Denise J. Montell - US grants

Cell movements are critical features of the development of all multicellular organisms, and loss of regulation of regulation of cell movement is an essential step in cancer metastasis. While some progress has been made in identifying cell and substrate adhesion proteins required for some cell movements, how the proteins mediate movement and how the timing of such movements is regulated remains unknown. This proposal focuses on the dramatic migration of six to ten "border cells" during Drosophila oogenesis as a model system for a genetic dissection of developmentally regulated cell movement. It deals primarily with one gene, slow border cells (slbo), whose function is required for initiation of migration at the proper time. Hypomorphic mutations at this locus cause delayed border cell migration, while stronger alleles cause failure of the migration, suggesting that a threshold level of slbo+ product is required to initiate the migration. A molecular characterization of this gene is proposed, including cloning the locus using existing single P-element insertion alleles, deriving a transcript map for the region, rescuing the mutant phenotype by germline transformation of slbo+ into a mutant background, and determining the deduced amino acid sequence of the slbo product. New alleles will be generated both by excision of transposons currently inserted in the gene and by EMS mutagenesis. DNA sequencing of point mutants will provide structure/function information, and production of antibodies to the protein will allow its subcellular localization and developmental distribution to be determined. Finally, genetic screens are proposed to identify other loci whose products are required for this migration and/or interact with the slbo product.

DESCRIPTION (appended verbatim from investigator's abstract): Epithelial to mesenchymal transitions are critical features of the development of most organs and tissues, however the molecular basis of this phenomenon is not well understood. In addition, most tumors are of epithelial origin and acquisition of an invasive phenotype is a critical, but relatively poorly understood step in metastasis. We have developed a model system for a systematic genetic approach to the study of epithelial to mesenchymal transitions. Studies from my lab and others indicate that the conversion of border cells in the Drosophila ovary from epithelial cells to migratory cells is a multistep process that depends upon changes in gene expression, cell adhesion and cytoskeletal organization. The first gene identified to play a role in border cell migration, slow border cells (slbo), encodes a Drosophila homolog of the C/EBP family of transcription factors. SLBO regulates the expression of a number of target genes including those that code for an FGF receptor homolog and E cadherin. In addition, the regulated activity of the GTPase Rac, but not Rho or Cdc42, is required during border cell migration. We have recently discovered a SLBO independent pathway, defined by three loci, taiman (tai), slow motion (slmo) and stuck in place (sip) that were identified in a screen for mutations that cause cell migration defects in mosaic clones. These loci exhibit dominant genetic interactions, indicating that they may act in a common biochemical pathway. The tai gene encodes a protein homologous to steroid hormone receptor coactivators, the first such protein identified in an invertebrate. In tai mutants the expression and subcellular localizations of a number of border cell proteins are unchanged, whereas Armadillo (ARM) protein levels remain unusually high, suggesting a possible explanation for the migration defect. The specific aims of this proposal are to 1) test the hypothesis that failure to down regulate ARM expression contributes to the tai migration defect; 2) test the hypothesis that TAI is a hormone dependent transcriptional regulator; 3) define the roles of slmo and sip in border cell migration; and 4) identify and characterize proteins that act in the Rac pathway to affect border cell migration.

DESCRIPTION (provided by applicant): Cell migration is a fascinating feature of embryonic development, and improperly regulated cell migration contributes to birth defects and tumor metastasis. We have developed a simple model system for a forward genetic approach to the study of cell motility in vivo, the migration of a subset of follicle cells, known as border cells, in the Drosophila ovary. We have established that multiple extracellular signals regulate their movement. 1) a global steroid hormone signal, ecdysone, acting through the ecdysone receptor and a transcriptional coactivator called Taiman; 2) a highly localized cytokine signal, which activates the JAK/STAT pathway; and 3) several growth factors, which signal through the EGFR and PVR receptor tyrosine kinases to guide the cells to their destination. In addition we have studied a variety of cytoskeleton-associated proteins and cell adhesion molecules that function in border cell migration. This proposal explores the mechanisms by which cell adhesion is dynamically regulated in migrating border cells, an important aspect of cell motility that is not well understood for any cell type. We propose three specific aims. The first is to investigate the mechanisms that govern trafficking and stability of E-cadherin, a homophilic cell-cell adhesion molecule that is required in border cells and in the cells upon which they migrate. To test whether E-cadherin is turned over more rapidly in border cells than in non-migrating follicle cells, we will employ a previously characterized variant of the red fluorescent protein that changes color over time, fused to E-cadherin. We will investigate whether EGFR and PVR signaling destabilizes cell adhesion by phosphorylation of specific tyrosine residues on beta-catenin/Armadillo. We will also determine whether endocytosis is important for regulating cell surface E-cadherin in migrating border cells. We will test whether Drosophila moesin contributes to E-cadherin dynamics. And we will investigate the mechanisms by which Myosin VI contributes to E-cadherin dynamics in border cells. In the second specific aim we propose to investigate in detail the mechanisms by which RhoGAP93B contributes to border cell migration by studying its biochemical activity, its expression pattern, subcellular localization, lethal phenotype and its regulation. Finally we propose to study a putative downstream target of Rho in border cells, rhophilin by characterizing the mutant phenotype, epistasis analysis with Rho and by identifying and characterizing interacting proteins.

The overall goal of this Initiative is to expand the current Migration Knowledgebase through the identification of cellular genes that regulate cell shape, adhesion and motility using high throughput RNA interference screens in cell- or organism-based assays. In addition, large scale expression screens will be employed in the fruit fly to identify candidate genes that display patterns of expression and intracellular localization consistent with a role in migration. Through coordinated cross-species analyses we will identify a set of genes that play a central and conserved role in cell adhesion and migration.

DESCRIPTION (provided by applicant): Cell migration is a major driving force in embryonic development, wound healing, and tumor metastasis. Therefore, understanding the molecular mechanisms that control cell motility is significant for human health. My laboratory has developed a relatively simple and genetically tractable model for the study of cell migration, the movement of a small group of cells in the Drosophila ovary known as the border cells. In the previous funding period we have identified and characterized a number of new border cell migration genes. We also overcame a major technical obstacle and succeeded in time-lapse, live-imaging of migrating border cells. We propose to build upon these advances to address a major unsolved question in the field of cell migration: how cell polarity and cytoskeletal dynamics are integrated to achieve coordinated, directed migration of a group of cells. To address this general question, we will combine genetic approaches, live-imaging and biochemistry with two specific aims. The first is to test the hypothesis that a protein we have named FAV, and its mammalian homologs, amplify EGFR-dependent cell polarity and integrate EGFR signaling, trafficking and cell polarity with actin dynamics. The second aim is to test the hypothesis that a newly identified protein, Tiarin, and its human homolog, are conserved regulators of actin dynamics and cell motility. Together these studies will provide insights into the mechanisms controlling border cell migration, correlate the findings with the broader field of actin dynamics, and use this model to identify and characterize genes important in human health and disease. PUBLIC HEALTH RELEVANCE: The ability to move is a property of cells from virtually all animals, from simple ones such as flies and worms, all the way to humans. The proposed research focuses on the movement of a small group of cells in the fruitfly, to learn more about how specific molecules orchestrate when, where and how they move. Since cell motility contributes to wound healing and tumor metastasis, these studies could lead to the discovery of new drug targets that could promote healing or inhibit the spread of cancer cells.

DESCRIPTION (provided by applicant): Properly regulated cell migrations are essential to human health. Genetic defects that impair cell motility cause birth defects such as brain malformations and immune deficiencies. On the other hand, the ability to migrate and invade converts curable tumors into incurable, metastatic disease. In addition, in order to achieve a major goal of regenerative medicine, which is the creation of artificial organs and tissues, it is necessary not only to specify all of the appropriate cell types, but also to control their organization, communication, and movements. Therefore it is of great importance that we understand and harness the mechanisms controlling tissue morphogenesis in general, and cell migration in particular. These are the long-term goals of our studies. Decades of research have revealed the molecules and mechanisms that control the movements of single cells in tissue culture dishes. How cells move through their intricate natural environments is less well-understood. In vivo cells often move in interconnected sheets, tubes, strands, and clusters. Despite their ubiquity and importance, such collective cell behaviors are not as well-studied as those of single cells. The shapes of cells moving through complex environments can differ greatly from the morphology of a cell migrating, unobstructed, on glass. These observations raise numerous questions. For example, how do the mechanisms of collective cell movement resemble or differ from single cell motility, and how is the great diversity of cell shapes achieved? One major difference between single and collective cell migration is that cells moving collectively maintain cell-cell adhesion even as they move. While we now know many of the molecules that are important for cell movements, we know far less about how the activities of these proteins are coordinated in space and time. To address these questions we have developed a relatively simple and genetically tractable model for the study of collective cell migration: the border cells in the Drosophila ovary. We propose to use new methods that we have developed to measure and even manipulate protein activities and mechanical forces in vivo with light. Our specific aims are to: 1) test the hypothesis that cell-cell adhesion serves multiple, critical functions in collectively migrating cells, including cluster organization, direcion sensing, and stabilization of protrusions. We will also compare directly the mechanisms of single and collective cell migration in vivo. 2) test the hypothesis that feedback between Rac and a tyrosine kinase coordinates polarity, protrusion, and adhesion during collective migration. Here we also propose to identify functional substrates of the tyrosine kinase. 3) test the Tropomyosin (Tm) code hypothesis, which postulates that the diversity of cell shapes and behaviors can be attributed to the diversity of dynamic F-actin structures, which in turn depend upon the combination of Tm isoforms present in a cell.

DESCRIPTION (provided by applicant): Cell movements are critical features of normal embryonic development and tissue homeostasis. Cell migration also drives tumor dissemination and contributes to autoimmune disease. In addition, successful regenerative medicine will require that we know how to control the assembly of cells into functional three-dimensional (3-D) architectures. It is therefore important that we uncover and harness the mechanisms that govern cell movements. Our basic understanding of how cells move derives from studies of cultured cells moving on hard surfaces in vitro. However cells appear and behave differently when cultured in more complex and compliant, 3-D environments. Moreover, in vivo, many cells move as interconnected groups. To elucidate the mechanisms regulating such a collective cell migration in vivo, my laboratory has developed a genetically tractable model: the migration of the border cells in the Drosophila ovary. Over the last >20 years we identified the molecular pathways that control the timing and direction of movement, as well as the critical contributions of key regulators of the actin cytoskeleton, such as the 21kD GTPase Rac. More recently we developed organ culture and live imaging approaches, which enable us to use innovative tools, such as photo-activatable analogs of Rac (PA-Rac) and F?rster Resonance Energy Transfer (FRET). Using these techniques we discovered novel properties of Rac and its relative Cdc42, including unexpected synergistic effects of the two proteins, and the instructive role of Rac in collective cell behaviors. Here we propose to build upon this foundation of genetic screening, live imaging, PA proteins and FRET probes, to test iconoclastic hypotheses concerning the relationships between Rac, Rho, and Cdc42 in migrating cells. In Aim 1, we propose to test the hypothesis that Rac and Cdc42 have redundant effects, in addition to their well-characterized non-redundant and newly discovered synergistic effects. In Aim 2 we will elucidate the molecular mechanisms underlying their synergy. In Aim 3 we propose to investigate the relationship between Rac and Rho. In Aim 4 we propose to elucidate the molecular mechanisms by which cells of a migrating group sense and communicate directional information between the migratory cells, to achieve coordinated collective chemotaxis. Each of these aims is founded upon substantial published and unpublished preliminary data. This work is significant because Rho, Rac and Cdc42 are key nodes in the signaling and cytoskeletal networks that control cell shape and movement. So understanding their mechanisms of action and inter-relationships in vivo is of fundamental importance to cell and developmental biology. Yet there remain key unanswered questions concerning how they function in concert to coordinate collective cell migration. In addition, our studies enhance our ability to understand and control cell movements in therapeutic settings, such as tumor metastasis and tissue engineering. The proposed projects are innovative because they take advantage of a unique combination of cutting edge tools that we have developed, to test new ideas concerning the overlapping and synergistic roles of Rho family proteins in collective cell migration in vivo.

DESCRIPTION (provided by applicant): Apoptosis plays essential roles in development and homeostasis in multicellular organisms by sculpting tissues, deleting unwanted structures, and eliminating abnormal, injured or dangerous cells. In addition, targeting apoptotic pathways is an important strategy for treatment of intractable diseases such as cancer, whereas limiting apoptosis may be beneficial for treating ischemic injury and degenerative disorders. Although loss- or gain-of-function of apoptotic regulators can artificially allow cells to survive beyond normal checkpoints, apoptosis is generally assumed to be an intrinsically irreversible process. However, we recently discovered a natural reversibility of late-stage apoptosis in human and mouse cells. Dying cells can reverse apoptosis and survive, despite having passed through checkpoints previously believed to be the point of no return, including caspase-3 activation and DNA damage. Simply washing away apoptotic inducers is sufficient to allow the majority of dying cells to survive and most hallmarks of apoptosis to vanish, indicating that reversal of apoptosis is an endogenous cellular mechanism. Notably, while most cells recover completely, a small fraction of cells that reverse apoptosis retain genetic alterations and undergo oncogenic transformation at a higher frequency than control cells. We propose that reversal of apoptosis may be a physiological mechanism that can serve several beneficial functions. Arrest of apoptosis at the execution stage could in principle promote survival of cells, such as neurons and heart muscle cells, which are difficult to replace. Alternatively or in addition, this recovery process, which we have named anastasis (Greek for rising to life), could promote genetic and phenotypic diversity in response to environmental or physiological stresses that initiate apoptosis. A negative side effect of this otherwise beneficial process is oncogenic transformation. We have developed and tested a biosensor to detect cells that have undergone anastasis in vivo in Drosophila melanogaster. In specific aim 1 we will test the hypothesis that anastasis functions to salvage cells that are difficult to replace, thus limiting permanent tissue damage following transient insults. We also propose to develop a similar biosensor for use in mammalian cells. In specific aim 2 we propose to initiate studies of the molecular mechanisms controlling anastasis. The proposed work has the potential to lead to a new understanding of and treatments for degenerative diseases and cancer.

We recently discovered a new biological phenomenon, which we call anastasis (Greek for ?rising to life?). Overturning the current dogma that cell death is irreversible, we found that a variety of normal and cancer cell types can reverse the process, survive, and proliferate. This reversibility takes place even after cells experience events widely believed to be points of no return, including activation of caspase enzymes and widespread DNA damage. Notably, while most cells fully recover and repair their damaged DNA, some cells retain mutations, and this increases the frequency of oncogenic transformation. The discovery of anastasis has at least five paradigm-shifting implications. First, we suggest that anastasis represents a previously unknown cause of cancer, so inhibiting anastasis should prevent cancer. Anastasis could also offer an explanation for the longstanding observation that repeated injury increases the incidence of cancer. Second, we propose that anastasis allows tumor cells to escape chemotherapy and evolve drug resistance. Therefore, inhibiting anastasis may enhance the effectiveness of chemo- and radiation therapies and prevent relapses. Third, salvaging cells on the brink of death via anastasis may limit permanent tissue injury due to transient environmental stresses or toxin exposures. Consequently, enhancing anastasis may promote tissue regeneration. Fourth, we posit that anastasis is a cell survival mechanism that protects cells that are difficult to replace such as neurons in the adult brain or heart muscle cells, so promoting anastasis could prevent or slow degenerative diseases. Fifth, we propose that the survival of germ cells with mutations acquired through anastasis provides a mechanism to enhance genetic diversity precisely when animals are exposed to stressful environmental conditions. This could accelerate adaptation to changing environments during evolution. Here we propose to test these ideas. We designed a biosensor that will allow us to identify and track cells that undergo anastasis in vivo by creating permanent expression of a reporter such as GFP in cells that survive caspase activation. Using this biosensor in mice we propose to test the hypotheses that transient injuries and stresses induce anastasis, that anastasis causes cancer and allows tumor cells to evade therapies and develop drug resistance. Using the biosensor in Drosophila, we will test the hypothesis that anastasis enhances genetic diversity in the population. In addition, we propose to decipher the molecular mechanisms that allow cells to reverse the dying process and survive and identify molecular approaches to inhibit or enhance anastasis. The successful completion of this project offers the potential to develop revolutionary new therapies for cancer, neurodegenerative diseases, and heart failure, and provide new insight into the mechanisms of evolution by natural selection.

DESCRIPTION (provided by applicant): The goal of the Gordon Research Conference on Direction Cell Migration is to provide a stimulating forum for the dissemination and discussion of new research, concepts and opportunities at the forefront of cell migration. Peter Devreotes and Jeff Segall, who organized the first meeting held in 2005, envisioned developing a forum fostering interactions among scientists with a strong interest in gradient sensing and directed cell migration. After 10 years, the conference is considered to be the top research conference in cell migration. The program for the 2015 conference, to be held January 24-30 at Hotel Galvez, Galveston, TX, is aimed at bringing together cell migration scientists from diverse disciplines, including molecular and cell biologists, engineers and physicists. A strong emphasis will be placed on (1) basic mechanisms of cell migration at the levels of signal transduction and actin assembly, (2) inclusion of researchers that work with model systems as well as in vivo systems, (3) the study of cell migration during development, leukocyte trafficking, wound healing, and cancer invasion and metastasis and (4) comparing and contrasting single-cell versus collective cell migration in 2- and 3-dimensional environments. The aims of the conference are to: (1) provide a forum for the discussion of cutting edge research in the area of directed cell migration, with a particular focus on multidisciplinary approaches and collaborative science, (2) promote both formal and informal scientific exchanges among scientists investigating directed cell migration, including young scientists, women and scientists from underrepresented minority backgrounds, and (3) discuss recent advances especially relevant to the biology of cancer invasion and metastasis. To accomplish these aims, the 2015 Conference will bring together an international cadre of young and senior scientists from academia that will interact in an atmosphere conducive to the free exchange of ideas. As in 2013, the 2015 GRC will be preceded by a GRS on Directed Cell Migration, organized by young researchers (Ritankar Majumdar and Shirin Pocha), with focus on using multidisciplinary approaches in the study of cell migration. This event will help nurture junior researchers towards a successful and rewarding career in the field of cell migration. The GRS will be opened by Andrew Ewald (JHU), who will discuss new paradigms in collective breast cancer invasion. The closing GRS lecture will be given by Xavier Trepat (Institute for Bioengineering of Catalonia, Spain) on the control of collective cell migration by intercellular adhesome. Eight talks selected from the abstracts and two poster sessions will allow for engaged interactions and discussions. In addition, an informal Ask the PI seminar with the Chairs and Keynote Speakers on career development in the field of cell migration will be included. For both, the GRC and GRS, financial support will assure that the meeting will be successful and provide needed sponsorship for travel and registration of invited speakers and other selected participants.

Project Summary The goal of the Gordon Research Conference on Direction Cell Migration is to provide a stimulating forum for the dissemination and discussion of new research, concepts, controversies and opportunities at the forefront of cell migration. Peter Devreotes and Jeff Segall, who organized the first meeting held in 2005, envisioned developing a forum fostering interactions among scientists with a strong interest in gradient sensing and directed cell migration. After 12 years, the conference is considered to be the top research conference in cell migration. The program for the 2017 conference, to be held January 21-27 at Hotel Galvez, Galveston, TX, brings together cell migration scientists from diverse disciplines, including molecular and cancer biologists, immunologists, developmental biologists, cell biologists, molecular biologists, engineers and physicists. A strong emphasis will be placed on (1) basic mechanisms of cell migration at the levels of signal transduction and actin assembly, (2) inclusion of researchers that work with model systems as well as in vivo systems, (3) the study of cell migration during development, leukocyte trafficking, wound healing, and cancer invasion and metastasis and (4) comparing and contrasting single-cell versus collective cell migration in 2- and 3- dimensional environments. The aims of the conference are to: (1) provide a forum for the discussion of cutting edge research and controversies in the area of directed cell migration, with the goal of bridging molecular/biochemical, cellular and organismal scales, (2) promote both formal and informal scientific exchanges among scientists investigating directed cell migration, including young scientists, women, and scientists from underrepresented minority backgrounds, and (3) discuss recent advances especially relevant to the biology of cancer invasion and metastasis. To accomplish these aims, the 2017 Conference will bring together an international cadre of young and senior scientists from academia that will interact in an atmosphere conducive to the free exchange of ideas. As in 2015, the 2017 GRC will be preceded by a GRS on Directed Cell Migration, organized by young researchers (Joseph Campanale and Brian Graziano). This event will help nurture junior researchers towards a successful and rewarding career in the field of cell migration. The GRS will be opened by Dr. Paul Kulesa (Stowers Institute), who will discuss neural crest migration. The closing GRS lecture will be given by Dr. Fanny Jaulin (Gustave Roussy) on imaging human colon cancer spread in ex vivo culture. Eight talks selected from the abstracts and two poster sessions will allow for engaged interactions and discussions. In addition, an informal ?Ask the PI? seminar with the Chairs and Keynote Speakers on career development in the field of cell migration will be included. For both, the GRC and GRS, financial support is needed to assure that the meeting will be successful and will provide needed sponsorship for travel and registration of invited speakers and other selected participants.

Cell migration is a critical feature of normal development and disease. For >25 years, we have been developing an in vivo model in which we combine sophisticated genetic and optogenetic manipulations with quantitative live imaging analyses to probe molecular mechanisms underlying collective cell migration: the border cells in the Drosophila ovary. When we began, we did not know a single gene that was required, and live imaging was impossible. Now many molecular pathways are known. We were the first to show that the small GTPase Rac controls protrusion and migration in vivo. We went on to use a photo-activatable form of Rac to show that activation in a single cell is sufficient to steer the entire cluster. Now Rac and its relatives Rho and Cdc42 are well known as key nodes in the signaling and cytoskeletal pathways that govern cell polarity and migration. Recently it has become clear that tumor cells disseminate in groups that resemble border cell clusters in several key aspects. So it is more relevant and interesting than ever to investigate the underlying mechanisms. Here we propose to continue our longstanding pattern of technical and conceptual innovation to address a key outstanding question. How do the molecular mechanisms of cell motility derived from studies of single cells migrating unobstructed on glass applies to the more diverse morphologies and behaviors of cells traveling in groups in vivo through 3D, cell-rich terrains. In cells migrating individually in vitro, mutually inhibitory interactions between Rac and Rho set up distinct protruding and contractile domains. It is unclear how this model applies to groups of cells moving in vivo. In Aim 1 we will address the following key open questions: In collectively moving cells, do Rac and Rho inhibit one another? Do they do so cell autonomously, non- cell-autonomously, or both? Is Rac only required in the lead cell for protrusion? Is Rho specifically required in following cells? Individually migrating cells can also switch from a Rac-dominated/protrusive mode of migration to a Rho-dominated, contractile mode. Do collectively moving cells exhibit comparable plasticity? In Aim 2, we propose to decipher the roles of apical/basal polarity complexes in collective chemotaxis. Individually migrating cells lack apical/basal polarity but collectively migrating cells require it. We propose to tease apart the autonomous and non-autonomous contributions of apical and basolateral protein complexes to the coordination of collective cell motility. Finally, in Aim 3 we propose to unify seemingly disparate observations into a common conceptual framework to test the hypothesis that multiple downstream effectors of Cdc42, all of which contribute to border cell migration, form an integrated network. These studies will lead to a more precise and comprehensive understanding of the intracellular signaling networks that provide cells with 3D coordinates. Our work will produce a paradigm for us to test in collaboration with our colleagues who study collective cell dissemination in metastasis.

Epithelial dynamics are critical during embryonic development and wound healing and are hijacked by cancer cells during the process of metastasis. We have developed a genetically tractable in vivo model to study a group of epithelial cells, the border cells of the Drosophila ovary, which exhibit dynamic cell behaviors including acquiring motility, detaching from an epithelium, migrating through neighboring tissue, and adhering to new cells at a distant site. While most studies of cell movements have focused on individual cells in vitro, cells frequently move in groups in vivo. Recently it has become clear that dissemination of clusters of cells is a common source of metastases in cancer. Most studies of both individual and collective cell motility focus on the intermediate step as cells migrate from one place to another. Little is known of the mechanisms by which cell collectives break away from their initial neighbors in the process of delamination. Even less is known about how cells make new connections upon arrival at their ultimate destination. To be concise we name this process neolamination. Here we propose to use the border cells to study the mysterious process of neolamination: attaching to a new site. In our first aim we build on a strong foundation of preliminary data describing the process by which border cells, after detaching from one epithelium and migrating for several hours, connect up to two new cell types: the oocyte and centripetal follicle cells. We report the identification of genes required for the process, providing the first clues to the molecular mechanism. In Aim 1, we propose to combine opto- and thermo-genetic approaches that have revolutionized neuroscience and state-of-the-art methods for imaging direct protein- protein interactions in living tissue, to study this essential, yet essentially unstudied, process. In Aim 2, we propose to study the cell biological processes and molecular mechanisms operating within the oocyte during the neolamination process. In Aim 3, we propose to use the same set of highly innovative approaches to study how border cells initially leave their epithelium of origin, in the process of delamination. Having described the delamination process at unprecedented resolution, we propose to study the underlying cellular and molecular mechanisms. The overarching goal is to build a conceptual model describing how the border cells integrate multiple extracellular signals to execute collective delamination and neolamination and establish a paradigm for the study of these critical dynamic cellular behaviors.

Across many species, including humans, one of the many consequences of aging is a reduction in fertility, particularly female fertility. Intriguingly some species can preserve fertility for longer than they otherwise would, under specific environmental conditions. For example, various species of Drosophila enter a state called adult reproductive diapause when they experience low temperatures and short day length. Under these conditions they can double their lifespan while maintaining fertility. Previous work has implicated a few genes in positive or negative regulation of adult reproductive diapause in Drosophila. For example, the insulin pathway negatively regulates diapause, suggesting mechanisms that are conserved with the regulation of metabolism, fertility, and lifespan in other species including C. elegans, mice, and humans. However, our mechanistic understanding of diapause is extremely limited, and how fertility is preserved is unknown. We have taken advantage of powerful genetic tools in Drosophila to carry out a genome-wide association study of diapause. This approach appears to be highly successful, as the few known genes emerged from the analysis, such as the insulin receptor. In addition, this screen revealed that the most highly enriched networks of genes associated with diapause include those involved in neuronal development and female reproduction. The neuronal development genes are striking, as they have not previously been associated with diapause and thus offer to provide new molecular and cellular insights. Here we propose to: 1) identify the genes controlling specific steps in the diapause program such as entry, maintenance, exit, preservation of fecundity, and lifespan; 2) test whether diapause genes are required during development to prepare the animals for diapause, or function specifically in adulthood; and 3) investigate the molecular mechanisms of germline stem cell preservation during diapause. We anticipate that just as studies of C. elegans dauer formation illuminated general pathways regulating metabolism, growth, reproduction and aging in animals ranging from worms to humans, studies of Drosophila diapause offer the exciting potential to uncover novel and general mechanisms of stem cell preservation, fertility maintenance, and lifespan extension.