Margaret T. (Minx) Fuller - US grants

The structural protein tubulin is involved in a variety of cellular processes important in morphogenesis, including cell division and flagellar assembly. In many cases, tubulin involved in different processes is found in structurally different microtubule arrays. The regulation of tubulin assembly into microtubule arrays with different functions may utilize discrete structural domains of the tubulin molecule and may be mediated by interactions between tubulin and different sets of microtubule associated proteins. The goal of our research is to determine how tubulin structure regulates tubulin function in vivo and to identify gene products which interact with tubulin during its several functions and which may regulate its assembly. As a model system, we propose a genetic, biochemical, and ultrastructural analysis of the function of tubulin and the molecules with which it interacts during spermatogenesis in Drosophila. We propose to identify genes encoding proteins which interact with the testis-specific Beta tubulin (Beta2) by isolation and characterization of mutations which fail to complement mutant alleles of Beta2 tubulin but which map to other sites on the genome. We have already identified two such "second-site" recessive male sterile mutations. We plan to identify the mutant gene products by 2D gel analysis or recombinant DNA techniques. We will investigate the function of the mutant gene products in spermatogenesis and the nature of their interaction with Beta2 tubulin by light and electron microscopic analysis of defects in spermatogenesis caused by "second-site" mutations both as homozygotes and in heterogyzous combination with Beta2 tubulin mutants. We also propose to analyze changes in tubulin structure caused by mutations in the Beta2 tubulin gene which affect some, but not all, of the functions of Beta2 tubulin during spermatogenesis. Changes in mutant tubulin structure will be assayed using monoclonal antibodies specific for different determinants on the tubulin molecule as probes of molecular structure. Structural changes will be correlated with changes in function of mutant tubulins in vivo and in vitro to determine if discrete structural regions of the tubulin molecule function uniquely in different kinds of microtubule dependent processes.

The goals of the proposed experiments are twofold: to understand the genetic control of key regulatory steps in the program of spermatogenesis and to elucidate the molecular mechanisms that mediate changes in cellular and subcellular morphology. We have identified several intriguing male sterile mutants that cause defects in spermatocyte development or early spermatid differentiation in preliminary screens of male sterile mutants induced by mobilization of a single marked P-element. Three mutants (ms(3)sa, can, and aly) identify potential regulatory genes required for the cell cycle transition from G2 to the male meiotic divisions in primary spermatocytes. To determine if these spermatocyte arrest mutants effect known cell cycle control mechanisms, we will assay the expression pattern and modification state of Drosophila cell cycle control genes in mutant testes using molecular and antibody probes. We will clone the three genes, starting with ms(3)sa, and determine if their products resemble kinases, phosphatases, or oncogenes consistent with their proposed role in cell cycle control during male meiosis. To determine if the genes act directly to control entry into meiotic division, we will test if male germline cells are driven into meiosis prematurely by expression of ms(3)sa, can or aly from a heterologous promotor, and we will establish order of action of the three genes by molecular epistasis tests. Two other mutations (fwd and neb) identify potential effector genes involved in specific cytoskeletal-based morphological events. The fwd mutation causes failure of cytokinesis, a microfilament-based event, during male meiosis. We will determine if the contractile ring is formed in fwd homozygotes, and if the product of the fwd gene is a component of the contractile ring in wild type by immunofluorescence microscopy. We will clone the gene and sequence corresponding cDNA(s) to determine if the fwd product resembles a known component of microfilament based motility systems. The neb mutation, which affects the microtubule-based elongation of the mitochondrial derivative, maps near a potential member of the kinesin heavy chain superfamily in Drosophila. We will determine if the neb product is a kinesin-like protein by molecular analysis. To determine if neb encodes a microtubule binding protein or motor, we will test the ability of expressed neb protein to bind to and/or move along microtubules in vitro. Two other genes that appear to affect the same process as neb could encode other components of the machinery that mediates elongation of the mitochondrial derivative, such as kinesin light chains or the proteins that connect the motor to the mitochondrial membrane. We will clone the genes identified by the neb-like mutants, determine the molecular identity of their gene products, and test if they bind to and/or alter the in vitro biochemical properties of the neb protein. Finally, we propose to identify and characterize mutants in additional regulatory and morphogenetic effector genes that act to control cellular and subcellular morphogenesis during spermatogenesis.

Meiosis I maturation arrest accounts for a significant fraction of idiopathic male infertility cases in humans. We propose to investigate the causes and molecular mechanism of meiosis I maturation arrest using Drosophila and mouse animal models. Five Drosophila genes, spermatocyte arrest, cannonball, always early, meiosis I arrest, and blockout, are required for progression of male meiosis and onset of spermatid differentiation. Mutations in these genes cause defects also characteristic of human meiosis I maturation arrest; early stages of spermatogenesis appear normal, spermatocytes with partially condensed chromosomes accumulate, and testes lack post-meiotic spermatids. This striking phenotypic similarity strongly suggests a crucial control point at the onset of meiotic division in males that is conserved from flies to man. We propose to identify the molecular nature and probe the mode of action of three of these genes and test the hypothesis that mammalian homologues of the Drosophila genes serve an analogous function in meiosis I maturation arrest. Identification of cause(s) of meiosis I maturation arrest could lead to preventive regimens, while understanding of its molecular mechanism could guide development of therapeutic interventions. To elucidate how the spermatocyte arrest type genes act to ensure normal spermatogenesis, we will determine the molecular nature of the Drosophila gene products, isolate mouse homologues, and determine in what cell types and stages of spermatogenesis the genes are expressed and the sub-cellular location of their protein products. To test if mutations in these genes cause meiosis I maturation arrest in mammals we will construct and phenotypically analyze mouse knockout mutations. If homozygous mutant mice also display meiosis I arrest, then the causes and mechanism of meiosis I maturation arrest are probably conserved from Drosophila to man and the basis mechanisms we elucidate in Drosophila should apply to understanding the condition in man. We will determine whether progression through male meiosis requires a permissive signal from somatic testis cells by germ cell transplantation and assess the role of the spermatocyte arrest type genes in the signaling process. Finally, we will use the power of genetic analysis in Drosophila to identify additional genes required for arrest at the meiosis I control point by screening for suppressors of the Drosophila mutants.

Many differentiated but renewable cell types, including blood, skin, and sperm, are derived from dedicated precursor cells, or stem cells, which maintain the essentially unlimited capacity for continued division. Despite their biological and medical importance, the mechanisms that control stem cell behavior are poorly understood. We propose to identify molecular mechanisms that specify stem cell identity, self-renewal, and commitment to differentiation, using the Drosophila germ line as a model tissue. We have identified three genes required for normal germ line stem cell behavior and fate: zonder kloten (zk) acts in establishment of male germ line stem cells, one shot (osho) is required for male germ line stem cell self-renewal, and stem cell tumor (stet) is required in both sexes for commitment to or initiation of differentiation by stem cell daughters. To pinpoint at which step(s) in stem cell establishment and self-renewal the genes act, we will examine embryonic and adult gonads for the earliest defects in the mutants using a variety of available markers, plus double mutant analysis. To elucidate the mechanism of action of these genes in specifying tissue renewal from dedicated stem cells, we will construct germ line/soma chimeras by pole cell transplantation to determine whether zk, osho, and stet function in the germ line itself or in somatic support cells and we will identify the zk, osho, and stet transcription units, sequence corresponding cDNAs, and examine the predicted protein products for structural motifs and homology to proteins of known function. Finally, we will use powerful first generation genetic selections for suppressors of osho to identify additional genes in the pathway of stem cell self- renewal. Variations of the mechanisms we uncover may govern stem cell behavior in other organisms and tissues, leading to applications in cancer, therapeutics, and genetic engineering.

The molecular mechanisms that specify stem cell identity and regulate stem cell self-renewal and differentiation are crucial for realizing the vast potential of stem cells for medicine. We propose to identify molecular mechanisms that regulate stem cell behavior in the Drosophila male germ line as a model system. During the last funding period, we demonstrated that somatic support cells provide a crucial microenvironment that regulates both stem cell self renewal and differentiation. We will probe the possible stem cell intrinsic role of the upd receptor domeless, negative regulators of JAK/STAT pathway signaling, and the Zn-finger transcription factor escargot in regulating stem cell self-renewal. We will test whether the upd-JAK-STAT signaling pathway that specifies stem cell-self-renewal in the male germ line regulates stem cell behavior in other stem cell types. We will identify gene products expressed preferentially in stem cells by microarray analysis and test their possible function in stem cell self-renewal or differentiation by mutant and over-expression studies. We will test genes implicated in precursor cell self renewal vs. differentiation in blood for roles in regulating male germ line stem cell behavior, including the AML1 homolog lozenge (lz), and homologs of Bmi1, implicated in maintenance of mouse hematopoietic stem cells. Finally, we will phenotypically characterize newly identified mutants that affect stem cell specification, division, self renewal and differentiation and molecularly clone the most promising genes, placing their mode of action in the context of pathways already known to regulate male germ line stem cell behavior by double mutant and molecular epistasis experiments.

DESCRIPTION (adapted from the application) Many differentiated cell types are derived from populations of relatively undifferentiated stem cells which retain capacity for continued proliferation and self-renewal. Stem cell populations are maintained by a crucial balance between alternate cell fates: daughter cells must choose between stem cell identity and self-renewal capacity vs. commitment to differentiate after a limited number of amplification divisions. Alteration of this crucial balance can lead to stem cell tumors or loss of tissue renewing capacity. During embryogenesis, stem cells are the principal units of organogenesis and tissue formation. Stem cells retained in adulthood are the units of regeneration, for example hematopoietic stem cells in bone marrow transplantation. Despite their biological and medical importance, the molecular mechanisms that specify stem cell fate and regulate self-renewal vs. differentiation are poorly understood. Recently developed functional assays allow isolation of several stem cell types. At the same time, work in genetically manipulatable model systems has begun to identify both cell intrinsic and extrinsic molecular mechanisms that specify asymmetric stem cell-like divisions leading to daughter cells with different fates. The goal of this meeting is to bring together scientists working on stem cells with scientists probing the genetic control of asymmetric cell division and cell fate to discuss the mechanisms that might control stem cell specification, self-renewal and commitment to differentiation. The topics presented and discussed at this meeting will inform students and postdoctoral fellows across disciplinary lines and give them a strong foundation from which to undertake the fundamental research required to realize the potential benefits in tissue regeneration and disease treatment promised by recent advances in stem cell research.

DESCRIPTION (applicant's description): Cytokinesis is a fundamental process in all dividing animal cells. However, the mechanisms that regulate and mediate the localized assembly of the F-actin contractile ring, its stable attachment to the cell cortex, and its coordinated disassembly are not known. We have identified mutations in 29 new genes required at different specific stages of cytokinesis during male meiosis in Drosophila. This mutant collection presents an unparalleled opportunity to elucidate the molecular mechanisms that mediate and regulate cytokinesis in animal cells. Assembly of the F-actin contractile ring requires james bond and at least eight other genes. frodo functions to maintain linkage between the constricting acto-myosin ring and anillin at the cell cortex. ftvs, fsco, fun, onr, and bns are required for both cytokinesis and polarized cell outgrowth during spermatid elongation, suggesting a common underlying mechanism. To investigate their stage and mode of action we will determine if the genes are required for assembly or localization of other contractile ring proteins, mid-spindle components, and key actin regulatory molecules such as CDC42 or Arp2/3 complex subunits. To investigate the common mechanism required for cytokinesis and flagellar elongation, we will determine if the fws, fsco, etc. mutations similarly affect F-actin assembly, disassembly, or reorganization or the localization of key actin-associated proteins during cytokinesis and at the tip of elongating spermatids. To elucidate molecular mechanisms of cytokinesis, we will clone selected genes, determine if their protein products are components of the contractile ring, central spindle, or spermatid flagella, test how their localization depends on wild type function of known cytokinesis genes and each other, and test whether they can bind anillin, septins, microtubules or actin, or bundle, sever, or cap microfilaments, or nucleate F-actin assembly in vitro.

DESCRIPTION (provided by applicant): We request partial funding for a FASEB conference on "Transcriptional Regulation During Cell Growth, Differentiation, and Development" to be held at Saxtons River, Vermont, June 29-July 4, 2002. Gene Regulation through transcription is fundamental for cell fate specification, cell differentiation, and the control of cell proliferation. Transcriptional regulation is critical for the development of multicellular organisms and for genesis of specific organs. Cell type specific transcription programs govern production of signature gene products such as insulin in the pancreas, neuropeptides in the brain, and the contractile machinery in heart and skeletal muscle. Transcription programs also set critical aspects of cell behavior including proliferation, response to environmental signals, cell adhesion, senescence and cell death. This conference will bring gether researchers studying the fundamental molecular mechanisms of transcriptional regulation with those studying cell fate decisions, developmental programs, cell proliferation, and differentiation to work toward a dtailed understanding of the molecular mechanisms by which the developmental program regulates granscription and transcriptional mechanisms regulate development. The sessions will communicate recent advances in understanding fundamental mechanisms of transcriptional activation and repression, chromatin structure, and long-range and epigenetic effects on gene expression and their relationship to the biology of cell fate decisions and response to extracellular signals during development, establishment of cell type specific gene expression programs during differentiation, and cell-cycle control. The conference will explore the important role of transcriptional regulation in normal differentiation and development and how aberrant gene expression is involved in cancer and disease. This conference differs from other transcription meetings in that does not focus simply on molecular mechanisms, but rather emphasizes how transcription is used by cells to specify development and differentiation, culminating in global changes in transcription during development in multicellular organisms. The meeting will cover problems encountered and solved across a wide range of species, including yeast, worms, flies, and mammals, highlighting both the similarities and differences. This conference will provide an up-to-date overview of one of the most fundamental and rapidly moving fields in bology and generate new directions for future research.

DESCRIPTION (provided by applicant): This proposal requests support for the purchase of a Leica TCS SP2 AOBS Vis-UV Confocal Microscope. This microscope, which will be a shared resource, will be crucial for the ongoing research of investigators working in several departments at the Stanford University School of Medicine. The projects of the major users investigate a range of NIH supported topics, including: i) cell polarity and asymmetric cell division in bacteria (Shapiro), epithelial cells (Nelson) and stem cells (Fuller), ii) intercellular signaling and cell fate in bacteria (Kaiser), mouse embryonic and neural stem cells (Nusse), and gametogenesis (Tazuke), iii) mechanisms of localized intracellular signaling by small molecule second messengers (Conti), iv) transcriptional regulation of neuronal and cardiovascular development (Crabtree), v) mechanisms of cytoskeletal function and membrane trafficking during cell division (Fuller), and vi) mechanisms of chromosome pairing and synapsis during meiosis (Villeneuve). All of these projects require precise localization of proteins or other cellular components in cultured cells or complex tissues. The ability to precisely localize proteins to specific cellular compartments and to infer protein interactions by colocalization provided by the requested shared Laser Scanning Confocal microscope will be crucial to elucidating the normal and pathogenetic mechanisms that regulate and mediate the important developmental and cellular events under investigation. The above projects are being undertaken by senior, intermediate, and recently recruited faculty members, who will cooperate in supporting this confocal facility. The enhanced resolution and sensitivity of this microscope, as well as its ability to effectively image several different fluors simultaneously in both single cells and in thicker tissue, will be crucial to the success of these NIH funded projects.

DESCRIPTION (provided by applicant): The gene regulatory mechanisms that program cell differentiation from proliferating precursor cells are fundamentally important for development, tissue homeostasis and cancer. Using the dramatic cellular differentiation program of spermatogenesis as a model stem cell lineage, we seek to understand the unique, cell type specific transcription program initiated in the spermatocyte stage that programs cells for terminal differentiation. We discovered that testis-specific TAFs, homologs of components of the general PolII transcription machinery, turn on expression of spermatid differentiation genes, in part by counteracting silencing by the Polycomb machinery in precursor cells. The immediate next challenge is to discover how the testis TAFs act - both directly at promoters to activate robust expression of terminal differentiation genes and in the spermatocyte nucleolus by sequestering the Polycomb transcriptional silencing complex. We will test whether the tTAFs form a testis-specific SAGA-like complex that recruits a histone deubiquitinating enzyme to promoter proximal stalled polymerases, allowing elongation of target transcripts, or instead act in a TFIID-like complex with TBP. We will investigate the role of candidate cofactors that may act with the tTAFs we have identified in a pilot RNAi screen, the mediator subunit Med27 and the orphan nuclear hormone receptor Hr51. Because the tTAFs play such a key role, the mechanisms that program the exquisitely cell type and stage specific expression of the tTAFs themselves are central for understanding the developmental regulatory pathway for cell differentiation. We will investigate the role and mode of action of the BTB-Zn finger transcriptional regulator lola, required for expression of tTAFs in spermatocytes, and its binding partner the Jil1 H3S10 kinase, which is required, like lola, for proper meiotic cell cycle progression and spermatid differentiation. In addition, in a directed genetic screen, we will test candidate transcription regulators expressed at the switch from spermatogonia to spermatocyte to identify additional cofactors that act with the tTAFs and elucidate the regulatory machinery that controls tTAF expression. Our work on the mechanism of action and mode of regulation of the tTAFs in the Drosophila male germ line may provide a paradigm for understanding how development programs cell-type specific terminal differentiation by counteracting repression by the PcG at specific target genes. In addition, understanding the mode of action of the meiotic arrest genes of Drosophila may illuminate mechanisms underlying meiosis I maturation arrest infertility in man. PUBLIC HEALTH RELEVANCE: Short statement of relevance: The goal of this project is to understand the gene regulatory mechanisms that program differentiation of male gametes. In both embryonic and adult stem cell lineages, terminal differentiation genes that are repressed by the Polycomb transcriptional silencing machinery in precursor cells must be robustly activated in a cell type specific and gene- selective manner as cell differentiate. Results of our analysis of the mode of action and regulation of the testis specific TAFs of Drosophila will establish paradigms for how cell type specific alternate forms of core gene regulatory complexes act to control cell differentiation, and how these mechanisms are themselves controlled by the germ line developmental program. Because mutations in the tTAFs cause meiosis I arrest and failure of spermatid differentiation, understanding their regulation and mode of action may illuminate mechanisms underlying the male infertility syndrome meiosis I maturation arrest.

DESCRIPTION (provided by applicant): Developmental programs must impose cell type specific controls on cell cycle progression for normal embryonic development, cell differentiation, tissue renewal and repair, and prevention of cancer. A striking case of developmental^ regulated cell cycle control is the meiotic cell cycle essential for all sexual reproduction. Meiosis features an extended G2 phase, meiotic prophase, during which many genes required for gamete differentiation are transcribed. The duration of meiotic prophase is controlled differently in males than in females. We are investigating the mechanisms that regulate timing of the G2/M transition of meiosis I in males in Drosophila as a model system. We found that developmental^ programmed translational control regulates timing of the G2/M transition of male meiosis I and coordinates meiotic cell cycle progression with the transcription program for spermatid differentiation by two independent pathways. Translational repression delays expression of cyclin B protein, and translational controls link expression of boule protein to the transcriptional program for spermatid differentiation. In turn, boule (a homolog of human BOULE and D/\Z), regulates translation of the cell cycle phosphatase cdc25/twine. We will investigate the molecular mechanisms of how Boule acts to relieve translational repression of cdc25/twine and how translational repression of Cyclin B is relieved in mature spermatocytes, dependant on elF4G2, a novel homolog of the translational initiation machinery component elF4G. We will investigate the mechanisms that delay translation of cyclin B protein in immature spermatocytes, including the mode of action of the RNA binding protein Tsr. To discover the regulatory mechanisms that make meiotic cell cycle progression depend on successful expression of spermatid differentiation genes in primary spermatocytes, we will identify c/s^ acting sequences responsible for translational repression of boule in tTAF mutant spermatocytes, screen for possible translational regulators that bind, and test their role in vivo. To discover how translation of boule is derepressed in response to tTAF function, we will identify candidate translational activators expressed under tTAF control, and determine whether heterologous expression allows boule translation in tTAF mutant spermatocytes. Our findings will illuminate the mode of action and regulation of the conserved RNA binding protein Boule and suggest mechanisms to test for similar function during mammalian spermatogenesis.

DESCRIPTION (provided by applicant): Regulation of Stem Cell Self-renewal and Differentiation NIH 1 R01 GM080501 Adult stem cells are required throughout life to replenish differentiated cells and repair damaged tissue. The molecular mechanisms that maintain and keep in check adult stem cell populations are key for harnessing the potential of adult stem cells for regenerative medicine as well as understanding the genesis and biology of cancer. We propose to investigate how interactions with the local environment of the stem cell niche maintain populations of germ line stem cells in the Drosophila testis, a powerful system for study of adult stem cells in vivo in the context of their niche. In previous funding cycles, we discovered that somatic support cells in the testis stem cell niche provide a crucial microenvironment that regulates both stem cell self renewal and differentiation, and that germ line stem cells (GSCs) orient toward this niche to set up a stereotyped mitotic spindle, ensuring the normally asymmetric outcome of GSC divisions. We showed that a cytokine like signal from the somatic hub activates the transcription factor STAT in GSCs and their partner somatic cyst stem cells (CySCs) and that activated STAT is critical for maintenance of CySC identity and GSC attachment to the hub. CySCs are an important component of the GSC niche and can maintain GSCs in ectopic sites away from the hub. We also found that germ cells require a "go differentiate" signal from somatic cyst cells to exit limitless stem cell proliferation and enter the spermatogonial program of limited transit amplifying (TA) divisions then differentiation. These findings highlight a new model for how signals from the niche regulate stem cell self- renewal, in which timely transition from stem to TA cell is choreographed by a balance between counteracting self-renewal and differentiation signals. We now propose to utilize the powerful system and tools we have established to identify the molecular circuitry that regulates stem cell behavior in response to cues from the niche. We will investigate how GSCs attach to and orient toward the hub and how this normal behavior is controlled by the Upd signal from the hub through activation of the transcription factor STAT. We will investigate whether CySCs maintain GSCs by sending a "self renew" signal or blocking a "go differentiate" signal and test candidate signaling mechanisms and regulators to understand how the niche regulates stem cell fate and how the actions of two stem cell types within the same niche are coordinated. Finally, we will test the model that activation of the EGFR in somatic cyst cells by a signal from cystoblasts downregulates the CySC program, allowing a timely switch from stem cell to progenitor state in both the germ line and somatic lineages. PUBLIC HEALTH RELEVANCE: The results of the proposed studies will establish paradigms for how the tissue microenvironment regulates self-renewal and differentiation of adult stem cells, which are centrally important for tissue maintenance and repair for many cell types in the human body. Understanding how support cell niches regulate adult stem cell behavior may illuminate how tumor stroma support cancer stem cells and how stem cells maintained in their normal environment may be restrained from uncontrolled proliferation by signals from support cells that trigger differentiation.

Meiosis is the signature event of the germ cell developmental program, absolutely required for sexual reproducfion eukaryotes. Proper regulafion of meiotic progression is crucial for producfion of funcfional gametes. A key aspect of meiosis is a germ cell specific cell cycle, in which a final round of DNA synthesis (premeiotic S) is followed by a greafiy extended G2, termed meiofic prophase, prior to the two meiotic divisions. This signature cell cycle delay provides critical fime for homologous chromosomes to pair, synapse and recombine, as well as for major biosynthetic events that drive gamete differentiation. In oocytes, the transcriptional, translational and morphogenetic changes that produce the egg and prepare for eariy embryogenesis are largely accomplished during meiofic prophase. In male germ cells, although dramafic morphological changes that produce mature gametes occur after the meiofic divisions, a major part of the gene expression program that sets up spermiogenesis takes place during meiotic prophase. In both sexes, meiotic onset, progression, maturation, and acfivafion of the meiotic divisions are key regulatory points. Understanding how these events are regulated is key for understanding the molecular basis of meiotic arrest infertility, for designing effective strategies for differentiating germ cells from embryonic precursors, and for developing and maturing competent gametes in vitro. The analysis proposed in this subproject of the U54 Cooperative Center for Reproductive and Stem Cell Biology will elucidate fundamental mechanisms that control the cell cycle for meiotic prophase, including the crifical control circuits that first pause the cell cycle, then finally acfivate the G2/M transition for meiosis I once proper expression of the program for gametogenesis has been achieved.

Meiosis is the signature event of the germ cell developmental program, absolutely required for sexual reproducfion eukaryotes. Proper regulafion of meiotic progression is crucial for producfion of funcfional gametes. A key aspect of meiosis is a germ cell specific cell cycle, in which a final round of DNA synthesis (premeiotic S) is followed by a greafiy extended G2, termed meiofic prophase, prior to the two meiotic divisions. This signature cell cycle delay provides critical fime for homologous chromosomes to pair, synapse and recombine, as well as for major biosynthetic events that drive gamete differentiation. In oocytes, the transcriptional, translational and morphogenetic changes that produce the egg and prepare for eariy embryogenesis are largely accomplished during meiofic prophase. In male germ cells, although dramafic morphological changes that produce mature gametes occur after the meiofic divisions, a major part of the gene expression program that sets up spermiogenesis takes place during meiotic prophase. In both sexes, meiotic onset, progression, maturation, and acfivafion of the meiotic divisions are key regulatory points. Understanding how these events are regulated is key for understanding the molecular basis of meiotic arrest infertility, for designing effective strategies for differentiating germ cells from embryonic precursors, and for developing and maturing competent gametes in vitro. The analysis proposed in this subproject of the U54 Cooperative Center for Reproductive and Stem Cell Biology will elucidate fundamental mechanisms that control the cell cycle for meiotic prophase, including the crifical control circuits that first pause the cell cycle, then finally acfivate the G2/M transition for meiosis I once proper expression of the program for gametogenesis has been achieved.

DESCRIPTION (provided by applicant): This proposal requests funds to purchase a Nikon A1Rsi resonant spectral confocal microscope with high spatial and temporal resolution, focus-maintenance and environmental control for imaging both live and fixed biological samples. This advanced state-of-the-art confocal microscope will be a shared resource, located in a well-established, multi-user, light and electron microscopy facility at Stanford University: the Cell Sciences Imaging Facility (CSIF). This facility is accessible to Stanford University's entire research community. The requested confocal microscope will support NIH funded projects from 9 major users. These projects investigate a wide range of NIH supported research applied to both live and fixed biological samples, including: (1) Regulation of stem cell renewal and differentiation (Drosophila, Fuller); (2) Molecular mechanisms of viral replication, assembly and egress from infected cells (mice, human, Arvin); (3) Analysis of neural development in the visual pathway (mice, rat, Shatz); (4) Pulmonary hypertension in genetically modified mice (mice, Rabinovich); (5) Molecular mechanisms underlying epithelial cell rearrangements in gastrulation (fish, Nelson); (6) Engineering 3D in-vitro niches to reveal fundamentals of cellular biomechanics (human, Heilshorn); (7) Primary cilium biogenesis and membrane trafficking in ciliopathies (human, Nachury); (8) Characterization and modeling of the neuronal mechanisms underlying the development of epilepsy (mice, rat, Prince); (9) Dynamic analyses of stem cell division and fate (human, Blau). These studies investigate critical functional and structural questions in a variety of model organisms and human tissues. This biomedical research impacts diverse aspects of human health and disease, ranging from stem cell development to understanding the cellular and molecular basis of sensory biology. All require state-of-the-art confocal instrumentation for imaging both fixed and live samples which is most effectively provided by the requested Nikon A1Rsi resonant spectral laser-scanning confocal microscope equipped with spectral detector for signal unmixing, resonant scanner and piezo z-drive for fast imaging, environmental control with focus maintenance for drift free time lapse imaging of samples.

Meiosis is the signature event of the germ cell developmental program, absolutely required for sexual reproduction eukaryotes. Proper regulation of meiotic progression is crucial for production of functional gametes. A key aspect of meiosis is a germ cell specific cell cycle, in which a final round of DNA synthesis (premeiotic S) is followed by a greatly extended G2, termed meiotic prophase, prior to the two meiotic divisions. This signature cell cycle delay provides critical time for homologous chromosomes to pair, synapse and recombine, as well as for major biosynthetic events that drive gamete differentiation. In oocytes, the transcriptional, translational and morphogenetic changes that produce the egg and prepare for early embryogenesis are largely accomplished during meiotic prophase. In male germ cells, although dramatic morphological changes that produce mature gametes occur after the meiotic divisions, a major part of the gene expression program that sets up spermiogenesis takes place during meiotic prophase. In both sexes, meiotic onset, progression, maturation, and activation of the meiotic divisions are key regulatory points. Understanding how these events are regulated is key for understanding the molecular basis of meiotic arrest infertility, for designing effective strategies for differentiating germ cells from embryonic precursors, and for developing and maturing competent gametes in vitro. The analysis proposed in this subproject of the U54 Cooperative Center for Reproductive and Stem Cell Biology will elucidate fundamental mechanisms that control the cell cycle for meiotic prophase, including the critical control circuits that first pause the cell cycle, then finally activate the G2/M transition for meiosis I once proper expression of the program for gametogenesis has been achieved.

DESCRIPTION (provided by applicant): The objective of our Center is to promote scientific excellence in translational science via well-designed studies of human germ cell development, based on a foundation of knowledge in model systems, and culminating with applications that address critical clinical need. To accomplish our objective, we propose three projects, a pilot project, and four cores. We also present several opportunities for a translational pilot project. The projects are: Project I) Germ Cell Differentiation from Human iPSCs and hESCs (Reijo Pera); Project 2) Translational Regulation of Meiotic Cell Cycle Onset and Progression in the Male (Fuller); Project 3) Derivation of Mature Human Oocytes from Primordial Follicles (Hsueh); and Pilot Project) Regulation of Translational Control in the Oocyte to Embryo Transition (Yao). We also present a translational pilot project for future consideration on primary ovarian insufficiency. Supporting the research are four cores: A) Administration; B) Outreach and Education; C) Microanalysis, Sequencing and Informatics; D) Reproductive Database. This application is a culmination of our reorganization and planning over the last several years and presents our vision for Reproductive and Stem Cell Biology based on outstanding basic and translational science. The application is put forth by a collaborative team that shares common interests in terms of genes and germ cell differentiation and maturation, pathways, developmental systems and overall educational, outreach and research goals. Each project consists of a strong basic component; in addition, three of the five projects and pilots have an equally-strong clinical component encompassing genetic analysis of human germ cell development from pluripotent stem cells to probe fundamental aspects and potential therapies, deriving mature oocytes to remedy primary ovarian insufficiency, and establishing the first registry of women with POI. Each project is relevant to the health of infertile women and men and each is informed by the elegant genetic systems of Drosophila and the mouse. The central theme of our Center is novel, forward-looking and built on a firm foundation of scientific and clinical inquiry with an innovative outreach and educational component with hopes of reaching out to other scientists, healthcare professionals.

DESCRIPTION (provided by applicant): The gene regulatory mechanisms that program cell differentiation from proliferating precursor cells are fundamentally important for development, tissue homeostasis and cancer. Using the dramatic cellular differentiation program of spermatogenesis as a model stem cell lineage, we seek to understand the unique, cell-type specific transcription program initiated in the spermatocyte stage that sets cells up for terminal differentiation. Here we will investigate how cell-type specific transcriptional activators and repressors we have discovered in Drosophila, and the chromatin modifying complexes and transcription machinery they recruit, together specify the transcription program required for male gamete differentiation. We will investigate how both somatic specific and spermatid differentiation transcripts are kept silent in proliferating precursor cells and probe how testis-specific TAFs (homologs of components of the general Pol II transcription machinery), and a testis specific form of the normally repressive MuvB/dREAM complex work with the transcriptional co-activator Mediator to activate expression of meiotic cell cycle and spermatid differentiation genes. We will investigate how a testis-specific Rest-like Zn Finger protein expressed at the onset of spermatocyte differentiation acts in this context to block inappropriate spermatogonial and somatic differentiation programs in spermatocytes. Defects in either of these latter two gene regulatory mechanisms results in arrest of spermatogenesis at the G2/M transition of meiosis I and failure to initiate spermatid differentiation. Our work on the mechanism of action of cell-type specific transcription activating complexes and master transcriptional repressors in the Drosophila male germ line will provide paradigms for understanding how development programs terminal differentiation of specialized cell types. In addition, understanding the mode of action of the meiotic arrest gene circuitry of Drosophila may illuminate mechanisms underlying meiosis I maturation arrest infertility in man.

DESCRIPTION (provided by applicant): The long-term goal of this project is to discover gene regulatory mechanisms that control differentiation of male gametes, critical for understanding the genetic and molecular basis of male infertility. We have identified a novel mechanism of regulation of gene product expression that may play a key role in the switch from spermatogonial proliferation to spermatocyte growth, meiosis and terminal differentiation. In a pilot study mapping 3' ends of transcripts by high throughput RNA-3'end-Sequencing from staged testis samples, we discovered many mRNAs expressed with long 3' UTRs in spermatogonia but short 3'UTRs due to selection of alternative polyadenlyation (APA) sites in spermatocytes and differentiating Drosophila male germ cells. Similar shortening of 3'UTRs occurs in mammalian spermatogenesis, indicating a deeply conserved regulatory mechanism. Because 3'UTRs can house important information for translational repression, stage-specific alternative 3' end selection may provide a novel mechanism to coordinately regulate cohorts of proteins for the next stage of male germ cell differentiation. In this R21, we propose to investigate the extent and role in control of stage-specific protein expression of 3'UTR shortening by APA during male germ cell differentiation, taking advantage of powerful genetic tools available in Drosophila. Our innovative strategy combines three approaches. We will use directed in vivo reporter assays to test the hypothesis that sequences in the extended 3'UTR of specific transcripts repress translation in spermatogonia, but are removed by APA to allow translation in spermatocytes, starting with the example of LolaF protein, which is not translated in spermatogonia but appears abruptly in early spermatocytes. In parallel we will use high throughput RNA-3'end- Seq to identify genome wide the transcripts subject to 3'UTR shortening as spermatogonia differentiate and identify shared sequence motifs that may suggest coordinate regulatory mechanisms. We will test the role of such cis-acting motifs in vivo using reporter constructs as above and investigate the role of trans-acting factors predicted to bind them by knocking down expression of candidate regulators using germ cell stage-specific RNAi or anti-miRNA sponges. Third, we will employ a novel method we developed to induce spermatogonia to differentiate in synchrony to determine if 3'UTR shortening by APA occurs at one discrete time in male germ cell differentiation or if different mRNAs are subject to APA at different steps. The approaches we propose are technically feasible, and if successful, our study may reveal a novel switch mechanism where germ cell differentiation is primed by expression of transcripts that are kept silent in spermatogonia by translational repression, until developmentally regulated cell type specific 3'UTR shortening relieves the repression, allowing abrupt and rapid onset of expression of proteins that may then drive subsequent stages of male germ cell differentiation. Our results will provide paradigms to investigate in spermatogenesis in mammals and may uncover new target mechanisms for male contraceptive strategies.

Developmental programs must impose cell-type-specific controls on cell cycle progression for normal embryonic development, tissue renewal and repair, and to prevent cancer. A striking case is meiosis, the signature event of germ cell development. Meiosis requires a specialized cell cycle (meiotic prophase) in which progression through G2 is slowed or arrested to allow time for the chromosomal events and gene expression programs that set the stage for formation of haploid gametes. We are using the powerful genetic tools and accessible cell biology of Drosophila spermatogenesis to investigate the regulatory mechanisms that specify and control progression through meiotic prophase in males, which is regulated differently than in females. We found that developmentally programmed translational control regulates expression of core cell cycle machinery proteins during male meiotic prophase by two independent pathways: translational repression by a male germ- cell-specific hnRNP A homolog Rbp4 in young spermatocytes delays expression of Cyclin B protein, and translational repression by as yet unknown mechanisms and activation by the RNA binding protein Boule (homolog of human BOULE and DAZ) regulate translation of the cell cycle phosphatase cdc25/twine. To discover the underlying mechanisms through which the germ cell program regulates meiotic cell cycle progression, we will investigate how Rbp4 and its partner Fest repress translation of cycB, testing models suggested by interacting proteins we have identified by mass spectrometry. To understand how the translational repression of cycB by Rbp4 is reversed in mature spermatocytes, we will investigate how interactions among the repression complex proteins and with the cycB 3'UTR change as spermatocytes progress from early to late meiotic prophase, taking advantage of a novel technique to trigger spermatogonia to initiate the spermatocyte program and progress through meiotic prophase in synchrony in vivo, and probe how this activation depends on the hnRNP Q homolog Syncrip (Syp). On the other arm of the pathway, we will investigate how the RNA binding protein Boule, conserved from flies to man, relieves translational repression of cdc25/twine to activate the G2/M transition for meiosis I and probe the role of Boule-interacting proteins in activation and action of Boule. We will map the RNA regulatory regions that specify translational repression of the cell cycle phosphatase cdc25/twine in young spermatocytes and test whether the hnRNP L homolog Smooth blocks premature expression of cdc25/twine indirectly by sequestering Boule to the nucleus, or acts directly on the mRNA to repress translation in immature spermatocytes, and whether other candidates, such as the Pumilio family member CG11123, act as translational repressors. Our findings will illuminate the mode of action and regulation of conserved RNA-binding proteins and hnRNP homologs in cell cycle control and suggest mechanisms to test for similar function during mammalian spermatogenesis.

Project Summary / Abstract Conserved regulation of the switch from proliferation to differentiation in the germ line stem cell lineage The switch from proliferation to differentiation is a key regulatory point in adult stem cell lineages, crucial for tissue maintenance and repair. Failure of this switch may contribute to genesis of cancer. Here we study the molecular mechanisms underlying a developmentally programmed transition from proliferation to differentiation in the germ cell lineage, where the switch from mitosis to meiosis is a defining event. Drosophila bgcn encodes an RNA binding protein required cell autonomously for spermatogonia to stop proliferation and enter meiosis. We created mice null mutant for the mouse homolog of bgcn and discovered that genetic mechanisms that underlie a clean switch from mitosis to meiosis are deeply conserved from Drosophila to mammals. Male germ cells in Bgcn mutant mice execute the spermatogonial mitotic divisions and initiate meiosis, turning on Stra8 and undergoing premeiotic DNA replication, but fail to stably implement the program for meiotic prophase. Instead the nascent spermatocytes show only low or transient expression of number of meiotic markers, fail to properly turn off several mitotic transcripts, prematurely enter an ectopic mitosis, then die. We propose to capitalize on these discoveries to map the regulatory circuitry controlling the switch from mitosis to meiosis in Drosophila males and explore how a homologous RNA binding protein, acting with different partners, enforces a clean switch from the mitotic program to the meiotic program in mammals. Using the short life span and powerful genetic tools in Drosophila, we identified the main target of repression by Bgcn and its co-factor Bam for the switch to meiosis as the RNA binding protein HOW, homolog of mammalian Quaking. We will investigate if Drosophila Bgcn and Bam directly regulate HOW mRNA translation or stability, mapping the HOW mRNA sequences required for these regulators to bind. To discover how HOW maintains the spermatogonial state, we will test the hypothesis that HOW prevents expression of a TGFB-class ligand required to signal to somatic cells that the germ cells are ready to progress. In parallel, in an unbiased approach to identify candidate substrates of HOW we will analyze RNA-Seq data to identify transcripts alternatively spliced in spermatogonia vs spermatocytes that have conserved HOW binding sites, test whether they co-immunoprecipitate with HOW from spermatogonia, then assess function in spermatogonia by genetic assays in Drosophila. To discover how mammalian Bgcn ensures an effective switch from mitosis to meiosis, we will investigate whether mBgcn targets mitotic transcripts that we discovered it binds for destruction or translational repression in young spermatocytes. To investigate how Bgcn, with its partner Meioc, may act indirectly to allow stable accumulation of meiotic transcripts, we will test a model based on our discovery that mBgcn binds certain spermatocyte specific piRNA precursors and is required for their accumulation. Our results will reveal molecular mechanisms that underlie the surprising discovery that RNA binding proteins play key roles in ensuring a clean switch from proliferation to differentiation in the male germ line stem cell lineage.

PROJECT SUMMARY (of Parent Project): Regulation of proliferation and differentiation in the male germ line adult stem cell lineage The switch from proliferation to differentiation is a key regulatory point in both embryonic development and the adult stem cell lineages that underlie tissue maintenance and repair. Failure of this switch may contribute to genesis of cancer. My laboratory has long used the Drosophila male germ line as a model to investigate how self-renewal, proliferation and differentiation are regulated in adult stem cell lineages. Several lines of our inquiry have recently begun to converge on the molecular mechanisms underlying the developmentally programmed transition from mitotic proliferation to onset of meiosis and differentiation, implicating a number of molecular and cellular mechanisms in regulating this critical switch. We find that RNA binding proteins involved in translational control and alternative splicing act cell autonomously to regulate the cessation of proliferation and that progression of differentiation requires communication from associated somatic support cells. We discovered that a developmentally regulated switch in the site at which specific nascent transcripts are cut to form 3? ends, leading to production of novel mRNA isoforms with shortened 3?UTRs, controls dramatic changes in the suite of proteins expressed in differentiating spermatocytes compared to proliferating spermatogonia. We found that dramatic changes in chromatin open over 2000 new promoters with novel core sequence structure to turn on the new cell type specific transcription program when cells initiate spermatocyte differentiation. Some of the earliest genes turned on in this differentiation program encode chromatin associated proteins that prevent spurious opening of normally cryptic promoters, thus preventing massive misexpression of genes associated with the wrong cell type. Other transcripts upregulated with differentiation onset encode cell type-specific translational regulators that delay production of core G2/M cell cycle machinery to program the extended G2 phase of meiotic prophase. Over the next 5 years, we propose to map how these processes collaborate to form the regulatory circuitry that initiates then executes the switch from mitosis to meiosis. We will investigate how the RNA binding proteins Bam and Bgcn trigger the switch from mitosis to differentiation by repressing expression of the alternative splice factor HOW, identify candidate substrates of HOW by immunoprecipitation followed by RNA- Seq, and assess their function in vivo, including whether they communicate with adjacent somatic support cells. We will investigate how the switch in proteins expressed due to alternative 3? end cut site selection on nascent transcripts is regulated and influences differentiation. We will investigate how cell-type specific chromatin regulators and proteins that recruit them to specific loci set up the new transcription program for differentiation. To elucidate how the developmental program remodels fundamental cellular processes like the cell cycle to set up differentiation of specialized cell types, we will investigate how cell-type specific RNA binding proteins first repress, then activate translation of cyclin B during meiotic prophase and how the DAZ homolog Boule regulates progression into the meiotic divisions.