Charles B. Kimmel, Ph.D. - US grants

Funds are awarded for support for the 26th Annual Meeting of the Northwest Regional Developmental Biology Conference to be held Thursday through Saturday May 6 - May 8, 1993, in the conference facilities at the University of British Columbia, Vancouver B.C. The main theme for the conference will be transition between developmental states. This theme will be introduced by the opening plenary lecturer (Gerold Schubiger) and expanded upon by the symposium speakers (George Sprague, Ry Meeks-Wagner, William Harris, and Barbara Wakamoto). Presentations by participants will be scheduled for a poster session or one of four platform sessions. Emphasis is on graduate student attendance and participation, including awards for the best student platform and poster presentations. Attendance in the range of 100-150 is expected. The award will be used exclusively to lower the cost of room and board for the estimated 55-75 graduate students expected to attend the conference.

zebrafish; confocal scanning microscopy; developmental neurobiology; mesoderm; head; cell migration; rhombencephalon;

vertebrate embryology; neurogenesis; developmental genetics; gene expression; neural plate /tube; cell differentiation; early embryonic stage; gene targeting; alternatives to animals in research; tissue mosaicism; developmental neurobiology; cell cell interaction; cell migration; neural crest; mesoderm; cell transplantation; embryo /fetus culture; genetically modified animals; fluorescent dye /probe; embryo /fetus transplantation; transfection; confocal scanning microscopy; zebrafish;

DESCRIPTION: Understanding the early development of patterning of the nervous system and its underlying genetic control is essential for progress in the alleviation of genetic-determined mental health disorders, of congenital diseases, and of cancers. In this proposal, Dr. Kimmel proposes to study neural patterning in the small and simple nervous system of the zebrafish embryo. This species has many attributes for both developmental and genetic study, and can serve as a model system for understanding the neural development in all vertebrates, including humans. The proposed experiments address the nature of segmental patterning of the hindbrain and the connections of the hindbrain segments with the jaw and pharyngeal segments of the head periphery. The specific aims proposed by Dr. Kimmel are as follows: (1) Mutations will be collected and characterized that disrupt the earliest stages of segmental patterning. The genes identified by these mutations might act specifically in the neural plate or in head mesoderm as predicted by alternative hypotheses. Molecular analysis and genetic mosaic analyses will establish in which tissue these early-acting genes function. (2) The fate map positions of functionally interacting cells in the hindbrain and head periphery will be compared. This analysis will test a prediction that at the early stage when segmental identities are specified in the head primordium, the progenitors of particular skeletal components, of muscle cells that insert on these components, and of motoneurons that control this muscle are precisly aligned in the head fate map. (3) The cellular basis of hindbrain and pharyngeal arch segment formation will be analyzed. Methods for cell lineage analysis and for time-lapse analyses of morphogenesis in vivo will be used to examine predictions that segments develop as lineage-restricted compartments, and to learn when segment borders form. Transplantation of single identified postmitotic young neurons in the early brain segments will be used to examine the molecular-genetic nature of cellular commitment to segmental identity.

Craniofacial malformations arise by errors in development, as can occur when genes that pattern the embryo do not function correctly. Learning about how the facial skeleton forms, as well as the genes that control its formation, is essential for the eventual understanding of the bases of inherited craniofacial disorders. The zebrafish provides a useful animal model for such studies: The zebrafish embryo is amenable for developmental and genetic investigation. Its embryonic head skeleton shares considerable similarity with that of humans. Genes that pattern zebrafish and human skeletal development have been highly conserved during evolution, hence understanding gained from zebrafish can apply directly to human development. This proposal is to examine critically a period of about a day of embryonic development in the zebrafish when cells form the rudiments of the facial skeleton. A "morphogenetic cascade" hypothesis proposes that, under genetic control, neural crest-derived cells in the pharyngeal arch primordia undergo a short series of rearrangements that, at each step, refine skeletal prepatterning. The first specific aim is to identify the cells, by fate mapping at successive steps of the cascade, that will form specific facial skeletal elements. The second aim is to watch the cells in the intact embryo directly, by making time-lapse recordings with a confocal microscope (4d-analysis), to learn whether and precisely how the predicted cellular rearrangements occur. Accomplishment of these two aims will reveal the cellular behaviors that build individual elements of the head skeleton. The third specific aim is to study the cascade genetically, by analyzing craniofacial mutants and patterning genes cloned by homology. Hierarchically upstream regulatory genes have already been identified by these techniques and the principal focus will be to identify and characterize new genes that execute that later, downstream, steps in the cascade.

We will continue our Program Project to learn the mechanisms of cell signaling, specification and commitment during embryonic development. We propose three component projects, each addressing one of these three issues. We will carry out these studies in the zebrafish, Brachydanio rerio, because of the advantages it offers as an experimental system, including labeling and transplanting single cells, genetic analysis of developmental pathways, and production of transgenic animals. Specifically, we propose to examine induction of the nervous system and the development of two cell lineages -- primary neurons that form the earliest neural circuitry in the embryo and neural crest cells that form the peripheral nervous system and structures in the head and heart. Induction of these lineages involves cell signaling by inducing (growth/survival) factors, and their receptors, and depends on cell position in the embryo. Before overt differentiation, cells become specified. While these specifications may involve phenotypic changes, they do not represent irreversible commitments that restrict the cells to a single developmental course. We propose to examine whether these "conditional" specifications are temporally separable from commitment in neuronal and neural crest lineages. Our main long-term objectives are to identify the regulatory molecules and their genes that underlie signaling, specification and commitment during vertebrate development, and to discover their functional interactions. Disruption of such mechanisms during development of the human central and peripheral nervous systems results in numerous diseases or syndromes, including (a) hereditary dysplasias and neuropathies such as Familial Dysautonomia, Hirschprung's disease (aganglionic megacolon), and orthostatic hypotension; (b) hereditary metaplasias, such as von Recklinghausen's disease (Neurofibromatosis); (c) numerous neoplasias, such as gliomas, neuroblastomas, and pheochromocytomas; and (d) congenital defects such as spina bifida, cleft lip/palate, and craniofacial defects associated with heart malformation (e.g. Pierre Robin syndrome). A detailed understanding of the regulatory mechanisms of normal neural development in vertebrates embryos will help elucidate such disease processes.

[unreadable] DESCRIPTION (provided by applicant): For all children to have the opportunity to achieve their full potential for healthy lives, free from disease, it is essential to understand mechanisms underlying developmental patterning and how this patterning can go awry in human disease. The goal of this program project is to elucidate one such mechanism - the function of reciprocal intercellular signaling that specifies embryonic cells to traverse particular developmental pathways and express restricted fates. This goal will be achieved for three sets of cell fates in three component projects, using the zebrafish, a widely-utilized animal model organism pioneered by this group at the University of Oregon. The projects take advantage of the attributes of the zebrafish for developmental genetic analyses, including gene expression studies, genetic mosaic investigations, and loss- and gain-of-function experiments that will establish the nature of the interactions. The projects include screens for new mutations and genes important in these signaling pathways, facilitated by the unique Zebrafish Facility, one of four core services. Project I, "Reciprocal signaling in skeletogenesis", tests hypotheses about the functioning of signaling molecules in patterning the shape of the palatal skeleton and the pathway of chondral bone development. Results will improve understanding of signaling pathways between cranial epithelia and mesenchyme, and between cartilage and bone progenitors. They will thereby inform our understanding of cleft palate, one of the most common human birth defects, and osteoarthritis that will affect nearly one in five Americans during the coming decade. Project II, "Reciprocal signaling in synaptogenesis", tests a novel hypothesis that Usher genes encode proteins that interact in a complex mediating reciprocal signaling between sensory cells and neurons with which the sensory cells form synaptic connections. The analyses will identify the critical components of the Usher gene network and provide an integrated understanding of Usher syndrome, the most frequent cause of deafness and blindness. Project III, "Reciprocal signaling in gastrointestinal tract development", explores the hypothesis that gut microbiota influence cell fate decisions in gut epithelium and enteric nervous system by modulating a highly conserved molecular signal, Notch. The work will elucidate reciprocal signaling and how it goes awry in disorders such as inflammatory bowel disease and related disorders that together affect more than 10% of the U.S. population. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): In response to RFA DE-03-001 "Gene Discovery for Craniofacial Disorders" this application is for a unique program of efficient gene discovery in zebrafish to find candidates for human disease genes: Our specific aims are to discover zebrafish genes that when mutated result in (1) craniofacial bone dysgenesis, and (2) retinal degeneration. For both, a novel combination of powerful approaches toward gene discovery will be utilized. Lesions in genes will be introduced by viral-mediated insertional mutagenesis. Because the viral insertions serve to tag the mutated genes, the genes can be identified and cloned extremely rapidly, often within a few days. The mutants will be identified in highly efficient, directed, first generation (G1) screens, using embryos and larvae obtained by parthenogenesis and vitally stained with fluorescent dyes to reveal the phenotypes. Parthenogenesis and G1 screening eliminates months of time and saves rearing hundreds of thousands of fish required in the more usual forward-genetic strategies of raising families of mutants and screening F3 progeny. The new strains will be made rapidly available to other researchers using community resources, the Zebrafish Information Network to advertise the mutants and the Zebrafish International Resource Center to distribute them. Various favorable attributes of the zebrafish embryo and larva will permit detailed phenotypic studies and rapid characterization of the functions of the genes in facial bone and eye development. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The investigators are applying for funding to help support a small, topical and intense scientific meeting that they are organizing on the neural crest. The meeting will be held June 25-27, 2003 at a site on Mt. Hood, near Portland, Oregon. Nineteen speakers, a mixture of established world-class scientists and beginning investigators representing eight countries have been invited and have agreed to participate. There will be poster sessions and we expect the total size of the meeting to be about 150.

A grant has been awarded to Dr. LeBlanc (North Carolina State University) to elucidate the basic endocrine control of sexual differentiation in a snail species (mud snail: Ilyanassa obsoleta) and to determine the mechanism by which the biocide tributyltin causes sexual ambiguity in marine snails. Tributyltin is an antifoulant used in marine paints. The material is ubiquitous in the marine environment and has been causally associated with the global occurrence of reproductive system abnormalities in marine snails. Studies will be performed to identify the hormones that are responsible for the development of the snail reproductive system. Initially, field surveys will be performed to establish relationships between hormone or hormone receptor levels and development of the snail reproductive system over the seasonal reproductive cycle. These investigations will be followed by laboratory experiments where levels of tentatively important hormones will be manipulated and consequences to reproductive system development will be established. Once the relevant hormones have been conclusively identified, the effect of tributyltin on those hormone levels and activity will be established. Results from this study will significantly advance our understanding of basic molluscan endocrinology. Many Asian and South American countries are developing snail culture as a sustainable food source for local consumption as well as export. Understanding the regulation of the reproductive cycle of snails would greatly enhance the economic feasibility of such operations. In addition, the International Maritime Organization has implemented a phase-out of tributyltin in marine paints owing to its adverse effects on marine ecosystems. The economic incentive to minimize drag on ocean-going vessels through the use of antifoulants will necessitate the development and use of tributyltin alternatives. Elucidation of the mechanism by which tributyltin interferes with sexual development of snails will help ensure that alternative antifoulants do not share this insidious property.

DESCRIPTION (provided by applicant): For the nervous system to function properly, it is crucial that embryonic neurons acquire appropriate identities so they can develop the properties necessary for their later functions. The longterm goal of our laboratory is to understand the mechanisms underlying these processes. These mechanisms will be investigated by studying development of individually identified primary motoneurons in embryonic zebrafish. Our hypothesis is that the identities of primary motoneurons are specified by a series of signals that regulate expression of transcription factors that control neuronal identity and later features of development, such as axonal pathfinding. A series of experiments to test aspects of this hypothesis are proposed. Zebrafish primary motoneurons initially express a specific transcription factor, islet1, which is then downregulated; later islet1 or a related gene, islet2 is expressed in each specific primary motoneuron. The roles of the distinct phases of islet gene expression in motoneuron identity and axonal pathfinding will be tested using mutants and morpholino antisense oligonucleotides to knock down gene function. Retinoic acid, a well-known teratogen that can have devastating effects on human embryos, is synthesized in zebrafish during the time that primary motoneurons are being specified. Exposure to exogenous retinoic acid alters motoneuron numbers and patterning. The role of endogenous retinoic acid in motoneuron specification, and its interactions with other signals including the Hedgehog and Delta/Notch pathways, will be tested by a series of genetic gain- and loss-of-function experiments. The effects of retinoic acid on regulation of somite-derived signals that affect motoneuron identity will also be examined; potential signals will be identified using differential molecular screens. Interactions between specific primary motoneurons and the muscle fibers they innervate regulate their identities and survival. The role of the Delta/Notch signaling pathway in this process will be tested using a genetic approach. Although many genes regulating motoneuron development are known, many remain undiscovered. A mutagenesis screen will be undertaken to identify new genes involved in specifying motoneuron identities.

[unreadable] DESCRIPTION (provided by applicant): In response to RFA DE-03-001 "Gene Discovery for Craniofacial Disorders" this application is for a unique program of efficient gene discovery in zebrafish to find candidates for human disease genes: Our specific aims are to discover zebrafish genes that when mutated result in (1) craniofacial bone dysgenesis, and (2) retinal degeneration. For both, a novel combination of powerful approaches toward gene discovery will be utilized. Lesions in genes will be introduced by viral-mediated insertional mutagenesis. Because the viral insertions serve to tag the mutated genes, the genes can be identified and cloned extremely rapidly, often within a few days. The mutants will be identified in highly efficient, directed, first generation (G1) screens, using embryos and larvae obtained by parthenogenesis and vitally stained with fluorescent dyes to reveal the phenotypes. Parthenogenesis and G1 screening eliminates months of time and saves rearing hundreds of thousands of fish required in the more usual forward-genetic strategies of raising families of mutants and screening F3 progeny. The new strains will be made rapidly available to other researchers using community resources, the Zebrafish Information Network to advertise the mutants and the Zebrafish International Resource Center to distribute them. Various favorable attributes of the zebrafish embryo and larva will permit detailed phenotypic studies and rapid characterization of the functions of the genes in facial bone and eye development. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Craniofacial malformations arise when development does not work correctly. Learning about head skeletal development, including the regulatory genes that control development, is essential for our understanding of what goes wrong in human inherited disorders. Developmental mechanisms are broadly shared among animals. The jaws of diverse vertebrates come from the same embryonic tissues, and depend on correct function of the same genes. E.g., mutation of the Endothelin 1 gene, encoding a cell-cell signal, causes a prominent reduction of the jaw in mice and in zebrafish, a valuable animal model on which this proposal focuses because of favorable attributes for study. Three projects are proposed to test predictions of hypotheses about the mechanisms underlying formation of bones and cartilages. The first investigation is to use precise cell marking procedures to learn arrangements of embryonic facial skeleton-forming cells, and to learn by time-lapse recordings in intact embryos, how the cells rearrange as they develop cartilages that differ from one another in shape. A further goal is to learn, by the same methods, how the cell arrangements and rearrangements are disrupted when critical pattern-determining genes, Hox genes and the Endothelin 1 gene, do not function. The second investigation is to use molecular methods to examine the cellular responses to Endothelin 1 signaling. This will be accomplished by learning what gene activities change in skeleton-forming cells receiving an Endothelin 1 signal, and what are the functional consequences of these changes. The third investigation is use a skeletal screen with larvae derived from mutagenized fish to discover new genes required for craniofacial patterning, and, by analyzing the mutants, to learn how the genes function.

Cells that form the skeleton first become patterned into specific shapes, and then differentiate to form stiff, supporting, structural elements. Cleft palate, one of the most common birth defects, arises from disruptions in the patterning phase of skeletogenesis, and osteoarthritis, which will affect nearly one in five Americans during the coming decade, stem from disturbed control of skeletal tissue differentiation and histogenesis. Both disorders can appear with improper functioning of developmental regulatory genes that control reciprocal signaling between cells that govern skeletal development. Our project investigates early developmental stages of zebrafish to understand reciprocal signaling in skeletogenesis. We hypothesize that a pathway patterning the palatal skeleton involves specific interactions among Sonic hedgehog, Platelet- derived growth factor, and other signals. We hypothesize that a pathway controlling histogenesis of cartilage and cartilage-replacement bone involves specific interactions among Bone morphogenetic proteins, Indian hedgehog, and Parathyroid hormone related protein. To test these hypotheses, we will examine the detailed abnormal developmental and skeletal phenotypes that arise by perturbing functions of genes in these pathways. We also propose to carry out forward and reverse genetic screens to identify new skeletal mutants. Our findings will improve our knowledge of signaling pathways between cranial epithelia and mesenchyme, and between cartilage and bone progenitors, and thereby inform our understanding of the pathogenesis of cleft palate and osteoarthritis.

Biologists have made great progress in understanding the genetic basis of simple traits, from the study of induced mutations in model organisms. However, most traits are complex, and their development is directed by many genes that are influenced by environmental conditions. The Cresko laboratory will examine natural populations of threespine stickleback fish to understand the developmental genetic basis of variation in complex head and jaw traits. These structures vary tremendously among individuals, populations and species. Despite this diversity, the development of head and jaw structures occurs through conserved genetic interactions and will prove highly informative about the proper development of similar structures in other vertebrates such as humans. This research will provide a much better understanding of the genetic basis of complex traits, be they characters important for stickleback, or the most common types of human diseases that afflict tens of millions of people. Dr. Cresko's group has an outstanding of outreach to elementary students and also of undergraduate training. They are also active contributors to the resources of the research community.

Biologists are just beginning to understand the genetic basis of variable aspects of or-ganisms such as size, shape and color, to name a few. However, most traits are com-plex, their development directed by many genes whose effects are influenced by environmental conditions. These combined effects on trait variation must be mediated through the identity and behavior of cells such that, for example, some cells proliferate more to make larger cartilages, or excrete additional matrix to make stronger bones. However, little is presently known about the precise mechanisms of cellular integration, and filling this gap in knowledge is a fundamental problem in biology and is the primary focus of this project. Natural variation in threespine stickleback fish provides an excel-lent opportunity to address this question. The goal of this research is to understand how cells integrate genetic and environmental information and lead to variation in complex head and jaw traits among populations of stickleback. These structures vary tremen-dously among individuals, populations and species. Despite this diversity, all vertebrates share conserved genetic interactions for the development of head and jaw struc-tures. Thus, research on these traits is useful for understanding the situation in stickle-backs, and is also highly informative about the proper development of similar structures in other vertebrates such as humans. These conditions are the product of many genes and their interactions with the envi-ronment, and lead to variations in cellular identities or behaviors (i.e. uncontrolled cell growth in cancer). This research will provide a much better understanding of the genetic and cellular basis of complex traits, be they characters important for stickleback, or the most common types of human diseases. In addition, Alaskan stickleback populations are studied in collaboration with laboratories at the University of Alaska Anchorage, which has a large Alaska Native student population, and the unique opportunity exists to include individuals in this underrepresented group into research and educational activi-ties. Furthermore, an educational website is maintained that uses stickleback to provide educators and researchers around the world with knowledge and skills necessary to learn about and perform research with stickleback.

Imaging the complete three-dimensional architecture of living, growing organisms with sub-cellular resolution and sufficient speed to capture cellular dynamics has long been an unattainable goal. Traditional approaches are sufficient for a few hundred cells, but are too slow and cause too much photodamage to probe larger systems, such as whole embryos. Recently, researchers have developed a new methodology, "light sheet microscopy" (LSM), in which illumination by a scanned sheet of light that excites fluorescent probes allows rapid, high resolution 3D imaging with minimal photodamage. This technique can acquire 3D images of entire developing zebrafish embryos with sub-cellular resolution for over 24 hours, creating one embryo-spanning 3D image every 60-90 seconds. This approach promises to revolutionize biological imaging.

This award is supporting a project with two main goals. The first goal aims to build a light sheet microscope at the University of Oregon, the first of its kind in the United States. This instrument will be invaluable to researchers studying zebrafish and other models of biological phenomena. It will illuminate the connections between cell migration and skeletal development in early embryogenesis; allow organism-wide mapping of interactions between bacterial populations and host immune cells, a key factor in normal as well as diseased physiology; enable studies of how particular mutations affect the development of the enteric nervous system over large length and time scales; and more. The second goal aims to extend the capabilities of light sheet microscopy, creating the next generation of light sheet microscopes. This project will develop (1) an instrument capable of 3D dark-field imaging, which will be especially important for visualizing non-fluorescent nanoparticle distributions in whole, living organisms, crucial to addressing concerns about nanotechnological toxicology; and (2) an instrument capable of combined fluorescence LSM and differential interference contrast microscopy, which will reveal the local context in which cells expressing particular fluorescent proteins act.

The broader impacts of this proposal have three facets. First, rapid 3D imaging will enable unprecedented advances in fields spanning the biological sciences, as noted above, and also the physical sciences, for example the structure and dynamics of soft materials. Second, the project will provide valuable cross-disciplinary training for postdoctoral researchers, graduate students, and undergraduates. Third, the visually striking nature of the 3D data resulting from LSM imaging are ideally suited to education and outreach, as they transform the illustration of topics like embryogenesis and self-assembly from static or schematic cartoons to vibrant and "real" 3D movies. Data from the proposed project will be incorporated into a new course on biophysics for non-science-major college undergraduates and a week-long day camp for socioeconomically disadvantaged high school students.

The project outcome-a functional light sheet microscope with dark-field and differential interference contrast imaging capabilities-will be available at the University of Oregon (Eugene, OR).

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) Enabling Technologies, and specific Challenge Topic 06-DE-102 Structural and Molecular Atlases of Craniofacial Development. We propose to construct a developmental atlas for the craniofacial skeleton of zebrafish, a widely used model organism for understanding conserved features of development of the skull, and human disorders such as cleft palate. The atlas will provide a key resource for scientists wishing to discover how the complex anatomy of the zebrafish craniofacial skeleton develops. It will serve as a critical reference for learning the defects of the many zebrafish craniofacial mutants now available for study, as well as new mutants now being generated through genome-wide initiatives. It also will be useful for understanding development of the zebrafish skull compared with other organisms, such as mouse. The atlas will be web-based and freely available on FaceBase, a web resource rapidly becoming the standard for understanding craniofacial development, and on ZFIN, the specific informatics resource for zebrafish development and genetics. It will include a series of developmental stages beginning with the 1-day postfertilization embryo when the skeletal primordia are present, but bones and cartilages have not yet formed. It will continue with selected stages through the 3-week-old juvenile stage when nearly all of the elements of the skull are present, and shaped approximately as in the adult, but of miniature size. It will be 3-dimensional and interactive: The user will be able to virtually dissect the skull - calling up particular subsets of elements of interest, to rotate the image and observe a set of elements from any point of view, to section through the structure in any plane, and to examine how the structure changes at different developmental stages. A key innovation is that the atlas will be based entirely on images taken of the living, developing organism expressing transgenic fluorescent proteins. These markers will be driven by the promoters of key skeletal regulatory genes, including fli1, expressed in all craniofacial mesenchyme, and dlx5a, expressed in a subset of this mesenchyme that is restricted to the ventral elements of the skull, including the lower jaw. During stages of skeletal morphogenesis and growth, the cells making the skeleton will be revealed specifically by expression of markers including sox10 and foxp2a for cartilage, and osterix for bone. The fish expressing one of these markers also will be vitally counterstained with a fluorescent dye such as Alizarin red to reveal the mineralized bone. The atlas will feature, for every stage, high magnification, through-focus, 2-color image stacks made by confocal microscopy. Such imaging takes full advantage of the many currently available transgenic constructs, the optical clarity of the zebrafish, and of its relatively small size and the relatively small number of cells that build the skull. These attributes combine to provide cell-level resolution with unsurpassed specificity and detail throughout an extended period of development. The atlas will also include, especially for the later developmental stages, medium-resolution images at lower magnification made by optical projection tomography (OPT). The user will be able to interactively manipulate these OPT stacks in the same way as for the confocal stacks, and will allow better 3-d understanding of the more global features of skull organization and development. PUBLIC HEALTH RELEVANCE: The zebrafish craniofacial atlas that will come from this project will provide a resource for skeletal anatomy in an animal model that is valuable for understanding human craniofacial disorders such as cleft palate. The resource will provide for understanding normal morphological developmental in zebrafish as well as being a reference for diagnosing the nature of craniofacial mutant phenotypes.

Safe, effective therapies generally target specific disease-related molecules that appear only in disease-related cell types. The problem: Gaps in our knowledge include a comprehensive definition of cell types in any vertebrate species over developmental time and knowledge of which genes each cell type expresses at what levels. Genes currently known only by sequence might provide unique targets for cell therapies if we knew which cell types express them. A way forward: It has recently become possible to identify the transcriptional profile of individual, single cells with unprecedented molecular precision using single-cell RNA sequencing (scRNA-seq) coupled with powerful highly dimensional-reducing software that groups cells into bioinformatically identified clusters containing cell types with closely related gene expression profiles. The goals of this project are first, the comprehensive identification of transcriptionally unique cell types over developmental time in zebrafish, a major medical model, and second, the release of these data as a resource to the research community in a convenient searchable format through the Zebrafish Information Network (ZFIN). Approach: Aim 1 is to define single cell transcriptome phenotypes for various stages of wild-type zebrafish embryos, larvae, and juveniles and to locate these annotated cell types by in situ hybridization experiments displaying the expression of cell type-specific marker genes on whole mounts and histological sections. Aim 2 is to define the single cell transcriptome phenotype for all major organs in wild-type zebrafish adult males and females and to identify prominent cell types in vivo by in situ hybridization for cell type-specific marker genes on histological sections. Aim 3 is to develop an automated bioinformatic pipeline to identify cell types in scRNA-seq clusters by comparing gene expression profiles to existing resources, including ZFIN, other model organism databases (AGR, Alliance of Genome Resources), and human gene expression data. Aim 4 is to develop an interface in ZFIN to enable the research community to easily query zebrafish scRNA- seq data. Innovation: No animal species currently has a comprehensive compendium of cell types organized by gene expression patterns on a genome-wide scale during development. Significance: This R24 application will develop resources and related materials that will 1) enhance, further characterize, and improve a critical animal model for the investigation of human disease mechanisms; 2) facilitate access to data generated from the use of animal models of human disease; and 3) address the research interests of many categorical NIH Institutes and Centers that focus on various organ systems and disease types. This resource will identify previously unknown cell types, thus facilitating the precision targeting of cell types for potential therapies; will associate previously unknown genes with specific cell types, thus increasing potential molecular targets for drug therapies; and will suggest hypotheses for gene expression networks, thus improving our knowledge of cellular mechanisms in health and deepening our understanding of gene interaction webs in disease etiology.