PLATFORMS: Immune System and Cell Death 83

1 Immune response to tumors in Drosophila. José C. Pastor-Pareja, Ming Wu, Tian Xu. Howard Hughes Medical Institute, Dept. of Genetics, Yale University School of Medicine, New Haven, CT. We use Drosophila to study cancer biology. In our laboratory, several genome-wide screens for mutations that promote tumor progression and metastasis have been performed. Fly tumors mutant for the polarity determinant scribble that express an oncogenic form of Ras at the same time (RasV12/scrib-/-) exhibit malignant behavior similar to that observed in human metastatic cancers. Features of these tumors include accelerated growth, loss of cell adhesion, basement membrane degradation, migration and invasion, as well as secondary tumor formation. Recently, we have discovered that flies are able to mount an immune response to RasV12/scrib-/- tumors. We are investigating this response and have found so far that tumors activate hemocytes and that hemocytes, in turn, adhere to tumors and affect their growth. We are currently adressing the molecular mechanisms underlying these phenomena and will present our initial findings regarding signaling between tumors and hemocytes and how hemocytes recognize tumors. This Drosophila model provides a tool for dissecting the genetic, molecular and cellular mechanisms of tumor-immune system interactions.

2 The bacterial symbiont Wolbachia confers resistance to viruses in Drosophila melanogaster. Luís Teixeira, Álvaro Ferreira, Michael Ashburner. Department of Genetics, University of Cambridge, Cambridge, United Kingdom. Many microorganisms colonize animals and establish interactions that range from mutualism to parasitism. These symbionts frequently influence their host’s physiology. We found that in Drosophila melanogaster the presence of tetracycline-sensitive bacteria confers resistance to Drosophila C virus (DCV). This resistance is maternally transmitted and the bacteria are intracellular. Using molecular markers we identified the bacteria as Wolbachia pipientis. Wolbachia infects a wide range of invertebrates, including several Drosophila species, and may be the most abundant strain of intracellular bacteria. The lethality induced by DCV infection is reduced in Wolbachia infected flies. Resistance is the result of the viral titre being much lower in Wolbachia infected flies than in those either uninfected or cured by tetracycline treatment. We are now investigating whether or not Wolbachia protects Drosophila from other viruses, and what the mechanism of protection is. To our knowledge this is the first report of a bacterial infection inducing resistance to a viral infection.

3 SENSING OF GRAM POSITIVE BACTERIA IN DROSOPHILA IMMUNITY. Lihui Wang, Petros Ligoxygakis. Dept Biochemistry, Univ Oxford, Oxford, United Kingdom. The genetic tractability of Drosophila melangaster has been proved invaluable in our understanding of host and pathogen interaction. Genetic screenings have identified three putative receptors in the defence against Gram positive bacteria: a glucan binding protein GNBP1, and Peptidoglycan Recognition Proteins: -SA and -SD (PGRP-SA and -SD). Nevertheless, the picture of how these three molecules work in sensing Gram positive invasion is largely sketchy. Using purified recombinant GNBP1, PGRP-SA and -SD, an in- depth investigation was performed on their roles in Gram positive peptidoglycan (PG) sensing from a molecular and biochemical perspective. From this study, it was found that GNBP1 and PGRP-SA form an essential protein complex in recognition of certain species of PG. In addition, GNBP1 exhibited an endomuramidase-like activity releasing smaller PG fragments to enable their recognition by PGRP-SA. PGRP-SD, not a potent PG receptor itself, augmented the binding capacity of GNBP1 to an extended family of PG via protein protein interaction. Furthermore, a high molecular weight complex containing all three proteins could be detected in solution. More importantly, addition of a highly purified PG fragment induced the occurrence not only of the ternary complex but also of dimeric formations among the three proteins. Taken together, these data indicate a synergistic mechanism of GNBP1, PGRP-SA and PGRP-SD in PG sensing to efficiently recognise a broad spectrum of Gram positive bacteria. 84 PLATFORMS: Immune System and Cell Death

4 The Infection-Induced Proteolysis of The Receptor PGRP-LC Activates the IMD Pathway and Melanization Cascades in Drosophila. Amy Tang1,2, Rebecca Schmidt2, Francesca Rinaldo1, Shayla Hesse1, Theodore Trejo1, Zachary Ortiz1, Masakazu Hamada2, Timothy Plummer1, Andrea Page-McCaw3, Jeffery Platt1, Amy Tang1,2. 1) Department of Surgery, Mayo Clinic College of Medicine, Rochester, MN 55902; 2) Department of Biochemistry and Molecular Biology, Mayo Clinic Cancer Center, Mayo Clinic College of Medicine, Rochester, MN 55902; 3) Department of Biology and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180. The Drosophila immune deficiency (IMD) pathway, homologous to the mammalian tumor necrosis factor (TNFa) signaling pathway, initiates antimicrobial peptide (AMP) production in response to infection by Gram-negative bacteria. A membrane-spanning peptidoglycan recognition protein, PGRP-LC, functions as a receptor for the IMD pathway. This receptor is known to be activated via pattern recognition and binding of monomeric peptidoglycan (DAP-type PGN) through the PGRP ectodomain. Here we report that treatment of Drosophila with proteases activates the IMD pathway and protease-dependent IMD activation requires the receptor PGRP-LC. PGRP-LC expression is down-regulated upon Gram-negative bacterial infection but is not affected by chemically-fixed bacteria or protease-deficient E.coli in S2 cells and in vivo. An ectodomain (PGRP)-deleted PGRP-LC receptor is functional and constitutively activates both AMP production and melanization. Our results suggest a model in which pathogen invasion and tissue damage may be monitored through the structural integrity of sentinel receptors such as PGRP-LC after host and pathogen are engaged by pattern recognition. We propose that the irreversible cleavage or down-regulation of innate immunity receptors/ligands may provide an additional cue for host recognition of pathogenic microbes and activation of multiple innate defense systems in Drosophila, thereby effectively enhancing its combat of bacterial infection and initiating tissue repair.

5 Developmentally-regulated cell death of Drosophila salivary glands utilizes ER stress-linked apoptosis. Robert Farkas1, Lucia Mentelova1,2, Peter Low3, Gabor Juhasz3, Miklos Sass3. 1) Institute of Experimental Endocrinology, Slovak Academy of Sciences, 83306 Bratislava, Slovakia; 2) Department of Genetics, Comenius University, Bratislava, Slovakia; 3) Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest, Hungary. Developmentally-associated programmed cell death (PCD) is a genetically encoded form of cell suicide that is required to remove superfluous or potentially dangerous cells. The Drosophila salivary glands (SGs) are highly specialized secretory organs, that serve as an useful model to study PCD as they respond to the steroid hormone ecdysone to undergo programmed histolysis during metamorphosis. Here we present data demonstrating that this tissue dies by an apoptosis-prone mechanism in which endoplasmic reticulum (ER) becomes fragmented into vesicles. Although a low level of autophagy can be seen in the SGs, inducing autophagy with rapamycin does not cause nor accelerates the cell death process. Although the genetic removal of Atg1 function or the RNAi silencing of other Atg genes inhibits the development of autophagy, it does not prevent the vesiculation and subsequent death of the SGs. The Drosophila SG cells exhibit externalization of phosphatidylserine and attract phagocytic macrophages, both pivotal hallmarks of apoptosis. We also show that SG apoptosis is linked to ER disintegration via an ER-stress mechanism. Genetic manipulation of ER-resident proteins (Ca-P60, Crc, Cnx99A,), chaperones and co-chaperones (Cct5, Hop, hsc3, P58IPK) resulted in a widespread and fast vesiculation of the ER, typical of that seen during the final apoptotic stage. In contrast, the genetic removal of the SERCA pump prevented tissue apoptosis. Thapsigargin, an inhibitor of endoplasmic reticular Ca2+-ATPase SERCA causing depletion of ER calcium stores, induces very effective vesiculation and death of SG cells almost instantly. The results presented here describe for the first time the importance of cytosolic chaperones in cell death-associated ER stress. Moreover, activation of Xbp1 implicates the involvement of unfolded protein response (UPR) signaling.

6 Wts function in autophagic cell death of salivary glands. Sudeshna Dutta1,2, Eric Baehrecke1. 1) Cancer Biology, University of Massachusetts Medical School, Worcester, MA; 2) Molecular & Cell Biology Program, University of Maryland, College Park, MD 20742. Proper coordination between cell growth, cell proliferation and cell death is crucial to maintain homeostasis in multicellular organisms. Disruption of these processes can cause disorders including cancer. Previous studies have shown that the Hpo signaling pathway regulates both cell proliferation and apoptotic cell death. We are investigating autophagic cell death of salivary glands, and hope to identify novel mechanisms that distinguish this form of cell death from apoptosis.A high-throughput proteomic screen resulted in the identification of many proteins that are expressed in dying salivary glands including Wts, a serine threonine protein kinase in the Hpo signaling pathway. Either RNAi knockdown of Hpo or loss-of-function mutations in wts, prevent degradation of salivary glands. Surprisingly, wts mutant salivary glands possess markers of both caspase activity and autophagy, which are hallmarks of cell death. The only cellular defect observed in these wts mutant salivary gland cells was their failure in growth arrest. These mutant cells have defects in class I PI3K signaling including altered phosphatidylinositol products and phospho-Akt localization. Although previous studies have shown that Hpo signaling pathway influences apoptosis and cell cycle, our study provides a novel mechanistic link between cell growth and the regulation of autophagic cell death during development. PLATFORMS: Immune System and Cell Death 85

7 Physiological apoptosis in the Drosophila ovarian polar cell lineage involves Hid-mediated activation of a Diap1/Dronc/ Drice cascade. Anne-Marie Pret, Asma Khammari, François Agnès, Pierre Gandille, Elisabeth Boissonneau. Centre de Genetique Moleculaire UPR2167, CNRS/Pierre and Marie Curie University Paris VI, Gif-sur-Yvette, France. Organ morphogenesis involves the strict control of cell number that in some cases has been shown to occur via elimination of excess cells by apoptosis. The underlying mechanisms allowing the precise counting of cells are not well understood. During Drosophila ovarian follicle formation, we have shown that selection of pairs of polar cells, specialized follicular cells present at each follicle extremity, occurs via apoptotic elimination among a small group of approximately 6 precursors produced during early oogenesis. The reduction in polar cell number plays a physiological role during oogenesis since prolonged survival of excess polar cells (via expression of the anti-apoptotic p35 protein) disturbs the differentiation pattern of adjacent follicle cells, including border cells which are necessary for formation of the micropyle, the sperm entry point into the oocyte. We have characterized the cell death machinery responsible for apoptosis of excess early polar cells. We show that it involves the functions of the pro-apoptotic regulator, Hid, the initiator caspase Dronc and its adaptor Dark/Apaf-1 and the effector caspase Drice. In addition, survival of the two mature polar cells requires the function of Diap1. Since we found expression of hid specifically in polar cells destined to die, we are presently testing whether general signaling pathways (Notch, EGFR, Jak-Stat) are responsible for regulation of hid. Finally, we are performing an RNAi-based screen using the GAL4/UAS system to identify regulators of the cell death program in polar cells.

8 Different caspases control distinct mechanisms of apoptosis-induced compensatory proliferation. Yun Fan, Andreas Bergmann. Department of Biochemistry & Molecular Biology, UT MD Anderson Cancer Center, Houston, TX. In multi-cellular organisms, apoptotic cells are capable of inducing compensatory proliferation of neighboring cells to maintain tissue homeostasis. In the Drosophila wing imaginal disc, it has been shown that dying cells trigger compensatory proliferation through secretion of the mitogens Decapentaplegic (Dpp) and Wingless (Wg). This process is under control of the initiator caspase Dronc, but not effector caspases. Here, by taking advantage of the developing eye disc, we show that distinct mechanisms control apoptosis-induced compensatory proliferation depending on the proliferating vs. differentiating status of the tissue. In response to apoptotic activity, Dpp and Wg signaling is preferentially induced in proliferating eye tissue similar to wing discs. However, in apoptotic differentiating eye tissue, effector caspases DrICE and Dcp-1 are required redundantly to trigger the activation of Hedgehog (Hh) signaling for compensatory proliferation. Interestingly, effector caspases in photoreceptor neurons stimulate Hh signaling which triggers cell cycle re-entry of cells that had previously exited the cell cycle. In summary, dependent on the developmental potential of the affected tissue, different caspases trigger distinct forms of compensatory proliferation in an apparent non-apoptotic function. 86 PLATFORMS: Neurophysiology and Behavior

9 Molecular mechanisms of odor receptor expression and function. Anandasankar Ray1, Wynand van der Goes van Naters2, John R Carlson2. 1) Department of Entomology, University of California, Riverside, CA; 2) MCDB, Yale University, New Haven, CT. Olfactory receptor neurons (ORNs) must select, from a large repertoire, which odor receptors to express. This process produces a highly stereotyped receptor-to-neuron map, thereby posing a remarkably complex problem of receptor gene regulation. We have used phylogenetic analysis amongst multiple Drosophila species to identify positive and negative regulatory elements. Mutational analysis shows that this formidable problem is solved via three classes of mechanisms: by elements that specify the expression of Or genes in the correct olfactory organ, by positive elements that activate Or genes in a subset of ORN classes within an organ, and by negative elements that restrict expression to only one ORN class. This highly precise regulatory program is conserved across species, and the odor response spectra of the ORNs have been remarkably well-conserved for several million years. The odor response spectrum of individual neurons depends upon individual Or genes that are expressed in them. Interestingly, receptors that respond to similar odors can share low sequence identity, which makes it a challenge to investigate mechanisms of receptor-odor interactions. Using a powerful bioinformatic method, we have identified two classes of signature amino acid motifs in the odor receptors; one that is enriched in receptors responding to the same odor, and another that is shared by a large number of receptors. Functional analysis of a signature motif that is present in most receptors reveals a role in signal transduction. On the other hand, the odor-specific class of signatures enables us to predict odor responses of receptors not only from Drosophila but a variety of other insects whose genomes have been sequenced.

10 The Drosophila sex peptide receptor mediates the post-mating switch in female reproductive behaviour. Nilay Yapici, Young- Joon Kim, Carlos Ribeiro, Barry J. Dickson. Molecular Biology and Genetics, Institute of Molecular Pathology, Vienna,Austria. Mating in many species induces a dramatic switch in female reproductive behaviour and physiology. In most insects, this switch is triggered by factors present in the male’s seminal fluid. How these factors exert such profound effects in females is unknown. Here, we identify the receptor for the Drosophila melanogaster sex peptide (SP), the primary trigger of the post-mating response in this species. The sex peptide receptor (SPR) is a G-protein coupled receptor that is specifically activated by low nanomolar concentrations of SP. It is expressed in the female’s reproductive tract, and in the brain and ventral nerve cord of both sexes. Females that lack SPR function, either entirely or only in the nervous system, fail to respond to SP. Such females continue to show virgin behaviours even after mating. SPR is highly conserved structurally and functionally across the insect order, opening up the prospect of novel strategies to control the reproductive and host-seeking behaviours of important agricultural pests and human disease vectors.

11 A novel tetraspanin required for synaptic endocytosis. Chi-Kuang Yao1,3, Yong Qi Lin1,3, Tomoko Ohyama1, Cindy Ly2, Patrik Verstreken1, Karen L. Schulze1,3, Hugo J. Bellen1,2,3. 1) Dept Molecular & Human Gen, Baylor Col Medicine, Houston, TX; 2) Department of Neuroscience, Baylor Col Medicine, Houston, TX; 3) Howard Hughes Medical Institute, Baylor Col Medicine, Houston, TX. Completion of neurotransmission relies on repeated synaptic vesicle release and retrieval. To identify new players in synaptic transmission, we performed an unbiased forward genetic screen using the eyFLP system. We identified a mutation that affects the morphology of the NMJs and causes a sprouty phenotype. We therefore named the gene flower. More importantly, we observe an obvious defect in endocytosis in these mutants as they exhibit a reduction in FM1-43 dye uptake; a rundown of synaptic transmission at 10Hz but not at 1 Hz stimulation; a severe depletion of vesicle number based on electron microscopy, and an significant increase in endocytic intermediates. flower is required in neurons and the protein localizes to vesicles and periactive zones. The protein contains four transmembrane domains, has not been characterized in any species, and is evolutionarily conserved from worms to man. In addition, most of the conserved amino acids map to the transmembrane (TM) domains, including key negatively charged amino acids in the middle of the TMs. The primary structure of the protein suggests that it may be a channel or transporter. However, the phenotype observed in flower mutants shows striking similarities with synapses that have been treated with La3+, a Ca2+ channel blocker (Kuromi et al., 2004). Based on preliminary data we speculate that flower encodes a novel Ca2+ channel. PLATFORMS: Neurophysiology and Behavior 87

12 Genetic Analysis of AMP-Activated Protein Kinase. Jay Brenman1, Nevzat Kazgan1, Paul Medina1, Vincent Mirouse2, Daniel St Johnston2, Linsay Williamson3, Erik Johnson3. 1) UNC Chapel Hill School of medicine, Chapel Hill, NC USA; 2) University of Cambridge, UK; 3) Wake Forest University, NC USA. AMP-activated protein kinase (AMPK) activity is constituted by a serine-threonine kinase catalytic subunit (α) and two regulatory subunits (β and γ). AMPK is proposed to broadly function as a cellular energy sensor inhibiting energy consuming activities while activating energy producing ones. Outside of a handful of predominantly metabolic enzymes, in vivo downstream targets of AMPK are largely uncertain. Interestingly, however, AMPKα is most homologous to kinases with known roles in cell polarity including SAD kinases and Par-1. During a forward screen searching for genes regulating larval neuronal dendrite development we identified the first know mutations in AMPKα. Animals with mutations in AMPKα contain neuronal dendrites with greatly enlarged dendritic swellings that could be rescued by autonomous expression of a wild type transgene. Mutations in AMPKγ produce nearly the same phenotype. One proposed upstream activator of AMPK activity is LKB1 (Par-4), which has a role in epithelial polarity. When we examined the role of AMPK in epithelial polarity, we found an energy-dependent requirement of AMPKα for maintenance of epithelial cell architecture. In the adult brain loss of AMPK function leads to neurodegenerative phenotypes. Surprisingly, the adult eye phenotypes of different molecules implicated in an AMPK signaling pathway suggest that the current vertebrate signaling pathway may not reflect the in vivo situation. Through structure/function analysis we identify previously unnoticed protein motifs required for AMPKα function in vivo and examine motifs required for subcellular localization. We also identify energy-sensitive behavioral phenotypes in flies expressing different AMPKα mutant transgenes.

13 Functional and Genomic Analyses of Genes Regulated by Fruitless in Adult Head and Central Nervous System Tissues. Thomas Goldman, Michelle Arbeitman. Molecular and Compuational Biology, USC, Los Angeles, CA. In Drosophila, all aspects of somatic sexual differentiation, including differences in adult physiology and behavior, are controlled by the sex determination hierarchy, an alternative pre-mRNA splicing cascade which results in the production of sex-specific transcription factors encoded by fruitless (fru). fru specifies the potential for all aspects of the male courtship ritual, an innate behavior. We have identified genes regulated as a consequence of male-specific FRU (FRU-M) using whole-genome microarray expression analyses. Surprisingly, by comparing genes regulated by FRU-M in all tissues of the adult head to just those in the central nervous system tissues, we found that FRU-M regulates gene expression in non-CNS system tissues, such as those of the adult head fat body. We also present molecular-genetic and behavioral analyses of one FRU-regulated gene, defective proboscis extension response (dpr), which encodes a putative cell adhesion molecule. We show that dpr is co-expressed with FRU-M in cells just below the medial region of the antennal lobe of the brain, and in the first thoracic segment of the ventral nerve cord. A strain homozygous for a hypomorphic allele of dpr, or a strain in which fru expression is reduced in the cells which express dpr, showed reduced latency in male courtship initiation and attempted copulation. Based on the expression patterns and behavioral analyses, we propose a model for how dpr functions in the FRU-M circuit to regulate courtship behaviors.

14 Dynamic range compression by feedback inhibition in the olfactory system. Cory M. Root1, Kaoru Masuyama1, Lina Enell2, Dick R. Nässel2, Jing W. Wang1. 1) Neurobiology Section, Div. of Biological Sciences, University of California, San Diego, La Jolla, CA; 2) Department of Zoology, Stockholm University, Svante Arrhenius vag 14S-106 91 STOCKHOLM, Sweden. The dynamic range of environmental cues is much larger than that of olfactory sensory neurons; therefore, a mechanism to alter sensitivity is required. We have investigated signal compression in a simple neural circuit, the Drosophila olfactory system. Olfactory receptor neurons (ORNs) of the same type converge onto a single glomerulus in the antennal lobe where they synapse onto uniglomerular projection neurons (PNs) and multiglomerular interneurons (INs). All olfactory information is delivered to higher brain centers by PNs and ORNs are the main drivers of PN output. Based on the results of this study, we propose that feedback inhibition is a mechanism to expand the olfactory dynamic range in Drosophila.

In mammalian cortex it is well established that activation of the G protein coupled receptor, GABAB, in presynaptic nerve terminals inhibits voltage gated calcium channels via the βγ complex to suppress neurotransmitter release. Here we report that Drosophila

ORNs express the GABAB receptor that mediates feedback inhibition of ORN presynaptic terminals. Using two-photon imaging of calcium and synaptic transmission, we find that GABAB signalling affects presynaptic calcium and synaptic transmission. Using a promoter fusion transgenic line, as well as RT-PCR and immunohistochemistry, we find that ORNs express the GABAB receptor.

Furthermore, blocking GABAB receptors reduces the dynamic range of ORN synaptic transmission that is matched by similar modulation of PN response, suggesting a presynaptic mechanism to alter the dynamic range of a sensory system. Strikingly, we find that different ORN channels have different levels of GABAB expression that is correlated with physiological sensitivity to GABAB modulation. Our findings reveal that different sensory channels have different levels of presynaptic inhibition that may permit heterogeneous modulation of dynamic range for different environmental cues. 88 PLATFORMS: Neurophysiology and Behavior

15 A novel vesicular neurotransmitter transporter expressed in the mushroom bodies and central complex is required for learning and female receptivity. Elizabeth S. Brooks1, Bac T. Nguyen1, Christopher J. Tabone2, J. Steven de Belle2, David E. Krantz1. 1) Semel Institute for Neuroscience, UCLA, Los Angeles, CA; 2) Department of Biological Sciences, UNLV, Las Vegas, NV. The mushroom bodies (MBs) are a distinct central brain structure required for olfactory learning and memory in Drosophila melanogaster. To date, the neurotransmitters used for signaling by the Kenyon cell neurons intrinsic to the MBs have not been clearly identified. However, we postulate that transporters must exist to move these neurotransmitters across cellular membranes. Here we report the identification of a cDNA for a predicted gene (CG10251) that functions as a vesicular neurotransmitter transporter in a subset of Kenyon cells. The CG10251 protein is similar in structure to, but distinct from, other known vesicular transporters. mRNA expression is low during development and high in the adult head. An antiserum raised against the carboxy terminus of the protein detects expression in a few neurons in the larval ventral ganglion, robust expression in the larval MB Kenyon cells as well as a large extrinsic neuron with projections into the MB lobes. In the adult brain, we similarly find robust protein expression in the MBs in a subset of Kenyon cell fibers in the peduncle and all 5 MB lobes. In addition, projections to both the optic ganglia and central complex become visible in the adult. Biochemical fractionation reveals the CG10251 protein localizes to synaptic vesicles, consistent with its proposed role as a vesicular transporter. We have generated a mutant through imprecise excision of a P-element just upstream of CG10251. Behavioral studies indicate this mutant has reduced fertility as well as altered copulatory behavior including reduced female receptivity. Olfactory learning assays reveal a modest but significant learning deficit in these mutants. We suggest that the CG10251 protein may be responsible for the storage of neurotransmitter in the MBs and the central complex and thus critical for both learning and sexual behavior function in D. melanogaster. We propose to rename the CG10251 gene portabella.

16 Regulation of Drosophila male courtship by complex integration of sensory information. Werner Boll1, Dimitrije Krstic1,2, Markus Noll1. 1) Institute of Molecular Biology, University of Zürich, Zürich, Switzerland; 2) Ph.D. Program in Molecular Life Sciences, Zürich, Switzerland. Courtship in Drosophila melanogaster is performed as a stereotyped and robust sequence of innate behavioral steps. A male perceives the presence of a potential mate through his visual, olfactory, and gustatory senses that direct him to initiate courtship, which in turn elicits a response from the courted animal. These different senses control courtship behavior and provide to courter and courtee a plethora of information about gender, conspecificity, receptivity, and sexual fitness. Understanding how the various sensory cues are interpreted by a male encountering a potential mate appears essential to comprehend the logic of the neuronal network that directs this complex behavior. To this aim, we utilized three mutations that inactivate different sensory modalities, either separately or in combinations, without affecting the functions of the CNS. Our results do not only describe the interplay and relative importance of the different sensory modalities in the male during courtship, but also show that the neural circuitry is reconfigured in the dark to compensate for the absence of visual cues. Analysis of male-male courtship reveals that the integration of visual, gustatory, and behavioral, but not olfactory cues determines the sexual orientation of the courting male. Based on these studies, we propose a model illustrating how the brain of a naïve Drosophila male integrates the various sensory stimuli during courtship. PLATFORMS: Organogenesis 89

17 Control of stem cell self-renewal and differentiation in the adult Drosphila intestine. Allison Bardin, Carolina Perdigoto, François Schweisguth. Dept Biol, Ecole Normale Superieure, Paris, France. A major challenge in stem cell research is to understand how different strategies are utilized in different biological contexts to allow both self-renewal and proper differentiation of stem cell progeny. Drosophila melanogaster provides several models of stem cell systems and has extensively developed genetic tools with which to address mechanisms of self-renewal and differentiation. The adult Drosophila melanogaster instestine harbors stem cells (ISCs) that provide newly differentiated cells throughout the adult lifetime, likely replacing those which turnover (Micchelli and Perrimon, Nature, 2006; Ohlstein and Spradling, Nature, 2006). The ISCs have been shown to employ the Notch signaling pathway to control the differentiation of their progeny. The Notch pathway is known to play important roles in many stem and progenitor cell systems (reviewed in Wilson and Radkte, Febs Lett., 2006). We are using the ISC to address how the Notch pathway is regulated during homeostasis in the adult intestine. In particular we are trying to understand how asymmetric fate of the daughter cells is controlled and what regulators of the Notch pathway contribute to this. We will present our ongoing work to understand the maintenance of stem cell identity, self-renewal and proliferation in the adult Drosophila intestine.

18 FoxK mediates TGF-beta signalling during midgut differentiation. Sergio Casas-Tinto1,2, Melisa Gomez-Velazquez1, Begoña Granadino-Goenechea2, Pedro Fernandez-Funez1. 1) Dept Neurology, UTMB, Galveston, TX; 2) Centro de Investigaciones Biologicas, CSIC, 28040 Madrid (Spain). The genetics and molecular mechanisms of endoderm differentiation are the least known of the three germ layers. It is clear, though, that inductive signals across germ layers play a key role in the development of the endoderm. In flies, the visceral mesoderm secretes TGF-beta/Dpp along with other signaling molecules that diffuse into the underlying midgut endoderm to control tissue- specific differentiation. TGF-beta signaling induces Mad phosphorylation, while the nuclear translocation of pMad regulates the expression of the Hox protein Labial. We report here the molecular and functional characterization of Drosophila FoxK, a transcription factor of the fork head box (Fox) family that responds to TGF-beta signalling and mediates labial regulation in the embryonic midgut endoderm. Specific antibodies raised against FoxK indicate a remarkable accumulation in the developing endoderm. The analysis of newly generated FoxK mutant alleles revealed that the embryos failed to generate midgut constrictions and lacked expression of Labial, a Hox protein critical for midgut differentiation. Our studies suggest that TGF-beta signalling directly regulates FoxK in midgut endoderm through functional Smad/Mad binding-sites on its 5’ region. Interestingly, FoxK can induce ectopic Labial expression only when co-expressed with the transcription factor Dfos/AP-1. Thus, FoxK and Dfos/AP-1 cooperatively regulate labial expression in midgut endoderm. Moreover, FoxK and Dfos/AP-1 can induce Labial expression even in the absence of pMad, indicating that the main role of Mad is to activate FoxK and Dfos/AP-1 in the endoderm. Then, FoxK and Dfos/AP-1 directly regulate labial to promote differentiation of parasegment 7 of the endoderm. Thus, we describe here a novel regulator of endoderm differentiation. Also, we propose a new mechanism for TGF-beta signalling that involves the sequential activation of tissue-specific transcriptional regulators during midgut endoderm differentiation.

19 The combinatorial control of muscle identity. Jonathan Enriquez, Laurence Dubois, Virginie Daburon, Michèle Crozatier, Alain Vincent. centre de biologie du developpement, Toulouse, France. Specification of muscle identity is a multistep process : Early positional information defines competence groups, from which muscle progenitors are selected, followed by asymmetric division of progenitors into muscle founder cells (FCs). Each FC seeds the formation of an individual muscle whose morphological and functional properties have been proposed to reflect the combination of transcription factors expressed by its founder. However, it is still unclear how early patterning and muscle-specific differentiation are linked. We addressed this question, using Collier (Col) expression as both a determinant and read-out of DA3 muscle identity. Characterisation of the col upstream region driving DA3 muscle-specific expression revealed the existence of two separate phases of cis regulation, correlating with conserved binding sites for different mesodermal transcription factors. Loss-of-function and gain- of-function experiments further showed that both Nautilus and Col were required for col activation in the myoblast nuclei that fuse to form the DA3 myofiber, thereby ensuring that all express the same identity program. Overexpression of Col and Nau, but neither Col nor Nau alone, can induce the formation of two DA3 muscles. Together, our results indicate that separate sets of cis-regulatory elements are required for expression of muscle identity factors in muscle progenitors and myofiber nuclei and support the concept of combinatorial control of muscle identity (Dubois et al., Development 2007, in press). Further analysis of col and nau mutant phenotypes showed that Col is required for the fusion process while Nau was required for the correct shape, orientation and epidermal attachment of the DA3 muscle, indicating that Nau and Col regulate, at least partly, different sets of genes. Finally, the role of homeotic genes in the final size of the DA3 muscle will be discussed. 90 PLATFORMS: Organogenesis

20 A key role of Pox meso in somatic myogenesis of Drosophila. Cheng Zhang1,5, Hong Duan1,3,5, Jianming Chen1,4, Helen Sink2, Erich Frei1, Markus Noll1. 1) University of Zurich, Institute of Molecular Biology, Zurich, Zurich, Switzerland; 2) Skirball Institute of Biomolecular Medicine, New York University Medical Center, 540 First Avenue, New York, NY 10016, U.S.A; 3) Present address: Sloan-Kettering Institute, Department of Developmental Biology, 1275 York Avenue, New York, NY 10021, U.S.A; 4) Present address: Department of Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, U.S.A; 5) These authors contributed equally to this work. Making a muscle in a vertebrate is both similar and different from that in Drosophila. Similar elements are deployed differently in flies and vertebrates. Are these differences real divergence of different developmental programs or rather variations of a common program is an interesting, yet presently unanswerable question. In vertebrates, Pax3 and Pax7 are important for the proper development of myogenic progenitor cells as well as for skeletal muscle development and regeneration. In Drosophila, the Pax gene Pox meso (Poxm), which belongs to the Pax1/9 family, was the first and so far only gene whose initial expression was shown to occur specifically in the anlage of the somatic mesoderm, yet its role in somatic myogenesis remained unknown. By studying the temporal and spatial expression patterns of Poxm and its loss- and gain-of-function phenotypes, we can demonstrate that it is one of the crucial genes regulating the development of the larval body wall muscles in Drosophila. It has two distinct functions expressed during different phases of myogenesis. The early function, partially redundant with the function of lethal of scute, demarcates the ‘Poxm competence domain’, a domain of competence for ventral and lateral muscle development and for the determination of at least some adult muscle precursor cells. The late function is a muscle identity function, required for the specification of muscles DT1, VA1, VA2, and VA3. Our results led us to reinterpret the roles of l(1)sc and twi in myogenesis and to propose a solution of the ‘l(1)sc conundrum’.

21 Two distinct progenitor populations remodel the Drosophila tracheal system during metamorphosis. Molly Weaver1,2, Mark Krasnow1,2. 1) Department of Biochemistry, Stanford University School of Medicine, Stanford, CA; 2) Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA. Drosophila larvae are composed of differentiated larval cells and undifferentiated progenitor cells called imaginal cells. At metamorphosis, most larval cells die whereas imaginal cells proliferate and differentiate into pupal and adult tissues. We find that there are two distinct populations of tracheal (respiratory system) progenitors : a classical population of imaginal cells that are set aside early in development in spiracular branch nests (SB tracheoblasts), and a novel population of differentiated larval dorsal branch (DB) stalk cells that transform into multipotent progenitors (DB tracheoblasts) late in larval life, and subsequently differentiate into three cell types under control of Breathless FGFR signaling. DB stalk cells are the first differentiated Drosophila cells found to naturally reenter the cell cycle, and provide a model for facultative stem cells in mammals, differentiated cells in the lung and other organs that are thought to replenish tissues after damage or disease.

22 Dynamic Regulation of the Eye Specification Network. Claire Salzer, Justin Kumar. Dept Biol, Indiana Univ, Bloomington, IN. The eye specification network, as it is currently modeled, consists of ten nuclear proteins that are woven into a complicated regulatory labyrinth. The regulatory relationships that are described for this cascade are based a number of criteria including in vivo molecular genetic analysis, in vitro biochemical and molecular assays as well as bioinformatic analysis of promoter sequences. Much of the in vivo data is focused on the effect that the removal of a network member has on the expression of the remaining genes. This analysis has led to the assignment of positive and inhibitory arrows throughout the cascade. We have generated retinal mosaic clones of several components of the network and have reexamined the regulatory relationships that exist amongst the pathway members. In particular we have focused on the relationship between sine oculis (so), eyes absent (eya) and dachshund (dac). The current model implies that the So-Eya protein complex promotes the expression of dac. We find that this is true only ahead of the morphogenetic furrow and in the first few columns of developing ommatidia. In more posterior regions of the retina both so and eya cooperate to repress dac. The implication is that the So-Eya complex can switch between the activation and repression states. Our results also demonstrate that several other regulatory relationships in the eye are dependent upon the geographical position within the developing eye field. We suggest that the eye specification network is much more dynamic than is currently envisaged by extant models. We will present and discuss our findings as well as present a more dynamic model that incorporates geographical positions. PLATFORMS: Organogenesis 91

23 Regulation of sexually dimorphic gonad development in Drosphila melanogaster. Nicole Camara, Mark Van Doren. Department of Biology, Johns Hopkins University, Baltimore, MD. The creation of sexual dimorphism during development is required for successful reproduction of the species, and is dependent on proper sex determination. The Drosophila gene doublesex (dsx) encodes a transcription factor that is required for all aspects of male and female somatic development outside the nervous system. DSX is a member of a family of DM containing proteins which are involved in sex-determination from vertebrates to invertebrates. Although dsx is understood to be a key sex-determination gene in Drosophila, little is known about how it functions to control sexually dimoprhic development. Here we investigae the role of dsx in gonad development in order to address remaining questions about dsx function. Is it required early or late for sex specific gonad development? Is it required autonomously or non-autonomously? What are the targets of DSX? We find that embryonic gonad development begins along a male pathway as evidenced by the presence of 3 male specific cell types in dsx mutant embryos. We propose that DSX may not be required for the initial specification of these cells, but may be required for their proper maitenance or function. Analysis of dsx mutant adult phenotypes indicates that both XX and XY dsx mutants can form either a male-like or female- like germline stem cell niche. Female germ cells appear more receptive to a male-like soma, than male germ cells to a female-like soma. We also present preliminary analysis of a new gene, CG5149, that may act downstream of dsx to control sexual dimorphism. CG5149 is expressed in the female soma and is absent from the male soma after gonad formation In later stages, CG5149 is also expressed in cells which become the male germline stem cell niche. This suggests that CG5149 may have distinct roles in male and female gonad development. CG5149 adults have a severe gonad defect with reduced germline in both males and females. CG5149 encodes a highly conserved protein of unknown function, and we are investigating its role in gonad development.

24 The Regulation and Function of Cad74A in Drosophila Oogenesis. Jeremiah J Zartman, Nir Yakoby, Chris A Bristow, Stanislav Y Shvartsman. Lewis Sigler Institute and Department of Chemical Engineering Princeton University, Princeton, NJ. During Drosophila oogenesis, a two-dimensional follicular epithelium encapsulating the developing egg gives rise to an elaborate three-dimensional eggshell, which includes tubular structures called dorsal appendages that project out from the main body. Egg morphogenesis and dorsal appendage formation has been studied extensively, but few connections have been made between signaling pathways, pattern formation, and the physical implementation of the morphogenetic program in this tissue. In an effort to address the gap between pattern formation and organogenesis, we have characterized the regulation and function of the non- classical cadherin, Cad74A, which is expressed starting in mid-oogenesis in all the columnar follicle cells contacting the oocyte except for two dorsolateral patches. The dorsolateral patches correspond to the cells that form the roof of the dorsal appendages. Using an antibody to Cad74A, we show that Cad74A repression in the dorsolateral patches is mediated by high levels of the transcription factor Br, which in turn is regulated by the integration of EGFR and Dpp signaling, the two pathways that are essential for patterning of the follicle cells. We also report the functional analysis of Cad74A which shows subtle phenotypes when expression levels are perturbed. On the basis of these results, we propose a model for Cad74A regulation and function during egg development. 92 PLATFORMS: Regulation of Gene Expression

25 The zinc finger protein Zelda is a key activator of the zygotic genome in Drosophila. Hsiao-Lan Liang1, Chung-Yi Nien1, Hsiao- Yun Liu1, Mark Metzstein2, Nikolai Kirov1, Christine Rushlow1. 1) Biology, New York Univ, New York, NY; 2) Human Genetics, Univ. of Utah, Salt lake city, UT. The control of embryogenesis is initiated by maternal gene products and later transferred to the zygotic genome in a process called the maternal to zygotic transition (MZT). Such processes involve the degradation of maternal transcripts and the activation of transcripts (Newport & Kirschner, 1982). Although general activators of the early zygotic genes have been postulated for years, none have actually been identified, in any organism. A recent finding that many early zygotic genes in Drosophila share a cis- regulatory heptamer motif, CAGGTAG and related sequences, collectively referred to as TAG-team sites (ten Bosch et al., 2006; De Renzis et al., 2007) suggests that a dedicated transcription factor may interacts with these sites to activate the zygotic genome. Here we report the discovery of a zinc-finger protein, Zelda (Zld), that binds specifically to TAG-team sites in the zen regulatory region, and is capable of activating transcription in transient transfection assays. Mutants lacking zld transcripts are defective in specific aspects of the cellularization process and fail to activate transcription of a large fraction of early zygotic genes, including zld and dpp, a sample of key cellularization genes, and the sex determination genes. These results suggest that Zelda may be a key activator of the genome activation in Drosophila.

26 Application of a cis-regulatory grammar for functional characterization of transcriptional regulatory elements in the Drosophila embryo. David N. Arnosti1, Ahmet Ay2, Chichia Chiu2, Evan Dayringer2, Rupinder Sayal1, Walid Fakhouri1. 1) Dept Biochemistry & Molec Biol, Michigan State Univ, East Lansing, MI; 2) Dept of Mathematics, Michigan State Univ, East Lansing, MI. To understand the specific and general features of cis regulatory elements controlled by key patterning regulators of the early embryo, we have quantitatively analyzed and modeled the activity of synthetic and semi-synthetic transcriptional modules regulated by short-range repressors Knirps, Giant, and Kruppel in the blastoderm embryo. Confocal laser imaging allows us to quantitatively “map” the gene regulatory surfaces associated with particular arrays of regulatory sequences. We assess a database of over 600 quantitatively analyzed embryos representing two dozen permutations of enhancers, and use this information to pursue two “bottom up” mathematical models. We have developed a three-tier quantitative mathematical model that associates potential functions with subelements of the enhancer, and combine them to provide predictions of the functional output of novel transcriptional elements. We have also adapted a fractional occupancy model that considers specific features of spacing and cooperativity in predicting gene activity. Using these approaches, we have accurately predicted the effects of spacing and stoichiometry between activators and repressors, a key feature of short-range repression, and are applying them to analyze the output of endogenous regulatory sequences. Our work complements “top down” approaches, and is aimed at informing and extending the power of current models of endogenous cis-regulatory elements to develop powerful bioinformatic tools applicable to population and evolutionary studies.

27 Enhancer identification by comparative genomics relies on abundant non-conserved DNA. Brant Peterson1, Emily Hare1, Venky Iyer1, Rick Kurashima3, Eric Jang3, Michael Eisen1,2. 1) Dept Molecular & Cell Biol, Univ California, Berkeley, Berkeley, CA; 2) Lawrence Berkeley National Lab, Berkeley, CA; 3) USDA Agricultural Research Service - PBARC, Hilo, HI. The identification of regulatory sequences in animal genomes is a significant challenge for computational and experimental genomics. The comparative genomic methods that have worked so well in vertebrates - in which unusually conserved non-coding sequences are often active in functional assays - have not worked well for members of the family Drosophilidae and other invertebrates. Here we apply these methods to three species of true fruit flies (Tephritidae) with genomes four to five times larger than D. melanogaster. We show that there are many discrete non-coding sequences conserved between these Tephritid species, and that nearly all (12/ 13) of the sequences we tested have regulatory activity in transgenic D. melanogaster embryos. We propose that the success of these methods in Tephritids, after their failure in Drosophila, is due to the presence of additional rapidly evolving (presumably largely non-functional) sequence between functional elements in the larger Tephritid genomes. Intriguingly, the expression pattern of one tested gene (giant) displays substantial divergence in embryos of the Tephritid B. dorsalis as compared to D. melanogaster, and furthermore the Tephritid enhancer is sufficient to reproduce the Tephritid pattern in the D. melanogaster embryo, indicating that the primary mechanism of expression evolution in this case is change in cis. We also demonstrate that mapping these conserved elements to the D. melanogaster genome clearly and accurately predicts functional regulatory elements from Drosophila using only primary sequence data, thus marking the first unambiguous success of a purely comparative approach to enhancer finding in D. melanogaster. We hypothesize that the general failure of comparative genomic methods in invertebrates to date is largely due to a bias for sequencing small genomes, and demonstrate that the sequencing of large genomes will greatly advance the identification of regulatory sequences. PLATFORMS: Regulation of Gene Expression 93

28 Sepsid even-skipped enhancers are functionally conserved in Drosophila despite lack of sequence conservation. Emily Hare1, Brant Peterson1, Venky Iyer1, Rudolf Meier2, Michael Eisen1,3. 1) Dept Molecular & Cell Biol, Univ California, Berkeley, Berkeley, CA; 2) Department of Biological Sciences, National University of Singapore, Singapore; 3) Genomics Division, Ernest Orlando Lawrence Berkeley National Laboratory, Berkeley, CA. Although changes in developmental gene expression play an important role in the evolution of organismal form and function, the molecular basis for such changes is poorly understood. To characterize the evolutionary dynamics of regulatory sequences and constraints on their function, we have sequenced and characterized the regulatory activity of the even-skipped locus from five fly species in the acalyptrate family Sepsidae. We visualized the expression patterns of key regulators of anterior-posterior patterning in the sepsid Themira minor and found them to be qualitatively similar to their Drosophila counterparts, suggesting that this regulatory network is conserved between the families. As in Drosophila, there is considerable non-coding sequence similarity between even the most distantly-related sepsid species. There is, however, very little non-coding sequence similarity between sepsids and drosophilids. We used within- and between-family comparisons, as well as binding site content, to identify cis-regulatory regions in the sepsid eve loci and characterized their activities in transgenic D. melanogaster embryos. The RNA expression patterns driven by these enhancers largely overlap the endogenous D. melanogaster eve pattern with several interesting differences. That this functional conservation occurs despite the nearly complete absence of primary sequence or transcription factor binding site conservation demonstrates that the constraints on enhancer organization are fairly loose. Strikingly, each eve enhancer contains a small number of highly-conserved elements that overlap known functional sites in D. melanogaster. These highly-conserved elements are highly enriched for pairs of binding sites that overlap or neighbor each other, suggesting that functional constraint acts on a larger unit than the single binding site.

29 Cell fate specification in the blastoderm embryo involves developmental regulation of transcription elongation. Peter Gergen1, Lisa Prazak1, Xiaoling Wang1, Kevin Celestrin1, Hyowon Choi2, Giorgio Medranda1. 1) Department of Biochemistry & Cell Biology, Stony Brook University, Stony Brook, NY; 2) Mount Holyoke University, South Hadley, MA. The simple combinatorial rules responsible for the metameric expression of sloppy-paired (slp1) provide several advantages for investigating the mechanisms of transcriptional regulation during cell fate specification in the Drosophla embryo. Recent studies show that the initial repression of slp1 in response to the pair-rule transcription factors occurs at a step downstream of PolII recruitment and transcripton inititation and involves the developmental regulation of transcription elongation. Biochemical experiments indicate that the negative elongation factor NELF participates in this process. Results of genetic experiments that investigate the in vivo function of NELF will be presented. We have also undertaken studies to identify the cis-regulatory determinants that contribute to the establishment and regulated elongation of a paused PolII complex on the slp1 promoter. These studies use PhiC31 integrase mediated site-specific integration of different reporter genes into the same chromosomal location in order to maximize the ability to reliably detect subtle and quantitative differences in gene expression. The results of experiments that investigate the functional interactions between two pair-rule responsive upstream enhancer elements and the slp1 promoter will be reported.

30 A comprehensive catalog of homeodomain DNA-binding specificities from D. melanogaster. Michael Brodsky1, Marcus Noyes1, Ryan Christensen2, Atsuya Wakabayashi1, Gary Stormo2, Scot Wolfe1. 1) Program in Gene Function & Expression, Univ Massachusetts Medical School, Worcester, MA; 2) Department of Genetics, Washington University, School of Medicine, St. Louis, MO. Homeodomain-containing proteins represent the second-largest family of transcription factors in animal genomes. A full description of homeodomains binding specificities is critical to understand how they regulate the expression of distinct target gene sets. We have characterized the DNA binding specificities of all 84 independent homeodomains from Drosophila, the first comprehensive characterization of homeodomain specificities from a metazoan genome. To visualize the range of DNA-binding specificities within this set, we performed a hierarchical clustering of transcription factors based on the similarity of their DNA recognition motifs. The majority of Drosophila homeodomains can be organized into 11 specificity groups. Some groups contain a large number of members (25 homeodomains are part of the “En group”), whereas others contain only 2 to 5 members. An additional 5 homeodomains have unique specificities that are not associated with any group. Thus, while many homeodomains recognize related DNA sequences, the homeodomain architecture can support a wide range of different DNA binding specificities. Correlation of DNA-binding specificities with residues at the protein-DNA interface, along with previous structural and biochemical analysis of homeodomains, has allowed us to construct a recognition code describing mechanisms of sequence discrimination by this family. We have confirmed the predictive power of this code with mutagenesis experiments in which one or a few amino acid changes predictably alter the DNA binding specificity of a homeodomain, including one example in which the preferred binding site is altered at four of six positions. By combining this recognition code and analysis of overall sequence similarities, we can use the Drosophila data to predict the specificities of the majority of human homeodomain proteins. These studies provide a framework to broadly predict the specificity of homeodomains from all metazoans. 94 PLATFORMS: Regulation of Gene Expression

31 Regulation of muscle identity by homeodomain transcription factors. Brian Busser1, Aditi Singhania1, Savina Jaeger2, Anton Aboukhalil2, Michael Berger2, Caitlin Gamble1, Stephen Gisselbrecht2, Martha Bulyk2, Alan Michelson1. 1) LDSB, NHLBI/NIH, Bethesda, MD; 2) Division of Genetics, Brigham & Women’s Hospital, Boston, MA. Homeodomain (HD) transcription factors (TFs) have been proposed to control the unique gene expression programs of individual muscle founder cells (FCs). We have investigated this hypothesis with an integrated genomics approach that combines genome- wide expression profiling, TF binding site determination, in silico evaluation of combinatorial TF codes, empirical testing of candidate enhancers, and both cis and trans tests of target gene regulation. We first showed by in situ hybridization that a small set of previously characterized FC genes is differentially responsive to over-expression of the muscle HD TFs Slouch, Muscle segment homeobox and Apterous. FC gene expression was activated and/or repressed by these TFs and responsiveness correlated with TF co-expression in wild-type embryos. Next, we extended the identification of HD-responsive genes on a genome-wide scale by expression profiling purified mesodermal cells from embryos in which an individual HD TF is over-expressed. These experiments revealed that different FC genes can be activated, repressed or remain unaffected by ectopic HD TFs. These effects are directly mediated at FC enhancers, as mutagenesis of HD binding sites inactivates known FC cis-regulatory modules (CRMs). Computational searches for combinations of TF motifs that are overrepresented in the noncoding regions of HD-responsive FC genes defined a HD-containing set of TFs. Predicted CRMs conforming to this TF code were functional FC enhancers. Unexpectedly, HD TFs also activated genes expressed in fusion competent myoblasts, which do not normally express these TFs. These results suggest that HD TFs regulate two distinct temporal waves of myogenic gene expression, one in the developing muscle FC, and a second in the mature multinucleated myotube. These studies provide new insights into the role of individual HD TFs in specifying cellular identity and into the transcriptional codes that regulate muscle gene expression.

32 Sequential developmentally programmed steps at target promoters reverse repression by Polycomb for terminal differentiation in a stem cell lineage. Xin Chen, Jose Morillo, Chenggang Lu, Margaret Fuller. Dev Biol, Stanford Univ, Stanford, CA. Emerging evidence indicates that repression by the Polycomb group (PcG) machinery may maintain terminal differentiation genes in a silenced state in precursor cells in embryonic and adult stem cell lineages. A major question remains how this epigenetically silenced state, capable of being maintained through many cell generations, is reversed to allow expression of differentiation genes appropriate to distinct cell types and developmental stages. Here we show that a series of events, orchestrated by the developmental program, reverses PcG repression to allow expression of terminal differentiation genes in the Drosophila male germ line adult stem cell lineage. In precursor cells, PRC2 components are expressed and terminal differentiation genes are transcriptionally silent, lack RNA PolII at the promoters and carry the H3K27me3 histone modification made by the E(z) enzyme. After the transition from proliferating spermatogonia to differentiating spermatocyte, the PRC2 components E(z) and Su(z)12 are downregulated and the testis TAF homologs (tTAFs) are expressed. However, action of tMAC, a testis specific version of the MIP/DREAM complex, is required for the recruitment of tTAFs and displacement of Polycomb (Pc) from the promoters of target differentiation genes. The tMAC is also required for relocalization of PRC1 components and tTAFs to the nucleolus. In addition, the PolII appears to arrive at the promoters of differentiation genes upon the switch from spermatogonia to spermatocyte, and is present even in tMAC or tTAF mutants, in which the targets are not expressed, suggesting that the PolII may be stalled at the promoter by Pc. These observations suggest terminal differentiation genes are turned on through a series of cell type and stage specific developmentally regulated events, in which repression by PcG set up in precursor cells initially blocks progression of RNA polymerase. After the cell fate switch to spermatocyte state, subsequent actions of cell-type specific transcriptional machinery then transform the chromatin landscape to allow terminal differentiation.

33 dHCF is required for Myc-dependent transcription regulation in Drosophila. Michael Furrer, Mirjam Balbi, Peter Gallant. Zoological Institute, University of Zürich, Zürich, Switzerland. dMyc is the Drosophila homolog of the vertebrate Myc onco-proteins, and it controls growth, proliferation and apoptosis during normal development. In order to exert its biological function, dMyc (in a complex with its partner protein dMax) binds to specific DNA sequences and activates or represses the expression of nearby genes. While a large number of dMyc targets have been identified, the mechanism by which dMyc controls their expression is not fully understood, although it seems to rely on the recruitment of chromatin modifiers. In order to identify transcriptional co-activators for dMyc, we have carried out an RNAi screen in S2 cells, and identified the Drosophila homolog of human host cell factor-1 (HCF-1) as an essential co-factor for dMyc. dHCF physically associates with dMyc and is required for the correct expression of dMyc targets in S2 cells. Downregulation of dHCF in vivo interferes with dMyc dependent growth and development, whereas dHCF overexpression synergizes with dMyc in the control of growth. These data suggest that dMyc controls the expression of its targets in part via recruitment of dHCF. This is in accordance with the role of dHCF as a component of several chromatin modifying complexes. dHCF physically interacts with the ATAC histone acetyltransferase complex, as well as with the trithorax-related Set1/Ash2 methyltransferase and the Sin3A containing histone deacetylase complexes. Taken together dHCF might act both as a positive and negative transcription regulator, by selectively modulating chromatin structure and, in addition, connect these chromatin-modifying activities to the transcription factor dMyc. PLATFORMS: Regulation of Gene Expression 95

34 Integration of inputs from multiple modular elements generates a gradient of transcriptional repression in response to the Dpp morphogen. Rahul Warrior1, Yao Li-Chin1, Phin Sopheap1, Rushlow Christine2, Arora Kavita1. 1) Dept Developmental & Cell Biol, Univ California, Irvine, Irvine, CA; 2) Department of Biology, New York University, New York, NY 10003. Morphogen gradients play fundamental roles in patterning and cell specification during development by eliciting differential transcriptional responses in target cells. In Drosophila, Decapentaplegic (Dpp), the BMP2/4 homolog, downregulates transcription of brinker (brk) in a concentration-dependent manner to generate an inverse graded distribution of the nuclear repressor. Both Dpp and Brk are critical for directing Dpp target gene expression in defined domains and the consequent execution of distinct developmental programs. Thus determining the mechanism by which the brk promoter interprets the Dpp activity gradient is essential for understanding both Dpp-dependent patterning, and how graded signaling activity can generate different responses through transcriptional repression. We have uncovered several key features of the brk promoter that suggest it utilizes a complex enhancer logic not represented in current models. First, we find that the regulatory region consists of multiple, dispersed, compact modules that can independently drive brk-like expression patterns. Second, we show that each module contains binding sites for the Schnurri/Mad/Medea (SMM) complex, which mediates Dpp-dependent repression, linked to regions that direct activation. Third, we find that the SMM repression complex acts through a distance-dependant mechanism that uses the canonical co-repressor C-terminal Binding Protein (CtBP). Finally, we provide evidence that the brk promoter employs a modular organization in which multiple enhancer inputs acting in parallel are integrated to generate the final pattern. We suggest that this specialized architecture underlies the ability of brk to respond to the Dpp gradient in a precise and robust fashion.

35 The Mechanism of Dscam Mutually Exclusive Splicing. Brenton Graveley1, Sara Olson1, Marco Blanchette2,5, Jung Park1, Yiannis Savva1, Gene Yeo3, Joanne Yeakley4, Donald Rio2. 1) Dept Genetics & Dev Biol, Univ Connecticut Health Ctr, Farmington, CT; 2) Department of Molecular and Cell Biology, Center for Integrative Genomics, University of California, Berkeley, CA 94720-3204 USA; 3) 4Crick-Jacobs Center for Theoretical and Computational Biology, Salk Institute, LaJolla, CA 92037 USA; 4) Illumina Inc., 9885 Towne Centre Drive, San Diego, CA 92121-1975 USA; 5) Stowers Institute for Medical Research, Kansas City, MO 64110 USA. The Down Syndrome Cell Adhesion Molecule (Dscam) gene plays essential roles in neural wiring and pathogen recognition in Drosophila. Dscam encodes 38,016 distinct isoforms via extensive alternative splicing. The 95 alternative exons in Dscam are organized into clusters that are spliced in a mutually exclusive manner. We have focused on understanding the mechanism of mutually exclusive splicing in the exon 6 cluster which contains 48 variable exons. Using comparative genomics we have discovered a complex system of competing RNA structures that act to ensure that only one variable exon is spliced to the upstream constitutive exon. Here we show that the hnRNP protein hrp36 acts specifically within, and throughout, the exon 6 cluster to prevent the inclusion of multiple exons. Moreover, hrp36 prevents SR proteins from promoting the ectopic inclusion of multiple exon 6 variants. Thus, the fidelity of mutually exclusive splicing in the exon 6 cluster is governed by an intricate combination of alternative RNA structures and a globally acting splicing repressor.

36 The role of site accessibility in microRNA target recognition. Nicola Iovino1, Michael Kertesz2, Ulrich Unnerstall1, Eran Segal2, Ulrike Gaul1, (NI,MK,ES,UG)=equal contributions. 1) Laboratory of Developmental Neurogenetics, Rockefeller University, New York, NY; 2) Weizmann Institute, Rehovot, Israel. MicroRNAs, genomically encoded small RNAs of 21-24 bases, have recently emerged as an important class of post-transcriptional regulators, but the precise mechanisms underlying their interaction with their mRNA targets are still poorly understood. Most studies so far have focused on the quality of the sequence match between microRNA and target and ignored the secondary structure of the target mRNA. We decided to systematically explore the importance of target site accessibility in microRNA-mRNA interactions. Using microRNAs expressed in S2 cells, we developed a sensitive linear reporter assay, in which neither microRNA nor target mRNA are overexpressed and the measured reductions in reporter activity are solely attributable to translational repression. We used this assay to measure over 60 microRNA-target interactions, spanning a wide range of target types and mutation designs, and found that mutations diminishing target accessibility significantly reduce microRNA-mediated translational repression, with effects comparable to those of mutations that disrupt sequence complementarity. Based on these observations, we devised a parameter- free thermodynamic model for microRNA-target interaction that computes the difference between the free energy gained by formation of the microRNA-target duplex, and the energetic cost of unpairing the target to make it accessible to the microRNA. We integrated site accessibility into a genome-wide target prediction program (PITA) and applied it to all 3’UTRs of fly, worm, mouse and human. We find that, in all four organisms, microRNAs sites show a marked preference for highly accessible regions, suggesting that genomes accommodate site accessibility by preferentially positioning targets in highly accessible regions. Thus, target accessibility proves to be a key factor in microRNA function. 96 PLATFORMS: Regulation of Gene Expression

37 Mextli, a novel eIF4E-binding protein from Drosophila. Greco Hernandez1,2, Gritta Tetweiler1,2, Mathiu Miron1,2, Paul Lasko1, Nahum Sonenberg2. 1) Dept. of Biology, McGill University; 2) Dept. of Biochemistry. McGill University. Eukaryotic translation initiation factor eIF4E is a key regulator of translation. Regulation of eIF4E is central to overall control of protein synthesis. eIF4E-binding proteins (4E-BPs) play a critical role in the regulation of eIF4E. They bind the dorsal convex surface of eIF4E to form a translationally inactive eIF4E/4E-BP complex. Upon stimulation of cells with hormones or growth factors, 4E-BPs become phosphorylated and dissociate from eIF4E, rendering eIF4E available to interact with eIF4G to form the translationally active eIF4G/eIF4E complex. Recently, the interaction of eIF4E with other proteins, including 4E-transporter (4E-T) and maskin, has been shown to regulate specific cellular or developmental processes. For these reasons, we are searching for additional eIF4E binding proteins in Drosophila. From a far-western genomic screen on a 20-22 h-old embryonic cDNA library, using 32P-labeled FLAG-HMK-eIF4E1 as a probe, we identified a novel eIF4E-binding protein with no similarity to other eIF4E-binding proteins such as eIF4G, 4E-T and the 4E-BPs. It possesses a canonical eIF4E binding site YXXXXLL at its carboxy-terminal end. When this motif is mutated to AXXXXAA, the interaction with eIF4E1 is abrogated. The novel eIF4E binding protein has a predicted molecular weight of 70.1 kDa and is encoded by three alternatively-spliced mRNAs encoded by a single gene. Interaction data between this protein and other eIF4E-related cap-binding proteins present in Drosophila, as well as other functional features of this protein will be presented.

38 Dorsal interacting protein 3 potentiates activation by Drosophila Rel-homology domain proteins. Girish Ratnaparkhi, Albert Courey. Chemistry & Biochemistry, UCLA, Los Angeles, CA. Dorsal interacting protein 3 (Dip3), identified as a Dorsal (DL) interactor in a yeast two-hybrid screen, contains both MADF and BESS domains (Bhaskar & Courey, Gene, 2002, 16:173). The BESS domain of Dip3 binds to the DL Rel homology domain, and we show that Dip3 synergizes with DL, Dorsal-like immunity factor (Dif) and Relish (Rel), three Rel family transcription factors encoded in the Drosophila genome. Less than 1% of eggs laid by homozygous viable dip31 flies show defects in dorso-ventral (D/V) patterning. However, Dip3 mutations enhance the patterning defects that result from dl haploinsufficiency, indicating a dispensable role for Dip3 in D/V patterning. Since Rel family factors play prominent roles in innate immunity, we examined the role of Dip3 in the immune response. dip31 larvae and adult flies are sensitive to immune challenge. Adult flies challenged with a mixture of gram positive (Micrococcus luteus) and gram negative (Escherichia coli) bacteria but not fungus (Beauveria brassiana) have shortened lifetimes- about half of the flies die within a month of immune challenge, while wild-type flies show little mortality during the same period. Quantitative RT-PCR was used to demonstrate that dip31 larvae show significant reduction of expression of the antimicrobial defense genes drosomycin, diptericin, defensin and cecropin-A. Chromatin immunoprecipitation experiments in S2 cells containing activated Toll and Immune deficient signaling pathways indicates the presence of Dip3 at the promoters of these genes. Binding requires the presence of Rel proteins at these promoters. On polytene chromosomes, Dip3 co-localizes in DAPI deficient regions to a subset of the bands occupied by RNA Polymerase II confirming its role in transcriptional activation. Immunolocalization of Dip3 at the chromocenter of polytene chromosomes suggests that in addition to its role in activation, Dip3 may also have a role at the centromeric region of cleavage stage chromosomes. PLATFORMS: Evolution and Quantitative Genetics 97

39 Concerted cis-regulatory evolution at multiple neuroectodermal loci. Justin Crocker, Albert Erives. Biology, Dartmouth College, Lebanon, NH. An outstanding question in evolutionary developmental biology is how the genetic mechanisms underlying developmental rates co-evolve with changes in life-history. This question is particularly relevant for Drosophilid embyrogenesis in which precise, spatio- temporal morphogen gradients pattern embryos that differ both by size (1-2x) and timing (1-2x) in different species. Evolutionary compensation of the production and decay rates of both maternal morphogens and early zygotic genes must somehow be achieved in order to progress through the equivalent embryonic stages in each species. Here, we compare the activities of multiple neurogenic ectodermal enhancers (NEEs) in Drosophila melanogaster and Drosophila virilis. Early syncytial Drosophila virilis embryos take 4 hours to complete early cell divisions, culminating in cellularization, and corresponding to a developmental rate that is twice as slow as Drosophila melanogaster. Nonetheless, the NEE-driven Drosophila virilis loci are expressed in a similar position and span along the dorsal-ventral axis of the stage 5 embryo. However, we show that the activities of virilis NEEs in transgenic melanogaster are more robust as measured by span of expression along the D/V axis compared to melanogaster NEE-driven transgenes. We trace this enhanced expression to configurations of Dorsal and Twist binding sites present in the set of NEEs and demonstrate that by mimicking the spatial configurations of virilis NEEs in melanogaster NEES, the melanogaster NEEs faithfully recapitulate the dorsal- ventral domains of expression of virilis NEEs in melanogaster embryos. We conclude that concerted cis-regulatory evolution across the NEEs has occurred in both D. virilis and D. melanogaster lineages in order to compensate through stabilizing selection evolutionary changes occurring in trans.

40 Estimating the fraction of sites under positive and negative selection via explicit population genetic hidden Markov models. Andrew Kern, David Haussler. Center For Biomolecular Science and Engineering, UC Santa Cruz, Santa Cruz, CA. Patterns of genomic variation within and between species are jointly determined by stochastic forces, such as drift and mutation, along with deterministic forces, such as selection. A long-standing question in the field of population genetics is to what extent does selection shape population genetic variation within the genome. Furthermore, the genomic targets of natural selection are scarcely known from natural populations. Fusing Hidden Markov Models (HMMs) with the classical population genetic theory of genic selection, we have developed and implemented a fully probabilistic population genetic HMM (popGenHMM), which allows for inquiry into the genomic targets of selection. In particular we use 2-state (selected and neutral states) and 3-state (neutral, positively selected, and negatively selected states) models whereby allele frequencies are emitted from each state with probabilities determined by sampling from the stationary distribution of allele frequencies as determined via diffusion approximations. Transition probabilities between states are assumed to be constant per base pair, thus allowing for a biologically reasonable reduction in the correlation between states over physical distance. Training of the models proceeds via an expectation maximization (EM) algorithm, followed by model selection using the Bayesian information criterion (BIC), Viterbi decoding, and subsequent scoring of predicted selected elements. We present results of our popGenHMM when applied to early release data from the Drosophila Population Genomics Project (http:/ /dpgp.org), consisting of roughly 7Mb of data resequenced from chromosomes 2L and X in 50 lines of Drosophila melanogaster. 3- state models are shown to fit the data significantly better than 2-state models, and large differences in estimates of selection parameters are observed between the X chromosome and 2L. Correlations to genomic annotations and global parameter estimates of the models are discussed.

41 cis-regulatory variation is typically poly-allelic in Drosophila. Jonathan Gruber, Anthony Long. Dept Ecology & Evol Biol, Univ California, Irvine, Irvine, CA. The expression of a particular suite of genes is necessary for most organismal traits, though both traits and expression levels vary heritably in populations. As a preliminary inquiry toward determining whether heritable variation in gene expression may be the general source of heritable phenotypic variation, we describe standing variation of cis-regulation among 16 strains of Drosophila melanogaster. With robust biological replication and multiple assays, we obtained precise Allelic Expression (AE) quantification from the Oligo Ligation Assay high-throughput SNP genotyping platform. We observed significant Differential Allelic Expression through at least one assay in 17/18 genes. Moreover, a novel crossing design allows us to combine AE estimates of the entire panel across assays. Of the differentially expressed genes queried with multiple and concordant assays, a majority show statistical support for three or more alleles. Our results suggest that variants affecting cis-regulation are a common feature of Drosophila genes, and that this variation is often the result of more than one segregating, cis-acting polymorphism. 98 PLATFORMS: Evolution and Quantitative Genetics

42 Evolutionary constraint and adaptation in the metabolic network of 12 Drosophila species. Anthony Greenberg, Sarah Stockwell, Andrew Clark. Dept Molec Biol & Genetics, Cornell Univ, Ithaca, NY. Taking advantage of the newly-available whole-genome sequences of 12 Drosophila species, we examined how protein function and metabolic network architecture influence rates of enzyme evolution. We found that despite high overall constraint, there were significant differences in rates of amino acid substitution among functional classes of enzymes. This heterogeneity arises because proteins involved in the metabolism of foreign compounds evolve relatively rapidly, while enzymes that act in “core” metabolism exhibit much slower rates of amino acid replacement, suggesting strong selective constraint. Network architecture also influences enzymes’ rates of amino acid replacement. In particular, enzymes that share metabolites with many other enzymes are relatively constrained, although apparently not because they are more likely to be essential. Our analyses suggest that this pattern is driven by strong constraint of enzymes acting at branch points in metabolic pathways. We conclude that metabolic network architecture and enzyme function separately affect enzyme evolution rates. We are currently extending these findings by experimentally estimating fluxes through energetic pathways in 92 wild-caught D. melanogaster lines.

43 Population transcriptomics of host shifts in the cactophilic Drosophila mojavensis. Luciano Matzkin, Therese Markow. Dept Ecology & Evolutionary Biol, Univ Arizona, Tucson, AZ. In the presence of environmental change, natural selection can shape the transcriptome. Those genotypes that are better able to modulate gene expression to maximize fitness will be favored. It is imperative to examine gene expression at the population level as it responds to an environmental shift in order to distinguish random or neutral gene expression variation from the pattern produced by natural selection. Drosophila mojavensis is a cactophilic fly endemic to the deserts of North America. This species contains four genetically isolated cactus host races each individually specializing in the necrotic tissues of different cactus species. The necrosis of each cactus species provides each of the resident D. mojavensis populations with a distinct chemical environment. Using a partial genome cDNA array we previously investigated the role of transcriptional variation in the adaptation of D. mojavensis to its hosts using one isofemale line. That dataset produced a set of candidate loci that were differentially expressed in response to host shifts, some of which have a non-neutral pattern of evolution. Using the recently sequenced and annotated genome of D. mojavensis, we recently developed a new gene expression array for all the annotated genes. This new microarray consists of 69,997 60-oligonucleotide probes representing 17,504 predicted genes. We now have employed this new array to reveal the effects of genotype, environment and their interaction on gene expression by exposing eight recently collected isofemale lines of D. mojavensis from Sonora to organpipe (native host) and agria (alternative host). In addition we quantify the amount of covariation of gene expression between genes.

44 The evolution of non-coding RNAs from insect Hox complexes. Matthew Ronshaugen. Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom. Large non-coding RNAs (ncRNAs) were identified in the Drosophila melanogaster Hox complex more than 20 years ago. Further study has ultimately led to the identification of more than 10 distinct non-coding transcription units. The majority of these ncRNAs are associated with previously described enhancers, Polycomb/Trithorax response elements that regulate Hox expression or miRNAs that attenuate Hox function. Recently, the human Hox complexes were shown to contain numerous ncRNAs, some of which similarly modulate Hox expression or function. With the exception of the Hox miRNA genes little is known about the evolution and conservation of these ncRNAs. It is not even clear if they are a general feature of Hox complexes outside of flies and humans or if any aspect of their expression or function is conserved. To begin to address this question I have used a comparative tiling microarray approach focusing on three developmentally diverse insects, the fruit fly Drosophila melanogaster, the honeybee Apis mellifera, and the flour beetle Tribolium castaneum. Tiling microarrays show that during embryonic development large ncRNAs are common to all three of the Hox complexes. I find that global patterns of intronic and intergenic transcription differ between the short germ beetle and long germ fruit fly. In situ analyses of these ncRNAs led to the surprising observation that a number of these ncRNAs overlap and are transcribed antisense to another gene. This investigation establishes that ncRNAs are common to many metazoan Hox complexes and may play a role in coordinating global Hox gene expression. It also begins to address whether variation in these ncRNAs has contributed to morphological evolution of the metazoa. PLATFORMS: Evolution and Quantitative Genetics 99

45 Structure and evolution of gene network involved in specification and function of extraembryonic membranes in insects. Yury Goltsev1, Gustavo Rezende1,2, Denise Valle2, Gregory Lanzaro3, Mike Levine1. 1) UC Berkeley; 2) Instituto Oswaldo Cruz, Brazil; 3) UC Davis. In many insects the activity of early D/V patterning gene network serves two important goals. 1-It provides keys for specification of various tissues in embryo proper. 2-It outlines and subdivides the territory dedicated for extraembryonic membranes (EM), which assist morphogenesis and play protective role during development. Using An. gambiae (two EMs - amnion and serosa) and D. melanogaster (one EM - amnioserosa) we study the evolution of D/V network as well as downstream effector genes partaking in EM function. In course of our studies in mosquito we have found significant rearrangement of DV network as it is known from fly embryo. Thus for example the patterns of dorsal genes such as of tup and doc clearly indicate specification of two dorsal lineages in mosquito as opposed to one in fly. Moreover reduced affinity of Dorsal sites in enhancer of mosquito sog gene leads to ventralization of sog expression and expansion of dorsal dpp signaling domain which largely correspond to overall zone occupied by the progenitors of two EMs. We went further and by microarray screen identified and examined the genes specifically enriched in mosquito serosa. We found genes belonging to wax production, chitin synthesis and crosslinking pathways which are apparently involved in production of additional (synthesized after oviposition) eggshell layer - serosal cuticle (SC). SC is described in other insects and is shown to have composite two-layered structure. The most outbound is a wax layer and more proximal is chitin-containing layer. Our desiccation resistance assays revealed that SC plays unique and primary role in protection of mosquito egg against arid conditions. Specifically we see that the mosquito egg gains desiccation resistance only after the SC is synthesized. Altogether we describe genetic basis of evolution and variability in specification of dorsal tissues in Dipterans and shed light on the molecular mechanisms of desiccation resistance in An. gambiae eggs.

46 Engineering the genomes of wild insect populations. Bruce Hay1, Chun-Hong Chen1, Haixia Huang1, Catherine Ward1, Jessica Su1, Ming Guo2. 1) Deptment of Biology, MC156-29, California Institute of Technology, Pasadena, CA; 2) Departments of Neuology and Pharmacology, The David Geffen School of Medicine at UCLA, Los Angeles, CA 90095. There are many contexts in which it would be useful to be able to manipulate the genomes of wild populations. For example, mosquito-borne diseases such as malaria and dengue fever infect hundreds of millions of people each year, and current approaches for prevention, which often focus on vector suppression, are not adequate. An alternative strategy for controlling the transmission of these diseases involves replacement of the wild insect population with transgenic animals unable to transmit disease. An essential component of such a strategy is the presence of a drive mechanism that will ensure the rapid spread of genes conferring disease refractoriness, which may carry a fitness cost, throughout the wild population. Maternal-effect selfish genetic elements (Medea elements) are attractive candidates to mediate drive. Medea elements select for their own survival by inducing maternal-effect lethality of all offspring not inheriting the element-bearing chromosome from the maternal and/or paternal genome. This behavior is predicted to lead to rapid spread of the element within the population even if it carries an associated fitness cost. Medea elements have been shown to exist in the flour beetle Tribolium castenaeum, but their molecular nature is unknown (Beeman et al., Science 256: 89-92). We have developed synthetic Medea elements that can drive population replacement in Drosophila and that are resistant to recombination-mediated dissociation of drive and effector functions (Chen et al., Science 316: 597-600). The genetic and cell biological principles utilized should be applicable to a number of other insect species, and have the potential to allow for iterative cycles of population replacement. We will discuss the development of Medea elements, as well as our progress in engineering reproductive isolation.

47 Genetic and genomic analyses of the wing polyphenism and polymorphism in the pea aphid (Acyrthosiphon pisum). Jennifer Brisson1, Greg Davis2, David Stern2, Sergey Nuzhdin3. 1) Section of Ecology & Evolution, University of California, Davis, Davis, CA; 2) Princeton University; 3) University of Southern California. The pea aphid, an emerging genomic model system, exhibits dramatically different adult phenotypes of winged or unwinged morphs that are induced by environmental conditions in asexual females (a polyphenism) and by a single unidentified genetic locus in males (a polymorphism). Specifically, during the spring and summer months, females reproduce parthenogenetically, producing clonal daughters for ten to twenty generations. These females typically lack wings. Under stressful conditions, however, such as when the host-plant becomes overcrowded, mothers produce daughters that develop wings and are capable of dispersing to other host plants. Males also exhibit winged and unwinged morphs, although in contrast to females, the male wing polymorphism is determined by a single unidentified locus on the X chromosome called aphicarus (api). Winged and unwinged male siblings are genetically identical except for their X-chromosomes, which carry either the api-unwinged or api-winged allele (named for their effect on males). Interestingly, genetic variation for the female wing polyphenism segregates with the male wing polymorphism suggesting that the developmental networks underlying the polyphenism and polymorphism are not independent. Further, this linkage is in reverse phase such that clones that produce winged males in the sexual phase of the life cycle are less likely to produce winged females during the asexual phase of the life cycle. Here we present our ongoing efforts to characterize the female wing polyphenism and male wing polymorphism using genetic and genomic approaches. 100 PLATFORMS: Evolution and Quantitative Genetics

48 Do insecticides alter natural host-parasitoid interactions in Drosophila? Neil Milan, Todd Schlenke. Dept. of Biology, Emory University, Atlanta, GA. Our previous work has shown that natural host plant toxins can have an effect on the interaction between fruitflies and their parasitoid wasps. Resistance to host plant toxins can help flies escape wasp parasitism in one of two general ways: volatile toxins can repel wasps from the toxic food (thus reducing the attack rate), and some toxins appear to limit growth of wasps inside the flies (thus increasing fly immune success). We now extend this work to two man-made toxins that many natural Drosophila melanogaster populations have evolved resistance against, the insecticides DDT and malathion. We are interested in whether Drosophila’s natural parasites have evolved similar insecticide resistance capabilities as their hosts, or whether evolution of resistance against DDT and malathion by D. melanogaster has allowed resistant fly populations a temporary reprieve from normal parasitoid-induced mortality. This work should shed light on a potential drawback of insecticide use to control pests: that natural parasites may suffer worse than the hosts the toxins are meant to control.

49 Multiple infections of Spiroplasma bacteria in Drosophila. Tamara S. Haselkorn, Therese A. Markow, Nancy A. Moran. Dept Ecology & Evolutionary Biol, University of Arizona, Tucson, AZ. Bacterial endosymbionts are common in insects and interact with their hosts in myriad ways, ranging from mutualistic to parasitic, and can have dramatic effects on their host’s evolution. Drosophila harbor far fewer heritable bacterial symbionts than other insects, namely only Wolbachia and Spiroplasma. While the incidence and effects of Wolbachia have been studied extensively, the prevalence and significance of Spiroplasma infections in Drosophila are far less clear. These small, gram-positive, helical bacteria infect a diverse array of arthropod hosts, conferring a variety of fitness effects, and are also well-known plant pathogens. Spiroplasma is a male-killer in certain Drosophila species, such D. melanogaster and species of the willistoni group. In other species of Drosophila, however, it is not a reproductive manipulator, and its effect is unclear. Previous studies have identified different Spiroplasma haplotypes circulating in Drosophila populations. We used a multi-locus sequence analysis to reconstruct a robust Spiroplasma endosymbiont phylogeny, assess genetic diversity, and look for evidence of recombination. Seven loci, many of which are bacterial housekeeping genes located in different areas of the Spiroplasma genome, were sequenced from over 70 Spiroplasma-infected individuals from seven different Drosophila species. Results from this intensive sampling and sequencing effort reveal at least five separate introductions of Spiroplasma into Drosophila. At least three phylogenetically distinct Spiroplasma haplotypes have infected Drosophila.

50 The phenotypic effects of interspecific cytonuclear interactions. Colin Meiklejohn, Kristi Montooth, Dawn Abt, David Rand. Dept EEB, Brown Univ, Providence, RI. Despite the extensive amount of comparative sequence data that has been generated for mitochondrially encoded genes, functional consequences of mitochondrial sequence evolution are known from only a handful of systems. In order to better understand the significance of mtDNA divergence for organismal phenotypes, we created a panel of D. melanogaster lines that contain mtDNA from multiple strains of D. melanogaster (both cosmopolitan and Zimbabwe races), D. simulans, and one haplotype of D. mauritiana. The performance of four X chromosomes relative to a standard in two autosomal backgrounds was measured in combination with these nine mtDNAs in order to identify X chromosome-mtDNA interactions affecting fitness. Despite the fact that hundreds of mutations have fixed between the D. melanogaster and D. simulans mitochondrial lineages, the foreign mitochondria show very slight effects on X chromosome segregation or any other assayed phenotypes, with one exception. A single D. simulans mtDNA haplotype causes a two and a half day delay in egg to adult development time, reduces female fecundity by 50%, lowers the activity of the cytochrome c oxidase complex, and produces short, thin macrochaete, but only in one D. melanogaster autosomal background. The sequence of this mitochondrial haplotype is virtually identical to another D. simulans mtDNA that shows no phenotypic effects, implicating a single mitochondrial mutation in this severely disruptive mito-nuclear epistatic interaction. PLATFORMS: Evolution and Quantitative Genetics 101

51 Satellite sequence de-condensation as a cause of hybrid lethality. Patrick M. Ferree, Daniel A. Barbash. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY. Haldane’s Rule states that deleterious phenotypes observed in interspecific hybrids are more likely to occur in the heterogametic sex (XY and ZW). A well-known exception to this rule is the embryonic lethality of (XX) hybrid females from D. simulans mothers and D. melanogaster fathers. A mutation suppressing this lethality, zygotic hybrid rescue (zhr), was previously mapped to the pericentric heterochromatin of the D. melanogaster X chromosome. This mutant line contains few copies of the pericentric 359 base pair (bp) repeat, a member of the 1.688g/cm3 satellites, suggesting that this non-coding satellite DNA may be a major component of the zhr locus that causes hybrid embryonic lethality. The 359 bp satellite is a particularly interesting candidate because D. simulans does not contain this repetitive sequence. We conducted a detailed cellular analysis to determine the underlying mechanism(s) causing embryonic lethality. Our studies show that during nuclear cycles 10-13 hybrid embryos exhibit major defects in nuclear spacing and mitotic synchrony. We also observe lagging chromatin at the metaphase plate. This defect does not reflect a general problem of chromatin condensation in hybrids. Rather, we have used fluorescence in situ hybridization (FISH) analysis to show that this lagging chromatin derives exclusively from the D. melanogaster X. Furthermore, we find that this lagging chromatin includes the 359 bp satellites, which appear severely under-condensed. To demonstrate the specificity of this defect we show that a translocation containing the 359 bp satellites to the Y chromosome results in Y-specific condensation defects and reverses the lethality from females to males. Our results suggest a direct role of the D. melanogaster X pericentric region, and specifically the 359 bp satellites, in the developmental failure of hybrid female embryos. Previous studies have identified several protein-coding genes as causing hybrid lethality. Our data suggest that rapidly evolving satellite sequences also play a major role in driving hybrid incompatibility between diverging species.

52 Adaptive evolution of the aging gene Insulin-like Receptor. Annalise Paaby1, Mark Blacket2, Ary Hoffmann2, Paul Schmidt1. 1) Department of Biology, University of Pennsylvania, Philadelphia, PA; 2) Centre for Environmental Stress and Adaptation Research, University of Melbourne, Melbourne, Australia. Several candidate genes for aging have been identified by extended longevity mutant phenotypes, including two members of the insulin signaling pathway: the Insulin-like Receptor (InR) and its substrate, chico. Research in the biology of aging has made important advances in our understanding of how such aging genes, and the insulin signaling pathway in particular, determine aging and longevity. However, it is unknown whether these aging loci actually contribute to genetic variance for lifespan in natural populations. For example, a gene under strong constraints may affect lifespan in its function but provide no genetic variation of functional significance. Here we present polymorphism data for InR and chico from natural populations of D. melanogaster across a wide latitudinal geography. InR shows evidence of adaptive evolution on both long and short timescales, exhibiting a history of divergent protein evolution since D. melanogaster shared a common ancestor with D. simulans two million years ago and a pattern of amino acid polymorphism by geography that suggests a response to contemporaneous selection pressures. These patterns are evident across reciprocal latitudinal clines on two continents, making isolation by distance an unlikely explanation. Our results suggest that two members of the insulin signaling pathway, both identified as candidate genes for aging by extended longevity mutant phenotypes, experience different selection pressures and contribute unequally to natural variation in lifespan phenotypes. 102 PLATFORMS: Cytoskeleton and Cell Biology

53 The role of cell surface internalization in epithelial polarity and proliferation control. Sarah L. Windler, David Bilder. Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA. In flies and mammals, loss of cell polarity is correlated with the transformation from normal to neoplastic tissue, but the link between polarity and proliferation is not understood. In order to learn more about the mechanisms governing epithelial cell polarity and proliferation control, we have developed an efficient genetic screen to identify fly neoplastic tumor suppressor genes (TSGs). From this screen, we isolated mutations that disrupt the endocytic adaptor complex, AP-2. In addition, we found that mutations in other genes that control the internalization step of endocytosis - including clathrin heavy chain (chc), and shibire (shi) - also function as neoplastic TSGs. Using a live trafficking assay, we demonstrate that cells mutant for AP-2α, chc, or shi have defects in Notch endocytosis. Even though these genes all function at the internalization step of endocytosis, different mutants show surprisingly different Notch localization, indicating that Notch may be internalized in an AP-2- and Clathrin-independent as well as -dependent manner. We further investigated the mechanism(s) by which these endocytic internalization mutants cause neoplastic transformation. Surprisingly, though accumulation of the apical polarity determinant Crumbs appears sufficient to drive neoplastic transformation, it does not appear to be necessary for the neoplastic phenotype. Alternative mechanisms responsible for polarity and proliferation defects in the endocytic mutants will be discussed.

54 Centrosomin is regulated by multiple kinases at mitotic centrosomes during development in D. melanogaster. Robert Eisman, Lei Gong, Melissa Phelps, Thomas Kaufman. Dept Biol, Jordan Hall A505, Indiana Univ, Bloomington, IN. Progression of the cell cycle, and the changes in protein composition of the centrosome during this process, are regulated by the phosphorylation/dephosphorylation of proteins. Several of the key regulatory kinases and phosphatases required for cell cycle progression are partially characterized, but the identity and the effects of phosphorylation on the function of specific target proteins remain largely unknown. We have shown that Centrosomin (Cnn), an essential core component of the D. melanogaster mitotic centrosome, is phosphorylated during embryogenesis. To understand the mitotic regulation of Cnn during development, we have begun an in-depth analysis of the phosphorylation state of Cnn at different stages of development and in different tissues and Drosophila cell lines. Two-dimensional western analysis reveals that Cnn is phosphorylated at multiple residues, and the pattern of phosphorylation varies depending on Cnn isoforms present, stage of development, and tissue or cell type. We have used both genetic and molecular techniques to identify phosphorylated residues within Cnn isoforms and to identify the kinases that regulate Cnn function during syncytial development. Additionally, we have systematically mutated specific phosphorylated sites in Cnn and have characterized the phenotypic consequences of constitutive phosphorylation and dephosphorylation at these residues during mitosis in D. melanogaster. This work has begun to elucidate the complex posttranslational regulation of Cnn at the mitotic centrosome and provides new insight into the molecular function of Cnn during centrosome replication and maturation throughout development.

55 In vivo quantitative imaging of coordinate cell movements within developing Drosophila embryos. Amy McMahon1, Willy Supatto2, Scott Fraser2, Angela Stathopoulos1. 1) Biology, California Institute of Technology, Pasadena, CA; 2) Beckman Imaging Center, California Institute of Technology, Pasadena, CA. Gastrulation is a conserved yet highly complicated embryonic process combining cell migration and morphological changes, which results in the establishment of different germ layers. In the Drosophila embryo, gastrulation involves dynamic cell movements of presumptive mesoderm cells: (I) invagination of these cells into the embryo, (II) an epithelial-to-mesenchymal transition, and (III) spreading of mesoderm cells within the embryo to form a monolayer. To date, the most authoritative studies of mesoderm migration have relied on observations from fixed embryos, leaving many questions regarding this process unanswered. To resolve this, we are performing in vivo analysis of embryos with 2-Photon Laser Scanning Microscopy. We have optimized this technique to image up to 90 microns within an embryo, allowing us to capture the entire process of gastrulation with sufficient spacial and temporal resolution. To our knowledge, this is the first time that mesoderm migration in its entirety has ever been observed in Drosophila. Furthermore, by using 3D cell tracking software, we can collect quantitative data regarding the behavior of individual or groups of cells as they move. We have also devised new methods for analyzing large data sets and decoupling different types of movement within embryos. By disassembling the migration into its key components, we have uncovered new insights: 1) that the dorsal spreading of the mesoderm is directed, while anterior-posterior movement is passive, and 2) that cells at the leading edge originate from a particular position within mesoderm cell collective and remain at the edge until the entire migration is complete. This approach of combining live cell imaging with quantitative analysis will be extended to study mutant backgrounds, providing insights into the mechanisms controlling mesoderm migration. We contend that live imaging of embryos will provide novel insights into the coordination of collective cell migration. PLATFORMS: Cytoskeleton and Cell Biology 103

56 Drosophila APC2 APC1 null epithelial clones exhibit wingless pathway dependent cell shape changes and epithelial misfolding. Sandra G. Zimmerman, Lauren M. Thorpe, Carolyn A. Mallozzi, Vilma R. Medrano, Brooke M. McCartney. Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA. Mutations in the colon cancer tumor suppressor, Adenomatous polyposis coli (APC), contribute to cancer initiation through APC’s role as a negative regulator of Wnt/Wingless (Wg) signaling and through APC’s role in cytoskeletal functions. The precise mechanisms by which APC proteins affect signaling and cytoskeletal function are not well understood. To determine the cellular consequences of complete loss of APC function, we have generated APC2 APC1 double null clones (APC2g10 APC1Q8) in the Drosophila wing imaginal epithelium. APC2g10 APC1Q8 tissue begins to exhibit morphological abnormalities, including smooth clone borders, apical constrictions, and slight invagination approximately 45 hours after clone induction. Smooth clone borders suggest changes in adhesion, but we did not observe significant changes in levels or localization of DE-Cadherin, Armadillo, or α-catenin at the adherens junctions. Throughout larval and pupal development, APC2g10 APC1Q8 clones develop progressively more dramatic invaginations and misfolding, resulting in outpocketing of the mutant tissue from the surrounding epithelium. In addition to these morphological changes, outpocketing clones overproliferate and are largely restricted to areas of the disc that do not normally activate Wg signaling. Consistent with the hypothesis that the outpocketing is due to ectopic Wg pathway activation, we have shown that blocking the Wg pathway in APC2g10 APC1Q8 clones suppresses the outpocketing. Further, activation of the Wg pathway independent of APC function is sufficient to induce the outpocketing. Our current model is that activation of the Wg pathway in the APC2g10 APC1Q8 clones induces apical constriction, resulting in inappropriate invagination of the epithelium. We are currently investigating the relationship between Wg pathway activation and the regulation of actin dynamics and apical constriction.

57 Hook-like is a negative regulator of endocytic trafficking. Adam Haberman, Sanchali Ray, Helmut Krämer. Cntr Basic Neurosci, UT Southwestern Med Cntr, Dallas, TX. In a screen to identify genes required for transport of proteins to lysosomes and lysosome-related pigment granules, we identified a mutation in hook-like (hkl). Hkl mutants have reduced eye pigmentation, but enhanced degradation of the endocytosed proteins Boss and Delta. Regions of Hkl have strong homology to the apoptotic protein Acinus, including 81% similarity to the region of Acinus that has been proposed to fragment chromatin during apoptosis. However, no apoptotic phenotypes have been identified in hkl mutants. Hkl mutants disrupt localization of the early endocytic protein Avalanche and Hook (Hk). Hk mutants also cause enhanced lysosomal degradation of endocytosed proteins, however hk and hkl phenotypes differ in two significant ways. Hkl does not cause the hooked bristles that are characteristic of hk mutants, and hkl modifies developmental signaling while hk does not. Loss of a single copy of hkl enhances the wing notch phenotype of a partial loss-of-function N allele and rescues the rough eye phenotype of the gain-of-function Ellipse allele of EGFR. Hkl trafficking and signaling phenotypes are opposite of those seen in mutations of genes required for uptake of activated receptors into MVBs, including hrs, erupted, and VPS25. Mutations in these genes cause accumulation of endocytosed proteins and an enhancement of N and EGFR signaling. Hkl mutants cause a reduction of endocytosed proteins and impair N and EGFR signaling. Thus, Hkl may function to slow or restrict endocytic trafficking at or upstream of MVBs. Interestingly, both hk and hkl are required for starvation-induced autophagy, as has also been seen for many positive regulators of MVB internalization, underscoring the importance of endosomes in autophagy.

58 Diaphanous, a regulator of cell contractility and motility. Catarina Homem, Mark Peifer. Dept Biol, Univ North Carolina, Chapel Hill, NC. Embryonic development requires coordinated tissue movements and cell shape changes. In order for all these processes to occur normally, cells must maintain adhesiveness, yet their underlying cytoskeleton has to be very dynamic. Since adhesion, cytoskeletal dynamics and cell contractility are interrelated, it is increasingly important to study these processes in whole animals during morphogenesis. It is unclear how changes in the actin cytoskeleton are coordinated with contractility and altered cell adhesion. We identified a novel mechanism for coordinate regulation of adhesion with the actomyosin cytoskeleton during embryogenesis. Diaphanous-related formins like Drosophila Diaphanous (Dia) are important regulators of actin polymerization. We examined Dia’s role during morphogenesis using both gain- and loss-of-function approaches. We used constitutively-active Diaphanous to examine its roles in morphogenesis and its mechanisms of action. This revealed an unexpected role in regulating myosin levels and activity at adherens junctions during cell shape change, suggesting that Diaphanous helps coordinate adhesion and contractility of the underlying actomyosin ring. These data are consistent with regulated Dia activity playing a normal role in amniooserosal cell apical constriction and epidermal cell shape changes. We tested this hypothesis by reducing Diaphanous function, revealing striking roles in stabilizing adherens junctions and inhibiting cell protrusiveness. These effects also are mediated through coordinated affects on myosin activity and adhesion, suggesting a common mechanism for Diaphanous action during morphogenesis. We are currently extending this work by examining the role Dia plays in migrating cells in both oogenesis and embryogenesis. 104 PLATFORMS: Cytoskeleton and Cell Biology

59 DRhoGEF2 regulates contractile force during segmental groove morphogenesis. Shai Mulinari, Mojgan Padash Barmchi, Udo Häcker. Experimental Medical Science, Lund University, Lund, Sweden. Morphogenesis of the Drosophila embryo is associated with dynamic rearrangement of the Actin-based cytoskeleton mediated by small GTPases of the Rho family. These GTPases act as molecular switches that are activated by guanine nucleotide exchange factors (RhoGEFs). One of these exchange factors, DRhoGEF2, has been shown to play an important role in the constriction of Actin filaments during pole cell formation, blastoderm cellularization and invagination of the germlayers at gastrulation. Here we show that DRhoGEF2 is equally important during morphogenesis of segmental grooves, which become morphologically distinguishable as tissue infoldings during mid-embryogenesis. Formation of segmental grooves is associated with apico-lateral accumulation of F- Actin and the Rho1-effector Diaphanous (Dia) in groove founder cells. At groove regression, DRhoGEF2, Myosin II and F-Actin but not Dia are enriched in cells posterior to the groove that undergo apical constriction. Examination of DRhoGEF2-mutant embryos indicates a role for DRhoGEF2 in the control of cell shape changes that occur during segmental groove formation and subsequent regression. DRhoGEF2-dependent groove formation requires the segment polarity genes engrailed and hedgehog. Overexpression of DRhoGEF2 in epidermal cells is sufficient to induce cortical Myosin II accumulation and cell contraction resulting in a deepening of segmental grooves that can be suppressed by Rho1 inactivation. Expression of activated DiaCA induces Myosin II accumulation and deepening of segmental grooves similar to DRhoGEF2-overexpression. However, unlike DRhoGEF2, DiaCA induces F-Actin polymerization, filopodia formation and strengthens cell-cell contacts. Our morphological analysis furthermore suggests, that Dia regulates cell shape in a way distinct from DRhoGEF2. We propose that DRhoGEF2 acts through Rho1 to regulate acto-myosin constriction but not Diaphanous-mediated F-Actin nucleation during segmental groove morphogenesis. Our data suggest that DRhoGEF2 may contribute to establish a balance of activity between different Rho1-effector pathways.

60 The golgi SNARE, Gos28, is essential for rhodopsin transport and photoreceptor survival. Erica E Rosenbaum, Natalia S Rozas, Nansi Jo Colley. Dept. of Ophth. & Vis. Sci., Dept. of Genetics Univ. Wisconsin, Madison, WI. In sensory neurons, successful transport of signaling molecules through the secretory pathway is essential for cell function and survival. SNARE proteins play a critical role in the final docking and fusion events of vesicular transport by forming distinct SNARE core complexes typically comprised of one v-SNARE motif (associated with the vesicle) and three t-SNARE motifs (associated with the target compartment). Based on in vitro studies, the Golgi SNARE protein, Gos28, has been implicated as a t-SNARE in anterograde transport from the ER to Golgi, transport within the compartments of the Golgi and retrograde transport of vesicles from the recycling endosome back to the Golgi. Here, we demonstrate a role for Gos28 in the vesicular trafficking of the major rhodopsin (Rh1) during its biosynthesis in Drosophila photoreceptor cells. Two mutant alleles of gos28 were identified, an EMS-generated allele and an allele containing a transposable P-element insertion. Antibodies directed to the Gos28 protein recognize a 24kD band in wild-type flies that is absent in the gos28 alleles. Mutations in Drosophila gos28 lead to severe defects in Rh1 levels and cause Rh1 to accumulate throughout the secretory pathway. Very little Rh1 reaches its final destination in the light-sensitive rhabdomeres, which normally contain rhodopsin and the other components of the phototransduction cascade. In the gos28 mutants, all other photoreceptor cell proteins tested are expressed normally, suggesting that Gos28 is specifically required for the transport of Rh1-containing vesicles. Mutations in gos28 also lead to a severe late-onset retinal degeneration. Our results illustrate a critical role for Gos28 in Rh1 trafficking and provide genetic evidence that defects in gos28 lead to inappropriate accumulation of Rh1 throughout the secretory pathway and subsequent retinal degeneration. Our gos28 mutants offer a powerful model for studying the in vivo role of Gos28 in vesicular transport. Drosophila Gos28 displays 48% homology with human Gos28, making it a candidate gene for retinal degeneration in humans.

61 Eating yourcellf into shape: Atg1 and autophagy in cell shape changes. Pavan Kadandale, Amy Kiger. Dept. of Cell & Developmental Biology, UCSD, La Jolla, CA. Atg1 is a conserved Ser/Thr kinase that is a key regulator of autophagy, the critical process by which cells target long-lived proteins and organelles to the lysosomes for degradation. Intriguingly, independent studies have established a role for Atg1 in cellular morphogenesis. Previous work shows that Atg1 is required for the proper elongation and branching of neurites. We show that Atg1 function is required for the ecdysone-induced elongation of Kc cells, as well as the spreading response of primary, dissected larval hemocytes. This shared requirement for Atg1 in both cellular morphogenesis and autophagy suggests a link between these two critical processes and a novel role for autophagy in the regulation of cell shape. We are, therefore, delineating the molecular mechanisms of Atg1-mediated functions and testing whether cell shape changes require autophagy. We show that Atg1 is required for both cytoskeletal remodeling and membrane trafficking in cells undergoing shape changes. Atg1 function also impacts phosphoinositide phosphate (PIP) regulation, as shown by a genetic interaction between Atg1 and mtm (myotubularin, a phosphoinositide phosphate phosphatase), and changes in the phosophoinositide 3-phosphate (PI3P) pools in Atg1 mutant cells. Blocking autophagy, either using a known inhibitor (3-Methyladenine) or by mutating another autophagy-related gene (atg3), also prevents the spreading response of primary larval hemocytes. In conjunction with others’ work, our results establish a link between Atg1 and PI3P, both of which are regulators of autophagy and cytoskeletal and membrane reorganization. Our data also indicates a requirement for autophagy in cell shape changes. Taken together, this suggests that interactions between Atg1 and PI3P pools are required for cytoskeletal and membrane remodelling via autophagy, ultimately resulting in the change of cell shape. Further work will establish the mechanisms by which the complex interplay between Atg1 functions, PI3P regulation and autophagy control cell shape changes. PLATFORMS: Cytoskeleton and Cell Biology 105

62 Dynamics of sarcomere assembly in the flight muscles. John Sparrow, Zacharias Orfanos. Dept Biol, Univ York, York, United Kingdom. Differentiation of striated muscle involves the organized assembly of many different muscle proteins into sarcomeres, the large regular macromolecular contractile complexes. Sarcomeres of the Drosophila indirect flight muscles (IFM) have an extremely regular structure compared to vertebrate striated skeletal muscle, providing a unique system for genetic studies of striated muscle differentiation and disease. Using a GFP protein trap of the Z-disc protein Salimus we have confirmed earlier EM studies that once the initial Z- body arrays (proto-sarcomeres) appear in the muscles at 30-35h after puparium formation (APF) they develop in synchrony to mature sarcomeres in all indirect flight muscles (DLM and DVM). Importantly no new sarcomeres are added after 40h APF. Proto- sarcomeres initially elongate, but then at 65-70h APF the Z-discs and sarcomeres begin to widen laterally, while sarcomere elongation continues to reach mature sarcomere length at 90-100h APF. In the absence of IFM-specific ACT88F actin (Act88F6 null mutation) Z-body/actin filament arrays also appear at 35h APF, suggesting that another actin isoform is responsible, but these structures are no longer apparent by 50h APF, confirming that ACT88F actin is required even for early sarcomere development. In the absence of sarcomere myosin heavy chain (Mhc7; IFM-specific Mhc null) some early, delayed, sarcomere elongation occurs, and, although mature sarcomere lengths are never achieved, the widening of Z-discs does occur. This argues that thick filament assembly is required for sarcomere elongation, but not for Z-disc widening. Using antibodies and sarcomeric protein GFP traps we have studied the dynamics of protein assembly into developing sarcomeres. Our results show that myosin assembles into the thick filaments at the M-line and that binding of flightin, an IFM-specific myosin binding protein, follows myosin incorporation, but with a detectable delay. Based on these and other data we will present a model of sarcomere development in IFM.

63 Genetic control of cell morphogenesis during formation of the Drosophila cardiac tube. Caroline MEDIONI1, Martine ASTIER1, Monika ZMOJDZIAN2, Krzysztof JAGLA2, Michel SEMERIVA1. 1) CNRS-UMR6216, IBDML, MARSEILLE, France; 2) INSERM U384, Faculté de Médecine, Clermont-Ferrand, France. Tubulogenesis is an essential component of organ development, yet the cellular mechanisms controlling tube formation are poorly understood. As a model of tubulogenesis, we analyzed the formation of the Drosophila cardiac lumen which arises from the migration and subsequent coalescence of bilateral rows of cardioblasts. Our detailed 3D and time lapse imaging of cell behaviour and the distribution of cell polarity markers reveals that lumen formation occurs by repulsion of pre-patterned secondary basal membrane domains rather than by fusion of apical membrane vesicles to the luminal compartment. Cardioblasts forming the lumen are devoid of an apical domain. Lumen formation is therefore driven by cell shape remodeling in contrast to all previously described models of tubulogenesis. In support of these findings, blocking the Slit/Robo pathway prevents cardioblast cell-shape changes and inhibits lumen formation. Our data reveal a new mechanism of tube formation which may be conserved during vertebrate heart tube formation and in vasculogenesis.

64 steamer duck inhibits epidermal cell-cell fusion in Drosophila larvae. Yan Wang, Michael Galko. The Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, TX. During cell-cell fusion pairs or groups of cells merge their plasma membranes to form a single cytoplasm that accommodates all their intracellular components, including multiple nuclei. This event is critical for fertilization and the subsequent development of diverse tissues, such as muscle, placenta and bone. Cell-cell fusion is also important for physiological and pathological events such as macrophage engulfment of foreign bodies, tissue repair and regeneration, and cancer progression. Despite its importance, the proteins directly executing cell-cell fusion remain obscure and its molecular basis is not well understood. When third instar Drosophila larvae are wounded, epidermal cells near the wound fuse with each other. We are exploiting this inducible cell-cell fusion assay to identify genes required for cell-cell fusion. We constructed a reporter line whose epidermal cell membranes and nuclei are fluorescently labeled and can be visualized in live larvae. Using this reporter we conducted a targeted pilot screen of 30 candidate wound healing genes in which gene function is conditionally knocked down by UAS-RNAi within the larval epidermis. This screen identified a negative regulator of epidermal cell-cell fusion called steamer duck (stck). stck encodes the protein PINCH, a known component of integrin-dependent cell adhesion structures at embryonic muscle attachment sites and between the adult wing epithelial layers. Immunofluorescence with an anti-PINCH antibody showed that PINCH is localized to epidermal cell membranes and that UAS-stck RNAi efficiently depleted epidermal PINCH protein. The hyperfusion phenotype caused by stckRNAi is not wound-dependent because it also was observed in unwounded larvae. However, we observed that PINCH and its known binding partner, integrin-linked kinase (ILK) are relocalized from the plasma membrane upon wounding. We are currently testing a model whereby destablization of epidermal cell-cell adhesion, either through wounding or loss of epidermal PINCH protein, leads to activation of a latent cell-cell fusion machinery in these cells. 106 PLATFORMS: Cytoskeleton and Cell Biology

65 The Frizzled and Fat/Dachsous pathways control wing topography. Simon Collier, Kristy Doyle, Justin Hogan, Eric Aten. Dept Biological Sci, Marshall Univ, Huntington, WV. The adult wing membrane is characterized by ridges that have an anteroposterior (A-P) orientation in the anterior wing and a proximodistal (P-D) orientation in the posterior wing. We have found that the formation and orientation of both A-P and P-D ridges is controlled by the Frizzled Planar Cell Polarity (Fz PCP) signaling pathway. The Fz PCP pathway also specifies P-D hair polarity in both the anterior and posterior wing. This raises a question; if the Fz PCP pathway polarizes wing cells for both hair polarity and ridge orientation, why is there a different relationship between hairs and ridges in the anterior wing compared to the posterior wing? We will present evidence that the specification of A-P and P-D wing ridges occurs at different times during pupal development. In an early phase of Fz PCP signaling (prior to 18 hours a.p.f.), P-D ridges are specified in both the anterior and posterior wing. In a late phase of Fz PCP signaling (prior to 32 hours a.p.f.), anterior ridges are reoriented to A-P and P-D hairs are specified across the wing. Our data suggest that the two phases of Fz signaling are characterized by differential use of Prickle protein isoforms. The Sple isoform is primarily active in the early phase and the Pk isoform in the late phase. A significant feature of this two-phase model is that only the anterior ridges are reoriented to A-P in the late phase. This implies that there is some activity preventing the reorientation of ridges in the posterior wing. We propose that the Fat/Dachsous signaling pathway plays this role. Loss of Fat, Dachsous or Four- jointed activity during wing development allows the formation of A-P ridges in the posterior wing. We will present data from timed knockdown and over-expression experiments to support this conclusion.

66 Stripe non-autonomously controls the orientation of actin-based protrusions. Stacie Dilks, Stephen DiNardo. University of Pennsylvania, Philadelphia, PA. The ability of cells to organize actin filaments into stable structures is vital to many cellular functions such as nutrient absorption and sensory input. Although the actin-based protrusions that underlie these processes often have elaborate shapes, it is unknown how cells form shaped protrusions. In the embryo, actin-based protrusions (called denticles) are produced by seven rows of ventral epidermal cells, and differ in shape and hooking polarity from row to row. Specifically, cell rows 1 and 4 are the only two rows in the denticle field that hook to the anterior. This leads us to wonder what is unique about denticle rows 1 and 4 that causes them to reverse their polarity and hook to the anterior. To date, no cell fate determinant or transcription factor is known to be unique to these two cell rows that might provide clues to their polarity reversal. Stripe (sr), a transcription factor required for larval muscle attachment, is expressed in a row-specific pattern in the epidermis. Interestingly, sr is expressed in denticle rows 2 and 5, immediately posterior to the two anterior-facing denticle rows. We now show that sr expression is required, non-autonomously, for the anterior denticle polarity seen in cell rows 1 and 4. Stripe is a downstream target of both the Hh and EGFR signaling pathways, and is the relevant target for anterior denticle polarity. Global overexpression of sr also results in a loss of anterior polarity, indicating that anterior polarity is not conferred on a cell simply via its proximity to a stripe- expressing cell, but through a more complex mechanism. By ectopically expressing stripe in a row-specific manner, we show that cells assign hooking polarity by integrating information from both neighboring cell rows. Finally, preliminary data indicates that sr may mediate its effect on denticle polarity via the cytoskeletal linker protein shortshop, as mutants in this sr target gene exhibit polarity defects that mimic stripe. PLATFORMS: Gametogenesis 107

67 Transition of male primordial germ cells to functional germline stem cells. Matthew Wawersik1, Rebecca Sheng2, Trevor Posenau1, Juliann Gumulak-Smith1, Erika Matunis2, Mark Van Doren3. 1) Biology Dept., College of William & Mary, Williamsburg, VA; 2) Dept. of Cell Biology, Johns Hopkins School of Medicine, Baltimore, MD; 3) Dept. of Biology, Johns Hopkins University, Baltimore, MD. Stem cells maintain tissue homeostasis by producing differentiating daughters that replace mature cells lost to injury or turnover. Adult germline stem cells (GSCs) are descendants of primordial germ cells (PGCs) that are specified during embryogenesis. While studies have examined the molecular requirements for PGC specification, migration, and coalescence, very few have examined the timing and regulation of GSC establishment. We have characterized the transition from PGCs to functional GSCs in the developing male Drosophila gonad. We find that anterior PGCs adjacent to the embryonic hub express GSC markers and behave functionally like GSCs by the end of embryogenesis, while PGCs in the posterior of the gonad appear to differentiate. In contrast to female PGCs that remain quiescent until the late third instar larval stage, GSCs in the male gonad produce transit amplifying daughter cells as mid first instar larvae. We also find that GSC establishment is concurrent with hub formation, suggesting that niche formation is a key step that initiates the PGC to GSC transition. Finally, analogous to adult GSCs, our data indicate that newly formed GSCs require JAK/STAT signaling to be maintained, while ectopic activation of the JAK/STAT pathway prevents germ cell differentiation. Together, these data indicate that the JAK/STAT pathway plays an important role in regulating the establishment of function GSCs in late- stage male embryonic gonads.

68 Live imaging of asymmetric centrosome migration and spindle orientation in Drosophila male germline stem cells. Jun Cheng1, Nahid Hemati2, Yukiko M. Yamashita2,3, Alan J. Hunt1. 1) Department of Biomedical Engineering; 2) Center for Stem Cell Biology, Life Sciences Institute; 3) Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI. Proper orientation of the mitotic spindle is essential for establishing the axis of asymmetric stem cell division. However, the mechanism and regulation of spindle orientation are poorly understood. The Drosophila male germline stem cells (GSCs) is one of the best studied model systems in which centrosome positioning and spindle orientation, leading to asymmetric stem cell division, are well documented, but mostly inferred from observations on fixed-cell preparations. We have established conditions for time- lapse live-cell imaging, allowing direct observation of centrosome movement and spindle formation, allowing the behavior to be tracked in detail. Using fluorescent protein-tagged α-tubulin and centrosomal proteins, the mitotic spindle orientation and centrosome migration during interphase were examined by time-lapse live-imaging. The results clearly confirmed that GSCs underwent asymmetric mitosis with the mitotic spindles orthogonal to the adjacent hub cells (niche cells). The somatic cyst progenitor cell (CPC) divisions were also recorded, which indicated that their mitotic spindles were not oriented during mitosis, suggesting that distinct cellular/molecular mechanisms are involved in the control of asymmetric divisions of GSCs and CPCs. Moreover, in our preliminary study, we observed that GSCs with misoriented centrosomes did not undergo mitosis for a prolonged time period, and entered mitosis only after the misoriented centrosomes rotated back into normal orientation, which implies that the centrosome orientation may be monitored in GSCs to ensure the asymmetric stem cell division.

69 Live Imaging of Drosophila Spermatogonia Dedifferentiating into Germline Stem Cells. Xuting Rebecca Sheng, Crista Brawley, Erika Matunis. Dept Cell Biol, Johns Hopkins Univ, Baltimore, MD. In the Drosophila testis, germline stem cells (GSCs) and somatic stem cells (SSCs) adhere to a group of stromal hub cells that comprise the stem cell maintaining microenvironment, or niche. Both stem cell populations produce daughter cells that are displaced away from the niche. Cellular differentiation within a stem cell lineage is commonly considered irreversible. However, if stem cells are depleted, their daughters (transit-amplifying cells) may revert back into stem cells using a process called dedifferentiation. Dedifferentiation occurs in both the Drosophila testis and ovary, but the mechanism is poorly understood. To establish a novel system where GSC daughters (spermatogonia) dedifferentiated into GSCs, we first ectopically expressed the differentiation factor Bag-of-marbles (Bam) in the testis. Bam expression resulted in GSC loss through differentiation, while SSCs appeared unaffected. We then withdrew ectopic Bam expression from flies depleted of GSCs and saw that GSCs were regenerated by the remaining spermatogonia. To bypass limitations of resolving specific cellular changes in fixed tissue, we cultured and imaged GFP-labeled germline cells during GSC recovery. This revealed that both single and interconnected clusters of spermatogonia near the hub make actin-rich protrusions resulting in hub contact. Upon initial contact with the hub, spermatogonia were able to increase the amount of hub contact despite the presence of SSCs adjacent to the hub. Furthermore, we observed occasional spermatogonia with protrusions that appeared to move closer towards the hub indicating that normally non-motile germline cells may become motile in order to replace lost stem cells. This suggests that dedifferentiating cells can actively displace SSCs when re-establishing occupancy of the niche and that signals from the hub may cause dedifferentiating spermatogonia to “hone” into the niche. 108 PLATFORMS: Gametogenesis

70 Asymmetric activation of Rac in germ line stem cells of the ovary controls both the plane of division and the response to BMP signals. Wen Lu1, M. Olivia Casanueva2, Chip Ferguson1,2,3. 1) Committee on Genetics; 2) Committee on Developmental Biology; 3) Dept. of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL. How adult stem cells maintain the balance between self-renewal and differentiation is a key question in stem cell biology. The germ line stem cells (GSCs) of the ovary reside at the anterior tip of each ovariole adjacent to somatic cells that form a “niche” responsible for GSC maintenance. Bone Morphogenetic Proteins (BMPs) secreted by the surrounding somatic tissues act as niche signals necessary for GSC maintenance. Our data support the hypothesis that interactions between the niche cells and the GSCs polarize the GSCs leading to the asymmetric activation of the small GTPase Rac at the GSC - niche interface. Activated Rac both controls the plane of GSC division, most likely by directing the localization of the cytoskeletal-associated protein APC2, and promotes BMP signaling in the GSC by elevating the activity of the JNK pathway. Together, these two activities of Rac cooperate to ensure a robust pattern of asymmetric self-renewal divisions. The future goals of this research are to determine how Rac is asymmetrically activated at the GSC - niche interface, and to explore the mechanisms by which elevated JNK signaling promotes BMP signal transduction in the GSC.

71 Insulin signals regulate GSC maintenance via the control of niche size. Hwei-Jan Hsu, Daniela Drummond-Barbosa. Department of Cell and Developmental Biology, Vanderibilt Univerisity Medical Center, Nashville, TN. Ovarian germline stem cells (GSCs) reside within a specialized microenvironment (or niche) that provides signals required for GSC maintenance. GSC activity is also modulated by external factors, such as diet, but this process is less well understood. Our previous work documented that neural-derived insulin-like peptides (Dilps) directly control ovarian GSC division in response to diet; however, it was not know whether insulin signals are also involved in the regulation of GSC maintenance. As female flies age, they progressively lose GSCs, and this is paralleled by a reduction in their niche size. Interestingly, we found that flies kept under a protein-poor diet lose their GSCs faster with age than flies kept under a protein-rich diet, suggesting that insulin signaling may affect GSC number and niche size. To specifically address if insulin signaling regulates GSC maintenance, we examined the GSC number and niche size in insulin receptor mutants and found that these mutants have fewer GSCs and smaller niche size than control females. To test if insulin signaling directly controls GSC maintenance, we performed insulin receptor mutant mosaic analysis in GSCs. Our results show that insulin receptor mutant GSCs exhibit a half-life comparable to that of control GSCs, suggesting that insulin signals regulate the survival of niche cells to indirectly promote the maintenance of GSCs. Remarkably, overexpression of Dilp2 maintains a higher number of GSCs and niche cells in aging females, suggesting that reduced insulin signaling underlies the gradual reduction in niche size and GSC loss that occurs naturally with age.

72 Stem Cell Maintenance Through Competition in the Follicle Stem Cell Niche. Todd Nystul, Allan Spradling. Embryology Dept, Carnegie Inst, Baltimore, MD. Follicle stem cells (FSCs) reside in the germarium of the Drosophila ovary, and are the progenitors of the follicular epithelium that surrounds the developing germline. We have used the FSCs as a model for understanding the behavior and maintenance of an epithelial stem cell in its native, in vivo environment (1). Using a newly developed dual-marked clonal system, we confirmed that there are exactly two FSCs per germarium, and found that they reside in separate, non-adjacent niches. This differs from the male and female germline stem cell niches, where multiple stem cells are contained within a fixed, cellular niche. Surprisingly, though the FSC niche is also fixed in place, it lacks a fixed stromal-cell component. We found that FSCs can be reliably identified without clonal analysis by their distinctive shape and consistent location in the germarium, which allowed us to analyze the behavior of early FSC daughters. Approximately half of the FSC daughters move away from the niche toward the posterior, while the other half migrate laterally and contact the opposite FSC niche before integrating into the growing epithelium. FSCs are regularly lost and replaced during adult life, and we observed that the laterally migrating FSC daughters facilitate this replacement by competing with resident stem cells for niche occupancy. We are currently screening for mutations that affect FSC function, and have identified several candidate genes which may alter the competitiveness of FSCs and their daughters for niche occupancy. 1. Nystul, TG and Spradling, AC. (2007). An Epithelial Niche in the Drosophila Ovary Undergoes Long-Range Stem Cell Replacement. Cell Stem Cell 1, 277-285. PLATFORMS: Gametogenesis 109

73 A Bam complex mediates a cap-dependent translational switch to promote stem cell differentiation. Jean Maines, Yun Li, Dennis McKearin. Department of Molecular Biology, UT Southwestern Medical Center, Dallas, TX. The balance between germline stem cell (GSC) self-renewal and differentiation in Drosophila ovaries is mediated by the antagonistic relationship between the Nanos-Pumilio (Nos-Pum) translational repressor complex, which promotes GSC self-renewal, and the expression of Bam, a key differentiation factor. We explored the mechanistic basis of Bam’s function promoting germ cell differentiation. We found that bam behaves genetically as a negative regulator of nos. Furthermore, Bam and Nos proteins are expressed in reciprocal patterns in young germ cells and sequences in the nos 3’ UTR are critical for Nos elimination from Bam expressing cells. Ectopic Bam expression causes differentiation of germline stem cells, and strikingly, this activity also acts through the nos 3’-UTR, since expression of a nos transgene with a tubulin 3’-UTR prevents Bam-induced GSC loss. Bam forms a complex with Bgcn, a member of the RNA helicase family, suggesting that the Bam-Bgcn complex may associate directly with RNA. Finally, we found that Bam is a component of an mRNA cap-binding complex, suggesting that a Bam complex blocks translation of nos, and perhaps other target RNAs, via cap-mediated translational repression. We conclude that Bam promotes germ cell differentiation by translational repression of nos, and declining Nos levels, in turn, promote differentiation by derepressing translation of differentiation-promoting factors. These findings emphasize the importance of translational repression in regulating the balance between stem cell self- renewal and differentiation, particularly in the germ cell lineage.

74 Role of Drosophila Ime4 and Ime2 in the Initiation of Meiosis. Cintia Hongay, Gerald Fink, Terry Orr-Weaver. Fink and Orr- Weaver Labs, Whitehead Inst, Cambridge, MA. Meiotic cell division is restricted to specialized cells of sexually reproducing eukaryotes and is required to preserve the species- specific chromosomal number upon fertilization. The early events that commit cells from a mitotically dividing population of germ stem cells to the unique divisions of meiosis are mostly unknown. In yeast, a unicellular organism, we have found that IME4 (Inducer of Meiosis 4, putative RNA methyltransferase) is a mitotic:meiotic switch: it is required in diploids to enter meiosis and, if expressed in haploids, it is sufficient to license them to initiate meiosis (Hongay et al., 2006). Expression of IME4 in haploid cells is followed by expression of IME2 (encoding a protein kinase), an event recognized as a hallmark of commitment to meiosis and subsequent sporulation in budding yeast. There are highly conserved homologs of these two genes in D. melanogaster, M. musculus, H. sapiens, and other metazoans, but their functions are presently unknown. We have detected gonadal expression of dIME2 and dIME4 in Drosophila suggesting a role in meiosis and thus evolutionary conservation of their functions in metazoans. In situ hybridization shows that dIME4 RNA is localized to the apical region of the testes, coinciding with the area where the decision to undertake a meiotic cell fate occurs. In addition, we have obtained two ime4 hypomorphs using a P-element excision strategy that are male and female sterile, a phenotype that we corroborated with RNAi against dIME4. These results support a role for dIME4 and potentially dIME2 in Drosophila meiosis. Hongay, C. F., Grisafi, P. L., Galitski, T., and Fink, G. R. (2006). Antisense transcription controls cell fate in Saccharomyces cerevisiae. Cell 127, 735-745. 110 PLATFORMS: Signal Transduction

75 Specificity of Signaling by Drosophila Fibroblast Growth Factors. Angelike Stathopoulos, Phoebe Tzou, Snehalata Kadam. Div Biol, MC 114-96, Caltech, Pasadena, CA. FGF signaling is used reiteratively throughout development of animals to control a diverse set of cellular processes including migration and differentiation. Over 120 FGF-FGF receptor (FGFR) combinations are possible in vertebrates, and we will present evidence that only 3 such combinations function in Drosophila. The Pyramus (Pyr) and Thisbe (Ths) genes from Drosophila encode the first pair of invertebrate FGFs that bind to the same FGFR isoform. We aim to determine whether different FGF ligands are required to effect qualitatively different responses downstream of FGFR activation and have a unique advantage using the simpler Drosophila model system. To this end, we have isolated single mutants affecting either Pyr or Ths genes and compare them with a deficiency mutant that we have previously characterized [DfBSC25], removing both genes. We find that both FGF genes are required to control migration of the mesoderm during gastrulation. Furthermore, the unique phenotypes of these single mutants suggest that both ligands work cooperatively to coordinate this process by different mechanisms. In addition, FGF signaling is required for regulating cell differentiation, as in the specification of pericardial, dorsal somatic mesoderm, and longitudinal visceral mesoderm founder (LVMF) cells occurring during embryogenesis. In pyr single mutants, pericardial and dorsal somatic mesoderm cells are mostly absent, whereas in ths mutants these same cells are indeed specified; both pyr and ths mutants affect LVMF specification. Our results indicate that Ths and Pyr have differential roles in cell migration and specification and that, in some biological contexts, they cannot substitute for one another. We have furthered our study mechanistically by defining the functional domains of these two FGFs. Unexpectedly, our preliminary results suggest that specificity is not imparted by the FGF ligand homologous regions of the proteins. Implications of these results toward specificity of FGF signaling in Drosophila compared with vertebrates will be discussed.

76 Endosomal Entry Regulates Notch Receptor Activation in Drosophila. Thomas Vaccari1, Han Lu1, Ritu Kanwar2, Mark Fortini2, David Bilder1. 1) Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA; 2) Center for Cancer Research, National Cancer Institute, Frederick, MD. Signaling through the transmembrane receptor Notch is widely utilized throughout animal development and is a major regulator of cell proliferation and differentiation. During canonical Notch signaling, internalization and recycling of Notch ligands controls signaling activity, but the involvement of endocytosis in activation of Notch itself is not well understood. To address this question, we have systematically assessed Notch localization, processing and signaling in a comprehensive set of Drosophila mutants that block access of cargo to different endocytic compartments. We will present data that indicate that processing and signaling of endogenous Notch is reduced in mutants that impair entry into the early endosome but is enhanced in mutants that increase endosomal retention. This dramatic activation switch does not involve Notch ligands and indicates that endosomal access of the Notch receptor promotes signaling. In mutants that block endosomal entry we also uncover an alternative, low-efficiency Notch trafficking route that can contribute to signaling. Our data suggest that altered residence in distinct endocytic compartments could underlie pathologies involving aberrant Notch pathway activation.

77 Pentagone, a novel BMP/Dpp target gene, involved in patterning and growth in Drosophila. Robin Vuilleumier1, Markus Affolter2, George Pyrowolakis1. 1) Developmental Biology Unit, Biology I, University of Freiburg, Freiburg, Germany; 2) Dept. of Cell Biology, Biozentrum, University of Basel, Basel, Switzerland. In Drosophila, patterning and growth of appendages is controlled by a small number of highly conserved signaling molecules. Among them, Decapentaplegic (Dpp), the ortholog of the BMP2/4 ligands in vertebrates, activates a conserved signal transduction pathway to transcriptionally regulate target genes. Recently, we uncovered a branch of the BMP/Dpp-signaling pathway which culminates in direct repression of a number of key developmental genes. This process depends on a highly conserved 16 bp long cis-regulatory element, the Silencer Element (SE). We use the consensus sequence of the SE as bait to perform genome-wide, in silico, screens to identify genes that are potentially repressed by the BMP/Dpp-signaling pathway. Here we report the identification and characterization of a novel BMP/Dpp target gene, pentagone (pent). In embryos and larval imaginal discs, pent expression is restricted to regions with low or absent BMP/Dpp signaling activity. We present in vitro and in vivo evidence that pent is directly repressed by the BMP/Dpp signaling pathway through the SEs present in the regulatory region of the gene. In addition, loss- and gain-of-function studies demonstrate that pent plays a crucial role for normal patterning and growth of lateral regions of the Drosophila wing. Interestingly, pent mutants display a comparable phenotype to mutants in components of the BMP/Dpp-signaling pathway. Because of these observations and the fact that pent encodes a secreted, multi-domain protein, we are currently testing the hypothesis that Pent regulates the activity of the Dpp-morphogen by modulating ligand distribution, availability and/or reception. PLATFORMS: Signal Transduction 111

78 A translational block to HSPG synthesis permits BMP signalling in the early Drosophila embryo. Douglas Bornemann1, Sangbin Park2, Sopheap Phin1, Rahul Warrior1. 1) Dept Developmental & Cell Biol, Univ California, Irvine, Irvine, CA; 2) Department of Developmental Biology, Stanford University School of Medicine, Stanford, California. Heparan sulfate proteoglycans (HSPGs) are extracellular macromolecules found on virtually every cell type in eumetazoans. HSPGs are composed of a core protein covalently linked to glycosaminoglycan (GAG) sugar chains that bind and modulate the signaling efficiency of many ligands including Hedgehog (Hh), Wingless (Wg), and Bone Morphogenetic Proteins (BMPs). Here we show that in Drosophila, loss of HSPGs differentially affects embryonic Hh, Wg and BMP signaling. We find that a stage-specific block to GAG synthesis prevents HSPG expression during establishment of the BMP activity gradient that is critical for dorsal embryonic patterning. Subsequently proteoglycan synthesis is initiated coincident with the onset of Hh and Wg signaling which require HSPGs. This temporal regulation is achieved through translational control of HSPG synthetic enzymes through Internal Ribosome Entry Sites (IRES). IRES-like features are conserved in GAG enzyme transcripts from diverse organisms arguing that this represents a novel evolutionarily conserved mechanism for regulating GAG synthesis and modulating growth factor activity.

79 Interactions between the retinal determination protein Eyes absent and the Abelson tyrosine kinase suggest a novel function for Eya in cytoskeletal regulation during axonogenesis. Wenjun Xiong, Noura Dabbouseh, Ilaria Rebay. Ben May Dept. for Cancer Research, The Univ. of Chicago, Chicago, IL. A fundamental question in developmental biology is how multiple signaling pathways converge on downstream transcriptional effectors to initiate and ensure proper developmental programs. The discovery that Eyes absent (Eya), originally described as a transcriptional cofactor, also functions as a protein tyrosine phosphatase, adds further complexity to the interplay between different signaling inputs and the retinal determination gene network. Although both activities of Eya are required for Drosophila eye development, the details of their regulation have yet to be revealed. In particular, little is known about the functions and regulation of Eya phosphatase activity. In a genetic screen designed to identify tyrosine kinases upstream of Eya, the Abelson tyrosine kinase (Abl) was implicated as a positive regulator of Eya’s function in retinal determination. The genetic synergy between Eya and Abl contributes to multiple developmental programs, including axon pathfinding in the embryonic CNS and the axon targeting process in the larval visual system. In biochemical assays, Abl directly phosphorylates Eya at multiple tyrosine residues, and can increase Eya tyrosine phosphorylation in vivo. As a result of this tyrosine phosphorylation, Eya relocates from the nucleus to the cytoplasm where we hypothesize it participates in phosphotyrosine-mediated signaling networks that regulate cytoskeleton dynamics. Supporting this model, we have found that nuclear-restriction of Eya compromises its function in vivo, and that coexpression of membrane- tethered Eya can reconstitute full Eya activity. Together our data suggest a model in which Eya, in addition to operating as a nuclear transcription factor, also participates as a phosphatase in cytoplasmic phosphotyrosine signaling networks that regulate neuronal morphogenesis.

80 A cell autonomous requirement for the Dally-like core protein in Hedgehog signaling. Elizabeth H. Williams1, William N. Pappano2, Philip A. Beachy1. 1) HHMI, Dept of Developmental Biology, Stanford Univ Sch Med, Stanford, CA; 2) HHMI, Dept of Molecular Biology & Genetics, Johns Hopkins Univ Sch Med, Baltimore, MD. The Drosophila glypicans Dally and Dally-like (Dlp) have well characterized roles in cell non-autonomous movement of morphogens such as Hedgehog (Hh) and Wingless (Wg). A genomic RNAi screen in cultured cells revealed that Dlp, but not Dally, also is required for cell autonomous Hh response. This role and specificity similarly are observed during embryonic patterning. While the heparan sulfate proteoglycan (HSPG) moieties of mature Dlp are necessary for its role in morphogen movement, we have found that these sugar modifications are dispensable for its role in cell autonomous Hh signaling. In a cultured cell Hh signaling assay, a Dlp variant DlpΔGAG, which lacks detectable HS modification, fully rescues RNAi depletion of endogenous dlp transcript. The Dlp core protein also is sufficient for some of the essential in vivo roles of Dlp during embryonic and larval development since expression of a DlpΔGAG transgene rescues the zygotic larval lethality of the dlpA187 null allele comparably to expression of a wild-type Dlp transgene. In addition, segment polarity in the larval cuticle, which requires both intact Hh and Wg signaling, is partially rescued in dlpA187 germline clones by DlpΔGAG expression. This cell autonomous activity for Dlp in Hh signal transduction maps to the N- terminal globular domain and requires membrane association but not specifically the glycosyl phosphatidylinositol (GPI) linkage characteristic of glypicans. The most closely related mammalian glypicans fully rescue RNAi depletion of endogenous dlp transcript in a cultured cell Hh signaling assay, demonstrating conservation of this activity through evolution. Beyond the established roles of the HS moieties of HSPGs in signal response and movement through tissues, our studies define a conserved activity for the core protein of the Dlp HSPG in cell autonomous Hh signal transduction. 112 PLATFORMS: Signal Transduction

81 Sequential actions of feedforward and feedback loops pattern Drosophila egg: genetic experiments and computational modeling. Nir Yakoby1,2, Jessica Lembong1,2, Trudi Schüpbach3, Stanislav Y. Shvartsman1,2. 1) Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ; 2) Dept. of Chemical Engineering, Princeton University, Princeton, NJ; 3) Howard Hughes Medical Institute and Dept. of Molecular Biology, Princeton University, NJ. During Drosophila oogenesis, the EGFR and Dpp signaling pathways specify several subpopulations of the follicle cells which give rise to dorsal eggshell structures. The roof of dorsal appendages is formed by the follicle cells that express Br, a Zn-finger transcription factor regulated by both pathways. EGFR induces Br in the dorsal follicle cells. This inductive signal is overridden in the dorsal midline cells, which are exposed to high levels of EGFR activation, and in the anterior cells, by Dpp signaling. We show that the resulting changes in the Br pattern affect the expression of Dpp receptor thick veins (tkv). By controlling tkv, Br controls Dpp signaling in late stages of oogenesis and, as a result, regulates its own repression in a negative feedback loop. We synthesize these observations into a mathematical model, whereby the dynamics of Br expression is driven by the sequential action of feedforward and feedback loops. The feedforward loop controls the spatial pattern of br expression, while the feedback loop modulates this pattern in time. Using a computational approach to systematically explore the feasibility and robustness of this mechanism, we show that it can successfully predict the dynamics of the eggshell patterning network in multiple mutant backgrounds.

82 Analysis of synthetic interactions in Drosophila by RNAi. Thomas Horn1, Elin Axelsson2, Wolfgang Huber2, Michael Boutros1. 1) Signaling & Functional Gen, German Cancer Research Ctr, Heidelberg, Baden-Wuerttemberg, Germany; 2) EMBL/EBI - European Bioinformatics Institute, Wellcome Trust Genome Campus, Cambridge CB10 1SD, UK. RNA interference (RNAi) has been used as a powerful technique to systematically deplete transcripts on a genome-wide scale. Due to the efficiency of RNAi in invertebrates, model organisms such as Drosophila and C. elegans have contributed important insights with the identification of novel components of diverse biological pathways. The transduction and integration of signals within and between pathways are highly complex, often requiring the interaction of multiple genes. Forward and reverse genetics have been a powerful approach to identify non-redundant components of signaling pathways, however, it is likely that redundant factors have escaped identification and will require combinatorial approaches. RNAi provides a powerful tool to probe redundancy in signaling networks and also allows addressing this problem in a systematic manner. In order to probe for functional redundancy in signaling pathways, we generated subsets of functional groups encoded by the Drosophila genome. For example, kinome RNAi libraries with two independent dsRNAs targeting all splice variants of the target transcripts were designed and synthesized. All possible double knock-down combinations were screened for synthetic effects using homogenous and high-content phenotypic readouts in Drosophila cells. This data set is being used to model genetic interaction networks in Drosophila. PLATFORMS: Pattern Formation 113

83 From parts to pattern: deciphering pair rule seven stripe formation with a completed cis-regulatory blueprint. Mark Schroeder, Ulrike Gaul. Rockefeller University, New York, NY. During blastoderm segmentation, the transition from the aperiodic gap gene patterns to the periodic pair rule gene patterns is particularly striking. Previous studies distinguished two steps in this transition: The primary pair rule genes (h, eve, run) establish their 7 stripe pattern piecemeal by interpreting maternal and gap patterns through a set of stripe-specific enhancers, while the secondary pair rule genes (ftz, odd, prd, slp) generate their pattern wholesale by interpreting the patterns of both primary and secondary pair rule genes through a single 7-stripe enhancer. Revisiting this problem with computational and experimental methods, we found that this model required substantive revision. By comparing the temporal evolution of the pair rule patterns with the known enhancers we found that early emerging stripes are driven by maternal/gap input and that, based on this reasoning, stripe-specific elements were missing for 10 stripes. Using the Stubb algorithm and known transcription factor binding preferences, we predicted the missing elements and validated them through lacZ reporter assays. Surprisingly, stripe specific elements generate all 7 ftz and run and 4 odd stripes, revealing pervasive maternal/gap input into 5 of the 7 patterned pair rule genes. With this complete set of pair rule enhancers, we reexamined the regulatory interactions between pair rule genes, which, through strong cross repression, produce two pairs of mutually exclusive 7-stripe patterns and, through weaker differential repression, create gradients and single-cell offsets in the expression patterns. We find that pair rule input is not restricted to 7-stripe elements but also required for proper pattern formation of several stripe-specific elements. Our findings suggest a new network structure which emphasizes the importance of eve, ftz and odd, the pair rule genes whose patterns are in register with the parasegments of the embryo.

84 Reading between the lines: pair-rule regulation of cell adhesion molecules. W. Ray Anderson1,2, Leslie Pick2. 1) Dept. of Cell Biology and Molecular Genetics and; 2) Dept. of Entomology, University of Maryland, College Park, MD 20742, USA. Transcriptional regulation plays a key role in development, yet transcription factors alone cannot build an embryo. Decades of study in the model organism Drosophila melanogaster have identified cascades of selector genes responsible for patterning the body plan in the early embryo, yet few realizator genes have been found. The seven striped expression pattern of the pair-rule genes is among the earliest specification events of the zygotic genome. It has long been known that the pair-rule system establishes the segment polarity gene network along the anterior-posterior axis. The expression of engrailed subdivides each segment into an anterior and posterior compartment. However, it remains an open question: are pair-rule genes, in addition to positioning stripes of engrailed, also “writing between the lines”? Using bioinformatics, RNA expression data, and more recently, transcriptional profiling of tightly staged wild type and ftz factor 1 (ftz- f1) pair-rule mutant embryos, we have identified additional genes in the transcriptional cascade downstream of fushi tarazu (ftz). This microarray analysis identified tartan (trn) as an early target of Ftz-F1. Tartan is a cell surface protein implicated in cell sorting and establishment of compartmental boundaries in the wing disc. Early trn expression overlaps with ftz and is dependent upon ftz and ftz-f1. The cross regulatory nature of the pair-rule system led us to look for genes encoding cell adhesion molecules having complementary transcriptional expression patterns. This identified Toll-6, which is structurally related to Tartan. Further analysis identified a set of structurally related genes implicated in cell adhesion with expression patterns matching additional pair-rule genes. This suggests a pair-rule regulated cell surface code capable of sorting individual cells into appropriate compartments. We are characterizing the expression patterns of these genes in pair-rule mutants as well as the cell sorting properties of combinations of these proteins in embryos and cell culture.

85 Regulation of Ft-Ds signaling by the DHHC transmembrane protein Approximated. Hitoshi Matakatsu, Seth Blair. Zoology, University of Wisconsin-Madison, Madison, WI. The protocadherins Fat (Ft) and Dachsous (Ds) are required for several processes in the development of Drosophila, including planar cell polarity (PCP) and the proximodistal patterning of appendages such as wings and legs. Work from our own and other laboratories indicates that some or all of these effects are mediated by a signaling pathway that is modulated by binding between Ft and Ds. Although recent studies show that Ft regulates growth control via the Hippo signaling pathway, it is unclear what other molecules mediate Ft-Ds signaling. We have therefore been analyzing mutations that show similar phenotypes for proximodistal patterning. approximated (app) mutants have a proximodistal patterning defect, and we have obtained new app mutants by EMS screening that show PCP defects in wing and abdomen. We found that app encodes a multipass transmembrane protein containing a DHHC-CRD (cysteine-rich domain). DHHC proteins have been recently found to add palmitate fatty acids to cytoplasmic proteins, thereby regulating their association with cell membranes. Most DHHC proteins are localized to, and thought to act in, the ER and Golgi. However, we found that App is concentrated at the apical cell cortex, overlapping the region where Ds and Ft are concentrated. We will present data that App regulates the membrane localization of select components of the Ft-Ds signaling signaling pathway. 114 PLATFORMS: Pattern Formation

86 Drosophila glypican Dally-like acts in FGF-receiving cells to modulate FGF signaling during tracheal morphogenesis. Dong Yan1,2, Xinhua Lin1,2. 1) Div Developmental Biol, Cincinnati Children’s Hosp, Cincinnati, OH; 2) The Graduate Program in Molecular and Developmental Biology, University of Cincinnati College of Medicine, Cincinnati, OH. Previous studies in Drosophila have shown that heparan sulfate proteoglycans (HSPGs) are involved in both breathless (btl)- and heartless (htl)-mediated FGF signaling during embryogenesis. However, the mechanism(s) by which HSPGs control Btl and Htl signaling is unknown. Here we show that dally-like (dlp, a Drosophila glypican) mutant embryos exhibit severe defects in tracheal morphogenesis and show a reduction in btl-mediated FGF signaling activity. However, htl-dependent mesodermal cell migration is not affected in dlp mutant embryos. Furthermore, expression of Dlp, but not other Drosophila HSPGs, can restore effectively the tracheal morphogenesis in dlp embryos. Rescue experiments in dlp embryos demonstrate that Dlp functions only in Bnl/FGF receiving cells in a cell-autonomous manner, but is not essential for Bnl/FGF expression cells. To further dissect the mechanism(s) of Dlp in Btl signaling, we analyzed the role of Dlp in Btl-mediated air sac tracheoblast formation in wing discs. Mosaic analysis experiments show that removal of HSPG activity in FGF-producing or other surrounding cells does not affect tracheoblasts migration, while HSPG mutant tracheoblast cells fail to receive FGF signaling. Together, our results argue strongly that HSPGs regulate Btl signaling exclusively in FGF-receiving cells as co-receptors, but are not essential for the secretion and distribution of the FGF ligand. This mechanism is distinct from HSPG functions in morphogen distribution, and is likely a general paradigm for HSPG functions in FGF signaling in Drosophila.

87 The homeotic genes labial and Deformed regulate decapentaplegic expression restricted to the peripodial epithelium and required for the formation of the adult head. Brian Stultz1, Mark Mortin2, Deborah Hursh1. 1) DCGT, FDA/CBER, Bethesda, MD; 2) LMG, NICHD/NIH, Bethesda, MD. Expression of decapentaplegic (dpp) in the lateral peripodial epithelium of the eye/antennal disc is necessary for correct morphogenesis of ventral structures of the adult head of the fly. This expression overlaps significantly with the expression of both labial (lab) and Deformed (Dfd) in the peripodial epithelium of the eye/antennal disc. A dpp mutation whose mutant phenotype is limited to the adult head, dpps-hc1, is a 15 bp deletion in the 5’ enhancer region of dpp which removes a HOX consensus binding site, as well as sites predicted to bind the HOX co-factors homothorax (hth) and extradenticle (exd). A 600 bp region around this deletion is rich in putative HOX, hth, and exd binding sites, and produces spatially correct peripodial dpp expression in β-galactosidase reporter constructs. Production of loss of function clones in eye/antennal discs indicates that lab and hth are required for activation of the dpp enhancer, while Dfd negatively regulates lab expression. Putative Lab, Hth, and Exd sites in and adjacent to the 15 bp deletion are required for correct enhancer function in β-galactosidase reporter constructs. Thus the morphogenesis of the adult head from the bilayer eye/antennal disc is regulated by a genetic network of HOX proteins and the dpp signal transduction pathway, restricted to the peripodial epithelium of this structure.

88 The anterior-posterior gradient of microtubule organization in the oocyte depends on Par-1-induced Tau phosphorylation. Ai-Guo Tian, Wu-Min Deng. Dept Biological Sci, Florida State Univ, Tallahassee, FL. Specification of the anterior-posterior (AP) axis in Drosophila oocytes requires posterior enrichment of the serine/threonine kinase Par-1 and proper organization of the microtubule cytoskeleton, but the mechanism by which Par-1 regulates the microtubule cytoskeleton in the oocyte has been unknown. The work reported here demonstrates that it does so through phosphorylation of the microtubule-associated protein Tau. Our results show that excessive overexpression of Par-1 or the Par-1 kinase domain in the germ-line cells disrupts microtubule organization in the oocyte. This phenotype is similar to that of the egg chambers taken from females fed the microtubule-depolymerizing drug colcemid. Interestingly, germ-line clones of tau also showed similar microtubule defects in the oocyte, suggesting a critical role for Tau in oocyte polarity. Using both biochemical and immunocytochemical approaches, we found that Tau is phosphorylated by Par-1 in the oocyte. This phosphorylation prevents active Tau from localizing to the posterior of the oocyte, thus destabilizing the microtubule-organizing center in the posterior and therefore causing the establishment of an AP gradient of microtubule distribution, which is essential for AP axis specification. PLATFORMS: Pattern Formation 115

89 Drosophila EGFR signaling is modulated by differential compartmentalization of Rhomboid intra-membrane proteases. Shaul Yogev, Eyal Schejter, Benny Shilo. Molecular Genetics, Weizmann Institut , Rehovot, Israel. EGF Receptor (EGFR) signaling is repeatedly employed throughout development to coordinate cellular decisions. In Drosophila, the precursor form of the cardinal EGFR ligand, Spitz, associates with the chaperone Star in the ER and is transported to a late compartment of the secretory pathway. There, cleavage by the intra-membrane protease Rhomboid-1 (Rho-1) releases the mature ligand and inactivates the chaperone. How versatility in signaling may arise from such a conserved core machinery is an open question. We now show that two additional Rhomboid proteases, Rho-2 and Rho-3, expressed in the germline and the developing eye, respectively, are localized and active in the ER as well as in the late compartment. While late-compartment cleavage by Rho- 2 and Rho-3 leads to EGFR activation, their ER activity plays an attenuating role, mainly due to premature cleavage and inactivation of the chaperone Star. Thus, differential compartmentalization of Rhomboids is used to modulate the levels of active ligand secretion in developmental settings where a tight control over EGFR activation range is required.

90 Regeneration genes affect the position, time and amount of blastema formation. Anne Sustar1, Kim McClure1,2, Gerold Schubiger1. 1) Dept Biology, Univ Washington, Seattle; 2) Dept Anatomy, UC San Francisco. Regeneration has been a curiosity for millennia and experiments have been performed for over 200 years. However a molecular and genetic understanding of the process is still in its infancy. Drosophila imaginal discs, like appendages in lower vertebrates, initiate regeneration by wound healing followed by localized proliferation of the regeneration blastema. In many systems the Wingless (Wg) signaling pathway is activated during wound healing and is necessary and sufficient for blastema formation. Progress in this field requires the identification of Wg target genes and their functions in regeneration. Our genome-wide search identified 143 candidate regeneration genes. We focus on candidate genes that are not expressed during leg disc development but are activated at wound sites and in the regeneration blastema by wg expression. In functional tests with this class of genes, we find that all of them dominantly modify regeneration, including: augmenter of liver regeneration (alr, CG12534), regeneration (rgn, CG6014), and Matrix Metalloproteinase 1 (Mmp1). These three genes are conserved in mammalian regeneration. Homozygous Drosophila mutants of rgn and Mmp1 have imaginal discs. We find that mutations in these genes delay, reduce or increase blastema growth, and later modify the discs’ regeneration capacity. We conclude that these genes function in the regeneration process of the organ but not in its normal development. 116 PLATFORMS: Drosophila Models of Human Diseases

91 A prion-like domain in Drosophila fragile X protein is essential for regulating synaptic plasticity. Paromita Banerjee1, Brian P. Schoenfeld2, Sean M.J. McBride2, Thomas C. Dockendorff1. 1) Dept Zoology, Miami Univ, Oxford, OH; 2) Molecular Cardiology, Albert Einstein College of Medicine, Bronx, NY. Fragile X mental retardation proteins regulate translation at synapses and their activity is vital for cognitive processes and synaptic plasticity in all species in which their function has been examined. The Drosophila fragile X protein (dFMR1) has a domain enriched in glutamine and asparagine residues that is similar to prion-like domains described in fungi and Aplysia. The dFMR1 prion-like domain confers protease resistance to green fluorescent protein and induces its aggregation in both yeast cells and Drosophila tissues, indicating that this prion-like domain can modulate the conformation of proteins. We have generated transgenic animals where the sole allele of dfmr1 that is present lacks the ability to code for the prion-like domain. The resulting mutant protein is stable and has no obvious anomalies in spatial expression as judged by Western blotting and whole-mount stains of adult brains. These mutant animals were examined for several neural development and behavior phenotypes that are associated with null alleles of dfmr1. Axon guidance and germline development phenotypes resulting from null alleles of dfmr1 are rescued by the mutant transgene, indicating that dFMR1 protein lacking the prion-like domain has some in vivo function. Notably, the dFMR1 prion-like domain is essential for proper regulation of synapse development at the larval neuromuscular junction, for normal circadian locomotion activity, and for naive courtship behavior and memory after conditioned courtship training. The circadian and courtship anomalies exhibited by the mutants show that the biochemical activity of the prion-like domain establishes the neural circuitry necessary for execution of these behaviors. Our results demonstrate a direct role by a prion-like domain in mediating behavior and synaptic plasticity within an animal model, and underscore the possibility that prion-like domains are vital for regulation of many physiologic processes in metazoans.

92 De novo CoA biosynthesis is required to maintain DNA integrity in a Drosophila model of Pantothenate Kinase-Associated Neurodegeneration. Ody Sibon, Floris Bosveld, Anil Rana, Harm Kampinga. Dept Cell Biol, Univ Groningen, Groningen, Netherlands. In humans, mutations in the PANK2 gene, coding for the first enzyme in the CoA biosynthesis pathway, are associated with Pantothenate Kinase-Associated Neurodegeneration (PKAN). Currently, the pathogenesis of this devastating neurodegenerative disorder is poorly understood. From a forward genetic screen in Drosophila, aimed to identify genes involved in surviving induced DNA damage, we isolated dPPCS (the second enzyme of the CoA biosynthesis pathway) as a novel gene required to maintain DNA integrity. The complete Drosophila CoA synthesis route was dissected, annotated and additional mutants that carry mutations in CoA enzymes were obtained (dPANK/fumble) or generated (dPPAT-DPCK) and used for further investigation. Drosophila CoA mutants are hypersensitive to ionizing radiation, suffer from altered lipid homeostasis, and the larval brains display increased apoptosis and elevated levels of damaged DNA. In addition, disruption of CoA synthesis in general provokes neurodegeneration in adults. Our data provide the first comprehensive analysis of the physiological implications of mutations in the entire CoA biosynthesis route in an animal model system. Surprisingly, our findings reveal a major role of this conserved metabolic pathway in maintaining DNA and cellular integrity during development of the central nervous system, and the data explain how impairment of CoA synthesis during development can lead to neurodegeneration in Drosophila. The presented Drosophila model will be of help to understand the consequences of impaired de novo CoA synthesis in higher eukaryotes and may provide insights in the pathogenesis of PKAN.

93 Using Drosophila to probe the activities of anthrax toxins. Annabel Guichard1, Shauna Mc Gillivray2, Beatriz Cruz-Moreno1, Victor Nizet2, Ehan Bier1. 1) Division of Biological Sciences, UCSD, La Jolla, CA; 2) Division of Pediatric Pharmacology & Drug Discovery UCSD School of Medicine La Jolla, CA. Anthrax is a severe and widely distributed disease caused by Bacillus anthracis, and is still a significant threat in underdeveloped countries. Anthrax primarily affects cattle, and occasionally humans, when contact occurs with sick animals, or, as in the case of the 2001 attack, when weaponized spores are inhaled. B. anthracis achieves infectivity mainly through the secretion of three toxins, PA (Protective antigen), EF (Edema Factor), and LF (Lethal Factor). After binding to surface receptors present on most mammalian cells (TEM8 and CMG2), PA gets endocytosed and permits the entry of EF and LF into the cytoplasm. EF is a potent Calmodulin- dependent Adenylate cyclase, and LF is Zinc metalloprotease that cleaves and inactivates most human Mitogen Activated Protein Kinase Kinases (MAPKK), and possibly unknown targets. B. anthracis is not known to infect insects, which lack homologs to the known receptors necessary for EF and LF entry. However, when expressed intracellularly in transgenic flies, LF and EF induce developmental phenotypes reflective of their established activities. For example, we found that LF inhibits dorsal closure during embryogenesis, and most likely acts at the level of Hemipterous, the Drosophila MAPKK acting in the JNK pathway essential to this process. EF also induces expected effects such as a hedgehog-like phenotype in the wing, consistent with the known role of cAMP- dependent PKA in inhibiting hedgehogh signaling. Here we show that LF and EF induce additional phenotypes suggesting that they cooperate to inhibit signaling pathways not known previously to be affected by these toxins. Importantly, we find that EF and LF act in the same capacity in human endothelial cells, suggesting a novel activity for anthrax toxins relevant to their pathogenicity. PLATFORMS: Drosophila Models of Human Diseases 117

94 Drosophila easily-shocked: phosphatidyl-ethanolamine metabolism and cardiac disorders. Hui-Ying Lim1, Robert J. Wessells2, Rolf Bodmer1. 1) Burnham Inst.for Medical Research, La Jolla, CA; 2) U.Mich,Ann Arbor,MI. We study the role of phosphatidyl-ethanolamine (PE) homeostasis in cardiac function using Drosophila easily-shocked (eas) mutants as a model. Loss-of-eas-function causes the fly to exhibit an increased risk of heart failure under conditions of stress induced by electrical pacing. Conversely, cardiac-specific expression of eas results in fly hearts being more resistant to pacing- induced stress. Moreover, the replenishment of wild-type Eas in the hearts of young eas flies afforded rescue of pacing-induced heart failure to a level close to the wild-type controls, thereby establishing the cardiac phenotypes as a consequence of the eas mutation. The eas mutant harbors a defect in PE metabolism, a fly model in epilepsy. Coincidentally, epileptic patients appear to experience an increased incidence of heart abnormalities that may be significant contributors to causing sudden death. The eas fly serves as an ideal system to explore the molecular basis of PE homeostasis underlying both neuronal and cardiac syndromes. To further examine heart function of eas flies, we analyzed the dynamic properties in semi-intact heart preparations, including heart rate, rhythmicity and contractility. In addition to alteration in cardiac performance, eas mutants displayed myocardial heart tube thinning, which could be rescued with heart-specific expression of eas. Previous studies in S2 cells showed that excess PE feedback inhibits the dSREBP pathway and downregulates the expression of target genes involved in fatty acid and phospholipid synthesis. To determine whether dSREBP is a potential effector of PE signaling involved in cardiac physiology, we expressed a dominant- negative form of dSREBP in eas mutants and found an alleviation of the eas heart phenotypes under both physiological and stressed conditions. Moreover, overexpression of constitutively-active dSREBP partially mimics the cardiac eas phenotype. Collectively, these results suggest that a defect in PE homeostasis of eas mutants may lead to excessive activity of the dSREBP pathway, and thereby resulting in cardiac abnormalities.

95 Regulation of ER stress induced apoptosis by the Unfolded Protein Response. Min-Ji Kang, Hyung Don Ryoo. Department of Cell Biology, NYU, School of Medicine, New York, NY. Proteins that fail to properly fold in the endoplasmic reticulum(ER), activate a signaling network known as the unfolded protein response (UPR), which leads to an increase in the capacity of ER to fold its client proteins or facilitate their degradation. A stress level in the ER beyond a certain threshold leads to apoptosis. Although activation of this cell death pathway has been implicated in certain degenerative disorders, its mechanisms remain controversial and unclear. We found that the UPR pathway involving the endonulease ire-1 and its mRNA target xbp1 is also conserved in Drosophila. The activity of this pathway can be measured through our xbp1-GFP sensor, which in response to ER stress, undergoes splicing and GFP activation. The xbp1-GFP is also activated in the retina of a retinal degeneration disease model caused by mutated Rhodopsin-1 alleles, ninaEG69D/+, and ninaERH27/+ flies. When ninaE is prematurely over expressed during eye disc development, it triggers a strong UPR reaction as evidenced by xbp1-GFP and an eye ablation phenotype due to excessive apoptosis. Mutation in ire-1 further enhances the extent of apoptosis, indicating that the phenotype is associated with ER stress. This has prompted us to examine the link between ER stress and apoptosis. Apoptosis induced by ninaE over expression is suppressed by p35, an effector caspase inhibitor. Unexpectedly, our initial evidence suggests that the initiator caspase Dronc is not involved in ER-stress triggered apoptosis. One of the ER associated degradation (ERAD) factors, sip3 suppresses the eye ablation by lowering the levels of the misfolded protein. These results establish a new model to study ER-stress induced apoptosis and identify targets for therapeutic intervention in ER stress-related diseases.

96 Genetic Interaction between Survival of Motor Neuron Gene (SMN) and BMP Signaling Pathway. Howard Chang1, Yokokura Takakazu1, Dimlich Douglas1, Kankel Mark1, Mukherjee Ashim2, Walker Amy3, Harris Jevede3, Duckworth April1, Hart Anne3, Van Vactor David1, Artavanis-Tsakonas Spyros1. 1) Dept Cell Biology, HMS, Harvard Medical School, Boston, MA; 2) Department of Molecular and Human Genetics Banaras Hindu University Varanasi-221005 India; 3) MGH Cancer Center, Building 149,Charlestown, MA. Spinal Muscular Atrophy (SMA) is a human neurodegenerative disease caused by mutations in the Survival of Motor Neuron (SMN) gene. Loss-of-function SMN leads to clinical manifestation, including neuron degeneration, muscle atrophy and lethality. SMN protein is ubiquitously expressed and is involved in RNA processing. Drosophila However, the tissue specific nature of SMN in neuron and muscle remains elusive. Here, we established a Drosophila model to investigate the SMN genetic circuitry, particularly in neuron and muscle. By using lethality and neuromuscular junction (NMJ) as markers, we found that both neuron and muscle are sensitive to SMN knockdown. Most importantly, the severity of both lethality and NMJ phenotypes correlate to the dosage of SMN in flies. We also investigated the SMN expression pattern at larval NMJ and found SMN is mostly localized at the post-synaptic structure. Finally, we performed a genome-wide genetic screen for SMN modifiers. We identified wit, a component of BMP signaling pathway, enhances SMN dependant lethality and NMJ phenotype. In addition, the BMP signal is reduced in the SMN knockdown background. Moreover, null mutation of dad, an antagonist of BMP signaling pathway, rescued the SMN dependant NMJ phenotypes. We conclude that we have found links between SMN and BMP signaling pathway at neuromuscular junction, which might open new avenues for SMA therapy. 118 PLATFORMS: Drosophila Models of Human Diseases

97 Regulation of phosphoinositide phosphates in Drosophila morphogenesis. Inês Ribeiro, Jared Dennis, Amy Kiger. Div Biological Sciences, Univ California, San Diego, La Jolla, CA. We are investigating the mechanisms that mediate subcellular spatial regulation important for cell shape changes. Phosphoinositide phosphates (PIPs), the phosphorylated forms of phosphatidylinositol, control localized and dynamic cellular processes through the recruitment of specific PIP-binding proteins. The association of mutations in PIP regulators with human diseases further demonstrates that PIP regulation is crucial. However, little is known about the identities and developmental requirements of PIP response in vivo. To address roles for PIPs in morphogenesis, I am taking two approaches. First, I adapted a collection of fluorescently-tagged PIP biosensors for systematic analysis of dynamic PIP subcellular distribution in Drosophila development. The specificities of the PIP biosensors were tested by functional assays in cell cultures, and verified constructs were used to generate transgenic fly lines. I am using these PIP reporter flies to determine the temporal-spatial distribution of specific PIPs during major morphogenetic events of wildtype embryogenesis, including dorsal closure and muscle development. The PIP reporters will be insightful in mutant backgrounds. Secondly, I am using Drosophila genetic mutant analysis to characterize developmental roles for specific PI(3)P regulators implicated in morphogenesis. We previously demonstrated that antagonistic function of the mtm phosphoinositide phosphatase and the class II PI3-kinase, PI3K68D, regulate a PI(3)P-dependent cell shape change. We therefore generated mutant alleles to study mtm and PI3K68D in development revealing that both are essential genes. Importantly, tissue-specific knockdown of mtm suggests its role in adult muscle development, reminiscent of mutations in the highly conserved human MTM1 responsible for X-linked myotubular myopathy. I am currently addressing the underlying role for mtm in muscle morphogenesis, as well as the muscle-requirements for PIP subcellular distribution.

98 Modeling human brain cancer in Drosophila. Renee D. Read, John B. Thomas. Molecular Neurobiology Laboratory. Salk Institute for Biological Studies. San Diego, CA 92037. Gliomas, neoplasms of glial cells and their precursors, are the most common and deadly malignant tumors of the central nervous system (CNS). These tumors diffusely infiltrate the brain and grow rapidly, properties that render them largely incurable. Formation of these tumors is a complex process involving accumulation of mutations in many genes, only some of which are known. In particular, constitutive activation of the EGFR-Ras and PI-3 kinase signaling pathways is a common feature in gliomas and is sufficient to cause glioma in mouse models. Yet, how these pathways specifically regulate glioma pathogenesis is unknown. To understand the molecular basis for this disease, we have developed a novel model in Drosophila for the purpose of carrying out large-scale genetic analyses to identify genes involved in glioma invasion and proliferation. The Drosophila CNS contains multiple glial cell types that are strikingly similar to their vertebrate counterparts in terms of function, development, and gene expression. Using techniques that target gene expression in glia and glial precursors, we have found that co-activation of both the EGFR-Ras and PI-3 kinase pathways in Drosophila glia gives rise to proliferative, invasive cells that create tumor-like growths in the fly brain, mimicking the human disease. We are now performing genetic screens for enhancers and suppressors of the EGFR/Ras-PI-3 kinase phenotype in order to identify new regulators of glial neoplasia. Furthermore, we are performing misexpression screens for additional loci that cause glioma-like phenotypes in the fly brain. The genes already identified in these screens represent excellent candidates for genes directly involved in pathogenesis.

99 Mutations in the gene clueless cause mitochondrial mislocalization and Parkinson-like phenotypes in the Drosophila ovary and muscle. Rachel Cox1,2, Megan Kutzer1, Shelley Paterno1,2, Allan Spradling1,2. 1) Dept Embryology, Carnegie Inst of Washington, Baltimore, MD; 2) HHMI. Mutations in mitochondrial DNA and in nuclearly encoded mitochondrial proteins are responsible for a large number of diseases, both spontaneous and inherited. A hallmark of mitochondrial and aging diseases is neuromuscular degeneration. While many neurodegenerative diseases such as Parkinson’s are associated with decreased mitochondrial function, it is becoming increasingly clear that poorly functioning mitochondria may be a primary cause of the disease, and not simply a result. We found the previously unstudied fly gene clueless (clu) causes severe effects on mitochondria in both muscle and ovarian cells. Mutations in clu are semi- lethal and clu mutants are short-lived. The mutant adults that are able to eclose are very uncoordinated, suggesting neuronal problems. clu also has a striking effect on flight muscle fibers and their mitochondria, causing indistinct sarcomere banding and enlarged, empty mitochondria. The muscle phenotype, lack of coordination, and aging defects are reminiscent of fly mutants in the parkin pathway, the homolog of a gene linked to familial Parkinson’s disease in humans. In the ovary, clu mutants exhibit mislocalized mitochondria clustered to one side of the cell in germline stem cells and germ cells, as well as in a subset of ovarian somatic cells. Mutant females exhibit reduced fertility and males are sterile. Clueless’ molecular function is unknown. The high degree of sequence identity from humans to yeast suggests Clueless is an important conserved mediator of mitochondrial movement and function. Studying clu function offers the opportunity to elucidate the link between mitochondrial mislocalization and function and neuromuscular degeneration. PLATFORMS: Drosophila Models of Human Diseases 119

100 ubiquilin antagonizes presenilin, stabilizes APP and promotes neurodegeneration. Ming Guo, Atish Ganguly, Renny Feldman. Dept Neurology & Pharmacology, Univ California, Los Angeles, Los Angeles, CA. Mutations in the Amyloid Precursor Protein (APP) and Presenilin, the catalytic component of the gamma-secretase complex, mediate familial Alzheimer’s disease (AD). Therefore, identifying regulators of Presenilin and APP is crucial for understanding AD pathogenesis. Recently, a splicing variant, in the ubiquilin 1 gene (UBQLN1) was reported to associate with an increased risk for late-onset AD. Previously, UBQLN1 was found to bind Presenilin in mammalian cells; however, the functional significance of this interaction in vivo remains unclear. Moreover, whether the disease-associated variants in UBQLN1 have altered function is unknown. Drosophila contains a single homolog of UBQLN1, Ubiquilin (Ubqn), which is a protein with a ubiquitin-like (UBL) domain and a ubiquitin associated (UBA) domain. We show that Drosophila Ubqn binds to Drosophila Presenilin (Psn) via its UBA domain, and that loss of ubqn function suppresses phenotypes that arise from loss of psn function during development. Furthermore, overexpression of ubqn in the eye results in adult-onset, age-dependent retinal degeneration, which is at least partially apoptotic in nature. The degeneration associated with ubqn overexpression can also be suppressed by psn overexpression and enhanced by expression of a dominant negative version of Psn. Remarkably, expression of the human AD-associated variant of UBQLN1 leads to significantly more severe and earlier onset of eye degeneration than does comparable expression of the human wildtype UBQLN1. In addition to interact with Psn, we find that Ubqn also physically binds APP via its UBA domain. Loss of ubqn function leads to a decrease in the steady state levels of APP, whereas ubqn overexpression results in an increase in the APP levels. Together, these data identify Ubqn as a regulator of Psn and APP, support an important role for UBQLN1 in AD pathogenesis, and suggest the possibility that expression of a human AD-associated variant can cause neurodegeneration independent of amyloid production. (AG & RF contributed equally.)

101 Genomic regulation of lipid storage. Mathias Beller1,2, Carole Sztalryd1,3, Herbert Jaeckle2, Brian Oliver1. 1) Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda MD 20892, USA; 2) Max-Planck-Institut fuer biophysikalische Chemie, Abt. fuer Molekulare Entwicklungsbiologie, Am Fassberg 11, 37077 Goettingen, Germany; 3) GRECC/Geriatrics , Veterans Affairs Medical Center 10, North Greene Street, Baltimore MD 21201, USA. Lipid droplets are the universal lipid storage organelles and crucial for maintaining organismic as well as cellular lipid and energy homeostasis. Given an emerging importance of lipid droplets in major diseases such as obesity, diabetes and atherosclerosis, the need to understand the regulation and function of this organelle has become urgent. To identify the range of biochemical pathways involved in lipid droplet function as well as specific regulators, we performed a genome-wide RNA interference screen in Drosophila Kc167 cells. Our data reveal that about 3% of the genome is required for cellular lipid droplet deposition or utilization. Most of the few known lipid metabolism regulators were identified. Importantly, the majority of Drosophila genes required for lipid droplet deposition/ utilization were previously not associated with lipid storage. They belong to pathways acting on distinct levels of cell function including gene expression, signaling, oxidative phosphorylation and vesicle-mediated transport. Secondary RNAi screening targeting orthologs of 100 selected Drosophila genes in a mammalian liver cell line showed similar phenotypes and demonstrated evolutionary conservation of function. Our findings suggest a major, evolutionary conserved role of COPI-mediated retrograde vesicle transport and mitochondrial fatty acid beta-oxidation for lipid droplet homeostasis. Further analysis of these and additional identified gene- functions such as signaling components and transcription factors will aid understanding cellular lipid storage regulation and may reveal novel approaches and targets for future therapeutic treatments.

102 Genetic modifiers of MeCP2 function in Drosophila. David Mittelman1, Holly Cukier1, Ann Collins1, Alma Perez1, Zhaolan Zhou1, Huda Zoghbi1,2, Juan Botas1. 1) Departemnt of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 2) Howard Hughes Medical Institute, Baylor College of Medicine, Houston TX. Rett syndrome (RTT) and some cases of autism are caused by a reduction of methyl-CpG-binding protein 2 (MeCP2), while duplication or triplication of the MECP2 locus cause a progressive neurological disorder characterized by mental retardation, seizures, and motor abnormalities. Similar phenotypes are recapitulated in mice that either lack or overexpress MECP2, thus underscoring the importance of properly controling MeCP2 levels. The tight regulation of this protein currently negates gene therapy as a treatment. To identify molecular mechanisms capable of compensating for altered MeCP2 function, we generated transgenic Drosophila producing human MeCP2. We find that MeCP2 associates with chromatin and is phosphorylated at serine 423 in Drosophila, as is found in mammals. Tissue-specific MeCP2 expression leads to anatomical (i.e., disorganized eyes, ectopic wing veins) and behavioral (i.e., motor dysfunction) abnormalities. Interestingly, these phenotypes are modified by Drosophila homologs of MeCP2 protein interactors Sin3a, REST and N-CoR. Furthermore, we identified novel genetic modifiers of MeCP2 including factors that function in chromatin remodeling. 120 PLATFORMS: Drosophila Models of Human Diseases

103 Generation of Neurotoxic Prion Protein Isoforms and the Role for Hsp70 in Prion Protein Conversion. Sergio Casas-Tinto, Melisa Gomez-Velazquez, Claudio Soto, Pedro Fernandez-Funez, Diego E. Rincon-Limas. Department of Neurology, University of Texas Medical Branch, Galveston, TX 77555, USA. Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrPC) converts into a misfolded and pathogenic isoform (PrPSc) whose unique biochemical and structural properties correlate with disease. In humans, prion disorders such as Creutzfeldt-Jakob disease have typically a sporadic origin, where wild type PrP spontaneously misfolds and aggregates in the brain. Unfortunately, major gaps still exist in the knowledge of how wild type PrP undergoes conformational changes and what are the cellular components involved in this process. To shed light on these issues, we studied the conformational dynamics of wild type PrP from Syrian hamster in transgenic flies. In fly neurons PrP progressively misfolds and acquires biochemical and structural properties of PrPSc, such as insolubility in non-ionic detergents, resistance to denaturing agents and recognition by PrPSc-specific conformational antibodies. The PrP isoform generated in flies is highly neurotoxic as it readily induces spongiform degeneration of brain neurons. Unlike mammalian PrPSc, fly-produced PrP is not resistant to protease digestion, a key feature of infectious PrP. This difference indicates that the neurotoxic conformer in prion diseases might be an intermediary conformer in the process of PrPSc formation. In an attempt to interfere with PrP misfolding in flies, we found that co-expression of PrP and the molecular chaperone Hsp70 protects against PrP-dependent neurodegeneration. Moreover, Hsp70 colocalizes and co- immunoprecipitates with abnormal PrP conformers. Additionally, we also found that Hsp70 inhibits amplification of mammalian PrPSc in a cell-free conversion system in vitro. Therefore, the direct interaction of Hsp70 and PrP prevents PrP misfolding. These results provide new and valuable insight into the mechanisms of spontaneous accumulation of neurotoxic PrP and uncover the potential therapeutic role of Hsp70 in treating these devastating disorders.

104 Rhomboid-7 and Omi are components of the Pink1/Parkin pathway which affects mitochondrial membrane dynamics. Alexander J Whitworth1, Jeffrey Lee2, Angela Poole3, Ruth Thomas3, Venus Ho1, Leo Pallanck3, Angus McQuibban2. 1) Dept of Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom; 2) Dept of Biochemistry, University of Toronto, Toronto, Ontario, Canada; 3) Department of Genome Sciences, University of Washington, Seattle, USA. Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by prominent loss of dopaminergic neurons in the substantia nigra and the presence of Lewy body inclusions, however, the pathogenic causes remain unclear. Mitochondrial dysfunction has long been implicated in the pathogenesis of PD but has recently been highlighted as a key pathologic mechanism. Genes identified in heritable forms of PD have provided a compelling link to mitochondria, however, the exact function of these genes and the events that lead to mitochondrial dysfunction are unknown. Targeted mutations in the Drosophila homologues of these PD-related genes has begun to reveal important insight into the their biological function. Recently, it was shown in Drosophila that the PD-linked genes pink1 and parkin act in a common pathway that maintains mitochondrial integrity by an unknown mechanism. Here, we identify the mitochondrial rhomboid protease, Rhomboid-7, as a novel upstream regulator in this pathway. We also show the mitochondrial protease Omi/HtrA2, along with Parkin, is a downstream effector of this pathway. Our biochemical data demonstrate a functional molecular link between Rhomboid-7 and both Pink1 and Omi, and genetic epistasis data place these factors in a genetic hierarchy. Furthermore, we present evidence that Pink1 and Parkin genetically interact with components of the mitochondrial fission and fusion machinery. Our findings indicate the Pink1/Parkin pathway likely functions to promote mitochondrial fission. These findings greatly further our understanding of the pathologic mechanisms of PD, firmly linking two new factors in a common pathway that regulates mitochondrial function. Furthermore, they raise the possibility that regulated intra-membrane proteolysis by Rhomboid-7 is a potential therapeutic target for the treatment of PD. PLATFORMS: RNA Biology 121

105 Global analysis of mRNA localization reveals a prominent role in the organization of cellular architecture and function. Eric Lecuyer1, Hideki Yoshida1, Christina Alm1, Neela Parthasarathy1, Tomas Babak1, Pavel Tomancak2, Henry Krause1. 1) Donnelly CCBR, University of Toronto, Toronto, Canada; 2) Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. The localization of mRNA molecules is an important regulatory mechanism for targeting proteins to specific cellular compartments, although the overall prevalence and variety of transcript localization events remains unknown. To characterize subcellular mRNA localization dynamics during early Drosophila embryogenesis, we conducted a high-throughput Fluorescent In Situ Hybridization (FISH) screen of over 4,000 distinct mRNAs, and show that the majority of expressed mRNAs (71%) are subcellularly localized. Many novel varieties of subcellular localization patterns were identified, implicating localized mRNAs in the assembly and regulation of diverse cellular modules and processes. Analysis of the localization data, which has been organized within a publicly available database (http://fly-fish.ccbr.utoronto.ca), reveals that transcripts with similar localization dynamics are enriched for specific gene functions and putative regulatory elements. This work establishes mRNA localization as a widespread gene regulatory mechanism and underscores the predictive value of transcript localization phenotypes in assigning gene functions. We have begun dissecting the localization mechanisms and biological functions of various classes of mRNAs and the results of these ongoing studies will be presented.

106 Fragile X Protein controls the efficacy of mRNA transport in neurons. Daniela C. Zarnescu, Patty Estes, Michelle O’Shea. Dept Molecular & Cell Biol, Univ Arizona, Tucson, AZ. Fragile X syndrome (FraX) is the most common form of inherited mental retardation and affects 1/4,000 males. The disease is caused by loss of function for the FMR1 gene, and patients affected by this disorder display cognitive deficits as well as attention deficit and hyperactivity, anxiety, and autism. FMR1 encodes a sequence specific RNA binding protein, FMRP, which is ubiquitously expressed and is thought to function in synaptic plasticity by controlling the localization and translation of target mRNAs in neurons. Using a biochemical purification approach and Affymetryx microarrays we have identified a subset of the Drosophila FMRP associated mRNAs. To determine if FMRP is required for the localization of these target mRNAs, we have developed a genetically encoded system for mRNA tracking in living cells. To visualize FMRP associated mRNAs in Drosophila neurons we are using a GFP tagged MS2 phage protein, which has the ability to bind with very high affinity a specific, stem-loop forming RNA sequence, referred to as the MS2 binding site. We cloned 12 MS2 binding sites downstream of a number of FMRP associated mRNAs, which can now be visualized via the MS2-GFP protein. Using the bipartite GAL4-UAS system we expressed these constructs in primary neuronal cultures and imaged live mRNA trafficking in wild-type and dFmr1 mutant neurons. Using this imaging system we have obtained evidence that mRFP-FMRP colocalizes in motile particles with target mRNAs in living neurons. Quantitative analyses of various trafficking features, including speed, net movement and directionality show that FMRP regulates the efficacy of mRNA transport in neurons. These results are supported by Fluorescent Recovery After Photobleaching experiments, which demonstrate that FMRP controls the dynamic exchange between the cellular mRNA pool and RNA granules. Taken together these data support a model whereby defects in FraX may be due to inefficient mRNA transport to synapses.

107 A genetic screen for asymmetrically localized RNAs in Drosophila tracheal cells. Jayan N Nair1, Maria Leptin1, Paolo Filardo2, Veit Riechmann2, Elizabeth R. Gavis3. 1) Institute for Genetics, University Of Cologne, Cologne, NRW, Germany; 2) Institute for Developmental Biology,University of Cologne,NRW, Germany; 3) Dept. of Molecular Biology, Princeton University, Princeton 08544, USA. Asymmetrical localization of mRNAs and localized protein synthesis have an important role in establishing and maintaining polarity in cells such as neurons or the Drosophila oocyte. Such localized protein synthesis provides a means for the regulation of developmental plasticity. In Drosophila a subset of highly branched cells of the respiratory system exhibits both a high degree of polarity and developmental plasticity. These tracheal cells respond to the need for oxygen in the surrounding tissue by outgrowth of branches, often at sites very distant from the nucleus. On the assumption that some of the proteins required at the site of outgrowth are synthesized locally rather than near the nucleus we have performed a screen for mRNAs with asymmetric subcellular localization. We tagged mRNAs with GFP in vivo and screened the Drosophila genome for asymmetrically localizing RNAs. Briefly, we have combined the MS2-GFP labeling system with the EP transposon technique. A fusion protein of GFP with the RNA binding protein MS2 is used to visualize RNAs carrying MS2 binding sites (RNA stem loop recognized by MS2 protein). The EP transposon, which we have modified by incorporating MS2 binding sites, can be used to generate transgenic lines harboring the EP-MS2 transgene. Each of these lines has a different gene tagged with MS2 binding sites and can be tested in different tissues using tissue specific GAL4-MS2-GFP lines. We have performed a pilot screen in Drosophila tracheal cells, oocytes and neurons and have identified 12 candidates exhibiting specific localization. We are currently analyzing and characterizing these candidates. In a broader perspective these candidates will be useful to understand the organization and function of the signals in mRNAs essential for their asymmetric localization. 122 PLATFORMS: RNA Biology

108 RNA silencing influences gypsy chromatin insulator function and nuclear organization. Elissa P. Lei, Nellie Moshkovich, Patrick J. Boyle. Laboratory of Cellular and Developmental Biology, NIDDK, NIH, Bethesda, MD. Chromatin insulators influence gene expression by establishing chromatin domains subject to distinct transcriptional controls, likely through alteration of their spatial organization. Several lines of evidence suggest that insulator proteins bridge distant DNA sequences dispersed throughout the genome, causing looping of the DNA and the creation of a distinct chromatin domain. Nuclear insulator complexes termed insulator bodies are tethered stably to the nuclear matrix possibly forming chromatin loops. Our research provides evidence that RNA silencing, a gene regulation mechanism known to act on the level of chromatin, is involved in gypsy insulator function and higher order chromatin organization. We suggest that RNA may promote the multimerization of insulator complexes and/or their ability to interact with a nuclear scaffold. Several new observations have been documented, which provide a more thorough mechanistic understanding of how RNA silencing contributes to gypsy insulator function. First, Argonaute proteins Piwi and Argonaute2 interact physically with the insulator protein CP190 similarly to the DEAD-box RNA- dependent helicase Rm62 but in an RNA-independent manner. In addition, Piwi can be found to colocalize with insulator bodies. Extensive analysis of known RNA silencing mutants has uncovered a role for two additional helicases, Armitage and Spindle-E. Like Rm62, Armitage and Spindle-E function as negative regulators of gypsy insulator function. Small scale purification of RNAs associated with the gypsy insulator complex has enriched a heterogeneous but specific class of RNA. Our current efforts are focused on thorough cataloguing of this pool of RNAs by deep sequencing and gaining mechanistic insight into this process using both biochemical and genetic approaches.

109 Analysis of the role of splicing and cis-acting elements in oskar mRNA localization in Drosophila oocyte. Sanjay Ghosh, Virginie Marchand, Anne Ephrussi. Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany. Localization of maternal mRNAs coupled with spatio-temporal control of their translation is a notable feature of early development of many organisms. In Drosophila, oskar mRNA, encoding the posterior determinant of the embryo, is localized in ribonucleoprotein (RNP) complexes, in a multi-step process, to the posterior pole of the oocyte, and its expression is regulated by localization- dependent translation control. Recent studies have demonstrated that splicing of the first intron of oskar and proteins of the exon junction complex (EJC) are required for posterior transport, suggesting interaction(s) between the EJC deposited at the first exon- exon junction upon splicing and trans-acting factor(s) binding to specific cis-acting elements in oskar mRNA. In order to test the sufficiency of splicing at the first intron and to uncover potential cis-acting localization elements in the oskar coding region, we have performed a systematic analysis in vivo involving transgenic deletion and replacement constructs. This study has identified a region within the oskar coding region that, together with splicing, is necessary for posterior localization of the mRNA. Furthermore, by using in vitro splicing and RNase H assays, we are identifying trans-acting factors bound specifically within this region that may affect splicing and/or EJC formation/deposition. The functional significance of the cis-element in formation of localization-competent oskar RNPs will also be discussed.

110 Control of alternative splicing by regulatory networks in Drosophila. Britta Hartmann1, R. Castelo1, S. Boue1, M. Blanchette2, E. Peden3, R. RioSingh3, D. Rio2, J. Valcarcel1. 1) Centre de Regulació Genòmica, Barcelona, Spain; 2) University of California, Berkeley, USA; 3) University of Colorado, Boulder, USA. Alternative splicing (AS) is a major contributor to the complexity of higher eukaryote proteomes. Paradoxically, however, little is known about how regulatory networks influence this process. To address this question we employed whole genome splicing-sensitive microarrays to explore the extent and biological impact of AS in two very different regulatory networks. Sex determination has served as a prime example for AS regulation. In contrast, though signaling pathways have been thoroughly studied in their impact on transcriptional regulation, virtually nothing is known if (and how) they regulate AS. Surprisingly, we identified over 400 genes exhibiting sex-specific AS in adult flies. Subsequent qRT-PCR analysis confirmed our microarray data and uncovered vast differences in isoform levels between sexes. Furthermore, we analyzed the AS pattern of over 40 candidates in the adult body, head and in embryonic cell-lines uncovering interesting tissue-specific patterns of sex-specific AS. For example bicoid possesses a strong sex- biased use of an alternative 3‘splice-site only in the adult head changing the translational activity of the protein. Surprisingly, binding sites of the known sex determinants Tra and Sxl were found only in a small portion of AS events. Overall, these results imply that other regulatory factors are involved in sex-specific splicing regulation. Indeed, RNA binding proteins are overrepresented in our list and could be candidates. The impact of two signaling pathways, Wingless and Insulin, on AS in Drosophila cells was also explored. Activation of both pathways resulted in extensive AS changes. Interestingly, similar functional groups of genes are affected at the level of AS and by gene expression. We observed many signaling components to be regulated suggesting another layer of crosstalk to other pathways. A computational screen among co-regulated AS events revealed new regulatory motifs, which we are currently validating. PLATFORMS: RNA Biology 123

111 Mir-3 and mir-318 regulate Drosophila nautilus gene expression. Anandarao Ravulapalli, Wei Qin, Bruce Paterson. Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. The Drosophila MyoD homolog, nautilus, is essential for normal myogenesis since its function is required to seed the correct founder myoblast pattern that prefigures the muscle fiber arrangement during embryonic development (Wei Q, et al., Proc. Natl. Acad. Sci. U.S.A. 104: 5461-6, 2007). The 3‘UTR of the nautilus gene is predicted to bind at least four microRNAs: mir-3, mir-309, mir-318 and mir-306*. To experimentally test this prediction, the nautilus 3‘UTR was sub-cloned directly downstream of the firefly luciferase ORF (F-luc-na-3‘UTR) and was then co-transfected into Drosophila Schneider S2 cells along with individual expression plasmids for the aforementioned miRNAs. Both mir-3 and mir-318 were found to substantially reduce luciferase activity when compared to either the control luciferase ORF without the nautilus 3‘UTR, or to the luciferase ORF with a nautilus 3‘UTR containing mutated microRNA binding sites. To try and understand the role of these microRNAs in vivo, we generated transgenic lines expressing mir- 3 and mir-318 using the gal-4/UAS system. Over expression of mir-3 under the control of the ubiquitously expressed pAct gal-4 driver resulted in a reduced and/or altered nautilus expression pattern and instances of severely disrupted embryonic muscle. However, similar expression of mir-318 did not affect either the embryonic nautilus or muscle pattern, even though mir-3 and mir- 318 are predicted to bind the same site in the nautilus 3’ UTR. This could reflect the fact that mir-3 expression initiates in stage 9 embryos just when nautilus expression is first noted, whereas mir-318 is expressed in the adult so prior site occupation by mir-3 may limit a mir-318 effect. We have initiated a transgene rescue of nauGFP, a nautilus null, using a genomic fragment lacking the mir-3/ 318 binding sites to determine if mir-3/318 regulation of nautilus expression during development is important. The results to date suggest that mir-3 “fine-tunes” nautilus expression in the embryo while mir-318 has a similar role in the larval and adult stages. 124 PLATFORMS: Genome and Chromosome Structure

112 Efficient identification of Drosophila Y-chromosome sequences by short-read sequencing. Bernardo Carvalho1, Andrew Clark2. 1) Dept de Genetica, Univ Fed Rio de Janeiro, Rio de Janeiro, Brazil; 2) Molecular Biology and Genetics, Cornell University, USA. Despite the completion of the genome sequence of 12 species of Drosophila, their Y chromosomes remain poorly known. A major obstacle has been the identification of Y chromosome sequences: due to its high content of repetitive sequences, the Y chromosome has been represented as highly fragmented in most genome projects, hidden within a large number of small, unmapped contigs. Identification of Y-chromosome sequences among these fragments has yielded important insights regarding the origin and evolution of the Y chromosome, but the process of testing Y-linkage remains labor intensive, precluding an exhaustive study of this chromosome. Parallel, short-read sequencing methods hold the promise of revolutionizing the identification of Y-linked contigs. A rapid application of this approach to three available Drosophila genomes (D. melanogaster, D. pseudoobscura and D. virilis), has more than doubled the number of known Y-linked genes in these species.

113 Evolution of nested genes in Drosophila and vertebrates. Fyodor A Kondrashov1, Raquel Rassis2, Alexey Kondrashov2, Eugene Koonin3. 1) Section of Ecology, Behavior & Evolution, UCSD, La Jolla, CA; 2) Center for Computational Medicine and Biology and the Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, 48109; 3) National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda MD, USA. Genomes with introns ofter contain nested genes, i.e. genes that are coded inside the introns of other genes. In particular, the human genome contains ~150 while the Drosophila melanogaster genome codes more than 800 of functional protein-coding nested genes. Many of these nested genes are functionally characterized, especially those in the human genome, however, the evolution of the nested state has not been considered. We reconstructed the evolution of the simplest and the commonest nested state, where the entire internal gene was contained within a single intron of the external gene. By comparing this gene structure in two sister species and an outgroup we were able to quantify the rate of gain and loss of nested genes. We find that the rate of gain of nested gene is much higher than the rate of loss in both Drosophila and Vertebrate clades, with various types of transpositions being the most common mechanism of acquisition. Thus, genome structure is increasing in complexity by loosing unidimensionality of gene organization through the accumulation of nested genes. Preliminary results find no support to suggest the role of selection in this process of nested gene accumulation.

114 Repetitive Elements and the Rise of Chimeric Genes in the D. melanogaster Subgroup. J Roman Arguello1, Shuang Yang2,3, Xin Li2,3, Yun Ding2,3, Qi Zhou2,3, Ying Chen1, Yue Zhang2, Ruoping Zhao2, Frédéric Brunet4, Lixin Peng2, Manyuan Long1, Wen Wang2. 1) Dept Ecology & Evolution, Univ Chicago, Chicago, IL; 2) Kunming Institute of Zoology, Kunminng, China; 3) Graduate School of Chinese Academy of Science, Beijing, China; 4) Ingénieur de Recherche en Bioinformatique Equipe Génomique Evolutive des Vertébrés IGFL, France. The use of chimeric genes presents a unique system with which to study the origin and evolution of genetic diversity. However, many aspects of their advents, for example the mutational mechanisms generating them, the frequency with which they arise, and the novel functions that they evolve, remain intriguing questions. This uncertainty is in part due to a lack of a data set comprised of experimentally verified young duplicates involved in the formation of chimeras. To address this, we use a battery of methods (expression studies, gene structure studies, sequence analyses) to identify chimeric genes over the D. melanogaster subgroup. In addition, we investigate the flanking regions of the paralog pairs to gain insights about the mutational mechanism giving rise to them. We identify 17 chimeric genes, most of which appear to be functional and have evolved divergent expression profiles. Interestingly, we find that repetitive elements, in particular DNAREP_1, are enriched near or at the boundaries the paralogs, consistent with either models of nonallelic recombination and possibly the shuffling of DNA through the movements of transposable elements. PLATFORMS: Genome and Chromosome Structure 125

115 Highly asymmetrical rates of evolution between paralogs following the duplication of testes expressed genes from neo-X- chromosomes to autosomes. Richard Meisel1, Mira Han2, Matthew Hahn2. 1) Penn State University; 2) Indiana University. Drosophila genomes consist of five major chromosome arms and a dot chromosome. In the ancestral karyotype, four of the major arms are telocentric autosomes, while one arm makes up the telocentric X chromosome. Throughout the evolution of the genus multiple independent fusions of chromosome arms have occurred, including some in which an ancestral autosome was fused to the X chromosome giving rise to a neo-X chromosome. These neo-X chromosomes provide us with a unique opportunity to study how the chromosomal context of a gene influences its function, especially in regards to sex bias and intragenomic conflict between sexes. We identified lineage specific duplicated genes in the sequenced Drosophila genomes for the purpose of comparing patterns of gene duplication between autosomes, ancestral X chromosomes, and neo-X chromosomes. The ancestral X chromosome acts as disproportionate source of inter-arm duplicated genes in some lineages, but not all lineages show this pattern. Two species with neo-X chromosomes (D. pseudoobscura and D. willistoni) have an excess of genes duplicated from the recently X-linked region to other chromosome arms. Many of these duplicated genes arose around the time of the X-autosome fusion event that gave rise to the neo-X chromosomes. Additionally, the derived copies of the neo-X to autosome paralogs tend to have amino acid sequences that evolve at much faster rates than the ancestral copies. Furthermore, the ancestral autosomal copy of many of the genes is expressed in the testis of adult males. We hypothesize that the autosomal derived copies have been specialized for the male functions of the gene, while the neo-X-linked copies have retained the majority of the ancestral suite of functions. The ancestral X chromosome probably also experienced this phenomenon when it became a sex chromosome, but it is currently much closer to equilibrium - in regards to male-biased and female-biased gene content - than the neo-X chromosomes and does not show such a striking pattern of recent duplication of male-expressed genes onto autosomes.

116 Age-related changes in double-strand break repair. William Engels, Christine Preston, Dena Johnson-Schlitz, Carlos Flores. Dept Genetics, Univ Wisconsin, Madison, WI. A recent surprise in the study of how double-strand breaks are repaired in the germline is that the process changes markedly as the organism ages. We monitored three alternative outcomes of I-SceI-induced breaks: SSA: Single-strand annealing NHEJ: Non-homologous end-joining HR-h: Homologous repair using the homolog as template (i.e., gene conversion) Young flies use mostly SSA with NHEJ as a strong second choice and HR-h a distant third. These parameters change steadily with age until HR-h finally becomes the dominant outcome in older flies. In recent work we have broadened these observations in several directions. First, we find that the repair outcomes and the age effect can depend on the genomic location of the break. A particularly oddball location occurs at site 86D where the repair process appears to be NHEJ-phobic in both young and old flies. We also compared the process between the male and female germlines. The dramatic rise of HR-h with age occurs in both sexes, but there are some unexpected differences. Females at all ages had much-reduced usage of NHEJ and an excess of aberrant repair events. We attempt to explain the phenomenon of age-dependent break repair in terms of the “antagonistic pleiotropy” model.

117 Evolutionary genetics of hybrid sterility in Drosophila. Nitin Phadnis, Allen Orr. Dept Biol, Univ Rochester, Rochester, NY. Recent studies in the genetics of speciation have identified several genes that cause intrinsic postzygotic reproductive isolation; almost all of them evolve rapidly. However, the evolutionary forces that drive the rapid changes in these genes remain unclear. The idea that genetic conflict, such as meiotic drive, may cause the evolution of intrinsic postzygotic reproductive isolation is intuitively appealing, but empirical evidence has been scant. Previously we showed that, “sterile” F1 hybrid males between the USA and Bogotá subspecies of Drosophila pseudoobscura become very weakly fertile when aged and produce all daughters, reflecting sex chromosome segregation distortion. Mapping studies showed that the same regions on the Bogotá X-chromosome underlie both hybrid male sterility and hybrid segregation distortion. The critical question is whether the same genes cause both hybrid segregation distortion and hybrid male sterility. We begin by focusing on a region linked to the visible mutation sepia on the D. pseudoobscura XR, which is known to play an essential role in both phenomena. We introduced 200 independent copies of the sepia region from USA into an otherwise Bogotá genome and backcrossed to Bogotá for 28 generations. So far, the genes causing hybrid male sterility from those causing hybrid segregation distortion have proved to be meiotically inseparable. We have further fine-mapped the genes responsible for both hybrid phenomena to an interval containing five genes, including a rapidly evolving candidate gene. Ongoing transgenic experiments may provide the first clear example of genetic conflict driving the evolution of reproductive isolation. 126 PLATFORMS: Genome and Chromosome Structure

118 Drosophila histone variant H2Av localizes to centromeres and regulates normal localization of centromeric histone H3 variant CENP-A/CID. Weiguo Zhang1,2, Gary H. Karpen1,2. 1) the Life Sciences Division, the Lawrence Berkeley National Laboratory, Berkeley, CA; 2) Department of Molecular and Cellular Biology, University of California-Berkeley, Berkeley, CA. CENP-A (CID in Drosophila) is the centromere-specific histone H3 variant and a primary epigenetic regulator for centromere identity and kinetochore formation. Drosophila centromeres are comprised of alternative chromatin domains containing CID or canonical histone H3 nucleosomes. It has been previously indicated that the centromeric H3 nucleosomes have a characteristic pattern of histone modifications distinct from both euchromatin and heterochromatin. Here we identified H2Av, the histone H2A variant in Drosophila, as another important epigenetic regulator of centromeric chromatin. By RNA interference (RNAi) experiments targeting H2Av in Drosophila S2 tissue culture cells, we demonstrated that H2Av is required for normal localization of CID and another essential inner centromeric protein CENP-C. We studied the impact of H2Av on loading of newly synthesized CID versus stability of CID proteins at centromeres. We found that the centromeric protein levels for both CID and CENP-C are significantly reduced after H2Av RNAi. We analyzed the distribution of H2Av in different phases throughout the cell cycle and found that H2Av directly localize to centromeres and that the majority of centromeric H2Av localize to H3 nucleosomes alternative to the CID nucleosomes. Our results provide evidence that the alternative H3 nucleosomes at centromeres are pivotal to normal centromere propagation. This work is supported by the Susan Komen Breast Cancer Foundation Postdoctoral Fellowship to W.Z. and NIH grant GM66272 to G.H.K. PLATFORMS: Techniques and Functional Genomics 127

119 A toolkit for high-throughput gene engineering in flies. Radoslaw K Ejsmont1, Mihail Sarov2, Pavel Tomancak1. 1) Tomancak Lab, MPI-CBG, Dresden, Germany; 2) BAC Facility, MPI-CBG, Dresden, Germany. To study gene regulation in vivo it is necessary to create faithful reporters of gene activity. The randomized nature and insertion bias of transposon mediated ‘gene trapping’ approach prevents systematic genome wide gene tagging. Therefore we developed a reverse genetic strategy to generate gene expression reporters systematically, by high-throughput recombineering of large genomic clones and subsequent P[acman]/phiC31 mediated transgenesis. Our intermediate goal is to establish a collection of live mCherry based gene expression reporters for marking tissues during Drosophila embryogenesis. We introduce mCherry at the N-terminus of selected genes, within large genomic clones, to preserve intact gene regulatory sequences. To allow gene tagging in high-throughput we adapted a recombineering pipeline developed to systematically tag C. elegans proteins. This procedure, based on Red/ET system, performs all recombineering steps in a single bacterial strain, in liquid culture, in 96-well format, within one week. We modified the C. elegans pipeline for fly transgenesis by introducing attB sites and 3xP3-DsRed fly selectable marker. We follow two distinct strategies to achieve high-throughput gene tagging. 1) We replicate the two-step C. elegans approach of first tagging the gene and then subcloning part of the BAC clone. This strategy can be seamlessly applied to any existing BAC library. 2) We generate and sequence a new fosmid library that incorporates attB, 3P3-DsRed and OriV in the backbone. This fosmid library enables highly efficient Red/ET tagging in 96-well liquid culture format and direct phiC31 mediated transgenesis of flies. We will present results of a pilot experiment where we performed tagging of 12 genes expressed during early embryogenesis with different fluorescent proteins (GFP and mCherry) to assess the benefits of different tags for monitoring early zygotic gene expression.

120 PhiC31-mediated cassette exchange in Drosophila. Jack R. Bateman, Anne M. Lee, Lillian Merriam, Laura Stadelmann, C.-ting Wu. Department of Genetics, Harvard Medical School, Boston, MA. We have developed a method for transgenesis that targets constructs to predetermined genomic sites and permits the integration of sequences lacking a phenotypic marker (1). Importantly, this method will facilitate transgenic studies by controlling for undesired position effects from flanking genomic regions and/or from a second transcription unit in the transformation vector. Using the phiC31 integrase system (2) in conjunction with Recombinase Mediated Cassette Exchange (RMCE), we have targeted a variety of donor cassettes, including those that do not carry visible markers, to eight defined loci in the Drosophila genome. Targeting is achieved by exchanging a donor cassette flanked by two attB sites with a previously integrated target cassette carrying the mini-white gene and flanked by two attP sites (also see (3) and (4) for RMCE driven by other recombinases). Because RMCE-mediated integration of the donor cassette is necessarily accompanied by loss of the target cassette, we were able to identify successful integrants simply by loss of mini-white eye color. Furthermore, the use of Drosophila lines that carry an endogenous source of the phiC31 integrase (5) can produce integration efficiencies of 50% and greater. We are continuing to develop new reagents to expand the utility of the RMCE system for different types of analyses, and are currently exploring strategies using RMCE to systematically alter a gene of interest at its natural locus. This work was supported by the National Institutes of Health via a fellowship to J.R.B. (1 F32 GM67460) and a grant to C.-t.W. (1 RO1 GM61936), and by Harvard Medical School. 1. Bateman et al., 2006. Genetics 173: 769. 2. Groth et al., 2004. Genetics 166: 1775. 3. Oberstein et al., 2005. Nat. Methods 2: 583. 4. Horn et al., 2005. PNAS 102: 12483. 5. Bischof et al., 2007. PNAS 104: 3312.

121 The Drosophila ORF Collection: a high quality resource for proteomic and functional genomic studies. Mark Stapleton, Charles Yu, Ken Wan, Soo Park, Bhaveen Kapadia, Bayan Parsa, Joseph W Carlson, Susan E Celniker. Genome and Computational Biology, Lawrence Berkeley National Lab, Berkeley, CA. The identification of all expressed genes and the structure(s) of their transcripts are prerequisites for many structural and functional genomic studies. One of the major goals of the Berkeley Drosophila Genome Project is to experimentally define the transcribed portions of the genome by producing a collection of fully sequenced cDNAs. To accomplish this goal, we have produced the Drosophila Gene Collection (DGC) which is the product of sequencing a collection of 263,617 ESTs. The project currently has 15,369 full-insert sequenced clones and contains 7,256 cDNAs whose translation agrees 100% with Release 5.3 FlyBase annotations. This set of clones, the DGC Gold Collection, represents the starting point in generating a high quality resource for functional genomic studies. To facilitate the use of the Gold Collection, we are in the process of cloning open reading frames (ORFs) from 6,662 cDNAs into the CreatorTM (Clontech) universal cloning system. This is a directional PCR cloning system that generates a proteomics-ready, easily transferable set of donor ORFs. The donor vector allows for the subsequent transfer of these ORFs into a wide variety of acceptor expression vectors using Cre recombinase. We are making two types of each ORF that will allow for the expression of three forms of a given protein: the first type of ORF contains its native stop codon which can be used to generate amino-terminal fusion proteins as well as the capacity to express native, untagged proteins; the second type of ORF does not contain its native stop codon which can be used to generate carboxy-terminal fusion proteins. Our goal for the initial phase of the project is to produce a total of 5,000 high-quality donor clones for each of the two types of ORFs. To date, we have made a total of 8,659 high-quality donor clones. This system and a core set of acceptor expression vectors and their uses will be discussed along with preliminary experiments demonstrating the system’s utility. 128 PLATFORMS: Techniques and Functional Genomics

122 Genome-wide mapping and annotation of protein expression and interaction in Drosophila melanogaster, using a hybrid PiggyBac/P-element YFP gene trap system with tandem affinity tags. Ed Ryder1, Helen Spriggs1, Emma Drummond1, Laura Harris1, Jane Webster1, Glynnis Johnson1, John Roote1, Nick Lowe3, Kathryn Lilley2, Svenja Hester2, Julie Howard2, Johanna Rees2, Steve Russell1,2, Daniel St. Johnston3. 1) Dept Genetics, Cambridge Univ, UK; 2) Cambridge Systems Biology Centre, Cambridge Univ, Cambridge, UK; 3) Gurdon Institute, Dept Genetics, Cambridge Univ, Cambridge, UK. We have initiated a screen to generate and characterise protein trap lines in Drosophila using a PiggyBac transposon-based strategy. The ability to generate in vivo tagged proteins has tremendous potential for furthering our understanding of developmental processes by allowing the characterisation of sub-cellular protein localisation and facilitating the isolation of multi-protein complexes. This large project involves collaborations with over thirty UK laboratories. Our Pig/P transposons are tagged with yellow fluorescent protein (YFP) incorporated into endogenous genes via an exon-trapping strategy, thus facilitating the visualization of trapped proteins in living embryos and larvae. As correct incorporation of the fluorescent tag is a rare event we employ an automated embryo sorter to select the insertions. Putative lines are mapped by iPCR and sequencing, and custom software predicts whether the insertion is in the correct frame for a functional YFP fusion. The transposed exons also contain two protein affinity tags that allow the protein to be isolated in its native complex by tandem affinity purification. Complex components are identified by tandem mass-spectrometry with spectra assigned to the fly proteome via the MASCOT search engine, and analysed using ProteinCenter (Proxeon). To aid in the characterisation of YFP-trap lines, we have developed web-based software which allows detailed annotation of protein expression at all stages of development and tissue types (including sub cellular location and spatial descriptors), using GO and FlyBase controlled vocabulary. The system allows multiple groups to work in collaboration and share uploaded images and annotation, whilst still protecting the original data.

123 Identification of compounds that modulate lipid droplet storage in S3 cells using qHTS. Douglas Auld1, Ya-Qin Zhang1, Noel Southall1, Mathias Beller3, James Inglese1, Christopher Austin1, Brian Oliver2. 1) NIH Chemical Genomics Center, NIH, Rockville, MD; 2) National Insitutes of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda MD, 20892; 3) Max-Planck-Institut for Biophysical Chemistry, Göttingen, Germany. The National Institutes of Health Chemical Genomics Center (NCGC) was formed in 2004 as part of the NIH Roadmap funded Molecular Libraries Initiative. The NCGC aims to enable scientists with useful chemical probes by providing expertise in assay development and industrial scale technologies for HTS of chemical libraries. The NCGC has developed a screening paradigm where libraries >100K in size are screened as seven-point concentration-response (CR) curves (qHTS). This approach has been applied to an assay for lipid droplet accumulation in S3 cells where we used both a fluorescence lipid droplet stain (BODIPY 493/503) and a fluorescent cytoplasmic stain (Invitrogen, CellTracker) to develop a cell-based assay for lipid drop formation following feeding with oleic acid. Laser scanning imaging was use to enumerate the amount of green and red fluorescence for cells seeded in 1,536-well plates enabling the rapid evaluation of potency values for ~10K compounds. The chemical libraries included both known bioactives as well as diverse compounds of unknown function. The types of compounds identified will be discussed. As well, the expansion of this technology platform to enable qHTS of large (>200 K) compound collections will be also be shown.

124 Systems model of ATP-generating metabolic network in Drosophila flight muscle. Jacob D. Feala1, Laurence Coquin2, Andrew D. McCulloch1, Giovanni Paternostro1,2. 1) Dept. of Bioengineering, University of California - San Diego, La Jolla, CA; 2) Burnham Institute for Medical Research. Computational models of biological systems are useful for generating predictions and testing hypotheses in-silico. We have reconstructed the reaction network of ATP-generating central metabolism in Drosophila flight muscle, in a stoichiometric matrix suitable for flux-balance simulations and constraint-based analysis. The network, built previously [1] using the annotated genome and the KEGG database of genes and reactions, was made specific for flight muscle by integrating absolute gene expression data for fly thorax, which is composed mostly of flight muscle. Each enzymatic reaction is included only if validated against the literature, unless required to fill a pathway gap. Boundary conditions were integrated into the model for the special case of steady-state hypoxia, based on metabolite measurements gathered in our lab. Quantitative 1H spectroscopy and biochemical assays were used to measure accumulation of products, substrate depletion, and ATP concentration over 4 hours of hypoxia, for wild-type and several enzyme deletion strains. This refined version of the model can be used to investigate chronic hypoxia in muscle tissue, or the generic model can be similarly tailored to other specific contexts of interest. PLATFORMS: Techniques and Functional Genomics 129

125 A Morphology and Gene Expression Atlas of Drosophila Embryogenesis. Cris L. Luengo Hendriks1, Soile V.E. Keränen1, Pablo Arbelaez2, Gunther H. Weber1, Charless C. Fowlkes3, Clara N. Henriquez1, David W. Kaszuba1, Bernd Hamann4, Jitendra Malik2, Mark Biggin1, David W. Knowles1. 1) Lawrence Berkeley National Laboratory, Berkeley, CA; 2) University of California, Berkeley, CA; 3) University of California, Irvine, CA; 4) University of California, Davis, CA. The Berkeley Drosophila Transcription Network Project (http://bdtnp.lbl.gov) has established a suite of live and fixed embryo imaging and image analysis methods that have provided the first quantitative three-dimensional description of Drosophila blastoderm morphology and gene expression at cellular resolution. This atlas has revealed a wealth of previously undetected biological results and is being used in our system-wide analysis of the early transcript network. During the 10 hours after blastoderm formation, large cell motions and complex patterns of differentiation generate late stage embryos with over 70 cell types and all major larval organs. Using an improved imaging technology that allows an entire embryo to be captured in three dimensions with high fidelity, we are developing computational methods to produce a quantitative, cellular-resolution atlas of all of embryo development. High-resolution fluorescence images of whole, fixed embryos are being acquired and used to develop segmentation methods to locate the individual nuclei and cells. Classification methods, using annotated morphological features and tissue-specific markers, are being developed to recognize specific body-plan and tissue structures. Registration methods are being developed to assemble multiple embryo images into average morphological maps, onto which measured, per-cell mRNA and protein expression levels can be added. To link these stage-specific maps, temporal movements and mitotic events will be tracked using live cell imaging of embryos expressing nuclear, cell membrane and tissue-specific fluorescent proteins. The goal is to produce an expandable computational, morphology and gene expression atlas of Drosophila embryogenesis at cellular resolution. 130 PLATFORMS: Chromatin and Gene Expression

126 Understanding the function of insulators in the Drosophila genome. David J. Marion1, Alexey A. Soshnev2, Kate Appleton3, Xingguo Li3, Misty D. Wehling3, Ryan M. Baxley2, Pamela K. Geyer1,2,3. 1) Genetics Program; 2) Molecular and Cellular Biology Program; 3) Department of Biochemistry, University of Iowa, Iowa City, IA 52242. Eukaryotic chromosomes contain many adjacent genes that show different spatial and temporal regulation. This implies that the long-distance action of transcriptional regulatory elements is restricted in eukaryotic genomes. Insulators block interactions between enhancers, silencers, and promoters in a position-dependent manner, but the mechanisms are poorly defined. A Su(Hw)-dependent insulator, 1A-2, resides in the intergenic region between the yellow and achaete genes, which show differences in temporal and tissue-specific expression. To address whether 1A-2 regulates independent transcription at the endogenous site, this region was deleted by homologous recombination and mRNA levels were assayed by RT-PCR. Our data suggest that the 1A-2 deletion has a minimal impact on yellow and achaete transcription patterns, but surprisingly, has a large effect on the expression of an intergenic non-coding (nc) RNA. Subsequent studies are underway to characterize this ncRNA and to define the mechanism of transcriptional regulation by 1A-2. To gain insights into mechanisms of insulator function, we are using the LacI tethering system developed by Belmont and colleagues, who established multiple transgenic lines that carry a tandem array of lac operator sequences inserted randomly in the genome. Our lab has generated expressor lines carrying insulator proteins fused to the LacI repressor. Tethering these proteins to distinct binding sites will address the following questions. 1) Do insulator proteins direct the formation of chromatin loops between distant sites? 2) Do insulator proteins affect local chromatin condensation and associated histone modifications? These studies permit a comparison of the properties of insulator proteins and will provide insights into how insulators affect chromosome organization.

127 Bhringi, a highly conserved regulator of Twist transcription factor activity. Scott J. Nowak1, Katie Gonzalez2, Mary K. Baylies1. 1) Dept. of Developmental Biology, Sloan-Kettering Institute, New York, NY; 2) Scripps Research Institute, La Jolla, CA. A yeast double interaction screen designed to recover novel Twist interaction partners lead to the identification of CG8580, a gene we have named bhringi (bhr). The phenotypes of bhringi mutants reveal a role for bhr during muscle development: loss of bhr during embryogenesis results in muscle loss, severely altered muscle morphology and defective muscle attachments. bhr encodes a 201 residue protein that is highly conserved across multiple species, from flies to humans. Immunohistochemistry indicates that bhringi is localized to the nucleus and is expressed broadly throughout the embryo during all phases of embryogenesis. Bhringi interacts with Twist both genetically and physically by GST-pulldown and co-immunoprecipitation in vitro. Further, loss of specific Twist- dependent target gene expression is observed in bhr mutants. Bhringi is localized to gene-rich regions of the genome and is also capable of interaction with subunits of the Brahma chromatin remodeling complex. Taken together, these results indicate a mechanism whereby Bhringi interacts with transcription factors and chromatin remodeling machinery to facilitate proper expression of Twist- dependent genes during Drosophila development.

128 Genetic analyses of cofactors that cooperate with the Brahma (SWI/SNF) chromatin remodeling complex in the regulation of target genes. Chhavi Chauhan1, Claudia Zraly1,2, Manuel Diaz1,2, Andrew Dingwall1,2,3. 1) Molecular Biology Program, Loyola University Chicago, Stritch School of Medicine, Maywood, IL; 2) Oncology Institute; 3) Department of Pathology. The Brahma (SWI/SNF) ATP-dependent chromatin remodeling complex uses energy to drive changes in contacts between chromosomal DNA and histones. These changes impact gene expression, either positively or negatively and contribute to the control of cell growth and patterning. We found that the hormone response genes were among the most sensitive to the loss of Brm complex functions during development. To gain insight into the mechanism of target gene regulation, we have focused on identifying additional cellular cofactors important for Brm complex functions on the hormone response genes. A dominant enhancer/suppressor screen was employed to identify these cofactors. One of the candidates, which we named cara mitad (cmi), encodes a protein related to a component of the mammalian MLL2/ALR nuclear receptor coregulator complex. This complex contains a histone methyltransferase activity that specifically modifies histone lysine residues to affect hormone signaling. All of the known components of this complex have orthologs in Drosophila, though few have been well characterized. We have generated null mutations in the cmi gene and we will present our genetic analyses of cmi function during development, as well as genetic interactions with components of the Brm complex. We hypothesize that a conserved nuclear receptor coactivator complex helps to direct Brm complex functions in regulating the hormone response during Drosophila development. PLATFORMS: Chromatin and Gene Expression 131

129 Paused Polymerase in the Drosophila Embryo. Michael S Levine1, Julia Zeitlinger2, Joung-Woo Hong1, Jess Piel1, Dave Hendrix1, Richard A Young3. 1) MCB, UC Berkeley, Berkeley, CA; 2) Stowers Institute for Medical Research, Kansas City, MO; 3) Whithead Institue, MIT, Cambridge, MA. The analysis of ~30 different Dorsal target enhancers suggests that those mediating gene expression in response to high levels of the Dorsal gradient contain a series of disordered low-affinity Dorsal and/or Twist activator binding sites. In contrast, enhancers mediating expression in response to low levels of the gradient (the “type 2” response) contain an ordered arrangement of optimal Dorsal and Twist binding sites. This organization is highly conserved in evolution and is likely to foster cooperative occupancy of linked operator sites. We suggest that type 2 enhancers have some of the properties of a transistor: they amplify weak and unstable signals to produce a constant output of gene activity. The accurate representation of genetic circuit diagrams depends on the designation of this special subset of enhancers. The conventional view of gene activation is that it depends on the recruitment of RNA polymerase II (Pol II) to the core promoter. However, there are a few documented examples of gene activation via Pol II elongation, whereby Pol II is bound, but paused at a fixed site just downstream of the transcription start site. It is not known to what extent this mechanism is used to establish differential patterns of gene expression in development. To investigate this issue, we performed ChIP-chip assays using antibodies directed against Pol II. At least 1,000 genes contain a paused or stalled form of Pol II prior to their activation in the Drosophila embryo. These include many developmental control genes, such as Hox genes and components of the FGF, Wnt, Hedgehog, TGF?, and Notch signaling pathways. Altogether, these results suggest that the regulation of Pol II elongation, not recruitment or initiation, might be a critical mechanism for gene activation in development.

130 Chromatin-remodeling by Kismet in Transcription and Development. Kristel M. Dorighi1, Shrividhya Srinivasan1,2, John W. Tamkun1. 1) MCD Biology, UC Santa Cruz, Santa Cruz, CA; 2) Developmental Biology, Stanford University, Stanford, CA. Factors that regulate chromatin structure play important roles in development. Members of the Polycomb group (PcG) of repressors and their antagonists, the trithorax group (trxG) of activators, act at the level of chromatin to maintain patterns of gene expression and cellular identities in multicellular organisms. Kismet is an ATP-dependent chromatin-remodeling factor of the trxG that facilitates an early step in transcriptional elongation. We are currently investigating how Kismet interacts with other trxG proteins to counteract PcG-mediated transcriptional repression. Using Drosophila melanogaster as a model organism, we found that Kismet promotes the recruitment of two histone H3 lysine 4 methyltransferases of the trxG family - ASH1 and TRX to chromatin. In addition, Kismet opposes the methylation of histone H3 on lysine 27, a repressive histone modification catalyzed by PcG proteins that is required for stem cell self-renewal and the maintenance of pluripotency. Since the function of PcG and trxG proteins has been highly conserved during evolution, these findings suggest the human counterpart of Kismet - CHD7 - regulates stem cell development by counteracting PcG repression. We are currently testing this model in the human embryonic stem cell-like cell line Ntera2. Even if our hypothesis is incorrect, characterization of the function of CHD7 will shed light on the molecular nature of CHARGE syndrome, a serious developmental disorder linked to mutations in human CHD7.

131 Regulation of Myc-induced cell growth by the histone H3K4 demethylase Lid. Julie Secombe, Ling Li, Robert Eisenman. Div Basic Sci, Fred Hutchinson Cancer Res Ctr, Seattle, WA. The Myc family of mammalian transcription factors control cell growth and cell cycle progression and are implicated in the genesis of a wide range of cancers when misregulated. To gain insight into the mechanism by which Myc functions, we are characterizing dMyc, the sole Drosophila ortholog of Myc. dMyc is functionally similar to mammalian Myc proteins and can regulate cell cycle progression and cell growth during Drosophila development. To identify genes required for dMyc-mediated cell growth, we carried out a genetic interaction screen for dose-sensitive enhancement or suppression of a rough eye phenotype generated by overexpression of dMyc. One suppressor identified in this screen was the Trithorax group gene, little imaginal discs (lid). We have shown that Lid binds to dMyc and is required for dMyc-induced expression of the growth regulatory gene Nop60B. In addition, we have demonstrated that Lid is histone H3 lysine 4 demethylase and that Lid’s enzymatic activity is negatively regulated by dMyc, which binds to Lid’s catalytic JmjC domain. We are currently determining the mechanism by which Lid functions in Myc-mediated transcriptional activation and cell growth. 132 PLATFORMS: Chromatin and Gene Expression

132 P element repression by an epigenetic telomeric trans-silencing involving RNA silencing and heterochromatin formation. Stéphane Ronsseray, Thibaut Josse, Laure Teysset, Anne-Laure Todeschini, Augustin de Vanssay, Clara Sidor, Valérie Delmarre, Dominique Anxolabéhère. Dynamique du Génome et Evolution, Inst Jacques Monod, Paris, France. The study of P element repression in Drosophila melanogaster led to the discovery of the telomeric Trans-Silencing Effect (TSE), a repression mechanism by which a transposon or a transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequences, “TAS”) has the capacity to repress in trans, in the female germline, a homologous transposon or transgene located in euchromatin. We will present the properties of this new silencing. TSE shows variegation among egg chambers in ovaries when silencing is incomplete. It displays an epigenetic transmission through meiosis which involves an extrachromosomal maternally- transmitted factor. This silencing is highly sensitive to mutations affecting both heterochromatin formation (Su(var)205 encoding HP1 and Su(var)3-7) and the repeat associated small interfering RNA (rasiRNA) silencing pathway (aubergine, homeless, armitage and piwi). In contrast, TSE is not sensitive to mutations affecting r2d2 which is involved in the canonical RNA interference (siRNA) pathway, nor to a mutation in loquacious which is involved in the micro RNA (miRNA) silencing pathway. These results, taken together with the recent discovery of TAS homologous small RNAs associated to PIWI proteins, support the proposition that TSE involves a rasiRNA pathway linked to heterochromatin formation. Therefore, the study of TSE provides insight into the genetic properties of a germline-specific small RNA silencing pathway. We will also present data showing that TSE behaves as a cellular repression mechanism; this mechanism would have been coopted by the P element to establish repression of its own transposition after its recent invasion of the D. melanogaster genome. PLATFORMS: Physiology and Aging 133

133 The TOR pathway couples nutrition and developmental timing in Drosophila. Pierre Leopold, Sophie Layalle. CNRS/University of Nice, Nice, France. In metazoans, final adult size depends on both the rate and the duration of growth. These two parameters, which together determine the range of animal growth, are modified by nutritional cues. Molecules of the insulin/IGF family control the growth rate, while the duration of juvenile growth is controlled by pulses of steroid hormones. Here we show that in Drosophila, nutrition modifies developmental timing through a sensor tissue called the prothoracic gland (PG) that secretes the moulting hormone ecdysone. When activity levels of the Target of Rapamycin (TOR) kinase, which represents the main nutrient-responsive pathway, are experimentally reduced in the PG, the peak of ecdysone that marks the end of larval development is abrogated. This abrogation extends the duration of larval growth and thereby increases animal size, but without changing the larval growth rate. Conversely, the extension of larval development that normally follows nutrient deprivation can be reversed by specifically reactivating the TOR pathway in PG cells. The sensitivity of the PG to TOR inhibition is temporally restricted to a narrow window occurring towards the end of larval development, defining a nutritional checkpoint that acts before the commitment to maturation. Our data indicate that the PG uses TOR as a molecular sensor to couple nutritional inputs to the production of a hormonal signal that controls the duration of the juvenile growth period.

134 Stem cell aging is controlled both intrinsically and extrinsically in the Drosophila ovary. Lei Pan1,2, Ting Xie1. 1) Stowers Inst, Kansas City, MO; 2) Inst of Biophysics,Chinese Academy of Sciences,Beijing,China. Adult stem cells have the ability to self-renew and generate differentiated cells for replenishing lost cells due to normal ageing, diseases or physical injury. Even though adult stem cells remain in adult tissues throughout an organism’s lifetime, functions of organs and tissues still decline with age. It is widely postulated that ageing could be, at least partially, caused by reduced stem cell number, activity or both. However, it largely remains a mystery as to how stem cell ageing is controlled. Here, we have used Drosophila ovarian germline stem cells (GSCs) as a model to demonstrate that both stem cell intrinsic ageing and decline of niche functions contribute to overall stem cell ageing. Partial reduction of niche BMP signals, Dpp and Gbb, speeds up stem cell ageing, while the increase in BMP signaling by overexpression of Gbb in the niche or an activated type I receptor in the GSCs can prolong their lifespan and promote their proliferation, indicating that age-related reduction of BMP signaling contributes to GSC ageing. Interestingly, overexpression of SOD, a gene that is known to help eliminate free oxygen species, in germline and niche cells can prolong GSC lifespan and increase GSC proliferation. Additionally, strengthening interactions between stem cells and the niche by overexpression of the cell adhesion molecule E-cadherin can also prolong GSC lifespan since the cadherin mediate cell adhesion is known to be important for keeping the GSC in the niche. Therefore, this study demonstrates that stem cell ageing is likely caused by a combination of intrinsic and niche ageing.

135 Crosstalk between the Insulin and Toll pathways shifts nutrient metabolism in response to infection. Justin DiAngelo1, Sara Cherry2, Morris Birnbaum1. 1) Institute for Diabetes, Obesity and Metabolism, Univ Pennsylvania/HHMI, Philadelphia, PA; 2) Department of Microbiology and The Penn Genomics Institute, Univ Pennsylvania, Philadelphia, PA. During nutrient abundance, animals increase their size and nutrient stores as regulated by the peptide hormone insulin. However, in response to environmental stresses animals conserve their energy by downregulating insulin signaling, which subsequently decreases animal size and metabolic reserves. One situation where stress-induced energy conservation has been well described is nutrient depletion; however, the effect of a different stress, infection, on energy preservation is less understood. Here, we demonstrate that activating critical mediators of the immune response in the fruit fly suppresses insulin signaling, leading to a decrease in both triglyceride and body size. This interaction is specific for the Toll pathway, as overexpressing the Imd pathway transcription factor relish does not show these phenotypes. These Toll-dependent phenotypic changes are due to decreased insulin signaling activity as the phosphorylation of dAkt, one of the major kinases in the insulin signaling pathway, is decreased in response to Toll activation. Epistasis reveals that Toll antagonizes the insulin signaling pathway at or downstream of PI3-kinase and this involves transcription as overexpressing the NFκB transcription factor dif also decreases dAkt phosphorylation. These genetic data reveal the evolutionarily conserved communication between the innate immune response and insulin action. Moreover, they suggest that this crosstalk evolved as a means to divert energy in times of stress from organismal growth to the acute requirement of combating infection. 134 PLATFORMS: Physiology and Aging

136 Rescue of the flightless phenotype of a Glutathione S-transferase S1 (GstS1) null mutant. Oksana Litvinova1, Sarah Dauback2, Ashis Mondal3, Piotr Zimniak2, Helen Benes1. 1) Neurobiol & Develop Sci; 2) Pharmacol & Toxicol; 3) Internal Medicine, Univ of Arkansas for Med Sci, Little Rock, AR. The products of lipid peroxidation, such as the electrophilic aldehyde 4-hydroxynonenal (4-HNE), may be important mediators of the pathological effects of uncontrolled oxidative stress. Previously, we showed that Drosophila GstS1-1 (DmGstS1-1) is the most abundant enzyme catalyzing 4-HNE conjugation in the fly, with primary localization in the Indirect Flight Muscles (IFMs). Yet, earlier studies of DmGstS1-1 proposed its main role to be a sensor for stretch-activated contraction of the IFMs (Clayton et al., J. Muscle Res. Cell Motil. 19: 117, 1998). Exceptionally among insect GSTs, GstS1-1 has an N-terminal extension which anchors it to tropomyosin-H in the IFMs. To elucidate how GstS1-1 may play a critical role in preventing oxidative toxicity in muscle or in sensing stretch in the contractile apparatus, we isolated mutant alleles of GstS1. Newly eclosed null mutant flies exhibited rapid degeneration of the IFMs, leaving other thoracic muscles intact. We observed no difference in level of tropomyosin-H between wild-type control and null mutant flies, indicating that GstS1-1 is not necessary for the localization of the IFM-specific tropomyosin. We are using the UAS/GAL4 system to determine if the importance of GstS1-1 for IFM preservation or function is due either to its enzymatic activity (in conjugating lipid peroxidation products) or to its structural/mechanical role in the contractile apparatus. Targeting of GstS1-1 to muscle (with the Mhc-Gal4 or Myosin Heavy Chain “driver”) restores flight to null mutant flies, whereas expression of a mouse GST (mGSTA4-4) with particularly high 4-HNE conjugating activity may not be sufficient. Further studies with other “drivers” will determine the role of motoneurons or other tissues in preserving IFM function. Our studies should provide insight into the role of oxidative stress and 4-HNE metabolism in neural and muscular degeneration underlying a number of human diseases.

137 The regulation of Drosophila lifespan by falafel. Brian Sage1, Xi Lou2, Li Qian3, Rolf Bodmer3, Heinrich Jasper2, Marc Tatar1. 1) Dept Ecol & Evol Biol, Brown Univ, Providence, RI; 2) Dept of Biology, Univ of Rochester, Rochester, NY; 3) Center for Neurosciences and Aging, The Burnham Institute, La Jolla, CA. Conserved pathways contribute to the regulation of aging. The C. elegans gene Suppressor of Map Kinase (SMK-1) is required for longevity extension upon reduced insulin signaling. To elucidate how SMK-1 participates in aging regulation we investigated its homolog in D. melanogaster, falafel. Here we show that falafel, a regulatory subunit of the protein phosphatase 4 complex, is a positive regulator of organisimal aging; overexpression of falafel extends lifespan in low nutrient conditions. This interaction between falafel and diet restriction is additive. Furthermore, preliminary data shows that dietary restricted flies with reduced levels of falafel are still long-lived, suggesting that longevity extension mediated by diet restriction doesn’t require falafel. Additional to the organismal aging regulation, flies overexpressing falafel in adult heart are more resistant to cardiac stress and have extended lifespan, indicating that falafel participates in autonomous processes of functional aging and cardiac stress resistance. By genetic epistasis interactions, we show that falafel is required for dFoxo and JNK mediated apoptosis in the eye. We are currently studying if, as in apoptosis, falafel works together with dFoxo regulating longevity. We are also analyzing possible physical interactions between Falafel, dFoxo, JNK, and other proteins that affect longevity. Our current goal is to understand the mechanism by which falafel regulates aging.

138 Genetic and environmental regulation of metabolism, behavior and lifespan by specific nutrient components. Danielle A. Skorupa1,3, Azra Dervisefendic1, Scott D. Pletcher1,2,3. 1) Huffington Center on Aging, Baylor College of Medicine, Houston, TX; 2) Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, TX; 3) Interdepartmental Program in Cell & Molecular Biology, Baylor College of Medicine, Houston, TX. Systems regulating energy storage and utilization are fundamental to organisms and strongly evolutionarily conserved. Paradoxically, disruption of normal energy intake or its sensing and signaling networks can lead to dramatic extension of organismal lifespan. The practice of dietary restriction, for example, promotes longevity by enacting an adaptive response that increases metabolic efficiency, reduces fecundity and enhances stress resistance. The exact nutrient or environmental cues that signal these beneficial adaptations remains elusive. By dissecting the macronutrients of the diet and altering environmental perception, we have gained a further understanding of the mechanism by which nutrients signal to affect lifespan. We find that particular mutants (i.e., FOXO) are deficient in enacting the appropriate nutrient response. In addition, behavioral (i.e., reproduction and feeding rates) as well as metabolic (i.e., obesity) phenotypes can be induced by the presence or absence of specific nutrients with total caloric intake playing only a minor role in phenotypic determination. Likewise, the mere perception (e.g., taste) of specific nutrients is sufficient to alter Drosophila physiology and behavior regardless of net energy alterations. Taken together, these data suggest that by understanding the specific signals emanating from the environment, lifespan extension as well as desirable metabolic adjustments resulting from dietary restriction regimes can be implemented even in the presence of high overall nutrient consumption while low caloric intake does not guarantee longevity. PLATFORMS: Physiology and Aging 135

139 Regulation of metabolism, organ senescence, and lifespan by the nutrient sensing TOR pathway. Sean Oldham, Claire Davies, Nancy Luong, Ryan Birse, R. J. Wessells, Suzanne Graham, Rolf Bodmer. Burnham Institute for Medical Research, La Jolla, CA, 92037. Drosophila has an evolutionarily conserved insulin/IGF system functionally analogous in the metabolic and growth aspects to the mammalian insulin/IGF system. Drosophila contains functionally conserved components of the insulin/IGF system, including the insulin ligands (Drosophila insulin-like peptides, DILPs). Loss of insulin signaling results in increased lipid levels, increased longevity, female sterility, and stress resistance. The Tuberous Sclerosis Complex (TSC1-2)/Target of Rapamycin (TOR) pathway responds to changes in insulin/IGF levels, amino acid levels, energy charge, lipid status, mitochondrial metabolites, and oxygen tension by adjusting cell growth. Using an allelic series of Drosophila TOR mutants, we show that TOR has additional roles regulating metabolism and lifespan. These phenotypes include lowered glucose and lipid levels, increased longevity, and a block in the age-dependent decline in heart function. We will describe the effects of altering TOR signaling on lifespan, organ senescence, and metabolism.

140 A genetic screen implicates the ER translocon and TRiC/CCT cytoplasmic chaperone complex as novel regulators of autophagy. Andrew M. Arsham, Thomas P. Neufeld. Dept of Genetics, Cell Biology & Development, University of Minnesota, Minneapolis, MN. Autophagy, the starvation-induced process of cellular bulk waste removal and recycling, has recently been implicated in neurodegenerative disorders, cancer, and longevity, and is tightly linked to pathways of cell growth, division, metabolism, and energetics. Autophagy is also thought to suppress cell damage and senescence by eliminating damaged organelles and macromolecules. We have used a mitotic mosaic approach to screen 383 lethal p-element insertions on chromosome 2L for dysregulation of autophagy—to the best of our knowledge, this is the first forward genetic screen for metazoan regulators of autophagy. One class of genes that emerged from the screen is involved in protein quality control and transport. These genes include the Sec61α subunit of the ER translocon and a subunit of the CCT/TRiC cytoplasmic chaperone complex. Subsequent RNAi knockdown experiments confirmed these genes’ involvement in regulating autophagy—knockdown of any single CCT subunit is sufficient to induce autophagy and suggests that defects in cytoplasmic protein quality control activate autophagy as a defense response. These results have important implications both in terms of the basic cell biology of cross regulation between autophagy and protein folding, and for autophagy’s role in suppressing neurotoxic protein aggregates such as polyglutamines in a variety of neurodegenerative diseases. 136 PLATFORMS: Neurogenetics and Neural Development

141 dfezl encodes a novel regulator of neural stem cell self-renewal in Drosophila. Mo Weng1,2, Shufen Situ2, Caitlin Gamble2, Cheng-Yu Lee1,2. 1) Department of Cell and Developmental Biology, University of Michigan School of Medicine, Ann Arbor, MI 48109; 2) Center for Stem Cell Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109. Asymmetric cell division provides an efficient mechanism for precise regulation of stem cell identity and potential (self-renewal) and generation of cellular diversity (differentiation) during development, maintenance of homeostasis, tissue regeneration and likely tumor suppression. Despite many seminal studies reporting a large number of genes that promote stem cell identity, regulation of stem cell self-renewal remain largely undefined. Drosophila neural stem cells (neuroblasts) divide asymmetrically to self-renew a neuroblast and to generate a differentiating ganglion mother cell, and serve as a model system to study regulation of self-renewal vs. differentiation in the context of development. To better understand regulation of self-renewal, we conducted a genetic screen to identify mutations that resulted in either increased or decreased neuroblast self-renewal. We discovered a novel mutation that showed a dramatic increase in neuroblasts at the expense of neurons. We mapped this mutation to cytological location 22B5-8, and sequence analysis of the mutant allele identifies a single nucleotide change resulting in an amino acid substitution in a previously uncharacterized gene CG31670. CG31670 encodes a novel zinc-finger transcription factor with strong homology in amino acid sequence to mammalian Fez and Fezl (forebrain embryonic zinc-finger and zinc-finger-like) proteins, and is renamed dfezl. Single dfezl mutant neuroblasts generate multiple neuroblast progeny and many post-mitotic neurons. dfezl mutant neuroblasts maintain normal cortical cell polarity. Thus, Dfezl likely functions either downstream or in parallel of the polarity proteins. dfezl appears to represent the most direct known regulator of neuroblast self-renewal. Latest progress on detailed characterization of the dfezl mutants and analysis of the dfezl protein will be discussed.

142 Dephosphorylation of Bazooka by PP2A is required for proper apical-basal polarity in embryonic neuroblasts. Michael P. Krahn, Andreas Wodarz. Department of Stem Cell Biology, University of Goettingen, Germany. Components of the PAR/aPKC (partitioning-defective/atypical protein kinase C) protein complex are essential for the establishment and maintenance of cell polarity in epithelial cells and in neuroblasts (NBs). The underlying mechanisms appear to be highly conserved throughout evolution, from worms to mammals. Research in Drosophila revealed that in the asymmetric division of NBs the determination of the distinct fates of the two daughter cells, the ganglion mother cell (GMC) and the NB, is dependent on asymmetric localization of the PAR/aPKC complex, consisting of Bazooka (Baz; the Drosophila homolog of PAR-3), PAR-6 and aPKC. Phosphorylation of Bazooka by the serine threonine kinases PAR-1 and aPKC is required for the proper localization and function of Baz. In contrast, little is known about the dephosphorylation mechanisms that counteract the activities of PAR-1 and aPKC. We found that PP2A, one of the major ubiquitous protein phosphatases involved in various processes like cell cycle control and cytoskeletal regulation, directly interacts with Baz via its structural A subunit. PP2A dephosphorylates Bazooka at two conserved serine residues and thereby antagonizes the kinase activities of PAR-1 and aPKC. Loss of PP2A phosphatase function leads to complete reversal of polarity in NBs. Proteins localized to the apical NB cortex in wild type, such as Baz, aPKC, Insc, Pins and G?i localize to the basal cortex in PP2A mutants, while Miranda and Prospero, which normally localize to the basal cortex, form a crescent at the apical cortex. We are currently investigating whether the reversed NB polarity is exclusively caused by compromised dephosphorylation of Baz or whether additional targets of PP2A are also relevant in this context.

143 From stem cell to unique neuron: Subdivision of the Castor temporal window by a feedforward loop involving Squeeze, Nab and Collier/Knot. Magnus Baumgardt1, Daniel Karlsson1, Javier Terriente2, Fernando J. Díaz-Benjumea2, Stefan Thor1. 1) Dept. of Clinical and Experimental Medicine, Linköping University Medical School, Linköping, Sweden; 2) Centro de Biología Molecular- Severo Ochoa/C.S.I.C., Universidad Autónoma-Cantoblanco, Madrid, Spain. In the developing Drosophila ventral nerve cord ~100 neurons express the LIM-HD transcription factor Apterous (Ap). The six thoracic Ap clusters consists of 4 Ap neurons of at least three different cells types; the Tvb neuron, that expresses the neuropeptide Nplp1, two ‘generic’ Ap cluster neuron (Tvx), and the Tv neuron, that expresses the neuropeptide FMRFamide. Several genes have been identified that specify Ap cluster neurons. These include the transcription factors ap, col, squeeze and dimmed, the transcriptional co-factors dac, eya and nab, as well as TGFb/BMP retrograde signaling. Genetic analysis reveals that these genes act in two different regulatory cascades to dictate Tvb and Tv cell identity. Both cascades are initially triggered by the expression of col, but importantly, downregulation of col is critical for the Tv cell fate. Previous studies reveal that all Ap cluster neurons are generated from NB 5-6t, and that the initial Ap cluster specification event is col-mediated. But how is this specification event channeled into two distinct cascades, which in turn dictate two distinct terminal cell fates; Tvb/Nplp1 and Tv/FMRFa? We find that the Ap neurons are generated from four consecutive GMCs (Tvb→Tvx→Tvx→Tv) during the last part of a large castor (cas) temporal window. Intriguingly the latter part of this cas window is subdivided into at least two ‘sub-windows’ by a feedforward mechanism, where 1) cas activates col, 2) cas activates sqz, 3) cas/sqz activates nab, and 4) where sqz/nab finally act together to downregulate col, in the last 3 Ap cluster neurons generated. This feedforward-mediated timing mechanism allows for col to perform its critical early postmitotic role - activation of ap and eya - but downregulates col in later born Ap cluster cells, thereby allowing for the specification of the Tv/FMRFa cell fate. PLATFORMS: Neurogenetics and Neural Development 137

144 Temporal transcription factors schedule the end of neurogenesis via cell cycle exit or apoptosis. Louise Cheng, Cèdric Maurange, Alex Gould. national institute for medical research, london, United Kingdom. The developmental stage at which postembryonic neurogenesis in the CNS terminates depends upon anterior-posterior position. In abdominal neuromeres, it is known that neural proliferation shuts down via Hox-dependent neuroblast apoptosis. In most other regions of the CNS, the mechanism by which neuroblasts stop dividing has been unclear. Here we examine the time course of neuroblast disappearance in the central brain and thoracic region during pupal stages. Using time-lapse movies, we observe that the final division of thoracic neuroblasts produces two-equal sized daughters that undergo cell cycle exit. The process executing the switch from size asymmetric-to-symmetric division involves the delocalisation of asymmetric determinants, resulting in the translocation of Prospero from the cell cortex into the neuroblast nucleus. The upstream timing mechanism triggering this Prospero nuclear translocation requires two members of the embryonic temporal transcription factor system, Castor and Seven Up, which are re- expressed in sequential bursts in postembryonic neuroblasts. Several ways of disrupting Castor or Seven Up expression lead to persistent neuroblast divisions in the adult CNS and consequent neural overproliferation. Together, these studies show that the temporal transcription factor system provides a global timing mechanism for shutting down neurogenesis, regardless of whether this is executed via cell cycle exit (thorax) or apoptosis (abdomen). They also provide evidence that the temporal transcription factor system coregulates cell fate and cell proliferation within the CNS.

145 Structural Basis for Robo Receptor Control of Lateral Positioning: An Unexpected Role for Robo Extracellular Domains. Timothy A. Evans1, Barry J. Dickson2, Greg J. Bashaw1. 1) Dept of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, PA; 2) Research Institute of Molecular Pathology, Vienna, Austria. Drosophila Roundabout (Robo) receptors regulate a number of fundamental axon guidance choices throughout development. Robo and Robo2 cooperate to mediate midline repulsion in response to Slit, while Robo2 and Robo3 specify lateral position of longitudinal axons within the CNS. Although the three Robo receptors share a high degree of structural and sequence similarity throughout their extracellular regions, their cytoplasmic domains are more divergent. Robo2 and Robo3 lack the CC2 and CC3 motifs that mediate interactions with downstream Robo signaling components, suggesting that Robo2 and Robo3 signaling may be qualitatively distinct from Robo. Consistent with this idea, forced expression of Robo2 or Robo3 in medial ipsilateral axons forces them into more lateral positions, while increased Robo expression does not. To investigate the structural basis for this activity, we have analyzed a series of chimeric receptors in which the extracellular and cytoplasmic domains of Robo, Robo2, and Robo3 have been swapped. Remarkably, the extracellular portions of Robo2 or Robo3 are sufficient to shift axons laterally, regardless of which Robo cytoplasmic domain is present. Further, we have determined that the Slit binding region of Robo2 is required for dictating lateral position. Using more restricted domain swaps, we have uncovered a bipartite contribution to lateral shifting activity involving the Slit binding region of Robo2 along with a second extracellular region. These observations suggest that the differential roles of Robo receptors in lateral positioning are caused in part by differential response to Slit, and indicate that multiple ectodomain modules contribute to this response.

146 Down Syndrome Cell Adhesion Molecules as multifunctional Netrin receptors required for midline crossing. Gracie L. Andrews, Thomas Kidd. Biology Department/ms 314, University of Nevada, Reno, NV 89557, USA. A major challenge in the field of axon guidance is to identify extracellular factors and receptors that guide axons. We have identified Down syndrome cell adhesion molecules (Dscams) as novel Netrin receptors necessary for midline axon crossing in the central nervous system (CNS). A reverse genetic screen in Drosophila revealed that mutations in Dscam and Dscam3 affected midline axon crossing, and were spatiotemporally expressed coincident with pioneer commissural axons. Dscam mutants exhibited defects in Bolwig’s nerve projection and salivary gland guidance. The defects in Bolwig’s nerve and the salivary gland were recapitulated in Netrin mutants. frazzled mutants, the previously characterized Drosophila Netrin attractive receptor, exhibit midline crossing defects that are more subtle than those found in netrin deletions, suggesting that there may be at least one more Netrin receptor. Disruption of frazzled and each Dscam (Dscam and Dscam3) in double mutants caused a significant worsening of midline crossing defects. In the Dscam-frazzled double mutant a specific subset of commissural axons (connectin positive) showed guidance defects beyond that of frazzled alone and approaching defects found in netrin deletions. Triple mutants, in which Dscam, Dscam3 and frazzled were mutated, exhibited a commissureless-like phenotype in which most axons were unable to cross the midline. These phenotypes led us to predict that Dscam and Dscam3 encode Netrin receptors. Cell culture binding assays show that Netrin binds to Dscam in evolutionarily conserved association. Kd values of Netrin and Dscam binding are statistically the same as Netrin and its receptor DCC (deleted in colorectal cancer.) We believe Dscam is an entirely attractive receptor. First, Dscam mutations in conjunction with mutations in the frazzled Netrin receptor gene, lead to a dramatic failure of axons to cross the CNS midline. Second, gain of function experiments lead to increased axon crossing of the midline. Finally, Dscam mutants can suppress mutations in the roundabout (robo) pathway that repels axons from the midline. 138 PLATFORMS: Neurogenetics and Neural Development

147 DOUBLESEX establishes sexual dimorphism in the Drosophila central nervous system in an isoform-dependent manner by directing cell number. Laura Sanders, Michelle Arbeitman. Molecular and Computational Biology, University of Southern California, Los Angeles, CA. doublesex (dsx) encodes sex-specific transcription factors that act at the top of one branch of the somatic sex determination hierarchy. dsx is responsible for directing all aspects of somatic sexual differentiation outside the nervous system. Here we show that the number of DSX-expressing cells in the central nervous system is sexually dimorphic during both pupal and adult stages. The number of DSX-expressing cells is established by both the sex-specific DSX isoform present and the amount of DSX activity in the cell. We demonstrate that in males, DSX is present in a portion of the neural circuitry in which the male-specific product of fruitless (fru) is produced; this circuit underlies all aspects of male courtship behaviors. We show, however, that the number of DSX-expressing cells is established independent of male-specific FRU and that the number of male-specific FRU-expressing cells is established independent of DSX. Additionally, we demonstrate that for one cluster of DSX-expressing cells in the ventral nerve cord, the sexual dimorphism in cell number is due to cell death. Therefore, in addition to its known role in establishing sexual dimorphism outside the CNS, our data demonstrate that DSX establishes sex-specific differences in neural circuitry by regulating the number of neurons.

148 Expression of vestigial in the Drosophila embryonic central nervous system. Kirsten Guss1, Hemlata Mistry1,2, James Skeath2. 1) Dept Biol, Dickinson College, Carlisle, PA; 2) Dept Genetics, Washington University School of Medicine, St. Louis, MO. The Drosophila central nervous system is an excellent model system in which to resolve the genetic and molecular control of neuronal differentiation. Here we show that the wing selector vestigial (vg) is expressed in discrete sets of interneurons. We track the axonal trajectories of a subset of VG-expressing cells in the ventral nerve cord and show that these cells descend from neuroblasts 1-2, 5-1, and 5-6. This work provides the necessary descriptive foundation for functional studies regarding the role of vestigial during interneuron differentiation. PLATFORMS: Cell Division and Growth Control 139

149 Insulin Receptor pathway regulates cell division through miRNAs and p21/dacapo in the Drosophila germ line stem cells. Jenn-Yah Yu, Steve Hatfield, Halyna Shcherbata, Karin Fischer, Hannele Ruohola-Baker. Department of Biochemistry, University of Washington, Seattle, WA. p21/dacapo (dap) is a potential target of miRNAs in regulating cell cycle of germ line stem cells (GSCs) in Drosophila melanogaster. Tissue-extrinsic signals, such as insulin, have also been shown to affect cell division of GSCs. Here, we explored the possibility that insulin receptor (InR) pathway may interact with the miRNA/dap regulatory circuit, therefore control GSC cell division. By using luciferase assay, we demonstrated that dap 3’UTR can be directly targeted by several miRNAs, including miR-7, miR-8, and miR- 309. By using a GFP sensor with dap 3’UTR, we showed in vivo that the dap 3’UTR does not only respond to miRNA activities but also responds to the InR signaling activities in GSCs. These results suggest that the InR activity may regulate dap expression through dap 3’UTR. By using genetic approaches, we demonstrated that the InR and miRNA/dap may act in the same pathway; reduction of dap partially rescued the cell cycle defect of InR deficient GSCs. Furthermore, decrease of InR signaling by restricting protein diet does not affect either dap or dicer-1 mutant GSCs. These data suggest that InR signaling may regulate GSC cell cycle through miRNA/dap.

150 The anaphase promoting complex / cyclosome (APC/C) is required for re-replication control in endoreplication cycles. Norman Zielke1,2, Silvia Querings2, Frank Sprenger2,3. 1) Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA., Select Country; 2) University of Cologne, Institute for Genetics, Zuelpicherstrasse 47, 50674 Koeln, Germany; 3) University of Zuerich, Institute of Zoology, Winterthurerstrasse 190, CH-8057 Zuerich, Switzerland. The endocycle is a cell cycle variant that is applied in various tissues of Drosophila and certain human cell types. Endoreplicating cells undergo multiple rounds of DNA replication that are separated by distinct G-phases, but never enter mitosis. Contrary to the current view of endoreplication, we have found that oscillation of APC/C-Fzr activity is essential for endocycle progression. In salivary gland endocycles, the APC/C-Fzr complex regulates the abundance of the Geminin protein, which is crucial to prevent relicensing in S-phase. Inactivation of the APC/C-Fzr complex in larval salivary glands results in Geminin stabilization and as a consequence DNA replication is inhibited. Geminin only accumulates in cells with high CycE/Cdk2 activity, suggesting that APC/C- Fzr activity is inhibited by CycE/Cdk2. Consistent with this idea we found that Geminin is stabilized upon CycE overexpression. Moreover, we provide evidence that CycE/Cdk2 and E2F1 cooperate in an autoregulaory feedback loop that drives the endocycle. Impaired DNA replication prevents E2F1 turnover in salivary glands and subsequently results in accumulation of E2F1 and its target genes, including CycE. This mechanism ensures that CycE/Cdk2 activity accumulates prior to S-phase and subsequently drops after completion of DNA replication. The peak of CycE/Cdk2 activity simultaneously triggers S-phase and prevents relicensing by Geminin stabilization through inhibition of APC/C-Fzr activity. In the following G-phase, when CycE/Cdk2 activity is low, the APC/C- Fzr complex is released and targets Geminin for proteasomal degradation. This allows licensing of replication origins and prepares for the next round of DNA replication. Thus, we have identified a novel APC/C regulated mechanism that prevents unscheduled DNA licensing in endoreplication cycles.

151 The role of Misato in Drosophila spindle assembly. Violaine Mottier, Giovanni Cenci, Fiammetta Vernì, Maurizio Gatti, Silvia Bonaccorsi. Genetica Biol Molecolare, Univ di Roma La Sapienza, Rome, Italy. The Drosophila misato (mst) gene encodes an evolutionary conserved protein that contains motifs shared among tubulins and myosin. Previous studies have shown that mst mutants die at the larval/pupal boundary and exhibit frequent polyploid cells in larval brains. However, the function of the Mst protein was not well defined. To determine the mitotic role of mst we stained mutant brains squashes for tubulin and Centrosomin (Cnn). We found that most mitotic cells were arrested in metaphase. In addition, 70% of these metaphase figures were monopolar and displayed abnormally long microtubules (MTs) emanating from the two unseparated centrosomes. Recruitment of centrosomal proteins such as DPLP, γ-tubulin, DGrip91, CP190, and D-TACC was not affected in mst mutants, consistent with a normal nucleating ability of the centrosomes. To elucidate the mechanisms underlying monoplar spindle formation we assayed MT regrowth following cold-induced depolymerization. In wild type larval brain cells, release from cold treatment resulted in rapid formation of MTs around both the chromosomes and the centrosomes. In contrast, in mst mutant cells, MTs were nucleated almost exclusively by the centrosomes. Immunostaining with an antibody to Mst showed that mitotic cells exhibit a strong, homogeneous signal that persists from prophase to telophase. Conversely, interphase cells consistently displayed a diffuse and weak Mst staining. Collectively, these results indicate that the mst function is primarily required during mitosis for chromatin-induced MT nucleation, and highlight the importance of this process in mitotic spindle assembly. 140 PLATFORMS: Cell Division and Growth Control

152 FUNCTIONNAL ANALYSIS OF A NOVEL FAMILY OF ERM PROTEIN PARTNERS DURING CELL DIVISION. Sébastien Carreno1,2, Hélène Foussard2, Jérome Miailhe2, Cédric Polesello2, Philippe Valenti2, Pierre Ferrer2, François Payre2. 1) Université de Montréal, IRIC, Montreal, QC, Canada; 2) Centre de Biologie du Développement, CNRS University ToulouseIII, Toulouse France. Cell division involves a stereotyped sequence of changes in cell morphology, regulated by localized cortical acto-myosin contractions. While the microtubule spindle is well known to influence location of the cleavage furrow, how cell shape transformations are coordinated with spindle reorganization throughout mitosis remains largely elusive. ERM (Ezrin, Radixin, Moesin) proteins, which are deregulated in several cancers, are known to link cortical actin to membrane, upon signal-mediated activation. We have previously shown that localized activation of Moesin, the unique drosophila ERM, is required both for cell shape changes and mitotic spindle positioning during cell division. We have isolated GIM a Moesin genetic interactor which defines a novel evolutionarily conserved protein family. Having shown that GIM binds specifically to the Moe protein, we explored a putative function of GIM during cell division. We show that GIM specifically co-localizes with activated Moe, at the cortex in pro/metaphase and at the cleavage furrow in ana/telophase. Interestingly, GIM also localizes at centrosomes and midbody. Similarly to Moe inactivation, depletion of GIM in S2 cells leads to severe defects in mitotic cell shape, with large cytoplasmic bulges that deform the cortex. Phylogenetic analysis allowed identification of 4 GIM orthologs in mammals. We characterized human GIM-like genes and analyzed the distribution of one out the 4 hGim proteins. During cell division, we show that it localizes both at the cleavage furrow and centrosomes/midbody. Taken together, our results show that the GIM family of ERM partners plays an important role in the control of cell division in Drosophila. Its functional conservation in humans may be relevant to understand regulation of ERM proteins during cell division, in normal and pathological situations.

153 Drosophila short neuropeptide F signaling regulates growth by ERK mediated insulin signaling. Kweon Yu1, Kyu-Sun Lee1, Kyung-Jin Min2, Marc Tatar2. 1) Centre for Regenerative Medicine, Korea Res Inst of Bioscience & Biotechnology, Daejeon, Korea; 2) Dept of Ecology & Evolutionary Biology, Brown University, Providence, RI. Insulin/insulin growth factor signaling plays a central role in growth, metabolism, and aging in animals. However, up-stream regulatory signaling for the insulin transcription is not well known. Here we present that Drosophila short neuropeptide F (sNPF) signaling regulates expression of Drosophila insulin like peptides (Dilps) through ERK activation in insulin producing cells and controls growth by regulating insulin receptor/dFOXO signaling in the fat body insulin target tissue. The gain-of-function sNPF or sNPFR1 receptor mutant produced a big body size through ERK mediated Dilps expression. When Drosophila CNS cells or rat pancreatic cells were treated with sNPF or NPY peptide, a sNPF vertebrate ortholog, Dilps or insulin expression was increased by the activation of ERK. In the fat body cells of sNPF mutants, expression of translational inhibitor 4E-BP was increased by the down-regulation of Akt and nuclear localized dFOXO, resulting in the reduction of cell size. Moreover, in sNPF mutants, high glucose levels of larval hemolymph were detected and lifespan was extended. Drosophila sNPF and evolutionary conserved mammalian NPY appear to regulate ERK mediated insulin expression and to thus systemcally modulate growth, metabolism, and lifespan.

154 GTRs - small GTPases implicated as novel regulaTORs of growth. Pankuri Goraksha, Thomas Neufeld. Molecular, Cellular, Developmental Biology & Genetics, University of Minnesota, Minneapolis, MN. Gtr1 & Gtr2 are evolutionary conserved members of a unique subfamily of Ras-like small GTPases. In mammals and S.cerevisiae, Gtr1 & Gtr2 form a stable heterodimeric complex. In S. cerevisiae, the Gtr1-Gtr2 complex is involved in regulation of exit from rapamycin-induced growth arrest and microautophagy1, and general amino acid permease sorting2, a function negatively regulated by TOR activity. In mammalian cell culture, the conserved GTPases Gtr1 and Gtr2 positively affect TOR activity as measured by phosphorylation of its well-known targets S6K and 4EBP3. In a collaborative approach to dissect their significance, we analyzed the requirement of Gtr1 and Gtr2 in the TOR pathway in a higher evolved multicellular organism, namely, Drosophila. We find that both Gtr1 and Gtr2 act as novel positive regulators of TOR pathway in Drosophila by regulating organismal growth, individual cell size, and starvation-induced autophagy. Gtr1 is positively affected by nutrient availability, and expression of its constitutively active form offers cells a growth advantage under nutrient limiting conditions. Gtr-complex activity is independent of Rheb and these effectors act in parallel with each other to enable growth. Thus, our investigation reveals one more novel Ras-like GTPase family complex, in addition to Rheb GTPase, to positively regulate TOR activity, thereby leading to growth and development in Drosophila. 1.Dubouloz F et al., Mol Cell. 2005; 19(1); 15-26 2.Gao M & Kaiser CA, Nat. Cell Biol. 2006; 8(7): 657-67 3.Guan KL (unpublished data). PLATFORMS: Cell Division and Growth Control 141

155 alpha-Endosulfine is a conserved protein required for meiotic maturation that interacts with an E3 ubiquitin ligase and regulates Twine/CDC25 levels. Jessica Von Stetina, Susanne Tranguch, Sudhansu Dey, Ethan Lee, Laura Lee, Daniela Drummond- Barbosa. Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN. Meiosis is tightly coupled to the development of gametes and must be well regulated to prevent chromosome segregation errors. During meiotic maturation, Drosophila oocytes progress from the initial prophase I arrest to an arrest in metaphase I, which is maintained until egg activation. The extrinsic and intrinsic factors controlling oocyte maturation and the metaphase I arrest, however, are poorly understood. Our recent work shows that Drosophila alpha-endosulfine (dendos) plays a key role in this process. dendos mutant oocytes remain in prophase I longer and, instead of progressing into metaphase I, they fail to form a meiotic spindle and their DNA becomes dispersed. This phenotype is remarkably similar to that of twine, the meiotic homolog of cdc25. The CDC25 phosphatase is known to activate cdk1/cyclinB, which is required for meiotic maturation, as shown in other systems. We also find that the levels of a Twine-LacZ fusion protein are reduced in dendos mutants, suggesting that Dendos is required for normal levels of Twine expression and presumably, cdk1/cyclinB activity. Intriguingly, from an in vitro binding screen, we identified the CG17033-encoded E3 ubiquitin ligase as a strong Dendos interactor. We generated null alleles of CG17033 and found that, in these mutants, the transition into metaphase I occurs prematurely. These results are consistent with the model that upon stimulation of the oocyte by a meiotic maturation signal, the rise in cdk1/cyclinB activity is limited by the degradation of Twine, and that Dendos binds to and inhibits this E3 to promote the transition into metaphase I. We are currently testing if overexpression of twine rescues the meiotic phenotype of dendos mutants. Finally, germline-specific expression of either Dendos or human alpha-Endosulfine rescues this defect, and we can also detect alpha-Endosulfine expression in mouse oocytes, which suggests evolutionary conservation of the meiotic function of alpha-Endosulfine.

156 Mutation in the Ubiquitin Activating Enzyme, E1, can cause tissue overgrowth by upregulating Ras pathway activity by both cell-autonomous and non-autonomous means. Hua Yan1, Mei-Ling Chin2, Cathie Pfleger1. 1) Department of Oncological Sciences, Mount Sinai School of Medicine, New York, NY; 2) Department of Molecular, Cell, and Developmental Biology, Mount Sinai School of Medicine, New York, NY. The Ras proteins are important regulators of signal transduction that can promote proliferation, cell survival, and differentiation. Mutations in Ras/Raf are found in 30% of solid tumors and mutations in components of the pathway are found in developmental disorders such as Noonan’s syndrome. In Drosophila, Ras85D represents a single homologue to human K-Ras, H-Ras, and N-Ras. It has recently been demonstrated that H-Ras is constitutively di-ubiquitinated and that a non-ubiquitinatable form of H-Ras demonstrates increased activity in terms of its ability to activate ERK. The biological consequences of H-ras ubiquitination in an organism are still unclear. In this report we demonstrate that cells containing hypomorphic mutation in E1, the most upstream enzyme in the ubiquitin pathway, display an upregulation of Ras/ERK activity and Ras-dependent ectopic division. Complete inactivation of E1 leads to a similar cell-autonomous increase in Ras/ERK activity, and the non-autonomous activation of Ras/ERK in adjacent tissue. Our findings may suggest that loss of Ras ubiquitination itself leads to growth-relevant Ras/ERK activation. Furthermore, different levels of inactivation in E1 lead to cell-autonomous and non-autonomous Ras activation. We propose that maintaining proper levels of E1 function in general, and Ras ubiquitination in particular are crucial to normal development and that the loss of Ras ubiquitination may play a role in developmental disorders and cancer.