Headwaters of the Zebrafish

PERSPECTIVES into grant applications, could serve as a way T. P. Cryopreservation of Musca domestica (Diptera: mutants have provided models of human Muscidae) embryos. Cryobiology 41, 153–166 (2000). 5,6 to collect supplemental funding from the 3. Leopold, R. A., Wang, W. B., Berkebile, D. R. & dysmorphologies , stimulating further researchers to whom stock centres bring the Freeman, T. P. Cryopreservation of embryos of the New efforts to achieve medical insights from the World screworm Cochliomyia hominovorax (Diptera: most direct benefit. Calliphoridae). Ann. Entomol. Soc. Am. 94, 695–701 zebrafish into physiology, behaviour and (2001). 7–9 Networks represent the future of the 4. Glenister, P. H. & Thornton, C. E. Cryoconservation — chemical dependency . Work with the worldwide animal model stock resource archiving for the future. Mamm. Genome 11, 565–571 zebrafish is also yielding insights into the (2000). (BOX 4). We need to work together in larger 5. Balling, R. ENU mutagenesis: analyzing gene function in relationship between single gene changes conglomerates, avoiding duplication of mice. Annu. Rev. Genomics Hum. Genet. 2, 463–492 and structural adaptations that occur dur- (2001). activity, ensuring economies of scale, and 6. Nadeau, J. H. Modifier genes in mice and humans. ing the course of evolution, as well as the cooperating in securing long-term funding Nature Rev. Genet. 2, 165–174 (2001). kinds of change in gene function that 7. Balmain, A. Cancer as a complex genetic trait: tumor platforms. If successful, these collective con- susceptibility in humans and mouse models. Cell 108, accompany the evolution of multigene fam- sciousness-raising efforts will serve as exam- 145–152 (2002). ilies10,11. The significance of the insights 8. Bucan, M. & Abel, T. The mouse: genetics meets ples for the support of other model organ- behaviour. Nature Rev. Genet. 3, 114–123 (2002). anticipated from genetic work with the 9. Paigen, K. & Epping, J. T. A mouse phenome project. isms. An expanded funding of stock centres Mamm. Genome 11, 715–717 (2000). zebrafish has spurred an international pub- will be necessary for the effective exploita- 10. Eppig, J. T. Algorithms for mutant sorting: the need for lic effort to complete the sequencing of its phenotype vocabularies. Mamm. Genome 11, 584–589 tion of the genetic and genomic informa- (2000). genome and its embracement by the tion on the organisms they protect. 11. Nadeau, J. H. et al. Sequence interpretation. Functional biotechnology industry. annotation of mouse genome sequences. Science 291, Nadia Rosenthal is at the Mouse Biology 1251–1255 (2001). The rise in prominence of research with Programme, European Molecular Biology 12. Eppig, J. T. & Strivens, M. Finding a mouse: The the zebrafish has occurred only in the past International Mouse Strain Resource (IMSR). Trends Laboratory, via Ramarini 32, 00016 Genet. 15, 81–82 (1999). decade (TIMELINE). The first international Monterotondo (Rome), Italy. conference that focused on this organism Acknowledgements Michael Ashburner is at the Department of The helpful comments of K. Matthews, S. Brown, N. Jenkins, was convened in 1990 (REF. 12). Sponsored by Genetics, University of Cambridge, Downing J. Eppig and M. Hrabe de Angelis are gratefully acknowledged. the US National Institutes of Health (NIH) Street, Cambridge CB2 3EH, UK. and National Science Foundation (NSF), Online links and hosted by the consortium of ‘Oregon Correspondence to N.R. zebrafish laboratories’,the gathering of ~40 e-mail: [email protected] FURTHER INFORMATION Gene Ontology: http://www.geneontolgy.org scientists from the United States and Europe doi:10.1038/nrg891 Michael Ashburner’s lab: 1. Ashburner, M. Europe must grant crucial funds for http://www.gen.cam.ac.uk/dept/ashburner.html sought to appraise the potential of research biological research. Nature 402, 12 (1999). Nadia Rosenthal’s lab: http://www.embl-monterotondo.it with this organism. Here, we retrace the aus- 2. Wang, W. B., Leopold, R. A., Nelson, D. R. & Freeman, Access to this interactive links box is free online. picious origins of the zebrafish field that were recognized at that meeting, and the subsequent innovations that transformed the zebrafish into a leading model organism. TIMELINE Our perspective is personal and therefore necessarily incomplete. We highlight the sequential contributions of individual scien- Headwaters of the zebrafish — tists and reflect on the shifting cultural views in the scientific community that both pro- emergence of a new model vertebrate pelled and retarded the ascent of this new model system.

David Jonah Grunwald and Judith S. Eisen Fashioning a genetic system The ability to carry out classical forward The understanding of vertebrate The zebrafish, a robust tropical fish that has genetic analyses rendered the zebrafish development has advanced considerably long been a common feature in home unique among vertebrate model organisms in recent years, primarily due to the study aquariums, has recently attained a pre-emi- and still continues to be largely responsible of a few model organisms. The zebrafish, nent position in biomedical research. for its power as a tool for studying vertebrate the newest of these models, has risen to Zebrafish researchers have amassed some- biology. The idea of applying mutational prominence because both genetic and thing that was previously thought to be analysis to study zebrafish embryonic devel- experimental embryological methods can impossible in a vertebrate — a vast store- opment originated with George Streisinger be easily applied to this animal. The house of mutations selected only on the (FIG. 1), who began working with the fish in combination of approaches has proven basis of how they affect the living organism. the late 1960s. Streisinger had been among powerful, yielding insights into the Hundreds of mutations that perturb basic the principal contributors to the dawn of the formation and function of individual developmental processes have been modern era of molecular genetics. Having tissues, organ systems and neural described, including those that affect the trained with Salvatore Luria and Max networks, and into human disease establishment of the shape of the embryo, Delbrück, Streisinger was at the core of the mechanisms. Here, we provide a personal the generation of germ layers, complex historic phage group throughout the 1950s, perspective on the history of zebrafish organ systems and specific cell types, the working at Caltech, Cold Spring Harbor, and research, from the assembly of the first organization of distinct brain regions Cambridge, UK. Streisinger’s phage work genetic and embryological tools through to and vascular architecture, and the establish- showed that the genetic code deduced in sequencing of the genome. ment of defined neural circuits1–4.The vitro coincided with the code in vivo, and

Timeline | Landmarks in zebrafish research Late 1990s to early 2000s Mutations are cloned and several genes that The method for affect common Streisinger producing clonal processes are woven produces haploid lines of homozygous into molecular pathways. Late 1960s embryos from zebrafish is 1993 The Trans-NIH Zebrafish Streisinger eggs activated published. Systematic large-scale Initiative is launched. obtains with ultraviolet- Gynogenetic screens for embryonic- The results of the ‘Big Establishment of a The beginning zebrafish from irradiated, procedures and their lethal mutations begin in Screen’ are published in a centralized, web-based of whole- commercial genetically potential applications Tübingen, Germany, and single issue of database, ZFIN, and a genome suppliers. impotent sperm. are described. Boston, USA. Development, volume 123. stock centre, ZIRC. sequencing

1960 1970 1972 1980 1981 1988 1990 1993 1994 1996 1997 1998 2000

Mid-1970s Mid-1980s The first The first conference on The First Cold Mid-to-late 1990s one-eyed Homozygous diploid Establishment of a research description of zebrafish is convened in Spring Harbor Development of pinhead is the embryos derived only from community focused on an induced Eugene, Oregon. The Conference on linkage map and first mutation the maternal genome are developmental and genetic embryonic-lethal fate map of the zebrafish Zebrafish Genetics genomic to be used to reveal recessive studies with the zebrafish. mutation in the gastrula reveals that and Development resources. positionally mutations present in the Cell-lineage studies in the zebrafish is organization of the early is held. Three Insertional cloned. germ line of a female. early embryo, visualization published. zebrafish embryo is simi- hundred and fifty mutagenesis is Gynogenetic procedures of neurite outgrowth in a lar to that of other verte- international established and are developed for living zebrafish embryo, and brates. Cell transplanta- researchers attend large-scale tion to generate geneti- mapping and gene- mutagenesis and mapping — the zebrafish is screens for cally mosaic embryos is linkage studies. regimes are reported. no longer just a insertional used to test autonomy promising model. mutants begin. of gene function.

no tail is the first mutation to be identified molecularly, using a candidate-gene approach.

yielded insights into the molecular nature of Then, as today, the workings of the nervous ticular target sites”(G. Streisinger, 1974 supple- induced mutations and the genetic structure system and the basis of thinking and behav- mental grant application to the National of bacteriophage T4 (REFS 13–16). It was a iour were regarded as the exceptional, almost Science Foundation). The stumbling block, as heady time, focused on the primacy of the mystical, problems still to be conquered by he saw it, was the efficiency with which selected gene, its structure, and its capacity to encode biologists. Brenner attacked this problem by mutant phenotypes could be recovered in a dynamic molecular responses to a changing bringing forth the new field of Caenorhabditis diploid vertebrate. There were bound to be lots cellular environment and to endure across elegans genetic research. Seymour Benzer of genes in these higher organisms, and very generations without becoming consumed or advocated harnessing the fruitfly to study few of them would be key to any single process distorted. By the mid-1960s, the fundamen- nervous system function and behaviour. under study. The trick was to identify interest- tal questions associated with the Streisinger aspired to unravel the genetic logic ing phenotypes associated with rare recessive DNA–RNA–protein story were well on their of neural development in a vertebrate. He mutations and to propagate those mutations way to a satisfying resolution. As Sydney began to tinker with a highly fecund tropical efficiently in an unseen state — the heterozy- Brenner wrote in his now-famous 1963 letter fish, the entire embryonic development of gous carrier. Streisinger was not alone in his to Max Perutz, it was just a matter of eluci- which — from egg to swimming larva — conviction that obviating what Bill Dove called dating “the chemical details of replication could unfold in a Petri dish. “the embarrassment of diploidy”was crucial and transcription […]. The future of molec- Immersed as deeply as he had been in the for the success of the genetic approach. C. ele- ular biology [lay] in the extension of wonders of phage, Streisinger was wedded to gans profited from its hermaphroditic lifestyle research to other fields of biology, notably the idea that mutational analysis was needed in in that single heterozygous carriers could pro- development and the nervous system”17.The vertebrates. His goal from the beginning was to duce both homozygous and heterozygous sib- focus on the gene and the success of the “study features of the organization and embry- ling progeny. Moreover, hermaphroditism mutational approach for understanding ological development of the vertebrate nervous freed the geneticist from limitations imposed physiological regulation in bacteria18 crystal- system through the use of mutant strains. [He by debilitating phenotypes that might have lized a commitment to the idea that both the was] particularly interested in the mechanisms restricted mating in other organisms. components and the logic of increasingly leading to the formation of specific synaptic Drosophila had a toolbox of genetic tricks that complex systems could be deconstructed connections and in the nature had been amassed over more than half a cen- using mutation-based genetic analysis. of the signals that guide specific axons to par- tury, including marked and balancer chromo-

718 | SEPTEMBER 2002 | VOLUME 3 www.nature.com/reviews/genetics © 2002 Nature Publishing Group PERSPECTIVES somes, which made it particularly easy to genetic contribution from sperm, thereby pro- distal to the centromere tended to be in het- monitor the inheritance of newly found muta- ducing haploid embryos. By the end of 1976, erozygous condition. Each of the gynogenetic tions. But, in vertebrates, tracking chromo- Streisinger and his associate Charline Walker procedures met Streisinger’s initial goal of some regions of interest was a great challenge, had transformed this simple beginning into recovering recessive phenotypes from mater- owing to the paucity of genetic markers. So, gynogenetic methods for producing wholly or nal genomes in a single generation. In addi- Streisinger focused initially on developing new partially homozygous diploid offspring19.To tion, these procedures could also be used to tools that would allow him to recover recessive generate a diploid embryo that was homozy- carry out genetic mapping and complementa- mutations efficiently from the germ line of gous at all loci, an egg was activated by ultra- tion analyses with extreme efficiency20. zebrafish and to identify quickly the few gems violet light-irradiated (genetically impotent) of interest. sperm, its haploid set of maternal chromo- Against all odds… somes was allowed to replicate and the initial The work to develop the zebrafish as a Why zebrafish? Streisinger initially brought segregation of chromosomes into daughter model organism was an immense gamble. several species of tropical fishes into his labo- cells was prevented by suppression of the first There was no history of genetic work with ratory, including medaka, which had an estab- mitotic cleavage of the zygote. To generate a this organism. Moreover, in the 1970s, lished history of genetic experimentation. In gynogenote that was partially homozygous, as Streisinger prepared the foundation the absence of any apparent record of these the second meiotic division was inhibited in an for the zebrafish system, there was wide- preliminary investigations, we can only sur- activated egg, producing an embryo whose spread scepticism as to whether his results mise his motivations for choosing to develop a diploid genetic composition was wholly could be translated into general principles. genetic methodology with the zebrafish. Four derived from sister chromatids (one half- More than a century after the publication of factors seem to have contributed to his ratio- tetrad). As genetic information between non- Darwin’s On the Origin of Species by Means nale. First, as the zebrafish bred prodigiously sister chromatids is recombined before the sec- of Natural Selection, there was little theoreti- in the laboratory, it was well suited for stan- ond meiotic division, this gynogenetic cal appreciation of the degree to which dard genetic analyses. Adults could be main- procedure yielded offspring that were hetero- vastly diverged species would share the regu- tained in breeding condition on a year-round geneous in genotype: genes proximal to the latory pathways that govern cell behaviour basis and individual females would give rise to centromere tended to be in homozygous and embryonic development. Before the era hundreds of progeny. Second, because of condition on the sister chromatids and genes of gene cloning, there were no data that external fertilization in zebrafish, gametes could be harvested separately, and the condi- tions of fertilization and ploidy could be manipulated for the purpose of genetic analysis. Third, because all embryonic development proceeded in full view of the researcher, screening for specific developmental phenotypes or early vision-dependent behaviours was feasible. Last, Streisinger had a passion for tropical fish. As a teenager, he had worked with fish at the American Museum of Natural History in New York City, and later, as an accomplished phage geneticist, his family vacations would be punctuated with detours to local fish hobbyist stores (L. Streisinger, personal communication).

New tools and methods. As indicated by his early grant applications (with an initial grant from the NSF and later grants from the NIH), during the first ten years of work with the zebrafish, Streisinger’s unwavering focus was to develop methods for rapidly uncovering recessive germ-line mutations. He wanted to free himself of the need to propagate each mutation through male and female heterozygous partners to produce homozygous offspring for screening. He reasoned that recessive phenotypes could be generated quickly by producing offspring derived solely from the maternal germ line (gynogenesis). His first accomplishment, which formed the basis of his 1973 grant application to the NSF, Figure 1 | Principal architects of zebrafish developmental genetics. From left to right, top row: was to establish a highly efficient method for George Streisinger (provided by the University of Oregon Archives), Charles Kimmel, Christiane activating the development of eggs without Nüsslein-Volhard; bottom row: Marc Fishman, Wolfgang Driever and a pair of adult zebrafish.

NATURE REVIEWS | GENETICS VOLUME 3 | SEPTEMBER 2002 | 719 © 2002 Nature Publishing Group PERSPECTIVES addressed whether genetic programmes that Streisinger’s zebrafish research programme.) regulate development were conserved. As a Commitment to answering a question was result, Streisinger was constantly embattled the only thing that mattered, and day-to-day to secure federal funding for his zebrafish progress was not measured closely. In this project. His efforts endured only through the environment, the zebrafish work proceeded prescient and persistent intervention of a in a set of converted army barracks on the handful of scientists, who by chance were edge of the University of Oregon campus. involved in the peer review and funding Enveloped with corrugated metal, the bar- process at NIH (M. C. Capecchi, G. Lark and racks were cooled by a constant supply of P.von Hippel, personal communications). water trickling down from the roof and Streisinger’s commitment to developing heated with judiciously placed small electric the tools for a new genetic system was heaters and fans, which occasionally would nurtured both by where he had come from ignite small fires in the laboratory. Progress and where he was. The phage world was accrued incrementally: first, methods were small and intimate, populated and mentored established to produce haploid and diploid by physicists who had eschewed the poten- gynogenotes; second, a breeding programme tially destructive applications of their was instituted to create strains that were research and redirected their research focus devoid of confounding lethal mutations in into biology with a special vigour. The early their background; and third, methods were phage period was remarkable for the rate developed to induce new germ-line muta- with which profound insights into basic tions. The first paper that described all the biological mechanisms emerged. Changing genetic procedures and announced the Figure 2 | The debut publication. Reproduction of the cover of the 1981 issue of Nature research direction was accepted, and pur- establishment of lethal mutation-free clonal containing the landmark paper by Streisinger et al. 19 suing the breakthrough experiment was lines was published in 1981 in Nature and that described genetic procedures for producing expected. In addition, confidence that a was heralded on the cover (FIG. 2).The homozygous diploid clones of zebrafish. The method of analysis was the key to asking accomplishment also received notice in sev- photo depicts sibling homozygous diploid golden questions was widespread.‘Hershey Heaven’ eral public venues, including a news com- (clear) and wild-type (pigmented) zebrafish — a term referring to the phage work of mentary cartoon published in the Chicago embryos. Al Hershey — was coined to describe the Tribune and US television coverage. process of using a single, powerful analytical Subsequent work on mutagenesis was pub- in embryos were described26,27. Finally, the technique to probe several important ques- lished in 1983 in Genetics21,22. lab began to screen on a regular basis for tions. Streisinger found himself in unusual embryonic mutants with interesting pheno- environments that were shaped by physi- The first fruits types. Among the first mutants to be isolated cists-turned-biologists of exceptional moral Although the tools for analysis were in place, was one that was later discovered to be defi- character: first, as a postdoc with Delbrück little was understood of the embryology cient in a growth factor needed for axis at Caltech, and later, as a faculty member of the zebrafish and the question remained determination, a second deficient in myofib- in the Institute of Molecular Biology as to the general biological insights that ril organization and a third in which a spe- founded by Aaron Novick at the University could be derived from studying a fish. cific portion of its nervous system failed to of Oregon. In both places, individuals were Influenced by contemporary studies on the form28–30. allowed or even expected to delve into their role of cell lineage in embryogenesis, own interests,“people lived with mistakes” Streisinger harnessed the only genetic trait Emergence of a research community (G. Lark’s description of Delbrück’s policy he had in hand — a recessive mutation that Meanwhile, the zebrafish was embraced at at Caltech), and the community as a whole affected pigmentation — to analyse whether Oregon — first by Charles Kimmel (FIG. 1), and was willing to underwrite, financially and individual cells acquired restricted develop- later by Monte Westerfield and Judith Eisen — otherwise, the efforts of individual col- mental fates at early stages of embryo- as a model organism that was spectacularly leagues. (F. Stahl and P.von Hippel, personal genesis. As Beatrice Mintz and Anne amenable to the study of nervous system communication. Despite the NIH policy to McLaren had shown in the mouse, development in particular, and vertebrate award funding to individual projects, the Streisinger found that early zebrafish blas- embryology in general. Neurobiologists had Institute of Molecular Biology, since its tomeres did not express a determinate cell never been fettered by concerns of “relevance”. inception, had pooled all equipment, media lineage, but instead contributed on a sto- They found all nerve cells of potential interest, facilities and administrative support into chastic basis to any specific differentiated tis- focusing principally on the suitability of a centralized facilities. It was through such sue23. He collaborated with others to deter- preparation for addressing a particular ques- devices that the Institute supplemented mine whether the nervous systems of tion. Kimmel quickly recognized the value of genetically identical animals were less het- the zebrafish embryo itself. In the mid-1970s, erogeneous than those of outbred animals. well before Streisinger’s work had come to He advocated the use of genetically deter- fruition, Kimmel initiated a series of neuro- “Changing research mined stocks of zebrafish as sentinels anatomical studies of the zebrafish embryo direction was accepted, and for genotoxic (able to cause damage to that uncovered the segmental structure of the DNA) agents in the environment24,25. brain. By the time Streisinger’s landmark pursuing the breakthrough Morphological and behavioural landmarks paper arrived, Kimmel and colleagues had experiment was expected.” of visual development that could be assayed described more identifiable neurons in the

a AP b European Drosophila geneticists had recognized the potential of the zebrafish, but beyond the boundaries of the Oregon labora- Epidermis Nervous system tories the project was largely viewed in the spadetail United States as Streisinger’s personal quest. Somites Consequently, US medical research funding Blood Pronephros Notochord institutions were reluctant to invest in its V D Wild type continuation. In a remarkably uncommon move in US academic institutions, where the indepen- Yolk cell dence of each research group is defended with Wild type resolve, the groups in Oregon worked in concert to maintain and expand Streisinger’s programme of genetic research with the c zebrafish. They established an informal course on zebrafish husbandry, genetics and embryonic anatomy, hosting several visiting scientists and sending representatives to other labs. Westerfield devised a primer on methods that was widely distributed33. Kimmel — an insightful character who was known among his Oregon colleagues for both his attention to detail and his homespun music and artistry 5 somites 30 somites — quietly laid out a road map to focus attention on the important biological questions Figure 3 | Determining cell autonomy of gene function. a | Fate map of the early gastrula zebrafish that could be asked in the zebrafish. embryo. The blastoderm of the early gastrula embryo is shaped like an inverted cup overlying the large Intrigued by the striking organization of yolk cell. Territories of future tissue fates are indicated on the picture of an early gastrula with respect to the identified neurons of the zebrafish brain, the dorsal (D) and ventral (V) midlines of the embryo and the animal pole (AP). Note that mesoderm fates arise from cells located near the outer margin of the blastoderm. b,c | Schematic illustration of how in 1982 Kimmel embarked on an ambitious transplantation chimaeras were used to test cell autonomy of the spadetail mutation. Cells removed programme, which was to continue for more from the blastoderm margin of dye-labelled wild-type and spadetail mutant late-blastula-stage embryos than ten years, to illuminate the developmen- were intermixed and implanted into a wild-type host embryo (b). Whereas transplanted wild-type cells tal steps that led to the origin and organiza- (green) integrate into their host environment, participating in the normal formation of somitic muscle, tion of distinct tissue types in the zebrafish the transplanted spadetail cells (orange) behave independently of their environment, failing to gastrulate embryo. Kimmel’s work defined a crucial normally and accumulating at the posterior of the embryo (c). Panel a is adapted from REF. 34. stage, just at the onset of gastrulation, when Panels b,c are adapted from REF. 37 © (1989) Macmillan Magazines Ltd. the prospective organization of the entire embryonic body plan emerged. This extended zebrafish than had been recognized in any were now to “investigate the genetic contribu- series of studies had two powerful effects other vertebrate31. The morphology and tion of the zygote to early developmental on zebrafish research. First, it provided both arrangement of the brain neurons led decisions in the zebrafish and to identify spe- the conceptual framework and the tools Kimmel to suggest that they arose as part of a cific developmental defects in photoreceptor needed to explore the regulation of cell fate in repeated developmental programme, and he development in fish homozygous for induced the zebrafish embryo. Second, it placed the began to probe the role of cell lineage in the mutations” (G. Streisinger, unpublished lab- zebrafish in the context of vertebrate develop- production of these anatomically related, seg- oratory documents). mental biology. Kimmel’s description of the mentally iterated brain neurons. Soon after- zebrafish fate map34 (FIG. 3a) came on the heels wards, Westerfield, Eisen and their colleagues of analogous studies in other vertebrate described a stereotypic arrangement of “the fish is a frog… is a model systems at a time when many were distinct spinal-cord motor neurons in adult looking for common principles to unify the and embryonic zebrafish, and showed that chicken… is a mouse” understanding of metazoan development in outgrowth of the axons of these motor neu- general and vertebrate development in partic- rons could be visualized with astounding clar- ular. One illustration of this quest was the ity in living embryos32. It was now clear that a As the first embryonic mutants emerged, burgeoning effort in the mid-1980s to dis- detailed analysis of nervous-system organiza- Streisinger and Kimmel began to plan collab- cover and understand the functions of Hox tion, differentiation of specific cell types and orative screening efforts on the basis of their genes in vertebrates. In his highly influential establishment of neural circuits could be shared interests in the patterning and differ- summary of the zebrafish lineage work, assayed in the zebrafish. If mutants that entiation of the nervous system. Then, with Kimmel announced “the fish is a frog… is a perturbed neural development could be gen- unexpected suddenness, efforts to test the chicken… is a mouse”35. erated, they would be recognized and har- promise of a mutational analysis of zebrafish Kimmel and his colleagues at Oregon vested for all their worth. In a 1984 summary development were thwarted by the death of mounted a tour-de-force effort to show the of his laboratory’s principal objectives, Streisinger in August 1984. By this time, depth and range of developmental questions Streisinger wrote that their primary goals Christiane Nüsslein-Volhard (FIG. 1) and other that could be addressed with the few

broader contexts, elucidating the regulatory work with Wieschaus and Gerd Jurgens logic and cell behaviours that are involved in were proving to be of compelling interest. executing a developmental programme. As Campos-Ortega recalls (personal com- These ideas were reinforced by the spectacu- munication), by the late 1980s,“Drosophila lar success of research with C. elegans. researchers had obtained a thorough Moreover, the link between phenotype and description of embryonic development the action of individual genes had been from the points of view of embryology, gen- bridged in Drosophila by molecular biology, etics and molecular biology. The synthesis of and many felt confident that it was only a biological disciplines had [almost] been hurdle of technology that separated mutant achieved, only cell biology was missing. A phenotypes in zebrafish from an understand- new way of working out biological prob- ing of their molecular nature. lems, using a multidisciplinary approach, Second, a new appreciation of the conse- had been firmly established in everyone’s quences of evolution slowly emerged with minds thanks to the work in Drosophila”. the demonstration that genetic and cell- The zebrafish system was intriguing because biological pathways were highly conserved it offered the potential of good embryology among all existing life forms. This idea was and good genetics with the prospect that illustrated by many kinds of experiment. cellular resolution could be achieved in a Yeast cell-cycle mutants could be rescued way that would yield insights into vertebrate with human genes. Homologous genes that development in particular, and cell biology Figure 4 | Results of the the ‘Big Screen’ are had been identified initially only by sequence more generally. announced. Reproduction of the cover of the similarity had analogous expression pat- issue of Development, volume 123. This issue included 37 papers that reported results from the terns, as revealed by RNA in situ hybridiza- The ‘Big Screen’ first large-scale screens for developmental mutants tion. A zebrafish embryonic mutant, no tail, By the late 1980s, a handful of prominent in the zebrafish. Approximately 4,000 embryonic- and the historic mouse developmental Drosophila developmental geneticists had lethal mutants were recovered and characterized. mutant Brachyury not only resembled each established research programmes with the other phenotypically, but also were subse- zebrafish, although the revolutionary impact quently discovered to have defects in orthol- of these programmes could scarcely have zebrafish embryonic mutants that were in ogous genes39. The neurobiologists had been been foreseen. As noted by Nüsslein-Volhard hand. Most notable was work published in right all along. It did not matter which ani- (personal communication),“At the time, the 1989 and 1990 on the spadetail mutant, mal you chose — fundamental processes fields of vertebrate and fly biology were miles, recently shown to be defective in a regula- were fundamentally conserved. miles apart […] fly people would speak of tory T-box transcription factor36. Kimmel The significance of Streisinger’s work to genes [only], frog people of factors […] showed that spadetail governed the morpho- establish the framework for genetic manip- Nobody yet believed in what was later felt to genetic behaviour of embryonic cells during ulation of a vertebrate had been immedi- be the incredible conservation of molecular gastrulation, and therefore it was crucial to ately grasped by several of the leading mechanisms.” the formation of the vertebrate body plan37. Drosophila developmental geneticists, who Nüsslein-Volhard’s plan was by far the Then, in a highly influential experiment, were already convinced of the insights to most ambitious. She would recapitulate the Kimmel’s colleagues, Robert Ho and Don be gleaned from a concerted mutational Drosophila screen for embryonic pattern Kane, showed that chimeric embryos, which analysis of development. Nüsslein-Volhard mutations in a vertebrate at the Max Planck had been generated using very simple cell- (personal communication) and Jose Institute in Tübingen, Germany. At the same transplantation methods, could be used to Campos-Ortega (personal communication) time, Marc Fishman recruited her gifted stu- identify the specific cells that required spade- each recall excited discussion of Streisinger’s dent, Wolfgang Driever, to Massachusetts tail function38 (FIG. 3b,c). The cell-autonomy 1981 paper in journal clubs that took place General Hospital to establish a parallel effort analyses that they introduced can be carried soon after its publication. After Streisinger’s (FIG. 1). Initially, aspects of husbandry that out with an ease and precision that is unri- death, Nüsslein-Volhard set a small aquar- would be required for a large-scale genetic valled in any other model organism used at ium of zebrafish on a windowsill and pon- analysis of embryonic development needed present, and have become de rigeur in the dered how to accomplish a study of the to be conquered. New procedures for muta- zebrafish field. magnitude that she deemed necessary genesis were developed and a standard The work done in Oregon in the 1980s (C. Nüsslein-Volhard, personal communi- multi-generational breeding protocol was highlighted the synergy that grew out of cation). Perhaps more than anyone else, she instituted to allow recovery of recessive blending classical embryological and genetic realized that all of the ~120 Drosophila mutations40,41. A core of brilliant young approaches in the zebrafish to ask a range of mutant genes that had come out of her postdoctoral associates and students was developmental and neurobiological ques- assembled. The ‘Big Screen’ for embryonic tions. Concurrently, two profound advances mutants was begun in 1993 and brought to in the Geist of developmental biology readied a conclusion exactly two years later. Between world science to embrace the zebrafish. “It did not matter which Tübingen and Boston, ~4,000 embryonic- Foremost, the work of Nüsslein-Volhard and animal you chose — lethal mutant phenotypes were recovered. Eric Wieschaus revolutionized the study of all To be of ultimate value to the general biology, by showing that collections of fly fundamental processes were research community, the mutants needed to mutant phenotypes could be woven into fundamentally conserved.” be described, sorted into complementation

722 | SEPTEMBER 2002 | VOLUME 3 www.nature.com/reviews/genetics © 2002 Nature Publishing Group PERSPECTIVES groups and preserved in a way that would genes for the zebrafish mutations. make them accessible to others. A decision “…the emergence of the Independently, the Hopkins laboratory was made that proved historic: description developed tools for insertional mutagenesis of the mutant phenotypes would not trickle zebrafish as a prominent and undertook a massive effort to cover out as tantalizing anecdotal individual ele- biological tool required much of the zebrafish genome with inser- ments — instead, they would be published tional mutations that could be rapidly together once the genetic and preliminary open-mindedness and cloned50,51. Antisense methods that had phenotypic characterizations of the entire tenacity by researchers with been mistrusted by molecular biologists for group had been completed. This effort took extraordinary vision.” decades were proved by Stephen Ekker and another year and culminated in the publica- his colleagues to be highly informative for tion of 37 papers gathered together in a sin- studying gene function in the zebrafish52.In gle issue (volume 123) of the journal In this procedure, marker segregation is 2000, the Sanger Centre initiated a project Development, devoted entirely to the analysed in pooled groups of wild-type or to sequence the zebrafish genome. Most zebrafish mutants (FIG. 4). mutant offspring that are produced from a importantly, to facilitate the rapid exchange The impact of the ‘Big Screen’ has been single pair of heterozygotes. For a polymor- of genomic data and genetic resources, a assessed in many reviews. The zebrafish had phic locus that is not linked to a mutation, centralized web-based database (see online been catapulted to the forefront of develop- all alleles will be found in both mutant and link to ZFIN) and a zebrafish stock centre mental biology research. The model for wild-type pools, whereas for a linked (Zebrafish International Resource Center) genetic analysis of development and physiol- marker, only one allele will be present in the were established by Monte Westerfield. ogy that had been established in Drosophila pool of mutant offspring. So, using bulk seg- had been extended fruitfully to new verte- regant analysis and a small set of DNA Conclusions brate problems. Several mutants that affect markers that are distributed across the map, Thirty years after George Streisinger began common processes helped to identify inter- only a very limited initial analysis is required his solo voyage to develop a new avenue of acting genetic pathways3,4,42,43. Hypomorphic to assign a new mutation to a chromosomal genetic research with a vertebrate, nearly mutations had a special role for modelling location. Furthermore, using standard meth- 800 scientists gathered at the Fifth human disease states6. In an immediate sense, ods of gene mapping, the vast numbers of International Conference on Zebrafish the ‘Big Screen’ provided researchers with an progeny that can be recovered from het- Development and Genetics (in June 2002) array of mutants that were relevant to many erozygous mating partners means that thou- to discuss with animation and exuberance aspects of vertebrate embryonic develop- sands of meioses can be scored readily, and their latest findings on the genetic basis of ment. It also provided impetus for the estab- therefore mutations can be mapped with a zebrafish development, physiology and lishment of numerous mutational analyses fine-scale precision not approached in other behaviour. In retracing the origins of the focused on specific developmental pro- vertebrate models. zebrafish field, we ask whether elements that grammes in the zebrafish44 and made it clear With the tools for forward genetic analy- were crucial to its success can be used to fos- that such methods could be applied in sis tested, and the pathway towards posi- ter similar leaps in the future. Certainly, the the mouse45. The reverberating effects of the tional cloning established, it was clear that emergence of the zebrafish as a prominent effort are still being felt. the development of supportive resources biological tool required open-mindedness would transform the zebrafish into a widely and tenacity by researchers with extraordi- Classical genetics to the molecular era embraced model. Under the guidance of nary vision. In addition, it required a Interest in the mutant phenotypes immedi- Harold Varmus, then Director of the NIH, considerable investment of time and an ately focused attention on the molecular and prompted by the persistent advocacy of environment that was permissive to the nec- identification of the defects. Years earlier, Len Zon, Marc Fishman and Nancy essarily slow maturation of new ideas and John Postlethwait anticipated the need for Hopkins, the NIH chose to transcend its technologies. It is incredible to realize in molecular landmarks scattered across the traditional institutional boundaries and today’s entrepreneurial scientific environ- zebrafish genome and initiated work to cre- invest significantly in the development of ment that Streisinger worked for more than ate a linkage map. The early mapping work, genomic resources for the zebrafish. This nine years on the project before its debut carried out largely by a cadre of undergradu- decision reflected a new confidence of the publication. His decision to wait until he ate students at the University of Oregon, NIH in the relevance of simple model had developed a robust network of genetic relied on the particular strengths of the organisms to medical research — a view tools, much like the decision by Nüsslein- zebrafish genetic system and introduced two that is substantiated, for example, by the Volhard and Driever to publish their mutant methodologies that are now commonplace successful use of the zebrafish as a bioassay strains as a comprehensive set, had an in the mapping of zebrafish mutations. First, for the function of human genes involved immense impact on the future of the field. construction of the linkage map depended in establishing left–right asymmetries49.As In both cases, unveiling work at a relatively wholly on Streisinger’s gynogenesis meth- a result of the Trans-NIH Zebrafish mature stage broadcast a clear outline of the ods, as segregation of polymorphic DNA Initiative, the zebrafish map was quickly potential trajectory of the field and thereby markers was assessed among sibling haploid consolidated and became densely popu- attracted many newcomers. Without invest- progeny46. Even today, analysis of marker lated with anonymous molecular markers, ment in such long-term ventures, a luxury pro- segregation among half-tetrad gynogenotes as well as newly discovered genes and vided to Streisinger by his unique environ- is used to assign new mutations rapidly to expressed sequence tags. Mutations were ment, we might not today harbour the linkage groups47. Second, a technique called positionally cloned. Syntenic relationships conviction that genetic studies with a little ‘bulk segregant analysis’ was introduced as between the zebrafish and mammalian tropical fish will reveal treasures of under- an initial mapping device for new mutants48. species were used to identify candidate standing for the basis of human diseases.

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