Eric S. Witze, Jodie J. Kasmir, and Joel H. Rothman

916 Genes acting with vab-8 for the posterior-directed migration of the can cell. Fred W. Wolf, Peter Yen, and Gian Garriga

917 Insights into the function of an insulin-like pathway in C. elegans: Regulation and outputs of age-1 Catherine A. Wolkow, Jason Z. Morris and Gary Ruvkun

918 Functions of C. elegans cell fate determining genes in C. briggsae Amanda J. Wright and Craig P. Hunter

919 egl-44 and egl-46, Two Negative Regulators of Touch Cell Fate Ji Wu, Anne Duggan and Marty Chalfie

920 A screen for loci that facilitate ionotropic glutamate receptor activity Zhi-Liang Wu, Dat Nguyen, Ross Francis, Jenny Kopczynski, and Allen Ebens

921 Developmental Regulation of C. elegans HCF Phosphorylation. J. Wysocka, Y. Liu, S. Lee, R. Kobayashi and W. Herr

922 Regulation of protein secretion into the perivitelline fluid of developing nematode ’eggs’ Hong Xiao, John Plenefisch and Richard Komuniecki

923 Functions of lin-6 in DNA replication and the S-phase checkpoint Huihong Xu, Bob Horvitz and Sander van den Heuvel

924 More about MES Proteins Lei Xu, Susan Strome

925 Adaptive response in C.elegans : Oxidative stress-responsive genes to aging increase life span Sumino Yanase and Nao Ishii

926 lep-1, a gene involved in male tail tip morphogenesis Ying Yang, Can Q. Nguyen, David H. Hall and David Fitch

927 Characterization of the DES-2/DEG-3 receptor, an ancient member of the nAChR family Lina Yassin, Boaz Gillo, Margalit Eshel, Millet Treinin

928 Effects of temperature and oxygen on life span in mev-1 Kayo Yasuda, Phil Hartman and Nao Ishii

929 Reproductive Isolation in Caenorhabditis Wei-Chih Yen and Scott Everet Baird

930 Mutations that confer aspects of the molting defects seen with lrp-1 mutations or sterol starvation John Yochem

931 A neural RNA-binding protein MSI-1 is involved in turning behavior during mating Akinori YODA, Hitoshi SAWA and Hideyuki OKANO

932 Isolation and characterization of protruding vulva sterile mutants John H. Yoder and Min Han

24 May 1999 15:50 50 50 Poster Session #3 Saturday, June 5, 5:00 - 8:00 p.m.

933 Green fluorescent protein as an epitope tag to purify fusion proteins and associating molecules Aki Yonezumi, Keiko Gengyo-Ando, Terumi Sakamoto and Shohei Mitani

934 The C. elegans sex-determining gene tra-1 may be translationally regulated by its 3’UTR Young Yoo, Eric Jan, and Elizabeth B. Goodwin

935 Components of the SNARE complex exhibit nonallelic noncomplementation Karen Yook, Stephen R.Proulx and Erik Jorgensen

936 Isolation and Characterization of a gene acting downstream of sma-2 signaling Satoru Yoshida, Makoto Mochii, Kiyokazu Morita, Miho Shimizu, Yuji Kohara, and Naoto Ueno

937 Isolation of target genes in daf-2 pathway Hui Yu and Pamela L. Larsen

938 Cloning and characterization of C. elegans ram-5: strucutral organization and expression pattern of the gene Yu, Raymond Y.L. and Chow, King L.

939 Dosage of ram-5 gene is critical for sensory ray morphogenesis in C. elegans Yu, Raymond Y.L. and Chow, King L.

940 Imaging of neuronal activity in olfactory circuits in C. elegans Tim Yu and Cori Bargmann

941 nSLO2, A Novel Cl - Activated K + Channel A. Yuan, M. Dourado, Z.W. Wang, A. Butler, and L. Salkoff

942 sup-39 mutations alter the use of cryptic splice sites in unc-73(e936) Alan M. Zahler

943 Expression pattern of a worm homologue of phogrin, a mammalian dense cored granule protein of neuroendocrine cells Tobias Zahn, Peg MacMorris, Christina Wasmeier, John C. Hutton

944 Analysis of several mutations that disrupt vulval development Weiming Zhang, Amanda Mapes and Min Han

945 Phenotypic analysis of two C. elegans endocytosis mutants rme-1 and rme-8 Yinhua Zhang, Barth Grant, Laura Pedraza and David Hirsh

946 The mechanisms of synaptic localization of sng-1, the C. elegans homologue of vertebrate synaptogyrin Hongjuan Zhao, Liping Wei and Michael L. Nonet

947 Identification and Analysis of Three New Mutations Involved in the Control of Cell Polarity During C. elegans Development Xiaojun Zhao, Thomas M. Ratliff and Michael A. Herman

948 glr-3 Encodes a non-NMDA Type Glutamate Receptor that is Widely Expressed in the Nervous System of C. elegans Yi Zheng and A. V. Maricq

949 Neuronal Timing Behavior in C. elegans can be Modified by a Dominant Mutation in the GLR-1 Ionotropic Glutamate Receptor Yi Zheng, Penelope J. Brockie and Andres V. Maricq

950 Characterization of the cul-4 gene Weiwei Zhong, Edward T. Kipreos

951 Parameters in transgenic rescue of spe-4, a germline gene involved in spermatogenesis Guang-dan Zhu and Steven W. L’Hernault

24 May 1999 15:50 51 51 Poster Session #3 Saturday, June 5, 5:00 - 8:00 p.m.

952 A Probabilistic Method for Accurate Gene Function Prediction using Phylogenetic Inference Christian M Zmasek and Sean R Eddy

24 May 1999 15:50 52 52 Accessing genome project data and the state of ACeDB

1999 International Worm Meeting abstract 1 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Accessing genome project data and the state of ACeDB Members of the Genome Sequencing Consortium and the ACeDB collaboration The Sanger Centre There are multiple ways of accessing the information from the genome project. First, you can search the genomic sequence on the blast server at either the Sanger Centre or GSC St Louis web site. The data sets searched on these servers are complete and updated every night. If you get a hit, you can retrieve the corresponding full sequence by web or ftp. Second, sequences are submitted to Genbank/EMBL. Finished sequence submissions contain some annotation including the gene predictions. Current unfinished sequence is also submitted to the HTG division of Genbank, so all sequences have an accession number, even if not finished. Finally, all the annotation information we have is made available via ACeDB, which also joins adjacent sequences and provides a wide variety of other information. The web version of ACeDB is most up to date. The state of the annotation and the specific information that is available will be described. Gene predictions are best efforts, and are liable to be wrong in the absence of supporting data. Please refer to them using the ’clone’.’number’ identifier from ACeDB or Genbank, unless they have a three letter gene name. If you are responsible for assigning a gene name to a sequence, or identifying the sequence for a gene, please let us know. Please also email corrections to gene predictions or other information - these do result in fixes to our databases and hence ACeDB and Genbank. St Louis and Sanger each curate the sequence annotation for the parts of the genome they sequenced (email [email protected] and [email protected]) but information sent to either site will be forwarded to the other as required. ACeDB itself is built from the sequence annotation databases at each site, the current state of the physical map, and a database of genetic and literature information maintained in conjunction with the CGC (see abstract by Hodgkin et al.). We have also been developing a web interface to ACeDB (see abstract by Stein et al.). The Sanger Centre version of this web site uses a database that is rebuilt each week. As well as providing access by names of objects and text search, we have recently been exploring using the AltaVista search tool and interface to access the database. Other plans for ACeDB will also be discussed.

24 May 1999 15:50 53 53 AcePerl: A New Face for Ace

1999 International Worm Meeting abstract 2 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. AcePerl: A New Face for Ace LD Stein 1 , J Thierry-Mieg 2 , R Durbin 3

1 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 2 CNRS, CRBM, Montpellier, France. 3 The Sanger Centre, Hinxton, UK.

The ACEDB database is used for the storage and retrieval of biological data in C. elegans and in other organism. However it has been hampered by a monolithic design and a user interface that is difficult to customize. We have created a software library called AcePerl that provides a simple programmer’s interface to ACEDB databases. With this library, programmers and savvy biologists can use the popular Perl language to write scripts to fetch objects from ACEDB databases, to manipulate and modify them, and to write them back into the database. The interface can also be used to combine information from multiple databases, making it possible to integrate data from different sources and interconnect multiple ACEDB servers. On top of AcePerl we have written an extensible Web browsing system for ACEDB databases called AceBrowser. It does away with many of the more complex ACEDB graphics, and substitutes instead simpler HTML pages that are easier to understand and more accessible to non-specialists. Using AceBrowser, we have created a new public access Web site for C. elegans information. This site provides access to the annotated C. elegans genome, clones, physical maps, genetic maps, and mutant information. In addition, the site provides access to a complete bibliography of nematode papers and abstracts. The system can be queried in a variety of ways, and provides a mechanism for sending comments directly to the database curators. AcePerl, AceBrowser and the ACEDB database itself are all distributed in full source code form, under a license that allows them to be used and redistributed freely: http://stein. shl.org/AcePerl ftp //ncbi.nlm.nih.gov/repository/acedb The C. elegans Web site is mirrored at: http://stein.cshl.org/elegans/ (New York) http://alpha.crbm.cnrs-mop.fr/cgi-bin/ace/simple/elegans (Montpellier) http://wormsrv1.sanger.ac.uk/cgi-bin/ace/simple/elegans (Cambridge)

24 May 1999 15:50 54 54 Expressed Genes in C. elegans

1999 International Worm Meeting abstract 3 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Expressed Genes in C. elegans Jean Thierry-Mieg 1,2 , Danielle Thierry-Mieg 1,2 , Tadasu Shin-I 1 , Yuji Kohara 1

1 NIG, Mishima, Japan. 2 NRS, Montpellier, France.

A large normalized set of C.elegans cDNAs has been constructed and the two ends of each cDNA have been sequenced in Mishima. The resulting 118300 traces together with 6000 other cDNA sequences available from GenBank were aligned over 95.8 megabases genomic DNA available from the C.elegans consortium. The expressed genes were reconstructed, and the exact splicing established. As of march 99, there are hits to 9100 genes, among which 320 are in yet unsequenced genomic holes. That is about half of the 19000 genes predicted by the consortium, but not an exact subset, because the actual transcripts merge or split or differ in some finer way from the predictions in almost half the cases. To fully exploit the data, we hand edit the basecall of the traces in view of the genomic sequence. About 4000 genes have been edited so far. Based on that set, a minimum of one third of the genes show alternative splicing and should generate more than one product. Alternative polyadenylation also frequently occurs. Transplicing to SL1 is apparent in only 35% of the probably complete genes. This may be an underestimate, but we do see SL1 in 12/14 of the complete genes directly upstream from the rare genes transpliced to SL2. Because we have both the cDNA and the genomic sequence, we derive accurate data on the distribution of the genes along the genome, the exact structure of the introns, the transplicing and the 3’ untranslated regions. Based on these independent sequence data, we confirm the high quality of the genomic sequence which presents less than one error per 40 kb and very few rearrangements. We expect that 3.2 Mb of the genome is still missing. We will present our methods and results and explain how to access the data on our servers http://alpha.crbm.cnrs-mop.fr or http://watson.genes.nig.ac.jp:8080/db/. We plan to finish editing all the data, but if your pet gene has not yet been edited, please don’t hesitate to ask, and we will edit it in priority.

24 May 1999 15:50 55 55 Post genomics strategies and resources in C.elegans

1999 International Worm Meeting abstract 4 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Post genomics strategies and resources in C.elegans Yuji Kohara, The Kohara Lab, Collaborators CREST, JST and Genome Biology Lab, National Institute of Genetics, Mishima 411-8540, Japan We will present our plans for genome biology of the worm based on our cDNA project and wish to discuss about possible coordination in the worm community. 1) More cDNA clones and full-length libraries: Thus far, we have analyzed some 65,000 cDNA clones and classified them into about 10,000 unique cDNA groups. Since the consortium has made the prediction of most of genes, our effort has been shifted to expression and function analysis rather than extension of the EST analysis. However, our recent analysis indicated that EST information is still essential to determine the gene structures (see Thierry-Mieg et al.). Thus, we are planning to continue the EST analysis together with full-sequencing of the clones that showed alternative splicing. In parallel, we plan to construct full-length cDNA libraries using the oligo capping method developed by Sugano’s group at the University of Tokyo. 2) Expression analysis by in situ hybridization: We have done in situ hybridization with 3,500 gene probes mostly from LG3 and LGX, and will finish the remaining 7,000 gene within a year. However, since giving of full annotation of the expression patterns is a time consuming task, we intend to release typical images and their brief description at our NEXTDB WWW site before adding full annotation. 3) Promoters for specific transcription: The in situ analysis has identified a lot of genes that show specific mRNA distribution. The promoters of these genes should be very useful for ectopic gene expression, cell/lineage/stage specific RNAi and so on. Therefore, we plan to identify such promoter fragments systematically. 4) Microarray and systematic RNAi: We selected some 8,000 representative clones (1-2Kb insert clones suitable for PCR) and are producing microarrays on glass slides (version 1). Preparation of the remaining 2,000 representative clones is also in progress to add the microarray (version 2). The same set could be applied to systematic RNAi. We are preparing to make these resources available to the community. 5) Others: Antibodies, deletion mutant bank (in collaboration with Shohei Mitani at Tokyo Women’s Medical College), computer simulation of development, and so on.

24 May 1999 15:50 56 56 A Gene Knockout Service for C. elegans

1999 International Worm Meeting abstract 5 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A Gene Knockout Service for C. elegans Gary Moulder 1 , Nathan Cook 1 , Brooke Toland 1 , Jenny Medelberg 1 , Robert Barstead 1 , David Hughes 2 , Chris Johnson 2 , Ngoc-Sung Ly 2 , Richard Durbin 2 , Alan Coulson 2 , Erin Gilchrist 3 , Greg Mullen 3 , Blazej Szczygielski 3 , Ann Rose 3 , Donald Moerman 3 , Steven Jones 4 , Stefan Eimer 5 , Christine Goebel 5 , Bianca Wiesinger 5 , Ralf Baumeister 5

1 The Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104. 2 The Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge, U.K. 3 The Biotechnology Laboratory, UBC, Vancouver, B.C., Canada. 4 Genome Sequence Centre, British Columbia, Canada. 5 LMB/Genzentrum, Munich, Germany.

The technology to knock out genes in C. elegans is sufficiently robust to be within the capabilities of most investigators. Nevertheless, the technology requires a significant investment of time and money such that most investigators are reluctant to undertake more than a few gene knockouts. Further, this de facto limitation translates to an appropriately conservative choice of targets for knockout, with only the most evolutionarily conserved genes making the list. The mission of the members of C. elegans Gene Knockout Consortium is to relieve labs of the burden of knocking out genes. The Consortium service likely will expand the range and numbers of genes that individual labs are willing to analyze. Ultimately, the Consortium intends to knockout all genes in C. elegans, thereby facilitating the initial study of even those genes that lack obvious orthologs in other species, but which may yet lead to interesting, broadly relevant biology. Our initial list of targeted genes will be based on requests from outside investigators. Initially we are restricting requests to five targets per year. This restriction will be eliminated as we develop greater capacity. To date we have eliminated the function of approximately 150 genes. Our goal in the coming year is to knockout approximately 1500 genes using chemical mutagens coupled with population PCR to induce and detect gene deletions. Requests for gene knockouts can be submitted through a form at the following web site: http://www.cigenomics.bc.ca/elegans.

24 May 1999 15:50 57 57 The complete family of G protein genes of Caenorhabditis elegans

1999 International Worm Meeting abstract 6 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The complete family of G protein genes of Caenorhabditis elegans G Jansen, KL Thijssen, P Werner, M van der Horst, E Hazendonk, RH Plasterk Division of Molecular Biology, The Netherlands Cancer Institute, Center for Biomedical Genetics, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands One of the new possibilities in post-sequence genetics is the analysis of complete gene families at once. We studied the family of heterotrimeric G proteins. C. elegans has 20 Ga, 2 Gb and 2 Gg genes. There is one clear homologue of each of the four mammalian classes of Ga genes, Go/Gi, Gs, Gq, G12, and there are sixteen new a genes. While the conserved Ga subunits are expressed in many neurons and muscle cells, GFP fusions indicate that 14 of the new Ga genes are expressed almost exclusively in a small subset of the amphid cells, sensory neurons in the head of the nematode, or other putative sensory neurons. We isolated null alleles for all Ga genes, using target selected gene inactivation. Furthermore gain-of-function alleles were generated by introducing the wild type genes as transgenes (XS). None of the amphid expressed genes are essential for viability. Based on the specific expression of many Ga subunits in chemosensory neurons, all mutants were tested for chemotaxis to water soluble and volatile attractants and repellents. Only four loss-of-function alleles show chemotaxis defects. In contrast, nine gain-of-function alleles show altered behavior in the various chemotaxis assays. The lack of loss-of-function phenotypes can be explained by functional redundancy, as has been described for gpa-2 and odr-3 (Roayaie et al., Neuron 20: 55-67, 1998). Alternatively, the Ga genes may be involved in the perception of compounds that have not yet been tested. Furthermore, they may have negative regulatory functions, as was found for gpa-5 and gpa-6. We are further testing these hypotheses by combining several of loss-of-function alleles. Based on functional analysis the 20 Ga genes can be divided into two groups: subunits that affect muscle activity (homologues of Gi/Goa, Gsa and Gqa), and 14 new Ga genes that are probably all involved in perception (Jansen et al., Nature Gen. In press).

24 May 1999 15:50 58 58 Using DNA microarrays to identify targets of the Ras/MAP kinase signaling pathway

1999 International Worm Meeting abstract 7 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Using DNA microarrays to identify targets of the Ras/MAP kinase signaling pathway Stuart K. Kim, Carrie B. Van Doren, Rebecca R. Begley Stanford University, Department of Developmental Biology, Stanford University Medical Center, Stanford, CA 94305 We are entering a new age in molecular genetics in which we can use sequence data from genome projects to dissect cell, developmental and disease pathways more completely and more sensitively than ever before. We need to develop new experimental approaches to analyze the vast amounts of data that are now available from genome projects. C. elegans is the only animal model system with a complete genome sequence, and thus will play a leading role in establishing approaches that make use of the full genome sequence. One key functional genomics approach is to use DNA microarrays to define changes in gene expression patterns during development and during the onset of disease. DNA microarrays could be used to identify genes that are regulated by specific transcription factors, by specific cell signaling pathways, by expression of homologs of human disease genes in transgenic animals or by addition of various pharmaceutical drugs. We are currently producing DNA microarrays that contain 11,401 genes from the C. elegans genome. To demonstrate that C. elegans microarrays are powerful tools to dissect developmental pathways, we are using these DNA microarrays to identify targets of the RTK/Ras/MAP kinase signaling pathway during vulval development. To globally detect differences in transcription patterns due to Ras signaling, a cDNA microarray is simultaneously hybridized with Cy3-labelled cDNA (red) (from reverse transcription of polyA mRNA) from let-23(lf) mutants, and Cy5-labelled cDNA (green) from let-60(gf) mutants. Genes that are expressed at similar levels in the two strains hybridize equally with both probes, and appear yellow on the microarray. Genes that are expressed at higher levels in the first strain appear red, and genes that are expressed at higher levels in the second strain appear green. The image intensities of the cDNA microarray are digitized, and the expression ratios between the two strains are calculated for every cDNA clone. The Ras target genes identified by the DNA microarrays will play a critical role in elucidating the molecular basis for Ras signaling. Once we have developed full genome C. elegans microarrays, we can make our DNA microarrays available to the entire C. elegans academic community.

24 May 1999 15:50 59 59 Identification of the 3’ splice site by both subunits of U2AF

1999 International Worm Meeting abstract 8 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification of the 3’ splice site by both subunits of U2AF D Zorio 1,2 , T Blumenthal 2

1 Department of Biology, Indiana University, Bloomington, IN 47405. 2 Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO 80262.

How are introns and outrons recognized in C. elegans? The 5’ splice sites on introns are identified by base pairing with U1 snRNA, as they are in other animals. However, C. elegans introns lack both the polypyrimidine tract and the branchpoint consensus sequences found in other animals. Instead, they have a highly conserved 3’ splice site octamer, U4 CAG/R, that is required for both intron and outron recognition. In other animals, the polypyrimidine tract is bound by the two-subunit splicing factor, U2AF, but it has never been determined how the invariant AG at the 3’ splice site is recognized. Here we report a probable solution to this problem for C. elegans. We have previously shown that C. elegans has both U2AF subunits and both are required for viability. We also showed that the large subunit gene contains an alternatively spliced exon containing multiple copies of the U4 CAG/R consensus. We proposed that these octamers in the alternative exon serve as sites for autoregulation of U2AF levels by alternative splicing. To test whether U2AF does in fact bind to this consensus, we performed in vitro cross-linking experiments with crude embryo extracts from transgenic worms that overexpress both U2AF subunits in response to heat shock (with the small subunit epitope tagged). We then used immunoprecipitation to identify U2AF-cross-linked bands. We found that both subunits cross-link to an RNA oligonucleotide containing the complete consensus (U4 CAG/G), but that only the large subunit cross-links to an RNA containing the 5 pyrimidines, but lacking the AG (U4 CGAA). Furthermore, the presence of the AG-less RNA causes dissociation of the two subunits from each other. An RNA lacking 3 of the pyrimidines, but retaining the remainder of the consensus (UA3 CAG/G), does not cross-link to either subunit. Our results suggest that U2AF small subunit may be responsible for recognition of the invariant AG at the 3’ splice site, and that simultaneous recognition of the short polypyrimidine tract by U2AF large subunit and the AG/R by U2AF small subunit may be the key event in 3’ splice site identification.

24 May 1999 15:50 60 60 An alternatively spliced U2AF65 exon capable of keeping a reporter gene mRNA from leaving the nucleus

1999 International Worm Meeting abstract 9 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. An alternatively spliced U2AF 65 exon capable of keeping a reporter gene mRNA from leaving the nucleus P MacMorris 1 , D Zorio 1,2 , T Blumenthal 1

1 Dept. of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver CO 80262. 2 Biology Dept., Indiana University, Bloomington IN 47405.

U2AF65 is an essential splicing factor that recognizes the 3’ splice site (UUUUCAG/R) in C. elegans; its production is controlled by alternative splicing in a novel way. The 1.7 kb product that lacks exon 3 encodes U2AF65 , whereas the 1.9 kb RNA, containing exon 3, is not a functional mRNA. It contains a premature stop codon, yet makes no detectable truncated protein. Although we expected that the relatively low levels of the 1.9 kb RNA could be due to Smg RNA surveillance, its level did not increase in smg-2 mutants. We hypothesized that the Smg machinery, which recognizes premature stop codons in the cytoplasm, would not be able to detect and degrade the 1.9 kb RNA if it remained nuclear. Exon 3 contains 10 repeats of the UUUUCAG/R octamer; these could be responsible for preventing transport out of the nucleus by binding U2AF without any subsequent splicing. The net effect of such a mechanism could be down-regulation of U2AF65 levels by accumulation of this product in the nucleus. To test this idea, we studied the expression in transgenic worms of a vit/gfp reporter with exon 3 inserted into its 3’UTR (gfpex3). Even though correctly spliced and polyadenylated intestinal gfpex3 mRNA levels were high, GFP was almost undetectable. Furthermore, in situ hybridization demonstrated dramatically that while control vit/gfp mRNA was cytoplasmic, the gfpex3 mRNA was almost entirely nuclear. If U2AF itself binds to the octamer repeats in exon 3 to prevent transport, a reduction in U2AF levels should allow the release of some mRNA, and thus an increase in GFP levels. We used RNAi to reduce U2AF levels in the intestines of injected worms and found a small burst of GFP in these animals. We conclude that U2AF binding of exon 3 prevents the gfpex3 mRNA from leaving the nucleus. U2AF65 could autoregulate similarly by binding to exon 3 of its own pre-mRNA, causing inclusion of this exon. It could then remain bound to the 1.9 kb alternatively spliced product, and keep it in the nucleus to serve as a source of U2AF when the need arises.

24 May 1999 15:50 61 61 Natural targets of mRNA surveillance in C. elegans

1999 International Worm Meeting abstract 10 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Natural targets of mRNA surveillance in C. elegans QM Mitrovich, P Anderson Department of Genetics, University of Wisconsin, 445 Henry Mall, Madison, WI 53706 The process whereby messenger RNAs containing premature translation termination codons are selectively and rapidly degraded has been conserved throughout eukaryotic evolution. The roles that this nonsense-mediated mRNA decay (NMD) play in wild-type populations, however, remain largely unknown. One proposed role for NMD is "mRNA surveillance," the selective elimination of aberrant mRNAs arising from errors in gene expression. To explore this and other possibilities, we performed a cDNA-based representational difference analysis to identify mRNAs whose abundance increases in smg(-) mutants, in which NMD has been eliminated. Such mRNAs should include the natural targets of NMD. One class of natural targets is alternatively spliced mRNAs. Pre-mRNAs from each of four large-subunit ribosomal protein genes are spliced to generate two message forms. The normal, protein-coding mRNAs are unaffected by smg(-) mutations. Alternative mRNAs arise from use of splice sites located within introns of the normal mRNAs. The retained intron fragments have in-frame stop codons and render the mRNAs substrates for NMD. In wild-type worms, the alternative mRNAs are undetectable by standard Northern blot analysis, while in smg(-) mutants they accumulate to levels comparable to those of the normal mRNAs. Although these alternative mRNAs are efficiently eliminated by NMD, they appear to be byproducts of "normal" gene expression, rather than the result of aberrant gene expression. Unlike most introns, including other introns from the same genes, these alternatively spliced introns are remarkably well-conserved among C. elegans, C. briggsae and C. remanei. In fact, the alternatively spliced introns can be more highly conserved than the surrounding coding sequences. Furthermore, we have detected intron fragment-containing mRNAs in wild-type C. elegans, C. briggsae and C. remanei, albeit at very low levels. We are currently investigating the role alternative splicing plays in ribosomal protein gene expression. One intriguing possibility is that these alternative mRNAs are the byproducts of feedback regulation that modulates levels of ribosomal protein mRNA. The smg surveillance system would serve to degrade the non-coding remnants of such regulation.

24 May 1999 15:50 62 62 Inherited and controllable interference by dsRNA

1999 International Worm Meeting abstract 11 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Inherited and controllable interference by dsRNA NN Tavernarakis 1 , SL Wang 1 , M Dorovkov 2 , A Ryazanov 2 , MA Driscoll 1

1 Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, New Jersey. 2 Department of Pharmacology, Robert Wood Johnson Medical School University of Medicine and Dentistry of New Jersey, New Jersey.

Double-stranded RNA interference (RNAi) is an effective and convenient method for disrupting specific gene expression in Caenorhabditis elegans. Nonetheless, this excellent tool has certain limitations. For example, because dsRNA is not stably inherited, "fresh" phenocopies must be continuously generated. Also, it is difficult to generate large populations of RNAi-induced ’mutants’, restricting possibilities for genetic and biochemical studies. Furthermore, although temporal requirements for essential genes can be tested by stage-specific injections, such manipulations are labor intensive. To extend the utility of RNAi, we developed and tested a method for expression of inherited inducible RNAi. We used the hsp16 heat-shock inducible promoter to drive the expression of snap-back transcripts. The template used for in vivo snap-back production was an inverted repeat of the gene of interest cloned downstream of the promoter. We have found that such snap-back genes are straightforward to clone into worm expression vectors. Upon heat-shock, a double-stranded RNA that can efficiently interfere with the expression of the cognate gene is generated. In all test cases, RNAi phenocopies faithfully reflected known mutant phenotypes. We have been successful in using RNAi-induced "mutants" for biochemical assay. This method should expand reverse genetic possibilities for C. elegans.

24 May 1999 15:50 63 63 RNA interference depends on genes required for mRNA surveillance

1999 International Worm Meeting abstract 12 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. RNA interference depends on genes required for mRNA surveillance DP Morse 1 , ME Domeier 2 , M Portereiko 2 , BL Bass 1 , SE Mango 2

1 Department of Biochemistry/HHMI, University of Utah, Salt Lake City, UT 84112. 2 Huntsman Cancer Institute Center for Children and Department of Oncological Sciences, University of Utah, Salt Lake City, UT 84112.

Double-stranded RNA (dsRNA) injected into C. elegans inhibits expression of genes homologous to the injected RNA1,2 . This phenomenon, called RNA interference (RNAi) in worms, may be related to homology-dependent gene silencing observed in other organisms. Seven smg genes are known to be involved in the degradation of aberrant transcripts, a process known as mRNA surveillance3 . Since there is evidence that RNAi works through mRNA degradation2 , we tested whether the smg pathway is required for RNAi. We observed that worms mutant in particular smg genes recovered rapidly from the effects of RNAi. In wild-type worms, the paralyzed phenotype resulting from injection of unc-54 dsRNA persisted throughout the lifetime of the affected progeny (unc-54 encodes a myosin heavy chain). The progeny of smg-2 worms injected with unc-54 dsRNA displayed the paralyzed phenotype at early time points similar to what has been observed previously2 . However, the smg-2 animals showed dramatically improved movement as they aged. Interestingly, while smg-3,4,5 & 6 worms also recovered from RNAi to varying extents, smg-1 animals did not show this pattern of recovery and remained paralyzed. Quantitative RT-PCR showed that unc-54 mRNA levels correlated well with the observed phenotypic patterns. We are currently targeting other genes by RNAi to test the generality of these observations. In summary, our data has (1) identified the first trans-acting factors required for RNAi; (2) linked the phenomenon of RNAi to the process of mRNA surveillance; (3) revealed phenotypic distinctions among the smg genes. One possible interpretation of our results is that RNAi proceeds through a two-step mechanism: mRNA is initially degraded in a smg-independent step and low RNA levels are maintained by a process that depends on a subset of smg genes.

1. Guo & Kemphues Cell 81, 611 2. Fire et al. Nature 391, 806; Montgomery & Fire PNAS 95, 15502. 3. Hodgkin et al. Genetics 123, 301; Pulak & Anderson Genes Dev 7, 1885; Cali et al. Genetics 151, 605.

24 May 1999 15:50 64 64 Genetic analysis of RNA interference in C. elegans

1999 International Worm Meeting abstract 13 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic analysis of RNA interference in C. elegans H Tabara 1 , M Sarkissian 1 , L Timmons 2 , A Fire 2 , CC Mello 1

1 Univ. of Massachusetts, Worcester, MA 01605, USA. 2 Carnegie Institution of Washington, Baltimore, MD 21210.

RNA interference (RNAi) has become a major tool for the targeted inhibition of gene function in C. elegans. New observations continue to increase the utility and ease with which interference can be achieved. For example RNAi can now be introduced by feeding or soaking. Despite these breakthroughs there are still numerous questions. For example, what is the in-vivo function of RNAi? How does interference occur? How is RNA taken up and transmitted, and is the interference effect amplified? In order to address these questions we have begun screens for mutations that diminish RNAi. We first optimized a bacterial system that efficiently drives dsRNA expression for an essential gene (pos-1). From an EMS screen of 100,000 F2 animals, 80 strains were found that were able to grow on the pos-1 bacteria. 17 of 26 strains tested are also resistant to RNAi by injection. Two of these strains define one complementation group on LGV near unc-42. This locus has been tentatively named rid-1, for RNAi deficient. The rid-1 mutants appear strongly resistant to RNAi for all genes tested and exhibit no other phenotype. In order to facilitate cloning of RNAi genes we next searched for transposon induced alleles. Surprisingly, we found that both the mut-2 and mut-7 high hopper strains are themselves resistant to RNAi, although the RNAi resistance of mut-7 is primarily specific to maternal genes. RNAi resistance and the mutator activities co-map suggesting that they have a common genetic cause. In addition, several but not all, of the new mutants exhibit sterility and mild him phenotypes correlated with mut-2 and mut-7 strains. These findings raise the possibility that suppression of transposon mobilization may be an in-vivo function of RNAi. Alternatively, activation of transposons and loss of RNAi may be secondary to a more general defect in gene regulation. Another interesting possibility is that the transposons themselves (once mobilized) are actively suppressing RNAi in the high hopper strains. We will present our genetic analysis of the RNAi deficient loci and describe our efforts at cloning.

24 May 1999 15:50 65 65 The Epigenetics of Germline Maintenance

1999 International Worm Meeting abstract 14 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Epigenetics of Germline Maintenance B Kelly, A Fire Department of Embryology, Carnegie Institution of Washington, Baltimore, MD We are interested in characterizing the mechanism(s) by which the germline escapes the narrowing of developmental potential that is characteristic of somatic differentiation. There appear to be several processes specific to the germline that it likely employs to maintain its totipotency. These include the pie-1 mode of global transcriptional repression, which is effective in the early embryo1 . pie-1-mediated repression, however , is absent after the middle stages of embryogenesis; indeed, the larval germline is both proliferative and transcriptionally active. Therefore, the maintenance of totipotency in the larval and adult germline must allow for full transcription of numerous genes with concomitant repression of somatic differentiation. The mechanisms that restrict germline gene expression appear to coincide with mechanisms that prevent the efficient expression of many transgenes in the germ lineage. Mutations that disrupt transgene silencing in the germline disrupt germline viability; conversely, a subset of sterile mutations, including the mes (maternal effect sterile) mutants identified by the Strome lab, disrupt germline silencing. Other mutations that disrupt germline silencing include a subset of mutator (germline transposon activation) and him (chromosome segregation) mutants; these mutants also exhibit various degrees of germline defects. Our working hypothesis is that these mutations disrupt some aspect of germline chromatin organization. The processes by which germline silencing is maintained have an epigenetic nature-- that is, both the silenced and desilenced states are passaged, or "remembered" through multiple cell divisions and, in some cases, multiple generations. This "memory" is evidenced by a lag period between the disruption of components of the process, and manifestation of the phenotype. Hence, as in the case of the mes mutations, sterility is delayed for a full generation after homozygosity of the mutation. Acetylation of nucleosomal histones has been shown to play a role in epigenetic inheritance in many systems. We are currently investigating the functional significance of correlations between histone under-acetylation and germline silencing.

1Seydoux, G., Mello, C. C., Pettitt, J., Wood, W. B., Priess, J. R., Fire, A. (1996). Nature 382: 713.

24 May 1999 15:50 66 66 Transposon silencing in C.elegans.

1999 International Worm Meeting abstract 15 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Transposon silencing in C.elegans. René F. Ketting, Henri G.A.M. van Luenen, Miriam T. Smits, Ronald H.A. Plasterk Div. Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands The genome of C.elegans contains many different transposable elements. The most active of these is the Tc1 element. Although active in somatic cells, this element does not jump in the germline of wildtype Bristol N2 worms. To understand this, we searched for mutants in which the Tc1 element is activated in the germline of Bristol N2. We performed two screens, with two different markers to score for Tc1 activation: unc-54(r323::Tc1) and unc-22(st136::Tc1). In the normal Bristol background these alleles are completely stable. However, when Tc1 is activated reversion of the associated phenotype will be observed. We found 42 mutants, at a frequency of around 3*10-3 , indicating that the mutants are regular loss of function alleles. One of the mutants, mut-7(pk204), was analysed in further detail. Mut-7(pk204) was found to act semidominantly; transposition frequencies of heterozygous mut-7 worms are around 10% of that of the homozygous worms. Given the mutation frequency this is likely to reflect haplo-insufficiency of the gene product. We mapped the locus to LGIII, in the area between unc-16 and unc-47. We hope to have the gene identified by the time of the meeting. Apart from transposon activation, mut-7(pk204) worms display chromosome non-disjunction, visualised by a Him phenotype, and sterility. Like the transposition frequency, both these phenotypes are temperature sensitive. At 25°C we find almost 4% males and almost 100% sterility. All other mutant alleles tested, including mut-2, mut-4, mut-5 and mut-6 are also temperature sensitive sterile. This suggests that they all act in the same pathway. Complementation analysis has shown that 9 out of 29 of our mutants do not complement mut-7(pk204), suggesting we are dealing with a limited set of genes. Besides Tc1, also Tc3, Tc4 and Tc5 were found to be activated by mut-7(pk204). How can one pathway activate such diverse transposons? A clue comes from our observation that gene expression from simple repetitive arrays can be seen in the germline of mut-7 worms1) . This suggests that mut genes regulate transposons by repression of their transposase genes in the germline.

1) Transgenic strain kindly provided by Bill Kelly and Andy Fire

24 May 1999 15:50 67 67 UNC-43 CaMKII regulates the density of central glutamatergic synapses in vivo

1999 International Worm Meeting abstract 16 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. UNC-43 CaMKII regulates the density of central glutamatergic synapses in vivo Christopher Rongo, Joshua Kaplan Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200 Synaptic connections undergo a dynamic process of stabilization or elimination during development. To determine how central synapses change during development, we observed neuron-neuron synapses in C. elegans that contain the AMPA-type GluR GLR-1. We previously showed that chimeric receptors tagged with the GFP (GLR-1::GFP) can be used to visualize glutamatergic synapses. In late L1 animals, GLR-1::GFP becomes localized to synapses along the lengths of axons in both the ventral cord and nerve ring processes. During larval growth, the density of GLR-1 synapses is held constant despite dramatic increases in neurite length. Here we describe the role of the UNC-43 type II calcium and calmodulin dependent protein kinase (CaMKII; D. Reiner, E. Newton, & J. Thomas, pers. comm.) in regulating formation of glutamatergic synapses in C. elegans. The coupling of synapse number to neurite length requires both UNC-43, and the UNC-2 and EGL-19 voltage-gated calcium channel subunits. Mutants lacking UNC-43, UNC-2, or EGL-19 accumulate high levels of perinuclear GLR-1::GFP in their cell bodies. Constitutive activation of UNC-43 also resulted in reduced numbers of GLR-1-synapses; however, GLR-1::GFP did not accumulate in the cell bodies of neurons. A GFP::UNC-43 protein containing a mutation that generates a constitutively active kinase was diffusely distributed in axons, unlike wild-type GFP::UNC-43, which was localized to punctate structures in axons. Thus, constitutively activated CaMKII fails to localize to synaptic sites and decreases the density of GLR-1 synapses, suggesting that synaptically localized CaMKII is required to add or maintain GLR-1 at synaptic sites. Our results suggest that CaMKII regulates GLR-1 by two distinct mechanisms: regulating transport of GLR-1 from cell bodies to axons, and regulating the addition or maintenance of GLR-1 to synapses.

24 May 1999 15:50 68 68 A C. elegans liprin protein SYD-2 regulates presynaptic active zones

1999 International Worm Meeting abstract 17 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A C. elegans liprin protein SYD-2 regulates presynaptic active zones M Zhen 1,2 , Y Jin 1

1 Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064. 2 e-mail: [email protected]

Chemical synapses allow information flow between neurons and their target cells. At synaptic junctions, specialized cellular structures are developed in both pre- and post-synaptic cells to facilitate synaptic transmission. Presynaptic structure is characterized by the accumulation of synaptic vesicles and an electron-dense membrane structure called the active zone. To elucidate the molecular mechanisms underlying the presynaptic architecture, we carried out a genetic screen for mutants with abnormal 1,2 presynaptic morphology of GABAergic motoneurons using the Punc-25 -SNB-1::GFP marker . From 5,000 haploid genomes, we identified 12 new genes, called syds (synapse defective), which are required for expression and localization of the SNB-1::GFP marker1 . We report here the cloning and functional analysis of the syd-2 gene. In syd-2 mutants axonal outgrowth is normal but the SNB-1::GFP marker and other presynaptic proteins are diffusely localized at the nerve termini, suggesting that syd-2 specifically affects synapse formation. syd-2 encodes a C. elegans liprin. Liprins have been shown to interact with the second phosphatase domain of LAR-type receptor-tyrosine phosphatases (RPTPs) and localize RPTPs to focal adhesions in mammalian cell cultures3 . SYD-2 protein is expressed in the presynaptic region independent of synaptic vesicles, indicating that it might be a structural component in the presynaptic termini. We analyzed the ultrastructure of chemical synapses in syd-2 animals, and found that the total synaptic vesicle number per synapse was not altered in syd-2 mutants, whereas the length of active zones was significantly increased. We propose that SYD-2 plays a role in synapse formation by serving as an anchor protein to regulate active zone formation. SYD-2 might regulate a tyrosine phosphorylation signaling cascade at synaptic junctions by recruiting RPTPs.

1. Zhen and Jin (1997) 11th International Worm Meeting Abstract 682. 2. Nonet (1999) J. Neurosci. Methods (in press) 3. Serra-Pagès et al. (1998) J. Biol. Chem. 273: 15611-15620.

24 May 1999 15:50 69 69 Synaptic choice is regulated by UNC-4 and UNC-37-dependent repression of motor neuron-specific genes.

1999 International Worm Meeting abstract 18 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Synaptic choice is regulated by UNC-4 and UNC-37-dependent repression of motor neuron-specific genes. AR Winnier, JY Meir, JM Ross, KM Lickteig, DM Miller Dept. of Cell Biology, Vanderbilt Univ. Med. Ctr, Nashville, TN 37232 Animal movement depends on axial arrays of motor neurons that transmit signals from the brain to muscles. Although it is clear that the function of these circuits is defined by the creation of synapses between specific sets of cells, the factors that control these choices are largely unknown. In C. elegans, synaptic input to VA motor neurons is defined by the cell autonomous function of the UNC-4 homeobox protein. unc-4 mutants are unable to crawl backward because VA motor neurons are miswired with inputs from an interneuron (AVB) normally reserved for their sister cells, the VB motor neurons. UNC-4 contains a C-terminal domain that resembles the Engrailed eh1 repressor domain. Missense mutations in the UNC-4 eh1 lead to ectopic expression in the VAs of the VB-specific genes, acr-5 and del-1. Therefore, we hypothesize that UNC-4-dependent repression of VB-specific genes prevents the VAs from adopting VB-type synaptic inputs. Here we show that UNC-4 repressor activity depends on physical contact with the Groucho-like co-repressor UNC-37 and that this interaction is mediated by the UNC-4 eh1 domain. The backward movement defect of UNC-4 eh1 mutants is suppressed by a dominant unc-37 missense mutation (E580K) in the WD-40 protein-protein interaction domain. We have shown that the restoration of normal locomotion in these unc-4-unc-37 double mutants is correlated with repression of acr-5 and del-1 GFP reporter genes in the VAs. Furthermore, yeast two-hybrid assays have shown that eh1 missense mutations disrupt UNC-4 affinity for UNC-37 and that these interactions are restored by the E580K WD-40 point mutation. Therefore, our data indicate that UNC-4 eh1 mutations perturb UNC-4 function by blocking interaction with the co-repressor protein UNC-37 and that these interactions, as well as UNC-4 repressor activity, can be restored by a specific missense mutation in UNC-37. We propose that unc-4 specifies synaptic input to the VA motor neurons solely by preventing the expression of VB-type genes. This conclusion argues that there must exist an unc-4-independent mechanism that actively promotes the establishment of VA-type inputs and that derepression of VB-type genes is dominant to this pathway.

24 May 1999 15:50 70 70 A C. elegans JNK signal transduction pathway regulates coordinated movement via type-D GABAergic motor neurons

1999 International Worm Meeting abstract 19 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A C. elegans JNK signal transduction pathway regulates coordinated movement via type-D GABAergic motor neurons M Kawasaki, N Hisamoto, J Ninomiya-Tsuji, K Matsumoto Division of Biological Science, Graduate School of Science, Nagoya University, and CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya 464-8602, JAPAN The c-Jun NH2-terminal kinase (JNK) of the mitogen-activated protein kinase superfamily is activated in response to a variety of cellular stresses and is involved in the apoptosis in neurons. However, the roles of JNK signaling pathway in the nervous system are unknown. The genes for the C. elegans homolog of JNK, jnk-1, and its direct activator, jkk-1, were isolated based on their abilities to function in the yeast Hog1 MAP kinase pathway. JKK-1 is a member of the MAP kinase kinase superfamily and functions as a specific activator of JNK. Both jnk-1 and jkk-1 are expressed in most neurons. jkk-1 null mutant animals (km2) exhibit defects in locomotion that can be rescued by the conditional expression of JKK-1 in mutant adults, suggesting that the defect is not due to a developmental defect. Mosaic analysis suggested that ventral nerve cord motor neurons are important for coordinated movement in the mutant animals. To investigate whether expression of jkk-1 in D-type motor neurons is important for its effect on locomotion, we expressed the jkk-1 cDNA under the control of the promoter for the unc-30 gene, whose expression is restricted primarily to the DD and VD neurons. The plasmid containing a Punc-30::jkk-1 transgene was introduced as an extrachromosomal array into jkk-1(km2) mutant animals. When we examined locomotion phenotypes of ectopic JKK-1-expressing animals, we found that the Punc-30::jkk-1 transgene could rescue the jkk-1 movement defects. The unc-25 gene, which encodes the GABA biosynthetic enzyme glutamic acid decarboxylase, also effects locomotion via its effect on D-type motor neurons. To investigate the functional relationship between unc-25 and jkk-1, double unc-25(e156); jkk-1(km2) mutant was constructed and characterized. unc-25(e156); jkk-1(km2) double mutants moved similar to that of unc-25(e156) mutants. Thus, the phenotype in jkk-1 mutants requires unc-25 activity. These data suggested that the C. elegans JNK pathway functions in type D GABAergic motor neurons and thereby modulates coordinated locomotion.

24 May 1999 15:50 71 71 vab-7 controls the DB neuron fate

1999 International Worm Meeting abstract 20 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. vab-7 controls the DB neuron fate B Esmaeili 1 , J Ross 2 , C Neades 1 , D Miller 2 , J Ahringer 1

1 Wellcome/CRC Institute, Cambridge, UK. 2 Vanderbilt University, Nashville, TN.

The C.elegans even-skipped homologue, vab-7, is required for posterior muscle and epidermal patterning. vab-7 mutants are also forward Unc. VAB-7 is expressed in DB motorneurons, which innervate the dorsal body muscles and are required for proper forward movement. To examine the morphology of DB neurons, we used an unc-129::gfp reporter gene, which labels DA and DB processes and cell bodies. Both DA and DB neurons send circumferential commissures to the dorsal nerve cord (DNC), but on reaching it, DAs send axons anteriorly and DBs posteriorly. We found that in vab-7 mutants DB neurons have the same axonal polarity as the DAs (anteriorly directed) in the DNC, suggesting that they have DA characteristics. To investigate this further, we examined the expression of UNC-4, a homeodomain protein expressed in the DAs; unc-4::gfp is ectopically expressed in the DBs in vab-7 mutants. unc-4 is responsible for the change in polarity of DBs in vab-7 mutants, since their polarity is restored to wild type in unc-4; vab-7 double mutants. Furthermore expression of the DB marker, acr-5::gfp (a nicotinic acetylcholine a-receptor subunit) is lost in vab-7 mutants, also because of ectopic unc-4 expression. These data indicate that vab-7 is required for the DB fate. To investigate the sufficiency of vab-7 for DB fate determination we ectopically expressed vab-7 under the control of the unc-3 promoter, which drives expression in the DAs and DBs and some postembryonic neurons (AS, VA, VB). This causes DAs to have DB characteristics: DA axonal polarity in the DNC is reversed, unc-4::gfp expression is repressed, and acr-5::gfp is ectopically expressed. Furthermore, ectopic expression of vab-7 induces ectopic unc-129::gfp expression in postembryonic neurons, suggesting a possible DB fate acquisition by these neurons. In support of this model, the postembryonic AS neurons, which have anteriorly directed axons in the DNC, have reversed (posterior) polarity when vab-7 is ectopically expressed. Therefore, vab-7 is sufficient to induce at least some aspect of DB fate in both embryonic and postembryonic neurons. Interestingly, even-skipped in Drosophila also controls the guidance of neurons that innervate dorsal muscles, so there is conservation in the function of these proteins.

24 May 1999 15:50 72 72 Genes involved in the formation of ciliated endings of sensory neurons in C. elegans

1999 International Worm Meeting abstract 21 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genes involved in the formation of ciliated endings of sensory neurons in C. elegans P Swoboda, JH Thomas Department of Genetics, University of Washington, Seattle, WA 98195, U.S.A. Cilia are an important cellular differentiation of sensory neurons for receiving information from the environment. 60 of the 302 C. elegans neurons have ciliated endings. Mutations in a large number of genes have been identified that affect the structure of these sensory cilia. Of these, daf-19 mutations are unique in completely lacking all sensory cilia. This was shown by electron microscopy and by analysis of transgenic animals expressing GFP in different sensory neurons. Aside from the absence of sensory cilia, the neurons seem to be morphologically normal. We previously reported that daf-19 encodes an RFX-type transcription factor. This class of proteins has been found in humans, mice and yeast. From work with mammalian cell cultures the RFX-binding sites (x-boxes; 14-16 bp long) for some members of the family are known. We found x-box-like sequences in the promoter regions of genes known to be expressed or to function in ciliated sensory neurons. As of now we have tested whether daf-19 transcriptionally regulates three of these genes in vivo by fusing their promoters to GFP (che-2, osm-1, osm-6; containing either wild-type or mutated x-box sequences, respectively). We found that these promoters drove GFP expression only in ciliated sensory neurons and that expression was dependent on daf-19 function and on the x-box sequence. Using these C. elegans x-box sequences as a guide, we searched the C. elegans genome database and identified 20-30 additional x-boxes which, by sequence similarity and position within the promoter region, define new genes that are potential daf-19 regulatory targets. Our results strongly suggest that DAF-19 regulates the differentiation of sensory cilia by activating the transcription of a battery of genes whose products form the sensory cilium.

24 May 1999 15:50 73 73 Cyclic GMP Signaling via ODR-1, DAF-11 and PKG-1

1999 International Worm Meeting abstract 22 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cyclic GMP Signaling via ODR-1, DAF-11 and PKG-1 ND L’Etoile, Y Zhang, CI Bargmann Dept. Anatomy and HHMI, UCSF A wide variety of attractive odors are sensed by just two pairs of sensory neurons: AWA and AWC. The rudiments of olfactory signaling within AWC have been sketched out: G-protein coupled receptors initiate a signaling cascade which may end with the opening of the heteromeric cGMP liganded cation channel comprised of TAX-2 and TAX-4. How is specificity of signaling insured so that individual odors can be distinguished and how is the animal able to adapt to an ever-present odor? ODR-1 and DAF-11 are receptor guanylyl cyclases (RGCs) required for AWC and AWB mediated chemotaxis and repulsion (Birnby et al. 1995 IWM abs. #65; L’Etoile and Bargmann 1997 IWM abs. #335). These molecules are postulated to provide the second messenger cGMP for olfactory signaling. In other systems cGMP production is stimulated by an extracellular ligand’s binding to the receptor domain. Does ODR-1 act as a receptor for odorants? Probably not: an ODR-1 molecule that does not have a receptor domain is still able to effect chemotaxis towards odorants. Interestingly, worms that overexpress ODR-1 have normal chemotaxis but are defective in odorant discrimination and sensory information processing: these worms are no longer able to adapt to butanone or isoamyl alcohol and are also unable to distinguish between butanone and benzaldehyde in behavioral assays. The overexpression phenotypes of odr-1 may be a consequence of exuberant cGMP production. Alternatively, overexpressed ODR-1 may bind to and sequester a protein (e.g. OSM-9) required for adaptation. We are testing the predictions of these models. Preliminary immunological studies indicate that both proteins are localized in the cilia of AWC. We characterized a mutant, ky95, that suppresses the chemotaxis defect of a daf-11 mutation but does suppress odr-1. This phenotype indicates that ODR-1 can act independently of DAF-11 to effect chemosensation. ky95 animals exhibit normal chemotaxis, but like ODR-1 overexpressers they have defects in olfactory discrimination and they do not adapt to any of the AWC responsive odorants tested. Preliminary identification of ky95 as encoding a cGMP dependent protein kinase leads (PKG-1) to the speculation that cGMP is used as a signal to both initiate and extinguish the chemosensory pathway that leads to olfaction.

24 May 1999 15:50 74 74 The LIM homeobox gene lim-4 distinguishes between two olfactory neuron fates

1999 International Worm Meeting abstract 23 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The LIM homeobox gene lim-4 distinguishes between two olfactory neuron fates A Sagasti 1 , O Hobert 2 , G Ruvkun 3 , C Bargmann 1

1 UCSF and HHMI, San Francisco, CA 94143. 2 Columbia University, New York. 3 Mass. General Hospital, Boston.

The promoter region of the C. elegans candidate olfactory receptor str-2 drives expression of GFP in a single AWC olfactory neuron (see abstract by E. Troemel). The AWC neurons are required for attractive chemotaxis behaviors in response to a specific subset of attractive volatile odorants. In order to investigate how the unique features of olfactory neurons are determined, we performed a screen for misexpression of str-2::GFP. Three mutants in one gene were isolated that express the reporter in an ectopic pair of sensory cells, the AWB olfactory neurons, which mediate repulsive olfactory responses. Many features of the AWB cells have been transformed towards an AWC fate in this mutant. Not only is the str-2::GFP AWC marker ectopically expressed in AWB, but str-1::GFP, an AWB marker, is turned down in the mutants and the distinct cilia and axon morphologies of the AWB neurons are transformed into more AWC-like morphologies. AWB function is also transformed--rather than mediating repulsive behavioral responses, the AWB cells in this mutant mediate attractive responses, like AWC. Rescue of these mutant phenotypes was obtained with a clone of the LIM homeobox gene lim-4, and genetic lesions have been found in all three alleles of the gene. LIM-4 is expressed in AWB, as well as a few other head neurons, and is required for its own expression in AWB. Ectopic expression of LIM-4 in the AWC neuron pair is sufficient to force those cells to adopt an AWB fate. LIM-4 thus acts as a switch that distinguishes between alternative sensory neuron fates.

24 May 1999 15:50 75 75 A Forkhead Homolog Involved in Specifying Chemosensory Neuron Cell Fate

1999 International Worm Meeting abstract 24 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A Forkhead Homolog Involved in Specifying Chemosensory Neuron Cell Fate TR Sarafi-Reinach, P Sengupta Dept. of Biology; Brandeis University; Waltham, MA 02454 We are interested in understanding how the diverse functions of chemosensory neurons are specified during development. To address this question, we have identified and characterized mutants defective in sensory neuron development. We expressed GFP specifically in the AWA neurons, which sense a subset of volatile attractants. Worms carrying this GFP construct were mutagenized, and mutants showing aberrant marker expression were identified. One mutant, previously referred to as sns-3(oy10), shows ectopic expression of the AWA marker odr-7::GFP in an additional pair of neurons. These neurons also express ODR-7 protein and other AWA markers including odr-10::GFP and gpa-5::GFP. The ciliary morphology of these neurons resembles that of AWA as well. The ectopically expressing neuron is in the correct position to be AWA’s lineal sister ASG. Indeed, preliminary evidence suggests that in sns-3(oy10) animals, expression of the ASG marker unc-30::GFP is frequently lost. This suggests that in sns-3 mutants, ASG has been transformed into AWA. This perturbation of cell fate seems to be specific for the AWA/ASG lineage in the head. We have used markers to examine the fate of other head neurons including AWB, AWC, ASH, AFD, and AIY. While some of these neurons show minor abnormalities, none are perturbed to the same degree as AWA/ASG. We have cloned sns-3(oy10) and found that it is an allele of unc-130 , a forkhead homolog previously cloned by Nash and Culotti. unc-130 has distal tip cell migration defects similar to defects seen in unc-5, unc-6, and unc-40 mutants. We found no changes in AWA/ASG cell fate in these other mutants. unc-130(oy10) has a missense mutation in a conserved residue of the forkhead domain. We will be examining additional unc-130 alleles generously provided by Nash and Culotti, to determine the extent of their AWA/ASG defects. Preliminary evidence indicates that unc-130 is not expressed in AWA or ASG in adults; however, it does show broad embryonic expression which we are currently investigating. These results indicate that mutation of a forkhead homolog results in a cell fate conversion, in which ASG adopts an AWA-like cell fate. Further experiments are underway to test this model.

24 May 1999 15:50 76 76 unc-130 is required to maintain the correct expression pattern of unc-129 in C. elegans.

1999 International Worm Meeting abstract 25 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. unc-130 is required to maintain the correct expression pattern of unc-129 in C. elegans. B Nash, JG Culotti Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto and Department of Medical and Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. unc-130 is required for the dorsoventral guidance of pioneer growth cones and distal tip cells during development in C. elegans. The gene encodes a putative transcription factor with a highly conserved forkhead domain. In order to characterize the role of unc-130 in circumferential guidance, we have constructed double mutants between unc-130 and other mutants known to be required for this process. unc-5, unc-6 and unc-40 are all required for dorsalward guidance of distal tip cells and pioneer growth cones (Hedgecock et al,1990. Neuron 4, 61-85). Double mutants between null alleles of unc-130 and either unc-5 or unc-6 both have more penetrant dorsalward guidance defects than the respective single mutants, suggesting that unc-130 acts, at least partially, in a parallel pathway. unc-129, a TGF-beta, is also required to guide pioneer growth cones along the dorsoventral axis (Colavita et al, 1998. Science 281, 706-709). unc-129 mutants do not, however, have defects in the dorsalward migration of the distal tip cell. Strikingly, unc-129; unc-130 double mutants have much less penetrant distal tip cell migration defects than the unc-130 alleles. unc-129::GFP is expressed in dorsal body wall muscle but not ventral muscle. This pattern of expression is important for unc-129 function (Colavita et al). However, in unc-130 mutants unc-129::GFP can be detected in both dorsal and ventral muscles. Thus, forkhead UNC-130 appears to be required to maintain the correct spatial expression pattern of the TGF-beta UNC-129. We have recently discovered that mutations in unc-130 also disrupt the morphology of sensory rays in the male tail. The defects are most similar to those seen in mab-21 mutants. The male tail defects appear to act through a separate pathway, as unc-129 mutants do not suppress this phenotype. An unc-130::GFP fusion construct is expressed in hypodermal cells, intestinal cells and in ventral body wall muscle. We are presently investigating where unc-130 is required by mosaic analysis.

24 May 1999 15:50 77 77 A Screen for Suppressors of unc-6DC: Interactions between UNC-6 and NPR-1, a Neuropeptide Y Receptor

1999 International Worm Meeting abstract 26 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A Screen for Suppressors of unc-6DC: Interactions between UNC-6 and NPR-1, a Neuropeptide Y Receptor Q Wang, WG Wadsworth Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway NJ 08854 The expression of unc-6DC transgenes cause the longitudinal motoneuron axons of the ventral nerve cord to migrate circumferentially (see Lim et al, this meeting). To better understand the mechanisms involved in this process, we undertook a genetic screen for suppressors of the ventral nerve cord phenotype. We took advantage of one transgenic line that exhibits the ventral nerve cord phenotype in the unc-6(+) background and causes the animals to be slightly Unc. In the screen, we first selected animals that reverted to wild type movement. These were then checked for the expression of the transgene and suppression of the ventral nerve cord phenotype. From a screen of 40,000 mutagenized haploid genomes, 6 mutations were recovered. We have found by non-complementation test and DNA transformation that one of the suppressors, ur89, is an allele of npr-1, which encodes a neuropeptide Y receptor (de Bono et al, Cell 94, 1998)*. npr-1 (ur89) and npr-1(ad609), suppress the unc-6DC phenotype while a weak allele, npr-1(n1353), does not. The expression of unc-6DC suppresses the clumping phenotype of npr-1 mutants. We are currently testing other alleles of npr-1 and are sequencing the ur89 allele. The genetic interactions between NPR-1 and UNC-6 are intriguing, we speculate that there are receptor complexes on the surface of the ventral cord interneurons and motoneurons that regulate how the axons respond to UNC-6 and fasciculation cues. We have tested three of the other mutations and they do not have the npr-1 phenotypes. One mutation complements npr-1 and others have not been tested.

*We are grateful to Mario de Bono and Cory Bargmann for providing npr-1 alleles and DNA.

24 May 1999 15:50 78 78 Mechanisms of axon pathfinding by SAX-3

1999 International Worm Meeting abstract 27 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Mechanisms of axon pathfinding by SAX-3 J Hao, J Zallen, D Lee, K Prehoda, M Tessier-Lavigne, W Lim, CI Bargmann UCSF, San Francisco, CA 94143. We are studying the mechanisms by which the axon guidance receptor sax-3 directs axonal outgrowth. sax-3 mutants exhibit a variety of axon phenotypes, including anterior misrouting of axons in the nerve ring, abnormal midline crossing in the ventral cord, and defects in ventrally-directed guidance (e.g. AVM, HSN). sax-3 encodes a transmembrane protein with five immunoglobulin and three fibronectin type III repeats, and has close homologs in flies (roundabout) and vertebrates (rat and human Robo1, Robo2). Expression of sax-3 in AVM under the mec-7 promoter rescues its ventral defect, arguing that sax-3 acts as a receptor. What is the signal to which sax-3 responds? One candidate is slit, a secreted molecule first identified in a screen for midline CNS mutants in flies. Experiments with a worm homolog of slit demonstrate that high levels of slit expression are found in regions avoided by sax-3-dependent axons. In particular, slit is expressed strongly in dorsal, but not ventral, body wall muscle, and in epidermal cells that lie at the anterior boundary of the nerve ring. This expression pattern suggests a model in which sax-3 is a repulsive receptor that directs axons away from sources of slit. In support of this model, misexpression of slit in ventral muscles is sufficient to disrupt AVM ventral growth, an effect which is dependent upon the presence of sax-3. How does sax-3 elicit changes in growth cone behavior? Binding studies show that a conserved motif in the cytoplasmic domain of sax-3 interacts with enabled, a protein implicated in actin assembly, Listeria motility and cell protrusion. In support of a role of enabled in sax-3 guidance, mutations in unc-34, a worm homolog of enabled, show strong genetic interactions with sax-3, in which weak unc-34 alleles dominantly enhance axon defects of a weak sax-3 allele. We have found that a second conserved motif in SAX-3 binds to a C. elegans homolog of dreadlocks, a SH2/SH3 adapter molecule first identified in flies in a screen for axon guidance mutants in the visual system. Currently we are focusing on obtaining more functional evidence for role of unc-34/ena and dock in sax-3 guidance. These studies may provide a link between guidance events at the membrane and known modulators of the cytoskeleton.

24 May 1999 15:50 79 79 UNC-53 is involved in growth cone steering and co-localises with microtubule plus-ends

1999 International Worm Meeting abstract 28 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. UNC-53 is involved in growth cone steering and co-localises with microtubule plus-ends Marc Van de Craen 1 , Luc Maertens 1 , Nina Cromheecke 1 , Marleen Brunain 1 , Peter Verhasselt 2 , Marc De Raeymaeker 1 , Walter Luyten 2 , Christ Platteeuw 1 , Joel Vandekerckhove 3 , Johan Geysen 2 , Thierry Bogaert 1

1 Devgen N.V. Technologiepark 9, Zwijnaarde, Belgium. 2 Functional Genomics, Janssen Research Foundation, Turnhoutseweg 30, Beerse, Belgium. 3 Flanders Interuniversity Institute for Biotechnology, Dept Medical Protein Chemistry, Ledeganckstraat 35, Gent.

Growth cone steering is a cyclic process which starts with filopodia outgrowth at the leading edge of the cell followed by the reading and integration of multiple directional signals that are translated into local stabilisation of the F-actin cytoskeleton in one of the growth cone spikes. Subsequent interaction of the F-actin cytoskeleton with microtubule plus-ends leads to permanent stabilisation of a part of the growth cone in the new direction. Finally, a new cycle of filopodia outgrowth is initiated in the new direction. UNC-53 (Steerin) is a novel nematode signal transduction molecule that integrates extracellular signals into cellular responses which determine the direction of migration of hypodermal cells, neurons, myoblasts and the excretory cell in C. elegans. Based on homology, Steerin is predicted to contain an N-terminal alpha-actinin-like F-actin binding domain and a C-terminal nucleotide-binding domain. The middle region predominantly consists of alpha helical structures comprising two proline rich helices and three coiled coils. A family of structurally and functionally related human Steerins has been cloned in a collaborative effort. Recombinant Steerin was shown to bind recombinant GST-SEM-5 and GST-GRB-2, suggesting that Steerin converts receptor tyosine signalling into directional signals. Steerin can also physically associate with the cytoskeleton via F-actin and microtubule plus-ends. Biochemical studies illustrated that Steerin can directly interact with F-actin. Steerin was shown to co-localise with microtubules with preference for the microtubule (+)-ends using monoclonal antibody post-fixation immunofluorescence and transient overexpression of GFP-Steerin fusion proteins in living mammalian cells. Overexpression of Steerin and a number of deletion mutants in mammalian cells has a positive effect on neuronal outgrowth, filopodia production, lamellipodia formation and cell motility.

24 May 1999 15:50 80 80 How the EGL-20/Wnt protein specifies two different migratory behaviors in the Q cell lineage

1999 International Worm Meeting abstract 29 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. How the EGL-20/Wnt protein specifies two different migratory behaviors in the Q cell lineage JS Whangbo, CJ Kenyon Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143-0448, U. S. A. The QL and QR neuroblasts are bilateral homologues that give rise to cells that migrate in opposite directions along the anteroposterior (A/P) body axis. These cells undergo identical division patterns, yet they have opposite migratory behaviors. QL and its descendants migrate posteriorly, whereas QR and its descendants migrate anteriorly. We have investigated the role of a C. elegans Wnt homolog, egl-20, in regulating these differences. This gene has two very different roles in Q cell migration. First, egl-20 leads to the expression of the Hox gene mab-5 in QL, but not QR. mab-5 then acts as a developmental switch that programs the QL descendants to migrate into the posterior body region. Interestingly, egl-20 has a very different function in the QR lineage, where it promotes migration toward the anterior. How does egl-20 have such different functions in the QL and QR lineages? By examining the expression pattern of a rescuing egl-20::GFP fusion, we find that EGL-20 is evenly distributed on the left and right sides of the animal. Thus, the different effects of egl-20 on the QL and QR lineages cannot be caused by left-right asymmetric expression of EGL-20. EGL-20 is expressed only in the posterior body region; however, we find that misexpression of the gene in the anterior (using a myo-2::egl-20-GFP fusion) can fully rescue the Egl-20 mutant phenotype, indicating that its asymmetric localization along the A/P body axis is not responsible for the different effects it has on the QL vs. QR lineages. Finally, we asked whether the response of the QL and QR cells to EGL-20 might be dose dependent . By varying the duration of heat-shock in animals carrying a hs::egl-20 fusion, we found that it is. High levels of EGL-20 activate mab-5 expression, whereas low levels promote anterior migration. In addition, we found that although EGL-20 is capable of triggering either response in either Q cell, QL is more sensitive than QR. Thus, the ability of EGL-20 to elicit these different responses resides in mechanisms that allow different levels of EGL-20 to initiate different migratory behaviors, as well as mechanisms that make QL and QR sensitive to different levels of EGL-20.

24 May 1999 15:50 81 81 A C. elegans ror receptor tyrosine kinase regulates cell motility and cell polarity

1999 International Worm Meeting abstract 30 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A C. elegans ror receptor tyrosine kinase regulates cell motility and cell polarity WC Forrester 1 , M Dell 2 , E Perens 2 , G Garriga 2

1 Dept. of Biology, Indiana University, Bloomington, IN 47405. 2 MCB, University of California, Berkeley, CA 94720.

Final destinations of migrating cells often are precisely regulated. Our analysis of cam-1 mutants suggests that cam-1 plays a role in this process. In cam-1 mutants, ALMs, CANs and ccmLs fail to migrate posteriorly to their normal destination. In contrast, anterior-directed migrations of HSN and BDU neurons often extend beyond their normal destinations. To gain insight into its function, we cloned cam-1 . We find that cam-1 encodes a Ror receptor tyrosine kinase (RTK). Rors are a family of RTKs of unknown function identified in Drosophila and humans. Although CAM-1 is expressed globally, mosaic analysis indicates that it functions cell autonomously in CAN cell migration. Overexpression of cam-1 results in incomplete migrations of HSN and BDU neurons, a phenotype opposite to that produced by loss of cam-1 function. CAM-1 also is required to orient cell polarity. Six V cells divide asymmetrically in L1s. In cam-1 mutants, 10% of V1 divisions were reversed. Like V cells, six Pn.aap neuroblasts divide asymmetrically in L1 males to generate CA(n-2) and CP(n-2). CAs appear to accumulate low levels of serotonin. CPs express high levels of serotonin and extend an axon posteriorly to the tail. Instead of extending posteriorly, the CP1 axon of 35% of cam-1 mutants extended anteriorly. We also often detected a weakly staining serotonergic neuron just posterior to CP1, suggesting that polarity of the P3.aap division was reversed. cam-1 interacts with egl-20 to regulate cell migrations and asymmetric cell divisions. egl-20 encodes a WNT signaling molecule (Maloof et al. Develop. 126: 37-49) that acts in V cell polarity (Whangbo et al. WBG 14:58) and HSN migration (Desai et al. Nature 336:638-46); V5 polarity often is reversed and HSNs migrate incompletely. The migration and V cell polarity defects stimulated us to analyze cam-1; egl-20 double mutants. We found that the mutations suppressed each other for HSN migration defects and enhanced each other for V cell polarity defects. Our results suggest that CAM-1 responds to positional information along the A-P axis to guide migrating cells and orient asymmetric cell divisions, and that CAM-1 regulates these processes with EGL-20.

24 May 1999 15:50 82 82 Control of DAF-7 TGF-b expression and neuronal process development by a receptor tyrosine kinase KIN-8 in C. elegans

1999 International Worm Meeting abstract 31 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Control of DAF-7 TGF-b expression and neuronal process development by a receptor tyrosine kinase KIN-8 in C. elegans M Koga 1 , M Take-uchi 2 , T Tameishi 1 , Y Ohshima 1

1 Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan. 2 Department of Genetics, Graduate University for Advanced Studies, National Institute of Genetics, Mishima 411, Japan.

KIN-8 in C. elegans is highly homologous to human ROR-1 and 2 receptor tyrosine kinases of unknown functions. These kinases belong to a new subfamily related to the Trk subfamily. A kin-8 promoter::gfp fusion gene was expressed in ASI and many other neurons as well as in pharyngeal and head muscles. A kin-8 deletion mutant was isolated and showed constitutive dauer larva formation (Daf-c) phenotype: about half of the F1 progeny became dauer larvae when they were cultivated on an old lawn of E. coli as food. Among the cells expressing kin-8::gfp, only ASI sensory neurons are known to express DAF-7 TGF-b, a key molecule preventing dauer larva formation. In the kin-8 deletion mutant, expression of daf-7::gfp in ASI was greatly reduced, dye-filling in ASI was specifically lost and ASI sensory processes did not completely extend into the amphid pore. The Daf-c phenotype was suppressed by daf-7 cDNA expression or a daf-3 null mutation. ASI-directed expression of kin-8 cDNA under the daf-7 promoter or expression by a heat shock promoter rescued the dye-filling defect, but not the Daf-c phenotype, of the kin-8 mutant. These results show that the kin-8 mutation causes the Daf-c phenotype through reduction of the daf-7 gene expression and that KIN-8 function is cell-autonomous for the dye-filling in ASI. KIN-8 is required for the process development of ASI, and also involved in promotion of daf-7 expression through a physiological or developmental function.

24 May 1999 15:50 83 83 SDC-2 triggers hermaphrodite sexual development and targets dosage compensation machinery to X chromosomes

1999 International Worm Meeting abstract 32 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. SDC-2 triggers hermaphrodite sexual development and targets dosage compensation machinery to X chromosomes HE Dawes, DM Lapidus, JD Lieb, BJ Meyer HHMI and Department of Molecular and Cell Biology University of California, Berkeley, CA 94720, USA In many sexually reproducing organisms, switch genes coordinately regulate all sex-specific aspects of development. We have shown SDC-2 (Sex and Dosage Compensation) to be the pivotal hermaphrodite-specific factor that initiates sexual differentiation and X-chromosome dosage compensation in C. elegans. SDC-2 acts as both a strong gene-specific repressor and a more modest chromosome-wide repressor to control these two distinct aspects of development. To induce hermaphrodite (XX) development, SDC-2 represses transcription of the male-specific sex gene her-1 by associating with its promoter. In an in vivo assay in which extrachromosomal arrays were tagged with lacO/LacI::GFP, we showed that SDC-2 associates specifically with the her-1 promoter region. This association is prevented by sdc-3(Tra) mutations, which disrupt the sex-specific repression of her-1 but do not affect dosage compensation. To activate dosage compensation, SDC-2 localizes to hermaphrodite X chromosomes and triggers assembly of the dosage compensation complex on X. This complex includes mitotic and meiotic factors that are required in both sexes. SDC-2 appears to associate with X in the absence of other dosage compensation proteins, suggesting a role for SDC-2 in recognizing X chromosomes and thereby conferring chromosome-specificity to dosage compensation. As further evidence for SDC-2’s targeting ability, SDC-2 recruits all members of the dosage compensation complex to her-1 even though many of these proteins are not required for her-1 repression. SDC-2 is not expressed in males. Ectopic expression of SDC-2 in XO embryos triggers assembly of the dosage compensation complex on the male X and causes extensive XO-specific lethality from underexpression of X-linked genes. All these XO animals are rescued by dosage compensation mutations and many develop as fertile hermaphrodites, indicating that SDC-2 can also activate hermaphrodite differentiation in XO animals. Thus, SDC-2 is the key hermaphrodite switch in C. elegans, acting exclusively in XX animals to confer hermaphrodite-specificity to dosage compensation and to induce hermaphrodite sexual development.

24 May 1999 15:50 84 84 Molecular Similarity of Sex Determining Genes In Worms, Flies, and Perhaps Mammals

1999 International Worm Meeting abstract 33 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Molecular Similarity of Sex Determining Genes In Worms, Flies, and Perhaps Mammals C Raymond 1 , J Kettlewell 1 , E Parker 2 , D Zarkower 1,2

1 Biochemistry, Molecular Biology and Biophysics Graduate Program. 2 Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455 USA.

Different phyla employ diverse molecular mechanisms to control sex determination. Recently we found that mab-3 of C. elegans encodes a protein related to the products of the doublesex (dsx) gene of Drosophila. mab-3 is required in males for V ray formation and repression of yolk protein synthesis. MAB-3 and DSX contain a novel DNA binding motif that we have named the DM domain. Both genes are downstream sexual regulators that direct formation of sex-specific peripheral sense organs and repress yolk protein synthesis. Furthermore, the male DSX isoform can replace MAB-3 in the male tail. The similarity of mab-3 and dsx may represent the first example of evolutionary conservation of sex determining mechanisms between phyla. To test the relationship of DSX and MAB-3 further, we compared DNA binding and transcriptional regulatory properties of the two proteins. MAB-3 and DSX bind similar DNA sequences in vitro. All six C. elegans vitellogenin (vit) genes have potential MAB-3 binding sites. A MAB-3 site in vit-2 is required in vivo for repression of vit-2 reporter expression in males, suggesting that MAB-3, like DSX, is a direct transcriptional repressor of yolk protein genes. This is the first connection of regulatory genes to structural genes in C. elegans sex determination. mab-3 mRNA levels are repressed in XX animals by tra-1. A 1500 bp segment of mab-3 upstream DNA, which is sufficient for rescue of mab-3 mutants, directs mostly male-specific reporter gene expression in the intestine and a number of neurons. Mutation of a potential TRA-1 binding site results in elevated reporter gene expression in the intestine of XX animals, suggesting that TRA-1 directly represses mab-3 transcription in that tissue. DM domain proteins may be involved in mammalian sexual development. We have identified five DM domain genes from mouse and human. Two genes, Dmrt1 and Dmrt2, are expressed in embryonic gonad in the mouse. The human homologues of these genes map to a short (250 kb) region deleted in human XY sex reversal. To test the roles of Dmrt1 and Dmrt2 directly, we are making targeted mouse mutants.

24 May 1999 15:50 85 85 The TRA-1 sex-determination protein regulates sexually dimorphic programmed cell death by transcriptionally repressing the egl-1 cell-death activator gene

1999 International Worm Meeting abstract 34 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The TRA-1 sex-determination protein regulates sexually dimorphic programmed cell death by transcriptionally repressing the egl-1 cell-death activator gene B Conradt 1,2 , B Horvitz 2

1 MPI for Neurobiology, 82152 Martinsried, Germany. 2 HHMI, Dept. Biology, MIT, Cambridge, MA 02139, U.S.A.

The cell-death activator gene egl-1 (egl, egg-laying defective) acts as a negative regulator of the cell-death inhibitor gene ced-9 and is the most upstream acting component of the central pathway required for programmed cell death in the C. elegans soma. egl-1 encodes a BH3-containing protein, which has been proposed to activate cell death by binding to and thereby negatively regulating the Bcl-2-like CED-9 protein (1). A loss-of-function mutation in egl-1 blocks most if not all somatic cell deaths that occur during development. This mutation is a five bp deletion in the coding region of egl-1 and results in the formation of a truncated EGL-1 protein (1). Dominant gain-of-function mutations in the egl-1 gene, by contrast, result in ectopic cell death: egl-1(gf) mutations cause the activation of the cell-death pathway in the HSNs (HSN, hermaphrodite-specific neuron) not only in males but also in hermaphrodites, in which the HSNs normally survive (2). The egl-1(gf) mutations are single-base changes within a putative binding site for TRA-1 (TRA, transformer), the terminal and global regulator of somatic sex (3). This site is located 5.6 kb downstream of the egl-1 transcription unit. TRA-1 binds to this site in vitro, and this binding is disrupted by the introduction of the egl-1(gf) mutations. We propose that TRA-1 acts as a repressor of egl-1 transcription in the HSNs to ensure the survival of these neurons in hermaphrodites. This hypothesis is supported by our finding that the tra-1 gene determines the cell-death fate of the HSNs in an egl-1-dependent manner.

1. Conradt, B. and H. R. Horvitz. (1998). Cell 93, 519-529. 2. Trent, C., Tsung, N., and H. R. Horvitz. (1983). Genetics 104, 619-647. 3. Zarkower, D. and Hodgkin, J. (1992). Cell 70, 237-269.

24 May 1999 15:50 86 86 TRA-1 regulates the cellular distribution of the tra-2 mRNA in C. elegans

1999 International Worm Meeting abstract 35 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. TRA-1 regulates the cellular distribution of the tra-2 mRNA in C. elegans LE Graves, SP Scott, EB Goodwin Department of Cell and Molecular Biology and the Lurie Cancer Center, Northwesern University Medical School tra-1 is the terminal regulator in the C. elegans sex-determination pathway, and the activity of the gene is necessary and sufficient for female cell fate specification. TRA-1 is a member of the Kruppel family of zinc-finger transcription factors. Other family members include Ci from Drosophila and the vertebrate GLI proteins, which mediate the hedgehog and sonic hedgehog signals, respectively. The mechanism by which these factors act is poorly understood, and no post-transcriptional activities have previously been identified for these proteins. We now have evidence, however, that TRA-1 may post-transcriptionally regulate the levels and sub-cellular distribution of the tra-2 mRNA. tra-2 is upstream of tra-1 in the genetic hierarchy, and functions to promote female cell fates. Previous reports indicated that tra-1 positively feeds back to regulate tra-2. To explore this interaction, we used RNA in situ analysis to compare the tra-2 mRNA patterns between wild-type and tra-1(null) backgrounds. We found that the tra-2 mRNA is predominately cytoplasmic in the wild-type, but nuclear in the tra-1(null) animals, suggesting that TRA-1 promotes nuclear export of the tra-2 mRNA. This effect of TRA-1 on the tra-2 mRNA is mediated by the tra-2 3’untranslated region (3’UTR), as lack of TRA-1 activity decreases the expression of reporter transgenes containing the tra-2 3’UTR. Furthermore, consistent with the endogenous tra-2 mRNA, the transgene RNA is retained in the nucleus in the absence of TRA-1 activity. This retention is dependent upon a sequence of approximately 30 nucleotides within the tra-2 3’UTR, termed the nuclear localization element. Finally, TRA-1 may be directly affecting the tra-2 mRNA, as TRA-1 binds a sequence within the tra-2 3’UTR which partially overlaps the localization element. These data indicate a post-transcriptional role for TRA-1, and support a model in which the binding of TRA-1 to the tra-2 3’UTR relieves nuclear retention of the tra-2 mRNA.

24 May 1999 15:50 87 87 The Brachyury-related gene mab-9 and cell fate determination in C.elegans: a tale of tails.

1999 International Worm Meeting abstract 36 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Brachyury-related gene mab-9 and cell fate determination in C.elegans: a tale of tails. AC Woollard, JA Hodgkin MRC Laboratory of Molecular Biology, Cambridge, CB2 2QH, UK. Mutants in the mab-9 (male abnormal) gene have grossly deformed male tails lacking most internal structures. This phenotype results from a spatial transformation of the lineage of the major tail-specific blast cell B towards that of its anterior neighbour, Y, and a transformation of the blast cell F lineage towards that of its anterior neighbour U1 . The role of mab-9 during development therefore is to distinguish the fate of B from that of Y, and likewise F from U, giving these cells their spatial identity. In the hermaphrodite, B forms part of the structure of the rectum: mab-9 mutant hermaphrodites have rectal abnormalities and are constipated. Therefore, mab-9 can be viewed as functioning both in the development of the hermaphrodite hindgut and the male tail in C. elegans. We have cloned mab-9 by fine mapping and transformation rescue and find that mab-9 encodes a member of the T-Box family of transcription factors, the prototype of which, Brachyury, was first identified as a mutation disrupting tail development in mouse2 . The Drosophila homologue, brachyenteron, is required for hindgut formation3 . mab-9::GFP localises to the nucleus and is expressed in B and F lineages during larval development. Inappropriate expression of mab-9 can result in embryonic lethality, suggesting that the expression of mab-9 has to be tightly controlled during development. Progress towards the identification of mab-9 regulators and targets will be presented and the possible evolutionary significance of mab-9 will be discussed.

1 Chisholm and Hodgkin, Genes Dev., 3: 1413-1423, 1989

2 Dobrovolskaia-Zavadskaia, C R Sceances Soc. Biol., 97: 114-116, 1927

3 Kispert et al., Genes Dev., 8: 2137-2150, 1994. Singer et al., Development, 122: 3707-3718, 1996

24 May 1999 15:50 88 88 Genetic analysis of male mating behavior: What’s lov got to do with it?

1999 International Worm Meeting abstract 37 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic analysis of male mating behavior: What’s lov got to do with it? MM Barr, PW Sternberg California Institute of Technology, HHMI Vulva location behavior is an attractive model system for studying sensory perception in that information processing may be followed from stimulus (the hermaphrodite vulva) to sensory receptor (the male hook and post cloacal sensilla [pcs]) and ultimately to behavioral response (stop). We have delimited the vulva location signal to the cellular level. Hook neurons sense the 2° lineage-derived vulva cells while the pcs respond to the 1° lineage-derived cells. Ablation studies, SEM analysis, and interspecific behavioral observations suggest mechanosensation and chemosensation may be involved in vulva location behavior. We have initiated a genetic analysis of vulva location behavior, first examining the mating behavior of existing sensory mutants. Only cilium structure mutants were vulva location defective. The observation that only mutants defective in many sensory behaviors exhibit vulva location defects while males with specific sensory defects (such as Mec, Odr, and non-cilia defective Osm and Che mutants) behave essentially as wild type suggests a different set of gene products/molecules mediate vulva location behavior. We next screened for mutants defective specifically in vulva location behavior. lov-1 (for location of vulva defective) is required for two male sensory behaviors, response and vulva location. lov-1 encodes a putative membrane protein with a mucin-like, serine-threonine rich amino terminus followed by two blocks of homology to human polycystins encoded by the autosomal dominant polycystic kidney disease (ADPKD) genes. lov-1 is specifically expressed in adult male sensory neurons of the rays, hook, and head, mediating response, vulva location, and potentially chemotaxis to hermaphrodites, respectively. Polarized localization of LOV-1::GFP to ciliated sensory endings is consistent with a role in sensory reception and signaling.

24 May 1999 15:50 89 89 Genes utilized in protracting the male C. elegans spicules

1999 International Worm Meeting abstract 38 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genes utilized in protracting the male C. elegans spicules LR Garcia, PW Sternberg HHMI & California Institute of Technology, Division of Biology,Pasadena, CA 91125 U.S.A. Insertion of the male spicules into hermaphrodites requires acute control of the spicule muscles. The spicule insertion motor program initially entails a series of rapid contractions of the spicule protractor muscles that allow the spicules to pry apart the vulval lips. When the spicules partially penetrate the vulva, the protractor muscles fully contract, and the spicules extend into the hermaphrodite; further spicule movements cease. Mutations and pharmacological agents that induce spicule protraction are used to dissect this behavior. Four spc (spicule protraction constitutive) genes that regulate the behavior have been identified. Males containing spc-1(sy557), spc-2(sy559), spc-3(sy574), or spc-4(sy558) protrude their spicules in the absence of mating signals. The mutant males are morphologically normal, but ~12 hours into adulthood their spicules spontaneously protract. The spc-1(sy557) and or spc-4(sy558) defects require both the anal depressor muscle and the spicule motor neurons. Ablation of both cell types suppresses the defect, whereas single ablations have no effect; thus 2 circuits, one involving the motor neurons and one involving the anal depressor, potentially regulate the contraction of the spicule muscles. Mutations in unc-36, egl-30, and egl-19 also suppress the sy557 and sy558 defects; thus these mating essential genes are involved in protracting the spicules. Mutation in cha-1 also suppresses sy557, implicating that acetylcholine promotes protraction. The acetylcholine agonists levamisole and arecoline induce spicule protraction. Males missing the spicule motor neurons and the anal depressor still protract when exposed to these agents. Mutations in unc-38, unc-29, and unc-68 completely suppress levamisole-, but not arecoline-induced protraction. These genes are not essential for mating. In contrast, mutation in egl-19 suppresses arecoline-, but not levamisole-induced protraction. Males mutant in both unc-38 and egl-30 are also suppressed for arecoline-induced protraction. Thus we entertain the hypothesis that acetylcholine activates two pathways that promote spicule protraction: one involving unc-38, unc-29, and unc-68, and another involving egl-30 and egl-19; mutations in spc-1 and spc-4 interfere with the regulation of the egl-30-egl-19 pathway.

24 May 1999 15:50 90 90 The PGL Family of P-granule-associated Proteins Interact and Function Redundantly in C. elegans Germline Development

1999 International Worm Meeting abstract 39 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The PGL Family of P-granule-associated Proteins Interact and Function Redundantly in C. elegans Germline Development I Kawasaki, A Amiri, Y Fan, S Strome Indiana University, Bloomington, IN 47405 P granules are distinctive ribonucleoprotein complexes observed in the cytoplasm of germ cells at all stages of development (with the exception of sperm). By molecular genetic analysis of a mutant defective in antibody staining of P granules, we identified PGL-1 as being a protein component of P granules1 . PGL-1, a novel protein, is associated with P granules at all developmental stages. The presence of an RNA-binding motif, an RGG box, at its C-terminus predicts that PGL-1 is an RNA-binding component of P granules1 . pgl-1 mutants contain defective P granules, which lack several P-granule epitopes, and are sterile, with both a maternal and a zygotic component to the sterility1 . Sterility is the result of defects in proliferation and gametogenesis1 . Interestingly, the sterility caused by null alleles of pgl-1 is highly sensitive to temperature1 , suggesting either that PGL-1 functions as a molecular chaperone whose function is required at elevated temperature, or that other partially redundant proteins function with PGL-1 and at low temperature are sufficient for fertility. Using a yeast two-hybrid screen for proteins that interact with PGL-1, we identified PGL-1 itself and two related proteins, which are termed PGL-2 and PGL-3. PGL-3 has 60% identity (74% similarity) with PGL-1 and contains an RGG box at its C-terminus. PGL-3, like PGL-1, is associated with P granules at all stages of development in wild-type worms. However, PGL-3 shows a different distribution than PGL-1 and is present in a distinct gradient in the adult germline. PGL-3 remains associated with P granules in pgl-1 mutant worms. RNAi was used to eliminate PGL-3’s function and to address whether it functions redundantly with PGL-1. pgl-1(bn102); pgl-3(RNAi) worms show significantly enhanced sterility at low temperature, compared to either single mutant alone. Our findings suggest that PGL-1 and PGL-3 proteins interact and function redundantly in P granules during C. elegans germline development. We are investigating whether PGL-2 also is in P granules and participates with PGL-1 and PGL-3 in insuring fertility.

1 Kawasaki et al. (1998) Cell 94: 635-645.

24 May 1999 15:50 91 91 EPS-1 Prevents P-Granule Expression in the Soma

1999 International Worm Meeting abstract 40 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. EPS-1 Prevents P-Granule Expression in the Soma Y Unhavaithaya, T Shin, C Mello U. Mass. Med. Center, Worcester Perhaps the oldest question in developmental biology is what mechanisms distinguish the germline from the soma. Here we describe a new gene, eps-1 (ectopic P-granules in soma), required for this process in C. elegans. eps-1 was identified through a yeast 2-hybrid screen using the germline specific protein, PIE-1, as bait. eps-1(RNAi) causes early larval lethality, characterized by somatic cells that exhibit abnormal germline-like cellular morphology. Consistent with a soma to germline transformation, the arrested larvae show a striking punctate perinuclear expression of P-granule epitopes, K76 (PGL-1), OICID4, GLH-2, and GLH-3, identical to that observed for germline P-granules. This phenotype appears to reflect de-novo zygotic synthesis of P-granules, first the maternal P-granules are segregated properly, second the first ectopic P-granules appear only after the 2-fold stage and third a PGL-1::GFP transgene is activated zygotically in somatic cells with similar timing. Thus EPS-1 appears to function to block the de-novo synthesis of P-granules in the soma. To look for zygotic specific activities associated with eps-1 we used a new mutant strain that lacks maternal RNAi (see abstract by Tabara et al.). In this background eps-1(RNAi) results in viable offspring that bypass the larval arrest and instead form sterile adults with a marked under-proliferation of the germline. Antisera raised against EPS-1 recognize an abundant nuclear protein present in all cells (including the germline) at all stages of development. This staining was nearly completely abolished in eps-1(RNAi) embryos suggesting that this nuclear protein is EPS-1. The EPS-1 protein contains five zinc-fingers similar to those of the Drosophila Su(Hw) (Suppresser of hairy wing) protein. SUHW acts as a transcriptional insulator in Drosophila, blocking the long-range influences of enhancer elements. Perhaps EPS-1 functions as a barrier between somatic and germline specific transcriptional domains in the C. elegans genome. Conceivably this insulating function could work in both directions preventing the spread of germline enhancer activity into somatic genes and vice versa. Understanding the role of PIE-1 and/or other germline factors in regulating EPS-1 should lead to new insights into the mechanisms that distinguish the soma from the germline.

24 May 1999 15:50 92 92 FBF, NANOS-3 and the regulation of germline fates

1999 International Worm Meeting abstract 41 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. FBF, NANOS-3 and the regulation of germline fates Sarah Crittenden 1 , Brian Kraemer 2 , Andrei Petcherski 1 , Shanping Wang 1 , Beilin Zhang 2 , Maria Gallegos 1 , Gary Moulder 3 , Robert Barstead 3 , Marvin Wickens 2 , Judith Kimble 1

1 HHMI and UW-Madison, Madison, WI 53706. 2 UW-Madison, Madison, WI 53706. 3 Oklahoma Medical Research Foundation, Oklahoma City, OK 73104.

Germline fate decisions include mitosis or meiosis, survival or death, and spermatogenesis or oogenesis. FBF-1 and FBF-2 (FBF), nearly identical proteins in the Puf family (for Pumilio and FBF), regulate the hermaphrodite sperm-oocyte switch by repressing the fem-3 3’UTR (1). We now find that FBF also regulates germline proliferation. In fbf(dsRNAi) and fbf-1 fbf-2 double mutants, all germline cells enter meiosis and differentiate as sperm. Genetic experiments place fbf upstream of the gld-1 gld-2 control over meiosis. In gld-1 gld-2 double mutants, all germline cells remain mitotic (2); the same is true of fbf-1 fbf-2; gld-1 gld-2 quadruple mutants. Experiments to determine the regulatory relationships between fbf and glp-1 are in progress. In addition, we have found that fog-1, which originally was identified by its role in sperm specification (3), also plays a role in germline proliferation. Therefore, FBF and FOG-1 provide key links between controls of proliferation and sex determination. Using two-hybrid screens with FBF as bait, we identified NOS-3. The C. elegans genome harbors three nanos homologs. NOS-3 interacts specifically with both FBF-1 and FBF-2, but NOS-1 or NOS-2 do not interact with either. To explore the functions of the three NOS proteins, we used RNAi and isolation of a nos-3 deletion mutant to show that the three nos genes influence the sperm/oocyte switch, and also all three are required for germline survival. The nos effect on germline survival does not require ced-3, and therefore is unlikely to trigger the programmed cell death pathway. In contrast to the nos genes, fbf is not required for germline survival. Therefore, FBF and NOS act together for the sperm/oocyte switch, but have distinct roles in germline proliferation (FBF) and survival (NOS). We propose that distinct partners target FBF and other Puf proteins to different mRNAs, which in turn leads to diverse biological outcomes.

1. Zhang and Gallegos et al. (1997) Nature 390, 477-484. 2. Kadyk and Kimble (1998) Development 125, 1803-1813. 3. Barton and Kimble (1990) Genetics 125, 29-39.

24 May 1999 15:50 93 93 daz-1, a C. elegans homologue of DAZ (Deleted in Azoospermia), is required for the progression of meiosis in oogenesis

1999 International Worm Meeting abstract 42 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. daz-1, a C. elegans homologue of DAZ (Deleted in Azoospermia), is required for the progression of meiosis in oogenesis T Karashima, A Sugimoto, M Yamamoto Department of Biophysics & Biochemistry, Graduate School of Science, University of Tokyo, Tokyo 113, Japan The DAZ family is composed of proteins with a conserved RNP-type RNA-binding motif. Members of this family appear to be involved in meiosis in various organisms. For example, deletion of the DAZ gene cluster on the human Y chromosome correlates with azoospermia. A mutation in the Drosophila homologue of DAZ, boule, results in complete male sterility due to a defect in spermatogenesis. From the genome sequence data, we identified a single C. elegans homologue of DAZ, and named it daz-1. To elucidate the function of this gene, we isolated a deletion allele of daz-1, termed tj3, by the PCR-oriented screen. The daz-1(tj3) homozygous hermaphrodite was sterile with complete penetrance. The gonad of the daz-1 adult hermaphrodite was full of small nuclei, with no cells displaying the characteristic morphology of developing or mature oocytes. However, apparently normal sperm were seen in the spermatheca. By DAPI staining, the majority of the germ nuclei showed morphology similar to those of the pachytene stage, and no diplotene-diakinesis nuclei were found. On the other hand, daz-1 males produced fully-functional sperm. Thus, unlike other DAZ genes, C. elegans daz-1 appears to be despensable for spermatogenesis but essential for meiosis during oogenesis. The effect of daz-1(tj3) was examined in several germ-cell sex determination mutants. In feminized XX animals of fem-3(lf); daz-1 and fog-3(lf); daz-1, defective oogenesis was observed. X0 animals of fog-3(lf); daz-1, which had male soma and female germline, showed similar pachytene arrest of the presumptive oocytes. In contrast, the XX animals of fem-3(gf); daz-1, which had female soma and male germline, produced apparently normal sperm. Taken together, the consequence of the daz-1(tj3) mutation depends on the sexual identity of the germline, regardless of the somatic sex. Northern analysis and in situ hybridization showed that, in hermaphrodite, daz-1 mRNA is present exclusively in germline, starting to emerge at L2 stage. A much weaker signal was detected in adult male gonad. From these observations, we speculate that daz-1 function is essential during oogenesis, especially for the progression of prophase of meiosis I.

24 May 1999 15:50 94 94 Receptor-Mediated Endocytosis in C. elegans

1999 International Worm Meeting abstract 43 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Receptor-Mediated Endocytosis in C. elegans B Grant 1 , Y Zhang 1 , L Pedraza 1 , DH Hall 2 , D Hirsh 1

1 Columbia University College of P&S, Dept. of Biochemistry New York, NY 10032. 2 Albert Einstein College of Medicine, Center for C. elegans Anatomy Bronx, NY 10461.

Endocytosis, the vesicle-mediated internalization of membrane or membrane bound macromolecules, mediates many key aspects of cellular function and development. These functions include uptake of nutrients, synaptic recycling, membrane turnover, and regulation of growth-factor receptor signaling. Although the basic kinetics and morphology of endocytic processes have been well described, many questions remain about the identity and function of the proteins which mediate endocytosis. We have identified C. elegans mutants (receptor-mediated endocytosis genes) in which endocytosis of a YP170(yolk protein)::GFP fusion by oocytes is defective. We describe here two of these genes: rme-2, which encodes a putative receptor for yolk proteins, and rme-1, which encodes a novel endosome associated protein, which may mediate endocytic trafficking in all cells. rme-2 mutants produce slightly small oocytes which appear morphologically normal but are refractory to the uptake of YP170::GFP and endogenous yolk. We found that rme-2 mapped near a predicted member of the LDL-receptor superfamily T11F8.3, and that each of our 3 rme-2 alleles was associated with specific lesions within the T11F8.3 gene. Furthermore, T11F8.3(RNAi) closely phenocopied the rme-2 mutant phenotype. Affinity purified antisera against the predicted intracellular and extracellular domains of RME-2 specifically stained the oocyte plasma membrane. Under certain circumstances RME-2 and YP170::GFP can co-localize to the same endocytic vesicles within oocytes. Furthermore, muscle cells forced to ectopically express RME-2 bound YP170::GFP, unlike wild-type muscle. These findings indicate that RME-2 is the C. elegans yolk receptor. We have also analyzed the rme-1 gene, which encodes a member of a new class of EH domain protein conserved through evolution. rme-1 mutants display reduced yolk uptake by oocytes, and accumulate large vacuolar structures in intestine and hypodermis. We find that RME-1 is a widely expressed cytoplamic protein associated with endosomal membranes. Experiments are underway to analyze rme-1 function in the endosomes of the intestine, and to study the rme-1 phenotype by electron microscopy.

24 May 1999 15:50 95 95 Identification of RNA Targets of GLD-1

1999 International Worm Meeting abstract 44 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification of RNA Targets of GLD-1 M Lee 1 , B Grant 2 , D Hirsh 2 , T Schedl 1

1 Genetics, Washington University School of Medicine. 2 Biochem & Mol. Biophysics, Columbia University.

The gld-1 gene has multiple functions in C. elegans germline development. It has an essential function in female meiotic prophase progression and oocyte differentiation. In gld-1 null hermaphrodites, female germ cells enter meiotic prophase and progress to early pachytene but then inappropriately leave meiotic prophase and proliferate mitotically producing a germline tumor. gld-1 also has a nonessential, redundant function to initiate meiotic development and functions to promote male sex determination in the hermaphrodite germline. GLD-1 is a cytoplasmic germline protein containing a KH RNA binding motif. GLD-1 may thus bind and regulate the translation or stability of germline mRNAs. To identify in vivo RNA targets of GLD-1, a biochemical approach has been employed. A FLAG-tagged GLD-1 (GLD-1/FLAG) transgene was used to rescue a gld-1 null mutant. Cytoplasmic lysates were prepared from adult hermaphrodites and GLD-1/FLAG was immunoprecipitated (IP) with either mouse anti-FLAG Ab or mouse IgG and the bound material eluted with FLAG peptide. RNA was then purified from the eluate and converted to cDNA (FLAG cDNA and IgG cDNA). The IgG cDNA was subtracted from the FLAG cDNA to remove cDNAs corresponding to nonspecifically trapped RNAs. After two independent subtractions, about 250 clones from each were sequenced. 17 clones were identified at least once in each subtraction. RT-PCR analysis with gene specific primers suggest the majority are specifically enriched in FLAG IPed RNA. rme-2, a yolk protein receptor which is required for uptake of yolk protein by oocytes (Grant et al., 1998 ECWM Abstract 31), is one of the identified target RNAs. Antibody staining of wild-type hermaphrodite gonads shows a reciprocal distribution for RME-2 and GLD-1: RME-2 is absent from the distal region where GLD-1 is abundant while RME-2 increases in abundance in plasma membranes of growing oocytes when GLD-1 levels fall precipitously. Consistent with GLD-1 functioning as a translational repressor, RME-2 prematurely accumulates in the distal region of gld-1 null hermaphrodites. In situ staining shows that rme-2 RNA is present in the distal region of both wild-type and gld-1 null. GLD-1 thus functions to restrict yolk protein receptor translation to growing oocytes.

24 May 1999 15:50 96 96 GLD-1 represses tra-2 translation through a poly (A) tail-dependent mechanism

1999 International Worm Meeting abstract 45 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. GLD-1 represses tra-2 translation through a poly (A) tail-dependent mechanism E Jan, EB Goodwin Northwestern University Medical School and the Robert Lurie Cancer Center, Chicago, Il 60611 The C. elegans sex determination gene, tra-2, is translationally regulated by elements in the 3’untranslated region (3’UTR) called TGEs. tra-2 activity is required for female development. Male development in both somatic and germline tissues requires that tra-2 translation is repressed via the TGEs. The TGE control is a conserved mechanism present in invertebrates and vertebrates that controls the translation of other mRNAs. Recently, we have demonstrated that the C. elegans GLD-1 protein binds specifically to the TGEs and represses translation in a TGE-dependent manner in vitro and in vivo. GLD-1 is a cytoplasmic germline specific protein and is a member of the STAR family of RNA binding proteins. GLD-1 is essential for oogenesis and is also necessary for inhibiting premeiotic proliferation and promoting hermaphrodite spermatogenesis. In general, the mechanism of 3’UTR regulation and the function of STAR proteins are not well understood. Polysome analysis suggest the TGEs repress translation by inhibiting ribosome initiation in C. elegans. To better understand the mechanism of GLD-1 repression, we developed a yeast in vitro translation assay. We find that addition of GLD-1 protein to yeast extracts represses translation of reporter RNAs in a TGE-dependent manner. We also find that this repression occurs only when reporter RNAs carry a poly (A) tail, suggesting that GLD-1 represses translation through a poly (A) tail-dependent mechanism. Furthermore, deadenylation is not necesssary for GLD-1 repression, since GLD-1 can inhibit translation of RNAs carrying a stretch of 30 nucleotides 3’ to the poly (A) tail which blocks deadenylation. Lastly, the 5’cap of an mRNA is not required for GLD-1 repression suggesting that the mechanism of TGE control is not acting through the cap-binding protein, eIF4E. Our results suggest a model by which binding of GLD-1 to the TGEs represses translation by influencing the effect of the poly (A) tail on ribosome initiation.

24 May 1999 15:50 97 97 FOG-1 is a Cytoplasmic Polyadenylation Element Binding protein

1999 International Worm Meeting abstract 46 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. FOG-1 is a Cytoplasmic Polyadenylation Element Binding protein SW Jin 1 , J Kimble 2 , RE Ellis 1

1 Department of Biology, University of Michigan, Ann Arbor MI 48109. 2 HHMI and University of Wisconsin, Madison, WI 53706.

The fog-1 and fog-3 genes are required to specify that germ cells differentiate as sperm. Although mutations in either gene cause germ cells to develop as oocytes instead, they have no effect on somatic fates. Thus, one simple model is that fog-1 and fog-3 act in response to the sex-determination genes to regulate germ cell fates. We cloned fog-1 to test this model. First, we used RFLP mapping and deletion mapping to define a region of the YAC Y54E10 that contains fog-1. Second, we used sequence data from this region to identify candidate genes, and tested these by Southern analysis for lesions associated with fog-1 alleles. We found that q241 is a deletion of most of fog-1 and q492 is a small deletion located within the gene. Third, we used transformation rescue experiments to confirm our identification of fog-1. Northern analyses, RACE and RT-PCR experiments show that fog-1 produces four transcripts. These transcripts fall into two groups -- two long transcripts of 2138 and 2237 nucleotides differ in their polyadenylation sites, and two short transcripts of 1612 and 1711 nucleotides lack the first four exons. All four messages are transpliced to SL1. Using RNA-mediated inactivation, we found that the large transcripts are necessary for germ cells to become sperm. Furthermore, Northern analyses show that the quantity of large transcripts is regulated by the sex-determination genes. Finally, the promotor for the large transcripts contains three potential TRA-1A binding sites. Thus, we propose that the sex-determination genes act through TRA-1A to control expression of fog-1. The large transcripts encode a protein of 619 amino acids, which resembles Cytoplasmic Polyadenylation Element Binding proteins. These proteins regulate translation of target messages by controlling the lengths of their poly-A tails. Genetic and molecular tests show that nonsense mutations behave like null alleles of fog-1, but that missense mutations in the conserved RNA-binding domains have a strong dominant negative effect. Thus, we propose that FOG-1 acts as part of a complex that controls translation of key messenger RNAs in the developing germ line.

24 May 1999 15:50 98 98 CPEBs: a family of related RNA-binding proteins involved in distinct steps of spermatogenesis.

1999 International Worm Meeting abstract 47 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. CPEBs: a family of related RNA-binding proteins involved in distinct steps of spermatogenesis. CM Luitjens, JE Kimble, MP Wickens UW-Madison. Translational control via 3’UTRs is critical in the germline of many species. In Xenopus, an RNA-binding protein, CPEB (Cytoplasmic Polyadenylation Element Binding Protein), binds to 3’ UTRs of specific maternal mRNAs and is thought to regulate their translation. The C. elegans genome contains 4 putative CPEB homologs. The similarity is greatest in the region that contains 2 RNA Recognition Motifs (RRMs) and a novel C/H-containing Zn-binding domain, both of which are needed for Xenopus CPEB to bind RNA. Using RNAi, we find that one CPEB homolog, cpb-1, is required for spermatogenesis in both hermaphrodites and males; cpb-1(RNAi) animals have arrested primary spermatocytes and lack secondary spermatocytes or mature sperm. Wild-type males produce viable cross-progeny when mated to the cpb-1(RNAi) hermaphrodites; thus cpb-1 is not required for oogenesis. cpb-1 mRNA is germline-specific. CPB-1 protein is detected in the meiotic region of the L4 germline, but not in adults. Using CPB-1 as bait in the two-hybrid system, we recovered FBF, a repressor of fem-3 required for the sperm/oocyte switch; the interaction’s significance is being investigated. A second homolog encodes fog-1, a gene required to specify sperm fate. The homolog maps to the fog-1 locus, and RNAi directed against the gene results in a feminized (Fog) germ line. We sequenced this gene from a fog-1(q253) mutant and found an amino acid substitution in a residue highly conserved among CPEBs, confirming that it is the fog-1 gene (also see Jin et al abstract). Using FOG-1 as bait in a two-hybrid screen, we’ve found one specific partner to date; analysis of this partner will be presented. In every species examined, CPEBs are implicated in oogenesis. However, in C. elegans, animals subjected to RNAi against all CPEB homologs simultaneously are Fog. Moreover, oocytes of these animals support embryogenesis and produce viable cross-progeny. Thus CPEBs are dispensable for oogenesis in C. elegans. This is the first report of CPEBs having a role in spermatogenesis, and contrasts with the roles of Xenopus CPEB and Drosophila orb. Worm CPEBs are required for two steps in spermatogenesis: fog-1 specifies the sperm fate, and cpb-1 implements that decision. We suggest that CPB-1 and FOG-1 bind to and regulate specific mRNAs to execute these functions.

24 May 1999 15:50 99 99 Getting Intimate with the Right Partner

1999 International Worm Meeting abstract 48 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Getting Intimate with the Right Partner AF Dernburg, AM Villeneuve Developmental Biology, Stanford University A unique and ubiquitous feature of sexual reproduction is the pairing between homologous chromosomes that occurs during meiosis. This intimate association allows partner chromosomes to undergo genetic exchange, which is in turn essential for accurate chromosome segregation at the first meiotic division. Although this process has intrigued biologists for decades, many fundamental questions about its mechanism remain; e.g., how do chromosomes first encounter their homologs? Once physical contact is made, how is their homology recognized? Using FISH and high-resolution 3-D microscopy, we can visualize chromosome pairing at specific chromosomal loci within intact gonads. Since the entire temporal sequence of meiosis is present within each adult worm, this approach quickly revealed the stage at which meiotic pairing occurs. Combined with reverse genetics, these cytological methods have resolved another crucial question: Conflicting results from yeast and Drosophila had fueled a debate as to whether recombination is essential for meiotic chromosome pairing. By knocking out a gene, spo-11, whose function is necessary to initiate recombination (in collaboration with Mike Dresser, Gary Moulder, and Bob Barstead) we abolished all meiotic exchange, but found that pairing and synapsis were unperturbed in this mutant. This study demonstrated pairing can occur in the absence of recombination in a metazoan organism, and that this paradigm is probably extremely widespread. We are now focusing on the role of "pairing centers" in meiotic chromosome pairing and synapsis. Cytological examination of worms lacking X-chromosome pairing centers has revealed that these distal regions are indeed essential for intimate association between the Xs. We are testing whether synaptonemal complex components can associate with defective X chromosomes. Two additional approaches have enabled us to perturb normal homolog pairing: First, genetic screens for Him mutants have uncovered trans-acting factors involved in pairing (see MacQueen abstract). Another strategy is to study rearrangements, which require the pairing machinery to contend with chromosomes that do not share homology along their entire lengths. We are currently testing the long-standing assumption that balancer chromosomes suppress recombination by disrupting pairing between homologous sequences.

24 May 1999 15:50 100 100 Meiosis, Mitosis and Their Relationship to Dosage Compensation

1999 International Worm Meeting abstract 49 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Meiosis, Mitosis and Their Relationship to Dosage Compensation A Chan, D Pasqualone, B Meyer HHMI and Dept. of Molecular and Cell Biology, University of California, Berkeley, CA 94720 Meiosis reduces the number of chromosomes in reproductive cells, forming haploid gametes. It involves several cellular processes: homologous chromosome synapsis, recombination, sister-chromatid cohesion, and chromosome segregation. Members of the highly conserved SMC (Structural Maintenance of Chromosomes) protein family are central for diverse chromosome dynamics, including mitotic chromosome condensation and sister-chromatid cohesion as well as X-chromosome dosage compensation. We therefore explored whether SMC proteins are also involved in meiosis. Using a reverse genetic approach, we found that one C. elegans SMC protein plays a critical role in meiosis and is encoded by the him-1 gene (High Incidence of Males). A weak him-1 allele causes abnormal X-chromosome nondisjunction, producing 16% males. Our results indicate that at elevated temperatures, weak him-1 mutants have up to ten univalents at diakinesis, indicating a defect in homologous chromosome pairing. By molecular criteria, h134 and h55 (Rose lab) are null alleles. Maternally rescued null mutants develop into adults but are sterile with defects in germline chromosome structure, including aberrant pachytene. In wild-type animals our HIM-1 antibodies localize between homologous chromosomes during pachytene and between homologs and sister chromatids during diakinesis, further indicating a role for HIM-1 in homologous chromosome synapsis and a possible role in sister-chromatid cohesion. FISH analysis suggests that him-1 mutants are defective in germline mitosis. Thus, SMC proteins are crucial for both mitosis and meiosis. Does him-1 interact with dosage compensation genes involved in meiosis, such as dpy-28? By immunofluorescence, DPY-28 and HIM-1 colocalize during pachytene. At diakinesis, the two antibodies show a similar staining pattern between homologs, but DPY-28 staining is greatly reduced between sister chromatids. Strikingly, DPY-28 fails to localize along meiotic chromosomes in him-1 null mutants, indicating that it depends on HIM-1 for proper localization. In contrast, localization of DPY-28 to X during dosage compensation depends on another SMC protein, DPY-27, implying that the roles of DPY-28 in meiosis and dosage compensation are mediated by different SMC proteins.

24 May 1999 15:50 101 101 Synapsis and Chiasma Formation in C. elegans Require HIM-3, a Component of the Axial Element That Functions in Meiotic Chromosome Segregation

1999 International Worm Meeting abstract 50 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Synapsis and Chiasma Formation in C. elegans Require HIM-3, a Component of the Axial Element That Functions in Meiotic Chromosome Segregation MC Zetka 1 , I Kawasaki 2 , S Strome 2 , F Müller 1

1 Institute of Zoology, University of Fribourg, CH-1700 Fribourg, Switzerland. 2 Dept. of Biology, Indiana University, Bloomington, IN 47405.

Meiotic chromosomes are organized about a proteinaceous core, usually referred to as an axial element, that polymerizes between replicated sister chromatids. Axial elements have been implicated in a number of meiotic processes. However, functional information is lacking for the most promising structural candidates. We present the molecular and genetic analyses of a meiosis-specific chromosome component, HIM-3, that functions in three distinct processes: crossing over, synapsis, and segregation. Mutations in him-3 result in a reduction of crossing over and an increase in the frequency of nondisjunction of all chromosomes. We have cloned the him-3 gene and found that both existing alleles of him-3 contain missense mutations. The him-3 protein shares 31% similarity to Hop1p of S. cerevisiae, a nonstructural component of meiotic chromosomes that is essential for synapsis. To directly test the role of HIM-3, RNAi was used as a tool to eliminate the function of the endogenous gene. Injected mothers segregated a large number of arrested embryos and a high frequency of males, diagnostic of a defect in chromosome segregation. DAPI staining revealed the presence of unsynapsed chromosomes at pachytene and 12 univalents at diakinesis, indicating that in the absence of HIM-3, chromosomes fail to synapse and form chiasmata. Antibodies raised against HIM-3 show that the protein is localized to condensing chromosomes early in prophase and to the cores of both synapsed and desynapsed chromosomes later in prophase. During the metaphase - anaphase transition of meiosis I, HIM-3 is released from the chromosomes, suggesting a role for HIM-3 in promoting sister chromatid arm cohesion, which is released at this time. This interpretation is supported by the recovery of nondisjunction events from him-3 (e1256) oocytes. To test if HIM-3 has a role in sister chromatid cohesion, we are performing FISH experiments to determine if premature sister chromatid separation occurs in him-3 mutants.

24 May 1999 15:50 102 102 him-4 encodes an extracellular matrix protein required for cell adhesion and germ-line chromosome segregation

1999 International Worm Meeting abstract 51 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. him-4 encodes an extracellular matrix protein required for cell adhesion and germ-line chromosome segregation BE Vogel, EM Hedgecock Johns Hopkins University, Dept. of Biology, Baltimore MD 21218 Defects in X chromosome segregation during meiosis result in a high incidence of male progeny from him mutant hermaphrodites. Among the 14 known him genes, him-4 is unique since mutant animals also exhibit defects in cell adhesion. Specialized hemidesmosome and intermediate filament mediated attachments that anchor the uterus and 4 mechanosensory neurons to the body wall are defective in him-4 mutants. As a result, the uterus everts through the vulva shortly after egg-laying begins, resulting in a dramatic decrease in mutant brood size. Mutant animals also exhibit mild defects in mechanosensory function. him-4 encodes hemicentin, a 5198 amino acid secreted protein primarily composed of 48 tandem Ig modules followed by 3 EGFs. Unique sequences at both ends of the molecule suggest that this protein could form a 200nm rod with specialized adaptors at either end that could span the basement membrane between uterine cells (or mechanosensory neurons) and epithelial cells. GFP tagged hemicentin localizes to sites where cells make elongated attachments to the epithelium that are resistant to mechanical stress. These sites include the attachment of a specialized uterine cell (utse) to epithelial seam cells, to many neurons (including mechanosensory neurons ALM and PLM), at the anterior ends of the pharynx and intestine, but not to body wall muscles, which make punctate attachments to the epithelium at dense bodies. In the germ-line, hemicentin-GFP is located between ’T’ shaped plasma membranes of syncytial germ cells, localizing preferentially to the top of the ’T’, close to the rachis. Loss of hemicentin may result in defects in synthesis or stabilization of the plasma membranes separating germ cells, accounting for the multinucleate germ cells and large numbers of aneuploid gametes observed in him-4 mutants.

24 May 1999 15:50 103 103 Mutants with post-fertilization meiotic progression defects

1999 International Worm Meeting abstract 52 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Mutants with post-fertilization meiotic progression defects A Golden 1 , P Sadler 2 , G Holt 2 , M Wallenfang 3 , G Seydoux 3 , D Shakes 2

1 NCI-FCRDC, Frederick, MD. 2 Biology Dept., College of William and Mary, Williamsburg, VA. 3 Johns Hopkins University, Baltimore, MD.

Ovulated oocytes in C. elegans have two choices. Unfertilized oocytes enter an endomitotic cell cycle and are laid as unshelled, polyploid oocytes. Fertilized oocytes complete meiosis, form eggshells, and develop into embryos. While much is known about the cellular events of post-meiotic 1-cell embryos, little is known about the signaling cascades and cell cycle machinery required to resume meiosis after fertilization. Our recent genetic and RNA-mediated interference (RNAi) studies have revealed several likely components of this process. For instance, emb-27 mothers at 25o C produce 1-cell embryos which lack polar bodies and arrest with condensed sperm and oocyte chromosomes. We have generated similarly arrested 1-cell embryos during RNAi studies of the C. elegans cdc2 homolog, the cdc-25 triple, and various anaphase promoting complex (APC) components. To identify other genes required for meiotic progression following fertilization, we performed a temperature-sensitive embryonic lethal screen, similar to that described by Kemphues et al (1988). We isolated 30 temperature-sensitive embryonic lethal mutants that produce 1-cell arrested embryos when homozygous mothers are shifted to 25o C as L4s. Of these 30, 23 are defective in oocyte meiotic progression. The other seven continue to cycle in the absence of cell division. Of the meiotic progression mutants, four are new emb-27 alleles, and the other 19 fall into three other complementation groups. In our analysis of the 19 meiotic progression mutants, we are focusing on meiotic defects in oocytes and sperm and mitotic defects in germline proliferation. The mutant oocytes examined to date all exhibit a strong meiosis I metaphase-to-anaphase transition block. Furthermore, when shifted to 25o C as L1s, most of the mutants give rise to sterile adults with moderate to severe germline proliferation defects. In addition to our phenotypic analysis, we are mapping the genes so that we can determine their molecular identities. We anticipate identifying components of APC itself and of the signaling pathway that triggers the post-fertilization resumption of meiosis.

Research sponsored by the Jeffress Foundation and the NCI, DHHS, under contract with ABL.

24 May 1999 15:50 104 104 Genes Involved in Meiotic and Mitotic Spindle Formation

1999 International Worm Meeting abstract 53 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genes Involved in Meiotic and Mitotic Spindle Formation MA Srayko, MR Dow, PE Mains Department of Biochemistry and Molecular Biology, University of Calgary, Canada The C. elegans oocyte cytoplasm supports the formation of both meiotic and mitotic spindles. However, these two structures differ greatly in their size, structural morphology, and position within the embryo. We have genetically identified genes that are involved in the assembly of the meiotic spindle and the transition to mitotic spindle formation. mei-1, a gene encoding a member of the AAA family of ATPases, is required for the assembly of the meiotic spindle. However, mei-1 must be inactivated prior to mitosis, otherwise mitotic spindles are small and misoriented. mei-2 and tbb-2 are genetic activators of mei-1. mei-2 encodes a novel protein that, like MEI-1, localizes to the meiotic spindle and polar bodies, but unlike MEI-1, also strongly stains condensed meiotic chromatin and the condensed sperm pronucleus. Despite these differences in immunolocalization, the entire MEI-2 staining pattern is dependent on mei-1(+). MEI-1 and MEI-2 staining disappears at the completion of anaphase II. A mutation in a b-tubulin gene, tbb-2, was identified as a suppressor of the mitotic-defective mei-1(gf). tbb-2(sb26) has a missense mutation within the isotype-specific C-terminus. tbb-2(sb26) homozygotes are wild-type, suggesting that this sequence alteration specifically lowers, but does not eliminate, the activity of mei-1. tbb-2(sb26) enhances the weak meiotic defects associated with mei-1(ct103) and mei-2(ct98) mutations, also indicating a role in meiotic spindle assembly. mel-26 is the post-meiotic inhibitor of mei-1. Mutations in mel-26 result in mitotic spindle defects due to ectopic mei-1 activity during mitosis. The function of mel-26(+) seems very specific for turning off mei-1 since it is not required in the absence of mei-1: mitosis is normal in mei-1(0);mel-26 double mutants (although meiosis is abnormal). Ectopic MEI-1 and MEI-2 are concentrated at the mitotic centrosomes in mel-26 mutants. MEL-26(+) localizes to the centrosomes during wild-type mitosis, suggesting a possible role in protecting mitotic spindles from mei-1/mei-2 activity after meiosis is complete.

24 May 1999 15:50 105 105 Secretion is Required to Complete Cytokinesis in Caenorhabditis elegans

1999 International Worm Meeting abstract 54 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Secretion is Required to Complete Cytokinesis in Caenorhabditis elegans AR Skop 1 , D Bergmann 2 , WA Mohler 1 , JG White 1

1 University of Wisconsin-Madison, Madison, WI 53706. 2 University of Colorado-Boulder, Boulder, CO 80309.

Cytokinesis is the fundamental process of cleaving a cell into two. We have shown that the final pinching off and separation of daughter cells in the C. elegans embryo requires a functional secretory pathway. We inhibited secretion in early C. elegans embryos by treatment with Brefeldin A and by suppressing expression of Rab11, a GTPase specifically required for Golgi to plasma membrane vesicle targeting. By using multifocal plane, time-lapse recording of treated embryos, we showed that these treatments produced similar failures in cytokinesis: the furrow initiates and ingresses normally yet does not complete. Instead, after a time-lag, the furrow fully regresses leaving an uncleaved multinucleated cell. We have used multiphoton microscopy in conjunction with an FM143 membrane probe to show that the furrow does not completely pinch off during these abortive cleavages. The midzone of the mitotic spindle has been shown to be defective in mutants that exhibit failures in the terminal phase of cytokinesis. We suggest that spindle midzone microtubules are necessary for vesicle targeting to the late cleavage furrow in a manner analogous to the function of the phragroplast microtubules of plant cell cytokinesis. This leads to the conclusion that plant and animal cell cytokinesis are probably fundamentally similar processes.

24 May 1999 15:50 106 106 Depletion of syntaxins in the early C. elegans embryo reveals a role for membrane fusion events in cytokinesis

1999 International Worm Meeting abstract 55 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Depletion of syntaxins in the early C. elegans embryo reveals a role for membrane fusion events in cytokinesis V Jantsch-Plunger, M Glotzer Research Institute of Molecular Pathology, Dr. Bohr-gasse 7, 1030 Vienna Austria During cytokinesis, the plasma membrane of the parent cell is resolved into the two plasma membranes of the daughter cells. Membrane fusion events mediated by the machinery that participates in intracellular vesicle trafficking may contribute to this process. In order to test this hypothesis, we sought to inhibit various types of membrane fusion events and to determine whether any specific fusion pathway is essential for cytokinesis. The early embryo of the nematode Caenorhabditis elegans was chosen as a model system since the early divisions can be observed in real time by nomarski optics, the genome sequencing project is essentially complete, and an efficient technique is available to produce embryos lacking specific gene products. Two classes of molecules that are required for membrane fusion are the t- and v-snares. The t-snares (syntaxins) comprise a multigene family and each t-snare is thought to play a role in selective membrane fusion events in the cell. We have analyzed the genome of C. elegans and identified eight genes that have the sequence hallmarks of the syntaxin gene family. Embryos depleted of individual syntaxins were produced by RNA-mediated interference (RNAi). Embryos deficient for one syntaxin, termed syn-1, become multinucleate due to defects in three processes: polar body extrusion, karyomere fusion, and cytokinesis. In the adult, SYN-1 protein is found primarily in the gonad where it is localized to the incomplete membranes between germ cell nuclei and to the plasma membrane of developing oocytes. In the embryo, SYN-1 is localized to ingressing cleavage furrows as well as exhibiting a punctate distribution in the cytoplasm, suggesting a vesicle localization. Consistent with the defect in karyomere fusion, SYN-1 staining spots are concentrated around the decondensing chromatin.

24 May 1999 15:50 107 107 HKP-1, a kinetochore-associated protein is assembled onto mitotic chromosomes during prophase and is important for chromosome segregation in Caenorhabditis elegans

1999 International Worm Meeting abstract 56 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. HKP-1, a kinetochore-associated protein is assembled onto mitotic chromosomes during prophase and is important for chromosome segregation in Caenorhabditis elegans LL Moore 1 , M Morrison 2 , MB Roth 2

1 Division of Basic Sciences and Molecular and Cellular Biology Program. 2 Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109.

Mitotic chromosomes in Caenorhabditis elegans posses sister kinetochores which extend the lengths of each chromosome. These kinetochores resemble those present on mammalian chromosomes at least at the ultrastructural level, suggesting that C. elegans might be an ideal system to study the kinetochore. We sought to learn more about how kinetochores are assembled and function during mitosis by generating a monoclonal antibody, 6C4, that recognizes the poleward face of the holocentric chromosomes at metaphase. Further evidence that mAb 6C4 recognizes the kinetochore is provided by co-localization with both an antibody directed against the C. elegans homolog of the centromere protein, CENP-A, (see abstract by Buchwitz et al.) and the MPM-2 antibody which identifies a kinetochore-associated phosphoepitope present in a variety of organisms. Early in mitosis mAb 6C4 stains dots along the chromosomes which later in prophase resolve into two structures present on opposing faces of chromosomes suggesting that mAb 6C4 can be used to study the assembly of the kinetochore in C. elegans. Expression screening using mAb 6C4 identified a protein that we named HKP-1, HoloKinetic Protein-1. We also identified a second protein from the C. elegans genome sequence database, HKP-2, that is 54% similar to HKP-1. When expression of HKP-1 is reduced by RNA interference (RNAi), staining with mAb 6C4 is eliminated, indicating that hkp-1 encodes a mAb 6C4 antigen. RNAi with hkp-1 and hkp-2 separately did not result in any significant phenotype, however when both were used for RNAi aberrant anaphases were observed as early as the first mitotic division. hkp-1(RNAi);hkp-2(RNAi) embryos arrest at approximately 100 cells with different amounts of DNA in individual nuclei. These results indicate that HKP-1 is a kinetochore-associated protein that is involved in the fidelity of chromosome segregation.

24 May 1999 15:50 108 108 The role of lin-5 in chromosome segregation.

1999 International Worm Meeting abstract 57 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The role of lin-5 in chromosome segregation. M Lorson 1 , M Walhout 1 , M Vidal 1 , B Horvitz 2 , S van den Heuvel 1

1 MGH Cancer Center, Charlestown, MA 02129. 2 HHMI, MIT, Cambridge, MA 02139.

Once a cell has duplicated its DNA, it must separate sister chromatids to opposite poles and then undergo cytokinesis. Failure to segregate chromosomes faithfully into daughter cells can result in aneuploidy. To understand the requirements for successful chromosome segregation, we focused on the C. elegans gene lin-5. Cells in lin-5 mutants initiate mitosis but chromosomes fail to align at a metaphase plate and cytokinesis does not occur. Despite these defects, cells exit mitosis without delay and initiate subsequent rounds of DNA replication followed by abortive mitoses. We have cloned the lin-5 gene and shown that it encodes a novel coiled-coil protein. LIN-5 protein associates with the spindle in meiosis and is predominantly located at the centrosomes during mitosis. LIN-5 is also detected at the mitotic spindle during metaphase and at the cell membrane. The localization of LIN-5 to the centrosomes and spindle is microtubule dependent. Using a conditional allele of lin-5, we showed that partial inactivation of lin-5 can cause an arrest in chromosome segregation and delay exit from mitosis implicating a secondary role for lin-5 in checkpoint control. We have also studied several mitotic processes: lin-5 function appeared non-essential for the periodic activation and inactivation of cdc-2(ncc-1), centrosome duplication or formation of a bipolar spindle. However, spindle rotation and chromosome movements are dependent on lin-5.Together, these results strongly suggest that lin-5 promotes spindle force likely through an effect on spindle dynamics or motor protein function. To understand how lin-5 functions with other components of the mitotic machinery, we performed a two-hybrid screen. Five interactors were identified multiple times, including LIN-5 itself and ZC8.4 which is predicted to encode a Parascaris univalens PUMA1 homolog. PUMA1 is associated with the spindle apparatus in Parascaris (Esteban et al., 1998). RNAi with the two-hybrid interactors did not result in defects in meiosis or mitosis. However, staining with PUMA1 antibodies indicated that ZC8.4 localizes to the same mitotic structures as LIN-5. This localization was disturbed in embryos following lin-5(RNAi). We are investigating if and how LIN-5 and ZC8.4 act together in mitosis.

24 May 1999 15:50 109 109 Regulation of post-embryonic G1 cell cycle progression by a cyclin D/cyclin dependent kinase-like complex in C. elegans

1999 International Worm Meeting abstract 58 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Regulation of post-embryonic G1 cell cycle progression by a cyclin D/cyclin dependent kinase-like complex in C. elegans M Park, MW Krause Laboratory of Molecular Biology, National Institutes of Health, Bethesda, MD 20892 The formation of a complex multicellular organism requires the control of cell proliferation within the context of environmental and developmental signaling. Much of our understanding of the cell cycle has come from studies in yeast, in marine invertebrate and amphibian eggs, and in mammalian tissue culture. These studies have defined the basic machinery involved in the initiation of cell cycle entry and progression. The cell cycle is controlled by the activity of cyclin dependent kinases (CDKs) in association with a regulatory cyclin subunit. A distinct set of cyclin/CDK complexes act during each phase of the cell cycle (G1, S-phase, G2, and Mitosis). D-type cyclins in association with specific CDKs are responsible for initiating progression from G1 to S-phase. The synthesis of cyclin D and the assembly of cyclin D with its catalytic CDK partner are regulated by mitogen induced signal transduction cascades. We have identified C. elegans homologs of cyclin D ( cyd-1 ) and its potential cognate cyclin dependent kinase CDK4/6 ( cdk-4 ). In order to understand the role of these G1 cell cycle regulators in C. elegans development, we have analyzed the defects caused by depletion of these factors using a deletion mutant ( cdk-4 ) and by RNA-mediated interference ( cyd-1 and cdk-4 ). We show that cyd-1 and cdk-4 are necessary for proper division of the post-embryonic blast cells. The post-embryonic blast cells undergo temporary and prolonged G1 arrest during larval development. These cells re-enter the cell cycle upon hatching and at each larval molt. We show these cells fail to divide after hatching and are arrested in G1 in animals that are depleted of cyd-1 and cdk-4 function. This is consistent with the role of the cyclin D/CDK4/6 complex in the regulation of G1 to S-phase progression. Even though these genes appear to be expressed during late embryogenesis, as well as larval development, the defects that we observe are restricted to post-embryonic development. Our results suggest that the C. elegans cyclin D/CDK4/6 complex is involved specifically with controlling the initiation of cell divisions and the coordination point for many signaling pathways to regulate G1 cell cycle decisions during post-embryonic development.

24 May 1999 15:50 110 110 Genes, mdf-1 and mdf-2, encoding mitotic checkpoint components are essential in C. elegans

1999 International Worm Meeting abstract 59 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genes, mdf-1 and mdf-2, encoding mitotic checkpoint components are essential in C. elegans R Kitagawa, A Rose Department of Medical Genetics, University of British Columbia, Vancouver, BC Canada V6T 1Z3. In Saccharomyces cerevisiae, several genes, which encode components of the mitotic checkpoint, such as MAD1, 2, 3 or BUB1, 2, 3, are required for cell cycle arrest in response to defects in the chromosome segregation machinery. We investigated the function of mitotic checkpoint genes in multicellular organisms. We have identified a gene encoding 203 amino acids, having 37% and 50% sequence identity with budding yeast Mad2p and hMAD2, respectively, and named the gene mdf-2 after mitosis arrest deficiency. The mdf-2 gene product is capable of rescuing the benomyl sensitive phenotype of S. cerevisiae mad2 mutants. Using a yeast two-hybrid system, we identified a gene encoding an MDF-2 interacting protein, designated mdf-1 which is predicted to encode a coiled-coil protein sharing weak sequence similarity with the S. cerevisiae gene, MAD1. Antibody to MDF-2 localized to the centrosomes and the central spindle region in the early cleaving embryonic cells in a cell cycle dependent manner. In addition, we found the prominent staining of MDF-2 in the nuclei of gut cells destined for endreduplication, and in the germ cells at prophase of meiosis I. Silencing of mdf-1 and mdf-2 genes by RNAi resulted in larval arrest, adult sterility, or mature adults with incorrect chromosome number at meiotic prophase, giving few viable offspring and a high frequency of XO males. In addition, we obtained a deletion mutant of the mdf-1 locus (isolated by the C. elegans gene knockout facility, University of British Columbia). The deletion, gk2, removed 2724 bases of DNA in the mdf-1 locus, including nucleotides that encode amino acids 3-679 of MDF-1. The mdf-1 deletion homozygotes look normal, but produced a range of mutant phenotypes including unhatched embryos, arrested L1 larvae, fertile hermaphrodites that give few progeny and a high incidence of males, as well as sterile adults. Mutant phenotype and RNAi phenocopy results are identical. The strain cannot be maintained as a homozygote beyond the F2 generation, making it an unconditional maternal effect sterile. These results revealed the essential function of mdf-1 and mdf-2 for normal development and fertility as well as the maintenance of proper chromosome number. mdf-1 and mdf-2 for normal development and fertility as well as the maintenance of proper chromosome number.

24 May 1999 15:50 111 111 Two C. elegans mutants with defects in telomere replication

1999 International Worm Meeting abstract 60 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Two C. elegans mutants with defects in telomere replication S Ahmed, J Hodgkin MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, UK The germline is an immortal cell lineage which is passed from one generation to the next indefinitely. To identify genes which are required for germline immortality, we looked for C. elegans mutants with mortal germlines - worms which could reproduce for several healthy generations but eventually become sterile. A number of mortal germline (mrt) mutants were identified which become effectively sterile between generations F4 and F16. Two have been studied in detail: mrt-1 and mrt-2 accumulate end-to-end chromosome fusions in later generations and exhibit telomere shortening, indicating that both mutants become sterile as a result of defects in telomere replication. Studies in yeast and mouse suggest that the late-onset chromosome fusion phenotype observed in mrt-1 and mrt-2 probably results from a loss of in vivo activity of the telomere replication enzyme telomerase (1, 2). mrt-1, mrt-2 and the mrt-1,mrt-2 double mutant all have the same telomere shortening rates, indicating that MRT-1 and MRT-2 act in the same telomere maintenance pathway. However, mrt-2 has additional phenotypes - it is hypersensitive to X-rays and has mutator activity. mrt-2 mapped near a candidate gene Y41C4A.o that encodes a homolog of the S. pombe RAD1 checkpoint protein which when mutated results in X-ray hypersensitivity (3). The mrt-2 allele has a splice site mutation in Y41C4A.o which eliminates most of this highly conserved gene product. In addition, high copy number arrays of Y41C4A.o produce a phenocopy of mrt-2 X-ray hypersensitivity - possibly via cosuppression silencing of this gene in the germline. Therefore the MRT-2 checkpoint protein may be required for proper processing of both abnormal double-strand breaks caused by X-rays and normal double-strand breaks present at telomeres. These results are consistent with the notion that telomeres are replicated as a special kind of DNA damage.

1. Nakamura TM, et al. (1998), Science 282: 493-496. 2. Blasco MA, et al. (1997), Cell 91: 25-34. 3. Sunnerhagen P, et al. (1990), Mol Cell Biol. 10: 3750-3760.

24 May 1999 15:50 112 112 The heterochronic gene lin-42 and circadian rhythms: is there a connection?

1999 International Worm Meeting abstract 61 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The heterochronic gene lin-42 and circadian rhythms: is there a connection? M Jeon 1 , HF Gardner 2 , AE Rougvie 1,2

1 Department of Biochemistry, University of Minnesota, St. Paul, MN 55108. 2 Department of Genetics and Cell Biology, University of Minnesota, St. Paul, MN 55108.

Mutations in the heterochronic gene lin-42cause the hypodermal seam cells to terminally differentiate one stage earlier than wild type, during the L3 molt. To understand how lin-42 functions relative to other known heterochronic genes in the timing of seam cell terminal differentiation, we have cloned lin-42 and studied its expression during development. A distinguishing feature of LIN-42 is the presence of a PAS domain. The PAS domain has been found in a variety of proteins, notably in several key proteins which form the molecular clock of circadian rhythms*. In Drosophila, one of these key clock factors is Period (dPER). Interestingly, database searches reveal that LIN-42 is most similar to the PER family of proteins. Conversely, searches of the C. elegans genome reveal that LIN-42 is the best match to dPER. A hallmark of PER is its oscillating expression pattern, which is critical to its function as a molecular clock. To test if the LIN-42/PER similarity reflects functional conservation, we looked at lin-42 message levels during development. We performed RT-PCR experiments, using cDNA pools isolated every 2 hours during post-embryonic development (generous gift from Iain Johnstone). Quantitation relative to a control mRNA shows that lin-42 message levels oscillate relative to molting cycles, with peaks during the intermolt periods. A rescuing GFP fusion construct shows hypodermal LIN-42::GFP expression from late embryogenesis into adulthood. Quantitative western blots are being used to evaluate levels of LIN-42::GFP during development. In Drosophila, the PAS domain is a site of interaction for a second clock protein, Timeless. We have sequenced Kohara cDNAs predicted to encode a protein, TIM-1, with high similarity to the Drosophila Timeless. Unlike PER and TIM in flies, we find that lin-42 and tim-1 have distinct mRNA accumulation patterns. We are investigating TIM-1 expression patterns and LIN-42 and TIM-1 interactions to see if their mechanism of action has been conserved through evolution.

* Nambu et al. (1991) Cell 67:1157; Huang et al. (1993) Nature 364: 259.

24 May 1999 15:50 113 113 The heterochronic gene lin-41 encodes a temporally regulated RING finger protein that controls the timing of appearance of LIN-29 protein.

1999 International Worm Meeting abstract 62 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The heterochronic gene lin-41 encodes a temporally regulated RING finger protein that controls the timing of appearance of LIN-29 protein. FJ Slack, G Ruvkun Department of Genetics, Harvard Medical School, and Department of Molecular Biology, Massachusetts General Hospital, Blossom St, Boston, MA, 02114, USA The heterochronic pathway controls the temporal progression of C. elegans development by temporally regulating the abundance or activities of a succession of heterochronic genes. lin-41 null mutant animals precociously express adult characteristics at earlier larval stages. In contrast, overexpression of lin-41 causes a retarded phenotype: some cells reiterate late larval fates at the adult stage. Thus lin-41 is necessary and sufficient for the delay of adult-specific fates during earlier larval stages. lin-41 negatively regulates the expression of lin-29, a transcription factor required for adult fates. lin-29 is translated at the L4 and adult stages from an mRNA expressed from the L2 stage. In a lin-41 mutant, LIN-29 protein appears as early as the L2 stage. Thus LIN-41 may regulate the timing of lin-29 mRNA translation. lin-41 is alternatively spliced and encodes two members of the RBCC (RING finger, B-Box, Coiled coil) family, a diverse family of regulatory proteins, that includes the RNA binding protein SSA/Ro. lin-41 is widely expressed. In the hypodermis its expression is down-regulated during the L4 stage, which is when translation of the lin-29 mRNA is initiated. A fusion of GFP to a functional LIN-41 protein reveals that the protein is predominantly cytoplasmic. Based on precedents from the RBCC gene family, LIN-41 may regulate lin-29 expression by binding to the mRNA to control its translation. We isolated lin-41 mutations as suppressors of let-7 mutations. let-7 encodes a small untranslated RNA that is expressed at the L3 and later stages (B. Reinhart, personnel communication) and is complementary to two sites in the 3’UTR of lin-41. This complementarity and the temporal regulation of lin-41 expression at the time when let-7 RNA is up-regulated suggests that let-7 inhibits lin-41 expression by binding to the 3’ UTR. Thus activation of the let-7 regulatory RNA during the L4 and adult stages may downregulate LIN-41 expression to then relieve the translational inhibition of the lin-29 mRNA. In this way, lin-41 may transduce temporal information from a regulatory RNA to a transcriptional output.

24 May 1999 15:50 114 114 Caenorhabditis Genetics Center

1999 International Worm Meeting abstract 63 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Caenorhabditis Genetics Center TL Stiernagle 1 , SD Martinelli 2 , JA Hodgkin 3 , L Avery 4 , RK Herman 1

1 University of MN, St. Paul, MN 55108. 2 The Sanger Centre, Cambridgeshire CB10 1SA England. 3 MRC-LMB, Cambridge CB2 2QH, England. 4 University of Texas Southwestern Medical School, Dallas, TX 75235.

The Caenorhabditis Genetics Center (CGC), supported by NIH NCRR, supplies Caenorhabditis strains and information to researchers. The St. Paul team is responsible for acquiring, maintaining and distributing worm stocks, generating and maintaining a C. elegans bibliography, and publishing the Worm Breeder’s Gazette (WBG). The Cambridge team acts as a clearing house for C. elegans genetic nomenclature and maintains the genetic map. The Dallas team maintains the C. elegans web page. The CGC now has over 3300 different strains. We strive to have at least one allele of every published gene and all chromosome rearrangements, duplications and deficiencies. In addition, we have several strains of species closely related to C. elegans. Strains are available upon written request, which should include a brief statement of the intended use of the strains. Email requests (to [email protected]) are satisfactory. The CGC bibliography currently includes over 3300 research articles and book chapters. The WBG is published 3 times each year and currently has over 800 subscribers. Various types of information from the CGC are available electronically. By gopher you can get current strain lists, the WBG subscriber directory, WBG Tables of Contents and the CGC bibliography. This information, along with the Worm Breeder’s Gazette, is also made available by Leon Avery at http://elegans.swmed.edu/ .

We like to be acknowledged in papers for providing strains. We also like to receive reprints of worm papers.

24 May 1999 15:50 115 115 unc-13 mutants accumulate synaptic vesicles and are defective in evoked neurotransmitter release

1999 International Worm Meeting abstract 64 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. unc-13 mutants accumulate synaptic vesicles and are defective in evoked neurotransmitter release JE Richmond, WS Davis, EM Jorgensen Dept. of Biology, University of Utah, Salt Lake City, Utah 84112 Synaptic vesicles are released rapidly in a calcium-dependent manner. Several lines of evidence suggest that UNC-13 is important for synaptic transmission. First, unc-13 mutations produce paralysis, neurotransmitter accumulation, and aldicarb resistance. Second, the mammalian homologue, Munc-13, is localized to active zones and interacts with syntaxin (a component of the synaptic release machinery) and DOC2 (a putative regulator of neurotransmitter release). Third, overexpression of Munc-13 enhances phorbol ester-induced synaptic potentiation. Finally, the molecular structure of UNC-13, with two calcium binding domains, suggests that UNC-13 might be important in sensing rises in intracellular calcium required for neurotransmitter release. These data pose several unanswered questions: Is UNC-13 required for the release of synaptic vesicles in the cleft, is it a modulator of release or is it required for some other process? First we asked whether synaptic vesicle densities were affected by unc-13 mutations. Serial electron micrographs of unc-13(e1091) revealed an 80% increase in vesicle density over wild type at morphologically defined active zones. The average intersynaptic vesicle density was also increased. These data suggest that the synaptic defect is not the result of insufficient vesicle numbers at the synapse. Second we asked whether vesicles were being released. We have developed an electrophysiological preparation that allows us to measure evoked neurotransmitter release. Analysis of C. elegans neuromuscular synaptic function revealed a large reduction in evoked release in both unc-13(e1091) and unc-13(e51) to less than 7% of wild type, as well as a significant reduction in the frequency, but not amplitude, of spontaneous neurotransmitter release. Taken together, these results indicate that UNC-13 is an important presynaptic regulator of calcium-dependant neurotransmitter release and is a potential calcium sensor.

24 May 1999 15:50 116 116 The Rab3 GTP/GDP exchange factor homologue AEX-3 is a regulator of two different pathways for presynaptic activities

1999 International Worm Meeting abstract 65 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Rab3 GTP/GDP exchange factor homologue AEX-3 is a regulator of two different pathways for presynaptic activities K Iwasaki 1 , EM Jorgensen 2 , R Toyonaga 1

1 Natl. Inst. of Bioscience, Tsukuba Japan. 2 Univ. of Utah, Salt Lake City, UT USA.

We previously identified aex-3 as a regulator of synaptic transmission1 . aex-3 mutants show a variety of behavioral defects including those in defecation, pharyngeal pumping, and male mating. aex-3 encodes a protein highly homologous to Rat Rab3 GTP/GDP exchange factor (GEF). RAB-3, a small GTP binding protein implicated in synaptic transmission, normally localizes to presynaptic terminals but is mislocalized in aex-3 mutants to neural cell bodies. This suggests that AEX-3 regulates presynaptic activities through the RAB-3-dependent pathway.

Unlike aex-3 mutants, rab-3 mutants show no defecation motor defects2 . This suggests that AEX-3 may have functions in addition to that as a Rab3 GEF. Furthermore AEX-3 was recently identified as a homologue of the human protein MADD [MAP kinase activating protein containing the Death Domain3 ]. MADD interacts with the Tumor Necrosis Factor receptor through Death Domains (DD). Therefore, we hypothesized that AEX-3 may also interact through its C terminus or DD region with another protein(s) and that this interaction might be important for defecation control. To investigate this possibility, we used the yeast two hybrid system to identify AEX-3 C-terminal binding proteins. From a library of 106 clones, 62 positive clones were isolated of which 41 were derived from a single gene, which now we call cab-1 (C terminus of AEX-3 binding). CAB-1 has no homology to other known proteins. cab-1 is expressed in various neurons, consistent with the notion that AEX-3 and CAB-1 interact in vivo. cab-1 mutants have defects in the defecation motor program, suggesting that CAB-1 functions in the aex-3 pathway. However, unlike aex-3 and rab-3 mutants, cab-1 mutants show no defects in other behaviors including pharyngeal pumping and male mating. These observations indicate that unlike other GEFs, AEX-3 is a regulator of two different pathways (the rab-3 and cab-1 pathways) for presynaptic activities.

1) Iwasaki et al. Neuron 18:613 2) Nonet et al. J Neurosci. 17:8061 3) Schievella et al. J Biol Chem. 272:12069

24 May 1999 15:50 117 117 Gqa and Goa Pathways Act Antagonistically to Regulate Synaptic Transmission in C. elegans

1999 International Worm Meeting abstract 66 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Gqa and Goa Pathways Act Antagonistically to Regulate Synaptic Transmission in C. elegans KG Miller, MD Emerson, JB Rand Program in Molecular and Cell Biology Oklahoma Medical Research Foundation Oklahoma City, OK 73104 An understanding of the signaling pathways that regulate neurotransmitter secretion is crucial for understanding how the nervous system establishes, maintains, and modifies behavior. A common class of signaling pathways in neurons begins with neurotransmitter receptors coupled to heterotrimeric G-proteins of the Gq/G11 class. We investigated the EGL-30 (Gqa) pathway in C. elegans by using genetic screens to identify genes that confer neuronal phenotypes similar to egl-30 mutants. We found that one such gene, egl-8, encodes a homolog of phospholipase Cb (PLCb). PLCb, a known downstream effector of Gqa, functions to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) into the small signaling molecules diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). Immunostaining revealed that EGL-8 is localized to processes and cell somas throughout the nervous system, as well as near cell junctions in intestinal cells. The intestinal localization may be related to EGL-8’s function in the pboc step of the defecation cycle. The aldicarb resistance and reduced locomotion rate of egl-30 and egl-8 mutants are in contrast to mutants in the GOA-1 (Goa) pathway, which exhibit aldicarb hypersensitivity and hyperactive locomotion. A genetic analysis suggests that the GOA-1 pathway acts antagonistically to EGL-30 and EGL-8, because strains containing reduction of function mutations in both pathways often had wild type aldicarb sensitivity and/or locomotion rate. The results support a model in which production of DAG by the Gqa pathway promotes synaptic transmission, while removal of DAG by the Goa pathway inhibits synaptic transmission.

24 May 1999 15:50 118 118 egl-8 and egl-30 define a pathway that modulates cholinergic neurotransmission

1999 International Worm Meeting abstract 67 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. egl-8 and egl-30 define a pathway that modulates cholinergic neurotransmission MR Lackner, SJ Nurrish, JM Kaplan Dept. of Molecular and Cell Biology, University of California, Berkeley, CA 94702. G proteins modulate many of the behaviors exhibited by C. elegans, including locomotion, foraging, and egg laying. We have previously reported that egl-8 encodes a phospholipase C beta that may be an effector of the Gq alpha subunit encoded by egl-30. egl-8 mutants exhibit many phenotypes that resemble those caused by egl-30 mutations, including sluggish locomotion, feeble head foraging movements, and defective egg laying. We will present several lines of evidence suggesting that EGL-8 and EGL-30 may be important components of a pathway important for release of acetylcholine from motorneurons, thus modulating locomotion. First, both egl-8 and egl-30 are expressed in motorneurons but not in body wall muscle. Second, loss-of-function mutations in either gene cause resistance to the cholinesterase inhibitor aldicarb, suggesting that these mutants release lower than normal levels of acetylcholine. The resistance of egl-8 mutants is abolished by expressing egl-8(+) specifically in motorneurons, suggesting a site of action in these cells. Conversely, transgenic egl-30(gf) strains exhibit marked hypersensitivity to aldicarb, suggesting that these worms are releasing too much acetylcholine. A potential target of this pathway is the synaptic release protein UNC-13, which encodes a high affinity Diacyl glycerol (DAG) receptor. Our working model is that activation of EGL-8 by EGL-30 results in production of DAG, which binds UNC-13 and results in increased release of acteylcholine. Supporting this model are the observations that addition of exogenous phorbol esters (DAG analogs) abolishes the aldicarb resistance seen in egl-8 mutants, and that transgenic worms expressing only a form of UNC-13 that is unable to bind DAG are markedly less susceptible to egl-30(gf) induced aldicarb hypersensitivity. A potential ligand for the egl-30/egl-8 pathway, as well as the relationship of this pathway to the serotonin/goa-1 pathway will also be considered.

24 May 1999 15:50 119 119 Signaling by Go and Gq : suppressors of activated Goa

1999 International Worm Meeting abstract 68 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Signaling by Go and Gq : suppressors of activated Goa WJ Chen, YM Hajdu-Cronin, Pw Sternberg HHMI/California Institute of Technology, Pasadena, CA 91125, U.S. A. Goa is involved in several C. elegans behaviors including egg-laying, feeding and movement. We isolated 24 independent suppressors of a Goa gain of function mutation under the control of a heat shock promoter (syIs17). These define 8 complementation groups and can be divided into 3 classes according to behavior. Class I (sag-1, sag-2 aka. eat-16) mutants are hyperactive and egg-laying constitutive, mimicking the phenotype of Goa (lf). Class II includes sag-3, sag-5 and sag-7, they are egg-laying deficient. Class III includes sag-4, sag-6 and sag-8, they are nearly wild type in appreance and behavior. Double mutants constructed between sags of different classes suppress syIs17 better than single mutants, indicating the three classes of genes suppress syIs17 in different mechanisms. Western analysis indicates that Class II and III reduce the level of heat-shock induced GOA-1. sag-4 was positionally cloned and it encodes a cyclin K homolog which is associated with TFIIH involved in RNA polymerase II transcription. Genes in Class II and III might act as transcription factors or protease inhibitors to suppress syIs17 in a general or heat shock-specific way. Class I mutants do not affect the heat-shock induction of GOA-1. We positionally cloned eat-16 and found it encodes a RGS7 (Regulators of G protein Signaling) homolog. Double mutants between eat-16 and egl-30 (Gqa ) suggest that eat-16 acts as a Gq GAP since eat-16 suppresses egl-30 partial loss of function alleles but not the candidate null allele. Transfection of eat-16 into COS-7 cells reduces the PLC activity caused by endogenous mammalian Gq/G11. sag-1 encodes a diacyl glycerol kinase (aka dgk-1, Nurrish et al, personal comm.). Animals defective in both eat-16 and sag-1 are inviable, but reducing Gqa function restores their viability, suggesting that lethality is due to excessive production of second messengers downstream of Gqa such as diacyl glycerol. These suppressor analyses suggest that Go and Gq pathways function antagonistically in C. elegans, both sag-1 and eat-16 act downstream of or parallel to Goa and negatively regulate Gq signaling, and Goa might negatively regulate the Gq pathway via EAT-16, SAG-1 or other unknown effectors.

24 May 1999 15:50 120 120 Serotonin inhibits Acetylcholine release via depletion of Diacylglycerol at the neuromuscular junction.

1999 International Worm Meeting abstract 69 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Serotonin inhibits Acetylcholine release via depletion of Diacylglycerol at the neuromuscular junction. SJ Nurrish 1 , L Segalat 2 , JM Kaplan 1

1 361 LSA, UC Berkeley, Berkeley, CA 94720. 2 CNRS-IPMC, 660 route des lucioles, Valbonne 06560, France.

Serotonin modulates a number of behaviours in C. elegans including locomotion. We show that serotonin inhibits locomotion by inhibiting release of acetylcholine (ACh) by motor neurons. Mutants lacking GOA-1 (a Gao subunit) or DGK-1 [a diacylglycerol (DAG) kinase] have hyperactive locomotion, which is resistant to exogenous serotonin, suggesting that this pathway mediates the serotonin response. Epistasis experiments suggest that DGK-1 acts downstream of or in parallel to GOA-1. Several results suggest that the focus of serotonin regulation of locomotion is the ventral cord motor neurons. First, goa-1 and dgk-1 are expressed in many neurons (including the ventral cord motor neurons) but not in the body wall muscles. Second, 2 candidate serotonin receptors are expressed in the motor neurons (Tim Niacaris WBG 15(2),1998 WCM). Third, exogenous serotonin made animals resistant to the acteylcholinesterase inhibitor aldicarb whereas mutants with reduced serotonin levels, cat-4, were hypersenstive to aldicarb. Gain and loss of function mutations in DGK-1 and GOA-1 also had reciprocal effects on aldicarb sensitivity - increased and decreased gene activity caused resistance and hypersensitivity, respectively. Since DGK-1 phosphorylates diacylglycerol (DAG), it was possible that the effects of the serotonin pathway were to promote destruction of DAG in the motor neurons. Consistent with this idea, we found that exogenous phorbol esters also caused aldicarb hypersensitivity, and could reverse the aldicarb resistance caused by expression of constitutively active forms of GOA-1. These results suggest that the serotonin pathway inhibits locomotion by decreasing the levels of DAG in motor neurons, thereby inhibiting release of ACh. One important target in this process is the DAG binding protein UNC-13, which is required for ACh release. GOA-1 regulated the accumulation of UNC-13 at ventral cord neuromuscular junctions, but had no effect on the distribution of a mutant form of UNC-13 that does not bind DAG. Our current working model is that serotonin reduces DAG levels at neuromuscular synapses thereby decreasing the abundance of UNC-13 at ACh release sites.

24 May 1999 15:50 121 121 An Ionotrophic Serotonin Receptor and a Serotonin Reuptake Transporter Are Involved in Experience-Dependent Modulation of Behavior

1999 International Worm Meeting abstract 70 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. An Ionotrophic Serotonin Receptor and a Serotonin Reuptake Transporter Are Involved in Experience-Dependent Modulation of Behavior R Ranganathan 1 , SC Cannon 2 , HR Horvitz 1

1 HHMI, Dept. Biology, MIT, Cambridge, MA 02139. 2 Dept. Neurobiology, Harvard Medical School, Boston, MA 02114.

Hermaphrodites respond to the presence of a bacterial lawn by slowing their locomotion rate. Animals deprived of bacteria for 30 minutes exhibit enhanced slowing when they encounter a bacterial lawn. This modulatory response is mediated by serotonin (5-HT)1 . Mutations in mod-1 and mod-5 (modulation of locomotion defective) affect both the modulatory response and 5-HT neurotransmission1 , but in opposite manners. mod-1(n3034) mutants display a dominant phenotype of reduced slowing in the modulation assay and 5-HT resistance in liquid assays of locomotion (exogenous 5-HT inhibits locomotion of wild-type animals). We have cloned mod-1 and found that it encodes a ligand-gated ion channel. Electrophysiological studies of MOD-1 in Xenopus oocytes show that the MOD-1 channel is an ion channel gated specifically by 5-HT and has a pharmacological profile distinct from its mammalian counterparts. Channels carrying the mod-1(n3034) missense mutation behave in a dominant-negative manner in oocytes, as predicted by the phenotype of two mod-1 deletion mutants. mod-5 encodes a protein similar to mammalian 5-HT reuptake transporters, which are the proposed sites of action of the tricyclic antidepressants and the selective 5-HT reuptake inhibitors (SSRIs), such as Prozac. A defect in 5-HT re-uptake is consistent with the characteristics of mod-5 mutants: defective 5-HT loading of the NSM neurons2 , more pronounced slowing than the wild type in the modulatory response, and hypersensitivity to exogenous 5-HT in liquid assays of locomotion. An understanding of how mod-1 and mod-5 function within a neural circuit should provide insights into how the processes of ionotrophic 5-HT neurotransmission and reuptake are involved in experience-dependent modulation of C. elegans behavior. Small molecules that manipulate 5-HT neurotransmission include drugs of major importance in the clinic. Our studies of the 5-HT pathway in C. elegans may define analogous human neural circuits and may suggest novel means of manipulating such circuits.

1. Sawin, E., (1996), Ph. D. Thesis, MIT. 2. Trent, C., (1982), Ph. D. Thesis, MIT.

24 May 1999 15:50 122 122 Fluoxetine (prozac) resistant mutants identify a novel family of multipass transmembrane proteins

1999 International Worm Meeting abstract 71 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Fluoxetine (prozac) resistant mutants identify a novel family of multipass transmembrane proteins RK Choy 1 , JH Thomas 1,2

1 Program in Molecular and Cellular Biology, University of Washington. 2 Department of Genetics, University of Washington.

Although over 20 million people have taken fluoxetine for a wide range of mental disorders, its molecular mechanism of action remains unproven. Fluoxetine is a member of the selective serotonin reuptake inhibitor (SSRI) class of antidepressants, which inhibit the presynaptic serotonin reuptake transporter. However, it is still unclear whether this inhibition is responsible for their antidepressant action. In C. elegans, SSRIs induce contraction of nose, body-wall and egg-laying muscles. Two pieces of evidence suggest that SSRIs induce nose and body muscle contraction by acting on a non-serotonergic target: 1) exogenous serotonin does not induce nose and body muscle contraction, and 2) serotonin deficient cat-4 mutants are still sensitive to fluoxetine-induced nose and body muscle contraction. We have previously reported the isolation and characterization of several mutants that are nose resistant to fluoxetine (Nrf). These mutants are cross-resistant to other SSRIs, but are fully sensitive to levamisole, an unrelated cholinergic agonist that also causes nose muscle contraction. Therefore, these Nrf mutations may identify novel genes relevant to antidepressant action. Mutations in three of the Nrf genes (nrf-5, nrf-6 and ndg-4) have a common secondary phenotype of producing pale eggs (Peg). These mutants are defective in yolk transport and accumulate yolk in the pseudocoelomic space. nrf-6 and ndg-4 both encode homologous transmembrane proteins and define a novel gene family of over a dozen members in C. elegans. This family encodes proteins that are predicted to have a large N-terminal extracellular domain of about 200 amino acids, followed by 12 transmembrane domains. nrf-6 and ndg-4::gfp fusions are both expressed strongly in hypodermal cells at the tip of the nose, in intestinal cells, and at lower levels in other hypodermal cells. We are attempting to determine the site of action for the Nrf and Peg phenotypes by expressing nrf-6 and ndg-4 under tissue-specific promoters. We have identified several members of the nrf-6/ndg-4 family in Drosophila and Bombyx mori and are also attempting to identify mammalian homologs.

24 May 1999 15:50 123 123 Pharyngeal Calcium Transients and Prospects for Excitable Cell Imaging

1999 International Worm Meeting abstract 72 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Pharyngeal Calcium Transients and Prospects for Excitable Cell Imaging RA Kerr 1 , V Lev-Ram 2 , RY Tsien 2,3 , WR Schafer 1

1 Department of Biology, UC San Diego, 9500 Gilman Drive, La Jolla, CA 92093. 2 Department of Pharmacology, UC San Diego, 9500 Gilman Drive, La Jolla, CA 92093. 3 HHMI, UC San Diego, 9500 Gilman Drive, La Jolla, CA 92093.

C. elegans offers many advantages as a neurobiological model, but physiological studies, especially in living animals, have been difficult. We are using in vivo calcium imaging as an alternative approach. Specifically, we have been testing whether the FRET-based, ratiometric calcium-sensitive protein cameleon, developed in Roger Tsien’s lab, can be used to visualize neuronal and muscle calcium transients in live animals. Our initial focus, while developing the technique, has been on detecting and measuring the calcium influx accompanying contraction of the pharyngeal muscle. We expressed various cameleons under control of the pharyngeal-specific promoter myo-3, and imaged the fluorescence ratio emitted by the pharyngeal muscle cells. We observed prominent peaks in these ratiometric traces characteristic of calcium transients, which precisely accompanied muscular contractions and which did not appear under conditions where pumping was inhibited. To determine whether cameleon could be used to quantify calcium transients, we analyzed the behavior of mutants in the pharyngeally expressed voltage-gated calcium channel EGL-19. Thus far, we have found that the inactivation-deficient gain-of-function allele ad695 increases the duration but not magnitude of the calcium influx, a result consistent with the electrical recordings made in the Avery lab. We have also investigated the effects of several potential calcium-channel regulators. For instance, the G-proteins GOA-1 and EGL-30 are expressed in the pharynx, and recessive mutations in either gene reduce pharyngeal pumping. We found that egl-30 transients fell within the range of wild-type, whereas two different goa-1 mutants showed a significant decrease in the magnitude and duration of their pharyngeal calcium transients, suggesting that GOA-1 may act as a positive regulator of calcium channels in the pharynx, whereas EGL-30 may modulate muscle activity through a different target. These results indicate the viability of the technique in muscles and also provide encouragement that cameleon may be usable as an indicator of neuronal activity; we are working on overcoming technical barriers to making useful neuronal recordings.

24 May 1999 15:50 124 124 EXP-2 is a K+ channel that repolarizes the pharyngeal muscle

1999 International Worm Meeting abstract 73 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. EXP-2 is a K + channel that repolarizes the pharyngeal muscle MW Davis 1,2 , R Fleischhauer 3 , JA Dent 1 , R Joho 3 , L Avery 1

1 Department of Molecular Biology and Oncology, University of Texas Southwestern Med. Center, Dallas TX 75235-9148. 2 [email protected] 3 Center For Basic Neuroscience, University of Texas Southwestern Med. Center, Dallas TX 75235-9111.

We have found that exp-2 encodes a K+ channel involved in the repolarization of pharyngeal muscles. Electropharyngeograms and intracellular current clamp recordings show that exp-2 gain-of-function pharyngeal muscles have short action potentials. Loss-of-function muscles have long action potentials with very slow repolarization. These electrophysiological defects are what we expect if exp-2 encodes a subunit of the C. elegans negative spike channel (NSC), a K+ current in Ascaris pharyngeal muscles that is responsible for the fast repolarization at the end of the action potential (Byerly and Masuda, 1979, J Physiol 288, 263). We have expressed the EXP-2 channel in Xenopus oocytes. Its properties closely resemble those of the NSC. Upon depolarization, the channel activates slowly (t=56ms at +20mV) and inactivates rapidly. Repolarization then rapidly removes inactivation and allows K+ current to flow until the channel deactivates. These properties result in a channel that conducts rapidly when the voltage drops below a threshold after a long depolarization and make EXP-2 well suited to repolarize the muscle quickly at the end of the action potential. The exp-2 gain-of-function mutation is a cys to tyr mutation in the sixth transmembrane segment of the protein. It causes the channel to activate at potentials at least 100mV more negative than the wild-type channel. This results in a substantial fraction of open channels at -80mV, and would explain many of the phenotypes of the gain-of-function mutant.

24 May 1999 15:50 125 125 EAT-2 is a beta subunit of a nicotinic acetylcholine receptor involved in neurotransmission from MC to pharyngeal muscle

1999 International Worm Meeting abstract 74 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. EAT-2 is a beta subunit of a nicotinic acetylcholine receptor involved in neurotransmission from MC to pharyngeal muscle JP McKay, DM Raizen, L Avery UT Southwestern Medical Center 5323 Harry Hines Blvd. Dallas Texas 75235 Pharyngeal pumping speeds up in response to bacteria. This behavior requires pharyngeal motor neuron, MC (Neuron 3:473). Mutations in eat-2 prevent rapid pharyngeal pumping. Two results implicate eat-2 in MC neurotransmission. First, electrical recordings from the pharynx of an eat-2 mutant are similar to recordings from an MC- worm. Second, ablating MC has no effect on the pumping rate of eat-2 mutants. We have identified fourteen independent recessive mutations in eat-2 that result in a reduced pumping rate. There is complex intragenic complementation between some of the eat-2 alleles as well as allele-specific interactions between eat-2 and a semidominant allele of another slow pumping mutant, eat-18 (Genetics 141: 1365). eat-18 is MC- by the same criteria as eat-2. A model consistent with these data is that EAT-2 and EAT-18 interact in a protein complex that is involved in acetylcholine neurotransmission from MC. We cloned eat-2 and it encodes a b-subunit of a nicotinic acetylcholine receptor. Expression of an eat-2::GFP fusion construct is restricted to pharyngeal muscle. Also, an eat-2 cDNA expressed under the control of the pharyngeal muscle specific myo-2 promoter rescues the slow pumping eat-2 mutant phenotype. These results indicate that EAT-2 functions in pharyngeal muscle. We sequenced the eat-2 alleles that demonstrate intragenic complementation or allele specific interaction with eat-18 and found that all are missense mutations. The amino acid changes in the eat-2 alleles that show intragenic complementation cluster in the amino-terminal extracellular region of the protein. This region has been shown previously to be important for receptor assembly and ligand binding. These mutations may identify specific amino acids that are important for these processes.

24 May 1999 15:50 126 126 Analysis of UNC-49 reveals two structural determinants for steroid modulation of GABA receptors

1999 International Worm Meeting abstract 75 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of UNC-49 reveals two structural determinants for steroid modulation of GABA receptors BA Bamber, T Rutar, EM Jorgensen Dept. Biol., Univ. of Utah, Salt Lake City, UT 84112.

GABAA receptors are important inhibitory neurotransmitter receptors. A multitude of compounds act as allosteric modulators of GABAA receptors. Many of these have proven to be useful anticonvulsant and anxiolytic drugs, agricultural pesticides or anti-nematocidal agents. However, the biological significance of allosteric modulation of GABAA receptors is not clear because, for the most part, candidates for endogenous modulators have not been found. For this reason, neurosteroids are an interesting class of modulator. Neurosteroids can be isolated from brain tissue, and they seem to influence behavior and cognitive function. In vitro, neurosteroids can either enhance or inhibit GABA receptor activity, depending on the structure of the steroid. Exogenously-administered neurosteroids are powerful enough to induce general anaesthesia. Neurosteroids with useful drug properties could conceivably be designed, however a structural understanding of how they interact with GABA receptors is a prerequisite. Unfortunately, the study of neurosteroids has lagged behind studies of other allosteric regulators. Our characterization of the C. elegans GABAA -like receptor has provided a new approach to study neurosteroid function.

The unc-49 gene produces two GABA receptor subunits, UNC-49B and UNC-49C, which function at the C. elegans neuromuscular junction. UNC-49B can form a homomeric GABA receptor which is relatively insensitive to the inhibitory neurosteroid pregnenolone sulfate (PS). UNC-49B can also co-assemble with UNC-49C to form a heteromeric GABA receptor which is effectively inhibited by PS. By constructing chimeric UNC-49B/C subunits, we demonstrated that the UNC-49C transmembrane domains are required for PS sensitivity. With a second set of chimeric subunits, we demonstrated that PS acts through two discrete sites, one of which impairs GABA binding and the other impairs receptor activation. Finally, we have narrowed down both sites to one or two amino acids by site-directed mutagenesis. These results identify, for the first time, residues within a GABA receptor subunit which are critical for neurosteroid modulation. These results also hint that the answers to many other receptor structure-function questions may lie within the C. elegans genome.

24 May 1999 15:50 127 127 The Genetics of Ivermectin Resistance

1999 International Worm Meeting abstract 76 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Genetics of Ivermectin Resistance J Dent 1,2 , D Vassilatis 3 , M Smith 3 , L Avery 1

1 Department of Molecular Biology and Oncology, UT Southwestern Medical Center, Dallas, TX 75235-9148. 2 Department of Biology, McGill University, Montreal, QC H3A 1B1. 3 Department of Biochemistry, Merck Co. Rahway N.J.

Because ivermectin activates glutamate-gated chloride channels (GluCls), it has been proposed that this class of channels is the relevant nematocidal target of ivermectin. However, the mutations in the genes avr-14, avr-15 and glc-1, which encode a-type GluCl subunits in C. elegans, do not confer substantial ivermectin resistance. Carl Johnson reported that an avr-14; avr-15 double mutant is substantially resistant to ivermectin. We have extended his result by constructing all combinations of avr-14(ad1302), avr-15(ad1051) and glc-1(pk54). Only the avr-14(ad1302); avr-15(ad1051) double exhibited substantial resistance (~10-fold increase in EC50). However, the avr-14(ad1302); avr-15(ad1051) glc-1(pk54) triple mutant exhibited an ~4,000-fold increase in resistance. Thus all three GluCl genes contribute significantly to ivermectin sensitivity in C. elegans. We have also extended Carl’s observation that two other types of genes modulate ivermectin resistance. The innexin-encoding genes unc-7 and unc-9 showed no resistance as single mutants or in combination with any of the GluCl-encoding genes except for avr-15. In contrast, the Dyfs decrease ivermectin sensitivity by ~2.5-fold as single mutants. Furthermore , the resistance conferred by a representative Dyf, osm-1(ad1307), adds to the resistance conferred by all other combinations of GluCl or Unc genes tested. However, osm-1 seems to preferentially affect the avr-14-mediated sensitivity because the sensitivity of osm-1 avr-14 = osm-1 but the sensitivity of osm-1 avr-15 osm-1. We propose a model of the mechanisms of ivermectin resistance whereby Dyf mutants inhibit cuticle permeability through amphids which preferentially reduces ivermectin’s concentration in the nervous system. avr-14 and probably glc-1 are expressed in the nervous system and mediate nervous system sensitivity to ivermectin. avr-15 mediates pharyngeal sensitivity. unc-7 and unc-9 mutations probably reduce nervous system sensitivity by restricting the spread of ivermectin-induced hyperpolarization from neurons expressing GluCls to neurons with which they are normally electrically coupled.

24 May 1999 15:50 128 128 Mutations in the AMPA-type glutamate receptor, glr-1, block olfactory associative and non-associative learning in C. elegans.

1999 International Worm Meeting abstract 77 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Mutations in the AMPA-type glutamate receptor, glr-1, block olfactory associative and non-associative learning in C. elegans. GE Morrison, D van der Kooy Department of Anatomy & Cell Biology, University of Toronto, Toronto, Canada M5S 1A8 The AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazole propionate) type ionotropic glutatmate receptor mediates fast excitatory neurotransmission in the vertebrate brain and is important for synaptic plasticity and the initial induction of long-term potentiation (LTP). Neurotransmission through glutamate also occurs in C. elegans. The putative C. elegans AMPA receptor gene, glr-1, is expressed in the post-synaptic targets of the ASH sensory neuron and plays a role in mechanosensory avoidance but not osmotic avoidance, two behaviours mediated by ASH. Here we show that neurotransmission through glr-1 plays a significant role in experience-dependent behaviour in C. elegans. glr-1 mutant worms are deficient in olfactory associative learning in which worms learn not to track the previously attractive conditioned cue, diacetyl (DA), after it has been paired with an aversive acetic acid (AA) solution. glr-1 mutant worms acutely sense and respond to DA and AA just as wild type worms do, yet are unable to form an association between the two stimuli. Moreover, glr-1 mutant worms are impaired in non-associative learning (habituation) after long exposures to the same DA stimulus. The C. elegans learning mutants, lrn-1 and lrn-2, are impaired in chemosensory associative learning and have no deficits in habituation. The results suggest that although associative and non-associative learning can be genetically dissociated (lrn-1 and lrn-2) they also share some common molecular processes including glr-1.

24 May 1999 15:50 129 129 NMDA Receptors in C. elegans: Their Role in Effective Locomotion

1999 International Worm Meeting abstract 78 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. NMDA Receptors in C. elegans: Their Role in Effective Locomotion PJ Brockie, DM Madsen, AV Maricq Department of Biology, University of Utah, Salt Lake City, Utah 84112 A number of well defined behaviors of C. elegans have been described. Almost all require both coordination and modulation of locomotion. We have shown that a mutation in a C. elegans ionotropic glutamate receptor subunit, nmr-1, appears to impair the efficiency of certain behaviors by interfering with the modulation of locomotion. nmr-1 encodes a protein with greatest identity to rat NR1, a member of the NMDA class of glutamate receptors. NMR-1 is expressed in a small subset of the nervous system, including the interneurons AVA, AVE, AVD and PVC. Chalfie et al., have shown that these command interneurons form part of the Touch Response Circuit that regulates locomotion. By characterizing the effect of a deletion mutation in nmr-1, we have shown that it functions within this circuit to coordinate the switch between forward and backward movement. On average, wild-type worms spend approximately 20 seconds moving in a forward direction before making a brief reversal. In contrast, nmr-1(ak4) mutants modulate their direction of movement less often and spend twice as long moving forward before reversing. Interestingly, the deletion mutation does not affect either coordination or velocity of movement. Using standard assays, nmr-1(ak4) worms show no defects in chemotaxis towards the attractant diacetyl. In order to explore the possibility that movement defects in nmr-1(ak4) worms affect the efficiency of chemotaxis we designed a worm maze assay. Using CuSO4 as a repellent we tested the ability of wild-type and nmr-1(ak4) worms to navigate through a maze of CuSO4 barriers towards a source of diacetyl. Preliminary data suggest that nmr-1(ak4) worms do not perform as well as wild-type in the maze assay. After 2 hours about 35% of the wild-type worms have moved from the origin, through the maze to the diacetyl on the opposite side. nmr-1(ak4) worms have less success with only 12% reaching the attractant. The phenotype can be rescued by introducing wild type NMR-1. Furthermore, recent data indicate that an N-terminal NMR-1::GFP full length fusion, but not a C-terminal fusion, can rescue the phenotype. This suggests that the PDZ domain recognition motif (FAV) at the C-terminus of NMR-1 is required for localization, a function impaired when GFP is fused immediately downstream of FAV.

24 May 1999 15:50 130 130 PAR-6/PAR-3/PKC-3, a PDZ-mediated protein complex important for establishing early embryonic polarity

1999 International Worm Meeting abstract 79 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. PAR-6/PAR-3/PKC-3, a PDZ-mediated protein complex important for establishing early embryonic polarity TJ Hung, E Lee, KJ Kemphues Field of Genetics and Development, Cornell University, Ithaca, NY 14853 The par-6, par-3 and pkc-3 genes are required to establish polarity in the C. elegans embryo. Mutant embryos of par-3, par-6 and pkc-3(RNAi) exhibit similar phenotypes. The colocalization of PAR-3, PAR-6 and PKC-3 at the anterior periphery of one-cell embryos and their dependence for peripheral localization on one another have led us to propose that these three proteins may exist in a complex. To test this hypothesis, we examined the interactions between PAR-3, PAR-6 and PKC-3. We discovered that PAR-3, PAR-6 and PKC-3 can physically associate with each other in vitro. PAR-6 can bind to PAR-3 via PDZ mediated interaction. We also identified PKC-3 as one of two proteins that interact with PAR-6 in a yeast two hybrid screen we performed. Furthermore, GST-PAR-3 can bind to PKC-3 and PAR-6 in embryo extracts. Finally, using anti-PAR-6 antibodies, we were able to co-precipitate PAR-3 and PKC-3 from embryo extract. Together, these data suggest the existence of a PDZ-mediated tripartite protein complex at the anterior periphery of one-cell embryos that is important for establishing embryonic polarity.

24 May 1999 15:50 131 131 pod-1 links embryonic polarity to cellular architecture

1999 International Worm Meeting abstract 80 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. pod-1 links embryonic polarity to cellular architecture C Rappleye 1 , C Smith 1 , A Paredez 1 , K McDonald 2 , R Aroian 1

1 U.C. San Diego. 2 U.C. Berkeley.

Cell polarity is critical to the early C. elegans development. The first division of the embryo is asymmetric, giving rise to the large anterior cell AB and the small posterior cell P1 . Genetic screens identified par mutants (Kemphues et al., 1988) that perturb this anterior-posterior (a-p) polarity. Early experiments indicated that actin also played a role in a-p polarity (Hill and Strome, 1988). To explore this further, we isolated actin-binding proteins (ABPs) present in embryos (Aroian et al., 1997). One of these, CABP11, was polarized to the anterior of 1-cell embryos. Here we characterize CABP11, now called POD-1, with surprising results. POD-1 is an unusual protein that encodes two degenerate copies of the ABP Coronin. POD-1 protein localizes to the anterior cortex of P cells in a cell-cycle dependent manner. We knocked pod-1 out by generating a deletion allele. pod-1 is required only maternally, suggesting a specialized role in embryogenesis. Loss of pod-1 leads to loss of asymmetry similar to par mutants-- the first cleavage is often symmetric, leading to synchronous and parallel second divisions. Germline granules are missegregated, and both PAR-3 and PAR-1 protein lose their asymmetry. Thus, pod-1 is required for a-p polarity. Reciprocally, some par genes are required for POD-1 asymmetry. These data suggest pod-1 functions in same pathway as par genes. However, unlike other polarity mutants, loss of pod-1 leads to dramatic defects in the physical organization of the early embryo, i.e., formation of blebs, formation of abnormal endocytic compartments, formation of unstable cell divisions, and impaired eggshell function. Cortical actin is not perturbed in the mutant and the cell-cycle progresses normally. To achieve higher resolution, we compared wild-type and mutant embryos at the ultrastructural level. Although the eggshell appeared normal, abnormal plaque material was found on the surface of mutant embryos. These data suggest pod-1 represent a new class of developmental gene in the early embryo. We believe that pod-1 influences embryonic polarity via a cellular mechanism also required for cell architecture and that pod-1 links the generation of embryo polarity to the cytoskeleton. Our results allow us to hypothesize on the mechanism involved in generating polarity.

24 May 1999 15:50 132 132 mes-1, a gene required for unequal divisions of the germline in early C. elegans embryos, encodes a membrane protein localized to the boundary between the germline and gut cells.

1999 International Worm Meeting abstract 81 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. mes-1, a gene required for unequal divisions of the germline in early C. elegans embryos, encodes a membrane protein localized to the boundary between the germline and gut cells. LA Berkowitz, S Strome Indiana University Bloomington, IN 47405 One mechanism for generating different cell types is asymmetric cell division. The mes-1 gene is required for the asymmetric divisions of the germline blastomeres in embryos. Mutations in this gene cause the offspring of homozygous mutant mothers to develop into sterile adults. Mutant offspring are sterile because the primordial germ cell P4 develops instead as a muscle precursor, like its sister cell D (1). This cell fate transformation is the result of loss of asymmetry during the divisions that generate P4 and D. In wild-type embryos, asymmetry is generated in P2 and P3 by rotation and migration of the centrosome-nucleus complex to an asymmetric position within the cell. In mes-1 mutant embryos, this complex in P2 and P3 fails to rotate and migrate, resulting in a loss of asymmetry (2). Thus, MES-1 participates in generating division asymmetry and distinct daughter cells. MES-1 is predicted to be a 106 kD transmembrane protein with overall structural similarity to receptor tyrosine kinases (RTKs). However, the absence of highly conserved residues essential for kinase activity suggests that MES-1 probably does not function as a kinase. In wild-type embryos MES-1 is present as a thin crescent between each germline blastomere and the adjacent gut cell (i.e. between P2 and EMS, then between P3 and E). This position correlates with the region of P2/P3 to which the centrosome-nucleus complex migrates. The pattern of MES-1 staining is disrupted in mutants that affect division patterns in the early embryo (some of the par mutants and let-99), but appears normal in mutants that alter the fate of either P2 (pie-1) or EMS (pop-1, mom-2, mom-4). Ectopic staining is seen in mex-1 embryos, which, among other defects, mislocalize P granules. The role of MES-1 may be to attract intracellular components to an asymmetric site on the plasma membrane or to provide an orientation signal for P-cell-specific events. We hypothesize that, in addition to the well known P2 induction of correct EMS fates, an interaction between P2 and EMS also influences the fate of P2, and that MES-1 is a component of this EMS-P2 interaction.

(1) Strome et al. Dev. 121, 2961-72 (1995) (2) Hird et al. Dev. 122, 1303-12 (1996)

24 May 1999 15:50 133 133 PIE-1 localization to the germ lineage depends on two distinct mechanisms that act on separate domains in the PIE-1 protein

1999 International Worm Meeting abstract 82 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. PIE-1 localization to the germ lineage depends on two distinct mechanisms that act on separate domains in the PIE-1 protein KJ Reese, MA Dunn, G Seydoux Mol. Biology & Genetics, Johns Hopkins U., School of Medicine, Baltimore, MD 21205 During embryonic cleavages, maternal PIE-1 is segregated to the germ lineage where it inhibits mRNA production and maintains germ cell fate. The mechanisms that localize PIE-1 to the germline are not known, but may be related to those that localize P granules. Like P granules, PIE-1 is uniformly distributed in newly fertilized eggs, but becomes localized to the posterior before the first cleavage, resulting in most of PIE-1 segregating to the P1 germline blastomere. This asymmetric segregation is repeated at each unequal P cell cleavage. We have constructed a PIE-1:GFP fusion which faithfully reproduces PIE-1 localization in vivo. We first used this fusion to assess the role of non-coding sequences. We found that these sequences are neither necessary nor sufficient for localization, suggesting that the mechanisms that localize PIE-1 primarily act on the PIE-1 protein rather than the pie-1 RNA. We next analyzed coding sequences and identified two domains required for localization. A first domain near the carboxyl terminus of the protein is necessary and sufficient for localizing PIE-1 to the posterior prior to cell division. This domain includes PIE-1’s second CCCH finger, which on its own can target GFP to P granules. We also identified a second domain necessary and sufficient for degrading PIE-1 in somatic blastomeres. This domain corresponds to PIE-1’s first CCCH finger, and is required to remove any PIE-1 left over in the somatic daughter after cell division. These observations strongly suggest that PIE-1 localization is regulated by two distinct mechanisms: a first mechanism which localizes PIE-1 to the posterior prior to cell division, and a second mechanism which destabilizes PIE-1 in somatic cells. Consistent with the idea that these two mechanisms are mediated by different factors, we have found that par-1 activity is required for the first mechanism but not the second. Additionally, we have shown that segregation of PIE-1 to the posterior depends on the presence of an intact actin cytoskeleton, but does not absolutely depend on PIE-1 binding to P granules. These results suggest that the mechanisms that generate A/P asymmetry in the early embryo can act directly on PIE-1 to promote its segregation.

24 May 1999 15:50 134 134 Studies on POS-1 interacting proteins.

1999 International Worm Meeting abstract 83 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Studies on POS-1 interacting proteins. Ken-ich Ogura, Yuji Kohara CREST, JST, and Genome Biology Lab., National Institute of Genetics, Mishima 411-8540, Japan. The pos-1 gene encodes a TIS11 type zinc finger protein similar to PIE-1 and MEX-1. pos-1 mutants show maternal effect embryonic lethality with either reduced or excess pharynx, almost no intestine, no germ cell and extra hypodermis. POS-1 proteins localize to P-lineage, being a temporary component of P granules. Unlike PIE-1, POS-1 presents in cytoplasm but not in nuclei. POS-1 is suggested to function in translation of maternal mRNAs such as apx-1, however, the molecular process in which POS-1 functions is largely unknown. Aiming at understanding the molecular process, we have identified the proteins that interact with POS-1 by using the yeast two hybrid system. These included POS-1 itself, MEX-3 (a KH type RNA binding protein), a novel RNA binding protein (named PIP-1 after Pos-1 Interacting Protein), a serine/threonine protein phosphatase 2A catalytic subunit (PP2AC). We have confirmed that POS-1 directly bound to these proteins in vitro. The results indicated that POS-1 could form homodimer or homocomplex. MEX-3 appears to regulate the translation of maternal pal-1 mRNA, POS-1 may function in the translation of maternal mRNA in cooperation with MEX-3. RNAi experiments with pip-1 yielded phenotypes similar to the pos-1 mutants. The pip-1 mRNA was detected abundantly in early embryos, and showed its localization to posterior blastomeres after 4 cell stage. Immunostaining with anti-PIP-1 antibodies showed that PIP-1 was present equally in the cytoplasm of both AB and P1 blastomeres at 2 cell stage, and began to localize to the posterior blastomeres at 4 cell stage; the staining was strong in P2 and EMS but weak in ABa and ABp. Interestingly, this staining pattern is in contrast with MEX-3. PIP-1 was suggested to be a temporary component of P granules. Furthermore, using the two hybrid system, we found that PIP-1 interacted with MEX-3 and also MEX-1. Finally, we revealed that the PP2AC homolog is encoded by let-92, a gene required for early larval development. Our analysis showed that LET-92 plays essential roles in the coordination of nuclear division and cytokinesis and P granule localization. It is interesting that POS-1 has putative phosphorylation sites of casein kinase 2 and PKC. We will discuss about the possible mechanisms and functions of the interactions among these proteins.

24 May 1999 15:50 135 135 Non-destructive, 3-D imaging of GFP-tagged proteins throughout C. elegans embryogenesis

1999 International Worm Meeting abstract 84 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Non-destructive, 3-D imaging of GFP-tagged proteins throughout C. elegans embryogenesis JA Waddle 1 , RH Waterston 2

1 University of Texas Southwestern Medical Center at Dallas. 2 Washington University School of Medicine.

A powerful feature of C. elegans research is the ability to determine mutant phenotypes at the resolution of single cells. The advent of green fluorescent protein (GFP) provides means to carry the single cell analysis one step further, that is, to characterize critical events that take place during a short interval within a cell cycle. Improvements in computers and imaging devices motivated us to develop a 3-dimensional image acquisition system capable of merging traditional embryonic cell lineage analysis with the latest GFP technology. To gauge the utility of our microscope setup, we imaged a transgenic line carrying a fusion between GFP and an isoform of histone H1 (kindly provided by Melanie Dunn and Geraldine Seydoux). The most important outcome of our efforts is the ability to non-destructively (e.g the embryos hatch and are fertile) collect a 3-dimensional time lapse fluorescence image sequence starting at first cleavage and spanning more than 8 hours of development. The histone H1::GFP images greatly facilitate the tracking of nuclear movements and cell divisions in the bottom half of later stage embryos. The application of computational methods, to reduce the effects of out of focus light, greatly improves image detail and the signal-to-noise ratio. Current efforts are focused on custom software capable of tracking the 3-dimensional distribution and behavior of the histone H1::GFP labeled nuclei, that is, automated embryonic cell lineage analysis. In summary, several technologies have coalesced to provide non-damaging means to track the 3-dimensional distribution of important molecules during embryogenesis. Such technology will complement existing methods for phenotypic analysis in an era when both the embryonic cell lineage and the genome descriptions are complete.

24 May 1999 15:50 136 136 Regulation of POP-1 asymmetry

1999 International Worm Meeting abstract 85 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Regulation of POP-1 asymmetry Rueyling Lin Dept. Mol. Biol. & Oncol., UT Southwestern Med. Cntr., Dallas, TX POP-1 functions downstream in the P2/Wnt signaling pathway that regulates the fate difference between a-p sisters MS and E. I have shown that a monoclonal antibody to POP-1 (MabRL2) stains the MS nucleus more intensely than the E nucleus, consistent with the genetic finding that pop-1(+) activity is required for MS cell fate. Furthermore, POP-1 is detected at higher levels in the anterior sister for all a-p divisions throughout early embryogenesis. We have previously shown that differences in POP-1 activity between sister cells are required for the anterior cell to adopt a fate different from that of its posterior sister. Based upon POP-1 staining, mutants defective in POP-1 asymmetry fall into either of two classes. Mutants in Class I are defective in POP-1 asymmetry in only MS and E blastomeres. These are mutations in genes involved in P2/Wnt signaling, such as mom-1 and mom-2. Mutants in Class II are defective in POP-1 asymmetry throughout the embryo - i.e. all blastomeres stain equally for POP-1. These include lit-1(t1512), wrm-1(RNAi), and a newly characterized mutant in my laboratory, pie-3(zu405). All mutants that affect global POP-1 asymmetry also affect POP-1 asymmetry between MS and E. pie-3(zu405) is a ts maternal-effect lethal mutation. At 250 C, 35% of the embryos have equal POP-1 staining throughout the entire embryo, consistent with the percentage of embryos having MS to E fate transformations as determined by ablation analyses. Mutations in pie-3 also result in C producing EMS-like tissue types. Lineage analyses will be performed to address whether C adopts the fate of EMS in pie-3 mutants. I have mapped pie-3 to an interval of approximately 100 kb on LG IV and am currently trying to identify the gene. I propose a model whereby global embryonic a-p polarity is maintained by a mechanism involving the genes wrm-1, lit-1, pie-3 and pop-1. This mechanism is required for all successive a-p divisions instead of for a single, early asymmetry that sets the a-p pattern for all subsequent divisions. However, in selected a-p divisions (e.g. EMS dividing to MS and E), the global a-p polarity is modulated by an extra layer of regulation. In such cell divisions, an additional signal(s), such as Wnt signaling, is integrated with the global mechanism to fine-tune the polarity of these selected a-p sisters.

24 May 1999 15:50 137 137 The LIT-1 and MOM-4 Protein Kinases Control Cell-Fate in Response to Anterior/Posterior Polarity Signals

1999 International Worm Meeting abstract 86 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The LIT-1 and MOM-4 Protein Kinases Control Cell-Fate in Response to Anterior/Posterior Polarity Signals T Shin, C Rocheleau, J Yasuda, CC Mello U. Mass. Med. Center, Worcester, MA Previous work by several groups suggests that Wnt/Wg signaling components and an APC related protein function to couple cell fate decisions to relative anterior/posterior (a/p) position of sister cells. The target of this polarity signaling pathway appears to be the downregulation of POP-1, a TCF/LEF1 related transcription factor. Here we show that a/p polarity signaling in C. elegans involves two proteins with homology to known protein kinases. MOM-4 is similar to the vertebrate TGFb-activated kinase TAK1, which appears structurally and functionally similar to Map kinase kinase kinases such as MEKK1. The C. elegans polarity gene lit-1 (Kaletta et al., 1997) encodes a protein related to the Drosophila tissue polarity protein NEMO. We show that WRM-1, a b-catenin related protein, binds to LIT-1 both in vivo and in vitro, and can activate a LIT-1-dependent kinase activity in mammalian cells. This activity in turn promotes the phosphorylation of POP-1 protein. The WRM-1/LIT-1 kinase activity is further stimulated by co-expression of two MOM-4 related vertebrate kinases, MEKK1 and TAK1. Finally, we show that the WRM-1/LIT-1 kinase activity reduces the nuclear localization of the POP-1 protein in mammalian cells, and thus mimics the effect of signaling in the C. elegans embryo. In Drosophila and vertebrates, the Wnt/Wg signal is thought to increase the available pool of the WRM-1 related protein b-catenin/Armadillo, which in turn is thought to bind to the POP-1 related protein TCF/dTCF and to enter the nucleus where this complex activates Wnt/Wg responsive genes. The current findings suggest a new function for WRM-1/b-catenin as an activating subunit for the LIT-1/NEMO kinase. These findings suggest a possible biochemical explanation for the reversed genetic relationship of WRM-1 and POP-1 with respect to their fly and vertebrate homologs. Finally, it is interesting to speculate that WRM-1 and LIT-1 may be independently activated by distinct upstream signals perhaps accounting for some of the apparent complexity observed in polarity signaling in this and other organisms.

TS, CR, and JY contributed equally to this work.

24 May 1999 15:50 138 138 MAP Kinase and Wnt signaling Pathways converge to directly down-regulate HMG-domain containing transcription factors in C. elegans and vertebrates

1999 International Worm Meeting abstract 87 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. MAP Kinase and Wnt signaling Pathways converge to directly down-regulate HMG-domain containing transcription factors in C. elegans and vertebrates MD Meneghini 1 , JC Carter 1 , T Ishitani 2 , N Hisamoto 2 , J Ninomiya-Tsuji 2 , S Nagai 2 , M Nishita 2 , H Shibuya 2 , Christopher J. Thorpe 1 , Danielle R. Hamill 1 , K Matsumoto 2 , B Bowerman 1

1 Institute of Molecular Biology, University of Oregon, Eugene, OR 97403. 2 Department of Molecular Biology, Graduate School of Science, Nagoya University, Chkusa-ku, Nagoya 464-0814, Japan.

Wnt signaling pathways regulate the activity of HMG-domain containing transcription factors such as TCF to direct cell fate decisions during animal development. In C. elegans, the HMG-domain containing transcription factor POP-1 distinguishes anterior/posterior (a-p) daughter fates throughout development, and Wnt signaling is known to down-regulate POP-1, specifying the fate of one posterior daughter cell called E. We show that the genes lit-1 and mom-4 also are required to down-regulate POP-1 in E and in other posterior cells during embryogenesis. Consistent with acting in a common pathway, mom-4 and lit-1 mutant embryos exhibit similar phenotypes and encode a MAPKKK homologous to vertebrate TAK1 and a MAPK-related protein homologous to Drosophila NEMO respectively. Thus, Wnt signaling and MAP Kinase-related pathways converge to down regulate POP-1. Rather than converging at the level of the pop-1 promoter influencing pop-1 transcription, the regulation of POP-1 by Wnt signaling and the mom-4/lit-1 pathway seems to be direct as pop-1 function for the a-p decisions we investigate is provided maternally. Analysis in vertebrate systems confirms that this convergence is direct as the LIT-1 homolog NLK binds to and phosphorylates the HMG-domain protein TCF-4, blocking its ability to function as a transcriptional activator in conjunction with ß-catenin. Moreover, microinjection of NLK into dorsal blastomeres can block axis induction in Xenopus, and NLK also blocks the ability of ectopically expressed ß-catenin to induce a second body axis in Xenopus. Thus, the convergence of the mom-4/lit-1 MAP Kinase-related pathway with Wnt signal transduction machinery is conserved in vertebrates.

24 May 1999 15:50 139 139 src-1 interacts with a Wnt signaling pathway and an APC-related gene to control cell fate decisions in the early C. elegans embryo.

1999 International Worm Meeting abstract 88 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. src-1 interacts with a Wnt signaling pathway and an APC-related gene to control cell fate decisions in the early C. elegans embryo. J Hogan 1 , Y Bei 2 , C Mello 2 , J Collins 1

1 Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, NH 03824. 2 Cancer Center, Department of Cell Biology, University of Massachusetts Medical Center, Worcester, MA 01604.

The vertebrate proto-oncogene Src is a protein-tyrosine kinase that has been implicated as a component of receptor-mediated signal transduction pathways important for cell growth and differentiation. Activated Src mutants induce neoplastic transformation and lead to tumorigenesis in laboratory animals. Despite years of intensive investigation the biological role of Src still eludes us. To understand it role in nematode development, we took a reverse genetic approach, knocking-out src-1, a C. elegans Src ortholog. We isolated a 4.5 kb deletion, src-1(cj293), using a standard PCR screen of ENU mutagenized populations. Molecular analysis confirms that cj293 is a loss-of-function allele: we detect a shortened transcript via RT-PCR which could encode a truncated protein lacking the entire SH2 and kinase domains. As we previously reported, src-1(cj293) confers a fully penetrant maternal effect lethal phenotype. This lethality can be rescued at low levels by zygotic expression of src-1. These experiments have uncovered a maternal effect sterile phenotype for src-1 that is currently being investigated. Arrested src-1(cj293) embryos exhibit a lack of morphogenesis and appear to contain excess endoderm. Ablation studies are in progress to confirm this observation and determine the source of the extra gut cells. Defects in ABar spindle orientation are also observed. These phenotypes resemble those observed in mom mutants, suggesting that src-1 functions with these genes in a Wnt pathway that directs E blastomere fate in the early embryo. Consistent with this idea, src-1(cj293) enhances penetrance of the Mom phenotype observed in these mutants. Further, this effect requires expression of pop-1. Based on evidence from vertebrate studies indicating beta-catenin may be a common target of Src and Wnt signaling, we are investigating whether SRC-1 mediates its effects on this Wnt pathway through an interaction with WRM-1, a C. elegans beta-catenin homolog.

24 May 1999 15:50 140 140 Spatiotemporal control of end-1 expression: positive regulation by the Wnt pathway and lineage-specific repression

1999 International Worm Meeting abstract 89 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Spatiotemporal control of end-1 expression: positive regulation by the Wnt pathway and lineage-specific repression JJ Kasmir, ES Witze, JL Sumerel, J Zhu, JH Rothman University of California- Santa Barbara The E lineage is specified by the combined action of the maternal Wnt pathway and maternal SKN-1. These factors regulate E-specific expression of end-1 and end-3, GATA factor-encoding genes expressed during a short interval (E2 to E8 cell stage). To elucidate the maternal control of these zygotic genes, we are investigating elements and factors that regulate end-1. The end-1 regulatory region appears to be modularized, with a cluster of SKN-1 binding sites in a distal region (region III), a more proximal region (region II) containing elements conserved between end-1, end-3, and the C. briggsae end-1, and a proximal-most region (region I) containing putative Wnt pathway-responsive elements, including a site resembling the consensus site for Lef-1, the target of the Wnt pathway. While regions II and III appear to enhance end-1 expression, the 264 bp most proximal portion of region I confers proper spatial and temporal expression of end-1. Within region I, a 49 bp interval is essential for repression in non-E lineages and presumably identifies a target for a repressor acting outside of the E lineage. Just proximal is a 54 bp segment (region Ib), containing the apparent Wnt-responsive elements, which we find is essential for end-1 expression in all lineages. The Wnt pathway in C. elegans was thought to act negatively, i.e., by inhibiting the Lef-1-like POP-1 protein, which represses endoderm development in the MS lineage. Our results suggest that Wnt-affected POP-1 may also activate end-1 and endoderm development. Indeed, while depletion of maternal POP-1 derepresses endoderm and end-1 expression in the MS lineage, it strongly enhances the impenetrant gutless phenotype of skn-1(-), demonstrating a positive role for POP-1 in endoderm specification. Thus, the Wnt signal apparently acts both to inhibit the MS-repressive activity of POP-1, and to cause POP-1 to be a transcriptional activator of the end genes in the E lineage. Both the repressive and activating functions of POP-1 may act through the Lef-1 site in region Ib of end-1. We have found that early embryonic extracts bind to region Ib in interactions that depend on the Lef-1 consensus site (see also abstract by Witze et al.).

24 May 1999 15:50 141 141 Specification of endomesoderm by the MED-1 and MED-2 GATA factors: similarity of endoderm and mesoderm specification

1999 International Worm Meeting abstract 90 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Specification of endomesoderm by the MED-1 and MED-2 GATA factors: similarity of endoderm and mesoderm specification MF Maduro, JH Rothman Dept. MCD Biology, UC Santa Barbara, Santa Barbara, CA 93106 E and MS, though sisters, undergo highly different developmental programs. While the E cell gives rise only to gut (endoderm), MS produces primarily mesodermal tissue, including posterior pharynx and somatic gonad. The maternal skn-1 gene is required for EMS fate: in skn-1 mutants, both E and MS can adopt a C-like fate. end-1 and end-3 encode redundant GATA factors that appear to act downstream of SKN-1 in the E lineage to promote endoderm development. These genes are repressed in the MS lineage by maternal POP-1. Until recently, no zygotic factors were known that directly act to specify MS fate. We have found that an end-3 neomorphic mutation (zu247) identified by Russell Hill and Jim Priess causes E to adopt an MS fate and alters a single residue in the zinc finger of END-3. This observation suggested that the altered END-3 protein might function like an MS-promoting GATA factor. Indeed, we identified a pair of redundant GATA factors, MED-1 and MED-2, that specify MS. med-1 and med-2 are on separate linkage groups and are 98% identical. RNAi of med-1 and med-2 results in embryos lacking the MSaa and MSpa-derived posterior pharynx, and blocks expression of MSap-specific lag-2. This defect in MS specification is accompanied by ectopic expression of vab-7, a C-lineage-specific marker, suggesting that MS adopts a C fate. In addition, ~1/3 of mutant embryos also lack gut, suggesting a role in E specification. Reporter fusions of both med genes show SKN-1-dependent expression in the early E and MS lineages. However, ectopically expressed med-1 appears to promote only ectopic MS fate, while suppressing E fate. This suggests that the med genes act in parallel with the Wnt pathway in E specification, promoting E fate only when both the med and end genes are expressed. We postulate that MS fate is specified by the med genes, driven by SKN-1, coupled with the repression of the end genes. In E, the Wnt signal activates the end genes, and the med/end combination promotes E fate. These findings also indicate a remarkable similarity in the mechanisms that specify E and MS: both blastomeres require redundant pairs of GATA factors that dictate their identities in a cell that otherwise adopts a C-like cell fate.

24 May 1999 15:50 142 142 Does Wnt signaling distribute HAM-1?

1999 International Worm Meeting abstract 91 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Does Wnt signaling distribute HAM-1? N Hawkins, G Garriga Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720. Both intrinsic and extrinsic mechanisms polarize asymmetrically dividing cells. Many asymmetrically distributed intracellular molecules have been described that segregate fate to daughter cells during mitosis, and in C. elegans, Wnt signaling has also been shown to polarize asymmetrically dividing cells. However, it is not known whether cell interactions are involved in distributing intracellular factors, or the mechanism by which Wnts polarize asymmetrically dividing cells. We have found a potential link between the asymmetrically distributed protein HAM-1, that segregates fate in certain neuroblast divisions, and Wnt signaling. We are focusing on the asymmetric divisions that generate the HSN and PHB neurons. In this lineage, an HSN/PHB neuroblast divides asymmetrically to generate an anterior daughter cell that dies and a posterior daughter cell, the HSN/PHB precursor. This precursor then divides to produce the HSN and PHB neurons. In ham-1 mutants, the HSN/PHB neuroblast frequently divides symmetrically producing two HSN/PHB precursors. The HAM-1 protein is asymmetrically localized to the posterior periphery in the HSN neuroblast and segregated to the HSN/PHB precursor upon division. In a yeast two-hybrid screen to identify proteins that interact with HAM-1, a dishevelled (dsh) homolog, C34F11.9, was isolated. DSH is a cytoplasmic protein, that upon Wnt stimulation becomes hyperphosphorylated and recruited to the cell membrane. In C. elegans, Wnt signaling orients the polarity of several asymmetrically dividing cells. The interaction of DSH with HAM-1, an asymmetrically localized protein, suggests that Wnt signaling may direct the asymmetric distribution of intracellular proteins. Although we were unable to generate an RNAi phenotype with C34F11.9, RNAi with a second dsh homolog, C27A2.6, resulted in a Ham-1 phenotype in the HSN/PHB lineage. Overexpression of C27A2.6 at high concentrations is lethal, while at lower concentrations we observe a weakly penetrant Ham-1 phenotype in the HSN/PHB lineage. A dsh::GFP transcriptional fusion is strongly expressed in embryos at stages coincident with HAM-1 expression. Experiments are in progress to isolate mutations in C27A2.6 and to determine if dsh is required for the localization of HAM-1.

24 May 1999 15:50 143 143 A new Abd-B homolog is required for posterior patterning of the embryo

1999 International Worm Meeting abstract 92 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A new Abd-B homolog is required for posterior patterning of the embryo KM Van Auken, D Weaver, B Robertson, LG Edgar, WB Wood MCDB, U. Colorado, Boulder 80309 The four previously described C. elegans Hox genes appear to control only anterior embryonic or postembryonic patterning, raising the question of how posterior patterning in the embryo is dictated. The nob-1 locus, mapping about 15 cM right of the known Hox cluster, is defined by three mutations that disrupt this patterning: a viable mutation, ct230, causing variable tail abnormalities, and two L1-lethal mutations, ct223 and ct351, causing severe posterior morphological abnormalities (the Nob phenotype). nob-1 is predicted to encode a Hox protein with strong similarity in the homeodomain to products of the posterior Hox paralog (PHP) group genes, including egl-5, Drosophila Abd-B (72% similarity) and the vertebrate Hox9-13 genes. Further sequencing in this region revealed a third PHP group homolog php-3, (89% similar to Abd-B), beginning just ~200 bp from the 3’ end of nob-1. nob-1 transcripts are present in pregastrulation embryos, all larval stages, and the adult soma, but appear absent from the germ line. php-3 transcripts appear to be rarer but can be detected by RT-PCR in embryos and later stages. Both transcripts are trans-spliced to SL1, suggesting that nob-1 and php-3 do not constitute an operon. Analysis of the mutant alleles revealed that all are deletions. ct230 removes only small parts of exon 2 and the adjacent intron in nob-1; ct351 removes the homeobox of nob-1 and all of php-3; and ct223 removes at least 10 kb of flanking sequence 5’ of nob-1, but no coding sequence. To understand the respective functions of the two genes, we have performed dosage and RNAi experiments. Whereas most ct230 embryos develop into fertile adults, few ct230/eDf2 embryos do so, suggesting that ct230 is a hypomorph. Only about 45% of nob-1(RNAi) embryos develop into fertile adults, which have variably abnormal tails like those of ct230. In contrast, 95% of php-3(RNAi) embryos develop into fertile adults. The double nob-1(RNAi); php-3(RNAi) phenotype is no more severe than that of nob-1(RNAi) alone. We conclude that C. elegans has a split Hox cluster (like flies) including three PHP group genes (unlike flies, more like vertebrates). The role of php-3 is still uncertain, but nob-1 appears to be required for normal posterior patterning in the embryo.

24 May 1999 15:50 144 144 The cadherin-catenin complex stabilizes filopodial adhesive contacts during hypodermal morphogenesis

1999 International Worm Meeting abstract 93 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The cadherin-catenin complex stabilizes filopodial adhesive contacts during hypodermal morphogenesis WB Raich 1 , C Agbunag 2 , JR Priess 2 , J Hardin 1

1 University of Wisconsin, Madison, WI 53706. 2 FHCRC and HHMI, Seattle, WA 98109.

The sealing of epithelial sheets via the formation of adherent cell-cell junctions is an important morphogenetic event during animal development, yet little is known about how migratory epithelia modulate their junctional connections. The three dimensional shape of embryonic epithelia makes their analysis difficult, and perturbation of junctional molecules via mutation or RNA interference typically disrupts epithelia prior to sheet sealing. Mutations in the C. elegans cadherin hmr-1, a-catenin hmp-1, and b-catenin/armadillo hmp-2 are a valuable exception, as these molecules are not essential for general cell adhesion (M. Costa et al., 1998. J. Cell Biol. 141, 297-308). We have used time-lapse multiphoton laser scanning microscopy to analyze the mechanisms of migration and sealing of the C. elegans hypodermis during ventral enclosure at the subcellular level. Cell-cell junctions were visualized using two reporters: HMP-1-GFP and JAM-1-GFP (for "junction associated molecule"; JAM-1 is recognized by the MH27 monoclonal antibody). HMP-1-GFP is distributed uniformly throughout filopodia extended by the first ventral hypodermal cells to reach the midline ("leading cells"). By 5 min after their contact at the ventral midline, HMP-1 a-catenin rapidly accumulates at future sites of junction formation between contralateral cells. JAM-1 is inserted at ventral midline junctions 15 min following a-catenin accumulation. Cadherin-mediated adhesive strengthening is specifically required to stabilize initial adhesion events between filopodia; in embryos lacking hmr-1 or hmp-1 mRNA, leading cells approach the midline, establish filopodial contact, but do not form stable junctional connections. In addition, in the absence of HMR-1, ectopic and precocious cell-cell fusions occur between hypodermal cells that reach the ventral midline. In contrast, posterior ventral hypodermal cells, which do not display long filopodia, can make correct attachments at the ventral midline in the absence of HMR-1 or HMP-1. Our results indicate that embryonic epithelial cells that use filopodia for migration require recruitment of a-catenin at sites of nascent junction formation to correctly regulate mature junction assembly.

24 May 1999 15:50 145 145 The C. elegans type XVIII collagen/endostatin homologue is involved in cell migrations and axon guidance

1999 International Worm Meeting abstract 94 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The C. elegans type XVIII collagen/endostatin homologue is involved in cell migrations and axon guidance BD Ackley, JM Kramer Northwestern University Medical School We have identified cle-1 as a C. elegans homologue of vertebrate type XVIII collagen, which contains the endostatin domain identified as an inhibitor of angiogenesis in mammals. cle-1 encodes three protein isoforms that contain the following domains: a Gly-X-Y domain and a C-terminal domain with 50% similarity to mouse endostatin (CLE-1A, B, C); a thrombospondin related domain similar to that found in the N-termini of type XV and XVIII collagens (CLE-1A, B); fibronectin type III repeats most similar to the DCC/unc-40/frazzled family of netrin receptors (CLE-1A). The three isoforms have different temporal and spatial expression patterns. Form A is detected by RT-PCR only during embryogenesis. GFP reporter constructs for Form B express in dorsal motor neurons and some nerve ring neurons from threefold through larval development. Form C GFP expression starts at L2 and continues through adult stages in the excretory cell and the canal associated neurons (CANs). Antiserum made to a common domain shows strong protein localization on the nerve ring, along dorsal and ventral nerve cords and CAN axons and weaker accumulation present in other basement membranes. A deletion that removes the endostatin domain, cle-1(cg120), results in partial loss of gene function with variably penetrant phenotypes, including Egl, slight Unc and gonad and distal tip cell migration defects. Using a neuronal GFP reporter we observed missing HSNs, defasciculations in dorsal and ventral nerve cords, and defects in dorso-ventral axon projections and fasciculations in cg120 mutants. Cell migration and axon guidance defects were seen in cells that do not express cle-1, suggesting this protein does not function cell autonomously. Although single heterozygotes are essentially wild-type, some trans-heterozygotes of the cg120 deletion with mutations in the netrin receptors, unc-5 or unc-40, show Unc and Egl phenotypes, suggesting that cle-1 may interact with the netrin guidance pathway. The cg120 deletion over a chromosomal deficiency results in embryonic and larval lethality suggesting that the cle-1 null phenotype may be lethal. Our results suggest a role for endostatin in neurogenesis in C. elegans and strengthen the suggestion of a functional link between angiogenesis and neurogenesis.

24 May 1999 15:50 146 146 Nidogen is non-essential for basement membrane assembly but synthetic lethal with a dystroglycan-like gene

1999 International Worm Meeting abstract 95 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Nidogen is non-essential for basement membrane assembly but synthetic lethal with a dystroglycan-like gene SH Kang, J Johnson, S Wang, JM Kramer CMB Dept., Northwestern University Medical School, Chicago, IL 60611 Nidogen can act as a link between laminin and type IV collagen in basement membranes (BM), binding to both with high affinity, and possibly directing collagen IV assembly. The single C. elegans nidogen gene, nid-1 (V, F54F3), generates three splice isoforms that all have carboxyl laminin and amino collagen binding domains, but differ in the number of EGF repeats in the central rod. Only the longest form is expressed in embryos, the shortest form predominates post-embryonically. nid-1 is expressed in body wall muscle (bwm) at the start of morphogenesis. At L2, expression begins in the intestine and restricts to bwm in the head. Distal tip cells, spermatheca, accessory muscles, and touch neurons also express nid-1. Anti-NID-1 antisera show localization to the same sites as collagen IV, all BMs except the pseudocoelomic face of bwm and the hypodermis between bwm quadrants. An in-frame Tc1 excision deletion, cg118, removes the collagen binding domain and has an ~WT phenotype. The truncated NID-1 shows normal localization and no apparent defect in collagen IV assembly. A second deletion, cg119, is a molecular null that is viable and fertile, and has normal collagen IV assembly. The fecundity of both nid-1 mutants is reduced to 60-70% of WT, but they show no other obvious phenotype. Nidogen is not essential for assembly of BMs in C. elegans. RNAi into the nid-1 null background identified a dystroglycan-like gene, dgn-1, as a synthetic lethal. dgn-1 (X, T21B6) has strong similarity to vertebrate dystroglycan around the a/b cleavage site and transmembrane domain, but not in the laminin or dystrophin binding regions. DGN-1 may link different matrix and intracellular molecules than does vertebrate dystroglycan. RNAi of dgn-1 into N2 results in vulval and gonadal defects, but in the nid-1 null background it results in highly penetrant embryonic lethality with herniation of the hypodermis. An EMS generated deletion of dgn-1 is sterile, with frequent protruding vulva and Muv phenotypes. Preliminary analyses of arrested nid-1; dgn-1 embryos show hypodermal defects consistent with the RNAi results. NID-1 and DGN-1 may interact directly or indirectly, possibly through laminin.

24 May 1999 15:50 147 147 The gon-1 gene is a metalloprotease required for gonad morphogenesis

1999 International Worm Meeting abstract 96 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The gon-1 gene is a metalloprotease required for gonad morphogenesis R Blelloch 1 , S Santa Anna-Arriola 1,2 , J Hodgkin 2 , J Kimble 1

1 University of Wisconsin-Madison. 2 MRC-LMB, Cambridge, UK.

Development of the C. elegans gonad involves several morphogenetic processes. First is arm extension, which is controlled by the gonadal leader cells (distal tip cell, DTC, in hermaphrodites). Second is formation of the somatic gonadal primordium (SGP), which involves coalescence of somatic gonadal precursor cells to generate a structure that prefigures the adult somatic gonad. Third is transformation of the SGP and its descendants into the complex tubular organs (e.g. uterus, spermatheca) of the adult gonad. The gon-1 gene is required for all three processes: gon-1 null mutants do not extend gonadal arms, do not form an SGP and do not generate organized tubular structures. However, cell lineage patterns are unaffected in gon-1 mutants and tissue differentiation occurs normally. We find that laser ablation of the leader cells in wild-type animals phenocopies gon-1, although the effects are less severe. Therefore, we propose that many, but not all, of the gonadal defects in gon-1 mutants can be ascribed to a loss of leader cell function. The gon-1 gene encodes a protein containing a metalloprotease domain followed by numerous thrombospondin type-1 repeats. It belongs to a small gene family that includes a bovine collagenase. A gon-1 promoter fusion, gon-1 5’::GFP, drives expression in leader cells and in muscle cells. When the gon-1 coding region is placed under control of a DTC promoter (lag-2::GON-1) and is expressed in gon-1(null) mutants, arm extension occurs, but the arms are thin. Expression from a muscle promoter promotes gonadal expansion along all axes. We postulate that gon-1 is normally expressed in the leader cells to allow proximal-distal extension of the gonadal arms and in muscle cells to achieve the dorsal-ventral and left-right expansion of the gonad. We also suggest that the GON-1 metalloprotease achieves these ends by remodeling the basement membrane to permit and direct gonadal growth.

24 May 1999 15:50 148 148 MIG-17, an adam family metalloprotease required for dtc migration in C. elegans

1999 International Worm Meeting abstract 97 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. MIG-17, an adam family metalloprotease required for dtc migration in C. elegans K Nishiwaki 1 , N Hisamoto 2 , K Matsumoto 2 , N Suzuki 2

1 PRESTO, JST and Fundamental Research Labs., NEC Corporation. 2 Dept. of Biological Science, Faculty of Science, Nagoya Univ. and CREST, JST.

The distal tip cells (DTCs) migrate on the body-wall basement membrane in a U-shaped pattern during gonadal development. They initially move along the ventral muscles, turn dorsally, and migrate on the lateral hypodermal cells. They turn again over the dorsal muscles and migrate along them. We have isolated mutants whose DTCs meander on the lateral hypodermal cells after the first turn. It is possible that the guidance cue for DTC migration or recognition of such a cue by DTCs is abnormal in these mutants. So far, eight genes have been identified by analyzing 20 isolated mutations. Many double mutant combinations displayed enhanced meandering phenotypes, suggesting that they affect the same or parallel pathways. Mutations in one of these genes mig-17 cause abnormal attachment of DTCs to the body wall. The DTCs migrate erratically on the body wall or are detached from it and migrate along the gonad or the intestine. We have cloned mig-17 and found that it encodes a novel member of the ADAM family metalloproteases. MIG-17 seems to have four distinct domains: following the signal peptide are a pro-domain, a metalloprotease domain, a disintegrin-like domain, and a cys-rich domain. The transmembrane domain conserved in conventional ADAMs was not detected in MIG-17, suggesting that it is secreted. A mig17::GFP fusion gene was functional and fully rescued a mig-17 null mutant. The GFP fluorescence was observed around the gonad and on the internal surfaces of the body wall muscles, consistent with localization of MIG-17 on the basement membranes. Deletion or point mutation analysis revealed that any of metalloprotease, disintegrin-like, or cys-rich domains are essential for the rescue of the mig-17 null mutant. The MIG-17 metalloprotease may function in the basement membranes to remodel their components, and this remodeling may ensure the attachment of DTCs to the body wall and their directional migration along the migration cue.

24 May 1999 15:50 149 149 The Ephrin VAB-2 interacts with the VAB-1 Eph receptor to regulate neural and epidermal morphogenesis.

1999 International Worm Meeting abstract 98 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Ephrin VAB-2 interacts with the VAB-1 Eph receptor to regulate neural and epidermal morphogenesis. ID Chin-Sang, SE George, M Ding, AD Chishlom Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064, USA Mutations in the genes vab-1 and vab-2 cause similar defects in neural and epidermal morphogenesis. vab-1 encodes an Eph receptor tyrosine kinase (RTK) that is expressed in the developing nervous system, and is required in neurons for proper epidermal morphogenesis (George et al., Cell 92:633). Eph receptors bind membrane bound ligands (ephrins) to regulate a variety of processes, including embryonic pattern formation, angiogenesis, and axon guidance. vab-2 encodes a member the Ephrin family of ligands for Eph receptors (1997 International Worm Meeting Abstracts, p.386). The predicted VAB-2 protein contains a potential GPI attachment signal sequence, but is more similar to transmembrane Ephrins in its receptor binding domain. Three lines of evidence suggest that VAB-2 is a ligand for VAB-1. First, we have expressed VAB-2 on mammalian 293T cells and shown that VAB-2 is GPI anchored and that VAB-2 specifically binds a soluble VAB-1:Alkaline Phosphatase fusion protein with high affinity. Missense alterations in the predicted receptor binding domain of VAB-2 abolish this interaction. Second, VAB-1 and VAB-2 are expressed in adjacent sets of neurons in worms supporting that VAB-1 and VAB-2 may physically interact during embryogenesis. Furthermore, pan-neuronal expression of vab-2 can partially rescue the epidermal defects of vab-2 null mutants. Third, we have examined the phenotype of vab-1; vab-2 double mutants and shown that vab-2 null mutations do not enhance the phenotypes of vab-1 null mutants implying that vab-2 functions mostly in a vab-1 pathway. Mutations affecting the kinase domain of VAB-1 cause weak phenotypes, indicating VAB-1 may have both kinase-dependent and kinase-independent signaling functions. Interestingly, vab-2 mutations synergise with vab-1 kinase mutations but not with a vab-1 extracellular domain mutation, suggesting that VAB-2 may mediate a kinase-independent function of VAB-1, perhaps through a reverse signaling mechanism. Our results suggest that VAB-1 and VAB-2 signaling operates between neurons and is required for normal neural and epidermal morphogenesis in C. elegans.

24 May 1999 15:50 150 150 Multiple ephrins control cell organization in C. elegans through kinase-dependent and kinase-independent functions of the VAB-1 Eph receptor

1999 International Worm Meeting abstract 99 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Multiple ephrins control cell organization in C. elegans through kinase-dependent and kinase-independent functions of the VAB-1 Eph receptor X Wang 1 , PJ Roy 1,2 , SJ Holland 1 , LW Zhang 1 , JG Culotti 1,2 , T Pawson 1,2

1 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Ave., Toronto, ON, M5G 1X5. 2 Department of Molecular and Medical Genetics, University of Toronto, ON, M5S 1A8, Canada.

Vertebrates contain a large number of Eph receptors and their ephrin ligands, complicating analysis of their relative functions. However, the C. elegans genome contains only four ephrin genes (vab-2, efn-2, efn-3, and efn-4) which encode potential GPI-modified proteins, and a single Eph receptor gene (vab-1). Deletion mutations in the previously uncharacterized efn-2 and efn-3 ephrin genes were isolated by reverse genetics. The vab-2, efn-2 and efn-3 ephrins act in a combinatorial fashion to activate the single VAB-1 tyrosine kinase in vivo. Single, double and triple ephrin mutants reveal distinct but overlapping functions in cellular organization, that in aggregate mirror those of the VAB-1 receptor. To varying extents, ephrin mutants exhibit head morphology defects and failure of the epidermis to enclose the embryo. In particular, reciprocal ephrin-Eph receptor signaling is required for the correct alignment of specific epidermal cells in the tail. Phenotypic characterization of mutants and expression pattern analysis suggests that the VAB-1 receptor signals through both the kinase-dependent and kinase-independent pathways, both of which are dependent on multiple ephrins. Ephrins and their receptor therefore establish a complex pattern of interactions that specify cell organization during development. To further study the kinase-dependent signaling properties of the VAB-1 receptor, a biochemistry approach was undertaken to partially purify proteins that associate with the autophosphorylated receptor.

24 May 1999 15:50 151 151 The soc genes in FGF receptor signaling in C. elegans

1999 International Worm Meeting abstract 100 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The soc genes in FGF receptor signaling in C. elegans JL Schutzman, MJ Stern Yale University, New Haven CT 06520, USA Fibroblast growth factor receptors (FGFRs) mediate a diverse set of biological processes. Thus, understanding the different signaling mechanisms used by FGFRs is of considerable importance to our understanding of developmental biology and human disease. We are characterizing EGL-15 FGFR signaling pathways to elucidate FGFR mediated signal transduction in C. elegans. EGL-15 is required for multiple events during development. Modulating EGL-15 signaling strength can have very different phenotypic effects. Complete loss of EGL-15 activity results in a larval arrest phenotype while hypomorphic alleles can confer a Scrawny body morphology. Hyperactive EGL-15 signaling due to constitutive activation of EGL-15 or loss of its negative regulator, the CLR-1 receptor tyrosine phosphatase, results in a distinctive Clr (Clear) phenotype. We use suppression of the Clr phenotype of clr-1 mutants to identify genes involved in EGL-15 signal transduction. Genetic screens for suppressors of clr-1 (soc genes) identified four major genes: egl-15, sem-5, soc-1 and soc-2. SEM-5, a GRB2 homolog, functions as an adaptor molecule downstream of multiple receptor tyrosine kinases. soc-2 encodes a novel leucine rich repeat protein also identified as a positive regulator of LET-60 RAS signaling during vulval development. FGFR signaling in other systems involves the SH2 domain-containing phosphatase SHP2 and an SOS-like guanine nucleotide exchange factor. In C. elegans, orthologous components are encoded by ptp-2 and possibly let-341, respectively. We have tested null alleles of each of these genes and found that both confer a Soc phenotype, implicating them in EGL-15 signaling. By characterizing null alleles of all of these soc genes, we have been able to assess the relative importance of these components for EGL-15 signaling. The molecular analysis of soc-1 now allows us to assemble these components into a pathway by which we can begin to trace EGL-15 signaling. We show that soc-1 encodes a novel protein composed of an N-terminal pleckstrin homology domain and multiple potential SH2 and SH3 domain binding sites. We are currently testing the hypothesis that SOC-1 acts to recruit other components required for EGL-15 signaling into a signaling complex.

24 May 1999 15:50 152 152 sop-1, -2 and -3: New Components That Regulate C. elegans Hox Gene Expression

1999 International Worm Meeting abstract 101 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. sop-1, -2 and -3: New Components That Regulate C. elegans Hox Gene Expression H Zhang, SW Emmons Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 During male tail development, the Hox genes mab-5 and egl-5 are required for the seam cell V6 to generate five pairs of sensory organs, called rays. How mab-5 and egl-5 expression is initiated in V6 and how this expression pattern is dynamically regulated throughout development is largely unknown. pal-1, which encodes a Drosophila caudal homolog, is required for mab-5 expression in V6. Neighboring seam cells send inhibitory signals to inhibit mab-5 expression. The nature of these inhibitory signals is unknown, but Wnt signaling, as well as pal-1, can overcome them (1). To understand how Hox genes are spatially and temporally regulated, we performed a screen for suppressors of pal-1(e2091), a regulatory mutant, and identified three new genes, sop-1, -2 and -3 (suppressor of pal-1), that are involved in regulating mab-5 and egl-5 expression in V6 and elsewhere. Mutations in all three genes are recessive and strongly suppress the pal-1(e2091) ray defect in a mab-5-dependent manner. By mosaic analysis, we found that sop-1 cannot suppress the ray phenotype of pal-1(ct224), a null allele. This suggests that sop-1(bx92) may suppress pal-1(e2091) by restoring pal-1(+) activity in V6. We have cloned sop-1 by transformation rescue, and found that it encodes a 3520-AA protein that contains a Q-rich region at its C terminus. Mutations in the human sop-1 homolog, HOPA, have been implicated in mental retardation (2). sop-2 mutants have pleiotropic ray and vulval phenotypes that might be explained by the ectopic expression of Hox genes. By using mab-5::gfp and egl-5::gfp reporter genes, we have found that Hox genes are ectopically expressed in sop-2 mutants, including massive expression in the head neurons, possibly accounting for a lethal phenotype at 25°C. The molecular characterization of sop-2 is in progress. bar-1, a b-catenin homolog, is required for ray production in sop-3; pal-1 mutants. This suggests that sop-3 acts to prevent inappropriate expression of the Wnt pathway. No previously known components of the Wnt pathway are present in the small genetic interval containing sop-3.

(1) CP Hunter et al. (1999) Development 126, 805-814 (2) RA Philibert et al. (1998) Molecular Psychiatry 3, 303-309.

24 May 1999 15:50 153 153 Vulval pattern formation revisited

1999 International Worm Meeting abstract 102 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Vulval pattern formation revisited M Wang, PW Sternberg HHMI and Division of Biology, 156-29, Caltech, Pasadena, CA 91125 A lin-3-encoded EGF-like growth factor produced by the anchor cell (AC) induces the vulval precursor cells (VPCs) to form a 3°-3°-2°-1°-2°-3° pattern during early L3. The granddaughters of the 1° VPCs form an EFFE pattern; the granddaughters of the 2° VPCs form an ABCD/DCBA pattern. These cells then undergo distinctive cell divisions and morphogenesis at late L3 and L4 to form a vulva. The precise formation of the final pattern is a multiple step process requiring the proper functioning of complex gene networks. We have first focused on the 1° lineage pattern. At least two steps are involved in 1° fate determination and patterning: first, induction to ensure a vulval fate and turning on early genes like egl-17; second, turning off early genes and differentially regulating the expression of late genes, such as cdh-3, in E and F cells. By testing the response of VPCs to LIN-3 as a function of time, we demonstrate that VPC daughters are competent to respond to LIN-3. Truncation of LIN-3 signaling by AC ablation or overexpression of dominant negative RAS at different times indicates that a LIN-3 signal from the AC is required for proper induction at least until the end of the first VPC cell cycle or even later. If the level of LIN-3 signal is reduced by a lin-3 hypomorph mutation, the AC is required beyond the first VPC cell cycle. In addition to the induction step, AC signaling is also involved in the second step of 1° patterning. Manipulation of LIN-3 signaling after the induction step (the early 4-cell stage of the VPCs) results in misspecification of the presumptive E and F cells. We are trying to decipher the signaling pathways that connect the initial VPC patterning to VPC progeny patterning. lin-39 is one connection between the ras signaling pathway and the downstream gene expression, cell fusion and cell cycle regulation. Careful examination of the second step of 1° patterning suggests that it can be divided into substeps that are temporally and spatially regulated by a transcription factor network. For example, while turning off early gene expression requires lin-11 (LIM), keeping off early gene expression and regulating the differential expression of late genes require lin-11 and cog-1 (homeobox) in both E and F and particularly egl-38 (PAX) in F cells.

24 May 1999 15:50 154 154 A modular system of docking sites mediates ERK map kinase recognition of substrate proteins such as LIN-1 and KSR-1

1999 International Worm Meeting abstract 103 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A modular system of docking sites mediates ERK map kinase recognition of substrate proteins such as LIN-1 and KSR-1 Dave Jacobs, Kerry Kornfeld Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110 MAP kinases are signaling molecules that phosphorylate S/TP motifs in a wide variety of substrate proteins. The mechanisms that enable MAP kinases to identify substrate proteins and phosphorylate particular residues are not well characterized. We are investigating how MAP kinases interact with substrate proteins. We have focused on two C. elegans substrates, LIN-1 and KSR-1. LIN-1 contains 441 amino acids and is likely to be a DNA-binding transcription factor, since it contains an N-terminal ETS domain. lin-1(lf) mutations cause a multivulva phenotype, and lin-1(gf) mutations cause a vulvaless phenotype, suggesting lin-1 functions as a switch and lin-1 activity prevents Pn.p cells from adopting the 1° vulval fate. Genes in the Ras signaling pathway promote the 1° fate, and epistasis analyses indicate lin-1 functions downstream of these genes, including mpk-1 ERK MAP kinase, suggesting lin-1 is negatively regulated by the action of this signaling pathway. KSR-1 is a protein kinase that positively mediates Ras signaling.We showed that the C-terminal region of LIN-1 was sufficient to function as a high-affinity substrate for ERK MAP kinase in vitro. We analyzed six lin-1(gf) mutations; each alters or eliminates the C-terminal sequence FQFP, suggesting this motif is required for negative regulation of LIN-1. This motif is conserved in other ETS proteins that also mediate Ras signaling. Biochemical experiments demonstrated that FXFP is an evolutionarily conserved docking site that mediates ERK MAP kinase binding to these substrates. FXFP and the D box, a different docking site that is conserved in these ETS proteins, form a modular recognition system, since they can function independently or in combination. FXFP is specific for ERK, whereas the D box mediates binding to ERK and JNK MAP kinase, suggesting that the partially overlapping substrate specificities of ERK and JNK result from recognition of shared and unique docking sites. These findings enabled us to predict new ERK substrates, such as KSR-1, which contains a FXFP motif and is a high-affinity substrate for ERK. Furthermore, this information was used to design peptide inhibitors of ERK that functioned in vitro and in vivo.

24 May 1999 15:50 155 155 lin-55 DP and an E2F-like gene act in the lin-35 Rb pathway to antagonize let-60 ras signaling during vulval induction

1999 International Worm Meeting abstract 104 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. lin-55 DP and an E2F-like gene act in the lin-35 Rb pathway to antagonize let-60 ras signaling during vulval induction CJ Ceol, HR Horvitz HHMI, Dept. Biology, MIT, Cambridge, MA 02139 U.S.A. The Ras signaling pathway of vulval induction is antagonized by the synthetic multivulva (synMuv) genes. On the basis of genetic interactions, the synMuv genes have been grouped into two classes, A and B. An animal is Muv if it has mutations in both a class A gene and a class B gene, whereas an animal has a wild-type vulva if a mutation is present in a gene or genes of a single class. lin-35, a class B synMuv gene, encodes a protein similar to the mammalian tumor suppressor pRb and the related proteins p107 and p130 (Lu and Horvitz, 1998, Cell 95: 981-991). A well-characterized binding partner of pRb is the DP/E2F heterodimeric transcriptional activator. pRb/DP/E2F complexes are thought to repress transcription of DP/E2F-responsive genes, including genes required for S phase cell-cycle progression. We are determining the roles of DP and E2F proteins in the specification of vulval cell fates. We cloned the class B synMuv gene lin-55 and have found that it encodes a protein similar to the DP family of transcription factors. To investigate the null phenotype of lin-55, we isolated a deletion allele, n3316. In addition to a synMuv phenotype, lin-55(n3316) causes maternal-effect lethality. We have also identified two C. elegans E2F-like genes. A deletion allele, n3318, of a gene we are temporarily calling C.e.E2F-1 behaves like a class B synMuv mutation. n3318 also causes maternal-effect lethality. We are currently comparing the lethal phase of n3318 embryos with that of lin-55(n3316) embryos and are investigating the role of the other E2F-like gene in vulval development and embryogenesis. We propose that LIN-55 and C.e.E2F-1 form a complex with LIN-35 Rb and other class B synMuv proteins and that this complex represses transcription of genes that promote vulval development.

24 May 1999 15:50 156 156 abi-1, a gene required for vulval morphogenesis, encodes a nuclear hormone receptor homolog

1999 International Worm Meeting abstract 105 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. abi-1, a gene required for vulval morphogenesis, encodes a nuclear hormone receptor homolog Z Chen, M Han HHMI, Dept. of MCDB, Univ. of Colorado at Boulder Vulval cell fate specification causes three out of six vulval precursor cells (VPCs) to undergo some characteristic divisions, followed by specific cell migration and fusion events. Here we report the study of abi-1(ku217) and another gene defined by the ku285 allele that disrupt these processes. In a WT vulva, the induced VPCs, P(5-7).p, divide twice in a longitudinal orientation. In the third and final round of division, the four granddaughters of P5.p undergo longitudinal(L), longitudinal(L), transverse(T) and no(N) divisions respectively. P7.p divides in a mirror-symmetrical way to P5.p, and the granddaughters of P6.p undergo only transverse(T) divisions. In ku217 animals, the "T" divisions of vulval cells are disrupted specifically, resulting in no division. However, the cell fates of these nondivided "T" cells may not be the same as those of the "N" cells, based on the study of the expression of some vulval cell-specific marker genes. In addition to the division defects, cell migration and fusion processes are affected in abi-1(ku217) animals. These morphogenetic defects are also displayed in the pseudovulvae of let-60(gf);abi-1 and lin-1(lf);abi-1 double mutants, indicating that the abi-1 gene functions in an event downstream of the vulval cell fate induction. The abi-1(ku217) is a TS, recessive mutation, and dosage analysis suggests that it is a loss-of-function mutation. We have cloned this gene using DNA-mediated microinjection transformation. The ABI-1 protein, which is 572 a.a. long, is homologous to members of the nuclear hormone receptor superfamily. A missense mutation (L32F) in the DNA-binding domain is associated with the ku217 allele. An abi-1::gfp translational fusion gene is expressed extensively in the worm, including in the 12 Pn.p cells and hypodermal cells, but not in the AC or any other somatic gonadal cells. However, RNAi of the abi-1 gene displays a highly penetrate gonadal phenotype. The ku285 is another mutant which disrupts the transverse divisions of the vulval cells. Lineage studies of ku285 animals show that, unlike in the abi-1(ku217) mutant, there is a transformation from transverse division to longitudinal division, with no defects in other division patterns. The ku285 is also a loss-of-function allele, and the molecular cloning is in process."

24 May 1999 15:50 157 157 Patterning and differentiation of the ventral uterine cells that connect to the vulva

1999 International Worm Meeting abstract 106 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Patterning and differentiation of the ventral uterine cells that connect to the vulva AP Newman, HN Cinar Department of Biochemistry, Baylor College of Medicine, Houston TX 77030 Differentiated cells acquire their identities by a combination of intrinsic and extrinsic factors acting on intermediates in the cell lineage and the terminal descendants they produce. In the ventral uterus, a lin-12-mediated signal from the AC induces adjacent VU intermediate precursor cells to adopt the p cell fate and produce daughters that connect to the vulva. One set of p daughters, called utse, forms the thin laminar process dorsal to the vulva, while the other p daughters, called uv1 cells, attach the vulva to the utse [1]. p daughters become uv1 in response to LIN-3 EGF signaling from the vulva and utse in the absence of this signal [2]. In screens for egg-laying-defective mutants with uterine defects, we have isolated alleles of sel-12 (which encodes a presenilin [3]) and cog-2 (which encodes a SOX domain transcription factor [4]) with p cell lineage defects. Specifically, an additional round of cell division indicative of failure to properly specify or maintain the p cell fate was observed in 46/60 sel-12(ty11) and 48/60 cog-2(ty3, ty8) p cell daughters followed. Differences in pattern formation suggest that sel-12(ty11) gives a partial loss of function in p cell induction (consistent with the previously demonstrated role of SEL-12 in LIN-12/Notch signaling [3]) whereas cog-2(ty3, ty8) mutants are defective in maintenance of the p cell fate and differentiation of its daughters. The LIN-11 LIM domain transcription factor is required for p daughter differentiation and responds to lin-12 activity. We propose that lin-12 activity in the p cells leads to the following genetically separable events: specification of the p cell fate, maintenance of this fate, and production of daughters that connect to the vulva. The LIN-11 and COG-2 transcription factors may have related functions in the latter events. The activity of the lin-11 and cog-2 genes might help to make p daughters competent to differentiate as uv1 in response to LIN-3 signaling and as utse in its absence.

[1] Newman, White, and Sternberg (1995, 1996). Development 121: 263-271; 122: 3617-3626. [2] Chang, Newman, and Sternberg (1999). Current Biology 9: 237-246. [3] Levitan and Greenwald (1995). Nature 377: 351-354. [4] Hanna-Rose and Han (1999). Development 126: 169-179.

24 May 1999 15:50 158 158 Who said you need two to tango? The development of the real and pseudo vulvae of a let-60 gf mutant

1999 International Worm Meeting abstract 107 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Who said you need two to tango? The development of the real and pseudo vulvae of a let-60 gf mutant G Shemer 1 , R Sharma-Kishore 1,2 , B Podbilewicz 1

1 Department of Biology, Technion-Israel Institute of Technology, Haifa, 32000, Israel. 2 Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia.

We have recently characterized the complete cellular events including migrations, remodeling of adherens junctions, cell fusions and ring formation during organogenesis of the wild-type vulva*. Ras/let-60 in C. elegans has been studied extensively and much is known about the pathways and the genetic interactions during the early phases of vulva development. However, very little is known about its role in the cellular processes mentioned above. To analyze the role of ras during vulva formation and to investigate the specific cellular events involved, we have used confocal reconstructions of the let-60 (n1046) gf mutant. By observing larvae from different stages of development stained with MH27 monoclonal antibody that recognizes the adherens junctions of the cells, we have characterized for the first time the cellular events of the evolving real and pseudo vulvae characteristic to this mutant. In wild-type, cells generated by the vulva lineage migrate towards the center until they meet their symmetrical homologues and form a stack of seven rings*. We have shown that in n1046 these migrations are delayed. This gives rise to asymmetric vulvae in size, orientation and presence of primordial cells. The vulvae primordia do form concentric rings followed by fusions of the cells within them but in the absence of specific cells, other cells adopt their structural fate so as to form a stack of only five or six rings. The primordial cells of the pseudovulvae, even in the absence of symmetric counter cells, create full "self" rings with successive fusions within the rings. We propose that the precursor cells of the real and pseudo vulvae compete between themselves, resulting in the formation of bilateral asymmetric vulvae as opposed to the wild-type. The two halves of the vulva develop autonomously and in the absence of one half, its counter half takes its structural fate to form a functional vulva. We will discuss our proposed new nomenclature (instead of LLTN) and compare the structural cell fates in wild-type and n1046 vulvae. *Sharma-Kishore et al. (1999) Development 126, 691-699.

24 May 1999 15:50 159 159 The even-skipped homolog Ppa-vab-7 is involved in the phylogenetic restriction of the vulva equivalence group in Pristionchus pacificus

1999 International Worm Meeting abstract 108 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The even-skipped homolog Ppa-vab-7 is involved in the phylogenetic restriction of the vulva equivalence group in Pristionchus pacificus B Jungblut 1,2 , R Sommer 1

1 Max-Planck Institut für Entwicklungsbiologie, Department for Evolutionary Biology, Spemannstr. 35, 72076 Tübingen, Germany. 2 E.mail: [email protected]

It has been proposed that equivalent cells with multiple potentials are intermediate steps in the evolution of cell diversity. One example is the vulva equivalence group in nematodes. In C. elegans, 6 cells P(3-8).p are initially equipotent to form vulval tissue but only P(5-7).p respond to RAS signaling to form vulva in wild-type. During nematode evolution, various alterations of the vulva equivalence group have been observed. In Pristionchus pacificus, the vulva equivalence group is no longer present and phylogenetic analyses indicates this to be a derived character. The two anterior cells P(3,4).p die of apoptosis, whereas the posterior cell, P8.p is no longer competent to adopt vulval cell fate after fusion with the surrounding hypodermis. P7.p is limited in its developmental competence and can only adopt one of the two alternative vulval fates. To study the evolution of this process we screened for new mutations that reconstitute the vulva equivalence group in the posterior body region by preventing cell fusion of P8.p. We isolated two mutations tu113 (TMP/UV screen) and tu125 (standard EMS screen). Complementation analysis showed both mutants to be allelic. Both mutations also shift the vulva from P(5-7).p to P(6-8).p. Molecular analysis indicates that this phenotype is caused by mutations in the even-skipped homolog Ppa-vab-7. Cel-vab-7 mutants do not influence the vulval competence of P8.p. This indicates that Ppa-vab-7 has adopted a new biological function that increases the working load of this gene and enables the evolution of cell diversity.

24 May 1999 15:50 160 160 Genetic analysis of vulva development in Oscheius sp. CEW1

1999 International Worm Meeting abstract 109 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic analysis of vulva development in Oscheius sp. CEW1 MA Félix 1,2 , M Viney 1 , ML Dichtel 2 , S Louvet 2 , PW Sternberg 1

1 Biology 156-29, HHMI, Caltech, Pasadena CA91125, USA. 2 Institut J. Monod, CNRS/Univ. Paris 6 et 7, Tour 43, 2 pl. Jussieu, 75251 Paris Cedex 05, France.

In C. elegans, the 3 fates of the Pn.p vulva precursor cells are specified around the same time by a graded induction by the anchor cell and lateral signaling among the precursors. This mechanism is not conserved in other nematodes. Considerable variations are found within the Rhabditidae (family including C. elegans), Panagrolaimidae and Cephalobidae. One other vulva patterning mechanism relies on 2 successive nested inductions by the anchor cell: the first induction specifies vulval vs. non-vulval fates, the second specifies inner vs. outer vulval fates in the Pn.p daughters. We have started a genetic analysis in such a species, Oscheius/Dolichorhabditis CEW1. One advantage of this species is its simple vulva lineage. We have isolated about 60 vulva mutants in an F2 Egl screen. We found several Hyperinduced mutants affecting the first induction only, in which P4.p and P8.p adopt an outer vulval fate (2°), but no Multivulva mutants with an alternation of 1° and 2° fates. Many mutants affect the number of vulval cell divisions, irrespective of fate induction. Some have an additional third round of division for P5.p and P7.p; some have a fourth round in the P6.p lineage (unknown phenotype in C. elegans). In other mutants conversely, P(4-8).p, or P4.p and P8.p only, divide too few times. Laser ablation studies indicate that the number of divisions of P(5-7).p depends on a gonadal signal, but can be uncoupled from fate specification. Some of these mutants also have fate induction defects, but we did not find Vulvaless mutants defective in fate induction only. Other frequent phenotypic categories are: i. P(4-8).p, the competence group, behave like non-competent Pn.ps (lin-39 or bar-1 phenotype in C. elegans). ii. The competence group is enlarged to P3.p. iii. The vulva is miscentered on P7.p. This spectrum of vulva mutations is very different from that of C. elegans. It reflects the 2-step mechanism of vulval induction, and also suggests that the relation between vulval cell cycle and fate differs from that of C. elegans. We are currently analysing the role of the Ras pathway in Oscheius (see abstract by Delattre and Félix).

24 May 1999 15:50 161 161 Genetic Dissection of Radiation-Induced Apoptosis and Cell Cycle Arrest in C. elegans

1999 International Worm Meeting abstract 110 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic Dissection of Radiation-Induced Apoptosis and Cell Cycle Arrest in C. elegans A Gartner, S Milstein, M Hengartner Cold Spring Harbor Laboratory; 1 Bungtown Road; Cold Spring Harbor, New York 11724, USA; Tel: 516-367-8363; Fax: 516-367-8461. In higher organisms, little is known about the checkpoint mechanisms that trigger cell cycle arrest and programmed cell death, largely due to the absence of a genetic system. Our current research centers on the development of such a genetic system to study DNA damage induced cell cycle arrest and apoptosis in a multicellular system. Towards that direction we found that the germ line of C. elegans has ideal properties. In contrast to the somatic tissues of the worm, the germline continues to proliferate throughout life. In addition to giving rise to differentiated gametes a high proportion of germ cells are eliminated by apoptosis. To determine whether germ cell death can be used as a model system to study the mechanism(s) underlying DNA damage-induced apoptosis and cell cycle arrest, we analyzed the effects of genotoxic agents on germ cells. We found that germ cells can both halt cell cycle progression and induce apoptosis following exposure to gamma radiation. Interestingly, these two responses are spatially separated. Mitotic germ cells arrest proliferation, whereas pachytene stage meiotic germ cells undergo apoptosis following genotoxic stress. Radiation-induced germ cell death requires the same apoptotic core machinery as is needed for somatic cell death, confirming that it is apoptotic in nature. However, based on the differential genetic behavior of certain cell death mutations, radiation-induced germ cell death, physiological germ cell death, and somatic cell death are genetically distinct. To elucidate the pathways leading to radiation induced death we isolated two classes of mutants: The first class of mutants is resistant to radiation induced cell death, the second class is hypersensitive to damage-induced apoptosis. Our best characterized radiation resistant-mutant is rad-5(mn159), which we found upon screening through a panel of existing candidate mutations. Currently, we aim at cloning rad-5 , which does not map close to any conserved checkpoint genes. Furthermore, we also want to take advantage of reverse genetic approaches to determine whether homologs of checkpoint genes known to function in yeast systems affect radiation-induced cell death and cell cycle arrest.

24 May 1999 15:50 162 162 CED-9 and EGL-1 regulate the subcellular localization of CED-4

1999 International Worm Meeting abstract 111 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. CED-9 and EGL-1 regulate the subcellular localization of CED-4 B Hersh, F Chen, B Conradt, Z Zhou, B Horvitz HHMI, Dept. Biology, MIT, Cambridge, MA 02139 EGL-1, CED-9, CED-4, and CED-3, the core components of the C. elegans pathway for programmed cell death, are conserved in mammalian apoptosis. EGL-1, a BH3-domain protein that may act by negatively regulating CED-9 activity, has been shown to bind CED-9 protein. CED-9 is a member of the Bcl-2 family of cell-death regulators and negatively regulates the activities of CED-4 and CED-3. CED-4 is similar to mammalian Apaf-1, which activates caspases, cysteine proteases that effect cell killing. CED-3 encodes a caspase. Various physical interactions among these components have been demonstrated in vitro, in yeast and in mammalian cells. We generated polyclonal antibodies against CED-9 and CED-4 and used them to determine the expression patterns and subcellular localizations of these two key cell-death proteins. Endogenous CED-9 and CED-4 proteins are localized to mitochondria in wild-type embryos, in which the majority of cells survive. However, in embryos in which most cells have been induced to die, such as embryos homozygous for loss-of-function mutations in ced-9 or overexpressing the cell-death activator EGL-1, CED-4 is released from mitochondria and assumes a perinuclear localization. CED-4 redistribution induced by EGL-1 can be blocked by a gain-of-function mutation in ced-9 but not by a loss-of-function mutation in ced-3, suggesting that it precedes cell-death execution. Missense mutations within the CED-4 protein itself also can disrupt CED-4 localization. These findings suggest that the subcellular localization of CED-4 may correlate with the life-or-death decision of a cell. Cells that survive maintain CED-4 localization at mitochondria, possibly through interaction with CED-9. Cells in which programmed cell death has been activated release CED-4 to redistribute to nuclear membranes.

24 May 1999 15:50 163 163 Identification and Characterization of Downstream Targets of the Cell-Death Protease CED-3

1999 International Worm Meeting abstract 112 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification and Characterization of Downstream Targets of the Cell-Death Protease CED-3 DA Ledwich, CE Duffy, CK Lau, HE Metters, PT Huynh, D Xue MCDB, Univ. of Colo., Boulder, CO 80309 Analysis of downstream pathways of important enzymatic biomolecules (e.g., kinases and proteases) which have multiple substrates has always been a difficult challenge. Targets of these enzymes could be difficult to identify through conventional genetic screens since elimination of one of the multiple targets of an enzyme may fail to cause any visible phenotype that can be scored in genetic screens. Here we report a novel genetic screen that will allow us to identify most of the downstream targets of the CED-3 death protease. The cell-death gene ced-3 encodes a cysteine protease that executes programmed cell death by cleaving critical protease targets. To identify the downstream targets and pathways that mediate CED-3 cell-killing activity, we carried out a novel and sensitive genetic screen to isolate mutations that partially suppress or delay cell death caused by constitutively activated CED-3 death protease. In this screen, we co-expressed CED-3 with GFP in six touch receptor neurons and used GFP fluorescence as a sensitive cell-existence marker to detect the presence of touch receptor neurons whose death is either partially suppressed or delayed by mutations. So far, we have isolated more than sixty such mutations. Five of them are alleles of the ced-1 gene, suggesting that it is a plausible strategy to identify genes that act downstream of ced-3 (ced-1 acts downstream of ced-3 to mediate cell corpse engulfment). The other mutations define at least five new genes, which we named sop genes (suppressor of death protease). Importantly, mutations in these sop genes not only affect CED-3-mediated ectopic cell-death but also affect normal programmed cell death in nematodes. Specifically, mutations in sop-1 and sop-2 weakly suppress general cell death, while mutations in sop-3, sop-4 and sop-5 act like ced-8 mutations by causing the delay of cell-killing (1). Epistasis analysis suggests that five sop genes act downstream of ced-3 but upstream of the engulfment ced genes. This novel genetic screen should help us identify most of the components and pathways that act downstream of the CED-3 death protease to execute cell death, which currently is the missing link of the cell death pathway.

1. Stanfield et. al. (1997) 11th International C. elegans Meeting Abstract, page 229.

24 May 1999 15:50 164 164 A CED-2/Crk-II, CED-5/DOCK180, CED-10/Rac pathway controls cell-corpse engulfment and cell migration

1999 International Worm Meeting abstract 113 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A CED-2/Crk-II, CED-5/DOCK180, CED-10/Rac pathway controls cell-corpse engulfment and cell migration PW Reddien, HR Horvitz HHMI, Dept. Biology, MIT, Cambridge, MA 02139 The engulfment of cells undergoing programmed cell death is controlled by at least seven genes that define two parallel pathways. Mutations in genes of one pathway, including ced-2, ced-5, ced-10, and ced-12, result in defects both in cell-corpse engulfment and in the migration of the gonadal distal tip cells. ced-5 is known to encode a protein similar to human DOCK180 (Downstream of Crk 180KD) (1). DOCK180 has been found to interact with the the oncoprotein Crk and the Rac GTPase. We have now cloned the ced-2 and ced-10 genes and found them to encode proteins homologous to Crk-II and Rac, respectively. That ced-2/ced-5/ced-10 exist together in a pathway strongly indicates that Crk-II, DOCK180, and Rac all functionally interact in vivo. CED-2 contains an N-terminal SH2 (Src-Homology 2) domain followed by two SH3 domains, suggesting that CED-2 functions as an adaptor protein to mediate protein-protein interactions. Rac is a member of the Ras-like GTPase family. A subgroup of these GTPases, consisting of Rho, Rac, and Cdc42, regulate cell morphological changes by controlling the organization of the cytoskeleton. An analysis of the cell-migration defects in ced-2 and ced-10 mutant animals demonstrates that the distal tip cells, while competent for migration, are defective in maintaining correctly polarized pathfinding. Ectopic expression experiments suggest that CED-2 and CED-10 can act outside of dying cells for cell-corpse engulfment. We propose that CED-2/Crk-II localizes CED-5/DOCK180 to the plasma membrane, leading to the activation of the CED-10/Rac GTPase within engulfing and migrating cells. Such activation presumably regulates the polarity of cytoskeletal extensions in response to apoptotic or migrational cues. We are analyzing predictions made by this model, such as protein-protein interactions and protein localization.

1. Wu, Y. C. and Horvitz, H. R. (1998). Nature 392, 501-504.

24 May 1999 15:50 165 165 The Cell-corpse Engulfment Gene ced-1 Encodes A Transmembrane Receptor That May Act To Recognize Dying Cells

1999 International Worm Meeting abstract 114 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Cell-corpse Engulfment Gene ced-1 Encodes A Transmembrane Receptor That May Act To Recognize Dying Cells Z Zhou, B Horvitz HHMI, Dept. Biology, MIT The engulfment of cells by their neighbors is an evolutionarily conserved process that serves to remove unwanted or potentially harmful cells. The molecules that mediate this fundamental process are only now beginning to be elucidated. In C. elegans, mutations in seven genes that define two partially redundant pathways cause cell corpses to persist abnormally. ced-2, 5, 10, and 12 are part of a Crk/DOCK180/Rac signaling pathway proposed to mediate cytoskeletal reorganization (see abstract by Reddien and Horvitz). ced-6 encodes an adaptor-like protein and ced-7 encodes an ABC transporter. We propose that these two genes act in one of the pathways together with ced-1 to control cell-corpse recognition and to initiate phagocytosis. We have cloned ced-1 and found that it encodes a transmembrane receptor-like protein. CED-1 is localized on the surface of many cells in embryos and early larvae. Induced expression of ced-1 can rescue the engulfment defects of ced-1 mutants hours after the corpses have formed, suggesting that the activity of CED-1 in engulfing cells, rather than in corpses, is responsible for its engulfment function. We hypothesize that CED-1 acts as a receptor that recognizes corpses. Currently we are attempting to identify the ligand(s) for CED-1, which may be corpse-specific cell surface molecules, and proteins that interact with the cytoplasmic domain of CED-1, which may be downstream signaling molecules. We have also performed a large scale genetic screen for new engulfment mutants. Using a sem-4 mutant background, we examined F2 bags of worms for F3 embryos with persistent corpses. We isolated 68 potential engulfment mutants. These mutants define distinct phenotypic categories, including viable mutants with strong or weak engulfment defects; viable mutants that have persistent abnormal-looking corpse-like bodies; and mutants that are both engulfment-defective and embryonic lethal. We hope the further characterization of these mutants will both help us understand the known engulfment genes and identify additional genes involved in this process.

24 May 1999 15:50 166 166 A Common Set of Engulfment Genes Mediates the Removal of Both Apoptotic and Necrotic Corpses in C. elegans

1999 International Worm Meeting abstract 115 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A Common Set of Engulfment Genes Mediates the Removal of Both Apoptotic and Necrotic Corpses in C. elegans S Chung 1 , T Gumienny 2 , M Hengartner 2 , M Driscoll 1

1 Department of Molecular Biology and Biochemistry, Rutgers University, Piscatway, NJ 08854. 2 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724.

In C. elegans, necrotic-like cell death (NCD), characterized by cell swelling, can be initiated by a hyperactive ion channel mutation (for example, MEC-4(A713V)). We investigated the requirements for programmed cell death (PCD) genes in NCD caused by a toxic MEC-4 subunit. We find that neither a gain-of-function allele of PCD inhibitor ced-9 (a Bcl-2 homolog) nor a loss-of-function mutation in egl-1 (a gene that encodes a BH3 domain protein that antagonizes CED-9) affects the kinetics of necrosis onset or absolute corpse numbers. Likewise, strong loss-of-function alleles of the ced-3 caspase and its regulator, ced-4, do not alter initiation or extent of degeneration. These data support that activating mechanisms for necrotic and apoptotic cell death are distinct. In striking contrast, we find that all genes required for phagocytotic removal of PCD corpses (the engulfment ced genes) are also required for efficient NCD corpse elimination. Moreover, a screen for additional genes required for NCD corpse removal identified an allele of a novel gene, ced-12, that causes persistence of both NCD and PCD corpses, underscoring our conclusion that irrespective of the mode of cell death, a common mechanism eliminates cell corpses. Interestingly, although ced-2, ced-5, ced-10, ced-6, and ced-7 mutations show maternal effect rescue for PCD corpse removal, only ced-7 shows maternal effect rescue for elimination of NCD corpses. Zygotically supplied products of ced-1, ced-2, ced-5, ced-6, and ced-10 can suffice for NCD corpse removal. We are testing whether ced-5, ced-6, and ced-7 act in NCD dying cells or engulfing cells. In summary, ced-1, ced-2, ced-5, ced-6, ced-7, ced-10, and ced-12 are the first C. elegans genes identified to affect removal of necrotic-like cell death corpses. Since phagocytotic mechanisms appear conserved from nematodes to humans, our findings raise the possibility that necrotic cell death corpses can be eliminated via a controlled process in higher organisms.

24 May 1999 15:50 167 167 Mutations That Suppress Degenerative Cell Death Can Extend Lifespan

1999 International Worm Meeting abstract 116 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Mutations That Suppress Degenerative Cell Death Can Extend Lifespan LA Herndon, M Driscoll Rutgers University Several genetic "conditions" including mec-4(u231), deg-3(u662) and activated GaS induce swelling and degeneration of neurons. This death occurs independently of the programmed cell death pathway. mec-4(u231)[the example we study most] specifies a hyperactive channel subunit and may kill cells via a mechanism similar to mammalian excitotoxic cell death. To learn more about the degenerative death mechanism, we screened for mutations that block mec-4(d)-induced death.

An integrated array of punc-8 mec-4(u231) causes swelling and degeneration of some interneurons and many ventral cord neurons, resulting in severe paralysis. Using this behavioral phenotype, we screened for suppressor mutations that block the deleterious effects of mec-4(u231): suppressors restore normal or near normal locomotion by preventing cell death. The genes identified by these mutations are called des genes (degeneration suppressor). In a screen of 45,000 haploid genomes, we isolated 30 independent death suppressors. 14 strong suppressors appear to affect novel genes. Three of the strongest suppressors on chromosome V, defined by alleles bz29, bz30 and bz31, are particularly interesting because they suppress degeneration induced by multiple insults. We have identified a candidate rescuing cosmid for bz29 and bz30, and are in the process of cloning this gene. Aging nematodes accumulate vacuolar structures that look similar to mec-4(d)-induced necrosis. This led us to wonder whether animals in which degenerative cell death is blocked by mutation might age differently. We hypothesized that if degenerative cell death plays a critical role in C. elegans aging, and if des mutations suppress aging-associated vacuolation, our des mutants might have extended lifespans. Indeed, both bz29 and bz30 (alleles of the same gene) have an extended lifespan, similar to what is seen in age-1 (hx546) animals. At least one more des allele, affecting a different gene, has an extended lifespan. We are testing the long-lived lines to determine the extent of vacuole formation during aging, general locomotor activity and resistance to heat and oxidative stress. In addition, we are determining whether there is an interaction between the des mutations and other known mutations that affect lifespan.

24 May 1999 15:50 168 168 MEC-6 directly interacts with the mechanosensitive channel subunits, MEC-4 and MEC-10

1999 International Worm Meeting abstract 117 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. MEC-6 directly interacts with the mechanosensitive channel subunits, MEC-4 and MEC-10 DS Chelur, L Chen, M Chalfie Department of Biological Sciences, Columbia University, New York, NY 10027 Response to gentle touch in Caenorhabditis elegans is mediated by six touch receptor neurons and the products of at least 12 genes. The mec-6 gene was initially identified in a search for touch insensitive mutants. Subsequently, mutations in mec-6 were found to suppress the degeneration producing mutations in four degenerin genes, mec-4 and mec-10 (which are thought to encode subunits of the mechanically-gated ion-channel in the touch cells), deg-1, and unc-8. Thus, although the most pronounced phenotype produced by mutations in mec-6 is touch insensitivity, the suppression phenotype suggests that mec-6 is more generally required for degenerin function. We cloned mec-6 by refining its map position on chromosome I and then, narrowed down the rescuing activity to the cosmid W02D3. Using genomic sequence, we generated PCR products corresponding to nine of the predicted genes on this cosmid from wild type and several mec-6 mutants. The amplified DNA in the region of one of the genes was altered in animals with the mec-6(u450) and mec-6(e1609) mutations. We have identified defects in twenty-three mec-6 alleles (including 4 deletion mutants). Subsequent transformation rescue experiments confirmed that this gene was mec-6. A 1.2 kb cDNA isolated from Peter Okkema’s library contains the complete mec-6 coding sequence and encodes a predicted protein of 377 amino acids with limited sequence similarity to the mammalian enzyme paraoxonase. mec-6 is widely expressed in many cell types including the touch receptor neurons consistent with its requirement for functioning of several degenerin channels. Most if not all of MEC-6 is extracellular since the protein has a single N-terminal transmembrane domain and is glycosylated when synthesized in vitro in presence of microsomes. In in vitro experiments, MEC-6 immunoprecipitates the mechanosensitive channel subunits MEC-4 and MEC-10. Furthermore, we and Monica Driscoll’s lab have shown that the stability of MEC-4 protein is affected in mec-6 mutants. Based on these observations we postulate that either (1) MEC-6 is a subunit of the channel, in which case its interaction with MEC-4 and MEC-10 stabilizes the channel or (2) MEC-6 is required for proper localization of the channel subunits.

24 May 1999 15:50 169 169 ins-14, one of many insulin-related genes in C.elegans, can regulate dauer formation

1999 International Worm Meeting abstract 118 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. ins-14, one of many insulin-related genes in C.elegans, can regulate dauer formation SB Pierce 1 , R Wisotsky 2 , L Liu 2,3 , G Ruvkun 1

1 Dept. of Molecular Biology, Massachusetts General Hospital, Boston MA. 2 Axys Pharmaceuticals, NemaPharm Group, South San Francisco, CA. 3 Current address: Cambria Biosciences, Waltham, MA.

The activity of daf-2, which encodes a homolog of insulin and insulin-like growth factor I (IGF-I) receptors (1), is required for non-dauer development and normal adult life span, and is likely to be regulated by one or more insulin-like ligands. By searching the C. elegans genome database, we identified 30 ins genes predicted to encode insulin-like peptides, that could be grouped into several subfamilies on the basis of structural similarities. One gene, ins-14 on F13B12, contains likely C-peptide cleavage sites typical of mammalian insulins. To investigate whether this family of insulin-related genes could play a role in dauer formation, several members were overexpressed by injecting worms with the appropriate PCR-amplified genomic regions. We hypothesized that overexpression of putative DAF-2 ligands might suppress the Daf-c phenotype of weak daf-2 mutants or mutants in the parallel daf-7 TGF-ß pathway, which synergizes with the daf-2 insulin-like signaling pathway (2). None of the tested genes suppressed daf-2(e1365), a non-null allele, although these transgenic worms may not have produced enough ligand to overcome the impaired DAF-2 receptor. However, the ins-14, but not the M04D8.2 or T10D8.a&b, transgene maternally suppressed the Daf-c phenotype of daf-7(e1372). Similar results were seen with an ins-14 transgene in which the coding region was replaced with the cDNA for human insulin. An ins-14::GFP transgene is expressed in many neurons, intestine, and vulval muscles. Finally, we isolated a deletion mutant, ins-14(nr2091), in which the entire coding region is removed. ins-14(nr2091) had no dauer phenotype as a single mutant, but enhanced the Daf-c phenotype of daf-7 in the ins-14(nr2091);daf-7(e1372) double mutant. The suppression of the daf-7(e1372) Daf-c phenotype by ins-14 overexpression and its enhancement by the ins-14(nr2091) mutation is consistent with ins-14 acting as a DAF-2 agonist. Other members of the ins gene family may act either redundantly as agonists or possibly as antagonists of DAF-2.

(1) Kimura et al, Science 1997;277:942 (2) Ogg et al, Nature 1997;389:994

24 May 1999 15:50 170 170 Multiple Layers of Cell Non-autonomy in the Control of C. elegans Lifespan

1999 International Worm Meeting abstract 119 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Multiple Layers of Cell Non-autonomy in the Control of C. elegans Lifespan J Apfeld, C Kenyon University of California, San Francisco, CA 94143-0448 The insulin/IGF-receptor homologue daf-2 regulates aging in C. elegans. Decreasing daf-2 activity causes fertile adults to remain active much longer than normal and to live more than twice as long. A more severe decrease in daf-2 function causes young larvae to enter a state of diapause rather than progressing to adulthood. The lifespan extension and dauer formation phenotypes observed in daf-2 mutants are dependent on the activity of daf-16, a transcription factor of the HNF-3/winged-helix family. We have asked which cells require daf-2 gene activity in order for the animal to develop to adulthood and to age normally. We found that daf-2 functions cell non-autonomously in both processes. Our findings imply that the lifespan of C. elegans is determined by a signaling cascade in which daf-2 acts in multiple cell lineages to regulate the production or activity of a secondary signal (or signals), which, in turn, controls the growth and longevity of individual tissues in the animal. C. elegans obtains information about its external environment through sensory cilia. We have found that animals with defective sensory cilia live longer than animals with intact sensory cilia. This lifespan extension is dependent on the activity of daf-16, and mutations that disrupt cilium structure do not further increase the lifespan of daf-2 mutants. The long-lived cilium structure mutants feed normally, suggesting that sensory perception itself, rather than an associated effect on food intake, influences lifespan. The lifespan of C. elegans is also under the control of signals from the germline; laser ablation of the germline precursors leads to a lifespan extension that is dependent on the activity of daf-16 (1). We have found that a number of mutations that interfere in different ways with the development of the germline also extend lifespan. Together, our results indicate that the C. elegans insulin/IGF-1 like signaling pathway may integrate two different types of information in setting the lifespan of the animal: internal signals that reflect the state of the germline, and external signals that may convey information about the animal’s surrounding environment.

(1) Hsin and Kenyon, Nature, in press.

24 May 1999 15:50 171 171 Cell-Type Specific age-1 Expression Reveals Multiple Outputs for Insulin-like Signaling

1999 International Worm Meeting abstract 120 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cell-Type Specific age-1 Expression Reveals Multiple Outputs for Insulin-like Signaling CA Wolkow 1 , MS Lee 2 , K Kimura 3 , G Ruvkun 1

1 Dept. of Mol. Biology, Mass. General Hospital and Dept. of Genetics. 2 Dept. of Pathology, Harvard Medical School, Boston, MA. 3 Div. of Biol. Sci., Nagoya Univ., Nagoya, Japan.

Dauer arrest and longevity in C. elegans are controlled by an insulin-like pathway containing daf-2, an insulin receptor-like gene, and age-1, a p110-like subunit of PI(3)K. To identify cells where this pathway functions, transgenic animals with cell-type restricted age-1 expression were constructed by expressing an age-1 cDNA from tissue-specific promoters: in neurons from the pan-neuronal unc-14 promoter; in mechanosensory (plus a few other) neurons from the mec-7 promoter; in muscle from the unc-54 promoter; in the intestine using a gut-specific synthetic promoter (Marshall, S. and J. McGhee, in prep). Transgenic animals with extrachromosomal arrays of the promoter-cDNA fusions were examined for the ability of cell-type restricted age-1 expression to rescue the dauer-constitutive, aging and metabolic phenotypes of age-1(mg44). Dauer arrest was rescued by age-1 expression in any of the above tissues, showing that age-1 action in a variety of different cells is sufficient, but not necessary, for non-dauer development. These results suggest that age-1 regulates a hormonal output from any of several sources for non-dauer development, as suggested by the genetic mosaic analysis of daf-2 (Apfeld, J. and C. Kenyon, 1998). The extended lifespan of age-1(mg44) was only rescued by pan-neuronal age-1 expression. In agreement, pan-neuronal expression of a daf-2 cDNA rescued the extended lifespan of daf-2(e1370). Expression of age-1 from the mec-7, muscle, or gut promoters was unable to restore normal lifespan to age-1(mg44). These results suggest that control of aging may be limited to the nervous system. The metabolic effects of age-1 also appear to be controlled in a specific subset of cells. Sudan Black staining of age-1(mg44) reveals enhanced fat accumulation in the intestine. Muscle age-1 expression was able to rescue the fat accumulation defects of age-1(mg44), while this phenotype was not rescued by age-1 expression in any other tissues. We conclude that dauer formation, longevity and metabolism are controlled cell non-autonomously by age-1, but that these age-1 outputs are controlled independently in different cell types.

24 May 1999 15:50 172 172 The tkr-1 life-extension gene, which responds to starvation and environmental stress, is regulated by the daf-16 forkhead transcription factor.

1999 International Worm Meeting abstract 121 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The tkr-1 life-extension gene, which responds to starvation and environmental stress, is regulated by the daf-16 forkhead transcription factor. S Murakami, TE Johnson University of Colorado, Institute for Behavioral Genetics. Gerontogenes are genes whose alteration causes life extension. Previous studies have identified several C. elegans gerontogenes, including age-1 (phosphatidylinositol 3 OH kinase) and daf-2 (insulin-like receptor), which are negative regulators of longevity and resistance to environmental stress. age-1 and daf-2 have been suggested to form an insulin-like signal transduction pathway dependent on daf-16 (forkhead transcription factor). We have identified a new class of gerontogenes that extend life span when up-regulated. The first such gerontogene was designated tkr-1 (tyrosine kinase receptor-1) (Murakami and Johnson, 1998). Overexpression of tkr-1 extends life span 40-100% and confers increased resistance to heat and UV stress. Purified TKR-1 has tyrosine kinase activity. Mutagenesis analysis suggests that the TKR-1 kinase activity is essential for increased longevity and stress resistance. The cytoplasmic tkr-1 kinase domain shows similarity to the mammalian fibroblast growth factor receptors (FGFR) and can be functionally substituted with that of human FGFR-1. Inactivation of tkr-1 by RNAi and by dominant negative mutations caused developmental delay, decreased reproduction, short life and stress sensitivity. These data strongly indicate that tkr-1 regulates longevity and stress resistance. The overexpression phenotypes of tkr-1 require daf-16 function, suggesting that tkr-1 signaling interlocks with the insulin-like age-1/daf-2 signal transduction pathway. Moreover, TKR-1::GFP analysis suggests that tkr-1 is induced after exposure to UV light, heat, starvation and perhaps oxidative stress. This induction and the expression of tkr-1 during aging is suppressed by a reduction of function mutation in daf-16. In conclusion, tkr-1 is a stress responsive RTK that also regulates longevity and is likely to take part in a daf-16 dependent signal transduction pathway in a way distinct to that of age-1 and daf-2.

24 May 1999 15:50 173 173 A Cytosolic Catalase, CTL-1, Is Necessary For daf-c- and clk-1-dependent Extension of Adult Life-span in C. elegans

1999 International Worm Meeting abstract 122 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A Cytosolic Catalase, CTL-1, Is Necessary For daf-c- and clk-1-dependent Extension of Adult Life-span in C. elegans James Taub, Joe Lau, Charles Ma, Jang Hee Hahn, Rafaz Hoque, Jonathan Rothblatt, Martin Chalfie Department of Biological Sciences, Columbia University, New York, NY 10027 We have found that C. elegans is the first animal species to have a cytosolic catalase (encoded by the ctl-1 gene) in addition to having the usual peroxisomal catalase (encoded by the ctl-2 gene). The ctl-1 and ctl-2 genes are arranged tandemly on chromosome II near unc-52. mRNA for ctl-1, but not for ctl-2 is upregulated in daf-2 dauers and in age-1 adults. This increase accounts for the increase previously noted in the catalase activity in age-1 animals, and suggests a role for the ctl-1 catalase in age-1- and daf-2- dependent life-span extension. The ctl-1(u800) mutation was identified fortuitously during a subtractive cDNA screen because it resulted in at least a 10-fold reduction of ctl-1 mRNA. The mutation, a single base pair deletion that introduces a stop codon at amino acid 50, results in a reduction of total catalase activity of more than 50%. Animals carrying the ctl-1(u800) mutation have approximately 30% shorter life-spans than do wild-type controls; dauer life-span is correspondingly reduced. ctl-1 is genetically downstream of daf-c genes and clk-1 with respect to adult life-span. The ctl-1 mutation eliminates the life-span extension seen in daf-2, age-1, and daf-2; daf-12 animals, yet does not affect dauer formation. The ctl-1(u800) mutation also eliminates the extension of adult life-span produced by the clk-1(e2519) mutation, yet does not affect the slowing of larval development seen in clk-1 animals. These epistasis results indicate that ctl-1 is necessary for the life-span extension seen in long-lived daf-c and clk-1 animals. The extension of adult life-span that these animals exhibit, may be due to the inappropriate expression of ctl-1 (and other antioxidant enzymes) in adults. ctl-1may have evolved to cope with oxidative stress during the extended period of pre-reproductive dormancy in dauer larvae or may function as part of a more general response to limited food availability.

24 May 1999 15:50 174 174 Long-lived C. elegans mutants have reduced metabolic rates.

1999 International Worm Meeting abstract 123 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Long-lived C. elegans mutants have reduced metabolic rates. Wayne A. Van Voorhies, Samuel Ward Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721 Numerous mutations increase the longevity of the soil nematode C. elegans. In principle these mutations could define genes involved in a process specific for aging. Alternatively, these mutations could alter a general process, such as metabolic rate, and increase longevity as a consequence. Consistent with this hypothesis, environmental conditions that reduce C. elegans metabolic rate also extend longevity. Many of the mutations that increase worm life span may simply act by depressing the animals’ metabolic rates, mimicking the response that is typically induced by stressful environmental conditions. We tested this hypothesis by comparing the metabolic rates between the various long-lived mutant worms to determine if long-lived mutants have reduced metabolic rates. Previous measurements of C. elegans metabolic rate involved placing unfed worms in liquid and monitoring changes in the dissolved oxygen levels. C. elegans is a terrestrial, not an aquatic nematode and in liquid culture worms grow more slowly, have an altered morphology, cannot mate, and have reduced fertility and metabolic rate. Recent advances in the sensitivity of carbon dioxide gas analyzers and in analysis software allow the measurement of worm metabolic rates of a small number of worms maintained on solid medium and fed E. coli. Using such a system we compared the metabolic rate of wild-type C. elegans grown at various temperatures to the metabolic rates of the long-lived mutants, age-1, clk-1, clk-2, daf-2, and clk-1;daf-2. We found that long-lived mutants have significantly lower metabolic rates than wild-type worms. When the increased life span of daf-2 mutants is suppressed by combining it with a daf-16 mutation, the metabolism also returns to normal. These results indicate that the increased longevity of some long-lived C. elegans mutants may be a consequence of a reduction in their metabolic output rather than an alteration of a genetic pathway specific for aging. These results are consistent with theories of aging based on damage caused by oxidative damage, but the mechanism responsible for the inverse correlation between metabolic output and longevity remains unknown.

24 May 1999 15:50 175 175 Signals from the Reproductive System that Regulate the Lifespan of C. elegans

1999 International Worm Meeting abstract 124 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Signals from the Reproductive System that Regulate the Lifespan of C. elegans H Hsin, CJ Kenyon Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143-0448 Understanding how the aging process can be regulated is a fascinating and fundamental problem in biology. We have found that signals from the reproductive system influence the lifespan of the nematode C. elegans. If the cells that give rise to the germline are killed with a laser microbeam, the lifespan of the animal is extended (Hsin and Kenyon, Nature, in press). Our findings suggest that germline signals act by modulating the activity of an insulin/IGF-1-like pathway known to regulate the aging of this organism. Mutants with reduced activity of the insulin/IGF-1 receptor homolog DAF-2 have been shown to live twice as long as normal (1,2,3), and their longevity requires the activity of DAF-16, a member of the forkhead/winged-helix family of transcriptional regulators (1,2,4,5). We find that in order for germline ablation to extend lifespan, DAF-16 is required, as well as a putative nuclear hormone receptor, DAF-12 (6,7). In addition, our findings suggest that signals from the somatic gonad have an opposite effect on lifespan, and that this effect appears to require DAF-2 activity. Together our findings imply that the C. elegans insulin/IGF-1 system integrates multiple signals to define the rate of aging of the animal. This study demonstrates an inherent relationship between the reproductive state of this animal and its lifespan, and may have implications for the co-evolution of reproductive capability and longevity.

1. C. Kenyon, et al., Nature 366, 461-4 (1993). 2. P. Larsen, P. Albert, D. Riddle, Genetics 139, 1567-83 (1995). 3. K. Kimura, H. Tissenbaum, Y. Liu, G. Ruvkun, Science 277, 942-6 (1997). 4. K. Lin, J. Dorman, A. Rodan, C. Kenyon, Science 278, 1319-22 (1997). 5. S. Ogg, et al., Nature 389, 994-9 (1997). 6. W-H. Yeh. Ph.D. Thesis, University of Missouri, Columbia (1991). 7. D. Riddle, P. Albert, in C. elegans II D. Riddle, T. Blumenthal, B. Meyer, J. Priess, Eds. (Cold Spring Harbor Laboratory Press, 1997) pp. 739-68.

24 May 1999 15:50 176 176 Illicit Sex in Georgia: inter-species reproductive interactions and speciation in the genus Caenorhabditis

1999 International Worm Meeting abstract 125 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Illicit Sex in Georgia: inter-species reproductive interactions and speciation in the genus Caenorhabditis KL Hill, SW L’Hernault Emory University Department of Biology, Atlanta, GA 30322 Speciation is both caused by and results in the reproductive isolation of related populations. We and others have found that aspects of reproductive interaction are affected by speciation. We have studied reproductive interactions among the Caenorhabditis species C. elegans, C. briggsae and C. remanei. Nematode reproduction consists of: mating, sperm activation, sperm migration, stimulation of ovulation, sperm competition and fertilization. Sperm migration to the spermatheca is an essential step in reproduction. A live fluorescent-labelling technique demonstrated that, in all inter-species pairwise mates, male-derived sperm successfully migrated to the spermatheca in the recipient hermaphrodite or female, showing that the attractant signal is conserved. Prior work of others has shown that spermless, feminized C. elegans hermaphrodites ovulate at low rates, and insemination via mating increases the ovulation rate irrespective of fertilization. We found that in inter-species C. briggsae male X C. remanei or C. elegans (fem-1) female, C. remanei male X C. elegans (fem-1) female and C. elegans male X C. remanei female mates, mated females ovulate at a significantly higher rate than unmated controls. In sperm competition, male-derived sperm take precedence in fertilization over resident hermaphrodite-derived sperm or other male-derived sperm. The large size of male-derived sperm in C. elegans is important for successful competition. Competitive ability is independent of fertilization, since mutant spe-9 sperm compete successfully, but do not fertilize. C. remanei sperm, which localize within the spermatheca correctly and are ~4X larger than C. elegans sperm, do not compete with C. elegans hermaphrodite-derived sperm. Therefore, factors besides sperm size and spermathecal targeting are required for sperm competition. These and other data show that all reproductive interactions examined, but not normal development, are conserved between C. remanei and C. briggsae. In contrast, only the steps preceding gamete interaction have been conserved between C. elegans and either C. remanei or C. briggsae. This suggests that species divergence in Caenorhabditis has occurred by both pre-zygotic and post-zygotic mechanisms.

24 May 1999 15:50 177 177 C. elegans: A Model Organism for Teaching?

1999 International Worm Meeting abstract 126 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. C. elegans: A Model Organism for Teaching? SJ Aamodt LSU-Shreveport, Shreveport, LA 71115 The need to integrate research and curriculum was recently addressed by Norman Fortenberry, the Director of the Division of Undergraduate Education, National Science Foundation (http://www.cur.org/governmt.html). He states, "The new challenge is to integrate research into the curriculum so that it is truly available to ALL." The idea is that the stimulation, skills and understanding of process that come with doing research must be developed in all students, not just the undergraduates who participate in research projects. The models that exist for involving all students include research-based undergraduate courses, inquiry-based laboratory exercises and courses structured around problem solving. Currently, my use of C. elegans in teaching undergraduates involves the supervision of undergraduate research. I also provide mutants for observation in the genetics laboratory course. I am planning to expand my use of C. elegans by incorporating research-based exercises into our curriculum. My colleagues and I hope to implement exercises with C. elegans in the laboratory courses for genetics, developmental biology and animal behavior. Although the drawbacks to using C. elegans in undergraduate education include the small size (a good microscope is required), the need to learn manipulation, and the fact that the databases are currently relatively hard to use, the advantages of using C. elegans include the wealth of information, the availability of mutants and the ease of culture and storage.

24 May 1999 15:50 178 178 Using C. elegans to Teach Embryonic Development to Undergraduates

1999 International Worm Meeting abstract 127 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Using C. elegans to Teach Embryonic Development to Undergraduates RV Aroian, D Johnson, G Wienhausen U.California San Diego We have begun to use C. elegans as a model system in our undergraduate embryology laboratory class at the University of California, San Diego. In the past, this 10 week class has used Sea Urchin, Xenopus, Chick, and other non-genetic systems to demonstrate to students embryonic development. In this poster we will outline the 6 new weeks of C. elegans modules. These modules allow the students to use advanced techniques to explore widely applicable principals of embryonic development. These techniques include wild-type lineage analysis, mutant lineage analysis, use of specific inhibitors to alter embryonic development, immunofuorescence analysis of wild type and mutants using antibodies to various cell biology and developmental markers, genome searching, and knocking out genes using RNA-mediated interference. Our goal is to one day publish an article on our results in the Worm Breeder’s Gazette. The embryonic development of more advanced model organisms such as Xenopus and chick are later used to compare and contrast to C. elegans. This work is greatly enhanced by the cooperativity of the C. elegans community in the sharing of antibodies, mutants, and other reagents such as GFP strains. Last year we used protocols, stains, and antibodies from to Geraldine Seydoux, Craig Mello, Pete Okkema, Michel Labouesse, Bruce Bowerman and the CGC. Thanks to them and others that have generously shared their reagents.

24 May 1999 15:50 179 179 C. elegans in Introductory Biology Projects

1999 International Worm Meeting abstract 128 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. C. elegans in Introductory Biology Projects EA De Stasio Lawrence University, Appleton, WI 54911 Thanks to relatively small class sizes and a commitment to hands-on science education, all students in our introductory biology course undertake 5-week research projects in groups of 2-4 students each with any one of the biology faculty. Worm biology works great. I have used and adapted ideas for projects from previous worm meetings, papers, and my own research. Thanks to all who see your work in the projects below. In 5 weeks, motivated undergraduates can do more than you think. I have arranged the projects below in order of difficulty and student time commitment. In all projects we intend students to learn the general method of scientific research, the importance of controlled observation, and approaches to data analysis, as well as learning the ambiguities inherent in interpreting biological data. Students are also expected have a role in designing experiments. 1. Motility assays - Students quantitatively compare motility of paralyzed, suppressed and wild type worms. Data analysis includes descriptive statistics and t-test. Students learn the importance of positive and negative controls. 2. Complementation tests - Students can use any mutant collection. Dealing with suppressors adds a level of complexity to the conceptual portion. Again, positive and negative controls are used. 3. Chemotaxis experiments - Students measure the attraction of worms to certain molecules. Data can be quantitative or qualitative and experiments must be carefully controlled. Students begin to see the difficulty of controlling all variables. 4. Worm learning - An extension of chemotaxis experiments; by pairing food or other attractant with non-attracting substances, one can test whether worms have memory. Students have fun choosing substances to test. Worms are repulsed by garlic, for example. 5. Worm metabolism - We have recently tried measuring oxygen consumption of C. elegans. Again, controls are vital and quantitative data are produced.

24 May 1999 15:50 180 180 Worms in class at NYU

1999 International Worm Meeting abstract 129 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Worms in class at NYU D Fitch 1 , EJ Hubbard 1 , S Clark 2

1 Department of Biology, New York University, New York, NY. 2 Molecular Neurobiology Program, Skirball Institute, NYU School of Medicine, New York, NY.

We use worms as teaching models in labs and lectures for our courses in Developmental Genetics, Molecular Genetics, Advanced Genetics, Cell Biology, and Principles of Evolution. In the laboratory component of Developmental Genetics, students test hypotheses and gain hands-on experience with models systems to reinforce lecture topics. In a demonstration of the "forward" approach to identify genes, students test for linkage with a cross between wild type males and hermaphrodites homozygous for 3 markers. Students use germline transformation with dominant markers or RNAi to demonstrate "reverse" approaches to understanding gene function. To test the functions of the distal tip cell, students perform laser ablations, replicating the classic experiments of Kimble and White (1981). The utility of GFP as a marker is demonstrated as students use epifluorecence microscopy to examine axon guidance mutants and mosaic animals. In the graduate genetics courses, we use primary worm literature for small-group discussions to explore topics as basic as 3-factor mapping and as complex as integration of multiple signaling pathways. Formal debates are used to augment students’ skills in presentation and persuasion. Primary research papers are discussed in depth to understand current biological questions and approaches as well as how to design control experiments, discriminate among alternative hypotheses, uncover assumptions, and write papers and proposals. In Molecular Genetics, students write mock grant proposals about problems of their choice, incorporating approaches covered in the course; in Advanced Genetics, students write in-depth reviews of a "heavy" genetics paper. Worm literature is also used to illustrate principles and experimental approaches in the Cell Biology and Evolution courses (open to undergraduates). In Cell Biology, worms are used almost exclusively as a model for understanding mechanisms of programmed cell death. In the Evolution course, heterochronic mutants are discussed for their relevance to macroevolutionary changes. Papers by Sommer et al. (1994-1995) are used to illustrate how model systems may be applied to address important evolutionary principles such as canalization and co-option.

24 May 1999 15:50 181 181 Using PCR in an undergraduate lab course to detect deletions in the unc-93 gene

1999 International Worm Meeting abstract 130 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Using PCR in an undergraduate lab course to detect deletions in the unc-93 gene JL Lissemore, LL Lackner, GD Fedoriw Biology Department, John Carroll University, University Heights, OH 44118. PCR is a powerful and common molecular biology technique with many applications. We have developed an exercise for an undergraduate molecular biology lab course using PCR to detect deletions in the unc-93 gene of C. elegans. unc-93 encodes a putative transmembrane protein muscle protein of unknown function. Wildtype and three different unc-93 deletion mutant strains (carrying alleles lr12, lr28, and lr81) were grown, harvested, and frozen at -20 C by the instructor. Students isolate genomic DNA from each strain using a genomic DNA isolation kit from Gentra Systems, Inc. (Minneapolis, MN). We have used this kit for several years and it seems to be almost foolproof in the hands of undergraduates. Following proteinase K and RNase A treatment, proteins in the worm extract are precipitated by high salt and the genomic DNA is recovered by isopropanol precipitation. PCR reactions using two sets of primers (A and B) from two different regions of the unc-93 gene are carried out on the genomic DNA from wildtype and mutant strains and the results analyzed by agarose gel electrophoresis. Primer pair A yields a 789 bp fragment, while primer pair B yields a 728 bp fragment. The A primers detect a 173 bp deletion in lr28 and a 78 bp deletion in lr81 while the B primers detect a 517 bp deletion in lr12. The use of wildtype DNA and primers for amplifying two different regions of the unc-93 gene provide internal controls. Five student groups carried out this exercise during the Spring 1999 semester. While there was variation in the yield of the products, especially with primer pair B, all five groups detected the presence of deletions of the expected size in the expected strains.

We gratefully acknowledge the assistance of Dr. Beth DeStasio, Biology Dept., Lawrence University, Appleton, WI, who provided the unc-93 strains and primers used in this exercise. LLL and GDF made equivalent contributions to this work.

24 May 1999 15:50 182 182 A project-based laboratory course using C. elegans

1999 International Worm Meeting abstract 131 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A project-based laboratory course using C. elegans LM Miller Santa Clara University This NSF-sponsored project integrates research and education by developing an intensive new laboratory course in which undergraduate students participate in a supervised research project. This research project is directly related to ongoing research in the laboratory of the principal investigator. The course is called "Project Lab" and is taught for one quarter each year with a class size of 7-16 students. Each year, the course focuses on research activities appropriate for that particular stage of the research project. The addition of this course to our biology curriculum enhances the science education of undergraduate students at Santa Clara University by providing hands-on laboratory experience and exposure to real research problems. It also enhances the research program of the principal investigator by training and motivating students for research. Specifically, students isolate and study mutations in genes that are required for cell fate determination in the process of vulval development in the nematode, C. elegans. The identification and analysis of these genes will set the stage for future experiments in cell fate determination. C. elegans is ideally suited for an undergraduate laboratory course. With appropriate training, undergraduate students can quickly learn to manipulate nematodes for use in genetic and/or molecular experiments. The first two Project Lab classes have made considerable progress on two different projects. For the first project, Project Lab students isolated and characterized over 30 new mutations that represents genes involved in the proper choice of cell fate during C. elegans vulval development. For the second project, several Project Lab students have been involved in a structure/function analysis of a transcription factor required for proper patterning of cell fate in C. elegans vulval development. There is a manuscript in preparation for this project, which will be submitted for publication within the next year. Three Project Lab students will be authors on this paper. I have also successfully used C. elegans in my upper division genetics laboratory course. This is a 10-week course in which students use nematodes to study inheritance patterns and map genes (using STS mapping).

24 May 1999 15:50 183 183 Lecture/lab combo: P-granule antibody staining

1999 International Worm Meeting abstract 132 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Lecture/lab combo: P-granule antibody staining MK Montgomery Macalester College, Biology Dept., 1600 Grand Ave., St. Paul, MN 55105 P-granules are cytoplasmic components that segregate with the germ lineage in C. elegans. They have been used in developmental biology courses to illustrate the classical concept of a cytoplasmic determinant, although P-granules alone are insufficient to bestow a germline fate on cells containing them. Nonetheless, P-granule staining is relatively simple and can lead to discussions encompassing segregation of maternal components, the nature of the germ line versus soma, and the technique of antibody staining. Also, strains such as the par mutants that are defective for proper P-granule segregation are available through the CGC, and are nice for doing side-by-side comparisons with wild type. The initial characterization of P-granules was done by Susan Strome (U. of Indiana) and through her efforts there are two monoclonal antibodies commercially available, OIC1D4 and K76, both from the Developmental Studies Hybridoma Bank at the U. of Iowa. I have personal experience with only OIC1D4, which in my students’ hands has worked remarkably well. Because immunostaining is a procedure that requires more time than the typical 3-hour lab period, I have students continue the protocol during scheduled lecture hours, which I call "Lecture/Lab Combos." There is a significant amount of "down time" during staining, in which students are simply transferring slides from one solution to another. I use the down time to cover lecture material on cytoplasmic determinants and related topics. Students do the initial freeze cracking through application of primary antibody during the scheduled lab period; washes and overnight application of secondary antibody during the next two lecture periods. I use a CY2-goat anti-mouse secondary antibody and thus an epifluorescent microscope is required for viewing stained embryos. I have found that the trickiest part of the protocol for the students is at the beginning; namely, the application of just enough pressure to burst gravid adults without oversquashing the embryos. Still, last semester 8 out of 9 students in my Developmental Biology class obtained really nice results. A detailed protocol for P-granule staining (based on protocols worked out by S. Strome and Geraldine Seydoux) can be found under the "Course Pages" at my website: http://www.macalester.edu/~montgomery/

24 May 1999 15:50 184 184 Use of C. elegans in Undergraduate Biology Courses.

1999 International Worm Meeting abstract 133 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Use of C. elegans in Undergraduate Biology Courses. William R. Morgan Department of Biology, College of Wooster, Wooster, OH 44691 Many of the features that make C. elegans an excellent research organism -- its small size, rapid life cycle, and ease of cultivation -- also make it useful for undergraduate instruction, including courses in genetics, developmental biology, and molecular biology. I have used C. elegans most extensively in an undergraduate genetics course. Here, as part of a hypothetical genetic dissection to investigate neurobehavior (modeled on Brenner’s original work), students perform a multi-week gene mapping experiment in preparation for complementation analysis (see http://www.wooster.edu/biology/bio306/C.elegans_Overview.html) Recently, an STS-based gene mapping exercise was added to our introductory biology laboratory investigations. I have also used C. elegans in an undergraduate developmental biology course to demonstrate tissue-specific gene expression and the use of reporter genes. In a molecular biology course, C. elegans and related nematodes are the basis of a semester-long research project to screen for gene orthologs (see http://www.wooster.edu/biology/bio309/Bio309_Lab_Project.html). While the use of C. elegans has been advantageous overall, it does present some unique impediments to novices (including the difficulty of distinguishing sexes and transferring individuals) that must be considered for its successful use in the undergraduate teaching laboratory.

24 May 1999 15:50 185 185 Biology Biology 478: Research & Seminar on the Molecular Biology of Model Organisms

1999 International Worm Meeting abstract 134 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Biology Biology 478: Research & Seminar on the Molecular Biology of Model Organisms JG Pelliccia Bates College, Department of Biology, Lewsiton ME 04240 The fruit fly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans, have served as useful model organisms for cellular and molecular research. The genome sequencing projects for these organisms have given us an unprecedented insight into what it takes to code for the myriad functions that make a multicellular animal. A diversity of molecular genetic techniques makes the production and analysis of transgenic animals routine, and basic developmental and neurobiological processes first described in these model organisms have served as a starting point for understanding the function of homologous processes in more complex animals. In this Seminar & Research course, students perform laboratory, literature, and genome database research on current problems in the molecular biology of these model organisms

24 May 1999 15:50 186 186 Development of an Open-ended Muscle Physiology Experiment with C. elegans

1999 International Worm Meeting abstract 135 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Development of an Open-ended Muscle Physiology Experiment with C. elegans J Sulcove, T Allen Biology Dept, Oberlin College, Oberlin, OH 44074 Given the broad range of mutant strains that have deficits in behaviors relevant to an undergraduate course in physiology, we sought to develop a discovery-based experiment with C. elegans that would both amplify concepts being learned in class and permit students to participate in the experimental design. The created experiment spans two to three laboratory sessions and requires a minimal amount of equipment. The cyclic interaction of myosin with actin is the basis of tension development in muscle cells, as well as eukaryotic cells generally, and the mechanical events are coupled to the hydrolysis of MgATP by myosin. The experiment challenges students to characterize quantitatively the deficits in muscular function arising from selected missense mutations in unc-54 (myosin heavy chain B). The experiment exploits the availability of unc-54 mutations (e.g., s74 and s95 alleles) that have salient functional deficits without disruption of muscle structure. Following discussion of Reynold’s numbers, students generally develop swimming assays in which tail-beats per minute are examined as a function of bulk solution viscosity. The concepts that the students consider when designing their experiments include the following: osmolarity and its measurement; bulk solution viscosity and its measurement; muscular fatigue and endurance; and force-velocity relations and their interpretation in the context of muscular efficiency. Students have found the experiment engaging, because of the linkage between mutations in human cardiac myosin and familial hypertrophic cardiomyopathy, a lethal heart disease. Moreover, the accessibility of the crystalline structure of the myosin head (containing the catalytic domain and the actin-binding regions) permits students to correlate their results with the emerging structure-function relationship of myosin. The experiment can also be extended to the analysis of other mutations affecting muscular function (e.g., unc-27/Troponin I-2; please see abstract presented at this meeting by Burkeen et al., In Vivo Analysis of Troponin I Domains in Muscle Function).

24 May 1999 15:50 187 187 Surfing the Genome: Using the C. elegans Genomic Sequence to Teach Molecular Genetics

1999 International Worm Meeting abstract 136 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Surfing the Genome: Using the C. elegans Genomic Sequence to Teach Molecular Genetics B Wightman Muhlenberg College, Allentown, PA, 18104. There is considerable interest in inquiry-based learning in current undergraduate science pedagogy. The idea is that students learn science better by participating in the process, rather than taking endless notes in a large lecture hall. This approach conveys the notion that science is not static, but rather an ongoing process. Moreover, it is widely-believed that students retain information more effectively if they have experienced science first-hand. The completion of the C. elegans genomic sequence provides a unique opportunity to engage students in the process of science. In my Advanced Molecular Genetics class five students (all sophomores and juniors) learned molecular genetics and protein structure through an independent project approach. Each student was provided with a "roadmap" for designing their own project. Their assignment was to identify a poorly-characterized gene from the C. elegans database that was a member of an orthologous gene family, research their gene and the gene family, perform RT-PCR to isolate clones of their gene, characterize their clones, and finally investigate gene function by RNAi. Current technology has advanced to the point where such a project is not as unrealistic as it might sound. In the process of performing their projects, students learn about genomics, protein structural motifs and functional domains, computer-based sequence analysis and retrieval, and experimental design. It is my hope that this approach will create better-trained future scientists and physicians. This project was funded in part by an ILI grant from the National Science Foundation.

24 May 1999 15:50 188 188 Conservation of sequence and intron/exon structure between the homologous pag-3 genes from C. elegans and C. briggsae

1999 International Worm Meeting abstract 137 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Conservation of sequence and intron/exon structure between the homologous pag-3 genes from C. elegans and C. briggsae EJ Aamodt, L Shen, B Rose, J McDermott Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, LA 71130-3932 The pag-3 gene encodes a zinc finger protein involved in controlling neuron specific gene expression. pag-3 mutants were first isolated based on misexpression of touch receptor genes in the BDU interneurons, the lineal sisters to the ALM touch neurons. pag-3 mutants are lethargic, kink when trying to move backward (reverse kinker Unc) and have lineage defects in the P-neuroblast lineages (see abstract by Cameron et al., this meeting). We identified a fosmid containing the C. briggsae homologue of the C. elegans pag-3 gene and the Washington University Genome Sequencing Group sequenced it. When transformed into a C. elegans pag-3 mutant, the C. briggsae pag-3 gene rescued the pag-3 reverse kinker and lethargic phenotypes. The C. elegans pag-3 gene fused to lacZ was expressed in the same pattern in C. elegans and C. briggsae. Unlike many gene homologues compared between C. elegans and C. briggsae, extensive sequence conservation was found in the noncoding regions upstream of the pag-3 exons, in several of the introns and in the downstream noncoding region. Furthermore, the splice acceptor and splice donor sites were conserved and the size of the introns and exons was surprisingly similar. Based on the genomic sequence, the sequence of the C. briggsae PAG-3 protein was predicted. The predicted protein sequence was 85% identical to the sequence of C. elegans PAG-3. These results showed the evolutionary conservation of the pag-3 gene sequence, its expression and function. Because so much of the non-coding regions of pag-3 are conserved, the control of pag-3 may be quite complex, involving the binding of many transacting factors.

24 May 1999 15:50 189 189 Characterization of the heterochronic gene lin-57, a gene hypostatic to lin-4

1999 International Worm Meeting abstract 138 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of the heterochronic gene lin-57, a gene hypostatic to lin-4 JE Abrahante 1 , EA Miller 1 , AE Rougvie 1,2

1 Dept. of Genetics & Cell Biology, University of Minnesota, St. Paul, MN 55108. 2 Dept. of Biochemistry, University of Minnesota, St. Paul, MN 55108. lin-57is a novel gene identified in a screen for heterochronic mutants that exploits the adult specific nature of the rol-1phenotype. rol-1(e61)mutants exhibit a stage-specific locomotion defect: larvae swim in the normal sinusoidal pattern but adults roll. This adult specific rol-1phenotype depends upon the synthesis of adult cuticle and is temporally altered by mutations in the heterochronic gene pathway. For example, lin-4and lin-29mutants never synthesize adult cuticle and consequently lin-4 rol-1and rol-1 lin-29double mutants never roll. Conversely, lin-14and lin-28mutants synthesize adult cuticle early and rol-1double mutant combinations with these genes result in animals that roll as larvae. In attempts to identify additional heterochronic genes, we mutagenized rol-1(e91)animals and screened for animals that either roll precociously or fail to roll as adults. Among the mutants identified in this screen was a precocious roller that defined lin-57. In lin-57mutants, seam cell terminal differentiation and adult cuticle synthesis occur one stage early, during the L3 molt. In this respect lin-57is similar to the other known precocious mutants lin-14, lin-28, lin-42and lin-58. However, lin-57is unique because it is the first known heterochronic gene to which lin-4is epistatic. In lin-4; lin-57double mutants the seam cells fail to terminally differentiate. To better understand the role of lin-57in the heterochronic gene pathway, we initiated its molecular cloning. lin-57resides on the X chromosome, in a 1.5 mu interval between mec-2and stP33. A single cosmid from this region, F13D11, fully rescues the lin-57 phenotype. Genefinder predicts four ORFs in F13D11: 1) a tyrosine phosphatase, 2) a zinc finger protein, 3) a histidine phosphatase and 4) a dihydroflavonol 4-reductase (DFR). We narrowed the lin-57rescuing activity to a 12Kb fragment containing the histidine phosphatase and DFR. To determine which ORF encodes lin-57,we have created additional constructs, including frameshifts in the predicted ORFs. In addition, we have generated a second allele and are sequencing both alleles. A full report on the identity, expression patterns and genetic interactions of lin-57will be presented.

24 May 1999 15:50 190 190 Pharmacological analysis on unc-68 suppressor animals of Caenorhabditis elegans

1999 International Worm Meeting abstract 139 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Pharmacological analysis on unc-68 suppressor animals of Caenorhabditis elegans R Adachi, Y Sakube, H Kagawa Department of Biology, Faculty of Science, Okayama University, Okayama 700-8530, JAPAN unc-68 encodes the C. elegans ryanodine receptor consists of 5,071 amino acid residues. Mutation sites of isolated unc-68 mutants are e540 (Stop by frame-shift in exon 22), x14 (Trp153 to Stop) and r1161 (Tc1 induced deletion), respectively. Only one missense unc-68(kh30) is isolated as a ketamine-response abnormal (kra-1) showing convulsions with intermittent paralysis in 30 mM ketamine, an anesthetic for clinical use. The unc-68(kh30) mutation produced a full-sized protein having an amino acid substitution at Ser1,444 to Asn which is a putative phosphorylation site of protein kinase C. To investigate the molecules interacting with the ryanodine receptor, we tried to isolate suppressor animals of unc-68(kh30). unc-68(kh30) animals were treated with 50 mM of EMS, and F2 progeny were screened with 30 mM ketamine. Six suppressor animals were isolated by choosing a paralyzed phenotype like as that of the wild-type. We observed responses of suppressor animals against some anesthetics, caffeine, ouabain and ryanodine. In 50 mM caffeine, wild-type and all isolated worms showed transition from convulsion to paralysis. From motility on NGM plate and the response to caffeine, we assigned 6 suppressor animals to 3 groups. There were two of each suppressors showing slow movement and high sensitivity to caffeine, low sensitivity and others, respectively. We are mapping these suppressors by three-factor and two-factor crosses. Our aim of this approach is to isolate a gene controlling the ryanodine receptor through protein kinase C.

24 May 1999 15:50 191 191 adm-2 has an essential role during early embryogenesis in C. elegans

1999 International Worm Meeting abstract 140 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. adm-2 has an essential role during early embryogenesis in C. elegans V Adir, B Podbilewicz Department of Biology, Technion- Israel Institute of Technology, Haifa 32000 Israel The ADAMs gene family encodes transmembrane proteins, which contain both A Disintegrin and A Metalloprotease domains. Members of this protein family have been shown to be involved in cell-cell interactions: fertilin a and b (involved in sperm-egg fusion), meltrin a (appears to be required for myotube formation in mice), TACE (a TNF a converting enzyme), the kuzbanian gene product essential for neural fate in Drosophila, and sup-17 a suppressor of lin-12 which plays a role in gonad and vulva development in C. elegans. adm-1 , was the first gene from this family found in C. elegans, and is expressed in cells that undergo cell fusion. However, adm-1 lacks the catalytic site consensus sequence for a zinc dependent metalloprotease. adm-2, which encodes for an additional ADAM in C. elegans, was found by sequence homology searching. We have performed a series of experiments designed to try and determine the role of this gene in C. elegans: 1) Interference by injection of adm-2 dsRNA showed embryonic lethality at about the 100 cell stage, in 50% of eggs laid. Nomarski microscopy revealed that the cells of these embryos appeared detached and disorganized compared to normal embryos. 2) UV/TMP mutagenesis followed by PCR screening identified a mutant with a 1.5Kb deletion in the adm-2 gene. Homozygocity to this mutation lead to a maternal effect embryonic lethal phenotype similar to that found in the RNAi experiment with embryonic lethality at about the 100 cell stage. 3) The GFP gene was fused to the adm-2 promoter, and its expression was used to follow the cell specific expression of adm-2. We found that GFP was expressed from 3 fold embryos to adults, mainly in the nerve ring, in the ventral nerve cord and in the CAN cells. The experiments described above suggest that inhibition of adm-2 expression (either by RNAi or mutagenesis) results in a defect in cell organization, at an early embryonic stage. Since ADM-2 is predicted to be a transmembrane protein containing both disintegrin and metalloprotease domains, this implies that ADM-2 could act either as integrin ligand or as protease or both, and function as a regulator of cell-cell interactions by changing the adhesivisity between cells, in the early embryo and later in the nervous system in postembryonic stages.

24 May 1999 15:50 192 192 T-box genes involved in embryonic patterning

1999 International Worm Meeting abstract 141 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. T-box genes involved in embryonic patterning Julie Ahringer, Michael Mitsch Wellcome CRC Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, England T-box genes are transcription factors, some of which are have a role in mesoderm formation and/or organisation in vertebrates (e.g. Brachyury, VegT, and Eomesodermin). We previously showed that vab-7 is required for patterning the posterior mesoderm, and we would like to find additional genes involved. Therefore, we have begun to investigate whether any C. elegans T-box genes have a function in mesodermal patterning. There are 17 T-box homologues in the C. elegans genomic sequence. So far, we have inactivated the functions of 11 singly using RNAi. None has an embryonic lethal phenotype. However, when two divergently transcribed T-box genes (T07C4.2 and T07C4.6) are inactivated together, the resulting progeny are lethal. These embryos have grossly abnormal body morphology, with severe muscle and epidermal patterning defects. The intestine is also abnormally shortened. To ask whether these genes have a function in patterning with vab-7, we examined vab-7 expression in the RNAi embryos. We found that mesodermal expression of vab-7 is abolished, but epidermal expression is normal. The number of muscle cells is normal in these embryos, so this suggests that vab-7 might be a target of these genes in the mesoderm. Consistent with this, there is a consensus brachyury binding site in the vab-7 promoter. When T07C4.6 is expressed under the control of a heat shock promoter, weak ectopic vab-7 expression is induced in some cells of early embryos (100 cell stage), but later embryos do not have ectopic vab-7. Therefore, T07C4.6 is not generally sufficient for vab-7 expression, but may be sufficient in particular cells during a small time window. A T07C4.6::gfp reporter gene is first expressed in nuclei at the 28-cell stage in Ea and Ep. Later expression is in diverse cell types, including the epidermis, muscles and intestine. The pharynx and most neurons do not express the reporter gene, and expression is gone by the end of embryogenesis. The results show that T07C4.2 and T07C4.6 have overlapping mesodermal and ectodermal patterning functions, and may play a role in intestinal development as well. We are currently inactivating the remaining T-box genes, singly and in groups to find if any others also play a role in embryonic patterning.

24 May 1999 15:50 193 193 Analysis of a regulator of body length in C. elegans

1999 International Worm Meeting abstract 142 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of a regulator of body length in C. elegans M Aili, S Tuck UCMP, Umeå University, SE-901 87 Umeå, Sweden. Body length in C. elegans is regulated by a conserved signalling pathway activated by the TGFb superfamily member, DBL-1/CET-1a,b,c,d,e . DBL-1/CET-1c,e is thought to function by binding the receptors, SMA-6d and DAF-4a , which in turn appear to activate the SMAD transcription factors, SMA-2, SMA-3 and SMA-4b . Loss-of-function mutations in genes encoding any of the components of the pathway cause a marked decrease in body size, the Sma phenotype. Conversely, constitutive activation of the pathway caused by overexpression of DBL-1/CET-1, results in a dramatic increase in body lengthc,d . A mutation in thelon-3 gene, e2175, confers a Lon phenotype similar to that caused by overexpresion of DBL-1/CET-1. We have mapped lon-3 to within 0.1 m.u. of myo-3 and rescued the lon-3(e2175) phenotype using a cosmid that contains the myo-3 gene. Neighbouring cosmids fail to rescue so it seems likely that lon-3 lies on the myo-3 cosmid. Hermaphrodites homozygous for lon-3(e2175) are Lon as adults but are not perceptibly longer than WT at earlier developmental stages. e2175/+ hermaphrodites are considerably shorter than e2175 homozygotes but, as older adults, are slightly longer than WT hermaphrodites of the same age. e2175/Df animals are Lon whereas +/Df are WT in length. Thus e2175 appears to reduce gene activity and may be weakly antimorphic. daf-4; lon-3 double mutants display the Sma phenotype. lon-3 is therefore a negative regulator of TGFb signalling that functions, genetically, upstream of daf-4. We are presently conducting a mosaic analysis of lon-3 and investigating interactions between lon-3 and another regulator of body length, lon-2. We also plan to examine interactions between lon-3 and genes encoding components of the TGFb signalling patways in C. elegans regulating dauer development and axonal guidance. b

a Estevez et al. (1993). Nature 365: 644-649.

b Savage et al. (1996). PNAS 93: 790-794.

c Suzuki et al. (1999). Development 126:241-250.

d Krishna et al. (1999). Development 126:251-260.

e Morita et al. (1999). Development 126: 1337-1347.

24 May 1999 15:50 194 194 27° in the life of wild and mutant worms

1999 International Worm Meeting abstract 143 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. 27° in the life of wild and mutant worms M Ailion 1 , JH Thomas 2

1 Program in Molecular and Cellular Biology, Univ. of Washington, Seattle, WA 98195. 2 Department of Genetics, Univ. of Washington, Seattle, WA 98195.

Dauer formation in C. elegans is strongly induced at 27°, a temperature just below the highest temperature that permits growth and reproduction. At this temperature, dauer formation is much more strongly induced by pheromone than at 25° and even in the absence of exogenous pheromone, N2 makes some dauers at 27°. This basal level of dauer formation is probably not dependent on endogenous pheromone since daf-22 mutant animals also form dauers at a similar frequency at 27°. All twenty-one natural isolates of C. elegans that we’ve tested have virtually the same thermal limit and all induce dauer formation at 27°. Dauers formed at high temperatures have normal fertility if recovered at lower temperatures, suggesting that the strong induction of dauer formation at stressful temperatures was selected for as a protection against sterility or death. In contrast to the low wild-type level of dauer formation at 27°, many mutants are strongly Daf-c at 27° but are not Daf-c at 25°. Mutants with this phenotype include unc-64, unc-31, unc-3, daf-3 and the dyf mutants. We have performed screens for Daf-c mutants at 27° and isolated 100 mutants. We isolated twenty-one alleles of known Daf-c genes (many weak alleles), fifteen alleles of dyf or Daf-d genes, five alleles of unc-31 or unc-3 and 13 alleles of new Daf-c genes. Many other mutants have only been partially characterized. Of the new Daf-c mutations, two are alleles of pdk-1 and one is an allele of aex-6. The Daf-c phenotype of many of the mutants is fully suppressed by mutations in daf-16, suggesting that these genes act in the daf-2/age-1 (insulin-receptor /PI3 kinase) pathway. These include unc-64 and unc-31, which encode neurosecretory proteins that may be involved in insulin secretion, and pdk-1 which encodes a PI3-dependent protein kinase that acts downstream of age-1. The aex-6(sa699), sa573 and sa691 mutations also appear to affect this pathway and may encode novel components of the insulin pathway. In addition to their Daf-c phenotype, the aex-6(sa699) and sa691 mutants also have defects in movement and defecation. We have mapped sa691 to a narrow region of the X chromosome between lin-32 and unc-2 and are attempting to clone it.

24 May 1999 15:50 195 195 Identification and characterization of long-lived mutants in C. elegans

1999 International Worm Meeting abstract 144 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification and characterization of long-lived mutants in C. elegans J Alcedo, D Garigan, N Arantes-Oliveira, N Libina, B Albinder, J Apfeld, H Hsin, B Tsung, C Kenyon Dept. of Biochemistry and Biophysics, University of California San Francisco, 513 Parnassus Ave., San Francisco, CA 94143-0448 C. elegans is an excellent organism in which to study the regulation of lifespan. Genetic analyses have already shown that an insulin/IGF-like hormonal control system, the DAF-2 pathway, regulates lifespan in the worm. However, many components of the pathway, whose existence has been inferred from previous studies (see abstracts by Apfeld and Kenyon and by Hsin and Kenyon, International Worm Meeting 1999), have not been identified. To search for more genes that would help to elucidate mechanisms that control lifespan in the worm, our group has done an EMS mutagenesis screen for long-lived worms and found 29 independent long-lived mutants. Two mutants fail to complement daf-2, while one mutant fails to complement age-1. Some of the remaining mutants form dauers at 27 °C but complement daf-2 or age-1, and may identify new genes involved in regulating lifespan. Some of the other mutants may have defects in the sensory cilia as determined by the inability of their amphid sensory neurons to take up DiI (see abstract by Apfeld and Kenyon, International Worm Meeting 1999); and this defect has been linked to the long-life phenotype of at least one mutant. In addition, some of the mutants have a slow-growing phenotype and might be candidate clk alleles. We are currently mapping these mutations using a PCR-mapping strategy.

24 May 1999 15:50 196 196 Structure/ function of C-terminus motor KLPs in C.elegans.

1999 International Worm Meeting abstract 145 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Structure/ function of C-terminus motor KLPs in C.elegans. MY Ali, T Harada, ST Khan, SS Siddiqui Toyohashi University of Technology Kinesins are intracellular transport motors found in all eukaryotes. Here we describe a special subgroup of kinesin like proteins in C. elegans that are distinguished by location of their motor domain in the carboxyl terminal of the protein. First such atypical C-terminal motor in C. elegans, CeKLP-3 was identified by sequencing a rare cDNA clone (Khan et al., 1997). CeKLP3, encoded by the klp-3 gene (Cosmid T09A5.2) is located on LGII, as one of the C-terminus kinesins, and belongs to the yeast Kar3 (Meluh and Rose, 1990) and Drosophila ncd kinesins (Endow et al., 1990), that are retrograde motors, mediate chromosmal movement during mitotic cell divisions. In a PCR amplification based screening assay, using degenerate primers to the conserved ATP and MT binding sites in the motor domain as probes, we have identified three new cDNA clones that reveal new C-terminus kinesins in C. elegans ( KLP-15, KLP-16, and KLP-17). Northern blot analysis suggest that these cDNA clones are close to full length, deduced from genomic sequences. Phylogenetic relationship of these motor proteins suggests that KLP-15, KLP-16, and KLP-17, are members of the ncd/kar3 subfamily, primarily involved in chromosome segregation. However secondary structure of these new members is different in the non-motor domain, i.e. the stalk and amino terminus cargo binding domains have different size and charge distribution. The CeKLP-3, is involved in chromosome separation, as the overexpression of the klp-3 in transgenic animals increases the incidence of males caused by nondisjunction of the X-chromosome, and results in polyploid embryos that are arrested in early development. Similarly, an anti-sense construct of the klp-3 cDNA expressed under the heat shock promoter affects chromosomal segregation (Khan et al.,1997). Our data on embryonic insitu hybridization suggest that klp-15 and klp-16 express at a high level in early embryogenesis in the cytoplasm, but the klp-17 expresses strongly in the cell nuclei. We are in the process to identify the role of of all three genes using dsRNAi and the promoter fusion expression using gfp.

We thank A. Coulson, A. Fire, Yuji Kohara, Y. Ohshima , T. Stiernagle for materials, and Monbusho for a research grant to SSS.

24 May 1999 15:50 197 197 Multiple kinesins play critical role in chromosomal movement during embryonic cell divisions in C.elegans.

1999 International Worm Meeting abstract 146 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Multiple kinesins play critical role in chromosomal movement during embryonic cell divisions in C.elegans. MY Ali, ST Khan, AS Mohammed, SS Siddiqui Toyohashi University of Technology A superfamily of genes klp-1 - klp-20 encoding kinesin proteins has been identified in C. elegans (see abstract by Siddiqui et al.), that has representative members belonging to all nine groups of kinesins characterized in mammals. A major question in kinesin biology is how many kinesins are expressed in a single cell type, and if they are unique, or have overlapping functions? Early embryogenesis in C. elegans is well characterized and offers a good opportunity to elucidate kinesin motor function at genetic, cellular and molecular levels. For example, the unc-116 KHC has been previously shown to affect first mitotic cell division (Hall and Hedgecock, 1991; Patel et al., 1993, Ali et al. 1999). Here we have charcterized several members of the kinesin family, such as klp-3 (Khan et al., 1997) klp-7, klp-12, and klp-14 , which function in chromosome movement during early embryogenesis. Cloning of a C.elegans cDNA of the klp-7 gene reveals that it encodes a CeKLP7 of 689 amino acids. which is a member of MCAK/KIF2 subfamily. Members of this group have a distinct primary and secondary structure, as their motor domain is located in the middle of the protein sequence. MCAK kinesin that lacks the ATP binding motor domain, binds centromeres but not the microtubules and can disrupt chromosome segregation during anaphase (Hunter et al. 1998). Our insitu hybridization result suggests that CeKLP-7 functions in early embryogenesis. In a parallel experiment, klp-7 gene inactivation with the double stranded RNAi in adult animals, produced dead eggs, arrested in different developmental stages, suggesting a requirement of the klp-7 gene in embryogenesis. Similar to the klp-7, we have analysed the role of CeKLP12, an ortholog of the chicken chromoskinesin, and the CeKLP-14, an ortholog of the BimC family of kinesins. Results on in situ hybridization, RNAi, immunocytochemistry, and fusion gene expression suggest that CeKLP-12, and CeKLP-14 are directly involved in early cell divsions in C. elegans during embryogenesis.

We thank J. Sulston, Y. Kohara, A. Fire and Y. Ohshima for support. Funds from Monbusho, Japan were provided for grant in aid for basic research to SSS.

24 May 1999 15:50 198 198 In vivo function of kinesins in C elegans neuromuscular system

1999 International Worm Meeting abstract 147 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. In vivo function of kinesins in C elegans neuromuscular system MY Ali, F Hori, AS Mohammed, SS Siddiqui Toyohahsi University of Technology,Japan Kinesins are ubiquitous microtubule based ATPase motors in eukaryotes involved in the transport of a variety of cellular cargo. Although recently much has been known about the crystal strucure and biophysical properties of kinesin proteins; there is a paucity of understanding about the in vivo function of kinesins. Since the C. elegans is well suited for a molecular genetic analysis we have cloned and characterized cDNA clones encoding twenty or so kinesin related proteins (Khan et al., 1997;). Mutations in four previously known genes encoding kinesins produce behavioral deficit. These include the unc-116, unc-104, osm-3 and the vab-8 (Otsuka et al., 1991, Hall and Hedgecock, 1991; Patel et al., 1993; Shakir et al., 1993, Tabish et al., 1995, Wolf et al., 1998; Ali et al., 1999). Using in situ hybridization technique, and a lacZ reporter fusion with unc-116 promoter, we have determined the pattern of expression of the khc , during development. Our data suggest a 341 base sequence in the unc-116 promoter that is required for the expression in the ventral cord axonal processes and motor neurons, and muscles in the body wall, vulva, and pharynx. Expression of unc-116 is observed during embryonic and postembryonic development, remains high in late larval and adult stages, suggesting khc requirement through entire development. The khc is believed to be involved in the transport of mitochondria and other membrane bound vesicles, and moves from minus end to the plus end of the microtubules. Promoter fusion experiments using the unc-104::lacZ gene yielded sterile transgenic animals, and animals that were completely paralysed and uncoordinated. These results suggest that overexpression of the unc-104 affects coordinated behaviour and development and is required for the normal neuromuscular development. Members of the UNC-104 kineisn family include the mouse KIF1A, involved in the fast axonal transport, and is a monomeric kinesin that does not require a second kinesin motor head to move along the MT tracks (Nonaka et al., 1999). We have also discovered two new memebrs of the UNC-104 family, KLP-4, and KLP-6, that are respectively 1576, and 928 aa long monomeric kineisns. In situ hybridization suggest that klp-4 gene is also expressed in the nervous system. We are now studying gene inactivation using RNAi, and deletion mutant analysis to further explore the in vivo function of these novel KLPs.

We thank A. Otsuka and D. T. Mieg for sharing unpublished results and support.

24 May 1999 15:50 199 199 Determining the roles of octopamine and CREB in worm behavior

1999 International Worm Meeting abstract 148 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Determining the roles of octopamine and CREB in worm behavior m alkema, b horvitz HHMI, Dept. Biology, MIT, Cambridge, MA 02139, USA To study processes that may underlie behavioral plasticity, we are analyzing the roles of the biogenic amine octopamine and the CREB protein in C. elegans. Exogenous octopamine inhibits egg-laying, pharyngeal pumping and defecation. Octopamine has been identified in C. elegans extracts but has not been localized to specific cells. Octopamine biosynthesis requires a tyramine ß-hydroxylase (TBH) activity to convert tyramine to octopamine. We isolated a deletion of a putative C. elegans tyramine ß-hydroxylase gene (tbh-1) by screening a chemical deletion library. tbh-1 mutants are viable and have no obvious abnormalities in brood size, egg-laying, chemotaxis, mechanosensation, thermotaxis or dauer formation. tbh-1 mutants move more slowly than wild-type animals and are hypersensitive to exogenous serotonin in locomotion assays. Using immunohistochemistry we have shown that TBH-1 is localized to the cell bodies and neuronal processes of the two RIC interneurons, suggesting that the RIC neurons use octopamine as a neurotransmitter. We are further analyzing the role of octopamine and the RIC neurons in locomotion rate. The cyclic AMP-response element binding protein CREB seems to play a central role in long-term memory in Aplysia, Drosophila and mice. We characterized a C. elegans CREB gene (crb-1). The similarity of CRB-1 to mammalian and Drosophila CREB family members is particularly striking in the predicted DNA-binding bZIP domain and cAMP-dependent kinase site. CRB-1 can bind to cyclic AMP-response element (CRE) sites in vitro. A crb-1::GFP transgene is ubiquitously expressed during early embryogenesis and is specifically expressed in several sensory neurons from the L1 stage to adulthood. We isolated a deletion allele of crb-1 from a chemical deletion library. crb-1 mutant animals are healthy and are being analyzed for behavioral defects.

24 May 1999 15:50 200 200 Biological Roles of Troponin T

1999 International Worm Meeting abstract 149 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Biological Roles of Troponin T T Allen 1 , L Hong 2 , J Ward 2 , A Burkeen 1 , EA Bucher 2

1 Biology Dept, Oberlin College, Oberlin, OH 44074. 2 Dept of Cell and Dev. Biol., U. of Pa., Phila., PA 19104.

The in vivofunctions and interactions of TnT in muscle contraction are poorly understood. Thus, we are studying the four C. elegans TnT genes, which collectively give rise to at least eight distinct transcripts, by comparisons of their encoded proteins, their expression patterns, the individual and combined effects of TnT gene RNAi loss of function, and consequences of site-directed mutagenesis. The mup-2/TnT-1gene codes for a single isoform expressed in embryonic/larval body wall muscles and in the hermaphrodite oviduct. RNAi of TnT-1 phenocopies the mup-2 null embryonic phenotype. The TnT-2gene encodes the major larval and adult body wall isoform. The TnT-3 gene, which is expressed in early embryonic body wall muscle and in pharyngeal muscle and other minor muscle groups, generates at least four distinct transcripts. The TnT-4 gene, which is active in pharyngeal muscle, gives rise to two isoforms differing in the N-terminal region. RNAi of TnT-4 causes L1 arrest, consistent with a likely defect in pharyngeal function (starvation). Comparisons of C. elegans and vertebrate TnTs reveals two highly conserved regions. One is a cluster of charged residues located in the N-terminal region of TnT believed to interact in a calcium-independent manner with tropomyosin. Mutations near this charged cluster in human cardiac TnT are linked with familial hypertrophic cardiomyopathy. There is also a highly conserved heptad repeat of leucines in the C-terminal region, which is thought to allow interaction with troponin I and to be essential for TnT’s role in regulating muscle contraction. Surprisingly, one of the TnT-3 isoforms replaces, through alternative RNA splicing, the heptad repeat with a sequence of about 70 amino acids having some similarity to filaggrin, which binds intermediate filaments, as well as to the Wolf-Hirschhorn Syndrome Critical Region 1 protein (WHSC1), which is expressed in rapidly growing embryonic tissues. The effects in vivo of site-directed mutations within the conserved charged cluster and the heptad repeat of TnT-1 are being examined, and our current data support that mutation of these domains causes dominant hypercontracted phenotypes, consistent with an essential role of these regions in inhibition of tension development.

24 May 1999 15:50 201 201 Analysis of C. elegans host defense response to bacterial pathogen Pseudomonas aeruginosa

1999 International Worm Meeting abstract 150 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of C. elegans host defense response to bacterial pathogen Pseudomonas aeruginosa G Alloing, R Feinbaum, S Mahajan-Miklos, M Tan, FM Ausubel Department of Molecular Biology, Massachusetts General Hospital , Boston, MA Pathogenic bacteria are one of the major causes of disease in both animals and plants. Despite the specificity of the interaction between a bacterial pathogen and its host, there appear to be a number of common mechanisms underlying the process of disease (1,2). On the host side, it has been demonstrated that plants and animals share a set of defense mechanisms involved in "innate immunity". A signal transduction pathway with similarity to the Drosophila developmental regulatory Toll pathway appears to be critical for innate immunity in flies, mammals and plants (1). The underlying similarities between host/bacterial pathogen interactions suggested that a genetically tractable host/pathogen model could greatly improve our understanding of bacterial diseases in mammalian systems. A host/pathogen model system has been developed utilizing C. elegans and PA14, a clinical isolate of P. aeruginosa that is also virulent in Arabidopsis and mouse models. Depending on the medium used for bacterial growth, PA14 kills worms within 24 hours (Fast Killing or FK) or after a few days (Slow Killing or SK). FK is mediated at least in part by diffusible toxins whereas SK requires an infection-like process (3,4). Characterization of several PA14 mutants that are less pathogenic showed that the two killing processes require different bacterial virulence factors. We are interested in understanding the host response to infection by PA14. Towards this end, we have identified the C. elegans homologs of the Drosophila Toll receptor and Pelle kinase and are studying the role of these proteins in response to PA14 in both FK and SK. In addition, six C. elegans mutants have been isolated that are resistant to FK. We are beginning to map and characterize these mutants.

(1) Belvin M.P. and Anderson K.V. (1996) Ann. Rev. Cell Dev. Biol. 12, 393-416 (2) Finlay B.B and S. Falkow, S. (1997). Mol. Biol. Microbiol. Rev. 61, 136-169 (3) Mahajan-Miklos, S. et al (1999). Cell 96, 47-56 (4) Tan, M.-W. et al (1999). PNAS 96, p715-720

24 May 1999 15:50 202 202 Regulation of Cell fusion in C. elegans

1999 International Worm Meeting abstract 151 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Regulation of Cell fusion in C. elegans SD Alper, C Kenyon University of California, San Francisco Cell fusion is a common process in C. elegans. The C. elegans hypodermis consists of several multinucleate syncytia that are generated by the fusion of cells throughout development. The largest such syncytium is hyp7, which spans most of the length of the worm and which contains more than 100 nuclei. We are carrying out two screens to identify mutations affecting cell fusion. First, we screened for mutations that prevent fusion of the Pn.p cells with hyp7 at the end of L1. Mutations identified in this screen affect the decision of these cells to fuse. Second, in order to identify mutations in genes that carry out the cell fusion process, we are screening for mutations that affect the fusion of the seam cells that line the lateral surface of the worm. We identified several mutations that affect the pattern of Pn.p cell fusion and have been characterizing two mutations that affect Pn.p cell fusion by altering Hox protein activity. The fusion decision of the 12 Pn.p cells is controlled by two Hox genes, lin-39 and mab-5. lin-39 is expressed in P(3-8).p and in hermaphrodites prevents fusion of these cells. mab-5 is expressed in P(7-11).p in both sexes, but is not active in hermaphrodite Pn.ps. In ref-1(mu220) (REgulator of Fusion) hermaphrodites, P9.p and P10.p fail to fuse with hyp7. This is due, at least in part, to inappropriate activation of MAB-5 in ref-1 hermaphrodites. ref-1 maps to the center of chromosome II; transformation rescue experiments with cosmids in that region are in progress. In males, lin-39 and mab-5 each individually prevent Pn.p cell fusion in P(3-6).p and P(9-11).p, respectively. However, in P7.p and P8.p, where both Hox genes are expressed in the same cell, they somehow neutralize one another’s activities, so that P7.p and P8.p fuse with hyp7. In ref-2(mu218) males, P7.p and P8.p fail to fuse with hyp7, perhaps because LIN-39 and MAB-5 fail to cancel each others activities. ref-2 maps to the center of the X chromosome. Descendants of the seam cells fuse with hyp7 at all larval stages and ultimately the seam cells fuse with each other during L4. We are using a membrane localized gfp fusion expressed in the seam cells to identify mutants in which seam cell fusions fail to occur. Results of this screen will be presented at the meeting.

24 May 1999 15:50 203 203 Temporal coordination of cell cycle with development in C. elegans larvae.

1999 International Worm Meeting abstract 152 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Temporal coordination of cell cycle with development in C. elegans larvae. VR Ambros 1 , Y Hong 1 , R Lee 1 , R Roy 1,2

1 Department of Biological Sciences, Dartmouth College, Hanover NH 03755. 2 Current address: McGill University, Montreal PQ.

cki-1 encodes a cyclin-dependent kinase inhibitor (CKI) of the p21/p27 family. Analysis of cki-1::GFP expression patterns and cki-1(RNAi) phenotypes suggest that cki-1 mediates the developmental control of the G1/S transition in certain larval cell lineages. Deletion analysis of cki-1 upstream sequences indicates that the developmental expression pattern of cki-1::GFP reflects transcriptional control of cki-1 during the cell cycle. An 8kb segment of cki-1 5’ sequence contains at least five cell type-specific elements that drive expression of cki-1::GFP during G1 in diverse cell types. cki-1 appears to mediate the developmental control of cell cycle progression in many of these cells, including the vulva precursor cells (VPCs). We have investigated how VPC cell cycle progress and cell fate commitment are temporally coordinated. In VPCs, the heterochronic gene pathway, including lin-14, maintains the uncommitted state of VPCs, and acts via cki-1 to hold the VPCs in G1 until the L3. Removing lin-14 activity specifically abolishes cki-1::GFP expression in VPCs. Elevated lin-14 activity (either as a result of a lin-4 mutation, or of VPC-specific expression of transgenic lin-14) causes prolonged cki-1::GFP expression in VPCs and delayed cell division. lin-14(lf) or cki-1(RNAi) animals execute VPC cell divisions in the L2, indicating that wild type VPCs execute G1/S when LIN-14 and CKI-1 decrease sufficiently. Then, as the VPCs re-enter the cell cycle, they acquire competence to express one of three cell fates (1o , 2o or 3o ) that are subsequently determined by Ras and LIN-12 signaling pathways. We examined how the selection of these vulval fates is affected by progression through the VPC cell cycle. Our results suggest that VPCs execute two developmental decisions separable by cell cycle phase: Ras signaling controls a 1o vs 2o cell fate choice before VPC S phase in cells receiving an inductive signal; by contrast, in uninduced VPCs, LIN-12 signaling acts after S phase to control a 2o vs 3o cell fate choice. Coupling developmental decisions to cell cycle transitions may provide a mechanism for prioritizing or ordering choices of cell fates for multipotential cells.

24 May 1999 15:50 204 204 tbx-9 encodes a transcription activator

1999 International Worm Meeting abstract 153 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. tbx-9 encodes a transcription activator Y Andachi Genome Biology Lab., National Institute of Genetics, Mishima 411-8540, Japan tbx-9 is one of T-box family genes that share a DNA binding motif, T-box. Genes of the family are found widely in metazoans, and many of them are involved in important events for development. Three T-box genes, Brachyury (T), VegT and tbx-2, have been shown to encode transcription factors. In C. elegans, the whole genome sequence predicts that this organism has about 20 T-box genes, and cDNA clones corresponding to the predicted genes are being isolated by the cDNA project. As I am interested in the regulation of gene expression, I have been studying one of the T-box genes, CELK02736 = tbx-9, which is the first one among T-box genes whose cDNA clones have been isolated. I previously showed that tbx-9 is expressed in only a few cells of early-stage embryos and tbx-9 deletion mutants generated by gene disruption have defects in morphogenesis of the posterior body region of the embryo. To test whether the tbx-9 protein has a sequence-specific DNA binding activity, the tbx-9 DNA binding sequence was determined as consensus among sequences of DNA fragments that were selected from a mixture of DNA fragments of random sequence by forming complex with the tbx-9 protein. The determined tbx-9 binding sequence was 18 bases in length and had a palindromic structure. The T binding sequence determined by a similar method has been reported to be palindromic, and the binding sequences of T and tbx-9 are almost identical. To know whether the tbx-9 protein is a transcription factor, transactivation of a reporter gene depending on the tbx-9 binding sequence was examined. To this end, the following plasmids were constructed. For forced expression of tbx-9, the tbx-9 gene was ligated downstream of the hsp16-41 heat shock promoter. For assay of transactivation, the tbx-9 binding sequence was inserted upstream of the pes-10 minimal promoter and the reporter lacZ gene. Transgenic lines containing the both plasmids were produced by co-injection. Ectopic expression of tbx-9 by subjecting transgenic embryos to heat shock caused activation of the lacZ gene, indicating that the tbx-9 protein functions as a transcription activator. As the next goal, I am aiming at identifying target genes of tbx-9.

24 May 1999 15:50 205 205 Molecular genetic analysis of daf-12 and related receptors

1999 International Worm Meeting abstract 154 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Molecular genetic analysis of daf-12 and related receptors A Antebi, C Kober-Eisermann Max-Planck-Institut fuer Molekulare Genetik, D-14195 Berlin, Germany. daf-12 encodes a nuclear receptor that regulates the dauer diapause and developmental age. In general, nuclear receptors have a modular structure that includes a variable N-terminus with transactivation function, a pair of C2 Zn fingers comprising the DNA binding domain (DBD), a hinge region of variable length, and a C-terminal ligand binding domain (LBD) that contains dimerization and transactivation helixes. At least three daf-12 isoforms are identified by RT-PCR, including one isoform which contains the LBD but lacks the DBD. Most daf-12 alleles are lesions in DBD or LBD, but mutants can be grouped into 6 phenotypic classes based on their dauer defective phenotypes and their heterochronic phenotypes in gonadal and extragonadal tissues. Candidate molecular null alleles, which disrupt DBD and LBD, are dauer defective and have impenetrant heterochronic phenotypes. By contrast, recessive gain-of-function alleles, which retain the DBD but truncate the LBD, are dauer defective and have penetrant heterochronic phenotypes. We propose that products from gain-of-function alleles interfere with the activity of some other locus. Interestingly, two other nematode receptors related to daf-12,F33D4.1 andZK662.3, are found in the C. elegans genome, and are potential candidates for the proposed redundant activity. All three nematode receptors are homologous to the Drosophila DHR96, revealing that this branch of the nuclear receptor family is phyletically conserved. Like daf-12, F33D4.1 and ZK662.3 give rise to various alternately spliced products. To investigate the function of these genes, we are generating mutations by reverse genetics and constructing GFP fusion proteins.

24 May 1999 15:50 206 206 Three mutants that affect vulva development in C. elegans

1999 International Worm Meeting abstract 155 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Three mutants that affect vulva development in C. elegans I Antoshechkin, R Amaria, M Han HHMI, Department of MCDB, University of colorado, Boulder, CO 80309 Using temperature-sensitive screens, we identified a number of mutations that disrupt vulval development. Here we describe three of these mutations. ku250 is a mutation that disrupts vulva induction at 20 °C. At 25 °C, ku250 worms have severely reduced brood size and a significant sterility. Lineage analysis indicates that the first VPC division is normal, however, the second and third divisions of P5.p, P7.p, or both usually do not occur. In some cases, P6.p also fails to undergo the second and third round of divisions. ku250 can efficiently suppress the Muv phenotype caused by the gain-of-function allele of let-60, n1046, but cannot suppress the Muv phenotype caused by a lin-31 allele indicating that ku250 acts downstream of or in parallel to let-60 and upstream of lin-31. Epistasis analysis with other members of the Ras pathway is in progress. ku250 appears to be a partial loss of function mutation since both vulva lineage and viability defects are significantly stronger when ku250 is placed over a deficiency. ku250 has been mapped to LGIII between unc-32 and sma-3. We are in the process of injecting cosmids to rescue the mutation. ku252 has a phenotype similar to that of ku250. P5.p and P7.p are the most commonly affected lineages, and they are underinduced. Penetrance of the lineage defect, however, is only about 40%, compared to more than 80% in ku250. ku252 worms are also significantly more sick at 15 and 20 °C and practically inviable at 25 °C. At the lower temperatures, worms have a very small brood size and significant sterility. We are currently performing an epistasis analysis to determine ku252 relationship with the Ras pathway. ku252 maps to the right arm of LGI. Fine mapping is in progress. ku254 mutation is not temperature sensitive. Most of ku245 worms undergo normal induction and generate 22 cells that contribute to the vulva. However, the morphogenesis stage in about 60% of worms is abnormal. Many worms fail to make an opening that connects vulva to the uterine lumen. Other animals have a grossly abnormal invagination without a recognizable "christmas tree". High percentage of the animals has gonad migration defects. These do not correlate with the severity of the vulval defects. All ku254 animals are slightly lethargic. ku254 maps to LGX between dpy-6 and unc-18.

24 May 1999 15:50 207 207 Identification and tissue expression of CETMIV, the fourth isoform of the tropomyosin gene in Caenorhabditis elegans

1999 International Worm Meeting abstract 156 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification and tissue expression of CETMIV, the fourth isoform of the tropomyosin gene in Caenorhabditis elegans A Anyanful, Y Sakube, H Kagawa Faculty of Science, Okayama University, 3-1-1 Tsushima Naka, Okayama 700-8530, Japan. The Caenorhabditis elegans tropomyosin gene (tmy-1) locates on chromosome I in the region of the lev-11 gene and encodes three isoforms; CeTMI, CeTMII and CeTMIII by two promoters and alternative splicing. CeTMI and CeTMII are expressed in body wall, anal, vulva and male tail muscles and share a common 5’ upstream promoter. CeTMIII is expressed in the pharyngeal muscles with a promoter region in the third intron. With the elucidation of the fourth isoform (CeTMIV) by the Kohara project, it deemed necessary to also determine its genomic sequence and tissue pattern for similarities or differences with the other isoforms. We used Rapid Amplification of cDNA Ends and microinjection with lacZ and gfp fusion plasmid assay techniques respectively to identify the genome structure and tissue specific expression. We report similarities of transspliced leader RNA, initial ATG and amino acid residues between CeTMIII and CeTMIV and a difference of two exons as a result of change in pattern of alternative splicing. CeTMIV was expressed in muscle cells of the pharynx and gut. Deletion analysis showed that the essential sequence for expression was between 108 - 846 bp upstream. We also show the presence of both the myo-2 enhancer and ges-1 like sequence and propose that the B and C subelements activation of the myo-2 gene may be utilized for pharyngeal expression and the ges-1 like sequence for gut expression. These results indicate the additional expression of CeTMIV in the gut making it quite similar to PHA-4 protein and is the only tropomyosin isoform to be implicated with gut expression. We conclude that C.elegans has only one tropomyosin gene encoding four isoforms and utilizes two each for body muscles. Currently, we are reassessing the tissue expression of CeTMIII with an inclusion of an additional intron and exclusive exon to establish its primary promoter region and an additional tissue expression since this new construct also emcompasses the ges-1 like region.

24 May 1999 15:50 208 208 The UNC-45 protein is a component of muscle thick filaments and co-localizes with myosin heavy chain B, but not myosin heavy chain A.

1999 International Worm Meeting abstract 157 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The UNC-45 protein is a component of muscle thick filaments and co-localizes with myosin heavy chain B, but not myosin heavy chain A. D Pilgrim Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 Mutants in the Caenorhabditis elegans unc-45 gene have disorganized muscle thick filament and reduced thick filament number in body wall muscles. The UNC-45 protein contains three tandem tetratrico-peptide repeats (TPR) motifs and a CRO1/SHE4 domain. Genetic evidence indicates that UNC-45 protein may play a key role in the muscle thick filament assembly. Green-fluorescent protein (GFP) reporter fusion shows expression in all the muscle cells examined and limited only to muscle cells. In the body wall muscles, a functional UNC-45-GFP fusion protein is localized to the A-bands of thick filaments compared with the pattern under polarized light microscopy. To confirm this, polyclonal antibodies against UNC-45 were generated and immunofluorescence studies using these antibodies demonstrated that UNC-45 protein co-localizes with myosin heavy chain B, but not myosin heavy chain A, in the thick filaments in both wildtype and unc-45 (ts) mutant nematodes. Moreover, UNC-45 protein has also been shown to be expressed in the premorphogenesis embryos and may be assembled to the thick filaments after myosin heavy chain isoforms. Based on these results, we propose that the UNC-45 protein is a structural component of muscle thick filaments and co-localizes with myosin heavy chain B in the body wall muscles of the nematode.

We would like to thank Dr. D. Miller for providing antibodies against myosin heavy chain isoforms and Dr. A. Fire for GFP vectors.

24 May 1999 15:50 209 209 Nucleoside transporters in C. elegans

1999 International Worm Meeting abstract 158 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Nucleoside transporters in C. elegans PJ Appleford 1,2 , M Griffiths 2 , SA Baldwin 2 , EG Chomey 3 , SY Yao 3 , D MacGregor 1 , RE Isaac 1 , D Coates 1 , CE Cass 3 , JD Young 3

1 School of Biology, University of Leeds. 2 School of Biochemistry & Molecular Biology, University of Leeds. 3 Depts. Physiology and Oncology, University of Alberta.

Nucleoside transporters (NTs) are essential for nucleotide synthesis by salvage pathways in cells which lack de novo biosynthetic pathways and have important roles in adenosine-mediated processes in mammals (e.g. neurotransmission, platelet aggregation and coronary vasodilation) by modulating levels of extracellular adenosine. NTs are classified into equilibrative (ENT) and concentrative (CNT) transporter families. ENTs are widely distributed in mammalian tissues and can be further divided into two subclasses on the basis of their sensitivity to the transport inhibitor nitrobenzylthioinosine (NBMPR). Recently, cDNAs encoding ENTs of both NBMPR-sensitive and NBMPR-insensitive subclasses have been cloned from human and rat tissues. The ENT proteins are predicted to contain 11 membrane-spanning alpha helices and constitute a novel family of transporters. A search of the C. elegans genome has identified 6 putative nematode ENT genes encoding proteins with between 18-29% sequence identity to mammalian ENTs and 11 predicted membrane-spanning alpha helices. Five of the predicted proteins contain the sequence motif G-X-G-T/X-L-L/M-P-W-N, characteristic of the ENT family, in the first membrane-spanning domain. To date, cDNA for one of the C. elegans genes has been expressed in Xenopus oocytes and shown to mediate saturable uptake of a broad range of purine and pyrimidine nucleosides, confirming the identity of this nematode protein as a functional ENT.

24 May 1999 15:50 210 210 Molecular investigation of smg-4, required for mRNA surveillance in C. elegans

1999 International Worm Meeting abstract 159 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Molecular investigation of smg-4, required for mRNA surveillance in C. elegans R Aronoff 1 , R Baran 2 , J Hodgkin 3

1 Max-Planck Inst. for Med. Res., Molecular Neurobiology, Heidelberg, D-69120 GERMANY. 2 Dept. of Biology, Sinsheimer Laboratories, UCSC, Santa Cruz, CA 95064, USA. 3 MRC-LMB, Hills Road, Cambridge CB2 2QH, ENGLAND.

Premature translational stop codons in messenger RNA can trigger a process in all eukaryotes known as nonsense mediated decay (NMD) or mRNA surveillance. Studies in C. eleganshave shown that this system can prevent the accumulation of potentially toxic, truncated peptides (Pulak and Anderson, 1993, Genes and Dev. 7,1885-1897). At least seven genes in the nematode, smg-1 through smg-7, are required for mRNA surveillance. We have cloned smg-4by physical mapping and functional rescue assays. Gene truncation and disruption experiments have defined a minimal rescuing fragment. This includes a sequence encoding a predicted protein of 368aa, with a possible bipartite nuclear localization signal and no significant homologs in current databases. Disruption of this protein coding region within the second exon, by a frameshift or deletion of just two amino acids, abolishes rescue activity. Analysis of cDNAs shows that the gene can undergo alternative splicing, leading to the production of a truncated protein lacking a carboxy-terminal domain with limited sequence similarity to a microtubule associated protein. Transcripts from the gene are transpliced with SL2, consistent with its genomic location as the downstream member of an operon. A puzzling feature of smg-4is its very low forward mutation frequency, both in general smg screens (Cali et al., 1999, Genetics 151:605-616) and in our non-complementation screens. Molecular changes have been found in all three known alleles, one of which (r1169) is a large deletion that affects both the gene described above, and the upstream gene in the operon. This raises the possibility of functional redundancy within the locus. The two other alleles (r1181and ma116) affect respectively the donor and acceptor splice sites flanking the first intron of the downstream gene; however, additional changes in the upstream gene have not yet been excluded. RNA from this first intron could form a stable secondary structure, with a loop that exhibits complementarity to stop codons. Possible explanations to unify these findings will be presented.

24 May 1999 15:50 211 211 RNA and Predicted Ionotropic Glutamate Receptors in the C. elegans Genome

1999 International Worm Meeting abstract 160 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. RNA and Predicted Ionotropic Glutamate Receptors in the C. elegans Genome R Aronoff 1 , S Binnenbose 1 , C Petersen 2 , P Seeburg 1 , R Sprengel 1

1 Max-Planck Institute for Medical Research, Molecular Neurobiology, Jahnstrasse 29, D-69120, Heidelberg, Germany. 2 Max-Planck Institute for Medical Research, Cell Physiology, Jahnstrasse 29, D-69120, Heidelberg, Germany.

The genes glr-1, nmr-1 and nmr-2 are only a few of the predicted ionotropic glutamate receptor (GluR) -related sequences in the worm genome. We have begun to characterize more of these predicted genes with the aim of not only learning about the vertebrate genes by examining these orthologues in this simple genetic system, but going from gene to behavioral phenotype in the animal. Ultimately we hope to use smg -dependent dominant negative and RNAi strategies to achieve this end. Initially, we are examining predicted genes with defined ests which are most similar to vertebrate AMPA or Kainate receptors. In particular we have focused on sites of these which are analogous to those which control ion permeability and channel kinetics in the vertebrate GluRs, the ’editing’ sites, modified post-transcriptionally by RNA editing. Similar to the glr-1 cDNA, we have found by RT-PCR that the major cDNAs for two of these genes (K04G7.2/B0280.12, tentatively denoted glr-2 and C06A8.9/.10, glr-3) exhibit no editing of the ’Q/R’ site that modulates ion permeability (1). The significance of GluR channels in the worm which might allow unimpeded influx of divalent cations remains to be determined. The kinetically important R/G site is currently being investigated. These studies have brought to light the fact that glr-2 is the host gene for an alternative trans-spliced leader RNA operon. This gene cluster is intronically encoded, associated with an extended inverted repeat, and possibly transcribed from promoters on either genomic strand (2). In addition, glr-2 may itself be alternatively spliced, based upon alignments of genomic and est sequences. This putative alternative splicing does not include the region carrying the trans-spliced leader genes, and is not analogous to the ’flip/flop’domain of the vertebrate GluRs. Rather, it would alter a more N-terminal domain, perhaps affecting ligand binding or other functions of this molecule. Further analysis of the genomic organization and cDNAs expressed from glr-2 will be presented.

1)Sommer, B. et al. (1990) Science 249: 1580-1585. 2)Ross, L. H. et al. (1995) JBC 270: 22066-22075.

24 May 1999 15:50 212 212 Homologues of claudin, integral protein of mammalian tight junction, are present and important in C. elegans

1999 International Worm Meeting abstract 161 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Homologues of claudin, integral protein of mammalian tight junction, are present and important in C. elegans A Asano 1 , K Asano 1 , M Furuse 2 , H Sasaki 2 , S Tsukita 1,2

1 Tsukita Cell Axis Project, JST. 2 Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto, Japan.

Recently, new integral proteins of tight junction were discovered in mouse and human (Furuse et al., J. Cell Biol., 141, 1539, 1998; Morita et al., PNAS, 96, 511, 1999). These claudin family proteins are members of tight junction strands. Although presence of tight junctions in C. elegans is not reported, septate junctions and septate-like junctions seem to play similar functions instead. We searched the gene database of C. elegans, and found two homologues of claudin family proteins (claudin-CE1 and -CE2) with four-transmembrane domains, conserved two Cys in the first loop, and similar molecular weight. Interestingly, a protein (claudinD) was also found that has molecular weight about twice of claudin-CE1, and other characteristic structures are likely to have two claudin molecules tandemly repeated. These 3 proteins are coded from nearby sites on chromosome X. Claudin-CE1::GFP with 1.2kb upstream promoter region was expressed in spermatheca which is known to have septate junctions, and gut. Expression of claudin-CE2::GFP was much less, but tissue distribution was similar. RNAi experiments using dsRNA mixture of claudin-CE1, claudin-CE2 and Exon1-4 fragment of claudinD were performed. About 40% of F1 of the injected worms have decreased F2 production (in average 48% decrease), whereas 22% of F1 have almost normal numbers of F2’s. Thus, these proteins seem to be important for reproduction of the worms. When expressed in MDCK-II epithelial cells, Claudin-CE1::GFP was localized at cell-cell junctions. Electron microscopic studies are under way.

We are grateful to Miss. Akiko Kamamoto whose technical assistance make this work possible.

24 May 1999 15:50 213 213 A Cell Cycle Regulator in the Germ Line

1999 International Worm Meeting abstract 162 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A Cell Cycle Regulator in the Germ Line NR Ashcroft, A Golden ABL-Basic Research Program, FCRDC, Frederick, MD 21702 Cyclin dependent kinases control cellular events throughout different stages of the cell cycle. One of these kinases, CDC2, regulates the cell’s entry into mitosis. CDC2 itself is regulated by a dual specificity phosphatase called CDC25. This phosphatase removes inhibitory phosphates from CDC2, thus driving the cell into M-phase. Whereas other organisms are known to have between one and three cdc25 genes, four cdc25 homologues have been identified in the C. elegans genome (named cdc-25.1, cdc-25.2, cdc-25.3 and cdc-25.4). We have described the expression pattern of cdc-25.1 using an affinity purified antibody. CDC-25.1 was localized to maturing oocytes, and was observed in the nuclei and cortical membranes of young embryos. CDC-25.1 was also found in the larval germ line precursor cells, Z2 and Z3. Disruption of cdc-25.1 expression using RNAi resulted in the majority of embryos from injected hermaphrodites failing to hatch. These embryos were aneuploid. Further analysis suggested that this aneuploidy was due to aberrant meioses after the oocytes were fertilized. However, the few embryos treated with RNAi that did hatch developed into agametic Glp-like adults. This same sterile phenotype was observed in a molecular null cdc-25.1 mutant (nr2036 allele generated by Nemapharm, Inc.). Both hermaphrodites and males were sterile. The somatic gonad appeared to develop normally, but the proliferating germ cells died after the L3 larval stage - a time when wild type germ cells enter meiosis. Transgenes containing wild type copies of cdc-25.1 weakly rescued the sterility. Point mutations were introduced into the CDC-25.1 phosphatase domain and these transgenes behaved like dominant negatives. Further analysis of the cdc-25.1 mutant will be presented at the meeting.

Research sponsored by the National Cancer Institute, DHHS, under contract with ABL.

24 May 1999 15:50 214 214 Identification of fork head transcription factors expressed during C. elegans embryogenesis.

1999 International Worm Meeting abstract 163 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification of fork head transcription factors expressed during C. elegans embryogenesis. S Aslam, A Mounsey, P Bauer, C Royall, IA Hope School of Biology, University of Leeds, Leeds, LS2 9JT, UK. The pes-1 gene was identified in a promoter trapping screen for C. elegans genes expressed in specific cell lineages during early embryogenesis. The predicted PES-1 proteins have homology to the fork head family of transcription factors which includes members from vertebrates, Drosophila and yeast. Most amino acid identity is in a 60 amino acid domain which has been shown to have DNA-binding activity. This suggests that the fork head gene family encodes transcription factors involved in the developmental control of other genes. We have sought to identify fork head transcription factors which could function to control events in early C. elegans embryogenesis by identifying those expressed at this stage of development. The C. elegans genome sequencing project has provided the entire complement of fork head genes in the genome. Eleven novel fork head genes have been revealed in addition to the four previously identified. Reporter gene constructs have been made for ten of these eleven genes, five of which show embryonic expression or an embryonic component in their expression pattern. These embryonic expression patterns are now being characterized in terms of the cell lineage. We plan to examine the function of the embryonically expressed fork head genes using RNA interference.

24 May 1999 15:50 215 215 Does the ortholog of the brain development genes ems/EMX have a function in C. elegans?

1999 International Worm Meeting abstract 164 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Does the ortholog of the brain development genes ems/EMX have a function in C. elegans? G Aspöck, TR Bürglin Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel The Drosophila empty spiracles (ems) and the vertebrate Emx1 and Emx2 genes define brain regions in a way that brain parts expressing these genes are largely missing in loss-of function mutants. Additionally, ems is involved in tracheal development, and Emx2 knockout mice completely lack a urogenital system. Bürglin et al. (1989) first identified ceh-2 in a screen for C. elegans homeobox genes. With the ems/Emx genes ceh-2 shares a hexapeptide, 80% identity in the homeodomain and flanking regions as well as an intron at a conserved position. However, the function of ceh-2 does not seem to be comparable to ems/Emx: Anti-peptide antibody staining and expression of gfp/lacZ reporter constructs are restricted to a subset of cells in the pharynx, muscle m2, epithelial e2, and the neurons I3, M3 l/r, and NSM l/r. In an EMS mutagenesis screen we isolated ceh-2(ch4), a 2.5 kb deletion that covers the hexapeptide and homeodomain. The mutant is perfectly viable and fertile and shows no obvious phenotype. What might ceh-2 do? Among the ceh-2 expressing cells, M3 and NSM have been described in more detail. M3 l/r are inhibitory glutamatergic neurons that controls pharynx muscle relaxation. Laser ablation of M3 causes the pharynx muscle to relax more slowly after contraction, a pump cycle is about 10% longer and the electropharyngeogram lacks several inhibitory post-synaptic potentials (Avery, 1993; Raizen and Avery, 1994). Although ceh-2(ch4) mutants do not look starved as M3 ablated animals do, it may be useful to test for M3 activity in ceh-2(ch4). NSM l/r contain serotonine and stimulate pumping; we will test ceh-2(ch4) for corresponding defects. In addition we will express ceh-2 in Drosophila ems mutants to test whether ceh-2 can rescue some of the fly phenotypes.

References: Avery (1993) J. Exp. Biol. 175:283-297 (and many WBG abstracts) Bürglin et al. (1989) Nature 341:239-243 Raizen and Avery (1994) Neuron 12:483-495

24 May 1999 15:50 216 216 A second look at warthog and groundhog genes

1999 International Worm Meeting abstract 165 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A second look at warthog and groundhog genes G Aspöck, H Kagoshima, TR Bürglin Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel The hedgehog family of signaling glycoproteins have many essential functions in animal development. They are secreted by "posterior" cells in fly parasegments, wing discs, leg discs and vertebrate limb buds and also define subgroups of neurons in the central nervous systems of flies and vertebrates. Hedgehog proteins consist of two domains: the carboxyterminal autoprotease/ cholesterol transferase cleaves off the secreted amino-terminal signal and anchors it to a cholesterol moiety, thus to the membrane. C. elegans apparently does not have a hedgehog homolog (although there is a slim chance that it has not been sequenced yet). However, several C. elegans genes contain the carboxy-terminal autoprotease domain. We call it hog domain; it is also referred to as Hint domain because of the similarity to inteins, bacterial self-splicing proteins (Porter et al.,1996). The hog domains are associated with novel amino-terminal sequences that are up to now unique to nematodes. Using wart and ground as names for these domains C. elegans has five warthog and three goundhog genes. One gene has an orphan amino-terminus (quahog), and there is a presumed pseudo-gene, consisting only of a hog domain. Additionally there are 5 wart-only and 12 ground-only genes, 2 ground-derived genes, and 28 ground-like genes which all lack a hog domain. wart, warthog, ground, and ground-like homologues are also present in parasitic nematodes. Here we present sequence comparisons, gfp expression patterns and some RNAi experiments of wrt and grd genes. Several wrt gfp reporter constructs are expressed in the hypodermal syncytia; expressions of other wrt genes include seam cells, socket and sheath cells of anterior and phasmid sensilla, gonad sheath cells, spermatheca, uterus, the pharyngeal gland cell g1, the excretory cell, pharyngeo-intestinal valve cells and rectal cells. Only three grd reporter constructs are expressed; we find gfp in rectal cells, motor and other neurons.

References: Porter et al. (1996). Cell 86:21-34. Bürglin (1996) Curr. Biol. 6:1047-1050

24 May 1999 15:50 217 217 Complete closure of the anterior and posterior hypodermis needs the C. elegans homeobox gene ceh-43.

1999 International Worm Meeting abstract 166 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Complete closure of the anterior and posterior hypodermis needs the C. elegans homeobox gene ceh-43. G Aspöck, TR Bürglin Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel The genome sequencing project sequenced a close ortholog of the Drosophila Distalless (Dll) and the vertebrate Dlx genes with 73% amino acid identity and a conserved intron position in the homeodomain (Stock et al.,1996; Wilson et al., 1994). Distalless/Dlx genes function in nervous system development and the distal outgrowth of appendages (antennae, limbs, legs etc.) in many species (Panganiban et al., 1997 and references therein). ceh-43::gfp reporter constructs are first expressed in some (not all) ABxxxxx cells and later in neuronal precursors and neurons of the head and tail, occasionally also in syncytial hypodermis. The expression matches with the staining of the antibody against the Drosophila DLL protein (Panganiban et al., 1997). RNAi using a cDNA clone kindly provided by Yuji Kohara is 100% embryonic or L1 lethal. Embryos develop normally until the 1.5x stage; subsequently, cells emerge from the buccal cavity and sometimes the tail, resulting in rupture of the embryo. In the few embryos that manage to hatch the pharynx and intestine are completely detached from the hypodermis. ceh-43::gfp expressing neurons and axons look normal in these animals. We believe that the RNAi phenotype is specific because two deficiencies that cover the gene show the described phenotypes. We think that the embryonic phenotype may either be due to incomplete closure of the anterior and posterior hypodermis, or because these points are too weak to withstand the internal pressure exerted during morphogenesis. jam-1::gfp (kindly provided by Jeff Hardin) expression shows that the hypodermal cell sheet looks normal before embryos rupture. The larval attachment problem might arise from a lack or weakness of the same cell-cell or cell-matrix adhesion molecules or mechanisms.

References: Panganiban et al.(1997). Proc Natl Acad Sci USA 94:5162-6 Stock et al.(1996). Proc Natl Acad Sci USA 93:10858-63 Wilson et al.(1994). Nature 368:32-38

24 May 1999 15:50 218 218 The regulator hypothesis revisited: CAMs, IgSFs, ZIGs and tissue patterning

1999 International Worm Meeting abstract 167 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The regulator hypothesis revisited: CAMs, IgSFs, ZIGs and tissue patterning ON Aurelio, J Zhu, O Hobert Columbia University, College of Physicians & Surgeons, New York, NY10032 The regulator hypothesis proposed by G.Edelman 15 years ago posits that a defined and rather limited number of cell adhesion molecules (CAMs) provides an area code for tissue-specific morphogenetic event (Edelman, 1984). This hypothesis was later expanded to include pattern formation in the brain, including the generation of specific neural circuits. With the completed genome sequence we are now in the unique position to analyze the full complement of CAM genes in an organism and can address whether the expression of CAM genes follows the prediction of the regulator hypothesis. We have begun to analyze one CAM superfamily, the immunoglobulin-domain (IgSF) superfamily. An implicit posit of the regulator hypothesis is its requirement of only a very small set of CAMs, presumably from the same family, so that similar, yet cell-specific recognition mechanisms can be utilized in a temporally and spatially tightly regulated manner. As such, any small subfamily of putative CAMs are attractive candidates to provide an address code for patterning events. Within the IgSF superfamily we indeed find a small subfamily, containing 8 members, which we termed zig genes. Their protein products are composed exclusively of 2 Ig domains, a signal sequence, a GPI anchor site and no transmembrane domain. We have generated GFP fusions to all 8 zig genes and found most of them to be strongly expressed in both overlapping and distinct tissues types; e.g. zig-1 is expressed exclusively throughout most of the cells of the nervous system; zig-7 is expressed almost exclusively in muscles. Yet the expression of other zig genes, such as zig-4 and zig-5 is restricted to a very limited set of head and tail neurons. Several aspects of these expression pattern, i.e. complete tissue type expression, are in agreement with the ZIG proteins being involved in delineating tissue types and confering a tissue-wide identity to the cells surface; other aspects of expression however argue for more cell-specific roles of ZIG proteins.

24 May 1999 15:50 219 219 Genetic Mapping of Multiple Loci Determining Life Span in C. elegans

1999 International Worm Meeting abstract 168 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic Mapping of Multiple Loci Determining Life Span in C. elegans S Ayyadevara, JJ Thaden, RJ Shmookler Reis Depts. of Medicine, Geriatrics, and Biochemistry & Molec. Biology, Univ. of Arkansas for Med. Sci. and VA Medical Ctr., Little Rock, AR 72205 We conducted two new inter-strain crosses, between C. elegans strains Bergerac-BO and either CL2a or RC301, and created a heterogeneous recombinant inbred (RI) population from each, for a total of four crosses constructed and analyzed to date. The longest-lived 1% of worms in each RI population and comparable numbers of unselected young worms were genotyped. Data were then analyzed by maximum-likelihood scanning using both composite interval mapping and nonparametric interval mapping. We have now observed a total of 12 loci with highly significant LOD scores (10 in the present two crosses), each well above the p<0.05 threshold for genome-wide scans. Since genetic mapping in interstrain crosses can only detect loci which are dimorphic in the parental strains, each cross can identify a subset of the total life-span affecting loci. From the number of loci shared by multiple inter-strain crosses, we estimate that C. elegans harbors only a few dozen polymorphic genes with a comparable effect on life span to the 12 QTLs we have found. We have now initiated back-crosses to construct multiple congenic (near-isogenic) lines for each QTL region in a parent of contrasting phenotype. Several of these back-crossed lines have been tested for longevity and differ significantly from the recurrent parent.

Supported by NIH grant AG-09413.

24 May 1999 15:50 220 220 A quantitative trait locus for body size

1999 International Worm Meeting abstract 169 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A quantitative trait locus for body size RB Azevedo, CG Knight, AM Leroi Department of Biology, Imperial College, Silwood Park, Berks SL57PY, UK Bristol N2 is about 40% larger than Bergerac RW7000 in adult hermaphrodite body length. We have measured body size at 96h in 73 recombinant inbred lines (RILs), resulting from a cross between N2 and RW7000 performed by T. Johnson. These data were analysed for the presence of quantitative trait loci QTLs by interval mapping on the genotypes for 31 Tc1 markers. The analyses revealled one highly significant QTL for body size on chromosome IV, accounting for 25% of the phenotypic variance among RILs between N2 and RW7000. We present evidence that this QTL affects final adult body size, not growth rate. The 95% confidence interval for the body size QTL calculated by bootstrapping ranges from +1.7 to +9.0cM, a region spanning approximately 4.5Mb. This confidence interval for the QTL overlaps with that of a published QTL for fertility. We have identified six candidate body size loci, affected by Tc1 transposon insertions in RW7000 but not N2, within the QTL region. These include unc-30 and deb-1. A cloning strategy for the QTL is discussed.

24 May 1999 15:50 221 221 Analysis of a temperature sensitive muation in zen-4 kinesin like protein reveals a requirement during ventral enclosure

1999 International Worm Meeting abstract 170 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of a temperature sensitive muation in zen-4 kinesin like protein reveals a requirement during ventral enclosure TM Baas 1 , W Raich 2 , D Hamill 3 , JD Hardin 1

1 Department of Zoology, University of Wisconsin at Madison. 2 Program in Cellular and Molecular Biology, University of Wisconsin at Madison. 3 Institute of Molecular Biology, University of Oregon.

ZEN-4 is a member of the MKLP-1 subfamily of kinesin motor proteins which bundle antiparallel microtubules and localize to the equatorial region of the spindle midzone in mitotic cells. Embryos homozygous for the null allele the zen-4 (w35) fail to complete ventral enclosure, during which the hypodermis normally migrates bilaterally to meet at the ventral midline. zen-4(w35) homozygotes produce the normal number of hypodermal cells but lose bilateral symmetry of the migrating epithelial sheet; posterior hypodermal cells migrate concurrently with or in advance of anterior leading edge cells. Using the temperature-sensitive allele zen-4(or153) (kindly supplied by D. Hamill and B. Bowerman, Univ. of Oregon) we determined when the protein is required during ventral enclosure. Embryos were shifted from the permissive temperature (15° C) to the restrictive temperature (20° C) and observed four hours later. Embryos shifted before initiation of the migration of the leading edge cells fail to enclose, whereas those shifted after migration has begun enclose and continue to develop normally through hatching. To determine the kinetics of inactivation of the temperature-sensitive protein, embryos at the two-cell stage were shifted and observed. All cell divisions following the shift were incomplete and the cells became mulinucleate indicating that the protein is inactivated within one to two minutes after the shift to the restrictive temperature. These results indicate that ZEN-4 is required for the initiation of ventral enclosure but not subsequent hypodermal morphogenesis in the embryo.

24 May 1999 15:50 222 222 Cloning and Characterization of the Cytokinesis mutant stu-4.

1999 International Worm Meeting abstract 171 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cloning and Characterization of the Cytokinesis mutant stu-4. AS Badrinath, JG White University of Wisconsin, Madison Madison, WI 53706 We are characterizing the temperature-sensitive mutant stu-4(e2406) (originally isolated by David Livingstone). When gravid adults are shifted to the non-permissive temperature 100% of the embryos die due to maternal effect lethality. We examined the cause of this lethality using 4-D time-lapse (Nomarski) microscopy. The polar bodies fail to be extruded resulting in several ooctye ’pronuclei’ migrating toward the posteriorly located sperm pronucleus where they generally meet at the center of the embryo. Spindle formation, and reformation of the nuclei following chromosome segregation appears to be normal, but at no time point is there any sign of furrowing at the cortex. This results in a multi-nucleated single celled embryo. When L2 or L3 larval stage e2406 animals are shifted to the non-permissive temperature 100% of them become sterile and exhibit an uncoordinated phenotype to a lesser extent. These defects are presumably due to failures in cell divisions in the post-embryonic lineages giving rise to the gonad and ventral nerve cord respectively. We have mapped stu-4to - 3.20 on LG I between ace-2and unc-11. The deficiency qDf3 uncovered stu-4whereas qDf4 did not. We previously reported that the cosmid K03E5 was able to rescued stu-4(WBG 15(3): 26). Currently, we are testing this result more rigorously as well as checking other YACs and cosmids in the region. To confirm that this gene corresponds to stu-4we would like to sequence e2406, and further alleles which we are currently in the process of recovering. In a non-complementation screen singled F1 males from EMS mutagenized him-8hermaphrodites were crossed to stu-4(e2406) dpy 5(e61)/szT1 hermaphrodites and we looked for the presence of non-dpy sterile progeny. From 740 haploid genomes screened we have obtained one additional allele which we are currently in the process of backcrossing.

24 May 1999 15:50 223 223 Associatiions of Caenorhabditis species with terrestrial isopods.

1999 International Worm Meeting abstract 172 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Associatiions of Caenorhabditis species with terrestrial isopods. SE Baird Department of Biological Sciences, Wright State University, Dayton OH Many rhabditid nematodes use other soil invertebrates for transport between microenvironments. These phoretic associations form as dauer larvae and typically have no ill-effects on the host animal. Caenorhabditis remanei has been reported as a phoretic associate of terrestrial isopods (Armadillidium vulgare and A. nasatum) and of snails (Oxychilus cellarius). However, these associations were reported from anthropogenic environments (compost heaps) and may not accurately reflect the natural history of C. remanei. Here, associations of C. remanei and with woodland isopods are reported. Five species of isopods, Trachelipus rathkii, Cylisticus convexus, Porcellio scaber, Porcellio spinicornis, and A. nasatum, have been obtained from woodland habitats in and around Dayton Ohio. C. remanei has been found in association with four of these species, T. rathkii, C. convexus, P. scaber, and A. nasatum. In all cases, associated nematodes were dauer larvae. The absence of infesting nematodes in in P. spinicornis is significant. These isopods were found intermingled with infested isopods of other species and the probability of not detecting infesting nematodes in P. spinicornis due to sampling error is less than 0.01. Experiments to identify the cause of this host preference are under way as are additional collections to determine the geographic extent of C. remanei - isopod associations. et tu elegans? In the course of these studies, various isopods were obtained from commercial sources. From two different sources, C. elegans-infested populations of P. scaber were obtained. Local populations of P. scaber are being examined for C. elegans infestations.

24 May 1999 15:50 224 224 Genetic and electrophysiological analysis defines the composition of a native GABA receptor in C. elegans

1999 International Worm Meeting abstract 173 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic and electrophysiological analysis defines the composition of a native GABA receptor in C. elegans BA Bamber, J Richmond, EM Jorgensen Dept. Biol., Univ. of Utah, Salt Lake City, UT 84112. What is the subunit composition of a neurotransmitter receptor at a synapse? This question has been difficult to answer because the necessary electrophysiological and genetic experiments are often impossible to perform in the same organism. We have combined genetic and electrophysiological analyses in C. elegans to define the structure of a native GABA receptor. The unc-49 locus encodes three full-length GABA receptor subunits. We hypothesized that two these subunits, UNC-49B and UNC-49C, form a heteromeric GABA receptor in vivo because 1) UNC-49B and UNC-49C GFP translational fusions co-localize at the neuromuscular junction; and 2) UNC-49C::GFP localization to synapses requires UNC-49B, suggesting a physical interaction between the two subunits. One prediction of our hypothesis is that UNC-49B and UNC-49C should form a heteromeric receptor when co-expressed in heterologous cells. To test this prediction, we expressed UNC-49B in Xenopus oocytes either alone or in combination with UNC-49C. Our results showed that the two subunits could form a heteromeric receptor: UNC-49B alone formed a homomeric GABA receptor which showed strong competitive block by picrotoxin (PTX), and weak inhibition by the neurosteroid pregnenolone sulfate (PS). By contrast, UNC-49B and UNC-49C together formed a GABA receptor which showed weak, non-competitive PTX block and strong PS inhibition. Electrophysiological analyses of the GABA receptor in vivo indicate that UNC-49B and UNC-49C comprise the native GABA receptor. First, patch clamp analysis of wild-type C. elegans muscle cells revealed a GABA receptor with PTX and PS sensitivities which correspond to the UNC-49B/C heteromer. Second, we demonstrated that the pharmacological properties of the wild-type receptor require both UNC-49B and UNC-49C. Muscles of null mutants were completely unresponsive to GABA. Animals expressing UNC-49B alone, though phenotypically wild-type, showed GABA-evoked currents which resembled the UNC-49B homomer. Only when UNC-49B and UNC-49C were expressed together was wild-type PTX and PS responsiveness restored. Taken together, these results define the GABA receptor at the C. elegans neuromuscular junction as an UNC-49B/C heteromer.

24 May 1999 15:50 225 225 Transcriptional regulation of sex specific bi-directional promoter between male tail collagen gene and sperm specific protein gene in Caenorhabditis elegans

1999 International Worm Meeting abstract 174 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Transcriptional regulation of sex specific bi-directional promoter between male tail collagen gene and sperm specific protein gene in Caenorhabditis elegans T Bando, I Tatsuji, H Kagawa Department of Biology, Faculty of Science, Okayama University, Okayama 700-8530, JAPAN Gene expression control in tissue, stage and sex specificities is one of the most interesting subjects. We found SPE-17-like protein gene (designated spe-TI) and one of cuticle collagen gene (designated col-TI) in the cosmid clone C46C8 mapped in the center of chromosome V of C. elegans. SPE-TI is a Ser/Thr rich hydrophilic protein. COL-TI belongs to the cuticular collagen family similar to SQT-3 (COL-1) at the rate of 84%. Both genes are separated by 1.3 kb and transcribed bi-directionally. It is of interest to know how and what kind of factors control the gene expression of both. Using the promoter/lacZ fusion plasmid techniques, we analyzed the promoter activity of each genes. spe-TI expressed in 80 cells of spermatheca after 4th larva stage of hermaphrodite and col-TI expressed only in the male tail tip of adult stage. Heat shock promoter driven spe-TI antisense RNA reduced the brood size at about half compared with heat shocked wild type(N2). Unidirectional deletion analysis indicated that the essential sequence of spe-TI expression was over 672bp from spe-TI initial codon, and male and cell lineage-specific sequence of col-TI expression was over 610bp from col-TI initial codon but essential sequence was in 510bp. These results suggest that both promoters overlap to each other. We also found the MAB-18 (vertebrate Pax-6 homologue) binding sequence on 1.0 kb upstream of col-TI and some MAB-3 (Drosophila dsx homologue) and vertebrate spermatogenesis specific transcription factor SRY/SOX-5 (C. elegans cog-2 homologue) binding sequence between both genes. Interestingly col-TI/lacZ fusion gene was expressed in transformed animal of mab-5 and lin-29 but not in mab-18 mutant background. These results suggest that CeSRY/SOX-5 and MAB-18 stimulate spe-TI and col-TI respectively together with MAB-3, and these factors regulate the transcriptional directions by sex-specific style. Now we would like to try binding experiments between MAB-18 and col-TI promoter in vitro. We believe that the study of transcriptional regulation in this bi-directional promoter is a gateway into understanding the sex-specific morphological changes in terminal differentiation.

24 May 1999 15:50 226 226 Characterization of Calcineurin, a Ser/Thr protein phosphatase, in C. elegans

1999 International Worm Meeting abstract 175 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of Calcineurin, a Ser/Thr protein phosphatase, in C. elegans J Bandyopadhyay, J Lee, J Ahnn Dept. of Life Science, Kwangju Institute of Science & Technology, Kwangju 500-712, Korea Calcineurin, a major CaM binding protein in brain, is a heterodimer of a catalytic subunit (58-64 kDa), calcineurin A (CnA), and a Ca2+ binding regulatory subunit (19 kDa), calcineurin B (CnB). It is classified as protein phosphatase 2B and is the only serine/threonine protein phosphatase under the control of Ca2+ /CaM. It plays a critical role in the coupling of Ca2+ signal to cellular responses, and as well as, in the regulation of neuronal function. While the overall sequence of CnA from different organisms is more divergent, phylogenetic studies reveal that the protein structure of CnB and the regions of CnA that play a critical role in the activity of the phosphatase, are conserved from yeast to man. Since we were interested to identify and characterize C. elegans calcineurin and study its function, we searched the worm genome database which revealed the presence of a predicted CnA and CnB (we termed CeCnA and CeCnB) on the cosmids, C02F4 (LGIV) and F55C10 (LGV), respectively. CeCnA displays approximately 77% sequence identity to mammalian CnA, and CeCnB has about 80% sequence identity with mammalian and Drosophila homologues. Interestingly, the predicted CeCnB is approximately 40 kDa in contrast to the 19 kDa CnB in other organisms. We identified CeCnA expressing strongly in neuronal cells, body wall muscles and vulva muscles as evidenced by the GFP expression pattern. Through Kohara’s EST bank, several clones that represent CeCnA cDNAs were found which will be used to confirm the gene structure by northern and Southern analysis, and as well as for antibody preparation. To understand the biological role of calcineurin in C. elegans, RNAi technology will be utilized to understand the phenotypes associated with CnA/CnB disruption. Currently, we are also screening C. elegansEMS-mutagenized library by PCR. So far, one candidate mutant has been identified for CeCnA after the second round of sib selection. Future work involving the regulation of other cellular proteins by calcineurin, such as intracellular calcium channels (ryanodine receptor or IP3 receptor) will help us to elucidate the exact role of calcineurin in C. elegans.

24 May 1999 15:50 227 227 A Screen for Components of the GOA-1 Signaling Pathway

1999 International Worm Meeting abstract 176 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A Screen for Components of the GOA-1 Signaling Pathway IA Bany, MR Koelle Yale University Neurotransmitters can mediate neural communication by activating heterotrimeric G proteins that signal through poorly understood pathways. GOA-1, a G protein expressed in the C. elegans nervous system, is 80% identical to human Goa, the major G protein of the brain. Neural signaling defects in goa-1 mutants result in hyperactivity of several behaviors, including feeding, locomotion, and egg laying. To identify components of the GOA-1 signaling pathway, I am conducting a saturation-scale screen for mutants that have a phenotype similar to that of goa-1 loss-of-function mutants. In the past it has been difficult to identify mutants with this phenotype (thin and empty of eggs) because they resemble both young adults and the partially sterile animals that appear at a high frequency following mutagenesis. I have now developed an efficient selection for hyperactive egg-laying mutants that overcomes the previous difficulty. My selection takes advantage of the fact that the time delay between fertilization and laying of an egg is decreased in these mutants resulting in the eggs being laid at unusually early stages of development. Since eggs adhere to the surface they are laid on, washing worms off a plate leaves the eggs behind. The eggs laid by worms not hyperactive in egg laying will hatch ~12.5 hours after being laid, whereas the eggs laid at early developmental stages by hyperactive mutants will remain unhatched for an additional ~1.5 hours. If the hatched worms are rewashed off the plate 12.5 hours after an initial wash, the eggs left on the plate are highly enriched for hyperactive egg-laying mutants. By thus selecting for early-laid eggs and then visually rescreening for animals that resemble goa-1 mutants, I am identifying mutants with neurotransmitter signaling defects due to altered function of proteins either upstream or downstream of GOA-1. This screen does isolate mutants with the desired phenotype; to date I have screened approximately 2500 mutagenized haploid genomes, about 10% of the projected total. I have collected 25 mutants that demonstrate varying degrees of increased egg-laying activity. Five mutations seem strong enough to warrant further investigation, and interestingly are semi-dominant. I plan to clone and characterize the genes identified in my screen in order to gain a molecular understanding of GOA-1 signaling.

24 May 1999 15:50 228 228 Characterization of syd-5, a gene that may be involved in synaptogenesis.

1999 International Worm Meeting abstract 177 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of syd-5, a gene that may be involved in synaptogenesis. R Baran, Y Jin University of California, Santa Cruz The GABAergic DD and VD motor neurons provide a simple system for studying synaptic development and polarity. The DD and VD neurons have similar axonal morphology, but opposite direction of synaptic transmission. Both classes of neurons extend an axon along the ventral nerve cord, branch to form a commissure, and branch again to extend along the dorsal nerve cord. In adults the DDs receive inputs on the ventral side and form synapses onto dorsal body wall muscles. VDs exhibit the opposite synaptic polarity, receiving input on the dorsal side and innnervating ventral muscles. syd-5was identified in a screen for mutations that alter D neuron synapses using a synapse-specific marker Punc-25-SNB-1-GFP1,2 . In wild-type worms, Punc-25-SNB-1-GFPappears as regularly spaced puncta along the dorsal and ventral nerve cords, corresponding to neuromuscular junctions made by the DD and VD neurons. In syd-5(ju89)mutants, the Punc-25-SNB-1-GFPpuncta are dispersed unevenly, especially along the dorsal nerve cord of adult syd-5animals where the DD neurons synapse with dorsal muscles. The pattern of the SNB-1-GFPmarker in syd-5(ju89)mutants closely resembles the SNB-1-GFPphenotype of loss-of-function syd-3mutants, however, ju89behaves genetically as a weak gain-of-function mutation. syd-3encodes a novel presynaptic protein containing a guanine exchange factor (GEF) domain, suggesting it functions in a G-protein signaling pathway required for the adult synaptic pattern of the D neurons3 . syd-5may function in the same pathway, or in a parallel pathway for D neuron synaptogenesis. syd-5maps between egl-33and lin-11on chromosome I. We are currently using cosmid clones from this region in transformation rescue experiments to determine the molecular identity of syd-5.

1 Zhen & Jin, 1997 International Worm Meeting Abstract

2 Nonet, M., 1999. J. Neurosci. Methods.

3 Huang et al., 1999 International Worm Meeting Abstract

24 May 1999 15:50 229 229 UCS Domain Functions as a Myosin Assemblase

1999 International Worm Meeting abstract 178 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. UCS Domain Functions as a Myosin Assemblase JM Barral 1 , HF Epstein 1,2

1 Department of Biochemistry, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. 2 Department of Neurology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.

Muscle thick filaments have served as models to study biological multi-protein assemblies. In C. elegans, body wall thick filaments are tubules with a core of paramyosin subfilaments coupled by filagenins, and a surface layer of two differentially localized myosin isoforms: myosin A in the center and myosin B in the polar regions (Liu et al. 1998. JCB;140:347-353). Early work suggested that the product of the unc-45 gene may function as a myosin assemblase (Epstein and Thomson. 1974. Nature;250:279-280). We have found that temperature sensitive (ts) mutations in the UNC-45 protein lead to a 50% decrease in the amount of thick filaments in muscle cells at the restrictive temperature. Furthermore, the filaments that assemble have abnormal structure, as evidenced by immunofluorescence and immunoelectron microscopy. Myosins A and B are scrambled throughout the filament length and upon isolation, the filaments display marked in vitro instability. Three ts mutations are amino acid substitutions in conserved residues within a predicted "UCS" domain similar to three fungal proteins: CRO1 (P. anserina), She4p (S. cerevisiae), and Rng3p (S. pombe ) (Barral et al. 1998. JCB;143:1215-1225). These proteins were found in screens searching for mutants defective in septation (CRO: Berteaux-Lecellier et al. 1998. EMBOJ;17:1248-1258), the segregation of molecules during budding and endocytosis (SHE: Jansen et al. 1996. Cell;84:687-697; Wendland et al. 1996. JCB;135:1485-1500), and contractile ring formation (RNG: Wong and Balasubramanian. 1998. MBC;9:474a). These processes all require the assembly of competent acto-myosin structures. At its amino-terminus, UNC-45 contains three TPR motifs. These are present in proteins implicated in protein folding and assembly (Frydman and Hohfeld. 1997. TIBS;22:87-92). Our current work is directed towards testing the model that the UCS domain of UNC-45 binds myosins A and B differentially, and that the TPR domain interacts with molecular chaperones, which keep myosin in an assembly-competent state until it is properly placed in the filament. UCS proteins may operate through a common mechanism, recognizing specific myosin isoforms and assembling them into structures appropriate for their particular cellular function.

24 May 1999 15:50 230 230 Polarized Localization of PAR-3 and PAR-2 in C. elegansEmbryos Requires ooc-5 and ooc-3

1999 International Worm Meeting abstract 179 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Polarized Localization of PAR-3 and PAR-2 in C. elegansEmbryos Requires ooc-5 and ooc-3 SE Basham, LS Rose University of California, Davis CA 95616 USA Division of a cell containing a polar distribution of fate determinants can produce daughters with distinct cell fates. For this process to work efficiently, polarity and spindle orientation must be coordinated. In C. elegansembryos, divisions in the AB lineage are symmetric and occur in an orthogonal pattern of cleavage. Polarized cells in the P lineage undergo asymmetric divisions in which the mitotic spindle is repeatedly oriented on the same axis due to a rotation of the nuclear-centrosome complex. The pargenes are required for both normal polarity of the 1-cell embryo and division pattern in the 2-cell embryo. Genetic and molecular analyses suggests that PAR-3 protein plays a role in preventing nuclear rotation in the AB cell while PAR-2 functions to allow nuclear rotation in P1 by restricting PAR-3 localization in this cell (reviewed in Rose and Kemphues, Ann. Rev. Genet. 1998. 32). Hermaphrodites homozygous for mutations in ooc-5or ooc-3produce reduced sized embryos that fail to undergo nuclear rotation in the P1 cell. We examined the distribution of PAR proteins in oocmutant embryos. In almost all 1-cell ooc-5and ooc-3embryos examined, localization of PAR-1, PAR-2 and PAR-3 was normal. However, in the majority of 2-cell mutant embryos examined, PAR-3 was present around the entire P1 periphery or extended abnormally far posteriorly. PAR-2 localization was reciprocally reduced or absent from the P1 periphery. In addition, nuclear rotation can occur in ooc-5;par-3and ooc-3;par-3double mutant embryos, suggesting that failure of P1 nuclear rotation in oocembryos is due, at least in part, to the mislocalization of PAR-3 in this cell. PAR-1 is asymmetrically localized in wild-type embryos in a pattern similar to PAR-2. PAR-1 localization in the P1 cell of ooc-5embryos parallels that of PAR-2, being reduced or absent from the periphery. To our knowledge, these are the first mutations described that specifically affect re-establishment of PAR domains in the P1 cell. We have begun a molecular analysis of both the ooc-5and ooc-3genes. Using germline transformation, we have identified a single cosmid that rescues the ooc-5phenotype and a single cosmid that rescues the ooc-3phenotype. We are currently using RNAi and injection of cosmid fragments to identify the ooc-5and ooc-3genes.

24 May 1999 15:50 231 231 Cellular and genetic analysis of Gq alpha mediated signaling pathways in C. elegans

1999 International Worm Meeting abstract 180 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cellular and genetic analysis of Gq alpha mediated signaling pathways in C. elegans CA Bastiani, S Gharib, PW Sternberg, MI Simon Division of Biology, California Institute of Technology, Pasadena, CA 91125 U.S.A. egl-30 encodes the C. elegans homologue of the mammalian heterotrimeric G protein alpha subunit, Gq. Reduction-of-function and loss-of-function mutations in egl-30 affect a diverse array of behaviors in C. elegans. These include: viability, egg-laying, pharyngeal pumping, movement, and spicule protraction. In mammals, the Gq family is coupled to a wide variety of receptors, including receptors that respond to acetylcholine and serotonin. Upon activation, mammalian Gq family members can directly activate all isoforms of phospholipase C beta. In worms, acetylcholine is the only neurotransmitter required for viability and both acetylcholine and serotonin directly regulate egg-laying, pharyngeal pumping, and movement. It is possible that EGL-30 couples to receptors that can respond to both of these neurotransmitters in C. elegans. In order to ultimately dissect egl-30 mediated signaling pathways at both a cellular and molecular level, we have taken approaches to identify genes which interact in a pathway with egl-30 and have examined the cellular localization of EGL-30. Six mutations were identified which can dominantly suppress mutant phenotypes conferred by egl-30(md186), a reduction-of-function allele of egl-30. One of these mutants phenocopies overexpression of egl-30 with respect to movement and egg-laying. These worms move with exaggerated body bends, lay early eggs, and have an empty uterus. Genetic ablation of the HSN neurons suppresses the Egl-c phenotype of this suppressor mutant. These mutants are also partially resistant to the serotonin agonist alpha-methyl 5-HT. However, genetic ablation of the HSN neurons suppresses partial resistance to alpha-methyl 5-HT in egg-laying assays, indicating that this gene might function via the HSN neurons. We have also examined EGL-30 localization using polyclonal antibodies specific for EGL-30. Antibody staining indicates that egl-30 is expressed in both the nervous system and in muscle cells. Further studies are being carried out to identify cells in which egl-30 expression is sufficient in order to rescue egl-30 mutant behavioral defects with respect to egg-laying, pharyngeal pumping, and movement. egl-30md186C. elegans

24 May 1999 15:50 232 232 A genetic screen for inhibitors of the vulval induction pathway

1999 International Worm Meeting abstract 181 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A genetic screen for inhibitors of the vulval induction pathway GR Battu, A Hajnal Div. Of Cancer Research, Dept. Of Pathology, Schmelzbergstr. 12, University of Zürich, 8091 Zürich, Switzerland. The vulva of the hermaphrodite is made from the descendants of three of the six equivalent Vulval Precursor Cells P3.p to P8.p (VPCs). In response to the inductive anchor cell signal, P6.p adopts the 1o fate which is to divide thrice to give 8 descendants that form the inner vulval tissue. A lateral signal from P6.p induces P5.p and P7.p to adopt the 2o fate which is to divide thrice to give 7 descendants each that form the outer vulval tissue. The remaining VPCs (P3.p, P4.p and P8.p) adopt the 3o fate which is to divide once and give 2 descendants that fuse with the hyp7 cell of the hypodermis. The anchor cell signal activates in P6.p a highly conserved RTKase/Ras/MAP kinase pathway that specifies the 1o fate. While many genes that are required for the transduction of the inductive signal have been isolated and characterized in detail, less is known about genes that negatively regulate vulval induction. Known inhibitors include sli-1, unc-101, sur-5, gap-1 and the class A and B Syn Muv genes. We have performed genetic screens to isolate and characterize novel inhibitors of vulval induction. Since animals containing a single mutation in an inhibitor usually do not exhibit excess vulval induction, we performed genetic screens in a sensitized gap-1(0)background for recessive mutations that exhibit a multivulva (Muv) phenotype. We thus isolated 9 new mutations (termed meg for multivulva enhancer of gap-1). Three of these mutations, meg-1(ga145), meg-2(ga139), meg-2(ga153),define two genes that map to chromosomal regions that do not contain known inhibitors of vulval development. Single mutants in meg-1 and meg-2 exhibit a wild type vulval phenotype. meg-1; gap-1 animals exhibit a 70% penetrant Muv phenotype but no other obvious defects. In addition to a 100% penetrant Muv phenotype, meg-2; gap-1 animals exhibit partial sterility, suggesting an involvement of meg-2 in oogenesis. Using a combination of three-factor crosses and deficiency mapping, we have located meg-1 on LGII between fer-15 and zyg-9 . To clone the gene, we are currently injecting YAC and cosmid clones that span this region into meg-1; gap-1 animals and are looking for rescue of the Muv phenotype. Similar studies with meg-2 are in the offing.

24 May 1999 15:50 233 233 Quantification of Transcriptome Distortion After Amplification Using cDNA Micro-Arrays

1999 International Worm Meeting abstract 182 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Quantification of Transcriptome Distortion After Amplification Using cDNA Micro-Arrays LR Baugh, CP Hunter Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138 Complete genomic sequences and micro-array based technologies allow for genome-wide parallel analysis of gene expression. Such transcriptional profiling makes it possible to map clusters of coordinately regulated genes and in turn, in combination with computational and traditional molecular and genetic techniques, to ask questions about the regulation and significance of such clusters. How many determinants define a given cluster? Do those determinants act in cis or trans? How many clusters are regulated by a given determinant? To what extent do master regulators specify unique fates through conserved sets of target genes?

Due to the relatively large amount of Poly A+ RNA required to hybridize a single micro-array and thus the virtual impossibility of attaining ample, synchronous starting material, the total message pool, or transcriptome, of single embryos or cells will have to be amplified to address such questions. However, the amplification process should not significantly distort the relative abundance of sequences in the transcriptome. Using a yeast cDNA micro-array, we have quantified the distortion imparted on a transcriptome during different amplification procedures, and we plan to use the results of this analysis in the design of experiments employing a C. elegans cDNA micro-array.

24 May 1999 15:50 234 234 A C. elegans homolog of the human gene for X-linked kallmann syndrome

1999 International Worm Meeting abstract 183 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A C. elegans homolog of the human gene for X-linked kallmann syndrome P Bazzicalupo 1 , A Facciolli 1 , MA Hilliard 1 , E Di Schiavi 1 , A Ballabio 2 , E Rugarli 2

1 International Institute of Genetics and Biophysics, Via G. Marconi 10, 80125, Napoli, Italy. 2 Telethon Institute of Genetics and Medicine, TIGEM, via Olgettina 58, 20132, Milano, Italy.

Kallmann syndrome (KS) is an inherited disorder defined by the association of anosmia and hypogonadism. The human, chicken and quail genes have been identified. They code for novel secreted proteins, KALs, which contain a new combination of domains: the four-disulfide-core domain together with fibronectin type III domains. The vertebrate genes are expressed in the olfactory bulb and are required for proper neuronal connectivity and cell or axonal migration in this region both during development and in adult life. The C. elegans gene K03D10.1, identified by the Sequencing Consortium, codes for a protein with significant homology and colinear domain topology to vertebrate KALs. We named the gene kal-1. A corresponding cDNA clone from the Kohara collection has been obtained and sequenced. The C. elegans protein presents at its C-terminus a hydrophobic trans-membrane domain which is absent in the vertebrate counterparts. RT-PCR and experiments with reporters indicate that the gene is transcribed beginning with late embryos and during larval stages. Specific antibodies to fragments of CeKAL-1 have been generated. The nematode protein is detected in worm extracts as a cleaved C-terminal fragment with an apparent mass of 42 kD. Similarly the vertebrate KALs, secreted by COS cells, are present in the medium as cleaved C-terminal fragments of 42-45 kD. We have isolated a deletion mutant of kal-1 using TMP + UV as mutagen and screening by PCR. The deletion is 2.2 Kb long and eliminates 440 terminal aminoacids. Transcriptional analysis and the use of specific antibodies indicates that the deletion mutant (gb503) is a functional null. We are characterizing the phenotype of kal-1(gb503) worms which show no major visible abnormality. Mutants appear to chemotax normally to several odorants to avoid soluble repellents and respond to mechanic stimuli. We will present further data on the behavioral characterization of kal-1(gb503) and on experiments with GFP constructs in which the path followed by several neurons in this strain has been studied.

24 May 1999 15:50 235 235 GSK-3 plays a positive role in Wnt signaling in C.elegans embryogenesis

1999 International Worm Meeting abstract 184 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. GSK-3 plays a positive role in Wnt signaling in C.elegans embryogenesis Yanxia Bei, Kevin Fitzgerald, Craig C. Mello Umass Medical Center GSK-3 is a highly conserved serine/threonine kinase implicated in many signaling pathways in diverse organisms. In Drosophila and vertebrates GSK-3 is thought to be downregulated in response to Wnt signaling. When active, GSK-3 antagonizes signaling in these systems by promoting the proteolysis of ß-catenin, a component of the Wnt responsive transcriptional machinery. When GSK-3 activity is blocked, ß-catenin levels are high, allowing ß-catenin to complex with its transcriptional co-factor (TCF/dTCF), enter the nucleus and drive the expression of Wnt responsive genes. During C.elegans embryogenesis, a Wnt signaling system is implicated in endoderm induction. This pathway also employs both a ß-catenin and a TCF related factor (WRM-1, and POP-1 respectively). Surprisingly however, the POP-1 protein does not function as an effector but rather is downregulated by signaling. We have recently shown that WRM-1 binds to and activates the LIT-1 kinase, which in turn phosphorylates POP-1. In COS cells this results in the exclusion or export of POP-1 protein from the nucleus, reproducing the effects of signaling in-vivo (See abstract by Shin and Rocheleau et al.,). To identify the role of GSK-3 in this process we have analyzed the phenotype of gsk-3 (RNAi) and of a gsk-3 deletion (obtained from Nemapharm). Laser ablation studies on gsk-3 depleted embryos indicate that 56% of E cells fail to make intestine and instead produce extra pharyngeal mesoderm. This phenotype is similar to those of mom-2/Wnt and wrm-1 suggesting that GSK-3 functions positively in the induction of endoderm. One attractive model is that GSK-3 collaborates with WRM-1/LIT-1 to promote nuclear exclusion and perhaps proteolysis of POP-1. Further in-vivo and in-vitro studies will address this question. Paradoxically, removing GSK-3 activity also reveals a latent ability of the Cp blastomere to undergo a Wnt dependent induction of intestine. Thus GSK-3 is a positive regulator of Wnt signaling and gut formation in the E-cell lineage but also has an additional gut repressive function in the Cp blastomere. Clearly there is much to learn about the in-vivo functions of GSK-3.

24 May 1999 15:50 236 236 Cellular, molecular, and genomic analysis of the novel matrix receptor gene mua-3

1999 International Worm Meeting abstract 185 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cellular, molecular, and genomic analysis of the novel matrix receptor gene mua-3 M Bercher 1 , J Wahl 1,2 , B Vogel 2 , E Hedgecock 2 , J Plenefisch 1

1 Department of Biology, University of Toledo, Toledo, OH 43606. 2 Department of Biology, Johns Hopkins University, Baltimore MD 21218.

Normal locomotion of the nematode Caenorhabditis elegans requires transmission of contractile force from the myofibrillar lattice of the body wall muscles through a series of mechanical linkages between the muscle cells, the surrounding extracellular matrix, the hypodermis (epidermis), to the collagenous cuticle (the exoskeleton). Mutations in mua-3 result in a progressive flaccid paralysis of all or part of the body due to detachment of the body wall muscles from the cuticle, thus this gene is apparently required for the normal maintenance of mechanical linkage between muscle and cuticle as the animal grows in size postembryonically. mua-3 has been cloned and encodes a predicted 3623 amino acid protein encoded on 30 exons as confirmed by RT-PCR. In addition a smaller protein missing the first 36 amino acids is predicted to exist due two alternate SL1 spice variants. MUA-3 is a novel matrix receptor with a large extracellular domain, a single transmembrane helix, and a smaller cytoplasmic domain. The extracellular domain contains four distinct protein-folding motifs, i.e., either 4 or 5 LDL, 52 EGF, 1 vWFA and 2 SEA modules. These motifs suggest it may help tether the muscle or hypodermal cells to the collagenous structures of the basal lamina and/or cuticle. A search of GenBank revealed a second C. elegans gene, initially designated mrp-1, homologous to mua-3, and a nearly identical C. briggsae ortholog, cbmua-3. It has been subsequently demonstrated by E. Bucher that the mrp-1 sequence is the same as the muscle attachment gene mup-4 (E. Bucher, personal communication). In order to determine the cellular localization and expression patterns of MUA-3, monoclonal antibodies to the cytoplasmic domain have been raised, and the patterns of MUA-3 expression are being determined by indirect immunoflorescence in both wild-type and mua-3 mutant animals. In addition, MUA-3 expression patterns in animals mutant for several other muscle mutations are being examined. The results and our interpretations of these localization studies will be presented.

24 May 1999 15:50 237 237 Molecular characterization of the C2H2 zinc finger protein MUA-1

1999 International Worm Meeting abstract 186 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Molecular characterization of the C2H2 zinc finger protein MUA-1 MR Bercher, X Zhou, J Plenefisch Department of Biology, University of Toledo, Toledo, OH 43606 MUA-1 is required for postembryonic muscle development in C. elegans. Although normal in appearance, the mechanical links between muscles and exoskeleton(cuticle) are exceptionally weak in MUA-1 mutant animals resulting in a progressive detachment of the body wall muscles from each other and from the hypodermis. The predicted MUA-1 protein contains three C-terminal C2H2 zinc finger domains that have high sequence homology to zinc finger transcription factors of the EKLF/BTEB2 family. While the majority of proteins containing C2H2 zinc fingers are transcription factors that bind GC rich enhancer sequences. A putative N-terminal activation domain in MUA-1 remains uncharacterized. It has some compositional similarity but no sequence homology to other activation domains. Based on the zinc finger homologies and the presence of a putative activator domain, MUA-1 is predicted to act as a transcription factor driving muscle specific protein expression. A fusion protein containing the MUA-1 activation domain fused to the GAL-4 DNA binding domain was constructed. When expressed, the GAL-4-DNA binding domain of this fusion protein binds to a 17mer enhancer sequence upstream of a CAT gene in the reporter construct. If the MUA-1 activation domain is an authentic activation domain, it will drive transcription of CAT in a Cos cell line. CAT expression was assayed in the Cos cell extracts, showing that MUA-1 has modest transcriptional activity. Further experimentation is now needed to further characterize the transcriptional effects of MUA-1. We are also examining MUA-1 expression during muscle development and will be ßattempting to identify the target genes activated in vivo.

24 May 1999 15:50 238 238 Establishing the handedness of left/right asymmetry in C. elegans

1999 International Worm Meeting abstract 187 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Establishing the handedness of left/right asymmetry in C. elegans DC Bergmann, WB Wood Dept. of MCD Biology, U. Colorado, Boulder The invariant dextral handedness of C. elegans embryos, larvae and adults is generated by orienting the divisions of the ABa and ABp blastomeres at the 4-cell-stage, such that their left daughters lie to the anterior of their right daughters. A screen for maternal-effect mutations that resulted in adults with reversed left/right asymmetry identified one, defining the spn-1 locus, that could produce significant numbers (40% of surviving adults) of left/right reversed animals. Weak alleles of other genes required for embryonic cleavage plane orientation produced no more than 2% left/right reversed adults. Analysis of the spn-1(it143) hypomorphic allele has shown that the early cleavage plane orientation dictates all subsequent left/right body asymmetries during development. Recordings of embryos from spn-1 mutant mothers showed spindle orientation defects in the AB cells responsible for generating handedness, and relatively minor aberrations in division planes of other lineages. A screen for suppressors of spn-1 lethality identified at least 8 independent loci, some of which map to regions without previously known polarity or spindle orientation mutants. All these suppressors prevent both lethality AND handedness reversal. The spindle orientation of the ABa and ABp cells is the first observable left/right asymmetry in the embryo, and it appears that the ability of these cells to divide correctly is cell autonomous. Experiments with isolated wild-type blastomeres (Goldstein, JCB 129:1071-1080, 1995) and our laser ablation experiments have shown that the early AB spindle orientations are not dictated by external signals, nor is the skewed orientation of the cleavages a response to spatial constraints from other blastomeres within the eggshell. In blastomeres isolated from spn-1 mutant embryos, we observed that the AB blastomeres lose their normal helical pattern of division and instead divide with random orientations, suggesting that spn-1 acts autonomously in the dividing cells, possibly as a signpost or anchor for the mitotic spindle. spn-1 maps to the most distal portion of LG I left, telomeric to mex-3, and its molecular identity has continued to elude us. We are attempting to find RC301/N2 polymorphisms to allow more precise positioning of spn-1 in this marker-poor region.

24 May 1999 15:50 239 239 Olfactory Plasticity in C. elegans: A Concentration-dependent Separation of Habituation and Adaptation.

1999 International Worm Meeting abstract 188 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Olfactory Plasticity in C. elegans: A Concentration-dependent Separation of Habituation and Adaptation. N Bernhard, D van der Kooy University of Toronto, Toronto CANADA M5S-1A8 Continuous or repeated presentation of a single stimulus causes a decrement in an animal’s behavioural response to that stimulus. Most studies investigating this phenomenon fail to differentiate between habituation (a reversible decrease in the behavioural response which is a form of non-associative learning) and adaptation (a decrement in response that is likely due to sensory or effector fatigue, which eventually recovers but can not be dishabituated). Previous studies investigating this type of behavioural plasticity in C. elegans have suggested that any response decrement after exposure to high concentrations of a volatile odorant is due to adaptation. Work from our lab has demonstrated, however, that the decrement in chemotaxis observed with exposure to pure benzaldehyde can be dishabituated, suggesting that the response decrement is due to a non-associative learning process, as opposed to adaptation. We have also shown habituation and dishabituation at low concentrations of diacetyl (DA) in a single worm paradigm. Thus, we set out to determine if both olfactory adaptation and habituation can be observed in the same paradigm, and if these two processes can be differentiated. After exposure to very high concentrations of DA (100%), worms showed a decrement in chemotaxis to a point source of DA. Furthermore, despite the presentation of various strong or noxious potentially dishabituating stimuli (ranging from centrifugation at 1000g to vortexing and cold shock) this response decrement did not return to baseline levels. However, after exposure to low concentrations of DA (0.001%), observed habituation of the chemotaxic response was reinstated to naive chemotaxis levels by exposing worms to a dishabituating stimulus (centrifugation at 250g). Surprisingly, exposure to intermediate concentrations of DA (25% and 0.01%) did not cause worms to exhibit a response decrement at all. These concentration specific effects reveal a distinct boundary between the two processes: adaptation at high concentrations versus habituation at low concentrations of the odorant.

24 May 1999 15:50 240 240 TEG-1 Functions in the GLD-1 Pathway to Initiate Meiotic Development

1999 International Worm Meeting abstract 189 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. TEG-1 Functions in the GLD-1 Pathway to Initiate Meiotic Development LW Berry, D Hansen, T Dang, DF Schneider, T Schedl Genetics, Washington Univ. School of Medicine The germline is a polar tissue with distal cells proliferating and more proximal cells initiating meiosis. The DTC - GLP-1/Notch receptor signaling pathway (1) promotes proliferation of distal germ cells by negatively regulating two redundant parallel pathways, one containing GLD-1 and the other GLD-2, that each promote initiation of meiosis (2,3). Germ cells in gld-1 and gld-2 single mutants enter meiosis normally, but fail to do so in the double mutant resulting in tumorous germlines analogous to constitutive activation of GLP-1 (3,4). Two screens were conducted to identify other genes necessary for entry into meiosis. First, three genes, teg-1, -2 & -4, where identified as recessive tumorous enhancers of a weakly activated glp-1 allele. TEG-2 & -4 likely are negative regulators of GLP-1/Notch receptor signaling in both the germline and soma as teg-2 & -4 mutations, but not teg-1, enhance the Muv phenotype of a weakly activating mutation in a second Notch receptor, lin-12. TEG-1 functions in the GLD-1 pathway to initiate meiosis as teg-1 single mutant germ cells enter meiosis normally while the gld-2; teg-1 double mutant has a synthetic tumorous phenotype. In contrast, the gld-1; teg-1 double shows the gld-1 null phenotype. Second, to identify genes acting in the GLD-1 pathway for entry into meiosis, recessive mutations that are synthetic tumorous in a gld-2 background were isolated. This screen yielded gld-1 mutations, a teg-1 mutation and mutations in at least three other loci which have a synthetic tumorous phenotype with gld-2 in both hermaphrodite and male germlines. teg-1 also functions in the hermaphrodite germline sex determination pathway; teg-1 promotes the female fate while gld-1 and fog-2 promote the male fate. teg-1 negatively regulates gld-1 and fog-2 as both are epistatic to the teg-1 masculinization of the germline (Mog) phenotype. teg-1 is thus distinct from mog-1 to -6 genes that act downstream of gld-1 and fog-2 in sex determination. Furthermore, sex determination genes downstream of gld-1/fog-2 do not appear to be involved in the decision to initiate meiosis as none of the tested gene mutations show a synthetic tumorous phenotype with gld-2 or suppress gld-1;gld-2.

1) Kimble & Simpson ’97; 2) Francis et al ’95; 3) Kadyk & Kimble ’98; 4) Berry et al ’97

24 May 1999 15:50 241 241 RNA interference of SMN, the gene causing Spinal Muscular Atrophy in humans, leads to embryonic lethality and to germ cell apoptosis in the nematode Caenorhabditis elegans.

1999 International Worm Meeting abstract 190 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. RNA interference of SMN, the gene causing Spinal Muscular Atrophy in humans, leads to embryonic lethality and to germ cell apoptosis in the nematode Caenorhabditis elegans. S Bertrandy 1 , S Lefebvre 1 , Y Kohara 2 , A Munnich 1 , D Thierry-Mieg 3

1 INSERM U393, Hôpital Necker, 149 rue de Sèvres, 75743 Paris Cedex 15, France. 2 NIG, Mishima, Japan. 3 CRBM-CNRS, Montpellier, France.

Spinal Muscular Atrophy (SMA) is a frequent autosomal recessive neuromuscular disorder characterized by degeneration of spinal motoneurons. The disease results form alterations of Survival Motor Neuron (SMN) gene encoding a 294 aa protein of unclear function. SMN protein interacts with RNA and RNA-binding proteins and is involved in spliceosomal snRNP biogenesis and pre-mRNA splicing. Recently, we have shown that SMN proteins from C. elegans and Danio rerio bind RNA in vitro (HMG, in press). At the meeting, we will present in situ RNA hybridization and immunofluorescence data in C.elegans. Preliminary results indicate that this ubiquitous gene is highly expressed in gonads. In very early embryos, the protein localizes mainly to cytoplasmic granules and later in the nucleolus. RNA interference experiments were carried out by injection of SMN ds-RNA in wild type animal that generated a progeny with various defects. Two-days post-injection, there was sudden burst of late embryonic lethality that affects 100% of late progeny, indicating SMN requirement for survival in C. elegans. Furthermore, the day after injection, many healthy adult progeny had gonads with few germ cells and signs of germ line apoptosis. This defect was completely suppressed when injecting the ced-4(n1162) mutant. This epistasis result indicates that SMN has a protective effect on germ line cells and acts before ced-4 in the caspase/ICE cascade. Interestingly, the ced-4 mutant did not suppress the embryonic or larval lethality. Unlike what seems to happen in the mouse, where SMN inactivation led to massive apoptosis in early embryos, here the cause for lethality is not just ced-4 dependent apoptosis. We would like to know if SMN controls alternative splicing of ced-4 in germ line and if it plays a role in maturation, splicing, protection or transport of specific RNAs especially of maternal one.

24 May 1999 15:50 242 242 Genetic analysis of neuromuscular junction formation in C. elegans.

1999 International Worm Meeting abstract 191 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic analysis of neuromuscular junction formation in C. elegans. J Bessereau, E Jorgensen Dept. Biology, University of Utah, Salt Lake City, UT 84112 C. elegans body wall muscle cells contact the motor axons in the ventral or dorsal cords either by sending to the cord a cytoplasmic process called a muscle arm or by making en-passant synapses with the adjacent neurites. In the cord, each muscle receives dual innervation from both cholinergic excitatory motor neurons and GABAergic inhibitory motor neurons. We would like to study how the neuromuscular junctions develop. Specifically, we would like to identify the genes involved in muscle-arm targeting, recognition between pre- and post-synaptic partners and differentiation of the post-synaptic apparatus. To characterize the basic rules of C.elegans neuromuscular junction formation, we used an UNC-49::GFP translational fusion as a post-synaptic marker and examined its distribution in known mutant backgrounds. unc-49 encodes a muscle GABAA receptor and is concentrated at the post-synaptic domain of inhibitory neuromuscular junctions. We established that: 1) presynaptic inputs are required to achieve post synaptic differentiation: in lin-6 mutants the ventral muscles are not innervated and do not exhibit UNC-49::GFP clustering. 2) GABA transmission per se is not required to build GABAergic synapses: in unc-25 mutants GABA is not synthesized but UNC-49::GFP distribution is wild-type. 3) A few neuromuscular contacts are sufficient to polarize the muscle cells and target the GABA receptor to the cord: in unc-30 mutants UNC-49::GFP is in clusters along the nerve cord despite the paucity of GABAergic synapses. To identify the genetic functions required for neuromuscular junction formation, we are screening for mutants with altered UNC-49::GFP distribution. From a pilot screen of a 1,000 genomes, we identified 14 mutants that affect UNC-49::GFP distribution. To more accurately characterize the phenotype of these mutants we have generated reagents to analyze the distribution of the components of neuromuscular junctions. To visualize acetylcholine receptors, we built an UNC-38::GFP translational fusion and also made antibodies to UNC-29. To visualize GABA receptors we made antibodies to UNC-49. To examine the alignment of pre- and postsynaptic elements of the GABA synapse we labeled the synaptic vesicle protein VAMP with CFP and the UNC-49 GABA receptor with YFP.

24 May 1999 15:50 243 243 Development of a mutagenesis strategy based on heterologous transposition in C. elegans.

1999 International Worm Meeting abstract 192 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Development of a mutagenesis strategy based on heterologous transposition in C. elegans. J Bessereau, A Wright, E Jorgensen Dept. Biology, University of Utah, Salt Lake City, UT 84112 The insertion of transposons into genes provides a molecular tag for the rapid cloning of mutated genes. Unfortunately the background of endogenous transposons in C.elegans makes this strategy difficult. The mobilization of a heterologous transposon in the C. elegans germline could provide a unique tag to facilitate gene cloning from mutants. Using PCR techniques, it would be possible to identify the genomic sequences that flank the transposon. The mutated gene could be identified by simple comparison of these sequences with the genome sequence. We initiated this project using the Himar transposon. Himar is a member of the mariner family that was identified in the horn fly Haematobia irritans. It encodes a single protein, the HIMAR1 transposase, that binds the inverted terminal repeat sequences present at the end of the transposon. Like other mariner elements, the transposase seems to be the only factor required to catalyze transposition, and thus Himar should be capable of transposition in C. elegans. To test whether the HIMAR1 transposase could be expressed in C.elegans, we engineered the Himar1 coding sequence, placed it under the control of the myo-3 promoter and generated transgenic worms. Using anti-HIMAR1 antibodies, we confirmed that a full-length transposase was properly expressed and targeted to cell nuclei. To be used for mutagenesis, the transposase must function in the germ line. To test for germ line expression we first integrated an array containing the Himar transposon and the rol-6(sd) marker to provide a target for the transposase. Transposase expression in the germ line was achieved using the glh-2 promoter. We observed that the glh-2-Himar1 construct causes excision of the Himar transposon and loss of the whole [Himar; rol-6] integrated array from approximately 50% of the haploid genomes. We also built a germ line inducible expression system using a heat shock promoter. The low basal rate of excision observed at room temperature is increased 10 to 50 times after heat shock. Although we are able to cause very efficient germline excision of Himar, we have not been able, up to now, to identify de novo insertions of the transposon in the genome. Parallel experiments using another transposon of the mariner family are in progress.

24 May 1999 15:50 244 244 Behavioral Effects of Exposure to Ethanol

1999 International Worm Meeting abstract 193 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Behavioral Effects of Exposure to Ethanol JC Bettinger, SL McIntire Gallo Center, UCSF, San Francisco, CA 94110 USA Exposure to ethanol interferes with complex behaviors in many model systems, but it has been difficult to correlate effects of ethanol on behavior with observations of its effects on specific molecular targets. Recently, studies of Drosophilademonstrated a link between ethanol sensitivity and a learning pathway1 : A screen for mutations that cause flies to be hypersensitive to the effects of ethanol on postural control yielded an allele of amnesiac, a putative neuropeptide known to be involved in learning2 . After exposure to ethanol,C. elegansdisplay uncoordinated movement (characterized by a decreased amplitude of the sine waveform and lethargy), and decreased rate of pumping and egg-laying (SLM, unpublished observations). After several hours of exposure, worms develop an acute tolerance to ethanol, and recover to resemble untreated controls. We are interested in determining whether or not exposure to and development of tolerance to ethanol alter any of the more complex behaviors exhibited by worms, including chemotaxis. Our preliminary experiments on the effect of ethanol on chemotaxis suggest that brief or prolonged exposure to moderate concentrations of ethanol (100-200 mM) does not prevent chemotaxis to the volatile odorant benzaldehyde. With extended exposure, worms become insensitive to chemoattractants in a process termed adaptation. Worms that have adapted to a particular chemoattractant will not climb a gradient of that chemoattractant3 . Given that worms are able to chemotax in the presence of ethanol, we can test the effect of ethanol on adaptation. We are determining whether or not exposure to low-to-moderate concentrations of ethanol interferes with the process of adaptation to benzaldehyde and other odorants.

1 Moore, M.S.; DeZazzo, J.; Luk, A.Y.; Tully, T.; Singh, C.M. and Heberlein, U. (1998). Cell 93: 997-1007.

2 Feany, M.B. and Quinn, W.G. (1995). Science 268: 869-873.

3 Colbert, H.A. and Bargmann, C.I. (1995). Neuron 14: 803-812.

24 May 1999 15:50 245 245 Investigating the sex-specific expression and function of the putative chemosensor srd-1.

1999 International Worm Meeting abstract 194 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Investigating the sex-specific expression and function of the putative chemosensor srd-1. M Bickle 1 , D Hughes 2 , J Hodgkin 1

1 Laboratory of Molecular Biology, MRC, Hills Road, Cambridge CB2 2QH, UK. 2 Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

We are analysing the function and expression of the putative chemosensor encoded by srd-1. srd-1 was identified as a predicted seven transmembrane receptor by Troemel et al in 1995 1 . A fusion of 3750 bp of promoter region of srd-1 to GFP was expressed in the ASI amphid neurons in hermaphrodites whereas in males it was expressed in the ASI, ADF and what appeared to be the R8/9 ray neurons of the tail. We have identified five putative TRA-1A binding sites in the promoter of srd-1 at approximately -810, -910, -2020, -2600 and -3480. TRA-1A is the zinc-finger protein at the end of the sex-determination pathway which is believed to control sex-specific transcription 2 . We mutated these binding sites in a full-length srd-1::GFP fusion kindly provided by Cori Bargmann. Mutation of the TRA-1A binding site at -2600 resulted in the expression of GFP in the ASI and ADF amphid neurons in hermaphrodites, suggesting that TRA-1A might bind directly to the promoter of srd-1. We are testing this hypothesis by gel-shift assays. We are also investigating the function of srd-1. We have isolated in collaboration with the Sanger Center a deletion at the srd-1 locus. The deletion covers -583 to +1017 eliminating the first two exons and parts of the third exon thus representing a likely null mutant. The mutant srd-1 is healthy and shows no morphological abnormalities in males or hermaphrodites. It forms dauers and stays on or near the bacterial lawn suggesting it is not impaired in any gross way for chemotaxis and males can mate. We are performing quantitative dauer formation, chemotaxis and mating assays. srd-2 is a close homolog of srd-1 with 48% identity. We will perform RNAi with srd-2 in a srd-1 mutant and test for behavioral alteration.

1.Troemel, E.R., Chou, J.H., Dwyer, N.D., Colbert, H.A. & Bargmann, C.I. Divergent seven transmembrane receptors are candidate chemosensory receptors in C. elegans. Cell 83, 207-218 (1995). 2.. Hodgkin, J. A genetic analysis of the sex-determining gene, tra-1, in the nematode Caenorhabditis elegans. Genes Dev 1, 731-745 (1987).

24 May 1999 15:50 246 246 Analysis of the UNC-44 Ankyrins

1999 International Worm Meeting abstract 195 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of the UNC-44 Ankyrins BR Bill, SS Chan, DA Darwis, MH Domanus, B McFaul, A Khan, K Stergulz, S Windler, N Velamparampil, AJ Otsuka Department of Biological Sciences, Illinois State University, Normal, Il 61790-4120 Ankyrins are cortical cytoskeletal proteins involved in anchoring the cytoskeleton to the plasma membrane. Mutations in the unc-44 ankyrin gene result in axon outgrowth and guidance defects. We have taken several approaches to characterize the unc-44 ankyrins. Polyclonal antibodies prepared against the spectrin-binding domain (AO271 Ab) and the STEP domain (AO346 Ab) were affinity purified against recombinant antigen on Western blots. In freeze-fractured worms, AO346 Ab stained the hermaphrodite nervous system, including the nerve ring, nerve cords, commissures, and ganglia. Staining occurred along nerve processes and at the periphery of the cell bodies. In males, the nervous system stained, including tail ray neurons. In comma stage embryos, staining was observed in the animal anterior. In later stage embryos, strong staining was observed in the nerve ring and nerve cords. Using the AO271 Ab, staining was observed at the periphery of many cell types, which is consistent with expected localization of ankyrins in the cortical cytoskeleton. Staining was also observed in the oocyte cytoplasm. While it cannot be ruled out that the interaction may be non-specific or due to residual anti-yolk Abs not removed during affinity purification, it is intriguing to note that there are abundant cytoplasmic ankyrins lacking ankyrin repeat domains in vertebrate kidney cells. In order to understand the biochemical properties of UNC-44 ankyrins, we started to purify C. elegans ankyrins. Starting from protocols for the purification of vertebrate red blood cell or brain ankyrins, attempts were made to purify UNC-44 ankyrins as membrane-associated proteins, followed by differential dissociation from the membrane fraction. The various ankyrin isoforms displayed weak partitioning by differential dissociation. The large AO13 ankyin isoform generally fractionated into the pellet and required 7 M urea for partial solubilization. The current strategy is to purify the ankyrins by affinity chromatography on a bovine spectrin column. Because vertebrate brain ankyrin is glycosylated, we are testing the ability of worm ankyrins to bind to a wheat germ-affinity column.

24 May 1999 15:50 247 247 Preliminary Studies of the ets Gene Family in C. elegans

1999 International Worm Meeting abstract 196 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Preliminary Studies of the ets Gene Family in C. elegans AG Blaszczak , MB Smith , BJ Graves Department of Oncological Sciences and the Huntsman Cancer Institute, University of Utah School of Medicine 50 North Medical Drive Salt Lake City, UT 84132 USA The ets proteins have become a good model system to study the molecular mechanisms of eukaryotic transcriptional control. These regulatory transcription factors share a highly conserved, ~85 amino acid DNA binding domain termed the ETS domain. Genes encoding ets proteins are found in many different species, throughout the metazoa. Vertebrate ets genes include at least 18 paralogs in the human genome. Because all ets proteins bind similar DNA sequences and are often found in the same cells/tissues, an important question is how can an individual ets protein regulate a unique set of target genes. Mechanisms undoubtedly have evolved that provide specific biological roles for individual ets proteins. For example, mouse knockouts of particular ets genes have distinct phenotypes, implying different functional targets. Our goal is to provide insights into the mechanism(s) that constitute biological specificity in the ets transcription factor family by employing the model organism, C. elegans. Ten ets genes in the C. elegans genome can be identified by sequence conservation of the ETS domain. Only lin-1 has been identified genetically as a transcription factor necessary for vulva differentiation. We have constructed a distance-matrix-based phylogenetic tree of all known ets genes including these ten C. elegans homologs. Most, but not all C. elegans ets genes are closely related to known vertebrate genes. We are analyzing the expression patterns of GFP-ets fusion proteins and the phenotypes from RNAi and genetic deletion experiments. Our preliminary studies of two C. elegans ets genes have localized their expression patterns to cell types that are distinct from the vulva, the location of lin-1 expression. We hope these approaches can guide the development of forward genetics strategies for the study of the regulation of ets proteins in biological contexts.

24 May 1999 15:50 248 248 The mut-2 mutator of C. elegans

1999 International Worm Meeting abstract 197 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The mut-2 mutator of C. elegans QF Boese, J Collins University of New Hampshire The mut-2 mutator plays multiple regulatory roles in the C. elegans germline. In addition to regulating activity of at least four distinct transposon families it is implicated in chromosome segregation, gametogenesis, germline gene silencing and DNA double-strand break repair. Elucidating the function of mut-2 will enhance the utility of transposons for functional genomic studies in this organism and shed light on mechanisms that maintain structural and functional integrity of eukaryotic genomes. Using the Him phenotype conferred by mut-2(r459), we mapped the gene between dpy-14 and sem-4 on LGI. However, efforts to identify a molecular clone of the gene were hampered because the available phenotypes were unsuitable for standard transformation rescue approaches. This roadblock led us to examine the temperature-sensitive (ts) behavior of mut-2: the original mut-2 isolates TR674 and TR679 are inviable at 25o C. Analysis of ancestors and recombinants derived from these isolates demonstrated that the ts phenotype cosegregates with mut-2 and is unrelated to the Bergerac ancestry of these strains. Closer inspection (with assistance from Eric Lambie) revealed that sterility results from a defect in gametogenesis. Gonads of mut-2 animals raised at 25o C appear normal but have a dearth of sperm which exhibit variable morphology. Oocytes show no gross abnormalities but may be affected as well. DAPI staining reveals no obvious chromosomal defects. We are testing if wild-type sperm can rescue sterility and determining the temperature-critical stage of sperm (and oocyte?) development. The ts sterile phenotype is rescued by sDp2, a large free duplication that covers the dpy-14 sem-4 interval. This suggests it may be a suitable phenotype for identifying a molecular clone of mut-2. Two smaller free duplications that bisect this interval, hDp62 and hDp65, were tested; hDp65 rescues, hDp62 does not. This positions mut-2 between +1.57 and +1.66, a region covered by three cosmids, containing ~20 ORFs. Work is in progress to clone mut-2 by rescuing sterility. Recent evidence suggests mut-2 strains may be resistant to RNAi (1). We are exploring the use of this phenotype in our cloning efforts as well. 1) Tabara, H and Mello, C. 1999. (this meeting)

24 May 1999 15:50 249 249 Embryonic development of a mononchid nematode

1999 International Worm Meeting abstract 198 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Embryonic development of a mononchid nematode G Borgonie, A Coomans Department Biology, University Gent, Ledeganckstraat 35, 9000 Gent, Belgium As part of our effort to study the evolution of the nematode embryonic lineage, we have started to analyse nematodes considered more primitive then rhabditids. Mononchids are primitive, carnivorous, terrestrial nematodes exhibiting a fundamentally different embryonic development then C. elegans. Embryos, who take 4-5 days to develop, are not transparent enough to allow use of standard 4D microscopy. The first division producs two equal blastomeres. Subsequent divisions produce wave after wave of equal blastomeres. At this early stage it is impossible to determine body axes or identify the germ line precursors. Analysis is done using transmission electron microscopy for identifying the germ line precursors and P-granule like structures. Micro injection of (fluorescent) tracers helps in identifying subsequent fates later in development. So far, data indicate development in these primitive nematodes is much more variable and regulated than in C. elegans.

24 May 1999 15:50 250 250 The Clr Phenotype and FGFR Signaling

1999 International Worm Meeting abstract 199 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Clr Phenotype and FGFR Signaling CZ Borland, MJ Stern Yale University Hyperactivation of the EGL-15 mediated fibroblast growth factor receptor (FGFR) signaling pathway results in a distinctive Clr phenotype: the pseudocoelom fills with fluid, the internal organs are compressed, and the animals develop into sterile adults. This Clr phenotype can result from inactivation of CLR-1, a receptor tyrosine phosphatase that inhibits signaling through this pathway, or by constitutive activation of EGL-15 itself. We have used the Clr phenotype to identify genes encoding other components of the EGL-15 FGFR signaling pathway. Mutations that can suppress the Clr phenotype of clr-1(e1745ts) have identified components that positively mediate EGL-15 signaling (soc mutants, suppressors of Clr). Mutations that cause a Clr phenotype could be either inactivating mutations in negative regulatory genes such as clr-1, or they could be activating mutations in positive mediators. We have identified a mutation in the let-60 ras gene, ay75, that confers a late-onset Clr phenotype. ay75 appears to have gain-of-function characteristics, consistent with RAS being a positive mediator of FGFR signal transduction in other systems. First, the molecular lesion in ay75, G60R, corresponds to substitutions found in transforming alleles of human RAS genes. Second, ay75 confers a partially penetrant Muv phenotype, characteristic of other gain-of-function let-60 ras alleles such as n1046. Third, ay75 confers a temperature sensitive dominant sterile phenotype which can be reverted by intragenic mutation. Eisenmann and Kim1 have identified another let-60 ras mutation, ga89, which has temperature-sensitive gain-of-function characteristics and displays a Clr phenotype similar to that of ay75. To understand the relationship of RAS with hyperactive EGL-15 signaling, we have used these let-60 ras alleles to conduct epistasis experiments with soc mutants as well as with mutants which block RAS signaling in other processes. Our data support a role for LET-60 RAS in this pathway that is consistent with its role in other receptor tyrosine kinase signaling pathways, and with the MAPK cascade being required for the Clr phenotype of RAS gain-of-function mutants.

1 Genetics 146: 553-565 (1997).

24 May 1999 15:50 251 251 RNA interference targets the pre-mRNA of the lir-1/lin-26 operon to prevent expression of both genes

1999 International Worm Meeting abstract 200 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. RNA interference targets the pre-mRNA of the lir-1/lin-26 operon to prevent expression of both genes JM Bosher, P Dufourcq, S Sookhareea, M Labouesse IGBMC, CNRS/INSERM/ULP, BP163, 67404 Illkirch, CU de Strasbourg, FRANCE We have been investigating the complex genomic organisation of three genes: lir-2, lir-1 and lin-26 which are organised in two overlapping operons (Dufourcq et al., Genetics, in press). All three of these genes encode homologous putative transcription factors which define a new C2H2 motif related to TFIIIA zinc fingers. Initially we used double-stranded RNA interference (RNAi) to investigate the function of lir-1 and lir-2. We found that lir-1(RNAi) leads to severe hypodermal defects, which are reminiscent of various lin-26 mutant phenotypes, resulting in embryonic or larval lethality. We account for these lin-26 like phenotypes by showing that lin-26 expression is severely down-regulated in lir-1(RNAi) arrested embryos. We have excluded the possibility that this is due to cross-interference between homologous sequences because injection of lir-2 dsRNA, which shows greater homology to lin-26 than lir-1, fails to give any phenotype. Recently we isolated a lir-1 null mutant which is viable and since there is no lir-1 maternal contribution we exclude the possibility that the lethal phenotypes observed in lir-1(RNAi) embryos are attributable directly to lir-1 function. Because lir-1 and lin-26 are organised in a transcriptional operon we conclude that injection of dsRNA corresponding to lir-1 specifically interferes with expression of lin-26 which is the downstream gene in the operon. We have tested two other operons (lin-15B/lin-15A and ppp-1/tra-2) to see if they show the same effect. For both of these operons RNAi against the upstream gene has no effect on expression of the downstream gene demonstrating that this is not a general feature of all operons. We hypothesise that the pre-mRNA of the lir-1/lin-26 operon is not processed efficiently enough such that it becomes a target for RNAi. This is demonstrated by the fact that injection of dsRNA corresponding to lir-1 and lin-26 intron sequence also results in the lin-26 like phenotypes. This provides direct evidence that RNA interference prevents gene expression by targeting nuclear transcripts. It also highlights that caution is sometimes necessary when interpreting RNA interference results without the benefit of mutant alleles.

24 May 1999 15:50 252 252 Investigating apico-basal polarity in the embryonic gut of C. elegans

1999 International Worm Meeting abstract 201 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Investigating apico-basal polarity in the embryonic gut of C. elegans O Bossinger 1,2 , A Klebes 1 , C Theres 1 , C Segbert 1 , E Knust 1

1 Genetics Institute, Heinrich Heine University, 40225 Düsseldorf, GERMANY. 2 [email protected]

The generation and maintenance of epithelial polarity relies on the correct reception of signals and cues from apical and baso-lateral sources (1). Thus, in Drosophila the Crumbs protein (2, 3) is localized apically to the zonula adherens (ZA; 4) and plays a key role in maintaining the polarized phenotype of the ectoderm and its derivatives (5). We have started to investigate epithelial polarization in the embryonic gut of C. elegans, a simple tube of 20 cells, arranged in a series of nine rings (6). Using the sequence of the Drosophila Crumbs cytoplasmic domain, we identified two C.e. homologues of CRB. We applied RNA interference technique (cDNA clones kindly provided by Dr. Kohara), in order to disturb normal development, but no visible phenotypes could be detected so far. Immuno-staining during morphogenesis of C.e. demonstrates expression of CeCRB1 in the gut and later in the pharynx, while at the same time intestinal staining is reduced. Double-labelling against phosphotyrosine reveals localization of CeCRB1 apical to the ZA in the embryonic gut of C.e. We are now concentrating on basal cues necessary for polarization of the gut epithelium and have begun to study C.e. mutants with defects in major constituents of basement membranes (e.g. let-2, emb-9, unc-52), beta-integrin (pat-3) or vinculin (deb-1). The phenotype of ZA organization in the above mutants was analyzed by confocal microscopy. It ranges from fragmentation to complete absence of the ZA. While elements of the Wnt/pop-1 signalling pathway have been postulated to act autonomously within the E-lineage for anterior-posterior patterning (7), we suggest that processing of extracellular matrix information is a prerequisite for establishing correct apico-basal polarity in the embryonic gut of C.elegans.

(1) Eaton and Simons, (1995). Cell 82: 5-8; (2) Tepass et al., (1990). Cell 61: 787-799; (3) Wodarz et al., (1995). Cell: 82, 67-76; (4) Tepass, (1996). Dev. Biol. 177: 217-225; (5) Tepass and Knust, (1990). Roux´s Arch. Dev. Biol. 199: 189-206; (6) Sulston et al., (1983). Dev. Biol. 100: 64-119; (7) Schroeder and McGhee, (1998). Development 125: 4877-4887.

24 May 1999 15:50 253 253 Developmental regulation of the cell cycle in C. elegans

1999 International Worm Meeting abstract 202 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Developmental regulation of the cell cycle in C. elegans M Boxem, S van den Heuvel MGH Cancer Center, Charlestown MA 02129 The mechanisms by which developmental signals interact with the general cell-cycle machinery remain poorly understood. To address this issue in C. elegans, we first characterized components of the basic C. elegans cell-cycle machinery. We focused on a family of key cell-cycle regulators, the Cyclin-dependent kinases (Cdks). By molecular biological approaches and BLAST database searches, we identified six putative C. elegans Cdks. In RNAi experiments, only one of these kinases blocked cell division completely. This kinase, NCC-1 (first identified by Mori et al., 1994), could act to promote passage through a single cell-cycle transition, analogous to Cdks in higher eukaryotes, or could promote passage through multiple cell-cycle transitions, like CDC28/cdc2 in yeasts. To differentiate between these two possibilities, we characterized ncc-1 loss-of-function mutations isolated in a genetic screen based on the phenotype of rare larval arrested ncc-1(RNAi) animals (Boxem et al., in press). We found that ncc-1 is required for M phase in meiotic and mitotic divisions, but not for progression through G1 and S phase. Our results indicate that C. elegans uses multiple Cdks to regulate cell-cycle transitions, similar to higher eukaryotes, and that ncc-1 is the C. elegans ortholog of Cdk1/Cdc2 in other metazoans. None of the six identified kinases were found to be essential for DNA replication by RNAi experiments. Therefore, we addressed the possibility of redundancy between these kinases. Injection of dsRNA from K03E5.3 (an ORF ~40% identical to Cdk1/Cdc2, Cdk2 and Cdk3) into ncc-1/+ animals resulted in more severely mutant progeny than injection into N2, indicating a possible redundancy between these two kinases. To identify genes involved in linking developmental processes to the intrinsic cell-cycle machinery, we initiated screens to identify mutants that arrest early in the cell cycle, when external signals are most likely to affect proliferation. We are analyzing four mutants isolated in a screen for conditional alleles that arrest cell division in L1. For this screen hydroxyurea was used to select against animals that entered S-phase at the nonpermissive temperature. In addition, we have initiated a screen based on absence of the S-phase marker RNR::GFP (R. Roy and V. Ambros) to identify mutants that arrest cell division in G0 /G1 .

24 May 1999 15:50 254 254 Complex alteration of respiration, metabolic potential and ATP stores in long-lived Clk mutants of Caenorhabditis elegans

1999 International Worm Meeting abstract 203 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Complex alteration of respiration, metabolic potential and ATP stores in long-lived Clk mutants of Caenorhabditis elegans BP Braeckman, K Houthoofd, A De Vreese, JR Vanfleteren Department of Biology, University of Gent, Ledeganckstraat 35, B-9000 Gent, Belgium Clk mutants of Caenorhabditis elegans are characterised by an overall slow down of temporal processes and increase in life span. It was hypothesised that Clk mutations slow down the pace of many cellular functions and lower the rate of energy metabolism, possibly resulting in slower production of reactive oxygen species which in turn could result in slower ageing. We tested this hypothesis by measuring respiration rates, light production capacities (a measure of metabolic potential) and ATP levels in various strains harbouring mutant alleles of the Clk genes clk-1 and gro-1, and of three other genes that interact with the Clk genes. We found a mild reduction of oxygen consumption rates and little alteration of metabolic capacities in the single Clk mutants during the first 4-5 days of their adult lives, relative to the wild-type strain. This difference tended to fade away with increasing age, however, and aged Clk mutants eventually retained higher metabolic capacities than the wild-type control strain N2. These profiles are suggestive of physiological time being retarded, relative to chronological time in Clk mutans. Ageing clk-1 and gro-1 mutants also retained substantially elevated ATP levels relative to N2, and the simultaneous presence of the daf-2(e1370) or age-1(hx546) alleles boosted this effect. Thus, energy production and consumption appear to be uncoupled in these mutants. Mutation in daf-16 suppressed the Age and ATP phenotypes, but not the reduction of respiration rate imparted by mutation in clk-1.

24 May 1999 15:50 255 255 Further Analysis of the Gonad-Dependent Mechanism of Sex Myoblast Migration

1999 International Worm Meeting abstract 204 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Further Analysis of the Gonad-Dependent Mechanism of Sex Myoblast Migration CS Branda, MJ Stern Yale University, New Haven, CT 06520 During hermaphrodite development, two sex myoblasts (SMs) are generated in the posterior of the animal and migrate anteriorly to final positions flanking the precise center of the developing gonad. The gonad is responsible for attracting the SMs to their precise positions; this attraction requires the activities of the genes egl-17, which encodes a fibroblast growth factor (FGF), and egl-15, which encodes a fibroblast growth factor receptor (FGFR). We have previously presented evidence that EGL-17/FGF can act as an instructive guidance cue for the SMs; egl-17 is visibly expressed in P6.p in a gonad-dependent manner and can attract the SMs from this source1 . However, in the absence of the vulval precursor cells (VPCs), the SMs still reach their precise positions, suggesting that there is an additional source of egl-17 expression outside of P6.p that we have not detected using our original egl-17 reporters. The gonad-dependence of precise SM positioning suggests that the gonad itself is likely to be the additional source. To explore the possibility that our original rescuing constructs do not contain enhancer regions essential for gonad-dependent expression of egl-17 outside of the VPCs, we have tested the capability of egl-17 genomic fragments to restore precise SM positioning in lin-39; egl-17 double mutants. Rescuing activity must come from a source other than the VPCs in this double mutant since lin-39 is required for the VPCs to express egl-17. In this background, the original egl-17 rescuing fragment restores precise SM positioning only partially, whereas a larger construct with an expanded upstream regulatory region can fully rescue. This larger fragment drives expression of GFP in additional cells, including a subset of cells within the somatic gonad. In addition to being a source of an attractive guidance cue, the gonad also appears to be the source of a repulsive cue since the SMs are actively repelled from the gonad in egl-17 and egl-15 mutants. In the process of testing candidate genes for their potential role in mediating this gonad-dependent repulsion, we found that sax-3/robo does not mediate the repulsion, but does affect the dorsal/ventral localization of the SMs in the absence of the gonad-dependent attraction.

1 Burdine, R. et al. (1998) Development 125:1083-93

24 May 1999 15:50 256 256 Isolation and Characterization of Suppressors of the C. elegans timing gene clk-1

1999 International Worm Meeting abstract 205 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Isolation and Characterization of Suppressors of the C. elegans timing gene clk-1 R Branicky, J Feng, S Hekimi Dept. of Biology, McGill University, 1205 Dr. Penfield Ave., Montreal Quebec, Canada H3A 1B1 Mutations in the maternal effect gene clk-1 affect developmental, cellular and behavioural timing. They result in a mean lengthening of embryonic and post-embryonic development, cell cycle period, and lifespan as well as the defecation, swimming and pharyngeal pumping cycle periods. CLK-1 has structural and functional homologues from yeast (Coq7p) to humans. In yeast, coq7 mutants cannot respire because they cannot manufacture ubiquinone. However, unlike coq7 mutants which cannot respire at all, the phenotype of clk-1 mutants is only a mild (~10%) reduction in respiration, suggesting that clk-1 is involved in the regulation of energy metabolism. However, it is unclear how such a mild defect in respiration would result in the dramatic slowdown of most timed processes in the worm. In order to study the pleiotropy of clk-1 mutations, we are studying clk-1 at its interface with one of the features that it affects, the defecation cycle. We have designed a screen for suppressors of the slow defecation phenotype of clk-1 worms that is efficient and highly specific. We have identified a number of mutations (9 out of ~1500 haploid genomes screened) that can suppress clk-1. Some of these mutations can speed up defecation in clk-1 mutants to even faster than wild-type rates. Together with the prevalence of clk-1 suppressors, these observations suggest that the general slowdown in behaviour exhibited by clk-1 worms is not due to a simple energy limitation. Rather, they lend support to the theory that clk-1 is involved in the regulation of a number of distinct biological clocks.

24 May 1999 15:50 257 257 Identification of promoter elements of parasitic nematode genes in transgenic C. elegans

1999 International Worm Meeting abstract 206 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification of promoter elements of parasitic nematode genes in transgenic C. elegans C Britton 1 , DL Redmond 2 , DP Knox 2

1 Wellcome Centre for Molecular Parasitology, University of Glasgow, 56 Dumbarton Road, Glasgow, UK. 2 Moredun Research Institute, Pentlands Science Park, Midlothian, UK.

Very little is known of gene regulatory mechanisms and transcription factors in parasitic nematodes. Studies in parasitic species are limited by the complex life cycles and absence of a suitable transformation system. As many aspects of gene expression and regulatory mechanisms are likely to be conserved in different nematode species, we have made use of C. elegans as a heterologous transformation system to examine parasite promoter activity. So far we have observed strict tissue-specific lac Z reporter gene expression following transformation of C. elegans with three different parasite promoter constructs. Constructs containing 2 kb of upstream sequence of the parasite genes pep-1, a pepsinogen gene and ac-2, a cysteine protease gene, both of which are expressed in the gut of the sheep parasitic nematode Haemonchus contortus, direct expression exclusively in gut cells of transgenic C. elegans. Using upstream sequence of a parasite cuticular collagen gene, expression is observed exclusively in hypodermal cells. By deletion and mutation analysis we have begun to identify the putative parasite regulatory motifs important for this tissue-specific expression and aim to characterise the trancription factors involved.

24 May 1999 15:50 258 258 G protein signaling pathways in C. elegans

1999 International Worm Meeting abstract 207 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. G protein signaling pathways in C. elegans LA Brundage, PW Sternberg, MI Simon Division of Biology, California Institute of Technology, Pasadena CA 91125 In mammals, heterotrimeric G proteins are classified into four families, Gi/Go, Gq, Gs and G12, based on similarity of their sequence and function. C. elegans egl-30 encodes a G protein alpha subunit which is more than 80% identical to two members of the mammalian Gq alpha family(1). We are interested in genes which act in the egl-30 pathway, as they may encode new regulators of G protein signaling. eat-11 is one such gene. eat-11 animals arrest at hatching in the presence of low levels of the cholinergic agonist arecoline(2) and mutations in egl-30 strongly suppress this arecoline effect(1). We have found that eat-11 animals exhibit behaviors which are opposite to egl-30(r.f) animals in several respects. egl-30(r.f.) animals move slowly and with reduced flexations, and lay few eggs in the presence of serotonin. By contrast, eat-11 animals move more quickly than wild type animals and with exaggerated flexations. In addition, eat-11 animals respond dramatically more strongly than N2 to serotonin in an egg laying assay. We hope to clarify the relationship between egl-30 and eat-11 by determining the molecular identity of the EAT-11 protein. To elucidate the G12 pathway in C. elegans, we are studying the gene gpa-12, encoding a G12 alpha subunit on cosmid F18G5. In contrast to the other G protein families, the receptors and the second messengers involved in transduction of signals via the G12 family are largely unknown. Reexamination of expression patterns has shown that gpa-12::gfp is expressed most strongly in the pharyngeal muscle and hypodermus. We have engineered a constitutively active allele of gpa-12 under hsp-16 control. When this allele is induced by heat shock during embryogenesis, animals hatch into uncoordinated, developmentally arrested larvae which eventually recover. We plan on exploiting this phenotype to identify gpa-12 effectors in a genetic screen.

(1) Brundage, L., Avery, L., Katz, A., Kim, U-J., Mendel, J.E., Sternberg, P. W., and Simon, M.I. (1996). Mutations in a C. elegans Gqa gene disrupt movement, egg laying, and viability. Neuron 16: 999-1009. (2) Avery, L. (1993) The genetics of feeding in Caenorhabditis elegans. Genetics 133: 897-917.

24 May 1999 15:50 259 259 ceh-13 is involved in the anterior organization of the C. elegans embryo

1999 International Worm Meeting abstract 208 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. ceh-13 is involved in the anterior organization of the C. elegans embryo K Brunschwig 1 , C Wittmann 1 , R Schnabel 2 , TR Bürglin 3 , H Tobler 1 , F Müller 1

1 Institute of Zoology, University of Fribourg, Perolles, CH-1700 Fribourg. 2 Institut für Genetik, TU Braunschweig, Spielmannstr. 7, D-38106 Braunschweig. 3 Department of Cell Biology, Biozentrum, CH-4056 Basel.

The remarkable conservation of Hox genes among metazoans has led to the proposal that these genes may be fundamental to the control of axial patterning in all animals. ceh-13, which is the labial ortholog, belongs to the C. elegans Hox cluster. By using specific antibodies and reporter constructs, we have shown that the ceh-13 gene has a complex expression pattern and that the rostral boundary of its expression domain is anterior to those of the other Hox genes. In order to link the expression pattern with the function of ceh-13, we have isolated a ceh-13 loss-of-function allele, sw1, by transposon-mediated mutagenesis. The ceh-13(sw1) homozygotes exhibit a Vab phenotype characterized by incompletely penetrant zygotic lethality. On average, 97% of the ceh-13(sw1) mutants arrest during embryogenesis or at early larval stages. From the onset of elongation, lineage analyses and immunostainings revealed defects in the organization of the anterior epidermis and anterior body wall muscle cells, nevertheless, the identity of these cells is correctly specified. In addition, ceh-13(sw1) mutant embryos show fusion and adhesion defects in ectodermal cells. Interestingly, at this stage, CEH-13 is present in anterior hypodermal cells as well as in anterior dorsal body wall muscle cells and in cells of the prospective ventral nerve cord. To further analyze how positional information is generated and maintained during morphogenesis, we are currently trying to identify upstream and downstream interacting genes. Our preliminary results suggest that adhesion molecules may be regulated by the ceh-13 gene.

24 May 1999 15:50 260 260 A C. elegans histone H3-like protein localizes to the centromere of both holokinetic and monokinetic chromosomes.

1999 International Worm Meeting abstract 209 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A C. elegans histone H3-like protein localizes to the centromere of both holokinetic and monokinetic chromosomes. BJ Buchwitz, LL Moore, K Ahmad, S Henikoff, MB Roth Division of Basic Sciences and Molecular and Cellular Biology Program, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109 The proper segregation of sister chromatids during mitosis is mediated by a specialized region of the chromosome known as the centromere. Recent studies have shown that specialized nucleosomes, containing histone H3-like proteins (Cenp-A in mammals and Cse4p in budding yeast), reside at the centromere of monokinetic chromosomes and are important for the proper segregation of sister chromatids. We are examining this phenomenon using C. elegans holokinetic chromosomes. A search of the C. elegans genome identified CeCenp-A as a potential homolog of Cenp-A and Cse4p. RNAi with CeCenp-A resulted in chromosome segregation defects and embryo lethality. To examine the wildtype distribution of the protein, we raised a rabbit polyclonal antibody against CeCenp-A. The antibody recognizes interphase nuclei and condensed chromosomes. By late prophase, there are two distinct, longitudinal bands of reactivity on each chromosome. At metaphase, the reactivity is localized to the poleward faces of the metaphase plate. We can eliminate staining by either blocking with the antigen used to generate the antibody or by RNAi with CeCenp-A. These observations are consistent with CeCenp-A being a centromeric component of the holokinetic C. elegans chromosome and suggest that CeCenp-A is a homolog of Cenp-A and Cse4p. To investigate the relationship between holokinetic and monokinetic chromosomes, we expressed CeCenp-A as a GFP fusion protein in Drosophila melanogaster Kc167 cells. The fusion protein was localized to the centromeres of the monokinetic D. melanogaster chromosomes, as demonstrated by co-localization with POLO kinase, a kinetochore marker. This suggests a conserved mechanism for segregation of holokinetic and monokinetic chromosomes.

24 May 1999 15:50 261 261 Inositol Phosphate Signaling in C. elegans

1999 International Worm Meeting abstract 210 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Inositol Phosphate Signaling in C. elegans YK Bui, M Wang, PW Sternberg HHMI/Division of Biology, Cal Tech 156-29, Pasadena, CA 91125 USA The LET-23 receptor tyrosine kinase activates a ras dependent signaling pathway to mediate several tissue specific functions in C. elegans. We showed previously,(Clandinin et al. Cell 1998. 92: 523-533.)that LFE-1/ITR-1, an inositol 1,4,5-triphosphate receptor (IP3R) homologue, acts as a ras independent positive effector of LET-23, while LFE-2/IP3 kinase acts as a negative effector to mediate fertility. Cytosolic calcium levels are known to encode signals in excitable and non-excitable cells to mediate various cellular functions. The levels of IP3 and possibility IP4 are important for regulating intracellular calcium levels which mostly likely control spermethecal dilation and hence ovulation. We are working on microscopy techniques to image cytosolic calcium levels during ovulation in the worm. We are also conducting screens to identify more components of this fertility pathway to increase our understanding of the regulatory network of proteins that control calcium levels, as well as the mechanism by which tyrosine receptor kinases can activate distinct signaling pathways in different tissues. Presently, we have identified potential candidate suppressors of the sterile lfe-1; lfe-2double mutant, two of which are extragenic suppressors and two of which are possibly intragenic lfe-1 revertants. To investigate whether altered membrane potential may affect IP-3 induced calcium release, we are also constructing double mutants of lfe-1with other regulators of cytosolic ion flux. Because IP3- induced increases in intracellular calcium concentrations are important for a number of receptor signaling pathways in many cell types (Berridge, Nature 1993. 361: 315), studying how these components regulate calcium mobilization should increase our general understanding of calcium signaling.

24 May 1999 15:50 262 262 Mutations that Affect Synaptic Localization of GLR-1

1999 International Worm Meeting abstract 211 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Mutations that Affect Synaptic Localization of GLR-1 M Burbea, CG Rongo, JM Kaplan Department of Molecular and Cellular Biology, 361 Life Sciences Addition, UC Berkeley, Berkeley, CA 94720-3200 The orderly flow of information in the nervous system requires that proteins are specifically targeted to pre- and post-synaptic specializations. We are interested in identifying the molecules that target and maintain proteins to synapses. We have used the GLR-1 AMPA type glutamate receptor as a marker for post-synaptic localization. A wild type animal expressing a GLR-1::GFP fusion exhibits punctate spots along the ventral nerve chord representing clustering of the glutamate receptor at specific synaptic terminii. To identify genes involved in synaptogenesis, we are screening mutagenized GLR-1::GFP worms for defects in GLR-1 localization. We predict classes of mutants to represent trafficking, targeting, localization and stabilization molecules necessary to initiate and maintain synaptic architecture. Approximately 3500 genomes have been clonally screened so that sterile or inviable mutants may be recovered as heterozygotes. Eight mutants have been isolated which are aberrant in GLR-1::GFP localization and their phenotypes will be described.

24 May 1999 15:50 263 263 Analysis of C. elegans non-coding regions: a bioinformatics approach

1999 International Worm Meeting abstract 212 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of C. elegans non-coding regions: a bioinformatics approach M Burke, EF Ryder Worcester Polytechnic Institute, Dept. of Biology and Biotechnology, Worcester, MA 01609 With the C. elegans genome now completely sequenced, and predicted coding regions well-documented, it seems reasonable to try to gain more information about the upstream non-coding regions of predicted genes. It would be useful to be able to document these regions in a way similar to the coding regions, so that eventually we could predict when and where a gene of interest would be turned on, just by looking at the associated upstream sequence. The initial goal of this project is to use the genome sequence to find consensus sequences in non-coding regions that may direct expression of genes to specific sets of cells. We used a database of about 100 expression patterns of genes that are expressed in the nervous system, kindly provided by Shawn Lockery (University of Oregon). We performed pair-wise comparisons of the upstream regions of 6 genes that are expressed in the ASI neurons, and 5 genes that are expressed in the ASK neurons. We located an 11 base pair consensus sequence that was found as a perfect match in the upstream regions of 4 out of 6 genes expressed in ASI neurons, but in none of the regions upstream of genes expressed in ASK neurons. As another control, we searched for this consensus sequence in randomly generated DNA sequence containing the same percentage composition of each base as was found in the upstream region of genes expressed in ASI neurons. Only partial matches of the consensus were found in 5 of the 10 randomly generated sequences. We are continuing this analysis to try to locate other consensus sequences. We plan to test the predictive value of these sequences by determining the expression patterns of genes whose upstream regions contain such consensus sequences.

24 May 1999 15:50 264 264 In Vivo Analysis of Troponin I Domains in Muscle Function

1999 International Worm Meeting abstract 213 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. In Vivo Analysis of Troponin I Domains in Muscle Function A Burkeen 1 , J Sulcove 1 , EA Bucher 2 , T Allen 1

1 Biol. Dept, Oberlin College, Oberlin, OH 44074. 2 Dept of Cell and Developmental Biology, U. of Pa., Phila., PA 19104.

In vitro biochemical experiments have led to predictions about the role of particular regions of TnI in regulation of contraction of muscle. To test whether these predictions hold in vivo, we have initiated a study of the unc-27 gene in C. elegans, which is represented by three available alleles (e155, su142sd, and su195sd). Mutants for the unc-27 gene show either sluggish or no movement on bacterial lawns and display structural abnormalities in body wall muscle cells (Brenner. Genetics 77:71-94, 1974; Zengel & Epstein. Cell Motil. 1:73-97, 1980); transformation with a cosmid (ZK721) containing the C. elegans TnI-2 gene rescues the strain from its mutant phenotype (pers. com., R.J. Barstead). Analysis of swimming behavior of wild-type and unc-27 homozygous adult hermaphrodites in nonviscous (S-basal buffer; rel. viscosity 1.0) and viscous (S-basal with 1% methyl cellulose; rel. viscosity 4.5) media revealed that su142sd is the most deleterious and that su195sd is the least. Sequencing showed that the mutant alleles code for prematurely terminated TnI-2: the recessive e155 at residue Gln10, the semi-dominant su142sd at Gln122 (aligns with Lys87 of rabbit fast TnI), and the semi-dominant su195sd at Glu207 (aligns with Met170 of rabbit fast TnI). In situ localization of mRNA of TnI-2 and the three other C. elegans TnI genes explained the late onset of muscular defects associated with unc-27 mutations, as well as the sparing of muscle groups other than the body wall. TnI-2 is expressed in larval and adult body-wall muscle, whereas TnI-1 is expressed in embryonic/larval body-wall muscle, as well as larval/adult reproductive muscles. TnI-4 is expressed in the pharynx. The rigid paralysis caused by su142sd confirms in vivo that the central region of TnI is needed for inhibition of tension development. The semi-dominance of the allele and the severity of paralysis it causes, relative to that caused by e155 (a presumed null), suggest that TnI truncated just before the "inhibitory region" (aa 96-118 of rabbit fast TnI) is capable of competing against wild-type TnI-1 and TnI-2 for binding sites on the thin filament and of causing disinhibition/activation of contraction. Supported by Templeton Medical Research Foundation (TA) and NIH (EAB).

24 May 1999 15:50 265 265 Molecular characterization of mutations in dig-1, a gene involved in sensory map formation.

1999 International Worm Meeting abstract 214 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Molecular characterization of mutations in dig-1, a gene involved in sensory map formation. CT Burket, S Hubbard, EF Ryder Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA 01609. We are interested in the development of topographic maps. Topographic maps coordinately map spatial information from the environment to the brain. We are studying the maps formed by two sets of head sensory neurons in C. elegans, the IL1’s (visualized by an unc-5::GFP construct kindly provided by J. Culotti) and the IL2’s (visualized by DiO staining; C. Bargmann, pers. comm.). Currently, we are studying mutations in the gene dig-1 that affect the formation of these maps. In dig-1 mutant animals, IL1 and IL2 sensory processes are aberrant, and sometimes fail to reach their normal destinations in the nose (WBG 14(1):49). Two dig-1 alleles show changes by PCR and Southern analysis in a very large candidate cell adhesion molecule found on cosmid K07E12. One allele, dig-1(n1480), was created by gamma ray mutagenesis, and shows a small rearrangement in the 3’ end of the candidate gene. A second allele, dig-1(nu336), occurred during an EMS mutagenesis of a strain containing the unc-5::GFP marker as a non-integrated array. The array integrated during the mutagenesis, and segregated with the dig-1 mutation, suggesting that the integration caused the mutation. We have now determined by PCR that the change we observed in the candidate gene was not due to this insertion, but instead is due to a point mutation resulting in an RFLP near the 5’ end of the candidate gene. We are now separating the GFP insertion from the RFLP to determine whether the RFLP is solely responsible for the phenotype of nu336. We will then sequence the affected regions of both this allele and n1480 in order to determine the precise molecular basis of the mutations. In addition, we are conducting RNAi experiments to phenocopy the mutant phenotype, as well as Northern analysis to determine cDNA structure and expression patterns of the candidate gene.

24 May 1999 15:50 266 266 Suppression of the neuropeptide gene flp-1 in Caenorhabditis elegans.

1999 International Worm Meeting abstract 215 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Suppression of the neuropeptide gene flp-1 in Caenorhabditis elegans. ER Bush, H Field Dept. of Biology, Boston University, 2 Cummington Street, Boston, MA02215. The neuropeptide gene flp-1 is one of at least 20 genes encoding FMRFamide-related neuropeptides (FaRPs) in the nematode Caenorhabditis elegans. flp-1 can be alternatively spliced to produce two transcripts, which encode seven distinct FaRPs, six of which have been biochemically isolated from C. elegans (Rosoff et al, 1992). The NY2009 strain of C. elegans carries a construct containing a flp-1 cDNA under the control of an inducible heatshock protein (hsp16) promoter (Phsp::flp-1). Heatshock of these animals induces overexpression of flp-1 and results in a locomotory defect characterized by sluggish movement and a flattened waveform (Nelson et al, 1998). The transgene is inserted into chromosome IV. We have chemically mutagenized these worms to isolate a mutation that suppresses the sluggish phenotype induced by overexpression of flp-1. 624 haploid genomes have thus far been screened. Two individuals from the same haploid parent suppress the sluggish phenotype. These animals both contain the same allele (yn7), and have been designated NY44 and NY44A. The suppressor mutation is on chromosome IV. Animals have a defecation phenotype independent of their suppression of flp-1 overexpression-induced sluggishness. The defecation cycle period of yn7 animals (NY44, 103.3±6.1 seconds; n=12) is significantly longer than that of wildtype (5.4±2.7 seconds; n=15; P<0.05). Wildtype defecation period (77.2±3.9 seconds; n=15) is lengthened at 16°C, but this is not true for the suppressor allele (NY44, 102.2±1.7 seconds; n=12). NY44 and NY44A defecation periods are significantly different from wildtype at all temperatures (P<0.05). This suppressor mutation may affect a gene that acts downstream of flp-1, such as a protease, receptor or signaling molecule. Alternatively, it may affect one of the other flp genes in C. elegans.

Nelson, L.S., Rosoff, M.L. & Li, C. Science 281 1686-1690 (1998). Rosoff, M.L., Burglin, T.R. & Li, C. Journal of Neuroscience 12 2356-2361 (1992).

24 May 1999 15:50 267 267 Studying human disease genes in C. elegans

1999 International Worm Meeting abstract 216 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Studying human disease genes in C. elegans EA Buttner, HR Horvitz HHMI, Dept. Biology, MIT, Cambridge, MA 02139 USA We are generating worm models of hereditary neurological diseases to study the genetic mechanisms underlying these disorders. Our current efforts are focused on models of the gain-of-function diseases familial amyotrophic lateral sclerosis (FALS) and Huntington’s Disease (HD) and the loss-of-function diseases spinal muscular atrophy (SMA) and lissencephaly. To attempt to model ALS we have generated transgenic worms carrying extrachromosomal arrays of the human copper/zinc superoxide dismutase cDNA driven by conditional or constitutive promoters. We employed a similar strategy to attempt to model HD. We are currently performing western blot analyses to assay expression of the human proteins in our transgenic strains. We have used RNAiand PCR-based deletion screening to study the loss-of-function phenotypes associated with disruption of C. eleganshomologs of the known lissencephaly disease genes LIS1 and DCX and the LIS1-interactor nudC. RNAiof the LIS1-like gene produced an embryonic-lethal phenotype. We have isolated two deletion mutants of the worm LIS1 homolog and are now characterizing their phenotypes. Studies of Aspergillusand mice indicate that LIS1 interacts with a gene similar to the Aspergillus nudCgene. RNAiof the C. elegans nudChomolog yields an embryonic-lethal phenotype; escapers die at the L3 and L4 stages or become sterile adults. We have also obtained a candidate deletion within the worm nudChomolog and are now isolating worms containing this deletion. Mutations of DCX (doublecortin) in humans produce X-linked subcortical laminar heterotopia and lissencephaly syndrome. RNAiof the DCX homolog produced a variety of defects at low penetrance, including embryonic lethality, rollers, and severely malformed bodies. We are currently screening for deletions of the worm DCX homolog. RNAi of the SMA disease gene Survival of Motor Neurons (SMN) homolog yielded sterile progeny, many of which moved with abnormally deep body bends. Abnormalities of the reproductive system included morphologically misshapen gonads and defective oocyte maturation. We have obtained a candidate deletion of the SMN homolog and are isolating worms with this deletion.

24 May 1999 15:50 268 268 unc-16 May Play a Role in DD Motorneuron Remodeling in C. elegans

1999 International Worm Meeting abstract 217 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. unc-16 May Play a Role in DD Motorneuron Remodeling in C. elegans DT Byrd, M Walcoff, Y Jin University of California, Santa Cruz, Dept. Biol., Sinsheimer Labs, CA 95064 The DD motorneurons are born embryonically, innervate ventral body wall muscles in L1 animals, and remodel to innervate dorsal body wall muscles in L2 through adult animals. We visualize the process of DD remodeling in vivo with Punc-25-SNB-1::GFP, a synaptic vesicle marker expressed by the DDs and other GABAergic neurons 1 . Using this marker, Hallam and Jin found that lin-14, a heterochronic gene, regulates the timing of DD remodeling 2 . lin-14 loss-of function mutant L1s express Punc-25-SNB-1::GFP precociously along the dorsal cord, indicating that the DDs remodel early. In order to identify additional molecular components involved in DD neuron remodeling, we screened F2 L1 progeny of EMS mutagenized animals for precocious dorsal expression of Punc-25::SNB-1::GFP. We isolated ju79 from 500 haploid genomes. Linkage, three-factor, and deficiency mapping placed ju79 close to ced-7 on LGIII. Complementation analysis with candidate genes revealed that ju79 is a fifth allele of let-981, a locus identified by Dr. Ann Rose’s lab in screens for essential genes. In the process of refining the genetic mapping, we found that unc-16(e109) failed to complement ju79. We also found that unc-16/sDf110 L1s express Punc-25-SNB-1::GFP precociously and are larval lethal, indicating that, contrary to earlier map data, sDf110 fails to complement unc-16. ju79 and alleles of unc-16 and let-981 form an allelic series in which ju79, unc-16(e109 and n730), and let-981(s1072) are partial loss-of-function alleles and let-981(s453, s529, and s222) are genetic null alleles. The loss-of-function phenotypes range from sluggish and egg-laying defective in ju79 and unc-16(e109 and n730) to low penetrance larval lethal or sterile in let-981(s1072). The null phenotype, exhibited by let-981(s453, s529, and s222), is larval lethal. We have rescued unc-16 with a single cosmid and will report our molecular identification of the gene.

1. Nonet, M. 1999. J. Neurosci. Methods. In press. 2. Hallam and Jin. 1998. Nature 395:78-82.

24 May 1999 15:50 269 269 mec-14 Encodes a Novel Member of the Oxido-reductase Superfamily That May Modulate Mechanosensory Channels

1999 International Worm Meeting abstract 218 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. mec-14 Encodes a Novel Member of the Oxido-reductase Superfamily That May Modulate Mechanosensory Channels GA Caldwell, L Chen, N Hom, S Gangadharan, M Huang, M Chalfie Department of Biological Sciences, Columbia University The product of the C. elegansmec-14gene is required for the function of the six touch receptor neurons. Mutant animals are touch insensitive, yet have differentiated touch receptor neurons. Genetic interactions between mecgenes have suggested that mec-14encodes a protein that modulates the proposed mechanosensory apparatus. The mec-14gene had been previously mapped to a region on Chr. III. We have cloned mec-14using a combination of transposon-tagging and cosmid/plasmid rescue. Sequence analysis of all 9 existing alleles of mec-14indicates that these mutations lie within the same predicted open-reading frame. mec-14produces a 1.5 Kb transcript that disappears in mec-3mutants and is truncated in a partial deletion of the mec-14locus. MEC-14 is a protein of 453 amino acids that exhibits some similarity to aldo-keto reductases and the b-subunits of Shaker-type potassium channels. Although this homology is weak, it supports a regulatory role for the MEC-14 protein in mechanotransduction. The dendrogram of a converged set of all members of the oxido-reductase family in the GenBank database suggests the mec-14gene product lies on a distinct branch of this family tree. We have generated both GFP and lacZ fusions to the C-terminal codon of mec-14that restore touch sensitivity in mutants with a mec-14 (u82)deletion allele. Transformed animals display a bright and punctate staining/ fluorescence in both the cell body and along the length of the axons that is restricted to the six touch cells. This pattern of expression may represent the distribution of mechanosensory channels or associated proteins in touch cells. Further analysis of this expression pattern in mecmutant animals indicates that MEC-14 may be linked to the cytoskeleton since this punctate expression becomes diffuse in a mec-12 e1605(a-tubulin defective) mutant, but remains present in other mecmutants. We have also developed a novel expression cassette in which stop codons were placed in all frames just upstream of a SL-2 splice signal from the deg-3gene and subcloned into a polylinker enables simultaneous rescue of mutant phenotypes and reporter gene expression - without an a priorirequirement of direct gene fusion to GFP.

24 May 1999 15:50 270 270 C. elegans Genes Representing a Link Between Nuclear and Neuronal Migration

1999 International Worm Meeting abstract 219 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. C. elegans Genes Representing a Link Between Nuclear and Neuronal Migration GA Caldwell, AL Dawe, NR Morris, M Chalfie Columbia University. Nuclear migration is essential for proper growth, development, and cellular function in all eukaryotes. In the filamentous fungus Aspergillus nidulansfour genes required for nuclear migration have been cloned. Because they encode cytoplasmic dyneins, the nudAand nudGgenes are directly implicated in nuclear movement. The nudFgene product encodes a protein that is most similar to human LIS-1. Mutations in LIS-1cause Miller-Dieker lissencephaly - a severe disorder in which arrested neuronal migration results in the underdevelopment of the cerebral cortex. The nudCgene encodes a protein of unknown function, however, since a nudCmutant exhibits a temperature-sensitive deficiency of NUDF protein, NUDC appears to regulate NUDF. We previously identified the worm homologs of nudCand nudFand named these nud-1and lis-1,respectively. Expression of nud-1cDNA rescues the nuclear migration defect of a nudCmutant in A. nidulans,thereby indicating a conserved function between these two proteins. In contrast, neither human nor C. elegans lis-1rescues nudF. A nud-1::gfpfusion indicates this gene is strongly expressed in the amphid and phasmid sensory neurons and in embryos, and is transiently expressed in the gonadal primordium and gut. Likewise, expression of a C. elegans lis-1::gfpfusion was primarily observed in neuronal processes such as the dorsal and ventral nerve cords and commissures. Additional expression of lis-1is localized to multinucleated cell-types or syncitia including the spermathecal valve and lateral seam cells (in adults). Whereas differences between nud-1and lis-1GFP expression indicate these genes may function independently in certain tissues, RNAi experiments on both nud-1and lis-1yield similar phenotypes (including embryonic lethality, sterility, and altered vulval morphology), supportive of a joint role in gonadal and germline development. These observations provide the basis for investigating mechanisms by which nuclear migration may function in various aspects of C. elegansdevelopment.

24 May 1999 15:50 271 271 Molecular mechanisms of pop-1 transcriptional regulation

1999 International Worm Meeting abstract 220 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Molecular mechanisms of pop-1 transcriptional regulation D Calvo, Y Shi Harvard Medical School, Department of Pathology, 200 Longwood Ave, Boston, MA, 02115, USA. POP-1 is a putative DNA-binding protein containing a HMG box motif related to the TCF/LEF-1 family of transcription factors. In Drosophila and mammals, these molecules play a critical role in the Wnt signaling pathway. TCF/LEF-1 proteins can function as activators or repressors depending on the cellular millieu and/or promoter context. In C. elegans, POP-1 has been shown to be essential in cell fate determination during early embryogenesis. Mutations in the genes encoding components of the wnt pathway result in E and MS both adopting MS-like fates, suggesting a role of this pathway in endoderm specification. In contrast, pop-1 genetic mutants display a phenotype opposite to that of the wnt-related molecules in gut induction, as both E and MS cells adopt an E-like fate, indicating that POP-1 activates MS-specific differentiation, represses E fate, or both. However, very little is known about the biochemical role of this molecule and the molecular mechanisms that underlie its activity. To elucidate the role that POP-1 plays in these processes, we investigated the molecular mechanisms by which POP-1 carries out its biological activities in cell fate determination and differentiation. We fused different domains of pop-1 in frame to the DNA-binding domain of GAL-4 and assayed these chimeras for their transcriptional activity in mammalian cells. Full length GAL4-POP-1 represses transcription of a reporter gene, consistent with the genetic data. We have identified a hydrophobic region that is necessary for transcriptional repression and are in the process of identifying amino acid residues that are critical for this repression activity. With the repression-defective mutants, we will use both biochemical and genetic approaches to identify co-repressors involved in POP-1-mediated repression. We have also identified an activation domain located at the N-terminal region of POP-1. The corresponding region in TCF and LEF-1 (POP-1 homologs from Drosophila and mammals) interacts with b-catenin. Work is in progress to determine interaction of b-catenin with this region of POP-1 and the functional importance of such an interaction. The repression- and activation-defective POP-1 mutants will be analyzed for their ability to rescue the phenotypes associated with the pop-1 genetic mutations.

24 May 1999 15:50 272 272 pag-3 may specify both neuroblast cell fate and terminal fates during development of the ventral cord

1999 International Worm Meeting abstract 221 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. pag-3 may specify both neuroblast cell fate and terminal fates during development of the ventral cord S Cameron 1,2 , JB McDermott 3 , E Aamodt 3 , B Horvitz 1

1 HHMI, Dept. Biology, MIT, Cambridge, MA 02139. 2 Children’s Hospital/Dana-Farber Cancer Institute, Boston, MA 02115. 3 LSU Medical Center, Shreveport, LA 71130.

Pathways controlling the programmed deaths of specific cells in C. elegans are likely to be evolutionarily conserved, and human homologues of genes in these pathways are candidate disease genes. We recovered two alleles of the gene pag-3 as mutations that result in increased numbers of cell corpses in the ventral cord (see abstract by Cameron, Tsung, and Horvitz). pag-3 is a 336 amino acid Zn-finger protein with extensive identity to the mammalian proto-oncogene Gfi-1. We have shown that in pag-3 animals the Pn.aaa neuroblast reiterates the fate of its mother, Pn.aa, generating supernumerary Pn.aap cells. These cells can undergo programmed cell death, resulting in an increased number of cell corpses in the ventral cord. These findings establish a role for pag-3 in determining neuronal cell fate by regulating cell lineage. pag-3 may also function to regulate terminal aspects of neuronal differentiation. To begin to test this idea we used an affinity-purified antiserum to define the expression pattern of PAG-3 and, in particular, to determine whether it is present during terminal differentiation. We found that PAG-3 protein is present in the touch neurons, the BDU neurons, and many neurons in the head and tail of embryos and adults. In the ventral cord, protein is first detected in the Pn.aa neuroblast, consistent with the lineage findings that descendants of this cell are abnormal. PAG-3 protein remains present through the birth of the mature VA, VB, and VC motor neurons derived from the Pn.aa neuroblast, then rapidly disappears from all but six cells in the mature ventral cord. Expression persists in adults in VA11 and VA12 as well as in four cells of the retrovesicular ganglion. PAG-3 expression during terminal differentiation of ventral cord neurons and its persistence through adulthood in specific subsets of ventral cord neurons suggest that pag-3 may regulate both neuroblast lineage and specific aspects of neuronal differentiation.

24 May 1999 15:50 273 273 A screen for mutations that affect programmed cell death in the ventral cord

1999 International Worm Meeting abstract 222 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A screen for mutations that affect programmed cell death in the ventral cord S Cameron 1,2 , N Tsung 1 , B Horvitz 1

1 HHMI, Dept. Biology, MIT, Cambridge, MA 02139. 2 Division of Pediatric Hematology/Oncology, Children’s Hospital/Dana-Farber Cancer Institute, Boston, MA 02115.

We have developed an efficient screen for mutations that affect the survival or death of specific ventral cord cells during normal development. A lin-11::gfp reporter construct is expressed in the six VC motor neurons that arise from the P3-P8 lineages as Pn.aap cells. The lineally equivalent Pn.aap cells from the W, P1, P2 and P9-12 lineages die by programmed cell death. In ced-3 animals, in which cell death is prevented, all the "undead" Pn.aap nuclei express GFP and fluoresce brightly. We are using this reporter to screen for mutations that result in the survival of Pn.aap cells in lineages in which they normally die, or the death of Pn.aap cells in lineages in which they normally survive. Hermaphrodites are mutagenized with EMS, and the F2 progeny are screened using a stereomicroscope for animals with an abnormal number or pattern of fluorescing nuclei. To date, 20,000 haploid genomes have been screened, and 70 mutants isolated. Mutants with abnormal survival of Pn.aap cells include new loss-of-function mutations in ced-3, ced-4, and egl-1. New pag-3 alleles have been recovered (see abstract by Cameron et al.). Mutants with abnormal death of Pn.aap cells include alleles of at least one gene not previously identified as affecting Pn.aap survival. As the Pn.aap cells are sexually dimorphic, weak, self-fertile Tra mutations are also recovered. Mutants isolated in this screen will be characterized with the goal of identifying the factors that determine whether individual Pn.aap cells survive or die.

24 May 1999 15:50 274 274 Serotonin Modulation of Locomotion by Regulated Neurotransmission

1999 International Worm Meeting abstract 223 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Serotonin Modulation of Locomotion by Regulated Neurotransmission T Carey, SJ Nurrish, JM Kaplan UC Berkeley, MCB Dept , 361 LSA, Berkeley CA 94720 Defects in serotonin (5HT) signaling produce hyperactive locomotion. A hyper screen done in the Kaplan lab identified three components of a potential 5HT signal transduction cascade modulating locomotion: CAT-1 is a vesicular monoamine transporter (1,2); GOA-1 is a Gao subunit (3,4); and DGK-1 is a diacylglycerol kinase (2). Serotonin appears to modulate locomotion by inhibiting synaptic release of acetylcholine (ACh). Treatment with exogenous 5HT makes animals resistant to a higher endogenous level of ACh produced by treatment with aldicarb (Ric), an inhibitor of acetylcholinesterase. As mutants with defective exocytosis machinery are also Ric, these results suggest that 5HT inhibits ACh release at neuromuscular junctions. goa-1 and dgk-1 mutants are hypersensitive to aldicarb (Hic), presumably due to increased ACh release. A direct relationship between 5HT signal transduction and regulation of synaptic vesicle exocytosis could occur by DGK-1 altering the levels of DAG. DAG binds to the phorbol ester binding protein UNC-13, and the mammalian homolog Munc-13 interacts with syntaxin to bind part of the exocytotic core complex (VAMP, SNAP25, and syntaxin) and stimulate vesicle release (5,6). The phenotype of unc-13 fits this model, as mutants are defective in neurotransmission and Ric (7,8). The proposed model for 5HT modulation of locomotion is missing several components likely to be represented by some of the remaining hyper mutants. We are analyzing the phenotypes, aldicarb testing, and mapping these mutants. One expectation is to find a metabotropic serotonin receptor that activates goa-1. In addition, the link between goa-1 and dgk-1 is unclear, with several possible intermediate steps. Finally, G-proteins modulate neurotransmitter release in other systems by regulating Ca++ and K+ channels, as well as the secretory machinery.

1.Duerr, J. S. et al. J Neurosci 19:72-84 (1999) 2.Nurrish and Kaplan, in preparation 3.Segalat, L et al. Science 267:1648-1651 (1995) 4.Mendel, J. et al..Science 267:1652-1655 (1995) 5.Betz, A. et al. J Biol Chem 272:2520-6 (1997) 6.Betz, A et al. Neuron 21:123-126 (1998) 7.Miller, K. et al. Proc. Natl. Acad. Sci. USA 93:12593-12598 (1996) 8.Hosono, R et al. Zool. Sci. 6:697-708 (1989)

24 May 1999 15:50 275 275 tab-1, a Gene Involved in Response to Anterior Touch

1999 International Worm Meeting abstract 224 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. tab-1, a Gene Involved in Response to Anterior Touch L Carnell 1 , B Harfe 2,3 , A Fire 2 , M Chalfie 1

1 Columbia University. 2 Carnegie Institution of Washington. 3 Emory University.

Thetab-1u271mutation was identified in a screen for genes required for touch cell function. tab-1animals are Tab (touch abnormal) only in the head. Forward moving animals will stop upon touch to the anterior region of the body, but fail to initiate backward movement. This response implies the anterior touch cells are functional and that the mutant defect probably lies downstream in their connections to postsynaptic targets. tab-1animals during later stages of development gain sensitivity to prodding with a wire (Tab), but not to a hair (Mec). This event coincides with additional connections that are produced in the anterior touch cell circuit by the postembryonic touch cell, AVM. The tab-1animals are not Unc, Not, or Osm. However, they do show an exaggerated degree of nose-lifting from the agar as seen with animals lacking CEP neurons (Kaplan and Horvitz, IWM, 1991). We cloned tab-1and found that it encodes a homeodomain protein. The u271allele is a nonsense mutation within the homeodomain. Since only a single allele of tab-1has been identified, we performed RNAi to confirm that the u271mutation was a loss of function. Injection of RNAi resulted in animals that were largely touch insensitive. The TAB-1 protein shows the greatest homology to the product of the Drosophila gene bsh(brain-specific-homeobox) with 85% identity in the homeodomain region. Both the tab-1::gfpprotein fusion and the tab-1::lacZrescuing fusion showed similar patterns of expression. tab-1is expressed very early in the embryo in about 20-30 cells, but decreases to about 6-8 cells in later stages. Six of these neurons have been identified as AIB, AVJ, and RIV. In a tab-1mutant background, only the expression of the AIB neurons is maintained suggesting that TAB-1 is required for maintenance of its own expression. In an unc-42background, tab-1expression in AIB is missing and the axon of the AVJ fails to extend down the ventral cord suggesting a role for UNC-42 both in maintaining expression of tab-1and in controlling axon guidance. Because AIB, AVJ, and RIV neurons have not been previously identified to be part of the touch cell circuit, we are in the process of determining the function of these neurons and their role in anterior touch by laser ablation.

24 May 1999 15:50 276 276 ts emb mutants: a retrospective (on the g-set) & future uses II: recent news on some neurobiological collaborations

1999 International Worm Meeting abstract 225 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. ts emb mutants: a retrospective (on the g-set) & future uses II: recent news on some neurobiological collaborations R Cassada University of Freiburg In the age of knockouts, temperature sensitive mutants still have some clear-cut advantages, which will be considered, as well as attempting to debunk some of the assumed disadvantages. Can a ts mutant have a null phenotype? Yes, even more than one! But how to prove it’s the/a null-phenotype? And a knockout often may not manifest an interesting early embryonic null-phenotype because of rescue through perdurance/persistence of maternal wild-type product (of course, germline mosaics might help get around this - or NOT). Looking at the set I know best (Goettingen set, genes emb-11 to 35), some points can be made in retrospect: Both technically and strategically, most of the protocols chosen were favorable in terms of efficiency and results: Mutants in some interesting genes turned up (as also shown by others), and they could be usefully characterized thanks to their conditional phenotypes. And ts mutants remain very valuable and useful to have in many situations. Part II: Plans and the latest results of 3 small joint projects on various aspects of C.elegans developmental neurobiology in collaboration with non-C.elegans groups will be described and discussed.

24 May 1999 15:50 277 277 Excitation-contraction coupling in C. elegans muscle cells as monitored by multiphoton excitiation FRET imaging of calcium indicator proteins.

1999 International Worm Meeting abstract 226 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Excitation-contraction coupling in C. elegans muscle cells as monitored by multiphoton excitiation FRET imaging of calcium indicator proteins. V Centonzel 1 , E Maryon 2 , D Wokosin 1 , B Saari 2 , P Anderson 2

1 Integrated Microscopy Resource, UW Madison, Madison WI 53706. 2 Dept of Genetics, UW Madison, Madison WI 53706.

Muscle contraction is initiated by membrane depolarization followed by a rapid elevation of cytoplasmic Ca2+ . This process, called excitation-contraction coupling, is mediated by voltage gated Ca2+ channels in the plasma membrane and intracellular Ca2+ channels in the sarcoplasmic reticulum. Using a system similar to that described by Kerr, et al. (WCWM ’98) we are characterizing Ca2+ transients during pharyngeal pumping in wild-type animals, which we will compare to transients in animals having mutations in genes such as Ca2+ channels.

Multiphoton excitation is being used to elicit Ca2+ -dependent fluorescence resonance energy transfer (FRET) emissions from a cameleon (ycam2) protein (provided by R. Tsien). ycam2 consists of two mutant GFP moieties having differing excitation and emission spectra, connected by a Ca2+ -binding linker peptide. When ycam2 binds Ca2+ it undergoes a conformational change that allows FRET between the 480nm-emitting moiety (cyan fluorescent protein-CFP) and the 535nm-emitting moiety (yellow fluorescent protein-YFP). Ca2+ increases are thus detectable as an increase in the YFP/CFP emission ratio. We have expressed ycam2 in pharyngeal muscle cells using the myo-2 promoter. Imaging is performed on a prototype multidimensional multiphoton microscope. The instrument allows wide field direct detection of 3 channels: 1) bright field image; 2) YFP emission (535-550nm); 3) CFP emission (380-485nm). ycam2 fluorescence is imaged with a Ti-sapphire laser tuned to 800nm to primarily excite the CFP moiety. Time series are collected and data is subsequently analyzed using NIH-image.

We have observed Ca2+ transients during pharyngeal pumping by dissecting pharynxes in serotonin containing saline and imaging the terminal bulb. We observe 10% changes in YFP/CFP emission ratio from "baseline" to "peak" which correlate with opening of the terminal bulb lumen. Although our system does not yet allow "real time" resolution of Ca2+ transients, we can correlate pharyngeal muscle contraction with calcium elevation in wild-type and mutant animals.

This work was supported by RR00570.

24 May 1999 15:50 278 278 How are anterior cell migrations guided by mig-13?

1999 International Worm Meeting abstract 227 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. How are anterior cell migrations guided by mig-13? Q Ch’ng, L Moore, M Sym, C Kenyon Department of Biochemistry, UC San Francisco, CA 94143-0448 The development of the Q neuroblasts in C. elegans provides an excellent opportunity for studying the intricate processes controlling cell migration. Two bilaterally homologous cells, QL and QR, are born near the posterior region of the worm. These cells and their descendents migrate in opposite directions either posterior (QL lineage) or anterior (QR lineage) to their site of origin. mig-13 was previously identified as a guidance factor that promotes migrations in the anterior direction and is specifically required for the anterior migrations of the QR descendants. We are taking several approaches to understand further how the anterior migrations of the QR descendants are guided by mig-13. A rescuing mig-13::GFP fusion was previously found to be expressed in the anterior and mid-body ventral cord motor neurons, which cross the migratory track of the QR descendants. Consistent with this expression pattern, mosaic analysis revealed that mig-13 acts non-autonomously to direct the migrations of the QR lineage. We are expressing mig-13 in all neurons, as well as subsets of ventral cord neurons to confirm that expression of mig-13 in the ventral cord is sufficient for controlling the QR migrations. These experiments will further pinpoint where mig-13 expression is required for anterior migrations of the QR lineage. mig-13 is predicted to encode a novel transmembrane protein containing a CUB domain and LDL-receptor repeat in the extracellular region as well as a proline-rich domain in the intracellular region. Thus, we are also initiating structure/function studies of MIG-13 to determine the functional roles of these domains. It is of particular interest to determine if the intracellular region is required for mig-13 function, as this might suggest that a signal transduction pathway outside the Q lineage is required for directing their anterior migrations. A Wnt homolog, egl-20, and a Hox gene member, lin-39, are also required for the anterior migrations of the QR descendants. We are investigating mig-13’s role(s) in positioning the QR descendants in relation to these genes through epistasis analyses. Lastly, we are initiating genetic screens to isolate additional components of this migratory pathway.

24 May 1999 15:50 279 279 Developmental patterning in the C. elegans hindgut

1999 International Worm Meeting abstract 228 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Developmental patterning in the C. elegans hindgut Helen M. Chamberlin, James H. Thomas Department of Genetics, University of Washington, Seattle WA In C. elegans, the posterior hindgut is comprised of four cells (F, U, B and Y) that each adopt a distinct cell fate in both males and hermaphrodites. The genes mab-9, egl-38 and lin-48 play an important role in specifying the fates of these cells. mab-9 functions to make the two dorsal cells (F and B) different from their ventral neighbors (U and Y; Chisholm and Hodgkin, 1989, G & D 3: 1413). egl-38 encodes a Pax transcription factor that functions to make the two anterior cells (F and U) different from their posterior neighbors (B and Y; Chamberlin et al., 1997, Dev. 124: 3919-3928). lin-48 encodes an Ovo-like zinc finger transcription factor required for normal development of F and U, and functions to make the left/right lineal homologues, U and B, different from each other. We are interested in how these genes function together to specify cell fate. We have found that lin-48::gfp transgenes are expressed in the two anterior cells, F and U, as well as in a small number of other cells. In egl-38 mutants the hindgut expression is greatly reduced, whereas expression in other cells is unaffected. In contrast, lin-48::gfp expression is not affected in lin-48 mutants. This suggests that egl-38 is genetically upstream of lin-48, and required for its expression in the hindgut. egl-38 is required for the normal development of both the hindgut and the hermaphrodite egg-laying system. In contrast, genetic and expression data for lin-48 indicate it does not function in the egg-laying system. Our results are consistent with a model that lin-48 is a tissue-specific target for egl-38. We plan to test whether the lin-48 promoter is a direct target for EGL-38, as well as to identify other factors that contribute to the tissue-specific functions of egl-38.

24 May 1999 15:50 280 280 Immunoaffinity purification of the C. elegans dosage compensation complex

1999 International Worm Meeting abstract 229 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Immunoaffinity purification of the C. elegans dosage compensation complex RC Chan, M Albrecht, C Tsai, BJ Meyer HHMI and Dept. of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3204 Dosage compensation (DC) in C. elegans equalizes X expression between males (XO) and hermaphrodites (XX) by reducing transcription from both hermaphrodite X chromosomes. A large protein complex specifically localizes to the X chromosomes of XX animals to implement DC. Members of this DC complex include the SMC (structural maintenance of chromosome) proteins DPY-27 and MIX-1, and a non-SMC protein DPY-26. The latter two proteins also function in chromosome segregation during mitosis or meiosis, demonstrating the recruitment of chromosome segregation proteins to the regulation of gene expression. A second class of genes (sdc) that coordinately controls sex-determination and DC is required for the X localization of the DC complex. To gain an in-depth view of the interactions among these various DC proteins and to identify other DC proteins, we developed an immunoaffinity purification scheme to enrich for the DC proteins localized on DNA. To characterize the DC complex, peptide antibodies were raised against the carboxyl terminal (c-ter) sequences of DPY-26, MIX-1 and DPY-27, and were used to precipitate the complex from crude embryonic extracts. The benefit of this approach is that the precipitate can be eluted with c-ter peptides under non-denaturing conditions to allow further fractionation and biochemical analysis. Our analysis provided the following findings: (1) The product of the genetically characterized DC gene dpy-28 was shown to be a member of the complex. Besides DC, DPY-28 also functions in meiosis (see Chan et al.), further reinforcing the model that DC evolved by recruiting factors essential to chromosome segregation. The addition of DPY-28 to the DC complex revealed a striking overall similarity between the DC complex and the Xenopus 13S condensin, required for mitotic chromosome condensation in vitro. This result suggests that DC may be mechanistically similar to mitotic chromosome compaction. (2) The SDC-2 and SDC-3 proteins were also present in the precipitate, suggesting that SDC proteins directly recruit the DC complex to X (see Dawes et al.) (3) Two other proteins of 165- and 80-kD were found. Work is in progress to identify these two proteins and to further purify the DC complex for biochemical analysis.

24 May 1999 15:50 281 281 SOS-1, a C. elegans homolog of SOS, mediates vulval induction, viability, fertility and spicule development.

1999 International Worm Meeting abstract 230 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. SOS-1, a C. elegans homolog of SOS, mediates vulval induction, viability, fertility and spicule development. C Chang 1 , NA Hopper 2 , PW Sternberg 1

1 HHMI and Division of Biology, Caltech, Pasadena, CA 91125. 2 MRC-Laboratory of Molecular Biology, Cambridge CB2 2QH, UK.

The EGF RTK signaling pathway is utilised multiply during development of animals. The components involved in this pathway and the mechanism of signaling are highly conserved among species. It is believed that RAS is activated by the stimulation of guanine nucleotide exchange following the recruitment of SOS to the plasma membrane. Recruitment of SOS follows receptor activation and is dependent upon the adaptor protein GRB-2. Two negative regulators of EGFR signaling, SLI-1 and ROK-1, both have proline-rich domains that can interact with the SH3 domain of SEM-5. Competition between SOS and negative regulators for binding to the SEM-5 adaptor is a potential mechanism of direct negative regulation. Alternatively, the SH3 domains of SEM-5 may, in addition to the recruitment of SOS, also recruit negative regulators. The testing of these models requires the identification of C. elegans SOS. Thus far, the C. elegans homolog of SOS has not yet been identified. However, the sequencing consortium has recently identified an incomplete genomic sequence which genefinder predicts to encode a C. elegans homolog of SOS. By using RT-PCR, we have been able to obtain a partial cDNA of this gene and confirm that it encodes a SOS homolog (SOS-1). The identification of a full length cDNA is underway. Analysis of SOS function using double-stranded RNA interference (RNAi) reveals that SOS participates in those functions mediated by RAS. Progeny of hermaphrodites injected with sos-1 dsRNA showed a low penetrance of L1-L2 lethality. For those escapers, the uterine lineage is abnormal such that uterine cells are generally mis-positioned or mis-specified. During oogenesis, a significant retardation of pachytene exit is observed. let-60(n1046) but not lin-15(n765) suppresses the lethality conferred by the sos-1 RNAi, which places SOS-1 function downstream of LET-23 and upstream of LET-60. Hyperinduced vulval phenotypes in both let-60(n1046) and lin-15(n765) are suppressed by the sos-1 RNAi with less effect on the let-60(n1046) mutants. In addition, adult males generally exhibit crumpled spicules. Since all the above phenotypes are also exhibited by mutants in RAS signaling pathway, we conclude that SOS-1 acts on RAS during larval development.

24 May 1999 15:50 282 282 Control of Early Germ Line Proliferation

1999 International Worm Meeting abstract 231 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Control of Early Germ Line Proliferation A Chaudhary, J Hubbard Department of Biology, New York University, New York, NY Kimble and White (1981) found that ablation of Z1 and Z4, the cells that give rise to the somatic gonad, influences the fate of Z2 and Z3, the cells that give rise to the germ line. In the absence of Z1 and Z4, the germline progenitor cells neither divide nor enter meiosis. This experiment suggests that a cell-cell communication event may be required to initiate the proliferation and/or the meiotic competence of Z2 and Z3. The resulting effect of the ablation experiment differs from the phenotype of a glp-1 null mutant in which Z2 and Z3 divide one or two times, enter meiosis early and form sperm.1 This effect also differs from the phenotype of certain germ line proliferation mutants in which Z2 and Z3 divide several times but do not enter meiosis (e.g., glp-4 2 and glp-33 ). We are screening for mutants that affect germline development. We have obtained a variety germline phenotypes including a phenotypic class (Nog for no germ line) that is currently represented by nine mutants. In these mutants, the adult somatic gonad appears normal but is devoid of germ nuclei. Genetic and phenotypic analysis of this class of mutants is in progress. There are many developmental mishaps that could cause a Nog phenotype. For example, the germline precursors (Z2 and Z3) may divide but later die. Alternatively, Z2 and Z3 may not be generated, or they may be generated but may not arrive in the gonad primordium, or they may arrive in the gonad primordium but fail to proliferate. Interference with cell-cell communication between the somatic and germ lineages suggested by the Kimble and White experiment could account for the last scenario. We have mapped one allele of this class, ar228, to the left arm of LGV. ar228 mutant animals look normal in the early L1: the gonad primordium contains Z1, Z2, Z3 and Z4. As the L1 progresses, however, Z2 and Z3 take on a slightly abnormal morphology and fail to produce a germ line. Therefore, the ar228 allele could provide a means to explore a potential cell-cell communication event necessary to initiate the proliferation of Z2 and Z3.

1.Austin and Kimble, 1987 2.Beanan and Strome, 1992 3.Kadyk et al., 1997

24 May 1999 15:50 283 283 ARM-1 is a novel body wall muscle protein that binds UNC-44

1999 International Worm Meeting abstract 232 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. ARM-1 is a novel body wall muscle protein that binds UNC-44 L Chen, V Bennett HHMI, Duke University Medical Center, Durham, NC27710 Ankyrins are a family of spectrin-binding proteins associated with the cytoplasmic surface of the plasma membrane. They bind and cluster several different integral membrane proteins to the spectrin cytoskeleton; these proteins include voltage-gated sodium channels, Na/K ATPase, and the L1CAM family of neuronal cell adhesion molecules. The ankyrin family is represented by Ankyrin R, G, and B in vertebrates, and a single gene in both Drosophila and C. elegans; the ankyrin gene in C. elegans is encoded by unc-44. Mutations in unc-44 result in animals with multiple defects that include axon guidance and cell migration defects. Ankyrin can be divided into four domains: 1) the ankyrin repeats which bind membrane proteins, 2) the spectrin binding domain, 3) the death domain, and 4) the C terminus, which is the least conserved domain among the ankyrins. While the first two domains are well studied, little is known about the death domain and the C terminus. As a first step to characterizing these two domains, we carried out a yeast two hybrid screen (using Barstead’s libraries) to identify proteins interacting with the UNC-44 death domain and C terminus. Only one death domain interacting protein was identified in the screen: the spectrin binding domain of UNC-44. This result is likely to have implications on the mechanism of ankyrin’s ability to cluster associated membrane proteins. The C terminus, on the other hand, was found to bind several different proteins, one of which is a novel protein of approximately 1,000 kD, and has low homology to another C. elegans protein (cosmid ZK783) and a Trypanosome EST. The gene encoding this novel protein is arm-1 (Ankyrin-binding Repeat Muscle protein), and is closely linked to lin-11 on chromosome I. As the name suggests, immunostaining shows that arm-1 is expressed in body wall muscles. The staining pattern shows ARM-1 to be in dense bodies. A striking feature of ARM-1 is that it contains at least 40 highly conserved repeats, each consisting of 76 amino acids. Interestingly, this level of conservation occurs also at the nucleotide level, with very little wobble. We are currently carrying out biochemical studies on the repeats, as well as performing RNAi to interfere with endogenous ARM-1 function.

24 May 1999 15:50 284 284 A Study of LAD-1, the C. elegans Homologue of the L1 Family of Neuronal Adhesion Molecules

1999 International Worm Meeting abstract 233 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A Study of LAD-1, the C. elegans Homologue of the L1 Family of Neuronal Adhesion Molecules L Chen 1 , A Otsuka 2 , V Bennett 1

1 HHMI, Duke University Medical Center, Durham, NC27710. 2 Dept of Biological Sciences, Illinois State University, Normal, IL.

A cell’s interactions with other cells and ability to adapt to its environment are dependent on proteins associated on its plasma membrane. Ankyrin, an intracellular protein that couples several integral membrane proteins to the spectrin cytoskeleton, is instrumental in organizing many membrane proteins into specialized domains. Recently, the L1 family of neuronal cell adhesion molecules, was shown to bind ankyrin. These proteins, which include L1, NrCAM, Ng CAM, neurofascin, and CHL1, are believed to be involved in neurite outgrowth and axonal fasciculation and targeting during development. In Drosophila, a single L1 family homologue, neuroglian, was identified and characterized. Loss of function mutations in neuroglian result in animals that arrest as larvae. Interestingly, there is no obvious defect in the development of their nervous system. A homologue of the family has been identified in C. elegans (Yuji Kohara and genome sequencing), and has been designated lad-1 (L1-like ADhesion). lad-1 is closely linked to unc-24 on chromosome IV. As with the L1 family members, LAD-1 possesses variable extracellular domains composed of six Ig and five FNIII domains, a transmembrane domain, and a cytoplasmic tail that contains a highly conserved ankyrin-binding domain. We are interested in determining LAD-1’s function and studying its interaction with UNC-44, the C. elegans ankyrin homologue. As a first step, we compared LAD-1 expression with that of UNC-44. lad-1 is expressed in the nervous system, with the nerve ring, and the ventral and dorsal nerve cords showing robust staining. lad-1 is also expressed in body wall muscles, hypodermal cells, and the gonad. While LAD-1 and UNC-44 appear to colocalize in the same tissues, we will further test their interaction using biochemical means. We used both RNAi and dominant negative constructs to interfere with endogenous LAD-1 function. Both approaches resulted in animals that were Vab and Unc, with variable lethality. Apparently, lad-1 function is required for viability and proper morphogenesis. Screens for a genetic lad-1 mutant are currently being carried out, which will be important in determining the role of lad-1.

24 May 1999 15:50 285 285 Transcriptional control of germ cell fate

1999 International Worm Meeting abstract 234 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Transcriptional control of germ cell fate PJ Chen, RE Ellis Department of Biology, University of Michigan, Ann Arbor, MI 48109 The fog-1 and fog-3 genes are required to specify that germ cells differentiate as sperm. One possible model is that fog-1 and fog-3 act in response to the sex-determination genes to regulate germ cell fates. Although many regulatory interactions in the germ line occur at the translational level, fog-3 appears to act at the nexus of a set of transcriptional controls. A region of less than 800 nucleotides is required to drive expression of fog-3 on extra-chromosomal arrays. This promotor contains five potential TRA-1A binding sites, more than are found near any other C. elegans gene. Furthermore, this region of the fog-3 promotor binds TRA-1A in gel-shift assays, and this binding is disrupted by mutations in these five sites. We used quantitative RT-PCR to show that tra-1 acts downstream of the fem genes to regulate fog-3. We suspect that tra-1 controls transcription of fog-3, rather than stability of the messages, since the levels of partially processed fog-3 transcripts are also affected by mutations in the sex-determination genes. The TRA-1A binding site most proximal to fog-3 might overlap sequences required for the primary fog-3 transcript; this interference could explain in part how TRA-1A represses fog-3. Mutations in the fem genes have a weak effect on expression of fog-3 in a tra-1(e1099) mutant background; thus we suspect that the fem genes not only inhibit TRA-1A, but also promote expression of fog-3 in a separate manner. In addition, tra-1; fem double mutants that produce wild-type levels of fog-3 transcripts nonetheless make only oocytes, which indicates that the fem genes regulate another activity necessary for spermatogenesis. The amino-terminal 116 amino acids of FOG-3 are similar to the Tob, BTG1 and BTG2 proteins of vertebrates. We find that 6 out of 8 missense mutations in FOG-3 map to this domain, and several of these alter conserved residues. In vertebrates, this domain of BTG1 and BTG2 mediates binding to CAF-1, which is part of a transcriptional regulatory complex. RNA-mediated inactivation shows that caf-1 is essential for both embryogenesis and germ-line function in C. elegans. We are now testing FOG-3 for direct interactions with CAF-1 and other regulatory proteins.

24 May 1999 15:50 286 286 Are Complex N-glycans Essential for the Development of the Nematode C. elegans?

1999 International Worm Meeting abstract 235 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Are Complex N-glycans Essential for the Development of the Nematode C. elegans? S Chen 1 , S Zhou 1 , M Sarkar 1 , AM Spence 2 , H Schachter 1

1 Dept. of Biochem., Hospital for Sick Children, 555 University Ave., Toronto, ON, M5G 1X8. 2 Dept. of Mol. and Med. Genetics, University of Toronto, King’s College Circle, Toronto, ON, M5S 1A8, Canada.

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I) is a key enzyme in the synthesis of hybrid and complex N-glycans. Disruption of the GnT I gene in the mouse results in arrest of embryogenesis. Defects in the synthesis of complex N-glycans have been shown to be associated with several human congenital diseases. We are investigating the role of complex N-glycans in C. elegans. Three C. elegans genes homologous to mammalian GnT I have been identified by searching the genome databases. They are designated gly-12, gly-13 and gly-14. cDNAs encoding these predicted genes have been cloned and their expression patterns determined. Both gly-12 and gly-14 encode active enzymes while gly-13 does not, towards the substrate tested [Chen et al., 1999]. To better understand its biological function, we wanted to isolate a GnT I null mutant. We screened worms that were UV irradiated in the presence of trimethylpsoralen(TMP) and isolated a mutant with a 1.6kb deletion of gly-12. The deletion covers from intron 6 to exon 12, a DNA fragment encoding most of the GnT I C-terminal catalytic domain. The mutants have no obvious phenotypic defects. A Tc1 insertion has been detected within intron 9 of gly-14. Screening for Tc1 derived deletions is under way. We will create double mutants that lack both gly-12 and gly-14 to test the redundancy of these two GnT I genes.

Chen, S., Zhou, S., Sarkar, M., Spence, A. M., and Schachter, H. (1999) J. Biol. Chem. 274, 288-297.

24 May 1999 15:50 287 287 Dissecting the Meiotic Recombination Pathway

1999 International Worm Meeting abstract 236 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Dissecting the Meiotic Recombination Pathway GM Chin, KA Kelly, KJ Hillers, AM Villeneuve Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305 During meiosis, the faithful segregation of homologous chromosomes relies upon the formation of chiasmata between homologs. Chiasmata result from crossovers formed during meiosis I. We have identified crossover-defective mutants by screening for meiotic chromosome segregation defects, using a xol-1:GFP reporter to identify worms with green (XO) eggs in the uterus. Mutants with defects in the recombination machinery itself exhibit cytologically normal pairing and synapsis during early and midprophase but lack chiasmata later in prophase. Consequently, such mutants produce 96% dead aneuploid embryos with a few euploid survivors. An assay using gamma-irradiation to induce meiotic recombination allows us to differentiate between crossover-defective mutants blocked at different steps in the crossover pathway. Radiation-induced DNA breaks can bypass the requirement for SPO-11, the enzyme that initiates meiotic recombination by generating double-strand breaks (DSBs) (Dernburg et al.,1998). In contrast, radiation cannot bypass the requirement for HIM-14 and MSH-5, germline-specific MutS family members that are proposed to act late in the recombination pathway to promote crossover resolution. A distinct response to irradiating the germline of me41 mutant hermaphrodites is seen: radiation not only fails to rescue the me41 mutant but also severely decreases survivorship. This suggests that repair of meiotic DSBs may be defective in the me41 mutant. The me41 allele is a missense mutation that changes the charge at a conserved residue in the worm ortholog of the highly conserved MRE11 protein. In yeast, this multifunctional protein is required for two distinct steps in meiotic recombination and is involved in vegetative DSB repair and telomere length maintenance. Biochemical studies in mammalian systems have also implicated MRE11 in DNA repair, but analysis of its in vivo role has been thwarted by inviability of cells lacking MRE11. The me41 mutation provides an entry point to studying the in vivo role(s) of this pivotal recombination/repair protein in a metazoan system.

24 May 1999 15:50 288 288 Functional Characterization of C. elegans calsequestrin, a high capacity calcium-binding protein

1999 International Worm Meeting abstract 237 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Functional Characterization of C. elegans calsequestrin, a high capacity calcium-binding protein J Cho, K Park, D Kim, J Ahnn Dept. of Life Science, Kwangju Institute of Science & Technology, Kwangju 500-712, Korea A putative calsequestrin gene (csq-1) was found in the nematode Caenorhabditis elegans. The csq-1 was predicted to encode a 417 amino acid-long protein which showed the highest similarity (50% similarity, 30% identity) to the rabbit skeletal calsequestrin. Indeed C. elegans calsequestrin, when recombinantly expressed in E. coli, demonstrated its calcium binding activity. Immunostaining revealed that the calsequestrin protein was expressed in sarcomeric structures of body-wall muscle. Northern analysis and in situ mRNA hybridization data showed the csq-1 began to be expressed in body-wall muscle cells during embryogenesis and the expression was maintained through the adult stage. In order to study body-wall muscle specific regulation of calsequestrin gene expression, approximately 2 kilobase upstream sequences of calsequestrin gene were analyzed. Expression of green fluorescent protein was observed in body-wall muscle of live transgenic animals. Deletion analyses of upstream sequences have revealed a putative promoter sequence and a regulatory element which appeared to enhance reporter gene expression. Several possible binding sites for transcription factors were identified including sites for YY1 and NF-W2, a muscle specific zinc finger transcription factor and a ubiquitous enhancer binding protein, respectively. RNA mediated interference (RNAi) blocked calsequestrin protein expression in the body-wall muscle but not in the pharyngeal muscle or vulval muscle. However, no defective phenotype of body-wall muscle was observed suggesting calsequestrin may not be essential for body-wall muscle contraction.

24 May 1999 15:50 289 289 Gene and chromosome specific localization of SDC-1 directs sexual fate and implements dosage compensation.

1999 International Worm Meeting abstract 238 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Gene and chromosome specific localization of SDC-1 directs sexual fate and implements dosage compensation. DS Chu, DM Lapidus, BJ Meyer HHMI and the Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA The SDC-1 protein coordinately regulates the sex determination and dosage compensation pathways in C. elegans. Genetic evidence indicates that SDC-1 works in concert with SDC-2 and SDC-3 to direct sexual fate and reduce X expression in hermaphrodites. SDC-1 is a 139 kD protein that contains seven putative zinc finger DNA binding domains. Mutations in sdc-1 cause partial masculinization of most XX animals but have no effect on males (XO). This defect is caused by aberrant regulation of the male-specific her-1 gene (Villeneuve and Meyer 1987, Cell, 48:25-37) and results in elevated her-1 transcript levels in sdc-1 mutant hermaphrodites (Trent et al. 1991, Mech. Dev., 34:43-56). We asked if SDC-1 binds directly to the her-1 promoter to repress its transcription. To do so, we created extrachromosomal arrays carrying multiple copies of (1) the her-1 promoter region, (2) transgenes encoding a GFP-LacI fusion protein and (3) lacO, the binding site for the LacI repressor protein. Arrays were visualized by GFP fluorescence through confocal microscopy. Using anti-SDC-1 antibodies, we found that SDC-1 colocalizes to the her-1 promoter. SDC-2 and SDC-3 also localize to the her-1 promoter (see abstract by Dawes, H. et al.) indicating that SDC-1 works in combination with SDC-2 and SDC-3 to repress her-1 transcription directly. sdc-1 mutants also disrupt dosage compensation, causing elevated X-linked gene transcripts and dumpy and egg-laying defective phenotypes. Interestingly, though mutations in all other dosage compensation genes affect the abundance or localization of the dosage compensation complex, sdc-1 mutations fail to perturb the complex. By immunostaining, we found that SDC-1 localizes to the X chromosome and colocalizes with other dosage compensation complex members such as SDC-3 and DPY-28. Therefore, SDC-1 may directly modulate the activity of the dosage compensation complex once assembled on X rather than contributing to its assembly or localization.

24 May 1999 15:50 290 290 Investigating the function of the SMA-1 SH3 domain in C. elegans morphogenesis

1999 International Worm Meeting abstract 239 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Investigating the function of the SMA-1 SH3 domain in C. elegans morphogenesis AI Cid, J Austin University of Chicago, Chicago, IL Spectrin is a cytoskeletal actin binding protein made up of two subunits each of the proteins a-spectrin and b-spectrin. We have identified an unusual member of the b-spectrin family, SMA-1, that is required for proper morphogenesis of the C. elegans embryo. SMA-1 is a homolog of Drosophila bH -spectrin. Like other b-spectrins, SMA-1 and bH -spectrin contain an N-terminal actin binding domain, a series of spectrin repeats and a C-terminal pleckstrin homology domain. However, SMA-1 and bH -spectrin contain more spectrin repeats than other b-spectrins, and have an SH3 domain not found in other b-spectrins. SMA-1 and bH -spectrin also differ from other b-spectrins in their localization within the cell: b-spectrin is found laterally, while SMA-1 and bH -spectrin are found apically.

SH3 domains are protein-protein interaction motifs which bind ligands containing proline-rich regions. We would like to know the function of this domain within SMA-1. The SMA-1 SH3 domain may play a number of roles in SMA-1 function: it could be responsible for SMA-1 localization within the cell, or it could interact with proteins that regulate SMA-1 function. We would like to determine if the SMA-1 SH3 domain plays a role in SMA-1’s intracellular localization or more directly in its function in morphogenesis. We are using a yeast two-hybrid screen to identify SMA-1 SH3 domain ligands. We have identified several positives from our screen which we are in the process of sequencing. We also plan to do more in depth analysis of the SMA-1 SH3 domain, making specific mutations in the SMA-1 SH3 and examining loss-of-function phenotypes of potential SMA-1 SH3 ligands by RNA interference. To address whether the SH3 domain is responsible for SMA-1 protein localization we have begun to make GFP protein fusions containing the SMA-1 SH3 domain. By transforming worms with these SH3-GFP fusion constructs we will be able to determine where the SMA-1 SH3 domain localizes within the cell. With these methods we can attempt to understand what role the SMA-1 SH3 plays in C. elegans morphogenesis.

24 May 1999 15:50 291 291 Transgenic Strains of the Nematode Caenorhabditis elegans as Biomonitors of Environmental Metal Contamination

1999 International Worm Meeting abstract 240 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Transgenic Strains of the Nematode Caenorhabditis elegans as Biomonitors of Environmental Metal Contamination LK Cioci, JH Freedman, L Qiu Nicholas School of the Environment, Duke University, Durham, North Carolina 27708 Transition metal contamination poses a serious environmental and human health threat. The bioavailability of transition metals in environmental samples can only be assessed with living organisms. A transgenic strain of C. elegans has been engineered that can be used to monitor the bioavailability of metals. A reporter transgene consisting of a fragment of the promoter from the C. elegans metallothionein-2 gene (mtl-2) that controls the transcription of a b-galactosidase reporter (lacZ) has been integrated into the genome of this organism. By using these transgenic C. elegans, the toxicological response to metals in soil and water samples can quickly be measured with a simple histochemical staining assay. C. elegans that contain the mtl-2/lacZ transgene provide a more sensitive assay of cadmium, mercury, zinc and nickel exposure than LC50 assays or those using nematodes that contain heat-shock protein-based reporter transgenes. Soil exposure assays also indicated a clear response of the transgenic C. elegans. These studies demonstrate that C. elegans, which contain mtl-2:lacZ transgenes, can function as sensitive toxicological indicators of metal contamination in environmental samples.

24 May 1999 15:50 292 292 GFP transgene expression pattern based mutant screens to identify genes involved in endoderm development in Caenorhabditis elegans.

1999 International Worm Meeting abstract 241 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. GFP transgene expression pattern based mutant screens to identify genes involved in endoderm development in Caenorhabditis elegans. C Clucas, I Johnstone Wellcome Centre for Molecular Parasitology, University of Glasgow, Glasgow G11 6NU. The C. elegans gut is exclusively derived from the E lineage. At hatch, the gut consists of 20 mononucleate cells. Although the gut cells do not divide post embryonically, DNA replication and nuclear division occurs in most gut cells during L1 producing bi-nucleate cells, and endoreduplication of all gut nuclei occurs during all larval stages. We have recently performed forward genetic screens on worms containing an integrated gut specific GFPlacZ reporter gene to identify genes involved in various aspects of gut development. The pattern of GFP fluorescence was used to detect mutants and for preliminary phenotype characterisation. Various interesting classes of phenotypes were obtained including mutants with extra gut cells, mutants with multi-nucleate cells (possibly tetra-nucleate), mutants with fewer gut nuclei (but correct number of cells) and mutants with an altered GFP/gut nuclei integrity. We have so far characterised one mutation belonging to the class with extra gut cells (as indicated by MH27 cell boundary staining). lin-60 (ij048) maps 0.5MU to the right of dpy-5 on chromosome I. Further experiments are underway to investigate the nature of alterations to the E lineage in this mutant. lin-60 (ij048) displays some interesting genetics. Current data would suggest both maternal and zygotic components to function. ij048 is at least partly dominant.

24 May 1999 15:50 293 293 Molecular Characterization of the Nuclear Pore Protein, gp210

1999 International Worm Meeting abstract 242 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Molecular Characterization of the Nuclear Pore Protein, gp210 Merav Cohen, Yosef Gruenbaum Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel The nuclear envelope is a complex structure with multiple functions. It is composed of outer and inner lipid bilayer membranes, nuclear pores and nuclear lamina. The two nuclear membranes are separated by a 20-40 nm perinuclear space and are connected at the nuclear pore complexes (NPCs). The NPCs are passageways for transport of macromolecules between the nucleoplasm and the cytoplasm. The NPC is a large protein complex composed of 100 different proteins, termed nucleoporins, with an estimated molecular weight of 120 MDa. The membrane domain of the NPC has a unique content of both integral and peripheral proteins. In mammalian species the only known integral proteins of this membrane domain are gp210 and POM121. A gp210 cDNA clone was previously isolated from rat, and anti-gp210 antibodies were previously obtained in rat and Drosophila, and were used to map the transmembrane domain and to show that the large amino part of gp210 is embedded in the perinuclear space. The gp210 protein was found to be involved in nuclear transport since anti-gp210 antibodies can interfere with transport through the nuclear pores. Previous studies, using cell free systems from Drosophila and Xenopus also suggested a role for gp210 in nuclear pore assembly. Computer search in Drosophila databases revealed EST clones with significant homology to the rat gp210. These clones were used to obtain the complete sequence of the Drosophila gp210 and to detect a homologue in Caenorhabditis elegans. The C. elegans gp210 protein is 25% and 34% identical and 44% and 55% similar to the rat and the Drosophila gp210 proteins, respectively. More strikingly is the conservation of the general structure of the protein and the location of the transmembrane domain in all gp210 homologues. This evolutionary conservation of the protein indicates a functional conservation of gp210 in nuclear pore assembly and activity. In order to gain more information about the role of gp210 in both nuclear pore assembly and nuclear transport, we have started double stranded RNA mediated interference experiments and we are currently screening for deletions in the gp210 gene. In addition, we have produced a fusion protein of gp210 and are currently making antibodies against the protein.

24 May 1999 15:50 294 294 Acetylcholinesterase genes in C. elegans. I. ace-1 and ace-2 encoding classes A and B AChEs (ACE-1 and ACE-2)

1999 International Worm Meeting abstract 243 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Acetylcholinesterase genes in C. elegans. I. ace-1 and ace-2 encoding classes A and B AChEs (ACE-1 and ACE-2) D. Combes, E. Culetto, M. Grauso, Y. Fedon, J.-P. Toutant, M. Arpagaus Différenciation cellulaire, INRA, Montpellier, France Three genes encoding distinct pharmacological classes of acetylcholinesterase (AChE of classes A, B and C) had been defined by genetics: ace-1, ace-2 and ace-3. We first cloned and sequenced ace-1. A 2.4 kb fragment of 5’ region and the coding sequence of ace-1 were sufficient to rescue the uncoordinated phenotype ace-1;ace-2. GFP expression driven by this 5’ region was used to define the tissue specific pattern of ace-1 transcription: body wall and anal muscle cells, pm5 pharyngeal muscle cells and only a few neurons in the head and retrovesicular ganglia. A comparison of 2.4 kb of promoter regions of ace-1 in C. elegans and C. briggsae showed four blocks of conserved sequences. One of these blocks was shown to behave as a body wall muscle cells enhancer and another was found necessary for pharyngeal localization. We cloned and sequenced ace-2 (encoding AChE of class B). ACE-2 possesses all the residues characteristic of the cholinesterase family. Whereas the C-terminus is hydrophilic in ACE-1 with a strong similarity with C-terminal sequences of T subunits of vertebrate AChEs, the C-terminus of ACE-2 is similar to that of H subunits of vertebrate AChEs, which is cleaved and exchanged to a glycolipid anchor during post-translational maturation. C-terminal sequences of ACE-1 and ACE-2 are thus totally compatible with their molecular forms described previously. It is noteworthy that in vertebrates, a single AChE gene gives rise to H or T transcripts through alternative splicing of the two last exons, whereas in C. elegans two ace genes are required for generating such a diversity. ace-2 is located on chromosome I (Y44E3 and Y52G11). We sequenced ace-2 in the mutant strain g72: we found a transition G---A that resulted in the change G441E. This introduces a negative charge in immediate vicinity of the positively-charged H440 and might disrupt the normal charge relay (E327, H440, S200) that activates S200 during catalysis. GFP expression driven by 3.7 kb of 5’ region showed that ace-2 was transcribed in a large number of neurons in the head and anal ganglia and in pm5 cells of the pharynx. In contrast with ace-1, ace-2 was not expressed in body wall muscle cells.

24 May 1999 15:50 295 295 Acetylcholinesterase genes in C. elegans.II. Tandem organization of the third and fourth genes.

1999 International Worm Meeting abstract 244 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Acetylcholinesterase genes in C. elegans.II. Tandem organization of the third and fourth genes. D. Combes, E. Culetto, M. Grauso, Y. Fedon, J.-P. Toutant, M. Arpagaus Différenciation cellulaire, INRA, Montpellier, France In an attempt to clone ace-3, a sequence (clone 48) was obtained by RT-PCR on total RNAs from C. elegans ace-1 null mutants. This clone hybridized to YACs Y48B6 and Y59C8 indicating a site on chromosome II near unc-52, the expected location of ace-3. Blast examination of genomic sequences covering this region in C. briggsae showed that two sequences on chromosome II in this species were related to clone 48. One was the homologous of clone 48 and the other had also the characteristics of an ace gene. Those two sequences were located in very close proximity on chromosome II with only 372 bp between the stop of the upstream ORF and the ATG of the downstream ORF. A similar tandem organization of these two ace genes was also found in C. elegans by PCR (with 356 bp between the two ORFs and 200 bp separating the polyadenylation site of the 5’ gene and the trans-splicing site of the 3’ gene). Due to the close proximity of the two genes on chromosome II it was not possible to decide which sequence corresponded to ace-3 (defined as encoding AChE of class C) and those genes were provisionally named ace-x (upstream ORF) and ace-y (downstream ORF). Using RT-PCR, both genes were shown to be transcribed in both species. In both C.elegans and C. briggsae, ace-x contains 16 introns and ace-y 7 introns. We found FGQSAG (ace-x) and VGESAG (ace-y) for sequences around the active serine: both sequences suggest a low catalytic activity. The C-terminal sequences are of the H type suggesting that ACE-X and ACE-Y are glycolipid-anchored membrane proteins. We then sequenced ace-x and ace-y in the mutant ace-3 (strain dc2) in order to identify ace-3. We found that the only change was a deletion (580 nt-long) covering the non coding region between ace-x and ace-y as well as two small portions of coding sequence including the stop codon of ace-x and the initiator ATG of ace-y. The deletion did not shift the reading frame, so that a single long ORF was present in the mutant dc2. A single large mRNA was found by RT-PCR in dc2 instead of two distinct mRNAs in N2 strain. It is likely that the folding of the large encoded protein prevents catalytic activity of ace-x and ace-y. Thus the dc2 mutant did not allow the identification of ace-x or ace-y to ace-3.

24 May 1999 15:50 296 296 A C. elegans-based study of Huntington Disease-associated pathways

1999 International Worm Meeting abstract 245 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A C. elegans-based study of Huntington Disease-associated pathways J Connolly 1 , S Holbert 1 , I Denghien 1 , C Philippe 1 , S Morinière 1 , M Chalfie 2 , C Néri 1

1 Fondation Jean Dausset - CEPH, 75010 Paris, France. 2 Biological Sciences, Columbia University, New-York, NY 10027.

Polyglutamine (polyQ) expansion in huntingtin (htt) underlies Huntington’s disease (HD). The mechanisms which cause neuron dysfunction and degeneration in HD are unknown. To identify HD-associated pathways, we have undertaken a C. elegans-based study that relies on two approaches: screening for proteins able to interact with htt, and screening for genetic suppressors of mutated htt-dependent phenotypes in transgenic worms. We used the mec-3 promoter (mec-3p) to express GFP fusions which contain a short N-terminal fragment of htt with a normal (17 units) or expanded (84 units) polyQ tract. The mec-3 gene is expressed in 10 neurons including the six touch receptor neurons AVM, ALML, ALMR, PVM, PLML and PLMR. Worms expressing the long polyQ construct were less than 30% and 50% Mec at the tail at Day 1 and 7, respectively. Worms expressing the short polyQ construct or animals with GFP expression driven by mec-3p were less than 7% and 30% Mec at the tail over the same time-frame. Cell death was not observed in these experiments. There was an imperfect correlation between the perinuclear aggregation of GFP fusions in PLM cells and the Mec phenotypes observed. Our preliminary data suggest that N-terminal htt fragments with a long polyQ induce touch insensitivity when expressed in touch receptor neurons, and that transgenic worms expressing mutated htt might be useful for screening for genetic suppressors of behavioural phenotypes. We have screened a C. elegans cDNA library (R. Barstead, OMRC, OK) by using two-hybrid selection in yeast. The screening with a N-terminal htt fragment containing 15 Glns allowed the identification of a new Htt Interacting Protein (wHIP3). WHIP3 shows variation of interaction between normal and mutated htt, and is homologous with a human protein (hHIP3) encoded by a gene expressed in the brain. Additional studies are being performed in order to test whether hHIP3 might be involved in HD. We will present the results of experiments based on both approaches.

24 May 1999 15:50 297 297 CeTwist mutants have defective non-striated muscle development

1999 International Worm Meeting abstract 246 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. CeTwist mutants have defective non-striated muscle development AK Corsi 1 , S Kostas 2 , E Jorgensen 3 , A Fire 2 , M Krause 1

1 Laboratory of Molecular Biology, NIDDK/NIH, Bethesda, MD 20892. 2 Dept. of Embryology, CIW, Baltimore, MD 21210. 3 Dept. of Biology, Univ. of Utah, Salt Lake City, UT 84112.

We are interested in understanding how the bHLH (basic helix-loop-helix) transcription factor CeTwist influences the development of the worm. Homologs in other organisms have revealed a role for Twist in mesodermal development. Twist in D. melanogaster is required to set aside the entire mesoderm, however in M. musculus the Twist homolog is required for the development of only a subset of mesodermal structures such as the complete formation of the neural tube. Elucidation of Twist function in a third model organism, C. elegans, might establish a pattern for Twist function in development. The CeTwist protein is expressed in a subset of mesodermal cells: in the embryonic non-striated tail muscles involved in defecation and in the postembryonic mesoblast M cell and its descendants that become all of the larval-derived mesodermal cells including body wall muscles, coelomocytes, and the sex muscles (vulva and uterine) which are needed for egg-laying. We have been characterizing two alleles of hlh-8, the gene that codes for CeTwist. One allele is a putative null allele that is missing the HLH domain required for dimerization. The second (semidominant) allele has a point mutation in an invariant amino acid codon in the basic domain required for DNA binding. The two hlh-8 alleles do not complement one another. Both mutant strains have similar gross morphological phenotypes; the animals are egg-laying defective (EGL) and constipated (CON). The CON and EGL defects in the mutants are likely to reflect the absence of differentiated non-striated muscles in the body as these animals lack intestine-associated defecation muscles and sex muscles. Our recent efforts in characterizing the hlh-8 mutant defects have been focused on the use of various GFP reporters in the mutants to follow the divisions of the M cell, to see if CeTwist target genes are turned on, and to quantify the number of differentiated M cell descendants that are formed. Differences in the phenotypes between the putative null and the semidominant mutant highlight potential CeTwist functions, and we hope to use these alleles for genetic screens that will elucidate other proteins that function with CeTwist.

24 May 1999 15:50 298 298 Complex cuticle patterning is revealed with GFP-tagged struts.

1999 International Worm Meeting abstract 247 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Complex cuticle patterning is revealed with GFP-tagged struts. JR Crew, JM Kramer Northwestern University Medical School The adult C. elegans cuticle contains a unique medial layer with structures called struts. Struts are noteworthy because they are very highly ordered extracellular structures oriented perpendicularly to the other cuticle layers. TEM shows that animals mutant in any of the six bli genes lack struts in otherwise normal cuticles, suggesting that they function together to form struts. We cloned the bli-1 and bli-2 cuticle collagen genes. BLI-1 is unusual, with large and highly charged amino- and carboxy-terminal domains. Some bli-1 mutations alter these domains, demonstrating their importance. BLI-1::GFP fusion proteins incorporate into struts that show patterned distributions such as more dense incorporation of BLI-1::GFP into struts over muscle cells. In hermaphrodites, a three-row pattern of dim-bright-dim repeats across each annular segment. In males, the pattern is different, repeating as bright-dim -bright. Animals mutant in bli genes either fail to secrete BLI-1::GFP or secrete and incorporate it into abnormal struts. bli-4 mutants appear not to secrete BLI-1::GFP, providing evidence that the BLI-4 protease can process a cuticle collagen in vivo. To understand strut morphogenesis, TEM was used to study adult cuticle formation in L4 animals. Adult cuticle assembly begins by mid-L4 and is essentially complete by the L4-adult lethargis. The cortical layer is secreted first; soon after, round electron dense structures arrayed near the annular furrows are seen. These are early struts. Next, the fiber layers and basal layer are secreted. Finally, the external cortical layer is added and the medial layer expands. During these assembly steps, the animal is able to move, although what the muscles are pulling against is unclear. Connections between the L4 cuticle and the muscles appear to be maintained for some of this period. Because the hypodermis directly contacts the assembling cuticle, we propose that the hypodermis acts as a template. The hypodermis contains hemi-adherens junctions that appear to correlate spatially with struts, and may pattern the cuticle. This model implies that junctional and cytoskeletal proteins may play roles in strut patterning. We plan to examine the roles these molecules play in this process using the BLI-1::GFP as a marker system.

24 May 1999 15:50 299 299 T04C9 encodes a novel protein related to centaurin and oligophrenin GTPase Activating Proteins

1999 International Worm Meeting abstract 248 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. T04C9 encodes a novel protein related to centaurin and oligophrenin GTPase Activating Proteins JR Cross, N Zahedi, HA Baylis, LS Harrington, TR Jackson Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, UK Small GTPases of the Ras, Rho, Rab and Arf families play key roles in the regulation of fundamental cellular processes such as exo- and endocytosis, cell morphology, movement and cell division. Formation of the GTP bound ’active’ state of these proteins is promoted by interaction between the GTPase and specific Guanine nucleotide Exchange Factors (GEF’s). Similarly formation of the ’inactive’ GDP bound form of the protein can be promoted by interaction with specific GTPase Activating Proteins (GAP’s). These GAP proteins may function simply as terminators of GTPase signalling, however there is some evidence that they may also act as effectors small GTPase mediated signal transduction cascades. Our laboratory has characterised a family of inositol phospholipid binding proteins termed the centaurins which contain a region of homology to known Arf-GAP proteins. Whilst searching for C. elegans homologues of mammalian centaurin b’s we identified a number of potential exons in T04C9 sharing considerable identity with the N-terminal portion of centaurins b1 and b2 as well as with oligophrenin a mammalian Rho-GAP protein associated with X-linked mental retardation. Utilising PCR and RACE procedures we have isolated cDNA clones for this gene from C. elegans libraries. These encode a protein whose N-terminal contains a PH domain very similar to those found in mammalian a- and b-centaurins suggesting that it too may provide a target for PI 3-kinase generated second messengers. We are now seeking to identify biochemical activities associated with this protein by expression in mammalian cell culture.

24 May 1999 15:50 300 300 Cholesterol Functions Enantiospecifically in C. elegans

1999 International Worm Meeting abstract 249 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cholesterol Functions Enantiospecifically in C. elegans CM Crowder 1,2 , L Metz 1 , AS Kumar 2 , DF Covey 2

1 Dept. of Anesthesiology, Washington University, St. Louis, MO. 2 Dept. of Molecular Biology/Pharmacology, Washington University, St. Louis, MO.

Cholesterol is present as a single enantiomer in metazoan cells and functions to alter membrane properties such as fluidity. A fundamental unanswered question in membrane biology is the degree of enantioselectivity of cholesterol’s function. In order to address this question, we have synthesized the unnatural enantiomer (ent-chol) of cholesterol. ent-chol has the exact same physical properties (lipophilicity, melting point, etc.) as natural cholesterol (nat-chol) but is its mirror image at all 8 chiral centers. We then tested in C. elegans whether ent-chol can substitute in vivo for nat-chol. We grew synchronized cultures of N2 for several successive generations on three types of plates: 1) NGM plates + 5 µg/ml nat-chol; 2) NGM plates with no added cholesterol (no-chol); 3) NGM plates + 5 µg/ml ent-chol. No difference in fecundity, developmental rate, morphology, or behavior was seen in the first generation of animals grown on the three different plates. However, starting in the second generation, development was slowed on both the no-chol and ent-chol plates. Also, movement of ent-chol worms was markedly slower and uncoordinated and mildly affected in the no-chol worms. Speed of movement on unseeded agar was quantitated by analysis of digitized movies: ent-chol worms = 29.6+/-23.8 µm/sec; no-chol worms = 116+/-17 µm/sec; nat-chol = 133 +/- 28.9 µm/sec. By the third generation, the brood size and developmental rate of the ent-chol worms was decreased drastically although a few worms managed to grow to adulthood and lay viable eggs even after eight generations on ent-chol. Starting at generation three, we saw some embryonic and larval lethality on ent-chol plates. Many dead larvae appeared to have separated from their cuticle. For all endpoints measured, ent-chol worms were more severely affected than no-chol worms. Our results demonstrate that the mirror image of cholesterol is incapable of substituting for natural cholesterol in vivo; thus, the absolute configuration of cholesterol is essential for its normal function. We are currently performing experiments to determine how much ent-chol is incorporated into worm membranes and/or the extent to which it blocks uptake or trafficking of residual nat-chol.

24 May 1999 15:50 301 301 sad-1, sad-2: Cloning Progress, Branching Digress

1999 International Worm Meeting abstract 250 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. sad-1, sad-2: Cloning Progress, Branching Digress G Crump, C Bargmann HHMI and UCSF, Box 0452, San Francisco, CA 94143 U.S.A. We conducted a screen for sensory neuron synaptic architecture mutants, using a fusion between the synaptic vesicle protein SNB-1 (VAMP) and GFP expressed specifically in the amphid sensory neuron ASI. SNB-1::GFP is expressed in six or seven clusters of vesicles in ASI, consistent with the White EM data showing six or seven synapses per ASI neuron. We recovered six complementation groups that affected the pattern of SNB-1::GFP: three known genes, unc-11, unc-51, and unc-104, and three novel sad genes that seem to have specific defects in synaptic architecture. These mutants; sad-1, sad-2 and sad-3; display a variably disorganized vesicle cluster phenotype; often regions of axons lack vesicle clusters whereas other regions hyperaccumulate clusters. Our favorite hypothesis is that vesicle clusters fail to anchor to points of cell-cell contact in the presynaptic neuron or that cell-cell contacts fail to form between the pre- and post- synaptic neurons. We have mapped sad-1 and sad-2 to small regions on X. We are injecting cosmid pools in an attempt to get transformation rescue. In sad-1 and sad-2 mutants a subset of sensory neurons’ axons develop auxiliary branches in the nerve ring. Whereas ASI and AWC display low penetrance axon branching and, for AWC, rare lack of termination phenotypes, ASE and ASJ axons are normal. This is in contrast to the posterior-directed axon branches seen in activity and cilium structure mutants in ASE and ASJ but only rarely in AWC and ASI (work in our lab by Peckol et. al.). This suggests that different populations of neurons may regulate their axon morphologies in different ways, with some neurons relying on electrical activity and others on proper synaptic adhesion or signaling for axon maintenance.

24 May 1999 15:50 302 302 Not all nematodes have constant cell lineages

1999 International Worm Meeting abstract 251 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Not all nematodes have constant cell lineages Ana S. Cunha, Ricardo B. R. Azevedo, Armand M. Leroi Department of Biology, Imperial College, Silwood Park, Ascot, Berks SL5 7PY, United Kingdom Nematodes are widely thought to be ’eutelic’, that is, each adult tissue is made up of an invariant number of cells. We wondered whether this was always true, and so counted the numbers of cells in the hypodermal syncytium (hyp-7) in 15 different free-living species (Rhabditids, Panagrolaimids, and Cephalobids). Among species, mean hypodermal cell number varies 3-fold, but cellular constancy (as measured by among-individual variance in cell number) varies by 3 orders of magnitude. Furthermore, there is a strong positive correlation between the mean and variance in cell number. Thus, Panagrellus redivivus has approximately 3x as many hypodermal cells as C. elegans, but is 300x as variable. We reasoned that there are two basic ways in which one species might come to be more variable than another. First, it could be some species are more variable just because they have more complex lineages. Second, it could be that some species are more variable because they have a higher error rate during particular cellular events. To test among these alternatives, we first determined the canonical V cell lineages of two species, Oscheius myriophila and Rhabditella octopleura. Then we modelled these lineages (and those of C. elegans, and P. redivivus) computationally, introducing plausible kinds of variation in cell divisions and fates into the simulation. These simulations generate predictions, for a given canonical lineage, of the relationship between cellular error rates and variance in adult cell number. We found that the enormous variance observed in P. redivivus (and in some other species such as Rhabditoides regina) can only be explained by far higher error rates in V cell lineages than are found in C. elegans. Notably, Sternberg and Horvitz, when lineaging P. redivivus, observed variant V cell lineages of a sort that have never been found in C. elegans.

24 May 1999 15:50 303 303 Characterization and Cloning of pag-1

1999 International Worm Meeting abstract 252 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization and Cloning of pag-1 M Cunningham, G Xie, Y Jia, J McDermott, E Aamodt Louisiana State University Medical Center Two new genes, pag-1 and pag-2 were identified by mutations that result in enhanced mec-7lacZ expression. Five mutant alleles of pag-1 and two mutant alleles of pag-2 were identified in a screen of 40,000 animals. All of these mutations were recessive to the wild-type alleles and acted in trans. pag-1 was mapped to the center of chromosome III close to clk-1. Mutations in pag-1 resulted in a twelve- to eighteen-fold increase in mec-7lacZ expression and mutations in pag-2 resulted in a three- to six-fold increase. Neither pag-1 nor pag-2 caused phenotypes other than overexpression of transgenes. pag-1(ls2) increased the level of mec-7lacZ mRNA and did not appear to increase its translational efficiency. The pag-1 mutations affected other reporter genes in transgenic arrays independent of promoter, coding or untranslated sequences but did not affect the expression of the corresponding endogenous genes. Furthermore, pag-1(ls2) increased the expression of transgenic mec-7 with short upstream and downstream sequences but did not affect the expression of transgenic mec-7 from a cosmid with at least 10 kb of 5’ and 3’ noncoding sequences. Thus, pag-1 acted to increase expression from multicopy arrays in C. elegans independent of sequence, perhaps by affecting chromatin structure. We hope to use a mec-7gfp reporter to clone pag-1. mec-7gfp fluorescence is brighter in a pag-1 mutant than in wild-type. We are currently using the mec-7gfp fluorescence assay to attempting to rescue the pag-1 gene. We are crossing the pag-1; mec-7gfp mutant with strains transformed with cosmids in the region of clk-1. We hope to find a cosmid that results in decreased mec-7lacZ expression. If successful, we will attempt rescue with cosmid subclones until the pag-1 gene has been identified.

24 May 1999 15:50 304 304 Heterotrimeric G proteins of C. elegans: cDNA sequence analysis and interaction studies

1999 International Worm Meeting abstract 253 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Heterotrimeric G proteins of C. elegans: cDNA sequence analysis and interaction studies E Cuppen, G Jansen, RH Plasterk The Netherlands Cancer Institute, Division of Molecular Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Heterotrimeric G proteins are signal transduction molecules situated at the interface between extracellular signals received via G protein-coupled receptors (over 600 predicted in the C.elegans genome) and intracellular second messenger systems. We set out to study the function of all heterotrimeric G protein genes in C.elegans. Thus far, two Gb subunit genes (gpb-1 and 2), two Gg genes (gpc-1 and 2) and 20 Ga genes have been identified in the genomic sequence (Jansen et al., Nature Gen. in press). Besides one member (gsa-1, goa-1, egl-30 and gpa-12) of each of the mammalian Ga classes (Gs, Go/Gi, Gq, and G12) there are 16 new C.elegans specific genes (gpa-1 to 11, 13 to 16 and odr-3). We have cloned the cDNA’s for all the G protein subunits. RT-PCR and sequence analysis gave no indications for alternative splicing, but we found that about 10% of the exons of this highly conserved protein family were predicted incorrectly by Genefinder. We will employ all cDNA’s in yeast two- and three-hybrid interaction studies to test for interaction specificity between the different subunits. Furthermore, we will screen cDNA libraries for interacting proteins. The interaction specificity of novel proteins can easily be tested using mating assays, because all C.elegans heterotrimeric G protein subunits are available. Since we know that multiple Ga subunits are expressed in particular cells (Jansen et al., Nature Gen. in press), specificity in interactions mediated by these subunits may provide an efficient mechanism regulating signaling via G protein coupled receptors.

24 May 1999 15:50 305 305 The IP3 Receptor is a Timekeeper for the Defecation Cycle Rhythm

1999 International Worm Meeting abstract 254 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

The IP 3 Receptor is a Timekeeper for the Defecation Cycle Rhythm P Dal Santo 1 , MA Logan 1 , AD Chisholm 2 , EM Jorgensen 1

1 Department of Biology, University of Utah, Salt Lake City, UT 84112. 2 Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064.

The C. elegans defecation cycle is characterized by the activation of three distinct muscle contractions every 45-50 seconds. How is cycle time maintained? To address this question we identified and characterized mutants with abnormal cycle times. Specifically, we cloned the dec-4 gene and demonstrated that it encodes the inositol trisphosphate receptor (IP3 receptor).

Mutations in the IP3 receptor alter the timing of the defecation cycle. Null mutations eliminate the cycle, hypomorphic mutants have a slow cycle, and overexpression of the protein results in a fast cycle time. The IP3 receptor is expressed in several different tissues including the pharynx, the somatic gonad, and the intestine. Using mosaic analysis we demonstrated that IP3 receptor expression in the intestine is necessary and sufficient for normal timing of the cycle. This indicates that the periodic muscle contractions can be controlled non-autonomously from the intestine.

IP3 receptors are localized to the endoplasmic reticulum and are known to function as calcium release channels. Activation of the channel by a phospholipid signaling pathway leads to the production of temporally and spatially regulated calcium oscillations. Our current data predicts a model in which calcium oscillations in the intestine trigger the defecation motor program. To test this model we injected calcium-sensitive dyes into the posterior intestinal cells of adult animals. Our results demonstrate that calcium oscillations occur in the intestine. The frequency of calcium oscillations are consistent with the timing of the defecation cycle and peak calcium levels occur just before the onset of the muscle program. We conclude that periodic calcium release in the intestinal cells initiates the muscle contractions of the defecation cycle and that the IP3 receptor is a central component of the timekeeping mechanism that regulates this behavioral rhythm.

24 May 1999 15:50 306 306 Toward the cloning of vab-6, a gene involved in axon guidance and morphogenesis

1999 International Worm Meeting abstract 255 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Toward the cloning of vab-6, a gene involved in axon guidance and morphogenesis G Dalpe, PJ Roy, J Culotti Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada, M5G 1X5. C.elegans morphogenesis is a complex process requiring numerous short-range migrations, rearrangements, and concerted cell movements. Several existing C.elegans mutants disrupt essential morphogenic events since they result in severe body deformities, classically described as a vab (variable-abnormal) phenotype. Several studies have demonstrated that molecules shown to play cell and axon guidance roles in other model systems have homologues in the worm that play broad roles in hypodermal morphogenesis (George et al., 1998, Cell 92, 633-643; Wang et al., 1999, submitted). For example, mab-20 mutants that have disruptions in the C.elegans homologue of Drosophila Semaphorin II, have severe morphological deformities, axon guidance defects and fusion of sensory rays in the male tail. Ectopic hypodermal cell contacts and other observations in mab-20 mutants suggest that Semaphorin II is required to repel cell extensions (Roy et al., 1999, in preparation). Another previously isolated mutant called vab-6 has several phenotypic traits that are strikingly similar to those observed in mab-20 mutants, including ectopic hypodermal seam cell contacts, male tail sensory ray fusions and misguided axons. Although mab-20 and vab-6 mutants may affect the same developmental events, a double mutant between a putative mab-20 null mutation (ev574) and vab-6(e697) could not be made on several attempts. This suggests that mab-20 and vab-6 may be in parallel pathways. Consistent with this hypothesis, double mutants containing a temperature sensitive allele of mab-20(bx61) (Baird et al., 1991, Dev. 113, 515-526) and vab-6(e697) display embryonic lethality and severe morphological defects at restrictive temperatures. vab-6 genetically maps to position -27.17 on LGIII near unc-45 (-27.13) (Dave Pilgrim, personal communication). To better understand the function of VAB-6, we are presently trying to clone the gene. We show that the morphological abnormalities of vab-6 can be rescued with the cosmid F10C5 that resides to the left of unc-45. F10C5.2 is a predicted transmembrane protein and F10C5.1 has a region highly homologous to the TPR domains found in yeast CDC16, CDC23 and CDC27. We are now trying to identify a smaller rescuing DNA fragment within that cosmid.

24 May 1999 15:50 307 307 Characterization and Mapping of odr-9/egl-4

1999 International Worm Meeting abstract 256 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization and Mapping of odr-9/egl-4 SA Daniels, P Sengupta Department of Biology, Brandeis University, Waltham, MA 02454 We identified two olfactory mutants, ky27 and ky185, in two independent screens for mutants defective in chemotaxis towards the volatile odorant diacetyl. Complementation tests showed that ky27 and ky185 are allelic to the known mutant egl-4. egl-4 has five previously identified alleles: n477, n478, n479, n579 and n612, four of which are available for analysis. All six odr-9/egl-4 alleles we tested exhibit strong defects in diacetyl chemotaxis. All alleles also show widespread defects in other olfactory responses mediated by both AWA and AWC neurons, although the diacetyl response is the most severely affected. odr-9/egl-4 mutants also display several other pleiotropies. They are egl-d, with mutants holding ~3 X as many eggs in utero as wildtype, and ~30% of these eggs being past the 1 1/2-fold stage. odr-9/egl-4 mutants are also hyperforaging, male mating defective and hypersensitive to dauer pheromone. The dauer pheromone hypersensitivity may contribute to the weak daf-c phenotype of odr-9/egl-4. Mutants are 17%-26% longer in body length than wildtype and have dark intestines. The many pleiotropies of odr-9/egl-4 mutants suggest that ODR-9/EGL-4 functions in multiple pathways. Interestingly, although all mutant alleles tested show all the above phenotypes, they do not fall into a perfect allelic series; for example, the mutant with the strongest defect in pyrazine chemotaxis also has the weakest defect in diacetyl chemotaxis. Generally, n478 and n479 have strong defects in most of the categories tested, whereas n477 and ky185 have weaker defects. Data compiled suggest n478 is the most likely candidate for a null allele of odr-9/egl-4. Currently we are focusing on further characterization of odr-9/egl-4 alleles, double mutant analysis and cloning efforts. We are using PCR-based polymorphism mapping to narrow down the region of interest to facilitate identification of candidate genes. We hope that the molecular cloning of odr-9/egl-4 will help us to understand more about not only olfactory signaling in worms, but also about the relationships among diverse processes, such as chemoattraction, dauer formation, egg-laying and body size determination, all of which seem to require odr-9/egl-4.

24 May 1999 15:50 308 308 Characterisation of sigma class glutathione s-transferases of C.elegans

1999 International Worm Meeting abstract 257 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterisation of sigma class glutathione s-transferases of C.elegans J Daulby, D Coates, RE Isaac School of Biology, University of Leeds The Glutathione S-Transferases (GST) are a superfamily of enzymes that catalyse the nucleophilic addition of the tripeptide glutathione (GSH) to endogenous and xenobiotic electrophilic substrates. GSTs are principally detoxification enzymes, but have been implicated in the development of resistance to a range of pesticides, herbicides and a number of drugs. These enzymes also serve as non-catalytic carrier proteins (ligandins). Recently, a sigma class related GST isolated from Ascaridia galli, displayed a high level of specific activity in the GSH dependent isomerisation of prostaglandin H to prostaglandin E1 . This suggests that certain sigma GSTs may be involved in eicosanoid metabolism. We are investigating the possible functions of members of the sigma (s) class GST of C.elegans. 20 s class GST-like genes have been identified in C.elegans. We have amplified the corresponding cDNA for 7 of these genes by PCR from a cDNA library, and successfully expressed six of the seven cDNA sequences in Escherichia coli. The recombinant protein product of the R03D7.6 gene has GSH binding properties, allowing purification by affinity chromatography. This protein also shows high levels of activity towards the universal GST substrate 1-chloro-2,4-dinitrobenzene (CDNB). Antibodies raised to recombinant R03D7.6 have been used to immunoblot GST protein in crude extracts of mixed stage C. elegans and in column purified native GST, demonstrating that R03D7.6 is expressed at detectable levels. The antibody is being used to validate the expression pattern obtained with nematodes transformed with a R03D7.6::lacZ promoter construct.

1 Meyer D.J., Muimo R., Thomas M., Coates D. and Isaac R.E. (1996) Biochem. J. 313, 223-227

24 May 1999 15:50 309 309 gut-2 and gut-4 Encode Proteins with Similarity to Sm-like Proteins

1999 International Worm Meeting abstract 258 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. gut-2 and gut-4 Encode Proteins with Similarity to Sm-like Proteins A Davies, M Moseley, J Shaw Department of Genetics and Cell Biology, University of Minnesota, St. Paul, MN 55108 Maternal effect lethal mutations in gut-2 and gut-4 lead to very similar embryonic defects including a very penetrant failure to produce differentiated gut cells and pharyngeal muscle cells, a failure of E cells to elongate their cell-cycle at the 26-cell stage of embryogenesis and a failure to gastrulate. Embryos arrest development with an approximately wild-type number of cells and differentiated hypodermal, body-wall muscle and neuronal tissues. Laser ablation of early blastomeres in embryos from mutant mothers indicate that E and MS produce hypodermal, body-wall muscle and neuronal cells. These tissues are normally produced by the C blastomere; however, the number of neuronal cells derived from the mutant E and MS blastomeres suggests that they do not take on precisely a C-like fate. Deletion mutations of gut-2 have a slow-growing, sterile phenotype and are thus stronger than the maternal effect lethal mutations. Cloning of gut-2 and of gut-4 has revealed that both of these genes encode proteins with similarity to Sm-like proteins, which are related to core spliceosomal snRNP proteins. A functional GUT-2::HA protein is localized to nuclei and is expressed in most cells, including the germline. The lesions in two gut-2 alleles and four gut-4 alleles have been analyzed; all six mutations affect the same conserved glycine residue in the GUT-2 and GUT-4 proteins. Thus the maternal effect lethal mutations are special alleles of these genes. It is possible that these special alleles affect a particular function of gut-2 and gut-4 leading to the observed defects in E- and MS-derived cell fate. Alternatively, these special alleles may reduce the efficiency of general splicing so that the production of mature mRNA is reduced for a few transcripts whose levels are critical for specifying the fates of these cells.

24 May 1999 15:50 310 310 Functional Overlap between the mec-8 Gene and Five sym Genes in C. elegans

1999 International Worm Meeting abstract 259 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Functional Overlap between the mec-8 Gene and Five sym Genes in C. elegans AG Davies 1,2 , CA Spike 1 , JE Shaw 1 , RK Herman 1

1 Department of Genetics and Cell Biology, University of Minnesota, St. Paul, MN 55108. 2 Present address: Gallo Clinic and Research Center, San Francisco, CA.

Inactivating mutations in a majority of the 19,000 protein-coding genes of C. elegans may result in wild-type or nearly wild-type phenotypes. Some of these mutationally-silent genes may encode essential functions redundantly. In an attempt to identify such genes, we have screened for mutations that are synthetic lethal with a mec-8 loss-of-function mutation. We have so far identified five independent mutations in four genes, sym-1 through sym-4, where sym stands for synthetic lethal with mec-8. The phenotype conferred by each sym mutation by itself is essentially wild-type. Mutation in sym-1 or sym-2 in combination with any mec-8 loss of function mutation leads to embryonic arrest at the twofold stage of elongation, with high penetrance. Mutation in sym-3 or sym-4 in combination with any mec-8 loss-of-function mutation gives arrested hatchees with bulbous noses. Earlier work showed that mec-8 encodes a regulator of alternative RNA splicing and that mec-8 null mutants have defects in sensory neurons but are generally viable and fertile. The mec-8; sym-1 arrested embryos exhibit severe defects in attachment of body muscle to extracellular cuticle. sym-1 can encode a protein containing a signal sequence and 15 contiguous leucine-rich repeats. Our one sym-1 mutant allele is a nonsense mutation in the middle of the coding sequence. A fusion of sym-1 and the gene for green fluorescent protein rescued the synthetic lethality of mec-8; sym-1 mutants. The fusion protein was secreted from the apical hypodermal surface of the embryo. We propose that SYM-1 helps to attach body muscle to extracellular cuticle. Neither northern blots nor cDNAs gave any evidence for alternative splicing of sym-1 transcripts. We favor the idea that sym-1 provides a function that is redundant with that of a gene whose transcripts are processed by MEC-8. RNA-mediated interference experiments indicated that a close relative of sym-1 functionally overlaps both sym-1 and mec-8 in affecting muscle attachment. We speculate that sym-2 may act in the same pathway as sym-1 and that sym-3 and sym-4 act in an unrelated pathway.

24 May 1999 15:50 311 311 Mutants with altered sensitivity to the intoxicating effects of ethanol

1999 International Worm Meeting abstract 260 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Mutants with altered sensitivity to the intoxicating effects of ethanol AG Davies, CL Eastman, NG Coufal, H Kim, SL McIntire Gallo Center, UCSF, San Francisco, CA 94110 USA Understanding the action of ethanol on nervous system function and behavior is complicated by the existence of multiple molecular targets for ethanol, which include GABAA and NMDA receptors. We are interested in identifying novel neuronal targets of ethanol and understanding the mechanisms that regulate sensitivity to ethanol. Toward this goal we seek to identify mutants with altered responses to ethanol in C. elegans. Exposure, on a plate, to pharmacologically relevant concentrations of ethanol results in modified behavior of wild-type animals within minutes. With increasing concentrations of ethanol, N2 animals decrease their rates of egg laying, pharyngeal pumping and locomotion in addition to reducing the amplitude of the body sine waveform during movement. At high exogenous concentrations (400-500 mM) the animals become essentially paralyzed. Over several hours, wild-type worms adapt to continued exposure to the same concentration of ethanol; the rates of movement, egg laying and pharyngeal pumping of the adapted animals approach that of untreated animals. We are screening for mutants that are altered in their initial sensitivity and the subsequent development of tolerance to ethanol. This screen includes the isolation of new mutations, further characterization of previously isolated mutations with altered sensitivities to ethanol (Morgan and Sedensky, Alcohol. Clin. Exp. Res. 19:1423-29) and examination of previously characterized mutations in genes with known roles in nervous system function. At high concentrations, the volatile anesthetics (VAs) halothane and isoflurane produce locomotor effects similar to those of ethanol on wild-type animals. Crowder et al. (Anesthesiology 85: 901-912) generated isogenic recombinant strains from wild-type Bristol and Bergerac strains to identify quantitative trait loci responsible for sensitivity to halothane and isoflurane. To determine if the ethanol response pathway overlaps with the VA response pathways, we are testing these strains for altered sensitivity to ethanol. In plate assays, preliminary data indicate that there is no correlation between a strain’s response to halothane and isoflurane and its response to ethanol.

24 May 1999 15:50 312 312 Annotation of uncharacterized C. elegans proteins by transferring knowledge from the Yeast Proteome Database (YPD).

1999 International Worm Meeting abstract 261 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Annotation of uncharacterized C. elegans proteins by transferring knowledge from the Yeast Proteome Database (YPD). BP Davis, KJ Roberg-Perez, PE Hodges, MC Costanzo, AH McKee, ME Cusick, WE Payne, JE Brooks, JI Garrels Proteome, Inc. Proteome, Inc. is constructing a C. elegans proteome database that presents properties and functions for the entire set of C. elegans proteins, predicted to be approximately 19,000 in number (1). This database is built by expert curators who are reviewing the entire C. elegans literature and contains essentially all available knowledge for the characterized proteins of C. elegans (2). Here we describe use of the Yeast Proteome Database (YPD), a well-developed proteome database for S. cerevisiae, to annotate many of the uncharacterized proteins of C. elegans. Examples will be presented of knowledge transfer from well-characterized proteins in YPD to create informative C. elegans Protein Reports for as yet uncharacterized proteins. The knowledge transferred is the exhaustive biochemical, genetic and cell biology information on S. cerevisiae contained in the YPD, distilled by expert curation from the research literature about yeast proteins. The mechanism of transfer is the BLAST similarity of C. elegans proteins to S. cerevisiae proteins, and to Drosophila and human proteins. Since highly similar proteins in different organisms have like functions, uncharacterized C. elegans proteins can be predicted to have like functions of similar, well-characterized proteins in yeast. Protein similarities have also been used to build protein families in yeast and worm (3) and are also the basis for domain databases (e.g., Pfam). The C. elegans proteome database has expanded upon these sources to add additional protein family knowledge to the C. elegans Protein Reports. Functional genomic information from YPD can be used to predict potential functions of C. elegans proteins. Many transcript profile experiments from the yeast community are presented in YPD, and we plan, in the same manner, to make the C. elegans proteome database a repository for public functional genomic data. Tools for transcript profile analysis that already exist in YPD will be used for analysis of profiling experiments in C. elegans.

1. The C. elegans Sequencing Consortium, Science 282, 2012 (1998). 2. Roberg-Perez et al., this meeting. 3. Chervitz, et al., Science 282, 2022 (1998)

24 May 1999 15:50 313 313 dpy-23 encodes a component of a clathrin adaptor complex and is required for the endocytosis of synaptic vesicles

1999 International Worm Meeting abstract 262 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. dpy-23 encodes a component of a clathrin adaptor complex and is required for the endocytosis of synaptic vesicles WS Davis 1 , P Baum 2 , G Garriga 2 , EM Jorgensen 1

1 Department of Biology, University of Utah, Salt Lake City, UT 84112. 2 Dept. Molecular & Cell Biology, University of California, Berkeley, CA 94720.

Endocytosis of synaptic vesicle membrane has long been thought to involve clathrin and its associated adaptins. Adaptins are heterotetrameric complexes comprising two large subunits, a medium chain and a small subunit. The adaptin complex binds to a ligand present on the membrane and recruits clathrin triskelions, which assemble into a lattice, thereby imparting a characteristic size and shape to the budding membrane. Presently three adaptin complexes are known: AP-1 has been shown to be involved in budding membrane from the trans-Golgi; AP-2 is involved in endocytosis from the plasma membrane; the function of AP-3 has not been thoroughly characterized. The precise role of clathrin and the adaptins in the endocytosis of synaptic vesicle components has not yet been determined. Although clathrin mediated endocytosis occurs at the synapse, a number of other mechanisms have been proposed. We have cloned dpy-23 and found that it encodes m2, the medium chain of the AP-2 complex. dpy-23(e840), an allele generated by X-ray mutagenesis, is a large deletion encompassing 100kb of genomic DNA, and dpy-23(gm17) is a point mutation that disrupts a splice site. We examined the role of the AP-1 and AP-2 adaptin complexes in synaptic vesicle endocytosis using electron microscopy. Mutations in the m2 subunit of AP-2 (dpy-23) severely deplete synaptic vesicles at the synapse. Mutations in the m1 subunit of AP-1 (unc-101) do not affect the density of synaptic vesicles at the neuromuscular junction. We conclude that AP-2 is essential for the proper recycling of synaptic vesicle membrane.

24 May 1999 15:50 314 314 Cloning and characterization of the class A synthetic multivulva genes

1999 International Worm Meeting abstract 263 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cloning and characterization of the class A synthetic multivulva genes EM Davison, B Horvitz HHMI, Dept. Biology, MIT, Cambridge, MA 02139 The receptor tyrosine kinase/Ras pathway essential for vulval induction is negatively regulated by two redundant pathways. Hermaphrodites mutant in only one of these two pathways (A or B) appear wild-type. Hermaphrodites mutant in both pathways (A and B) exhibit the synthetic Multivulva (synMuv) phenotype. Recent studies have shown that the class B synMuv genes inhibit Ras-mediated vulval development via an Rb/E2F/DP-mediated pathway (1,2). The class A synMuv genes function in parallel to this Rb pathway, but the molecular mechanism by which they inhibit vulval development is not known. Various screens for multivulva animals have defined four genes in the synMuv class A pathway: lin-8, lin-15A, lin-38, and lin-56. Of these genes, only the lin-15A locus has been cloned previously (3,4). lin-15A encodes a novel protein with no recognizable functional or structural motifs. To elucidate the mechanism by which the class A synMuv genes inhibit the Ras pathway, we have cloned lin-56 and lin-8. Both appear to encode novel proteins. We are currently working toward characterizing the expression pattern of lin-56. Progress in the mapping of lin-38 will also be reported.

(1) Lu, X. and H.R. Horvitz. (1998). Cell 95: 981-991. (2) Ceol, C. and H.R. Horvitz. (1999). 12th International C. elegans Meeting. (3) Clark, S.G., Lu, X., and H.R. Horvitz. (1994). Genetics 137: 987-997. (4) Huang, L.S., Tzou, P., and P.W. Sternberg. (1994). MBC 5: 395-411.

24 May 1999 15:50 315 315 The Genetics of C. elegans social behavior

1999 International Worm Meeting abstract 264 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Genetics of C. elegans social behavior MG de Bono, CI Bargmann HHMI/Dept. of Anatomy, UCSF, San Francisco CA 94143, USA Natural isolates of C. elegans behave in one of two distinct ways on food. Solitary strains (e.g. N2) slow down in response to a bacterial lawn, disperse evenly across the lawn, and forage alone. Social strains accumulate where bacteria are thickest, aggregate together, and do not slow down on food until they join a clump. Behavioral differences between social and solitary strains are seen only in the presence of food. By a combination of genetic and molecular studies* we have shown that the behavioral differences between social and solitary wild strains are due to a single amino acid substitution in a single gene called npr-1 (pka bor-1, and renamed by kind permission of Randy Cassada and the CGC). npr-1 encodes a G-protein coupled receptor that belongs to the neuropeptide Y family of seven transmembrane receptors. Predicted null mutations in npr-1 (neuropeptide receptor resemblence) make the solitary N2 strain take on social behavior. We are attempting to define the molecular and cellular circuitries that regulate social and solitary behaviors. We know that social behavior is not disrupted by mutations that abolish previously described sensory responses in C. elegans (e.g. che-3, che-13, odr-3, mec-3, ttx-3) but requires the cyclic nucleotide gated ion channel encoded by tax-2 and tax-4, and the capsaicin receptor homolog osm-9. tax-2 and tax-4 are expressed in ten overlapping sensory neurons. We are currently determining which of these neurons require tax-2/tax-4 activity for social behavior. To identify the circuit which represses social behavior in solitary animals, we are searching for the ligand for npr-1. Based on homology, we predict that npr-1 will have a neuropeptide ligand. In collaboration with Marc Caron at Duke University we are attempting to set up an assay system in mammalian tissue culture cells that would allow us to test candidate ligands or C. elegans extracts for their ability to stimulate npr-1. *de Bono and Bargmann, Cell, 94, 679-689

24 May 1999 15:50 316 316 spn-2 and spn-3: two genes involved in spindle orientation

1999 International Worm Meeting abstract 265 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. spn-2 and spn-3: two genes involved in spindle orientation LR DeBella, LS Rose Section of Molecular and Cellular Biology, University of California, Davis CA 95616 USA Orientation of cell division is critical for the partitioning of cytoplasmic factors and setting up cellular interactions necessary for proper development. In C. elegans, the first cleavage bisects the long axis of the embryo, resulting in an anterior AB cell and posterior P1 cell. Subsequent cleavages in the AB lineage are symmetrical and follow an orthogonal pattern of division. Divisions in the P1 lineage are asymmetric and occur repeatedly on the same axis, due to a 90o rotation of the nuclear-centrosome complex. We are using time-lapsed video microscopy to study a number of maternal effect lethal mutations that alter spindle orientation in early embryos. The spn-2 gene affects spindle orientation at the 2 and 4-cell stage. spn-2/sDf130 worms have defects in oocyte formation. Together these data suggest a continuing role for spn-2 in spindle orientation during embryogenesis, as well as a possible role earlier in the germline. Some spn-3 embryos misorient their spindle in the 1-cell, while others show defects at the 2 or 4-cell stage. spn-3/sDf127 embryos all have orientation defects during the first division. These data suggest that spn-3 plays a role in spindle orientation during the first three divisions after fertilization. Variability in the phenotypes of spn-2 and spn-3 mutants, coupled with the stronger defects of each allele over a deficiency, suggest that we do not have null alleles for these genes. Future characterization of the null phenotypes, using either stronger alleles or RNAi, will provide more information as to the role these genes are playing in spindle orientation. Mutations in spn-1 also cause defects in spindle orientation at the second and third cleavages. Double mutant spn-1; spn-3 embryos have stronger defects in spindle orientation than mutations in either gene alone, suggesting that these genes are either acting in the same pathway or in partially redundant pathways. Mapping of spn-2 and spn-3 is being performed as a first step towards cloning. The spn-2 gene has been placed within sDf130, to the left of sDf121, on chromosome III. spn-3, also on chromosome III, has been placed within sDf127 between sDf125 and sDf135. Once cloned, a more detailed examination of these genes will be completed to elucidate their role in cell division orientation.

24 May 1999 15:50 317 317 Evolution of vulval development and P11/12 cell migration in nematodes

1999 International Worm Meeting abstract 266 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Evolution of vulval development and P11/12 cell migration in nematodes M Delattre, MA Félix Institut J.Monod. CNRS/Univ.Paris 6 et 7. 2 place Jussieu. 75251 Paris Cedex 05.FRANCE The nematode vulva is formed by a set of precursors called Pn.p cells. In C. elegans, each Pn.p cell is specified by an inductive signal (lin-3/let-23/let-60 signaling pathway) coming from the gonadal anchor cell (AC), and a lateral signaling between the Pn.p cells. In other nematode species, like Oscheius sp. CEW1 (family Rhabditidae), patterning of the Pn.p cells is specified not by several simultaneous cellular interactions but by two successive inductions by the AC. Our aim is to understand the molecular nature of the two signals coming from the AC and of the transduction pathway in CEW1. The let-60 /Ras gene has been cloned by hybridization of cDNA and genomic libraries of CEW1. Overexpression and RNAi experiments are underway to see if Ras is involved in the first, the second or both inductions by the AC in CEW1. We are also trying to clone the CEW1 lin-3 gene. At hatching, the parents of the Pn.p cells (12 Pn cells) are aligned along the A/P body axis in six left/right pairs. Their nuclei then migrate into the ventral cord. For most left/right pairs, the chirality of migration is random. But in the P11/12 pair in C. elegans, the left cell usually migrates anteriorly and becomes P11, whereas the right cell becomes P12. In Panagrellus redivivus (family Panagrolaimidae), conversely, the right cell becomes P11. We wonder how this chirality of migration evolves in nematodes. I have looked at P11/12 migration in species of 3 differents families. There is at least one inversion within the Rhabditidae, one between Cephalobidae and Panagrolaimidae and it is unbiased in Pellioditis (family Rhabditidae). In addition, the chirality of P11/12 migration is inverted in the sinistral A. bodenheimeri compared to the closest dextral species. So it seems that inversion of chirality is linked to inversion of whole body handedness. Now, I am looking systematically at cells close to P11/12 to understand if a local inversion of symmetry leads to an inversion in migration. In C. elegans, P11/12 migration is specified independently of P12 fate as it is unaffected in P12 specification mutants (lin-3, lin-44, egl-5).

24 May 1999 15:50 318 318 WASP and Disabled work in parallel to UNC-34, a C. elegans homolog of enabled

1999 International Worm Meeting abstract 267 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. WASP and Disabled work in parallel to UNC-34, a C. elegans homolog of enabled N Hawkins 1 , E Kong 1 , W Forrester 2 , G Garriga 1

1 Department of Molecular and Cell Biology, U. of California, Berkeley, CA. 2 Biology Department, Indiana University, Bloomington, IN.

Mutations in unc-34 partially disrupt the migrations of cells and axon growth cones and the fasciculation of axons into nerve bundles. We have found that unc-34 encodes a homolog of the Drosophila Enabled (Ena) and mouse Mena proteins that have been implicated in regulating cytoskeletal dynamics though F-actin assembly (Dell et al. 1998 WCWM Abstract). In Drosophila, ena is necessary for axon outgrowth and fasciculation. Our results show that in C. elegans Ena also functions in cell migration. The weak effects of unc-34 null mutations on cell and growth cone migrations suggest that additional genes act to regulate actin assembly. In RNAi experiments, we have found that a gene similar to Drosophila disabled (dab) and a gene encoding a homolog of the Wiskcott-Aldridge Syndrome Protein (WASP) act in parallel to unc-34 to regulate cell motility. In Drosophila, Disabled (Dab) is a negative regulator of Ena. In C. elegans, in contrast, we have found that a dab-like gene appears to act in parallel to unc-34. RNAi experiments with dab result in cell migration defects, and dab(RNAi) in an unc-34 null mutant results in cell migration defects that are more severe than in either an unc-34 or dab(RNAi) background. WASP has also been implicated in cytoskeletal dynamics. In addition, Ena and WASP have an EVH1 domain at their amino termini which binds vinculin and zyxin. wasp(RNAi) animals display no obvious phenotypes, but wasp(RNAi) in unc-34 null mutants results in synthetic lethality. Most animals die as embryos, and the few embryos that hatched were too disorganized to score the positions of migratory cells. To test whether wasp was involved in cell motility, we injected wasp RNA into unc-34(e315ts). At the permissive temperature many of the embryos hatched and these animals were found to have severe cell migration defects. We are currently screening for mutations that are synthetically lethal with unc-34.

24 May 1999 15:50 319 319 Expression of LIN-15a, a negative regulator of vulval induction

1999 International Worm Meeting abstract 268 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Expression of LIN-15a, a negative regulator of vulval induction JA DeModena, LS Huang, PW Sternberg HHMI/Division of Biology 156-29, Caltech, Pasadena, CA 91125 During vulval development, an inductive signal from the anchor cell induces three of the six vulval precursor cells to adopt vulval fates. lin-15 acts non-autonomously to negatively regulate the activity of the LET-23 (receptor tyrosine kinase/Ras) pathway, which mediates vulval induction from the anchor cell. lin-15 is part of a larger group of loci which are involved in this negative regulation pathway. These loci are known as synthetic Multivulva genes (synMuvs) and comprise two functionally redundant pathways, A and B, which act together as negative regulators of let-23 signaling. Single mutations in either class A or B genes result in a wild type phenotype, but homozygous mutations in both class A and B genes result in a Muv phenotype (Ferguson & Horvitz [1989] Genetics, 123, 109-121). lin-15 encodes two novel and hydrophilic polypeptides. These products account for the previously known, separable, A and B functions of lin-15. We previously reported that LIN-15B is nuclear and broadly expressed. Antibodies generated to lin-15A demonstrate a similar expression pattern. We are now testing whether lin-15A expression is regulated by other A class synMuv genes.

24 May 1999 15:50 320 320 Mitochondrial DNA mutation rate in Caenorhabditis elegans

1999 International Worm Meeting abstract 269 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Mitochondrial DNA mutation rate in Caenorhabditis elegans DR Denver 1 , K Morris 1 , L Vassilieva 2 , M Lynch 2 , WK Thomas 1

1 Department of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110. 2 Department of Biology, University of Oregon, Eugene, OR 97403.

The mutation rate of mitochondrial DNA (mtDNA) has previously been measured by two basic approaches: phylogenetic comparisons among closely related species with estimated divergence times and pedigree analyses. In humans, estimations of mutation rate based on phylogenetic analyses have been orders of magnitude lower than some estimates based on pedigree analysis. Resolution of this discrepancy is necessary for a clear understanding of the mechanisms of mtDNA mutations and for the proper use of mitochondrial sequences in molecular phylogenetic and population analyses of diverse organisms. To investigate the rate and pattern of mitochondrial mutations we have utilized a set of artificially evolved C. elegans lines. These lines have been propagated through single hermaphrodites for over 200 generations. We are currently in the process of sequencing the entire mitochondrial genome from 74 of these mutation accumulation lines. Preliminary data from three protein-coding genes (COII, ND1, ND5) has yielded multiple mutations of varying types.

24 May 1999 15:50 321 321 Analysis of the C. elegans homolog of the FHIT tumor suppressor gene

1999 International Worm Meeting abstract 270 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of the C. elegans homolog of the FHIT tumor suppressor gene WB Derry, S van Kreeveld, JH Rothman Department of Molecular, Cellular & Developmental Biology, University of California, Santa Barbara, CA 93016 USA Alterations in the FHIT gene occur frequently in the development of several human cancers (1). The Fhit protein is a diadenosine P1 ,P3 -triphosphate hydrolase and is a member of the histidine triad superfamily of nucleotide binding proteins (2). The cellular mechanism of Fhit activity and the relationship between Fhit signaling and tumorigenesis are presently unknown. The C. elegans and Drosophila FHIT genes encode a fusion protein in which the Fhit domain is fused with a novel domain showing homology to bacterial and plant nitrilases, and are referred to as NitFhit(3). We are interested in understanding the role of NitFhit in development and programmed cell death. RNAi of C. elegans NitFhit causes an embryonic arrest phenotype, suggesting an essential role for this gene in development. We are currently analyzing the loss-of-function phenotype and the effect of ectopicNitFhit expression on viability and programmed cell death in the worm.

(1) Huebner, K., Garrison, P.N., Barnes, L.D. & Croce, C.M. (1998). Ann. Rev. Genet.,32: 7-31. (2) Barnes, L.D., Garrison, P.N., Siprashvili, Z., Guranowski, A, Robinson, A.K., Ingram, S.W., Croce, C.M., Ohta, M. & Huebner, K. (1996). Biochemistry, 35: 11529-11535. (3) Pekarsky, Y., Campiglio, M., Siprashvili, Z, Druck, T., Sedkov, Y, Tillib, S., Draganescu, A., Wermuth, P., Rothman, J.H., Huebner, K., Buchberg, A.M., Mazo, A., Brenner, C. & Croce, C.M. (1998). Proc. Natl. Acad. Sci. USA, 95: 8744-8749.

24 May 1999 15:50 322 322 Characterization of two ras-signaling components in C. elegans

1999 International Worm Meeting abstract 271 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of two ras-signaling components in C. elegans EJ DesJardins, DW Sherbenou, M Han The University of Colorado at Boulder, Department of Molecular, Cellular and Developmental Biology, Boulder, CO 80309 The induction of vulval cell fates in C. elegans is controlled by a RAS signal transduction pathway. In an attempt to identify components within this pathway, our lab has used genetic suppressor screens to identify genes which, when mutated, suppress the multivulval (Muv) phenotype caused by a let-60(n1046)/ras gain-of-function mutant allele. These screens have defined RAF, MEK and MAP kinase as intermediates acting within this pathway, in addition to a number of novel genes. Two of the novel genes identified in these screens are studied in this work. One novel gene identified in these screens is sur-2 (Genes & Dev. (1995) 9;2251-2265). Worms homozygous for sur-2 loss-of-function alleles are vulvaless (Vul). Epistasis analysis verified that SUR-2 acts downstream of the let-60/RAS pathway and possibly downstream or in parallel to sur-1/MAP kinase. The SUR-2 gene product bears no significant homology to known signaling proteins; hence, its biochemical activity remains unknown. It is our hope that identifying SUR-2 interacting proteins in a yeast two-hybrid interactor screen may yield clues to SUR-2 function or regulation. A SUR-2:GAL4 DNA binding domain fusion protein was used as bait in the screen. Six positive clones have been identified using a C. elegans mixed stage random primed GAL4 activation domain library (courtesy of Robert Barstead). The cDNA inserts in these six clones encode four vitellogenin genes, 5’ nucleotidase, and one novel gene. We have chosen to pursue analysis of the novel cDNA, and our analysis is presented here. A second gene, sur-9, was also identified by the ability of a mutant allele, sur-9(ku258), to suppress the Muv phenotype caused by a let-60/ras gain-of-function allele. The sur-9(ku258) mutation alone produces a worm capable of VPC induction, albeit with vulval and other developmental defects. Linkage analysis places the sur-9 gene on LG-III. Three point mapping has localized the sur-9 gene within an interval bounded by unc-32 and emb-9. Cosmid rescue to clone the sur-9 gene is currently underway. Our analysis of sur-9 is presented.

24 May 1999 15:50 323 323 The MADS box containing factor CeMef-2 is not required for normal myogenesis and development

1999 International Worm Meeting abstract 272 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The MADS box containing factor CeMef-2 is not required for normal myogenesis and development D Dichoso 1 , T Brodigan 1 , JS Lee 2 , KY Chwoe 2 , R Llacer 1 , S Kostas 3 , A Fire 3 , JH Ahnn 2 , M Krause 1

1 Laboratory of Molec. Biol., NIH, Bethesda, USA. 2 Dept. Life Sciences, Kwangju Inst. of Sci & Tech., Kwangju, KOREA. 3 Dept. of Embryology, Carnegie Institution, Baltimore, USA.

Mef-2 is an evolutionarily conserved, MADS-box family transcription factor. Mef-2 was originally identified by its affinity for a muscle-specific cis-acting regulatory sequence and historically it has been considered an important myogenic factor. In vertebrates, Mef-2 has been shown to activate muscle-specific target genes and to interact with MyoD family members. However, because there are four Mef-2 genes in the mouse that are differentially spliced to generate multiple isoforms, it has been difficult to analyze the in vivo function of Mef-2 in mammals. The single Mef-2 homolog in Drosophila has been knocked out and shown to be essential for the differentiation (but not specification) of all muscle types during development. C. elegans has a single gene encoding a protein similar to Mef-2. Our interest in muscle determination prompted us to investigate if CeMef-2 played a role in myogenesis during worm development. Initial efforts using chromosomal deficiencies and reporter genes were unable to demonstrate any myogenic role for CeMef-2. More recently we have isolated two independent deletions of the mef-2 gene using the PCR based stately describe by Moulder and Barstead. At least one of these deletions is likely to be a null allele because it removes most of the DNA binding and dimerization motif. Animals homozygous for either deletion of mef-2 develop normally and have no discernable defects. These results suggest that CeMef-2 is not essential for myogenesis and development in C. elegans. Given previous observations with Drosophila, these experiments suggest an evolutionarily divergent role for MEF-2.

24 May 1999 15:50 324 324 Nicotine Adaptation is PKC Dependent

1999 International Worm Meeting abstract 273 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Nicotine Adaptation is PKC Dependent KA Dickinson UCSD 9500 Gilman Drive La Jolla, CA 92093 We are interested in studying nicotine adaptation in C. elegans. When initially exposed to nicotine, worms become paralyzed but gradually adapt to the drug and regain their ability to move. Another consequence of nicotine adaptation is a change in egg-laying behavior. Wild type worms display a dose dependent stimulation of egg-laying under inhibitory conditions (M9 solution) by the nicotinic acetylcholine receptor agonist levamisole. However, long term nicotine treatment results in adapted worms that are able to move around but unable to be stimulated to lay eggs by levamisole in M9. We found that worms begin to lose their responsiveness to levamisole after one hour on nicotine and completely fail to respond to levamisole after three hours on nicotine. We also found that the effects of nicotine adaptation are long-lasting, with adapted worms failing to respond to levamisole after a 24 hour incubation on drug free medium following exposure to nicotine. To identify molecules involved in the nicotine adaptation process, we performed the egg- laying assay for nicotine adaptation on C. elegans mutants with known defects in neuronal signaling molecules. This assay produced interesting results when we tested a strain of worms with a mutation in the tpa-1 gene, a gene that encodes protein kinase C. Naïve tpa-1 worms respond to levamisole with a similar dose response to wild type. However, nicotine treated tpa-1 worms display no reduction in levamisole-induced egg-laying even after overnight exposure to nicotine. This suggests that the long-term effects of nicotine on egg-laying behavior are dependent on PKC. To identify more molecules involved in nicotine adaptation, we are screening for adaptation defective mutants. Ultimately, such studies may elucidate the molecular mechanisms behind nicotine adaptation in worms and eventually lead to an understanding of nicotine addiction in humans

24 May 1999 15:50 325 325 Genetic characterization and molecular cloning of the defecation gene aex-1

1999 International Worm Meeting abstract 274 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic characterization and molecular cloning of the defecation gene aex-1 M Doi, K Iwasaki National Institute of Bioscience and HumanTechnology, 1-1, Higashi, Tsukuba, Ibaraki, 305-0046 Japan The C. elegans defecation motor program is achieved by periodic activation of a stereotyped sequence of three specific muscle contractions: posterior body muscle contraction (pBoc), anterior body muscle contraction (aBoc) and expulsion (Exp)(1) . Our current focus is to understand the regulatory mechanism of the aBoc and Exp execution. Ablation by a laser microbeam of two GABAergic motor neurons, AVL and DVB, eliminate these contraction (2) . GABA is necessary at least for Exp since unc-25 mutants, which are deficient in GABA, lack this contraction (2) . These observation suggest that, unlike the inhibitory function of classical GABAergic neurons, AVL and DVB might be excitatory GABAergic neurons for the aBoc and Exp steps. To understand this unusual GABAergic pathway, we characterize defecation motor program mutants and we are currently cloning aex-1. We localized the aex-1 region to a 100 kb interval between unc-13 and gld-1 on chromosome 1. Although rescue of the aex-1 mutant by injection of cosmid DNAs was possible, the transformation frequency with these DNAs was extremely low. Therefore, we attempted to clone aex-1 through identification of RFLPs. We isolated 9 new aex-1 alleles from 26,000 haploid genomes by non-complementation. In these alleles, one was the deletion uncovering gld-1 through goa-1. We identified RFLPs in this region by genomic southern hybridization and restriction analysis of PCR products. We are currently confirming that these RFLPs correspond to the aex-1 locus.

(1) Thomas, J. H. (1990) Genetics 124: 855-872. (2) Mclitire, S. L, et al. (1993) Nature 364: 337-341.

24 May 1999 15:50 326 326 Isolation and characterization of new class A synthetic multivulva genes

1999 International Worm Meeting abstract 275 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Isolation and characterization of new class A synthetic multivulva genes JM Doll, E Davison, HR Horvitz HHMI, Dept. Biology, MIT, Cambridge, MA 02139 The study of vulval induction in C. elegans provides a useful model for the analysis of the genetic and molecular mechanisms involved in cell-fate determination. Previous work has defined two classes of genes (A and B) that act redundantly to negatively regulate the adoption of vulval cell fates by vulval precursor cells. The elimination of a member of each gene class results in a synthetic multivulva (synMuv) phenotype. The B class of synMuv genes includes genes homologous to members of the mammalian Rb signaling pathway. By contrast, the class A genes remain relatively poorly understood. Previous screens to isolate class A genes have revealed, but have been unable to recover, sterile or maternal-effect lethal synMuv mutants. Therefore, beginning with a strong lin-15B background (n744), we are now undertaking a clonal screen to identify previously unknown class A synMuv genes as well as new alleles of those genes already recovered.

24 May 1999 15:50 327 327 Three RGS proteins appear to control Goa signaling in C. elegans

1999 International Worm Meeting abstract 276 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Three RGS proteins appear to control G oa signaling in C. elegans M Dong, MR Koelle Yale University RGS proteins (regulators of G protein signaling) inhibit G protein signaling by directly converting active G proteins (GTP-bound) to their inactive GDP-bound forms. Since almost all RGS proteins tested act similarly to each other in vitro, we would like to know why there are so many (19 in humans). We overexpressed 11 of the 12 C. elegans RGS genes in transgenic worms and found that only three of them, egl-10, C05B5.7, and F16H9.1, stimulated egg laying. This suggests that overexpressing any of these three RGS genes inhibits GOA-1, the C. elegans homolog of the human brain G protein Go , since GOA-1 signaling inhibits egg laying. Is this the normal function of these RGS genes or merely an artifact of overexpression? In fact, EGL-10 has previously been shown to genuinely function as an inhibitor of GOA-11 . Our current interest is in understanding the roles of C05B5.7 and F16H9.1. Expression of C05B5.7::GFP was seen in most or all neurons, resembling the expression patterns of both EGL-10 and GOA-1. F16H9.1::GFP was expressed in a large subset of neurons, the uterine muscles, and the spermatheca. We obtained putative null mutants for C05B5.7 and F16H9.1 by PCR screening mutant libraries for deletions in these genes. Phenotypic analysis of these mutants shows that egl-10, C05B5.7, and F16H9.1 play different roles in the regulation of egg laying. The C05B5.7 mutant has a weak egg-laying defect, the egl-10 mutant has a strong egg-laying defect, and the effects of the two mutations are additive in the double mutant. Overexpression of C05B5.7 can completely rescue the egg-laying defect of an egl-10 null mutant. A goa-1 null mutation suppresses the effect of the C05B5.7 mutation. Therefore, C05B5.7 appears to be a weak inhibitor of GOA-1, while EGL-10 is a strong inhibitor of GOA-1. The egg-laying defect of the egl-10 single mutant as well as that of the C05B5.7; egl-10 double mutant can be further enhanced by the F16H9.1 deletion, suggesting that F16H9.1 may also contribute to control of GOA-1. However, because effects of F16H9.1 on egg production complicate analysis of its effects on egg laying it is difficult to fully assess its role in GOA-1 signaling.

1 Koelle, M.R. and Horvitz, H.R. (1996) Cell 84, 115-125

24 May 1999 15:50 328 328 Structure/function analysis of LIN-31, a winged-helix transcription factor involved in Caenorhabditis elegans vulval development

1999 International Worm Meeting abstract 277 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Structure/function analysis of LIN-31, a winged-helix transcription factor involved in Caenorhabditis elegans vulval development DB Doroquez, H Hess, N Andrews, LM Miller Department of Biology, Santa Clara University The lin-31 gene encodes a winged-helix transcription factor involved in specifying vulval cell fates (Miller et al, Genes and Dev., 7:933, 1993). The LIN-31 protein contains a DNA-binding domain, an acidic region, a serine-rich region, and a proline-rich region. To elucidate possible functions of these domains, the 4 kb genomic domain spanning the lin-31 coding region was sequenced in fourteen lin-31 mutant alleles. This sequencing revealed 6 nonsense mutations, 3 deletions, 2 transposon insertions, 1 frameshift mutation, and 2 missense mutations. The two missense mutations are located at conserved residues in the DNA-binding domain. It is important to determine if the amino acid substitutions result in a destabilized protein or if their encoded mutant proteins are defective because they are no longer able to bind their DNA target. We have ruled out the first possibility by using antibody binding experiments to show that the LIN-31 protein is present in the proper cells at the proper stage of development. To confirm that the LIN-31 proteins encoded by these two mutant alleles are defective in DNA binding, we are performing in vivo extrachromosomal array binding experiments that utilize lac operator/repressor recognition. We are constructing transgenic strains whose extrachromosomal arrays carry multiple copies of the rat transthyretin (TTR) promoter (a known HNF-3 winged-helix transcription factor binding target), lac operator (lacO), and transgenes encoding GFP-tagged lac repressor (lacI::GFP). Binding of tagged lac repressor to lacO sequences allows us to tag the TTR promoter in vivo. Failure of marked TTR and antibody-stained mutant LIN-31 to co-localize to the array may suggest that these LIN-31 proteins fail to bind their DNA target. In summary, we have learned that (1) the null phenotype of lin-31 is the phenotype displayed by most of the existing alleles, (2) direct screens for Muvs and Vuls may only yield null alleles of lin-31, and (3) the DNA-binding domain plays a critical role in LIN-31 function.

24 May 1999 15:50 329 329 Genetic and molecular characterization of sel-8, a suppressor of lin-12 gain-of-function mutants

1999 International Worm Meeting abstract 278 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic and molecular characterization of sel-8, a suppressor of lin-12 gain-of-function mutants TG Doyle, I Greenwald HHMI, Columbia University, New York, NY 10032 We have been studying sel-8, a suppressor of lin-12(d) mutations that also shows genetic interactions with glp-1 (1). The one existing sel-8 allele, sa54, suppresses lin-12(d) in an allele-nonspecific and dose-dependent way. In an otherwise wild-type background, sel-8(sa54) causes an incompletely penetrant early embryonic lethality at 15C. The lethality can be partially rescued by zygotic sel-8 activity. We used three-factor mapping to locate sel-8 in an interval of LGIII between ben-1 and unc-93 that contains seven predicted ORFs. We sequenced these ORFs from a sel-8(sa54) mutant, and found a nonsense mutation in one ORF, C32A3.1. Over 95% of the other ORFs in this region have been sequenced and no other mutations have been found. The nonsense mutation cosegregates with the sel-8 suppression and cold-sensitive embryonic lethality phenotypes. In addition, RNAi using dsRNA from C32A3.1 produces embryonic lethality in 95% of F1s in an N2 background. Surviving F1s show defects characteristic of loss of lin-12 activity as well as germline proliferation defects reminiscent of loss of glp-1 activity. Based on the RNAi phenotype and on the presence a single nonsense mutation in sel-8(sa54) animals, we conclude that C32A3.1 is sel-8. C32A3.1 is predicted to encode a glutamine-rich 468 amino acid protein with no similarity to any proteins in the database. It does not appear to contain a signal sequence or transmembrane domains. The nonsense mutation occurs relatively late in the sequence and this observation in combination with the RNAi data suggests that sel-8(sa54) is probably not a null allele. We are in the progress of analyzing sel-8 further genetically and molecularly, and we will report on our progress at the meeting.

1. Tax, F.E., J.H. Thomas, E.L. Ferguson, and H.R. Horvitz, Genetics 147:1675-1695 (1997).

24 May 1999 15:50 330 330 Function and expression of ceh-32, a sine oculis homologue.

1999 International Worm Meeting abstract 279 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Function and expression of ceh-32, a sine oculis homologue. C Dozier, G Cassata, G Niklaus, H Kagoshima, TR Bürglin Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel ceh-32 is a C.elegans homeobox gene of the six/sine oculis (so) gene family. It belongs to the six3 subclass of genes which include mouse six3, chicken, mouse and Drosophila optix. From an embryonic cDNA library (kindly provided by P. Okkema) we cloned a ceh32 cDNA. The sequence of this 1860 bp cDNA clone revealed an open reading frame of 439 amino acids. The locus is present on the cosmid W05E10. RNAi of ceh-32 caused a Vab phenotype with the typical "notched head" aspect, reminiscent of the Vab-3 (Pax-6) phenotype. The alae reach the anterior part of the head, whereas in normal animals they do not. This is also similar to the Vab-3 phenotype, where some of posterior structures reach more anterior locations. We also observed gonadal defects: the gonads are smaller and/or misplaced. To examine the expression pattern of ceh-32, promotor gfp fusion constructs were generated. Analysis of the gfp transgenic lines showed a dynamic embryonic and post-embryonic expression. In embryonic stages, the expression in the anterior H0, H1 lineage overlaps with vab-3. In post-embryonic stages ceh-32 is expressed in e1, e2 and e3 epithelial cells of the pharynx, in M3 myoblast of the pharynx, in g1 and g2 gland cells of the pharynx, as well as in Z1 and Z2 somatic gonad precursor cells and later in the gonadal sheath cells. By Northern blot analysis of mixed worm stages a decreased ceh-32 expression was observed in a vab-3 genetic background. We do not know whether this is due to the lack of transcriptional control via vab-3, or to the lack of ceh-32 expressing cells. This is under investigation. We also investigated the role and expression of ceh-33, 34 and 35, three other six/so homeobox genes. gfp reporters for ceh-33 and 34 showed very weak expression in the pharynx of late embryos and early larvae. For ceh-35::gfp we saw no expression. For ceh-33 and 35 we were able to amplify gene specific cDNA fragments from the 3’ and 5’ end, respectively, while we were not able to amplify ceh-34. RNAi of ceh-33 and ceh-35 caused gonadal defects in the injected worms with a small amount of normal oocytes produced.

24 May 1999 15:50 331 331 Teneurin, a novel morhogenetic factor in C. elegans

1999 International Worm Meeting abstract 280 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Teneurin, a novel morhogenetic factor in C. elegans K Drabikowski, R Chiquet-Ehrismann Friedrich Miescher Institute, P.O. Box 2543 CH-4002 Basel, Switzerland The ten-m gene was identified by Baumgartner et. al. [1994] in Drosophila. It is the first pair rule gene, which is not a transcription factor but an extracellular matrix protein. It is also expressed in the developing nervous system. Members of the Ten-m protein family were also identified in vertebrates and named "teneurins" [Minet et. al., in press]. Teneurins are differentially expressed in the nervous system. One of the human teneurins is mapped to an X-linked non-specific mental retardation syndrome. Teneurins are type II transmembrane proteins. The N-terminal intracellular part is followed by a single transmembrane domain. The extracellular domain is around 2500 aa and contains EGF-like repeats. The C. elegans teneurin is located at chromosome III and is mapped to the cosmid R13F6. By 5’-RACE analysis we identified an alternative trascription start, 8 kb upstream located on the cosmid F28F5. This alternative transcript increases the length of the intracellular domain of teneurin from 39 to 220 aa. We have analysed the expression pattern of teneurin by transfecting worms with promoter-GFP fusion constructs. Protein from the downstream promoter is expressed early in embryogenesis. In larvae and adult worms it is expressed in a subset of neurons including lateral and ventral ganglia, the lumbar ganglion and motor neurons. RNA interference aimed against the 5’ alternative extension did not result in an obvious phenotype and needs to be investigated in more detail. RNAi aimed against the transcripts from both promoters revealed a complex phenotype. Most larvae burst during hatching and the survivors often burst at later stages of development. The gut is not fully developed and often bifurcates. Adult hermaphrodites have very little or no sperm. The gonad is often displaced. Teneurin is one of the candidates for the Dig-1 phenotype. The teneurin RNAi phenotype is more severe than that of the Dig-1. It cannot be excluded, however, that Dig-1 carries a minor mutation responsible for partial loss- or malfunction of teneurin. The expected neuronal phenotype might be masked by the dramatic tissue malformations. The complex RNAi phenotype suggests that teneurin is crucial for cell migration and morphogenesis.

24 May 1999 15:50 332 332 A Novel Suppresor of unc-93(e1500)-induced paralysis in C. elegans

1999 International Worm Meeting abstract 281 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A Novel Suppresor of unc-93(e1500)-induced paralysis in C. elegans AM Dude, J Donohue, J Tennessen, EA De Stasio Lawrence University The C. elegans mutation unc-93(e1500) confers a characteristic paralyzed phenotype, denoted "rubberband". Worms carrying this allele are flaccidly paralyzed and egg-laying defective. The e1500 allele produces a missense trans-membrane protein that appears to interfere with the control of muscle contraction rather than contraction itself (Greenwald and Horvitz, 1980). Certain mutations in any of five genes (unc-93, sup-9, sup-10, sup-11, and sup-18) can suppress the rubberband phenotype, resulting in wild type motility (Greenwald & Horvitz, 1980,1982,1986; Levin & Horvitz, 1993). Three novel suppressor mutations were isolated after ENU mutagenesis of unc-93(e1500) animals (De Stasio et al., 1997). These alleles are assumed to be mutations in a new gene in the unc-93 family or they are novel alleles of one of the known suppressors, as they: 1. complement null alleles of sup-9, sup-10, and unc-93 2. are not fully dominant suppressors as are sup-11 alleles 3. suppress to wild type motility, unlike sup-18 alleles. Unlike other e1500 suppressors, the new suppressor alleles are recessive suppressors of e1500 homozygotes and dominant suppressors of e1500 heterozygotes. Suppression to wild type motility is complete in either case, though males of homozygous suppressed strains have little or no mating ability. The suppressor alleles confer no visible phenotype of their own. All three mutations appear to be alleles of the same gene based on non-complementation of a slightly Dpy-Rol phenotype seen in a dpy-17 genetic background. Linkage and mapping experiments have indicated that sup-new alleles are linked to lin-31 on chromosome II, on which sup-9 is also located. Three factor analysis using a second marker gene, bli-2, is being used to determine the relative order of sup-9, lin-31, bli-2, and sup-new.unc-93(e1500)sup-9sup-10unc-93e1500bli-2dpy-17unc-93(e1500)bli-2unc-93(e1500)

24 May 1999 15:50 333 333 Chromogranin A Immunoreactivity in C. elegans

1999 International Worm Meeting abstract 282 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Chromogranin A Immunoreactivity in C. elegans J Duerr 1 , J Gaskin 1 , L Eiden 2 , J Rand 1

1 Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK. 2 Section on Molecular Neuroscience, NIMH, Bethesda, MD.

Chromogranin A (CGA) is the most abundant protein in chromaffin granules, catecholamine-containing vesicles of the mammalian adrenal medulla. It is also widespread in the nervous system and in a number of other endocrine tissues. CGA is believed to be a pro-hormone, comprising more than one peptide (neuro)hormone. In addition, its biochemical properties have led to the suggestion that it may also have intracellular roles. Antibodies to CGA have identified a related sequence in at least one invertebrate, the lobster (Rieker et al.). Antibodies against a portion of CGA have also been used on Ascaris suum (Brownlee et al.), where immunoreactivity was found in the ventral and lateral nerve cords. However, no sequence highly homologous to CGA has yet been described by the sequencing consortium. We have used antibodies against two peptides derived from vertebrate CGA to examine the distribution of related peptides in C. elegans. Antibodies to both CGA peptides recognize a number of bands on Westerns, including a shared band of the appropriate size for CGA. Like bonafide CGA, this protein is resistant to boiling. However, antibodies against the two peptides give different patterns on fixed worms. An antibody against the N-terminal peptide strongly labels a subset of neurons in the head ganglia, including some sensory neurons. unc-104 mutants, in which synaptic vesicles and proteins are mislocalized in cell bodies, show an enrichment of anti-CGA in a subset of neuronal somas. This is consistent with the CGA-like immunoreactivity being enriched in synaptic vesicles, as would be expected for a neuropeptide. On the other hand, antibodies to a peptide from the middle of the protein label most neurons in a uniform way and this staining is not altered in unc-104 mutants. We are currently trying to understand this difference in pattern and to determine whether the antigens recognized by the anti-CGA antibodies reflect the distribution of proteins or peptides with CGA-related functions.

This work was supported by grants from OCAST and NIH.

24 May 1999 15:50 334 334 Development of Neurotransmitter Phenotypes in the VC Motor Neurons

1999 International Worm Meeting abstract 283 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Development of Neurotransmitter Phenotypes in the VC Motor Neurons J Duerr, J Gaskin, D Frisby, J Rand Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK The VC neurons are a class of six post-embryonic motor neurons with synapses on the vulva muscles, other body muscles, and a few other motor neurons (White et al.). In late L4, all six VCs develop FMRFamide-like immunoreactivity (Schinkmann and Li) and some are induced to branch by the vulva (Li and Chalfie). We have used specific antibodies to determine that some of the VCs probably use acetylcholine and a monoamine as well as a FMRFamide-like peptide as neurotransmitters. The antibodies we used were generated against synaptic proteins, so they only weakly label cell bodies (whose position aids greatly in cell identification). Therefore, we used unc-104 mutants (in which synaptic proteins are mislocalized in somas) as well as over-expressing transgenic lines (in which ’excess’ protein is found in somas) to identify neurons. In adults, all of the VCs are usually labeled with antibodies to two cholinergic markers, the acetylcholine synthetic enzyme choline acetyltransferase (ChAT, encoded by cha-1) and the vesicular acetylcholine transporter (VAChT, encoded by unc-17). VC4 and VC5, but not the other VCs, are also labeled with antibodies to a monoaminergic marker, the vesicular monoamine transporter (VMAT, encoded by cat-1). Antibodies to ChAT, VAChT, and VMAT label the VC motor neurons in slightly different patterns spatially and temporally. When the VCs are born in late L1, they do not exhibit detectable cholinergic or aminergic immunoreactivity. This is consistent with the expression pattern of other post-embryonic cholinergic neurons. During larval development, the intensity of cholinergic immunoreactivity increases fairly uniformly in most post-embryonic ventral nerve cord cholinergic neurons. However, some of the VCs continue to lack detectable immunoreactivity or express very low and variable levels. As the vulva develops, ChAT and VMAT immunoreactivity greatly increase in VC4 and VC5 and increase slightly in some of the other VCs. This is followed by expression of VMAT in VC4 and VC5 (but not in the other VCs). We are studying the temporal and spatial patterns of these and other late VC markers to try and understand their regulation and relevance. This work was supported by grants from OCAST and NIH.

24 May 1999 15:50 335 335 Analysis of p130, a component of the P/CAF complex, in C. elegans

1999 International Worm Meeting abstract 284 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of p130, a component of the P/CAF complex, in C. elegans P Dufourcq 1 , Y Nakatani 2 , M Labouesse 3 , Y Shi 1

1 Havard Medical School, Dept. Pathology, Boston, MA 02115. 2 Laboratory of Molecular Growth Regulation, N.I.H. Bethesda, MD 20892. 3 IGBMC, 1 rue Laurent Fries, BP163, 67404 Strasbourg Cedex, France.

Recent studies show that proteins involved in chromatin remodeling such as histone acetylases play essential roles in differentiation, and these proteins are conserved from yeast, C. elegans to human. The mammalian transcriptional cofactors p300 and CBP are histone acetylases and their C. elegans homolog cbp-1 has been to be a critical regulator of cell fate and differentiation (1). Other histone acetylases such as P/CAF and TAFII250 are also conserved in C. elegans, but their biological functions are unknown. The P/CAF complex is composed of more than 20 proteins including previously identified proteins, such as TBP Associating Factors (TAFs), as well as novel proteins of unknown function (2). We find that components of the P/CAF complex such as p/CAF, the TAFs and p130, a protein related to UV damaged-induced protein (DDB1), are conserved in C. elegans. The high level of sequence similarity suggests that these proteins are likely to share similar biological activities. Thus, insight into the biology and mechanisms of action of the p/CAF complex in C. elegans will be directly relevant to our understanding of the functions of mammalian p/CAF in cell growth and differentiation. We examined the role of p130 in C. elegans by RNAi and identified an essential function for p130 in C. elegans early embryogenesis. p130 (RNAi) embryos arrest between 200 and 250-cells stage, as opposed to cbp-1 (RNAi) embryos which arrest with an increased cell number (~1000 cells). Morphological and immunological analyses suggest that these embryos lack endoderm, mesoderm and hypodermal differentiation. Cell lineage analysis shows that the intestinal founder cells (Ea and Ep) fail to gastrulate, indicating an early defect associated with the failure of the p130 (RNAi) to undergo endoderm differentiation. We are currently investigating the molecular mechanisms that underlie the role of p130 in C. elegans embryogenesis.

1. Shi, Y., and Mello, C.C. (1998). A CBP/p300 homolog specifies multiple differentiation pathways in C. elegans. Gen. Dev. 12, 943-955. 2. Ogryzko,V., et al. (1998). Histones-like TAFs within the PCAF histone Acethylase Complex. Cell. 94, 35-44.

24 May 1999 15:50 336 336 Genome-Wide RNAi-Based Screen to Identify Genes Required for Cell Division Processes in C. elegans.

1999 International Worm Meeting abstract 285 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genome-Wide RNAi-Based Screen to Identify Genes Required for Cell Division Processes in C. elegans. C Echeverri 1 , P Gönczy 1 , K Oegema 1 , S Pichler 1 , M Kirkham 1 , M Brehm 1 , A Coulson 2 , S Jones 2 , A Hyman 1

1 EMBL, Heidelberg, Germany. 2 Genome Sequence Centre, BC, Canada.

We are initiating a genome-wide screen to identify genes required for cell division using RNA-mediated interference. Phenotypes will be scored by time-lapse DIC microscopy, thus permitting a detailed analysis of the distinct cellular processes underlying the first two embryonic divisions. The following methodology will be applied for each gene. First, an algorithm is used to select optimal PCR primer pairs for each of the ~18,450 predicted genes in the worm genome. Using these primers, a DNA template is generated by PCR for each gene. Two complementary ssRNAs are then made from this template by in vitro transcription and annealed together to yield a dsRNA (~1mg/ml). The dsRNA is injected into the gonads of adult N2 hermaphrodites, embryos are dissected out 24 hrs later, and the first two cell divisions are recorded. In order to maximize our throughput, we plan to inject dsRNAs in pools of 8, and confirm positive candidates by injections of individual dsRNAs. Using this approach, a first group of 64 genes was revealed to contain 3 dsRNAs yielding defects in chromosome segregation, P1 rotation and P1/AB nuclear position, respectively. In parallel, we are also testing a panel of positive control dsRNAs, to test whether dilution in pools of 8 dsRNAs would affect the penetrance of the phenotypes. As seen for the 3 newly identified genes, RNAi phenotypes for cytoplasmic dynein and zyg-9 remained fully penetrant when diluted 8-fold in other dsRNAs. In contrast, however, RNAi phenotypes for the par-3 and cyk-1 dsRNAs tested were lost after only 3-fold dilutions with other dsRNAs. When diluted in buffer alone though, the same two dsRNAs lost their potency only after 1024-fold and 256-fold dilutions, respectively. Therefore, the mixing of multiple dsRNAs significantly decreases the RNAi potency of certain dsRNAs. We are currently further investigating the nature of this effect to determine whether a lowering of our pool sizes may be warranted. We also expect that this work will offer new insights into the mechanistic basis of the RNAi effect. By the worm meeting, we will have chosen our pool size, and initiated the screen of all predicted genes on chromosome III.

24 May 1999 15:50 337 337 Embryonic pal-1 expression in the AB lineage is required for late gastrulation and rectal formation.

1999 International Worm Meeting abstract 286 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Embryonic pal-1 expression in the AB lineage is required for late gastrulation and rectal formation. LG Edgar, WB Wood U. Colorado, Boulder CO 80309 During C. elegans embryogenesis the caudal homolog pal-1 is expressed in small subsets of many lineages in a dynamic temporal pattern. In the AB lineage, it first appears in ABplpappp and ABplppppp at the 350-cell stage. These cells form the left posterior border of the ventral cleft in late gastrulation, when lateral ridges of mesectodermal cells move to the ventral midline. pal-1 expression is observed during this cell movement, before ABplppppp meets its lineal homolog, ABprppppp, to begin the closure of the cleft. After closure pal-1 expression persists in the daughters of ABplppppp, and also appears in Abprppppp daughters. These cells divide once again, move posteriorly, and differentiate as hypodermal cells of the tail (tail spike and hyp 10) and internal cells associated with the rectum (left intestinal muscle, anal depressor muscle, anal sphincter, and the single AB body wall muscle). In 20% of embryos homozygous for the presumed null allele pal-1(ct224), closure of the ventral cleft is never completed. Ventral hypodermal enclosure is also delayed or abortive. ct224 embryos that terminate with a Nob phenotype (~80%) show delayed ventral cleft closure but eventually complete hypodermal enclosure. In wild-type embryos, laser ablation of one or two of the cells bordering the posterior ventral cleft, just before its closure, delayed or prevented closure, often producing phenocopies of the unenclosed or Nob pal-1 phenotypes, and confirming a key role for these cells in late gastrulation. Most embryos in which only one cell was ablated developed to L1, but were consistently missing rectal structures. Expression of pal-1 in these AB cells and in two MS cells which migrate to the posterior appears to be controlled by 5’ elements more than 1 kb upstream from the SL1 splice site. A reporter construct with 1.2 kb of upstream sequence is not expressed in the migrating MS cells and the AB cells. An integrated pal-1 genomic construct with 1.0 kb of upstream sequence rescues the Nob phenotype, but although they hatch, these embryos, like those with single AB ablations, have no rectum. Thus expression of pal-1 in AB cells appears to have two required functions, first during ventral cleft closure and later for rectal morphogenesis.

24 May 1999 15:50 338 338 The Chromosome II Genetic Toolkit

1999 International Worm Meeting abstract 287 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Chromosome II Genetic Toolkit ML Edgley, CA Turner, DL Riddle Division of Biological Sciences, University of Missouri, Columbia, MO 65211 We have generated a set of tools to aid genetic and molecular studies over most of chromosome II, consisting of an inversion that acts as a balancer (mIn1), new marked lethal mutations distributed across the balanced region, a better-characterized set of existing deficiencies, and new deficiencies isolated in lethal mutant screens. Analysis of the deficiencies and mutations has greatly increased the resolution of the genetic map in some areas and helped refine the genetic/physical map alignment. The inversion mIn1 balances a large part of the chromosome from let-552 to rol-1, including all of the gene-dense cluster. We generated several genetic variants of mIn1 to broaden its utility for mutational analysis, and we have used this balancer in mutant screens to isolate lethal mutations that identify new genes, as well as deficiencies that provide new endpoints useful for mapping. Screens of 10,000 mutagenized chromosomes yielded approximately 150 recessive lethal mutations. These were analyzed by complementation with existing deficiencies and inter se complementation to map them to deficiency endpoint zones and establish complementation groups. This work resulted in mutations in 28 genes not previously identified by mutation, new alleles of seven existing genes, and six new deficiencies. Endpoints of five existing deficiencies were refined. Over the region left of dpy-10, the number of zones defined by deficiency endpoints increased from five to 13. Refinement of the genetic/physical map alignment is being approached in two ways, making use of DNA sequence generated by the C. elegans genome project: (1) localization of deficiency endpoints by PCR analysis of deficiency homozygotes, and (2) three-factor mapping of Tc1 polymorphisms relative to deficiency endpoints. These tools should help speed genetic and molecular analysis by providing mutant alleles of genes predicted from DNA sequence, and by providing a means to rapidly narrow the range of coding regions that might correspond to existing mutations. This work is supported by the NIH National Center for Research Resources (NCRR).

24 May 1999 15:50 339 339 Genetic Variants and Structural Characterization of the Chromosome II Inversion mIn1

1999 International Worm Meeting abstract 288 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic Variants and Structural Characterization of the Chromosome II Inversion mIn1 ML Edgley, DL Riddle Division of Biological Sciences, University of Missouri, Columbia, MO 65211 The chromosomal rearrangement mIn1 (previously called mC6) virtually eliminates recombination in heterozygotes over the large interval covering the gene cluster on LG II from let-552 to rol-1. We have generated a set of marked and unmarked variants of this balancer to maximize its utility; and used the variants to map a transgene inserted into the balancer and to demonstrate that mIn1 is an inversion, the second such rearrangement identified in C. elegans. In addition, DNA dimorphisms were used to map the right breakpoint of mIn1 to a small region on the physical map to the right of rol-1. Variants of mIn1 include versions that are unmarked; singly and doubly marked with different combinations of recessive mutations including dpy-10, unc-4, rol-1 and several unmapped lethals; and marked with the integrated semi-dominant GFP-expressing element mIs14. These variants suppress recombination over the same interval and to the same degree as the original isolate. Thus, they provide a number of useful phenotypes for moving and following the balancer in different genetic backgrounds. Unmarked and doubly-marked mIn1 chromosomes were used in three-factor mapping experiments to demonstrate that the order of dpy-10 and unc-4 in the rearrangement is reversed relative to wild type, showing that mIn1 is an inversion. Similar experiments demonstrated that rol-1 lies within the inverted region, and that mIs14 lies left of dpy-10 on the normal genetic map. The position of the right breakpoint of mIn1 was refined by three-factor mapping experiments using DNA dimorphisms between the wild strains N2 and RC301. The mIn1 breakpoint was found to lie between rol-1 and jsP301, a region spanned by four cosmids and a YAC bridge. Our collection of mIn1 variants is a useful, well-characterized set of tools for analyzing mutations over most of chromosome II, including most of the chromosome to the left of the region balanced by mnC1. Furthermore, the structural analysis of the inversion may lead to insights into the mechanisms of pairing and recombination. This work was supported by the NIH National Center for Research Resources (NCRR).

24 May 1999 15:50 340 340 Cell fusion as an alternative fate for surviving non-vulval Pn.p cells in Pristionchus pacificus.

1999 International Worm Meeting abstract 289 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cell fusion as an alternative fate for surviving non-vulval Pn.p cells in Pristionchus pacificus. A Eizinger 1,2 , R Sommer 1

1 Max Planck Institute for Developmental Biology, Dept. for Evolutionary Biology, Spemannstrasse 35, D-72076 Tübingen Germany. 2 E.mail: [email protected]

We are studying evolutionary changes during vulva development between Caenorhabditis elegans and Pristionchus pacificus. Homologous cells of the ventral epidermis, some of which give rise to the vulva, differ in their cell fate between these two species. Non-vulval cells, P(1-4,8-11).p, fuse in C. elegans with an epidermal syncytium, whereas in P. pacificus P(1-4,9-11).p die of programmed cell death (PCD). From the four surviving cells, P(5-8).p, the three anterior form the vulva, whereas P8.p remains epidermal. We used the monoclonal antibody MH-27, which stains adherens junctions of non-fused epidermal cells, to study the cell fate of P8.p in P. pacificus. The antibody staining revealed that P8.p fuses with the syncytium soon after hatching. We previously identified mutations in the cell death gene Ppa-ced-3, in which the 12 Pn.p cells survive. MH-27 antibody staining showed that with the exception of P(5-7).p all ventral epidermal cells fuse with the surrounding hypodermis. Therefore, Pn.p cells that die in wild-type enter a cell fusion pathway, if cell death cannot be executed. One simple hypothesis is that cell fusion and PCD are related, alternative cell fate pathways, which might even posses some similar molecular aspects. The observation of Pn.p cell fusion in Ppa-ced-3 is in contrast to Ppa-ped-6, a mutation described previously, that enables P(3,4).p to specifically survive, whereas P(1,2,9-11).p still die of PCD. Alltogether nine mutants have been isolated, which show a similar phenotype to Ppa-ped-6. Complementation analysis revealed that they are all allelic to Ppa-ped-6. P(3,4).p not only survive, but they start to differentiate and form ectopic vulva-like protrusions. Cell ablations of the gonad precursors, Z(1,4), showed that all surviving Pn.p cells except P8.p have the potential to differentiate, resembling a multivulva phenotype. Molecular analysis of the Ppa-ped-6 mutation is ongoing.

24 May 1999 15:50 341 341 mup-4, a Gene Required for Epidermal Morphogenesis and Muscle Position, is a Member of a Novel Family of Transmembrane Proteins.

1999 International Worm Meeting abstract 290 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. mup-4, a Gene Required for Epidermal Morphogenesis and Muscle Position, is a Member of a Novel Family of Transmembrane Proteins. T Elbl, L Hong, J Ward, A Franzini-Armstrong, EA Bucher Univ. of Pennsylvania, Dept. of Cell and Dev. Biology, Philadelphia, PA 19104-6058. mup-4 is a member of a new family of genes hypothesized to mediate cell-cell and/or cell-matrix interactions gene in C. elegans. mup-4 and mua-3 have recently been cloned and are homologues. Mutants for these genes show related, but distinct phenotypes: mup-4 mutants exhibit defects in muscle position at 3-fold 1 , whereas mua-3 mutants display muscle position defects during larval stages2 . In addition, mup-4 mutants have epidermal defects in embryonic enclosure and vulval morphogenesis (in genetic mosaics). mup-4 and mua-32 encode large transmembrane proteins having novel, largely unrelated, intracellular domains, and similar extracellular domains with EGF-like, type XII collagen, and novel, shared, motifs. The phenotypic and molecular data are consistent with functions in cell-extracellular matrix (ECM) or cell-cell adhesion and/or signaling pathways. We are currently testing to distinguish among possible models for MUP-4 function by: (i) determining the temporal, spatial and subcellular localization of MUP-4; (ii) examining the ultrastructural defects in mup-4 mutants; and (iii) testing for interacting partners with yeast 2-hybrid and genetic interaction studies. Thus far, our ultrastructural studies demonstrate that muscle detachment is associated with a lesion between the hypodermis and the exoskeletal cuticle, and our expression studies demonstrate that mup-4 is expressed in the hypodermis overlying muscles. Furthermore, consistent with the proposed role in morphogenesis, we find that mup-4 is also expressed in embryonic hypodermal cells, prior to embryo enclosure, and in the anchor cell, a cell involved in vulval morphogenesis. One possible hypothesis to explain these phenotypic and expression data is that MUP-4 functions: i) in hypodermal adhesion or polarity and thus influences epidermal morphogenetic events and ii) by adhering the apical membrane of the hypodermis to the cuticular ECM and transmitting the force of muscle contraction to the exoskeleton.

1 Gatewood, B. K., and E. A. Bucher. 1997. Genetics 146:165-83.

2 J. Plenefisch and E. Hedgecock, pers. com.

24 May 1999 15:50 342 342 A Nematode Drug Screen For Presenilin Inhibitors In Alzheimer’s Disease

1999 International Worm Meeting abstract 291 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A Nematode Drug Screen For Presenilin Inhibitors In Alzheimer’s Disease BR Ellerbrock, EM Thomas, TG Geary, KA Greenlee, NC Stratman, JR Brashler, GW Melchior, AE Buhl, DB Carter, ME Gurney Pharmacia & Upjohn, 301 Henrietta, Kalamazoo MI 49007 The genome of C. elegans contains three orthologs of the Alzheimer’s disease presenilin genes. When mutated in C. elegans, loss of presenilin function causes a defect in egg-laying. In human cells, loss of presenilin-1 (PS1) function blocks amyloid-beta peptide processing from the amyloid protein precursor (APP). Thus, we sought to screen in worms for compounds that block egg laying, and hence phenocopy presenilin loss of function. Egg laying in C. elegans was assessed by measuring the release of chitinase. Chitin makes up approximately 5% of the nematode eggshell and helps maintain the structural integrity of the egg. Chitin is degraded in concert by chitinase and N-acetylglucosaminidase (Mol. Biochem. Parasitol. 58:317-324, 1993). An assay for egg laying by measuring chitinase was tested in wildtype C. elegans (N2 strain), sel-12 presenilin mutants (provided by Dr. Iva Greenwald, Columbia Univ.) and 2 strains of Egl-36 Kv3 potassium channel mutants. The sel-12 mutations causes a nearly complete defect in egg-laying while the Egl-36 mutants are partially defective. Release of chitinase from the Sel-12 and Egl-36 strains was significantly (p<0.001) reduced in comparison to the wildtype strain. Approximately 11,000 compounds were screened at 10uM for suppression of egg laying by wildtype worms. A subset of these inhibitors may block presenilin function. Potential hits in the chitinase assay caused 50% inhibition of chitinase activity in replicate samples. Thirty-seven potential hits were identified in the primary screen. All of these compounds were evaluated in a secondary assay looking at the dose response (0.03-10uM) of the compound. Sixteen real hits with no cellular toxicity for C. elegans were identified. These compounds were also evaluated for inhibition of APP processing. The real hits and 1700 compounds at the 10uM are currently being evaluated in the lin-12 (notch) C. elegans for reversal of the egg-laying defect. These secondary screens make use of knowledge regarding the genetic interaction of C. elegans presenilin (sel-12) and notch (lin-12). To date, a single compound has been identified which inhibits egg laying in C. elegans and also amyloid-beta peptide processing in human cells.

24 May 1999 15:50 343 343 Integration of lineal, spatial, temporal and sexual coordinates by a C. elegans hox gene promoter

1999 International Worm Meeting abstract 292 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Integration of lineal, spatial, temporal and sexual coordinates by a C. elegans hox gene promoter SW Emmons 1 , HB Ferreira 2

1 Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY. 2 Centro de Biotecnologia and Departamento de Biologia Molecular e Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.

Cells make developmental decisions following cues that provide them with information about their lineal ancestry, their position in the body or the identities of their neighbors, developmental stage, and sex. Regulatory pathways are known that convey each of these developmental coordinates to cells. These multiple inputs are integrated by the promoters of regulatory genes, so that cells make correct choices of cell fate at each point in the cell lineage. The C. elegans Hox gene egl-5 is such an integrating regulatory transcription factor in the cell lineages leading to the male rays. In posterior seam cell lineages, transcription of egl-5 is initiated at a specific time in a subset of lineage branches only in the male. We would like to define the factors that act on the egl-5 promoter to specify each of these conditions. egl-5 expression in the male seam, studied by antibody staining, first occurs in V6.ppp, progenitor of rays 4, 5, and 6, during the L2 larval stage. During L3, expression is found in lineage branches leading to rays 3-6. In egl-5(0), rays 3-5 assume abnormal positions, and ray 6 is lacking. Seam cell expression is dependent on mab-5, a second gene of the Hox gene cluster. mab-5, which may be considered to convey lineal information, is expressed long before egl-5 transcription is initiated, is expressed in lineage branches where egl-5 transcription is not initiated, and is expressed in hermaphrodites as well as males. We postulate that a heterochronic gene may trigger egl-5 expression at the correct time, a Wnt pathway may limit transcription to posterior lineage branches, and TRA-1 may limit transcription to males. We are using egl-5::gfp reporter genes to study these possibilities. Continued reporter gene activity is dependent on the endogenous egl-5 locus, suggesting that once initiated, egl-5 expression is maintained by a positive feedback loop. We plan to sequence the egl-5 promoter region from additional nematode species to identify conserved regulatory sequences, which will be tested by altering them in reporter genes.

24 May 1999 15:50 344 344 or148 and or209: Two genes involved in cell division timing in early C.elegans embryogenesis

1999 International Worm Meeting abstract 293 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. or148 and or209: Two genes involved in cell division timing in early C.elegans embryogenesis SE Encalada, DR Hamill, B Bowerman Institute of Molecular biology, University of Oregon, Eugene, OR 97403 In early C. elegans embryos, cells are produced by asymmetric divisions and appear to be born with characteristic cell cycle times. For example, the first cleavage of the embryo produces a smaller posterior daugher blastomere called P1 that divides more slowly than the anterior daughter, called AB. These cell cycle differences may be important for proper regulation of pattern formation. To understand how these differences in cell cycle timing arise, and to determine if they are important for pattern formation, we have screened for mutants with alterations in the cell cycle timing of early embryonic cells. In a screen for temperature sensitive embryonic lethal mutants, we have identified three mutations, or148, or209, and or182, that result in cell cycle delays in the early embryo. These three mutations define three separate genes based on genetic mapping and complementation tests. For one of these genes, defined by or148, we also have identified three non-conditional mutations that are allelic (or16, or17, and or27). A common phenotype of these mutants is a prolonged three cell stage due to a delay in the division of P1. In at least one of these mutants (or182, see abstract by Kathryn Swan), the delay of P1 is predominantly observed in this lineage. Preliminary observations of the terminal phenotype of these mutants suggest that these embryos frequently lack pharynx and intestine and instead make extra hypodermis. The two genes identified by alleles or148, and or209 map to linkage group III. The position of or148 is approximately at 2.0mu, just to the left of unc-50, and mapping of or209 is underway. We are now microinjecting cosmids to obtain rescue of the mutant phenotype of or148. Cloning of these genes will give a more complete pictuire of their roles in cell division timing.

24 May 1999 15:50 345 345 UNC-13 and Synaptic Neurotransmission

1999 International Worm Meeting abstract 294 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. UNC-13 and Synaptic Neurotransmission RJ Eustance, J Duerr, I Maruyama, H Maruyama, A Duke, J McManus, J Rand Oklahoma Medical Research Foundation, Oklahoma City, OK, and Scripps Institute, La Jolla, CA unc-13 is essential for normal neuronal function and is involved in neurotransmitter release; C. elegans mutants are uncoordinated and resistant to the anti-cholinesterase aldicarb. UNC-13 has several regions homologous to PKC regulatory domains conferring it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88 , 1991). These binding properties indicate that UNC-13 may regulate neurotransmitter release in response to calcium and as part of a signal transduction pathway. A 5.9kb transcript coding for a 200kDa protein was initially identified (Maruyama and Brenner, PNAS 88, 1991). We identified two additional types of transcripts using cDNA library screens and RACE. One transcript includes a 1kb alternatively spliced exon and another transcript lacks the 5’ region included in the other two transcripts. All three transcripts are identical at the 3’ end. 21 of the 40 unc-13 alleles in our collection are sequenced. Mutations in the 5’ end alter two types of transcripts resulting in an uncoordinated coily phenotype. A 2.8kb deletion at the 3’ end removes all unc-13 transcripts, resulting in a lethal phenotype. This 2.8kb deletion was identified by Bob Barstead using PCR analysis. Antibodies recognizing different regions of UNC-13 indicate that protein products from the three transcripts have different expression and localization patterns. Antibodies recognizing the N-terminal region label synapses, but not synaptic vesicles, of most or all neurons. Many unc-13 alleles have decreased staining with this antibody. Antibodies against the region coded for by the alternatively spliced exon stain some sensory neurons and some neuronal processes. Antibodies recognizing the C-terminal region, common to all predicted UNC-13 proteins, label synapses and neuronal processes of most or all neurons. This staining pattern decreases in only 3 alleles, all of which are lethal or partially lethal. The existence of several unc-13 transcripts, the alternate antibody staining patterns, and the phenotypes resulting from elimination of some or all transcripts suggest that unc-13 protein products have different expression and localization patterns and possibly different functions.

24 May 1999 15:50 346 346 The sequence of the SL2 RNA spliced leader is not required for transcription or trans-splicing

1999 International Worm Meeting abstract 295 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The sequence of the SL2 RNA spliced leader is not required for transcription or trans-splicing D Evans 1,2 , T Blumenthal 2

1 Department of Biology, Indiana University, Bloomington, IN 47405. 2 Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO 80262.

C. elegans utilizes a specialized spliced leader RNA, SL2 RNA, to process the products of downstream genes in operons. By comparative phylogenetic analysis of the 22 known SL2 RNA genes, we have identified conserved sequences: the SL 5’ end, the trans-splice site, the Sm-binding site, and the top of the third stem-loop. To study the importance of these regions on expression and trans-splicing specificity, we expressed SL2 RNA variants in transgenic worms and measured trans-splicing to gpd-3 mRNA. Unlike SL1 RNA, SL2 RNA has no promoter element located within the SL sequence. Rather, expression is dependent on a proximal sequence element, similar to those of the snRNA genes. It had been hypothesized that base-pairing within the SL2 RNA, between the SL and the trans-splice site and bases just downstream, mimics that found between the 5’ splice site and U1 snRNA, which plays no role in trans-splicing. To test this idea in SL2 trans-splicing, we mutated the 20 nt at the 5’ end of the 22 nt SL. Since the predicted secondary structure is conserved among the natural variants of SL2 RNA, we expected that this large mutation would prevent trans-splicing due to an RNA structural change. Therefore, we also made mutations with compensatory changes that would allow formation of this first stem. Interestingly, the first mutation, which mutates nearly the entire SL, was nevertheless trans-spliced. Even more striking was the trans-splicing of mutant SL2 RNAs with changes in most of the first stem and loop, demonstrating that large mutations to the ’intron’ sequences of SL2 RNA are also tolerated. In contrast, mutations to the conserved top of the third stem-loop do prevent trans-splicing, even though the mutant SL2 RNA is expressed at a high level, demonstrating the importance of this conserved sequence to SL2 RNA function. The top of the third stem-loop of SL1 RNA is not conserved, suggesting that SL2 RNA may have evolved a key function at this location.

24 May 1999 15:50 347 347 A biochemical search for factors that localize glp-1 translation in early development.

1999 International Worm Meeting abstract 296 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A biochemical search for factors that localize glp-1 translation in early development. TC Evans, S Bellamy U. of Colo. Health Sci. Cntr. In the early C. elegans embryo, asymmetric cell division leads to the localized expression of cell fate regulators. Because these regulators are mostly maternal factors that are transcribed in germ cells, they must be regulated post-transcriptionally in oocytes and embryos. The membrane receptor, GLP-1, is localized to anterior cells of the early embryo where it regulates anterior fates. GLP-1 localization is achieved by translational control of glp-1 mRNA through its 3’ untranslated region (3’ UTR). What are the trans-acting factors that directly control translation of glp-1 mRNA? As one approach to this problem, we are attempting to identify and isolate these factors biochemically. Analysis of reporter mRNAs in oocytes and embryos suggests that the glp-1 3’ UTR contains several cis-acting elements. An oocyte control region contains two or more partially redundant elements that repress translation in oocytes. An embryonic control region contains two apparent regulatory elements; one that represses translation in posterior cells of embryos, and one that promotes translation (or mRNA stability) in early embryos. GLP-1 localization could be achieved either by localized repression or activation of mRNA translation. Using UV cross-linking techniques, we have detected several RNA binding proteins from crude extracts that specifically associate with an RNA containing the embryonic control region of the glp-1 3’UTR. A 50 kd protein is detected in adult extracts; this protein shows reduced binding to an RNA with a mutation that abolishes posterior repression in the embryo, but is not affected by mutations that perturb translational activation. Two polypeptides of 75-80 kd are detected in extracts from early embryos; these polypeptides fail to bind RNAs containing either a mutation that abolishes posterior repression, or a mutation that prohibits translational activation (or mRNA stability). Further mutational analysis is underway to more precisely correlate these binding activities with translational regulation in vivo. Our plan is to purify these factors and identify their genes using mass spectrometry.

24 May 1999 15:50 348 348 We do it, bees do it, even educated fleas do it, (plants as well), but how do worms fight infections?

1999 International Worm Meeting abstract 297 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. We do it, bees do it, even educated fleas do it, (plants as well), but how do worms fight infections? JJ Ewbank 1 , D Ferrandon 2 , P Bulet 2 , JA Hoffmann 2

1 Centre d’Immunologie de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France. 2 I.B.M.C., 15 rue Rene Descartes, 67084 Strasbourg Cedex, France.

Over the last few years, insights into the antimicrobial defenses of the fruit fly Drosophila melanogaster have revealed remarkable parallels between insect and mammalian innate immunity (1). Relatively little is known about the immune responses of C. elegans. In Drosophila, an infection provokes the rapid sysnthesis of powerful antimicrobial peptides and opsonising proteins (2). While seven distinct anti-microbial peptides have been identified in flies, thus far, only two have been characterised from C. elegans, encoded by abf-1 and abf-2 on C50F2 (3; see also 4). Searching for conserved peptide motifs has yielded two further family members, abf-3 and abf-4, (on F54B8 and Y38H6C, respectively) the study of which we are undertaking. Further, we are using MALDI-TOF mass spectrometry to investigate whether other antimicrobial peptides are induced upon infection of C. elegans. In Drosophila, the expression of anti-fungal peptides is controlled by the Toll pathway (5). A search of the genome reveals that C. elegans possesses structural homologues of most of the components of this signalling cascade (for Toll, pelle, tube, cactus, on cosmids C07F11, K09B11, F45G2 and C04F12, respectively). As a first step, we are using Northern analysis to determine whether this putative pathway is implicated in worm defense mechanisms. The results of these investigations will be presented.

1. Medzhitov and Janeway, 1998, Curr Opin Immunol, 10, 12-5 2. Hoffmann and Reichhart, 1997, Trends in Cell Biology, 7, 309-316 3. Kato, 1997, International Worm Meeting abstract 296 4. Kato and Komatsu, 1996, J. Biol. Chem., 271, 30493-30498 5. Lemaitre et al., 1996, Cell, 86, 973-983

24 May 1999 15:50 349 349 Analysis of polyglutamine-mediated cellular dysfunction in C. elegans.

1999 International Worm Meeting abstract 298 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of polyglutamine-mediated cellular dysfunction in C. elegans. PW Faber 1,2 , JR Alter 1,2 , JM Spoerke 3 , B Westlund 3 , ME MacDonald 4 , AC Hart 1,2

1 Massachusetts General Hospital, Cancer Center, Boston MA 02129. 2 Harvard Medical School, Department of Pathology; Boston MA 02129. 3 Axys Pharmaceuticals, South San Francisco CA 94080. 4 Massachusetts General Hospital, Neurogenetics Unit; Boston MA 02129.

Huntington’s Disease (HD) is one of 8 dominant human neurodegenerative disorders caused by expansion of an unstable CAG-trinucleotide repeat encoding polyglutamine (polyQ). How expanded polyQ stretches cause disease is unknown. We generated a C. elegans model for polyQ-mediated cellular dysfunction by expressing fragments of huntingtin, the protein product of the HD locus, containing polyQ stretches of 2, 23 (control) or 95 and 150 (toxic) residues in the ASH sensory neuron. Expression of Htn-Q150, but not Htn-Q2, Htn-Q23 or Htn-Q95 causes an ASH dye-filling defect, but no ASH cell death, in 8-day-old animals. Despite the absence of cell death this dye-filling defect is dependent on ced-3 function, suggesting that Htn-Q150 activates the apoptotic cell death pathway. Htn-Q150 coexpression with subthreshold levels of a second toxic (but non-apoptotic!) transgene, an OSM-10::GFP fusion protein, causes enhanced dye-filling defects and cell death in 3- and 8-day-old animals. This cell death depends on ced-3 function, supporting the notion that Htn-Q150 activates the apoptotic cell death pathway. Htn-Q150 and OSM-10::GFP coexpression (but neither alone) leads to a strong defect in nose touch response (Faber et al., 1999 PNAS 96, 179-184). Our current efforts include 1) Targeting Htn-Q150 expression to other cell types to create phenotypes more amenable to high throughput screens. 2) Screening (10,000 animals to date) for recessive enhancers of the Htn-Q150 mediated ASH dye-filling defect in 3d animals (unenhanced <1% defective). So far we have identified one enhancer mutation (>rt13) and one integrated array (rtIs12) that maintain their enhancing potential in 4x backcrossed (4x) animals. Another putative enhancer has just been isolated. rt13 enhancement is array dependent; 79% (n = 160) and 1% (n = 150) of rt13 (4x) ASH neurons are dye-filling defective at 3d in the presence or absence of an Htn-Q150 array, respectively. Importantly, two new Htn-Q150 arrays generated in rt13 (4x) animals by microinjection reproduce the ASH dye-filling defect of 73% (n = 162). Further analysis of rt13 is underway.

24 May 1999 15:50 350 350 Molecular Characterization of mes-4

1999 International Worm Meeting abstract 299 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Molecular Characterization of mes-4 Y Fang, S Strome Department of Biology, Indiana University We are studying maternal-effect sterile (mes) mutants in an effort to understand maternal control of C. elegans germline development. Four genes, mes-2, mes-3, mes-4, and mes-6, all give a similar mutant phenotype: progeny of mes/mes mothers undergo apparently normal embryogenesis and develop into healthy appearing adults but fail to produce a functional germline. MES-2 and MES-6 are the C.elegans homologs of Enhancer of zeste and Extra sex combs, both members of the Polycomb group in Drosophila, and MES-3 is a novel protein. mes-4 was mapped to a gap in cosmid coverage. Transformation rescue of mes-4 mutants was accomplished by injecting DNA prepared from YAC Y2H9. A cDNA clone in Y. Kohara’s library, yk35f6, encodes one of the genes in this region. RNAi disruption of the function of yk35f6 resulted in production of sterile progeny. The proof that yk35f6 encodes part of mes-4 came from sequencing the seven mutant alleles of mes-4. Four alleles have single missense mutations, and three alleles have small deletions. Northern blot analysis showed that mes-4 RNA is essentially restricted to the germline. The predicted MES-4 protein contains three zinc fingers (one RING finger and two PHD fingers), a 150 aa motif known as the SET domain, and a nuclear localization signal. RING fingers and PHD fingers are found in many proteins with diverse functions and are thought to mediate protein-protein interactions. The SET domain is common to chromatin-binding proteins. A SET domain is also found in MES-2. MES-4 shows the highest degree of overall similarity to NSD1 and ASH1. Murine NSD1 has five PHD-type zinc fingers, a SET domain, and two distinct nuclear receptor interaction domains not found in MES-4. The function of NSD1 is not known. ASH1 is a member of the trithorax group in Drosophila. It contains a PHD-type zinc finger and a SET domain. Trithorax group proteins are thought to be responsible for long-term maintenance of transcriptional activation of specific genes; they also participate in transcriptional repression of some genes as well. Current thinking is that Polycomb and Trithorax group proteins form multimeric protein complexes and control chromatin structure and gene expression at specific sites on chromosomes. We hypothesize that MES-4 forms complexes with other MES proteins and modifies chromatin structure in the germline of C.elegans.

24 May 1999 15:50 351 351 Biochemical and Genetic Studies of the ETS-Domain Protein, LIN-1

1999 International Worm Meeting abstract 300 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Biochemical and Genetic Studies of the ETS-Domain Protein, LIN-1 DA Fantz, K Kornfeld Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110 The conserved Ras signaling pathway regulates multiple cell fates during C. elegans development. Genetic analyses suggest that a major function of this pathway during vulval development is to negatively regulate lin-1. lin-1 encodes a 441 amino acid protein with a C-terminal ETS domain and N-terminal docking sites and phosphorylation sites for ERK MAP kinase. ETS-domain proteins typically function as transcription factors and are conserved mediators of Ras signaling. We are using biochemical and genetic approaches to determine how LIN-1 is regulated by Ras signaling and how LIN-1 controls cell fates. Electrophoretic mobility shift assays (EMSA) were used to measure the DNA-binding activity of the ETS domain of LIN-1. The purified LIN-1 ETS domain can bind a consensus ETS-binding site, and competition experiments with mutant oligonucleotides indicated that the binding is sequence specific. To determine the transactivation potential of the LIN-1 C-terminus, we generated a fragment of LIN-1 fused to a heterologous DNA-binding domain from the yeast GAL4 protein and assayed the transactivation potential of the fusion protein in vertebrate cultured cells. The C-terminus of LIN-1 functioned as a transcriptional activator and that activity was increased by growth factor induction of the Ras pathway. Mutations that eliminate MAP kinase docking sites in the C-terminus of LIN-1 reduced the transactivation potential of LIN-1 in response to Ras signaling. This result is surprising, since genetic data suggest that the Ras pathway negatively regulates lin-1 activity. Taken together, these results indicate that LIN-1 is a DNA-binding transcription factor. To identify genes that interact with lin-1, we screened for suppressors of the Muv phenotype caused by lin-1 (n383), a non-null but nonetheless strong loss-of-function allele. After screening about 20,000 haploid genomes, we identified 18 suppressor mutations. One mutation causes partially penetrant recessive lethality; mutants display a rigid, rod-like morphology. The same phenotype is caused by mutations that completely inactivate the Ras pathway, indicating that the gene identified by this mutation plays a crucial role in Ras signaling during vulval formation and at an earlier developmental stage.

24 May 1999 15:50 352 352 Genetic and Molecular Analysis of sel-5

1999 International Worm Meeting abstract 301 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic and Molecular Analysis of sel-5 H Fares, I Greewnald Department of Biochemistry and Molecular Biphysics, Columbia University. Howard Hughes Medical Institute. Two alleles of sel-5 were initially identified as suppressors of the egg-laying defect of the gain of function lin-12(n302) mutation (Tax et al., 1997). In the same study sel-5 was also shown to function, by mosaic analysis, in the presumptive VU cell (same as lin-12). We have extended that analysis with several lin-12 mutants and show that the suppression by sel-5 is dependent on the severity of the lin-12 mutation. We also show that sel-5 is unable to suppress the activated lin-12(intra) allele, suggesting that sel-5 functions upstream of the activating cleavage event. Consistent with this, sel-5 does not seem to affect the transcriptional feedback regulation in Z1.ppp and Z4.aaa that establishes one as a signaling cell and the other as receiving. We also do not see an effect of sel-5 on lin-12 in other tissues where the latter is known to function. We have cloned sel-5 and shown that it encodes two forms of a protein that share an amino-terminus with homology to ser/thr kinase catalytic domains. Initial subcellular localization experiments suggest that the proteins are cytoplasmic. Furthermore, both forms are able to rescue sel-5 mutants. During the genetic analysis of sel-5, we discovered that while mutants in this gene do not interact with lin-12 in the vulval precursor cells (VPCs), these mutants did partially suppress the vulvaless phenotype of some primary pathway mutants. So far, we have only seen suppression of let-23(sy1), lin-2(e1309) and lin-7(e1413), but not of let-23(n1045), sem-5(n2019), or lin-10(n1390). We will present data on the localization of LET-23 in these mutants and will speculate on what this all means.

24 May 1999 15:50 353 353 Identification and Analysis of Vulval Morphogenetic Mutants

1999 International Worm Meeting abstract 302 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification and Analysis of Vulval Morphogenetic Mutants DS Fay, M Han The University of Colorado at Boulder, Dept MCDB and HHMI, Boulder, CO 80309-0347 While the inductive signaling events leading to the adoption of vulval cell fates have been well characterized, the subsequent steps during which committed vulval precursor cells execute the correct division patterns and undergo morphogenesis are not well understood. We have previously reported the use of temperature-sensitive Egl screens to identify mutants defective in vulval development. During the course of such screens we observed a high frequency of sterile animals with protruding vulva (Pvl), which have been previously reported to display a variety vulval defects1 . In order to identify genes required for both vulval development and fertility, we have undertaken a large-scale screen to isolate Pvl sterile mutations. Of the eight alleles reported here, two alleles (ku276, ku279) appear to be primarily defective in the morphogenetic steps following cell divisions. In contrast, six other alleles (ku277, ku278 ku256, ku280, ku281, and ku282) display varying defects in the execution of the proper vulval lineage pattern. In addition, many mutants possess abnormal gonads with defects observed in germline cells, the somatic gonad, and gonad arm migration. Mapping data indicate the above eight lesions to represent distinct loci. We have obtained single cosmid rescue for ku256 which maps just left of dpy-14 on LGI, and have narrowed down the rescuing fragment to a region likely to contain a single open reading frame. ku256 animals appear principally defective in executing the final round of vulval cell divisions. Mid-L4 animals contain 9-14 vulval cells (avg.=12) that have invaginated and reside in asymmetric positions. In addition about 15% of ku256 animals display a small posterior invagination containing 2-3 cells. Germ cells are also aberrant in ku256 mutants appearing large and irregular. Complementation tests indicate that ku256 is allelic to the previously identified Pvl sterile mutation evl-10 (ar95) 1 . Experiments are underway to further characterize the ku256 phenotype and to define the molecular lesion. In addition, rescue experiments are currently being carried out for several of the alleles described above.

1. Seydoux et al., 1993. Dev. Biol. 157, 423-436.

24 May 1999 15:50 354 354 vav Function in C. elegans

1999 International Worm Meeting abstract 303 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. vav Function in C. elegans RT Fazzio, MC Beckerle, AV Maricq Department of Biology, University of Utah, Salt Lake City, UT 84112 The proto-oncoprotein Vav is a guanine nucleotide exchange factor (GEF) for members of the Rho/Rac family of monomeric GTPases. Vav homologs have been identified in humans, mice, rat and C. elegans. Extensive studies in mammalian tissue culture cells and chimeric mice have implicated Vav in signal transduction pathways that lead to the reorganization of the actin cytoskeleton. Vav comprises several functional domains that are highly conserved in known signaling molecules. At the amino acid level, the C. elegans homolog, VAV-1, shares 33% identity with the human VAV protein and displays each of the domains thought to be important for signaling. VAV-1 contains a calponin-homology (CH) domain and an acidic domain (AD). Selective disruption of these regions of the mammalian proteins results in oncogenic transformation. VAV-1 also contains a Dbl homology (DH) domain, two SH3 domains and one SH2 domain. In humans, the DH domain is responsible for the GEF activation of Rho, Rac-1 and Cdc-42, and the Src-homology regions are known to interact with identified signaling molecules. To determine the function of VAV-1, in vivo, we used a Tc1 transposon-based mutagenesis approach to generate a genomic disruption of the vav-1 locus. This resulted in the deletion of the carboxy-terminal 80% of VAV-1, including the DH, SH3 and SH2 domains. The putative null was outcrossed multiple times to remove the Tc1 background, and the region was sequenced to confirm the presence of a genomic deletion. Phenotypic characterization of this mutant indicated that the disruption of vav-1 results in larval lethality. Experiments designed to rescue the vav-1 phenotype and better define the developmental basis for lethality are currently in progress. Future experiments will focus on the identification of proteins that interact with VAV-1. To complement the functional analysis, VAV-1 expression was studied using several constructs that fused regions of the vav-1 gene to the reporter gene encoding Green Fluorescent Protein (GFP). Transgenic lines expressing these constructs indicated the presence of VAV-1 in several neurons, body wall muscle, gonad, and vulval cells. We are currently generating monoclonal antibodies to this protein to further characterize vav-1 expression.

24 May 1999 15:50 355 355 Genetic and Phenotypic Analysis of spe-32, a Gene Required for Spermatogenesis with an Associated Larval Lethal Phenotype

1999 International Worm Meeting abstract 304 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic and Phenotypic Analysis of spe-32, a Gene Required for Spermatogenesis with an Associated Larval Lethal Phenotype Frederick Fell, Daniel Halperin, Diane Downing, Samuel Ward Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA The gene spe-32 is required for Caenorhabditis elegans spermatogenesis. When a temperature sensitive mutation of spe-32 is grown at restrictive temperature the mutants produce defective spermatozoa that fail to fertilize oocytes. The mutation affects both hermaphrodites and males and is partially reversible if the worms are shifted from the restrictive to the permissive temperature. Temperature-shift experiments have shown that the temperature sensitive period is during the late L4 stage, corresponding to the time of spermatogenesis. Worms also display a temperature sensitive lethality which affects the offspring of homozygous spe-32 mutants when their eggs are shifted to the restrictive temperature. The worms hatch and die as L1 larvae. The lethality seems to be maternally inherited, but experiments are continuing to see if it also has a zygotic effect. spe-32 has been mapped between elt-1 and dpy-20 and fails to complement both eDf19 and mDf7, which locates it between 4.45 and 4.59cM on LGIV. It is currently being mapped more precisely prior to cloning. While mapping we were unable to separate the two phenotypes. We are continuing efforts to determine whether the lethal and spermatogenesis-defective phenotypes result from pleiotropic effects of one gene or from mutations in two tightly-linked genes.

24 May 1999 15:50 356 356 cul-2 and the vhl Tumor Suppressor Gene: A genetic study

1999 International Worm Meeting abstract 305 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. cul-2 and the vhl Tumor Suppressor Gene: A genetic study H Feng 1 , G Moulder 2 , R Barstead 2 , ET Kipreos 1

1 Department of Cellular Biology, University of Geogia, Athens, GA 30602. 2 Oklahoma Medical Research Foundation, Oklahoma City, OK 73104.

Inactivation of the Von Hippel-Lindau tumor suppressor gene (VHL) causes cancers of the kidney, brain and retina in humans. The VHL protein associates with Elongin B and Elongin C in a VBC complex. VHL was initially thought to inhibit transcription by sequestering Elongin B and C from the Elongin (SIII) complex, composed of Elongins A, B and C. However, the concentration of Elongin B and C is reported to be 100 to 1000-fold higher than that of VHL in cells (Conaway et al., 1998, BBA 1377: M49) making it unlikely that VHL can sequester them. Currently, how VHL functions to suppress tumor formation is unknown. Recently, VHL was found to bind to human CUL-2 (Pause et al., 1997, PNAS 94: 2155). cul-2 is a member of the cullin gene family. Cullin homologs Cdc53 and CUL-1 are involved in the degradation of Cyclins and CKIs to regulate cell cycle progression. In cul-2 (ek1) mutants, germ cells are arrested in G1 phase, and this is correlated with a higher level of CKI-1, a member of the CIP/KIP family of cyclin-dependent kinase inhibitors. Our results suggest that cul-2 is required for the G1 to S phase transition, potentially functioning by degrading CKI-1. Interestingly, overexpression of wild type VHL in human cells also induces a G1 phase cell cycle arrest and increases the level of the CKI p27KIP1 (Kim et al., 1998, BBRC 253: 672). Based on these observations, we propose a model in which VHL negatively regulates CUL-2, which negatively regulates CKIs, which negatively regulates the cell cycle. Since a VHL homolog is present in C. elegans we are undertaking a genetic study of cul-2 and vhl . Both the vhl(ok161) allele and the cul-2(ek1) allele were obtained by PCR screening for deletions in EMS mutagenized animals, by G.M./R.B. and E.T.K., respectively. We are trying to gain insights into the (potential) genetic interaction of vhl and cul-2 by making a strain homozygous for vhl(ok161) and heterozygous for cul-2(ek1) and analyzing the resulting homozygous progeny. We are in the final stage of making this strain and we will present the double mutant phenotype at the meeting.

24 May 1999 15:50 357 357 Functional Analysis of peb-1 during Pharyngeal Development

1999 International Worm Meeting abstract 306 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Functional Analysis of peb-1 during Pharyngeal Development AP Fernandez, PG Okkema Dept. of Biological Sciences, University of Illinois at Chicago, Chicago, IL The C. elegans pharynx is a neuromuscular organ consisting of five distinct cell types. We have been characterizing how gene expression is regulated in one of these cell types, the pharyngeal muscles, by examining the pharyngeal muscle-specific myosin gene, myo-2. myo-2 expression is regulated by a combination of cell type- and organ-specific signals. The organ-specific signals target a site in the myo-2 enhancer, termed the C subelement. We and others have shown that the C subelement contains binding sites for multiple factors, including the Forkhead family transcription factor PHA-4 and the Smad protein DAF-3. We have identified another DNA-binding protein, PEB-1, that binds the C subelement and is strongly expressed in the pharynx (see abstract for this meeting by Thatcher, etal.). PEB-1 is a novel factor that does not share apparent similarity with any known DNA-binding protein. To learn more about PEB-1 function during pharyngeal development, we are following three experimental approaches. First, we are producing antibodies against PEB-1 to determine its tissue distribution and sub-cellular localization. Two PEB-1 fusion proteins have been used to immunize mice and rabbits. Polyclonal ascites fluid has been isolated from the mice and antibodies recognizing both PEB-1 fusion proteins are detectable on Western blots. Second, we are using a PCR-based screen to identify a peb-1 deletion mutant (following the protocols of G. Moulder and R. Barstead). peb-1 maps on the left arm of the X chromosome in a region containing no pharyngeal defective mutants, and RNAi has not produced an informative phenotype. We hope to use a deletion mutant to understand peb-1 function in the pharynx. Third, we are examining the effect of expressing peb-1 ectopically in C. elegans. Strains expressing the peb-1 cDNA under control of the hsp 16-2, hsp 16-41, unc-54, or mec-7 promoters have been generated. Thus far we have not seen ectopic expression of pharyngeal-specific markers in these strains. However, the hsp::peb-1 strains exhibit a heat-inducible lethal phenotype, and the unc-54::peb-1 strain displays an adult onset Unc phenotype. These phenotypes suggest we are expressing ectopic peb-1 in these strains, and we will verify this using antibodies against PEB-1.

24 May 1999 15:50 358 358 Structural requirements for the trigger in RNA-mediated interference

1999 International Worm Meeting abstract 307 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Structural requirements for the trigger in RNA-mediated interference A Fire 1 , J Fleenor 1,2 , S Parrish 1,3

1 Carnegie Institution of Washington, Department of Embryology. 115 West University Parkway, Baltimore, Md. 21210. 2 present address for SX: Department of Genetics, Washington University. 3 Biology Graduate Program, Johns Hopkins University, Baltimore Md. 21218.

This poster will describe in vivo interference activities for a series of modified double-stranded RNAs that are based on segments of the genes unc-22 and gfp. Double stranded RNAs have been shown to act as potent inducers of gene-specific molecular silencing in C. elegans [a]. This process apparently reflects a well conserved control mechanism: recent reports have confirmed the effectiveness of dsRNA-triggered interference mechanisms in a variety of additional species including plant, insect, and protozoan systems [b-e]. By characterizing structural requirements (on the triggering side) for these two well defined segments in C. elegans , we hope to illuminate general features of the interference mechanism. Our experiments involve a variety of manipulations to produce dsRNAs with sequence or chemical alterations, followed by injection into C. elegans and assays for genetic interference. The manipulations are designed to address the following questions: 1. How precise and extensive are requirements for homology with the target gene? 2. What features distinguish the incoming RNA as "foreign"? 3. What chemical groups on the incoming RNA participate in interference? 4. Do the incoming sense and antisense strands have distinct roles in triggering interference?

a. Fire, Xu, Montgomery, Kostas, Driver, Mello. Nature 391, 806 b. Waterhouse, Graham, Wang, PNAS 95, 13959 c. Ngô, Tschudi, Gull, Ullu, PNAS 95, 14687 d. Kennerdell and Carthew, Cell 95, 1017 e. Misquitta and Paterson, PNAS 96, 1451

24 May 1999 15:50 359 359 The Role of C. elegans lin-5 in Mitosis

1999 International Worm Meeting abstract 308 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Role of C. elegans lin-5 in Mitosis R Fisk, M Walhout, M Vidal, S van den Heuvel MGH Cancer Center, Charlestown, MA 02129 Faithful segregation of sister chromatids in mitosis is critical for cell survival. To better understand chromosome segregation, our lab has focused on the lin-5 gene. In lin-5 mutants, chromosomes fail to align properly at the metaphase plate, and cells do not undergo anaphase or cytokinesis. Nevertheless, cells exit mitosis and continue rounds of DNA replication and abortive mitoses, resulting in polyploid cells. We have shown that the lin-5 gene encodes a novel coiled-coil component of the spindle apparatus. To further elucidate the role of lin-5, we have initiated genetic and two-hybrid screens. We screened for dominant suppressors of the lin-5(ev571ts) allele. The ev571ts mutants develop normally at 15°C but are sterile at 25° due to failure in cell division. We screened for suppressors of the sterile phenotype at 25°C. We isolated 68 suppressor mutations (about 1/20,000 haploid genomes) which were all found to be linked to lin-5. We are currently using these candidate intragenic suppressors for structure function analysis of LIN-5. We are also initiating screens for suppressors of the lin-5(e1457) allele. This allele contains a single missense mutation in the 5’ end of the open reading frame, yet it genetically behaves as a null. In a two-hybrid screen, LIN-5 was shown to interact with itself and several other coiled-coil proteins. As a way of validating the in vivo relevance of these interactions, we are using the ev571ts allele, whose product contains a 3 amino acid insertion in the coiled-coil domain. We have shown that the ev571ts product is expressed and localizes properly at 25°C. Therefore, loss of lin-5 function in ev571ts may reflect a failure of LIN-5 to interact with key partners. We are examining whether any of the previously identified two-hybrid interactions are lost by the ev571ts mutation. If so, we will determine whether suppressors identified in our genetic screen may restore lin-5 function by restoring interaction with a specific target. The e1457 allele will also be introduced into the two-hybrid system to test whether this mutation affects any of the previously identified interactions.

24 May 1999 15:50 360 360 Evolution of Rhabditidae, a family for C. elegans

1999 International Worm Meeting abstract 309 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Evolution of Rhabditidae, a family for C. elegans D Fitch 1 , N Eakin 1 , K Kiontke 2 , C Nguyen 1 , J Vanfleteren 3 , A Vierstraete 3 , P De Ley 3 , R De Wachter 4 , M Sutherlin 5 , W Sudhaus 2

1 New York University, New York, NY, USA. 2 Freie Universitaet Berlin, Germany. 3 University of Gent, Belgium. 4 University of Antwerp, Belgium. 5 University of Nebraska, Lincoln, NE, USA.

C. elegans provides a model for discovering how genes produce form. Applying this model to discern how changes in these genes have produced different forms first requires knowledge about the evolution of species closely related to C. elegans. We have traced the evolution of some important features such as vulval and male tail pattern and morphogenesis, characteristics of the stoma and gonads, hermaphroditism, and copulatory behavior. To trace these evolutionary changes, we have inferred phylogenetic relationships among the major species groups in family Rhabditidae, of which C. elegans is a member. Our data show that the vulva was originally located midbody and posteriad VPC migrations occurred independently at least twice, correlating with changes to one-armed gonads. In the male tail, some rays have independently changed positions, suggesting a degree of mosaicism, whereas some seam cells share fate changes, consistent with coordinate regulation. Male tail tip morphogenesis has undergone several changes between "leptoderan" and "peloderan" in different ways (e.g., peloderan tails have been formed by tail hypodermis retraction with or without cell fusion, revealing separable mechanisms). Both losses and gains of coiling to stabilize copulation have occurred, correlating with some aspects of male tail form. The glottoid part of the stoma has been reduced or lost more than once, explaining a difference from previous phylogenetic hypotheses which assumed absence is primitive. Hermaphrodites derived from females in independent lineages, but no unambiguous case exists for the reverse. Relationships were inferred from nearly complete 18S ribosomal RNA genes aligned by modeling secondary structures. Although our data show that some intraspecific polymorphism exists, most relationships are well resolved. Increasing taxon representation (50 Rhabditidae) substantially improves the resolution.

Other contributors: C. Pomilla and J. Chiu (New York University) and A. Duhachek (University of Nebraska).

24 May 1999 15:50 361 361 Evidence for cospeciation between entomopathogenic nematode Heterorhabditis and its bacterial symbiont, Photorhabdus

1999 International Worm Meeting abstract 310 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Evidence for cospeciation between entomopathogenic nematode Heterorhabditis and its bacterial symbiont, Photorhabdus A Fodor 1 , E Szállás 1 , BJ Adams 2

1 Department of Genetics, Eötvös University, Budapest, Hungary. 2 Department of Nematology, University of California, Davis, CA 95616-8668.

Entomopathogenic nematode (EPN) Steinernema and Heterorhabditis genera are "located" to left and right to C. elegans on the phylogenetic tree of Phylum Nematoda based on comparative sequence analysis of the 18S rRNA gene (Blaxter et al.,1998). Both genera forms obligate, mutualistic extracellular endosymbiosis with their respective bacteria Xenorhabdus and Photorhabdus. The cospeciation between Steinernema and Xenorhabdus is evident. Due to comparative phylogenetic analysis of ITS1 DNA sequences, 6 phylogenetic species are identified. Due to comparative sequence analysis of 16S rRNA gene of 90 Photorhabdus strains 10 subtaxa of 1 heterogeneous species, P.luminescens (P.l) is identified. Although host-symbiont systems are predicted to show extensive parallel cladogenesis, studies of phylogenetic relationships among P. l. symbionts associated with different species of nematodes have shown little, if any congruence with recovered evolutionary history. Using the P.l. 16S and Heterorhabditis ITS1 ribosomal gene sequences in Gen-Bank, as well as some new sequence data representing two Hetero’ species, phylogenies are reconstructed for symbiotic bacteria and their nematode hosts. Clinical P.l. strains were probably evolving from a shared common ancestor with the H.megidis symbiont. Unrooted trees of the symbiont were identical in topology to previous results revealing evidence of coevolution among some nematodes and their symbionts including subspecific relationships among different populations of nematodes and their hosts.But by re-rooting these same trees, even more compelling reconstructions of coevolution can be recovered. Molecular clock is rejected, but a comparison of the two phylogenies suggests a correlation in the timing of cladogenic events between the two mutualistic organisms. This comparison reveals synchronous/preemptive cladogenesis of the endosymbiont relative to the timing of differentiation of its host. Ongoing research shows a high degree of host specificity among endosymbionts precluding host-switching as a common phenomenon.

Adams et al., J. Nematol. 1-39 (1998) Blaxter et. al., Nature 392, 71-75, Szállá s et al., IJSB 47, 402-407.

24 May 1999 15:50 362 362 A screen for mutants with defects in asymmetric cell division

1999 International Worm Meeting abstract 311 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A screen for mutants with defects in asymmetric cell division C. Andrew Frank, Nancy Hawkins, Gian Garriga Dept. of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3204, USA. The C. elegans protein HAM-1 participates in the asymmetric divisions of at least five neuroblasts. In particular, we are studying its role in the asymmetric division of the HSN/PHB neuroblast, which divides to produce a daughter cell fated to die and the HSN/PHB precursor cell. HAM-1 is asymmetrically distributed in this neuroblast and inherited by the HSN/PHB precursor. Loss-of-function ham-1 mutants can cause the daughter cell that normally does not inherit HAM-1 to assume an HSN/PHB-like fate instead of a cell death fate. This can result in duplications of the HSN and PHB neurons. Since the cell that does not inherit HAM-1 is the cell whose fate is altered by mutations, our current model is that HAM-1 acts as a tether for cell fate determinants rather than as a cell fate determinant itself. We have conducted genetic screens to identify genes that participate in the same process as HAM-1. To perform these screens, we have utilized the gmIs12 strain, which carries an integrated srb-6-gfp fusion, causing GFP to be expressed in several cells, including the PHA and PHB phasmid neurons. We mutagenized this strain and simply looked for abnormal phasmid neuron phenotypes in the F2 generation. From a non-clonal screen of approximately 18,000 genomes, we identified three mutants with the ham-1 phenotype of extra phasmid neurons: ham-1(gm214), egl-5(gm224), and lin-32(gm239). As these mutations were in genes already implicated in HSN/PHB development, we elected to screen further. We next attempted an F2 clonal screen. From a screen of approximately 2600 genomes, we have identified four mutants with extra phasmid neurons, and these mutants still require further characterization. The mutants that produce extra phasmid neurons are most relevant to our study of HAM-1, but in addition, we have identified other mutants as well. A few of our mutants occasionally produce fewer than normal phasmid neurons. One can hypothesize that mutations resulting in fewer phasmid neurons affect genes that may be involved in determining the HSN/PHB precursor fate. We have also found some mutants with abnormal phasmid neuron axon pathfinding.

24 May 1999 15:50 363 363 Evaluation of Excitation-Contraction Coupling in C. elegans Muscle.

1999 International Worm Meeting abstract 312 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Evaluation of Excitation-Contraction Coupling in C. elegans Muscle. CJ Franks, ET Claverol, C Rogers, JE Chad, RJ Walker, L Holden-Dye University of Southampton, SO16 5PE, UK. Electrophysiology is a vital technique for the phenotypic analysis of C. elegans muscle (Bessou et al. 1998). However, excitation-contraction coupling consists of a series of phenomena, only some of which manifest themselves as measureable change in the electrical properties of the cells. For example, systems that modulate realease of calcium from intracellular compartments, or calcium sensitivity of the contractile apparatus, can profoundly affect muscle function. We are currently developing an automated system for the correlation of electrophysiological and imaging data that provides a robust measure of the latency between action potentials and the muscle contractions with which they are associated. Video images of the muscle preparation are acquired using a standard black and white CCD camera and the data are recorded to hard disk via a proprietary frame grabber. Single electrode, membrane potential recordings are made from pharyngeal muscle in the manner described by Franks et al. (1997) and data are recorded using an Axon instruments Digidata multichannel acquisition system. One channel is used to record the electrophysiological data while the second records the output from a circuit designed and built ’in-house’. This circuit is connected between the CCD and the frame grabber. It essentially splits the video signal in two. The first output from the circuit, contains the full video signal and is recorded by the frame grabber. The second output contains only the vertical sync signal, stripped from the original video feed. This timing signal is recorded on channel two of the acquisition system producing a timestamp for each frame of the video. We have also designed an image processing algorithm that makes it possible to quantify the degree of muscle contraction in each video frame. Thus we have a standardised and automated system that can correlate electrophysiological and video data with a temporal resolution limited only by that of the CCD. We are using the system to analyse disruption of E-C coupling by pharmacological agents and to assess phenotypic variation between wild-type and mutant C. elegans. We will report on our progress at the meeting.

Bessou, et al. (1998). Neurogenetics. In Press. Franks et al. (1997) J. Physiol. Lond. 504P: 15.

24 May 1999 15:50 364 364 C. elegans Inhibitor of Apoptosis Protein (IAP) homologue BIR-1 plays a conserved role in cytokinesis, not apoptosis

1999 International Worm Meeting abstract 313 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. C. elegans Inhibitor of Apoptosis Protein (IAP) homologue BIR-1 plays a conserved role in cytokinesis, not apoptosis AG Fraser 1 , CL James 2 , GI Evan 2 , MO Hengartner 1

1 Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA. 2 Biochemistry of the Cell Nucleus Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK.

Inhibitor of Apoptosis Proteins (IAPs) suppress apoptotic cell death in several model systems and are highly conserved between insects and mammals. All IAPs contain at least one copy of the ~70 amino acid Baculovirus IAP Repeat (BIR) domain; BIR domains are essential for the anti-apoptotic activity of the IAPs. However, both the marked structural diversity of IAPs and the identification of BIR-containing proteins in yeasts have led to the suggestion that BIR-containing proteins (BIRPs) might play roles in other, as yet unidentified cellular processes besides apoptosis. We have identified and characterised two C. elegans BIRPs, BIR-1 and BIR-2; these are probably the only BIRPs in C. elegans. Over-expression of BIR-1 is unable to inhibit developmentally-occurring cell death in C. elegans and inhibition of bir-1 expression by RNAi does not increase cell death. Instead, embryos lacking bir-1 are unable to complete cytokinesis and become multinucleate. This cytokinesis defect can be partially suppressed by transgenic expression of survivin, the mammalian BIRP most structurally related to BIR-1, suggesting a conserved role for BIRPs in the regulation of cytokinesis. Indeed, survivin is upregulated 40-fold at G2/M-phase and binds mitotic spindles although its role at the spindle is still unclear. We conclude that BIR-1 is probably not involved in the general regulation of apoptosis, but is required for embryonic cytokinesis. We suggest that BIRPs may regulate cytoskeletal changes in diverse biological processes including cytokinesis and apoptosis.

24 May 1999 15:50 365 365 gon-4 encodes a novel protein required for timing of somatic gonadal cell divisions

1999 International Worm Meeting abstract 314 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. gon-4 encodes a novel protein required for timing of somatic gonadal cell divisions LC Friedman 1 , S Santa Anna-Arriola 1,2 , J Hodgkin 2 , J Kimble 1

1 University of Wisconsin-Madison. 2 MRC-LMB, Cambridge, UK.

During gonadogenesis, somatic gonadal blast cells undergo an essentially invariant pattern of cell divisions, migrations and differentiation. We have isolated four recessive, loss-of-function alleles of the gon-4 locus. All gon-4 mutants are sterile with variable defects in gonad formation. In most gon-4 adults, the gonad is smaller than normal and gonadal structures are not properly organized. By cell lineage analysis, we found that the initial defect in gon-4 mutants is a dramatic delay in the timing of somatic gonadal cell divisions. For example in gon-4 mutants, Z1 or Z4 often divide in L2 instead of L1. Furthermore, the synchrony of Z1 and Z4 cell divisions is lost, resulting in a loss of symmetry in the hermaphrodite gonad. Although fewer cells are made than normal, GFP reporters for specific somatic gonadal tissues are expressed at the correct times in gon-4 mutants, suggesting that delayed divisions and an incomplete lineage do not block cell differentiation. By contrast, the germ line precursor cells divided normally in L1 and L2 (later germ line defects might reflect DTC defects), the M and B lineages were normal during L1 and L2 (they were not followed further), and the timing of larval molts was unaffected. Therefore, the cell division defects appear to be specific to the somatic gonadal lineage. The gon-4 locus maps to linkage group IV near unc-43. Using transformation rescue, RNAi, and sequence analysis, we have established that gon-4 encodes a novel protein (transcript K04D7.5). All four gon-4 alleles have nonsense mutations in the coding region. Developmental Northern blots suggest that gon-4 mRNA is expressed mainly in the germ line. A gon-4::GFP transgene rescued gon-4(q519) and expressed GFP in somatic gonadal cells and in the germ line. Preliminary results indicate that aGON-4 antibodies co-localize with a marker of mitotic cells (phosphohistone H3) in the germ line, consistent with the idea that gon-4 is required for normal progression through the cell cycle.

24 May 1999 15:50 366 366 Origin and evolution of microsatellites in C. elegans

1999 International Worm Meeting abstract 315 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Origin and evolution of microsatellites in C. elegans LM Frisse 1 , L Vassilieva 2 , M Lynch 2 , WK Thomas 1

1 School of Biological Sciences, University of Missouri - Kansas City, Kansas City, MO 64110. 2 Department of Biology, University of Oregon, Eugene, OR 97403.

Microsatellites, small direct repeats, are a ubiquitous feature of eukaryotic genomes. The high mutation rate of microsatellite loci has made them useful in genetic mapping and evolutionary studies. Microsatellites have also been recognized as the causal agents of numerous human genetic diseases and are used as indicators for tumor formation. Despite their widespread use, little is known about the origin of microsatellites, factors that affect microsatellite frequency and distribution, and the rates and patterns of microsatellite mutation. Understanding the rates and patterns of microsatellite evolution is critical for their use in studies of evolution, genetic mapping and mutational mechanisms. The recent completion of the Caenorhabditis elegans genome allows the testing of many of the hypotheses concerning microsatellite evolution. We have identified 953 microsatellites with 2-5 bp repeats, which are at least 10 perfect repeat units in length. These loci have been physically mapped allowing us to investigate the frequency and distribution of microsatellites across an entire metazoan genome. The majority of the loci identified are dimers. There is no clear pattern for microsatellite distribution within the genome. Microsatellite frequencies are not consistent with base composition or dinucleotide sequence composition; however, there is some evidence that microsatellites may be seeded by a telomeric activity. In order to understand the rates and patterns of microsatellite mutations, we assayed 29 microsatellite loci in a set of 80 mutation accumulation lines of the nematode C. elegans propagated for 140 generations. Investigation of mutation rate included dimer loci over a wide range of allele sizes. This study clearly demonstrates that the mutation rate increases with increased repeat number and that the pattern of mutation is biased towards small additions.

24 May 1999 15:50 367 367 The APC-related gene apr-1 is required for morphogenesis of the embryo and generation of the vulval equivalence group

1999 International Worm Meeting abstract 316 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The APC-related gene apr-1 is required for morphogenesis of the embryo and generation of the vulval equivalence group E Froehli, A Hajnal Div. of Cancer Research, Dept. of Pathology, University of Zurich, Schmelzbergstr. 12, CH-8091 Zurich, Switzerland apr-1encodes the worm homolog of the human Adenomatous Polyposis Colon (APC) tumor suppressor gene. The APC mutations that are commonly found in human colon cancer delete the ß-cateninbinding site of APC and result in the constitutive activation of ß-cateninin a winglesssignaling pathway. These observations have suggested that APC may act as a negative regulator of winglesssignaling. To study the role of apr-1in worm development, we have isolated an EMS-induced deletion of 1414 base pairs (apr-1(zh10)) that removes the first, second and most of the third exon of apr-1. apr-1(zh10)embryos arrest at about the comma stage and fail to undergo elongation. The ventral hypodermal cells do not reach the ventral midline, resulting in defective ventral closure. A similar phenotype has been observed in hmp-2(a ß-catenin) mutant embryos (Costa et al., 1998). In addition, the anterio-lateral hypodermis is disorganized and expression of the HOX gene ceh-13is down regulated in hypodermal and mesodermal cells of the anterior body region. During larval development, APR-1 is expressed in the six vulval precursor cells (VPCs) and in the seam cells. To study the role of apr-1in vulval development, we have used two different approaches: (A) Tissue-specific RNAi by expressing antisense apr-1cDNA under control of the Pn.p cell-specific lin-31promoter. (B) Mosaic analysis by introducing wild-type apr-1on an extrachromosomal array into an apr-1(zh10)mutant background and screening for animals that have lost the apr-1transgene in the VPCs or their P cell precursors. VPCs that lack apr-1activity loose their adherens junctions and adopt an undivided, non-vulval cell fate like Pn.p cells that are outside of the vulval equivalence group. Mutations in bar-1(another ß-catenin,Eisenmann et al., 1998) and in the HOX gene lin-39(Clark et al., 1993 and Wang et al., 1993) cause a similar phenotype. Furthermore, RNAi of apr-1results in the down-regulation of lin-39expression in the ventral cord of L1 larvae. In summary, our results suggest that wild-type apr-1may function as a positive effector of the winglesssignaling pathway to induce expression of the HOX genes ceh-13in the embryo and lin-39in the larva.

24 May 1999 15:50 368 368 An analysis of the 26F4 gene which is expressed in the nuclei of seam cells and a subset of neurons

1999 International Worm Meeting abstract 317 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. An analysis of the 26F4 gene which is expressed in the nuclei of seam cells and a subset of neurons T Fujii, G Shioi, Y Shibata, H Fujisawa, S Takagi Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan By promoter trapping using GFP as a reporter, we have isolated a clone, p26F4, which expressed GFP in a subset of neurons. The 26F4 gene, corresponding to p26F4, was physically mapped to cosmid T19C3 on LGIII. Worms transformed with p26F4 and expressing GFP intensely displayed a kinker-like Unc phenotype, suggesting that the gene may play an important role in the nervous system. We have isolated cDNA for the 26F4 gene by RT-PCR and determined the sequence. The cDNA encodes a novel protein of 92 amino acid residues, which is hydrophilic and proline-rich. A comparison of the cDNA and genomic sequences revealed that the 26F4 gene consisted of 3 exons. We have analyzed the expression pattern of the 26F4 gene in details. As p26F4 contains only a part of the 26F4 gene and 800 bps of the 5’-upstream region of the gene, we have generated a construct containing the 6.5kb-upstream region and the entire 26F4 gene with gfp fused to the C-terminal of the product. Worms transformed with the construct expressed GFP in all seam cells in addition to the same subset of neurons as the original p26F4. In the cells, GFP is localized to nuclei. By EMS-mutagenesis we have generated a mutation for the 26F4 gene in which the 1st exon was deleted completely. Worms homozygous for the mutation did not show any marked abnormality. Further analysis of the mutant phenotype is underway.

24 May 1999 15:50 369 369 Analysis of an RNA-binding protein ceHuD in C. elegans

1999 International Worm Meeting abstract 318 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of an RNA-binding protein ceHuD in C. elegans M Fujita, T Kawano, H Sakamoto Department of Biology, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nadaku, Kobe 657-8501, Japan RNA recognition motif (RRM) is found in various RNA-binding proteins of diverse function. This motif can be readily identified because of its relatively high conservation of the amino acid sequence. As the first step to uncover the biological roles of RNA binding proteins of the RRM family, we searched the C. elegans genome database for the RRM-containing protein and found a gene encoding a protein, referred to as ceHuD, which shares an extensive similarity with the human HuD. HuD was initially identified as an autoimmune antigen in human paraneoplastic neurologic disorders and is expressed in a neuron-specific manner. The questions of whether ceHuD is expressed neuron-specifically like HuD and what role it plays in C. elegans have not yet been addressed. Here we show, using transgenic worms, that ceHuD is expressed in specific neurons in the nerve ring and the ventral nerve cord, and that this expression pattern is observed in all developmental stages after hatching. We also show that ectopic expression of ceHuD has an irreversible and pleiotropic effect on adult worms, such as uncoordinated, egg laying-defective and clear phenotypes. Since these phenotypes are often observed with mutants of neuronal dysfunction, it is suggested that ceHuD plays an important role in maintenance and/or proper function of mature neurons. Disruption of the ceHuD gene is currently in progress.

24 May 1999 15:50 370 370 A novel WD40 protein, CHE-2, acts cell-autonomously in the formation of sensory cilia.

1999 International Worm Meeting abstract 319 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A novel WD40 protein, CHE-2, acts cell-autonomously in the formation of sensory cilia. M Fujiwara, T Ishihara, I Katsura National Institute of Genetics Mutations in the C. elegans gene che-2 cause defects in the cilium structure of sensory neurons and defects in behaviors that are mediated by these neurons, such as chemotaxis and osmotic avoidance (J.A.Lewis and J.A.Hodgkin 1977, T.A Starich et al 1995). We visualized the morphology of the cilia with GFP under a promoter specific to a few sensory neurons, and traced their formation during the development of the wild-type and che-2 mutant animals. The results showed that the abnormality of cilia in che-2 mutants is due to a defect in extension and not due to degeneration. We cloned che-2 gene by the cosmid rescue method. It encodes a novel protein of 760 amino acids with four WD40 repeats in the N-terminal region. WD40 repeat is known to be involved in protein-protein interaction and formation of multiprotein complexes. che-2 (mn395) which has a missense mutation in the WD40 repeats, shows mild defects in various behaviors such as chemotaxis and osmotic avoidance. The other alleles which show severer behavioral defects, were revealed to have nonsense mutations in the C-terminal region, although this region has no similarity to any known protein. The genetical analysis of over-deficiency suggests that these severer alleles are candidates of null alleles. CHE-2::GFP is expressed in almost all the ciliated sensory neurons, and localized at cilia. The expression starts at the late embryonic stage when cilia are formed and continues to the adult stage. We demonstrated that che-2 acts cell-autonomously, by expressing che-2 in a subset of sensory neurons. The function and morphology of only the neurons that expressed che-2 could be restored. We also expressed che-2 cDNA under the control of a C. elegans heat shock promoter. Expression of che-2 at the larval stage or even the adult stage was sufficient for the formation of sensory cilia although it occurs at the late embryonic stage in the normal development. Furthermore, the cilia formed in the adult stage were enough for the animal to restore the ability of osmotic avoidance.

24 May 1999 15:50 371 371 Watching the elt-2 GATA factor binding to its own promoter inside gut nuclei of developing C. elegans embryos

1999 International Worm Meeting abstract 320 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Watching the elt-2 GATA factor binding to its own promoter inside gut nuclei of developing C. elegans embryos T Fukushige, MJ Hendzel, DP Bazett-Jones, JD McGhee University of Calgary, Calgary Alberta, CANADA In working out transcriptional networks, one would ideally like to determine if a particular factor binds directly to a candidate target promoter inside the unperturbed embryo. Properties of the gut-specific elt-2 gene have allowed us to develop such a method. We have previously shown, by means of ectopic expression studies, that elt-2 autoregulates its own promoter. Since there are 20 WGATAR sites in the 4 kb elt-2 promoter, it seems likely that this regulation is direct. To test this, we fused GFP close to the C-terminus of elt-2 in the context of the full elt-2 promoter; this construct rescues the elt-2 null mutant. When multicopy transgenic arrays (~ 100 copies) of this rescuing elt-2::GFP construct were integrated into the genome, transgenic embryos (examined at the 4 - 8 E cell stage) show two discrete intense foci of fluorescence in the majority of their gut nuclei. These foci are not observed using a different elt-2::GFP construct lacking the DNA binding domain. We interpret these fluorescent foci as direct evidence of elt-2::GFP binding to its own promoter in the multicopy transgenic arrays, inside the living unperturbed embryo. The genetic behaviour of the fluorescent dots support this interpretation: the number of dots decreases to roughly one in embryos heterozygous for the array; the number of dots reverts to two when a second array consisting of only the elt-2 promoter is introduced. Dots are not seen in vector only controls. When multiple copies of the lac operator are introduced into the same array as the elt-2:GFP construct, co-localizing fluorescent foci are observed using exogenous lac repressor and GFP antibody; (this experiment is similar to that of Carmi et al, 1998, Nature 396, ...used to detect binding of the sex-1 protein). We hope to extend this approach to other transcription factors binding to other target promoters, all inside the living embryo.

24 May 1999 15:50 372 372 The elt-2 Gene and The Regulatory Network Controlling C. elegans Gut Development.

1999 International Worm Meeting abstract 321 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The elt-2 Gene and The Regulatory Network Controlling C. elegans Gut Development. T Fukushige, JD McGhee University of Calgary, Calgary Alberta, CANADA How is gut specific gene expression controlled and how is a functioning intestine produced from the single E cell progenitor? The elt-2 gene encodes a single zinc finger GATA transcription factor that is expressed in the gut from the 2E to adult stage. elt-2 is necessary for correct gut cell differentiation, and elt-2 null mutants die at the L1 stage, due to malformed intestines. Although ectopic expression of ELT-2 in the embryo can activate ectopic expression of the gut specific esterase gene (ges-1), gut granules (GG), and a gut specific antigen (MH33), these gut markers are still expressed in the elt-2 mutant. The end-1 gene (described by Rothman lab) also encodes a GATA transcription factor and is expressed in the gut from the 1E to 4E cell stage. Thus, end-1 is a good candidate to compensate for the lack of elt-2. Ectopic expression of end-1 in elt-2 null embryos can indeed activate genes producing MH33 and ICB4 antigens; however, ges-1 is not activated. Thus, some other factor in the early gut of the elt-2 mutant compensates for the lack of elt-2. A possible candidate is a new GATA transcription factor (called elt-4) located 5 kb upstream of elt-2. Elt-2 and elt-4 have highly conserved zinc finger domains as well as N -terminal region. An elt-4 reporter gene construct containing 5 kb of 5’ flanking region is expressed in all gut cells as well as 6 cells in the posterior pharynx. We have detected elt-4 mRNA by RT-PCR and are now assembling the entire gene. We will test the function of the elt-4 gene in the gut by using RNAi, ectopic expression experiments and gene knockout. What factors control elt-2 gene expression in the very early gut lineage? We have shown that end-1 can activate elt-2 gene expression through a 700 bp region on the elt-2 promoter. We have also shown that elt-2 autoregulates its own promoter and, by using an in situ GFP assay, have shown that this interaction is direct (See abstract by Fukushige et al.). We are extending our analysis to other genes that may lie upstream of elt-2 as well as to downstream genes that are direct elt-2 targets.

24 May 1999 15:50 373 373 Embryonic roles of the cki-1 and cki-2 genes

1999 International Worm Meeting abstract 322 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Embryonic roles of the cki-1 and cki-2 genes M Fukuyama 1 , SB Gendreau 1,2 , WB Derry 1 , JH Rothman 1

1 Department of MCD Biology, UC Santa Barbara, Santa Barbara, CA 93106. 2 Present address: Exelixis Pharmaceuticals, Inc. 260 Littlefield Ave., South San Francisco, CA 94080. cki-1 and cki-2 are adjacent genes that encode C. elegans members of the mammalian p21Cip1 /p27Kip1 family of cyclin-dependent kinase inhibitors (CKIs) (1, 2). Hong et al. (2) showed that RNAi of cki-1, but not cki-2, causes extra cell divisions in a number of larval cell lineages. We found that embryos homozygous for a deficiency deleting both genes show hyperplasia in many lineages, elongation defects, and excess cell corpses. A cosmid containing the cki genes can apparently rescue these phenotypes, suggesting that the cki genes are required for timely exit from the embryonic cell cycle, morphogenesis and suppression of programmed cell death (PCD). However, the rescued deficiency embryos contain fewer nuclei than wild type, perhaps indicating that overexpression of the cki gene(s) from the cosmid causes precocious cell-cycle arrest or that a positive cell cycle regulator is deleted by the deficiency. Previous studies using GFP reporter constructs suggested that cki-1 is expressed when embryonic and post-embryonic cells become post-mitotic. Surprisingly, cki-2 is ubiquitously expressed in proliferating embryonic cells as well as in post-mitotic cells (1, 2). Although CKIs have been found in proliferating cells in other animals, the significance of this expression has yet to be established. Despite the embryonic expression of the cki genes in many cells, previous RNAi experiments failed to show an embryonic phenotype for either cki gene alone or in combination. However, we recently found that RNAi directed against an extended region of cki-2 causes a phenotype reminiscent of that of the deficiency, perhaps indicating that cki-2 per se is critical for embryonic cell-cycle exit. We are currently attempting to generate knockouts of the cki genes and are characterizing the early embryonic expression of the CKI proteins in an effort to elucidate their roles in cell proliferation, cell-cycle exit, and PCD during embryogenesis.

1. Gendreau, S.B. and Rothman, J.H. (1997). 11th International Worm Meeting. 2. Hong, Y., Roy, R. and Ambros, V. (1998). Development 125, 3585-3597.

24 May 1999 15:50 374 374 emb-30 Encodes a Novel Protein Required for Germline Proliferation and Completion of the Meiotic Divisions

1999 International Worm Meeting abstract 323 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. emb-30 Encodes a Novel Protein Required for Germline Proliferation and Completion of the Meiotic Divisions T Furuta, B Koch, R Auty, J Kirchner, D Greenstein Dept. of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232 USA How is cell cycle control different between the germ line and soma? We isolated a strong temperature-sensitive allele of the emb-30 locus (Cassada et al., Dev. Biol. 84: 193-205, 1981), tn377ts III, that blocks the meiotic divisions and germline proliferation. emb-30 mutant oocytes undergo normal nuclear envelope breakdown, ovulation, and fertilization. They are able to set up a meiotic spindle but apparently do not progress through meiotic M-phase. As a result, no polar bodies are produced, pronuclei do not form, and embryonic development does not occur. If emb-30(tn377ts) mutant embryos are shifted to the non-permissive temperature, they grow into sterile adults. This Sterile phenotype results when Z2 and Z3 block during mitosis of their first attempted division during the L1 larval stage. By contrast, somatic development is apparently normal. When emb-30(tn377ts) adults are shifted to the non-permissive temperature, mitotic germ cells accumulate in the distal mitotic zone. Thus, emb-30 is likely to be required continually for completion of germline mitosis. We isolated 14 additional alleles in unbiased non-complementation screens. The strongest alleles are Sterile/Evl and appear to significantly reduce or eliminate emb-30 gene function by genetic and molecular criteria. Further analysis of the new alleles suggests that emb-30 has both germline and somatic functions and that the germline functions are separately mutable. Genetic mapping placed emb-30 within the emb-9-sod-4 interval. A left boundary was provided by the deficiency tnDf2 that fails to complement emb-30 and genes on its right (e.g. ced-7 and sqv-3), but complements emb-9. We localized the left breakpoint of tnDf2 to the cosmid T26G10. RNAi directed against a predicted gene from an adjacent cosmid (F54C8) resulted in an identical one cell arrest phenotype seen in several emb-30 alleles. Therefore, emb-30 appears to encode a novel 117 kdal protein. This identification was confirmed by sequencing the 15 chemically generated emb-30 alleles. The sequence of the emb-30 mutant alleles identifies residues that are critical for emb-30 function.

24 May 1999 15:50 375 375 Sensory Neuron Polarity and Interneuron Function

1999 International Worm Meeting abstract 324 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Sensory Neuron Polarity and Interneuron Function ME Gallegos, JA Zallen HHMI, Dept. of Anatomy, UCSF, San Francisco, CA 94122 Most sensory neurons in C. elegans are bipolar, extending one dendrite and one axon process from the cell body. While axon and dendrite polarity is established normally in sax-1 and sax-2 mutants, ectopic neurites often emerge from the cell bodies of certain neurons. A similar phenotype has been seen in a variety of mutants that disrupt neuronal activity, including mutants with defective cilium structure in the sensory dendrite and mutants with defective ion channels (E. Peckol, in press). These results suggest that ectopic neurite outgrowth may be secondary to improper neuron function. In contrast, neurons in sax-1 and sax-2 mutants seem to function normally, suggesting that sax-1 and sax-2 either act downstream of neuron function or in a parallel pathway that does not involve neuronal activity. sax-1 encodes a serine/threonine kinase related to the Ndr protein kinase in humans (62% identity) and flies (60 % identity).While the function of the Ndr kinases is unknown, other closely related kinases have been shown to play a role in cell shape and polarity. Interestingly, sax-1 and sax-2 mutants also have a cell shape defect. The sax-1; sax-2 double mutant resembles the single mutant phenotype suggesting that these two genes may function in the same pathway. To identify other likely components of this kinase pathway I am in the process of cloning sax-2, which maps to the unc-79/lon-1 interval on chromosome III. I am also interested in understanding the function of the interneurons and motorneurons that interpret sensory signals required for chemotaxis. Preliminary laser ablations by Cori Bargmann and EM reconstructions by John White suggest that elimination of more than one interneuron may be required to completely eliminate chemotaxis to a given odorant. It is also likely that different sensory neurons connect with distinct and overlapping sets of interneurons. I hope to take advantage of this cellular redundancy in order to design genetic screens that should isolate mutants specifically defective in interneuron function. I am now in the process of confirming and extending the interneuron ablations to characterize the neural circuit required for chemotaxis behavior.

24 May 1999 15:50 376 376 Genetic and molecular analyses of ced-8, which controls the time of appearance of cell corpses

1999 International Worm Meeting abstract 325 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic and molecular analyses of ced-8, which controls the time of appearance of cell corpses BD Galvin, HR Horvitz HHMI, Dept. Biology, MIT, Cambridge, MA 02139, USA In C. elegans development, the timing of programmed cell deaths in the soma is essentially invariant. The molecular mechanisms that control this timing are poorly understood. Mutations in ced-8 result in the delayed appearance of cell corpses. These mutations were originally identified in screens for mutations that cause the presence of cell corpses in late-stage embryos. ced-8 has been cloned (G.Stanfield, M. Hengartner, and Bob Horvitz; 1995 International Worm Meeting abstract 486) and appears to encode a multiple-pass transmembrane protein. To determine the role that ced-8 plays in programmed cell death, we intend to use both genetic and molecular approaches. We will screen for suppressors of the ced-8 phenotype. Specifically, using a sem-4; ced-8 mutant background, we will screen for F2 bags that contain F3 embryos wild-type in the timing of cell deaths. This screen may elucidate the role of ced-8 in programmed cell death and identify additional genes involved in cell-death execution. We also will generate antibodies for immunohistochemistry and perform biochemical assays to address the function of CED-8.

24 May 1999 15:50 377 377 vex-2 mutations perturb vulval fate execution at the first vulval-specific cell division

1999 International Worm Meeting abstract 326 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. vex-2 mutations perturb vulval fate execution at the first vulval-specific cell division DS Garbe, RS Kishore, MV Sundaram University of Pennsylvania School of Medicine, Philadelphia PA 19104 We have identified a mutation, ku233, that causes defects in the first vulva-specific cell division. We identified the ku233 mutation based on the egg-laying defective (Egl) phenotype of the mutants. ku233 hermaphrodites also have an incompletely penetrant sterile phenotype, and ku233 males have crumpled spicules. The mutants otherwise appear normal and vigorous. ku233 is a recessive, gamma-induced, temperature-sensitive allele, and maps to the far left end of chromosome III. This is a relatively gene-poor region of the genome, and ku233 complements all other known mutations in the region. Thus ku233 defines a previously undescribed locus. We followed the Sternberg lab lead in naming this locus vex-2, for vulval execution defective. We used Nomarski microscopy and MH27 immunostaining to observe different stages of vulval development in ku233 mutants. We found that the Pn.p and Pn.px cells appear normal, but that defects appear at the first vulval-specific cell division, when Pn.px cells divide to generate Pn.pxx cells. The Pn.pxx cells derived from P5.p, P6.p or P7.p often have abnormally small nuclei, and these abnormal Pn.pxx cells do not adopt organized positions beneath the gonadal anchor cell. The Pn.pxx cells then divide to generate Pn.pxxx cells that also have abnormally small nuclei and fail to undergo the proper morphogenetic movements, such that the vulval invagination appears grossly abnormal by the L4 stage. Vulval fate induction is controlled by a number of signaling pathways, including an RTK/Ras/ERK pathway. The vex-2(ku233) mutant defects suggest that VPCs can respond to these signaling pathways by entering the next cell cycle, but then are either unable to properly execute the division, or generate daughter cells of an inappropriate type. We hypothesize that vex-2 could encode a factor that responds to or cooperates with ERK signaling to execute specific downstream events. To make possible a test of such models, we are cloning vex-2, and conducting screens for additional vex-2 alleles.

24 May 1999 15:50 378 378 lin-42, a molecular link between circadian rhythms and developmental timing

1999 International Worm Meeting abstract 327 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. lin-42, a molecular link between circadian rhythms and developmental timing HF Gardner, M Jeon, AE Rougvie Department of Genetics and Cell Biology, University of Minnesota, St. Paul, MN 55108. Department of Biochemistry, University of Minnesota, St. Paul, MN 55108. Cells of a developing animal must incorporate cues of developmental time with those of position and sexual identity in order to adopt the appopriate cell fate. The heterochronic genes of C. elegans specify temporal information during post-embryonic development. Mutations in these genes alter the timing of the terminal differentiation of the lateral hypodermis. lin-4, lin-14(gf), and lin-29 mutant animals undergo retarded development; seam cells fail to terminally differentiate and instead reiterate larval fates. lin-14(lf) and lin-28 mutant animals develop precociously; seam cells express adult fates abnormally early. Our lab has cloned lin-42, another gene that controls post-embryonic developmental timing in the lateral hypodermis. Seam cells in lin-42 mutants terminally differentiate at the L3 molt, one stage earlier than in wild type. The predicted lin-42 protein contains a PAS domain known to mediate protein-protein interactions in other proteins. The LIN-42 PAS domain is most similar to the PAS domains found in the PERIOD (PER) class of circadian rhythm proteins. The PAS domain is the first identified motif shared between members of these two classes of biological timing mechanisms. Our goal is to understand how lin-42 controls stage-specific developmental events. To this end, we are characterizing the temporal and spatial expression patterns of lin-42. We are interested in learning the extent to which the sequence similarity of LIN-42 to Drososphila PER reflects a functional similarity. Given the known oscillation of per gene transcripts, we tested lin-42 mRNA levels for cycling behavior. We compared the levels of lin-42 mRNA to levels of a control message of 20 timepoints during post-embryonic development, and found that lin-42 transcript levels oscillate. Unlike per mRNA levels which flucuate with a 24-hour period, lin-42 transcript levels oscillate relative to the molts. We are also preparing to identify factors that interact with LIN-42 using a yeast two-hybrid system. We will test for interactions with known heterochronic gene products and with worm homologues of known PER-interacting proteins.

24 May 1999 15:50 379 379 duf-1(zu316cs) - Dorsal UnFused

1999 International Worm Meeting abstract 328 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. duf-1(zu316cs) - Dorsal UnFused T Gattegno, B Podbilewicz Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel. During embryonic development of C.elegans, hypodermal cells undergo programmed cell fusion to create multi-nucleated cells. Though the temporal and spatial patterns of the fusion events in the wild type have been characterized the molecular mechanism that induces cell fusion is unclear(1). Analysis of mutants affected in the fusion process provides an opportunity to clone the genes involved. duf-1(zu316cs) has fewer fusion events in the dorsal hypodermis compared to wild type(2) and it was isolated during a screen for elongation defects(3). Embryonic elongation occurs simultaneously with a series of fusion events at the hypodermis. We have characterized zu316cs as a zygotic lethal cold sensitive mutation and mapped it to the left arm of chromosome X, between -15.81 and -17.26 in the genetic map. The arrest phenotype was characterized using Nomarski optics; arrested embryos continue to move and have a twisted tail(2). Fusion analysis was done using the monoclonal antibody MH27 that recognizes the apical boundaries of hypodermal cells(1); a strain expressing MH27-GFP fusion protein(4) and carrying zu316cs enables efficient characterization of living embryos. MAbs indicative for muscles, intestine, pharynx, glial-like cells and hypodermis were used to characterize zu316cs. Staining patterns were fundamentally normal implying that zu316cs is specifically affected in the hypodermal fusion process. We further analyzed the number and identity of the unfused cells by counting junctions in arrested embryos (7.6±3.2; n=61) in comparison to wild type embryos at similar elongation stages (1.5±0.5; n=15). zu316cs arrest phenotype at the restrictive temperature can be artificially created by growing wild type embryos at 5°C(5). These results suggest conditional relationship between elongation and fusion; elongation does not proceed beyond 2 fold when hypodermal fusion is affected. Phenotypic and molecular characterization of duf-1 will contribute to a better understanding of the association between elongation and fusion processes.

[1] Podbilewicz B. and White J.G. (1994) Dev. Biol. 161, 408-424. [2] Gattegno T. and Podbilewicz B. (1997) IWM Abstract, p.546. [3] Costa M. and Priess J. (1995) IWM Abstract, p.165. [4] Mohler W.A., et al. (1998) Curr Biol. 8, 1087-1090. [5] Podbilewicz B. (1999) IWM Abstract.

24 May 1999 15:50 380 380 Cloning and characterization of C. briggsae and C. remanei homologs of fem-1

1999 International Worm Meeting abstract 329 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cloning and characterization of C. briggsae and C. remanei homologs of fem-1 JP Gaudet, AM Spence Dept. of Molecular and Medical Genetics, University of Toronto, Toronto, ON, M5S 1A8, CANADA fem-1 is one of three fem genes required for male development in Caenorhabditis elegans. Its product, FEM-1, is a 656 amino acid protein that contains seven copies of the ankyrin (ANK) repeat near its N-terminus, and the equivalent of 1.2 copies of the tetratrico peptide repeat (TPR) near its C-terminus. To investigate the function and evolution of fem-1, we cloned fem-1 homologs from the closely related species C. briggsae and C. remanei. The two genes are 71% and 69% identical, respectively, to C. elegans fem-1. This is higher than the overall conservation of other C. briggsae homologs of C. elegans sex determination proteins1,2,3 , suggesting that there are stronger evolutionary constraints on fem-1 than on the other sex determination genes. Cb-fem-1 can partially rescue a null mutation in Ce-fem-1, indicating that the sequence conservation reflects a functional conservation. However, a Cb-fem-1 transgene also dominantly feminizes males that lack maternal Ce-fem-1 (+/fem-1 [m-z+] XO), indicating that the sequence conservation is not complete. Because the ANK repeats show a higher overall conservation than the rest of FEM-1, we hypothesized that the dominant negative effect of Cb-fem-1 was mediated through the non-ANK portions of the protein. Consistent with this, a chimeric FEM-1 protein containing the ANK repeats of Cb-FEM-1 can fully rescue a Ce-fem-1 null mutation and shows no dominant feminization. This demonstrates that the ANK repeats of Cb- and Ce-FEM-1 are functionally interchangeable, and that interactions mediated by this region may also have been conserved. To examine the role of Cb-fem-1 in C. briggsae, we performed RNA-mediated interference using Cb-fem-1 RNA. Injection of this RNA into C. briggsae causes dominant feminization of male soma, demonstrating that Cb-fem-1 is involved in C. briggsae sex determination. However, Cb-fem-1 RNA injections do not affect the germline of C. briggsae, possibly reflecting the rapid divergence of germline sex determination mechanisms.

1 Kuwabara (1996). Genetics 144(2): 597-607.

2 de Bono and Hodgkin (1996). Genetics 144(2): 587-595.

3 Hansen and Pilgrim (1998). Genetics 149(3): 1353-1362.

24 May 1999 15:50 381 381 The sex determination protein FEM-3 accumulates in nuclei

1999 International Worm Meeting abstract 330 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The sex determination protein FEM-3 accumulates in nuclei JP Gaudet, AM Spence Dept. of Molecular and Medical Genetics, University of Toronto, Toronto, ON, M5S 1A8, CANADA Adoption of the male sexual fate requires the activity of all three fem genes, which negatively regulate tra-1 in somatic tissue. In XXs, fem activity is inhibited via an interaction between the predicted transmembrane protein TRA-2A and the novel protein FEM-31 . This allows the transcription factor, TRA-1A, to promote the female fate. In XOs, FEM-3 is presumably released from TRA-2A to act with the other FEMs to permit male development. Since TRA-1A is a genetic target of FEM activity, the FEMs appear to be involved in transducing a signal from the membrane to the nucleus. To understand the role of the FEMs in this signaling pathway, we examined the subcellular localisation of the FEM proteins. FEM-1 and FEM-2 both appear to be predominantly cytoplasmic in both XX and XO animals, based on cell fractionation studies and antibody staining with epitope-tagged versions of the proteins. When expressed under the control of a heat shock promoter, a GFP::FEM-3 fusion initially shows a strong cytoplasmic signal and weak nuclear signal. However, the cytoplasmic signal disappears by 6-8 hours after heat-shock, whereas the nuclear GFP::FEM-3 remains. In addition, expression of reduced levels of GFP::FEM-3 results in a reduction of cytoplasmic signal with no apparent loss of nuclear signal. We therefore believe that nuclear FEM-3 may be biologically relevant. To pursue the role of nuclear FEM-3, we analysed an N-terminal portion of the protein that encodes a putative nuclear localisation signal (NLS)2 . Deletion of this region eliminates nuclear retention of GFP::FEM-3, while the fusion of the putative FEM-3 NLS to GFP was sufficient to promote nuclear accumulation of GFP. Hence, this sequence is both necessary and sufficient for nuclear localisation, suggesting that it is a bona fide NLS and may be important for FEM-3 activity. Unfortunately, deletion of the NLS abolishes FEM-3’s ability to interact with both TRA-2A and FEM-2 in yeast two hybrid tests. We are therefore making point mutations in the NLS to disrupt the nuclear localisation of FEM-3 without affecting other aspects of the protein’s function. In this way, we hope to determine the phenotypic consequence of disrupting nuclear localisation of FEM-3.

1 Mehra et al. (submitted)

2 J. Ahringer et al. (1992). EMBO 11(6): 2303-2310.

24 May 1999 15:50 382 382 Two Distinct Importin a’s, IMA1 and IMA2, Differentially Regulate Germ Line Cell Fates

1999 International Worm Meeting abstract 331 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Two Distinct Importin a’s, IMA1 and IMA2, Differentially Regulate Germ Line Cell Fates KG Geles, SA Adam Dept. of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611 Metazoans express multiple nuclear localization signal (NLS) receptors, or Importin a’s, some within the same cell type. The ability of distinct Importin a’s to regulate the nuclear localization of cell fate determinants during development has not been elucidated. In order to identify specific functions for different Importin a homologs during development and cell fate specification, C. elegans was chosen as a model system. Nuclear protein import requires a heterodimer of the classical Importins, a and b. Members of the Importin a family bind classical-type NLSs, while Importin b mediates their association with the nuclear pore. Sequence data from the C. elegans genome project has identified three Importin a (IMA) proteins which we have termed IMA-1, -2 and -3. Based on phylogenetic analysis, IMA-2 and -3 are novel members of the Importin a family. IMA-1 is the C. elegans homolog of the human Qip1 protein. As evidenced by northern blot analysis and immunofluorescence, IMA-1 is expressed in multiple cell types including the germ line, while IMA-2 is exclusive to the adult germ line. IMA-1 and -2 are expressed in all embyronic cells until L1 and have a dynamic subnuclear localization during the cell cycle. IMA-1 and -2 associate with the Nuclear Pore Complex (NPC) during G1 as evidenced by co-localization with the anti-NPC antibody, MAb414, and are found within the nucleus during mitosis. RNAi was used to investigate the role of a specific IMA during development. Surprisingly, distinct germ line defects were observed in the injected animals. RNAi of ima-1 results in Po germ lines that lack nuclei in pachytene and diakinesis of meiosis I, but instead contain more condensed nuclei from the distal to proximal region. Interestingly, germ lines from ima-2 RNAi’ed adults contain an abnormal mitotic region containing meiotic-like nuclei. Based on tissue expression and nuclear localization, these results suggest distinct roles for two Importin a’s during germ line cell fate specification. We hypothesize that IMA-1 and IMA-2 transport a specific subset of nuclear proteins within the syncytium of the adult germ line.

24 May 1999 15:50 383 383 An enhanced gene disruption method is suitable for reverse genetics of Caenorhabditis elegans

1999 International Worm Meeting abstract 332 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. An enhanced gene disruption method is suitable for reverse genetics of Caenorhabditis elegans Keiko Gengyo-Ando 1 , Shohei Mitani 1,2

1 Department of Physiology, Tokyo Women’s Medical University School of Medicine, 8-1, Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan. 2 PRESTO, Japan Science and Technology Corporation.

The genome project of the nematode Caenorhabditis elegans is completed. It is advantageous and informative to disrupt nematode genes to study their function. Recently, the deletion mutagenesis for reverse genetics in C. elegans has been often done with chemical mutagens instead of "transposon insertion and excision". However, efficiency of gene disruption method reported previously was still too low for large-scale isolation of deletion mutants (at most about one mutant in 400,000 genomes). To develop an efficient mutagenesis procedure, we first isolated mutant alleles of a single locus ben-1, a b-tubulin gene, from mutagenized worms with EMS, UV of short wavelength or various conditions of TMP plus UV of long wavelength. We isolated a total of 170 independent alleles from the ben-1 locus (77 by EMS, 2 by short-wavelength UV and 91 by TMP/UV) by phenotypic screenings as previously, and then analyzed DNA lesions of these alleles by PCR and sequencing. Although the probabilities for mutant isolation by EMS were higher than those by short-wavelength UV and TMP/UV, in contrast to previous assumption, the probabilities for detectable deletion mutations were very low among EMS-treated mutants (none out of 77 alleles). We also failed to find deletion mutants derived from short-wavelength UV-treatments. However, the probabilities for detectable deletion mutations were high among TMP/UV treated mutants (a total of 30 out of 91 alleles). Furthermore, we found that the deletion sizes depend on the concentration of TMP. Thus, hereafter we focused our efforts to TMP/UV mutagenesis. To improve the sensitivity and specificity of mutant detection we have optimized PCR protocol by using these deletion mutants. Finally, we could isolate deletion mutants about 50 to 100-folds as efficiently as before by an appropriate condition of TMP/UV mutagenesis and improved PCR protocol. This highly efficient procedure enabled us to do sib-selection of genomic DNAs derived from single-cultured mutant library. The method, optimized this way, is so efficient that isolating deletion mutants on the whole-genome scale is now realistic.

24 May 1999 15:50 384 384 Still Searching for components of the VAB-1 pathways

1999 International Worm Meeting abstract 333 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Still Searching for components of the VAB-1 pathways SE George, AD Chishom University of California at Santa Cruz We are identifying the mechanisms by which the C. elegans Eph receptor tyrosine kinase, VAB-1, signals in neural and epithelial morphogenesis. VAB-1 has both kinase-dependent and kinase-independent functions during C. elegans development (George et al, Cell 92:633). The kinase-dependent (forward signaling) function of VAB-1 is mediated by its cytoplasmic kinase domain, possibly through tyrosine phosphorylation of the juxtamembrane domain. Proteins that interact with the cytoplasmic domain of Eph receptors have been identified in vertebrates using the yeast two-hybrid system. Candidate molecules include a low molecular weight protein tyrosine phosphatase and adaptor proteins such as Nck, Grb2, and Grb10. We are using a modified yeast two-hybrid screen to identify genes that function in VAB-1 forward signaling. Initial progress and methodology of the screen will be presented. The nature of the VAB-1 kinase-independent function is unknown but may involve the extracellular domain. Our genetic and biochemical studies indicate that VAB-2 (a C. elegans ephrin) is a ligand for VAB-1 and that it appears to mediate the kinase-independent function of VAB-1 (see Chin-Sang et al abstract). vab-2 mutations display synthetic lethality with vab-1 kinase mutations. We also observe synthetic lethality between vab-1 kinase mutations and a mutation in the receptor protein tyrosine phosphatase, PTP-1, suggesting that PTP-1 may be acting in a parallel pathway to the VAB-1 kinase activity (see Harrington et al abstract). To identify genes that are acting in the VAB-1 kinase-independent pathway or in parallel pathways we are performing screens to identify mutations that are synthetic lethal with vab-1. Progress of the screen will be presented.

24 May 1999 15:50 385 385 Phenotypic and molecular analysis of mig-8

1999 International Worm Meeting abstract 334 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Phenotypic and molecular analysis of mig-8 B Gerisch, C Weitzel, A Antebi Max-Planck-Institut für Molekulare Genetik, D-14195 Berlin The mig-8 locus affects various aspects of gonadogenesis. In hermaphrodites, the distal tip cells fail their dorsal and centripetal reflexions, and instead migrate into head and tail on the ventral muscle bands. Miscued oocytes and loose germ cells often are found in the body cavity of adults; these late stage phenotypes, however, may reflect earlier delays in L3 gonadoblast divisions. In males, the linker cell usually arrests migration on the dorsal muscle bands. What unites these various gonadal phenotypes is their appearance by larval stage L3. We therefore interpret mig-8 phenotypes as a heterochronic delay or arrest of L3 and later stage programs within the somatic gonad. Several other features suggest that mig-8 is a heterochronic gene. First, gonadal phenotypes are fully suppressed by development through the dauer stage, a characteristic of many heterochronic loci. Second, mig-8 phenotypes resemble those of another heterochronic locus, daf-12, particularly those daf-12 alleles which confer gonadal heterochrony only. In double mutants with these daf-12 alleles, no synergistic effect is seen. Moreover, mig-8 is partially suppressed by daf-12 (lf) alleles which have no gonadal phenotype on their own. Therefore, mig-8 and daf-12 could act in the same pathway. Relatedly, both mig-8 and daf-12 fail to express the unc-5 dorsal guidance receptor on schedule or at all (Su et al., WBG: 14(1): 54). Conceivably, mig-8 and daf-12 interact, and unc-5 is a possible target gene. mig-8 maps on the X-chromosome between mec-2 and mup-2. To identify the molecular product of mig-8, we are now injecting overlapping cosmids from this region to rescue the mutant phenotype. Because there is only one mig-8 allele, we are also generating additional alleles in non-complementation screens.

24 May 1999 15:50 386 386 More Crossed Wires in unc-37 Ventral Cord

1999 International Worm Meeting abstract 335 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. More Crossed Wires in unc-37 Ventral Cord EA German 1 , DM Miller 2 , DH Hall 1

1 Center for C. elegans Anatomy, Albert Einstein Col. of Med., Bronx, NY 10461. 2 Dept. of Cell Biology, Vanderbilt Univ., Nashville, TN 37232.

Uncoordinated movement in unc-37(e262) adults is superficially similar to the impairment observed in unc-4 animals. In unc-4, loss of coordination results from a specific wiring deficit between interneurons and motorneurons in the ventral nerve cord; VA motor neurons receive inputs normally reserved for their VB sister cells. We have reconstructed the detailed synaptic pattern in an adult unc-37 nerve cord from serial thin sections (1). We have identified the major interneurons and 21 motorneurons in the anterior ventral nerve cord, and their synaptic interactions. In both unc-37 and unc-4, AVB interneurons form inappropriate gap junctions onto class A motor neurons, while AVAs fail to make normal junctions onto these targets. This specific change may explain the similarity in their gross behavioral phenotypes. This finding is also consistent with the proposal that normal synaptic input to VA motor neurons depends on UNC-4 and UNC-37 dependent repression of VB-specific genes (2). There are also additional wiring changes and morphological differences in unc-37 ventral cord, however, which seem significant. The major interneurons are diminished in axon caliber and form very few chemical synapses. Many ventral cord motor neurons show minor changes in branching or axon caliber. At the muscle plate, the synaptic outputs of class A and class B motorneurons seems to be subject to high degrees of crosstalk: at each contact to muscle, there is a 50% probability that another motorneuron axon of the opposite class will insert at the site of contact, much the way that the DD dendrite generally does in the ventral cord, or the VD dendrite does in dorsal cord. Thus, it seems likely that class A motorneurons will simultaneously signal onto both the muscle arms and class B axons, and vice versa. Such crosstalk is practically unknown in wild type cord and rare in unc-4 ventral cord. We especially thank John White for giving us access to the original prints from wild type and unc-4 reconstructions, from which we are currently confirming these differences in axon caliber and opportunities for crosstalk.

1. German, Hall and Miller (1998) East Coast Worm Meeting. 2. Winnier et al. (1999) this meeting.

24 May 1999 15:50 387 387 The role of the GATA factor elt-3 in hypodermal development

1999 International Worm Meeting abstract 336 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The role of the GATA factor elt-3 in hypodermal development JS Gilleard, JS McGhee Department of Biochemistry and Molecular Biology, University of Calgary, Alberta, Canada We have previously shown, by reporter gene analysis, that the GATA transcription factor elt-3 is expressed in all hypodermal cells during embryogenesis with the exception of the lateral seam cells and H1, H2 and H3 cells of the head. Staining with specific ELT-3 antibody confirms the reporter gene expression pattern and shows the endogenous protein is localised to the cell nucleus consistent with its proposed role as a transcriptional regulator. Expression begins immediately after the final cell division that gives rise to these cells in the embryo suggesting elt-3 may function in the early stages of hypodermal cell differentiation. We have used the heat shock promoter to misexpress elt-3 and elt-1. Forced expression of either gene early in embryogenesis (up to approximately 120 minutes) produces arrested embryos of approximately 250-350 cells. Arrested embryos express the hypodermal cell markers lin-26, MH27 and dpy-7 in a large number of cells. The effects of elt-1 and elt-3 expression are similar but the effect of elt-1 is consistently more dramatic. For example the mean number of DPY7::GFP positive cells in elt-1 arrested embryos is 93 (n=15) whereas for elt-3, it is 72 (n=15). We are currently investigating the ability of elt-1 and/or elt-3 to transform non-hypodermal to hypodermal-like fates by forced expression in isolated blastomeres. We have used imprecise Tc1 transposon excision to isolate a deletion that completely removes the predicted DNA binding domain of the elt-3 gene. Animals homozygous for the deletion are viable, show no gross visible defects and have a normal brood size. The hypodermis appears normal and the hypodermal markers lin-26, MH27 and dpy-7 are all expressed in their wild type pattern. There are now 10 members of the GATA transcription factor family known in C.elegans (Clarke and Berg 1998) and at least two of these are expressed in the hypodermis (Page et al 1997; Koh et al 1998 wcwm abstract 153). We are performing RNAi of these different GATA factors in the elt-3 mutant background to investigate the possibility that loss of elt-3 function is compensated for by other GATA factors.We are also planning synthetic lethal screens for mutations that show a visible or lethal phenotype in combination with the elt-3 mutation.

24 May 1999 15:50 388 388 The Nuclear Receptor Superfamily: Conservation of Sequence and Function

1999 International Worm Meeting abstract 337 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Nuclear Receptor Superfamily: Conservation of Sequence and Function CR Gissendanner, T Lindblom, L Hipsher, AE Sluder Dept. of Cellular Biology, University of Georgia, Athens, GA The nuclear receptor (NR) superfamily of transcription factors includes the steroid, thyroid, and retinoid receptors in vertebrates and the ecdysone receptor in insects. The C.elegans genome sequence has revealed 260 NR genes, 5-fold more than is known from any other species. This makes NRs the most abundant transcription factor gene family in the worm. Our recent analysis of the C.elegans NR genes demonstrated that the NR family has undergone extensive diversification in nematodes (Sluder et al (1999) Genome Research 9:103). While most of these NR genes appear to be nematode-specific, 14 belong to conserved NR classes also found in both vertebrates and insects. Members of these conserved classes of NRs should have an important function in the nematode. Seven NRs are currently known to have a function in C.elegans. Of these seven, five are members of conserved classes and two of these five receptors have clear orthologs in vertebrates and insects. We are now investigating the function of the remaining 9 conserved NRs by examining their expression patterns and RNAi phenotypes as well as attempting to correlate them to known genetic loci. We have obtained expression data for all and have documented functions for most of these genes. We have preliminary data correlating two of these genes with known genetic loci (CG & AS abstract). Two of the more intriguing conserved NR genes are nhr-8, a member of the PXR/DHR96 NR class and nhr-41, a member of the TR4/DHR78 NR class. nhr-8 is required for gut function (TL & AS abstract) and nhr-41 functions in molting. The role of nhr-41 in molting is interesting as its insect ortholog, DHR78, may direct the onset of metamorphosis in insects, upstream of the ecdysone response (Fisk & Thummel (1998) Cell 93:543). Two other C.elegans NR genes, nhr-23 (Kostrouchova et al (1998) Development 125:1617) and nhr-25 (CG & AS abstract), also have phenotypes that affect molting and cuticle shedding, respectively. Orthologs of these genes are also involved in molting and metamorphosis in insects, raising the possibility that nematodes and insects share a homologous "molting gene cassette".

24 May 1999 15:50 389 389 nhr-25, the C. elegans ortholog of Ftz-F1, is required for hypodermal and somatic gonad development

1999 International Worm Meeting abstract 338 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. nhr-25, the C. elegans ortholog of Ftz-F1, is required for hypodermal and somatic gonad development CR Gissendanner, AE Sluder Department of Cellular Biology, University of Georgia, Athens, GA The Ftz-F1 family is a phylogenetically conserved class of nuclear receptors (NRs) that functions in insect metamorphosis and embryonic patterning and in vertebrate adrenal and gonad development. C. elegans also has a Ftz-F1-like gene, nhr-25, and we have initiated an analysis of its function. nhr-25 is 81% and 76% identical to the DNA binding domains of insect and vertebrate Ftz-F1 genes, respectively. This makes nhr-25 the likely nematode ortholog of these Ftz-F1 genes. nhr-25 has two SL1-transpliced mRNA transcripts that code for two different protein isoforms. Interestingly, one isoform does not have a DNA binding domain. nhr-25 is expressed in later stage embryos and all larval stages but not in adults. An NHR-25::GFP fusion protein is expressed exclusively in hypodermal cells of embryos and larvae. Disruption of nhr-25 function by RNAi generates a high percentage of embryonic lethality. Embryos arrest at two-fold when hypodermal integrity is lost during morphogenesis. Larvae that hatch are dumpy, uncoordinated and arrest at L1 or L2. These larvae have a Vab phenotype with a fully penetrant posterior patterning defect and incompletely penetrant anterior patterning defect. Larvae also have difficulty shedding molted cuticle. These phenotypes are consistent with defective hypodermal development. A few nhr-25(RNAi) animals escape to become adults and these animals are Vul, sterile and fail to form a uterus and spermatheca. nhr-25(RNAi) adults do make oocytes but their gonads become tumorous. We have not yet seen NHR-25::GFP expression in the developing somatic gonad but we are continuing to analyze the expression of this reporter. We have also correlated nhr-25 with a known genetic locus. An nhr-25 genomic subclone can complement emb-15(g15), which maps to the nhr-25 region on the X-chromosome.

24 May 1999 15:50 390 390 The netrin UNC-6 can redirect axons in vivo

1999 International Worm Meeting abstract 339 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The netrin UNC-6 can redirect axons in vivo Z Gitai, M Tessier-Lavigne, CI Bargmann Dept. of Biochemistry and HHMI, UCSF, San Francisco, CA 94143-0452 The netrin UNC-6 is involved in both the attraction and repulsion of circumferentially migrating cells and axons in C. elegans. Attraction to netrin/UNC-6 is mediated by the UNC-40 receptor, while repulsion is mediated mainly by the UNC-5 receptor. Culotti et al. have performed extensive characterization of the genetic pathways for UNC-5-dependent netrin repulsion, but less is known about UNC-40-dependent netrin attraction. In wild type animals UNC-6 is expressed ventrally, and the HSN axon is directed ventrally via the UNC-40 receptor, which can act at least partially cell autonomously within the HSN neuron to mediate ventral guidance. This ventral migration is severely disrupted in unc-6 and unc-40 mutants. We used the muscle-specific fragment of the UNC-129 promoter (kindly provided by Drs. A. Colavita and J. Culotti) to misexpress UNC-6 in the dorsal muscle. In an unc-6 mutant background, HSN axons can be redirected toward the dorsal UNC-6 source. This demonstrates that axons can be attracted to UNC-6 in vivo. The attractive migration toward both endogenous and ectopic UNC-6 requires the UNC-40 receptor. To identify additional genes required for UNC-6 attraction, we are currently screening for mutations that suppress the HSN redirection to dorsal UNC-6. These genes are expected to define components of the UNC-40 pathway. Axonal growth cones are exposed to many different cues that act in concert to direct their trajectories. Guidance receptors such as SAX-3/Robo normally act in parallel to UNC-40 to direct ventral HSN axon guidance. In animals with ectopic dorsal expression of UNC-6, the dorsal migration of the HSN axon is directed solely by UNC-6. Other pathways that direct HSN ventral guidance are not expected to be affected by the dorsal UNC-6, and continue to direct the HSN axon ventrally. Thus, these pathways would serve to antagonize HSN attraction to the ectopic dorsal UNC-6. Mutations in components of these pathways should present themselves as enhancers of the redirection to dorsal UNC-6. The dorsal UNC-6 strain therefore provides a useful genetic background to identify and study molecules required for the reception and transduction of the UNC-6/UNC-40 signal, as well as parallel pathways that specify ventral axon guidance.

24 May 1999 15:50 391 391 let-21, a Homolog of the ect2 Proto-oncogene, is Necessary for Germline Development and Mitotic Cell Cycle Progression

1999 International Worm Meeting abstract 340 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. let-21, a Homolog of the ect2 Proto-oncogene, is Necessary for Germline Development and Mitotic Cell Cycle Progression A Godbey 1 , P Kuwabara 2 , E Lambie 3 , R Francis 1 , T Schedl 1

1 Genetics, Washington Univ School of Medicine. 2 MRC Laboratory of Molecular Biology. 3 Biology, Dartmouth College.

An important control point in meiotic development is the transition from pachytene to diplotene/diakinesis (Pachytene EXit) which involves the nuclear events of disassembly of the synaptonemal complex, chiasma formation, chromosome condensation and the differentiation events of oocyte cellularization and growth. Mutations that disrupt this transition show a Pex phenotype where nuclei arrest in pachytene and clump, and there is a loss of the hexagonal packing of cells along the gonadal surface distal to the loop. Previous work demonstrated that mutations in RAS/MAP kinase pathway genes show Pex and vulvaless phenotypes (1). Genetic screens have identified additional Pex mutations. Two mutations, with no obvious somatic defects, were found to be allelic to let-21(e1778), originally identified by E. Hedgecock. Positional cloning identified let-21 as T19E10.1, a homolog of the proto-oncogene ect2. LET-21 and ect2 both have two N-terminal BRCT related domains, first described in the breast cancer protein BRCA1 and certain cell cycle checkpoint genes (2). Both have a centrally located DH domain (~200aa) and its associated PH domain (~100aa) that function as guanine nucleotide exchange factors (GNEF) for Rho-family GTPases which have been implicated in control of the cytoskeleton (3). Truncation of ect2, removing both BRCT domains, results in transformation of fibroblasts in vitro (4) suggesting that the BRCT domains may in some way negatively regulate the GNEF activity. A role for let-21 in cell cycle progression is suggested by the sterile unc (Stu) phenotype of e1778. Two internal deletion alleles were identified that are likely null and show a more pronounced Stu phenotype where only rudimentary gonads are formed. A role for let-21 in cytoskeletal organization is suggested by the cellular and cytoplasmic disorganization observed in mutant germlines. LET-21 may function to coordinate nuclear and cytoplasmic activities during both the mitotic and meiotic cell cycle.

1) Church et al. ’95, Dev 121:2525; 2) Bork et al. ’97, FASEB J 11:68; 3) Soisson et al. ’98, Cell 95:259; 4) Miki et al. ’93, Nature 362:462

24 May 1999 15:50 392 392 New Genes Involved in Ras-mediated Induction of Vulval Cell Fates

1999 International Worm Meeting abstract 341 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. New Genes Involved in Ras-mediated Induction of Vulval Cell Fates JL Goldstein, K Kornfeld Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110 The development of the C. elegans vulva requires the activity of the Ras signaling pathway. To identify components of this pathway, we screened for mutations that suppress the multivulva phenotype caused by a let-60 (n1046gf) mutation that constitutively activates the ras gene. Forty-three alleles comprising 21 complementation groups were identified. These genes include conserved members of the Ras signaling cascade, such as Raf (lin-45), Ksr (ksr-1), MEK (mek-2), MAP kinase (mpk-1), and an ETS transcription factor (lin-1). We are now analyzing additional complementation groups that are represented by single alleles: n2528 maps to LGX and n2517 maps to LGIV. We are using high resolution mapping relative to single nucleotide polymorphisms to positionally clone the genes affected by these mutations, and genetic analysis to establish how these genes regulate the Ras pathway.

24 May 1999 15:50 393 393 FMRFamide-related peptides and egg-laying

1999 International Worm Meeting abstract 342 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. FMRFamide-related peptides and egg-laying S Golik, L Hardaker, W Schafer Department of Biology, UCSD, La Jolla, CA 92093-0349 The egg-laying motorneurons release multiple neurotransmitters, each of which appears to control a specific aspect of egg-laying behavior. Under favorable conditions, wild-type worms fluctuate between alternative behavioral states for egg-laying: an active state, during which eggs are laid in clusters, and an inactive state, during which eggs are retained. Switching between states, as well as egg-laying within the active state, are stochastic, Poisson-like processes. We showed previously that serotonin, released primarily from the HSNs, facilitates the switch from the inactive to the active state, while acetylcholine, released from both the HSNs and VCs, promotes egg-laying within the active state. Li and Chalfie have shown that both the HSNs and VCs contain one or more FMRFamide-related peptides, and provided pharmacological evidence that these neuropeptides may control of egg-laying behavior. To investigate the roles of these peptides in more detail, we have analyzed the egg-laying behavior of deletion mutants (kindly provided by Chris Li) that are defective in production of specific FaRP precursors. We have found that one of these mutants, defective in the gene flp-1, have dramatically lengthened inactive phases, but completely normal egg-laying within the active phase. Moreover, although these mutants are stimulated to lay eggs by serotonin with approximately normal dose responses, their rate and pattern or egg-laying on serotonin are abnormal. Thus, we hypothesize that the FLP-1 peptides and serotonin act in parallel to affect the same step in egg-laying behavior--inducing the switch between the inactive and active phases. Experiments to investigate the cellular and molecular mechanisms for the FLP-1 peptides’ affects on egg-laying will be described.

Thanks to Chris Li for discussions and for providing strains.

24 May 1999 15:50 394 394 zyg-8 controls anaphase spindle positioning in the one-cell stage embryo

1999 International Worm Meeting abstract 343 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. zyg-8 controls anaphase spindle positioning in the one-cell stage embryo P Gönczy 1 , T Kaletta 2 , R Schnabel 2 , H Schnabel 2 , T Hyman 1

1 European Molecular Biology Laboratory, Heidelberg. 2 Institut für Genetik, TU Braunschweig, Braunschweig.

In animal cells, the position of the spindle at the end of anaphase determines placement of the cleavage furrow, which forms equidistant from the spindle poles and at right angle to the spindle axis. In the wild-type one cell stage C. elegans embryo, the spindle is displaced slightly towards the posterior during the course of anaphase. As a result, the one cell stage embryo cleaves into a larger anterior blastomere and a smaller posterior one. We searched for mutants in anaphase spindle positioning in the one cell stage embryo by screening maternal-effect embryonic lethal mutants by time-lapse DIC video microscopy. We identified a novel class of loci whose function is required for proper anaphase spindle positioning; one of these loci is zyg-8. In most zyg-8 mutant embryos, the spindle sets up as in wild-type, but is then displaced excessively towards the posterior during anaphase, resulting in aberrant cleavage furrow placement. Excess posterior displacement is observed in 5/7 zyg-8 alleles, and when a zyg-8 mutation is put over deficiency, suggesting that this represents a strong loss-of-function phenotype. Why does the loss of zyg-8 function result in excess posterior displacement? One possibility is that overall antero-posterior (A-P) polarity is affected. However, like in wild-type, PAR-3 protein is restricted to the anterior cortex and P granules are segregated to the posterior of zyg-8 mutant embryos, suggesting that A-P polarity is, in fact, not affected. Moreover, excess posterior displacement is suppressed in zyg-8 par-3 and zyg-8 par-2 double mutants, showing that zyg-8 mutant embryos respond to changes in overall A-P polarity. Another possibility is that astral microtubules are affected, since they are required for aspects of spindle positioning in other systems. Indeed, we found that astral microtubules during anaphase in zyg-8 mutant embryos are shorter than in wild-type, usually not reaching all the way to the cortex. Based on these observations, we propose that zyg-8 normally restricts the extent of posterior spindle displacement during anaphase by ensuring proper anchorage of astral microtubules to the anterior cortex.

24 May 1999 15:50 395 395 Autonomy vs. non-autonomy of lin-15 action

1999 International Worm Meeting abstract 344 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Autonomy vs. non-autonomy of lin-15 action AS Gonzalez-Serricchio, PW Sternberg California Institute of Technology lin-15 encodes two novel hydrophilic proteins, LIN-15A and LIN-15B. lin-15 is one of the synthetic multivulva (synmuv) family of genes that in certain combinations of mutations result in a multivulva (muv) phenotype. The muv phenotype is the result of mutations in each class, referred to as A and B, which signify two functional redundant pathways in vulval induction. We used both, the GFP-LacI + lacO256 system (that was described at previous meeting) and the ncl-1 gene, to determine the site of action of lin-15A and lin-15B . Both markers indicate that lin-15A gene function is required within the vulval precursor cells (VPCs) to prevent an excessive number of VPCs from generating vulval progeny. Previous mosaics with a free duplication and a mutation that eliminates function of both lin-15A and B (lin-15(n309) ) [Herman and Hedgecock, Nature 348, 1990] indicated that at least one lin-15 function acts cell non-autonomous. lin-15B has a broad focus, i.e., no single site of action. Further mosaics for lin-15B are currently underway using the SUR-5::GFP(Yochem et al., 1998) as an additional mosaic marker.

Herman, R.K., and Hedgecock, E.M. (1990). The size of the Caenorhabditis elegans vulval primordium is limited by lin-15 expression in surrounding hypodermis. Nature 348: 169-171 Yochem, J., Gu, T., and Han, M (1998). A new marker for mosaic analysis in Caenorhabditis elegans indicates a fusion between hyp6 and hyp7, two major components of the hypodermis. Genetics 149: 1323-1334

24 May 1999 15:50 396 396 Ionic Currents in C. elegans Mechanosensory Neurons

1999 International Worm Meeting abstract 345 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Ionic Currents in C. elegans Mechanosensory Neurons MB Goodman 1 , SR Lockery 2 , D Lenzi 2 , M Chalfie 1

1 Columbia University, New York, NY. 2 University of Oregon, Eugene, OR.

As a prelude to testing how the electrical properties of mechanosensory neurons are altered in mechanosensory abnormal animals, we recorded membrane current in PLMR and PLML. The PLM cells are a pair of biopolar neurons required for the response to gentle touch in the tail1 . Both anterior and posterior neurites remain attached to the exposed cell body and can be partly filled with dye from the recording pipette. We used dyes known to permeate vertebrate gap junctions (Lucifer Yellow, Cascade Blue and sulforhodamine 101). While we observed dye-coupling between other neurons in the head and tail, dye-coupling was not routinely observed between PLM and other neurons. Steady-state current in PLM consisted of a small hyperpolarization-activated current, an inward current between -45 and -30 mV, and a rapidly-decaying outward current at voltages greater than 5 mV. Outward current was carried by K+ ions, since it was absent from recordings obtained with K+ -free pipettes. In addition, we frequently observed large, single-channel openings. We are currently studying the pharmacology of these ionic currents in order to characterize them more fully and to identify candidate genes that encode these channels.

1 Chalfie, et al (1985) J. Neurosci. 5: 956

24 May 1999 15:50 397 397 Structure/Function Analysis of the EGL-15 FGF Receptor

1999 International Worm Meeting abstract 346 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Structure/Function Analysis of the EGL-15 FGF Receptor SJ Goodman, MJ Stern Yale University, 333 Cedar St., SHM I352, New Haven, CT 06520 The fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases play important roles in many aspects of normal development. To understand their function better, we have assembled an FGFR signaling pathway in C. elegans using the Clr/Soc phenotypes that can result from various modulatory effects on the EGL-15 FGFR. A Clr phenotype can result from hyperactivation of this pathway caused either by compromising the function of the CLR-1 receptor tyrosine phosphatase that negatively regulates the EGL-15 signaling pathway or by constitutively activating EGL-15 itself. Conversely, mutations that reduce signaling through this pathway can suppress the hyperactive Clr phenotype, resulting in a Soc (suppression of Clr) phenotype. To compare the signal transduction mechanisms utilized by the human type 1 FGFR (hFGFR1) and EGL-15, we are determining whether portions of the hFGFR1 can function in place of similar regions of EGL-15 in EGL-15/hFGFR1 chimeras and testing their function using two assays. In one assay, we are testing whether chimeric transgenes can rescue the Soc phenotype of egl-15(n1783) in clr-1(e1745ts); egl-15(n1783) mutants. The second assay tests whether the chimeric receptors can confer a dominant Clr phenotype when hyperactivated by the neu* transmembrane domain that has been used to hyperactivate EGL-15. Our initial focus has been on the intracellular portion of these FGFRs. We have shown that the hyperactive EGL-15/hFGFR1 chimera can confer a dominant Clr phenotype similar to that of hyperactivated EGL-15, but that the non-hyperactivated chimera does not rescue the Soc phenotype of egl-15(n1783). We will report on further analyses that probe the functional similarity of these FGFRs.

24 May 1999 15:50 398 398 odr-7 and the AWA olfactory neurons

1999 International Worm Meeting abstract 347 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. odr-7 and the AWA olfactory neurons G Gopinathrao, P Sengupta Dept. of Biology, Brandeis University, Waltham, MA 02454 Worms respond to volatile attractants using the AWA and AWC olfactory neurons. We have previously shown that odr-7 determines the sensory functions of the AWA neurons by regulating the AWA-specific expression of the olfactory receptor odr-10 and the putative channel encoding gene osm-9. odr-7 encodes a unique member of the nuclear hormone receptor superfamily and is expressed only in the AWA neurons. Our goal is to further characterize the role of odr-7 in the determination of AWA function. To date, a total of four odr-7-regulated genes in the AWA neurons have been identified. Comparison of the promoters of these genes has identified conserved sequence elements that may be required for odr-7-mediated regulation. These sequences are being further examined. We are also identifying the minimal odr-7-responsive elements in the promoter of odr-10. DNA binding experiments with these sequences will be carried out to determine if ODR-7 regulates its targets directly or indirectly. Expression of target genes is also being examined in multiple alleles of odr-7, since our results suggest that these genes are regulated differentially in these odr-7 alleles. Although odr-7 is necessary for the specification of AWA function, is it also sufficient? Ectopic expression of odr-7 using the heat-shock promoter does not result in ectopic expression of AWA-specific genes. Expression of odr-7 in the AWB and AFD neurons also fails to cause expression of target genes. Currently, we are expressing odr-7 in the AWC olfactory neurons as well as in the ASG neurons (lineal sisters of AWA). It is likely that ODR-7 functions with other gene products to specify AWA function. In order to identify interacting gene products, we have carried out a two-hybrid screen using ODR-7 as bait. Several candidate genes have been isolated including one encoding a putative protein phosphatase and another with limited homology to homeobox genes. The phosphatase homolog is expressed in most neuron types, while the homoebox homolog is expressed in a limited subset of head and motor neurons. We plan to carry out a more extensive screen for ODR-7 -interacting proteins in collaboration with Marc Vidal at MGH. These experiments and others (to be described on the poster), will allow us to fully examine and characterize the role of odr-7 in the determination of AWA sensory function.

24 May 1999 15:50 399 399 Control of cell division axis choice in C.elegans

1999 International Worm Meeting abstract 348 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Control of cell division axis choice in C.elegans M Gotta, J Ahringer Wellcome/CRC Institute, Tennis Court Road, Cambridge CB2 1QR, United Kingdom The orientation of the cleavage plane is a critical aspect of development. The cleavage plane is established by the position and orientation of the mitotic spindle, which is set by the position of the centrosomes. In C. elegans embryos, cell division planes follow a fixed pattern, allowing abnormalities to be easily analysed. In previous work, we found that gpb-1, which encodes the beta subunit of a heterotrimeric GTP-binding protein, has a crucial role in spindle orientation. In embryos lacking GPB-1 activity, mitotic spindles in early cell divisions are formed properly but they are randomly oriented instead of following a fixed pattern. Lack of GPB-1 does not appear to affect the polarity of the zygote. Interestingly, GPB-1 localises to the asters during cell division. To further characterise the G protein involved in spindle orientation, we sought the Gg partner. There are two Gg genes in the genome, gpc-1 and gpc-2. RNA interference to gpc-2, but not to gpc-1, causes embryo lethality. Staining of early embryos with anti-tubulin antibodies shows that cleavage planes in early cell divisions are abnormal. However, the first cleavage is asymmetric and P granules are correctly localised to the germline precursor cells. This suggests that GPC-2 is probably the partner of GPB-1 in controlling spindle orientation. We are currently trying to find interactors of Gbg by a three hybrid screen. As a second approach, we are trying to identify other factors involved in spindle orientation by RNAi to candidate genes based on the homology with yeast polarisation during mating which is controlled by a heterotrimeric G protein. Upon activation by the receptor, the Gbg dimer activates downstream factors such as Cdc42p and Ste20p. Injection of dsRNA for two of the many Ste20p homologues show no phenotype, although this may reflect a redundant function. RNAi to the C. elegans Cdc42p homologue causes embryo lethality and the early embryos are osmotically sensitive. In these embryos cleavage planes are abnormally oriented. In addition the first cleavage can be symmetric and P granules are not properly localised. We have undertaken a two hybrid screen with CDC-42 and we are currently analysing the interacting clones by RNAi.

24 May 1999 15:50 400 400 Can orthologous homeotic lin-39 genes from Pristionchus and Caenorhabditisfunctionally replace each other?

1999 International Worm Meeting abstract 349 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Can orthologous homeotic lin-39 genes from Pristionchus and Caenorhabditisfunctionally replace each other? K Grandien 1,2 , R Sommer 1

1 Max-Planck Institut für Entwicklungsbiologie, Abteilung für Evolutionsbiologie, Spemannstr 35, 72076 Tübingen. Tel: 07071-601372, Fax: 07071-601449. 2 Email: [email protected]

We use the nematode development as a model to study molecular alterations occurring during evolution of animal development. The work presented here focuses on a molecular comparison of the expression and function of the Hox gene lin-39 during the development of the vulva in the two species Caenorhabditis elegans and Pristionchus pacificus. Previous studies have demonstrated important changes in the function of lin-39 in these two species with regard to vulval development. In C.elegans, lin-39 inhibits the vulval precursor cells to undergo cell fusion whereas in P.pacificus, this gene instead inhibits the corresponding cells from undergoing programmed cell death. Additionally, in P.pacificus, lin-39 is only needed during the early stage of vulval development whereas in C.elegans it is required later during anchor cell-derived induction of the vulval precursor cells. In contrast to these species-specific differences in the role of lin-39 this gene seems to perform the same function in both species with regard to the specification of the Pn.aap neuroblast cells. One of the aims of our study is to compare the regulatory regions required for expression of the lin-39 genes in the two different species. To begin with we started to characterize the regulatory regions of the C.elegans lin-39 gene. The results presented here indicate the prerequisite of multiple different regulatory elements in promoter, intronic and 3’ untranslated regions of the C.elegans lin-39 gene for establishment and maintenance of lin-39 expression. In addition, we are currently performing experiments where we express P.pacificus lin-39 in C.elegans lin-39 mutants and analyze the extent of mutant phenotype rescue. Preliminary results indicate that there might be functional differences of the lin-39 genes in the two species with regard to vulval development.

24 May 1999 15:50 401 401 Axonemal transport proteins in nematodes

1999 International Worm Meeting abstract 350 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Axonemal transport proteins in nematodes WN Grant 1 , A Dubowsky 1 , R Wisotzkey 2 , CD Johnson 2

1 Flinders University, Adelaide, Australia. 2 Axys Pharmaceuticals, San Francisco.

We have previously reported the cloning of che-3 and shown that it encodes a dynein heavy chain (DHC). Comparison with the available (mostly partial) DHC sequences shows that CHE-3 is a member of the novel 1b isotype of DHCs, most closely related to Chlymandomonas, sea urchin and mammalian genes implicated in axonemal and intraflagellar transport during cilia synthesis and regeneration. To explore this apparent sequence and perhaps functional homology further we obtained partial sequence of the che-3 homologues in other nematodes (Caenorhabditis briggsae, Ostertagia circumcincta, Onchocerca volvulus & Parastrongyloides trichosuri). Alignment of these sequences with typical cytoplasmic and isotype 1b DHCs reveals several sites where all of the 1b sequences display the same amino acid substitution relative to the cytoplasmic dynein heavy chain sequences. This, together with the defective amphid phenotype of che-3 mutants suggests that che-3 defines a novel, conserved class of DHC that is required for the generation of cilia in C. elegans chemosensory neurones. There are mammalian nervous system EST’s with strong homology to che-3, raising the possibility that che-3 homologues function in the mammalian sensory nervous system.

24 May 1999 15:50 402 402 SEM-4 regulates vulval cell fate and expression of lin-39 hox

1999 International Worm Meeting abstract 351 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. SEM-4 regulates vulval cell fate and expression of lin-39 hox Kelly Grant, Wendy Hanna-Rose, Min Han MCD Biology, HHMI, University of Colorado, Boulder, CO 80309 Both lin-39 hox gene and Ras pathway signaling activity are important components of vulval cell fate specification. Ras activity affects LIN-39 expression (Maloof and Kenyon, 1998 and Clandinin, et al, 1998) through a mechanism that has yet to be determined. We have identified a candidate transcriptional regulator of lin-39 and are investigating whether this factor may provide a link between Ras signaling and lin-39 regulation. In a screen for vulval defective mutants, we identified multiple mutants with abnormal vulval cell lineages, including 3 alleles of sem-4. sem-4 encodes a zinc-finger protein with recognized roles in fate specification of sex myoblasts, coelomyctes and several neurons (Basson and Horvitz, 1996), but sem-4 was not previously reported to affect vulval cell lineages. Approximately 60% of worms with sem-4 mutations exhibit an abnormal P7.p lineage and a less penetrant P5.p lineage defect. To examine the role of SEM-4 in vulval formation, we analyzed its interactions with other genes that affect vulval differentiation. Analysis of animals carrying mutations in both sem-4 and either let-60ras or lin-45raf suggested that sem-4 does not function directly in the Ras pathway. However, we observed a strong genetic interaction between sem-4 and lin-39. A sem-4; lin-39(n709ts) double mutant displays a significant decrease in induction of both P5.p and P7.p. Additionally, we have observed that expression of lin-39::lacZ (gift of Maloof and Kenyon) is reduced in the vulva in a sem-4 mutant background. Furthermore, over-expression of LIN-39 from a heat shock promoter can partially rescue a sem-4 mutant. These data suggest that lin-39 acts downstream of sem-4 to regulate vulval differentiation, and the molecular identity of SEM-4 suggests that it may regulate the transcription of lin-39. We have determined that a SEM-4::GFP translational fusion (gift of M. Koelle) is expressed in the vulva precursors cells just after the first cell division and persists until after cell division in the vulva lineage is complete. Expression is stronger in P7.p and its descendants, consistent with sem-4 mutations having a greater effect on P7.p. We are currently investigating whether SEM-4::GFP is regulated by signaling through the Ras pathway to determine if SEM-4 acts between Ras signaling and lin-39.

24 May 1999 15:50 403 403 Investigations of the Low Density Lipoprotein Receptor-Related Protein (LRP) in Caenorhabditis elegans

1999 International Worm Meeting abstract 352 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Investigations of the Low Density Lipoprotein Receptor-Related Protein (LRP) in Caenorhabditis elegans JL Grimsby, ME Roemer Northern Michigan University, Marquette, MI 49855 The low density lipoprotein receptor-related protein (LRP) is a receptor found on the cell surface. It is a member of the low density lipoprotein (LDL) receptor family. This family of receptors functions to remove molecules from the blood, including lipoprotein molecules. This function is important because these lipoprotein molecules are thought to be a factor in the progression of atherosclerosis and Alzheimer’s disease. Studies on the clearance of lipoprotein molecules from the blood by LRP could lead to advances in the treatment of these diseases. This research project investigates the availability of an alternate source of LRP for these studies and seeks to characterize the LRP protein. Regulation of the LRP gene in Caenorhabditis elegans will also be explored. Characterization of the LRP will be done using ligand binding assays. Regulation of the LRP gene will be explored through the investigation of mRNA. Preliminary studies have detected an LDL receptor family-like molecule present in Caenorhabditis elegans.

24 May 1999 15:50 404 404 SMG-5 and SMG-7 Interact Directly and are Required to Regulate SMG-2 Phosphorylation

1999 International Worm Meeting abstract 353 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. SMG-5 and SMG-7 Interact Directly and are Required to Regulate SMG-2 Phosphorylation A Grimson, M Page, K Anders, P Anderson Laboratory of Genetics, UW Madison, Madison, WI 53706 Nonsense-mediated mRNA decay, or NMD, functions to degrade mRNAs containing premature stop codons and has been conserved throughout the Eukarya. In C. elegans, function of the seven smg genes is required for NMD. smg-5 and smg-7 encode proteins of 549 and 459 amino acids, respectively. Both are novel genes, though smg-7 contains two TPR (tetratricopeptide repeats) domains, which are known in other proteins to mediate protein-protein interactions. We have previously demonstrated by co-immunoprecipitation studies that SMG-5 and SMG-7 are members of a protein complex. Two-hybrid technology was utilized to determine whether these proteins interact directly. A two-hybrid screen using smg-7 as bait recovered multiple smg-5 clones. To delineate the interaction domains, we undertook a series of two-hybrid tests using a series of smg-5 and smg-7 subclones. SMG-7 requires both TPR domains for an interaction with SMG-5. The amino terminal third of SMG-5 is not required for an interaction with SMG-7. Further mapping of interaction domains is in progress. We have discovered a probable cycle of SMG-2 phosphorylation / dephosphorylation that is important for NMD. In smg-5(-), smg-6(-) or smg-7(-) mutants, a phosphorylated isoform of SMG-2 accumulates to abnormally high levels. This accumulation is dependent on the wild-type function of smg-1, smg-3 and smg-4. For example, a smg-1(-) smg-5(-) double mutant does not accumulate phosphorylated SMG-2. Significantly, smg-1 encodes a probable protein kinase, although we do not yet know whether SMG-1 is the direct kinase of SMG-2. The smg genes may be classified into two groups, those required to phosphorylate SMG-2 and those that ensure no phosphorylated SMG-2 accumulates. We have surveyed smg-2 alleles for those that may be defective in dephosphorylation / phosphorylation. Of seven smg-2(-) alleles that encode full length protein, two accumulate phosphorylated SMG-2. These alleles may encode proteins that are incapable of dephosphorylation. We believe the genetic evidence argues that phosphorylation and dephosphorylation of SMG-2 is central to NMD.

24 May 1999 15:50 405 405 Properties of RNAi inheritance in C. elegans

1999 International Worm Meeting abstract 354 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Properties of RNAi inheritance in C. elegans A Grishok, H Tabara, T Shin, C Mello University of Massachusetts Medical School, Worcester, MA One of the most interesting features of RNAi in C. elegans is its heritability. In most cases interference is inherited by the F1 progeny of the injected animal but in rare cases can also be inherited in subsequent generations and possibly indefinitely. The fact that RNAi is heritable gives rise to numerous questions: does injected material persist in some stable form up to the next generation of the injected worm? Is long-term inheritance of RNAi different from the short-term? Does persistence of interference require chromatin modifications at the locus? To address these questions we have undertaken a genetic analysis of the RNAi inheritance process. We first asked if interference can be transmitted in crosses with both the sperm and egg. For several genes tested, injected hermaphrodites, but not injected males were able to transmit interference. Interestingly, however, we found that male progeny of an injected hermaphrodite can readily transmit interference via their sperm. These observations suggest that males are defective for the establishment but not the transmission of heritable interference. The ability to perform genetic crosses with strains bearing RNAi allowed us to ask if the targeted gene is needed for inheritance. In both the F1 and F2 generations we found that interference can be inherited in the absence of the target locus implying an extrachromosomal or at least unlinked mode of inheritance. We also noticed that occurrence of long-term inheritance correlates with the expression of target genes in the germline suggesting possibility of target RNA dependent catalytic amplification of RNAi. Could such a process explain the very long-term inheritance observed in some instances? Alternatively is there a second distinct mechanism such as the mysterious germline silencing effect that is induced or selected to maintain gene silencing in later generations? To address these questions we are now asking if mutants defective in RNAi (see abstract by Tabara et al.,) are also defective in long-term inheritance of the interference process.

24 May 1999 15:50 406 406 Localization pathways of the glutamate-gated ion channels NMR-1 and GBR-2

1999 International Worm Meeting abstract 355 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Localization pathways of the glutamate-gated ion channels NMR-1 and GBR-2 ME Grunwald, C Rongo, JM Kaplan Dept. of Mol. & Cell Biol., Univ. of California, LSA 361, Berkeley, CA 94720-3200 Glutamate plays a major role in the signal transduction of ASH neurons. ASH neurons connect to interneurons involved in the response to nose touch, osmotic shock and volatile repellents (Hart et al., 1995; Maricq et al., 1995). The glutamate receptor GLR-1 has previously been shown to be expressed in synaptic target neurons of ASH and to mediate nose touch sensitivity (Hart et al., 1995; Maricq et al., 1995). We have recently shown that the PDZ protein LIN-10 is responsible for localizing GLR-1 to the synapses between ASH and interneurons (Rongo et al., 1998). The role of LIN-10 is particularly interesting since this protein is also part of a complex of PDZ proteins required for the localization of the epithelial LET-23 receptor (Simske et al., 1996; Kaech et al., 1998). We are now interested whether the localization of other glutamate receptors is similarly controlled. We are focussing on the putative NMDA receptor subunit NMR-1 and glutamate-gated chloride channel subunits GBR-2a and GBR-2b encoded for by avr-14. NMR-1 encodes a protein bearing 33 % identity to the vertebrate NMDA receptor subunit NR1. GBR-2a and GBR-2b encode proteins of 416 and 430 amino acids, respectively; both proteins are homologous to the previously identified a-subunit of a glutamate-gated ion channel which forms ivermectin-sensitive channels (Cully et al., 1994). We have generated translational fusion proteins of NMR-1, GBR-2a and GBR-2b with GFP (expressed using the glr-1 promoter) and examined their subcellular localization in neurons. GBR-2a was restricted to cell bodies whereas GBR-2b was also localized in a punctate pattern in the ventral nerve cord. Current experiments aim to identify which synapses utilize GBR-2b and whether pathways similar to those for GLR-1 are involved.

24 May 1999 15:50 407 407 Simulation of the Embryogenesis of C. elegans in a Computer Model: Necessity of Active Cell Movements to Establish the Premorphogenetic Regions

1999 International Worm Meeting abstract 356 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Simulation of the Embryogenesis of C. elegans in a Computer Model: Necessity of Active Cell Movements to Establish the Premorphogenetic Regions M Gumbel 1 , R Schnabel 2 , HP Meinzer 1

1 Deutsches Krebsforschungszentrum Heidelberg, Germany. 2 Institut für Genetik, Technische Universität, Braunschweig, Germany.

Our aim is to create a tool which enables the modeling of the embryogenesis of C. elegans. Computer simulations should be useful to determine the minimal requirements necessary to arrange cells into the specific regions found in the premorphogenetic embryo. It was the classical view that cells are mainly placed in their final position by mitoses. This view has been challenged by observing the embryogenesis with a 4D-microscope which suggests that active cell migrations contribute to the assortment of cells. To determine the contribution of mitoses to the arrangement of cells we modeled embryos where cells are placed only by mitoses. As blastomeres, up to 250 min. in development, are more or less spherical, they are represented as spheres in the simulation. The model mimics the elasticity of cells by applying repellent forces if cells are pushed together. Cell adhesion is modeled by attractive forces. Upon division cell volumes are attributed to the daughters as measured in embryos. Cell cycle times and the directions of mitoses were calculated from the observation of 3 normal embryos using a 4D-microscope. This data is fed into simulations starting at the 4- or 12-cell stage. None of the simulations produced a pattern similar to that observed in vivo which suggests that mitoses per se do not cause the arrangement of cells in the embryo. A tendency in cells to stay localized at the original position of the founding blastomere was observed. For instance, the ABpla descendants, which normally spread over the entire length of the embryo, stayed at anterior position. No gastrulation occurred in the simulations, thus this process is not driven by the mitotic pattern. A rotation of the embryo similar to that in normal development was sometimes observed. A test with strong cell-cell adhesion improved the establishment of regions by preventing the mixing of cells derived from different blastomeres.

24 May 1999 15:50 408 408 Further studies of programmed germ cell death

1999 International Worm Meeting abstract 357 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Further studies of programmed germ cell death TL Gumienny 1 , E Lambie 2 , MO Hengartner 1

1 Program in Genetics at SUNY Stony Brook and Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA. 2 Dartmouth College, Hanover, NH 03755, USA.

Germ cells in C. elegans can either remain mitotic stem cells or enter meiosis and undergo spermatogenesis or oogenesis. We have previously reported that germ cells can also undergo programmed cell death (PCD). PCD occurs extensively in the germ line as a part of or as a by-product of oogenesis, and requires the known cell death genes. Loss-of-function mutations in the cell death repressor gene ced-9cause excess germ cell death, while mutations in cell killer genes ced-3 and ced-4 prevent germ cell death. The genes involved in engulfment and nuc-1 are also required to remove and degrade germ cell deaths. Known cell-specific cell death regulators (egl-1, ces-1, and ces-2) have no apparent effect on physiological germ cell death (however, see abstract by Gartner et al. for effects of cell death genes on radiation-induced germ cell death). Germ cells function as nurse cells before becoming oocytes. We suggest that the number of nurse cells generated exceeds the number of oocytes that can be accommodated, and that the superfluous nurse cells are removed through PCD. If cell death is blocked (as in ced-3(lf) and ced-4(lf) animals), then the syncytium expands in older animals to accommodate the extra germ nuclei. We have found that germ cell death can occur only after the Ras-mediated commitment to exit pachytene. Mutations in the Ras pathway that block meiotic cell cycle progression also block germ cell death. Pachytene-arrested cells have the capacity to die, but are prevented from doing so through a ced-9-dependent mechanism. This finding suggests that activation of the Ras pathway may allow progression of germ cells from an early, cell-death resistant stage, to a later, cell-death sensitive stage of meiosis. (See abstract by Milstein et al. describing a screen to identify additional factors regulating germ cell death.)

24 May 1999 15:50 409 409 A putative nuclear factor of the inductive signaling pathway specifying vulval cell fates in C. elegans

1999 International Worm Meeting abstract 358 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A putative nuclear factor of the inductive signaling pathway specifying vulval cell fates in C. elegans BP Gupta 1 , J Liu 2 , PW Sternberg 1

1 Division of Biology, California Institute of Technology, Pasadena, CA 91125. 2 Dept of Neurobiology, Stanford Univ School of Medicine, Stanford, CA 94305.

The inductive signaling pathway mediated by LIN-3, an epidermal growth factor homolog, and its receptor LET-23 is known to specify different cell fates during C. elegans development. The best characterized one is the development of vulva in hermaphrodites which involves LET-60 ras, LIN-45 raf and MAP kinase as downstream components. Activation of these factors leads to the expression of nuclear target genes which specify vulval cell fates. The known nuclear factors are LIN-1 (ETS domain protein), LIN-31 (Winged Helix transcription factor), LIN-25 (Novel), SUR-2 (Novel) and LIN-39 (Homeodomain containing protein). We are characterizing the role of a putative nuclear factor, SLI(sy341), in the development of hermaphrodite vulva. sli(sy341) was identified in a genetic screen for the suppressors of lin-3 egg-laying defective phenotype. Genetic epistasis experiments with lin-25, sur-2, lin-1 and lin-31 suggest that it is the most downstream gene. sy341 appears to be a weak gain of function allele since a single copy of the mutation weakly suppresses lin-3 vulvaless defect. Three factor and deficiency mapping experiments suggest that the gene is located between unc-4 and bli-1 on chromosome II. We are in the process of cloning the gene to better understand its function in vulval cell fate specification.

24 May 1999 15:50 410 410 Quantitative Trait Loci for Chemotaxis in C. elegans : Repulsion Mutants

1999 International Worm Meeting abstract 359 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Quantitative Trait Loci for Chemotaxis in C. elegans : Repulsion Mutants MC Gurganus, PC Phillips Department of Biology, University of Texas at Arlington, Arlington, TX 76019 USA Chemotaxis in C. elegans is influenced by numerous genes and complex biochemical pathways. Worms are attracted to low concentrations of benzaldehyde but repulsed by high concentrations. Several attraction mutants have been identified but little work has been done with repulsion mutants. In this study, genetic factors responsible for repulsion to high concentrations of benzaldehyde during chemotaxis have been detected in C. elegans subjected to mutagenesis. Independent repulsion mutant lines were generated by EMS mutagenesis of N2 worms. Offspring of mutagenized worms were assayed for chemotactic response at 2 ml benzaldehyde (a repulsive concentration). Single worms attracted to benzaldehyde at 2 ml were selected, allowed to self, and their offspring were measured for chemotactic response. In 14 of the lines, the mutant phenotype (attraction when there should have been repulsion) seemed hereditary. Repulsion response of the mutant lines differed from control N2 worms by as much as 23 standard deviations. However, the response was quantitative. Mutants were subjected to 10 generations of selfing and selection of single hermaphrodites to eliminate any heterozygosity introduced during mutagenesis while maintaining the mutant phenotype through chemotaxis assays. Repulsion responses of G10 mutant worm lines were compared with that of N2 and initial mutant isolates. Benzaldehyde attraction response at low concentrations was also measured. Standard genetic tools including mapping, complementation testing, and RNAi will be employed to locate and characterize the effects of these QTL. A more detailed characterization of these mutants will help to elucidate the biochemical pathways in chemotaxis and the nature of variation in quantitative traits.

24 May 1999 15:50 411 411 Screening Transacting Factors for RNA-duplex Mediated Temporal Down-regulation of LIN-14 Protein.

1999 International Worm Meeting abstract 360 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Screening Transacting Factors for RNA-duplex Mediated Temporal Down-regulation of LIN-14 Protein. G Ruvkun Dept. of Molecular Biology, Mass. General Hospital, Boston, MA 02114 In C. elegans, the lin-14 protein (LIN-14) temporal gradient specifies stage-specific cell fate during development. This temporal regulation occurs at a post-transcriptional step. We have demonstrated that lin-4/lin-14 RNA duplex formation is essential for the generation of the LIN-14 temporal gradient, and specific bulged C residues of the lin-4 RNA in the duplex are required for down-regulation of LIN-14. We have proposed that transacting factor(s) recognizes the bulged C of the lin-14/lin-4 duplex in vivo and stabilizes the duplex for down-regulation of LIN-14. To identify transacting factors that may recognize bulged C in the RNA duplex we performed two different screens.

In one approach, the lin-14 3’ UTR was fused to a marker gene rol-6mut whose semi-dominant mutation makes worms rolling (Rol). The appendage of the lin-14 3’ UTR to the rol-6mut gene down-regulates ROL-6mut after the first larval stage and allows transgenic worms to move normally (non-Rol). The mutations disrupting down-regulation of ROL-6mut via the lin-14 3’ UTR would suppress the non-Rol phenotype of the transgenic worm and make it Rol. From 20,000 genomes of transgenic worms (non-Rol) we found 43 suppressors (Rol) and we have selected 8 candidates which are in at least two linkage groups for further characterization. None of the suppressors have phenotype as strong as lin-4(null). We are currently mapping and characterizing these suppressors. In the second approach we screened for suppressors of lin-4(ma161), a C to U point mutation of LIN-4(ma161) RNA which results in a bulged U instead of C in the lin-4/lin-14 RNA duplex. We screened more than 200,000 genomes and found 14 suppressors which suppress both vulvaless and long body size of lin-4(ma161). Two of them fail to suppress lin-4(null) suggesting that they may recognize the bulged U of the mutant LIN-4(ma161) RNA in the duplex. Both have been mapped to LG I. We are currently testing the possibility that the suppressors are the heterochronic gene lin-28 (LG I) and lin-41 (LG I) by injecting cosmids of lin-28 and lin-41.

24 May 1999 15:50 412 412 tra-2 regulation in Caenorhabditis remanei, a gonochoristic worm

1999 International Worm Meeting abstract 361 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. tra-2 regulation in Caenorhabditis remanei, a gonochoristic worm ES Haag, JE Kimble HHMI & Univ. of Wisconsin, Madison, WI USA The C. elegans hermaphrodite is essentially identical to females of related gonochoristic species, except for spermatogenesis in its otherwise female body. This androdioecious mode of sexual reproduction is rare in both plants and animals, but has evolved independently from gonochoristic ancestors multiple times in different Rhabditid nematode species. We would like to discover the genetic controls that distinguish sex determination in hermaphrodites and females. To this end we are characterizing tra-2 in the closest gonochoristic relative of C. elegans, C. remanei. As in C. briggsae (Kuwabara and Shah, 1994), a divergent C. remanei tra-2 homologue exists as part of a conserved operon. RNAi demonstrates that the function of Cr-tra-2 in repressing male fates in both germ line and soma is conserved. Given that downregulation of Cr-tra-2 by RNAi results in sperm, we assessed the conservation of two known negative-regulatory sites required for hermaphrodite spermatogenesis in C. elegans. One, the mx region of TRA-2A and TRA-2B (Kuwabara et al., 1998), is as well conserved in C. remanei as in C. briggsae, suggesting that this control is not unique to hermaphroditic species. Another, the TGE element in the 3’ UTR of the tra-2 mRNA (Goodwin et al., 1993; Jan et al., 1997), appeared, by inspection, to be missing in C. remanei. However, RNA gel-shift assays demonstrate that there is a factor in C. remanei that specifically binds both the C. elegans and C. remanei 3’ UTRs. This suggests that TGE-mediated downregulation of tra-2 is also conserved in C. remanei, a hypothesis we are now testing. Several researchers have proposed that the mx and/or TGE controls may be novel features of hermaphroditic species. However, our data suggest that while they are necessary for hermaphrodite spermatogenesis, they are general and do not constitute the actual "switch mechanism" per se. androdioecy: Mating system with hermaphrodite and male sexes. Used in nematodes, shrimp, and a few odd flowers. gonochorism: (a.k.a. dioecy) Mating system with male and female sexes; very popular.

Goodwin, E.B., et al. (1993) Cell 75: 329-339. Jan, E., et al. (1997) EMBO J. 20: 6301-13. Kuwabara, P.E. and Shah, S. (1994) Nucl. Acids Res. 22: 4414-4418. Kuwabara, P.E., et al. (1998). Dev. Biol. 204: 251-262.

24 May 1999 15:50 413 413 Characterization of an SMC-like protein in C. elegans

1999 International Worm Meeting abstract 362 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of an SMC-like protein in C. elegans KA Hagstrom, D Pasqualone, M Albrecht, J Lieb, BJ Meyer University of California at Berkeley, Berkeley, CA 94720 Members of the highly conserved SMC (structural maintenance of chromosomes) protein family are involved in several aspects of chromosome dynamics. From yeast to man, SMC proteins are essential for processes such as mitotic chromosome condensation, sister chromosome cohesion, and recombinational DNA repair. We are investigating the C. elegans homologs of this chromosomal protein family, and have found that they play critical roles in dosage compensation, mitosis, and meiosis. The SMC proteins DPY-27 and MIX-1 are part of the dosage compensation protein complex that binds to hermaphrodite X chromosomes and downregulates their transcription. In addition, MIX-1 associates with all mitotic chromosomes in both sexes and is essential for their proper segregation. A third SMC family member, HIM-1, appears to function in meiosis, perhaps for sister chromatid cohesion (see abstract from A. Chan, D. Pasqualone, T. Wu, and B.J. Meyer.) Characterization of the only other C. elegans SMC homolog, SLP-2 (SMC-like protein 2) is presented here. SLP-2 RNA interference produces dead embryos with abnormal DNA bodies, most likely resulting from defective mitotic chromosome segregation. Transgenic worms carrying extra wild type copies of the SLP-2 gene exhibit lethality, with a high incidence of males among the survivors. This Him phenotype implies that misexpression of SLP-2 also disrupts chromosome segregation during meiosis. Preliminary immunolocalization studies indicate that the SLP-2 protein is not specifically localized to the X, but rather is associated with all chromosomes in both the germline and in embryos. To investigate potential associations between SLP-2 and other SMC proteins, co-immunoprecipitation studies were performed. In embryonic extracts, SLP-2 co-immunoprecipitates with MIX-1, but not with DPY-27. Based on these results, we speculate that while DPY-27 is a partner for MIX-1 within the dosage compensation protein complex, SLP-2 is the partner for MIX-1 in a complex that executes mitotic chromosome segregation. We hope that continued study of the composition and function of SMC protein complexes will provide a greater understanding of their influence on chromosome mechanics.

24 May 1999 15:50 414 414 Regulation of cell-cell associations in the C. elegans male tail hypodermis

1999 International Worm Meeting abstract 363 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Regulation of cell-cell associations in the C. elegans male tail hypodermis AC Hahn, SW Emmons Dept. of Molecular Genetics, Albert Einstein College of Medicine, Bronx NY 10461 The hypodermis of the C. elegans male tail is a dynamic two-dimensional array of cells that gives rise to the sensory rays. Each ray is derived entirely from its respective precursor cell, named Rn (where n corresponds to the number of the ray), which executes a stereotyped sublineage to generate the three cells that comprise each ray. The descendants of a given Rn cell cluster together and separate from descendants of other Rn cells to form a ray at a characteristic position within the hypodermis. Certain mutants fail to maintain this separation, resulting in fusions between rays. By molecular criteria, genes affected by these mutations fall into two classes. The first class includes the Hox genes mab-5 and egl-5, the Pax-6 homolog mab-18 and components of a TGF-b family pathway. These regulatory genes may determine the associative specificity of ray component cells by controlling expression of a second class of effector genes that act directly on adhesion. Potential effector genes include mab-20, a homolog of the secreted axon guidance molecule Semaphorin II (P.J. Roy and J. Culotti, personal communication). We are identifying additional genes that regulate cell-cell associations in this tissue by examining the ray phenotype of mutants in known adhesion molecules, and by further characterization of existing ray fusion mutants. mab-26 has a phenotype very similar to mab-20 and hence may act in the same or in a parallel pathway. We are studying the effects of mutations in regulatory genes on expression of effector genes in order to identify regulatory connections. Finally, we are using mosaic analysis to determine the focus of action of effector genes.

24 May 1999 15:50 415 415 Go May Regulate Behavior by Antagonizing Gq

1999 International Worm Meeting abstract 364 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Go May Regulate Behavior by Antagonizing Gq YM Hajdu-Cronin, WJ Chen, PW Sternberg HHMI and Division of Biology, California Institute of Technology sag-1 and eat-16 (pka sag-2) were isolated as suppressors of the paralysis of syIs17[hsp::goa-1(Q205L)] (1). eat-16 encodes a Regulator of G protein Signaling similar to RGS7, RGS9 and EGL-10. EGL-10, the presumed RGS for Go (2), and EAT-16, which regulates EGL-30 (Gqa), have similar expression patterns (Patikoglou and Koelle, pers. comm.), implying that RGS7 homologs can distinguish between major families of Ga subunits in vivo. sag-1 encodes a diacyl glycerol kinase (aka dgk-1, Nurrish et al, pers. comm.); we found that SAG-1 also negatively regulates Gq. eat-16(sy438); sag-1(sy428) double mutants arrest at L1-L2 stage (99% penetrance), but egl-30(md186) eat-16; sag-1 triple mutants are healthy, suggesting that EAT-16 and SAG-1 synergistically reduce the effect of EGL-30 signaling. To further understand the cause of death and identify more components in the pathway, we are seeking other suppressors of eat-16; sag-1 lethality. Triple mutant construction is underway with some of the "usual suspects," and a suppressor screen is planned. That genes in the Gq pathway were identified in a screen designed to mutate downstream targets of GOA-1 suggests that Go modulates behavior by antagonizing Gq signaling, possibly via EAT-16 or SAG-1. For simplicity, we are focusing on egg-laying behavior in our analysis of this G protein network. Expression studies of goa-1 using reporter constructs, subject to the usual caveats, show Go in the HSN, VC neurons and vulval muscles (1, 3). Because it is unknown if EAT-16 and/or SAG-1 are Go effectors, we want to see if Go, Gq, EAT-16 and SAG-1 are all required in the same cells in the egg-laying apparatus. Therefore we are starting mosaic analysis for rescue of the egg-laying constitutive phenotype of eat-16 and sag-1. Two mutations recovered as double mutants with eat-16 and sag-1 and later isolated, seem to primarily affect locomotion. sy552 animals leave tracks with low amplitude, and sy415 animals leave tracks with high amplitude compared to wild type. We have mapped sy415 to a small interval on LGV and are starting transformation rescue experiments.

1. Mendel et al., Science 267, 1652-1655 (1995) 2. Koelle and Horvitz, Cell 84, 115-125 (1996) 3. Segalat et al., Science 267, 1648-1651 (1995)

24 May 1999 15:50 416 416 ric-3, a gene required for acetylcholine receptor function

1999 International Worm Meeting abstract 365 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. ric-3, a gene required for acetylcholine receptor function S Halevi 1 , J McKay 2 , M Treinin 1

1 Department of Physiology, The Hebrew University Hadassah School of Medicine, Jerusalem, Israel. 2 Dept. of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas TX.

The ric-3 mutation was isolated via two separate screens: for suppressors of the neuronal degeneration caused by the mutated nicotinic acetylcholine receptor (nAChR) subunit deg-3(u662)1 , and in a screen for mutants resistant to the acetylcholinesterase inhibitor aldicarb2 . As the ric-3 mutants do not show reduced levels of acetylcholine and are resistant to the nAChR agonist levamisole, the aldicarb resistance is likely to originate with a defect in postsynaptic transmission. The specificity of the ric-3 mutation to cholinergic signalling is indicated by study of the slow pharyngeal pumping observed in ric-3 mutants. Electropharyngeograms (EPGs) of ric-3 mutants show that while the glutamergic M3 spikes are unaffected, the cholinergic MC spike is absent. The defect in the MC spike is more severe in the ric-3 mutants than in mutants lacking the pharyngeal nAChR subunits EAT-2 or EAT-18, suggesting that ric-3 may affect the muscarinic AChR as well. The association of ric-3 with the function of such heterogenous acetylcholine receptors as the levamisole receptor, the pharyngeal nAChRs, and DEG-3, which is evolutionarily ancient, suggests that ric-3 does not code for an nAChR subunit. We are in the process of cloning ric-3 via transposon tagging and cosmid transformation rescue, having been fortunate in receiving a set of ric-3 alleles, including transposon insertion alleles, from James Rand. Ric-3 maps between unc-8 and unc-24 on chromosome IV. We think it of great interest to discover how RIC-3 acts as a general modulator of acetylcholine receptor function. RIC-3 may be involved in receptor synthesis, targetting or clustering, or may be a factor which alters regulation of the channel via postranslational modifications.

1.Treinin, M. and M. Chalfie (1995). Neuron 14, 871-877. 2. Miller, K. G. et al. (1996). Proc. Natl. Acad. Sci. USA 93, 12593-12598.

24 May 1999 15:50 417 417 Ultrastructural Features of the Adult Hermaphrodite Gonad: Relations Between the Germ Line and Soma

1999 International Worm Meeting abstract 366 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Ultrastructural Features of the Adult Hermaphrodite Gonad: Relations Between the Germ Line and Soma DH Hall 1 , VP Winfrey 2 , G Blaeuer 2 , LH Hoffman 2 , T Furuta 2 , KL Rose 2 , O Hobert 3 , D Greenstein 2

1 Center for C. elegans Anatomy, Albert Einstein College of Medicine, Bronx, NY. 2 Vanderbilt University School of Medicine, Nashville, TN. 3 College of Physicans and Surgeons, Columbia Univ., New York, NY.

New views of the adult hermaphrodite gonad using multiple EM and fluorescent microscopic techniques have uncovered several dynamic structures that are likely to underlie soma-germline signaling and the reproductive functions of the adult gonad. SEM has been helpful in judging the shape of the distal tip cell and the extent of its tentacle-like processes. In SEM, the distal edge of sheath cell 1 displays finger-like filapodia extending into the bare region, often intercalated between the germ cells, at their outer edge. Distal sheath filapodia can be followed in serial thin sections and can also be visualized by confocal microscopy, using a LIM-7::GFP reporter construct. We hypothesize that the filapodia aid the extension and spreading of the distal sheath over new germ cells in the distal arm. Without such action, the distal border of sheath cell 1 might gradually drift proximally, leaving more germ cells exposed. SEM, TEM, and freeze fracture indicate that the proximal sheath cells are fenestrated with multiple small pores forming conduits from the gonadal basal lamina to the surface of the oocyte. In most instances where pores occur, extracellular yolk particles penetrate the gonadal basal lamina to directly touch the underlying oocytes. The primary route of yolk transport apparently proceeds from the intestine into the pseudocoelom, then through sheath pores to the surface of the oocyte, where endocytosis occurs. Both sheath/oocyte and sheath/sheath gap junctions were observed in the proximal gonad. Actin/myosin bundles are anchored to the proximal sheath cell plasma membrane at plaque-like structures termed hemi-adherens junctions, which in turn are closely associated with the gonadal basal lamina. Gap junctions and hemi-adherens junctions are likely to function in the coordinated series of contractions required to ovulate the mature oocyte.

24 May 1999 15:50 418 418 ram mutations in C. elegans cause hypertrophy of structural and hypodermal cells in the male sensory rays

1999 International Worm Meeting abstract 367 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. ram mutations in C. elegans cause hypertrophy of structural and hypodermal cells in the male sensory rays DH Hall 1 , CQ Nguyen 1,2 , KL Chow 2

1 Center for Caenorhabditis elegans Anatomy, Department of Neuroscience, Albert Einstein College of Medicine, Pelham Parkway, Bronx, NY 10461, USA. 2 Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.

In C. elegans, morphogenesis of ray cells after their cell fate commitment concludes the process of sensory ray differentiation. The process takes place within two hours with dramatic cell migrations and specific cell-cell associations. Mutant screens uncovered at least ten different genetic loci involved in this tail retraction event. The Ram phenotype presented by these mutations is having broadened stubby sensory rays observable by light microscopy. We have examined the cellular defects in a few representative mutant strains by electron microscopy. Defects were highly variable even among individuals of the same strain. In general, it was found that ray neurons were normal with proper dendritic differentiation and opening to the exterior. On the other hand, abnormalities could be observed in the structural cells, hypodermal tissue or both. The structural cell process was often bloated or vacuolated at the distal end of a ray, where it wraps around the dendritic endings. Large spaces sometimes occurred between structural and hypodermal cells, as if proper adhesion failed. The hypodermis itself was often highly disorganized, vacuolated with large membranous whorls and subjected to hypertrophy within the fan, with random incidence of trapping multiple nuclei in the fan and rays. Its border with the fan cuticle was sometimes highly irregular. Acellular material, probably remains left behind from the retracted hypodermis, was often detected in the fan forming "brushes" and gave the fan a rough appearance under light microscopy. We have utilized a ram-5-GFP marker transgene to label the structural cells in all the ram mutants for examining the ray assembly defect. The preliminary results were in line with that of the EM analyses. This reporter will be instrumental to analyzing abnormal features of these mutants. By defining the foci of gene action, it will help in our mechanistic dissection of the role of ram gene products in shaping the sensory rays.

24 May 1999 15:50 419 419 Bridging the GAP: New Insights into SYD-1 Structure and Function

1999 International Worm Meeting abstract 368 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Bridging the GAP: New Insights into SYD-1 Structure and Function SJ Hallam, Y Jin Dept. Biol., University of California, Santa Cruz CA 95064 We previously reported the isolation and cloning of syd-1 (1). Type D GABAergic neurons in syd-1 loss-of-function mutants form abnormal synaptic outputs. In wild-type L1s, DD motoneurons innervate ventral body wall muscles. In late L1, DDs remodel their presynaptic zones, forming synapses with dorsal body wall muscles and eliminating synapses with ventral body wall muscles. During this process, newly born VD motoneurons form synapses with ventral body wall muscles. Prior to remodeling, DDs of syd-1 mutants incorrectly innervate both ventral and dorsal body wall muscles. In later larval and adult stages syd-1 mutants exhibit a three-fold reduction in VD synaptic outputs, although dorsal synaptic outputs arising from the remodeled DDs appear normal. To determine whether syd-1 might play a temporal role in the remodeling process we examined syd-1(ju2); lin-6(e1466) and syd-1(ju2); unc-55(e402) strains in which VDs are either absent (lin-6) or mis-specified (unc-55). We found that DDs in these double mutant strains correctly remodeled their remaining ventral synaptic outputs in late L1. To further examine the role of syd-1 in synapse formation we drove expression of functional SYD-1::GFP in DDs and VDs using the unc-25 promoter. Punc-25 SYD-1::GFP was predominantly localized to ventral processes of DDs and VDs in all larval and adult stages. Taken together, our results suggest that syd-1 may provide positional information specifying the formation of ventral synaptic outputs. syd-1 encodes a protein containing an N-terminal PDZ domain, a Pleckstrin Homology (PH) domain, and a C-terminal GTPase Activating (GAP) domain that is similar to rhoGAP family members. To examine whether the GAP domain of SYD-1 has in vivo function, we generated missense alterations in conserved positions identified in the crystal structure of related rhoGAP to be essential for G-protein binding. We found that these point mutants failed to rescue both L1 and adult pre-synaptic defects. rhoGAP family members have been implicated in regulating actin cytoskeletal dynamics in cultured neurons (2). Our observations suggest that the GAP domain of syd-1 is required for function within DDs and VDs and may provide a link between SYD-1 and the actin cytoskeleton.

(1) Hallam and Jin, WBG14(5): 41 (2) Nobes, C and Halll, A (1995) Cell 81, 53-63

24 May 1999 15:50 420 420 Epitope-based functional analysis on the ryanodine receptor of Caenorhabditis elegans

1999 International Worm Meeting abstract 369 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Epitope-based functional analysis on the ryanodine receptor of Caenorhabditis elegans T Hamada, Y Sakube, H Kagawa Department of Biology, Faculty of Science, Okayama University, Okayama 700-8530, Japan The ryanodine receptor of C. elegans (CeRYR) is encoded by only one gene unc-68/ryr-1 and consists of 5,071 amino acid residues. Structure of RYRs are composed of large N-terminal hydrophilic region, high conserved transmembrane segments and C-terminus. Potential calcium-, ATP-, and calmodulin-binding sites locate in N-terminal region. Mutation sites of isolated unc-68 mutants are as follows; e540 (Stop by frame-shift in exon 22), x14 (Trp153 to Stop) and r1161 (Tc1 induced deletion). unc-68(kh30) isolated as a ketamine response abnormal (Kra) phenotype has the mutation site at Ser1444Asn, which is putative phosphorylation site of PKC. We have reported that the promoter/lacZ fusion plasmids expressed in muscles and non-muscles under the control of 5’ upstream sequences. To know the real expression tissue and functional domains of CeRYR, we raised seven region-specific antibodies against CeRYR fusion peptides. In seven antisera, R21, R16 and R22, which raised against amino acid residues of 945-1315, 1315-1615 and 1874-2164, specifically reacted with CeRYR. It suggests that these regions may be exposed to the cytoplasmic surface. Affinity-purified R16 antibody stained the body wall, pharyngeal and vulval muscles of the wild-type and kh30, but not those of unc-68 null mutants. R16 staining was also observed in muscle cells of the wild-type during muscle development. Double staining with anti-paramyosin antibody or Rhodamine-phalloidin indicated that CeRYR was localized in thin filament containing regions (I-bands) in body wall muscles. Radio-labeled calcium overlay assay confirmed that calcium bound strongly to the fusion protein of two EF-hand motifs and weakly to two additional regions of residues 1874-2164 and C-terminus. Interestingly the fusion protein of two EF-hand motifs shows a calcium-dependent motility shift on SDS-PAGE analysis. This result means that two EF-hand motifs may play a role for calcium-induced calcium release with conformational changes.

24 May 1999 15:50 421 421 Characterization of temperature-sensitive embryonic-lethal mutants with early cell cycle defects

1999 International Worm Meeting abstract 370 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of temperature-sensitive embryonic-lethal mutants with early cell cycle defects DR Hamill, T Kurz, BA Bowerman Institute of Molecular Biology, University of Oregon, Eugene, OR 97403 We are conducting a screen for temperature-sensitive embryonic-lethal mutants, focusing our analysis on early cell cycle mutants. The phenotypes of the mutants can be subdivided roughly into the following categories: 1) one-cell arrest; 2) centrosome function / spindle assembly; 3) spindle orientation; 4) cell cycle timing; and 5) cytokinesis defects. We will present our work on three mutants with phenotypes that fall into three of these categories. (See also the abstracts of Swan and Bowerman, and Encalada and Bowerman for a description of more of these mutants.) The primary defect in or195ts appears to be in spindle assembly, but there are additional defects in cytokinesis and perhaps DNA segregation. During the first cell cycle, the mitotic spindle is short and unorganized, and although a cleavage furrow begins to pinch in, it regresses. Additionally, when membranes reform around the DNA, the newly reassembled nuclei are usually small and supernumerary. In a second mutant, or207ts, embryos have a penetrant defect in cytokinesis. Two processes functionally related to cytokinesis, pseudocleavage and polar body extrusion, appear normal indicating that the defect is highly specific for cytokinesis. Cytokinesis is also defective in approximately 50% of 1-cell stage or198ts embryos. In embryos where the first cell division is successful, nuclei positioning is abnormal at the 2-cell stage, and spindle orientation is often incorrect in either or both the AB and P1 blastomeres. We currently are mapping each of these mutants: or195ts maps just to the left of dpy-5 on LGI, or207ts is at approximately +1.5 mu on LGI, and or198ts maps to approximately -0.7 on LGIII. Our goal is to clone the genes responsible for these mutations and eventually to gain a better understanding of how the cell cycle is regulated.

24 May 1999 15:50 422 422 UNC-70 is ß-Spectrin

1999 International Worm Meeting abstract 371 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. UNC-70 is ß-Spectrin M Hammarlund, EM Jorgensen Dept. of Biology, University of Utah, SLC UT 84112 We cloned unc-70 and found that it encodes the C. elegans homolog of b-spectrin. b-spectrin is an essential component of the membrane skeleton, a protein network that lines the plasma membrane. Animals homozygous for recessive alleles of unc-70 have morphological, developmental and behavioral defects: they are dumpy, slow growing and paralyzed. These multiple defects suggest that the membrane skeleton is required for a variety of cellular processes, as previously suggested by the large number of biochemical interactions in which it participates. We assembled the full-length cDNA sequence of two splice variants of the unc-70 gene that differ only at their extreme N termini. Both variants are SL1-spliced and contain all the canonical b-spectrin domains, which include an N-terminal actin-binding region, a middle region containing 17 spectrin repeats, and a C-terminal PH domain. We found that unc-70::GFP reporter transgenes are expressed in neurons, muscles and the intestine. While all the recessive alleles share the phenotypes described above, we have established an allelic series that suggests some of these alleles retain some b-spectrin function. The most severe alleles are early larval lethal when homozygous, and are completely recessive. Animals homozygous for less severe alleles are viable. These weak alleles are semidominant: heterozygotes are morphologically normal but uncoordinated. Sequence data shows that the severity of the phenotype is inversely correlated with the length of the truncated mutant protein. We are examining these alleles in a smg background to establish how much b-spectrin function is retained by the various truncations. One striking defect in unc-70 mutants is in neural development. We expressed GFP in the GABAergic nervous system of unc-70 animals and assayed neural morphology. We found severe defects in cell migration, fasciculation and axon pathfinding. These defects probably account for the paralyzed phenotype of unc-70 mutants. We are testing whether the requirement for b-spectrin in neuronal development is cell-autonomous. The membrane skeleton may function by creating local microdomains at the plasma membrane. To test this model we will use a GFP fusion to assay the subcellular distribution of b-spectrin, and we will test whether unc-70 mutations affect the localization of various synaptic proteins.

24 May 1999 15:50 423 423 cog-4/egl-26 encodes a novel protein involved in vulva morphogenesis

1999 International Worm Meeting abstract 372 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. cog-4/egl-26 encodes a novel protein involved in vulva morphogenesis W Hanna-Rose, M Han Department of MCD Biology and HHMI, University of CO, Boulder, CO 80309-0347 An important aspect of vulva morphogenesis involves forming a proper connection with the uterine lumen. In screens for mutants defective in vulva morphogenesis, we have isolated multiple mutants in which the uterus and the vulva fail to make a proper connection, resulting in an Egl- phenotype. Two alleles with this connection of gonad defective (Cog) phenotype define the gene cog-4. In animals carrying lesions in the cog-4 gene, a thick layer of tissue appears at the apex of the vulva where the thin process formed by the uterine seam cell is normally visible. This tissue appears to be of vulval origin, and it blocks the entrance to the vulva from the uterus. The vulval lineages in cog-4 mutant animals are normal. Uterine seam cell fate also appears normal as assayed by the expected fusion of the anchor cell and the uterine seam cell. However, during the early part of the fourth larval stage, vulval morphology becomes abnormal when some vulval cells become disorganized and take up aberrant positions at the apex of the vulva. We have cloned cog-4 using microinjection transformation rescue. cog-4 is predicted to encode a novel protein of 317 amino acids. COG-4 displays significant similarity to a protein encoded by a C. briggsae EST, but no other homologies have been identified. Our cog-4alleles are allelic to two previously identified mutations in egl-26. The four cog-4/egl-26 alleles carry missense mutations in the second or fourth exons of the gene. A functional translational fusion of COG-4 to GFP is expressed transiently in the anchor cell cytoplasm during the third larval stage. More interestingly, COG-4::GFP expression is later visible around the edges of the uterine and vulval lumens. Expression is also observed in areas corresponding to the lumens of the pharynx, the excretory duct, and the rectum. Mosaic analysis is ongoing to determine the site of action of cog-4. We have already determined that cog-4 activity is not required in the P1 lineage, which gives rise to the uterus, in order to specify a proper vulval-uterine connection, and further details of the mosaic analysis will be described at the meeting.

24 May 1999 15:50 424 424 Genes involved in axonal branching of an amphid neuron

1999 International Worm Meeting abstract 373 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genes involved in axonal branching of an amphid neuron JC Hao, M Tessier-Lavigne, CI Bargmann Department of Anatomy and HHMI, UCSF, San Francisco, CA 94143-0452, USA In both the developing and mature nervous system, neurons can innervate multiple targets by sprouting secondary axon collaterals, or branches, from a primary axon shaft. Although positive and negative regulators of primary axonal growth cone guidance have been identified, little is known about the molecular mechanisms mediating axonal branching morphogenesis. In order to elucidate genes that may control axonal branching, we are performing a screen to identify mutants defective in either branch formation or elongation by using a reporter expressed in ADL, a chemosensory neuron. Unlike other amphid neurons, whose axons enter the nerve ring via the amphidial commissures, the axon of ADL projects into the nerve ring laterally, where it then branches into both a dorsal and a ventral process. These processes may represent one primary axon and one branch, or alternatively, the axon may bifurcate once it enters the nerve ring, giving rise to both processes. As a first step, we have examined the effects of known axon guidance genes on ADL axonal morphology. One molecule of particular interest is Slit, a secreted factor that has been shown to promote axon collateral formation and elongation of vertebrate sensory axons in vitro (K. Wang et al., Cell, in press). Slit may be a ligand for the SAX-3/Robo receptor, and sax-3 mutants exhibit a variety of axonal branching morphologies, ranging from loss of both branches to multiple ectopic branches. Since sax-3 deranges the nerve ring, at this point we cannot distinguish whether these represent primary branching defects, or secondary consequences of disruptions caused by sax-3 guidance defects. We are currently using reverse genetic techniques to obtain a slit loss of function mutant in C. elegans. Interestingly, the unc-6/unc-40 pathway may mediate ADL ventral branch formation. In unc-6 or unc-40 mutants, about 70% of ADL axons have an unbranched axon that extends dorsally in the nerve ring. This may be a primary branching defect since most other aspects of ADL guidance are normal. These results propose a role for the unc-6/unc-40 and slit/sax-3 pathways as regulators of ADL axonal branching.

24 May 1999 15:50 425 425 in vitro analysis of C. elegans synaptobrevin in the synaptic vesicles docking to the plasma membrane

1999 International Worm Meeting abstract 374 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. in vitro analysis of C. elegans synaptobrevin in the synaptic vesicles docking to the plasma membrane S Harada 1 , T Sassa 2 , R Hosono 2

1 Center for Biomedical Research and Education, Faculty of Medicine, Kanazawa University, Kanazawa 920-8640 JAPAN. 2 Department of Physical Information, Faculty of Medicine, Kanazawa University, Kanazawa 920-8640 JAPAN.

Synaptobrevin and syntaxin are crucial components of exocytosis in neurons. Despite evidence linking syntaxin, SNAP-25 and synaptobrevin to exocytosis, it is unclear how these proteins are involved in the sequence of events leading to vesicle docking and membrane fusion. To further clarify the role of synaptobrevin, we cloned synaptobrevin cDNA into the pQE vector that includes 6xHis-tag coding sequence. Our study focused on the interactions among synaptobrevin, UNC-18 and Cesyntaxin. We previously showed that UNC-18 binds to syntaxin with high affinity. We examined the binding ability of synaptobrevin to UNC-18 and syntaxin. In addition, we cloned cDNA encoding C. elegans homolog of yeast SEC10 (CeSec10) and SEC15 (CeSec15) which interacts with SEC1, a yeast homolog of UNC-18. The gene encoding CeSec10, sec10-1 consists of 4 exons, whose open reading frame consists of 1980 bp. The 659 amino asid sequence identity is about 30% to yeast Sec10p and about 40% to human Sec10p. The length of CeSec15 cDNA is a 4kb and exhibits 30% identity to rat Sec15p in amino acid level. We are interested in investigating the association of UNC-18 with CeSec10 and CeSec15.

24 May 1999 15:50 426 426 Role of the Caenorhabditis elegans homologs of cdk5 and p35

1999 International Worm Meeting abstract 375 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Role of the Caenorhabditis elegans homologs of cdk5 and p35 TE Harbaugh, G Garriga University of California, Berkeley While cdk5 was originally identified in vertebrates based on its homology to the cell cycle regulated cdc2, further study has demonstrated a function not in the cell cycle but instead in post-mitotic neurons. Mouse knockouts of cdk5 and its activator, p35, result in defects in the pattern of neuronal migration in the cortex (1,2). Studies in cultured rat neurons demonstrated that the cdk5/p35 kinase activity can promote neurite outgrowth (3). At this point, however, little is known about the upstream activators and downstream targets of the cdk5/p35 kinase. The Caenorhabditis elegans homolog of cdk5 is 74% identical to mouse, and the p35 homolog contains a 159 aa region that is 54% identical. Translational GFP reporter constructs for cdk5 and p35 are expressed in the cytoplasm of most neurons beginning around the two-fold stage of embryogenesis and continuing into adulthood. To produce a loss of function phenotype, we injected dsRNA for either cdk5, p35, or both into N2 worms. In each case, most of the progeny had no gross visible phenotype, although a few Dpy Unc worms were produced. To identify loss of function mutations in cdk5 and p35, we will also screen a deletion library by PCR. To generate an overexpression phenotype, DNA containing the cdk5 or p35 genomic region was injected into N2 worms to produce extrachromosomal arrays. Worms carrying a cdk5 array (40 µg/ml) have no visible phenotype, and worms carrying a p35 array (40 µg/ml) are occasionally slightly uncoordinated (Unc). Worms carrying an array with both cdk5 and p35, however, are strongly Unc. These worms have defects in fasciculation and axon pathfinding. Along with the expression pattern, this strongly suggests a role for cdk5 and p35 in neuronal development. We will screen for suppressors of this Unc phenotype to identify other genes that are acting in the cdk5/p35 pathway.

(1) Gilmore EC, Ohshima T, Goffinet AM, Kulkarni AB, Herrup K, J Neurosci, 18:6370-7 (1998) (2) Chae T, Kwon YT, Bronson R, Dikkes P, Li E, Tsai LH, Neuron, 18:29-42 (1997) (3) Nikolic M, Dudek H, Kwon YT, Ramos YF, Tsai LH, Genes Dev, 10:816-25 (1996)

24 May 1999 15:50 427 427 Genome project Data in the EMBL Nucleotide Sequence Database.

1999 International Worm Meeting abstract 376 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genome project Data in the EMBL Nucleotide Sequence Database. DT Harbison, P Sterk, G Stoesser European Bioinformatics Institute EMBL Outstation The EMBL Nucleotide Sequence Database is produced maintained and distributed at the European Bioinformatics Institute (EBI) in collaboration with NCBI (USA) and DDBJ (Japan). Nucleotide sequence data are collected mainly through direct submission by researchers and genome sequencing groups. To facilitate the submission and processing of the dramatically increasing volume of genome project data, the EBI has developed two submission mechanisms: 1. Submission to genome project accounts. Genome sequencing centers submit formatted sequence data to their accounts. Automated daily processing ensures rapid inclusion of new and updated sequence records into the EMBL database. 2. Automated inclusion of unfinished sequence (HTG). To make ’unfinished’ genomic sequence data rapidly available to the scientific community, We scan a number of FTP servers (e.g. Sanger Centre FTP server)on a daily basis and automatically generate and update database records when new sequence data in FastA format is encountered. Genome sequencing centers that wish to make use of these submission protocols should contact the EBI. The email address for enquiries is [email protected] The EBI maintains a genome monitoring table which presents weekly updated progress statistics for the major genome projects, such as the C.elegans project and can be accessed via http://www.ebi.ac.uk/~sterk/genome-MOT. In an effort to make complete genome and contig information available, we have set up an experimental ftp site (ftp.ebi.ac.uk/pub/databases/embl/genomes). In collaboration with GenBank and DDBJ, we plan to include such data in a new database division (CON) in 1999.

24 May 1999 15:50 428 428 C. elegans Centaurins: Components of a PI 3-Kinase Signalling pathway?

1999 International Worm Meeting abstract 377 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. C. elegans Centaurins: Components of a PI 3-Kinase Signalling pathway? LS Harrington, HA Baylis, TR Jackson Laboratory of Molecular Signalling, Department of Zoology, Downing Street, Cambridge, CB2 3EJ, UK. The gene age-1 encodes a homologue of the mammalian phosphoinositide 3-OH kinase (PI 3-kinase) catalytic subunit. AGE-1 functions on the same pathway as DAF-2, an insulin-like receptor homologue, regulating lifespan and dauer formation. Signal transduction through an analagous pathway in mammals via the insulin receptor may involve members of the centaurin protein family. Centaurins are characterised by containing a PH domain, an Arf-GAP homology domain including a potential zinc finger and a number of ankyrin repeats. C. elegans centaurin homologues were detected using BLAST searches against the genomic database with the protein sequences of human centaurins. The results indicated the existence of one potential centaurin b homologue and one potential centaurin g homologue. These were subsequently cloned from C. elegans cDNA libraries. Two splice variants of the C. elegans centaurin b homologue (cnt-1), located on chromosome II, were identified and are both trans-spliced to SL1. The predicted proteins are 826 and 742 amino acids long and are 33% and 30% identical at the amino acid level to human centaurin b2 respectively. The C. elegans centaurin g homologue (cnt-2), located on chromosome III, has at least 3 splice variants. Two full length splice variants identified so far are predicted to encode proteins of 951 and 1107 amino acids with 33% and 30% identity to human centaurin g at the amino acid level. All 4 predicted C. elegans centaurins contain the defining features of a PH domain, Arf-GAP homology domain and ankyrin repeats. Centaurin a, the founding member of this protein family has been shown to bind phosphatidylinositol 3,4,5-trisphosphate (PIP3 ) with high affinity and specificity. Human centaurin a and b1 translocate to the plasma membrane in response to growth-factor stimulated PIP3 production. A GFP-CNT-1A fusion protein shows similar behaviour in cultured mammalian cells. I am now seeking to investigate the role that this PI 3-kinase-PIP3 -centaurin signalling pathway may play in C. elegans.

24 May 1999 15:50 429 429 Genetic interaction of the VAB-1 receptor tyrosine kinase with the PTP-1 receptor tyrosine phosphatase

1999 International Worm Meeting abstract 378 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic interaction of the VAB-1 receptor tyrosine kinase with the PTP-1 receptor tyrosine phosphatase RJ Harrington 1 , MJ Gutch 2 , MO Hengartner 2 , NK Tonks 2 , I Grinberg 1 , AD Chisholm 1

1 Dept. of Biology, University of California, Santa Cruz CA 95064. 2 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724.

Mutations in the C. elegans Eph receptor tyrosine kinase, VAB-1, and the ephrin, VAB-2, cause defects in neural and epidermal morphogenesis. Mutations in the VAB-1 kinase domain do not cause complete loss of function, suggesting that vab-1 may have kinase dependent and kinase independent functions (George et al., Cell 92: 633). vab-1 null mutations cause incompletely penetrant defects, suggesting that VAB-1 signaling might be redundant with parallel pathways. To identify components of such parallel pathways we investigated whether vab-1 mutations displayed synthetic lethality with other morphogenetic mutants. A mutation in a C. elegans receptor tyrosine phosphatase, ptp-1(op147), was isolated by M. Gutch, M. Hengartner, and N. Tonks, and was reported to cause mild morphogenetic defects similar to those of weak vab-1 alleles. We have found thatptp-1(op147) strongly synergizes with mutations affecting the VAB-1 kinase domain but not with mutations affecting the VAB-1 extracellular domain or with mutations in the VAB-2 ephrin. These data suggest that PTP-1 and the VAB-1 kinase activity function in parallel redundant pathways to regulate morphogenesis. We are characterizing ptp-1 genetically and molecularly to understand the basis for this synergistic interaction with VAB-1. PTP-1 is most similar to LAR-like receptor protein tyrosine phosphatases, characterized by globular domains and multiple fibronectin type III repeats in the extracellular domain and two tyrosine phosphatase domains in the cytoplasmic domain. We have found that the ptp-1 locus encodes two to four different transcripts. Studies are in progress to define the ptp-1 gene structure, determine the ptp-1 expression pattern, and characterize the ptp-1; vab-1 interaction.

24 May 1999 15:50 430 430 Mutations in unc-26, the C. elegans synaptojanin homolog, disrupt synaptic vesicle recycling and result in defects in cytoskeletal organization.

1999 International Worm Meeting abstract 379 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Mutations in unc-26, the C. elegans synaptojanin homolog, disrupt synaptic vesicle recycling and result in defects in cytoskeletal organization. TW Harris 1 , E Hartwieg 2 , HR Horvitz 2 , EM Jorgensen 1

1 Department of Biology, University of Utah, Salt Lake City, UT 84112 USA. 2 Department of Biology, MIT, Cambridge, MA 02139 USA.

Synaptojanin is a phosphatidylinositol-4 and 5-phosphatase found in synaptic regions. Because synaptojanin binds to proteins required for endocytosis, it has been proposed to function in synaptic vesicle recycling. The C. elegans synaptic function mutant, unc-26, encodes a homolog of the vertebrate synaptojanin protein. We have tested the hypothesis that synaptojanin functions in synaptic vesicle recycling by characterizing the synaptic ultrastructure of unc-26 mutants. First, mutants show a significant depletion in the number of synaptic vesicles at the synapse, indicating a disruption in the recovery of vesicles from the plasma membrane. Second, intermediate endocytic structures accumulate in the plasma membrane near synapses. Third, coated vesicles accumulate in the cytoplasm near synapses, indicating that shedding of the clathrin coat following fission of the vesicle from the membrane requires synaptojanin activity. Finally, endosomal compartments accumulate and enlarge, implicating a role for synaptojanin in the budding of vesicles from the endosome. We conclude from these results that synaptojanin facilitates multiple steps in synaptic vesicle endocytosis, specifically in the endocytosis of plasma membrane and the uncoating of vesicles after fission. We also find pleiotropic defects in the organization of the cytoskeleton and in vesicle transport from the cell body and from Golgi stacks. These defects are not readily reconciled with our current understanding of synaptojanin function, but suggest that the phospholipid composition of membranes plays a critical role in the regulation of these processes and structures.

24 May 1999 15:50 431 431 Oxygen Mutagenesis in C. elegans

1999 International Worm Meeting abstract 380 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Oxygen Mutagenesis in C. elegans PS Hartman 1 , B Ponder 1 , H Kasai 2 , N Ishii 3

1 Biol. Dept., TCU, Fort Worth, TX 76129. 2 Inst. Occup.& Environ. Health, Japan. 3 Tokai School of Medicine, Isehara, Kanagawa 259-11, Japan.

Oxygen-induced mutation frequencies in C. elegans were determined using the fem-3 gene as a target. Rare loss-of-function mutations can be detected in fem-3 by virtue of their ability to relieve the temperature-sensitive sterility imposed by a gain-of-function mutation. Frequencies were measured in wild-type, rad-8 and mev-1 genetic backgrounds. As expected, increases in oxygen (up to 90%) resulted in increased recovery of mutations in the wild-type background. Mutation frequencies were significantly elevated (ca. 5-10X) by the mev-1 mutation at all oxygen concentrations tested (from 10-50%). Conversely, the mutation frequencies were unaffected by inclusion of the rad-8 mutation. There are two reasons why a mutation in mev-1 could confer hypermutability to oxygen. First, since mev-1 encodes a subunit of the mitochondrial enzyme succinate dehydrogenase [Ishii et al.1998. Nature 394,694). mutation likely resulted in excess superoxide anion production in the mitochondria. This, in turn, could have easily translated into more oxidative damage to the nucleus. Second, SOD levels are reduced in mev-1 animals, making them less able to eliminate that superoxide anions that are generated. The presence of 8-oxoguanine has been measured in wild-type, rad-8 and mev-1 animals. This DNA damage is generated at high frequencies by oxidative damage and is considered to be its primary mutagenic lesion [Grollman and Moriya, 1993, Trends Genet. 9, 246]. It was somewhat surprising that 8-oxoguanine levels were experimentally identical in the three strains when reared under atmospheric oxygen. It is possible that significantly more oxidative damage was induced in mev-1 animals but not reflected in steady-state levels, which were measured. For example, senescent human fibroblasts excised almost four times more 8-oxoguanine than did young cells yet accumulated only 35% more damage (Chen et al., 1995. Proc. Natl. Acad. Sci 92, 4337). This represents the first example in which a mitochondrial defect that alters free radical production impacts mutation frequency. It therefore provides strong support for the theory that free radicals generated in the mitochondria can cause DNA damage in the nucleus, which can lead to aging.

24 May 1999 15:50 432 432 Developmental switches in the life-cycle of the parasitic nematode Strongyloides ratti.

1999 International Worm Meeting abstract 381 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Developmental switches in the life-cycle of the parasitic nematode Strongyloides ratti. SC Harvey 1 , ME Viney 2

1 Division of Biological Sciences, University of Edinburgh, Edinburgh, EH9 3JT, UK. 2 School of Biological Sciences, University of Bristol, Bristol, BS8 1UG, UK.

In common with many other parasitic nematodes, the life-cycle of Strongyloides ratti is complex. There are two distinct adult generations: one parasitic and female only and the one free-living and dioecius. The progeny of the parasitic females develop into three morphs: infective third stage larvae (iL3s), free-living adult males and free-living adult females. The proportion of larvae that develop into each morph is affected by host immune status and by environmental conditions outside the host. Here we use genetical and parasitological analyses to show that in the life-cycle of S. ratti there are two discrete developmental switches. The first is chromosomal sex determination occurring inside the host. The second is a form of phenotypic plasticity that affects only the genetically female progeny of the parasitic females. These can develop into two morphs: free-living adult females or iL3s. These findings clarify the developmental basis of the complex life-cycle of S. ratti and show how such life-cycles can result from simple, binary switches. Comparison of this life-cycle with that of C. elegans indicates how a number of simple changes can produce marked changes in a life-cycle.

24 May 1999 15:50 433 433 Role of cysteine protease(s) in C. elegans embryogenesis/development

1999 International Worm Meeting abstract 382 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Role of cysteine protease(s) in C. elegans embryogenesis/development S Hashmi 1 , S Lustigman 2

1 Laboratory of Molecular Parasitology, New York Blood Center, 310 East 67 th Street, New York, NY 10021-6204. 2 Lindsley F. Kimball Res. Inst., New York Blood Center, NY 10021.

The nematode, Onchocerca volvulus is one of the leading sources of infectious blindness and severe chronic dermatitis in human. The worm is transmitted by bites of Simulium black flies. As in other species of nematodes O. volvulus grows and develops through four molts. In our previous studies we have shown that cysteine protease(s) (cp) and their endogenous inhibitor, onchocystatin are probably involved in the process of larval molting and in the development of the microfilariae (L1) from egg. Homologs of O. volvulus cysteine protease cathepsin L-like and cathepsin Z-like, were identified in the C. elegans cosmids T03E6 and F32B5 respectively, and named the corresponding genes cpl (cathepsin L-like) and cpz (cathepsin Z-like). Study of the functional activities of these gene products in C. elegans could lead to a better understanding of the function of cysteine protease genes in other nematodes as well. In an initial attempt to study the role of cysteine protease in the development of C. elegans we used an RNA interference approach. dsRNA prepared from yk146d10 cDNA clone (obtained from Y Kohara) corresponding to T03E6.7 gene (cathepsin L) was injected in the wild type N2 hermaphrodites gonad and assayed the phenotype. The observed phenotype was surprising. The injected animals survived 5 days after injection but at the same time produced very few eggs (<5). We could classify the phenotype lethal for egg production. Although this data is preliminary it proposes that cathepsin L-like cysteine protease (cpl) has a role in the formation of the embryos. We are in the process of analyzing RNAi for cathepsin Z, as well as studying the expression patterns of cpl:: gfp and cpz :: gfp fusion constructs during development of all stages. We hope to be able to distinguish roles of both enzymes in C. elegans.

24 May 1999 15:50 434 434 Does the leucine zipper protein ZIP-1 function as an activator of gene expression in pharyngeal muscle?

1999 International Worm Meeting abstract 383 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Does the leucine zipper protein ZIP-1 function as an activator of gene expression in pharyngeal muscle? C Haun, JD Thatcher, PG Okkema Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL Expression of the pharyngeal muscle-specific myosin gene myo-2 is regulated by cell type-specific signals that target a site in the myo-2 enhancer called the B subelement. This regulatory sequence can enhance reporter gene expression specifically in the pharyngeal muscles. We have previously shown that activity of the B subelement requires binding of the NK-2 family homeodomain factor CEH-22, and that CEH-22 is a key regulator of B subelement cell type specificity. However, mutational analyses of the B subelement indicate additional factors are likely to be necessary for its enhancer function. To identify these factors we have continued our screen of a C. elegans cDNA expression library for proteins binding the B subelement and have identified one new factor that binds a site distinct from CEH-22. This factor, which we call ZIP-1, shares weak similarity to basic-leucine zipper family transcription factors. zip-1 maps on chromosome III near pie-1, and its genomic structure predicted by Genefinder is designated Y75B8A.35. We are currently examining the expression pattern and function of zip-1 during C. elegans development. zip-1 mRNA is detectable by Northern blot in all C. elegans developmental stages. To determine the activity pattern of the zip-1 promoter, we have examined expression of a zip-1::lacZ fusion in transgenic C. elegans. zip-1::lacZ is expressed strongly in the pharynx and a limited set of non-pharyngeal tissues, which we have provisionally identified as the seam cells, vulval muscles, and a number of cells near the rectum. We are now generating antibodies against ZIP-1 to verify this expression pattern and to examine the subcellular localization of ZIP-1. As a preliminary characterization of its function we have attempted RNAi with zip-1; however we have not yet observed an informative phenotype. We are planning to continue our analysis of zip-1 by isolating a mutant as well as by examining the effect of ectopic zip-1 expression.

24 May 1999 15:50 435 435 exc-7 Encodes a Homologue to the RNA-Processing Protein ELAV

1999 International Worm Meeting abstract 384 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. exc-7 Encodes a Homologue to the RNA-Processing Protein ELAV D Hawkinson, KV King, M Buechner Univ. Kansas, Dept. Molecular Biosciences, Lawrence, KS, 66045 Mutations in nine exc genes, and in let-4, let-653, and sma-1, cause the excretory canals to swell into fluid-filled cysts of varying size and placement. The exc-7 allele rh252 characteristically exhibits smaller septate cysts along the canal terminating in a bolus of cysts. The length of these mutant canals varies from about 1/3 to 1/2 the length of wild-type canals. Mutant worms also often exhibit a slightly deformed tail whip. We previously mapped the exc-7 gene between the left ends of mnDf58 and mnDf59 on LGII, an area spanned by 13 cosmids. Both canal and tail whip phenotypes were rescued by microinjection of cosmid E02H1, in which GeneFinder predicts 10 genes. The position was narrowed by microinjection of cosmid restriction fragments, and located by microinjection of a PCR product containing the promoter and coding regions of the predicted gene F35H8.5. F35H8.5 encodes a homologue of the paraneoplastic encephalomyelitis HuD antigen. This protein and its homologues have been characterized in Drosophila, Xenopus, and mammals as RNA-binding proteins containing multiple 80-amino-acid RNA recognition motifs. The predicted EXC-7 protein is most closely homologous to Drosophila Elav14, which is expressed early in neuronal cell development. Although the excretory cell acts as the C. elegans osmoregulatory organ, it has many neuronal characteristics: It is an ectodermally-derived cell whose sister is a neuron; in addition, extension of canal processes requires neural guidance cues such as UNC-6, UNC-53, and UNC-73. Our previous electron micrographs (with Ed Hedgecock and Dave Hall) of canals in exc-7 mutants show abnormal concentrations of endosomes at the tips of the cells, which could reflect abnormal accumulations of incorrectly processed RNA. Surprisingly, although the phenotype of ELAV mutants in other creatures shows strong neural defects, our RNAi results, as well as the phenotype of the rh252 allele placed over a deficiency, suggest that the null phenotype shows strong effects in the excretory canal and the tail whip, with negligible effects on neuronal processes. We are currently making GFP constructs to identify the location of EXC-7 expression, and are sequencing the rh252 mutation in order to understand the molecular basis of the Exc-7 phenotype.

24 May 1999 15:50 436 436 DIE-1, an apparent zinc-finger protein, is required for intercalation and actin organization in the dorsal hypodermis

1999 International Worm Meeting abstract 385 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. DIE-1, an apparent zinc-finger protein, is required for intercalation and actin organization in the dorsal hypodermis PJ Heid 1 , WB Raich 1 , SB Gendreau 2 , JH Rothman 2 , JD Hardin 1

1 University of Wisconsin, Madison, WI 53706. 2 University of California at Santa Barbara, Santa Barbara, CA 93106.

Cell rearrangements are ubiquitous to all organisms and are often vital for normal morphogenesis. The dorsal hypodermal cells of C.elegans undergo cell rearrangements referred to as dorsal intercalation. The physical movements of these cells have been described and are essentially invariant in wild-type animals; however, little is known about the molecular control of intercalation. It is also unclear what impact intercalation has on the subsequent morphogenesis of the organism. To address the mechanism by which hypodermal cells rearrange as well as the role of intercalation in C.elegans development we have undertaken analysis of a recessive zygotic lethal mutant which fails to undergo normal intercalation called die-1 (dorsal intercalation and elongation defective). In addition to defective intercalation, die-1(w34) mutants fail to elongate and display muscle organization and pharynx attachment defects. w34 was mapped to LGII and cloned; the smallest rescuing fragment encodes a C2H2 zinc finger protein. Phenotypes of embryos obtained from germ line mosaic animals or by RNA interference, as well as the DNA sequence of the lesion in die-1(w34) suggest that w34 is a null allele and that there is no maternal requirement for DIE-1. Confocal imaging of embryos expressing DIE-1::GFP or stained with DIE-1 antibody show that DIE-1 is present in nuclei of dorsal cells prior to and during intercalation, in ventral cells during enclosure, in body muscle cells, and in pharynx cells during attachment. Imaging of phalloidin stained embryos indicates that actin filaments do not organize properly in the dorsal hypodermis of die-1 mutants. Preliminary mosaic analysis suggests that individual dorsal hypodermal cells must express die-1 in order for intercalation to occur normally. We propose that die-1 encodes a transcription factor that acts as a general regulator of morphogenesis by activating expression of factors required for cell movement. In the dorsal hypodermis, we propose that die-1 is required for intercalation; in the absence of intercalation actin cannot organize properly, which disrupts the subsequent process of elongation.

24 May 1999 15:50 437 437 Forkhead genes in C.elegans

1999 International Worm Meeting abstract 386 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Forkhead genes in C.elegans M Hellqvist Greberg, D Baillie IMBB, Simon Fraser University, Burnaby, B.C, V5A 1S6, Canada Forkhead genes encode transcription factors characterized by a highly conserved region of 100 amino acids necessary for DNA binding. Members of the forkhead gene family have been identified in eukaryotic organisms ranging from yeast to mammals. They have proved to be an important gene family involved in development and tumorigenesis. We want to study this gene family in the nematode c.elegans and compare our results to the human genes. We have identified at least 15 members of the forkhead gene family in the recently finished sequence of C.elegans. 4 of them have known mutants, daf-16, pha-4, pes-1 and lin-31. By doing comparisons between the C.elegans genes and known members of the forkhead gene family we have identified two genes that has possible human orthologs. Another gene show high homology to a known Drosophila gene, Slp-2, which is involved in early development. The Bailllielab have recently performed an essential gene analysis in a region on chromosome three defined by the duplication sDp3. One forkhead gene is located in this area. We have requested knockouts for these four from the C.elegans knockout facility at the University of B.C. RNAi projects have been started for the four genes. To determine when the genes are expressed we have started to clone the promoter regions of these genes into GFP fusion vectors. A long-term goal is to knockout all the genes in the gene family and do double knockout. We also want to find interacting proteins to understand how extracellular signal can reach the nucleus and influence transcription. The double knockouts are very useful because it has been shown in mouse that the different members can sometimes compensate for each other and thereby giving rise to no phenotype. Because the worm is a simpler organism it is also possible that the worm has just one gene when mammals have two or more.

24 May 1999 15:50 438 438 Initiation of epithelial polarity during C. elegans intestinal morphogenesis

1999 International Worm Meeting abstract 387 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Initiation of epithelial polarity during C. elegans intestinal morphogenesis GJ Hermann, BH Leung, JR Priess Fred Hutchinson Cancer Research Center, Seattle, WA 98109 and Howard Hughes Medical Institute Model systems such as yeast have provided a great deal of insight into how polarity develops in single cells. However, comparatively little is known about how cellular polarity is coordinated during embryonic development to organize large numbers of cells into the various organs of the body. Morphogenesis of the C. elegans intestine provides an excellent system to study the acquisition of cell polarity. The mature intestine is a bilaterally symmetric tube comprised of polarized epithelial cells whose surfaces are subdivided into apical and basal-lateral domains. All of the cells that form the intestine derive as a clone from the embryonic precursor called E. E undergoes a series of anterior/posterior, and one left/right, cell divisions to produce two groups of precursors; one on the left side and one on the right side of the embryo. These epithelial precursors polarize so that their apical surface is positioned at the region of contact between the left and right groups of cells. The first sign of intestinal cell polarity visible in the light microscope occurs when nuclei within the intestinal precursors become asymmetrically positioned adjacent to the future apical membrane. In order to identify the cues that initiate polarization of intestinal cells we have performed an ultrastructural characterization of this process. We have found that centrosomes migrate to the side of the nucleus facing the future apical membrane at the time of nuclear positioning. Microtubules are found running along the future apical and lateral cell membranes at this time. In preliminary experiments, we have found that asymmetric nuclear positioning is disrupted by microtubule destabilizing drugs. We are currently investigating whether there are mechanistic similarities between nuclear positioning in the post-mitotic intestinal cells and nuclear/spindle positioning in early embryonic blastomeres of C. elegans.

24 May 1999 15:50 439 439 Contributions of degenerative cell death and a WRN-related helicase to C.elegans aging

1999 International Worm Meeting abstract 388 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Contributions of degenerative cell death and a WRN-related helicase to C.elegans aging LA Herndon, M Driscoll Rutgers University Cell death is a highly regulated process that plays important roles in normal development and in response to cellular injury. Whether injury-induced cell death, also known as necrosis, contributes in a critical way to the aging of an organism is unknown. Since aging worms exhibit many vacuolar structures that look similar to degenerative cell deaths (for example, as induced by mec-4(d) mutations), we wondered about a possible relationship between necrotic-like cell death and aging. We identified genes required for degenerative cell death, called des genes (degeneration suppressors), by screening for mutations that block necrosis. We hypothesized that if these mutations also suppress aging-associated vacuolation and if degenerative cell death plays a critical role in C. elegans aging, the des mutants might have extended lifespans or exhibit increased vitality late in life. We show that animals mutant in one gene, identified by alleles bz29 and bz30, have an extended lifespan. We are characterizing these lines to determine extent of random vacuole formation, general locomotor activity, resistance to heat and oxidative stresses, and interaction with the dauer formation mutants that are known to affect the lifespan of adult animals. In another line of experimentation, we are attempting to identify mutations that may accelerate senescence in C. elegans. Mutations in the human Werner’s syndrome helicase (WRN) and in the related yeast SGS1 helicase produce features of accelerated aging. There are four C. elegans genes that exhibit sequence similarity to the RecQ family of DNA helicases. We have generated a deletion within the C. elegans gene most similar to the Werner’s gene (F18C5.2). Preliminary results indicate that these animals appear to have a normal lifespan and genetic stability. As there may be redundancy in the activity of these helicases, we are currently characterizing animals carrying both the Cewrn deletion and a mutation in another helicase family member, him-61 . We are screening for deletions in the other two C. elegans DNA helicase family members. Establishment of a nematode model for accelerated aging disorders should extend understanding of the action of helicase family members in senescence.

1. Wicky, C., A. Rose and F. Mueller. 1998. European Worm Meeting Abstract.

24 May 1999 15:50 440 440 n3194, identified in a screen for suppressors of ced-9(n1950), may define an ion channel required for cell viability

1999 International Worm Meeting abstract 389 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. n3194, identified in a screen for suppressors of ced-9(n1950), may define an ion channel required for cell viability B Hersh, E Hartwieg, B Horvitz HHMI, Dept. Biology, MIT, Cambridge, MA 02139 To identify genes that may interact with the cell-death protector gene ced-9, we screened for suppressors of the Ced phenotype of ced-9(n1950gf) animals by looking for the reappearance of corpses in ced-9(n1950) embryos. We identified one candidate, n3194, with a large number of refractile bodies in embryos. n3194 maps to LGIII, complements ced-9(lf), and causes a recessive maternal-effect lethal phenotype. A second allele, n3264, was isolated in a screen for engulfment-defective mutants (see abstract by Z. Zhou) and does not cause maternal-effect lethality. A third allele, zu223, was isolated in an unrelated screen for maternal-effect lethals (J. Priess, personal communication). Neither the accumulation of refractile bodies nor the lethality of n3194 is suppressed by loss-of-function mutations in ced-3 or ced-4. The extra dying cells are acridine-orange positive but not TUNEL-positive, distinguishing them from normal programmed cell deaths. We examined the morphology of the refractile bodies in n3194 mutants using electron microscopy and determined that the bodies resemble degenerative deaths rather than apoptotic deaths. n3194 resides in the gene R13A5.1, which encodes a protein highly similar to the products of human and mouse ESTs of unknown function with weak similarity to K+ and Ca2+ channels. We identified nonsense mutations in n3194 and zu223 and a missense mutation altering a conserved residue in n3264. An R13A5.1-GFP fusion capable of rescuing n3194 is expressed on the plasma membrane of all cells in early embryos. Later the GFP marker becomes restricted to the excretory canals and several neurons in the head. These neurons have cell bodies directly posterior to the anterior bulb of the pharynx and extend processes into the nose. R13A5.1 appears to be required for cell viability during embryogenesis. Its observed localization in the excretory canal and its homology to ion channels suggest a role in maintaining ionic homeostasis.

24 May 1999 15:50 441 441 The control of cell polarity and cell migration in C. elegans by WNT signaling and a protein similar to a metastasis-associated factor

1999 International Worm Meeting abstract 390 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The control of cell polarity and cell migration in C. elegans by WNT signaling and a protein similar to a metastasis-associated factor SM Hettenbach, MA Herman Division of Biology, Kansas State University, Manhattan, KS 66506 In C. elegans, the anteroposterior polarities of a pair of cells in the tail, called TL and TR, are controlled by a WNT signal encoded by lin-44. Mutations in lin-44 cause the reversal of T cell polarity. Mutations in lin-17, which encodes a Frizzled-like protein, cause the loss of T cell polarity, suggesting that LIN-17 is a receptor of the LIN-44 WNT signal. Mutations in egl-27 also cause the loss of T cell polarity. In addition, the descendants of the migratory QL neuroblast migrate incorrectly in egl-27 mutants (Herman et al., 1999). Components of the conserved Wnt pathway also control the migration of the QL descendants by regulating the expression the Hox gene mab-5. Furthermore, certain egl-27 mutants and egl-27 RNAi animals display embryonic patterning defects. The predicted EGL-27 protein contains a region of similarity to MTA1, a factor overexpressed in metastatic cells. Further analysis of the EGL-27 sequence has revealed several regions of similarity to proteins involved in chromatin remodeling (Solari, et al., 1999). MTA1 has been shown to be a subunit of a novel complex displaying ATP-dependent chromatin-remodeling and histone deacetylase activities. Specifically, MTA1 appears to function as a transcriptional repressor (Xue, et al., 1998). Overlaps in the phenotypes of egl-27 and Wnt pathway mutants suggest that EGL-27 interacts with Wnt signaling pathways in C. elegans. Similarities to a component of a chromatin-remodeling and histone deacetylase complex suggest that EGL-27 may function, in part, to modify chromatin structure to control gene expression in response to Wnt signaling pathways. One result of Wnt signaling is the accumulation of b-catenin in the nucleus, where it interacts with TCF/LEF-1 to regulate transcription of target genes. The level of POP-1, a C. elegans homolog of TCF/LEF-1, is higher in the anterior daughters of many anteroposterior asymmetric cell divisions (Lin et al., 1998). We found that the level of POP-1 is also higher in the anterior T cell daughters in wild-type animals, but is higher in the posterior T cell daughters in lin-44 animals, suggesting that the LIN-44 WNT signal controls cell polarity by affecting the distribution of POP-1.

24 May 1999 15:50 442 442 POP-1 expression in mirror symmetric lineages.

1999 International Worm Meeting abstract 391 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. POP-1 expression in mirror symmetric lineages. RJ Hill 1 , R Lin 2 , JR Priess 1

1 HHMI and FHCRC, Seattle, WA USA. 2 Dept. of Mol. Biology and Oncology, U. T. Southwestern Medical Center, Dallas TX USA.

The vast majority of anterior/posterior divisions in the early C. elegans embryo are polarized, resulting in an anterior sister with a high level of the transcription factor POP-1 in the nucleus and a posterior sister with a low level of POP-1 in the nucleus. This consistent pattern of POP-1 expression by sister cells suggests that precursor cells have a defined polarity that is uniformly aligned with respect to the anterior/posterior body axis. We have previously reported that differences in POP-1 activity between embryonic sister cells direct sister cells to become different cell types. The level of POP-1 is also unequal between anterior/posterior cells born during larval development. For example, after the division of a seam cell, the anterior sister has a higher level of POP-1 than the posterior sister. One exception to the general POP-1 expression pattern occurs in organs such as the vulva that have mirror symmetry along the anterior/posterior axis. The vulva is formed from three precursor cells called P5.p, P6.p and P7.p. The cell lineages of these precursors have mirror symmetry along the anterior/posterior axis with the point of symmetry being the center of the P6.p lineage. We have found that POP-1 expression follows the mirror symmetry in the vulval lineages. Among the descendants of P5.p, anterior sisters have a higher level of POP-1 than their posterior sisters. In contrast, the descendants of P7.p express POP-1 in the reversed pattern such that anterior sisters have a lower level of POP-1 than their posterior sisters. We are investigating the genetic basis for this naturally occurring reversal in the POP-1 expression pattern. Mutations in the gene lin-17, a component of the Wnt signaling pathway, are known to disrupt the polarity of the cell lineage of P7.p. We have found that lin-17 mutations disrupt the expression of POP-1 in the descendants of P7.p We propose that Wnt signals act to reverse the polarity of POP-1 expression in a subset of vulval lineages.

24 May 1999 15:50 443 443 Avoidance of water soluble repellents

1999 International Worm Meeting abstract 392 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Avoidance of water soluble repellents MA Hilliard 1,2 , P Bazzicalupo 1

1 International Institute of Genetics and Biophysics, Naples, Italy. 2 University of Naples, Federico II, Naples, Italy.

The mechanisms underlying water soluble sensitivity (taste) are poorly understood both in vertebrates and in invertebrates. We have developed a new assay to study, in C. elegans, the avoidance of water soluble repellents. This assay, the drop test, consists in delivering a small drop of the chosen solution behind a worm freely moving on the plate surface. By capillarity the solution reaches the anterior end of the animal where the amphids are located. If the solution contains a repellent, the worm immediately stops its forward movement and starts backward. If no repellent is present, the worm continues moving forward. With this test we have identified 16 repellents and the list of them will be presented. We also developed a variation, the dry drop test, in which the drop is delivered just in front of the worm but far enough that the animal reaches the drop only when it dries out. Quinine is one of the substances that induces in the worms a strong avoidance response. Humans perceive it as tremendously bitter; in the channel catfish it induces responses in neurons of the oropharyngeal taste system. In order to discover the genes involved in its sensitivity we used quinine, with the drop test, to screen for mutants. We isolated 15 mutants and two of them gb 404 and gb 408, that do not complement, have been better characterized phenotypically. They show a partial specificity in that they continue to respond to a subset of substances; their amphids cilia appear normal when stained with DiO or FITC. Genetic data locate them on the right arm of the LGIV between dpy-4 and stP35 or immediately past stP35 but very close to it. So far initial rescue attempts have failed. We have tested the existing mutants of the osm series with some of the repellent substances and we found a partial overlap between the drop test and the ring test (barrier test). Finally, we are attempting to identify the cells responsible for the avoidance of different substances in the drop test by genetic manipulations. We are using different sensory neurons specific promoters to drive mec-4(d) as a dominant cell autonomous killer gene (M. Driscoll). We are also trying to rescue the function of specific cells in an osm-6 mutants background (thanks to C. Bargmann for the suggestion and to J.Shaw for osm-6 cDNA).

24 May 1999 15:50 444 444 Systematic RNAi experiments with maternal genes.

1999 International Worm Meeting abstract 393 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Systematic RNAi experiments with maternal genes. K Hirono, S Onami, Y Kohara CREST, JST and Genome Biology Lab., National Institute of Genetics The accumulation of gene expression patterns that are being produced in this lab provides unique sets of limited numbers of genes that possibly participate in specific stages, lineages, tissues and so on. Such sets of genes could be subjected to systematic analysis to elucidate the molecular mechanisms governing the biological processes. Since we are particularly interested in early embryogenesis, we have focused on a set of genes whose mRNA are maternally supplied and disappear before 18cell-stage or localize to specific cells in early embryos. We selected 100 such genes out of 2000 genes based on our in situ hybridization analysis, and have analyzed them with respect to RNAi phenotype as well as protein distribution by immunostaining (see also Onami et al.). We micro-injected double-stranded RNA into the gonads of ten N2 worms per gene. Phenotypic analyses were performed on the injected worms themselves, F1 embryos, larvae and adults that survived (called "escapers") and F2 embryos, with respect to embryogenesis, larval growth, sterility, morphogenesis, the expression of several differentiation markers and so on. Out of the 100 genes, 45% showed F1 embryonic-lethality or the reduction of the number of F1, 34% showed various phenotype with only escapers, 18% showed sterility, and 2% did not show any phenotype. The phenotypes of some gene were consistent with the results of protein distribution. For example, RNAi with the cDNA group CELK02786 caused no or very fragile egg shell, and the antibody against the gene product stained the surface of entire embryo, probably underneath egg shell, suggesting that the gene is involved in the process of egg shell formation. RNAi of 4 out of 11 genes whose immunostaining patterns looked P-lineage specific caused germ line defect. We believe that this approach of the combination of the analysis of expression patterns and RNAi phenotypes provides highly useful information. We are preparing to release these data on our WWW site (http://watson.genes.nig.ac.jp:8080/db/).

24 May 1999 15:50 445 445 Novel functions of the Ras-MAPK signal in neurons of C. elegans

1999 International Worm Meeting abstract 394 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Novel functions of the Ras-MAPK signal in neurons of C. elegans T Hirotsu, S Saeki, Y Iino Molecular Genetics Research Laboratory, The University of Tokyo, Tokyo 113-0033, JAPAN The Ras-MAPK signal transduction pathway plays key roles in vulval induction and several other developmental events in C. elegans. However, the function of the pathway in the nervous system is not known. We will report our results indicating that the pathway functions in neurons involved in olfaction and in locomotion. Chemotaxis to volatile odorants is mediated by two pairs of chemosensory neurons in C. elegans : AWA and AWC. The gain-of-function mutant of let-60ras showed a severe defect in chemotaxis to odorants sensed by AWC. This phenotype was suppressed by loss-of-function mutations in ksr-1, mek-2 or mpk-1, which function downstream of let-60 in vulval induction. let-60(lf) and lin-45(lf) mutants showed moderate but significant defects in chemotaxis to both AWA- and AWC-sensed odorants. The expression of wild type and a mutant form of let-60 by a heat shock promoter revealed that the pathway functions in mature neurons and is not involved in neural development. The cell-specific expression of let-60 using the gcy-10 promoter, which drives expression in AWC, AWB and I1, altered response to isoamylalcohol(which is sensed by AWC), but not to diacetyl(AWA). Thus the proper functioning of the Ras-mediated signaling pathway is probably required in the mature AWC neurons. We analyzed the expression of let-60 using a reporter construct employing GFP, and observed its expression in AWC, consistently with the above observations. Loss-of-function mutants affected in this pathway also exhibited another interesting phenotype. A large fraction (25%-60%) of the sem-5(lf), let-60(lf) and lin-45(lf) mutants showed unidirectional turns resulting in circular locomotion on bacteria-free plates. The phenotype of let-60(lf) was rescued by the induced expression of let-60(+) during embryogenesis, or by the neuron-specific expression using the H20 promoter. These results suggest that the circular locomotion is due to developmental defects of nervous systems.

24 May 1999 15:50 446 446 Ventral enclosure in C. elegans: getting to the molecules

1999 International Worm Meeting abstract 395 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Ventral enclosure in C. elegans: getting to the molecules JS Simske, JD Hirsch, DT Locke, RA Smith, JD Hardin University of Wisconsin. Madison, WI 53706 We are interested in the molecules that regulate the behavior of epithelial cells as they migrate, change shape and adhesiveness, and ultimately generate three-dimensional shape during tissue morphogenesis. We have conducted a general screen for enclosure defective mutants. Enclosure involves the wrapping of the six rows of epidermal, or hypodermal cells (two each of dorsal, seam and ventral hypodermis) around the embryo and the subsequent formation of adhesive junctions along the ventral midline. Since enclosure takes place midway through embryogenesis, we reasoned that the genes required for enclosure would be zygotically expressed; therefore we concentrated on identifying zygotic lethal mutants (Z) in the progeny of mutagenized animals. The screen was carried out in the strain lin-7(e1449)II; jcIs1(jam-1::GFP)IV so that cellular junctions could be visualized in arresting or arrested embryos. Since we were also able to score for arrested embryos in the F3, this screen allowed the recovery of maternal effect mutations (M) or "incompletely penetrant homozygous viable mutations (H)" (like Vab, but with a required embryonic phenotype). We screened through approximately 9,000 haploid genomes and have recovered 16 alleles (2 H, 4 Z, 9M and one semi-dominant allele). Ten are mapped to the following chromosomes: LG I (1 allele), II (1),IV(4) and V(4). So far only one complementation group has multiple alleles (jc2 (H) and jc3 (Z)). The screen is far from saturation. We have identified several representative phenotypes while screening. The most severe phenotype is complete failure of enclosure and full retraction of the hypodermis back to its original dorsal position. Less severe are partial enclosures, during which anterior or posterior regions may remain unenclosed, resulting in the extrusion of embryonic contents through head and tail ruptures respectively. Finally, embryos may enclose completely, but irregularly, resulting in body shape defects during elongation. Mutants typically display a range of all three arrest phenotypes but often one arrest phenotype is most common. We are close to cloning jc1 (LGIV) jc11 (LGII) and jc2/jc3 (LGIV) and will report on their molecular identities.

24 May 1999 15:50 447 447 Effect of overexpressing mab-21, isolation and identification of mab-21 interacting cellular components in C. elegans by epitope tagging

1999 International Worm Meeting abstract 396 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Effect of overexpressing mab-21, isolation and identification of mab-21 interacting cellular components in C. elegans by epitope tagging YM Wong, KL Chow Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong In C. elegans male tail, the peripheral sensory structures develop during the late larval stages. They comprise of nine pairs of sensory rays embedded in a acuticular fan. These rays play a crucial role in the mating behavior of male nematodes. Each ray has its own specific morphological and functional identities. A set of genes has been defined to regulate ray differentiation. Among them mab-21 regulates ray 6 formation. Since its isolation, mab-21’s cellular role is by large unknown. To establish the role of mab-21 in normal development, we have overexpressed mab-21 cDNA with a variety of promoters. The results showed that mab-21 expression could perturb the identity determination of a number of rays. Most dramatically, tail spike could be induced in male animals. The relationship of the tail spike phenotype and mab-21 expression in the tail hyp cells will be examined. These dominant phenotypes once established in stable transgenic lines will be useful for screening of suppresser mutations to identify the regulatory targets of mab-21. To further establish mab-21’s role in the signaling process, attempts were made to study the signaling molecules interacting with MAB-21. While multiple attempts to generate antibody against MAB-21 did not yield good reagent with high specificity, we have switched to protein tagging. Expression vector with fusion proteins gene expressing MAB-21 tagged with glutathione S-transferase (GST) or consecutive histidine residues (His tag) were constructed. Bacterial expressed protein was purified by affinity chromatography specific for the tags. Using these protein reagents, isolation of other signaling molecules involved in the ray formation process by immunoprecipitation or affinity chromatography is underway. Similar tagged constructs in worm expression vector have been made and will also be introduced into animals for parallel in vivo interaction study. These transgenic animals will be useful for subcellular localization of the mab-21 gene product, and to identify interacting components in worms.

24 May 1999 15:50 448 448 ceh-10 regulates ttx-3: An initiation-maintenance switch paradigm

1999 International Worm Meeting abstract 397 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. ceh-10 regulates ttx-3: An initiation-maintenance switch paradigm O Hobert 1 , Z Altun-Gultekin 1 , G Ruvkun 2

1 Columbia University, College of Physicians & Surgeons, New York, NY10032. 2 Mass.General Hospital, Boston, MA 02114.

It seems intuitive that cell fate specification should be subdivided into an initiation step and a subsequent maintenance step which ensures the preservation of specific differentiation features. This simple model has indeed been proven true for many developmental processes. The differentiation of the AIY interneuron appears to follow this rule as well. We define a genetic cascade of two transcription factors that are required in consecutive steps to initiate and then maintain AIY cell fate. ceh-10 is a homeobox gene that is expressed during early proliferative embryonic stages in a few head neurons, including AIY (Svedsen and McGhee, 1995). ttx-3 encodes a LIM homeobox gene that is expressed in AIY and also in a few other neurons (Hobert et al., 1997; this poster). We define a 243 bp intronic fragment of the ttx-3 gene that is sufficient to drive GFP expression in AIY; its expression is then maintained throughout adulthood. Given that ttx-3 is potentially involved in synpatic maturation of AIY (see abstract by Hall et al.), the ttx-3::GFP reporter gene fragment can be considered to be a marker for the terminal differentiation of AIY. We find that ceh-10 is both required and sufficient for ttx-3::GFP expression in AIY. In ceh-10 null mutants, a neuron that is in the correct position for AIY is formed (Forrester et al., 1998), yet ttx-3::GFP expression is not initiated. On the other hand, ectopic expression of ceh-10 using the heat shock promoter induces ectopic expression of ttx-3::GFP reporter gene constructs. Notably, ceh-10 expression disappears after hatching , yet ttx-3 expression is maintained. This process appears to rely on an autoregulatory mechanism since in ttx-3 null mutant animals, ttx-3::GFP expression is initiated but not as strongly maintained past the embryonic stage. Taken together these data suggest that ceh-10 is required to initiate the AIY differentiation program by initiating the expression of certain AIY differentiation marker(s). After ceh-10 has performed this initial task, the protein disappears, yet one of the proteins that is induced by CEH-10, namely TTX-3, now take over the job of maintaining its own expression and presumably that of other AIY cell fate markers as well.

24 May 1999 15:50 449 449 ttx-3 affects neurite branching and neural connectivity

1999 International Worm Meeting abstract 398 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. ttx-3 affects neurite branching and neural connectivity O Hobert 1 , G Ruvkun 2 , DH Hall 3

1 Dept. of Biochemistry, Columbia University, New York, NY 10032. 2 Mass.General Hospital, Boston, MA 02114. 3 Center for C. elegans Anatomy, AECOM, Bronx, NY 10461.

ttx-3 is a LIM homeobox gene that is required for AIY interneuron function in the thermoregulatory circuit (Mori et al., 1995, Hobert et al., 1997). In ttx-3 mutant animals, AIY is generated and several aspects of terminal AIY differentiation are executed, such as expression of FMRFamide or the 7TM gene sra-11. However, the neuron is functionally defective and displays subtle neurite sprouting defects. We have investigated the neuroanatomy in more detail by analyzing the synaptic connectivity in a ttx-3(ks5) null mutant animal by serial section EM reconstruction. The major axon of each AIY resides in the normal neighborhood demonstrating that ttx-3 is not required for the basic features of AIY differentiation or for axonal process outgrowth and placement; however, excess branches were noted at several points, generally at likely turning points along its normal trajectory. This result suggests that the cell may either fail to retract some filapodia when executing a turn or re-initiate sprouting at previous turning points later in development. While other interneurons also adopt normal neighborhoods, some amphid sensory axons are apparently misguided and fail to find the AIY axon within the ventral ganglion neuropil. These defects could either be due to to a defective AIY interneuron or could reflect ttx-3 function in a few additional neurons that we have recently found to express ttx-3. Most strikingly, both AIYs reveal severe synaptic connectivity defects along their main process; although the main axon is in close proximity to many of its normal synaptic connectivity partners, most synaptic contacts to pre- and postsynaptic neurons are absent. These results are consistent with a model in which TTX-3 regulates the expression of cell surface molecules that "tag" AIY to allow its recognition by pre- and postsynaptic partners. Differences between the left and right AIY neural defects in the ttx-3 mutant also implicate an activity dependent model: the AIY which receives some sensory inputs shows fewer errors in downstream synapse formation than the AIY which receives no inputs. Given the small sample size we have analyzed (n=2), further work will be needed to confirm these defects.

24 May 1999 15:50 450 450 The neural function of lim-6, a new LIM homeobox genes, suggests a common theme in LIM homeobox gene function

1999 International Worm Meeting abstract 399 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The neural function of lim-6, a new LIM homeobox genes, suggests a common theme in LIM homeobox gene function Oliver Hobert 1 , Kristin Tessmar 2 , Gary Ruvkun 2

1 Columbia University, College of Physicians & Surgeons, New York, NY 10032. 2 Harvard Medical School, MGH, Boston, MA 02114.

We describe the function of the lim-6 homeobox gene and compare its mutant phenotype to those of other LIM homeobox genes in the worm. lim-6, the worm ortholog of the vertebrate lmx-1a/b genes, is expressed in several head and tail neurons, the excretory gland cell and the developing uterus. Deletion of most of the coding region of lim-6 causes defects in the terminal differentiation of lim-6 expressing cells. In the uterus, a specific type of endothelial cells, the uterine toroid cells display adhesive defects. In the chemosensory neuron ASEL, lim-6 propagates asymmetric information; lim-6, which is expressed in ASEL, but not ASER, is required to determine the asymmetric repress expression of the putative sensory receptor gcy-5 in ASEL. All the neurons that express lim-6 are generated in lim-6 mutant animals and adopt several correct terminal differentiation features; yet several lim-6 expressing GABAergic neurons are functionally defective. Specifically, the ultradian defectation rhythm mediated by the GABAergic AVL and DVB motorneurons is defective and AVL and DVB display axonal defects. We find that expression of unc-25, the gene necessary for GABA synthesis, is partially dependent on lim-6. We show a GABAergic input into the dauer program and suggest that lim-6 is necessary for this input. The comparison of the mutant phenotypes of all the LIM homeobox that we have studied reveals striking similarities. Although the LIM homeobox genes ttx-3, lin-11 and lim-6 are expressed in non-overlapping sets of neurons, the neural phenotypes of loss of each LIM homeobox gene functions reveals a common theme: In each case, the neurons are generated, adopt several terminal differentiation features yet display subtle neurite sprouting defects. This observation suggests that each of the LIM homeobox genes regulates the expression of a similar set of target genes in distinct set of neurons.

24 May 1999 15:50 451 451 A new bacterial pathogen of C.elegans, and the isolation of resistant mutants

1999 International Worm Meeting abstract 400 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A new bacterial pathogen of C.elegans, and the isolation of resistant mutants J Hodgkin MRC-LMB, Hills Road, Cambridge CB2 2QH, England Two spontaneous variant strains of C. elegans, originally isolated on the basis of a Dar (Deformed Anal Region) phenotype, have been found to suffer from a disease rather than a genetic alteration. They are infected by slow-growing gram-positive bacteria, which belong to a species of the large coryneform genus Aureobacterium/Microbacterium. The bacteria adhere to the surface of the rectum and post-anal region of worms, causing a striking localized swelling of the underlying hypodermal tissues. The bacteria do not appear to invade across the cuticle. The swelling leads to interference with defecation, and hence to constipation and slower growth rate in infected worms, but the infection does not usually kill them. Response by the worm has been investigated first by surveying a variety of known mutants for alteration in the infection, either to resistance or to hypersensitivity. Mutants known to be altered in susceptibility to killing by virulent Pseudomonas aeruginosa (Mahajan-Miklos et al., 1999, Cell 96: 47-56) still develop a standard Dar phenotype on infection. Mutations affecting possible components of the Toll pathway were also tested (see abstract by Link et al.), with negative results. Mutants representing about 200 other genes have been tested, and most were found to exhibit a standard Dar response. Mutations in three of eight tested srf genes (affecting surface antigenicity) lead to resistance, with no tail swelling and no reduction in growth rate. The slow growth caused by the bacteria permits an efficient selection for mutants that are resistant to infection. These turn out to be relatively frequent. Of the first 30 resistant mutants to be analysed, a few proved to be new isolates of srf-2, srf-3 or srf-5, but most define new genetic loci, termed bus (for Bacterially Un-Swollen). So far, at least eight distinct bus genes have been defined and genetically mapped. Some probably mediate resistance by altering bacterial adherence to the cuticle, but others may affect different steps in the progress of infection and induced swelling. The processes of infection and resistance provide promising opportunities for the investigation of several areas of C. elegans biology, such as response to infection, host-parasite interaction, cuticle structure and hypodermal morphogenesis.

24 May 1999 15:50 452 452 Genetic mapping and genetic nomenclature: the CGC subcontract

1999 International Worm Meeting abstract 401 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic mapping and genetic nomenclature: the CGC subcontract J Hodgkin 1 , R Durbin 2 , S Martinelli 2

1 MRC-LMB, Hills Road, Cambridge CB2 2QH, England. 2 The Sanger Centre, Hinxton, Cambridge CB10 1SA, England.

Since 1992, genetic mapping and genetic nomenclature for C. elegans have been supervised as a subcontract of the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources. Activities of the subcontract fall under four headings: 1. Genetic Map. Data on the properties and map location of genes defined either by mutation or by sequence analysis are solicited from the C. elegans research community or extracted from publications, and entered into the database ACeDB. Genetic and physical maps are updated periodically, and a printed genetic map booklet is distributed every two years. Possible changes to the presentation of the genetic map, both electronically and as a hard-copy document, will be presented. 2. Genetic Nomenclature. A summary of recommendations is maintained at: http://elegans.swmed.edu/Genome/nomen.html. Recently added recommendations will be listed and explained. 3. Laboratory Designations. Each laboratory engaged in active genetic research on C. elegans is provided with unique strain and allele designators. The number of registered laboratories has grown linearly, with about 25 new P.I.s each year. In contrast, the number of publications on C. elegans has grown exponentially. 4. Gene Naming. The CGC subcontract provides a clearing house for gene names, so that proposed new gene names can be checked for acceptability and added to the gene name registry, when appropriate. It is desirable that new names be registered before publication, in order to avoid duplicated, unnecessary or misleading gene names. So far, fewer than 15% of the 20,000 genes predicted by the genomic sequence have been given standard 3-letter names. Some possible strategies for increasing the consistency and comprehensibility of gene naming, both within C. elegans and between the worm and other popular genetic organisms, will be proposed and discussed. We welcome corrections and suggestions for improvement in the management and presentation of the map and nomenclature.

24 May 1999 15:50 453 453 A CLR example of redundancy: cdh-3 and enc-1 share a redundant function in excretory duct morphogenesis

1999 International Worm Meeting abstract 402 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A CLR example of redundancy: cdh-3 and enc-1 share a redundant function in excretory duct morphogenesis LA Hodgson, J Pettitt Department of Molecular and Cell Biology, University of Aberdeen, Aberdeen, UK. cdh-3 encodes a protocadherin required for morphogenesis of the hyp10 cell. We have extended earlier phenotypic analyses of the cdh-3(pk87) loss-of-function mutation, and have found that it confers a weakly penetrant Clr phenotype. Analysis of the 1-2% of pk87 homozygotes that die with a Clr phenotype reveals that in many of the animals the excretory duct is misshapen and appears refractile, suggesting that defects in the morphogenesis of the duct may be the cause of the Clr phenotype. Indeed, fluid accumulation is first apparent in the region surrounding the duct. A wild-type cdh-3 gene on an integrated array is able to rescue this phenotype, confirming that it is caused by loss of cdh-3 function. The low penetrance of the Clr phenotype suggests that under laboratory conditions other gene(s) are able to compensate for the loss of cdh-3 function, and that these gene(s) share a redundant function with cdh-3. To identify such genes we screened for mutations that enhanced the penetrance of the Clr phenotype. We have screened through 1500 F2 animals and identified a mutation, fe2, that defines a gene which we have termed enc-1 (enhancer of cdh-3). fe2 is a maternal-effect, temperature sensitive mutation. At 20o C, about fifty percent of the progeny of enc-1(fe2) cdh-3(pk87) double mutants arrest with a Clr phenotype. At 25o C, all the progeny arrest with defects in embryonic morphogenesis, with a high proportion displaying enclosure and elongation defects. The presence of a cdh-3 rescuing transgene effectively reduces the penetrance of the Clr phenotype to zero, demonstrating that cdh-3 and enc-1 encode redundant overlapping functions with regard to excretory duct morphogenesis. However, restoring cdh-3 function has no effect upon the phenotype displayed at 25o C, indicating that enc-1 has an additional function that is not compensated by cdh-3. We are currently investigating the cellular defects responsible for the embryonic lethality at 25o C. Preliminary evidence indicates that the hypodermis becomes disorganised during morphogenesis, consistent with the observed enclosure and elongation phenotypes.

24 May 1999 15:50 454 454 Toward Understanding Cell-Type-Specific Cell Death Regulation

1999 International Worm Meeting abstract 403 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Toward Understanding Cell-Type-Specific Cell Death Regulation DJ Hoeppner, MO Hengartner Cold Spring Harbor Laboratory and Program in Genetics SUNY StonyBrook. 1 Bungtown Road, Cold Spring Harbor, NY 11724 Our goal is to understand how different cell types regulate programmed cell death in C. elegans. Toward this end, we are defining the genes necessary for Hermaphrodite-Specific Neuron (HSN) death. The HSNs are a pair of motor-neurons that are required for egg-laying. The HSNs die by programmed cell death in males, presumably in response to masculine signals from the sex determination pathway. The HSNs also die in hermaphrodites carrying gain-of function alleles of the egl-1 gene. Hermaphrodites lacking HSNs are defective in egg-laying (Egl), but are viable and display no pleiotropic defects. Thus, egl-1(gf) provides a good background to identify mutations in genes that are specifically required for HSN death, as restoration of HSN function must be achieved by blocking its death. In a screen for mutants that revert the egg-laying defect of egl-1(n1084), we isolated two mutations, op181 IV and op166 X, that are required for HSN death, but not, as far as we can tell, any other cell death. In addition to suppressing HSN death, op181 and op166 have a common set of pleiotropic defects including uncoordinated movement, lethality with a terminal Clr phenotype, egg-laying defects and male mating defects. We speculate that op181 and op166 are mutations in genes required for complete differentiation in the nervous system.

24 May 1999 15:50 455 455 A functional analysis of the role of UNC-11, a nematode AP180 homolog, in synaptic vesicles endocytosis.

1999 International Worm Meeting abstract 404 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A functional analysis of the role of UNC-11, a nematode AP180 homolog, in synaptic vesicles endocytosis. A Holgado, A Alfonso Department of Biological Sciences, University of Illinois at Chicago. Endocytosis of synaptic vesicle (SV) membranes via clathrin coated pits involves adaptor/assembly proteins such as AP-2 and AP180. unc-11 encodes an AP180 homolog and has at least two functions: 1) specifying the size of SVs during recycling and 2) sorting of the SV protein synaptobrevin (Nonet et al., 1999). In vitro , UNC-11 binds to clathrin and promotes assembly of clathrin coats (Golan, Prasad, Lafer and Alfonso, unpublished). Thus loss of unc-11 function results in both exo and endocytosis defects. In our study we were interested in dissecting the contribution of unc-11 function to endocytosis of SV membranes. We used vital fluorescent probes to learn more about the role of this clathrin adaptor/assembly protein in neuronal SV recycling. An optical FM4-64 assay, originally developed by Betz and Bewick (1992), facilitates the direct visualization of endocytosis by the activity-dependent SV uptake of the lipophilic fluorescent dye. Further stimulation of the synapse results in the activity-dependent destaining of the synaptic terminals, which can be attributable to exocytosis of the dye loaded SVs. To study the role of UNC-11 in SV recycling, we induced vesicular membrane internalization in presence of FM4-64, so only loaded SVs are detectable. Finally, we quantify the signal at the presynaptic terminals after staining and following a depolarization-dependent destaining. We have measured dye uptake in mutants of different unc-11 alleles, mutants defective in endocytosis (dyn-1), exocytosis (unc-18, snb-1), as well as the unc-11 double mutant combinations. Our data from unc-11 mutants suggest that UNC-11 is not essential for endocytosis of synaptic membranes. In unc-11(e47), a null allele, we still see FM4-64 uptake, but there is a significant decrease in this uptake (18% of that of wild type). The decrease could be due to a direct effect in endocytosis, or an indirect defect in endocytosis as a result of a defect in exocytosis. Since we are using experimental conditions in which the ready releasable pool of SVs have been exhausted, exocytosis is not a limitation in the SV loading. And in absence of unc-11 function we still see a decrease in dye uptake, therefore we conclude that UNC-11 must play an important role in endocytosis.

24 May 1999 15:50 456 456 The life-extension, oxidative stress resistance and antioxidant enzyme gene expression

1999 International Worm Meeting abstract 405 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The life-extension, oxidative stress resistance and antioxidant enzyme gene expression Y Honda, S Honda Tokyo Metropolitan Institute of Gerontology The free radical theory of aging is attracting considerable attention. Reactive oxygen species (ROS) are generated during cellular metabolism. These ROS, in turn, cause oxidative damage to DNA, and to proteins. This theory of aging proposes that, although the defense systems that enzymatically detoxify these ROS have evolved, not all ROS are detoxified; oxidative damage caused by the ROS not caught by the defense systems accumulates to cause deleterious consequences in senescence. We have tried to find the genetic pathway(s) which alleviate oxidative stress in the longevity gene network of C. elegans to see if incresed capacity to detoxify ROS is involved in the life extension. We have screened the life-extension (Age) mutants that are associated with the oxidative stress resistance phenotype, referred to as Oxr phenotype. The Oxr phenotype may be determined by pathways involved in the detoxification of ROS. These include superoxide dismutase (SOD) and catalase. SOD is a major enzyme which protects against oxidative stress by catalyzing the removal of O2 -, a central ROS involved in the generation of various toxic ROS. Similar to the Age phenotype, the Oxr phenotype was regulated by the genetic pathway of insulin-like signaling from daf-2 via daf-18 and age-1 to the daf-16 gene. The Oxr phenotype in the daf-2 mutation was enhanced by the mutation in clk-1, although clk-1 mutant did not display the Oxr phenotype. We will demonstrate the pattern of expression of antioxidant defense enzyme genes, and its association with the Oxr phenotype among the Age mutants of C. elegans. The results suggest that the gene network controls longevity by regulating expression of the antioxidant defense system. We have investigated the role of these genes in determining life span of C. elegans.

24 May 1999 15:50 457 457 Isolation and Characterization of Genes Responsible for Ethanol Sensitivity in Caenorhabditis elegans

1999 International Worm Meeting abstract 406 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Isolation and Characterization of Genes Responsible for Ethanol Sensitivity in Caenorhabditis elegans M Hong, J Lee Yonsei University The mechanisms and sites of action of volatile anesthetics and ethanol are not fully understood. In the hope of understanding the mechanisms of general anesthetics, we identified genes that control sensitivity to ethanol and anesthetics in the invertebrate system Caenorhabditis elegans. Because the response of C. elegans to ethanol and anesthetics is similar to that of mammals, elucidating the action mechanism of ethanol and anesthetics in the nematode will help understanding their mechanisms in higher organisms including humans. As a start, we wanted to identify genes responsible for ethanol sensitivity by isolating mutations that confer ethanol resistance. About ten thousand wild-type nematode individuals (N2) were subject to mutagenesis, and the offspring were screened in the second generation for resistance to ethanol exposure. We found 9 independent mutations so far. ys9, the first ethanol-resistant mutant isolated in our screen, was not different from N2 in shape and behavior, but showed resistance to 7 vol% ethanol treatment. ys10, a second mutation, was also wild type in shape and behavior. ys11 animals displayed other phenotypes as well as ethanol resistance. The ys11 animals are medium dumpy (Dpy). In contrast to the results of ethanol test, ys9, ys10, and ys11 were more sensitive than N2 in halothane test. Interestingly, none of the ys9, ys10, and ys11 mutant animals survived after freezing and thawing, indicating that there may be alteration in membrane properties. From the results of thin layer chromatography and gas chromatography, composition of membrane phospholipids and steroids of ys9 was different from that of N2. Other mutations did survive freezing and thawing. We are currently performing genetic and molecular characterization of the genes for those mutations, and will present up-to-date results.

24 May 1999 15:50 458 458 Inositol monophosphatase is required for AWC-mediated chemotaxis to volatile odorants

1999 International Worm Meeting abstract 407 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Inositol monophosphatase is required for AWC-mediated chemotaxis to volatile odorants L Honigberg, M Sutherlin, CD Johnson Axys Pharmaceuticals, NemaPharm Group, South San Francisco CA Inositol signaling has been implicated in olfaction in Drosophila (1) and other species. Among the deletion mutations generated at Axys is imp-1(nr2060), a 3 kb deletion in the C. elegans homolog of inositol monophosphatase. This enzyme is involved in recycling phosphatidylinositol bisphosphate (PIP2 ) breakdown products back into PIP2 . The nr2060 deletion, which removes all imp-1 coding sequences, allowed us to test the role of inositol signaling in C. elegans olfaction. imp-1 mutants had no obvious growth or movement defects, but were almost completely defective for chemotaxis to benzaldehyde and isoamyl alcohol, two odorants detected by the AWC neurons. In contrast, imp-1 mutants chemotax normally to diacetyl, an odorant detected by the AWA neurons. Thus, inositol signaling may specifically be required for AWC neuron development or function. Given that AWC-mediated chemotaxis involves signaling through cGMP (2), the proposed role of imp-1 in AWC function raises the question of how cGMP- and inositol-dependent pathways might interact to transduce odorant signaling.

(1) Riesgo-Escovar et al. (1995) Proc Natl Acad Sci USA 92:2864; (2) Birnby et al. (1995) International Worm Meeting, abstract 65; Coburn and Bargmann (1996) Neuron 17:695; Komatsu et al. (1996) Neuron 17:707; L’Etoile and Bargmann (1997) International Worm Meeting, abstract 335.

24 May 1999 15:50 459 459 A region of the myosin rod critical for interaction with paramyosin

1999 International Worm Meeting abstract 408 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A region of the myosin rod critical for interaction with paramyosin PE Hoppe, RH Waterston Washington University St. Louis, MO 63110 Although models have been proposed for the precise intermolecular interactions that occur in paramyosin assembly and myosin assembly, little is known about myosin-paramyosin contacts. Paramyosin forms the filament core upon which myosin assembles. We have exploited a genetic interaction between myosin heavy chain A (MHC A) and paramyosin to define a region of the MHC rod that is critical for interaction with paramyosin and for filament stability. Animals homozygous for the missense paramyosin allele e73 exhibit highly disorganized muscle that contains brightly birefringent aggregates containing mutant paramyosin. Increased expression of MHC A improves motility and structure in e73 by increasing incorporation of the mutant paramyosin into normal thick filaments (Otsuka, 1986).We have used transgenes to demonstrate that increased MHC B does not suppress e73. Using chimeric MHCs which contain portions of MHC A and B, we have mapped the critical MHC A residues to a 322-aa region at the C-terminus of the rod. e73 suppression and MHC A-specific filament initiation function, which is required for viability, do not co-map, suggesting that suppression occurs during filament elongation rather than initiation. The 322-aa domain corresponds to the predicted region of overlap if MHC and paramyosin assemble in parallel with the same overlap proposed for paramyosin (Gengyo-Ando and Kagawa, 1991). Because activity is lost upon division of this region, the effective intermolecular contacts appear to require the summing of interactions along relatively large domains of the 2 molecules. Chimeric MHCs which contain the N-terminal MHC A rod but have MHC B sequences in the 322-aa active region can rescue mutants lacking either MHC A or B, but cause a dominant synthetic lethality in e73 and other severe paramyosin mutants. Our model is that the MHC A sequences in the N-terminal rod allow the constructs to invade the MHC A-only region. When paramyosin function is compromised, the presence of C-terminal MHC B sequences at the filament center produces an unstable filament. In contrast, the chimera that contains MHC A sequences only in the 322-residue active region also invades the MHC A-only domain, but can support contraction, arguing that this region of MHC A participates in interactions important for thick filament stability.

24 May 1999 15:50 460 460 Negative regulation of egfr signalling in C. elegans.

1999 International Worm Meeting abstract 409 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Negative regulation of egfr signalling in C. elegans. NA Hopper 1,2 , C Chang 2 , GM Lesa 2,3 , PW Sternberg 2

1 MRC-Laboratory of Molecular Biology, Cambridge, CB2 2QH, UK. 2 HHMI/Division of Biology, 156-29, California Institute of Technology, Pasadena, CA91125. 3 current address: ICRF, London, WC2A 3PX, UK.

let-23 (a member of the EGFR family of receptor tyrosine kinases) is required several times during larval development to mediate inductive signalling through activation of the RAS/MAP kinase pathway. In the adult hermaphrodite, LET-23 activity but not RAS activity is also required for the process of ovulation. Genetic screens have identified sli-1 and rok-1 (among others) as negative regulators of LET-23 mediated signalling. sli-1 and rok-1 encode a cbl homologue and a non-receptor tyrosine kinase related to the Ack sub-family, respectively. Mutations in sli-1 are able to strongly suppress a severe hypomorph of let-23 for those functions mediated through RAS, such as vulval induction and viability, but are unable to suppress the fertility defect of let-23 (rf). In contrast, rok-1 weakly suppresses all let-23 phenotypes examined, including sterility. Animals bearing a single mutation in either sli-1 or rok-1 alone are wild-type with respect to vulval induction, whereas doubly mutant animals are hyperinduced. Genetic analyses suggest that rok-1 may be dependent upon sem-5 for its activity, and that sli-1 most likely acts at the receptor/sem-5 step. Transgenic experiments where mutant let-23 is expressed in a let-23(0) background have identified Y1225 of LET-23 as a negative regulator of those functions of LET-23 mediated through RAS activation. Vulval induction is largely wild-type in transgenic animals expressing let-23Y1225F in a let-23(0) background. Interestingly, expression of let-23Y1225F synergises with rok-1, but not sli-1, to cause greater than wild-type vulval induction. An interpretation of this result is that SLI-1 exerts its negative regulation of LET-23 through Y1225. This interpretation is strengthened by the report that Cbl (homologue of sli-1) interacts with a negatively acting tyrosine in ZAP-70. This interaction occurs between a divergent SH2 domain conserved in sli-1 and a phospho-tyrosine of ZAP-70 in a very similar amino acid context as Y1225 of let-23.

24 May 1999 15:50 461 461 The C. elegans Amyloid Protein Precursor-Related Gene Has An Essential Function

1999 International Worm Meeting abstract 410 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The C. elegans Amyloid Protein Precursor-Related Gene Has An Essential Function AM Hornsten Boston University, Department of Biology The early-onset form of Alzheimer’s Disease has been linked to mutations in several genes, including the Amyloid Protein Precursor gene (APP). APP-related genes have been found in humans, mice, and Drosophila. The function(s) of APP and APP-related proteins, however, have not been clearly elucidated. We are studying the function(s) of the APP-related gene, apl-1, in C. elegans. apl-1 encodes a putative single pass transmembrane protein which shares many similarities with the APP family of proteins. Since other members of this family are cleaved to release the extracellular domain from the membrane, it is likely that APL-1 will be processed similarly. apl-1 is expressed in over 50 cells, including neurons, muscles, and supporting cells, in the head region, ventral cord, and tail region. To generate a mutation in the apl-1 gene, we obtained a Tc1 transposon insertion strain (apl-1::Tc1) from Ron Plasterk. Tc1 insertion animals were screened for improper repair of a Tc1 excision. Two deletion alleles, yn10 and yn5, were isolated. yn10 animals contain a deletion that removes most of the apl-1 coding region. Homozygous yn10 animals show a larval lethal phenotype. These data suggest that yn10 represents a complete loss-of-function allele. The yn10 larval lethality is rescued by microinjection of the apl-1 genomic region and by expression of APL-1 in neurons. We will be attempting to rescue the yn10 larval lethality with the human APP gene. We are currently doing an EMS screen to generate other mutations in apl-1 that cause larval lethality. We are also performing genetic mosaic analysis on yn10 animals to determine the specific cells in which apl-1 gene function is necessary. yn5 animals contain a deletion which removes sequences encoding the cytoplasmic and transmembrane regions of the APL-1 protein. yn5 animals show developmental, egg-laying, and motor-related defects. apl-1::Tc1 insertion animals show similar phenotypes, but to a lesser degree. By analyzing the yn5 phenotypes, we have determined that resistance to serotonin-induced egg-laying and a decreased defecation rate are due to a partial loss-of-function and a gain-of-function mutation, respectively. These data suggest that APL-1 acts both as a membrane-bound protein and as a secreted protein.

24 May 1999 15:50 462 462 Central role of UNC-13 and UNC-18 in synaptic vesicle docking

1999 International Worm Meeting abstract 411 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Central role of UNC-13 and UNC-18 in synaptic vesicle docking R Hosono 1 , S Harada 2 , JB Rand 3 , IN Maruyama 4 , T Sassa 1

1 Dept. Physical Information, Kanazawa U., Kanazawa. 2 Center Biomed. Res. Edu., Kanazawa U., Kanazawa. 3 Program Mol.Cell Biol.,Oklahoma U. Oklahoma, OK 92037. 4 Dept Cell. Biol. Scripps Res. Inst. La Jolla, GA 73104.

An essential step for the synaptic transmissions is that synaptic vesicles dock to the presynaptic terminal. The components necessary for the docking including synaptobrevin, syntaxin, synaptotagmin and SNAP-25 are well understood at the molecular level. However, the regulation remains unclear. We studied the contribution of UNC-13 and UNC-18 in the regulation. In an in vitro sedimentation assay, UNC-18 bound to both Ce syntaxin (unc-64 gene product) and the N-terminal region of UNC-13 (UNC-13N) with an ED50 of 0.08 and 0.04 mM, respectively. The mild behavioral mutant md1401 showed the amino acid substitution I 133 V, while the severe mutant b403 had the amino acid substitution E549K. The UNC-18 protein from the md1401 mutant bound normally to both UNC-13 N and Ce syntaxin, whereas UNC-18 from b403 was defective in binding to Ce syntaxin and showed greatly reduced binding ability to UNC-13N. These findings indicate that the C-terminal region of UNC-18 is more important for the binding. The three proteins do not form the ternary complex. Moreover, the binding of UNC-18 to GST-UNC-13N was not interrupted in the presence of Ce syntaxin. However, the amount of UNC-18 that bound to GST-Ce syntaxin promptly decreased during incubation with UNC-13N. UNC-13N preferentially bound to UNC-18 in the UNC-18-Ce syntaxin complex and dissociated UNC-18 from the complex. All unc-18 mutants isolated were defective in the neurotransmitter release. Based on these results, we proposed a model concerning the regulatory role of UNC-13 and UNC-18 for synaptic vesicle docking to the plasma membrane. That is, the binding of UNC-18 probably induces a conformational change in Ce syntaxin which acquires the binding ability to synaptic vesicles following the UNC-13 dependent release of UNC-18 from the complex.

24 May 1999 15:50 463 463 him-10 encodes a novel protein involved chromosome segregation

1999 International Worm Meeting abstract 412 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. him-10 encodes a novel protein involved chromosome segregation M Howe 1 , DG Albertson 2 , BJ Meyer 1

1 HHMI and MCB UCB, CA 94720. 2 Cancer Research Institute, UCSF, CA 94143.

Faithful chromosome segregation through mitosis is essential to all eukaryotic species. However, it is not known whether the mechanisms involved in chromosome segregation are conserved among species. The mitotic chromosomes of C. elegans are holocentric, meaning that the kinetochores are dispersed along the length of the chromosome. Understanding this atypical mitotic chromosome structure may help to identify those features of the segregation apparatus that are strictly conserved through evolution. him-10(e1511ts) was the first gene in C. elegans to be implicated in mitotic chromosome segregation. Initial observations suggested that the mutation causes defects in both germline and somatic mitosis (1,2). Our data suggest that him-10 may be required during sperm meiosis. Specifically, the L4 TSP for sterility can be rescued by mating with N2 males. We cloned him-10 and determined e1511ts to be a Pro to Ser change at a.a. 109. The gene is predicted to encoded a novel protein, and the mutation is likely to cause only a partial loss of function. We conducted dsRNAi experiments to learn more about the null phenotype of him-10. Progeny from injected hermaphrodites die as embryos. This lethality is not likely due to gamete aneuploidy, because in the injected hermaphrodites, oocytes in diakinesis are euploid, and sperm meiosis was complete prior to injection. Instead, the lethality is likely due to severe defects in embryonic mitosis. DAPI and tubulin antibody staining of the embryos revealed displaced metaphase chromosomes, unipolar chromosome attachments, bifurcated spindles, irregularly sized nuclei, and multinucleate cells. The wide array of aberrant structures are all consistent with HIM-10 playing a direct role in chromosome segregation. We determined the subcellular localization of HIM-10 using polyclonal antibodies. HIM-10 associates with the kinetochore face of mitotic chromosomes and encases entire chromosomes at sperm meiosis. We have not detected HIM-10 antibody staining in the mitotic region of gonad. The pattern of localization of HIM-10 and loss of function phenotypes of him-10 are consistent with HIM-10 functioning at the chromosome in spindle assembly.

1. VILLENEUVE, A.M. (1994) WBG 12.4:58 2. HEDGECOCK, E.M. and HERMAN, R.K. (1995) Genetics 141: 989-1006.

24 May 1999 15:50 464 464 Myotactin, a novel adhesion molecule involved in muscle-hypodermal signaling.

1999 International Worm Meeting abstract 413 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Myotactin, a novel adhesion molecule involved in muscle-hypodermal signaling. MC Hresko, LA Schriefer, P Shrimankar, RH Waterston Wash. Univ. Med. Sch., St Louis, MO. 63110 A physical link between the bodywall muscle and the hypodermis is required for locomotion in C. elegans. This linkage is mediated in part by hemidesmosome-like structures, called fibrous organelles (FO; Francis and Waterston, 91), found in regions of the hypodermis adjacent to muscle. We are interested in understanding the signals required to establish and maintain the correct spatial relationship between muscle and the FO. The localization of a protein we have named myotactin suggests it may participate in muscle-hypodermal communication. Myotactin is expressed by dorsal and ventral hypodermal cells and in adults localizes in regions of the hypodermis known to contain high concentrations of FO. However, during part of embryogenesis, the position of myotactin mirrors that of the contractile apparatus in the forming muscle. Myotactin is a novel transmembrane protein with at least 32 fibronectin type III repeats extracellularly and a unique cytoplasmic tail, consistent with a role in muscle-hypodermal linkage. Analysis of let-805 mutants st456 and s2706, which we believe to be myotactin loss-of-function mutants, suggests myotactin is involved in muscle cell adhesion and in maintaining the connection between muscle and FO. Like known muscle (pat class) mutants, the myotactin mutants arrest elongation at the 2-fold stage, display little or no movement, and die before hatching. Further examination of the mutants shows the muscle forms as in wild type, but the muscle cells detach from the hypodermis when contractions begin, consistent with a role in muscle cell adhesion. A second aspect of the myotactin mutant phenotype is delocalization of FO. Initially, the FO appear to form and localize as in wild type. However, after the muscle cells detach, FO are seen all through the dorsal and ventral hypodermis. Confocal and immunoelectron microscopy results demonstrate a close spatial relationship between myotactin and FO. However, a molecular link between myotactin and FO has not been shown, and therefore it’s not clear how myotactin is involved in maintaining the connection between muscle and FO. To investigate in more detail the role myotactin plays in FO development, we are performing a 2-hybrid screen to identify potential myotactin-interacting proteins.

24 May 1999 15:50 465 465 Structure-function analysis of LIN-14

1999 International Worm Meeting abstract 414 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Structure-function analysis of LIN-14 M Hristova, VR Ambros Department of Biological Sciences, Dartmouth College, Hanover, NH 03755 The heterochronic genes control the timing of certain post-embryonic cellular events in the nematode Caenorhabditis elegans. In the lateral hypodermal seam cells, for instance, the heterochronic gene lin-14 regulates the division pattern that each seam cell executes during the first and second larval stages. lin-14 also controls the timing of cell cycle progression for certain cells, including the vulva precursor cells, and the timing of dauer larva arrest. We are interested in determining the molecular mechanisms by which the lin-14 gene product affects cell fates. LIN-14 is a novel nuclear protein expressed late in embryogenesis and during the first and second larval stages in C. elegans. The C-terminus of LIN-14 , fused to GFP, is sufficient to rescue the lin-14 phenotype in seam cells, and associates with mitotic chromosomes in vivo (Hong and Ambros, 1997 IWM abstract # 486). We have found that the same C-terminal fragment of LIN-14, expressed in bacteria, displays nucleic acid binding activity in vitro, supporting the hypothesis that LIN-14 may function through an interaction with DNA or nuclear RNA. Experiments are underway to test whether LIN-14 also associates with chromatin in interphase nuclei, and to identify and characterize complexes of LIN-14 with nucleic acid and/or other nuclear proteins. We are employing immunohistochemistry to test various domains of LIN-14 for their roles in the developmental and cell cycle dynamics of LIN-14 chromatin binding.

24 May 1999 15:50 466 466 Context-dependent gene silencing in the soma of C. elegans

1999 International Worm Meeting abstract 415 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Context-dependent gene silencing in the soma of C. elegans J Hsieh 1,2 , S Kostas 1,2 , A Fire 1

1 Department of Embryology, Carnegie Institution, Baltimore, Maryland 21210. 2 Biology Graduate Program, Johns Hopkins University.

The activity patterns of somatic transgenes provide a means to study gene silencing in C. elegans. Transgene silencing phenomena have been observed in a variety of organisms, and are often associated with repetitive contexts. In both the soma and germline of C. elegans, certain transgenes present in long tandem arrays are silenced or show mosaic expression (i.e. many cells contain the transgene but lack detectable expression). Silencing can be relieved when the constructs are in a non-repetitive context: in F1 assays or imbedded in arrays with excess of genomic carrier DNA (1). In order to identify trans-acting factors that modulate the levels of context-dependent gene silencing, we have carried out a number of genetic screens. In one screen, we looked for a reduction of expression from a uniformly expressing bodywall muscle transgene. Three alleles of a single chromosomal gene were isolated. Mutations in this gene, designated tandem-array-modifier-1 (tam-1) have similar effects on a number of different reporter transgenes with diverse expression patterns. Other tam-1 mutations were picked up independently in a screen by Liu, Chang, and Sternberg (2,3). The TAM-1 protein is nuclear and contains two cysteine-rich domains, a "RING" finger and a "B-box". Although the molecular function of these domains are unknown, roles in context-dependent gene expression have been suggested for several RING-finger proteins. One biological role of context-dependent silencing is suggested by the unexpected observation that tam-1 behaves as a class B SynMuv gene. The inhibition of Ras signalling during vulval induction is controlled by two redundant pathways, the class A and class B SynMuv genes. Further investigating this correlation, we see a silenced-transgene phenotype in non-vulval tissue in several other class B mutants, suggesting that the role of class B genes is more widespread than just in the inhibition of Ras signalling during vulval development. Our present studies are focused on understanding the connection between repetitive contexts and gene silencing and on the role of tam-1 in these processes.

1 Kelly et al Genetics 46: 227,

2 Chang et al 1997 C. elegans Meeting: 619,

3 Liu, J. CalTech Thesis (1998)

24 May 1999 15:50 467 467 Each C. elegans Laminin a Subunit Mediates Distinct Aspects of Morphogenesis

1999 International Worm Meeting abstract 416 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Each C. elegans Laminin a Subunit Mediates Distinct Aspects of Morphogenesis C Huang, PD Yurchenco, WG Wadsworth Pathology Dept., RWJ Med. School, Piscataway NJ Laminin is a heterotrimeric glycoprotein composed of a, b, and g subunits. The genome sequencing project reveals two a, one b and one g laminin subunit genes. Based on sequence analyses, major features and known binding sites are conserved. The predicted a chains, laminin aA and laminin aB, are most similar to the vertebrate a1/a2 and a5 chains respectively, while the b and g resemble the b1/b2 and g1 chains. Thus, there are two predicted laminin trimers, aAbg and aBbg. Mutations in epi-1, which encodes laminin aB, have been isolated and characterized (abstract ECWM 98). They cause defects that are consistent with the developmental roles for basement membranes (BMs). We report the expression patterns of laminin aA and aB. By immunostaining, both first appear at gastrulation between germ layers. However, laminin aA is heavily deposited anteriorly, surrounding pharygeal precursors, whereas laminin aB is more posteriorly localized. Laminin aB becomes localized to the BMs associated with pharynx, intestine, gonad, body wall, body wall muscle, and muscles throughout development. In contrast, laminin aA accumulates in pharyngeal BM, intestinal BM and body wall muscle BM during elongation and its level in intestinal BM and body wall muscle BM gradually decreases. In larvae and adults, laminin aA is only sometimes detected weakly in pharyngeal BM and body wall muscle BM. It is primarily localized in the spermatheca. Injection of dsRNA directed to epi-1 produces phenotypes similar to those observed in epi-1 alleles and the injection of dsRNA directed to lam3, which encodes laminin aB, causes L1 larval arrest with severe pharyngeal defects. Double dsRNA-mediated interference directed to both genes produces embryos that arrest at morphogenesis with phenotypes more severe than those caused by the RNAi of either epi-1 or lam3 alone, indicating that both genes contribute to morphogenesis. Together, our RNAi and immunostaining results indicate that laminin aB has a broad role in maintaining the structural integrity of BMs and for regulating many aspects of morphogenesis, while laminin aA is restricted to specialized membranes and is required for the morphogenesis of specific tissues.

24 May 1999 15:50 468 468 A Screen for Mutations that Affect C. elegans Aging

1999 International Worm Meeting abstract 417 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A Screen for Mutations that Affect C. elegans Aging C Huang, K Kornfeld Department of Molecular Biology & Pharmacology, Washington University School of Medicine, St. Louis, MO 63110 How aging is controlled is a fundamental biological question that remains largely unanswered. C. elegans promises to be a powerful model system for studying the genetic basis of aging, as it ages rapidly and mutations in genes such as age-1 and daf-2 have been shown to significantly extend the lifespan. We have initiated a screen for mutations that delay the aging process. We characterized the average time for N2 worms to cease egg-laying, display significantly reduced body movement and pharyngeal pumping, and die. Using EMS mutagenesis, we are conducting an F2 clonal screen for mutants with a persistence of the youthful condition for one or more of these phenotypes. We have screened about 500 (haploid) genomes and isolated some potentially interesting mutations.

24 May 1999 15:50 469 469 Identification of proteins that may interact with MEX-3 to regulate pal-1 translation

1999 International Worm Meeting abstract 418 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification of proteins that may interact with MEX-3 to regulate pal-1 translation NN Huang 1 , M Walhout 2 , M Vidal 2 , CP Hunter 1

1 Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138. 2 Massachusetts General Hospital Cancer Center, Building 149, 13th Street, Charlestown, MA 02129.

We are conducting a yeast 2-hybrid screen to identify proteins that interact with the KH domain protein MEX-3. Molecular and genetic experiments have identified the RNA-binding protein MEX-3 as key to the spatial and temporal control of PAL-1 translation. pal-1 encodes a homeodomain protein that is required to specify the identity of somatic P2 descendents. pal-1 mRNA is present throughout the embryo but PAL-1 protein is expressed only in the P1 descendents. In mex-3 mutants, PAL-1 is expressed throughout the embryo. mex-3 activity is also required to restrict translation of reporter mRNAs that contain pal-1 3’-UTR sequences. Since translational regulation by the 3’-UTRs of mRNAs appears to be a common theme in early embryogenesis, we are conducting a yeast 2-hybrid screen with MEX-3 as bait in order to identify additional factors that are involved in translational regulation of maternal mRNAs. We have identified a number of proteins that interact with MEX-3 in the 2-hybrid screen that may reflect interactions in the embryo. For example, MEX-3 is present in P-granules and we have identified an interaction with a zinc finger protein that is a known P-granule component. We have also identified interactions with several proteins with homology to known RNA-binding proteins, a ribosomal protein, another zinc finger protein, a protein with an SH3 domain, two novel proteins, and two particularly intriguing interactions. We are currently in the process of continuing the 2-hybrid screen and determining whether the interactions identified are biologically relevant. We are specifically interested in proteins that interact with MEX-3 to regulate pal-1 translation.

24 May 1999 15:50 470 470 SYD-3, a giant protein with a GEF domain, functions in synapse formation

1999 International Worm Meeting abstract 419 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. SYD-3, a giant protein with a GEF domain, functions in synapse formation X Huang, M Zhen, Y Jin Dept. Biol., University of California, Santa Cruz, CA 95064 The presynaptic terminus is specialized for synaptic vesicle docking and release. Although much has been learned about neurotransmitter release, how the presynaptic terminus is organized is poorly understood. Our lab has used a presynaptic GFP marker (Punc-25-SNB-GFP) to study synapse formation in D neurons. In wild type animals, GFP fluorescent puncta that represent the presynaptic termini of VD and DD neurons are evenly distributed along ventral and dorsal cords. From a genetic screen, a number of mutants were isolated with abnormal morphology of the presynaptic termini, defining 12 genes named syd, for synapse defective (1). In syd-3 mutants, the GFP puncta in the dorsal cord, corresponding to synapses of DD neurons, are sparse and irregular in size; in the ventral cord the puncta from VD neurons are slightly diffused. We mapped syd-3 between dpy-11 and unc-42 on chromosome V, and cloned syd-3 by germline transformation rescue. syd-3 encodes a 3766 amino-acid residue protein with sequence similarity to human PAM (protein associated with Myc). PAM was identified based on interaction with Myc and is abundant in human brain tissue (2). Both SYD-3 and PAM have a guanine exchange factor (GEF) domain and a highly conserved C-terminal zinc finger cluster domain. A rescuing GFP tagged syd-3 is expressed throughout the nervous system and localized to the presynaptic region. This synaptic localization is unchanged in unc-104 mutants, suggesting that SYD-3 localization is independent of synaptic vesicle transport. Using a series of SYD-3-GFP translational fusions, we have identified a region in SYD-3 that may be required for synaptic localization of SYD-3. syd-3 mutants alone do not exhibit obvious visible phenotypes. However, syd-3;syd-1 and syd-3;syd-2 double mutants are severely uncoordinated and display strong SNB--GFP defects. syd-1 encodes a putative GAP protein (3), and syd-2 encodes a Liprin (1). To identify other components in syd-3 signaling, we performed a suppressor screen on syd-3;syd-1 double mutants and isolated a number of suppressors for syd-3. We will report molecular analysis of syd-3 and characterization of the suppressors.

1. Zhen and Jin (1999) International meeting abstract 2. Guo Q. et al. PNAS 95: 9172 (1998) 3. Hallam and Jin (1999) International meeting abstract

24 May 1999 15:50 471 471 A Novel Protein, UNC-79, Controls Anesthetic Sensitivity.

1999 International Worm Meeting abstract 420 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A Novel Protein, UNC-79, Controls Anesthetic Sensitivity. JA Humphrey, MM Sedensky, PG Morgan Departments of Genetics and Anesthesiology, Case Western Reserve University, Cleveland, OH. Introduction: The gene unc-79 has a pharmaceutical profile which indicate it is important in determining volatile anesthetic sensitivity. Binding studies by Eckenhoff have shown a difference in the binding of halothane in the unc-79 mutant compared with the wildtype. Here we report the cloning and further characterization of the unc-79 gene. Methods: Mutant rescue was done by the technique of Mello. RT PCR, Northern blots, subcloning and DNA sequencing were done by standard protocols. Each mutation was confirmed by sequencing in two directions and in two independent PCR reactions. Blast algorithm was used to analyze homologies between sequences of unc-79 and other known genes. Results: The smallest fragment of DNA which rescues the unc-79 phenotype was predicted to encode a single protein E03A3.6. Northern blots have shown that the predominant message from this gene is about 10KB in size. Two transposon induced unc-79 alleles show a shift in size of this message consistent with insertion of a transposon in the message. RT PCR reveals that at least two splice forms exist for E03A3.6 but that the majority of the message is in an unspliced form. Sequencing has identified mutations in E03A3.6 in 3/5 EMS induced unc-79 alleles. The proteins encoded by the E03A3.6 gene are not homologous to any currently known gene. One allele of unc-8, n491n1193 has a phenotype identical to unc-79. Other alleles of unc-8 have diverse genetic interactions with unc-79, acting either as a suppressor or to give anovel phenotype. Discussion: unc-79 has a phenotype identical to an allele of the degenerin subunit, unc-8. Its anesthetic sensitivity, and that of unc-8, is suppressed by unc-1, a gene highly homologous to stomatin. unc-79 has no known homologies; however, the genetic data support the hypothesis that unc-79 is involved with unc-1 and unc-8 in ion channel regulation. It is unclear why the majority of the unc-79 message is unspliced but this raises the possibility that splicing may be important in determining the expression pattern of the protein. ACKNOWLEDGEMENTS: The authors are indebted to Monica Driscoll and Nektarios Tavernarakis for ongoing critical discussions and for sharing unpublished data. MMS and PGM were supported by NIH grant GM45402.

24 May 1999 15:50 472 472 Suppressors of syntaxin reduction-of-function regulate general anesthetic sensitivity

1999 International Worm Meeting abstract 421 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Suppressors of syntaxin reduction-of-function regulate general anesthetic sensitivity S Hunt 1 , B van Swinderen 1 , C Liu 1 , A Hawasli 1 , CM Crowder 1,2

1 Department of Anesthesiology, Washington University School of Medicine. 2 Department of Molecular Biology/Pharmacology, Washington University School of Medicine.

We have previously shown that mutations in the neuronal syntaxin gene unc-64 profoundly alter the volatile anesthetic (VA) sensitivity of C. elegans. We found that two hypomorphic unc-64 alleles confer hypersensitivity to the VAs isoflurane and halothane but that a third unc-64 hypomorph is VA resistant. The difference between the isoflurane EC50 ’s (the concentration where the effect on locomotion is half maximal) of the hypersensitive and resistant alleles is over 30-fold. In order to understand the molecular mechanisms of syntaxin’s regulation of anesthetics, we are searching for genes that interact with the syntaxin gene. Thus, we initiated a screen for suppressors of the locomotion defect of unc-64(e246lf). The F2 progeny of EMS treated unc-64(e246) were screened for better moving animals. We have screened 14,400 F1 genomes and have established seven independent strains that clearly move better than e246. The suppressor strains moved between 6-8 times faster than e246 as measured from movies taken of them crawling on agar without food. Outcrossing of the seven strains revealed three segregating phenotypes, loopy movers (4 strains), jerky movers (1 strain), and long worms (1 strain); one strain segregated no obvious visible phenotype. We are currently determining whether these phenotypes are in fact those of the e246 suppressors. A similar screen has been performed by Owais Saifee in Mike Nonet’s lab. They also isolated loopy e246 suppressors. We tested the anesthetic sensitivity of two of their loopy strains, js126 and js127e246. Both strains were anesthetic resistant with EC50s more than twice that of N2. This confirms that this is a reasonable approach towards finding "anesthesia genes". We are currently determining the anesthetic sensitivity of the suppressors isolated in our screen.

24 May 1999 15:50 473 473 Neuromodulation in C. elegans and unc-34 suppressors

1999 International Worm Meeting abstract 422 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Neuromodulation in C. elegans and unc-34 suppressors Melissa Hunter-Ensor, Bob Horvitz HHMI, Dept. Biology, MIT, Cambridge, MA 02139 C. elegansalters its behavior in response to environmental changes. How is this behavioral plasticity achieved? In many invertebrates, neuromodulators such as histamine help to achieve behavioral flexibility. Using an anti-histamine antibody we identified histamine-like immunoreactivity in adult C. elegans. We also identified a putative histidine decarboxylase (HD), an enzyme that synthesizes histamine from histidine. A transcriptional GFP fusion of this HD gene colocalizes with histamine-staining in head neurons, which we have tentatively identified as RIH, URX and AFD. We also observed GFP expression in the HSN neurons. We have tested histamine antagonists, including H1, H2 and H3 antagonists, for their effects on C. elegansbehavior. H1 antagonists inhibit pharyngeal pumping and egg-laying, suggesting that histamine may modulate feeding and egg-laying in C. elegans. To determine whether the putative histidine decarboxylase synthesizes histamine, we will ectopically express HD in the mechanosensory neurons and will test for histamine-staining in these neurons. We will also examine the behavioral effects of ablating the HD-GFP expressing head neurons. We are also interested in understanding how the neural networks that generate behavior are assembled and in identifying key molecules involved in the assembly process. unc-34 mutants display defects in axonal outgrowth and fasciculation. unc-34 is a homolog of Drosophila enabled(G. Garriga, personal communication). First identified as a genetic modifier of abl, enabled functions in axonal outgrowth1 . Six suppressors of the locomotor abnormalities of unc-34 animals were previously identified2 . We are presently mapping these suppressors and screening for additional suppressors.

1. Gertler, F.B., Doctor, J. S., Hoffmann, F. M. Science. 248:857-60, 1990. 2. Bloom, L. Ph.D. Thesis, MIT, 1993.

24 May 1999 15:50 474 474 Finding the role of PAR-4 in embryonic asymmetry

1999 International Worm Meeting abstract 423 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Finding the role of PAR-4 in embryonic asymmetry DD Hurd, DG Morton, KJ Kemphues Cornell University, Ithaca, NY 14853 We are investigating the role of the PAR-4 protein in early embryogenesis. The par-4 gene, like other par genes, is required to establish cytoplasmic asymmetry in the one-cell embryo. par-4 mutations result in synchronous early cleavages, failure to localize P granules, and alterations in cell fates. We cloned the par-4 gene (J. Watts and D.Morton, J. Bestman and K. Kemphues, manuscript in preparation), and found that it encodes a serine-threonine kinase. Several par-4 alleles have mutations that alter conserved amino acids within the kinase domain, suggesting that kinase activity is essential for PAR-4 function. Using an antibody to the amino-terminus of the protein we found that PAR-4 is expressed in hermaphrodite and male germ lines and in oocytes and embryos. The protein is present in the cytoplasm of one-cell embryos, with some enrichment at the cell cortex. By the two-cell stage, protein is clearly enriched around the cortex of both cells. This cortical distribution continues in all cells throughout early embryogenesis. In contrast to the asymmetric distributions of PAR-1, PAR-2, PAR-3, and PAR-6, PAR-4 is symmetrically distributed in all early embryonic blastomeres. The PAR-4 distribution is not affected by mutations in the other par genes. To further understand the role of the PAR-4 protein, we are taking two approaches to identify potential substrates and other interacting proteins. First, we are using full-length, truncated and mutant forms of PAR-4 in a two-hybrid screen. In our initial screens we identified several candidates that we are testing for embryonic effects by RNAi. Second, we are constructing kinase-active PAR-4 fusion proteins to use in screening a cDNA expression library for potential phosphorylation substrates.

24 May 1999 15:50 475 475 Supramolecular Organization of Coiled-Coil Proteins By Novel Coupling Proteins

1999 International Worm Meeting abstract 424 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Supramolecular Organization of Coiled-Coil Proteins By Novel Coupling Proteins AH Hutagalung, F Liu, CC Bauer, I Ortiz, HF Epstein Baylor College of Medicine The assembly of coiled-coil proteins into cytoskeletal filaments, including multiple types of intermediate filaments and the myosin II thick filament, is critical for cell structure and function. The biological mechanism in assembly process is the focus of our research. The C. elegans thick filament contains two types of coiled-coil proteins, myosin and paramyosin. The core of the filament containing paramyosin but not myosin permits direct structural analysis of the arrangement of the coiled-coils. Structural modeling, based on 3-D reconstruction of electron microscopic images, suggests that the core consists of seven loosely arranged paramyosin sub-filaments coupled by internal ring-like structures. Three novel proteins, the filagenins, have been identified as potential coupling proteins. a-, b-, and g-filagenins are small, basic proteins with calculated isoelectric points of 10.52, 10.61 and 11.49, respectively. a- and b-filagenins contain high percentages of aromatic amino acids. Western blotting shows that the filagenins are associated with the core. Immunofluorescent microscopy localizes them to the A-band mainly in the body wall muscle. Immuno-EM shows that b-filagenin asembles with paramyosin in the core with a periodicity matching the 72 nm repeat of paramyosin assemblages. In CB1214 mutants where paramyosin is absent, myosin assembles in its place to form core-like tubular filaments. In these abnormal filaments, b-filagenin appears with a periodicity identical to that in the wild-type core. These results support our 3-D model of the core and suggest that the filagenins may stabilize and direct filament assembly. Another line of research in our lab is the possible relationship between g-filagenin and the C. elegans orthologue of zyxin (CeZyxin), a vertebrate protein found at focal adhesion plaques. The 2.2 kb cDNA for g-filagenin encodes CeZyxin further downstream in an alternate reading frame. Northern blot analysis with a g-filagenin specific probe shows that the major transcript for g-filagenin is 2.2 kb. We are currently testing whether both proteins can be translated from the same mRNA and if they interact with each other.

24 May 1999 15:50 476 476 Genes required for axonal fasciculation/target recognition

1999 International Worm Meeting abstract 425 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genes required for axonal fasciculation/target recognition H Hutter 1,2 , EM Hedgecock 1

1 Johns Hopkins University, Baltimore, MD. 2 current address: Max-Planck-Institute for Medical Research, Heidelberg, Germany.

One of the central problems in developmental neurobiology is the question of how the growing axons of neurons find their way through the complex environment of the developing embryo to their target areas, where synaptic partners are choosen. Since synapses in C. elegans are made en passant between neighbouring axons in an axon bundle the correct fasciculation of axons even within a bundle is crucial for the correct wiring of the nervous system. To study the molecular basis of this axon-axon recognition mechanism, we have begun screens for mutants with fasciculation defects in a defined subset of axons, the interneuron axons in the ventral cord (visualized in a strain expressing a glr-1::GFP marker). Thus far, we have recovered mutants with defects ranging from rather minor defasciculations over short distances to a severe disorganization of the ventral cord that extends to other classes of neurons as well. The interneuron axons affected in the mutants are part of the motor circuit, yet not all of the mutants show obvious movement defects as might be expected from a disruption of the motor circuit. This indicates a certain amount of redundancy in the motor circuit. Four of the mutants defining four different genes are being studied in more detail. rh299 and rh300 both show strong fasciculation defects of interneuron axons and no obvious defects in the outgrowth of other axons as judged by various GFP markers. These mutants might define genes specifically involved in the target recognition of the interneurons of the motorcircuit. In rh309 and rh315 mutants interneuron axons not only are defasciculated in the ventral cord, they sometimes don’t reach the cord at all and extend at abnormal lateral positions. Many other axons are also affected and show variable outgrowth defects. Mutant animals are Unc. These mutants define genes that are more generally required for axonal outgrowth. Based on their map position and phenotype these mutants seem to define previously unidentified genes: rh299 is close to sem-4 on chromosome I, rh300 is between dpy-10 and unc-104 on II, rh309 is not far to the right of unc-68 on IV and rh315 is near daf-4 on III.

24 May 1999 15:50 477 477 Inactivation of a VHA-2 gene in Caenorhabditis elegans with the dsRNAi technique

1999 International Worm Meeting abstract 426 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Inactivation of a VHA-2 gene in Caenorhabditis elegans with the dsRNAi technique O Hüttmann, E Schierenberg Zoologisches Institut, Universität Köln, D-50923 Köln, Germany

We injected double-stranded RNA of vha-2, a 16kDa proteolipid subunit of the vacuolar H+ -ATPase into the gonads of adult C. elegans hermaphrodites in order to inhibit the formation of a functional gene product. We found that more than 80% of the embryos generated by the injected specimens arrest before hatching, most of them at one of two different developmental stages. During the first 10 h after injection 90% of the embryos arrest as late morphogenesis stages with abnormal gut cells. Most embryos produced thereafter arrest as 1-cell stages. The presence of giant vacuoles in the intestinal cells and a much decreased gut-specific endocytosis (visualized with fluorescently marked transferrin) in the late arresting phenotype indicates the presence of defective lysosomes. The early arrests demonstrate that the VHA-2 gene product is required from the very beginning of embryogenesis. The different stages of embryonic arrest probably reflect the presence or absence of the necessary maternal vha-2 mRNA.

24 May 1999 15:50 478 478 Regulation of the C. elegans epidermal growth factor homolog LIN-3

1999 International Worm Meeting abstract 427 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Regulation of the C. elegans epidermal growth factor homolog LIN-3 B Hwang, N Moghal, K Verbrugghe, J Liu, P Sternberg Department of Biology, California Institute of Technology, Pasadena, CA LIN-3, an epidermal growth factor in Caenorhabditis elegans, is involved in multiple developmental processes. Much is known about the downstream pathways from LIN-3 and its receptor LET-23, but little is understood about the mechanisms that regulate LIN-3. We are taking several approaches to understand the mechanisms regulating LIN-3. First, anti-LIN-3 antibodies and epitope-tagged LIN-3 have been generated to study its tissue-specific expression and putative regulated processing events. Second, the cis-acting elements of lin-3 which govern its anchor cell-specific and vulval cell-specific expression are being sought by deletion analysis of lin-3 regulatory regions. Finally, a genetic screen looking for mutations that suppress the vulvaless phenotype caused by decreased LIN-3 function has identified a potential candidate regulator of lin-3 as well as downstream signaling molecules. One mutation, rok(sy340) suppresses lin-3 in a semi-dominant manner and maps to LG X. sy340 is epistatic to let-23(sy97) and let-60(n2034), but not to lin-45(sy96), suggesting that rok(sy340) may encode a product working between Ras and Raf. Another mutation, prl-1 (positive regulator of lin-3), has been characterized by three dominant gain of function alleles. The spectrum of suppression by prl-1 suggests that prl-1 acts at the same level or upstream of let-23. Gonad ablation abolished the suppression by prl-1, suggesting that PRL-1 acts through the anchor cell to induce vulval cells. Intragenic revertants of prl-1 are being sought to isolate loss of function mutants of prl-1, which will be valuable for addressing the function of prl-1 in the regulation of lin-3.

24 May 1999 15:50 479 479 Nucleotide-sugar biosynthesis and glycosylation are involved in vulval morphogenesis

1999 International Worm Meeting abstract 428 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Nucleotide-sugar biosynthesis and glycosylation are involved in vulval morphogenesis H Hwang, HR Horvitz HHMI, Dept Biology, MIT, Cambridge, MA 02139 Eight sqv (squashed vulva) genes (sqv-1 to 8) have a mutant vulval morphogenetic phenotype that is defined by reduced separation between the anterior and posterior halves of the vulva in the L4 (1). SQV-3 and SQV-8 are glycosyltransferase-like proteins, and SQV-7 is similar to a putative nucleotide-sugar transporter (2). We have previously reported the cloning of sqv-1 and sqv-4 (3). Both SQV-1 and SQV-4 are similar to enzymes involved in nucleotide-sugar metabolism. These molecular identities suggest that glycosylation is important in shaping the vulva during development. We have now determined biochemically that SQV-4 is a UDP-glucose dehydrogenase. Recombinant SQV-4 expressed in E. coli can reduce NAD in the presence of UDP-glucose, suggesting that UPD-glucose is being converted to UDP-glucuronate. Like known UDP-glucose dehydrogenases, SQV-4 appears to act specifically on UDP-glucose, as it fails to reduce NAD using any of the many other nucleotide-sugars tested. Mutant recombinant SQV-4 derived from either of the two genetically identified mutant alleles does not show detectable activity. Preliminary antibody staining using a rabbit polyclonal antibody directed against a GST::SQV-4 indicates that SQV-4 is localized to the vulval cells and several other tissues, including seam cells. This result agrees with the expression of SQV-4::GFP fusion protein and supports a model in which SQV-4 acts in the vulval cells during vulval development. We also are trying to determine the functions of the other four cloned sqv genes using biochemical assays and heterologous complementation. Lastly, we are mapping and cloning the three remaining sqv genes.

1. Herman, T. et al. (1999). PNAS 96: 968-973. 2. Herman, T. and Horvitz, H.R. (1999). PNAS 96: 974-979. 3. Hwang, H. and Horvitz, H.R. (1998) East Coast C. elegans meeting, p. 103.

24 May 1999 15:50 480 480 Characterization of suppressors of daf-1, daf-8 and daf-14.

1999 International Worm Meeting abstract 429 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of suppressors of daf-1, daf-8 and daf-14. T Inoue, JH Thomas Dept. of Genetics, University of Washington, Seattle, WA, USA Dauer formation is controlled in part by a TGF-beta related signal transduction pathway. Mutations in some of these components (daf-1, -4, -7, -8, and -14) lead to constitutive dauer formation under dauer non-inducing conditions. In order to identify additional components of the dauer pathway, we isolated 36 independent mutations that suppress the Daf-c phenotype of daf-1, daf-8 or daf-14. We tentatively classify the mutations into three categories: 1) alleles of daf-3, daf-5 and daf-12. These genes were previously identified in screens for suppressors of daf-4 and daf-7. Many of these alleles suppress dauer formation completely, even under dauer inducing conditions. 2) sa315. This unique mutation suppresses the Daf-c phenotype strongly, with a small fraction of animals forming partial dauers. Interactions of this mutation with various Daf-c genes are somewhat similar to those of daf-16, daf-18, and akt-1(dm), perhaps indicating a defect in the daf-2/age-1 insulin receptor pathway. Curiously, sa315 maps to the same genetic interval as age-1. We are currently testing to see if this is an unusual allele of age-1. The third class of mutations suppress the Daf-c phenotype of daf-1, -8 or -14 incompletely (90-99% suppressed at 25C), and where tested, also weakly suppress daf-4 and/or daf-7. So far, mapping and complementation tests among these suppressors have revealed three new genes. These genes were not found in previous screens probably because the suppression is relatively weak. Currently, we are characterizing further these suppressors. As part of this analysis, we are cloning scd-1, one of the incomplete suppressor genes. scd-1 suppresses all pleiotropies of daf-8, -7, and -14 but does not suppress daf-11, suggesting that it may be a novel component of the TGF-beta-related signal transduction mechanism.

24 May 1999 15:50 481 481 Analyses of the Interaction of Two Sensory Signals on the Behavior under Well-fed and Starved Conditions

1999 International Worm Meeting abstract 430 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analyses of the Interaction of Two Sensory Signals on the Behavior under Well-fed and Starved Conditions T Ishihara 1,2 , I Katsura 1,2

1 Struct. Biol. Cent., Natl. Inst. Genet. 2 Dept. Genet., Grad. Univ. Adv. Stud., Mishima, Shizuoka, 411-8540 Japan.

Among many environmental stimuli, C. elegans seems to respond to only a few stimuli simultaneously. To elucidate how the neural circuit processes many sensory signals to respond properly, we have designed a new assay system to analyze interaction between two responses: chemotaxis to odorants and avoidance from copper ion. In this assay, the behavior of C. elegans depends on the concentration of both two stimuli, which are sensed by different neurons. This observation suggests that these two responses repress each other. We can argue that this mutual interaction takes place in a defined part of the neural circuit, which consists of about 10 pairs of neurons. We have identified several mutants that show abnormality in this interaction. One of the mutants, ut236 prefers to avoid copper ion rather than to go to odorants, while glr-1 mutants show the opposite phenotype. However, dose responses to the odorants or the copper ion of these mutants are indistinguishable from those of the wild type. We mapped the ut236 mutation between stP33 and unc-18 on the chromosome X and have identified a 15 kb DNA fragment of the cosmid C36B7 that can rescue the ut236 phenotype. Further analysis of the predicted genes in this region is underway. When worms are starved for several hours, their behavior in this assay is changed. They prefer to go to odorants rather than to avoid copper ions as compared with worms in well-fed conditions. This behavioral change is due, at least partially, to weaker avoidance behavior from copper ion by starved animals. Furthermore, in the presence of serotonin, this behavioral change by starvation is not observed. We also isolated mutants that show abnormality in this behavioral change. One of the mutants, ut235 does not change the response to copper ion by starvation, although this mutant behaves almost like the wild type in the change of movement phenotype by starvation and by serotonin. In addition, the ut235 ut236 double mutant entirely prefers to avoid copper ions than to go to the odorants even if they are starved. By characterizing these mutants, we hope to clarify the modification mechanism of the neuronal activity.

24 May 1999 15:50 482 482 An update on the analysis of the ttx-2 gene involved in thermotaxis

1999 International Worm Meeting abstract 431 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. An update on the analysis of the ttx-2 gene involved in thermotaxis K Ishii 1 , I Mori 2 , Y Ohshima 1

1 Department of Biology, Kyushu University. 2 Division of Biological Science, Nagoya University.

C. elegans responds to various environmental signals by moving either toward or away from the stimulus. When the wild type animals were grown at a temperature ranging from 15-degree to 25-degree and placed on a thermal gradient without food, they migrate toward their growth temperature and then move isothermally around that temperature. We are focusing on this thermotaxis behavior as a model to study neuronal plasticity. Many thermotaxis-defective (ttx or tax) mutants had been isolated by EMS-mutagenesis. Thermotaxis-defective mutants are divided into three major phenotypic classes: athermotactic (non-temperature-responsive), cryophilic (cold-seeking) and thermophilic (heat-seeking) classes. We genetically mapped ttx-2(ks4) mutation, one of the cryophilic mutations, on chromosome III into the region of about 0.5 mu between dpy-17 and lon-1. We introduced cosmid DNAs in this region of the wild type into the ttx-2 mutant to determine if they rescue the mutation by monitoring thermotaxis behavior of the transgenic animals. In these experiments, W03A5 cosmid weakly rescued thermotaxis abnormality of the ttx-2 mutant on a radial temperature gradient. We also examined the rescue activities of PCR fragments derived from W03A5 cosmid DNA. A 11.2kb fragment seems to show weak, but significant rescue activity. Further works are in progress to identify and characterize the ttx-2 gene.

24 May 1999 15:50 483 483 Oxidative Stress and Aging in C.elegans: What lessons learned from mev-1?

1999 International Worm Meeting abstract 432 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Oxidative Stress and Aging in C.elegans: What lessons learned from mev-1? N Ishii 1 , P Hartman 2

1 Molec. Life Sciences, Tokai University School of Medicine, Isehara, Kanagawa 259-1193 Japan. 2 Biol. Dept., Texas Christian University, Fort Worth, TX 76129.

By measuring the life spans of various recombinant inbreds, Johnson and Wood estimated that genetic variability accounted for 20% to 50% of C. elegans life span.1 We measured life span of animals over two successive generations and found that, while some variation exists in from generation to generation, the life span of a parent had little effect on that of its progeny. What are the environmental factors that control the balance of life span? Studies with the mev-1 gene provide evidence that reactive oxygen species (ROS) generated during respiration are key environmental factors in this process. Mev-1 mutants are hypersensitive to oxygen and methyl viologen (a singlet oxygen generator) and age precociously, particularly at high oxygen concentrations.2 They also accumulate various aging markers more rapidly than does wild type.3 The mev-1 mutation is in the cytochrome b subunit of succinate dehydrogenase (cyt-1), which compromises enzymatic activity of the mitochondrial complex II.4 It is likely that, as a result of this mutation, significantly higher levels of ROS are generated during respiration. Mev-1 animals shown hypermutability to oxygen, which represents the first example of a mitochrondrial dysfunction impacting nuclear mutation frequencies. In addition, temperature affects life span differently in mev-1 and wild type, likely through these metabolic differences. Electron microscopy reveals inclusions in the mitochondria of mev-1 animals, particularly under elevated oxygen concentrations. In addition, mev-1 embryos contain abnormally high numbers of ced-3-dependent apoptotic cells. Collectively these data muster substantial support for the theory that ROS-induced damage is important in biological aging.

1 Johnson and Wood 1982 PNAS 79, 6603

2 Ishii et al., 1990 Mutat. Res. 237, 165.

3 Hosokawa et al., 1994. MAD 74, 161; Adachi et al., 1998 J. Geront. 53A, B240

4 Ishii et al., 1998. Nature 394, 6694

24 May 1999 15:50 484 484 Toward four-dimensional database of gene expression in C. elegans

1999 International Worm Meeting abstract 433 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Toward four-dimensional database of gene expression in C. elegans Masahiro Ito, Tomoko Motohashi, Yohei Minakuchi, Yuji Kohara CREST, JST and Genome Biology Lab, National Institute of Genetics, Mishima, Japan One of the most challenging targets in the post genomics era is computer simulation of development. We feel C.elegans is the most suitable system for this purpose, since the complete parts list of cells has been identified and the expression patterns and functions of all genes are being analyzed systematically. Aiming at the final goal and at integrating such information, we are constructing a computer graphics (CG)-based four-dimensional (3D + time course) database of gene expression in C.elegans. Thus far, we have made a CG for early embryogenesis up to 100 cell stage based on 4D Nomarski image data including the data kindly supplied by Kevin O’Connel at the University of Wisconsin at Madison. Since it was very hard for conventional image processing software to extract the contour of cells from the Nomarski images, we adopted a ’low-tech’ procedure; firstly we traced the contour of individual cells and nuclei by hand-writing on thousands of the original images of the 4D microscopic data and then reconstructed 3D structures by piling up and making relationship among the contour data from different focal planes. The resulting 3D structures of various stage embryos were arranged along the time course of development and connected each other by an interpolating method to finally generate the CG of early embryogenesis. The graphics is faithful to the real embryogenesis, thus, it provides various information such as the volume of individual cells, the extent of cell-cell contact and so on. To incorporate the information of gene expression patterns into the 4D graphics database, we have developed a procedure to superimpose a fluorescent-confocal data of the distribution of mRNA and proteins onto the CG. In the procedure, we use triple color staining, namely, DAPI and Cy-3 for nuclei and POS-1 (P-lineage specific) as internal markers for superimposing, respectively, and Cy-5 for a query gene product. By this fashion, we are incorporating the immunostaining patterns of maternal gene products. We are planning to make the 4D database accessible over the internet.

24 May 1999 15:50 485 485 Cloning and characterization of dinhr-6, a putative homolog of the Drosophila ecdysone response gene E75 , in a parasitic nematode.

1999 International Worm Meeting abstract 434 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cloning and characterization of dinhr-6, a putative homolog of the Drosophila ecdysone response gene E75 , in a parasitic nematode. MC Jackson 1 , CV Maina 2 , K Crossgrove 1

1 Loyola College in Maryland, 4501 N. Charles St., Baltimore, MD 21210. 2 New England Biolabs, Inc., 32 Tozer Rd., Beverly, MA 01915.

Nuclear hormone receptors (NHRs) are known to be essential for developmental processes in a wide variety of multicellular organisms, including C. elegans. One extensively studied example of the developmental role of NHRs is in the control of molting and metamorphosis in Drosophila. Molting and metamorphosis in Drosophila are regulated by the steroid hormone 20-OH ecdysone, hereafter referred to as ecdysone. The ecdysone signal is transduced by NHRs. Nematodes also undergo a series of molts during development. Molting from the third to the fourth larval stage of the filarial parasite Dirofilaria immitis(the causative agent of dog heartworm disease) can be prematurely induced in vitro by ecdysone, suggesting that ecdysone, or a related compound, may play a role in the development of this nematode. Putative homologs of the EcR and usp genes, which encode NHRs that form the functional ecdysone receptor in Drosophila, have been identified in D. immitis. The ecdysone signal in Drosophila is modulated by a number of transcription factors that are induced as a primary response to ecdysone. We have identified a putative D. immitis homolog of the Drosophila E75 ecdysone primary response gene. E75 encodes multiple isoforms and is essential for metamorphosis in Drosophila. The protein encoded by the D. immitis E75 homolog, dinhr-6, is 83% identical to E75A in the DNA binding domain. We cloned the 5’ end of dinhr-6 and determined that the transcript uses the SL1 spliced leader. We are currently using a variety of techniques to clone the 3’ end of the gene and have identified the complete DNA and ligand binding domains. Northern blot analysis suggests that dinhr-6 encodes multiple isoforms and is female-specific in adults. A putative E75 homolog, nhr-85, was recently identified in C. elegans (Ann Sluder, personal communication). We discuss the potential of using C. elegans to help determine the function of parasite proteins.

24 May 1999 15:50 486 486 A method for high resolution mapping relative to single nucleotide polymorphisms used to positionally clone cdf-1, a gene involved in vulval development

1999 International Worm Meeting abstract 435 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A method for high resolution mapping relative to single nucleotide polymorphisms used to positionally clone cdf-1, a gene involved in vulval development J Jakubowski, K Kornfeld Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110 A conserved receptor tyrosine kinase-Ras-MAP kinase signal transduction cascade is required for specification of the C. elegans vulva. To identify genes involved in Ras-mediated signaling, we screened for mutations that suppressed the multivulva (Muv) phenotype caused by a constitutively active let-60 ras gene. We isolated 43 mutations that define 21 complementation groups, including the mutation n2527. n2527 reduces the penetrance of the lin-15(lf) Muv phenotype but not the lin-1(lf) Muv phenotype. If these genes act in a linear pathway, the gene affected by n2527 functions downstream of lin-15 and let-60 ras and upstream of lin-1. Three factor mapping experiments positioned n2527 between mup-2 and unc-6 on chromosome X. To positionally clone the gene affected by n2527, we developed a method of high resolution mapping relative to polymorphisms identified by DNA sequencing. We took advantage of the complete genomic sequence to search for polymorphisms at defined genomic positions in the RC301 strain. We found about one polymorphism per 1.4 kb in predicted intergenic regions of RC301; most are single nucleotide polymorphisms (SNPs). By alternating between identifying polymorphisms and mapping, we positioned the n2527 mutation in a 9.6 kb interval. The molecular lesion was identified by sequencing the interval, and the candidate gene was confirmed by transformation rescue. This approach should be generally applicable, and particularly useful for mutations that cannot be readily rescued by transformation. The n2527 mutation affects a previously uncharacterized gene. The predicted protein has 519 amino acids and is similar to members of the cation diffusion facilitator (CDF) family of proteins. We named this gene cdf-1. CDF-1 is most similar to mammalian ZnT-1 and yeast COT1 which function to reduce the intracellular concentration of Zn2+ and Co2+ ions, respectively. Thus, CDF-1 may regulate the intracellular concentration of a heavy metal ion. CDF family members have not been reported to regulate Ras signaling, suggesting cdf-1(n2527) may reveal a previously uncharacterized function for these proteins.

24 May 1999 15:50 487 487 Characterisation of a Neurotransmitter Transporter Gene (T25B6.7) in C.elegans

1999 International Worm Meeting abstract 436 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterisation of a Neurotransmitter Transporter Gene (T25B6.7) in C.elegans C James, D Coates, RE Isaac School of Biology, University of Leeds Termination of signals at the synapse is mainly carried out by members of the sodium- and chloride- coupled neurotransmitter transporter protein (NTP) family, which take neurotransmitter into presynaptic and glial cells by linking it to a sodium concentration gradient. They have a putative structure of 12 transmembrane domains linked by hydrophilic loops. To date over 40 members of the NTP family have been cloned and charaterised mainly from vertebrates. T25B6.7 was predicted to encode a member of the NTP family by the genome sequencing consortium. The promoter driven LacZ expression pattern of T25B6.7 is observed in embryonic, larval and adult stages. In the adult hermaphrodite staining is seen in neuronal processes in the pharyngeal region, lateral neuronal processes, vulval D cells, anal sphincter, and nuclei posterior to the anus. The adult male has a similar pattern, with staining in the copulatory apparatus. In the L1 stage, expression occurs in nuclei along the lateral aspect of the worm and in the ~400 min. embryonic stage there are nuclei staining in the dorso-lateral aspect in the posterior of the embryo. The larval and embryonic patterns of expression suggest the V and T lineages some of which go on to form neurones, socket cells and sheath cells. Deletions were carried out in the T25B6.7::LacZ construct to investigate promoter/enhancer elements. A deletion leaving 796bp of 5’ flanking sequence reduced the level of expression of the T25B6::LacZ construct. Sequence upstream of this site is homologous to 5’ flanking sequence of other genes in the cosmids F21G4 and R03G8. A full length cDNA for this gene has been isolated. Sequence analysis reveals that this is an atypical member of the family. Secondary structure prediction programs suggest an 11 transmembrane domain protein flanked by long carboxy and amino termini. dsRNA was transcribed from the cDNA and injected into C.elegans, this produced late embryonic arrest. Vacuoles were visible in the arrested embryo. The expression pattern and RNA experiments suggest that T25B6.7 is important in developmental stages of C.elegans and may be involved in blast cell fate inductions. It is difficult to speculate on the role of the gene in adult stages as its DNA sequence and expression pattern offers no obvious clues.

24 May 1999 15:50 488 488 Molecular analysis of daf-9, a gene controlling C. elegans larval development

1999 International Worm Meeting abstract 437 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Molecular analysis of daf-9, a gene controlling C. elegans larval development K Jia, PS Albert, IM Caldicott, DL Riddle University of Missouri, Columbia, MO 65211, USA TGF-ß-like and insulin-like signals control the decision between dauer formation and continuous development to the adult. Other than dauer-specific collagen expression and genes associated with stress resistance, little is known about the ultimate targets of these signal transduction pathways or how these targets are regulated. Mutations in the daf-9gene result in constitutive formation of dauer-like larvae even in abundant food. Morphogenesis of daf-9"dauer larvae" is incomplete: some tissues exhibit dauer-specific morphology, whereas others do not. Our previous genetic studies placed daf-9at the end of the genetic pathway for dauer formation. We postulate that DAF-9 is required for non-dauer morphogenesis in certain tissues. We mapped daf-9between two DNA markers (pKP943and stp33)on the X chromosome, and we cloned a candidate gene in this region by Tc1 transposon tagging. Our preliminary RNAi results suggest that this gene could be daf-9. Currently, we are analyzing other mutant daf-9alleles. Molecular analysis of daf-9may reveal a tissue-specific regulatory component targeted by TGF-ß and insulin signalling pathways.

24 May 1999 15:50 489 489 An essential role for aha-1, a PAS-domain-containing regulatory gene in C. elegans

1999 International Worm Meeting abstract 438 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. An essential role for aha-1, a PAS-domain-containing regulatory gene in C. elegans H Jiang, R Guo, JA Powell-Coffman Department of Zoology and Genetics, Iowa State University, Ames, IA 50011 We are studying the cellular and developmental functions of C. elegans ahr-1 and aha-1, the orthologs of the mammalian aryl hydrocarbon receptor and ARNT, to better understand the function and regulation of genes containing basic helix-loop-helix and PAS domain motifs. This class of transcriptional regulatory genes has been shown to mediate several important developmental processes in other organisms, including central midline formation, tracheal cell specification, and circadian clock function. The bHLH-PAS proteins function predominantly as heterodimers, and their activities are regulated by nuclear translocation. To analyze the cellular and subcellular localization of AHR-1 and AHA-1, we have generated transgenic lines carrying lacZ and GFP reporters. We have also affinity-purified AHA-1-specific antibodies, and we are in the process of refining the staining conditions. Using our current protocols, the polysera recognizes overexpressed AHA-1-GFP in situ, and we can detect endogenous AHA-1 in some tissues, such as the rectum. AHA-1 is expressed in several cell types that do not express AHR-1-GFP (1), and we hypothesize that AHA-1 has other dimerization partners. By analyzing genomic data, we have identified additional PAS-domain-containing genes, and we are taking two approaches to determine whether they interact with ahr-1 or aha-1. First, we have constructed GFP reporter constructs for these genes and are comparing their expression patterns to those of aha-1 and ahr-1. Second, we have cloned full-length cDNAs, and we will determine which gene products co-immunoprecipitate with AHA-1 in vitro. We have isolated two Tc-1 mediated deletions in the aha-1 gene, and they are likely null alleles. aha-1 (ia01) homozygotes die during early larval development, perhaps due to intestinal tract defects. We are initiating mosaic analysis to better understand the developmental requirements for aha-1 function. We are also screening for mutations in ahr-1.

(1) Powell-Coffman, J.A., Jiang, H., Cary, C.L., and Wood, W.B. 1998 Midwest C. elegans Mtg.

24 May 1999 15:50 490 490 Mutations in sof-1 and sof-2 can suppress fog-1(q253ts)

1999 International Worm Meeting abstract 439 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Mutations in sof-1 and sof-2 can suppress fog-1(q253ts) SW Jin, RE Ellis Department of Biology, University of Michigan, Ann Arbor MI 48109 The fog-1 and fog-3 genes specify that germ cells differentiate as sperm rather than as oocytes. To identify genes whose products regulate or interact with FOG-1, we screened for suppressors of fog-1(q253ts). Hermaphrodites that are homozygous for q253 produce sperm and oocytes at 15°C, but only oocytes at 25°C, preventing self-fertilization. We raised fog-1(q253) animals at the permissive temperature, treated them with EMS, and shifted their F2 progeny to 25°C to select for self-fertile individuals. From a screen of approximately 40,000 haploid genomes, we isolated 19 suppressor mutations. Most of the suppressors are recessive, and none are completely penetrant. The majority of these suppressors fall into two complementation groups. Eight alleles fail to complement each other and map near sma-3 on LGIII. We named this locus sof-1, for suppressor of fog-1. Because the sof-1 mutations are recessive and arose at a rate of approximately 1/5000 haploid genomes, they are likely to cause a loss-of-function; we are carrying out a non-complementation screen to test this hypothesis. Although most alleles of sof-1 are good suppressors of fog-1(q253ts), they have little effect on sperm production in a wild-type background. Four other alleles fail to complement each other, and map to LGII, a few map units to the left of dpy-10. We named this locus sof-2. These mutations are also recessive and might cause a loss-of-function. As with sof-1, most alleles of sof-2 are good suppressors of fog-1(q253ts), but have little effect on sperm production in a wild-type background. However, preliminary results suggest that hermaphrodites produce significantly more sperm in sof-2; sof-1 unc-32 strains than in unc-32 controls. Thus, these genes might play a redundant role in the regulation of germ cell fates. We are now isolating null alleles of sof-2 to use in genetic tests of this hypothesis. Finally, the dominant mutations v19 on LGI and v12 on LGX also suppress fog-1(q253ts), and five recessive mutations have a very weak effect on fog-1 activity, and have not been studied further.

24 May 1999 15:50 491 491 A diet of double-stranded RNA allows simple male/female genetics

1999 International Worm Meeting abstract 440 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A diet of double-stranded RNA allows simple male/female genetics SW Jin, PJ Chen, D Salinsky, RE Ellis Department of Biology, University of Michigan, Ann Arbor, MI 48109 Although male/hermaphrodite genetics has given us many blessings, it also creates one major problem -- distinguishing between cross-progeny and self-progeny. Traditionally, this problem has been solved in one of three ways: (1) using a marker mutation to distinguish between self- and cross-progeny, (2) using mutations to create true females, which produce only cross-progeny, or (3) using hermaphrodites that have been purged of sperm. The first two methods require extra crosses, and occasionally create problems because of the effects of the marker mutation on the phenotype being studied. The final method requires careful handling of worms to identify purged hermaphrodites, which usually yield few progeny. We have found that double-stranded RNA homologous to fog-1 creates female worms with high efficiency. Almost 100% of the progeny of worms injected with a 1 mg/ml solution of fog-1 dsRNA develop as females. To simplify administration of the double-stranded RNA, we developed a procedure for raising large numbers of larvae in a 1 µl drop. This method yields female animals rapidly, easily, and with high efficiency; these females can be used for any desired cross. At our poster we will provide information and materials for production of fog-1 dsRNA, and for producing female worms by feeding this dsRNA to larvae.

24 May 1999 15:50 492 492 Marker Rescue of Srf-2

1999 International Worm Meeting abstract 441 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Marker Rescue of Srf-2 WA Jobling, SM Politz Dept. of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA 01609 Mutations in several genes, including srf-2, affect surface composition in C. elegans by unmasking hidden antigenic determinants in the cuticle. The mechanism by which this unmasking occurs is unknown; however, it appears to involve loss of surface components. Because nematode surface coat proteins appear to be mucin-like O-linked glycoproteins, unmasking could involve changes in glycosylation as well as changes in surface protein expression. By molecular cloning, we hope to identify the genetic function of such surface defective genes. Srf-2 I was mapped in multi-factor crosses with visible markers and a Tc1 polymorphism to a region of IR spanned by a few cosmid clones and one YAC clone. A mixture of two cosmids from the candidate region was co-injected into srf-2(yj262) mutants along with rol-6(su1006) plasmid DNA. Rescue was assessed by staining Rol individuals from transformed lines with FITC-soybean agglutinin, which stains the surface of srf-2 but not wild type worms. Three of seven lines tested showed rescue of the srf-2 phenotype (reduction in fraction of fluorescent individuals compared with wild type). We currently are testing individual cosmids and smaller fragments to further narrow the srf-2 candidate region.

24 May 1999 15:50 493 493 Genetic Amenability of Chromosome I left

1999 International Worm Meeting abstract 442 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic Amenability of Chromosome I left RC Johnsen 1 , SJ Jones 2 , AM Rose 1

1 Department of Medical Genetics. 2 BC Genome Sequence Centre.

We have completed the analysis of lethals recovered using the duplication, sDp2. 250 essential genes have been identified in the left half of chromosome I by mutational analysis. 600 lethals were recovered after mutagenesis of 31,600 marked chromosomes balanced by sDp2. We have added 133 previously unidentified let genes, to our published collection of 117. In addition, lethal alleles of five known genes with visible mutants were recovered. The 250 essential genes are represented by 502 mutations. For 88 of the genes, more than one allele exists. Statistical analysis would indicate that we have hit approximately two-thirds of the targets (essential genes) in the region. The mutations were generated using ethylmethane sulfate (EMS) at a dosage of either .017 or .025 M. In our analysis, in addition to the lethal alleles, seven EMS-induced deletions were recovered. We have estimated an average forward mutation rate of 5 x 10-5 for lethals under these conditions used in this analysis. We examined the mutations for stage of developmental arrest. Eighteen embryonic, 167 larval, and 42 sterile adult arrests were found. The mutants in this analysis were isolated using the same balancer under similar screening conditions, and recovery conditions should be equal for all subintervals of the sDp2 region. We have compared the mutability of genes within the genetically defined cluster region to that outside. As a result of the sequencing and annotation of the C. elegans genome, we have a precise estimate of the number of predicted coding regions. We have compared structural and functional characteristics of the genes in different regions of chromosome I left, and of genes on other chromosomes, to their mutational accessibility.

24 May 1999 15:50 494 494 Automated sorting and dispensing of C. elegans to wells of microtiter plates

1999 International Worm Meeting abstract 443 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Automated sorting and dispensing of C. elegans to wells of microtiter plates CD Johnson 1 , R Clover 1 , B Reardon 1 , PB Krauledat 2 , AA Ferrante 2 , WP Hansen 2

1 Axys Pharmaceuticals, NemaPharm Group, South San Francisco, CA. 2 Union Biometrica, Inc., Somerville, MA.

For generating a large number of separate populations of worms, liquid cultures in the wells of microtiter plates use space more efficiently than do standard cultures on agar plates. At NemaPharm, we routinely grow worms in microtiter plates for a wide variety of procedures including both forward and reverse genetics experiments as well as for high-throughput chemical screening. To automate distribution of worms into microtiter plate wells, we have constructed a machine that efficiently sorts and dispenses live nematodes. The mechanism used for measuring nematodes is based, in principle, on the nematode counting machine built by Lou Byerly, Randy Cassada and Dick Russell in the early 1970’s (1). Nematodes are suspended in liquid and passed though a narrow nozzle into the center of a rapidly flowing sheath current. The resultant shear forces straighten the animals and orient them parallel to the direction of flow. The straightened animals are then passed at high speed (~1 m/sec) through an orthogonal sheet of laser light where, in the current model, an in-line detector measures the attenuation of the laser light and an attached computer calculates the length of the nematode based upon time-of-flight through the beam. Below the detector, the worm-containing stream is deflected to waste by a air jet controlled by the computer that shuts off momentarily to deliver a selected animal to a well (distribution volume is 0.25 - 1 µl per worm). The current sorter/dispenser is capable of distributing 10 worms to the wells of 96-well plates in 2 minutes with a precision of ±0.7 worms. Distribution to 384 and 1536 well plates is also feasible with the current model. Machines with more sophisticated detecting and measuring capabilities are planned for the future.

(1) Byerly, et. al. (1975) Rev Sci. Instrum., 46:517-522

24 May 1999 15:50 495 495 unc-110, egl-23 and a newly recognized unc-93 family

1999 International Worm Meeting abstract 444 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. unc-110, egl-23 and a newly recognized unc-93 family DB Johnstone 1 , M Kunkel 2 , L Salkoff 2 , JH Thomas 1

1 MCB and Genetics, Box 357360, UW, Seattle, 98195. 2 Dept of Anatomy and Neurobio; Genetics, WashU, St. Louis, MO 63110.

The K+ channel superfamily includes recently discovered subunits with four TM (TM) domains and two pore segments called TWIKs. Two major unanswered questions about TWIK subunits are: how do they form channels (e.g. are other subunits required?), and once formed, what are the properties of these channels? The cloning of sup-9 (Perez de la Cruz, 1997 IWM) provided the first clue: sup-9 is a TWIK, and based on gene interactions, SUP-9 subunits may form heterotrimeric channels with UNC-93 and SUP-10 (Greenwald and Horvitz, 1980; Levin and Horvitz, 1993). But what of other TWIKs? This question is intriguing because the sequencing consortium revealed about 50 genes for TWIK subunits in worms. We have recently found mutations in two more TWIK subunits that may help answer these questions. As previously reported (Johnstone, et al. 1998 WCWM), we found that unc-110 encodes a TWIK subunit. unc-110(e1913)/+ mutants have a flaccid, rubber-band-Unc phenotype, as does a partial intragenic revertant, e1913 sa859, which is homozygous viable. Translational unc-110::gfp fusions express GFP only in body-wall muscle. We hypothesize that e1913sd causes inappropriate opening of the UNC-110 K+ channel in body wall muscles, which would oppose muscle excitation (see related abstract, Kunkel et al.). A putative null mutant with a nonsense mutation in exon 2 (sa954) appears grossly wild-type. We find no genetic interactions between unc-110 and either unc-93 or sup-10 (data not shown), suggesting that UNC-110 does not require the same auxilliary subunits as SUP-9 to form channels. Instead, we noticed an overlooked family of over a dozenunc-93-like genes in the genome. In order to identify genes that interact with unc-110 we screened 105,000 haploid genomes for supression of the Unc phenotype of sa859. We have recently found that egl-23 also encodes a TWIK. Both dominant egl-23 alleles, n601 and n2579, reduce egg-laying and enteric muscle contraction. A putative null mutant with a 213 bp deletion starting in exon 2 is grossly wild type. egl-23 does not appear to interact genetically with the unc-93 group, and we have screened 25,000 haploid genomes for suppression of the Egl phenotype.

24 May 1999 15:50 496 496 pvl-5 is involved in the early development of the vulva in C. elegans

1999 International Worm Meeting abstract 445 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. pvl-5 is involved in the early development of the vulva in C. elegans PM Joshi, DM Eisenmann Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD 21250. The C.elegans vulva is formed from the descendants of the P cells. The 12 P cells, born early in embryogenesis, migrate into the ventral midline by the L1 larval stage where they divide to give an anterior Pn.a neuroblast and a posterior Pn.p hypodermal cell. Of the 12 Pn.p cells, six (P3.p - P8.p) form the vulval equivalence group and are called the vulval precursor cells. On induction by the anchor cell, P5.p, P6.p and P7.p adopt induced cell fates and divide to generate the 22 cells that form the vulva. pvl-5 (ga87) was isolated in a screen for genes affecting the development of the vulva (WBG 13.1, p69). In ga87 animals, there is an average of 7.1 Pn.p cells present in the ventral midline as opposed to 12 Pn.p cells in wildtype. Most probably ga87 is a null mutation as the phenotype of ga87/mnDf39 is no worse than ga87 alone. An additional allele of pvl-5 has been isolated in an F1 non-complementation screen and is being characterized. pvl-5 has been mapped to a small region covered by the deficiency mnDf39 on LGII. The cloning of pvl-5 by cosmid rescue is underway. We do not know why there are too few Pn.p cells in ga87. It could be either due to a loss of P cells or Pn.p cells by cell death, a failure of P cells to migrate into the ventral midline or a Pn.p to Pn.a cell fate transformation. To distinguish between these possibilities I will use unc-4::gfp which is expressed in the neurons derived from the Pn.a cells (David M. Miller III and Charles J. Niemeyer, Development 121, 2877-2886). In the case of a hypodermal (Pn.p) to a neuroblast (Pn.a) cell fate transformation there will be more unc-4::gfp expressing cells in the ventral nerve cord. If there is a loss of P cells or a failure to migrate there might be fewer unc-4::gfp expressing cells in the VNC. If pvl-5 affects the maintenance of Pn.p cells alone there would be no change in the number of cells expressing the marker. I will also study the P and Pn.p cells using jam-1::gfp (gift of Jeff Simske). jam-1 codes for the protein recognized by the MH27 antibody and hence JAM-1::GFP outlines the boundaries of hypodermal cells. The fate of P cells and their lineage will be followed in a pvl-5(ga87); jam-1::gfp strain. Further results from these experiments will be presented.

24 May 1999 15:50 497 497 Regulatory sequence for body wall muscle specific expression of Caenorhabditis elegans

1999 International Worm Meeting abstract 446 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Regulatory sequence for body wall muscle specific expression of Caenorhabditis elegans H Kagawa, H Ohta, T Bando Department of Biology, Faculty of Science, Okayama University Okayama 700-8530 JAPAN C. elegans has mainly two muscle tissues; pharynx for feeding and body wall for locomotion. Two of each myosin heavy chain genes are expressed in pharynx and body wall respectively. One paramyosin gene, unc-15 expresses in both. Two of each tropomyosin isoforms are expressed in both from one tropomyosin gene, tmy-1/lev-11 by two promoters and alternative splicing1 . Okkema and Fire find that pharynx specific muscle gene is controlled by ceh-222 . It is interesting to know how muscle protein genes express in body-wall muscles3 . By microinjection analysis on the promoter/lacZ fusion plasmid genes we found hundred base pairs of 5’untranslated sequences is essential for the troponin C gene, pat-10 expression in body-wall muscle4 . We identified in this region a sequence similar to a enhancer of hlh-1, a MyoD homologue of the worm3 . Interestingly together with this enhancer other enhancers were in the proximal promoter of tmy-1 which control body wall expression. Search of consensus sequences of Sp1 and MyoD recognition and enhancers in many muscle genes suggests that there may be some transcription mechanism in body-wall muscle. Combination of many enhancers may control the specificity and quantity of the gene products. On the assumption of this idea we found some genes express in body-wall muscles. One additional finding from genome sequence search indicates that an unique sequence in the troponin C gene, pat-10 locus was found 15 places in the genome. Interestingly the other troponin C gene tnc-2 has also had a different sequence appeared in 18 places. Our final goal is to find some key sequences which could be important for a gene expression or gene evolution.

1 Kagawa et al, 1995 J. Mol. Biol. 251, 603-613.

2 Okkema and Fire, 1994 Development, 120, 2175-2186.

3 Krause et al, 1994 Dev. Biol. 166,133-148.

4 Terami et al, J Cell Biol.

24 May 1999 15:50 498 498 The LIM homeobox gene ceh-14 is required for sensory neuron differentiation

1999 International Worm Meeting abstract 447 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The LIM homeobox gene ceh-14 is required for sensory neuron differentiation H Kagoshima, G Cassata, TR Burglin Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel The LIM homeobox gene ceh-14 is expressed in a number of sensory neurons and interneurons in the head and tail ganglion, which we identified using GFP reporters. We are particularly interested in the thermosensory AFD neurons and the phasmid neurons PHA and PHB in the tail. We isolated three ceh-14 deletion mutant alleles from a Tc1 insertion line (kindly provided by Dr. Andachi and Dr. Kohara) of this gene locus. The largest deletion allele, ceh-14(ch3), is a putative null mutation and showed no morphological defects and most behaviors were normal (i.e., locomotion, chemo- & touch-sensation, pumping etc.). However, we found thermosensory defects and phasmid dye-filling defects. We could rescue these two phenotypes using full-length ceh-14::gfp reporter constructs. As control, reporter constructs with gfp fused to the 1st exon of ceh-14 did not rescue. The thermosensory defect is not due to missing AFD neurons, since several promoter::gfp reporters (ceh-14, gcy-8, nhr-38, rph-5 and tax-2 (1)) revealed that in a ceh-14(ch3) background the AFD neurons are still present and have dendrites extending to the tip of the nose. However, EM sections suggest mild abnormalities in the AFD fingers (missing fingers, asymmetric dendrite placement). It is known that two other LIM homeobox genes, ttx-3 and lin-11, are expressed in AIY and AIZ interneurons, respectively (2,3). These two interneurons are involved in integration of the thermal information. It suggests that these three LIM homeobox genes define a "functional code" for the thermosensation circuitry, similar yet different from the LIM code for motorneurons in the vertebrate CNS. We showed that the C. elegans LIM domain binding protein, LDB-1, interacts with the LIM domain of CEH-14 in a specific manner in yeast assays (WBG 15(3)). Expression studies of a gfp reporter construct showed that ldb-1 is expressed in many neurons, apparently mainly those ones known to express LIM homeobox genes.

1) reporter constructs were kindly provided by Dr. L. Doolittle, Dr. D. Garbers (gcy-8), Dr. J. Satterlee, Dr. P. Sengupta (nhr-38), Dr. M. Koga, Dr. Y. Oshima (rph-5), Dr. S. Grantner, Dr. C. Bargmann(tax-2) 2) Hobert O., et al. (1998) J. Neuroscience 18: 2084-2096 3) Hobert O., et al. (1997) Neuron 19: 345-357

24 May 1999 15:50 499 499 A screen for new genes involved in osm-9 signaling

1999 International Worm Meeting abstract 448 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A screen for new genes involved in osm-9 signaling AH Kahn, D Tobin, CI Bargmann Howard Hughes Medical Institute and University of California, San Francisco. 513 Parnassus Avenue, San Francisco, CA 94143-0452. osm-9 encodes a predicted cation channel that is involved in multiple C. elegans sensory modalities. Loss of osm-9 function eliminates chemotaxis to the AWA-sensed odorant diacetyl, compromises adaptation to AWC-sensed odorants, and diminishes responses to ASH-sensed noxious stimuli (high osmolarity solutions, nose touch, volatile repellents). osm-9 is homologous to sensory channels in other species, including the Drosophila TRP/TRPL phototransduction channels, and is most similar to the mammalian nociceptive capsaicin receptor, VR1. Although Ca2+ signaling through the TRP/TRPL channels has been well-studied, relatively little is understood about the mechanisms of osm-9 signaling. To that end, we performed a visual screen to identify molecules which interact genetically with osm-9. The screen employed a missense allele of osm-9, which contains a substitution in a conserved residue of an ankyrin protein-protein interaction motif. In anticipation of identifying gain-of-function mutations, we decided to screen for dominant suppressors of osm-9. Approximately 69,000 adult F1 progeny were obtained from mutagenized hermaphrodites, and subjected to a behavioral enrichment for AWA chemotaxis. The enrichment yielded roughly 1,400 F1s. Those animals were then screened for expression of an activity-sensitive Green Fluorescent Protein (GFP) fusion gene that is down-regulated in osm-9 mutants, yielding 61 candidate mutants. Of those, 16 bred true for high-penetrance expression of the GFP reporter. Several of those suppressors also show varying degrees of rescue of the Osm behavioral phenotype. Overall amphid morphology is unperturbed, as evidenced by normal dye-filling of amphid sensory neurons. We are currently mapping these mutants using conventional markers as well as a polymorphism-based approach. We are also characterizing their cellular and behavioral phenotypes in further detail. Ultimately, we anticipate that these mutants will uncover novel elements of sensory signal transduction, and that lessons learned from C. elegans can inform our understanding of sensory processing in other species.

24 May 1999 15:50 500 500 Anteroposterior Axon Guidance Genes

1999 International Worm Meeting abstract 449 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Anteroposterior Axon Guidance Genes B Kahoussi, C Chiu, T Smith, SG Clark Molecular Neurobiology Program, Skirball Institute, NYU School of Medicine, New York, NY 10016 USA The formation of the C. elegans nervous system requires the directed migration of cells and growth cones along the dorsoventral and anteroposterior body axes. To identify longitudinal axon guidance genes, we are conducting several screens for mutants with defects in axon outgrowth and pathfinding along the anteroposterior body axis. Transgenic strains expressing GFP under cell-type-specific promoters were generated to visualize individual neurons and their axons in living animals. PVQ is a pair of bilaterally symmetric neurons (PVQL and PVQR) located in the lumbar ganglia that can be detected in a sra-6::gfp transgenic strain. Each extends a single axon that enters the ventral nerve cord and initially runs along the right side of the hypodermal ridge. At the end of the pre-anal ganglion, PVQL crosses to the left side of the cord and continues to the nerve ring in the head, while PVQR remains on the right side and also extends to the nerve ring. To identify mutants defective in the growth of the PVQ axons, we treated sra-6::gfp animals with the mutagen EMS, isolated behavioral and morphological mutants in the F2 generation, and then examined their progeny for defects in axon extension and pathfinding. We recovered sixty-five mutants from screening 18,000 haploid genomes. Thirty-five mutants lacked or had a reduced expression of GFP in PVQ, suggesting a defect in PVQ cell fate. These mutants had a variable penetrance and, in some cases, also had axon outgrowth defects. Fifteen mutations caused the PVQ axons to terminate their growth prior to reaching the nerve ring. Fifteen mutations caused PVQ axon pathfinding defects, such as the inappropriate extension along the dorsal nerve cord and lateral midline or across the ventral midline. We are currently characterizing these mutants further and continuing our screen for new mutants. Additional screens to identify genes needed for the posteriorly directed growth of the AVA, AVB and AVD axons and the growth of the ventral cord pioneer axons AVG and PVP are also underway. We hope to identify genes needed specifically for longitudinal axon outgrowth and pathfinding as well as genes required generally for all axon extension and guidance.

24 May 1999 15:50 501 501 The cytokinesis defective mutant cyk-3 makes a contractile ring, but it fails to ingress

1999 International Worm Meeting abstract 450 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The cytokinesis defective mutant cyk-3 makes a contractile ring, but it fails to ingress S Kaitna 1 , P Gönczy 2 , T Kaletta 3 , H Schnabel 3 , R Schnabel 3 , T Hyman 2 , M Glotzer 1

1 Research Institute of Molecular Pathology, Dr. Bohr-gasse 7, 1030 Vienna Austria. 2 European Molecular Biology Labs, 69117 Heidelberg, Germany. 3 Institut für Genetik, TU Braunschweig, Spielmannstr. 7 D-38106 Braunschweig, Germany.

Cytokinesis divides one cell into two daughter cells. This process is not well understood at the molecular level. In our lab we are interested in identifying proteins required for cytokinesis and to understand how they contribute to the assembly and function of the contractile ring. We are currently using forward genetics in Caenorhabditis elegans to help identify additional key players. cyk-3(t1525) is a mutant originally identified in a screen for maternal effect embryonic lethal (MEL) mutants on linkage group III (LGIII). cyk-3 homozygous hermaphrodites are viable, but produce only dead embryos. The embryonic lethality is due to a severe defect in cytokinesis. cyk-3 mutant embryos successfully complete karyokinesis, but fail to form a cleavage furrow that separates the daughter cells. Time-lapse analysis of early embryos reveals that neither pseudocleavage nor any furrowing reminiscent of cytokinesis occurs. However, we have examined the distribution of actin throughout the first cell cycle and found that an actin ring assembles at the correct time and place. We have also found that this mutant fails to reorganise actin into an "anterior cap" during pronuclear migration. These observations suggest that this gene might be involved in reorganising actin-based structures. Likewise, its function during cytokinesis might be to allow for the disassembly of the contractile ring which is known to accompany furrowing. The positional cloning of this gene is well underway. The mutation maps to position -1.4cM on LGIII. As this mutant has some features in common with nop-1, we crossed these strains and found that they are not allelic. We are currently trying to rescue the embryonic lethality of cyk-3 by injecting cosmids from this region.

24 May 1999 15:50 502 502 Different expression patterns and activities of the three forms of PHA-4

1999 International Worm Meeting abstract 451 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Different expression patterns and activities of the three forms of PHA-4 JM Kalb, JD McGhee University of Calgary, Calgary, Alberta T2N 4N1 CANADA pha-4 encodes the worm ortholog of the Drosophila Forkhead and mammalian HNF-3 transcription factors and is essential for pharynx and rectum development (1,2). The gene produces three major transcripts that start with exons 1, 2 and 3 and encode three predicted protein products differing at their N-terminal ends (called PHA-4 A, B and C respectively). Embryos immuno-stained with antibodies raised against regions common to all the pha-4 products show that PHA-4 is expressed at high levels in pharynx and rectum nuclei and also at low levels in gut nuclei. Since it is proposed that PHA-4 functions by participating in all transcriptional activation necessary for pharynx development, we are interested in determining if any of the PHA-4 products are specifically expressed in the pharynx and if there are any differences in transcriptional activating abilities of the three products. To address expression patterns, we have now raised two new antibodies to the N-terminal regions of PHA-4 specific to PHA-4 A and to PHA-4 A and B. We have not yet detected expression of these products in N2 embryos. However, when embryos transformed with a PHA-4 reporter gene are stained with these new antibodies, we detect PHA-4 A and B expression in the gut, but not in the pharynx or rectum (the reporter fusion is near the C-terminus of PHA-4 and is expressed in the pharynx, gut and rectum). This suggests that the shortest product, PHA-4 C, is the only product expressed in the pharynx and rectum. The promoter of the pharyngeal myosin myo-2 gene contains an element, called the C-subelement, that is capable of driving reporter gene expression in all cell types of the pharynx and is likely regulated through PHA-4. Using a yeast one-hybrid system, we have now shown that PHA-4 A, B and C all bind to the C-subelement to activate gene expression. However, PHA-4 C appears to be a much weaker activator than either PHA-4 A or B. We are planning to exploit this system to screen for proteins that cooperate with PHA-4 C to increase expression of pharyngeal regulated genes.

1) Horner et al. (1998) Genes Dev 12:1947-52. 2) Kalb et al. (1998) Development 125:2171-80.

24 May 1999 15:50 503 503 tlp-1 encodes a variant TATA-binding protein that is essential for embryogenesis

1999 International Worm Meeting abstract 452 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. tlp-1 encodes a variant TATA-binding protein that is essential for embryogenesis LS Kaltenbach, M Domeier, SE Mango Huntsman Cancer Institute Center for Children and Department of Oncological Sciences, University of Utah, Salt Lake City, Utah, 84112 One component of the basal transcription machinery is a 700kD complex called TFIID. TFIID is composed of TATA-binding protein (TBP; CeTBP in C. elegans1 ) and several TBP-associated factors. The prevailing view is that this complex is expressed in all cells, at all stages, and that it plays an important role in promoter recognition and recruitment of additional components of the basal machinery to target genes. We have identified a variant of TBP (called tlp-1 for TBP-like protein) in a yeast two-hybrid screen designed to identify proteins that interact with conserved C-terminal sequences of the pharynx identity factor PHA-42 . TBP variants have also been found in flies, humans and rodents3 . Given the ubiquitous and fundamental role of TBP during transcription, why do animals need a second TBP-like protein? Our data suggest that tlp-1 has an essential function during embryogenesis. First, we examined tlp-1 expression. We defined the tlp-1 gene structure and constructed a tlp-1::GFP chimera. This gene is expressed widely from early embryogenesis until adulthood. By contrast, CeTBP::GFP expression does not initiate until the 3-fold stage. Second, we used RNA interference to determine the loss-of-function phenotypes of tlp-1 and CeTBP. The phenotype of most tlp-1(RNAi) embryos mimics the effects of blocking RNA polymerase II4 : expression of an early zygotic GFP reporter construct is extinguished, E and MS blastomeres fail to gastrulate normally, and embryos arrest at about the 100-cell stage with little overt differentiation. CeTBP(RNAi) embryos, on the other hand, progress through embryogenesis and arrest as fully differentiated L1 larvae. In summary, we propose that i) PHA-4 activates transcription by targeting the basal transcription machinery via the PHA-4 C-terminus and ii) variant TBP-like proteins play an essential role during embryogenesis to mediate RNA polymerase II-dependent transcription.

1. Lichsteiner and Tjian, 1993. 2. Thanks to Bob Barstead and Phillip James for yeast two-hybrid reagents. 3. Crowley et al., 1993; Hansen et al., 1997; Ohbayashi et al., 1999. 4. Edgar et al, 1994; Powell-Coffman et al., 1996. Thanks to Spencer Wright for technical assistance.

24 May 1999 15:50 504 504 Expression of Dominant Negative Forms of Laminin b: Evidence for a Functional Role for Polymerized Laminin in Basement Membranes.

1999 International Worm Meeting abstract 453 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Expression of Dominant Negative Forms of Laminin b: Evidence for a Functional Role for Polymerized Laminin in Basement Membranes. G Kao, X Ren, W Wadaworth Robert Wood Johnson Medical School, Piscataway NJ Laminin, a major component of all basement membranes (BM) is a heterotrimer of a, b and g subunits which trimerize along their long arms to form a cruciform with three short arms. There are pleiotropic effects on survival, reproduction and behavior when any laminin subunit is missing. Here we address the issue of BM assembly and function. Our understanding of the roles of the subunits during development, allows us to address a long standing issue of whether polymerized laminin is important for a functional BM. Biochemical studies have shown that trimers can polymerize via short arm interactions to form a 2-dimensional network. This can be blocked by adding trimers lacking short arms or by adding short arm fragments to compete for polymerization sites. It is not clear to what extent polymerization takes place in vivo and how important it is for a functional BM. If important, the biochemical studies predict that transgenes expressing short arm deletions in any subunit or over expressing a short arm ought to act in a dominant negative fashion. We tested this hypothesis by using the hsp16-2 promoter to drive expression of truncated versions of lam-1(b subunit) that encode only the short arm or have deletions in the short arm to varying extents. Transgenic embryos exhibit embryonic and L1 lethality with phenotypes similar to those caused by strong epi-1 (a subunit) alleles. Heat shocked L1 animals survive and grow, but show defects in gonad migration and germline development. We see gonad arms without sperm or oocytes, with reduced germ cell proliferation, and with sperm but no oocytes. Gonadal sheath cells provide cues for germline development. In laminin mutants they often peel away from the arms due to defects in the BM. It is likely that perturbation of the BMs by these transgenes has the same effect on the sheath and hence on germline development. We are now examining the integrity of the BM in the heat shocked animals by electron microscopy and by a functional b:GFP fusion which labels all the BMs. These results provide the first evidence that laminin polymerizes in vivo and that polymerized laminin is required for BM functions b.

24 May 1999 15:50 505 505 Characterization of Laminin beta and gamma Genes.

1999 International Worm Meeting abstract 454 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of Laminin beta and gamma Genes. G Kao, C Huang, WG Wadsworth Robert Wood Johnson Medical School, Piscataway NJ 08854 We have characterized laminin b (lam-1) and laminin g (lam-2) genes. The sequencing project has uncovered genes for one isoform each of the two subunits and two isoforms of laminin a, epi-1 and lam-3. (See abstract by Cheng et al.). This indicates that two types of trimers likely are form in basement membranes : aAbg and aBbg. A hypomorphic mutation (rh219) exists in lam-1, whose phenotypes we have described in previous meetings (IWM 1997). Both lam-1(RNAi) and lam-2(RNAi) animals arrest development during morphogenesis. Significantly, this phenotype is identical to the arrest seen in double knockout of epi-1 and lam-3, and is more severe than either knockout alone. We examined the expression patterns of lam-1 and lam-2 using promoter:GFP fusions. Both genes are expressed in body wall muscles, pharynx, vulval muscles, the rectal muscle group and the distal tip cells. The expression patterns of b and gsubunit genes overlap with and are more widespread than either epi-1 or lam-3 alone. Taken together these results suggest that lam-1 and lam-2 are found in all basement membranes and indicate that trimers aAbg and aBbg are formed.

24 May 1999 15:50 506 506 fzr-1, a homologue of fizzy-related/HCT1, appears to be required for cell division control in various tissues

1999 International Worm Meeting abstract 455 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. fzr-1, a homologue of fizzy-related/HCT1, appears to be required for cell division control in various tissues T Karashima, M Sawa, A Sugimoto, M Yamamoto Department of Biophysics & Biochemistry, Graduate School of Science, University of Tokyo, Tokyo 113, Japan Progression through mitosis requires activation of the anaphase promoting complex (APC), which triggers ubiquitin-dependent degradation of specific proteins such as mitotic cyclins. Recent studies have shown that two related WD40 repeat proteins, CDC20/Fizzy and HCT1/Fizzy-related, might be substrate-specific activators of APC. The C. elegans genome sequencing project has identified a C. elegans homolog of fizzy-related (fzr), which we named fzr-1. To understand the role of fzr-1 in the course of development, we have isolated a deletion allele of fzr-1 by the PCR-oriented screen. fzr-1 homozyogotes showed variable phenotypes: 64% of them were arrested at L1-L2, 14% arrested at L3-L4, and the rest (12%) became sterile adults. In the sterile worms, gonad arms were hardly elongated, and no differentiation of uterus and spermathecae was observed. In these unelongated gonads, germ cells appeared to undergo proliferation to some extent, but they failed to complete gametogenesis, producing no mature oocytes. Most fzr-1 sterile adults were associated with the Unc phenotype, suggesting a defect in ventral-cord motor neurons. Missing vulvae and protruded vulvae were also often observed. The apparent defects in somatic gonad, germ cells, ventral-cord neurons and vulva suggest that fzr-1 is required for postembryonic lineage in various tissues. Similar Stu (Sterile and Uncordinated) phenotype has been observed in general cell cycle mutants, such as lin-5, lin-6 and stu mutants, supproting the model that fzr-1 is involved in cell cycle regulation. Notably, whereas loss of fzr function in Drosophila results in an extra cycle of cell division in the epidermis, the fzr-1 defect in C. elegans appears to cause premature arrest of cell division. Developmental stage-specific Northern analysis revealed that expression of fzr-1 is most abundant in embryo, gradually decreases toward L4, and then increases again in the adult stage. The high expression observed in adults is likely to correspond to its expression in oocytes and fertilized embryos. The mRNA level of fzr-1 apparently correlates with the cell cycle activities at each stage, again supporting its involvement in cell cycle regulation.

24 May 1999 15:50 507 507 PLC210, a conserved Ras/Rap1A-associating phospholipase C is required for fertility in C. elegans

1999 International Worm Meeting abstract 456 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. PLC210, a conserved Ras/Rap1A-associating phospholipase C is required for fertility in C. elegans K Kariya, M Shibatohge, Y Liao, C Song, X Gao, T Kataoka Department of Physiology II, Kobe University School of Medicine, Kobe 650-0017, Japan We have isolated a 210 kDa C. elegans phospholipase C (Ce-PLC210) through a two-hybrid screening for LET-60/Ras-binding proteins (WBG 14 #5, 34, 1997, J. Biol. Chem. 273, 6218, 1998). We have recently isolated its human homolog (Hs-PLC210) as well. Both of them possessed the X, Y and C2 domains highly homologous to those of the phosphoinositide-specific PLC (PI-PLC) and hydrolyzed phosphatidylinositol 4,5-bisphosphate in vitro. However, PLC210 possessed two additional functional domains unseen in any known PI-PLCs. One was the C-terminal Ras-associating (RA) domain bearing a structural homology with those of RalGDS and AF-6. This domain associated in vitro with human Ha-Ras and Rap1A in a GTP-dependent manner and competed with yeast adenylyl cyclase for binding Ha-Ras. The binding was abolished by specific mutations within the effector region of Ha-Ras. The other was the N-terminal CDC25-like domain, which possessed a structural homology to guanine nucleotide exchange proteins for Ras such as S. cerevisiae CDC25 and D. melanogaster Sos. Deletion mutations of the PLC210 gene in C. elegans resulted in sterility similar to that of a mutation of the gene for LET-23/EGF receptor, and the phenotype was rescued by transgenic expression of the wild-type PLC210 gene. These results suggest that PLC210 belongs to a novel class of PI-PLC, which is a putative effector of Ras or Rap1A conserved through evolution.

24 May 1999 15:50 508 508 Investigation into the lin-12 mediated feedback loops regulating the AC/VU decision

1999 International Worm Meeting abstract 457 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Investigation into the lin-12 mediated feedback loops regulating the AC/VU decision X Karp 1 , I Greenwald 2

1 Department of Genetics and Development, Columbia University College of Physicians and Surgeons, New York, NY 10032. 2 HHMI and Department of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York, NY 10032.

The AC/VU decision provides a simple model system to study lateral specification during development (reviewed in 1). Two cells of the somatic gonad, Z1.ppp and Z4.aaa, have equivalent developmental potential in that either one may become the anchor cell (AC) or a ventral uterine precursor cell (VU). LAG-2, a ligand of the DSL family, and LIN-12, a receptor of the LIN-12/Notch family, mediate interactions between Z1.ppp and Z4.aaa, so that only one AC is formed. Genetic mosaic analysis has shown that the cell with higher lin-12 activity will always take on the VU fate, and suggested that a feedback mechanism amplifies a difference in lin-12 activity (2). Expression studies have further shown that feedback occurs at the level of transcription during the AC/VU decision. Specifically, before the decision, both Z1.ppp and Z4.aaa express lin-12 and lag-2. As the decision progresses lin-12 transcription becomes restricted to the presumptive VU whereas lag-2 transcription becomes restricted to the presumptive AC (3). The expression studies suggest the existence of two types of feedback loop within the receiving cell: the downregulation of lag-2 transcription and the upregulation of lin-12 transcription, both occurring only upon lin-12 activation. We are interested in identifying other components of these feedback loops. We are beginning by examining candidate genes based on homology to genes in Drosophila, where a negative feedback loop that represses Delta expression upon Notch activation has been described (1). In the future, we hope to undertake genetic screens for new genes that are involved in feedback loops during the AC/VU decision.

1. I. Greenwald, Genes & Development 12, 1751-1762 (1998). 2. G. Seydoux, and I. Greenwald, Cell 57, 1237-1245 (1989). 3. H. A. Wilkinson, K. Fitzgerald, and I. Greenwald, Cell 79, 1187-1198 (1994).

24 May 1999 15:50 509 509 The EGL-3 prohormone convertase modulates ASH-interneuron synapses

1999 International Worm Meeting abstract 458 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The EGL-3 prohormone convertase modulates ASH-interneuron synapses J Kass, P Kim, JM Kaplan UC Berkeley MCB LSA 361-3200 Berkeley, CA 94720 How do animals distinguish between sensory stimuli? We are studying a neural circuit in C. elegans in which the ASH sensory neuron, detect noxious chemical and mechanical stimuli and signal to the worm to move backward. Three stimuli are detected by the ASH neurons: nose touch, high osmolarity and volatile repellants. Genetic and molecular evidence suggests that different signals are produced at the ASH-interneuron synapses in response to the different stimuli. GLR-1 glutamate receptors are expressed in interneurons, are clustered at ASH-interneuron synapses, but are required specifically ASH-mediated touch sensitivity. We isolated egl-3(nu349) as a suppressor of glr-1(n2461) touch insensitivity, Alleles of egl-3 cause three phenotypes: they are egg-laying defective, insensitive to gentle touch to the body, and they suppresses the nose touch defect of glr-1. The egl-3 mutation restores function of the ASH-interneuron synapses because glr-1(n2461); egl-3(nu349) double mutants became nose touch defective after ablation of ASH. The egl-3 mutants were also defective for habituation of the nose touch response. We have cloned egl-3, and found that it encodes a homolog of Prohormone Convertase 2, a neuroendocrine-specific protease (C51E3.7). Two egl-3 alleles correspond to mutations in predicted exons of C51E3.7: n150ts contains two missense mutations, in the catalytic domain and the homoB domain; nu349 contains a missense mutation in the homoB domain. The homoB domain has been shown to be required for maturation and secretion of the protease (Taylor et al., 1998). Both alleles are recessive. These results suggest that a neuropeptide (processed by EGL-3) inhibits ASH-interneuron signaling, and is involved in habituation to repeated sensory stimuli.

24 May 1999 15:50 510 510 C. elegans homologue of Sra-1, a target of Rac GTPase, is involved in hypodermal morphogenesis

1999 International Worm Meeting abstract 459 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. C. elegans homologue of Sra-1, a target of Rac GTPase, is involved in hypodermal morphogenesis K Kasuya 1 , H Qadota 1 , M Soto 2 , C Mello 2 , K Kaibuchi 1

1 Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0101, Japan. 2 University of Massachusetts Medical Center, Worcester, MA 01605, USA.

The Rho family small GTPases (Rho, Rac, Cdc42) regulate cell motility and cell polarity through the rearrangement of actin cytoskeleton and through cell adhesion. We have previously identified Sra-1 as an effector for Rac1 GTPase in mammalian cells. Sra-1 directly interacts with GTP-bound Rac1 in vitro, but little is known about the physiological functions of Sra-1. Here, we examined the physiological functions of Sra-1 using C. elegans. We identified CeSra-1 as a C. elegans homologue of Sra-1 (51% identical to human Sra-1). To study CeSra-1 functions, we used RNA-mediated interference. We found that CeSra-1(RNAi) shows about 100% embryonic lethality with failure of the hypodermis to enclose embryos. In CeSra-1(RNAi) embryos visualized by MH27 immunostaining, differentiation of the hypodermal cells seems to be normal, but the dorsal intercalation and ventral enclosure did not occur. To investigate further the function of CeSra-1, we observed the RNAi phenotype of C. elegans homologues of the possible Sra-1 associated proteins previously identified in mammalian cells. Among them, only CeHEM-2(RNAi) shows a similar phenotype to that of CeSra-1(RNAi). Since the mutant allele of CeHEM-2, inx-1, was isolated by Soto et al. (this meeting), we named CeSra-1 inx-2. We raised antibodies to both proteins. Based on immunostaining results, both CeSra-1/INX-2 and CeHEM-2/INX-1 are expressed at a minimum in the hypodermis during the embryogenesis. Furthermore, we found that CeSra-1/INX-2 interacts with CeHEM-2/INX-1 in the two-hybrid system. Taken together, these results suggest that CeSra-1/CeHEM-2 complexes are involved in hypodermal morphogenesis.

24 May 1999 15:50 511 511 Immune defense of C.elegans: abf operon, ASABF type antimicrobial peptide genes

1999 International Worm Meeting abstract 460 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Immune defense of C.elegans: abf operon, ASABF type antimicrobial peptide genes Y Kato National Institute of Sericultural and Entomological Science, Tsukuba, Ibaraki 305-8634, Japan The immune defense system of C. elegans remains to be elucidated. To find a clue, we are searching for the immune effector molecules in C. elegans , combined with the biochemical analysis of the big nematode, Ascaris suum . At least four immune effector proteins have been identified in Ascaris body fluid: a peptide that inhibits bacterial growth (ASABF), a bacteriolytic protein (ASBLF), and two proteins that agglutinate bacteria (ASAGF-1 and 2). The antimicrobial peptide, ASABF, was purified and its complete amino acid sequence was determined. A cDNA encoding ASABF was also cloned, and further screening of a cDNA library indicated that ASABF forms a family of at least four members (ASABF-alpha, beta, gamma, and delta). ASABF is a basic peptide with four intramolecular disulfide bridges and is effective especially against Gram positive bacteria. Database searches revealed two ASABF homologs ( abf-1 and 2 ) in C. elegans . abf-1 and 2 seem to form a typical operon, and their polycistronic RNA was experimentally detected. The transcript of the downstream gene, abf-2 , was exclusively trans-spliced with SL1, although a spacer sequence with 78 bp was found between the poly A additional site of abf-1 and the SL1 acceptor site of abf-2 . The abf operon was expressed in the pharynx and pharyngeal neurons. Since live bacteria are concentrated in the pharynx due to normal feeding, we speculate that ABF might defend the pharyngeal tissue against bacterial infection from the lumen, and/or help with the digestion of ingested bacteria.

24 May 1999 15:50 512 512 Structure of Neural Network of C. elegans Observed by a Random Walker

1999 International Worm Meeting abstract 461 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Structure of Neural Network of C. elegans Observed by a Random Walker K Kawamura 1 , S Morita 2 , K Oshio 2 , Y Funabashi 1 , Y Osana 3 , K Oka 4

1 Department of Physics, Faculty of Science and Technology, Keio University, Hiyoshi 3-chome, Kohoku-ku, Yokohama, 223-8522 Japan. 2 "Future Research" Project, Faculty of Science and Technology, Keio University, Hiyoshi 3-chome, Kohoku-ku, Yokohama, 223-8522 Japan. 3 Department of Electrical Engineering, Faculty of Science and Technology, Keio University, Hiyoshi 3-chome, Kohoku-ku, Yokohama, 223-8522 Japan. 4 Department of System Design Engineering, Faculty of Science and Technology, Keio University, Hiyoshi 3-chome, Kohoku-ku, Yokohama, 223-8522 Japan.

A database of synaptic connectivity of 302 neurons of the C. elegans has been constructed[1] from the observations of Albertson and Thomson[2] and White et al.[3] by some of the present authors. A network formed by 302 neurons of the C. elegans is represented on a computer by a network which consists of 302 dots combined by (arrowed) bonds. To analyse the structure of the neural network, behavior of a random walker on it is studied. The walker is displaced among dots which represent neurons over bonds which model synaptic connection. In terms of walking distance defined by minimum time steps which is necessary for the random walker to be displaced between neurons, distances among all neurons, whose synaptic connectivity are described by the above authors, have been determined. Almost all neurons are located within the walking distance of three time steps but walking distance among phalingeal neurons and somatic neurons are more than four time steps. The network is extended in a (more than) nine dimensional space around three nanohedra which are mutually combined by manifolds of less dimension. Each nanohedron consists of nine dots representing interneurons mutually connected by synapses and these nanohedra are located near the center of the network. The lattice is biased by the rectification of the chemical synapse in the sence that a random walker prefers to be displaced from sensory neurons to motor neurons.

[1] K. Oshio, S. Morita, Y. Osana and K. Oka: C. elegans connectivity data, Technical report of CCEP, Keio Future No.1 (1998) [2] D. G. Albertson and J. N. Thomson: Phil. Trans R. Soc. Lond. B. 275 (1976) 299 [3] J. G. White, E. Southgate, J. N. Thomson and S. Brenner: Phil. Trans. R. Soc. Lond. B 314 (1986) 1.

24 May 1999 15:50 513 513 A new mutant which affects longitudinal and circumferential migrations of distal tip cells

1999 International Worm Meeting abstract 462 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A new mutant which affects longitudinal and circumferential migrations of distal tip cells T Kawano, JG Culotti Samuel Lunenfeld Research Inst., Mount Sinai Hosp., Toronto Ont. Canada M5G1X5 The U-shaped gonad arm of C. elegans hermaphrodite is created by the sequence of longitudinal-dorsal-longitudinal migrations of distal tip cells (DTCs). Previously known circumferential axon guidance mutants such as unc-5, 6, and 40 affect specifically the dorsalward guidance of DTC. We have identified a new mutant which exhibits a semi-dominant defect on dorsal migrations of DTCs. Interestingly homozygotes of the mutant show various patterns of supernumerary turns of gonad suggesting disruption of both longitudinal and circumferential guidance. The linker cell of homozygote males also makes supernumerary turns and often fails to reach the tail. The mutant animals are not Unc and no abnormal axon trajectories have been observed. The pathway involving the mutant may be specific for guiding DTC. Homozygotes give a small brood size and lay dead eggs, suggesting some extent of maternal effect embryonic lethality.

24 May 1999 15:50 514 514 Analysis of SR protein genes in C. elegans

1999 International Worm Meeting abstract 463 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of SR protein genes in C. elegans T Kawano, M Fujita, H Sakamoto Department of Biology, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nadaku, Kobe 657-8501, Japan SR proteins contain one or two copies of RNA recognition motif (RRM) as RNA-binding domain and an arginine/serine-rich (RS) domain. Previous in vitro studies have revealed that mammalian SR proteins have essential roles in constitutive splicing of pre-mRNAs. In addition, some SR proteins have been shown to play an important role in splice site selection. To investigate the in vivo function of SR proteins, we first searched the C. elegans genome database and found five genes potentially encoding SR protein (ceSRp20, ceSC35, T28, W12.2 and W12.3). We next examined, using transgenic worms, the expression of these SR proteins fused with green fluorescent protein (GFP) under the control of their native promoters. All of these SR-GFP fusion proteins were localized in the nuclei of almost all somatic cells, consistent with that SR proteins are constitutive splicing factors as in mammalian cells. However, simultaneous suppression of two or more SR protein-coding genes by dsRNA-mediated interference resulted in drastic phenotypes, such as embryonic lethality, abnormal gonadal formation and decrease of fertility. These results raise the possibility that SR proteins also play important roles in the development and the germ cell differentiation in C. elegans. Disruption of genes encodong SR proteins and a search for target genes whose mRNA expression are affected in such mutants are currently in progress.

24 May 1999 15:50 515 515 CDC-42 is Required for Polarity in the Early Embryo

1999 International Worm Meeting abstract 464 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. CDC-42 is Required for Polarity in the Early Embryo AJ Kay, CJ Hunter Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138 To identify genes important for divisions of polarized cells in early C. elegans embryos, we are using RNA interference (RNAi) to investigate the function of conserved cytoskeleton regulators. The rho GTPase CDC-42 regulates cytoskeletal organization in several systems, including T-cells and budding yeast. We have found that RNA inhibition of cdc-42 disrupts polarization in the early embryo, including the extent of pseudocleavage and spindle orientations at the two-cell stage. In wild-type 2-cell embryos, the smaller P1 blastomere divides along the long axis of the embryo while the larger AB cell divides transverse to this axis. In 50% of cdc-42(RNAi) embryos, the spindle orientations in the larger and smaller blastomeres are reversed. Other spindle orientations also occur. Molecular and genetic studies have shown that PAR-3 protein is primarily localized to AB and is required to inhibit spindle rotation. In par-3 2-cell embryos both spindles are arranged longitudinally. We find that in cdc-42(RNAi) embryos, PAR-3 localizes to P2 rather than AB. To test whether PAR-3 is required for the variable spindle orientations in cdc-42 inhibited embryos, we used RNAi to inhibit both cdc-42 and par-3. To our surprise, many of the symmetrically divided embryos (par-3 phenotype) displayed cdc-42(RNAi) phenotypes for spindle orientations. Spindle orientations were also investigated in par-1(RNAi) cdc-42(RNAi) embryos. In both par-1 mutant embryos and par-1(RNAi) cdc-42(RNAi) embryos the spindle orientations are as in wild type. This result shows that par-1 is required for the reverse spindle orientations observed in cdc-42(RNAi) embryos. Together these results indicate that cdc-42(RNAi) may disturb spindle orientations both through mislocalizing PAR-3 and through the activity of PAR-1.

24 May 1999 15:50 516 516 Do Impaired Mitochondria of the Nervous System Cause Hypersensitivity to Volatile Anesthetics in C.elegans?

1999 International Worm Meeting abstract 465 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Do Impaired Mitochondria of the Nervous System Cause Hypersensitivity to Volatile Anesthetics in C.elegans? EB Kayser, PG Morgan, MM Sedensky Depts. of Genetics and Anesthesiology, Case Western Reserve University and University Hospitals, Cleveland OH 44106 USA The mutant gas-1(fc21) is hypersensitive to volatile anesthetics. In air, the worm moves normal but has a reduced brood size and life span. gas-1 has been identified by sequence homology to encode a subunit (49kDa IS-protein) of the mitochondrial respiratory chain complex I (NADH dehydrogenase). The mutation changed an evolutionarily conserved amino acid probably impairing mitochondrial function. Here, we present two attempts to localize where mutant GAS-1 potentiates anesthetic action. The most plausible tissues are the nervous system or body wall muscles. Since the GAS-1 protein functions intracellularly the target tissues for volatile anesthetics are expected to be among those expressing gas-1. The expression pattern was studied in transgenics which express the fluorescent reporter EGFP under the control of the gas-1 promoter. Strong fluorescence was found in nerves, pharyngeal muscle, body wall muscle, and the posterior end of the gut indicating gas-1 is normally expressed in these tissues. An EGFP::GAS-1 fusion rescued the mutant indicating the anesthetic sensitive tissue was covered by the transgenic expression. Finding broad expression of gas-1 was not trivial because an isogene exists encoding a nearly identical protein suggesting differential expression of the two genes. In C. elegans two drugs have been employed to distinguish between pre- and postsynaptic sites of genetic or pharmacological actions. Resistance to aldicarb alone indicates a presynaptic block while resistance to both aldicarb and levamisole points to a postsynaptic block. gas-1(fc21) tolerates twice the concentration of aldicarb than N2 but does not react differently to levamisole. Thus the mutation has a stronger effect on the presynaptic nerves than on the postsynaptic muscles. The expression of gas-1 does not rule out any tissue as the place where mutant GAS-1 potentiates anesthetic action. However, the mild resistance to aldicarb but indifference to levamisole indicates that it mediates hypersensitivity to volatile anesthetics in the nervous system rather than in muscle.

24 May 1999 15:50 517 517 Innexins play a role in modulating the pharyngeal pumping rate of dauer larvae.

1999 International Worm Meeting abstract 466 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Innexins play a role in modulating the pharyngeal pumping rate of dauer larvae. JP Keane, L Avery Dept. of Molecular Biology and Oncology, UT Southwestern Med. Center, 6000 Harry Hines Blvd, Dallas, TX, 75235-9148 Dauer larva pharynxes pump at a rate of 0.3 pumps/minute compared to 200 pumps/min in adults (nervous system mediated pumping-neurogenic) and 45 pumps/min in adults wherein the pharyngeal nervous system has been ablated (muscle mediated pumping-myogenic). To elucidate how neurogenic and/or myogenic pumping are regulated we initiated a forward genetic screen to find dauer larvae with elevated pharyngeal pumping rates. One resulting mutant was found to be an allele of the previously cloned innexin gene unc-7. unc-7 mutant dauers pump 3-5 times faster than wild type under normal conditions but can be induced to pump up to 10 times faster when the animal is tapped on its posterior. When unc-7 dauers are placed in exogenous serotonin the pumping rate can be increased to 80 pumps/min whereas wild type levels are not increased under these conditions. A number of observations suggest that unc-7 mutations affect the neurogenic regulation of pumping. Firstly, EPG recordings reveal a large number of I-phase spikes, which are believed to arise through motor neuron MC activity. Secondly, combinations of unc-7 with eat mutants which reduce MC neurotransmission to the pharynx (eat-2 and eat-18) reduce the pumping rate. Neurogenic involvement is currently being tested by laser ablating MC and examining the pumping rate of operated animals. Reduction of function mutations of goa-1 have also been observed to have dauer pumping rates similar to those of unc-7 (both in exogenous serotonin) and we are examining the possibility that both might function in the same pathway by constructing double mutants. There are two main proposals regarding how an innexin may be involved in the neurogenic modulation of dauer pumping rates. The first draws from observations that unc-7 mutations can result in synapse formation between inappropriate neurons in the locomotory circuit. MC may therefore be synapsing with a neuron which cannot suppress MC activity in the dauer. The second proposal is that in wild type dauers MC is clamped in an "off" state, either electrically or through messenger molecules diffusing through the gap junction. In the unc-7 mutant, which presumably has reduced coupling, MC remains partially active. Myogenic involvement has not been ruled out.

24 May 1999 15:50 518 518 Two of the five isoforms of protein synthesis initiation factor eIF4E, which distinguish between mono- and trimethylated mRNA caps, are essential for viability in C. elegans

1999 International Worm Meeting abstract 467 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Two of the five isoforms of protein synthesis initiation factor eIF4E, which distinguish between mono- and trimethylated mRNA caps, are essential for viability in C. elegans BD Keiper 1 , BJ Lamphear 1 , EJ Aamodt 1 , M Jankowska 2 , A Deshpande 3 , T Blumenthal 3 , RE Rhoads 1

1 Biochemistry and Molecular Biology, LSU Medical Center, Shreveport, LA 71130. 2 Dept. of Chemistry, Univ. of Warsaw, Warsaw, Poland. 3 Biochemistry and Molecular Genetics, Univ. of Colorado HSC, Denver, CO 80262.

The recognition of the 5’-cap structure of mRNA by the eukaryotic translation initiation factor 4E (eIF4E) is a critical step in the recruitment of most mRNAs to the ribosome. In C. elegans, mRNAs from 70% of protein-encoding genes contain an unusual 2,2,7-trimethylated GTP (TMG) cap structure, while the remaining 30% contain a canonical 7-methylated GTP (MMG) cap. TMG-capped mRNAs arise from trans-splicing of small nuclear RNAs (either SL1 or SL2) onto the 5’ end of the processed transcripts. We have identified five expressed genes encoding eIF4E in the C. elegans genome (ife-1, ife-2, ife-3, ife-4 and ife-5) and cloned and sequenced corresponding cDNAs. Proteins IFE-1 and IFE-5 bind both TMG and MMG caps, while IFE-3 and IFE-4 bind only MMG caps. IFE-2 shows a strong affinity for MMG and a weak affinity for TMG. Sequences of the TMG-binding proteins, IFE-1 and IFE-5, are highly homologous and similar to IFE-2. Each of the strictly MMG-binding proteins, IFE-3 and IFE-4, are closely related to different human cap-binding proteins [eIF4E-1 and eIF4E-3 (also called 4EHP)] and therefore quite divergent from one another. Systematic knockout of each of the IFE’s using RNAi indicates that only IFE-1 and IFE-3 are required for the continued viability of worms and suggests that one eIF4E of each binding specificity is essential for translation of C. elegans mRNAs. Together, these five isoforms may provide a means to differentially regulate the translation initiation of mRNAs bearing different cap structures and/or splice leaders. Interestingly, most of the ife mRNAs are themselves trans-spliced, as indicated by the presence of SL sequences. IFE-4, which is the most divergent from the others, appears to be encoded by an SL2-containing mRNA, indicating that its gene may reside in a polycistronic gene cluster.

24 May 1999 15:50 519 519 A class VI myosin has a role in fertility

1999 International Worm Meeting abstract 468 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A class VI myosin has a role in fertility JF Kelleher 1 , GG Moulder 2 , R Barstead 2 , MA Titus 1

1 University of Minnesota, Minneapolis, MN. 2 Oklahoma Medical Research Foundation, Oklahoma City, OK.

Class VI myosins are essential for hearing in mammals and are thought to be vesicle motors in Drosophila, where they play roles in spermatogenesis and cellularization during embryogenesis. Myosin VI localizes to the brush border of epithelial cells in mammalian kidney and to the leading edge and Golgi network in fibroblasts. The C. elegans genome contains two class VI myosins encoded by hum-3 (heavy chain of unconventional myosin, J. Molec. Biol. 272: 523) and hum-8. We have used chemical mutagenesis of large populations of animals coupled with PCR detection using gene-specific primers and multiple rounds of sib selection to isolate single animals with deletion mutations within the class VI myosin genes. The deletion in hum-3 removes 2 kb from the core of the motor domain and is most likely a loss-of-function allele. The 1kb hum-8 deletion is also within the motor domain. These strains are presently being outcrossed to remove background mutations. Preliminary evidence indicates that homozygous hum-3 mutant animals are partially to completely sterile, producing 0 to 4 progeny while heterozygous hum-3/+ siblings produce normal broods. DIC observation of sterile animals reveals that oocytes are packed tightly in the gonad but do not divide and thus are unlikely to have been fertilized. Morphology of the spermatheca is abnormal. The hum-3 promoter was used to drive expression of the green fluorescent protein (GFP) in cells which normally express hum-3. GFP is expressed in a small number of cells which appear to be neurons based on the long thin processes they extend along the length of the animal, as well as in a few cells within the pharynx and in the tail. We are in the process of identifying these cells. No expression was evident in the gonad, where the transgene may be silenced. Complex array injections are in progress to look for germ line expression of hum-3. hum-8 promoter fusions are expressed predominately in the gut of the worm, as well as in the pharynx and in other, as yet unidentified, cells. Phenotypic analysis of hum-8 mutant animals is in progress. These mutants are not sterile. The hum-3 and hum-8 mutants provide a unique opportunity to determine the full range of class VI myosin function in a multicellular organism.

24 May 1999 15:50 520 520 A search for genes that interact with unc-31 or act in the same pathway

1999 International Worm Meeting abstract 469 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A search for genes that interact with unc-31 or act in the same pathway LA Khan, K Iwasaki Lab of Molecular Neurobiology, National Institute of Bioscience and Human-Technology, 1-1 Higashi, Tsukuba-305, Japan We are interested to study synaptic transmission by focusing on unc-31 in C. elegans . Several lines of evidence suggest that unc-31 plays a role in synaptic transmission. Firstly, unc-31 animals are defective in a variety of behaviors including locomotion, egg-laying, pharyngeal pumping and dauer recovery. Secondly, unc-31 is expressed in a variety of neurons. Thirdly, UNC-31 protein is the C. elegans homolog of mammalian CAPS (Calcium-dependent Activator Protein for Secretion): in PC12 cells, CAPS is necessary for DCV (Dense Core Vesicles) exocytosis but not for SV (Synaptic Vesicle or Small Clear Vesicles) exocytosis. DCVs contain catecholamins, neuropeptides or hormones, whereas SVs contain classical small neurotransmitters such as acetylcholin . These observations suggest that unc-31 may play a role in DCV exocytosis. To elucidate the possible mechanism of unc-31 action we are using both molecular and genetic approaches to identify molecules that potentially interact with UNC-31 or function in the same pathway. Using chemical mutagenesis we isolated 45 revertants of the unc-31(e928) mutant; all revertants can reverse the lethargic locomotory phenotype of unc-31 mutants. Some revertants are hyperactive while others resemble the wild type locomotory phenotype. Using the yeast two-hybrid system we isolated a novel gene (C18B2.5) from a C. elegans cDNA library using unc-31 cDNA as bait. Presently, we are characterizing the unc-31 revertants and this novel gene.

24 May 1999 15:50 521 521 OSM-3 Subfamily of the Kinesin Motors in C.elegans

1999 International Worm Meeting abstract 470 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. OSM-3 Subfamily of the Kinesin Motors in C.elegans ST Khan, MY Ali, SS Siddiqui Toyohashi University of Technology Previously our group has cloned C. elegans osm-3 gene by transformation rescue of the osm-3 mutants (Shakir et al., 1993), and shown that it shares homology with the mouse KIF3A/KIF3B (Hirokawa, 1996) and sea urchin KRP85/95 (Scholey, 1996) heteromeric kinesins. We also showed using an osm-3:: lacZ reporter gene that it expresses in an exclusive set of 26 chemosensory neurons, that are exposed to the outside environment through a hole in the animal cuticle (Tabish et al.; 1995). In a search for heterotrimeric kinesin proteins, Kinesin-II, as proposed by Scholey, 1996; we have identfied and sequenced new cDNA clones which encode KLP-10, KLP-11, and KLP-18, kinesins which share high homology with OSM-3 in the motor domain. Following the suggestion of Wademan et al., 1996, we have also cloned and sequenced a cDNA encoding nematode KAP-1, that harbors ten repeats of the armadillo motif (Khan et al., 1999; submitted). Here we report that the OSM-3 homolog KLP-11 on Northern blot analysis reveals two distinct bands, the larger of the two bands is about to 4.5 kb and the other is about 2.6 kb, suggesting mutiple transcripts of the klp-11 gene. On the other hand, kap-1 cDNA encodes a transcript of 2.1 kb and appears to encode a single class of mRNA. Gene expression studies using the embryonic in situ hybridization reveals that the osm-3 and kap-1 display similar pattern of expression in the head and tail ganglia, corresponding to the sensory neurons. Post-embryonically this expression remains high in the head and tail of the developing larvae. However, expression of klp-11 is much broader, it expresses in motor neurons and other cells, in addition to the osm-3 expressing sensory cells. The other two members of this family, KLP-10 and KLP-18, are highly homologous to each other (96%), but differ significantly from OSM-3 and KLP-11 kinesin, sugggesting a subfamily within the OSM-3 family. Data on promoter gene fusion and immunocytochemistry of some of these members will be presented.

We thank M.L. A. Khan, Y. Kohara, J. Miwa, Y. Ohshima, A. Otsuka, D. T-Mieg, and S. Jones for support. Funds were provided by Monbusho in support of basic research to SSS.

24 May 1999 15:50 522 522 Genetic analysis of lj22, lj21 and lj10 genes involved in nicotine adaptation in C. elegans

1999 International Worm Meeting abstract 471 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic analysis of lj22, lj21 and lj10 genes involved in nicotine adaptation in C. elegans J Kim, W Schafer Depts. of Neuroscience and Biology, UC San Diego, La Jolla, CA 92037 Long-term nicotine treatment causes adaptation, characterized by desensitization, attenuation, and functional inactivation of receptors while levels are increased. We wish to use genetic analysis in C. elegans to study the molecular mechanism behind nicotine adaptation. Our lab has identified two ways to characterize nicotine adaptation in C. elegans: by a muscle phenotype mediated by nAChRs in body muscle, and an egg-laying phenotype mediated by receptors in both the vulval muscles and the VC neurons (see L. Waggoner poster). Acute treatment of wild type worms with nicotine or levamisole causes complete paralysis and muscle contraction, while long-term treatment renders them tolerant, restoring their muscle function and normal body length. Acute treatment with agonist also stimulates egg-laying in wild type worms, while long-term treatment diminishes this response. The lev-1 mutant shows weak resistance to the effects of nicotine. By using the muscle phenotype, our lab has identified mutants that suppress the lev-1 resistance and restore nicotine sensitivity. lj21, lj22 and lj10 are such lev-1 suppressor mutants. The lev-1 background has been recently crossed out of these mutants, and we see that lj21 and lj22 display hypersensitivity to nicotine and defects in ability to adapt to its muscle phenotypes. We plan to study next the egg-laying phenotype of all three mutants. Of particular interest is lj22 because it has been shown that this mutant displays defects in both the muscle and egg-laying behaviors. Additionally, lj22 has a very interesting movement phenotype: it generates motion, yet is unable to travel over a distance. This phenotype is mostly observed during larval and early-adult stages while eggs are being laid, and is no longer observed in older adults. lj22 is also characterized by a shorter length and dumpier body, and males show defects in mating. This gene has been shown to be linked to the X-chromosome, and further mapping studies will be performed with the intent to clone the gene. Hopefully, this will enable us to further our understanding of nicotine adaptation on the molecular level.

24 May 1999 15:50 523 523 Function of flp neuropeptide gene family

1999 International Worm Meeting abstract 472 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Function of flp neuropeptide gene family K Kim Department of Biology, Boston University, MA 02215 FMRFamide-related peptides (FaRPs) are short neuropeptides that have been found throughout the animal kingdom. FaRPs have been shown to have many general functions, including cardioregulation, muscle control, pain modulation and learning. In C. elegans, at least 50% of the neurons are FMRFamide-like immunoreactive. To date, at least 20 genes, flp-1 through flp-20, encoding these peptides have been identified in C. elegans. To determine the cell specific expression of the flp genes in C. elegans, transgenic animals expressing a gfp (green fluorescent protein) reporter construct under the control of each flp promoter were generated. To date, flp-3, flp-5, flp-8, and flp-12 cell specific expression has been determined. flp-3-expressing cells include three pairs of neurons (IL1D, OLL, URB), and one unpaired neuron (PQR). flp-5-expressing cells include two pairs of neurons (ASE, RMG) and one unpaired neuron (I4). flp-8-expressing cells include two pairs of neurons (ASE, URX) and one unpaired neuron (PVM). flp-12-expressing cells include a set of four motorneurons (SMB) and two unpaired neurons (AVM, PVM). flp-2, flp10, and flp-15 cell specific expression patterns are being determined. To determine the function of the flp genes in C. elegans, several flp deletion mutants, including flp-3, flp-7, flp-8, and flp-10, were isolated by screening Ron Plasterk’s C. elegans EMS-mutagenized library. The size of the deletion in flp-3 or flp-8 deletion mutants is 2.6 kbp or 1.4 kbp, respectively, and deletes the entire coding region of both genes. Both flp-3 and flp-8 deletion mutants have not shown any phenotype so far, suggesting that these genes have overlapping functions with other flp genes. A flp-10 deletion mutant is being backcrossed. In addition, transgenic worms containing a flp-1, flp-3, or flp-8 cDNA under the control of the neural specific promoter were generated. These transgenic worms are being characterizing. To identify genes that may regulate expression of the flp genes, we are using transgenic animals expressing a gfp reporter construct under the control of flp-1 or flp-12 promoter in modifier screens. These worms are being mutagenized, and mutants showing altered expression of these markers are being isolated.

24 May 1999 15:50 524 524 alt-1, a Gene Required for Patterning Longitudinal Axon Tracts at the Midlines

1999 International Worm Meeting abstract 473 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. alt-1, a Gene Required for Patterning Longitudinal Axon Tracts at the Midlines S Kim, WG Wadsworth Robert Wood Johnson Medical School, NJ 08854 During development, a simple scaffold of longitudinal and circumferential axon tracts forms. We undertook a genetic screen to identify genes required for this patterning and we have isolated 11 new mutants. One of these, alt-1 (ur41), was identified because dorsal sublateral (DSL) nerves form along the dorsal midline and the ventral nerve cord is abnormally symmetric. By light or electron microscopy, there are no other obvious defects in morphology. The alt-1(ur41) animals have several novel features. First, alt-1 is required for patterning only a subset of neurons along the midlines. Second, mispositioned axons do not wander in alt-1(ur41), instead they generally stay in the wrong tract. For example, DSL nerves are positioned to either the sublateral position or along side of the dorsal midline. In 45% of the mutants, either the left or right tract is mispositioned and in 45%, both are mispositioned. These results suggest that alt-1 is required to prevent these nerves from forming along the dorsal midline. Third, alt-1 (ur41) affects the ability of axons to initially select the right or left side of the midline, however it does not affect their ability to respond to the signals that keep axons to either side. At the ventral midline, the PVQL axon abnormally crosses to the right tract and the left fascicle from the nerve ring sometimes fails to cross to the right ventral cord tract. Ventral midline motoneurons often send their longitudinal axons to the left side of midline instead of to the right side. Furthermore, at the dorsal midline, the motor axons from the right side often fail to immediately cross to the left side and migrate instead along the right side of the midline. As a result of these defects, nerve cords become abnormally symmetric. This suggests that alt-1 is required for the asymmetric distribution of longitudinal axon tracts at the midlines. Interestingly, unc-40 and sax-3 partially suppress alt-1(ur41) phenotypes, indicating possible involvement of these genes in the same process. The alt-1(ur41) phenotypes are rescued by germ line transformation with a single cosmid DNA that is predicted to contain 3 genes.

24 May 1999 15:50 525 525 A Telomere Binding Protein in the Nematode C. elegans.

1999 International Worm Meeting abstract 474 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A Telomere Binding Protein in the Nematode C. elegans. S Kim, J Lee Yonsei University Telomeres are multifunctional elements that shield chromosome ends from degradation and end-to-end fusions, prevent activation of DNA damage checkpoints, and modulates the maintenance of telomeric DNA by telomerase. In normal cells, telomeres shorten with successive cell divisions, probably due to the terminal sequence loss that accompanies DNA replication. Telomeric DNA has been found to interact with proteins in many organisms. For example, hTRF1, a human telomeric-repeat binding factor, is involved in the telomere length regulation. We wanted to identify and elucidate functions of telomere binding proteins in the nematode C. elegans. Telomeric repeat of C. elegans consists of a long stretch of (TTAGGC)n. However, the mechanism that modulates telomere length in C. elegans has not been investigated. We have isolated Ceh-37, a novel homeotic protein in C. elegans, as double-stranded telomere binding factor by yeast one-hybrid screening. We found that Ceh-37 specifically binds C. elegans telomere in vitro. We generated and examined series of deletion constructs by gel retardation assay and yeast one-hybrid assay. We found that the N-terminal domain and the homeotic domain is sufficient for telomere binding. Since the homeodomain alone nor the N-terminal domain alone was not able to bind the telomere and the N-terminal domain could compete with the binding of telomere by the full length Ceh-37 protein, we purpose that Ceh-37 binds the telomere as dimers or higher complex, and that the N-terminal is required for the dimerization. Ceh-37 is expressed in the nuclei of embryos as shown by visualizing GFP reporter protein. We plan to generate dominant-negative mutant animals by overexpressing the N-terminal domain, and examine any phenotypes associated with the disruption of Ceh-37 function. We will present updated results.

24 May 1999 15:50 526 526 cDNA cloning and functional analysis of the C. elegans dna topoisomerase IIIa gene

1999 International Worm Meeting abstract 475 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. cDNA cloning and functional analysis of the C. elegans dna topoisomerase IIIa gene Y Kim, H Koo Dept. of Biochemistry, College of Science, Yonsei university, Seoul 120-749, Korea Eukaryotic DNA topoisomerase III which is classified as type I DNA topoisomerase catalyzes the partial removal of negative DNA supercoils. The enzyme is required to maintain genome stability, and its null mutation blocks yeast sporulation and the embryogenesis of mice. cDNA clones of C. elegans DNA topoisomerase IIIa were obtained by performing RT-PCR using degenerate deoxynucleotides encoding amino acid sequences conserved in the human and yeast enzymes and then screening a C. elegans cDNA library using the RT-PCR product as a probe. The exact 5’-end cDNA clone was obtained by carrying out RT-PCR using a SL1 primer, which produced a 2.4 kb long cDNA sequence in combination with the clone from the cDNA library. C. elegans DNA topoisomerase IIIa polypeptide overexpressed in E. coli showed a catalytic activity of relaxing negatively supercoieled DNA in vitro. When the DNA topoisomerase IIIa expression in C. elegans was inhibited by microinjecting the double-stranded RNA, blockages in the oocyte formation and early embryogenesis were observed. The strong mRNA expression in the gonad coincided with the RNA interference phenotype. These results suggest that the DNA topoisomerase III a plays an important role in the gametogenesis and also embryogenesis. The specific reaction which the enzyme catalyzes in these developmental progressions remains to be explained.

24 May 1999 15:50 527 527 An approach toward imaging activities of the neural network

1999 International Worm Meeting abstract 476 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. An approach toward imaging activities of the neural network K Kimura 1 , K Matsumoto 1 , I Mori 1,2

1 Div. of Biol. Sci., Nagoya Univ. 2 PRESTO, JST, Japan.

All neural connections have been revealed through the extensive EM analysis1 , which should be the structural basis of all neural functions. Further, as demonstrated for the touch response and thermotaxis behavior, laser ablation studies in some cases lead to identify the neural circuit consisting of a set of neurons, which is required for the particular behavior2, 3 . We still do not know, however, how actual neural signals are conveyed in the network. For example, it remains to be elucidated as to how different sensory signals are transmitted in the network to generate appropriate behaviors. To address these questions, we aim to monitor the activities of the neural network using an optical method. The optical method allows monitoring multipoint simultaneously, and it is not destructive. Among optically detectable changes shown to associate with neuronal excitation, the calcium change is one of the best studied because of its massive magnitude, relative to other changes. Already, cameleon, a GFP-based calcium sensor4 , has been successfully used to monitor calcium transients associated with pharyngeal pumping5 . We are currently setting up an optical system for cameleon to monitor calcium changes in neurons in the thermotaxis circuit. In the neural model for thermotaxis, the counterbalancing regulation by the two downstream interneurons was proposed to be critical for the migration to a preferred temperature3 . It is still unknown, however, whether signals transmitted between the connected neurons in the circuit are excitatory or inhibitory. Recently, we have shown that thermotaxis can be used to study memory and learning (see the abstract by Mohri et al). What kind of changes is occurring in the network during learning? With the availability of the neural model, we believe that the thermotaxis circuit provides an ideal simple network to apply the cameleon-based imaging. Through this analysis, we hope not only to understand the actual neural regulation of thermotaxis, but also to help develop a system for imaging neuronal activities of the C. elegans nervous system as a whole. We thank Dr. Miyawaki for cameleon constructs.

1. CGC Bibliography #938, 2. CGC #765, 3. CGC #2214, 4. Miyawaki et al., Nature 388 (1997), 5. Kerr & Schafer, ’98 WCWM

24 May 1999 15:50 528 528 Ca2+/CaM-dependent protein kinases (CaM-K)-mediated signal cascade is conserved in C. elegans

1999 International Worm Meeting abstract 477 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Ca 2+ /CaM-dependent protein kinases (CaM-K)-mediated signal cascade is conserved in C. elegans Y Kimura, K Eto, M Muramatsu, H Tokumitsu Helix Research Institute, Inc.

In mammals, Ca2+ /CaM protein kinases (CaM-K) is thought to be important for Ca2+ -dependent signal transduction. This cascade consists of the upstream CaM-kinase kinase (CaM-KK) and downstream CaM kinase I (CaM-KI) and CaM-kinase IV (CaM-KIV). In the present study, we have identified CaM-KK and CaM-KI orthologue of C. elegans (named CeCaM-KK and CeCaM-KI, respectively) and analyzed their biochemical activities. CeCaM-KK has two unusual features in the catalytic domain which is conserved between species, that is, a lack of acidic residues important for substrate recognition and an Arg-Pro rich insert (RP-domain) which is essential for the recognition and activation of CaM-KIV and CaM-KI. Indeed, CeCaM-KK activated mammalian CaM-KIV in vitro. CeCaM-KI is highly homologous to mammalian CaM-KI and is activated by CeCaM-KK through phosphorylation of Thr179 in a Ca2+ /CaM-dependent manner. Truncation at residue 295 in CeCaM-KI generates a constitutively active enzyme, suggesting that COOH-terminal at residue 295 contains autoinhibitory and CaM-binding domains. Unlike mammalian CaM-KI, CeCaM-KI mainly localized in the nucleus of transfected mammalian cells by the virtue of the NH2 -terminal six residues containing a functional nuclear localization signal. Taken together, these results suggest that CaM-KK/CaM-KI cascade in C. elegans is conserved and operated both in vitro and intact cells. Current research aims to the physiological functions of CeCaM-KK and CeCaM-KI cascadein vivo. Analyses for the expression patterns and isolation of mutant worms for both genes are underway.

24 May 1999 15:50 529 529 How are guidance and motility coupled in cells and axons?

1999 International Worm Meeting abstract 478 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. How are guidance and motility coupled in cells and axons? RM Kindt, CJ Kenyon Department of Biochemistry, UCSF, San Francisco, CA, 94143-0448 Cells and axons respond to extrinsic guidance cues by directed movement, but how guidance information actually regulates the cytoskeletal components that control movement is not known. The anterior migrations of the QR descendants are controlled cell-extrinsically by a novel transmembrane protein, MIG-13; in mig-13 mutants, these migrations are shortened (work of Mary Sym). We found that the QR cell descendants migrate more slowly in mig-13 mutants. This result suggests that the cell’s "motility machinery" is indeed altered by the presence or absence of guidance information. The Rho GTPase mig-2 is a good candidate for a component of the motility machinery: in null or putative activated mig-2 mutants the migrations of the QR descendants are shortened, but the direction of migration is never altered. Since QR descendants in mig-2 mutants migrate farther than in mig-13 mutants, we were surprised to find that in mig-13 mig-2(activated or null) double mutants the cells moved a shorter distance than in mig-13 alone. This finding suggests that migrating cells determine their final positions by somehow integrating the available guidance information (which influences both direction and speed of their migration), and then using the motility machinery to execute the decision. Integrin receptors containing the pat-3 beta integrin subunit are likely to act with mig-2, based on the phenotypes of a new, viable pat-3 allele, and of transgenic worms in which the embryonic lethality of pat-3(null) mutants is rescued by muscle-specific expression of PAT-3. Axonal growth cones may use mechanisms similar to those of migrating cells to select and execute their direction of movement. However, the role of mig-2 and pat-3 appears to be different in growth cones: in both activated mig-2 mutants and animals carrying our new pat-3 allele, the HSN axon grows but is not correctly guided. In contrast, in the cell migrations described above, these mutations alter movement but not guidance. This finding suggests that the wild-type function of these genes in the HSN axon may be to transmit guidance signals (perhaps mediated by unc-40 and sax-3) rather than to permit movement per se.

24 May 1999 15:50 530 530 Sensory axon guidance defects in C. elegans

1999 International Worm Meeting abstract 479 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Sensory axon guidance defects in C. elegans SA Kirch, GC Crump, CI Bargmann HHMI and Department of Anatomy; UCSF; San Francisco, CA 94143 Worms depend on taste, touch and smell to sense and explore their environment. Appropriate responses to information received through these different modalities depends on precise connectivity between sensory neurons and downstream effector neurons in the nerve ring. Little is known about how extending axons migrate through this nerve ring neuropil during development to find their partners. How is positional information interpreted by an extending axon so that desired synaptic partners are found? We focused on the guidance of the ASI chemosensory neurons to their appropriate positions in the nerve ring. A mutant screen led to the isolation of 16 candidate mutant strains that appeared to have ASI axon guidance and termination phenotypes. These mutations define at least five new genes which we have named sax-10-14 (sensory axon guidance). We have observed four main phenotypic classes of ASI axon defects. These defects include: 1) termination shortly after the axon enters the nerve ring or half-way in its dorsal trajectory within the ring, 2) axons that appear to thicken, branch or wander often crossing the ventral midline, 3) axons that project anteriorly instead of ventrally, terminate prematurely and bifurcate and 4) a dye-filling defective class, indicating cilium structure or amphid pore defects in addition to the abnormal ASI morphology. Mutants display either one or a combination of these phenotypes with a penetrance that ranges form 13% to 100%. Dye filling of sensory neurons with the fluorescent dye DiI, and examination of neurons with other cell-specific GFP constructs in these mutants reveals that the overall morphology of the nerve ring is normal in many of these animals, and that the mutations disrupting ASI are probably in genes specifically required by ASI or a subset of neurons. To facilitate genetic analysis and cloning of the sax genes, we have mapped the genes and are currently trying to rescue the sax-10 phenotype by cosmid injections and germline transformation. We are also analyzing known mutants for effects on axon guidance in the nerve ring. We will conduct another visual screen for defects in chemosensory neurons in an effort to identify more molecules required for axon guidance and nerve ring development.

24 May 1999 15:50 531 531 Elucidating the roles of GLH-1 and GLH-2, two constitutive P-granule components, in germline development

1999 International Worm Meeting abstract 480 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Elucidating the roles of GLH-1 and GLH-2, two constitutive P-granule components, in germline development J Kirchner 1 , K Kuznicki 2 , P Smith 2 , K Bennett 2 , S Strome 1

1 Department of Biology, Indiana University, Bloomington, IN 47405. 2 MMI Dept. University of Missouri, Columbia, MO, 65203.

GLH-1 and GLH-2 are members of a family of Vasa-like germline helicases inC. elegans. Both proteins contain eight conserved RNA helicase motifs, glycine-rich motifs, and retroviral-like CCHC zinc fingers. Based on antibody staining, both GLH proteins are associated with P granules at all stages of germline development. We have used reverse genetics to study the function of these genes in germline development. Injection of dsRNA to bothglh-1andglh-2caused the injected worms to produce sterile progeny, with a higher percent sterile progeny at elevated temperature than at lower temperature. The sterile progeny had stunted gonad arms, lacked gametes, and lacked detectable accumulation of GLH-1 and GLH-2. In addition, they failed to show P-granule staining for PGL-1 and for four other P-granule epitopes. Similar levels of sterility were induced by injection of dsRNA specific forglh-1,but not by dsRNA specific toglh-2. To address whether the sterility is maternal-effect or zygotic and the issue of redundancy, we sought mutants. We isolated a Tc1 insertion inglh-1from Michael Hengartner’s transposon library. The Tc1 insertion is in exon 4 ofglh-1. glh-1::Tc1mutants show both maternal-effect and zygotic sterility, both of which are sensitive to temperature -glh-1::Tc1homozygous mutants produce 100% sterile offspring at elevated temperature, but only 10-15% sterile offspring at low temperatures. These phenotypes are very similar to the phenotypes seen inpgl-1 mutants, a gene encoding another P-granule component. In addition, P-granules inglh-1::Tc1homozygotes fail to stain for GLH-1, PGL-1, and four other P-granule epitopes, but do stain positively for GLH-2, MEX-1, and MEX-3. Western analysis on this strain revealed that GLH-1 protein accumulation was not detectable while PGL-1 protein was present. This suggests that PGL-1 protein does accumulate but is not localized to P granules in this mutant. These results suggest that at leastglh-1function is required for germline development mainly at high temperatures.

24 May 1999 15:50 532 532 Mutant Myosin Expression in Transgenic Lines of Caenorhabditis elegans

1999 International Worm Meeting abstract 481 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Mutant Myosin Expression in Transgenic Lines of Caenorhabditis elegans A Kirmizis, SK Olson, AM Peregrine, EA De Stasio Department of Biology, Lawrence University, Appleton, WI 54911, USA Many previous studies of muscle assembly in the nematode C. elegans have shown that the rod domain of myosin heavy chain B (MHC B), encoded by the unc-54 gene, is both necessary and sufficient for myosin and thick filament assembly. More recent studies have investigated the role of the myosin head domain in thick filament and sarcomere assembly. Four dominant nonsense mutations in the head domain of MHC B, which result in a muscle disruptive phenotype when the animals are viewed under polarized light, have been characterized. These four mutations are located close to the myosin rod/head junction in the unc-54 suggesting that the presence of deformed myosin heads are toxic to normal muscle assembly (Pulak & Anderson, 1993). Four transgenic lines of worms, which contain truncated versions of the unc-54 gene, have been created to test the hypothesis that myosin heads confer the disruptive muscle phenotype. The transgenic lines were constructed in a temperature-sensitive smg-1 genetic background to allow conditional expression. RT-PCR detected mutant unc-54 mRNA in the three transgenic lines examined. These animals have normal motility and do not show any reproductive defects. The results suggest either that the presence of mutant myosin has no effect in C. elegans striated muscle assembly or the truncated protein is not synthesized. To distinguish between these possibilities, we are constructing GFP and epitope-tagged versions of mutant myosin gene.

24 May 1999 15:50 533 533 Searching for Regulators of Cell Fate Specification

1999 International Worm Meeting abstract 482 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Searching for Regulators of Cell Fate Specification M Kirouac, PW Sternberg HHMI and Division of Biology, 156-29, Caltech Pasadena, CA 91125 Pasadena, CA 91125 lin-3 encodes the inductive signal for vulval formation, and it is expressed in the AC at the time of vulval indution. Two transcripts of lin-3 are made in the worm, LIN-3A and LIN-3B (Liu, in prep). It is hypothesized that the two transcripts work as redundant signals to pattern the vulva in a precise manner. LIN-3A is thought to be secreted and to act at a distance to induce VPCs that are not in direct contact with the anchor cell. LIN-3B is thought to be a locally-acting form of LIN-3 which indirectly induces P6.p (Liu, 1997 thesis). Little is known about how LIN-3 is regulated either temporally or spatially or if individual VPCs respond to LIN-3 using different mechanisms which shape cellular fate specification. In order to investigate the role of LIN-3 in cell fate specification, I have been conducting a F2 screen using a reduction of function lin-3(n378) strain which contains an integrated transgene that carries a mutation that knocks out lin-3B splicing and should, therefore, only produce lin-3A. The line carrying the integrated transgene is fertile and 100% multivulval. Animals that lack lin-3A are sterile (Liu, personal communication). To potentiate rescuing any lines that are sterile because they lack lin-3A, lfe-1 was incorporated into the strain background.lfe-1(sy290) is a gain of function allele that partially rescues the sterility caused by let-23 loss of function animals. The strain is fertile and 100% multivulva. From these screens, I have been looking for animals which are non-Muv. I expect to find several catagories of mutants: Intercellular negative regulators of the inductive signal; Intracellular desensitization of the lin-3 signal; temporal regulators of LIN-3 signal; genes involved in the production/ processing of LIN-3; and general trascriptional regulators. I have identified 6 mutants with a substantial decrease in induction, and I am currently characterizing them.

24 May 1999 15:50 534 534 KSR-1::GFP expression, and effects of ksr-1 on LIN-12::GFP

1999 International Worm Meeting abstract 483 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. KSR-1::GFP expression, and effects of ksr-1 on LIN-12::GFP RS Kishore, MV Sundaram University of Pennsylvania School of Medicine, Philadelphia. PA 19104 ksr-1 encodes a putative kinase that positively influences vulval induction. ksr-1 mutations do not significantly affect vulval development in an otherwise wild-type background, but they do suppress the Muv phenotype of let-60(n1046gf) and enhance the Vul phenotype of other Ras pathway mutants. The mechanism of action by which KSR-1 acts is poorly understood. It may stimulate the activity of a known Ras pathway component (such as Raf or Mek) or it may act in parallel to Raf/Mek/Erk to control more downstream targets. We have currently initiated studies to dissect the molecular mechanism of ksr -1 action. We are examining whether ksr-1 mutations affect the expression of certain molecular markers for vulval fates. One of the first markers we’ve examined is LIN-12::GFP, which is normally downregulated in P6.p at the time of Ras signalling (1). Most ksr-1(n2526) mutants fail to downregulate LIN-12::GFP at this time; instead, LIN-12::GFP expression persists in P6.p and its descendants until the L4 stage. Since most ksr-1(n2526) mutants have normal vulva development, this argues that downregulation of LIN-12 is not absolutely necessary for normal vulval patterning. However, failure to downregulate LIN-12 could potentially contribute to the suppressor and enhancer effects of ksr-1 mutations seen in sensitized genetic backgrounds. We have also looked at in vivo expression and localization patterns of KSR-1::GFP. It is expressed in most cells of the embryo, appearing both cytoplasmic and membrane-associated. In larvae, KSR-1::GFP is most highly expressed in neurons of the head, ventral cord and tail, as well as in the canal-associated neurons. It is seen in both cell bodies and axons. We were most interested in examining KSR::GFP expression in the developing vulva. KSR-1::GFP appears diffuse at early Pn.p stages, but is clearly associated with cell peripheries starting from the Pn.px stage and persisting into the L4 stage. It appears both membrane-associated and cytoplasmic but is excluded from the nucleus at all stages. It also appears to localize more to the basal membrane than to the apical membrane.

1. Levitan, D. and Greenwald, I. (1998) Development , 3101-3109.

24 May 1999 15:50 535 535 Difference in habituations induced by mechanical stimuli in Caenorhabditis elegans

1999 International Worm Meeting abstract 484 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Difference in habituations induced by mechanical stimuli in Caenorhabditis elegans K Kitamura 1 , S Amano 2 , R Hosono 1

1 Department of Physical Information, Faculty of Medicine, Kanazawa University, Ishikawa 920-0942, JAPAN. 2 Cancer Research Institute, Kanazawa University, Ishikawa 920-0934, JAPAN.

C. elegans shows decrease in a response to a given stimulus after repeated mechanical stimuli, such as a tap or touch. The habituation behavior following stimuli and the neural circuitry have been closely examined. However, the neuron constituting the circuit which is directly related to the habituation and the molecular mechanism in the neuron involved in the habituation remained unknown. One approach to elucidate the habituation is to compare different types of habituations induced by mechanical stimuli. We therefore, compared habituations induced by three types of mechanical stimuli: touch on the head (head-touch), touch on the anterior body (anterior-touch), and mechanical tapping of the Petri dish, all of which evoke backward movement. In initial responses, touch stimuli evoked about two times larger responses than tapping. Worms habituated by 30 times of repetitive stimuli are still responsible to the stimuli in the order of head-touch, anterior-touch and tapping. Habituation generated by repetitive mechanical stimuli recovered gradually in 60 min. in our system. Can a worm habituated by one type of mechanical stimulus respond to another one? Worms habituated by 50 times of head-touch slightly responded to anterior-touch. Worms habituated by anterior-touch, however, respond strongly to head-touch. Worms habituated by head-touch or anterior-touch responded little to tapping, but worms habituated by tapping responded to touch stimuli. These results show that habituated state is not same but different depending on the types of mechanical stimuli. Habituation was tested in worms whose AVA interneurons were ablated by the laser microbeam. Habituation behavior of the treated animals were normal, indicating that intact neural circuitry is not essential for the habituation behavior. The hierachial relationship of habituation induced by head-touch and anterior-touch was also observed. Therefore, it is less likely that the neural circuitry brings about the difference of habituation. Rather we propose that the difference is caused by the strength of stimuli (or sensitivity of worms).

24 May 1999 15:50 536 536 Genetic analysis of C. elegans elongation using sma-1 suppressors

1999 International Worm Meeting abstract 485 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic analysis of C. elegans elongation using sma-1 suppressors SL Kniss, J Austin University of Chicago, Chicago, IL SMA-1 spectrin is required for changes in cell shape that occur as the C. elegans embryo elongates from a ball of cells into a long, thin worm. Animals lacking SMA-1 are shorter at hatching than wild type animals due to a decreased rate of embryonic elongation. Normal elongation depends on circumferential actin bundles in the embryonic epidermal cells; organization of these bundles is disrupted in sma-1 mutants suggesting that SMA-1 secures the actin bundles to the apical membrane during elongation (V. Praitis, unpublished results). The sma-1 alleles isolated in our lab vary in their effect on elongation rate suggesting that SMA-1 may play a regulatory as well as a structural role during elongation. To identify proteins that act with SMA-1 during morphogenesis, we isolated phenotypic suppressors of the sma-1 elongation defect. We have identified both dominant and recessive suppressors using a screen based on the inability of sma-1 L1 larvae to chemotax to food. The suppressing mutations vary in their effect on elongation as well as other phenotypic characteristics including viability, egg laying and mobility. We are currently generating loss-of-function alleles of the genes containing dominant suppressor mutations in order to map and clone these genes. There are several mechanisms by which mutations in the suppressed animals may result in increased hatchling length. First, suppressing mutations may strengthen a weak association between SMA-1 and an interacting protein such as protein 4.1 or a-spectrin. Second, proteins that act in parallel to SMA-1 to link the actin cytoskeleton to the plasma membrane may be overexpressed allowing them to substitute for defective SMA-1 in the mutants. Third, suppressing mutations may alter the strength of contraction of the actin fibers. Finally, our mutations may increase the stability of sma-1 mRNA since the sma-1 allele used in our screens is suppressible by smg mutations. To distinguish between these possibilities, we are comparing the organization of the actin bundles in our suppressed mutants to that in the original sma-1 mutant. By examining suppressor phenotypes and identifying the genes containing suppressing mutations, we hope to understand the role of these genes in C. elegans morphogenesis.

24 May 1999 15:50 537 537 Growth cone migration in C. elegans

1999 International Worm Meeting abstract 486 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Growth cone migration in C. elegans KM Knobel, D Warner, EM Jorgensen, MJ Bastiani University of Utah, Department of Biology, 257 South 1400 East, Salt Lake City, UT 84112 Neuronal processes are guided to their targets by a growth cone at the tip of the extending axon. The growth cone explores its environment using specialized cytoplasmic extensions called filopodia and lamellipodia. We are interested in the molecules regulating these growth cone activities. To visualize growth cones we expressed GFP in a subset of neurons and are using time-lapse confocal microscopy to observe growth cone behavior in living, intact C. elegans larvae. Previously, we characterized the GABA VD motor neuron growth cones as they migrate from the ventral nerve cord toward the dorsal nerve cord (Knobel, IWM ’97, WCWM ’98). We are embarking on experiments to define temporal aspects of outgrowth in the embryo. We are also studying growth cones in unc-119 mutants. unc-119 encodes a novel protein (cloned and characterized by the Pilgrim lab). The unc-119 mutant has severe outgrowth defects. Although most of the GABA commissures reach the dorsal nerve cord, many are branched. The axons often prematurely truncate in the dorsal cord and the ventral nerve cord is often defasciculated. We are interested in the branching phenotype and have determined that the amount of branching increases over time. Specifically, most of the embryonic DD commissures look normal at hatching but as these animals age, their DD commissures become more branched. These branches occur because axonal sprouts, normally retracted in wild-type worms, form functional growth cones that extend secondary branches to the dorsal cord. In addition, we see new growth cones sprout from the DD cell bodies during VD growth cone migration. The axon outgrowth phenotype is rescued by expressing a wild-type copy of the UNC-119 protein in GABA neurons in unc-119 mutants. Our current hypothesis is that there is an intrinsic mechanism maintaining growth cone primacy in wild-type neurons. This prevents the formation of multiple growth cones at the tip of an extending axon. In the unc-119 mutant this mechanism is defective resulting in supernumerary growth cones and axonal processes. We are now directly screening for mutants with abnormal outgrowth. Three classes of mutants were identified, those with branched axons, those with premature truncation of axons, and those with disorganized processes. C. elegansunc-119

24 May 1999 15:50 538 538 Comparative genomics applications of a comprehensive, non-redundant protein sequence registry

1999 International Worm Meeting abstract 487 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Comparative genomics applications of a comprehensive, non-redundant protein sequence registry RG Knowlton, JS Aaronson Bioinformatics Dept., SmithKline Beecham Pharmaceuticals, King of Prussia, PA To aid in ascribing functions to protein sequences obtained through genomic methods, we have developed a protein sequence database and a web-based query tool which facilitate cross-species sequence comparisons and protein characterization. One of the general problems encountered in database searching is sequence redundancy. BLAST searches of even a single database often return multiple hits which are actually different database entries for the same gene, if not the same sequence, and the problem is magnified when sequences are drawn from combined data sources. We have addressed the redundancy issue in a Protein Sequence Registry (PSR), a relational database which centers around a list of protein sequences which is both comprehensive and non-redundant. The PSR is currently compiled from all protein sequences in SwissProt, PIR, GenPept, NR, TREMBL, SGD, YPD, Wormpep and the complete set of predicted C. elegans ORFs [Science, 282:2012(1998)]. Every distinct protein sequence in the union of these sets is assigned a permanent, unique identifier. The database also identifies sequences which are subsequences or almost identical. In separate tables, essential information from each database entry (e.g., species, description, accessions, links to other sources) is recorded and cross-referenced to the corresponding protein sequence in the PSR. The PSR makes identification of newly reported protein sequences straightforward, thereby minimizing the calculations needed for new pairwise alignments and making it practical to maintain up-to-date BLAST comparisons of the proteomes of model organisms. For a given sequence, the database retrieves the most similar sequences in human, mouse, rat, Drosophila, S. cerevisiae, S. pombe, and C. elegans, and displays extensive information associated with each hit.

24 May 1999 15:50 539 539 Synthesis of glycoprotein is important for molting and male tail sensory ray morphogenesis in Caenorhabditis elegans

1999 International Worm Meeting abstract 488 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Synthesis of glycoprotein is important for molting and male tail sensory ray morphogenesis in Caenorhabditis elegans KL Chow Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong C. elegans male tail is a complex structure specialized for reproductive function. The morphogenetic events occurring at the late L4 larval stage represents a process of tissue differentiation. Active cellular movement occurs and new cell-cell association need to be established. It offers an excellent paradigm for the study of tissue remodelling. Recent characterization of mutants with ray morphological defects revealed that epitope recognized by FITC-WGA was detected at high level in mutants with lumpy ray (Ram) phenotype. We postulated that overexpression of this epitope might be a result of the mutations, blocking the synthesis of these glycoprotein epitope would revert the phenotype. This hypothesis was tested by treatment of mutant worm with glycosylation inhibitors. Failure of reverting the mutant phenotype in these experiments suggests that Ram phenotype is not caused by overexpression of the GlcNAc containing molecules. On the contrary, blocking of glyco-conjugate synthesis in wild-type animal generated Ram-like phenotype. We defined the window of the inhibitor effect on ray morphogenesis to be around late L4 stage, at the time of male tail retraction, where active cell migration and cell-cell association took place. In addition, glycosylation inhibitor blocked the molting of L4 cuticle implicating a role of glycoprotein in this molting process. Previous studies have suggested that WGA binding antigen is masked by components of the cuticle. srf mutants lose the cuticular components and give to high WGA staining signals on the body. We have further characterized these ram mutants by protein analysis to ascertain the role of glycosylation. Our result suggests that glyco-conjugates are constantly expressed beneath the surface cuticle. Similar to the features of the srf genes, ram genes may represent another group of cell surface glycoproteins, the presence of which ensure normal matrix-cell and cell-cell association for an intact body outer layer. When they are mutated, cuticular matrix and cell-cell interaction will be disrupted revealing the glyco-antigens beneath the surface.

24 May 1999 15:50 540 540 T19E10.1 protein, a putative RhoGEF related to the mouse oncoprotein Ect2, is involved in versatile developmental processes.

1999 International Worm Meeting abstract 489 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. T19E10.1 protein, a putative RhoGEF related to the mouse oncoprotein Ect2, is involved in versatile developmental processes. Y Kodama 1 , A Sugimoto 1 , J Rothman 2 , M Yamamoto 1

1 Dept. Biophys. Biochem., Grad. Schl. Sci., Univ. Tokyo, Japan. 2 Univ. of California-Santa Barbara, USA.

Mutations in the cdl-1(cell death lethal) gene were isolated in a zygotic embryonic lethal screen. In cdl-1 mutants, several embryonic defects were observed: 1) excess cell corpses in late embryogenesis, 2) defects in elongation, 3) failure in the attachment of pharynx to buccal-cavity. By transformation assay, we found that a cosmid T19E10 could rescue these cdl-1 phenotypes but an overlapping cosmid R06F6 could not. These rescue data made the predicted ORF T19E10.1 a good candidate for cdl-1, and thus we studied it further. The deduced T19E10.1 protein showed overall similarity to a mouse oncoprotein, Ect2. T19E10.1 and Ect2 share two conserved domains: the BRCT domain,which is found in several cell cycle-related proteins, and the DH domain, which represents the catalytic domain of guanine nucleotide exchange factors for Rho-type small G proteins(RhoGEFs). To determine the function of T19E10.1, we performed an RNAi experiment for this ORF. T19E10.1(RNAi) worms showed various defects. Most F1 progeny had multinucleate blastomeres. Moreover, dsRNA-injected P0 worms eventually produced large multinucleate oocytes. These multinucleate cells appeared to be generated due to the absence of cytokinesis. Some F1 embryos showed morphological defects similar to those of the cdl-1 mutants, such as poor elongation and detachment of pharynx from buccal-cavity. In addition we observed probable defects in either postembryonic cell division or gonadal migration in some progeny. We also performed RNAi for rho-1, which might encode a downstream effector of T19E10.1, and found that rho-1(RNAi) worms showed similar defects to those of T19E10.1(RNAi). These results suggest that T19E10.1 protein may regulate various events such as cytokinesis, determination of cell shape and cellular migration through RHO-1 activity in vivo. Since not all the phenotypes caused by the loss of function of T19E10.1 agreed with those of the original cdl-1 alleles, we are currently determining the mutation sites of the cdl-1 mutants to prove that the two genes are identical. We will also characterize a deletion allele of T19E10.1, which we recently obtained by the UV/TMP method.

24 May 1999 15:50 541 541 Site-Specific Recombination Tools in C. elegans

1999 International Worm Meeting abstract 490 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Site-Specific Recombination Tools in C. elegans K Komachi, D Blinder, O Hughes Eon Current transformation techniques for C. elegans, while effective, produce unpredictable transgene expression (due to context and copy number effects) - and are not readily scaleable to the screening of large numbers of transformants. Additionally, the ability to generate promoter/enhancer traps, transposon tagged mutants, and predictable somatic recombination events would be useful in numerous endeavors. While site-specific recombination systems for genetic manipulation have been well demonstrated in other systems, the experimental advantages of C. elegans may make these recombination systems uniquely powerful. We are developing two classes of site-specific recombination tools: 1) inversion system derivatives (lox/Cre from E. coli P1 phage and FLP from the yeast 2µ), and 2) exogenous transposons (HOBO and MOS - both from Drosophila). So far, we have concentrated on two uses of lox/Cre recombination: lox/Cre facilitated chromosomal integration of a transgene, and lox/Cre mediated rearrangement of a transgene to control expression. The lox/Cre facilitated transformation system involves three elements: 1) a strain containing a chromosomally integrated lox site, 2) an expression vector containing a lox site, and 3) Cre recombinase protein. Cre protein can be supplied either directly by co-injection along with the lox site containing expression vector, or expressed endogenously. To take full advantage of lox/Cre and transposon facilitated transformation or screening, germ-line expression of the recombinase is desirable. Through problematic, we are having increasing success with germ-line expression from the following three systems: 1) a Seydoux/Fire like pie-1 promoter vector and excess genomic DNA, 2) the Mello lab pie-1 YAC, and 3) Eon’s integrated single copy lox/Cre or transposon based expression vectors. This last germ-line expression system extends the strategy of expressing germ-line promoters in complex environments by bypassing the formation of an extra-chromosomal array, and directly integrating transformed lox or transposon vectors at single or nearly single copy number. Finally, we hope to show single cell induction of recombination via laser induced recombinase expression from a heat shock promoter leading to single cell induction or elimination of expression.

24 May 1999 15:50 542 542 Toxicity Testing in C. elegans

1999 International Worm Meeting abstract 491 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Toxicity Testing in C. elegans K Komachi, M Galenko, D Blinder, O Hughes Eon Genomics, combinatorial chemistry, and high throughput screening have converged to enable pharmaceutical and biotech companies to produce potential new drugs at an unprecedented rate. Unfortunately, parallel advances in the safety and toxicity evaluation of these drugs have not matched the advances in producing and identifying new drug leads. Cost and time concerns severely limit the range of toxicity testing practical at early stages in drug development. After narrowly focused in vitro toxicity assays, drug leads proceed directly into animal tests. In many ways C. elegans seem tailor-made for bridging the gap between cell based in vitro assays and rodent based animal tests. C. elegans is perhaps the only complex multi-cellular animal that offers the experimental convenience of a single-celled organism. We have developed a number of toxicity tests by mapping early rodent-based tests onto C. elegans - while concentrating on high throughput analysis. Specifically, we are examining developmental, neuronal, and genetic toxicity of drugs - as well as questions of absorption and metabolism of the drug. In one example, we have taken advantage of the XX/XO hermaphrodite/male life cycle of C. elegans to develop a screen for compounds that promote chromosomal miss-segregation and aneuploidy. Although aneuploidy is known to play a significant role in major human health problems such as birth defects and cancer, we know little the about the possible aneugenicity of our chemicals and drugs. Using a male specific GFP transgene, we can rapidly quantify the aneugenicity of a compound by the number of males and/or amount of GFP signal produced. We are currently focused on determining and improving the predictive value of C. elegans based toxicity tests. We are examining methods of increasing drug sensitivity by increasing absorption (using ABC transporter mutants, electroporation, and permeabilizing agents or carriers), by "humanizing" key components, and by combining a "first hit" mutations in the system being examined with drug exposure to produce synthetic (synergistic) phenotypes. Finally, we have examined the effect of pre-incubating the drugs to be screened with mammalian microsomal liver extracts.

24 May 1999 15:50 543 543 The role of jam-1 during C. elegans embryogenesis

1999 International Worm Meeting abstract 492 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The role of jam-1 during C. elegans embryogenesis M Köppen 1 , G Moulder 2 , JS Simske 1 , A Radice 3 , R Barstead 2 , J Hardin 1

1 Zoology Research, University of Wisconsin, Madison, WI 53706. 2 Oklahoma Medical Research Foundation, Oklahoma City, OK 73104. 3 Kimball Research Institute, New York, NY 10021. jam-1 (junction-associated molecule, formerly ’mh-27’) encodes the antigen recognized by the MH27 antibody. JAM-1 protein localizes to the adherens junctions of all epithelia of C. elegans (D. Hall, pers. comm.). We are interested in its function during epithelial morphogenesis, in particular its role in the hypodermis during embryogenesis. Northern analysis, RT-PCR and sequencing of Kohara cDNAs identified 3 splice forms of jam-1, likely containing alternative 3’ ends. Additional Northern blots will address this definitively. Sequence analysis suggests that JAM-1 is a novel protein with a predicted coiled-coil motif. RNAi analysis targeting the predicted common domain of the protein yields embryos that enclose and initially elongate normally but then arrest at the 2-3-fold stage, typically with a vacuole forming inside the embryo. RNAi specifically targeting alternative splice variants produces no phenotypes, suggesting that only the shortest transcript is essential. We have isolated 2 mutant alleles of jam-1; jc18 was isolated in a linked lethal screen in our laboratory, ok160 was isolated in a PCR-based screen in the Barstead laboratory. ok160 is a deletion of most of the essential domain of the gene and likely a null. Embryos homozygous for either allele look identical to RNAi embryos. As shown previously, the localization of a-catenin and JAM-1 are mutually independent and the cadherin/catenin complex appears to function normally in the absence of JAM-1, suggesting a separate role for JAM-1 in the junctions. Since the hypodermis does not have morphologically distinct adherens and occluding junctions, we hypothesize that JAM-1 is required to control paracellular flow of solutes through the hypodermis. In the absence of the protein, junctions would be leaky, allowing fluid from the exterior to enter the embryo, leading to elongation arrest and the formation of vacuoles. To test this, we will assess the permeability of the hypodermis of wild-type and jam-1 embryos to size-specific dextran conjugates. In addition, we will assess the integrity of the adherens junctions in the absence of JAM-1 at the ultrastructural level using TEM.

24 May 1999 15:50 544 544 A genetic screen for genes involved in gut development and differentiation

1999 International Worm Meeting abstract 493 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A genetic screen for genes involved in gut development and differentiation JD Kormish, JD McGhee Department of Medical Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, T2N 4N1, CANADA To investigate genes potentially involved in gut development and differentiation, we are developing a genetic screen for gut obstructed (gob) mutants. The gut specific elt-2 GATA factor appears necessary for correct gut cell development. elt-2 null mutants arrest as L1s with a blocked intestinal lumen and abnormal brush border (1). When elt-2 null mutants are fed on a mixture of fluorescent beads and Escherichia coli, the beads collect as a large "gob" in the posterior pharynx and anterior gut that can be easily detected under the dissecting microscope. We are now using this gut obstructed phenotype to screen for mutants that should potentially identify genes involved in gut development. Preliminary results of this screen are promising, as several candidates have already been isolated.

1) Fukushige T, Hawkins MG and McGhee JD (1998) Dev Biol 198(2):286-302

24 May 1999 15:50 545 545 Approaches to Identify Mutants of the C. elegans wee-1.3 Gene

1999 International Worm Meeting abstract 494 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Approaches to Identify Mutants of the C. elegans wee-1.3 Gene ME Kosinski, A Golden NCI-FCRDC, Frederick, MD 21702 The G2 to M phase progression of the cell cycle is controlled by the universal regulator, CDC2. The WEE1 kinase family negatively regulates CDC2 by phosphorylation of amino terminal threonine and tyrosine residues. These phosphorylations prevent the progression of the cell cycle from G2 to M phase. As reported earlier, three WEE1 homologs were identified in the C. elegans genome database. We showed previously that one of the three homologs, wee-1.3, produced a novel phenotype when its expression was perturbed by the reverse genetic technique, RNA-mediated interference (RNAi). When expression is perturbed, the injected animal becomes sterile. This sterility is caused by the most proximal oocytes exiting from diakinesis prematurely and thus clogging up the gonad arm. This data, along with epistatic experiments with cdc25 and cdc2 RNAi, has allowed us to construct a model in which WEE-1.3 is required to keep CDC2 inactive in ooyctes until fertilization occurs. Once CDC2 is activated by CDC25, upon fertilization, meiosis resumes and normal development occurs. In addition to the above RNAi phenotype seen in injected animals, a few of the F1 progeny laid by the injected animal before it went sterile also have a sterile phenotype. Sterility in the F1 progeny is caused by the absences of a germline. To further characterize this phenotype we would like to obtain a mutant of wee-1.3. To accomplish this goal we are performing both a reverse and a forward genetic screen. Our first approach consists of a PCR based deletion screen in which a library of mutagenized animals is screened for a deletion in the wee-1.3 gene. The second approach is a linked sterile screen. The wee-1.3 gene is located 0.1 map units from unc-4, (uncoordinated movement Unc). This close proximity allows us to use the unc-4 gene as a linked marker, thus we screen for sterile mutants linked to unc-4. To then further narrow down the candidates to a wee-1.3 mutant we can cross them into several deficiencies that map to this region of chromosome II and score for a sterile phenotype. Once a mutant has been scored under the smallest deficiency, we can then determine whether it is a wee-1.3 mutant by performing cosmid rescue and sequencing the wee-1.3 gene from the sterile mutants.

This research was sponsored by the National Cancer Institute, DHHS, under contract with ABL.

24 May 1999 15:50 546 546 Postembryonic Muscle Patterning

1999 International Worm Meeting abstract 495 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Postembryonic Muscle Patterning SA Kostas 1,2 , AZ Fire 1

1 Carnegie Institution of Washington, Department of Embryology, 115 West University Parkway, Baltimore, MD 21210. 2 Biology Graduate Program, Johns Hopkins University, Baltimore, MD 21218.

We are interested in understanding the genetic and molecular mechanisms required for muscle patterning. The worm has a variety of muscle types that arise through a combination of lineage-specific programs and cell-cell interactions. A subset of bodywall muscles, all of the sex muscles (uterine and vulval) and two non-muscle mesodermal cells are born postembryonically. All of these cells are derived from the lone postembryonic mesodermal blast cell, M. Characterization of several genes active in the M lineage and their mutant phenotypes (e.g. 1, 2, 3, 4) has indicated the usefulness of studying this lineage as a paradigm of mesodermal development and myogenesis. Using green fluorescent protein (gfp) reporters that are active in cells derived from the M lineage we examined alterations in cell pattern in a number of characterized mutant strains, and screened for new mutants with altered muscle lineages. Preliminary analysis of these mutants has indicated roles in specific cell-fate decisions within the M lineage. One surprising observation from this analysis is that mutations in mog-1, 2, 4, 6 result in a uterine to vulval muscle transformation. This phenotype is identical to that of three novel mutants isolated from our screens. An analysis of the mog-x focus of action will be described, as will the efforts to map and clone the mutants with similar phenotypes. We are interested in determining the target(s) for Mog action in sex muscle patterning. We will also describe the efforts to clone mutants involved in other M lineage cell fate decisions. Eventually we hope to apply this information to an overall understanding of mesodermal and muscle development.

1. Greenwald, I. et al., Cell 34:435-444 1983. 2. Kenyon, C. Cell 46: 477-487 1986. 3. Harfe, B. et al., Development 125: 421-429 1998. 4. Harfe, B. et al., Genes Dev. 12: 2623-2635 1998.

24 May 1999 15:50 547 547 Genetic dissection of molting pathway

1999 International Worm Meeting abstract 496 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic dissection of molting pathway M Kostrouchova 1 , M Krause 2 , Z Kostrouch 1 , JE Rall 2

1 LMBG, Ctr. Inherited Metab. Dis. & LMP,1st Inst.Pathol., Charles University, Prague, Czech Republic. 2 Lab.Mol.Biol. & Diabetes Branch, NIDDK, NIH, Bethesda, MD 20892.

CHR3, a C. elegans nuclear hormone receptor similar to Drosophila DHR3 and the human ROR/RZR group, is important for regulation of molting. The CHR3 loss of function (RNAi) leads to a characteristic phenotype including an inability to completely shed the old cuticle during molting(1). There are only a few genes known to be involved in proper molting. These include let-454, let-462 and let-473(2) and in lrp-1 genes(3). To find additional genes involved in the molting pathway we performed a mutant screen to look for molting defects that were similar to the CHR3(RNAi) phenotype. We have examined the progeny from 1000 F1 animals (2000 haploid genomes) and found 4 mutations with molting defects; we continue to investigate one of them. The majority of these mutant worms do not complete the L4/adult molt and some animals are also affected in earlier stages. The worms do not shed and degrade the cuticle from the head area and they die several days later because of starvation. The mutant animals are also shorter and DPY. The adult hermaphrodites have only a few developing embryos inside that often do not hatch. We are now in the process of mapping the mutation.

1. Kostrouchova, M., Krause, M., Kostrouch, Z. & Rall, J. E. (1998) Development 125, 1617-26. 2. Johnsen, R. C. & Baillie, D. L. (1991) Genetics 129, 735-52. 3. Yochem, J., Tuck, S., Greenwald, I. & Han, M. (1999) Development 126, 597606. C. elegans, Drosophila

24 May 1999 15:50 548 548 Characterization of mutants that mis-traffic synaptic vesicle proteins in touch neurons

1999 International Worm Meeting abstract 497 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of mutants that mis-traffic synaptic vesicle proteins in touch neurons SP Koushika, AM Schaefer, ML Nonet Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110 Mutants that mis-accumulate vesicle tagged GFP in touch cells were isolated in a screen for synaptogenesis defects (refer to A. M. Schaefer and M. L. Nonet’s poster for greater detail). The screen utilized pmec-7::snb-1::GFP where synaptobrevin (SNB-1) is an integral membrane protein that is present on synaptic vesicles. Four mutants (js121, js319, js320 and js351) mis-accumulate SNB-1::GFP at the ends of all touch cells where no synapses have been described. All other domains of pmec-7::snb-1::GFP expression and SNB-1::GFP localization are unaltered in these mutants. These mutants fall into three complementation groups where js121 and js319 are allelic to each other. js319 and js320 also mis-accumulate synaptotagmin (SNT-1), another synaptic vesicle protein. In unc-104(e1265), a kinesin-like gene necessary for transport of synaptic vesicles, the mis-accumulations are absent. These observations suggest that the defects in js319 and js320 are likely to arise due to mis-trafficking/ or release of vesicles rather than mis-targeting of the SNB-1 protein. Both js121 and js351 are sterile and have a protruding vulva, perhaps indicating their role in a common pathway. Further phenotypic characterization and mapping of these mutants will be presented.

24 May 1999 15:50 549 549 Core 2 N-acetylglucosaminyltransferase Homologues in C. elegans: Preliminary Characterization.

1999 International Worm Meeting abstract 498 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Core 2 N-acetylglucosaminyltransferase Homologues in C. elegans: Preliminary Characterization. A Krizus, CE Warren, E Partridge, JW Dennis Samuel Lunenfeld Research Institute, 600 University Ave, Toronto, ON M5G 1X5, Canada. A search of the C. elegans genome database has revealed a total of 6 core 2 N-acetylglucosaminyltransferase (Core 2 GlcNAc-T) homologues. Of these six, no single homologue appears to be more significantly similar to its mammalian counterpart than any other. We have undertaken studies to determine the function in C. elegans of all of the members of this family. To this end, cDNA clones have been isolated by RT-PCR for all six homologues. Overall, the observed splice junctions are in good agreement with those predicted by Gene Finder. cDNA fragments have been used to conduct RNA interference experiments for C54C8.11, F22D6.11 and F22D6.12. gld-1 injected in parallel was used as the positive control. The core 2 GlcNAc-T RNAi’s have yielded no obvious aberrant phenotypes. Fully coding cDNA constructs of all 6 family members are being derived and transgenic lines developed that express these gene-products under the control of the heat-shock promoter. Induced protein will be immunopurified and tested for enzyme activity. Tc1 mediated mutagenesis has been used to isolate a 2 bp frameshift allele (ev686) of F44F4.6 (gly-1). This causes premature termination of the polypeptide prior to the putative catalytic domain and is presumably null. Phenotypic characterization of this strain has revealed a ray 1 defect in male tails. The defect shows incomplete penetrance and is temperature sensitive with respect to its penetrance. Work is in progress to determine if the defect is due to a mispositioning, fusion or loss of the ray.

24 May 1999 15:50 550 550 Structure-Function Analysis of UNC-73

1999 International Worm Meeting abstract 499 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Structure-Function Analysis of UNC-73 T Kubiseski, R Steven, M Rosen, J Culotti, T Pawson Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON The protein UNC-73 was identified in C.elegans upon mutation to exhibit axon fasciculation defects with premature terminations in the nerve cords as well as defects in pioneering growth of commissures (1). Two forms of UNC-73 arising from alternative splicing have been identified, and contain Dbl homology (DH) domains, which have been implicated as exchange factors by regulating guanine nucleotide exchange of GDP to GTP for the RHO/RAC/CDC-42 family of the small GTPases. Previous work had demonstrated the first DH domain as having specific exchange factor activity towards the RAC GTPase, and that this function was eliminated by introducing the Ser 1216 Phe mutation of the unc-73 (rh40) into this DH domain (2). This residue is either Ser or Thr in virtually all known DH domains. The structure of another DH domain from the protein beta-PIX indicate that this residue is located on the exposed face of helix A, and appears to play a direct role in binding and/or catalysis for activating the RAC GTPase. The role of the SH3 domain was also investigated. A minigene expressing the UNC-73B form was demonstrated to rescue the e936, gm33 and gm40 alleles of unc-73. To determine if the SH3 domain of UNC-73B was required for this rescue, constructs of the minigene with the SH3 replaced with GFP were generated and tested for rescue in worms homozygous with the rh40 and gm40 alleles. Results indicate that mutant worms expressing this construct as an extrachromosomal array were virtually wild-type with respect to movement, suggesting that the SH3 domain of UNC-73B is not required to rescue the unc phenotype of the rh40 and gm40 alleles.

(1) Cullotti, J.G. (1994) Curr. Opin. Genet. and Develop., 4, 587. (2) Steven, R., et al. (1998) Cell, 92, 785.

24 May 1999 15:50 551 551 How is ceh-22 expression regulated in pharyngeal muscle?

1999 International Worm Meeting abstract 500 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. How is ceh-22 expression regulated in pharyngeal muscle? CA Kuchenthal, W Chen, T Vilimas, PG Okkema Department of Biological Sciences, University of Illinois-Chicago, Chicago, IL The NK-2 family homeodomain factor CEH-22 is an important regulator of gene expression in pharyngeal muscle, where it binds the enhancer and activates expression of the pharyngeal muscle-specific myosin gene, myo-2. ceh-22 is expressed exclusively in pharyngeal muscle beginning at the bean stage of embryogenesis and is the earliest marker of pharyngeal muscle differentiation. A 1.9 kb promoter fragment is sufficient to direct reporter gene expression in a pattern identical to the endogenous ceh-22 gene. We are characterizing regulatory sequences in the ceh-22 promoter region to elucidate early steps in pharyngeal muscle development. We have identified two restriction fragments from the ceh-22 promoter region that function as transcriptional enhancers and have designated them the distal and proximal enhancers. When assayed in transgenic lines, these enhancer fragments activate reporter gene expression in distinct temporal and spatial patterns. The distal enhancer fragment activates gene expression in both muscle and non-muscle cells in the pharynx, as well as in a subset of non-pharyngeal tissues. The earliest we have observed distal enhancer activity in the pharynx is at the bean stage. In contrast, the proximal enhancer fragment primarily activates gene expression in pharyngeal muscles. However, the earliest we observe proximal enhancer activity is at the 1-1/2 fold stage. We are interested in understanding how activity of both the proximal and distal enhancers is regulated. We have identified a 143 bp region near the 5’ end of the proximal enhancer fragment that contributes substantially to pharyngeal muscle enhancer activity. Within this region we have found two distinct segments sufficient to activate reporter gene expression primarily in the same subset of pharyngeal muscles that express ceh-22. Interestingly, one of the segments contains a candidate CEH-22 binding site, suggesting the proximal enhancer may be a site of ceh-22 autoregulation. Similarly, within the distal enhancer fragment we have localized a 119 bp fragment sufficient to enhance reporter gene expression specifically in pharyngeal muscle and marginal cells. We are analyzing both ceh-22 enhancers and plan to use these sequences to identify factors regulating ceh-22 expression.

24 May 1999 15:50 552 552 The cloning and characterization of smg-3, a gene required for mrna surveillance in C. elegans

1999 International Worm Meeting abstract 501 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The cloning and characterization of smg-3, a gene required for mrna surveillance in C. elegans SL Kuchma 1 , BM Cali 2 , P Anderson 1

1 Department of Genetics, University of Wisconsin, Madison, WI 53706. 2 Microbia Incorporated, Cambridge, MA.

Eukaryotic mRNAs containing premature termination codons are rapidly degraded relative to their wild-type counterparts, a phenomenon termed "nonsense-mediated mRNA decay" (NMD). In C. elegans, the functions of seven smg genes, smg-1 through smg-7, are required for NMD. While nonsense mutant mRNAs are unstable in smg(+) genetic backgrounds, such mRNAs exhibit normal stability in smg(-) mutants. Thus, the smg genes function as a cellular mRNA surveillance system which prevents the accumulation of nonsense mutant messages. Such messages can be generated by somatic or germline mutations, or by errors in transcription, RNA processing, or transport. Nonsense mutant messages encode truncated polypeptides that can be deleterious to the cell. By eliminating such messages, NMD prevents the synthesis of potentially disruptive nonsense fragment polypeptides. We have cloned smg-3 in order to identify its role in NMD. smg-3 localizes to a cosmid gap in the physical map. This gap is spanned by a single 600 kb YAC, Y73B6, that has been shown by DNA transformation experiments to rescue smg-3 mutants. Using DNA sequence from Y73B6, BLAST searches reveal a region of homology to UPF2/NMD2, a gene required for NMD in Saccharomyces cerevisiae. When used as a probe, DNA from this smg-3 candidate gene detects Southern blot polymorphisms associated with certain smg-3 mutations. For example, genetic evidence suggests that smg-3(r930) is a deletion mutation. In r930 mutants, DNA from the smg-3 region is undetectable on Southern blots. Northern blot analysis detects a message in wildtype that is absent in r930 mutants and altered in other smg-3 mutants. To confirm that this candidate gene is smg-3 and to uncover additional information about this gene, the following experiments are in progress: 1) DNA transformation experiments to complement smg-3 mutants; 2) Sequencing wild-type smg-3 cDNA to obtain the entire predicted protein sequence; and 3) Sequencing DNA from smg-3 mutants to identify causative mutations.

24 May 1999 15:50 553 553 Overexpression of a Novel Gene Causes Meiotic Nondisjunction

1999 International Worm Meeting abstract 502 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Overexpression of a Novel Gene Causes Meiotic Nondisjunction LM Kuervers, DL Janke, DL Baillie Simon Fraser University Burnaby, B.C. Canada Our lab has generated a cosmid transgenic library in order to rescue a number of essential genes on chromosome III (Janke et al 1997). We reported the discovery of a him (high incidence of males) phenotype exhibited by two separate transgenic lines containing either overlapping cosmids F56D2 or C05D2. We have isolated a single locus, C05D2.5 which when present on an extrachromosomal array results in a him phenotype. C05D2.5 has no significant homologies in the database but does have four cDNAs in Yuji Kohara’s database indicating that C05D2.5 is an expressed gene. We are interested in discovering the role of this gene in chromosome segregation. Depending on the copy number of C05D2.5, between 4% and 27% self-progeny males are produced. In accordance with the increase in self-progeny males, there is an increase in embryonic lethality indicating autosomal death. We have also shown that nondisjunction occurs in the oocyte line and most probably the spermatocyte line. However, the transgenic progeny of a mating using a transgenic male and a Dpy-Unc hermaphrodite does not exhibit nondisjunction until the F2 stage indicating: 1) a build-up of the product is required and 2) there is not enough of the product in the sperm of the male passed on to the F1 progeny. Interestingly, this nondisjunction effect is carried on for at least three generations subsequent to losing the extrachromosomal array in transgenic animals. There is a decrease in male production and an increase in brood size with each successive generation. We have performed a RNAi experiment and found the result to be a significantly reduced nondisjunction effect of 4.1% males and a brood size of 166. We have been unable to detect any cytological defect in prophase of meiosis I, therefore the disruption in chromosome segregation appears to occur after diakinesis. In accordance with this we have found no disruption in recombination. As of yet we are unable to rule out either possibility of the overproduction of the RNA or protein product causing the effect. We are further characterizing the role of this novel gene in the process of chromosome segregation in C. elegans.

Janke, D. L. et al. (1997) Interpreting a Sequenced Genome: Toward a Cosmid Transgenic Library of Caenorhabditis elegans. Genome Research 7974-985.

24 May 1999 15:50 554 554 Mutations in a two-P domain potassium channel cause uncoordinated movement in c. elegans.

1999 International Worm Meeting abstract 503 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Mutations in a two-P domain potassium channel cause uncoordinated movement in c. elegans. MT Kunkel 1 , DB Johnstone 2 , ML Nonet 1 , JH Thomas 2 , L Salkoff 1

1 Dept. of Anatomy and Neurobiology and Dept. of Genetics, Washington Univ. School of Medicine, St. Louis, MO 63110. 2 Program in Molecular and Cellular Biology and Dept. of Genetics, Box 357360, Univ. of Washington, Seattle, WA 98195.

As many as 50 genes encoding potassium (K+ ) channel subunits that contain two pore-associated regions (2P channels) are present within the C. elegans genome (http://nt-salkoff.wustl.edu). We have identified two mutants, unc-110(e1913) and mah-2(cn110), that map to a single gene encoding a 2P channel subunit, n2P18 (also see related abstract from Johnstone et al.). These mutants act semi-dominantly to confer uncoordinated movement in C. elegans. unc-110(e1913) animals display uncoordinated movement at room temperature, whereas mah-2(cn110) animals are affected only at elevated temperatures at which they become paralyzed. Expression of GFP under the channel promoter in C. elegans indicates that this channel subunit is expressed only in body-wall muscle, consistent with the phenotype of defective body-wall muscle contraction. By sequencing PCR products from wild-type and mutant C. elegans, we have identified two missense mutations within the predicted coding region of this 2P channel subunit. Both wild-type and mutant channels express outward K+ currents in Xenopus oocytes, but the magnitude of the whole cell current from the mutant channels is much larger than that observed from the wild type channel (UNC-110MAH-2WT). Thus, gain-of-function mutations in this gene may result in greater K+ conductance in body-wall muscle; this would inhibit muscle membrane excitation and thereby prevent contraction. Increasing temperature results in a pronounced increase in whole cell currents from both wild-type and mutant channels. We believe the temperature sensitivity seen with mah-2(cn110) animals is due to a large increase in K+ conductance above a critical level. The activity of the wild-type channel is low such that this level is not reached (up to 35°C), whereas this threshold is crossed even at room temperatures in unc-110(e1913) animals.

24 May 1999 15:50 555 555 VAB-17, a novel WD-40 protein involved in morphogenesis

1999 International Worm Meeting abstract 504 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. VAB-17, a novel WD-40 protein involved in morphogenesis PE Kuwabara 1,2 , R Benton 2 , JA Hodgkin 2

1 Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge UK CB10 1SA. 2 MRC Laboratory of Molecular Biology, Hills Road, Cambridge, UK CB2 2QH.

cr7 was identified as a viable mutant displaying a highly penetrant dorsal head bulge and a less penetrant tail bulge, somewhat similar to vab-1,-2, and -3 mutants. The gene represented by cr7 is named vab-17. Several lines of evidence indicate that vab-17 is essential for embryogenesis, and that cr7 is hypomorphic. vab-17 was genetically mapped to LGV and its physical location was refined using PCR to map deficiency breakpoints. None of the candidate cosmids rescued the vab-17 defect, but rescue was achieved using fosmids covering a cosmid gap. A 10 kb minimal vab-17(cr7) rescuing fragment was found, which led to the identification of several Kohara cDNAs. These cDNAs correspond to alternatively spliced variants of vab-17. Additional cDNA clones were obtained by RT-PCR, and it was found that vab-17 expresses multiple transcripts of different sizes that are alternatively spliced and have alternative 5’ ends. cr7 is a nonsense mutation in an alternative exon. The largest predicted protein encoded by the vab-17 locus has 7-12 WD-40 repeats and a molecular mass of 180 kDa. WD-40 motifs are believed to mediate the assembly of macromolecular complexes. To obtain a preliminary picture of VAB-17 expression, an in-frame GFP fusion was constructed using the minimal vab-17(cr7) rescuing fragment. The fusion protein localises to the cytoplasmic face of adherens junctions, but unlike the adherens junction staining displayed by MH27, the VAB-17 fusion is not associated with embryonic hypodermal adherens junctions. In the course of genetically mapping vab-17, it was found that mutations in members of the GABAergic pathway exhibit synthetic lethality with vab-17(cr7). We have taken advantage of this lethality to identify a series of potential vab-17 intragenic revertants, which are weakly Vab, yet suppress the synthetic lethality.

24 May 1999 15:50 556 556 The C. elegans ptc genes play essential, distinct and apparently Hh-independent developmental roles

1999 International Worm Meeting abstract 505 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The C. elegans ptc genes play essential, distinct and apparently Hh-independent developmental roles Patricia E. Kuwabara Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge UK CB10 1SA; MRC Laboratory of Molecular Biology, Hills Road, Cambridge, UK CB2 2QH Patched-dependent cell signalling is widespread in the animal kingdom, playing roles in developmental patterning, cell proliferation, and carcinogenesis. The first patched (ptc) gene was identified in Drosophila because of its role in establishing segment polarity. In humans, Ptc is a tumour-suppressor protein in basal cell carcinomas, and mutations in PTCH are implicated in Gorlin’s syndrome. Biochemical evidence indicates that Ptc and the serpentine protein Smoothened (Smo) form a receptor complex for the morphogen Hedgehog (Hh). Current models suggest that Ptc inhibits Smo, and that Hh relieves this inhibition by binding to Ptc. In turn, Smo promotes the transcription of TGF-b and Wnt family members. In C. elegans, three genes encode proteins with strong sequence similarity to Drosophila and vertebrate PTC proteins. In addition, a number of other predicted C. elegans proteins comprise a subfamily of distant PTC relatives. Here it is reported that ptc-1 and ptc-3 are the C. elegans genes most closely related to the ptc genes so far identified in other phyla; ptc-2 appears to be a pseudogene that arose through a recent duplication of ptc-1. Genomic and cDNA sequence analyses have been performed for both ptc-1 and ptc-3. Similar to other Ptc proteins, the predicted PTC-1 and PTC-3 proteins each have 12 potential membrane spanning domains and a cholesterol sensing domain. PTC-1 and PTC-3 share approximately 33% identity with Ptc proteins from other phyla. Inactivation of ptc-1 or -3 by RNAi reveals that each gene is essential for development: ptc-1 is needed for cellularisation during gametogenesis, whereas ptc-3 is required for late embryogenesis. To corroborate the ptc-1(RNAi) and ptc-3(RNAi) phenocopies, mutants have been obtained that delete regions of either ptc-1 or ptc-3 (thanks to B. Barstead and L. Liu). Each of the deletion mutants displays phenotypic properties similar to those observed by RNAi. Surprisingly, both Hh and Smo appear to be absent from the C. elegans genome. Therefore, these findings suggest that the control of ptc activity may have diverged during evolution, and further raise the possibility that PTC may have functions that are both Hh- and Smo-independent.

24 May 1999 15:50 557 557 Unwinding the P granules: evidence from RNAi and glh mutants

1999 International Worm Meeting abstract 506 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Unwinding the P granules: evidence from RNAi and glh mutants KA Kuznicki 1 , JW Kirchner 2 , PA Smith 1 , KL Bennett 1

1 University of Missouri, Columbia, MO 65212. 2 Indiana University, Bloomington IN 47405.

The Bennett laboratory studies the four germline RNA helicases (GLHs), components of the C. elegans P granules. The GLHs are members of the DEAD-box family of RNA helicases and contain retroviral-like CCHC zinc fingers and uncharged glycine-rich repeats (Roussell and Bennett 1993 PNAS 90:9300-9304, Gruidl et al. 1996 PNAS 93:13837-13842). GLH-3 and GLH-4, more recently discovered helicases, differ from GLH-1 and GLH-2; GLH-3 lacks the uncharged glycine-rich repeats of the others, while GLH-4 is the most unique. RNAi implies that each GLH has a different role. RNAi of glh-1 or glh-2 produces sterility in a small percentage of the F1 generation at the restrictive temperature. By contrast, glh-3 RNAi causes smaller brood sizes in the injected worm at the restrictive temperature. Using a combination of the helicase regions of glh-1, glh-2, and glh-3 results in a more dramatic temperature sensitive phenotype. Approximately 75% of the F1 generation is infertile. Other combinatorial glh RNAi injections will further determine if the glhs function synergistically. A glh-3- strain with a 1.7 kb deletion was identified in our laboratory from a TMP/UV library we generated. Preliminary analysis of the glh-3- strain indicates that it has a mild temperature sensitive fertility defect, with brood size approximately 60-70% of that seen with wild type worms at 25-26°C. This result is consistent with that seen in the glh-3 RNAi experiments. Ongoing experiments include glh-4 RNAi and screening for additional glh mutants.

24 May 1999 15:50 558 558 Transcriptional Mediators in the Nematode Caenorhabditis elegans

1999 International Worm Meeting abstract 507 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Transcriptional Mediators in the Nematode Caenorhabditis elegans J Kwon 1 , J Park 2 , B Kim 2,3 , Y Kim 2 , J Lee 1

1 Yonsei University, Korea. 2 SamSung Biomedical Research Institute, Korea. 3 KAIST, Korea.

Mediators are the transcriptional cofactors originally isolated from the yeast. We are interested in the functions of mediators in C. elegans. MED6 is one of the components of the mediator complex. In yeast, MED6 is required for activated transcription of subset of inducible genes. However, the biological significance of mediators in metazoan development or transcription system has not been examined yet. In the hope of elucidating the functions of mediators in metazoa, we started examination of med-6 functions in the nematode Caenorhabditis elegans. We cloned a med-6 homolog in C. elegans by database search and RT-PCR. We then investigated the expression patterns of Ce-MED6 by antibody staining. Ce-MED6 is expressed in the peripheral region of the nuclei of all cells at all developmental stages, where actively transcribed portions of the genome are supposedly localized. For the functional analysis of Ce-MED6, we performed RNA interference experiments. The phenotypes of the progeny from the wild-type animals injected with double-stranded RNA were embryonic lethality, arrested larvae, and sterility, indicating that Ce-MED6 is required for many aspects of development including embryogenesis and germ cell development. It is crucial to test whether the transcription of any tissue- or stage-specific genes is affected by Ce-MED6 malfunction in vivo. To this end, we looked into the effect of RNAi on the transcription levels of ceh-13 and nhr-2. The expression of these genes are lineage- or stage-specific and were down-regulated by our Ce-Med6 RNAi. In contrast, ribosomal protein and sur-5 that are constitutively expressed didn’t show remarkable differences from normal expression levels. Let-425 may be identical to the Ce-Med6, because a nonsense mutation is found in the coding region of Ce-Med6 from the let-425 mutant. We found that Ce-MED6 colocalizes with RNA polymerase II in a big complex in vivo by biochemical methods. We are also trying to identify and characterize more mediators in C. elegans, for example, Ce-Med7, Ce-Med10, and Ce-Srb7, and will also present the functional analysis of these genes.

24 May 1999 15:50 559 559 Characterization of a C. elegans homologue of band 4.1 protein

1999 International Worm Meeting abstract 508 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of a C. elegans homologue of band 4.1 protein SJ Kwon, SK Jung, JH Ahnn Department of Life Science, K-JIST, 1 Oryong-dong Puk-ku Kwang-ju, 506-712, Korea Band 4.1 is a cytoskeleton protein found in human erythrocyte. It has been reported that Band 4.1 regulates membrane mechanical properties through modulation of interaction between spectrin and actin, and also regulates band 3-ankyrin interaction. However, the exact biochemical and pyshiological function of this protein has not been elucidated. When we searched the Yuji Kohara’s in situ database for genes specially expressed in body-wall muscle, we came across a clone, yk21h2. This clone appeared to be expressed only in body-wall muscle during embryogenesis. We found a gene, F43d9.1, which shows high homology with band 4.1 protein. This protein is highly conserved in the other organisms. It has 83% identity with band 4.1 protein. We are interested in the facts that C. elegans does not have erythrocytes but this protein is so highly conserved that we can suggest it in an evolutional aspect. According to In situ data(Yuji Kohara), it is transcripted along the body-wall muscle during embryogenesis. So we expect that this protein may be associated with muscle development. Now we are trying to characterize it in C. elegans. To investigate the pyshiological role of this protein, we are trying to see the expression pattern in the C. elegans, and to get the deletion mutant using EMS mutagenesis.

24 May 1999 15:50 560 560 Processing of ROP-1, degradation of CeY RNA and formation of dauer larvae.

1999 International Worm Meeting abstract 509 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Processing of ROP-1, degradation of CeY RNA and formation of dauer larvae. JC Labbé 1 , LA Rokeach 1 , S Hekimi 2

1 Dépt. biochimie, Université de Montréal, Montréal, Qc, H3C 3J7, Canada. 2 Dept. Biology, McGill University, Montréal, Qc, H3A 1B1, Canada.

The Ro ribonucleoprotein complex (RoRNP) has been initially described as an autoimmune target in human diseases such as systemic lupus erythematosus and Sjögren’s syndrome. Its general structure is composed of one protein of 60 kDa, Ro60, that binds to one of four small RNA molecules, designated Y RNAs. Although no function has been assigned to the RoRNP, Ro60 has been shown to bind mutant 5S rRNA molecules in Xenopus oocytes, suggesting a role for Ro60 in 5S rRNA biogenesis. In order to identify the function of RoRNP, we have taken a genetic approach in C. elegans. As such, we cloned the gene rop-1, encoding the homologue of the human Ro60 protein. ROP-1 has been shown to bind to a small RNA, designated CeY RNA, that folds in a similar manner than its human and Xenopus counterparts. We previously demonstrated that the ribosomes of a rop-1(-) strain contain a higher percentage of mutant 5S rRNA molecules, thereby strengthening the notion of a link between ROP-1 and 5S rRNA quality control. Here we demonstrate that ROP-1 is processed during C. elegans larval development. While ROP-1 migrates with an apparent molecular weight of 66.0 kDa in embryo, L1 and L2 nematode extracts, it shifts to 62.5 kDa in L3 extracts, and remains in this form in the L4 and adult extracts. Furthermore, this mobility shift is absent in dauer and post-dauer larvae extracts. The activity responsible for this processing was reconstituted in vitro and displayed a requirement for a pH 6.0 reactional buffer. This activity could be specifically inhibited by 1 µM pepstatin, a potent inhibitor for members of the aspartic proteinase family. These results indicate that an aspartic proteinase is activated at the L3 stage of C. elegans larval development and is involved in the processing of ROP-1. Moreover, the processing of ROP-1 was concomitant with the degradation of its associated CeY RNA. We mapped one of the degradation sites on the CeY RNA molecule in the loop at about half its length, near a consensus polypyrimidine stretch. Taken together, these results suggest a link between ROP-1 and an RNA degradation pathway and support the hypothesis that Ro60 might be involved in the quality control of 5S rRNA.

24 May 1999 15:50 561 561 A fate map of organs and tissues at the early gastrula stage

1999 International Worm Meeting abstract 510 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A fate map of organs and tissues at the early gastrula stage M Labouesse, R Legouis IGBMC, BP163, 67400 Illkirch, France Most organs and tissues have polyclonal origins in C. elegans.This raises the question of whether specification of a given tissue/organ identity relies on a unique global regulator or on multiple regulators that would act in different lineages. Recently, work from several labs has shown that the genes elt-1 (Page et al., Genes Dev. 11:1651-1661) and pha-4 (Horner et al., Genes Dev. 12:1947-1952; Kalb et al., Development 125:2171-2180) establish epidermis and pharynx identity, respectively, during early gastrulation. We have reexamined wild-type development to see if, at the time when elt-1 and pha-4 first act, the precursors of organs and tissues are organised in a salt and pepper fashion or if they are clustered together according to the ultimate fate of their progeny. Using a 4D-microscope we recorded several wild-type embryos and observed that starting between the 50- and 80-cell stages, pharynx, gut, epidermis, muscle and nervous system precursors are located in distinct and non-overlapping positions, independently of their lineal origin. Specifically, we found that pharyngeal precursors reproducibly occupy the ventral-anterior part of the embryo, muscle precursors the ventral-posterior part, epidermal precursors the dorsal part extending posteriorly, gut precursors the interior part, while nervous system precursors form a large stripe that runs centrally from the anterior tip of the embryo to almost its posterior tip. These observations raise several questions. If there are defined fields of precursors at the early gastrula stage, what are the genes that define these fields? Are maternal genes involved in defining them? To what extent does this fate map provide new cues to understand early embryogenesis? Has the fate map been evolutionarily conserved? To address these questions, we are currently examining the positions of organ and tissue precursors in other species, as well as in various maternal and early zygotic mutants known to affect cell fates such as skn-1,or elt-1. In conclusion, our results suggest that despite its invariant lineage C. elegans embryos develop much like other animals by defining prospective organ and tissue regions during early gastrulation.

24 May 1999 15:50 562 562 C. elegans dynamin-related protein drp-1 controls severing of the mitochondrial outer membrane

1999 International Worm Meeting abstract 511 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. C. elegans dynamin-related protein drp-1 controls severing of the mitochondrial outer membrane AM Labrousse, MD Zappaterra, DA Rube, AM van der Bliek Department of Biological Chemistry, UCLA School of Medicine, Box 951737, Los Angeles, CA 90095 Recent evidence from yeast and mammals suggest that dynamin-related proteins affect mitochondrial morphology. We analyzed C. elegans drp-1, which is a structural and functional homologue of yeast and mammalian dynamin-related proteins. The injection of C. elegans drp-1 inhibitory RNA (RNAi) into adult hermaphrodites was lethal to ensuing progeny, suggesting that the drp-1 gene is required during embryogenesis. High levels of expression in the neurons and muscle cells of adult worms suggested that drp-1 was also important at later stages in life. We investigated the function of C. elegans drp-1 using transgenic animals that overexpress mutant drp-1. Mutant drp-1 disrupted the mitochondrial distributions in muscle cells, but the ER morphologies in those same cells were not affected. To investigate the nature of the mitochondrial defect, we labeled the mitochondrial matrix and the mitochondrial outer membrane with different GFP variants. Double labeling showed that the collapsed blebs of mitochondrial matrix were connected by thin tubules of mitochondrial outer membrane. Therefore, mutant drp-1 could block scission of the mitochondrial outer membrane, while scission of the mitochondrial inner membrane was unaffected. GFP-tagged mutant drp-1 was localized to the connecting tubules of outer membrane, suggesting that the block in scission of the outer membrane was a direct consequence of interference by mutant drp-1. In contrast, GFP-tagged wild type drp-1 was localized in spots along normal mitochondria where it might be primed for mitochondrial severing. Together, these results suggest that the drp-1 protein controls the severing of the mitochondrial outer membrane, analogous to the role of dynamin in vesicle formation.

24 May 1999 15:50 563 563 Negative regulation of LET-23 mediated signaling

1999 International Worm Meeting abstract 512 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Negative regulation of LET-23 mediated signaling CJ Lacenere, PW Sternberg Mail Code 156-29, Division of Biology, Caltech, Pasadena, CA 91125 Although many positive components of the LET-23 mediated signaling pathway have been identified and characterized, negative regulation of this pathway is less understood. Several negative regulators have been identified including sli-1, rok-1, gap-1 and unc-101. A mutation in any one of these negative regulators results in a wild-type vulva. However, certain double mutant combinations of these negative regulators results in a Multivulva phenotype. We are currently characterizing sli-2. sli-2 was originally isolated as a semi-dominant suppressor of the reduction of function receptor allele let-23(sy1). sli-2 appears to act upstream of let-60 RAS and downstream or in parallel with let-23. sli-2 has no apparent phenotype on it’s own, but double mutants with any of the negative regulators listed above results in a Multivulva phenotype. Duplication data suggests that sli-2 may be a gain-of-function allele. We are currently attempting to clone sli-2 and are performing screens to isolate new alleles.

Supported by the U.S. Army Breast Cancer Research Program

24 May 1999 15:50 564 564 Luminescent Caenorhabditis elegans: a novel eukaryotic biosensor

1999 International Worm Meeting abstract 513 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Luminescent Caenorhabditis elegans: a novel eukaryotic biosensor C Lagido, J Pettitt, LA Glover Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK Biosensors produce a measurable response when exposed to an environmental signal. The intensity of their response is often correlated to that of the signal, enabling its detection and quantification. Bioluminescent microbial biosensors have been developed and applied to detect xenobiotics in a wide variety of situations. However, since eukaryotic and prokaryotic cells very likely differ in their sensitivity to xenobiotics, we decided to investigate the use of C. elegans as a biosensor. We transformed C. elegans with the luc gene, from the firefly Photinus pyralis, under the control of the let-858 promoter and obtained an integrated line. The luc gene encodes an enzyme, firefly luciferase, which catalyses the oxidation of luciferin in a reaction that consumes ATP and produces light. The light emitted by cells expressing luc is thus a measure of their ATP levels and therefore of their metabolic status. Exposure to toxic compounds will disrupt the general metabolism of the cells and this can be measured as a decrease in bioluminescence. Exposure of luc expressing C. elegans to various concentrations of copper resulted in decreased light output. There was good correlation between the decrease in light and the copper concentration. This demonstrates the use of luminescent C. elegans as a novel and rapid biosensor. Experiments are currently underway to test the effects of several other toxicants on C. elegans bioluminescence. In addition to its usefulness as a biosensor, bioluminescent C. elegans provide us with a very convenient tool for studying the regulation of C. elegans metabolism in vivo.

24 May 1999 15:50 565 565 Dominant Spermatogenesis-defective mutants in C. elegans

1999 International Worm Meeting abstract 514 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Dominant Spermatogenesis-defective mutants in C. elegans Todd Lamitina, S.W. L’Hernault Emory University During differentiation, asymmetric cellular divisions are a common feature and result in non-equivalent daughter cells. This process of asymmetric cytoplasmic partitioning is central to development, yet its mechanism is poorly understood. All spermatogenesis defective mutants (spe mutants) studied to date have been recessive. However, I have chosen to focus on a rare class of spe mutants that are dominant. Dominant mutants allow study of processes that might be affected by two or more genes acting in a redundant fashion. Such genes can be difficult or impossible to detect by studying only recessive mutants. There are 15 mutants in this class and they were obtained in an unbiased manner so they sample the entire genome. Ultrastructural analyses of two of these mutants have been performed and they have a startling phenotype not observed in any of the more that 40 analyzed spe mutants defined by recessive alleles. Both the e1947 and q89 mutants arrest as primary spermatocytes yet contain many intracellular features of a haploid spermatid. It appears that both mutants allow aspects of sperm differentiation to occur in the absence of any nuclear and cytoplasmic divisions. As such, they affect a key molecule in the coordination of events required for sperm differentiation. A traditional complementation test between q89 and e1947 is not possible since both are completely penetrant dominant sterile mutants. Based on their identical EM phenotypes and three-point mapping data, q89 and e1947 are probably allelic and have tentatively been assigned to the new gene, spe-37. Heterozygous dominant sterile mutants will become fertile if a null mutation that eliminates the dominance is induced in cis. Such a null mutant will reveal if either q89 or e1947 has a loss of function phenotype and if it is spe, lethal or wild type. A recessive phenotype would then allow complementation tests between e1947 and q89 to confirm allelism. Such null mutants should also aid in determining the DNA sequence of the spe-37 gene and how it is altered by dominant and recessive mutations.

24 May 1999 15:50 566 566 PKL is targeted to F-actin via a PDZ domain

1999 International Worm Meeting abstract 515 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. PKL is targeted to F-actin via a PDZ domain M Land, C.S. Rubin Albert Einstein Col. Med., Bronx, NY 10461 PKL1 and PKL2 are large C. elegans S/T protein kinases. Catalytic domains of PKL1, PKL2 and MAST205, a mammalian protein kinase, are 70% identical. Each kinase contains a conserved PDZ domain. N and C termini of PKL and MAST205 are divergent. Clues regarding the functional significance of these kinases were obtained. Immunostaining revealed that PKL1 is aligned along filaments of body wall and vulva muscles, adjacent to actin. PKL1 is also abundant in spermatheca and the oocyte plasma membrane. PKL2 is enriched in pharyngeal muscle (adjacent to F-actin), the apical surface of intestine and the gonad. Both PKLs are associated with punctuate vesicular structures in many worm tissues. The PKL promoter is active in cells identified by immunostaining. Screening via the yeast 2-hybrid system revealed that SKN-1, WRM-1 and DSH interact with the PKL PDZ domain. Binding was verified in vitro and recombinant WRM-1, SKN-1 and DSH were phosphorylated by PKL1. Partial co-localization of PKL2 and SKN-1 in embryos suggests that these two proteins interact in vivo. MAST205 is expressed in testis, skeletal muscle and heart. Radiolabeled PDZ domains from MAST205 and PKL bound a 33 kDa skeletal muscle protein. Functional screening of a mouse muscle cDNA expression library yielded cDNAs encoding tropomyosinB. cDNAs encoding C. elegans tropomyosin cDNAs were obtained from Y. Kohara and expressed in E. coli. Nematode tropomyosins bind to the PDZ domain of PKL. Mammalian tropomyosins are phosphorylated in vivo; their abilities to modulate myofibril assembly and contractile force are thought to be regulated by this post-translational modification. PKLs may be targeted to actin filaments in body wall and pharyngeal muscles via interaction with tropomyosin. Anchored PKL/MAST205 may catalyze phosphorylation of tropomyosin and/or troponin subunits, thereby regulating contraction. PKL2 is detected in the actin-containing "midbody" in embryos. Microinjection of PKL antisense RNA generated embryos containing binucleate cells, consistent with a failure in cytokinesis. Transgenic nematodes expressing anti-sense PKL RNA in muscle are paralyzed at L1 and move slowly at other developmental stages. PKL2 may be involved in asymmetric segregation of developmental factors. For example, SKN-1, a PKL binding partner, accumulates to high levels in P1 blastomeres of two cell embryos. C. elegans

24 May 1999 15:50 567 567 Identification of a gene required for pharyngeal elongation and deleted by mnDf90.

1999 International Worm Meeting abstract 516 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification of a gene required for pharyngeal elongation and deleted by mnDf90. SK Lange, SE Mango Department of Oncological Sciences and Hunstman Cancer Institute Center for Children. University of Utah, Salt Lake City, UT, 84112 Midway during embryogenesis the pharyngeal precursors assemble into a ball of cells that comprises the pharyngeal primordium. Over the next 1-2 hours the primordium elongates to form a tube that is connected to the buccal cavity. We have analyzed the phenotypes of homozygous deficiencies to identify loci involved in this process (see abstract by Portereiko, Domeier and Mango). We found that mnDf90 embryos fail to undergo the initial stages of pharyngeal elongation and never attach to the buccal cavity. Other aspects of pharynx development appear normal: the correct number of pharyngeal cells are present, the pharynx appears to differentiate normally, and MH27 staining indicates that the pharyngeal muscles are properly organized. To isolate the gene(s) responsible for the mnDf90 pharynx defect we screened 3100 haploid genomes for lethal mutations that map to the mnDf90 region. We identified three alleles: px3, px4 and px5. The most severe allele is px4, which arrests as a 2-fold embryo with an unelongated but differentiated pharynx. Homozygous px3 embryos arrest at the 3-fold or L1 stages. The pharyngeal phenotype of px3 resembles that of px4. Embryos mutant for the third allele, px5, have increased numbers of cell corpses in addition to the unelongated pharyngeal phenotype. Our immediate goals are to determine whether these alleles define one or two different genes and to isolate additional alleles.

24 May 1999 15:50 568 568 Developmental determination of AWA and AWB chemosensory neurons

1999 International Worm Meeting abstract 517 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Developmental determination of AWA and AWB chemosensory neurons AE Lanjuin, TJ Melkman, P Sengupta Department of Biology, Brandeis University, Waltham, MA 02454 Our lab is interested in understanding how the fate and functions of chemosensory neurons are determined. We have been looking for factors involved in the determination of AWA and AWB sensory neuron fate. The strategy we used is to identify mutants that misexpress GFP driven by the AWA specific promoter odr-7, or the AWB specific promoter str-1. AWA:Mutants in which AWA specific expression is altered fall into two categories. One class of mutants shows reduced expression of odr-7::GFP in the AWA neurons. At least one of these mutations is in the odr-7 gene. odr-7appears to be required for maintenance but not initiation of its own expression. Other mutants in this class are currently being further characterized. A second class includes a mutant that shows odr-7::GFP expression in only one of the bilaterally symmetric AWA cells. Preliminary experiments show that this mutant has defects in AWA mediated behavior, consistent with its possible involvement in AWA specification and function. Further characterization of these mutants is underway, as well as a continuing search for additional mutants. AWB: To date, we have screened more than 8000 haploid genomes using str-1::GFP to follow AWB fate. These screens have isolated three classes of mutants. One class shows reduced str-1::GFP expression. Among mutants in this class we isolated several alleles of lim-4. Recent work by Sagasti et al has elucidated its role in AWB specification. In a lim-4 mutant, AWB is converted to AWC (Sagasti, Bargmann, pers. comm). We have also isolated two alleles of sns-8 X, which appears to function downstream of lim-4. Recent evidence suggests that sns-8 may have a role in the maintenance of AWB fate in adults. We have also found a mutant with ectopic str-1 expression. 29% of sns-9 II mutants have an ectopic str-1::GFP expressing cell when reared at 25.5°C. This ectopic cell appears to have AWB-like cilia. In the third class of mutants str-1 expression is seen variably in one, both or none of the AWB neurons. The cloning and characterization of sns-8, sns-9, and of the other mutants isolated, should provide insight into how AWB fate is initiated, restricted and maintained.

24 May 1999 15:50 569 569 Sodium Azide Induces Thermotolerance In C. elegans By A Mechanism Similar To The Heat Shock Response

1999 International Worm Meeting abstract 518 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Sodium Azide Induces Thermotolerance In C. elegans By A Mechanism Similar To The Heat Shock Response MR Massie, KD Boggs, KE Stine, GE White Ashland University, Ashland, Ohio 44805 Sodium azide, which inhibits cytochrome oxidase c and ATP synthase, creates a chemical hypoxia in cells and can induce certain heat shock proteins. It is a worm anesthetic but the mechanism by which C. eleganscan survive prolonged exposure to this chemical is not known. We hypothesized that chemicals affecting energy metabolism induce the heat shock response in C. elegansand produce a thermotolerant worm.

For control (no azide or heat exposure) worms taken directly to 37 o C, their survival probability (SP) = 0.07, while for worms exposed to 10 mM azide and then taken to 37 o C, their SP = 0.68 (p<0.05), thereby indicating azide induced thermotolerance. This compares well with worms exposed to 33 o C and then taken to 37 o C, SP = 0.92 (p<0.05). We then hypothesized that the molecular mechanism of the response to azide involves induction of the heat shock proteins. Using SDS-PAGE, Coomassie Brilliant Blue staining, and Western Blot analysis, we observed induction of hsp70 and hsp16, but the levels of induction were low when compared to worms exposed to 33 o C. To further elucidate this mechanism, we tested the only known hsp mutant, daf-21 (p673). Using our standard protocol, when grown at the permissive temperature, it demonstrated azide induced thermotolerance. At the restricted temperature, however, there was no azide induced thermotolerance. These results suggest that while hsp90 is not induced by sodium azide, it is an essential part of the worm’s response to stress. Azide induces thermotolerance in C. elegansby a mechanism similar to the heat shock reponse but at reduced levels of hsp induction. In light of the daf-21results, we examined other dauer mutants for their ability to undergo azide induced thermotolerance (see Lapoczka et al, this meeting), in addition to identifying intrinsically thermotolerant substrains of N2s (see Smith et al., this meeting).

24 May 1999 15:50 570 570 Altered Response Of Dauer Mutants To Sodium Azide Exposure And The Acquisition Of Thermotolerance.

1999 International Worm Meeting abstract 519 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Altered Response Of Dauer Mutants To Sodium Azide Exposure And The Acquisition Of Thermotolerance. EM Lapoczka, KE Stine, GE White Ashland University, Ashland, Ohio 44805 We have demonstrated that exposure to low levels of sodium azide confers thermotolerance in C. elegansby a mechanism similar to the heat shock response (see Massie et al., this meeting). As a part of that study, we found that daf-21(hsp90), a constitutive dauer forming mutant, acquired sodium azide induced thermotolerance when grown at the permissive temperature, but not at the restricted temperature, implying an important role for hsp90 in the worm’s response to stress. Since the dauer state represents a significant response to external stresses, we wanted to determine whether those results reflected the importance of hsp90 in the response to stress, or was this a general characteristic of all dauers. We tested four different dauer mutants: daf-2(m41, an insulin/IGF-I receptor), daf-7(m62, a member of the TGF-b superfamily), daf-14(m77, encodes dwarfin/MAD/DPC-4), and daf-21(p673, hsp90). We demonstrated the acquisition of sodium azide induced thermolerance in all mutants tested at the permissive temperature; however, our initial studies failed to demonstrate the acquisition of thermotolerance in any mutants grown at the restricted temperature. We noted that dauers took longer to anesthetize with sodium azide (15-30 mins vs. <5 for controls); therefore, we increased the exposure from 1 to 2 hours. In addition, we lengthened the post heat shock recovery. This allowed us to demonstrate azide induced thermotolerance in all of these daf mutants, including daf-21. These results demonstrate that it is not the dauer state alone which would prevent a worm from obtaining sodium azide induced thermotolerance, and that hsp90 may not be a critical part of the worm’s response to stress.

24 May 1999 15:50 571 571 Evolutionary & ecological aspects of aging in C. elegans

1999 International Worm Meeting abstract 520 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Evolutionary & ecological aspects of aging in C. elegans S Le Comber, L Partridge, D Gems Department of Biology, University College London, London NW1 2HE, U.K. Traditionally, most work on aging in C. elegans has adopted the methods of classical genetics. Our aim is to extend this approach to include evolutionary biology, and in particular to address the question of the effect of life span extension on fitness. We are currently investigating this question in the laboratory, using a range of mutant types. Initially, we are measuring two components of fitness: the intrinsic rate of increase, r, which is frequently used as a global measure of fitness (1), and the ability of starved populations to survive over long periods. These give an indication of the effects of mutations on fitness in "boom" and "bust" conditions, respectively. Evolutionary optimality theory predicts the occurrence of trade-offs between different fitness traits including fecundity and life span (2). In C. elegans, reproduction by self-fertilization does not reduce life span (3), nor does increasing egg production (4), and mutation of daf-2 can extend life span without reducing brood size (5). This argues against a trade-off between fecundity and longevity in C. elegans. However, it is unclear whether the Age phenotype affects the length of the reproductive period and overall fecundity, where sperm number is not limiting. Furthermore, trade-offs between fecundity and longevity may not be detected under replete nutritional conditions. These issues are under investigation. One problem with laboratory analysis of fitness is that it is unclear how well laboratory measures of fitness correlate with the organism’s fitness in the conditions to which it is adapted. Unfortunately, little is known about the ecology of C. elegans in the wild. For instance, the degree to which wild populations of C. elegans experience "boom and bust" cycles, or relatively stable populations, is unknown. Equally, the importance of males in wild populations, or whether they occur at all, is unknown. For this reason, we are also setting out to investigate the ecology of C. elegans in the wild.

(1) Roff (1992) The Evolution of Life Histories; (2) Stearns (1992) The Evolution of Life Histories; (3) Friedman and Johnson (1988) Genetics 118: 75; (4) Gems and Riddle (1996) Nature 379: 723; (5) Gems et al. (1998) Genetics 150: 129.

24 May 1999 15:50 572 572 Programmed cell death in the ventral epidermis of Pristionchus pacificus

1999 International Worm Meeting abstract 521 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Programmed cell death in the ventral epidermis of Pristionchus pacificus KZ Lee 1,2 , R Sommer 1

1 Max Planck Institute for Developmental Biology, Dept. for Evolutionary Biology, Spemannstrasse 35, D-72076 Tuebingen Germany. 2 E.mail: [email protected]

We are studying the evolution of developmental processes by comparing vulva development between C. elegans and Pristionchus pacificus. In P. pacificus, seven of the twelve ventral epidermal cells undergo programmed cell death during late embryogenesis. We have previously shown that mutations in the ced-3 homolog of P. pacificus prevent apoptosis of P(1-4,9-11).p and that Ppa-ced-3 mutants suppress the vulvaless phenotype of the homeotic gene Ppa-lin-39. We have used other genetic screens to isolate new components of the cell death machinery in the ventral epidermis: in a Nomarski F2 screen, we have isolated two mutations that block programmed cell death, like Ppa-ced-3. Complementation tests revealed these two mutations to be allelic to each other, but not allelic to Ppa-ced-3. We are currently testing the hypothesis that these mutations, originally called Ppa-ipa-2, correspond to the Ppa-ced-4 gene. To clone the ced-4 homolog of P. pacificus we want to use the syntenic organization of the C. elegans and P. pacificus genome, that has been shown to be present in at least some areas of the genome. Cel-ced-4 is closely linked (appr. 40 kb) to Cel-pal-1. We have cloned the Ppa-pal-1 by polymerase chain reaction and have isolated a genomic Lambda clone of this gene. We are now using a chromosomal walking strategy to find Ppa-ced-4. In addition, we are using new genetic strategies to isolate more cell death defective mutants in P. pacificus.

24 May 1999 15:50 573 573 Integrin signaling in C. elegans

1999 International Worm Meeting abstract 522 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Integrin signaling in C. elegans M Lee, J Schwarzbauer Department of Molecular Biology, Princeton University, Princeton, NJ 08544 Integrins are a large family of adhesion receptors that are essential for mediating cell-extracellular matrix interactions in multicellular organisms. Integrins also function as signaling receptors that integrate bidirectional information between the extracellular environment and the inside of the cell. Binding of matrix proteins induces clustering of integrins into protein complexes called focal adhesions, initiating intracellular signaling and protein phosphorylation involved in cell adhesion, migration, and proliferation. Structurally, integrins are heterodimers composed of one a and one b subunit. Integrin-induced phosphorylation events depend on the b cytoplasmic domain. Expression of chimeric proteins consisting of the b1 integrin cytoplasmic tail connected to a heterologous extracellular and transmembrane (TM) domain in cultured cells results in reduced cell adhesion and migration, suggesting that an unpaired b cytoplasmic domain can interfere with the functions of endogenous integrins. In C. elegans, integrins apat-2 and bpat-3 are required for body wall muscle function and null alleles cause paralysis and lethality during embryogenesis. Expression of a chimeric subunit consisting of the bpat-3 TM and cytoplasmic domains (HA-btail) connected to a heterologous signal sequence and extracellular domain in body wall and sex muscles of transgenic nematodes has a dominant negative effect. Transgenic animals show phenotypes of varying severity including paralysis at embryonic or larval stages, uncoordinated movement, and defects in egg-laying. Immunofluorescence staining shows localization of chimeric integrins to dense bodies, complexes in body wall muscle that are similar to focal adhesions. Severely disorganized muscle filaments as seen by rhodamine-conjugated phalloidin staining indicate that the chimeric integrin is inhibiting endogenous integrin functions. Mutations in the HA-btail are currently being analyzed to identify sequences important for cytoskeletal connections and signaling. Further characterization of phenotypes will help us to understand the molecular interactions required for integrin function in vivo.

24 May 1999 15:50 574 574 Expressional control of the C. elegans dna topoisomerase i gene

1999 International Worm Meeting abstract 523 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Expressional control of the C. elegans dna topoisomerase i gene M Lee, H Koo Yonsei university To investigate the expressional regulation of C. elegans DNA topoisomerase I, the mRNA expression was probed by in situ mRNA hybridization, and the transcriptional control was analyzed in transgenic C. elegans using a reporter gene. The DNA topoisomerase I mRNA began to appear in the early embryos, was maximized in the expression level at the 200-cell stage, and then disappeared rapidly afterwards. When the 5’-upstream DNA sequence ranging from the trans-splicing site to the -1.0 kb site was linked with the lacZ coding DNA, the reporter gene expression was observed in the embryos, remained strong until the L1 stage, and then decreased gradually. The strong reporter gene expression in the embryos coincided with the mRNA expression of native DNA topoisomerase I, suggesting that the 1.0 kb long 5’-upstream DNA sequence was sufficient to direct the topoisomerase I gene expression. The discrepancy between the relative variations of the DNA topoisomerase I mRNA and reporter protein levels after the embryonic stage is likely to be due to the instability of the DNA topoisomerase I mRNA. With serial deletions from both ends of the 5’-upstream DNA sequence, several DNA regions affecting the transcriptional activation were localized. To study in vivo function of the C. elegans DNA topoisomerase I, the antisense RNA was microinjected into C. elegans gonads. The RNA-mediated interference of gene expression caused abnormal oogenesis and embryogenesis.

24 May 1999 15:50 575 575 Extension of C. elegans lifespan by a mutation in daf-21/hsp90

1999 International Worm Meeting abstract 524 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Extension of C. elegans lifespan by a mutation in daf-21/hsp90 R Lee, G Ruvkun Department of Molecular Biology, Massachusetts General Hospital, 50 Blossom Street, Boston, MA 02114 Longevity often associates with resistance to environmental stress. In C. elegans, long-lived (Age) mutants such as age-1 and daf-2 are more resistant to oxidative, UV-radiation, or thermal stresses. To get at the proximal causes for this longevity-associated stress resistance, we are focusing on the worm stress-response gene hsp90 because it is a molecular chaperone; in contrast to many other ’house-keeping’ genes, its mRNA level is dramatically increased in the dauer compared to other developmental stages (Dalley & Golomb 1992, Dev Biol 151: 80). The Thomas lab found that daf-21(p673) has a missense mutation in the worm hsp90 gene (Malone and Thomas, WBG14.4: 52). daf-21(p673) has a temperature-sensitive dauer constitutive (Daf-c) phenotype. Based on this phenotype, genetic analysis showed that p673 is a recessive, possibly neomorphic mutation that encourages dauer formation by affecting chemosensory neuron function (Malone et al, IWM93: 297). We found that daf-21(p673) mutant lived about 40-50% longer (mean and maximal adult lifespan) than wild-type animals when shifted from the permissive (15°) to the restrictive (25°) temperature at the L4 or young adult stages. Whereas either daf-16(mgDf47) or daf-12(m20) efficiently suppressed the Daf-c phenotype (Thomas et al 1993, Genetics 134: 1105), only daf-16 suppressed Age, suggesting that daf-21 may affect dauer formation and lifespan independantly. daf-16 is a member of the Forkhead class of transcription factors; and it mediates all of the effects caused by daf-2 insulin-like signaling, including lifespan (Ogg et al 1997, Nature 389: 994; Lin et al 1997, Science 278: 1319). One possible explanation for the observation that a daf-16 null mutation is epistatic to daf-21 is that daf-21 expression is positively regulated by daf-16. We are testing this hypothesis. We are also asking if Age mutants in general have an elevated hsp90 expression or function.

We thank Jim Thomas for providing daf-21 mutant strains.

24 May 1999 15:50 576 576 Genes that are involved in controlling morphogenesis of the embryo.

1999 International Worm Meeting abstract 525 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genes that are involved in controlling morphogenesis of the embryo. R Legouis 1 , JM Bosher 1 , DL Baillie 2 , A Rose 3 , M Labouesse 1

1 IGBMC, BP163, 67404 Illkirch Cedex, France. 2 Dept of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada. 3 Dept of Medical Genetics, University of British Columbia, Vancouver, Canada.

One of our interests is to identify genes involved in the control of embryonic morphogenesis during development. This process consist of four main steps: 1) generation of a patch of hypodermal cells in a posterior/dorsal position; 2) migration/extension of these hypodermal cells ventrally and anteriorly to fully enclose the embryo; 3) elongation of these cells, which in turn drives embryonic elongation; 4) synthesis of the cuticle. To identify morphogenesis mutants we relied on two parallel approaches. First, a deficiency screen using MH27 and LIN-26 antiserum as markers showed us that we can identify unique regions affecting enclosure/elongation (Dev. Dyn. 210: 19-32). Starting from there, we examined pre-existing mutations that map to deficiencies of interest to see if they recapitulate the deficiency phenotypes. Second, we induced new mutations using EMS or a transposon mutator background as mutagens. Combining the two approaches, we found one mutation which affects ventral enclosure, and nine mutations which affect elongation but not enclosure. let-169(h1483)LGIR (one allele) mutants are ventral enclosure defective; this defect subsequently leads to rupturing of the embryos and extrusion of the pharynx and gut. let-344LGVL (two alleles) mutants fail to elongate beyond the comma stage. let-413 LGV (four alleles) mutants arrest at the 1.5 to 2 fold stage with an abnormal punctate MH27 staining pattern. let-?(mc36)LGIVR mutants arrest between the 11/2 and 2 fold stages. mc34 mutants were initially picked as enclosure defective, but after backcrossing they rather displayed a Pat phenotype. Indeed, we found that mc34is a new allele of pat-2. mcDf2 LGX, which was previously named eld-1(mc13), turns out to be a 550kb deficiency with a very strong elongation defect. We are currently concentrating our efforts on the cloning of let-169(h1483) and let-344(s376)which we have positioned in a region of 150 to 200 kb.

24 May 1999 15:50 577 577 Organizational Cues for Intestinal Morphogenesis in C. elegans

1999 International Worm Meeting abstract 526 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Organizational Cues for Intestinal Morphogenesis in C. elegans BH Leung 1,2 , GJ Hermann 2,3 , JR Priess 1,2,3

1 Molecular and Cellular Biology Program, FHCRC/UW, A3-013, P.O. Box 19024, 1100 Fairview Ave., Seattle, WA 98109-1024. 2 Fred Hutchinson Cancer Research Center, A3-013, P.O. Box 19024, 1100 Fairview Ave., Seattle, WA 98109-1024. 3 HHMI, A3-013, P.O. Box 19024, 1100 Fairview Ave., Seattle, WA 98109-1024.

We are studying intestine formation in the embryo of Caenorhabditis elegans as a model system for organ morphogenesis. The intestine consists of 20 cells that form a bilaterally symmetric tube connecting the pharynx and rectum. The cells are highly polarized, with apical brush borders and basal basement membranes. All 20 intestinal cells are derived from a single early blastomere, E. To ask if extrinsic signals from the surrounding tissue are required for organizing the intestine, we have physically isolated E and allowed it to develop in culture. In culture, E forms a cluster of polarized cells surrounding a central lumen. Similar to developing intestine in the normal embryonic context, these intestinal cells have their nuclei positioned apically, near the lumenal surface. Thus establishment of apicobasal polarity in the intestine does not appear to require extrinsic signals. The bilateral symmetry of the intestinal cells raises the possibility of differences between the left and right groups of intestinal precursors. The left/right groups are created by a single left/right division in the E lineage. To test if differences in cell polarity are established after the left/right division, either the left or right cells were killed immediately following the division. In both cases, a single row of uniformly polarized cells develops after laser ablation. When the left intestinal cells were ablated, the right intestinal cells polarized contralaterally to the left; when the right cells were ablated, the left intestinal cells polarized right. In contrast, ablation of anterior or posterior precursors at the same stage results in bilaterally symmetric polarized tubes. These results suggest anterior and posterior halves have equivalent information regarding intestinal organization, while left and right halves contain different organizational information.

24 May 1999 15:50 578 578 mig-5, a dsh Homologue, Controls Cell Migration and Cell Fate Determination in C. elegans

1999 International Worm Meeting abstract 527 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. mig-5, a dsh Homologue, Controls Cell Migration and Cell Fate Determination in C. elegans C Guo, E Hedgecock Department of Biology, The Johns Hopkins University, Baltimore, MD 21218 mig-5 mutants exhibit various phenotypic defects related to cell migration and cell fate determination. In mig-5 mutant hermaphrodites, one or both DTCS are often missing or not functioning which results in the loss of one or two of gonadal arm, respectively. The descendants of the QL neuroblast fail to migrate posteriorly but migrate anteriorly instead. The loss of some vulval precursor cell(s) results in an asymmetric vulva or mini vulva. In the preanal equivalence group, the P12 cell often adopts a P11 cell fate. The mig-5 gene has been positionally cloned on chromosome II. mig-5 encodes an nematode ortholog of dsh which is in the wingless signal transduction pathway. The MIG-5 protein contains three conserved domains including a PDZ domain (also known as Discs-Large homology repeats[DHRs]). PDZ domain has been identified in a diverse set of proteins. Some functions of the PDZ domain are involved in clustering of membrane proteins and linking signaling molecules in a multiprotein complex at specialized membrane sites by protein-protein binding. We have investigated the expression pattern of mig-5 protein using GFP tagging method. MIG-5::GFP is expressed in the migrating DTCs , in P cells and interestingly, in the nerve ring. MIG-5::GFP is also expressed in embryos. The expression pattern suggests that mig-5 function may not be limited to the wingless signal transduction pathway. Currently, we are employing yeast two-hybrid system to find protein(s) which might interact with MIG-5. We have obtained several clones for MIG-5 binding protein and are in process of evaluating these candidates.

24 May 1999 15:50 579 579 Involvement of mag-1, a homolog of Drosophila posterior group gene mago nashi, in hermaphrodite germ-line sex determination

1999 International Worm Meeting abstract 528 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Involvement of mag-1, a homolog of Drosophila posterior group gene mago nashi, in hermaphrodite germ-line sex determination R Boswell, B Wood MCD Biology, U. Colorado, Boulder CO 80309 In D. melanogaster, the posterior group gene mago nashi is involved in oocyte patterning during oogenesis, including dorso-ventral determination as well as posterior germ plasm assembly. The mag-1 gene is the most convincing homolog of a Drosophila posterior group gene in C. elegans, since it shares 80% identity and 88% similarity with mago nashi and can functionally replace it in flies (1). In attempts to understand the possible function(s) of mag-1 in worms, we have found using RNA-mediated interference that mag-1(RNAi) animals display a masculinization-of-germ-line (Mog) phenotype in hermaphrodites, suggesting a role in hermaphrodite germ-line sex determination. Epistasis analysis showed that mag-1 acts upstream of fog-2, which promotes temporary masculinization of the hermaphrodite germ line during the L4 stage. This result suggests that mag-1 is required for maintaining oogenesis in adult hermaphrodites, possibly by negatively regulating fog-2 activity. The mag-1 gene also appears to act upstream of the gld-1 gene. Consistent with these results, ectopic sperm production caused by mag-1(RNAi) was found to depend on the fog-1 and fem-1, -2 and -3 genes, which act downstream of fog-2 and gld-1 in the germ-line sex determination pathway. In addition to germ-line masculinization, mag-1(RNAi) caused lethality of progeny embryos when the Mog phenotype was suppressed in a fog-2(lf) background, suggesting an essential role for mag-1 during embryogenesis. These embryos were arrested during morphogenesis with an apparent elongation defect. The expression pattern of JAM-1(MH27)::GFP, which localizes to adherens junctions, showed that hypodermis is disorganized in these embryos. Embryonic expression of the mag-1 gene prior to and during morphogenesis is consistent with a role in embryogenesis.

1. Newmark, P. A., Mohr, S. E., Gong, L. and Boswell, R. E. (1997). mago nashi mediates the posterior follicle cell-to-oocyte signal to organize axis formation in Drosophila. Development 124: 3197-3207.

24 May 1999 15:50 580 580 Molecular characterization of the novel cadmium-inducible gene, cdr-1

1999 International Worm Meeting abstract 529 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Molecular characterization of the novel cadmium-inducible gene, cdr-1 V Liao, JH Freedman Nicholas School of the Environment, Duke University, Durham, NC 27708 The transition metal cadmium is a pervasive and persistent environmental contaminant that has been shown to be both a human toxicant and carcinogen. To inhibit cadmium-induced damage, cells respond by increasing the expression of genes encoding stress-response proteins. In most cases, the mechanism by which cadmium affects the expression of these genes remains unknown. We have identified multiple novel cadmium-responsive genes in C. elegans using differential display technique. One of the genes, designated cdr-1, was selected for further analysis. The cdr-1 cDNA encodes a 32 kDa, integral membrane protein that is identical to the C. elegans F35E8.11 predicted gene product. The rate of cdr-1 mRNA accumulation following cadmium exposure is significantly greater than either of the C. elegans metallothioneins. This suggests that CDR-1 may function in the defense against cadmium-induced toxicity. Several putative upstream regulatory elements (URE) are identified in the cdr-1 promoter. Potential UREs include a metal-responsive element, a heat-shock element, cAMP-response element, and AP-1 and GATA elements. Transgenic C. elegans carrying cdr-1/lacZ reporter transgene and whole-mount in situ hybridization were used to determine the cell-specific pattern of cdr-1 gene expression. When nematodes were treated with cadmium, cdr-1 is exclusively transcribed in intestinal cells throughout post-embryonic stages of developmental. In the absence of cadmium, cdr-1gene expression is not observed at any developmental stage. Together, these results suggest that cdr-1 is regulated in a cadmium-inducible and cell-specific fashion. The effects of other stressors including transition metals (e.g., zinc, copper, mercury, lead), oxidative stress, and heat shock on cdr-1 transcription was evaluated using transgenic C. elegans. Exposure of transgenic C. elegans to various stressors failed to induce reporter transgene expression. These results suggest that cdr-1 specifically responds to cadmium, indicating that cdr-1 transgene can serve as a sensitive biomonitor of cadmium contaminant.

24 May 1999 15:50 581 581 Regulation of vesicular proteins by UNC-4 and UNC-37

1999 International Worm Meeting abstract 530 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Regulation of vesicular proteins by UNC-4 and UNC-37 KM Lickteig 1 , J Duerr 2 , D Frisby 2 , J Rand 2 , DM Miller 1

1 Dept. of Cell Biology, Vanderbilt University Medical Center, Nashville, TN 37232. 2 Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104.

Transmission of chemical neuronal impulses depends on fusion of neurotransmitter vesicles with the presynaptic membrane. Here we present evidence that expression of vesicular proteins is regulated in specific cholinergic motor neurons by unc-4 and unc-37. The transcription factors UNC-4 (homeobox protein) and UNC-37 (Groucho) are required for proper differentiation of DA and VA motor neurons ("unc-4 motor neurons"). In unc-4 and unc-37 loss-of-function mutants, we observe decreased levels of vesicular proteins in unc-4 motor neurons. unc-17 encodes the vesicular acetylcholine transporter (VAChT) and cha-1 encodes choline acetyltransferase (ChAT). Both of these proteins are associated with synaptic vesicles and are required for cholinergic function. unc-17 and cha-1 are coordinately transcribed from a common promoter. In unc-4 and unc-37 mutants, antibody staining of UNC-17 and ChAT is substantially reduced in unc-4 motor neurons. However, unc-4 and unc-37 mutations do not decrease expression of a GFP reporter gene driven by the unc-17-cha-1 promoter. Since the levels of UNC-17 and ChAT proteins are decreased in unc-4 and unc-37 mutants whereas unc-17-cha-1 transcription is not affected, we hypothesize that unc-4 and unc-37 are regulating unc-17 and cha-1 via a post-transcriptional mechanism. This model is also consistent with the observation that unc-4 and unc-37 are required for maintaining normal levels of the vesicular proteins, synaptotagmin, VAMP, and RAB-3. The concomitant effects of unc-4 and unc-37 mutations on at least five different vesicular proteins may mean that UNC-4 and UNC-37 regulate expression of gene products involved in general aspects of vesicle synthesis, assembly, and/or stability. Since UNC-4 and UNC-37 are hypothesized to function as transcriptional repressors, we further propose that UNC-4 and UNC-37 may negatively regulate a gene that destabilizes vesicular proteins in unc-4 motor neurons. A major goal of this work is to define the mechanism whereby unc-4 and unc-37 regulate neurotransmitter vesicle proteins in cholinergic motor neurons.

24 May 1999 15:50 582 582 UNC-3 regulates cholinergic motor neuron differentiation.

1999 International Worm Meeting abstract 531 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. UNC-3 regulates cholinergic motor neuron differentiation. KM Lickteig 1 , D Frisby 2 , J Duerr 2 , D Miller 1 , J Rand 2

1 Dept. of Cell Biology, Vanderbilt University Medical Center, Nashville, TN 37232. 2 Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104.

Muscle contraction is stimulated by impulses from motor neurons. The excitatory signal in most cases is transmitted by the neurotransmitter acetylcholine which is released into the synaptic cleft when neurotransmitter vesicles fuse with the presynaptic membrane. Cholinergic function depends on choline acetyltransferase (ChAT) which synthesizes ACh and the vesicular acetylcholine transporter (VAChT) which mediates ACh uptake into vesicles. In C. elegans, VAChT (unc-17) and ChAT (cha-1) share a common promoter and are coordinately expressed. Here we show that UNC-3, a homolog of the vertebrate OLF-1/EBF (O/E) zinc finger transcription factor, regulates expression of UNC-17 and ChAT in ventral cord motor neurons (DA, DB, VA, VB, AS). UNC-3 is normally expressed in these cholinergic motor neurons. In unc-3 mutants, both UNC-17 and CHAT are missing from these cells as assayed by antibody staining. A parallel decrease in unc-17-cha-1-GFP reporter gene expression is also observed in unc-3 mutants. However, expression of another vesicular protein, synaptotagmin, is not affected. These data are consistent with a model in which UNC-3 functions as a transcriptional activator of unc-17-cha-1 but is not required for expression of other vesicular proteins. Intriguingly, UNC-17 and ChAT expression in ventral cord motor neurons is selectively eliminated by a point mutation ("Beta" mutation) in a strong consensus O/E binding site in the unc-17-cha-1 promoter (See Frisby et al., this meeting). Experiments are currently underway to determine if UNC-3 binds to this site. The striking conservation of the vertebrate VAChT and ChAT genomic structure and the expression of O/E transcription factors in the mammalian spinal cord may indicate that the UNC-3-dependent expression of VAChT and ChAT is also conserved.

24 May 1999 15:50 583 583 Molecular Studies on a Putative Potassium Channel Gene, F08B12.3 Found in Caenorhabditis elegans Genome

1999 International Worm Meeting abstract 532 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Molecular Studies on a Putative Potassium Channel Gene, F08B12.3 Found in Caenorhabditis elegans Genome HH Lim, CS Park, J Ahnn Kwang-ju Institute of Science & Technology (K-JIST), 500-712 Kwang-ju, Korea Two putative homologues of large conductance Ca2+-activated K+ channel alpha-subunit gene (slowpoke or slo) were found by C. elegans genome sequencing project. These genes (Y51A2D.h and F08B12.3) bear similar structural features of Slo family K+ channels previously identified ranging from Drosophila to human. One of the two genes, F08B12.3, show somewhat a lower sequence similarity and lacks the key functional domains (Calcium-bowl and charged S4 segment) found in all Slo proteins. Thus, C. elegans F08B12.3 may encode a subfamily of Slo channels with distinct functional characteristics or even a member of a new K+ channel family. We first investigated the expression pattern of F08B12.3 using a reporter gene, green fluorescence protein (GFP). A construct containing in-frame fusion of gfp with about 2 kb 5’- upstream region and 1 kb of F08B12.3 coding sequence was made and wild type worms (N2) were transformed with the reporter construct. Green fluorescence was detected in various neuronal cells, especially those at the nerve ring, the ventral tract of the body, and the tail, suggesting some functional roles of this gene product in C. elegans nervous system. A full-length cDNA of F08B12.3 was cloned by screening cDNA library for functional studies. The gene encodes a protein containing six putative transmembrane segments with a putative K+-selective pore region (GYG motif) and a large C-terminal cytosolic domain as predicted by genome sequencing. The C-terminal region of F08B12.3 was over-expressed in bacteria and the antibody against this region was obtained from rats. Whole mount immunostaining further confirmed its expression in these neuronal cells detected by GFP experiments. Currently, we endeavor to find a deletion mutant based on chemically mutagenized worm library. A putative mammalian counterpart of C. elegans F08B12.3 was found from a human brain EST database. Deduced amino acid sequences of human EST #180888 have 50 % identities and 70 % similarities to the C-terminal sequences of F08B12.3. Northern blot and mRNA dot blot analysis using the human EST clone as a prove revealed a strong and specific expression of this gene in the brain and the skeletal muscle.

24 May 1999 15:50 584 584 Domain C of netrin UNC-6 is Required to Inhibit the Circumferential Migration of Longitudinal Motoneuron Axons in the Ventral Cord

1999 International Worm Meeting abstract 533 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Domain C of netrin UNC-6 is Required to Inhibit the Circumferential Migration of Longitudinal Motoneuron Axons in the Ventral Cord Y Lim, S Mallapur, G Kao, X Ren, WG Wadsworth Graduate School of Biomedical Sciences, Department of Pathology, Robert Wood Johnson Medical School In the C.elegans embryo, some ventral midline motoneurons extend a process circumferentially to the dorsal midline and a process longitudinally along ventral nerve cord interneurons. Circumferential migrations are guided by netrin UNC-6, which repels the motoneuron axons dorsally. We show that expression of unc-6DC, which encodes UNC-6 without domain C, rescues circumferential migration defects in unc-6 null animals. This activity depends on the netrin receptors, UNC-5 and UNC-40. These results indicate that the guidance activities of UNC-6 in vivo do not require netrin domain C. We also show that expression of unc-6DC causes longitudinal motoneuron axons to migrate circumferentially from the ventral nerve cord. This activity is also UNC-5 and UNC-40 dependent. Since longitudinal motoneuron axons migrate along UNC-6-expressing interneurons in the ventral nerve cord, we propose that local interactions mediated by domain C inhibit the ventral nerve cord axons from responding to the repellent UNC-6 cue.

24 May 1999 15:50 585 585 Specification of the Command Interneurons: A Genetic Approach to Identify Genes Required for Cell Fate Determination

1999 International Worm Meeting abstract 534 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Specification of the Command Interneurons: A Genetic Approach to Identify Genes Required for Cell Fate Determination Chingju Lin, P. J. Brockie, S. Poulson, Andres V. Maricq Dept. of Biology, University of Utah, Salt Lake City, UT 84112 Of the neurotrsnamitter receptors, the NMDA-type glutamate receptor has received considerable attention due to its pivotal roles in many processes such as synaptic plasticity and neuronal excitotoxicity. In C. elegans, we have identified two genes that encode putative NMDA receptors, nmr-1 and nmr-2. Using the reporter molecule GFP, we have shown that NMR-1 and NMR-2 are co-expressed in a small number of neurons, including the command interneurons AVA, AVD, AVE, and PVC. These neurons are known to be required for the control of locomotion (Chalfie et al.). A deletion mutation in nmr-1 causes defects in the timing of locomotory activity (see abstract by Brockie et al.). Although NMDA receptors are of central importance in all nervous systems, we do not understand what genes are required for the expression of these receptors, nor do we understand how neurons that are destined to express NMDA receptors differentiate into their terminal fate. We have undertaken a visual-based screen for mutations that affect the development and differentiation of the command interneurons. The transgenic strain akIs3 expresses GFP under the control of the nmr-1 promoter. GFP expression can be easily observed using a fluorescence equipped dissecting microscope. We mutagenized the akIs3 transgenic strain and screened roughly 20,000 haploid genomes for aberrant GFP expression. We focused on mutations that affected GFP expression in only a subset of the command interneurons. These genes are expected to be required for differentiation of neurons that express nmr-1. We have found many mutants with aberrant GFP expression, but have focused on 2 of these mutations: one shows a striking decrease in GFP expression in PVC alone. By DIC optics, this neuron is still present, suggesting that the mutation affects a late step in differentiation. The second mutant shows faint GFP expression in PVC and lacks GFP expression in AVD. Again, PVC was present, but have yet to determine whether AVD is present. We have mapped both mutants: one lies near dpy-24 on LG I, the other near unc-18 on LG X. To clone these two genes, we are now pursuing cosmid rescue experiments. In addition, we are characterizing the behavioral defects associated with both mutants.

24 May 1999 15:50 586 586 isolation OF ADDITIONAL DAF-2 SUPPRESSORS

1999 International Worm Meeting abstract 535 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. isolation OF ADDITIONAL DAF-2 SUPPRESSORS K Lin, C Kenyon Department of Biochemistry & Biophysics, University of California at San Francisco, San Francisco, CA 94143-0448 The wild-type C.elegans ages rapidly, undergoing development, senescence, and death in less then 3 weeks. In contrast, mutants with reduced activity of daf-2, a homolog of the insulin and insulin-like growth factor (IGF-1) receptors (Kimura et al., 1997), age more slowly and live more than twice as long (Kenyon et al., 1993). Wild-type DAF-2 activity also promotes growth to adulthood and prevents dauer formation, since severe loss of daf-2 function causes the animals to become dauers in the presence of food. Both the lifespan extension and dauer-constitutive phenotypes caused by daf-2 mutations are dependent on the activity of daf-16, which encodes an HNF-3/forkhead family member (Ogg et al., 1997; Lin et al., 1997). Previous screens for suppressors of the dauer-constitutive phenotype of daf-2 in our lab have resulted in isolation of just additional alleles of daf-16 (Lin et al., 1997). In order to overcome this problem and isolate additional daf-2 suppressors, we created daf-2(e1370) animals with extra copies of the wild-type daf-16 gene, by injecting a rescuing genomic daf-16 construct and integrating the arrays into the genome. The integrated array fully rescued dauer formation in the daf-16(null);daf-2(e1370) background. We screened a total of 100,000 genomes for suppressors of the dauer constitutive phenotype of daf-2(e1370), and we’re now screening for suppressors of the lifespan extension phenotype. We will report our progress in characterizing the suppressors we isolated.

24 May 1999 15:50 587 587 Acid Sphingomyelinase Regulates RAS Signaling Functions

1999 International Worm Meeting abstract 536 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Acid Sphingomyelinase Regulates RAS Signaling Functions X Lin 1 , MO Hengartner 2 , RN Kolesnick 1

1 Sloan-Kettering Institute, NY, NY. 2 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

The sphingomyelin (SM) pathway, initiated by hydrolysis of plasma membrane SM to ceramide, is an ubiquitous signaling system linking diverse environmental stresses and cell surface receptors to intracellular effector pathways. To evaluate the SM pathway in C. elegans, we cloned two distinct acid SMase genes, asm-1 and asm-2 [Lin et al., J. Biol. Chem. 273, 14374 (1998)]. These genes are 30% identical with each other and the human gene. When expressed in COS-7 cells, ASM-1 is entirely secreted whereas ASM-2 is only 20% secreted. Further, ASM-2, but not ASM-1, is Zn2+ -dependent. Using the Tc1 transposon insertion/deletion approach, an asm-1 null allele (kk1) was isolated. Asm-1(kk1) homozygotes display no detectable phenotype. Our laboratory investigates ionizing radiation (XRT) signaling. C. elegans when irradiated with 10-400 Gy develop dose-dependent vulval malformations. Low dose XRT [lte]50 Gy induces a protruding vulval phenotype, whereas at 50 Gy a vulvaless phenotype also occurs. These vulval malformations occur exclusively after XRT in L1 or early L2, and at higher doses are manifest in 75% of adults. lf mutations in genes of the RAS-MAP kinase pathway including ksr-1(ku68), mek-2(n1989) and sur-1(ku1) enhanced sensitivity to XRT. Similarly, asm-1(kk1) animals exhibit increased XRT sensitivity. These effects appear apoptosis-independent, as ced-3(n717) does not affect radiosensitivity. Animals expressing an asm-1 transgene (kkEx1) are radioresistant and even at 400 Gy display almost no vulvaless. These data indicate that the MAP kinase pathway provides a signal protecting developing vulva from XRT damage. Epistasis analysis revealed that KSR-1 is likely to be downstream of ASM-1. Subsequent investigations assessed developmental signaling through the MAP kinase pathway. Asm-1(kk1) reduced penetrance of the Muv phenotype caused by gf let-60(n1046) by 50%. Furthermore, asm-1(kk1) synergistically enhanced the abnormal vulva phenotype caused by failure of P6.pp division in sur-1(ku1). These investigations show that asm-1, like ksr-1, is not required for normal vulval development but becomes important when signaling is disturbed by mutation or XRT stress. ASM-1 appears to molecularly order downstream (or parallel) to LET-60 and upstream of KSR-1.

24 May 1999 15:50 588 588 Isolation of the pat-6and pat-12genes

1999 International Worm Meeting abstract 537 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Isolation of the pat-6and pat-12genes X Lin, BD Williams University of Illinois at Urbana Champaign Caenorhabditis elegansbody-wall muscle cell development is an advantageous system for the study of integrin signal transduction in vivo. Mutations in the pat-2and pat-3genes, which code for integrin alpha and beta subunits, respectively, severely disrupt early cytoskeletal assembly events that occur at the muscle cell membrane. The mutant embryos arrest development with the Pat phenotype (Paralyzed, Arrested-elongation at Two-fold), characterized by non-functional body wall muscle cells. The pat-2and pat-3genes were identified in a previously reported genome-wide screen for mutants with the Pat phenotype (Williams and Waterston, 1994). We have recently shown that a third gene identified in this screen, pat-4, codes for the C. eleganshomologue of Integrin-Linked Kinase, a serine-threonine kinase implicated in integrin-mediated signal transduction (see abstract by Mackinnon and Williams). With the goal of identifying other proteins involved in the integrin-mediated events of muscle cell development, we are pursuing the positional cloning of the pat-6and pat-12genes, which were also defined in our original screen for Pat mutants. pat-6maps genetically to the right end of chromosome IV. We are attempting to molecularly isolate pat-6by transformation rescue and are currently injecting pools of cosmid DNA from the corresponding region of the physical map. We are taking a similar approach with pat-12. We will present our progress at the meeting. We will also present additional characterization of the muscle assembly defects in pat-6and pat-12mutants as revealed by immunostaining experiments with an existing panel of antibodies to many different muscle cell proteins.

24 May 1999 15:50 589 589 nhr-8 is Required for Gut Function and May Respond to the Food Signal

1999 International Worm Meeting abstract 538 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. nhr-8 is Required for Gut Function and May Respond to the Food Signal TH Lindblom, AE Sluder Dept. of Cellular Biology, University of Georgia, Athens, GA Among the conserved nematode members of the nuclear receptor (NR) superfamily (see abstract by Gissendanner et al) is a subfamily comprised of daf-12, nhr-48, and nhr-8. These NRs are most closely related to the vertebrate vitamin D and SXR (steroid and xenobiotic) receptors and Drosophila DHR96. We have undertaken an analysis of nhr-8. The nhr-8 mRNA has been detected from embryo to L4 with a peak of expression in L3. An NHR-8::GFP fusion is expressed in all gut cells from late stage embryos to adults. Intriguingly, expression of the reporter is strong in well fed larvae but substantially reduced in those starved of food. Experiments are underway to determine if the nhr-8 promoter responds to food or if this expression pattern is simply a reflection of global repression of gene expression during starvation. nhr-8(RNAi) animals progress through L1 and L2 with no obvious defects and then arrest as L3s. Arrested animals have enlarged gut lumen that are often bloated with intact bacteria. On occasion, we have observed the defecation of live bacteria. We are currently testing nhr-8(RNAi) animals for gut function by assaying for the presence of gut enzymes such as the pho-1 acid phosphatase (Beh et al 1991 Dev. Biol. 147: 133). We have performed epistasis analysis of nhr-8(RNAi) and several alleles affecting dauer development. nhr-8(RNAi) causes a significant increase in dauer formation in the temperature sensitive Daf-C background of daf-7(e1372) at the permissive and restrictive temperatures. nhr-8(RNAi); daf-2(m41 or e1370) animals arrest as L1 or L2s with defects reminiscent of the nhr-8(RNAi) arrested L3s. So far, we have been unable to detect a genetic interaction between nhr-8 and daf-12 or daf-16. There are five larval lethals in the genetic interval of nhr-8 and we are currently testing them for nhr-8 allelism by transformation rescue. The nhr-8(RNAi) phenotypes seen in the Daf backgrounds may indicate a direct role for nhr-8 in the dauer decision or may simply be another reflection of an inability to digest food. The vertebrate SXR is expressed in the liver and intestine and regulates the expression of several cytochrome P450 genes in response to a diversity of compounds. NHR-8 may also control the expression of gut genes in response to ingested ligands or metabolic by-products.

24 May 1999 15:50 590 590 Progress of cloning spe-10

1999 International Worm Meeting abstract 539 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Progress of cloning spe-10 Wesley C. Lindsey, Steven W. L’Hernault Emory University During spermatogenesis, the ER/Golgi derived, fibrous body-membranous organelles (FB-MOs) play a central role in the proper segregation of cytoplasmic components into the spermatids. Normally, FB-MO’s arise in the developing spermatocyte. The FB first appears as short fibers of Major Sperm Protein (MSP) in the body of the MO. The FB-MO complexes increase in size as the spermatocyte develops and then segregate to the budding spermatid, transporting material required for these spermatids to undergo spermiogenesis. After the spermatids bud off of the residual body, the FB-MO complex disassociates. MSP fibers depolymerize to reassemble in the pseudopod, and the MO’s fuse with the plasma membrane, releasing its contents onto the cell surface and forming a permanent fusion pore. Mutation of spe-10 prevents the proper function of the FB-MOs and spermatogenesis is disrupted. The FB-MO complexes break down prematurely, the fibrous bodies are left behind in the residual body and do not disassemble as in wild type spermatids. The membranous organelles segregate to spermatids, but then become swollen and vacuolated. Associated with these defects is improper localization of the nucleus in spermatids. So, resulting spermatozoa have vacuolated MO’s, short pseudopods, and they fail to translocate. Previous work positioned spe-10 on chromosome V, covered by deficiency sDf35. Three factor mapping places it between unc-42 and sma-1. (Shakes, D.C., and Ward, S. 1989. Dev. Biol. 134, 307-316.) On the physical map, this region is well represented by overlapping YACs and cosmids. Therefore, we are attempting to clone spe-10 by transgenic rescue. We have successfully rescued the spe-10 mutant phenotype by YACs containing wild-type DNA that covers the region between unc-42 and sma-1. We are now attempting cosmid rescue, in order to narrow down this region. Progress towards the cloning of spe-10 will be reported.

24 May 1999 15:50 591 591 Genetic analysis of beta amyloid toxicity in transgenic C. elegans

1999 International Worm Meeting abstract 540 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic analysis of beta amyloid toxicity in transgenic C. elegans CD Link, A Smith, A Fluet, CJ Johnson Institute for Behavioral Genetics, University of Colorado, Boulder, CO 80309 The beta amyloid peptide (Abeta) forms deposits in the brains of Alzheimer’s disease patients and is believed to be central to the pathology of this disease. We have generated transgenic C. elegans animals expressing relatively high levels of human Abeta in hopes of modeling some aspects of this disease. Animals with muscle-specific expression of Abeta have amyloid deposits and show a distinctive, temperature-dependent progressive paralysis. Our recent studies suggest that this phenotype is a result of specific Abeta toxicity, and not due to non-specific effects of foreign protein expression or toxic properties of the transgenic array itself (e.g., titration of muscle-specific transcription factors). Specifically, we find that transgenic lines expressing equivalent or higher levels of the putative non-toxic met35cys or single chain dimer Abeta variants do not show the progressive paralysis. In addition, RNA inhibition of transgenic (wild-type) Abeta expression also completely blocks the paralysis phenotype. We have therefore undertaken initial screens to identify mutations that block Abeta toxicity, in order to identify genes involved in this process. From our initial small-scale screens, we have identified four mutations that significantly reduce the paralysis phenotype in transgenic animals. One extragenic mutant, dv54, dramatically reduces transgene expression, and though intriguing, is presumably not relevant to Abeta toxicity. However, the other three mutants, dv53,55, and 56, express Abeta at parental levels and have amyloid deposits, yet still show suppression of the paralysis phenotype. These mutants are being further characterized. In collaboration with Stuart Kim, we are complementing this genetic analysis by examining gene expression profiles in amyloid and control animals using microarray hybridization.

24 May 1999 15:50 592 592 C. elegans immunity? A reverse genetic analysis of components of the Toll signaling pathway

1999 International Worm Meeting abstract 541 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. C. elegans immunity? A reverse genetic analysis of components of the Toll signaling pathway EM Link 1,2 , L Nelson 1 , G Hardiman 1,3 , J Hodgkin 4 , LX Liu 1,2

1 Axys Pharmaceuticals, NemaPharm Group, South San Francisco, CA. 2 Cambria Biosciences, Waltham, MA. 3 Axys Pharmaceuticals, La Jolla, CA. 4 MRC-LMB, Cambridge, UK.

The Toll signaling pathway is involved in innate immunity in both flies and mammals. The Drosophila pathway, including the Toll receptor, Pelle kinase, and Dorsal transcription factor, regulates both dorsal-ventral polarity in the embryo and antimicrobial peptide production in adult flies. The human inflammatory cytokine IL-1 signals through the IL-1 receptor (whose cytoplasmic domain is homologous to that of Toll) and the Pelle homolog IRAK, ultimately activating NFkB, a rel protein homologous to Dorsal. Human Toll homologs also signal to NFkB(1). To investigate this pathway in C. elegans, we identified orthologs of four genes and generated deletion mutations for each: tol-1 = Toll receptor, pik-1 = Pelle/IRAK kinase, trf-1 = TRAF, and ikb-1 = IkB. Worms with a deletion affecting all of tol-1 except the N-terminal leucine-rich repeats arrest as small, deformed larvae, a phenotype rescued by the tol-1 cosmid. However, a second tol-1 deletion in the intracellular domain seems wild-type. Deletion mutants of the other three genes show no obvious phenotype. To test whether these deletion alleles affect putative C. elegans host defenses, we exposed worms to an Aureobacterium strain that causes anal infection and local tissue swelling (2). The swelling phenotype was not altered in the four viable mutants. We also examined the expression of abf-1 and abf-2, which encode homologs of Ascaris antibacterial peptides (3). abf-1 and abf-2 expression was not altered in the four mutants compared to wild type, following either usual culture or Aureobacterium infection. Orthologs for other downstream elements of the Toll pathway, including NIK, IKK and NFkB (1), are conspicuously absent from the complete worm genome, suggesting that C. elegans has evolved an alternative transcriptional output for tol-1. Although our preliminary assays were negative, we cannot rule out a possible role for tol-1 in worm immunity, given the dearth of known microbial pathogens of C. elegans.

(1) Kopp ER & Medzhitov R, Curr Opin Immunol 1999; 11:13. (2) Hodgkin J & Corneliussen B, WBG 1997; 15(1):73. (3) Kato Y, WBG 1996; 14(4):37.

24 May 1999 15:50 593 593 Patterning of dopaminergic identity in C. elegans ray neurons by a DBL-1 signaling pathway and a Hox gene

1999 International Worm Meeting abstract 542 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Patterning of dopaminergic identity in C. elegans ray neurons by a DBL-1 signaling pathway and a Hox gene R Lints, SW Emmons Albert Einstein College of Medicine, Department of Molecular Genetics, Bronx, NY 10461 As a model for understanding how neurotransmitter identity is specified, we are studying the genetic basis for dopaminergic (DA) cell fate in the male rays. In wild type males, dopamine is synthesized in the A-type neurons of rays 5, 7 and 9, as well as in 8 neurons common to both sexes. We have found that tyrosine hydroxylase, which is essential for dopamine biosynthesis, is encoded by the gene cat-2, and that cat-2::gfp reporters are specifically expressed in all dopaminergic cells. We are using these reporters, together with staining for dopamine, to understand how DA fate is specified in the rays. The TGFb-family ligand DBL-1 plays a critical role in DA fate specification. In the absence of the signal, DA expression in rays 5, 7 and 9 is dramatically reduced, while, unexpectedly, rays 4 and 6 display low-frequency ectopic expression of DA fate. Conversely, heat shock-induced expression of DBL-1 causes the normally non-dopaminergic rays 3, 4, 6 and 8 to express DA with a frequency of 5 to 70%. Heat shock, however, does not induce expression in rays 1 and 2. Temperature shift experiments using a ts allele of daf-4, the type II DBL-1 receptor, indicate that the DBL-1 pathway functions in the ray neuroblast (Rn.a), its daughter, and its granddaughter, the A-type neuron itself. Taken together, these results suggest that the DBL-1 pathway acts to refine a prepattern that biases rays 5, 7 and 9 toward DA fate. In wild type, we propose that regulated expression of DBL-1 ensures that rays 5, 7 and 9 receive appropriate levels of ligand to enhance the prepatterned expression of DA fate; other competent rays are exposed to levels that act to suppress expression of this fate or are possibly inhibited by a secondary signal. The prepattern requires the Hox gene egl-5. EGL-5 is present in V rays 3 to 6 and is necessary for these rays to respond to the DBL-1 signal. However, egl-5 alone is not sufficient to explain the prepattern, as it is not expressed in the T rays (rays 7, 8 and 9). Taken together, these studies suggest that in wild-type males, the robust pattern of DA fate results from the combined activities of the prepattern, established in part by egl-5, and the DBL-1 signal.

24 May 1999 15:50 594 594 Specification of Neuronal Connectivity in the C. elegans Male Tail

1999 International Worm Meeting abstract 543 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Specification of Neuronal Connectivity in the C. elegans Male Tail J Lipton, SW Emmons Dept. of Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461 Male mating behavior requires several specialized sex-specific neural structures including the nine bilateral pairs of sensory rays. Each ray has a distinct identity characterized by its morphology, transcription factor expression profile, and neurotransmitter phenotype and may play a specific role in the control of mating behavior. This has led us to ask whether neurons sharing certain identity determinants such as neurotransmitter phenotype, might also share patterns of connectivity. Conversely, ray neurons that do not share similar roles in behavior might have different synaptic partners. In order to examine these possibilities we have focused on the three dopaminergic ray neurons (R5A, R7A, R9A). We are using a reporter specific to dopaminergic neurons, cat-2::gfp, to examine the axonal patterns of the dopaminergic ray neurons. The axons of R5A and R7A follow similar paths but appear to synapse on distinct cells in the preanal ganglion. The axon of R9A synapses in a complicated pattern also in the preanal ganglion but has a trajectory different from that of R5A or R7A. Sulston et al. (1980) have tentatively identified the EF interneurons of the preanal ganglion as targets of the ray neurons. In order to help identify the targets of the dopaminergic neurons, we have constructed reporters for two putative dopamine type-1 receptors and a dopamine type-2 receptor, identified by sequence similarity. These reporters will serve to label the postsynaptic targets of the dopaminergic neurons. By examining reporters that label non-dopaminergic ray axons, we can determine whether neurons with differing neurotransmitter phenotypes have the same or different targets. We are also examining mutants of the TGF-b pathway and Hox genes which result in transformations of dopaminergic identity to test whether the genetic control of neurotransmitter phenotype might also extend to synaptic connectivity. These experiments could shed light on the role of dopamine in mating behavior by elucidating the relationship between neurotransmitter usage and synaptic specificity.

24 May 1999 15:50 595 595 Phylogenetic analysis of vertebrate and invertebrate Delta/Serrate/LAG-2 (DSL)

1999 International Worm Meeting abstract 544 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Phylogenetic analysis of vertebrate and invertebrate Delta/Serrate/LAG-2 (DSL) JL Lissemore 1 , WT Starmer 2

1 Department of Biology, John Carroll University, University Heights, OH 44118. 2 Department of Biology, Syracuse University, Syracuse, NY 13244.

Delta/Serrate/LAG-2 (DSL) proteins are putative transmembrane signaling molecules that regulate cell differentiation in metazoans. DSL proteins are characterized by the presence of a motif unique to these proteins, the DSL motif, and a variable number of tandemly repeated copies of an epidermal growth factor-like (EGF) motif. We have completed a phylogenetic analysis of fifteen DSL proteins from eight species. Our findings reveal that at least one gene duplication occurred prior to the divergence of the D. melanogaster and vertebrate lineages, with subsequent duplications in vertebrates. The three known C. elegans proteins likely arose by two independent duplications in the nematode lineage. Analysis of EGF repeats suggests that EGF 2 has been conserved among DSL proteins in vertebrates and D. melanogaster. The sequences of two EGF repeats have been perfectly conserved in vertebrate orthologs: EGF 2 in Delta and EGF 15 in Jagged/Serrate. Finally, the linear order of EGF repeats has been conserved in the vertebrate Jagged/Serrate orthologs and vertebrate Delta orthologs.

24 May 1999 15:50 596 596 Hox factors and other components in patterning of embryonic and postembryonic myogenic lineages

1999 International Worm Meeting abstract 545 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Hox factors and other components in patterning of embryonic and postembryonic myogenic lineages J Liu, AZ Fire Carnegie Institution of Washington, Department of Embryology, Baltimore, MD 21210 Bodywall muscles are derived from four of the embryonic founder cells as well as the postembryonic M blast cell. We are studying patterning of both the embryonic and postembryonic muscles in order to understand how myogenic cell fates are specified during development. As an entry point to study muscle patterning during embryonic development, we characterized enhancer elements from the hlh-1 regulatory region that are able to drive reporter expression in muscle precursors of the MS, D and C lineages. Analyses of these elements suggested regulatory roles of the Hox genes and the hlh-1 gene itself. These analyses also suggested regulatory mechanisms involving unknown bZIP, bHLH and novel transcription factors. We are currently in the process of genetically identifying these factors. The postembryonic M lineage provides an alternative model to study myogenic fate specification. The M lineage gives rise to 14 bodywall muscles, 2 coelomocytes and 16 sex muscles. A number of genes had previously been identified to function in patterning the M lineage, including the Hox gene mab-5 and the bHLH transcription factor twist (1, 2, 3). The role of the Hox factors in patterning the M lineage has been something of a mystery: mab-5 is expressed during the entire M lineage, but mab-5 mutants cause only limited lineage transformation. We will describe a series of experiments indicating a more central role for the Hox genes in activating twist and specifying the M lineage. We found that lin-39 mab-5 double mutants fail to activate twist and completely lack products of the M lineage. Expression (either ectopic or in a normal pattern) of either Hox gene is sufficient to activate twist expression. However, twist activation is not sufficient for specification of the M lineage. Current efforts are directed towards identification of other factors involved in specifying the M lineage.

1. Kenyon, C. (1986), Cell 46: 477-487 2. Harfe B. D., Vaz Gomes A., Kenyon C., Liu J., Krause M., Fire A. (1998) Genes & Dev. 12: 2623-2635 3. A. Corsi, S. Kostas, E. Jorgensen, A. Fire, and M. Krause (poster)

24 May 1999 15:50 597 597 CDK-Activating Kinase (CAK): A genomic search

1999 International Worm Meeting abstract 546 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. CDK-Activating Kinase (CAK): A genomic search J Liu, ET Kipreos Cellular Biology, University of Georgia The major cell cycle regulator, cyclin-dependent kinases (CDKS) require phosphorylation by a CDK-Activating Kinase (CAK) to be active. Cell cycle regulatory proteins are highly conserved in eukaryotes, therefore, one might expect that the central regulator CAK would also be conserved. In human, xenopus and clams, the protein CDK7 was proposed to be a CAK by biochemical experiments. However, those experiments didn’t show that CDK7 functions as a CAK in vivo. Recently, a Drosophila cdk7 temperature sensitive mutant was obtained and was shown to be defective in CAK activity in vivo, but only for Cdc2/CyclinA and Cdc2/CyclinB complexes and not for the Cdk2/CyclinE complex (Larochelle et al., 1998, Genes & Dev. 12: 370). This result raises two questions. First, what is the CAK for the Cdk2/cyclinE complex? Second, since CDK7 is also the RNA polymerase II CTD kinase, was the cdk7 phenotype merely due to impaired transcription of a real CAK? In S. cerevisiae, in contrast, another protein, CAK1/CIV1, has been shown convincingly by genetic and biochemical experiments to be the essential CAK. CAK1 is a diverged CDK family member. At present no CAK1 homolog has been found. The sequenced C elegans genome enabled us to search for a CAK1 ortholog. We identified 12 C. elegans CDKs by using BLAST and PROFILE searches of the C. elegans genomic database with the six budding yeast CDKs and 16 human CDKs. We then used maximum likelihood, parsimony, and neighbor-joining methods to study the phylogeny of these 34 CDKs. All thre approaches produced similar trees. We determined that C. elegans has a CDK7 ortholog, but no obvious CAK1 ortholog; other major categories of human CDKs have their counterparts in C. elegans. One C. elegans CDK, H01G02.2, could not be definitively grouped with other CDKs. Although H01G02.2 is more similar to other yeast CDKs than to CAK1, it is theoretically possible that CAK1 and H01G02.2 are orthologs that have diverged by unequal rates of evolution. We are currently employing an RNAi approach to determine the loss-of-function phenotypes of H01G02.2 and CDK7. If our phylogeny results are confirmed, it would indicate that one of the most central aspects of cell cycle regulation, the CDK-Activating Kinase, has not been conserved from yeast to higher eukaryotes.

24 May 1999 15:50 598 598 Molecular, functional and behavioral characterization of unc-11 mutant alleles.

1999 International Worm Meeting abstract 547 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Molecular, functional and behavioral characterization of unc-11 mutant alleles. K Liu, EB Landies, A Kurani, A Holgado, R Golan, A Alfonso Department of Biological Sciences, University of Illinois at Chicago, M/C 066, 845 W. Taylor Street, Chicago, IL 60607 unc-11 encodes the nematode homolog of the mammalian neuronal specific clathrin assembly protein AP180 (Nonet et al., 1999). Five alternatively spliced cDNAs and three protein isoforms can be detected in the wild type strain N2. Like its mammalian counterpart, the UNC-11 protein(s) is(are) enriched in the nervous system. However, they are also expressed in the intestine, the coelomocytes and the gonad. unc-11 has at least two functions in the nervous system: 1) to sort/recycle the synaptic vesicle protein synaptobrevin and 2) to regulate clathrin cage sizes (Nonet et al., 1999). Therefore, UNC-11 functions affect both exocytosis and endocytosis of synaptic membrane components and consequently mutants are defective in synaptic transmission (Nonet et al., 1999, Holgado and Alfonso, 1999 C. elegans meeting). To address the biological relevance of the individual cDNAs and determine whether the proteins they encode have different functions and subcellular localizations in vivo, we have created transgenic lines expressing individual isoforms. We present our findings on the functional, biochemical and subcellular characterization of three different isoforms. To determine structure-function correlations, the available 15 mutant alleles were sequenced. The molecular null alleles are viable, supporting the conclusion that unc-11 function is not essential for synaptic transmission (Nonet et al., 1999). Most of the mutations result in frameshifts and these early truncations affect all putative isoforms derived from the gene. Since UNC-11 is expressed in the intestine, gonad and the coelomocytes, we are interested in determining possible functions of UNC-11 in tissues other than the nervous system. We present data on several mutant alleles defective in neuronal function, in which mutant proteins are detectable and their immunolocalization suggests interesting novel functions.

Supported by NS32449 to A.A. Nonet, Holgado, Brewer, Serpe, Norbeck, Holleran, Wei, Hartweig, Jorgensen and Alfonso, 1999 (submitted). UNC-11, a C. elegans AP180 homolog, regulates the size and protein composition of synaptic vesicles.

24 May 1999 15:50 599 599 The C. elegans Engulfment Protein CED-6 Might be Conserved Across Species

1999 International Worm Meeting abstract 548 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The C. elegans Engulfment Protein CED-6 Might be Conserved Across Species Qiong A. Liu 1,2 , Michael O. Hengartner 1

1 Cold Spring Harbor Laboratory, 1 Bungtown Rd, Cold Spring Harbor, N. Y. 11724. 2 [email protected]

The rapid engulfment of apoptotic cells is a specialized immune response used by organisms to remove the "nonself", apoptotic cells, and it is an inevitable second part of apoptosis. In vivo, the engulfment process is a swift and efficient process and usually takes place at a very early stage of apoptosis, it prevents the release of potentially harmful contents from dying cells to injure the surrounding tissue. In C. elegans, seven genes, ced-1, -2, -5, -6, -7, -10 and-12 have been identified to affect the engulfment process. The genetic and the phenotypic studies have put ced-1, -6, and -7 into one group, and ced-2, -5, and -10 into another. Genetic studies also demonstrated that ced-6 might act downstream of both ced-1 and ced-7. We have previously cloned ced-6. This gene encodes a novel protein that contains a phosphotyrosine binding (PTB) domain at its N-termini and a potential SH3 binding sites at its C-terminal half. By genetic mosaic analysis ced-6 has been demonstrated to act within engulfing cells. CED-6 might be an adaptor molecule that acts in a tyrosine kinase pathway that is specifically required for engulfing cells to remove apoptotic cells. To understand whether the CED-6 engulfment pathway is conserved in species, we have isolated potential ced-6 homologues from other species including human. These CED-6 homologues are very similar to C. elegans CED-6 structurally. Overexpression of hCED-6 in C. elegans can rescue the engulfment defect in the ced-6 mutant animals, suggesting that hCED-6 might be a functional homologue of C. elegans CED-6. Thus, CED-6, and the CED-6 signal transduction pathway is likely to be conserved from C. elegans to man.

24 May 1999 15:50 600 600 Identification and Analysis of Proximal Proliferation (Pro) Mutants

1999 International Worm Meeting abstract 549 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification and Analysis of Proximal Proliferation (Pro) Mutants J Hubbard Department of Biology, New York University, New York, NY The adult C. elegans hermaphrodite gonad is organized in a distal to proximal axis that is reflected in the maturation state of the germ nuclei. Mitosis occurs distally and is followed proximally by meiosis and gametogenesis. Mutations that cause a proximal proliferation (Pro) phenotype alter the pattern of germline development without affecting the ability of the cells to execute their normal fates (Schedl, 1997). Specifically, in addition to the normal distal-to-proximal pattern of mitosis, meiosis and gametogenesis, a second cluster of mitotic germ cells is observed proximal to mature gametes. The identification and analysis of genes that, when mutant, can cause this phenotype should reveal mechanisms by which the germline pattern is normally generated and maintained. As part of a genetic screen to identify loci involved in germline development, we have thus far identified eight mutants that display a Pro phenotype but have no obvious somatic gonad defects. Our screen is designed to obtain mutations that, in addition to affecting the germ line, may cause embryonic or early larval lethality. All eight alleles are temperature sensitive for the Pro phenotype. Three of the Pro mutants map to the same region of LGIII, and sequence analysis revealed lesions in the glp-1 coding sequence. Activity of the well-characterized GLP-1 receptor, a member of the LIN-12/Notch family, is associated with maintenance of mitosis and/or inhibition of meiosis in the germ line (Austin and Kimble, 1987; Schedl, 1997). The LIN-12/Notch signaling pathway is well conserved throughout metazoans (Greenwald, 1998). Therefore, alleles that can offer new insight into GLP-1 function or new information about the signaling pathway are useful. We believe that our glp-1(Pro) alleles reveal an intrinsic difference in the regulation of glp-1 activity within the germ line, and that they may offer valuable insight into the mechanisms regulating the spatial and temporal control of proliferation during germ line development. Our on-going genetic and phenotypic analysis is aimed at determining the nature of the alleles, their dependence on known ligands and effectors, and the origin of the proximally located proliferating germ cells. Genetic and phenotypic analysis of the other (non-glp-1) Pro alleles is also underway.

24 May 1999 15:50 601 601 Continuing characterization of serotonergic marker genes and serotonin-deficient mutants

1999 International Worm Meeting abstract 550 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Continuing characterization of serotonergic marker genes and serotonin-deficient mutants CM Loer, GP Merritt Department of Biology, University of San Diego, 5998 Alcala Park, San Diego, CA 92110 We are interesting in the regulation of genes used by serotonergic neurons in C. elegans. We and others have identified several genes that are expressed or are likely to be expressed in serotonergic neurons, including genes encoding serotonin synthetic enzymes Tryptophan Hydroxylase (TrpH) and Aromatic L-Amino Acid Decarboxylase (AAADC), and the biopterin cofactor synthetic enzyme GTP Cyclohydrolase I (GCH). These genes may correspond to the mutationally-identified genes cod-5, bas-1, and cat-4, respectively. We have previously rescued the serotonin- and dopamine-deficient mutant bas-1 by injecting DNA containing two predicted AAADC genes (thanks to Fred Wolf, Garriga lab, for this clone). One or both of these ORFs may be needed for serotonin synthesis. We plan to test this by injecting clones mutated in each of the ORFs. The two predicted genes are separated by only 369 bp; we are also testing whether they are expressed together as an operon. The mutant cat-4 (e1141)V is serotonin- and dopamine-deficient and appears to have a leaky cuticle. We are currently comparing the cuticles of cat-4 mutants and wild type worms using EM to see whether any structural differences are apparent. Sequencing through the region in which cat-4 maps genetically identified a candidate gene with homology to GCH, which catalyzes the first step in biopterin cofactor synthesis. Biopterin is a cofactor used by all aromatic amino acid hydroxylases, which include tryptophan hydroxylase (serotonin synthesis), tyrosine hydroxylase (dopamine synthesis) and phenylalanine hydroxylase (tyrosine synthesis/phenylalanine catabolism). We are also injecting the F32G8 cosmid into cat-4 mutants to see whether the clone will rescue.

24 May 1999 15:50 602 602 Using RNA interference to screen for new factors involved in the control of glp-1 translation and early embryonic polarity

1999 International Worm Meeting abstract 551 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Using RNA interference to screen for new factors involved in the control of glp-1 translation and early embryonic polarity AL Lublin, SA Barbee, TC Evans University of Colorado Health Science Center, Denver, Colorado, 80262 In early C. elegans development, asymmetric cell division leads to the localization of maternal factors to specific cells in the embryo. GLP-1 is a notch-like receptor that is localized to anterior cells of early embryos, where it regulates anterior fates. GLP-1 localization results from translational control of glp-1 mRNA through its 3’ untranslated region (3’ UTR). Our hypothesis is that a regulatory system functioning through the 3’ UTR of glp-1 contributes to the generation of early polarity. Of the 20 or so maternal genes identified from conventional genetic screens, only a few (e.g. par genes) are required for GLP-1 localization. Several additional factors must exist. RNA interference (RNAi) is a powerful tool to screen for specific early embryonic phenotypes and identify new genes that play important roles in embryonic patterning. Using RNAi, we have initiated a screen to identify new genes involved in glp-1 regulation and the establishment of embryonic asymmetry in the early embryo. A normalized cDNA library enriched for 1-8 cell stage embryos has been constructed. Double stranded RNAs are transcribed in pools from PCR amplified templates of randomly selected cDNA clones. To screen embryos for defects in early embryonic polarity, a transgenic strain has been established that carries a modified PIE-1:GFP germline expression vector (kindly provided by Geraldine Seydoux). This transgene expresses GFP mRNA under control of the 3’ UTR of glp-1. Similar to endogenous glp-1, GFP fluorescence is localized to anterior cells in early embryos. Following injection of dsRNA pools into this strain, embryos are screened for defects in GFP and P-granule localization. Preliminary screening of this library indicates that most RNA pools can be screened for both GFP expression and P-granule localization. Further, a known gene required for GLP-1 localization can be recovered with this screen.

Authors ALL and SAB contributed equally to this work.

24 May 1999 15:50 603 603 TRA-1 is a Phosphoprotein and Interacts with FEM-2, a Protein Type 2C Phosphatase

1999 International Worm Meeting abstract 552 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. TRA-1 is a Phosphoprotein and Interacts with FEM-2, a Protein Type 2C Phosphatase DH Lum 1 , D Zarkower 2 , AM Spence 1

1 Dept. of Molecular and Medical Genetics, University of Toronto, ON, M5S 1A8, CANADA. 2 Institute of Human Genetics, Minneapolis, MN 55455, U.S.A.

In XO animals the three fem genes, fem-1, fem-2, and fem-3, act to negatively regulate tra-1, the terminal regulator of somatic sex determination. To understand the mechanism of tra-1 regulation by the fems we have attempted to detect physical interactions between these proteins. We have identified an in vitro interaction between FEM-2, a Type 2C protein phosphatase, and the product of its genetic downstream target, TRA-1. The TRA-1 protein is a putative transcription factor with five zinc fingers related to those of the vertebrate GLI proteins. Deletion analysis suggests the phosphatase domain of FEM-2 interacts with the first two zinc fingers of TRA-1. These results are consistent with a model where FEM-2 negatively regulates TRA-1 activity through a dephosphorylation event. A prediction from this model is that TRA-1 may be phosphorylated in a sex dependent manner. Over-expression of Myc-TRA-1 has enabled the detection of at least two MycTRA-1 isoforms that can be collapsed to a single band upon phosphatase treatment. We are examining Myc-TRA-1 in both male enriched and hermaphrodite lysates in an attempt to detect sex specific phosphoisforms. To further characterize domains important for the interaction we are assaying the effects of various point mutations in TRA-1 and FEM-2 on their binding in vitro. Mutations disrupting or enhancing the interaction in vitro can then be tested in vivo for any effects on the proper regulation of TRA-1 by FEM-2. We will report on these results as well as the phosphorylation state of TRA-1 in various fem mutant lysates.

24 May 1999 15:50 604 604 The Cytoplasmic Domain of TRA-2 Localizes to the Nucleus and Interacts with TRA-1

1999 International Worm Meeting abstract 553 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Cytoplasmic Domain of TRA-2 Localizes to the Nucleus and Interacts with TRA-1 DH Lum 1 , P Kuwabara 2 , D Zarkower 3 , AM Spence 1

1 Dept. of Molecular and Medical Genetics, University of Toronto, ON, M5S 1A8, CANADA. 2 MRC Laboratory of Molecular Biology, Hills Road, Cambridge , CB2 2QH. 3 Institute of Human Genetics, Minneapolis, MN 55455, U.S.A.

In XX animals the tra-2 gene negatively regulates the activity of the three fem genes. The absence of fem activity in XX animals frees tra-1, the terminal regulator of somatic sex determination, to promote female development. The tra-2 gene encodes two proteins TRA-2A and TRA-2B. The larger of the two proteins, TRA-2A, is a predicted transmembrane protein with a large cytoplasmic domain. TRA-2B is expressed in the hermaphrodite germline and is predicted to be a soluble protein consisting of just the C-terminal domain of TRA-2A. The negative regulation of the FEMs is mediated via a direct interaction between the novel protein FEM-3 and the cytoplasmic domain of TRA-2. Overexpression of the C-terminal domain of TRA-2A (TRA-2Ac) is sufficient for FEM negative regulation and the transformation of XO animals into females. To try to determine the mechanism of TRA-2Ac feminizing activity we overexpressed various TRA-2Ac fragments. Surprisingly, a C-terminal fragment of TRA-2Ac, incapable of interacting with FEM-3 in the yeast 2-hybrid system, maintained weak feminizing activity. Both yeast 2-hybrid and biochemical data demonstrate that this C-terminal domain of TRA-2Ac interacts with TRA-1. Consistent with a TRA-2/TRA-1 interaction in vivo is the colocalization of both GFP::TRA-2Ac and Myc-TRA-1 to the nucleus of both XX and XO animals. Deletion analyses has defined binding regions in both TRA-2Ac and TRA-1. We are testing point mutations in the interacting domains for effects on the interaction and biological activity. Genetics indicates that TRA-2A is the major activity in the XX soma responsible for inhibition of the FEMs and thereby indirectly activates TRA-1A. It is possible that a TRA-2Ac/TRA-1 interaction is important for direct and or indirect regulation of TRA-1 in XX animals. This raises the intriguing possibility that TRA-2A is cleaved to allow a TRA-2Ac-like domain to enter the nucleus and interact with TRA-1A.

24 May 1999 15:50 605 605 Ethanol Tolerance in C. elegans

1999 International Worm Meeting abstract 554 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Ethanol Tolerance in C. elegans K Lundin, PC Phillips Biology Department, Box 19498, University of Texas at Arlington, Arlington, TX 76019-0498 It is thought that increased ethanol tolerance in humans leads to increased risk of alcoholism. Unfortunately, not much is known about the genetics and physiology of ethanol tolerance, and therefore we have begun a set of preliminary experiments on this topic using Caenorhabditis elegans as a model system. Individuals were tested for initial tolerance over a wide range of ethanol concentrations, yielding a determination of the lethal concentration. Initial tolerance to ethanol after five minutes of exposure was surprisingly high, with a tolerance threshold of approximately 36% with an LD50 of 19.5%. The tolerance considered here is of an extreme type (lethality) compared to an intoxication response such as lack of movement (Morgan and Sedensky 1995) or mating ability (Crowder et al. 1996), but is very straightforward to assay and is compatible with many similar studies in Drosophila. Determination of the lethal concentration of wild type worms allows for a simplified screening method for obtaining genetic mutants with higher ethanol tolerances. We are currently in the process of searching for such mutants. We also hope to study the influence of previous exposure to ethanol by contrasting tolerance of naive individuals to that of individuals exposed as larvae.

Crowder, C. M., Shebester, L. D., Schedl, T. 1996. Behavioral effects of volatile anesthetics in Caenorhabditis elegans. Anesthesiology 85:901-912. Morgan, P. G., Sedensky, M. M. 1995. Mutations affecting sensitivity to ethanol in the nematode, Caenorhabditis elegans. Alcoholism: Clinical and Experimental Research 19:1423-1429.

24 May 1999 15:50 606 606 UNC-115 and AXM-1 Define a New Axon Guidance Signal Transduction Pathway

1999 International Worm Meeting abstract 555 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. UNC-115 and AXM-1 Define a New Axon Guidance Signal Transduction Pathway EA Lundquist, CI Bargmann University of California-San Francisco Growth cones sense and respond to extracellular cues that provide axon guidance information. Guidance signals are translated to the actin cytoskeleton, which underlies growth cone outgrowth and steering. UNC-115, a putative cytoskeletal linker, is composed of a C-terminal actin-binding villin headpiece domain and three N-terminal LIM protein interaction domains. unc-115 mutants have specific defects in axon guidance and unc-115 is required cell-autonomously in neurons to guide their axons. Specific aspects of axons’ trajectories are affected by unc-115 mutation indicating that unc-115 is not required for general axon elongation. UNC-115 might function downstream of specific guidance events involving the guidance receptors unc-5, unc-40 and sax-3, as mutations in these genes cause axon phenotypes similar to unc-115.We have identified other cytoplasmic molecules that interact with unc-115 in axon guidance. unc-73 encodes a molecule with two GTP exchange factor domains (GEFs) that activate Rho-family GTPases (Steven et al.) and mig-2 encodes a Rho-family GTPase (Zipkin, et al.). unc-115 mutations enhance the CAN axon termination of the weak unc-73(e936) allele and do not enhance the strong unc-73(gm40) allele, indicating that unc-115 and unc-73 function in the same pathway to mediate CAN axon outgrowth. unc-115 enhances unc-73(rh40), a mutation that inactivates the first UNC-73 GEF domain, suggesting that UNC-115 and the GEF1 domain are in parallel pathways and that UNC-115 might cooperate with a different domain of UNC-73. unc-115 and null mutations in mig-2, a Rho GTPase, display synthetic CAN axon termination. This redundancy suggests that UNC-115 and MIG-2 function in parallel, perhaps in parallel pathways downstream of UNC-73.Using the yeast two-hybrid system, we have identified a new molecule called AXM-1 (for axon morphology-defective) that associates with the UNC-115 LIM domains. AXM-1 is a member of a conserved family of WD-40 repeat-containing proteins. RNAi with axm-1 causes axon phenotypes similar to unc-115 mutants, suggesting that the biochemical UNC-115/AXM-1 interaction might mediate axon guidance events. UNC-115 and AXM-1 might form a complex that modulates the actin cytoskeleton of a growth cone in response to guidance signals.

24 May 1999 15:50 607 607 C. elegans homologues of MuSK and rapsyn interact to control motor neuron function

1999 International Worm Meeting abstract 556 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. C. elegans homologues of MuSK and rapsyn interact to control motor neuron function G Caldwell, L Carnell, M Chalfie Department of Biological Sciences, Columbia University Synapse formation at the mammalian neuromuscular junction requires at least two postsynaptic proteins, the receptor tyrosine kinase MuSK and the acetylcholine-receptor associated protein rapsyn. These proteins interact with presynaptically expressed agrin. C. elegans has genes encoding proteins similar to all three of these proteins. The MuSK homologue, the product of the kin-8 gene (a.k.a cam-1), is expressed in neurons and muscle. Animals that expressed a translational fusion in which the kinase domain of KIN-8 was replaced by the green fluorescent protein (GFP) or that have been injected with RNAi were uncoordinated (Unc) and egg-laying defective(Egl). Furthermore, the level of kin-8 mRNA was reduced in unc-4 and unc-37 mutant animals (these latter genes encode transcription factors that control the choice of presynaptic partners for the VA motor neurons of the ventral cord). In addition, the number of ventral cord motor neurons expressing a kin-8::lacZ fusion was reduced in an unc-4 background. These results suggest that kin-8 may regulate neuronal activity, and possibly synapse formation, in C. elegans. Similar findings were found with the C. elegans rapsyn homologue, the product of the rap-1 gene. RAP-1 was expressed in many cells that also express KIN-8 and although RNAi with rap-1 did not produce a pronounced phenotype in wild-type animals, it yielded a phenotype identical to that of kin-8 RNAi in a hypomorphic kin-8 mutant (gift from M. Koga and Y. Oshima). In contrast, RNAi for agr-1, the gene for the C. elegans agrin homologue, did not produce the same phenotype. We also examined the synapses formed by the interneurons (AVA, AVD, AVE) and the A-type motorneurons in the ventral cord using a vamp::gfp construct (provided by M. Nonet) driven by a sek-1 promoter (the sek-1 gene is expressed in AVA,AVD,AVE and some other tissues, M. Tanaka et al. WBG 15(4):30). The sek-1::vamp::gfp is expressed in a similar pattern as reported by Tanaka et al. and punctated staining can be seen along the ventral cord. In the unc-4(e120) and unc-37(e262) backgrounds, the ventral cord staining become weak and diffuse, indicating loss of synapse formation. Similar results were obtained when kin-8 RNAi was injected into wild-type animals carrying the sek-1::vamp::gfp construct.

24 May 1999 15:50 608 608 PAT-4 is a homologue of integrin-linked kinase and is required for assembly of the myofilament lattice in body-wall muscle cells

1999 International Worm Meeting abstract 557 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. PAT-4 is a homologue of integrin-linked kinase and is required for assembly of the myofilament lattice in body-wall muscle cells AC Mackinnon, BD Williams University of Illinois at Urbana Champaign A previous genetic screen for mutants with the Pat phenotype (Paralyzed, Arrested elongation at Two-fold) yielded a subset of mutants with severely disrupted sarcomere assembly in embryonic body-wall muscle cells (Williams, 1994). Mutants in this subset have defined the pat-2 and pat-3 genes which code for the a and b integrin subunits expressed in body-wall muscle cells, and identified several new genes likely to work in conjunction with pat-2 and pat-3 during muscle cell differentiation. Here we report that we have molecularly isolated pat-4, one of these newly defined genes. We mapped pat-4 to the left end of chromosome III and identified the gene C29F9.7 as a promising candidate. C29F9.7 is the C. elegans homologue of Integrin-Linked Kinase (ILK), a serine/threonine kinase. Overexpression of ILK in vertebrate tissue culture cells suggests that ILK plays a role cell cycle regulation, confers anchorage-independent cell growth, stimulates both focal adhesion and ECM assembly, upregulates the Wnt signaling pathway, and regulates GSK-3 and PKB/AKT activities. We rescued pat-4 homozygous animals with the genomic cosmid clone DE10 and subsequently with a PCR product containing C29F9.7. We sequenced two pat-4 mutant alleles and detected polymorphisms which either introduced a stop codon or altered a splice site. In addition, myosin organization is severely disrupted in pat-4 embryos. These results demonstrate that pat-4 codes for C. elegans ILK homologue, and ILK is therefore required for assembly of the myofilament lattice in body-wall muscle. We are taking several approaches to better understand the in vivo function of pat-4. We are determining the localization of the PAT-4 protein by constructing a pat-4::gfp fusion gene. We are also staining pat-4 embryos with antibodies that recognize different components of dense bodies and M-lines. These structures form the contractile apparatus and link it to the underlying ECM. Our preliminary results indicate integrin polarization to the basal membrane is not affected by mutations in the pat-4 gene. However, cytoskeletal organization is disrupted. These preliminary results support the hypothesis that pat-4 functions as a downstream regulator of integrin-mediated signaling required for muscle assembly.

24 May 1999 15:50 609 609 Mutations Affecting Meiotic Pairing and Synapsis

1999 International Worm Meeting abstract 558 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Mutations Affecting Meiotic Pairing and Synapsis AJ MacQueen, AM Villeneuve Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305 Successful meiotic chromosome segregation relies on a prior association between homologous chromosomes. Our goals are to understand how the homologs recognize one another, and then maintain functional associations throughout prophase of meiosis. Using high resolution cytological methods developed in the lab (Dernburg and Villeneuve abstract) we have been screening through a collection of meiotic chromosome segregation mutants to identify mutations that specifically disrupt pairing and synapsis of homologous chromosomes. We have identified mutations in three complementation groups that cause defects in homolog pairing. In the me18 mutant, chromosomes are clearly asynapsed in the pachytene region of the germline, where partner chromosomes are normally aligned in parallel tracks with one another. Fluorescent In Situ Hybridization (FISH) confirms an absence of homolog pairing. Further, chromatin in me18 nuclei entering meiosis does not undergo the distinct morphological transition that normally accompanies the onset of pairing, suggesting that me18 disrupts an early step required for initiation of pairing. Mutations in a second class (me17, me19), also cause extensive asynapsis, but pachytene region nuclei have a chromatin distribution that is distinct from me18 nuclei. Chromatin is asymmetrically distributed, similar to the nuclear organization usually confined to the "transition zone", where nuclei enter meiosis. FISH analysis on the me17 mutant revealed a high frequency of unpaired chromosomes in pachytene region nuclei, but preliminary analysis suggests that FISH signals may be more frequently paired in the transition zone. The morphology of the meiotic nuclei together with the FISH data suggest that me17 chromosomes may initiate, but fail to maintain a paired state. Our analysis demonstrates that homolog pairing and synapsis can be subdivided into discrete steps using this approach. The data further show that homolog pairing is dispensable for successful completion of meiosis and gametogenesis in C. elegans.

24 May 1999 15:50 610 610 Electrophysiologic analysis of neuromuscular transmission

1999 International Worm Meeting abstract 559 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Electrophysiologic analysis of neuromuscular transmission JM Madison, JM Kaplan 361 LSA, University of California, Berkeley, CA 94720 In worms, serotonin (5-HT) modulates the rate of locomotion. Neurotransmitters like 5-HT can modulate neuromuscular function by altering the activity of both voltage dependent ion channels and the transmitter release machinery. To better understand the molecular and cellular mechanisms that underlie 5-HT modulation of locomotion, we have developed methods for electrophysiologic analysis of the body wall neuromuscular junction (NMJ). We have developed a dissection that permits access to the ventral body wall muscles (similar to a dissection described by J. Richmond and E. Jorgensen). Expression of VAMP::GFP and GLR-1::GFP translational fusions in the ventral cord and GFP in body wall muscles suggests that these filleted animals maintain cellular and synaptic structure similar to intact worms. We have used this fillet and whole cell voltage clamp analysis to study voltage dependent currents and synaptic currents at the body wall NMJ. Voltage clamp analysis of ventral body wall muscle reveals a number of delayed outward rectifier potassium currents that are differentially sensitive to the K+ channel blockers 4-aminopyridine (4-AP) and tetraethyl ammonium (TEA). Furthermore, these potassium currents show differential sensitivity to steady state inactivation. Application of the K+ channel blockers 4-AP, TEA, and cesium reveals a high voltage activated calcium current that is blocked by cadmium, a general calcium channel blocker. In addition to voltage dependent currents, we have also recorded macroscopic currents activated by the cholinergic agonist levamisole, and miniature excitatory post-synaptic currents (mEPSCs). mEPSC amplitude histograms show a normal distribution with a mean amplitude of 5-6 picoamps. Further experiments analyzing serotonin modulation of body wall neuromuscular function will be discussed. The electrophysiologic analysis of neuromuscular function and neuromodulation will help further our understanding of the molecular and cellular basis of behavior.

24 May 1999 15:50 611 611 A Zyxin-like Molecule in C. elegans

1999 International Worm Meeting abstract 560 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A Zyxin-like Molecule in C. elegans DM Madsen, RT Fazzio, MC Beckerle, AV Maricq Department of Biology, University of Utah, Salt Lake City, UT 84112 Neuronal cells depend on cues from their environment to guide them to their correct targets. Focal adhesions are sites of contact between a cell and its substrate. These sites contain many specialized proteins that control and regulate cellular motility and shape. Amongst these is zyxin, a multifunctional LIM domain containing protein that colocalizes with cytoskeletal proteins. Zyxin may function to recruit components of the actin assembly machinery to specific sites in the cell and to stimulate spatially restricted actin polymerization. In mammalian cells, zyxin can shuttle between adhesion plaques and the nucleus, suggesting a mechanism by which changes in cell adhesion can trigger changes in gene expression and cellular differentiation. To gain a mechanistic understanding of signaling at adhesion plaques we have undertaken a study of a zyxin homologue in C. elegans. We isolated a cDNA that encodes a zyxin-like molecule in C. elegans. This gene, zyx-1, has the characteristic features of zyxin, including a proline-rich N-terminal region believed to be important for protein-protein interactions, as well as 3 LIM domains, each with a canonical LIM consensus sequence. Using various fusions of zyx-1 to the reporter gene Green Fluorescent Protein (GFP), we observe expression in both neurons and muscle. We find localization to dense bodies and in the spermatheca. We are also generating antibodies to ZYX-1 to better define its subcellular localization. We anticipate that ZYX-1 functions as part of a protein complex that transduces substrate adhesion into changes in cytoskeleton function. To perturb the function of zyx-1, we mapped its genomic structure and used a PCR-based strategy to identify two strains containing Tc1 transposable elements in the zyx-1 gene. Imprecise excision of the Tc1 elements led to deletion mutations that disrupt either the N-terminal or C-terminal regions. These strains are viable and phenotypic analysis is underway.

24 May 1999 15:50 612 612 Genetic hierarchies and GATA factor specificity in ems development

1999 International Worm Meeting abstract 561 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic hierarchies and GATA factor specificity in ems development MF Maduro, JH Rothman Dept. MCD Biology, UC Santa Barbara, Santa Barbara, CA 93106 As one approach to revealing genetic hierarchies at work in EMS development, we are dissecting the role of the END-1/3 and MED-1/2 GATA factors by exploiting their ability to alter the fates of non-EMS blastomeres when expressed ectopically. For example, our results show that med-2::GFP responds to ectopically expressed SKN-1, consistent with our finding that med-2::GFP expression is greatly reduced in skn-1(RNAi) embryos. Our preliminary data suggest that such embryos can contain an excess of E or MS descendant cell types, consistent with a role for SKN-1 in both E and MS determination. To our surprise, while ectopic med-1 results in repression of gut development and widespread mesoderm development, it can promote ectopic expression of end-1::GFP. These data suggest the regulatory hierarchy: skn-1 - med-1/2 - end-1/3, which is further supported by the temporal order of expression of these genes. However, this pathway is likely not to be strictly linear, as genetic and biochemical evidence suggest that SKN-1 can also act directly on end-1/3. We are using a similar approach to assay for GATA factor specificity. Unlike the two-finger ELT-1 protein, all of the 10 other C. elegans GATA factors contain a single zinc finger and basic domain. Limited similarities between GATA factors acting in similar lineages suggest the possibility that regions outside the DNA binding domain may confer specificity during trans-activation. For example, END-1 and END-3 share only very limited similarity in their zinc fingers but appear to activate the same sets of target genes both within the E lineage (as shown by rescue of gut formation in embryos deleted for both genes) and outside E (shown by heat-shock). In contrast, ectopic MED-1 causes embryonic arrest and ectopic expression of markers found in MS descendants. To elucidate the basis for the specificity of these genes in heat-shock experiments, we are analyzing GATA factor chimeras in which the DNA-binding domains of END-3 and MED-1 have been reciprocally exchanged.

24 May 1999 15:50 613 613 An RNAi screen for embryonic patterning genes

1999 International Worm Meeting abstract 562 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. An RNAi screen for embryonic patterning genes MF Maduro, J Sumerel, JH Rothman Dept. MCD Biology, UC Santa Barbara, Santa Barbara, CA 93106 We are taking advantage of RNAi as a tool for identifying genes with potentially important roles in the developing embryo. This approach circumvents the extensive effort required to molecularly identify the relevant genes identified from mutagenesis or deficiency screening and facilitates identification of genes that might be required both maternally and zygotically. In our screen, we obtain RNA synthesis templates from an extant cDNA library. The templates are pooled in groups of eight and used to synthesize RNA. Although the library we are using has not been normalized, this has not been a major limitation; in preliminary experiments, we found that approximately 35% of pools cause penetrant embryonic arrest. For any phenotype recovered from a pool, we inject or soak individual RNAs and identify the gene from a small amount of cDNA sequence. To expedite analysis of mutant phenotypes, we are using a set of GFP markers, including a translational fusion of the gene encoding the MH27 antigen (a gift from Jeff Simske), which marks the adherens junctions of epithelia and allows us to recover embryos with abnormal epidermal patterning. For example, one pool produced viable L1 animals with a penetrant hypodermal protrusion near the posterior pharyngeal bulb; the MH27::GFP marker revealed a bilateral alteration in the local pattern of hypodermal seam cells. The gene responsible for the phenotype, which has previously been named nmy-1, encodes a non-muscle myosin; we are currently investigating the role of this gene in seam cell development. We will report our progress with nmy-1 and other genes identified in the screen.

24 May 1999 15:50 614 614 lon-1, a putative downstream target of TGFb signaling

1999 International Worm Meeting abstract 563 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. lon-1, a putative downstream target of TGFb signaling LL Maduzia 1 , P Shetgiri 1 , S Krishna 1 , C Savage-Dunn 2 , H Wang 1 , RW Padgett 1

1 Waksman Institute, Rutgers University, Piscataway, New Jersey 08855 USA. 2 Department of Biology, Queens College, CUNY, Flushing, New York 11367 USA.

TGFb signaling is involved in numerous developmental processes. In C. elegans, distinct TGFb-like signals regulate body size and male tail development (Sma/Mab pathway) as well as dauer formation (dauer pathway). Our lab is interested in studying body size regulation with a focus on the Sma/Mab pathway. Mutations in identified pathway components such as dbl-1 (ligand), daf-4 and sma-6 (receptor kinases) and sma-2, sma-3 and sma-4 (Smads) result in worms that are phenotypically small. In contrast, mutations in lon-1, lon-2 and lon-3 result in worms that are 1.5 times greater in length than wildtype worms. Due to their effects on body size, it is possible the lon genes work in conjunction with the sma genes to coordinate proper body size formation. In an effort to elucidate the functions of the lon genes, and their relationship with the Sma/Mab pathway we cloned lon-1. lon-1 encodes a novel protein containing signal sequences for secretion and GPI linkage. Preliminary results suggest that lon-1 is expressed in the posterior gut, the hermaphrodite tail, and throughout the length of the animal in the hypodermal regions. Our results reveal that the sma genes may have similar expression patterns in the hypodermis. These data, along with our findings that lon-1 is epistatic to the pathway components, suggests that lon-1 is a target of the Sma/Mab pathway.

24 May 1999 15:50 615 615 Isolation and characterization of novel TGFb-like components

1999 International Worm Meeting abstract 564 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Isolation and characterization of novel TGFb-like components LL Maduzia 1 , H Wang 1 , S Cohen 1 , C Savage-Dunn 2 , RW Padgett 1

1 Waksman Institute, Rutgers University, Piscataway, New Jersey 08855 USA. 2 Department of Biology, Queens College, CUNY, Flushing, New York 11367 USA.

Our lab has identified and characterized several components of a TGFb-like pathway in C. elegans. This pathway, referred to as the Sma/Mab pathway, when mutated, results in worms which are smaller in size and display ray fusions and crumpled spicules in the male tail. Currently, this pathway comprises a ligand, dbl-1, two serine/threonine kinase receptors, sma-6 and daf-4, and the Smads, sma-2, sma-3 and sma-4. To isolate novel genes in the pathway, we performed two screens for small animals and identified sma-10, sma-11, sma-12 and sma-13. These mutants do not display the male tail defects characteristic of the pathway, and hence may act only to regulate body size morphogenesis. We have evidence to place at least one of these genes in the pathway. Firstly, lon-1, a putative TGFb target gene that is epistatic to all existing Sma/Mab pathway components, is also epistatic to sma-10, demonstrating that sma-10 behaves similarly to existing pathway components. Secondly, sma-10 mutants suppress the long phenotype caused by dbl-1 overexpression, placing it downstream in the pathway. Further experiments will clarify whether these sma genes are involved in TGFb-like signaling. In addition, using a PCR based screen, we have isolated a deletion mutant in tig-2, a TGFb ligand most similar to the BMP5-7/60A class of ligands. Three C. elegans TGFb ligands including dbl-1, daf-7 and unc-129, have been characterized and found to define distinct pathways. tig-2 may define a separate pathway or may behave like its Drosophila homolog, partially functioning with the BMP-like genes, dbl-1 or daf-7. To address this issue we have begun to characterize this tig-2 deletion mutant. Homozygous tig-2 animals are healthy, viable, wildtype in body size, do not possess male tail ray fusions and exhibit no obvious dauer phenotype. tig-2 dbl-1 mutants are small with ray fusions similar to dbl-1 mutants. We are currently examining the expression pattern of tig 2 as well as performing additional experiments to determine its role in C.elegans development.

24 May 1999 15:50 616 616 RNAi Screening with a non-redundant cDNA set

1999 International Worm Meeting abstract 565 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. RNAi Screening with a non-redundant cDNA set I Maeda 1 , Y Kohara 2 , M Yamamoto 1 , A Sugimoto 1

1 Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Tokyo 113, JAPAN. 2 Gene Network Lab, National Institute of Genetics, Mishima 411, JAPAN.

RNA interference (RNAi) is a powerful tool for the transient inactivation of specific gene expression. We aim to establish a new screening method in which RNAi is performed systematically, to identify genes involved in specific developmental processes. To maximize the efficiency, we use a tag-sequenced, non-redundant cDNA set (made by Y. K.) as templates for preparing dsRNA. To deliver dsRNA into worms, we use the "soaking method" (in which worms are soaked in dsRNA solution) instead of microinjection, so that multiple RNAi can be performed concurrently. Although the soaking method has been known to be less potent and to give variable results, we optimized the condition to achieve a satisfactory efficacy to be used for the screen. In the control experiments, all nine genes tested (unc-22, glp-1, mei-1, mes-3, fem-1, glh-1, glh-2, pgl-1 and emo-1) with this method exhibited expected phenotypes with high penetrance. We are currently screening for the RNA species that cause sterility or embryonic lethality, however, we will record any other noticeable phenotypes. We plan to make the RNAi data available to the public at the NEXTDB WWW server at NIG (See the abstract by Shin-i et al.). In the pilot screen, 10/64 gave embryonic lethality of the F1s, and 6/64 resulted in sterility of the P0s. Progress on the screen will be presented.

24 May 1999 15:50 617 617 A Role for fem-2 During Embryonic Elongation

1999 International Worm Meeting abstract 566 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A Role for fem-2 During Embryonic Elongation PE Mains Department of Biochemistry & Molecular Biology, Univiversity of Calgary, Calgary, Alberta, Canada An actin-mediated contraction of the epidermal cells elongates the spherical embryo into a long, thin worm1 . let-502 mutants fail to elongate while mel-11 mutant embryos hypercontract2,3 . Genetic results and analogies with smooth muscle contraction suggest that myosin phosphatase (MEL-11) inhibits contraction. At the appropriate time, Rho-binding kinase (LET-502) inactivates MEL-11, allowing elongation to proceed. We found that fem-2 enhances let-502 and suppresses mel-11, suggesting a role for fem-2 during morphogenesis. Effects of fem-2 on embryonic elongation were unexpected. However, when fem-2(0) hermaphrodites were selfed, 4% of the progeny arrested as unelongated embryos and another 20% had morphological phenotypes (Dpy, Vab, or Rol) suggestive of embryonic morphogenesis defects (because of maternal-effects, first generation fem-2 homozygotes are self-fertile). fem-2 function during elongation is likely independent of its role in sex determination since genetic interactions were not observed when let-502 or mel-11 were combined with fem-1, fem-3, tra-1, tra-3 or fog-2. We noticed no changes in sexual phenotype with let-502 or mel-11 alone. fem-2 and mel-11 encode subunits of unrelated types of phosphatases2,4,5 . These phosphatases cannot be interchangeable because while fem-2(+) potentiates morphogenesis, mel-11(+) inhibits it. The triple mutant suggests that fem-2 is part of a parallel system that can elongate the embryo in the absence of the let-502/mel-11 pathway: while let-502;mel-11 double mutants undergo near normal morphogenesis, let-502; mel-11; fem-2 triple mutants fail to elongate.

1 Priess & Hirsh (1986) Dev. Biol. 117:156;

2 Wissmann et al. (1997) Genes Dev. 11:409;

3 Wissmann et al.(1999) Dev. Biol. (in press);

4 Pilgrim et al. (1995) Mol. Biol. Cell 6:1159;

5 Chin-Sang & Spence (1996) Genes Develop. 10:2314

24 May 1999 15:50 618 618 Characterization of the role of rho-1 and nuclear migration during P cell migration

1999 International Worm Meeting abstract 567 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of the role of rho-1 and nuclear migration during P cell migration CJ Malone 1 , S Orita 1,2 , M Han 1

1 MCDB, University of Colorado, Boulder CO 80309 USA. 2 present address: Shionogi and Co. Osaka 553-0002 JAPAN.

The small GTPase rhoA has been implicated in a variety of cellular movements. To address the role of rhoA during C. elegans development we have performed a reverse genetics approach. RNAi of the C. elegans rhoA homolog, rho-1, produces embryonic lethality, indicating that it is an essential gene. To identify specific requirements for rho-1 we expressed dominant negative (dn) and gain-of-function (gf) rho-1 under the control of the col-10 promoter, which drives expression in hypodermal cells. Expression of this rho-1(dn) construct causes less than the normal 12 P cells to occupy the ventral cord. Analysis of these animals suggests that P cells are missing from the ventral cord due to failed P cell migration. unc-73, which encodes a protein capable of acting as an exchange factor for the mammalian small GTPase, Rac1, also causes a P cell migration defect. To test whether UNC-73 acts through rho-1 during P cell migration, we expressed rho-1(gf) in animals homozygous for a weak unc-73(e936) allele. This construct was able to rescue the P cell migration defect of unc-73, suggesting that UNC-73 may act as an exchange factor for RHO-1, activating it during P cell migration. We have shown previously that the nuclear envelope protein UNC-84 is required for nuclear migration during P cell migration. To identify other genes that may be involved in the P cell nuclear migration, we have undertaken RNAi studies of the C. elegans homologs of NUDC and NUDF, two proteins required for Aspergillus nuclear migration. Both C. elegans genes appear to be essential, because RNAi of either can cause complete embryonic lethality in the progeny of injected animals. Although severely deformed, these embryos form a pharynx, develop gut granules and express the MH27 epitope. Interestingly, escapers from the RNAi injections are Unc, Pvl and sterile. The Unc and Pvl phenotypes are similar to those observed in animals mutant for unc-83 or unc-84, which cause P cell nuclear migration defects. We are currently determining if a P cell nuclear migration defect is the cause of the Unc and Pvl phenotypes of the NUDC/F RNAi animals.

24 May 1999 15:50 619 619 Analysis of glutamate transporter knockouts in the worm

1999 International Worm Meeting abstract 568 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of glutamate transporter knockouts in the worm I Mano, M Driscoll Department of Molecular Biology and Biochemistry, Rutgers University, 604 Allison Rd., Piscataway, NJ 08854. Glutamate (Glu) is the major excitatory neurotransmitter in the mammalian brain. Glu is involved in a wide spectrum of developmental, physiological and pathological processes. Glu exerts these activities by activating ionotropic and metabotropic receptors (GluRs) that assemble via PDZ-domain containing proteins into complexes with other membrane and cytoplasmic proteins that modulate the specific signal. A central determinant of Glu action is its rate of removal from the synaptic cleft, mediated by specific Glu transporters (GluTs). Molecular analysis in C. elegans and physiological analysis in A. suum suggest that many of the molecules and functional aspects of glutamatergic neurotransmission are conserved from nematodes to humans. The C. elegans genome includes six genes that are highly homologous to mammalian GluTs. In order to gain insight into the mechanisms of normal and abnormal glutamatergic neurotransmission in the worm, we are conducting systematic screens of deletion libraries for strains harboring knockouts of worm GluTs. We have isolated a knockout of the K08F4.4 GluT gene and we are currently characterizing its phenotype. We are screening additional libraries to isolate knockouts in the other worm GluTs. We plan to determine expression patterns and RNAi phenotypes of all worm GluTs. Our long-term goal in elaborating phenotypes of GluT knockouts is to elucidate the mechanisms of action of known and novel gene products that participate in normal and pathological processes induced by Glutamatergic neurotransmission.

24 May 1999 15:50 620 620 The unc-13 Puzzles in Neurotransmission

1999 International Worm Meeting abstract 569 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The unc-13 Puzzles in Neurotransmission Ichi Maruyama Department of Cell Biology, The Scripps Research Institute, 10550 N.Torrey Pines Rd, La Jolla, CA 92037 Putative null alleles of unc-13 have severely impaired (almost paralyzed) locomotion, slow irregular pharyngeal pumping, and are resistant to acetylcholinesterase (AChE) inhibitors. They also accumulate abnormally high levels of acetylcholine (ACh). Motor and sensory neurons are misplaced in the mutants. Furthermore, abnormal connections between major interneurons through gap junctions as well as abnormal development of motor neurons have been found. The unc-13 gene encodes a protein with the second messenger diacylglycerol, Ca2+ and phospholipid-binding domains. We are currently analyzing a role for unc-13 in neurotransmission by making double and triple mutants between unc-13 and other genes, including ace-1, ace-2, ace-3, dyn-1, rab-3, snt-1, unc-18, unc-17, unc-64 and unc-104, involved in neurotransmission. The unc-13 ace double mutants had better locomotive activities than the unc-13 single mutants. Hypersensitivity of the ace mutants against AChE inhibitors was also suppressed by unc-13 mutations. These results suggest that the neurotransmitter ACh is deprived in neuro-neural and neuro-mascular junctions. However, unc-13 ace double mutants accumulate ACh at markedly elevated levels, compared to unc-13 or ace single mutants, suggesting that only regulated ACh release but not its constitutive release is impaired in unc-13 mutants. Double mutants between unc-13 and rab-3 had behavior phenotypes resembling unc-13 but the level of accumulated ACh was intermediate between the levels of unc-13 and rab-3. Mutants doubly defective in unc-13 and snt-1 had similar phenotypes to unc-13 but ACh accumulation was the same as snt-1. These results are consistent with evidence that RAB-3 and SNT-1 are involved in visicular transport and recycling, respectively. Based on the results, we propose at least two parallel pathways in Ca2+-regulated neurotransmitter release and UNC-13 is involved in the major pathway. Constitutive release of neurotransmitter seems to be unimpaired and, therefore, UNC-13 may function at the very last step of synaptic vesicle exocytosis as a major Ca2+ sensor. This proposed role for UNC-13 is consistent with its molecular structure of the protein described above.

24 May 1999 15:50 621 621 Revisiting Ryanodine Resistance in Caenorhabditis elegans

1999 International Worm Meeting abstract 570 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Revisiting Ryanodine Resistance in Caenorhabditis elegans E Maryon, L Sowinski, P Anderson Dept. of Genetics, UW-Madison, Madison WI, 53706. We are investigating the biological roles of ryanodine receptor (RyR) channels in regulating contraction of C. elegans muscle cells. RyRs are Ca++ channels located in the sarcoplasmic reticulum (SR), a modified endoplasmic reticulum specialized for rapid uptake and release of Ca++ . Muscle depolarization induces RyRs to open, elevating cytoplasmic Ca++ and causing contraction of the myofilament lattice. Ryanodine is a plant alkaloid that binds with high affinity to RyRs, gating the channels open. Ryanodine thus induces a constitutive Ca++ leak from the SR, and causes contractile paralysis in striated muscle. When exposed to ryanodine, C. elegans exhibits hypercontraction and paralysis of bodywall muscle. Paralysis is dependent on unc-68 function, the only RyR gene in the C. elegans genome.

To identify genes involved in Ca++ regulation of contraction, we have isolated ryanodine resistant mutants. We picked F2 progeny of EMS-mutagenized animals having improved motility in liquid cultures containing paralyzing concentrations of ryanodine. In addition to previously described (WBG 12(4):69) screens done in 100-300 µM ryanodine, we have done similar screens in 1mM ryanodine. We have also picked resistant animals after several generations of growth in liquid cultures containing 0.5-1.0 mM ryanodine.

From a total of 4 x 10 5 mutagenized haploid genomes we have isolated 38 resistant mutants that comprise at least 8 complementation groups. Many of the mutants are dominant or semi-dominant with respect to ryanodine resistance. Mapping and complementation tests are underway to identify genes corresponding to the mutant alleles. In addition to a large number of unc-22 alleles isolated in these screens, we have isolated at least two alleles of unc-68. We are further characterizing the most resistant mutants using electron microscopy and ryanodine binding assays. In addition to our mutant hunts, we have investigated ryanodine resistance of mutants with defects in the myofilament lattice. A number of these mutants (unc-22, lev-11, unc-80, unc-82, unc-89, unc-98, unc-112) exhibit some degree of ryanodine resistance. We infer that alterations of the myofilament lattice in these mutants reduces the sensitivity of the contractile apparatus to Ca++ .

24 May 1999 15:50 622 622 C. elegans unc-119 mutants define novel neurite development defects

1999 International Worm Meeting abstract 571 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. C. elegans unc-119 mutants define novel neurite development defects WP Materi 1 , MF Maduro 2 , D Pilgrim 1

1 University of Alberta, CW405 Biological Sciences Centre, Edmonton, Alberta T6G 2E9, Canada. 2 UC Santa Barbara, Department of MCD Biology, Biological Sciences II, Santa Barbara, CA 93106.

We are characterizing the role of unc-119 (one of a large number of uncoordinated mutants) in the development of the worm’s nervous system. unc-119 mutants are defective in nervous system organization. Using confocal microscopy and transgenic worms expressing GFP under the control of neural-specific promoters, we have viewed the axons and dendrites of selected subsets of neurons. By comparing the morphology of these selected neurons in mutant versus wild-type worms, we have been able to show that unc-119 mutants define a novel class of nervous system defects. UNC-119 may be important in transducing many signals that affect growth cone elongation, fasciculation (nerve fiber bundling) and turning. Thus it may have a more general role than other known axon guidance proteins, such as UNC-6 - the worm netrin homologue - and a more specific role than other known growth cone signal transduction proteins, such as UNC-33 - a collapsin response mediator homologue. The predicted amino acid sequence of UNC-119 is novel although homologs can be found in Drosophila, the zebra fish and in mammals. A human homolog is enhanced in retinal tissue and has also been found in fetal brain tissue. Because it has no apparent enzymatic or structural domains, we have performed a yeast two-hybrid analysis looking for proteins that might interact with UNC-119 and give some clue as to its function. Two different interacting proteins have been identified to date, one of which is a transcription factor homologous to the Drosophila gene zfh-1. This gene is known to be crucial for correct development of both the central and peripheral nervous systems in the fly.

24 May 1999 15:50 623 623 Regulation of the expression of mab-21: a candidate Hox target gene

1999 International Worm Meeting abstract 572 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Regulation of the expression of mab-21: a candidate Hox target gene Rie Matsui 1 , Daihachiro Tomotsune 2 , Naoki Takahashi 1

1 Nara Institute of Science and Technology, Takayama, Ikoma, Nara 630-1010, Japan. 2 Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan.

The Hox genes determine cell fates along the anterior-posterior body axis of many organisms. Hox gene products are believed to be transcription factors that regulate expression of target genes. We identified several candidate Hox gene targets by immunopurification of DNA sequences bound to a Hox protein in native chromatin (1). Recently, we isolated a mouse orthologue of C. elegans mab-21 as a candidate target of HoxC4. In C. elegans, mutations in mab-21 result in loss of the ray 6 morphology and fusion this ray with ray 4. This mutant phenotype is similar to that of the egl-5 (Hox). It is implicated that mab-21 acts in the same pathway as egl-5 in specification of ray morphology by genetic analysis (2). These results suggest that mab-21 gene is a candidate target of Hox gene products. The expression pattern of mab-21 has been studied using GFP reporter gene fusion. The last fifth exon of mab-21 was fused in frame with GFP (using Andy Fire vector pPD. 95.75) and 3.2Kb of 5’ flanking sequence was included. mab-21::gfp expression is first seen in the hypodermal cells in pre-comma stage. In adult animals, mab-21::gfp is expressed in the hypodermal cells, neurons, head muscles and ray6. We identified the elements necessary and sufficient for mab-21 gene expression using deletion constructs. The deletion analysis indicated that a 450bp region upstream of the transcription start site is sufficient for hypodermal cell expression. A 300bp region in the fourth intron of the mab-21 is necessary for neuron expression and a 450bp region in the intron of the mab-21 is necessary for ray 6 expression. We are currently testing whether egl-5 can drive ectopic expression from a reporter construct containing ray 6 control element.

(1). Tomotsune et al., (1993) Nature 365: 69-72 (2). Chow et al., (1994) Development 120: 2579-2593

24 May 1999 15:50 624 624 Progress on the cloning of spd-2, a gene involved in centrosome function and the establishment of embryonic polarity

1999 International Worm Meeting abstract 573 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Progress on the cloning of spd-2, a gene involved in centrosome function and the establishment of embryonic polarity KN Maxwell, KF O’Connell, JG White Laboratory of Molecular Biology, University of Wisconsin-Madison, Madison, WI 53706 The events that occur shortly after fertilization are crucial to ensuring the proper execution of developmental programs in embryogenesis and, thus, to the survival of the embryo. These events include establishment of the anterior-posterior axis and construction of a bipolar mitotic spindle. Using mutations in the spd-2 gene, we have shown that these two events are dependent on the presence of a functional centrosome. b-tubulin immunofluorescence shows that spd-2 embryos lack the organized arrays of microtubules normally associated with the sperm pronucleus after fertilization; foci of g-tubulin are also absent from early spd-2 embryos. As a result, spd-2 embryos show a disruption of all aspects of embryonic polarity that have been investigated. Furthermore, the lack of a centrosome prevents the establishment of a bipolar mitotic spindle, causing the majority of spd-2 embryos to fail in the first division. (see abstract by O’Connell et al). In order to further understand the function of the spd-2 gene product in early development, we have initiated cloning of the spd-2 gene. The spd-2 gene is defined by two temperature-sensitive alleles, oj29 and oj42. Most likely the stronger allele, oj29 shows 100% embryonic lethality when L4 animals are shifted to the restrictive temperature; furthermore, spd-2(oj29) shows a completely penetrant sterile, uncoordinated phenotype when animals are exposed to the restrictive temperature during postembryonic development. Standard genetic mapping techniques were used to position spd-2 between let-89 and unc-29 on chromosome I. Transgenic lines were established with six of the cosmids that span this genetic region, and one cosmid, F43G9, gives embryonic rescue. We are currently testing this transgenic line for further indication of both embryonic and post-embryonic rescue. Furthermore, in order to determine which predicted open-reading frame on F43G9 corresponds to the spd-2 gene, we are injecting double-stranded RNA corresponding to these ORFs into wild-type worms to look for phenocopy of spd-2.

24 May 1999 15:50 625 625 Development of molecular markers that migrate across synapses in the C. elegans nervous system

1999 International Worm Meeting abstract 574 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Development of molecular markers that migrate across synapses in the C. elegans nervous system Steven McCarroll 1,2 , Cori Bargmann 1

1 U.C. San Francisco and Howard Hughes Medical Institute 2 [email protected]

The synaptic connectivity of the adult C. elegans nervous system has been determined from electron micrographs (1). This way of analyzing synaptic connectivity is accurate but labor-intensive. The ability to visualize synaptic connectivity more easily would allow us to probe the effects of development, experience, and mutations on synaptic circuitry. To some extent this can be done by examining pre- and postsynaptic markers in specific neurons (2). We would like to develop a general method to label a given neuron’s synaptic targets or inputs. To that end, we are working to develop molecular markers that will specifically migrate across synapses in the C. elegans nervous system. As a first step we are pursuing molecules which migrate across synapses in other organisms. Our approaches have included fusions of reporter genes to genes encoding wheat germ agglutinin (WGA) and tetanus toxin. The ASER-specific promoter Pgcy-5 was used to express a WGA:GFP fusion protein. This fusion protein migrated from ASER into several other neurons, labeling a stereotyped set of neurons which extend processes in the nerve ring. Migration of WGA-GFP from ASER to these neurons requires UNC-104 and UNC-18 but not UNC-31, suggesting an involvement of synapses in the process of WGA:GFP release or uptake. However, the set of neurons labeled by ASER::wga:gfp does not correspond to the ASER synaptic contacts identified by EM reconstruction (1). We are trying to identify the mechanism of WGA:GFP translocation and to modify WGA:GFP so that its transcellular translocation is specific for synaptic sites. A goal of these experiments is targeting secretion spatially: getting neurons to secrete a protein specificallly at synaptic sites. One approach we are using involves fusion to synaptically-localized transmembrane proteins via a cleavage sequence that will be recognized by extracellular proteases such as furin.

(1) White et al. Phil Trans Roy Soc. 314: 1. (2) Rongo et al. Cell 94: 751.

24 May 1999 15:50 626 626 Searching for Targets of pag-3

1999 International Worm Meeting abstract 575 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Searching for Targets of pag-3 JB McDermott, EJ Aamodt Louisiana State University Medical Center, 1501 Kings Highway, Shreveport, LA 71130 Mutations in pag-3 cause pleiotropic defects including a lethargic phenotype, a reverse kinker uncoordinated phenotype, lineage defects (see abstract by Cameron et al.), and misexpression of touch neuron-specific genes in the BDU interneurons. pag-3 encodes a zinc finger protein related to the vertebrate proto-oncoprotein Gfi-1. Gfi-1 functions as a transcriptional repressor in transfected cells, and is expressed in lymphoid tissues. pag-3 is expressed in several neural cell types, including the touch cells, the BDU cells, and cells of the VA, VB and VC motor lineages. We are taking three strategies to identify targets of PAG-3. First, we are trying to identify authentic PAG-3 binding sites in touch neuron genes such as mec-7. Second, we are using DNA chip technology to identify genes whose expression is altered in pag-3 mutants. Third, we are screening for suppressers of the pag-3 uncoordinated phenotype. To identify PAG-3 binding sites we have focused on the mec-7 gene. mec-7 is one of the touch neuron genes misexpressed in the BDU neurons of pag-3 mutants. PAG-3 expressed in bacteria binds well to the consensus Gfi-1 binding site and to mec-7 sites containing the core (AAT/GC) of the Gfi-1 consensus site. To correlate the binding of PAG-3 to changes in target gene expression, we transformed worms with mec-7lacZ mutated at the strongest PAG-3 binding sites. Since PAG-3 may act directly or indirectly on mec-7, we have transformed worms with deletions of mec-7lacZ to identify the element responsible for misexpression in the BDU neurons. For the DNA microarray screen, we have prepared mRNA from pag-3(ls64) and N2 L1 larvae. The ls64 allele contains a nonsense mutation just upstream of the zinc fingers. Northern analysis will be used to confirm that the abundance of specific mRNAs is altered in pag-3 mutants. Finally, we are looking for suppressers of the uncoordinated phenotype by screening for improved motility in mutagenized pag-3(ls20) worms.

24 May 1999 15:50 627 627 Constitutively activated integrin during muscle development

1999 International Worm Meeting abstract 576 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Constitutively activated integrin during muscle development KA McDonald, JZ Schwarz, BD Williams Univ. of Illinos, Urbana, IL 61801 Integrins are heterodimeric transmembrane proteins that bind components of the extracellular matrix (ECM). Recent advances have shown that integrin-mediated signaling can control fundamental cell properties such as motility, cytoskeletal remodeling, differentiation, and gene expression. We are investigating integrin function during muscle cell development in C. elegans. Body wall muscle cells express the integrin a subunit PAT-2 and b subunit PAT-3, both of which are located in regularly patterned cytoskeletal anchorage structures analogous to vertebrate muscle Z and M lines. pat-2 and pat-3 mutations disrupt cytoskeletal assembly, resulting in the Pat developmental arrest phenotype. The integrin heterodimer can modulate its affinity for ECM ligands and the ability of its cytoplasmic domain to interact with the cytoskeleton and associated signaling molecules. The "non-activated" heterodimer has a low affinity for extracellular ligands and does not show cytoskeletal association and related signaling. Conversely, the "activated" heterodimer has high affinity for ligand and does associate with the cytoskeleton. In our working model of body wall muscle development, integrin is activated by binding to an ECM ligand, and in response, initiates cytoskeletal assembly. To test this model, we have introduced a mutation into a functional pat-3::gfp construct that is predicted to constitutively "activate" the integrin heterodimer (Hughes et al., 1996, JBC 271: 6571). This mutation disrupts a highly conserved potential salt bridge between the a and b subunits. When expressed transgenically in a wild-type background the "activated" PAT-3::GFP protein localizes normally to muscle cell attachment structures. We have observed mispositioned sex muscle insertions in these animals, indicating dominant effects on cell migration and/or insertion. Surprisingly, the "activated" pat-3::gfp transgene rescues pat-3 loss-of-function homozygotes, but only at very low frequency. Most animals expressing the transgene arrest as Pats. We are currently characterizing the defects in muscle cell development that occur in these animals. The relatively few rescued adults often show examples of mispositioned body wall and sex muscles, again consistent with migration/insertion defects.

24 May 1999 15:50 628 628 Whole Genome Analysis of C. elegans Using DNA Microarrays

1999 International Worm Meeting abstract 577 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Whole Genome Analysis of C. elegans Using DNA Microarrays JS McDowall, S Jones, T Freeman, A Coulson, P Kuwabara Sanger Centre, Wellcome Trust Genome Campus, Hinxton, England We have recently received funding to produce C. elegans whole genome DNA microarrays, which will be used to investigate gene expression patterns on a global scale. We are taking advantage of the C. elegans genome sequence and the software developed at the Sanger Centre to predict PCR primer pairs that will specifically amplify 1 kb coding regions from each of the predicted 18,000 C. elegans genes. The development of primers is being performed in collaboration with Stuart Kim (Stanford, CA). The DNA microarrays will be produced following protocols originally developed in the laboratory of P. Brown (Stanford, CA) and used by S. Kim to develop a partial worm DNA microarray. The microarrays will be generated by robotically gridding each of the 18,000 1kb DNA fragments onto poly-L-lysine coated glass slides. The microarrays will then be used to compare steady-state RNA populations expressed by animals with different genetic backgrounds or physiologies by differential hybridisation of cDNAs. These cDNAs will carry either a Cy3 or Cy5 fluorescent tag to be incorporated during reverse transcription. Following hybridisation, the resulting fluorescent signals will be detected by confocal laser scanning and expressed as two-colour ratios of differential expression. Bioinformatic methods for archiving and analysing these data will be extended during the course of these studies. The power of DNA microarray technology is two-fold: DNA microarrays yield functional information on genes in the form of expression profiles, and they enable an entire genome to be searched in a single reaction to identify primary and secondary response genes. Quantitative data can be obtained on both up- and down-regulated genes to help decipher genetic pathways. The technology is sufficiently sensitive to detect 2-fold differences in gene expression. We plan to use the microarrays to address a series of key questions in developmental biology. For example, we plan to compile a profile of downstream targets for each of the predicted homeodomain proteins in the worm. Finally, we will make this technology available to the members of the research community.

24 May 1999 15:50 629 629 LIN-13, a zinc-finger protein that acts in vulval development

1999 International Worm Meeting abstract 578 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. LIN-13, a zinc-finger protein that acts in vulval development A Melendez, IS Greenwald HHMI and Columbia University The synthetic Multivulva (synMuv) genes comprise two classes of genes that act in the determination of vulval precursor cell (VPC) fates (1,2). Under certain conditions, alleles of lin-13 display some of the genetic properties of a Class B SynMuv gene (2; our observations, which we will present). We have found that lin-13 encodes a large nuclear protein of 2,249 amino acids with 14 zinc-fingers of the C2-H2 class. Both of the existing lin-13 alleles are nonsense mutations. lin-13(n387) is a TCA to TGA (S525stop) change that disturbs the first zinc-finger and would encode a predicted protein of 524 amino acids. lin-13(n388) is a CGA to TGA (R857stop) change right after the second zinc finger and would encode a predicted protein of 856 amino acids. Thus, both nonsense mutations would result in greatly truncated LIN-13 protein products. The molecular nature of the lin-13 alleles was surprising, since Ferguson and Horvitz (1) showed that lin-13 (n387)/nDf16 is lethal which suggested that lin-13(n387) was hypomorphic, and the lin-13 null phenotype is zygotic lethality. To explore the issue of the lin-13 null phenotype, we used RNAi (3). We assayed the phenotype of the progeny of N2 hermaphrodites injected with dsRNA derived from lin-13 cDNA genomic clones. Injected mothers were incubated at 15°C or at 25°C. Progeny raised at 25°C were Muv and sterile, whereas progeny raised at 15°C were sterile but not Muv. Thus, the phenotypes caused by RNAi resemble those caused by the existing lin-13 alleles, and raise the distinct possibility that the null phenotype of lin-13 is the heat sensitive phenotype displayed by the two existing alleles. We have also begun a genetic mosaic analysis to determine the cellular focus of lin-13 in vulval development (lin-13 reporter constructs suggest that it is expressed mainly in hyp7 and possibly in the VPCs). We will report on our progress and we will also speculate about the connection between lin-13 and the class B synMuv gene lin-35Rb.

1. Ferguson, E.L. and Horvitz, H.R. (1985) Genetics 110, 17-72. 2. Ferguson, E.L. and Horvitz, H.R. (1989) Genetics 123, 109-121. 3. Fire, A., et al. (1998) Nature 391, 806-811.

24 May 1999 15:50 630 630 Electrophysiological Analysis of C. elegans Ionotropic Glutamate Receptors

1999 International Worm Meeting abstract 579 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Electrophysiological Analysis of C. elegans Ionotropic Glutamate Receptors JE Mellem, Y Zheng, PJ Brockie, AV Maricq Department of Biology, University of Utah, Salt Lake City, Utah 84112 USA A fundamental problem in neurobiology is to understand how neuronal circuits function to control behavior. In C. elegans, a simple circuit that controls movement has been identified. Coordinated movement and escape from tactile stimuli requires the command interneurons AVA, AVB, AVD, AVE, and PVC (Chalfie et al., Wicks and Rankin). Our lab has shown that many ionotropic glutamate receptor subunits are expressed in the command interneurons and that perturbation of individual subunits leads to altered locomotion (see abstracts by Zheng et al. and Brockie et al.). To better understand how receptor subunits contribute to neuronal function, we are undertaking an electrophysiological analysis of glutamate receptors. When expressed in Xenopus oocytes, GLR-1 does not function as a glutamate-gated receptor, although a constitutively active variant of GLR-1 forms a leaky, ligand-independent channel. Perhaps other receptor subunits are required to form a functional receptor? Six ionotropic glutamate receptors (glr-1 - glr-4, nmr-1, and nmr-2) are expressed in the neuron AVA. We will determine whether these subunits form glutamate-gated homomeric receptors and which combinations of receptor subtypes form functional receptors. We have now started an in vitro analysis of glutamate receptor subunits, expressed alone or in combinations, in Xenopus oocytes and HEK 293 cells. We are also continuing our efforts towards an in vivo analysis of the command interneuron circuitry. Using patch-clamp technology we have successfully obtained on-cell and whole-cell recordings from a small number of command interneurons. Our initial efforts have focused on identifying the voltage-dependent currents in these neurons. We will present data we have gathered from in vitro analysis of the glutamate receptor subunits, our initial in vivo data, and the details of the dissection procedure used to isolate the command interneuron circuitry.

24 May 1999 15:50 631 631 him-5 and him-8

1999 International Worm Meeting abstract 580 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. him-5 and him-8 Philip Meneely, Toni Amato, Alex Ensminger, Maria Robinson, Marnie Gelbart Dept of Biology, Haverford College, 370 Lancaster Ave., Haverford, PA 19041 Mutations in many different genes result in an unusually high incidence of male self-progeny due to meiotic loss of the X-chromosome. Of these, the most extreme are mutations in him-8 (40% male self-progeny) and him-5 (up to 35% male self-progeny). Genetic and cytological evidence indicates that him-8 affects the X chromosome exclusively during meiosis, whereas him-5 affects the X much more strongly than the autosomes, but does affect the autosomes. Both him-5 and him-8 greatly reduce the number of crossovers on the X chromosome, and affect the location of the residual crossovers. In other organisms, these have been termed "pre-condition" mutants or "crossover control" mutants but the molecular mechanism is obscure. We have been carrying out a molecular analysis of the him-8 and him-5 loci, beginning with transformation rescue experiments, and including cDNA isolation, RT-PCR, northerns, sequencing wild-type and mutant strains, and most recently RNAi experiments (which we hope to have done by the meeting). The molecular structure of the him-8 locus is complex, frustrating, and unresolved at this point. Although much less studied than him-8, the him-5 locus appears to be much simpler, and the predicted protein is very small (143 amino acids) and very basic (predicted pI of 10.5) with no homology to other known proteins. Some models for genes involved in crossover control predict that they might affect the structure of meiotic chromosomes, and the putative HIM-5 protein would not discourage such speculation.

24 May 1999 15:50 632 632 Interference during Meiosis on the X Chromosome and an Autosome

1999 International Worm Meeting abstract 581 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Interference during Meiosis on the X Chromosome and an Autosome Philip Meneely, Tate Kauffman, Kathryn Strain, Laura Lehnhoff, Anna Farago Dept of Biology, Haverford College, 370 Lancaster Ave., Haverford, PA 19041 In all organisms examined, including C. elegans, the number and location of exchanges during meiosis is highly regulated. The aspect of this that is usually recalled from undergraduate genetics courses is interference, in which the presence of one crossover on a chromosome reduces the probability of a second crossover occuring nearby. The molecular mechanisms of interference, or more broadly, crossover control, are poorly understood but the extensive genetic and physical map data suggest that worms might be a suitable model for studying this. Several investigators, using a variety of methods, have proposed that the X chromosome during oogenesis has very high interference. With STS polymorphisms that allow us to assay the entire chromosome as a unit, we find that for most of the length of the X chromosome, interference is complete and that there is exactly one crossover per X chromosome. The STS polymorphisms also allow us to examine interference on the autosomes during oogenesis, which had not been tested previously. For LGIII, we again find interference is extremely high, and may be complete. From among more than 80 recombinant chromosomes, we found no unambiguous examples of two or more crossovers; approximately 8 double crossovers would have been seen if there were no interference. These data suggest that there is exactly one crossover per chromosome during oogenesis. We are extending the analysis to look at autosomal recombination and interference during spermatogenesis where double crossovers have previously found. The very small number of crossovers per chromosome and the presence of double crossovers on autosomes during spermatogenesis suggested to us that spontaneous males may arise from occasional X chromosomes that do not receive an exchange during hermaphrodite spermatogenesis. Consistent with this, we have confirmed that there is no detectable X chromosome non-disjunction during oogenesis suggesting that the X chromosome non-disjunction most likely occurs during hermaphrodite spermatogenesis.

24 May 1999 15:50 633 633 An Astounding Variety of Neuropeptides

1999 International Worm Meeting abstract 582 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. An Astounding Variety of Neuropeptides LA Messinger, AO Stretton Department of Zoology, University of Wisconsin-Madison Peptides are the original signaling molecules in metazoan nervous systems. They are also ubiquitous in nervous systems that employ ’classical’ neurotransmitters where they serve as modulators, hormones and transmitters. Cataloging peptide diversity is an essential step in comprehending their range of function and their evolution. The first recognition of nematode neuropeptide diversity came from Ascaris suum through the isolation and sequencing of 20 peptides. In C. elegans 14 reported precursor genes predict 43 peptides. Eighteen of the A. suum and all but one of the C. elegans peptides end in RFamide. Does this represent the true complement of nematode peptides or is it a sampling artifact?. A cursory search of sequences from the Genomic Sequencing Consortium has identified a multitude of genes with the hallmarks of peptide precursors. These include repeats with shared C-terminal sequences that end in the amide donating Gly. The repeats are separated by basic residues serving as processing sites and there is a conserved segment near the N-terminus that may be the signal sequence. The C-termini of these predicted peptides include SFa, RIa, MYa, RPa, NRa, GLa, GFa, AFa and QFa as well as some previously unreported ones with RFa. This more than doubles the number of predicted peptides that have been reported. The search was likely to recognize only sequences with multiple amidated peptides and at least one dibasic processing site thus there may be many more peptide encoding sequences awaiting discovery with a more sophisticated search. It is a damn good thing this is a simple nervous system. Evidence for the expression of some of these new sequences includes ESTs and PCR amplifications of total RNA from mixed-staged worms. As with the previously reported genes, the evolutionary origins of these genes are unexplained. No orthologs are apparent. That indicates they are evolving through a mechanism other than duplication and divergence or they are all ancient enough to have lost traces of similarity.

24 May 1999 15:50 634 634 che-14encodes a distant member of the PATCHED receptor family required for differentiation of ectodermal epithelial cells

1999 International Worm Meeting abstract 583 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. che-14encodes a distant member of the PATCHED receptor family required for differentiation of ectodermal epithelial cells G Michaux, S Sookhareea, G Belliard, C Hindelang, M Labouesse IGBMC, BP163, F-67404 Illkirch Cedex To find genes required for differentiation of non-neuronal ectodermal cells, we have looked for mutations displaying similar defects as mutations in lin-26(n156),a gene known to specify and/or maintain the fates of hypodermal and glial-like cells. Both che-14(e1960)(Perkins et al,Dev Biol 56: 110-156 1986) and lin-26(n156)(Labouesse et al,Development 122: 2579-2588 1996) lead to ultrastructural defects in glial-like cells. Using a dye-filling test as an assay, we isolated one new che-14mutation, mc16,in a non-complementation screen against e1960after examining 10000 genomes. We found by electron microscopy that mc16,as e1960,affects chemosensory organs by closing the amphid and phasmid channels. Both alleles induce a weakly penetrant embryonic lethality reminiscent of lin-26(mc2)and a partial rod-like larval lethality probably due to hypodermal defects. We have cloned che-14by complementation rescue of the dye filling defect due to e1960.che-14encodes a protein, with 10 to 12 transmembrane domains, which displays 45% similarity/20% identity with PATCHED, the HEDGEHOG receptor. The two che-14mutations are G-A transitions that affect splice donor sites located towards the 3’ end of the gene. To look at the expression pattern of che-14we inserted the gfpin frame at the 3’ end of che-14.The fusion protein is expressed in all epithelial ectodermal cells, and is found mostly at the apical membrane but also in the cytoplasm. To find CHE-14 partners, we screened for mutations displaying both the rod-like larval lethality and the dye-filling defect (Rdy phenotype). After TMP/UV mutagenesis of 13000 genomes, we kept 10 mutants, one of which is a new che-14allele. The mc35mutation is an insertion combined with a deletion which could produce a CHE-14 protein half its normal size. Together our data suggest that che-14is required to maintain the polarity of epithelial cells in the ectoderm. We are currently examining the specific function of the different potential functional domains found in CHE-14 by deletion analysis in the che-14::gfpfusion. Injection in che-14mutants will allow us to follow both rescue and protein localisation.

24 May 1999 15:50 635 635 Specifying cell fates in the C lineage

1999 International Worm Meeting abstract 584 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Specifying cell fates in the C lineage KM Mickey, JR Priess MCB Program and HHMI, FHCRC, Seattle, WA, 98109 The daughters of the C blastomere, Ca and Cp, have nearly identical lineage patterns. However, Ca produces two neurons and has high levels of the POP-1 protein, while Cp does not make any neurons and has low levels of POP-1. We wondered if pop-1 plays a role in the Ca/Cp difference since pop-1(+) is required to specify many other a/p differences in the early embryo. Using a neuronal GFP marker (provided by D. Pilgrim), we find that neither Ca nor Cp produces neurons in embryos lacking POP-1 (pop-1RNAi). In embryos with high levels of POP-1 (such as mom-2(wingless);apr-1(apc)RNAi double mutants), both Ca and Cp can produce neurons. Epistasis analysis indicates that pop-1(+) activity is required for Cp to make neurons in the mom-2;apr-1 double. These results indicate that high levels of POP-1 are required for the ability of Ca to make neurons and that the asymmetry of POP-1 between Ca and Cp is important for making these sisters different. When Ca and Cp divide, they each produce an anterior daughter that has high levels of POP-1 and makes hypodermis, and a posterior daughter that has low levels of POP-1 and makes muscle. Does pop-1(+) play a role in this a/p difference? When Caa or Cpa, the hypodermal precursors, are isolated in pop-1(RNAi) embryos, they each make muscle and appear to lack hypodermis, suggesting that pop-1(+) is required. We noticed that when we lineaged Cpa in an intact pop-1(RNAi) embryo, it produced hypodermal cells. This surprising result suggests a possible cell-cell interaction influencing cell fate decisions in the pop-1 mutant embryos. We currently are investigating the biological relevance of such an interaction. We are interested in identifying factors that may act with POP-1 to specify cell fates in the daughters and granddaughters of the C blastomere. We have isolated a mutant, zu352, that is defective in specifying the fate of Cp. In zu352 mutant embryos, Cp makes intestine while Ca produces hypodermis and muscle. We are interested in how the cell fates of Ca and Cp in zu352 are specified, and if pop-1(+) plays a role in this Ca/Cp difference.

24 May 1999 15:50 636 636 Characterization of a C. elegans orthologue of the human SMN gene mutated in spinal muscular atrophy

1999 International Worm Meeting abstract 585 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of a C. elegans orthologue of the human SMN gene mutated in spinal muscular atrophy I Miguel-Aliaga 1 , E Culetto 2 , DS Walker 3 , HA Baylis 3 , DB Sattelle 2 , KE Davies 1

1 Department of Human Anatomy and Genetics, University of Oxford, UK. 2 The Babraham Institute, Laboratory of Molecular Signalling, Cambridge, UK. 3 Department of Zoology, University of Cambridge, UK.

Spinal Muscular Atrophy (SMA) is a common autosomal recessive disorder characterized by the loss of lower motor neurons in early stages of development. The aethiology of this genetic disorder is not yet completely understood. The Survival Motor Neuron (SMN) gene is responsible for this disease (1). The SMN protein is ubiquitously expressed and seems to be involved in RNA biogenesis (2). The specific effect on motor neuron degeneration therefore remains to be explained. An orthologue of the Human SMN gene has been identified in C. elegans (CeSMN) on chromosome I (3). The full length cDNA has been isolated using 5’RACE and RT-PCR. Northern blotting identifies a single transcript of about 800 bp, which is present in embryos, during larval development and in adulthood. A full length CeSMN gene::GFP fusion construct is expressed in the nuclei of neurons, body wall and vulva muscle cells, hypodermal cells, gut cells and the excretory canal cell. GFP expression has been also observed in the gonad. The endogenous CeSMN protein has been localized using a polyclonal antibody. The staining in embryos shows that CeSMN is mainly nuclear and is present at very early developmental stages and remains detectable in all blastomeres throughout embryonic development. RNA interference experiment indicate a crucial role for CeSMN in embryonic viability, as its knockdown leads to a significant decrease in the progeny of injected worms. Surviving worms are uncoordinated, amorphous, lack muscle tonicity, have a protruding vulva and are sterile. Preliminary results from yeast two-hybrid experiments suggest similar protein interaction properties to human SMN. These results demonstrate an evolutionary conservation of function of this important protein. We are now looking for a stable SMN knockout mutant, which may allow us to find SMN-interacting genes (supressor mutants) that may contribute to a better understanding of this genetic disorder.

(1) Lefebvre et al. (1995) Cell 80, 155-165. (2) Pellizzoni et al. (1998) Cell 95, 615-624. (3) Talbot et al. (1997) Human Mol. Gen. 6, 497-500.

24 May 1999 15:50 637 637 Observing Basement Membrane Structure and Dynamics in C.elegans with epi-1:GFP

1999 International Worm Meeting abstract 586 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Observing Basement Membrane Structure and Dynamics in C.elegans with epi-1:GFP NB Miliaras, E Hedgecock Department of Biology, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218 The basement membrane is a thin sheet of extracellular proteins that can form a barrier to separate cells of different tissue types and also form a substrate across which cells communicate. For example, at the neuromuscular junction it lies between the axon terminal of the neuron and the muscle, and within the kidney glomerulus it forms a molecular filter between the capillaries and the nephron. In C.elegans, basement membrane surrounds body wall muscle, the gonad, intestine, and the basal surface of the hypodermis. It also plays a critical role during animal morphogenesis; in addition to providing a surface for cells to attach to as they organize into different tissues, it functions as a substrate for cell migration and helps to organize cell polarity through its interactions with cell membrane proteins and the cytoskeleton. Cells attach to the basement membrane through receptors such as the integrins, these interactions may influence cell fate decisions since they can activate signaling pathways that regulate gene expression. Consistent with this diverse role of the basement membrane, the molecular composition and structure of the basement membrane varies among different tissues and within the same tissue at different times during development. The laminins are the major basement membrane proteins that attach to cells. They are large (400-900 kDa) heterotrimers consisting of an a, b, and g chain There are two known laminin heterotrimers in C.elegans each with a different a chain: aA and aB. The aB laminin chain is encoded by the epi-1 gene which has been cloned and sequenced. We have shown that epi-1 is expressed predominantly by muscle cells. In order to observe the dynamic changes in basement membrane structure and composition during C. elegans development, we have created a transgene whereby the aB laminin chain is fused at its C -terminus to a green fluorescent protein (GFP) marker. The epi-1: GFP distribution within the basement membrane will be presented.

24 May 1999 15:50 638 638 Two-color GFP expression in C. elegans

1999 International Worm Meeting abstract 587 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Two-color GFP expression in C. elegans D Miller 1 , N Desai 1 , D Hardin 1 , D Piston 2 , G Patterson 2 , J Fleenor 3 , A Fire 3

1 Dept. of Cell Biology, Vanderbilt Univ.Med. Ctr, Nashville, TN. 2 Dept. of Molecular Physiology.and Biophysics, Vanderbilt Univ.Med. Ctr, Nashville, TN. 3 Carnegie Inst. of Washington, Baltimore, MD.

The Green Fluorescent Protein (GFP) is now widely used for imaging cells and organelles in living animals. GFP variants with distinct excitation and emission spectra have been developed and should prove highly useful for simultaneous imaging of separate proteins labeled with different colored GFPs. Here we describe expression vectors and fluorescence filter sets that can be used to obtain bright, distinct images of "Cyan" GFP (CFP) and "Yellow" GFP (YFP) in C. elegans (1). GFP vectors specifically designed for expression in C. elegans (2) were modified to include amino acid substitutions appropriate for CFP (Y66W, N146I, M153T, V163A) or YFP (S65G, V68A, S72A, T203Y) (see www.ciwemb.edu ). Previously described promoter regions and localization signals were employed to create trangenic animals expressing CFP and YFP either in different cells or in separate intracellular compartments. In these experiments, CFP appears "Blue" and the YFP signal is "Green." For example, an unc-4 promoter element drives expression in VA motor neurons whereas a del-1 upstream region is specific for the adjacent VB motor neurons. Transgenic animals expressing both unc-4::CFP and del-1::YFP display side-by-side VA (Blue) and VB (Green) motor neurons in the ventral nerve cord. In another experiment, cytoplasmically localized YFP and nuclear-localized CFP were expressed under the control of the muscle-specific unc-54 promoter to produce Green bodywall muscle cells with Blue nuclei. It should now be possible to utilize these vectors for a wide variety of two-color labeling experiments. The fluorescence filter sets used in the work were developed in collaboration with Chroma Technology Corp.

For CFP: excitation = 436/10 nm; dichroic = 450 nm; emission = 485/50 nm For YFP: excitation = 500/20 nm; dichroic = 515 nm; emission = 520 nm long pass. (1) Miller, et al. (1999) BioTechniques, in press. (2) Fire, et al. (1998). p. 153-168. In M. Chalfie and S. Kain (Eds.), GFP Strategies and Applications. John Wiley and Sons, NY

24 May 1999 15:50 639 639 GOA-1 (Goa) and RIC-8 interact to Regulate Synaptic Transmission in Adults as well as Centrosome and Nuclear Positioning in Early Embryogenesis

1999 International Worm Meeting abstract 588 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. GOA-1 (Goa) and RIC-8 interact to Regulate Synaptic Transmission in Adults as well as Centrosome and Nuclear Positioning in Early Embryogenesis KG Miller, JB Rand Program in Molecular and Cell Biology Oklahoma Medical Research Foundation Oklahoma City, OK 73104 Molecular and genetic analyses suggest that the GOA-1 (Goa) pathway interacts with the EGL-30 (Gqa) pathway to regulate synaptic transmission.1 To identify other components of this complex regulatory network, we screened for additional genes that confer phenotypes similar to egl-30 (rf) mutants. Mutants in one such gene, ric-8, share many phenotypes with egl-30 (rf), including strong aldicarb resistance, reduced body flexion, and strongly reduced rates of locomotion and egg laying. A genetic analysis suggests that, like EGL-30, RIC-8 also interacts with the GOA-1 pathway, although the precise relationship of RIC-8 to the EGL-30 and GOA-1 pathways is not yet clear. In addition to the neuronal phenotypes of ric-8 mutants, both alleles of ric-8 exhibit an incompletely penetrant embryonic lethality (15% for ric-8 (md1909) and 28% for ric-8 (md303)). Both the neuronal and the embryonic defects disappear in an intragenic revertant of md303. The embryonic lethality of both alleles is enhanced to 100% when the parental genotype is goa-1/+; ric-8/ric-8. This suggests that maternally supplied RIC-8 and GOA-1 function together in early embryogenesis. To identify the function(s) disrupted in these mutants, we observed mutant embryos as they progressed from fertilization through the 8-cell stage. ric-8 mutants showed occasional defects in cleavage plane orientation that were secondary to improper centrosome localization and, in addition, showed delayed nuclear positioning. These defects were strongly enhanced in embryos derived from a goa-1/+; ric-8/ric-8 parent. We conclude that GOA-1 and RIC-8 are involved in the production of a signal that regulates the position of centrosomes and nuclei in the large cells of young embryos. We cloned the ric-8 gene and found that it encodes a novel protein that is related to vertebrate EST sequences of unknown function. Immunostaining revealed that the RIC-8 protein is localized throughout the nervous system and in germ cells.

1 Miller, Emerson, and Rand, 1999. C. elegans meeting abstract.

24 May 1999 15:50 640 640 ETR-1, an elav-type RNA-binding protein with homology to human CUG-bp, is essential for muscle development in C. elegans.

1999 International Worm Meeting abstract 589 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. ETR-1, an elav-type RNA-binding protein with homology to human CUG-bp, is essential for muscle development in C. elegans. CA Milne, J Hodgkin MRC-LMB, Cambridge, UK Post-transcriptional processing of gene products by RNA-binding proteins has been increasingly recognised as an key means of regulating gene expression. A family of RNA-binding proteins with similarity to Drosophila ELAV is one group that has been identified as important for regulating developmental events. Family members have three RNP-type RNA binding motifs, the first two separated from the third by a tether region of approximately 100 amino acids. We previously identified C. elegans ETR-1 in a two-hybrid screen which used FOX-1, an RNP containing protein involved in sex determination, as bait. ETR-1 contains the characteristic arrangement of 3 RNP motifs but has an unusually large, glutamine-rich tether region of 376 amino acids. An etr-1:GFP reporter construct reveals that etr-1 is expressed in muscle precursor cells in the early embryo and in several muscle types including body wall muscle and vulval muscle in the adult. No expression is observed in pharyngeal muscle. RNAi experiments using the unique tether region result in embryonic lethality. Arrested embryos fail to elongate beyond the two-fold stage and have a phenotype similar to that observed for Pat mutant embryos. Pat mutants which have been cloned are known to encode structural components of muscle. Interestingly, the most closely related protein to ETR-1 is human CUG-bp which has been implicated in myotonic muscular dystrophy (DM). CUG-bp is known to bind a CUG-rich region in the intron of chicken TroponinT to regulate alternative splicing and is thought to bind to the CUG expansions in the 3’UTR of DM-protein kinase that are associated with DM. Expression of etr-1 in muscle and the appearance of a Pat phenotype with RNAi suggests that ETR-1 is required for proper muscle development, perhaps by regulating the post-transcriptional processing of one or more of the structural components. The biological relevance of the original physical interaction between ETR-1 and FOX-1 in the two-hybrid screen remains unclear as there is no obvious affect of etr-1 on sex determination. The embryonic expression patterns of the two proteins overlap however and an in vivo interaction cannot be ruled out. Further characterisation of potential targets and the mode of action of C. elegans ETR-1 is underway.

24 May 1999 15:50 641 641 A screen for genes that control programmed cell death in the germ line

1999 International Worm Meeting abstract 590 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A screen for genes that control programmed cell death in the germ line S Milstein 1,2,3 , A Gartner 2 , M Hengartner 2

1 Program in Genetics, SUNY Stony Brook. 2 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724 USA. 3 [email protected]

During somatic development in C. elegans , 131 cells undergo programmed cell death (PCD) in a highly reproducible and stereotyped way. In contrast, PCD in the germ line results in many more deaths, which are not temporally stereotyped. Although death is the fate of about half of the potential gametes, the genes that regulate the decision of these cells to die, and the reasons for their deaths are unknown. In order to characterize the genes involved in the control of PCD in the germ line, we are performing a genetic screen for mutants that show excessive germ cell death. Our screening strategy takes advantage of our observation that in adult hermaphrodites, the vital dye Acridine Orange specifically stains apoptotic germ cells. Because of the large number of cells present in the adult germ line, we can screen for mutants with altered staining patterns using a high power dissecting microscope. To date we have screened over 45,000 genomes and recovered 20 mutants. Strong mutations were mapped to linkage groups, and complementation analysis preformed on those mapping near each other. These mutations fall into seven complementation groups, most of which have a single allele. In all cases tested, germ cell apoptosis was suppressed when placed in a ced-3(lf) background. However, we often observe phenotypes that were not seen in either of the single mutants, such as necrotic germ cell death, or a reduction in the number of oocytes. We are continuing to characterize these mutants to determine if they are directly involved in germ cell death, or if they result in catastrophic damage that activates the PCD pathway.

24 May 1999 15:50 642 642 spf-1 is required for DTC specification and SGP formation during gonadogenesis

1999 International Worm Meeting abstract 591 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. spf-1 is required for DTC specification and SGP formation during gonadogenesis Jennifer Miskowski 1 1 , Yongjing Li 2 2 , Ron Ellis 3 3 , Judith Kimble 1,2 1,2

1 Univ. of Wisconsin-Madison, Madison, WI 53706. 2 HHMI, Madison, WI 53706. 3 Dept. of Biology, Univ. of Michigan, Ann Arbor, MI 48109 USA.

To address how cell proliferation, differentiation, and morphogenesis are coordinated during organogenesis, we are studying gonadogenesis in C. elegans. In wild-type hermaphrodites, 12 somatic gonadal cells are generated by early L2. By L3, ten of these cells coalesce into the gonad center to form the somatic gonadal primordium (SGP), while the two distal tip cells (DTC) remain at the gonad ends. The DTCs and anchor cell (AC) differentiate at this stage, while the remaining cells divide further during L3 and L4 to make adult tissues. The spf-1 gene is defined by three recessive, loss-of-function mutations that map to the right arm of LGI. spf-1 mutants have two dramatic effects on gonadogenesis. First, specification of the DTCs does not occur, as demonstrated by a lack of gonadal arm extension and absence of markers of DTC fate. By lineage analysis of spf-1 gonads, the cells that normally would differentiate as DTCs, Z1.aa and Z4.pp, divide in mid-L2. In wild-type, these cells never divide. Furthermore, the timing of other somatic gonadal cell divisions is aberrant in spf-1 mutants. Therefore, spf-1 is required for the normal specification of DTCs and for the normal pattern of somatic gonadal cell divisions. Second, in spf-1 mutant hermaphrodites, the somatic gonadal cells fail to form the SGP. Instead, they position themselves at the periphery of the gonadal mass and encapsulate the germline cells. Therefore, spf-1 is required for the morphogenetic rearrangement to make the hermaphrodite SGP. (By contrast, the male SGP forms essentially normally, and males can produce cross-progeny.) Despite these early defects, the somatic gonadal cells make differentiated gonadal tissues later, although these tissues are not organized into typical structures. We cloned spf-1 and found it to encode a novel cytoplasmic protein. The spf-1 cDNA detects a single transcript by Northern analysis. A SPF-1::GFP construct is expressed in all somatic gonadal cells just prior to SGP formation and remains on in all somatic gonadal cells that continue to divide until late L4 when they terminally differentiate. The role of spf-1 is being tested further by misexpression, laser ablation and antibody staining.

24 May 1999 15:50 643 643 Members of a divergent nuclear hormone receptor family are expressed in multiple cell types in C. elegans

1999 International Worm Meeting abstract 592 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Members of a divergent nuclear hormone receptor family are expressed in multiple cell types in C. elegans T Miyabayashi 1,2 , MT Palfreyman 1 , F Slack 3 , P Sengupta 1

1 Dept. of Biology, Brandeis University, Waltham, MA 02454. 2 Fundamental Research Laboratory of Life Science, Asahi Chemical Industry, JAPAN. 3 Dept. of Genetics, Harvard Medical School and Dept. of Molecular Biology, MGH, Boston, MA 02114.

The C. elegans genome is predicted to encodes 270 putative nuclear hormone receptors (NHRs). The functions of only a few of these genes have been defined. Interestingly, over 200 of the NHRs are members of a non-phylogenetically conserved class. The function of only one gene in this class, odr-7, has been established. We are interested in investigating the functions of this large and relatively understudied class of divergent NHRs. As odr-7 (odorant response abnormal) plays an important part in the functional specification of an olfactory neuron type (AWA), we are especially interested in the functions of these divergent NHRs in neurons. We have initiated an expression survey using fusions to the reporter gene GFP as a first step towards defining the roles of odr-7 related NHRs. To date we have examined the expression patterns of 28 NHRs unique to nematodes. Five of the examined genes are specifically expressed in neuronal cells while eight others are expressed in a variety of tissue types. In addition, we have found eight genes which are strictly expressed in the lateral hypodermal (seam) cells. These eight genes are also tightly clustered in a neighbor joining tree of NHR DNA-binding sequences, and hence may identify a subgroup of tissue specific NHRs. We also find that three GFP-tagged NHR proteins are localized to the nucleus, while a fourth is localized to the cytoplasm. Preliminary functional analysis using RNAi, dominant negative mutations and VP-16 activation has been undertaken on a subset of the 28 genes. Experiments suggest that the seam cell-specific NHRs play a role in the development of the hypodermis. The genome project is also currently generating deletion in the five neuronally expressed NHRs. Experiments are also underway to define when these NHRs are activated during development. This may allow us to initiate a search for possible ligands. These studies should help elucidate the roles of this divergent class of nematode-specific NHRs.

24 May 1999 15:50 644 644 sdf-13/Ce-tbx-2 mutants have abnormality in odorant-specific adaptation.

1999 International Worm Meeting abstract 593 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. sdf-13/Ce-tbx-2 mutants have abnormality in odorant-specific adaptation. K Miyahara 1 , N Suzuki 1,2 , T Ishihara 1 , I Katsura 1

1 NIG, Yata, Mishima, Shizuoka 411-8540, Japan. 2 present adress: Div. Biol. Sci., Nagoya Univ., Nagoya, Japan.

We have isolated mutants in sdf (synthetic abnormality in dauer formation) genes, which convey a dauer-constitutive phenotype in unc-31 (e169) background (Sdf-c phenotype). At least 13 new genes are definrd by these mutations. Some of these mutants show abnormality also in chemotaxis, defecation, and adaptation. To investigate genes function at a molecular level, we are cloning genes for these mutations. Mutants (ut180, ut192) in one of the sdf mutants, sdf-13, showed a high penetrance of Sdf-c phenotype. sdf-13 maps between dpy-17 and lon-1 on LGIII. We injected cosmid pools from this region in mutant animals and obtaine rescue. Further analysis showed that sdf-13 gene is identical with the Ce-tbx-2 gene which encodes a T-box protein (Agulnik at al.: Genomics 25: 214-219, 1995). SDF-13/CE-TBX-2 is a direct ortholog of Drosophila Omb and mouse TBX2, and is thought to function as a transcriptional factor. Mutations in sdf-13 gene caused defects in adaptation to some AWC-sesed odorants. After prolomged exposure to any one of these odorants, wild-type animals show decreased responsiveness to some of these odorants for several hours (odrant-adaptation), but sdf-13 amimals showed almost normaly chemotaxis even after such treatment. These data suggest that sdf-13/Ce-tbx-2 may play critical role in the control of odorant-adaptation through gene expression.

24 May 1999 15:50 645 645 Conservation of the C. elegans Dpy-20 transcription regulator in Drosophila.

1999 International Worm Meeting abstract 594 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Conservation of the C. elegans Dpy-20 transcription regulator in Drosophila. AM Mohamed, MY Ali, SS Siddiqui Toyohashi University of Technology In C. elegans the dpy-20 gene was previously cloned by David Baillie group. They showed that it encodes a novel non-collagen protein, (Clark et al., 1995). Our group later reported that the dpy-20 overexpresion suppresses transcription of alpha tubulin genes in the ventral cord motor neurons without affecting tubulin expression in other neurons located in the head and tail ganglia (Fukushige and Siddiqui, 1995). Northern blot analysis and dpy-20::lacZ reporter gene expression in transgenic wild type animals showed that dpy-20 is expressed not only in the hypodermal cells but also in a set of ventral cord neurons, and this expression is observed only during larval stages. Subsequent genetic experiments using loss of function dpy-20 alleles and a number of mutants, such as those affected in the ventral cord motor neuron development shows that indeed dpy-20 interacts with these genes. Similarly when dpy-20::lacZ reporter gene was expressed in different mutant backgrounds (such as unc-13, unc-25, unc-30, and unc-119), expression of dpy-20 is clearly affected. These observations has lead us to propose that DPY-20 is a novel transcription regulator specific to motor neurons. Significant homology exists between the LIN-39 homeotic gene and the DPY-20 primary sequence. We have therefore sought to uncover dpy-20 orthologs in Drosophila and mouse, and have isolated two independent genomic clones from a Drsophila genomic library in EMBL vector (kindly provided by Bob Holmgren). In situ hybridization of these clones show that one of them is distributed at two different locations in the fly genome, whereas the other clone hybridizes to a unique site. We are also screeningDrosophila cDNA libraries made from larval and embryonic stages to identify fly cDNAs corresponding to the dpy-20 gene. In a parallel experiment we have identified that mouse genome also contains dpy-20 hybridizing sequences, which show a single band on Southern blot. We hope to identify the dpy-20 homolog from Drosophila and study its role in motor neuron development. Sydney Brenner kindly encouraged the idea of searching for the dpy-20 homologs, for which we remain grateful.

We thank D. Baillie, D. Janke, H. Kose, Y. Hotta, Y. Kohara, R. Holmgren, Y. Yoshifumi, Y. Nishida, T. Matsuo, and Y. Ohshima for support.

24 May 1999 15:50 646 646 Hypodermal cell fusions are not required for gross morphogenesis in C. elegans

1999 International Worm Meeting abstract 595 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Hypodermal cell fusions are not required for gross morphogenesis in C. elegans WA Mohler, JG White University of Wisconsin - Madison The onset of elongation of the C. elegans embryo coincides with a dramatic series of stereotyped cell fusions giving rise to syncytia within the hypodermis. As circumferential cytoskeletal contraction within the hypodermis has been proposed to drive elongation, cell fusion might be expected to contribute to the process of elongation by somehow enabling more efficient contraction or by enhancing the strength of the integral hypodermal epithelium. However, a recently discovered mutation indicates that morphogenesis does not depend upon formation of these syncytia. In a screen for mutations affecting hypodermal cell fusion, we have employed a live "clonal" plating approach to assess phenotype by high-power epifluorescence. Using a JAM1(MH27)::GFP transgene in the parental strain, F2 progeny of mutagenized worms are examined for residual adherens junctions between cells ordinarily fused during embryogenesis. We have isolated a recessive allele, oj55, which yields viable homozygous mutants with unfused hypodermal cells. In some individuals, embryos undergo morphogenesis and hatching without any of the normal cell fusions between precursors of the large hypodermal syncytia hyp6 and hyp7. In later larval stages a multi-rowed lateral seam forms, presumably because of failed fusions of H, V, and T descendents into hyp7. The oj55 allele can be propagated in a homozygous strain, although experiments are underway to determine whether severely affected individuals are fertile, and to assess effects on cell fusions in other syncytial tissues, such as the vulva and uterus. The discovery of oj55 clearly establishes that the elongation that makes C. elegans a worm does not require formation of hypodermal syncytia. Furthermore, the apparently normal function of hypodermal cells in migration, morphogenesis and cuticle formation suggests that the gene affected by the oj55 mutation plays a specific role in the process of cell membrane fusion.

24 May 1999 15:50 647 647 Genetic analysis of cultivation temperature avoidance induced by starvation

1999 International Worm Meeting abstract 596 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic analysis of cultivation temperature avoidance induced by starvation A Mohri, M Koike, I Mori Division of Biological Science, Nagoya University, Nagoya. Thermotaxis is an important behavior that C. elegansuses to ensure its survival and reproduction in the natural habitat. After cultivation with food for several hours, C. elegansmigrates toward its cultivation temperature and then moves isothermally on a temperature gradient. This positive thermotaxis turns into negative thermotaxis after cultivation under conditions that involve starvation and/or overcrowding (1). How does C. elegansassociate memorized cultivation temperatures with environmental conditions? We demonstrated that starvation alone can induce negative thermotaxis. The animals were cultivated with food or without food at 25- or 15-degree for 3 hours, and then their thermotaxis behaviors were examined in either single animal assay or population assay. Most of the well-fed animals migrated to the cultivation temperatures, whereas most of the starved animals showed several migration patterns, all of which are indicative of cultivation temperature avoidance behavior. As already reported for other behaviors such as pharyngeal pumping and egg laying, we also obtained the evidence to suggest that exogenous serotonin and octopamine mimic well-fed and starved state, respectively, in thermotaxis. For example, most animals that had been placed on unseeded plates containing serotonin at 25-degree for 3 hours migrated to 25-degree on a temperature gradient, and most animals that had been placed on seeded plates containing octopamine at 25-degree for 3 hours migrated away from 25-degree. To identify genes and cells required for associating temperature memory with the feeding state, we are attempting to isolate mutants that can respond to feeding states normally, but are defective in the reversal of thermotaxis behavior. We so far screened 1,500 genomes for animals that constitutively migrate to cultivation temperatures regardless of the starvation experience. We so far obtained one mutant, designated aho-1for abnormal hunger orientation, which exhibits the aimed behavioral abnormality. The genetic, behavioral and pharmacological analysis of the aho-1mutant is in progress. We are also continuing to isolate more ahomutants, in order to dissect memory and learning as well as neuroendocrine system in C. elegans.

(1) Hedgecock and Russell (1975), PNAS 72, 4061-4065.

24 May 1999 15:50 648 648 Identification of upstream regulatory elements and transcription factors responsible for cell-specific expression of the C. elegans metallothionein genes

1999 International Worm Meeting abstract 597 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification of upstream regulatory elements and transcription factors responsible for cell-specific expression of the C. elegans metallothionein genes LH Moilanen 1 , T Fukushige 2 , JH Freedman 1

1 Nicholas School of the Environment, Duke University, Durham, NC. 2 Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada.

Metallothioneins (MT) are small, cysteine-rich proteins that function in metal detoxification and homeostasis. Although virtually every eukaryotic organism expresses MT or MT-like proteins, cell and tissue-specific patterns of transcription are observed. The nematode C. elegans provides an excellent system for the investigation of mechanisms that control cell-specific MT transcription in vivo. The inducible expression of the C. elegans MT genes, mtl-1 and mtl-2, occurs exclusively in intestinal cells. Sequence comparisons of the MT genes with other C. elegans intestinal cell-specific genes identified multiple repeats of GATA-transcription factor-binding sites (i.e., GATA-elements). In vivo deletion and site-directed mutation analyses confirm that one GATA-element in mtl-1 and two in mtl-2 are required for transcription. Electrophoretic mobility shift assays show that the C. elegans GATA-transcription factor ELT-2 specifically binds to these elements. Ectopic expression of ELT-2 in non-intestinal cells of C. elegans activates mtl-2 transcription in these cells. Likewise, mtl-2 is not expressed in nematodes in which elt-2 has been disrupted. These results indicate that cell-specific transcription of the C. elegans MT genes is regulated by the binding of ELT-2 to GATA-elements in these promoters. The evolutionary conserved nature of GATA-elements and transcription factors suggests that similar regulatory processes may be present in higher eukaryotes.

24 May 1999 15:50 649 649 Comparison of gene expression in C. elegans and C. briggsae

1999 International Worm Meeting abstract 598 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Comparison of gene expression in C. elegans and C. briggsae LF Molin, IA Hope School of Biology, Leeds University, Leeds LS2 9JT Previously a collection of maternal effect embryonic lethal mutations was screened for their effects on the expression pattern of a pes-1::lacZ fusion gene. Ectopic expression was observed in mutants for the gene emb-46 and, because of the nature of the alteration to the expression pattern, this gene was selected for further study. Using the markers available in the region (+7.2 mu on chromosome II), location of emb-46 was restricted to a region covered by 2 YACs and 8 overlapping cosmids but phenotypic rescue experiments to locate the gene have been unsuccessful so far. Furthermore, using the cosmids covering the region as probes, no RFLPs were detected for any of the seven mutant alleles isolated for this gene. A new project has been started to compare sequence and gene expression pattern data between C. elegans and C. briggsae to identify the location and function of gene regulatory regions. A pilot study was started with 12 genes for which a reproducible and specific expression pattern was described in C. elegans. Plasmids carrying C. elegans gene promoter regions fused to the lacZ reporter gene were injected into C. briggsae germline with the C. elegans rol-6 marker and transmitting lines were established. Expression patterns were found to be similar in both species for ten genes. For the other two genes, F54D5.1 and pes-1, expression in C. briggsae only partially matches the expression observed in C. elegans. No C. briggsae homologue has been sequenced so far for these two genes. The pes-1::lacZ reporter gene is strongly expressed in C. briggsae, as in C. elegans, in the D lineage. In C. briggsae, weak staining is observed in a cluster of a few cells anteriorly that could define a restricted subset of AB descendant cells as compared to the pattern observed in C. elegans. No staining is observed in C. briggsae in the cells Z1 and Z4. In order to isolate the C. briggsae homologue of pes-1, a C. elegans pes-1 cDNA probe has been used to screen a C. briggsae DNA library. Several clones have been identified and are currently been analysed by degenerate PCR and Southern blot experiments.

24 May 1999 15:50 650 650 Molecular and functional diversity of the nicotinic acetylcholine receptor gene family of C. elegans

1999 International Worm Meeting abstract 599 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Molecular and functional diversity of the nicotinic acetylcholine receptor gene family of C. elegans NP Mongan, E Culetto, N Gower, V Raymond, DB Sattelle The Babraham Institute Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, UK. Several strategies have been combined to study nicotinic acetylcholine receptors (nAChR) in C. elegans. Genetic screens have isolated levamisole resistant mutants leading to the molecular cloning of 4 nAChR subunit genes unc-29, lev-1, unc-38 and unc-63. The obtention of resistant mutant to the anticholinesterasic drug aldicarb defined ric genes and one of them, ric-3 is a good gene nAChR candidate. Also the isolation of mutant worms with pharyngeal pumping defects lead to the identification of eat-2 and eat-18 mutants which are also putative nAChR. Searching the C. elegans genome consortium database we have characterized at the molecular level novel nAChR subunits. 23 cDNA subunits have been partially cloned and sequenced (acr genes). We estimate the final number to be in the range of 30-40. By homology the subunits fall into five different classes, three of which have no vertebrate counterpart. The genomic organization of the subunits will be presented. Our major current goal is to determine the expression pattern of the known nAChRs in order to understand the combination of subunits which forms native receptors. To address this problem we have used quantitative PCR on staged cDNA, GFP tagging and in situ hybridization. Preliminary results using quantitative RT-PCR have shown that acr-16 is expressed at the young adult stage, whereas unc-38 is expressed from L1 to adult stages. This work should lead to the construction of an in vitro expression library of recombinant nAChRs allowing screening of new cholinergic anthelmintic drugs and a more extensive characterisation of those drugs for wich activity on nAChR has previously been shown. We have undertaken such study on expressed recombinant acr-16 and shows that oxantel was an agonist on the vertebrate nAChR but an antagonist on ACR-16. Finally the combination of in vitro expression results, cellular localization and the use of dsRNAi will provide more insight into the physiological role of each member of this multi-gene family.

24 May 1999 15:50 651 651 Genetic study of Gas-coupled signal transduction in C. elegans

1999 International Worm Meeting abstract 600 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Genetic study of G as -coupled signal transduction in C. elegans C Moorman, HC Korswagen, AM van der Linden, RH Plasterk Department of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands To get more insight in signaling via the heterotrimeric G protein alpha subunit Gas in C. elegans, a constitutively active Gas mutant was made (Korswagen et al, 1997). Expression of this constitutively active form of Gas from an inducible promoter resulted in hypercontraction of body-wall muscle cells and in induction of neuronal degeneration. Using mutagenesis, extragenic suppressors that inhibit the neuronal degeneration were identified. Until now, these suppressors fell into two complementation groups, sgs-1 and sgs-2 (suppressor of activated Gs). 45 new suppressors are now being classified in complementation groups. sgs-1 was cloned, and encodes an adenylyl cyclase, that is most similar to mammalian adenylyl cyclase type IX. An sgs-1::gfp reporter construct shows a general neuronal expression pattern, demonstrating that sgs-1 is expressed in the neurons that are susceptible to activated Gas-induced degeneration. The mutations in sgs-1 that suppress the neuronal degeneration change conserved residues in the two catalytic domains or in the first transmembrane domain, suggesting that these mutations do not represent complete loss-of-function alleles of sgs-1. A library of chemically-induced deletion mutants will be screened to isolate an sgs-1 null allele. The C. elegans sequence database contains a second predicted adenylyl cyclase, acy-2. An acy-2::gfp reporter construct is expressed in the canal-associated neurons (CAN cells) (Korswagen et al, 1998). acy-2 null mutant animals have a phenotype that is reminiscent of clr-1 mutant animals, suggesting that acy-2 null mutants have a defect in the CAN cells. Gas null mutant animals also have this clr-1 like appearance. Taken together, these results suggest that Gas and ACY-2 perform an essential function in the CAN cells. sgs-2 was mapped to chromosome V, in an interval between him-5 and unc-76. Rescue with cosmids from this region is ongoing.

24 May 1999 15:50 652 652 The C. elegans Spectrin Based Membrane Skeleton

1999 International Worm Meeting abstract 601 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The C. elegans Spectrin Based Membrane Skeleton S. Moorthy, L. Chen, V. Bennett HHMI & Depts of Biochemistry & Cell Biology , Duke Univ , NC 27710 USA The spectrin based membrane cytoskeleton was first characterized in red cells where it forms a visualizable polygonal network on the cytoplasmic surface of cell membranes. Its principle component is the heteromeric protein spectrin, made up of alpha & beta subunits. It provides mechanical support & flexibility to the membrane, enabling survival of red cells in circulation. Proteins related to spectrin are expressed in almost all metazoan cells. We identified a C. elegans homolog of betaG spectrin on LGV (wm97e421). Injection of its ssRNA into syncytial gonads of hermaphrodites resulted in a variety of phenotypes in progeny, from arrested larvae to dpy unc adults, but near normal protein levels. However using dsRNA mediated interference (RNAi) we almost completely eliminate the protein and then 90% of progeny arrest as uncoordinated L1 larvae with neuronal defects. Hammarlund et al previously reported unc-70 alleles with similar phenotypes and proposed that unc-70 was a regulator of synaptic transmission (wcwm98p129). They now report that unc-70 is the C. elegans betaG spectrin and we concur. Our own results show that UNC70 is highly expressed in synapse rich structures of the nervous system, though it expresses throughout development and associates with membranes of most cell types. Hence unc-70 probably has multiple roles during development. C. elegans betaH sma-1 and alpha imo-1 spectrins have been previously identified (cgc3094, wcwm98p30). SMA1 and UNC70 have mutually exclusive subcellular localizations in gut and hypodermal cells (J. Austin pers. comm., our data). Using RNAi we found sma-1(RNAi) worms are viable and small with head morphogenetic defects; unc-70(RNAi) worms arrest as early L1 larvae of normal morphology; imo-1(RNAi) worms arrest as early L1 larvae that are shorter with head morphogenetic defects; and finally sma-1(RNAi);unc-70(RNAi) double mutant animals are morphologically indistinguishable from imo-1(RNAi) progeny, even though the latter have normal levels of UNC70 protein. These data suggest SMA1 and UNC70 play complementary roles in development and IMO1 is required for each alpha-beta spectrin complex.

24 May 1999 15:50 653 653 Identification of cis- and trans- acting factors controlling the translation of maternal pal-1 mRNA

1999 International Worm Meeting abstract 602 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification of cis- and trans- acting factors controlling the translation of maternal pal-1 mRNA DE Mootz, CH Hunter Dept. of Molecular and Cellular Biology Harvard University, Cambridge, MA 02138 Proper patterning of the C.elegans embryo relies on the posterior localization of PAL-1. Two observations suggest this posterior localization is due to the regulated translation of maternal pal-1 mRNA. First, pal-1 mRNA is present in oocytes and all cells of the early embryo, whereas PAL-1 protein is not detected until the 4-cell stage. Second, the pal-1 3’ UTR can restrict the translation of a reporter RNA (lacZ::pal-1 3’ UTR) to posterior blastomeres. We are interested in characterizing the molecular mechanisms underlying the spatial and temporal control of pal-1 translation. Towards this end, we are trying to identify the translational control elements in the pal-1 message, as well as the factors that bind these elements. To identify control elements, we are performing deletion analysis of a lacZ reporter RNA bearing the pal-1 5’ and 3’ UTRs. To complement this analysis, we are looking for regions of pal-1 5’ and 3’ UTRs that are evolutionarily conserved. Thus far, we have cloned the pal-1 3’ UTR in C.remanei and C.briggsae, and have found a 36 nucleotide element that is highly conserved between all three species. We are testing the functional significance of this element using the lacZ reporter RNA assay, and we intend to use this element as bait in genetic and biochemical schemes to identify proteins that specifically interact with pal-1 mRNA. To date, our efforts to identify translational regulators that bind pal-1 mRNA have focused on MEX-3. mex-3 encodes a putative RNA binding protein containing two KH domains (Draper et al., 1996), and genetic studies have suggested that it functions through the pal-1 3’ UTR to repress pal-1translation. In order to determine whether MEX-3 is a direct regulator of pal-1 translation, we are testing whether MEX-3 specifically associates with pal-1 mRNA, either alone, or in a complex with other proteins. We have shown that recombinant MEX-3 is capable of binding the pal-1 3’UTR in vitro, and we are testing the specificity of this interaction. In addition, we are performing both in vitro and in vivo experiments to test for the presence of MEX-3 in a pal-1 mRNA binding complex.

Draper, B.W., C.C.Mello, B.Bowerman, J.Hardin, and J.R.Priess (1996). Cell 87: 205-216.

24 May 1999 15:50 654 654 Search of in vivo targets of Hox gene products

1999 International Worm Meeting abstract 603 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Search of in vivo targets of Hox gene products T Morimitsu, K Murayama, R Matsui, N Takahashi Nara Institute of Science and Technology, Takayama, Ikoma, Nara 630-0101, Japan The Hox genes encode homeodomain transcription factors that determine cell fates during development, but the Hox gene targets are poorly understood. To identify Hox gene targets we developed a procedure. The procedure consists of two steps; isolation of the DNA sequences bound by Hox protein in native chromatin, and identification of the gene surrounding the sequence bound by Hox protein. We isolated several candidate targets of mouse Hox gene products by this procedure(1). Since C. elegans has the advantages(e.g. small genome size and completion of genome sequencing) for this procedure. We are currentry trying to isolate in vivo binding sequences of C. elegans Hox gene products(MAB-5 and EGL-5) by affinity purification of tagged (FLAG epitope and (His)6)protein-DNA complex. We injected tagged Hox(mab-5 and egl-5) genes into mutant animals. These genes rescued mutant mail tail phenotype as well as wild type genes. These results suggest that tagged Hox gene products recognize target sequences similar to that of wild type gene products. A preliminary analysis of in vivo bindig sequences of Hox gene products will be presented.

(1) Tomotsune, T. et al., (1993) Nature 365, 69-72

24 May 1999 15:50 655 655 CeBRAM-1A and CeBRAM-2B, new signal mediators of TGF-b signaling pathways in the C. elegans

1999 International Worm Meeting abstract 604 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. CeBRAM-1A and CeBRAM-2B, new signal mediators of TGF-b signaling pathways in the C. elegans K Morita, M Shimizu, S Yoshida, M Mochii, H Shibuya, N Ueno Division of Morphogenesis, Department of Developmental Biology, National Institute for Basic Biology,Okazaki 444-8585, JAPAN TGF-b family of growth factors regulate many aspects of cellular function. The signaling is common in diverse animal species from vertebrates to C. elegans. We have been interested in the signaling pathway of the TGF-b family. We reported about cloning of a full-length cDNA (0.9 kb) of CeBRAM-1A (We previously called CeBRAM-2 ), a DAF-1 binding protein (WBG 15 (3): 38). HA-tagged CeBRAM-1A transiently expressed in COS7 cells was found to bind type I receptors, both ALK-3 (mammalian) and DAF-1 (C. elegans). CeBRAM1A;;GFP fusion protein was found to be expressed dominantly in amphid and pasmid neurons (e.g. ASI, ASK, ASJ, etc.). Subsequently, the null mutant was created by Tc1 deletion method. The CeBRAM-1A null mutant had the genetic interaction with mutants in daf-7 TGF-b pathway by examining dauer formation of the double mutants. These results suggest that CeBRAM-1A is involved in the daf-7 TGF-b pathway in C elegans as a negative regulator. We have now identified another human BRAM-1 homologue, CeBRAM-2B, in C. elegans. CeBRAM-1A and CeBRAM-2B have 66% amino acid identity and are conserved extremely in the carboxy-terminal region which seems to be necessary for binding to the type I receptor. Interestingly, double strand RNA interference (dsRNAi) of CeBRAM-2B showed Lon phenotype. These results indicate the possibility that CeBRAM-2B is involved in the cet-1/dbl-1/sma pathway of another TGF-b signaling. We are currently trying to identify the CeBRAM-2B null mutant by TMP-UV method.

24 May 1999 15:50 656 656 The Stressed Worm Project

1999 International Worm Meeting abstract 605 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Stressed Worm Project James F Morley, Sanjeev Satyal, Josh Segal, Richard I. Morimoto Dept of Biochemistry Molecular and Cell Biology, Northwestern University, Evanston, IL Genes encoding molecular chaperones are activated in response to a variety of stresses and developmental events. The study of chaperones and co-chaperones has traditionally focused on experiments in bacteria, yeast and mammalian tissue culture cells. We have begun to investigate the regulation of the stress response and the function of molecular chaperones in the nematode, C. elegans. These studies will help us to understand the relationship of the stress response to processes such as cell death and development. We have performed a molecular evolutionary analysis of chaperone genes by reciprocal BLAST searches of the C. elegans and S. cerevisiae genomes, focusing on the HSP40, HSP70, and HSP90 families. Dendrogram analysis of predicted protein sequences reveals: a) 13 HSP70s, consisting of orthologs of the soluble cytoplasmic SSAs, two orthologs of the ER KAR2/BiP, and orthologs of mitochondrial HSP70 b) a single ortholog of the co-chaperone BAG-1 c) three HSP90s, comprised of one direct ortholog of the cytoplasmic HSC82 and HSP82 chaperones, an ortholog of the ER protein GRP94, and a ~70kD protein which is not directly related to any yeast HSP90 d) seven HSP40 orthologs. This analysis has served as a basis for our rationale in selecting genes for deletion. The transcriptional regulation of chaperone genes involves an interplay between a heat shock transcription factor and various positive and negative regulators. We have generated a deletion mutation in heat shock factor binding protein (hsb-1), a negative regulator of the heat shock response. A deletion in hsb-1 gives rise to no gross morphological abnormalities, however, hsb-1 mutant animals have a significantly shorter lifespan than N2 animals. To study the in vivo regulation of chaperone activity, we have generated a deletion mutation in the co-chaperone bag-1. BAG-1 is a negative regulator of Hsp70 and interacts with important signaling molecules including BCL-2, RAF-1, and steroid receptors. Transgenic strains expressing a bag-1 promoter::LacZ/GFP fusion exhibit X-gal staining in the intestine, head and tail neurons. Deletion of bag-1 does not affect timing of developmental stages, embryonic and larval viability, or lifespan. However, bag-1 mutant animals exhibit a significant increase in brood size compared to the N2 strain.

24 May 1999 15:50 657 657 Quantitative Complementation Testing Reveals Variation in Chemotaxis Caused by the odr-1 Locus in Natural Isolates of C. elegans.

1999 International Worm Meeting abstract 606 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Quantitative Complementation Testing Reveals Variation in Chemotaxis Caused by the odr-1 Locus in Natural Isolates of C. elegans. JJ Morphew 1 , PC Phillips 1,2

1 Department of Biology, University of Texas at Arlington, Arlington, Tx 76019 USA. 2 [email protected]

Chemotactic behavior toward the volatile odorant, benzaldehyde, has been characterized in a number of different isolates of C. elegans . One of the genes underlying this behavior, odr-1 , has previously been characterized as a guanalyl cyclase present in the G-protein regulated pathway of the response (Troemel and Bargmann, 1997 WM Abstract 41). Odr-1 mutants, deficient in their response to benzaldehyde, were crossed with different isolates to generate F1 individuals. The heterozygotes generated from an odr-1 male crossed with a wild type hermaphrodite (RC301, KR314, DH424, CB4855, CB4856, CB4932, BO and AB3) were scored across concentrations. Testing the various wild type alleles against the mutant can be used to reveal variation either in the allele itself or with other interacting loci. This quantitative complementation test revealed variation due to the presence of the mutant allele at both attractive and repulsive concentrations. While quantifying this effective variation, it was also noted that the expression patterns of the F1 heterozygotes varied when the parental origin of the mutant allele was reversed, suggesting a maternal component to this regulated response.

24 May 1999 15:50 658 658 Time-series analysis of pirouettes in C. elegans chemotaxis.

1999 International Worm Meeting abstract 607 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Time-series analysis of pirouettes in C. elegans chemotaxis. TM Morse 1 , JT Pierce 1 , SE Owens 1 , ML Moravec 1 , AB Bates 1 , TC Ferree 2 , SR Lockery 1

1 Institute of Neuroscience, University of Oregon, 97403-1254. 2 EGI, 1900 Millrace Dr., Eugene, OR 97403.

Chemotaxis in C. elegans involves a series of abrupt turns (pirouettes) triggered by movement down a gradient of chemical attractant* . Analysis of the time series of concentration change experienced by a worm, together with its pirouette record, suggests a three-stage chemotaxis mechanism in which chemical concentration (C(t)) is differentiated (dC(t)/dt), smoothed (q(t)), and converted into pirouette probability by a nonlinear function (P(q[t])). To test the plausibility of this mechanism, we constructed a computer model in which the smoothing filter and the nonlinearity were estimated from the time series of dC(t)/dt and the pirouette records of real worms. Pirouettes were modeled by sampling randomly, via a Poisson process with probability P(q[t]), from the distribution of direction changes associated with pirouettes in real worms. The average chemotaxis index (time-average of normalized C(t)) of model worms (0.47 ± 0.05 SD, n = 2000) closely matched the average chemotaxis index of real worms (0.45 ± 0.04 SD, n = 45), indicating that the three-stage mechanism is quantitatively sufficient to account for C. elegans chemotaxis. To determine the form of the smoothing filter and nonlinearity directly, we have devised a new behavioral assay that subjects unrestrained worms to negative-going impulses in dC/dt as they swim across a sharp border between high and low concentrations of attractant. Preliminary results show that immediately after a border crossing, large impulses make P(t)~= 1, while small impulses make 0 t) ><<1, as predicted by the three-stage chemotaxis mechanism. The model provides a well-defined theoretical framework for the neuronal analysis of chemotaxis in >C. elegans.

* Pierce, J.T., and Lockery, S.R., J. Neurosci. (Submitted)

24 May 1999 15:50 659 659 The heterochronic gene lin-46 encodes a non-essential member of the moeA family and may be a target of lin-28 regulation

1999 International Worm Meeting abstract 608 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The heterochronic gene lin-46 encodes a non-essential member of the moeA family and may be a target of lin-28 regulation EG Moss 1,2 , RL Lee 2 , JE Johnstone 1 , D Au Yeung 2 , V Ambros 2

1 Fox Chase Cancer Center, Philadelphia, PA 19111. 2 Dept. of Biology, Dartmouth College, Hanover, NH 03755.

The heterochronic genes lin-14 and lin-28 coordinate the timing of developmental events in early larval stages. If either activity is reduced then precocious expression of L3-specific events occurs in the L2. Because these genes act in diverse cell types to control a variety of cell division and differentiation events, they may act on diverse targets or on just a few that in turn have diverse affects. Mutations in lin-46 completely suppress lin-28 loss-of-function alleles and lin-14 hypomorphs. To our surprise, null mutations in lin-46 suppress null mutations in lin-28 to the wildtype phenotype. This immediately raises two questions: Might lin-46 be one of several targets of lin-28? and What can lin-46 tell us about the relationship between lin-14 and lin-28? Several experiments have delivered more questions than answers. By GFP fusion assay, lin-46 appears to be expressed exclusively in two bilaterally symmetric AV interneurons and this expression is not temporally regulated. This is in contrast to lin-14 and lin-28, which are expressed ubiquitously in the L1 and then repressed. A precocious phenotype can result from lin-46 trangenes in a wildtype background, indicating that the level of lin-46 is important in heterochronic regulation. lin-46 mutations can suppress all alleles of lin-28, hypomorphs of lin-14, but not null alleles of lin-14. Mutant combinations suggest that lin-46 does not act by raising the level of an activity upstream of lin-14 or lin-28. Curiously, a lin-46 mutation appears unable to suppress the double mutant lin-42; lin-14(ts), two mutations it can suppress separately, suggesting there is an important relationship among these genes. lin-46 encodes a member of the moeA family, which includes vertebrate gephyrin and a molybdopterin synthesis pathway component. These homologies suggest nothing more than the LIN-46 protein may interact with other proteins to carry out its function. Perhaps LIN-46 is a component of a multi-protein complex, whose components are targets of lin-28 negative regulation, so that a null mutation in lin-46 can reduce the output of the pathway without eliminating it.

24 May 1999 15:50 660 660 Systematic analysis of mRNA distribution by whole mount in situ hybridization

1999 International Worm Meeting abstract 609 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Systematic analysis of mRNA distribution by whole mount in situ hybridization T Motohashi, T Ohba, I Sugiura, M Obara, S Kitayama, T Suzuki, T Shin-i, Y Kohara CREST, JST and Genome Biology Lab., National Institute of Genetics, Mishima 411-8540, Japan In this lab, C.elegans EST project has been performed, which has identified 10,955 cDNA species based on the comparison of tag sequences of some 65,000 clones. The distribution of the cDNA species among the six chromosomes is quite even; LG1: 1856, LG2:1855, LG3:1670, LG4:1645, LG5:1713, LGX:1424 (see Shin-i et al. and Thierry-Mieg et al.). To clarify their expression patterns, we have established a efficient protocol for in situ hybridization on whole mount embryos, larvae and adults (http://watson.genes.nig.ac.jp.8080/db/method), and have applied the procedure to the classified cDNA groups. Thus far, we have done the in situ analysis for 3500 cDNA groups mostly from an autosome LG3 and the sex chromosome LGX; LG1:6, LG2:522, LG3:1448, LG4:190, LG5:49 and LGX:1281. Analysis of the remaining 7000 cDNA groups is also in progress and will be finished within a year. Although the experimental part of the in situ hybridization is very efficient, analysis of the results including taking photos and giving annotation is very time consuming process. Technical information on our in situ methods and statistics of the results of the expression patterns will be presented. All the data will become available at (http://watson.genes.nig.ac.jp:8080/db/).

24 May 1999 15:50 661 661 The Unexpected Phenotype of Alpha-actinin Nulls

1999 International Worm Meeting abstract 610 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Unexpected Phenotype of Alpha-actinin Nulls Gary Moulder, Janet Duerr, Steve Fields, Robert Barstead Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma The actin binding protein alpha-actinin is a prominent component of the Z-disks of vertebrate striated muscle. In other cell types alpha-actinin is found at adherens junctions. The molecule can be divided into three domains: an N-terminal actin binding domain, four internal 120 residue repeats, and a C-terminal domain with EF hand Ca++ binding motifs. Both the actin-binding domain and the repeat sequences are homologous to those found in beta-spectrin and dystrophin. alpha-actinin is purified as a homodimer in which the repeat sequences pack in an antiparallel orientation. In C. elegans alpha-actinin is found at adherens junctions in the gut and at the dense bodies in the body wall muscle. Other proteins found with alpha-actinin in dense bodies include vinculin and integrin. The dense bodies, therefore, have the properties of a focal adhesion, a type of adherens junction found in cultured vertebrate cells. Focal adhesions serve to link the actin cytoskeleton to the extracellular matrix. Mutations that eliminate vinculin or integrin in C. elegans cause a Pat phenotype; such mutants are paralyzed as embryos and arrest elongation at the two-fold stage. Based on the known biochemical interactions of these proteins in vertebrate models, we expected that alpha-actinin would be the major actin binding protein in the dense body and, therefore, that it would be as critical to the function of the dense body as vinculin and integrin. We were surprised to discover that mutations eliminating alpha-actinin did not lead to a Pat phenotype. Such mutants have nearly normal looking muscle as assayed by polarized light microscopy, nearly normal dense body arrays as assayed by immunofluorescence microscopy using antibodies to integrin, talin and vinculin, and normal motility. The mutants, however, show abnormal accumulations of actin at the ends of the muscle cells and, as assayed by electron microscopy, have dense bodies that are shorter and broader at the base. We conclude that other dense body proteins can substitute for the actin binding function of alpha-actinin at the dense body.

24 May 1999 15:50 662 662 Expression Pattern Data For Genes Predicted In The C. elegans Genome.

1999 International Worm Meeting abstract 611 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Expression Pattern Data For Genes Predicted In The C. elegans Genome. A Mounsey, P Bauer, D McCarroll, IA Hope School of Biology, University of Leeds, Leeds, LS2 9JT, UK. Fundamental to an understanding of the C. elegans genome are expression patterns for the thousands of predicted genes. To this end, we have systematically subcloned promoter regions of predicted genes into lacZ reporter plasmids. At present 250 predicted genes have been analysed using reporter gene fusions, with 47% directing ß-galactosidase expression to observable levels. There is a slight positive correlation with EST data. 56% of reporter gene fusions showing expression have ESTs, compared to 45% for reporter gene fusions with no expression. For predicted genes showing expression, 35% express in pre-comma-stage embryos and 75% in adult worms, with expression in other life stages seen in 60-65% of instances. Expression in neurons is observed for 33% of predicted genes showing expression, in hypodermis for 28%, and in the pharynx for 24%. The ultimate aim of our generating expression pattern data, is to provide part of the vehicle through which we will understand how the genome, via the developmental programme, generates the worm. However, to do this we require many more gene expression patterns. The completion of the genome sequencing project has allowed us to embark on a new phase of expression pattern data generation that should generate hundreds (or optimistically thousands) of expression patterns. Libraries of 5-7kb random genomic DNA fragments, inserted into a modified pPD lacZ reporter plasmid, have been constructed. We intend to construct similar libraries in GFP vectors, so that microinjection can be made more efficient through simultaneous injection of multiple gene fusions. Sequencing of individual clones (thanks to Amanda McMurray at the Sanger Centre) determines if a gene fusion has been formed. Our reporter plasmid modification allows trivial correction of out-of-frame fusions. Using this method we aim to analyse 15-20 genes per week, and generate 700-1000 new expression patterns over the next two years. Expression pattern data are made publicly available in ACeDB (thanks to the efforts of Sylvia Martinelli and Erik Sonnhammer) and via the World Wide Web (http:// www.personal.leeds.ac.uk/~acedb/Hope/epa.htm). The aim is to develop a database of expression pattern information of use to the entire C. elegans community.

24 May 1999 15:50 663 663 Isolating genes asociated with longevity

1999 International Worm Meeting abstract 612 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Isolating genes asociated with longevity MJ Munoz, DL Riddle University of Missouri, Columbia MO 65211 Dauer larvae can live for as long as six months, whereas the life span of animals that develop continuosly to the adult is only about two weeks. Temperature-sensitive daf-2 mutants enter the dauer stage constitutively at 25ºC, and when grown at permissive temperature their adult life span is doubled (Kenyon et al. 1993). Mutations in daf-16, encoding a forkhead transcription factor (Ogg et al. 1997, Lin et al. 1997), suppress the increased daf-2 longevity. Thus, expression of some of the genes that are targets of DAF-16 activation may be responsible for increased longevity. We are using the differential display PCR method (Liang and Pardee 1997) to look for differences in gene expression between a daf-2 mutant (long life span) and a daf-2; daf-16 double mutant (wild-type life span). We have identified two differentially expressed genes that have a higher level of expression in the daf-2 mutants. The sequence of these clones predicted two different proteins with unknown function. We have also looked for differences in gene expression between a daf-2; daf-12 double mutant (four times wild-type life span) and a daf-2; daf-12; daf-16 triple mutant (wild-type life span) (Larsen et al. 1995). In this case we have found eleven differentially expressed genes. Seven of them are expressed at higher levels in the triple mutant. We have sequenced two of these and both encode proteins homologous to lectins. The other four genes are expressed at higher levels in the daf-2; daf-12 double mutant. We have cloned and sequenced one, which encodes a protein with unknown function. We are cloning and sequencing the remaining genes. We plan to integrate this information with that obtained from other methods (SAGE analysis, subtraction libraries and microarrays) to select candidates for genes that enhance longevity. We then plan test the effect of their (1) overexpression and (2) loss of function on life span.

Kenyon et al. (1993) Nature 366:461-464 Larsen P. et al. (1995) Genetics 139:1567-1583 Liang P. and Pardee, A. B. (1997) Science 257:967-971 Lin K et al. (1997) Science 278:1319-1322 Ogg et al. (1997) Nature 389:994-999

24 May 1999 15:50 664 664 Temporal expression of collagen genes in Caenorhabditis elegans during development.

1999 International Worm Meeting abstract 613 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Temporal expression of collagen genes in Caenorhabditis elegans during development. JM Muriel, IL Johnstone WCMP, Anderson College, University of Glasgow, 56 Dumbarton Rd, Glasgow, G11 6NU, Scotland (UK) The C. elegans cuticle (exoskeleton), is an extracellular matrix consisting predominantly of small collagen-like proteins that are extensively cross-linked. The first cuticle is produced during late embryogenesis and a new one is synthesised by the underlying hypodermis prior to each of the four moults. In previous research we demonstrated that cuticular collagen genes are expressed in a temporal series during cuticle synthesis and repeated during each synthetic phase. We hypothesised that the temporal control of collagen gene expression during each synthetic phase may exist to facilitate correct assembly of individual collagens into the multi-protein complex. We have been using the cloned wild type copies of various collagen genes for which visible mutants exist, to repair the phenotype of corresponding mutants. This is a good assay for function of the individual collagen. We have swapped the promoters between genes that are expressed at a variety of times to investigate the significance of timing of expression with respect to function. Our data indicates that timing plays a significant role in function. To further test the effect of expressing collagens at different times, we have epitope tagged several genes. Transgenically expressed tagged molecules detected by Western blotting show evidence of different higher order interactions (multimerisation) dependant on timing of expression.

24 May 1999 15:50 665 665 Characterization of the Shank, a novel PSD-family protein, in C. elegans.

1999 International Worm Meeting abstract 614 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of the Shank, a novel PSD-family protein, in C. elegans. J Ahnn Dept. of Life Science. Kwangju Institute of Science & Technology, Kwangju 500-712, Korea Shank is a novel family of PSD(post-synaptic density) protein complex. It was found in rat brain and contains PDZ(PSD-95, Disk-Large, ZO-1) domain that mediates binding to GKAP, ankyrin repeats, SH3 domain, SAM domain that mediates multimerization, and a proline-rich domain that binds cortactin. It was reported that these multiple protein-interactions cause shank to function as a scaffold protein in the PSD, cross-linking receptor/PSD-95 complexes and coupling them to regulators of the actin cytoskeleton. A C. elegans homologue of shank (C33B4.3) was found in the C. elegans genome data base and showing 40% identity over 1,000 amino acids. Shank in C. elegans shows relatively high sequence identity in the regions of ankyrin repeats and PDZ domain, but little homology in the SH3 domain. A partial cDNA clone(yk481a4) was found that can be used to confirm the gene structure by Northern blot.In order to study expression pattern of Shank in C.elegans,the Green-Fluorescent Protein reporter system was used. The green-fluorescent protein was expressed in vulva epitherial cells, head sensory neurons and tail region. Specially, the vulval expression of this GFP was stronger than others. We are preparing antibodies of shank and trying to elucidate its biological role in C. elegans by RNAi experiment.Screening for deletion mutant by EMS-mutagenesis is also under way.

24 May 1999 15:50 666 666 Isolation of novel body size mutants of C.elegans

1999 International Worm Meeting abstract 615 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Isolation of novel body size mutants of C.elegans Y Nakano, Y Ohshima Dept. of Biology, Fac. of Science, Kyushu Univ. How is the size of a multicellular animal decided ? We are attempting to study the mechanisms of the body size determination and finally we want to make a giant nematode. Mutants with an abnormal body size like sma or lon had previously been identified. Several of them have mutations in a TGF-b signaling pathway and some in cytoskeleton. To get more information about body size, many new mutants must be isolated. First, we isolated several smaller and thinner mutants from a mutator strain (RW7097) by observation with a stereomicroscope. They were back crossed with wild type N2 and examined for phenotypes. The estimated volumes of their adults are 40-20% smaller than N2. More recently, we have tried to isolate individuals which have an increased body size from EMS mutagenized N2. We have obtained six larger size mutants. Some of them have approximately twice as much volume as the wild type. They have been back crossed three times with N2. The original and back crossed mutants are being examined as to their body size and other phenotypes. The back crossed mutants are also subjected to genetic mapping.

24 May 1999 15:50 667 667 An analysis of C.elegans plexin genes

1999 International Worm Meeting abstract 616 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. An analysis of C.elegans plexin genes F Nakao, T Fujii, F Suto, H Fujisawa, S Takagi Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, 464-8601, Japan Plexin is a family of transmembrane proteins the extracellular segment of which posesses three repeats of a cysteine-rich domain similar to the domain found in the c-Met proto-oncogene protein product. Xenopus plexin has the Ca++ -dependent homophilic cell adhesion activity. In mice, each member of plexin is expressed in a distinct subset of neurons, suggesting the possibility that plexins play roles in the formation of particular neural circuits. Recently, it was shown that a Drosophila plexin is a receptor for a member of semaphorin, and mutations in the plexin gene and the semaphorin gene cause similar morphological defects in the nervous system. To examine the function of plexin further, we have been analyzing plexin genes in C.elegans. By blast search we have identified two plexin genes in C.elegans, which we tentatively named cep-1 and cep-2. cep-1 is on cosmid K04B12, which is mapped to the right arm of chromosome II, and cep-2 is on YAC Y55F3 mapped to the left arm of chromosome IV. A sequence analysis of cDNAs revealed that the cep-1 gene consists of 16 exons. The predicted sequence of CEP-1 bears homology of around 20% to vertebrate plexins. An expression analysis by using GFP showed that cep-2 appears to be expressed in neurons. The similarity in the structure and the expression pattern of vertebrate and C.elegans plexins might reflect conserved functions of this class of proteins.

24 May 1999 15:50 668 668 Functional analysis of rapsyn in AchR clustering

1999 International Worm Meeting abstract 617 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Functional analysis of rapsyn in AchR clustering S Nam, J Lee Yonsei University Nicotinic acetylcholine receptors(nAChRs) are clustered at high density in the postsynaptic membrane of neuromuscular junction. Rapsyn(achR Associated protein at SYNapse) is a peripheral membrane protein that is required for the AChR clustering at the neuromuscular junction. Rapsyn is conserved in many species including C. elegans. To understand rapsyn-mediated AChR clustering at the neuromuscular junction, we have examined various constructs of rapsyn fused to green fluorescent protein in transgenic animals. In wild-type C. elegans, rapsyn was expressed specifically in muscle synapse and neuron. When a putative dominant negative mutant rapsyn gene in which histidine residue of zinc finger motif was substituted by glutamine was examined, expression in muscle cells was altered and the transgenic animals showed uncoordinated phenotype, suggesting that the zinc finger motif of rapsyn is essential for AChR clustering. Examination of the transgenic animals bearing either wild type rapsyn or dominant negative mutant rapsyn in the genetic background of unc-29(AChR mutant), and ric-4 (md1088) showed that both the functional presynaptic activity and functional receptors are required for normal localization and function of rapsyn. We will present results from examination of various deletion derivatives of rapsyn. We are currently dissecting the cis-acting regulatory elements required for expressing rapsyn specifically in the muscles and neurons. We will also present data from this experiment.

24 May 1999 15:50 669 669 SPE-12 may function as a plasma membrane signal amplifier inducing spermatids to become spermatozoa

1999 International Worm Meeting abstract 618 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. SPE-12 may function as a plasma membrane signal amplifier inducing spermatids to become spermatozoa J Nance, E Davis, S Ward The University of Arizona Before fertilizing oocytes, haploid spermatids must extend a pseudopod which enables them to maneuver within the reproductive tract. Four mutants (spe-8, spe-12, spe-27, and spe-29 ) that prevent this process, known as spermiogenesis, are unlike other spe genes in that spermatids are defective only in virgin hermaphrodites; male sperm and self-sperm in mated animals can successfully complete spermiogenesis. To explain this phenotype, a model was created in which spermatids can begin spermiogenesis after receiving hermaphrodite- or male-derived signals. Mutations in these genes would result in an ineffective response to hermaphrodite signal, allowing sperm to differentiate only after the male signal is introduced by mating. Since spe-12(hc76) is likely a null mutant, we have examined its Spe phenotype in more detail. Few spe-12(hc76) self-sperm are rescued after mating even though mutants make normal numbers of sperm. Thus SPE-12 function is needed even when the male signal initiates spermiogenesis. As predicted, spe-12 males are also less fertile than wild-type. This defect is subtle, however, and is detected only when males mate hermaphrodites that have self-sperm. Since mutant male sperm still outcompete wild-type self-sperm, this fertility defect may result from an increased ovulation rate (stimulated by self-sperm). Oocytes could expel spe-12 spermatids before they complete spermiogenesis and crawl. We now favor a model in which SPE-12 functions to increase the strength of the signal. Differences in the signal itself could explain the Spe-12 phenotype: a weak hermaphrodite signal would fail to initiate spermiogenesis without SPE-12, while a stronger male signal could initiate spermiogenesis, albeit inefficiently, even in SPE-12’s absence. We have cloned several of these genes to understand how they may direct spermiogenesis initiation. Though novel proteins, SPE-12 and SPE-29 each contain a single predicted transmembrane domain. SPE-12 is localized to the spermatid plasma membrane, and a SPE-12::GFP fusion localizes to spermatids as well. We are currently investigating the localization of SPE-29. As both spe-12 and spe-29 interact genetically with spe-27 , perhaps all three gene products function in a signaling complex at the spermatid plasma membrane.

24 May 1999 15:50 670 670 Pharmacology and Regulation of the C. elegans Dopamine Transporter

1999 International Worm Meeting abstract 619 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Pharmacology and Regulation of the C. elegans Dopamine Transporter Richard Nass 1 , Janet Duerr 2 , Jim Rand 2 , David M. Miller 3 , Randy D. Blakely 1

1 Dept of Pharmacology and Center for Molecular Neuroscience, Vanderbilt U. Medical Center, Nashville, TN 37232. 2 Program in Molecular and Cell Biology, OMRF, Oklahoma City, OK 73104. 3 Dept of Cell Biology and Center for Molecular Neuroscience, Vanderbilt U. Medical Center, Nashville, TN 37232.

The neurotransmitter dopamine (DA) plays a significant role in a number of physiological processes including motor control, cognition, emotion, and reward. Abnormal DA signaling has been implicated in a variety of neuropathological illnesses such as Parkinson’s disease, schizophrenia, depression, and drug addiction. A primary mechanism for regulating the strength and duration of dopamine signalling involves the Na+ -dependent dopamine transporter (DAT). Following DA release at the synapse, DAT mediates the reuptake of DA into the presynaptic neurons. DATs are targeted by psychoactive drugs such as methylphenidate (Ritalin), cocaine and amphetamines. Although DAT pharmacology has been extensively characterized, endogenous regulators of DAT expression, localization, and activity are as yet unknown. DA is an important neurotransmitter in C. elegans . To provide an opportunity for genetic evaluation of DAT regulators, we have cloned the C.elegans dopamine transporter (CeDAT) 1 . The CeDAT gene encodes a 615 amino acid polypeptide with high sequence identity to the mammalian dopamine transporters (43%). Mammalian cells expressing CeDAT exhibit saturable and high affinity Na+ and Cl - dependent DA transport, and are inhibited by the mammalian DAT antagonist cocaine and amphetamines, as well as the antidepressant imipramine. These structural and pharmacological profiles indicate that CeDAT is likely to function as the dopamine transporter in C. elegans and is similar to its mammalian counterpart. We are currently exploring the expression of CeDAT using CeDAT-GFP and peptide antibodies. Several transgenic lines have been developed which target CeDAT transgenes to dopaminergic neurons and processes. We are also investigating the role of CeDAT in response to cocaine and amphetamines in N2 and CeDAT knockout worms. These studies as well as psychostimulant-induced phenotypes suitable for genetic screens of CeDAT regulators will be presented.

1. Jayanthi et al. (1998) Mol. Pharm. 54 :601-609.

24 May 1999 15:50 671 671 Analysis of the mode of bar-1 function in vulval cell fate specification

1999 International Worm Meeting abstract 620 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of the mode of bar-1 function in vulval cell fate specification L Natarajan, DM Eisenmann Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD 21250. Vulval development in C.elegans is an excellent model system to study the role of extracellular signaling in cell fate determination. Six Pn.p cells, P3.p to P8.p, form the vulva precursor cells (VPCs) and can adopt one of three cell fates denoted 1°, 2° and 3°. The vulva is made from the descendants of P5.p, P6.p and P7.p which adopt 2°, 1°, 2° fates respectively. Previous studies show that inductive signals are transduced by evolutionary conserved RTK/Ras and Notch signaling pathways in the VPCs. In a screen for protruding vulva (Pvl) mutants, the bar-1 gene was identified and was shown to encode a protein related to b-catenin and Armadillo, which are well established components of Wnt signaling pathways. Work from our lab and others suggests that bar-1 is involved in Wnt pathway regulation of Hox gene expression in three processes; a) Cell fate determination in VPCs, b) QL neuroblast migration, and c) P12 hypodermal cell fate specification.1,2,3 To identify factors interacting with bar-1 in vulval development, we are taking two approaches: a) To identify proteins interacting with BAR-1, we are using the Gal4 yeast two hybrid assay. A bar-1 cDNA encoding Armadillo repeats 1-9 is used as a bait to screen a worm cDNA library. Interacting clones will be sequenced and analyzed to determine the nature of the proteins they code for and their expression pattern in the vulva will be observed. b) A mutation in the egl-18 gene, ga97, was identified in the Pvl screen and causes the same vulval defect as a bar-1 mutation. Therefore, we would like to characterize the egl-18 gene product. egl-18 is mapped between dpy-9 and unc-17 on LG IV and we are using RFLP mapping to localize the gene. Further understanding of the genetic interactions of egl-18 with other mutants, the egl-18 pattern of expression, and the effects of egl-18 on Hox gene lin-39 expression will allow us to determine how egl-18 functions in vulval development.

References: 1. Eisenmann et al (1998), Development 125, 3667-3680. 2. Maloof et al (1999), Development 126, 37-49. 3. Jiang et al (1998), Development 125, 2337-234

24 May 1999 15:50 672 672 Identification and Analysis of Novel C. elegans Neuropeptide Genes

1999 International Worm Meeting abstract 621 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification and Analysis of Novel C. elegans Neuropeptide Genes A Nathoo 1 , R Moeller 2 , A Hart 2,3

1 Harvard University. 2 MGH Cancer Center, Dept. of Pathology, 149-7202 13th Street, Charlestown, MA 02129. 3 Harvard Medical School, Dept. of Pathology, 149-7202 13th Street, Charlestown, MA 02129.

Neuropeptides play critical roles in neurotransmission in vertebrates and invertebrates. We are interested in identifying the neuropeptides used by the ASH sensory neurons. Genetic and molecular evidence suggests that ASH neuropeptides may play critical roles in differential synaptic signaling of sensory information. Previous analysis in C. elegans (CGC#3193) has focused on the FMRFamide related peptide (flp) neuropeptide genes. The ASH neurons do not contain FMRFamide immunoreactivity. We have identified and are characterizing 21 putative neuropeptide like protein (nlp) genes of C. elegans. nlp genes were identified using a structure-based (not homology-based) C. elegans database searching strategy. Neuropeptides are encoded as preproproteins that contain a signal sequence and mono- or dibasic endopeptidase sites which flank the active neuropeptides. Multiple repeats of virtually identical, active neuropeptides are frequently found within the same gene. Standard database searching programs were used to identify C. elegans nlp genes: those which encode proteins with a signal peptide and 2 or more repeats of related putative neuropeptides flanked by di- or monobasic amino acids. This strategy eliminates the need for significant sequence identity with previously identified neuropeptides. Six of the predicted genes correspond to ESTs. The neuropeptides encoded by the nlp genes vary in their relationship to neuropeptides identified in other species. For example, nlp-1 and nlp-2 (C01C4.1 and T24D8.5-3) predicted peptides have approximately 40% identity with buccalin and myomodulin neuropeptides from other invertebrates, respectively. In contrast, predicted peptides encoded by nlp-10 (F37A8.4) are not homologous to any known neuropeptide. To address if nlp genes are expressed in neurons, we are generating 21 nlp::GFP reporter constructs. So far, all 19 reporter constructs tested express GFP exclusively or predominantly in neurons. Two nlp genes are expressed in ASH sensory neurons, 5 in the pharynx, 5 in the ventral nerve cord and 2 in the HSN neurons. We will address nlp gene function using immunohistochemical, genetic and molecular techniques.

24 May 1999 15:50 673 673 Interaction between PIE-1 and RCK/p54, a conserved RNA helicase

1999 International Worm Meeting abstract 622 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Interaction between PIE-1 and RCK/p54, a conserved RNA helicase RE Navarro 1 , E Shim 1 , A Walker 1 , Y Shi 2 , TK Blackwell 1

1 Center for Blood Research, Harvard Medical School. 2 Department of Pathology, Harvard Medical School.

In embryos produced by homozygous pie-1 mutants, germline blastomeres adopt somatic fates. The PIE-1 protein is localized to oocytes and early embryonic germline cells, in which it present in the cytoplasm but also accumulates in nuclei and appears to block all mRNA transcription. This repression seems to involve a PIE-1 domain that can inhibit transcription in human cells, suggesting an conserved target. Using a fusion of this repressor domain with GST, we have affinity-purified a PIE-1-interacting protein from a human cell extract. Microsequencing has shown that this protein is RCK/p54, an evolutionarily-conserved DEAD box RNA helicase. The C. elegans RCK homolog (CeRCK) binds specifically to PIE-1, through part of the repressor domain. RCK/p54 is most closely related to the translation factor eIF-4A, which is essential for recruitment of capped mRNAs to the ribosome. RCK/p54 is predominantly cytoplasmic, but has also been detected in nuclei. In Drosophila and Xenopus, it is expressed specifically during oogenesis and early embryonic stages, and in Xenopus it is abundant in maternal mRNA-protein granules. In mice, RCK/p54 is expressed in oocytes, but also is present in self-renewing tissues in the adult. We have begun to analyze the function of CeRCK in vivo by performing RNAi. Injected animals initially produce eggs that give rise to animals which appear normal through the L4 stage, but as adults are sterile. In these CeRCK-RNAi progeny, the morphology of the gonad does not appear grossly abnormal, but the uterus is empty. Within less than 24 hours, injected animals cease to produce any embryos, and their gonads resemble those of their sterile progeny. Our findings suggest that CeRCK plays an essential and perhaps evolutionarily conserved role during oogenesis. Its similarity to eIF-4A and the RCK/p54 expression patterns in other organisms suggest that it might function in controlling translation or packaging of maternal mRNA. We are investigating how CeRCK is involved in oogenesis and is expressed during development, and how its functions and subcellular localization might relate to those of PIE-1.

24 May 1999 15:50 674 674 Evidence for the involvement of END-2, a member of the steroid hormone receptor family, in endoderm and mesoderm formation.

1999 International Worm Meeting abstract 623 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Evidence for the involvement of END-2, a member of the steroid hormone receptor family, in endoderm and mesoderm formation. ED Newman-Smith, JH Rothman Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106 In embryos which lack a region of chromosome V, the endoderm determining region (EDR), the E founder cell adopts a C-like fate. Two genes in the EDR, end-1and end-3, rescue this phenotype and encode GATA factors that are expressed specifically in the endoderm lineage. A third EDR gene, end-2, encoding a member of the steroid hormone receptor family can also restore the correct fate to the E cell in EDR mutants. However, the role of end-2 in early development is apparently complex. While end-2 rescues the gutless phenotype of an EDR deficiency mutant, it can also promote mesoderm and repress endoderm development under different circumstances: ectopic expression of end-2 inhibits production of endoderm and results in an apparent excess of MS descendants. In addition, RNAi depletion of end-2 function suppresses the phenotype of a mutation [end-3 (zu247)] that causes E to adopt an MS-like fate, suggesting a role for end-2 in promotion of MS fate and repression of E fate. A potential dual role for end-2 in E and MS development is also reflected in its expression pattern: while apparently maternal end-2 transcripts are present in the early germline lineage and are lost in somatic lineages, apparent zygotic transcripts are detectable in both the endoderm and pharynx. Analysis of the function of the putative ligand binding domain suggests that a steroid-like ligand may be involved in endomesoderm differentiation. Ectopic expression of END-2 lacking this domain results in the apparent loss of MS-derived tissues, with little or no effect on endoderm. Similar experiments with mammalian steroid hormone receptors result in constitutively active receptors; however, the phenotype observed with end-2 suggests that this construct may be a dominant negative. These results suggest that a steroid hormone receptor-like transcription factor, possibly regulated by a ligand, may mediate endomesoderm differentiation in C. elegans, perhaps by collaborating with both the MS-specifying and E-specifying GATA factors that we have identified (see abstracts by Kasmir et al. and Maduro et al.).

24 May 1999 15:50 675 675 The unc-43 Ca2+/Calmodulin-dependent kinase II (CaMKII) is a critical neuronal regulator in C. elegans

1999 International Worm Meeting abstract 624 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The unc-43 Ca 2+ /Calmodulin-dependent kinase II (CaMKII) is a critical neuronal regulator in C. elegans EM Newton, JH Thomas University of Washington unc-43 encodes the C. elegans CaMKII homologue. CaMKII is a protein kinase responsible for orchestrating many cellular responses to the second messenger Ca2+ . CaMKII has been extensively studied biochemically and many potential targets have been identified by in vitro kinase assays. However, due to the lack of specificity of these assays, it remains unclear what proteins are phosphorylated by CaMKII in vivo. We determined the localization of UNC-43 by in situ antibody staining and GFP fusion analysis. A monoclonal antibody to rat CaMKII a stains the nerve ring and the ventral and dorsal nerve cords of wild-type worms. An unc-43 promoter-gfp fusion is expressed in ventral cord neurons, a variety of sensory neurons and interneurons throughout the animal and the HSN motorneurons. Expression is also observed in body-wall and vulval muscles and in the intestine. This expression pattern suggests that unc-43 functions in neuronal regulation throughout the animal consistent with the diverse phenotypes seen in unc-43 mutants. A kinase-activating mutation causes defects in egg laying, defecation expulsion and timing, locomotion and body muscle tone. Loss of function mutations cause generally opposite phenotypes in each case. We are currently determining the site of action for each phenotype using tissue specific expression of wild-type or kinase-activated unc-43 cDNAs in an unc-43 null background. We are also testing a peptide inhibitor of CaMKII for the ability to cause loss of function phenotypes when expressed in vivo. unc-43(lf) mutants have a repetition in their defecation motor program that we refer to as the "echo" phenotype with a consistent latency of 11 seconds. Double mutants between unc-43 and defecation cycle (dec) mutants were constructed to test whether they would alter the latency of the echo. Instead, strong suppression of the echo was observed for all double mutants examined (4 in total). Two dec mutants (flr-1 and flr-4) have been cloned and both are expressed in the intestine, suggesting that the location of the defecation "clock" may be in the intestine. Experiments are currently underway to test if the observed expression of unc-43::gfp in the intestine is responsible for its regulation of the defecation clock.

24 May 1999 15:50 676 676 Diplogasteroides berwigi: A model for the further phylogenetic analysis of nematode vulval evolution

1999 International Worm Meeting abstract 625 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Diplogasteroides berwigi: A model for the further phylogenetic analysis of nematode vulval evolution H Nguyen 1,2 , B Jungblut 1 , R Sommer 1

1 Max-Planck Institut für Entwicklungsbiologie, Department for Evolutionary Biology, Spemannstr. 35, 72076 Tübingen, Germany. 2 E.mail: [email protected]

Our lab is studying the evolution of developmental processes by comparing vulva development between C. elegans and Pristionchus pacificus. This cellular, genetic and molecular comparison links changes in cell fate specification to differences in the molecular mechanisms during development. Ultimately, comparative developmental biology has to consider not only the mechanistic but also the phylogenetic perspective. The identification of genes that have changed their function over long evolutionary timescales, such as C. elegans and P. pacificus can only be the first step. More detailed comparison at the phylogenetic level have to follow by studying more closely related species. Fortunately, molecular and morphological analyses provide us with detailed phylogenetic trees of nematodes. Based on the current phylogeny, several species can be selected for functional analysis. Here we describe our initial genetic and molecular work in Diplogasteroides berwigi, an ancestral member of the Diplogastridae. This hermaphroditic strain has a 5 day generation time and is accessible to genetic analysis. One of the aims of comparative genetics in this species is the functional comparison of mab-5, a homeotic transcription factor, that has changed its function dramatically between P. pacificus andC. elegans. mab-5 cloning from D. berwigi and first mutant analysis is reported.

24 May 1999 15:50 677 677 New mutants defective in postembryonic morphogenesis of the male tail tip

1999 International Worm Meeting abstract 626 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. New mutants defective in postembryonic morphogenesis of the male tail tip C Nguyen, Y Yang, T Del Rio, D Fitch Department of Biology, New York University, New York, NY 10003, USA We are interested in elucidating the genes and mechanisms involved in morphogenesis. We are using the male tail tip as a model to identify genes involved in this process. During the late L4 stage in wild type males, the four tail tip hypodermal cells (hyp8-11) fuse and retract, generating the blunt-ended ("peloderan") tail tip of the adult. In some species related to C. elegans, the tail tip cells fail to retract, producing a pointy ("leptoderan", Lep) tail tip. This suggests that tail tip morphogenesis can be uncoupled from general male tail morphogenesis. Discovering the genes responsible for tail tip morphogenesis in C.elegans will also point to candidate loci involved in the evolution of different tail tip morphologies. To identify such genes, we are performing Normarski screens for mutants in which the tail tip cells fail to retract, resulting in adult males with tails resembling the leptoderan tails of other species. We isolated fifteen new mutants from a screen of 30,000 haploid genomes using TMP/UV as a mutagen. The Lep phenotype is highly penetrant in all these mutants; expressivity is variable in most. Despite high penetrance of the Lep phenotype, males of mutants ny8, ny13, ny17 and ny18 can mate well. Our preliminary mapping results place ny8 and ny17 on LG I and ny13 on LGIII. We are also working with the DEB-induced mutant bx85, kindly provided by Scott Emmons. The males are sterile, the tail tip is unretracted and the fan encompasses the tail tip. Our three-factor data and deficiency complementation tests place bx85 near dpy-20 on LG IV.

24 May 1999 15:50 678 678 unc-61Locus Encodes for C. elegans Septin That Plays a Critical Role in Post-embryonic Cytokinesis

1999 International Worm Meeting abstract 627 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. unc-61Locus Encodes for C. elegans Septin That Plays a Critical Role in Post-embryonic Cytokinesis TQ Nguyen 1 , H Sawa 2 , JG White 1

1 Laboratory of Molecular Biology, University of Wisconsin, Madison, WI 53706. 2 Department of Neuroanatomy, Osaka University Medical School, Osaka, Japan.

The mechanisms that regulate cytokinesis remain poorly understood. Over the past few years, a class of gene called septins has been identified and shown to play a critical role in cytokinsis in several organisms including S. cerevisiae and D. melanogaster. We have identified, cloned, and characterized the C. elegans unc-61locus that encodes for a C. elegansseptin, sep-2. sep-2 gives a 2.1 kb transcript that encodes a polypeptide of 568 amino acids with a predicted molecular weight of 65 Kd. When translated, SEP-2 has significant homology to other known septins including C. elegansSEP-1. Like most septins, SEP-2 has the GTPase domain at the N-terminus and a predicted coiled-coiled domain at the C-terminus Phenotypically, unc-61(e228and n3169)have vulva protrusion, egg-laying defects, gonad extrusion, and male abnormality. These phenotypes are classical characteristics of a mutant that has post-embryonic failures in cytokinesis. Genetically, unc-61maps to 6.49 map unit to the right arm of chromosome V and is covered by a cosmid C18G4 that contains the full length sep-2. Cosmid C18G4 and its subclones rescued unc-61mutants. Furthermore, RNAi using sep-2 cDNA phenocopies all aspects of unc-61 mutants. Sequencing analysis reveals that both alleles of unc-61(e228and n3169)have a premature stop codon resulting in truncated protein. We generated antibodies against SEP-2 peptides and performed indirect immunostaining to determine the localization of this gene product during cytokinesis in early embryos. SEP-2 localized to the anterior cortex in the early embryos and became localized in all cleavage furrows at early telophase. It formed a ring-like structure in close association with an actomyosin contractile ring. Eventually, SEP-2 localized to the persisting intercellular bridge remaining there until the next cell division. Further characterization showed that SEP-2 was not detected in unc-61mutants. In addition, SEP-2 also failed to localize in unc-59(e261and e1005),mutant alleles of sep-1. These and the reciprocal observations suggest that SEP-1 is required for SEP-2 localization and vice versa.

24 May 1999 15:50 679 679 Serotonin and Feeding

1999 International Worm Meeting abstract 628 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Serotonin and Feeding TR Niacaris, L Avery University of Texas Southwestern Medical Center Serotonin increases the rate of pharyngeal pumping and egg laying, decreases foraging behavior and is required for proper male mating behavior. Serotonin can increase pharyngeal pumping by stimulating MC, a motor neuron that stimulates pharyngeal muscle contraction, or by acting directly on pharyngeal muscle. To determine the receptor or receptors that mediate these responses, we have searched the genomic sequence for candidate genes having sequence identity to known vertebrate and invertebrate serotonin receptors. We found several candidate metabotropic serotonin receptors using this method: ser-1, ser-2, ce-5HT-1, C09B7.1, C52B11.3, F01E11.5, F14D12.6, M03F4.3, T02E9.3, K02F2.6 and F15A8.5. Three of these receptors (ser-1, ce-5HT-1, and M03F4.3) have been determined to be responsive to serotonin in heterologous expression systems (Ribeiro and Hamdan IWM97, p278 and Olde and McCombie, 1997). In addition, we found two candidate ionotropic (5-HT3 ) receptors in the genomic sequence (C31H5.3 and C40C9.2.) We have created GFP fusion constructs to ten of the thirteen candidate genes using overlap extension PCR (C52B11.3, T02E9.3 and F15A8.5 are in progress). Six of the genes, ser-1, ser-2, C09B7.1, K02F2.6, C31H5.3 and C40C9.2 show pharyngeal expression of the fusion construct. ser-1 expresses strongly in pharyngeal muscle cells of the procorpus (pm3), isthmus (pm5), and terminal bulb (pm6, pm7 and pm8) (S. Nurrish pers. com. and my observations). ser-2 pharyngeal muscle expression is specific for metastomal flap muscles (pm1), and anterior terminal bulb muscles, pm6. The ser-2::GFP construct also expresses in muscles of the head, the pharyngeal serotonergic neuron NSM, the ventral nerve cord and unidentified neurons in the nerve ring, head and tail regions. In males, ser-2::GFP expresses in dorsal and ventral body muscles of the tail region. ser-2::GFP also expresses in the male-specific diagonal muscles and serotonergic CP neurons. The expression pattern of ser-2 appears to be consistent with several known effects of serotonin. We are currently working to more carefully determine the pharyngeal cells that express C09B7.1, K02F2.6, C31H5.3 and C40C9.2. In addition, we are attempting to knockout the pharyngeal-expressing candidate receptors.

24 May 1999 15:50 680 680 Electrophysiological Recording from Identified C. elegans Chemosensory Neurons

1999 International Worm Meeting abstract 629 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Electrophysiological Recording from Identified C. elegans Chemosensory Neurons WT Nickell 1 , RK Pun 1 , CI Bargmann 2 , SJ Kleene 1

1 University of Cincinnati College of Medicine, Cincinnati, Ohio. 2 The University of California, San Francisco, California.

The simple nervous system of C. elegans provides an attractive model for the study of sensory transduction and neural integration. In particular, two pairs of chemosensory neurons (AWA and AWC) mediate chemosensation to different identified sets of odorants; for each of these neurons, strains have been developed in which these neurons express Green Fluorescent Protein. Unfortunately, electrophysiological recordings have proven difficult: the worm is too small for conventional microsurgical tools and is surrounded by a tough cuticle. The only published technique for exposing the C. elegans nervous system involves immobilizing the worm with cyanoacrylate adhesive and puncturing the cuticle with a glass needle attached to a micromanipulator. We report here a simpler approach. Approximately 100 worms were transferred from low-tonicity saline to a pool of modified "Dent’s Solution" confined on a ice-cold metal plate. Worms were immobilized at this temperature. Under 60X magnification, the worms were bisected with a #11 scalpel blade at the terminal bulb of the pharynx. Den’t Solution was modified by addition of 100 mM sucrose and 200 µM carbachol: increased tonicity reduced the effect of explosive release of internal pressure during cutting; the cholinergic agonist carbachol blocked spontaneous movements of the heads. In about 10% of these heads, fluorescent neurons were intact and exposed. These heads were immobilized and oriented with a suction pipette. The neurons were patched using an 8 Mohm pipette under a 40X objective. We do not find fast voltage-gated sodium currents. Most patches contain one of two distinct channel types. An outwardly rectifying channel was activated at positive potentials. This channel has distinct openings, a unit conductance of 28 pS and a reversal potential near -63 mV. An inwardly rectifying channel opens at potentials more negative than -50 mV. In contrast with the outward rectifier, this channel does not exhibit distinct conductance states.

24 May 1999 15:50 681 681 An interspecies comparison of lin-25

1999 International Worm Meeting abstract 630 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. An interspecies comparison of lin-25 L Nilsson, T Tiensuu, S Tuck UCMP, Umeå University, Sweden. lin-25 lies downstream of let-60 ras in the genetic pathway for induction of vulval fates and encodes a novel 130 kDa protein. LIN-25 is expressed first in all six VPCs independently of genes in the ras-MAPK cascade but later becomes restricted to the descendents of cells adopting vulval fates, P5.p, P6.p and P7.p. In order to help elucidate how LIN-25 functions biochemically we sort to identify parts of the protein most important for its activity. To identify evolutionary conserved domains we cloned lin-25 from the related nematode C. briggsae. We identified a fosmid clone, G05D12, by DNA hybrization which the C. briggsae sequencing consortium kindly sequenced for us. The C. briggsae lin-25 gene is predicted by Genefinder to have a similar arrangement of exons and introns to that of C. elegans lin-25. The sequence of the predicted product of the C. briggsae gene has however, diverged significantly from that of C. elegans LIN-25 (57% identity). The amino acid identities are fairly evenly distributed throughout the entire length of the two proteins but three domains can be identified, the largest at the C-terminus, within which the identity is much higher. The C. briggsae gene appears to be an orthologue of C. elegans lin-25 since it is able to rescue the C. elegans lin-25 phenotype. 10 alleles of lin-25 have been isolated to date, 7 that behave as null mutations by genetic criteria. Characterization of these alleles reveals that the C-terminus of LIN-25 is required for stability. The null allele, ku78, is predicted to encode a truncated protein lacking the last 121 amino acids (of 1139). ku78 homozygotes contain only very low levels of LIN-25 protein. In extracts from WT worms, multiple polypeptides are detected with anti-LIN-25 antibodies that have lower apparent molecular weights than the full-length protein. Tagging of the WT protein with the HA epitope reveals that these smaller species result from removal of the C-terminus. lin-25’s position in the genetic pathway suggested that the protein might be a target of the MPK-1 MAPK. To examine this possibility we generated mutants in vitro encoding proteins lacking the potential MAP kinase phosphorylation sites. A gene encoding a protein lacking all four sites rescues the lin-25 phenotype with almost WT efficiency. Thus LIN-25 does not appear to be a direct target of MPK-1.

24 May 1999 15:50 682 682 Expression of carbohydrate antigens in the nematode Caenorhabditis elegans

1999 International Worm Meeting abstract 631 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Expression of carbohydrate antigens in the nematode Caenorhabditis elegans K Nomura 1 , KH Nomura 1 , S Mizuguchi 1 , S Ichikawa 2 , Y Hirabayashi 2

1 PRESTO (JST) and Kyushu University, Department of Biology, Faculty of Science 33, Kyushu University, Fukuoka 812-8581, Japan. 2 Frontier Research Program, Institute of Physical and Chemical Research (RIKEN), Saitama 351-0198, Japan.

Glycoproteins and glycolipids are positioned mainly on the outside layer of cell membranes. They are forming frontier layer of cells, where incoming information and molecules are first received and processed. Important roles of carbohydrate antigens in this frontier layer have been revealed recently in various organisms, and their critical roles in development and cell-cell recognition have been clearly shown. We previously reported that among human ABO blood group antigens, only blood-group-B antigens are expressed on frog (Xenopus laevis) early blastomere membranes. We also reported that the blood-group-B antigens and lectins recognizing them are playing essential roles in Ca2+ dependent cell-cell adhesion of frog blastomeres (Dev Genes Evol, 208,9-18, 1998). Blood-group-B active carbohydrates are indispensable components of frog Ca2+ dependent cell-cell adhesion system. This is the first report showing blood-group-B active carbohydrates are playing any physiological roles in multicellular organisms, and the result will be helpful to gain insights on evolution of blood group antigens in multicellular organisms. In this study, we used various antibodies to examine what kinds of carbohydrate antigens are expressed in glycoproteins and glycolipids of the worm. Neutral glycosphingolipids (GSL) and the activity of their synthesizing enzyme ceramide glucosyltransferase(GlcT-1) were detected. GlcT-1 gene has been isolated and identified. Although neutral GSL were detected in worms, no acidic GSL including gangliosides have been detected. Among several metabolites of GSL, sphingomyelin (SM) was detected and especially strong staining by anti-SM antibody was found in spermatheca region of adult worms. Blood-group-B active GSL and glycoproteins and a lectin recognizing them were found, but blood-group-A (or H) active antigens were not detected. HNK-1 active glycoproteins were detected but HNK-1 active GSL were not found. We are currently examining the expression patterns of these antigens and are studying functions of these glycoconjugates in developing worms.

24 May 1999 15:50 683 683 Effectors of the RAB-3 synaptic vesicle-associated small GTP protein

1999 International Worm Meeting abstract 632 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Effectors of the RAB-3 synaptic vesicle-associated small GTP protein M Nonet 1 , J Staunton 1,2 , G Hadwiger 1 , L Wei 1 , B Ganetsky 2

1 Washington University School of Medicine. 2 University of Wisconsin. rab-3 encodes a small-GTP protein that reversibly associates with synaptic vesicles. Rab3 function is thought to be regulated by its cycling between a soluble GDP-bound form and a vesicle-associated GTP-bound form. Here we characterize the C. elegans mutants of two candidate rab-3 effectors first identified in vertebrates; rabphilin and RIM. The vertebrate proteins bind rab3 specifically in the GTP-bound state. These two large proteins contain multiple domains including a zinc finger domain that mediates rab3 binding and C2 domains that share homology to the calcium binding motifs of synaptotagmin. The C. elegans genes share the general organization of these domains and only exhibit similarity within these regions. Both C. elegans genes are expressed in the nervous system. Vertebrate rabphilin associates with synaptic vesicles; localization of worm rabphilin (RBF-1) in wild type animals and mislocalization of the protein in unc-104 mutants suggests RBF-1 associates with synaptic vesicles. However, by contrast with the vertebrate homolog, C. elegans RBF-1 is not mislocalized in rab-3 mutants. Rabphilin deletion mutants exhibit no detectable synaptic defects when examined using behavioral, pharmacological, physiological or ultrastructural assays. Vertebrate RIM is associated with the active zone at vertebrate terminals. C. elegans unc-10 encodes the vertebrate RIM homolog. A functional worm unc-10::GFP fusion is localized in tight puncta in synaptic-rich regions, consistent with localization to the active zone. However, this localization has yet to be confirmed with antibodies, and thus remains preliminary. unc-10 deletion mutants exhibit moderate behavioral, pharmacological and physiological defects. Surprisingly, these defects are stronger than those of rab-3 mutants suggesting that RIM functions in pathways independently of RAB-3 signaling. We are assessing if dominant unc-10 mutants (isolated in E. Jorgensen’ s lab) function independently of rab-3 to provide further evidence of a RAB-3-independent pathway transduced through RIM. We are also characterizing the domains required for punctate localization of UNC-10 as a step towards identifying components of the presynaptic active zone.

24 May 1999 15:50 684 684 RNAi: how low can you go?

1999 International Worm Meeting abstract 633 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. RNAi: how low can you go? J Norman, K Galey, MK Montgomery Macalester College, Biology Dept., 1600 Grand Ave., St. Paul, MN 55105 We are aiming to determine the minimal sequence required to obtain effective disruption of gene function with the reverse genetic technique RNAi (RNA interference)1 . We are approaching this problem by cloning homologs of C. elegans genes with known mutant phenotypes from the closely related C. briggsae. Subclones with known length and percent sequence identity are used to create templates for double-stranded RNA (dsRNA). The dsRNAs from the C. briggsae subclones are then introduced into C. elegans by either injection or soaking. Progeny are examined for the appropriate mutant phenotypes and processed for in situ hybridization to determine loss of the endogenous message. The effectiveness of relatively short sequences (60-300 bp) between 70 and 90% sequence identity is then compared with the effectiveness of the same sequences from C. elegans (thus, same size but close to 100% sequence identity between the interfering RNA and the target). We are primarily focusing on maternally expressed genes (ama-1, glp-1, skn-1), but are also testing zygotic genes (e.g. hlh-1) to assess stage-related differences. Our results should be useful for designing effective RNAi strategies for both C. elegans and other nematode species. Ultimately, we hope to use RNAi as a tool to study gene function in genetically less tractable species, such as Cephalobus, in order to do comparative studies of maternal genes controlling embryonic cell fates.

1 Fire et al. 1998 Nature 391: 806-11

24 May 1999 15:50 685 685 Analysis of alpha spectrin function during muscle development in C. elegans.

1999 International Worm Meeting abstract 634 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of alpha spectrin function during muscle development in C. elegans. K.R. Norman, D.G. Moerman Department of Zoology, University of British Columbia, Vancouver, BC, Canada Assembly, maintenance, and growth of the myofilament lattice in developing muscle tissue is a complex set of events that is not well understood. However, these events are crucial for proper muscle function. It has previously been demonstrated that nascent sarcomeres initiate assembly at the membrane. In order to better understand these processes we have chosen to study the role in muscle development of the major membrane cytoskeletal protein, alpha spectrin. We isolated a mutation in the C. elegans alpha spectrin gene, called spc-1 (spectrin) in a mutagenesis screen designed to identify animals with muscle defects in early development. The C. elegans alpha spectrin protein is 61% identical and 76% similar in its entirety to the Drosophila alpha spectrin and it is 57% identical and 72% similar in its entirety to the vertebrate non-erythroid alpha spectrin, indicating that nematode alpha spectrin is the ortholog of vertebrate non-erythroid alpha spectrin. We have localized the alpha spectrin protein to the dense bodies (Z-line analog) and the I bands in the body wall muscle of C. elegans. Remarkably, this is the same localization pattern that is observed in vertebrate muscle. Animals lacking functional alpha spectrin die just after hatching and have defects in myofilament organization. More specifically, when compared to myofilaments in wild type animals, the myofilaments in the mutant animals are abnormally oriented relative to the longitudinal axis of the embryo. In cross section, the myofilaments appear to be loosely associated with the sarcolemma as compared to wild type. Also, we have observed defects in the musculature of the pharynx. This muscle tissue is abnormally shaped, has a disorganized arrangement of myofilaments and labors during contraction. From this work, we conclude that alpha spectrin is an important component of muscle and that it is involved in myofilament organization. An in depth description of the mutant phenotype and alpha spectrin expression and localization will be presented.

24 May 1999 15:50 686 686 Habituation modulates the interaction of multiple response mechanisms mediating olfactory chemotaxis

1999 International Worm Meeting abstract 635 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Habituation modulates the interaction of multiple response mechanisms mediating olfactory chemotaxis W Nuttley, D van der Kooy Department of Anatomy and Cell Biology, University of Toronto, 1 King’s College Circle, Toronto, ON M5S 1A8 Canada Analysis of the kinetics of chemotaxis in response to 100% benzaldehyde revealed an initial attractive response which was followed by a strong aversion. We show that this behaviour is mediated by two genetically separable, attraction- and aversion-mediating chemotactic response mechanisms. The attractive response, but not the aversion, is disrupted in odr-3(n2150) animals and presumably mediated by the AWC sensory neurons. Although initially strong, the attraction in wild-type worms habituated within an hour allowing an aversive response mechanism to dominate the behaviour of the animals. Analysis of the attraction deficient odr-3 mutants indicated that the aversive response is weak in naive animals and exposure to benzaldehyde strengthens this response. The aversive response was not disrupted by the odr-3(n2150) mutation, nor by the osm-9(ky10), odr-1(n1936), glr-1(n2461) or deg-1(u38) alleles, indicating that this chemotactic aversion operates independent of the ASH sensory neurons and is therefore distinct from the previously described, short-range, reflexive aversion to benzaldehyde (Colbert et al., 1997, J. Neurosci. 17, 8259-8269). The putative olfactory adaptation mutant adp-1(ky20) was examined for it’s response kinetics based on our assumption that adaptation of the attractive response is required for expression of the aversion. This strain was, however, indistinguishable from wild-type in kinetic assays. A re-examination of benzaldehyde adaptation in adp-1 mutants revealed that the phenotype observed in this line is due to a super-sensitivity to a dishabituating stimulus, rather than a defect in the adaptation to odorants, per se. Additionally, the olfactory adaptation observed in wild-type N2 worms was also shown to be subject to dishabituation, whereby the attractive response is re-established within a few minutes, rather than the few hours typically seen for spontaneous recovery.

24 May 1999 15:50 687 687 A genetic mosaic screen for mutations affecting the P1 lineage

1999 International Worm Meeting abstract 636 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

A genetic mosaic screen for mutations affecting the P 1 lineage J Nyström 1 , H Stewart 2 , L Nilsson 1 , D Baillie 2 , S Tuck 1

1 UCMP, Umeå University, SE-901 87 Umeå, Sweden. 2 Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, B.C., Canada, V5A 1S6.

It is likely that many genes required for cell fate determination and morphogenesis during the later stages of C. elegans postembryogenesis are also required during embryonic development. Some of these genes may be difficult to identify in conventional genetic screens either because viable, non-null alleles of them are very rare or because the embryonic phenotype they give rise to is ameliorated by partial maternal rescue. In order to try to circumvent these problems we have designed a genetic mosaic screen to examine the effect on postembryogenesis of mutations causing growth arrest of the embryo or early larva. To begin with, in order to assess the feasibility of the approach, we are examining the effect of a group of lethal mutations under sDp3 III on the development of cells within the P1 lineage. We have linked an extrachromosomal array containing the sur-5::gfp marker to sDp3 and generated 80 strains of the genotype dpy-17(e164) ncl-1(e1865) let-x unc-32(e189); sDp3-svEx[unc-4(+)-sur-5::gfp] . Worms segregating from these strains lacking the duplication in the descendants of E are identified by screening plates for viable worms that fail to express SUR-5::GFP in the intestine. These worms are analyzed to determine the point at which the duplication was lost (E, EMS or P1 ). Those in which the duplication was lost in EMS or P1 are then examined in detail under the compound microscope. Presently we are scoring mosaic worms for a variety of different defects including those in dtc migration, Z1.ppp/Z4.aaa fate determination, sex myoblast migration, gonad development, morphogenesis of the uterus, gametogenesis and generation of the ceolomocytes. In addition, for P1 mosaics that are fertile, the phenotype of embryos they give rise to is also examined. To date we have analyzed 42 strains in this way. Mosaic worms have - - been found for all but six of them. Eight strains give rise to P1 or EMS mosaics in which the gonad fails to develop and three in which the dtc migrations are markedly abnormal. We are presently characterizing the phenotypes of worms segregating from these strains in more detail and screening through other strains for mosaics.

24 May 1999 15:50 688 688 The zyg-1 Gene Encodes a Serine/Threonine Kinase Required for Bipolar Spindle Formation

1999 International Worm Meeting abstract 637 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The zyg-1 Gene Encodes a Serine/Threonine Kinase Required for Bipolar Spindle Formation KF O’Connell 1 , C Caron 2 , KJ Kemphues 2 , JG White 1

1 Laboratory of Molecular Biology, University of Wisconsin, Madison, WI 53706. 2 Section of Genetics and Development, Cornell University, Ithaca, NY 14853.

Duplication of the centrosome, the cell’s microtubule organizing center, is a critical event in the morphogenesis of the bipolar mitotic spindle. This event, which occurs precisely once per cell cycle, must be tightly regulated to avoid the presence of too few or too many centrosomes and the subsequent formation of mono- or multipolar spindles. At the molecular level, centrosome duplication is poorly understood; a more complete description of the process will require the identification and analysis of all pertinent genes. To this end, we have been studying a gene implicated in centrosome duplication in C. elegans. We have analyzed the embryonic lethal phenotypes of six zyg-1 alleles and found that they fall into two classes: class I is composed of three ts alleles that give rise to blastomeres containing monopolar spindles, and class II is composed of three nonconditional alleles that give rise to blastomeres with mono or multipolar spindles. Genetically, the monopolar phenotype is a strict maternal effect, and immunocytochemical charaterization of the monastral microtubule arrays in these embryos indicates the presence of a single pair of centrioles consistent with a defect in centrosome duplication. In contrast, genetic and cytogical data indicate that the multipolar phenotype arises from defects in spermatogenesis, however, the nature of these defects is presently unclear. As the embryo inherits its centrosome from the sperm, we believe that the spermatogenesis defects of class II mutants result in the production of fetilization-competent sperm containing multiple centrosomes. To further define its function, we have cloned the zyg-1 gene and determined the complete cDNA sequence. Conceptual translation of the open reading frame indicates that zyg-1 encodes a 79 kDa polypeptide with an amino terminal serine/threonine kinase domain. Interestingly, all class I alleles are missense mutations that map immediately C-terminal of the kinase domain, while all class II alleles result in small C-terminal truncations. These results indicate that zyg-1has multiple functional domains and plays a complex role in centrosome duplication.

24 May 1999 15:50 689 689 Role of Astral Microtubules in the Establishment of Anterior-Posterior Polarity and Pronuclear Migration

1999 International Worm Meeting abstract 638 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Role of Astral Microtubules in the Establishment of Anterior-Posterior Polarity and Pronuclear Migration KF O’Connell, KM Maxwell, JG White Laboratory of Molecular Biology, University of Wisconsin, Madison, WI 53706 In the C. elegans zygote, asymmetry is established along the A-P axis during a period known as pseudocleavage (PC). During PC, the cell cortex and underlying cytoplasm are dramatically remodeled, a process that involves the movement of cytoplasm in the form of a fountain flow; in the cortex, material such as actin foci are transported anteriorly, while internally, a counter flow of deeper cytoplasm transports constituents such as P granules to the posterior. The events of PC give rise to discreet cortical and cytoplasmic domains: PAR-3 is localized to the anterior cortex, PAR-2/PAR-1 to the posterior cortex, and P granules to the posterior cytoplasm. The sperm pronucleus and/or its associated astral microtubules play a major role in this process as their position determines the orientation of A-P polarity and the direction of the cytoplasmic/cortical flows. We have isolated mutations in a gene, spd-2, that delay and attenuate the formation of sperm asters until after PC. Analysis of mutant zygotes reveals an abnormal PC and the complete absence of A-P polarity: the fountain flow of cytoplasm and the associated asymmetric contractions of the cell cortex are absent, cortical domains of PAR-2 and PAR-3 are not established, and P granules do not localize to the posterior. Our results allow us to elaborate upon an earlier model, and explain how the sperm asters might interact with the PAR proteins to polarize the embryo along the A-P axis. In addition, we find that later developmental events such as pronuclear migration are affected in spd-2 mutants In wild-type embryos, the oocyte pronucleus migrates in two phases; initially, it travels at a slow (3.6 ± 1.1 m/min) rate as it approaches the cell center, then as it passes the equator, it accelerates and travels at a rapid (33.5 ± 7.4 m/min) rate before meeting the sperm pronucleus in the posterior. The sperm pronucleus exhibits only a slow (3.4 ± 1.1 m/min) anteriorly-directed movement. In the absence of sperm asters, the fast phase of oocyte pronuclear migration is absent and both pronuclei migrate at the slow rate to the cell center. These observations indicate that the fast phase involves interactions between the astral microtubules and motor proteins on the oocyte pronucleus.

24 May 1999 15:50 690 690 The Representation of C.elegans Genome Sequencing Data in SWISS-PROT

1999 International Worm Meeting abstract 639 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Representation of C.elegans Genome Sequencing Data in SWISS-PROT C O’Donovan European Bioinformatics Insitute The data generated from the genome sequencing projects has led to a dramatic increase in the amount of translated protein sequences and we at SWISS-PROT are working to incorporate all of this protein sequence data with proper sequence analysis and annotation. In order to achieve this, we have selected a number of model organisms for priority annotation. One of those selected is Caenorhabditis elegans. The Caenorhabditis Genome Sequencing Laboratories in St. Louis, Missouri, USA and Hinxton, Cambridge, UK have currently sequenced and made available more than 80% of the genetic material contained in this free-living organism. At present, there are approximately 1,963 C.elegans entries in SWISS-PROT(Release 37) and 12,763 C.elegans protein sequence entries in SP-TREMBL (Release 9). The SP-TREMBL entries will be annotated by a curator before integration into the SWISS-PROT database. We try to include as much annotation information as possible in SWISS-PROT. This is particularly important in the case of the protein sequences obtained from genome sequencing projects as these are obtained mainly from CDS prediction programs and therefore are hypothetical. We use various sequence alignments against SWISS-PROT and TREMBL, transmembrane and signal prediction programs, the PROSITE pattern database and other programs to identify potential functionality by similarity. These procedures also enable the detection of additional reports of this sequence from biochemical sources. We try as much as possible to merge all these data to provide as much information as possible and minimise redundancy. This has proved to be of advantage to the genome sequencing groups, enabling them to assess their data. Each SWISS-PROT entry contains not only protein sequence information but also citation references, sequence features and cross-references to other databases, such as EMBL, PROSITE, Medline, WormPep and ENZYME which enables the user to retrieve additional information related to the protein sequence. Textfiles on the status of the sequencing and identification of genes and proteins (http://www.ebi.ac.uk/ebi_docs/swissprot_db/gp_html) have been created and/or maintained by SWISS-PROT to provide a useful reference for the user.

24 May 1999 15:50 691 691 Analysis of Essential Genes in the sDf125 Region of Chromosome III

1999 International Worm Meeting abstract 640 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of Essential Genes in the sDf125 Region of Chromosome III NJ O’Neil, D Chan, GP Vatcher, LM Kuervers, DL Baillie Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada Our lab has generated 142 essential gene mutations defining 112 genes under sDp3, on chromosome III. We have divided the region between dpy-17 and dpy-19 into 12 zones that are defined by overlapping deficiencies and duplications. These zones were determined physically by PCR testing the deficiencies to determine endpoints. We have mapped the 112 essential gene mutations into these zones. One such region is deleted by sDf125. This region spans 420 000 base pairs and is completely covered by sequenced cosmids. Within this zone there are 107 predicted coding elements, many of which with similarities to mitochondrial and ribosomal-associated proteins. To date, we have mapped 17 essential genes into this zone. Using transgenics from our lab’s cosmid transgenic library, we positioned 13 of the 17 genes to cosmids. 12 of the 13 genes lie in the rightmost 250 kB of the region. This distribution could be due to the large number of olfactory receptors in the left half of this region. We have begun subcloning the rescued genes in this essential gene rich region and have positioned several of them to single predicted coding elements. let-716 encodes the worm orthologue of RRP5 (C16A3.3). RRP5 is an essential rRNA biogenesis protein in yeast. It is required for the processing of at least three of the four ribosomal RNA molecules (18S, 5.8S and 28S). This processing occurs in the nucleolus of the yeast cell. Both alleles of let-716 block as L1 larvae. As mentioned above, this region is rich in ribosome-associated proteins and we are looking at other lethal mutations which may be part of ribosomal biogenesis. let-721 encodes electron transfer flavoprotein-ubiquinone oxidoreductase (C05D11.12). This protein is essential for proper mitochondrial electron transfer. C05D11.12 encodes one of many electron transport proteins found in this region. Two NADH ubiquinone oxidoreductases (C16A3.4 & T26A5.3) are also in this region. We have two alleles of let-721, one arrests as a sterile adult while the other is a maternal effect lethal. The Mel allele is male rescueable. We have made a LET-721:GFP fusion protein and are currently looking at its localization.

24 May 1999 15:50 692 692 The PTEN homologue DAF-18 acts in the DAF-2 insulin receptor-like metabolic signaling pathway

1999 International Worm Meeting abstract 641 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The PTEN homologue DAF-18 acts in the DAF-2 insulin receptor-like metabolic signaling pathway S Ogg, G Ruvkun Department of Molecular Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School, Boston, MA 02114. An insulin-like signaling pathway, from the DAF-2 insulin receptor homologue, the AGE-1 phosphoinositide 3-kinase, the AKT-1/AKT-2 serine/threonine protein kinases to the DAF-16 Fork head transcription factor, regulates the metabolism, development and life span of Caenorhabditis elegans. DAF-2 insulin receptor is likely to signal via the AGE-1 PI 3-kinase which generates lipid second messengers (PI-3,4-P2 and PI-3,4,5-P3 ) that activate the Akt kinases. The Akt kinases act redundantly to antagonize the activity of DAF-16 FKHR (Paradis and Ruvkun, 1998). AKT-1 can directly phosphorylate DAF-16 in vitro and AKT-1/AKT-2 are likely to directly antagonize DAF-16 FKHR in vivo. Inhibition of daf-18 gene activity bypasses the normal requirement for AGE-1 PI 3-kinase and partially bypasses the need for DAF-2 insulin receptor signaling. Using RNA interference, we have demonstrated that the suppression of age-1 mutations by a daf-18 mutation depends on AKT-1/AKT-2 signaling. This suggests that DAF-18 acts between the AGE-1 PI 3-kinase and the Akt kinases. Our molecular characterization revealed that daf-18 encodes a homologue of the human tumor suppressor PTEN (MMAC1/TEP1). PTEN has both tyrosine phosphatase activity and 3-phosphatase activity towards PI-3,4-P2 and PI-3,4,5-P3 (Maehama and Dixon, 1998). DAF-18 PTEN likely antagonizes AGE-1 PI 3-kinase and limits AKT-1 and AKT-2 activation by dephosphorylating AGE-1 PI 3-kinase generated lipid second messengers. DAF-18 PTEN may act to modulate or down regulate DAF-2 insulin receptor/AGE-1 PI 3-kinase signaling and/or isolate distinct PI 3-kinase signaling complexes. The action of daf-18/PTEN in this metabolic control pathway suggests that PTEN may modulate mammalian insulin signaling and may be variant in diabetic pedigrees. These results also suggest a mechanism by which human PTEN suppresses tumor formation by limiting cell growth and survival signals through Akt.

Maehama, T. and Dixon, J.E. (1998). J. Biol. Chem. 273:13375-13378. Paradis, S. and Ruvkun, R. (1998). Genes Dev. 12:2488-2498.

24 May 1999 15:50 693 693 KEL-1, a homologue of Drosophila Kelch, is essential for larval development

1999 International Worm Meeting abstract 642 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. KEL-1, a homologue of Drosophila Kelch, is essential for larval development M Ohmachi 1 , A Sugimoto 1 , Y Iino 2 , M Yamamoto 1

1 Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo, 113-0033, Japan. 2 Molecular Genetics Research Laboratory, University of Tokyo, Hongo, 113-0033, Japan.

We isolated C. elegans cDNAs that could trans-complement the fission yeast mes1 mutation, which causes a defect in the second meiotic division in the microbe. Here we report characterization of one of these cDNAs and its chromosomal gene, which we named kel-1. The kel-1 gene encodes a 618-amino-acid polypeptide that shows similarity to Kelch-related proteins, including Drosophila Kelch, which is known to be essential for oogenesis, and mammalian NRP/B, which has been implicated in neuronal differentiation. Kelch-related proteins carry two conserved motifs, which are named the BTB/POZ and the kelch repeat domains, respectively. KEL-1 contains both of them. To investigate the function of the kel-1 gene in C. elegans, animals mutagenized with UV-trimethylpsoralen were screened for kel-1 deletion alleles. One deletion allele, kel-1(pe201), was isolated successfully. Loss of kel-1 function was found to cause growth arrest at an early larval stage, most likely at the beginning of L2. The kel-1 deletion mutant appeared to be normal in morphology, locomotion and pumping action, at least for a short while after hatching. However it failed to convey foods effectively to intestine, hardly increased in size, and died within ten days. Analyses using immunostaining and reporter gene expression indicated that kel-1 are expressed almost exclusively in the g1 pharyngeal gland cells during late embryogenesis and at all developmental stages thereafter. These gland cells have been suggested to secrete materials aiding digestion. Taken together, we conclude that KEL-1 protein fulfills an essential function for feeding in pharyngeal g1 gland cells.

24 May 1999 15:50 694 694 flr-2 gene, which controls the growth rate of flr-1, flr-3 and flr-4 mutants, encodes a putative TGF-b antagonist.

1999 International Worm Meeting abstract 643 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. flr-2 gene, which controls the growth rate of flr-1, flr-3 and flr-4 mutants, encodes a putative TGF-b antagonist. A Oishi 1 , M Take-uchi 1,2 , T Ishihara 1,2 , I Katsura 1,2

1 Struct. Biol. Cent., Natl. Inst. Genet., Mishima, Shizuoka 411-8540, Japan. 2 Dept. Genet., Grad. Univ. Adv. Stud., Mishima, Shizuoka 411-8540, Japan.

flr (fluoride-resistant) mutations are classified into two categories. Class 1 mutations, which map in flr-1, flr-3 and flr-4 genes, show pleiotropic phenotypes, such as strong resistance to fluoride ion, slow growth, short defecation cycle periods, synthetic abnormality in dauer formation, etc. (Katsura et al.: Genetics 136: 145, 1994). These genes encode an ion channel of the DEG/ENaC superfamily, a kinase-like molecule, and a novel Ser/Thr protein kinase, respectively, which seem to constitute a regulatory system in the intestine (Take-uchi et al.: PNAS 95: 11775, 1998; our unpublished results). Class 2 mutations, which map in flr-2 and flr-5, confer weak resistance to fluoride ion and suppress the slow growth and synthetic dauer abnormality (but not the defecation abnormality) of class 1 mutations. To elucidate how the class 1 flr regulatory system controls diverse functions, we are cloning class 2 flr genes, which are thought to act downstream of class 1 genes in the regulatory cascade. We cloned flr-2 gene by transformation rescue, and found that the hypothetical gene product has homology to some TGF-b antagonists such as DAN, Gremlin, and Cerberus. This may be reasonable, because suppression of the slow growth phenotype of class 1 flr mutants by flr-2 mutations can be explained if FLR-2 antagonizes a growth factor. Two mutations in flr-2 were located at conserved Gly and Cys residues, respectively. We are now investigating which cells synthesize and secrete FLR-2, which of the four TGF-b homologs in C. elegans might interact with FLR-2, and how class 1 flr genes regulate the synthesis or activity of FLR-2 or those of a protein that is antagonized by FLR-2.

24 May 1999 15:50 695 695 Isolation of new thermotaxis-defective mutations using the interaction with dafmutations in TGF-b-like signaling pathway

1999 International Worm Meeting abstract 644 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Isolation of new thermotaxis-defective mutations using the interaction with dafmutations in TGF-b-like signaling pathway Y Okochi, M Okumura, I Mori Laboratory of Molecular Neurobiology, Division of Biological Science, Nagoya University. We are interested in understanding the underlying molecular mechanisms of thermotaxis. Toward this end, we want to isolate and characterize as many thermotaxis-defective mutants as possible, and then identify molecular components involved in different aspects of thermotaxis, including thermosensation, temperature memory formation, and learning. Recently, thermotaxis-defective ttx-3mutation was found to affect the temperature-sensitive daf-c phenotype of daf-7mutants, by enhancing recovery from dauers at 25-degree as well as dauer formation at 15-degree (1). The ttx-3gene encodes a LIM homeodomain-containing transcription factor and is expressed in AIY (1), an essential interneuron for thermotaxis. This result implicated a new screening method for isolating thermotaxis-defective mutants (2). Since the daf-7gene encodes a TGF-b-like ligand, do other daf-c mutations that disrupt TGF-b-like signaling pathway interact with the ttx-3mutation, and if so, do other ttxmutations affect those daf-c mutations? We first addressed these questions, and investigated the interactions between daf-7 and ttx-3, daf-1 and ttx-3, daf-7and ttx-1, and daf-1and ttx-1. The ttx-1mutation causes animals to seek cold temperatures as does ttx-3 (cryophilic phenotype), and is likely to disrupt the function of AFD (see abstract by Sasakura et al.), a thermosensory neuron for thermotaxis. The daf-1gene encodes a TGF-b-like Type I receptor. We demonstrated that both ttx-1and ttx-3mutations indeed interact with both daf-7and daf-1mutations. We then conducted a genetic screen to isolate new thermotaxis-defective mutations that could possibly interact with the daf-1(ts)mutation. Several candidate mutants were isolated so far. Of these, ttx(nj2)mutation causes athermotactic (non-temperature-responsive) phenotype, and ttx(nj1) and ttx(nj3) mutations both cause thermophilic (heat-seeking) phenotype. Also, these three mutations were found to cause abnormal chemotactic response to NaCl. The genetic analysis of these mutations and further isolation of thermotaxis-defective mutants using the daf-7 mutation are underway.

(1) Hobert et al., 1997, Neuron 19, 345-357. (2) Hobert et al., 1997, ICM.

24 May 1999 15:50 696 696 Chemosensory Control of Surface Antigen Switching

1999 International Worm Meeting abstract 645 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Chemosensory Control of Surface Antigen Switching DP Olsen, LJ Miceli, SM Politz Dept. of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA 01609 USA Nematodes restrict specific surface molecules to a particular developmental time or stage and can switch surface molecules in response to environmental shifts, such as entry of a parasite into a new host. We identified a surface antigen switch in which wild-type C. elegans is induced to display an L1 surface antigen at a later larval stage (inducible larval display or ILD) when hatched and grown on agar containing an extract of liquid culture supernatant. We also identified mutations that result in nonconditional display of this antigen on all four larval stages (constitutive larval display or CLD). These include mutations in srf-6 and in previously identified dauer-constitutive genes involved in signal transduction during dauer larva formation. These facts support the idea that environmental signals induce surface antigen switching. Although the CLD mutations appear to affect a facultative switch similar to dauer formation, surface antigen switching and dauer formation can be separately blocked or induced, suggesting that they are controlled differently. For example, srf-6 mutations apparently do not affect dauer formation. To help define a distinct role for srf-6, we examined double mutants combining srf-6(yj13) with mutations in the ts daf-c genes daf-1, daf-4, daf-7, or daf-14. Striking increases in the fraction of individuals forming dauer larvae at 16°C (a permissive temperature for these daf-c mutants when tested alone) were observed with all of these double mutants. These results suggest that srf-6 acts in parallel with the TGF-b signaling pathway that includes these daf-c genes. By testing ILD in mutants, we have demonstrated that this response requires intact ciliated sensory nerve endings. Che-3 mutations resulted in greatly reduced ILD, but daf-6 or osm-3 mutants showed ILD responses similar to wild type. All sensory cilia are abnormal in che-3 mutants, while in daf-6 and osm-3 mutants, only the detection of water-soluble substances by a subset of amphid neurons is affected. Taken together, these results suggest that ILD requires some ciliated sensory nerve endings, but not the exposed amphid neurons that detect water-soluble chemicals. We are currently testing additional mutants to further define functional requirements for ILD.

24 May 1999 15:50 697 697 Developmental regulation and mechanism of action of the lin-4 translational repressor RNA.

1999 International Worm Meeting abstract 646 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Developmental regulation and mechanism of action of the lin-4 translational repressor RNA. P Olsen 1 , R Feinbaum 2 , F Liu 1 , HS Silverman 1 , E Moss 1,3 , V Ambros 1

1 Dartmouth College Department of Biology Hanover NH 03755. 2 Mass General Hospital Department of Molecular Biology, Boston, MA. 3 Fox Chase Cancer Center, Philadelphia, PA. lin-4 encodes a small RNA that is complementary to sequences in the 3’ untranslated regions of lin-14 and lin-28 mRNAs and that acts to down-regulate LIN-14 and LIN-28 during the L1 and L2. This down-regulation is essential for the proper timing of events in C. elegans larval development. The temporal profile of lin-4 RNA accumulation determines the timing of LIN-14 and LIN-28 protein down-regulation, and thereby controls the timing of postembryonic developmental events. To explore the role of lin-4 upstream sequences in the timing of lin-4 expression, we tested lin-4 deletion constructs for their expression in transgenic worms. These experiments have identified elements essential for lin-4 transcription, and also some apparent negative regulatory elements. A one-hybrid screen for C. elegans proteins that bind to essential lin-4 upstream sequences in yeast cells has identified a zinc-finger DNA binding protein that specifically recognizes lin-4 DNA. We are testing for potential developmental roles for this protein in lin-4 regulation. We have investigated the mechanism of lin-4 RNA action by examining the fate of lin-14 mRNA in vivo during the time that lin-4 RNA is expressed. Our results suggest that the rate of synthesis, the state of polyadenylation, the abundance in the cytoplasmic fraction, and the polysomal sedimentation profile of lin-14 mRNA do not change in response to the accumulation of lin-4 RNA. We conclude that lin-4 RNA represses LIN-14 protein production by acting after initiation of lin-14 mRNA translation. Surprisingly, lin-4RNA seems to be required for translational repression of LIN-14 and LIN-28 only in the presence of lin-14 and lin-28 activities, suggesting that lin-4RNA represses translation by modulating a mutual translational feedback circuit between lin-14 and lin-28. The potential roles of heterochronic genes other than lin-4 in this feedback pathway are being explored.

24 May 1999 15:50 698 698 Habituation of C. elegans for the touch sensitivity

1999 International Worm Meeting abstract 647 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Habituation of C. elegans for the touch sensitivity K Omata, F Yonezawa, K Kawamura Faculty of Science and Technology, Keio University, Shin-Kawasaki Mitsui Bldg. W.3F, 890-12 Kashimada, Saiwai-ku, Kawasaki 211-0958, Japan In order to study the habituation of C. elegans for the touch sensitivity, we carry out computer simulations, in which the neural circuit is formed by making use of the data table constructed recently by Oshio et al [1]. The i-th neuron is connected with the neighboring j-th neuron through the coupling strength Kij , which is varied dynamically by the Hebb rule. Note that Kij is not necessarily equal to Kji because there are one-way connections between the neurons by chemical synapses. As a reference state, we first deal with the neural circuit consisting only of the neurons ALM, AVM, PLM, PVM, AVA, AVB, PVC, AVD, A and B, that are related to the forward and backward movement directly. We give periodic stimuli to the sensory neurons PLM, PVM, and monitor the response of the motor neuron A. We find that the frequency of the response decreases with time, which indicates that the habituation to the touch sensitivity actually takes place. As one deviation from the reference state, we kill the inter-neuron AVD, and perform the same analysis described in the above. There is a tendency that the decay of the response curve becomes faster, and the habituation is enhanced. As the other deviations, there are several possibilities of killing the inter-neurons AVA, AVB, PVC and/or AVD. We discuss the enhancement of the habituation in relation with the recent experimental results by Hosono.

[1.] K. Oshio, S. Morita, Y. Osana and K. Oka; ‘‘C. elegans synaptic connectivity data’’, Technical Report, CCEP, Keio Future No.1 (1998)

24 May 1999 15:50 699 699 Characterization of the neural circuit of C. elegans: model analysis of the touch response

1999 International Worm Meeting abstract 648 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of the neural circuit of C. elegans: model analysis of the touch response K Omata, F Yonezawa, K Kawamura Faculty of Science and Technology, Keio University, Mitsui Bldg. W.3F, 890-12 Kashimada, Saiwai-ku, Kawasaki 211-0958, Japan In order to characterize the neural circuit of C. elagans, we construct a simple model by making use of the data table completed recently by Oshio et al . [1]. We assume that the signal of a neuron is calculated by the product of the signals from the neighboring neurons, and we investigate the touch sensitivity to continuous stimuli described by sinusoidal functions as defined in the rage from 0.0 to 1.0. We calculate the responses of the motor neurons by changing the frequencies of the stimuli. In our calculations, we change only the frequency wPLM for the input signal to the sensory neuron PLM, while the frequency for the other sensory neurons ALM, AVM and PVM is fixed to be a same value w0 . We show that the output signals from the motor neurons A and B oscillate in time. We measure the minima of the oscillation for each wPLM value. The plot of the minima versus wPLM shows different hehaviors for the case of the neuron A and B. As for the signals from the neuron A, the values of the minima are widely distributed between 0.0 and 1.0 for all wPLM . As for the signals from the neuron B, on the other hand, the features are different for different wPLM values. (a) In the high frequency region of wPLM / w0 0.4, the oscillation is simple harmonic and there exists only one minimum value (Imin = 0.0). (b) As wPLM / w0 is decreased, another minimum appears at a certain frequency, and the bifurcation takes place discontinuously. This behavior is different from usual continuous bifurcation observed in nonlinear systems. After a few discontinuous branching occur, signals with five periods can be seen in the intermediate frequency region of 0.3 wPLM / w0 <0.4. (c) In the low frequency region of >wPLM / w0 <0.3, the minimum values of the signal are widely distributed as in the case of the output signals from the motor neuron A. The similar behaviors are also observed even when the input-signal frequencies of the four sensory neurons are varied. From the analysis of these results, we discuss qualitative features of the circuit.

[1] K. Oshio et al.; ‘‘C. elegans synaptic connectivity data’’, Technical Report, CCEP, Keio Future No.1 (1998).

24 May 1999 15:50 700 700 A screen for mutations that affect the localization of CED-4

1999 International Worm Meeting abstract 649 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A screen for mutations that affect the localization of CED-4 D Omura, B Hersh, F Chen, B Horvitz HHMI, Dept. Biology, MIT, Cambridge, MA 02139 In the programmed cell death pathway, ced-4 is a key activator of theC. elegans caspase ced-3. Genetically, ced-4 is upstream of ced-3 and is necessary for normal programmed cell death to occur. egl-1 and ced-9 act upstream of ced-4 in the genetic pathway and are involved in activating and inhibiting ced-4 activity, respectively. Previous work has shown that the localization of ced-4 seems intimately tied to its activity. In cells that are not undergoing programmed cell death, CED-4 is localized to mitochondrial membrane surfaces. When these cells are induced to die by overexpressing egl-1 or by ced-9 loss-of-function, CED-4 is localized to the perinuclear membrane1 . CED-4 localization appears to correlate with the life-or-death decision of a cell, but how and why the localization occurs is unknown. When overexpressed, a CED-4::GFP fusion protein appears to localize to the mitochondria and perinuclear region of cells that are not undergoing programmed cell death. To identify factors important for the localization of CED-4, we will screen for mutants in which this CED-4::GFP fusion protein fails to localize to the perinucleus of viable cells.

1 Hersh, B., Chen, F., Conradt, B., Zhou, Z., and Horvitz, HR (1999) 12th International C. elegans Meeting

24 May 1999 15:50 701 701 Protein Expression Pattern Analysis of Maternal mRNAs.

1999 International Worm Meeting abstract 650 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Protein Expression Pattern Analysis of Maternal mRNAs. S Onami 1 , T Nagaoka 1 , Y Kohara 1,2

1 CREST, JST. 2 Genome Biology Lab., National Institute of Genetics, Mishima 411-8540, Japan.

Our cDNA/expression pattern project has identified many maternal mRNAs whose function is unknown. To study their function, we have started protein expression pattern analysis. Our main interest is cell fate determination in early embryogenesis. Thus, we focus on genes whose mRNA is maternally supplied, and 1) disappears before 18cell-stage or 2) localizes to specific cells. We picked up 100 such genes out of 2000 genes based on our in situ hybridization results. We are raising rat antisera against all of these genes using bacterially expressed partial proteins (100-150 amino acids). Until now, we have obtained them for 54 genes and stained embryos and gonads. Fifty antisera (93%) showed detectable staining. The sub-cellular staining patterns are classified into perinuclear cytoplasm and plasma membrane (8), cytoplasmic granules (7), nucleus and plasma membrane (6), P-granules (6), cytoplasm (5), nucleus (5), nuclear membrane (5), plasma membrane (5), and egg shell (3). Thirty-one antisera (57%) showed tissue-specific staining before hatching and 9 of them showed cell-specific staining before 18cell-stage. Twelve antisera (22%) stained specific regions of the gonad. We also analyzed RNAi phenotype for those 100 genes (see Hirono et al.). We found 4 genes whose RNAi resulted in germ line defects out of 11 genes whose immunostaining pattern looked like P-cell specific. We believe that this approach provides fruitful information about maternal mRNAs. We are preparing to release these data on our WWW site (http://watson.genes.nig.ac.jp:8080/db/).

24 May 1999 15:50 702 702 Genetic and biochemical studies on modulators of unc-60 in body wall muscle

1999 International Worm Meeting abstract 651 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic and biochemical studies on modulators of unc-60 in body wall muscle S Ono 1 , DL Baillie 2 , KB Mercer 1 , GM Benian 1

1 Depts of Pathology and Cell Biology, Emory Univ., Atlanta, GA 30322, USA. 2 Inst. of Molecular Biology and Biochemistry, Simon Fraser Univ., Vancouver, B.C. V5A 1S6, Canada.

The C. elegans unc-60 gene encodes two functionally distinct isoforms of ADF/cofilin. We have recently shown that one of the gene products, UNC-60B, is specifically required for proper assembly of actin into myofibrils. Biochemical analysis of UNC-60B has suggested that it enhances actin filament turnover by accelerating both polymerization and depolymerization and by severing actin filaments. However, during myofibril assembly, many other factors are involved and coordinately regulate the assembly of highly organized functional structures. We present our genetic and biochemical analysis of candidate modulators for UNC-60B. The sup-12 is a recessive suppressor of unc-60. Therefore, myofibrils of sup-12;unc-60 double mutants appear normal. Although sup-12 itself does not show an obvious phenotype in muscle, we found that sup-12(st89) causes a remarkable reduction in the protein level of UNC-60B but not of UNC-60A. In wild-type muscle, UNC-60B was found in the myofibrils, while, in sup-12 mutant, UNC-60B was largely dissociated from the myofibrils. These results suggest that the sup-12 gene product is a modulator of the function of UNC-60B or other actin-binding proteins. In addition to the currently available sup-12 alleles, st89 and st203, we have recently isolated 4 new alleles of sup-12 that should help understand the function of the sup-12 gene. The identification of the sup-12 gene by transgenic rescue is in progress. Tropomyosin (TM) has been reported to compete with ADF/cofilin for actin binding in other systems. Since TM is a major component of thin filaments of myofibrils, we tested the possibility of competition between UNC-60B and TM in vitro. Using actin and TM from wild-type C. elegans, we found that TM tightly bound to F-actin and inhibited binding of UNC-60B to F-actin. However, partial depolymerization of F-actin by UNC-60B was not inhibited by TM. These results suggest that, in the presence of TM, access of UNC-60B to the side of F-actin is inhibited, while UNC-60B can enhance actin dynamics from the filament ends. We are in the process of characterizing the effects of mutations in the TM gene, tmy-1/lev-11 on Unc-60 phenotypes.

24 May 1999 15:50 703 703 Behavioral, Structural and Genetic Analyses of unc-jd19: A Gene Affecting the Development of the DD Motoneurons.

1999 International Worm Meeting abstract 652 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Behavioral, Structural and Genetic Analyses of unc-jd19: A Gene Affecting the Development of the DD Motoneurons. K Oommen, B Walthall Department of Biology, Georgia State University, Atlanta, GA 30303 We are interested in characterizing genes that contribute to developmental and functional features of the type D locomotory neurons. The DD and VD motoneurons have similar morphologies and functions, however, they have different synaptic patterns and arise at different developmental stages from distinct lineages. The embryonic DD motoneurons receive dorsal inputs and innervate ventral muscle during the L1 stage. However during the L1-L2 molt, they reverse their polarity so that they receive ventral input and innervate dorsal muscle. During this molt a large number of postembryonic motoneurons, including the VD motoneurons are incorporated into the circuit. The VD motoneurons receive dorsal input and innervate ventral muscle, the initial synaptic pattern of the DD motoneurons. From this point on the DD and VD motoneurons form a reciprocal inhibitory network onto dorsal and ventral body wall muscles that contributes to the animal’s sinuous pattern of locomotion. unc-jd19 mutants coil ventrally upon hatching and after the L1-L2 molt they coil dorsally. The stage specific change in the uncoordination suggests a defect in the DD motoneurons. This is supported by epistasis tests in which the "shrinker" phenotype of unc-30 and unc-25, two mutants that result in the absence of GABA in the DD and VD motoneurons, masks the coiling phenotype of unc-jd19. Examination with Nomarski optics has shown a disruption in the stereotyped positions of cell bodies of ventral cord motoneurons in L1 animals as well as a reduction in motoneuron number. Results using GABA immunohistochemistry in adult animals suggest that some, if not all, of the missing neurons are DD motoneurons. In addition, we will present our map data for this gene, which apparently does not correspond to any previously mapped uncoordinated gene on chromosome III.

24 May 1999 15:50 704 704 Neural Network Model for Touch Sensitivity in Caenorhabditis elegans

1999 International Worm Meeting abstract 653 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Neural Network Model for Touch Sensitivity in Caenorhabditis elegans Y Osana 1 , K Oshio 1 , S Morita 2 , Y Funabashi 1 , K Oka 1 , M Hagiwara 1 , K Kawamura 1

1 Faculty of Science and Technology, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Japan. 2 Faculty of Engineering, Shizuoka University, 3-5-1, Johoku, Hamamatsu, Shizuoka, 432-8561, Japan.

In this research, we propose an artificial neural network model for touch sensitivity in Caenorhabditis elegans. The neural circuit for touch sensitivity in C.elegans has been examined in detail and is constituted by 66 neurons grouped in 14 classes. The proposed neural network model is directory inspired from the real connectivity data in C. elegans and is based on one of the suitable artificial neural networks, Boltzmann machine. This model has visible units (input units and output units) and invisible units (hidden units). In the nerve system of C. elegans, we considered that the sensory neurons which receive touch stimulus correspond to the input units, the interneurons correspond to hidden units, and the motor neurons correspond to output units. In this research, for simplicity, each unit in the proposed neural network model corresponds to a neuron class instead of a neuron. That is, the neural network model is composed of four input units (ALM, AVM, PLM, PVM), five hidden units (AVA, AVB, AVD, LUA, PVC) and five output units (AS, DA, DB, VA, VB). The values of connection weights which correspond to synapses are determined under the constraint of the connectivity observed in C. elegans by Hebbian-like learning algorithm as same as in the conventional Boltzmann machine . We carried out a series of computer simulations and showed that the proposed model replicates the similar behaviors observed in the real C.elegans. In the simulations, after the values of connection weights in the proposed model are determined, when the sensory neurons for anterior touch ALM and AVM are stimulated, the motor neurons for backward movement AS, DA and VA are excited. In the same way, when the sensory neurons for posterior touch PLM and PVM are stimulated, the motor neurons for forward movement DB and VB are excited. We also compared the lesion test in the proposed model with the laser ablation test in the real C.elegans. As in real C.elegans, the simulation results show that AVD and PVC interneurons are essential for backward and forward movement, respectively.

24 May 1999 15:50 705 705 Analysis of neuronal connectivity of C. elegans using random walker

1999 International Worm Meeting abstract 654 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of neuronal connectivity of C. elegans using random walker K Oshio 1 , Y Funabashi 1 , S Morita 2 , Y Osana 1 , K Oka 1 , K Kawamura 1

1 Faculty of Science and Technology, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Japan. 2 Faculty of Engineering, Shizuoka University, 3-5-1, Johoku, Hamamatsu, Shizuoka, 432-8561, Japan.

From the detailed report of White et al. and Albertson et al., we have almost complete knowledge about the synaptic connectivity of C. elegans with the type of synapse (electric junction or chemical synapse). However, the type of each chemical synapse (excitatory or inhibitory one) is not described. Conventional electrophysiological methods for C. elegans is difficult because the size of the neurons is too small to penetrate the intracellular electrode. On the other hand, computational studies of neuronal circuit are now possible by virtue of the above mentioned elaborate experimental studies of neuroanatomists. We have built a new data base of the whole neuronal circuit including pharyngeal neurons only from the article of White et al. and Albertson et al. There exist two other data bases to the knowledge of the present authors. The first data base was constructed by Achacoso and Yamamoto who also analyzed the properties of the network. Another data base was constructed from the article of White et al. by Durbin, which is available on the internet homepage. To begin understanding signal processing on the nervous system, we have investigated the neuronal connectivity by putting random walkers on certain neurons of the network. Here we ignore internal structure of neurons. Random walker is a particle which moves among neurons randomly, and can be considered to be transmitted signal. In our simulation, random walkers are put on certain sensory neurons at each time step, this corresponds to stimulation which sensory neurons accept. We removed random walkers at certain motor neurons, this means, for instance, signals are transmitted to muscles which cause normal action. As a result, we have found that the degree of relation of each neuron for input neurons can be known from the probability to find random walker, although the difference between excitatory and inhibitory of chemical synapses is not taken into account. The simulational results will be shown comparing with real function such as the touch sensitivity.

E-mail: [email protected]

24 May 1999 15:50 706 706 lin-18, a gene necessary for vulval cell orientation, encodes a Ryk-like predicted transmembrane tyrosine kinase

1999 International Worm Meeting abstract 655 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. lin-18, a gene necessary for vulval cell orientation, encodes a Ryk-like predicted transmembrane tyrosine kinase ML Goodson 1 , CJ Goodwin 1 , S Gharib 2 , PW Sternberg 2 , WS Katz 1

1 Department of Biochemistry, University of Kentucky College of Medicine, Lexington KY. 2 Howard Hughes Medical Institute and Division of Biology, California Institute of Technology, Pasadena CA.

Mutations in lin-18 affect two aspects of cell asymmetry during vulval development. They reverse the orientation of a pair of sister cells that have different fates, and they reverse the direction of cell migration and asymmetric shape changes in cells descended from the affected sister cells. We have cloned lin-18 by germline transformation experiments. It encodes a predicted receptor tyrosine kinase, similar to Ryk of mammals and Derailed of Drosophila. We have sequenced three lin-18 alleles. e620 and n1051 have nonsense codons in the predicted extracellular domain. ga75 contains a short internal deletion that is predicted to generate a truncated protein lacking the intracellular domain. Analysis of LIN-18 expression and localization is in progress. lin-18 has a synergistic phenotype with the predicted Wnt-receptor lin-17, and with lin-45 raf, indicating that multiple signals are involved in achieving correct cell interactions in the vulva.

24 May 1999 15:50 707 707 A Muv connection to early embryogenesis

1999 International Worm Meeting abstract 656 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A Muv connection to early embryogenesis BD Page, DA Waring, JR Priess Howard Hughes Medical Institute and FHCRC FHCRC, Seattle, WA In screens for maternal-effect lethal mutants defective in specifying early cell fates, we isolated one allele each of genes we call mex-2 and mex-4. The mex-2 and mex-4 mutants have very similar early embryonic phenotypes. In both mex-2 and mex-4 mutant embryos, the AB blastomere produces tissues similar to those normally generated by the EMS blastomere. In wild-type embryos, an isolated AB produces neurons and hypodermal cells. In contrast, an isolated AB from a mex-4 embryo produces body wall muscle (90%), pharyngeal (90%) and intestine (10%) cells. Such a phenotype is suggestive of misregulation of SKN-1, a protein important for specifying these three tissue types. Indeed both mex-2 and mex-4 embryos have a higher than wild-type level of SKN-1 protein in AB and its daughters. This result suggests that mutations in these genes affect expression of maternal transcripts. The mex-4 gene encodes a homologue of the vertebrate DP-1 protein. DP-1 is a member of a transcription heterodimer complex and its dimerization partner is E2F. Multiple E2F homologues are encoded in the C. elegans genome; one of these genes is very close to the genetic position of the mex-2 gene. I have found that the mex-2 mutant has a missense mutation in the E2F gene. How do E2F and DP-1 affect early cell fate in the embryo? In other organisms, DP-1 and E2F-1 cooperate to link cell cycle progression with transcription. We have not observed any cell cycle defects in mex-4 mutant embryos, and the gonad of a mex-4 hermaphrodite appears normal. Thus, we think that these genes are not affecting cell fates by influencing the cell cycle. Both mex-2 and mex-4 genes have roles in postembryonic development. These genes were independently isolated by the Horvitz lab based on their role in the synthetic multivulva pathway. Since ras mutations can suppress the synMuv phenotype, I tested whether ras mutations could also suppress the embryonic defect of mex-4 mutants. Preliminary experiments indicate that suppression occurs. This result suggests that parallels exist between vulva induction and embryogenesis.

24 May 1999 15:50 708 708 Characterization of bac-1 (big anchor cell)

1999 International Worm Meeting abstract 657 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of bac-1 (big anchor cell) RE Palmer, KB Brown, BP Gupta, PW Sternberg California Institute of Technology, Pasadena, CA, 91125 We are using the C. elegans hermaphrodite vulva as a model system to study the mechanism of cell signaling and fate specification. During development, the Vulva Precursor Cells (VPCs) create an invariant pattern (3°-3°-2°-1°-2°-3°). There is machinery in place to make each VPC different from its companions. A new mutation, bac-1(sy329), was identified in an EMS mutagenesis screen of 12,000 haploid chromosome sets for mutants with exploding vulvae. sy329 is completely recessive and shows incomplete penetrance (40-75%) for vulval defects in the homozygotes. Approximately 40% of sy329 hermaphrodites have exploding vulvae. An additional 15% of the worms have protruding or multi-vulvae. Vulval lineage analysis of sy329 reveals that 40% of the hermaphrodites have extra primary-derived cells. Often, additional Pn.p cells also form vulval tissue. These vulval defects were suppressed by mutations in the well-characterized signaling pathway required for vulval formation. Homozygote hermaphrodites and males of sy329 are completely sterile and exhibit several somatic gonadal defects. There is no germ cell differentiation in either hermaphrodites or males. Microscopic examination of sy329 hermaphrodites with Nomarski optics revealed that the germ cell nuclei fuse. DAPI staining of sy329 gonads showed that these large ’germ’ cells contained a massive excess of chromosomal DNA. In sy329 hermaphrodites, the distal tip cells (DTC) and the anchor cell (AC) are noticeably enlarged. The enlarged DTC phenotype is highly penetrant whereas the enlarged AC phenotype is less penetrant. In addition, it is not uncommon to observe two DTC per gonad arm, and in some animals, two AC. Since the AC is required for the formation of the vulva, we postulate that the enlarged AC may be responsible for the extra vulval induction observed in sy329. Linkage analysis placed the sy329 mutation on LG I. Deficiency and three-factor mapping placed the sy329 between unc-13 and lin-10. sy329 in trans to the deficiencies nDf24 or nDf25 are indistinguishable from sy329 homozygotes. We are presently in the process of cloning the gene to understand its function in vulval cell fate.

24 May 1999 15:50 709 709 Phylogenetic analysis of entomopathogenic nematode / bacterium symbiotic complexes by using PhastSystem PAGE PCR-RFLP of rDNA spacer sequences

1999 International Worm Meeting abstract 658 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Phylogenetic analysis of entomopathogenic nematode / bacterium symbiotic complexes by using PhastSystem PAGE PCR-RFLP of rDNA spacer sequences H Pamjav 1 , D Triga 1 , E Szállas 1 , A Fodor 1 , Z Buzás 2

1 Department of Genetics, Eötvös University, Budapest, Hungary. 2 Agrobiotechnology Research Center, Gödöllõ, Hungary.

Entomopathogenic nematode (EPN)species belonging to theSteinernema and Heterorhabditis genera are closely related to C. elegans (Blaxter et al., 1998). They form an intimate symbiotic complex with bacteria Xenorhabdus spp. and Photorhabdus spp., respectively, which are closely related to E. coli. The symbiosis is developmentally regulated: there is a direct cell-cell contact between the gut cell of the (infective) dauerlarva and the primary phase variant of the symbiotic bacteria. The symbiosis is taxon specific. Individual Xenorhabdus species used as symbionts by each Steinernema species and individual strains of Photorhabdus luminescens used as symbionts by each Heterorhabditis strains belonging to different species. The phylogenetic relations between species and strains have been analysed by RFLP of the internally transcribed spacer region of the respective rRNA operon. The PCR-amplified DNAs were digested by different restriction enzymes and the restriction patterns obtained by PhastSystem PAGE were compared. On the basis of the comparative analysis of the ITS1 - ITS2 patterns nematodes could be identified at species level. Xenorhabdus species could also be identified on the basis of the comparative analysis of the spacer region of the 16S - 23S rRNA operon. P. luminescens strains, belonging to different subclusters (Szállás et al., 1997) could be separated on the basis of the RFLP pattern of the spacer region of the 16S - 23S rRNA operon.

Adams, B.J. (1998). Species conception and the evolutionary paradigm in modern nematology. J. nematol. 30, 1-21. Adams, B.J., Burnell, A.M. @ Powers,T.O. (1998). A phylogenetic analysis of Heterorhabditis based on ITS 1 DNA sequence data. J. nematol. 30, 22-39. Blaxter,R. et al.(1998).Molecular evolutionary framework for Phylum nematoda. Nature 392,71-75. Szállás E., Koch, H., Fodor,A.,Szentirmai, A., Nealson,K.H. Stackebrandt, E.(1997).Phylogenetic evidence for taxonomic heterogeneity of P.luminescens. IJSB 47, 402-407.

24 May 1999 15:50 710 710 4D Imaging System to Study C. elegans Development

1999 International Worm Meeting abstract 659 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. 4D Imaging System to Study C. elegans Development A Papp 1 , M Palopoli 2

1 Tritech Research, 2961 Veteran Avenue, Los Angeles, CA 90064. 2 Biology Department, Bowdoin College, 6500 College Station, Brunswick, ME 04011.

Whereas C. elegans wild-type development is relatively invariant, mutations can alter development in many ways. We have developed a flexible, lower-cost 4-dimensional imaging system to aid in the observation of development, toward an understanding of its basis in cell-intrinsic processes and cell-cell interactions. A microscope system with Differential Interference Contrast optics and video capabilities has been fitted with computer automation equipment to permit automatic optical sectioning (3D) over time (4D).

The Tritech RoboScopeTM system provides a simple user-programmable environment and hardware to control the following microscope functions: Focus Control for optical sectioning; Lamp On/Off Control so as not to damage specimens by heating between image acquisition points; UV Lamp Shutter-open/Shutter-close Control so as not to damage specimens between image acquisition points, and not to damage the UV source by frequent on/off cycles; and Serial Image Capture with storage to disk. The programming environment also provides for Pausing, Looping, and for the user to change program parameters while the program is running to make any desired adjustments. A computer-controlled X-Y stage is also available, making it possible to collect 4D data from more than one specimen in a single experiment.

24 May 1999 15:50 711 711 Characterization of Calreticulin (Crt-1), a calcium binding protein, in Caenorhabditis elegans

1999 International Worm Meeting abstract 660 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of Calreticulin (Crt-1), a calcium binding protein, in Caenorhabditis elegans B Park, D Lee, J Ahnn Department of Life Science, Kwangju Institute of Science & Technology, Kwangju, 500-712, KOREA C. elegans crt-1 is mapped to the chromosome V and encodes a multi-functional calcium binding protein, calreticulin. It consists of 395 amino acids and has the N-terminal signal sequence as well as C-terminal ER retention sequence, HDEL. C. elegans crt-1 shows 61 % amino acid sequence identity with mouse calreticulin. We characterized the spacio-temporal pattern of the crt-1 gene expression by northern, in situ hybridization, western, whole-mount immunostaining. The mRNA transcripts were abundant during early embryonic stages and mostly restricted to intestinal precursor cells during later stages. These results were further confirmed by whole-mount immunostaining with anti-recombinant calreticulin antibody. Calcium overlay with 45 Ca and stains-all experiments showed that the recombinant calreticulin binds to calcium. The blocking of calreticulin gene function by RNA interference (RNAi) showed little or no effect on the survival of the worms. However, the protein level reduced, but not disappeared completely, as evidenced from the western blot experiment and the whole-mount immunostaining. We are currently investigating the function of crt-1 gene by screening for the deletion mutant using EMS mutagenesis.

24 May 1999 15:50 712 712 Studies of C. elegans HIP1

1999 International Worm Meeting abstract 661 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Studies of C. elegans HIP1 A Parker, A Rose Department of Medical Genetics, University of British Columbia, Vancouver, B.C, Canada We are interested in determining the function of a gene in C. elegans, CeHIP1, which shares sequence identity with human HIP1 (Huntingtin Interacting Protein 1) and Sla2 of yeast. CeHIP1, HIP1, and Sla2 also share sequence identity with the C-terminus of vertebrate talin. Specifically, these proteins contain a conserved protein domain, known as an I/LWEQ module. This module has been shown to bind F-actin (McCann and Craig, PNAS, 1996). We wish to study CeHIP1’s role in developmental and tissue specificity in C. elegans. We have characterized the gene structure of CeHIP1 and have determined it to be trans-spliced to SL1. We cloned the putative promoter region into a GFP expression vector. Fluorescence could be detected in pharyngeal neurons, possibly the M4 and I3 interneurons. CeHIP1 maps between dpy-19 and sma-2 on LGIII, and maps to cosmid ZK370 on the physical map. The genetic map indicates that nDf17 should uncover CeHIP1(ZK370.3) Using PCR, primers specific to CeHIP1 amplified DNA from nDf17 homozygotes. The control, primers for sma-2 (ZK370.4), do not amplify. Thus, it appears that nDF17 does delete DNA in the region corresponding to cosmid ZK370, but does not delete the CeHIP1 sequence tested (i.e. CeHIP1 appears to be skipped in nDf17). In addition, three deletion candidates have been identified from PCR screens of mutagenized libraries, but none of the deletion bearing animals could be recovered. We have used RNAi to study the effects of a reduction of CeHIP1. The progeny of injected animals have an increased rate of embryonic lethality in comparison to wild type. A number of unshelled oocytes were also present on the plates. We have conducted a yeast two-hybrid screen to identify interacting protein partners of CeHIP1. Two clones were isolated from the screen. The first shows identity to a transcription initiation factor type IIB. The second clone shows identity to the C-terminal portion of the Drosophila hedgehog protein. In collaboration with the Michael Hayden laboratory (University of British Columbia), we are characterizing nematode-human protein interactions. There is evidence that overexpression of HIP1 in human cells cause cell death through an apoptotic pathway (A. Hackam, personal communication). We are working to determine if overexpression of CeHIP1 causes a similar condition in C. elegans.

24 May 1999 15:50 713 713 Investigating the fates of trigger RNAs and target transcripts in RNA-mediated interference

1999 International Worm Meeting abstract 662 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Investigating the fates of trigger RNAs and target transcripts in RNA-mediated interference SN Parrish 1,2 , AZ Fire 1

1 Carnegie Institution of Washington, Department of Embryology. 115 West University Parkway, Baltimore, Md. 21210. 2 Biology Graduate Program, Johns Hopkins University, Baltimore Md. 21218.

We seek to explore the molecular mechanisms responsible for RNA-mediated genetic interference (RNAi). In nematodes, introduction of double-stranded RNA corresponding to a segment of an endogenous genetic locus can result in specific silencing of that locus, essentially producing a knock out phenotype [1]. To date, evidence indicates that this interference reflects a post-transcriptional mechanism, resulting in the loss of the endogenous transcript [2]. Only a few molecules of dsRNA are required per cell to mediate interference, suggesting either an amplification or catalytic aspect of the process [1]. To gain an understanding of the mechanism of RNAi, we are examining the fates of the two key players in this pathway, the endogenous target RNA and the dsRNA effector molecule. First, we are attempting to follow alterations in the endogenous transcripts after the introduction of dsRNA. As a start, we are trying to map possible cleavage events or potential chemical modifications through primer extension and RT PCR of the target transcript. In a complementary set of experiments, we are also examining potential changes in the dsRNA triggering molecule. Through the characterization of the target and effector RNA molecules, we hope to acquire some insight into the mechanism of RNA-triggered silencing. With this knowledge, in conjunction with genetic identification of components in the pathway, it may be possible to unravel the events and intermediates essential for RNAi.

1. Fire, Xu, Montgomery, Kostas, Driver, Mello. Nature 391, 806 2. Montgomery, Xu, and Fire. PNAS 95, 15502

24 May 1999 15:50 714 714 Egls: a new growth control pathway?

1999 International Worm Meeting abstract 663 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Egls: a new growth control pathway? MN Patel, AM Leroi Imperial College of Science, Technology and Medicine It is well known that some egls have large adult body sizes due to the bloating effect of egg retention. But we thought that this bloating - which is trivial - might conceal more interesting body size effects, and to pick these up we measured adult worms before the onset of egg production. We looked at mutant alleles from 47 known egl loci and measured their body-size at 36 and 48 hours after hatching (at 20 C). At 48 hours, before they bloat, 10/47 of the surveyed alleles were larger by volume (about +33%) and length (about +10%) compared to the wildtype. Why? There are, roughly, two kinds of egls: those that affect HSN migration and fate, and those that affect muscle form or function. We knew that egls of the latter kind were sometimes large. For example, egl-19 hypomorph alleles grow very quickly and become 50% larger than wt at adulthood; egl-19 encodes an ion channel subunit. To our surprise, however, many of the HSN defective egls (egl-1, -15, -17, -46, -49, -50) are giant as well. We suspect that these genes also affect the fate of some other cell critical to growth control. It is interesting to note that two of our giant egls, egl-17 and egl-15, are respectively a FGF ligand and receptor. Is this a new growth control pathway independent of the well-known TGF-b pathway?

24 May 1999 15:50 715 715 Two conserved domains of the EGL-10 RGS protein cooperate to inhibit G protein signaling in the C. elegans nervous system

1999 International Worm Meeting abstract 664 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Two conserved domains of the EGL-10 RGS protein cooperate to inhibit G protein signaling in the C. elegans nervous system GA Patikoglou, PB Sigler, MR Koelle Dept. of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06536 RGS (Regulator of G protein Signaling) proteins were identified through genetic studies in yeast and C. elegans as negative regulators of G protein signaling. In vitro studies using mammalian RGS and Ga proteins show that RGS proteins are potent GTPase Activating Proteins (GAPs), accelerating the slow intrinsic rate of GTP hydrolysis by Ga proteins and thus converting them to their inactive GDP-bound forms. While all RGS proteins contain an "RGS domain" that in some experiments is sufficient to function as a GAP, a subset of RGS proteins also contain an additional N-terminal conserved region of unknown function called the RGS-N domain. In C. elegans, one member of this subset is EGL-10, which was the first C. elegans RGS protein to be genetically characterized and was shown to inhibit the neural Ga protein GOA-1 to control egg-laying behavior. To analyze the function of the two conserved RGS protein domains, we used a neural-specific promoter to express EGL-10 and its subdomains in C. elegans and assessed their effects on egg laying. Although full-length EGL-10 rescued the egl-10 egg-laying defect, the EGL-N domain alone did not, and an RGS domain fragment gave only partial rescue. Surprisingly, the RGS-N and RGS domain fragments completely rescued the egl-10 egg-laying defect when coexpressed as separate polypeptides. Therefore, the RGS-N domain appears to assist the RGS domain in inhibiting G-protein signaling and the two domains need not be covalently attached to function together. Like EGL-10, EAT-16 is an RGS-N containing RGS protein in C. elegans , but EAT-16 appears to inhibit a different neural Ga protein, EGL-30. Neural promoter-driven overexpression of EAT-16 does not rescue the egl-10 egg-laying defect but does rescue the defects seen in an eat-16 mutant. We are conducting domain-swapping experiments between EAT-16 and EGL-10 to find out where the Ga protein target specificity determinants in these proteins lie. To extend our genetic findings we have purified GOA-1 and various EGL-10 protein fragments for use in biochemical assays and X-ray crystallographic studies.

24 May 1999 15:50 716 716 ImmunoEM Localization of YP170::GFP Reveals Yolk Transport

1999 International Worm Meeting abstract 665 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. ImmunoEM Localization of YP170::GFP Reveals Yolk Transport M Paupard, A Miller, DH Hall Center for Caenorhabditis elegans Anatomy, Department of Neuroscience, Albert Einstein College of Medicine, Pelham Parkway, Bronx, NY 10461, USA. We have tested a variety of antibodies in post-embedding protocols to localize proteins in thin sections of C. elegans. Despite recent improvements, about 50% of tested antibodies fail to work satisfactorily. Those that fail apparently involve antigens that are denatured during alcohol dehydration prior to LR Gold embedment. Fixation of chilled whole worms within a microwave oven provides more uniform, rapid fixation than worms fixed by immersion or cut open in fixative by a blade (1,2). Just as for immunofluorescence, it is still necessary to compare fixative strengths to obtain optimal preservation while retaining antigenicity. Here we compare the merits of several monoclonal and polyclonal antibodies against GFP to localize yolk protein (YP170::GFP strain) in thin sections, using a gold-linked secondary Ab. Commercial anti-GFP antibodies were obtained from Clontech, Quantum and RDI. Until now, it has been difficult to prove which organelles contain yolk, or to trace the exact path by which yolk travels from intestine to gonad and embryos (3). The YP170::GFP label has also been used by light microscopy to monitor endocytosis of yolk in wild type and mutant tissues (4). By immunoEM, GFP label in YP170::GFP animals is concentrated in two classes of dark-staining organelles in the intestine. Labeled yolk particles are found in the pseudocoelom. Labeled granules and free label above background levels are seen in the cytoplasm of oocytes in the proximal gonad and egg chamber. We will present TEM evidence for yolk passage through sheath pores and endocytosis into oocytes separately (5). We thank Barth Grant (Columbia U.) for providing the YP170::GFP strain used in this study.

1. Li and Kimble, International C. elegans Meetings, 1995, 1997 2. Miller and Hall, Worm Breeder’s Gazette 15: 15, 1998 3. Bossinger and Schierenberg, Int. J. Dev. Biol. 40: 431-9, 1996. 4. Grant, Zhang, Pedraza, Hall and Hirsh, this meeting. 5. Hall, Winfrey, Blauer, Hoffman, Furuta, Rose, Hobert and Greenstein, this meeting.

24 May 1999 15:50 717 717 SUP-9 and SUP-18 may be components of a K+ channel involved in the regulation of muscle contraction

1999 International Worm Meeting abstract 666 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. SUP-9 and SUP-18 may be components of a K+ channel involved in the regulation of muscle contraction I Perez de la Cruz 1 , SC Cannon 2 , HR Horvitz 1

1 HHMI, Dept. Biology, MIT, Cambridge, MA 02139. 2 Dept. Neurobiology, Harvard Medical School, Boston, MA 02114, USA.

Rare altered-function mutations in the genes unc-93, sup-9, or sup-10 result in the abnormal regulation or coordination of muscle contraction (e.g., Levin, J. and Horvitz, H.R. Genetics 135, 53-70, 1992). These mutants move sluggishly, are unable to lay eggs, and exhibit the rubber-band phenotype: when worms are prodded on the head, they contract and relax along their entire body without moving backwards. Genetic studies suggest these three genes act at the same step, possibly by encoding subunits of a protein complex. Loss-of-function mutations in a fourth gene, sup-18, completely suppress the rubber-band phenotype caused by sup-10(n983) and partially suppress the unc-93(gf) and sup-9(gf) rubber-band phenotypes. unc-93 and sup-10 encode novel putative transmembrane proteins.

We have found that sup-9 encodes a member of the TWIK family of K+ channels. We have injected sup-9, unc-93, and sup-10 cRNAs into Xenopus oocytes but have not detected K+ selective currents. We are currently expressing sup-9 singly or with unc-93 and sup-10 in HEK cells in an attempt to reconstitute a functional channel complex. To better understand the structure-function relationship of TWIK K+ channels we are determining the sequences of the approximately 100 alleles of sup-9. We have also raised antibodies against SUP-9 to determine its in vivo localization. We have cloned sup-18 and found that it encodes a putative membrane protein. Interestingly, SUP-18 is distantly related by sequence to a family of bacterial NADH oxidases. We are currently expressing MBP-SUP-18 fusion proteins in E. coli to assay nucleotide binding and dehydrogenase activities. We have raised anti-peptide antibodies against SUP-18 to test whether it colocalizes with SUP-9 in vivo.

24 May 1999 15:50 718 718 Assays for interactions between HER-1 and TRA-2

1999 International Worm Meeting abstract 667 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Assays for interactions between HER-1 and TRA-2 Marc D. Perry, Roxana N. Sultan, Nimerta Rajwans Department of Molecular and Medical Genetics, University of Toronto,Toronto, ON M5S 1A8, CANADA We are interested in how her-1 interacts with downstream genes in the sex determination pathway to specify cellular sexual fates. Given that the relative levels of HER-1 and TRA-2 affect the sexual fate decision of a cell (Kuwabara and Kimble, 1995 M.B.C. 121: 9), and that dominant tra-2(eg) alleles are insensitive to negative regulation by her-1 (Kuwabara, 1996 Devel. 122: 7), we wish to discern whether or not the large transmembrane protein pTRA-2A is the direct target of HER-1’s masculinizing activity. We have engineered Sf9 insect tissue culture cells to express a construct encoding HIS-tagged HER-1 (Perry and Rajwans, 1997 11th Int. Worm Meeting) and are using the secreted protein in binding assays with mammalian COS cells which have been engineered to express a TRA-2-HA fusion protein. We have generated antibodies against HER-1 and are using these in conjunction with anti-HA antibodies and fluorescent secondary antibodies to detect co-localization of the two proteins. In parallel, we are using Kunkel mutagenesis to generate expression constructs encoding the tra-2(eg) allele in order to test the effects of this mutation on HER-1 binding. To correlate tra-2 function in vivo with its ability to bind HER-1 in vivo we will delimit the portions of TRA-2 which are essential to the interaction, by using truncated TRA-2 variants in binding assays with HER-1.

24 May 1999 15:50 719 719 Identification of cis-acting elements and trans-acting factors responsible for her-1 regulation

1999 International Worm Meeting abstract 668 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification of cis-acting elements and trans-acting factors responsible for her-1 regulation Marc D. Perry, Michael K. Garroni, Nimerta Rajwans Dept. of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8 Expression of the cell non-autonomous gene her-1 is required for male development in XO animals. her-1 activity in XX animals is low due to negative regulation by the three sdc genes located directly upstream in the genetic pathway controlling sex determination and dosage compensation. We have previously shown that transgenic F1 XX animals carrying extrachromosomal arrays containing either of the two male-specific promoter regions of her-1 can be variably sexually transformed, Dpy, sick, or dead (Perry et al. 11th International C.elegans meeting p. 20). These phenotypes resemble sdc(lf) mutations (Meyer, 1997 C. elegans II: p. 219) and support a model in which her-1 regulatory regions on the arrays are titrating away not only factors necessary for her-1 negative regulation, but also factors necessary for X chromosome dosage compensation. We are interested in defining those regions in the promoters responsible for the regulation of her-1 and the trans-acting factors which bind them. To assay this a series of smaller fragments comprising the two promoters have been inserted into extrachromosomal arrays and microinjected into gonadal syncitia to create transgenic lines carrying multiple arrays. Many of these lines have shown the "sdc phenocopy" of variable masculinization, Dpy, sickness or death of XX animals. These lines have been crossed with xol-1 mutant lines and assayed for their ability to rescue XO specific lethality similar to that of sdc(lf) alleles ability to rescue XO lethality (Miller et al. 1988 Cell 55: 167-183 ). In order to identify trans-acting factors which are binding the regulatory regions, minimal fragments which are able to sdc phenocopy are used in electrophoretic mobility shift assays with nuclear extracts prepared from embryos. Positive shifts from whole nuclear extract are compared to shifts carried out with in vitro translated proteins. In addition to this the binding proteins identity can be confirmed through supershifts with antisera against suspected participants. Further fine mapping of the occupied regions of the her-1 promoters will be achieved through DNAse1 footprinting using both whole nuclear extracts and in vitro translated proteins. Specific mutations in the regulatory regions can also be assayed for their effect on binding of identified factors.

24 May 1999 15:50 720 720 Isolation of RNAi resistant mutants from C. elegans

1999 International Worm Meeting abstract 669 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Isolation of RNAi resistant mutants from C. elegans AG Petcherski, J Kimble HHMI and University of Wisconsin-Madison, Madison, WI 53706 dsRNA can specifically inhibit gene activity in C. elegans. A similar phenomenon may exist in plants, fungi, and flies. The use of this phenomenon, called RNA-mediated interference or RNAi, is widely applied in C. elegans to analyze gene functions. However, the mechanism underlying RNAi remains a mystery. To begin to address this mechanism, we have begun to isolate RNAi defective mutants. Briefly, P0 animals were mutagenized with EMS. F1 progeny was allowed to develop to adulthood and then bleached to collect F2 embryos. We soaked F2 animals in dsRNA derived from genes (e.g. lag-1) expected to either sterilize or kill the animals. We reasoned that mutants resistant to RNAi would live and be fertile, while other animals would die or produce no progeny. Because "dsRNA soaking" was not 100% efficient, we repeated the selection procedure over several generations for a total of 10 times. From approximately 20,000 F2 animals we isolated several mutants resistant to RNAi. At present, we are mapping the mutants and trying to group them into complementation groups. In summary, preliminary results indicate that we have isolated mutation(s) in at least one gene required for RNA interference, indicating that RNAi has a genetic component. Further progress will be reported at the meeting.

24 May 1999 15:50 721 721 Influence of temperature on reproductive rate in C. elegans: r versus Ro

1999 International Worm Meeting abstract 670 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Influence of temperature on reproductive rate in C. elegans: r versus R o PC Phillips 1 , BL Armstrong 1 , CP Coucke 1 , RB Huey 2

1 Biology Department, Box 19498, University of Texas at Arlington, Arlington, TX 76019. 2 Department of Zoology, University of Washington, Seattle, WA 98195.

Temperature is one of the primary determinants of ectotherm performance and fitness. Because of its quick generation time, easy maintenance, and powerful genetics, C. elegans provides an ideal model for testing many of the central theories of thermal ecology. As a precursor to a general set of experiments in this area, we have been characterizing thermal performance curves for C. elegans (N2) over a wide range of temperatures. There are potentially many ecologically relevant measures of performance. Two standard measures of fitness, the net reproductive rate (Ro ) and the intrinsic rate of increase (r), differ in their sensitivity to the rate of reproduction. Both measures varied by several fold as the temperature moved from 12C to 26C. As an interesting side-note, we found that the temperature of the agar was often several degrees lower than the thermostat temperature of the incubator or of enclosed flasks of water within the incubator, probably because of evaporative cooling at higher temperatures. Because of this, we report agar temperatures measured directly with a digital thermocouple thermometer. Net reproductive rate (total offspring production) displayed a large plateau in optimal performance from roughly 15-22C. The intrinsic rate of increase peaked at higher temperatures with a narrower plateau from 22-24C. The observation that r peaks at higher temperatures than Ro appears to be a general phenomenon in ectotherms and is probably caused by r’s sensitivity to the rate of offspring production, which is substantially higher at higher temperatures. Before reaching a plateau, there is a roughly 40% increase in the rate of offspring production for every degree increase in temperature. Worms raised at 23C produce offspring over three times faster than worms raised at 12C. If selection for rapid reproduction is as important as it appears to be in C. elegans, then the effects of temperature on r should generate strong selection on many other aspects of the thermal ecology of these nematodes.

24 May 1999 15:50 722 722 RNAi screening for polarity genes

1999 International Worm Meeting abstract 671 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. RNAi screening for polarity genes F Piano, AJ Schetter, KJ Kemphues Section of G&D, Cornell University, Ithaca NY 14853 We are interested in identifying genes involved in embryonic polarity by RNAi based screening. In addition to the obvious advantage of screening cloned genes, this approach can potentially find genes not seen in classic genetic screens due to their pleiotropic effects. We constructed a cDNA library from dissected ovaries to enrich for maternally loaded transcripts and then tested random cDNAs from this library for RNAi effects. This unbiased cDNA selection can identify genes beyond those predictable via heuristic methods, and could be used in other nematode species to identify genes involved in homologous developmental processes. We addressed two questions: what is the most efficient way to perform such a screen, and what types of RNAi effects would arise from our ovary library? We chose to analyze 200 cDNAs singly for frequency of embryonic lethality and relate phenotypes classes to partial DNA sequence. Surprisingly, we found that out of the 195 clones tested so far, 71 gave rise to embryonic lethality and almost half of these had one-cell defects or reduced fecundity. Regarding throughput, these data lead us to conclude that microscopy, not injection, is rate limiting and that screening using pools of more than 3 cDNAs would be inefficient. We have sequenced 53 lethals and found that 6 are predicted to encode pioneer proteins. With respect to redundancy, 38 clones are represented once and 15 clones encode 6 genes. Thus far no known par gene has been recovered. To determine the range of embryonic defects, we are characterizing the lethal phenotypes by time lapse video microscopy. Many RNAi effects could fit expectations from sequence information. For example, genes encoding putative cell cycle regulatory proteins resulted in altered cell cycle timing and multiple karyosomes in early blastomeres; components of the transcriptional machinery gave rise to late embryonic lethality whereas those involved in protein synthesis or degradation caused one-cell arrest. Other clones, in addition to the pioneer genes, elicited less predictable defects. For example, an enolase gave rise to dramatic blebbing suggesting a potential new role for an old gene and topoisomerase II affected cytokinesis. Finally, we identified a putative RNA binding protein that elicited a Par-like phenotype.

24 May 1999 15:50 723 723 ooc-3, a novel gene required for rotation of the centrosome-nucleus complex in P1

1999 International Worm Meeting abstract 672 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. ooc-3, a novel gene required for rotation of the centrosome-nucleus complex in P 1 SC Pichler 1 , P Gönczy 1 , H Schnabel 2 , R Schnabel 2 , AA Hyman 1

1 European Molecular Biology Laboratory, D-69117 Heidelberg. 2 Institut für Genetik, TU Braunschweig, D - 38023 Braunschweig.

During the asymmetric cell division of the P1 blastomere, proper positioning of the mitotic spindle along ° the antero-posterior axis relies on a 90 rotation of the centrosome-nucleus complex. This P1 rotation depends on astral microtubules, actin and a site in the anterior cortex enriched in actin, dynactin and the actin-capping protein (CP). During rotation, astral microtubules emanating from one centrosome attach at the cortical site und subsequent microtubule shortening places this centrosome close to the cortical site leading to a 90° rotation of the centrosome-nucleus complex. To elucidate the mechanisms underlying such rotation we searched for mutants defective in P1 rotation by screening an extensive collection of maternal-effect embryonic lethal mutations on chromosome II by time-lapse DIC videomicroscopy. We identified one mutant, t1308 in which rotation in P1 is defective in 100% of the embryos and in P0 in 50% of the embryos. In addition, this mutation causes non-penetrant meiotic and mitotic defects leading to an abnormal number of female pronuclei in P1 and multiple nuclei in both AB and P1 . By investigating the localisation of the polarity markers PAR-3 and the P-granules we found that the overall embryonic polarity is properly established in ooc-3 mutant embryos. This suggests that ooc-3 is a gene more specifically required for rotation, possibly for the organization of the cytoskeleton. Tubulin staining, however, showed that astral microtubules are unaffected in mutant embryos. We are currently investigating whether the anterior cortical site is properly assembled by performing actin-, CP- and dynactin stainings. Complementation analysis revealed that ooc-3 is allelic to the previously identified locus ooc-3 (mn241). ooc-3 had been mapped by deficiency between unc-53 and age-1. We have identified a candidate gene by rescuing the phenotype with entire cosmids and cosmid fragments. RNAi and sequencing of the two mutant alleles confirmed the candidate gene to be ooc-3. Sequence analysis revealed that ooc-3 is a novel gene coding for a putative transmembrane protein. We are currently preparing antibodies to investigate the localisation of OOC-3.

24 May 1999 15:50 724 724 The role of Rho-dependent kinase (LET-502) and myosin phosphatase (MEL-11) in embryonic elongation

1999 International Worm Meeting abstract 673 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The role of Rho-dependent kinase (LET-502) and myosin phosphatase (MEL-11) in embryonic elongation AJ Piekny 1 , AK Wissmann 1,2 , PE Mains 1

1 Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, T2N 4N1. 2 Axys Pharmaceuticals, Inc., South San Francisco, CA, 94080.

C.elegans morphogenesis occurs when actin-mediated hypodermal cell-shape changes alters the embryo from a ball of cells into a tube-like structure (1). let-502 and mel-11 play key roles in this process (2,3). Dominant-negative let-502 mutants fail to elongate while null mutants of mel-11 hyperelongate. let-502 alleles isolated as mel-11 suppressors show a range in phenotypes from early arrest (dominant-negatives), to wild type (hypomorphs). There is no correlation between the molecular nature of the mutation and the phenotype, although almost all mutations are in the kinase domain. Early arrest does correlate strong suppression of mel-11. We are unsure of the exact null let-502 phenotype, and we are performing RNAi experiments to clarify this. Sequence homology searches in the C.elegans database revealed several genes that are potentially responsive to Rho or Rac/Cdc42-like molecules, which have been implicated in regulating cytoskeletal rearrangements. Some of these genes will be examined for any potential roles in embryonic elongation by RNAi. The pattern of LET-502 and MEL-11 expression support our model for their roles in morphogenesis. Antibodies to LET-502 and MEL-11 show they are both expressed in adult gonads, and in hypodermal cells in embryos of varying stages. However, while LET-502 expression is stable, MEL-11 expression appears to be more transient and correlates with hypodermal cell-shape changes during elongation. Western blots and immunostaining with let-502 and mel-11 mutant strains are being performed.

1. Priess, J.R. & D.I. Hirsh. 1986. Dev. Biol. 117: 156-173. 2. Wissmann, A. et al. 1997. Genes Dev. 11: 409-422. 3. Wissmann, A. et al. 1999. Dev. Biol. (in press).117et al.

24 May 1999 15:50 725 725 The chemosensory neuron ASER regulates the initiation but not the execution of pirouettes in C. elegans chemotaxis.

1999 International Worm Meeting abstract 674 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The chemosensory neuron ASER regulates the initiation but not the execution of pirouettes in C. elegans chemotaxis. JT Pierce-Shimomura, MR Gaston, SR Lockery Institute of Neuroscience, 1254 University of Oregon, Eugene, OR 97403. The behavioral strategy underlying C. elegans chemotaxis involves sharp turns (pirouettes) triggered by a sustained period in which the animal moves down the chemical gradient1 . As a first step in determining the role of individual neurons in this strategy, we ablated the single chemosensory neuron ASER in 19 worms and followed each animal with a high resolution tracking system during chemotaxis in a gaussian gradient of ammonium chloride, previously shown to be sensed by the ASE neurons2 . Most ASER- animals failed to reach the peak of the gradient in the 20 min assay period, while ASER+ (anaesthetized and recovered but not ablated; n = 13) reached the peak, consistent with previous results2 . ASER- animals (n = 6) were able, however, to reach the peak of a gradient of diacetyl, sensed by a different chemosensory neuron. To determine the chemotaxis deficit more precisely, the tracking data were analyzed by noting the animal’s bearing with respect to the peak of the gradient immediately before (Bbefore ) and after (Bafter ) pirouettes. The distribution of Bbefore was flat, indicating that pirouettes in ASER- animals were triggered randomly. In contrast, the distribution of Bbefore values for ASER+ animals showed that pirouettes in this group were triggered by movement down the gradient, as expected1 . Ablation of ASER did not affect the execution of pirouettes, since the distributions of bearing change (Bafter - Bbefore ) were similar in the ASER- and ASER+ animals. Thus, ablating ASER disrupts the initiation but not the performance of pirouettes, consistent with the sensory role of ASER.

1. Pierce & Lockery (1998) West Coast Worm Meeting abstract 59 2. Bargmann & Horvitz (1991) Neuron 7:729-42

24 May 1999 15:50 726 726 C32E8.7, a C.elegans homolog of the diabetes autoantigen ICA69

1999 International Worm Meeting abstract 675 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. C32E8.7, a C.elegans homolog of the diabetes autoantigen ICA69 M Pilon 1 , AM Spence 2 , HM Dosch 1

1 Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8. 2 Dept. Molecular and Medical Genetics, University of Toronto, Ontario, Canada M5S 1A8.

Type I diabetes is an autoimmune disease in which the immune system targets antigens in the insulin-producing pancreatic beta-cells. The Islet Cell Antigen of 69 Kd (ICA69) was discovered by screening islet cell expression libraries with serum from diabetic rats or human patients. The protein exhibits no significant homology with proteins of known function, except for the presence of a putative coiled coil motif possibly implicated in protein-protein interactions. Based on its amino acid sequence, ICA69 is predicted to be a cytosolic soluble protein. Only one ICA69 gene seems to exist in mouse (478 aa), rat (480 aa) and human (483 aa); these exhibit 87% overall identity. In mouse, the ICA69 protein is expressed in most organs, but is most abundant in tissues rich in neuroendocrine or secretory cells such as brain, pancreas and stomach mucosa. The C32E8.7 (418 aa) predicted gene encodes a C.elegans homolog of ICA69 (33% overall identity with human ICA69, including a stretch of 280 aa with 58% conservation). Using a GFP reporter in transgenic C.elegans, we found that the C32E8.7 promoter is specifically expressed in all C.elegans neurons; the timing of promoter activity coincides with the appearance of differentiated neurons during embryogenesis (~400 minutes). Because: 1) most diabetes autoantigens are components of the insulin secretory granules; 2) The high levels of ICA69 expression in neuroendocrine/secretory tissues; and 3) the C.elegans homolog of ICA69 is expressed in differentiated neurons, we hypothesized that ICA69 is involved in regulated exocytosis which mediates, for example, insulin secretion by pancreatic beta-cells and neurotransmitter release by neurons. In collaboration with Prof. Plasterk (Amsterdam, NL), we have isolated a C32E8.7 mutant (NL2003) in which nucleotides -395 to +2276 (262 codons) have been deleted. The NL2003 mutant exhibits no overt phenotype. However, we have observed a slight shortening of life span, and preliminary experiments suggest that the mutant is slightly resistant to aldicarb, an inhibitor of acetylcholinesterase. This later observation would support our hypothesis that ICA69, and its C.elegans homolog, is involved in exocytosis.

24 May 1999 15:50 727 727 A JAVA Based 4D Lineage Analysis Application

1999 International Worm Meeting abstract 676 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A JAVA Based 4D Lineage Analysis Application JN Pitt 1,2 , RJ Hill 1,2 , B Leung 1,3 , JR Priess 1,2,3,4

1 Fred Hutchinson Cancer Research Center,Seattle 98109. 2 Howard Hughes Medical Institute. 3 MCB Program,University of Washington,Seattle 98195. 4 Dept. of Zoology,University of Washington, Seattle 98195.

We are developing a JAVA based variation of the IMR’s "4D Viewer" software package(1), that runs on both Macintosh and Windows 95/98/NT machines. The program imports 4D data sets created by the IMR’s "4D Grabber" application and can also construct 3D stacks from sets of individual image files. The program allows the user to label cells in a 4D stack and construct rotatable 3D models that retain cell identifications. This function allows the user to rotate a previously analyzed 4D set so that it corresponds to the 3D orientation of a newly collected second data set; this aids in the identification of cells viewed from a different orientation. The program also simultaneously constructs fully editable lineage diagrams that are linked to the 3D models and the 4D image data. The 3D viewer provides depth fading, zooming, rotation and has tools for color coding cells and coupling cells together with lines. The program will be available free of charge after the meeting, and will be available for download on our website.

(1)Thomas, C, P DeVries, J Hardin and J White (1996) Four-Dimensional Imaging: Computer Visualization of 3D Movements in Living Specimens. Science 273: 603-607.

24 May 1999 15:50 728 728 Cold sensitive fusion: kinetic dissection of cell fusion during morphogenesis in N2 and duf-1

1999 International Worm Meeting abstract 677 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cold sensitive fusion: kinetic dissection of cell fusion during morphogenesis in N2 and duf-1 B Podbilewicz 1 , M Glickman 1 , Y Rabin 2 , T Gattegno 1

1 Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel. 2 Department of Mechanical Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel.

Cell fusion is an essential process in development conserved throughout evolution. We have analysed the sequence of epithelial cell fusions in C. elegans[1] and have isolated mutations that disrupt cell fusion in the embryo. To elucidate the molecular mechanisms of cell fusion in situ we arrested or slowed down this process by lowering the temperature. We used confocal microscopy and a custom-made cooling stage to record 4D-timelapse movies of developing embryos expressing MH27-GFP fusion protein in the adherens junctions[2] at different temperatures. At temperatures below 4o C, comma stage embryos did not elongate and epithelial fusions did not occur. However, at 5o C embryos elongated to 2-fold at a rate of about 20-fold slower than that at 20o C and dorsal cell fusions did not occur. Moreover, this treatment phenocopies the arrest of duf-1(zu316cs) embryos at 15o C[3]. The fusion rate measured from timelapse recordings as disappearance of MH27-GFP staining varied from 50 nm/min at 6o C to 1000 nm/min at 27.5o C. Thus, we show that the cell fusion rate is strongly dependent on temperature. Using a combination of kinetic and genetic analyses we have initiated the dissection of the multi-step process involving the opening and expansion of fusion pores necessary for morphogenesis and the formation of organs in C. elegans and other organisms. We propose that cell fusion in C. elegans can be dissected into at least three steps: (1) transient hemifusion; (2) rearrangements of two lipid bilayers into one (fusion pore formation arrested at 5o C); and (3) expansion of fusion pore (estimated fusion rates at 6o C). Using Arrhenius plots for N2 and duf-1 we can get information on the mechanisms of pore expansion during cell fusion in vivo. The energy of activation can be estimated and compared between wild-type and mutant.

[1] Podbilewicz B. and White J.G. (1994) Dev. Biol. 161, 408-424. [2] Mohler, W.A et al (1998) Curr. Biol. 8, 1087-1090. [3] Gattegno T. (1999) IWM Abstract.

24 May 1999 15:50 729 729 In Search of Phenotypes for ptl-1

1999 International Worm Meeting abstract 678 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. In Search of Phenotypes for ptl-1 P Polk 1 , E Aamodt 2 , S Aamodt 1

1 Louisiana State University, Shreveport, LA 71115. 2 Louisiana State University Medical Center, Shreveport, LA 71130-3932.

In mammals, the microtubule-associated protein tau is located in the axons of neuronal cells and is believed to stabilize the microtubule cytoskeleton and function in neurite outgrowth. A C. elegans homologue of tau, ptl-1 (protein with tau-like repeats), was identified by the C. elegans Genome Sequencing Consortium and characterized by us (McDermott et al, 1996, Biochemistry 35: 9415-9423) and Goedert et al. (1996, J. Cell Sci. 109:2661-2672). We obtained a C. elegans strain with a deletion of the repeat region from the NemaPharm Group at Axys Pharmaceuticals. The ptl-1 deletion strain has no apparent phenotype and we have not detected differences between N2 and the ptl-1 deletion strain in sensitivity to colchicine, benomyl or cytochalasin D. Work done by N. Hirokawa and colleagues has shown that in mice, tau functions redundantly with microtubule-associated protein MAP1B. Mice homozygous for a null allele of tau (Harada et al., 1994, Nature 369: 488-491) or MAP-1B (Takei et al., 1997, J. Cell Biol. 137: 1615-1626) have mild phenotypes, but double mutants have severe defects in brain development as a result of defects in neurite outgrowth (Takei et al., 1998, Mol. Biol. of the Cell 9:394a). Because of this precedent for redundancy in the function of tau and MAP-1B, we searched for C. elegans genome for a MAP1B homologue but no candidate was found. To attempt to identify proteins that act cooperatively or are redundant to ptl-1, we have begun a screen for synthetic mutations. We crossed the ptl-1 deletion into a strain (obtained from David Baillie) that has cosmid F42G9 (containing ptl-1) and rol-6 as an extrachromosomal array. We will mutagenize the ptl-1 deletion strain containing the F42G9 array to screen for genes that are synthetic to ptl-1.

24 May 1999 15:50 730 730 Families of C. elegans tyrosine kinase receptors (RTK): evidence for continuing evolution of metazoan multigene families

1999 International Worm Meeting abstract 679 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Families of C. elegans tyrosine kinase receptors (RTK): evidence for continuing evolution of metazoan multigene families C Popovici, R Roubin, F Coulier, P Pontarotti, D Birnbaum INSERM U.119, 27 Bd. Leï Roure, 13009 Marseille, France. The availability of the complete genomic sequence of the C. elegans allowed the identification of new putative RTK genes. We describe here the genomic organization of two gene families that encode putative RTKs. One family encodes two already characterized proteins, KIN-15 and KIN-16, and 9 new proteins. The other nine proteins (encoded by the cosmids C08H9, M01B2, R09D1, T01G5, ZK938 and W04G5) have not yet been characterized. The putative extracellular region of proteins of this family is very short. A second family (F59F3.1, F59F3.5, T17A3.1 and T17A3.8) has a large extracellular region containing Ig domains (Popovici et al., abstract book). It has not been described as functional in C. elegans. Analysis of the phylogenic tree built on the alignment of the tyrosine kinase shows that the two novel RTK families constitute a monophyletic group that does not branch clearly with one of mammalian RTK families. In addition, it seems that the two RTK families described here have evolved from a common ancestor containing a large extracellular region. The loss of exons encoding the majority of the extracellular region followed by cis or trans duplications may be the mechanism of apparition of the family with short extracellular region. RTKs with short extracellular region, in association with chitinase-like genes, are mostly found on chromosome II in two clusters. Among the 33 chitinase and chitinase-like genes, 24 are located in the two clusters. These clusters are separated by a 0.5 Mb region containing 2.5 kb of repetitive sequences that may enhance the unequal crossing-over and thus the high number of RTK copies and chitinase genes. The phylogenic analysis of the chitinases showed that all those located on chromosome II branched together and distinctly from the chitinases of genes located on the other chromosomes and from chitinases from other species. In this branch, two families are again recognized corresponding to the two clusters of RTKs-chitinases identified on the chromosome II. The mechanisms and driving forces conducting to an independent and specific evolution of these various families of genes in C. elegans will be discussed.

24 May 1999 15:50 731 731 C. elegans genes encoding Tyrosine Kinase Receptors share structural features with mammalian Vascular Endothelial Growth Factor Receptors (VEGFRs)

1999 International Worm Meeting abstract 680 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. C. elegans genes encoding Tyrosine Kinase Receptors share structural features with mammalian Vascular Endothelial Growth Factor Receptors (VEGFRs) C Popovici, MJ Santoni, D Birnbaum, R Roubin INSERM, U119, 27 Bd Leï Roure, 13009, Marseille, France The availability of the complete C. elegans genomic sequence has allowed the identification of a family with new putative tyrosine kinase receptor (RTK) genes (see Popovici et al. in the abstract book) and our aim is to describe functionally the four members which compose this family. The main structural feature of this family, which includes F59F3.1, F59F3.5, T17A3.1 and T17A3.8, is the presence of seven immunoglobulin-like domains in the putative extracellular region of each of these molecules. This conservation of domain architecture resembles the mammalian VEGFRs. F59F3.1 and F59F3.5 are situated on chromosome X and although in tandem do not appear to belong to the same operon. T17A3.1 and 8 are in an opposite orientation on chromosome III and separated by what appears to be a pseudogene in which only part of the immunoglobulin-like domains containing extracellular region persists. Only F59F3.1 has the corresponding cDNAs in the Y. Kohara cDNA library collection. We examined the RNA expression of these RTK genes by RT-PCR. We detected the presence of F59F3.5 and T17A3.8 transcripts in embryo and L1 enriched populations, whereas the F59F3.1 and T17A3.1 transcripts were evidenced in the mixed stage populations. Transcriptional silencing of the F59F3.1 gene by RNAi led to a drastic phenotype combining early larval arrest with clear and scrawny larvae. Thus at least some of these genes appear to be important for the worm development. The existence of VEGFR like in the worm is intringuing and we hope getting and discussing more data during the poster session.

24 May 1999 15:50 732 732 Pharyngeal Morphogenesis

1999 International Worm Meeting abstract 681 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Pharyngeal Morphogenesis MF Portereiko, ME Domeier, SE Mango Department of Oncological Sciences and Huntsman Cancer Institute Center for Children, University of Utah, Salt Lake City, UT 84112 Our lab is interested in studying pharyngeal morphogenesis, the process whereby cells of the pharyngeal primordium elongate to form a tube that connects to the buccal cavity. We have taken two approaches towards understanding this process. First, we analyzed the movements associated with wildtype pharyngeal morphogenesis. We built a chimera composed of a digestive tract-specific promoter controlling GFP expression, and we targeted GFP to the plasma membrane by including the ’CAAX’ sequence from mig-21 . We introduced the GFP chimera into wildtype animals and observed the behavior of pharyngeal cells using time-lapse microscopy2 . Our preliminary studies suggest that pharyngeal morphogenesis depends in large part on directed cell shape changes, and not on intercalation (e.g. convergence/extension) nor cellular migration. The cumulative effects of anterior pharyngeal cells increasing their longitudinal dimension appears to be sufficient to bridge the distance between the pharyngeal primoridum and the nascent buccal cavity. These data suggest that directed changes in the actin cytoskeleton may be important for pharyngeal elongation. Second, we searched for loci involved in pharyngeal morphogenesis using a deficiency screen. We surveyed approximately a third of the genome for deficiencies with pharyngeal elongation defects and chose two for further analysis. i) mnDf90 embryos form a pharyngeal primordium that fails to attach to the buccal cavity (see abstract by Lange and Mango); ii) uDf1 embryos attach to the buccal cavity similar to wildtype embryos. However, these mutants form a bifurcated tube with two lumens3 . This phenotype could reflect a defect in cellular positioning within the primordium. Alternatively, the cell shape or polarity changes that normally occur during morphogenesis may be disrupted. These embryos contain the wildtype number of pharyngeal cells, and appear to differentiate normally. Our current goals are to follow cellular behaviors in the deficiency embryos as well as identify single point mutations that mimic the deficiency phenotypes.

1. Zipkin et al., 1997 2. Special thanks to Bill Mohler, John White and the IMR for their help. 3. see also Terns et al., 1997

24 May 1999 15:50 733 733 The bHLH proteins LIN-32 and HLH-2 function together in multiple aspects of ray development in C. elegans

1999 International Worm Meeting abstract 682 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The bHLH proteins LIN-32 and HLH-2 function together in multiple aspects of ray development in C. elegans DS Portman, SW Emmons Dept. of Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461 In the L3 male tail, the nine pairs of Rn.a cells begin to execute the ray sublineage, a program that generates the structural cell and two neurons of each ray. This process requires lin-32, an atonal-like bHLH gene that is expressed in each Rn cell. We would like to understand how both intermediate (neuroblast) and terminal (neuronal and structural) cell fates are established during this process. To address this issue, we screened for modifiers of the partial ray-loss phenotype of lin-32(e1926). We recovered two missense alleles of hlh-2, the worm E/daughterless ortholog, both of which strongly enhance ray loss, eliminating all rays. Consistent with findings from other organisms, this phenotype suggests that HLH-2 functions as a heterodimerization partner for LIN-32. Notably, Krause et al. (Development (’97) 124:2179) have shown that hlh-2 expression is restricted to a small number of cells during postembryonic hermaphrodite development (in contrast to the ubiquitous expression of E/da proteins in other organisms), suggesting that regulated hlh-2 expression may be important. Our antibody staining and GFP reporter studies suggest that hlh-2 is not turned on in the seam until the Rn.a stage, one cell division later than the point at which expression of a lin-32::gfp reporter is first detectable. hlh-2 might therefore be a target of a LIN-32 homodimer, raising the possibility that the target specificity of lin-32 could change depending on its dimerization state. lin-32 has previously been shown to be required for the neuroblast fate of Rn.aa and Rn.ap; these cells take hypodermal fates in males carrying a strong lin-32 allele. We have found that weaker alleles of lin-32 and hlh-2;lin-32 double mutants can also display defects in the terminal step of the ray sublineage, particularly in Rnst (the ray structural cell) and Rn.aap, which normally undergoes cell death. In the hlh-2;lin-32 double mutant, "complete" ray sublineages are sometimes executed, yet rays are never made. These results suggest that multiple aspects of ray development, both early and late, rely on lin-32 and hlh-2 function. We hope to identify target genes and interacting loci to better understand how lin-32 and hlh-2 control both intermediate and terminal cell fates.

24 May 1999 15:50 734 734 Function of the Receptor Tyrosine Kinase cam-1/kin-8.

1999 International Worm Meeting abstract 683 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Function of the Receptor Tyrosine Kinase cam-1/kin-8. S Poulson, DM Madsen, CS Walker, AV Maricq Department of Biology, University of Utah, Salt Lake City, UT 84112 Many developmental processes, including the development of synapses, are mediated by signal transduction through receptor tyrosine kinases (RTK). For example, in mice the formation of cholinergic synapses has been shown to be dependent on the function of the RTK Musk. Thus it seems likely that RTKs similar to Musk should also play a role in synaptic or neural development in C. elegans. One RTK gene, kin-8, was identified by Koga and Ohshima and is most similar to the vertebrate Ror and Musk RTKs. Ma and Chalfie showed that kin-8 may be a downstream target of unc-4 and also showed that a KIN-8::GFP fusion had dominant negative effects causing a reverse kinking and egl phenotype. Forrester and Garriga showed that a mutation causing abnormal migrant of the CAN neurons mapped to kin-8 (renamed cam-1). Independently, we had generated both a dominant-negative allele and a deletion mutation in kin-8 and observed a phenotype similar to that described by Ma and Chalfie. Using a ceh-23::GFP strain provided by Jen Zallen and Cori Bargmann, we have also shown that our cam-1 deletion strains show a profound defect in CAN cell migration (this was kindly confirmed by Wayne Forrester). We are most interested in the uncoordinated phenotype, which is unlikely to be accounted for by a defect in CAN cell migration. Our approach has been to examine transgenic strains that express neural or muscle specific GFP reporter constructs in a cam-1/kin-8 background. For example, using a nmr-1::GFP (see abstract by Brockie et al) construct we have now shown that neuronal processes in the nerve ring are mislocalized. This may contribute to the uncoordinated phenotype. Our previous characterization of the expression pattern of cam-1/kin-8 used fusions to GFP that were based on a cDNA that we subsequently discovered was not full-length. Using 5 prime RACE we have identified coding exons that are far removed from where we previously believed the gene to start. This has prompted us to re-analyze the expression pattern of kin-8/cam-1::GFP. RTKs of the Ror type have been shown to posses an intracellular ATP binding domain essential for phosphorylation. To confirm that kin-8/cam-1 encodes a functional RTK we have undertaken studies in tissue culture cells to demonstrate phosphorylation on tyrosine residues.

24 May 1999 15:50 735 735 X Chromosome Counting Mechanisms in C. elegans

1999 International Worm Meeting abstract 684 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. X Chromosome Counting Mechanisms in C. elegans JR Powell, K Amonlirdviman, BJ Meyer Howard Hughes Medical Institute and Department of Molecular and Cell Biology, University of California at Berkeley 94720 Sex is determined in C. elegans by an X chromosome counting mechanism. The developmental switch gene xol-1 assesses the number of X chromosomes by responding to the dose of a small number of X-linked regulatory genes called signal elements. XX animals have a double dose of these signal elements, allowing xol-1 to be repressed and hermaphrodite development to ensue. XO animals have a single dose of X signal elements, allowing xol-1 to be activated and male development to ensue. Two X signal elements have been extensively characterized: the nuclear hormone receptor SEX-1, which represses xol-1 transcriptionally, and the RNA binding protein FOX-1, which represses xol-1 post-transcriptionally. XOL-1 expression and activity are dependent on proper splicing of its sixth intron. Transgenic studies by M. Nicoll have shown that this sixth intron is both necessary and sufficient for FOX-1 regulation of xol-1 expression. Increased doses of FOX-1 correlate with aberrant splicing of the sixth intron in vivo. We have begun investigating the detailed molecular mechanisms of FOX-1 activity. Preliminary data suggest that FOX-1 binds directly to the xol-1 sixth intron. In addition to this detailed study of the known X signal element fox-1, we are characterizing a putative X signal element defined by the mutation y323. This mutation was found in a screen for suppressers of the total XO lethality caused by duplicating two regions on the left end of X known to contain other signal elements. y323 is not located on the duplicated portion of X or in any of the other regions known to contain X signal elements. It has no phenotype on its own, but exhibits strong synergistic sex determination and dosage compensation defects in combination with fox-1. We have mapped y323 to a 1.2 cM interval on the left half of X and are further characterizing this mutation in order to learn more about the regulation of xol-1 activity by the X signal.

24 May 1999 15:50 736 736 Identification and characterization of kinesins required for early embryogenesis

1999 International Worm Meeting abstract 685 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification and characterization of kinesins required for early embryogenesis JA Powers, DJ Rose, O Bossinger, S Strome, WM Saxton Dept. of Biology, Indiana Univ., Bloomington, IN 47405 A search of the C. elegans genome, using the Kinesin heavy chain gene (unc-116), identified 17 kinesin family proteins. These proteins share a conserved globular "motor" domain that can bind microtubules and hydrolyze ATP, but other domains responsible for organelle binding and other unknown functions are highly divergent. RNAi analysis indicates that at least six of these worm kinesins are required for normal embryogenesis. Two are described here. CeMKLP1, which is encoded by the zen-4 gene, concentrates in the spindle interzone during anaphase (Powers et al., Curr. Biol. 8, 1133-1136, 1998; Raich et al. MBC 9, 2037-2049, 1998). Orthologs in other species have been implicated in assembly of the metaphase spindle, the anaphase interzone, and the cleavage furrow. Surprisingly, in CeMKLP1(RNAi) embryos, mitosis and spindle structure appear normal until late anaphase. Microtubule bundles form in the early anaphase interzone, and the cleavage furrow assembles and initiates normal inward movement. However, interzone bundles do not gather into a stable midbody, and furrow ingression always fails before complete closure. These and additional results identify CeMKLP1 as a specific component of interzone microtubule bundles that is required to encourage the advance and stable closure of the cleavage furrow. A Chromokinesin homologue is required for correct chromosome segregation. In CeChromoK(RNAi) embryos, the first mitotic division takes much longer than normal and often results in multiple micronuclei. Anti-tubulin and DNA staining of RNAi embryos shows that a fairly normal bipolar spindle assembles but that chromosomes do not align tightly on the metaphase plate. During anaphase, some chromosomes are lost from the spindle and chromosome bridges often stretch across the interzone. The lost chromosomes and micronuclei suggest that CeChromoK is important for chromosome spindle interactions. The anaphase bridges further suggest that it may be important for preventing or resolving bipolar chromatid attachment. The molecular mechanisms and protein assemblies that mediate motility processes in early embryogenesis are largely unexplored. C. elegans provides a unique opportunity to use RNAi, genetics, cytology and biochemistry to address such questions.

24 May 1999 15:50 737 737 SMA-1 is an actin-binding protein required for epithelial cell morphogenesis

1999 International Worm Meeting abstract 686 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. SMA-1 is an actin-binding protein required for epithelial cell morphogenesis V Praitis, J Austin University of Chicago, Chicago, IL sma-1 encodes an unconventional member of the b-spectrin family of actin-binding proteins, that has a specific role in morphogenesis. While conventional b-spectrins localize to the lateral membrane in epithelial cells, SMA-1 localizes to the apical membrane of epithelial cells and is twice the size of conventional b-spectrins. sma-1 mutant embryos have morphogenetic defects in multiple epithelial tissues: there is a decreased rate of epidermal elongation, resulting in decreased body length at hatching, and both pharynx and excretory cell are abnormally shaped. sma-1 is expressed in the epidermis, pharynx and excretory cell, for a brief period at the start of morphogenesis, suggesting it may act autonomously in each of these tissues. Multiple lines of evidence indicate that embryonic elongation is due to contraction of apically-located epidermal actin fibers. In the sma-1 null mutant, which has an out-of-frame deletion in the SMA-1 N-terminal actin-binding domain, actin fibers are disorganized and appear detached from the apical membrane. Based on this phenotype and the known function of other b-spectrins, we believe SMA-1 binds to both actin fibers and apical membrane proteins, forming a connection that allows cytoskeletal contraction to be converted into changes in cell shape. Sequencing non-null sma-1 alleles confirmed the importance of the SMA-1 actin-binding domain. 10/11 sequenced alleles contain premature stop codons. sma-1 alleles whose predicted proteins contain little more than the N-terminal actin-binding domain have only a weak effect on elongation. Surprisingly, sma-1 alleles with longer predicted proteins have more severe elongation phenotypes. The 11th sequenced allele, sma-1(ru7), has a missense mutation in the actin-binding domain and a phenotype that suggests an active role for SMA-1 in cell morphogenesis. In wild-type embryos, there is an evenly distributed layer of SMA-1 associated with the apical membrane of epidermal cells. In sma-1(ru7) embryos, SMA-1 is mislocalized within the lateral epidermal cells, forming an apical membrane-associated SMA-1 ’ring’ in each cell with a corresponding actin ’ring’. The result is a striking example of ectopic morphogenesis: in these worms, lateral bulges form at the site of the spectrin/actin rings, creating worms that appear to have wings.

24 May 1999 15:50 738 738 The unc-3 locus encodes an O/E transcription factor, CeO/E, which is required for axonal guidance and chemosensory function.

1999 International Worm Meeting abstract 687 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The unc-3 locus encodes an O/E transcription factor, CeO/E, which is required for axonal guidance and chemosensory function. BC Prasad 1 , RR Reed 1,2

1 Department of Molecular Biology and Genetics, Johns Hopkins University, 725 N. Wolfe Street, Baltimore MD 21205. 2 HHMI, Johns Hopkins University, 725 N. Wolfe Street, Baltimore MD 21205.

The O/E family of rHLH transcription factors has been implicated in neurogenesis, axonal pathfinding, muscle formation and B cell maturation. The murine O/E proteins are expressed transiently in the developing CNS and PNS during times of axonogenesis and are expressed in olfactory neurons throughout development where they may regulate components of the olfactory signaling cascade. We have identified a C. elegans O/E homologue (CeO/E) that is encoded by the unc-3 locus. Unc-3 mutants display an uncoordinated phenotype attributed to a severe defasiculation and miswiring of the ventral cord (VNC). Using a GFP fusion to the unc-3 promoter and specific antibodies, we observed that CeO/E is expressed transiently in the excitatory VNC motor neurons and throughout development in a pair of chemosensory neurons (ASI amphid neurons). Several observations suggest that the protein may have distinct roles in the two cell types. Although the axonal and dendritic projections of the ASI appear to be normal when labeled with DiI, unc-3 mutants enter the dauer pathway under inappropriate conditions. Although CeO/E possesses highly conserved domains associated with DNA binding in the mammalian homologue, identification of DNA binding sites for the C. elegans protein has not been achieved. We have shown that CeO/E protein can homodimerize in vitro, although the DNA binding specificity of CeO/E is distinct from the mammalian O/E proteins. Several unc-3 target genes (daf-7, unc-17/cha-1, unc-4) have been identified by other laboratories and each contains a mammalian binding site in the promoter region. Remarkably, we have been unable to demonstrate CeO/E binding at these sites. We are generating chimeras of O/E-1 and CeO/E to understand the DNA binding specificity of the CeO/E protein and utilizing a yeast-2-hybrid screen with both O/E-1 and CeO/E to identify interacting proteins in C. elegans. These studies should reveal the mechanism of CeO/E transcriptional activation and target gene regulation.

24 May 1999 15:50 739 739 The C. elegans sex determination gene mog-6 encodes a cyclophilin

1999 International Worm Meeting abstract 688 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The C. elegans sex determination gene mog-6 encodes a cyclophilin P Pugnale, AR Puoti Dept. of Zoology. University of Fribourg, Fribourg, Switzerland Germ cells in the C. elegans hermaphrodite can differentiate either into sperm or oocytes. While sperm is produced in the late L4 larva, all the germ cells in the adult differentiate into oocytes. The switch from spermatogenesis to oogenesis is dependent on the repression of the fem-3 mRNA. Although the precise molecular mechanisms leading to fem-3 repression are still unknown, several lines of evidence indicate that the expression of the fem-3 mRNA is controlled through a cis-acting element located in its 3’untranslated region (UTR). In order to understand the mechanisms that regulate fem-3, we are searching for trans-acting factors that interact with the fem-3 3UTR. The mog-6 gene is an excellent candidate for encoding such a regulator, since the Mog-6 loss-of-function phenotype is similar to that of fem-3 gain-of-function mutants, and since the expression of a reporter gene flanked by the fem-3 3’UTR is stronger in a mog-6 mutant background that in wild type worms. We have mapped the mog-6 locus to a four-cosmid region. Transient but consistent rescue of mog-6 could be obtained with a 4 kb fragment that is predicted to encode a cyclophilin. Cyclophilins represent a family of proteins that bind to the immunosuppressive drug Cyclosporin A. Cyclophilins have been shown to be involved in different cellular processes including RNA processing. Consistent with its role in germline cell fate specification, mog-6 is mainly expressed in the hermaphrodite germ line. We are currently studying the expression pattern and the molecular function of MOG-6 in order to understand its role in C. elegans development and more specifically, in the regulation of the fem-3 mRNA.

24 May 1999 15:50 740 740 unc-53 expression pattern and phenotype

1999 International Worm Meeting abstract 689 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. unc-53 expression pattern and phenotype N Pujol 1,2 , T Bogaert 1

1 Devgen N.V. Technologiepark 9, Zwijnaarde, Belgium. 2 present address IBDM, Case 907, 13288 Marseille, France.

We have characterised the expression of unc-53 using a GFP reporter strategy. The unc-53 gene spans over 30 kb, contains very large introns in its 5’ region, and has several SL1-spliced transcripts. We identified two different promoter regions, each associated with a distinct SL1. Each promoter element leads to GFP expression in a subset of pioneer neurons, sex muscles, sensory socket cells, distal tip cells and the excretory cell. The expression begins at the early comma stage and is present during process outgrowth. We found an expression in a majority of the cells known to be affected in unc-53 mutants (e.g. DA and AS motoneurons, vulval, diagonal and spicule retractor muscles, the excretory cell) suggesting that the observed expression patterns accurately reflect the in vivo expression pattern of the gene. During the morphogenesis of the sex muscles, we observed that in wild type, while the sex myoblasts undergo their three divisions, they migrate longitudinally, sending longitudinally processes along the edge between the seam and muscles to reach the hypodermis. Those processes could serve to sense migration cues. In unc-53 mutants, these structures don’t form and the A/P migration is dramatically reduced. Moreover, while in unc-53 mutants the mature vulval muscles have an organized and attached myofilament cytoskeleton, they make a morphologically different adhesive structure with the hypodermis, characterized by a rounded appearance. We used the defined promoters fused to the cDNA of unc-53 to construct minigenes directing the expression of unc-53 in specific cells. In unc-53 mutants, the axons of the paired neurons ALN, which normally run along the entire animal on the sub-lateral cords, stop before the vulval region and turn to join the dorsal cord. By expressing UNC-53 in those neurons in the mutant, we were able to rescue the mutant phenotype, suggesting that unc-53 acts cell autonomously in those cells. Moreover, when we over-expressed UNC-53 in the mec cells, which are normally bipolar, these neurons send ectopic filopodia-like outgrowths in different directions. Together with our previously reported work, these data suggest that expression levels or activation of the UNC-53 protein must be highly regulated to set up the normal direction and levels of outgrowth.

24 May 1999 15:50 741 741 Axonal pathfinding defects in CePhox2/ceh-17 mutants

1999 International Worm Meeting abstract 690 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Axonal pathfinding defects in CePhox2/ceh-17 mutants N Pujol 1 , J Ewbank 2 , R Terranova 2 , JF Brunet 1

1 IBDM, Luminy, 13288 Marseille, France. 2 CIML, Luminy, 13288 Marseille, France.

We are studying the C. elegans homologue of two vertebrate prd-like homeobox genes, Phox2a and Phox2b, involved in neuronal-subtype specification (1,2,3,4). The predicted amino acid sequence of CEH-17 is 88% identical to murine Phox2 proteins within the homeodomain, a level of similarity often associated with functional conservation between homeobox genes of vertebrates and invertebrates. An antibody raised against the C-terminus of CEH-17 reveals an expression restricted to 5 neurons located in the head ganglia: ALA and 4 ventral neurons tentatively identified as SMB. Interestingly, all those neurons send posteriorly directed axons along the lateral and sublateral cords. The expression is strongest from the 3-fold stage to L1 and decreases thereafter to a faint level in adults. In addition, different GFP fusions show a variable expression in other neurons not substantiated by immunohistochemistry and whose significance is therefore unclear. We have isolated a deletion allele in ceh-17. The 1.3 kb deletion extends from 0.6 kb upstream of the transcription start site to the first helix of the homeodomain and is thus expected to create a null allele. Indeed, immunostaining of the mutant reveals no CEH-17 expression. The hermaphrodite and male homozygous mutants are viable and fertile. Crosses between the mutant strain and a strain in which GFP is expressed under the control of a unc-53 promoter (see abstract by Pujol and Bogaert) allowed ALA axonal pathfinding to be assessed in the mutant. In the wild type, the two ALA axons migrate on the lateral cords as far as the tail, beginning at the 3 fold stage until early L1 stage (i.e. a period corresponding to the strongest expression of CEH-17). In the ceh-17 mutant, the axons of 80% of the scored ALA neurons stop at the level of the vulva. In 5% of the cases, the axon stop at the vulva but then returns half way along the same lateral cord. Therefore, CePhox2/ceh-17 is required for the posteriorly directed growth of ALA axons in the second half of their trajectory, from the gonad to the tail. We are now ectopically expressing CEH-17 and assessing the functional conservation of CEH-17 and murine Phox2.

(1) Morin et al. 97 Neuron 18 411-423 (2) Pattyn et al. Nature 99 in press (3) Ewbank et al. 98 WBG 15,3:17 (4) Pujol et al. 99 WBG 15,5:29

24 May 1999 15:50 742 742 CUL-2 is required for mitotic chromatin condensation

1999 International Worm Meeting abstract 691 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. CUL-2 is required for mitotic chromatin condensation G Punkosdy, H Feng, W Zhong, ET Kipreos Department of Cellular Biology, University of Georgia, Athens, GA 30602 cul-2 is a member of the cullin gene family in which two members, cul-1 and CDC53, function as E3s in the ubiquitin-mediated proteolysis of cell cycle regulators. cul-2(ek1) homozygous progeny of heterozygous parents have normal embryonic and postembryonic somatic cell lineages. cul-2 germ cells undergo a late onset G1 phase arrest that correlates with increased level of CKI-1 protein. The late onset of germ cell arrest as well as the observation that cul-2 dsRNA induces an immediate embryonic arrest, suggests that cul-2 is supplied as maternal product that perdures through the larval stages. cul-2 homozygotes lay only a small number of eggs, which all arrest with approximately 24 cells. These embryos have defective cytoskeletal movements with numerous cytoplasmic projections. The length of mitosis, and prometaphase in particular, is greatly increased. cul-2 embryos have a seemingly normal mitotic spindle, however, the chromosomal DNA in cul-2 mutants is not condensed. This uncondensed DNA appears to contribute to other embryonic cul-2 phenotypes: unequal DNA segregation, chromosome bridging, and multinuclei formation. cul-2 mutants have defective meiosis II in approximately 2/3 of cases, however, we observe uncondensed chromosomes at the first mitotic division even when the correct 4n DNA content is present upon pronuclei fusion. Other mutants that have defective meiosis also have multinuclei formation, the classic example is loss-of-function mei-1 alleles. We found that mei-1(b284) embryos with multinuclei have normal chromosome condensation although they have 30 or more chromosomes, suggesting that, in contrast to cul-2, mei-1 multinuclei form due to excessive chromosome numbers. In cul-2 oocytes in diakinesis, chromosomes have normal size and shape, suggesting that cul-2 is not required for meiotic chromosome condensation. However, in late cul-2 homozygote adults (4-5 days post-larvae), the 6 bivalents separate to produce 12 univalents. This effect is not a result of the old age of the cul-2 adults as young adult cul-2 homozygotes from germline mosaic parents also have univalents. The observed chromatin condensation defect as well as the separation of bivalents suggests that cul-2 has role(s) promoting chromosome condensation/integrity.

24 May 1999 15:50 743 743 DEAH-box proteins and sex determination in C. elegans

1999 International Worm Meeting abstract 692 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. DEAH-box proteins and sex determination in C. elegans AR Puoti 1 , JE Kimble 2

1 Dept. of Zoology, University of Fribourg, 1700 Fribourg, Switzerland. 2 University of Wisconsin, Madison 53706, USA.

The adult C. elegans hermaphrodite switches to oogenesis after a short burst of spermatogenesis. This dramatic change of cell fate involves the regulation of the fem-3 mRNA at a posttranscriptional level. Genetic screens have identified six mog genes that are required for the repression of fem-3 through its 3’UTR, and consequently, for the switch from spermatogenesis to oogenesis. We have cloned three mog genes, mog-1, mog-4 and mog-5. All three mog genes encode proteins of a distinct subfamily of DEAH-box proteins. This subfamily is characterized by a well-conserved central domain that is also found in yeast and mammalian pre-mRNA splicing factors. In addition to the three MOG proteins, three other DEAH-box proteins of this specific subfamily have been identified throughout the C. elegans genome. Using RNA interference, we have tested whether the three additional DEAH-box proteins might function as MOG proteins. While RNA interference with mog RNAs leads to close to 100% of dead embryos that are arrested at a specific stage during early development, this particular phenotype has not been observed with RNAs corresponding to the three additional DEAH-box proteins. Our data suggest that out of the six DEAH-box proteins of a specific subfamily found in C. elegans, MOG-1, MOG-4 and MOG-5 are the only ones that correspond to MOG proteins. We propose that MOG-1 and MOG-5 are the C. elegans homologues of the yeast and human splicing factors PRP16 and PRP22, and hPRP16 and HRH1 respectively, while MOG-4 bears most similarity with the yeast splicing factor PRP2. We are investigating the role of MOG-1, MOG-4 and MOG-5 in the regulation of the fem-3 by searching for factors they might interact with.

24 May 1999 15:50 744 744 cDNA libraries enriched in clones representing rare mRNAs

1999 International Worm Meeting abstract 693 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. cDNA libraries enriched in clones representing rare mRNAs AT Puzyrev, DL Riddle Division of Biological Sciences, University of Missouri, Columbia, MO 65211-7400 We have developed a method to remove abundant sequences from cDNA libraries based on second-order kinetics of reassociation of double-stranded cDNAs. First, the same set of dauer larva-specific cDNAs (unnormalized cDNAs) was cloned into two vectors - lambda gt11 and lambda ZapII. Second, the cDNA inserts of recombinant lambda ZapII molecules were amplified by PCR and used for preparation of an abundant cDNA pool. The preparation of this pool involved denaturation of the double-stranded cDNA inserts, partial reassociation, and selection of the double-stranded fraction. Single-stranded unnormalized cDNAs were prepared from the lambda gt11 library and mixed with an excess of the abundant cDNA pool. After denaturation and partial reassociation, the single-stranded fraction was selected. This fraction included single-stranded cDNAs generated from recombinant lambda gt11 molecules that did not hybridize with abundant cDNAs, as well as nonhybridized single-stranded cDNAs generated from the abundant cDNA pool. Using primers specific for lambda gt11, the single-stranded cDNAs were amplified by PCR and cloned. We have prepared and characterized two rare cDNA libraries, differing in the degree to which abundant sequences have been depleted. One of them exhibited a 50-100x increase in concentration of daf-1, daf-4, and daf-7 cDNAs along with a 3-fold decrease in concentration of the abundant Hsp90 cDNA (daf-7 is expressed at low levels in only two cells), relative to the unnormalized cDNA library. The second, more heavily depleted cDNA library exhibited a 3-6x increase in concentration of daf-1, daf-4, and daf-7 cDNAs and at the same time a 35-fold decrease in concentration of Hsp90 cDNA. In this latter cDNA library, the daf sequences were sufficiently abundant not to be enriched as much as in the former cDNA library. We would expect that cDNAs more rare than daf cDNAs should be more highly enriched. We have sequenced 10 random clones of the latter rare cDNA library. Half of these correspond to genes that are not represented by any ESTs in the C. elegans EST database. Such cDNA libraries enriched in rare cDNA sequences can serve as a resource for finding new ESTs and for construction of subtractive cDNA libraries to investigate dauer-specific gene expression.

24 May 1999 15:50 745 745 Functional analysis of C. elegans PKN homologue

1999 International Worm Meeting abstract 694 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Functional analysis of C. elegans PKN homologue H Qadota, T Miyauchi, K Kasuya, K Kaibuchi Division of Signal Transduction, Nara Institute of Science and Technology, The Rho family GTPases (Rho, Rac, Cdc42) are involved in the regulation of actin reorganization, cell polarity, cell growth, and cell-cell adhesion. The Rho family GTPases have two interconvertible forms; the GTP-bound active and GDP-bound inactive forms. The GTP-bound form of Rho family GTPases interacts with their specific effectors. The GTP-bound form of RhoA binds preferentially to Protein Kinase N (PKN), Rho-kinase (also called ROK), myosin binding subunit of myosin phosphatase (MBS). Besides proceeding of functional analysis of Rho-kinase and MBS, the physiological functions of other effectors of Rho GTPase including PKN remain to be clarified. Here we examined physiological functions of PKN using C. elegans as a genetic model system. We have identified the C. elegans homologue of mammalian PKN in genome sequencing database and Yuji Kohara’s EST sequence library. The cDNA clone, yk345c10, which we call pkn-1 (C. elegans PKN), maps to cosmid F46F6 on the right arm of LGX. The predicted protein shows significant homology to mammalian PKN. E. coli expressed NH2 -terminus of PKN-1 fused with maltose binding protein binds preferentially to GTPgS GST-CeRhoA, but not to GDPGST-CeRhoA, suggesting that the C. elegans PKN homologue also interacts with Rho GTPase in C. elegans. Based on expression studies using the pkn-1::gfp fusion construct, pkn-1 is preferentially expressed in the muscle cells. Overexpression of the catalytic domain of PKN-1 under the control of heat shock promoter caused abnormal movement, suggesting that CeRhoA and PKN-1 are involved in the regulation of the muscle contraction.

24 May 1999 15:50 746 746 Promoter analysis of the lin-26 gene.

1999 International Worm Meeting abstract 695 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Promoter analysis of the lin-26 gene. S Quintin, A Davies, BG den Boer, M Labouesse IGBMC, BP163, 67404 Illkirch cedex, France. The gene lin-26is involved in the specification and/or maintenance of hypodermal and glial-like cell identity and is also required for somatic gonad development. How is regulated an early specification gene? To answer this question, we began an analysis of the lin-26promoter, taking advantage of its strong conservation in the C. briggsaehomologue. So far we have identified four major cis-elements that activate lin-26expression in particular cells. This is based on three criteria: 1) the specific enhancer activity of these elements when put in front of a minimal promoter linked to GFP -pPD97.82, from Andy Fire; 2) the complementary lack of expression when the corresponding element is deleted from a lin-26::GFP fusion and 3) the rescuing activity of deleted constructs, tested in a null background. A gonad element (760bp): when the gonad element is deleted, animals are sterile with gonad defects. Conversely, this element drives GFP expression in Z1 and Z4, the precursors of the somatic gonad in which lin-26is normally expressed. A head and tail element (530bp): it drives GFP in two regions of the embryo starting at the comma stage: in about 20 cells in the head (mostly glial-like cells and a few hypodermal cells) and in 4 cells in the tail (hyp10 and the spike cells). Deletion of this element leads to dye filling defects in amphids and phasmids. A hypoderm element (4kb): it is only defined by the first criterion in that this element expresses GFP in all cells of the major hypodermis as well as in a few cells of the minor hypodermis. This expression requires the activity of elt-1since in elt-1(zu180)mutants the number of GFP-expressing cells is dramatically reduced. Rescue experiments show that this element is dispensible suggesting redundancy in hypoderm-specific promoter elements. A rectum element (3.5kb): it is again only defined by enhancer properties, turning on GFP in rep cells. Expression of lin-26in these cells is not essential for viability. Future experiments will focus on the definition of smaller regions responsible for these patterns and on the identification of regulators that bind to these target sequences.

24 May 1999 15:50 747 747 Analysis of C05C12.3 and F54D1.5, two genes for gon-2-like putative cation channels

1999 International Worm Meeting abstract 696 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of C05C12.3 and F54D1.5, two genes for gon-2-like putative cation channels CD Ragnauth, HA Baylis Department of Zoology, University of Cambridge, Downing Street, Cambridge, CB2 3EJ, UK In order to understand how the highly structured nature of calcium signals is achieved it is necessary to identify and characterise the channels responsible for calcium flux in animal cells. However the proteins responsible for a wide range of observed calcium fluxes in cells remain to be identified. We are therefore interested in identifying novel calcium and/or cation channels in animals. C05C12.3 and F54D1.5 are members of a family of three potential calcium channel proteins in C. elegans. The third member is GON-2 (E. Lambie pers. comm.). Mammalian relatives of these channels include melastatin. Further homologues have been identified in the genome project databases of Drosophila, mouse and humans. Thus this family of channels is widely distributed in animals. We have used 5’ RACE to identify the 5’ ends of the C05C12.3 and F54D1.5 mRNAs, in both cases these differed from those previously predicted. We used this information together with that from ESTs characterised by Yuji Kohara to clone full length cDNAs for each gene. These have been sequenced. In situ hybridisation to larval and adult worms suggested that C05C12.3 might be expressed in the gut. We have constructed C05C12.3::GFP fusions using a 4.7 kb region of the genomic DNA extending from exon 2 of the C05C12.3 gene to 4.5kb upstream. Preliminary experiments suggest GFP expression in the gut. We will present further analysis of these constructs. Experiments to investigate the role of C05C12.3 in C. elegans are underway. We are currently performing RNAi experiments and the gene has been submitted to the Gene Knockout Project. These experiments will help to understand the functions of these putative channels in animals.

24 May 1999 15:50 748 748 Characterization of unc-23 and its suppressors

1999 International Worm Meeting abstract 697 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of unc-23 and its suppressors sP Rahmani, DG Moerman Department of Zoology, University of British Columbia. 6270 University Blvd. Vancouver, BC. Canada. V6T1Z4 Animals homozygous for mutations in the unc-23 gene exhibit progressive dystrophy of the anterior body wall musculature and have a diagnostic bent-head phenotype. This phenotype is not observed when animals are grown in liquid culture (Bullerjahn and Riddle, pers. Comm.). Neither muscle cell positioning nor myofilament assembly is affected in liquid grown unc-23 animals. Muscle cell attachment, however, is affected since a small amount of stress applied results in detachment of the muscle cells from the hypodermis. The results of immunological staining of unc-23 animals with antibodies to basement membrane and hypodermal components suggests that the primary defect inunc-23 animals is located within the hypodermis. We have used PCR/deficiency mapping to molecularly map unc-23 to a region of nine cosmids. Our transformation rescue attempts, using the cosmids within this region, has to date been unsuccessful. We are now adding other methods such as YAC injections and RNAi to our list of approaches in order to rescue unc-23. To further characterize the role of unc-23 in muscle cell attachment and identify other interacting proteins, we screened for suppressors of unc-23(e25) animals. We have identified 7 dominant and 5 recessive suppressors. We have determined that four of these are intergenic suppressors. We will report on the mapping and further characterization of these suppressors.

24 May 1999 15:50 749 749 UNC-1 and UNC-8 interact to control anesthetic sensitivity in C. elegans

1999 International Worm Meeting abstract 698 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. UNC-1 and UNC-8 interact to control anesthetic sensitivity in C. elegans S Rajaram, MM Sedensky, PG Morgan Departments of Anesthesiology and Genetics, University Hospitals and Case Western Reserve University, Cleveland, OH INTRODUCTION: We have undertaken a study of genes that define a pathway controlling sensitivity to volatile anesthetics. Mutations in unc-1 cause widely differing changes in anesthetic sensitivity. The sequence of unc-1 shows homology to stomatin, an integral membrane protein found in essentially all tissues in mammals. Here we report 1. the further characterization of unc-1 2. the cellular and temporal expression of unc-1 and 3. the genetic interactions between unc-1 and unc-8. RESULTS: Mutations affecting anesthetic sensitivity were distributed primarily in the cytoplasmic portion of stomatin. Temperature shift experiments with temperature sensitive mutations indicated that the unc-1 function is required throughout the life of the animal. It is expressed in several classes of C. elegans neurons with little staining of non-neuronal cells. The UNC-1 protein interacts with a variety of other proteins, including the degenerin UNC-8. Like unc-1, unc-8 alleles have a wide range of altered sensitivities to volatile anesthetics. CONCLUSION: In humans, stomatin is believed to regulate an associated ion channel, most likely a sodium channel in a ’ball & chain’ fashion. The localization of most mutations to the cytoplasmic domain of the protein suggests abnormal gating of a channel in these unc-1 mutants. Since mutations in unc-1 interact with mutations in unc-8, it is possible that the ENaC-like protein is a subunit of the channel gated by unc-1. This is particularly interesting since ENaC-like channels and stomatin like messages have recently been found in mammalian brain. Studies with GFP reveal extensive expression of the unc-1 gene in both sensory and motor neurons, but not in body wall muscles. In addition, genetic studies indicate that mutations in unc-1 and in unc-8 directly affect anesthetic sensitivity. We therefore postulate that volatile anesthetics directly affect the ability of UNC-1 and UNC-8 to control ion flux across membranes which results in a direct effect on mobility in C. elegans. ACKNOWLEDGEMENTS: The authors are indebted to Monica Driscoll and Nektarios Tavernarakis for ongoing critical discussions and for sharing unpublished data. MMS and PGM were supported by NIH grant GM45402.

24 May 1999 15:50 750 750 The RdgC Homolog CePPEF: A Protein Phosphatase Found in Interneurons and the Cilia of Several Sensory Neurons.

1999 International Worm Meeting abstract 699 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The RdgC Homolog CePPEF: A Protein Phosphatase Found in Interneurons and the Cilia of Several Sensory Neurons. PY Ramulu, J Nathans Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, PTCB 804, 725 N. Wolfe St., Baltimore, MD 21205 The PPEF (protein phosphatase with EF-hands) family consists of rdgC, a gene causing retinal degeneration in D. melanogaster, mPPEF-1 and mPPEF-2 in mice, and cePPEF in C. elegans. RdgC, mPPEF-1, and mPPEF-2 have been shown to be localized to sensory neurons, with rdgC and mPPEF-2 found in the Drosophila and mouse eye respectively, and with mPPEF-1 localized to dorsal root ganglia and inner ear neurons in mice. We show here that the CePPEF protein is also a neuron-specific phosphatase. Using GFP fusions, we identify approximately 20 head neurons, including 4 ciliated neurons, and one tail neuron which express cePPEF. This data is confirmed by preliminary antibody staining with serum raised against the recombinant cePPEF protein. Moreover, GFP fusions to the full-length protein indicate that ciliated neurons expressing cePPEF concentrate the protein in the cilium. The N-termini of cePPEF, mPPEF-1, and mPPEF-2 all contain consensus myristoylation signals of M-G-X-X-X-S/T and, consistent with this finding, NLS-GFP fusions of as few as 8 of the N-terminal amino acids of CePPEF lead to nuclear exclusion, and efficient filling of the dendritic and axonal processes. In contrast, fusions with only 5 N-terminal CePPEF residues, which lack the consensus serine/threonine, are localized primarily to the nucleus. Analysis of GFP fusions which mutate the consensus N-terminal glycine of CePPEF are in progress. Experiments are also underway to test if the native PPEFs, or PPEFs exogenously expressed in C. elegans or tissue culture, are myristoylated, and if myristoylation is a calcium-dependent process.

24 May 1999 15:50 751 751 Behavioral Analyses of nmr-1: Does the Mutation Affect Learning in the Worm?

1999 International Worm Meeting abstract 700 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Behavioral Analyses of nmr-1: Does the Mutation Affect Learning in the Worm? C. H. Rankin 1 , E. Phillip 1 , J. Wells 1 , P.J. Brockie 2 , D.M. Madsen 2 , A.V. Maricq 2

1 Department of Psychology, University of British Columbia, Vancouver, BC, Canada. 2 Dept. of Biology, University of Utah, Salt Lake City, Utah.

We have shown that C. elegans is capable of both long and short-term habituation in the response to tap, as well as context conditioning in habituation of the tap response. We have also determined that the neural circuit underlying the response to tap is composed of the 5 touch cells (2 ALMs, 2 PLMs and 1 AVM), one putative stretch receptor (PVM) and 5 pairs of interneurons (2 each of AVA, AVB, AVD, PVC and DVA). Recent work with a mutant strain, eat-4, deficient in a gene involved in glutamate transmission (expressed in the sensory neurons of this circuit) has led to the hypothesis that the touch cells are glutaminergic. This hypothesis has been supported by the finding that some or all of the interneurons of the circuit express genes that code for a number of putative glutamate receptor subunits including subunits with significant homology for AMPA-type receptors (glr-1) and for NMDA-type receptors (nmr-1), and that glr-1 worms show greatly reduced responses to tap and nmr-1 worms show slightly reduced responses to tap. In these experiments we report the effects of a deletion mutation of a gene that shows 33% homology to the vertebrate NR1 subunit of the NMDA-type glutamate receptor. This mutant strain (nmr-1) was isolated by Brockie and Maricq in Utah and supplied to us for behavioral testing. In addition we report preliminary observations on glr-1 worms response to tap. The nmr-1 worms’ response to tap is slightly smaller and of longer latency than responses of N2. We found essentially normal habituation and recovery from habituation at 10 and 60 s ISI in nmr-1 worms. We also tested whether the mutation affected long-term memory for habituation training as well as the associative aspect of context conditioning in which a chemical cue is paired with habituation to tap.

24 May 1999 15:50 752 752 Characterisation of C.elegans C-terminal kinesin KLP-3

1999 International Worm Meeting abstract 701 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterisation of C.elegans C-terminal kinesin KLP-3 Rao Susmitha, Manjari Mazumdar National Center for Biological Sciences,UAS-GKVK Campus,Bangalore-560065,Karnataka, India KLP-3 is a C-terminal kinesin-like protein in nematode C.elegans, which shares extensive homology with yeast Kar3 and Drosophila ncd motor. These kinesins have previously been shown to mediate chromosomal movement and segregation during mitosis and meiosis. Overexpression of klp-3 gene causes production of dead eggs and reduction in the number of males due to non-disjunction of the X-chromosome, suggesting, that this kinesin may be involved in chromosome movement and segregation and specially in the movement of the sex chromosome during meiosis. Antisense klp-3 RNA expression causes many developmental abnormalities in the progeny, suggesting again a critical role for the klp-3 function in chromosome segregation. Determination of the rate and direction of motility and steady state kinetic properties of KLP-3 may be important for elucidating its function in vivo. The klp-3 full-length cDNA was subcloned in a bacterial expression vector and expressed as 6xHis-tag protein and a truncated version as GST-fusion protein in E.coli cells (BL-21 pLysS). The 6x His-tagged full-length protein obtained from the bacterial cell lysate was purified over Ni-NTA (Qiagen) affinity column and Mono-Q ion exchange column (Pharmacia). Microtubule binding assay was carried out with the full-length and the truncated GST-fusion protein of KLP-3. It was found that both the fragments bound specifically to the taxol-polymerised tubulin and released from MT at 10mM ATP but required additional 100mM KCl, which is consistent with the data of other C-terminal kinesin, ncd, in Drosophila. Work is in progress to determine the local conformation of ATP binding site, the MT-activated steady state ATPase activity and the motor properties of KLP-3.

24 May 1999 15:50 753 753 Identification of EGL-27 Molecular and Genetic Interactions

1999 International Worm Meeting abstract 702 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification of EGL-27 Molecular and Genetic Interactions TM Ratliff, MA Herman Division of Biology, Kansas State University, Manhattan, KS 66506 We are interested in the control of cell polarity during C. elegans development. Our approach has been to identify mutations which disrupt the polarity of the T cell in the hermaphrodite tail. Mutations in the Wnt gene lin-44 and in the frizzled-related gene lin-17 have been shown to cause a reversal and loss of T cell polarity, respectively (Herman et al., 1995; Sternberg and Horvitz, 1988). Mutations in egl-27 also cause a loss of T cell polarity as well as defects in QL daughter cell migration (Herman et al., 1999). These two processes are known to involve Wnt signals and pathways. In addition, certain egl-27 mutants and egl-27 RNAi animals display embryonic patterning defects. The amino terminus of the predicted egl-27 protein contains a region of similarity to MTA1 (Herman et al., 1999), a mammalian factor overexpressed in metastatic cells (Toh et al., 1994). Further analyses of the EGL-27 amino terminal sequence has revealed several regions of similarity to proteins involved in chromatin remodeling (Solari, F., Bateman, A. and Ahringer, J., pers. comm.). MTA1 has been shown to be a subunit of a novel complex displaying ATP-dependent chromatin-remodeling and histone deacetylase activities. Specifically, MTA1 appears to function as a transcriptional repressor (Xue et al., 1998). The carboxy terminus of EGL-27 is glutamine-rich, suggesting it may be involved in protein-protein interactions. We propose that EGL-27 is a bifunctional protein having both a transcriptional repression function and an as yet identified function. To better understand EGL-27 function we are determining those proteins which interact with EGL-27 at both the molecular and genetic levels. We are using the yeast two-hybrid system to screen for proteins that interact with either the amino terminal domain, carboxy terminal domain or full length EGL-27 protein. To identify genetic interactions, we are constructing and analyzing strains doubly mutant for a loss-of-function mutation in egl-27 and a mutation in specific genes involved in specifying cell polarity, Wnt signaling or chromatin remodeling.

24 May 1999 15:50 754 754 Actions of cholinergic anthelmintics and ivermectin on recombinant homomeric nicotinic acetylcholine receptors chicken a7 and C. elegans ACR-16

1999 International Worm Meeting abstract 703 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Actions of cholinergic anthelmintics and ivermectin on recombinant homomeric nicotinic acetylcholine receptors chicken a7 and C. elegans ACR-16 V Raymond, NP Mongan, DB Sattelle The Babraham Institute, Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, UK. Two homomer-forming nicotinic acetylcholine receptor subunits (nAChRs) with 47% identity in their amino acid sequences have been used to compare aspects of the pharmacology of expressed vertebrate and nematode nAChRs of defined molecular composition. Functional recombinant receptors were studied in Xenopus oocytes following nuclear injection of cDNA encoding either chicken a7 or C. elegans acr-16 (1) nAChR subunits. Voltage-clamp electrophysiology was used to compare the actions of cholinergic anthelmintics on these recombinant homomeric nicotinic AChRs. Butamisole (an imidazothiazole), morantel (a tetrahydropyrimidine) and methyridine (a pyrimidine) were without agonist effects on either the chicken a7 or the C. elegans ACR-16 in the range 10nM-1mM. However, for both receptors butamisole and morantel antagonised responses to acetylcholine (ACh) whereas at high concentrations (1mM), methyridine did not affect responses to ACh of either receptor. Oxantel, a tetrahydropyrimidine structurally related to morantel, was without agonist actions on ACR-16, but was an ACh antagonist. In contrast, it was found to have partial agonist actions on chicken a7 at concentrations in the range 10µM-300µM. In agreement with a previous study (2), the macrocyclic anthelmintic compound ivermectin, which acts primarily on L-glutamate gated chloride channels, was found to enhance the amplitude of responses of chicken a7 AChRs to ACh and nicotine. However, ivermectin did not potentiate the ACh induced responses of ACR-16 but diminished slightly ACh responses of the nematode ACR-16. This points to differences in the modulation of these similar homomeric nAChRs. This study has revealed differences in pharmacology of vertebrate and nematode recombinant nAChRs with respect to the action of cholinergic anthelmintics and ivermectin. Further studies employing chimeras and site-directed mutagenesis should increase our understanding of the molecular basis of these pharmacological differences and may eventually assist in the design of novel anthelmintics with enhanced selectivity.

(1)Ballivet et al. (1996) J. Mol. Biol. 258, 261-269 (2)Krause et al. (1998) Mol. Pharmacol. 53, 283-294

24 May 1999 15:50 755 755 The engulfment of dying cells contributes to the killing process of programmed cell death

1999 International Worm Meeting abstract 704 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The engulfment of dying cells contributes to the killing process of programmed cell death PW Reddien, JS Cameron, HR Horvitz HHMI, Dept. Biology, MIT, Cambridge, MA 02139 The engulfment of a cell corpse by a neighboring cell typically occurs rapidly after the generation of a cell programmed to die. This engulfment can initiate before the completion of cell division, as shown by electron microscopy (1). Because cell corpses are generated in mutants defective in engulfment, the proposed function of engulfment has long been solely the removal of unwanted apoptotic cell bodies. We have discovered, however, that in addition to functioning in cell-corpse removal, engulfment assists in the killing of dying cells. Animals with strong loss-of-function mutations in the killer gene ced-3 lack most if not all programmed cell deaths. However, weaker ced-3 mutants exist that lack only a small percentage of programmed cell deaths. We found that such a partial block in the execution of programmed cell death is enhanced by mutations in genes that are involved in the engulfment process (ced-1, ced-2, ced-5, ced-6, ced-7, and ced-10). Furthermore, mutations in these engulfment genes alone are sufficient to result in a low-penetrance survival of some cells that normally die in the ventral cord. Lineage analysis shows that cells that fail to die initially show some morphological characteristics of programmed cell deaths but ultimately appear morphologically indistinguishable, using Nomarski optics, from living cells. Surviving Pn.aap cells in these ventral cord lineages are capable of expressing the cell-type specific reporter lin-11::gfp (see Cameron, Tsung, and Horvitz, this meeting), suggesting that they are capable of differentiating. We are analyzing the relative contributions of the different engulfment genes to cell killing, how the engulfment role in killing interacts genetically with ced-8 and ced-9, and the contribution of the killing role of engulfment to the death of different cell types.

1. Sulston, J.E., Albertson, D.G., and Thomson, J.N. (1980). Dev. Biol. 78, 542-576.

24 May 1999 15:50 756 756 Does mab-19 encode an hepatocyte growth factor receptor-related gene?

1999 International Worm Meeting abstract 705 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Does mab-19 encode an hepatocyte growth factor receptor-related gene? DJ Reiner, BJ Meyer HHMI and Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA The mab-19 gene is defined by two hypomorphic alleles originally characterized by Sutherlin and Emmons (Genetics 138: 675). Both mab-19 mutations were shown to cause defects in male sensory ray morphology, the T-lineage, and migration of the distal tip cell, all with incomplete penetrance. Both mutations in trans to a deficiency of the region cause embryonic lethality due to failure of ventral hypodermal closure. Therefore, mab-19 mediates cell fate determination, migration and morphogenesis. We further mapped mab-19 to a small genetic interval containing a predicted receptor tyrosine kinase. The cytoplasmic domain of this kinase is most related to the hepatocyte growth factor receptor, also known as the c-met proto-oncogene, while the cysteine-rich extracellular domain is novel. In mammals, HGFR also mediates diverse developmental events like mab-19. Therefore, we hypothesize that mab-19 encodes the HGFR-like protein. A PCR product containing only the HGFR-like gene rescues the incompletely penetrant male tail defects of mab-19. However, no mutations were found in the entire cDNA sequence from both hypomorphic alleles. A GFP transcriptional fusion is widely expressed in different tissue types, but appears to be subject to transcriptional silencing after the first generation. We plan to identify a null mab-19 mutation, find the molecular lesions in mab-19, and raise antibodies to determine protein localization.

24 May 1999 15:50 757 757 Gene Expression Profiles in the Germline Using Microarrays

1999 International Worm Meeting abstract 706 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Gene Expression Profiles in the Germline Using Microarrays V Reinke, J Wang, CB Van Doren, RR Begley, SK Kim Stanford University Medical Center, Stanford, CA 94305 Microarray technology, in combination with the completion of the entire genomic sequence of C. elegans, provides the means to investigate the expression of every gene in the genome in response to a particular condition or mutation. Additionally, microarray analysis allows for comparison across multiple experiments, and can form higher-order expression patterns for groups of genes. Members of the laboratory have generated a C. elegans microarray containing genomic PCR products of approximately 13,000 genes, with plans to create a "whole genome" microarray in the near future. Experiments in the lab are directed in part toward investigation of gene expression patterns in the germline. The germline is amenable to microarray analysis as it comprises a large portion of the worm, and germline-specific transcripts will be well represented. Several aspects of gene regulation can be explored in this tissue. Meiosis and gametogenesis are regulated both spatially and temporally; additionally, mutants with germline phenotypes can be useful in probing the functional significance of gene expression patterns that are seen. To establish a baseline for genes expressed in the germline, wild type (N2) germline formation will be compared with that of a glp-4(bn2ts) mutant, in which Z2 and Z3 do not proliferate at 25°C and are unable to fill the somatic gonad with germ cells (Beanan and Strome, 1992). Transcripts present in N2 that are absent or greatly reduced in glp-4(bn2ts) indicate genes with germline-restricted expression. Further microarray analysis utilizing several germline mutants will aid in dividing the list of germline-specific genes into several subcategories. Mutants that affect the switch from mitotis to meiosis can be used to identify those genes regulated in mitosis but not meiosis, or vice versa. Mutants affecting sex determination can be studied to identify transcripts that are regulated in a sex-specific manner. Additionally, mpk-1(lf) results in arrest of meiotic prophase at the pachytene stage and can be used to identify genes acting downstream of map kinase to promote progression through pachytene. Such experiments will give insight into the gene expression patterns that underlie the mechanisms of germline formation and function, and provide an example of the power of the new genomic technologies available today.

24 May 1999 15:50 758 758 Effects of Heat Shock on Early Embryogenesis and Gut Development

1999 International Worm Meeting abstract 707 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Effects of Heat Shock on Early Embryogenesis and Gut Development K. Resendes, D.C. Shakes Dept. of Biology, College of William and Mary, Williamsburg, VA 23187 Heat is a recognized teratogen in animals and there is string evidence that it causes significant damage to human embryos.(1) To determine if C. elegans could serve a good model system for analyzing such effects, we began studying the effect if heat shock on early C. elegans embryogenesis . In earlier studies (2), we showed that C. elegans embryogenesis is affected by heat shock. In particular, we found that early embryos respond to heat shock immediately halting cell division. After a temporary developmental arrest, these embryos resume cell proliferation but subsequently fail to undergo morphogenesis. To further investigate this embryonic heat shock response and to determine if the observed defects resulted from the disruption of specific cell-cell interactions during development, we began to examine the specific effects of heat shock on gut induction. We chose to study gut induction because it is easy to score gut differentiation by the presence of gut specific markers and because gut induction has been specifically pin-pointed to a cellular interaction between the P2 and EMS blastomere during the four-cell stage (3,4). Our recent studies have revealed a correlation between heat shock during the four cell stage and gut induction. In the first set of experiments, we shocked embryos for twelve to fifteen minutes at 37C. If the heat shock was done at the four cell stage, the embryos failed to develop gut granules, however if the shock was carried out after the EMS cell had already divided, the treated embryos consistently produced gut granules. In follow-up experiments, we have attempted to pin-point the period within the 14-15 minute EMS cell cycle which is critical for gut induction. Our results show that heat shocks initiated during the first six minutes of the EMS cell cycle prevent gut formation whereas later shocks are permissive for gut development. This result is consistent with findings that gut induction is thought to occur 5-6 minutes into the cell cycle (5). Thus our results suggest that heat related birth defects may be linked to the disruption of specific cell-cell interactions during early development and that C. elegans will be a good model system for such studies.

1)Edwards et al (1997) Int J Dev Biol 41(2):345-58. 2)Dichoso et al (1995) Int WM. 3)Goldstein (1993) Nature 357:255-257. 4)Thorpe et al (1997) Cell

24 May 1999 15:50 759 759 Characterization of Ets Transcription Factors in C. elegans

1999 International Worm Meeting abstract 708 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of Ets Transcription Factors in C. elegans RD Reventar, A Hart, L Zhang, J Culotti, A Bernstein Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute of Mount Sinai Hospital, 600 University Ave., Toronto, Ontario, Canada, M5G 1X5 We are investigating C. elegans homologs of the Ets family of transcription factors. In particular, we are focusing on members of the Elf subfamily. In humans, elf genes have been implicated in regulatory roles including development, immune function and tumorigenesis. For instance, the human elf-1 gene induces T cell-specific gene expression during T cell activation and maturation. Moreover, elf-2 has been implicated in T cell leukemia, and the amplification or up-regulation of the epithelial-specific, elf-3 gene may be involved in breast cancer. By developing a simple genetic model, we aim to study the biological function of members of this interesting elf subfamily and to identify interacting factors. In a reverse genetic approach, we used a PCR-based screen of a frozen worm library to isolate worms carrying specific genomic insertions of the Tc1 transposon. Our screen identified the presence of a Tc1 insertion between the pointed and ets domains of the putative C. elegans homolog of human elf-3, designated F22A3.1. We have since isolated a line homozygous for the Tc1 insertion that will next be amplified to construct a sublibrary of insertion worms. Using a similar PCR assay, this sublibrary will be screened for deletions in F22A3.1. Deletions are produced at a low frequency, when DNA flanking the Tc1 transposon is lost as it excises from the gene. An F22A3.1 mutant may provide insight into the function of F22A3.1 in vivo and may allow enhancer and suppressor screens for interacting genes. Furthermore, in expression studies, we made green fluorescent protein (GFP) reporter constructs driven by the predicted promoters of F22A3.1 and another elf-related gene, C33A11.4. To date, we generated transgenic worm lines from the first construct. Our results suggest that F22A3.1 expression is restricted to hypodermal precursor cells during the developing larval stages and is specific to hypodermal tissues in the adult worm. Sequencing of cDNA’s and 5’ RACE was performed to determine the exon/intron structure of F22A3.1 and C33A11.4. For future studies we are preparing reagents for dsRNA interference, overexpression and mutant rescue.

24 May 1999 15:50 760 760 Ce-hcr-51 gene, the RAD51/DMC1 homolog in Caenorhabditis elegans: a functional characterization

1999 International Worm Meeting abstract 709 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Ce-hcr-51 gene, the RAD51/DMC1 homolog in Caenorhabditis elegans: a functional characterization C Rinaldo 1 , P Bazzicalupo 1 , S Ederle 2 , M Hilliard 1 , M Mastrogiacomo 1 , A La Volpe 1,3

1 IIGB - CNR, - 10, Via Marconi - 80125 Naples, Italy. 2 Dipartimento di Genetica e Biologia Generale e Molecolare, Università "Federico II", - 8, Via Mezzocannone - 80134 Naples, Italy. 3 [email protected]

We have cloned a RAD51/DMC1 homolog in Caenorhabditis elegans, we named Ce-hcr-51 for homolog of Saccharomycescerevisiae RAD51. This locus, we mapped on chromosome IV on the left of the mec-3 gene on fosmid H36F17, is transcribed into two alternative mRNAs potentially coding for proteins of 395 (Hcr51L) and 357 (Hcr51S) aminoacids in length (Rinaldo et al.1998). Experiments of RNA interference show that the inactivation of the locus results in adult hermaphrodite sterility of the F1. The F2 embryos show gross abnormality, develop to different stages, but they do not hatch, suggesting a meiotic defect leading to aneuploidy. We are also analysing the gene expression pattern by injecting the promoter sequences upstream the GFP/LacZ coding sequences, and by RNA analysis. We are using a two hybrid system assay to study the ability of the putative products of hrc-51 to form homodymers and heterodymers in vivo. We identified two regions of the Hrc51S protein involved in the homodymerization process, one at the aminoterminal corresponding at the homodymerization domain already mapped in the yeast Rad51 protein (Donovan et al.1994), and another located in the carboxiterminal half of Hrc-51 that seems to play a role either in the self-association activity or in stabilizing the protein. We are also studying proteins interacting with Ce-hcr-51 products.

Donovan J. W., Milne G. T., and Weaver D. T. (1994) Genes Dev 8 : 2552-62. Rinaldo C., Ederle S., Rocco V., and Volpe A. L. (1998) Mol. Gen. Genet. 260 : 289-294.

24 May 1999 15:50 761 761 Isolation and Identification of Suppressors of an Activated Ca++/Calmodulin-Dependent Protein Kinase II Homolog in C. elegans

1999 International Worm Meeting abstract 710 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Isolation and Identification of Suppressors of an Activated Ca ++ /Calmodulin-Dependent Protein Kinase II Homolog in C. elegans M Robatzek, JH Thomas University of Washington, Department of Genetics, Seattle, WA 98195

Ca++ /calmodulin-dependent protein kinase II (CAMKII) is thought to participate in mammalian learning based on transgenic and knockout mouse studies1 however, the mechanism of CAMKII function in this process has not been elucidated. The phosphorylation of many gene products has been attributed to CaMKII based on in vitro assays, but in vivo evidence for these interactions is minimal. UNC-43, the C. elegans homolog of CAMKII, is 83% identical in the kinase domain to the mammalian CAMKII, suggesting that the targets of this kinase will also be conserved. We are using a genetic approach to identify in vivo targets of unc-43. A kinase-activated allele of unc-43, n498, causes defects in egglaying, locomotion, and defecation. We reasoned that loss-of-function mutations in targets of unc-43 would suppress one or more of the unc-43(n498) phenotypes. From a screen of 28,000 EMS-mutagenized haploid genomes, 19 recessive extragenic suppressors were isolated and placed into four complementation groups. The mutations in these four groups affect goa-1(4 alleles), the alpha subunit of an heterotrimeric Go protein2 , dgk-1/sag-1(4 alleles), a diacylglycerol kinase3 , eat-16/sag-2(4 alleles), an RGS protein4 , and eat-11(7 alleles)5 . As single mutants, these genes cause hyperactive locomotion and early egglaying. Mutations in each of these genes suppress the unc-43(n498) locomotion and egg-laying defects partially, consistent with unc-43 acting directly on two or more of the genes, or with unc-43 acting in parallel to these genes in the control of egglaying and locomotion. We are currently attempting to distinguish between these two models for unc-43 function.

1 Silva, A. et al 1992 Science. Mayford, M. et al 1995 Cell.

2 Lochrie, M.A. et al 1991 Cell Regulation. Mendel, J.E. et al 1995 Science. Segalat, L. et al 1995 Science.

3 Nurrish, S. et al 1997 International Worm Meeting Abstracts. Chen, W. et al 1997 International Worm Meeting Abstracts.

4 Avery, L. 1993 Genetics. Chen, W. et al 1998 West Coast Worm Meeting Abstracts and Personal Communication.

5 Avery, L. 1993 Genetics.

24 May 1999 15:50 762 762 The C. ELEGANS and Yeast Proteome Databases: An integrated knowledge system.

1999 International Worm Meeting abstract 711 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The C. ELEGANS and Yeast Proteome Databases: An integrated knowledge system. KJ Roberg-Perez, BP Davis, ME Cusick, PE Hodges, MC Costanzo, AH McKee, WE Payne, JI Garrels Proteome Inc. 100 Cummings Center, Suite 435M Beverly, Ma 01915 The C. elegans Proteome Database will be released later this year. It will comprise protein reports for each of the approximately 19,000 characterized and uncharacterized open reading frames identified by the C. elegans sequencing project (1). The C. elegans Proteome Database will have a structure and scope similar to the Yeast Proteome Database (YPD), which has proven to be an effective integrated research tool for researchers studying protein function in all organisms. Like YPD, each protein report will contain an extensive tabulation of free-text annotations, protein properties, and references in a convenient one-protein, one-Web-page format (see http://www.proteome.com/YPDhome.html). In its final form the C. elegans Proteome Database will provide protein properties and free-text annotations derived from a review of the entire C. elegans literature. The same types of protein information found in YPD will be presented, as well as new types relating to expression in a multicellular organism throughout development. The yeast and C. elegans databases will integrate seamlessly through links between related genes, common pathways, conserved protein interactions, etc. Researchers in both the yeast and worm fields should be able to use this resource to quickly understand the relevance of a new finding in one organism to the biology of the other, and often to generalize the function to higher animals. The common database format, and associated multispecies search and presentation tools, will constitute a Biological Knowledge System that can help biologists from all communities to access, compare, and utilize information from model organisms.

1. The C. elegans Sequencing Consortium, Science 282, 2012 (1998).

24 May 1999 15:50 763 763 Genetic analysis of genes involved in hypodermal function in Caenorhabditis elegans

1999 International Worm Meeting abstract 712 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic analysis of genes involved in hypodermal function in Caenorhabditis elegans AB Roberts, C Clucas, IL Johnstone Wellcome Centre for Molecular Parasitology, University of Glasgow, Glasgow G11 6NU The C. elegans cuticle is a complex multilayer stucture consisting predominately of a group of small collagen-like proteins known as cuticular collagens. The collagen genes show differential regulation during the nematode life cycle with different genes being expressed at specific stages e.g. dpy-7 is an early collagen gene and is expressed about 4 hours earlier than the col-12 collagen gene. The cuticle plays a central role in the maintenance of post-embryonic body shape and is synthesised and secreted by the hypodermal cells. Previously, a forward genetic screen using strains carrying either col-12/GFP or dpy-7/GFP fusion transgenes had generated several embryonic lethal mutants with phenotypes suggesting hypodermal abnormalities. Genetic and molecular characterisation of 2 of these mutant alleles - ij013 and ij017 is currently underway. ij013 does not express col-12/GFP. It elongates and then collapses, has functional body wall muscle and apparently normal gut development. ij017 shows an abnormal pattern of col-12/GFP expression. It elongates and then collapses back with herniations appearing on the hypodermis. Both mutants have normal dpy-7/GFP expression patterns which, with the evidence of elongation, suggests the presence of a dysfunctional cuticle. Mapping of the mutant genes has been performed using STS mapping and classical genetic crosses. ij013 has been assigned to Chromosome V between unc-51 and pha-4. Complementation tests show that ij013 is distinct from pha-4. ij017 has been placed between lin-31 and stP101 on Chromosome II. Phenotypic rescue using cosmids covering these regions is currently being performed.

24 May 1999 15:50 764 764 Searching for POP-1 target genes by single-embryo subtractive hybridization

1999 International Worm Meeting abstract 713 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Searching for POP-1 target genes by single-embryo subtractive hybridization S Robertson, M Reuben, R Lin Dept. Mol. Biol. & Oncol., UT Southwestern Med. Cntr., Dallas, TX POP-1 protein is similar to the Wnt pathway transcription factor TCF-1/LEF-1, which has been shown in other systems to function as either an activator or a repressor. We have previously shown that POP-1 protein is higher in MS than in E in 8-cell embryos and that Wnt signaling regulates a POP-1 dependent switch between MS and E cell fate. In pop-1 mutant embryos the MS blastomere adopts the fate of its sister, E, and produces endoderm. POP-1 could therefore function as either a repressor of E fate or an activator of MS fate. Complicating the design of a genetic screen for POP-1 targets is the fact that POP-1 may independently regulate multiple genes, mutations in which individually generate phenotypes different from pop-1. Therefore, we have undertaken a molecular screen to identify zygotic genes transcribed in the 8-12 cell embryo, on the assumption that POP-1 targets should be amongst these new transcripts. We reasoned that subtraction of cDNA prepared from a 12-cell wild-type embryo against cDNA prepared from a 4-cell embryo would enrich for newly synthesized zygotic transcripts over this 30-minute time period while at the same time removing the highly abundant maternal transcripts shared by both embryos. We have used an extremely sensitive RT-PCR protocol to prepare total 3’ EST cDNA from individual staged embryos. The integrity, yield and developmental stage of each single-embryo total cDNA have been confirmed using 3’-end probes from two ubiquitous maternal transcripts (tba-2 and ncc-1) and two early zygotic transcripts (end-1 and pes-10) (1,2). A subtraction has been carried out with 12-cell cDNA as tracer and 4-cell cDNA as driver, and efficiency of subtraction measured by the removal of ubiquitous sequences concomitant with the relative elevation of 12-cell specific sequences. We are using 3’ cDNAs prepared from individual staged embryos from mutants that either have an MS to E fate transition (pop-1), an E to MS transition (mom-2; lit-1), or that lack MS and E (skn-1) to assign subtraction products to particular blastomeres. In addition, we are sequencing the subtraction clones. Finally, RNAi will be utilized as a final screen to assay the function of selected clones in MS and E blastomeres.

1. Development 120: 2823. 2. G&D 11: 2883.

24 May 1999 15:50 765 765 Towards the cloning of two genes involved in sex myoblast migration

1999 International Worm Meeting abstract 714 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Towards the cloning of two genes involved in sex myoblast migration MK Robinson, MJ Stern Dept. of Genetics, Yale University, New Haven, CT 06520 In the Caenorhabditis elegans hermaphrodite a pair of M derived cells, known as the sex myoblasts (SMs), migrate anteriorly during the second larval stage to take up positions that precisely flank the center of the developing gonad. The SMs subsequently divide and differentiate to give rise to the egg-laying muscles. The migrations of these cells are guided by multiple, genetically separable mechanisms. These include a gonad-independent mechanism which is required for the coarse positioning of the SMs and a gonad-dependent mechanism necessary for the precise positioning of the SMs.1 To better understand the mechanisms underlying SM migration guidance we are working to determine the molecular identities of two genes, unc-71 and sem-3, that both affect the migrations of the SMs when mutated. Mutations in unc-71 affect both the gonad-independent mechanism of SM migration and axon outgrowth and fasciculation. unc-71 maps to the right arm of linkage group III and was rescued by germline transformation with either of two overlapping YACs, Y37D8 and Y52B8.2 This region of overlap is not spanned by cosmids. We have determined the extent of overlap between these two YACs and are using fragments derived from this region to perform transformation rescue experiments. We are also performing a non-complementation screen in an attempt to identify deletions in unc-71 that will facilitate the identification of this gene by Southern blot or PCR-based methods. sem-3(n1655) was identified as a spontaneous mutation that caused subtle defects in sex myoblast migration and vulval muscle attachment. This mutation maps to linkage group IV and is allelic to let-654. We are interested in cloning sem-3 to characterize its roles in SM migration and vulval muscle attachment as well as to understand its essential function. sem-3 maps between the endpoints of the deficiencies sDf62 and sDf8.3 We have performed transformation rescue experiments with cosmids from this region and have preliminary results suggesting that we have rescued the Egl defect of sem-3(n1655). Work to confirm these results and to determine the identity of the open reading frame corresponding to the sem-3 gene will be discussed.

1. Chen & Stern, TIGS 14:322. 2. L. DeLong, personal communication. 3. Clark & Baillie, Mol Gen Genet 232:97

24 May 1999 15:50 766 766 UNC-86: The POUer of Interaction

1999 International Worm Meeting abstract 715 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. UNC-86: The POUer of Interaction S Roehrig, I Roeckelein, R Donhauser, R Baumeister Genzentrum, Feodor-Lynen-Str. 25, 81377 Munich, Germany The POU transcription factor UNC-86 is necessary for the determination and differentiation of 57 neurons. These neurons differ in function, position, lineage and their time of expression of unc-86, suggesting that unc-86 regulates different downstream genes in these distinct neural cell types. In order to understand how unc-86 determines cell fates we pursued two strategies. In the first approach, mutations in partial loss of function (u5, u168) and null alleles (n412) of unc-86 were identified. These mutations were assayed for their effect on the DNA and protein interaction of the UNC-86 protein in vivo using a modified one-hybrid yeast system. In the second approach this system was also used to identify novel proteins that interact with UNC-86. By the first approach we found that single amino acid substitutions in the first helix (u168) and in the linker region (u5) between the third and fourth helix of the POU specific domain eliminate the interaction between UNC-86 and the LIM homeodomain protein MEC-3 in the mechanosensory neurons. This and other data show that specific residues in the POU specific and homeodomain mediate protein interactions of UNC-86. A single amino acid substitution in the recognition helix of the POU homeodomain (n412) leads to a failure in DNA-binding. However, it was previously shown that unc-86 regulates its own expression and that the UNC-86 protein levels in n412 are similar to wild-type. This suggests that the autoregulation of unc-86 is independent of the ability of UNC-86 to bind DNA. Using the modified one-hybrid system six candidate interactors were identified. One candidate gene, which encodes a cAMP dependent protein kinase, is especially promising, since we were able to show that UNC-86 is phosphorylated by PKA in vitro. Furthermore, UNC-86, which is mutated in a serine residue known to be phosphorylated in other POU proteins, shows altered DNA-binding and a dominant phenotype when expressed in wild-type animals. Other candidates include nuclear proteins, whose mammalian homologues have been shown to interact with POU proteins. In addition at least two of the candidates are expressed in some of the unc-86 expressing neurons. We propose that UNC-86 is regulated by phosphorylation and is able to determine cell fates by interacting with different proteins in different cells.

24 May 1999 15:50 767 767 New mutations involved in growth cone steering of the excretory cell

1999 International Worm Meeting abstract 716 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. New mutations involved in growth cone steering of the excretory cell IG Roelens 1 , R Feichtinger 2 , I Maillet 2 , T Bogaert 2

1 Dept. Of Genetics,University of Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium. 2 Devgen N.V., Technologiepark 9, B-9052 Zwijnaarde, Belgium.

During the development of an organism, migratory growth cones encounter guidance cues for both dorso-ventral and antero-posterior axes (e.g. the excretory canals, neuronal axons). The excretory cell is an excellent model to study both D-V and A-P migrations. This cell sends out two lateral projections, which, after growing along the D-V axis, split to form an anterior and a posterior branch (the excretory canals). The phenotype of these canals in unc-53, unc-5 and unc-71 mutants shows that they are very sensitive to changes in the signaling pathways responsible for directional migration. Based on the results of prior genetic and molecular research, we propose the following model for the migration of the excretory canals. 1. together with unc-5 and unc-71, unc-53 is important for the dorso-ventral migration of the lateral branches of the excretory canals (Buesa et al., EWM 1996) 2. for the migration from the lateral branching point to the vulva the excretory canal integrates a gonad-dependent signal as well as a signal from the unc-53 pathway (Chen et al.,1997, Dev Biol 182, 88-100) 3. unc-53 is needed for the migration of the excretory canals from the vulva into the tail To identify new components of the pathways involved in the integration of dorso-ventral signals, we designed an F2 screen in an unc-53 mutant. This provides us with a sensitized background to find animals with excretory cells that send out ectopic ventral projections. Up to date, we isolated seven mutants. Three mutants (bg2, bg4 and bg6) map on the right arm of chromosome III. We are performing complementation analysis with these mutants. We are also performing PCR-based mapping of two more mutants, one of which lies on the left arm of chromosome II(bg5).

1 as research assistant at the Fund for Scientific Research-Flanders

24 May 1999 15:50 768 768 The unc-112 and dim-1 genes encode novel proteins required for myofilament lattice assembly and stability

1999 International Worm Meeting abstract 717 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The unc-112 and dim-1 genes encode novel proteins required for myofilament lattice assembly and stability T Rogalski, D Devenport, M Gilbert, G Mullen, D Moerman Department of Zoology, University of British Columbia, Vancouver, B.C. Canada Embryos homozygous for null mutations in the unc-112 gene exhibit a Pat terminal phenotype. They arrest at the two-fold stage of embryogenesis and have severely disorganized body wall muscle. In contrast, animals carrying the r367 allele are viable, although they do have disorganized body wall muscle and are paralyzed as adults. We have isolated several extragenic suppressors of unc-112(r367) by selecting well moving animals from the progeny of mutagenized hermaphrodites. All of the suppressor mutations characterized are alleles of the dim-1 (disorganized muscle) gene positioned between dpy-7 and dpy-6 on the X chromosome. These alleles are null mutations and are able to suppress the paralyzed phenotype of unc-112(r367) when either heterozygous or homozygous. Thus, reduction or loss of dim-1 gene function results in suppression of unc-112(r367). Hermaphrodites homozygous for a dim-1 mutation alone exhibit wild-type movement, however, the body wall muscle appears disorganized when viewed under polarized light. The dim-1 gene encodes a novel 640 amino acid protein that is presumably required for myofilament lattice stability in the body wall muscle. We have identified the sequence alterations for three dim-1 alleles, and are in the process of generating a polyclonal antibody to the DIM-1 protein. The unc-112 gene corresponds to the C47E8.7 ORF, and encodes a novel, membrane associated, intracellular protein that co-localizes with perlecan and integrin at muscle cell dense bodies and M-lines. The 720 amino acid UNC-112 protein is most similar to a human protein of unknown function called Mig-2 (Wicks et al., 1994, J. Cell Sci. 107:227), but also shares a short, ~50 amino acid region of homology with Talin, Band 4.1 and Ezrin that may be important for attachment to the plasma membrane. Our analysis of embryos homozygous for unc-112 null alleles reveals that this protein is required for the proper localization of PAT-3/integrin in the muscle cell membrane. In contrast, the absence of the UNC-112 protein has no effect on the localization of UNC-52/perlecan in the ECM.

24 May 1999 15:50 769 769 Localisation and function of the FMRF-amide-like neuropeptides in C.elegans .

1999 International Worm Meeting abstract 718 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Localisation and function of the FMRF-amide-like neuropeptides in C.elegans . C Rogers 1 , CJ Franks 1 , RJ Walker 1 , JF Burke 2 , L Holden-Dye 1

1 School of Biological Sciences, University of Southampton, Southampton S016 7PX. 2 Dept. Biochemistry, University of Sussex, Brighton BN1 9QG.

C.elegans contains FMRFamide-like neuropeptides, encoded by at least 18 different genes (Nelson et al., 1998). These genes are expressed in different subsets of neurones and development stages and are likely to play a key role in neuronal function. Using functional and biochemical analysis, we aim to use C.elegans to define principles of peptidergic signalling applicable across the phyla. Analysis of peptide content: Tissue was dissected from wild-type and gene deletion mutant C.elegans for Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectroscopy (MALDI-TOF) (Kellett et al.,1996). Molecular masses were calculated for neuropeptides predicted from the completed genome sequence. Accuracy was within 1 Da (0.1%). Two of the peptides detected in pharyngeal dissections, SAEPFGTMRFamide and GAKFIRFamide, are products of the genes flp-3 and flp-5, respectively. Biological actions of the neuropeptides: Intracellular voltage recordings were made from the terminal bulb and the actions of bath-applied peptides, determined. AF1 (KNEFIRFamide) is the most potent excitatory peptide tested to date (threshold 30 nM). GAKFIRF-amide, is also excitatory, but with a higher threshold. SAEPFGTMRF-amide, is the only peptide, so far, with an inhibitory action. We are extending these protocols to test for the actions of peptides on specific subsets of neurones and on somatic muscle. In summary, two of the peptides detected by MALDI-TOF in the pharyngeal muscle also have biological activity. By further refinement of this approach we expect to delineate the neuropeptide content of identified neurones and to examine the consequences of a physiologically relevant peptide ’cocktail’ for cell signalling, in this simple and tractable model system.

Acknowledgements: Thanks to Ann Hart for sharing unpublished information and to Chris Li for provision of gene deletion animals. Kellett et al.,1996 J.Neuroscience 16, 4949-4957 Nelson et al., 1998. Mol. Brain Res.58, 103-111

24 May 1999 15:50 770 770 Feeding For Phenotypes

1999 International Worm Meeting abstract 719 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Feeding For Phenotypes J Rogers, J Hubbard Department of Biology, New York University, New York, NY We were intrigued by the recent results of Timmons and Fire (1998) suggesting that RNAi-induced phenotypes could be observed by simply feeding worms with bacteria that are producing the dsRNA of interest. Given the potency of RNAi in the germ line, we wondered if this technique could be used to produce germline-defective phenotypes of interest to our lab. In addition, we sought to apply the technique to identify genes involved in germline function that may otherwise cause a loss-of-function embryonic lethal phenotype. To test this possibility, we collected several cDNAs from genes that produce loss-of-function germline-defective phenotypes (some of which also cause embryonic or larval lethatity) and introduced them into the "double T7" vector described by Timmons and Fire. We are testing these constructs by feeding synchronous populations of L1 larvae with bacteria that express dsRNA and scoring them in the adult for germline defects. Preliminary data suggests that germline phenotypes can be obtained in this manner, but that they may be restricted to events that occur relatively late in germline development. For example, gld-1 dsRNA fed to L1 larvae produced an abnormal oocyte phenotype characteristic of partial loss-of-function alleles of gld-1 (class E and F; Francis et al., 1995) at 60% penetrance. Progeny of unaffected adults were then grown on RNAi-producing bacteria. In these animals we observed the more severe tumorous phenotype in which progression through meiotic prophase is altered and oogenesis does not occur (Francis et al., 1995).

24 May 1999 15:50 771 771 The Caenorhabditis elegans Vps34 Class of PI 3-kinase

1999 International Worm Meeting abstract 720 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Caenorhabditis elegans Vps34 Class of PI 3-kinase L Roggo 1 , M Zetka 1 , V Bernard 1 , A Rose 2 , F Müller 1 , MP Wymann 3

1 Institute of Zoology, University of Fribourg, CH-1700 Fribourg, Switzerland. 2 Department of Medical Genetics, University of British Columbia, Vancouver, Canada V6T 1Z3. 3 Institute of Biochemistry, University of Fribourg, CH-1700 Fribourg, Switzerland.

Phosphoinositide (PI) 3-kinases are a family of enzymes catalyzing the phosphorylation of PIs at the D-3 position and have been shown to play an important role in surface receptor signalling. In yeast the activity of the Vps34p subtype is indispensable for correct vesicular protein sorting, however, the role of Vps34p in multicellular organisms has not yet been elucidated. We have isolated a 2.9 kb cDNA in Caenorhabditis elegans containing a putative ORF of 901 amino acids with strong homology to the Vps34 family. The VPS34 transcript is SL2 trans-spliced and has been shown to be part of a polycistronic transcription unit located on the left half of chromosome I. By using polyclonal antisera raised against the Vps34p homolog, a wortmannin-sensitive PI 3-kinase activity could be immunoprecipitated. By microinjection transformation rescue we demonstrated that the C. elegans homolog of Vps34p is encoded by the gene let-512, of which all five alleles arrest at mid-stage larval. Sequencing of the five alleles found two missense mutations in the catalytic domain and three nonsense mutations that result in a premature stop codon. The three nonsense alleles lack the putative core catalytic domain. Preliminary expression studies indicate that there is strong expression of let-512(VPS34) in early developmental stages from egg to L2 and that this expression decreases in later stages. Analysis of the mutants is consistent with antisense injection results; loss-of-function mutations in let-512(VPS34) result in the formation of vacuoles within different cell types and tissues, consistent with the null phenotypes observed in other organisms, as well as slow growth.

24 May 1999 15:50 772 772 Functional Organization of the Nuclear Envelope in C. elegans

1999 International Worm Meeting abstract 721 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Functional Organization of the Nuclear Envelope in C. elegans T Rolef Ben-Shahar 1 , J Liu 2 , D Reimer 3 , M Treinin 4 , P Spann 1 , M Cohen 1 , A Fire 2 , K Weber 3 , Y Gruenbaum 1

1 Dept. of Genetics, The Hebrew University, Jerusalem. 2 Carnegie Institution of Washington, Dept. of Embryology, Baltimore. 3 Department of Biochemistry, MPI for Biophysical Chemistry, Göttingen. 4 Dept. of Physiology, The Hebrew University, Jerusalem.

In eukaryotic cells, DNA replication, RNA processing and ribosome assembly all occur in the nucleus, while protein synthesis occurs in the cytoplasm. These activities are physically separated by the nuclear envelope. The nuclear envelope is a complex structure composed of outer and inner lipid bilayer membranes, nuclear pores and nuclear lamina. The nuclear lamina is involved in nuclear organization, nuclear assembly, DNA replication, differentiation and apoptosis. Lamins are the major and best-studied proteins of the nuclear lamina. Caenorhabditis elegans has a single 64 kDa B-lamin homologue, termed CeLam-1. Anti-CeLam-1 antibodies were used to show that CeLam-1 is present in the nuclear envelope of essentially all cells during development, including all germ cells except mature sperm. Antibody staining also revealed a patchy pattern of lamin in the nuclear envelope and, in many cases, staining in the nuclear interior. Transgenic lines expressing GFP fused to the entire coding region of CeLam-1 were prepared and used to show a similar GFP localization pattern to that observed using antibody staining. RNA-mediated genetic interference of CeLam-1 activity resulted in abnormal nuclei and early embryonic lethality, demonstrating that CeLam-1 is an essential gene and is required for nuclear organization. Another gene that we study is the glycoprotein gp210, known in other systems to be involved in organization and function of the nuclear pore. Caenorhabditiis elegans has a single gp210 homologue, Ce-gp210. Genetic experiments are currently being performed in order to study the role of the gp210 gene in vivo.

24 May 1999 15:50 773 773 UNC-4 and UNC-37 repress VB-specifying genes to define VA traits

1999 International Worm Meeting abstract 722 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. UNC-4 and UNC-37 repress VB-specifying genes to define VA traits JM Ross 1 , E German 2 , DH Hall 2 , DM Miller 1

1 Dept. of Cell Biology, Vanderbilt University Med. Ctr., Nashville, TN 37232. 2 Dept. of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461.

VA and VB motor neurons arise as sister cells, but adopt different morphologies and patterns of synaptic input. VA-type synaptic inputs define a circuit for backward movement, while VB-type synaptic inputs define a circuit for forward movement. The UNC-4 homeoprotein is required to specify the VA pattern of synaptic input; in unc-4 mutants VAs receive the VB pattern of synaptic input. We have proposed a model in which UNC-4 acts in the VAs to repress genes that normally specify VB-type inputs (Winnier et al., this meeting). Two observations support the hypothesis that UNC-4 acts as a repressor: 1) UNC-4 requires the activity of UNC-37, a homolog of the Drosophila co-repressor Groucho. 2) The UNC-4 C-terminus contains an Engrailed repressor domain (eh1). We have identified two targets of UNC-4/UNC-37 repression. del-1::gfp is normally expressed in VB motor neurons, but is ectopically expressed in VA motor neurons in unc-4 and unc-37 mutants. acr-5::gfp is normally expressed in VB and DB motor neurons, but is ectopically expressed in VA and DA motor neurons in unc-4 and unc-37 mutants. As cell surface proteins, either DEL-1 (degenerin-like Na channel subunit) or ACR-5 (nicotinic acetylcholine receptor alpha subunit), could specify VB synaptic inputs or define other VB traits. If our model of negative regulation by UNC-4 and UNC-37 is correct, then ectopic expression of UNC-4 in the VBs should repress VB-specific gene expression, and may be sufficient to respecify the VBs with VA-type inputs. To test this hypothesis, we expressed UNC-4 in the VBs using the del-1 promoter. Ectopic expression of UNC-4 in the VBs represses acr-5::gfp, while del-1::gfp expression remains unchanged. Worms harboring an integrated del-1::unc-4 array (wdIs2) display a phenotype which is consistent with the VBs receiving VA-type synaptic inputs and functioning in backward movement: wdIs2 worms are impaired in forward movement and coil ventrally when backing. EM reconstruction of the wdIs2 ventral nerve cord will determine whether this wdIs2 Unc phenotype is a result of miswiring of the VBs with VA-type inputs.

24 May 1999 15:50 774 774 Regulation of dauer larva development in Caenorhabditis elegans by daf-18, a homologue of the tumour suppressor PTEN

1999 International Worm Meeting abstract 723 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Regulation of dauer larva development in Caenorhabditis elegans by daf-18, a homologue of the tumour suppressor PTEN Jean Pierre Rouault 1 , Patricia E. Kuwabara 2 , Olga M. Sinilnikova 3 , Laurent Duret 4 , Danielle Thierry-Mieg 5 , Marc Billaud 3

1 INSERM U453, Centre Leon Berard, Lyon, France. 2 LMB, Cambridge, UK. 3 CNRS UMR 5641, UCBL1, Lyon, France. 4 CNRS UMR 5558, UCBL1, Villeurbanne, France. 5 CRBM CNRS, Montpellier, France.

The tumour suppressor gene PTEN (also called MMAC1 and TEP1) is somatically mutated in a variety of cancer types. In addition, germline mutation of PTEN is responsible for two dominantly-inherited related cancer syndromes called Cowden disease and Bannayan-Ruvalcaba-Riley syndrome. The PTEN gene encodes a dual-specificity phosphatase which inhibits cell spreading and migration partly through the inhibition of the integrin-mediated signalling. Furthermore, PTEN regulates the levels of phosphatidylinositol 3, 4, 5-trisphosphate (PIP3) by specifically dephosphorylating position 3 on the inositol ring. We report here that the dauer formation gene daf-18 is the Caenorhabditis elegans homologue of PTEN. DAF-18 is a component of the insulin-like signalling pathway controlling entry into diapause and adult longevity that is regulated by the DAF-2 receptor tyrosine kinase and the AGE-1 PI3 kinase. We found that the daf-18 (e1375) mutation within the PTEN homologue results in the generation of a premature stop codon. Others have shown that the daf-18 mutation suppresses the life-extension and constitutive dauer formation associated with daf-2 or age-1 mutants. Similarly, we show that inactivation of daf-18 by RNA-mediated interference mimics this suppression, and that a wild type daf-18 transgene rescues the daf-18 dauer defect. These results indicate that the PTEN/DAF-18 antagonizes the DAF-2/AGE-1 pathway, perhaps by catalyzing the dephosphorylation of PIP3 generated by AGE-1. These data further support the notion that mutations of PTEN contribute to the development of human neoplasia through an aberrant activation of the PI3K-Akt/PKB signalling cascade.

24 May 1999 15:50 775 775 Cloning and characterization of aex-2 and aex-4, two genes required for defecation

1999 International Worm Meeting abstract 724 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cloning and characterization of aex-2 and aex-4, two genes required for defecation EK Round, JH Thomas Department of Genetics, University of Wahington, Seattle, WA 98195 C. elegans follows a stereotyped series of muscle contractions during defecation: first a posterior body-muscle contraction (pBoc), followed by an anterior body-muscle contraction (aBoc) and finally contraction of the enteric muscles which results in expulsion of the gut contents (Exp). In the presence of food, this defecation motor program occurs every 45 seconds. The neurotransmitter GABA specifically activates the expulsion since mutants lacking GABA have enteric muscle contractions in only 15% of defecation cycles and have no defect in aBoc or pBoc. In contrast, mutations in seven defined genes cause defects in both the aBoc and Exp steps of the defecation motor program (Aex phenotype)(1). Colin Thacker and Ann Rose have shown that aex-5 is a proprotein convertase in the kexin/subtilisin family(2), suggesting a role for peptide signaling in the process of defecation. With the hope of identifying molecules that act in the same pathway as aex-5, we are mapping and cloning two other Aex genes. From a large number of recombinants, we obtained a precise map position for aex-2 between the anchor genes unc-110 and unc-115 on the X chromosome. These data were used to choose cosmids for transgenic rescue experiments. We have rescued the aex-2 phenotype with a pool of two cosmids and are currently working to identify the exact gene responsible for this rescue. We have mapped aex-4 between unc-78 and lin-18 on the X chromosome and are currently mapping aex-4 relative to polymorphisms in this region.

1. J.H. Thomas, 1990. Genetics 124: 855-872. 2. C. Thacker and A.M. Rose, West Coast Worm Meeting Abstract 1998, #29.

24 May 1999 15:50 776 776 Extra distal tip cells in cki-1(RNAi) animals do not result from duplication of existing distal tip cells.

1999 International Worm Meeting abstract 725 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Extra distal tip cells in cki-1(RNAi) animals do not result from duplication of existing distal tip cells. RD Roy 1 , E Lambie 2 , V Ambros 2

1 Department of Biology, McGill University. 2 Department of Biological Sciences, Dartmouth College, Hanover NH, 037552.

Cyclin-dependent kinase inhibitors (CKIs) play a key role in arresting cell cycle in response to various physiological, environmental, and developmental cues. RNA-mediated interference of cki-1 activity (cki-1(RNAi) results in pleiotropic phenotypes. Misregulation of cell division control following cki-1(RNAi) permits progenitor cells to divide before being appropriately determined. This is likely the basis of some of the observed vulval patterning defects (extra VPCs and anchor cells), the germline defect (extra distal tip cells (DTCs)) and the inability to form differentiated dauer alae (hypodermal cell divisions). To address whether extra DTCs arise from duplications of existing cells or whether they are produced from an ectopic source in cki-1(RNAi), the production of extra DTCs was examined in lag-2::GFP; cki-1(RNAi) animals. The cki-1(RNAi)-induced ectopic DTCs do not appear before the late L3 stage as per lag-2::GFP expression. Therefore laser ablation of distal tip cells in the early L3 (from picking L2 lethargus animals) should eliminate the production of DTCs in the adult if the extra DTCs result from duplication of endogenous DTCs. DTCs are observed in nearly all ablated animals indicating that DTCs arise earlier and simply are not determined until later, or they stem off from another lineage later in development. To test if these cells are born earlier, similar ablations were performed in the somatic gonad progenitors in L1 animals. Removal of Z1.a and Z4.p resulted in the production of DTCs in the adult despite removal of these progenitor cells. Removal of Z1.p and Z4.a lead to only 2 DTCs per animal suggesting that the extra DTCs arise from these cells and not from duplications of the DTCs later. Despite the appearance of a cki-1(RNAi)-induced cell fate transformation, a more consistent interpretation would lie in the ability of cki-1 to block cell divisions until cells are appropriately determined. Premature divisions that occur in cki-1(RNAi) may permit daughter cells to retain mother cell fates and hence give rise to lineage modifications. This possibility will be confirmed in chromosomal mutants of cki-1.

24 May 1999 15:50 777 777 New Alleles of mec-4

1999 International Worm Meeting abstract 726 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. New Alleles of mec-4 D Royal, MA Royal, S Narayan, M Driscoll Rutgers University, Nelson Labs A223, 604 Allison Rd., Piscataway NJ 08854 USA mec-4 encodes a member of the DEG/ENaC ion channel superfamily that is hypothesized to be a subunit of a mechanosensitive ion channel in the touch receptor neurons. The MEC-4 protein has two membrane spanning domains (MSDI and MSDII) that flank a large extracellular domain. The N and C-termini project into the cytoplasm. Rare dominant mec-4 alleles specify hyperactive channels that cause touch cell swelling and degeneration. Structure/function studies suggest that steric affects in mutant proteins prevent the channel from closing properly. To learn more about both normal channel function and degenerative cell death mechanisms we looked for suppressors of mec-4(d)-induced cell death. We expressed GFP from the mec-4 promoter, labeling the 6 touch cells, mec-4(u231) was crossed into the mec-4p/GFP line, causing the six touch cells to die. We then mutagenized this strain with EMS and screened for animals with restored fluorescence. We isolated 11 alleles that mapped to the right arm of the X chromosome. 10 of these mapped to the right of unc-7, in the vicinity of mec-4. Here we describe the result of sequencing mec-4 in candidate intragenic alleles. One of the alleles, bz46, is a deletion that results in the loss of the last 98aa. Interestingly, bz46 exhibits allele-specific interactions with the other isolated suppressor alleles. Our results should provide insight into the structure/function of the mechanosensory ion channel apparatus. mec-4

24 May 1999 15:50 778 778 New Investigations of the Phosphatidylinositol Signaling Pathway

1999 International Worm Meeting abstract 727 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. New Investigations of the Phosphatidylinositol Signaling Pathway D Royal, MA Royal, N Sirotin, M Driscoll Rutgers University, Nelson Labs A223, 604 Allison Rd., Piscataway NJ 08854 USA The rdgB homologues have been implicated in neurodegeneration in flies and mice. In Drosophila, rdgB disruption causes olfactory deficits and light-exacerbated retinal degeneration. In mice mutations effecting the related PITP (phosphatidylinositol transfer protein) causes the vibrator phenotype and neuronal cell death. The C. elegans genome encodes one member of the PITP superfamily and several homologues of yeast SEC14. SEC14 like PITP transfers phosphatidyl choline for phosphatidylinositol between donor and acceptor lipid membranes. To learn more about the identity of these genes in C. elegans we are determining their expression patterns and mutant phenotypes. A fusion of the C. elegans rdgB homologue promoter to GFP is expressed in the germline, the spermatheca and 3 pairs of head neurons. The membrane surrounding the egg also fluoresces. We plan to characterize the mutant phenotypes using an RNAi approach. As a second inroad toward investigating mechanisms of PI signaling we are probing lithium sensitivity. Lithium is thought to inhibit two enzymes involved in the PI pathway, inositol polyphosphate 1-phosphatase and inositol monophosphate phosphatase. At high concentrations lithium causes embryonic arrest (Tabuse WBG 14(4):26). However, at lower concentrations animals become uncoordinated. The UNC phenotype is dosage dependant and reversible. In this report we will discuss the effects of lithium on known mutants affecting the PI pathway and we will give preliminary results of a screen for lithium-resistant mutants.

24 May 1999 15:50 779 779 Probing the functions of c. elegans dyn-1 binding proteins

1999 International Worm Meeting abstract 728 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Probing the functions of c. elegans dyn-1 binding proteins DA Rube, AM Labrousse, K Kong, AM van der Bliek Department of Biological Chemistry, UCLA School of Medicine, Box 951737, Los Angeles, CA 90095 Dynamin is a 100 kDa GTPase that forms a collar around the necks of budding clathrin-coated vesicles. Constriction of the dynamin collars might sever the vesicles from the plasma membrane. C. elegans dynamin, encoded by the dyn-1 gene, is expressed at high levels in neurons and at lower levels in other cell types. Mutations in the dyn-1 gene cause uncoordinated movement by blocking the recycling of synaptic vesicles by endocytosis. Our laboratory studies the mechanism of dynamin assembly and constriction. It is likely that dynamin-binding proteins assist the assembly of dynamin collars and perhaps regulate the constriction process. In mammals, the principal binding partners of dynamin are members of the amphiphysin family. C. elegans has two amphiphysin-like proteins, which might be homologous to the mammalian amphiphysins. We gave the C. elegans homologues the provisional names amp-1 and shp-1, because phylogenetic analysis suggests that the two C. elegans proteins represent each of the two mammalian subclasses (amphiphysins and SH3P4/8/13). Amphiphysin might recruit dynamin to the coated pit where it can then assemble into a collar. To investigate the potential interactions between SH3P4/8/13 proteins and dynamin, we have begun an analysis of C. elegans shp-1. Using promoter GFP fusions, we found that C. elegans amp-1 is expressed at high levels in muscles and neurons, while shp-1 expression was limited to the nervous system. We are currently conducting a deficiency screen for shp-1 mutants. We are attempting to co-immunoprecipitate shp-1 and dyn-1 proteins from lysed C. elegans and we are testing shp-1 RNAi. Based on the expression pattern and the homology with amphiphysin, we expect that loss of shp-1 function might result in uncoordinated movement, similar to the defects caused by mutations in C. elegans dyn-1.

24 May 1999 15:50 780 780 Evolution of glp-1 and lin-12 genes in Caenorhabditis species

1999 International Worm Meeting abstract 729 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Evolution of glp-1 and lin-12 genes in Caenorhabditis species David Rudel 1 , Lisa Friedman 1 , Judith Kimble 1,2

1 Department of Biochemistry, University of Wisconsin-Madison. 2 Howard Hughes Medical Institute, Madison, WI 53706.

The C. elegans glp-1 and lin-12 genes encode Notch-like transmembrane receptors that regulate cell fates and patterning during development. glp-1 is important for germline and early embryonic fates, while lin-12 is more critical for post-embryonic somatic cell fate decisions. The GLP-1 and LIN-12 proteins appear interchangeable, suggesting that the regulation of these two genes governs their distinct biological roles. We have begun to analyze the evolution of the glp-1 and lin-12 genes. To this end, we have cloned glp-1 and lin-12 from both C. briggsae and C. remanei. To test the biological roles of the glp-1 orthologs, we used RNAi. We found that Cb-glp-1(RNAi) and Cr-glp-1(RNAi) into C. briggsae and C. remanei respectively leads to dead embryos that lack an anterior pharynx, similar to the C. elegans Glp-1 embryonic phenotype. Furthermore, escapers that survive embryogenesis can exhibit a typical C. elegans Glp-1 germline phenotype. Therefore, the biological roles of glp-1 appear to be the same in C. briggsae and C. remanei. To ask whether the regulation of the glp-1 orthologs was conserved, we raised antibodies to Cb-GLP-1, and have compared 3’UTR sequences. We find that Cb-GLP-1 is restricted to the AB descendants of C. briggsae early embryos, and that the 3’UTRs of Cb-glp-1 and Cr-glp-1 harbor a highly conserved (80% identity) region corresponding to the part of the 3’UTR that is critical for translational regulation in Ce-glp-1. The coding region, by contrast, is only 55%-65% identical. Therefore the regulation of maternal glp-1 mRNA appears to be conserved in C. briggsae and C. remanei. We conclude that glp-1 and lin-12 adopted their distinct roles prior to the radiation within Caenorhabditis. To shed light on the ancestral gene, our next step is to identify a homolog from a more distant species (e.g. Dolichorhabditis, Pristionchus) that may have diverged before the duplication.

24 May 1999 15:50 781 781 Identification of new loci required for sensitivity to specific odorants

1999 International Worm Meeting abstract 730 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification of new loci required for sensitivity to specific odorants R Rutherford, K Roayaie, CI Bargmann Department of Anatomy, UCSF and HHMI, 94143-0452 Seven-transmembrane (7TM) chemoreceptors compose the single largest gene family in the genome of Caenorhabditis elegans(1). However, with the exception of the diacetyl receptor ODR-10, the functional ligands and greater biological importance of these 7TM chemoreceptors remain unknown. Analysis of these molecules can inform our understanding not only of C. elegans behavior and development, but also of the rapid evolution of eukaryotic gene families. We are employing a forward genetic approach to expand our understanding of the biological functions of the 7TMs and other genes involved in olfactory signaling. We have conducted screens to identify mutants with selective loss of sensitivity to two specific odorants: 2,3-pentanedione and pyrazine. From the latter screen we identified three mutants with reduced sensitivity to pyrazine that respond normally to diacetyl. Because pyrazine and diacetyl are sensed by the same olfactory neuron pair (AWA), these mutants may affect specific olfactory recognition or signaling. Further genetic and behavioral characterization of these mutant lines is underway and will be reported.

(1) C. elegans seq. consortium. (1998) Science 292: 2015.

24 May 1999 15:50 782 782 The role of the STAR family in tra-2 3’UTR translational regulation

1999 International Worm Meeting abstract 731 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The role of the STAR family in tra-2 3’UTR translational regulation L Saccomanno, EB Goodwin Department of Cell and Molecular Biology and the Lurie Cancer Center, Northwesern University Medical School The C. elegans sex determination gene, tra-2, encodes a large transmembrane protein that is required for female sexual development. Repression of tra-2 translation is necessary for male cell fate development in both somatic and germ line tissues. Two elements located in the 3’UTR of the tra-2 mRNA, called TGEs, are required for tra-2 translational regulation. GLD-1 is a member of the STAR family of RNA binding proteins and regulates tra-2 translation in the hermaphrodite germline to permit spermatogenesis. However, translational regulation of tra-2 also occurs in the soma. The STAR family contains an approximately 200 amino acid domain (STAR domain) that is highly conserved, raising the possibility that another STAR protein represses tra-2 in the soma. The C. elegans sequencing project has identified at least five predicted open reading frames that have strong similarity to GLD-1. We have preliminary evidence that suggests at least one of these five STAR proteins, T21G5.5, can regulate translation through the tra-2 3’UTR. T21G5.5 is 67% identical to GLD-1 at the amino acid level within the STAR domain. I have performed RNA-mediated interference (RNAi) of T21G5.5 in transgenic C. elegans animals that carry a reporter transgene encoding the lacZ gene with the wild type tra-2 3’UTR. The F1 progeny following RNAi of T21G5.5 had an increased level of b-gal staining in the soma compared to wild type transgenic animals. 22% of the RNAi animals had b-gal activity in intestinal cells compared to 12.5% of non-injected. In addition, animals that stained for b-gal after injection with T21G5.5 also showed an increase in the number of b-gal stained intestinal cells (2-8 cells) compared to wild type (1-2 cells). Disruption of T21G5.5 activity also resulted in animals with interesting morphological phenotypes. F1 animals had a dumpy phenotype and an altered tail morphology. In addition, the gonads of these animals were sexually transformed. These preliminary data suggest that T21G5.5 is a likely candidate for a TGE-dependent translational repressor. However, these results do not eliminate the possibility that other STAR proteins are also 3’UTR-dependent translational regulators. We are currently testing this possibility.

24 May 1999 15:50 783 783 Anucleate C. elegans sperm can crawl, fertilize oocytes, and direct anterior-posterior polarization of the 1-cell embryo.

1999 International Worm Meeting abstract 732 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Anucleate C. elegans sperm can crawl, fertilize oocytes, and direct anterior-posterior polarization of the 1-cell embryo. PL Sadler 1,2 , DC Shakes 2

1 Dept. of Biology, University of Houston, Houston, TX. 2 Dept. of Biology, College of William and Mary, Williamsburg, VA.

Although it has long been appreciated that spermiogenesis, the cellular transformation of spherical haploid spermatids into bipolar, motile spermatozoa, occurs in the absence of new DNA transcription, few studies have addressed whether the physical presence of a sperm nucleus is required either during spermiogenesis or for subsequent sperm functions during egg activation and early zygotic development. To determine the role of the sperm nucleus in these processes, we analyzed two C. elegans mutants whose spermatids lack both nuclei and DNA. Here we show that these anucleate sperm not only differentiate into mature functional spermatozoa, but they can also crawl towards and fertilize oocytes. Furthermore, we show that these anucleate sperm induce both normal egg activation and A-P polarity in the one cell C. elegans embryo. The latter finding demonstrates for the first time that although the anterior-posterior embryonic axis in C. elegans is specified by sperm (Goldstein and Hird, 1996), the sperm nucleus itself is not required. Also unaffected is the completion of oocyte meiosis, the formation of an impermeable eggshell, the process of cytoplasmic rearrangement, and the migration/rotation/centration of the oocyte pronucleus. Our investigation of these mutants confirms that in C. elegans, neither DNA nor a sperm nucleus is required for spermiogenesis, proper egg activation, or the induction of A-P polarity.

24 May 1999 15:50 784 784 Getting to anaphase: emb-27 is required for chromosome segregation in the germline of C. elegans

1999 International Worm Meeting abstract 733 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Getting to anaphase: emb-27 is required for chromosome segregation in the germline of C. elegans PL Sadler 1,2 , DC Shakes 2

1 Dept. of Biology, University of Houston, Houston TX. 2 Dept. of Biology, College of William and Mary, Williamsburg, VA.

The mitotic metaphase to anaphase transition in both yeast and vertebrates is driven by a multimeric ubiquitin ligase known as the anaphase promoting complex, APC. While the mitotic functions of APC are being actively investigated, almost nothing is known about APC’s meiotic functions, a question which is particularly amenable to analysis in C. elegans. To address this question, we are studying both metaphase to anaphase defective (MAT) mutants and RNAi embryos depleted in maternal APC components (see Abstracts by Golden et al & Wille et al.). Of the five known MAT mutants, emb-27 is our most fully characterized to date. Our analysis indicates that EMB-27 is required for germline chromosome segregation during both meiosis and mitosis. Both emb-27 primary oocytes and spermatocytes mature normally but arrest development in meiotic metaphase I; thus they fail to either segregate homologs or progress to anaphase. Consequences of this metaphase arrest prove different for each type of gamete. emb-27 oocytes arrest development at the meiotic one cell stage. The mutant primary spermatocytes are also blocked in metaphase I; as a result, secondary spermatocytes are never formed. However because this cell cycle block is unlinked to gamete differentiation, anucleate spermatids still bud from the arrested primaries. In addition to their aforementioned meiotic defects, upshifted emb-27 adults also accumulate abnormally high numbers of metaphase figures within their mitotic zones. We believe that these excess metaphase figures arise from mitotic metaphase blockage rather than tumorous overproliferation because the mutants also exhibit abnormally small gonads and variably sized pachytene nuclei. However to further test the requirement for EMB-27 during germline mitosis, we examined the germline proliferation defects in mutants shifted to restrictive temperatures as L1 larvae. Our most severe alleles proved to be GLP. These combined results indicate that EMB-27 is essential for both mitotic germline proliferation and maintenance. Taken together, our results indicate that EMB-27 is specifically required to promote anaphase within the germline. To determine the molecular nature of this critical gene, emb-27 has been physically mapped to a two cosmid region on LGII. Of the potential gene candidates in this region, only APC6(cdc16) yields an RNAi phenotype which mimics the one-cell arrest seen in emb-27 mutants. We are currently sequencing this gene in the mutant alleles.

Research funded, in part, by the Jeffress Foundation and NSF.

24 May 1999 15:50 785 785 Another form of associative learning in chemotactic behavior of C. elegans

1999 International Worm Meeting abstract 734 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Another form of associative learning in chemotactic behavior of C. elegans S Saeki 1,2 , M Yamamoto 1 , Y Iino 3

1 Department of Biophysics and Biochemistry, The University of Tokyo, Tokyo 113-0033, Japan. 2 Present address: Tokyo Research Laboratories, Kyowahakkokogyo Co. Ltd., Machida, Japan. 3 Molecular Genetics Research Laboratory, The University of Tokyo, Tokyo 113-0033, Japan.

An associative learning paradigm based on chemotactic behavior has previously been reported by van der Kooy’s group (Wen et al., 1997, Behav. Neurosci. 111:354-368). In this paradigm, chemotaxis to sodium ion and chloride ion were differentially conditioned by paired presentation of one of the ions with food, and the other without food. We have found another form of associative learning in chemotaxis. When worms were starved on plates including NaCl, their chemotaxis to NaCl was dramatically reduced. This conditioning required both presence of NaCl and absence of bacteria, indicating that it is not a mere adaptation or habituation. In contrast, decrease in chemotaxis to the volatile chemoattractant isoamylalcohol after continuous exposure to the same odorant (Colbert and Bargmann, 1995, Neuron. 14:803-812) occurred irrespective of the presence or absence of food. While chemotaxis to isoamylalcohol did not significantly change after conditioning with NaCl, chemotaxis to other water-soluble attractants also decreased. This suggests that altered response of a cell(s) that is specifically involved in chemotaxis to water-soluble chemoattractants is responsible for the behavioral change. Decrease in chemotaxis occurred slowly over 3-4 hours of conditioning and returned quickly to the original level when either of the conditioning stimulus, NaCl or starvation, was removed. Application of serotonin partially blocked this change in chemotaxis, consistent with the proposed function of this neurotransmitter in food signaling. We have isolated several mutants with reduced plasticity as assayed in this paradigm. This form of behavioral plasticity expands the opportunities in which one can study molecular and cellular mechanisms of learning using C. elegans.

24 May 1999 15:50 786 786 Identifying synaptic regulators by screening for suppressors of a syntaxin hypomorph

1999 International Worm Meeting abstract 735 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identifying synaptic regulators by screening for suppressors of a syntaxin hypomorph O Saifee, ML Nonet Department of Anatomy & Neurobiology, Washington University School of Medicine, St. Louis, MO 63110, USA Fusion of neurotransmitter-laden vesicles at active zones is the central event underlying synaptic transmission at chemical synapses. Although recent studies have suggested that the minimal molecular machinery for release may consist of syntaxin, synaptobrevin, and SNAP-25, additional components are likely required to achieve the tightly regulated, calcium-dependent release characteristic of neuronal exocytosis. In an attempt to identify regulators capable of modulating release we are using the reduced synaptic transmission of the hypomorphic syntaxin allele, unc-64(e246), as a sensitized background to screen for suppressors that enhance transmission. The suppressors increase movement of this nearly paralyzed syntaxin allele and when outcrossed from e246 they generally appear hyperactive with increased sensitivity to the acetylcholinesterase inhibitor aldicarb. Of 14 candidate suppressors, the largest group maps to slo-1, a large conductance voltage- and calcium-sensitive potassium channel (see abstract by ZW Wang, et. al.). Another suppressor appears to disrupt the calcium/calmodulin-dependent protein kinase II encoded by unc-43, while preliminary evidence suggests that a third class may be gain-of-function alleles of egl-30, which encodes a Gq alpha subunit. Additionally, a dominant suppressor that restores the greatest level of activity to e246 remains under investigation. The challenge ahead is to uncover the mechanism by which these proteins are acting to modulate synaptic transmission. Our initial attempts to determine if the regulation of syntaxin is direct or indirect have focused on identifying the site of action of the suppressors. Are they acting at the presynaptic nerve terminal, where syntaxin functions, or at the postsynaptic target, in muscle at the neuromuscular junction? To address this question, we tested the ability of these suppressors to also suppress the dauer constitutive phenotype of e246 at 25°C. Suppression of this phenotype suggests that some suppressors may have action outside of muscle. Further analysis aimed at discerning how these modulators are acting should further our understanding of how synaptic transmission is regulated.

24 May 1999 15:50 787 787 Novel Screens for Regulators of G1 Phase of the Cell-cycle in C. elegans

1999 International Worm Meeting abstract 736 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Novel Screens for Regulators of G1 Phase of the Cell-cycle in C. elegans RM Saito 1 , D Srinivasan 2 , S van den Heuvel 1

1 MGH Cancer Center, Charlestown, MA. 02129. 2 Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA. 02115.

We are studying the regulation of entry into and progression through the G1 phase of the cell-cycle in C. elegans. Progression through the eukaryotic cell-cycle is regulated by the activities of cyclin-dependent kinases and their cyclin regulatory subunits. In higher eukaryotes, entry into G1 has been suggested to depend on induction of cyclin D expression by mitogenic signals and the subsequent assembly with CDK4 or CDK6. Progression through G1/S also requires the activity of CDK2/cyclin E. Here, we describe a novel screen designed to identify genes that regulate cell-cycle entry and progression through G1 during C. elegans development. To identify genes that regulate G1 events, we are screening for mutants that display inappropriate temporal expression of the G1-specific marker, cyd-1::GFP (a gift from M. Park and M. Krause), or the S phase marker, rnr::GFP (a gift from R. Roy and V. Ambros). cyd-1 encodes the C. elegans cyclin D homolog. The cyd-1::GFP is an ideal marker for G1 entry since its induction corresponds to the predicted time of G1 entry in the postembryonic blast cells. Similarly, the temporal expression of rnr, a C. elegans ribonucleotide reductase homolog, coincides with S phase. Thus, the time of entry into and progression through G1 can be determined by the observed expression from the cyd-1::GFP and rnr::GFP transgenes, respectively. We are currently performing test screens for precocious expression of the GFP markers in semi-clonal, synchronized L1 populations. Briefly, F1 progeny of the mutagenized transgenic strains are allowed to lay eggs on a plate before they are re-isolated and harvested for their unlaid eggs. These F2 progeny are hatched in the absence of food, fed to synchronize development and then screened for precocious GFP expression. The details and our progress will be presented at the meeting.

24 May 1999 15:50 788 788 Progress in cloning mua-6: gene required for post embryonic muscle attachment

1999 International Worm Meeting abstract 737 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Progress in cloning mua-6: gene required for post embryonic muscle attachment K Saiyasisongkhram, W Omotosho, S Patel, N Penning, J Plenefisch Department of Biology, University of Toledo, Toledo, OH 43606 Normal locomotion of C. elegans results from the transmission of motor force produced by the myofilament lattice of the bodywall muscles to the cuticle. A chain of specialized cytoskeletal- plasma membrane - matrix linkages in both the muscle and the basaly apposed hypodermal cells have been implicated in this role. Many of the genes that encode for known matrix ligands and receptors (e.g. integrins, collagen type IV, perlecan) have been shown to be absolutely required for embryonic assembly of muscle adhesions, and when mutated result in embryonic paralysis. Mutations in genes that might regulate the assembly of these linkages in response to post-embryonic growth and physiological use may show a more subtle phenotype, for example, a dystrophic paralysis. Indeed, a novel class of genes, in which mutations results in late onset of paralytic phenotype and are clearly required for the integrity of muscle-cuticle attachments during post-embryonic growth, have been recovered and designated mua, for muscle attachment defective. mua-6 has been mapped to mec-4 sup-10 interval on the X-chromosome. Homozygous mua-6(rh85) animals form normal embryonic muscle attachments but show a progressive failure of the adhesive sites resulting in muscle cells detaching from each other and from the hypodermis as the worm grows post-embryonically. The gross symptoms range from mild paralysis to severe paralysis and death. We are interested in the molecular identity of mua-6. The genetic interval containing mua-6 is spanned by 14 cosmids. We are microinjecting DNA prepared from these cosmids into let-2 +/+mua-6 animals to test for rescuing activity. To date we have eliminated the genomic region closest to sup-10. We are now concentrating our efforts on the remaining two-thirds of this interval.

24 May 1999 15:50 789 789 Identification and Characterization of Proteins Associated with The Ryanodine Receptor in Caenorhabditis elegans

1999 International Worm Meeting abstract 738 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification and Characterization of Proteins Associated with The Ryanodine Receptor in Caenorhabditis elegans Y Sakube, H Kagawa Okayama University, Department of Biology, Faculty of Science, Okayama 700-8530 JAPAN The C. elegans ryanodine receptor (CeRYR) is encoded by the single gene, unc-68 on chromosome V and consists of 5,071 amino acid residues having 40% identity with those of other animals. Non-sense mutant animals of unc-68 alleles (e540 and x14) are viable with a slower motility. The kh30 mutation, which was originally isolated as ketamine response abnormal phenotype, has a mutation Ser1,444 to Asn, at a putative phosphorylation site by PKC. We aim to isolate interacting molecules with CeRYR especially at the most hydrophilic region likely exposed to the cytoplasmic surface. By using yeast two-hybrid system, we searched for interacting proteins which could potentially regulate the RyR’s function, especially at the cytoplasmic region surrounding kh30 mutation. Isolated two interacting proteins in F57F4.3 and F57F4.4, on chromosome V were almost identical except for their C-terminal end and encoded by adjacently located ORFs on cosmid F57F4. This candidate protein consists of about 2,000 amino acid residues with ten times repeated structure of 200 amino acid sequences motif which had weak similarity with LY6/UPAR domain or EGF-like repeat. We also searched for a hydrophobic region near the N-terminal end of CeRYR at where a hot spot for human malignant hyperthermia mutations occurred. We found that some proteins interacted with this region. About half of them were newly identified proteins having poor similarity with any previously identified proteins, but the remainder contained some transcription factor-like proteins, a mitcondorial enzyme, type-IV collagens (LET-2 and EMB-9) and others. The physiological functions of these proteins are remained to be addressed. Now we focus on F57F4.3 and F57F4.4 proteins to know their expression pattern. Further experiment such as gene silencing will allow us to solve their intact role for muscle contraction.

24 May 1999 15:50 790 790 EHS-1, the C.elegans homologue of mammalian Eps15, acts with DYN-1 in synapic vesicle recycling.

1999 International Worm Meeting abstract 739 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. EHS-1, the C.elegans homologue of mammalian Eps15, acts with DYN-1 in synapic vesicle recycling. AE Salcini 1 , MA Hilliard 2 , PP Di Fiore 1,3 , P Bazzicalupo 2

1 Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy. 2 Istituto Internazionale di Genetica e Biofisica, CNR, 80125 Naples, Italy. 3 Istituto di Microbiologia, Universiy of Bari, 70124 Bari, Italy.

The C.elegans gene ehs-1 (zk1248.3) codes for the homologue of mammalian Eps15, a protein involved in endocytic processes. The binding to AP-2 complex, its localisation at the rim of coated pits, the presence in the N-terminal part of three EH domains, a protein-protein interaction domain shared with yeast proteins involved in endocytosis, the inhibition of receptor internalisation by dominant negative mutants of Eps15, are the prominent features of the mammalian protein. We will show that EHS-1 retains the biochemical properties of Eps15 and, as its mammalian counterpart, it is involved in a specializated endocytic process. Immunofluorescence analysis with specific antibodies in wild type worms reveals that EHS-1 localizes exclusively to synaptic-rich regions. Its mislocalisation in unc-104 mutant worms indicates that EHS-1 is transported to synapses by the same machinery used by synaptic vesicles. Reporter analysis confirms that ehs-1 is expressed in most or all neurons,beginning in late embryos and that expression in other cells is absent or below detection. Transformation of worms with constructs producing full length or truncated forms of EHS-1 fused to GFP have helped identify the domains of the protein necessary for its transport to the synapses. RNA interference and transformation with a truncated form of EHS-1 producing a dominant negative effect, result in a temperature-sensitive, reversible uncoordinated phenotype, reminiscent of that of dyn-1 mutants. We will discuss data indicating that ehs-1 acts in the synaptic vesicle recycling pathway and that it interacts genetically with dyn-1.

24 May 1999 15:50 791 791 Investigating A Role For Cholinergic Signaling In The Dauer Formation Pathway

1999 International Worm Meeting abstract 740 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Investigating A Role For Cholinergic Signaling In The Dauer Formation Pathway GM Sandoval, G Ruvkun Massachusetts General Hospital and Harvard Medical School Animals with mutations in the daf-7 TGF-b gene (Ren at al. Science 274, 1996) arrest at the dauer larval stage in the presence of favorable conditions for reproductive growth. Exogenous application of the muscarinic receptor agonist oxotremorine rescues dauers allowing them to enter the reproductive life cycle (Heidi Tissembaum, personal communication) suggesting a potential role for muscarinic signaling in the dauer/nondauer developmental switch. To address more directly the role of cholinergic signaling pathways we made double mutants between daf-c genes and animals mith mutations that have decreased cholinergic signaling. We observed a synthetic L1 lethality in the double mutant of the insulin-like receptor daf-2( e1370) and a vesicular transporter of acetylcholine unc-17 (e245) (Alfonso, A. et al. Science 261, 1993). We are conducting a screen to suppress this lethality to isolate mutations in components of the cholinergic signaling pathway which may mediate dauer formation. We are also using a reverse genetic approach to analyze components of the cholinergic signaling pathway. By sequence comparisons in the C. elegansgenome, we have identified three muscarinic receptor homologues. We have analyzed the expression of a GFP promoter fusion of two of these genes. We are currently analyzing the expression of the third. We have not observed expression of our constructs in neurons identified as having a role in the dauer signaling pathway. Both constructs are expressed in a small set of neurons with one construct also exhibiting expression in intestinal cells. Deletion mutants of these genes will be generated and analyzed for dauer phenotypes to address whether there is muscarinic input into the dauer pathway.

24 May 1999 15:50 792 792 A mut-6 screen for RNAi deficient mutants

1999 International Worm Meeting abstract 741 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A mut-6 screen for RNAi deficient mutants M Sarkissian, H Tabara, CC Mello University of Massachusetts, Worcester, MA 01605, USA. To investigate the mechanism of RNAi, we have undertaken genetic screens for RNAi deficient mutants (tentatively named rid-loci). EMS mutagenesis has already yielded at least several RNAi resistant mutant strains (see the abstract by Tabara et al.). rid-mutants arise at knock out frequency and most appear to be recessive in nature. We therefore decided to search for transposon induced alleles that might be useful in cloning the rid- mutant genes. 100,000 mut-6, lin-2 worms were allowed to grow on E.Coli expressing dsRNA for a maternal gene (pos-1). One round of enrichment was carried out and the worms were replaced on the bacteria expressing the dsRNA. Thus far 14 viable strains have been obtained and a secondary microinjection based screen is now underway. So far one mut-6 strain appears to exhibit complete resistance to both maternal and zygotic RNAi. We hope to report on further genetic mapping and clonng of these mutants.

24 May 1999 15:50 793 793 Differential expression of CeCRMP/DHP-1 and CeCRMP/DHP-2, novel members of CRMP/DRP/DHP/UNC-33 family in C. elegans.

1999 International Worm Meeting abstract 742 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Differential expression of CeCRMP/DHP-1 and CeCRMP/DHP-2, novel members of CRMP/DRP/DHP/UNC-33 family in C. elegans. Y Sasaki 1 , T Takemoto 2 , H Kimura 2 , M Nonaka 3 , Y Goshima 1

1 Dept. Pharmacol., Yokohama City Univ. Sch. Med. 2 Dept. Exp. Radiol., Shiga Univ. Med. Sci. 3 Grad. Sch. Sci., Tokyo Univ.

Collapsin is recognized as a member of a semaphorin/collapsin gene family which is thought to be involved in axonal guidance. We isolated a collapsin response mediator protein (CRMP) from chick dorsal root ganglion, using Xenopus laevis oocyte expression system. In rodents, 4 subtypes of CRMP are expressed mainly in the nervous system during development. Independently, we cloned 4 human dihydropyrimidinase (DHP)-related proteins (DRP-1-4), human homologues of CRMP, by their homology to DHP. DHP is the second enzyme in the pyrimidine degradation pathway. However, all CRMP/DRPs have replacement at one or more essential residues for DHP activity, and CRMP/DRP purified from brain does not show any DHP activity. Therefore, a role of CRMP/DRPs in axonal guidance remains to be elucidated. Interestingly, chick CRMP/DRP shares 33% amino acid identity with the C. elegans UNC-33. unc-33 nematodes move in an uncoordinated fashion, and axon outgrowth is aberrant. In database of C. elegans Genome Project, we here identified two additional genes of CRMP/DRP/DHP/UNC-33 homologues. These two gene products shared similar amino acid identities with chick CRMP/DRP (40-46%) and rat DHP (49-55%), and thus these genes were termed CeCRMP/DHP-1 and -2. To examine the expression of these related genes, we constructed CeCRMP/DHP::GFP (green fluorescent protein) transgenes, which were driven under control of CeCRMP/DHP native upstream genomic sequences. The CeCRMP/DHP-1::GFP was expressed in some neuronal cells and hypodermis in larval stage, but was downregulated in adult stage. On the other hand, the expression of CeCRMP/DHP-2::GFP was seen predominantly in body wall muscle cells at 180-cell stage and continued throughout life.

24 May 1999 15:50 794 794 Cellular and molecular analysis of thermotaxis-defective mutants

1999 International Worm Meeting abstract 743 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cellular and molecular analysis of thermotaxis-defective mutants H Sasakura, A Kuhara, H Komatsu, I Mori

1 Division of Biological Science, Nagoya University. C. elegans can sense a range of temperatures and migrates toward the temperature at which it was cultivated with food for several hours on a temperature gradient. To date, several genes required for this thermotaxis behavior have been identified. The tax-4 and tax-2 genes encoding alpha and beta subunits of cyclic nucleotide-gated channel, respectively, are expressed in the AFD thermosensory neuron. The ttx-3 gene encodes a LIM homeodomain protein that is expressed in the AIY interneuron. Like ttx-3 mutants, ttx-1 and ttx-2 mutants show cryophilic phenotype. These cryophilic phenotypes are mimicked by killing AFD or AIY neurons, which is consistent with the possibility that ttx-1 and ttx-2 genes function in AFD or AIY. To observe any defects in AFD or AIY neurons, we introduced AFD-specific gcy-8::GFP and AIY-specific ttx-3::GFP markers into ttx-1 and ttx-2 mutants as well as into ttx-3, tax-2, tax-4 and tax-6 mutants. We found that the expression of gcy-8::GFP in AFD was strongly down-regulated in the ttx-1 mutant. Since gcy-8 encodes a membrane-bound type guanylyl cyclase, we next investigated whether an AFD-specific, GFP-tagged H13 transcription factor is affected in ttx-1 mutants. The expression of H13::GFP was almost completely undetectable in ttx-1 mutants. These results are consistent with the hypothesis that ttx-1 gene encodes a transcriptional regulator. In addition, this study enabled us to detect abnormal microvillus-like structures in the AFD sensory endings of ttx-1 mutants, as previously reported through the EM analysis. The unc-101 gene encodes a clathrin adaptor, a molecule that is thought to be important for intracellular transport. We observed structural abnormalities in the AFD sensory endings of unc-101 mutants, which is quite similar to those of ttx-1 mutants. Altogether, our results suggest that the ttx-1 gene functions in AFD. Besides ttx-1 mutants, our study also revealed that the expression of gcy-8::GFP was significantly down-regulated in tax-2 and tax-4 mutants. Our current model is that abnormal signaling accompanied by tax-2 or tax-4 mutation leads to the reduced overall activity of the AFD neuron, which in turn down-regulates the transcriptional level of gcy-8.

We thank T. Ishihara for H13::GFP prior to publication.

24 May 1999 15:50 795 795 A novel C. elegans gene encodes a nicotinic acetylcholine receptor a subunit is capable of forming a functional hetero-oligomeric receptor in vitro when co-expressed with lev-1 and unc-29

1999 International Worm Meeting abstract 744 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A novel C. elegans gene encodes a nicotinic acetylcholine receptor a subunit is capable of forming a functional hetero-oligomeric receptor in vitro when co-expressed with lev-1 and unc-29 DB Sattelle, E Culetto, NP Mongan, K Matsuda, JT Fleming, MD Squire, JA Lewis, HA Baylis The Babraham Institute, Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, UK. Nicotinic acetylcholine receptors (nAChRs) mediate the fast action of the neurotransmitter acetylcholine at cholinergic synapses in the nematode C. elegans. An extensive and diverse family of nAChRs has been described in this organism (1). Genetic screens for mutants resistant to the cholinergic anthelmintic drug levamisole (2, 3) led to the molecular characterization of nAChRs subunit genes unc-29, lev-1 and unc-38 (4). Of the eleven levamisole resistant genes so far characterized genetically, the molecular identity of five, unc-63, unc-74, lev-8, lev-9 and lev-10, remains to be determined. A new nAChR has been cloned using cross hybridization with unc-38 and unc-29 cDNAs and RT-PCR. This nAChR is a putative a subunit as it has the typical YxCC motif in loop C of the acetylcholine binding site. We have shown that this subunit is encoded by unc-63. Three different mutant alleles of unc-63 have also been sequenced. The allele x37 confers on worms high resistance to levamisole and a very slow locomotion. Sequencing shows that a stop codon in the extracellular loop interrupts the open reading frame in the x37 allele. Worms with two rare alleles b404 and x26 have a slight levamisole resistance and nearly normal movement. The sequencing of b404 allele shows a deletion in the large intracellular loop whereas the x26 allele have a C to Y mutation in the highly conserved dicysteine loop in the N-terminal region. We used voltage clamp electrophysiology to study heterologous expression in Xenopus oocytes injected with cDNA expression constructs for lev-1, unc-29 and unc-63. This recombinant nAChR gives a robust current which is dose-dependent to acetylcholine (EC50 = 30 µM). This value agrees well with EC50 obtained for native nematode nAChRs. This recombinant heteromeric receptor is blocked reversibly by mecamylamine.

(1) Mongan et al. (1998) Receptors & Channels 6, 218-228. (2) Brenner, S. (1974) Genetics 77, 71-94. (3) Lewis, J.A. et al. (1980) Genetics 95, 905-928. (4) Fleming, J.T. et al. (1997) J. Neurosci 17, 5843-5857.

24 May 1999 15:50 796 796 The AFD Thermosensory Neurons: Specification of Cell Fate and Function

1999 International Worm Meeting abstract 745 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The AFD Thermosensory Neurons: Specification of Cell Fate and Function JS Satterlee, P Sengupta Department of Biology, Brandeis University 415 South St. Waltham, MA 02454 The AFD neurons are required for thermosensation in C. elegans. This pair of ciliated sensory neurons has a unique morphology and presumably expresses a specific suite of genes which confer thermosensory function. For example, the promoters of both a nuclear hormone receptor homolog (nhr-38) and a receptor guanylate cyclase homolog (gcy-8) drive GFP expression specifically in this neural pair. We are examining the mechanism by which the fates and functions of the AFD thermosensory neurons are specified, by identifying genes that work in a cascade to direct expression of these AFD-specific markers. Mutations in such genes are predicted to result in absent or reduced gene expression in AFD, or possibly ectopic expression of these markers in other cells. In a pilot screen (6800 haploid genomes), we identified two mutants with reduced/absent expression of nhr-38::GFP in the AFD neurons. These mutants show defects in AFD, AWC and ASE sensory neuron functions. The sns-4(oy16) mutant was found to be allelic to tax-4, while the sns-5(oy17) mutant is allelic to tax-2. The tax-2/tax-4 genes encode subunits of a cyclic nucleotide-gated channel which functions in some sensory neurons, including AFD. It is possible that the tax-2/tax-4 channel is required for activity-dependent expression of some AFD specific genes. We have also performed a genetic screen to identify mutants with reduced/absent expression of gcy-8::GFP in the AFD neurons (25,900 haploid genomes). Four robust mutants were isolated, and these were placed into two complementation groups, sns-6 and sns-7, each with two alleles. sns-6 has been mapped to a 2 cM region of LG IV. Radial thermal gradient assays show that sns-6 and sns-7 are both thermotaxis defective, however we have not yet determined whether the animals are athermotactic or thermophilic. These mutants are not defective in behaviors mediated by the AWA, AWC, ASE, and ASH neurons, and exhibit no other defects. The sns-6 and sns-7 gene products are therefore likely to be important in the specification of AFD function. It is also possible that sns-6 and sns-7 function in the thermosensory signaling pathway.

24 May 1999 15:50 797 797 Chaperones to the rescue of misfolded (proteins in) worms?

1999 International Worm Meeting abstract 746 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Chaperones to the rescue of misfolded (proteins in) worms? Sanjeev Satyal 1 1 , Enrico Schmidt 1 1 , Kazunori Kitagawa 1 1 , Neal Sondheimer 2 2 , James M. Kramer 3 3 , Susan Lindquist 2 2 , Richard I. Morimoto 1 1

1 Dept. of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208. 2 HHMI, Dept. of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637. 3 Department of CM Biology, Northwestern University, Evanston, IL 60208.

Expansion of CAG trinucleotide repeats encoding polyglutamine stretches have been implicated in neurodegenerative diseases, such as dentatorubral-pallidoluysian atrophy (DRPLA). The expression of the expanded CAG repeats mutant DRPLA proteins in human tissue culture cells results in formation of peri- and intranuclear aggregates. Similar aggregates were also observed in worms upon expression of the mutant DRPLA proteins in body wall muscle cells using the unc54 promoter, whereas the expression of the wild type DRPLA protein does not lead to aggregate formation. Aggregate formation will be assessed in the context of the various chaperone deficient strains generated in our laboratory and in animals with a compromised stress response resulting from the overexpression of CeHSB-1, a negative regulator of the stress response. Additional protein substrates (from S. cereviseae and D. discoideum) that could have a propensity to form aggregates were selected by scanning the protein sequence databases for proteins with high glutamine or asparagine content. Overexpression of these proteins in yeast cells does indeed cause the formation of aggregates and their ability to form similar aggregates in C. elegans is being investigated. The worms that display the DRPLA aggregates represent a tool to study the role of chaperones in preventing aggregate formation. Hsp104 has been demonstrated to have a potent ability to mediate the resolubilization of high molecular weight protein aggregates in concert with Hsp70 and Hsp40. This feature of Hsp 104 is unique among various heat shock proteins and therefore we are in the process of co-expressing Hsp104 along with other chaperones, like Hsp40 and Hsp70, to determine their effects on the ability of DRPLA mutants and other proteins to form aggregates in vivo.

24 May 1999 15:50 798 798 Protein fold assignment and functional analysis of the C. elegans genome

1999 International Worm Meeting abstract 747 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Protein fold assignment and functional analysis of the C. elegans genome JM Sauder, RL Dunbrack Institute for Cancer Research, Fox Chace Cancer Center Three-dimensional protein structures can provide powerful insights into protein functions that are not attainable from sequence information alone. While the atomic structures of fewer than a dozen C. elegans proteins are known, it is possible to identify proteins in the genome that have homologues in the Protein Data Bank (PDB) of experimentally determined structures and build 3D protein structure models of the C. elegans sequences based on these homologues. We have used profile-based and intermediate-sequence search methods to identify structural homologues for protein sequences encoded by the C. elegans genome and are able to assign folds to almost 30% of the non-membrane proteins of C. elegans. Our results indicate that the top 8 non-membrane protein folds represent 10% of the genome sequences, and that the most common folds in the genome are protein kinase domains, ferredoxin-like folds, and the DNA-binding domain of glucocorticoid receptor-like proteins. Our methods are sensitive enough to detect many distant homologues which may share only 10-20% sequence identity. We will give several examples of sequences in C. elegans where the resulting protein structure models are useful in interpreting mutagenesis studies. Correlating genotype and phenotype relationships through protein structure is particularly useful in these cases given the role of C. elegans in understanding development. We propose the creation of a C. elegans mutation database as a central repository for genotype/phenotype changes, which can be expanded by on-line with input from scientists worldwide. These mutation data can be correlated with our models of the protein structures automatically, where the positions of mutated residues can be highlighted on a molecular graphics representation of the structure model available on the World Wide Web.

24 May 1999 15:50 799 799 Mutations affecting asymmetric T cell division

1999 International Worm Meeting abstract 748 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Mutations affecting asymmetric T cell division H Sawa, H Kouike, H Okano Department of Neuroanatomy, Osaka University Medical School, Suita, 565-0871, Japan. Asymmetric cell division is a fundamental mechanism for the production of cellular diversity during development. In C. elegans, the asymmetry of a number of cell divisions is affected by mutations in lin-17 or lin-44. lin-17 encodes a receptor molecule that belongs to the Frizzled family. lin-44 encodes a signaling protein that belongs to the Wnt family. We have proposed that the polarities of cells undergoing asymmetric divisions are regulated by the LIN-44 signal which act through the LIN-17 receptor. How the signal is transduced from the LIN-17 receptor and how the consequent cell polarities are established are unknown. The polarities of the T cell division is regulated by lin-17 and lin-44. In wild type, anterior daughter of the T cell produces hypodermal cells and posterior one makes neural cells including phasmid socket cells. In lin-17 mutants, both daughters produce hypodermal cells. To identify genes involved in asymmetric cell divisions, we are screening for mutants that lack phasmid socket cells (the phenotype is called Psa for phasmid socket cell absent). We have so far identified more than 10 mutants with Psa phenotype which are not lin-17, lin-44 nor egl-27. Lineage analyses have shown that the T cell division can be symmetric in at least 5 of the mutants (psa-1 through psa-5). psa-1 and psa-2 are temperature-sensitive maternal effect embryonic lethal. psa-2 specifically affects the elongation of embryo. Temperature shift experiments have shown that psa-1 is required for the T cell polarity before the T cell division. Molecular analyses of these mutants are underway. We have also found that a lit-1 mutation can disrupt the T cell polarity. lit-1 is involved in asymmetric cell divisions of the EMS blastmere that also requires mom-2/wnt and mom-5/frizzled. The result suggests that a signaling pathway similar to the one that controls the EMS polarity regulates the T cell polarity.

24 May 1999 15:50 800 800 Mutations that Alter Interneuronal Synaptogenesis

1999 International Worm Meeting abstract 749 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Mutations that Alter Interneuronal Synaptogenesis AM Schaefer, ML Nonet Washington U. School of Med. To study molecular mechanisms of interneuronal synapse formation, we have screened C. elegans for mislocalization of tagged synaptic vesicles. The template strain carries the integrated construct p mec-7::snb-1::GFP. This construct is designed to express only in the six touch cells, and to fuse GFP to the synaptic vesicle membrane protein SNB-1. Wild type integrants show a consistent and relatively simple pattern of fluorescence. Touch cell bodies are labeled, axonal processes are visible, and there are discrete fluorescence accumulations in the nerve ring and ventral nerve cord where synapses have been observed by serial reconstruction. There is no predicted behavioral consequence for loss or alteration of the touch neurons’ chemical synapses, so we have screened random F2 animals for aberrations in the fluorescence pattern. Screening of more than 2000 haploid genomes indicates thirteen complementation groups, falling into three phenotypic classes. The first class, containing nine complementation groups, includes animals lacking all fluorescence accumulations along the ventral nerve cord and a subset of accumulations in the nerve ring. These animals generally have no phenotype other than an altered GFP pattern: axonal outgrowth is normal, the animals move well, and they respond to touch. We are focusing on the two complementation groups with multiple alleles, including a locus on LG V which maps to a four-cosmid region. In the second phenotypic class are two non-complementing mutants, which show a long strip of fluorescence along the ventral nerve cord, rather than the discrete accumulations seen in the wild type. In addition to an altered GFP pattern, these animals are Egl, and slightly slower to develop. Their altered fluorescence pattern phenocopies the pattern in wild type animals where post-synaptic targets have been ablated; nevertheless, the mutants’ behavior suggests that post-synaptic cells in these animals have differentiated. We have mapped this locus to the right arm of Chrom. II. These first two classes suggest deficits in synapse formation. In contrast, the third class contains animals which show the wild type fluorescence pattern, but have additional fluorescence accumulations at the ends of the touch cell processes. For further description of this third class, see poster of S. Koushika, A. M. Schaefer, and M. L. Nonet.

24 May 1999 15:50 801 801 Investigating the role of P granules in germ cell development

1999 International Worm Meeting abstract 750 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Investigating the role of P granules in germ cell development JA Schisa, J Pitt, JR Priess Fred Hutchinson Cancer Research Center P granules are associated with the germ cell lineage throughout development, and therefore have been hypothesized to have some function in germline development. The components of P granules include several proteins, and there are many indications P granules also contain RNA. For example, we and others have identified several proteins with putative RNA-binding motifs that associate with P granules in embryos and/or larvae and adults. Seydoux and Fire (1994) showed that P granules in the embyro appear to contain SL1 RNA and poly (A)+ RNA. Finally, we and others have observed punctate staining, similar to the appearance of P granules, in the germline blastomeres of embryos after in situ hybridization experiments for various maternally-provided RNAs. We are interested in determining the significance of RNA in P granules. To develop a better understanding of what RNAs might be in P granules, we want to look at various classes of maternally-expressed RNAs by in situ hybridization in gonads, oocytes, and embryos. These classes include mRNAs that are translated throughout the gonad, mRNAs that are translated in oocytes, and mRNAs that are translated only in embryos. Our primary effort thus far has been to improve in situ hybridization conditions. However, we have not yet observed significant P granule association in the gonads with any mRNAs examined to date. Specific RNAs might not be abundant in gonadal P granules if those RNAs were rapidly processed through P granules in normal development. Mutants with abnormally large P granules could be defective in processing, and thus provide a valuable reagent for our in situ hybridization experiments. We did a pilot screen to ask if such mutants could be isolated and found 5 sterile or maternal-effect lethal mutants with larger than normal P granules. We hope to report whether these mutants have any RNA localization defects.

G. Seydoux and A. Fire (1994) Development 120, 2823-2834.

24 May 1999 15:50 802 802 unc-120 encodes a SRF transcription factor involved in muscle development

1999 International Worm Meeting abstract 751 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. unc-120 encodes a SRF transcription factor involved in muscle development LA Schriefer, IM Caldicott, RH Waterston Department of Genetics, Washington University Medical School, St. Louis MO, 63110 Our previous studies have shown that the ts allele unc-120(st364) has a reduced number of muscle A and I bands as compared to N2 and is paralyzed at 25°C. Rescue by cosmid transformation reveals a 8kb region that confers wt muscle structure and movement to unc-120(st364). unc-120 encodes a protein of 327 a.a. that has a 60 a.a. domain near the n-terminus that is highly conserved in, and diagnostic of, MADS transcription factors. This domain is most homologous to drosophilia Serum Response Factor (SRF), a type of MADS transcription factor that has a role in muscle development. The sequence of the homologous C. briggsae clone shows complete conservation in this region and high conservation throughout the gene. An unc-120:GFP promoter fusion reveals expression in body-wall and vulva muscles but is not expressed in the pharynx. unc-120 is expressed continually in the body-wall specific cells of the embryo from 215 minutes on. This is earlier than other body-wall muscle components such as myosin. RNAi experiments suggest that the null phenotype of unc-120 is temperature independent and is a very sickly, largely paralyzed animal with poorly organized body-wall muscle birefringence and sharply reduced levels of expression of muscle components such as myosin and actin. The mutation in st364, the sole unc-120 allele, alters the trans-splice leader splice site. Reversion analysis, rtPCR and sequencing indicate that st364 primarily uses a downstream out-of-frame splice site. Intragenic wt revertants of st364 destroy this site and use another downstream in-frame splice site that encodes a protein missing the n-terminus and part of the MADS domain but is presumably functional. Extragenic revertants have been isolated that may alter the trans-splice mechanism.

24 May 1999 15:50 803 803 mex-5 and ah6.5 act redundantly to establish cell fates in the early embryo

1999 International Worm Meeting abstract 752 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. mex-5 and ah6.5 act redundantly to establish cell fates in the early embryo CM Schubert 1 , R Lin 2 , J Priess 1,3

1 Department of Zoology, University of Washington. 2 Department of Molecular Biology and Oncology, UT Southwestern Medical Center. 3 Fred Hutchinson Cancer Research Center and Howard Hughes Medical Institute, Seattle, Washington.

The distinct identities of germline and somatic blastomeres requires the asymmetric expression of proteins normally localized predominantly to germline blastomeres. A group of three proteins, PIE-1, POS-1 and MEX-1 is expressed predominantly in germline blastomeres. Each of these proteins is required for the development of germline, and high levels of PIE-1 are deleterious to somatic blastomeres. In embryos from mothers mutant for the gene mex-5, somatic blastomeres have increased levels of proteins found predominantly in germline blastomeres, including PIE-1, POS-1 and MEX-1. Germline blastomere levels of these proteins, however, appear normal in mex-5 mutant embryos. I have generated a monoclonal antibody specific to the MEX-5 protein, and have found that MEX-5 is expressed in blastomeres which normally lack PIE-1, POS-1 and MEX-1 protein. These results suggest that MEX-5 acts in somatic blastomeres to negatively regulate the expression of proteins normally found in germline blastomeres. The MEX-5 predicted protein contains two copies of a CCCH "finger" motif, and shows 66 percent identity with a protein encoded for by the gene with the cosmid name AH6.5. A strain containing a deletion in AH6.5 was obtained from Corry de Vries in the lab of Ron Plasterk. This strain appears wild-type. Embryos from worms doubly mutant for ah6.5 and mex-5 show an enhancement of the mex-5 phenotype. These embryos express PIE-1, POS-1 and MEX-1 in somatic blastomeres at levels higher than in the mex-5 mutant embryos alone. However, ah6.5;mex-5 double mutant embryos also have reduced levels of these proteins in the germline. I am currently performing experiments which address the mechanism of mex-5 and ah6.5 function.

24 May 1999 15:50 804 804 The Great Divide: Analysis of single-cell cytokinesis-defective mutants in C. elegans

1999 International Worm Meeting abstract 753 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Great Divide: Analysis of single-cell cytokinesis-defective mutants in C. elegans JM Schumacher 1 , M Wallenfang 2 , G Seydoux 2 , A Golden 1

1 ABL-Basic Research Program, NCI-FCRDC, Frederick, MD 21702. 2 Dept. of Mol. Biol. and Genetics, Johns Hopkins Univ. School of Med., Baltimore, MD 21205.

Mitosis culminates in the equal segregation of sister chromatids to each of two newly- formed cells. Genetic instability can be caused not only by defects in the segregation process itself, but also by failures in cytokinesis that can lead to the formation of polyploid cells. Previous studies in our lab have focused on two kinases that are directly involved in each of these events (Schumacher et al. 1998a,b). The C. elegans AIR-1 protein is required for proper assembly and function of the mitotic spindle, whereas the highly related AIR-2 protein is required for the stabilization of the cleavage furrow and the completion of cytokinesis. In an attempt to isolate mutations in the AIR-2 locus as well as identify new components in the AIR-2 pathway, we have recovered thirty temperature-sensitive maternal-effect lethal mutants that arrest at the one-cell stage. DAPI staining of all thirty mutants revealed that seven were highly polyploid at the restrictive temperature and essentially had the same phenotype as air-2(RNAi) cytokinesis-defective embryos (Schumacher et al. 1998b). Genetic mapping of and complementation tests between six of the mutants have shown that all six map to distinct genetic intervals and represent six independent loci. The seventh has not yet been mapped. Three of the mutants map to Chr. I, two map to Chr. V, and the remaining locus maps to a chromosome arm. All six appear to represent new mutants in that they do not map to genetic intervals that contain previously described cytokinesis-defective mutations. Finer mapping data as well as the analysis of candidate genes within the relevant intervals will be presented.

Schumacher, J., Ashcroft, N., Donovan, P. and A. Golden (1998a) Development 125:4391-4402. Schumacher, J., Golden, A. and P. Donovan (1998b) J Cell Biol. 143:1635-1646. Research sponsored by the National Cancer Institute, DHHS, under contract with ABL.

24 May 1999 15:50 805 805 A screen for mutants defective in the specification of programmed cell death in the postdeirid lineage

1999 International Worm Meeting abstract 754 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A screen for mutants defective in the specification of programmed cell death in the postdeirid lineage H Schwartz, B Horvitz HHMI, Dept. Biology, MIT, Cambridge, MA 02139 During wild-type hermaphrodite development, 131 somatic cells undergo programmed cell death. While many genes involved in the execution of cell death have been identified, the mechanisms that control the commitment of specific cells to undergo programmed cell death are poorly understood. To date, mutations in three genes, ces-1, -2, and -3 (cell death specification), have been found that affect specifically the deaths of particular cells. ces-1 and ces-2 have been cloned and shown to encode transcription factors. We intend to perform a genetic screen for ces genes involved in the deaths of the sisters of the PVD neurons in the postdeirid lineage. We chose this lineage because the deaths occur during postembryonic development, making the deaths easy to identify using Nomarski optics, and because reporters exist that seemed likely to be expressed in these cells if their deaths were prevented by a mutation affecting programmed cell death. In ced-3 mutants that appear to be completely defective in programmed cell death, roughly half of the "undead" PVD sisters contain dopamine, like their "aunt" the PDE cell, as seen by formaldehyde-induced fluorescence. We have found that GFP reporters for dopaminergic neurons are expressed in 60% of undead PVD sisters in mutants defective in programmed cell death. We are beginning a screen using cat-2::GFP (kindly provided by Robyn Lints) to identify mutants in which the PVD sisters survive.

24 May 1999 15:50 806 806 Classical and reverse thermosensory genetics in C. elegans

1999 International Worm Meeting abstract 755 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Classical and reverse thermosensory genetics in C. elegans EM Schwarz, M Chalfie Dept. of Biol. Sci., 1212 Amsterdam Ave., Columbia Univ., New York, NY 10027 Metazoan thermosensation is imperfectly understood. We are using both classical and reverse genetics to clarify thermosensation in C. elegans. Thermal uncoupling of a daf-c mutation: The daf-7(e1372) mutation renders dauer formation highly temperature-sensitive (100% dauers at 25 deg. C.; ~1% dauers at 15 deg. C.; refs. 1-2). This phenotype is not due to DAF-7 thermolability, since null mutations have similar sensitivity (3). Hobert et al. (2) demonstrated that ttx-3 uncouples this thermosensitivity, inducing daf-7(e1372) to produce ~30% dauers at 15 deg. C. and ~50% non-dauers at 25 deg. C. Thermosensory mutations should thus be obtainable by first screening mutagenized daf-7(e1372) at 25 deg. C. for dauer suppressors, and then examining each suppressed strain for 1% dauer production at 15 deg. C. We have screened ~24,000 mutant genomes in F2 and ~190,000 in F1, obtaining 21 independent lines which showed dauer suppression at 25 deg. C. but dauer or pseudodauer production at 15 deg. C. Work to assign them to complementation groups is underway. gcy-8: This gene, encoding a guanylate cyclase receptor, was found by Yu et al. (4) to be expressed solely in the thermoreceptor AFD. However, it is unclear whether this gene actually mediates thermal signals in AFD. We are using reverse genetics to assay loss and gain of gcy-8 function in daf-7(e1372) animals. Ablation of function is being tried by RNAi. Increase of function is being tried through a gcy-8 transgene with a mutation homologous to E974A in atrial natriuretic peptide, which induces constitutive activity in cell culture (5). If GCY-8 actually directly carries thermal signals, we predict that loss- and gain-of-function gcy-8 mutations should have opposite effects on dauer formation of daf-7(e1372) animals at 15 deg. versus 25 deg. C.

1. Swanson and Riddle (1981), Dev. Biol. 84, 27-40. 2. Hobert et al. (1997), Neuron 19, 345-357. 3. Ren et al. (1996), Science 274, 1389-1391. 4. Yu et al. (1997), Proc. Natl. Acad. Sci. USA 94, 3384-3387. 5. Wedel et al. (1997), Proc. Natl. Acad. Sci. USA 94, 459-462.

24 May 1999 15:50 807 807 Studies of PAR-2 localization by ectopic expression in somatic cells

1999 International Worm Meeting abstract 756 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Studies of PAR-2 localization by ectopic expression in somatic cells P Schweinsberg, L Boyd University of Alabama-Huntsville, Huntsville, AL 35899 The par genes are required for the establishment of proper anterior-posterior polarity in C. elegans zygotes. Most PAR proteins are localized to the cortical region of early blastomeres. We are interested in understanding how this cortical localization is achieved. Our studies focus on the PAR-2 protein which has an asymmetric cortical localization in cells of the P lineage. After fertilization, PAR-2 is localized to the posterior cortex in the P0 cell and continues to have an asymmetric distribution at the cortex in the P1, P2 and P3 cells. This distribution most likely allows for the unequal partitioning of PAR-2 to the daughter cells at division. In order to understand the mechanism for localizing PAR-2, we are using an overexpression vector obtained from the Fire lab to achieve ectopic expression of a PAR-2:GFP fusion protein (GFP: green fluorescent protein). Using this system we see that the fusion protein is expressed in somatic cells of embryos after the 18 cell stage. GFP is detected at the cortex in these cells indicating that all embryonic cells have the capacity for cortical association by PAR-2. We have begun a deletion analysis of the protein to determine what region of PAR-2 is required for this cortical localization. In addition to the structural analysis of the PAR-2 protein, we are interested in identifying other factors that are involved in localizing PAR-2. Work from the Kemphues lab has shown that the par-3 and par-6 genes are required for the asymmetric distribution of PAR-2 in the P cells but not for the cortical localization. Recently, Steve Basham and Lesilee Rose informed us of two mutants, ooc-5 and ooc-3, with reduced levels of cortical PAR-2 in the P1 cell (1998 West Coast worm meeting abstract #12). We were interested to test if this might be due to a reduced ability of PAR-2 to interact with the cortex or, conversely, if the abnormal PAR-2 distribution might be due to some indirect effect such as the expanded cortical distribution of PAR-3 in the mutants. To distinguish between these two scenarios, we placed our PAR-2:GFP transgene in the mutant strains. Our initial results indicate that GFP is cortical in the mutant embryos. Thus, it appears that the affect of ooc-3 and ooc-5 mutation on PAR-2 localization is indirect.

24 May 1999 15:50 808 808 Towards a genome-wide collection of transposon insertions

1999 International Worm Meeting abstract 757 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Towards a genome-wide collection of transposon insertions L Ségalat 1 , C Bessou 1 , F Schurr 1 , O Hauser 1 , T Alvarez 1 , S Sookhareea 2 , R Legouis 2 , M Labouesse 2

1 CNRS-IPMC, 660 route des lucioles, 06560 Valbonne, France. 2 IGBMC, BP163, 67400 Illkirch, France.

With the genome sequence being complete, it is essential to determine the function of each predicted gene. To facilitate the cloning of genes, it is also important to create as many polymorphisms as possible. With these goals in mind, we have launched a large scale project whose long-term objective is to isolate insertions of transposable elements in most C. elegans genes. To do so, we have chosen to use techniques based on the random insertion of natural worm transposons, an approach which has been pioneered in the Plasterk lab. Starting from mutator backgrounds, we generate clones and determine the position of new insertions by a modification of the transposon insertion display protocol described in the WBG (Vol 14, n°4 page 20), in which DNA flanking transposons can be amplified by anchored PCR, and sequenced. Assuming ideal statistical conditions (non-biased insertion sites, independence of insertion events and no intergenic regions), the Poisson distribution would predict that 30,000 independent sequence reads would be enough to hit 80% of the estimated 19,000 C. elegans at least once. In practice, since the last of these three conditions cannot be met (and assuming the other two are), 30,000 insertions should lead to an insertion every few kilobases, which would give a polymorphism coverage of the genome much higher than the current one. It is expected that approximately a quarter of these insertions will be in coding sequences and UTRs (representing. potential mutations). This project is a complementary alternative to the gene-directed PCR-based search for deletions. We also believe that this project (currently estimated at $2-4 Million for 30,000 sequence reads) is competitive for cost and labor compared to gene targeting. Furthermore, transposon insertions should potentially provide a wider spectrum of alterations, which will be needed as genetic tools besides the knock-outs. We are currently running small-scale pilot experiments on a few hundred clones to optimize the protocols and validate the approach.

24 May 1999 15:50 809 809 Translational regulation of the heterochronic gene lin-28 by the lin-4 RNA and a lin-14-dependent positive feedback loop

1999 International Worm Meeting abstract 758 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Translational regulation of the heterochronic gene lin-28 by the lin-4 RNA and a lin-14-dependent positive feedback loop K Seggerson, JE Johnstone, EG Moss Fox Chase Cancer Center, Philadelphia, PA 19111 The heterochronic gene lin-28 is one of several regulators that control the stage-specificity of a variety of postembryonic developmental events throughout the animal. Two other heterochronic genes, lin-4 and lin-14 regulate lin-28. lin-4 encodes a short RNA that represses lin-28 expression after the L1. Without this repression, retarded development results. However, lin-28 can be repressed after the L1 in the absence of lin-4 if the lin-14 activity is reduced. lin-14, therefore, is a positive regulator of lin-28. lin-14 expression is likewise dependent on lin-28 after the L1 (Arasu, et al. 1991). Thus there is a mutual positive regulatory feedback loop between lin-14 and lin-28, two genes that are both repressed by lin-4. We’ve found that, like lin-14, lin-28 repression by lin-4 occurs by a post-transcriptional mechanism. Further, we’ve found that the lin-14 dependence of lin-28 expression is also post-transcriptional. By sucrose gradient analysis we’ve found that the repression via each regulatory circuit does not prevent the lin-28 mRNA from associating with polyribosomes. This is consistent with a mechanism of repression that acts after translation initiation. Also, these observations are consistent with a model proposed by V. Ambros in which a single regulatory mechanism affects lin-28, and the lin-4 and lin-14 regulatory circuits impinge oppositely on that mechanism. We are conducting experiments to test this model. A lin-4 complementary element (LCE) in the 3’ UTR of lin-28 is required for the repression by lin-4. We mapped the lin-14-dependence also to the 3’ UTR of lin-28 and found that the LCE is not required for lin-14-dependence. We have constructed lin-28:GFP transgenes bearing alterations of the 3’ UTR and tested these for down-regulation of GFP and lin-28 activity in two genetic backgrounds that reveal each regulatory circuit. The lin-14-dependence and the lin-4 regulation appear to be mediated by separate cis-acting elements. We would like to know whether these elements could confer repression on a heterologous gene. We are also seeking factors that interact with these elements in order to learn how lin-28’s repression takes place.

24 May 1999 15:50 810 810 The role of Cyt-1 in oxdative stress-induced mitochondrial dysfunction and apoptosis in C.elegans

1999 International Worm Meeting abstract 759 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The role of Cyt-1 in oxdative stress-induced mitochondrial dysfunction and apoptosis in C.elegans N Senoo-Matsuda 1 , S Yoshimura 2 , R Muto 1 , A Akatsuka 1 , P Hartman 3 , N Ishii 1

1 Tokai University School of Medicine, Isehara, Kanagawa. 2 Tokyo Research Laboratory, Kyowa Hakko Kogyo Co. Ltd. 3 Biol. Dept., TCU, Fort Worth, TX 76129.

The mev-1(kn1) mutant of C. elegans is hypersensitive to oxygen. Unlike the wild type, its lifespan dramatically decreased as oxygen concentration was increased. Recently, we have shown that mev-1 encodes a subunit of the enzyme succinate dehydrogenase cytochrome b (Cyt-1), which is a component of complex II of the mitochondrial electron transport chain [Nature 394:694(1998)]. We have also reported that the ability of complex II to catalyze electron transport from succinate to ubiquinone is compromised in mev-1 animals. To further elucidate the effects of the mev-1 mutation, we examined the expression of cyt-1 in wild type and mev-1 mutants using a number of different experimental approaches. Histological methods were employed to show that, in young adults, succinate dehydrogenase activity was clearly present in wild type but was undetectable in mev-1 animals. Using Western and Northern analyses, cyt-1 expression was found to be similar in mev-1 and wild type, with relatively constant amounts of mRNA but increasing amounts of protein accumulating throughout development. Transmission electron microscopy revealed that mev-1 mitochondria had morphological abnormalities, particularly at high (50%) oxygen concentrations. In young adults, these included the presence of electron-dense inclusions and disorganized cristae. In embryos, mev-1 nuclei had an appearance characteristic of those undergoing chromosome fragmentation. These EM data are consistent with Nomarski DIC microscopy, which showed that the number of apoptotic cells was significantly higher in mev-1 than in wild-type embryos. Elevated apoptosis was suppressed by ced-3, indicating that the abnormal signal in mev-1 embryos triggered the normal ced-3/ced-9 apoptotic pathway. Unlike with wild type, many mev-1 embryos failed to hatch when subjected to high oxygen concentrations. Taken together, these data indicate that Cyt-1 plays a key role in the regulation not only in aging but throughout development of C. elegans. In addition, the data reveal new pathological manifestations of the mev-1 mutation.

24 May 1999 15:50 811 811 Cytokinesis in C. elegans embryos: analysis of cyk-1 function

1999 International Worm Meeting abstract 760 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cytokinesis in C. elegans embryos: analysis of cyk-1 function AF Severson 1 , JC Carter 1 , DR Hamill 1 , DL Baillie 2 , BA Bowerman 1

1 University of Oregon, Eugene, OR. 2 Simon Fraser University, Burnaby, B.C., Canada.

cyk-1 encodes an FH protein required for cytokinesis in C. elegans embryos. Embryos homozygous for weak alleles of cyk-1 form cleavage furrows which frequently ingress extensively through the cytoplasm before failing. CYK-1 protein localizes cortically in cleavage furrows, but is detectable only after substantial ingression. We have proposed a model in which CYK-1 functions late in cytokinesis to stabilize cleavage furrows through interactions with the contractile ring and mitotic spindle, and that CYK-1 requires these interactions for its localization. We have tested this model in three ways: analysis of a stronger cyk-1 allele, double mutant analysis, and immunolocalization experiments. Worms homozygous for cyk-1(s2833), our strongest allele, are sterile, so the cytokinesis defect observed in weak alleles may not be the null phenotype. Embryos from cyk-1(s2833)/cyk-1(t1568) mutant worms fail to furrow. Thus, cyk-1 may also be required for an early step in cytokinesis. Further evidence that cyk-1 functions early in cytokinesis comes from analysis of cyk-1; zen-4 double mutant embryos. ZEN-4 is a mitotic kinesin-like protein that localizes to the spindle interzone. zen-4(or153ts) mutant embryos produce extensive furrows that ultimately regress, similar to the weak cyk-1(or36) phenotype. In contrast to either single mutant, cyk-1(or36); zen-4(or153ts) double mutant embryos fail to form cleavage furrows. We conclude that cyk-1 interacts with the ZEN-4 kinesin-like protein, and these genes function early in cytokinesis. To further test our model, we examined CYK-1 localization in zen-4 mutant embryos. CYK-1 accumulates normally in zen-4 mutant embryos; thus, function of this spindle-associated protein is not required for CYK-1 localization. Additionally, inactivation of mlc-4, a non-muscle myosin II regulatory light chain required for cleavage furrow ingression, does not prevent cortical CYK-1 localization. Thus, proximity between the cleavage furrow and the spindle are not required for CYK-1 localization. We are currently performing experiments to identify requirements for CYK-1 localization and to understand the genetic interaction between cyk-1 and zen-4.

24 May 1999 15:50 812 812 Genetic Analysis of Signaling Pathways that Determine the Deaths of Sex-Specific Neurons

1999 International Worm Meeting abstract 761 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic Analysis of Signaling Pathways that Determine the Deaths of Sex-Specific Neurons MS Shah, J Nyugen, D Xue MCDB, University of Colorado Boulder, CO 80309 In C. elegans 131 of the 1090 cells generated during development of the hermaphrodite adult animal undergo programmed cell death. We are interested in identifying new genes involved in the life vs. death decisions of normally dying cells. Specifically, we are interested in studying the signaling pathways that regulate the deaths of hermaphrodite specific neurons (HSNs) and male specific chemosensory neurons (CEMs) in different sexes. HSNs in C. elegans control egg-laying in hermaphrodite animals and normally undergo programmed cell death in males in which they are not needed. In contrast, CEM neurons mediate male chemotaxis towards the hermaphrodite during mating but are programmed to die in hermaphrodites where their function is not needed. The life vs. death decisions of the HSN and CEM neurons present an interesting paradigm for studying sex-specific cell death specification. Mutations in several genes (her-1, tra-2, and egl-1) have been identified that cause inappropriate death of HSNs in hermaphrodites. her-1 encodes a novel secreted molecule whose activity is high in males to promote male somatic cell fate, while tra-2 encodes a putative transmembrane receptor for the HER-I protein. The egl-1 gene is generally required for the cell-killing in nematodes but is not involved in sex determination. Thus her-1 and tra-2 mediate a novel signal transduction pathway that integrates into the cell-death pathway through the egl-1 gene to control HSN cell death. In order to identify new components in this signaling pathway, we have performed several genetic screens to isolate suppressors of inappropriate HSN cell death caused by loss-of-function mutations in the tra-2 gene. We expect to isolate mutations in genes that control general sex determination, genes that control general programmed cell death, and genes that are involved in specifying the death of HSN neurons in males. So far, we have isolated more than forty mutations. One of them is an allele of the ced-4 gene, a general cell-death gene. Eleven mutations result in fem phenotypes and are likely to be alleles of fem genes. Several mutations seem to affect only HSN cell death and are likely to affect genes that are involved in HSN death specification. We are focusing on studying these HSN-specific mutations.

24 May 1999 15:50 813 813 The Cloning of laf-1: A gene involved in the translational regulation of tra-2

1999 International Worm Meeting abstract 762 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Cloning of laf-1: A gene involved in the translational regulation of tra-2 S Shah, LE Graves, EB Goodwin Northwestern University Medical School, Lurie Cancer Center, Chicago IL 60611 tra-2 is required for development of female cell fates in C. elegans. It encodes a transmembrane protein that must be repressed in order for male cell fate specification to occur in males and hermaphrodites. tra-2 is translationally regulated via two regulatory elements (TGEs) in its 3’ untranslated region. It has been shown that a factor, DRF, binds specifically to the TGEs, and is thought to negatively regulate translation of tra-2. The sex determining gene, laf-1, is required for negative regulation of tra-2 translation and may encode a component of DRF. laf-1(lf) /+ mutations result in the feminization of hermaphrodite and male animals, and disrupt the control of a reporter transgene by the TGEs. laf-1 homozygous animals die, suggesting that laf-1 may regulate other mRNAs besides tra-2. The molecular identification of laf-1 should help to elucidate its role in the translational control of tra-2 and give insight into other potential regulatory functions of laf-1 in C. elegans development. We are currently using germline transformation rescue of the laf-1 lethal phenotype to clone laf-1. We have mapped laf-1 to chromosome III by linkage analysis. An 8.5kb cosmid, T02B2, was found to rescue the laf-1 lethal phenotype. Furthermore, transgenic animals containing a 2.4kb subclone of T02B2 also rescues the laf-1 lethality. Interestingly, this region of DNA does not appear to contain any large open reading frames or sequence homology to any known genes. Northern Analysis has yielded preliminary evidence that laf-1 may correspond to a 1.2kb transcript. RT-PCR and sequencing of laf-1 null embryos is currently underway to identify the molecular lesion.

24 May 1999 15:50 814 814 Identification of novel C. elegans caspases, their regulators and targets

1999 International Worm Meeting abstract 763 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification of novel C. elegans caspases, their regulators and targets S Shaham, I Herskowitz, CI Bargmann UCSF, 513 Parnassus Avenue, San Francisco, CA 94143 Programmed cell death (PCD) in metazoans is mediated by proteases of the caspase family. These proteases are synthesized as proproteins that are processed, often by other caspases, to form the mature protease. Mutations in the C. elegans caspase gene ced-3 prevent normally occurring PCD, suggesting that this gene plays an essential role in this process. To understand how CED-3 promotes cell death we are interested in defining regulators and targets of CED-3 activity. Using sequences from the C. elegans genome project and standard hybridization experiments we identified three C. elegans caspase-related genes in addition to ced-3. These genes encode alternate transcripts, suggesting that worms may contain a repertoire of at least seven caspase-related proteins (CSP-1A, CSP-1B, CSP-1C, CSP-2A, CSP-2B, CSP-3, and CED-3). Since mammalian caspases can act in proteolytic cascades, we examined whether worm caspases can process each other in vitro. We found that active CSP-1B cleaved proCSP-1B, proCED-3 and proCSP-2B, and that active CED-3 cleaved proCED-3 and proCSP-2B. These experiments suggest that CSP-1B may regulate proCED-3 activation and that CSP-2B may be a target of CED-3. To further understand the functions of csp-1, csp-2, and csp-3 we are in the process of generating mutations in these genes. We have also been searching for CED-3 regulators and targets using a novel screen employing the yeast S. cerevisiae. We found that CED-3 expression in yeast results in growth arrest that is dependent on CED-3 protease activity and is blocked by known caspase inhibitors. Thus, we have constructed a C. elegans yeast expression library to look for genes that inhibit CED-3-mediated growth arrest of yeast. Such genes could represent inhibitors of CED-3 activity or processing, or targets of CED-3 activity. We have isolated 15 different cDNAs so far. Further characterization of the respective genes is ongoing.

24 May 1999 15:50 815 815 cip-1, a C. elegans gene that can activate the programmed cell death pathway

1999 International Worm Meeting abstract 764 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. cip-1, a C. elegans gene that can activate the programmed cell death pathway S Shaham, CI Bargmann UCSF, 513 Parnassus Avenue, San Francisco, CA 94143 Programmed cell death (PCD) is a common metazoan cell fate. In C. elegans 12% of the somatic cells that are born undergo PCD and half of the germ cells born have a similar fate. Excess or insufficient PCD can lead to a variety of human diseases. Three gene families regulate PCD. In C. elegans these are represented by ced-9 (a bcl-2 family member), ced-4 (similar to human Apaf-1), and ced-3 (a caspase protease). Although much is known about the machinery that executes PCD, only in a few instances have the signal transduction pathways leading to activation of this machinery been elucidated. We are interested in defining molecular signaling events that trigger PCD activation in worms. As a first step we identified a novel gene (cip-1) whose protein product interacts with the CED-9 protein. CIP-1 (CED-9 interacting protein) binds to CED-9 in a two-hybrid assay. Precipitation experiments using GST-CED-9 and 35 S-labelled CIP-1 also indicate that these proteins may interact. Reminiscent of C. elegans EGL-1, CIP-1 may contain a BH3 domain (bcl-2 homology domain 3) mediating interaction with CED-9. To understand CIP-1 function we expressed the protein using C. elegans heat-shock promoters. Animals expressing HS-CIP-1 are inviable. This inviability is rescued by ced-3 mutations. Thus, CIP-1 may act upstream of the PCD machinery to regulate PCD activation. In addition to characterizing cip-1, we are screening for new genes and characterizing known genes required for the death of the male linker cell. This cell undergoes PCD in the L4/Adult molt after its role in gonadogenesis. Ablation experiments suggest that adjacent cells may trigger this death. Thus, mutations in genes required for signal transduction events that trigger linker cell death should result in its survival in the adult. To score for such survival we are using a lag-2::GFP reporter gene (generously provided by J. Kimble) that is expressed in the linker cell.

24 May 1999 15:50 816 816 Regulation of Body Size by TGF-beta-like Signaling in C. elegans

1999 International Worm Meeting abstract 765 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Regulation of Body Size by TGF-beta-like Signaling in C. elegans AN Shajahan, SE Baird Department of Biological Sciences, Wright State University Body size in C. elegans is regulated by DBL-1, a BMP homolog (Suzuki et al., Development 126: 241-250, 1999). DBL-1 signals through DAF-4 and SMA-6, the type II and the type I receptors, respectively, and the extracellular signal is transduced into the nucleus via SMA-2, SMA-3 and SMA-4, SMAD proteins (Savage et al., PNAS 93: 790-794, 1996; Krishna et al., Development 126: 251-260). Therefore, in vivo expression patterns of the proteins are expected to indicate when and where the TGF-b-like signaling is required to regulate body size. DBL-1 and DAF-4 are expressed primarily in the neurons in late embryos, larvae and adults (Patterson et al., Gen. & Dev. 11: 2679-2690, 1997; Suzuki et al., 1999). Accordingly, in a temperature shift experiment, where daf-4(m592);daf-3 hermaphrodites were shifted between permissive (15 °C) and restrictive (25 °C) temperatures at various stages of development, we have found that DAF-4 is required from late embryonic stages through adulthood. To investigate the expression pattern of SMA-2, we shall construct a functional sma-2::gfp reporter plasmid and rescue wildtype body size in sma-2 mutants. In order to be consistent with with its role as a signal transducer, the requirement for SMA-2 expression is expected to be cell autonomous. The temporal and spatial expression of SMA-2, as compared to that of DBL-1 and DAF-4 will help locate the signaling and responding tissues. Therefore, our investigation attempts to demonstrate which tissues in C. elegans affect or is affected by regulation of body size by the TGF-b-like signaling.

24 May 1999 15:50 817 817 Progress towards cloning smg-6

1999 International Worm Meeting abstract 766 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Progress towards cloning smg-6 H Shang, P Anderson Department of Genetics, University of Wisconsin-Madison Eukaryotic transcripts that contain premature stop codons are less stable than their wild-type counterparts, a phenomenon termed nonsense-mediated mRNA decay (NMD). NMD in C. elegans requires functions of seven smg genes. smg-6 is unique among described smg genes, in that it is likely to be an essential gene. Among eight smg-6 alleles isolated in a "non-complementation" screen, three are lethal when homozygous. Thus, smg-6 may have functions in addition to its role in NMD, which is known to be a nonessential process. We have mapped smg-6 to an approximately 4 map unit interval between dpy-1 and rP18 by analyzing 200 crossovers in the dpy-1 to laf-1 region. rP18 is contained in the middle of YAC Y71H2, whose sequence has not yet been assembled. laf-1 is located near the right end of Y71H2. dpy-1 is approximately 8 map units to the left of laf-1, but it has not yet been placed on the physical map. Based on our mapping data, smg-6 is most likely near the left end of Y71H2, in the region of overlap with YAC Y54H10. Southern blot analysis using cosmids from this region detect a DNA rearrangement in smg-6(r1217), one of the lethal smg-6 alleles. Experiments to pinpoint smg-6 within this promising region by DNA transformation are currently in progress.

24 May 1999 15:50 818 818 Studies of the mechanisms and possible roles of LIN-12 post-transcriptional downregulation during VPC specification.

1999 International Worm Meeting abstract 767 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Studies of the mechanisms and possible roles of LIN-12 post-transcriptional downregulation during VPC specification. DD Shaye 1 , I Greenwald 2

1 Dept. of Genetics and Development, Columbia University, New York, NY 10032. 2 HHMI and Dept. of Biochemistry, Columbia University, New York, NY 10032.

In wild-type hermaphrodites, each vulval precursor cell (VPC) adopts one of three distinct fates, 1°, 2° or 3°, in a precise and invariant spatial pattern (reviewed in 1). Analysis of mutants defective in vulval development have suggested that at least three different signaling events are involved in the correct specification of VPC fates. An inhibitory signal originates in the hypodermal syncytium. An inductive signal originates in the gonadal anchor cell and allows VPCs to overcome the inhibitory signal leading to the induction of vulval fates. Finally, a lateral signal originates in the presumptive 1° cell and is received by the presumptive 2° cells through LIN-12. Recent studies using a lin-12::gfp fusion have shown that one output of the inductive signal is to influence LIN-12 protein accumulation (2). In wild-type hermaphrodites, all six VPCs initially express LIN-12::GFP. However, LIN-12::GFP accumulation is reduced specifically in P6.p at the time of vulval induction, although expression of the lin-12::lacZ reporter remains constant (3). The inductive signal seems to be necessary and sufficient for this downregulation to occur. These results have led to the proposal that the downregulation of LIN-12 in the presumptive 1° cell is at least one component of the mechanism by which the inductive signal imposes a bias on lateral signaling. We are interested in elucidating the mechanism by which the inductive signal controls the downregulation of LIN-12 in P6.p and whether this downregulation plays a role in correctly generating a lateral signal. In order to answer these questions we have taken two approaches. First, we are examining the effects of mutations in the inductive pathway and in the known targets of this pathway on LIN-12::GFP downregulation. Second, we will determine whether the control of LIN-12::GFP downregulation occurs at the translational or posttranslational level by identifying the region of lin-12 (e.g., 3’ UTR or protein sequences) that targets LIN-12::GFP for downregulation in P6.p.

1) I. Greenwald, in C. elegans II. CSH Press, Cold Spring Harbor, NY, (1997). 2) D. Levitan and I. Greenwald, Development 125, 3101 (1998). 3) H. Wilkinson and I. Greenwald, Genetics 141, 513 (1995).

24 May 1999 15:50 819 819 The Nonmuscle Myosin Regulatory Light Chain Gene mlc-4 is Required for Cytokinesis, Anterior-Posterior Polarity, and Body Morphology during C. elegans Embryogenesis

1999 International Worm Meeting abstract 768 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Nonmuscle Myosin Regulatory Light Chain Gene mlc-4 is Required for Cytokinesis, Anterior-Posterior Polarity, and Body Morphology during C. elegans Embryogenesis CA Shelton, JC Carter, BA Bowerman Institute of Molecular Biology, University of Oregon, Eugene, OR. 97403-1229 Using RNA-mediated genetic interference (RNAi) in a phenotypic screen, we identified the sole conserved nonmuscle myosin II regulatory light chain (nmRLC) gene in Caenorhabditis elegans, which we name mlc-4. Maternally-supplied mlc-4 function is required for cytokinesis during both meiosis and mitosis, and for establishment of anterior-posterior (a-p) asymmetries after fertilization. Reducing the function of mlc-4 or nmy-2, a nonmuscle myosin II gene (1), also leads to a loss of polarized cytoplasmic streaming in the C. elegans zygote, supporting models in which cytoplasmic flow may be required to establish a-p differences (2). However, germline P-granules are normally localized to the posterior despite these defects. Because P-granule localization requires an intact actin cytoskeleton (3), these results suggest that an actin-dependent mechanism other than cytoplasmic flow or mlc-4/nmy-2 activity generates some anterior-posterior asymmetries in the C. elegans zygote. To determine if mlc-4 has essential zygotic functions, we isolated a deletion allele. We show that removing zygotic mlc-4 function results in a partial elongation phenotype during embryogenesis. Moreover, A mlc-4::GFP transgene is expressed in lateral rows of hypodermal cells, and these cells fail to properly change shape in mlc-4 mutant animals during elongation. This phenotype is similar to that obtained from let-502 mutations and we suggest that the previously described let-502/mel-11 regulatory pathway (4) may interact with MLC-4 to generate contractile forces required for proper elongation of the embryo.

1. Guo, S. & Kemphues, K.J. Nature 382:455-458 (1996). 2. Goldstein, B. & Hird, S.N. Development 122:1467-1474 (1996). 3. Strome, S. & Wood, W.B. Cell 35:15-25 (1983). 4. Wissmann, A., Ingles, J., McGhee, J.D. & Mains, P.E. Genes Dev 11: 409-422 (1997).

24 May 1999 15:50 820 820 Genetic characterisation of lon-4 gene

1999 International Worm Meeting abstract 769 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic characterisation of lon-4 gene ZZ Shen, MN Patel, AM Leroi Imperial College of Science, Technology and Medicine The genetic specification of body size is not well understood in any metazoan. Recent studies revealed that the TGF-b signalling pathway is involved in the control of body size in Caenorhabditis elegans. Mutations in the components of the TGF-b pathway, which result in a small body size, have been identified in daf-4, sma-2, sma-3, sma-4, sma-6, dbl-1/cet-1 (Estevez et al., 1993; Savage et al., 1996; Suzuki et al., 1999; Morita et al., 1999). Evidence from other studies has suggested that lon genes are also involved in this signalling pathway. For example, when the ligand dbl-1/cet-1 is overexpressed, a lon phenotype occurs. Further, epistasis studies have indicated that lon-1 is epistatic to sma-2, sma-3, sma-4 and daf-4, while lon-2 and lon-3 are upstream to the sma genes (Savage et al., pers.comm.). We conducted a mutational screen to collect different body size mutants. Out of 50,000 genomes screened, 21 lon mutants were obtained after backcrossing. Twelve of these were allelic to known lon loci, lon-1, lon-2 and lon-3. Nine mutants complemented these known lon loci and were assigned to five new loci. Four mutants were in the same locus on chromosome II, which we called lon-4. Two-factor and three-factor crosses indicated that lon-4 is in the right side of chromosome II (genetic position is 1.44 ±0.44). Epistasis studies have shown that sma-2 and daf-4 are epistatic to lon-4. Molecular cloning of lon-4 is in progress.

24 May 1999 15:50 821 821 Global analysis of ABC transporters in C. elegans

1999 International Worm Meeting abstract 770 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Global analysis of ABC transporters in C. elegans JA Sheps 1 , M Yeung Chau 1 , V Ling 1 , DL Baillie 2

1 British Columbia Cancer Research Agency, 610 West 10th Ave. Vancouver, BC, Canada. 2 Insitiute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada.

ABC transporters are a group of ATP Binding Cassette proteins highly conserved through all kingdoms of life. ABC transporters may be grouped into several distinct structural classes on the basis of amino acid sequence and domain organisation. ABC transporters play a role in the high level of resistance of C. elegans to a wide range of environmental toxins. Among these is the P-glycoprotein (Pgp) subclass, whose members are involved in transport of hydrophobic compounds and as such function in defence of the body from toxic natural products in the diet. P-glycoproteins are also a critical component of the mammalian blood-brain barrier and their function is necessary for tolerance of drugs that are normally minimally toxic to mammals, such as ivermectin. Strains of worm carrying Pgp mutants have demonstrated enhanced sensitivity to natural pathogens of C. elegans such as Pseudomonas aeruginosa (Mahajan-Miklos et al., (1998) Cell 96:47-56). We estimate that there are more than 60 ABC transporters in the Caenorhabditis elegans genome. The functions of most of these are unknown. We have completed a phylogenetic analysis of all these ABC transporter genes and we find that the P-glycoprotein subclass in C. elegans contains 15 genes. Previously only 4 Pgps had been named in C. elegans, and only two of these have been knocked out (Broeks et al., (1995) EMBO J. 14:1858-66). Production of knockout mutations in all 15 genes is in progress through the auspices of the international C. elegans gene knockout consortium. We are screening the P-glycoprotein knockout worms for potential drug sensitivity phenotypes using an assay which monitors the uptake and distribution of fluorescent drugs. Results so far indicate that the pgp-1 knockout, previously described as lacking a phenotype, does alter the distribution of the fluorescent drug rhodamine 123 within the body of the worm, and is thus a candidate drug resistance gene also. This approach, when extended to all ABC transporters, is expected to ultimately identify all such genes involved in the resistance of C. elegans to environmental insult.

24 May 1999 15:50 822 822 Involvement of cog-1 in uterine-vulval attachment

1999 International Worm Meeting abstract 771 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Involvement of cog-1 in uterine-vulval attachment DR Sherwood, RA Palmer, PW Sternberg California Institute of Technology, HHMI/Division of Biology 156-29, Pasadena, CA 91125 We are interested in understanding general cellular and molecular mechanisms underlying tissue remodeling and morphogenesis. Towards this goal, we are examining the attachment of the uterus and vulva during C. elegans development. Uterine-vulval connection requires the breakdown of basement membranes, the process of cellular invasion, cell fusions and the formation of de novo adhesions between cells. We have previously identified a mutant, cog-1 (connection of gonad), in which there is a complete lack of attachment between the uterus and vulva. The first observable defect in uterine-vulval attachment in cog-1 mutant hermaphrodites is the failure of the uterine anchor cell to penetrate and initiate contact with the vulval epithelium. The cog-1 gene encodes a homeodomain transcription factor that likely regulates the expression of other genes necessary for uterine-vulval attachment. A transgenic cog-1::GFP reporter protein is expressed in both uterine and vulval cells during the connection process. To better understand the role of COG-1 in uterine-vulval attachment we are examining cellular aspects of anchor cell invasion into the vulval epithelium in both wild-type and cog-1 mutant hermaphrodites. Furthermore, we are performing a genetic mosaic analysis to determine whether COG-1 function is required in the uterus, vulva or both tissues for a proper connection to form.

24 May 1999 15:50 823 823 Possible involvement of octopamine in the hyperactivity caused by feeding defects

1999 International Worm Meeting abstract 772 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Possible involvement of octopamine in the hyperactivity caused by feeding defects Y Shibata, T Fujii, H Fujisawa, S Takagi Division of Biological Science, Nagoya University Graduate School of Science, Nagoya, 464-8602, Japan In C.elegans, starvation is known to alter several behaviors, including olfactory adaptation and feeding. Feeding-defective mutants can be useful tools to analyze behaviors affected by starvation. We have been analyzing locomotory activity of eat mutants. We have identified a novel eat locus, eat-20, by a reverse genetic approach. In eat-20 mutants, the pharyngeal pumping rate was slowed and bacterial transport was inefficient, and the mutants exhibited the starved appearance typical of eat mutants. We have noticed that eat-20 mutants were hyperactive in locomotion. Most of previously identified eat mutants, such as eat-2, 4, 10, 11, 13 and 15, were also hyperactive in the presence of food. The extent of increase of the locomotory activity generally correlated with the strength of Eat phenotype of the mutants. These results suggest that hyperactivity is caused by feeding defects. Octopamine was reported to have effects that were similar to those of the depletion of food on several behaviors of worms. We have revealed that exogenous octopamine, in the presence of food, enhanced the activity of wild type animals to the level similar to that of strong Eat mutants. Octopamine did not further enhance the activity of strong Eat mutants. Phentolamine, which is known to block an octopamine receptor in the locust, did not affect the activity of wild type worms in the presence of food, but decreased the activity of eat mutants to the level of untreated wild type animals. We propose a model that , in eat mutants, starvation promotes the release of octopamine which in turn activates a neural pathway controlling locomotion. Together with the previously reported observations, our results suggest that octopamine transmits a starvation signal in C.elegans.

24 May 1999 15:50 824 824 Molecular Interaction of Myotonic Dystrophy Protein Kinase: The Structure and Function of DMPK Family Members in C. elegans.

1999 International Worm Meeting abstract 773 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Molecular Interaction of Myotonic Dystrophy Protein Kinase: The Structure and Function of DMPK Family Members in C. elegans. M Shimizu 1 , HF Epstein 2

1 Department of Developmental Biology, National Institute for Basic Biology, Okazaki, JAPAN. 2 Department of Neurology, Baylor College of Medicine, Houston, Texas, USA.

Myotonic Dystrophy Protein Kinase (DMPK) is a Ser/Thr protein kinase and it represents a new group of proteins called "the DMPK family". We are interested in the molecular interactions of DMPK in several model systems, including the structure and function of DMPK family members in C. elegans. There are two C. elegans homologues with respect to the catalytic domain have been identified through sequencing the entire C. elegans genome. The closest homologue for DMPK is the predicted product of a locus on chromosome V that we call CeDMPK which has 53.5% identity in its catalytic domain to that of human DMPK. In addition to its DMPK-like ser/thr kinase domain, CeDMPK contains four other functional domains: a large coiled-coil region, a cysteine-rich zinc-finger domain (CRD), a pleckstrin-homology motif (PH), and a Cdc42/Rac binding domain (CRIB). To study the temporal and spatial expression pattern of CeDMPK in C. elegans, a transgenic line was established by injecting the gfp expression vector which contains a 1.9 Kb upstream of the start codon through a part of exon 3 of CeDMPK genomic DNA fused in frame to the GFP protein. We found GFP expression at different cell types through the developmental stages including at pharyngeal muscles at all developmental stages from late embryos to adults, in body-wall muscles at L2-L3, and the vulval precursor descendants (hypodermal cells) at L3-L4. Since the expression pattern is similar to egl-19 (a1 subunit of an L-type voltage-activated calcium channel), this result maybe consistent with a possible involvement of the the human DMPK in the calcium homeostasis. Potential knockout phenotypes obtained by RNA-mediated inhibition include protruding vulva, abnormal morphology at pharynx, production of a few progeny, and Lumpy-Dumpy phenotype which suggest the possible roll of CeDMPK in morphological development of the worm. Isolation of CeDMPK null mutant is underway to apply the powerful genetic study of C. elegans to clarify the regulators of DMPK in the cell signaling cascade.

24 May 1999 15:50 825 825 Characterization of Flectin -like protein in C.elegans

1999 International Worm Meeting abstract 774 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of Flectin -like protein in C.elegans Jiyeon Shin, Sunja Kim, Jungsu Lee, Joohong Ahnn Dept. of Life Science. Kwangju Institute of Science and technology, Kwangju 500-712, Korea Flectin is a new extracellular matrix protein which was identified from eye extract of chick embryo. Homologous proteins were found in many other vertebrates suggesting that flectin may be an evolutinary conserved protein. A flectin-like gene (ZK783.4) was found in C. elegans genome data base, showing approximately 40% similarity (20% identity) to chick flectin. In order to examine the localization of this gene, reporter gene fused with this gene were expressed in germ-line transformed transgenic animals. Green Fluorescent protein was expressed in various neurons, hypodermis and distal tip cells from early embryonic stage throughout larval and adult stages. Interestingly, strong expression patterns were observed in neuronal cells and hypodermal cells which compose the ectodermal tissue during early embryogenesis. This observation suggest that flectin-like protein may be important for the development of hypodermis and neurons. In addition, majority of cells expressing reporter gene are migrating cells during development. In order to study its function , we are preparing C.elegans flectin antibodies. We have screened for deletion mutant by treating worm with EMS and isolated a deletion mutant candidate. We are currently following this deletion mutant candidate in order to characterize its function in C.elegans.

24 May 1999 15:50 826 826 Zinc-finger and KH domain Proteins in C. elegans Embryogenesis

1999 International Worm Meeting abstract 775 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Zinc-finger and KH domain Proteins in C. elegans Embryogenesis T Shin, CC Mello U. Mass. Med. Center, Worcester, MA In the early embryo, the expression of maternally encoded proteins is controlled both spatially and temporally. Two groups of maternal proteins have been implicated in this process. The first consists of Cys3-His zinc-finger proteins including PIE-1, MEX-1, MEX-5/AH6.5 and POS-1. The second class consists of KH domain proteins including ALP-1, MEX-3 and GLD-1. Mutations in most of these genes cause embryonic cell fate transformation, and in some cases, are correlated with clear misexpression of maternal proteins. Both Cys3-His zinc-fingers and KH domains are conserved in a wide variety of organisms and in some cases are implicated in direct RNA binding, raising the possibility that these two families of proteins directly regulate maternal mRNA translation. We identified ALP-1 as a two-hybrid interacter of PIE-1. This interaction requires a part of ALP-1 KH domain and a C-terminal region in PIE-1 outside of its zinc-fingers. We also found that while ALP-1 binds only to PIE-1 among all zinc-finger proteins tested, other KH domain and zinc-finger proteins can interact with multiple partners. For example, GLD-1 can bind strongly to POS-1 and weakly to PIE-1, and POS-1 can interact with MEX-3. These data suggest that zinc-finger/KH domain proteins may function together as a complex and/or regulate each other’s activity. Consistent with this view, gld-1(RNAi) produces a no-gut defect very similar to that caused by pos-1 mutations. Furthermore, alp-1 interacts genetically with pos-1 and gld-1, completely suppressing the no-gut phenotype in both. Thus, the two protein families appear to regulate each other in a complex manner. How do zinc-finger/KH domain proteins function in controlling maternal mRNA translation? Do they directly recognize RNA? What are the biochemical bases for their apparently complex interactions both physically and genetically? Finding answers for these questions will require in vivo biochemical analyses. Toward this end, we have generated transgenic animals that stably express functional HA-tagged PIE-1 protein. We are currently examining in vivo protein-protein interaction between HA-PIE-1 and other members of the two protein families. These studies should shed light on biochemical and physiological roles for the evolutionarily conserved zinc-finger/KH motif proteins.

24 May 1999 15:50 827 827 NEXTDB: The nematode expression pattern map database.

1999 International Worm Meeting abstract 776 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. NEXTDB: The nematode expression pattern map database. T Shin-i, Y Kohara CREST, JST and Genome Biology Lab., National Institute of Genetics, 1111 Yata Mishima 411-8540, Japan We have developed a WWW-based database "NEXTDB" to integrate all the information of ESTs and gene expression patterns of C.elegans which are being produced and analyzed in this laboratory. Raw data of tag sequencing are processed by removing ambiguous regions which contain larger rate of "N" (ambiguous base), cloning vectors, and poly-A tails. The process 3’-tags are classified unique cDNA groups applying a cumulative method by use of FASTA. Both 5’- and 3’-tag sequences are mapped to cosmid sequences by use of BLASTN, then the results are compared to the cDNA groups to confirm the positions of the groups on the genome. BLASTX searches are also done to confirm homologies of predicted CDSs to genes of other organisms. All the above processes are done automatically. Images of whole mount in situ hybridization which show the expression patterns of individual cDNA groups are loaded to NEXTDB. Then they are annotated with respect to developmental stages and expression patterns on the database. Once annotated, the images are arranged properly along development on the screen. In order to integrate NEXTDB, we applied a hierarchical model to arrange all the clones and cDNA groups; 1) chromosome, 2) cosmid clone, 3) CDS, 4) cDNA group, and 5) cDNA clone. The cosmid map which connects 1) and 2) are obtained from AceDB. The relations about cosmids and their CDS are retrieved from the annotations of the Sanger Centre sequence data. All of the information are linked each other in NEXTDB and displayed visually by use of JAVA applets. The latest version is available through the following URL. http://watson.genes.nig.ac.jp:8080/db/index.html

24 May 1999 15:50 828 828 A Screen for Mutants with Abnormal Neural Morphology

1999 International Worm Meeting abstract 777 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A Screen for Mutants with Abnormal Neural Morphology G Shioi 1 , M Shoji 1 , M Nakamura 1 , T Ishihara 2 , I Katsura 2 , H Fujisawa 1 , S Takagi 1

1 Laboratory of Neuroscience, Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan. 2 Structural Biology Center, National Institute of Genetics, Mishima, 411-8540, Japan.

To identify genes involved in the morphogenesis of the nervous system, we have been screening for mutants with morphological defects in the nervous system. In this study, larval lethal mutants with neural defects were screened to identify efficiently novel genetic loci. For the purpose of easily visualizing the neural morphology, we made a transgenic strain which expressed GFP in almost all neurons. We have mutagenized the strain(P0) with EMS, and have screened F2 progenies of singled F1 progenies. We have isolated 25 mutants which had abnormal ventral nerve cords. The ventral cords of these mutants often ran in abnormal paths, defasciculated, and sometimes were detached from the body wall when mutant worms moved. Motoneurons on the ventral cord were sometimes misplaced on the lateral body. In addition to the neural defects, some mutant worms also had defects in other tissues. The excretory canals extended in abnormal paths and were meandering. Body wall muscles were detached from the body wall. Most mutant worms could not move, and lay in the shape of coils or circles on the plates. We suspect that the detachment of the body wall muscles causes mutant worms to show Unc phenotype. The mutants that we have isolated were classified into at least six non-complementation groups by genetic mapping and complementation tests. Some mutations were mapped to the known loci such as mua-1, but others were mapped to two novel loci which we tentatively named vec-1(ventral nerve cord abnormal) and vec-2. We are now trying to rescue vec-1 and vec-2 mutatnts with cosmid clones.

24 May 1999 15:50 829 829 C.elegans Kinesin Superfamily: Subfamilies and Surprises

1999 International Worm Meeting abstract 778 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. C.elegans Kinesin Superfamily: Subfamilies and Surprises SS Siddiqui 1,2 , MY Ali 1 , ST Khan 1 , ML Khan 3 , F Hori 1 , T Harada 1 , K Nishikawa 4 , KF Kobayashi 4 , ZK Siddiqui 1

1 Toyohashi University of Technology, Mishima 411, Japan. 2 Pharmacology Dept, UIC Medical College, Chicago, IL 60612. 3 National Instt. of Biosci.and Human Tech., Tsukuba,J apan. 4 National Institute of Genetics, Mishima 411, Japan.

Movement defines life. In the life of a eukaryote two major systems provide intracellular transport, using actin based myosin and microtubule based kinesin motors. The hallmark of kinesins is a 350 residue globular motor domain that contains the highly conserved ATP binding and micortubule binding sites. We have generated degenerate primers corresponding to these conserved sites and used PCR amplification to screen nematode cDNA and genomic libraries to clone and characterize cDNAs encoding more than 20 C. elegans kinesins (Khan et al., 1997; 1999). Phylogenetic tree analysis shows that the nematode kinesins can be grouped into nine distinct groups. Group-I (KHC, UNC-116); Group-II (UNC-104, KIF-1); Group-III (OSM-3, KIF3A/B); Group-IV (CEKLP-12, Chromokinesin); Group-V (CEKLP-14, DmKRP130); Group-VI (CEKLP-3; DmNcd); Group-VII (CEKLP-7, MCAK); Group-VIII (CEKLP-8, MKLP1), Group-IX (VAB-8; outgroup), representing all major classes of kinesins found in mammals. Using cellular and molecular approaches we have deduced that most of the kinesins are required for embryogenesis, such as in chromosomal movement. Post-embryonically tissue expression becomes more specific, e.g. osm-3 is specific to a class of chemosensory neurons (Tabish et al., 1995), khc unc-116 is expressed in motor neurons and musculature (Patel et al., 1993; Ali et al., 1999), unc-104 is involved in synaptic vesicle transport (Hall and Hedgecock, 1991; Otsuka et al., 1991), and the vab-8 is involved in axonal migration, and morphogenesis (Wolf et al., 1998). Analysis of the double mutants constructed with various kinesin mutant alleles reveal specific genetics interactions between nerve specific kinesins . These results allow us to propose a novel hypothesis regarding the in vivo function of different kinesins during development. which will be discussed.

We thank S. Endow, L.S. B.Goldstein, G. Garriga, D. Hall, N. Hirokwa, D. T. Mieg, A. Otsuka, J. Scholey, S. Strome, J. White, for encouragement in this project. Funds were provided by Monbusho, Japan to SSS.

24 May 1999 15:50 830 830 A PP2A regulatory subunit positively regulates Ras-mediated signaling during C. elegans vulval induction

1999 International Worm Meeting abstract 779 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A PP2A regulatory subunit positively regulates Ras-mediated signaling during C. elegans vulval induction DS Sieburth 1 , M Sundaram 2 , M Han 1

1 HHMI and Department of MCD Biology, University of Colorado, Boulder, CO 80309. 2 Department of Genetics, UPenn School of Medicine, Philadelphia, PA 19104-6145.

We describe evidence that a regulatory B subunit of PP2A (PP2A-B), encoded by the sur-6 gene, positively regulates a RTK-Ras-MAP kinase signaling cascade during Caenorhabditis elegans vulval induction. Although there is no phenotype on an otherwise wild type background, loss of sur-6 PP2A-B function suppresses the phenotype caused by an activated Ras mutation. Furthermore a loss-of-function sur-6 PP2A-B mutation enhances vulval induction defects associated with mutations in lin-45 raf, sur-8 or mpk-1 MAP kinase. However, a sur-6 PP2A-B mutation, unlike a sur-8 mutation, fails to enhance vulval cell fate specification defects in a ksr-1 mutant background. Genetic epistasis analysis suggests that, like ksr-1, sur-6 PP2A-B acts downstream of or in parallel to ras and upstream of raf. Interfering with PP2A catalytic subunit expression has the same qualitative effect on ras signaling as sur-6 mutations, suggesting that PP2A has a positive regulatory role in ras-mediated signal transduction. Therefore, we propose that SUR-6 PP2A-B stimulates Ras pathway signaling by specifically regulating PP2A catalytic function and that PP2A acts together with KSR-1 to possibly up-regulate LIN-45 Raf activity.

24 May 1999 15:50 831 831 The gonad as fertile ground for genetic studies of organogenesis

1999 International Worm Meeting abstract 780 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The gonad as fertile ground for genetic studies of organogenesis KR Siegfried 1 , F Markussen 1 , LD Mathies 1 , LC Friedman 1 , P Kroll-Conner 1,2 , J Kimble 1,2

1 University of Wisconsin-Madison, Madison, WI 53706. 2 HHMI, Madison, WI 53706.

We have focused on the early events of gonadogenesis in C. elegans to understand how organ primordia are established and how organ polarity can be specified. Early gonadogenesis occurs in three phases. During embryogenesis, the somatic gonadal precursors migrate and embrace the germline precursors to form the 4-celled gonad primordium. During L1 and early L2, cell divisions generate 12 somatic gonadal cells in hermaphrodites and 10 in males. Finally, during either L1 (males) or late L2 (hermaphrodites), the somatic gonadal cells migrate and rearrange themselves to establish a somatic gonadal primordium (SGP), which prefigures the pattern of the adult gonad. The cells of the 4-celled gonadal primordium are arranged with two-fold rotational symmetry in both sexes. In the hermaphrodite, SGP formation requires migration of the somatic gonadal cells into a central cluster that retains two-fold rotational symmetry. This symmetry is maintained throughout hermaphrodite development generating the two-armed gonad. In males, symmetry is broken, and formation of the male SGP involves migration of the somatic gonadal cells into an asymmetric cluster at the anterior end. Thus, in both sexes, cell rearrangements define the final polarity and structure of the organ. We have set out to dissect the genetic controls of these early events of gonadogenesis. To this end, we have isolated 55 mutants with defects in early gonadogenesis. Two mutants affect primordium assembly during embryogenesis, one mutant fails to break gonadal symmetry in males, and the rest have defects in SGP formation in hermaphrodites. We will report on our progress in analyzing these mutants.

24 May 1999 15:50 832 832 Analysis of the ventral enclosure mutant ct254

1999 International Worm Meeting abstract 781 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of the ventral enclosure mutant ct254 KA Simokat 1 , JS Simske 1 , L Edgar 2 , WB Wood 2 , JD Hardin 1

1 Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, WI 53706. 2 Department of MCD Biology, University of Colorado, Boulder, CO 80309.

We are interested in the mechanisms of epithelial morphogenesis, with the goals of understanding how specific molecules affect the shape, migration, and adhesiveness of epithelial sheets and thereby generate three-dimensional structure. In particular, we would like to identify the molecules that play a direct role in ventral enclosure. Ventral enclosure is the process by which hypodermal cells originating on the dorsal surface of the embryo migrate along the exterior, and eventually form adhesive junctions along the ventral midline. We have identified a mutant, ct254, that shows defects in the process of ventral enclosure. ct254 is a psoralen induced mutant identified in a screen for lethal mutations on LG III that affect early posterior development. 4D time lapse recordings show that this mutant displays variable embryonic lethal phenotypes, which include: 1) completely unenclosed embryos in which the hypodermis retracts to the dorsal surface, 2) head enclosure defects as a result of incomplete enclosure of the anterior hypodermis, and 3) a ventral midline rupture due to imperfect junction formation. The variability of phenotypes is reminiscent of the range of phenotypes observed in mutants isolated in an ongoing screen in our laboratory for ventral enclosure specific mutants (See abstract by J Simske et al). Antibody staining of ct254 embryos with MH27 (epithelial junctions), LIN-26 (hypodermal nuclei), R224 (paramyosin) show that homozygotes contain the normal complement of cells and that muscle quadrant organization appears normal. These results suggest the mutant may act in a hypodermis-specific manner. The organization of seam cells in ct254 embryos appears disorganized based on analysis of expression of JAM-1-GFP (the junctional protein identified by MH27 and GFP). Further analysis with this reporter and HMP-1-GFP (alpha-catenin GFP) are underway to determine how the migration and shape changes of the hypodermal cells during ventral enclosure are abnormal in this mutant. Currently ct254 is being mapped, and is located on the left of unc-32. The map location will be further refined in an attempt to clone the mutated gene.

24 May 1999 15:50 833 833 VAB-9 is a junction associated, putative membrane-spanning protein required for coordinated epithelial morphogenesis

1999 International Worm Meeting abstract 782 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. VAB-9 is a junction associated, putative membrane-spanning protein required for coordinated epithelial morphogenesis JS Simske, JD Hardin University of Wisconsin. Madison, WI 53706 We are interested in mutations that affect morphogenesis through the alteration of specific cell behaviors (e.g. cell motility and cell shape changes) rather than those that alter cell lineage or pattern. One such mutation is vab-9; since vab-9 mutants have tail, body and vulval morphology defects but no alterations in cell lineage. vab-9 has been cloned by cosmid rescue, cDNAs have been recovered by RT-PCR, and the molecular lesions in e1744 and ju6 ( kindly provided by A. Chisholm) have been identified. A full length vab-9 cDNA expressed under the control of a heat shock promoter rescues vab-9(e1744) phenotypes. VAB-9 is a 221aa protein that bears no significant homology to any known gene in the database but has four strongly predicted transmembrane spanning domains. A rescuing VAB-9-GFP protein localizes to cellular junctions with a pattern that bears a striking resemblance to that of JAM-1 (for junction associated molecule 1, the antigen recognized by the monoclonal antibody MH27), suggesting that VAB-9 is an integral membrane junctional protein. VAB-9-GFP does not appear to be expressed in the intestine and expression in the uterus and the male tail is currently being characterized. Based on JAM-1 expression, vab-9 larvae with body shape defects display uneven elongation. In wild-type, the seam cells of larvae normally elongate along the anterior-posterior axis and appear rectangular in shape. In contrast, the seam cells in vab-9 animals often elongate along the dorsal-ventral axis or display a circular shape. Where such defects are observed, there is a direct correlation between seam cell shape and body shape abnormalities. Thus seam cells in vab-9 animals are able to elongate, but the coordination or polarity of seam cells along the anterior-posterior axis is impaired. We propose that VAB-9 is involved in cell-cell communication during elongation, and that this communication is required for the smooth coordinated elongation of the larvae. Since the predicted topology of VAB-9 is similar to connexins or the tight junction component occludin it is possible that VAB-9 may couple hypodermal cells chemically or adhesively. We are currently testing these possibilities.

24 May 1999 15:50 834 834 A screen for extragenic suppressors of spe-9

1999 International Worm Meeting abstract 783 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A screen for extragenic suppressors of spe-9 AW Singson, SW L’Hernault Emory University, Department of Biology, Atlanta, GA 30322 Mutations in the spe-9 gene lead to the production of sperm with wild type morphology and motility that cannot fertilize eggs even after contact between gametes. The spe-9 gene encodes a sperm transmembrane protein with an extracellular domain that contains ten epidermal growth factor (EGF)-like repeats. A common feature of proteins that include EGF-like motifs is their involvement in extracellular functions such as adhesive and ligand-receptor interactions. Additionally, the overall structure of the predicted SPE-9 protein is similar to that of ligands for the Notch/LIN-12/GLP-1 family of transmembrane receptors. We have designed a screen to identify the oocyte receptor for SPE-9 and other genes required for fertilization. There are several important features of this screen. First, we are screening for new mutations that restore fertility to spe-9 mutant worms. The restoration of self-fertility to self-sterile hermaphrodites is an extremely powerful selection tool. Second, the use of temperature sensitive alleles of spe-9 permit the culture and screening of very large numbers of worms, thus increasing the chance of isolating very rare mutations. Moreover, this approach should allow the recovery of mutations not obtainable by previous screening strategies. Finally, the use of specific alleles of spe-9 with point mutations in their extracellular domains will be more likely to detect compensatory mutations in the egg receptor for spe-9. We will report our latest progress with this screen.

24 May 1999 15:50 835 835 EGO-1, required for germline development, is related to plant rna-directed rna polymerases

1999 International Worm Meeting abstract 784 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. EGO-1, required for germline development, is related to plant rna-directed rna polymerases A Smardon, JM Spoerke, SC Steven, N Mackin, EM Maine Dept. of Biology, Syracuse University, Syracuse, NY 13244 ego-1 has been described as a gene required for various aspects of germline development, including efficient DTC-to-germ line signaling (Qiao et al. ’95). More recently, we reported the cloning of what appeared to be a distinct, although very tightly linked gene, ego-6 (Smardon et al. ’98 WBG). Based on molecular studies, we can now say that ego-1 and "ego-6" are in fact the same gene. The loss of ego-1 gene function is associated with premature meiosis, occasional abnormal nuclei in the distal (mitotic) region, an expanded mitosis-to-pachytene transition zone, defects in gametogenesis, and a slight reduction in germ cell numbers. Identified by their ability to enhance a mild glp-1(ts) phenotype in the germ line, ego-1 alleles also enhance a mild lag-1(ts) phenotype in the germ line. ego-1 was placed on the physical map by identification of a deletion associated with ego-1(om84). This deletion disrupts a gene who structure subsequently was determined by recovery and sequencing of cDNAs and RT-PCR products. The gene includes 15 exons and is trans-spliced to SL1. RNAi and mutation analyses confirmed the identity of this gene as ego-1. It is predicted to encode a 1632 amino acid protein with limited homology to the products of several other eukaryotic genes (see below). Two strong mutant alleles, om84 and om97, contain extremely premature stop codons and thus are likely to be null. The weaker om71 allele contains a Pro-Ser substitution at position 930; this residue is conserved among all known EGO-1 family members. A single ego-1 transcript is detected in L4 and adult animals. It is present predominantly, if not entirely, in the germ line. Anti-EGO-1 antibodies are being generated to examine the protein expression pattern. EGO-1-related proteins include a set of RNA-directed RNA polymerases recently identified in tomato and four other plant species (Schiebel et al. ’98). Their limited homology to EGO-1 may indicate a common domain, such as one that binds RNA, rather than conserved overall function. Functions of the three ego-1 paralogs are being examined using RNAi.

Qiao et al. (1995) Genetics 141, 551-569; Smardon et al. (1998) WBG 15, 31; Schiebel et al. (1998) Plant Cell 10, 2087-2102.

24 May 1999 15:50 836 836 Genome-wide analysis of C. elegans sperm development

1999 International Worm Meeting abstract 785 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genome-wide analysis of C. elegans sperm development HE Smith, EB Davis, JF Nance, S Ward University of Arizona, Tucson, AZ 85721 Our lab is interested in the developmental processes that direct sperm formation in Caenorhabditis elegans. Prior genetic screens have identified 60 genes required for proper sperm development, and several of these fer (fertilization-defective) or spe (spermatogenesis-defective) genes have been cloned by cosmid rescue. However, analyzing the function of these genes has been hampered by the length of time required to clone each gene, and the lack of homology to genes of known function. Because all of the previously cloned fer/spe genes exhibit sperm-specific expression, the identification of those genes expressed only in the developing sperm would speed cloning and identify a subset of genes with homologs. The recent completion of the genome sequence offers the opportunity for genetic analysis on a global scale. Therefore, we are attempting to identify all of the estimated 400 genes expressed solely during sperm development by 1) sequencing clones from a sperm-specific subtracted cDNA library and 2) differential screening of microarrays (in collaboration with the laboratory of Stuart Kim). Both strategies utilize temperature-sensitive mutant hermaphrodites that produce only oocytes (fem-1) or sperm (fem-3gf) at the restrictive temperature. Because the somatic tissues are unaffected, any difference in gene expression between these two mutants is due to differences between sperm and oocyte production. We have constructed two high-quality cDNA libraries from mRNA isolated from fem-1 and fem-3gf mutants. Both libraries have been normalized to equalize the relative representation of abundant vs. rare transcripts. The fem-1 library has been subtracted from the fem-3gf library to generate a library enriched to ~20% sperm-specific genes. Reverse Northern blots with fem-1 and fem-3gf probes have been used to identify sperm-specific clones, which are being sequenced by the Genome Sequencing Center at Washington University. By comparing these results to Stuart Kim’s differential microarray screening with fem-1 and fem-3gf probes, we can assay the sensitivity and specificity of these two methods. We will also present data from our current attempts to improve germline expression of transgenic arrays as well as increased efficacy of germline RNA inactivation.

24 May 1999 15:50 837 837 When humans lead the way: uncovering the role of C27H5.1

1999 International Worm Meeting abstract 786 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. When humans lead the way: uncovering the role of C27H5.1 Jessica Smith, Michelle Chambers, Dave Pilgrim University of Alberta C27H5.1 is a weak homologue of UNC-119, a gene involved in axon guidance and outgrowth. We first became interested in C27H5.1 because of its similarity to UNC-119. We have since learned that there is a mammalian homologue of C27H5.1; rod phosphodiesterase delta subunit PDE6D. These two proteins are very highly conserved exhibiting 70% identity and 85% similarity. UNC-119 also has a close mammalian homologue; retinal protein HRG4/RRG4. Both C27H5.1 and UNC-119 are pan-neuronal in C. elegans while HRG4 and PDE6D are expressed in the mammalian retina. The conservation of these genes has led us to speculate that they may constitute a new protein family. While UNC-119 has been fairly well characterized, very little is known about C27H5.1. Based upon its conservation with UNC-119 and PDE6D, we suspect that C27H5.1 is playing an important role, perhaps in nervous system development. However, C27H5.1 may play a more general role. Unlike UNC-119, C27H5.1 expression is not limited to the nervous system. There is also expression in pharyngeal and vulval muscle. To determine this role, we have performed RNA interference against C27H5.1. Unfortunately, no phenotype was observed. Subsequently we performed RNAi against GFP under the control of the C27H5.1 promoter. There was no reduction in fluorescence suggesting that the expression of C27H5.1 is not affected by RNAi. We are currently screening for a deletion in C27H5.1. Although we have not yet been successful in determining the C27H5.1 mutant phenotype, studies with its mammalian homologue, PDE6D, suggest a possible role for this protein. PDE6D has been shown to bind to and solubilize rod phosphodiesterase (PDE). Since PDE6D expression is not limited to the retina, PDE d may regulate the membrane binding of a variety of proteins. Interestingly, C27H5.1 also binds human rod phosphodiesterase, suggesting that there is at least some functional conservation between PDE d and C27H5.1.

24 May 1999 15:50 838 838 Identification And Characterization Of Intrinsically Thermotolerant Substrains Of Caenorhabditis elegans

1999 International Worm Meeting abstract 787 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification And Characterization Of Intrinsically Thermotolerant Substrains Of Caenorhabditis elegans ND Smith, KE Stine, GE White Ashland University, Ashland, Ohio 44805 The response to external stresses has been studied in a number of organisms including C. elegans. The heat shock response is a classic example. We reported previously that wild type worms, normally maintained at 25o C, when exposed directly to 37o C, show a low survival probability (0.05) when compared to N2s pretreated with either 33o C or 10 mM sodium azide exposure (see Massie et al., WBG 15(3): 22, 1998, and this meeting). Because we consistently observed this 5% surival rate, we wished to determine whether it resulted from variations in the "microenvironment" of the petri dish, or if it was intrinsic to these worms.

An "unstressed" population of N2s was exposed to 37o C for 2 hours, followed by a four hour recovery period at 25o C. The plates were then examined for the presence of living worms. Consistent with our previous results, about 5% of the worms survived. Five young animals, tentatively identified as N2IT# (intrinsically thermotolerant) were transferred to individual plates and allowed to self. We observed that these animals took approximately 96 additional hours before producing a significantly reduced F1 (<50). 3/5 strains had enough F1s to retest at 37o C. Survival probabilities varied in each of the substrains, but were significantly higher than 0.05, with N2IT6 demonstrating the highest survival probability. We then examined N2IT6 for evidence of heat shock protein induction. Coomassie G250 stained Laemmli gels indicated significantly elevated hsp70 levels, while Western Blot analysis indicated significantly elevated hsp16 levels when compared to unshocked controls which is consistent with our previous observations (Massie et al., 1998). We are currently testing all substrains to determine how many generations can maintain this thermotolerance and are examining them for heat shock protein induction. The question of how this subset of worms became thermotolerant remains unclear.

24 May 1999 15:50 839 839 The GLHs: antibodies, protein biochemistry, and a few mutants.

1999 International Worm Meeting abstract 788 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The GLHs: antibodies, protein biochemistry, and a few mutants. PA Smith, KA Kuznicki, WA Leung, A Estevez, KL Bennett Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65212 Four germline-specific RNA helicases (GLHs) have been identified in the free living nematode C. elegans. With antibodies specific to each, it has been determine that all four GLHs localize to the germline P granules, dense aggregates of RNAs and proteins that may be determinants of the germ cell lineage. The GLHs are present in P granules throughout the life of the worm. The GLH proteins are most closely related to the D. melanogaster Vasa germline specific RNA helicase and its homologs in other organisms; however, the GLHs differ from most other RNA helicases in having CCHC zinc fingers. In addition, GLH-1, GLH-2, and GLH-4 have N-terminal non-charged glycine rich repeats. These repeats have some homology to the repeats found in the nucleoporins and loricins and may participate in the aggregation of the proteins in P granules. In order to determine the biochemical role of the GLH proteins in the P granules, 6-His tagged GLH proteins have been designed to be expressed in recombinant baculovirus. GLH-1, GLH-2, GLH-3 and GLH-4 expressed proteins have been successfully isolated using metal-affinity resin. All four proteins have been shown to bind RNA in vitro by filter binding assays. GLH-1 and GLH-2 have been tested and demonstrate weak ATPase activity. The regions of GLH-1 responsible for RNA binding are being tested using various truncated fusion proteins. The ultimate goal of this line of research is to determine if the GLHs bind specific RNAs. To assess the in vivo function of the glh genes, RNAi and mutant isolation have been initiated. RNAi results imply the GLHs are critical for fertility in the worm. A glh-3- strain with a 1.7 kb deletion was isolated by screening a TMP/UV library generated in our laboratory. Initial examination of the glh-3- strain indicates it possesses a mild temperature sensitive defect in fertility; glh-3- animals have 60-70% fewer progeny than wild type worms at 25-26° C. The mutant glh-3 phenotype is consistent with the glh-3 RNAi experiments. Isolation of glh-2 and glh-4 mutant strains as well as combinatorial RNAi of the glh genes is in progress.

24 May 1999 15:50 840 840 Expression of the daf-12 gene

1999 International Worm Meeting abstract 789 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Expression of the daf-12 gene MI Snow, PL Larsen Molecular Biology Program and Division of Biogerontology, University of Southern California, Los Angeles, CA 90089 During early larval development, a cascade of signaling molecules directs the decision to either promote reproductive growth or form the alternative dauer larvae. In the dauer formation genetic pathway, two branches converge on daf-12 and a third branch interacts with daf-12. To initiate molecular studies on the regulation of daf-12 function, we performed Northern analysis of mRNA from daf mutant strains that were grown under dauer inducing conditions. The mRNA was hybridized with a segment of the daf-12 gene that is shared by the three transcripts. We observed a decline in steady-state mRNA levels for each of the three daf-12isoforms in daf-3, daf-7 and daf-16 compared to wild type animals. Thus, daf-12 mRNA levels are regulated by TGF-ß and insulin-like signaling components of the dauer pathway. The molecular analysis of the daf-12 cDNAs has shown three transcripts of different lengths due to differential splicing, termed isoforms A1, A2 and B. Northern analysis showed that the daf-12 isoforms are expressed throughout development. It is possible that the three isoforms function in different tissues at distinct times in development. Precedence exists in the Ecdysone receptor, a Drosophila melanogaster hormone receptor involved in metamorphosis in which the isoforms are expressed in different combinations in different tissues during development (Talbot, et al., Cell, 1993). Based on the genomic and cDNA structures of the daf-12 gene, two regions are being tested for promoter activity to assess where the gene is expressed. One possible promoter region is 5 prime of the A1 isoform and the other is located in a 14 kbp intron that is upstream of the A2 and B isoforms. We have created transgenic animals carrying different fragments fused to the GFP and are currently determining their expression patterns.

24 May 1999 15:50 841 841 Characterisation of TRA-3, a predicted calpain protease involved in sex determination

1999 International Worm Meeting abstract 790 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterisation of TRA-3, a predicted calpain protease involved in sex determination SB Sokol 1 , R Benton 1 , PE Kuwabara 1,2

1 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. 2 Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK

The tra-3 gene promotes XX female development: XX tra-3(m-z-) mutants are transformed from hermaphrodite to male, but XO tra-3 males are unaffected. Barnes and Hodgkin (EMBO J. 15:4477-4484,1996) have shown that tra-3 encodes a predicted homologue of calpain, a calcium-activated regulatory cysteine protease. The predicted TRA-3 protein shows sequence similarity to vertebrate calpains, but where other calpains have Ca2+ -binding EF-hand motifs, TRA-3 has a novel domain. Two mammalian homologues of TRA-3 have been identified; we propose that TRA-3 is the founding member of a new subfamily of calpains. We are studying how TRA-3 functions in sex determination. We have rescued all aspects of the tra-3 mutant phenotype with a full-length tra-3 cDNA driven by its native promoter. Regions essential for TRA-3 activity have been defined by a series of truncation experiments. To provide evidence that TRA-3 may have proteolytic activity, we have shown that a transgene generating TRA-3 protein with its putative catalytic cysteine changed to a serine is no longer capable of rescue. tra-3 activity appears to be controlled post-transcriptionally because a tra-3::GFP promoter fusion is expressed in both XX and XO animals. We have also found that overexpression of tra-3 mildly feminises XO males, but does not feminise the germline of XX hermaphrodites. In order to examine the proteolytic activity of TRA-3, we have expressed recombinant TRA-3 protein for proteolysis assays, and we are analysing potential TRA-3 interaction partners generated from a two-hybrid screen. In vertebrates, calcium regulation of calpains is mediated through EF-hands. Although TRA-3 lacks EF-hands, we have shown that it carries an active Ca2+ -binding C2 domain. This domain is not essential for tra-3 activity; however, a tra-3 allele with a premature stop codon in the C2 domain yields XX hermaphrodites with a mildly masculinised germline. We propose that the C2 domain may regulate TRA-3 activity. We have purified recombinant TRA-3 C2 domain and are examining its Ca2+ -binding properties in order to understand whether Ca2+ plays a role in regulating TRA-3 activity.

24 May 1999 15:50 842 842 The C.elegans genes egl-27 and egr-1 are similar to MTA1, a member of a chromatin regulatory complex, and are redundantly required for embryonic patterning.

1999 International Worm Meeting abstract 791 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The C.elegans genes egl-27 and egr-1 are similar to MTA1, a member of a chromatin regulatory complex, and are redundantly required for embryonic patterning. F Solari, A Bateman, J Ahringer Wellcome CRC Institute, Tennis Court Road, Cambridge CB2 1QR, and The Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, ENGLAND. We previously showed that vab-7 encodes a homeodomain protein of the even-skipped family that patterns posterior muscle and epidermal tissues. To find additional genes implicated in patterning, we screened for enhancers of a weak vab-7 allele. One of the enhancers isolated, we3, is a new allele of the gene egl-27. Like previously isolated alleles of egl-27, egl-27 (we3) mutants have defects in egg laying, phasmid dye filling and male tail morphology. However egl-27 (we3) additionally have defects in the organisation of muscle and epidermal cells during embryogenesis. Genetic and molecular data suggest that egl-27 (we3) may be a null allele of the locus. We cloned egl-27 and found that it is similar to MTA1, a human gene with elevated expression in metastatic carcinomas. Sequence analysis of the predicted EGL-27 protein identified four conserved domains: a GATA like zinc finger domain, a myb-like DNA binding domain and two newly defined domains ELM1 and ELM2, for "EGL-27 and MTA1 homology". MTA1 has recently been shown to be a component of a protein complex with histone deacetylase and nucleosome remodelling activities, suggesting that EGL-27 is involved in chromatin remodelling. One further C.elegans gene, egr-1, is also similar to EGL-27 and MTA1. We found that egl-27 and egr-1 are functionally redundant, and have a fundamental role in embryonic patterning. When both are inactivated using RNAi, cells in essentially all regions of the embryo fail to be properly organised during gastrulation. However, tissue type determination appears normal and tissues become well-differentiated in these embryos. We assayed the expression of a number of embryonically expressed zygotic patterning genes (vab-7, lin-44, and HOX genes) to see if their loss could explain the disorganised phenotype, but all are expressed normally. However, hlh-8, a target of the HOX gene mab-5, is not expressed, suggesting that mab-5 requires egl-27 to activate hlh-8. We propose that EGL-27 and EGR-1 function as part of a chromatin regulatory complex required for the function of regional transcription factor involved in embryonic patterning.

24 May 1999 15:50 843 843 inx-1 is required for body morphogenesis and encodes C. elegans HEM-2, a putative Rac GTPase associated protein

1999 International Worm Meeting abstract 792 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. inx-1 is required for body morphogenesis and encodes C. elegans HEM-2, a putative Rac GTPase associated protein M Soto 1 , K Kasuya 2 , H Qadota 2 , K Kaibuchi 2 , C Mello 1

1 University of Massachusetts Medical Center, Worcester, MA 01605, USA. 2 Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara, 630-0101, Japan.

The inx-1 and inx-3 mutants (intestine on the exterior) were found in maternal effect screens for mutations affecting embryonic gut development. Inx mutant embryos arrest development with their intestinal cells spread over the ventral surface of the embryo. Lineage studies show that the E cells gastrulate properly, however the hypodermal cells fail to migrate over and enclose the embryo and instead contract up into a tight cluster on the dorsal surface. inx-1 was cloned by testing candidate genes in a small genetic interval for the presence of a Tc1 insertion. One gene, named CeHEM-2 by the Genome Project, contains a Tc1 insertion in the inx-1 mutator induced allele zu196. CeHEM-2 (RNAi) induces the inx-1 phenotype, further supporting the identity of inx-1 as a mutation in CeHEM-2. INX-1 is a highly conserved but novel protein. Using column chromatography, mammalian HEM-2 was found to be associated with Rac1, a small GTPase implicated in rearrangements of the actin cytoskeleton. INX-1 antibodies recognize the cytoplasm and cell cortex of many if not all cells in the C. elegans embryo, but may be enriched at hypodermal junctions. inx-1 and inx-3 also have zygotic phenotypes. The vulva of Inx worms forms properly (Wendy Hanna-Rose, personal communication), yet homozygotes become severely Egl leaking only a few eggs. This phenotype is reminiscent of the Egl defect of unc-73, a guanine exchange factor which activates the Rac GTPase in vitro. UNC-73 is involved in the gonad independent mechanism for sex myoblast migrations. Two hybrid assays suggest that INX-1 interacts with CeSra-1, an effector of Rac1 GTPase in vertebrates. RNAi with CeSra-1 has the same embryonic phenotype as inx-1 (see abstract by Kasuya et al). We are investigating the model that INX-1,CeSra-1/INX-2 and perhaps inx-3 are effectors of a small GTPase in C. elegans. This pathway may reorganize the actin cytoskeleton to control cell shape changes necessary for the migration of the hypodermal cells in the embryo and migration of the sex myoblasts post-embryonically.

24 May 1999 15:50 844 844 C. elegans Neurexin-1

1999 International Worm Meeting abstract 793 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. C. elegans Neurexin-1 J Soutschek, J Scheel Max-Planck-Institute for Developmental Biology, Spemannstr. 35/1, 72076 Tübingen, Germany Neurexins, a family of highly diverse transmembrane proteins, have been implicated in formation and functions of chemical synapses. We have identified and characterised the neurexin, nrx-1, from the nematode C. elegans to elucidate its function. C. elegans NRX-1 is highly similar to mammalian Neurexins I-III. Extensive alternative splicing of the nrx-1 gene gives rise to dozens of protein isoforms. Two major isoforms, a-neurexins and N-terminally truncated versions, the b-neurexins, are present in C. elegans. RNA splicing is developmentally regulated; distinct isoforms are upregulated during periods of synaptogenesis. NRX-1 proteins are expressed during late, but not early stages of axonal outgrowth in the nervous system of C. elegans. Whereas b-neurexins are distributed throughout the neuronal plasma membrane, a-neurexins are localised specifically to presynaptic terminals. Thus, differential expression of neurexins seems to occur at the subcellular rather than the cellular level. a-neurexins are localised to synaptic sites in unc-104 mutants, suggesting that a-neurexins anticipate presynaptic sites. Thrashing rates of nrx-1(a) are reduced to 75% of wildtype animals. Whereas synaptic connectivity seems not to be altered in these mutants, extra neuronal branches and fasciculation defects are observed. Transgenic overexpression of truncated versions of NRX-1 in wildtype animals results in the formation of additional neuronal branches and processes, ending in growth cone- or blob-like structures, stopping and deviation of tracts, and fasciculation defects. The extra branches and growth cone-like structures are dynamic. The thrashing rate of transgenics with these structural changes is reduced to 50% of the wildtype rate. The various overexpression phenotypes and the changes observed in the mutant indicate that neurexins are not involved in synapse specification, but seem essential for establishing and maintaining contact in neuronal fascicles and synaptic regions.

24 May 1999 15:50 845 845 Characterization of smu-2, a Gene Implicated in RNA Splicing

1999 International Worm Meeting abstract 794 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of smu-2, a Gene Implicated in RNA Splicing AK Spartz, RK Herman, JE Shaw Department of Genetics and Cell Biology, University of Minnesota, St. Paul, MN 55108 The investigation of unc-52 pre-mRNA alternative splicing has led to the identification of genes, such as smu-2, that may be involved in RNA processing. UNC-52 is the C. elegans homolog of perlecan, a basement membrane protein essential for embryonic development. Viable alleles of unc-52 bear mutations within the alternatively spliced region between exons 15 and 19 (Rogalski et al. Genetics 139: 159). Previous work showed that 15-19 and 16-19 splices are dependent on the putative RNA binding protein MEC-8 (Lundquist et al. Development 122:1601). The smu-2 gene was identified in a screen for suppressors of mec-8(ts); unc-52(ts) conditional synthetic lethality. Recessive, loss-of-function smu-2 mutations suppress, in addition to the mec-8; unc-52 synthetic lethality, other defects conferred by mec-8, as well as the uncoordination conferred by nonsense mutations within exon 17 of unc-52. smu-2 mutations by themselves are mildly deleterious but otherwise confer no obvious phenotype. We propose that smu-2 has a role in RNA processing that may be fairly general. To test this idea, we have have used RT-PCR to analyze the effect of smu-2 mutation on the relative abundance of alternatively spliced unc-52 transcripts. We have found that the abundance of transcripts containing 16-18 splices compared to transcripts containing 16-17-18 splices is increased in smu-2 mutants. These data suggest that mutation in smu-2 may compensate for the loss of some unc-52 transcripts caused by mec-8 mutation by increasing the abundance of others. We are now working on a more quantitative comparison of unc-52 alternative splicing patterns. To characterize smu-2 further, we are cloning the gene. Previously smu-2 was mapped 2.8 cM to the left of unc-85 on chromosome II. smu-2 complements the deficiencies ccDf1 and ccDf2 but not ccDf11. Two-factor mapping places smu-2 0.95 cM to the left of the cloned gene lin-31. We have found five dimorphisms between N2 and RC301 strains that have helped us to localize smu-2 to a 40-kb region. We are currently attempting to rescue smu-2 using cosmid and YAC clones from this region.

24 May 1999 15:50 846 846 The inhibitor-of-apoptosis (IAP)-like protein BIR-1 is required for cytokinesis

1999 International Worm Meeting abstract 795 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The inhibitor-of-apoptosis (IAP)-like protein BIR-1 is required for cytokinesis EK Speliotes 1 , A Uren 2 , D Vaux 2 , HR Horvitz 1

1 HHMI, Dept. Biology, MIT, Cambridge, MA 02139. 2 WEHI, Melbourne, Australia.

Determining whether common mechanisms mediate aspects of mitosis and apoptosis is fundamental to understanding both normal biology and disease. We are studying bir-1 and bir-2, two genes that encode proteins with sequence similarity to the evolutionarily conserved IAP family of proteins, some of which protect against programmed cell death (PCD) when overexpressed. The normal functions of IAPs and how these functions relate to the ability of this family of proteins to protect against PCD, if at all, remain to be determined. bir-1(RNAi) embryos have defects in a late stage of cytokinesis and arrest with multinucleate cells. In bir-1(RNAi) embryos, meiotic divisions are completed, but polar bodies are not extruded; multiple nuclei within a common cytoplasm may stay separate from one another and divide asynchronously or may fuse to form large polyploid nuclei. During mitosis, condensed chromosomes from polyploid nuclei are separated by multipolar spindles. Other events including pronuclear migration, pseudocleavage, and the timing and position of the first mitotic division appear to be unaffected. bir-1(RNAi) embryos generated by ced-3 and ced-4 mutant mothers also arrest, suggesting that this phenotype is not caused by ectopic activation of a cell death program. Animals homozygous for the bir-1 deletion n3329 produce multinucleate embryos consistent with the RNAi results. bir-1(n3329) homozygotes are also variably thin, Unc, Mig(DTC), Pvl, Ste and can burst at the vulva, as can animals that escape the embryonic lethality following RNAi, suggesting that bir-1 may be needed for normal development of many tissues. Animals homozygous for a deletion of bir-2 (provided by R. Barstead) and bir-2(RNAi) animals do not show any gross defects. bir-1 is most similar to human survivin, a G2/M-upregulated, spindle-associated protein that protects against PCD and is expressed in high-grade tumors. We are currently testing whether survivin can substitute for bir-1 in worms, characterizing single and double bir mutants, determining the expression patterns of BIRS, and analyzing BIR two-hybrid interacting proteins. We hope our studies of birs will help us to better understand cytokinesis and the mechanism(s) of action of the IAPs.

24 May 1999 15:50 847 847 smu-1 Affects the Accumulation of Alternatively-Spliced Transcripts from the unc-52 Gene

1999 International Worm Meeting abstract 796 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. smu-1 Affects the Accumulation of Alternatively-Spliced Transcripts from the unc-52 Gene CA Spike, RK Herman, JE Shaw Department of Genetics and Cell Biology, University of Minnesota, St. Paul, MN 55108 smu-1 is a recessive suppressor of the synthetic lethal interaction between certain alleles of mec-8 and unc-52 (Genetics 138: 83-101). smu-1 mutations can also suppress other phenotypic effects of mec-8 and unc-52 mutations. Previous experiments demonstrated that mutations in the mec-8 gene, which encodes a putative RNA binding protein, affect the accumulation of certain alternatively spliced unc-52 messenger RNAs (Development 122: 1601-1610). Since smu-1 suppresses the phenotypes of mutations in the mec-8 and unc-52 genes individually, as well as in combination, we have proposed that its gene product functions in pre-mRNA splicing. Our hypothesis is that inactivation of the smu-1 gene product by loss-of-function mutations affects the alternative splicing patterns of mec-8targets (such as unc-52) and results in a small amount of bypass suppression of the mec-8 mutations. Consistent with this hypothesis, we have found that the smu-1 gene encodes a conserved WD-repeat protein, suggesting that smu-1 may function in a multi-protein complex. To test the hypothesis directly, we are studying the accumulation of unc-52 mRNA splice isoforms in smu-1 mutant animals. We are focusing on splice isoforms that skip exon 17, because smu-1 can suppress the unc-52 alleles e669 and e669su250ts, which have the nonsense mutation e669 in exon 17. We have shown by RT-PCR experiments that the level of the alternatively spliced mRNA isoform that skips only exon 17 (exon 15-16-18-19 isoform) increases in smu-1 loss-of-function mutants. These experiments suggest that SMU-1 protein is required to minimize the formation of the this mRNA isoform. We are currently using RNase protection to confirm these results. We have also been studying the expression pattern of smu-1. A smu-1::GFP transgene containing the smu-1 promoter and the first third of the SMU-1 protein is ubiquitously expressed in somatic cells and localizes to the nucleus. This localization pattern is also consistent with the hypothesis that SMU-1 is involved in pre-mRNA splicing.

24 May 1999 15:50 848 848 Creating a C. elegans cell line

1999 International Worm Meeting abstract 797 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Creating a C. elegans cell line DG Srinivasan, S van den Heuvel MGH-East Cancer Center, Charlestown, MA 02129 Powerful genetic and cell biological approaches can be used to study gene function in C. elegans; however, biochemical characterization of such functions is troublesome. To facilitate biochemical studies, we are attempting to establish a C. elegans cell line. As potential sources, we are using embryonic, germline and somatic tissues. Embryonic cells are used for their proliferative capacity. Moreover, culture conditions for embryonic cells have been established previously. We treat embryos with chitinase-chymotrypsin to obtain suspensions of single cells and small cell clumps. The cells are plated in a Schneider’s-based medium yet are viable for only 1-3 days, and continuous divisions are rare. We are combining these methods with random mutagenesis to create cells with improved proliferative potential. Expression of rnr::gfp, an S-phase reporter (R. Roy and V. Ambros), will serve as a marker for viability and proliferation. The second approach utilizes germline tissue in combination with well characterized mutations: gld-2(dx32) gld-1(q485) double mutants contain a tumorous, mitotic germ line, epi-1(rh191ts) mutants grown at 20° C form cellularized germ cells, and cells in ced-3(n717) animals do not undergo apoptosis. Combination of these mutations may allow us to culture an autonomously proliferating germ cell line. Germ cells, dissected from these mutant gonads, will be monitored for continuous proliferation. As a final approach, we are attempting to culture adherent somatic cells. Mammalian culture methods often select for adherent cells following digestion of tissue samples. Using a similar strategy, mixed stage worms are dissected, briefly trypsinized and plated in a Schneider’s-based medium. Cell masses that tend to grow out from the cut sites and free cells are examined for proliferation and adherence. This technique will be combined with random mutagenesis and putatively oncogenic mutations to create cells with proliferative advantages. These techniques may allow us to obtain a cell line from at least of one of these tissue sources. Because of its proliferative capacity and totipotent nature, we consider the germ line the optimal source for a tissue culture cell line. Successful establishment of a cell line will aid us in probing the molecular nature of genetic and cellular interactions.

24 May 1999 15:50 849 849 Regulation of cell death vs. non-cell death in the ventral epidermis of Pristionchus pacificus.

1999 International Worm Meeting abstract 798 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Regulation of cell death vs. non-cell death in the ventral epidermis of Pristionchus pacificus. J Srinivasan 1,2 , R Sommer 1

1 Max Planck Institute for Developmental Biology, Dept. for Evolutionary Biology, Spemannstrasse 35, D-72076 Tübingen Germany. 2 E.mail: [email protected]

We are studying the evolution of cell fate specification, using vulva development as a model system and compare C. elegans with Pristionchus pacificus. In P. pacificus, seven ventral epidermal cells P(1-4,9-11).p undergo programmed cell death during late embryogenesis, whereas P(5-8).p survive and participate in vulva formation. We have previously shown that the lin-39 homolog of P. pacificus prevents apoptosis of P(5-8).p and that mutations in Ppa-lin-39 result in a generation vulvaless phenotype. We used various genetic screens, primarily TMP/UV mutagenesis, to isolate new generation vulvaless mutants and have upto now identified four additional mutations not allelic to Ppa-lin-39. These new mutations have a more severe vulvaless phenotype and show additional pleiotropic defects. Further genetic characterization indicated some of these mutants to be haplo-insufficient i.e. heterozygous animals show a protruding vulva phenotype and are egg-laying defective. Complementation tests among these mutations is ongoing. As these generation vulvaless mutants have a phenotype similar to Ppa-lin-39, we want to test whether potential cofactors for homeotic transcription factors are mutated in these mutants. In Drosophila, Extradenticle and Homothorax have been identified to be important co-factors for the activity of homeotic genes. In C. elegans, ceh-20 and ceh-25, respectively, are the homologs of these genes. We cloned both genes from P. pacificus, and are currently checking for linkage between Ppa-ceh-20/Ppa-ceh-25 and the candidate mutants. Incase no linkage is obtained between the candidate mutants and Ppa-ceh-20/Ppa-ceh-25, we plan to use RDA to identify the genetic loci causing these mutant phenotypes. RNA interference studies on Ppa-ceh-20 and Ppa-ceh-25 is also ongoing.

24 May 1999 15:50 850 850 Characterisation of neprilysin (NEP) and endothelin-converting enzyme (ECE) from C. elegans

1999 International Worm Meeting abstract 799 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterisation of neprilysin (NEP) and endothelin-converting enzyme (ECE) from C. elegans PR Stancombe, M Sajid, D Coates, RE Isaac School of Biology, University of Leeds, Leeds LS2 9JT, UK Cell-surface peptidases participate in the postsecretory processing and metabolism of neuropeptides and peptide hormones. Neutral endopeptidase-24.11 (NEP) is the prototype of a mammalian subfamily of zinc metallopeptidases that also includes endothelin-converting enzyme (ECE), the product of the PEX gene and KELL, an erythrocyte cell-surface antigen. These enzymes have central roles in a variety of physiological and disease processes, including cardiovascular function, cartilage and bone metabolism, inflammation and embryogenesis. Human zinc metalloproteinases belonging to this subfamily are important targets for therapeutic drugs. C. elegans has in the region of 90 genes with the zinc-binding consensus motif HEXXH of the superfamily of zinc metalloproteinases. Cluster analysis of predicted amino acid sequences reveals a group of 8 genes with closer similarity to mammalian NEPs/ECEs than the others. Hydrophobicity plots of the predicted protein sequences for these 8 genes revealed 5 that gave the expected domain structure seen in mammalian homologues; a short NH2 -terminus intracellular region, a single transmembrane domain and a large extracellular region with a COOH-terminus active site. Expression patterns of beta-galactosidase and GFP have been obtained for 4 of these genes. T16A9.4 has a largely neuronal expression pattern with a pharyngeal and vulva muscle component; ZK970.1 and T05A8.4 are expressed in body wall muscle cells; ZK20.6 is pharynx specific. RNA interference experiments are being conducted with these genes to determine any null phenotypes.

24 May 1999 15:50 851 851 Analysis of downstream events in the pathway for programmed cell death

1999 International Worm Meeting abstract 800 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of downstream events in the pathway for programmed cell death GM Stanfield, E Hartwieg, HR Horvitz HHMI, Dept. Biology, MIT, Cambridge, MA 02139 We are genetically and molecularly analyzing genes that may be involved in the downstream events of programmed cell death, such as the adoption of cell-corpse morphology and the engulfment and degradation of cell corpses. To facilitate phenotypic analyses of cell-death mutants, we are interested in developing markers that identify different stages in the cell-death process. Toward this end, we have developed methods for performing TUNEL (TdT-mediated dUTP Nick End Labeling) assays on C. elegans. Our analyses of wild-type, DNA degradation-defective and engulfment-defective embryos indicate that there are at least three steps of DNA degradation in cell corpses: an initiation step in which TUNEL-reactive ends are generated, a nuc-1-mediated step in which TUNEL-reactive ends are degraded (or masked) and an engulfing cell-mediated step (Hedgecock et al., Science 220, 1277-9, 1983) in which DNA degradation is completed. ced-8 and ced-11 are candidate downstream cell-death genes. In ced-8 mutant embryos, refractile cell corpses appear later than in wild-type embryos. However, TUNEL detects more dying cells at early embryonic stages in ced-8 embryos than are visible using Nomarski microscopy, suggesting that DNA degradation is less delayed than is the onset of refractility. Thus, ced-8 may couple cell killing to morphological changes in the dying cell. In ced-11 embryos, dying cells do not assume the refractile, electron-dense characteristic of normal cell corpses. TUNEL assays indicate that ced-11 embryos have slightly increased numbers of TUNEL-reactive nuclei relative to the wild type. Thus, mutations in ced-11 might result in slowed DNA degradation in addition to altered cell-corpse morphology.

24 May 1999 15:50 852 852 Cloning and analysis of the cGMP-dependent protein kinase CGK-1

1999 International Worm Meeting abstract 801 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cloning and analysis of the cGMP-dependent protein kinase CGK-1 J Stansberry 1 , E Baude 1 , RE Ellis 2 , MD Uhler 1

1 Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109. 2 Department of Biology, University of Michigan, Ann Arbor, MI 48109.

In mammals, cyclic GMP (cGMP) has been shown to regulate smooth muscle contraction, platelet aggregation and calcium channel function. Furthermore, in some neurons cGMP has been implicated in the modulation of synaptic plasticity, long-term potentiation and long-term depression. It appears that cGMP functions predominately by activating the cGMP dependent protein kinase (cGK), a member of the serine/threonine kinase family. To learn how this kinase controls such a wide variety of cellular functions, we are studying this protein in C. elegans. We used degenerate PCR to isolate a cDNA fragment with similarity to mammalian cGMP-dependent kinases. To obtain the complete cDNA, we screened a library with this fragment, and isolated a clone with a 2.6 kb insert. Although this clone lacked the 5’ end of the message, we isolated these sequences using an SL1 primer and reverse primers from within the gene. Northern analysis of RNA prepared from a mixed population of males and hermaphrodites confirmed that this gene produces a single transcript of the predicted size. Because our cDNA encodes a protein that resembles mammalian and Drosophila cGKs, we named it cgk-1. To determine if CGK-1 behaves like a cGMP-dependent protein kinase, we expressed it in Sf-9 cells and purified it by affinity chromatography. We find that CGK-1 has a 700 nM Ka for cGMP, and a 7 µM Ka for cAMP, which shows that it preferentially binds cGMP. Furthermore, CGK-1 undergoes autophosphorylation and can phosphorylate known cGK target peptides. Thus, in vitro assays show that CGK-1 functions much like other cGMP-dependent protein kinases. Northern analyses reveal that cgk-1 is expressed throughout development. We do not yet have antibodies to CGK-1, so we used a fusion gene to begin elucidating where cgk-1 is expressed. In this construct, transcription of GFP is driven by 2.4 kb of DNA from the cgk-1 promoter. Our reporter construct is strongly expressed in the marginal cells of the pharynx, the intestine, body wall muscles and sex muscles, the spermatheca, and a small set of neurons that includes cells in the amphids and phasmids. We are now using RNA-mediated inactivation to learn what functions cgk-1 might play.

24 May 1999 15:50 853 853 Localization of Gap Junction Proteins

1999 International Worm Meeting abstract 802 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Localization of Gap Junction Proteins TA Starich 1 , A Miller 2 , DH Hall 2 , JE Shaw 1

1 Dept. Genetics and Cell Biology, University of Minnesota, St. Paul, MN 55108. 2 Dept. Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461.

The innexins (invertebrate connexins) represent gap junction (GJ) proteins in C. elegans and Drosophila. The strongest evidence for this is borne out of experiments showing that the Drosophila Shaking-Bvit protein is able to mediate electrical coupling when expressed in paired Xenopus oocytes (Phelan et al., Nature 391:181, 1998). Similar experiments have now shown that the C. elegans INX-3 protein is also capable of mediating electrical coupling in paired Xenopus oocytes (Landesman, White, Paul and Goodenough, submitted). To investigate the function of GJs during embryogenesis and establishment of neural wiring we are using GFP fusions and specific affinity-purified antibodies to determine which innexins are expressed early in development and in the nervous system. By these criteria at least three innexins, including INX-3, are expressed in 2-cell embryos, although one of these does not appear to localize to plaques at this stage. Two innexins, including UNC-7, are strongly expressed in the nerve ring, and UNC-7 is also detected in the ventral and dorsal nerve cords, pharyngeal nervous system and other process bundles. In the hypodermis, expression of at least three innexins has also been observed. RNAi experiments and mutant screens are ongoing for these innexin genes; the RNAi phenotype for INX-3 suggests a role in maintaining hypodermis integrity during embryogenesis and attachment of the anterior pharynx in the head. To verify that innexins are associated with GJs, polyclonal antibodies raised to the carboxyl terminus of INX-3 were used in immuno-EM localization studies. A post-embedding immunogold technique (Miller and Hall, WBG 1998; Hall, Methods in Cell Biology, 1995) was employed after animals were immersed in fixative in a microwave oven to promote rapid fixation and to improve penetration of the embedding resin. Virtually all GJs could be labelled in the pharynx in adult, larval or late embryonic stages. This binding was specific since pre-mixing of the antibody with the fusion peptide reduced or eliminated labelling. In early embryos, very small GJ plaques showed labelling in cells which were not highly differentiated (i.e. prior to expression of pharyngeal markers).

24 May 1999 15:50 854 854 The roles of unc-83, unc-84, and anc-1 in nuclear migration and anchorage

1999 International Worm Meeting abstract 803 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The roles of unc-83, unc-84, and anc-1 in nuclear migration and anchorage DA Starr, CJ Malone, M Han University of Colorado, Department of MCD Biology, Campus Box 347, Boulder, CO 80309 We have undertaken a molecular analysis of three genes to better understand the basic processes of nuclear migration in the context of cellular migrations during development. Mutations in two of these genes, unc-84 and unc-83, block the nuclear migrations of hypodermal P cells, which normally migrate from a lateral position to the ventral cord, and of hyp7 precursor cells, which normally elongate circumferentially across the dorsal midline (2, 3). Mutations in unc-84 and anc-1 disrupt the proper anchorage of hypodermal nuclei (1, 2). unc-84 has been cloned and encodes for a novel protein with a predicted trans-membrane domain and a C-terminal domain with high similarity to the S. pombe spindle pole body component Sad1. An UNC-84:GFP protein is detected at the nuclear envelope (3). To confirm this finding, we are in the process of raising antibodies against UNC-84. We have further mapped unc-83 on chromosome V and have obtained single cosmid rescue of the hyp7 nuclear migration defect with the cosmid W01A11. We are currently attempting to rescue unc-83 with subclones of W01A11. Finally, we are in the process of cloning anc-1. It has been linked to the physical map to the right of unc-11 (which is within cosmid C32E8) and to the left of hDf7 (which has a left breakpoint in cosmid C24G7). We are currently injecting cosmids in this region.

1. Hedgecock, E. M., and J. N. Thomson. 1982. Cell. 30:321-330. 2. Malone, C. J., W. D. Fixsen, H. R. Horvitz, and M. Han. 1999. Submitted to Development. 3. Sulston, J., and H. R. Horvitz. 1981. Dev. Biol. 82:41-55.

24 May 1999 15:50 855 855 Identification of mutants affecting mitotic chromosome segregation in the embryo

1999 International Worm Meeting abstract 804 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification of mutants affecting mitotic chromosome segregation in the embryo JH Stear, BJ Buchwitz, LL Moore, MB Roth Division of Basic Sciences and Molecular and Cellular Biology Program, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109 We are interested in characterizing the centromere in C. elegans. As part of this work we have screened a collection of 250 temperature sensitive, embryonic lethal mutants by staining with DAPI. A large percentage (10%) of these mutants displayed alterations in the distribution of DNA in the embryo. We have used high resolution Deltavision microscopy to determine that twelve of these mutants exhibit defects in mitotic chromosome segregation. To further characterize these mutants we stained them with several different centromere antibodies. One class of the mutants shows appropriate individualization of mitotic chromosomes during prophase and congression to the metaphase plate, yet fails to exhibit appropriate deposition of the histone H3 variant ceCENPA. Another class shows defects in the transition from metaphase to anaphase. We have begun to map and clone a subset of these mutants, and are also in the process of performing a saturating screen to isolate additional mutants with similar phenotypes.

24 May 1999 15:50 856 856 Characterization of Muscarinic Acetylcholine Receptors in the Pharynx

1999 International Worm Meeting abstract 805 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of Muscarinic Acetylcholine Receptors in the Pharynx KA Steger, L Avery Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd Dallas, TX 75235-9148 Acetylcholine signaling helps control feeding in C. elegans. While pharyngeal nicotinic pathways have been identified, evidence for muscarinic signaling remains circumstantial. One clue is that worms that do not produce acetylcholine fail to pump, while worms lacking nicotinic responses can pump, at a reduced rate. In addition, worms lacking nicotinic signaling in the pharynx are sensitive to the cholinesterase inhibitor aldicarb- an effect that can be diminished by atropine, a muscarinic antagonist. We are using molecular and pharmacological approaches to characterize pharyngeal muscarinic receptors. The C. elegans genome contains three ORF’s with significant homology to known muscarinic receptors. By creating GFP-gene fusion constructs, we have examined the expression patterns of these genes, and found one, C15B12.5, expressed in anterior pharyngeal muscle. We are now attempting to knock out this gene by isolating mutants from a deletion library. We have also begun to characterize the responses of wild type and mutant worms to muscarinic agonists and antagonists. Dissected pharynxes of wild type worms exhibit rapid pumping in the presence of 100 mM bethanechol (a specific muscarinic agonist), without a dramatic increase in pharyngeal motor neuron activity. This observation suggests that muscarinic agonists can act directly on the pharyngeal muscle. In addition, eat-2 and eat-18 mutant worms, which lack nicotinic signaling through the control neuron MC, exhibit similar rapid pumping in the presence of bethanechol. Since some muscarinic receptors act through Gaq/ Phospholipase C/ calcium signaling, we are examining the response of Gaq (egl-30) mutants to bethanechol. Muscarinic receptors also function through inward rectifying G-protein-coupled (IKAch) potassium channels; we are examining the C. elegans genome for candidate channels. Using these genetic and pharmacological approaches, we hope to characterize the mechanisms of action and role of muscarinic signaling in pharyngeal pumping.

24 May 1999 15:50 857 857 Regulation of the actin cytoskeleton: The identification of genes that interact with the Rac activator unc-73

1999 International Worm Meeting abstract 806 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Regulation of the actin cytoskeleton: The identification of genes that interact with the Rac activator unc-73 RM Steven, JG Culotti, T Pawson Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Canada unc-73 mutants have a variety of defects in axon guidance and cell migration. The axons of many neurons fail to reach their targets and often travel along abnormal pathways. The strongest loss of function allele of unc-73 exhibits maternally rescued embryonic and larval lethality. The largest UNC-73 isoform of 2,463 a.a. contains, in order, eight spectrin-like repeats, two dbl homologous (DH)/ pleckstrin homologous (PH) tandem domain elements that flank an SH3 domain, an Ig domain and a FnIII domain. Smaller UNC-73 isoforms also exist. Published experiments suggest that UNC-73 function involves signaling through the Rac pathway to regulate the actin cytoskeleton in migrating cells and growth cones. The unc-73(rh40) mutation (S1216F) is located in a highly conserved region of the DH-1 domain. This mutation eliminates the RacGEF and actin polymerization activities of the DH-1 domain. unc-73(rh40) animals were screened for suppression of the Unc phenotype following mutagenesis with EMS. A single nonUnc rh40 suppressor was identified in a screen of 200,000 F1 animals and 120,000 haploid genomes in the F2 generation. This suppressor is dominant and tightly linked to unc-73. Sequencing of the unc-73 gene in the suppressor strain revealed that the suppressor mutation (L1269F) is located close to the rh40 mutation within the DH-1 domain of unc-73. Examination of the 3D structure of the DH domain indicates that the suppressor mutation may structurally compensate for the defect caused by the rh40 mutation. The effect of the suppressor mutation on RacGEF and actin polymerization activity was tested in vitro. The introduction of the (L1269F) suppressor mutation into a DH-1 domain containing the (S1216F) rh40 mutation restored the actin polymerization activity of the DH-1 domain as tested in Rat2 cells. The in vitro RacGEF activity of the S1216F/L1269F DH-1 domain, however, was not restored. Additional genetic screens are underway in the hope that genes that interact with unc-73 will be identified. Studies of the human UNC-73 homologue, Trio, have revealed a potential interaction between Trio and the PTPase LAR. Genetic evidence of an interaction between unc-73 and a C. elegans LAR-like gene, ptp-1 (M. Hentgartner, p.c.), will be presented.

24 May 1999 15:50 858 858 How does the pharyngeal primordium generate different pharyngeal cell types?

1999 International Worm Meeting abstract 807 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. How does the pharyngeal primordium generate different pharyngeal cell types? JB Stevenson 1 , ME Domeier 1 , A Chisholm 2 , SE Mango 1

1 Dept. of Oncological Sciences, Huntsman Cancer Institute Center for Children, University of Utah, Salt Lake City, UT, 84112, USA. 2 Dept. of Biology, Sinsheimer Laboratories, UC Santa Cruz, Santa Cruz, CA, 95064.

Formation of the pharynx consists of three phases: i) specification of pharyngeal precursors during early embryogenesis, ii) assembly of these precursors into a pharynx primordium, and iii) terminal differentiation of the five pharyngeal cell types and morphogenesis of the pharynx into its adult form. Establishment of the pharyngeal precursors requires the pha-4 gene, which encodes a transcription factor expressed in all cells of the pharynx primordium. In a pha-4 mutant background, no or few pharyngeal cells are generated1. How are five different pharyngeal cell types produced from an apparently homogeneous population of pharyngeal precursor cells? And what role does pha-4 play in this process, given its pan-pharyngeal expression pattern? To address these questions we are studying how one pharyngeal cell type, the marginal cell, is established during development. We have identified an early marker of marginal cells, namely the paired domain protein PAX-9. C. elegans pax-9::GFP is expressed in all nine marginal cells as well as three other pharyngeal cells. Deletion analysis has tentatively identified a region between -424 and -155 from the translational start site that is essential for appropriate pax-9 expression. Further deletion analysis is underway to identify potential cis elements within this region that are required for expression of pax-9. In addition, we are characterizing the phenotypes associated with ectopic pax-9 and pax-9(RNAi) to learn the function of pax-9 during marginal cell development.

1. Mango et al., Development 1994; Horner et al., Genes and Development 1998, Kalb et al., Development 1998.

24 May 1999 15:50 859 859 NHR transcription factors and cuticle synthesis in C.elegans

1999 International Worm Meeting abstract 808 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. NHR transcription factors and cuticle synthesis in C.elegans EJ Stewart, IL Johnstone WCMP, Anderson College, University of Glasgow, 56 Dumbarton Rd, Glasgow, G11 6NU, Scotland The C.elegans hypodermis manufactures 5 cuticles throughout the life cycle of the animal. The collagens required for cuticle assembly are produced in distinct waves of expression during embryogenesis and subsequently each larval stage. We predict that this co-ordinated and reiterative process is under tight transcriptional regulation. Phylogenetic analysis has indicated that all moulting organisms probably share a single common ancestor. Molecular data from Drosophila Melanogaster, the best studied of these organisms, has shown that nuclear hormone receptors (NHR’s) have an important role to play in the moulting cycle. The C.elegans genome contains approximately 170 predicted cuticular collagen genes and 230 NHR like genes. We are investigating the possibility that some of the NHR’s are involved in regulating the transcriptional patterns of collagen gene expression that occur during the moulting cycle. We have tested the temporal pattern of expression of mRNA abundance for several NHR genes. Using this approach, we have identified some that share a similar pattern of fluctuations in mRNA abundance during each larval stage to those detected for the collagen genes. This is strong circumstantial evidence of a link between these NHR genes and the control of collagen gene expression.

24 May 1999 15:50 860 860 Cilium Structure Genes osm-1, daf-10, che-11 and che-13

1999 International Worm Meeting abstract 809 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cilium Structure Genes osm-1, daf-10, che-11 and che-13 S Stone, A Davies, T Ricke, J Shaw Department of Genetics and Cell Biology, University of Minnesota, St. Paul, MN 55108 Mutations in the cilium structure genes, including osm-1 , daf-10 , che-11 , che-13 and osm 6 , lead to defects in osmotic avoidance, chemotaxis, response to dauer-inducing signals and uptake of fluorescent dyes by the amphid and phasmid neurons. Analysis of these mutants by electron microscopy (Perkins et al. Dev. Biol. 117 :456-487; Albert et al. J. Comp. Neurol. 198 :435-451) demonstrated structural defects in their sensory cilia, which were severely shortened. To further our understanding of processes involved in the assembly of sensory cilia, we are continuing to characterize the cilium structure genes. Analysis of the location of a functional OSM-6::GFP (Collet et al. Genetics 148 :187-200) in several cilium structure mutants has indicated that none of the above mutants appears to affect the expression osm-6::gfp ; however, each affects the localization of OSM-6::GFP within the amphid neurons. We have also cloned osm-1 , daf-10 , che-11 and che-13 . The carboxyl-terminal half of OSM-1 is predicted to be composed primarily of a repeating motif of approximately 44 amino acids, with unknown function. Interestingly, both DAF-10 and CHE-11 contain weakly similar repeats. The amino acid sequences of OSM-1, CHE-11 and CHE-13 (as well as OSM-6) have significant similarity to partial peptide sequences of polypeptides that are hypothesized to form protein complexes involved in intraflagellar transport (IFT) in Chlamydomonas (Cole et al. J. Cell. Biol. 141 :993-1008). The IFT complexes are proposed to move along the outer doublet microtubules of the axoneme, next to the plasma membrane, and to be involved in the assembly of the motile flagella of Chlamydomonas . Database searches indicate that these proteins also have vertebrate homologues. Thus these proteins may have widely conserved function in transporting components required for assembly and/or function of both motile and non-motile cilia and flagella.

24 May 1999 15:50 861 861 The Caenorhabditis sex determining protein FEM-2 evolves faster than its paralogs

1999 International Worm Meeting abstract 810 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Caenorhabditis sex determining protein FEM-2 evolves faster than its paralogs PM Stothard, DD Hansen, DB Pilgrim Department of Biological Sciences, University of Alberta, Edmonton AB, T6G 2E9, Canada We characterized the orthologs of two C. elegans genes from the related nematode C. remanei. Both genes are predicted to encode proteins that belong to the protein phosphatase type 2C (PP2C) superfamily. One of the genes encodes FEM-2, a sex determining protein, while the other (T23F11.1, a gene predicted from the genomic sequence of C. elegans) has no known sex determining role. Comparison of the C. remanei sequences with their C. elegans orthologs indicates that FEM-2’s PP2C domain is much more divergent than the same domain in T23F11.1 (68% identity vs. 95% identity), and that the entire FEM-2 protein is just 59% identical between C. elegans and C. remanei. PP2C sequences were also isolated from the zebrafish, Danio rerio, and compared to their previously identified mouse orthologs. Surprisingly, the zebrafish/mouse PP2C domains are more conserved than FEM-2’s PP2C domain (74%-80% identity), even though the mouse and zebrafish diverged around 400 million years ago, while C. elegans and C. remanei are thought to have diverged between 20 and 50 million years ago. Together these findings show that the PP2C domain of the sex determining protein FEM-2 evolves more quickly than the same domain in other PP2Cs. The rapid evolution of FEM-2 could be the result of strong selection driving divergence, or weak selection allowing for more rapid divergence. Comparison of the full length C. remanei FEM-2 to its C. elegans and C. briggsae orthologs reveals that the FEM-2 protein contains a large amino terminal domain and a highly charged carboxy terminal domain in all three species. These accessory domains are not found in other PP2Cs, and their presence in all three orthologs suggests that FEM-2 has similar molecular roles in male/hermaphrodite species (C. elegans and C. briggsae) and male/female species (C. remanei). Indeed, we show that a C. remanei fem-2 transgene is able to function as a promoter of the male sexual fate in C. elegans somatic cells.

24 May 1999 15:50 862 862 Homologs of the C. elegans masculinizing gene her-1 in C. briggsae and the filarial parasite B. malayi

1999 International Worm Meeting abstract 811 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Homologs of the C. elegans masculinizing gene her-1 in C. briggsae and the filarial parasite B. malayi A Streit 1,2 , B Robertson 2 , J Schein 3 , IH Kamal 4,5 , M Marra 3 , B Wood 1

1 Dept. of MCD Biology, U. Colorado, Boulder CO 80309. 2 Present address: Institute for Zoology, University of Zurich, Winterthurerstr. 190, 8057 Zurich, Switzerland. 3 Genome Sequencing Center, Washington U. School of Medicine, St. Louis MO 63108. 4 Filarial Genome Project Resource Center, Smith College, Northampton MA 01063. 5 Present address: Department of Biochemistry, Ain Shams University, Abassiah, Cairo 11566, Egypt.

The masculinizing gene her-1 in C. elegans (Ce-her-1) encodes a novel protein, HER-1A, which is required for male development. To identify conserved elements in her-1 we have cloned and characterized two homologous nematode genes: one by synteny from the closely related free-living species C. briggsae (Cb-her-1), and the other, starting with a fortuitously identified EST, from the distantly related parasite Brugia malayi (Bm-her-1). The overall sequence identities of the predicted gene products with Ce-HER-1A are only 57% for Cb-HER-1, which is considerably lower than has been found for most homologous briggsae genes, and 35% for Bm-HER-1. However, conserved residues are found throughout both proteins, and like Ce-HER-1A, both have putative N-terminal signal sequences. Ce-her-1 produces a larger masculinizing transcript (her-1a) and a smaller transcript of unknown function (her-1b); both are present essentially only in males. By contrast, Cb-her-1 appears to produce only one transcript, corresponding to her-1a; it is enriched in males but present also in hermaphrodites. Although this suggests different regulatory mechanisms, comparisons of upstream and intron-2 sequences identified elements common to both Caenorhabditis species. Injection of dsRNA transcribed from Cb-her-1 into C. briggsae hermaphrodites caused XO animals to develop into partially fertile hermaphrodites. Introducing a Cb-her-1 construct as a transgene under control of the C. elegans unc-54 myosin heavy chain promoter caused strong masculinization of both C. briggsae and C. elegans hermaphrodites. Introduction of a similar Bm-her-1 construct into C. elegans caused weak masculinization. Therefore, in spite of considerable divergence the Cb gene is likely to be a functional ortholog of Ce-her-1, and the distantly related Bm gene may be as well.

24 May 1999 15:50 863 863 Beyond the ENDs: the network of genes regulating gut development

1999 International Worm Meeting abstract 812 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Beyond the ENDs: the network of genes regulating gut development K Strohmaier 1 , K Koh 2 , I Hope 3 , JH Rothman 1,2

1 Program in Biochemistry and Molecular Biology. 2 Dept. of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA. 3 Dept. of Biology, University of Leeds, Leeds LS2 9JT, U. K.

In an effort to understand how differentiation and organogenesis of the intestine are regulated, we are analyzing the network of genes acting downstream of the maternal and zygotic factors that specify the identity of the E blastomere. One such gene, eel-1, encodes an ODD-SKIPPED-like protein. An eel-1 reporter is expressed in the endoderm (midgut) lineage beginning at the 4E cell stage. EEL-1 may therefore perform a homologous function to that of Drosophila BOWEL, an ODD-SKIPPED family member which is expressed throughout the entire digestive tract (i. e., foregut, midgut, and hindgut) and which has been implicated in hindgut differentiation. We identified an apparent null allele of eel-1 in a deletion knockout screen. This mutation, which removes the entire coding region, results in arrested L1s with a normal-looking gut. We do not yet understand the reason for L1 lethality. While the predicted EEL-1 open reading frame codes for a protein containing 159 amino acids with 3 C2 -H2 -zinc fingers, 5’-RACE suggests a product that that starts farther upstream, but which is still within the region removed by the knockout deletion. The absence of a conspicuous gut phenotype in the eel-1 knockout suggests either that it performs a role in gut differentiation (e.g., activation of digestive functions) that is not evident in the structure of this organ or that there exists a gut differentiation factor that acts redundantly with eel-1. To test for such redundancy, we have begun to analyze genetic interactions between eel-1 and other genes expressed at similar stages in the gut lineage. We found that RNAi of the hnl-1 gene, which encodes an HNF-4-like transcription factor expressed in the early E lineage, does not yield a discernible phenotype alone or in combination with the eel-1 knockout mutation. We are further investigating genetic and regulatory interactions between these and other genes expressed in the early gut lineage.

24 May 1999 15:50 864 864 nos-1 and nos-2, two genes related to Drosophila nanos, are required for primordial germ cell development.

1999 International Worm Meeting abstract 813 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. nos-1 and nos-2, two genes related to Drosophila nanos, are required for primordial germ cell development. K Subramaniam 1 , T Brodigan 2 , M Krause 2 , G Seydoux 1

1 Dept. of Mol. Biol. and Genetics, Johns Hopkins U., School of Med., Baltimore, MD 21205. 2 NIH/NIDDK/LMB, Building 5, Room B1-04, 5 Center Drive, MSC 0510, Bethesda, MD 20892.

The mechanisms that guide the development of primordial germ cells (PGCs) during embryogenesis are still largely unknown. In Drosophila, the maternal factor Nanos was shown recently to be required for PGC migration into the somatic gonad1 . We have investigated whether a similar function may be performed by nanos-related genes in C. elegans. We identified three nanos-related genes in the database and have shown that two of them, nos-1 and nos-2, regulate the development of PGCs. Both genes encode proteins specific to the germline. NOS-2 is expressed in oocytes, and in P4 and Z2 and Z3 in early embryos. Fluorescent in situ experiments indicate that maternal nos-2 RNA is concentrated on P granules in early embryos. In contrast, NOS-1 is expressed zygotically in Z2 and Z3 and their progeny starting in mid-embryogenesis. Simultaneous disruption of nos-1 and nos-2 by RNAi results in sterility in 99% of treated animals. In contrast, 0% of nos-1(RNAi) and only 30% nos-2(RNAi) animals are sterile. Similar results were also obtained with nos-1(jv5), a deletion allele which lacks most of the nos-1 ORF. Immunostaining of the three "mutant" combinations with P granule antibodies reveals that NOS-2 is required for efficient incorporation of Z2 and Z3 into the somatic gonad, and that NOS-1 and NOS-2 are required redundantly to prevent premature proliferation of Z2 and Z3 and maintain germ cell viability in larvae. These results indicate that a requirement for nanos-like genes in PGC development has been conserved between flies and worms, and suggest that nanos homologs in vertebrates may also function in the germline.

1. Forbes, A. and Lehmann, R. Development 125, 679-690 (1998).

24 May 1999 15:50 865 865 osm-10, osm-16(rt6) and not(rt32): genes required for signal transduction in the ASH sensory circuit.

1999 International Worm Meeting abstract 814 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. osm-10, osm-16(rt6) and not(rt32): genes required for signal transduction in the ASH sensory circuit. H Sugimoto 1 , S Matsumoto 2 , R Moeller 3 , AC Hart 1,3

1 Harvard Medical School, Dept. of Pathology. 2 Kyoto University, Kyoto, Japan. 3 MGH Cancer Center, Charlestown, MA 02129.

Mechanosensation and osmosensation are essential, yet poorly understood processes. The ASH neurons in C. elegans detect mechanical, osmotic and chemical stimuli; laser ablation studies indicate that they are primarily responsible for detecting light touch to the nose, high osmolarity and volatile repellents (1-octanol) (CGC82; WBG 10(1):89; CGC2309 and CGC1374). We are studying the molecular pathways involved in detecting and distinguishing these stimuli using modality specific mutations - those defective in responding to only 1 or 2 of the ASH stimuli. OSM-10, a novel cytoplasmic protein with 38 putative phosphorylation sites, is required specifically for osmosensation (ECWM97 #226), as an osm-10 null allele, nr2076, disrupts osmosensation but not mchanosensation nor chemosensation. (Thanks NemaPharm). An E to K mutation in a putative tyrosine phosphorylation site of OSM-10 also disrupts osmosensation. Thus, osm-10 may play a role in intracellular signal transduction. To elucidate the function of osm-10 and to identify other components of the ASH-mediated osmosensory pathway, we are isolating putative OSM-10 interacting proteins using the yeast two-hybrid system. (Thanks to M. Walhout, H. Endoh and M. Vidal). The expression patterns and the gene disruption phenotypes of putative interactors will be determined to test the biological relevance of the interactions. We are also characterizing 2 new modality specific mutations osm-16(rt6ts) and not(rt32). rt6 animals are defective in their response to high osmolarity and 1-octanol, but are normal in nose touch avoidance. Thus, rt6 may encode a component of the signal transduction machinery shared by osmosensation and chemosensation, but not by mechanosensation. rt6 maps to the right end of the X chromosome near unc-7. Further mapping and cloning of rt6 is now underway. rt32 animals are defective in nose touch response, but are wild-type for other ASH-mediated responses. We are currently mapping rt32. Characterization of rt6 and rt32 mutants should lead us to the better understanding of mechanosensation and osmosensation, as well as the molecular mechanisms involved in distinguishing different stimuli.

24 May 1999 15:50 866 866 Possible role for a C. elegans homologue of protein phosphatase 4 in the replication and function of centrosomes

1999 International Worm Meeting abstract 815 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Possible role for a C. elegans homologue of protein phosphatase 4 in the replication and function of centrosomes E Sumiyoshi, A Sugimoto, M Yamamoto Dept. Biophys. Biochem., Grad. Schl. Sci., Univ. Tokyo, Japan Protein phosphatase 4 (PP4) is a conserved ser/thr protein phosphatase, which is known to localize to centrosomes. From the study using a Drosophila mutant in which expression of PP4 is reduced, this phosphatase has been implicated to be required for the nucleation of microtubules by centrosomes. In C. elegans, two possible homologues of PP4, namely Y75B8A.30 and Y49E10.3, have been identified by the genome project. They show 76% and 70% amino acid identity to rabbit PP4, respectively. To investigate the function of PP4 in C. elegans, we performed RNAi experiments for these PP4 homologues using cDNA clones kindly provided by Y. Kohara. RNAi for Y75B8A.30 caused embryonic lethality, while RNAi for Y49E10.3 caused larval lethality, indicating that the two PP4 homologues act at different stages of the life cycle. We further characterized Y75B8A.30 to understand its function during embryogenesis. Double staining with anti-tubulin antibody and DAPI revealed that some fraction of 1-cell embryos of Y75B8A.30(RNAi) had condensed chromosomes at the metaphase plate with no astral nor kinetochore microtubules, indicating that the aster and spindle formation was delayed compared to the nuclear event. Another fraction of Y75B8A.30(RNAi) embryos showed a multi-polar spindle with two sperm pronuclei at the first cleavage, which might be resulted from either multi-fertilization or over-replication of centrosomes. To distinguish these two possibilities, we performed RNAi for Y75B8A.30 in zyg-1 (oj7) mutant, in which centrosomes can not replicate at the restrictive temperature. In the pseudo-double mutant embryos, neither multiple pronuclei nor multi-polar spindle formation was observed. Thus, we speculate that the defects seen in Y75B8A.30(RNAi) embryos are likely to be caused by premature replication of centrosomes rather than multi-fertilization. In Y75B8A.30(RNAi) embryos, the spindle formation and/or the centrosomal replication cycle appear to be uncoordinated with the nuclear division cycle, suggesting that PP4 in C. elegans may play a key role in the regulation of the replication cycle of centrosome. For further characterization, we are currently screening for a deletion mutant of Y75B8A.30 by the UV/TMP method.

24 May 1999 15:50 867 867 Towards Cloning of daf-5

1999 International Worm Meeting abstract 816 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Towards Cloning of daf-5 Li Sun, Garth Patterson Rutgers University, Department of Molecular Biology and Biochemistry A TGF-ß-like signaling pathway is involved in the developmental determination of dauer/L3 fate in C. elegans. Dauer-formation defective mutations in daf-3 and daf-5 are epistatic to dauer-formation constitutive mutations in daf-7 (encoding TGF-ß ligand), daf-1 and daf-4 (encoding the likely receptors for DAF-7), and daf-8 and daf-14 (encoding Smad proteins). This epistasis relationship suggests that daf-3 and daf-5 are either negatively regulated by the TGF-ß pathway or acting in parallel. DAF-3 is a Smad transcription factor; daf-5 is not yet cloned. DAF-5 may function in a parallel subpathway, or act as a transcription cofactor or a modifier of DAF-3. We are pursuing cloning of daf-5 to allow us to better understand the developmental program of larvae in C. elegans. daf-5 is located on Chromosome II, close to unc-52, with very few useful mapping markers around. We are trying to get fine mapping with DNA polymorphisms according to the method described by Anneliese M. Schaefer et al. (Worm Breeder’s Gazette 15(4): 17), injecting cosmids for transformation rescue, and analyzing candidate genes in the region.

24 May 1999 15:50 868 868 A genetic screen for synthetic Vulvaless/Lethal mutations

1999 International Worm Meeting abstract 817 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A genetic screen for synthetic Vulvaless/Lethal mutations MV Sundaram, A Goldman, R Howard University of Pennsylvania School of Medicine, Philadelphia PA 19104 Vulval fate induction is controlled by several different signaling pathways, including an RTK/Ras/ERK pathway. Some genes that influence vulval development have wild-type or nearly wild-type null phenotypes, and their roles are only revealed in appropriate sensitized genetic backgrounds. For example, mutations in ksr-1 and sur-8 do not cause strong phenotypes singly, but do cause highly penetrant Vulvaless and Lethal phenotypes in combination with each other, or in combination with weak alleles of other Ras pathway genes such as lin-45 raf. We reasoned that we could identify additional genes acting with ksr-1 or sur-8 by screening for additional synthetic Vul/Let mutations. As a starting point for the screen, we used the weak lin-45 raf mutants ku51 or ku112, which have nearly wild-type phenotypes. In our initial screens of under 5000 EMS mutagenized genomes, we’ve identified five mutations that cause moderately penetrant Egl and Lethal phenotypes in these lin-45 mutant backgrounds, but little or no phenotype in a lin-45(+) background: cs1 is an allele of ksr-1, cs24 is an allele of sur-6, cs26 is a novel non-Vul allele of sur-2, and cs28 IV and cs30 X appear to define new genes (which we are cloning). In addition to our screen, we’ve also directly tested candidate genes for synthetic effects using existing mutations or RNAi, and we observed synthetic effects for mig-2 rac and a CNK homolog, among others. By testing for synthetic effects of different mutation/RNAi pairs, we are attempting to place these new genes into functional groups that may define different redundantly-acting pathways. We find that ksr-1 and sur-6 are members of the same functional group, since ksr-1(n2526) and sur-6(cs24) do not synergize with each other, although each synergizes strongly with either lin-45(ku112) or sur-8(ku167). These genetic interactions are consistent with a model in which ksr-1 and sur-6 function together to promote vulval fates. Derek Sieburth and Min Han previously demonstrated similar interactions between the ksr-1(ku68) and sur-6(ku123) alleles, and showed that sur-6 encodes a regulatory B subunit of protein phosphatase 2A (PP2A). Thus, KSR-1 might function to regulate PP2A, or might be regulated by PP2A. We are collaborating with Sieburth and Han to test such models.

24 May 1999 15:50 869 869 Mediators and Modulators of dbl-1 Functions in Body Size Control

1999 International Worm Meeting abstract 818 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Mediators and Modulators of dbl-1 Functions in Body Size Control Y Suzuki, GA Morris, WB Wood Department of MCD Biology, University of Colorado, Boulder, CO 80309-0347 Size regulation of body components is a poorly understood aspect of animal growth and development. We have previously shown that the dbl-1 gene, a C. elegans gene encoding a TGF-b ligand, is a dose-dependent positive regulator of cell size and body size increase during post-embryonic development of the worm (1). Identification of additional components that act upstream and downstream of dbl-1 could provide new insights into the mechanisms of size regulation. Studies of the epistatic relationships between known lon genes and components of the dbl-1 signal transduction pathway have placed lon-2 and lon-3 upstream of dbl-1, and lon-1 downstream of the Smad genes of the pathway (2, 3). More careful analysis has shown, however, that dbl-1 lon-3 double mutants are small but slightly longer than dbl-1 single mutants, leaving open the possibility that lon-3 acts independently of the dbl-1 pathway. We are in the process of cloning lon-3 to allow further analysis of its function. To identify potential targets of the body size control pathway, we are carrying out screens for suppressors of the Sma and Lon phenotypes caused by dbl-1(lf) mutations and dbl-1 overexpression, respectively. In a screen of 14,000 haploid genomes for suppressors of the Sma phenotype, we have isolated a number of candidate suppressors. Some isolates exhibit phenotypes similar to the Lon-1 phenotype, and others exhibit phenotypes that resemble the semi-Sma phenotypes of dbl-1 lon-3 double mutants or dbl-1 heterozygotes. We plan genetic and molecular characterization of genes defined by these suppressors.

1. Suzuki, Y., Yandell, M. D., Roy, P. J., Krishna, S., Savage-Dunn, C., Ross, R. M., Padgett, R. W. and Wood, W. B. (1999). Development 126: 241-250. Similar results have been obtained by Morita, K., Chow, K. L., and Ueno, N. (1999). Development 126: 1337-1347. 2. Savage, S., Padgett, R., and Baird, S. (1994) WBG 13(2): 38 3. Suzuki, Y., Morris, G., and Wood, B. (1999) WBG 15(5): 44

24 May 1999 15:50 870 870 or191ts and or182ts are Embryonic Lethal, Temperature-Sensitive Mutations that Disrupt Spindle Rotation and Cell Cycle Timing, Respectively, in Two-Cell Stage C. elegans Embryos.

1999 International Worm Meeting abstract 819 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. or191ts and or182ts are Embryonic Lethal, Temperature-Sensitive Mutations that Disrupt Spindle Rotation and Cell Cycle Timing, Respectively, in Two-Cell Stage C. elegans Embryos. KA Swan, DR Hamill, BA Bowerman University of Oregon, Institute of Molecular Biology, Eugene, OR 97403-1229 USA. In order to understand the processes governing the establishment of asymmetry within the early C. elegansembryo, our lab has begun an investigation of several conditional mutants isolated in a screen for temperature sensetive, embryonic lethal mutations (see abstract by D. Hamill). Two of these mutations, or191ts and or182ts disrupt different aspects of asymmetry normally observed at the two-cell stage. The or191ts mutation results in random spindle orientation within the two-cell stage blastomeres AB and P1 , which normally divide transversely and longitudinally, respectively.

In addition to dividing along different axes, the cell cycle times of AB and P1 are unequal in wild-type embryos, with AB dividing slightly ahead of P1 . A second mutation we have identified increases this asymmetry. The mutation or182ts, delays the cell cycle timing of both AB and P1 . The AB cell cycle time, however, is increased only slightly, while the time of the P1 cell cycle is doubled. or191ts maps to the center of chromosome V while or182ts maps to the far left arm of chomosome III. Phenotypic analysis of these mutants and progress on identifying the affected genes will be presented. For information concerning the analysis of other mutants affecting similar processes see posters presented by Danielle Hamill and Sandra Encalada in our laboratory.

24 May 1999 15:50 871 871 A C. elegans model of Niemann-Pick type C disease: two NPC1 homologs act redundantly in dauer formation

1999 International Worm Meeting abstract 820 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A C. elegans model of Niemann-Pick type C disease: two NPC1 homologs act redundantly in dauer formation M Sym, M Basson, CD Johnson Axys Pharmaceuticals, NemaPharm Group, 100 Kimball Way, South San Francisco, CA 94080 Niemann-Pick type C (NP-C) disease is a progressive neurodegenerative disease characterized by the inappropriate accumulation of cholesterol in lysosomes. Recently, putative loss-of-function mutations in the human gene NPC1 were found to be associated with many cases of NP-C disease; however, the molecular function of the NPC1 protein is not known. To create a C. elegans model of NP-C disease, we generated deletion mutations in two NPC1 homologs, designated npc-1 and npc-2. Both deletions are predicted to cause early truncations in the protein and are therefore good candidates for null mutations. npc-1(nr2022) mutant animals display several defects including slow growth, a modest reduction in brood size and an egg-laying constitutive behavior. By contrast, npc-2(nr2023) mutant animals do not display any obvious phenotypes. Unexpectedly, npc-1(nr2022); npc-2(nr2023) double mutant animals display a completely penetrant dauer-constitutive (Daf-c) phenotype not observed in either single mutant, suggesting that npc-1 and npc-2 play redundant roles in dauer formation. This synthetic Daf-c phenotype can be rescued by arrays containing wild-type copies of either npc-1 or npc-2. The Daf-c phenotype of the npc double mutant is unusual in that it is not temperature-sensitive and that the mutant animals recover from dauer within several days. How might the npc genes be acting to control dauer larva formation? The fact that neural degeneration is the major defect in NP-C patients suggests that NPC1’s role in promoting normal cholesterol trafficking is critical for neuronal function. Thus, the npc genes might act in neurons (e.g.,amphid neurons) that regulate entry into and exit from dauer. The availability of a disease model for NP-C disease provides an opportunity to identify other genes involved in cholesterol trafficking and to discover therapeutics to treat NP-C patients.

24 May 1999 15:50 872 872 The role of the ER membrane protein calnexin in programmed cell death

1999 International Worm Meeting abstract 821 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The role of the ER membrane protein calnexin in programmed cell death M Sym, M Basson, CD Johnson Axys Pharmaceuticals, NemaPharm Group, 100 Kimball Way, South San Francisco, CA 94080 The ER membrane protein calnexin is thought to help proteins fold properly within the lumen of the ER and may function to insure that only properly folded proteins proceed further along the secretory pathway. Recently, calnexin has been hypothesized to play a role in cell death based on experiments in yeast (1). To test the role of calnexin in programmed cell death, we isolated two deletion mutations in the C. elegans calnexin homolog cnx-1. Although both deletions are predicted to cause early truncations in the protein, the two deletions had different phenotypic effects. cnx-1(nr2009) animals exhibited a partially penetrant larval lethality and a mild reduction in the number of programmed cell deaths. By contrast, cnx-1(nr2010) animals appeared wild type. Although the lack of a mutant phenotype in nr2010 animals remains puzzling, these results suggest that calnexin plays roles in both a process essential for viability and in promoting cell death. In preliminary experiments to examine whether calnexin acts in the canonical cell death pathway, RNAi of cnx-1 did not suppress the lethality caused by a ced-9(lf) allele, suggesting that calnexin does not act directly downstream of ced-9. Calnexin’s role in promoting cell death was supported by examining transgenic animals carrying cnx-1 arrays. These animals exhibited a high frequency of embryonic lethality, but those embryos that progressed to the comma stage had an increased number of cell deaths compared to wild type. Thus, cnx-1 transgenic animals presumably over-expressing calnexin appear to have more cell deaths than normal, the opposite of the effect seen in nr2009 deletion mutants. These results raise the possibility that calnexin acts not only as a chaperone for protein folding within the ER, but also to relay information concerning misfolded proteins to the cell death machinery.

(1) Torgler et al. (1997) Cell Death Diff., 4:263.

24 May 1999 15:50 873 873 The role of serotonin in C.elegans

1999 International Worm Meeting abstract 822 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The role of serotonin in C.elegans J Sze 1 , M Victor 2 , Y Shi 2 , G Ruvkun 3

1 Dept. of Anatomy and Neurobiology, College of Medicine, UC Irvine, CA 92697. 2 Department of Pathology, Harvard Medical School, Boston, MA 02115. 3 Dept. of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114.

We are interested in identifying POU-factor unc-86 downstream genes that mediate neuronal function and animal behaviors. UNC-86 is expressed in the serotonergic neurons NSM, RIH, AIM, and HSN. In unc-86 null mutants, these neurons are generated, but RIH, AIM, and HSN do not accumulate serotonin. We have used a candidate gene approach and found that a putative tryptophan hydroxylase gene is regulated by unc-86. The enzyme tryptophan hydroxylase catalyzes the first and rate-limiting step of neurotransmitter serotonin biosynthesis. The complete genome sequence of C. elegans reveals the presence of one sequence homologous to tryptophan hydroxylase, zk1290.2, which we name tph-1. Analysis of tph-1 cDNA indicates that tph-1 has 41% identity over the entire coding region and 59% identity at the C-terminal 2/3 of the sequence corresponding to the catalytic domain to the human brain tryptophan hydroxylase. The tph-1::GFP is strongly expressed in the secretory motor neuron NSM, the amphid sensory neuron ADF, and the motor neuron HSN. Vary rarely, it is expressed in the interneurons AIM and RIH. In males, it is in addition expressed very strongly in the male-specific CP neurons and 4-5 cells in the tail. unc-86 is expressed in the serotonergic neurons NSM, AIM, RIH, and HSN. In unc-86(n846) null mutant animals, these neurons are generated, but do not express the tph-1::GFP, whereas the tph-1::GFP expression in ADF is not affected. ADF does not express UNC-86. tph-1 is located in the genetic region to which a male mating mutant cod-5 has been approximately mapped, a tph-1 intron mutation in cod-5(sy181) that we reported in the 1998 East Coast worm meeting abstract was not detected in subsequent analyses, so we are still unsure about the connection between cod-5 andtph-1. We generated a tph-1 deletion mutation by sib-screening mutagenized animals with PCR primers from the tph-1 gene. The resulting mutant allele, tph-1(mg280) is viable, but shows a variety of behavior defects. We are currently screening for other factors that regulate the tph-1::GFP expression, and mutations that suppresstph-1(mg280) phenotypes.

24 May 1999 15:50 874 874 Essential role of emb-1 and emb-8 in establishing early embryonic polarity

1999 International Worm Meeting abstract 823 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Essential role of emb-1 and emb-8 in establishing early embryonic polarity Y Tabuse 1 , J Miwa 2

1 Fundamental Research Laboratory. 2 R&D Group, NEC Corporation.

Asymmetric cell divisions critically important to generate diverse cell types in multicellular organisms require polarized distribution of cytoplasmic components and the proper alignment of the mitotic spindle. In early C. elegans embryos, various cellular components including P-granules become polarized along the anterior-posterior (A-P) axis, which is defined by an extrinsic cue provided by sperm. The first mitotic spindle posteriorly placed results in asymmetric first cleavage. Maternally expressed par genes are known to require the establishment of the A-P polarity in the early C. elegans embryo. Recently, we have reported that an atypical type protein kinase C (PKC), PKC-3, co-localizes with PAR-3 asymmetrically to the anterior cortex of one-cell embryos and plays an essential role in establishing the early embryonic polarity. In order to identify other genes necessary for polarity establishment, we began to screen for mutants that show abnormality in PKC-3 localization and early cleavage pattern. We have stained about 30 existent maternal-effect embryonic lethal mutants with an anti-PKC-3 antibody and so far found that several mutants show par-like defects in PKC-3 localization and early cleavage pattern. Among those emb mutants, we report here the phenotypic characteristics of emb-1 and emb-8 mutants. About 75% of one-cell embryos in both mutants show uniform PKC-3 distribution and symmetric first division at 23.5C. Both AB and P1 blastomeres divide transversely to the A-P axis at the second cell cycle in most of the affected embryos. These phenotypes are similar to those exhibited by par-2 and par-5 mutants. P-granules are mislocalized or undetectable in most of emb-1 and emb-8 mutant embryos, although the defect of P-granule-localization seems to be more severe in emb-8 than in emb-1 embryos. To understand the functions of these genes in polarity establishment, we are now investigating the relationship between emb-1 and emb-8 genes and pkc-3 or par genes, and also trying to clone emb-1 and emb-8 by cosmid rescue.

24 May 1999 15:50 875 875 CeHSF and HSB-1: The heat shock response regulators

1999 International Worm Meeting abstract 824 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. CeHSF and HSB-1: The heat shock response regulators Li-Jung Tai, Sanjeev Satyal, Sally McFall, Sue Fox, Catherine Airey, Tim Roytman, Richard Morimoto Northwestern University, 2153 Sheridan Road, Evanston, IL 60208 One of the key components of the stress response is the heat shock transcription factor whose activation leads to synthesis of heat shock proteins. We have analyzed the C. elegans HSF with the help of C. elegans genome project, and found the following features. First, the C. elegans genome appears to encode a single heat shock factor (CeHSF Y53C10A.12), unlike higher metazoans whose genomes encode multiple HSFs. Second, CeHSF encodes a 75 KD protein with a DNA binding domain (residues 87 to 196) and a trimerization domain composed of hydrophobic repeats (HR) A and B (residues 193 to 285). Although the overall identities of CeHSF to S. Cerevisiae (ScHSF) and human HSF (HsHSF) are 17 and 24% respectively, the identities of the DNA binding and trimerization domains to ScHSF and HsHSF are 23 and 40% respectively. Third, sequence analysis of CeHSF showed an apparent lack of HR-C, a regulatory motif of heptad repeats at the C terminus conserved in all known animal HSFs. HR-C has been shown to be important for keeping HSF in a latent monomeric state during unstressed condition. The fact that C. elegans heat shock response has been shown to be inducible, yet CeHSF lacks the HR-C to keep it inactive in normal condition indicates that the mechanism of CeHSF activation may be distinct from other known animal HSFs. Lastly, the EST of C. elegans HSF (EST yk485h11.5) binds specifically to the consensus heat shock element when expressed as a recombinant fusion protein in bacteria. Currently, we are making antibodies and deletion worms to study the expression and function of CeHSF. In the process of dissecting the regulatory mechanism of HSF, a heat shock binding protein (HSB-1) was cloned and shown to negatively regulate the heat shock response in both C. elegans and mammalian cell lines. The HSB-1 deletion worms have a 30% decrease in life span comparing to N2 wildtype worms. The role of HSB-1 in regulating the heat shock response and its interaction with HSF are being examined using overexpression and deletion worms, as well as mammalian tissue culture models.

Satyal SH et. al. Genes and Dev. 1998, 12(13):1962-74. Stringham, EG et. al. Molecular Biology of the Cell. 1992, 3:221-233. Wu, C. Annu. Rev. Cell Dev. Biol. 1995, 11: 441-69.

24 May 1999 15:50 876 876 Pathway analysis of the daf-12 nuclear receptor receptor

1999 International Worm Meeting abstract 825 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Pathway analysis of the daf-12 nuclear receptor receptor D Tait, A Ludewig, A Antebi Max-Planck-Institut für Molekulare Genetik, D-14195 Berlin daf-12 encodes a nuclear receptor, a class of transcription factors that typically depends on steroid or retinoid-like ligands to transactivate genes. daf-12 lies at the intersection of heterochronic and dauer pathways, regulating L3 larval stage options of reproductive growth and the dauer diapause. Mutants exhibit delayed heterochronic phenotypes and inappropriately regulate dauer formation. In addition, daf-12 mutants enhance the Age phenotype of daf-2 hypomorphic alleles. The molecular identity of daf-12 suggests that endocrine factors could regulate developmental age, diapause and perhaps aging in nematodes. We would like to identify the regulators, interacting proteins as well as downstream targets of daf-12. Potential inputs from the dauer pathway include components of TGF-beta and Insulin/IGF-like signaling. We have performed genetic epistasis experiments with daf-12 Daf-c alleles and Daf-d mutants which act at the terminal branches of these two pathways, daf-3 (encoding a SMAD from the TGF-beta branch) and daf-16 (encoding a Forkhead homolog from the Insulin branch). In double mutants, daf-12 Daf-c phenotypes are partially epistatic, placing daf-12 downstream or in parallel. However, daf-3 and daf-16 could modify daf-12 activity, or have daf-12-independent roles in dauer formation. To further understand daf-12 regulation, we are constructing daf-12::GFP gene fusions as well as generating polyclonal antibodies. We are also screening two-hybrid libraries for potential daf-12 interacting proteins.

24 May 1999 15:50 877 877 Cloning, characterization and gene disruption of CeCRMP/DHP-1and -2

1999 International Worm Meeting abstract 826 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cloning, characterization and gene disruption of CeCRMP/DHP-1and -2 T Takemoto 1 , H Kimura 1 , Y Sasaki 2 , N Hamajima 3 , Y Goshima 2 , M Nonaka 4

1 Dept. Exp. Radiol., Shiga Univ. Med. Sci. 2 Dept. Pharmacol., Yokohama City Univ. Sch. Med. 3 Dept. Pediat., Nagoya City Univ. Med. Sch. 4 Grad. Sch. Sci., Univ. Tokyo.

The vertebrate CRMP/DRP (collapsin response mediator protein/dihydropyrimidinase-related protein) family contains at least four members, which are implicated in the development of the nervous system. Dihydropyrimidinase (DHP), an amidohydrolase involved in the pyrimidine degradation pathway, shows about 60 % amino acid identity with CRMP/DRP. DHP is a zinc enzyme, and the five possible zinc-binding residues, four histidines and one aspartic acid, have been identified by aligning its amino acid sequence with other amidohydrolases. Interestingly, all CRMP/DRP members have replacement at one or more of these residues, and therefore, most probably lack amidohydrolase activity. To elucidate evolution of CRMP/DRP and DHP, we isolated two cDNA clones from C. elegans, which show a closer similarity than previously identified unc-33 to CRMP/DRP and DHP. The phylogenetic tree analysis indicated that the gene duplication and following differentiation between CRMP/DRP and DHP occurred in the vertebrate lineage after the divergence of nematodes. Therefore, these two sequences were termed CeCRMP/DHP-1 and -2, implying that they correspond to a common ancestor of CRMP/DRP and DHP. Although both CeCRMP/DHP-1 and -2 retained all the five zinc-binding residues, only CeCRMP/DHP-1 exhibited a significant DHP activity when expressed in COS cells. Exon/intron organizations of the CeCRMP/DHP-1 and -2 genes showed a closer similarity to human CRMP/DRP-2 than to human DHP. Targeted gene disruption of CeCRMP/DHP-1 and -2 by TMP/UV method was performed to investigate their in vivo function. We have already isolated one CeCRMP/DHP-2 deleted mutant strain, and the results of the detailed analyses will be presented.

24 May 1999 15:50 878 878 Effects of UNC-2, a a-1 Calcium Channel Subunit, on Neuronal Migration and Development

1999 International Worm Meeting abstract 827 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Effects of UNC-2, a a-1 Calcium Channel Subunit, on Neuronal Migration and Development TM Tam, WR Schafer Department of Biology, University of California at San Diego; La Jolla, CA 92093 Our lab is interested in the roles of voltage gated calcium channels in nervous system function and development. We have been studying the product of the gene unc-2, which encodes the homolgue of the vertebrate a-1 subunit of a non-L-type calcium channel. Typical unc-2 alleles display three main phenotypes: sluggishness, kinky uncoordinated movement and constitutive egg-laying (Egl-c). Mosaic analysis and RNA in situ hybridization have correlated the sluggish behavior to motorneurons and muscles, the kinky uncoordinated movement to motorneurons and Egl-c to egg-laying neurons. Recently, we have analyzed deletion and insertion alleles of unc-2, some of which are putative nulls. In these alleles, we have observed an additional phenotype: the final positions of the neurons descended from QR are abnormal. In particular, the dorsal/ventral positioning of the nuclei is often disrupted, suggesting that unc-2 is specifically required for the final dorsal/ventral migration of these cells. These defects are approximately 50% penetrance in the most severe deletion mutants (Mary Simms in Cynthia Kenyon’s lab first noticed a very low penetrance defect in the EMS-generated allele unc-2(e55)). The QL migration as well as the ALM’s appear normal. To determine the role of the calcium channel, we are testing expression pattern with GFP, and the cell autonomy of the migration defect by mosaic analysis. These experiments may indicate whether the UNC-2 calcium channel mediates a calcium influx directing the dorsal/ventral positioned Q cell migration. aunc a

24 May 1999 15:50 879 879 PGP and MRP homologs in C. elegans protect the nematode from Pseudomonas aeruginosa pathogenicity

1999 International Worm Meeting abstract 828 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. PGP and MRP homologs in C. elegans protect the nematode from Pseudomonas aeruginosa pathogenicity MW Tan 1 , A Broeks 2 , RH Plasterk 1 , FM Ausubel 2

1 Molecular Biology, Massachusetts General Hospital, Boston, MA 02114. 2 Molecular Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam.

The multidrug resistance P-glycoprotein (PGP) and the multidrug resistance-associated protein (MRP) are members of an evolutionarily conserved ABC transporter family. The C. elegans genome contains at least 14 PGPs and 4 MRPs. PGP-1, PGP-3 and MRP-1, but not PGP-4, protect C. elegans from heavy metal toxicity. Only PGP-3 is required for resistance to chloroquine and colchicine (1, 2). We tested the role of these genes in the C. elegans-P. aeruginosa pathogenesis model (3, 4). P. aeruginosa strain PA14 kills C. elegans by at least two distinct mechanisms. In low salt, minimal medium, PA14 kills worms in 2-3 days ("slow-killing", SK). When worms are exposed to PA14 grown in a high osmolarity, rich medium, they die within 4-24 hours ("fast-killing", FK). Characterization of bacterial mutants that have dramatically different effects in the two conditions provide evidence that FK and SK are mechanistically distinct (4). Deletion of any of the pgp and/or mrp genes tested results in increased worm mortality to P. aeruginosa under the FK conditions. One of the mediators of FK is pyocyanin (3). To determine the specificity of various MRP and PGPs proteins with respect to protecting C. elegans from killing by pyocyanin, we tested two P. aeruginosa mutants, one that synthesizes wild-type levels of pyocyanin and one that synthesizes reduced levels. Only PGP-1 and MRP-1 protect against pyocyanin-mediated killing. SK involves an infection-like process (4) and only the pgp-4 and mrp-1 deletion mutants are more sensitive. Because some defecation mutants are also more sensitive to SK by PA14, we tested pgp-4 and mrp-1 for defects in the defecation motor program. Interestingly, although both mutants are not constipated, they are defective in the expulsion (Exp) step. The above data suggest that ABC transporters have a protective role in pathogenesis and, in addition, may also be required for proper function of the C. elegans defecation motor program.

1. Broeks, A. et al (1995) EMBO J. 14, 1858-66 2. Broeks, A. et al (1996) EMBO J. 15, 6132-43 3. Mahajan-Miklos, S. et al (1999) Cell 96, 47-56 4. Tan, M.-W. et al (1999) PNAS 96, 715-20.

24 May 1999 15:50 880 880 A C. elegans SEK-1 MAP kinase kinase regulates egg-laying and male turning behaviors.

1999 International Worm Meeting abstract 829 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A C. elegans SEK-1 MAP kinase kinase regulates egg-laying and male turning behaviors. M Tanaka-Hino 1 , M Kawasaki 1 , K Nishiwaki 2 , N Hisamoto 1 , K Matsumoto 1

1 Division of Biological Science, Graduate School of Science, Nagoya Univ., CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya 464-8602, JAPAN. 2 Fund. Reseach Lab., NEC Corp., Miyukigaoka, Tsukuba, JAPAN.

JNK and p38 belong to a subgroup of the mitogen-activated protein kinase (MAPK) superfamily and are activated in response to a variety of stresses in mammalian cells. SEK1/MKK4 activates both JNK and p38 MAPKs as a MAPK kinase. We have identified C. elegans SEK1 homolog, SEK-1. In hermaphrodites, sek-1::gfp was expressed in many cells including excretory/secretory cells, all VCn motorneurons, all touch receptor neurons, and vulval epithelial cells. In males, sek-1::gfp was expressed in ventral nerve cord neurons, probably CPn motorneurons, which have the lineages same as VCn in hermaphrodites. To understand the role of sek-1,we generated a sek-1 disruption allele. The sek-1 null mutant hermaphrodites were defective in egg-laying (Egl-d) and formed bag-of-worms. Other behaviors including pharyngeal pumping and defecation were normal in sek-1 mutant animals. Trimeric G protein a subunits GOA-1 and EGL-30 are involved in regulation of egg-laying. Since goa-1 mutant and egl-30 overexpression animals show egg-laying constitutive (Egl-c) phenotype, we analyzed the genetic interaction of sek-1 with goa-1 and egl-30. The goa-1;sek-1 double mutant showed both Egl-d and Egl-c phenotypes, suggesting that goa-1 and sek-1 regulate egg-laying independently. Egl-30 overexpression in the sek-1 mutant exhibited the Egl-c phenotype, suggesting that SEK-1 functions upstream of EGL-30. We next analyzed the male phenotype in sek-1 mutant males. The sek-1mutant males were morphologically normal but showed a defect in turning behavior. These results suggest that the MAPK cascade mediated by SEK-1 regulates egg-laying and male turning behaviors via VCn and CPn neurons, respectively.

24 May 1999 15:50 881 881 Knockout of Elongation Factor-2 Kinase Extends Lifespan in C. elegans

1999 International Worm Meeting abstract 830 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Knockout of Elongation Factor-2 Kinase Extends Lifespan in C. elegans N Tavernarakis 1 , CE Mendola 2 , KS Pavur 2 , L Zhang 3 , Z Altun-Gultekin 2 , W Wadsworth 2 , M Driscoll 1 , AG Ryazanov 2

1 Dept. of Molecular Biology and Biochemistry, Rutgers Univ., Piscataway, NJ. 2 Dept. of Pharmacology and Pathology, UMDNJ-R.W. Johnson Medical School, Piscataway, NJ. 3 Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Canada.

Elongation factor-2 kinase (EF-2 kinase) is a ubiquitous protein kinase that regulates protein synthesis by phosphorylating and inactivating elongation factor-2 (EF-2), which catalyzes movement of the ribosome along mRNA during translation (Nature 334:170-173). EF-2 kinase belongs to an emerging new class of protein kinases called alpha-kinases that are structurally and evolutionarily unrelated to conventional eukaryotic protein kinases (Proc. Natl. Acad. Sci. 94: 4884-4889; Curr. Biol. 9: R43-R45). The physiological role of EF-2 phosphorylation remains a mystery. To investigate the role of EF-2 kinase, we isolated a TC1 insertion mutant for C. elegans EF-2 kinase. The TC1 insertion is in the middle of exon 2 which contains the EF-2 kinase catalytic domain. The TC1 insertion completely abolishes EF-2 kinase activity. Mutant worms lacking functional EF-2 kinase showed slightly decreased locomotion, pharyngeal pumping, defecation, and developmental rates. The mutant phenotypes were rescued by injection of the wild-type EF-2 kinase gene. Intriguingly, C. elegans eEF-2 kinase loss of function mutants live 25% longer than do wild type animals. These results provide the first direct evidence that regulation of protein synthesis and longevity may be intimately related.

24 May 1999 15:50 882 882 Cytoskeleton dynamics affect the function of membrane channels and interfere with mec-4(d)-induced cell death

1999 International Worm Meeting abstract 831 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cytoskeleton dynamics affect the function of membrane channels and interfere with mec-4(d)-induced cell death NN Tavernarakis, SL Wang, MA Driscoll Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, New Jersey In our analysis of mutations that block mec-4(d)-induced degeneration of the six touch cells, we identified lesions in a gene which encodes a novel protein, that shares a low level of similarity to microtubule-associated proteins. This gene spans cosmids F08B6 and C37A2 and was originally predicted by the Genome Sequencing Consortium as being two separate genes (F08B6.5 and C37A2.5, on the two cosmids). Deletions of the gene cause animals to arrest growth early in development. We tested for complementation of lethal mutations in the region and determined that this gene corresponds to let-398. It also appears to be allelic to vms-1 an essential gene that genetically interacts with the kinesin-like gene vab-8. Animals with lethal mutations in vms-1 can be rescued by introduction of multiple copies of vab-8. let-398/vms-1 is expressed broadly in the C. elegans nervous system including sensory neurons, interneurons and motor neurons of the ventral nerve cord. Additionally, strong expression is observed in the pharynx, the canal cell and some muscles. Interestingly, expression of the kinesin-like gene vab-8 extensively overlaps with let-398 expression. Our findings implicate a novel cytoskeletal component in biogenesis, function and/or stability of degenerin channels. Our working hypothesis is that let-398 is involved in the intracellular trafficking of proteins across long neuron axons to their destined sub-cellular localization. We are currently in the process of evaluating this model by directly examining the sub-cellular localization and stability of MEC-4 and UNC-8 in the absence of LET-398 and VAB-8.

24 May 1999 15:50 883 883 Distinct cytoplasmic and nuclear functions for the germline factor PIE-1?

1999 International Worm Meeting abstract 832 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Distinct cytoplasmic and nuclear functions for the germline factor PIE-1? C Tenenhaus, M Dunn, M Wallenfang, K Subramaniam Mol. Bio. and Genetics, Johns Hopkins U., S.O.M., Baltimore MD. PIE-1 is a maternal protein which segregates with the early germ lineage where it maintains germ cell fate. We showed previously that one function of PIE-1 is to inhibit mRNA transcription in germline blastomeres. Consistent with this, PIE-1 is present in nuclei, and can inhibit transcription when expressed in Hela cells (Batchelder et al., 1999). PIE-1, however, is also present in the cytoplasm of germline blastomeres and on P granules, raising the possibility that it has a function outside the nucleus. We obtained initial evidence in support of this possibility by analysing the effect of PIE-1 on nos-2 expression. nos-2 is a nanos-related gene which encodes a maternal RNA preferentially maintained in germline blastomeres and on P granules. NOS-2 protein is expressed in P4 and its daughters Z2 and Z3. This expression is dependent on PIE-1; in pie-1(null) embryos, NOS-2 protein is absent. This effect may be caused by a lack of maternal nos-2 RNA in P4, since in pie-1(RNAi) embryos nos-2 RNA is prematurely lost from the germ lineage. To test whether PIE-1’s effect on NOS-2 expression is dependent on PIE-1’s ability to repress transcription, we examined pie-1(zu154)smg-1(cc545ts) embryos. These embryos express a truncated form of PIE-1 missing the domain required for transcriptional repression in Hela cells. We find that this mutant is no longer able to repress transcription, but is still able to express NOS-2. This result suggests that PIE-1 has two independent activities: one required to inhibit transcription, and one required to express NOS-2 and possibly other maternal factors. Interestingly, germ cells in pie-1(zu154)smg-1(cc545ts) express the gut marker ELT-2, as do germ cells in pie-1(null) mutants, suggesting that PIE-1’s ability to repress transcription is essential to maintain germ cell fate. To further dissect pie-1 function, we have devised a method to efficiently express mutant pie-1 transgenes in pie-1(null) embryos and assay their ability to repress transcription, activate NOS-2 expression and maintain germ cell fate. We are currently using this method to determine which domains of PIE-1 are responsible for each of these activities.

24 May 1999 15:50 884 884 Functional analysis on the troponin C of the pat-10 mutant of Caenorhabditis elegans

1999 International Worm Meeting abstract 833 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Functional analysis on the troponin C of the pat-10 mutant of Caenorhabditis elegans H Terami, H Kagawa Okayama university

Troponin C is a Ca2+ -binding component of troponin complex involved in Ca2+ regulation of muscle contraction through the thin filament. We have identified two C. elegans troponin C genes, pat-10 and tnc-2. pat-10::lacZ fusion gene was expressed in body wall muscles. Mutation of pat-10(st575) showing a Pat(paralyzed arrest at embryonic two-fold stage) phenotype raised two point mutations in troponin C at D64N and W153*. The first mutation was in the second Ca2+ -binding site and the second caused a deletion of the C-terminal H-helix. To determine the function of the mutant troponin C, we constructed two mutant troponin C proteins PAT-10-M1(D64N) and PAT-10-M2(W153*) by using site-directed mutagenesis. PAT-10-M1 had no mobility shift in the presence and absence of Ca2+ on SDS-PAGE and was different from the wild type. PAT-10-M2 showed a Ca2+ -dependent mobility shift, although it could not bind to TNI-1 by West-western analysis. We found that the D64 site is important for Ca2+ -dependent conformation change and the C-terminal H-helix is essential for troponin I binding. This result was consistent with recent X-ray crystallographic structures. We also found the pat-10 (st575) embryo failed to stain with an antibody to troponin C. These results suggest the pat-10 (st575) could come from defects of Ca2+ -dependent conformation change and of troponin I binding. This is the first troponin C mutant analysis in any animal. We hope to understand the function of troponin C in the development of muscle by analyzing of pat-10 mutant. We are currently constructing animal with each of two different mutations to know what kind of phenotype appeared in the mutant animal. In addition, We also found that tnc-2 is only expressed in the pharyngeal muscles by injecting tnc-2::lacZ fusion plasmid. It will be of interest to know whether TNC-2 can function in the body wall muscles and can also rescue Pat phenotype.

24 May 1999 15:50 885 885 Comparative genomic analysis: the structure of a complex gene, bli-4

1999 International Worm Meeting abstract 835 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Comparative genomic analysis: the structure of a complex gene, bli-4 C Thacker 1 , MA Marra 2 , A Jones 1 , DL Baillie 3 , AM Rose 1

1 University of British Columbia. 2 Washington University. 3 Simon Fraser University.

Comparative genomic analysis was used to investigate the gene structure of the bli-4 locus from two related Caenorhabditis species. In C. elegans, bli-4 is a complex gene encoding a member of the kex2/subtilisin-like family of proprotein convertases. The C. briggsae gene was able to rescue both viable blistered and developmentally arrested mutants of C. elegans bli-4, demonstrating functional conservation. The conservation of the gene structure between the two species was determined. Both gain and loss of introns were observed. Sequence comparisons coupled with RT-PCR analysis identified four additional coding exons, which had not previously been identified using standard recombinant DNA techniques, bringing the total number to nine. This approach has been of particular value in the case of a gene such as bli-4 in which some transcripts are clearly under-represented in cDNA libraries. cDNAs and ESTs have only been identified for the bli-4A, B, C, D and E products. In addition, deletion analysis of sequences outside of coding regions, combined with phenotypic rescue experiments, identified regulatory elements that alter the expression of the bli-4 gene. These results demonstrate the power of genomic sequence comparisons for elucidating the functional organization of a complex gene.

24 May 1999 15:50 886 886 Kex2/subtilisin-like Proprotein Convertase activity is required for Cuticle Structure/Formation and Defecation.

1999 International Worm Meeting abstract 836 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Kex2/subtilisin-like Proprotein Convertase activity is required for Cuticle Structure/Formation and Defecation. C. Thacker, A.M. Rose Department of Medical Genetics, 6174 University Boulevard, U.B.C., Vancouver, B.C. V6T 1Z3, Canada. Many physiologically important proteins are synthesized as large inactive polypeptides. Activation commonly occurs by limited proteolytic cleavage at mono-, di or oligobasic residues, a reaction catalyzed by members of the kex2/subtilisin-like proprotein convertase family (kexins) of serine endoproteinases. C. elegans encodes four kexin family members including bli-4, Celfurin, CelPC2 and aex-5. We are interested in determining the developmental role of each of the kexins, and their substrate specificity. A comprehensive genomic comparison of the C. elegans and C. briggsae bli-4 locus now suggests that the gene encodes at least nine isoforms. Molecular and genetic studies of bli-4 mutants show that one role of the convertases is the processing of cuticle collagens during development. To further our understanding of how the convertase gene accomplishes this feat, we are using FLAG-epitope-tagged transgenes that encode candidate substrate proproteins to examine their spatial and temporal cleavage pattern in wild-type animals and bli-4 mutant strains. Candidate genes which may encode BLI-4 substrates, include bli-1, bli-2 and dpy-5, all of which encode cuticle collagens that contain the Arg-X-X-Arg kexin cleavage motif toward the N-terminus. dpy-5 mutations dominantly suppress blistering of the adult cuticle. Disruption of the DPY-5 R-X-X-R motif confers a dominant Dpy phenotype to transgenic animals. Arrested larvae and animals with a distinctive "sausage-link" phenotype are also observed. These results suggest that unprocessed DPY-5 may form abnormal cuticle complexes. We have recently shown that the defecation mutant aex-5 encodes a member of the kexin family and is expressed as part of an operon with unc-54 suggesting that the role of this convertase is restricted to muscle tissue. Worms homozygous for the candidate aex-5 null mutation, h1557, are thin, slow growing, weak Egl and severely constipated. Three additional alleles kindly, provided by Jim Thomas, are the result of missense mutations that presumably affect the catalytic activity or the dynamic localization of the endoprotease. We are currently examining the localization of AEX-5 in order to better understand the role of this enzyme in the defecation process.

24 May 1999 15:50 887 887 Effects on nitrogen and energy metabolism of a defective DAF-2 insulin receptor-like protein

1999 International Worm Meeting abstract 837 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Effects on nitrogen and energy metabolism of a defective DAF-2 insulin receptor-like protein John J. Thaden 1,2,3 , Kewen Jauss 2 , Heather S. Reis 4 , Henry J. Mroczkowski 5 , Daniel Owen 4 , Robert J. Shmookler Reis 1,2,3,5

1 Dept. of Geriatrics, Univ. of Arkansas for Medical Sciences. 2 Dept. of Medicine, Univ. of Arkansas for Medical Sciences. 3 J.L. McClellan Memorial Veterans Affairs Med. Ctr. Research-151, 4300 W. 7th St., Little Rock, Arkansas 72205. 4 no address 5 Dept. of Biochemistry & Molecular Biology, Univ. of Arkansas for Medical Sciences.

The insulin/IGF-1 receptor homolog DAF-21 must function in order for C. elegans to develop, reproduce, and age normally2 (unless the forkhead transcription factor DAF-16 also is defective3 ). A proline-to-serine mutation e1370ts in the DAF-2 kinase domain1 causes constitutive dauer formation in homozygotes grown at 25 C, and ~100% life extension in adults upshifted at L44 . Unlike dauers, daf-2 adults feed and reproduce, but like dauers, they do accumulate excess fat, suggesting that daf-2 regulates metabolism in adults1 . Might the DAF longevity phenotype be due to a metabolic equivalent of dietary restriction? That daf-2 double mutants with pharyngeal pumping defects have life spans further extended5 implies otherwise. Here, we report that CB1370 daf-2(e1370) incorporated as much tritium as N2 and JT7098 daf-2;daf-16(m26) when fed for 1h on OP50 previously grown with 3H-amino acids, whereas JT5488 daf-2;daf-12(m20) incorporated less. Also, life spans of both CB1370 daf-2 and JT7098 daf-2;daf-16 were extended 30% by dietary restriction. These results support the conclusion5 that DAF-2 longevity is not due to a genetically-induced caloric restriction. In spite of similar feeding, excretion of ammonia nitrogen was 61-67% less for four daf-2 strains, compared to a panel of other strains. In progress are measurements of excreted amino acids, apparently the other major nitrogen end product for nematodes. Reduced ammonia excretion indicates that nitrogen metabolism is also under daf-2 regulation. It may be that proteins and purines, like fats, are stored in large excess by dauer larvae and daf-2 adults; however, the latter was not evident in measures of nematode total protein.

Supported by NIH grant AG-09413. 1. Kimura et al. (1997) Science 277, 942-8. 2. Gems et al. (1998) Genetics 150, 129-55. 3. Ogg et al. (1997) Nature 389, 994-9; and Lin et al. (1997) Science 278, 1319-22. 4. Kenyon et al. (1993) Nature 366, 461-4; and Larsen et al. (1995) Genetics 139, 1567-83. 5. Lakowski & Hekimi (1998) Proc Nat Acad Sci USA 95, 13091-6.

24 May 1999 15:50 888 888 Molecular and genetic analyses of unc-69, a gene required for axon guidance.

1999 International Worm Meeting abstract 838 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Molecular and genetic analyses of unc-69, a gene required for axon guidance. S Tharin 1 , B Wightman 2 , N Tsung 3 , E Hartweig 3 , G Garriga 4 , HR Horvitz 3 , MO Hengartner 1

1 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 2 Muhlenberg College, Allentown, PA. 3 Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA. 4 University of California, Berkeley, CA.

The unc-69 gene is required for axon guidance, outgrowth and fasciculation in C. elegans . In unc-69 mutants many axons, including those of the HSN and DD/VD neurons, exhibit guidance defects. Furthermore, the axons of HSN, ALA, RID and AVKR terminate prematurely, and the dorsal and ventral nerve cords are defasciculated in these mutants. unc-69 encodes a novel 108 amino-acid protein with a predicted coiled-coil motif near its C-terminus. Rescuing unc-69::gfp fusion constructs are expressed predominantly in axons. This expression pattern, combined with the lack of a signal sequence, suggests that unc-69 acts cell-autonomously. To test this hypothesis, we are carrying out mosaic analysis. UNC-69 shows significant similarity to the predicted products of previously uncharacterized human, mouse and Drosophila genes ( hunc-69 , munc-69 and drunc-69 , respectively), identified by expressed sequence tags. The predicted vertebrate proteins also have coiled-coil structures near their C-termini and are 77% identical to UNC-69 over the coiled-coil regions. hunc-69 mRNA is enriched in human fetal brain, although its expression is not restricted to this tissue. A hunc-69 transgene rescues the uncoordinated phenotype of unc-69 mutants, suggesting that the two gene products are functionally homologous. The high degree of sequence conservation over the coiled-coil domain strongly suggests that protein-protein interactions mediated by this domain are required for UNC-69 function. We are currently carrying out a yeast two-hybrid screen to identify candidate UNC-69 interaction partners. We speculate that UNC-69 participates in a conserved signaling pathway that transduces extracellular guidance cues to the axonal cytoskeleton.

24 May 1999 15:50 889 889 PEB-1, A Novel DNA Binding Protein Involved In Pharyngeal Gene Expression

1999 International Worm Meeting abstract 839 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. PEB-1, A Novel DNA Binding Protein Involved In Pharyngeal Gene Expression JD Thatcher, C Haun, PG Okkema University of Illinois at Chicago Expression of myo-2, a pharyngeal muscle-specific myosin heavy chain gene, is controlled in part by organ-specific signals that target a sequence within the myo-2 enhancer called the C sub-element. The C sub-element is a unique regulatory sequence that enhances reporter gene expression in all pharyngeal cell types. To identify genes controlling pharyngeal gene expression, we performed a yeast one hybrid screen for C. elegans factors binding the C sub-element. Several clones were isolated for a gene we call peb-1, for pharyngeal enhancer binding protein (previously referred to as CAFa). peb-1 encodes a novel protein with no recognizable similarity to any known DNA binding proteins. We have demonstrated that PEB-1 specifically binds the C sub-element in vitro, and that it requires the hexanucleotide sequence TGCCGT. This PEB-1 binding site overlaps a binding site for PHA-4, suggesting PEB-1 and PHA-4 may interact. The DNA binding domain of PEB-1 maps to a 157 amino acid region, which is sufficient for specific binding. We have examined the peb-1 expression pattern using of a peb-1::lacZ reporter and by in situ hybridization. Robust expression was observed in the pharynx, predominantly in muscle and marginal cells. Pharyngeal expression initiates by the comma stage of embryogenesis, persists in larval stages and diminishes by the adult stage. Non-pharyngeal cells were also stained in many transformants, although this staining was typically less frequent and less intense than pharyngeal staining. Non-pharyngeal staining cells included the hypodermis, body wall muscle, ventral nerve cord, cells around the vulva and rectum, but not intestine. Taken together these results suggest that peb-1 defines a previously unidentified class of DNA binding protein, and that it may control gene expression during pharyngeal development.

24 May 1999 15:50 890 890 Analysis of a deletion mutation of del-1, a member of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily

1999 International Worm Meeting abstract 840 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of a deletion mutation of del-1, a member of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily HA Thieringer, N Tavernarakis, M Driscoll Rutgers University, Department of Molecular Biology and Biochemistry, Nelson Labs A220, Piscataway, NJ 08854 del-1(degenerin-like) encodes a protein that is a member of the DEG/ENaC superfamily. Reporter fusions to del-1 suggest that this protein is expressed in the V class of motor neurons (Neuron, 18: 107-119) and in SABVR and SABVL (David Miller pers. comm.). No degeneration-causing mutations in del-1 have been reported, and in fact there are no known mutations in this gene. del-1 is expressed in many of the same cells as another degenerin, unc-8. Null alleles of unc-8 cause subtle defects in locomotion, most notably there is a decrease in both the wavelength and amplitude of the worm tracks. We have postulated that both UNC-8 and DEL-1 are components of a stretch-sensitive ion channel in the motor neurons that modulates the sinusoidal movement of the worm. We screened a mutagenized C. elegans library for deletions within the del-1 coding region, and isolated a 1380 base deletion, del-1(bz33). This deletion begins at residue 256 just after the first cysteine-rich domain of the DEL-1 protein, and extends into the 12th intron. The mutant allele could at best produce a truncated protein with just one transmembrane domain. Our initial look at this mutant suggests del-1(bz33) does not have the same locomotion defect as unc-8(lf). Crosses of this allele with both gain-of-function and loss-of-function unc-8 alleles may help to answer questions about the role of these degenerins in nematode locomotion and proprioception.

24 May 1999 15:50 891 891 Cross-Platform Software Tools for Time-Lapse Imaging of 3D Samples (4D Imaging)

1999 International Worm Meeting abstract 841 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cross-Platform Software Tools for Time-Lapse Imaging of 3D Samples (4D Imaging) CF Thomas, JG White National Biophotonics Resource, Univ. of Wisconsin, Madison, WI 53706 In 1996, the NBR released a pair of software applications to visualize time-lapse data sets captured from dynamic 3D samples as they change over time (Thomas et al. (1996) Science 273, 603-607). The "4D Turnaround" application allowed 4D data sets to be processed into compressed digital movies, and the "4D Viewer" application allowed navigation through, visualization of, and annotation of these complex and extensive data sets. Although quite successful, these software tools were limited by their native-coded nature to being runnable only on the Macintosh computer platform. Utilizing the "Java" programming language Java from Sun Microsystems and the latest advances in Quicktime digital media technology from Apple Corporation, the NBR’s latest 4D Imaging products are improved, cross platform versions of the "4D Turnaround" and "4D Viewer" applications. These Java 4D applications will run on both the Macintosh and the Windows platforms, offering simplified 4D imaging to a much larger audience of researchers in the C. elegans community. Like its native-coded predecessor, "4D Turnaround/Java" allows users to process extensive 4D data sets into compressed Quicktime-movie-based data sets which are much more easily visualized and take up much less hard disk space than the unprocessed raw images. In addition to handling 8-bit and 16-bit TIFF files, and Bio-Rad’s PIC format, the software also handles color images, and multi-channel 4D data sets from Bio-Rad’s 1024 microscopes. An improved graphic user interface (GUI) makes "4D Turnaround/Java" even easier to use. "4D Viewer/Java" offers the ability to navigate through, place bookmarks in, and annotate extensive 4D data sets, not unlike like its Mac-only ancestor. However, the Java version of the software removes the built-in limitations on the number of bookmarks that can be placed, focal planes that can be viewed, overlay objects that can be created, etc. The new software also features an improved GUI for greater ease-of-use and offers increased flexibility and durability, making it a more powerful tool than ever before.

More information on the NBR’s 4D software can be found on our web page at: "http://www.bocklabs.wisc.edu/imr/facility/4D/4d.htm". This work supported by Grant DRR-570 to the National Biophotonics Resource.

24 May 1999 15:50 892 892 Comparison of Cytidine Deaminase in Brugia pahangi and C. elegans

1999 International Worm Meeting abstract 842 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Comparison of Cytidine Deaminase in Brugia pahangi and C. elegans F Thompson 1 , S Hunter 1 , C Britton 2 , E Devaney 1

1 Department Veterinary Parasitology, Glasgow University, Bearsden Road, Glasgow, Scotland. G61 1QH. 2 WCMP, Anderson College, Glasgow University, 56 Dumbarton Road, Glasgow, Scotland. G11 6NU.

Lymphatic filarial worms are nematode parasites which cause disease in many tropical regions. These parasites have a developmental cycle which involves both an insect vector and a mammalian host. The filarial homologue of cytidine deaminase (CDD) was cloned in a differential screen which sought to identify cDNAs which were up-regulated as the L3 of Brugia transfers between hosts. CDDs are a family of enzymes involved in the salvage pathway for pyrimidine nucleosides and the site-specific editing of mRNA in mammals. In Brugia, the expression of cdd is triggered by exposure to the mammalian environment and is largely restricted to the post-infective larval stages. This expression pattern is not consistent with that of a house keeping gene. Recombinant Brugia CDD was expressed and shown to possess the characteristics of a functional CDD enzyme. In a heterologous editing assay the recombinant protein was shown to exhibit a novel RNA binding activity but was unable to edit the RNA sequence. In order to determine the functional significance of these molecules in nematodes we have begun to characterise the CDDs in C. elegans. Two C.elegans homologues, Ce-cdd-1 and Ce-cdd-2 were identified. These genes have been cloned and the expression pattern throughout the life-cycle analysed. As in Brugia, the mRNAs are expressed in a stage-specific manner. Reporter constructs have been made and will be used to define the temporal and spatial expression patterns of these genes. Using RNA interference assays, a reproducible phenotype has been obtained; the worms are longer and thinner than wild type and display a head-lifting motion. RNA binding and editing assays using recombinant Ce-CDD-1 and Ce-CDD-2 are in progress. A recent search of ACeDB using a Prosite signature for the cytidine deaminase active site identified a third potential CDD on cosmid C29F5. The predicted protein contains an additional amino acid residue in the active site which may affect its catalytic activity. However the predicted size of this protein (31kDa) and the presence of several motifs which are all characteristics of the mammalian editing enzymes warrants further study of this gene.

24 May 1999 15:50 893 893 Functional analysis of UNC-51 and UNC-14 involved in axonal formation in C.elegans

1999 International Worm Meeting abstract 843 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Functional analysis of UNC-51 and UNC-14 involved in axonal formation in C.elegans HZ Tian, YM Ohshima Kyushu University It was known that mutations in the unc-51 or unc-14 genes of the nematode Caenorhabditis elegans result in various abnormalities in axonal elongation and axonal structures. The unc-51 and unc-14 genes were cloned by our group, and they are predicted to encode a novel serine/threonine protein kinase (Ogura et al., Genes Dev., 1994), and a novel protein that binds to UNC-51 (Ogura et al.,Genes Dev., 1997). To demonstrate the kinase activity of UNC-51, we prepared constructs in pUcD2SRÉøvector and pCAGGS vector to express full-size UNC-51, the kinase domain of UNC-51, full-size UNC-51 carrying a mutation in the kinase domain, or the C-terminal domain of UNC-51, all of which were tagged with c-Myc epitope. We transfected each of the constructs into COS-7 cells, examined their expression with western blots and an anti-Myc antibody. We also assayed kinase activity of an immunoprecipitated extract on MBP (myelin basic protein) and histone H1 in vitro. The kinase activities of the extracts transfected with full-size UNC-51 or the kinase domain on histone H1 or a few COS-7 proteins were suggested as compared to those transfected with the mutant UNC-51, the C-terminal domain or the vector. Results of western blotting detection of the tagged proteins was not reproducible. To increase the expression and detection efficiency, we plan to use another tag and antibody. Autophosphorylation activity and phosphorylation sites in the substrates with full-size UNC-51 will be identified. Furthermore, we plan to examine the effect of UNC-14 on the kinase activity of UNC-51.

24 May 1999 15:50 894 894 A genetic approach to identify the molecular target of SB-204269 in Caenorhabditis elegans

1999 International Worm Meeting abstract 844 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A genetic approach to identify the molecular target of SB-204269 in Caenorhabditis elegans M Tijsterman 1 , J Ahringer 2 , S Rastan 3 , H Herdon 3 , GP Livi 3 , RH Plasterk 1

1 Department of Molecular Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands. 2 Wellcome CRC Institute, University of Cambridge, Cambridge. 3 SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Harlow, Essex, UK.

The compound SB-204269 shows potent anticonvulsant activity in a range of animal seizure models without any neurological or cardiovasular side-effects1 . A stereospecific binding site has been identified in the rat brain2 but the molecular nature of this unique binding site remains to be elucidated. We have started to use the strength of C. elegans genetics in an attempt to identify the molecular target of SB-204269. Exposure of mothers to SB-204269 (micromolar range) results in embryonic lethality in this organism whereas its enantiomer SB-204268 does not. A C. elegans strain with a general increased sensitivity to drugs (pgp-1; pgp-3; mrp-1) are also more sensitive to SB-204269 than Bristol N2 and the obtained level of killing allows a large-scale selection for mutants that have gained resistance specifically to the compound. Besides the use of EMS as a source to introduce mutations into the C. elegans genome we crossed a mutator allele (mut-7) into this genetic background to allow a more easy detection of the causal mutation via a transposon display experiment. Both EMS- and "mutator"-screens are currently ongoing.

1 Upton et al. (1997) Br. J. Pharmacol., 121, 1679-1686

2 Herdon et al. (1997) Br. J. Pharmacol. 121, 1687-1691

24 May 1999 15:50 895 895 On the Systemic Nature of dsRNA-mediated Genetic Interference in C. elegans

1999 International Worm Meeting abstract 845 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. On the Systemic Nature of dsRNA-mediated Genetic Interference in C. elegans Lisa Timmons, Jamie Fleenor, Andrew Fire Carnegie Institution of Washington 115 West University Parkway Baltimore, MD 21210 We are investigating the mechanisms by which double-stranded RNA produces a systemic interference response. The ability of an exogenous dsRNA to interfere with the function of an endogenous gene has been described[1]. This was first observed in the progeny of animals whose germlines had been injected with dsRNA. Subsequent observations demonstrated the existance of a mechanism by which dsRNA injected into the body cavity of an adult (in which few somatic cell divisions occur) could affect the entire animal. In addition, C. elegans can respond in a gene-specific manner to dsRNA encountered in the environment. In particular, an RNAi effect can be observed when worms eat dsRNA expressed in bacteria[2]. This ability of cells outside the gut to be affected by dsRNA administered throught the gut has led us to the question of how dsRNA or some effect mediated by dsRNA spreads from the site of initial contact throughout the animal. We are currently investigating the extent of the "spreading effect" and are using genetic screens to identify genes that might mediate spreading. A second question related to systemic interference that we are addressing is that of differential tissue susceptibility of RNAi. We and other investigators have noted that some cells in the nervous system appear refractory to RNAi[3]. We are currently investigating the cellular pattern of this refractibility in the nervous system and are using genetic screens to identify components that enable neurons to become refractile to RNAi.

[1] Fire, A., Xu, S.-Q., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998) Nature. 391: 806-811. [2] Timmons, L., and A. Fire. (1998) Nature.Oct 29: 395(6705):854.[3] Fire, A., and J. Fleenor. (1998). Worm Breeder’s Gazette 15(3): 8.

24 May 1999 15:50 896 896 UNC-96: an intermediate filament protein of muscle?

1999 International Worm Meeting abstract 846 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. UNC-96: an intermediate filament protein of muscle? TL Tinley, KB Mercer, C Alberico, X Tang, R Santoianni, GM Benian Depts. of Path. and Cell Biol., Emory University, Atlanta, GA 30322 USA Viable alleles of unc-96 show normal movement but have a disorganized body wall muscle structure. By polarized light microscopy, there are highly birefringent "needles" near the ends of muscle cells, without any definite A or I bands. By EM, the most severe allele, su151 has extra collections of thin filaments or intermediate filaments (IF), and masses of thick and thin filaments in abnormal locations. We have identified 2 new alleles. The first, r291, was isolated as a suppressor of unc-105 and the second, sf18, by a non-complementation screen. To address whether the disorganization of unc-96 is due to deterioration of the lattice as a result of contraction, we made a double mutant of unc-96 with a mutation in the myosin heavy chain that decreases myosin activity (unc-54(s95)) but does not appreciably affect muscle structure. unc-54(s95); unc-96(su151) shows an increase in organization. This protection is also seen in unc-54(s95) / +; unc-96(su151). We are in the process of examining muscle structure of unc-96 mutants at early stages of development. unc-98, another muscle affecting gene, shows an identical polarized light phenotype to unc-96. We have constructed an unc-96 unc-98 double and it shows enhanced muscle structure disorganization. After more finely mapping unc-96, we inspected the genomic sequence for IF protein candidates using ACeDB. One candidate, encoded on cosmids M6 and T19D7, is homologous to IF Protein A, a muscle specific IF isoform purified from Ascaris. We were able to achieve rescue using these cosmids or a 12 kb genomic fragment containing the gene. Surprisingly, we have been unable to detect the mutation sites in this gene by sequencing all of the coding sequence, most of the introns, and 1.7 kb of upstream sequence from su151 and r291. A second IF protein is encoded by the cosmid F25E2 that lies only several cosmids to the right from M6. We were unable to rescue unc-96 with this gene, and in addition, sequencing of su151 failed to reveal a mutation site. A non-complementation screen is currently underway to better position the gene on the physical map using rearrangements. The ultrastructural phenotype and rescue with an IF protein gene suggests that unc-96 truly encodes an IF protein or a protein that interacts with IFs.

24 May 1999 15:50 897 897 UNC-89 participates in Rho-like signaling to organize thick filaments

1999 International Worm Meeting abstract 847 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. UNC-89 participates in Rho-like signaling to organize thick filaments TL Tinley 1 , R Kindt 2 , E Baraldi 3 , C Kenyon 2 , GM Benian 1

1 Depts. of Path. and Cell Biol., Emory Univ., Atlanta, GA 30322. 2 Dept. of Biochem. and Biophys., UCSF, San Francisco, CA 94143. 3 EMBL, Heidelberg, Germany 69012.

Mutants in unc-89 are thinner and more transparent, but move normally. The myofilament lattice is thinner than normal, with primary defects in the organization of thick filaments, and for most alleles, no M-line. UNC-89 is located in the middle of A-bands, a region that contains M-lines. unc-89 encodes a giant 732,000 polypeptide primarily composed of Ig domains, as well as SH3, GEF and PH domains. GEF domains (guanine nucleotide exchange factors) exchange GDP for GTP and thereby activate Rho-like GTPases. We hypothesize that unc-89 receives a signal from outside the muscle cell and then recruits other molecules to organize the myofilament lattice. By confocal microscopy UNC-89 is localized throughout the depth of the myofilament lattice, including near the muscle cell membrane. Upon injection of bacterially expressed GEF-PH (from UNC-89) into NIH3T3 mammalian fibroblasts, a reorganization of F-actin was observed similar to what was seen by injecting V12Rac. This was not seen with the PH domain or BSA controls. A fusion protein of GST with an UNC-89 peptide containing the SH3, DH and PH domains gave GEF activity for nematode MIG-2 but not for mouse Rab5.To obtain genetic evidence for this type of signaling, we have examined the phenotype of unc-89; mig-2 double mutants. mig-2 encodes a member of the Rho family and when mutant results in the failure of many migrating cells to complete their journeys during development. mig-2::GFP is expressed in many cells in embryos and early larvae, including body wall and pharyngeal muscle, and is localized at or near the cell membrane (Zipkin et al. 1997). mig-2 alleles have normal appearing muscle. When mu28 is combined with two hypomorphic alleles of unc-89, ad539 and st79, the Unc-89 phenotype is enhanced. When mu28 is combined with the unc-89 null allele, su75, the Unc-89 phenotype remains the same. This suggests that mig-2 and unc-89 are members of the same pathway. Interestingly, when mu28 is combined with the unc-89 hypomorphic allele, e1460, the phenotype is not worsened. Similar experiments with mig-2(rh17), an activated allele, gave the same results.

24 May 1999 15:50 898 898 Using model organisms to identify genes that affect life span

1999 International Worm Meeting abstract 848 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Using model organisms to identify genes that affect life span HA Tissenbaum, L Guarente Massachusetts Institute of Technology Previous work in C. elegans has identified a number of mutants that affect life span. We are trying to identify new components of the aging mechanism by isolating mutations in genes that are conserved in C. elegans and have previously been shown be important in the aging process of other eukaryotic organisms. 1) Werner Syndrome: People with Werner Syndrome (WRN) show many signs of premature aging (reviewed in Yu et al. 1997). The WRN protein is a member of the RecQ family of helicases. In yeast, mutations in the WRN homolog, SGS1, cause yeast to age prematurely (Sinclair et al. 1997). Four members of the RecQ family have been identified in the C. elegans database. We are currently isolating knockout mutations of all four of the family members. As well, we are generating GFP fusions and doing RNAi to help elucidate the different functions of these four genes. These studies will help to determine if any of the four genes is the true WRN homolog in C. elegans and whether they function to control aging. 2) SIR2 homologs: Studies from yeast (S. cerevisiae) have identified the SIR proteins (SIR2, 3, 4) as important components for yeast aging (reviewed in Sinclair et al. 1998). SIR2, however, is unique among the SIR proteins in several respects including that it is highly conserved from yeast to man. There are two SIR2 homologs identified in the C. elegans database. To understand the role of C. elegans SIR2 in aging, we are generating knockout mutations and GFP fusions as well as performing RNAi. We are also using the candidate gene approach to identify mutants in the SIR2 homologs.

References: Sinclair D.A., Mills K., and Guarente L. (1997). Science 277:1313-6 Sinclair D.A., Mills K., and Guarente L. (1998). Annu Rev Microbiol 52:533-60 Yu C.E., Oshima J., Wijsman E.M., et al. (1997) Am J Hum Genet 60:330-41

24 May 1999 15:50 899 899 Roles of osm-9/Capsaicin Receptor Family Members in Chemosensory Behavior

1999 International Worm Meeting abstract 849 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Roles of osm-9/Capsaicin Receptor Family Members in Chemosensory Behavior D Tobin 1 , D Madsen 2 , G Moulder 3 , R Barstead 3 , AV Maricq 2 , C Bargmann 2

1 HHMI, UCSF, San Francisco, CA, 94143. 2 University of Utah, Salt Lake City, UT 84112. 3 Oklahoma Medical Research Foundation, Oklahoma City, OK 73104.

The osm-9 gene is required for a wide range of sensory modalities in C. elegans; mutants exhibit defects in chemosensation, mechanosensation, osmosensation, and certain forms of olfactory adaptation. osm-9 encodes a putative ion channel with weak homology to the Drosophila transient receptor potential protein (TRP), a cation channel involved in visual sensation. More strikingly, osm-9 is the closest known relative of the vertebrate capsaicin receptor, a cation channel expressed in pain-sensing neurons and gated by the active component of chili peppers. OSM-9 is expressed in ten amphid neurons, three ciliated mechanosensory neurons, and in a few other cell types. In the amphid neuron AWA, OSM-9 localizes to sensory cilia, suggesting a direct role in sensory transduction. The vertebrate capsaicin receptor VR1 responds to heat, capsaicin, and acid. Channels in the TRP family are gated by molecules related to diacylglycerol. Yet little is understood about how OSM-9 is activated. Might OSM-9 respond to similar physiological cues? We asked if mammalian VR1 could substitute for OSM-9. By expressing VR1 under an AWA-specific promoter we could partially rescue osm-9’s AWA defects. Thus, the homology between the two proteins may have functional relevance. Three close relatives of osm-9 are each expressed in subsets of osm-9-expressing cells. We call these genes ocr (for osm-9/capsaicin receptor-related) genes. ocr-1 is expressed primarily in AWA while ocr-2 is expressed strongly in ASH. Most ion channels in vivo consist of two to five similar subunits encoded by one or more genes. Based on their expression patterns and similarity to osm-9, we hypothesized that the OCR channels might coassemble with OSM-9 to form heteromultimeric channel complexes. Behavioral data suggest that OSM-9 may play a primary role in sensory transduction for some behaviors, but a modulatory role for others. We propose that different combinations of subunits may account for the distinct functions of osm-9 in different sensory neurons. Knockouts of these relatives reveal roles for the ocr genes in chemosensation. We will present our characterization of these mutants.

24 May 1999 15:50 900 900 The TGFb Signaling Phenotypes

1999 International Worm Meeting abstract 850 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The TGFb Signaling Phenotypes R Tokarz, C Giannikas, C Savage-Dunn Department of Biology, Queens College, CUNY, Flushing, NY 11367. We are interested in studying TGFb signaling pathways. In C elegans, a type II receptor, daf-4, is a common mediator of two distinct TGFb signaling pathways, the Dauer branch, and the Small branch. The Dauer branch mutants are Dauer-constitutive as well as Egg-laying defective, while the Small branch mutants have markedly reduced body size, approximately 50% that of the wild type, as well as defects in the male tail. Each of these pathways uses distinct Smads, the TGFb signaling molecules. We are characterizing the developmental consequences of mutations in these pathways. First, we are studying the egg-laying defective genes that may function with the Dauer branch. Previous studies have shown that two egg-laying genes, egl-4 and egl-32, may be implicated in TGFb signaling by their suppression by daf-3, an antagonistic Smad of the Dauer branch which also suppresses the egg-laying phenotype of other upstream members of the Dauer pathway. We attempted to find other members of this pathway by testing other egg-laying mutants with similar pharmacological properties to egl-4 and egl-32 for suppression by daf-3. We’ve also mapped egl-32 using chromosomal deficiencies and are in the process of cloning the gene. There are no known TGFb components in the area to which we’ve mapped egl-32, which means we are probably dealing with a new component of the pathway. Second, we are beginning to address the question of why Small mutants have reduced body size. As a first step, we’ve constructed growth curves of N2 and all known TGFb components of the Small pathway, showing that they have a postembronic growth defect leading to their reduced body size. We’ve also measured cell sizes to determine whether only some or all cells are smaller. Third, we have characterized a sma-3 allelic series. These experiments show that sma-3 is not an essential gene, acts specifically in the Small but not the Dauer branch, and that other Smads do not function in the absence of SMA-3.

24 May 1999 15:50 901 901 Decreases in Body Size as a Function of Age and Proposal of Adulthood Phases

1999 International Worm Meeting abstract 851 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Decreases in Body Size as a Function of Age and Proposal of Adulthood Phases MK Tom 1,2,3 , EM Crimmins 3 , LH Smith 1 , PL Larsen 2,3

1 Department of Biology, University of Southern California, Los Angeles, CA 90089. 2 Molecular Biology Program, University of Southern California, Los Angeles, CA 90089. 3 Andrus Gerontology Center, University of Southern California, Los Angeles, CA 90089.

C. elegans is an exquisite developmental model system, but there are gaps in our knowledge of this system with regard to the aging adult. For example, it is unknown whether the 959 cells of the adult hermaphrodite change throughout adulthood. To refine adulthood events more than the generally used ones of cessation of reproduction and death, we have designed a method of following individuals as they age and made body size measurements. Size was determined for wild type and 12 long-lived mutant alleles of daf-2. Population level analysis demonstrated that individual strains exhibit differences in length and decrease in body size after the cessation of egg laying. This gross pattern could be the result of multiple patterns at the level of individuals. Longitudinal analysis of individuals of five selected strains, demonstrated that all individual worms decrease in size regardless of genotype. The strains do not differ significantly on the first day of adulthood in size. The strains do differ in maximum size, but they do not differ in size just before death. We hypothesize that increased nutrient uptake and/or assimilation efficiencies early in life, coupled with alterations in metabolic partitioning and increased maintenance functions combine to increase life span. In the long run, to understand how the daf-2/age-1 pathway functions in longevity, it will be necessary to model it in the context of the passage of time as an adult. Classification of stages within adulthood does not currently exist. The timing of events contributing to aging is not fixed as developmental events are, so we propose using a time frame that is relative to the individuals’ adult life span and call them phases. To follow convention for the larval stages, the series to be added to complete the life course of the animal are called M1 maturation, M2 maintenance, M3 moribundity, and M4 mortality. The current criteria for distinguishing the adulthood phases are reproduction, body size and motility.

24 May 1999 15:50 902 902 Cloning of the C. elegans ADAR

1999 International Worm Meeting abstract 852 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cloning of the C. elegans ADAR LA Tonkin, BL Bass Department of Biochemistry/HHMI, University of Utah, Salt Lake City, Utah, 84132, USA Adenosine deaminases that act on RNA (ADARs) are a class of enzymes that deaminate adenosines to inosines within double stranded regions of RNA molecules. ADAR substrates have been most extensively studied in mammals where they have been found by noticing A to G transitions when comparing genes with their cDNAs. Inosine pairs with cytidine and thus is converted to a guanosine during cDNA synthesis. The translation machinery reads inosine as guanosine, leading to codon changes that can be silent, specify a different amino acid, or alter a stop to a sense codon. C. elegans extracts have ADAR activity, and recently several ADAR substrates were identified in worms using a method for systematically identifying inosine-containing RNAs (Morse and Bass, submitted). Unlike previous substrates, deamination sites in these substrates are in non-coding regions. Deamination sites are mostly in 5’ and 3’ untranslated regions, suggesting ADARs may play a role in translational control, localization or stability. Previously, we cloned a candidate C. elegans ADAR (T20H4.4) that contained a single double stranded RNA binding motif (dsRBM) and conserved catalytic deaminase domain (Hough, Lingham, Bass, submitted). We have overexpressed this protein in several different systems, but have been unable to detect deaminase activity. All known active ADARs have 2 or 3 dsRBMs coupled to the catalytic domain. With the completion of the genome project a new ADAR candidate has been identified (H15N14.1a/b). This gene has all the hallmarks of known active ADARs: 3 dsRBMs accompanied by a largely conserved deaminase domain. Aside from the two minor 3’ splice site variants indicated by ESTs in the database, northern analyses indicated the existence of two major transcripts of 2.9 and 3.1 kb. We cloned several cDNAs by PCR and analyzed the cDNAs by restriction enzyme digests and sequencing. Our data indicate the presence of at least four splice variants. These are in N- and C-terminal regions that do not structurally affect the dsRBMs or deaminase domain. Similar active splice variants are present in mammals and frogs. Effects of these splicing variants may be more subtle in terms of regulation, substrate specificity or tissue specific expression. Work is currently underway to overexpress active protein for biochemical characterization.

24 May 1999 15:50 903 903 Characterization of str-2 asymmetric expression

1999 International Worm Meeting abstract 853 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of str-2 asymmetric expression ER Troemel, A Sagasti, CI Bargmann HHMI and UCSF C. elegans detects hundreds of chemicals with a small number of chemosensory neurons and a large number of chemosensory receptors. Most chemosensory neurons are found in bilaterally symmetric pairs, with a right neuron and a left neuron. While many candidate chemosensory receptors are expressed in both neurons of a pair, we have found that the candidate receptor, str-2, is expressed in a bilaterally asymmetric fashion. Half of the animals with an integrated str-2::GFP fusion express str-2 in the left AWC olfactory neuron, and the other half in the right AWC neuron. In order to determine the default state for str-2 expression in AWC, we have performed ablations of AWC precursors. When either the AWCL or AWCR precursor (ABplpaa or ABprpaa) is ablated, the remaining AWC neuron fails to express str-2. These results suggest that the default state is "off" for str-2 expression in AWC, and that both AWC neurons are required for str-2 expression. The two AWC neurons contact one another through their axons, which could be a site for communication between these two neurons. Indeed, in a number of axon guidance mutants neither AWC expresses str-2, consistent with a model in which an AWC neuron doesn’t express str-2 until it contacts its contralateral partner. Neuronal activity appears to play a crucial role in str-2 expression. Loss of function mutations in the unc-2 and unc-36 voltage-gated Ca++ channel subunits and in the unc-43 Ca++ /CaM kinase lead to both AWC neurons expressing str-2, while an unc-43 gain of function mutation leads to neither AWC expressing str-2. cGMP signalling also appears to be important for str-2 expression. Mutations in the guanylyl cyclases daf-11 and odr-1 and in the tax-2/4 cGMP-gated channel decrease str-2 expression. Epistasis analysis and laser ablations in these mutants leads to a working model for str-2 asymmetric expression: Ca++ entering the unc-2/36 channel stimulates unc-43 to inhibit str-2 expression in one neuron more than the other, which causes that neuron to be the "off" AWC. The "off" AWC then signals to the other AWC to instruct it to be "on", perhaps by inhibiting the activity of the unc-2/36 channel and unc-43. Signalling through guanylyl cyclases and the tax-2/4 channel then functions to maintain expression from the str-2 promoter.

24 May 1999 15:50 904 904 Using the C. elegans genome to search for the vesicular glutamate transporter

1999 International Worm Meeting abstract 854 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Using the C. elegans genome to search for the vesicular glutamate transporter M Trupp 1 , K Schuske 2 , M Hammarlund 2 , S McIntire 1 , E Jorgensen 2

1 Dept. of Neurology, UCSF, School of Medicine, San Francisco, CA 94110. 2 Dept. of Biology, U of Utah, SLC, UT 84112.

Previously, we cloned the unc-47 gene and showed that it encodes the vesicular GABA transporter which is required to transport GABA from the cytoplasm into synaptic vesicles. unc-47 identifies a new family of putative transport proteins present in C. elegans, yeast, and mammals that is most similar to amino acid permease proteins in plants. We believe that the vesicular glutamate transporter will be related to the vesicular GABA transporter for two reasons. First, the molecular structure of GABA closely resembles glutamate. Second, glutamate and GABA have similar bioenergetic properties for transport into synaptic vesicles. Specifically, GABA and glutamate transport depend more on the membrane potential component of the proton gradient rather than the pH component. We are screening through the thirteen C. elegans members of the family to determine if any is the vesicular glutamate transporter. The first method we used was to make GFP reporter constructs to determine whether any of these genes is expressed in glutamatergic neurons. Of the thirteen candidate transporters, eight are expressed in neurons, as well as in other tissues such as muscle and gut. It is possible that a vesicular glutamate transporter may be required in nonneuronal cells for transport of glutamate into other acidified compartments, and therefore expression may not be neuron specific. None of the genes are expressed exclusively in glutamatergic neurons, although two of the genes may be expressed in some. It is possible that the GFP expression does not accurately reflect the transporter genes’ expression since most of the constructs contain only upstream sequence driving expression of GFP. For these reasons we are taking another approach to determine whether any of these proteins might transport glutamate. We are isolating cDNAs for the eight candidates that are expressed in neurons. The cDNAs will then be expressed in tissue culture cells where transport activity can be assayed. Finally, we will identify mutations in candidate genes, and determine if they are defective for glutamatergic neurotransmission.

24 May 1999 15:50 905 905 Searching for new genes involved in dosage compensation

1999 International Worm Meeting abstract 855 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Searching for new genes involved in dosage compensation C Tsai, BJ Meyer HHMI and Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720 In C. elegans dosage compensation equalizes expression of X-linked genes between males (XO) and hermaphrodites (XX). This chromosome-wide regulatory process is essential for hermaphrodite development. A biochemically defined protein complex containing DPY-26, DPY-27, DPY-28 and MIX-1 has been shown to associate with X chromosomes in a sex-specific fashion and is required for global repression of hermaphrodite X-linked genes. Because MIX-1 is also required for mitotic chromosome segregation and mix-1 mutations cause lethality in both sexes, mix-1 was missed in our screen for sex-specific lethals. To identify other dosage compensation components that might have been missed, we used the following observation: mutations in all known dosage compensation genes suppress the sexual transformation caused by certain sex determination mutations in a dominant fashion, allowing recessive lethal mutations to be identified by their dominant suppression effect. For example, heterozygous mix-1 mutations can suppress the masculinization of XX animals caused by homozygous sdc-3(Tra) mutations. The suppression screen is shown below. sdc-3(Tra)/+ -- sdc-3(Tra) 99% Tra,1% hermaphrodite m/+; sdc-3(Tra)/+ -- m/+; sdc-3(Tra) 30-90% hermaphrodite -- isolate mutants So far we have screened 12,000 haploid genomes and isolated 9 mutants that show moderate to strong suppression: two are lethal mutations, two are maternal-effect lethal, one is a mix-1 allele, one is an sdc-3 null allele and three show no obvious phenotypes. The two lethal and one maternal-effect lethal mutations have been mapped extensively and their map positions indicate that they are mutations in three different genes that have not previously been implicated in dosage compensation. Preliminary immunofluorescence studies showed that all three mutations disrupt the association of DPY-26 but not DPY-27 with the X chromosome. DAPI staining of dead embryos revealed the presence of chromatin bridges and multinucleated cells, suggesting an essential role in chromosome segregation. These observations are reminiscent of defects caused by mix-1 mutations and suggest that the mutations isolated in the screen identify new genes that play a role in dosage compensation as well as chromosome segregation. Attempts are ongoing to clone the genes.

24 May 1999 15:50 906 906 Nematodes with Mitochondrial Diseases

1999 International Worm Meeting abstract 856 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Nematodes with Mitochondrial Diseases WY Tsang, BD Lemire Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada The mitochondrial respiratory chain (MRC) is composed of 5 protein complexes capable of generating ATP by oxidative phosphorylation. Defective mitochondrial function can result in a wide variety of mitochondrial diseases, including myopathies, heart disease, and diabetes. To better understand how a defective MRC is linked to mitochondrial dysfunction and disease manifestation, we are isolating and characterizing MRC mutations in the nematode, Caenorhabditis elegans. The formation of the MRC requires the expression of genes from both the nuclear and mitochondrial genomes. We produced a library of EMS-mutagenized N2 and screened the library for deletion mutations in both genomes using nested PCR. 3 mutations have been isolated and characterized. The first two mutations are nuclear deletions in the nuo-1 (C09H10.3) and in the atp-2 genes (C34E10.6) encoding the active site subunits of complexes I and V, respectively. In each case, a balanced heterozygote strain produces 1/4 homozygous mutant offspring which have an L3 arrest phenotype. RNAi experiments with atp-2 dsRNA result in significant embryonic arrest. We speculate that a maternal contribution of mRNA or protein is allowing the development to the L3 stage. We have detected the presence of a normal level of truncated, but probably mistargeted ATP-2 in the arrested animals. The third mutation (uaDf5) is a 3.1 kb mtDNA deletion which removes 4MRC and 7 tRNA genes. uaDf5 animals are heteroplasmic, having varying proportions of wildtype and mutant mtDNAs. We have maintained the heteroplasmy for over 30 generations and have demonstrated maternal inheritance of the mtDNA deletion. Moreover, we have shown a stochastic inheritance pattern of the uaDf5 mtDNA. uaDf5 animals appear to be aphenotypic. We speculate that the L3 arrest phenotype seen in our nuclear mutants is a consequence of insufficient energy production. In support of this hypothesis, we have determined that the passage from L3 to L4 is associated with a 3-fold increase in mtDNA copy number as well as a significant increase in the levels of ATP-2.

24 May 1999 15:50 907 907 Identification of Genes Required for Spindle Orientation in the Embryo

1999 International Worm Meeting abstract 857 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification of Genes Required for Spindle Orientation in the Embryo M Tsou, AP Hayashi, LS Rose University of California, Davis, CA 95616 USA The timing and orientation of cell division are critical for normal development. We have taken a genetic approach to identify genes involved in spindle orientation, and thus cell division pattern, in C. elegans (Rose and Kemphues, 1998. Devel. 125; Tsou and Rose, unpublished). In embryos, cells of the P lineage exhibit nuclear rotation events which orient the spindle successively on the same axis, while in cells of the AB lineage, spindles are oriented transverse to the previous axis. We isolated maternal effect lethal mutations and examined embryos from the mutant lines for alterations in the normal cleavage pattern. From screening approximately 10,000 haploid genomes, we obtained mutations that define six genes required for normal spindle orientation at the 2-cell stage. We obtained multiple alleles of let-99, ooc-5 and ooc-3 (at a rate comparable to that of some par mutants) suggesting that these genes may play a maternal specific role in spindle orientation. In contrast, we isolated only one allele of spn-1, spn-2 and spn-3. This and the behavior of the spn mutations in trans to deletions (see abstract by DeBella and Rose) suggests that the spn genes may have lethal or sterile null phenotypes. Mutations in let-99 and the spn genes cause defects in both the AB and P lineages without having obvious effects on one-cell polarity. let-99 embryos exhibit two major defects. First, there are reversals of the normal pattern of spindle orientations. Second, both the nuclear-centrosome complex and the spindle appear to be unstable in position. Spindle orientation defects and nuclear-centrosome position instability are also seen in gpb-1 maternally depleted embryos (Zwaal et al 1996. Cell 86; L. Rose, unpublished). We are currently exploring the relationship among let-99, the spn genes and gpb-1 by a variety of techniques, in addition to searching for LET-99 interacting proteins using the two-hybrid approach.

24 May 1999 15:50 908 908 Adapting a manual clonal screen to semi-automation

1999 International Worm Meeting abstract 858 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Adapting a manual clonal screen to semi-automation BT Tsung 1 , AA Ferrante 2 , WP Hansen 2 , PB Krauledat 2 , C Johnson 3 , CP Hunter 1

1 MCB, Harvard University, Cambridge MA, 02138. 2 Union Biometrica, 19 Ward St., Somerville, MA 02143. 3 Axys, Nemapharm Group, South San Francisco, CA 94080.

Clonal screens are an effective, but labor intensive, means to identify mutants with specific phenotypes. We are exploring the capabilities of a nematode flow-sorting system to assist with our continuing clonal screen for conditional lethal mutations (see abstract by Tsung et al., this meeting). The system (developed by Union Biometrica, Somerville MA. in collaboration with Axys, Nemapharm group) automatically analyzes and sorts individual nematodes by length (indicative of development stage or mutant type) from mixed populations and distributes them, along with a minimal volume, unharmed into microplates. Individual F2 L4 worms from a synchronized population were deposited into each well of a 96-well micro-titer culture plate seeded with 50 µl of diluted HB101. The current system accurately delivered a single nematode of the selected size (in our case 15% of the heavily mutagenized population were of the selected size) into 95/96 wells on 82 plates at about 3 minutes per plate. All worms were viable in test runs using non-mutagenized worms. Comparisons to our manual screen will be presented.

24 May 1999 15:50 909 909 A clonal screen to identify genes that control pal-1 expression.

1999 International Worm Meeting abstract 859 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A clonal screen to identify genes that control pal-1 expression. BT Tsung, A Kay, DM Mootz, N Huang, AJ Wright, MR Gerber, W Winston, K Cho, CP Hunter Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138 We are interested in identifying genes that control the pattern of PAL-1 expression in the early embryo. PAL-1 expression is controlled, at least in part, by the regulated translation of maternal pal-1 mRNA. By screening existing collections of maternal-effect lethal mutants we previously identified four genes that are required for the spatial and temporal pattern of PAL-1 expression. However, it is likely that many of the genes that regulate PAL-1 expression in the early embryo will also have essential functions at other times in development precluding their isolation in traditional maternal-effect screens. Therefore, we are isolating conditional, temperature-sensitive lethal mutations that cause embryonic arrest. Shifting fertile adults to the restrictive temperature should disable both maternal and zygotic gene products and thus reveal the earliest embryonic requirement for the mutant gene. So far, we have isolated 206 conditional lethal mutations from 49,322 fertile F2 animals from approximately 140 separate mutagenized pools. To identify interesting mutants we are determining the PAL-1 expression patterns as well as characterizing the early lineages, and terminal phenotypes.

24 May 1999 15:50 910 910 Characterization and cloning of the muscle activation gene unc-58

1999 International Worm Meeting abstract 860 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization and cloning of the muscle activation gene unc-58 M Tzoneva, JH Thomas Department of Genetics, University of Washington, Seattle WA 98115 unc-58 was first identified by dominant mutations that cause hypercontracted body-wall and egg-laying muscle in C. elegans. unc-58(dm) animals are rigidly paralyzed and egg-laying constitutive. Putative loss-of-function unc-58 alleles have been isolated as revertants of the unc-58(dm) phenotype. These alleles have no obvious phenotype on their own, suggesting that unc-58(dm) mutants result in an inappropriately activated gene product. unc-58(dm) stands out among other muscle activation mutations because it has the following unusual phenotypes. First, unc-58(dm) animals frequently flip around their longitudinal axis. This phenotype is, to our knowledge, not found in any other C. elegans mutant. Second, the unc-58(dm) phenotype is partially rescued by the drug endosulfan, an antagonist of GABA-gated chloride channels (B. Wightman and G. Garriga, personal communication; our unpublished data). Third, unc-58(dm) alleles exhibit a defect in the outgrowth of HSN neuron processes (B. Wightman and G. Garriga, personal communication). Because the unc-58(dm) mutation affects muscle excitability, it may encode an ion channel or a regulator of ion channel activity. We plan to clone the unc-58 gene and perform further genetic, pharmacological and molecular experiments to elucidate its mechanism and site of function. We have mapped unc-58 to a small region just to the left of unc-115. We have obtained putative Tc1 insertions in unc-58 by screening in a mut-6 background for revertants of the uncoordinated phenotype of an unc-58(dm) allele. We are now using cosmids from the unc-58 region as probes to identify the Tc1 insertions.

24 May 1999 15:50 911 911 Cloning of che-1 gene involved in chemotaxis of C. elegans

1999 International Worm Meeting abstract 861 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cloning of che-1 gene involved in chemotaxis of C. elegans O Uchida, M Koga, Y Ohshima Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan Caenorhabditis elegans provides a good model system for studying a variety of behaviors owing to its simple and identified network of 302 neurons. The animals respond to various chemical stimuli by moving either toward or away from the stimulus. che-1 mutants of C. elegans which exhibit erratic behavior to water-soluble attractants had been isolated previously. The che-1 mutants have several morphological defects in the sensory endings of neurons at the tip of the head. We precisely mapped a che-1 mutation genetically into the region between dpy-14 and bli-4 on the chromosome I. The che-1 gene was identified by a series of rescue experiments with cosmid clones or DNA fragments in this region. Mutation sites for five che-1 alleles were found in the predicted ORF, which lead to structural changes of the putative product. che-1 encodes a putative zinc finger protein that is highly homologous to those of glass and Krüppel genes of Drosophila, which are involved in photoreceptor differentiation and body segmentation, respectively.

24 May 1999 15:50 912 912 Molecular characterization of C. elegans protein phosphatase 1 homologue (CePP1)

1999 International Worm Meeting abstract 862 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Molecular characterization of C. elegans protein phosphatase 1 homologue (CePP1) H Ueda 1 , K Hirano 2 , M Nakamura 2 , S Harada 3 , T Sassa 2 , R Hosono 1,2

1 Gra. Sch. Natural Science and Technology, Faculty Med, Kanazawa Univ. Kanazawa, Ishikawa, Japan. 2 Dept. Physical Information, Faculty Med, Kanazawa Univ. Kanazawa, Ishikawa, Japan. 3 Center of Biomedical Research and Education, Faculty Med, Kanazawa Univ. Kanazawa, Ishikawa, Japan.

Protein phosphorylation is involved in many cellular processes. The steady state levels of these protein phosphorylations are dependent on the balance of activities of protein kinases and protein phosphatases. Serine/threonine protein phosphatase type1 (PP1) has a variety of functions including neural modulation and signaling events. We performed molecular analysis of the pp1-1 gene encoding C. elegans protein phosphatase 1 homologue (CePP1) . We found a region the C. elegans genomic DNA containing the deduced open reading frame with significant identity to the murine and Drosophila PP1. The region, found in cosmid F56C9 located on chromosome III was designated as pp1-1 consisting of 7 exons. The gene has a typical promoter sequence TATATTG which is located 71 bases upstream from ATG . CePP1 possesses a highly conserved amino acid sequence with the rat (89 % identical) and Drosophila (82% identical) PP1. Northern blotting analysis was carried out to pursue the expression of the pp1-1 gene transcript. The transcript was detected at the similar level throughout development, though transiently decreased at the L2 stage. We explored cells expressing the pp1-1 gene using a gfp and lacZ reporter construct. The gene was mainly expressed in pharyngeal and body muscles. Disruption of the pp1-1 gene by Tc1 insertion showed no visible influence on development or locomotory behavior. Furthermore ablation of the PP1 function by microinjection of RNAi is in progress to confirm the observed phenotype in Tc1 insertion mutants. Further detailed analyses to determine when and where the pp1-1 gene is expressed during the development are in currently progress using in situ hybridization and immunohistochemistry with antisera against CePP1.

24 May 1999 15:50 913 913 The ADM-1 protein: possible roles in development and interacting proteins.

1999 International Worm Meeting abstract 863 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The ADM-1 protein: possible roles in development and interacting proteins. C Valansi, G Shemer, B Podbilewicz Department of Biology, Technion-Israel Institute of Technology, Haifa 32000,Israel ADM proteins in C. elegans are multidomain membrane receptors that contain A Disintegrin and Metalloprotease domains similar to snake venoms. The members of this family are involved in growth factor and receptor shedding in different cell-cell and cell-matrix interactions. ADM-1 was the first gene from this family that was found in C.elegans. ADM-1 expression in cell membranes that participate in epithelial cell fusions and in sperm, suggests that it can be involved in cell fusion events of developmental importance. To investigate its putative role, we have done dsRNA interference experiments. About 20% of the progeny showed abnormal phenotypes which included degenerated gonads, arrested oocytes, vulvaless and uncoordinated with midbody backwards movement. In addition, 10% of the progeny died in early embryonic stages. It is known that members of the ADAMs family (fertilins) form heterodimers and undergo proteolytical processing. Thus, to investigate the processing and the putative biochemical and genetic interactions between ADM-1 and other proteins, we have used a monoclonal antibody that recognizes the cytoplasmic domain of ADM-1 to affinity purify interacting proteins. ADM-2 or other ADM proteins may interact with ADM-1 as occurs between fertilin a and b in mammals. Subcellular fractionation, detergent extractions and affinity purification verify the subcellular localization of ADM-1 and biochemical interactions between ADM-1 and cytoskeleton components. Using western blots we followed the proteolytical processing of ADM-1 from a precursor to the mature form. The amino termini of different proteins that copurify with ADM-1 will be sequenced and mass spectroscopy will be used to identify interacting proteins. The cellular localization of adm-1 products together with the RNAi experiments suggest that adm-1 can have different roles in embryonic and postembryonic development.

24 May 1999 15:50 914 914 A genetic screen for recessive mutations that suppress the egg-laying defect of lin-12 gain-of-function mutations

1999 International Worm Meeting abstract 864 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A genetic screen for recessive mutations that suppress the egg-laying defect of lin-12 gain-of-function mutations LG Vallier 1 , X Karp 2 , N Chen 3 , I Katric 2 , I Greenwald 1,3

1 Howard Hughes Medical Institute, Columbia University, 701 W. 168th St., New York City, NY 10032. 2 Department of Genetics and Development, Columbia University, 701 W. 168th St., New York City, NY 10032. 3 Department of Biochemistry and Biophysics, Columbia University, 701 W. 168th St., New York City, NY 10032.

During development of the hermaphrodite somatic gonad, signals between two equipotent cells, Z1.ppp and Z4.aaa, interact so that one becomes the anchor cell (AC) and the other becomes a ventral uterine precursor cell (VU). The activity of the receptor LIN-12 specifies this "AC/VU decision". Mutations that eliminate lin-12 activity lead to 2 ACs. Mutations in lin-12 that lead to a constitutively activated receptor have a 0 AC defect, so a vulva is not formed. We are interested in identifying other genes that influence the AC/VU decision. Previous genetic screens have identified several "sel" genes (suppressor or enhancer of lin-12) that affect lin-12 activity during the AC/VU decision (Sundaram and Greenwald, 1993; Tax et al, 1997). However, these screens were not performed to saturation. Thus, other genes are likely to be involved in this decision and are currently still unidentified. We are focusing on obtaining additional suppressors of the 0 AC defect caused by constitutive lin-12 activity (as in Tax et al., 1997). We are looking for extragenic mutations that suppress the egg-laying defect of a weak gain-of-function allele, lin-12(n302). L4 unc-36(e251) lin-12(n302) hermaphrodites were mutagenized with EMS using standard genetic methods. Plates containing F2 hermaphrodites were screened for the presence of eggs. These eggs were picked and examined as adults for egg-laying competence. Currently, over 200,000 F1 haploid genomes have been screened and 25 extragenic mutations that suppress the egg-laying defect of n302 have been identified. Extragenic mutations will be mapped by standard techniques and genetically characterized. Our goal is to saturate this screen.

Sundaram and Greenwald (1993) Genetic and phenotypic studies of hypomorphic lin-12 mutants of C. elegans. Genetics 135:755-763 Tax et al (1997) Identification and characterization of genes that interact with lin-12 in Caenorhabditis elegans Genetics 147:1675-1695

24 May 1999 15:50 915 915 Genetic analysis of signal transduction components involved in taste perception

1999 International Worm Meeting abstract 865 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic analysis of signal transduction components involved in taste perception C van den Berg, E Hazendonk, C de Vries, RH Plasterk Netherlands Cancer Institute Worms are able to respond to a wide variety of soluble and volatile compounds. We have developed a genetic screen to unravel signal transduction components in the avoidance of repulsive compounds. Mutagenised worms (generated both by EMS or by transposon insertions) were placed on a chemotaxis assay plate on one side on a thin line of CuSO4, with a volatile attractant on the other side. While wild type worms are unable to cross this boundary, mutants that are defective in sensing CuSO4 are able to pass the barrier. So far 90 mutant lines have been isolated, following EMS mutagenesis. All these mutants are also unable to sense 4M fructose and 2% SDS. 34 lines were rescreened for their proper expression of a GFP marker of amphid neuron structure (GPA15::GFP, which is normally expressed in the ASH, ADL and ASK neurons), in order to determine structural abnormalities. 3 of these mutants showed structural alterations in their cilia that resembles those previously seen in che-3 mutants (Wicks et al, in prep.). Complementation analysis confirmed allelism to the che-3 gene. Furthermore, 37 mutants were tested for the ability to take up fluorescent dye in their exposed amphid neurons. 25 mutants fail to dye fill and thus have structural alterations in their amphids. The remaining 12 mutants showed normal dye filling and are thought to have more subtle defects in sensory function. We are currently testing these mutants for odorant perception and water soluble attraction to determine whether they are specifically defective in aversion or in general perception. We have also isolated 21 transposon insertion mutants, which are also all generally defective in aversion to water soluble compounds. Among these, we have identified 2 more alleles of che-3, one allele of osm-6 and one allele of daf-10. We are currently isolating the transposon flanks of the remaining mutants to identify the gene mutated by the transposon.

24 May 1999 15:50 916 916 Mutations that suppress the lethargic and egg-laying defective phenotype of Gb overexpression

1999 International Worm Meeting abstract 866 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Mutations that suppress the lethargic and egg-laying defective phenotype of Gb overexpression AM van der Linden, F Simmer, HC Korswagen, RH Plasterk The Netherlands Cancer Institute, Division of Molecular Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands G proteins act as signal-transducing molecules that connect serpentine transmembrane receptors to various intracellular effectors. In C. elegansseveral G proteins have been identified. There are 20 Ga subunits, two Gb subunits (GPB-1 and GPB-2) and at least two Gg subunits (Jansen, pers. comm.). Previously, Zwaal et al.(1) studied GPB-1 in detail. GPB-1 is over 80% identical to mammalian Gb subunits and is required throughout development. Animals homozygous for the gpb-1deletion arrest in the first stage of larval development. This lethal phenotype is morphologically similar to gsa-1and acy-2null mutant animals, suggesting the absence of an essential Gs-coupled signaling pathway in early development (2). Progeny of mosaic animals that lost maternal expression of GPB-1 in the germline are embryonic lethal due to the inappropriate orientation of cleavage planes in early cell division. Overexpression of GPB-1 reduces locomotion and egg-laying and resembles the phenotype of goa-1(gf)mutants. The second G beta subunit (GPB-2) is not required for viability, but it is shown to modulate muscle activity (Simmer, pers. comm). Whereas GPB-1 and GPB-2 show similar expression patterns and are both involved in locomotion and egg-laying, they seem to have opposite functions in regulating muscle and neuron activity. Presently, we are investigating the role of these proteins during embryonic development. The effects of GPB-1 on behavior may represent either a direct signal function of the Gbg-subunit on downstream targets, or Gbg-levels may indirectly influence G alpha activity, or both. To identify downstream components of the Gbg-signaling pathway, we screened for mutations that suppress the lethargic and egg-laying defective phenotype of GPB-1 overexpression. We isolated 13 independent mutations that suppress this phenotype, named gbs(G beta suppressor). Two mutations are hyperactive (loopy) and egg-laying constitutive in the absence of GPB-1 overexpression, and are phenotypically similar to goa-1(lf)mutants. We mapped gbs-1to chromosome I. We are currently mapping and characterising these suppressors.

1) Zwaal et al.(1996) Cell 86; 2) Korswagen et al.(1998) EMBO J. 17

24 May 1999 15:50 917 917 World-wide worm SNPs: Dissecting the molecular evolution of the species using a high density SNP map

1999 International Worm Meeting abstract 867 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. World-wide worm SNPs: Dissecting the molecular evolution of the species using a high density SNP map H Van Luenen, R Koch, R Plasterk The Netherlands Cancer Institute, Division of Molecular Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands We would like to reconstruct the history of the C. elegans species at the genome level, therefore we sampled the genomes of four natural isolates (strain CB4857 isolated in Claremont, California, RC301 from Freiburg, Germany, TR403 from Madison, Wisconsin and AB1 from Adelaide, Australia) for single nucleotide polymorphisms (SNPs). Random genomic DNA fragments from the 4 strains were shotgun cloned and sequenced. There was no selection for transcribed or non-transcribed regions of the genome. In total we sequenced 1572 clones resulting in over 1 Mb of sequence information. The sequences are compared to the canonical Bristol N2 sequence to ask the question whether the clone maps to a unique sequence, and -if so- whether it contains polymorphisms. Once a SNP is identified we check other strains for the presence of the same polymorphism by PCR amplification and sequence analysis. In an initial experiment we found approximately one SNP per 3000 bp sequenced. The SNPs are randomly spread over the genome. Based on these observations we expect to find approximately 500 SNPs, one in every 200 kb. In the initial experiment we found, as expected, that several SNPs initially detected in one strain were also present in some but not all other strains. For example: a T in the Australian AB1, is a G at the same position in Bristol N2 in cosmid K10D2 at position 27946, and we found it to be like AB1 in the Californian CB4857 strain and the German RC301 strain, while the TR403 strain from Wisconsin resembles the Bristol N2 strain. Thus different patches of the genome have different ancestors. With our high density SNP map we will generate a genome map for each isolate which will show how each genome is patched together from a limited set of parental strains. The SNP’s will be added to ACeDB, and can also be used as markers on the genetic map. They can be recognised by PCR followed by sequencing, but we also found that the SNPs we looked at could be visualised by SSCP analysis. We thank Jane Rogers and Amanda McMurray for their assistance in sequencing the clones.

24 May 1999 15:50 918 918 Promoter analysis of mel-32(SHMT) using GFP expression and interspecies DNA comparison

1999 International Worm Meeting abstract 868 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Promoter analysis of mel-32(SHMT) using GFP expression and interspecies DNA comparison GP Vatcher, DL Baillie Institute of Molecular Biology and Biochemistry, Simon Fraser University, Vancouver, B.C. V5Z 1S6, Canada The mel-32 gene in Caenorhabditis elegans encodes a serine hydroxymethyltransferase isoform. SHMT is a ubiquitous, highly conserved enzyme that is the major source of the one-carbon units used in the biosynthesis of purines, pyrimidines, thymidylate, methionine, and lipids. Seventeen EMS induced mutant alleles of mel-32(SHMT) all give rise to a recessive maternal effect lethal phenotype. Sixteen of these alleles alter highly conserved amino acids, while one allele introduces a stop codon that truncates two thirds of the predicted protein. mel-32 genomic DNA was used to screen a genomic library from the closely related nematode Caenorhabditis briggsae. Computer analysis reveals that CbMEL-32(SHMT) is 92% identical (97% similar) to CeMEL-32(SHMT) at the amino acid level over the entire length of the protein (484 amino acids), while the coding DNA is 82.5% identical (over 1455 nucleotides). The C. briggsae genomic clone fully rescues the Mel-32 phenotype in C. elegans, indicating functional conservation and suggesting that the promoter and enhancer regions share enough homology to regulate the proper temporal and developmental control of mel-32(SHMT). The upstream and downstream regions of these genes share only 43% homology overall, but a dot matrix analysis reveals that there are stretches of highly conserved sequence. These conserved islands probably represent sequence elements which bind regulatory factors. The six regions with the highest homologies range in size from 11 to 44 nucleotides and share between 75% and 100% identity. To study the expression pattern of this gene in C. elegans a series of mel-32(SHMT)::GFP fusions were constructed, one with a wild-type promoter, and several with 5’ deletions that dissect the conserved regulatory regions. Preliminary analysis shows that the wild-type promoter displays extensive gut staining, while a deletion construct that removes a highly conserved GATA-binding dimer consensus sequence abolishes gut staining.

24 May 1999 15:50 919 919 Elucidating the function of a novel DNA polymerase

1999 International Worm Meeting abstract 869 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Elucidating the function of a novel DNA polymerase AI Vaz Gomes, EL Sonnhammer Center for Genomics Research, Karolinska Institutet, S-171 77 Stockholm Sweden The C. elegans genomic sequence has revealed the presence of a gene (W03A3.2) homologous to bacterial DNA polymerase I (PolA). This family was previously thought to be specific for bacteria but other genes clearly belonging to this family are now found in many eukaryotes, including Drosophila, maize, Plasmodium, Arabidopsis, mouse, and human. No such gene is present in yeast however. It thus appears that this is an ancient family of DNA polymerase which has been lost in some species. Functional studies on the Drosophila homolog (mus-308) have indicated a role in repair of crosslinked DNA (1). Both the Drosophila and Arabidopsis homologs posses an N-terminal helicase domain. Intriguingly, a similar helicase domain is encoded upstream of W03A3.2, denoted W03A3.3. They were not predicted as one gene by Genefinder because another gene, ceh-10, lies in between these domains the opposite strand. We are currently addressing the following questions: 1. Are the helicase and polymerase domains parts of one gene in the worm too? 2. What is the function of this or these genes? 3. What is the evolutionary relationship between the PolA-like sequences in different species? Indications that the domains may be transcribed separately include: Attempted RT-PCR from the helicase to the polymerase domain yielded no product; RNAi knockdown of the helicase domain induced degeneration of embryos "in utero" while targeting the polymerase domain gave no phenotype at all. Indications for one single gene include: No RT-PCR product was yielded between SL1 or SL2 and the polymerase domain; The fly and plant homologs have the two domains on a single protein chain. On the other hand, a recently cloned human homolog, named Pol eta (3), lacks the helicase domain. We have showed that the helicase domain is expressed (transspliced to SL1) and has a function essential for development. More work is needed to understand the polymerase domain. These studies are being pursued by simultaneous targeting, Northern analysis, RACE-PCR, expression studies, and antibody raising for immunodetection.

1. Harris et al (1996) Mol.Cell.Biol. 16, 5764-5771 2. Sonnhammer & Wootton (1997) Curr.Biol. 7, R463-465 3. Sharief, Ropp and Copeland, unpublished

24 May 1999 15:50 920 920 Transgenically improved entomophatogenic nematodes for pest control

1999 International Worm Meeting abstract 870 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Transgenically improved entomophatogenic nematodes for pest control T Vellai 1 , A Molnár 2 , L Lakatos 2 , Z Bánfalvi 2 , F Müller 3 , A Fodor 1

1 Eötvös University, Department of Genetics, Budapest, Hungary. 2 Agrobiotechnology Research Center, Gödöllõ, Hungary. 3 Institute of Zoology, University of Fribourg, Switzerland.

Several strains of the entomophatogenic nematode (EPN) species (Heterorhabditis spp. and Steinernema spp.) can be used efficiently against a wide spectrum of insect pests. However, the agricultural application of EPNs has been limited by their extreme sensitivity to desiccation due to high salinity and drought. We report here on an attempt to improve the desiccation tolerance of EPNs transgenically by transforming them with the gene tps1, coding for trehalose-6-phosphate synthase (TPS1), an enzyme that is involved in the biosynthesis of trehalose. The disaccharide trehalose, an osmoprotectant molecule of key importance, is accumulated in a number of organisms in response to desiccation and stabilises the dehydrated lipid membranes. In order to increase the desiccation tolerance by transgenic means, we have cloned tps1 from yeast into the nematode expression vector pPD49.78 (kindly provided by A. Fire), containing the C. elegans heat shock promoter hsp16-2. This tps1 construction (pAM-C02) was injected into C. elegans hermaphrodites. Upon heat induction, tps1 is expressed in almost all larval and many adult somatic cells. C. elegans transgenic for tps1 are highly resistant to osmotic stress, whereas wild type animals are not. The tps1 construction pAM-C02 was also injected into Steinernema feltiae females. Based on previous observations, the hsp16-2-driven tps1 was assumed to be expressed in S. feltiae as well. Because there is no transformation marker for selection in Steinernema spp. available, transgenic progeny were selected directly by their increased tolerance to high osmolarity and were checked for tps1 by a "single-animal"-based PCR. Like in C. elegans, a dramatic increase in desiccation tolerance due to trehalose overproduction was observed in transgenic S. feltiae adults. From our experiments we conclude that desiccation tolerance in nematodes can be induced without significant negative consequences by using an inducible promoter and the tps1 gene of the yeast.

24 May 1999 15:50 921 921 ’High density C. elegans screening’: a drug discovery platform applied to the steerin (unc-53) pathway

1999 International Worm Meeting abstract 871 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. ’High density C. elegans screening’: a drug discovery platform applied to the steerin (unc-53) pathway K Ver Donck 1 , I Mailliet 2 , I Roelens 2 , L Bols 1 , P Van Osta 1 , T Bogaert 2 , J Geysen 1

1 Janssen Research Foundation, Beerse, Belgium. 2 DeVGen, Gent, Belgium.

Green fluorescent protein (GFP) technology enables cellular resolution in the living animal. When combined with light sensitive cameras and with up-to-date automated stage driving, image acquisition and image analysis, microscopists can observe, analyse and derive objectively calculated parameters from composite digital images: a novel technology termed ’high density nematode screening’. We developed this technology platform for 96-well plate in vivo nematode phenotype drug screening, using a transformed C. elegans strain, expressing GFP in the vulval myoblasts under the control of the ceh-25 promoter. The vulval myoblasts were previously shown to be dependent on steerin (unc-53) for their correct positioning in larval develpment (Bogaert, unpublished). The screening was run on an inverted fluorescence microscope, equipped with an automated (XYZ) stage and an intensified camera. SCIL-image software (University of Amsterdam) on Silicon Graphics unix was used to write programs for stage driving, automated focussing (patent pending), seamless image grabbing, generation of composite images and semi-automated image picking for visual analysis of the wild type or compound-induced phenotype. Using this screening assay, a lead compound previously shown active in agar plate cultures was confirmed and novel compounds discovered as in vivo inhibitors of the steerin signaling pathway. The activity of this compound has been confirmed using mouse neuroblastoma cells overexpressing nematode steerin. Computer simulations using the crystal structure of GRB-2 C-terminal SH3-domain suggest that this compound may be a diproline mimetic capable of inhibiting SH3-binding to proline-rich helices.

24 May 1999 15:50 922 922 Steerins (UNC-53) in health and disease: from C. elegans gene to drug discovery

1999 International Worm Meeting abstract 872 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Steerins (UNC-53) in health and disease: from C. elegans gene to drug discovery C Buesa, P Verhasselt, M De Raeymaeker, L Maertens, C Platteeuw, J Geysen, K Ver Donck, M Vandecraen, E Stringham, T Bogaert Janssen Research Foundation, Beerse, Belgium & DeVGen, Gent, Belgium Genetic, cellular, biochemical and structural evidence suggested that nematode steerin is a central regulator of ’directional’ cell motility, by transducing receptor tyrosine-kinase signals into recruitment of F-actin and microtubuli in the nematode and in mammalian cells; indicating the existance of a conserved pathway in mammals and hence the existance human steerins (see also Vandecraen et al., 1999). A human steerin gene family has been cloned. Steerins comprise a N-terminal F-actin binding domain and a C-terminal predicted nucleotide binding domain and a middle domain of alpha-helical structure comprising a microtubule binding domain, coiled-coils I, II and III and 2 Pro-rich SH3 binding-like signatures. Human steerins display five more highly conserved boxes, discriminating them from nematode steerin. Funcional equivalence with nematode steerin was confirmed for human steerin-1 for activation of the F-actin cytoskeleton and binding to microtubule (+)-ends. Steerin-1 immunoreactivity localised to the microtubule (+)-ends in G361 melanoma cells (polyclonals). Expression of the human steerins is complex, both in terms of variants per tissue and tissue specificity (multiple tissue Northern blots). The chromosomal loci of both steerins are indicative of a putative role in neoplasms. A nematode screening platform was developed to search for inhibitors of the steerin/unc-53 pathway in the nematode. A first compound discovered induces pharmacologically an unc-53 (lf) phenotype in the sex myoblasts of wild type C. elegans (see Ver Donck et al., 1999, Celegans99 meeting abstract). In conclusion, animal steerins emerge as a novel structurally and functionally conserved animal protein family operational in a conserved pathway that transduces positional signals over e.g. receptor tyrosine kinase pathways into recruitment of the F-actin cytoskeleton. Steerins appear to perform this function from the microtubule (+)-end. Indicative evidence exists for a role of steerins in neoplastic disease and amenability of the pathway to pharmacology was shown using the nematode as a drug screening model.

24 May 1999 15:50 923 923 The function of Rap1 and its guanine-nucleotide-exchange factors in C. elegans

1999 International Worm Meeting abstract 873 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The function of Rap1 and its guanine-nucleotide-exchange factors in C. elegans MH Verheijen 1 , J Jansen 2 , RH Plasterk 2 , JL Bos 1

1 Laboratory for Physiological Chemistry and Centre for Biomedical Genetics, University Utrecht, Utrecht, the Netherlands. 2 Division of Molecular Biology and Centre for Biomedical Genetics, The Netherlands Cancer Institute, Amsterdam, the Netherlands.

RAP1 is a small, RAS-like GTPase that is activated by several extracellular stimuli via at least three different second messengers, namely diacylglycerol, calcium and cyclic AMP. In contrast to our knowledge about the function of RAS, only little is known about the function of RAP1. RAP1 was first identified as a suppressor of RAS-induced transformation, a feature of overexpressed RAP1 which is a result of interference with RAS-signaling pathways. However, recently we showed that activation of endogenous RAP1 does not interfere with RAS signaling (Zwartkruis et al., (1998) EMBO J. 17, 5905). To obtain insight into the biological function(s) of RAP1, we study the role of RAP1 and its guanine-nucleotide-exchange factors (RAPGEFs) in C. elegans. The genome sequencing project has revealed the existence of two RAP genes, which are the putative homologues of mammalian rap-1 and rap-2. In addition, four putative homologues of mammalian RAPGEFs were found: the C. elegans c3g, a calcium- and diacylglycerol-inducible GEF (caldaggef), the cAMP-inducible GEF (epac) (de Rooij et al., (1998) Nature 396, 474) and a PDZ-containing GEF (pdz-gef) (de Rooij et al., manuscript in prep.). The specificity of these putative RAPGEFs for RAP1 will be determined in vitro. We are currently investigating the expression patterns of these genes using GFP gene fusions. In addition, we are screening for loss-of-function mutants in a chemically mutagenized library and are determining the effect of overexpression and constitutively active variants on transgenic worms. Via this approach we hope to obtain insight into the physiological role of RAP1 signaling in C. elegans.

24 May 1999 15:50 924 924 CBP-1 in C. elegans embryonic development and differentiation

1999 International Worm Meeting abstract 874 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. CBP-1 in C. elegans embryonic development and differentiation M Victor 1 , C Mello 2 , Y Shi 1

1 Department of Pathology, Harvard Medical School, Boston, MA 02115. 2 University of Massachusetts, Cancer Center, Worcester, MA 01605.

Mammalian p300 and CBP are closely related transcriptional coactivators that possess histone acetyltransferase (HAT) activity and function as integrators of transcription and signaling events. Recently, we have shown that a C. elegans gene closely related to p300/CBP, cbp-1, is required to specify multiple differentiation events during embryogenesis. In endoderm differentiation, cbp-1 appears to promote differentiation by antagonizing the repressive activity of histone deacetylases, suggesting that a balance between histone acetyltransferase and deacetylase may be important for endodermal differentiation (1). To further investigate the mechanisms underlying the differentiation-promoting activities of CBP-1, we isolated a cbp-1 deletion mutant using a PCR-based approach. Antibody staining shows that this deletion resulted in a cbp-1 null allele. The majority of embryos homozygous for the cbp-1 deletion die around the 2-fold stage, suggesting that maternally provided CBP-1 is sufficient for the initiation of most differentiation events. We have begun to rescue the cbp-1 deletion mutant using a YAC that carries a wildtype cbp-1 gene. Preliminary results suggest that both maternal and zygotic defects can be rescued. Dead embryos segregated from the rescued animals morphologically resemble the cbp-1(RNAi) embryos. A detailed analysis of these embryos as compared to the cbp-1(RNAi) embryos and embryos derived from the cbp-1 deletion mutant will be presented. Work is in progress to use this YAC-based approach to determine the role of various functional domains of CBP-1 (i.e. the HAT domain of CBP-1) in differentiation.

(1) Shi, Y. and Mello, C. (1998) A CBP/p300 homolog specifies multiple differentiation pathways in Caenorhabditis elegans, Genes Dev. 12, 943-55. Martin Victor is supported by a postdoctoral fellowship from the DAAD, Germany.

24 May 1999 15:50 925 925 The Elusive Rec-1

1999 International Worm Meeting abstract 875 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Elusive Rec-1 V Vijayaratnam, A Rose Department of Medical Genetics, University of British Columbia, Vancouver, BC Canada V6T 1Z3 Recessive mutation in the Rec-1 gene alters the recombination pattern along the autosomes in C. elegans (Zetka and Rose, 1995). The authors concluded that the rec-1 mutation eliminates the meiotic recombination pattern and generates a genetic map proportional to physical distance. The autosomes have pronounced gene clusters that are not detectable on the X chromosome. We have tested the effect of rec-1 mutation on the X chromosome. Recombination frequencies between (I) Unc-1 Dpy-3 (II) Dpy-3 Unc-20 (III) Lon-2 Dpy-6 and (IV) Unc-7 Lin-15 were measured in wild type and the rec-1 mutant background. We observed an alteration in recombination frequencies in the rec-1 mutant background consistent with the altered pattern observed on the autosomes. The alterations were not as large as for the autosomes, and were in the direction predicted if the meiotic pattern were eliminated and the genetic distance proportional to physical distance. It has been shown that the him-1 mutation reduces the recombination frequency at the right end of the X chromosome (Broverman and Meneely, 1994), and the total length of the chromosome is one third that of wild type. We tested rec-1 recombination in the him-1 background. Our data show that the rec-1 mutation does not alter the him-1 pattern but does change the frequency. Candidates for the gene product of rec-1 are being analyzed. F33H2.1 gene was selected as the first candidate, however no sequence alteration was found in the rec-1 mutant (Wicky and Rose, unpublished data). Furthermore, immunostaining of the F33H2.1 protein showed localization in the muscle dense bodies. A second candidate, F33H2.5 is currently being investigated. RNAi of F33H2.5 gave an embryonic arrest phenotype. Our search for the sequence alteration responsible for the rec-1 mutant phenotype continues.

1) Zetka, M., and Rose, A. M., (1995). Mutant rec-1 eliminates the meiotic pattern of crossing over in C. elegans. Genetics, 141: 1339-1349. 2) Broverman, S. A. and Meneely, P. M. (1994). Meiotic mutants that cause a polar decrease in recombination on the X chromosome of C. elegans. Genetics 136, 119-127.

24 May 1999 15:50 926 926 Protein-Protein Interactions Involving the Sex Determining Protein, FEM-3

1999 International Worm Meeting abstract 876 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Protein-Protein Interactions Involving the Sex Determining Protein, FEM-3 U Vivegananthan, AM Spence Department of Molecular and Medical Genetics, Univeristy of Toronto, 1 King’s College Circle, Toronto, ON, Canada, M5S 1A1 Male development in C. elegans depends on the activities of the three fem genes. Conversely, female development requires that fem activity be negatively regulated. We are interested in how the sex determining protein FEM-3 functions and how its activity is regulated. We believe that a physical interaction between FEM-3 and FEM-2 is required to promote the adoption of male cell fates (Chin-Sang and Spence, 1996. Genes and Dev.10: 2314-25). We also have evidence that an interaction between FEM-3 and the C-terminus of TRA-2A is required for the negative regulation of fem activity in XX somatic tissues (Mehra, A., Gaudet, J., Heck, L., Kuwabara, P. and Spence, A. submitted). We want to use genetic assays to test our hypotheses about the biological significance of the two known FEM-3 interactions. We screened in vitro mutagenized fem-3 cDNAS, in a modified version of the two-hybrid system, to look for FEM-3 mutants that are able to bind to FEM-2, or to TRA-2A, but not both. We have identified two mutations that selectively disrupt the FEM-3/FEM-2 interaction and five mutations that selectively disrupt the FEM-3/TRA-2A interaction. We are currently testing the effects of these mutants on sex determination in vivo. We predict that FEM-2 non-binding mutants of FEM-3 will be incapable of supporting male development. In contrast, we predict that the TRA-2A non-binding mutants of FEM-3 will dominantly masculinize the XX soma, irrespective of the level of TRA-2A activity.

24 May 1999 15:50 927 927 Genetic analysis of the entomopathogenic nematode - bacterium symbiosis from bacterial side

1999 International Worm Meeting abstract 877 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genetic analysis of the entomopathogenic nematode - bacterium symbiosis from bacterial side A Völgyi 1,2 , A Fodor 1 , S Forst 2

1 Department of Genetics,Eötvös University, Budapest, Hungary. 2 Division of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI, USA.

The insect-pathogenic bacterium Xenorhabdus nematophilus(Enterobacteriaceae) is a symbiont of the entomopathogenic nematode Steinernema carpocapsae. X. nematophilus are carried into sensitive insect larvae by the infective dauer juvenile (IJ), released into the hemolymph and kill the insect. X. nematophilus proliferates in the and enter stationary phase. In stationary phase, X. nematophilus secretes products, supporting the nematode development. The nematodes complete several cycle and thannext generation IJ leave the cadaver with a limited number of phase I cells inside to find new host. Phase II cells arise spontaneously during prolonged culturing. During phase shift, several phenotypes change pleiotropically. Phase II cells do not take specific dyes up; are defective in production of antibiotics, crystal proteins, fimbriae and opnB membrane protein; can’t swarm on semisolid surface. The mechanisms leading to the simultaneous changes of pleiotropic phenotypes of different biochemical background must be based on the function of regulatory gene(s), activating and/or repressing several phase specific operons. Tn10 mutagenesis was adopted to tag them. Both phase II and phase I cells were mutagenized in order to obtain mutants of phase I and phase II phenotypes, and several putative mutant of phase II phenotype occurred amongst the mutagenized phase I cells. The Tn10 insertions were produced by using the conjugative and suicide mini-Tn10 system, S17-1/pLOFKmR. Kanamycin resistant colonies were initially screened for the loss of dye (bromothymol blue) uptake. The putative mutants were analyzed for appearance of phenotypes, characteristic for the opposit phase variant. Nucleotide sequence analysis of the flanking region around the insertion in one mutants revealed a partial open reading frame that was homologue with ybhE of E. coli (at amino acid level showed 47.8% identity). Due to the Blast Search, downstream to ybhE, ybhD is located within ybh operon in E. coli.The targeted gene seems to belong to the LysR transcription regulatory proteins family.

24 May 1999 15:50 928 928 A nicotinic acetylcholine receptor involved in regulation of egg-laying behavior.

1999 International Worm Meeting abstract 878 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A nicotinic acetylcholine receptor involved in regulation of egg-laying behavior. LE Waggoner, DS Poole, WR Schafer Department of Biology, University of California at San Diego, La Jolla, CA 92093 We are interested in understanding the role nicotinic acetylcholine receptors play in egg-laying behavior. The nicotinic receptor agonist levamisole has been known to stimulate egg-laying in C. elegans. We have found that response is dependent on three genes, unc-29, unc-38 and lev-1, which encode nAChR subunits. In addition, mutations in these genes confer a slower rate of egg-laying under nominal conditions, suggesting that an UNC-29-containing nAChR promotes normal egg-laying. Expression studies using GFP-tagged UNC-29 show that this receptor is localized to both the vulval muscles and the VC neurons; however, it is not found in the HSNs. In addition, mosaic analysis, performed in our lab, indicates that while expression in both muscle and neurons is necessary for optimal response, expression in either location is sufficient for some stimulation of egg-laying by levamisole. Ablation of VC neuronal precursor cells prevents response to levamisole, supporting the hypothesis that the receptor’s site of action is, in part, neuronal. Furthermore, cha-1 animals, which have low levels of endogenous ACh, are resistant to stimulation by levamisole, suggesting that the neuronal function of the receptor might be to facilitate of ACh, thus stimulating muscle contraction and egg-laying. Interestingly, we find that mutations in genes that confer weak resistance to levamisole in body wall muscle, including lev-8 and lev-9, also show resistance to stimulation of egg-laying. We are interested in characterizing these genes, which are thought to encode positive regulators of nAChR activity. Progress in cloning one of these genes, lev-9, will be presented.

24 May 1999 15:50 929 929 The C. elegans protein interaction mapping project: a test-case using proteins involved in vulval development

1999 International Worm Meeting abstract 879 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The C. elegans protein interaction mapping project: a test-case using proteins involved in vulval development AJM Walhout 1 , R Sordella 1 , N Thierry-Mieg 1 , M Brasch 2 , G Temple 2 , J Hartley 2 , M Lorson 1 , S van den Heuvel 1 , H Endoh 1 , M Vidal 1

1 MGH Cancer Center, Charlestown, MA. 2 Life Technologies Inc., Rockville, MD.

The nearly complete genome sequence of C. elegans predicts 19,099 protein-encoding genes. So far, at least 75% of the predicted genes have no function assigned: i) ~500 have been cloned and characterized functionally , ii) ~1,100 have been identified genetically but remain to be cloned, and iii) putative functions can be proposed for ~3,600 based on their homology to relatively well characterized S. cerevisiae gene products. The fact that three quarters of worm genes have no function assigned would justify efforts to help accelerate the pace of gene function discovery. To this end, several "functional genomics" projects are already under development for C. elegans with the goal of providing an interface between the genome sequence and conventional biology. Protein-protein interactions form the basis of many biological processes and their identification often suggests functional information. In this context, we are examining the possibility of generating a comprehensive protein interaction map for C. elegans. Our questions are centered on the feasibility of using the yeast two-hybrid system to identify and validate potential interactions using high-throughput strategies. Specifically, our current efforts focus on: i) the cloning of worm ORFs into a versatile vector using a novel automatable system referred to as "Gateway cloning" (these reagents should be valuable not only for two-hybrid selections but also for other functional genomics projects), ii) high-throughput two-hybrid selections, iii) data release into ACeDB and iv) the development of strategies for in vivo validation of potential interactions. We will present data on those four issues in the context of ~35 proteins involved in vulva development. The data suggest that it should be possible to generate a genome-wide protein interaction map for C. elegans. Acknowledgments. This project benefits greatly from the help, support, contributions, or reagents from the following labs: Chalfie, Coulson, Hart, Horvitz, Hunter, Hyman, Kim, Plasterk, Thierry-Mieg.

24 May 1999 15:50 930 930 Role of General Transcription Factors in Early C. elegans Gene Regulation

1999 International Worm Meeting abstract 880 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Role of General Transcription Factors in Early C. elegans Gene Regulation AK Walker, Y Shi, TK Blackwell Center for Blood Research, Department of Pathology, Harvard Medical School. Early zygotic transcription activates differentiation-specific genes necessary for blastomere specification and gene products needed for core biological functions. ama-1 (RNA polymerase II) RNAi embryos fail to initiate zygotic transcription, arresting at about 100 undifferentiated cells. We are evaluating the requirements for general transcription regulators in early transcriptional events. Biochemical and genetic analysis in yeast have identified many regulatory cofactors which function between the polymerase and gene specific activators. Some, such as the TAFIIs, have been predicted to coordinately regulate cell cycle genes or to interact with activators. However, their role in metazoan gene regulation is unclear. Our approach has been to (1) identify C. elegans homologues of regulatory cofactors and obtain corresponding clones (from Y. Kohara or the Sanger Centre), (2) evaluate the RNAi phenotype and (3) analyze the RNAi effect on reporter genes in vivo. RNAi of orthologs for human TAFII250, TAFII130, TAFII100, and TAFII31/32 produced an early embryonic arrest, similar to the ama-1 RNAi. We have also analyzed CeTAFII250 and CeTAFII100 RNAi in the end-1::gfp strain (obtained from J. Rothman). end-1 is expressed in the E2-E8 cells and is regulated by the maternal factor SKN-1. Ectopically expressed end-1 induces exogenous gut and adoption of the long Ea/Ep-like cell cycle (Zhu (98) G&D 12:3809). RNAi of skn-1 and ama-1 in end-1::gfp worms blocks gfp expression. RNAi of CeTAFII250 or CeTAFII100 does not affect gfp expression, even while the embryos show the early ama-1 like arrest. Thus, CeTAFII250 and CeTAFII100 are required for core biological functions, but not to express a differentiation specific gene. Lineage analysis of the CeTAFII250 RNAi embryos shows that the E daughters divide early, with the MS daughters. Our results show that RNAi of CeTAFII250 uncouples end-1 expression from the end-1 dependent cell cycle lengthening in the E daughters. We will continue to evaluate requirements for specific TAFIIs and other regulatory cofactors on early reporter genes in vivo, and use this as a model system to investigate how these biochemically defined factors are required for specific gene regulation in vivo.

24 May 1999 15:50 931 931 glr-5 and glr-7: Two Glutamate Receptor Subunits That May Have a Role in Thermotaxis.

1999 International Worm Meeting abstract 881 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. glr-5 and glr-7: Two Glutamate Receptor Subunits That May Have a Role in Thermotaxis. CS Walker, DM Madsen, PJ Brockie, AV Maricq Department of Biology, University of Utah, Salt Lake City, UT 84112 At least ten ionotropic glutamate receptors have been identified in C. elegans. Of these, two non-NMDA receptor subtypes show a limited expression in the nervous system. In transgenic strains that express chimeric GLR-5::GFP or GLR-7::GFP fusion proteins, fluorescence is observed in only the RIA neuron. This neuron functions downstream of AIY and AIZ and is required for normal thermotactic responses. We would like to determine the subcellular distribution of GLR-5 and GLR-7 and gain some understanding of how they may contribute to the function of RIA. An especially interesting opportunity for the study of localization of glutamate receptors is presented by our finding that GLR-5 and GLR-7 are co-expressed in only one neuron. This permits the non-ambiguous assignment of synaptic localization. We can now directly test whether GLR-5 and GLR-7 co-localize and determine whether they co-localize with presynaptic inputs from specific sensory neurons, e.g., ASH, AIY and AIZ using YFP, CFP and GFP constructs. We will present data from our localization studies. Also, we can directly test in Xenopus oocytes whether GLR-5 and GLR-7 form homomeric or heterometic receptors. We are in the process of screening the Barstead and Maruyama cDNA libraries in order to clone full-length cDNAs encoding these two receptors. Finally, we would like to understand the behavioral consequences of deleting these two genes. We are now attempting to generate deletion mutations in both glr-5 and glr-7 using a Tc1 based approach.

24 May 1999 15:50 932 932 Studying the function of inositol 1,4,5-trisphosphate receptors in C. elegans

1999 International Worm Meeting abstract 882 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Studying the function of inositol 1,4,5-trisphosphate receptors in C. elegans DS Walker, HA Baylis Department of Zoology, University of Cambridge, Downing Street, Cambridge, CB2 3EJ, UK By regulating intracellular calcium flux, Inositol 1,4,5-trisphosphate receptors (IP3Rs) are central to the organisation of calcium signals and are therefore fundamental to a wide variety of cellular processes. IP3Rs in C. elegans are encoded by a single gene, itr-1 (also called lfe-1). In previous work (Baylis H.A., Furuichi T., Yoshikawa F., Mikoshiba, K., Sattelle D.B., submitted), we have characterised the structure of this gene in detail and identified a number of splice variants. The gene encodes mRNAs with three alternative 5’ ends, originating from exons that are widely spaced in the genome. The C. briggsae homologue of itr-1 was identified by screening the fosmid library and this region of the C. briggsae genome sequenced by the GSC St. Louis. Comparison of the two genes has identified a number of conserved regions that may be important in the control of ITR-1 expression. Antibody staining of embryos, larvae and adults is revealing a wider pattern of ITR-1 expression than previously described. IP3Rs are present throughout embryogenesis. Itr-1::GFP fusions containing the region upstream of the most 5’ exon exhibit expression in the vulva. We are currently extending these studies. Clandinin et al. (1) have previously shown that itr-1 functions downstream of let-23 in the of control ovulation. We are further investigating the roles of IP3Rs by ablating the function of this gene by RNAi. With a view to determining the molecular basis of IP3R function, in terms of both cellular distribution and their integration into multiple signalling pathways, we are using Yeast Two Hybrid screens to identify proteins with which they interact.

Clandinin, TR, DeModena, JA, Sternberg, PW (1998) Cell 92, 523-533

24 May 1999 15:50 933 933 Identification and analysis of temperature sensitive embryonic lethal mutations that disrupt gene expression in early C. elegans embryos

1999 International Worm Meeting abstract 883 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification and analysis of temperature sensitive embryonic lethal mutations that disrupt gene expression in early C. elegans embryos MR Wallenfang, GC Seydoux Dept. of Mol. Bio. and Genetics, Johns Hopkins U. Sch. of Med., Baltimore, MD 21205 Transcription of mRNAs has been observed as early as the 3-cell stage in somatic, but not germline, blastomeres. This difference is due to an inhibitory mechanism which delays the onset of mRNA transcription in the germ lineage until the 100-cell stage (Seydoux and Dunn, 1997; Seydoux et al., 1996). The absence of newly synthesized mRNAs in germline blastomeres correlates with the absence in these cells of a specific phosphoisoform of the CTD of RNA polymerase II (RNAPII-H5). Germline blastomeres also have reduced levels of another RNAPII phosphoisoform, RNAPII-H14. In contrast, both of these isoforms are detected at high levels in somatic blastomeres. Although the exact function of these isoforms remains unclear, these observations have suggested that RNAPII may be inactive in germline blastomeres. We conducted a screen to identify embryonic lethal mutants that disrupt patterns of gene expression in early embryos. This screen made use of a pes-10:gfp reporter transgene that drives GFP fluorescence in all blastomeres, with the exception of the germline blastomeres. From approximately 1,000,000 EMS mutagenized genomes, we recovered 1000 Ts embryonic lethal mutants. Among these, we identified 7 mutants with GFP expression in all blastomeres, including 6 alleles of previously identified par-like genes (par-2, par-4, zyg-11, and let-99). One other mutant shows par-like and other pleiotropic defects and is being further characterized. We also identified 11 mutants which express no pes-10:GFP. Staining for RNAPII-H5 and RNAPII-H14 reveals that these mutants fall into 3 classes: those with wild-type H5 and H14 patterns (6 alleles), those with low levels of H5 and H14 in all blastomeres (3 alleles), and those with normal H5 and H14 levels early (<50 cells) and reduced H5 levels later (2 alleles). Further analysis of one allele (ax54) from this third class revealed that early embryonic mRNAs are transcribed in this mutant but remain trapped in the nucleus. In addition, expression of an intronless gfp reporter is unaffected in ax54 embryos, suggesting that there may be a defect in pre-mRNA processing. ax54 fails to complement let-307. Detailed analysis of the remaining H5/H14 defective mutants is under way.

24 May 1999 15:50 934 934 RHA-1: A conserved RNA helicase in C. elegans important for development

1999 International Worm Meeting abstract 884 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. RHA-1: A conserved RNA helicase in C. elegans important for development KM Walstrom 1 , KA Swan 2

1 Division of Natural Sciences, New College of USF, Sarasota, FL 34243. 2 Institute of Molecular Biology, University of Oregon, Eugene, OR 97403.

RNA helicases are important proteins involved in RNA metabolic reactions, such as splicing and transcription. Last year we discovered an RNA helicase (RHA-1) required for development in C. elegans. This protein is homologous to Drosophila maleless and human RNA helicase A (hRHA). Each of these proteins interact with DNA binding proteins and are proposed to regulate transcription. Specifically, hRHA is capable of mediating an association between CREB binding protein (CBP) and RNA polymerase II. This suggests that hRHA may play a role in mediating the effects of CBP on transcription (Nakajima et al. (1997) Cell 90, 1107). RHA is also required for proper gastrulation in mice (Lee et al. (1998) PNAS 95, 13709). RNA interference (RNAi) was used to deplete C. elegans embryos of RHA-1. We injected 800 bp of double-stranded RNA transcribed from DNA encoding the N-terminus of RHA-1 and observed a range of phenotypes culminating in dead embryos. Some dead embryos exhibited muscle development and most lacked gut granules. This suggests that endodermal development was compromised. Less severely affected progeny reached adulthood but were sterile or laid mainly oocytes. Others have shown that performing RNAi with RNA transcribed from C. elegans cbp-1 results in embryos lacking mesodermal, endodermal, and hypodermal cells (Shi & Mello (1998) Genes and Develop. 12, 943-955). Thus one explanation of our rha-1 RNAi results is a partial block of CBP function. Alternately, RHA-1 may be required for the function of other transcription factors, or it may be generally required for transcription or translation. Our current work involves biochemical experiments on the arginine-glycine-rich (RG) RNA binding region of RHA-1. This RG region binds specifically to single-stranded RNAs; therefore, it may have sequence specific RNA binding. The RG region of RHA-1 was subcloned from a previously isolated cDNA clone (from Yuji Kohara). This protein has been overexpressed in E. coli as a His-tagged polypeptide. RNA binding results will be discussed.

24 May 1999 15:50 935 935 Convergent Genetic Programs Create the Synaptic Pattern that Distinguishes the VD and DD Motor Neurons from One Another

1999 International Worm Meeting abstract 885 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Convergent Genetic Programs Create the Synaptic Pattern that Distinguishes the VD and DD Motor Neurons from One Another B Walthall, M Zhou Biology Department, Georgia State University, Atlanta GA 30303 How do genes orchestrate the many different features necessary to assemble and maintain functional neural circuits? One particularly important challenge is the establishment of diverse but specific synaptic patterns among neurons and their targets. The VD and DD motor neurons (mns) together form a cross-inhibitory network that maintains the proper phase relationship between the waves of dorsal and ventral muscular contraction that create the sinuous patterns of locomotion. The VD mns release the inhibitory neurotransmitter GABA onto ventral muscle as a result of dorsal cholinergic input from excitatory mns. Conversely, the DD mns inhibit dorsal muscle in response to input from ventral excitatory mns. The transcription factor Unc-30 is expressed shortly after the birth of both the VD and DD mns and is necessary for proper structural and functional features (McIntire, 1993; Jin, 1994, 1999). More than 15 genes are known to be regulated either directly or indirectly by Unc-30 and mutations in these genes produce identical defects in both mn classes. Reduction of inhibition on either the dorsal or ventral side (either surgically or genetically) produces asymmetric patterns of movement. Thus the analysis of asymmetric unc mutants could identify genes that effect either the synaptic specificity of the DD or the VD mns. unc-55 mutants exhibit a strong ventral asymmetric pattern of locomotion. In these mutants the VD mns adopt the DD mn synaptic pattern. Unc-55 is a transcription factor that is expressed in the postembryonic VD mns and their sisters, the AS mns, shortly after their birth (10 hrs after hatching). We report here experiments examining interactions among unc-30 and unc-55. To test whether Unc-30 regulates the expression of unc-55 in the VD mns we placed an unc-55:gfp reporter in an unc-30(e191) mutant background. We observed gfp expression in VD and AS mns suggesting that the two genes are regulated independently of one another. unc-55:gfp expression is characterized by rapid onset and dissipation in the ventral cord mns, suggesting temporal regulation. However, unc-55:gfp expression in an unc-55(jd8) mutant background was brighter and lasted longer than usual, indicating that Unc-55 negatively regulates its own expression.

24 May 1999 15:50 936 936 Expression of amyloid precursor and related protein in C. elegans

1999 International Worm Meeting abstract 886 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Expression of amyloid precursor and related protein in C. elegans H Wang Department of Biology, Boston University, 5 Cummington street, Boston, MA 02215 The major component of senile plaques in the brains of Alzheimer’s disease patients is the amyloid peptide, which is derived from a larger amyloid precursor protein (APP). Early-onset familial Alzheimer’s disease has been shown to correlate with mutations in three genes, including the APP gene. We are studying a APP-related gene, apl-1, in C. elegans. To identify genes that interact with apl-1, transgenic lines in which an apl-1 cDNA is under the control of a neural-specific promoter (VAMP) were generated. These transgenic strains were confirmed by Western blot analysis to overexpress APL-1. The transgenic animals have several phenotypes, including sluggish movement and a flattened waveform. To identify genes that interact with apl-1, we mutagenized transgenic animals with EMS, and looked for reversion of the sluggish phenotype. After screening about 3,000 haploid genomes, two strains that suppress the sluggish phenotype were isolated. The yn8 strain shows wild-type movement, while the yn9 strain shows hyperactive movement. These possible suppressor genes are currently being mapped. The putative suppressor mutation in the yn9 strain does not map to chromosome V or X. APL-1 does not contain the amyloid peptide. To determine whether production of amyloid peptide has any effect in C. elegans, transgenic strains containing a mouse APP cDNA under the control of the VAMP promoter were generated. The mAPP transgene is transcribed in transgenic animals as assayed by RT-PCR. Western blot analysis to determine whether the amyloid peptide is produced is underway. Several phenotypes have been characterized. The transgenic animals have an egg-laying defect, decrease in movement, and a longer defecation cycle. The huAPP cDNA was obtained recently, and similar studies will be performed.

24 May 1999 15:50 937 937 Identification of Potential mpk-1 Targets by DNA Microarray Analysis

1999 International Worm Meeting abstract 887 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification of Potential mpk-1 Targets by DNA Microarray Analysis J Wang, V Reinke, CB Van Doren, RR Begley, SK Kim Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305 DNA microarray technology permits large-scale analysis of gene expression. With the completion of the C. elegans genome project, analysis of gene expression differences on the genome-scale is now possible. The generation of a potentially large set of differential regulated genes should prove to be a powerful tool to complement the many resources available to C. elegans. Currently, we have a microarray consisting of approximately 13,000 elements, or about two-thirds of the genome, with the intent to generate a full-genome array soon. We would like to identify the transcriptional response to the activation of the Ras/MAP kinase signaling pathway, with the goal of identifying the specific response in the vulva. To this end we will be using a heat-shock MAP kinase (mpk-1) construct to drive expression throughout the animal. We reason that global over expression of MAP kinase by a heat-shock promoter will generate a robust and detectable transcriptional response. Some of the expression differences will be the core response to MAP kinase activation, while hopefully a subset will represent the vulval-specific targets of MAP kinase. Since MAP kinase expression is under the control of the heat-shock promoter and to control for heat-shock induced expression, we will also delineate the core heat-shock response in N2 worms. In order to subdivide the MAP kinase targets into a core response and the specific responses, we will make comparisons among microarray experiments from staged and mixed-staged worms. Expression differences common to all the experiments are likely to be the core MAP kinase response. Furthermore, those genes that are regulated only during a specific stage would represent stage-specific and potentially tissue-specific responses. These experiments will generate large amounts of data, while fruitful analysis of the large data sets is difficult. One way to begin dissecting the data is to group genes based on co-variance of gene expression across a diverse set of microarray experiments. Clustering the target genes into functional groups will refine the description of the MAP kinase dependent response and will provide a springboard for the identification of a gene or set of genes for further analysis.

24 May 1999 15:50 938 938 cMi-2, a component of histone deacetylase, is essential for C. elegans development

1999 International Worm Meeting abstract 888 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. cMi-2, a component of histone deacetylase, is essential for C. elegans development S Wang 1 , Y Zhang 2 , D Reinberg 2 , M Driscoll 1

1 Department of Molecular Biology & Biochemistry, Rutgers University, Piscataway, NJ 08854. 2 Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854.

Dermatomyositis is an autoimmune inflammatory disease. Antibodies against Mi-2 are regarded as a specific marker in dermatomyositis. Mi-2 is a 218 kd protein of 1912 amino acids belonging to the SNF2/RAD54 helicase family. It contains 2 PHD-zinc finger domains, 2 chromodomains and a helicase/ATPase domain. Biochemical studies demonstrated that Mi-2 is a component of a complex that has histone deacetylase and nucleosome remodeling activities. Homozygous mutants of Drosophila dMi-2 die as first or second instar larva. We identified two Mi-2 homologs in the C. elegans genome, F26F12.7 and T14G8.1. Mi-2 and F26F12.7 are 44% identical; Mi-2 and T14G8.1 are 44.8% identical. The helicase/ATPase domain is most highly conserved. To study the in vivo functions of these two C. elegans Mi-2 homologs, we screened for gene knockouts. We identified a deletion of 1616 bp between the third exon and the forth exon of F26f12.7, which creates a stop codon. The encoded truncated protein is 528 amino acids truncated between the two chromodomains. Homozygous mutants (derived from heterozygotes) can develop into adults but are completely sterile. We are further characterizing the mutant phenotypes and screening for a T14G8.1 deletion. We are also characterizing the human Mi-2 protein by biochemical approaches.

24 May 1999 15:50 939 939 Two zinc finger proteins interact with the TRA-2 intracellular domain

1999 International Worm Meeting abstract 889 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Two zinc finger proteins interact with the TRA-2 intracellular domain Shanping Wang, Judith Kimble Howard Hughes Medical Institute and Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706 The tra-2 gene promotes female fates in C. elegans. Loss of tra-2 activity causes XX animals to develop as males, whereas gain-of-function mutations of tra-2 transform hermaphrodites into females. The TRA-2A protein is predicted to be a trans-membrane protein with a cytoplasmic tail. To search for proteins that interact with this cytoplasmic tail, we screened a C. elegans Gal4 fusion activation library, using the cytoplasmic portion of TRA-2A as a bait. Out of 5 million transformants screened, two zinc finger proteins were identified that bind specifically to TRA-2. One interacting protein, isolated only a single time, was TRA-1, a key sex determining regulator in C. elegans. The other one, called TRP-1 (for TRA-2A reactive protein), was isolated multiple times in the pool of positive clones. BLAST search indicated that TRP-1 is one member of a zinc finger protein family that has homologs in Drosophila, mouse and human. Deletion analysis revealed that the TRP-1 interacting domain lies in the N-terminus of cytoplasmic region of TRA-2A. Investigation of the function of TRP and this network of proteins will further our understanding of the function of tra-2 in promoting female fates.

24 May 1999 15:50 940 940 A molecule containing a Hedgehog-intein (HINT) domain is required for molting in C. elegans.

1999 International Worm Meeting abstract 890 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A molecule containing a Hedgehog-intein (HINT) domain is required for molting in C. elegans. X Wang, P Beachy, G Seydoux The Johns Hopkins Univ. School of Medicine Hedgehog proteins undergo an autoprocessing reaction to yield an amino-terminal fragment (Hh-N) and a carboxy-terminal fragment (Hh-C). Hh-N functions in signaling, whereas Hh-C, which consists of a Hint domain and a sterol recognition region, mediates the processing reaction. This reaction proceeds via an internal thioester intermediate and results in the covalent linkage of cholesterol to Hh-N. This modification in turn causes Hh-N to remain tightly associated with the cell surface, thus effectively limiting its free diffusion and range of action. We are interested in determining whether similar mechanisms might be operating in the biogenesis of other secreted molecules. Towards this goal, we have started to characterize a C. elegans protein (T05C12.10) with sequence similarity to the Hint domain of Hedgehog (Porter, JA. et al.,1996a,b; Burglin, TR., 1996). In this protein, as in the Hedgehog family, the Hint domain is located near the carboxy-terminus, and is preceded by an amino-terminal domain bearing a signal sequence (but with otherwise no homology to Hh-N). We have shown that the Hint domain of T05C12.10 can lead to the formation of an internal thioester intermediate in vitro. We have begun to characterize the functions of the T05C12.10 using RNA-mediated interference. Our results suggest that T05C12.10 is essential for molting. Injection of T05C12.10 double-stranded RNA results in 100% larval lethality. The larvae apparently die from a failure to shed old cuticles during molts. This phenotype resembles the phenotype of worms starved for cholesterol and of mutants lacking the megalin homologue, lrp-1 (Yochem, J. et al. 1999). The amino-terminal domain of T05C12.10 contains several sequence motifs similar to certain extracellular matrix proteins. Consistent with this, T05C12.10 protein colocalizes with megalin in the apical surface of hypodermal cells during larval and adult stages. Western analysis indicates that there are two forms of T05C12.10 in vivo, and that the relative abundance of the smaller form increases during each molt. These observations suggest that processing of T05C12.10 coincides with, and may be regulated by, molting. We are currently determining the identity of the smaller form as well as testing its role in molting.

24 May 1999 15:50 941 941 PIE-1 Gets Help from CeUBC9 in Germline Protection

1999 International Worm Meeting abstract 891 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. PIE-1 Gets Help from CeUBC9 in Germline Protection X Wang, T Shin, CC Mello U. Mass. Med. Center, Worcester, MA Embryonic germ cells undergo a sequence of stem cell-like divisions in which one and only one daughter maintains germline potential at each division. The germline remains undifferentiated due to the presence of a small germline-specific protein PIE-1, which functions to block the activity of somatic differentiation factors in the germline. PIE-1 is thought to achieve this germline protection by blocking transcription altogether. However, the specific mechanism of transcriptional repression as well as the mechanisms that restrict PIE-1 to the germline remain unknown. In order to address these questions, we have used a yeast two hybrid screen to look for PIE-1 binding proteins. One protein identified in this screen is a C. elegans homolog of vertebrate UBC9 (CeUBC9). CeUBC9(RNAi) causes several embryonic defects, but most importantly, approximately 20% of the embryos produce extra pharyngeal tissue from the germline blastomere, P2, a defect also seen in pie-1 mutants. This observation suggests that CeUBC9 is required for efficient execution of PIE-1-dependent somatic fate suppression. Consistent with this view, CeUBC9 and pie-1 show an interesting genetic interaction. Whereas a pie-1 heterozygote normally makes 100% viable progeny, CeUBC9(RNAi) in a pie-1 heterozygote results in a nearly fully penetrant pie-1 (extra pharynx and intestine) phenotype. Vertebrate UBC9 is shown to catalyze the covalent linkage of SUMO-1 peptides (small ubiquitin-like molecules) to protein substrates, and this modification is correlated with 1) increased stability or 2) changes in subcellular localization of the recipient protein. Interestingly, we found that the levels of PIE-1 and of PIE-1::GFP appear to be reduced in the nuclei of CeUBC9(RNAi) embryos. Furthermore, prior to metaphase, PIE-1::GFP transiently localizes to numerous punctate foci at the nuclear periphery. Based on these observations, we hypothesize that CeUBC9 controls PIE-1 by modulating its nuclear localization, degradation or both. We are currently testing whether PIE-1 is a SUMO-1 decorated protein in vivo and whether UBC9 can modify PIE-1 in vitro. Through this and other experiments, we should be able to determine how PIE-1 is regulated by, or regulates, CeUBC9 and whether UBC9 functions similarly in C. elegans and in vertebrates.

24 May 1999 15:50 942 942 Molecular Cloning and Characterization of a STAT homologue in C. elegans

1999 International Worm Meeting abstract 892 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Molecular Cloning and Characterization of a STAT homologue in C. elegans Y Wang, DE Levy Department of Pathology and Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, New York 10016 USA Though characterized by simplicity and directness in mammalian systems, JAK/STAT signaling pathway is involved in many different regulatory events, such as innate immune responses to viral or bacterial infection, T cell functions, and embryonic development. In Drosophila, a STAT homologue has been identified and shown to be essential for embryonic development. The fact that Dictyostelium discoideum has a STAT-like molecule to regulate its pre-stalk cell differentiation suggests the existence of STAT(s) in a wide variety of organisms. Here we report a C. elegans homologue of STATs. Nucleotide sequence analysis reveals that the 2.4kb cDNA contains a single ORF about 2.1kb which encodes a 80KD protein. With human STAT5b, ceSTAT shares 29% identity in SH2 domain, 37% in DNA binding domain. It also contains a GYIQ tyrosine phosphorylation motif which is conserved in all the STATs. Surprisingly, ceSTAT does not have an N-terminal domain which is highly conserved in mammalian STATs as well as in Drosophila STAT. Northern analysis of total RNA from mix-staged N2 worms shows a single mRNA of 2.4kb. When expressed in cell culture, ceSTAT can be tyrosine phosphorylated by mammalian kinases, resulting in its ability to bind DNA probe containing GAS element. Further, the C-terminus contains a transactivation domain that will function when fused to the GAL4 DNA binding domain. Initial studies of the function of ceSTAT involved injection of dominant-inhibitory dsRNA. We saw no phenotype in either injected animals, F1 or F2 progeny. This suggests that ceSTAT is probably not involved in worm developmental processes. To further determine the function of ceSTAT, we are currently isolating mutagenized worms carrying genomic mutations.

24 May 1999 15:50 943 943 Function, Tissue Distribution, and Mutants of nSLO1

1999 International Worm Meeting abstract 893 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Function, Tissue Distribution, and Mutants of nSLO1 ZW Wang, O Saifee, T Jegla, M Nonet, L Salkoff Department of Anatomy and Neurobiology, Washington University, 660 S. Euclid Avenue, St. Louis, MO 63110

The slo-1 gene of C. elegans encodes a large-conductance, Ca2+ -activated K+ channel. Deduced amino acid sequence reveals a protein (nSLO1) of 1141-residues which shares a high level of homology with the SLO1 proteins of mammals and Drosophila. Similar to these homologues, the nematode slo-1 gene is widely expressed in the nervous system and in many muscle types. This was shown by the expression pattern of a slo-1 promoter::GFP transgene. When nSLO1 was expressed in Xenopus oocytes, both single-channel and macroscopic currents could be recorded in inside-out membrane patches. The activity of the channel was Ca2+ - and voltage-dependent; higher activity was seen with increasing cytoplasmic Ca2+ concentrations and at more positive membrane potentials. Mutants of slo-1 were discovered in a screen for suppressors of a syntaxin mutant unc-64 (e246). Homozygous e246 animals are behaviorally lethargic and uncoordinated. After subjecting e246 animals to EMS, animals showing a revertant phenotype were analyzed for new mutations. Six allelic mutants were thus identified and mapped to chromosome V in the vicinity of unc-80 where slo-1 resides. In the absence of e246, animals homozygous for the new mutants are phenotypically jerky, and aldicarb-hypersensitive. However, in the genetic background of the e246 mutation, these mutants partially suppress the e246 lethargy. Introduction of the wild-type slo-1 gene (minigene) into the double mutants reinstated lethargy. Sequencing of the js118 mutant allele revealed a 270-bp deletion in slo-1 that removed the splice acceptor site of exon 16. As a result, exon 16 was absent; frame shift and premature termination occurred in the 17th exon. A large portion of the C-terminus, including the region conferring Ca2+ -sensitivity, was therefore removed. These observations are in agreement with a hypothesis that the syntaxin mutation inhibits neuromuscular activity by reducing neurotransmitter release, whereas a loss of function mutation of the slo-1 gene counters this effect by decreasing K+ conductance (and therefore increasing membrane excitability) at presynaptic and/or postsynaptic sites. Our results suggest that nSLO1 plays an important role in neuromuscular activities in C. elegans.

24 May 1999 15:50 944 944 gly-2 is an N-acetylglucosaminyltransferase V and Can Rescue the Mammalian Lec4 Mutation.

1999 International Worm Meeting abstract 894 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. gly-2 is an N-acetylglucosaminyltransferase V and Can Rescue the Mammalian Lec4 Mutation. CE Warren, JW Dennis Samuel Lunenfeld Research Institute, 600 University Ave, Toronto, ON M5G 1X5, Canada. GlcNAc-TV is a key regulator of polylactosamino-N-glycan chain formation and is causally involved in the metastasis of tumours in mice and humans. We have characterised the C. elegans orthologue (gly-2) originally identified via yk126h8. Northern analysis indicates a single rare ~2.2kb species of mRNA. 5’ RACE indicates that the transcript is exclusively trans-spliced to SL1 at the acceptor site immediately upstream of the probable initiator methionine in the genomic sequence (C55B7.2). The deduced polypeptide is 669 residues with 37% identity and 60% overall similarity to mammalian homologues. Golgi glycosyltransferases are type II transmembrane protein, and as expected, the relatedness is notably stronger in the C-terminal portion of the alignment (catalytic domain) and hydropathy analysis indicated a single potential transmembrane domain at the N-terminal. Constructs driving the secretion of soluble forms of GLY-2 were transfected into Lec4 cells, a GlcNAc-TV deficient mutant CHO line. We found that the CHO-expressed gene-product is enzymatically active and the catalytic domain starts at I138 , after which there is high similarity with mammalian homologues. Interestingly, the genomic gene is split by an in-frame intron boundary between K137 and I138 .

We observed that gly-2 can rescue the cell surface phenotype of Lec4 cells which lack endogenous GlcNAc-TV. Lec4A (CHO) cells have active enzyme which is mislocalised due to a missense mutation. We introduced the equivalent mutation, GLY2-L116 R, and abolished the phenotype rescue of Lec4 cells. We infer that the nematode enzyme can be correctly localised to the medial Golgi and acceptor specificity is well conserved with mammalian enzyme. We have isolated a C. elegans line carrying an allele where a Tc1 transposon is inserted into an intron in the 3’ region of gly-2 and a sub-library is being screened for a deletion allele. GlcNAc-TV null mice have suppressed tumour induction and metastasis. We aim to exploit C. elegans as a genetic tool to analyse the pathways by which these phenomena are mediated.

24 May 1999 15:50 945 945 Attempts toward larger C. elegans by a sma transgene

1999 International Worm Meeting abstract 895 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Attempts toward larger C. elegans by a sma transgene N Watanabe, Y Ohshima Department of Biology, Faculty of Science, Kyushu University, 6-10-1, Hakozaki, Fukuoka, 812-8581, Japan How to make the nematode C. elegans larger? We pay attention to the transforming growth factor-b (TGF-b) pathway. TGF-b superfamily ligands have major regulatory effects on growth and differentiation in vertebrates. In C. elegans, daf-1 and daf-4 encode serine/threonine kinase receptors related to TGF-b receptors. daf-4 mutant animals reveal multiple roles for TGF-b-like signalling and they were reported to be smaller than the wild type. Genes sma-2, sma-3 and sma-4 encode Smad family transcription factors acting in a daf-4 signalling pathway, and their mutants were also reported to be smaller. Considering these results, we expect that wild type genes for sma-2, sma-3, sma-4 and daf-4 may have an ability to induce a larger body size phenotype. First we measured the body length and maximum diameter of wild type and those mutant animals and confirmed that they are smaller. We also estimated their body volume in an approximation. We injected each of the wild-type genes sma-3 and sma-4 at varied concentrations, and daf-4 to the corresponding mutants. Some of the transgenic lines with sma-4 or sma-3 wild-type gene are nearly completely rescued with respect to the body size (length, maximum diameter and estimated volume). We also injected the sma-4 wild-type gene into the wild-type animal, and obtained a transgenic line longer than the wild type. The effect of sma-3 or sma-4 transgenes on the body size varied. The body size of the transgenic lines partly depends on the concentration of the gene in the injected solutions, and also varied from line to line. For a similar purpose, we are interested in the sma-5 gene. sma-5 was mapped on X chromosome within a region of about 0.6 map unit, but has not been cloned so far. We are trying to clone it by cosmid rescue. If sma-5 is identified, we will inject it into the wild-type nematode to see whether the transgenic nematode is larger than wild type.

24 May 1999 15:50 946 946 Examining the role of polyunsaturated fatty acids in growth and development of C. elegans

1999 International Worm Meeting abstract 896 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Examining the role of polyunsaturated fatty acids in growth and development of C. elegans JL Watts, JA Browse Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340 Polyunsaturated fatty acids (PUFAs) play important roles in the functioning of membranes and in the synthesis of signaling molecules. C. elegans can synthesize a wide range of polyunsaturated fatty acids using only saturated and monounsaturated fatty acids from E. coli as precursors. In order to study the role of PUFAs in development we have isolated a number of mutant lines exhibiting altered fatty acid compositions. Most of these fatty acid profile differences can be attributed to mutations in known desaturase genes which encode enzymes responsible for inserting double bonds into a fatty acid chain, or in genes involved in the elongation of 18-carbon to 20-carbon fatty acids. One mutant, fat-2 (qa1017), is defective in D12-desaturase activity, resulting in a block in the pathway of long chain PUFA biosynthesis. Whereas about 40% of membrane fatty acids in wild-type worms consist of PUFAs, these fatty acids are undetectable in fat-2 worms. The fat-2 mutants can be maintained as a homozygous line, however they grow very slowly, produce small broods, and exhibit a high degree of embryonic lethality. They are also uncoordinated and slightly dumpy. Feeding fat-2 worms small amounts of arachidonic acid rescues these defects, resulting in wild type movement and appearance, greater than 90% hatch rate, and a faster growth rate. We are currently examining the developmental defects of this strain in more detail in order to understand the role of PUFAs in growth, reproduction, and the nervous system of C. elegans.

24 May 1999 15:50 947 947 egl-2 encodes an eag-like potassium channel blocked by imipramine in C. elegans

1999 International Worm Meeting abstract 897 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. egl-2 encodes an eag-like potassium channel blocked by imipramine in C. elegans A Wei 1 , D Weinshenker 2 , J Thomas 2 , L Salkoff 1,3

1 Dept. of Anatomy and Neurobiology, Washington Univ. Sch. of Med., St. Louis, MO 63110. 2 Dept. of Genetics, Univ. of Washington, Seattle, WA 98195. 3 Dept. of Genetics, Washington Univ. Sch. of Med., St. Louis, MO 63110.

Dominant gain-of-function (gof) mutations of egl-2 in the nematode Caenorhaditis elegans result in animals defective in egg-laying, enteric muscle contractions and chemotaxis. These behavioral defects are rescued by imipramine, a tricyclic antidepressant. The egl-2 gene was cloned and found to encode an eag-like potassium channel subunit. Injection of wild-type egl-2 cRNA in Xenopus oocytes produced slowly activating voltage-dependent potassium currents. Two gof alleles were identified as missense mutations substituting a highly conserved alanine (A478) to valine within S6. Expression of mutant egl-2(A478V) cRNA produced potassium channels shifted in voltage-dependence of activation towards hyperpolarized potentials (V50(WT)=-7 mV, V50(A478V)=-40 mV). Thus, mutant EGL-2(A478V) potassium channels could underlie behavioral defects observed in vivo, through inappropriate suppression of excitability in critical cells. Promoter fusion constructs with green fluorescent protein revealed expression in a subset of neurons and muscles consistent with this possibility. Imipramine at low concentrations, blocked wild-type and (A478V) EGL-2 channels expressed in Xenopus oocytes (Kd(WT)=16 micromolar, Kd(A478V)=2.4 micromolar). Suppression of behavioral defects due to the gof egl-2 mutation, thus most likely results from a direct block of mutant EGL-2 channels by imipramine. Similar in vitro properties were observed with a mouse brain eag homolog (meag). These results suggest that in addition to imipramine’s primary therapeutic action by blocking serotonin reuptake transporters at presynaptic terminals, secondary effects may result from the direct block of eag-like potassium channels.

24 May 1999 15:50 948 948 The C. elegans KQT-1 potassium channel is essential for normal pharyngeal pumping rhythm and viability: An animal model for long-QT syndrome.

1999 International Worm Meeting abstract 898 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The C. elegans KQT-1 potassium channel is essential for normal pharyngeal pumping rhythm and viability: An animal model for long-QT syndrome. AD Wei, LB Salkoff Dept. of Anatomy and Neurobiology, Washington University School of Medicine. KQT potassium channels are encoded by a multigene family related to KvLQT1, a human gene responsible for one form of long-QT syndrome, a cardiac arrhythmia risk factor. Additional human members of this gene family are associated with hereditary epilepsy (BFNC2) (KCNQ2, KCNQ3) and deafness (DFNA2) (KCNQ4). C. elegans possesses multiple kqt genes (kqt1-3). We have initiated analysis of kqt genes in C. elegans with the aim of establishing a useful animal model for the genetic analysis of in vivo functions of KQT channels. C. elegans KQT-1 shares approximately 70% amino acid identity with KvLQT1. Expression studies of KQT-1 in Xenopus oocytes revealed voltage-gated potassium currents, similar to those produced by KvLQT1, but with strikingly slower activation kinetics. A potential role for kqt-1 was suggested by a promoter-GFP fusion. kqt-1::GFP revealed prominent expression in pharyngeal bulb. This myogenic muscle shares many features with cardiac myocytes, including the use of broad action potentials to generate rhythmical contractions. The similarity of action potential waveforms between cardiac myocytes and pharyngeal muscles, as well as the shared expression of KQT-type genes, suggested an important conserved role for these channels in shaping the electrical properties of these cells. To test the role of kqt-1 in pharyngeal function, a dominant-negative KQT-1 subunit was created. This was made by introducing a missense mutation of a conserved S6 residue (A338E), corresponding to a human KvLQT1 allele associated with long-QT syndrome. Dominant-negative KQT-1 and wild-type (WT) KQT-1 subunits were overexpressed in pharyngeal muscles, using the pharyngeal muscle promoter myo-2. Transgenic worms overexpressing dominant-negative KQT-1(A338E) exhibited reduced viability, in marked contrast to worms overexpressing WT KQT-1. Electropharyngeograms revealed abnormally long pump durations. These results suggest that suppression of kqt-1 channel function in pharyngeal muscles compromises viability through the lengthening of action potentials, leading to prolonged contractions. Future experiments will focus on identification of genes that suppress or modify this mutant phenotype.

24 May 1999 15:50 949 949 ric-6 and ric-7, two genes involved in C. elegans neurotransmission

1999 International Worm Meeting abstract 899 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. ric-6 and ric-7, two genes involved in C. elegans neurotransmission RM Weimer, EM Jorgensen Department of Biology, University of Utah, Salt Lake City, UT 84112 USA Release of neurotransmitter into the synaptic cleft requires the coordinated action of numerous proteins at the presynaptic terminal. We have taken a genetic approach to identify proteins required for this process. To this end, we are in the process of characterizing two genes, ric-6 and ric-7, that were previously identified in a behavioral screen and appear defective in neurotransmission. Mutations in ric-7 disrupt both GABAergic and cholinergic neurotransmission. ric-7 animals lack enteric muscle contractions during defecation and display a weak shrinker phenotype, both features of a loss of GABAergic function. In addition, ric-7 animals are resistant to the acetylcholinesterase inhibitor aldicarb, although they are sensitive to the acetylcholine receptor agonist levamisole indicating a presynaptic cholinergic defect. The organization of ventral cord motor neurons and the density of neuromuscular junctions appear to be normal in ric-7 animals and thus, RIC-7 does not function in neuronal development. Taken together, these phenotypes suggest a role for RIC-7 in the release of neurotransmitter at synapses. The ric-7 phenotype is rescued by an 18kb fragment that is predicted to contain a single open reading frame encoding a 694 amino acid protein with no known homology. Expression of green fluorescent protein (GFP) under the control of the ric-7 promoter identifies a predominately neuronal expression pattern. This data further supports the hypothesis that ric-7 encodes a neuronal protein involved in presynaptic function. We are currently in the process of identifying other loci that genetically interact with ric-7. ric-6, like ric-7, also plays a role in both presynaptic cholinergic and GABAergic function. Mutations in ric-6 result in aldicarb resistant, levamisole sensitive animals that also display a weak shrinker phenotype. The ric-6 phenotype is rescued by a 5kb fragment that encodes the C. elegans homolog of the vacuolar ATPase B subunit. Vacuolar ATPase function is thought to be required for synaptic vesicle acidification, a process that is essential for loading neurotransmitter into vesicles. We will test this model using electrophysiological techniques recently developed in C. elegans.

24 May 1999 15:50 950 950 Information Coding in the C. elegans Olfactory System

1999 International Worm Meeting abstract 900 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Information Coding in the C. elegans Olfactory System PD Wes, CI Bargmann HHMI and Department of Anatomy. University of California, San Francisco, CA 94143-0452 Different signal transduction pathways coexist within the same cell, interacting in complex ways to produce novel responses, yet preserving fidelity. The olfactory system of C. elegans can be used to study the molecular basis of information processing within a single cell. C. elegans senses a broad array of attractive odors with just two pairs of sensory neurons, AWA and AWC. A number of odorants activate each neuron. Isoamyl alcohol (iaa), 2,3-pentanedione (pd), butanone (bt) and benzaldehyde (bz) all activate AWC, and, furthermore, each odorant utilizes the same cGMP signaling cascade. Nevertheless, animals are able to distinguish amongst some of these odorants. For instance, when placed in a uniform field of bt, animals will no longer chemotax toward a point source of bt (defined as saturation), yet will chemotax toward bz. A screen was conducted to identify mutants that responded normally to bz in the absence of bt, but failed to chemotax toward a point source of bz in a field of bt (defined as cross-saturation). 20,000 haploid genomes were screened, yielding 25 candidates. A range of phenotypes was observed, with the strongest mutant, ky542, displaying total cross-saturation of bz by bt. ky542 exhibits other defects in odor recognition or processing. It has reduced sensitivity to pd, though its sensitivity to all others odorants remains undiminished. ky542 also has enhanced discrimination between iaa and bz. Olfactory discrimination is also enhanced by starvation, a form of plasticity modulated by serotonin1 . In addition, ky542 and other cross-saturation mutants display a defect in olfactory adaptation, a long-lasting form of signal desensitization (see abstract by Noelle l’Etoile and C.B.). Several models that may shed light on these olfactory phenotypes are being tested. Understanding the principles behind olfactory discrimination may provide new insights into information coding within single cells, and may also elucidate some forms of behavioral plasticity that are modulated by C. elegans olfaction.

1 Colbert, H.A., Bargmann, C.I. 1997.

24 May 1999 15:50 951 951 A sensitized genetic screen to identify genes that act in basolateral membrane localization of the LET-23 EGF Receptor

1999 International Worm Meeting abstract 901 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A sensitized genetic screen to identify genes that act in basolateral membrane localization of the LET-23 EGF Receptor CW Whitfield, M Povelones, BJ Coffey, AF Hajnal, SK Kim Department of Developmental Biology, Stanford University, Stanford, CA 94305 The LET-23 EGF Receptor is localized to the basolateral membrane domain of the epithelial vulval precursor cells, where it acts through a conserved Ras/MAP kinase signaling pathway to induce vulval differentiation. lin-2, lin-7 and lin-10 act in LET-23 basolateral localization, since mutations in any of these three genes result in mislocalization of LET-23 to the apical membrane domain and cause a signaling defective (vulvaless) phenotype. Although null mutations in lin-2, lin-7 or lin-10 cause a ~95% vulvaless phenotype, reduced function mutations cause both a low penetrance vulvaless phenotype and a low penetrance multivulva phenotype. This multivulva phenotype is strongly enhanced by mutations in the gap-1 gene. For example, lin-10(n1509ts) animals are 7% vulvaless and 11% multivulva (15°), while lin-10(n1509ts); gap-1(lf) animals are 0% vulvaless and 81% multivulva (15°). gap-1(lf) animals are wild-type. Since gap-1(lf) can strongly enhance the multivulva phenotype associated with a weak lin-10 allele, we predicted that gap-1(lf) combined with new mutations that reduce (but do not eliminate) LET-23 at the basolateral membrane would cause a multivulva phenotype. We mutagenized gap-1(lf) animals, screened ~100,000 F1 genomes and isolated 91 mutant multivulva strains. Immunostaining with anti-LET-23 antibodies revealed that 37 of these strains are defective in basolateral membrane localization of LET-23 in the vulval precursor cells. Mutations that alter LET-23 localization fall into two major classes. In the first class (17 mutants) LET-23 is reduced at the basolateral membrane and concentrated at the apical membrane. Genetic complementation and mapping results indicate that this class includes new alleles of lin-2 and lin-10. We are currently testing whether any mutations in this class identify new genes that act in LET-23 basolateral localization. In the second class (20 mutants) LET-23 is reduced at the basolateral membrane and is largely intracellular. This staining phenotype could represent a LET-23 secretion defect. Mapping results indicate that two of these mutants are likely to be alleles of let-23, and may thus identify cis-acting elements that are important in LET-23 secretion.

24 May 1999 15:50 952 952 A strategy for the genetic analysis of the perception of taste sub-modalities.

1999 International Worm Meeting abstract 902 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A strategy for the genetic analysis of the perception of taste sub-modalities. SR Wicks, MA Essers, RH Plasterk Div. of Mol. Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands The worm responds to a wide variety of soluble compounds. However, the mechanisms of taste signal transduction are poorly understood. At least five taste modalities are recognised as distinct (sour, sweet, salty, bitter and umami), and each sub-modality may be detected and transduced independently. We have described a novel screen of soluble compound chemotaxis that we have successfully applied to genetic screens of taste perception. Using this assay we have isolated and cloned spontaneous alleles of che-2 (codes for a protein containing WD40 motifs, Fujiwara et al, 1998, WBG, 15(3):39) and che-3 (codes for an isoform of the dynein heavy chain, manuscript in preparation) from mutator strains. We used ammonium acetate as the attractant in these screens since it produces the most robust chemotaxis response that we have been able to characterise (a chemotaxis index in excess of 0.99 is not uncommon). One potential limitation of these screens is that the chemotaxis assay, when used as described above, is so robust that only very severe chemotaxis phenotypes are detected. Since our primary interest is in signal transduction, we have modified the assay such that more subtle phenotypes may be identified. One way to achieve this is to lower the concentration of attractant used. We have shown that worms respond to Na+Cl- at concentrations as low as 10 uM. Another way in which the quadrant assay may be made more sensitive is to pour agar containing two distinct attractants into pairs of opposite quadrants. We have tested many pairs of compounds to find a pair that results in clear, characteristic kinetics of population partitioning. Several candidate pairs have been identified, including NH4+Cl- vs. NH4+CH3COO-, and Na+Cl- vs. Glutamine. In each case, worms show an immediate (within 5 min.) preference for the former compound, but after 30 min., show a preference for the latter compound. These kinetics allow us to screen for loss of ability to taste either one compound or the other, and at the same time quickly identify the more common phenotype of complete loss of chemotaxis ability. Thus, we should be able to examine taste sub-modalities genetically. Such screens using salt and the amino acid glutamine are currently under way.

24 May 1999 15:50 953 953 Regulative Development in a Nematode Embryo: A Hierarchy of Cell Fate Transformations

1999 International Worm Meeting abstract 903 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Regulative Development in a Nematode Embryo: A Hierarchy of Cell Fate Transformations O Wiegner, E Schierenberg Zoologisches Institut, Universität Köln, D-50923 Köln, Germany Cell specification during embryogenesis of the model system C. elegans involves a combination of conditional and autonomous mechanisms. We have begun to study the development of other nematodes to investigate how well cell-specification mechanisms are preserved among closely related species. Here we report that the embryo of the soil nematode Acrobeloides nanus expresses a so far undescribed regulative potential. This allows - different to C. elegans- the development up to hatching and sometimes to fertile adults after elimination of early blastomeres, even if this means the loss of more than 50% of the embryos original volume. In our experiments eliminated cells are replaced in a sequential and hierarchical manner by specific neighbouring blastomeres. Thus, early somatic blastomeres in A. nanus are multipotent being capable to give rise to more than one somatic founder cell. Germline cells, however, cannot be replaced. A model is presented according to which in A. nanus cellular identities are assigned by a series of reciprocal inhibitory cell-cell interactions absent in C. elegans.

24 May 1999 15:50 954 954 The Roles of fax-1 and unc-20 in Nervous System Development

1999 International Worm Meeting abstract 904 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Roles of fax-1 and unc-20 in Nervous System Development B Wightman, N Carmean, B Ebert, H Kohrt, E Murphy, S Nguyen, G Radzievsky, K Klampert Biology Department, Muhlenberg College, Allentown, PA, 18104, USA. Specification of neuron identity requires the activation of a number of discrete developmental programs. Among these is pathway selection by growth cones. The fax-1 gene is required for pathfinding of the AVK axons that extend along the ventral nerve cord and nerve ring, and for normal expression of a FMRFamide-like neurotransmitter gene (flp-1) in the AVK neurons. The fax-1 gene encodes a member of the superfamily of nuclear hormone receptors, and has a DNA-binding domain related to the Drosophila Tailless protein, suggesting that it regulates the transcription of other genes. We observe FAX-1 expression in embryonic neurons, including the AVK interneurons, just prior to axon extension, but after neurogenesis. These data suggest that fax-1 coordinately regulates the transcription of genes that function in the selection of axon pathways and neurotransmitter expression during the specification of neuron identity. We are currently focusing our efforts on identifying the DNA binding site for FAX-1 and the downstream target genes that are regulated by fax-1. Mutations in unc-20 cause defects in axon pathfinding by various neurons, indicating that it also functions in nervous system development. Temperature-shift experiments suggest that unc-20 is required during embryonic development for normal HSN axon pathfinding during post-embryonic development. Phil Anderson’s laboratory has identified six alleles of unc-20 that are homozygous L1 lethal, suggesting that unc-20 also provides an essential function. In order to understand the function of unc-20 we are cloning the gene. We have mapped unc-20 to a relatively small region of about 25 kb in the middle of cosmid C15C7. Transformation rescue and RNAi experiments to locate the unc-20 gene are ongoing.

24 May 1999 15:50 955 955 The Anaphase-Promoting Complex is Required for Meiosis I Anaphase

1999 International Worm Meeting abstract 905 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The Anaphase-Promoting Complex is Required for Meiosis I Anaphase L Wille, M Abdolrasulnia, D Shakes Dept. of Biology, College of William and Mary, Williamsburg, VA The mitotic metaphase to anaphase transition in both yeast and vertebrates is driven by a multimeric E3 ubiquitin (Ub) ligase known as the anaphase promoting complex, APC. APC drives mitotic progression by sequentially targeting specific substrates for destruction, but its meiotic role is poorly characterized. To analyze APC’s role during C. elegans meiosis, we are depleting maternal stores of APC components by RNA-mediated interference. Our goal is to analyze all APC genes in C. elegans, with initial studies focusing on APC1(BIME) and APC2. APC1 is postulated to be a negative regulator of mitosis since bimE mutants enter mitosis prematurely and can bypass a G2 or S phase arrest (Osmani et al., 1988), subsequently arresting in mitotic metaphase. RNAi of the APC1 C.elegans homolog (W10C6.1) resulted in fertilized oocytes which arrest in meiosis metaphase I (Chase et al., 1998). Later studies using a different RNA preparation, resulted in "weaker" phenotypes, including embryos with cytokinesis defects and hatching larvae which develop into sterile adults. Such adults are often severely GLP, but others have abnormal vulvals and small, highly disorganized gonads filled with differentiated gametes. We are currently testing whether the observed variability is dosage dependent. APC2 shares a conserved cullin domain with the G1-S Ub ligase component cdc53 which suggests that, like cdc53, APC2 may interact directly with Ub conjugating enzymes (Yu et al., 1998). Consistent with its role as a core APC component, RNA-mediated interference of APC2 expression results in a strong 1-cell arrest phenotype in which maternal chromosomes lock in a prolonged meiosis metaphase I configuration. We have also observed weaker phenotypes, suggesting that APC2 RNAi effects are also dosage dependent. Our results suggest that APC function is not required for oocyte maturation, oocyte ovulation, fertilization, or the initial post-fertilization congression of the oocyte chromosomes. However APC function is critical during the meiosis I metaphase to anaphase transition, embryonic mitosis, and germline proliferation.

Chase et al. (1998) WBG 15(4):45 Yu et al. (1998) Science 279: 1219-22 Osmani et al. (1988) Cell 52: 241-251

24 May 1999 15:50 956 956 Identification and analysis of a new mutation affecting T cell polarity

1999 International Worm Meeting abstract 906 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification and analysis of a new mutation affecting T cell polarity KL Williams, MA Herman Division of Biology, Kansas State University, Manhattan, KS 66506 We are interested in how cell polarity is controlled during C. elegans development. Our approach is to identify and study genes involved in the control of cell polarity by identifying mutations that disrupt the polarities of individual cells. Mutations in lin-44 cause the polarities of certain cells in the tail, called TL and TR, to be reversed with respect to the body axis. LIN-44 is a WNT signaling protein that is made by the skin cells at the tip of the developing tail and functions to specify the polarity of more anterior tail cells. Mutations in lin-17 and egl-27 cause the loss of TL and TR cell polarities. LIN-17 is a Frizzled-like protein, suggesting that it is a receptor of LIN-44 WNT signal. EGL-27 contains a domain similar to MTA1, recently shown to be a subunit of a complex with chromatin-remodeling and histone deacetylase activities, and is required in the T cells for proper cell polarity. We want to learn how LIN-44 WNT signal from the tail skin cells influences the polarities of the receiving cells. The defects in T cell polarity observed in lin-44, lin-17 and egl-27 mutants cause the two neurons of the phasmid, a sensory structure in the tail, to fail to fill with fluorescent dyes. New genes that may interact with lin-44, lin-17 and egl-27 were identified by screening for additional mutations that result in a phasmid dye-filling defect caused by defects in T cell polarity. We are currently studying mn593, a mutation isolated by Claire Kari in a preliminary screen of this type. Subsequent screens have identified three other new mutations affecting T cell polarity (see Abstract by X. Zhao, et al.). mn593 complements mutations in all other known genes that affect T cell polarity. The mn593 mutation causes an incompletely penetrant phasmid dye-filling defect. Furthermore, one or both pairs of phasmid socket cells are missing in most mn593 animals, indicating a T cell lineage defect. Analysis of mn593 hermaphrodite T cell lineages is underway and will determine the exact nature of the defect. In addition, mn593 males have abnormal tail morphologies. We have mapped mn593 to the interval between dpy-5 and unc-63 on LGI. We are currently in the process of cloning the gene affected by mn593 by microinjection of the relevant cosmid and YAC clones into mn593 hermaphrodites.

24 May 1999 15:50 957 957 An Analysis of Cytokinesis in Early C. elegans Embryos by Electron Microscopy

1999 International Worm Meeting abstract 907 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. An Analysis of Cytokinesis in Early C. elegans Embryos by Electron Microscopy EM Williams-Masson, JG White University of Wisconsin, Madison, WI 53706 USA Animal cells generally undergo cytokinesis through the constriction of an equatorial band of actin microfilaments, followed by the pinching off and resealing of membranes in the daughter cells. We are using electron microscopy to study the interactions of the late cytokinetic furrow with the remnant of the spindle mid-zone, which forms the midbody in the cleaving cell. Cytokinesis is a dynamic process involving labile proteinaceous and membranous structures. Traditional chemical fixation methods act slowly to preserve cellular components and a large percentage are often extracted. As an alternative, we are using high-pressure freezing to rapidly preserve native cytological structure. Embryos are frozen in milliseconds at very low temperatures and high pressure, which allows the rapid cryoimmobilization of samples without damage from ice crystal formation. The samples are freeze-substituted at -70° to promote dehydration, slowly warmed to room temperature, lightly osmicated and embedded in resin. Select embryos are serial sectioned and examined by transmission electron microscopy. Examination of an embryo in the early stages of cytokinesis revealed a bundle of microtubules perpendicular to the future site of the cleavage furrow, approximately two microns from the leading edge of one side of the furrow. The bundle was bent in a U-shape, appearing to be pulled towards the nearest cleavage furrow, with long trails of microtubules sweeping away. Serial sections revealed the presence of microtubules parallel to the cleavage furrow in sections above and below the tubular bundle, and on both sides of the bundle. These circumferential microtubules also appeared to interact with the leading edge of the cleavage furrow. Vesicles were seen in the vicinity of the advancing cleavage furrow. An embryo in late cytokinesis exhibited an intercellular midbody that contained remnants of the mitotic spindle. This intercellular bridge was very lobular in nature, and these numerous lobes may function to seal the final breach between the cleaving cells. Examination of more embryos should further illuminate the interaction of the cleavage furrow with the midbody during cleavage in early C. elegansembryos, and gold immunolabelling studies are underway to identify key molecular players in the process of cytokinesis.

24 May 1999 15:50 958 958 K-Cl Cotransporters in C. elegans

1999 International Worm Meeting abstract 908 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. K-Cl Cotransporters in C. elegans J Willis, E Kipreos Department of Cellular Biology, University of Georgia, Athens In vertebrate cells the K-Cl cotransporter has the capacity to reduce cellular concentration of K and Cl. The value of this activity is unclear. The carrier is activated by hypotonic cell swelling in mammalian but not in fish red cells; in the latter it is activated only by isotonic swelling. In mammalian red cells the carrier is activated by slight warming and this insures balance of K influx and efflux at elevated temperature. Isoforms of the carrier have been implicated in downsizing of mammalian erythrocytes during differentiation and in reduction of cell chloride concentration in developing neurons. The gene sequences for two mammalian isoforms were identified in 1996 and at that time two similar sequences were recognized in the C. elegans database (found in cosmids KO2A2, "KO2", on chromosome 2, and R13A1, "R13", on chromosome 4). R13 exists as an EST, and using primers based on KO2 we have obtained a PCR product of the appropriate length using a C. elegans cDNA library as template. Hence both genes are expressed. We have made double-stranded RNA for segments of each of these genes and have injected these singly and in combination into ovaries of C. elegans. Screens for lethality, growth rate and productivity among the progeny of injected worms have not yielded differences. About a third of surviving worms injected with dsRNA for both KO2 and R13 have produced progeny that exhibit reduced survival at elevated temperature. Screens based on isosmotic and hypotonic cell swelling are being developed. These studies may help to establish the role of K-Cl cotransporters in a whole organism.

24 May 1999 15:50 959 959 Mutations in the L-type calcium channel EGL-19 affect UNC-4 function

1999 International Worm Meeting abstract 909 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Mutations in the L-type calcium channel EGL-19 affect UNC-4 function AR Winnier, DM Miller Dept. of Cell Biology, Vanderbilt University, Nashville, TN 37232 Wildtype movement depends on precisely defined connections between interneurons and motor neurons. In unc-4 mutant animals, the VA motor neurons, which are required for backward locomotion, are miswired to interneurons that are normally reserved for their sister cells, the VBs. As a result, unc-4 mutant animals cannot move backward. unc-4 encodes a homeoprotein that is required for the repression of VB-specific traits in the VA motor neurons. This UNC-4-mediated repression is dependent on interactions with the Groucho-like co-repressor protein UNC-37 (1). We would like to identify other genes which function with unc-4. Using genetic screens that resemble strategies used to study unc-4/unc-37 interactions, we have uncovered mutations in the EGL-19 calcium channel which appear to modify UNC-4 function. The semi-dominant wd29 (gf) allele was isolated in an Unc-4 suppressor screen. wd29 is an allele-specific suppressor of unc-4 missense mutations and maps to the stp44-unc-24 (IV) interval. A revertant allele, wd29wd45, results in the loss of wd29-dependent Unc-4 suppression. Homozygous wd29wd45 enhances the Unc-4 phenotype of unc-4(e2322ts) but does not result in a backward movement defect in a wildtype background. wd29wd45 fails to complement the egg-laying defective and long phenotypes of the n582, ad1015, and ad1006 egl-19(lf) mutants. Furthermore, the egl-19 mutations n582 and ad1006 enhance the unc-4(e2322ts) phenotype. Therefore, we propose that wd29wd45 represents an egl-19(lf) mutation. egl-19, which encodes an L-type calcium channel, is expressed in muscle and neurons although a function for EGL-19 in unc-4-expressing motor neurons has not been described (2). Currently, we are testing the idea that egl-19 is a downstream target of UNC-4 by evaluating egl-19::gfp in wildtype and unc-4 mutant backgrounds.

(1) Winnier, A. et al., this meeting. (2) Lee, R. et al. (1997) EMBO J. 16: 6066-6076.

24 May 1999 15:50 960 960 A Chemical Genetic Screen for Embryonic Lethality in C. elegans

1999 International Worm Meeting abstract 910 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A Chemical Genetic Screen for Embryonic Lethality in C. elegans WM Winston 1 , SJ Haggerty 1 , SL Schreiber 1,2 , CP Hunter 1

1 Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138. 2 Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138.

We are interested in how the polarity of the C. elegans early embryo is determined. Traditionally, mutants isolated in screens for maternal effect or temperature conditional lethal mutations are analyzed for polarity defects. However, not all genes or biological processes are susceptible to maternal effect or conditional inactivation by simple mutation. A different approach that can conditionally disrupt biological processes is the addition of an inhibitory small molecule. Just as genomes can be scanned for mutations that produce a desired phenotype, so can small molecules. We are planning large-scale phenotypic screens of small molecule combinatorial chemical libraries with the intent of finding molecules that have potent and specific effects on embryonic development. The first combinatorial library that will be screened contains approximately 16,000 molecules. Later screens will use libraries that contain up to 2.2 million members. We are currently developing strategies for high throughput screening and analysis. These include growing synchronized nematodes in 384 and 1536 well liquid culture, using vital reporters to monitor development, and sensitive scanners for identifying wells containing candidate molecules.

24 May 1999 15:50 961 961 Candidate catecholamine receptors in C. elegans

1999 International Worm Meeting abstract 911 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Candidate catecholamine receptors in C. elegans RF Wintle, W Kawczynski, NR Santos, HH Van Tol Depts. of Pharmacology, Psychiatry and Inst. of Medical Sciences, University of Toronto, and Division of Molecular Neurobiology, Centre for Addiction and Mental Health, Toronto, Canada Biogenic amine receptors in C. elegans have been implicated in various behaviors including egg laying, defecation, motility and food sensing.1 These receptors belong to the superfamily of G protein coupled receptors, which are structurally characterized by their transmembrane heptahelical structure. Analysis of the genome sequence revealed thirteen likely amine neurotransmitter receptors.2 Using the protein sequence of mammalian catecholaminergic receptors, we identified eight candidate receptors for catecholamines. Unlike the other putative amine neurotransmitter receptors from C. elegans, these receptors have the canonical conserved aspartate and serine residues in transmembrane domains three and five, which are critical for catecholamine binding. We have cloned full length cDNAs for these receptors. Analysis of our cDNA clones largely agrees with GeneFinder predictions and shows that all of these receptors contain numerous introns. The deduced gene structure for these receptors is reminiscent of the structure of dopamine D2-like, and several a-adrenergic receptors, but is not like the intronless D1-like amd b-adrenergic receptors. The amino acid sequences, as derived from the cloned cDNAs, display the characteristic seven hydrophobic transmembrane domains in a hydrophobicity analysis. The transmembrane domains have the highest degree of sequence conservation with the mammalian catecholaminergic receptor family, which is between 39 and 48% amino acid identity. None of the identified receptors display an obvious homology to one single mammalian catecholaminergic receptor subtype. These receptors are being analyzed by various approaches to identify their pharmacological profile, so that they can be classified accordingly (receptors for dopamine, octopamine, serotonin or other trace amines).

1. Weinshenker, D. et al. (1995). J. Neurosci. 15(10):6975-6985 2. Bargmann, C.I. (1998) Science 282(5396):2028-33

24 May 1999 15:50 962 962 ATP-binding cassette (ABC) transporters form a large and diverse gene family in C. elegans

1999 International Worm Meeting abstract 912 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. ATP-binding cassette (ABC) transporters form a large and diverse gene family in C. elegans RG Wisotzkey 1 , W Grant 2 , G Hardiman 1 , CD Johnson 1

1 Axys Pharmaceuticals, NemaPharm Group, South San Francisco, CA. 2 Flinders University, Adelaide, Australia.

ATP-binding Cassette (ABC) transporters are integral membrane proteins that couple the hydrolysis of ATP to the transport of a variety of substrates across the membrane (1). Members of the ABC transporter superfamily have either one or two ABC domains, which are composed of up to six transmembrane helices followed by a nucleotide-binding domain. ABC transporters have diverse functions in both normal cell biological processes and in protecting cells against noxious compounds. Members of the ABC transporter superfamily function in export of mating factor in yeast, the transport of eye pigment proteins in Drosophila, and antigen presentation in vertebrates. In C. elegans, the ABC transporter ced-7 is necessary for the engulfment of cell corpses (2), and other ABC transporters have been shown to affect sensitivity to natural toxins and heavy metal resistance (3). In humans, mutations of ABC transporters are associated with diseases including cystic fibrosis, progressive familial intrahepatic cholesterasis, Stargardt’s macular dystrophy and adrenoleukodystroyphy. ABC transporters are also potential targets for cancer treatment, as up-regulation of the multidrug resistance (mdr) class of ABC transporters in tumors causes resistance to multiple, chemically unrelated compounds. We have identified 53 ABC transporters in C. elegans by screening genomic DNA sequence generated by the Genome Sequencing Project. These transporters can be divided into seven subfamilies, three of which have two ABC domains similar to the vertebrate mdr transporters and the cystic fibrosis chloride channel, and four of which have a single ABC domain similar to HLA antigen transporters. Using gene chip technology, we are currently examining the expression of these transporters in normal development as well as in response to chemical treatment.

(1) Cole and Deeley (1998) BioEssays 20:931; (2) Wu and Horvitz (1998) Cell 93:951; (3) Broeks et al. (1995) EMBO J. 14:1858; Broeks et al. (1996) EMBO J. 15:6132

24 May 1999 15:50 963 963 Analysis of Muscle Function in C. elegans collagen IV mutants.

1999 International Worm Meeting abstract 913 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of Muscle Function in C. elegans collagen IV mutants. CM Witkowski, JM Kramer Dept of CMB, Northwestern University, 303 E. Chicago Ave., Chicago IL 60611 Analysis of myofilament organization in collagen IV mutants suggests multiple roles for C. elegans collagen IV. These roles include maintenance and stabilization of the myofilament lattice, cell growth and shape. We hypothesize that collagen IV provides elastic capacity to the basement membrane (BM) necessary for functioning of the body wall muscles and the pharynx. We crossed the transgene jam-1::gfp (MH27 antigen::gfp) that highlights hypodermal cell boundaries into an emb-9 (a1(IV) chain) null background. In normal animals, contraction of muscle cells causes an accordion-like deformation of the hypodermal cells and cell shape(rectangular) is restored upon relaxation. The emb-9 null embryos (lack BM collagen IV) arrest at 3-fold. They initially have a normal organization of hypodermal cells, but by the 2-2.5-fold stage the shape is rounded and JAM-1::GFP is unevenly distributed in the plasma membrane. The null herniated phenotype and the loss of restoration of hypodermal cell shape suggest that the myofilament lattice contracts and relaxes abnormally due to loss of BM elasticity provided by collagen IV. We have made an emb-9 null unc-54 double mutant strain to examine the effect of reduced muscle contraction on the myofilament organization. The unc-54(s95) mutation affects the contraction/relaxation cycle of muscle cells with near normal myofilament lattice ultrastructure. The contraction is reduced putting less strain on the dense body line, M-line and the cell boundaries of muscle cells. We can assess organization of the myofilament lattice under these conditions, and observe if reduced contractions allow the null animals to advance beyond the 3x arrest. We are using an emb-9 promoter::gfp fusion containing a stop transfer sequence to highlight the membrane of muscle cells to analyze cell shape and loss of cell positions in the arrested emb-9 null embryos. The null mutants initially have the normal position of the 81 muscle cells present in the embryo. C. elegans muscle cells do not fuse and maintenance of the cell-cell boundaries by attachment to the BM is required. The collagen IV network elasticity maintains muscle and sarcomere organization/positions which are required to distribute the force of contractions laterally for proper nematode movement. a

24 May 1999 15:50 964 964 Thermal Avoidance: a novel approach to study nociception in C. elegans

1999 International Worm Meeting abstract 914 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Thermal Avoidance: a novel approach to study nociception in C. elegans N Wittenburg, R Baumeister Genzentrum/LMU, Feodor-Lynen-Str. 25, 81377 München, Germany We have developed a laser based assay to study the thermal avoidance behavior in C. elegans. wild type behavior: Upon stimulation with a micro-focused laser 98% of wild type animals show a reflexive withdrawal response consisting of three characteristical phases: foraging animals first stop, then reverse for 1-2.5 body lengths, and finally turn away from the heat source to resume forward movement in a new direction. We are using this robust assay to analyze what parameters, mutations or pharmacological substances modulate Tav response. thermal nociceptors are responsive to capsaicin: As in other animal models, the thermal avoidance response is significantly increased after exposure to capsaicin, the pungent ingredient in hot chili peppers. This hyperalgesia was completely blocked by preincubation with the competitive inhibitor capsazepine. VR1 is a capsaicin- and heat-sensitive Ca2+-channel in rats. We have analyzed the thermal avoidance response in a C. elegans homologue of VR1. receptive fields: Animals are sensitive to heat in the head and the tail, with the tip of the nose being the most sensitive region. Sensitivity in the tail indicates that there must be at least one class of, yet unidentified, thermosensory cells in addition to AFDL/R neurons which are involved in thermotaxis. Tav differs significantly from thermotaxis: Thermotaxis mutants all displayed a wt Tav behavior, even mutants that prevent the generation of neurons in the neuronal circuit for thermotaxis. Vice versa, most of the Tav mutants we identified are wt for thermotaxis, suggesting that Tav and Ttx are genetically and neuronaly distinct. Tav mutants: From our mutant studies we developed a model suggesting (a) the types of sensory neurons involved in the Tav behavior, and (b) neurotransmitters and neuropeptides that modulate the response. These studies also allow us to differentiate thermal from mechanical and chemical nociception.

24 May 1999 15:50 965 965 Biochemical approaches to the identification of endoderm regulatory factors in the early embryo

1999 International Worm Meeting abstract 915 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Biochemical approaches to the identification of endoderm regulatory factors in the early embryo ES Witze, JJ Kasmir, JH Rothman Department of MCD Biology, University of California, Santa Barbara, CA end-1 and end-3 are major zygotic regulators of endoderm development that are specifically expressed in the endoderm lineage from the E to the E8 stage. These genes are among the best known candidates in C. elegans for zygotic genes directly regulated by a number of maternal cell fate-specifying factors including POP-1, the target of the maternal Wnt pathway, and the maternal SKN-1 transcription factor. Evidence from a number of studies suggests that regulation of end-1 and endoderm formation involves additional regulatory factors that have not been identified by genetic approaches. For example, there appear to be as yet unidentified factors that repress end-1 expression in non-endodermal lineages (see abstract by Kasmir et al.). To complement our genetic approaches, we are using biochemical methods to identify trans-acting factors that restrict end-1 expression to the E lineage. We have succeeded in isolating nuclear extracts from developing early embryos that reveal protein-DNA interactions with regions of the end-1 promoter. Using regulatory regions shown to be required for E-lineage specific regulation of end-1 (see abstract by Kasmir et al.), we have detected multiple protein-DNA interactions within the first 310 base pairs of the end-1 promoter. These include factors from early embryonic extracts that bind to a region containing a putative Lef-1 consensus binding sequence; this binding activity appears to be dependent upon the presence of the Lef-1 sequence. Although POP-1 is an apparent C. elegans homolog of Lef-1, we have not determined whether this factor is indeed POP-1. To characterize the binding activities we have identified, we have used avidin-conjugated magnetic beads to isolate factors from extracts that bind to biotin-linked DNA probes. We are using MALDI-TOF mass spectrometry, which allows us to obtain precise molecular weights of tryptic peptides obtained from the affinity-purified factors, in an effort to determine the identity of these factors. We hope that these methods will not only reveal the protein complexes involved in Wnt- and SKN-1-dependent regulation of endoderm, but might also identify regulatory proteins that genetic approaches inevitably miss.

24 May 1999 15:50 966 966 Genes acting with vab-8 for the posterior-directed migration of the can cell.

1999 International Worm Meeting abstract 916 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Genes acting with vab-8 for the posterior-directed migration of the can cell. FW Wolf, P Yen, G Garriga University of California, Berkeley vab-8 functions cell autonomously to promote the posterior directed migrations of cells and axon growth cones. How this novel intracellular protein acts to guide migrations posteriorly remains to be determined. Analysis of gene interactions with misexpressed vab-8 and loss-of-function vab-8 mutants indicates a role for a limited set of genes in vab-8 dependent migrations. Misexpression of vab-8 in the ALM cell results in reorientation of ALM axon outgrowth from anterior directed to posterior directed. Loss-of-function unc-73/TRIO and gain-of-function mig-2/Rac mutations suppressed ALM axon reorientation without grossly altering vab-8 expression, suggesting that signaling through GTPases is essential for vab-8 function. Additionally, unc-73 but not mig-2 loss-of-function mutations enhanced hypomorphic vab-8 mutant CAN migration defects. mig-2 may normally act redundantly with other GTPases for CAN migration. egl-15 and enu-1 mutations enhanced the penetrance of ALM axon reorientation. Although egl-15 mutations alone did not disrupt CAN cell migration, they enhanced CAN migration defects of vab-8. Mutations in egl-17, which encodes an FGF ligand that may bind the EGL-15 FGFR, also enhanced vab-8 CAN migration defects. CLR-1 is a receptor tyrosine phosphatase that antagonizes EGL-15. clr-1(e1745ts) suppressed the CAN cell migration defects of a vab-8 hypomorph. Thus, egl-17/FGF, egl-15/FGFR, and clr-1/RTP are required for proper CAN cell migration. Perhaps FGF signaling positions the CAN cells and the sex myoblasts act through similar mechanisms (Chen and Stern, TIG 14:322). enu-1 mutants have defects in axon pathfinding (Wightman et. al., Development 124:2571). enu-1(ev419) strongly enhanced uncoordination in both hypomorphic and null vab-8 mutants. Surprisingly, enu-1(ev419) suppressed the CAN cell migration defect of hypomorphic but not null vab-8 mutants, indicating that enu-1 is a negative regulator of vab-8. ENU-1 may either reduce VAB-8 levels or VAB-8 activity. We are attempting to clone enu-1.

24 May 1999 15:50 967 967 Insights into the function of an insulin-like pathway in C. elegans: Regulation and outputs of age-1

1999 International Worm Meeting abstract 917 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Insights into the function of an insulin-like pathway in C. elegans: Regulation and outputs of age-1 CA Wolkow 1 , JZ Morris 2 , G Ruvkun 1

1 Dept. of Molecular Biology, Mass. General Hospital and Dept. of Genetics, Harvard Medical School, Boston, MA 02114. 2 Skirball Institute, NYUMC, New York, NY 10016.

An insulin-like signaling pathway regulates diapause and life span in C. elegans. One component of this pathway, age-1, encodes a homolog of the p110 catalytic subunit of mammalian phosphatidylinositol 3-kinase, PI(3)K (Morris, et al., 1996 Nature 382(536-539)). In vertebrates, a regulatory subunit known as p85 or p55 is required to activate p110’s catalytic activity in response to signaling from upstream tyrosine kinase receptors. p85 and p55 each contain two SH2 domains flanking an inter-SH2 domain which binds to and activates p110 (Holt, et al., 1994 Mol Cell Biol 14(42-49)). Using the C. elegans genome sequence, we identified Cep55, a homolog of vertebrate p55. Genetic tests show that Cep55 acts in the same dauer-formation pathway as age-1 and daf-2, an insulin receptor-like gene (Kimura, et al., 1997 Science 277(942-946)). Furthermore, recombinant CEP55 binds to a fragment of AGE-1 containing the predicted p85/p55-binding domain. These results show that Cep55 encodes an age-1 regulatory subunit. To further understand the cellular basis of insulin-like signaling in worms, the subcellular localizations of a full-length CEP55::GFP fusion and a truncated AGE-1(1-596)::GFP fusion were examined. Cep55::gfp and age-1(1-596)::gfp are each expressed in many different, and sometimes overlapping, cells. Interestingly, in both cases, fluorescence can be found on intracellular vesicles in the soma of some neurons as well as diffusely in the cytoplasm. Mammalian PI(3)K has been suggested to play a role in regulating endosomal dynamics (Li, et al., 1995 PNAS 92(10207-10211)). We therefore propose that the CEP55- and AGE-1(1-596)-binding vesicles are endosomes. We have begun to examine whether daf-2/age-1 insulin-like signaling in C. elegans shares conserved outputs with vertebrate insulin pathways. In particular, vertebrate insulin can stimulate cellular glucose uptake via PI(3)K by triggering plasma membrane targeting of vesicles containing the GLUT4 glucose transporter (Tanti, et al., 1996 JBC 271(25227-25232)). We are investigating whether age-1 activity may similarly control glucose uptake in C. elegans.

24 May 1999 15:50 968 968 Functions of C. elegans cell fate determining genes in C. briggsae

1999 International Worm Meeting abstract 918 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Functions of C. elegans cell fate determining genes in C. briggsae AJ Wright, CP Hunter Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138 We are investigating the conservation of function of SKN-1 and PAL-1 between C. elegans and C. briggsae. In C. elegans, skn-1 is required to specify the EMS fate and pal-1 is required to specify the C and D fates. We have identified C. briggsae skn-1 and pal-1 homologues and are using RNA mediated interference (RNAi) to inhibit their function in C. briggsae. To assist in our phenotypic characterization we are identifying tissue specific antibodies that cross react with C. briggsae and ablating early blastomeres to ensure that C. briggsae early development does not differ substantially from that of C. elegans. Our preliminary results are encouraging. We have identified several monoclonal antibodies that cross react with C. briggsae antigens. These include 3NB12 which recognizes pharynx muscle, 5-6 which recognizes body wall muscle, J126 which recognizes intestinal cells and intestinal valve cells, and MH-27 which recognizes adherence junctions. Ablation experiments on early C. briggsae embryos have not yet indicated any obvious differences in cell fate between C. briggsae and C. elegans. Likewise blastomere interactions important in the early C. elegans embryo, like that of MS signaling to ABa at the 12 cell stage to promote pharynx formation in ABa, also appear to be required. C. briggsae pal-1(RNAi) produces dead embryos with an overall gross morphology different from that of C. elegans pal-1(RNAi) embryos. Preliminary characterization indicates that while C. elegans pal-1 function is restricted to specifying C and D blastomere fates, C. briggsae pal-1 function may be more broadly required. Future work will focus on investigating the C. briggsae pal-1(RNAi) phenotype more thoroughly and cloning and performing similar experiments on the SKN-1 homologue.

24 May 1999 15:50 969 969 egl-44 and egl-46, Two Negative Regulators of Touch Cell Fate

1999 International Worm Meeting abstract 919 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. egl-44 and egl-46, Two Negative Regulators of Touch Cell Fate A Duggan, M Chalfie Department of Biological Sciences, Columbia University, NY, NY10027 In wild-type C. elegans, touch cell fate is restricted to six cells. In egl-44 and egl-46 mutants, two other neurons, the FLP cells, express touch receptor-like features. egl-44 and egl-46 were first identified by egg-laying defective mutants with defects in the development of the HSN neurons (Desai et al., Nature 336: 638-646, 1988; Desai and Horvitz, Genetics 121: 703-721, 1989). Thus, egl-44 and egl-46 mutations affect several different neurons. We cloned egl-44 and egl-46 by transformation rescue, identified the mutations in the existing mutant strains, and recovered full-length cDNAs. egl-44 produces a 2.1 kb mRNA and egl-46 produces a 1.6 kb mRNA. egl-44 encodes a member of the TEF (transcription enhancer factor) family. TEF proteins, found from yeast to human, are involved in a variety of developmental processes. Like other family members, EGL-44 protein contains a specific DNA-binding domain (the TEA/ATTS domain) and a transcriptional regulation domain. egl-44::gfp reporter constructs are expressed in a few neurons in the head and tail and in some intestinal cells. Some staining in the region near FLP suggests that they may express egl-44. Although all existing mutations are missense changes, RNAi suggested that their phenotype is the null phenotype. egl-46 encodes a protein containing some Zn-finger motifs similar to those in the IA-1 protein, a protein expressed in many human neuroendocrine tumor and small cell lung cancer cell. egl-46::gfp fusions are expressed in the Q lineages, which undergo extra division in egl-46 mutant. Both the EGL-46 protein and the 3’ end of its mRNA contain sequences that may target the gene products to rapid degradation. Consistent with these sequence elements, expression of egl-46::gfp fusions is transient in HSN, FLP and ventral nerve cord neurons. Since both egl-44 and egl-46 are expressed in the FLP cells, yet mutation in either one changes FLP cell fate, both genes are needed for repression of touch cell characteristic. Indeed, an egl-46 promoter fusion with gfp is expressed in the ALM, AVM and PVM cells. Moreover, egl-46 expressed from mec-7 promoter has no detectable effect on touch cell function. We are currently examining the effects of coexpression of these two genes in the touch cells.

24 May 1999 15:50 970 970 A screen for loci that facilitate ionotropic glutamate receptor activity

1999 International Worm Meeting abstract 920 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A screen for loci that facilitate ionotropic glutamate receptor activity D Nguyen, R Francis, J Kopczynski, A Ebens Exelixis Pharmaceuticals, 260 Littlefield Ave., South San Francisco, CA 94080 Glutamate is the principal excitatory neurotransmitter in humans; excessive glutamate receptor signaling is responsible for much of the cell death that occurs during stroke and is also thought to play a significant role in several chronic neurodegenerative diseases. The C. elegans glutamate receptor glr-1 has previously been described to function in the command neurons that are essential for normal locomotion1, 2 . We have introduced an activating mutation discovered in the mouse Lurcher mutant into glr-1 and observed a profound effect on locomotion. Transgenic worms expressing the putatively activated glr-1 receptor have a compromised ability to translocate across a culture plate due to a five-fold increase in reversal frequency. Co-expression of a GFP reporter indicates that viability of the command neurons is not affected in this glr-1(lurcher) strain. Since an analogous amino acid substitution in the mouse Lurcher mutant produces a constitutively activated glutamate receptor, we believe that the worm "lurching" phenotype results from constitutive depolarization of the command neurons. Villu Maricq’s group at Utah has independently obtained similar results3 . To identify prospective drug targets that would suppress glutamate receptor activity when antagonized, we have screened for recessive mutations that can suppress the lurching phenotype of activated glr-1. From 300,000 genomes we recovered 61 suppressed individuals in the F2 generation. Of the 42 fertile animals, 24 appeared to have defects in the integrated array as judged by loss of a co-integrated GFP marker and failure to resume lurching when outcrossed. Of the remaining isolates, 15 were independent alleles and were placed into two complementation groups. We propose the name suppressor of lurcher, or sol-1 and sol-2, for these loci.

1. Maricq AV, et al., Nature. 1995 Nov 2;378(6552):78-81 2. Hart AC, et al., Nature. 1995 Nov 2;378(6552):82-5. 3. 1998 West Coast Worm Meeting abstract 79. Turning on neurons: A new genetic tool for studying neuronal circuit function in C. elegans. Yi Zheng, Penelope J. Brockie, Andres V. Maricq

24 May 1999 15:50 971 971 Developmental Regulation of C. elegans HCF Phosphorylation.

1999 International Worm Meeting abstract 921 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Developmental Regulation of C. elegans HCF Phosphorylation. J Wysocka 1 , Y Liu 2 , S Lee 1 , R Kobayashi 1 , W Herr 1

1 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724. 2 Department of Medicine, University of Southern California, Los Angeles, CA 90033.

HCF is a mammalian protein which is required for cell proliferation. During herpes simplex virus (HSV) infection the viral transactivator VP16 associates with HCF to initiate HSV-gene transcription. The same N-terminal domain in HCF that is required and sufficient for interaction with VP16 is also responsible for interaction with the cellular transcription factor LZIP. Mammalian HCF possesses a functional homolog in C. elegans that can associate with VP16 and the human LZIP. Although the C. elegans HCF (CeHCF) is much smaller than its human counterpart, the N-terminal and C-terminal regions of CeHCF share high sequence similarity with the corresponding regions of mammalian HCF. In contrast, the central region of CeHCF has no extensive similarity to any known protein. CeHCF is present in oocytes, embryos, and L1 larvae, but is not detected in other larval stages and adult somatic cells. CeHCF is phosphorylated and its phosphorylation status is developmentaly regulated: a hyperphosphorylated form is present in embryos, whereas a hypophosphorylated form is present in L1 larvae. Only the hyperphosphorylated form binds VP16, suggesting that HCF association with VP16 -- and presumably cellular effector molecule -- is developmentally regulated. When expressed in mammalian cells, CeHCF is also phosphorylated and the modification varies through the cell cycle.

In vivo 32 P phosphate labeling and two-dimensional phosphopeptide mapping reveal that the pattern of HCF phosphorylation in both C. elegans and mammalian cells is very similar, if not the same. We have identified phosphorylation sites by phosphopeptide sequence analysis of CeHCF isolated from worms. Consistent with cell-cycle regulation of phosphorylation when expressed in mammalian cells, the CeHCF phosphorylation sites match a consensus sequence for cyclin-dependent kinases. Unexpectedly, the phosphorylation sites map to the nonconserved central region. Together our results suggest (i) that the central region plays a regulatory role for association of N-terminal domain with putative cellular effectors and (ii) that, although the way CeHCF is recognized by kinases has been conserved in animals, the recognition sequences involved in its phosphorylation have not been conserved.

24 May 1999 15:50 972 972 Regulation of protein secretion into the perivitelline fluid of developing nematode ’eggs’

1999 International Worm Meeting abstract 922 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Regulation of protein secretion into the perivitelline fluid of developing nematode ’eggs’ H Xiao, J Plenefisch, R Komuniecki Department of Biology, University of Toledo, Toledo OH 43606 During the early development of the parasitic nematode, Ascaris suum, larva are bathed in perivitelline fluid (PF) within the eggshell. However, little is known about the secretion of the PF. A cDNA coding for one of the major PF proteins (As-p18) was cloned, sequenced and functionally expressed. Analysis of the predicted amino acid sequence of As-p18 indicated that As-p18 exhibited most identity to a family of putative C. elegans fatty acid binding proteins arranged in tandem on X chromosome. A similar gene cluster was also identified in C.briggsae although it also contained two psuedogenes. The potential C. elegans As-p18 homologues (LBP-1, LBP-2 and LBP-3) contained putative secretory peptides and predicted structural characteristics, in common with As-p18 and in contrast to all other LBPs identified. To characterize the synthesis and presumably the secretion of the LBPs, a vector was constructed fusing the individual promoter regions of lbp-1, lbp-2 and lbp-3 to sequence coding for a nuclear localization signal and GFP. These individual GFP fusion constructs and a plasmid marker pRF4(rol-6) were cotransfected into the gonads of hermaphroditic C. elegans. The expression of the lbp-1 fusion construct was localized to nuclei of hyp-7 cells during early embryogenesis, i.e., the comma and three -fold stages of early C. elegans development. This pattern of lbp-1 expression is completely consistent with the results obtained from Northern and immunoblots of As-p18 in A. suum and suggest that As-p18 is secreted by hypodermal cells into the perivitelline fluid prior to the formation of the cuticle. In contrast, lbp-2 and lbp-3 exhibited different expression patterns, i.e., lbp-2 was expressed in muscle, later in development and lbp-3 exhibited a expression pattern which appeared to be a composite of lbp-1 and lbp-2. These results suggested that As-p18 might also be secreted from somatic muscle latter in development and this hypothesis was confirmed by 2D gel analysis of perientiric fluid isolated from adult A. suum. These studies are continuing with the intention of identifying the promoter elements responsible for the stage and tissue-specific expression of the lbps.

24 May 1999 15:50 973 973 Functions of lin-6 in DNA replication and the S-phase checkpoint

1999 International Worm Meeting abstract 923 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Functions of lin-6 in DNA replication and the S-phase checkpoint Huihong Xu 1 , Bob Horvitz* 2 , Sander van den Heuvel 1

1 MGH Cancer Center, Charlestown, MA 02129. 2 HHMI, MIT Dept. Biology, Cambridge, MA 02139.

In homozygous lin-6 mutants, somatic cells fail to undergo DNA synthesis but continue nuclear and cytoplasmic divisions. Treatment with the DNA-replication inhibitor hydroxyurea (HU) delays onset of mitosis in wild-type animals but not in lin-6 mutants. Together, these results indicate that initiation of mitosis is normally dependent on completion of DNA synthesis and that interdependence of the S and M phases requires the function of lin-6. The lin-6 gene encodes a member of the MCM family of DNA-replication factors, sharing 54% amino-acid identity with human MCM4. Studies in yeast and Xenopus have shown that MCM proteins form part of a protein complex that assembles on DNA and is required for initiation of DNA replication. A role for lin-6 in formation of this "pre-replication complex" is consistent with its observed role in DNA synthesis. Based on the Lin-6 phenotype, this complex may also be required for activation of the S-phase checkpoint. We further examined lin-6 functions using LIN-6-specific antibodies and RNAi. LIN-6 was detected in embryonic cells during all stages of the cell cycle and first became apparent in the postembryonic blast cells shortly before DNA synthesis. The subcellular localization of LIN-6 changed from nuclear in interphase to diffusely cytoplasmic in mitosis. In late anaphase, coincident with inactivation of Cdk1/NCC-1, LIN-6 colocalized with chromatin. Thus, assembly of the DNA-replication complex may be initiated in late mitosis and depend on post-transcriptional regulation of LIN-6/MCM4. LIN-6 protein expression was reduced below detectable levels by RNAi. Following RNAi, fertilized oocytes completed meiosis but failed DNA replication. Nevertheless, they continued spindle duplications and cell divisions and fragmented their genome between the daughter cells. Alternative methods to inhibit DNA replication such as HU treatment or ribonucleotide reductase RNAi resulted in a checkpoint arrest without chromosome segregation. We have tested several additional genes that act in DNA replication in other species for a potential role in the S-phase checkpoint. Our results indicate that formation of the DNA replication pre-initiation complex is critical for the coupling of DNA replication and chromosome segregation in metazoans.

24 May 1999 15:50 974 974 More about MES Proteins

1999 International Worm Meeting abstract 924 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. More about MES Proteins S Strome Indiana University, Bloomington, IN 47405 Four maternal-effect sterile (mes) genes function in germline development in C. elegans. Homozygous mes mutants produce sterile but otherwise healthy progeny. Four of the genes, mes-2, mes-3, mes-4 and mes-6, likely function in the same biological pathway and are required for survival and proliferation of the germline (1,2). Sequence analyses showed that MES-2 and MES-6 are homologs of Polycomb Group (PcG) proteins in Drosophila (3,4), MES-3 is a novel protein (2), and MES-4 has motifs characteristic of PcG and Trithorax Group (TrxG) proteins in Drosophila (see abstract by Fang and Strome). PcG proteins in Drosophila localize as multiprotein complexes to specific sites on chromosomes and maintain repression of the expression of homeotic and other genes. Similarly, in C. elegans, MES-2 and MES-6 are localized in the nuclei of germ cells (3,4), and repress germline expression of transgenes from extrachromosomal arrays (5). Immunoprecipitation and sucrose gradient analysis have demonstrated that MES-2 and MES-6 associate with each other in vivo and are present in a complex. To find other components that might function with the MES proteins, we devised a novel test for whether eliminating the activity of candidate genes can enhance the phenotype of mes mutants. By searching the C. elegans genome sequence, we identified one additional PcG homolog and four TrxG homologs to test. Disruption by RNAi of the function of one of them, a TrxG homolog (on cosmid C26E6), enhanced the sterile phenotype of mes-3 or mes-4 (sterility seen in mes/mes progeny from mes/+ mothers, which is a generation earlier than normal), but not of mes-2 or mes-6. The gene on C26E6 encodes two transcripts, both of which are highly enriched in the germline, as are the mes gene mRNAs. The proteins encoded by both C26E6 transcripts are predicted to be localized in nuclei and may be components of MES complexes. We speculate that MES-3 and MES-4 serve critical roles in MES-mediated regulation of germline development, and that the combined loss of one of these MES proteins and C26E6 function results in zygotic-effect sterility.

(1) Capowski et al. (1991) Genetics 129, 1061-1072. (2) Paulsen et al. (1995) Genetics 141 1383-1398. (3) Holdeman et al. (1998) Development 125, 2457-2467. (4) Korf et al. (1998) Development 125, 2469-2478. (5) Kelly and Fire (1998) Development 125, 2451-2456.

24 May 1999 15:50 975 975 Adaptive response in C.elegans : Oxidative stress-responsive genes to aging increase life span

1999 International Worm Meeting abstract 925 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Adaptive response in C.elegans : Oxidative stress-responsive genes to aging increase life span S Yanase 1,2 , N Ishii 1

1 Dept. of Mol. Life Sci., Tokai Univ.Sch Med., Isehara, Kanagawa 259-1193 Japan. 2 Dept. of Med. Tech., Kanagawa Pref. Coll. of Nurs. & Med. Tech., Nakao 1-5-1, Asahi-ku, Yokohama, Kanagawa 241-0815 Japan.

Short-term exposure to high concentrations of oxygen (90%) produces an adaptive response to oxidative stress in C.elegans. Specifically, L1 larvae acquire resistance to subsequent X-irradiation if pre-exposed to oxygen. This suggests that the reactive oxygen species (ROS) present during high oxygen exposure induce genes that confer protection against X-irradiation. If these defensive systems have something in common, the adaptive response is also expected to be effective in slowing aging. We have found that the intensity of adaptive response appears to elongate life span. We identified some of the genes whose expression was induced by high-concentrations oxygen. In particular, the expression of hsp16-1 and hsp16-48 genes, encoding heat shock proteins, were rapidly and firmly elevated with the exposure. Furthermore, we compared the expression of oxidative stress-responsive genes in two mutants, age-1 and mev-1. age-1 is a long-lived strain and mev-1 is a short-lived strain. The difference in intensity of adaptive response to high oxygen concentrations in the two strains reflects their relative degrees of resistance to oxidative stress. Also, the expression of antioxidant genes encoding superoxide dismutase (SOD) and catalase may help determine life span and aging rate. We are measuring the quantity of expression of sod-1, 2, 3 and 4 genes and catalase-1 and 2 genes in age-1 and mev-1 strains.

24 May 1999 15:50 976 976 lep-1, a gene involved in male tail tip morphogenesis

1999 International Worm Meeting abstract 926 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. lep-1, a gene involved in male tail tip morphogenesis Y Yang 1 , C Nguyen 1 , DH Hall 2 , D Fitch 1

1 New York University, New York, NY, USA. 2 Center for C.elegans Anatomy, Albert Einstein College of Medicine, Bronx, NY, USA.

To understand what genes and mechanisms are involved in morphogenesis we are studying the 4-celled tail tip (hyp8-11). The cellular architecture of this structure is identical in both sexes until late L4 when the hyp cells fuse and retract in the male, changing the shape of the tail from pointy to round. In the hermaphrodites, these cells retain the pointy shape of the larval stages. Mutations at the lep-1 locus result in a pointy ("leptoderan", Lep) tail tip in adult males without pleiotropic effects on morphology or behavior in either sex. Our data suggest that lep-1 is required specifically for tail tip morphogenesis, although it might function redundantly elsewhere. Both TEM data and immunofluorescent staining with the MH27 antibody (which recognizes an epitope in belt adherens junctions) show that both fusion and retraction of the tail tip cells in lep-1 mutants are delayed. This delay is especially pronounced for the most posterior cell, hyp10, which sometimes fails to fuse altogether. These data are consistent with a trigger originating anterior to the tail tip and with a posteriad progression of fusions and changes in cell shape. Genetic analysis indicates that the two EMS-induced lep-1 alleles (bx37 and bx42) are semidominant. Penetrance of the Lep phenotype for these alleles is 95%; expressivity is variable. The results of segmental aneuploid analysis suggest that expression of the phenotype is sensitive to dosage of the wild type allele and that our alleles are hypomorphic. Our transformation rescue and mapping experiments place the lep-1 locus near mec-8 on LGI. We are currently isolating the gene to characterize it and its product at the molecular level.

24 May 1999 15:50 977 977 Characterization of the DES-2/DEG-3 receptor, an ancient member of the nAChR family

1999 International Worm Meeting abstract 927 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of the DES-2/DEG-3 receptor, an ancient member of the nAChR family L Yassin, B Gillo, M Eshel, M Treinin Department of Physiology, The Hebrew University - Hadassah Medical School, 91120 Jerusalem, Israel. DEG-3 is a subunit of a nicotinic acetylcholine receptor (nAChR) that can mutate to cause neuronal degeneration. deg-3 is part of an operon containing a second nAChR subunit, des-2. DES-2 is necessary for DEG-3 dependent channel formation in Xenopus oocytes, and for deg-3(u662) induced degenerations. Thus the two genes probably interact in forming a channel in-vivo (Treinin et al, PNAS 95:15492). Sequence analysis of both deg-3 and des-2 show that they are highly divergent members of the nAChR family. Both show similar low (30%) identity to different branches of the nAChR family, suggesting that they branched early from the family tree. Indeed, electrophysiological analysis of the DES-2/DEG-3 receptor expressed in Xenopus oocytes shows that it is a highly divergent nAChR, as application of choline leads to much higher responses than the application of acetylcholine (ACh). Choline was already shown to be an agonist of a-7, also a nAChR, but ours is the first demonstration of a receptor which is more sensitive to choline then to ACh. Choline being a metabolite is unlikely to play a role in synaptic transmission. Accordingly, DEG-3 is distributed all over the cell (as visualized by antibody staining), showing no preference to synaptic regions. As part of the analysis of this divergent nAChR we are also doing structure function analysis, using mutations in deg-3 which where identified in screens for suppressors of deg-3(u662) induced degeneration. Sequences of the mutations demonstrate structural conservation within the nAChR family, as all missense mutations affect conserved residues. Genetic analysis of the mutations has identified a small number of negative dominant suppressors. These mutations are likely to effect functional attributes of the receptors and will be further analyzed using electrophysiology.

24 May 1999 15:50 978 978 Effects of temperature and oxygen on life span in mev-1

1999 International Worm Meeting abstract 928 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Effects of temperature and oxygen on life span in mev-1 K Yasuda 1 , P Hartman 2 , N Ishii 1

1 Life Sciences, Tokai University School of Medicine, Ishehara, Kanagawa 259-1193, Japan. 2 Biol.Dept., Texas Christian University, Fort Worth, TX 76129.

Mutations in the mev-1 gene confer hypersensitivity to oxygen and methyl viologen (Ishii et al., 1990. Mutat. Res. 237, 165). mev-1 encodes a subunit of the mitochondrial enzyme succinate dehydrogenase (Ishii et al., 1998. Nature 394, 694). In order to determine the effects of temperature on aging in mev-1 animals, life spans were measured at 15C and 25C in wild type and the mev-1 mutant under three different oxygen concentrations (1%, 21% and 90%). As noted previously, oxygen modestly reduced the life spans of mev-1 animals at 1% and 21%; specifically, mev-1 lived about 80% as long as wild type at these two concentrations. Conversely, 90% oxygen reduced mev-1 lifespan over 50% relative to wild type. Temperature was found to modify this relationship in a subtle but interesting fashion. In wild type, effects of oxygen of life span were not particularly influenced by temperature. This can best be visualized by comparing the ratios of the life spans at 16C versus 25C: wild type lived 18% longer when reared at 16C versus 25C under 1% oxygen. It lived 29% and 11% longer under oxygen concentrations of 21% and 90%, respectively. Conversely, temperature substantially modulated the ability of oxygen to reduce life span in mev-1. Specifically, mev-1 animals lived 38.2% and 47.4% longer at 16C than at 25C when reared at 1% and 21%, respectively. However, the life span was only 4.6% longer at 16C as opposed to 25C when animals were incubated under 90% oxygen. These data can be explained by supposing that, at lower oxygen concentrations, the metabolic increases imposed by growth at 25C elevated celluar levels of reactive oxygen species (ROS). This effect should be particularly acute in mev-1 animals because most ROS are the consequence of respiration and the mev-1 defect in succinate dehydrogenase likely results in overproduction of ROS. Collectively these data add further substance to the notion that ROS impact aging in both mev-1 and wild-type animals.

24 May 1999 15:50 979 979 Reproductive Isolation in Caenorhabditis

1999 International Worm Meeting abstract 929 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Reproductive Isolation in Caenorhabditis WC Yen, SE Baird Department of Biological Sciences, Wright State University Reproductive isolation refers to all genetic mechanisms that prevent or limit gene flow between species. The combination of C. elegans (males)/CB5161 (females) is cross-fertile but hybrid animals arrest during embryogenesis. Arrested embryos displayed four distinct terminal phenotypes. 70% failed to gastrulate. Of these 42% arrested at the 28-30 cell stage and 28% continued to divide until approximately 100 cells were present. 30% of the hybrid embryos did gastrulate. Of these, 18% arrested at the onset of morphogenesis and 12% arrested at the two-fold stage. Embryonic lineages were consistent with this arrest profile. In eleven hybrid embryos, no lineage defects were observed prior to the division of the E founder cell. In seven hybrids, either E did not divide (1), or E.a and E.p did not divide (3), or E.a and E.p divided aberrantly along an A-P axis. These embyros failed to gastrulate and arrested either at the 28 cell stage or at the 100 cell stage. In four hybrids, E.a and E.p divided normally. These hybrids gastrulated and arrested either at the onset of morphogenesis (3) or at the two-fold stage (1). In C. elegans, gastrulation defects result from inhibition of zygotic transcription, from defects in endoderm specification, and from mutations in genes with direct effects on gastrulation. Two zygotic genes that exhibit gastrulation-defective phenotypes due to defects in endoderm specification are end-1 and rrs-1. We are testing these genes for dysgenic interactions in C. elegans/CB5161 hybrids. We also are scoring hybrid embryos for the presence of rhabditin granules as a marker for gut differentiation. Finally, we are using a pes-10::gfp reporter to assess zygotic transcription in hybrid embryos.

24 May 1999 15:50 980 980 Mutations that confer aspects of the molting defects seen with lrp-1 mutations or sterol starvation

1999 International Worm Meeting abstract 930 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Mutations that confer aspects of the molting defects seen with lrp-1 mutations or sterol starvation JJ Yochem University of Minnesota, St. Paul, MN 55108 USA Mutations that produce larvae that are completely stuck in old cuticle, that have rings of unshed cuticle that constrict regions of the body, or that have attached to the tail a train of old cuticle will be described. Such larvae can be detected with a dissecting microscope, permitting a semi-clonal screen. The search for such mutations was begun because a failure to complete molting is an aspect of the phenotype conferred by loss-of-function mutations in lrp-1, the product of which resembles mammalian gp330/megalin, a large member of the LDL receptor family of proteins [Yochem et al., Development 126, 597 (1999)]. It is hoped that new mutations will reveal genes whose products are required for the same process as LRP-1. The mutations also have the potential for increasing our understanding of molting, a process that has not been much studied genetically in nematodes. To date, mutations have defined eight candidate genes. Of particular note are mlt-3(sv8) and mlt-7(sv16), both of which closely resemble the lrp-1 mutant phenotype. Thanks to Simon Tuck, Min Han, and Bob Herman.

24 May 1999 15:50 981 981 A neural RNA-binding protein MSI-1 is involved in turning behavior during mating

1999 International Worm Meeting abstract 931 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A neural RNA-binding protein MSI-1 is involved in turning behavior during mating A Yoda, H Sawa, H Okano Dept. of Neuroanatomy, Osaka Univ. Medical School. Drosophila Musashi is one of neural RNA-binding proteins essential for neural development and is required for asymmetric cell divisions of sensory organ precursor cells. Mouse Musashi1, which was identified based on the homology to Drosophila Musashi, is highly expressed in CNS stem cells but not in fully differentiated neurons or glial cells. We identified a C. elegans Musashi homologue, MSI-1, which has two RNA-recognition motifs (RRM1 and RRM2). The RRMs of MSI-1 show extensive similarity to the RRMs of the Drosophila and the mouse Musashi proteins. We examined expression of a msi-1::GFP fusion gene which contained 20 kb msi-1 genome DNA fused to GFP DNA. GFP expression was observed in many cells in the nerve ring. At L1 stage, GFP was also expressed in six cells in the ventral nerve cord which was identified as GABAergic motor neurons. A msi-1 mutant was isolated using TMP/UV mutagenesis and a PCR screening. The msi-1 mutant has a deletion which includes RRM1 of MSI-1. The msi-1 mutant is fertile and is normal in appearance and movement, but we observed lower mating efficiency in the msi-1 mutant than in wild type. This phenotype was partially rescued by 20kb msi-1 genomic DNA. (Method: Five males of the appropriate genotype were mated with three Dpy Unc hermaphrodites for 24 hr. Hermaphrodites which produced cross progeny were counted. The efficiencies were 96% in wild type, 37% in msi-1, 88% in rescued msi-1 and 71% in another rescued msi-1 male.) We found that the msi-1 mutant males were defective at least in the turning behavior during mating. It has been reported that the serotonergic CP motoneurons play a role in male turning behavior. But in the msi-1 mutant male, the neurons were stained normally with anti-serotonin antibody. The msi-1 mutant male also displayed serotonin-induced tail curling at similar level to wild type. The defect in the msi-1 mutant could be due to sensory neurons or interneurons involved in the turning behavior. As the mating behavior depend on complex neural activity, the result suggests that msi-1 gene may play important roles for the neural development or neural function.

24 May 1999 15:50 982 982 Isolation and characterization of protruding vulva sterile mutants

1999 International Worm Meeting abstract 932 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Isolation and characterization of protruding vulva sterile mutants JH Yoder, M Han HHMI, Department of MCD Biology, University of Colorado at Boulder An F1 clonal screen of 3600 haploid genomes (EMS mutagenized) generated approximately 30 sterile mutants with a protruding vulva phenotype. These mutants fall into two general classes. The majority of the mutants exhibit gross lineage defects in the vulval precursor cells while a smaller number appear to have proper VPC lineages but morphological defects preventing formation of wildtype vulvae. We have mapped several of these mutants within small genetic intervals and are focusing our immediate attention on three of the most promising mutants, ku266, ku275 and ku267. ku266 and ku275 fail to complement and map to LG IV between unc-31 and unc-30. These alleles define a potential heterochronic gene with phenotypes reminescent of lin-14(gf) and lin-28(gf). For both alleles the length of the VPC cell cycle is extended and subsequent division in some lineages fails to occur generating a non-functional vulva comprising 8-11 cells. Additionally, both ku266 and ku275 males have severe tail defects. Both alleles have adult cuticle defects; alae have gaps and are absent in some animals. This cuticular defect is more severe in ku266 which exhibits a blister phenotype in a large percentage of animals. We are currently constructing double mutants between ku266/ku275 and lin-14(lf) and lin-28(lf), each of which exhibit precocious vulva and cuticle development, to determine epistatic relationships. Additionally, we are taking advantage of GFP reporter constructs expressed in specific vulval cells to aide in lineage analysis and cosimd injection rescue attempts are underway. ku267 is a morphogenesis defect mutant in which the VPC lineages appear wildtype in most worms (an occasional mutant is observed with two ventral protrusions) and the vulval invagination appears wildtype up to the mid-L4 larval stage. The X-mas tree stage of mid- to late-l4, however is never observed and subsequent eversion leads to the protruding phenotype. This allele maps to LG I near the hP6 physical marker. We are currently trying to determine to which side of hP6 ku267 lies and we will soon begin cosmid injection rescue attempts.

24 May 1999 15:50 983 983 Green fluorescent protein as an epitope tag to purify fusion proteins and associating molecules

1999 International Worm Meeting abstract 933 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Green fluorescent protein as an epitope tag to purify fusion proteins and associating molecules Aki Yonezumi 1,2 , Keiko Gengyo-Ando 1 , Terumi Sakamoto 1,3 , Shohei Mitani 1,3

1 Department of Physiology, Tokyo Women’s Medical University School of Medicine, 8-1, Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan. 2 Present address: Department of Internal Medicine, Japanese Red Cross Medical Center. 3 PRESTO, Japan Science and Technology Corporation.

GFP (green fluorescent protein) is a useful molecule which is applied for many biological analyses including observation of protein distribution expressed by transgenes while the host organisms are alive and fusion proteins are functional. We have generated a monoclonal antibody (mAb mFX73, formerly 65B12) which recognizes a higher conformation of the protein GFP. The antibody is specific to GFP and has very high affinity for the molecule. The antibody coupled to beads effectively recovered GFP from crude extracts of transgenic animals. To examine the possibility whether the molecules associated with this functional fusion protein could be purified, we tried to isolate downstream genes of a neuronal transcripiton factor UNC-86. A transgene which encodes an UNC-86/GFP fusion protein and expressed by a native unc-86 cis-element rescued unc-86 mutant phenotypes, suggesting that the fusion protein is functional. We cross-linked protein-DNA complex in vivo, then immunoprecipitated the UNC-86/GFP fusion protein together with its binding DNA fragments. A known target gene of UNC-86, mec-3 was specifically recovered when examined by PCR. We made a library by DNA fragments purified this way. By sequencing each clone and searching C. elegans genome sequence database, we are now examining what kind of DNA sequences are bound by UNC-86/GFP in vivo. Most common sequences found so far are autoregulatory elements of unc-86 gene itself. The experimental system using GFP as an epitope-tag should be useful to purify proteins and their associating molecules from cells and organisms expressing functional GFP fusion proteins.

24 May 1999 15:50 984 984 The C. elegans sex-determining gene tra-1 may be translationally regulated by its 3’UTR

1999 International Worm Meeting abstract 934 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The C. elegans sex-determining gene tra-1 may be translationally regulated by its 3’UTR Y Yoo, E Jan, EB Goodwin Department of Cell and Molecular Biology and the Lurie Cancer Center, Northwestern University Medical School, Chicago, IL 60611 tra-2, which is necessary for determining female cell fate in C. elegans, has been shown to be translationally regulated by two elements in its 3’UTR. These elements are also found in C. briggsae tra-2 and the human oncogene GLI. This suggests that these elements, termed TGEs for tra-2 and GLI elements, are involved in a more broadly conserved mechanism of translational regulation. We are currently investigating whether C. elegans tra-1, which is homologous to the human oncogene GLI and is the terminal regulator in the sex determination pathway, may also be regulated by TGEs. Translational regulation of tra-1 would suggest an additional form of regulation for determining sexual cell fate. Moreover, regulation of tra-1 at the translational level may also provide insight into the control of the human oncogene GLI in tumor growth. To determine whether tra-1 expression is controlled on a translational level through TGEs, we initially identified potential TGEs through sequence analysis. This search revealed one potential TGE in the 3’UTR of tra-1which later was shown to bind GLD-1 in RNA gel shift analyses. It has been previously established that GLD-1 recognizes and binds tra-2 TGEs in a specific manner. Presently, we are using reporter transgenes to functionally test this putative TGE and examine whether there are other TGEs in the 3’UTR. We are planning to use immunofluorescence and RNA in situ experiments to reveal whether indeed tra-1 is regulated in this manner.

24 May 1999 15:50 985 985 Components of the SNARE complex exhibit nonallelic noncomplementation

1999 International Worm Meeting abstract 935 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Components of the SNARE complex exhibit nonallelic noncomplementation K Yook, SR Proulx, E Jorgensen Dept.of Biology, Univ.of Utah, Salt Lake City, UT 84112 USA The failure of two recessive alleles to complement has been the established method of determining genetic identity. However, there are instances in which alleles of two separate genes exhibit noncomplementation. One interpretation of this genetic interaction is that the gene products of these noncomplementing loci are involved in the same process. Although nonallelic noncomplementation has potential use as a means to genetically dissect biological processes, attempts to use it as a strategy to identify interacting genes have had limited success. Defining the genetic principles behind nonallelic interactions may increase the success rate of this strategy. We have explored the rules of nonallelic noncomplementation by characterizing interactions among five synaptic function genes. Our results demonstrate that nonallelic noncomplementation requires the presence of an altered gene product. We tested the interactions among three synaptic function genes known to physically interact, unc-13, unc-64 and snb-1 (kindly provided by Mike Nonet). These three genes encode the C.elegans homologues of the exocytotic SNARE complex proteins Munc-13, syntaxin and synaptobrevin, respectively. We used resistance to an inhibitor of cholinesterase as an assay of synaptic efficacy. Null alleles of unc-13 and unc-64 fully complement one another implying that synaptic efficacy in C.elegans is not based on gene dosage. Hypomorphic alleles of unc-13, unc-64 and snb-1 exhibit robust nonallelic noncomplementation with one another. Surprisingly, in the presence of a hypomorphic synaptic function allele, the dosage of the other synaptic function gene becomes limiting. Moreover nonallelic noncomplementation may be specific to physically interacting gene products. Noncomplementation of unc-13(n2813) is not seen with alleles of unc-41, a protein likely to be involved in endocytosis (K.Grundahl and J.Rand per.com.) nor unc-17, the vesicular acetylcholine transporter. Both unc-41 and unc-17 are required in the synaptic vesicle cycle pathway during neurotransmission; however, they are not known to physically interact with unc-13. Therefore it is likely that nonallelic noncomplementation may be a phenomenon limited to interacting gene products. Results from our screen using this strategy support this hypothesis.

24 May 1999 15:50 986 986 Isolation and Characterization of a gene acting downstream of sma-2 signaling

1999 International Worm Meeting abstract 936 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Isolation and Characterization of a gene acting downstream of sma-2 signaling S Yoshida 1 , M Mochii 1 , K Morita 1 , M Shimizu 1 , Y Kohara 2 , N Ueno 1

1 Division of Morphogenesis, Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan. 2 National Institute of Genetics, Mishima 411-8540, Japan.

TGF-b signaling pathway is conserved in diverse animal species from vertebrate to nematode. The signaling is known to regulate many aspects of cellular function. Sma pathway, one of TGF-b signaling pathways in C. elegans, regulates the body length and the male tail patterning of the worm. We are interested in the genes acting downstream of TGF-b signaling pathway. To identify such genes, we performed differential hybridization between sma-2 mutant and wild type worms using high-density filter on which over 7000 cDNAs were arrayed. Through the comparison of expression profiles of the cDNAs in sma-2 and wild type, we identified several genes, whose expression level is different between the two, as the candidate targets of the TGF-b signaling pathway. We focus on a cDNA clone yk307b1, one of the genes whose expression level is high in sma-2. The predicted gene product is novel and has the putative N-terminal signal sequence. Northern blot analysis also confirmed increased level of expression of this gene in sma-2 than in wild type. In situ hybridization suggested the expression in two neural cells in the head and a cell in the tail through the late embryo to the adult. Additionally, from the data base search, we found three other related genes in C. elegans, which share the amino acid identity from 64 to 81 %. All four genes are on chromosome V and two of them are tandem. Further analysis with overexpression and GFP fusion construct is in progress. The screen for null mutant with TMP/UV method is also ongoing.

24 May 1999 15:50 987 987 Isolation of target genes in daf-2 pathway

1999 International Worm Meeting abstract 937 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Isolation of target genes in daf-2 pathway PL Larsen Molecular Biology Program and Division of Biogerontology, University of Southern California, Los Angeles, CA 90089. An insulin-like receptor daf-2 signal pathway regulates both dauer formation and adult life span in C. elegans. A search for genes downstream of daf-2 was performed by employing the differential display PCR method to compare the expression pattern of daf-2 mutant and N2 wild-type animals. With the primer sets tested so far, a total of thirty-four unique clones have been isolated from differentially displayed bands and sequenced. Database search for each clone gave the physical map location, the predicted open reading frame and the functional homologies. Eight of the clones are novel proteins. Northern analysis was used to confirm the differential expression pattern shown by DD-PCR. We named these verified genes as dao genes for daf-2 adjusted over- or under-expression. The dao-1 gene showed significantly reduced expression in daf-2 mutant adults but similarly high levels in N2 adults and daf-16; daf-2 mutant adults. Therefore dao-1 gene is positively regulated by DAF-2 and negatively regulated by downstream signaling component, DAF-16. Northern hybridization of dao-1 in aged samples indicates dao-1 expression correlates with wild-type life span. The dao-2 mRNA displayed a similar pattern as dao-1. The dao-3 gene was over-expressed in N2 young adults but not the daf-16; daf-2 mutant adults, suggesting this gene is regulated by the daf-2 gene but not the daf-16 gene. The patterns of both dao-4 and dao-5 expression were higher in daf-2 young adults and lower in N2 and daf-16; daf-2 double mutant adults. The sixth clone has relatively higher expression level in dauer samples compared to the adult samples and there is no difference in levels among adult samples. This gene is not regulated by daf-2 signaling and it is a false positive in our DD-PCR assay, though it may contribute to dauer formation or maintenance. Except for dao-3, which is either upstream of daf-16 or in a non-daf-16 branch, the dao genes are downstream of daf-16 and these target genes may contribute to the processes of dauer formation and of life span determination.

24 May 1999 15:50 988 988 Cloning and characterization of C. elegans ram-5: strucutral organization and expression pattern of the gene

1999 International Worm Meeting abstract 938 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Cloning and characterization of C. elegans ram-5: strucutral organization and expression pattern of the gene KL Chow Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong In C. elegans, nine pairs of bilateral sensory rays are formed during the late L4 larval stage. This morphogenesis process involves diverse cellular activities such as the formation of junctions with correct neighbors, cell migration, changes in cell-shape and cell-volume. A number of mutations were isolated to affect this morphogenetic process. The major group of these loci, the ram’s, showed a lumpy ray phenotype when they are mutated. In mutant ram animals, tight cylindrical morphology unique to individual ray is not maintained. Instead, rays are swollen with highly variable morphology. We attempted to characterize the morphogenesis process by cloning these ram genes. ram-5 mutation representing one of them was rescued by cosmid transformation. Three overlapping cosmids, H01A2, H02I07 and T24C2 could restore the wild type phenotype in ram-5 mutants. The result suggested that ram-5 gene was located in the overlapping region. Deletion analyses defined a minimal rescuing fragment of 9.5 Kb. More detailed deletion study suggested that the ram-5 gene was encoded in the minus strand, and the promoter was defined in a 2.5 Kb region on this genomic fragment. The identity of this locus will be further confirmed by genomic mapping of mutant alleles and RNA interference. It is interesting to note that a number of these deletion constructs caused a ray loss phenomenon when they carried high copy of the transgene (see the accompanying abstract by Yu and Chow). While no known cDNA was isolated in the library screened and the EST database, we used mutational strategy, inverse PCR, 5’ and 3’ RACE to define the transcript(s). Sequence analysis predicts that the encoded protein is a transmembrane glycoprotein with a short domain sharing similarity with cuticulin, a nematode cuticular protein. Promoter reporter constructs in transgenic animals revealed that ram-5 was expressed in ray structural cells in the male tail and a number of neuronal cells in the amphids, and pharyngeal region. These expression patterns suggest that this gene may have additional neuronal functions in worm.

24 May 1999 15:50 989 989 Dosage of ram-5 gene is critical for sensory ray morphogenesis in C. elegans

1999 International Worm Meeting abstract 939 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Dosage of ram-5 gene is critical for sensory ray morphogenesis in C. elegans KL Chow Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong During normal tissue morphogenesis, cell adhesion and repellent molecules have to be expressed in the right level at the right place and right time. Deviation from the normal expression level may not be tolerated for tissue function nor animal vitality. In our study of ram-5 mutation, it was noticed that ram-5 mutant animals rescued by transformation constantly threw progeny with missing ray phenotype. A strong correlation of missing ray phenotype and the rescue activity was established. We speculate that there was a causative relationship between the ram-5 gene dosage and missing ray phenotype and thus employed both genetic and molecular approaches to test this notion. In the genetic analysis, two stable transgenic lines, KC61 and KC62 were used. These stable lines carried truncated and wild type ram-5 genes in a him-5 background. They showed 18% and 100% ray loss respectively. When heterozygous mutant transgenic worms were generated from them to lower the transgene dosage, the percentage of ray loss dropped to 0% and 53% respectively. A positive correlation between ray loss and transgene copy number was established in these lines by Southern analysis. This result argues that high level of ram-5 product might interfere the regular ray assembly process. When truncated ram-5 gene product was expressed in him-5 worms, both swollen ray tip and lumpy ray phenotype were observed. However, when the WT ram-5 gene was injected into the him-5 worm, it did not give these phenotypes. The results suggest that truncated RAM-5 protein can compete with the WT RAM-5 protein for its interacting components to form inactive complexes. By lowering of functional complex level, swollen ray tip or lumpy ray phenotype was resulted. Further over-expression of the truncated protein would lead to severe ray loss. We propose that ram gene products function as a multi-protein complex in a specific stoichiometry. Alteration of the ratio of these proteins will adversely affect the formation of functional complexes. As a result, ray cell assembly cannot take place and rays fail to attach to the cuticle to establish proper morphology.

24 May 1999 15:50 990 990 Imaging of neuronal activity in olfactory circuits in C. elegans

1999 International Worm Meeting abstract 940 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Imaging of neuronal activity in olfactory circuits in C. elegans CI Bargmann HHMI and UCSF, San Francisco, CA 94143 [email protected] We are interested in studying the neural circuits underlying chemotaxis behavior in C. elegans. To this end, we are attempting to image neuronal activity in intact worms using cameleons -- fluorescent indicators, based on spectral variants of GFP and calmodulin, that allow ratiometric imaging of calcium concentrations (Miyawaki et al, Nature 388 882). To work out the best conditions for this experiment, we have attempted to record calcium changes in body wall muscle during locomotion. Under control of the myo-3 promoter, yellow cameleon-2 is abundantly expressed in body wall muscle. Dual-emission wavelength imaging of myo-3::ycam2 worms during sinusoidal locomotion reveals ratio (F535/F480) increases in body wall muscle preceding muscle contraction, consistent with calcium increases during excitation-contraction coupling. Initial attempts to express yellow cameleon-2 in chemosensory neurons resulted in low levels of expression, even when injected at high concentrations. We therefore engineered worm-optimized yellow cameleons by interrupting cameleon coding sequences with artificial C. elegans introns. Addition of five introns to yellow cameleon resulted in a robust increase in expression as assayed under the mec-7 promoter. Currently, we are expressing our modified yellow cameleon-2 in AWA, AWB and AWC, chemosensory neurons that have been shown to mediate attraction or repulsion from a variety of volatile odorants. The sensory transduction channels in these neurons are predicted to allow calcium entry after odorant stimulation. Progress on recording calcium changes in response to odorant application will be described.

24 May 1999 15:50 991 991 nSLO2, A Novel Cl- Activated K+ Channel

1999 International Worm Meeting abstract 941 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. nSLO2, A Novel Cl - Activated K + Channel A. Yuan 1 , M. Dourado 3 , Z.W. Wang 1 , A. Butler 1 , and L. Salkoff 1,2 1 , A Yuan 2 , M Dourado 3 , ZW Wang 2 , A Butler 2 , L Salkoff 2,4

1 1 Department of Anat. and Neuro. 2 Department of Genetics, Washington University, Saint Louis MO. 3 University of California, San Francisco 2 Department of Anat. and Neuro, Washington University, Saint Louis MO. 3 University of California, San Francisco. 4 Department of Genetics, Washington University, Saint Louis MO.

SLO1 channels are high conductance K+ channels activated by voltage and Ca2+ . These channels serve as a link between membrane excitability and changes in intracellular Ca2+ . We unexpectedly found a new SLO-like channel in C. elegans called nSLO2. Nslo2 (nematode slo-2) was subsequently cloned from a C. elegans embryonic cDNA library, and was expressed in Xenopus oocytes. Surprisingly, nSLO2 requires intracellular Cl- as well as Ca2+ for activation. This unique property has never been described for any - - other ion channel. Perhaps nSLO2 is an intracellular Cl sensor; changes in [Cl ]i may directly influence - membrane electrical behavior by modulating the activity of nSLO2. Changes in [Cl ]i have been observed - in different cells, for example the [Cl ]i decreases as mammalian neurons mature.

As the first step in elucidating the physiological role of nSLO2, we determined its tissue specific expression pattern in C. elegans. The slo-2 genomic sequence and its native promotor were fused to GFP. Nematodes transformed with this promotor:GFP fusion revealed an expression pattern in muscles and in some neurons. Interestingly, this expression pattern overlaps with that of nSLO1 (see related poster by Wang et al.), suggesting that the two subunits may form functional heteromeric channels in vivo. Preliminary results show that nSLO1 and nSLO2 are capable of forming functional heteromers when co-expressed in Xenopus oocytes.

24 May 1999 15:50 992 992 sup-39 mutations alter the use of cryptic splice sites in unc-73(e936)

1999 International Worm Meeting abstract 942 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. sup-39 mutations alter the use of cryptic splice sites in unc-73(e936) AM Zahler University of California, Santa Cruz sup-39 mutations have been identified as allele-specific dominant suppressors of the unc-73(e936) coordination defect (1). The e936 allele contains a mutation in intron 16 that changes the canonical G found at the first base of the intron to a U (2). Since sup-39 mutations are involved in the allele-specific suppression of a splice site defect, I tested whether mutations in sup-39 lead to changes in e936 splicing. In e936 animals, the splice donor mutation in intron 16 leads to activation of three cryptic 5’ splice sites. Two of these sites leads to splicing into a nonsense reading frame. The third splice site is unusual; the wild type splice donor is used even though the intron begins with U instead of the canonical G. In e936 worms, this splice site is used in 10% of the stable unc-73 mRNAs. This is consistent with the finding that e936 worms make a very low level of full length UNC-73 protein (2). Mutations in smg-3 do not affect the ratio of e936 cryptic splice sites. In e936;sup-39(je5) animals, the same three cryptic splice sites are used however their relative levels change; the in-frame splice site represents 35% of the stable unc-73 messages. The levels of unc-73 message in wild type, e936 and e936;je5 animals are equivalent. Thus sup-39 mutations may be functioning at the level of splicing to change the relative usage of the cryptic splice sites. Consistent with this, the amount of full-length UNC-73 protein in e936;je5 animals is higher than in e936 animals, but lower than in N2. A model for the mechanism of suppression of e936 by sup-39 mutations is that sup-39 mutations may lead to a change in the preference of the splicing machinery for e936 intron 16 cryptic splice sites. This leads to an increase in the amount of in-frame unc-73 message and an increase in UNC-73 protein leading to the development of a coordinated animal.

1. Run, J. Q. et al. (1996). Genetics 143: 225-36. 2. Steven, R. et al. (1998). Cell 92: 785-95.

24 May 1999 15:50 993 993 Expression pattern of a worm homologue of phogrin, a mammalian dense cored granule protein of neuroendocrine cells

1999 International Worm Meeting abstract 943 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Expression pattern of a worm homologue of phogrin, a mammalian dense cored granule protein of neuroendocrine cells T Zahn 1 , P MacMorris 2 , C Wasmeier 1 , JC Hutton 1

1 Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Denver, CO. 2 Department of Biochemistry & Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO.

In mammalian cells polypeptide hormones and neurotransmitters are stored in dense cored granules formed directly from the trans Golgi network (TGN) and transported to their site of exocytosis along microtubular pathways. Unlike synaptic vesicles, they cannot be refilled after exocytosis and the granule membrane proteins follow a distinct trafficking route in the cell. The dense cored granule membrane protein phogrin of the insulin-secreting pancreatic b-cell provides a convenient marker of insulin granule movement and biogenesis. It undergoes phosphorylation in response to secretagogues indicating that it may be involved in regulation of granule locomotion or exocytosis. Phogrin is also a major autoantigen in Type I Diabetes patients. The presence of a closely related C. elegans homologue B0244.2 provides an opportunity to study the function of the protein in a species that is tractable both in terms of genetic manipulation and visualization of cell trafficking. The full length cDNA of B0244.2 was sequenced and encodes a putative 86.7 kD transmembrane protein that is 55% identical in its C-terminal domain to rat pancreatic islet phogrin. This region is homologous (25-40% identity) to the larger family of protein tyrosine phosphatases (PTP) though neither B0244.2 nor phogrin appear enzymatically active. The tissue distribution of B0244.2 in C. elegans was examined by microinjection of a GFP construct driven by either a 2.4 or 7.6 kb upstream promoter of the C. elegans gene. The fluorescence tag decorated a series of 20-30 neuronal cell bodies in the head, tail and vulva of the worms with a lesser signal extending into axonal structures. The neuronal localization of B0244.2 in C. elegans parallels the neuroendocrine distribution of phogrin in mammals and indicates that the molecule has great potential for future cell biological studies of the function of these molecules in neurotransmission and disease processes.

24 May 1999 15:50 994 994 Analysis of several mutations that disrupt vulval development

1999 International Worm Meeting abstract 944 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Analysis of several mutations that disrupt vulval development W Zhang, A Mapes, M Han HHMI, Dept. of MCDB, University of Colorado at Boulder, CO80309 We have conducted ts and sterile mutant screens for mutations that disrupt various steps of vulval development. Analyses of several mutations will be reported. ku260 and ku272. Mutations have similar vulval phenotype likely due to defects in VPC generation. Mutants display either an everted vulva (Evl or Pvl) or fail entirely to differentiate vulval tissue. Nomarski analysis indicates that such mutants are often missing more than one Pn.p cell, resulting in either abnormal or no vulval invaginations. Using an unc-47::GFP as a marker, we observed that many GABAergic neurons in the ventral cord are absent (approximately 50% missing). ku260homozygotes are Unc, likely due to the missing VD neurons. Both ku260 and ku272 mutants have an abnormal somatic gonad and contain no oocytes, leading to a sterile phenotype. ku260 was mapped to LGIV between unc-24 and unc-129 and is uncovered by eDf19 . Pools of cosmids are being injected in order to rescue the mutant phenotype. ku260 fails to complement evl-24(ar124) which was previously isolated by Seydoux and Greenwald as a class III Evl mutation(1). ku272 has been mapped to right arm of LGI. ku259, ku255 and ku270. These mutations appear to cause defects in both vulval lineage and morphogenesis and display an Evl phenotype (subtle for ku270). We also detect a low to moderate percentage of double invaginations and protrusions in ku259 and ku255 mutant worms. Nomarski analysis indicates that the abnormal or double invaginations observed are at least partly due to lineage defects. All three mutations also cause a sterile phenotype (incomplete for ku270). ku259 was mapped to LGIII between dpy-1 and daf-2. DNA-mediated transformation is currently being performed to identify the affected gene. ku255 was mapped between unc-13 and nob-3 on LGI and failed to complement evl-17(ar94) (1). ku270 has been mapped to the right of lin-25 on LGV. The cloning of both genes is in progress. 20-30% of ku273 homozygous mutants are Egl and some contain two or three vulval protrusions. Nomarski analysis reveals that the Egl and Muv phenotype are caused by defects in vulval lineage and morphogenesis. We are currently mapping this gene to a chromosomal location.

(1) Seydoux, G., C. Savage, and Greenwald. I. (1993). Dev. Biol. 157,423-436.

24 May 1999 15:50 995 995 Phenotypic analysis of two C. elegans endocytosis mutants rme-1 and rme-8

1999 International Worm Meeting abstract 945 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Phenotypic analysis of two C. elegans endocytosis mutants rme-1 and rme-8 Y Zhang, B Grant, L Pedraza, D Hirsh Dept. of Biochemistry and Molecular Biophysics, Columbia University, New York, NY10032 Yinhua Zhang, Barth Grant, Laura Pedraza and David Hirsh, Dept. of Biochemistry and Molecular Biophysics, Columbia University, New York, NY10032 We wish to complement the classical cell biology studies on receptor-mediated endocytosis in animal cells with a genetic analysis in C. elegans. We isolated mutants defective in yolk-protein uptake using our YP170-GFP assay (see abstract by Grant et al). Here we describe two endocytosis mutants: rme-1 which appears to function in the endosome, and rme-8 which appears to function early in endocytosis. rme-1 mutants have a mild block in YP170-GFP uptake, but have normal brood size and viability. rme-1 worms also form large vacuoles in their gut cells and to a lesser degree in hypodermal cells. In wildtype gut cells, RME-1 is present on small vesicles with a punctate pattern. These vesicles take up a low level of GFP from the body cavity when it is secreted from muscle. In one missense mutant, RME-1 is also present on the surface of large vacuoles. The large vacuoles accumulate GFP to a higher level than do wild type vesicles. One model would be that the large vacuoles are derived from the small vesicles and reflect a defect in endosome sorting which results in a mild reduction of YP170-GFP uptake by rme-1 mutants. Currently we are testing this model. RME-1 contains a conserved region of 555 amino acids which has 75% identity to its mammalian homologs, indicating they function similarly in mammalian cells. rme-8 is a ts lethal with a strong block in YP170-GFP uptake at all temperatures. rme-8 is required at all developmental stages; shifting mutant larvae to 25C causes arrested development. Arrested larvae fail to shed their old cuticles. Shifting adult hermaphrodites to 25C causes endoreplication in oocytes and degeneration of the gonad and intestine. The yolk receptor (RME-2) and alpha-adaptin (a component in clathrin coated pit) co-localize with yolk in discrete dots at 15C and in large patches at 25C. This colocalization suggests that the rme-8 mutation blocks the internalization step of endocytosis at the plasma membrane. rme-8 maps to a 300kb genomic interval between gld-1 and lin-10; we are further mapping it in order to clone it and identify its function in endocytosis.

24 May 1999 15:50 996 996 The mechanisms of synaptic localization of sng-1, the C. elegans homologue of vertebrate synaptogyrin

1999 International Worm Meeting abstract 946 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. The mechanisms of synaptic localization of sng-1, the C. elegans homologue of vertebrate synaptogyrin H Zhao, L Wei, ML Nonet Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110, U.S.A. The mechanisms by which synaptic vesicle (SV) proteins are localized to SVs are poorly understood, with respect to the targeting signals within SV proteins and the molecules involved in their sorting pathways. To address these issues, we chose to study sng-1, the C. elegans homologue of vertebrate synaptogyrin, a four transmembrane domain protein of SVs with both the N- and C-terminus facing the cytoplasm. sng-1 encodes a 248 amino acid protein with 30% identity to rat synaptogyrin. Immunohistochemistry using an antibody raised against the C-terminal domain of SNG-1 was performed on wild type animals and transgenic animals expressing a C-terminal SNG-1-GFP fusion. This revealed a punctate staining pattern along the nerve cords and the SAB motor axons in the head, suggesting that both native SNG-1 and SNG-1::GFP are localized to nerve terminals. In addition, SNG-1::GFP colocalizes with another SV protein, synaptotagmin. Moreover, in unc-104 mutants, which are deficient in axonal transport of SVs, SNG-1::GFP accumulates in neuronal cell bodies. These results strongly suggest that SNG-1::GFP is targeted to SVs in C. elegans. Deletion analysis using SNG-1::GFP shows that the C-terminal, but not the N-terminal domain of SNG-1, is necessary for its synaptic localization. The required region has been mapped within 40 amino acids and we are currently attempting to further refine the localization signal. However, the C-terminal domain of SNG-1 is not sufficient to target other membrane proteins to SVs. To identify proteins that interact with SNG-1 and may mediate its synaptic localization, a yeast two-hybrid screen was performed using the C-terminal domain of SNG-1 as bait. Positive clones were obtained from two different C. elegans cDNA libraries and are currently being analyzed. In parallel work, we are assessing whether the localization signal is conserved between C. elegans and vertebrates by similar deletion analysis. Preliminary results suggest that synaptogyrin-GFP is targeted to SVs of ganglion cells in retina preparations of multiple species.

24 May 1999 15:50 997 997 Identification and Analysis of Three New Mutations Involved in the Control of Cell Polarity During C. elegans Development

1999 International Worm Meeting abstract 947 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Identification and Analysis of Three New Mutations Involved in the Control of Cell Polarity During C. elegans Development X Zhao, TM Ratliff, MA Herman Division of Biology, Kansas State University, Manhattan, KS 66506 We are interested in how cell polarity is controlled during metazoan development. Our approach is to identify and study genes involved in the control of cell polarity by identifying mutations that disrupt the polarities of individual cells. Mutations in three genes: lin-44, lin-17 and egl-27 have previously been found to affect the polarity of certain cells in the tail called TL and TR. lin-44 encodes a WNT signaling protein that is made by the skin cells at the tip of the developing tail and functions to specify the polarity of the more anterior T cells. lin-17 encodes a putative WNT receptor and egl-27 encodes a protein similar to a metastasis-associated factor, MTA1, and has been shown to be required within the T cells for normal polarity. We screened for additional mutants that affect T cell polarity to identify new genes that may interact with the genes known to control T cell polarity. We screened approximately 45,000 haploid genomes and isolated three new mutants: mh15, mh17 and mh21. A fourth mutation, mn593, was isolated in a preliminary screen of this type (see abstract by K. Williams and M. A. Herman). All three mutations are recessive and have been mapped to distinct intervals on LG IV. Phenotypic analyses indicate that mh15 and mh21 mutants display more severe T cell defects than do mh17 mutants. In addition, all three mutations cause males to have abnormal tail morphologies. Some mh15 and mh17 male tails appear to have fused rays. The T cell division pattern is defective in these mutants. Cell lineage analysis of mh15 indicates that several T cell daughters fail to divide and cell fates are not properly specified. These observations are consistent with a defect in T cell polarity. We are further analyzing the phenotypes of all three mutants and attempting to clone these genes by microinjection of relevant cosmid and YAC clones.

24 May 1999 15:50 998 998 glr-3 Encodes a non-NMDA Type Glutamate Receptor that is Widely Expressed in the Nervous System of C. elegans

1999 International Worm Meeting abstract 948 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. glr-3 Encodes a non-NMDA Type Glutamate Receptor that is Widely Expressed in the Nervous System of C. elegans Y Zheng, AV Maricq Department of Biology, University of Utah, Salt Lake City, Utah 84112 USA We have shown that a defect in a glutamate receptor subunit, glr-1, differentially affects the transmission of sensory input from the polymodal sensory neuron ASH (see also Hart et al, 1995). Mutants are defective in the backing response to tactile stimuli, but have normal responses to osmotic stimuli. One possible explanation for this interesting phenotype is that other glutamate receptors are required for the proper transmission of sensory input from ASH. One non-NMDA subtype of glutamate receptor that we have identified is glr-3. We are most interested in glutamate receptors found in the command interneurons. Based on transgenic strains that express fusions of glr-3 with GFP, we have shown that glr-3 is expressed in the command interneurons as well as many other neurons, including neurons in the ventral cord. We have isolated a full-length cDNA for glr-3 that encodes a 932 a.a. receptor with 4 predicted transmembrane (TM) domains. GLR-3 has many of the topological features of glutamate receptors and shows strong conservation in the predicted TM domains. It has some unusual features however, including a KG sequence in the highly conserved residues, (Q/R Q), that are found in TM II of most non-NMDA receptors. The GFP expression data obtained from transgenic strains shows that glr-3 is expressed in some of the same command interneurons that express glr-1, glr-2, glr-4, nmr-1, and nmr-2. To evaluate the contribution of this receptor to behavior we have intiated experiments designed to generate a null mutation. We have identified two strains that contain a Tc1 transposable element in the glr-3 gene: one inserted in an intron, the other in an exon. We are now in the process of generating deletion mutations by imprecise excision of these Tc1 elements. We will examine the behavior of these mutants as well as the behavior of strains with combinations of receptor mutations. How does glr-3 contribute to the function of these neurons? To begin to address this question we will determine the subcellular location of this receptor subunit. In particular, we are interested in determining whether glr-3 co-localizes with any of the other receptor subunits. This localization data will help predict which receptor subunits form functional heteromeric receptors.

24 May 1999 15:50 999 999 Neuronal Timing Behavior in C. elegans can be Modified by a Dominant Mutation in the GLR-1 Ionotropic Glutamate Receptor

1999 International Worm Meeting abstract 949 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Neuronal Timing Behavior in C. elegans can be Modified by a Dominant Mutation in the GLR-1 Ionotropic Glutamate Receptor Yi Zheng, Penelope J. Brockie, Andres V. Maricq Department of Biology, University of Utah, Salt Lake City, Utah 84112 We have developed a novel method to selectively activate neurons in C. elegans by targeted expression of a dominantly active form of the C. elegans ionotropic glutamate receptor, GLR-1. We engineered into glr-1 the A/T mutation first identified in the mouse d2 glutamate receptor (Zuo et al.). This mutation caused the d2 ion channel to be constitutively open or leaky. The mutated C. elegans receptor, GLR-1(A/T), also forms a leaky ion channel when expressed in Xenopus oocytes. By expressing GLR-1(A/T) in transgenic worms, we used the mutated channel to activate specific neurons in C. elegans, including the command interneurons required for coordinated directed movement (Chalfie, et al.). Transgenic worms show a dramatic phenotype -- they rapidly alternate between forward and backward movement. The phenotype could be partially suppressed by introducing a second mutation known to reduce the conductance of the ion channel. These data suggested that the leaky GLR-1(A/T) channel was depolarizing the command interneurons and that these interneurons controlled the frequency of reversals. Support for this was provided by genetic studies that showed that the phenotype was independent of sensory input from the touch cells. To identify neurons central to the control of the hyper-reversal phenotype we used genetic and optical perturbation techniques. Laser ablating the PVC interneurons in GLR-1(A/T) transgenic worms had no effect on the frequency of reversals. Furthermore, limiting GLR-1(A/T) expression to all command interneurons except AVB also resulted in a hyper-reversal phenotype. Collectively, these data indicate that GLR-1(A/T) acts primarily by depolarizing the backward command interneurons, AVA, AVD and AVE. The GLR-1(A/T) transgenic worms provide a sensitized genetic background to identify gene products that function within these command interneurons to control the pattern of locomotion. We mutagenized transgenic worms and screened 9000 haploid genomes for recessive mutations that either suppress (reduce reversals) or enhance (more severe movement defects) the hyper-reversal phenotype. We are in the process of characterizing the mutants obtained in this screen.

24 May 1999 15:50 1000 1000 Characterization of the cul-4 gene

1999 International Worm Meeting abstract 950 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Characterization of the cul-4 gene W Zhong, ET Kipreos Cellular Biology, University of Georgia cul-4 is a member of the cullin family. Cullins are components of ubiquitin-ligase assemblies that promote ubiquitin mediated degradation of specific target proteins. The functions of three cullins, CUL-1, CUL-2 and CDC53, are known and all are cell cycle regulators. Little is known about CUL-4. It has been reported that the human homologue of the cul-4 gene is amplified and overexpressed in primary breast cancers (Chen, L. C. et al. Cancer research, 38: 3677-3683, 1998), suggesting a role of CUL-4 in cancer progression. We used dsRNA microinjection to get information about the mutant phenotypes. With cul-4 dsRNAi, we observed that the majority of larvae arrested in the L2 stage. In these larvae, we saw huge hypodermal nuclei (up to 9 times bigger than normal nuclei) while other cells appeared to have normal sized nuclei. The huge hypodermal nuclei may result either from endoreplication, in which cells undergo extra rounds of S phase while bypassing mitosis, from fusion of synctial nuclei, or from G1 arrest. In certain L2 arrested animals, we observed that some germ cells had differentiated into sperm, suggesting that certain developmental programs, including germ cell differentiation, continued in cul-4 RNAi animals although cell division was arrested. Alternatively, cul-4 may normally function to inhibit sperm differentiation. The cul-4 RNAi results indicate that loss of cul-4 function is lethal. We have found that there are several lethal genes in the vicinity of cul-4. Among them, let-263 and let-236 are unidentified genes. We have started to test whether these lethals are cul-4. One strain, let-263 has a phenotype of L2 arrest and large hypodermal cells, similar to the cul-4 RNAi phenotype. We are currently determining whether let-263 strain has DNA mutations in the cul-4 gene with the chemical-cleavage-mismatch method developed by Cotton et al,1988. We are also in the process of generating deletion mutants by PCR screening animals mutated with EMS. Finally, we are in the process of making a GFP-tagged cul-4 to determine its expression profile.

24 May 1999 15:50 1001 1001 Parameters in transgenic rescue of spe-4, a germline gene involved in spermatogenesis

1999 International Worm Meeting abstract 951 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. Parameters in transgenic rescue of spe-4, a germline gene involved in spermatogenesis G Zhu, SW L’Hernault Graduate Program in Genetics and Molecular Biology, and Department of Biology, Emory University, Atlanta, GA 30322 Transgenes for many genes normally expressed in the C. elegans female germline either do not show or exhibit poor expression. This has been shown by others to be due to transcriptional silencing (Kelly W. G., et al. 1997. Genetics 146: 227-238). Such transcriptional silencing is potentiated by simple repeat transgenic arrays composed of multiple copies of only one or two recombinant plasmids. The situation seems somewhat different in the testes. Expression from simple repeat transgenes occurs at a sufficient level to allow phenotypic rescue of the self-sterile phenotypes associated with any of 10 spe mutants. Rescue of a self-sterile phenotype is both a sensitive and quantitative indicator of transgenic expression level. We have studied the spe genes to define parameters involved in transcriptional silencing of transgenes in the testes. Efficient rescue of spe-4 was achieved by simple transgenic arrays composed of spe-4 genomic sequence (which includes all seven introns). In contrast, simple arrays of intronless spe-4 constructs, driven by either the spe-17 or the spe-4 promoter, did not allow rescue of the self-sterile spe-4 phenotype. The expression level was elevated by putting back the first spe-4 intron or by co-injecting yeast genomic DNA (complex arrays) with the spe-4 constructs. The spe-4 construct with the first intron in a complex transgenic array restored self-fertility to fully penetrant self-sterile spe-4 mutants, and they produced broods as large as 40. However, the rescuing efficiency varied among individual transformants in the same transgenic line, and transcriptional silencing of this array became progressively more pronounced with each new generation. This was an especially serious problem when transgenes were propagated in spe-4/+ hermaphrodites because there was no selection for high-level expression. In a homozygous spe-4 hermaphrodite, the expression level of SPE-4 from the transgenic array determines the self-fertility of the worm, so silenced arrays tend not to be inherited. This explains why the silencing of spe-4 transgenes was not previously noticed.

24 May 1999 15:50 1002 1002 A Probabilistic Method for Accurate Gene Function Prediction using Phylogenetic Inference

1999 International Worm Meeting abstract 952 These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author. A Probabilistic Method for Accurate Gene Function Prediction using Phylogenetic Inference CM Zmasek, SR Eddy Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110 Computational sequence function prediction, including the analysis currently done for the C. elegans genome project, is usually accomplished by methods based on sequence similarity (e.g. BLAST). Unfortunately, sequence similarity does not guarantee functional similarity. A gene can evolve into descendants which fulfill different biological roles but which still retain a high degree of sequence similarity. Here we present an approach for sequence function prediction which incorporates both probabilistic and phylogenetic methods. Ultimately, this approach is based on the comparison of the inferred evolution of sequences with the inferred evolution of species. Briefly, the approach works as follows. First, based on sequence similarity, the sequence to be analyzed is putatively assigned to a Pfam protein family (Pfam: amino acid sequence database containing alignments of proteins and protein domains, at http://pfam.wustl.edu/). The N sequences of this Pfam alignment are used to construct a phylogenetic tree. Then, 2N-3 different tree topologies are constructed by placing the sequence to be analyzed on each branch of this tree. The likelihood of each of these 2N-3 different tree topologies, which only differ by the placement of the sequence to be analyzed, is calculated by a maximum likelihood algorithm. The most likely tree topology is compared with our current knowledge of species evolution. In case of agreement between the most likely tree topology and the species tree, a functional prediction (or a refinement of a previous prediction) is possible. In case of conflict, the sequence to be analyzed either belongs to another protein family or is a first representative of a novel class of proteins. Some examples from the C. elegans genome are shown and compared with the results of sequence similarity based methods.

24 May 1999 15:50 1003 1003