Monte Westerfield, Ph.D. - US grants

A central problem of neurobiology is to understand how specific connections are formed during development of the nervous system. Interactions between neurons and their targets and between presynaptic neurons competing for targets are known to be important for the establishment of proper synaptic connections, although the cellular details of these interactions are not well understood. The proposed experiments will provide a major advance in our understanding of these processes by characterizing cellular interactions between individual living motoneurons as they establish neuromuscular junctions during embryonic development. A system that we have recently developed will be used to observe living motoneurons in vivo, as their axons grow and make synaptic contacts with muscle fibers. Individual identified motoneurons in live zebrafish embryos will be labeled with fluorescent dyes and the growth of their axons will be monitored with computer enhanced video microscopy in these rapidly developing, optically clear embryos. The growth of particular motoneurons in a number of embryos will be characterized to learn if their axonal branching patterns are stereotyped and stable. These results will, for the first time, provide a detailed description of the extension and retraction of axonal branches and synaptic terminals by identified vertebrate neurons during the initial establishment of their axonal arbors. Then, the growth of motoneurons that innervate adjacent regions of muscle will be compared directly by labeling them with different colored dyes to see how individual axonal branches from different neurons interact. Finally, one or more motoneurons will be ablated with a laser and subsequent growth of the remaining motoneuron(s) will be monitored to see how competitive interactions affect the size and shape of a neuron's axonal arbor and the distribution of its synapses.

minority institution research support; secondary schools;

We propose to establish a DNA analysis facility with the purchase of several key instruments for the automated analysis of DNA molecules. The instrumentation requested is commercially available and represents the core of many genome and DNA sequencing centers at other major research universities. This type of facility is essential for research projects that require experiments to be done on a genome-wide scale. There are currently three such research programs at the University of Oregon; all of these absolutely require the establishment of the proposed DNA analysis facility if they are to achieve their stated goals. In addition, the acquisition of the requested instrumentation is likely to have significant ancillary benefits. Many other users of the instruments will be able to complete their projects more efficiently and economically by incorporating the automated methods into their research programs. The establishment of the DNA analysis facility should also greatly aid our recruitment of new faculty with research interests in developmental genetics, molecular neuroscience and molecular evolution. These are all areas in which we plan to hire new faculty in the near future. Specifically, funds are requested for: 1) an Integrated Separation Systems AutoGen 740 that can prepare up to 200 DNA samples of viral DNA, bacterial plasmid DNA, yeast artificial chromosomal (YAC) DNA or genomic DNA in a 24 hour period; 2) a Biomek 2000 robotic workstation and sideloader for robotic manipulations of clones and nucleic acids; and 3) an ABI 377 DNA sequencer and the necessary accessories to perform high throughput automated DNA sequence and microsatellite analysis. In addition, several small equipment items (including Macintosh PowerPC 8100, two GeneAmp 9600 thermocyclers, and two microfuges) are requested to equip the DNA analysis facility. Presently, there is no facility in the State of Oregon that can provide the research support of the proposed DNA analysis facility. Fundin g this proposal will allow the faculty to fully develop recently initiated projects that utilize emerging intellectual and technical advances in the genetic analysis of complex (quantitative) characters, and that take advantage of map-based cloning strategies to isolate and characterize genes of major effect. These projects all aim to elucidate the genetic and molecular basis of fundamental biological processes, and encompass a broad spectrum of biology, from evolution and population genetics, to developmental genetics, to behavioral genetics. For example, the plant genetics project that will supported by these instruments will identify genes within the plant genome that control the evolution of different strategies of plant reproduction within a single species in which individuals have been geographically isolated for many generations. The dog project uses the same experimental logic being applied in the plant project, but in this case, the plan is to take advantage of the striking morphological and behavioral differences between dog breeds to gain insight into the genetic and molecular basis for these differences. The zebrafish research efforts are concentrated on identifying genes important for embryonic development of the organism, including genes required for the proper development and functioning of the nervous system. An important approach which is being pursued by these investigators is to identify new mutations that effect developmental processes in question. The ability to place these new mutations onto a high quality genetic map will facilitate the cloning of the genes and eventual molecular understanding of how these genes function. It should be noted that many of the projects described in this proposal are collaborative in nature. This reflects the efforts of our faculty to combine our individual expertise in order to advance our fields of interest more rapidly. Establishing the DNA analysis facility with the acquisition of the requested instrumentati on will represent a major advance in these collaborative efforts, and will provide the infrastructure necessary for the success of our research programs.

9410460 Westerfield This three-year award supports U.S.-U.K. cooperative research in neurobiology between two research groups at the University of Oregon and University of London. The U.S. investigators are Monte Westerfield and Judith Eisen and the British investigators are Nigel Holder and Stephen Wilson. The cooperative effort extends recent research by both groups in anterior central nervous system of zebra fish embryos. Newly discovered genes, have been identified either by cloning or mutation, as candidates for regulating developmental processes in the central nervous system of zebra fish embryos. The objective of this new effort is to study the potential roles of these genes in the anterior central nervous system and brain segments of zebra fish. Zebra fish are ideal vertebrates for studying embryonic and genetic analyses since a large number of their regulatory genes have already been identified. The results from this project, when compared with complementary studies in mammals, will advance our knowledge about the evolution of the anterior regions of the vertebrate central nervous system in general, the genetic mechanisms that regulate patterning, and gene expression that demarcate brain subdivisions. The project takes advantage of complementary expertise of the U.S. and British investigators and provides access to mutant zebra fish and genetic sequencing techniques developed by both groups.

This grant supports the planning and initial development of a database for neurons of the zebrafish nervous system. Neurons are the integrative units of nervous systems. A common goal in many areas of neuroscience research is to understand nervous system organization and function in terms of the properties of individual neurons. The zebrafish nervous system contains large numbers of identifiable neurons, some of which have been characterized in terms of their detailed anatomical, physiological, developmental and genetic properties. The objectives of this grant are: to develop a prototype database system for neuronal, genetic and developmental zebrafish data which will provide meaningful ways of relating various neuronal properties (and thus help understand neuron functions) and to make that database publicly available over the Internet.

9420796 Weeks Hormones often exert dramatic effects on animal behavior, yet the mechanisms by which they act on the nervous system to alter behavior are not yet well understood, The identification of mechanisms for hormone-nervous system interactions is facilitated by studying animals with simple and accessible nervous systems. In this project, a group led by Dr. Weeks will utilize the hawkmoth, Manduca sexta, to study profound neural and behavioral changes in response to developmental alterations in steroid and peptide hormone levels. A specific behavior during the initial phase of metamorphosis in Manduca. pre-ecdysis behavior, is performed to loosen the old cuticle before it is shed at each molt. The performance of this behavior is attenuated at specific times during development. This work will correlate changes in hormones with functional changes in specific identified brain cells. The results of this research will be relevant to understanding neural mechanisms of hormonally-mediated behavioral changes in a wide variety of living organisms. ***

confocal scanning microscopy; image processing; biomedical facility; vertebrate embryology; computer program /software; bioimaging /biomedical imaging; cryostat; scanning electron microscopy; freeze etching; transmission electron microscopy;

embryo /fetus culture; embryo /fetus transplantation

DESCRIPTION (provided by applicant): Skeletal muscles are comprised of two major cell types, slow and fast, that have distinct physiological, biochemical, morphological and developmental properties. The long-term goals of this work are to understand the mechanisms that regulate the formation and patterning of these different muscle types and how these mechanisms are altered in muscular diseases. Previous studies of zebrafish embryos have suggested that Hedgehog (Hh) produced by notochord and floor plate acts locally to specify slow muscle. Even though muscle precursor cells throughout the somite are apparently competent to respond to Hh, only a single monolayer precisely adjacent to the source of Hh, forms slow muscle. The proposed studies will use embryological and genetic methods to test a new hypothesis that accounts for this remarkably precise patterning: Two mechanisms limit the inductive action of Hh to immediately adjacent cells. First, notochord induces a subset of muscle precursors, in an Hh independent manner, to form adaxial cells, an epithelial monolayer that blankets the notochord. Then, in response to Hh signaling, cells in this monolayer express high levels of Patched and Hedgehog interacting protein that bind Hh at high affinity and limit its action. Thus, adaxial cells adjacent to the notochord receive high levels of Hh that induces them to become slow muscle, while at the same time, they reduce Hh signaling to more lateral cells that consequently form fast muscle.

DESCRIPTION (provided by applicant): The long-term goal of the proposed studies is to understand how cells are specified to form the inner ear. The inner ear arises from a specialized set of cells, the otic placode that forms at the lateral edge of the neural plate adjacent to the hindbrain. The otic placode, like other cranial placodes, is thought to arise from a common precursor pool called the preplacodal ectoderm. Fgf signals from the early mesendoderm and later from the hindbrain are required to specify the otic placode, however it is currently unknown how cells are allocated to the preplacodal ectoderm or how they respond to the inductive signals that trigger their differentiation into the ear. Previous studies showed that Bmp signaling, in addition to Fgf signaling, is required for specification of the otic placode and that the Fgf and Bmp signaling pathways converge on a set of transcription factors, notably Dlx3b, Sox9a, and Foxi1, that mediate induction and differentiation of the otic primordium. Mutations or haploinsufficiency of FOXI1 or SOX9 in humans can lead to abnormal inner ear development and conductive hearing loss. The proposed study will test the hypothesis that these factors function as a molecular switch that triggers otic induction, changing the sensitivity of preotic cells from low to high Fgf sensitivity, and that this switch is regulated by a balance between Bmp and Fgf signals. The proposed studies will provide the first comprehensive explanation of the mechanisms underlying otic induction. 1. The proposed studies will test whether Bmp and Foxi1 form a positive regulatory loop to maintain Dlx3b expression in cells that form the preplacodal ectoderm. They will examine whether altering Bmp signaling or Foxi1 activity alters Dlx3b expression and subsequent preplacodal ectoderm formation. These experiments will elucidate the mechanism that underlies initial patterning of the preplacodal ectoderm. 2. The proposed studies will test whether Foxi1 directs cells to form the preplacodal ectoderm by differentially regulating their responses to Bmp and Fgf signaling. They will examine whether altering Foxi1 activity alters Fgf and/or Bmp receptor expression and downstream pathways. These experiments will explain how the balance between Fgf and Bmp signals is sensed and maintained. 3. The proposed studies will test whether Sox9a and Foxi1 function as a molecular switch that triggers otic induction by altering the activities of Foxi1 and Sox9a and examining the resulting effects on Fgf sensitivity and otic specification as indicated by marker expression and placode and vesicle formation. These experiments will elucidate the central mechanism of otic induction. The proposed studies of foxi1 and sox9a have direct relevance to human health. Enlarged vestibular aqueduct (EVA), the most common form of inner ear abnormality in humans, can be caused by digenic inheritance of a heterozygous mutation in the SLC26A4 gene and a heterozygous mutation in the FOXI1 gene. EVA is associated with fluctuating and sometimes progressive sensorineural hearing loss and disequilibrium. Haploinsufficiency in SOX9 leads to campomelic dysplasia with complications that include conductive hearing loss.

Previous studies implicate members of the Hedgehog (Hh) and Nodal signaling pathways in specification of the anterior central nervous system (CNS). The proposed studies are based on a specific hypothesis that postulates that Nodal and Hh signaling systems interact in a network. Experiments will test this hypothesis by analyzing loss and gain of function of components in both the Nodal and Hh pathways and by identifying the regulators of these pathways in a forward genetics mutagenesis screen.

DESCRIPTION: (provided by applicant) Discovering the functions of the tens of thousands of genes in the human genome is a required step for understanding human biology and disease. Genetic model organisms, including zebrafish, play a critical role in this discovery process, because genetic analysis can connect gene sequence and function. Model organism databases, like ZFIN, provide tools required to make this connection. Zebrafish has emerged as a premiere model organism because powerful techniques allow efficient generation and recovery of zebrafish mutations affecting genes that regulate developmental patterning, organogenesis, physiology and behavior. Recent advances make it easy to study gene function in transgenic zebrafish and with antisense oligonucleotides. The functions of many of these genes are conserved among vertebrate groups. Thus, analysis of zebrafish mutations provides insights into gene functions in other vertebrates, including humans. The long term goals for ZFIN are a) to be the community database resource for the laboratory use of zebrafish, b) to develop and support integrated zebrafish genetic, genomic and developmental information, c) to maintain the definitive reference data sets of zebrafish research information, d) to link this information extensively to corresponding data in other model organism and human databases, e) to facilitate the use of zebrafish as a model for human biology and f) to serve the needs of the research community. This project will expand curation of zebrafish phenotypes, implement Gene Ontology annotation of ZFIN gene records and incorporate the assembled zebrafish whole genome sequence into ZFIN. This work will provide a powerful means for researchers to associate gene sequence and function, thus facilitating cross-species analyses of genome organization and evolution as well as studies of vertebrate gene function and disease.

OVERALL PROJECT SUMMARY/ABSTRACT Zebrafish is a premiere organism to study vertebrate development, physiology, behavior, genetics and disease. Powerful techniques allow efficient generation and recovery of zebrafish mutations affecting genes that regulate developmental patterning, organogenesis, physiology and behavior. Recent advances make it easy to study gene function in transgenic zebrafish and with antisense oligonucleotides. The functions of many, if not most of these genes are conserved among vertebrate groups. Thus, analysis of zebrafish mutations provides insights into gene function in other vertebrates, including humans. The Zebrafish International Resource Center has been established as a repository that provides animals, materials and services to the research community. This proposal seeks continued funding 1) to serve as a central repository for zebrafish genetic stocks and research materials, 2) to provide consultation and pathology services, and 3) to develop a platform, based on a panel of PCR assays, to screen for the most prevalent pathogens of laboratory zebrafish. These materials, services and information will be made widely available to the research community.

epizootiology; genetic models; developmental genetics; animal population genetics; genetic registry /resource /referral center; communicable disease transmission; genetic strain; animal colony; biomedical resource; computer system design /evaluation; veterinary medicine; veterinary science; Internet; sperm; genetically modified animals; animal genetic material tag; mutant; information systems; handbook; cryopreservation; animal care; zebrafish;

Discovering the functions of the tens of thousands of genes in the human genome is a required step for understanding human biology and disease. Genetic model organisms, including zebrafish, play a critical role in this discovery process, because genetic analysis can connect gene sequence and function. Model organism databases, like ZFIN, provide tools required to make this connection. The zebrafish has emerged as a premier organism to study vertebrate biology. Powerful techniques allow rapid efficient generation and recovery of mutations affecting genes that orchestrate developmental patterning, organogenesis, physiology, and behavior. It is easy to study gene function by generating transgenic zebrafish, by knocking down gene function with morpholino antisense oligonucleotides, or by altering gene function by genome editing. The genome has been sequenced and about 50% of the protein coding genes have been mutated by targeted gene knockout technology. Large-scale projects are underway or planned that will produce functional data about almost all the genes and sequence-based functional elements in the genome. Multiple mutations and gene knockdowns can be combined in the same individual to study gene modifiers and other genetic interactions. The functions of most of these genes are conserved among vertebrate groups. Thus, analysis of zebrafish mutations provides insights into gene functions in other vertebrates, including humans. The long term goals for ZFIN are a) to be the community database resource for the laboratory use of zebrafish, b) to develop and support integrated zebrafish genetic, genomic, developmental, and physiological information, c) to maintain the definitive reference data sets of zebrafish research information, d) to link this information extensively to corresponding data in other model organism and human databases, e) to facilitate the use of zebrafish as a model for human biology, and f) to help serve the broad needs of the biomedical research community. This project will continue and expand curation of zebrafish research data, develop expanded support for zebrafish models of human disease, expand and integrate links to other databases, and maintain and update the zebrafish reference genome. This work will provide a powerful means for researchers to associate gene sequence and function, thus facilitating studies of human gene function and disease as well as cross-species analyses of genome organization and evolution.

The University of South Dakota Main Campus is awarded a grant to develop tools to integrate evolutionary and model organism phenotype data using ontologies in order to address questions about the genetic and developmental regulation of evolutionary morphological transitions. The project will develop and apply ontologies for taxa and phenotypes in a large clade of fishes (the Ostariophysi) and to develop a suite of database and web-interface tools that will enable researchers to investigate relationships between evolutionary phenotypes and those seen in genetically characterized developmental mutants of zebrafish (a species within Ostariophysi). Combining the wealth of genomic data on the numerous phenotypic mutants for zebrafish, with the span of corresponding anatomical diversity in ostariophysan species will allow researchers to discover previously unrealized connections between evolutionary change, genes, and the developmental processes in which these genes play a role. The team will demonstrate the feasibility of the approach by implementing a select number of use-case queries as a proof-of-concept. The queries will be implemented via a web-based user interface for searching and analyzing the data content. The research will be carried out in collaboration with the National Evolutionary Synthesis Center at the University of North Carolina.

DESCRIPTION (provided by applicant): Conducting experiments on model organisms is fundamental to biomedical research. Three of the most important are budding yeast (fundamental studies), rat (pharmacological, behavioral and neurological studies) and zebrafish (developmental, neurological and toxicological studies). Databases to capture and curate the wealth of data on these model organisms have been established and are known collectively as Model Organism Databases (MODs). Modern biology has resulted in the complete DNA sequence (`genome') of the human as well as these model organisms. In turn this has led to a new era of research in which experiments are carried out at the whole genome scale. The success of genomics has fuelled a challenge to integrate genomic datasets within the MODs in such a way that querying them and extracting data in a flexible fashion is possible for all scientists;not just specialists known as bioinformaticians, although it is also important to provide bioinformaticians with powerful tools. As part of previous work in support of another model organism, the fruitfly, InterMine software was developed to greatly increase the power and flexibility with which scientists can utilize genomic data. InterMine was designed to be applied easily to other areas of biology and organisms. Indeed it is currently being used to manage data from the NIH-funded modENCODE project which is experimentally characterizing the entire genomes of the fruitfly and nematode model organisms. The aim of this project is to apply the InterMine software to three MODs: budding yeast, rat and zebrafish. This provides a number of advantages to each database: functionality that their user communities demand but that are not yet available;a standard interface and set of functionality between MODs;an opportunity for the different MODs to inter-operate providing a tool to compare and contrast the behavior of genes and proteins between this set of organisms, a feature that is not generally available today. This project will be carried out as a collaboration between the team that developed InterMine, based in Cambridge UK, and the teams that develop and maintain the three MODs, based at Stanford University (yeast, SGD), the Medical College of Wisconsin (rat, RGD) and the University of Oregon (zebrafish, ZFIN). This proposal provides one staff member per site, and the resulting team will work together to transfer data into, and add analysis tools to, InterMine databases that will be integrated at each MOD site and within their user interfaces. A benefit of working together in this way is that developments at one site can immediately benefit the others. By the end of the project the MODs will be able to provide far greater functionality to their research communities, and improvements to the underpinning InterMine software will be freely available to the broader community. The proposed project is unique in its integration of experimental results across the major model organisms. This integration is essential for our advanced understanding of molecular genetics, cell biology, developmental biology, physiology, and most importantly, human health and disease. PUBLIC HEALTH RELEVANCE: The recent decoding of the human genome sequence has unprecedented implications for the future of human healthcare through improved understanding of human development, functioning, aging and disease. However, much of the experimental work that has to be done to fully understand these events cannot be done in humans and must therefore be carried out in so-called model organisms. The proposed project will address a pressing need to improve the efficiency with which the huge amounts of Model Organism data being generated can be integrated, analysed and compared, which will lead to improved understanding of humans and thus to better disease diagnosis, prognosis, prevention and cure.

DESCRIPTION (provided by applicant): Human Usher syndrome, the most frequent cause of deaf blindness, is characterized by congenital deafness, due to loss of sensory hair cells, and progressive retinal degeneration, due to retinitis pigmentosa. Twelve different chromosomal loci have been linked to Usher syndrome and nine of the genes have been identified to date. Identification of the missing Usher genes is crucial for diagnosis and patient counseling. The nine known genes encode a surprisingly broad range of different types of proteins. Although the roles of these proteins are poorly understood, recent studies suggest that they function together in a multimolecular complex. This project focuses on analysis of the two scaffold proteins that play a central role in organizing the complex, and a new potential member of this organizing scaffold. The project has three main aims: 1) to determine whether the newly discovered gene encodes an Usher scaffold protein, 2) to analyze the functions of the scaffold proteins in organizing the Usher proteins into a complex, and 3) to determine how the Usher protein complex functions in cells of the inner ear and retina. PUBLIC HEALTH RELEVANCE: Usher syndrome, the leading cause of deaf blindness, is a genetically heritable disorder that affects tens of thousands of Americans. Deafness in Usher syndrome is due to loss of inner ear sensory hair cells and can range from moderate to profound. Blindness is due to retinitis pigmentosa. Mutations in any one of at least a dozen different genes can cause Usher syndrome, only nine of the genes have been identified. Recent research suggests that the various proteins encoded by the Usher genes act together in a multimolecular complex, although the processes that lead to loss of inner ear and retinal cells when the complex is defective are unknown. This project will elucidate how and where the complex functions and the mechanisms by which it assembles. The project will also identify new members of the Usher gene family. This information is important for diagnosis, genetic counseling, and design of therapies for Usher patients.

DESCRIPTION (provided by applicant): Conducting experiments on model organisms is fundamental to biomedical research and underpins research that leads to healthcare advances. Five of the most important models are Mouse (developmental and behavioural studies), nematode (developmental and parasitological studies), budding yeast (fundamental molecular studies), rat (pharmacological, behavioural and neurological studies) and zebrafish (developmental, neurological and toxicological studies). Model Organism Databases (MODs), which capture and curate the wealth of data on these model organisms, have been established. Modern biology has resulted in the complete DNA sequence (genome) of the human as well as these model organisms. In turn this has led to a new era of research in which experiments are carried out at the whole genome scale. The success of genomics has fuelled a challenge to integrate genomic datasets within the MODs in such a way that querying them and extracting data in a flexible fashion is possible for all scientists as well as specialist bioinformaticians. As part of previous work in support of another model organism, the fruitfly, and more recently to manage the data from the multi-institutional NIH-funded modENCODE project, InterMine software was developed to greatly increase the power and flexibility with which scientists can utilize genomic data. InterMine was designed to be applied easily to other areas of biology and organisms. The aim of this project is to apply the InterMine software to the above five MODs: mouse, nematode, budding yeast, rat and zebrafish. This provides a number of advantages to each database: user-community driven functionalities that are not yet available; a standard interface common to all MODs; greater inter-operation between MODs to provide a common set of tools to compare and contrast the properties of genes and proteins within this set of organisms, a feature that is not generally available today. This project will be carried out as a collaboration between the team that developed InterMine, based in Cambridge UK, and the teams that develop and maintain the five MODs, based at the Jackson Laboratory (mouse, MGI), the Ontario Institute for Cancer Research (nematode, WormBase), Stanford University (yeast, SGD), the Medical College of Wisconsin (rat, RGD) and the University of Oregon (zebrafish, ZFIN). This proposal provides one staff member per site, and the resulting team will work together to transfer data into, and add analysis tools to, InterMine databases that will be integrated at each MOD site. A benefit of working together in this way is that developments at one site can immediately benefit the others. By the end of the project the MODs will be able to provide far greater functionality to their research communities, and improvements to the underpinning InterMine software will be freely available to the broader biological database community. The proposed project is unique in its integration of experimental results across the major model organisms. This integration is essential for our advanced understanding of molecular genetics, cell biology, developmental biology, physiology, and most importantly, human health and disease.

DESCRIPTION (provided by applicant): Human Usher syndrome, the most frequent cause of deaf blindness, is characterized by congenital deafness, due to loss of sensory hair cells, and progressive retinal degeneration, due to retinitis pigmentosa. Although nine of the genes responsible for Usher syndrome and one genetic modifier gene have been identified to date, we still lack an understanding of the normal functions of these genes or what goes wrong in the disease. This is primarily because the ten known genes encode a surprisingly broad range of different types of proteins, all with multiple isoforms, and we currently lack animal models and tools to study their functions. Due to recent advances in zebrafish genetic technology, it is now possible to isolate mutations in any gene and easily produce transgenic animals with altered gene expression. This project will develop libraries of mutants, transgenics, and antibodies to characterize all the genes known to contribute to Usher syndrome. Together, these libraries will provide the first resource ever generated to dissect the distinct functions of the complete set of known genes that underlie a human disease.

Collaborative grants are awarded to the University of South Dakota and the University of North Carolina to develop ontology-driven tools for machine reasoning over large volumes of phenotype data. Human-readable descriptions of "phenotypic" properties such as anatomy and behavior are not well-suited to computational analysis. Yet, in evolutionary biology, genetics and development, computational assistance is necessary to discover patterns within the enormous volumes of descriptive phenotype data that are being reported in the literature and in online databases. Ontologies are structured, controlled vocabularies that can be applied to collections of descriptive data to permit logical reasoning to be used. Using the evolutionary transition from fins to limbs as a test system, this project will develop ontologically-aware software that allows users to discover similar sets of phenotypes for different taxa or mutant genes within large and diverse datasets. A fast semantic similarity engine will be developed to allow searches for evolutionary transitions and mutant genes characterized by similar phenotypic profiles. An ontological framework for reasoning over homology will be developed to allow rigorous reasoning over evolutionary diverse lineages. Natural language processing tools will be developed to improve upon the efficiency of mining phenotype data from the literature and improving data consistency. This suite of tools will be tested on a large number of skeletal phenotypes from diverse fossil and modern vertebrates. Taxonomic and anatomical ontologies for vertebrates will be augmented and hypotheses of anatomical homology formally encoded. The ontologies and software tools, together with phenotypes extracted from the vertebrate systematic literature, will be integrated in the knowledgebase with genetic and phenotype data from three vertebrate model organisms: zebrafish (Danio rerio), African clawed frog (Xenopus laevis), and mouse (Mus musculus). The knowledge base will be exposed to generic reasoners using semantic web standards. The system will be validated by its success in retrieving candidate genes for the well-studied vertebrate fin-limb transition and other major events in skeletal evolution.

The evolutionary breadth of the test data requires the development of a rigorous framework for reasoning over hypotheses of homology. Another goal is to develop and evaluate natural language processing tools for efficiently capturing ontological descriptions of phenotype from the descriptions available in the published literature. The suite of tools will be validated by recovering developmental genetic pathways that underlie the evolutionary transition from fin to limb in vertebrates, and refined by iterative testing with domain bioinformaticians on the project and biologists from the broader user community.


A broad community of users will participate through the lifecycle of this project in the development of community standards and resources for the interoperability and computability of phenotypic knowledge. This will be achieved through workshops, usability testing sessions, and coordination with key research networks. Stakeholder ownership will be enhanced by rapid and open release of a variety of products that we anticipate to be of immediate and enduring value to the greater biology community, including tools for streamlining data curation and performing large-scale semantic similarity searches, high quality vertebrate taxonomy and anatomy ontologies, and standards for reasoning over homology. We will provide a unique training environment for students, postdocs and summer interns, including Native Americans through outreach at the University of South Dakota and minority and female students though a collaboration with Project Exploration at the University of Chicago. Project progress and outcomes will be disseminated through both traditional and online outlets for scholarly communication (including blog posts and mailing lists); the primary web presence will be at https://www.phenoscape.org/wiki/.

? DESCRIPTION (provided by applicant): This project will conduct functional studies in collaboration with the NIH Intramural Undiagnosed Diseases Program (NIH-UDP) to investigate the underlying genetics, biochemistry, cell biology, and pathophysiology of a novel disease that has been linked to a variant of the YPEL3 gene identified through the NIH-UDP.

PROJECT SUMMARY The proposed Phase II continuation of the Model Organism Screening Center (MOSC) for the Undiagnosed Diseases Network (UDN) builds on extensive tools, reagents, and pipelines that we established in Phase I. The leadership team represents international expertise in Drosophila, zebrafish, and human medical genetics. In Phase I, the MOSC developed an interface in the UDN Gateway that supports submission of cases, genes, and variants that the clinical sites propose for model organism studies. The MOSC also developed MARRVEL (www.MARRVEL.org), an online tool that integrates human and model organism data and helps to prioritize variants. The MOSC further established collaborations with independently funded collections of rare disease cohorts at Baylor College of Medicine (BCM) that we use to identify matches to UDN phenotypes and genetic variants. The MOSC holds regular conference calls with clinical sites and provides tools that help them to select variants. The MOSC will continue calls with the sites to review informatic analyses and then assign variants (estimated at 45 per year) for in-depth model organism studies of variants and genes. In the Drosophila Core at BCM, approximately 30 genes and variants per year are studied with innovative technology in flies. For 15 additional genes/variants that affect vertebrate-specific genes or biology, the Zebrafish Core at the University of Oregon will induce new mutations in zebrafish with CRISPR/Cas9 followed by high throughput phenotypic analyses. The MOSC will also develop tools and reagents for future variant studies in Drosophila and zebrafish for an additional 100 genes per year. We will share these additional resources with the broader research community through an innovative ModelMatcher service and will also prioritize genes for generation of mouse knock-outs in collaboration with the Knockout Mouse Phenotyping Project (KOMP). Thus, the proposed center will provide informatics selection, human genetics expertise, broad versatile model organism resources, and in-depth studies of UDN cases to aid in diagnosis. The MOSC employs the most innovative technologies in human genomics and Drosophila and zebrafish genetics, based on extensive previous experience modeling human disease.

PROJECT SUMMARY/ABSTRACT Management, Dissemination and Training Core Discovering the functions of the tens of thousands of genes in the human genome is a required step for understanding human biology and disease. Genetic model organisms, including zebrafish, play a critical role in this discovery process, because genetic analysis can connect gene sequence and function. Model organism databases, like ZFIN, provide tools required to make this connection. The zebrafish has emerged as a premier organism to study vertebrate biology. Powerful techniques allow rapid efficient generation and recovery of mutations affecting genes that orchestrate developmental patterning, organogenesis, physiology, and behavior. It is easy to study gene function by generating transgenic zebrafish, by knocking down gene function with morpholino antisense oligonucleotides, or by altering gene function by genome editing. The genome has been sequenced and about 50% of the protein coding genes have been mutated by targeted gene knockout technology. Large-scale projects are underway or planned that will produce functional data about almost all the genes and sequence-based functional elements in the genome. Multiple mutations and gene knockdowns can be combined in the same individual to study gene modifiers and other genetic interactions. The functions of most of these genes are conserved among vertebrate groups. Thus, analysis of zebrafish mutations provides insights into gene functions in other vertebrates, including humans. The long term goals for ZFIN are a) to be the community database resource for the laboratory use of zebrafish, b) to develop and support integrated zebrafish genetic, genomic, developmental, and physiological information, c) to maintain the definitive reference data sets of zebrafish research information, d) to link this information extensively to corresponding data in other model organism and human databases, e) to facilitate the use of zebrafish as a model for human biology, and f) to help serve the broad needs of the biomedical research community. This core will provide support for public access, dissemination, training, and outreach. We will maintain our administrative structure for management of ZFIN, including our Scientific Advisory Board. We will provide guidelines and tutorials for using ZFIN. We will provide general information about research activities, meetings, etc., and we will maintain a research community wiki. We will continue to develop user surveys, employ user-centered design, and provide training sessions at conferences.

? DESCRIPTION (provided by applicant): Discovering the functions of the tens of thousands of genes in the human genome is a required step for understanding human biology and disease. Genetic model organisms, including zebrafish, play a critical role in this discovery process, because genetic analysis can connect gene sequence and function. Model organism databases, like ZFIN, provide tools required to make this connection. The zebrafish has emerged as a premier organism to study vertebrate biology. Powerful techniques allow rapid efficient generation and recovery of mutations affecting genes that orchestrate developmental patterning, organogenesis, physiology, and behavior. It is easy to study gene function by generating transgenic zebrafish, by knocking down gene function with morpholino antisense oligonucleotides, or by altering gene function by genome editing. The genome has been sequenced and about 50% of the protein coding genes have been mutated by targeted gene knockout technology. Large-scale projects are underway or planned that will produce functional data about almost all the genes and sequence-based functional elements in the genome. Multiple mutations and gene knockdowns can be combined in the same individual to study gene modifiers and other genetic interactions. The functions of most of these genes are conserved among vertebrate groups. Thus, analysis of zebrafish mutations provides insights into gene functions in other vertebrates, including humans. The long term goals for ZFIN are a) to be the community database resource for the laboratory use of zebrafish, b) to develop and support integrated zebrafish genetic, genomic, developmental, and physiological information, c) to maintain the definitive reference data sets of zebrafish research information, d) to link this information extensively to corresponding data in other model organism and human databases, e) to facilitate the use of zebrafish as a model for human biology, and f) to help serve the broad needs of the biomedical research community. This project will continue and expand curation of zebrafish research data, develop expanded support for zebrafish models of human disease, expand and integrate links to other databases, and maintain and update the zebrafish reference genome. This work will provide a powerful means for researchers to associate gene sequence and function, thus facilitating studies of human gene function and disease as well as cross-species analyses of genome organization and evolution.

APPLIED RESEARCH ABSTRACT The Zebrafish International Resource Center has been established as a repository that provides animals, materials and services to the research community. The Applied Research Core will develop a platform, based on a panel of PCR assays, to screen for the most prevalent pathogens of laboratory zebrafish.

RESOURCE ABSTRACT The Zebrafish International Resource Center has been established as a repository that provides animals, materials and services to the research community. The Resource Core will 1) serve as a central repository for zebrafish genetic stocks and research materials and 2) provide consultation and pathology services.

Abstract We will expand and modernize the Zebrafish International Resource Center (ZIRC) to 1) improve the efficiency of acquisition, maintenance, and distribution of NIH funded resources, 2) promote biosafety, animal welfare, and work safety of animal care staff, and 3) expand the cryopreservation repository and laboratory spaces. ZIRC serves biomedical research as the central repository for wild-type, transgenic, and mutant zebrafish lines, other research materials, and diagnostic health services. ZIRC has outgrown the space of 500 genetic alleles designed for its program 20 years ago due to scientific advancements that vastly increased fish line generation and boosted the number of alleles at ZIRC to 44,937. This growth significantly impacted ZIRC?s quarantine procedures, biosecurity, management of live and cryopreserved lines, and molecular genotyping. Current operations, including animal support, are severely limited by space constraints and a floorplan that restricts workflow and operational support. We propose to improve animal life support, wash room equipment, quarantine, and laboratory areas. 1) We will expand the aquaculture room, modernize the water systems, and add support space. The new systems will be more biosecure, efficient, responsive to changes in water demand, and more accessible for maintenance and repair. 2) To enhance our efficiency and biosecurity, we will build a new wash room with a walk-in washer for parallel processing of equipment. 3) We will add a second quarantine space to enable faster, simultaneous processing of fish imports from facilities with varying biosecurity levels. 4) We will add a new information technology office and expand the cryogenic freezer and laboratory spaces. A new hallway will bypass fish housing for better biosecurity, improved workflow, and connection to the new building additions. 5) We will add a dedicated husbandry research space to expand ZIRC?s research capacity and flexibility. Additional stand-alone fish tank racks and undercounter washers in the fish housing and husbandry research spaces will separate fish with varying health status. In the new research and quarantine spaces, we will install combined flow-through and recirculating water systems to maximize operational flexibility. The proposed improvements will enhance research, personnel safety, operational flexibility, and genotyping capacity. Construction and fixed equipment will modernize the ZIRC?s infrastructure and improve efficiency, thereby limiting costs to researchers and enhancing ZIRC?s services to the biomedical research community.

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.