PROGRAM & ABSTRACTS
C. elegans Development, Cell Biology, & Gene Expression Meeting 2012
Thursday, June 7 – Sunday, June 10, 2012 University of Wisconsin-Madison Memorial Union 800 Langdon Street Madison, Wisconsin 53706
Meeting Organizers E. Jane Hubbard: Skirball Institute, NYU School of Medicine ([email protected]) Jeremy Nance: Skirball Institute, NYU School of Medicine ([email protected]) Ahna Skop: University of Wisconsin-Madison ([email protected]) Martha Soto: Robert Wood Johnson Medical School, UMDNJ ([email protected])
2012 Organizing Committee Jon Audya, U. Wisconsin, Madison (USA) Zhirong Bao, Sloan-Kettering Institute (USA) Ryan Baugh, Duke U. (USA) Rafal Ciosk, Friedrich Miescher Institute for Biomedical Research, (Switzerland) Monica Colaiacovo, Harvard U. (USA) Monica Gotta, U. de Geneve (Switzerland) Alla Grishok, Columbia U. (USA) Caroline Goutte, Amherst College, (USA) Kristen Hagstrom, U. Mass Medical Center (USA) Max Heiman, Harvard Medical School (USA) Steve L’Hernault, Emory U. (USA) Valerie Reinke, Yale U. (USA) Lesilee Rose, U.C. Davis (USA) Rick Roy, McGill U., Quebec (Canada) Jennifer Schisa, Central Michigan U. (USA) Asako Sugimoto, Tohuko U., Sendai (Japan) Xiaochen Wang, NIBS, Bejing (China)
iii SPONSORS
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ACKNOWLEDGEMENTS All Sponsoring Companies University of Wisconsin Memorial Union Conference Services
91 High Street ● Amesbury, Massachusetts 01913 USA ● Tel +1 978-388-7159 ● Fax: +1 978-388-7854 iv [email protected] ● www.kramerscientific.com C. elegans Development, Cell Biology, & Gene Expression Meeting
Thursday, June 7 – Sunday, June 10, 2012
Conference Program
Thursday, June 07, 2012
12 noon–7:30 pm Registration Check-In Annex Room
12 noon Poster Set up Great Hall, Reception Room, and Main Lounge
5:00–7:00 pm Opening Reception Tripp Commons 7:00 Opening Remarks 7:00–9:00 pm Platform Session #1 Union Theater Morphogenesis I and Polarity Chairs: Lesilee Rose and Asako Sugimoto 7:15 Keynote: Ken Kemphues Three pathways to polarity maintenance 7:45 Jessica L Feldman (Lab: Priess) A role for the centrosome and PAR-3 in the hand-off of microtubule organizing center function during epithelial polarization 8:00 Yelena Y Bernadskaya (Lab: Soto) Three Axonal Guidance Pathways Help Polarize the Actin Cytoskeleton During Embryonic Epidermal Cell Migration 8:15 Jessica Shivas (Lab: Skop) Arp2/3 mediates early endosome dynamics that participate in the maintenance of polarity in C. elegans 8:30 Hongjie Zhang (Lab: Gobel) Clathrin/AP-1 cooperate with sphingolipids to regulate apical polarity and lumen formation during C. elegans tubulogenesis 8:45 Vijaykumar S Meli (Lab: Frand) The Fibrillin-like fbn-1 Gene Regulates Epithelial Stem Cell and ECM Dynamics in Molts
v 9:00–11:00 pm Poster Session #1 & Refreshments Great Hall, Reception Room, and Main Lounge (ODD number posters present)
Great Hall & Reception Room (4th floor) Cell Biology 51 83 Cell cycle and cytokinesis 84 92 Cell Death 93 103 Cell Fate 104 121 Gene Regulation 122 145 Germline 146 184
Main Lounge (2nd floor) Morphogenesis 185 211 New Technologies 212 220 Polarity 221 231 Sex Determination 232 234
Friday, June 08, 2012
7:00 am Registration continues Annex Room
7:30–9:00 am Breakfast Buffet Inn Wisconsin
9:00–10:45 am Platform Session #2 Union Theater Morphogenesis II & Cell Death Chairs: Max Heiman and Caroline Goutte 9:00 Keynote: Shai Shaham (Lab: Shaham) A New C. elegans Cell Death Program: Implications for Neurodegeneration and Cancer 9:45 Yan Zhang (Lab: Wang) C. elegans NRF-5 Regulates Cell Corpse Engulfment By Mediating PS Appearance On Phagocytes 10:00 Sasha De HeBoldnau (Lab: Braeckman) Globin 12 of Caenorhabditis elegans Regulates the p38 and JNK MAPK Pathways through Redox Signaling to Control Germline Apoptosis 10:15 Michael Hurwitz (Lab: Hurwitz) sli-1 Cbl Inhibits the Engulfment of Apoptotic Cells 10:30 Matthias K Morf (Lab: Hajnal) MADD-2 Negatively Regulates Anchor Cell Invasion 10:45 Vida Praitis (Lab: Praitis) The C. elegans Hailey-Hailey Disease Homolog pmr-1 is Essential for Cell Migration During Gastrulation
11:00–11:15 am Refreshment Break Union Theater Lobby
vi 11:15 am–1:00 pm Platform Session #3 Union Theater Germline I and Gametogenesis Chairs: Steve L’Hernault and Rafal Ciosk 11:15 Keynote: David Greenstein (Lab: Greenstein) Control of Oocyte Meiotic Maturation: Links to Germ Cell Proliferation and Global Control of the Oogenic Program 12:00 Kari Messina (Lab: Shakes) Regulators of MSP Assembly and Dynamics in C. elegans Spermatocytes 12:15 Gunasekaran Singaravelu (Lab: Singson) The sperm surface localization of the TRP-3/SPE-41 Ca2+ permeable channel depends on SPE-38 function in Caenorhabditis elegans 12:30 Jun Takayama (Lab: Onami) Timely Generation of the Fertilization Calcium Wave by a Sperm TRP Channel 12 :45 Simona Rosu (Lab: Villeneuve) Regulation of Meiotic DSB Formation in C. elegans
1:00–2:30 pm Luncheon Buffet Inn Wisconsin
2:30–5:30 pm Platform Session #4 Union Theater Cell Cycle and Cell Biology Chairs: Jon Audhya and Richard Roy 2:30 Keynote: Karen Oegema (Lab: Oegema) Title: TBD 3:15 Marie Delattre (Lab: Delattre) Evolution of spindle shape and motion in one-cell stage nematode embryos 3:30 Jill M Schumacher (Lab: Schumacher) The Tousled-like Kinase TLK-1 is a Component of the Outer Kinetochore and Potentiates Mitotic Spindle Dynamics in the Early C. elegans Embryo 3:45 Asako Sugimoto (Lab: Sugimoto) Identification of unconventional components of the γ-tubulin complex in C. elegans 4:00 Elsa Kress (Lab: Gotta) The Cdc48/p97 cofactor UBXN-2 and its orthologues p47/p37 control centrosome maturation in prophase via Aurora A
4:15–4:30 pm Refreshment Break Union Theater Lobby
4:30 Anjon Audhya (Lab: Audhya) Regulation of COPII subunit recruitment to ER exit sites 4:45 Joshua N Bembenek (Lab: Chan) Condensin I: A New Component of the Abscission Checkpoint
vii 5:00 Matyas Gorjanacz (Lab: Mattaj) LEM-4 Coordinates Mitotic Signaling on BAF to Enable its Essential Function in Nuclear Envelope Formation 5:15 Ismar Kovacevic (Lab: Cram) Filamin is Required to Initiate Calcium Signaling and Maintain F-actin Organization in the Spermatheca
5:30–7:00 pm Dinner Buffet Inn Wisconsin
7:00–9:00 pm Poster Session #2 & Refreshments Great Hall, Reception Room, and Main Lounge (EVEN number posters present)
Great Hall & Reception Room (4th floor) Cell Biology 51 83 Cell cycle and cytokinesis 84 92 Cell Death 93 103 Cell Fate 104 121 Gene Regulation 122 145 Germline 146 184
Main Lounge (2nd floor) Morphogenesis 185 211 New Technologies 212 220 Polarity 221 231 Sex Determination 232 234 9:00–11:30 pm Late Night Poster Session Great Hall, Reception Room, and Main Lounge Open Viewing (All numbered posters present)
Saturday, June 09, 2012
7:00 am Registration Continues Annex Room
7:30–9:00 am Breakfast Buffet Inn Wisconsin
9:00 am–12:00 noon Platform Session #5 Union Theater Germline II, Meiosis, and Sex Determination/Dimorphism Chairs: Monica Colaiácovo and Jennifer Schisa 9:00 Keynote: Monica Colaiácovo (Lab: Colaiácovo) Germline maintenance and meiosis: mechanistic insights from C. elegans 9:30 Aaron Kershner (Lab: Kimble) Identification of Direct GLP-1/Notch Targets that Regulate Germline Stem Cells 9:45 Rafal Ciosk (Lab: Ciosk) Genome-wide Analysis of GLD-1 Mediated mRNA Regulation Uncovers a Role in mRNA Storage viii 10:00 E. Jane Albert Hubbard (Lab: Hubbard) In the C. elegans Germ Line, S6K promotes Cell Cycle Progression and the Proliferative Fate and mediates the Effects of Diet
10:50–10:30 am Refreshment Break Union Theater Lobby
10:30 Mara Schvarzstein (Lab: Villeneuve) Chromosome and centrosome inheritance in meiosis 10:45 Daniel Cortes Estrada (Lab: McNalley) Non-random Segregation of Unpaired X Chromosomes in C. elegans Female Meiosis (asbt. # 152) 11:00 Anna K Allen (Lab: Golden) Role of the Inhibitory Kinase WEE-1.3 in Regulating the Meiotic Cell Cycle and Fertility in C. Elegans 11:15 Michael J. White VanGompel (Lab: Rose) The Torsin Homolog OOC-5 is Required for Normal Nucleoporin Localization 11:30 Matthew Berkseth (Lab: Zarkower) Identification of Direct Targets of the Caenorhabditis elegans Global Sexual Regulator TRA-1 by Chromatin Immunoprecipitation 11:45 Te-Wen Lo (Lab: Meyer) Evolution of Caenorhabditis Dosage Compensation
12:00–2:00 pm Luncheon Buffet (posters down by 2:00 pm) Inn Wisconsin
2:30–4:00 pm Workshops Union Theater 4:00–4:30 pm Refreshment Break Union Theater Lobby 4:30–6:30 pm Platform Session #6 Union Theater Gene Regulation Chairs: Valerie Reinke and Ryan Baugh Introduction: Alla Grishok 4:30 Keynote: Craig Mello (Lab: Mello) RNAi and Immortality: Recognition of Self/non-Self RNA in the C. elegans Germline 5:15 Gyorgyi Csankovszki (Lab: Csankovszki) The onset of dosage compensation is linked to the loss of developmental plasticity 5:30 David J Katz (Lab: Katz) The Histone Demethylase SPR-5 and the Histone Methyltransferase MET-2 Comprise a Novel Epigenetic Reprogramming Switch 5:45 Shouhong Guang (Lab: Guang) Nuclear RNAi mediates silencing of repetitive sequences in C. elegans
ix 6:00 Xiao-Dong Yang (Lab: Lin) Dimerization of βCatenin/WRM-1 Allows Intermolecular Autophosphorylation of LIT-1 in the Activation Loop 6:15 Morris Maduro (Lab: Maduro) Organ defects in adults resulting from threshold blastomere specification
7:00–9:30 pm Banquet Union South
9:30–Midnight Dance Union South
Sunday, June 10, 2012
9:00–12:30 pm Platform Session #7 Union Theater Cell Fate and Emerging Technologies Chairs: Monica Gotta and Zhirong Bao 9:00 am Keynote: Julie Ahringer (Lab: Ahringer) Title: TBD 9:45 Hillel Kugler (Lab: Kugler) Modeling germline population dynamics 10:00 Julia L Moore (Lab: Bao) Dev-scape: An intuitive tool for automated phenotyping with single cell resolution 10:15 Abigail Cabunoc (Lab: Stein) WormBase 2012: Website Redesign 11:00 Scott Robertson (Lab: Lin) DSL-2 Mediates a Notch Signal From EMS Descendant(s) to ABp Descendants 11:15 Jennifer A Schumacher Tucker (Lab: Chuang) Intercellular Calcium Signaling in a Gap Junction Cell Network Establishes Left-Right Asymmetric Neuronal Fates 11:30 Colin Maxwell (Lab: Baugh) Nutritional control of mRNA isoform expression during developmental arrest and recovery in C. elegans 11:45 David J Reiner (Lab: Reiner) Ras and its Effector RalGEF Both Perform Dual, Antagonistic Functions during C. elegans Vulval Patterning 12:00 Allison L Abbott (Lab: Abbott) The microRNA miR-786 is Required for Rhythmic Calcium Wave Initiation in the C. elegans Intestine
12:30–2:00 pm Luncheon Buffet Inn Wisconsin
x TABLE OF CONTENTS
Thursday, June 07, 2012 - 7:00–9:00 pm Platform Session #1 - Union Theater Morphogenesis I and Polarity Abstracts 1 - 6 Chairs: Lesilee Rose and Asako Sugimoto 1 Keynote: Three pathways to polarity maintenance Ken Kemphues 2 A role for the centrosome and PAR-3 in the hand-off of microtubule organizing center function during epithelial polarization Jessica Feldman, James Priess 3 Three Axonal Guidance Pathways Help Polarize the Actin Cytoskeleton During Embryonic Epidermal Cell Migration Yelena Bernadskaya, Andre Wallace, Jillian Nguyen, William Mohler, Martha Soto 4 Arp2/3 mediates early endosome dynamics that participate in the maintenance of polarity in C. elegans Jessica Shivas, Ahna Skop 5 Clathrin/AP-1 cooperate with sphingolipids to regulate apical polarity and lumen formation during C. elegans tubulogenesis Hongjie Zhang, Ahlee Kim, Nessy Abraham, Liakot Khan, David Hall, John Fleming, Verena Gobel 6 The Fibrillin-like fbn-1 Gene Regulates Epithelial Stem Cell and ECM Dynamics in Molts Vijaykumar Meli, Alison Frand Friday, June 08, 2012 - 9:00–10:45 am Platform Session #2 - Union Theater Morphogenesis II & Cell Death Abstracts 7 - 12 Chairs: Max Heiman and Caroline Goutte 7 Keynote: A New C. elegans Cell Death Program: Implications for Neurodegeneration and Cancer Shai Shaham 8 C. elegans NRF-5 Regulates Cell Corpse Engulfment By Mediating PS Appearance On Phagocytes Yan Zhang, Haibin Wang, Xiaochen Wang
xi 9 Globin 12 of Caenorhabditis elegans Regulates the p38 and JNK MAPK Pathways through Redox Signaling to Control Germline Apoptosis Sasha De Henau, Lesley Tilleman, Francesca Germani, Caroline Vlaeminck, Jacques Vanfleteren, Luc Moens, Sylvia Dewilde, Bart Braeckman 10 sli-1 Cbl Inhibits the Engulfment of Apoptotic Cells Courtney Anderson, Shan Zhou, Emma Sawin, Bob Horvitz, Michael Hurwitz 11 MADD-2 Negatively Regulates Anchor Cell Invasion Matthias Morf, Ivo Rimann, Mariam Alexander, Peter Roy, Alex Hajnal 12 The C. elegans Hailey-Hailey Disease Homolog pmr-1 is Essential for Cell Migration During Gastrulation Vida Praitis, Rebecca Mandt, Leah Imlay, Charlotte Feddersen, Alexander Sullivan-Wilson, Tyson Stock, Walter Liszewski, Adityarup Chakravorty, Dae Gon Ha, Angela Schacht, Michael Miller, Lensa Yohannes, Juliet Mushi, Zelealem Yilma, Sarah Kniss, Jeff Simske Friday, June 08, 2012 - 11:15 am–1:00 pm Platform Session #3 - Union Theater Germline I and Gametogenesis Abstracts 13 - 17 Chairs: Steve L’Hernault and Rafal Ciosk 13 Keynote: Control of Oocyte Meiotic Maturation: Links to Germ Cell Proliferation and Global Control of the Oogenic Program David Greenstein 14 Regulators of MSP Assembly and Dynamics in C. elegans Spermatocytes Kari Messina, Marc Presler, Leah Towarnicky, Diane Shakes 15 The sperm surface localization of the TRP-3/SPE-41 Ca2+ permeable channel depends on SPE-38 function in Caenorhabditis elegans Gunasekaran Singaravelu, Indrani Chatterjee, Sina Rahimi, Marina Druzhinina, Lijun Kang, Shawn Xu, Andrew Singson 16 Timely Generation of the Fertilization Calcium Wave by a Sperm TRP Channel Jun Takayama, Shuichi Onami 17 Regulation of Meiotic DSB Formation in C. elegans Simona Rosu, Anne Villeneuve
xii Friday, June 08, 2012 - 2:30–5:30 pm Platform Session #4 - Union Theater Cell Cycle and Cell Biology Abstracts 18 - 26 Chairs: Jon Audhya and Richard Roy 18 Keynote: Title: TBD Karen Oegema 19 Evolution of spindle shape and motion in one-cell stage nematode embryos Aurore-Cecile Valfort, Soizic Riche, Reza Farhadifair, Daniel Needleman, Marie Delattre 20 The Tousled-like Kinase TLK-1 is a Component of the Outer Kinetochore and Potentiates Mitotic Spindle Dynamics in the Early C. elegans Embryo Jessica De Orbeta, Jason Ford, Gary Deyter, Tokiko Furuta, Jill Schumacher 21 Identification of unconventional components of the γ-tubulin complex in C.elegans Nami Haruta, Eisuke Sumiyoshi, Yu Honda, Masahiro Terasawa, Mika Toya, Asako Sugimoto 22 The Cdc48/p97 cofactor UBXN-2 and its orthologues p47/p37 control centrosome maturation in prophase via Aurora A Elsa Kress, Francoise Schwager, Rene Holtackers, Esther Zanin, Francois Prodon, Jonas Seiler, Annika Eiteneuer, Asako Sugimoto, Hemmo Meyer, Patrick Meraldi, Monica Gotta 23 Regulation of COPII subunit recruitment to ER exit sites Kristen Witte, Amber Schuh, Jan Hegermann, Ali Sarkeshik, Jonathan Mayers, Katrin Schwarze, John Yates III, Stefan Eimer, Anjon Audhya 24 Condensin I: A New Component of the Abscission Checkpoint Joshua Bembenek, Koen Verbrugghe, Gyorgyi Csankovszki, Raymond Chan 25 LEM-4 Coordinates Mitotic Signaling on BAF to Enable its Essential Function in Nuclear Envelope Formation Matyas Gorjanacz, Claudio Asencio, Iain Davidson, Rachel Santarella-Mellwig, Geraldine Seydoux , Iain Mattaj 26 Filamin is Required to Initiate Calcium Signaling and Maintain F-actin Organization in the Spermatheca Ismar Kovacevic, Erin Cram
xiii Saturday, June 09, 2012 - 9:00 am–12:00 noon Platform Session #5 - Union Theater Germline II, Meiosis, and Sex Determination/Dimorphism Abstracts 27 - 35 Chairs: Monica Colaiácovo and Jennifer Schisa 27 Keynote: Germline maintenance and meiosis: mechanistic insights from C. elegans Monica Colaiácovo 28 Identification of Direct GLP-1/Notch Targets that Regulate Germline Stem Cells Aaron Kershner, Heaji Shin, Judith Kimble 29 Genome-wide Analysis of GLD-1 Mediated mRNA Regulation Uncovers a Role in mRNA Storage Claudia Scheckel, Dimos Gaidatzis, Jane Wright, Rafal Ciosk 30 In the C. elegans Germ Line, S6K promotes Cell Cycle Progression and the Proliferative Fate and mediates the Effects of Diet Dorota Korta, Debasmita Roy, Simon Tuck, E. Jane Albert Hubbard 31 Chromosome and centrosome inheritance in meiosis Mara Schvarzstein, Anne Villeneuve 32 Role of the Inhibitory Kinase WEE-1.3 in Regulating the Meiotic Cell Cycle and Fertility in C. Elegans Anna Allen, Jessica Nesmith, Andy Golden 33 The Torsin Homolog OOC-5 is Required for Normal Nucleoporin Localization Michael White VanGompel, Lesilee Rose 34 Identification of Direct Targets of the Caenorhabditis elegans Global Sexual Regulator TRA-1 by Chromatin Immunoprecipitation Matthew Berkseth, Kohta Ikegami, Jason Lieb, David Zarkower 35 Evolution of Caenorhabditis Dosage Compensation Te-Wen Lo, Caitlin Schartner, Catherine Pickle, Barbara Meyer
xiv Saturday, June 09, 2012 - 4:30–6:30 pm Platform Session #6 - Union Theater Gene Regulation Abstracts 36 - 41 Chairs: Valerie Reinke and Ryan Baugh 36 Keynote: RNAi and Immortality: Recognition of Self/non-Self RNA in the C. elegans Germline Craig Mello 37 The onset of dosage compensation is linked to the loss of developmental plasticity Laura Custer, Gyorgyi Csankovszki 38 The Histone Demethylase SPR-5 and the Histone Methyltransferase MET-2 Comprise a Novel Epigenetic Reprogramming Switch Shana Kerr, Chelsey Chandler, Joshua Francis, Erica Mills, David Katz 39 Nuclear RNAi mediates silencing of repetitive sequences in C. elegans Fei Xu, Xufei Zhou, Hui Mao, Jiaojiao Ji, Shouhong Guang 40 Dimerization of βCatenin/WRM-1 Allows Intermolecular Autophosphorylation of LIT-1 in the Activation Loop Xiao-Dong Yang, Scott Robertson , Rueyling Lin 41 Organ defects in adults resulting from threshold blastomere specification Morris Maduro, Gina Broitman-Maduro, Leila Magistrado, Shruthi Satish Sunday, June 10, 2012 - 9:00–12:30 pm Platform Session #7 - Union Theater Cell Fate and Emerging Technologies Abstracts 42 - 50 Chairs: Monica Gotta and Zhirong Bao 42 Keynote: Title: TBD Julie Ahringer 43 Modeling germline population dynamics Hillel Kugler, E. Jane Albert Hubbard 44 Dev-scape: An intuitive tool for automated phenotyping with single cell resolution Julia Moore, Zhuo Du, Anthony Santella, Christian Pohl, Zhirong Bao
xv 45 WormBase 2012: Website Redesign Abigail Cabunoc, Norie de la Cruz, Adrian Duong, Maher Kassim, Xiaoqi Shi, Todd Harris, Lincoln Stein 46 DSL-2 Mediates a Notch Signal From EMS Descendant(s) to ABp Descendants Scott Robertson, Jessica Medina, Rueyling Lin 47 Intercellular Calcium Signaling in a Gap Junction Cell Network Establishes Left-Right Asymmetric Neuronal Fates Jennifer Schumacher Tucker, Chieh Chang, Chiou-Fen Chuang 48 Nutritional control of mRNA isoform expression during developmental arrest and recovery in C. elegans Colin Maxwell, Igor Antoshechkin, Nicole Kurhanewicz, Jason Belsky, L. Ryan Baugh 49 Ras and its Effector RalGEF Both Perform Dual, Antagonistic Functions during C. elegans Vulval Patterning Kimberly Monahan, Rebecca Whitehurst, Tanya Zand, Channing Der, David Reiner 50 The microRNA miR-786 is Required for Rhythmic Calcium Wave Initiation in the C. elegans Intestine Benedict Kemp, Erik Allman, Lois Immerman, Megan Mohnen, Maureen Peters, Keith Nehrke, Allison Abbott Poster Topic Cell Biology Abstracts 51 - 83 51 GLO-2 is a BLOC-1 Subunit that Functions in Gut Granule Biogenesis Alec Barrett, Olivia Foster, Annalise Vine, Greg Hermann 52 The Conventional Kinesin-1/UNC-116 Acts in PHB Phasmid Neurons to Mediate Proper Cell Body Position Ben Barsi-Rhyne, Kristine Miller, Chris Vargas, Miri VanHoven 53 Genetic Interaction and Structure/Function Studies of MEL-28, a Protein Required for Nuclear Envelope Function and Chromosome Segregation Anita Fernandez, Carly Bock, Allison Lai, Emily Mis, Fabio Piano 54 Oocyte Meiotic Spindle Assembly in C. elegans Amy Connolly, Sara Christensen, Valerie Osterberg, Josh Lowry, John Yochem, Bruce Bowerman 55 Identifying Proteins that Interact with the Serine/Threonine Kinase UNC-82 in Muscle Cells Christopher Duchesneau, April Reedy, Hiroshi Qadota, Guy Benian, Pamela Hoppe xvi 56 A LET-23 localization and expression screen identifies a novel mechanism of EGFR regulation through Ezrin/Radixin/Moesin proteins Juan Escobar Restrepo, Peter Gutierrez, Andrea Haag, Alessandra Buhler, Christina Herrmann, Maeva Langouet, David Kradolfer, Erika Frohli, Attila Stetak, Alex Hajnal 57 Growth of Muscle Adhesion Complexes During Postembryonic Development Brandon Fields, Nate Szewczyk, Lewis Jacobson 58 CDK-1 inhibits meiotic spindle shortening and dynein-dependent spindle rotation in C. elegans Jonathan Flynn, Marina Ellefson, Francis McNally 59 The C. elegans Uterine Seam Cell: a Model for Studying Nuclear Migration and Cell Outgrowth Srimoyee Ghosh, Paul Sternberg 60 Cadherin FMI-1 Maintains the Structure of the PVD Mechanosensory Neurons Julie Grimm, Benjamin Podbilewicz 61 Two Functional Domains in C. elegans Glypican LON-2 Can Independently Inhibit DBL-1 Growth Factor Signaling but Require Accessory Moieties Suparna Bageshwar, Tina Gumienny 62 Mutational Analysis of Residues Required for Activation the UNC-82 Serine-Threonine Kinase Jason Kintzele, Pamela Hoppe 63 Genetic Analysis of Calcium Regulation in the C. elegans Intestine Jocelyn Laboy, Kenneth Norman 64 The Tubulin Deglutamylase CCPP-6 Functions Exclusively in Ciliated Dopaminergic Neurons in C. elegans Ethan Landes, Brendan O’Flaherty, Elizabeth De Stasio, Peter Swoboda, Brian Piasecki 65 Protein Sequences Within the UNC-82 S/T Kinase that Affect Subcellular Localization in Pharyngeal Muscle Latrisha Lane, Chiyen Wong, Caitlyn Carter, Pamela Hoppe 66 Characterization of vh45, a Candidate Regulator of Early to Late Endosomal Maturation Fiona Law, Shang Xiang, Christian Rocheleau 67 cil-5 Mediates Ciliary Receptor Localization and Sensory Function in C. elegans Kara Braunreiter, Greg Fischer, Casey Gabrhel, Jamie Lyman Gingerich
xvii 68 Neuroligin has Cell-autonomous and Non-autonomous Functions in C. elegans Jacob Manjarrez, Greg Mullen, Ellie Mathews, Jerrod Hunter, Jim Rand 69 Genetic and Molecular Dissection of Novel Pathways Required for Nuclear Migration in the Model System C. elegans. Yu-Tai Chang, Shaun Murphy, Jonathan Kuhn, Minh Ngo, Daniel Starr 70 FLN-1/filamin is equiredr for spermathecal contractility Jose Orozco, Ismar Kovacevic, Erin Cram 71 Isolation of Mutations that alter Nile Red Staining in C. elegans Stephanie Burge, Anthony Otsuka 72 Epithelial Dynamics During the G1-to-G2 Pore Cell Swap in the Excretory System Jean Parry, Amanda Zacharias, Hasreet Gill, John Murray, Meera Sundaram 73 The Arp2/3 activator WAVE/SCAR Promotes Clathrin Mediated Endocytosis in the Polarized C. elegans Intestinal Epithelia Falshruti Patel, Martha Soto 74 Visualizing Dynamics of Meiotic Prophase Chromosome Structures Divya Pattabiraman, Marc Presler, Grace Chen, Anne Villeneuve 75 CRL2/LRR-1 E3-Ligase Prevents Progression Through Meiotic Prophase in the Adult C. elegans Germline Julien Burger, Jorge Merlet, Nicolas Tavernier , Benedicte Richaudeau, Asja Moerkamp, Rafal Ciosk, Bruce Bowerman, Lionel Pintard 76 Regulated Nucleocytoplasmic Shuttling of SPAT-1/BORA Coordinates CDK-1 and PLK-1 Activation For Proper Mitotic Entry in the Early C. elegans Embryo Nicolas Tavernier , Anna Noatynska, Julien Burger, Costanza Panbianco, Jorge Merlet, Benedicte Richaudeau, Emmanuelle Courtois, Thibaud Leger, Monica Gotta, Lionel Pintard 77 PPFR-1 Phosphatase 4 subunit is a regulator of MEI-1/Katanin activity during meiosis that is rapidly targeted for degradation by CRL-3/MEL- 26 E3-ligase in the transition to mitosis in C. elegans Jose-Eduardo Gomes, Benedicte Richaudeau, Etienne Formstecher, Paul Mains, Lionel Pintard 78 A Genetic Analysis of the Axon Guidance of the C. elegans Pharyngeal Neuron M1 Osama Refai, Evvi Rollins, Patrcia Rhos, Jeb Gaudet 79 Using C. elegans to Explore the Role of Presenilin in Calcium Signaling Shaarika Sarasija, Kenneth Norman
xviii 80 Novel Roles For A Cell Adhesion Protein DYF-7 In C. elegans Body Size Determination Robbie Schultz, Tina Gumienny 81 DAF-16 Promotes Developmental Growth in Response to Persistent Somatic DNA Damage Michael Muller, Maria Ermolaeva, Laia Castells-Roca, Peter Frommolt, Sebastian Greiss, Jennifer Schneider, Bjorn Schumacher 82 Purification and Characterization of Glyceraldehyde-3-Phosphate Dehydrogenase from Caenorhabditis elegans Valeria S. Valbuena, Megan Gautier, Justin Spengler, M. Banks Greenberg, M. Leigh Cowart, Katherine Walstrom 83 Three axonal guidance pathways differentially signal to the regulators of the actin cytoskeleton during axonal migration Andre Wallace, Yelena Bernadskaya, Martha Soto Poster Topic Cell cycle and cytokinesis Abstracts 84 - 92 84 Microtubules and Fertilization: The MEI-1/Katanin mediated cytoskeletal transition from meiosis to mitosis in the developing embryo Sarah Beard, Paul Mains 85 Understanding Proteasomal Regulation of SZY-20 in the Centrosome Assembly Pathway Michael Bobian, Mi Hye Song 86 Mitotic spindle proteomics reveals conserved Caenorhabditis elegans proteins potentially necessary for cytokinesis Mary Kate Bonner, Daniel Poole, Tao Xu, Ali Sarkeshik, John Yates III, Ahna Skop 87 Non-random Segregation of Unpaired X Chromosomes in C. elegans Female Meiosis Daniel Cortes Estrada, Francis McNally 88 Parallel mechanisms promote RhoA activation during polarization and cytokinesis in the early C. elegans embryo Yu Chung Tse, Michael Werner, Katrina Longhini, Jean-Claude Labbe, Bob Goldstein, Michael Glotzer 89 ATX-2, the C. elegans ortholog of ataxin 2, is necessary for cytokinesis. Megan Gnazzo, Ahna Skop
xix 90 Identification and Characterization ofmel-15 as a New Paternal-effect Lethal Mutant in C. elegans Aimee Jaramillo-Lambert, Kathryn Stein, Andy Golden 91 RNA-binding Proteins ATX-2/PAB-1 Regulate Centrosome Assembly and Size Sarah Mets, Kelly Haynes, Eric Vertin, Dongyan Zhang, Mi Hye Song 92 ubc-25 encodes a conserved ubiquitin-conjugating enzyme that is required for developmentally controlled cell cycle quiescence David Tobin, Sarah Roy, Mako Saito Poster Topic Cell Death Abstracts 93 - 103 93 NAD salvage biosynthesis and programmed cell death; a new model for investigating cell death mechanisms Matt Crook, Wendy Hanna-Rose 94 The Possible Role of Autophagic Cell Death in the Regulation of Excitotoxicity in C. elegans John Del Rosario, Itzhak Mano 95 Genes Required for Cell Shedding, a Caspase-Independent Mechanism of Programmed Cell Elimination Dan Denning, Bob Horvitz 96 Investigating the pro-apoptotic function of ced-9 Kaitlin Driscoll, Peter Reddien, Brad Hersh, Bob Horvitz 97 SPTF-3 SP1 and PIG-1 MELK Function in Distinct Pathways to Promote M4 Neuron Cell-Type Specific Programmed Cell Death Takashi Hirose , Bob Horvitz 98 Using HITS-CLIP to study mRNA targets of RNA-binding proteins involved in germ cell apoptosis in C. elegans Martin Keller, Deni Subasic, Kishore Shivendra, Michaela Zavolan, Micheal Hengartner 99 Utilization of Alternative mRNAs for CED-4/Apaf-1 During Germ Cell Apoptosis J. Kaitlin Morrison, Brett Keiper 100 A Small-Molecule Screen Identifies a Linker Cell Death Inhibitor Andrew Schwendeman, Shai Shaham 101 Wave Regulatory Complex Genes Are Involved in the Engulfment of Apoptotic Cells Elena Simionato, Michael Hurwitz xx 102 In Search of Genes that Regulate Germ Cell Apoptosis in C. elegans Angel Villanueva-Chimal , Carlos Silva-Garcia , Laura Lascarez-Lagunas, Rosa Navarro 103 let-70, an E2 Ubiquitin-Conjugating Enzyme, Promotes the Non- Apoptotic Death of the Linker Cell Jennifer Zuckerman Poster Topic Cell Fate Abstracts 104 - 121 104 Elucidating the let-7 Independent Role of lin-28 Jennifer Alaimo, Bhaskar Vadla, Kevin Kemper, Eric Moss 105 Regulation and function of SYS-1/beta-catenin during hypodermal stem cell divisions Austin Baldwin, Bryan Phillips 106 Germline Expressed GLP-1 Regulates Embryonic Endoderm Specification Ahmed Elewa, Takao Ishidate, Sandra Vergara, Tae-Ho Shin, Masaki Shirayama, Craig Mello 107 Investigating the Role of SEM-4/SALL in Development of the Postembryonic Mesoderm Vikas Ghai, Chenxi Tian, Jun Liu 108 A Screening To Find Suppressors Of The Wnt Pathway Eva Gomez-Orte, Begona Ezcurra, Beatriz Saenz-Narciso, Juan Cabello 109 MEX-5 regulates mRNA stability during germ cell development and asymmetric cell division Manoel Prouteau, Gilles Udin, Monica Gotta 110 A Screen for Mislocalization and Misexpression of LET-23 EGF Receptor during Vulval Development Andrea Haag, Juan Escobar Restrepo, Alex Hajnal 111 A Role of the LIN-12/Notch Signaling Pathway in Diversifying the Non- Striated Egg-Laying Muscles in C. elegans Jared Hale, Carolyn George, Nirav Amin, Zachary Via, Leila Toulabi, Jun Liu 112 UNC-62/Meis and CEH-20/Pbx proteins work together to control asymmetric cell divisions during C. elegans development by regulating WRM-1/β-catenin localisation Samantha Hughes, Charles Brabin, Alison Woollard
xxi 113 The Ras-ERK/MAPK Regulatory Network Controls Dedifferentiation In Caenorhabditis elegans Germline Dong Seok Cha, Udaya Sree Datla, Sarah Hollis, Judith Kimble, Myon-Hee Lee 114 A sma-9 Suppressor Screen to Identify New Players in the BMP-like Sma/Mab Pathway in C. elegans Lindsey Szymczak, Katharine Constas, Arielle Schaeffer, Sinthu Ranjan, Saad Kubba, Emad Alam, Dennis Liu, Chenxi Tian, Herong Shi, Jun Liu 115 Further evidence for the importance of the MED-1 and -2 GATA factors in endoderm specification Morris Maduro, Gina Broitman-Maduro, Shruthi Satish 116 Regulation and function of nhr-67/tailless in uterus development George McClung, Lauren Pioppo, Jenny Hall, Rachel Dordal, Catherine Ezzio, Evan Fletcher, Amanda Gavin, Sheila Clever, Bruce Wightman 117 Does lin-46 Tip the Balance of hbl-1 Activity in the Succession of Hypodermal Blast Fates? Eric Moss, Kevin Kemper, Bhaskar Vadla 118 Post-transcriptional Regulation of Maternally-supplied Wnt Ligand During Early Embryogenesis Marieke Oldenbroek, Scott Robertson, Tugba Guven-Ozkan, Rueyling Lin 119 Abstract withdrawn 120 Regulation of LET-23 EGFR signaling and trafficking by a putative Arf1- GEF Olga Skorobogata, Christian Rocheleau 121 Examining the Fate of Centrosomally Uncoupled SYS-1/Beta-catenin to Explore Spindle-Independent Roles of the Centrosome during Asymmetric Cell Divisions Setu Vora, Bryan Phillips Poster Topic Gene Regulation Abstracts 122 - 145 122 Function and evolution of the diverged NR2E nuclear receptors nhr-111 and nhr-239 Emily Bayer, G. Michael Baer, Christopher Alvaro, Katherine Weber, Ramzy Burns, Michael Lilly, Anvi Patel, Benjamin Perlman, Sheila Clever, Bruce Wightman 123 Redefining POP-1 Binding Sites in C. elegans Chandan Bhambhani, Ken Cadigan
xxii 124 In vivo Regulation of the Alternative Splicing of the Pro- and Anti- Apoptotic Gene ced-4 Anna Corrionero, Bob Horvitz 125 Identifying HLH-8/Twist Homodimer Target Genes Nirupama Singh, Peng Wang, Ann Corsi 126 Understanding the Role of Overlapping MicroRNA Networks During Nematode Development Jeanyoung Jo, Kimberly Breving, Kenya Madric, Aurora Esquela-Kerscher 127 Intracellular Trafficking and Endocytic Regulation of the DBL-1/BMP- like pathway in C. elegans Ryan Gleason, Adenrele Akintobi, Ying Li, Barth Grant, Richard Padgett 128 Identification and characterization of targets of the REF-1 family member, HLH-25 Raymarie Gomez, Han-ting Chou, Casonya Johnson 129 The Mediator Subunit CDK-8 Negatively Regulates EGFR-Ras-MAPK in Vulva Development Jennifer Grants, Stefan Taubert 130 A Lipid-Binding Protein that Modifies cGMP Signaling is Required for Host Odor Sensing and Body Morphology in Pristionchus pacificus Ray Hong, Jessica Cinkornpumin, Dona Roonalika Wisidagama, Veronika Rapoport 131 Elucidating The Role of Genetic Redundancy In The Wnt Signaling Pathway In Regulating Q Neuroblast Migration Ni Ji, Teije Middelkoop, Hendrik Korswagen, Alexander van Oudenaarden 132 Can the Rate of Transcription be Quantitatively Determined in Relation to Transcription Factor Binding Affinity? Brett Lancaster, James McGhee 133 Regulated Splicing of the Cholinergic Gene Locus Ellie Mathews, Greg Mullen, Jim Rand 134 Short Capped RNAs and Nuclear Run-On Reveal Pol II Pausing and Backtracking in C. elegans Colin Maxwell, William Kruesi, Nicole Kurhanewicz, Leighton Core, Colin Waters, Igor Antoshechkin, John Lis, Barbara Meyer, L. Ryan Baugh 135 The mRNA Splicing Regulator SPK-1 Is Required for Cell Polarity in One-Cell C. elegans Embryos Martin Mikl, Carrie Cowan
xxiii 136 The Transcriptional Repressor Protein CTBP-1 Regulates the Differentiation of DA Motor Neurons Hannah Nicholas, Duygu Yucel, Estelle Llamosas, Anna Reid, Aaron Lun, Sashi Kant, Merlin Crossley 137 The Role of C. elegans bHLH-29 Transcription Factor in Stress Response Thanh Quach, Casonya Johnson 138 Loss of the ubiquitin-specific protease usp-48 allows for direct conversion of a somatic tissue into neurons in Caenorhabditis elegans Dylan Rahe, Tulsi Patel, Oliver Hobert 139 Chromatin Structure and Genome Stability in C. elegans Valerie Robert, Cedric Rakotomalala, Cecile Bedet, Florence Couteau, Monique Zetka, Francesca Palladino 140 A New Attempt to Elicit an RNAi Phenotype with the LIM- homeodomain Transcription Factor LIM-7 Laura Vallier, John Coppola 141 The Histone Demethylase UTX-1 Is Essential for Normal Development, Independently of Its Enzymatic Activity Julien Vandamme, Lisa Salcini 142 A Conserved SBP-1/Phosphatidylcholine Feedback Circuit Regulates Lipogenesis in Metazoans Amy Walker, Rene Jacobs, Jenny Watts, Veerle Rottiers, Lorissa Niebergall, Anders Naar 143 HLH-29, REF-1 family protein functions in the spermatheca Ana White, Casonya Johnson 144 Promoter analysis of the GATA type transcription factor ELT-2 Tobias Wiesenfahrt, Jannette Berg, James McGhee 145 Genetic Screen for Novel Repair Genes Implicated in UV-induced DNA Damage Response Stefanie Wolters, Bjoern Schumacher Poster Topic Germline Abstracts 146 - 184 146 The eIF4E-binding protein IFET-1 is a broad-scale translational repressor and is required for normal P granule ultrastructure Madhu Sengupta, Lloyd Low, Joseph Patterson, Traude Beilharz, Jennifer Schisa, Peter Boag
xxiv 147 Spindle assembly checkpoint proteins monitor synapsis during meiosis in C. elegans Tisha Bohr, Piero Lamelza, Needhi Bhalla 148 A global genomic survey of genes that mediate LKB1/PAR-4- dependent germline stem cell quiescence in C. elegans Rita Chaouni, Richard Roy 149 VPR-1, a VAPB homolog required for germ line proliferation and differentiation Pauline Cottee, Jack Vibbert, Sung Min Han, Michael Miller 150 Paternal Mitochondria Elimination From the Germline in C. elegans Embryos Dominika Bienkowska, Sylvain Bertho, Carrie Cowan 151 CACN-1 is required for gonad and germline development Hiba Tannoury, Erin Cram 152 HIS-35, a histone H2A variant that differs from canonical H2A by one amino acid, functions in fertility Francisco Guerrero, Rodrigo Estrada, Meghann Shorrock, Margaret Jow, Diana Chu 153 SNF-10, an SLC6 transporter required for sperm activation by C. elegans males Kristin Fenker, Angela Hansen, Conrad Chong, Molly Jud, Gillian Stanfield 154 Putative protamines, SPCH-1/2/3, localize to mature sperm chromatin and may play a role in fertility Jennifer Gilbert, Dana Byrd, Diana Chu 155 Sperm Vs Sperm: Determining the Cellular Basis of Sperm Competition Jody Hansen, Daniela Chavez, Gillian Stanfield 156 Evaluating the Role of the V-ATPase B Subunit Utilizing C.elegans Sperm Melissa Henderson, Elizabeth Gleason, Ying Long, Taylor Walsh, Emily Wang, Steven L’Hernault 157 The RNA binding protein TIA-1.2 is essential for fertility in C. elegans Gabriela Huelgas Morales, Carlos Silva Garcia, Rosa Navarro Gonzalez 158 Germline Hexosamine Pathway Synthesis of UDP-GlcNAc is Regulated by SUP-46 Wendy Johnston, Aldis Krizus, Arun Ramani, Andrew Fraser, James Dennis 159 Role of Notch re-localization in establishing germline stem cell quiescence in C. elegans dauer larvae Pratik Kadekar, Nathan Navidzadeh, Patrick Narbonne, Emily Wendland, Richard Roy
xxv 160 Protein synthesis regulation in the germline: eIF4 factors promote selective mRNA translation for meiosis, differentiation, maturation or apoptosis. Melissa Henderson, Jacob Subash, Vince Contreras, Anren Song, Sara Labella, Andrew Friday, Monique Zetka, Robert Rhoads, Brett Keiper 161 P-TEFb—Independent Phosphorylation of RNA Polymerase II CTD- Ser2 in the C. elegans Germline Elizabeth Bowman, Bill Kelly 162 sacy-1 Links Somatic Control of Oocyte Meiotic Maturation, Germline Sex Determination, and Gamete Maintenance Seongseop Kim, J. Amaranath Govindan, Zheng Jin Tu, David Greenstein 163 Investigating the Role of SMC-5/6 in Preventing Germline Genomic Rearrangement Killeen Kirkconnell, Dane Session, Raymond Chan 164 The let-479 Gene Encodes a Homolog of SPE-42 and is Required for C. elegans Fertilization Tim Kroft, Luke Wilson, Lindsey Magnuson, Gabe Fall 165 Spindle Assembly Checkpoint Plays a Role In DNA-damage-induced Cell Cycle Arrest In C. elegans Male Germ Line Katherine Lawrence, JoAnne Engebrecht 166 Investigating the Role of Membrane Trafficking in Temperature- Sensitive Lethal Mutants with Defects in both Gonad Development and Embryonic Eggshell Production Josh Lowry, Amy Connolly, John Yochem, Bruce Bowerman 167 Genome destabilization and checkpoint activation during cell cycle reentry of the primordial germ cells Z2 and Z3 Ash Williams, Brendan Kramer, Matthew Michael 168 Sensory Regulation of the C. elegans Germ Line through TGF-β- Dependent Signaling in the Niche Diana Dalfo, David Michaelson, E Albert Hubbard 169 In Vitro Analysis of C. elegans H2A Variants Ahmad Nabhan, Geeta Narlikar, Diana Chu 170 ZHP-3 Regulates Meiotic Chromosome Dynamics Christian Nelson, Cate Paschal, Needhi Bhalla 171 Distinct roles for FBF-1 and FBF-2 in silencing meiotic mRNAs Alexandre Paix, Ekaterina Voronina, Geraldine Seydoux
xxvi 172 Natural Variants of C. elegans Demonstrate Defects in Both Sperm Function and Oogenesis at Elevated Temperatures Lisa Petrella, Susan Strome 173 Exploring Novel Features of Gametogenesis in a Non-C. elegans Clade Kathryn Rehain, Zechariah Dillingham, Ethan Winter, Diane Shakes 174 Nutritional Control of Germline Stem Cells Hannah Seidel, Judith Kimble 175 Characterization of SYGL-1, A Novel Regulator of Germline Stem Cells Heaji Shin, Aaron Kershner, Judith Kimble 176 Uncovering the Role of Condensin I during C. elegans Meiosis Margarita Sifuentes, Joshua Bembenek, Karishma Collette, Gyorgyi Csankovszki 177 The metazoan gene akirin is required for synaptonemal complex disassembly and bivalent structure during Caenorhabditis elegans meiosis Amy Clemons, Heather Brockway, Yizhi Yin, Yaron Butterfield, Steven Jones, Monica Colaiacovo, Sarit Smolikove 178 Chromatin Regulation in the Meiotic Germ Line Matthew Snyder, Xia Xu, Eleanor Maine 179 Global Control of the Oogenic Program by Components of OMA-1 Ribonucleoprotein Particles Caroline Spike, Donna Coetzee, David Greenstein 180 Early and Late Roles for Gonadal Innexins: Germ Cell Proliferation and Meiotic Maturation Todd Starich, David Hall, David Greenstein 181 Oocyte-to-embryo Transition: a Screen for mbk-2 Suppressors Yuemeng Wang, Harold Smith, Kevin O’Connell, Geraldine Seydoux 182 A Functional RNAi Screen Identifies Regulators of RNP Granule Assembly in Aging Oocytes Megan Wood, Kevin Gorman, Joseph Patterson, Jennifer Schisa 183 A Novel Function of MRE-11 in Caenorhabditis elegans Yizhi Yin, Sarit Smolikove 184 Illuminating the Formation and Regulation of Meiotic Crossovers with GFP:COSA-1 Karl Zawadzki, Rayka Yokoo, Anne Villeneuve
xxvii Poster Topic Morphogenesis Abstracts 185 - 211 185 exc-2 and Maintenance of Tube Structure of the Excretory Canals Hikmat Al-Hashimi, Matthew Buechner 186 C. elegans nuclear hormone receptor, nhr-25 regulates vulval terminal cell properties and migrations during development Nagagireesh Bojanala, Marek Jindra, Masako Asahina 187 Characterizing regulators of the C. elegans cytoskeleton Benjamin Chan, Simon Rocheleau, Paul Mains 188 The Morphological and Functional Alterations of the Anal Depressor Muscle in Male C.elegans Xin Chen, L. Rene Garcia 189 TMD-1 / Tropomodulin Regulates Intestinal and Excretory Cell Development Rachel Walker, Corey Hoffman, Elisabeth Cox-Paulson 190 Roles Of Heparan Sulfate Proteoglycans In Embryonic Morphogenesis Katsufumi Dejima, Suk-Ryool Kang , Andrew Chisholm 191 C. elegans body size is regulated by TGF-β signalling in multiple tissues. Aidan Dineen, Jeb Gaudet 192 Functional Dissection of SAX-7, a Homologue of Human L1CAM in C. elegans Dendritic Branch Formation Xintong Dong, Oliver Liu, Kang Shen 193 ani-1 is required for morphogenesis of C. elegans embryos and functions in parallel to the rho-1 pathway. Nellie Fotopoulos, Yun Chen, Alisa Piekny 194 A Genome-Wide RNAi Screen to Identify New Components of a Muscle-To-Epidermis Mechanotransduction Pathway Essential for Embryonic Elongation Christelle Gally, Agnes Aubry, Michel Labouesse 195 The EXC-1 RAS-Domain Protein Mediates Vesicle Movement in the Excretory Canals Kelly Grussendorf, Brendan Mattingly, Alex Salem, Matthew Buechner 196 A Screen For Genes Controlling Vulval Morphogenesis Qiutan Yang, Matthias Morf, Sarfarazhussain Farooqui , Juan Escobar, Alex Hajnal
xxviii 197 LEP-2/Makorin Promotes let-7 microRNA-mediated Terminal Differentiation in Male Tail Tip Morphogenesis R Antonio Herrera, Karin Kiontke, Samuel Ahn, David Fitch 198 pix-1 Generates a Gradient of Contraction Forces in Hypodermal Cells of Elongating Embryos in Caenorhabditis elegans Sharon Harel, Emmanuel Martin, Bernard Nkengfac, Karim Hamiche, Mathieu Neault, Sarah Jenna 199 Analysis of the Role of ENU-3 in Axon Outgrowth and Guidance in C. elegans Callista Yee, Karmen Lam, Anna Bosanac, Marie Killeen 200 Identifying Regulators of Gonadal Development in C. elegans by Cell- specific Transcriptional Profiling Mary Kroetz, David Zarkower 201 Caenorhabditis elegans DNA-2 Helicase/Endonuclease Plays A Vital Role In Maintaining Genome Stability, Morphogenesis, And Life Span Myon-Hee Lee, Sarah Hollis, Bum Ho Yoo, Keith Nykamp 202 The Role of LIN-3 During Morphogenesis of the Dorsal Lumen in the Vulva Louisa Mueller, Matthias Morf, Alex Hajnal 203 Somatic gonad precursor migration in C. elegans Monica Rohrschneider, Jeremy Nance 204 VAB-9 and Vertebrate Orthologue TM4SF10 Cooperate with Adherens Junction Proteins and Actomyosin to Regulate Epithelial Polarity and Morphogenesis Jeff Simske 205 The C. elegans DM domain genes dmd-3 and mab-3 function during the late stages of male gonad development Michele Smith, Alyssa Herrmann, Emily Kivlehan, Lauren Whipple, Douglas Portman, D. Adam Mason 206 Analysis of Non-Muscle Myosin II During Dorsal Intercalation in Caenorhabditis elegans Elise Walck-Shannon, Jeff Hardin 207 Establishing Caenorhabditis elegans as a Model for Neural Tube Defects Bridget Waller, Kassi Crocker, Timothy Walston 208 Anillin is required for Epidermal Morphogenesis during C. elegans Embryogenesis Denise Wernike, Alisa Piekny
xxix 209 What Causes Partial Penetrance of a Developmental Phenotype? Claire Williams, Maxwell Heiman 210 MIG-10 interacts with ABI-1 to induce asymmetric outgrowth- promoting activity in response to guidance cues Yan Xu, Christopher Quinn 211 Molecular characterization of maternally malformed 3 (mal-3) Yemima Budirahardja, Thang Doan, Ronen Zaidel Bar Poster Topic New Technologies Abstracts 212 - 220 212 A Semi-Automated Pipeline for the Identification of Novel Mutants with Cell Number Defects Peter Appleford, Alison Woollard 213 A Novel Fluorescence-Based Method to Visualize Protein-Protein Interactions in Living Caenorhabditis elegans Han Ting Chou, Casonya Johnson 214 Spectrum: Building Pathways to Biomedical Research Careers for Girls and Women of Color Diana Chu, Rebecca Garcia, Kimberly Tanner 215 Establishing and using a modified NGM (ENGM) to culture an manipulate the entomopathogenic nematode, Heterorhabditis bacteriophora Zsofia Csanadi, Abate Birhan Addise, Anita Alexa, Barnabas Jenes, Zsofia Banfalvi, Andrea Mathe-Fodor, Katalin Belafi-Bako, Andras Fodor 216 A MultiSite Gateway®-Compatible Three-Fragment Vector Construction Kit Using Galactose Selection Iskra Katic, Wolfgang Maier 217 Screening for C. elegans Mutants with Subtle Phenotypes with Microfluidics and Computer Vision Adriana San-Miguel, Matthew Crane, Peri Kurshan, Kang Shen, Hang Lu 218 Two Novel Staining Protocols Resolve Caenorhabditis elegans Cuticular Structures For Live Imaging And Transmission Electron Microscopy Robbie Schultz, E. Ann Ellis, Tina Gumienny 219 Improving the Sensitivity and Selectivity of Mutation Identification by Next-Generation Sequencing Sijung Yun, Michael Krause, Harold Smith
xxx 220 Worm Proteins Overtake Biochemistry Lab to Inspire Inquiry Katherine Walstrom Poster Topic Polarity Abstracts 221 - 231 221 Understanding temporal and spatial features of polarity establishment Simon Blanchoud, Felix Naef, Pierre Gonczy 222 PAR proteins regulate the localization of LET-99 during asymmetric division Eugenel Espiritu, Jui-Ching Wu, Lesilee Rose 223 On the Role of RGA-3/4 in Foci Formation of NMY-2 in C. elegans Masashi Fujita, Shuichi Onami 224 Isolation, Identification, and Characterization of Free-Living Nematodes Lauren Leister, Alan Massouh, Alexis Plaga, Ramon Carreno, Danielle Hamill 225 A Dominant Mutation in a C. elegans Splicing Factor Results in Reversed AP Polarity in the Early Embryo Reza Keikhaee, Bruce Nash, John Yochem, Bruce Bowerman 226 Identifying Mechanisms of Contact-Mediated Cell Polarization Diana Klompstra, Dorian Anderson, Jeremy Nance 227 ER Compartmentalisation and the Regulation of Polarity in the C. elegans Embryos Zuo Yen Lee, Monica Gotta, Yves Barral 228 A Cullin-5-RING Ubiquitin Ligase Regulates Asymmetric Cell Division in Early C.elegans Embryos Anne Pacquelet, Emeline Daniel, Gregoire Michaux 229 Evolution of GPR Regulation in the Control of Spindle Positioning for Two Cænorhabditis Species Embryos Soizic Riche, Francoise Argoul, Melissa Zouak, Alain Arneodo, Jacques Pecreaux, Marie Delattre 230 Coupling Centrosome Position And Cortical Polarity Sabina Sanegre, Carrie Cowan 231 GLD-3(S) Contributes to PIE-1 Asymmetry in Zygotes Jarrett Smith, Geraldine Seydoux
xxxi Poster Topic Sex Determination Abstracts 232 - 234 232 Phosphorylation State of a Tob/BTG Protein, FOG-3, Regulates Initiation and Maintenance of the Caenorhabditis elegans Sperm Fate Program Myon-Hee Lee, Kyung Won Kim, Clinton Morgan, Dyan Morgan, Judith Kimble 233 Molecular Analyses of FOG-1 and FOG-3, Terminal Regulators of the Sperm/Oocyte Cell Fate Decision Daniel Noble, Scott Aoki, Marco Ortiz Sanchez, Kyung Won Kim, Judith Kimble 234 RNA-Seq Analysis of Germline Sex Reprogramming Elena Sorokin, Judith Kimble
xxxii Three pathways to polarity maintenance Ken Kemphues Cornell University
Contact: [email protected] Lab: Kemphues
Keynote 1 1 A role for the centrosome and PAR-3 in the hand-off of microtubule organizing center function during epithelial polarization Jessica Feldman, James Priess Fred Hutchinson Cancer Research Center, Seattle, WA, USA The centrosome is the major microtubule organizing center (MTOC) in dividing cells and in many post-mitotic, differentiated cells. In other cell types, however, MTOC function is reassigned from the centrosome to non-centrosomal sites. Here, we analyze how MTOC function is reassigned to the apical membrane of C. elegans intestinal cells. After the terminal intestinal cell division, the centrosomes and nuclei move near the future apical membranes, and the postmitotic centrosomes lose all, or nearly all, of their associated microtubules. We show that microtubule-nucleating proteins such as γ-tubulin and CeGrip-1 that are centrosome components in dividing cells become localized to the apical membrane, which becomes highly enriched in microtubules. Our results suggest that centrosomes are critical to specify the apical membrane as the new MTOC. First, γ-tubulin appears to redistribute directly from the migrating centrosome onto the lateral, then apical membrane. Second, γ-tubulin fails to accumulate apically in wild-type cells following laser ablation of the centrosome. We show that centrosomes localize apically by first moving toward lateral foci of the conserved polarity proteins PAR-3 and PAR-6, and then move together with these foci toward the future apical surface. Embryos lacking PAR-3 fail to localize their centrosomes apically, and have aberrant localization of γ-tubulin and CeGrip-1. These data suggest that PAR proteins contribute to apical polarity in part by determining centrosome position and that the reassignment of MTOC function from centrosomes to the apical membrane is associated with a physical hand-off of nucleators of microtubule assembly.
Contact: [email protected] Lab: Priess
2 Platform Session #1 - Morphogenesis I and Polarity Three Axonal Guidance Pathways Help Polarize the Actin Cytoskeleton During Embryonic Epidermal Cell Migration Yelena Bernadskaya1, Andre Wallace1, Jillian Nguyen1, William Mohler2, Martha Soto1 1UMDNJ/RWJMS, Piscataway, NJ, USA, 2University of Connecticut, Farmington, CT, USA Migrating cells must integrate multiple guidance cues to direct their movements during embryonic development. Some of the best-studied regulators of cell migration and growth are the UNC-6/netrin, SLT-1/slit and VAB-2/Ephrin guidance cues and their receptors, UNC-40/ DCC, SAX-3/Robo and VAB-1/Eph. However, the mechanisms that interpret these signals downstream of the receptors and reorganize the actin cytoskeleton accordingly are not well understood. Using live imaging of F-actin in developing embryos we show three guidance receptors, UNC-40/DCC, SAX-3/Robo and VAB-1/Eph, differentially regulate the subcellular polarization and abundance of F-actin in migrating epidermal cells. Interestingly, our data suggests that high levels of F-actin are not essential for directed migration but that correct polarization of remaining F-actin is. Using genetic and molecular techniques we find that the three guidance receptors affect the localization of the WAVE/SCAR complex and its activator CED-10/Rac1, thus regulating formation of branched actin networks in the embryonic epidermis. Loss of any of these receptors results in defects in epidermal morphogenesis similar to those observed in the ced-10 and wve-1 mutants. Our results suggest that proper membrane recruitment and activation of CED-10/Rac1 and of WAVE/SCAR result in polarized F-actin that permits polarized movements and suggest how multiple guidance cues can result in distinct changes in actin nucleation during morphogenesis.
Contact: [email protected] Lab: Soto
Platform Session #1 - Morphogenesis I and Polarity 3 Arp2/3 mediates early endosome dynamics that participate in the maintenance of polarity in C. elegans Jessica Shivas, Ahna Skop University of Wisconsin-Madison The widely conserved Arp2/3 complex is crucial for the formation of branched actin networks. These networks play important roles in a variety of cellular processes, including endocytosis. In C. elegans, the actin cytoskeleton has been characterized in its role in the establishment of PAR asymmetry and cytokinesis. However, the contributions of actin to maintaining polarity, prior to the onset of mitosis, remain unclear. Endocytic recycling has been reported to function as an important mechanism in the dynamic stabilization of cellular polarity. We previously reported a role for the C. elegans ortholog of dynamin, DYN-1, in the stabilization of PAR asymmetry during maintenance phase through its participation in spatially and temporally regulated endocytosis. We now provide evidence that depletion of the Arp2 subunit of the Arp2/3 complex, ARX-2, disrupts the cortical formation and localization of short actin filaments and foci that are normally present during polarity maintenance phase. We also observe defects in the organization and dynamics of endocytic regulators and polarity proteins during this time. We detect actin in association with the early endosome and endosomes are significantly larger upon disruption of ARX-2 levels. Finally, we detect aberrant accumulations of cytoplasmic PAR-6 in association with the enlarged early endosomes for prolonged periods of time when ARX-2 levels are reduced. This is observed when PAR-6 occupies a slightly smaller cortical area, suggesting a disruption in the endocytic dynamics associated with PAR-6. We propose a mechanism in which Arp2/3 regulates actin dynamics at the early endosome that promote rapid recycling of internalized polarity cues during polarity maintenance phase.
Contact: [email protected] Lab: Skop
4 Platform Session #1 - Morphogenesis I and Polarity Clathrin/AP-1 cooperate with sphingolipids to regulate apical polarity and lumen formation during C. elegans tubulogenesis Hongjie Zhang1, Ahlee Kim1, Nessy Abraham1, Liakot Khan1, David Hall2, John Fleming1, Verena Gobel1 1Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA, 2Albert Einstein College of Medicine, Bronx, NY, USA Biological tubes are composed of polarized epithelial cells with apical membranes building the central lumen and basolateral membranes contacting adjacent cells and the extracellular matrix. We carried out a genome-wide morphological RNAi screen that examined the requirement of lethal genes for tube/lumen formation, using animals engineered with ERM-1::GFP-labeled apical/lumenal membranes. This screen identified a distinctive intestinal phenotype where the contiguous central lumen was transformed into multiple ectopic laterals lumens. Further analysis revealed that multiple-lumen formation was caused by a conversion of apicobasal polarity, with displacement of apical membrane components to the lateral membrane and/or cytoplasm and de novo formation of microvilli at lateral membranes. This polarity conversion appeared to occur independent of prior junction assembly defects, compatible with a trafficking defect disrupting the directional targeting of membrane components or polarity regulators. Among other molecules, the loss of several unrelated fatty-acid- and sphingolipid(SL)- biosynthetic enzymes was found to cause this phenotype. Follow-up biosynthetic pathway screens identified membrane glycosphingolipids (GSLs) as the underlying lipid compound, mediating the function of these enzymes. GSLs are presumed raft components that reside on vesicle membranes and on lumenal plasma membranes. They have a documented apical sorting function in mammalian cell lines, although have not yet been shown to define membrane domain identities in vivo. The loss of CHC-1, the clathrin heavy chain, and of several subunits of the clathrin AP-1 adaptor also caused a polarity/ectopic lumen phenotype, supporting the notion of an underlying trafficking defect. Clathrin, however, has a well-defined role in endocytosis, but its regulation of plasma-membrane-directed transport is thought to be limited to the basolateral membrane. Here, we demonstrate that CHC-1/AP-1 cooperate with SL-biosynthetic enzymes in apical sorting. We show that GFP::CHC-1 and BODIPY-ceramide vesicles associate perinuclearly and assemble asymmetrically at polarized plasma membrane domains, in a codependent and AP-1-dependent manner. Based on these findings, we propose a trafficking pathway for apical membrane polarity in tubulogenesis that implies: (1) a clathrin/ AP-1 function on an apically-directed transport route; and (2) the convergence of this route with a sphingolipid-dependent apical trafficking path.
Contact: [email protected] Lab: Gobel
Platform Session #1 - Morphogenesis I and Polarity 5 The Fibrillin-like fbn-1 Gene Regulates Epithelial Stem Cell and ECM Dynamics in Molts Vijaykumar Meli, Alison Frand University of California Los Angeles, Los Angeles, California, USA The molting cycle involves the periodic removal and deposition of extracellular matrices (ECM). The stem cell-like lateral seam cells contribute to the production of new matrices during the molts, but undergo asymmetric divisions early in every larval stage. In addition, successive transitions between seam cell temporal fates coincide with the molts. However, the molecular mechanisms that coordinate ECM and stem cell dynamics during the molts are not yet understood. Here, we describe FBN-1, a protein that is similar to human fibrillins, which are the major components of ECM fibers defective in Marfan Syndrome and other inherited disorders of skin and connective tissue. The fbn-1 gene emerged from a full-genome, RNAi- based screen for larvae unable to fully shed cuticles; fbn-1(tm290) mutants also exhibit molting defects. A transcriptional fbn-1::gfp-pest fusion gene is transiently but reiteratively expressed in the hypoderm during every molt. In addition, the expression of multiple splice variants of fbn-1 suggests substantial post-transcriptional gene regulation. To better define the function of fbn-1, we characterized the status of the cuticle in fbn-1 mutants, using cell and molecular biological approaches including TEM. A functional COL 19::GFP fusion protein was improperly deposited and disorganized in cuticles of fbn-1(lf) adults, and structural cuticle abnormalities were detected by TEM. Consistent with these findings, rearrangements in the actin cytoskeleton of the hypodermis were not obvious in fbn-1 mutants undergoing the fourth molt, but were readily detected in wild-type animals stained with Rh-phalloidin. Further, the lateral seam cells were detected using standard markers for the cell nuclei and margins. At the L4-to-adult transition, some seam cells failed to fuse or exit the cell cycle in approximately 35% of fbn-1(-) animals. The axis of seam cell division was also abnormal in fbn-1(RNAi) animals, suggesting de-regulation of the Wnt signaling pathway. Indeed, genetic analyses confirmed that mutations that affect the Wnt or other conserved cell-ECM signaling pathways modify the phenotypes of fbn-1(-) larvae. Taken together, our findings indicate that FBN-1 polymers likely serve as both structural and instructive components of matrices remodeled during the molts. We propose that the certain activities of FBN-1 macromolecules orchestrate stem cell and ECM dynamics in larval development.
Contact: [email protected] Lab: Frand
6 Platform Session #1 - Morphogenesis I and Polarity A New C. elegans Cell Death Program: Implications for Neurodegeneration and Cancer Shai Shaham Rockefeller (USA) Death is a vital developmental cell fate required to sculpt organs, eliminate harmful cells, and counter cell division. Apoptosis, an extensively studied cell death process, requires caspase protease activation, and is accompanied by chromatin compaction and cytoplasmic shrinkage. Mice lacking apoptotic effectors survive to adulthood, a surprising result given the prevalence of cell death during murine development. Thus, non-apoptotic cell death may play key roles in animal development. Genes dedicated to non-apoptotic developmental cell death have not been previously described. We study the programmed death of the linker cell, which leads gonadal elongation in Caenorhabditis elegans males. Strikingly, the linker cell dies independently of caspases and other apoptotic effectors. Moreover, dying linker cells display non-apoptotic ultrastructural features including nuclear envelope crenellation, uncondensed chromatin, and organelle swelling. We uncovered a novel program, unleashed within the linker cell to promote its demise. One component, PQN-41- a polyglutamine-repeat protein, promotes and is expressed at the onset of death. Regulators and co-expressed effectors have also been identified. Dying linker cells bear ultrastructural similarities to dying cells in normal vertebrate development and to degenerating cells in polyglutamine-induced diseases. Our results may, therefore, provide in-roads to understanding non-apoptotic cell death in metazoan development and disease.
Contact: [email protected] Lab: Shaham
Keynote 2 7 C. elegans NRF-5 Regulates Cell Corpse Engulfment By Mediating PS Appearance On Phagocytes Yan Zhang, Haibin Wang, Xiaochen Wang National Institute of Biological Sciences, Beijing, China Phagocytosis of apoptotic cells is crucial for tissue remodeling, suppression of inflammation, and regulation of immune responses. Phosphatidylserine (PS), which is confined to the inner leaflet of plasma membrane in living cells, is exposed on the surface of apoptotic cells, thus serving as an “eat me” signal for engulfment. How PS is externalized and recognized is not well understood. We recently identified C. elegans TTR-52 as an extracellular bridging molecule which links PS on apoptotic cells with the CED-1 receptor on phagocytes. However, whether additional extracellular proteins are involved in recognizing apoptotic cells remains to be determined. In this study, we identified NRF-5, a secreted lipid transfer/LPS-binding family protein, as a novel regulator of cell corpse engulfment. The NRF-5 protein is expressed in and secreted from body wall muscle cells and clusters around apoptotic cells. We found that recognition of cell corpses by NRF-5 is disrupted in ced-7(lf) mutants but not altered in tat-1(lf) mutants which cause ectopic exposure of PS on living cell surfaces. As loss of tat-1 results in appearance of TTR-52 on the surface of both dying and living cells, NRF-5 and TTR-52 may recognize apoptotic cells in different manners. We observed that PS, which is externalized to the outer leaflet of plasma membranes in apoptotic cells, is also detected on the surface of engulfing cells. Loss of NRF-5 function completely blocks PS appearance on engulfing cells, a phenotype observed in both ced-7(lf) and ttr-52(lf) mutants. Our data suggest that NRF-5 may function together with CED-7 and TTR-52 to mediate PS appearance on phagocytes, and thus promotes cell corpse engulfment.
Contact: [email protected] Lab: Wang
8 Platform Session #2 Morphogenesis II and Cell Death Globin 12 of Caenorhabditis elegans Regulates the p38 and JNK MAPK Pathways through Redox Signaling to Control Germline Apoptosis Sasha De Henau1, Lesley Tilleman2, Francesca Germani2, Caroline Vlaeminck1, Jacques Vanfleteren1, Luc Moens2, Sylvia Dewilde2, Bart Braeckman1 1Ghent University, Ghent, Belgium, 2University of Antwerp, Antwerp, Belgium Redox signaling is present in a wide range of cell biological processes, including cell proliferation, cell differentiation, cell migration, and apoptosis. This form of signaling is tightly controlled, compartmentalized and tissue-specific. Herein, we show that a globin ofC. elegans, globin-12 (GLB-12), functions through redox signaling and that it regulates multiple aspects of oogenesis, including germline apoptosis. The broad role of GLB-12 in oogenesis is demonstrated by the effects of glb-12 RNAi; it causes severely reduced fecundity, smaller gonads, increased levels of germline apoptosis, and several defects during oocyte development. A translational reporter shows that GLB-12 is membrane-bound and present in the distal gonadal sheath cells, the spermatheca-uterine valve and the uterus. By focusing on the increase in germline apoptosis, we found that GLB-12 signals through the JNK and p38 MAPK pathways; when one or both of these pathways is eliminated, the increase in germline apoptosis following glb-12 RNAi is no longer present. This finding is further supported by Western blot data, which shows that GLB-12 negatively regulates the p38 MAPK pathway. Furthermore, we demonstrate that the p38 MAPK pathway is specifically active in the distal part of the germline, overlapping the same region where GLB-12 is present. Our biochemical analysis of GLB-12 made clear that this globin functions in redox signaling. Unlike most other globins, GLB-12 cannot bind oxygen; instead, it will actively convert oxygen to superoxide by electron transfer. The relatively unstable superoxide can in vivo be converted into hydrogen peroxide by superoxide dismutases (SODs). This hydrogen peroxide can then act as a biological messenger in redox signaling. When we applied glb-12 RNAi in mutants for the five C. elegans sod genes, we found that fecundity is further reduced in the intracellular SOD-1 mutant and restored to almost normal levels in the extracellular SOD-4 mutant. This suggests that these two SODs modulate the redox signaling pathway that is used by GLB-12. Translational reporters for SOD-1 and SOD-4 confirm their presence in, or around, the gonadal sheath. Furthermore, we also show that GLB-12, SOD-1 and SOD-4 work together to regulate p38 MAPK activity and germline apoptosis levels. Based on our results, we present a model in which GLB-12 is part of a redox signaling pathway that is modulated by an intracellular and extracellular SOD. This pathway regulates p38 and JNK MAPK activity, and, consequently, germline apoptosis levels.
Contact: [email protected] Lab: Braeckman
Platform Session #2 Morphogenesis II and Cell Death 9 sli-1 Cbl Inhibits the Engulfment of Apoptotic Cells Courtney Anderson1, Shan Zhou1, Emma Sawin1, Bob Horvitz2, Michael Hurwitz1 1Yale University School of Medicine, New Haven, CT, USA, 2MIT, Cambridge, MA, USA The engulfment of apoptotic cells is required for normal metazoan development and tissue remodeling. In C. elegans, two parallel and partially redundant conserved pathways act in cell-corpse engulfment. One pathway includes the adaptor protein CED-2 CrkII and the small GTPase CED-10 Rac, and acts to rearrange the cytoskeleton of the engulfing cell. The other pathway includes the receptor tyrosine kinase CED-1 and might recruit membranes to extend the surface of the engulfing cell. Loss-of-function (lf) mutations in these genes cause the persistence of unengulfed cell corpses. Cbl, the mammalian homolog of the C. elegans signaling protein SLI-1, interacts with CrkL and Rac and modulates the actin cytoskeleton. Mutation of Cbl contributes to a wide range of human cancers. SLI-1 contains three domains, an N-terminal tyrosine kinase binding domain, a RING finger domain and a C-terminal proline-rich domain. SLI-1 inhibits LET-23 EGFR/LET- 60 Ras signaling by ubiquitinating LET-23 via its RING finger domain. We found that sli-1(lf) suppresses the engulfment defects of ced-1 pathway null mutants but not of ced-10 Rac pathway mutants, suggesting that sli-1 acts either upstream of the ced-10 Rac pathway, in parallel to both pathways or downstream of the ced-1 pathway. The ced-10 Rac pathway is also required for proper migration of the distal tip cells (DTCs) during the development of the C. elegans gonad. sli-1(lf) partially restores normal DTC migration in ced-10 Rac pathway null mutants. Thus, SLI-1 does not act by inhibiting the CED-10 Rac pathway and is unlikely to be downstream of the CED-1 pathway since the CED-1 pathway is not involved in DTC migration. Mutation of another inhibitor of engulfment, the tyrosine kinase gene abl-1, in combination with sli-1(lf) enhanced the effect on both engulfment and DTC migration, demonstrating that the two genes act independently of each other. Experiments using sli-1 transgene constructs lacking specific domains of sli-1 show that only constructs lacking the N-terminus fail to rescue the sli-1 DTC migration defect completely, suggesting that the role of SLI-1 in these processes requires the tyrosine kinase binding domain but is at least partially ubiquitin ligase-independent. Consistent with this finding, modulation of LET-60 Ras signaling had no effect on engulfment. We propose that SLI-1 opposes the engulfment of apoptotic cells via a pathway that is distinct from the two known engulfment pathways using a mechanism not yet identified for SLI-1 in worms.
Contact: [email protected] Lab: Hurwitz
10 Platform Session #2 Morphogenesis II and Cell Death MADD-2 Negatively Regulates Anchor Cell Invasion Matthias Morf1,2, Ivo Rimann1, Mariam Alexander3, Peter Roy3, Alex Hajnal1 1Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland, 2Molecular Life Sciences PhD program, Uni ETH Zurich, Switzerland, 3Department of Molecular Genetics, The Terrence Connelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Canada Cell invasion is a tightly regulated process, during which cells cross tissue borders. Uncontrolled invasion can lead to metastatic cancer growth. During C. elegans larval development, a specialized cell in the somatic gonad called anchor cell (AC) breaks two basal laminae and then invades the adjacent vulval tissue to form a connection between the developing vulva and uterus. Multiple signals from the vulval cells and the ventral nerve cord regulate AC invasion. How the AC can integrate these signals is largely unknown. We have identified MADD-2, a conserved RING finger and E3 ubiquitin ligase protein, as a regulator of AC invasion. madd-2 has independently been identified by both the P. Roy lab as a gene acting downstream of the UNC-40 Netrin receptor and controlling muscle arm extensions and by the Bargmann & Tessier-Lavigne groups that showed a similar role of madd-2 in axon branching. The human madd-2 homologue, Mid1, is mutated in most cases of Opitz syndrome, a disease characterized by ventral midline defects. Our analysis of basal laminae breaching in madd-2 mutants demonstrates that AC invasion is delayed, but to a lesser extend than in unc-6 mutants. Surprisingly, madd-2(lf) partially rescues the unc-6(lf) AC invasion defects, suggesting that during AC invasion madd-2 is epistatic to unc-6. Additional analyses of AC shape, polarity and dynamics demonstrate a loss of proper AC orientation and the formation of highly dynamic ectopic protrusions in madd-2(lf) mutants, indicating a loss of directed invasion. We thus tested whether madd-2(lf) could allow AC invasion in the absence of any guidance cues. To eliminate all guidance cues for the AC, we ablated the VPCs in madd-2(lf); unc-6(lf) double mutants and checked for signs of basal laminae breaching. While the AC detached from the basal laminae that remained intact in VPC ablated unc-6(lf) single mutants, we observed AC attachment and initial signs of basal lamina breaching but no invasion in VPC ablated madd-2(lf); unc-6(lf) double mutants. We propose that madd-2 prevents undirected AC invasion, thus allowing the AC to respond to guidance cues from the ventral nerve cord and the vulval cells. In summary, madd-2 is the first negative regulator of AC invasion identified.
Contact: [email protected] Lab: Hajnal
Platform Session #2 Morphogenesis II and Cell Death 11 The C. elegans Hailey-Hailey Disease Homolog pmr-1 is Essential for Cell Migration During Gastrulation Vida Praitis1, Rebecca Mandt1, Leah Imlay1, Charlotte Feddersen1, Alexander Sullivan-Wilson1, Tyson Stock1, Walter Liszewski1, Adityarup Chakravorty1, Dae Gon Ha1, Angela Schacht1, Michael Miller1, Lensa Yohannes1, Juliet Mushi1, Zelealem Yilma1, Sarah Kniss2, Jeff Simske3 1Grinnell College, Grinnell, IA, USA, 2U. of Chicago, Chicago, IL USA, 3Rammelkamp Ctr, Cleveland, OH, USA Hailey-Hailey disease or benign familial pemphigus (MIM# 169600) is a semi-dominant human disease marked by severe skin lesions and blistering, thought to be the result of altered cell adhesion in keratinocytes. The disease is caused by mutations in ATP2C1/ SPCA1, a Ca2+/Mn2+ ATPase that localizes to the golgi where it acts in protein processing, metal homeostasis, and Ca2+ signaling. Our laboratory has been characterizing the role of PMR-1, the C. elegans homolog of ATP2C1. We first identified alleles of pmr-1 in two genetic screens designed to identify conditional alleles of genes required during morphogenesis. Subsequent mapping, complementation, and sequencing analysis confirmed that the identified strains carried mutations in pmr-1. All four alleles of pmr-1 are temperature-sensitive, showing complete embryonic lethality at 25C, but with increased viability at lower temperatures. Embryos homozygous for pmr-1 loss-of-function alleles die with variable terminal phenotypes, including enclosure failures, head ruptures, body morphogenesis defects and pharynx unattached phenotypes, defects that look superficially similar to the human cell adhesion phenotypes. Analysis using GFP expression constructs, as well as antisera staining experiments with a variety of cell fate markers, indicate cell fates are normal in Pmr-1 embryos. Similarly, cell lineaging analysis using StarryNite and Acetree software indicates cell division timing is normal. However, using these same tools, and taking advantage of temperature-shift analysis, we were able to identify specific cells that become mis-positioned in Pmr-1 embryos during mid- gastrulation. Our analysis indicates that while ingression is normal, cells that migrate along the surface of the embryo, including ventral neuroblasts, C-derived blastomeres, and anterior cells that give rise to hypodermis and ring ganglia, exhibit significantly reduced rates of cell migration compared to controls. It is these failed migrations that cause the later enclosure and morphogenesis defects. To better understand the molecular pathways responsible for the cell migration defects we have begun gene interaction experiments. Our analysis indicates that the embryonic lethality caused by pmr-1 loss-of-function alleles can be partially suppressed by a gain-of-function allele of the inositol-sensitive Ca++ channel gene itr-1. The most parsimonious explanation for these data is that pmr-1 and itr-1 act in the same Ca++ signaling pathway during cell migration.
Contact: [email protected] Lab: Praitis
12 Platform Session #2 Morphogenesis II and Cell Death Control of Oocyte Meiotic Maturation: Links to Germ Cell Proliferation and Global Control of the Oogenic Program David Greenstein University of Minnesota, Minneapolis, MN, USA In sexually reproducing animals, oocytes arrest at diplotene or diakinesis and resume meiosis (meiotic maturation) in response to hormones. Chromosome segregation errors in female meiosis I are the leading cause of human birth defects; age-related changes in the hormonal environment of the ovary are a suggested cause. C. elegans serves as an incisive genetic model for studying the control of oocyte meiotic maturation by hormonal signaling and soma-germline interactions. The meiotic maturation processes in C. elegans and mammals share a number of molecular and biological similarities. Major sperm protein (MSP) and luteinizing hormone (LH), though unrelated in sequence, both trigger meiotic resumption using somatic Gαs-adenylate cyclase-PKA pathways and soma-to-germline gap-junctional communication. At a molecular level, the oocyte responses apparently involve the control of conserved protein kinase pathways and post-transcriptional gene regulation in the oocyte. At a cellular level, the responses include cortical cytoskeletal rearrangement, nuclear envelope breakdown, assembly of the acentriolar meiotic spindle, chromosome segregation, and likely changes important for fertilization and the oocyte-to-embryo transition. In my talk I will discuss our efforts to define oocyte meiotic maturation control mechanisms from signal reception to oocyte response. I will focus on recent results that establish links between the control of meiotic maturation and mechanisms needed for germ cell proliferation and the global control of the oogenic program.
Contact: [email protected] Lab: Greenstein
Keynote 3 13 Regulators of MSP Assembly and Dynamics in C. elegans Spermatocytes Kari Messina, Marc Presler, Leah Towarnicky, Diane Shakes College of William and Mary, Williamsburg, VA, USA In crawling spermatozoa, the major sperm protein (MSP) plays two key roles: as a cytoskeletal protein, its polymerization/depolymerization dynamics drive pseudopod motility, and as an extracellular signaling molecule, it triggers both oocyte maturation and ovulation. However during spermatogenesis, all transcription and most translation of this highly abundant protein (40% of the soluble protein in haploid sperm) occurs prior to the meiotic divisions. Thus in developing spermatocytes, MSP is sequestered into transient organelles called fibrous bodies (FBs) which both prevent MSP from interfering with chromosome segregation and cytokinesis and subsequently facilitate its segregation away from residual bodies and into spermatids. While biochemical studies have illuminated key details regarding the polymerization of MSP within pseudopods, the only factor previously known to be essential for the initial assembly of MSP into FBs was SPE-6, a spermatogenesis-specific member of the casein kinase I superfamily (Varkey et al., 1993; Muhlrad and Ward, 2002). Our laboratory has now identified SPE-7 as a key player in FB assembly. spe-7(mn252) spermatocytes fail to assemble FBs and exhibit subsequent cell cycle and cytokinesis defects. An anti-SPE-7 antibody reveals that wildtype SPE-7 dynamically localizes to FBs, segregates to budding spermatids, and then becomes undetectable in mature, haploid spermatids. We are currently analyzing SPE-7 dynamics in a variety of spermatogenesis-defective (Spe) mutants to understand its role in FB assembly and disassembly. Intriguingly, SPE-7 assembles into FB-like structures in the absence of spe-6, suggesting a model in which SPE-7 functions to nucleate FB assembly with kinase activity by SPE-6 required for MSP addition. On the other hand, the disappearance of SPE-7 does not coincide with FB disassembly; in gsp-3/4 mutant spermatids, MSP remains persistently locked in FB-like structures yet the timely disappearance of SPE-7 remains unaffected. Notably, SPE-7 is stabilized in mutants in which individual spermatids fail to separate from the central residual body, suggesting a link to the segregation and separation events of the budding division and perhaps to the subcellular relocalization of SPE-6, which also occurs at this time.
Contact: [email protected] Lab: Shakes
14 Platform Session #3 - Germline I and Gametogenesis The sperm surface localization of the TRP-3/SPE-41 Ca2+ permeable channel depends on SPE-38 function in Caenorhabditis elegans Gunasekaran Singaravelu1, Indrani Chatterjee1, Sina Rahimi1, Marina Druzhinina1, Lijun Kang2, Shawn Xu2, Andrew Singson1 1Waksman Institute, Piscataway, NJ, USA, 2University of Michigan, Ann Arbor, MI, USA Despite undergoing normal development and acquiring normal morphology and motility, mutations in spe-38 or trp-3/spe-41 cause identical phenotypes in Caenorhabditis elegans – mutant sperm fail to fertilize oocytesdespite direct contact. SPE-38 is a novel, four-pass transmembrane protein and TRP-3/SPE-41 is a Ca2+ permeable channel. Localization of both of these proteins is confined to the membranous organelles (MOs) in undifferentiated spermatids. In mature spermatozoa, SPE-38 is localized to the pseudopod and TRP-3/SPE-41 is localized to the whole plasma membrane. Here we show that the dynamic redistribution of TRP-3/SPE-41 from MOs to the plasma membrane is dependent on SPE-38. In spe-38 mutant spermatozoa, TRP-3/SPE-41 is trapped within the MOs and fails to reach cell surface despite MO fusion with the plasma membrane. Split-ubiquitin yeast-two-hybrid analyses revealed that the cell surface localization of TRP-3/SPE-41 is likely regulated by SPE-38 through a direct protein-protein interaction mechanism. We have identified sequences that influence the physical interaction between SPE-38 and TRP-3/SPE-41, and show that these sequences in SPE-38 are required for fertility in transgenic animals. We identified additional proteins that can bind with either SPE-38 or TRP-3/SPE-41 and a novel protein that can bind with both SPE-38 and TRP-3. Despite the mislocalization of TRP-3/SPE-41 in spe-38 mutant spermatozoa, ionomycin or thapsigargin induced influx of Ca2+ remains unperturbed. This work reveals a new paradigm for the regulated surface localization of a Ca2+channel.
Contact: [email protected] Lab: Singson
Platform Session #3 - Germline I and Gametogenesis 15 Timely Generation of the Fertilization Calcium Wave by a Sperm TRP Channel Jun Takayama, Shuichi Onami RIKEN Quantitative Biology Center, Kobe, Japan Fertilization calcium wave is a universal trigger for the egg activation that converts the egg to the embryo. Although egg activation occurs in a timely coordinated fashion among other processes, the mechanism that ensures the on-time generation of the calcium wave is unknown. Here we show that the timely generation of the calcium wave is controlled by TRP- 3, a sperm-specific calcium-permeable channel in C. elegans. First, we found that fertilization generates a biphasic calcium wave in the oocyte: the fast local wave and the slow global wave. The fast local wave emerged and disappeared near the sperm entry point, whereas the slow global wave traveled from that point to the opposite pole. The calcium response started at the moment of sperm entry, which was visualized either by a sudden bulge of the oocyte cytoplasm or by a dark “patch” appeared in the oocyte membrane labeled by GFP-PH. Next, we examined the calcium response of mutants defective in sperm or oocyte function. spe-9 mutants, whose sperm cannot enter into the oocyte, showed no calcium response. Egg activation factor mutant spe-11, whose sperm cannot activate the egg but can enter into the oocyte, showed a wildtype-like biphasic wave. Although most of the mutants of trp-3 cannot enter into the oocyte, we found that trp-3(sy693) escapers that entered into the oocyte showed no local wave but a wildtype-like global wave with delayed onset. Local calcium elevation could trigger the simultaneous generation of the global wave in computer simulation that assumes that the oocyte has calcium-induced calcium release (CICR) machinery. Approximately half of the embryos fertilized by trp-3 escapers did not hatch. On the other hand, egg factor mutant egg-3(tm1191), whose oocyte fails to become activated, showed a reduced amplitude in the global wave. These results suggest that (i) triggering factor(s) for the calcium wave and SPE-11 are independently transmitted from sperm and (ii) TRP-3 is required both for the generation of the local wave and the timely generation of the global wave. The global wave may be generated either spontaneously with delayed onset or in response to the local calcium elevation, as is the case with IP3 receptors that are stimulated by IP3. Therefore the local calcium wave mediated by sperm TRP-3 may guarantee the on-time generation of the calcium wave probably to coordinate with other processes during the oocyte-to-embryo transition.
Contact: [email protected] Lab: Onami
16 Platform Session #3 - Germline I and Gametogenesis Regulation of Meiotic DSB Formation in C. elegans Simona Rosu, Anne Villeneuve Stanford University, Stanford, CA Crossovers (COs) between the DNA molecules of homologous chromosomes provide the basis of physical links (chiasmata) that ensure proper segregation of homologs at the meiosis I division. Crossover recombination is initiated by formation of double strand DNA breaks (DSBs) by the SPO-11 protein. We identified a new gene (F26H11.6, defined by theme96 mutation) involved in promoting DSB formation during C. elegans meiosis. me96 mutant worms exhibit a defect in chiasma formation that becomes progressively more severe with age. Immunofluorescence experiments suggest the defect is a result of reduced DSBs; furthermore, providing exogenous breaks by irradiation rescues chiasma formation. The F26H11.6 protein localizes to chromatin in transition zone and early pachytene nuclei, corresponding to the presumed timing of DSB formation during meiotic prophase. This localization suggests that F26H11.6 may help create a chromatin environment that is competent for SPO-11 dependent DSB formation. F26H11.6 localization is dependent on CHK-2, a kinase required for early meiotic events including pairing, chromosome mobilization, and DSB formation. Furthermore, F26H11.6 localization is prolonged in mutants lacking synaptonemal complex (SYP) proteins (which exhibit persistent chromosome mobilization and elevated levels of DSB-dependent recombination intermediates) and in some mutants impaired in downstream steps of CO formation. These and other data suggest that F26H11.6 localization serves as a marker for DSB competence, providing a visual readout for the operation of feedback mechanisms that coordinate competence for DSB formation with the organizational state of the chromosomes and that couple cessation of DSB formation with the formation of CO-competent recombination intermediates.
Contact: [email protected] Lab: Villeneuve
Platform Session #3 - Germline I and Gametogenesis 17 Title: TBD Karen Oegema University of California - San Diego
Contact: [email protected] Lab: Oegema
18 Keynote 4 Evolution of spindle shape and motion in one-cell stage nematode embryos Aurore-Cecile Valfort1, Soizic Riche1, Reza Farhadifar2, Daniel Needleman2, Marie Delattre1 1LBMC, CNRS-University of Lyon1, Lyon, France, 2Harvard University, SEAS, MCB, Cambridge, MA, USA Although genomic, molecular, development and phenotypic evolution have been largely explored; there is little understanding of how cell biological processes evolve. Due to their fundamental role at the basis of developmental processes, cellular functions are highly constrained and are often thought to rely on very conserved underlying mechanisms. However, constraints on the final phenotype could hide a diversity of underlying mechanisms that remain cryptic, yet contributing to the flexibility of cellular processes. Most nematode embryos undergo a first asymmetric division that gives rise to a small posterior cell containing germ cell fate. We used this unique feature to ask 1) whether this strong phenotypic conservation reflects different underlying mechanisms of asymmetric spindle positioning, 2) which steps are the most constrained, 3) which steps can vary and allow the diversification of solutions retained in the course of evolution. To address these questions we recorded the first embryonic division of ~60 different nematode species (3 different strains per species, 15 embryos per strain) by DIC microscopy and found a large diversity of intermediate phenotypes such as nuclei centration, spindle position, centrosome size, spindle motion, spindle elongation, etc. We developed an automated DIC image analysis, enabling us to quantity the differences in thousands of embryos from hundreds of strains. Importantly, differences are also found on a microevolutionnary time scale, between species of the Caenorahbditis genus. Parameter measurement is underway and will be mapped on the well-known phylogeny to deduct the evolutionary trend of spindle shape and motion changes. In a subset of interesting species, in depth analysis will be done to identify parameters changes. We will present our detailed comparison between C. elegans and C. briggsae embryos. Through a combination of molecular and biophysical approaches we uncovered a new conserved mechanism in the control of anaphase spindle oscillations and identified interspecific changes in the control of the G-protein regulator GPR.
Contact: [email protected] Lab: Delattre
Platform Session #4 - Cell Cycle and Cell Biology 19 The Tousled-like Kinase TLK-1 is a Component of the Outer Kinetochore and Potentiates Mitotic Spindle Dynamics in the Early C. elegans Embryo Jessica De Orbeta, Jason Ford, Gary Deyter, Tokiko Furuta, Jill Schumacher University of Texas MD Anderson Cancer Center Tousled-like kinases are highly conserved and have been implicated in chromatin remodeling, transcription, and DNA replication and repair. We discovered that C. elegans TLK-1 also has an essential role in mitosis as a substrate and activator of the Aurora B kinase AIR-2. AIR-2 phosphorylates TLK-1 at S634 and while TLK-1 is highly expressed in interphase nuclei, a phospho-specific antibody revealed that pTLK-1(S634) is localized to kinetochores, as is a GFP-TLK-1 transgenic protein. To discern the functional role of TLK-1 at kinetochores, we interrogated the placement of TLK-1 in the kinetochore assembly hierarchy. This analysis revealed that TLK-1 is localized to the outer kinetochore downstream of BUB-1, and is independent of the NDC-80 or RZZ kinetochore complexes. Examination of BUB-1- dependent kinetochore proteins revealed that TLK-1 kinetochore localization is disrupted in hcp-1/cenp-f(RNAi) embryos but is not affected by loss of the highly related HCP-2 protein, or the CENP-F-dependent proteins LIS-1 and CLS-2/CLASP. Instead, our results revealed that TLK-1 is required for the kinetochore localization of both LIS-1 and CLS-2. While neither CLS-2 nor TLK-1-depletion results in severe mitotic defects in early embryos, embryos co-depleted for TLK-1 and CLS-2 display gross errors in chromosome congression and segregation, suggesting that these proteins are collaborating to enable functional kinetochore-microtubule attachments. We recently identified a second role for TLK-1 in mitotic spindle dynamics in the early C. elegans embryo. Live imaging of GFP-labeled embryos treated with control or tlk-1(RNAi) revealed that spindle rotation is significantly delayed in TLK-1-depleted one-cell embryos. While nuclear envelope breakdown (NEB), and chromosome condensation and congression occurred with similar kinetics, the nuclear/centrosome complex did not rotate until after NEB and congression in tlk-1(RNAi) embryos, resulting in metaphase spindles that were perpendicular to the anterior-posterior axis. Despite this delay, the spindles of the TLK- 1-deficient embryos completed rotation by mid-anaphase, essentially undergoing a very fast, albeit late rotation. Additional live imaging revealed that significantly fewer microtubules reach the cell cortex in one-cell tlk-1(RNAi) embryos, suggesting that TLK-1 potentiates microtubule dynamics in the early C. elegans embryo.
Contact: [email protected] Lab: Schumacher
20 Platform Session #4 - Cell Cycle and Cell Biology Identification of unconventional components of the γ-tubulin complex in C. elegans Nami Haruta1, Eisuke Sumiyoshi1, Yu Honda1, Masahiro Terasawa2, Mika Toya2, Asako Sugimoto1,2 1Graduate School of Life Sciences, Tohoku University, Sendai, Japan, 2Laboratory for Developmental Genomics, RIKEN Center for Developmental Biology,Kobe, Japan The γ-tubulin complexes (γTuC) play an important role in microtubule nucleation. Many organisms have two types of γTuCs,the γ-tubulin small complex (γTuSC) and the γ-tubulin ring complex (γTuRC).The γTuSC consists of γ-tubulin and two other components (GCP2and GCP3 in mammals), and γTuSCs and several additional components(such as GCP4~6) form γTuRCs that have higher polymerization activity than γTuSCs. In C.elegans, while the γTuSC components are known (γ-tubulin/TBG-1,GCP2/GIP-1, GCP3/GIP-2), γTuRC-specific components have not been identified in the genome. Here, we report the identification of two candidate proteins for γTuC components. These two proteins, tentatively named GTAP-1 and GTAP-2, were co-immunoprecipitated with FLAG-tagged γ-tubulin from C. elegans embryonic extracts. Although both proteins did not have detectable homologies with γTuRC-specific components of other organisms, yeast two-hybrid analysis showed the physical interaction between GTAP-2 and one of the γTuSC components, GIP-2, and sucrose gradient centrifugation showed that both GTAP-1 and-2 were cofractionated with γ-tubulin. Live imaging of GFP-tagged GTAP-1 and -2 in C. elegans embryo revealed that both proteins colocalized with γ-tubulin at centrosomes throughout cell cycle, and this centrosomal localization is dependent on γ-tubulin. On the other hand, RNAi depletion of GTAP-1 and/or -2 caused ~70% decrease of γ-tubulin at centrosomes, but surprisingly the amount of microtubules was unaffected and the RNAi-treated embryos were viable. Under the condition in which the amount of centrosomal γ-tubulin was reduced, depletion of GTAP-1/2 decreased centrosomal microtubules. Taken together, we propose that GTAP-1 and GTAP-2 are novel components of the γTuC, and that they contribute to the recruitment of γ-tubulin to centrosomes. Additionally, our results imply that in normal conditions only a minor fraction of the γTuC at centrosomes is used for microtubule nucleation.
Contact: [email protected] Lab: Sugimoto
Platform Session #4 - Cell Cycle and Cell Biology 21 The Cdc48/p97 cofactor UBXN-2 and its orthologues p47/p37 control centrosome maturation in prophase via Aurora A Elsa Kress1, Francoise Schwager1, Rene Holtackers2, Esther Zanin3, Francois Prodon1, Jonas Seiler4, Annika Eiteneuer4, Asako Sugimoto5, Hemmo Meyer4, Patrick Meraldi2, Monica Gotta1 1Faculty of Medicine, University of Geneva, Geneva, Switzerland, 2Eidgenossische Technische Hochschule (ETH), Zurich, Switzerland, 3Ludwig Institute for Cancer Research, University of California, San Diego, USA, 4Faculty of Biology, Universitat Duisburg-Essen, Essen, Germany, 5Graduate School of Life Sciences, Tohoku University, Sendai, Japan. Tight temporal regulation of mitotic events and the spatial coordination of the cleavage plane with the axis of chromosome segregation are essential prerequisites for a successful cell division. During asymmetric cell divisions, the axis of chromosome segregation must also be aligned with the axis of polarity to allow proper segregation of cell fate determinants. This is achieved by orienting the mitotic spindle along the axis of polarity. Here we show that premature centrosome maturation results in defects in spindle orientation and aberrant asymmetric cell division. We find that depletion of UBXN-2, a substrate adapter of the AAA ATPase cdc48/ p97, prevents alignment of the mitotic spindle with the polarity axis in C.elegans. Our data indicate that UBXN-2 limits Aurora A accumulation at centrosomes during early prophase. In ubxn-2(RNAi) embryos, Aurora A is recruited earlier than in wild type, centrosomes display enhanced accumulation of microtubule dynamics regulators and an increased growth rate of astral microtubules. Furthermore UBXN-2 and AIR-1 co-immunoprecipitate from embryonic extracts suggesting a close association of these molecules. The spindle defects observed in ubxn-2(RNAi) embryos are partially rescued by co-depletion of AIR-1. Therefore, we postulate that UBXN-2 regulates AIR-1 levels at centrosomes to couple centrosome maturation timing with mitotic progression. This pathway is conserved as we further find in human cells, that the two orthologues of UBXN-2, p37 and p47 regulate the timing of centrosome separation in prophase, by limiting the centrosomal recruitment of Aurora A.
Contact: [email protected] Lab: Gotta
22 Platform Session #4 - Cell Cycle and Cell Biology Regulation of COPII subunit recruitment to ER exit sites Kristen Witte1, Amber Schuh1, Jan Hegermann2, Ali Sarkeshik3, Jonathan Mayers1, Katrin Schwarze2, John Yates III3, Stefan Eimer2, Anjon Audhya1 1University of Wisconsin-Madison, Madison, WI, USA, 2European Neuroscience Institute and Center for Molecular Physiology of the Brain (CMPB), 37077 Goettingen, Germany, 3The Scripps Research Institute, La Jolla, California 92037, USA Export of proteins from the endoplasmic reticulum (ER) in COPII-coated vesicles occurs at defined sites, which contain the scaffolding protein SEC-16. Although SEC-16 has been shown to interact with multiple COPII subunits to mediate vesicle biogenesis at the ER, mechanisms by which COPII recruitment is regulated remain poorly defined. Using both functional genomics and biochemical approaches, we identify a new, conserved SEC-16-interacting protein named TFG-1 that is required for COPII subunit accumulation at ER exit sites. Consistent with this finding, depletion of TFG-1 inhibits secretion of multiple cargoes from the ER. Furthermore, using immuno-gold EM techniques, we demonstrate that TFG-1 localizes to a matrix between ER exit sites and the Golgi. We hypothesize that a TFG-1 matrix may serve as a molecular sink, which helps to retain COPII components locally and facilitate efficient vesicle egress from the ER.
Contact: [email protected] Lab: Audhya
Platform Session #4 - Cell Cycle and Cell Biology 23 Condensin I: A New Component of the Abscission Checkpoint Joshua Bembenek, Koen Verbrugghe, Gyorgyi Csankovszki, Raymond Chan University of Michigan, Ann Arbor, MI, USA During cell division, chromosomes must clear the path of the cleavage furrow before the onset of cytokinesis. If chromatin obstructs the furrow, it can be damaged and furrow regression can occur causing mutations and aneuploidy, defects common in cancer. The abscission checkpoint stabilizes the cleavage furrow in the presence of chromatin obstructions, thus preventing the cleavage furrow from regressing and allowing more time for resolving chromatin obstructions (Steigemann et al., Cell, 2009). Whether cells with chromatin obstructions in a developing embryo can suppress cytokinesis failure has not been determined. This is especially important because conventional checkpoints that minimize chromosome segregation defects are sometimes attenuated during early embryonic divisions. To investigate this, we inactivated several genes essential for segregation in C. elegans embryos. We found that the P0 blastomere robustly suppresses furrow regression following depletion of condensin, cohesin, HCP-3 (CENP-A) and TOP-2 (topoisomerase II). The robustness of this suppression is reduced in AB and P1 blastomeres and when condensin is depleted. This response correlates with activation of AIR-2 (Aurora B) at the spindle midzone, which is needed for the abscission checkpoint in other systems. Condensin I, but not condensin II, localizes to the spindle midzone in anaphase and to the midbody during cytokinesis. Interestingly, condensin I shows prominent accumulation in the spindle midzone and midbody region in the presence of chromatin bridges, in a SPD-1 and AIR-2 dependent manner. We postulate that condensin I is required for either sensing or responding to chromatin obstructions to prevent cleavage furrow regression.
Contact: [email protected] Lab: Chan
24 Platform Session #4 - Cell Cycle and Cell Biology LEM-4 Coordinates Mitotic Signaling on BAF to Enable its Essential Function in Nuclear Envelope Formation Matyas Gorjanacz1, Claudio Asencio1, Iain Davidson1, Rachel Santarella- Mellwig1, Geraldine Seydoux 2, Iain Mattaj1 1European Molecular Biology Laboratory, Heidelberg, Germany, 2Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, USA The nucleus is the most complex eukaryotic organelle. Its structure is defined by the nuclear envelope (NE); composed of the NE membranes, nuclear pore complexes and in metazoa the nuclear lamina. In higher eukaryotes NE disassembles and reassembles during every cell division in order to allow faithful segregation of condensed sister chromatids. These mitotic events are driven by spatiotemporally controlled reversible phosphorylation of key molecules. While multiple phosphorylation events have been already described to drive NE disassembly, it is almost entirely unknown how dephosphorylation is regulated to enable its reassembly. By screening Caenorhabditis elegans strains harboring temperature sensitive embryonic lethal mutations we have identifiedlem-4 as a mitotic regulator that is required for the function of both a mitotic kinase and a phosphatase that act on BAF, an essential factor of nuclear assembly. We found that during mitotic exit LEM-4 is required for dephosphorylation of BAF by simultaneously inhibiting BAF’s mitotic kinase, VRK-1, and by stimulating a protein phosphatase 2A (PP2A) complex that can dephosphorylate BAF. By coordinating VRK-1 and PP2A mediated signaling on BAF, LEM-4 enables postmitotic NE reformation in a function that is conserved from worm to humans.
Contact: [email protected] Lab: Mattaj
Platform Session #4 - Cell Cycle and Cell Biology 25 Filamin is Required to Initiate Calcium Signaling and Maintain F-actin Organization in the Spermatheca Ismar Kovacevic, Erin Cram Northeastern University, Boston, MA, USA Mechanosensation at the cellular and tissue levels is critical for normal development and organ function. We are using the C. elegans spermatheca as a model system to study how cells sense mechanical forces in vivo. The spermatheca is a simple myoepithelial tube that naturally experiences cycles of stretching and constriction. Ovulated oocytes stretch the spermatheca, and trigger directional constriction to propel embryos into the uterus. We identified the C. elegans filamin ortholog FLN-1 as being required for normal spermathecal transit. Filamin is a stretch-sensitive structural and signaling scaffold that binds F-actin, transmembrane receptors, and a variety of intracellular signaling proteins. FLN-1 is expressed in spermathecal and uterine cells, colocalizes with F-actin, and is required to maintain the actin cytoskeleton in the spermatheca and uterus. Filamin-deficient animals accumulate embryos in the spermatheca, and consequently lay damaged eggs and exhibit reduced brood sizes. PLC-1/phospholipase C-ε is also required for the exit of embryos from the spermatheca, and analysis of double mutant animals suggests that PLC-1 and FLN-1 act in the same pathway. Because PLC-1 is thought to be upstream of intracellular calcium release, we used GCaMP—a genetically encoded calcium indicator—to image calcium during ovulation and spermathecal transit. Entry of an oocyte into the spermatheca initiates a distinctive series of calcium oscillations. The calcium transients originate at the distal end of the spermatheca and travel towards the uterus, in the same direction as ovulated oocytes. This suggests the calcium waves are controlling the directional constriction of the spermatheca. Loss of FLN-1 results in delayed onset of calcium signaling, followed by abnormal calcium oscillations. As expected, loss of PLC-1 entirely eliminates calcium signaling in the spermatheca. Gain-of-function mutations in the ITR-1/IP3 receptor enhance calcium release in the spermatheca, and partially rescue the brood size defect of filamin-deficient animals. We hypothesize that filamin is required in the spermatheca to maintain the cytoskeleton, respond to increased tension, and initiate calcium oscillations via the phosphatidylinositol pathway. Current work is focused on understanding the dual roles of filamin as a signaling and structural scaffold, as well as uncovering other components of the pathway.
Contact: [email protected] Lab: Cram
26 Platform Session #4 - Cell Cycle and Cell Biology Germline maintenance and meiosis: mechanistic insights from C. elegans Monica Colaiácovo Harvard Medical School
Contact: [email protected] Lab: Colaiácovo
Keynote 5 27 Identification of Direct GLP-1/Notch Targets that Regulate Germline Stem Cells Aaron Kershner1, Heaji Shin1, Judith Kimble1,2 1University of Wisconsin-Madison, Madison, WI, USA, 2Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, WI, USA GLP-1/Notch signaling maintains germline stem cells (GSCs): its loss drives germ cells from mitosis to meiosis at any stage of development and in either sex. Downstream of GLP- 1/Notch, two PUF proteins, FBF-1 and FBF-2 (collectively FBF), are also required for GSC maintenance, but only in L4s and adults. Thus, GLP-1/Notch must act through other factors in addition to FBF. We reasoned that such other factors might be targets of both GLP-1/Notch and FBF, and therefore investigated 14 genes common to lists of likely GLP-1/Notch (1) and FBF targets (2). Interestingly, simultaneous loss of two of these genes had the glp-1 null phenotype: single lst-1 (lateral signaling target) or sygl-1 (synthetic Glp) mutants had virtually normal germlines, but lst-1 sygl-1 double mutants made only 4-8 germ cells that differentiated into sperm (also see Shin et al abstract). Moreover, lst-1 and sygl-1 were also required for GSC maintenance in males and adults. Thus, lst-1 and sygl-1 are redundantly required for GSC maintenance throughout development, and their deletion mimics glp-1 loss. LST-1 protein harbors one Nanos-like zinc finger motif, suggesting a role in RNA regulation, but SYGL-1 has no recognizable motifs. To gain clues to function, we placed lst-1 sygl-1 in the genetic pathway of GSC control and examined their expression. Epistasis placed lst-1 and sygl-1 downstream or parallel to GLP-1/Notch and upstream of the GLD meiotic entry regulators. In situs revealed abundant lst-1 and sygl-1 mRNAs in the distal germline where GSCs reside, but not more proximally until oogenesis. Consistent with the idea that they are GLP-1/Notch targets, lst-1 and sygl-1 mRNA distal expression depended on active GLP-1/Notch signaling. Moreover, removal of LAG-1 binding sites from the sygl-1 promoter abolished distal but not proximal expression. Therefore, lst-1 and sygl-1 are likely bona fide targets ofGLP-1/Notch signaling in the distal germline. Epitope-tagged LST-1 protein localized to the cytoplasm. Based on its cytoplasmic location and its putative zinc finger, we suggest that LST-1, and possibly SYGL-1, have a key role in RNA regulation. Most importantly, this work forges an essential link between GLP-1/ Notch signaling and its direct targets in the GSC self-renewal pathway.
(1) Yoo and Greenwald (2004), Science 303:637-8; (2) Kershner and Kimble (2010), PNAS 107:3936-41.
Contact: [email protected] Lab: Kimble
28 Platform Session #5 - Germline II, Meiosis and Sex Determination/Dimorphism Genome-wide Analysis of GLD-1 Mediated mRNA Regulation Uncovers a Role in mRNA Storage Claudia Scheckel, Dimos Gaidatzis, Jane Wright, Rafal Ciosk Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Translational repression is often accompanied by mRNA degradation. In contrast, many mRNAs in germ cells and neurons are ‘stored’ in the cytoplasm in a repressed but stable form. Unlike repression, the stabilization of these mRNAs is little understood. A key player in C. elegans germ cell development is the STAR domain protein GLD-1. By genome-wide analysis of mRNA regulation in the germ line, we observed that GLD-1 has a widespread role in repressing translation but, importantly, also in stabilizing a sub-population of its mRNA targets. Additionally, these mRNAs appear to be stabilized by the DDX6-like RNA helicase CGH-1, which is a conserved component of germ granules and processing bodies. Because many GLD-1 and CGH-1 stabilized mRNAs encode factors important for the oocyte-to-embryo transition (OET), our findings suggest that the regulation by GLD-1 and CGH-1 serves two purposes. Firstly, GLD-1 dependent repression prevents precocious translation of OET-promoting mRNAs. Secondly, GLD-1 and CGH-1 dependent stabilization ensures that these mRNAs are sufficiently abundant for robust translation when activated during OET. In the absence of this protective mechanism, the accumulation of OET-promoting mRNAs, and consequently the oocyte-to- embryo transition, might be compromised.
Contact: [email protected] Lab: Ciosk
Platform Session #5 - Germline II, Meiosis and Sex Determination/Dimorphism 29 In the C. elegans Germ Line, S6K promotes Cell Cycle Progression and the Proliferative Fate and mediates the Effects of Diet Dorota Korta1, Debasmita Roy1, Simon Tuck2, E. Jane Albert Hubbard1 1New York University, New York (NY), USA, 2Umea University, Umea, Sweden Proper coordination of cell cycle progression with the balance between proliferative and differentiated fates is crucial for normal development in all organisms. In addition, an organism’s nutritional environment can also influence development. However, mechanisms underlying this coordination remain poorly understood. We use C. elegans germline development as a model to understand the molecular basis for this coordination. Our recent results implicate several highly conserved signaling pathways in this regulation, including the Insulin (Michaelson et al., 2010), Target of Rapamycin (TOR) (Korta et al. 2012), and TGFβ (Dalfó et al., 2012) pathways, together with the previously known role of the Notch pathway (see Hansen and Schedl, 2006; Kimble and Crittenden, 2007 for reviews). Recently, we found that rsks-1/ p70S6-Kinase (S6K), a direct substrate of the TOR/RAPTOR complex, is required for the proper accumulation of proliferative germ cells (“progenitors”) during larval development. This accumulation is important for establishing an optimal adult germline progenitor pool. This role for rsks-1 is germline-autonomous and requires a conserved TOR phosphorylation site. We found a similar but more severe defect upon germline-depletion of let-363/TOR or daf-15/RAPTOR. In other organisms, the TOR/RAPTOR complex also positively regulates eIF4E. Consistent with a conserved pathway in worms, we found that ife-1/eIF4E is also required for optimal expansion of the larval progenitor pool and that loss of both rsks-1 and ife-1 together reduces the germline progenitor pool more severely than either single mutant, similar to the depletion of TOR. Further, we found that rsks-1/S6K both promotes overall cell cycle progression and inhibits larval germline progenitor differentiation, and its germline role is genetically distinct from its influence on lifespan. Finally, we showed that rsks-1 activity is an important mediator of the effects of diet, especially amino acids, on the expansion of the larval germline progenitor pool . Our current work explores differences and similarities between the roles of TOR/ RAPTOR, ife-1 and rsks-1, and the mechanisms by which these pathways regulate cell cycle, differentiation and response to diet.
Contact: [email protected] Lab: Hubbard
30 Platform Session #5 - Germline II, Meiosis and Sex Determination/Dimorphism Chromosome and centrosome inheritance in meiosis Mara Schvarzstein, Anne Villeneuve Stanford University Successful embryonic development requires that at fertilization each gamete provide complementary components to the zygote. In C. elegans, the sperm contribute both a haploid genome and a pair of engaged centrioles. Correct centriole organization during male meiosis is critical to ensure normal bipolar mitotic spindle in the zygote. We identify a new role for meiosis specific HORMA domain proteins in regulating centriole dynamics during spermatocyte meiosis. During male meiosis centrioles normally undergo two rounds of duplication, resulting in haploid sperm each containing a single tightly engaged centriole pair. In horma mutants, we observe inappropriate separation of centrioles during meiosis II, resulting in separated centrioles in sperm. Further, an extra pair of centrosomes is detected in a subset of zygotes, presumably reflecting a single additional round of centriole duplication that was enabled by precocious centriole separation. We showed previously that HORMAs HTP-1/2 prevent premature loss of sister chromatid cohesion in the meiotic divisions by preventing removal of cohesin complexes containing the meiosis-specific subunit REC-8. We find thatrec-8 spermatocytes have similar inappropriate centriole separation phenotypes to those observed in htp-1/2 mutants. Our mutant analysis also implicates separase and shugoshin in centriole separation. Our findings are consistent with HORMA proteins preventing centriole disengagement by antagonizing separase-dependent cohesin removal. These findings suggest that the same specialized meiotic mechanisms that evolved to prevent premature release of sister chromatid cohesion also function to inhibit centriole separation, thereby ensuring that the zygote inherits the appropriate complement of chromosomes and centrioles. Although premature separation of centrioles in the horma spermatocyte results in a subset of one-cell embryos having multipolar spindles, centriole separation is not sufficient to trigger multipolar spindle formation in the dividing mutant spermatocytes. Instead we found that chromosome structure is important for normal spindle organization in spermatocyte meiosis. Our analysis of meiotic mutant spermatocytes, uncovered that a subset of these mutants exhibited multipolar spindles. Presence or absence of aberrant spindles correlated with ability of chromosomes to biorient at meiosis I. Our analyses imply that the presence of a few chromosomes proficient for biorientation at meiosis I ensure the formation of bipolar spindles.
Contact: [email protected] Lab: Villeneuve
Platform Session #5 - Germline II, Meiosis and Sex Determination/Dimorphism 31 Role of the Inhibitory Kinase WEE-1.3 in Regulating the Meiotic Cell Cycle and Fertility in C. Elegans Anna Allen, Jessica Nesmith, Andy Golden National Institutes of Health, Bethesda, MD, USA Meiosis is a specialized cell cycle by which the haploid gametes, oocytes and sperm, are produced. It is of crucial importance for sexual reproduction and human health, as defects during the meiotic divisions have serious deleterious outcomes such as infertility, spontaneous miscarriages, birth defects, and tumorigenesis. Meiosis is controlled via dueling regulatory phosphorylation events on the cyclin-dependent kinase (Cdk1) component of maturation promoting factor (MPF). The Wee1/Myt1 family of kinases provides inhibitory phosphorylations that keep MPF inactive, halting the meiotic cell cycle until it is stimulated to resume and coordinate oocyte maturation with fertilization. We have previously shown in C. elegans that depletion of the Myt1 ortholog WEE-1.3 causes precocious oocyte maturation and a very penetrant infertility phenotype. To further investigate the function of WEE-1.3 during oocyte maturation, we generated WEE-1.3 fluorescently tagged transgenic fusion lines and found that the WEE-1.3 protein exhibited a perinuclear expression pattern throughout the germline and developing embryo. By quantitative PCR we demonstrated that WEE-1.3-depleted germlines, containing precocious oocytes, have begun to transcribe embryonic genes and exhibit inappropriate expression of proteins normally limited to fertilized eggs. In addition, we performed an RNAi suppressor screen of the infertile phenotype exhibited upon WEE-1.3 depletion to identify novel factors that when co-depleted with WEE-1.3 restore fertility to the animals. We screened ~1900 essential genes and identified 150 that are suppressors of the WEE-1.3 depletion phenotype. Currently we are investigating the mechanism of how one suppressor ETR-1, an RNA-binding protein with human homologs implicated in disease, is functioning. Notably, our data supports a novel role for ETR-1 in germline development and/ or function. Many of the genes identified in this screen have the potential to be important, previously unknown, players in both the meiotic and mitotic cell cycles due to their interaction with a known cell cycle inhibitor. These studies are providing valuable input not only into meiotic maturation, but also into how the cell cycle is appropriately regulated and potential ways to bring an abnormal cell cycle back under control.
Contact: [email protected] Lab: Golden
32 Platform Session #5 - Germline II, Meiosis and Sex Determination/Dimorphism The Torsin Homolog OOC-5 is Required for Normal Nucleoporin Localization Michael White VanGompel, Lesilee Rose UC Davis, Davis, CA, USA The AAA+ ATPase OOC-5 is required for the re-establishment of polarity and spindle rotation in the P1 cell of 2-cell embryos. OOC-5 localizes to the endoplasmic reticulum (ER) and contiguous nuclear envelope, and is a homolog of the human DYT1 gene encoding the TorsinA protein. Mutations in DYT1 lead to early onset dystonia, a neuromuscular disease that usually presents during adolescence. Recent work has identified nuclear envelope (NE) proteins as interacting partners of TorsinA, including the outer nuclear envelope KASH protein Nesprin-3. What the precise role of TorsinA at the NE is remains unclear. Interestingly, it has been shown that in C. elegans the depletion of certain nucleoporins, components of nuclear pore complexes (NPCs), leads to an ooc-5 like phenotype. This suggests a link between Torsins and nuclear pores in the worm. Using antibody staining and GFP reporter strains we found defects in nucleoporin localization in ooc-5 worms, though lamin appears normal. Nucleoporin localization is apparently normal in the mitotic zone of mutant gonads. Clustering of nucleoporins occurs in the transition zone, where germ cells enter meiosis, and persists through all stages of meiosis. Interestingly, mislocalization of the outer nuclear envelope KASH protein ZYG-12 is not present until pachytene, after the transition zone, and is less severe than NPC defects. We are currently investigating whether NPC defects precede ZYG-12 defects in ooc-5 worms, and whether nuclear exclusion is affected. Nucleoporin clustering is also apparent in ooc-5 intestinal cells, indicating that ooc-5-dependent localization of NPC components is not germ-cell specific. Furthermore, EM analysis ofooc-5 oocytes shows blebbing of the nuclear envelope similar to that reported in torsinA mutant mice. Our results suggest that OOC-5 plays an important role in nuclear pore biology, a function which is likely to be broadly conserved.
Contact: [email protected] Lab: Rose
Platform Session #5 - Germline II, Meiosis and Sex Determination/Dimorphism 33 Identification of Direct Targets of theCaenorhabditis elegans Global Sexual Regulator TRA-1 by Chromatin Immunoprecipitation Matthew Berkseth1, Kohta Ikegami2, Jason Lieb2, David Zarkower1 1University of Minnesota, Minneapolis, MN, USA, 2University of North Carolina, Chapel Hill, NC, USA The nematode Caenorhabditis elegans naturally occurs as two highly dimorphic sexes, the XX hermaphrodite and the XO male. Sex is determined by a genetic pathway culminating in the transcription factor TRA-1, the worm homologue of vertebrate GLI proteins. Null mutations in tra-1 result in hermaphrodite-to-male sex reversal, indicating that TRA-1 and its downstream targets are responsible for generating all sexual dimorphism in the worm. However only a few direct TRA-1 targets have been described, and additional biologically important targets likely remain to be identified. To identify TRA-1 target genes throughout the C. elegans genome, we have performed chromatin immunoprecipitations using an affinity-purified rabbit polyclonal TRA-1 antibody followed by deep sequencing (ChIP-seq). We have identified ~400 TRA-1 binding sites in C. elegans with this approach. This list includes most of the previously identified TRA-1 binding sites and is significantly enriched for close matches to the TRA-1 consensus binding sequence. We have performed ChIP-seq experiments in L2, L3, and young adult animals, and found that TRA-1 binding at some sites varies across developmental time. We have also performed TRA-1 ChIP-seq in mutant animals lacking a germline and identified ~40 peaks significantly reduced in germline-less animals, suggesting they may be bound by TRA-1 only in the germline. We have also performed ChIP-seq on L3 stage C. briggsae, and identified ~50 TRA-1 binding sites in this species, several of which have been conserved in C. elegans, suggesting that that the regulation of nearby genes by TRA-1 is likely to be functionally important. To examine what role putative TRA-1 targets may play in sexual development, we have generated reporters for many genes adjacent to TRA-1 binding sites, and identified several that are expressed in a largely male-specific manner. We are in the process of ablating TRA-1 binding sites in these reporters to determine whether their male-specific expression patterns are controlled directly by TRA-1. Surprisingly, several targets are genes known to function upstream of TRA-1 in the global sex determination pathway, including xol-1, fem-3, and sup- 26, suggesting that one function of TRA-1 is to feed back onto the sex determination pathway to reinforce the sex determination decision.
Contact: [email protected] Lab: Zarkower
34 Platform Session #5 - Germline II, Meiosis and Sex Determination/Dimorphism Evolution of Caenorhabditis Dosage Compensation Te-Wen Lo, Caitlin Schartner, Catherine Pickle, Barbara Meyer HHMI/UC Berkeley, Berkeley, CA, USA Comparative studies have shown remarkable divergence in the conservation of developmental mechanisms. Strategies to determine sexual fate and to compensate X-chromosome dosage between sexes have evolved particularly rapidly: mammals, flies, and worms use fundamentally different methods. Understanding such rapidly changing processes requires comparisons over shorter evolutionary time-scales, such as between C. briggsae and C.elegans (15-30 Myr). Comparison of sex determination and dosage compensation across nematode species using heritable, targeted mutagenesis protocols we developed has shown that key features of the dosage compensation complex (DCC) and the genetic pathway that coordinates sex determination and dosage compensation are conserved. Despite conservation of the DCC and its regulatory hierarchy, the mechanisms for targeting the DCC to X chromosomes have diverged. The cis-acting DNA recruitment elements on X (rex) and their motifs that attract the DCC are distinct. C. elegans rex sites ported to C. briggsae fail to bind the C. briggsae DCC. The reciprocal also holds: C.briggsae rex sites ported into C.elegans fail to bind the C. elegans DCC. Also, C. briggsae rex sites lack the X-enriched C. elegans DNA motifs pivotal for DCC recruitment. The divergence of DCC binding sites between C. elegans and C. briggsae prompted us to explore X targeting in C. sp.9, which is closer to C. briggsae than to C. elegans. C. sp. 9 proteins homologous to DCC subunits of both C. briggsae and C. elegans co-localize on X chromosomes of C. sp. 9 hermaphrodites and C. briggsae/C. sp. 9 hybrid hermaphrodites. On-going ChIP-seq experiments will reveal the level of divergence in X targeting mechanisms. Dosage compensation provides a unique opportunity to study the co-evolution of regulator proteins and their binding sites. The evolution of DCC binding sites followed a different pattern from that of binding sites for conserved regulatory proteins that control many unrelated cellular processes. For multi-functional proteins few significant changes have occurred in their DNA binding domains and cognate DNA binding motifs. In contrast, DCC complexes, which lack the constraints of multiple functions, exhibit robust divergence in binding sites. To facilitate our evolutionary studies, we are optimizing site-directed mutagenesis for other nematode species and devising protocols for integration of homologous DNA. The most recent success has been P. pacificus.
Contact: [email protected] Lab: Meyer
Platform Session #5 - Germline II, Meiosis and Sex Determination/Dimorphism 35 RNAi and Immortality: Recognition of Self/non-Self RNA in the C. elegans Germline Craig Mello University of Massachusetts Medical School, HHMI
Contact: [email protected] Lab: Mello
36 Keynote 6 The onset of dosage compensation is linked to the loss of developmental plasticity Laura Custer, Gyorgyi Csankovszki University of Michigan, Ann Arbor, MI, USA Dosage compensation is a specialized gene regulatory process to equalize X chromosome gene expression between sexes. The dosage compensation complex (DCC) localizes to both X chromosomes of hermaphrodites, which leads to a two-fold downregulation of gene expression. In adult somatic tissues this is accompanied by DCC-dependent depletion of histone H4 lysine 16 acetylation (H4K16ac), a mark of active transcription, and enrichment of H4K20 monomethylation (H4K20me1), a repressive mark, on the X chromosomes. The DCC first accumulates on the X chromosomes at the 30-cell stage, coinciding with the developmental transition from plasticity to differentiation. We found that dosage compensated X chromosomes acquire their distinguishing chromatin marks with different kinetics. H4K20me1 becomes X-enriched at the comma stage, several cell cycles after DCC accumulation, suggesting that it is a late mark in the dosage compensation process. By contrast, H4K16ac is depleted on the X chromosomes even before the onset of dosage compensation. As opposed to later in development, this early depletion of acetylation does not depend on the presence of the DCC. Instead, depletion requires the activities of MES-2 (a subunit of a complex similar to the Polycomb Repressive Complex), as well as MES-4, and is observed in both males and hermaphrodites, perhaps as a consequence of germline silencing of the X chromosomes. The MES proteins are also required for the timely loss of developmental plasticity, and mes mutant embryos exhibit a delay in this transition. Consistent with the hypothesis that the onset of dosage compensation is linked to differentiation, DCC localization and H4K20me1 accumulation on the X chromosomes are delayed in mes mutant hermaphrodite embryos. We propose that as embryonic blastomeres lose their developmental plasticity, the X chromosomes in hermaphrodites transition from a MES protein-regulated state to DCC-mediated repression.
Contact: [email protected] Lab: Csankovszki
Platform Session #6 - Gene Regulation 37 The Histone Demethylase SPR-5 and the Histone Methyltransferase MET-2 Comprise a Novel Epigenetic Reprogramming Switch Shana Kerr, Chelsey Chandler, Joshua Francis, Erica Mills, David Katz Emory University, Emory University Extensive epigenetic reprogramming is required at fertilization to re-establish a developmental ground state, but the mechanism of this reprogramming is poorly understood. We previously demonstrated that mutation of spr-5, the C. elegans ortholog of the histone demethylase LSD1/KDM1, results in progressive sterility over generations due to the transgenerational accumulation of the histone modification H3K4me2. Thus H3K4me2 can serve as a stable epigenetic transcriptional memory, and erasure of H3K4me2 by SPR-5 in the germline prevents the inappropriate transgenerational transmission of this memory. Intriguingly, mutation of met- 2, which encodes an H3K9 methyltransferase similar to SETDB1, also results in progressive sterility over generations. As H3K4me2 and K3K9me are thought to have opposing effects on transcription, these similarities are consistent with a role for MET-2 in reinforcing SPR- 5 mediated erasure of H3K4me2 through the addition of H3K9me. In order to investigate this possibility, we created spr-5;met-2 double mutant worms. spr-5;met-2 mutants exhibit a synthetic maternal effect sterile phenotype with germline defects that are reminiscent of late generation spr-5 worms. In addition, spr-5;met-2 mutants have an accumulation of H3K4me2 at SPR-5 germline targets that is well beyond that of the single mutants alone and this increase in H3K4me2 correlates with huge increases in the expression of these genes. These data suggest that erasure of the active histone modification (H3K4me2) is coupled to the acquisition of a repressive histone modification (H3K9me2) during reprogramming at fertilization and that this novel epigenetic reprogramming mechanism is critical to undergo the cell fate transition from gametes to the embryo.
Contact: [email protected] Lab: Katz
38 Platform Session #6 - Gene Regulation Nuclear RNAi mediates silencing of repetitive sequences in C. elegans Fei Xu, Xufei Zhou, Hui Mao, Jiaojiao Ji, Shouhong Guang Univ. of Sci. & Tech. of China Nuclear RNAi (Nrde) pathway has been shown to inhibit transcription elongation and mediate heritable gene silencing in C.elegans. The endogenous function of Nrde pathway remains unclear. We investigated the genetic requirements of RNAi-induced off-target gene silencing and surprisingly identified that the nrde mutants are resistant to off-target gene silencing. >dpy-13 is a collagen gene, which belongs to a large gene family that contains more than 150 members with high sequence similarity. dpy-13(e458) mutant lacks most of the coding region, likely being a null mutation. dpy-13(e458) animals exhibit a dumpy phenotype, with a length roughly half of wild type N2 animals. eri(-) and ergo-1(-) animals exhibit enhanced sensitivity to RNAi. Feeding eri(-) and ergo-1(-) animals targeting the dpy-13 sequence elicits a phenotype which is extremely more severe (worm-ball-like) than dpy-13(e458). This finding indicates that dsRNA targeting the dpy-13 gene is able to trigger an off-target silencing effect. We examined the genetic requirements for this off-target effect. Interestingly, nrde-3, but not its secondary Argonaute paralogues in C. elegans, is critical for this process,suggesting that the Nrde pathway preferentially silences repetitive sequence elements. Consistent with this, nrde genes are required to silence transgenes which form repetitive arrays. We examined the endogenous small RNAs bound small RNAs bound to NRDE-3. Unlike the other two secondary Argonaute proteins CSR-1 and WAGO-1, most of the NRDE-3 targets share extensive sequence similarity. The functionality of Nrde pathway in silencing repetitive sequences is discussed.
Contact: [email protected] Lab: Guang
Platform Session #6 - Gene Regulation 39 Dimerization of βCatenin/WRM-1 Allows Intermolecular Autophosphorylation of LIT-1 in the Activation Loop Xiao-Dong Yang, Scott Robertson , Rueyling Lin UT Southwestern Medical Center, Dallas, TX, USA Activation of Wnt target genes in C. elegans embryos requires the TCF protein, POP-1, and a coactivating β-catenin, SYS-1. In Wnt responsive cells, nuclear POP-1 is lowered as a result of nuclear export, a process requiring phosphorylation of POP-1 by the conserved MAP kinase LIT-1. We have shown previously that the diverged β-catenin /WRM-1 binds to both POP-1 and LIT-1, functioning as a substrate-binding subunit for the LIT-1 kinase. In addition, WRM-1 is required for LIT-1 kinase activity, independent of its substrate-binding capability. The molecular mechanism by which WRM-1 activates LIT-1 remains unknown. Despite being highly conserved, the mammalian homolog NLK differs from LIT-1 in that it can be activated when expressed by itself. It has been shown that NLK undergoes homodimerization, which is essential for intermolecular autophosphorylation of T286 in the activation loop and kinase activity. We show that, unlike NLK, LIT-1 does not oligomerize effectively, nor does it undergo intermolecular autophosphorylation when expressed by itself. Coexpression with WRM-1 resulted in LIT-1 self association and phosphorylation at T220, which corresponds to T286 in NLK. We identify the domain required for both LIT-1 self association and activation to be the C-terminal 150 amino acids of WRM-1, a domain distinct from that responsible for binding to LIT-1 (aa’s 1-150) or POP-1 (ARM repeats 3-5). The C-terminal domain of WRM-1 is predicted to contain two coiled-coil motifs, motifs that often mediate protein-protein interactions. Using artificial coiled-coil motifs that can oligomerize, we show that substituting the C-terminal domain of WRM-1 with a dimmerization motif restores the ability of WRM-1 to activate LIT-1. More importantly, fusing an oligomerization motif to full-length LIT-1 results in LIT-1 activation in the absence of WRM-1. Together, our results demonstrate a molecular mechanism by which WRM- 1 activates LIT-1. WRM-1 dimerizes via its C-terminal coiled-coil motifs, resulting in multiple molecules of LIT-1, bound to the N-terminal domain of WRM-1, to be juxtaposed, allowing intermolecular phosphorylation. The resultant phosphorylation of LIT-1 in the activation loop leads to kinase activity and POP-1 phosphorylation.
Contact: [email protected] Lab: Lin
40 Platform Session #6 - Gene Regulation Organ defects in adults resulting from threshold blastomere specification Morris Maduro, Gina Broitman-Maduro, Leila Magistrado, Shruthi Satish University of California, Riverside, Riverside, CA, USA The embryonic E cell generates the C. elegans gut. E specification results from the transient expression of end-1 and end-3 in the early E lineage, which results in activation of elt-2 and elt-7, which is maintained by positive autoregulation. A recent work (Raj et al., 2010) examined the correlation of expression of end-1 and end-3 with activation of elt-2 in a skn-1 mutant background, in which specification of endoderm occurs in approximately 20-30% of embryos. The results suggested that activation of elt-2 occurs when a threshold of end-1 and end-3 expression has been reached. What is not known is whether successful activation of elt-2 by a threshold amount of the ends is sufficient for normal endoderm development, and whether or not animals that have ‘just barely specified E’ develop otherwise normally due to the positive autoregulation of elt-2, and the ability of embryos to buffer early deficits in end-1 and/or end-3 expression. These questions have been difficult to address experimentally, as knockdown of the upstream regulators that affect activation of the ends (skn-1, pop-1, med- 1/2) results in arrested embryos due to their essential roles in other lineages; the existing null mutants in end-1 and end-3 have only subtle defects individually (95-100% of embryos make gut), and double end-1 end-3 mutants completely fail to make any endoderm at all; and overexpression of elt-2 is known to be sufficient to make endoderm, but this does not reflect threshold activation by the ends. By inserting single-copy transgenes in which the MED-1,2 binding sites have been mutated, we have generated strains in which activation of only end-1 and/or end-3 are compromised, resulting in a spectrum of endoderm specification phenotypes arising from otherwise isogenic animals. Many embryos contain no gut, some have a few or several gut cells, and others make a relatively normal gut of 20 or more gut cells. Adults derived from the latter have phenotypes that can be attributed to abnormalities in intestine differentiation, such as accumulation of significantly more lipids. We have performed RNA-Seq of intact L4s and dissected adult intestines in these strains and identified differences with wild-type. Our results suggest that threshold activation of elt-2 by itself is not sufficient for normal endoderm differentiation, even when an apparently normal intestine is made, and that the adult intestine can carry a memory of incomplete specification by the ends.
Contact: [email protected] Lab: Maduro
Platform Session #6 - Gene Regulation 41 Title: TBD Julie Ahringer Gurdon Institute, University of Cambridge
Contact: [email protected] Lab: Ahringer
42 Keynote 7 Modeling germline population dynamics Hillel Kugler1, E. Jane Albert Hubbard2 1Microsoft Research Cambridge, 2New York University School of Medicine, Skirball Institute, Kimmel Stem Cell Center In recently published work [1] we constructed a dynamic computer model of the C. elegans germ cell population and used it to analyze the interplay between Notch signaling, cell cycle control, and anatomical constraints. Our analyses of model simulations provided predictions that were validated by laboratory studies. For example, we showed that germ cell proliferation rate during larval stages can influence the differentiation pattern in the adult. In more recent work, we have extended the previous model and have overcome certain limitations. For example we can now capture the three dimensional structure of the gonad and represent cells “pushing’’ each other during division. We have developed new general web-based tools to make the model accessible and amenable to extension and in-silico experimentation. The development of these tools is based on the premise that while a model- building/model-testing cycle is part and parcel of experimental biology that could be facilitated by dynamic computer-based models, many existing computational methods and tools are not accessible to experimental biologists (e.g., large sets of differential equations or complex software code). We are building these tools with the hope that they will be amenable to modeling many aspects of C. elegans development.
[1] Y. Setty, D. Dalfo, D.Z. Korta, E.J. A. Hubbard, and H. Kugler (2012) A model of stem cell population dynamics: in-silico analysis and in-vivo validation. Development 139: 47-56.
Contact: [email protected] Lab: Kugler
Platform Session #7 - Cell fate and New Technologies 43 Dev-scape: An intuitive tool for automated phenotyping with single cell resolution Julia Moore1, Zhuo Du1, Anthony Santella1, Christian Pohl2, Zhirong Bao1 1Sloan-Kettering Institute, New York, NY, USA, 2Frankfurt Institute for Molecular Life Sciences, Frankfurt, Germany Advances in imaging technology have provided an experimental platform in which dynamic biological processes can be observed at high spatial and temporal resolution. These advances have facilitated studies directly observing the progression of C. elegans embryogenesis at the single cell level. New computational tools are needed to efficiently identify subtle but statistically significant abnormalities in this high resolution data. We addressed this problem by constructing an automated phenotyping pipeline to format, analyze and display the data in intuitive and biologically meaningful forms. Dev-scape takes advantage of C. elegans’ invariant lineage to carefully characterize each cell’s wild type behavior. 50 wild type embryos were used to define statistical distributions of each cell’s proliferation, differentiation and morphogenesis. Mutant and RNAi treated embryos are measured and compared to the wild type distributions to quantify the statistical significance of any abnormalities. The raw data and corresponding significance values are displayed in multiple ways to elucidate complex phenotypes. Dev-scape provides 1) unprecedented insights into the variability in normal development and 2) the ability to detect aberrant behavior in single cells. By pairing Dev-scape with whole genome RNAi libraries, we can investigate the landscape of possible perturbations of embryogenesis.
Contact: [email protected] Lab: Bao
44 Platform Session #7 - Cell fate and New Technologies WormBase 2012: Website Redesign Abigail Cabunoc, Norie de la Cruz, Adrian Duong, Maher Kassim, Xiaoqi Shi, Todd Harris, Lincoln Stein Ontario Institute for Cancer Research, Toronto, Canada WormBase (www.wormbase.org) has served the ever growing needs of the nematode research community since 2000. Initially created as a resource for the C.elegans genome and its biology, WormBase now includes a wider variety of data from 15 related species. This growth in data is mirrored by increased website use. In response to the constantly growing data and user traffic, the WormBase web team has redesigned both the underlying architecture and user interface of the website. In this interactive presentation, we will review the new WormBase website, walk through basic and advanced tasks using the new interface, and examine several features which take advantage of the nematode research community.
Contact: [email protected] Lab: Stein
Platform Session #7 - Cell fate and New Technologies 45 DSL-2 Mediates a Notch Signal From EMS Descendant(s) to ABp Descendants Scott Robertson, Jessica Medina, Rueyling Lin UT Southwestern Medical Center, Dallas, Texas, USA Specification of pharyngeal precursors during embryogenesis requires multiple Notch- mediated cell-cell interactions. In the 12-cell embryo, the MS blastomere contacts two of the four ABa descendants and induces them to produce pharyngeal muscle later. At this stage, all ABa- and ABp-derived blastomeres express the Notch receptor, GLP-1, and some ABp descendants contact MS. However, no pharyngeal muscle is made from ABp-derived cells. It has been shown that a Notch interaction occurring in the 4-cell stage between P2 and ABp renders ABp descendants unresponsive to the subsequent Notch signal from MS. The Notch ligand responsible for the interaction in 4-cell is APX-1, but that in MS is not known. apx-1 mutant embryos produce induced pharynx from both ABa and ABp descendants. We generated a transcriptional GFP reporter that expresses in all Notch responsive early blastomeres, in a manner dependent on GLP-1. We noted a high level of GFP expressed in two of the four ABp descendants, ABprp and ABplp, in 12-cell embryos. At this stage, ABprp and ABplp, but not their siblings are in close contact with EMS descendants. We show that the high level of GFP in ABprp and ABplp is dependent on the EMS-specific transcription factor, SKN-1. Via microarray analyses, we identified two SKN-1-dependent Notch ligands, DSL-1 and DSL-2, expressing in the 12-cell embryo. dsl-2, but not dsl-1, depletion reduces the GFP in ABprp and ABplp to a level similar to other ABp descendants. Furthermore, depletion of dsl-2 in apx-1(-) mutant embryos resulted in a decrease of pharyngeal tissue. We showed that the pharyngeal marker, PHA-4 is only abolished in ABp-derived, but not ABa-derived cells in apx- 1;dsl-2(RNAi) embryos. In summary, we have uncovered a Notch/DSL-2-mediated signal from the EMS descendant(s) to ABp descendants at the 12-cell stage that is distinct from the one inducing the anterior pharynx. Our results show that the P2-to-ABp Notch signal does not render ABp unresponsive to subsequent Notch signaling. Instead, it alters the way in which ABp descendants respond to subsequent Notch signaling.
Contact: [email protected] Lab: Lin
46 Platform Session #7 - Cell fate and New Technologies Intercellular Calcium Signaling in a Gap Junction Cell Network Establishes Left-Right Asymmetric Neuronal Fates Jennifer Schumacher Tucker, Chieh Chang, Chiou-Fen Chuang Cincinnati Children’s Hospital Research Foundation, Cincinnati, OH, USA Many highly specialized cells must derive from a limited number of progenitors during nervous system development. An example of neuronal diversification is establishment of asymmetric gene expression across the left-right (L-R) axis, which occurs in the C. elegans AWC chemosensory neuron pair. Expression of the odorant receptor str-2 is random: either the right or left AWC expresses str-2 to become AWCON, and the contralateral cell becomes AWCOFF. Intercellular communication between the AWCs and 34 other neurons through the NSY-5/innexin gap junction network is required for asymmetric str-2 expression, but the mechanism by which NSY-5 mediates communication is unclear. Ca2+ is a good candidate signal because it moves through gap junctions in other contexts, and Ca2+ signaling cascade components are required autonomously in AWC to promote AWCOFF. However, the role of Ca2+ flux in non-AWCs has not been investigated. To test the hypothesis that Ca2+ mediates intercellular communication by traveling through NSY-5 gap junctions, we expressed genetically encoded calcium buffer proteins and tissue-specific nsy-5 RNAi under the control of various promoters in the NSY-5 network. Using genetic mosaic analysis to modify intracellular Ca2+ or NSY-5 levels in specific cells, we show that Ca2+ within non-AWCs requires NSY-5 to influence str-2 expression, suggesting that Ca2+ may be the signal that passes through NSY-5 gap junctions. Our results reveal that the AWC neuron pair relies on a balance of autonomous and non-autonomous Ca2+ inputs to diversify cell fates.
Contact: [email protected] Lab: Chuang
Platform Session #7 - Cell fate and New Technologies 47 Nutritional control of mRNA isoform expression during developmental arrest and recovery in C. elegans Colin Maxwell1, Igor Antoshechkin2, Nicole Kurhanewicz3, Jason Belsky1, L. Ryan Baugh1 1Duke University, Durham (NC), USA, 2California Institute of Technology, Pasadena (CA), USA, 3University of North Carolina, Chapel Hill (NC), USA Nutrient availability profoundly influences gene expression. Many animal genes encode multiple transcript isoforms, yet the effect of nutrient availability on transcript isoform expression has not been studied in genome-wide fashion. When C. elegans larvae hatch without food they arrest development in the first larval stage (L1 arrest). Starved larvae can survive L1 arrest for weeks, but growth and post-embryonic development are rapidly initiated in response to feeding. We used RNA-seq to characterize the transcriptome during L1 arrest and over time after feeding. 27% of detectable protein coding genes were differentially expressed during recovery from L1 arrest, with the majority of changes initiating within the first hour, demonstrating widespread, acute effects of nutrient availability on gene expression. We used two independent approaches to track expression of individual exons and mRNA isoforms, and we connected changes in expression to functional consequences by mining a variety of databases. These two approaches identified an overlapping set of genes with alternative isoform expression, and they converged on common functional patterns. Genes affecting mRNA splicing and translation are regulated by alternative isoform expression, revealing post-transcriptional consequences of nutrient availability on gene regulation. We also found that phosphorylation sites are often alternatively expressed, revealing a common mode by which alternative isoform expression modifies protein function and signal transduction. Our results detail rich changes in C. elegans gene expression as larvae initiate growth and post-embryonic development, and they provide an excellent resource for ongoing investigation of transcriptional regulation and developmental physiology.
Contact: [email protected] Lab: Baugh
48 Platform Session #7 - Cell fate and New Technologies Ras and its Effector RalGEF Both Perform Dual, Antagonistic Functions during C. elegans Vulval Patterning Kimberly Monahan, Rebecca Whitehurst, Tanya Zand, Channing Der, David Reiner University of North Carolina, Chapel Hill The Ras small GTPase oncoprotein interacts with a spectrum of functionally diverse effectors to promote cancer growth. How effector utilization is dynamically regulated to achieve different cellular consequences remains poorly understood. This is a critical question since many anti- Ras drug discovery efforts focus on inhibition of a single effector, Raf, and clinical results suggest that multidrug therapies will be frequently required to avoid drug resistance or increase treatment efficacy. Developmental fate patterning of theC. elegans vulva and Drosophila R7 photoreceptor are classic model systems for studying Ras signaling. EGF induces the six C. elegans epithelial vulval precursor cells (VPCs) to form a highly reproducible 3°-3°-2°- 1°-2°-3° pattern of fates; 1° and 2° cells pursue specialized vulval development programs while 3° cells remain unspecialized epithelial cells. EGFR activates Ras and the canonical Raf-MEK-ERK effector pathway to regulate transcription-dependent induction of a single 1 cell, which subsequently produces DSL ligands that laterally induce Notch-dependent 2 fate in the two neighboring VPCs. Multiple signaling mechanisms are thought to mediate mutual antagonism between 1° and 2° fates. We described recently that Ras switches effectors during vulval patterning by, in presumptive 2° cells, engaging the noncanonical effector RalGEF. Ras thereby mediates an EGF pro-2° signal in support of the Notch pro-2° signal, and provides the molecular mechanism for interpretation of an EGF patterning gradient. RalGEF activates the Ral small GTPase to promote 2° fate through an unknown downstream pathway. We find that RalGEF has an additional, Ral-independent function that antagonizes its Ral-dependent pro- 2° function. Previous mammalian cell studies suggest that RalGEF can scaffold PDK and Akt as a component of the PI3K phospholipid-signaling cascade. We find that PI3K antagonizes 2° fate in a RalGEF-dependent manner, consistent with RalGEF functioning as a PDK-Akt scaffold. Furthermore, the PI3K cascade inhibits FoxO transcription factor activation, and our results suggest that FoxO promotes 2° fate. We therefore hypothesize that RalGEF toggles between activating Ral to drive 2° fate and scaffolding the PI3K cascade to inhibit 2° fate, and that these two RalGEF pathways compete to control FoxO pro-2° activity. Perhaps Ras and RalGEF dual signaling reinforces initial cell fate patterning and increases developmental fidelity.
Contact: [email protected] Lab: Reiner
Platform Session #7 - Cell fate and New Technologies 49 The microRNA miR-786 is Required for Rhythmic Calcium Wave Initiation in the C. elegans Intestine Benedict Kemp1, Erik Allman2, Lois Immerman3, Megan Mohnen1, Maureen Peters3, Keith Nehrke2, Allison Abbott1 1Marquette University, Milwaukee, (WI), USA, 2University of Rochester School of Medicine and Dentistry, Rochester, (NY), USA, 3Oberlin College, Oberlin, (OH), USA Rhythmic behaviors are ubiquitous phenomena in animals. The best studied rhythmic behavior in C. elegans is defecation, which involves three coordinated muscle contractions every ~50 sec. The execution of the defecation motor program depends on intercellular calcium waves that initiate in the posterior intestine. Thus, the posterior intestine functions as the pacemaker for this rhythmic behavior. However, the molecular mechanism for pacemaker activity is not well understood. We found that the microRNA mir-786 is necessary for the supremacy of the posterior cell in the rhythmic initiation of calcium waves. Loss of mir-786 results in long arrhythmic defecation cycles (Miska et al., 2007) with calcium wave initiation often observed in the non-posterior regions of the intestine. These calcium waves often fail to trigger a full defecation motor program. Genetic data indicates that mir-786 functions upstream
of IP3R dependent calcium release. mir-786 is expressed in the posterior-most ring of intestinal cells, int9. We identify elo-2, as a likely direct target for miR-786 in the posterior intestine. elo-2 encodes a fatty acid elongase previously demonstrated to regulate defecation cycling (Kniazeva et al., 2003). We propose that miR-786 regulates lipid composition in the int9 posterior cells thereby functioning to establish or maintain pacemaker activity for this rhythmic behavior.
Kniazeva, M.,Sieber, M., Mccauley, S., Zhang, K., Watts, J. L., and Han, M. (2003). Genetics 163, 159- 169. Miska, E. A., Alvarez-Saavedra, E., Abbott, A. L., Lau, N.C., Hellman, A. B., McGonagle, S. M., Bartel, D. P., Ambros, V. R., and Horvitz, H. R. (2007). PLoS Genet 3,e215.
Contact: [email protected] Lab: Abbott
50 Platform Session #7 - Cell fate and New Technologies GLO-2 is a BLOC-1 Subunit that Functions in Gut Granule Biogenesis Alec Barrett, Olivia Foster, Annalise Vine, Greg Hermann Lewis and Clark College, Portland, OR, USA Caenorhabditis elegans intestinal cells are characterized by the presence of gut granules, lysosome-related storage organelles that contain autofluorescent and birefringent material. Gut granule formation requires the activity of evolutionarily conserved genes that when disrupted result in the loss and/or mislocalization of birefringent material into the embryonic intestinal lumen (the glo phenotype). Here we present our phenotypic and molecular analysis of glo-2, which encodes a small cytoplasmically localized protein that is orthologous to mammalian Pallidin. Pallidin functions as part of the 8-subunit containing BLOC-1 complex in trafficking to lysosome related organelles and defects in BLOC-1 activity result in the human disease Hermansky-Pudlak Syndrome. Seven BLOC-1 subunit homologues exist in C. elegans and we show that they likely form a complex and function together in gut granule biogenesis. Our studies of intracellular trafficking are consistent with BLOC-1 acting in parallel to the AP-3 mediated pathway to gut granules. Notably, loss of BLOC-1 activity has only subtle effects on trafficking to conventional lysosomes. We find that glo-2(-) is partially suppressed by overexpression of RAB-7, suggesting overlap in trafficking pathways to lysosome-related and conventional lysosomal organelles, which co-exist in C. elegans intestinal cells. Phenotypic analysis and genetic interactions point to a role of RAB-7 in facilitating the movement of gut granule cargo from the conventional endolysosomal trafficking pathway to gut granules. We present results of a genetic enhancer screen to identify factors that function with, and parallel to, BLOC-1.
Contact: [email protected] Lab: Hermann
Poster Topic: Cell Biology 51 The Conventional Kinesin-1/UNC-116 Acts in PHB Phasmid Neurons to Mediate Proper Cell Body Position Ben Barsi-Rhyne, Kristine Miller, Chris Vargas, Miri VanHoven San Jose State University, San Jose,(CA), USA Cell migration in the nervous system is vital for developing proper nervous tissue structure and defects in this process have been implicated in mental retardation. Cell migration is an active process that begins with a migration promoting signal that leads to cell polarization and extension of membrane protrusions at the leading edge. Many pathways have been found to play a role in this process, but the downstream molecular mechanisms have yet to be thoroughly characterized. UNC-116/Kinesin-1 has been previously been shown to be required for many processes including trafficking of synaptic vesicle components to the active zone and growth cone migration. We have found that UNC-116/Kinesin-1 also plays a role in maintaining neuronal cell body position. To understand the role of UNC-116/Kinesin-1 and its potential pathway members in this process, we study the PHB phasmid neurons in the lumbar ganglion of Caenorhabditis elegans. Interestingly this class of neurons is born in approximately its final position. However, using a combination of dye filling and cell-specific expression of the mCherry fluorophore, we have found that UNC-116/Kinesin-1 is required to maintain the anterior-posterior position of the PHB cell body throughout development. In unc-116/kinesin-1 mutant animals, at least one PHB cell body is frequently found anterior of the anal valve. In addition, the number of animals with the mutant phenotype increases progressively with developmental stage from approximately 15% in L1s to 65% in adult animals. Furthermore, cell specific rescue experiments indicate that UNC-116/Kinesin-1 functions cell autonomously to mediate this process. Our preliminary results suggest that this process occurs partially through an UNC-6/Netrin attractive signal. In addition, we have found that KLC-2, one of two kinesin- light-chains, also plays a role in this process. Interestingly, our preliminary data indicates that this defect may be specific for the UNC-116/Kinesin-1, as OSM-3/ Kinesin-2 and several other kinesin-like proteins including KLP-11, ZEN-4, VAB-8, and UNC-104 do not have severe defects in this process. We believe that these results suggest a novel role for UNC-116/Kinesin-1 in maintenance of PHB cell body position. To further elucidate the function of UNC-116/kinesin-1 in this process, we will continue to test additional potential pathway members.
Contact: [email protected] Lab: VanHoven
52 Poster Topic: Cell Biology Genetic Interaction and Structure/Function Studies of MEL-28, a Protein Required for Nuclear Envelope Function and Chromosome Segregation Anita Fernandez2, Carly Bock2, Allison Lai2, Emily Mis3, Fabio Piano1 1New York University, New York, (NY), USA, 2Fairfield University, Fairfield, (CT), USA, 3Yale University, New Haven. (CT), USA We have been studying mel-28/elys, a gene required both for the structural integrity of the nuclear envelope during interphase and the proper behavior of the chromatin during mitosis. MEL-28/ELYS is a large conserved protein that shuttles between the nuclear envelope and the kinetochore during the cell cycle. We performed a mel-28 genetic interaction screen and identified ~50 genes that cause novel phenotypes when RNAi-depleted in a mel-28 mutant background. We found genes that encode proteins required for translation, components of dynein and its regulators, and nucleoporins. To determine which domains of MEL-28 are required for its function we are executing a structure/function approach. We have found that the N-terminal AT-hook domains are required for MEL-28 function, but that the conserved coiled-coil domain is not.
Contact: [email protected] Lab: Piano
Poster Topic: Cell Biology 53 Oocyte Meiotic Spindle Assembly in C. elegans Amy Connolly, Sara Christensen, Valerie Osterberg, Josh Lowry, John Yochem, Bruce Bowerman Institue of Molecular Biology, University of Oregon In contrast to the centrosome-based assembly of mitotic spindles, how acentrosomal oocytes assemble bipolar meiotic spindles in the absence of centrosomes remains poorly understood. With its powerful genetics and transparent anatomy, Caenorhabditis elegans is a powerful model system for investigating this fundamental process. We can analyze the dynamics of oocyte meiotic spindle assembly with live cell imaging using GFP and mCherry fusions to meiotic spindle proteins. Using these tools, we are investigating how the meiotic spindle assembly genes mei-1, klp-18 and aspm-1 interact to produce a bipolar meiotic spindle. We are also investigating two new temperature-sensitive mutants with meiotic spindle defects, called or1092ts and or1292ts. Mutant one-cell or1092ts zygotes have multiple maternal pronuclei after the completion of the oocyte meiotic cell divisions, but mitotic cell divisions appear roughly normal. Complementation tests show or1092ts is not an allele of mei-1, mei-2, klp-18, or aspm-1. Based on live cell imaging, the or1092ts meiotic spindleis a disorganized array of microtubules with unorganized chromosomes and fails to extrude a polar body. The or1292ts mutant exhibits defects in both meiotic and mitotic spindle function. The meiotic spindle often fails to segregate chromosomes properly, as evident by the anaphase bridges detected during meiosis and the presence of multiple maternal pronuclei during the one cell stage. During the one-cell stage in or1292 mutant zygotes, pronuclear migration is abnormal and while the mitotic spindle aligns along the long axis it fails to move toward the posterior pole. In addition, we have observed intriguing defects in the regulation of microtubule dynamics in or1292ts mutants. Dense arrays of microtubules appear throughout the cortex during meiosis, and cytoplasmic microtubules appear throughout the anterior end of the embryo during pronuclear migration. We are currently using whole genome sequencing methods to determine the mutation site in both or1092ts and or1292ts. We have so far learned that or1092ts maps approximately between 10.5 and 12 Mb on Linkage group III; or1292ts maps roughly between 2 and 4 Mb on Linkage group III. Once we identify the causal mutations, we will further investigate the requirements for the affected genes during meiotic spindle assembly, and the role of the or1292ts locus in regulating microtubule dynamics.
Contact: [email protected] Lab: Bowerman
54 Poster Topic: Cell Biology Identifying Proteins that Interact with the Serine/Threonine Kinase UNC-82 in Muscle Cells Christopher Duchesneau2, April Reedy2, Hiroshi Qadota1, Guy Benian1, Pamela Hoppe2 1Emory University, Atlanta, Georgia, U.S.A., 2Western Michigan University, Kalamazoo, Michigan, U.S.A. We are interested in the mechanisms underlying myosin filament organization in striated muscle. UNC-82 is a serine/threonine kinase orthologous to human ARK5 (NUAK1) and SNARK (NUAK2). Mutations in the unc-82 gene cause defects in thick filament organization in striated muscle cells (Waterston et al. 1980). A full-length UNC-82::GFP fusion protein rescues this phenotype and localizes at or near the M-line, which is the thick filament attachment structure. Defects in the localization of the thick filament proteins myosin and paramyosin as well as the M-line component UNC-89/obscurin are observed in all unc-82(e1323) null mutant worms by the three-fold stage of embryogenesis (Hoppe et al. 2010). To better understand the role of unc-82, we have analyzed the distribution of components representing different “complexes” at the M-line, which contains many structural and probably signaling components. We found that membrane-proximal proteins such as UNC-112/kindlin were unaffected while membrane-distal proteins such as UNC-98, a thick filament component, showed large abnormal accumulations similar to those observed with the M-line component UNC-89/obscurin. These results indicate that UNC-82 activity is required for the proper organization of membrane-distal M-line components and suggest that UNC-82 targets an M-line or thick filament protein. To test which proteins may interact with UNC-82 directly, adults with ectopically localized myosin, paramyosin, and UNC-82::GFP were examined using antibody staining to determine which other proteins were recruited to the ectopic accumulations of these three proteins. Colocalization has been observed between UNC-82::GFP, myosin, paramyosin, and UNC-98, which is a potential chaperone required for the incorporation of paramyosin into thick filaments (Miller et al. 2008). A full-length paramyosin::GFP fusion expressed through heat shock in adults localizes to the thick filaments in wild-type adults, but fails to completely incorporate into filaments in unc-82 null mutants. Instead, much of the paramyosin is localized in numerous aberrant accumulations within the cytoplasm. Taken together, these results suggest that UNC-82 physically interacts, either directly or indirectly, with one or more of these three proteins: myosin, paramyosin, and UNC-98. These closely associated proteins may be targets of UNC-82 kinase activity, regulators of UNC-82, or possibly effectors of UNC-82 function.
Contact: [email protected] Lab: Hoppe
Poster Topic: Cell Biology 55 A LET-23 localization and expression screen identifies a novel mechanism of EGFR regulation through Ezrin/Radixin/Moesin proteins Juan Escobar Restrepo1, Peter Gutierrez1, Andrea Haag1, Alessandra Buhler1, Christina Herrmann1, Maeva Langouet1, David Kradolfer2,1, Erika Frohli1, Attila Stetak3,1, Alex Hajnal1 1Institute of Molecular Life Sciences. University of Zurich, Zurich, Switzerland, 2Swedish University of Agricultural Sciences. Uppsala BioCenter. Sweden, 3University of Basel, Basel, Switzerland C. elegans let-23 encodes the sole member of the ErbB family of receptors and is required for the formation of the hermaphrodite vulva. LET-23 is expressed at the plasma membrane of the six epithelial Vulval Precursor Cells (VPC) where is retained at the baso-lateral membrane by interacting with its C-terminal PDZ binding motif to a ternary complex formed by the PDZ proteins LIN-2, LIN-7 and LIN-10. Baso-lateral localization of LET-23 in the VPCs is required for efficient interaction with LIN-3 EGF secreted by the anchor cell (AC) in the somatic gonad. We have screened an RNAi library of all genes exhibiting a Protruding Vulva (Pvl) phenotype for defects in receptor localization and/or expression using a functional LET-23::GFP reporter (poster A.Haag et. al). We have identified ERM-1, the homologue of mammalian Ezrin, Radixin and Moesin proteins that link plasma membrane proteins to the actin cytoskeleton, as a negative regulator of the EGFR/RAS/MAPK pathway possibly by sequestering and stabilizing LET-23 in an inactive compartment at or near to the baso-lateral plasma membrane of the VPCs. The following lines of evidence support our model: (1) erm-1 (lf) or RNAi treatment against erm-1 causes a reduction in the baso-lateral LET-23::GFP signal. (2) erm-1(lf) suppresses the Vulvaless phenotype in reduction-of-function mutations in the LET-23/LET-60/MPK-1 pathway and enhances the Multivulva phenotype in a gain-of-function mutation in let-60 ras. (3) An ERM-1::mCherry translational reporter co-localizes with LET-23::GFP at the baso-lateral plasma membrane of the VPCs. (4) Recombinant ERM-1::GST interacts with LET-23 from worm extracts. The interaction is independent of LIN-7 or the PDZ binding motif of LET-23, suggesting that ERM-1 interacts with LET-23 through a complex distinct from the LIN-2/LIN-7/LIN-10 complex. (5) Fluorescence recovery after photobleaching with erm-1(lf) mutants showed a significantly faster recovery of basal LET-23::GFP compared to the wild-type. Taken together, our results indicate that ERM-1 inhibits the internalization of LET-23::GFP from the baso-lateral plasma membrane and/or the lateral diffusion within the plasma membrane. We propose that ERM-1 retains a fraction of LET- 23 in an inactive compartment, thereby competing with the activating LET-23/LIN-2/LIN-7/LIN-10 complex. ERM-1 may act as a buffer to prevent the immediate activation of the entire pool of baso-lateral LET-23 by LIN-3 and thus allow a prolonged LET-23 signal
Contact: [email protected] Lab: Hajnal
56 Poster Topic: Cell Biology Growth of Muscle Adhesion Complexes During Postembryonic Development Brandon Fields1, Nate Szewczyk2, Lewis Jacobson1 1Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260 USA, 2MRC/Arthritis Research UK Centre for Musculoskeletal Ageing Research, University of Nottingham, Derby, DE22 3DT UK The integrin-containing transmembrane attachment complexes of C. elegans are highly homologous to human focal adhesion complexes. In C. elegans, these form muscle-muscle attachments and anchor muscle contractile fibers to the hypodermis (dense bodies). Knockdown via mutation (reduces protein function) of either of two genes (unc-112 or unc-52) or RNAi (reduces amount of normal protein) of any one of fourteen genes encoding members of this complex provokes protein degradation in muscle cytosol and a variety of structural defects. In an unc-112ts mutant, paralysis occurs within 24h after shift of adults to nonpermissive temperature. Worms heterozygous for the unc-112ts mutation move at rates comparable to wild type, showing that the mutant protein does not “poison” adhesion complexes. Furthermore, acute RNAi treatment of adults causes sarcomere disruption and soluble protein degradation in 24h. Taken together, these observations imply a continuing requirement for new dense body proteins to maintain structural integrity. Does this reflect addition of new dense bodies, or protein accretion to existing dense bodies? Confocal microscopy of an unc-95::gfp strain and morphometric analysis were used to show that the number of dense bodies per muscle cell remains constant as the worm nearly doubles in length from L4 to mid-adulthood, while the mean size of each dense body increases. This implies that dense bodies are dynamic structures to which new proteins are added during postembryonic development and growth.
Contact: [email protected] Lab: Jacobson
Poster Topic: Cell Biology 57 CDK-1 inhibits meiotic spindle shortening and dynein-dependent spindle rotation in C. elegans Jonathan Flynn, Marina Ellefson, Francis McNally University of California, Davis In animals, the female meiotic spindle is positioned at the egg cortex in a perpendicular orientation to facilitate the disposal of half of the chromosomes into a polar body. In Caenorhabditis elegans, the metaphase spindle lies parallel to the cortex, dynein is dispersed on the spindle, and the dynein activators ASPM-1 and LIN-5 are concentrated at spindle poles. Anaphase-promoting complex (APC) activation results in dynein accumulation at spindle poles and dynein-dependent rotation of one spindle pole to the cortex, resulting in perpendicular orientation. To test whether the APC initiates spindle rotation through cyclin B–CDK-1 inactivation, separase activation, or degradation of an unknown dynein inhibitor, CDK-1 was inhibited with purvalanol A in metaphase-I–arrested, APC-depleted embryos. CDK-1 inhibition resulted in the accumulation of dynein at spindle poles and dynein-dependent spindle rotation without chromosome separation. These results suggest that CDK-1 blocks rotation by inhibiting dynein association with microtubules and with LIN-5–ASPM-1 at meiotic spindle poles and that the APC promotes spindle rotation by inhibiting CDK-1.
Contact: [email protected] Lab: McNally
58 Poster Topic: Cell Biology The C. elegans Uterine Seam Cell: a Model for Studying Nuclear Migration and Cell Outgrowth Srimoyee Ghosh, Paul Sternberg California Institute of Technology, Pasadena, CA, USA Defects in nuclear migration and cellular outgrowth are hallmarks of degenerative disease. Determining the molecular mechanisms that govern proper nuclear migration and cellular outgrowth may shed light on the pathologies of these diseases. We hope to identify new complexes that are involved in these processes by studying a cell where both nuclear migration and cellular outgrowth are occurring -- the uterine seam cell. The uterine seam cell (UTSE) connects the uterus to the body wall. It is a syncytium composed of nine nuclei that move outward in a bidirectional manner. The UTSE cell body stretches outward faster than its nuclei move, indicating that these two processes are distinct from one another. We are using a two- prong approach to identify the molecular mechanisms necessary for proper nuclear migration and cell outgrowth in UTSE development. Initially, we looked for cues from other parts of the uterus that influence UTSE behavior. Using ablation experiments we saw that two types of epithelial cells that line the uterine lumen, uterine toroid 1 and uterine toroid 2, are necessary for proper UTSE development. We also used a candidate screen to identify genes involved in UTSE nuclear migration and cell outgrowth. Not surprisingly, the KASH protein UNC-83, the SUN protein UNC-84, and its corresponding nuclear anchoring protein, ANC-1, are necessary for proper UTSE nuclear migration. Genes involved in extracellular matrix formation, such as the alpha integrin INA-1, and the laminin EPI-1 also play a role. Based upon expression studies we tested astacins, a class of zinc metalloproteases with no known function in migration or outgrowth. Two of these astacins, NAS-22 and NAS-21, had effects on UTSE development. We are currently determining the mechanism by which these genes ensure proper UTSE nuclear migration and cell outgrowth.
Contact: [email protected] Lab: Sternberg
Poster Topic: Cell Biology 59 Cadherin FMI-1 Maintains the Structure of the PVD Mechanosensory Neurons Julie Grimm, Benjamin Podbilewicz Technion Institute of Technology, Haifa, Israel C. elegans arborized sensory neuron--the PVD--is complex but highly stereotyped. It must innervate a large area without leaving gaps that could endanger the survival of the nematode. On the other hand, the dendrites should not be overly elaborate or hindered by unnecessary connections. The process of growth, retraction and overall maintenance during the development and maturation of the PVD may provide insight into how more complex neuronal circuits function. Recently, the seven transmembrane cadherin FMI-1 was implicated in PVD morphology (Oren-Suissa, M. , PhD thesis, 2011). FMI-1 is known to be important for follower axon navigation in C. elegans (Steimel et. al, 2010) as well as synapse morphology (Najarro et. al, 2012). It was found that mutants lacking the extracellular domain of FMI-1 showed defects in follower axon navigation. However, the intracellular domain was crucial for pioneer axon migration, which suggests dual or even multiple functions for the cadherin. FMI-1’s Drosophila ortholog, flamingo (fmi-1), was also found to be crucial for neuronal development (Kimura et. al, 2006). Kimura et. al found that fmi-1 null mutants exhibited extensive dendritic branching--but only in their sensory neurons. Our findings suggest that FMI-1 may have the opposite effect in the PVD of C. elegans. Work on the null allele tm306 has shown that without FMI-1 the PVD is severely retarded in growth. It appears that this phenotype becomes more pronounced with age and may even result from excessive retraction or degeneration of branches; however this aspect needs further investigation. Our aim is to understand the role of FMI-1 in PVD development and maintenance, and also determine if and how it interacts with other known PVD modifying proteins. Live imaging of PVD development during different life stages and recovery from dendritic injury will help elucidate the importance of FMI-1 in neuronal maintenance. Furthermore, using the various alleles now available to us, as well as fmi-1 constructs containing the full gene as well as engineered constructs missing various domains, we can understand not only if fmi-1 is required, but also which aspects are required for what steps of dendritic maintenance. Finally, and to merge this work with previous studies of the lab, we plant to evaluate which pathway FMI-1 functions in: in the same pathway as dendritic sculptors identified in our lab EFF-1 or NHR-25, or in parallel?
Contact: [email protected] Lab: Podbilewicz
60 Poster Topic: Cell Biology Two Functional Domains in C. elegans Glypican LON-2 Can Independently Inhibit DBL-1 Growth Factor Signaling but Require Accessory Moieties Suparna Bageshwar, Tina Gumienny Texas A&M Health Science Center College of Medicine, College Station (TX), USA Glypicans are GPI-linked proteoglycans with regulatory roles in several intercellular signaling pathways. How their structural complexity specifies function, including regulating Transforming Growth Factor-β (TGF-β) signaling, is a significant, but unresolved question relevant to both developmental and disease biology. We have previously established that C. elegans glypican LON-2 negatively regulates body size signaling by DBL-1, a TGF-β superfamily member, and binds TGF-β members (Gumienny et al., Current Biology (2007) 17(2): 159-164). We examined the functional requirements for glypican regulation of body length by DBL-1, a C. elegans TGF-β superfamily member. We provide evidence that two parts of C. elegans glypican LON-2 can independently inhibit DBL-1 signaling in vivo: the N-terminal furin protease product and the heparan-sulfated C-terminal region. While these two parts are each sufficient for LON-2 activity, furin cleavage of LON-2 into two parts is not required for LON-2 to inhibit DBL-1 signaling. While the C-terminal protease product is dispensable for LON-2 minimal core protein activity, it does affect the localization of LON-2. The glycosyl-phosphatidylinositol (GPI) membrane anchor is also not absolutely required for LON-2 core protein activity, but is required for the heparan-sulfated C-terminus to function. Furthermore, we show that an RGD protein-protein interaction motif in the LON-2 N-terminal domain is necessary for LON-2 core protein activity. Our work supports a model that LON-2 inhibits TGF-β signaling by acting as a scaffold for growth factor and an RGD-binding protein. In the context of the native LON-2 glypican, the N-terminal protein core and heparan sulfate side chains may together specify growth factor regulatory activity, facilitated by the GPI anchor and the RGD protein-protein interaction accessory moieties.
Contact: [email protected] Lab: Gumienny
Poster Topic: Cell Biology 61 Mutational Analysis of Residues Required for Activation the UNC-82 Serine-Threonine Kinase Jason Kintzele, Pamela Hoppe Western Michigan University, Kalamazoo, Michigan, United States C. elegans UNC-82 is a member of the AMPK related kinase family (AMPK-RK), which contains at least 12 genes related to AMPK (reviewed by Bright, 2009). Although AMPK was originally identified as a kinase that monitored cellular energy levels, other members of the AMPK-RK family have several other roles in the cell, including establishment of cell polarity, cytoskeletal organization, and neuronal pathfinding (Kemphues et al. 1988, Kim et al. 2010, Chartier et al. 2011). The C. elegans gene unc-82 is required for myosin organization in the striated body wall muscles (Hoppe et al. 2010). The vertebrate orthologs of C. elegans UNC-82 are the AMPK-RK family members ARK-5/NUAK1 and SNARK/NUAK2, which in human cell lines have been implicated in cancer cell survival, cellular stress responses, cellular motility and metabolic disorders (Suzuki et al. 2004, Yamamoto et al. 2008, Legembre et al. 2004, Tsuchihara et al. 2008). The role of these proteins in normal development and physiology is unclear. In cell lines, both have been found to localize to the nucleus and to regulate the cytoskeleton (Kuga et al. 2008, Zagórska et al. 2010). Biochemical studies of the human enzymes (Lizcano et al 2004) suggest that most AMPK-RKs are activated by the upstream kinase LKB1 (the ortholog of C. elegans PAR-4). However, data from other cell lines suggest that an NDR kinase and or the AKT/PKB kinase may also be involved (Suzuki et al. 2005, Lizcano et al 2004). We are using C. elegans as a model system to determine the mechanism(s) of activation of kinase catalytic activity and to identify possible downstream targets of the UNC- 82/ARK-5/SNARK proteins. In current experiments, we have targeted conserved residues that have been implicated in regulation of kinase activity in vertebrate systems and are testing their requirement in proper patterning of myosin in body-wall muscle. The easily scoreable myosin disorganization phenotype of the unc-82 gene will also allow us to screen for possible upstream kinases required for UNC-82 activation.
Contact: [email protected] Lab: Hoppe
62 Poster Topic: Cell Biology Genetic Analysis of Calcium Regulation in the C. elegans Intestine Jocelyn Laboy, Kenneth Norman Albany Medical College, Albany NY USA Defecation in the nematode Caenorhabditis elegans is a readily observable ultradian behavioral rhythm that occurs once every 40-50 s and is mediated by rhythmic calcium oscillations in the intestinal epithelium. For this behavior, calcium release from the endoplasmic reticulum into the cell cytosol is dependent on the inositol-1,4,5-triphosphate receptor (IP3R). One proposed mechanism of action through which this occurs in the C. elegans intestine involves the PLC-gamma (PLC-3) mediated cleavage of phosphatidylinositol-4,5-bisphosphate (PIP2) into two products, diacylglycerol and IP3. Cleavage of PIP2 activates TRPM-family calcium channel, GON-2, and allows calcium ions into the cell (1). This calcium signal and the other cleavage product of PIP2, IP3, trigger the opening of IP3Rs to create a robust calcium transient. To further understand the mechanisms underlying intestinal calcium oscillations, we are investigating the role of two other mutants in this behavior, vav-1 and kqt-3. vav-1 encodes a conserved Rho/Rac-family guanine nucleotide exchange factor. VAV-1 is expressed in the C. elegans intestine, and the null mutant exhibits calcium related behavioral defects, such as lengthened defecation cycle period similar to plc-3 and gon-2 mutants. kqt-3 encodes a KCNQ- family potassium channel that is also expressed in the intestine and the null mutation results in an altered defecation cycle similar to plc-3 and gon-2. Using genetic and cell biological analyses, we are examining the hypothesis that kqt-3, vav-1, plc-3 and gon-2 act in a common signaling pathway to mediate IP3R calcium transients. Thus far, our preliminary analysis indicates that plc-3 and gon-2 act in a common pathway, which is consistent with a previous study (1); however, kqt-3 appears to act in a parallel pathway to regulate calcium oscillations in the intestinal epithelium. Since signaling pathways are well conserved, these studies should provide insight into the mechanisms underlying IP3R mediated calcium oscillations. 1. Xing J, Strange K., Am J Physiol Cell Physiol. 2010 Feb;298(2):C274-82.
Contact: [email protected] Lab: Norman
Poster Topic: Cell Biology 63 The Tubulin Deglutamylase CCPP-6 Functions Exclusively in Ciliated Dopaminergic Neurons in C. elegans Ethan Landes1, Brendan O’Flaherty1, Elizabeth De Stasio1, Peter Swoboda2, Brian Piasecki1 1Lawrence University, Appleton, (WI), USA, 2Karolinska Institute, Huddinge, Sweden Cilia are microtubule-based organelles that protrude from the cell surfaces of most eukaryotic cells. These complex organelles are utilized in a variety of sensory and motility- based processes, including olfaction, light perception, and fluid propulsion. InCaenorhabditis elegans, cilia are found exclusively at the terminal ends of the dendritic processes of 60 neurons in an adult hermaphrodite. These ciliated sensory neurons (CSNs) are completely non-motile and are utilized in a variety of behavioral processes including chemosensation, mechanosensation, and thermosensation. Recently, the tubulin modifying protein CCPP-1 has been implicated in the ciliogenic pathway of C. elegans (Curr Biol. 21: 1685-1694. 2011). CCPP- 1 and its paralog, CCPP-6, both function in the deglutamylation of a-tubulin, a posttranslational modification that affects the velocity of kinesin-II along ciliary microtubules. We are currently characterizing the ccpp-6 gene in C. elegans. A ccpp-6 gene to GFP translational fusion construct is expressed exclusively in cephalic neurons (CEMs), a class of CSNs that have been implicated in dopaminergic signaling in C. elegans.CCPP-6::GFP localizes to the cilium, dendrite, axon, and neuronal cell body but is excluded from the nucleus of these cells. We intend to identify how this gene affects the behavior of nematodes and the role that CCPP-6 plays in dopaminergic signaling.
Contact: [email protected] Lab: Piasecki
64 Poster Topic: Cell Biology Protein Sequences Within the UNC-82 S/T Kinase that Affect Subcellular Localization in Pharyngeal Muscle Latrisha Lane, Chiyen Wong, Caitlyn Carter, Pamela Hoppe Western Michigan University, Kalamazoo, Michigan, USA We are interested in the mechanisms underlying the establishment and maintenance of the contractile apparatus in muscle cells. In previous work we identified the UNC-82 serine/ threonine kinase, which is required for the organization of the myosin filaments and some M-line components of body-wall muscle. The human orthologs of UNC-82 are NUAK1/ARK5 and NUAK2/SNARK. In body-wall muscle, which is similar to skeletal muscle in vertebrates, UNC-82::GFP is located at or near the M-line, which is the site where myosin filaments attach (Hoppe et al., 2010). We are currently investigating the function of the UNC-82 kinase in pharyngeal muscle cells, which resemble cardiac muscle. In the pharynx muscle of an otherwise wild-type strain, UNC-82::GFP is detectable only near the apical plasma membrane, which is adjacent to the cuticle-lined pharyngeal lumen. Since these cells are single-sarcomere muscles that contain radially-arranged actin and myosin filaments, the UNC-82::GFP is distant from the myosin filaments, which lie in the central region of the cell. To investigate the protein sequences required for UNC-82 localization in pharyngeal muscle, we generated GFP fusion constructs driven by the pharynx-muscle-specificmyo-2 promoter. A GFP fusion that contained only the N-terminal region including the kinase domain showed diffuse cytoplasmic localization. In contrast, a fusion containing the remaining ~1300 C-terminal amino acids localized to the nucleus. Point mutation of conserved catalytic residues within the kinase domain of a full-length construct resulted in UNC-82::GFP appearing in radially-arranged filamentous structures. These data suggest that a full-length, catalytically active protein is required for protein localization in pharyngeal muscle cells. The regulatory pathway of the UNC-82 ortholog NUAK1 in some human cell lines involves both phosphorylation in the kinase domain as well as phosphorylation of a threonine residue well outside the kinase domain in a putative Akt site. In C. elegans, the putative Akt site is located in an alternatively spliced exon that is included in transcripts made in pharyngeal muscle. We hypothesize that the regulatory pathway of NUAK 1 is conserved in UNC-82, and that activation of UNC-82 kinase activity requires phosphorylation at this site. To test this, we are currently performing site-directed mutagenesis of the threonine within the phosphorylation motif to test if mutant constructs have altered subcellular localization similar to that observed with kinase-dead mutant constructs.
Contact: [email protected] Lab: Hoppe
Poster Topic: Cell Biology 65 Characterization of vh45, a Candidate Regulator of Early to Late Endosomal Maturation Fiona Law, Shang Xiang, Christian Rocheleau McGill University, Montreal, Canada Diseases such as cancer, metabolic disorders and neuronal degeneration can originate from endocytic trafficking defects whereby improper management of cargoes, such as transmembrane receptors, result in prolonged activation of signaling pathways or toxic accumulation of aggregates. To understand how these defects arise, functions of the components controlling endocytic trafficking need to be characterized and clarified. As internalized cargo progresses along the endocytic pathway, Rab GTPase proteins associate with the enclosing vesicular membrane. These proteins alternate between inactivated GDP and activated GTP bound forms through the actions of Guanine nucleotide Exchange Factors (GEFs) and GTPase Activating Proteins (GAPs). Rab GTPases provide directionality to endocytic traffic and recruit effectors for mediating downstream processes. A vesicle carrying cargo destined for degradation requires Rab5 to be exchanged for Rab7 GTPase as it matures from an early to late endosome. This Rab conversion involves a multistep process where Rab5-GTP first recruits Rab7 GEF to activate Rab7, and Rab7-GTP then recruits a GAP to inactivate Rab5. Yet how this event is regulated and the mechanisms necessary for this event to occur are not fully understood. Using C. elegans, our lab has identified TBC-2 as a regulator of RAB-5/RAB-7 conversion. From biochemical and genetic studies, we found that TBC-2 is a RAB-5 GAP and that it requires RAB-7 to localize onto endocytic membranes. Results suggest that RAB-7 recruits TBC-2 to inactivate RAB-5 and therefore facilitates the RAB-5/RAB-7 conversion. Loss of tbc-2 function or expression of constitutively active RAB-5 result in the formation of large RAB-7 positive endosomes in intestinal cells. To find regulators of TBC-2 function, a forward genetic screen was conducted to isolate mutations that exhibit a tbc-2-like endosomal phenotype. One mutant, named vh45, displays large GFP::RAB-7 positive vesicles in intestinal cells. Data from complementation tests indicate that vh45 is not a mutation in tbc-2, but represents a new candidate regulator of endosomal maturation. I aim to determine which gene is affected by the vh45 mutation using a combination of whole-genome-sequencing and SNP mapping. I will further characterize the vh45 large vesicular phenotype through genetic and cell biological approaches to determine how vh45 disrupts endosomal maturation and whether it regulates TBC-2 localization or function.
Contact: [email protected] Lab: Rocheleau
66 Poster Topic: Cell Biology cil-5 Mediates Ciliary Receptor Localization and Sensory Function in C. elegans Kara Braunreiter1, Greg Fischer2, Casey Gabrhel1, Jamie Lyman Gingerich1 1University of Wisconsin-Eau Claire, Eau Claire, WI, U.S.A., 2University of Wisconsin-Madison, Madison, WI Primary (non-motile) cilia act as sensory antennae enabling cells to perceive the extracellular environment and respond appropriately. We are investigating the role of cil-5, a gene originally identified by its role in ciliary localization of the male-specific PKD-2 receptor. In addition to the PKD-2::GFP mislocalization observed in males, both male and hermaphrodite cil-5(my13) mutant C. elegans exhibit defective uptake of lipophilic dyes in head and tail neurons. These phenotypes suggest that cilium structure and function may be affected. Both sensitivity to chemicals and regulation of fat storage have previously been shown to be dependent on functional cilia. In chemotaxis assays, cil-5 mutants show hypersensitivity to some, but not all, volatile chemicals. In addition, analysis of intestinal fat droplets suggests that cil-5 mutants do not properly regulate fat storage. We are currently examining the integrity of the sheath cells which support ciliated neurons, using a whole genome sequencing approach to clone cil-5, and employing RNAinterference to identify additional factors involved in ciliary receptor localization.
Contact: [email protected] Lab: Lyman Gingerich
Poster Topic: Cell Biology 67 Neuroligin has Cell-autonomous and Non-autonomous Functions in C. elegans Jacob Manjarrez, Greg Mullen, Ellie Mathews, Jerrod Hunter, Jim Rand Oklahoma Medical Research Foundation, Oklahoma City, OK Neuroligins are postsynaptic adhesion proteins originally identified by their binding to presynaptic neurexins. Studies suggest that neuroligins function primarily in the maturation and/or maintenance of synapses. There are four neuroligin genes in humans, and mutations in the genes encoding neuroligin-3 and neuroligin-4 are associated with autism spectrum disorders (ASDs) in some families. We had previously examined the expression, localization and biological functions of neuroligin in Caenorhabditis elegans. C. elegans has a single neuroligin gene (nlg-1), and we had shown that nlg-1 null mutants are viable and are not deficient in any major motor functions. However, they are defective in a subset of sensory behaviors and sensory processing. nlg-1 mutants are also hypersensitive to oxidative stress (i.e., exposure to paraquat); this is an unexpected phenotype for a synaptic mutant. Like many other stress- sensitive mutants, nlg-1 mutants also have a reduced lifespan and an increased level of oxidative protein damage (Hunter et al., 2010). All of these mutant phenotypes are rescued by transgenic expression of mammalian neuroligin (human neuroligin-4 or rat neuroligin-1). The C. elegans and mammalian neuroligins, therefore, appear to be functionally equivalent (including having the ability to prevent or counteract oxidative stress). NLG-1 is normally expressed in ~20% of C. elegans neurons, including the pair of AIY interneurons. AIY cells receive direct synaptic input from different types of sensory neurons (e.g., chemosensory, thermosensory, nociceptive), and have been shown to play an important role in integration of sensory information. We find that expressing NLG-1 only in the AIY interneurons is sufficient to rescue all of the sensory deficits as well as the elevated oxidative stress present in nlg-1 mutants. However, we find that expressing NLG-1 ectopically in the AWA or AFD sensory neurons or the RIA or RIM interneurons (neurons which do not normally express this protein) can also rescue some mutant phenotypes. It is both noteworthy and surprising that expression of NLG-1 in only the two AIY neurons is sufficient to rescue all of the mutant phenotypes we examined. Equally noteworthy and surprising is the phenotypic rescue observed when the only NLG-1 in the animal is expressed ectopically in AWA or RIM cells that normally do not express NLG-1 - clearly a cell-non-autonomous effect. Supported by the Simons Foundation and Autism Speaks.
Contact: [email protected] Lab: Rand
68 Poster Topic: Cell Biology Genetic and Molecular Dissection of Novel Pathways Required for Nuclear Migration in the Model System C. elegans. Yu-Tai Chang, Shaun Murphy, Jonathan Kuhn, Minh Ngo, Daniel Starr University of California, Davis, University of California, Davis Moving the nucleus to an intracellular location facilitates many cell and developmental processes including mitotic and meiotic cell divisions, fertilization, cell migration, differentiation, and establishment of cellular polarity. The (Linker ofNucleoskeleton and Cy to skeleton) complexof SUN (UNC-84) and KASH (UNC-83) nuclear envelope proteins are involved in conserved mechanisms of nuclear migration. However, many nuclear migration events rely on independent mechanisms. To investigate novel mechanisms of nuclear migration, we utilize the behavior of larval P-cell nuclei in C. elegans. Failure in nuclear migration leads to P-cell death resulting in uncoordinated (Unc) and egg-laying defective(Egl) animals missing P-cell derived lineages. Null mutations in unc-83 or unc-84 inhibit nuclear migration by disrupting interactions between the nucleoskeleton and the cytoskeleton at 25°C, but at 15°C, P-cell nuclear migration occurs similar to wild-type. We therefore hypothesize that additional pathway(s) function synthetically to the unc-83/unc-84pathway to move P-cell nuclei at 15°C. To test our hypothesis, we isolated eightemu (enhancer of the nuclear migration defect of unc-83 or unc-84) alleles. Compared to unc-84null animals, emu; unc-84 double mutants had significantly fewer UNC- 47::GFP-positive GABA neurons (that were derived from P-cell lineages) at all temperatures. Using whole-genome sequencing, we have determined that the yc20 allele is a lesion in toca-1. We also found that fln-2 is an emu gene. toca-1(RNAi) and toca-1(tm2056) phenocopied yc20.
Moreover, the P-cell-specific rescue by thep hlh-3::toca-1::gfptransgene suggested that TOCA-1 functions to move nuclei in a cell-autonomous manner. TOCA-1 and FLN-2 have both been shown to be involved in actindynamics. Thus, we are currently examining actin organization in toca-1 and fln-2 mutant P-cells. In summary, TOCA-1 and FLN-2 function in a novel pathway for nuclearmigration.
Contact: [email protected] Lab: Starr
Poster Topic: Cell Biology 69 FLN-1/filamin is required for spermathecal contractility Jose Orozco, Ismar Kovacevic, Erin Cram Northeastern University, Boston, MA, USA The ability of tissues to sense and respond to mechanical forces is critical in development and normal physiology. We use the C. elegans spermatheca as a model to study mechanosensation in vivo. The spermatheca is a simple contractile tube that stretches to accommodate oocytes following ovulation. Following the entry of an oocyte, the spermatheca constricts in the distal to proximal direction to propel the fertilized oocyte into the uterus. The spermathecal cells are characterized by circumferential F-actin filaments, which play a critical role during constriction of the spermatheca. Previous work by our group has shown that FLN-1/filamin is required for maintenance of the F-actin cytoskeleton and for normal spermathecal constriction. Loss of FLN-1 results in a progressive disorganization of the F-actin cytoskeleton, suggesting that FLN-1 is required to reinforce the F-actin cytoskeleton. FLN-1 is required to initiate calcium signaling in the spermatheca and may play a structural role during spermathecal constriction. Calcium release in contractile cells stimulates myosin contractility. NMY-1/non-muscle myosin appears to be the only myosin expressed in the spermatheca, suggesting that NMY-1 is the main force generator. Indeed, nmy-1 depletion via RNAi, like fln-1 depletion, results in spermathecal constriction defects, and an abnormal spermatheca-uterine valve. NMY-1 contractility is controlled by the phosphorylation state of MLC-4/myosin light chain. MLC-4 activity is negatively regulated by MEL-11/myosin light chain phosphatase. mel-11(RNAi) results in a robust and striking spermathecal rupture phenotype due to hyperconstriction of the spermatheca around the embryo. The mel-11(RNAi) rupture phenotype is suppressed by loss of FLN-1. Ongoing work is focused on understanding how calcium signaling impinges on the myosin regulatory network, and whether filamin serves a structural and/or signaling role in the spermatheca.
Contact: [email protected] Lab: Cram
70 Poster Topic: Cell Biology Isolation of Mutations that alter Nile Red Staining in C. elegans Stephanie Burge1, Anthony Otsuka2,1 1Illinois State University, Normal, IL, USA, 2University of Hawaii at Hilo, Hilo, HI, USA There has been considerable interest in changes in gene activity that alter Nile red staining (Asrafiet al. Nature, 421:268-271, 2003). Some of these genes are involved in insulin regulation and lipid accumulation. We employed standard ethylmethane sulfonate mutagenesis to identify mutants that alter Nile red staining. Snip-SNP mapping was used to position several mutations on the genetic and physical maps. Further characterization was conducted based on the Nile red phenotype, light scattering phenotype, confocal microscopy, and thin layer chromatographic analysis of lipids. Optical sectioning by confocal microscopy revealed different sizes and patterns of lipid droplets in the mutants. Thin layer chromatography of lipids from the mutants demonstrated altered lipid profiles. In studies on longevity, one mutant showed a small, but significant, increase in life span. Because of the power ofC. elegans genetics and the available molecular tools, this system is well suited to the study of lipid accumulation.
Contact: [email protected] Lab: Otssuka
Poster Topic: Cell Biology 71 Epithelial Dynamics During the G1-to-G2 Pore Cell Swap in the Excretory System Jean Parry, Amanda Zacharias, Hasreet Gill, John Murray, Meera Sundaram University of Pennsylvania, Philadelphia, PA, US Epithelial cells from the epidermis, rectum, and excretory system have all been shown to dedifferentiate and migrate away to form neurons during the course of normal C. elegans development (Jarriault et al., 2008, Sulston and Horvitz, 1977). These events resemble classic epithelial-to-mesenchymal transition (EMT) and provide simple models for investigating the genetic control of epithelial junction dynamics and cell fate plasticity. The excretory system is comprised of three unicellular epithelial tubes connected in tandem, the canal cell, duct cell, and pore cell. The excretory pore cell is initially formed by the G1 cell during embryonic development. During mid L1, the G1cell will lose its epithelial characteristics and migrate towards the head, eventually dividing to produce two neuronal daughters. As G1 withdraws, it is replaced by the neighboring G2 cell. This process involves loss of the G1 autocellular and intercellular junctions, and remodeling of duct cell junctions to connect to a new partner. Incredibly, this transition occurs several hours after the excretory system has begun functioning. Newly discovered markers for the excretory system allowed us to observe this programmed EMT-like event in vivo. By fluorescently tagging the duct and pore cytoplasm (dct-5p::mCherry), junctions (AJM-1::GFP), and canal and duct lumen (VHA-5::GFP), we can perform live imaging of the G1/G2 swap. This imaging has revealed a highly stereotyped sequence of events that occur with sharp temporal precision over the course of a single hour in development. A forward mutagenesis screen in our lab was recently performed to identify prospective mutants in which the G1 cell does not withdraw from the excretory system or in which other aspects of the pore swap go awry, giving us a molecular entryway into this dynamic process.
Contact: [email protected] Lab: Sundaram
72 Poster Topic: Cell Biology The Arp2/3 activator WAVE/SCAR Promotes Clathrin Mediated Endocytosis in the Polarized C. elegans Intestinal Epithelia Falshruti Patel, Martha Soto UMDNJ-RWJMS Cells must internalize proteins and other molecules from their surfaces to survive. Studies in single-celled yeasts demonstrate the essential role of the branched actin nucleator, Arp2/3, and its activating nucleation promoting factors (NPFs) in the process of invagination from the cell surface through Clathrin-Mediated Endocytosis (CME). However, some mammalian studies have disputed the role of F-actin and Arp2/3 in CME in multicellular organisms. We investigated the role of Arp2/3 during endocytosis in C. elegans, a multicellular organism with polarized epithelia. The Arp2/3 activator, WAVE/SCAR, is essential for C. elegans embryonic morphogenesis, which was attributed to its ability to promote cellular protrusions. However, depletion of WAVE/SCAR alters junctional maturation, suggesting processes beyond protrusion formation are disrupted. We have shown that loss of the WAVE complex components lead to progressive defects in intestinal lumen morphogenesis and altered distribution of Apical Junction proteins, which suggested a role for WAVE in maintenance of polarity. We show here that loss of WAVE complex components severely disrupts the distribution of Transferrin Receptor, a protein that is internalized via CME. We find that the WAVE complex components and proteins involved in CME are mutually dependent for proper enrichment at the apical region of the C. elegans intestine. Consistent with these observations of WAVE/SCAR’s role in CME, one of the components of WAVE complex, GEX-3, interacts with dynamin in yeast-two hybrid studies. Further, the TOCA/F-BAR endocytosis proteins biochemically interact with the WAVE/ SCAR complex in mammals and C. elegans. We propose that WAVE-Arp2/3 dependent actin nucleation promotes CME at the apical intestinal epithelium and that altered CME contributes to the apical morphogenesis defects of WAVE mutants.
Contact: [email protected] Lab: Soto
Poster Topic: Cell Biology 73 Visualizing Dynamics of Meiotic Prophase Chromosome Structures Divya Pattabiraman1, Marc Presler2, Grace Chen1, Anne Villeneuve1 1Stanford University, Stanford, California, USA, 2Harvard University, Cambridge, Massachusetts, USA The synaptonemal complex (SC) is a highly-ordered proteinaceous structure that assembles at the interface between aligned homologous chromosome pairs during meiotic prophase. Although EM images of SCs give the impression of a rigid, scaffold-like structure, recent studies suggest that the SC may be much more dynamic than previously appreciated. We are investigating the dynamics of the SC structure using FRAP (fluorescence recovery after photobleaching) to visualize exchange of SC components within assembled SCs during meiotic prophase. The C. elegans system is particularly well-suited for this analysis, as the dispersal of pachytene chromosomes around the periphery of the nucleus, surrounding a centrally located nucleolus, makes it possible to bleach a portion of the SCs within a given nucleus while leaving the remainder of the SCs unbleached, providing both internal controls and imaging landmarks. Further, several different nuclei can be bleached and monitored in a single experiment. We have developed a protocol for FRAP analysis in pachytene nuclei of intact worms, using a strain expressing a functional GFP-tagged version of SYP-3, a component of the SC central region. Using this approach, we have revealed a previously hidden dynamics of the SC structure. We detect significant recovery of GFP::SYP-3 within 10 minutes after photobleaching, and recovery approaches a maximal value by 1- 1.5 h. In many experiments, recovery in the bleached portion of a partially bleached nucleus occurs concomitant with diminishing of signal in the unbleached portion of the same nucleus (relative to adjacent unbleached control nuclei), implying exchange of subunits between SCs. The observed time scale of recovery is slower than that seen for an oocyte nucleoplasmic protein (on the order of a few seconds) or for microtubules in the first mitotic spindle (on the order of a few minutes). However, it is much faster than that observed for components of the nuclear pore scaffold, which do not turnover in differentiated post-mitotic cells. Moreover, the observed rate of subunit exchange raises the possibility that the SCs may undergo complete turnover of their subunits during the duration of the pachytene stage. Thus, this structure has the potential to undergo substantial remodeling and reorganizing in response to different ongoing events of meiotic prophase.
Contact: [email protected] Lab: Villeneuve
74 Poster Topic: Cell Biology CRL2/LRR-1 E3-Ligase Prevents Progression Through Meiotic Prophase in the Adult C. elegans Germline Julien Burger1, Jorge Merlet1, Nicolas Tavernier 1, Benedicte Richaudeau1, Asja Moerkamp2, Rafal Ciosk2, Bruce Bowerman3, Lionel Pintard1 1Institut Jacques Monod, CNRS, Paris, France, 2Friedrich Miesher Institute For Biochemical Research, Basel, Switzerland, 3Institut of Molecular Biology, University of oregon, Eugene, US Precise control of the transition from self-renewal to terminal differentiation in stem cells is critical to maintain a balance between cell populations: an excess of stem cell self-renewal can lead to tumourigenesis, whereas an excess of differentiation can deplete the stem-cell pool. In the adult Caenorhabditis elegans germline, Notch signals emanate from the somatic distal tip cell to maintain germline stem cells (GSCs) in a proliferative state by repressing the expression of meiotic promoting factors. In this study, we show that the ubiquitin-proteolytic system act synergistically with the Notch pathway to prevent meiotic entry. Using a novel temperature-sensitive allele of the cul-2 gene, we found that the CUL-2 RING E3 ubiquitin ligase in combination with the Leucine Rich Repeat 1 substrate recognition subunit (CRL2/ LRR-1) negatively regulates the transition from the mitotic zone of the germline to the meiotic programme of chromosome pairing, synapsis, and recombination.
Contact: [email protected] Lab: Pintard
Poster Topic: Cell Biology 75 Regulated Nucleocytoplasmic Shuttling of SPAT-1/BORA Coordinates CDK-1 and PLK-1 Activation For Proper Mitotic Entry in the Early C. elegans Embryo Nicolas Tavernier 1, Anna Noatynska2, Julien Burger1, Costanza Panbianco2, Jorge Merlet1, Benedicte Richaudeau1, Emmanuelle Courtois1, Thibaud Leger1, Monica Gotta2, Lionel Pintard1 1Institut Jacques Monod CNRS, Paris , 2CMU, University of Geneva Acquisition of lineage-specific cell cycle duration is critical for metazoan development. In early C. elegans two-cell stage embryos, the anterior AB blastomere divides systematically before the posterior P1 blastomere and this asynchrony of cell division appears critical for proper embryonic development. Previous work established that asymmetric localization of the polo-like kinase PLK-1 promotes precocious mitotic entry in AB but it remains unclear how PLK-1 is regulated. Here we identify a positive feedback loop that coordinates PLK-1 and CDK-1 that involves tight regulation of the PLK-1 activator SPAT-1/Bora. We show that SPAT-1 is a nucleocytoplasmic shuttling protein containing functional nuclear localization (NLS) and nuclear export (NES) sequences. CDK-1 phosphorylates SPAT-1 presumably in the nucleus on multiple phosphorylation sites including a polo-docking site (S251), which is adjacent to the nuclear localization signal (NLS). Phosphorylation of S251 residue has two functions: first it orients the shuttling of SPAT-1 towards the cytoplasm by inhibiting the NLS activity and second, it contributes to PLK-1 activation by promoting the interaction between SPAT-1 and PLK-1. Once activated in the cytoplasm, PLK-1 reinforces CDK-1 activation. In addition, PLK-1 phosphorylates and targets SPAT-1 for degradation, possibly to terminate the positive feedback loop and to recycle PLK-1. We propose that multisite phosphorylation of SPAT-1 might set the threshold of mitotic entry, and contribute to the robustness of cell cycle timing regulation in the early embryo.
Contact: [email protected] Lab: PINTARD
76 Poster Topic: Cell Biology PPFR-1 Phosphatase 4 subunit is a regulator of MEI-1/Katanin activity during meiosis that is rapidly targeted for degradation by CRL-3/MEL- 26 E3-ligase in the transition to mitosis in C. elegans Jose-Eduardo Gomes1, Benedicte Richaudeau1, Etienne Formstecher2, Paul Mains3, Lionel Pintard1 1Institut Jacques Monod, CNRS, Paris, France, 2HYBRIGENICS SA, Paris, France, 3Departments of Biochemistry & Molecular Biology/Medical Genetics University of Calgary, Canada Protein phosphorylation by kinases is one of the most widespread forms of post-translational modification in eukaryotes. Owing to the action of protein phosphatases, phosphorylation can be reversed. Whereas protein Kinases and their phosphorylation targets have received much attention, comparatively much less is known about the role and regulation of protein phosphatases. Here we present a thorough analysis of the function and regulation of PPFR-1, a regulatory subunit of a trimeric Protein Phosphatase 4 (PP4) complex during the meiosis to mitosis transition in C. elegans. We show that PPFR-1 positively regulates the microtubule- severing activity of the MEI-1/MEI-2 Katanin complex during meiosis. PPFR-1 dephosphorylates MEI-1 and thereby activates the complex, which facilitates the disassembly of the meiotic spindle during anaphase and proper extrusion of polar bodies. Importantly, like its target MEI- 1, PPFR-1 is degraded by the ubiquitin-proteolytic system after meiosis. PPFR-1 specifically interacts with MEL-26, the substrate recognition subunit of the CRL-3/MEL-26 E3-ligase and like MEI-1, accumulates at centrosomes during mitosis in mel-26(-) embryos. We conclude that CRL3/MEL-26 degrades both MEI-1 and its activating PPase presumably to ensure spatial regulation of the microtubule-severing activity of the katanin complex and its rapid inactivation during the meiosis-to-mitosis transition.
Contact: [email protected] Lab: Pintard
Poster Topic: Cell Biology 77 A Genetic Analysis of the Axon Guidance of the C. elegans Pharyngeal Neuron M1 Osama Refai, Evvi Rollins, Patrcia Rhos, Jeb Gaudet University of Calgary, Calgary, Alberta, Canada The guidance of axons to their correct targets within an organ is a critical step in development. During the pharyngeal development, the M1 motorneuron establishes an axon that spans the whole organ and encounter its different tissues. Thus, M1 is most likely receiving guidance and interacting with its neighbour cells e.g. muscles and glands. Electron micrographs show that the M1 axon bundles with the g1P gland projection through the anterior half of the pharynx (i.e. the procorpus). Ablation of glands results in defects of the M1 trajectory at the procorpus, suggesting that g1P is necessary for M1 guidance. Growth cone defective mutants e.g. unc-51, unc-119, unc-115 and unc-34 showed defects similar to these that was observed after killing the gland. Whereas, genes of the major guidance pathways e.g. unc-6, sax-3, vab-1, spm-1 and smp-2 appear to play a minor role in guidance of the M1 axon. To identify novel mutations that may affect M1 axon migration, we screened 5000 hapliod genomes in a forward genetic screen. We isolated 12 mutants with abnormal morphology of the M1 neuron including alleles for known genes such as unc-51 and rpm-1. Interestingly, we didn’t observe any guidance defect at the M1 trajectory within the isthmus, suggesting that this part of the axon is established in a non-guidance manner. Taken together, our results present a model where the M1 axon outgrowth involves two phases: a growth cone-independent phase in the isthmus, which is followed by a growth cone-dependent phase through the procorpus.
Contact: [email protected] Lab: Gaudet
78 Poster Topic: Cell Biology Using C. elegans to Explore the Role of Presenilin in Calcium Signaling Shaarika Sarasija, Kenneth Norman Albany Medical College, Albany, NY, USA Mutations in the genes encoding Presenilin-1 (PS1) and Presenilin-2 (PS2) occur in most early onset Familial Alzheimer’s Disease (FAD), a rare form AD. Despite the fact that altered presenilin activity has been known to have a role in Alzheimer’s disease pathology, the functional consequences of mutations in presenilins are controversial and hence not fully understood. In fact, mutations in presenilins have been implicated in such diverse functions as altered processing of beta-amyloid precursor protein, Notch signaling, calcium entry and calcium removal from the cytoplasm. Thus, the role of presenilins in Alzheimer’s disease has remained elusive. The classic hallmarks of Alzheimer’s disease pathology are the formation of amyloid plaques and neurofibrillary tangles. However, it is thought that altered cellular events, like unbalanced calcium signaling precedes the formation of these pathological markers. Importantly, the dysregulation of intracellular calcium signaling can lead to excitotoxicity and cell degeneration. To identify mechanisms that regulate intracellular calcium signaling, we are utilizingC.elegans to understand the role presenilins play in calcium regulation. The C. elegans genes sel-12 and hop-1 encode transmembrane domain proteins orthologous to human presenilins. We are interested in investigating whether mutations in sel-12 and/or hop-1 can alter calcium homeostasis in C. elegans. Thus far we have found that the sel-12 null mutant, ty11, is hypersensitive to the muscle cell acetylcholine receptor agonist, levamisole, and the acetylcholine esterase inhibitor, aldicarb, suggesting that the muscle of the sel-12 mutant is hyper-excitable. Additionally, we have found that the mitochondria in the muscle of sel-12 mutants are structurally disrupted. Mitochondria act as a significant cytosolic calcium buffer in cells and mitochondrial calcium overload can lead to their disruption. Furthermore, our preliminary data points to the rescue of the hypersensitivity of the ty11 mutant to levamisole when it is introduced into a ryanodine null mutant, unc-68 background, which further supports a role of SEL-12/presenilin in calcium regulation. To directly investigate calcium signaling in the muscle of sel-12 mutants, we will employ optogenetic tools to measure calcium transients upon muscle stimulation. Since signaling mechanisms are well conserved, using C. elegans as a model system to unravel the role presenilins have in calcium signaling should provide insight into the pathological conditions that arise in AD.
Contact: [email protected] Lab: Norman
Poster Topic: Cell Biology 79 Novel Roles For A Cell Adhesion Protein DYF-7 In C. elegans Body Size Determination Robbie Schultz, Tina Gumienny Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, TX, USA Cell adhesion is critical for all multicellular organisms. While gross disruption of cell contacts is lethal, mild or tissue-specific cell adhesion defects can lead to developmental disorders and contribute to diseases, including deafness and schizophrenia. One class of cell adhesion proteins is the zona pellucida (ZP)-domain proteins, a family of extracellular molecules defined by their ZP domain, a protein polymerization motif. My project’s goal is to establish C. elegans ZP-domain family member DYF-7 as a model to understand the molecular and cellular action of ZP-domain proteins during postnatal development. DYF-7 is a critical anchoring protein for adhering dendrites of sensory neurons during embryonic development [1]. Loss of dyf-7 function is non-lethal and leads not only to sensory perception defects, as the affected neuronal cells do not contact the environment properly, but also body size defects [2]. Body size development is strictly regulated in C. elegans through genetic, structural, and environmental components. The best studied of these genetic regulators is the TGF-β DBL-1 pathway. While previous studies have shown that DYF-7 is involved in neural tip anchoring, explaining its sensing defect, DYF- 7’s role in body size development remains unexplored. We found dyf-7 acts post-embryonically to regulate body size. dyf-7 is expressed in larvae and adults. To determine the mode of dyf-7’s regulation of body size, we performed epistasis analyses between dyf-7 and dbl-1 pathway members. Double mutants are significantly smaller than either single mutant, indicating dyf-7 regulates body length at least partially independent from the dbl-1 TGF-β pathway. However, we also showed that expression of a DBL-1 pathway reporter was altered in animals lacking endogenous dyf-7, suggesting that dyf-7 regulates dbl-1 pathway signaling. In addition to a role in DBL-1 signaling, we found that loss of dyf-7 affects the organization of the cuticle, a defect that could be caused by mild cell adhesion defects in the cells secreting the cuticle and could also contribute to the small body size phenotype. These results indicate that the cell adhesion protein DYF-7 regulates body size development post-embryonically through integration of independent and dependent mechanisms, including TGF-β DBL-1 pathway signaling, cuticle organization, and environmental sensation. 1. Heiman, M. and Shaham, S. (2009). Cell 137, 344-355. 2. Starich, T., et al. (1995). Genetics 139, 171-188.
Contact: [email protected] Lab: Gumienny
80 Poster Topic: Cell Biology DAF-16 Promotes Developmental Growth in Response to Persistent Somatic DNA Damage Michael Muller, Maria Ermolaeva, Laia Castells-Roca, Peter Frommolt, Sebastian Greiss, Jennifer Schneider, Bjorn Schumacher Cologne Excellence Cluster for Cellular Stress Responses in Aging- Associated Diseases (CECAD), Institute for Genetics, University of Cologne, Germany Congenital defects in genome maintenance systems cause complex disease phenotypes characterized by developmental failure, cancer susceptibility and premature aging. In contrast to well-characterized cellular DNA damage checkpoint mechanisms, it remains poorly understood how DNA damage responses affect organismal development and maintain functionality of tissues when DNA damage gradually accumulates with aging. Here we show that transcription- coupled repair defects that in human Cockayne syndrome patients lead to developmental growth defects and progeria, specifically impair somatic development upon UV damage in C. elegans. DNA repair proficient animals, in contrast, transiently arrest development. Employing comprehensive gene expression analysis we identified a network of insulin-like growth factor signalling (IIS) genes that responds to DNA damage during C. elegans development. We show that the FoxO transcription factor DAF-16 is activated in response to DNA damage during development while the DNA damage responsiveness of DAF-16 declines with aging. We demonstrate that DAF-16 alleviates DNA damage induced developmental arrest through differential activation of downstream target genes that contrasts its established role in the starvation response, and even in the absence of DNA repair promotes developmental growth and enhances somatic tissue functionality. We propose that IIS mediates developmental DNA damage responses and that DAF-16 activity enables developmental progression amid persistent DNA lesions and promotes tissue maintenance through enhanced tolerance of DNA damage that accumulates with aging.
Contact: [email protected] Lab: Schumacher
Poster Topic: Cell Biology 81 Purification and Characterization of Glyceraldehyde-3-Phosphate Dehydrogenase from Caenorhabditis elegans Valeria S. Valbuena, Megan Gautier, Justin Spengler, M. Banks Greenberg, M. Leigh Cowart, Katherine Walstrom New College of Florida, Sarasota, FL Glyceraldehyde-3-Phosphate Dehydrogenase (GPD) is the glycolytic enzyme that adds inorganic phosphate to its substrate so that net ATP production is possible later in the glycolysis pathway. C. elegans has four gpd genes. The genes gpd-1 and gpd-4 are nearly identical and mainly expressed in embryos, while the homologous gpd-2 and gpd-3 are expressed in postembryonic worms (Huang et al., 1989, JMB 206, 411). The postembryonic genes are involved in protection from anoxia (Mendenhall et al., 2006, Genetics 174, 1173) and are upregulated in dauers and long-lived daf-2 mutants (McElwee et al. 2006, Mech. Age. Dev. 127, 922). In this project, high yields of worm extracts were achieved by large-scale worm production in egg plates. GPD was purified from mixed populations of C. elegans using a new protocol that included gel filtration and Blue Sepharose CL-6B affinity chromatography. In comparison to the previous methods described by Yarbrough and Hetch (JBC 259, 14711, 1984), our purification resulted in a higher yield of enzyme. Based on the Yarbrough and Hetch results, we expect that our GPD sample consists mainly of the adult GPD-2 and GPD-3 enzymes. SDS-PAGE results showed that the affinity column fractions contained several bands, but control enzyme assays did not indicate the presence of contaminating activity. When stored at 2 °C, the partially purified enzyme retained its activity for over a week. The reaction conditions were optimized, and a pH near 8.5 was a critical condition for maximum GPD activity with the glyceraldehyde-3-phosphate (G3P) substrate. Kinetic assays with varying concentrations of G3P and NAD+ were performed, and the Km values were 0.3 mM and ~1 mM, respectively. * This project was funded by grants from the NCF Council of Academic Affairs and the NCF Dubois-Felsmann Student Research and Travel Endowment.
Contact: [email protected] Lab: Walstrom
82 Poster Topic: Cell Biology Three axonal guidance pathways differentially signal to the regulators of the actin cytoskeleton during axonal migration Andre Wallace, Yelena Bernadskaya, Martha Soto RWJMS-UMDNJ Neuronal development is controlled by multiple guidance cues which are responsible for orchestrating the directed growth and migration of axons and growth cones. In C. elegans, it is known that UNC-40/DCC, SAX-3/Robo and VAB-1/Eph are three of the main receptors governing this process but it is still not clear how they regulate the actin cytoskeleton. Recent work from our lab has shown that during embryonic morphogenesis, these axonal guidance receptors function to modulate the actin cytoskeleton through effects on the WAVE/SCAR complex. Subsequently, we hypothesize that during neuronal development, directed growth and migration of axons and growth cones are dependent on proper reorganization of the actin cytoskeleton by the WAVE/SCAR complex. To test this hypothesis, we employed gain- of-function mutations of these three axonal guidance receptors which are commonly used to identify downstream targets of these pathways. We examined how mutations of the WAVE/ SCAR complex affect axonal migration and cell body morphology of the AVM mechanosensory neuron. Depletion of WAVE proteins led to suppressed AVM defects in the SAX-3 gain-of- function mutant. On the other hand, AVM ventral migration defects in the myr::unc-40 gain- of-function were enhanced with the depletion of WAVE/SCAR proteins. Finally, depletion of WAVE/SCAR components had no effect on AVM defects in the VAB-1 gain-of-function mutation. These results suggest that SAX-3 signals through the WAVE/SCAR complex during neuronal development. In addition, our results propose that UNC-40 may be functioning in other WAVE/ SCAR-independent pathways to regulate neuronal development. Overall, these results provide a platform on which we can study how axonal guidance cues function in reorganization of the actin cytoskeleton during neuronal development.
Contact: [email protected] Lab: Soto
Poster Topic: Cell Biology 83 Microtubules and Fertilization: The MEI-1/Katanin mediated cytoskeletal transition from meiosis to mitosis in the developing embryo Sarah Beard, Paul Mains The University of Calgary, Calgary. AB, Canada During embryonic development, dramatic changes of the C. elegans cytoskeleton occur in the transition from meiosis to mitosis requiring precise regulation of molecules specific to each type of spindle. Defects in microtubule organization during development can result in tissue pathologies, aneuploidy or even cancer. The microtubule severing complex, MEI-1, is required in meiosis to keep the spindle small but is inactivated prior to mitosis. This inhibition of MEI-1 during mitosis is dependent on the MEL-26/CUL-3 E3 ubiquitin ligase complex targeting MEI-1 for degradation prior to mitosis. The first aim of the project is to measure anti-MEI-1 staining levels in several mutant strains to determine how known genes function relative to one another. We aim to establish a standardized method to measure antibody staining levels from images of the embryos. Another pathway, involving the anaphase promoting complex (APC) and the MBK-2/DYRK kinase, has been found to promote mitotic MEI-1 degradation in parallel to MEL-26 mediated degradation of MEI-1. We wish to decipher whether APC and MBK-2 act in parallel or sequentially relative to one other in this process. We are also interested in deciphering the exact role of CUL-2, another E3 ubiquitin ligase, that is previously known to prevent MEL-26 from accumulating during meiosis. Making double mutants should resolve whether CUL-2 is also the missing ligase for MBK-2 mediated MEI-1 degradation functioning in parallel to the MEL-26/CUL-3 pathway, or if CUL-2 acts sequentially as an upstream activator of MEL-26/CUL-3. The second aim of the project is to continue investigating potential regulators of the cytoskeleton in the transition from meiosis to mitosis. We will conduct targeted RNAi screens for missing components of the pathway such as kinases, ubiquitin ligases and substrate adaptors. For example, FEM-1, a substrate adaptor for CUL-2 E3 ubiquitin ligase involved in sex determination, could be potential candidate for MEI-1 regulation during embryonic development. This project will assist in decoding the key regulatory molecules of the developmental remodeling of the cytoskeleton and progressively work our way back to the initial triggers of the pathway at fertilization.
Contact: [email protected] Lab: Mains
84 Poster Topic: Cell cycle and cytokinesis Understanding Proteasomal Regulation of SZY-20 in the Centrosome Assembly Pathway Michael Bobian, Mi Hye Song Michigan Technological University, Houghton, MI, USA The centrosome is a critical mediator in the animal cell cycle and serves as the primary microtubule-organizing center. During the cell cycle, the centrosome orchestrates microtubule dynamics, and forms mitotic bipolar spindles that are critical for accurate chromosome segregation. In C. elegans, SZY-20 is a suppressor of ZYG-1, a functional homolog to the human kinase Plk4. SZY-20 plays a critical role in regulating centrosome size and duplication. This protein localizes both at the centrosome and in the cytoplasm in a cell cycle dependent manner. Centrosomal levels of SZY-20 are highest during prometaphase and metaphase. Loss of SZY-20 leads to increased centrosome size, abnormal cell divisions, a failure of polar body extrusion, cytokinesis failure, shortened metaphase spindles, and detached centrosomes from the nuclear envelope (Song et al., 2008). To further elucidate the role of SZY-20 in regulating centrosome assembly and cell cycle, we utilized proteomics to identify proteins complexed with SZY-20. Among them, we have identified proteasome components that are reproducibly pulled-down with SZY-20. Interestingly, the proteasome has been shown to function at the centrosome to regulate the cell cycle (Didier et al., 2007). Here, we seek to understand how the proteasome functions in association with SZY-20 to regulate centrosome assembly. We hypothesize that SZY-20 is negatively regulated by the proteasome. Since centrosomal levels of SZY-20 are antiphasic to those of proteasomal component levels during the cell cycle progression, the proteasome might influence SZY-20 levels to control centrosome size and assembly. We have begun to characterize the genetic interaction between SZY-20 and proteasomal components by RNAi-knockdown and confocal microscopy.
Contact: [email protected] Lab: Song
Poster Topic: Cell cycle and cytokinesis 85 Mitotic spindle proteomics reveals conserved Caenorhabditis elegans proteins potentially necessary for cytokinesis Mary Kate Bonner1, Daniel Poole1, Tao Xu2, Ali Sarkeshik2, John Yates III2, Ahna Skop1 1University of Wisconsin-Madison, Madison, WI, USA, 2Scripps Research Institute, La Jolla, CA, USA Cytokinesis is an important and fundamental process in the development of all organisms. The factors that establish the cleavage furrow have remained mysterious and have eluded many for over 130 years. In order to identify factors required for early steps in cytokinesis, mitotic spindles from synchronized Chinese Hamster Ovary (CHO) cells were isolated. Proteins enriched from isolated metaphase-enriched spindles were identified by multidimensional protein identification technology (MudPIT) in collaboration with the Yates Lab at Scripps. We identified 1155 proteins associated with the mitotic spindle at one or more peptide hit (Bonner et al., 2011). Comparison of our data to the previously published CHO midbody proteome (Skop et al., 2004) revealed 314 proteins in common and 841 proteins unique to the CHO spindle associated proteins. Gene Ontology (GO) analysis revealed that 27% of the spindle proteins were associated with membrane, microtubules, actin cytoskeleton or unknown classes. To identify factors necessary for the membrane-cytoskeleton remodeling during cytokinesis, we are currently screening the orthologs in Caenorhabditis elegans using feeding RNAi. We have identified cognate C. elegans genes that correspond to 71% of the CHO candidate proteins. Of these candidates, 30% were not assigned a phenotype in previous C. elegans cell division screens. Of the C. elegans candidate genes that had been identified in previous screens, 65% of these proteins had been attributed with an EMB phenotype. We are currently screening candidate genes by feeding RNAi. To do this, we are assaying for embryonic lethality and multi-nucleate phenotypes in early embryos. We will present the results from these screens.
Contact: [email protected] Lab: Skop
86 Poster Topic: Cell cycle and cytokinesis Non-random Segregation of Unpaired X Chromosomes in C. elegans Female Meiosis Daniel Cortes Estrada, Francis McNally UC Davis, Davis CA Attachment between homologous chromosomes during meiosis I is essential for accurate segregation of chromosomes. Surprisingly, humans with three X chromosomes (triploX) have normal fertility and give birth predominantly to children with a normal chromosome complement. If the unpaired X segregated randomly at meiosis I, triploX mothers would produce 50% triploX daughters. The low frequency of triploX offspring observed, around 11-14% of daughters, suggests that female meiosis possesses a mechanism that prevents the inheritance of the unpaired X chromosome. In the C. elegans him-8 mutant, which possesses two unpaired X chromosomes at meiosis I, a similar situation occurs. If the two univalent X chromosomes segregated randomly at meiosis I, him-8 mutants should produce 25% XO male, 50% XX hermaphrodite and 25% XXX progeny. Instead Hodgkin et al., reported 38% XO male, 56% XX hermaphrodite and 8% XXX progeny, suggesting that female meiosis in C.elegans also possesses a mechanism of selective removal of unpaired X chromosomes. We are currently testing the hypothesis that univalent X chromosomes are deposited into the first polar body with high frequency. Using live imaging and fixed immunofluorescence, we found that 95% of him-8 metaphase I spindles have 7 chromosomes whereas 100% of wild-type metaphase I spindles had 6 chromosomes; demonstrating that in him-8 worms both univalent X chromosomes are still present by metaphase I. At metaphase II, 100% of embryos had 6 chromosomes whereas 40% of him-8 metaphase II spindles had 5 chromosomes, 55% had 6 chromosomes and 5% had 7 chromosomes. These numbers suggest that univalent chromosomes are lost between metaphase I and metaphase II. 88% of him-8 anaphase I spindles had 1 or 2 lagging chromosomes whereas only 2% of wild-type anaphase I spindles had lagging chromosomes. Quantifying the fates of lagging chromosomes reveals that 65% of these are expelled into the polar bodyand 35% are retained in the embryo. Our results are consistent with a model in which univalent X chromosomes biorient at metaphase I but lag at anaphase I because cohesin between sister chromatids is not cleaved. The delayed, poleward movement of these bioriented univalents is biased toward the polar body end of the spindle. Preliminary data suggests that the contractile ring may be involved in resolution of lagging chromosomes through its normal polar body formation activity. We are currently testing the involvement of the contractile ring in biased resolution of lagging chromosomes.
Contact: [email protected] Lab: McNally
Poster Topic: Cell cycle and cytokinesis 87 Parallel mechanisms promote RhoA activation during polarization and cytokinesis in the early C. elegans embryo Yu Chung Tse1, Michael Werner1, Katrina Longhini1, Jean-Claude Labbe2, Bob Goldstein3, Michael Glotzer1 1University of Chicago, Chicago, IL, USA, 2Universite de Montreal, Montreal, Canada, 3University of North Carolina at Chapel Hill, Chapel Hill, NC, USA The GTPase RhoA is a central regulator of cellular contractility in a wide variety of biological processes. During these events, RhoA is activated by guanine nucleotide exchange factors (GEFs). These molecules are highly regulated to ensure that RhoA activation occurs at the proper time and place. During cytokinesis, RhoA is activated by the RhoGEF ECT-2. In human cells, ECT-2 activity requires its association with CYK-4, which is a component of the centralspindlin complex. In contrast, in C. elegans embryos, not all ECT-2 dependent functions during cytokinesis require CYK-4. Here, we identify a novel protein, NOP-1, that functions in parallel to CYK-4 to promote RhoA activation. We use mutations in nop-1 and cyk-4 to dissect cytokinesis and cell polarization. NOP-1 makes a significant, albeit largely redundant, contribution to cytokinesis. In contrast, NOP-1 is required for RhoA activation during the establishment phase of polarization.
Contact: [email protected] Lab: Glotzer
88 Poster Topic: Cell cycle and cytokinesis ATX-2, the C. elegans ortholog of ataxin 2, is necessary for cytokinesis. Megan Gnazzo, Ahna Skop University of Wisconsin-Madison Mutations in ataxin-2 give rise to the devastating neurodegenerative disease spinocerebellar ataxia type 2 (SCA2). In SCA2 an increased expansion of a CAG repeat encoding a polyglutamine tract in ataxin-2 is observed. The human gene ataxin-2 has also been implicated in an increased risk for amyotrophic lateral sclerosis (ALS). The reason by which the mutations in ataxin-2 lead to neurodegeneration are unknown, and the cellular functions of ataxin-2 remain unclear. Our lab identified the ataxin-2 gene from isolated mammalian midbodies and the corresponding C. elegans ortholog, ATX-2, displayed defects in cytokinesis (Skop et al, 2004). To determine why ATX-2 leads to cytokinesis defects, we are characterizing its role in the early C. elegans embryo. Bioinformatic analysis revealed that ATX-2 is very highly conserved. ATX-2 contains several RNA binding motifs suggesting a role for ATX-2 in the control of mRNA translation. Local control of mRNA translation has been proposed as a mechanism for regulating synapse plasticity. We hypothesize that ATX-2 may play a role in mediating the local translation of RNAs found in the midbody during cytokinesis. We have identified four ATX-2 isoforms and would like to know how these isoforms function throughout embryonic development. We are currently constructing GFP constructs to two of the identified isoforms (full-length and C-terminal) to determine the localization of these constructs in the early embryo. Live imaging analysis has revealed defects in both meiotic and mitotic cytokinesis. Here, the second polar body often fails to be extruded and during the first division late failures in cytokinesis are observed. We will present our current analysis of ATX-2 function in cytokinesis.
Contact: [email protected] Lab: Skop
Poster Topic: Cell cycle and cytokinesis 89 Identification and Characterization ofmel-15 as a New Paternal-effect Lethal Mutant in C. elegans Aimee Jaramillo-Lambert, Kathryn Stein, Andy Golden LBG/NIDDK/NIH During fertilization the oocyte and the sperm fuse, restoring the somatic chromosome number and initiating zygote development. Oocytes and sperm are both products of meiosis, however, they are highly differentiated with distinct characteristics unique to their roles in fertilization and early embryogenesis. Oocytes are large, sedentary cells that provide a haploid genome and large stockpiles of RNA and proteins necessary for early embryonic cell divisions until zygotic transcription is initiated. Sperm are small, motile cells streamlined for fertilization. Sperm are generally thought of as only providing a haploid genome, but sperm also supply centrosomes and the signal for the initiation of the embryonic program. There is evidence that sperm contain other factors required for embryogenesis. Absence of these paternally provided components results in embryonic lethality (paternal-effect embryonic-lethal). In C. elegans only a single paternal-effect lethal (PEL) gene has been characterized, spe-11. SPE-11 is a novel cytoplasmic protein supplied by the sperm. The absence of functional SPE-11 results in embryonic failure at early stages. Although important, the molecular details of the sperm’s contribution to early embryogenesis remain largely unknown. We are interested in identifying and characterizing other potential paternal-effect candidates. Previous genetic screens identified embryonic lethal mutants that could be rescued by wild-type males indicating a sperm defect. Currently, we are characterizing one of these mutants, mel-15. Initial analysis confirms that sperm from mel-15 mutant males produce dead embryos, even when fertilizing wild-type oocytes. In addition, preliminary characterization indicates that mel-15 male sperm lack DNA and may have aberrant tail development. We are currently in the process of determining the molecular identity of mel-15.
Contact: [email protected] Lab: Golden
90 Poster Topic: Cell cycle and cytokinesis RNA-binding Proteins ATX-2/PAB-1 Regulate Centrosome Assembly and Size Sarah Mets, Kelly Haynes, Eric Vertin, Dongyan Zhang, Mi Hye Song Michigan Technological University, Houghton, MI, USA Centrosomes are critical sites for controlling microtubule dynamics, and exhibit dynamic changes in size, promptly responding to changing cellular demands during the cell cycle. As cells progress to mitosis, centrosomes recruit more PCM (maturation) and nucleate more microtubules to form bipolar spindles. The szy-20 gene encodes a novel centrosome- associated RNA-binding protein that negatively regulates ZYG-1. szy-20 mutants possess enlarged centrosomes which lead to abnormal microtubule processes and embryonic lethality. Thus, SZY-20 limits centrosome size by negatively regulating the recruitment of centrosome components. SZY-20 contains putative RNA-binding domains; mutating these domains perturbs RNA-binding by SZY-20 in vitro and its capacity to regulate centrosome size in vivo. It has been shown that a number of RNA-binding proteins associate with centrosomes and microtubules, and that they function to assemble mitotic spindles. To further understand the roles of SZY-20 and RNA-binding proteins in the regulation of centrosome assembly and size, We used proteomics to identify factors complexed with SZY-20, and identified other known RNA-binding proteins. By RNAi knockdown, we found that many SZY-20 interacting factors affect cell cycle and centrosome behavior. Some of these factors also exhibit strong genetic interactions with szy-20 and/or zyg-1. We are currently characterizing RNA-binding proteins (ATX-2 and PAB-1) using the range of genetic, cell biological, biochemical and optical approaches, to understand how this RNA-binding protein complex coordinates with SZY-20 and ZYG-1 to achieve proper centrosome assembly and size.
Contact: [email protected] Lab: Song
Poster Topic: Cell cycle and cytokinesis 91 ubc-25 encodes a conserved ubiquitin-conjugating enzyme that is required for developmentally controlled cell cycle quiescence David Tobin1, Sarah Roy1, Mako Saito1,2 1Dartmouth Medical School, Hanover, NH, USA, 2Norris Cotton Cancer Center, Lebanon, NH, USA Temporal control of cell cycle regulation is crucial in developing multi-cellular organisms, however the mechanisms that coordinate this process are largely unknown. Forward and reverse genetic screens in our lab identified genes necessary for the quiescence of vulva precursor cells (VPCs) in C. elegans, which temporarily exit the cell cycle at their generation in L1 until resuming divisions in L3. Notably, this screen identified thecdc-14 phosphatase as a novel regulator of quiescence in several cell lineages, contrary to its requirement for mitosis in yeast. Over 100 genes were identified our screens, most of which have not been previously implicated in regulating cell cycle quiescence. The screen identifiedubc-25 , a gene encoding a conserved ubiquitin-conjugating enzyme, as a positive regulator of VPC quiescence. ubc-25(ok1732) mutants display hyper-proliferation of intestinal nuclei, indicating its requirement for quiescence of intestinal nuclei. Hyper- proliferation of ubc-25(ok1732) intestinal nuclei is enhanced by cdc-14(he141) or fzr-1(ku298) mutant alleles, or by lin-35 RNAi, suggesting that ubc-25 acts in a distinct genetic pathway from these known negative regulators of G1/S. In contrast, RNAi of the SCF component cul-1 produces excess intestinal nuclei, which is not enhanced by the ubc-25(ok1732) mutation, suggesting that ubc-25 and cul-1 act in a linear genetic pathway. Loss of ubc-25 activity partially restores intestinal nuclear divisions in cyd-1(he112) mutant animals, and is further rescued when combined with RNAi of either cdc-14 or lin-35. These data are consistent with the hypothesis that cyd-1 promotes cell cycle entry through inhibiting parallel negative regulators of G1/S such as lin-35, cdc-14, and ubc-25. Our genetic evidence places ubc-25 in the SCF mediated pathway to control G1/S. We propose that ubc-25 is involved in degrading CYE-1. Consistent with this hypothesis, RNAi of ubc-25 restores intestinal nuclear divisions in cye-1(eh10) null mutants, which may occur through stabilization of maternally contributed CYE-1 protein. We currently are using western blot analysis to directly test the role of ubc-25 in CYE-1 stability, and generating UBC-25::GFP expressing strains to determine its localization during development and cell division.
Contact: [email protected] Lab: Saito
92 Poster Topic: Cell cycle and cytokinesis NAD salvage biosynthesis and programmed cell death; a new model for investigating cell death mechanisms Matt Crook, Wendy Hanna-Rose The Pennsylvania State University, University Park, (PA), USA We have developed a new model for studying programmed cell deaths in response to insult in C. elegans. PNC-1 converts nicotinamide (NAM) to nicotinic acid (NA) and is the first enzyme in the salvage pathway for biosynthesis of NAD+. In pnc-1 mutants two cell types, uv1 and OLQ, die in response to distinct insults and using mechanisms distinct from each other and from mec-4-induced necrosis. The four uv1 cells, at the uterine-vulval junction, die with a typical necrotic morphology in response to excess NAM, a by-product of NAD+ consumption that is cleared by PNC-1 activity. In stark contrast to mec-4d induced touch cell necrosis, uv1 death is unaffected by gross manipulation of cytoplasmic Ca2+ concentration or knockdown of genes involved in autophagy and nutrient sensing. uv1 cell death is, however, rescued by overexpression of LIN-3 or overactivation of its receptor LET-23, which are also responsible for uv1 specification. let-60/ Ras signaling is necessary but not sufficient for this rescue and IP3 production is neither required nor sufficient for rescue. This suggests that let-23 signals via another, as yet unidentified, pathway to promote survival of the uv1 cells, an avenue under further investigation. In contrast to the uv1 cell death model, NAM accumulation is not a sufficient insult to cause death of the OLQ cells, which are mechanosensory cells involved in head withdrawal and foraging. OLQ cell death is also not rescued by NA, suggesting that both PNC-1 product depletion (and the subsequent effects on NAD production) and substrate accumulation contribute to the insult that kills the OLQ cells. OLQ cell death increases when animals are grown on dead food, an undefined yet nutritionally poor growth medium that has previously been shown to exacerbate phenotypes caused by loss of NAD biosynthesis. Our data suggests a role for Ca2+ signaling, autophagy and nutrient sensing in OLQ cell death, but in a very different way to their roles in mec-4d induced touch cell necrosis. Thus, it is becoming clearer that not only is necrosis very much a controlled form of cell death, but that there are a wide range of insults that triggers the necrotic cell death program and variety of underlying mechanisms by which it is carried out. We believe that our pnc-1 model presents a powerful and complimentary system to further increase our understanding of genetically programmed cell death, its causes and its execution.
Contact: [email protected] Lab: Hanna-Rose
Poster Topic: Cell Death 93 The Possible Role of Autophagic Cell Death in the Regulation of Excitotoxicity in C. elegans John Del Rosario, Itzhak Mano Physiology, Pharmacology & Neuroscience, Sophie Davis Biomedical School, City College, City University of New York, New York, NY, USA Stroke is a leading cause of death in the United States. One of the main causes of stroke is a blockage of blood supply to the brain. The ensuing lack of oxygen triggers a neurodegenerative cascade in a process called brain ischemia. The excitatory neurotransmitter L-Glutamate (Glu) is normally expelled out from the synaptic cleft by the Glu Transporters (GluTs). The malfunction of GluTs due to a shortage in energy causes Glu to accumulate in the synapses to abnormal levels and over stimulates the Glu receptors (GluRs) on the post-synaptic cell, leading to the degeneration of the post-synaptic neuron in a process called excitotoxicity. We use a model of excitotoxicity in the nematode Caenorhabditis elegans by knocking-out the GluT gene glt-3 in a sensitive background. Recent reports suggest that autophagy, an evolutionary conserved cell death process, is involved in related forms of neurodegeneration. We now examine the possible role of autophagy in excitotoxic neurodegeneration and elaborate on its mechanism. We have two main focuses: 1) we are testing if autophagy takes place by monitoring it through an autophagy fluorescent flag; 2) we are determining the probable role of autophagy in excitotoxic neurodegeneration by using mutations that block autophagy such as bec-1 and unc-51(master-regulatory genes for autophagy).We are following the effect of autophagy-related mutation on the extent of neurodegeneration in our model of excitotoxicity. Understanding the molecular cascade of excitotoxicity and the potential involvement of autophagy in this process in nematodes might help us suggest protective strategies to reduce brain damage caused by brain ischemia.
Contact: [email protected] Lab: Mano
94 Poster Topic: Cell Death Genes Required for Cell Shedding, a Caspase-Independent Mechanism of Programmed Cell Elimination Dan Denning, Bob Horvitz HHMI, Dept. Biology, MIT, Cambridge, MA 02139 USA Programmed cell death plays critical roles in metazoan development and in the removal of damaged, infected or cancerous cells. Although most developmental cell deaths in C. elegans require the CED-3 caspase, some cells die in mutants completely lacking ced-3 function. We have determined the identities of eight cells that can be eliminated via extrusion (or shedding) from ced-3 mutant embryos. In wild-type embryos, these cells undergo ced-3-mediated apoptosis followed by engulfment. Thus, the canonical programmed cell death pathway and cell shedding function redundantly to ensure the elimination of a subset of cells fated to die. One of the cells that can be shed from ced-3 embryos is ABplpappap, the sister cell of which produces the RMEV neuron and the excretory cell. We predicted that ABplpappap might survive and adopt the fate of its sister cell in animals defective in both canonical programmed cell death and cell shedding. To identify factors required for cell shedding, we screened mutagenized ced-3 animals for ectopic excretory cells, using the transgenic reporter Ppgp-12::gfp to facilitate visualization of excretory-like cells. Three of our isolates with ectopic excretory cells contain mutations in pig-1, which encodes an AMPK-related serine-threonine kinase. A null mutation of pig-1 reduces the number of shed cells in ced-3 embryos by 75%, indicating that pig-1 is required generally for the generation of shed cells. Most mammalian AMPK-related kinases are activated via phosphorylation by the LKB1:STRAD:MO25 tumor suppressor complex. Inactivation of par-4/LKB1, strd-1/STRAD or mop-25.1 and mop-25.2 (paralogs of MO25) also blocks cell shedding in ced-3 animals. Additionally, the conserved T-Loop threonine (T169) of PIG-1, the predicted phosphorylation target of PAR-4/LKB1, is required for PIG-1 function, suggesting that the PAR-4 complex directly activates PIG-1 to regulate cell shedding. We are currently using SNP mapping and whole-genome DNA sequence determination to identify the genes mutated in other mutant strains with ectopic excretory cells. Through biochemical, genetic and cell biological experiments, we will determine how these genes cooperate with pig-1. Our goal is a mechanistic understanding of the cell shedding process.
Contact: [email protected] Lab: Horvitz
Poster Topic: Cell Death 95 Investigating the pro-apoptotic function of ced-9 Kaitlin Driscoll, Peter Reddien, Brad Hersh, Bob Horvitz Massachusetts Institute of Technology, Cambridge, MA Programmed cell death is a fundamental process that is required for proper development and tissue homeostasis in many organisms. Genetic analyses of C. elegans led to the discovery of the core components of the apoptosis pathway. One component, ced-9, is known to have an anti-apoptotic function, as ced-9(null) animals are maternal-effect lethal due to massive amounts of cell death and gain-of-function mutations in ced-9 prevent normal programmed cell deaths from occurring. However, ced-9 also has a pro-apoptotic function, which has not been well characterized. This function was discovered because weak ced-3 loss-of-function animals have more extra cells in a ced-9(null) background, indicating that decreasing ced-9 function can decrease cell death. In a genetic screen for mutations that enhance a defect in programmed cell death of weak ced-3(n2472) mutants, we recovered a ced-9(n3377) allele that not only enhances the ced-3 cell-death defect but also has a recessive cell-death defect on its own. Evidence suggests that ced-9(n3377) has a loss of ced-9 killing function rather than a gain of protective function. First, ced-9(n3377) confers a recessive increase in cell survival, which is different from the dominant gain of protective function ced-9(n1950) allele. Second, ced-9(n3377) acts different from the gain-of-function allele in relation to CED-4 localization. CED-9 normally localizes to mitochondria, where it binds CED-4 and prevents CED-4 from activating CED-3. Upon EGL-1 binding to CED-9, CED-4 is released and localizes to the perinuclear membrane. In ced-9 gain-of-function animals CED-4 is localized to mitochondria, even when EGL-1 is over- expressed. By contrast, in ced-9(n3377); ced-3(n2427) animals CED-4 is localized to the perinuclear membrane, as it is in ced-9(null);ced-3(n2427) animals. This finding is consistent with the increase in cell survival in ced-9(n3377) animals being caused by a loss of ced-9 killing function rather than a slight gain of protective function. Currently we are performing genetic screens to obtain additional loss of ced-9 killing function alleles and to identify suppressors of the ced-9(n3377) cell-death defect. These screens might identify genes that regulate the ced-9 killing function as well as unknown components of the cell-death pathway. We are also using molecular and biochemical techniques with ced-9(n3377) to evaluate if the ced-9 pro-apoptotic function is mediated through the core apoptotic pathway components and/or its regulation of mitochondrial fusion and fission. Ideally, these experiments will lead to a better understanding of the apoptosis pathway and possibly novel therapeutic targets for diseases caused by misregulation of programmed cell death.
Contact: [email protected] Lab: Horvitz
96 Poster Topic: Cell Death SPTF-3 SP1 and PIG-1 MELK Function in Distinct Pathways to Promote M4 Neuron Cell-Type Specific Programmed Cell Death Takashi Hirose , Bob Horvitz Dept. Biology, MIT, Cambridge, MA 02139 USA In C. elegans, 131 somatic cells undergo programmed cell death during wild-type hermaphrodite development. While genes that cause programmed cell death have been well studied, less is known about how a particular cell is specified to survive or to die by programmed cell death. To identify pathways involved in cell-type specific programmed cell death, we screened for mutations that cause a defect in the death of the sister of the pharyngeal M4 motor neuron. The M4 neuron is generated during embryonic development and survives to regulate muscle contraction in the pharynx, while the M4 sister dies by programmed cell death. Using genetic screens, we identified seven genes required for M4 sister cell death:ceh-32 , ceh-34, eya-1, sptf-3, pig-1, gcn-1 and abcf-3. Here we describe our studies of the SP1 family transcription factor SPTF-3 and the AMPA-related protein kinase PIG-1. Reduction of sptf-3 function decreases expression of the pro-apoptotic BH3-only gene egl-1 in the M4 sister and does not enhance a defect in M4 sister cell death in ced-9 null mutants. By contrast, a loss of pig-1 function does not affect egl-1 expression in the M4 sister and enhances a defect in M4 sister cell death in ced-9 null mutants. Also, sptf-3; pig-1 double mutants have a stronger defect in M4 sister cell death than do either of the single mutants. These results indicate that sptf-3 acts through the canonical cell-death execution pathway, while pig-1 acts in a distinct pathway in the regulation of M4 sister cell death. We previously reported that the C. elegans Six family homeodomain protein CEH-34 and the Eyes absent homolog EYA-1 promote the death of the M4 sister through the transcriptional activation of egl-1 (Hirose et al., PNAS 107, 15479-15484, 2010). An sptf-3 deletion does not affect ceh-34 or eya-1 expression in the M4 sister. This result suggests that sptf-3 acts in a distinct pathway from that of ceh-34 and eya-1 to promote egl-1 expression in the M4 sister. Our findings indicate that M4 sister cell death is regulated by at least three different pathways, in which 1) ceh-34 and eya-1 promote egl-1 expression, 2) sptf-3 promotes egl- 1 expression via a pathway distinct from that of ceh-34 and eya-1, and 3) pig-1 functions independently of the canonical cell-death execution pathway.
Contact: [email protected] Lab: Horvitz
Poster Topic: Cell Death 97 Using HITS-CLIP to study mRNA targets of RNA-binding proteins involved in germ cell apoptosis in C. elegans Martin Keller1,3, Deni Subasic1,3, Kishore Shivendra2, Michaela Zavolan2, Micheal Hengartner1 1Institute of Molecular Life Science, University of Zurich, Switzerland, 2Biozentrum, University of Basel, Switzerland, 3Molecular Life Science PhD Programm, Life Science Zurich Graduate School, ETH/University of Zurich, Switzerland Post-transcriptional control of mRNAs by RNA-binding proteins (RBPs) has a prominent role in the regulation of gene expression. RBPs interact with mRNAs to control their biogenesis, splicing, transport, localization, translation and stability. Defects in such regulation can lead to a wide range of human diseases from neurological disorders to cancer. Many RBPs are known to regulate apoptosis in the adult C. elegans germline. How these RBPs control apoptosis is however largely unknown. To address this question, we set out to establish a method to identify the mRNA targets apoptosis-associated RBPs. In a proof of principle experiment, we selected GLD-1, as translational regulator already has many known targets, including CEP-1, the C. elegans homologue p53 tumor suppressor. Using HITS-CLIP (cross-linking and RBP immunoprecipitation coupled with high-throughput sequencing) we could identify many of these known targets and the known binding motif of GLD-1 demonstrating the value of our method. Interestingly, we found that GLD-1 strongly binds to several sites in the 3’UTR of its own mRNA, suggesting that GLD-1 undergo auto regulation. Moreover, mRNAs for many of the other germline apoptosis RBPs (CGH-1, CPB-3, DAZ-1 and GLA-3) were also bound by GLD-1, hinting at the possible existence of an RBP regulon that orchestrates germ cell apoptosis. We now plan to apply our method on the complete set of RBPs that regulate germ cell apoptosis in order to identify the pathway that links germline apoptosis RBPs to the core apoptosis machinery.
Contact: [email protected] Lab: Hengartner
98 Poster Topic: Cell Death Utilization of Alternative mRNAs for CED-4/Apaf-1 During Germ Cell Apoptosis J. Kaitlin Morrison, Brett Keiper Brody School of Medicine at East Carolina University Germ cell apoptosis is the process by which superfluous oocyte progenetor cells are eliminated by committing themselves to die via signaling through the cell death (ced) signaling pathway. Nearly half of all germ cells in the C. elegans gonad are fated for death before reaching maturity. These cells are believed to act as “nurse cells” providing cytoplasmic components needed by their sibling cells. During apoptosis changes in protein synthesis occur upon activation of caspases that cleave the translation initiation factor, eIF4G, which is involved in the cap dependent recruitment of mRNA to the ribosome. Our study focuses on the relative contribution of the C. elegans eIF4G (IFG-1) cap dependent and independent isoforms (p170 and p130) to shifts in the protein synthesis mechanism and the selection of germ cells to die. Specifically, we are assessing the effect of such mechanisms on the translation ofced-4 mRNA variants. In addition to previously known splice variants of ced-4, ced-4L and ced-4S that have opposing apoptotic activities we have identified several alternativeced-4 message variants by RT-PCR and RNase Protection mapping. The distribution of the message variants and their translational efficiency was assayed in wildtype worms and worms depleted of either IFG-1 p170, CED-9, CED-3 or the germ line protein GLA-3. Our findings suggest a physiological link between translational control by IFG-1 and the expression of CED-4 to induce germ cell apoptosis.
Contact: [email protected] Lab: Keiper
Poster Topic: Cell Death 99 A Small-Molecule Screen Identifies a Linker Cell Death Inhibitor Andrew Schwendeman, Shai Shaham Rockefeller University, New York, NY, USA Programmed cell death plays a central role in animal development and disease. Although apoptosis is the best characterized cell death mechanism, it does not seem to account for all vertebrate programmed cell death. Our lab has described molecular and morphological features of the death of the C. elegans male-specific linker cell at the L4-adult transition (Abraham et al., 2007; Blum et al., 2012). These studies show that linker cell death is independent of all known apoptotic genes, and is accompanied by ultrastructural characteristics distinct from those of apoptotic cells. This novel morphological signature, including uncondensed chromatin, nuclear envelope crenellation, and swollen mitochondria and endoplasmic reticulum, have been described in dying cells of the developing vertebrate nervous system and in pathologies such as Huntington’s disease. To isolate additional regulators of linker cell death, and to test conservation with vertebrate cell-death processes, we developed a high-throughput small- molecule screen to identify inhibitors of linker cell death. Animals carrying a mig-24::GFP reporter transgene, expressed specifically in the linker cell, are grown on E. coli OP50-seeded plates until the L4 stage, washed, and incubated in S Basal buffer with candidate compounds in 384-well plates. After animals have become adults, a fluorescence cytometer is used to count surviving linker cells in each well. From a pilot screen of 1269 compounds, we identified a single small molecule that inhibits linker cell death without obvious pleiotropies. The compound has an IC50 in the nanomolar range, and its characteristics and method of action are under investigation. We aim to screen larger libraries to identify additional inhibitors.
Contact: [email protected] Lab: Shaham
100 Poster Topic: Cell Death Wave Regulatory Complex Genes Are Involved in the Engulfment of Apoptotic Cells Elena Simionato, Michael Hurwitz Yale University, School of Medicine, New Haven, CT, USA The engulfment of apoptotic cells is a conserved process involving the cytoskeletal rearrangement of one cell to surround another cell. This process is mediated by two parallel and partially redundant signalling pathways (CED-1, CED-6, CED-7, DYN-1 and CED-2, CED-5, CED-10, CED-12 and ABI-1). CED-1, 6 and 7 activate the GTPase DYN-1 Dynamin, a regulator of membrane dynamics. CED-2, 5 and 12 activate the small GTPase CED-10 Rac, a regulator of the actin cytoskeleton. ABI-1 is also a cytoskeletal regulator found in several protein complexes in mammals. One such complex is the Wave regulatory complex (WRC), which mediates actin polymerization in response to activation by Rac signaling. The WRC genes in worms are abi-1, wve-1, gex-2, gex-3 and nuo-3. Prior work has suggested that these genes are also involved in engulfment (Soto et al., 2002; Patel et al., 2008). Because all WRC genes are essential to the development of the animal, we tested if wve-1 and gex-2 are involved in engulfment using partial knockdown by feeding RNAi. We analyzed the effects of wve-1 and gex-2 RNAi in strains that were already partially defective in dyn-1 and ced-10 pathway genes to look for engulfment defects. The partial knockdown of wve-1 and gex-2 enhance the engulfment defects of all genes tested. To further analyze the role of wve-1 and gex-2, we tested their effects on distal tip cell (DTC) migration during gonad development, a process regulated by the ced-10 pathway. wve-1 and gex-2 RNAi enhanced the DTC migration defect of all ced-10 Rac pathway genes, suggesting that wve-1 and gex-2 act downstream of or in parallel to CED-10 Rac pathway in this process. We also tested the effect of wve-1 and gex-2 RNAi on actin dynamics during engulfment in strains defective in dyn-1 and ced-10 pathway. wve-1 and gex-2 RNAi led to a decrease in the number of cell corpses surrounded by actin, consistent with their known effects on the actin cytoskeletal rearrangement. Since RNAi of all WRC genes tested enhanced the engulfment defects of both known engulfment pathways, the WRC might act in parallel to both pathways. Alternatively, the WRC could be downstream of CED-10 but also activated by other pathways in parallel to the CED-10 pathway. In either model, our data imply that proteins independent of CED-10 activate the WRC. To date no activators of the WRC have been found independent of Rac proteins. We are now working to identify new CED-10 Rac-independent regulators of the WRC.
Contact: [email protected] Lab: Hurwitz
Poster Topic: Cell Death 101 In Search of Genes that Regulate Germ Cell Apoptosis in C. elegans Angel Villanueva-Chimal , Carlos Silva-Garcia , Laura Lascarez-Lagunas, Rosa Navarro Departamento de Biologia Celular y Desarrollo, Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico. Mexico. Apoptosis is a common feature of metazoan germ line. In C. elegans, fifty percent of germ cells are eliminated by apoptosis during oogenesis (physiological apoptosis). Higher levels of germ line apoptosis can be triggered by stress conditions such as DNA damage, starvation, heat shock, oxidative and osmotic stress. DNA damaged-induced apoptosis requires the p53 protein and the DNA damage machinery repair. On the other hand, heat shock, oxidative and osmotic stresses increase germ cell apoptosis in C. elegans by the MAPKK pathway, and through an EGL-1 and CEP-1 independent mechanism. The mechanisms that regulate physiological apoptosis and starvation-induced germ cell apoptosis are still unknown. We are currently searching genes that control physiological apoptosis and/or starvation-induced germ cell apoptosis. We tested six DNA or RNA binding protein coding genes that change their expression levels when animals are starved for 6 h. We found that under normal growing conditions high levels of germ line apoptosis are detected when C54G4.6, C28H8.9, T23F6.4, M03C11.8 and C27A12.2 are silenced. Additionally, we found a regular response to starvation- induced germ cell apoptosis when F56D2.6, C54G4.6, T23F6.4, M03C11.8 were silenced. On the other hand, C28H8.9(RNAi) and C27A12.2(RNAi) animals did not respond to starvation- induced germ cell apoptosis. C28H8.9(RNAi) animals showed less oocytes per gonad than control animals and C27A12.2(RNAi) animals showed early embryonic and growing defects, oocyte accumulation, alterations in the transition of mitosis to meiosis and 40% less offspring than control animals.
Contact: [email protected] Lab: Navarro
102 Poster Topic: Cell Death let-70, an E2 Ubiquitin-Conjugating Enzyme, Promotes the Non- Apoptotic Death of the Linker Cell Jennifer Zuckerman The Rockefeller University, New York, NY, USA Cell death is essential for animal development.Though apoptosis occurs during vertebrate development,evidence suggests it may not be the only operative cell-death process.The C.elegans linker cell is a male-specific cell that guides gonad formation.It lives for two days and dies to allow fusion of the vas deferens to the cloaca.Our studies showed that the linker cell dies independently of all known C.elegans cell-death genes.Further,dying linker cells exhibit non-apoptotic features including nuclear-membrane invagination, decondensed chromatin and swelling of organelles- features characteristic of non-apoptotic developmental cell death in the vertebrate nervous system and of neurodegeneration promoted by polyQ expansions, as in Huntington’s disease. An RNAi screen for genes promoting linker cell death uncovered the ubiquitin-conjugating enzyme let-70.Wild-type males fed E.coli expressing let-70 dsRNA show a strong block in linker cell death.The final stage of linker cell migration is blocked, but is genetically separable from death.Linker cell-only RNAi using rde-1 rescue blocks death but not migration, supporting the idea that these processes are independent and that let-70 functions cell-autonomously to kill. let-70::GFP fusions are expressed in the linker cell as the cell dies.Similar expression is seen with PQN-41,a polyQ protein important for linker cell death(Blum et al., 2012),suggesting that a transcriptional program regulates its timing. pqn-41 expression is controlled by the SEK- 1 MAPKK.Epistasis studies show that let-70 RNAi in concert with a sek-1 mutation does not result in additive survival.Also, let-70::GFP is not expressed in sek-1(ag1)mutants,suggesting that sek-1 operates upstream of let-70.Similar results were obtained with RNAi against the MAPK scaffold tir-1,and the MLL complex genes swd-2.2 and set-16.By contrast,let-70::GFP expression remains high after RNAi of the nuclear hormone receptor nhr-67,and joint RNAi of nhr-67/let-70 produces additive survival,suggesting these genes act in parallel. To identify LET-70 targets,we screened for E3s required for linker cell death. We found that RNAi against the seven-in-absentia homolog siah-1 or the ring-box rbx-1 blocked linker cell death in 10% of animals.siah-1(tm1968);rbx-1(RNAi)animals show a 30% survival,indicating that these genes play partially-redundant roles.Furthermore,linker cell-only RNAi of proteasome subunits blocks linker cell death.Our studies suggest an important role for protein degradation pathways in linker cell death.
Contact: [email protected] Lab: Shaham
Poster Topic: Cell Death 103 Elucidating the let-7 Independent Role of lin-28 Jennifer Alaimo, Bhaskar Vadla, Kevin Kemper, Eric Moss UMDNJ-Graduate School of Biomedical Sciences, Stratford, New Jersey, United States of America lin-28 is a conserved regulator of cell fate succession in animals. In Caenorhabditis elegans, it is a component of the heterochronic gene pathway that governs larval developmental timing, while its vertebrate homologs promote pluripotency and control differentiation in diverse tissues. LIN-28 is an RNA binding protein that can directly inhibit let-7 microRNA processing by a novel mechanism that is conserved from worms to humans. We found that C. elegans LIN-28 protein can interact with four distinct let-7 family pre-microRNAs, but in vivo, can inhibit the premature accumulation of only let-7. Surprisingly, however, lin-28 does not require let-7 or its relatives for its characteristic promotion of second larval stage cell fates. We have shown that lin-28 acts in two steps: first, thelet-7 -independent positive regulation of hbl-1 through its 3’UTR to control L2 stage-specific cell fates; and second, alet-7- dependent step that controls subsequent fates via repression of lin-41. The transcription factor encoded by hbl-1 is known to be regulated by at least three let-7 family members, and is thought to be the most proximal regulator of the succession of L2 to L3 cell fates. However, in addition to the eight potential let-7 family binding sites, there are also two potential lin-4 sites in hbl-1’s 3’UTR. The role of these sites in hbl-1 regulation is currently unknown. Present work seeks to elucidate if lin- 28’s positive regulation of hbl-1 is direct through binding of the hbl-1 mRNA or indirect via a mechanism involving the lin-4 family of microRNAs.
Contact: [email protected] Lab: Moss
104 Poster Topic: Cell Fate Regulation and function of SYS-1/beta-catenin during hypodermal stem cell divisions Austin Baldwin, Bryan Phillips University of Iowa The stem cell-like asymmetric divisions of the epithelial seam cells are controlled by the Wnt/beta-catenin Asymmetry (WBA) signaling pathway. The activity of this pathway results in the anterior daughter fusing to hyp7 (WBA independent fate) and the posterior daughter retaining the seam cell fate (WBA dependent fate). POP-1/TCF is exported from the posterior nucleus by a known mechanism involving WRM-1/beta-catenin and LIT-1/nemo-like kinase. SYS-1/beta-catenin is regulated postranslationally by the WBA pathway in other tissues, but little is known of SYS-1 expression or regulation in the seam stem cells. To investigate the mechanism of seam cell specification, we analyzed SYS-1 localization in wild type larvae and manipulated SYS-1 levels during seam cell division. We show that SYS-1 displays dynamic localization during seam cell division. SYS-1 localizes in rapid sequence to the cell cortex, midbody, and both centrosomes in the dividing mother cell. After cytokinesis, SYS-1 is undetectable in the unsignaled hypodermal daughter, but accumulates in the nucleus of the signaled seam cell daughter. The SYS-1 localization pattern is under the control of upstream WBA pathway members. The resultant reciprocally asymmetric pattern of low SYS-1/high POP-1 in the anterior daughter nucleus and high SYS-1/low POP-1 in the posterior daughter nucleus is consistent with a role in Wnt target gene transactivation. We also find that elevating SYS-1 levels in both daughters at the time of seam cell division induces duplication of the stem cell fate at the expense of the hypodermal fate. These data position SYS-1 as a target of Wnt regulation during seam stem cell specification and suggest that SYS-1, like canonical beta-catenin, plays a conserved role in maintaining stem cell populations.
Contact: [email protected] Lab: Phillips
Poster Topic: Cell Fate 105 Germline Expressed GLP-1 Regulates Embryonic Endoderm Specification Ahmed Elewa1, Takao Ishidate1, Sandra Vergara1, Tae-Ho Shin2, Masaki Shirayama1, Craig Mello1 1UMass Medical School, Worcester, MA, USA, 2Baylor College of Medicine, Huston, TX, USA Mutations in the CCCH zinc finger gene pos-1 and RNAi of the KH domain gene gld-1 result in identical maternal-effect embryonic lethal phenotypes involving the misspecification of several embryonic cell fates: including transformation of ABp to ABa, failure to specify endoderm and failure to specify germ-line cell fates. POS-1 and GLD-1 have been shown to function together to suppress translation of the GLP-1 mRNA in posterior sister cells at the 4-cell stage of embryogenesis. However, GLP-1, a transmembrane receptor related to Notch, is not required for endoderm specification, and its mis-expression in earlypos-1 embryos has not been linked to the loss of endoderm fate. We were therefore surprised to find the mutation glp-1( as a temperature sensitive suppressor of the pos-1 endoderm defect. glp-1( exhibits an amino-acid substitution (G1031D) in the 4th (of 7) ankyrin repeats. Several other glp-1ts alleles exhibit amino acid substitutions in the same ankyrin repeat region (including e2141, bn18 and q231). We found that all of these apparent loss of function ts alleles also suppress the endoderm defect of pos-1(zu148).Interestingly, the temperature sensitivity point for endoderm restoration occurs in the gonad hours prior to fertilization suggesting that glp-1 activity in the distal gonad can interfere with endoderm specification in pos-1 mutants much later in the early embryo. This temporal displacement of suppression is reminiscent of that described between efl-1 and pos-1 (Page B et al Mol. Cell March 2001 and Tenlen J et al. Genetics December 2006). EFL-1 is a homologue of vertebrate E2F transcription factor and mediates the expression of a host of targets in the C. elegans germline (Chi W & Reinke V Development 2006). Coupled with our previous finding that translation regulator gld-3 suppresses the endoderm defect of pos-1 and gld-1, we are exploring a model where glp-1 and efl-1 activity in the distal gonad promotes the expression of a gut antagonizer dependent on GLD-3 for its translation and repressed by GLD-1 and POS-1 in wild-type embryos to permit gut development.
Contact: [email protected] Lab: Mello
106 Poster Topic: Cell Fate Investigating the Role of SEM-4/SALL in Development of the Postembryonic Mesoderm Vikas Ghai, Chenxi Tian, Jun Liu Cornell University, Ithaca, NY, USA The C. elegans postembryonic mesoderm lineage, the M lineage, is a unique system that provides a high degree of temporal and cellular resolution for studying development of mesodermal tissues. The M lineage is derived from a single pluripotent precursor cell, the M mesoblast, which undergoes two waves of proliferation and differentiation to produce 32 cells. These cells include 14 bodywall muscles, two coelomocytes (dorsally-derived), and two multi-potent sex myoblasts (SMs; ventrally-derived) that will give-rise to 16 vulval and uterine muscles. Using this system, we have elucidated the mechanisms of several highly conserved transcription factors and signaling pathways in mesoderm development. These include a rolefor LET-381/FoxF and CEH-34/Six2 in coelomocyte specification (Amin et al.,2010), a role for SEM-2/SoxC in promoting the proliferative SM fate (Tian etal., 2011), and input from BMP, Notch, and Wnt pathways (Foehr et al., 2006; Greenwald et al.,1983; Foehr and Liu, 2008; Amin et al. 2009). Interestingly, SEM-4, the sole C. elegans member of the Spalt/SALL family of C2H2 zinc finger transcription factors is required for both dorsal and ventral cell fates in the M-lineage, as both coelomocytes and SMs are transformed to BWM cells in sem-4 mutants (Basson and Horvitz, 1996). Here we investigate the role of SEM-4 during the development of the M-lineage. sem-4 is expressed in a pattern in the M-lineage overlapping with both LET-381 and SEM-2. The M-lineage expression of sem-2 does not change in sem-4(n1378)mutants, nor does the expression of sem-4 in sem-2(n1343) mutants. This suggests that while sem-2 and sem-4 do not regulate each other’s expression, they probably either directly interact with each other or regulate the same set of target genes. We are currently testing these possibilities. We are also performing experiments between sem-4 and let-381,as well as other genes in the M-lineage to determine their regulatory relationships.
Contact: [email protected] Lab: Liu
Poster Topic: Cell Fate 107 A Screening To Find Suppressors Of The Wnt Pathway Eva Gomez-Orte2, Begona Ezcurra1, Beatriz Saenz-Narciso1, Juan Cabello1 1CIBIR, FUNDACION RIOJA SALUD, 2Center for Biomedica Research of La Rioja (CIBIR), Logrono, Spain The Wnt pathway typically has been defined as a pathway involved in fate specification. However, in addition to this function, the Wnt pathway is involved in other processes such as cell migration, engulfment of apoptotic corpses or proper mitotic spindle orientation. Our lab works in understanding how this pathway is regulated and how different signals are integrated to produce a coordinated response. We have started a screening to find genetic suppressors of mutants in different components of the Wnt pathway. Towards this aim, we have generated a strain lit-1 (t1512); wIs84 (pJM66 elt-2::GFP::LacZ, pRF4 rol-6(su1006dm)) that carries an integrated intestinal GFP marker and a Temperature Sensitive mutation in lit-1. LIT-1 is a Nemo-like kinase that phosphorilates POP- 1, the transcription factor of the Wnt pathway. In the absence of LIT-1, the unphosphorilated POP-1 remains in the nucleus and avoid the expression of the endoderm specific genes. Thus, at 15 C, lit-1 (t1512); wIs84 is viable and has a fluorescent intestine; whereas at 25 C produces only dead embryos without intestine and hence without any fluorescence. We have performed a pilot EMS screening to findlit-1(t1512) TS worms that at 25 C, were able to produce embryos with intestine (GFP positive). After mutagenize 100000 haploid genomes, we have found two independent lit-1 (t1512) strains that develop intestine. These strains are currently being outcrossed. The nature of the suppressor mutation will be determined by deep sequencing. We expect to find regulators downstream or in parallel to LIT-1.
Contact: [email protected] Lab: Cabello
108 Poster Topic: Cell Fate MEX-5 regulates mRNA stability during germ cell development and asymmetric cell division Manoel Prouteau, Gilles Udin, Monica Gotta CMU, University of Geneva, Geneva, Switzerland Establishment and maintenance of cell polarity are essential for many biological processes such as asymmetric cell division, proliferation, differentiation, and morphogenesis. In the C. elegans embryo the conserved PAR proteins regulate the cytoplasmic asymmetric localization of CCCH tandem zinc finger proteins (CCCH-proteins), which specify somatic and germ cell fates. Two redundant CCCH-proteins, MEX-5 and MEX-6 (referred to as MEX-5/6), accumulate in the anterior half of the one-cell embryo and are important to regulate polarity and cell fate specification. In yeast and mammalian cells, CCCH proteins bind specific mRNAs and promote their degradation. This activity depends on their ability to shuttle in and out of the nucleus. Interestingly, in mex-6;mex-5 mutant embryos, the enrichment of certain mRNAs (called class II mRNAs) in the germline precursors is lost and these mRNAs are found in all cells. We therefore investigated whether MEX-5/6 regulate polarity processes also by controlling class II mRNA stability. We find that MEX-5 is in a complex with proteins of the mRNA decay machinery in the embryo. Consistent with a role in mRNA decay, the total levels of class II mRNAs are increased in mex-5 mutants. In addition, we show that MEX-5, as the yeast and mammalian homologues, is shuttling in the nuclei of somatic cells. In the germline lineage, where MEX-5 must be kept inactive to avoid mRNA degradation, nuclear shuttling is inhibited by PAR-1 phosphorylation. Taken together our data suggest a model in which MEX-5 contributes to germline development by controlling the segregation of mRNAs in the P lineage via their degradation in somatic blastomeres.
Contact: [email protected] Lab: Gotta
Poster Topic: Cell Fate 109 A Screen for Mislocalization and Misexpression of LET-23 EGF Receptor during Vulval Development Andrea Haag, Juan Escobar Restrepo, Alex Hajnal University of Zurich, Zurich, Switzerland In polarized epithelial cells, the apical and basolateral membranes are composed of distinct proteins and lipids that provide specific functions. The mammalian epidermal growth factor receptor (EGFR), a member of the ErbB family of receptors, is mainly localized to the basolateral cell membrane (Kuwada et al., 1998). Mislocalization of mammalian ErB family members to the apical surface can de-regulate signaling by the receptor and result in disease (Du et al., 1995). Similarly, the C. elegans EGFR homolog LET-23 is targeted to the basolateral plasma membrane in vulval precursor cells. Vulval development is impaired if LET-23 is mislocalized. Previously, a ternary complex consisting of LIN-7, LIN-2 and LIN-10 has been shown to play an important role in the retention of LET-23 on the basolateral surface (Kaech et al., 1998). Nonetheless, the exact mechanism of LET-23 localization and the control of the receptor dynamics remain poorly understood. To identify new regulators of LET-23 localization, we performed an RNAi feeding screen using a functional LET-23::GFP translational reporter. We analyzed over 700 RNAi clones that are known to cause a protruding vulva (Pvl) phenotype. By evaluating LET-23::GFP expression at different developmental stages, we were able to identify several genes regulating LET-23 localization or expression in the VPCs and their descendants. To investigate if receptor mislocalization alters LET-23 signaling, we performed RNAi against selected candidates in a sensitized let-60 ras(gf) background. By this approach, we have so far identified three genes that negatively regulate RAS/MAPK signaling and control LET-23 localization. To confirm the RNAi results, we are currently analyzing LET-23::GFP localization in the corresponding mutant strains. Further experiments aim at investigating how these candidate genes control LET-23 localization and/or expression. Kuwada SK, Lund KA, Li XF, Cliften P, Amsler K, Opresko LK, Wiley HS (1998) Differential signaling and regulation of apical vs. basolateral EGFR in polarized epithelial cells. Am J Physiol. 275; C1419-28 Du J, Wilson PD (1995) Abnormal polarization of EGF receptors and autocrine stimulation of cyst epithelial growth in human ADPKD. Am J Physiol. 269; C487-95 Kaech SM, Whitfield CW, Kim SK (1998) The LIN-2/LIN-7/LIN-10 complex mediates basolateral membrane localization of the C. elegans EGF receptor LET-23 in vulval epithelial cells. Cell. 94; 761-71
Contact: [email protected] Lab: Hajnal
110 Poster Topic: Cell Fate A Role of the LIN-12/Notch Signaling Pathway in Diversifying the Non- Striated Egg-Laying Muscles in C. elegans Jared Hale, Carolyn George, Nirav Amin, Zachary Via, Leila Toulabi, Jun Liu Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, 14853 Our lab aims to understand the mechanisms underlying the diversification of the postembryonic mesoderm lineage, which arises during embryogenesis from a pluripotent cell known as the M mesoblast. During postembryonic development in C. elegans, the M mesoblast divides to generate fourteen striated body-wall muscles, two coelomocytes, and two sex myoblasts (SMs). The SMs further divide and differentiate into sixteen non-striated egg-laying muscles: four each of type I and type II vulval muscles and uterine muscles, respectively. While the MyoD family of transcription factors has been shown to play an evolutionarily conserved role in specifying the striated/skeletal muscles, relatively little is known about how different types of non-striated/smooth muscles are specified. In an RNAi screen for transcription factors important for muscle development, we found that RNAi knockdown of lag-1, which encodes a component of the LIN-12/Notch pathway, led to the production of extra type I vulval muscles. Similar phenotypes were also observed in animals with reduced functions of the Notch receptor LIN-12 and its ligand LAG-2. The extra type I vulval muscles in animals with reduced LIN-12/ Notch signaling did not appear to be a result of extra SMs or extra SM proliferation, rather a fate transformation from type II vulval muscles to type I vulval muscles. Consistent with this, we observed nuclear localization of the functional LIN-12::GFP in the undifferentiated type II vulval muscles. Thus LIN-12/Notch signaling is used repeatedly in the M lineage, first for promoting the ventrally-derived SM fate (Greenwald et al., 1983; Foehr and Liu, 2008), and second for specifying the type II vulval muscles in diversifying the SM lineage.
Contact: [email protected] Lab: Liu
Poster Topic: Cell Fate 111 UNC-62/Meis and CEH-20/Pbx proteins work together to control asymmetric cell divisions during C. elegans development by regulating WRM-1/β-catenin localisation Samantha Hughes, Charles Brabin, Alison Woollard Oxford University, Oxford During larval development, stem cell-like seam cells undergo asymmetric divisions producing an anterior daughter cell that differentiates by fusing with the hypodermal syncytium and a posterior daughter that retains the seam fate and has the ability to undergo further proliferation. As a result of a genome wide RNAi screen, we identified two transcription factor genes,ceh-20 / Pbx and unc-62/Meis, that are required for these asymmetric cell fate decisions. Animals that lack ceh-20 and/or unc-62 display severe seam cell hyperplasia that is absolutely dependent upon the function of rnt-1 and bro-1. Intriguingly the hyperplasia is largely restricted to the anterior H lineages of the seam. Lineage analysis reveals that the hyperplasia is the result of complete symmetrisation of asymmetric divisions towards the posterior proliferative fate. We observed the dynamic nature of CEH-20 localisation whose nuclear distribution is regulated by UNC-62. In addition, we found that the distribution of WRM-1/β-catenin is perturbed in the absence of CEH-20 suggesting a molecular mechanism by which ceh-20/unc-62 may control the establishment of seam division asymmetry.
Contact: [email protected] Lab: Woollard
112 Poster Topic: Cell Fate The Ras-ERK/MAPK Regulatory Network Controls Dedifferentiation In Caenorhabditis elegans Germline Dong Seok Cha1, Udaya Sree Datla1, Sarah Hollis1, Judith Kimble2,3, Myon-Hee Lee1,4 1Brody School of Medicine at East Carolina University, Greenville, NC, USA, 2HHMI, 3University of Wisconsin-Madison, Madison, WI, USA, 4Lineberger Comprehensive Cancer Center, University of North Carolina-Chapel Hill, Chapel Hill, NC, USA. How a committed cell can be reverted to an undifferentiated state is a central question in stem cell biology. This biological event, called dedifferentiation, is capable of replacing stem cells as they get aged or damaged. Tremendous progress has been made, but the mechanisms are poorly understood. Here we demonstrate that activation of Ras-ERK/MAPK signaling promotes cellular dedifferentiation in the Caenorhabiditis elegans germline. To activate signaling, we removed two negative regulators, the PUF-8 RNA-binding protein and the LIP-1 dual specificity phosphatase. Removal of these two regulators caused spermatocytes to dedifferentiate and begin mitotic divisions. Interestingly, reduction of Ras-ERK/MAPK signaling, either by mutation or chemical inhibition, blocked the initiation of dedifferentiation. By RNAi screening, we identified
RSKN-1/P90RSK as a downstream effector of MPK-1/ERK and as critical for dedifferentiation: rskn-1 RNAi suppressed dedifferentiation of spermatocytes and induced meiotic cell divisions. Because these regulators are all broadly conserved, we suggest that similarly molecular circuitry may control cellular dedifferentiation in other organisms, including humans.
Contact: [email protected] Lab: Lee
Poster Topic: Cell Fate 113 A sma-9 Suppressor Screen to Identify New Players in the BMP-like Sma/Mab Pathway in C. elegans Lindsey Szymczak, Katharine Constas, Arielle Schaeffer, Sinthu Ranjan, Saad Kubba, Emad Alam, Dennis Liu, Chenxi Tian, Herong Shi, Jun Liu Cornell University, Ithaca, USA The bone morphogenetic protein (BMP) pathway plays essential roles in multiple developmental processes during metazoan development. In C. elegans, the BMP-like Sma/Mab pathway regulates body size and male tail patterning. We have previously shown that the Sma/ Mab pathway also regulates mesoderm development. In particular, mutations in the zinc finger containing protein SMA-9 cause a dorsal to ventral fate transformation in the postembryonic mesodermal M lineage. This M lineage phenotype of sma-9 mutants can be suppressed by mutations in the Sma/Mab pathway, suggesting that SMA-9 antagonizes the function of Sma/ Mab signaling in patterning the M lineage (Foehr et al., 2006). The suppression of the sma- 9 M lineage phenotype by Sma/Mab pathway mutants appears specific, as mutations in the TGFbeta-like dauer pathway or mutations affecting body size without affecting the Sma/Mab pathway do not suppress the sma-9 M lineage defect. Through the suppressor screen, we have identified a positive modulator of the Sma/Mab pathway named DRAG-1, which is a membrane-associated protein that belongs to the RGM (repulsive guidance molecule) family (Tian et al., 2010). Motivated by our findings described above, we carried out a large-scalesma-9 suppressor screen for additional players in the Sma/Mab pathway. By clonally screening through 5300 haploid genomes, we identified thirty seven single-locus, recessivesma-9 suppressor mutations. Complementation tests showed that these include two alleles of sma-2, three alleles of sma-3, one allele each of sma-4, sma-6, daf-4, dbl-1 and lon-1. The remaining mutations define at least eleven complementation groups. Through whole genome sequencing, RNAi and transgenic rescue experiments, we have identified the corresponding gene for one of the complementation groups. Molecular genetic studies of this gene suggest that it encodes a trans-membrane protein that functions as a positive modulator of Sma/Mab signaling. Current research aims to decipher the molecular mechanism on how this gene product modulates Sma/Mab signaling.
Contact: [email protected] Lab: Liu
114 Poster Topic: Cell Fate Further evidence for the importance of the MED-1 and -2 GATA factors in endoderm specification Morris Maduro, Gina Broitman-Maduro, Shruthi Satish University of California, Riverside, Riverside, CA, USA The E cell clonally generates the C. elegans intestine. A feed-forward transcription factor cascade involving SKN-1, MED-1,2 and END-1,3 drives specification of E in the pre-gastrulation embryo. We have previously reported that RNAi of med-1,2 results in some 50% of embryos lacking endoderm. A strain that is homozygous for putative null alleles of med-1 and med-2 also results in ~50% of gut(-) embryos when the mothers carry irDp1, a modified version of sDp3 that carries unc-32(+), unc-119::YFP and med-1(+). Curiously, when mothers of med- 1,2(-) embryos are heterozygous for one (or both) of the med genes, the resultant med-1,2(-) embryos fail to make gut only ~15% of the time. We have shown using in situ hybridization that there are maternal med transcripts in the germlines of wild-type hermaphrodites, but not irDp1(+) hermaphrodites, suggesting that a maternal contribution of the meds explains the difference in penetrance of the gut defect. Others have suggested that the unc-32 transgene, and not abrogation of a maternal contribution, is responsible for the difference, and that the higher RNAi phenotype results from non-specific knockdown of other genes. Using transgene reporters for gut fate, we find that unc-32 sequences in irDp1 have no detectable effect on whether or not gut cells contain gut granules in med-1,2(-) embryos. We further assessed the contribution of the MEDs to endoderm specification by removing the GTATACYYY MED binding sites from the end-1 and end-3 genes. We inserted single copies of wild-type and MEDsite-less end-1 and end-3 genes into other genomic contexts by MosSCI and microparticle bombardment. We introduced these into a double mutant end-1,3 background or a strain carrying the deficiency itDf2, which deletes end-1 and end-3 (as well as many other genes). We find that when the MED sites are mutated in end-1 and end-3, 30%-50% embryos fail to make intestine. This is significantly higher than the 15% that is seen due to zygotic loss of the meds alone, and consistent with our prior reports using med-1; med-2; irDp1 mothers and RNAi. We also find that adults derived from embryos in which the end genes lack MED sites have defects associated with gut differentiation (see other abstract by Maduro et al.). Our results add to our prior findings that the MEDs play a significant role in the specification of endoderm in C. elegans.
Contact: [email protected] Lab: Maduro
Poster Topic: Cell Fate 115 Regulation and function of nhr-67/tailless in uterus development George McClung, Lauren Pioppo, Jenny Hall, Rachel Dordal, Catherine Ezzio, Evan Fletcher, Amanda Gavin, Sheila Clever, Bruce Wightman Muhlenberg College, Allentown, PA USA The tailless family of nuclear receptors is highly conserved among animals. The C. elegans tailless ortholog, nhr-67, is expressed in a dynamic pattern in pre-uterine cells. nhr-67 is initially expressed in the 4 pre-VU cells during the L2 stage, and subsequently upregulated in the anchor cell (AC), in response to the lin-12/lag-2 reciprocal signaling system. During the L3 stage, nhr-67 expression is maintained at high levels in the AC and at low levels in the six π cells whose twelve progeny form cells of the adult ventral uterus. nhr-67 is also expressed in the male LC and in neurons and functions in these cells have been defined by the Hobert and Sternberg labs. Homozygous deletions of nhr-67 generally cause developmental arrest in embryogenesis or after hatching at L1, with tail morphology defects. Mutants homozygous for hypomorphic nhr-67 promoter mutations that were identified by Bernard Lakowski’s laboratory, are viable, but defective for the development of the π cells and the AC. nhr-67(lf) mutations are epistatic to lin12(gf) mutations in the π cells, indicating that lin-12 activity depends on nhr-67. Expression of lin-12::gfp in all four pre-VU cells and later in VU cells depends on nhr-67, indicating that nhr-67 is an important regulator of lin-12 and therefore VU response to lag-2 signal. nhr-67 is also required for expression of zmp-1 and lag-2 in the AC. Taken together, these data define an important regulatory role for nhr-67 in both AC and VU development (Verghese et al., 2011, Dev. Biol. 356:516). Phenotypic similarities suggest that egl-43 and hlh-2 might function upstream or in parallel with nhr-67 to regulate AC development. The nhr-67 promoter mutations define a 276bp region that is important for nhr-67 function in uterine development. Deletion of this region results in a loss of nhr-67 expression in pre-VU, AC, and VU cells. We have collaborated with the Walhout Lab to identify potential upstream transcription factors that may bind to six evolutionarily- conserved candidate cis-acting elements in the 276bp region. Two of the six elements are perfect matches to E boxes that are predicted to bind HLH-2/HLH-4 and/or HLH-2/HLH-10 heterodimers. We are evaluating several candidates using one-hybrid and EMSA approaches. This work was supported by a grant from the NSF.
Contact: [email protected] Lab: Wightman
116 Poster Topic: Cell Fate Does lin-46 Tip the Balance of hbl-1 Activity in the Succession of Hypodermal Blast Fates? Eric Moss, Kevin Kemper, Bhaskar Vadla UMDNJ-GSBS, Stratford, NJ, USA In the regulation of larval hypodermal cell fates, the heterochronic genes lin-28 and lin-46 appear to act oppositely (positively and negatively, respectively) on hbl-1. HBL-1 is a zinc- finger transcription factor of the Ikaros family and seems to be the most direct effector of the L2 seam cell lineage pattern, which is characterized by a symmetric division followed by an asymmetric one that produces a hyp7 syncytial nucleus and a blast cell. lin-28 is well known to block let-7 accumulation, but let-7 and its target lin-41 are not involved in the regulation of seam cell fates in the L2 (see abstract by J. Alaimo). Through lin-46 we expect to learn more about how lin-28 and hbl-1 work. lin-46 null alleles completely suppress lin-28 null alleles restoring the normal cell lineage succession throughout the animal. LIN-46 protein is similar to proteins of bacteria and eukaryotes involved in the biosynthesis of an enzyme cofactor, but it seems to have been exapted for a role in the heterochronic pathway. Although LIN-46 retains the overall folding and structure of its homologs, the certain critical residues and surface charges have changed. Somewhere on LIN-46 is a surface that binds specifically to a important pair of C-terminal zinc fingers of HBL-1. In all other Ikaros family members, these zinc fingers are for homo- and hetero-dimerization. HBL-1 is unique among Ikaros proteins for not homodimerizing. So we have several questions to answer: Is the sole purpose of HBL-1’s C-terminal zinc fingers to permit negative regulation by LIN-46? Do these zinc fingers bind another Ikaros family member or some other protein necessary for HBL-1 activity? Does LIN-46 block this binding or does it simply drag HBL-1 off to the trash? So far we have evidence that HBL-1 uses these zinc fingers for another purpose. Finally, lin-46 shows oscillating expression at both the protein and RNA levels slightly out of phase with the major oscillating gene in the heterochronic pathway, lin-42. lin-46 expression peaks just prior to the molt, suggesting a burst of HBL-1 inhibitory activity a few hours before cell fates are executed. If lin-46 is missing, hbl-1 appears to resist down-regulation for at least one more stage. Therefore, the role of lin-46 in the pathway may be to ensure repression of hbl-1 activity at a critical time and the “ticking over” of cell fates in proper succession.
Contact: [email protected] Lab: Moss
Poster Topic: Cell Fate 117 Post-transcriptional Regulation of Maternally-supplied Wnt Ligand During Early Embryogenesis Marieke Oldenbroek1, Scott Robertson1, Tugba Guven-Ozkan2, Rueyling Lin1 1UTsouthwestern, Dallas, Texas, USA, 2Scripps Research Institute, Jupiter, Florida, USA During C. elegans embryogenesis, blastomere fate specification requires critical cell-cell interactions that are under precise spatiotemporal regulation. The mRNA for most components of signaling pathways in embryos are provided maternally and distributed uniformly. Temporal and spatial expression patterns of most maternally supplied proteins are regulated through the 3’ UTR of their mRNA. One Wnt signaling pathway in early embryos specifies the endoderm precursor and orientates division axes. While the signaling cells for these Wnt-mediated interactions have been demonstrated by genetic analyses and chimeric experiments, the expression pattern of the Wnt ligand, MOM-2, has not been determined. We show here that a reporter containing GFP fused to H2B and the mom-2 3’UTR is expressed precisely in known Wnt signaling cells. The expression is first detected in the germline blastomere, P2, in 4-cell embryos, and continues in descendants of P2. Through in vitro RNA binding assays and genetic analysis, we identified proteins that bind to the mom-2 3’ UTR and regulate its expression. Repression of mom-2 in 1- and 2-cell embryos is dependent on two RNA binding proteins, MEX-3 and SPN-4. In 4-cell embryos, MEX-3 becomes localized primarily in somatic blastomeres where it continues to repress mom-2. In the germline blastomere P2, SPN-4 is present at a high level but does not repress mom-2 expression. Expression of MOM-2 in P2 requires two germline-blastomere-specific RNA-binding proteins, PIE-1 and MEX-1, which both outcompete SPN-4 for binding to the mom-2 3’ UTR, thereby alleviating its repressive effect in P2. Repression of mom-2 in oocytes depends on two other RNA binding proteins, OMA-1 and OMA-2. After fertilization, OMA-1 and OMA-2 are degraded, partly due to their phosphorylation by GSK-3. Failure to degrade OMA proteins results in a phenotype similar to mom-2 mutant embryos. GSK-3 is well established as a negative regulator in the canonical Wnt pathway in flies and vertebrates. However, previous genetic studies reveal a positive role for GSK-3 inC. elegans embryos. Our results show this discrepancy to be due to a negative feedback loop, in which reduced GSK-3 activity leads to OMA protein persistence and ectopic repression of MOM-2 expression. Overall, our findings show the importance of translational regulation in restricting the expression of the C. elegans Wnt ligand MOM-2.
Contact: [email protected] Lab: Lin
118 Poster Topic: Cell Fate Abstract withdrawn.
Contact: [email protected] Lab: Rothman
Poster Topic: Cell Fate 119 Regulation of LET-23 EGFR signaling and trafficking by a putative Arf1-GEF Olga Skorobogata, Christian Rocheleau McGill University, Montreal, QC, Canada Epidermal Growth Factor Receptor (EGFR)/Ras/Mitogen Activated Protein Kinase (MAPK) signaling regulates cell proliferation, migration and apoptosis and misactivation can lead to cancer. An important mechanism of signal downregulation involves EGFR endocytosis and trafficking to the lysosome. In C. elegans a highly conserved LET-23 EGFR signaling pathway is required for vulval cell fate specification.The LIN-2 CASK/ LIN-7 Veli/ LIN-10 Mint11 complex is required for basolateral localization of LET-23 in the Vulval Precursor Cells (VPCs). Mutations in lin-2 result in a strong Vulvaless (Vul) phenotype due to the apical mislocalization of LET-23. To identify new regulators of LET-23 signaling and trafficking, we conducted a forward genetic screen for essential suppressors of lin-2(e1309). One of the strong suppressor mutants identified,vh4 , is partial embryonic lethal, has secretory defects in several tissues and enlarged lysosomal compartments. We used SNP mapping with whole-genome sequencing, as well as RNAi and genetic complementation, to identify vh4 as a hypomorphic allele of agef-1. AGEF-1 is homologous to the human BIG1/2Arf Guanine nucleotide exchange factors that localize to the trans-Golgi and endosomes. The vh4 mutation leads to substitution of a conserved Glu to Lys within a conserved domain of unknown function. Consistent with AGEF-1 being a Class I and II Arf GEF, a deletion mutant of Class I Arf, arf-1.2(ok796), was able to suppress the lin-2 Vul phenotype, moreover RNAi-mediated depletion of ARF-3, a Class II Arf, in arf-1.2; lin-2 animals resulted in even stronger suppression. Genetic epistasis experiments suggest that agef-1 might function at the level of LET-23 EGFR. Thus AGEF-1, ARF-1.2, and ARF-3 might promote the degradation or antagonize signaling of LET-23 EGFR in the VPCs. We hypothesize that AGEF-1 might either regulate the secretion of a negative regulator of LET-23 EGFR or trafficking of lysosomal enzymes required for efficient degradation of LET-23 EGFR. <
Contact: [email protected] Lab: Rocheleau
120 Poster Topic: Cell Fate Examining the Fate of Centrosomally Uncoupled SYS-1/Beta-catenin to Explore Spindle-Independent Roles of the Centrosome during Asymmetric Cell Divisions Setu Vora, Bryan Phillips University of Iowa A specialized Wnt/β-catenin signaling pathway is responsible for carrying out reiterated asymmetric cell divisions, allowing cells of the developing embryo and larva to navigate through a multitude of lineages. Asymmetric divisions regulated by this pathway give rise to daughter cells that exhibit differential activity of Wnt signaling components, allowing them to take on distinct transcriptional profiles. SYS-1/β-catenin is a major transcriptional effector of this pathway and is expressed at higher levels in the signaled daughter cell after a given Wnt-regulated asymmetric division. SYS-1 is subject to strict post-translational regulation and shows dynamic patterns of subcellular localization during the cell cycle. We are studying how subcellular localization of SYS-1 contributes to its proper regulation. In a number of different systems, β-catenin localizes to the centrosomes and has previously been implicated to function in microtubule outgrowth and centriole splitting. Similarly, SYS-1 localizes to the pericentriolar material (PCM) of the centrosomes during C. elegans cell divisions but is dispensable for proper spindle formation and orientation. Thus, the functional or regulatory significance of SYS-1 centrosomal localization is not known. We have used a two-step screening process to identify SYS-1 centrosomal regulators. First, a split-ubiquitin based yeast two hybrid screen with a SYS- 1 bait was used to identify SYS-1 interactors. Second, RNAi knockdown of interactors identified specific factors required for SYS-1 centrosomal localization. As these treatments uncouple SYS-1 from the centrosomes, we can now evaluate the regulatory status of centrosomally localized SYS-1 and examine the resulting effects on SYS-1 expression and target cell fates. Because the centrosome has been implicated in protein processing and degradation during mitosis, we are using this experimental system to explore a potential spindle-independent role of the centrosome in housing and distributing regulatory molecules such as β-catenin during asymmetric cell divisions.
Contact: [email protected] Lab: Phillips
Poster Topic: Cell Fate 121 Function and evolution of the diverged NR2E nuclear receptors nhr- 111 and nhr-239 Emily Bayer, G. Michael Baer, Christopher Alvaro, Katherine Weber, Ramzy Burns, Michael Lilly, Anvi Patel, Benjamin Perlman, Sheila Clever, Bruce Wightman Muhlenberg College, Allentown, PA, USA The NR2E subclass of nuclear receptors is conserved from cnidarians to vertebrates. Phylogenetic analysis identifies at least three major clades: the NR2E1/2 clade (nhr-67 and tailless), the NR2E3/5 clade (fax-1 and PNR), and a new clade that includes nhr-239 of Caenorhabditis, the HR83 nuclear receptors of insects, and an ortholog in non-vertebrate deuterostomes. Therefore, the nhr-239 clade appears to have an ancient origin, but has been lost from vertebrates. An additional NR2E gene, nhr-111, has a fax-1-like ligand-binding domain (LBD), but so far appears only in C. elegans and C. brenneri genomes, indicating that it is a relatively recent evolutionary elaboration within Caenorhabditis. We performed swaps among the LBDs of NR2E genes, demonstrating that NHR-111 and NHR-67 could function in place of the FAX-1 LBD. This prompted us to test a simple deletion of the FAX-1 LBD; it too could provide robust function. These experiments suggest that the LBD is not required for at least some functions of an evolutionarily-conserved C. elegans NR—and by extension that fax-1 activity is not necessarily ligand-dependent. The ok2526 deletion removes 5’ flanking DNA and the first exon of nhr-239, plus the last exon of the neighboring feh-1 gene. We have found that this deletion generates a fusion transcript between feh-1 and nhr-239, which probably produces no functional NHR-239 product. ok2526 homozygotes display a behavioral phenotype that appears to be related to that of npr-1; they avoid lawns of OP50 bacteria often congregating a short distance outside of the lawn, creating a “halo.” This phenotype is not observed on UV-killed lawns, suggesting that it is not a simple physical response to bacteria. nhr-239 is expressed at low levels in two to three pairs of head neurons, at least one of which appears to be of a sensory type and is located in the vicinity of URX. The ok519 deletion of the nhr-111 gene produces no detectable nhr-111 transcript, but does not cause an obvious morphological or behavioral phenotype. nhr-111 is a major node in high-throughput one-hybrid analysis of neuronal and metabolic promoters, an observation that seems surprising for a relatively recent evolutionary elaboration. We have found that nhr-111 is expressed broadly in most nematode tissues. We are currently testing possible functions for nhr-111 in regulation of genes that function in metabolism. This work is supported by a grant from the NSF.
Contact: [email protected] Lab: Wightman
122 Poster Topic: Gene Regulation Redefining POP-1 Binding Sites inC. elegans Chandan Bhambhani, Ken Cadigan University of Michigan, Ann Arbor, MI, USA We are deciphering the rules for DNA binding used by POP-1, a member of the T-cell factor (TCF) family of transcription factors and a major mediator of Wnt/β-catenin signaling in C. elegans. While all TCFs contain a High Mobility Group (HMG) domain that can bind DNA specifically, most invertebrate and some vertebrate TCFs also possess another DNA binding domain termed the C-Clamp. In Drosophila, the C-Clamp is known to bind motifs known as Helper sites, which are found in the vicinity of functional HMG binding sites and are essential components of the cis-regulatory Wnt Response Elements (WREs). POP-1 contains both the HMG and C-clamp domains, and so to better understand the architectural make-up of worm WREs, we are exploring the role of Helper sites in this organism. Potential Helper sites were found near the functional HMG sites in the WREs of Wnt targets ceh-22, psa-3 and end-1. We find that Helper sites are essential for Wnt induced expression ofceh-22 in the distal tip cells of the somatic gonad. In vitro, Helper sites are required for binding of POP-1 to all three WREs, and their importance is being tested for psa-3 and end-1 in vivo. Our data thus far indicate a bias in the spacing and orientation of functional HMG and Helper sites, which has enabled us to identify additional putative WREs in a computational search. Our study highlights the importance of Helper sites in defining functional POP-1 binding sites, and a better understanding of how POP-1 locates Wnt targets should facilitate further in silico identification of new WREs in the genome.
Contact: [email protected] Lab: Cadigan
Poster Topic: Gene Regulation 123 In vivo Regulation of the Alternative Splicing of the Pro- and Anti- Apoptotic Gene ced-4 Anna Corrionero, Bob Horvitz HHMI, Dept. Biology, MIT, Cambridge, MA 02139 USA The processing of pre-mRNAs by alternative splicing provides a cell with the ability to generate multiple mRNAs from a single gene. Despite many efforts to study this process, the regulation of alternative splicing in vivo and in a tissue- or developmental stage-specific manner as well as the functional implications of alternative splicing are not well understood. The C. elegans CED-4 protein promotes the activation of the caspase CED-3 and is essential for canonical programmed cell death (PCD). However, the ced-4 transcript is alternatively spliced, giving rise to two different isoforms with antagonistic functions generated by use of alternative 3’ splice sites (ss) in exon 4. The main isoform, CED-4S, is pro-apoptotic, while CED-4L is anti-apoptotic. ced-4 is the only apoptotic gene known to be alternatively spliced in C. elegans. How ced-4 alternative splicing is regulated is largely unknown. To study the regulation of ced-4 alternative splicing in vivo we have generated fluorescent reporters so that expression of CED-4L will give rise to GFP, while expression of CED-4S will give rise to RFP. These reporters showed a higher levels of expression of the ced-4S isoform, consistent with the alternative splicing levels observed for the endogenous gene. A deletion analysis of the reporters indicated the presence of two sequences important for the regulation of ced-4 splicing. One, located upstream of the exon 4L 3’ss, contains possible binding sites for the Fox-1 family members FOX-1 and ASD-1 and for the muscle-specific splicing factor SUP-12. These proteins might prevent the recognition of exon 4L 3’ss. Deletion of the FOX- 1/ASD-1 but not of the SUP-12 binding site weakly increased CED-4L expression. However, single mutants of fox-1, asd-1 or sup-12 did not modify the ced-4 isoform ratio and did not exhibit PCD defects in the anterior pharynx or ventral cord. We are now testing the effects of double mutants of these genes as well as of other genes involved in alternative splicing, such those that encode the Serine/Arginine-rich proteins implicated in cell survival. We are also trying to identify factor/s that bind upstream of exon 4 using biochemistry. In addition, we are performing a genetic screen for mutants with an increased CED-4L signal. We hope that by studying ced-4 alternative splicing using our fluorescent reporter system we will identify not only factors involved in alternative splicing but also modulators of the apoptotic pathway itself.
Contact: [email protected] Lab: Horvitz
124 Poster Topic: Gene Regulation Identifying HLH-8/Twist Homodimer Target Genes Nirupama Singh, Peng Wang, Ann Corsi The Catholic University of America, Washington, DC, USA The basic helix-loop-helix (bHLH) transcription factor, Twist, plays an important role in mesoderm development. The bHLH factors influence transcription by binding to a consensus DNA sequence called an E box as either a homodimer or a heterodimer. In C. elegans, the Twist homolog HLH-8 is required for a subset of mesodermal development, including M lineage patterning and differentiation of vulval and enteric muscles. The only known heterodimeric partner for HLH-8 is the more broadly expressed bHLH factor HLH-2/Daughterless protein. Previous work in our laboratory has shown that distinct HLH-8-containing dimers function in C. elegans development. For example, HLH-8 homodimers play a major role in the undifferentiated M lineage cells whereas HLH-8/HLH-2 heterodimers function in differentiated cells derived from the M lineage, the vulval muscles. In order to understand the unique functions of these dimers in development, we wanted to identify a collection of target genes that are regulated by each dimer. To identify target genes of HLH-8 homodimers, we overexpressed the protein and looked for genes that were upregulated using Affymetrix Oligonucleotide microarrays. We screened though the most promising candidates by making gfp reporters and looking for mesodermal expression. This approach led to 5 genes whose expression was coincident with HLH-8. These reporters were also examined in hlh-8 mutants to validate that their expression depended on HLH-8 and examined in hlh-2 mutants since HLH-8 homodimer candidates should not depend on HLH-2 for expression. Based on these criteria, we identified one gene that is expressed in vulval muscles and depends on HLH-8 but not HLH-2 for expression. The promoter of this gene was examined by making 5’ deletion constructs and by site-directed mutagenesis. These approaches led to the identification of a single palindromic E box that is responsible for vulval muscle expression and makes the prediction that this site may be found in other HLH-8 homodimer target genes.
Contact: [email protected] Lab: Corsi
Poster Topic: Gene Regulation 125 Understanding the Role of Overlapping MicroRNA Networks During Nematode Development Jeanyoung Jo, Kimberly Breving, Kenya Madric, Aurora Esquela-Kerscher Eastern Virginia Medical School, Norfolk, Virginia, USA Developmental genetics has aided our understanding of the intricate regulatory programs that rapidly turn a fertilized egg into a fully-grown organism. MicroRNAs (miRNAs) constitute a major class of conserved ~22 nucleotide non-coding RNAs that play pivotal roles in developmental gene regulation. They are found to control a wide-range of processes in the embryo such as cellular differentiation, proliferation, and apoptosis. However, few physiologically relevant and developmentally important miRNA targets have been identified and in silico miRNA target predictions are often error-prone and unreliable. Work with the simple but genetically powerful Caenorhabditis elegans (C. elegans) nematode worm allows us to screen for biologically relevant miRNA-target interactions in vivo that direct essential events during development and are potentially conserved in mammalian systems. We have focused on the role of the lin-4 and let-7 miRNA families during the formation of the gonad and egg-laying structures (vulva) in worms. This study sought to determine if members within a miRNA family are biologically distinct due to their unique temporal and spatial expression patterns or if they are really functionally different despite possessing identical “miRNA seeds”. Our results indicated that 1) closely related miRNAs members of the lin-4 family could not functionally compensate for one another using an in vivo lin-4 loss-of-function rescue assay while 2) certain members of the let-7 family are functionally redundant with non-homologous lin-4 miRNAs. Specifically, our deletion studies revealed that the poorly characterizedlin-4 homologue, miR- 237, and the let-7 family members, miR-48 and miR-84 function in an overlapping network likely with chromatin remodeling genes to direct cell cycle progression in the germline as well as vulva morphogenesis. We are currently using unbiased RNAi suppressor screens to identify physiologically relevant miRNA targets and investigate if these miRNA-target interactions are conserved in mammalian cells. This dual system approach will provide novel insights into the biological roles of miRNAs during human development and disease. Funding: This work is supported by EVMS start-up funds and a grant from the Thomas F. and Kate Miller Jeffress Memorial Trust (to A.E-K.).
Contact: [email protected] Lab: Esquela-Kerscher
126 Poster Topic: Gene Regulation Intracellular Trafficking and Endocytic Regulation of the DBL-1/BMP- like pathway in C. elegans Ryan Gleason1, Adenrele Akintobi2, Ying Li1, Barth Grant2, Richard Padgett1 1Waksman Institute, Rutgers University, 2Rutgers University Endocytosis has long been known as simply a way to internalize and traffic nutrients and membrane associated molecules that cannot pass the plasma membrane of the cell. Recent evidence has led to an emerging model where endocytosis orchestrates the topological landscape of signal transduction pathways throughout the cell by regulating the availability and trafficking (degradationversus recycling) of transmembrane signaling proteins. Using C. elegans as a model to study the endocytic trafficking of two TGFβ transmembrane receptors, DAF-4 and SMA-6, we have identified various endocytic pathways that regulate receptor internalization, availability, and recycling. Interestingly, these two receptors function in the same signal transduction pathway, but are recycled disparately. This work was prompted by the identification of sma-10, which was discovered through a genetic screen in the Padgett Lab, and which we show also affects the trafficking of the type I and type II receptors, SMA-6 and DAF-4. Experiments were done to distinguish between a role in receptor secretion and/ or in internalization of the receptors. We show that sma-10 does not act in secretion of the receptors but acts after receptor internalization. Further data will be presented to show how TGFβ receptors traffic in C. elegans and what role sma-10 plays in this process.
Contact: [email protected] Lab: Padgett
Poster Topic: Gene Regulation 127 Identification and characterization of targets of the REF-1 family member, HLH-25 Raymarie Gomez, Han-ting Chou, Casonya Johnson Georgia State University Proteins in the REF-1 family are distinguished by the presence of two basic helix-loop helix domains. HLH-25 is one of six members of this family, and though the gene encoding hlh-25 is known to be expressed during embryonic development in response to Notch signaling, there is no genetic or molecular data about the role of this family member in embryonic or post-embryonic development. Recently, our laboratory completed a microarray analysis of gene expression changes in hlh-25 mutants compared to wild-type animals. This analysis has uncovered putative roles for HLH-25 in regulating genes required for cell division and cell cycle progression. The objective of the research presented here is to further characterize the role of HLH-25 in these processes. My aims are first to validate twenty HLH-25 target genes by monitoring changes in their expression in wild-type and hlh-25 mutant animals. Here, I report on my efforts to generate transcriptional fusions for four of the targets and to isolate and characterize transgenic lines for each reporter construct.
Contact: [email protected] Lab: Johnson
128 Poster Topic: Gene Regulation The Mediator Subunit CDK-8 Negatively Regulates EGFR-Ras-MAPK in Vulva Development Jennifer Grants, Stefan Taubert University of British Columbia, Vancouver, Canada The Mediator complex is a conserved coregulator of eukaryotic transcription. Certain Mediator subunits are required for transcription of all protein coding genes, whereas others regulate specific gene programs. Cyclin dependent kinase 8 (CDK-8) is one Mediator subunit that exerts gene-specific regulation. To identify genes and programs regulated by CDK-8, we performed microarray analysis comparing cdk-8 null mutants and wild-type worms. Intriguingly, CDK-8 regulates gene targets in common with LIN-35/pRB, a regulator of C. elegans vulva development that negatively affects signaling through an EGFR-Ras-MAPK pathway. We found that, like LIN-35, CDK-8 also regulates vulva development, as cdk-8 null mutants display a low-penetrance multivulva (Muv) phenotype. Furthermore, null mutation of a negative regulator of the EGFR-Ras-MAPK pathway enhances the Muv phenotype of cdk-8 mutants. Thus, CDK- 8 controls vulva development via negative regulation of an EGFR-Ras-MAPK pathway. As another Mediator subunit, SUR-2, is a critical positive regulator of transcription downstream of the EGFR-Ras-MAPK pathway, I will explore the possibility that antagonism between the SUR-2 and CDK-8 Mediator subunits may fine-tune the output of an important developmental signaling cascade.
Contact: [email protected] Lab: Taubert
Poster Topic: Gene Regulation 129 A Lipid-Binding Protein that Modifies cGMP Signaling is Required for Host Odor Sensing and Body Morphology in Pristionchus pacificus Ray Hong, Jessica Cinkornpumin, Dona Roonalika Wisidagama, Veronika Rapoport California State University Northridge, Northridge (CA) In Pristionchus pacificus, the cGMP dependent protein kinase Ppa-EGL-4 is an important genetic modifier of insect pheromone reception. Upregulation of Ppa-egl-4 transcripts depends on intracellular cGMP levels and is highly variable among wild isolates from diverse host ranges. In particular, chemoattraction to the Oriental beetle sex pheromone is very strong in the Washington isolate (PS1843) but is elicited in the reference isolate California (PS312) only after a brief exogenous cGMP treatment. To obtain additional cGMP signaling factors involved in insect pheromone attraction, we performed forward genetic screens in P. pacificus for mutants that do not show attraction toward the oriental beetle pheromone (Z-7-tetradecen-2-one). Out of the four Oriental Beetle pheromone Insensitive mutants (obi’s) that we have isolated, the obi-1 allele showed the strongest chemosensory phenotype specific for lack of attraction to ZTDO. In addition to chemosensation, obi-1 acts upstream and parallel to Ppa-egl-4 in regulating pharyngeal pumping and locomotion. We identified the molecular lesion of obi-1 and found a nonsense mutation in an uncharacterized ORF encoding a protein with a lipid-binding motif. The transcriptional reporter shows obi-1p::gfp expression in specialized hypodermal cells, including the seam and putative duct cells. More importantly, obi-1p::gfp is also expressed in the amphid sheath cells, the glial cells responsible for forming enclosed compartments around the chemosensory amphid neurons. We further analyzed the obi-1 ortholog expression in C. elegans and found overlapping expression patterns with obi-1 in P. pacificus, but lacking in amphid sheath expression. We hypothesize that OBI-1 proteins are secreted and interacting with chemosensory neurons in the fluid-filled amphid compartments to modulate cGMP signaling during chemosensation. OBI-1 belongs to a small, mostly uncharacterized, but highly conserved group of lipid-binding proteins found in both C. elegans and P. pacificus as well as most metazoans.
Contact: [email protected] Lab: Hong
130 Poster Topic: Gene Regulation Elucidating The Role of Genetic Redundancy In The Wnt Signaling Pathway In Regulating Q Neuroblast Migration Ni Ji1, Teije Middelkoop2, Hendrik Korswagen2, Alexander van Oudenaarden1 1Massachusetts Institute of Technology, Cambridge (MA), USA, 2Hubrecht Institute, Utrecht (Utrecht), The Netherlands The intriguing observation that functionally redundant genes are highly conserved despite little selective pressure has puzzled biologists for a long time. To explore the emergent function of genetic redundancy, we used the C. elegans Qneuroblasts to examine how multiple Wnt receptors function together to regulate mab-5/Hox, a key Wnt target gene necessary and sufficient to drive posterior migration. To identify the contribution of each receptor to mab- 5activation, we used single molecule Fluorescent In Situ Hybridization(smFISH) to profile the expression of Wnt receptor and target genes in a series of wild-type and Wnt signaling mutant strains. Specifically, we found three Frizzled-type receptors mig-1/Fz( , lin-17/Fz, and mom-5/ Fz) to be specifically expressed in QL, two of which (mig-1 and lin-17) exhibited strong but opposite correlation with mab-5. In single and compound Frizzledmutants, we found mab-5 expression to be progressively reduced and increasingly variable. The variability in mab-5 expression correlated strongly with the partially penetrant migration phenotype, consistent with our recent report (Raj et al., 2010). Surprisingly, we found that mutations in either the Frizzled receptors, mab-5 or the Wnt ligand egl-20, can induce changes in the expression of the (non- mutated) receptors. Applying a mathematical inference algorithm to our gene expression data, we arrived at amost-probable network topology that features two negative and one positive egl-20-dependent feedback loops targeting, respectively, mig-1, mom-5 and lin-17. Network modeling predicts a division of labor among the three receptors between the initialization and maintenance phase of mab-5 activation. In an ongoing effort, we express Q cell specific Frizzled transgenes to assess downstream consequences when feedback regulation on the receptors is perturbed. Reference: Raj A etal. (2010) Variability in gene expression underlies incomplete penetrance. Nature 463,913: 913-918.
Contact: [email protected] Lab: van Oudenaarden
Poster Topic: Gene Regulation 131 Can the Rate of Transcription be Quantitatively Determined in Relation to Transcription Factor Binding Affinity? Brett Lancaster, James McGhee University of Calgary, Calgary, Alberta, Canada Transcription factors (TFs) are proteins that bind to DNA, usually at a sequence motif <10 bps long, and thereby influence the transcriptional activity of a target gene. We wish to develop an experimental system to investigate how transcription factor binding affinity determines the rate of target gene transcription inside a living animal. In the nematode C. elegans, the zinc finger transcription factor ELT-2 binds to a core TGATAA sequence of DNA to regulate the majority (possibly all) of the genes transcribed in the post-specification intestine. The affinity with which ELT-2 binds the core TGATAA sequence is presumed to be 100% relative to all other sequences. ELT-2 is incapable of activating gene transcription when the TGATAA sites within the promoters of genes it regulates are replaced with GTCGCC “GATA knockout sites”. The asp-1 gene encodes the major intestinal-specific aspartic protease of the worm, has two TGATAA sites in its 1kb promoter and is under direct ELT-2 control. Our objective is to quantitatively measure the binding affinity of ELT-2 to a multitude of binding site variants (BSVs), and, at the same time, measure the transcriptional output (mRNA) of the target gene with these variants in the promoter, all inside the living worm. We have constructed two versions of the asp-1 gene that differ only in the location of a KpnI site introduced by silent mutations within the asp-1 coding sequences. One reporter is regulated by the wildtype asp-1 promoter whereas the second reporter is regulated by a mutant promoter in which a TGATAA site has been replaced by, for example, a CGATAA site. Equal amounts of the two constructs are introduced into worms as multicopy transgenic arrays. RNA isolation followed by RT-PCR will produce a pool of reporter cDNAs in a ratio that reflects the in vivo transcript levels. The products can undergo KpnI digestion and gel electrophoresis to yield distinct banding patterns for each reporter that can easily be quantitated. Binding affinity of purified ELT-2 protein to each binding site variant will be determined by fluorescence anisotropy. We are currently conducting control experiments to validate the system and assess its sensitivity and robustness.
Contact: [email protected] Lab: McGhee
132 Poster Topic: Gene Regulation Regulated Splicing of the Cholinergic Gene Locus Ellie Mathews, Greg Mullen, Jim Rand Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Acetylcholine (ACh) is a major neurotransmitter in both vertebrate and invertebrate nervous systems. A single conserved locus encodes both the ACh biosynthetic enzyme (ChAT; cha- 1) and the vesicular ACh transporter (VAChT; unc-17) proteins. The VAChT coding region is contained within the first intron of thecha-1 gene, and alternative splicing gives rise to separate ChAT and VAChT transcripts. We identified two sets of inverted repeat sequences (designated R1 and R2) in the non-coding sequences flanking the unc-17 coding region, which form the basis for a model of “hairpin-mediated” alternative splicing of unc-17 and cha-1 transcripts. To test the role(s) of these inverted repeat sequences, we engineered a dual reporter in which the unc-17 coding region was replaced with green fluorescent protein (GFP) and the cha-1 coding region with a red florescent protein (“wCherry”). The resulting construct contains all of the conserved non-coding sequences in the regions flankingunc-17 and cha-1, and transgenic animals containing this construct correctly express both GFP and wCherry in the appropriate neurons. We have shown that both R1 and R2 repeats are necessary for normal expression of wCherry (cha-1). We found that the R1 hairpin structure, but not the precise R1 sequence, was important for downstream splicing. In contrast, both the R2 sequence and the hairpin structure were important for splicing. Analysis of unc-17 deletions (p1156 and md1447) identified a cryptic R1-pairing sequence, designated R’. We believe that R1/R1’ pairing is sufficient for adequate splicing of cha-1 mRNA, and we are currently testing this assumption. We have also analyzed the cha-1 - unc-17 genomic regions from five Caenorhabditis species. There is considerable species-to-species variation in the sequences, yet in each case, the R1 sequences from each species are much better matches to each other than they are to any other species. We also found structural counterparts of the R2 sequences in the Drosophila genome, and counterparts of the R1 sequences in the mouse and rat genomes. We therefore believe that such “hairpin- mediated” alternative splicing of VAChT and ChAT transcripts may be a general feature of cholinergic regulation. Supported by NIH grant R21 NS072923.
Contact: [email protected] Lab: Rand
Poster Topic: Gene Regulation 133 Short Capped RNAs and Nuclear Run-On Reveal Pol II Pausing and Backtracking in C. elegans Colin Maxwell1, William Kruesi2, Nicole Kurhanewicz5, Leighton Core3, Colin Waters3, Igor Antoshechkin4, John Lis3, Barbara Meyer2, L. Ryan Baugh1 1Duke University, Durham (NC), USA, 2University of California at Berkeley, Berkeley (CA), USA, 3Cornell University, Ithaca (NY), USA, 4California Institute of Technology, Pasadena (CA), USA, 5University of North Carolina, Chapel Hill (NC), USA When C. elegans larvae hatch in the absence of food they enter a state of development arrest (L1 arrest). Starved larvae respond rapidly to feeding, quickly initiating growth and post- embryonic development. We used ChIP-seq to show that Pol II is poised at the 5’ ends of growth and development genes during L1 arrest (Baugh et al, Science 2009). Pol II binding patterns suggest that elongation is under nutritional control, with the polymerase increasing elongation at many genes during immediate recovery from arrest. mRNA abundance also increases disproportionately for these genes. However, from these experiments it is impossible to know the post-recruitment point of regulation; ie, pre-initiation or elongation complex. We therefore used an RNA-seq protocol specific for short, capped RNAs to determine if we could detect the 5’ end of nascent transcripts as hallmarks of a paused elongation complex. We conducted a series of controls to confirm the specificity of our protocol, and we analyzedDrosophila S2 cells to compare with published results. We complemented this approach with global nuclear run-on (GRO-seq). Our analysis also benefits from mRNA-seq data for mature transcripts during L1 arrest and recovery as well as published Pol II ChIP-seq. We detect short, capped RNAs from the 5’ end of approximately 2,000 protein coding genes in L1 larvae. Pol II accumulation is also observed in the promoter-proximal region of these genes, and GRO-seq confirms that Pol II is paused on these genes. 3’ sequencing reveals a short, capped RNA size distribution similar to what has been reported for Drosophila and mammals. Analysis of a TFIIS mutant alters the size distribution, consistent with a model that involves TFIIS- facilitated cleavage of the 3’ end of the nascent transcript and backtracking to relieve pausing. Our results clearly demonstrate that Pol II pausing occurs in C. elegans larvae and that it is mechanistically related to pausing in other systems. This is particularly noteworthy in light of the absence of NELF homologs and the presence of 5’ trans-splicing in the worm.
Contact: [email protected] Lab: Baugh
134 Poster Topic: Gene Regulation The mRNA Splicing Regulator SPK-1 Is Required for Cell Polarity in One-Cell C. elegans Embryos Martin Mikl, Carrie Cowan IMP Vienna, Austria Cell polarity is a prerequisite for asymmetric division, which gives rise to daughter cells with different developmental fates. In one-cell C. elegans embryos, cell polarity comprises distinct cortical domains of actomyosin contractility and PAR proteins. Polarity establishment is initiated by a centrosome-dependent cue. In an RNAi screen for polarity establishment defects we identified one gene, spk-1, that appeared to have normal centrosome assembly and only mildly reduced cortical activity but nonetheless failed to establish correct PAR protein localization. Consistent with defects in PAR polarity, spk-1(RNAi) embryos divided symmetrically to give rise to equivalent daughter cells. SPK-1 is a kinase targeting the SR protein family of mRNA splicing factors and thereby potentially influences splice site selection. A transcriptome-wide analysis of RNA from WT and spk-1(RNAi) worms by Illumina sequencing did not show a general splicing defect, but misregulation of transcript levels in a small number of genes. By analyzing splice form abundance of candidate polarity mediators in SPK-1 depleted worms, we identified a splicing change in the par-5 3’UTR that leads to a reduction in PAR-5 protein levels in the embryo. par-5(RNAi) embryos showed similar polarity establishment defects to spk-1(RNAi) embryos, namely a reduced and instable PAR-2 domain. Thus SPK-1 may facilitate polarity establishment by regulating PAR-5, which in turn controls PAR-2 availability and maintenance at the cortex.
Contact: [email protected] Lab: Cowan
Poster Topic: Gene Regulation 135 The Transcriptional Repressor Protein CTBP-1 Regulates the Differentiation of DA Motor Neurons Hannah Nicholas1, Duygu Yucel1, Estelle Llamosas1, Anna Reid1, Aaron Lun1, Sashi Kant1, Merlin Crossley2 1University of Sydney, 2University of New South Wales, Sydney, NSW, Australia The C-terminal binding proteins are a group of transcriptional co-repressor proteins that are conserved throughout the animal kingdom. Nematodes have a single CtBP gene, called CTBP-1, which we have found to be predominantly expressed in the nervous system of C. elegans. In ctbp-1 mutants, motor neurons of the DA class fail to express the important marker of terminal differentiation unc-4. This is of interest since mutation of zag-1, which encodes a critical neuronal transcription factor, leads to a similar defect in DA motor neurons. We discovered through yeast two-hybrid screens that ZAG-1 physically interacts with CTBP-1. These results suggest that ZAG-1 regulates neuronal gene expression at least in part by recruiting CTBP-1 as an essential co-repressor in vivo. Mammalian members of the CtBP family are recruited to promoters through interactions with DNA-bound transcription factors that contain amino acid motifs of the form PXDLS, and CTBP-1 is similarly able to interact with PXDLS-containing transcription factors, such as ZAG- 1. Interestingly, we have found that the C. elegans CTBP-1 protein also contains intrinsic DNA binding capacity the form of a THAP domain. Following in vitro site-selection experiments, we have used the CisOrtho program1 to identify promoters that contain putative CTBP-1-THAP binding sites, representing candidate CTBP-1 target genes. With reference to both our own and published2 microarray datasets comparing transcripts from wild type animals with those from ctbp-1 mutants, and to expression pattern data, we have defined a sub-set of these as likely in vivo targets of CTBP-1-mediated repression. Given the reported role of CTBP-1 in the regulation of lifespan and stress resistance2, our identification of CTBP-1 target genes will make an important contribution to understanding the function of this transcriptional regulator in a range of contexts. 1. Bigelow HR, Wenick AS, Wong A, Hobert O. 2004. BMC Bioinformatics 5: 27 2. Chen S, Whetstine JR, Ghosh S, Hanover JA, Gali RR, et al. 2009. Proc Natl Acad Sci U S A 106: 1496-501
Contact: [email protected] Lab: Nicholas
136 Poster Topic: Gene Regulation The Role of C. elegans bHLH-29 Transcription Factor in Stress Response Thanh Quach, Casonya Johnson Georgia State University, Atlanta, GA, USA Iron is an essential element that involves in many biological processes such as oxygen transport, electron transport, mitochondrial energy production, DNA synthesis, and heme synthesis. In excess, iron can become toxic due to free radicals generation when reacting with oxygen through Fenton reaction. Thus, ferritin plays an important role in iron homeostasis in both prokaryotes and eukaryotes. The basic helix-loop-helix transcription factor, HLH-29, a member of the REF-1 family, is found to affect the expression of the ferritin genes ftn-1 and ftn- 2. Our focus is to determine the role of HLH-29 transcription factor in the iron homeostasis and stress response through the regulation of ftn-1. We are also interested in how ftn-1 is regulated by HLH-29. We have confirmed with RT-qPCR that the ftn-1 is affected by hlh-29 knockout. To determine if excess iron would cause toxicity and thereby shortens the C. elegans life span, we exposed the worms to ferric ammonium citrate. However, no change in the life span was observed. We have also tried to depleted the iron with a metal chelator, 2,2’-dipyridyl, and observed if the development was affected. Our preliminary result was inconclusive.
Contact: [email protected] Lab: Johnson
Poster Topic: Gene Regulation 137 Loss of the ubiquitin-specific proteaseusp-48 allows for direct conversion of a somatic tissue into neurons in Caenorhabditis elegans Dylan Rahe, Tulsi Patel, Oliver Hobert Columbia University Multicellular organisms begin as a single cell, executing an intricate choreography of genetic and epigenetic regulation to eventually become a complete organism. As pluripotent cells divide and specialize, their potential to become different cell types becomes increasingly restricted. The conversion of one cell fate to another, either in a normal context (development) or in an artificial environment (cellular reprogramming), is dependent on cellular context and involves both genetic and epigenetic changes. Fate-specifying transcription factors are not only regulated by proper spatiotemporal expression, but also negative cues which are thought to be mediated at the chromatin level. This has been evidenced by the limited and context- dependent ability for key fate-specifying transcription factors such as MyoD to induce specific cell fates in other cell types. To understand this context dependency and approach a mechanistic understanding of these negative cues during development, we use an assay in which a single terminal-fate-specifying transcription factor, CHE-1, is overexpressed by heatshock in late- or post-developmental stages. In wild-type animals, very few cells adopt CHE-1-dependent fates upon ectopic induction, consistent with previous data; however, we have recently shown that the absence of lin-53, a component of many chromatin-remodeling complexes, results in efficient and direct conversion of mitotic germ cells to a CHE-1-dependent fate. To extend these studies, we undertook a forward genetic screen for mutants in which direct conversion of tissues is observed upon ubiquitous CHE-1 induction. In one such mutant, ot674, we observe the expression of CHE-1-dependent fate markers in hypodermal tissue of all developmental stages. ot674 is an early stop in the gene usp-48. usp-48 encodes a ubiquitin-specific protease, which shares homology with the human gene USP48 and the yeast UBP15, all of which have no known function. Here we present initial characterization of the usp-48 mutant and the reprogramming phenotype it exhibits.
Contact: [email protected] Lab: Hobert
138 Poster Topic: Gene Regulation Chromatin Structure and Genome Stability in C. elegans Valerie Robert1, Cedric Rakotomalala1, Cecile Bedet1, Florence Couteau2, Monique Zetka2, Francesca Palladino1 1CNRS UMR5239, ENS-Lyon, Lyon, France, 2Departement of Biology, McGill University, Montreal, Canada Methylation of histone H3 lysine 4 (H3K4me), a mark associated with gene activation, is mediated by SET1 and the related mixed lineage leukemia (MLL) histone methyltransferases (HMTs) across species. Caenorhabditis elegans contains one SET1 protein, SET-2, and one MLL-like protein, SET-16. In a previous study we demonstrated that SET-2 is required for both di-and tri-methyation of H3K4 in the germline. Loss of SET-2 results in progressive sterility over several generations, suggesting an important function for H3K4 methylation in the maintenance of a functional germ line. In this poster, we will present data from genetics and irradiation assays showing that SET-2 is essential for genome stability and might be involved in double-strand break repair (DSBR). To further investigate a putative link between chromatin structure and DSBR in the C. elegans germline, we examined the IR sensitivity of strains affected for two additional chromatin marks, H3K9 or H3K36 methylation. Interestingly, preliminary results indicate that the H3K36 methyltransferase MET-1 may be required for DSBR.
Contact: [email protected] Lab: Palladino
Poster Topic: Gene Regulation 139 A New Attempt to Elicit an RNAi Phenotype with the LIM- homeodomain Transcription Factor LIM-7 Laura Vallier, John Coppola Hofstra University The LIM-homeodomain family of transcription factors are required for a multitude of developmental processes and family members have been noted to contribute in T-cell leukemia and breast cancer. Proteins within this family are expressed in C. elegans in neuronal tissues and in the gonadal sheath and other somatic non-neuronal tissue types. The LIM-7 LIM- homeodomain protein is an Islet ortholog and is expressed in numerous neurons, the gonadal sheath, as well as other cells that are probably muscular in origin. An in-frame deletion of the lim-7 gene results in L1 larval lethality; prior to death mutant animals exhibit pleiotropy: they are uncoordinated and ~50% have unattached pharynges as the two most prevalent phenotypes. Rescue of the deletion mutation using extragenic arrays results in sterile hermaphrodites with a few non-viable embryos and loose sperm in the body cavity. Despite a lethal phenotype, numerous attempts to elicit a double-stranded RNA (dsRNA) interference phenotype have not resulted in robust differences from controls. Recently, a new sensitized background was developed to assist in the transport of dsRNA into neuronal tissues, which are notoriously resistant to the effect of dsRNA interference. In this system, the SID-1 transmembrane protein, which is essential for systemic RNAi (Winston et al 2002), is placed under the pan-neuronal
promoter unc-119 (Punc-119sid-1) to allow increased transport of dsRNA into the neurons (Calixto et al 2010). Since lim-7 is expressed in many neurons we hypothesized that the lack of RNAi phenotype may lie in failure to allow dsRNA to enter neuronal cells efficiently. Using the sid-1 technology we are testing various aspects of the lim-7 phenotype via dsRNAi to ascertain if lack of a phenotype using dsRNAi in previous attempts was due to the expression of LIM-7 within neurons. Early results indicate a slower growth in Punc-119sid-1 animals treated with lim-7 dsRNAi than in the controls.
Contact: [email protected] Lab: Vallier
140 Poster Topic: Gene Regulation The Histone Demethylase UTX-1 Is Essential for Normal Development, Independently of Its Enzymatic Activity Julien Vandamme, Lisa Salcini BRIC - University of Copenhagen, Denmark Eukaryotic DNA is packaged within the nucleus through its association with histone proteins to form chromatin. Histones are subject to a wide variety of post-translational modifications, these epigenetic modifications influence gene expression and provide a unique mechanism for fine-tuning cellular differentiation and development in multicellular organisms. In particular, histone lysine methylation (mono-, di- and tri- states) is a dynamic epigenetic mark, playing fundamental roles in chromatin organization and function. Proteins of the Jumonji family are able to demethylate lysines via their JmjC domain and are conserved from yeast to humans. Here we report on the biological functions of the JmjC-containing protein UTX-1, the C. elegans homologue of mammalian UTX, a histone demethylase specific for di- and tri-methylated lysine 27 of histone H3 (H3K27me2/3). We demonstrate that utx-1 is an essential gene that is required for correct embryonic and postembryonic development. Consistent with its homology to UTX, UTX-1 regulates global levels of H3K27me2/3. Surprisingly, we found that the catalytic activity is not required for the developmental function of this protein. Biochemical analysis identified UTX-1 as a component of a complex that includes: SET-16 (a histone methyltransferase for H3K4), PIS-1, F21H12.1, ASH-2 and WDR-5. This complex is identical to the one described in mammals. Genetic analysis indicates that the defects associated with loss of UTX-1 are likely mediated by compromised SET-16/UTX-1 complex activity. Taken together, these results demonstrate that UTX-1 is required for many aspects of nematode development but, unexpectedly, this function is independent of its enzymatic activity.
Contact: [email protected] Lab: Salcini
Poster Topic: Gene Regulation 141 A Conserved SBP-1/Phosphatidylcholine Feedback Circuit Regulates Lipogenesis in Metazoans Amy Walker3, Rene Jacobs4, Jenny Watts1, Veerle Rottiers2, Lorissa Niebergall3, Anders Naar2 1Washington State University, Pullman, WA, 2Harvard Medical School, Boston, 3UMASS Medical School Worcester, MA USA, 4University of Alberta, Edmonton, Canada Transcriptional regulation may be affected by developmental cues, signal transduction or stress response pathways. A growing body of evidence also suggests that some transcription factors are affected by metabolic cues to coordinate gene expression with nutrient availability. The SREBP family of transcription factors regulate genes for generating fatty acids and phospholipid in metazoans and cholesterol synthesis in vertebrates. These factors are inhibited by cholesterol in vertebrates, however the regulatory feedback mechanisms for lipogenic genes have been less clear. Using C. elegans and mammalian models, we have found that SBP-1 and mammalian SREBP-1 control the expression of genes in the 1-carbon cycle, which produces methyl groups necessary for protein and phospholipid methylation. In addition, lack of phospholipid methylation initiates a regulatory cascade that results in increased SBP-1/ SREBP-1 activity. Up-regulation of SBP-1/SREBP-1 activity when methylation capacity or phospholipid synthesis is diminished results in increased lipogenesis and may be relevant to the development of fatty liver in mammals. Finally, our discovery that SREBP transcription factors are linked to metabolic pathways controlling methylation opens additional avenues in understanding how gene regulation is linked to metabolic control.
Contact: [email protected] Lab: Walker
142 Poster Topic: Gene Regulation HLH-29, REF-1 family protein functions in the spermatheca Ana White, Casonya Johnson GSU HLH-29, a member of the REF-1 family of transcription factors (TFs), is a basic helix-loop- helix (bHLH) protein that contains two bHLH domains, domain A and domain B. HLH-29 is expressed throughout development and life of the animal. Loss of hlh-29 and its paralog hlh-28 produces various phenotypes which includes sever ovulation defects. Genetic data indicates that hlh-29 functions in the distal spermatheca valve and in the spermatheca-uterine valve in the IP3 signaling pathway to regulate ovulation. bHLH TFs are known to form homodimers and heterodimers. In order to find binding partners for HLH-29 our lab has performed a yeast-two-hybrid screening using the C. elegans transcription factor library. Results identified FKH-6 as a protein that interacts with HLH-29 domain A. Far-western and pull-down assays confirmed HLH-29/FKH-6 interactions. Comparing expression profiles of each reporter gene, HLH-29::gfp was found to be expressed in the developing spermatheca of L4 stage animals and in the adult animals. Whereas FKH-6 is expressed in precursors of the somatic gonad, the Z1/Z4 cells during the L1 stage, and then expressed again in the spermatheca of early L3 to adult stage animals. These profiles suggest that HLH-29 interacts with FKH-6 in the L4 or in the adult animals. We propose that they regulate genes required in the L4 stage for spermatheca development or at a later stage for proper ovulation and fertilization. To further establish molecular interactions in vitro and in vivo our goals are 1) identify downstream targets of HLH-29/FKH-6 using electrophoresis mobility shift assays (EMSA) and RT-qPCR; 2) to correlate morphological changes in single and double mutant animals. One candidate gene is a nuclear hormone receptor NR4A human homolog of C.elegans nhr-6 which causes similar loss of function phenotypes as hlh-29 mutants, including ovulation defects, abnormal egg morphology, oocyte fragmentation, emo (endomitoticoocytes), and spermathecal exit and entrance defects. Our previous data also suggest that nhr-6 transcriptional activity is affected by the loss of HLH-29. More recently we identified regulatory binding sequences for FKH-6 and HLH-29 in both the promoter region and the regulatory sequences within intron 5. Additionally, our preliminary EMSA show that FKH-6 can bind to the regulatory region of nhr-6, and that HLH-29 can supershift this complex. Finally, our morphological studies suggest that, unlike FKH-6, HLH-29 does not affect morphology of the somatic gonad.
Contact: [email protected] Lab: Johnson
Poster Topic: Gene Regulation 143 Promoter analysis of the GATA type transcription factor ELT-2 Tobias Wiesenfahrt, Jannette Berg, James McGhee University of Calgary, University of Calgary It has been suggested that the GATA type transcription factor ELT-2 is the major regulator of transcription in the C. elegans intestine after endoderm specification, both embryonically and post embryonically. Rothman and Maduro have shown that the redundant GATA factors END-1 and END-3 are necessary for endoderm specification. Ectopic expression of END-1 or END-3 can initiate ectopic expression of ELT-2, suggesting that END-1 and END-3 can activate elt-2 expression in the earliest endoderm lineage. Previous experiments also showed that ELT-2 can bind to its own promoter in vivo. To understand the molecular details of how elt-2 transcription is initiated during embryonic development and is maintained thereafter, we are analyzing the promoter region of elt-2 in C. elegans. Comparison of 5 kb upstream sequences of the elt-2 gene from 4 different Caenorhabditis species revealed three conserved regions (CRI-CRIII). Deletion series as well as analysis of reporter constructs containing different combinations of the CRs suggested that CRI contains the basal promoter and CRIII contains the main enhancer of elt-2. The function of CRII is not yet clear. To find potential binding sites for END-1, END-3 and ELT-2, we searched for GATA sites within the CRs of the 5kb upstream region of elt-2. We identified 3, 3 and 4 conserved GATA sites within CRI, CRII and CRIII respectively. Band shift assays showed that END-1 and ELT-2 can bind to at least one and all four GATA sites within CRIII in vitro respectively. This suggests that END-1 (END-3 has not yet been tested) can activate elt-2 expression directly. Mutating the GATA sites within CRIII individually and in different combinations, suggested that every CRIII GATA site contributes positively to elt-2 expression. Reporter expression was absent after mutating all GATA sites within CRIII and CRI, suggesting that elt-2 regulation is exclusively dependent on GATA factors. To test if ELT-2 can drive intestinal specification and differentiation in the absence of END-1/END-3, we expressed elt-2 under control of the end-1 and end-3 promoters in the end-1/end-3 double mutant (kindly provided by Morris Maduro). Indeed, the end-1p::elt-2 construct is able to rescue the end-1/ end-3 double mutant with reasonable penetrance, showing that the endoderm differentiation factor ELT-2 can drive endoderm specification and further supporting the hypothesis that ELT-2 is involved in the regulation of every gene expressed in the intestine.
Contact: [email protected] Lab: McGhee
144 Poster Topic: Gene Regulation Genetic Screen for Novel Repair Genes Implicated in UV-induced DNA Damage Response Stefanie Wolters, Bjoern Schumacher CECAD at the institut of genetics, Cologne, Germany Ultraviolet radiation of the sunlight represents an environmental carcinogen, which can lead to helix-distorting lesions in the genome and therefore cause cancer. To cope with this damaging influence cells developed a multistep “cut and patch” mechanism to repair the impaired DNA. The Nucleotide Excision Repair (NER) removes helix distorting lesions and is divided into two branches, which differ in damage recognition: while initiation factors of Global-Genome-NER (GG-NER) scan the whole genome for damage, Transcription Coupled Repair (TCR) initiates repair when RNA polymerase II encounters a lesion during transcription. The NER pathway is highly conserved between mammals and C. elegans. We identified UV hypersensitivity phenotypes of known C. elegans NER mutants distinct for GG-NER und TCR. While mutations in TCR lead to developmental arrest at L1 larval stage, defects in GG- NER lead to germ line arrest and sterility upon UV radiation of L1 larvae. We took advantage of these phenotypes to design a random mutagenesis based screening strategy. By performing this screening method we identified five different mutants that are hypersensitive to UV light. The respective mutations were mapped to the C. elegans genome via SNP mapping technique and whole genome sequencing was performed in order to identify the impaired genes. Non-complementation analysis revealed that mutations in structural maintenance of chromosomes-5 (smc-5) lead to the UV-sensitivity phenotype in two of the five mutants. Previous studies, which were performed mainly in yeast, showed that smc-5 plays a role in homologous recombination repair and might be necessary for DNA structure as well. In this work we analyze the role of smc-5 in repair of UV-B induced DNA damage and its interplay with NER by genetic analysis, biochemistry and immunohistochemistry.
Contact: [email protected] Lab: Schumacher
Poster Topic: Gene Regulation 145 The eIF4E-binding protein IFET-1 is a broad-scale translational repressor and is required for normal P granule ultrastructure Madhu Sengupta1, Lloyd Low1, Joseph Patterson2, Traude Beilharz1, Jennifer Schisa2, Peter Boag1 1Monash University, Melbourne, Victoria, Australia, 2Central Michigan University, Mount Pleasant, MI P granules are large cytoplasmic ribonucleoprotein complexes that associate with the nuclear pore complexes on developing germ cells. We have identified that the C. elegans homologue of the eIF4E-transporter, IFET-1, is required for normal P granule formation and translational regulation of many germ cell mRNAs. Ultrastructural analysis using Transmission electron microscopy (TEM) of ifet-1 null animals indicates that the electron dense “crest” and “base” of P granules are abnormally formed, suggesting that RNA may not be concentrated in the granule normally. In addition, although P granules are similar in size to wild-type, there is a significant reduction in the number of nuclear pores associated with P granules in ifet-1 null animals. In the absence of IFET-1, the P granule components CGH-1 and CAR-1 fail to localise normally and instead are diffusely spread throughout the cytoplasm. When grown at 25oC, ~25% ifet-1 null animals have a masculinised gonad containing only sperm, and this phenotype is enhanced when the general translational inhibitors CGH-1, CAR-1 or PATR-1 are knocked down by RNAi. We also found that IFET-1 is required for translational repression in the distal gonad for 7 out 7 GFP-tagged germline reporter constructs tested. Orthologues of IFET-1, CGH-1, CAR-1 and PATR-1, play a crucial role in decapping-mediated mRNA decay in yeast and mammalian cells, where they repress translation as a first committed step towards mRNA decay. We propose that IFET-1, CGH-1, CAR-1 and PATR-1 function collectively as broad-scale translational inhibitors that repress some maternal mRNAs by forming a repressive complex on mRNA in P granules.
Contact: [email protected] Lab: Boag
146 Poster Topic: Germline Spindle assembly checkpoint proteins monitor synapsis during meiosis in C. elegans Tisha Bohr, Piero Lamelza, Needhi Bhalla University California Santa Cruz, Santa Cruz (CA), USA In order to achieve proper meiotic chromosome segregation, homologous chromosomes must pair and synapse in prophase I to promote crossover recombination. Improper chromosome segregation can lead to aneuploidy, which is associated with miscarriages, birth defects and tumorigenesis. Cell cycle checkpoints ensure accurate chromosome segregation by monitoring key events during cell division. In C. elegans, the synapsis checkpoint monitors synapsis of homologous chromosomes and triggers cell death in the event of asynapsis. The synapsis checkpoint requires cis-acting sites near the end of each chromosome, termed Pairing Centers (PCs), for activation. PCs promote pairing and synapsis by establishing transient connections with the cytoplasmic microtubule network via attachment to the nuclear envelope, but how they activate the synapsis checkpoint is currently unknown. The mitotic spindle assembly checkpoint also uses cis-acting sites, centromeres, as platforms for checkpoint activation to monitor microtubule attachments and tension at kinetochores. I will show that components of the spindle assembly checkpoint are also required for the synapsis checkpoint during C. elegans meiosis. Moreover, two components, Mad2 and Zwilch, localize to prophase I nuclei of the germline. These data support a model in which spindle assembly checkpoint proteins localize to pairing centers to monitor tension between homologues and/or attachment to microtubules in order to satisfy the synapsis checkpoint. These and future experiments will not only dissect how the synapsis checkpoint works in C. elegans but will also provide insight into conserved mechanisms that monitor chromosome behavior during cell division to maintain genomic integrity.
Contact: [email protected] Lab: Bhalla
Poster Topic: Germline 147 A global genomic survey of genes that mediate LKB1/PAR-4- dependent germline stem cell quiescence in C. elegans Rita Chaouni, Richard Roy Department of Biology, DBRI, McGill University, Montreal, Quebec, Canada Upon encountering harsh environmental conditions, Caenorhabditis elegans larvae are able to alter their developmental program and enter the dauer diapause, an alternative developmental stage that enables larvae to endure long periods of starvation and stress. During this arrested state, the germline stem cells, which normally divide during reproductive development, halt their proliferation and are consequently rendered quiescent. Previous work has revealed that LKB1/par-4 and AMPK/aak-2 cooperate under such nutrient-deficient conditions in order to mediate this germline stem cell quiescence. The knockdown of either one of these genes causes aberrant germline hyperplasia in dauer larvae, while the inactivation of LKB1/par-4 in AMPK/aak-2 null mutants causes an enhanced hyperplasia phenotype. Thus, although LKB1/ par-4 is known to regulate AMPK/aak-2 in C. elegans, our genetic analyses suggest that it is unlikely that AMPK/aak-2 is the sole mediator of germline stem cell quiescence downstream of LKB1/par-4. LKB1/par-4 is a tumor suppressor protein kinase that is implicated in the rare, autosomal dominant disease Peutz-Jeghers syndrome (PJS). In order to better understand its function in tumorigenesis, we characterized its role in regulating cellular quiescence in developmentally arrested larvae using a genome-wide RNA interference-based screen to identify suppressors of PAR-4-mediated germline hyperplasia. We identified several genes whose loss-of-function was found to rescue the germline hyperplasia observed in par-4 dauer larvae, suggesting that their expression is misregulated in the absence of LKB1/par-4. Future endeavors include the characterization of key candidates, many of which impinge on the actin cytoskeleton and its regulation. Further understanding of the function of these genes will provide additional insight as to how LKB1/PAR-4 blocks tumorous growth by regulating cell cycle quiescence.
Contact: [email protected] Lab: Roy
148 Poster Topic: Germline VPR-1, a VAPB homolog required for germ line proliferation and differentiation Pauline Cottee, Jack Vibbert, Sung Min Han, Michael Miller University of Alabama at Birmingham, Birmingham, Alabama, USA The Major Sperm Protein (MSP) is an important protein for sperm motility and oocyte maturation. Secreted from motile sperm, MSP binds to the Eph receptor VAB-1 and other unknown receptors expressed on oocytes and sheath cells to induce oocyte maturation and ovulation. The MSP domain is an evolutionary conserved motif and is linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and late-onset spinal muscular atrophy (Nishimura et al., 2004). A point mutation (P56S) within the MSP domain of VAPB/ ALS8 gene causes reduced VAPB expression in humans and ALS mouse models, indicating VAPB plays a role in the pathogenesis of this disease. Previous studies have shown that the VAPB-MSP domain is cleaved and secreted, and acts as a ligand for Eph receptors. Further, the P56S point mutation causes VAPB proteins to aggregate and is failed to be secreted (Tsuda et al., 2008; Han et al., 2012). VPR-1 is the worm VAPB homologue. Loss of this protein in the worm recapitulates many of the pathologies observed in ALS patients, including mitochondrial dysfunction and lipid metabolism defects. Further, VPR-1 null mutant animals are sterile due to germ cells failing to proliferate and differentiate. Germ line proliferation and differentiation defects in VPR-1 null mutant animals were rescued with extrachromosomal arrays of fosmid DNA containing the complete vpr-1 genomic locus, indicating that VPR-1 has a role in germ line development. Examination of the distal tip cells of VPR-1 null mutant animals has shown that these cells were enlarged and have shorter processes in comparison those of wild-type animals. Additionally, visualization of the sheath cells in VPR-1 mutant hermaphrodites showed severe defects in these cells. Using a sheath cell driven CED-1::GFP transgene, we have also observed an increase in apoptosis in the germ line of vrp-1 mutant animals. Silencing of selected genes involved in the physiological and DNA-damage induced apoptosis pathways indicate that the increase in apoptosis in VPR-1 mutant animals is of a physiological basis rather than from DNA damage. My current goal is to use mosaic analysis to understand where VPR-1 functions in regulating germ line and somatic gonad development.
Contact: [email protected] Lab: Miller
Poster Topic: Germline 149 Paternal Mitochondria Elimination From the Germline in C. elegans Embryos Dominika Bienkowska, Sylvain Bertho, Carrie Cowan I.M.P., Vienna, Austria An almost universal feature of sexual reproduction is the strict uniparental inheritance of mitochondria in the zygote. While several theories have been postulated to account for why mitochondria should be inherited from only one parent, there is sparse evidence in support of these ideas. In C. elegans, paternal mitochondria are delivered to the oocyte during fertilization but are gradually degraded in the embryo through autophagy. Using long-term time-lapse imaging of paternal mitochondria during embryogenesis, we find that the germline lineage preferentially eliminates paternal mitochondria compared to the soma. Already in the first asymmetric cell division of the embryo P0, PAR polarity promotes segregation of paternal mitochondria away from the germline. Germline elimination of paternal mitochondria appears to be completed by division of P3. In many embryos, some paternal mitochondria are present in the soma as late as the 100-cell stage, suggesting paternal mitochondria may not be harmful to the organism per se but rather specifically prevented from entering the germline. We are now using genetics to identify the mechanisms downstream of PAR polarity that control germline- specific elimination of paternal mitochondria and to identify the effects of aberrant paternal mitochondria inheritance on germline development.
Contact: [email protected] Lab: Cowan
150 Poster Topic: Germline CACN-1 is required for gonad and germline development Hiba Tannoury, Erin Cram Northeastern University, Boston, MA, USA CACN-1 is a well conserved protein of unknown molecular function, required in C. elegans for larval development including DTC migration, somatic gonad and germline development and fertility. CACN-1 is expressed throughout the hermaphrodite reproductive system, and observations in somatic gonad marker strains treated with cacn-1 RNAi show that cacn-1 is required for the development of the somatic gonad. Observations in rrf-1 mutant animals, in which the somatic (but not germline) RNAi response is defective, suggest that cacn-1 is required in the germline for fertility. C. elegans, a self-fertile hermaphrodite, produces sperm late in larval development before switching to oocyte production in adulthood. This switch is regulated by a set of RNA binding proteins and splicing factors downstream of the germline sex determination gene fem-3. In cacn-1 RNAi treated animals, copious sperm, but few, if any, oocytes are produced. Genetic interaction studies indicate that cacn-1 normally functions upstream of the sperm-to-oocyte differentiation decision pathway by repressing the fem-3, fog-1, and fog-3 male-fate promoting genes. Transcriptome sequencing data analysis shows upregulation of terminal male-fate differentiation factors, fog-1 and fog-3, in cacn-1 depleted animals. Continued expression of FOG-1 and FOG-3 likely disrupts the germ cell decision to switch to production of oocytes. Therefore, CACN-1 functions similarly to the known sperm- to-oocyte regulatory RNA binding proteins/splicing factors to negatively regulate the male fate promoting genes of the fem-3 pathway.
Contact: [email protected] Lab: Cram
Poster Topic: Germline 151 HIS-35, a histone H2A variant that differs from canonical H2A by one amino acid, functions in fertility Francisco Guerrero, Rodrigo Estrada, Meghann Shorrock, Margaret Jow, Diana Chu San Francisco State University, San Francisco, CA, U.S. Histone variants are incorporated during germ cell development to execute transcriptional programs that specify cell fate. HIS-35, a histone H2A variant that we have found via proteomic analysis, is enriched on sperm chromatin in comparison to embryo chromatin. Because HIS-35 differs by only 1 amino acid from canonical S-phase histone H2A, it is unclear when or why HIS-35 is incorporated during spermatogenesis. We hypothesize that HIS-35 incorporation during sperm formation regulates germ cell differentiation. Consistent with this, we found his-35(tm1328) deletion mutant hermaphrodites only produce 49% of the total progeny of N2 control hermaphrodites and lay few unfertilized oocytes or dead embryos. They also exhibit slow growth and small size. Cytological analysis of his-35(tm1328) mutant male and hermaphrodite germlines reveal incompletely penetrant defects in germ cell formation, including reduced numbers of developing germ cells and occasional defects in chromosome segregation. This suggests HIS-35 incorporation is important for optimal germ cell formation. Because of the high degree of similarity to H2A, HIS-35 is resistant to traditional immunochemistry techniques, thus we are creating transgenes to investigate HIS-35 incorporation during germ cell development. We have constructed GFP::HIS-35 to follow HIS-35 incorporation during specific stages of spermatogenesis. We are also constructing HIS-35 fused to DNA methyltransferase to help us track HIS-35 incorporation throughout the genome. Defining HIS-35 expression and localization, as well as the genes it interacts with, will elucidate mechanisms for how HIS-35 functions in fertility.
Contact: [email protected] Lab: Chu
152 Poster Topic: Germline SNF-10, an SLC6 transporter required for sperm activation by C. elegans males Kristin Fenker, Angela Hansen, Conrad Chong, Molly Jud, Gillian Stanfield University of Utah, Salt Lake City, UT, USA A key step of spermatogenesis is the acquisition of cellular motility. In nematodes, sperm move by crawling, and they become motile during a regulated process termed sperm activation in which subcellular rearrangements lead to formation of a pseudopod and relocalization of proteins required for fertilization to the cell surface. In C. elegans, sperm activation is regulated differentially in males and hermaphrodites to promote the two sexes’ distinct requirements for reproductive success. Male sperm activation is regulated by a serine protease, TRY-5, which is transferred during mating in seminal fluid to couple the onset of male sperm motility to their entrance into the female reproductive tract. The coupling of these events is important, as early sperm activation leads to male infertility, while a delay in achieving motility could cause sperm to be displaced from the hermaphrodite’s reproductive tract. Protease treatment of sperm in vitro induces their activation, suggesting TRY-5 may act directly on targets on the sperm plasma membrane. To search for such targets, we performed a genetic screen for factors required for sperm activation by males and obtained several alleles of the solute carrier 6 (SLC6) family plasma membrane transporter snf-10. Like try-5, snf-10 is not required for fertility and acts in parallel to the spe-8 group hermaphrodite sperm activation genes. However, unlike try-5, snf- 10 is not required for males to transfer activating factor, and its function is required in sperm. snf-10 mutant sperm fail to activate in response to protease in vitro or TRY-5 in vivo, but they can activate upon treatment with another known activator, the weak base triethanolamine. We currently are analyzing the localization of SNF-10 in sperm cells, and preliminary results suggest that it is present on the plasma membrane. Taken together, this plasma membrane localization and the fact that it functions downstream of try-5 make SNF-10 a strong candidate for the target of TRY-5 cleavage.
Contact: [email protected] Lab: Stanfield
Poster Topic: Germline 153 Putative protamines, SPCH-1/2/3, localize to mature sperm chromatin and may play a role in fertility Jennifer Gilbert, Dana Byrd, Diana Chu San Francisco State University, San Francisco, (CA), USA During spermatogenesis, chromatin becomes highly compacted to ensure the efficient delivery of DNA to the oocyte. Compaction of sperm chromatin in most animals is facilitated by deposition of small nuclear basic proteins (SNBPs) called protamines, which bind in the major groove of DNA to allow bending of the DNA. While protamine incorporation to compact sperm chromatin is well conserved, the high variability of protamine gene and protein sequences across phyla has complicated the identification of these proteins across species. We hypothesize that C. elegans have SNBPs that share similar molecular features and functions as protamines. As such, we expect them to be small, highly basic proteins enriched in sperm chromatin, and localize to DNA during late stages of spermatogenesis. To identify such proteins, a proteomic approach was taken. We identified three nearly-identical proteins, SPCH-1/2/3 that were abundant in sperm chromatin samples and not found in embryo chromatin. SPCH-1/2/3 are only 22kD and they have a predicted isoelectric point of ~13.7. Also consistent with the amino acid content of SNBPs, SPCH-1/2/3 consist of a high percent of arginine (28%) and serine (29%) residues. Immunostaining using an antibody that recognizes all three SPCH proteins shows that SPCH-1/2/3 localize to DNA in late stages of spermatogenesis and around adult sperm. Immediately after fertilization, SPCH-1/2/3 mark the paternal pronuclei and then are removed as the sperm pronucleus decondenses. Using proteomic analysis of acid solubilized sperm chromatin, we find that SPCH-1/2/3 are highly phosphorylated. Interestingly, major sites of phosphorylation are found on amino acids that differentiate SPCH-1 from SPCH-2 and SPCH-3. Due to the importance of protamines in sperm DNA compaction, we anticipate that loss of SPCH function could lead to fertility defects. In fact, our preliminary progeny counts of single SPCH mutants suggest elimination of SPCH function reduces fertility. Thus, SPCH-1/2/3 appear to function as protamines and may play an important role in male fertility.
Contact: [email protected] Lab: Chu
154 Poster Topic: Germline Sperm Vs Sperm: Determining the Cellular Basis of Sperm Competition Jody Hansen, Daniela Chavez, Gillian Stanfield University of Utah In C. elegans, male sperm must compete with hermaphrodite self sperm to fertilize oocytes. Male sperm precedence, the differential fertilization success of male sperm over hermaphrodite self sperm, is nearly absolute and relies on intrinsic differences between male and hermaphrodite sperm. Sperm motility, but not fertilization competence, is required for male precedence. Thus, C. elegans sperm competition provides a robust system to study cell competition in the context of migrating cells. Male spermatid size is significantly larger than that of hermaphrodites, and this larger size is correlated with faster crawling speeds and preferential residence in the spermathecae, where fertilization occurs. We seek to understand the cellular and molecular mechanisms contributing to male sperm precedence. By identifying sperm-specific gene products required for this process, we will improve our understanding of sperm migration and how it relates to cell competition. We have identified a mutant,me69 , that displays reduced male sperm precedence. In addition, me69 mutant male sperm accumulate slowly in the spermathecae as compared to those of the wild type, suggesting a defect in some aspect of motility or directional migration. However, me69 mutant hermaphrodites have normal brood sizes, providing evidence that mutant sperm retain functional motility. Notably, me69 mutant spermatids are the same size as wild-type spermatids, suggesting that mechanisms in addition to size contribute to male sperm precedence. Our current goal is to identify the me69 gene and to determine whether the me69 defect is due to changes in adhesion, cell signaling, or other factors affecting cell migration. Ultimately, identifying the cellular basis of the me69 defect also will differentiate among models of sperm competition. Currently, we have preliminary data identifying a candidate gene for me69, and we are building strains to test for rescue and determine protein expression. We also are characterizing the me69 migration defect by tracking wild-type and mutant sperm as they travel in the hermaphrodite reproductive tract.
Contact: [email protected] Lab: Stanfield
Poster Topic: Germline 155 Evaluating the Role of the V-ATPase B Subunit Utilizing C.elegans Sperm Melissa Henderson, Elizabeth Gleason, Ying Long, Taylor Walsh, Emily Wang, Steven L’Hernault Emory University, Atlanta, GA, USA Secretory vesicles are used during spermatogenesis to deliver proteins to the cell surface prior to sperm-egg fusion. Many of these proteins are essential for fertilization to take place. In C.elegans the membranous organelles (MOs) fulfill the role of these important secretory vesicles. As MOs mature, they undergo acidification, which is similar to what occurs in the acrosome of mammalian sperm. Our recent publication demonstrated that the acidification on MOs results from the presence of the V-ATPase complex and this process can be disrupted with the drug bafilomycin. C. elegans encodes two V-ATPase subunits, vha-12 and spe-5. vha-12 is located on the X chromosome and transcriptionally silent during spermatogenesis leaving spe-5, which is located on chromosome I, as the only B subunit utilized in sperm. The B subunit has two distinct roles in the V-ATPase complex, the hydrolysis of ATP and the binding of actin, which occurs at a highly conserved actin-binding site. We are evaluating these roles by further analysis of several separation of function spe-5 mutants, including a transgenic MosSci line in which actin binding has been abolished. vha-12 expression is required in many somatic cells, so the existence of spe-5 allows analysis the sperm V-ATPase mutants in sterile, but otherwise viable, animals.
Contact: [email protected] Lab: L’Hernault
156 Poster Topic: Germline The RNA binding protein TIA-1.2 is essential for fertility in C. elegans Gabriela Huelgas Morales, Carlos Silva Garcia, Rosa Navarro Gonzalez Cellular Physiology Institute UNAM, Mexico City, Mexico RNA binding proteins, such as TIA-1 and TIAR, regulate RNA at different levels in a variety of organisms. In the nucleus, these proteins participate in alternative splicing while in the cytoplasm they regulate mRNA stability and/or translation rate. Along with these functions, under stress conditions, TIA-1/TIAR aggregate to form stress granules in a reversible manner to inhibit translation and protect mRNAs in harmful conditions. In C. elegans, the lack of one of the three homologs of TIA-1, C18A3.5, leads to several germ line defects, including sterility. This fact is interesting since Tia-1(-/-) and Tiar(-/-) knockout mice show a high percentage of sterility and embryonic lethality as well, by a mechanism that remains unknown. Our aim is to study the phenotype of the tia-1.2 mutant focusing in its germ line defects, and specifically to understand TIA-1.2’s role in oogenesis, ovulation and fertility. tia-1.2 mutants are temperature sensitive, showing p-vulva and sterility at 25°C. By Nomarski and fluorescence microscopy we observed that tia-1.2 (tm361) have a smaller distal region along with shorter mitosis and larger pachytene regions, suggesting that there could be a misregulation on this mechanism. Oocytes seem to go through all maturation steps, but later on, mutant worms show an EMO phenotype and oocytes accumulate in the uterus. Based on this phenotype, we are currently testing several candidate genes that could be regulated by TIA-1.2.
Contact: [email protected] Lab: Navarro Gonzalez
Poster Topic: Germline 157 Germline Hexosamine Pathway Synthesis of UDP-GlcNAc is Regulated by SUP-46 Wendy Johnston1, Aldis Krizus1, Arun Ramani2, Andrew Fraser2, James Dennis1 1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada, 2Department of Molecular Genetics, University of Toronto, Toronto, Canada Eukaryotic cells are encased in a sugar coating, in the form of secreted and membrane- associated glycoconjugates. Extracellular glycoconjugates are essential for cell-cell interactions, and play critical roles in modulating cell signaling. The hexosamine pathway synthesizes UDP- GlcNAc, an essential building block for N- and O-glycan-modified glycoproteins, proteoglycans and sugar polymers including chitin. Chitin and chitin-binding glycoproteins in the eggshell and extraembryonic matrix (EEM) are required for multiple events in the C. elegans oocyte- to-embryo transition, including generation of a polyspermy barrier at fertilization, high fidelity meiotic chromosome segregation, polar body extrusion, normal MBK-2 localization, and A-P polarization (reviewed in Johnston and Dennis, 2012, Genesis). Therefore, the C. elegans zygote and surrounding eggshell/EEM provide an excellent model system for investigating the role of extracellular matrix glycoconjugates in early developmental events. gna-2 and gna-1 encode a key enzyme in the hexosamine pathway. In a gna-2(qa705) suppressor screen, we isolated 4 alleles of sup-46. Suppression of gna-2(qa705) maternal effect embryonic lethality depends on GNA-1 for production of UDP-GlcNAc. SUP-46 is required for normal hatchling number, particularly at high temperature (26oC) where sup-46 mutant brood sizes are ~25% of control. Genome-wide profiling of transcripts insup-46(qa710) by RNA-Seq demonstrates increased abundance of gna-1. Moreover, sup-46(qa710, 708) have elevated levels of GNA-1::GFP in the germline. Together, these results identify SUP-46 as a regulator of GNA-1 in the hexosamine pathway synthesis of UDP-GlcNAc. Current studies are underway to determine the mechanism by which SUP-46 controls gna-1 transcript level, and to examine the role of SUP-46 and GNA-1 in stress resistance. Supported by a CIHR grant.
Contact: [email protected] Lab: Dennis
158 Poster Topic: Germline Role of Notch re-localization in establishing germline stem cell quiescence in C. elegans dauer larvae Pratik Kadekar, Nathan Navidzadeh, Patrick Narbonne, Emily Wendland, Richard Roy Department of Biology, DBRI, McGill University, Montreal, Quebec, Canada Stem cells are regulated and maintained by signals that emanate from their specific environmental niche. Similarly in C. elegans germline, the somatic distal tip cells (DTCs) located at the extremities of the gonad, form a niche for the germline stem cells (GSCs) and regulate their proliferation. GSCs express the Notch receptor/GLP-1 on their membrane and is activated by the Notch ligand/LAG-2 expressed in the DTCs. This activation instructs the GSCs to undergo mitosis while inhibiting them from executing the meiotic pathway. Interestingly, the Notch ligand/LAG-2, is expressed and is active in the quiescent C. elegans dauer germ line. This suggests that in dauers, mitotic quiescence is regulated at some point downstream of receptor activation. We have found that over the course of dauer diapause, the Notch receptor/GLP-1 undergoes subcellular re-localization from the membrane to the rachis. In the hyperplasic dauer germline of an AMPK or PAR-4 mutant, GLP-1 is still present around the membrane of the GSCs and fails to re-localize to the rachis. This re-localization of the Notch receptor may therefore contribute to the appropriate establishment of mitotic arrest in the GSCs. If AMPK or PAR-4 are compromised, the Notch receptor/GLP-1 does not appropriately re-localize and may therefore be associated with the observed germline hyperplasia in these mutants. We are currently trying to unravel the link between LKB1/AMPK signaling and this novel Notch response.
Contact: [email protected] Lab: Roy
Poster Topic: Germline 159 Protein synthesis regulation in the germline: eIF4 factors promote selective mRNA translation for meiosis, differentiation, maturation or apoptosis. Melissa Henderson1, Jacob Subash1, Vince Contreras1, Anren Song2, Sara Labella3, Andrew Friday1, Monique Zetka3, Robert Rhoads2, Brett Keiper1 1Brody School of Medicine at East Carolina University, Greenville, NC 27834, 2LSU Health Sciences Center, Shreveport, LA 71130, 3McGill University, Montreal, Quebec, Canada Translational control of mRNAs represents the most important mode of gene regulation in animal germ cells, and many specific examples have been described. Only recently has the role of translation initiation factors (eIFs) in such translational control been appreciated. Our labs are uncovering mRNAs specifically regulated by individual isoforms of eIF4E and eIF4G, two subunits of the cap-binding initiation complex, in C. elegans germ cells. Deficiency in individual isoforms of eIF4E (e.g. IFE-1, IFE-2, IFE-3) or eIF4G (IFG-1) changes the fate of oocyte and/or sperm differentiation, resulting in blocked maturation steps, inefficient meiotic recombination, gamete fate switching, or induced germ cell apoptosis. Each reduces fertility, but in surprisingly unique ways depending upon the translation factor type. Altering the balance of IFG-1 p170/ p130 isoforms triggers the physiological apoptotic cascade by inducing cap-independent synthesis of CED-4, the worm Apaf-1 homolog. For two IFE isoforms we have identified mRNAs that uniquely require that isoform for efficient translation. These mRNAs encode proteins of critical function in oocyte and/or spermatocyte differentiation. IFE-1 is required for late stage oocytes to efficiently translatepos-1, pal-1, mex-1, and oma-1 mRNAs. Spermatocytes lacking IFE-1 fail in the final budding/cytokinesis step, accumulating as multinucleated secondary spermatocytes. Translation of msh-4 and msh-5 mRNAs, on the other hand, requires IFE-2 to synthesize proteins for meiotic crossover, allowing those cells to complete proper meiotic chromosome segregation. Neither eIF4E deficiency results in loss of global protein synthetic activity nor general growth capacity of the gonad. Thus, germ cell translation initiation factors appear to drive mRNA selection for specific developmental functions. The evidence supports a positive regulatory network of eIF4E-eIF4G-mediated translational control directing gamete differentiation/survival and cell death.
Contact: [email protected] Lab: Keiper
160 Poster Topic: Germline P-TEFb—Independent Phosphorylation of RNA Polymerase II CTD- Ser2 in the C. elegans Germline Elizabeth Bowman, Bill Kelly Emory University, Atlanta, GA, USA Multiple phosphorylation events targeting the C terminal domain (CTD) of the catalytic subunit accompany the progression of RNA Polymerase II (Pol II) through different stages of transcription. For example, the elongation stage is associated with phosphorylation of Serine 2 (Ser2P) of the Pol II CTD. This phospho-epitope has long been considered to be the product of the P TEFb (CDK 9/Cyclin T) complex in metazoans. Surprisingly, we have found that Ser2P in the C. elegans germ line occurs independently of the P TEFb complex. Instead, the appearance of Ser2P is fully dependent on another CTD Ser2 kinase/cyclin complex, CDK 12/Cyclin K. Whereas CDK 9/Cyclin T knockdown results in complete loss of Ser2-P in somatic lineages, substantial Ser2P is still present in the germ line at all stages. In contrast, CDK 12 and/or Cyclin K knock down results in only a partial Ser2P decrease in somatic nuclei, but complete loss of Ser2P in germ cells at all developmental stages. In addition, we find that CDK 12/Cyclin K, rather than P TEFb, appears to be the Pol II Ser2 kinase complex regulated by the maternal transcriptional repressor, PIE 1, in the nascent embryonic germ line. Transgenic analyses suggest that although CDK-9, CDK-12, and Cyclin K are ubiquitously expressed, Cyclin T (the partner of CDK-9) appears to have reduced expression in the germ line. Interestingly, the Pol II elongation regulator and known target of CDK-9, DSIF, is also expressed in all lineages. The striking prominence of P TEFb-independent Pol II CTD phosphorylation in the germline suggests that there may be basic, separable differences between transcriptional processes operating in the germline cycle, and in those engaged in differentiating somatic lineages.
Contact: [email protected] Lab: Kelly
Poster Topic: Germline 161 sacy-1 Links Somatic Control of Oocyte Meiotic Maturation, Germline Sex Determination, and Gamete Maintenance Seongseop Kim, J. Amaranath Govindan, Zheng Jin Tu, David Greenstein University of Minnesota, Minneapolis, MN, USA All described MSP-dependent meiotic maturation events in the germline require the
activity of the Gas-adenylate cyclase-protein kinase A pathway in the gonadal sheath cells. We conducted genetic screens for mutations that suppress the sterility of acy-4 adenylate cyclase null mutations. We identified 66s uppressor of adenylate cyclase (sacy) mutations that define at least 17 genes. Using a combination of whole-genome sequencing and positional cloning, we identified the molecular identities of eight sacy genes, which include sacy-1, twk-1, uev- 1, tom-1, pde-6, spr-2, spr-4, and spr-5. Our screen appears to have recovered regulators of meiotic maturation that function in both the germline (sacy-1 and spr-5) and the gonadal sheath cells (twk-1). sacy-1, spr-5, and twk-1 function downstream of kin-1/pka and appear to affect fertility cumulatively. Here we introduce sacy-1 which, in addition to negatively regulating meiotic maturation, functions in the hermaphrodite sperm-oocyte switch, and is required for gamete maintenance. sacy-1 encodes a highly conserved DEAD-box helicase orthologous to the Drosophila translational regulator Abstrakt. A rescuing GFP::SACY-1 fusion is expressed in most or all cells, and is localized to the nucleus and cytoplasm. We isolated three viable and fertile hypomorphic missense alleles of sacy-1 as acy-4 suppressors. Surprisingly, we found that these sacy-1 mutant alleles could suppress germline feminization and self-sterility caused by null mutations in fog-2. A maternal wild-type copy of sacy-1 antagonizes the suppression of fog-2 feminization. sacy-1 functions upstream of fem-3 in the germline sex-determination pathway, possibly at the level of tra-2. A sacy-1 null allele is sterile in both sexes and exhibits a gamete degeneration phenotype–sperm and oocytes appear to undergo necrotic cell death. The sacy-1 null gamete degeneration phenotype is independent of ced-3 and ced-4, but partially suppressed by a mutation in UNC-68 ryanodine receptor. Interestingly, germline feminization markedly delays oocyte degeneration in sacy-1 null mutants. sacy-1 null females ovulate large numbers of unfertilized oocytes, indicating that sacy-1 is a strong negative regulator of meiotic maturation. Genetic epistasis analysis indicates that sacy-1 likely functions upstream of oma-1 and oma-2, which are redundantly required for meiotic maturation. SACY-1 might function widely in translational regulation in the germline.
Contact: [email protected] Lab: Greenstein
162 Poster Topic: Germline Investigating the Role of SMC-5/6 in Preventing Germline Genomic Rearrangement Killeen Kirkconnell, Dane Session, Raymond Chan University of Michigan, Ann Arbor, MI, USA De novo germline mutations can lead to spontaneous abortion and genetic defects in humans. A frequently observed class of germline mutation is genomic rearrangement, which includes deletions and duplications (Lupski, 1997). Study of recurrent genomic rearrangements in human sperm found evidence suggesting rearrangements can originate from aberrant inter-homolog and intra-chromatid homologous recombination (Turner, 2008). Surprisingly, rearrangements from inter-sister recombination are estimated to be extremely rare, which may indicate that inter-sister recombination normally acts to suppress germline genomic rearrangement. We have previously shown that the C. elegans smc-5 and smc-6 mutants had defects in meiotic homologous recombination repair, likely in sister-chromatid recombination (Bickel, 2010). Homozygous smc-5 and smc-6 mutants also exhibit a mortal germline phenotype and become sterile over several generations, which suggests an accumulation of germline mutations. This study addresses whether the smc-5 and smc-6 mutants accumulate a higher frequency of germline mutations, and determines the types of mutations occurring. We adapted a phenotypic assay to identify spontaneous germline mutations at three genetic loci in the smc-5 smc-6 mutant background. The unc-93(e1500) mutation produces a toxic protein which confers uncoordinated movement. An unc-93(e1500) worm can revert to wildtype movement with mutations that disrupt the expression of the mutant UNC-93 protein or two ancillary proteins, SUP-9 and SUP-10 (Greenwald,1980). We found that the smc-5 smc-6 mutant has an approximately 93-fold higher reversion rate than wildtype worms. The reversion phenotype is heritable, so these reversion events are the result of germline mutations. Genetic complementation tests and PCR analysis are performed to determine which of the three genes contains the mutation responsible for the reversion. Our preliminary results confirm that the majority of reversions are due to disruption ofunc-93, sup-9, or sup-10. More importantly, PCR analysis of the mutated genes indicates a bias towards deletion events, with many removing an entire gene. These data support the prediction that smc-5 and smc-6 are important for preventing chromosome structural variations. We are mapping and sequencing the rearrangement breakpoints, and we plan to conduct a genome-wide analysis of de novo rearrangements in the smc-5 smc-6 mutant.
Contact: [email protected] Lab: Chan
Poster Topic: Germline 163 The let-479 Gene Encodes a Homolog of SPE-42 and is Required for C. elegans Fertilization Tim Kroft, Luke Wilson, Lindsey Magnuson, Gabe Fall University of Minnesota Duluth, Duluth, MN, USA We analyzed our collection of spermatogenesis defective (spe) mutants for late acting spe-9 class mutants that are likely to be involved in sperm-egg recognition, binding, or fusion during fertilization. Both males and hermaphrodites homozygous for eb137 mutations produce sperm that appear normal but fail to fertilize oocytes. Sperm persist in the spermathecae of eb137 mutant hermaphrodites and the hermaphrodites produce large numbers of oocytes, suggesting that sperm activation and major sperm protein signaling to the developing oocytes and somatic gonad are not compromised. Following mating, eb137 male-derived sperm migrate to the hermaphrodite spermathecae and compete with hermaphrodite sperm despite their inability to fertilize oocytes. Complementation testing with known LG V spe genes demonstrated that eb137 is an allele of let-479. Positional cloning, transgenic rescue, and sequence analysis revealed that let-479 encodes a homolog of the SPE-42 protein, which we previously showed is also required for C. elegans fertilization. LET-479 is predicted to be a 6-pass sperm plasma membrane protein with membrane topology identical to that of SPE-42. Like SPE-42, LET-479 contains 3 domains of interest: a large extracellular hydrophilic domain between transmembrane spans 3 and 4 containing 6 conserved cysteine residues, a DC-STAMP domain that includes transmembrane spans 5 and 6, and a C-terminal cytoplasmic RING domain. The presence of two proteins that are so similar, function in the same process, yet are completely non-redundant raises intriguing questions regarding the roles LET-479 and SPE-42 play during fertilization. The presence of SPE-42 and LET-479 homologs in all species that use bona fide sperm and eggs for fertilization suggests what we learn in the C. elegans system will be useful in understanding the mechanism of fertilization in other species, including humans.
Contact: [email protected] Lab: Kroft
164 Poster Topic: Germline Spindle Assembly Checkpoint Plays a Role In DNA-damage-induced Cell Cycle Arrest In C. elegans Male Germ Line Katherine Lawrence, JoAnne Engebrecht University of California Davis, Davis, CA, USA Persistent DNA damage in germline stem cells leads to embryonic lethality, progeny inviability or germline tumors. Consequently, cells closely monitor genomic integrity and can delay their progress through the cell cycle so that repair precedes division. In C. elegans, genotoxic perturbations to proliferative cells in the distal tip of the gonad activate checkpoints that initiate a cell cycle arrest. When this arrest is in response to stalled replication forks induced by hydroxyurea (HU), it is characterized by enlarged nuclei and can be visualized cytologically. HU damage is sensed by the C. elegans homolog of ATR, a PI3-related protein kinase, and launches a signaling cascade that results in a G1/S phase arrest. The signal transducers and downstream effectors of this DNA-damage-response (DDR) pathway have been studied extensively in hermaphrodites, but have not been investigated fully in males. While RNAi knockdown of several of these genes disrupts checkpoint output in hermaphrodites, the same treatment does not prevent HU-induced arrest in males. Our preliminary results strongly suggest that not all components of the DDR are essential for male cell-cycle arrest in response to stalled replication forks. We next investigated functional redundancy between the DDR and the spindle assembly checkpoint (SAC), which is most often associated with regulating kinetochore attachment to spindles during prometaphase/metaphase of mitosis and meiosis. We found that RNAi knockdown of several SAC components alone did not affect HU-induced cell-cycle arrest in males; however, knockdown of both ATR and SAC resulted in a failure to arrest in the presence of HU. This result suggests that, in males, the DDR and SAC work together to elicit arrest in the presence of stalled forks. To analyze this differentially regulated HU-induced arrest, we identified markers that characterize the stages of the cell cycle. Preliminary data suggests that the SAC, like the DDR, mediates an S phase arrest not predictive of its expected role as an inhibitor of cdc20 at metaphase. Future work aims to understand this novel role for SAC components and investigate the mechanisms used by the SAC to induce an S phase arrest.
Contact: [email protected] Lab: Engebrecht
Poster Topic: Germline 165 Investigating the Role of Membrane Trafficking in Temperature- Sensitive Lethal Mutants with Defects in both Gonad Development and Embryonic Eggshell Production Josh Lowry, Amy Connolly, John Yochem, Bruce Bowerman Institute of Molecular Biology, University of Oregon, Eugene OR, USA Membrane trafficking plays a major role in gonad development and embryogenesis in C. elegans. For example, signaling between the Distal Tip cells and early germline cells relies on both secretory and endocytic functions. Eggshell components are delivered to the surface of fertilized zygotes through secretory pathways as well. In the course of a recent mutant screen, we have identified temperature-sensitive, embryonic lethal mutants with eggshell defects that, when shifted to 26?C at the L1 stage, also have gonad development defects as adults. Those with >50% adult sterility make up the Osm/Ste class of mutants, comprised of 39 strains. We hypothesize that the two phenotypes reflect different gene requirements at different stages of development, and that the causal mutations affect genes involved in membrane trafficking. To determine if these mutants do have defects in membrane trafficking, we have taken a high-throughput genomics approach, employing the method of simultaneous whole-genome sequencing and SNP mapping1, to identify the affected genes in these mutants. To do this, we outcross our mutant strains to the Hawaiian polymorphic strain CB4856. We then isolate ~50 F2 progeny homozygous for the mutation and allow them to reproduce. These populations are then pooled, using approximately equivalent numbers of individuals from each population, and genomic DNA is extracted and prepared for sequencing on an Illumina HiSeq 2000. The short-read alignment program MAQgene is used to both align the resultant data to the C. elegans reference genome and identify SNPs from each of the parent strains. Plotting the ratio of Hawaiian SNP reads to total read depth allows us to map the mutations to intervals ranging from ~1-3Mbp in length. We can then search through the sequencing pileup in the mapped interval to identify candidate mutations. We have performed a pilot study and obtained high resolution SNP mapping data for 8 Osm/Ste mutants and are now compiling a list of candidate mutations for each strain. In addition, we are also preparing the other 31 strains for sequencing and analysis. This work will provide us with a toolkit we can use to understand the molecular basis for the phenotypes we have observed. References: Doitsidou, M. et al. C. elegans mutant identification with a one-step whole- genome-sequencing and SNP mapping strategy. PLoS One 5(11): e15435 (2010).
Contact: [email protected] Lab: Bowerman
166 Poster Topic: Germline Genome destabilization and checkpoint activation during cell cycle reentry of the primordial germ cells Z2 and Z3 Ash Williams, Brendan Kramer, Matthew Michael University of Southern California, Los Angeles, CA, USA The primordial germ cells Z2/Z3 are born during early embryogenesis. After completing S phase, Z2/Z3 arrest their cell cycles at prophase and remain arrested for the remainder of embryogenesis. During this period of prophase arrest, RNA pol II transcription is largely quiescent. Upon L1 hatching, and only if nutrients are present, Z2/Z3 activate RNA pol II transcription and reenter the cell cycle. We have been studying the events that occur within Z2/Z3 during cell cycle reentry. We find that the genome is transiently destabilized soon after feeding, at the same time that RNA pol II transcription is activated, and that this destabilization event activates the cell cycle checkpoint protein CHK-1. After repair of DNA damage, CHK-1 is deactivated, and the cells proceed into mitosis. Importantly, this cycle of genome damage and repair does not occur in somatic nuclei. We have also observed that RNAi-mediated depletion of CHK-1 causes Z2/Z3 to enter mitosis precociously, about one hour ahead of schedule, and they do so with unrepaired DNA damage. Taken together, our results identify a germ line-specific genome destabilization event that is monitored by CHK-1 and that is part of the normal cell cycle reentry process for Z2/Z3. The results of more recent experiments designed to uncover the molecular basis for the genome destabilization event will also be reported at the meeting.
Contact: [email protected] Lab: Michael
Poster Topic: Germline 167 Sensory Regulation of the C. elegans Germ Line through TGF-β- Dependent Signaling in the Niche Diana Dalfo, David Michaelson, E. Jane Albert Hubbard New York University School of Medicine, New York, NY, USA Germline progenitors accumulate during larval development to form an adult pool from which gametes are produced. We found that, independent of its roles in the dauer decision and lifespan, TGF-β modulates the balance of proliferation versus differentiation in the larval C. elegans germ line in response to sensory cues that report population density (dauer pheromone) and food abundance. TGF-β ligand-producing ASI sensory neurons are required for TGF- β-mediated germ cell accumulation, and the TGF-β receptor and downstream transcription complex act in the distal tip cell, the germline stem cell niche. Our results implicate TGF-β neuroendocrine signaling as a mediator between environmental cues, sensory neurons and the stem cell niche to influence the balance of proliferation and differentiation of the germline stem/progenitor pool.
Contact: [email protected] Lab: Hubbard
168 Poster Topic: Germline In Vitro Analysis of C. elegans H2A Variants Ahmad Nabhan1, Geeta Narlikar2, Diana Chu1 1San Francisco State University, San Francisco, CA, USA, 2University of California, San Francisco, San Francisco, CA USA The introduction of histone variants enables eukaryotic cells to regulate access to DNA. How histone variants act to alter chromatin structure and perform their function remains a mystery. Studies have implicated variants in a plethora of activities, for example, HTZ-1 plays a role in gene regulation and repair. Also in some organisms, including C. elegans, sperm specific histone variants are incorporated during global transcriptional repression. In C. elegans, HTZ-1 and HTAS-1 are the only H2A variants showing significant variation from H2A (59% and 51% identical, respectively). HTZ-1 is enriched in the embryo while HTAS-1 is only found in sperm. The disparity these two proteins exhibit in localization is reflected structurally: HTZ- 1 has an extended C-terminus while HTAS-1 has an extended N terminus. This leads to an interesting question: what type of chromatin structure do histone variants enriched in vastly different environments create? To understand this, we will assess how HTZ-1 and HTAS-1 alter chromatin structure to modulate accessibility to genetic material. We hypothesize the two variants alter chromatin structure to meet the transcriptional needs of the cell. Therefore we expect HTZ-1 increase accessibility to genetic material and HTAS-1 to decreases accessibility. To this end, we have expressed recombinant HTZ-1 and HTAS-1 along with C. elegans H2A H2B, H3 and H4 in E. coli. Proteins were purified using size exclusion and ion exchange chromatography. They were then combined in equa-molar amounts to create canonical and variant nucleosome core particles (NCPs). The stability of these core particles, which is inversely proportional to accessibility, will be measured as a function of ionic concentration using electrophoresis. We have also labeled H2A and its variants with Cy5, which were refolded with Cy3 labeled DNA, which will be used in our FRET experiments to gain a quantitative understanding of the influence variants have on stability of the nucleosome. Another factor affecting chromatin accessibility, reversible nucleosome unwrapping, will also be monitored using restriction enzyme activity (REA) (Widom 1991). In vitro analysis of C. elegans H2A subtypes will reveal the intrinsic properties responsible for their funcion in vivo.
Contact: A nab Lab: Chu
Poster Topic: Germline 169 ZHP-3 Regulates Meiotic Chromosome Dynamics Christian Nelson, Cate Paschal, Needhi Bhalla University of California Santa Cruz, Santa Cruz, CA, USA During meiosis, homologs must pair with their unique partner and undergo crossover recombination, forming physical linkages that hold chromosomes together until the first meiotic division. Once homologs have recombined, chromosomes must be restructured to promote attachment of homologs to opposite spindle poles and to ensure proper segregation. In C. elegans, ZHP-3 is required for both genetic exchange as well as the accompanying large- scale changes in chromosome structure during meiotic prophase. ZHP-3 contains a RING finger domain and purified ZHP-3 possesses auto-ubiquitination activity in vitro, suggesting that ZHP-3 is a ubiquitin ligase and that it may ubiquitinate target substrates to accomplish its tasks. Moreover, the localization of ZHP-3 during meiotic prophase is highly dynamic, suggesting it is heavily regulated. My project aims to identify meiotic substrates of ZHP-3 taking both a candidate biochemical approach as well as an unbiased genetic approach. Additionally, purified ZHP-3 will be analyzed via mass spectrometry to identify co-purifying proteins, potential regulators, and post-translational modifications. Preliminary data shows that ZHP-3 is a MAP kinase target in vitro and that MAP kinase is required for changes in ZHP-3 localization, suggesting that phosphorylation may act to temporally target ZHP-3 to unique substrates as meiotic prophase progresses. Combined, these data will help us to elucidate how ZHP-3 coordinates genetic recombination with large-scale chromosome structure changes to ensure proper meiotic chromosome segregation.
Contact: [email protected] Lab: Bhalla
170 Poster Topic: Germline Distinct roles for FBF-1 and FBF-2 in silencing meiotic mRNAs Alexandre Paix, Ekaterina Voronina, Geraldine Seydoux Johns Hopkins University School of Medicine, Baltimore, MD, USA Many genes in the C. elegans germline are regulated by post-transcriptional mechanisms acting through 3’ UTR sequences. We are interested in how this regulation manifests itself at the level of RNA stability, transport, translation and/or localization. To address this question, we have examined the sub-cellular localization of mRNAs in the germline, particularly meiotic mRNAs that are silenced in the distal (mitotic) region by FBF-1 and FBF-2 (gld-1, him-3, htp- 1/2). We have found that meiotic RNAs are distributed in a low distal/high proximal gradient in the mitotic zone, as reported previously for gld-1 (Jones et al., 1996). This pattern is unaffected in fbf-2 mutants. In contrast, in fbf-1 mutants, meiotic RNAs accumulate with FBF-2 in large aggregates in the rachis of the mitotic zone. In fbf-1 fbf-2 double mutants, meiotic mRNAs are uniformly distributed throughout the mitotic zone and translated. These findings suggest that FBF-1 and FBF-2 silence meiotic mRNAs by different mechanisms, and support a role for FBF-1 in RNA transport or degradation.
Contact: [email protected] Lab: Seydoux
Poster Topic: Germline 171 Natural Variants of C. elegans Demonstrate Defects in Both Sperm Function and Oogenesis at Elevated Temperatures Lisa Petrella, Susan Strome UC Santa Cruz, Santa Cruz The temperature sensitivity of fertility is conserved from nematodes through mammals and is generally correlated with a loss of sperm function. In order to better understand the mechanisms that underlie high temperature sterility in worms, we are analyzing fertility and germline organization at elevated temperature in 15 different wild-type isolates of C. elegans from around the world. Previous studies in C. briggsae and Drosophila showed that strains originating from temperate latitudes lose fertility at a lower temperature than strains originating from tropical latitudes. We determined the fertility of temperate and tropical strains of C. elegans and observed no correlation between latitude of strain origin and fertility at high temperature. We observed a wide distribution of population fertility among wild-type isolates at 27°C, ranging from 7% to 56%. In order to better understand the causes of high temperature sterility, temperature shift experiments were performed. Males up-shifted to high temperature as L4/young adults maintain fertility, while males raised at high temperature lose fertility. Sterile animals contain a wild-type-appearing germ line with mature spermatids. Down-shifting males raised at high temperature does not restore fertility. These male results differ from those observed in Drosophila and suggest that in C. elegans spermatogenesis is irreversibly impaired in males that develop at high temperature. Mating and down-shift experiments with hermaphrodites were performed to investigate the relative contributions of spermatogenic and oogenic defects to high temperature loss of fertility. We identified isolates that demonstrate predominantly spermatogenic defects, strains that show a mixture of spermatogenic and oogenic defects, and one strain that shows predominantly oogenic defects. Interestingly, among strains, the ability to maintain hermaphrodite sperm function at hightemperature does not correlate with the ability to maintain male sperm functionat high temperature. Our studies have uncovered unexpectedly high variation in both the loss of fertility and underlying problems with sperm function in natural variants of C. elegans at high temperature. One variant provides a novel example of oogenic defects underlying loss of fertility. These variants can now be used to investigate the molecular mechanisms that underlie the buffering of fertility in the face of environmental change.
Contact: [email protected] Lab: Strome
172 Poster Topic: Germline Exploring Novel Features of Gametogenesis in a Non-C. elegans Clade Kathryn Rehain, Zechariah Dillingham, Ethan Winter, Diane Shakes College of William and Mary, Williamsburg, VA, USA Several features of the C. elegans germline make it an excellent and somewhat simplified system for studying gametogenesis: 1) the germline can be observed directly through the transparent body wall, 2) the entire linear timeline of gametogenesis can be observed within individual gonads, and 3) mitotically dividing germ cells have the capacity to develop into either oocytes or spermatocytes. However as other nematode species are equally well suited for comparative studies of gametogenesis, we have begun to characterize gametogenesis in a Rhabditid clade that phylogenetically includes both Rhabditella axei and Rhabditis. sp. SB347, species with diverse reproductive modes, and all of which can be easily cultivated in the lab (Kiontke and Fitch, 2005; Felix, 2004; Shakes et al. 2011). In comparison to C. elegans, nematodes within this clade have gonads with many fewer cells. More specifically, both the distal mitotic region and the pachytene regions are not only organized differently than in C. elegans, but are, in comparison, highly reduced in both size and cell number. In the proximal gonad, the developing oocytes have extremely unusual chromatin morphology which proves to correlate with the presence of extremely large nucleoli. One interesting feature of this clade is that the males exhibit a wide range of sperm sizes and their spermatocytes exhibit diverse patterns of meiotic and partitioning divisions which, in some cases, lead to the differential survival of their X and non-X-bearing sperm (Shakes et al., 2011; this study). In summary, analysis of gametogenesis within this non C. elegans clade is revealing “natural variations on a theme” which have important implications not only for both our general understanding of nematode gametogenesis but also for modifications in the program of spermatogenesis that may underlie the evolution of diverse reproductive modes.
Contact: [email protected] Lab: Shakes
Poster Topic: Germline 173 Nutritional Control of Germline Stem Cells Hannah Seidel, Judith Kimble Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, WI, USA The germline of C. elegans represents a tractable model for studying how nutritional cues regulate stem cell behavior. Previous work showed that food availability influences the proliferation of undifferentiated germ cells in the larval germline; this response is mediated in part by insulin/IGF-like signaling (Dev. 2010 137:671-80) and TGF-β signaling (Curr. Biol. 2012 Epub Apr 5). Likewise, in oogenic hermaphrodites, nutrient deprivation causes germlines to shrink, and this shrinkage is reversible upon re-feeding (Science 2009 326:954-8, PLoS One 2011 6: e28074). Here we show that proliferation of adult germline stem cells (GSCs) requires the presence of food: Under fed conditions, GSCs divide continuously, only ceasing division as they enter the meiotic cell cycle; upon food removal, however, GSCs stop dividing and become quiescent. This quiescence can last for several days, with cell division only resuming upon the reintroduction of food. In addition, we find that food availability may also influence the effect of GLP-1/Notch signaling. Under fed conditions, GLP-1 signaling from the niche maintains GSCs in an undifferentiated state, and loss of this signal causes all germ cells to differentiate. By contrast, under some conditions, GSCs do not enter the meiotic cell cycle when starved glp-1(q224ts) mutants are shifted to restrictive temperature. One interpretation of this result is that GLP-1 is dispensable for maintenance of the quiescent GSCs typical of starved animals; however other interpretations exist. We are currently investigating possible regulators of starvation-induced quiescence and stem cell maintenance.
Contact: [email protected] Lab: Kimble
174 Poster Topic: Germline Characterization of SYGL-1, A Novel Regulator of Germline Stem Cells Heaji Shin1, Aaron Kershner1, Judith Kimble1,2 1Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA, 2Howard Hughes Medical Institute, University of Wisconsin- Madison, Madison, WI, USA C. elegans germline stem cells (GSCs) are maintained by GLP-1/Notch signaling from the stem cell niche. We have identified two putative Notch target genes, lst-1 (lateral signaling target) and sygl-1 (synthetic germline proliferation defective), that act redundantly to maintain GSCs throughout development and in both sexes (see Kershner, Shin and Kimble abstract). Here we focus on characterization of sygl-1, which had not been studied previously. The sygl- 1 locus encodes a single transcript (T27F6.4) that is predicted to generate a protein of 206 amino acids length. Standard programs (NCBI conserved domain search, Pfam 26.0, Phyre2) revealed no predicted folded domains or motifs in the SYGL-1 amino acid sequence. The sygl-1 locus is found in closely-related nematodes (C. japonica, C. remanei, C. briggsae, and C. brenneri), but is not broadly conserved. A sygl-1 deletion mutant (tm5040) removes 720bp, including the first exon and part of the first intron. The sygl-1 deletion mutant is homozygous viable, but possesses a smaller than normal mitotic region, consistent with the idea that sygl-1 is required to maintain the normal number of germ cells within the mitotic region. To understand the molecular function of SYGL-1, we are in the process of making an epitope-tagged SYGL-1 transgene and screening for SYGL-1 partner proteins.
Contact: [email protected] Lab: Kimble
Poster Topic: Germline 175 Uncovering the Role of Condensin I during C. elegans Meiosis Margarita Sifuentes, Joshua Bembenek, Karishma Collette, Gyorgyi Csankovszki University of Michigan, Ann Arbor, Michigan Condensin complexes are key determinants of higher-order chromatin structure during meiosis and mitosis in eukaryotes. However the different roles of condensins I and II in meiosis are poorly understood and have yet to be elucidated in eukaryotes. Interestingly, analysis of condensin I in C. elegans, demonstrates it localizes to a ring shaped domain between chromosomes at the midbivalent during metaphase and to the meiotic spindle between separating chromosomes during anaphase. Other proteins such as the Chromosomal Passenger Complex, BUB-1, KLP-19, HCP-1/2, and CLS-2 also localize to the midbivalent, suggesting that condensin I may function with proteins at the ring shaped domain to promote chromosome orientation, alignment, and separation. Depletion of condensin I by RNAi interference leads to gross meiotic spindle defects and abnormal chromosome organization. Our data suggests that condensin I supports orientation and movement of chromosomes throughout meiosis. Additionally, condensin I depletion prevents successful chromosome resolution at anaphase. Future studies will determine how condensin I functions together with other proteins at the midbivalent to influence chromosomal orientation during meiotic congression and segregation in C. elegans.
Contact: [email protected] Lab: Csankovszki
176 Poster Topic: Germline The metazoan gene akirin is required for synaptonemal complex disassembly and bivalent structure during Caenorhabditis elegans meiosis Amy Clemons1, Heather Brockway1, Yizhi Yin1, Yaron Butterfield2, Steven Jones2, Monica Colaiacovo3, Sarit Smolikove1 1University of Iowa, 2British Columbia Cancer Research Centre, 3Harvard Medical School During meiotic prophase I homologous chromosomes associate via the synaptonemal complex (SC). This structure is essential for proper crossover formation and therefore required for accurate chromosome segregation during meiosis. At the late stages of meiotic prophase I, following the formation of crossovers, the SC disassemble concurrently with the remodeling of the newly formed bivalent. It is expected that both events are crucial for proper meiotic chromosome segregation. However, little direct evidence supports this claim and the mechanisms controlling SC disassembly remain unclear. Here we identify akir-1 as a novel gene involved in these key events of meiotic prophase I in Caenorhabditis elegans. AKIR-1 is a conserved protein in metazoans that lacks any known function in meiosis. We show that akir-1 mutants exhibit aberrant meiotic division accompanied by severe meiotic defects in late prophase I. These defects include improper disassembly of the SC and aberrant restructuring of the bivalent. Although initial steps of SC disassembly (asymmetric disassembly) progress normally, resolution of chromosome arms is severely impaired. This includes defects specific to central region proteins of the SC, that are still capable of bridging homologs in the absence of crossovers (spo-11 mutant background). Earlier meiotic events, such as homologous pairing, RAD-51 loading and crossover formation, are not affected in the akir-1 mutants. Our studies places AKIR-1 downstream from any other protein known to affect SC disassembly. Furthermore, defects in arm resolution lead to structural abnormalities of the bivalent. These are accompanied by failure to compact the bivalent, which is independent of the condensin complex. Overall our data indicates that akir-1 is involved in promoting proper bivalent formation by the timely disassembly of the SC and its proper restructuring, leading to sharpening of our understanding of these fundamental meiotic events.
Contact: [email protected] Lab: Smolikove
Poster Topic: Germline 177 Chromatin Regulation in the Meiotic Germ Line Matthew Snyder, Xia Xu, Eleanor Maine Syracuse University, Syracuse, NY, USA Meiotic silencing is a conserved phenomenon targeting unpaired chromosomes and chromosomal regions during prophase of meiosis I. Meiotic silencing in animals typically occurs at the chromatin level and involves accumulation of histone modifications thought to promote a closed chromatin configuration. This chromatin structure may contribute to transcriptional repression and meiotic chromosomal events such as chromosome disjunction (Bean et al 2004, Jaramillo-Lambert and Engebrecht 2010). During meiosis in C. elegans, non-synapsed chromosomes are enriched for H3K9me2 relative to synapsed chromosomes (Kelly et al. 2002; Bean et al. 2004). Such non-synapsed chromosomes include the male X, homologous chromosomes that fail to synapse due to mutation, and chromosomal translocations/ duplications. The pattern of H3K9me2 accumulation during meiosis depends on activity of a small RNA pathway (Maine et al. 2005; She et al. 2009), which may direct activity of the histone methyltransferase, MET-2, responsible for germline H3K9me2 (Bessler et al. 2010). Taking a combined biochemical/genetic approach, we are identifying additional factors important for regulating H3K9me2 distribution during meiosis. We will describe our latest findings.
Contact: [email protected] Lab: Maine
178 Poster Topic: Germline Global Control of the Oogenic Program by Components of OMA-1 Ribonucleoprotein Particles Caroline Spike, Donna Coetzee, David Greenstein University of Minnesota, Minneapolis, MN, USA The oocytes of most sexually reproducing animals arrest at diplotene or diakinesis and resume meiosis (meiotic maturation) in response to hormones. The defining feature of meiotic maturation is M-phase entry triggered by CDK/cyclin B. In wild-type C. elegans, M-phase entry is spatially restricted to the –1 oocyte by mechanisms that remain to be defined. Meiotic maturation requires the presence of sperm, Gas-adenylate cyclase-PKA signaling in the gonadal sheath cells, and germline function of two Tis11-like CCCH zinc-finger proteins, OMA-1 and OMA- 2. To elucidate the mechanisms by which the OMA proteins promote meiotic maturation, we purified OMA ribonucleoprotein particles (OMA RNPs) from oocytes and identified associated proteins and mRNAs using mass spectrometry and RIP-Chip, respectively. To distinguish core OMA RNP components from those tethered by RNA, we also purified OMA complexes after treatment with RNase A. Many protein and mRNA components of OMA RNPs are critically important regulators of the oogenic program. OMA RNPs contain a large number of germline- expressed RNA-binding proteins including translational activators and repressors. Core OMA RNP components include multiple subunits of the GLD-2 poly(A) polymerase and CCR4/NOT1 deadenylase complexes and many RNA-binding proteins, including, MEX-3, MEX-1, and LIN- 41. OMA RNP components that appear to be tethered primarily via RNA associations include the P-body proteins CGH-1/p54 and CAR-1/Rap55. To examine the role of OMA RNP components in translational regulation, we generated 3’UTR-reporter constructs for several mRNA components of OMA RNPs. OMA-1/2 repress the translation of most tested targets in oocytes. Depletion of core OMA RNP components identified the NHL-family protein LIN-41 as a regulator of at least two OMA targets. Strikingly lin-41 is required for normal oogenesis and meiotic progression. In lin-41 null mutants, pachytene- stage female germ cells fail to progress to diplotene, and instead enter M-phase. By contrast, oma-1; oma-2 mutant oocytes never enter M-phase. Interestingly, the OMA RNP components GLD-2 and LIN-41 exhibit epistasis and mutual suppression: double mutant oocytes arrest with a chromosome configuration resembling pachytene. We hypothesize that the OMA RNP complex promotes meiotic maturation in the –1 oocyte by balancing and spatially restricting the repression and activation of critical mRNA targets.
Contact: [email protected] Lab: Greenstein
Poster Topic: Germline 179 Early and Late Roles for Gonadal Innexins: Germ Cell Proliferation and Meiotic Maturation Todd Starich1, David Hall2, David Greenstein1 1Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, 2Albert Einstein College of Medicine, Yeshiva University, Bronx, NY 10461 The germline gap junction innexins inx-14 and inx-22 are negative regulators of meiotic
maturation that act downstream of Gαs. In addition, inx-14(0) animals are sterile, producing few germ cells. We have further investigated germline innexins and determined functional relationships between inx-8 and inx-9 in the somatic sheath and DTC, and inx-14, inx-21 and inx-22 in the germline; we have found that innexins have an early role in germ cell proliferation, as well as later roles including meiotic maturation. Through use of INX-8, -14, -21 and -22 specific antibodies, and INX-8, -9 and -14 GFP fusions, we have established a complex co-dependency for localization of innexin proteins to gap junction-like puncta. Within the germline, INX-14 requires either INX-21 or INX-22 to localize to puncta; INX-21 and INX-22 reciprocally require INX-14 to localize. A reciprocal dependency also exists between germline innexins and INX-8 and -9 for localization. inx-14(0) animals produce few germ cells; this phenotype is mimicked in inx-22(0) inx-21(RNAi-injected) animals, supporting dependence of INX-14 on INX-21 or INX-22 for function. This phenotype is also mimicked in inx-8 inx-9 double mutants (see below), which avg. <4 germ cells per gonad arm, confirming a functional co-dependence between germline and somatic innexins. Consistent with an early functional role, expression of these innexins is detected in their respective Z1-Z4 and Z2-Z3 compartments. Germline expression continues throughout development; in early stages inx-8 and -9 are expressed primarily in the DTC, and by late L4 and adult stages sheath expression predominates, especially proximally. In adults gap junctions between sheath and germ cells are detected by IF in both distal and proximal arms, an observation supported by TEM analysis of freeze fracture samples. To more easily manipulate these junctions genetically, we have focused on somatic innexins inx-8 and -9, which represent a recent tandem duplication. We carried out a genetic screen to isolate an inx-8 inx-9 double mutant based on the predicted resemblance to the inx-14(0) sterile phenotype. inx-8 inx-9 mutants can be rescued by expression of inx-8 alone. To unmask late defects from early, we have: 1) generated a dosage-dependent temperature-sensitive rescuing array with a site-directed mutation in inx-8, and 2) generated a Plag-2::inx-8::gfp construct that rescues the germ-cell proliferation defect but not inhibition of meiotic maturation.
Contact: [email protected] Lab: Greenstein
180 Poster Topic: Germline Oocyte-to-embryo Transition: a Screen for mbk-2 Suppressors Yuemeng Wang1, Harold Smith2, Kevin O’Connell2, Geraldine Seydoux1 1Johns Hopkins University School of Medicine, Baltimore, MD, USA, 2National Institutes of Health, Bethesda, MD, USA MBK-2 is a kinase required for the oocyte-to-embryo transition. MBK-2 is activated during the transition and phosphorylates oocyte proteins whose stability or activity must be modified in zygotes. To identify new factors that function with MBK-2 during the transition, we have conducted a screen for suppressors of dd5, a temperature sensitive mutation in the kinase domain of MBK-2 (Quintin et al, 2003). We isolated 21 suppressors in a screen of more than one million genomes. We are using whole genome sequencing after outcrossing to a Hawaiian strain to map and identify the suppressors (Doitsidou et al, 2010). So far we have found that four suppressors are intragenic, and four map to genes implicated in cell cycle regulation (emb-30, fzy-1, plk-1, such-1). We will present our progress at the meeting.
Contact: [email protected] Lab: Seydoux
Poster Topic: Germline 181 A Functional RNAi Screen Identifies Regulators of RNP Granule Assembly in Aging Oocytes Megan Wood, Kevin Gorman, Joseph Patterson, Jennifer Schisa Central Michigan University, Mount Pleasant, MI, US In many animal species, oocytes arrest in meiosis until they are fertilized. It is well established that fertility diminishes as oocytes age, and several cytological studies have reported changes in the cytoplasm of aging, arrested oocytes. In this study our goal is to better understand the regulation and function of large ribonucleoprotein (RNP) granules that assemble in the germlines of Caenorhabditis nematodes that are either stressed, or in which ovulation is arrested due to old age or an absence of sperm. The large RNP granules are hypothesized to regulate mRNA stability or translation in arrested or stressed oocytes when fertilization is delayed (Jud et al., 2008). We have performed a targeted, functional RNAi screen to identify genes that are required for the assembly of RNP granules in meiotically-arrested oocytes, and we have identified over 100 genes that are necessary for the RNA-binding protein MEX-3 to assemble efficiently into large granules. Preliminary analyses suggest the largest gene classes of the screen positives are: RNA-binding proteins that localize to the RNP granules including cgh-1, car-1, puf-5, and pie-1; MSP signaling pathway members including inx-14, ceh-18, and goa-1; RNAi regulators including dcr-1 and csr-1; nuclear pore complex components including npp-9, npp-10, and ran-4; and microtubule-related proteins including tbb-1, tbb-2, and dhc-1. Interestingly, in yeast and mammalian cells, disruption of microtubules stimulates the formation of P bodies (processing bodies), while such disruption prevents the assembly of stress granules. With the discovery of these novel regulators of RNP granule assembly, we are now testing our hypothesis for their function. In several cases, when the normal assembly of RNP granules is prevented, we observe that fertility is decreased, supporting the hypothesis that RNP granules may help maintain the integrity of oocytes when fertilization is delayed. We are currently determining if RNA stability is diminished or translation of maternal mRNAs is de-repressed in oocytes when RNP granule assembly is defective.
Contact: [email protected] Lab: Schisa
182 Poster Topic: Germline A Novel Function of MRE-11 in Caenorhabditis elegans Yizhi Yin, Sarit Smolikove Univ.of Iowa,Iowa,IA, USA Accurate chromosome segregation in meiosis requires interhomolog crossovers (COs) generated by repair of programmed meiotic double-strand DNA breaks (DSBs) via homologous recombination (HR) pathway. Mre11 is one member of the MRX (Mre11, Rad50, Xrs2/Nbs1) complex that is essential for the formation of interhomolog COs. Studies in yeast indicate the MRX complex plays dual roles in COs formation, the formation of DSBs and the resection of DSBs. However, in Caenorhabditis elegans the evidence for MRX complex’s role in DSBs resection is limited. The currently known mutants of genes in MRX complex are null, showing absence of chiasma, indicating lack of meiotic DSBs. Here we report the first separation of function allele of mre-11 in C. elegans, mre-11(iow1). This allele shows defects specific to resection. mre-11 mutants exhibit a phenotype of chromosomal fragmentation and aggregation at the diakinesis stage of meiotic prophase I. This is accompanied by 100% of embryonic lethality and high level of germline apoptosis. As expected, mre-11(iow1) is epistatic to mre- 11 null and msh-5 mutants, and acts downstream of spo-11. RPA-1 and RAD-51 are the single stranded DNA (ssDNA) binding proteins in C. elegans, which sequentially associate to ssDNA following resection. In mre-11(iow1) mutants, the loading of RPA-1 onto the meiotic chromosomes is severely decreased , while RAD-51 does not load to meiotic chromosomes. These results suggest failure to resect meiotic DSBs to form ssDNA required for formation of COs via HR pathway in mre-11(iow1) mutants. However, since the HR pathway is blocked in mre-11(iow1) mutants, meiotic DSBs can only be repaired by other DNA repair pathways that are not usually used in meiosis. One of these pathways is the non-homologous end joining (NHEJ) pathway. Blocking the NHEJ pathway by the addition of the cKu-80 (a gene essential for NHEJ pathway) null mutation to the mre-11(iow1) genetic background, partially rescues mre-11(iow1) phenotypes (measured by RAD-51 loading, chromosomal aggregation and fragmentation, embryonic lethality and germline apoptosis). These results suggest NHEJ partially attributes to the mutant phenotypes observed in mre-11(iow1) and plays a role in repairing the meiotic DSBs in mre-11(iow1) mutants. Compared to wild type worms, mre-11(iow1) mutants do not load RAD-51 on DSBs induced by γ-irradiation from onset of prophase I to early-mid pachytene and show a reduced RAD-51 localization in other stages. This indicates that the resection function of MRE-11 is mainly required in early to mid meiotic prophase and is replaced in the other stages by an alternative nuclease. Overall our analysis of our unique mutant establish a role or MRE-11 in resection of DSBs, restricted to a subset of meiotic stages, and in blocking the NHEJ pathway in these nuclei. MRE-11 therefore plays a central role in controlling modes of DSB rapier in meiosis through is function in DSB resection.
Contact: [email protected] Lab: Smolikove
Poster Topic: Germline 183 Illuminating the Formation and Regulation of Meiotic Crossovers with GFP:COSA-1 Karl Zawadzki, Rayka Yokoo, Anne Villeneuve Stanford University, Stanford, USA Faithful chromosome segregation during meiosis I requires crossover (CO) recombination events that form the basis of temporary links between homologous chromosomes. Rather than creating many COs, most organisms create only a small number of widely spaced COs while ensuring that each pair of homologs receives at least one. This regulation of CO number and placement is collectively termed “crossover control” but the underlying mechanisms are poorly understood. In C. elegans CO control is particularly robust, with each homolog pair receiving one CO. We are investigating the formation and regulation of meiotic COs, building on our discovery of COSA-1 (Crossover Site Associated), a conserved CO protein that is required for the formation of COs and localizes to CO sites during the late pachytene and diplotene stages of meiotic prophase. A functional GFP::COSA-1 fusion protein serves as a robust in vivo reporter for the CO control system, as GFP::COSA-1 reliably localizes to six foci per nucleus (i.e. one focus for each homolog pair), even in the context of a large excess of DSBs. These and other data suggest that GFP::COSA-1 foci represent a more reliable surrogate than COs per se for the events that are being distributed by the interference mechanism. Thus to investigate the basis of CO control, we are conducting a genetic screen for mutations that alter the number of GFP::COSA-1 foci. We have isolated over a dozen mutants with defects in different meiotic processes and are further characterizing those mutants that may be relevant to understanding CO control.
Contact: [email protected] Lab: Villeneuve
184 Poster Topic: Germline exc-2 and Maintenance of Tube Structure of the Excretory Canals Hikmat Al-Hashimi, Matthew Buechner University of Kansas, Lawrence, (KS), USA Long, narrow, single-celled tubes are found in many tissues in our body, such as capillaries and the Schwann cells that wrap neurons with a myelin sheath. The excretory canal cell provides a model for investigating the formation and maintenance of narrow tubes. We study nine exc genes, mutations of which allow the excretory canals to swell into fluid-filled cysts. The position of all these genes have been narrowed, but a few remain to be cloned. I am currently cloning the exc-2 gene. This locus was shown previously to be located on the X chromosome between mec-2 and dpy-8, a region of ~425 Kb that contains ~170 genes. We have microinjected 12 fosmids that cover the majority of this region; though the mutant has not been rescued yet, we have narrowed the locus to ~145KB. Additionally, in order to identify new genes that affect the maintenance of the tube structure of the excretory canal, we plan to carry out an RNAi screening via feeding. exc mutations are viable, but other mutations that affect structure of the canal lumen (such as erm-1 and cdc- 42) have lethal effects in other tissues. In order to overcome the possible extra-canal lethality of candidate genes, we have constructed transgenic worms that should be RNAi-sensitive within the excretory cell and not in other tissues, by adapting the method used by the Chalfie lab (Calixto et al., Nature Methods ‘10) to knock down genes in the touch neurons. We have integrated a construct that drives expression of sid-1 to high levels in the canal through the use of the vha-1 promoter, in a sid-1 (RNAi-deficient) mutant. Preliminary tests show loss of RNAi effects for a hypodermally expressed gene (dpy-8), and we are currently confirming the effects of knockdowns of exc and other genes on both canal and extra-canal phenotypes.
Contact: [email protected] Lab: Buechner
Poster Topic: Morphogenesis 185 C. elegans nuclear hormone receptor, nhr-25 regulates vulval terminal cell properties and migrations during development Nagagireesh Bojanala, Marek Jindra, Masako Asahina Biology Centre, ASCR/Univ. South Bohemia, Ceske Budejovice, Czech Republic C. elegans vulva has been intensively studied for cell fate specifications and morphogenesis during development and a number of signaling pathways are involved in this process. The mature vulva is composed of 22 cells formed from the daughters of 3 vulva precursor cells [VPCs, P(5-7).p] that undergo three rounds of cell divisions to differentiate into seven distinct cell types, Vul A to F and the proper migration of these cells is essential to form functional vulva. Previous studies have shown the role of nhr-25 (ftz-f1/sf-1 homolog) in vulval cell inductions and morphogenesis, yet its role in cell migration has not been fully explored. We followed the terminal differentiation properties of Vul A-F and their migrations in nhr-25(lf) animals. L2-nhr- 25(RNAi) was used in this study to bypass the initial VPC induction defects. Defects during the short range migrations of Pn.pxx cells were seen in our nhr-25(RNAi) animals; P6.pxx (1°) and P5/7.pxx (2°) cells failed to initiate their dorsal and lateral migrations respectively. At the time of vulva fate execution process, the presumptive Vul E and Vul C cells changed their division axis from transverse (T) to longitudinal (L). In addition, the outer most cell types Vul A, B1 and B2 often failed to reach the vulva midline. Similar defects have been reported when semaphorin and Rac/Rho pathway gene functions were compromised. Interestingly, SMP-1::GFP expression was affected in nhr-25 (RNAi) animals and the genetic interaction between nhr-25 and smp-1(ev715) or plx-1(ev724) revealed that nhr-25 works in parallel to the semaphorin pathway. We also observed abnormal toroidal fusions within Vul E/F, A/B1/B2 and B1/B2 in nhr-25(RNAi) animals. Altered expressions of terminal vulval differentiation markers indicate that non-homologous cells may adopt homologous fate in nhr-25 (lf) background and later fused with each other. In summary, NHR-25 regulates vulva morphogenesis at multiple levels; at the initial induction, at the terminal differentiation of Vul A-F cell types and at the time of migration of vulval cells where NHR-25 co-operates with semaphorin pathway. Since we observed the genetic interaction between nhr-25 and semaphorin pathway also in the epidermal seam cells, the co-operation of these pathways may be universal. Supported by 204/09/H058 and Z60220518.
Contact: [email protected] Lab: Asahina
186 Poster Topic: Morphogenesis Characterizing regulators of the C. elegans cytoskeleton Benjamin Chan, Simon Rocheleau, Paul Mains Genes and Development Research Group, University of Calgary, Calgary, AB, Canada All eukaryotic organisms begin as a roughly spherical ball of cells, but the final shape of a species is a precise and tightly regulated process. In Caenorhabditis elegans, a smooth muscle-like contraction of an actin/myosin network mediates the elongation of a worm embryo from a ball of cells into a long, thin worm. Here we will continue to characterize a gene known to regulate this process, and discuss a novel gene which may be also involved. Previous work has shown that non-muscle myosin is able to generate contractile force through two redundant pathways in C. elegans. Phosphorylation of myosin light chain (MLC-4) activates non-muscle myosins NMY-1/2 which drives contraction. In contrast, dephosphorylation of MLC-4 is regulated by MEL-11/myosin phosphatase. In one pathway, the small GTPase RHO-1 activates LET-502/ Rho-binding kinase which inhibits MEL-11, halting contraction. In a second parallel pathway, FEM-2/protein phosphatase 2c is involved in regulating contraction. In a suppressor screen of mel-11, an allele of a Rho GEF (guanine exchange factor) rhgf-2 was identified. Previous work has shown that rhgf-2 genetically acts upstream of let-502 and in parallel to fem-2. In addition, RHGF-2 is believed to act as a GEF for RHO-1. However, the cellular localization of RHGF-2 has yet to be determined, and our current work will use a RHGF-2 antibody to address this question. In our previous suppressor screen of mel-11, an allele of a novel gene, sb89, was also isolated. We will continue to characterize this gene through traditional mapping experiments and whole genome sequencing.
Contact: [email protected] Lab: Mains
Poster Topic: Morphogenesis 187 The Morphological and Functional Alterations of the Anal Depressor Muscle in Male C.elegans Xin Chen1, L. Rene Garcia1,2 1Department of Biology Texas A&M University College Station TX , 2Howard Hughes Medical Institute The anal depressor is a sexually dimorphic muscle in C. elegans. It is an H-shaped cell in hermaphrodites and larvae males and alters its shape and function significantly during L4 male development. In adult males, the anal depressor functions as an auxiliary spicule protractor muscle whereas in hermaphrodites and larvae males it participates in defecation. We are interested in exploring the mechanism through which the anal depressor alters its morphology and function. First, we observed the morphological change of the anal depressor throughout the larval and adult stages using fluorescent microscopy. We found that from L1 to L3 stage the myofilaments contained within the anal depressor run dorsal-ventrally and from L2 stage relative to the hermaphrodite, the male anal depressor does not increase in size. At early L4, a ventral slit forms at the anal depressor, demarcating the ventral attachment into anterior and posterior regions. During mid L4 stage the anterior region elongates dorsal-anteriorly to attach to the developing dorsal protractor muscle, whereas the posterior region remains attached to the rectum. The myofilaments contained within the anterior attachment begin to reorient to run anterior-posteriorly. From late L4 until adulthood, the myofilaments contained within the posterior region atrophies making the central region of the anal depressor extremely thin. Questions arise as to whether the morphogenesis responds to intrinsic or extrinsic signals, or a combination of both. Extrinsic signals might come from the cells that are adjacent to the anal depressor. To test this hypothesis, we laser-ablated muscle, neuronal and epithelial precursor cells and monitored anal depressor development. We found that the M cell ablation, which eliminated all the sex muscles, resulted in anterior movement defects and abnormal morphology of the anal depressor in the adult male. We performed EMS mutagenesis and identified three mutant lines where the males had a normal larval, but abnormal adult anal depressor.Two mutant lines have severe defects in spicule morphogenesis whereas the third line has a subtle alteration in the position of the spicules. In the third mutant line, the adult anal depressor took the late L4 morphology with the posterior myofilaments failed to reorganize anterior-posteriorly.
Contact: [email protected] Lab: Garcia
188 Poster Topic: Morphogenesis TMD-1 / Tropomodulin Regulates Intestinal and Excretory Cell Development Rachel Walker, Corey Hoffman, Elisabeth Cox-Paulson SUNY College at Geneseo, Geneseo, NY Tropomodulins are actin-binding proteins that regulate the stability of the slow-growing ends of actin filaments.C. elegans has a tropomodulin homolog, TMD-1/UNC-94 that encodes two isoforms with functional and sequence similarity to vertebrate tropomodulins. TMD-1 is involved in body wall muscle development (1,2), regulation of junctional actin in hypodermal cells (3), and we have also found that it is needed for proper morphology of the intestine and excretory cell. In the intestine, TMD-1 localizes to the terminal web, which is an actin and intermediate filament rich structure that underlies the apical, luminal membrane. Loss oftmd- 1 function results in flattened morphology of the intestinal lumen and a reduction in luminal volume. In worms homozygous for the strong loss-of-function allele, tmd-1(tm724), the terminal web is thinner, the concentration of F-actin is reduced, and the intermediate filament protein, IFB-2, shows a slightly abnormal distribution. This points to a role for TMD-1 in regulating the ultrastructure of the terminal web. Additionally, tmd-1(tm724) mutants exhibit an overabundance of gut granules, indicating a potential defect in vesicle trafficking. In addition to intestinal abnormalities, tmd-1(tm724) mutants have excretory cell defects; specifically the canals fail to extend anteriorly and posteriorly during development. Improper structure of the intestine and excretory cell may account for the larval lethality, accumulation of fluid, and slow growth exhibited by tmd-1(tm724) mutants. Together, the data supports a new role for a tropomodulin in the development of tubular epithelial tissues.
1. Yamashiro et al. (2008) J. Cell Sci. 121: 3867-77. 2. Stevensen et al. (2007) J. Mol. Bio. 374: 936-50. 3. Cox-Paulson et al. (2012) in revision at Current Biology.
Contact: [email protected] Lab: Cox-Paulson
Poster Topic: Morphogenesis 189 Roles Of Heparan Sulfate Proteoglycans In Embryonic Morphogenesis Katsufumi Dejima, Suk-Ryool Kang , Andrew Chisholm University of California, San Diego, La Jolla, CA, USA Heparan sulfate proteoglycans (HSPGs) are cell surface or extracellular matrix molecules that are comprised of a core protein modified with heparan sulfate (HS), a negatively charged linear polysaccharide. HSPGs have been known to be associated with various biological processes such as growth factor signaling and cell adhesion. In C. elegans, mutations in genes involved in HSPG synthesis lead to embryonic lethality with morphogenetic defects, including defective ventral enclosure (Refs. 1,2). However, the cellular roles of specific HSPGs during early embryogenesis prior to ventral enclosure are still unclear. To define the specific roles of HSPGs in early development we are taking several approaches. First, we are examining the expression pattern of HSPGs in early embryonic development. Immunostaining of embryos with the HSPG side chain antibody 3G10 reveals widespread cell surface expression. We are testing whether this expression pattern reflects early embryonic expression of the known core proteins such as syndecan/SDN-1 or glypican/GPN-1. Second, we are characterizing the phenotypes of mutants with severe defects in HS synthesis (rib-1, rib-2, hst-1) using semi-automated cell lineage analysis (ref. 3). While such HS synthesis mutants display fully penetrant early embryonic lethality, animals lacking multiple known HSPG core proteins are semi-viable, suggesting the existence of additional as-yet uncharacterized essential HSPGs in the embryo. We are biochemically purifying total embryonic HSPGs in the hopes of identifying the complete complement of C. elegans HSPGs.
1. Hudson ML et al. 2006. Dev Biol 294: 352-65. 2. Kitagawa H et al. 2007. J Biol Chem 282: 8533-44. 3. Giurumescu CA et al. 2011. Worm Breeder’s Gazette 18 No. 4 p5
Contact: [email protected] Lab: Chisholm
190 Poster Topic: Morphogenesis C. elegans body size is regulated by TGF-β signalling in multiple tissues. Aidan Dineen, Jeb Gaudet University of Calgary, Calgary, (Alberta), Canada The coordination of organ size and body size to mediate proportional growth is an interesting biological problem. In C. elegans , body size is partially regulated by a TGF-β signaling pathway that also functions in male tail development and is therefore termed the Sma/Mab pathway (for Small and Male abnormal). Loss of function mutations in pathway genes such as the ligand dbl-1 or downstream receptor regulated Smad (R-Smad) sma-3 result in decreased post-embryonic growth, with adult sma mutants only achieving ~60-70% the body size of wild type animals. This small phenotype is due to a decrease in cell size, rather than cell number. Previous work has demonstrated that the Sma/Mab pathway can function cell-autonomously in the hypodermis to positively regulate body size. However, many of the components of this signalling pathway are expressed in additional organs such as the intestine and pharynx, raising the question of a possible role for this pathway in these organs. We compared the pharynx size of sma-3 mutants to those of dpy mutants, a class of mutants that have small body sizes due to cuticle defects, not the Sma/Mab pathway. We found that while sma and dpy mutants have a similar reduction in body lengths, the pharynx size of sma mutants was significantly reduced compared to dpy mutants. This result indicates that Sma/Mab pathway signalling is required for normal growth of the pharynx in addition to its previously described role in body size regulation in the hypodermis. We further show that contrary to previous models, the Sma/Mab pathway functions in multiple tissues to control body size. In particular, we find that pharyngeal expression of the R-Smad protein SMA-3 is sufficient to partially rescue both pharynx size and body size defects of sma-3 mutants. These results suggest that the Sma/Mab signalling pathway can function non cell-autonomously to regulate cell size and therefore body size. In addition, we find that rescue ofsma-3 mutants improves as the number of sma-3 expressing tissues is increased (e.g. expression of sma-3 in pharyngeal muscles, marginal cells and the hypodermis provides better rescue than expression in any tissue alone). Overall, our results suggest a model where TGF-β signalling in multiple tissues activates one or more downstream secreted signals that act non cell-autonomously to regulate body size in C. elegans.
Contact: [email protected] Lab: Gaudet
Poster Topic: Morphogenesis 191 Functional Dissection of SAX-7, a Homologue of Human L1CAM in C. elegans Dendritic Branch Formation Xintong Dong, Oliver Liu, Kang Shen Stanford University, Stanford (CA), US Dendritic morphology is critical for neuronal information processing. The location, shape and size of dendritic arbors determine what signals a neuron receives and how they are integrated and further transmitted. However, the mechanisms by which neurons acquire elaborate type- specific dendritic arbors have not been completely elucidated. In particular, the interaction between dendritic branches and its growth substrate is poorly understood. Unlike most neurons in the nematode Caenorhabditis elegans that are simple in morphology, the mechanosensory neuron PVD forms elaborate branches that envelope the body of the worm. The architecture of these branches is stereotypical and beautifully organized, following general principles of dendritic development such as tiling and self-avoidance, thus providing a strong tool to study genetic regulation of dendritic branching. Immunoglobin superfamily cell adhesion molecules (IgCAMs) have been implicated in many important aspects of neurogenesis including axon guidance, cell migration and synapse formation. This study focused on SAX-7, a homologue of human L1CAM. We have shown that loss of SAX-7 results in truncated 1° branches, disorganized 2° branches and complete loss of 3° or 4° branches in PVD. SAX-7 functions in hypodermal cells and subcellularly localizes around hypodermal-SEAM cell junctions close to 1° branches and sublateral regions where 3° branches are formed. Forward genetic screen has revealed a putative receptor for SAX-7 which functions autonomously in PVD. These results suggest a role of interaction between cell surface molecules and pre-patterned extracellular cues in directing dendritic branch development.
Contact: [email protected] Lab: Shen
192 Poster Topic: Morphogenesis ani-1 is required for morphogenesis of C. elegans embryos and functions in parallel to the rho-1 pathway. Nellie Fotopoulos, Yun Chen, Alisa Piekny Concordia University, Montreal (Quebec), Canada Morphogenesis describes the changes in cell shape and movement that give rise to tissues and is crucial for the development of all metazoans. These changes are driven by the actomyosin cytoskeleton. Cytokinesis, the final stage of mitosis, also requires the cell to undergo extensive cell shape changes to form two daughter cells. This is controlled by RhoA, which regulates the polymerization of actin and activation of myosin to build and ingress the contractile ring. Anillin is a component of the contractile ring, which maintains stable furrow ingression by scaffolding RhoA, myosin and actin filaments. Some of the core regulators of cytokinesis also regulate epidermal cell shape changes during C. elegans morphogenesis, but roles for anillin in morphogenesis have not yet been described. Of the three C. elegans anillin homologues, ANI-1 has the highest overall homology to human anillin. ani-1 regulates myosin localization and asymmetric furrow ingression during cytokinesis and polar body extrusion during meiosis. We hypothesize that ani-1 may also regulate the cytoskeletal-driven cell shape changes and movements for morphogenetic events. We found that ani-1 depletion leads to a range of phenotypes, including ventrally-ruptured embryos and lumpy dumpy larva that could indicate problems in ventral enclosure or elongation. This requirement for ani-1 is not strictly maternal since rde-1(ani-1 RNAi) worms outcrossed to N2 also displayed lumpy dumpy larva. Time-lapse imaging AJM-1::GFP (ani-1 RNAi) embryos revealed both ventral enclosure failure due to failed migration and fusion of ventral epidermal cells, and elongation phenotypes due to failed seam cell shape changes. ani-1 RNAi enhanced alleles of genes known to regulate elongation (rho-1, mlc-4, mel-11, let-502, rhgf-2and nmy-1), suggesting that ani-1 functions in parallel to the rho-1-mediated actin-myosin seam cell shape changes that drive elongation. Furthermore, time-lapse imaging of the enhanced nmy-1(sb113); ani-1 RNAi phenotype revealed ventral enclosure defects. Collectively, these findings support roles for ani-1 during ventral enclosure and we are currently investigating genetic interactions between ani-1 and known regulators of ventral enclosure.
Contact: [email protected] Lab: Piekny
Poster Topic: Morphogenesis 193 A Genome-Wide RNAi Screen to Identify New Components of a Muscle-To-Epidermis Mechanotransduction Pathway Essential for Embryonic Elongation Christelle Gally, Agnes Aubry, Michel Labouesse IGBMC, Strasbourg, France C. elegans embryonic elongation is controlled by myosin II in the epidermis and muscle contractions. Indeed, loss of muscle activity results in elongation arrest at the 2-fold stage (Pat phenotype) for a reason that has long remained unclear. We recently unraveled a mechanotransduction pathway between muscles and epidermis that accounts to a large extent for the Pat phenotype (Nature, 471, 99-103, 2011). Specifically, during the second phase of embryonic elongation, muscle contractions trigger a remodeling of hemidesmosomes (HDs) in the epidermis through the activation of the serine/threonine p21-activated kinase PAK-1. HDs connect the apical and basal sides of the epidermis through intermediate filaments. They ensure epidermis integrity and anchor it to the extracellular matrix. We showed that PAK-1 is a HD component whose kinase activity controls intermediate filament phosphorylation and their anchoring to HDs. PAK-1 seems to relay activity of the adaptor protein GIT-1, with which it forms a highly conserved complex together with the RacGEF protein PIX-1 at the HDs. GIT-1 appears to be the most upstream target of the mechanotransduction pathway since its localization to the HDs is maintained by muscle contraction. However, since git-1(tm1962) mutants do not induce a Pat-like embryonic arrest, a prediction is that muscle contraction also triggers a second parallel pathway, probably to activate myosin II. In order to identify this putative parallel pathway, we have recently undertaken a genome-wide RNAi screen in the synthetic git-1(tm1962) mutant background. We are looking for enhancers that might lead to a Pat-like elongation arrest. As an approach, we adapted the protocol developed by the lab of Andrew Fraser (Nat Protoc, 1, 1617-20, 2006), where we use 96-well worm liquid cultures looking for late embryonic arrests. We will present our preliminary findings. To better understand the link between HDs biogenesis and embryonic elongation, we are also developing new tools to visualize different components of the myosin II pathway and of the HDs. One of them is VAB-10A, the homolog of human plectin. vab-10 is a 27kb gene that encodes several isoforms. VAB-10A is localized to the HDs. We developed a genomic fusion with the GFP to follow dynamics of VAB-10A localization during muscle contraction. We think that this new fluorescent tool will help us to complete our view of the mechanotransduction pathway that controls embryonic elongation.
Contact: [email protected] Lab: Labouesse
194 Poster Topic: Morphogenesis The EXC-1 RAS-Domain Protein Mediates Vesicle Movement in the Excretory Canals Kelly Grussendorf, Brendan Mattingly, Alex Salem, Matthew Buechner University of kansas, Lawrence, (KS), USA Tubulogenesis involves formation of tubule shape and diameter along both the apical (lumenal) and basal sides. Once formed, the lumen diameter must be maintained as the animal moves and grows. The single-cell excretory canal cell provides a simple model to study these processes. The cell is located near the terminal bulb of the pharynx, and extends two hollow processes to the left and right lateral side of the worm, where they bifurcate and extend anteriorly and posteriorly to form an H-shaped structure. A set of nine exc genes maintain the narrow diameter of the canal apical surface. Mutations in these genes allow formation of fluid- filled cysts in the lumen of the canal. The Exc-1 loss-of-function (lof) phenotype shows cysts in the canals that are often located at the ends of the canal. These cysts vary in size and number, from cysts not much wider than normal lumen to cysts expanded to the entire diameter of the worm. We have cloned the exc-1 gene, which encodes a homologue of the RAS GTPase family, specifically the family of Interferon-Inducible GTPases (IIGP). This protein is expressed in the canals, and also in the amphid sheath, a glial structure that surrounds the amphid neuron sensory endings. exc- 1 (lof) mutants show accumulation of recycling endosome marker EEA-1, and concomitant attenuation of recycling endosome marker RME-1 within the excretory canals, a phenomenon also seen for exc-5 mutants. Overexpression of exc-1 forms a tubule with a normal apical surface but defective in the formation of the basal surface. Epistasis experiments suggest that EXC-1 acts downstream of the EXC-9 LIM domain protein, and upstream of the EXC-5 guanine exchange factor. In addition, EXC-1 and EXC-9 bind directly to each other, as indicated via yeast two-hybrid assay. Our results suggest that these proteins function together to allow efficient movement from early endosomes to recycling endosomes. We are conducting further assays to assess binding of EXC-1 to other possible target proteins.
Contact: [email protected] Lab: Buechner
Poster Topic: Morphogenesis 195 A Screen For Genes Controlling Vulval Morphogenesis Qiutan Yang, Matthias Morf, Sarfarazhussain Farooqui , Juan Escobar, Alex Hajnal Institute of molecule life science, University of Zurich, Zurich, Switzerland The C. elegans egg-laying organ, the vulva, is an outstanding system to investigate the principles of organogenesis. Studies of vulval induction have led to a detailed molecular model of vulval fate specification, which is based on the concerted action of the conserved EGFR/ RAS/MAPK, NOTCH and WNT signaling pathways. However, the molecular mechanisms governing vulval morphogenesis are largely unknown. After the last of three rounds of cell divisions, the 22 vulval cells undergo morphogenesis, which involves several distinct aspects: (1) The formation of a lumen through invagination of the vulval cells, (2) the circumferential extension of the cells towards the vulval midline, (3) the formation of homotypic contacts and fusion between contralateral partner cells, which results in the formation of seven syncytial toroids, (4) the contraction of the ventral lumen, (5) the expansion of the dorsal lumen through invasion of the anchor cell (AC) invasion, and (6) the eversion of the vulval tissue. In order to systematically identify genes required for vulval morphogenesis, we are performing a RNA interference (RNAi) screen of all genes, which have been reported to exhibit a protruding vulva (Pvl) phenotype when mutated or upon RNAi treatment. Since a Pvl phenotype can be caused by defects at any stage of vulval development, we are using the AJM-1::GFP reporter to label the apical junctions, which allows us to examine the number and shape of the toroids, the size of the vulval lumen and to observe the cell fusions. In our screen, we are scoring the morphology of the vulva at the “Christmas tree” stage in mid L4 larvae, after the toroids have been formed but before vulval eversion begins. Since most of the genes affecting cell fate specification have been previously identified, we are concentrating on candidate genes that do not alter the cell fates or vulval lineage but act at a later stage during fate execution. We will present our classification of the different morphogenesis phenotypes observed so far and further explore the roles of promising candidates genes during vulval morphogenesis.
Contact: [email protected] Lab: Hajnal
196 Poster Topic: Morphogenesis LEP-2/Makorin Promotes let-7 microRNA-mediated Terminal Differentiation in Male Tail Tip Morphogenesis R Antonio Herrera, Karin Kiontke, Samuel Ahn, David Fitch New York University, New York, (NY), US In C. elegans, heterochronic genes regulate when stage-specific events occur during larval development. They interact in a pathway to ultimately schedule terminal differentiation at the last larval stage (L4). In males, heterochronic genes control when a sex-specific terminal differentiation program, tail tip morphogenesis (TTM), occurs. During TTM the tail tip cells (hyp8-11) change their larval cone-shape by cell fusion, retraction, and migration to produce the rounded dome found in adults. Heterochronic genes that specify L4 fates (lin-41 and let-7) schedule TTM to start at middle L4. When lost, lin-41 and let-7 cause TTM to occur earlier (in L3) or later (in adults), resulting in adult tail tip phenotypes that are over-retracted or unretracted and leptoderan-like (Lep), respectively. We found a new heterochronic gene, lep-2, which is required for TTM to occur during L4. With comparative genomic hybridization on a lep-2 deletion mutant, we identifiedlep-2 as the sole C. elegans Makorin (Mkrn). Mkrns are ancient eukaryotic genes which have conserved motifs; a RING domain flanked by four C3H-zinc fingers. However, the functional role of Mkrns during development is not well known. We found that lep-2 mutant males retain the larval tail tip into adulthood and shift the expression of the TTM master regulator, dmd-3, later than expected. lep-2 animals exhibit other developmental- delay phenotypes: they fail to exit the larval molting cycle or produce an “adult” cuticle and, in males, the Lep phenotype is suppressed after passage through the dauer larvae stage—all characteristics of a mutation in a heterochronic gene. Through epistasis analysis, we have determined that lep-2 resides in the heterochronic pathway downstream of lin-14 to promote let-7. In lep-2 mutants we observe elevated levels of heterochronic gene reporters that are downregulated prior to and during TTM (lin-28 & lin-41). Our genetic data suggest that the function of lep-2 is to negatively regulate lin-28, the let-7 repressor. Also, LIN-28 protein levels are elevated in lep-2 mutants. LEP-2/Mkrn, LIN-28 and let-7 are highly conserved genes that regulate differentiation in mammals in a manner consistent with C. elegans heterochronic development. This suggests that an ancient Mkrn function may be to promote let-7 during differentiation across eukaryotes.
Contact: [email protected] Lab: Fitch
Poster Topic: Morphogenesis 197 pix-1 Generates a Gradient of Contraction Forces in Hypodermal Cells of Elongating Embryos in Caenorhabditis elegans Sharon Harel, Emmanuel Martin, Bernard Nkengfac, Karim Hamiche, Mathieu Neault, Sarah Jenna UQAM, Montreal, Quebec, Canada Early stage of elongation is driven by the contraction of circumferential actin filaments (CAFs) in lateral hypodermal cells where myosin-light chains (MLC-4 and MLC-5) are phosphorylated by the Rho GTPases effectors LET-502, MRCK-1 and PAK-1. These kinases are antagonized by the MLC phosphatase MEL-11 which is active in ventral and dorsal hypodermal cells and inactive in the lateral cells were most of the contraction force occurs. The regulators of MLC phosphorylation are organized in two parallel pathways the let-502/mel-11/mrck-1 and the pak-1 pathways. We identified the Rac- and Cdc-42-GEF, pix-1, as a new component of the pak-1 pathway. We showed that pix-1controls early and late stages of elongation in parallel of let-502 and mel-11 and in parallel or upstream of the GTPases rac-2 and cdc-42. We also show that pix-1 activity during early elongation establishes a gradient of contraction forces along the anterior-posterior and the dorsal-ventral axes of the embryo. These contraction gradients are required to insure the appropriate morphology and elongation of the larvae.
Contact: [email protected] Lab: Jenna
198 Poster Topic: Morphogenesis Analysis of the Role of ENU-3 in Axon Outgrowth and Guidance in C. elegans Callista Yee1, Karmen Lam2, Anna Bosanac1, Marie Killeen1 1Ryerson University, Toronto, 2York University, 4700 Keele St., Toronto, Ontario During development, many cells including neurons migrate from their places of birth to their final destinations along defined and usually invariant pathways. Development of a properly patterned and functional nervous system relies on many guidance cues including Netrin/UNC- 6, Slit/SLT-1 and the Wnts that guide migrating axons to their final correct destinations and allow them to synapse with the correct targets. There are receptors for each of these cues expressed on the growth cones at the tips of the migrating axons in chordates and in many metazoa. C. elegans has proven to be a good model for analysis of these proteins due to its simple nervous system consisting of 302 neurons in the hermaphrodite that can be visualized in vivo using appropriate markers. We conducted a genetic enhancer screen in an unc-5(e53)background to find mutations that enhanced the axon guidance defects of the DA and DB classes of motor neurons and found five independent mutants. Mutations in enu-3 had very weak motor axon guidance defects and enhanced the short range migration defects of the motor neurons in the hypomorphic strain unc-5(e152). The mutations enhanced the motor axon outgrowth defects in worm strains lacking either functional Netrin/UNC-6 or its receptor UNC-5 (Yee et al., 2011). Strains lacking functional UNC-40 were not significantly affected in motor neuron outgrowth or guidance by mutations in enu-3. It is likely that the motor neuron axon outgrowth defects observed in the absence of UNC-5 are due to the presence of functional UNC-40. The involvement of UNC- 40 and ENU-3 in motor axon outgrowth the absence of UNC-5 has been further investigated. ENU-3 (H04D03.1) is a novel putative trans-membrane protein of unknown function with four close homologues in the C. elegans genome, all larger than ENU-3. All five proteins have putative signal peptides and are predicted to be trans-membrane proteins. ENU-3::GFP was expressed throughout the nervous system, particularly along the ventral cord. We found that enu-3(tm4519) had no significant defects in the migrations of the touch receptor neurons but enhanced the defects of an unc-40 mutant strain in an UNC-6 dependent manner. The defects observed suggest that the axons may not be properly adherent to the surfaces over which they migrate. Our current research is directed towards understanding the nature of the interactions between ENU-3 and UNC-40.
Contact: [email protected] Lab: Killeen
Poster Topic: Morphogenesis 199 Identifying Regulators of Gonadal Development in C. elegans by Cell- specific Transcriptional Profiling Mary Kroetz, David Zarkower University of Minnesota, Minneapolis, MN, USA The gonad of C. elegans originates in the embryo as a four-cell primordium composed of two somatic precursor cells (Z1 and Z4) that flank two germ line precursor cells (Z2 and Z3). The gonad primordium is morphologically identical in the two sexes, but soon after the animal completes embryogenesis it begins to develop via one of two distinct sex-specific programs of organogenesis. Despite the extensive sexual dimorphism and previously defined cell lineages of the gonad, the genetic pathways that direct the development of this organ, including the sex-specific development, remain largely unknown. The overall aim of this work is to define the genetic networks that regulate gonadal development in both sexes. To identify early gonadal regulators, we used cell-specific transcriptional profiling of Z1/Z4 during mid-L1 larval development, just prior to the first division of Z1/Z4 when gonadogenesis begins and the first sex-specific differences of the gonad arise. We used a Z1/Z4-specific gfp reporter to isolate these cells in hermaphrodites by FACS and profiled transcripts by RNA-seq. Of the eight transcripts that are known to be enriched in Z1/Z4 during L1, all of them showed Z1/Z4-enrichment. Among the 200 most enriched transcripts in Z1/Z4, we identified several unannotated transcripts that are highly specific to Z1/Z4. A number of the Z1/Z4-enriched transcripts have subsequently been validated by reporter analysis, confirming the effectiveness of this approach. We are determining loss of function phenotypes by RNAi depletion and mutant analysis. Work is currently underway to identify male-specific Z1/Z4-enriched transcripts from fully masculinized XX-pseudomales. Comparisons of male vs hermaphrodite Z1/Z4-enriched transcripts will help identify transcripts important for sex-specific gonadal development.
Contact: [email protected] Lab: Zarkower
200 Poster Topic: Morphogenesis Caenorhabditis elegans DNA-2 Helicase/Endonuclease Plays A Vital Role In Maintaining Genome Stability, Morphogenesis, And Life Span Myon-Hee Lee1,2, Sarah Hollis1, Bum Ho Yoo3, Keith Nykamp4 1Brody School of Medicine at East Carolina University, Greenville, NC, USA, 2Lineberger Comprehensive Cancer Center, University of North Carolina- Chapel Hill, Chapel Hill, NC, USA, 3Department of Biochemistry, Yonsei University, South Korea, 4Center for Molecular Diagnostics and BioBanking, Prevention Genetics, Marshfield, WI, USA In eukaryotes, highly conserved Dna2 helicase/endonuclease proteins are involved in DNA replication, DNA double-strand break repair, telomere regulation, and mitochondrial function. The Dna2 protein assists Fen1 (Flap structure-specific endonuclease 1) protein in the maturation of Okazaki fragments. In yeast, Dna2 is absolutely essential for viability, whereas Fen1 is not. In C. elegans, however, CRN-1 (a Fen1 homolog) is essential, but Dna2 is not. Here we explored the biological function of C. elegans Dna2 (Cedna-2) in multiple developmental processes. We find thatCedna-2 contributes to embryonic viability, the morphogenesis of both late-stage embryos and male sensory rays, and normal life span. Our results support a model whereby CeDNA-2 minimizes genetic defects and maintains genome integrity during cell division and DNA replication. These finding may provide insight into the role of Dna2 in other multi-cellular organisms, including humans, and could have important implications for development and treatment of human conditions linked to the accumulation of genetic defects, such as cancer or aging.
References: Lee et al. (2011) Caenorhabditis elegans DNA-2 helicase/endonuclease plays a vital role in maintaining genome stability, morphogenesis, and life span. Biochem Biophys Res Commun., 407(3): 495-500. Lee et al.(2003) Caenorhabditis elegans dna-2 is involved in DNA repair and is essential for germ-line development. FEBS Lett., 555(2): 250-256. Lee et al.(2003) Dna2 requirementfor normal reproduction of Caenorhabditis elegans is temperature- dependent. Mol Cells,15(1): 81-86.
Contact: [email protected] Lab: Lee
Poster Topic: Morphogenesis 201 The Role of LIN-3 During Morphogenesis of the Dorsal Lumen in the Vulva Louisa Mueller, Matthias Morf, Alex Hajnal Institute of Molecular Life Science, Zurich, Switzerland The hermaphrodite vulva is an excellent organ to identify and study the molecular mechanisms controlling tissue morphogenesis during development. Vulval development is initiated by the anchor cell (AC) in the somatic gonad, which secretes LIN-3 EGF and induces the vulval cell fates in three of the six adjacent vulval precursor cells. After vulval induction, the AC breaches two basal laminae and invades in-between the innermost 1°-fated VPC descendants (the VulF cells). AC invasion is important for proper morphogenesis of the dorsal lumen formed by the VulF toroids [1] and to establish the uterine-vulval connection. During vulval morphogenesis, LIN-3 is secreted from the VulF cells to specify the uv1 fate [2]. Here, we investigated another function of LIN-3 produced by VulF during dorsal lumen morphogenesis. Vulva-specific lin-3 RNAi using an rde-1(lf) mutant expressing rde-1(wt) in the Pn.p cells prevented the expansion of the dorsal lumen by the AC. A similar defect in dorsal lumen morphogenesis was observed in egl-38(lf) mutants that do not express LIN-3 in VulF cells [1,3]. Moreover, egl-38(lf) mutants displayed defects in AC polarization. Based on these results, we propose that LIN-3 expressed by the VulF cells controls dorsal lumen morphogenesis by polarizing the AC and thus enabling it to migrate in between the VulF cells and expand the dorsal lumen.
[1] Estes, K. A. and Hanna-Rose, W. (2009). Dev Biol 328, 297-304. [2] Chang, C., Newman, A. P. and Sternberg, P.W. (1999). Curr Biol 9, 237-46. [3] Rajakumar, V. and Chamberlin, H. M. (2007). Dev Biol 301, 240-53.
Contact: [email protected] Lab: Hajnal
202 Poster Topic: Morphogenesis Somatic gonad precursor migration in C. elegans Monica Rohrschneider, Jeremy Nance New York University School of Medicine, New York, NY, USA In many organisms, the somatic gonad precursor cells (SGPs) and the primordial germ cells (PGCs) are born at a distance from one another, and must migrate in order to coalesce and form the primordial gonad. In C. elegans, the SGPs migrate nearly half the length of the embryo to reach the PGCs. This migration is critical, as the SGPs are required for survival and proliferation of the germ cells. However, little is known about what drives the SGPs to migrate, and what triggers them to stop. As a first step in addressing these questions, we constructed fluorescent transgenic strains to characterize the migration of the SGPs and their interactions with neighboring cells. We observed three distinct phases of SGP migration—first the SGPs migrated posteriorly along the edge of the endoderm. When they reached the PGCs, the SGPs extended a single long projection around the posterior of the PGCs. And finally the SGPs wrapped completely around the PGCs. We used genetic transformation of the PGCs and of endoderm cells to test the hypothesis that these cells are required for the three phases of SGP migration. Surprisingly, SGP posterior migration was grossly normal in mes-1 mutants which lack PGCs, and in end-1 end-3 mutants, which lack endoderm, suggesting that PGCs and endoderm do not provide a long-range attractive cue to the SGPs. However, SGPs extended longer and more disorganized projections in mes-1 mutants, and continued to extend projections long after wild-type SGPs had wrapped around the PGCs, suggesting that PGCs direct SGP wrapping. In end-1 end-3 mutants, SGP projections and wrapping were partially disrupted, suggesting that endoderm development or morphogenesis may be required for normal SGP wrapping of PGCs. Because the SGPs migrate posteriorly between the endoderm and mesoderm cell layers, and it had previously been reported that basement membrane forms between the germ layers, we depleted laminin to investigate the role of the basement membrane in SGP migration. In lam-1(RNAi) embryos, the SGPs migrate farther posteriorly, while still extending relatively normal projections and eventually wrapping around the PGCs. Therefore, basement membrane may be required for the SGPs to stop migrating when they reach the PGCs. Our analysis of SGP migration provides a foundation for identifying the molecular mechanisms that promote each step of primordial gonad assembly.
Contact: [email protected] Lab: Nance
Poster Topic: Morphogenesis 203 VAB-9 and Vertebrate Orthologue TM4SF10 Cooperate with Adherens Junction Proteins and Actomyosin to Regulate Epithelial Polarity and Morphogenesis Jeff Simske Rammelkamp Center, Cleveland (OH), USA Regulation of morphogensis and cell polarity requires the coordinated interaction between the actomyosin contractile apparatus and cellular junctions. VAB-9 is an adherens junction protein belonging to the claudin/PMP22/EMP family of tetraspan integral membrane proteins. vab-9 mutants have variable defects in elongation. Elongation requires coordinated contraction of actomyosin cables in the embryonic epidermis following enclosure. Non-muscle myosin activity is regulated in part by phosphorylation and activation of the regulatory light chain by Rho kinase LET-502 and de-phosphorylation and inactivation by the phosphatase MEL-11. Prior to elongation, MEL-11 is present in the cytoplasm of epidermal cells, but becomes localized to cell junctions as elongation proceeds, suggesting that preventing MEL-11 inhibition of actomyosin contractility requires junctional re-localization (Piekney and Mains, 2003). VAB-9, MEL-11 and activated myosin light chain proteins all co-localize at the cell junctions of enclosing epidermal cells and remain at seam cell junctions during elongation. Co-localization between MEL-11 and activated myosin light chain at junctions suggests regulation of myosin activity by MEL- 11 is complex and highly dynamic. In vab-9 mutants, MEL-11 fails to localize to cell junctions, indicating that VAB-9 promotes elongation through MEL-11 sequestration at junctions. In vab-9 animals activated myosin light chain levels appear lower in the cytosol, possibly due to inactivation by mislocalized MEL-11. vab-9 mutations suppress reduction-of-function mel-11 enclosure defects and sterility; similarly, mel-11 activity is required for VAB-9 localization, and altering actomyosin activity modifies vab-9 phenotypes. The VAB-9 vertebrate orthologue TM4SF10 was examined in MDCK cells to determine whether function is conserved and to characterize TM4SF10 protein complexes. Since vab-9 can be functionally replaced by a GFP- tagged version of the vertebrate protein TM4SF10, it is likely that at least some functions are conserved. In MDCK cells, TM4SF10-GFP co-localizes with and co-IPs with adherens junction proteins. Overexpression of TM4SF10-GFP results in delay in reformation of cell junctions following calcium switch, reduced apical surface area, altered cell polarity, disorganized cell junctions and altered F-actin organization. SiRNA inactivation appears to generally result in the opposite phenotypes. Direct interaction between VAB-9/TM4SF10 and actomyosin proteins has not been demonstrated in either system.
Contact: [email protected] Lab: Simske
204 Poster Topic: Morphogenesis The C. elegans DM domain genes dmd-3 and mab-3 function during the late stages of male gonad development Michele Smith1, Alyssa Herrmann1, Emily Kivlehan1, Lauren Whipple1, Douglas Portman2, D. Adam Mason1,2 1Siena College, Loudonville, (NY), 12211, USA, 2University of Rochester, Rochester, (NY) USA The development of an organism from a fertilized egg into a fully formed adult is a spectacularly complex process. Complexity is further compounded by the fact that, in most animal species, the sex-determination pathway must modify developmental pathways in order to generate two sexually dimorphic body forms. Previous studies have demonstrated that DM-domain transcription factors control sex-specific development in diverse animal phyla, suggesting that these genes were part of a core sexual differentiation pathway in the common ancestor of all eumetozoans. We have been examining the function of two C. elegans DM-domain transcription factors, DMD-3 and MAB-3, in directing the development of male-specific structures. We have previously observed that, DMD-3 and MAB-3 play a central role in guiding the male-specific remodeling of the L4 tail that generates the sexually dimorphic blunt-ended adult male tail. The expression patterns of dmd-3 and mab-3 reporter transgenes suggested that these genes also play a role in directing male gonad development. Beginning in mid-L3 males, both dmd-3 and mab-3 are expressed in the male-specific linker cell, a cell that functions to lead the male gonad from the mid-body down to the hindgut. In addition, in early L4 males dmd-3 expression commences in a subset of vas deferens cells that lie directly behind the migrating linker cell. Finally, dmd-3 is expressed male-specifically in the hindgut cells that eventually engulf the linker cell in mid-L4 males. Consistent with this expression pattern, mab-3 ; dmd-3double mutant males show distinct defects in linker cell migration. We observe that the linker cell fails complete its migration down to the hindgut in some mab-3; dmd-3 double mutant L4 males. In addition, double mutant linker cells exhibit an aberrant morphology during migration. Specifically, we observe that a high percentage of double mutant linker cells project out long cellular processes in both the anterior and posterior direction during the final leg of their migration in early L4 males. This occasionally culminates in pieces of the linker cell becoming detached from the main cell body. dmd-3single mutant males exhibit a similar, but less severe defect in linker cell migration, while linker cell migration in mab-3 mutants appears indistinguishable from wild type. Earlier stages of linker cell migration appear normal in the single and double mutant males. In addition, we find that linker cells that do complete migration fail to be engulfed properly by the hindgut cells in mab-3 ; dmd-3 late-L4 males, resulting in the persistence of the linker cell into adulthood. The failure of the linker cell to reach and/or be engulfed by the hindgut should result in a gonad that does not properly connect to the cloaca. Together, these results demonstrate that DMD-3 and MAB-3 function together to direct the late stages of male gonad development.
Contact: [email protected] Lab: Mason
Poster Topic: Morphogenesis 205 Analysis of Non-Muscle Myosin II During Dorsal Intercalation in Caenorhabditis elegans Elise Walck-Shannon, Jeff Hardin University of Wisconsin-Madison Cell intercalation is a morphogenetic movement that is used throughout animal development to shorten a tissue along one axis and extend it along the orthogonal axis. Dorsal intercalation within the C. elegans embryonic epidermis is a simple model to study cell intercalation. During dorsal intercalation, two rows of ten epidermal cells converge into one row of twenty cells. Non-muscle myosin II is required for cell intercalation in multiple systems; however, its role during these processes is not well understood. In C.elegans there are at least two non-muscle myosin II isoforms expressed during embryonic development that differ in their heavy chains, which are encoded by nmy-1 and nmy-2. I find that non-muscle myosin II has isoform-specific requirements immediately after dorsal cell division that are essential for dorsal intercalation to complete in C. elegans. Loss of function for nmy-2 alone yields misshapen cells that still increase in length, while loss of both nmy-1 and nmy-2 function yields misshapen cells that do not elongate. As dorsal cells acquire their normal polarized morphology shortly after division, NMY-2::GFP filaments accumulate inthe apical cortex and show periodic changes in intensity that are inversely correlated to cell area. Together, this suggests that non-muscle myosin II has a role early in intercalation. In future studies, I plan to study both the regulation of contractile activity by the phosphorylation of myosin regulatory light chain and the regulation of non-muscle myosin II localization. Determining the normal regulation and function of non-muscle myosin II during cell intercalation could have broad implications for both its misregulation during disease and its role during normal development.
Contact: [email protected] Lab: Hardin
206 Poster Topic: Morphogenesis Establishing Caenorhabditis elegans as a Model for Neural Tube Defects Bridget Waller, Kassi Crocker, Timothy Walston Truman State University The exact causes of most neural tube defects (NTDs) remain unknown. The disabling birth defect spina bifida, however, may result from a combination of genetic and environmental risk factors, including alcohol consumption early in pregnancy. Morphogenesis in Caenorhabditis elegans involves similar cell movements to what is seen in the vertebrate neural tube and many similar molecular contributions. The ease of embryonic study makes C. elegans a tractable model to understand the mechanisms that affect cell migration. Therefore, the goal of this project is to establish C. elegans as a model for NTDs through studying the embryonic defects that result from alcohol exposure. In this experiment, C. elegans embryos exposed in utero to 300 mM alcohol, equivalent to a BAC of 0.075, experienced a lethality rate of 47.25% (n=3200). Current work is classifying the stage of failed embryogenesis and the morphogenetic movements that are sensitive to exposure to alcohol.
Contact: [email protected] Lab: Walston
Poster Topic: Morphogenesis 207 Anillin is required for Epidermal Morphogenesis during C. elegans Embryogenesis Denise Wernike, Alisa Piekny Concordia University, Montreal In the early embryo, anillin (ANI-1) serves as a scaffold for actin, myosin and membrane- binding septins during cytokinesis. Cytokinesis is the process when one mother cell gives rise to two genetically identical daughter cells. It requires the rearrangement of cytoskeletal components to form an actin-myosin contractile ring, which ingresses to pinch in the cytoplasm of the cell. Anillin crosslinks and controls the dynamics of contractile ring components to ensure that the ring closure occurs with high fidelity. Cytokinesis can be interpreted as a form of cell shape change comparable to morphogenetic events that take place during tissue formation in C. elegans embryogenesis. Epidermal cells that undergo these events must rearrange their cytoskeleton, using actin-myosin filaments for cell shape changes and migrations to enclose the embryo and to drive its shape change. However, it is not known if anillin fulfills functions outside of cell division, particularly during C. elegans embryonic morphogenesis. We recently determined that anillin is maternally and zygotically required throughout embryogenesis, and RNAi-depleted embryos display phenotypes consistent with roles for ani-1 in ventral enclosure and elongation. Live-imaging of ani-1 RNAi embryos during ventral enclosure revealed that epidermal cells failed to meet at the ventral midline. In order to further explore a role for ani-1 in ventral enclosure, genetic epistasis experiments were performed with known regulators of cell adhesion. Adherens junctions are believed to play a pivotal role during C. elegans ventral enclosure, where they tightly seal the migrating ventral cells along the ventral midline. Adherens junctions consist of an extracellular domain, termed cadherin, that connects adjacent epithelial cells to each other, and an intracellular region that directly links to the actin cytoskeleton through its interaction with α-catenin and β-catenin. We found that hypomorphic alleles of cadherin and catenin enhanced anillin loss-of-function phenotypes. In addition, overexpression of α-catenin significantly alleviated anillin phenotypes, suggesting that anillin and α-catenin are in the same pathway. Further experiments are being performed to elucidate the relationship between anillin and components of the cell adhesion machinery.
Contact: [email protected] Lab: Piekny
208 Poster Topic: Morphogenesis What Causes Partial Penetrance of a Developmental Phenotype? Claire Williams1,2, Maxwell Heiman1,2 1Children’s Hospital, Boston, MA, USA, 2Harvard Medical School, Boston, MA, USA Partial penetrance is a poorly understood phenomenon that hinders the accurate prediction of phenotype from genotype. From first principles, partial penetrance implies a variable activity and a thresholding mechanism that partitions that activity into an “all or none” phenotype. To address the question of which activities may be variable and subject to a threshold, we have turned to an example in C. elegans neurodevelopment. Amphid sensory neuron dendrites are normally anchored at the nose tip by DYF-7, an extracellular matrix protein with a zona pellucida domain. Individual animals bearing a partially penetrant dyf-7 allele, dyf-7(ns117), exhibit one of two phenotypes, either short or full-length dendrites, even within isogenic populations raised in a shared environment. This partial penetrance suggests that, in these mutants, a limiting activity in dendrite development is subject to stochastic variability. To determine if this limiting activity is that of dyf-7(ns117) itself, we increased the dosage of dyf-7(ns117) in an otherwise dyf-7-null genetic background. Indeed, higher dosages of dyf-7(ns117) led to a decrease in penetrance of the abnormal phenotype, implying that some aspect of dyf-7(ns117) expression is the limiting, variable activity. This variability could arise at the level of transcription, translation, or protein processing and trafficking: DYF-7 normally undergoes extracellular proteolytic cleavage to assemble a “cap” at the dendrite tip. To visualize DYF-7 processing and trafficking in vivo, we tagged DYF-7 with an extracellular GFP and a cytoplasmic mCherry and observed differential localization of the two tags, consistent with proteolytic cleavage. The extracellular tag assembled in caps at dendrite tips while the cytoplasmic tag spread diffusely throughout cell membranes. In contrast, the extracellular and cytoplasmic tags on DYF-7(ns117) showed extensive overlap and were both present throughout cell membranes, suggesting that defective protein processing underlies the partial penetrance of this phenotype. Previous examples of partial penetrance have involved transcriptional variability and a thresholding mechanism based on positive feedback loops in gene regulatory networks. Thus, dyf-7(ns117) presents a counterexample in which variability in protein processing is somehow thresholded to produce an “all or none” developmental phenotype.
Contact: [email protected] Lab: Heiman
Poster Topic: Morphogenesis 209 MIG-10 interacts with ABI-1 to induce asymmetric outgrowth- promoting activity in response to guidance cues Yan Xu, Christopher Quinn University of Wisconsin Milwaukee Actin regulatory proteins have been implicated in the control of growth cone morphology. However, little is known about how these proteins are spatially organized to orchestrate the directional response to axon guidance cues. A key to understanding this process may be provided by the asymmetric accumulation of the MIG-10 (lamellipodin) cytoplasmic scaffolding protein in response to guidance cues. However, the mechanism that links MIG-10 to actin polymerization is not known. Using an RNAi screen in C. elegans, we identified the actin regulatory protein ABI-1 (Abi1) as a partner for MIG-10. We find that MIG-10 binds to the SH3 domain of ABI-1 and dosage sensitive genetic interactions indicate that MIG-10, ABI-1 and WVE-1 function together to mediate axon guidance. Analysis of double mutants shows that these proteins function in both the attractive response to UNC-6 (netrin) and the repulsive response to SLT-1 (slit). Epistasis analysis reveals that ABI-1 and WVE-1 function downstream of MIG-10 to mediate its outgrowth-promoting activity. Moreover, experiments with cultured mammalian cells suggest that the interaction between MIG-10 and ABI-1 mediates a conserved mechanism that promotes formation of lamellipodia. Together, these observations indicate that ABI-1 and WVE-1 mediate the outgrowth-promoting activity of MIG-10 to spatially direct axon growth in response to the UNC-6 and SLT-1 guidance cues.
Contact: [email protected] Lab: Quinn
210 Poster Topic: Morphogenesis Molecular characterization of maternally malformed 3 (mal-3) Yemima Budirahardja1, Thang Doan1, Ronen Zaidel Bar1,2 1Mechanobiology Institute Singapore, Singapore, 2Department of Bioengineering, National University of Singapore, Singapore During C. elegans morphogenesis, the ovoid-shaped embryo elongates four fold into a worm-shaped larva before hatching. Defects in elongation result in larval/adult body shape defects or developmental arrest. Genetic screens for maternal effect morphologically abnormal worms are likely to uncover genes required during embryonic morphogenesis. We are using whole genome sequencing to identify the affected genes in several mutant strains isolated by Hekimi et. al. in 1995 in such a screen [1]. One of the strains, MQ466 (mal-3), shows specific defects in elongation. It is temperature sensitive with close to 5% embryonic lethality (Emb) at 15°C, over 50% Emb at 20°C, and approximately 90% Emb at 25°C. Time-lapse Nomarski imaging at 20°C showed the majority of Emb is due to elongation arrest, concomitant with the formation of vacuoles within the embryo. Visualizing the junctional protein alpha-catenin/ HMP-1 in mutant embryos revealed improper cell alignment during ventral enclosure and what appears like uneven pulling forces perpendicular to the seam cells during elongation. Whole genome sequencing suggests mal-3 most likely results from a single missense mutation in T09B4.1, a mannosyltransferase involved in the synthesis of glycosylphosphatidylinositol (GPI). Attachment of a GPI-anchor is a post-translational modification leading to anchoring of a protein to the outer leaflet of the plasma membrane. Recently, GPI synthesis was shown to be essential for germline development in the worm [2], but its role in morphogenesis is currently unknown. Among the candidate GPI-anchored proteins in C. elegans are several cell adhesion and signaling proteins. Ongoing work is aimed at validating the molecular nature of mal-3 and at identifying the presumed GPI-anchored proteins whose function is critical for elongation.
References: [1] Hekimi, S., Boutis, P., and Lakowski, B. (1995). Viable maternal-effect mutations that affect the development of the nematode Caenorhabditis elegans. Genetics 141, 1351-1364. [2] Murata, D., Nomura, K.H., Dejima, K., Mizuguchi, S., Kawasaki, N., Matsuishi-Nakajima, Y., Ito, S., Gengyo-Ando, K., Kage-Nakadai, E., Mitani, S., et al. (2012). GPI-anchor synthesis is indispensable for the germline development of the nematode Caenorhabditis elegans. Mol Biol Cell 23, 982-995.
Contact: [email protected] Lab: Zaidel-Bar
Poster Topic: Morphogenesis 211 A Semi-Automated Pipeline for the Identification of Novel Mutants with Cell Number Defects Peter Appleford, Alison Woollard University of Oxford, Oxford, UK C. elegans RNAi screens provide a rapid and convenient way to identify novel genes involved in cell and developmental processes. However, not all tissues are amenable to silencing by RNAi and the knock down effect itself is both transient and variable. With the advent of cheaper whole-genome resequencing, traditional forward genetic screens are once again becoming a more attractive proposition. Although the mapping of new alleles by Whole Genome Sequencing (WGS) has been demonstrated to work by at least two similar methodologies, the initial screening for interesting phenotypes still represents a significant bottleneck in terms of the labour and time required. Our lab is interested in the mechanisms controlling the balance between proliferation and differentiation in the stem-like seam cell lineage. We have used the Biosorter (essentially, a large particle flow cytometer) developed by Union Biometrica to test whether such a platform is suitable for counting seam cells in the worm, utilising expression of the seam-specificscm::gfp reporter. As proof of principle, we initially showed that the Biosorter is capable of scoring the expected 16 peaks corresponding to seam nuclei present in late L4 hermaphrodites and that worms with aberrantly high numbers of seam nuclei are also correctly identified. We have now conducted three pilot screens with the Biosorter and have isolated at least 12 mutant strains which exhibit a range of defects in seam cell development, including seam cell hyperplasia and seam cell spacing abnormalities. We are currently preparing mutant strains for sequence analysis and we will present our progress at the meeting.
Contact: [email protected] Lab: Woollard
212 Poster Topic: New Technologies A Novel Fluorescence-Based Method to Visualize Protein-Protein Interactions in Living Caenorhabditis elegans Han Ting Chou, Casonya Johnson GSU, Atlanta, Georgia, USA Molecular and genetic studies have shown that proteins work in multimeric complexes to direct cellular events, and that transcription factors, in particular, can use selective interactions to differentially regulate gene expression. Existing methods for detecting these interactions are limited in the information that they provide, usually because the method of detection is most appropriate for studies done in cell culture or in single-celled organisms. Here we developed a transformative method to detect transient interactions among transcription factors within the nuclei of the cells of living animals. The transparent nematode Caenorhabditis elegans was used as the model organism and fluorescence imaging was used to detect interaction between a transcription factor tethered to the nuclear membrane and a fluorescently-labeled partner. Experiments were carried to study interactions of the REF-1 family proteins of the basic helix-loop-helix (bHLH) transcription factor superfamily involved in a wide range of biological processes. Interactions between HLH-29 and its dimerization partners were monitored during the normal life-cycle of wild-type hermaphrodites. This novel technique of capturing dynamic protein-protein interactions complements genome-wide studies of transcriptional networks that have been critical for the mechanistic and systems-wide studies being performed today.
Contact: [email protected] Lab: Johnson
Poster Topic: New Technologies 213 Spectrum: Building Pathways to Biomedical Research Careers for Girls and Women of Color Diana Chu, Rebecca Garcia, Kimberly Tanner San Francisco State University, San Francisco, CA, USA While progress has been made in encouraging girls in science, women of color are still largely absent from the biomedical research community and few materials or models currently exist that are designed specifically to attract girls of color tothese careers. The Science Education Partnership and Assessment Laboratory (SEPAL) in the Department of Biology at San Francisco State University (SFSU) has developed the Spectrum effort to address the dearth of women of color in biology. Through Spectrum, biomedical scientists who are women of color – including SFSU undergraduate students, Masters students, alumni in local doctoral and biotechnology positions, and faculty – and middle and high school students and teachers collaborate to: 1) co-sponsor after-school science clubs targeted at girls of color in high needs public schools, 2) develop a mentoring community of women of color trainees in biomedical research, 3) develop a series of video biographies that highlight the research programs of women of color biomedical researchers and scientific trainees, and 4) partner with the local and national Expanding Your Horizons organizations to disseminate Spectrum activities. During its initial three years, Spectrum engaged 279 middle and high school girls (46% Latina, 12% African American, 21% Asian, 13% Unknown, 8%White) across five club sites providing ~20 hours of academic enrichment inbiomedical science for each girl, including two field trips to the laboratories of SFSU women of color biologists. Evaluation data shows increases in the percentage of participating girls who agree with the following statements: 1) I have heard a woman scientist talk about how she became a scientist (pre: 50%, post: 92%), 2) I have heard a woman scientist talk about why she likes science (pre:61%, post: 95%), and 3) I have met a woman scientist who is like me (pre: 33%, post: 62%). Spectrum is supported by the National Center for Research Resources and the Division of Program Coordination, Planning, and Strategic Initiatives of the National Institutes of Health through R25RR024307, Supplement R25RR024307-05S, and Supplement R25RR024307-03S1.
Contact: [email protected] Lab: Chu
214 Poster Topic: New Technologies Establishing and using a modified NGM (ENGM) to culture an manipulate the entomopathogenic nematode, Heterorhabditis bacteriophora Zsofia Csanadi1, Abate Birhan Addise2, Anita Alexa3, Barnabas Jenes4, Zsofia Banfalvi4, Andrea Mathe-Fodor5, Katalin Belafi-Bako1, Andras Fodor2 1Research Institute of Bioengineering, Membrane Technology and Energetics H-8200, Veszprem, (Egyetem), Hungary, 2Institute of Animal Science & Breeding, University of Pannonia, Keszthely, Deak F., Hugary, 3Department of Biochemistry, Eotvos Lorand University, Budapest, Pazmany, Hugary, 4Biotechnology Research Center, Godollo, Szent- Gyorgyi, Hungary, 5Molecular and Cellular Imaging Center, Ohio State University, Wooster, (OH), United States Entomopathogenic nematode (EPN) species belonging to Heterorhabditis and Steinernema genera similarly to C. elegans are feeding on bacteria. But they can grow only on their own symbionts, Xenorhabdus and Photorhabdus bacteria, respectively. NGM is not an appropriate medium for neither of the symbiotic partners. In order to make reliable genetics on EPN species we developed a new solid media called ENGM (Entomopathogenic Nematode Growth Media) on which both H. bacteriophora and C. elegans develop normally. They are visible on the plate under stereomicroscope throughout their life cycles. We compromised the advantages of NGM and Woots agar media, and tested different ingredients both qualitatively and quantitatively. A proof of the usefulness of the new (ENGM) media is that we managed produce RNAi phenocopies from both C. elegans and H. bacteriophora by feeding them with the appropriate construction. In case of H. bacteriophora it was a Photorhabdus strain transformed with C. elegans dpy-3 feeding construction. We have started a project to produce and breed transgenic H. bacteriophora expressing tps1 gene from the yeast. We found that the Gene Booster technique is amenable for producing transgenic Heterorhabditis expressing govern by heat-inducible (hsp2) promoter governed yeast tps-1 cloned into the appropriate Fire vector. The phenotype is high osmotic tolerance.
Contact: [email protected] Lab: Belafi-Bako
Poster Topic: New Technologies 215 A MultiSite Gateway®-Compatible Three-Fragment Vector Construction Kit Using Galactose Selection Iskra Katic, Wolfgang Maier Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Invitrogen’s Gateway system, which uses recombinational cloning through phage lambda integrase, offers speed and reliability in creating molecular constructs. MultiSite Gateway enables modular design of expression vectors containing promoters, genes, and 3’UTRs in a sequence- and orientation-specific manner. The efficiency of Gateway cloning relies strongly on a negative selection against the ccdB death gene, which kills cells transformed with non- recombined educt vector. We and others have observed occasional mutations in the ccdB counter-selection cassette causing many false positive clones in subsequent Gateway reactions. We have therefore replaced the ccdB cassette with a galactokinase (galK) cassette, which, in principle, allows for both positive and negative selection. This system offers several advantages over conventional Gateway cloning using ccdB selection, including easy maintenance of donor and destination vectors. We have created a MultiSite Gateway-compatible vector construction kit, which also contains MosSCI and MosSCI-biotic destination vectors.
Contact: [email protected] Lab: N/A
216 Poster Topic: New Technologies Screening for C. elegans Mutants with Subtle Phenotypes with Microfluidics and Computer Vision Adriana San-Miguel1, Matthew Crane1, Peri Kurshan2, Kang Shen2, Hang Lu1 1Georgia Institute of Technology, Atlanta, (GA), USA, 2Stanford University, Stanford, (CA), USA Genetic screens in C. elegans have led to understanding the function of many relevant genes by detecting animals with interesting phenotypes. The identification of mutants with significantly altered phenotypes, easily identifiable by simple visual inspection, has reached a saturation point. Aside from the difficulty of typical screens that require manual handling and inspection of a very large number of animals, the current challenge lies on the identification of mutants with very subtle phenotypes difficult to identify by eye. Performing screens based on fluorescent reporters of very small features, such as synapses, present an exceptionally difficult scenario. The challenges include extracting quantitative information regarding synapse size, intensity and distribution from images where lipid droplets and gutauto-florescence is present. While human eyes can distinguish between some of these, they are not sensitive to slight differences in size, intensity or size distribution of such small features. Here, we use computer vision algorithms to objectively quantify relevant information from a mutagenized animal population and thus, identify mutants with subtle phenotypes. Additionally, we utilize microfluidic devices as a platform for automated worm imaging, handling and sorting. The developed microfluidic device has several advantages over devices previously used for worm imaging: it is a robust single layer device which can be easily fabricated without the need of alignment or incorporation of valves in a second layer. Moreover, the device has been designed to orient the worms in a dorsal-down position with the purpose of imaging the posterior dorsal area in all worms, where synapses are located. Computer algorithms allow the identification of worms which are in the right orientation and are suitable for imaging, as well as those which have a slightly altered phenotype and are sorted as mutants. Integrating microfluidics and computer vision we have generated a platform for automated high-throughput imaging and sorting of thousands of worms based on quantitative synapse- related features. This method enables imaging, phenotyping and sorting about 100 times faster than manual handling. Not only does this method allow performing genetic screens in a simple, automated and fast manner, but it also provides a platform for discovery of mutants which would otherwise be overlooked in a typical manual screen.
Contact: [email protected] Lab: Lu
Poster Topic: New Technologies 217 Two Novel Staining Protocols Resolve Caenorhabditis elegans Cuticular Structures For Live Imaging And Transmission Electron Microscopy Robbie Schultz1, E. Ann Ellis2, Tina Gumienny1 1Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, TX, USA, 2Microscopy and Imaging Center, Texas A&M University, College Station, TX, USA The C. elegans cuticle is a transparent exoskeleton that surrounds the animal, protecting the organism from the environment and facilitating locomotion. The cuticle is composed of several layers, including multiple inner collagen layers and an outer lipid layer. Ultrastructure patterns the cuticle, where alae form longitudinal ridges that run the length of the animal and annuli form circumferential ridges around the animal. While this ultrastructure is of interest to many researchers using C. elegans, it is not easily distinguished using standard methods. We have developed two techniques to visualize C. elegans cuticle structures: staining living organisms using a vital lipophilic dye, DiI (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate), for compound or confocal microscopy studies, and staining and fixing tissues with malachite green for transmission electron microscopy (TEM) studies. For live animal imaging, DiI fluorescently stains the outermost lipid layer. This stain resolves both annuli and alae (when present) [1]. Also, finer structures of the adult male tail, including rays and the fan, are highlighted upon staining with DiI [1]. For TEM studies of adult C. elegans, malachite green binds to the lipid layer that surrounds the animal, including the outer layer of the alae. Malachite green also preserves and differentially stains the inner layers of cuticle, distinguishing the cortical, medial, and basal layers. We demonstrate DiI and malachite green dyes are useful tools to resolve the structure of the C. elegans cuticle for correlative studies using compound/confocal and TEM microscopy, respectively.
Reference [1] R. D. Schultz, T. L. Gumienny, JoVE (2012) 59: e3362.
Contact: [email protected] Lab: Gumienny
218 Poster Topic: New Technologies Improving the Sensitivity and Selectivity of Mutation Identification by Next-Generation Sequencing Sijung Yun, Michael Krause, Harold Smith NIDDK/NIH, Bethesda, MD Next-generation sequencing provides a rapid and powerful means for identifying mutations on a genome-wide scale. Advances in sequencing technology have sparked similar improvements in software for sequence analysis. We have developed a modular analysis pipeline for mutation identification that uses a combination of publicly available tools: BFAST (for alignment), SAMTools (for mutation calling), and ANNOVAR (for annotation). We compared our results to those obtained using MAQGene, the standard analysis pipeline for C. elegans mutation identification. We observed a large degree of overlap between the sets of single- nucleotide polymorphisms (SNPs) identified by the two pipelines. For SNPs common to both pipelines, validation by Sanger sequencing indicated a high degree of accuracy. Similar analysis of the SNPs unique to each pipeline revealed a high false-positive rate for both, as well as a modest false-negative rate for MAQGene. We defined additional criteria that allow us to discriminate false-positive from true-positive SNP calls. We also validated a number of small insertions and deletions (indels) that were not detected using MAQGene. Our pipeline provides advantages for mutation identification in terms of sensitivity (by recovering SNPs and small indels that were previously missed) as well as selectivity (by limiting the number of false-positive SNP calls).
Contact: [email protected] Lab: Krause
Poster Topic: New Technologies 219 Worm Proteins Overtake Biochemistry Lab to Inspire Inquiry Katherine Walstrom New College of Florida, Sarasota, FL During Spring 2012, I changed my Biochemistry Lab course from a mostly “cookbook” lab course to an inquiry-based course. I chose lactate dehydrogenase (LDH-1) and four C. elegans predicted alcohol dehydrogenases as enzymes the students could study. Each group chose an enzyme, and they subcloned the cDNA into a protein expression vector and purified the enzyme during the first half of the course. During the rest of the course, the students proposed research projects to perform with their enzyme (if it had detectable activity) or with a similar, purchased enzyme. All seven groups proposed very different research projects, and some were more sophisticated than others. Most of the projects could eventually be developed into an undergraduate thesis project, which is required for all students at our institution. The projects addressed two main topics. One group of projects involved site-directed mutagenesis to change the substrate specificity of LDH-1. The other projects involved removing the zinc ion(s) in the alcohol dehydrogenases and replacing them with other metal ions. Enzyme kinetics experiments will be performed with the original and modified enzymes. The experiments we performed could easily be adapted to other lab courses or for other undergraduate research projects. I am also taking suggestions for enzymes to study next year! The most successful projects will involve enzymes with fewer than ~500 amino acids and with a predicted enzyme activity that can be detected using UV-VIS spectroscopy.
Contact: [email protected] Lab: Walstrom
220 Poster Topic: New Technologies Understanding temporal and spatial features of polarity establishment Simon Blanchoud1, Felix Naef2, Pierre Gonczy1 1EPFL SV ISREC, Lausanne, Switzerland, 2EPFL SV IBI, Lausanne, Switzerland Even though polarity establishment in the one-cell stage C. elegans embryo has been studied qualitatively using forward genetic and RNAi-based functional genomics, how polarity components interact in space and time remains poorly understood. This is due in part to the lack of automated methods to gather quantitative information with subcellular precision. Consequently, data quantification is often performed manually, which is repetitive, inefficient and prevents the precise and consistent analysis of large datasets. To circumvent this limitation, we developed a multi-channel image analysis software coupled with a reference coordinate system that we termed ASSET (for Algorithm for the Segmentation and the Standardization of C. elegans Time-lapse recordings). By automatizing the segmentation, our algorithm enables us to combine the great spatial and temporal resolution achieved in live recordings with an efficient computational pipeline, permitting the fast and coherent processing of a large number of recordings. Consequently, ASSET provides an adequate platform for image-based automated quantifications of dynamical processes. We now use ASSET to precisely measure fluorescence intensities from time-lapse recordings of PAR fusion proteins, starting with the posterior GFP-PAR-2 fusion protein. Combined with an effective mathematical model [Goehring et al., Science, 2011], these recordings allow us to quantify precisely key spatio-temporal features of polarity establishment. Of particular importance, we can derive in this manner values for the parameters governing the mutual inhibition of the anterior and posterior polarity complexes, yielding important insights on the underlying molecular mechanisms.
Contact: [email protected] Lab: Gönczy
Poster Topic: Polarity 221 PAR proteins regulate the localization of LET-99 during asymmetric division Eugenel Espiritu, Jui-Ching Wu, Lesilee Rose University of California, Davis, CA, USA Mitotic spindle positioning is essential for asymmetric divisions, where the spindle must be aligned with the axis of cell polarity. In many systems, PAR polarity proteins establish polarization of the cell and regulate spindle movements via a complex including Gα, GPR and LIN-5. We previously showed that LET-99 is a key regulator of GPR asymmetry in C. elegans one-cell embryos. LET-99 is asymmetrically localized at the cortex in a lateral-posterior band pattern, where it inhibits GPR localization. Analysis of LET-99 localization in mutant backgrounds showed that PAR-3 inhibits cortical LET-99 at the anterior cortex, while a gradient of PAR-1 inhibits LET-99 at the posterior-most cortex. In addition, PAR-1, a Ser/Thr kinase, was found to associate with LET-99 in vitro. To gain further insight in the mechanism of LET-99 localization, we tested LET-99 for association with the C. elegans 14-3-3 protein, PAR-5. In other systems, phosphorylation of targets by PAR-1 and the PAR-3 associated kinase PKC-3 generates binding sites for 14-3-3 proteins, which alters the targets’ localization. We found that PAR-5 bound to His-LET-99 in wild-type embryo extracts, but PAR-5 binding was greatly diminished in extracts from par-1(RNAi) embryos. Computer predictions for 14-3-3 binding sites followed by yeast-two hybrid (Y2H) assays identified two LET-99 serine residues essential for PAR-5 binding. To determine the in vivo relevance of these sites, we introduced S-to-A mutations into an otherwise full-length rescuing LET-99 transgene (LET-99-AA). When transferred into a let-99 deletion mutant background, the transgene-encoded LET-99-AA protein mislocalized to the entire posterior cortex of the one-cell embryo, similar to what was observed for LET-99 in par-1 mutant embryos. These and other results support the model that PAR-5 binds to LET-99 to prevent association with the posterior-most cortex, and that this interaction is regulated by phosphorylation of LET-99 by PAR-1. To begin to determine how LET-99 localization is restricted from the anterior cortex, we analyzed LET-99 after depletion of anterior PAR components. We found that PAR-3 is not sufficient for normal LET-99 localization, but rather the PAR-3 associating proteins, PAR-6 and PKC-3, restrict LET-99 localization from the anterior. In the future, we will test the hypothesis that PAR-1 and PKC-3 directly phosphorylate LET-99 using in vitro kinase assays.
Contact: [email protected] Lab: Rose
222 Poster Topic: Polarity On the Role of RGA-3/4 in Foci Formation of NMY-2 in C. elegans Masashi Fujita, Shuichi Onami RIKEN Quantitative Biology Center, Kobe, Japan Cortical actomyosin network often forms dense foci in animal embryos. It is unknown what mechanism underlies these non-uniform distributions. In vitro studies have reported that reconstituted actomyosin network can form aggregates without help of additional biochemical regulation. However, it is unclear whether the same mechanism governs foci formation in embryos. During the polarity establishment phase of one-cell C. elegans embryos, nonmuscle myosin NMY-2 forms many foci at the cell cortex, and its contractility is regulated by small GTPase RHO-1, RhoGAP RGA-3/4, and RhoGEF ECT-2. Here we propose a hypothesis that de novo formation of NMY-2 foci is controlled by RhoGAP-mediated lateral inhibition. We observed that mCherry::RGA-3 has foci-like distribution at the cell cortex and colocalize with NMY-2::GFP during the polarity establishment phase. Based on this observation, we constructed a mathematical model, in which Rho diffuse laterally on the membrane surface. Foci were modeled to have RhoGAP activity, which would make Rho predominantly the GDP-bound inactive form in the neighborhood of pre-existing foci. Computer simulation of this model successfully generated alternate patterns of active Rho and RhoGAP. We will perform knockdown experiments and compare the results with theoretical predictions.
Contact: [email protected] Lab: Onami
Poster Topic: Polarity 223 Isolation, Identification, and Characterization of Free-Living Nematodes Lauren Leister, Alan Massouh, Alexis Plaga, Ramon Carreno, Danielle Hamill Ohio Wesleyan University Our lab is interested in cell division, polarity establishment, and other early developmental processes. Nematodes, which include the rhabditids, are among the most widespread phyla of animals, and Caenorhabditis elegans is the best studied of the rhabditids. We seek to understand the similarities and differences between C. elegans and other rhabditids. In this study, we traveled to southern Florida to collect rhabditid nematodes for further characterization. Free-living rhabditids are often found in the soil, and many of these are known to spend part of their life cycle in association with millipedes; for this reason, both soil samples and millipedes were collected. We were able to culture 27 isolates using reagents and techniques commonly applied to C. elegans. We are using a combination of phenotypic and molecular techniques to characterize and identify these worms. We have photographed and measured the worms, taken time-lapse videos of their embryos, and used immunofluorescence microscopy to visualize sub-cellular components including microtubules and DNA. We have observed differences in the overall size of the worms, in tail morphologies, in the gonads, and in embryonic development. Furthermore we have isolated genomic DNA from each strain, amplified the 18S rRNA gene using PCR, and sequenced the products. These sequences were compared to each other and to sequences from public databases. We believe that the worms we collected represent five species, three of which are from the genus Oscheius, and some of which may represent previously undescribed species. Additional phenotypic and DNA sequence analysis will be needed to confirm this. With respect to early embryonic development, the rhabditids we isolated share some similarities with C. elegans, but there are intriguing differences as well. There are double-nuclei in the blastomeres of one group of worms. We have also noticed differences in polarity, represented by variability in the meeting point of the sperm and egg pronuclei, the relative sizes of the cells at the two-cell stage, and the timing and orientation of cell division at the two-cell stage. Essential elements of early polarity in C. elegans are not observed in some of these rhabditid nematodes. We believe that comparative studies like this will not only help us better understand an important phylum of animals, but they will also help us to understand cell division and developmental patterns more generally.
Contact: [email protected] Lab: Hamill
224 Poster Topic: Polarity A Dominant Mutation in a C. elegans Splicing Factor Results in Reversed AP Polarity in the Early Embryo Reza Keikhaee, Bruce Nash, John Yochem, Bruce Bowerman Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA Cell polarity is a fundamental property of most cells and is critical to generate cell diversity during development. While many of the molecules required for anterior-posterior (AP) cell polarity are conserved across animals, the mechanisms that establish it remain unclear. We have identified a semi-dominant, temperature-sensitive (ts), embryonic-lethal allele of the C. elegans ortholog of human SF3a66 called or430ts. SF3a66 is one of three subunits composing SF3a (splicing factor 3a) that is involved in the processing of pre-mRNA, but recent studies have shown that SF3a66 may also act as a microtubule binding and bundling protein independent of its RNA splicing function. About one half of the embryos produced by or430ts/ or430ts worms exhibit a remarkable reversal of AP cell polarity at the one-cell stage when raised at the restrictive temperature. Using a combination of visible marker mapping and whole genome sequencing, we found that or430ts maps to LG IV near the C. elegans ortholog of SF3a66 (which we have named repo-1 for reversed polarity), and we identified a mis-sense mutation in this gene. Reducing repo-1 function with RNAi results in significant reduction in the penetrance of polarity reversal; hence we concluded that the repo-1 mis-sense mutation is responsible for this phenotype. In addition to the polarity reversal, we also have observed several microtubule related defects in or430ts one-cell zygotes, consistent with a possible role for a REPO-1 microtubule binding activity. As a partial reversal of polarity has previously been observed in mutants that arrest in Meiosis I, we examined meiotic spindle assembly but did not observe defects in spindle structure or cell cycle progression. Nevertheless, we have been able to partially eliminate the or430ts polarity reversal by reducing the function of unc- 116 and lin5 using RNAi, which was shown previously to result in a failure of the oocyte meotic spindle to move to the cortex. We then hypothesized that a reduction in PKC-3 activity, due to splicing defects in or430ts mutants,might make these mutant embryos sensitive to polarity reversal by a normal oocyte meiotic spindle. Consistent with this hypothesis, we have found that reducing the gene dosage of pkc-3 in or430ts mutant results in an increased frequency of polarity reversal. We conclude that one important role for anteriorly enriched PKC-3 is to prevent oocyte meiotic spindle microtubules from establishing a reversed posterior pole.
Contact: [email protected] Lab: Bowerman
Poster Topic: Polarity 225 Identifying Mechanisms of Contact-Mediated Cell Polarization Diana Klompstra, Dorian Anderson, Jeremy Nance Skirball Institute, NYU School of Medicine , New York, NY During gastrulation, cells move to a position in the embryo that is appropriate for the type of tissue or organ that they will form. The directional movements of gastrulation are facilitated by a polarity that early embryonic cells acquire that allows them to asymmetrically localize cytoskeletal components. The polarity of early embryonic cells is determined by cell-cell contacts, which restrict the PAR polarity proteins PAR-3, PAR-6, and PKC-3 to contact-free surfaces. The goal of my project is to determine how cell contacts induce the PAR protein asymmetries that polarize early embryonic cells, preparing them for gastrulation. We previously identified the RhoGAP protein PAC-1 as an upstream regulator that is required to exclude PAR proteins from contacted surfaces of early embryonic cells. PAC-1 itself is recruited specifically to sites of cell contact, and directs PAR protein asymmetries by inhibiting the Rho GTPase CDC-42. How PAC-1is able to sense where contacts are located and localize specifically to these sites is unknown. We identified an N-terminal fragment of PAC-1 that is sufficient for localization to cell contacts, and showed that localization of this fragment depends on HMR-1/E-cadherin. We show that HMR-1recruitment to cell contacts depends on the presence of HMR-1 in the adjacent cell, suggesting that HMR-1 homotypic interactions recruit or stabilize the protein at contact sites. Finally, we show catenins that interact with the HMR-1 cytoplasmic tail function redundantly to recruit the PAC-1 N-terminal domain. In contrast to the PAC-1 N-terminus, full-length PAC-1 can localize to cell contacts when HMR-1/E-cadherin is removed, indicating that a redundant signal functions with HMR- 1/E-cadherin to recruit PAC-1 to contacts. These findings provide insights into how a polarity regulator is spatially segregated to a subdomain of the cortex to polarize cells.
Contact: [email protected] Lab: Nance
226 Poster Topic: Polarity ER Compartmentalisation and the Regulation of Polarity in the C. elegans Embryos Zuo Yen Lee1, Monica Gotta2, Yves Barral1 1Institut fur Biochemie, ETH Zurich, Zurich, Switzerland, 2Centre Medical Universitaire, University of Geneva, Geneva, Switzerland Diffusion barriers are used to compartmentalise different domains in polarised and specialised cells, such as budding yeast, primary cilium, spermatozoa and neurons. They are important in maintaining the structure and the functions of the cells. In budding yeast, lateral diffusion barriers have been identified at the bud neck in the plasma, the nuclear envelope and the endoplasmic reticulum (ER) membranes. As an example, the lateral diffusion barrier in the nuclear envelope promotes asymmetric distribution of aging factors by retaining them in the mother cells while creating young buds. The C. elegans embryos are highly polarised entities, particularly in their first division where polarity markers and fate determinants are asymmetrically distributed. During the establishment of polarity, the cytoplasmic proteins MEX-5 and PIE-1 are enriched exclusively in the anterior and the posterior of the embryo, respectively. The mechanisms of MEX-5 maintenance in the anterior domain are not well-understood. In wild type embryos, it was found that the ER was reorganised and redistributed asymmetrically after the first division with anterior enrichment. Additionally, the dynamics of MEX-5 was found to be decreased in the anterior domain. Therefore, we hypothesise that MEX-5 could be retained by an organelle in the cell and our interest is to investigate if the ER barrier is conserved in the C. elegans embryo, the molecular nature of such barrier and subsequently how it may contribute to the regulation of polarity in the embryos. By using photobleaching technique, the data showed restricted diffusion of the membrane protein SP12::GFP between the anterior and posterior ER domains. In contrast, the luminal protein KDEL::GFP diffused rapidly. The results showed that the ER in the C. elegans embryo is compartmentalised and potentially contribute as a mechanism to the polarity maintenance in the embryo.
Contact: [email protected] Lab: Barral
Poster Topic: Polarity 227 A Cullin-5-RING Ubiquitin Ligase Regulates Asymmetric Cell Division in Early C.elegans Embryos Anne Pacquelet, Emeline Daniel, Gregoire Michaux Institut de Genetique et de Developpement de Rennes, Rennes, France The early C. elegans embryo undergoes several rounds of asymmetric divisions in the P lineage, which are essential to generate different embryonic cell types, including germline cells. These asymmetric divisions depend on the establishment of a polarity axis - defined by the asymmetric localization of the PAR proteins – prior to mitosis. They also require proper spindle positioning along this polarity axis as well as the asymmetric inheritance of cell fate determinants during mitosis. We are interested in understanding the role of protein degradation and in particular of Cullin-RING ubiquitin ligases (CRLs) in these processes. CRLs are multisubunit complexes containing a cullin family protein, a RING domain containing protein, RBX-1 or RBX-2, as well as a substrate recognition module. We are currently investigating the role of Cullin-5-RING ubiquitin ligase complexes (CRL5) whose functions are so far poorly understood. We found that cul-5 mutants strongly enhance the embryonic lethality due to partial par-2 and par-4 loss of function. Notably, par-2;cul-5 embryos divide similarly to par-2 embryos at the one-cell stage but show enhanced defects in P1 spindle orientation at the two-cell stage, suggesting that CUL-5 may have a specific role in regulating polarity in P1 cells. Surprisingly, while par-4 embryos divide asymmetrically, cul-5par-4 mutants divide symmetrically at the one- cell stage, giving rise to two equally sized cells. However, these embryos do not have strong defects in PAR protein polarity. Ongoing experiments will help us understanding whether the symmetric division observed in cul-5par-4 embryos is due to improper mitotic spindle and/or cytokinesis furrow positioning. Importantly, we found that loss of the RING domain containing protein RBX-2 gives rise to similar phenotypes as loss of CUL-5. Moreover, we are currently investigating which substrate specific adaptor is working together with CUL-5 in the regulation of asymmetric divisions. Altogether, our results indicate that a Cullin-5-RING ubiquitin ligase contributes to the asymmetric divisions of the P lineage, by regulating both the size asymmetry of the first embryonic division and the orientation of the mitotic spindle during P1 division. These results thereby uncover new mechanisms involved in regulating asymmetric divisions as well as so far unknown functions of Cullin-5-RING ubiquitin ligases.
Contact: [email protected] Lab: Michaux
228 Poster Topic: Polarity Evolution of GPR Regulation in the Control of Spindle Positioning for Two Cænorhabditis Species Embryos Soizic Riche1, Francoise Argoul2, Melissa Zouak1, Alain Arneodo2, Jacques Pecreaux3, Marie Delattre1 1Laboratory of Molecular Biology of the Cell, Lyon, France, 2Physic Laboratory, Lyon France, 3Institute of Genetics and Developmental biology of Rennes, Rennes, France Asymmetric cell division is a fundamental mechanism relying on proper mitotic spindle positioning. In C. elegans one-cell embryos, it gives rise to two daughter cells of unequal size and fate. After fertilization and pronuclei meeting at the posterior side of the cell, the nucleus- centrosome complex (NCC complex) migrates back to the cell center. This centrally located spindle is then displaced toward the posterior pole during anaphase. While it is displaced, the spindle undergoes transverse oscillations that are more pronounced for the posterior than the anterior pole. These movements are known to be controlled by pulling forces acting on astral microtubules and the number and the molecular nature of motors have been characterized: a complex made of Gα proteins, linked to GPR (a GoLoco containing protein), LIN-5 (the Numa homolog) and Dynein is thought to be anchored at the cortex and activated at the onset of mitosis to pull on the spindle during anaphase. Our comparative analysis between C. elegans and C. briggsae embryos shows that a same division is achieved through different movements of pronuclei and spindle. We found that the pronuclei migrate further towards the anterior cell side in a Gα/GPR/LIN-5 dependent manner. Furthermore, anaphase spindle oscillations are delayed, lower in amplitude, and shorter in duration in C. briggsae. Through a combination of microtubule laser destruction, mutant analysis and mathematical modelling, we revealed the existence of a conserved positional switch for oscillations superimposed on the time control for spindle positioning. This switch is linked to the localisation of GPR crescent at the posterior cell side, which is conserved between species. However, we uncovered a differential localisation of GPR at the anterior cortex of embryos between species, suggesting evolutionary changes in GPR regulation. Importantly, GPR proteins share only 67% of similarity between C. elegans and C. briggsae. To gain insights into GPR regulation, we performed gene replacement experiment of gpr between species. Our preliminary data suggest that the differential localisation of GPR could be explained by differences in the protein sequences. Experiments are underway to narrow down the important residues responsible for conserved and divergent GPR functions.
Contact: [email protected] Lab: Delattre
Poster Topic: Polarity 229 Coupling Centrosome Position And Cortical Polarity Sabina Sanegre, Carrie Cowan Institute of Molecular Pathology, Vienna, Austria In one-cell C. elegans embryos, centrosomes play a key role in polarity establishment. Coincident with the initiation of polarity, paternally contributed centrioles begin to recruit pericentriolar material (PCM). Delays in PCM assembly delay polarization, and depletion of core PCM components, such as the structural protein SPD-5, prevent polarization. The molecular mechanism by which centrosomes control polarity establishment, however, is still unknown. The centrosomal kinase AIR-1 has been shown to be required for correct polarization of one-cell embryos. We find that AIR-1 has two functions in polarity establishment: it inhibits spontaneous polarization, and it coordinates the site of polarity establishment with the position of the centrosomes. In embryos depleted of AIR-1, spontaneous polarization generated a functional anterior-posterior polarity axis, including proper segregation of cortical and cytoplasmic fate determinants, suggesting that AIR-1 may not be required for polarity itself but rather for positioning the polarity axis in response to centrosome position. Despite AIR-1’s established role in centrosome maturation, AIR-1 depletion had no effect on the initial recruitment of PCM, further supporting that AIR-1 acts downstream of PCM assembly to control polarity. AIR-1’s centrosomal localization appears to be mediated by direct interaction with SPD-5. Thus SPD-5- dependent recruitment of AIR-1 to centrosomes integrates the temporal and spatial information provided by PCM assembly with downstream signals that establish polarity.
Contact: [email protected] Lab: Cowan
230 Poster Topic: Polarity GLD-3(S) Contributes to PIE-1 Asymmetry in Zygotes Jarrett Smith, Geraldine Seydoux Johns Hopkins School of Medicine PIE-1is a germ cell fate determinant that is asymmetrically segregated to the posterior of the zygote before the first division. In a screen for temperature-sensitive lethal mutations, Yingsong Hao identified three alleles ofgld-3 that delayPIE-1 asymmetry in the zygote (Hao, Y.,2005 - Ph. D. Thesis, Johns Hopkins U.). gld-3 codes for two isoforms GLD-3S and GLD- 3L with different carboxy-termini (Eckmann et al.,2004). GLD-3S and GLD-3L share five KH domains and a region that interacts with the poly-A polymerase GLD-2. GLD-3L also contains an FBF-binding site (Eckmannet al., 2004). We sequenced the allele ax202 and found thatthis mutation maps to the carboxy-terminus of GLD-3S. gld-3S(RNAi),but not gld-3L(RNAi), lead to embryonic lethality. Yeast two-hybrid experiments showed that PIE-1 binds preferentially to GLD-3S, and that this interaction is attenuated by ax202. These results suggest that GLD-3S could play a direct role in promoting PIE-1 asymmetry in zygotes.
Contact: [email protected] Lab: Seydoux
Poster Topic: Polarity 231 Phosphorylation State of a Tob/BTG Protein, FOG-3, Regulates Initiation and Maintenance of the Caenorhabditis elegans Sperm Fate Program Myon-Hee Lee1,2, Kyung Won Kim2, Clinton Morgan2, Dyan Morgan2, Judith Kimble2 1Brody School of Medicine at East Carolina University, Greenville, NC, USA, 2University of Wisconsin-Madison, Madison, WI, USA FOG-3, the single Caenorhabditis elegans Tob/BTG protein, directs germ cells to adopt the sperm fate at the expense of oogenesis. Importantly, FOG-3 activity must be maintained for the continued production of sperm that is typical of the male sex. Vertebrate Tob protein shave antiproliferative activity and ERK phosphorylation of Tob proteins has been proposed to abrogate “antiproliferative” activity. Here we investigate FOG-3 phosphorylation and its effect on sperm fate specification. We found both phosphorylated and unphosphorylated forms of FOG-3 in nematodes. We then interrogated the role of FOG-3 phosphorylation in sperm fate specification. Specifically, we assayed FOG-3 transgenes for rescue of a fog-3 null mutant. Wild-type FOG-3 rescued both initiation and maintenance of sperm fate specification. A FOG- 3 mutant with its four consensus ERK phosphorylation sites substituted to alanines, called FOG-3(4A), rescued partially: sperm were made transiently but not continuously in both sexes. A different FOG-3 mutant with its sites substituted to glutamates, called FOG-3(4E), had no rescuing activity on its own, but together with FOG-3(4A) rescue was complete. Thus, when FOG-3(4A) and FOG-3(4E) were both introduced into the same animals, sperm fate specification was not only initiated but also maintained, resulting incontinuous spermatogenesis in males. Our findings suggest that unphosphorylated FOG-3 initiates the sperm fate program and that phosphorylated FOG-3 maintains that program for continued sperm production typical of males. We discuss implications of our results for Tob/BTG proteins in vertebrates.
Contact: [email protected] Lab: Kimble
232 Poster Topic: Sex Determination Molecular Analyses of FOG-1 and FOG-3, Terminal Regulators of the Sperm/Oocyte Cell Fate Decision Daniel Noble1, Scott Aoki2, Marco Ortiz Sanchez3,1, Kyung Won Kim1, Judith Kimble1,2 1University of Wisconsin-Madison, Madison, WI, USA, 2Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, WI, USA, 3Universidad Nacional Autonoma de Mexico, Cuernavaca, Mexico Signaling from somatic cells regulates germline sex determination in all animals tested (worms, flies, vertebrates), but the germ cell response is best understood in nematodes. C. elegans uses a divergent hedgehog signaling pathway to control sexual differentiation in both somatic and germ cells (1). Within germ cells, the fog-1 and fog-3 genes are essential for sperm fate specification in both sexes and are terminal regulators of the sperm/oocyte fate decision (2). Both FOG-1 and FOG-3 proteins are implicated in mRNA regulation: FOG-1 is an RNA-binding protein of the cytoplasmic polyadenylation element binding (CPEB) class, while FOG-3 is a putative Tob/BTG protein (2),which functions in vertebrates as an adapter within an RNA regulatory complex. We previously reported the generation of a functional epitope- tagged fog-3::FLAG transgene (3). We have now also generated a functional epitope-tagged FLAG::fog-1 transgene. Both fog-3::FLAG and FLAG::fog-1 are maintained as rescuing transgenes in strains that lack the corresponding endogenous gene. Our current work focuses on identification of interacting proteins as well as associated mRNAs for both FOG proteins. Our preliminary results suggest that FOG-1 and FOG-3 may interact with each other and control an overlapping set of mRNAs.
(1) Zarkower, D. (2006) Somatic sex determination, WormBook; (2) Ellis, R. and Schedl, T. (2007) Sex determination in the germ line, WormBook; (3) Lee, M.-H. et al (2011) PNAS 108, 9125-9130.
Contact: [email protected] Lab: Kimble
Poster Topic: Sex Determination 233 RNA-Seq Analysis of Germline Sex Reprogramming Elena Sorokin1, Judith Kimble1,2 1University of Wisconsin-Madison, Madison, WI, USA, 2Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, WI, USA The molecular basis of germ cell fate specification as sperm or oocyte remains poorly understood. Whereas cell fates in somatic tissues are typically regulated transcriptionally, only post-transcriptional regulators have emerged as key for sperm/oocyte specification (e.g. FOG-1, FOG-3: see abstract by Noble et al). We hypothesized that, if transcriptional factors are terminal regulators of the sperm/oocyte decision, they must not be not tractable genetically, either due to redundancy or pleiotropy. We therefore took a completely different approach, which takes advantage of our recent discovery that U0126, a MEK kinase inhibitor, transforms a puf-8; lip-1 masculinized germline to produce functional oocytes instead of sperm (Morgan et al., 2010). More recently we have learned that MAPK activity is lowered within 15 minutes of U0126 treatment, and reprogramming occurs within hours of drug treatment (C. Morgan and J. Kimble, unpublished). Here we sequenced mRNAs isolated from whole animals during an 18-hr time course of chemical treatment. Data were obtained from U0126-treated or DMSO vehicle-treated animals of three distinct genotypes: puf-8; lip-1, which begin spermatogenic but reprogram their germlines to oogenesis after U0126 treatment; N2, which are oogenic with and without drug; and puf-8; fbf-1, which are spermatogenic with and without drug. We are analyzing our data for changes in mRNA abundance and changes in mRNA isoforms. Our preliminary results reveal a strong drug response in all three strains, but few changes specific to reprogramming. We are now testing the few genes changed for effect on germline sex determination in sensitized mutant backgrounds. Our progress will be reported at the meeting. Morgan, C.T., Lee, M.-H., Kimble, J., 2010. Chemical reprogramming of Caenorhabditis elegans germ cell fate. Nat Chem Biol 6, 102-4.
Contact: [email protected] Lab: Kimble
234 Poster Topic: Sex Determination AUTHOR INDEX
A Bayer, Emily...... 122 Beard, Sarah M...... 84 Abbott, Allison L...... 50 Bedet, Cecile...... 139 Abraham, Nessy...... 5 Beilharz, Traude H...... 146 Addise, Abate Birhan...... 215 Belafi-Bako, Katalin...... 215 Ahn, Samuel...... 197 Belsky, Jason A...... 48 Ahringer, Julie...... 42 Bembenek, Joshua N...... 24, 176 Akintobi, Adenrele M...... 127 Benian, Guy M...... 55 Alaimo, Jennifer...... 104 Berg, Jannette...... 144 Alam, Emad...... 114 Berkseth, Matthew...... 34 Alexa, Anita...... 215 Bernadskaya, Yelena Y...... 3, 83 Alexander, Mariam...... 11 Bertho, Sylvain...... 150 Al-Hashimi, Hikmat...... 185 Bhalla, Needhi...... 147, 170 Allen, Anna K...... 32 Bhambhani, Chandan...... 123 Allman, Erik...... 50 Bienkowska, Dominika...... 150 Alvaro, Christopher...... 122 Blanchoud, Simon...... 221 Amin, Nirav M...... 111 Boag, Peter R...... 146 Anderson, Courtney...... 10 Bobian, Michael R...... 85 Anderson, Dorian...... 226 Bock, Carly...... 53 Antoshechkin, Igor...... 48, 134 Bohr, Tisha...... 147 Aoki, Scott...... 233 Bojanala, Nagagireesh...... 186 Appleford, Peter J...... 212 Bonner, Mary Kate...... 86 Argoul, Francoise...... 229 Bosanac, Anna...... 199 Arneodo, Alain...... 229 Bowerman, Bruce...... 54, 75, 166, 225 Asahina, Masako...... 186 Bowman, Elizabeth...... 161 Asencio, Claudio...... 25 Brabin, Charles...... 112 Aubry, Agnes...... 194 Braeckman, Bart P...... 9 Audhya, Anjon...... 23 Braunreiter, Kara...... 67 B Breving, Kimberly...... 126 Brockway, Heather...... 177 Baer, G. Michael...... 122 Broitman-Maduro, Gina...... 41, 115 Bageshwar, Suparna...... 61 Budirahardja, Yemima...... 211 Baldwin, Austin T...... 105 Buechner, Matthew...... 185, 195 Banfalvi, Zsofia...... 215 Buhler, Alessandra...... 56 Bao, Zhirong...... 44 Burge, Stephanie A...... 71 Barral, Yves...... 227 Burger, Julien...... 75, 76 Barrett, Alec...... 51 Burns, Ramzy...... 122 Barsi-Rhyne, Ben...... 52 Butterfield, Yaron...... 177 Baugh, L. Ryan...... 48, 134 Byrd, Dana...... 154
235 C Cortes Estrada, Daniel B...... 87 Cabello, Juan...... 108 Cottee, Pauline A...... 149 Cabunoc, Abigail...... 45 Courtois, Emmanuelle...... 76 Cadigan, Ken...... 123 Couteau, Florence...... 139 Carreno, Ramon A...... 224 Cowan, Carrie R...... 135, 150, 230 Carter, Caitlyn...... 65 Cowart, M. Leigh...... 82 Castells-Roca, Laia...... 81 Cox-Paulson, Elisabeth A...... 189 Cha, Dong Seok...... 113 Cram, Erin J...... 26, 70, 151 Chakravorty, Adityarup...... 12 Crane, Matthew M...... 217 Chan, Benjamin G...... 187 Crocker, Kassi...... 207 Chan, Raymond C...... 24, 163 Crook, Matt...... 93 Chandler, Chelsey N...... 38 Crossley, Merlin...... 136 Chang, Chieh...... 47 Csanadi, Zsofia...... 215 Chang, Yu-Tai...... 69 Csankovszki, Gyorgyi...... 24, 37, 176 Chaouni, Rita...... 148 Custer, Laura M...... 37 Chatterjee, Indrani...... 15 D Chavez, Daniela...... 155 Chen, Grace...... 74 Dalfo, Diana...... 168 Chen, Xin...... 188 Daniel, Emeline...... 228 Chen, Yun...... 193 Datla, Udaya Sree...... 113 Chisholm, Andrew D...... 190 Davidson, Iain F...... 25 Chong, Conrad...... 153 De Henau, Sasha...... 9 Chou, Han Ting...... 213 de la Cruz, Norie...... 45 Chou, Han-ting...... 128 De Orbeta, Jessica...... 20 Christensen, Sara...... 54 De Stasio, Elizabeth...... 64 Chu, Diana...... 152, 154, 169, 214 Degema, Karen...... 18 Chuang, Chiou-Fen...... 47 Dejima, Katsufumi...... 190 Cinkornpumin, Jessica K...... 130 Del Rosario, John S...... 94 Ciosk, Rafal...... 29, 75 Delattre, Marie...... 19, 229 Clemons, Amy...... 177 Denning, Dan...... 95 Clever, Sheila...... 116, 122 Dennis, James W...... 158 Coetzee, Donna...... 179 Der, Channing J...... 49 Colaiacovo, Monica...... 27, 177 Dewilde, Sylvia...... 9 Collette, Karishma...... 176 Deyter, Gary M...... 20 Connolly, Amy...... 54, 166 Dillingham, Zechariah...... 173 Constas, Katharine...... 114 Dineen, Aidan...... 191 Contreras, Vince...... 160 Doan, Thang...... 211 Coppola, John...... 140 Dong, Xintong...... 192 Core, Leighton J...... 134 Dordal, Rachel...... 116 Corrionero, Anna...... 124 Driscoll, Kaitlin...... 96 Corsi, Ann K...... 125 Druzhinina, Marina...... 15
236 Du, Zhuo...... 44 Fraser, Andrew G...... 158 Duchesneau, Christopher D...... 55 Friday, Andrew J...... 160 Duong, Adrian...... 45 Frohli, Erika...... 56 Frommolt, Peter...... 81 E Fujita, Masashi...... 223 Eimer, Stefan...... 23 Furuta, Tokiko...... 20 Eiteneuer, Annika...... 22 Elewa, Ahmed...... 106 G Ellefson, Marina L...... 58 Gabrhel, Casey...... 67 Ellis, E. Ann...... 218 Gaidatzis, Dimos...... 29 Engebrecht, JoAnne...... 165 Gally, Christelle...... 194 Ermolaeva, Maria...... 81 Garcia, L. Rene...... 188 Escobar Restrepo, Juan M...... 56, 110 Garcia, Rebecca E...... 214 Escobar, Juan MI...... 196 Gaudet, Jeb...... 78, 191 Espiritu, Eugenel B...... 222 Gautier, Megan K...... 82 Esquela-Kerscher, Aurora...... 126 Gavin, Amanda...... 116 Estrada, Rodrigo...... 152 George, Carolyn...... 111 Ezcurra, Begona...... 108 Germani, Francesca...... 9 Ezzio, Catherine P...... 116 Ghai, Vikas...... 107 Ghosh, Srimoyee...... 59 F Gilbert, Jennifer M...... 154 Fall, Gabe T...... 164 Gill, Hasreet...... 72 Farhadifar, Reza...... 19 Gleason, Elizabeth J...... 156 Farooqui, Sarfarazhussain...... 196 Gleason, Ryan J...... 127 Feddersen, Charlotte...... 12 Glotzer, Michael...... 88 Feldman, Jessica L...... 2 Gnazzo, Megan M...... 89 Fenker, Kristin...... 153 Gobel, Verena...... 5 Fernandez, Anita G...... 53 Golden, Andy...... 32, 90 Fields, Brandon...... 57 Goldstein, Bob...... 88 Fischer, Greg...... 67 Gomes, Jose-Eduardo...... 77 Fitch, David...... 197 Gomez, Raymarie...... 128 Fleming, John...... 5 Gomez-Orte, Eva...... 108 Fletcher, Evan...... 116 Gonczy, Pierre...... 221 Flynn, Jonathan R...... 58 Gorjanacz, Matyas...... 25 Fodor, Andras...... 215 Gorman, Kevin...... 182 Ford, Jason R...... 20 Gotta, Monica...... 22, 76, 109, 227 Formstecher, Etienne...... 77 Govindan, J. Amaranath...... 162 Foster, Olivia...... 51 Grant, Barth...... 127 Fotopoulos, Nellie...... 193 Grants, Jennifer M...... 129 Francis, Joshua W...... 38 Greenberg, M. Banks...... 82 Frand, Alison R...... 6 Greenstein, David...... 13, 162, 179
237 Greenstein, David...... 180 Holtackers, Rene...... 22 Greiss, Sebastian...... 81 Honda, Yu...... 21 Grimm, Julie...... 60 Hong, Ray L...... 130 Grussendorf, Kelly A...... 195 Hoppe, Pamela E...... 55, 62, 65 Guang, Shouhong...... 39 Horvitz, Bob...... 10, 95, 96, 97, 124 Guerrero, Francisco...... 152 Hubbard, E. Jane Albert.... 30, 43, 168 Gumienny, Tina L...... 61, 80, 218 Huelgas Morales, Gabriela...... 157 Gutierrez, Peter...... 56 Hughes, Samantha...... 112 Guven-Ozkan, Tugba...... 118 Hunter, Jerrod...... 68 Hurwitz, Michael...... 10, 101 H Ha, Dae Gon...... 12 I Haag, Andrea...... 56, 110 Ikegami, Kohta...... 34 Hajnal, Alex...... 11, 56, 110, 196, 202 Imlay, Leah...... 12 Hale, Jared J...... 111 Immerman, Lois...... 50 Hall, David...... 5, 180 Ishidate, Takao...... 106 Hall, Jenny...... 116 Hamiche, Karim...... 198 J Hamill, Danielle R...... 224 Jacobs, Rene L...... 142 Han, Sung Min...... 149 Jacobson, Lewis...... 57 Hanna-Rose, Wendy...... 93 Jaramillo-Lambert, Aimee...... 90 Hansen, Angela...... 153 Jenes, Barnabas...... 215 Hansen, Jody M...... 155 Jenna, Sarah...... 198 Hardin, Jeff...... 206 Ji, Jiaojiao...... 39 Harel, Sharon...... 198 Ji, Ni...... 131 Harris, Todd W...... 45 Jindra, Marek...... 186 Haruta, Nami...... 21 Jo, Jeanyoung...... 126 Haynes, Kelly...... 91 Johnson, Casonya M.... 128, 137, 143, Hegermann, Jan...... 23 213 Heiman, Maxwell G...... 209 Johnston, Wendy L...... 158 Henderson, Melissa A...... 156, 160 Jones, Steven...... 177 Hengartner, Micheal O...... 98 Jow, Margaret...... 152 Hermann, Greg...... 51 Jud, Molly...... 153 Herrera, R Antonio...... 197 Herrmann, Alyssa...... 205 K Herrmann, Christina...... 56 Kadekar, Pratik...... 159 Hersh, Brad...... 96 Kang, Alan SR...... 190 Hirose, Takashi...... 97 Kang, Lijun...... 15 Hobert, Oliver...... 138 Kant, Sashi...... 136 Hoffman, Corey...... 189 Kassim, Maher...... 45 Hollis, Sarah E...... 113, 201 Katic, Iskra...... 216
238 Katz, David J...... 38 L Keikhaee, Reza...... 225 Labbe, Jean-Claude...... 88 Keiper, Brett D...... 99, 160 Labella, Sara...... 160 Keller, Martin...... 98 Labouesse, Michel...... 194 Kelly, Bill...... 161 Laboy, Jocelyn T...... 63 Kemp, Benedict J...... 50 Lai, Allison...... 53 Kemper, Kevin...... 104, 117 Lam, Karmen...... 199 Kemphues, Ken...... 1 Lamelza, Piero...... 147 Kerr, Shana C...... 38 Lancaster, Brett...... 132 Kershner, Aaron...... 28, 175 Landes, Ethan...... 64 Khan, Liakot...... 5 Lane, Latrisha S...... 65 Killeen, Marie T...... 199 Langouet, Maeva...... 56 Kim, Ahlee...... 5 Lascarez-Lagunas, Laura I...... 102 Kim, Kyung Won...... 232, 233 Law, Fiona...... 66 Kim, Seongseop...... 162 Lawrence, Katherine S...... 165 Kimble, Judith.28, 113, 174, 175, 232, Lee, Myon-Hee...... 113, 201, 232 233, 234 Lee, Zuo Yen...... 227 Kintzele, Jason...... 62 Leger, Thibaud...... 76 Kiontke, Karin...... 197 Leister, Lauren W...... 224 Kirkconnell, Killeen S...... 163 L’Hernault, Steven W...... 156 Kivlehan, Emily...... 205 Li, Ying...... 127 Klompstra, Diana...... 226 Lieb, Jason...... 34 Kniss, Sarah...... 12 Lilly, Michael...... 122 Korswagen, Hendrik...... 131 Lin, Rueyling...... 40, 46, 118 Korta, Dorota Z...... 30 Lis, John T...... 134 Kovacevic, Ismar...... 26, 70 Liszewski, Walter...... 12 Kradolfer, David...... 56 Liu, Dennis...... 114 Kramer, Brendan...... 167 Liu, Jun...... 107, 111, 114 Krause, Michael...... 219 Liu, Oliver...... 192 Kress, Elsa...... 22 Llamosas, Estelle...... 136 Krizus, Aldis...... 158 Lo, Te-Wen...... 35 Kroetz, Mary B...... 200 Long, Ying...... 156 Kroft, Tim L...... 164 Longhini, Katrina M...... 88 Kruesi, William S...... 134 Low, Lloyd...... 146 Kubba, Saad...... 114 Lowry, Josh...... 54, 166 Kugler, Hillel...... 43 Lu, Hang...... 217 Kuhn, Jonathan A...... 69 Lun, Aaron...... 136 Kurhanewicz, Nicole...... 48, 134 Lyman Gingerich, Jamie...... 67 Kurshan, Peri...... 217 M Madric, Kenya...... 126
239 Maduro, Morris...... 41, 115 Moerkamp, Asja...... 75 Magistrado, Leila...... 41 Mohler, William A...... 3 Magnuson, Lindsey...... 164 Mohnen, Megan...... 50 Maier, Wolfgang...... 216 Monahan, Kimberly B...... 49 Maine, Eleanor...... 178 Moore, Julia L...... 44 Mains, Paul E...... 77, 84, 187 Morf, Matthias K...... 11 Mandt, Rebecca...... 12 Morf, Matthias...... 196, 202 Manjarrez, Jacob...... 68 Morgan, Clinton T...... 232 Mano, Itzhak...... 94 Morgan, Dyan E...... 232 Mao, Hui...... 39 Morrison, J. Kaitlin...... 99 Martin, Emmanuel...... 198 Moss, Eric G...... 104, 117 Mason, D. Adam...... 205 Mueller, Louisa...... 202 Massouh, Alan R...... 224 Mullen, Greg...... 68, 133 Mathe-Fodor, Andrea...... 215 Muller, Michael...... 81 Mathews, Ellie...... 68, 133 Murphy, Shaun P...... 69 Mattaj, Iain W...... 25 Murray, John I...... 72 Mattingly, Brendan C...... 195 Mushi, Juliet...... 12 Maxwell, Colin...... 48, 134 Mayers, Jonathan...... 23 N McClung, George...... 116 Naar, Anders M...... 142 McGhee, James...... 132, 144 Nabhan, Ahmad...... 169 McNally, Francis J...... 58, 87 Naef, Felix...... 221 Medina, Jessica...... 46 Nance, Jeremy...... 203, 226 Meli, Vijaykumar S...... 6 Narbonne, Patrick...... 159 Mello, Craig...... 36, 106 Narlikar, Geeta...... 169 Meraldi, Patrick...... 22 Nash, Bruce...... 225 Merlet, Jorge...... 75, 76 Navarro Gonzalez, Rosa E...... 157 Messina, Kari...... 14 Navarro, Rosa E...... 102 Mets, Sarah...... 91 Navidzadeh, Nathan...... 159 Meyer, Barbara J...... 35, 134 Neault, Mathieu...... 198 Meyer, Hemmo...... 22 Needleman, Daniel...... 19 Michael, Matthew...... 167 Nehrke, Keith...... 50 Michaelson, David...... 168 Nelson, Christian R...... 170 Michaux, Gregoire...... 228 Nesmith, Jessica...... 32 Middelkoop, Teije...... 131 Ngo, Minh...... 69 Mikl, Martin...... 135 Nguyen, Jillian...... 3 Miller, Kristine...... 52 Nicholas, Hannah R...... 136 Miller, Michael...... 12, 149 Niebergall, Lorissa J...... 142 Mills, Erica S...... 38 Nkengfac, Bernard...... 198 Mis, Emily K...... 53 Noatynska, Anna...... 76 Moens, Luc...... 9 Noble, Daniel...... 233
240 Norman, Kenneth...... 63, 79 Portman, Douglas...... 205 Nykamp, Keith...... 201 Praitis, Vida...... 12 Presler, Marc...... 14, 74 O Priess, James R...... 2 O’Connell, Kevin F...... 181 Prodon, Francois...... 22 O’Flaherty, Brendan...... 64 Prouteau, Manoel...... 109 Oldenbroek, Marieke...... 118 Onami, Shuichi...... 16, 223 Q Orozco, Jose M...... 70 Qadota, Hiroshi...... 55 Ortiz Sanchez, Marco...... 233 Quach, Thanh K...... 137 Osterberg, Valerie...... 54 Quinn, Christopher C...... 210 Otsuka, Anthony J...... 71 R P Rahe, Dylan P...... 138 Pacquelet, Anne...... 228 Rahimi, Sina...... 15 Padgett, Richard W...... 127 Rakotomalala, Cedric...... 139 Paix, Alexandre...... 171 Ramani, Arun K...... 158 Palladino, Francesca...... 139 Rand, Jim...... 68, 133 Panbianco, Costanza...... 76 Ranjan, Sinthu...... 114 Parry, Jean M...... 72 Rapoport, Veronika...... 130 Paschal, Cate R...... 170 Reddien, Peter...... 96 Patel, Anvi...... 122 Reedy, April R...... 55 Patel, Falshruti B...... 73 Refai, Osama M...... 78 Patel, Tulsi...... 138 Rehain, Kathryn...... 173 Pattabiraman, Divya...... 74 Reid, Anna...... 136 Patterson, Joseph R...... 146, 182 Reiner, David J...... 49 Pecreaux, Jacques...... 229 Rhoads, Robert E...... 160 Perlman, Benjamin...... 122 Rhos, Patrcia...... 78 Peters, Maureen A...... 50 Richaudeau, Benedicte...... 75, 76, 77 Petrella, Lisa N...... 172 Riche, Soizic...... 19, 229 Phillips, Bryan T...... 105, 121 Rimann, Ivo...... 11 Piano, Fabio...... 53 Robert, Valerie J...... 139 Piasecki, Brian...... 64 Robertson, Scott M...... 118 Pickle, Catherine S...... 35 Robertson, Scott...... 40, 46 Piekny, Alisa J...... 193, 208 Rocheleau, Christian...... 66, 120 Pintard, Lionel...... 75, 76, 77 Rocheleau, Simon K...... 187 Pioppo, Lauren...... 116 Rohrschneider, Monica R...... 203 Plaga, Alexis R...... 224 Rollins, Evvi...... 78 Podbilewicz, Benjamin...... 60 Rose, Lesilee S...... 33, 222 Pohl, Christian...... 44 Rosu, Simona...... 17 Poole, Daniel S...... 86 Rottiers, Veerle...... 142
241 Roy, Debasmita...... 30 Shakes, Diane...... 14 Roy, Peter...... 11 Shen, Kang...... 192, 217 Roy, Richard...... 148, 159 Shi, Herong...... 114 Roy, Sarah H...... 92 Shi, Xiaoqi...... 45 Shin, Heaji...... 28, 175 S Shin, Tae-Ho...... 106 Saenz-Narciso, Beatriz...... 108 Shirayama, Masaki...... 106 Saito, Mako...... 92 Shivas, Jessica...... 4 Salcini, Lisa...... 141 Shivendra, Kishore...... 98 Salem, Alex...... 195 Shorrock, Meghann...... 152 Sanegre, Sabina...... 230 Sifuentes, Margarita H...... 176 San-Miguel, Adriana...... 217 Silva-Garcia, Carlos G...... 102, 157 Santarella-Mellwig, Rachel...... 25 Simionato, Elena...... 101 Santella, Anthony...... 44 Simske, Jeff...... 12, 204 Sarasija, Shaarika...... 79 Singaravelu, Gunasekaran...... 15 Sarkeshik, Ali...... 23, 86 Singh, Nirupama...... 125 Satish, Shruthi...... 41, 115 Singson, Andrew...... 15 Sawin, Emma...... 10 Skop, Ahna...... 4, 86, 89 Schacht, Angela...... 12 Skorobogata, Olga...... 120 Schaeffer, Arielle...... 114 Smith, Harold E...... 181, 219 Schartner, Caitlin M...... 35 Smith, Jarrett...... 231 Scheckel, Claudia...... 29 Smith, Michele...... 205 Schisa, Jennifer A...... 146, 182 Smolikove, Sarit...... 177, 183 Schneider, Jennifer...... 81 Snyder, Matthew P...... 178 Schuh, Amber...... 23 Song, Anren...... 160 Schultz, Robbie D...... 80, 218 Song, Mi Hye...... 85, 91 Schumacher Tucker, Jennifer A...... 47 Sorokin, Elena P...... 234 Schumacher, Bjoern...... 145 Soto, Martha C...... 3, 73, 83 Schumacher, Bjorn...... 81 Spengler, Justin W...... 82 Schumacher, Jill M...... 20 Spike, Caroline...... 179 Schvarzstein, Mara...... 31 Stanfield, Gillian...... 153, 155 Schwager, Francoise...... 22 Starich, Todd...... 180 Schwarze, Katrin...... 23 Starr, Daniel A...... 69 Schwendeman, Andrew R...... 100 Stein, Kathryn K...... 90 Seidel, Hannah S...... 174 Stein, Lincoln D...... 45 Seiler, Jonas...... 22 Sternberg, Paul W...... 59 Sengupta, Madhu...... 146 Stetak, Attila...... 56 Session, Dane...... 163 Stock, Tyson...... 12 Seydoux, Geraldine.25, 171, 181, 231 Strome, Susan...... 172 Shaham, Shai...... 7, 100 Subash, Jacob J...... 160 Shakes, Diane C...... 173 Subasic, Deni...... 98
242 Sugimoto, Asako...... 21, 22 Vergara, Sandra...... 106 Sullivan-Wilson, Alexander...... 12 Vertin, Eric...... 91 Sumiyoshi, Eisuke...... 21 Via, Zachary...... 111 Sundaram, Meera V...... 72 Vibbert, Jack...... 149 Swoboda, Peter...... 64 Villanueva-Chimal, Angel E...... 102 Szewczyk, Nate...... 57 Villeneuve, Anne...... 17, 31, 74, 184 Szymczak, Lindsey...... 114 Vine, Annalise...... 51 Vlaeminck, Caroline...... 9 T Vora, Setu...... 121 Takayama, Jun...... 16 Voronina, Ekaterina...... 171 Tanner, Kimberly D...... 214 Tannoury, Hiba...... 151 W Taubert, Stefan...... 129 Walck-Shannon, Elise M...... 206 Tavernier, Nicolas...... 75, 76 Walker, Amy K...... 142 Terasawa, Masahiro...... 21 Walker, Rachel...... 189 Tian, Chenxi...... 107, 114 Wallace, Andre...... 3, 83 Tilleman, Lesley...... 9 Waller, Bridget...... 207 Tobin, David V...... 92 Walsh, Taylor A...... 156 Toulabi, Leila...... 111 Walston, Timothy...... 207 Towarnicky, Leah...... 14 Walstrom, Katherine M...... 82, 220 Toya, Mika...... 21 Wang, Emily...... 156 Tse, Yu Chung...... 88 Wang, Haibin...... 8 Tu, Zheng Jin...... 162 Wang, Peng...... 125 Tuck, Simon...... 30 Wang, Xiaochen...... 8 Wang, Yuemeng...... 181 U Waters, Colin T...... 134 Udin, Gilles...... 109 Watts, Jenny...... 142 Weber, Katherine P...... 122 V Wendland, Emily...... 159 Vadla, Bhaskar...... 104, 117 Werner, Michael...... 88 Valbuena, Valeria S. M...... 82 Wernike, Denise...... 208 Valfort, Aurore-Cecile...... 19 Whipple, Lauren...... 205 Vallier, Laura G...... 140 White, Ana...... 143 van Oudenaarden, Alexander...... 131 Whitehurst, Rebecca E...... 49 Vandamme, Julien...... 141 Wiesenfahrt, Tobias...... 144 Vanfleteren, Jacques R...... 9 Wightman, Bruce...... 116, 122 VanGompel, Michael White...... 33 Williams, Ash...... 167 VanHoven, Miri...... 52 Williams, Claire R...... 209 Vargas, Chris...... 52 Wilson, Luke D...... 164 Verbrugghe, Koen JC...... 24 Winter, Ethan...... 173 Wisidagama, Dona Roonalika...... 130
243 Witte, Kristen...... 23 Zhang, Yan...... 8 Wolters, Stefanie...... 145 Zhou, Shan...... 10 Wong, Chiyen...... 65 Zhou, Xufei...... 39 Wood, Megan P...... 182 Zouak, Melissa...... 229 Woollard, Alison...... 112, 212 Zuckerman, Jennifer A...... 103 Wright, Jane E...... 29 Wu, Jui-Ching...... 222
X Xiang, Shang...... 66 Xu, Fei...... 39 Xu, Shawn...... 15 Xu, Tao...... 86 Xu, Xia...... 178 Xu, Yan...... 210
Y Yang, Qiutan...... 196 Yang, Xiao-Dong...... 40 Yates III, John...... 23, 86 Yee, Callista...... 199 Yilma, Zelealem...... 12 Yin, Yizhi...... 177, 183 Yochem, John...... 54, 166, 225 Yohannes, Lensa...... 12 Yokoo, Rayka...... 184 Yoo, Bum Ho...... 201 Yucel, Duygu...... 136 Yun, Sijung...... 219
Z Zacharias, Amanda L...... 72 Zaidel Bar, Ronen...... 211 Zand, Tanya P...... 49 Zanin, Esther...... 22 Zarkower, David...... 34, 200 Zavolan, Michaela...... 98 Zawadzki, Karl A...... 184 Zetka, Monique...... 139, 160 Zhang, Dongyan...... 91 Zhang, Hongjie...... 5
244 C. elegans Development, Cell Biology, & Gene Expression Meeting Thursday, June 7 – Sunday, June 10, 2012
UNIVERSITY BUILDINGS, ACCOMMODATIONS, PARKING and POINTS OF INTEREST Conference Site Location Accommodation Location Parking Lot Location Point of Interest Location 1. Memorial Union C1 A. Lowell Hall F2 Lot 6 C1 University Bookstore D2 2 Union South & Hotel B2 B. Chadbourne Hall C2 Lot 29 C3 Walgreens D2 C. University Inn D2 Lot 46 D2 Walgreens Pharmacy D2 D. Doubletree Hotel E3 Lot 83 D2 Monona Terrace G2 E Dahlmann Campus Inn D2 Overture Center F2
A B C D E F G
W . IL R 1 1 LO D Lowell Hall W Memorial Union
OBSERVATORY P6 MLE 1 A D
.R Walgreens W. LINDEN DR. E Dahlmann TUNLAW Campus Inn State Fountain Capitol C Chadbourne University SUPMAC Bookstore University .RD Hall B Lake St. Inn YTISREVINU .EVA Parking Ramp Overture .RD 2 2 SUPMAC Center Union South Walgreens Pharmacy P83 and Hotel Walkway .EVA P46
YTISREVINU ESEERBENGINEERING .N
HIGHLAND AVE. 2 D Doubletree Camp Randall Hotel Stadium Monona Terrace LA NEL
HISE AVE. 3 RET 3 .
P29
.TS LAKE REGENT ST. MONONA A B C D E F G 2012 E N MU Floor Legend Hotel Rooms 4th Floor Floor Room in Use S W Fourth Third Second First Class of '24 MEMORIAL UNION Recept. Room Great Hall Elevator Stairs Stairs Elevator
Stairs Stairs Capitol View W Round M Beefeaters Table Stairs to studios Room Room 3rd Floor East NOTE: Accessible from East elevator/ East end of building only. Langdon Board Elevator Room Room Stairs Rosewood Room W/M O dl Madison Room Studio B 3rd Floor West NOTE: Studios are not handicapped accessible. Accessible from West stairs only. Studio A Tripp Deck Stairs to 2nd Floor 2nd floor Tripp Commons
Stairs CE Development Locations Registration Profile Room Browsing Main Lounge Art Gallery Inn WI Deck { Annex Room (2nd floor) W Stairs W Library M Elevator Oral Sessions Stairs Phone Phone w Union Theater (1st floor) To Union Theater • • Elevator Inn Wisconsin Room Poster Sessions Great Hall (4th floor) Stairs u Stairs v Reception Room (4th floor) Central Reservations z Main Lounge (2nd floor) Play Circle Stairs Main Entrance and Conference Services Theater Workshops Annex Room w Union Theater (1st floor) Conference Headquartersrs Slide Preview Room { Rosewood (3rd Floor West) Entrance 1st Floor Meal/Buffet Rooms Outside Terrace Area x Inn Wisconsin (2nd floor East) Lakefront on Langdon Phone Opening• Reception y Tripp Commons (2nd floor) Wisconsin Union To Terrace Banquet/Dance Theater M Union South-See Campus Map Rathskeller W Stairs ElevatorInformation Desk Elevator Phones (downstairs) Tyme To MU Games Room Front Stairs Box Daily Scoop Entrance W M Office Deli To Park Street Paul Bunyan Room Stairs
Park Street Entrance