Gary M. Wessel - US grants

The developmental success of an early embryo depends fundamentally on the efficacy of oogenesis. Developing oocytes are geared almost entirely to the segregation and storage of newly synthesized macromolecules. Dr. Wessel will examine the ontogeny of two classes of storage organelles in oocytes of the sea urchin - vesicles storing extracellular matrix molecules that are destined for the embryonic blastocoel, and cortical granules that are exocytosed during the fertilization reaction. Both of these organelles originate early in oogenesis and their contents are each secreted, yet each organelle contains a discreet, non- overlapping population of proteins in distinct membrane bound vesicles. Dr. Wessel will examine the ontogeny of these organelles using cDNA and antibody probes that he has made to specific constituent proteins. The ultimate goal is to understand the mechanism of specific protein "stockpiling" that developing oocytes use to facilitate the commitment of their embryos to a program of rapid, independent development.

The ability of blastomeres from an early embryo to acquire a distinct program of cell fate is fundamental to the development of a multicellular organism. The ultimate goal of this research is to understand how cell fate becomes specified by cell interactions during regulative embryogenesis. Specifically this proposal examines the mechanisms and origins of control during the ontogeny of the sea urchin endoderm lineage. This lineage is chosen for several reasons: 1) its specification and differentiation during embryogenesis is regulated by cell interactions, 2) genes responsive to the inductive interactions have recently been cloned, 3) embryology of the sea urchin is classically described and amenable to manipulation, and 4) endoderm differentiation is fundamental to gastrulation in establishing the basic body plan of the embryo. Three features of endoderm ontogeny will be examined: 1) Examine the regulation of endoderm differentiation. Presumptive endodern cells will be dissociated from the embryo and cultured in vitro to test the hypothesis that differentiation is regulated by exogenous signals. Molecules of the extracellular matrix and growth factors will be tested. Differentiation will be assayed by gene activity specific to endoderm cells. Results from these experiments will be tested in vivo and will be compared to blastomeres normally restricted to non-endodermal cell fates - but which can be induced to an endodermal fate. 2) Investigate the mechanism for endoderm specific gene expression. These experiments will examine where in the embryo the specific transcriptional machinery originates for an endoderm program. That is, are components of the transcriptional machinery maternally derived and then activated locally in presumptive endoderm blastomeres, or are they produced by restricted zygotic transcription. The hypothesis posed is that the nuclear factors involved in this lineage specific transcription are activated by early cell interactions. To test this hypothesis, LvNI.2, a gene expressed specifically by endoderm cells will be examined for cis- regulatory elements important for temporal and spatial transcriptional regulation. These experiments will utilize an embryonic gene transfer system coupled with lac Z and CAT reporter genes. Such cis-regions will be used to examine the ontogeny of functional trans-acting factors in normal embryogenesis, in response to the extracellular matrix, and in non-endodermal lineages induced to an endoderm fate. 3) Determine the origin of extracellular matrix molecules which influence endoderm differentiation. The regulated ontogeny of the extracellular matrix (ECM) will be examined using molecular cloning techniques. The hypothesis will be tested that the extracellular matrix derived from maternal stores is important in the stimulation of zygotic ECM synthesis. This study will include the characterization of several novel ECM molecules.

Cortical granules are unique to oocytes and function during fertilization by secreting their contents to form a permanent block to polyspermy. We will study the biology of cortical granules with two major goals: l) to understand the mechanisms used by oocytes to regulate organelle biogenesis and protein storage; and 2) to understand their regulated secretion at fertilization. The oocyte of choice for these goals is from the sea urchin, where approximately 15,000 cortical granules are poised at the cell surface to exocytose in response to either sperm binding. It is the only oocyte for which l) cDNA clones have been isolated that encode content and membrane proteins specific to the cortical granules; 2) the cortical granules can be isolated in a functional form, and 3) in vitro culture, maturation and direct visualization of cortical granules are each possible. Cortical granules are different from most other secretory vesicles in that they remain docked at the cell surface for weeks, they are non recycling, and they contain over a dozen different proteins which are subcompartmentalized within the vesicle. Three specific aims are proposed: 1. We will identify determinants on the content proteins of cortical granule that are required for targeting into cortical granules. We will utilize cDNAs to the cortical granule specific proteins SFE 9, ovoperoxidase and hyalin in a protein reporter system to determine what minimal sequence is necessary for appropriate organelle targeting. Because the vesicle contains two morphologically and biochemically distinct regions, we will also examine the signals necessary for protein subcompartmentalization within the vesicle using proteins specifically compartmentalized in each region. 2. We will examine the mechanism and regulation of cortical granule translocation to the oocyte cell surface. We will determine the basis for selectivity of this process, i.e. selectivity for cortical granules, for their timing of translocation, and for the pathways used in organelle movement. In addition, we will examine the mechanism used to create a perfect monolayer of cortical granules at the oocyte cell surface. 3. We will identify the mechanism(s) of regulated secretion of cortical granules at fertilization. We will test the hypothesis that cortical granules contain a unique population of membrane proteins to direct the specialized biology of this vesicle by use of monoclonal antibodies against cortical granule membranes and by testing homologues of somatic cell proteins that are involved in regulated secretory function.

Cortical granules are secretory vesicles unique to eggs and oocytes. At fertilization they secrete their contents to form both a permanent block to polyspermy and to provide protection for early embryonic development. In this application we will address three questions: What is in the cortical granule?, What does it do?, and How does it get in there? We will use sea urchin eggs and oocytes to answer these questions because in this animal we can obtain approximately 106 eggs and 1 ~ oocytes per female, and because the approximately 15,000 cortical granules in each oocyte are synchronous in biogenesis, in translocation to the surface, in docking to the plasma membrane, and in secretion in response to sperm or parthenogenic activation. This system offers a unique opportunity to examine the molecular mechanism of cortical granule biogenesis and flinction. The sea urchin oocyte is also the only oocyte in which 1) cDNA clones have been isolated that encode content and membrane proteins specific to the cortical granules; 2) the cortical granules can be isolated in a fimctional form; and 3) in vitro culture and maturation of oocytes and direct visualization of cortical granules is possible. In addition to the synchrony of vesicle biogenic steps, cortical granules are different from most other secretory vesicles in that they are nonrecycling, and contain over a dozen different proteins that are specific to cortical granules and are subcompartmentalized within the vesicle. Three specific aims are proposed:

1. Identify the contents of the cortical granules. We will focus on the cDNA cloning of the three fertilization envelope proteins: proteohaisin, p90 and p63. These are major envelope proteins that must quickly interact with each other and with the vitelline layer to form an impenetrable layer within seconds of fertilization.

2. Characterize the function and regulation of the cortical granule proteins. We will determine the mechanism of fertilization envelope construction, a specialized extracellular matrix, by identiiying domains of cortical granule protein interactions that are responsible for envelope construction. We will then use this information to examine the hierarchy, and identity of interactions with the vitelline layer to understand the molecular mechanism for the rapid condensation of the fertilization envelope.

3. Determine how the contents are packaged selectively into cortical granules. We will make use of the newly identified cDNA clones to determine the mechanism of cortical granule biogenesis. Selective protein targeting into cortical granules will be studied using recombinant, tagged cortical granule proteins. The tags will include the myc epitope and the green fluorescent protein, and we will follow the fates of wild-type and modified cortical granule protein sequences during cortical granule biogenesis. We will also use these tagged proteins, in conjunction with biotinylated amino acid markers, to assay cortical granule biogenesis. Because of our recent success with in vitro maturation of sea urchin oocytes, we will also be able to study cortical granule protein function in vivo.

Genetic studies both at the organismal and cellular levels demand a sophisticated downstream analysis of the phenotypes that result from the genetic manipulations. The creation of the transgenic and knockout animals proposed in this application, as well as the greatly increased capabilities of investigators using cell culture models proposed by the proposed flow cytometry core, will greatly increase the need for evaluating cellular and tissue organization by state of the art microscopic methods. The optical sectioning capabilities of a confocal fluorescence microscope are ideal for these studies because they provide image data that allows a three dimensional reconstruction of structures. To accommodate the needs of this diverse group of investigators it is therefore proposed to significantly upgrade an existing facility equipped with a Zeiss 4100 Laser scanning microscope. While the facility has been of great use to researchers, the LSM-410 is capable of far greater imaging contrast resolution and sensitivity, than the existing electronics, software and filters will permit. The limitations of the current system are now posing an impediment to current and potential users of the facility, and it is critically necessary to upgrade the LSM-410. The upgrade will provide a significant increase in imaging contrast resolution, sensitivity and data acquisition and handling. Central to this will be an upgrade of the now obsolete software and hardware currently in place. It is also proposed to install a UV laser line that will allow multi-channel labeling, provide calcium imaging capabilities, and expand the selection of useful fluorophores. The core will be supervised by Gary Wessel, Associate Professor, an expert in the field of microscopy, who has a long track record of publications using the proposed technologies. On a day to day basis the core will be run by a highly trained technician, who will aid users in imaging and sample preparation. Users of this core facility will have access not only to a state of the art confocal microscope, but also to guidance, expertise and training towards applying this technology to their own research needs.

DESCRIPTION: (provided by applicant) Cortical granules are unique to oocytes and secrete their contents at fertilization to form a permanent block to polyspermy. Our long-term goal is to understand conserved mechanisms of fertilization from a perspective of the functional contribution of cortical granules. Our oocyte of choice for this goal is the sea urchin, where approximately 15,000 cortical granules are synchronous in biogenesis, translocation to the surface, docking to the plasma membrane, and secretion at fertilization. We will make use of the sea urchin oocyte for 1) the millions available from each female; 2) the cDNA clones in hand that encode proteins specific to the contents and membranes of cortical granules; 3) the ability to isolate cortical granules and reconstitute function; and 4) the amenable culture and maturation of oocytes in vitro and direct visualization of cortical granules. Here we propose three major goals: 1. Determine the functional contribution of the cortical granule protease to fertilization. This protease cleaves plasma membrane proteins of the egg, modifies the egg extracellular matrix, and alters other cortical granule content proteins. We will identify the proteolytic targets, enabling us to identify and functionally characterize proteins important for fertilization. 2. Biogenesis and translocation of cortical granules. We will dissect the mechanisms used by cortical granules in vivo to get to the cell surface (translocation), to make a near perfect monolayer of cortical granules at the plasma membrane, and the function of cortical granule membrane proteins in this regulation. 3. Identify the molecular basis for regulated exocytosis at fertilization. We will exploit the pre-docked status of cortical granules in this egg to identify proteins that regulate their exocytosis. We will focus on proteins that interact with SNARE homologues and of rab 3, and address both the mechanism for stimulating exocytosis, as well as the molecular clamp that blocks exocytosis until fertilization.

0315657
Wessel

Success at fertilization requires both sperm-egg fusion and blockage of excess sperm fusions, or polyspermy. Polyspermy is lethal in most animals, and oocytes have evolved mechanisms to minimize its occurrence. The most conserved mechanism throughout phylogeny involves a rapid remodeling of the egg extracellular matrix following fertilization by secretion of cortical granules, which are stimulus-dependent secretory vesicles unique to eggs and oocytes. Cortical granule contents modify the nascent egg extracellular matrix to form both a permanent block to polyspermy and to provide protection for early embryonic development.

The general question addressed in this application is: How do eggs so rapidly and efficiently construct their new extracellular matrix? We will use sea urchin eggs for this project because: 1) we can obtain, on average, 106 eggs per female, enabling large-scale biochemical approaches; 2) each of the constituents of this process - the cortical granules, the fertilization envelope, and the vitelline layer, can be isolated en masse in a functional form; 3) cDNA clones have been isolated that represent each major functional protein of this process; and 4) sea urchin eggs undergo one of the most extreme modifications at fertilization. They invest 6% of their protein mass and nearly 20% of their mRNA to this transformation of the nascent extracellular, vitelline layer into an impervious fertilization envelope within 60 seconds.

The broader implications of this project are that undergraduate and graduate students will be trained in diverse contemporary methods of cell, molecular, and developmental biology, and that general principles of fertilization and extracellular matrix biology will be integrated in a classically described, tractable system for educational purposes. In addition, project results and other educational material for K-16 will be continually updated on the Educational Resources page of our lab web site (http://www.brown.edu/Research/Wessel_Lab/).

Three Specific Research Aims are proposed:
1. Identify the vitelline layer proteins essential for fertilization envelope construction.
2. Determine the domains in cortical granule proteins required for construction of the extracellular matrix and block to polyspermy at fertilization.
3. Identify the peroxide-generating mechanism essential for envelope cross-linking.

[unreadable] DESCRIPTION (provided by applicant): The research capabilities of NIH investigators here at Brown are compromised by limited confocal microscope access and technology. Over the past several years, researchers have had to postpone experiments, travel to other institutions outside of the region to perform experiments, minimize the experimental protocol to accommodate the existing limitations, and farm out the confocal imaging of projects. This deficiency has created greatly inefficient use of NIH research support of investigators here. The applications of microcopy have also expanded for many investigators and the available, limited instrumentation is incapable of meeting these demands. We see no other way of acquiring such instrumentation without receiving this support from the NCRR. Twelve-faculty members, identified as Major Users, have collaborated to create this request for funds to support the acquisition of an integrated confocal microscope system. The Major User Group consists of twelve different laboratories with 86 researchers. The proposed instrument system contains the following: 1. Zeiss LSM 510 META confocal microscope, 2. Imaged through a Zeiss Axiovert 200 MOT inverted microscope, 3. Equipped with Narishige micromanipulators and an Eppendorf microinjector, 4. Microscope stage temperature control system. The requested instrument would significantly enhance the research productivity of the broad, interdisciplinary community of researchers described here, and greatly improve the biological image capabilities in the region. [unreadable] [unreadable]

cell component structure /function; egg /ovum; granule; biomedical resource; sea urchins;

bioimaging /biomedical imaging; three dimensional imaging /topography

ABSTRACT WESSEL Proposal # IOB-0620607
Sexual reproduction requires the formation of specialized cells, called gametes. The precursors of these cells, referred to as primordial germ cells, are determined very early in the life of the organism - as an embryo. In this application Dr. Wessel will examine what molecules are important for primordial germ cell development and determination in the sea urchin embryo. This is an important process for several reasons including that these cells are stem cells and studying this process in the sea urchin may help in our fundamental understanding of stem cell biology. Dr. Wessel will also examine how other echinoderms with different developmental strategies make their primordial germ cells. This comparative study will enable investigators to study how the primordial gems cells arise depending on differences in developmental mechanisms of the embryo. This project will also train graduate and undergraduate students in a wide array of modern experimental techniques. The results will be broadly disseminated in peer reviewed publications and online for K-12 students.

DESCRIPTION (provided by applicant): If fertilized, all eggs undergo an egg-to-embryo transition. Within minutes or hours of insemination, depending on the animal, the egg shuffles its existing molecular machinery and transforms itself into a cell with vastly different developmental potential. The first mechanistic alteration of this egg-to-embryo transition establishes the block to polyspermy, which is followed by and integrated with the turnover of maternal mRNA and cytoplasmic proteins, altered signal transduction capabilities, and changes at the cell surface including the addition and removal of specific membrane proteins and lipids. This transition is independent of new transcriptional activity, and for technical reasons, a majority of recent research towards understanding the egg-to-embryo transition has focused on signal transduction mechanisms and changes in mRNAs. The sea urchin and starfish, however, offer an opportunity to examine the changes that occur specifically at the cell surface. While the details of this process differ among animals, as with much of the reproductive phenomena, all eggs undergo this general transition. We will take advantage of the sea urchin and starfish because 1) millions of oocytes, eggs and embryos are readily obtained;2) all the major proteins of the cortical granules have been defined;3) the egg and oocyte plasma membrane can be manipulated experimentally in many ways;and 4) the genome sequence and genomic resources from the sea urchin Strongylocentrotus purpuratus are available. Results of this study will elucidate conserved mechanisms of this major developmental transition in all animals. Furthermore, because the changes examined here are at the cell surface, they will help clinicians develop new methods to non-invasively assess developmental potential in IVF applications for humans. PUBLIC HEALTH RELEVANCE: Immediately after fertilization, the egg rapidly transforms into an embryo with many new biochemical, cellular, and developmental features. This essential change is programmed into the egg and is independent of new gene expression;i.e., they reflect changes of the existing molecular machinery. Our research focuses on the changes that occur on the cell surface of the egg to embryo transition. These are some of the most rapid and conserved changes that occur in embryos and are detectable from the outside of the cell. Thus, this research will lead to a better understanding of a major transition in fertilization and development of all animals, and to the identification of non invasive, early markers indicative of successful development. Our results will have particular significance to clinical IVF predictions in human reproductive health.

DESCRIPTION (provided by applicant): Brown has almost 600 faculty/researchers in the Division of Biology and Medicine;it received $112,884,965 in NIH support in 2008, it has a strong translational and clinical research group, and a basic research emphasis on genomic applications. Yet this institution has limited DNA sequencing capabilities i.e. a single Applied Biosystems 3130xl Genetic Analyzer capillary sequencer. The research frontier has changed tremendously so that now DNA sequencing is used for exploration, for detailed analysis, and for quantitation. It is even cost effective now compared to many microarray approaches for analysis of e.g. whole genome transcriptome arrays. Investigators at Brown University are experiencing a burgeoning need for rapid, deep, and cost-effective DNA sequencing. An assessment of needs has coalesced to the High Throughput DNA Sequencing instruments;such an instrument would meet the vast majority of research needs. Unfortunately, the queues for off-site facilities ranged from 4 weeks (providing the investigator made all their own libraries), to over 2 months for full-service facilities. An off-campus sample was also subject to consistently getting delayed by new on-campus projects. We therefore request support to acquire a high-throughput DNA sequencer to handle the burgeoning needs for DNA sequencing in the Division of Biology and Medicine at Brown University. Historically, the short read sequencers (Solexa, SOLID) have been attractive for the high throughput and relatively low cost of sequence. However, they have lagged behind in their potential because of the difficulties in interpreting and aligning short sequencing reads, 25-35 bases in length, of metazoan genomes with large sequence complexity. This detraction is minimized now with the Illumina GAII platform, achieving read lengths of over 100 bases on average, and with paired end read capabilities included in this next generation unit, the read lengths now approach the lengths of alternative sequencing formats, e.g. 454 lengths, but with much higher throughput and significantly lower cost. Indeed, with new sequencing chemistries, and advances in software capabilities, de novo sequencing is added to its list of capabilities, making this type of instrument meet the investigator need at Brown University. This application represents the collective DNA sequencing needs of 32 major users and 9 minor users, representing 14 different research departments, and over 70 NIH grants. It contains a mix of basic and clinical researchers, M.D.s and Ph.D.s. Importantly, it contains a strong contingent of computational molecular biologists interested in better, and more easily making use of the large data sets resulting from such an instrument. The University is committed to supporting this instrument through personnel, laboratory space, computer data storage capabilities, and extended service contracts to maximize the investment of the instrument and to facilitate the output of each investigator.

The reproductive potential of an adult depends on the embryo forming specialized stem cells, called primordial germ cells. They are the only cells of the body that are able to develop into the eggs or sperm of the adult. If this mechanism fails, or if those cells are lost, the adult will not be able to make eggs or sperm, and will be reproductively sterile. The process of developing these important primordial germ cells is not understood, but they are a type of stem cells with the ultimate in developmental capability. These investigators are using a simple animal as a model to explore how the process of primordial germ cell formation works. They are using echinoderms, i.e. sea urchins and sea stars, as a model to explore this mechanism with an understanding that they will reveal general and fundamental principles of this process. Embryos from these animals are easily manipulated, they develop outside of the adult, and advanced genomic resources are available. Echinoderms are also closely related to vertebrates, so these results will have broad ramifications for animals in which this process is difficult to study.The research support will also have a significant impact beyond the bench as our lab has an aggressive outreach program. We are regular hosts to URM faculty and students, Professor Wessel serves as a scientific advisor to several preK-12 schools, and has involved a variety of students in the lab in observation, hands-on research, authorship, and creative learning. Overall, our outreach activities over the past 3 years have involved directly over 300 citizens, and benefit both the members of the lab and society, hopefully changing us all for a healthy science outlook in the future.

DESCRIPTION (provided by applicant): Cancer cells and germ cells share many cellular and molecule features and the link is even more striking when considering the many tumors resulting from aberrant germ cells. Moreover, many tumors require germline determinants for tumorigenesis, even those tumors from a non- germ line origin. As one of the original germline determinants identified, Vasa have been used extensively as a germline marker in many organisms. Recently though Vasa has received more widespread attention for its' presence in many human cancer cells, its' essential nature in tumor formation, its' role in multipotent cell functions, and recently, it's' function in regulation of the cell cycle. Despite its' long history,the functions and regulation of Vasa in these important and widespread roles are not yet known. This project is designed to reveal the regulatory mechanisms of Vasa that contributes to its' essential function in health and disease.

DESCRIPTION (provided by applicant): We combine several new technologies and apply them to oocytes and embryos with the goal of quickly, efficiently, and effectively altering genomes of animals. We utilize oocytes and early embryos in a non-transgenic approach to alter specific gene function. Oocytes will be used for construction of stable transgenic lines at the F0 generation, and embryos for homozygous gene alteration for quick gene screening. This technology will open the door for the study of new organisms and to either modify a gene for functional analysis or correct a gene that is otherwise rendered sub-functional. Genome manipulation approaches have been performed mostly with cultured cells and genetically tractable embryos because of the capabilities to introduce exogenous DNA constructs. To create stable transgenic animals, however, these approaches require back-crosses to reduce genetic mosaicism or integration into the germ line and thus increase the cost of time and resources. These limitations minimize the effectiveness for broad utilization in organisms key for biological research, especially those whose genetic tractability is low. The use transcriptional activator like effector (TALE) functions linked to an enzymatic domain of DNA deaminase activity into oocytes is novel, and success of this project will allow researchers to construct gen alterations at the F0 generation. This project crafts a single nucleotide genome targeting system at a predetermined site by use of the catalytic domains of ADAR. Our recent preliminary results demonstrate DNA-deaminating activity of this domain and coupled with a demonstrable TALE-targeting mechanism increases feasibility of this project. The outcome of this project will be useful to all researchers, especially those working in genetically less-tractable organisms, and those wishing to rapidly screen gene functions without transgenic approaches. We believe this technology will remove the wording non-model organism from the genetic research vernacular, and in the future may be applicable for clinical use by correcting a point mutation related to a congenital disease state.

Project Summary Quiescence is a common character of many stem cells. Low metabolic activity in these cells may function to minimize the potential damaging effects of stress, minimize the number of cells needed for replenishment, and may occupy unique niches. These cells are found in many adult human tissues, even in the tissues of the central nervous system. These naturally occurring stem cells however, are rare and difficult to isolate so a mechanistic understanding of their integrated cellular activities is largely unknown. We recently learned that the primordial germ cells (PGC) of the sea urchin are quiescent. From a highly active early embryonic cell, they rapidly and dramatically change their metabolic pathways, decrease their protein synthesis, have low mRNA turnover and diminished transcription, and reduced mitochondrial activity. These cells are quiescent at a time when the remainder of the embryo multiplies to thousands of cells with many unique gene and protein expression patterns. Following the quiescent period, the PGCs rapidly return to normal cellular activity. Thus, the phenotype is prolonged, inclusive, highly penetrant, and transient. Resolution of the phenotype will require large numbers of naturally occurring, isolated PGCs, high throughput molecular manipulations, quantitative biochemical approaches, and excellent clarity for in vivo optics ? features ideally suited for sea urchin embryos. This application will interrogate the molecular mechanism of this transient quiescence and the dramatic phenotypic changes in the stem cells of the germ line. Stem cell quiescence is shared by many stem cell types, in and out of the germ line, but sparingly few models are present to explore such mechanisms in abundance, in naturally occurring cells, and in vivo. Overall the novelty and impact of this work includes: 1) germline stem cells using oxidative glycolysis but without proliferation ? distinct from the Warburg effect; 2) broad, quantitative quiescence characters; 3) integration of metabolism with molecular mechanisms for quiescence; 4) analysis of quiescence on developmental potential of the stem cell, and 5) the cells studied are primordial germ cells, a cell type found in all sexually reproducing animals. Coupled with a tractable model system for study, this proposal provides outstanding potential to add to an exciting field of both basic and clinical interest for generation of sperm and eggs in the adult.

Project Summary Sexual reproduction requires a germ line and mammals craft the beginning of their germ line early in development by secreted signaling molecules. This mechanism in vivo remains obscure, largely from the technical limitations of experimenting on embryos developing in utero. Germ line induction in a human embryo is even more remote and errors in the process are associated with a lack of germ line (infertility) and mis- placed specification of germ line cells (teratomas). How the primordial germ cells form by cell-cell interactions during early development is the focus of this application and makes use of a sister group to chordates ? the sea star embryo. While not a common organism for biomedical research, the sea star model system has many experimental and strategic attractions for revealing this process. Millions of synchronous embryos from a single male/female cross allow biochemical and metabolic analysis of the germ line, the resultant embryos have ideal transparency for in vivo longitudinal imaging, they develop rapidly, are easy to manipulate (single cell drop- mRNA-seq, optogenetic controls) and they respond well to complementary gene perturbation approaches (CRISPR, morpholino (MASO), and small molecule perturbations. The existing genomic and reagent resources for the sea star, coupled with the tractable experimental characteristics of the sea star embryo, yields a unique and surrogate system for understanding mammalian germ line induction. The approaches documented in this application will accomplish three main goals to advance the field in transformative ways. 1) Three signaling pathways are prioritized for integrative stimulating and repressing activities during a brief window in early development. 2) The mechanism of germ cell fate restriction is interrogated by a restriction map - germ cell factors are retained in the future germ line, whereas somatic factors are rapidly restricted from the same field. 3) Commitment to the germ line is hypothesized to include rapid insulation from local embryonic signaling and instead transitions to a committed cell type with a unique transcriptome, and response to signal activity. Single cell mRNA drop-sequencing and optogenetic control over signaling at single cell resolution are used by taking full advantage of the optical clarity and accessibility of the embryo. Overall, these accomplished goals will provide direct translation for understanding inductive germ line formation in mammals, and will reveal mechanistic explanations for germ cell-less conditions leading to human infertility, and mis-expression of germ cells leading to germ line tumors.

Non-Technical Paragraph
Everything in biology is connected. It is the job of life scientists to reveal important biological properties in the most impactful, efficient, and economical way. To do so they look for model organisms that are particularly tractable for studying complex biological processes and then apply what is learned to better understand other organisms. For more than a century, sea urchins have provided a valuable research model that has contributed significantly to understanding of many fundamental biological processes such as fertilization, embryonic development and cell division. Sea urchins have proven to be a valuable model due to their close genetic relationship to vertebrate animals and many features that make experimentation easier. The goal of this grant is to create the next generation of tools to enhance the utility of sea urchins as research models that will enable new areas of research and to make these tools widely available to the scientific community. Areas of biological research to be enhanced by the tools created from this grant include a better understanding of how eggs and sperm interact at fertilization, understanding the rules of embryo development, how nerve cells are made, how sex is determined, how animals protect themselves from environmental insults and from infection, and how tissues and organs can regenerate when they are damaged. An essential component of the grant is to reach beyond scientists to the public, students and teachers and to make the sea urchin a highly attractive and impactful research and education tool of the twenty-first century.

Technical Paragraph
Sea urchin researchers have long sought to leverage the experimental tractability of the embryo and adult with genetic approaches but, to date, manipulations have been limited largely to dependence on morpholinos or pharmacology. The overarching goal of this EDGE grant is to build tools that overcome major obstacles to testing gene functionality in echinoderms, opening up a new era of discovery for diverse and integrated studies across all life history stages of this valuable sister group to chordates. This goal will be realized by prioritized Grand Challenges as follows: (1) Targeted DNA insertion for gene tagging, conditional, and reversible gene control; (2) Rapid, standardized protocols for larval culturing, metamorphosis, juvenile development, and adult sexual maturation including nutritional and environmental optimization for each stage; (3) Rapid, simple sexing protocols of animals to maximize resources; (4) Simple and efficient protocols for culturing cells from the embryo and adult in vitro with routine genetic manipulations; (5) Routine archiving/cryopreserving genetically manipulated sperm, embryos and cells for long term sustainability. These integrated new technologies with controlled and heritable genetic manipulations and the ability to test gene function and regulation in in vitro cell-based systems will enable new avenues of investigation that fully exploit the important properties of echinoderms as a research organism. This grant will focus on a single species of sea urchin, Lytechinus variegatus, but the bottlenecks opened by tools developed herein will permeate the phylum enabling significant advances across echinoderms. Rapid dissemination of results to the larger community of sea urchin researchers is an integral part of the grant.

This project is jointly funded by the Developmental Systems Cluster (DSC) in the Division of Integrative Organismal Systems of the Biological Sciences Directorate and the Established Program to Stimulate Competitive Research (EPSCoR).

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Project Summary Sexual reproduction requires a germline, a lineage of cells formed in early embryos that ultimately develops into eggs or sperm in the adult. Lore has it that when you lose your germline in development, you become a sterile adult. In many animals, especially in the current favorites of lab animals, that is largely true. We use ?rule breakers? though and find such lore unfounded. Our work focuses on the biology of primordial germ cells, how they form during early development, and how they regenerate when the originals are removed. Our work leverages embryos from a sister group to chordates ? the sea star and sea urchin. While not common organisms for biomedical research, these echinoderms have many strategic benefits for revealing unique perspectives in the biology of germline formation and regeneration. Millions of synchronous embryos from a single male/female cross allow biochemical and metabolic analysis of the germline, the resultant embryos have ideal transparency for in vivo longitudinal imaging, they develop rapidly, are easy to manipulate (single cell drop-mRNA-seq, optogenetics, cell and tissue transplantations) and they are well suited to complementary gene perturbation approaches (CRISPR/Cas9, morpholinoantisense oligonucleotides, MASO), and small molecule perturbations. The existing deep genomic and reagent resources for these animals, coupled with their tractable experimental characteristics, yields a unique system for understanding primordial germ cell biology with defined molecular and morphological endpoints, in live embryos with longitudinal analysis, distinct metrics of quantitation, and transgenerational evaluations. We interrogate all levels of gene expression for this work, from chromatin modification to post- transcriptional processing and post-translational networks, because that is what the embryos are ?telling? us is needed to understand these complex, and deeply rooted events in sexual reproduction. Our work emphasizes longitudinal, in vivo analysis using high resolution optical imaging coupled with genomic perturbations, signal pathway manipulations and manual transplantations and expirations to leverage contrasting mechanisms in germ cell formation between closely related organisms. Sea urchins and sea stars have historically not been genetically manipulated, and this reason is precisely how germ line regeneration has been discovered in this and other animals seen to bear this trait. Relying on manual manipulations meant the genes needed for regeneration were not disturbed, revealing their germ cell regenerative abilities. With new state-of-the-art technologies, these animals can now be exploited with transgenerational analysis. Overall, our work interrogates important biological questions from unique experimental perspectives using rule-breaking models for innovation in the pursuit of new knowledge.