DEVELOPMENTAL DYNAMICS 228:490 – 496, 2003
REVIEWS–A PEER REVIEWED FORUM
Genetic and Genomic Tools for Zebrafish Research: The NIH Zebrafish Initiative Rebekah S. Rasooly,1* Deborah Henken,2 Nancy Freeman,3 Laurie Tompkins,4 David Badman,1 Josephine Briggs,1 A. Tyl Hewitt,2 and The National Institutes of Health Trans-NIH Zebrafish Coordinating Committee
The National Institutes of Health (NIH) has been a leading advocate of the zebrafish as a model organism for the study of vertebrate development, physiology, and disease. Genomic tools, developed with the support of NIH funding, have made zebrafish even more attractive as a genetic system and have stimulated research using this model. The NIH continues to provide support for new and existing community resources, such as the Zebrafish International Resource Center and the Zebrafish Information Network, research tool development and mutant screens, and a wide array of investigator-initiated studies that are using zebrafish to elucidate many aspects of vertebrate biology. Developmental Dynamics 228:490 – 496, 2003. Published 2003 Wiley-Liss, Inc.† Key words: zebrafish; Danio rerio; research resources; ESTs; cDNA; genomics; ZFIN; NIH Received 15 May 2003; Accepted 11 June 2003
NIH SUPPORT FOR ZEBRAFISH RESEARCH: A BRIEF HISTORY The use of zebrafish, Danio rerio, as a model of vertebrate development and disease has blossomed over the past 15 years, primarily because of its value in both experimental and genetic analyses (Fig. 1). The popularity of this model system has led the National Institutes of Health (NIH) to advocate for the resources needed to develop further the zebrafish system for genetic studies of vertebrate embryogenesis and disease. Genomic tools, developed with the support of NIH funding, have made zebrafish even more attractive as a genetic system and have stimulated research using this model (Fig. 2).
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In addition to supporting an active cadre of zebrafish researchers in its own laboratories through the NIH intramural program, the NIH also supports zebrafish investigators at other institutions through the NIH extramural program. Since its establishment in 1997, the Trans Zebrafish Coordinating Committee (TZCC), which discusses issues relating to extramural research, has played a major role in promoting the use of zebrafish and ensuring the efficient integration of NIH activities to provide critically needed tools for the zebrafish community. Composed of representatives from 18 NIH Institutes and Centers (ICs), the TZCC considers and prioritizes new recommenda-
tions from the community, initiates and coordinates the development of major initiatives, and oversees these activities once they are funded. By mobilizing the resources of many ICs, the TZCC is able to support large zebrafish projects that would be beyond the scope of any single IC. The TZCC also serves another valuable function: it ensures that all resources derived from federal funds are made available to the entire community. The zebrafish community continues to have a well-organized internal leadership base that is a vocal advocate for its members. It is in large part due to the efforts of this group of leaders that the TZCC was
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 4 National Institute of General Medical Sciences, National Institutes of Health, Bethesda, Maryland *Correspondence to: Rebekah S. Rasooly, Ph.D., Division of Kidney, Urologic, and Hematologic Diseases, National Institute of Diabetes and Digestive and Kidney Diseases/NIH, Two Democracy Plaza, 6707 Democracy Blvd., MSC 5458, Bethesda, MD 20892-5458. E-mail:
[email protected] 2 3
DOI 10.1002/dvdy.10366
Published 2003 Wiley-Liss, Inc. †This article is a US Government work and, as such, is in the public domain in the United States of America.
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formed. In February 1997, members of the zebrafish community led by Len Zon attended an NIH-supported workshop, Current Advances in Defining the Zebrafish Genome, to assess the state of the science of the zebrafish model genome system. In the weeks after the workshop, NIH staff worked with the organizers to formulate plans for implementing
the community’s recommendations. The workshop report containing the recommendations was coauthored by Nancy Hopkins, Len Zon, and John Postlethwait. It was presented to Harold Varmus, then NIH Director, who gave formal recognition to the TZCC later that year. One set of recommendations in that report sparked a Request for Ap-
Fig. 1. Zebrafish Research Projects Funded by the NIH, 1992–2002. During this period, the number of projects supported at institutions outside the NIH dramatically increased. In addition, initiatives developed by the Trans Zebrafish Coordinating Committee (TZCC) have resulted in a marked infusion of funds ($10.24 million in fiscal year 1997, before TZCC; compared with $42.40 million in fiscal year 2001) for zebrafish research and resources.
Fig. 2. Zebrafish by the numbers.
plications, or RFA (a one-time solicitation with a specific funding commitment), on “Genomic Resources for the Zebrafish.” Five zebrafish genomics projects designed to generate several types of genomic maps were funded through this RFA. The five projects were initiated in 1998 with cooperative funding from 13 Institutes. A sixth project was later added. They were overseen by the TZCC, with the advice of an External Zebrafish Advisory Panel, and were completed in 2002. Also in response to the 1997 zebrafish workshop report, the NIH published a Program Announcement (PA), an ongoing solicitation without a specific amount of committed funds, which highlighted the goals and interests of the participating NIH ICs in using the zebrafish as a model for development and disease research. The objective of this PA was to promote the use of the zebrafish animal model for new and innovative research and approaches that would identify genes and elucidate the molecular and genetic mechanisms responsible for normal and defective development and disease processes. This PA, which has been reissued, is still active (Fig. 3). In February 1999, a large workshop on nonmammalian models, includ-
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Fig. 3.
ing zebrafish, was held at the NIH. The following May, the TZCC sponsored a 2-day community workshop, Genomic and Genetic Tools for the Zebrafish, which was attended by more than 120 researchers. The workshop amplified and prioritized recommendations from a panel of zebrafish investigators chaired by Len Zon and John Postlethwait. The workshop’s report, written by an Executive Committee chaired by Igor Dawid, Nancy Hopkins, and Len Zon, with the assistance of the TZCC, outlined the community’s needs for genetic and genomic tools. Based on the above report, the NIH published an RFA in 2000 entitled “Mutagenesis Screens/Phenotyping Tools for Zebrafish.” The purpose of the RFA was to encourage research that exploits the power of zebrafish mutagenesis screening to identify and characterize genes, pathways, and phenotypes associated with development, organ formation, behavior, and disease processes. Applications proposing to advance the technologies associated with such phenotyping were also welcome. As a result of this solicitation, the participating ICs made 14 awards. In 2001, this initiative was republished as a PA, entitled “Development of Zebrafish Mutagenesis and Screening Tools,” and it remains active as a
Useful NIH web sites.
PAR, an ongoing solicitation with no specifically committed funds but with a special review panel (Fig. 3). In addition to funding the special initiative, ICs participating in the TZCC are assisting in the support and recent expansion of the Zebrafish International Resource Center (ZIRC) and of the Zebrafish Information Network (ZFIN), the zebrafish model organism database, both located at the University of Oregon and headed by Monte Westerfield (Fig. 3). ● Zebrafish International Resource Center (ZIRC). Overseen by NIH’s National Center for Research Resources, ZIRC is a stock center that maintains zebrafish mutants and distributes them to the scientific community. ZIRC also provides state-of-the-art informational resources by means of the Internet. ● Zebrafish Information Network (ZFIN). Overseen by the National Human Genome Research Institute, ZFIN serves as a community resource for the dissemination of integrated genetic, genomic, and developmental information, as well as the maintenance of definitive data reference sets of zebrafish research information. The expansion of both the ZIRC and the ZFIN was assisted by the important and substantial contribu-
tions and administrative support provided by the State of Oregon and the University of Oregon. Both of these resources will continue to be of great significance to the zebrafish community as new mutants become available and the sequence of the zebrafish genome by the UK’s Sanger Centre nears completion. In April 2002, as the first projects funded by the Genomic Resources for the Zebrafish RFA were nearing completion, the TZCC sponsored a meeting for the zebrafish research community to advise the NIH on priorities for the future. The major recommendation emerging from that meeting was to fund the production of a full-length cDNA collection. Accordingly, in August 2002, the NIH launched the Zebrafish Gene Collection (ZGC), which has a goal of sequencing 8,000 unique zebrafish full-length cDNAs. NIH’s support of and interaction with the TZCC is a reflection of the interest NIH has in promoting biomedical research using zebrafish as a model system. The success of the TZCC is in large part due to the strong, interactive zebrafish community, the open lines of communication between the community and the NIH, and the willingness of community leaders and the NIH to work together to continually re-evaluate
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and prioritize the needs of the community. It is also important for the community to understand the central role of the peer review system in the NIH funding process. As the number of applications using zebrafish continues to increase, it is crucial that members of the community participate in the review process. Active participation on study sections will not only enlighten colleagues who work with other model systems about the value of zebrafish but also ensure that expertise is available for the fair and informative review of zebrafish grant applications.
ZEBRAFISH GENOMICS The five zebrafish genomics projects that were initially funded in response to the RFA issued in 1998 (Genomics Resources for the Zebrafish) and the sixth project, which was added in 1999, exceeded the RFA’s stated goals of developing a high resolution genetic map, creating a physical (Radiation Hybrid) map of the zebrafish genome and generating a large number of Expressed Sequence Tags (ESTs; Fig. 2). Complementary genetic maps were created by research teams led by Mark Fishman and Will Talbot. The Fishman map, based on a panel of F2 animals from an AB ⫻ India cross, contains Simple Sequence Length Polymorphism (SSLP) markers and spans 2,351 cM, with an average marker distance of 0.56 cM. The Talbot map was constructed by using heat-shocked homozygous diploid embryos and contains nearly 4,000 gene, EST, and SSLP markers. The two maps are cross-referenced because many of the same markers have been mapped on both, along with other ESTs and genes provided by the research community. The Talbot map effort also included a comparative genomics component to elucidate syntenic relationships among zebrafish and mammalian genomes. In collaboration with Christiane Nusslein-Volhard and Robert Geisler’s lab, Len Zon and his colleagues have produced a Radiation Hybrid (RH) map by using the Goodfellow T51 panel and the Ekker LN54 panel.
SSLPs have been mapped onto this panel to reduce the number of linkage groups and to integrate the RH and genetic maps. Through the efforts of the Geisler and Zon labs, the RH map now has more than 10,000 markers, including more than 6,000 ESTs and genes. Both labs are also mapping the ends of BAC clones to the RH map to create a physical scaffold for the genomic sequence. Nearly 500 ends have been mapped to date. The functional annotation of the zebrafish genome has been advanced by the work of Steve Johnson’s project, which has deposited more than 200,000 EST sequences into GenBank. These sequences have been assembled into more than 18,000 gene clusters. Primer pairs from the clusters have been produced, which facilitates mapping of the genes onto the RH map. In another project, Marnie Halpern’s group has generated, characterized, and mapped more than 60 gamma-ray–induced deficiencies, including regions on 22 of the 25 linkage groups. Many of the deficiencybearing stocks have been deposited in the ZIRC. The newly launched ZGC of unique, full-length zebrafish cDNAs is another invaluable development for the zebrafish genome sequencing project for the following reasons: (1) the ZGC enumerates genes and improves the accuracy of gene identification; (2) full-length cDNAs are valuable reagents because they facilitate genetic manipulations such as complementation tests or overexpression analysis and allow development of microarrays and other tools for genome-wide analysis; (3) fulllength transcripts provide information about the 5⬘ untranslated or AUG regions of genes, which are necessary for the construction of morpholinos, the primary tool for gene expression “knockdown” in zebrafish. The ZGC approach is to produce a large number of 5⬘ EST sequences from a diversity of cDNA libraries and then use bioinformatic approaches to search among those sequences and existing 5⬘ EST sequences from zebrafish from the Washington University collection for novel full-length
cDNA candidates. Candidates that are likely to be full-length and that are not redundant with existing clones are selected for complete double-stranded sequencing. Run as an ancillary project of the NIH Mammalian Gene Collection, the ZGC is also partnering with a fulllength cDNA project run by the Genome Institute of Singapore to identify and sequence additional clones. All data and reagents produced by the ZGC are publicly available. Data are submitted immediately to GenBank. All clones that are 5⬘ sequenced to produce ESTs are collected by the IMAGE consortium and are commercially available, as are the ZGC-produced libraries and the full-length clones. The latter are catalogued and updated automatically to provide a searchable list of sequenced full-length clones (Fig. 3).
FUTURE PROSPECTS Zebrafish genetic tool development and mutagenesis projects will continue to be supported in the next few years by an active PAR established in 2001. The PAR also is expected to expedite research projects because it allows non– hypothesis-driven project proposals to be critically reviewed together by one special emphasis panel rather than having the proposals spread across several standing study sections that would review them separately. One research obstacle expressed in the 2002 TZCC advisory meeting continues to be a problem for the zebrafish community—the high cost of microarrays. However, it is likely that competition between product lines and increased use by the community will bring down costs. Even so, it may be beneficial for the community to develop purchasing consortia to make these tools more accessible to everyone in the community. Establishing purchasing consortia is just one example of the directions the zebrafish community may need to take in the future. Now that the federal government’s doubling of the NIH budget is complete, the zebrafish community must become even more creative and ingenious in developing and funding beneficial initiatives and in fostering productive interactions
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between the public and private sectors. In part, this process will entail the community taking advantage of NIH’s established mechanisms of support, such as the standing program announcements relating to zebrafish. In addition, the NIH just published a new announcement expanding the use of unsolicited R21 applications (Fig. 3). Several of the ICs that are members of the TZCC are participating in this program, which calls for “high risk/high impact” projects. Investigators should take advantage of this mechanism, particularly for technology development and other potentially high-impact, non– hypothesis-driven projects. As always, investigators are urged to contact program staff at the NIH to discuss their ideas [see TZCC contacts on NIH Zebrafish Model Organism Web site]. NIH funding resources virtually untapped by the zebrafish community are the Small Business Innovation Research (SBIR) Program and the Small
Business Technology Transfer (STTR) Program. Every year, the NIH ICs are obligated by law to use a specified portion of their budgets to support these mechanisms. Although they are primarily small business enterprises, these grants are ideally suited for the development of potentially marketable zebrafish-related tools and reagents. Perhaps some of the community’s more entrepreneurial investigators may decide to establish their own small businesses or to collaborate with already established enterprises. Regardless of the approaches taken, a key element for the advancement of zebrafish research will be the continuing collaboration between the zebrafish community and the TZCC. This collaboration will ensure that the needs of the community will be continually re-evaluated, re-prioritized, and kept at the forefront of discussions on NIH funding discussions. Collaboration has been fruitful in the past, leading to the generation of tools that have been
important for advancing the usefulness of the zebrafish as a genetic model organism. Future collaboration will continue to ensure the availability of resources needed to maximize the benefit derived from the zebrafish genome sequence. As exciting as past advances have been, truly significant findings are yet to come as the zebrafish model system enhances its role in the arena of biomedical research.
ACKNOWLEDGMENTS We thank Ms. Sondra Sheriff, NICHD, for assisting in preparation of the figures and technical support, and Ms. Carolyn Benson, writer/editor, NIDDK, DKUH, for thoughtful editing of this manuscript and design of figures. A list of the members of the National Institutes of Health Trans-NIH Zebrafish Coordinating Committee can be found at www.nih.gov/science/models/zebrafish.
THE ZEBRAFISH MODEL IN NIH-SUPPORTED RESEARCH Several institutes at the National Institutes of Health support zebrafish studies that are directly relevant to their missions. General research categories include developmental processes; specific organs and tissues and their diseases; sensory systems and the brain; aging, immunity, and susceptibility to toxins and carcinogens; and resource development.
Developmental Processes Several institutes support basic research on developmental processes, currently the most intensively studied aspect of zebrafish biology. The National Institute of Child Health and Human Development (NICHD) funds research using the zebrafish as an animal model to clarify the mechanistic causes of human birth defects by identifying the many factors that regulate developmental processes. Current studies funded by NICHD span the early developmental timeline from fertilization through organogenesis, specifically identifying pathways and developmental processes involved in axis specification and patterning of the somites, notochord, and central nervous system. The National Institute for Environmental Health Sciences (NIEHS) is funding investigators to perform zebrafish screens to identify maternally expressed genes that are important in development. Aspects of development being analyzed include germ layer development, embryonic axis formation, anterior–posterior pattern formation, egg activation, fertilization, primordial germ cell development, and migration and morphogenesis of the embryo. The National Institute of General Medical Sciences (NIGMS) has a zebrafish portfolio that encompasses genetic, molecular, and/or biochemical techniques that perturb a process of interest. Some NIGMS-funded investigators study developmental processes such as gastrulation and dorsal–ventral pattern formation, which are conserved throughout the vertebrate lineage. Other investigators are analyzing genes involved in the development and maintenance of the prominent black and white stripes that give zebrafish its name. NIGMS also funds large-scale efforts to identify genes involved in embryonic development and patterning, using mutant screens, microarray analyses, and screens based on morpholinos and other state-of-the-art techniques for manipulating gene expression.
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Specific Organs and Tissues The zebrafish model has proven to be remarkably versatile and relevant to a wide array of NIH-supported studies on various tissues and organs and the diseases that affect them. Research funded by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) uses zebrafish as a model for the study of development, homeostasis, and repair of muscle, bone, cartilage, and skin. Currently supported projects include studies of cell migration in wound healing, specification of muscle cell fates, and the genetics of skeletal development. The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) is especially interested in the potential of zebrafish for analyzing normal and disease physiology in obesity and in many different systems, including the gastrointestinal tract, genitourinary system, pancreas and endocrine systems, and hematologic system. Some current NIDDK-funded studies focus on pancreatic islet cell formation, comparative gene analysis of cystic kidney phenotypes in zebrafish and mouse, identification of phospholipid and cholesterol processing pathways, and characterization of mutations affecting hematologic development. Another institute involved in this research area is the National Institute of Dental and Craniofacial Research (NIDCR). NIDCR funds research that generates zebrafish models to study the genes and pathways that influence all aspects of normal and abnormal development and diseases affecting craniofacial structures. NIDCR is especially interested in fostering the development of imaging and biomimetics approaches and technologies for high-throughput genetic and protein screens. Research on the development and function of the cardiovascular system is the focus of the National Heart, Lung, and Blood Institute (NHLBI), which funds studies that use the powerful mutagenesis screening applications possible in zebrafish. Important mutants such as heartstrings have been identified from screens assessing cardiac function. Zebrafish studies have the potential to provide insight into genes relevant to human heart disease, such as cardiomyopathies, congenital heart disease, and heart regeneration.
Sensory Systems and the Brain As a developmental system, the zebrafish has yielded many insights into the development of the brain and sensory organs. More recently, the adult zebrafish has proved to be an excellent model for studying sensory systems and behavior. The external development and transparency of the zebrafish embryo have facilitated National Eye Institute (NEI) -funded studies of the development of the cornea, the lens, and the normal neuronal patterning of the retina. Zebrafish have been identified that have mutations analogous to those that cause retinal photoreceptor degeneration in human diseases, such as retinitis pigmentosa and age-related retinal degeneration. Other NEI-supported studies focus on imaging the neuronal pathway from the retina and optic nerve to the tectum, work which has broad implications for the study of molecular and cellular events governing central nervous system development and plasticity. Researchers supported by the National Institute on Drug Abuse (NIDA) have found the zebrafish model to be a powerful means for understanding the molecular basis of tolerance, sensitization, and addiction to drugs of abuse. Zebrafish show conditioned preferences to several drugs of abuse and contain neuronal circuitry similar to that underlying addiction in humans. NIDA-funded studies also use zebrafish to investigate developmental and teratologic effects of drugs of abuse on the nervous system and to investigate the normal development of the zebrafish brain regions and neurotransmitter systems that mediate the effects of drugs of abuse. In the auditory and vestibular research arenas supported by the National Institute on Deafness and Other Communication Disorders (NIDCD), the zebrafish is one of the most rapidly growing and research-productive animal models. The vertebrate inner ear is a complex, multiple sensory system composed of utricles, semicircular canals, and maculas. In zebrafish, ectodermal cavitation gives rise to the otic vesicle, from which all the subsequent membranous labyrinth structures arise. Historically, mammalian animal models have posed difficult challenges of auditory/vestibular organ accessibility and the lack of cellular markers. The advantages of zebrafish transparent embryos and the powerful battery of molecular tools, reagents, and mutants have led to the identification of new auditory and vestibular developmental markers and mutants. Other NIDCDsupported research includes the beginnings of zebrafish auditory/vestibular behavioral assays that will provide the critical and necessary means to test and analyze the importance of these new genes on sensory, motor, and central nervous system processes.
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Aging, Immunity, and Susceptibility to Toxins and Carcinogens In the past few years, NIH-funded researchers at three institutes have turned to zebrafish to study several aspects of vertebrate physiology, including aging processes, immunology, and toxicology. The National Institute on Aging (NIA) has just begun to fund research in zebrafish to investigate the genetic and cellular mechanisms of aging. Current studies under way involve characterizing the lifespan and documenting pathology at all stages of life. This project also investigates the hypothesis that heat shock protein’s ability to respond to stress declines with age. A pilot study involving large-scale mutagenesis in zebrafish is being funded by the NIA to address the hypothesis that deletions in mitochondrial DNA, which increase with age in many species, are associated with accumulation of oxidative stress damage with age. The National Institute of Allergy and Infectious Diseases (NIAID) conducts and supports research to understand, treat, and ultimately prevent infectious, immunologic, and allergic diseases. Zebrafish are being used to analyze the development of lymphoid cells and organs from their earliest appearance through adulthood. Recent studies include investigations of host immune responses to tuberculosis infections. These studies have threefold value: they will provide a clearer understanding of the regulation of innate immunity; they may lead to a better understanding of immune regulation to intracellular pathogens; and they may advance our knowledge of the pathogenesis of human tuberculosis. Finally, the National Institute of Environmental Health Sciences (NIEHS) funds studies that use zebrafish as mechanistic models for the action of environmental toxicants and for environmental screening. Environmental factors such as the pesticide chlorpyrifos, dioxin, and carcinogens are being used to identify genes involved in toxicity, bioactivation, and metabolism. The goal of this research is to develop lines in which changes in well-characterized gene expression patterns can be directly related to sensitivity to carcinogens and other toxic components. In addition, transgenic zebrafish have been developed that express yellow and/or green fluorescent protein as a marker of specific pathway activation. These zebrafish are being used both for mechanistic studies as well as for screening for toxicants that activate these pathways.
Zebrafish Resources One of the most important priorities of NIH is to maintain community resources that support the work of all investigators. Since 1998, The National Center for Research Resources (NCRR) funds the Zebrafish International Resource Center (ZIRC), which provides scientists with wild-type and mutant zebrafish stocks, develops and maintains an Internet-accessible computer database, and is establishing standards and procedures for generating and maintaining healthier and more vigorous strains of fish. In addition, NCRR supports studies at the ZIRC and other facilities to broaden the utility of the zebrafish model for cross-cutting biomedical research that is not encompassed within a single NIH Institute or Center.
