Medaka and zebrafish, an evolutionary twin study

Transcription

1 Mechanisms of Development 121 (2004) Review Medaka and zebrafish, an evolutionary twin study Makoto Furutani-Seiki a, Joachim Wittbrodt b, * a SORST, Kondoh research team, Japan Science and Technology Agency (JST), Kyoto, Japan b Developmental Biology Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany Received 17 May 2004; accepted 17 May Abstract Comparison of two related species is one of the most successful approaches to decipher general genetic principles in eukaryotes. This is best illustrated in yeast, where the model systems Saccharomyyces. cervisiae and Schizosaccharomyces. pombe have been examined. Powerful forward genetics in both species, species-specific differences in biological features and the phylogenetic distance between the two species, make them well suited for a comparative approach. Recent whole genome sequencing has also facilitated comparative genomics of these simple eukaryotes. It is now possible to go a step further using higher eukaryotes. A duplication of the genome at the base of the teleost radiation, facilitated evolution of almost 25,000 fish species, more than half of all vertebrate species together. Two teleost genetic model systems have emerged in the past few decades: zebrafish, in which large-scale mutagenesis has been successfully performed, and Medaka, a Japanese killifish with a century of history in genetics and now, as reported in this issue, many induced mutations. In this review we will illustrate how comparison of these two model species, Medaka and zebrafish, can reveal conserved and species-specific genetic and molecular mechanisms underlying vertebrate development. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Fossil taxa; Teleost; Genome duplication; rx genes 1. Introduction The wide range of existing fish species reflects a unique situation within vertebrates: the number of extant species exceeds fossil taxa. Within the past 350 million years (Myr) the almost 25,000 distinct species emerged. Part of the wide variability they exhibit has been attributed to a whole genome duplication event (Meyer and Schartl, 1999; Sidow, 1996; Suga et al., 1999; Wittbrodt et al., 1998), which occurred at the base of the teleost radiation (Fig. 1, Christoffels et al., 2004; Vandepoele et al., 2004). Gene and genome duplications have been described as the driving force of evolution (Ohno, 1970), and models have been proposed for how duplicated genes and genomes evolve (Force et al., 1999). A genome duplication generates paralogous groups of (duplicated) genes. Being essentially free from selective pressure, subsequently one paralog can acquire a new function (neo-functionalization), or become * Corresponding author. Tel.: þ ; fax: þ addresses: jochen.wittbrodt@embl-heidelberg.de (J. Wittbrodt), furutani@dsp.jst.go.jp (M. Furutani-Seiki). inactivated (Fig. 1). Another possibility is that one of the duplicates takes over only a part of the original function (complementing the other paralog). Through this process the initially complex function of a gene can be split into several sub-functions and distributed among different paralogs (sub-functionalization). Both sub-function partitioning and neo-functionalization are not limited to regulatory elements, but can also occur within the coding region of a duplicated gene. After the initial genome duplication the genomes of different teleost lineages evolved independently. It is conceivable that due to their independent evolution, different fish species will show significant differences with respect to the fate of duplicated genes: in different lineages one or the other paralog may have undergone neofunctionalization or inactivation, the sub-function partitioning may be distributed differently between paralogs, or a gene pair might have undergone sub-functionalization in one lineage and neo-functionalization of one paralog in the other. Therefore comparisons of divergent teleosts will provide insights into the evolution of the genetic and biochemical networks controlling development and the plasticity of these regulatory networks. Such studies will /$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi: /j.mod

2 630 M. Furutani-Seiki, J. Wittbrodt / Mechanisms of Development 121 (2004) Fig. 1. Evolutionary relationships of teleost model systems and hypothesized consequences of genome duplication at the basis of their radiation. As indicated on the left part the duplication of a gene/genome can lead to subsequent gene loss, to sub-functionalization or to neo-functionalization of the paralogs generated in the duplication event. Sub- and neo-functionalisation are not limited to the non-coding region (coloured symbols), but can occur in the coding region (orange, differences in red). The genome duplication at the basis of teleost radiation likely affected the genomes of teleost model systems differentially as reflected by the schematic gene representations. also answer questions concerning the mechanisms underlying the divergence of initially closely related species. The independent evolution of duplicated genes and the resulting sub-functionalization in fish can be useful for obtaining results that are impossible to get for the corresponding (non-duplicated) homologue in mammals. For example, the targeted inactivation of a single mouse gene may lead to haplo-insufficiency or early lethality, thereby making it impossible to study the complex function of the gene in the mouse. One notable example is the hedgehog gene. While targeted inactivation of the single murine Sonic hedgehog gene produces an early and severe phenotype (Chiang et al., 1996), the loss of function of one of the zebrafish hedgehog paralogs results in a less severe mutant phenotype (Schauerte et al., 1998) that corresponds to only part of the mouse defects. The other functions of the mouse Sonic hedgehog are encoded by additional zebrafish hedgehog paralogs and when general hedgehog signalling is disturbed in fish, the effect is as dramatic as the mouse phenotype (Ingham and McMahon, 2001; McMahon et al., 2003; Russell, 2003). Another example is the retina specific homeobox gene Rx that when inactivated in mouse leads to an early eye and forebrain defect (Mathers et al., 1997). Mutations in both the Medaka and zebrafish rx3 gene, one of the three rx paralogs found in fish, result in a less severe phenotype: absence of optic vesicle morphogenesis (Loosli et al., 2001, 2003). Thus in fish, mutant phenotypes can be studied that are not evident in the mouse due to earlier or severe defects. For obtaining the full benefit of a comparative approach utilizing fish as one model it is not sufficient to study a single species. Developmental biology studies profit from the characterization of complementary systems and, as outlined below, there are many reasons why Medaka is the appropriate complement to zebrafish. An additional advantage of choosing these two teleosts is their estimated evolutionary distance for genomic comparisons. Progress in understanding gene function is expected from the in-silico identification of regulatory sequences of developmental genes. Recent comparative analyses of the Sox2 enhancer from mouse and human (evolutionary distance: 80 Myr) did not reveal discrete functional elements due to the high overall similarity. However, in a comparison of the respective genomic regions of chicken and mouse (evolutionary distance: 300 Myr), conserved sequence blocks precisely mirrored regulatory elements that had been identified independently on the basis of their function (Uchikawa et al., 2003). Given the distant placement of medaka and zebrafish in the phylogenetic tree and an estimated time of about Myr since their lineages separated (Wittbrodt et al., 2002, Fig. 1),

3 M. Furutani-Seiki, J. Wittbrodt / Mechanisms of Development 121 (2004) these teleost species appear to be well suited for a comparative analysis of gene regulatory mechanisms. 2. Practical considerations: biology and technology 2.1. Well established experimental techniques for both Medaka and zebrafish Medaka and zebrafish can be easily raised side by side in one aquatic system because they both require the same conditions with respect to water quality, temperature and light cycle (Westerfield, 2000). Most of the standard experimental procedures can be applied to both species with slight modifications, including the observation of embryos, gynogenesis, sperm freezing and in vitro fertilization, cell transplantation, RNA and DNA injection, in situ hybridization using riboprobes and immunohistochemistry. Once dechorionated, handling of the softer Medaka embryos requires some practice. Medaka embryos tolerate a wide temperature range (4 35 8C until the onset of heart beating and C thereafter, compared to C in the case of zebrafish, Westerfield, 2000), enabling manipulation of developmental rate and the design of specific screens for isolation of cold temperature sensitive mutations (see below). Zebrafish, in contrast, is a tropical fresh water fish and therefore less tolerant to extreme temperatures, especially prior to the onset of gastrulation. Methods for the production of transgenic fish by injection of DNA constructs (Ozato et al., 1986; Stuart et al., 1988; Thermes et al., 2002) or transposon (Grabher et al., 2003; Kawakami et al., 2000) or viral vectors (Amsterdam et al., 1999) are well established for both species. The use of Green Fluorescent Protein (GFP) reporters allows comparative in vivo studies of gene expression. Morpholino based knock down experiments have also been successfully applied in both species (Carl et al., 2002; Nasevicius and Ekker, 2000) Biology: similarities and differences Similar to zebrafish, Medaka is an egg-laying fresh water fish whose transparent embryos develop inside a transparent chorion. Mating and spawning is tightly correlated to the light cycle. The female Medaka spawns between 20 and 40 eggs every day within an hour after the onset of light. Zebrafish females do not spawn every day and a maximum number of eggs (several hundreds) is obtained by mating at approximately one week intervals (Westerfield, 2000). Thus, the total number of eggs laid by females of Medaka or zebrafish per week is comparable. While zebrafish eggs are fertilized as they are laid and fall to the ground, Medaka eggs remain attached to the female body by attachment filaments. This allows instant identification of reproductively active females. In zebrafish, mating couples are usually not left for more than two nights in a mating cage, otherwise they might harm each other and fish sometimes are lost. Consequently, matings need to be set up more frequently. Medaka couples can be left together in a tank and the female will spawn daily for several months. The collection of eggs from the same mating pair over consecutive days allows a given mutant phenotype to be confirmed immediately in medaka, which might take several weeks in the zebrafish. Early development is rapid in Medaka and zebrafish, the latter developing even slightly faster. While zebrafish hatch out of the thin chorion after 2 3 days as swimming larvae, Medaka embryos, well supported by yolk, stay inside the tough, chorion that protects them in their natural habitat until they hatch as feeding young fish after seven days. The Medaka chorion is a barrier not only protecting the embryo from the environment, but also interferes with imaging and experimental manipulations. To overcome this problem, several methods have been devised to facilitate experimental embryology. Attachment filaments on the chorion can be easily removed by rolling eggs on a piece of Whatman filter paper or by proteinase K digestion. The chorion itself can be digested using a crude lysate of hatching stage embryos. Until the end of epiboly, dechorionated embryos have to be kept in agar coated dishes to prevent damage of the delicate yolk cell. Once epiboly is completed, dechorionated embryos can be incubated in standard bacterial dishes and will develop at a normal pace. Thus all major manipulations (e.g. cell transplantations, bead implantations, etc.) can be efficiently and successfully applied to Medaka embryos (Winkler et al., 2000) Development and mutants-complementary aspects Under laboratory conditions, the generation time for Medaka is between 6 and 8 weeks and between 8 and 10 weeks for zebrafish. The transparent Medaka eggs are roughly 1 mm in diameter. The yolk, on which the embryos feed for the first 7 days of their life, is contained within a single yolk-cell. Developmental stages and corresponding morphological characteristics have been described in detail by Iwamatsu (Iwamatsu, 1994; Iwamatsu, 2004). First cleavages take approximately 30 min at 28 8C, gastrulation starts after 8.5 h and the neural axis is visible after 15 h. The developing Medaka embryo is partially embedded into the big yolk cell. In comparison to zebrafish, the embryo/yolk ratio is lower due to their different biology. Consequently the observation of gastrulation movements is easier in zebrafish. After the onset of gastrulation rhythmic contractions of the yolk occur in Medaka, which can be efficiently blocked by the addition of n-heptanol to the medium without interfering with embryonic development (Rembold and Wittbrodt, 2004), thereby allowing extended observations and time-lapse video microscopy. Interestingly, and in contrast to zebrafish, Medaka brain morphogenesis and most of organogenesis occurs early relative to somitogenesis (see Tables 1 and 2). A possible explanation is that, as the young fish hatch late, motility is not the most immediate goal to reach during embryogenesis.

4 Table 1 Staging table comparing developmental landmarks between Medaka and zebrafish up to the end of gastrulation 632 Period Stage Medaka Iwamatsu staging h, 28 8C h, 338C Zebrafish h, C Zygote 1-cell 1 cell 1, cell 0.2 Cleavage 2-cells 2 cells cells cells 2 2 arrays of blastomeres arrays of blastomeres 1 8-cells 2 4 arrays of blastomeres arrays of blastomeres cells 4 4 arrays of blastomeres arrays of blastomeres cells 2 regular tiers (horizontal arrays of blastomeres 1.75 rows) 64-cells (zebrafish) 3 regular tiers of blastmeres 2 Blastula 64-cells (Medaka) Horizontal division also takes place in central blatomeres 8 early morula cells YSL begins to appear 5 blastomere tiers, cleavage planes irregular cells 9 late morula blastomeres tiers cells 9 blastomeres tiers, YSL 2.75 forms 1k-cells, early blastula 10 early blastula 11 blastomeres tiers, single row of YSL nuclei, cell cycle asynchronous, MBT 3 Early high blastula High, oblong stage;.11 tiers of blastomeres, 3.3 blastodisc flattening, multiple rows of YSL nuclei Late blastula Middle high blastula 11 late blastula Sphere, dome stage: yolk 4 cell bulging from flat border toward animal poles as epiboly begins Late flat blastula: The blastoderm has flattened down capping the yolk sphere %epiboly, blastoderm inverted cup if uniform in thickness 4.7 Gastrula Germ-ring 20% epiboly, gastrulation is about to begin. Germ ring and extra-embryonic membrane formed. 12 pre-early gastrula Germ-ring stage: germ-ring visible from animal pole; 50% epiboly Early-gastrulation 30%epiboly. Rhythmic contraction begins. 13, 14 early-mid gastrula Shield stage: Embryonic shield visible from animal pole, 50% epiboly Mid-gastrulation Late-gastrulation 50% epiboly, the embryonic shield increase in size. the hypoblast cells clearly visible. 75% epiboly, axis, neural plate and neural keel can be seen. 15 mid-gastrula % epiboly: Dorsal side distinctly thicker; epiblast, hypoblast, evacuation zone visible 16 late gastrula % epiboly, axis and neural plate are found 90% epiboly, brain rudiment, Kupffer s vesicle 17 Notochord, brain rudiment 0-somite, optic vesicle Bud stage M. Furutani-Seiki, J. Wittbrodt / Mechanisms of Development 121 (2004) Note that optic vesicles are morphologically distinct and visible in Medaka prior to the onset of morphogenesis.

5 M. Furutani-Seiki, J. Wittbrodt / Mechanisms of Development 121 (2004) Table 2 Staging table comparing developmental landmarks between Medaka and zebrafish from somitogenesis stages onwards Period Stage Medaka Iwamatsu h, 28 8C h, 33 8C Zebrafish h, C Segmen-tation 1-somite 2-somite 2-somite, 100% epiboly somite Otic placode, lens somite Optic vesicle, polster, Kupffer s vesicle, neural keel 6-somite Otic vesicle, neural keel somite Pronephros 9-somite Heart anlage, lens, hindbrain ventricle, notochord, midbrain/ hindbrain boundary somite somite Blood island somite Otic placode, brain neuromeres somite Heart beating, gut somite Yolk extension, anus somite Otolith, notochord, liver, hindbrain rhombomeres somite Lens, otic vesicle, hindbrain neuromeres 22-somite Eye pigmentation Blood island somite Pectoral fin bud somite Otholith, midbrain/hindbrain boundary somite Pancreas Prim-5, pigmentation, heart beat, 24 hindbrain rhombomeres 32-somite Air bladder Prim-15, retina pigmented, liver somite Prim-25, somite Intersegmental vessel blood 30 Hatching 48 flow starts, olfactory pit, gall bladder Anterior allocation of heart Hatching 39 Therefore somite numbers are only useful for comparing landmarks in somitogenesis, the brain develops at a different pace (see Table 1 and Table 2). This difference in early development is reflected by a slightly different timing in the expression of key regulatory genes (e.g. Pax2, Köster et al., 1997; Krauss et al., 1991). As mutants are now available in both species, the comparison of mutations in orthologous genes should elucidate how differential timing of gene expression causes the differential expression of morphological characters. Zebrafish was selected as a model system for the functional analysis of vertebrate development because it allows the power of large-scale forward genetics to be combined with elegant cellular analyses (Driever et al., 1994; Ho and Kimmel, 1993; Kane et al., 1992; Kimmel and Warga, 1987; Mullins and Nüsslein-Volhard, 1993; Westerfield et al., 1990). Large-scale mutagenesis screens were facilitated by the biological features of zebrafish: its fecundity, short generation time and easy husbandry. The externally fertilized, rapidly developing, transparent embryos are perfectly suited for the analysis of development at the cellular level. Medaka not only shares these features, but provides some additional benefits due to its long history as a genetic model system. Inbred strains from different populations are available, which show a high degree of genetic polymorphisms. This facilitates the generation of high resolution genetic maps and the genetic analysis of monogenic traits and quantitative trait loci (Hyodo-Taguchi and Egami, 1985; Ishikawa, 2000; Wittbrodt et al., 2002). Due to its temperature tolerance during early development, cold- and heat-sensitive mutations were identified (Winkler et al., 2000). Several mutants identified in small-scale pilot screens (Ishikawa, 2000; Loosli et al., 2000) exhibited phenotypes not yet recorded in zebrafish, showing that Medaka mutagenensis screens can effectively complement those in zebrafish.

6 634 M. Furutani-Seiki, J. Wittbrodt / Mechanisms of Development 121 (2004) Comparative approaches in Medaka and zebrafish 3.1. Uncovering conserved and divergent developmental mechanisms Since both Medaka and zebrafish embryos develop in a similar manner, the direct comparison of a mutant phenotype and its functional and evolutionary interpretation is relatively straightforward: Similar phenotypes do not necessarily imply that orthologous genes are affected by the respective mutations. Only the molecular identification of the mutated gene will reveal whether gene function is conserved or species specific. To unravel similarities and differences in developmental modules is one of the great potential benefits of this comparative analysis of Medaka and zebrafish Study of species-specific features Medaka and zebrafish have several species-specific features that are amenable to the genetic studies discussed here. Two particularly good examples are sex determination and adult pigment patterning, which are best studied in Medaka and zebrafish, respectively. In zebrafish, none of the many markers on the genetic map is sex-linked and little is known about sex determination. Conversely, Medaka has an XX, XY sex-determination system like mammals, with the male determining locus on the Y chromosome. Sex determination has been the target of intensive research since the late 1950 s (Yamamoto, 1965; Schartl, 2004). Recently, a member of the dmrt gene family was found to be the sex-determining gene in Medaka, representing the first detected non-mammalian sex-determination gene in vertebrates. Mutations that affect gonad formation and sex determination in Medaka are likely to shed light on the different modes of regulation of these processes in teleosts. The beautiful stripes of the adult zebrafish are an excellent system to gain insights into autonomous pattern formation of pigment cells in the skin (Haffter et al., 1996). Mutations in 7 loci, some of which are already genetically identified (Dutton et al., 2001), affect formation of the stripes in zebrafish. Medaka adults do not exhibit a particularly ordered body pigmentation, but show a simpler pattern of relatively homogeneously distributed pigment cells. Determining at the molecular level what is responsible for these profound differences in appearance will help to increase our understanding of pigment pattern formation in general, a phenomenon that is poorly understood in vertebrates. Moreover, if the selective value of the pigmentation differences will be uncovered (e.g. adaptation to different environmental niches), the molecular and developmental knowledge can be used to understand how evolutionary forces shape pigmentation genes and their regulation Towards functional saturation Identification of mutants is strongly dependent on the screening procedure, i.e. in a morphology-based screen the visibility of the tissue is critical. Consequently, the number of mutants affecting conspicuous organs or tissues in zebrafish, such as the notochord and the melanophores, were higher than those affecting organs like the liver (Haffter et al., 1996). In Medaka conversely, the liver is easier and the notochord more difficult to detect than in zebrafish. Thus, performing a mutagenesis screen in the species where the screening procedure for a certain mutant phenotype is more sensitive, will add significantly to a comparable mutagenesis screen in the other species. A benefit of the complementary mutagenesis approach is that sub-functionalization of gene paralogs is likely to be different in diverse teleost species. The analysis of two divergent fish species uncovers functions that are masked by genetic redundancy or were generated by neo-functionalization in one, but not the other species. The combination of mutagenesis approaches in Medaka and zebrafish will in part also overcome differential mutabilities due to potential differences in the genomic context (e.g. the presence of mutation hotspots, differences in region specific DNA repair, or chromatin structure). Therefore, the combined analysis of mutagenesis screens in Medaka and zebrafish will lead to the identification of more gene functions in the vertebrate genome. 4. Pairs of matching mutants The first example of the complementary approach in Medaka and zebrafish came from the study of paralogs of the retina specific homeobox gene rx (Mathers et al., 1997). Three rx genes are found in Medaka, zebrafish and fugu, and together their expression patterns resemble the expression of the single mouse ortholog (Loosli et al., 2001, 2003). The individual paralogs in Medaka show distinct spatiotemporal patterns of transcription that only partially overlap late in the evaginated optic vesicle. In zebrafish, the expression of all three rx paralogs temporally and spatially overlap earlier during the evagination of the optic vesicles (Chuang et al., 1999). Although this overlap could indicate a functional redundancy in zebrafish, the phenotypes of the respective rx3 mutants in Medaka (eyeless, el) and zebrafish (chokh) are strikingly similar, and result in the failure of optic vesicle evagination (Fig. 2, Loosli et al., 2001, 2003; Winkler et al., 2000). Rescue of the zebrafish mutant phenotype with the Medaka rx3 locus indicates that the regulation of rx3 is evolutionarily conserved (Loosli et al., 2003). In zebrafish, the paralogs rx2 and rx1 depend on rx3 function, while in Medaka their expression is not dependent on rx3 (Loosli et al., 2001, 2003). Therefore rx1 and rx2 cannot compensate for the loss of rx3 function in zebrafish leading to an equally strong phenotype in both species.

7 M. Furutani-Seiki, J. Wittbrodt / Mechanisms of Development 121 (2004) Fig. 2. Matching pairs of mutants. Mutations in the homeobox containing transcription factor Rx3 similarly affect optic vesicle morphogenesis and differentiation in Medaka eyeless (A) and zebrafish chokh (C) larvae 6 days after fertilization (C, D reprinted from Loosli et al., 2003 with the permission of EMBO reports). The fact that in zebrafish but not in Medaka the rx1 and rx2 genes depend on the expression of rx3 suggests that a novel regulatory function was acquired. This indicates a different transcriptional control of rx1 and rx2 in the two species that is predicted to result from divergent, species-specific regulatory elements. The pair of matching mutants in Medaka and zebrafish provides a good example of divergence of developmental modules: rx3 function is required in both Medaka and zebrafish for the development of the evaginated optic vesicle. However, the molecular pathway by which this is achieved diverged significantly during the independent evolution of Medaka and zebrafish after the establishment of the rx paralogous group. As more affected genes responsible for Medaka mutant phenotypes are cloned and analyzed, detailed comparisons with zebrafish orthologs will reveal more pairs of matching mutants. These will not only help to unravel the molecular mechanisms of development, but will allow the study of the plasticity of developmental modules and the ways modules diverged during evolution. There are several examples of genes that have been identified by a positional cloning approach and that have allowed functional annotation of genes only known by sequence so far, such as the one-eyed-pinhead gene (EGF- CFC domain protein) in zebrafish and the b gene (AIM1 transporter protein) in Medaka (Fukamachi et al., 2001; Zhang et al., 1998). For the efficient cloning of mutants well-established resources are an essential prerequisite. In Medaka these resources are generated through the coordinated activity of an international consortium, the Medaka genome initiative (MGI) (Shima et al., 2003). The recently finished whole-genome shotgun sequence of the Medaka genome ( will significantly accelerate the genome project that includes the generation of genetic, gene expression and physical mapping resources (Kimura et al., 2004; Martinez-Morales et al., 2004; Naruse et al., 2004; Quiring et al., 2004). 5. Perspective The availability of two fish model systems for genetics, experimental embryology and molecular biology, namely Medaka and zebrafish is unique among vertebrates. This is not only because their evolutionary distance is well suited for comparative functional genomics. More importantly the existence of the two systems transforms the apparent weakness of fish, namely the many paralogous groups of genes formed due to genome duplications, into an advantage: to study the evolution of gene function in vertebrates comparatively, under conditions that allowed rapid changes. The primary strength of zebrafish and medaka systems is their usefulness for developmental biological studies. In other disciplines of biology, fish are also providing excellent models (e.g. for behavioural biology several species of cichlids and poeciliids (Autumn et al., 2002), for evolutionary ecology Fundulus, the stickleback and the East African cichlids (Kocher, 2004; Peichel et al., 2001; Schulte, 2001), for cancer research the platyfish and swordtails (Kazianis and Walter, 2002; Wellbrock et al., 2002). With sequencing of the Medaka and zebrafish genomes nearing completion,

8 636 M. Furutani-Seiki, J. Wittbrodt / Mechanisms of Development 121 (2004) the already available genome sequences of fugu and tetraodon (Aparicio et al., 2002; Crollius et al., 2000; Porcel et al., 2004, Fig. 1), and the genomes of additional species in the pipeline (trout, salmon, cod, Xiphophorus, cichlids, stickleback and more to come), fish are swimming into a splendid future. Acknowledgements We would like to thank Manfred Schartl and colleagues in our labs for valuable input and Felix Loosli, Juan- Ramon Martinez-Morales, Robert Kelsh, Filippo DelBene and William Norton for critically reading the manuscript. We are grateful to Charles Kimmel, Monte Westerfield, Hiroshi Nishina, Tomomi Watanabe, Satoshi Asaka, Haruka Momose, Takahiro Negishi for their contribution to the Medaka -zebrafish comparative staging table. Research on Medaka was supported by the PRESTO grant from Japan Science and Technology Agency (JST) to M.F.-S. and has been generously supported over many years by ERATO/SORST grants of JST to Hisato Kondoh and the European Commission, the Deutsche Forschungsgemeinschaft and the Human Frontier Science Program to J.W. References Amsterdam, A., Burgess, S., Golling, G., Chen, W., Sun, Z., Townsend, K., et al., A large-scale insertional mutagenesis screen in zebrafish. Genes Dev. 13, Aparicio, S., Chapman, J., Stupka, E., Putnam, N., Chia, J.M., Dehal, P., et al., Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes. Science 297, Autumn, K., Ryan, M.J., Wake, D.B., Integrating historical and mechanistic biology enhances the study of adaptation. Q Rev Biol 77, Carl, M., Loosli, F., Wittbrodt, J., Six3 inactivation reveals its essential role for the formation and patterning of the vertebrate eye. Development 129, Chiang, C., Litingtung, Y., Lee, E., Young, K.E., Corden, J.L., Westphal, H., Beachy, P.A., Cyclopia and defective axial patterning in mice lacking Sonic Hedgehog gene function. Nature 383, Christoffels, A., Koh, E.G., Chia, J.M., Brenner, S., Aparicio, S., Venkatesh, B., Fugu Genome analysis provides evidence for a whole-genome duplication early during the evolution of ray-finned fishes. Mol. Biol. Evol. 21(6), Chuang, J.C., Mathers, P.H., Raymond, P.A., Expression of three Rx homeobox genes in embryonic and adult zebrafish. Mech. Dev. 84, Crollius, H., Jaillon, O., Bernot, A., Dasilva, C., Bouneau, L., Fischer, C., et al., Estimate of human gene number provided by genome-wide analysis using Tetraodon nigroviridis DNA sequence. Nat. Genet. 25, Driever, W., Stemple, D., Schier, A., Solnica-Krezel, L., Zebrafishgenetic tools for studying vertebrate development. Trends Genet. 10, Dutton, K.A., Pauliny, A., Lopes, S.S., Elworthy, S., Carney, T.J., Rauch, J., et al., Zebrafish colourless encodes sox10 and specifies nonectomesenchymal neural crest fates. Development 128, Force, A., Lynch, M., Pickett, F.B., Amores, A., Yan, Y.L., Postlethwait, J., Preservation of duplicate genes by complementary, degenerative mutations. Genetics 151, Fukamachi, S., Shimada, A., Shima, A., Mutations in the gene encoding B, a novel transporter protein, reduce melanin content in medaka. Nat. Genet. 28, Grabher, C., Henrich, T., Sasado, T., Arenz, A., Furutani-Seiki, M., Wittbrodt, J., Transposon-mediated enhancer trapping in medaka. Gene 322, Haffter, P., Odenthal, J., Mullins, M.C., Lin, S., Farrell, M.J., Vogelsang, E., et al., Mutations affecting pigmentation and shape of the adult zebrafish. Dev. Genes Evol. 206, Ho, R.K., Kimmel, C.B., Commitment of cell fate in the early zebrafish embryo. Science 261, Hyodo-Taguchi, Y., Egami, N., Establishment of inbred strains of the medaka Oryzias latipes and the usefulness of the strains for biomedical research. Zool. Sci. 2, Ingham, P.W., McMahon, A.P., Hedgehog signaling in animal development: paradigms and principles. Genes Dev. 15, Ishikawa, Y., Medakafish as a model system for vertebrate developmental genetics. Bioessays 22, Iwamatsu, T., Stages of normal development in the medaka Oryzias latipes. Zool. Sci. 11, Iwamatsu, T., Stages of normal development in the medaka Oryzias latipes. Mech. Dev. 121, Kane, D.A., Warga, R.M., Kimmel, C.B., Mitotic domains in the early embryo of the zebrafish. Nature 360, Kawakami, K., Shima, A., Kawakami, N., Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage. Proc. Natl Acad. Sci. 97, Kazianis, S., Walter, R.B., Use of platyfishes and swordtails in biological research. Lab. Anim. (NY) 31, Kimmel, C.B., Warga, R.M., Cell lineages generating axial muscle in the zebrafish embryo. Nature 327, Kimura, T., Jindo, T., Narita, T., Naruse, K., Kobayashi, D., Shin-I, T., et al., Large-scale isolation of ESTs from medaka embryos and its application to medaka developmental genetics. Mech. Dev. 121, Kocher, T.D., Adaptive evolution and explosive speciation: the cichlid fish model. Nat. Rev. Genet. 5, Köster, R., Stick, R., Loosli, F., Wittbrodt, J., Medaka spalt acts as a target gene of hedgehog signaling. Development 124, Krauss, S., Johansen, T., Korzh, V., Fjose, A., Expression of the zebrafish paired box gene pax[zf-b] during early neurogenesis. Development 113, Loosli, F., Koster, R.W., Carl, M., Kuhnlein, R., Henrich, T., Mucke, M., et al., A genetic screen for mutations affecting embryonic development in medaka fish (Oryzias latipes). Mech. Dev. 97, Loosli, F., Winkler, S., Burgtorf, C., Wurmbach, E., Ansorge, W., Henrich, T., et al., Medaka eyeless is the key factor linking retinal dertermination and eye growth. Development 128, Loosli, F., Staub, W., Finger-Baier, K., Ober, E., Verkade, H., Wittbrodt, J., Baier, H., Loss of eyes in zebrafish caused by mutation of chokh/ rx3. Eur. Mol. Biol. Org. Rep. 4, Martinez-Morales, J.-R., Naruse, K., Mitani, H., Shima, A., Wittbrodt, J., Rapid chromosomal assignment of Medaka mutants by bulked segregant analysis. Gene 329, Mathers, P.H., Grinberg, A., Mahon, K.A., Jamrich, M., The Rx homeobox gene is essential for vertebrate eye development. Nature 387, McMahon, A.P., Ingham, P.W., Tabin, C.J., Developmental roles and clinical significance of hedgehog signaling. Curr. Top. Dev. Biol. 53, Meyer, A., Schartl, M., Gene and genome duplications in vertebrates: the one-to-four (-to-eight in fish) rule and the evolution of novel gene functions. Curr. Opin. Cell Biol. 11,

9 M. Furutani-Seiki, J. Wittbrodt / Mechanisms of Development 121 (2004) Mullins, M.C., Nüsslein-Volhard, C., Mutational approaches to studying embryonic pattern formation in the zebrafish. Curr. Opin. Genet. Dev. 3, Naruse, K., Hori, H., Shimizu, N., Kohara, Y., Takeda, H., Medaka genomics: a bridge between mutant phenotype and gene function. Mech. Dev. 121, Nasevicius, A., Ekker, S.C., Effective targeted gene knockdown in zebrafish. Nat. Genet. 26, Ohno, S., Evolution by gene duplication, Springer Verlag, Berlin. Ozato, K., Kondoh, H., Inohara, H., Iwamatsu, T., Wakamatsu, Y., Okada, T.S., Production of transgenic fish: introduction and expression of chicken delta-crystallin gene in medaka embryos. Cell Differ. 19, Peichel, C.L., Nereng, K.S., Ohgi, K.A., Cole, B.L., Colosimo, P.F., Buerkle, C.A., Schluter, D., Kingsley, D.M., The genetic architecture of divergence between threespine stickleback species. Nature 414, Porcel, B.M., Delfour, O., Castelli, V., De Berardinis, V., Friedlander, L., Cruaud, C., et al., Numerous novel annotations of the human genome sequence supported by a 5 0 -end-enriched cdna collection. Genome Res. 14, Quiring, R., Wittbrodt, B., Henrich, T., Ramialison, M., Burgtorf, C., Lehrach, H., Wittbrodt, J., Large-scale expression screening by automated whole-mount in situ hybridization a technical note. Mech. Dev. 121, Rembold, M., Wittbrodt, J., In vivo time-lapse imaging in medaka n-heptanol blocks contractile rhythmical movements. Mech. Dev. 121, Russell, C., The role of hedgehogs and fibroblast growth factors in eye development and retinal cell rescue. Vis. Res. 43, Schartl, M., A comparative view on sex determination in Medaka. Mech. Dev. 121, Schauerte, H.E., van Eeden, F.J., Fricke, C., Odenthal, J., Strahle, U., Haffter, P., Sonic hedgehog is not required for the induction of medial floor plate cells in the zebrafish. Development 125, Schulte, P.M., Environmental adaptations as windows on molecular evolution. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 128, Shima, A., Himmelbauer, H., Mitani, H., Furutani-Seiki, M., Wittbrodt, J., Schartl, M., Fish genomes flying. Eur. Mol. Biol. Org. Rep. 4, Sidow, A., Gen(om)e duplications in the evolution of early vertebrates. Curr. Opin. Gen. Dev. 6, Stuart, G.W., McMurray, J.V., Westerfield, M., Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos. Development 103, Suga, H., Koyanagi, M., Hoshiyama, D., Ono, K., Iwabe, N., Kuma, K., Miyata, T., Extensive gene duplication in the early evolution of animals before the parazoan-eumetazoan split demonstrated by G proteins and protein tyrosine kinases from sponge and hydra. J. Mol. Evol. 48, Thermes, V., Grabher, C., Ristoratore, F., Bourrat, F., Choulika, A., Wittbrodt, J., Joly, J.-S., I-SceI meganuclease mediates highly efficient transgenesis in fish. Mech. Dev. 118, Uchikawa, M., Ishida, Y., Takemoto, T., Kamachi, Y., Kondoh, H., Functional analysis of chicken Sox2 enhancers highlights an array of diverse regulatory elements that are conserved in mammals. Dev. Cell. 4, Vandepoele, K., De Vos, W., Taylor, J.S., Meyer, A., Van de Peer, Y., Major events in the genome evolution of vertebrates: Paranome age and size differ considerably between ray-finned fishes and land vertebrates. Proc. Natl Acad. Sci. USA 101, Wellbrock, C., Gomez, A., Schartl, M., Melanoma development and pigment cell transformation in xiphophorus. Microsc. Res. Tech. 58, Westerfield, M., The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), The University of Oregon Press, Eugene. Westerfield, M., Liu, D.W., Kimmel, C.B., Walker, C., Pathfinding and synapse formation in a zebrafish mutant lackingfunctional acetylcholine receptors. Neuron 4, Winkler, S., Loosli, F., Henrich, T., Wakamatsu, Y., Wittbrodt, J., The conditional medaka mutation eyeless uncouples patterning and morphogenesis of the eye. Development 127, Wittbrodt, J., Meyer, A., Schartl, M., More genes in fish? BioEssays 20, Wittbrodt, J., Shima, A., Schartl, M., Medaka a model organism from the Far East. Nat. Rev. Genet. 3, Yamamoto, T.O., Estriol-induced, X.Y., females of the medaka (Oryzias latipes) and their progenies. Gen. Comp. Endocrinol. 5, Zhang, J.J., Talbot, W.S., Schier, A.F., Positional cloning identifies zebrafish one-eyed pinhead as a permissive Egf-related ligand required during gastrulation. Cell 92,