Establishment of a zebrafish inbred strain, M-AB, capable of regular breeding and genetic manipulation.

Inbred strains of organisms are genetically highly uniform and thus useful for life science research. We have previously reported the ongoing generation of the zebrafish IM strain from the India (IND) strain through full sib-pair mating for 16 generations. However, the IM fish laid a small number of offspring and had a short lifespan, implying the need for discreet care in breeding. Here, we report the subsequent establishment of IM strain as well as the generation of a new inbred zebrafish strain, Mishima-AB (M-AB). M-AB was derived from the *AB strain by full sib-pair mating for over 20 generations, which fulfills the general criterion for the establishment of an inbred strain. In contrast to the IM case, maintenance of the M-AB strain by sib-pair mating required almost no special handling. Genome sequencing of IM individuals from the 47th generation and M-AB individuals from the 27th generation revealed that SNP-based genomic heterogeneity across whole-genome nucleotides was 0.008% and 0.011%, respectively. These percentages were much lower than those of the parental IND (0.197%) and *AB (0.086%) strains. These results indicate that the genomes of these inbred strains were highly homogenous. We also demonstrated the successful microinjection of antisense morpholinos, CRISPR/Cas9, and foreign genes into M-AB embryos at the 1-cell stage. Overall, we report the establishment of a zebrafish inbred strain, M-AB, which is capable of regular breeding and genetic manipulation. This strain will be useful for the analysis of genetically susceptible phenotypes such as behaviors, microbiome features and drug susceptibility.

High-speed system to generate congenic strains in medaka.

The congenic strain, an inbred strain containing a small genomic region from another strain, is a powerful tool to assess the phenotypic effect of polymorphisms and/or mutations in the substituted genomic region. Recent substantial progress in the genetic studies of complex traits increases the necessity of congenic strains and, therefore, a quick breeding system for congenic strains has become increasingly important in model organisms such as mouse and medaka. Traditionally, more than ten generations are necessary to produce a congenic strain. In contrast, a quick method has been reported previously for the mouse, in which the use of genetic markers reduces the required number of backcross generations to about a half that of the traditional method, so that it would take around six generations to obtain a congenic strain. Here, we present an even quicker congenic production system, which takes only about four generations. The system can produce medaka congenic strains having part of the HNI-II (an inbred medaka strain derived from the northern Japanese population, Oryzias sakaizumii) genome in the HdrR-II1 (another inbred strain from the southern Japanese population, O. latipes) background. In this system, the availability of frozen sperm and genotype data of the BC1 male population makes it possible to start marker-assisted congenic production after obtaining the BC2 population. Our evaluation revealed that the system could work well to increase the percentage of recipient genome as expected, so that a congenic strain may be obtained in about one year.

Pattern of fin rays along the antero-posterior axis based on their connection to distal radials.

BACKGROUND: Teleost paired fins are composed of two endoskeletal domains, proximal and distal radials, and an exoskeletal domain, the fin ray. The zebrafish pectoral fin displays elaborately patterned radials along the anteroposterior (AP) axis. Radials are considered homologous to tetrapod limb skeletons, and their patterning mechanisms in embryonic development are similar to those of limb development. Nevertheless, the pattern along the AP axis in fin rays has not been well described in the zebrafish pectoral fin, although several recent reports have revealed that fin ray development shares some cellular and genetic properties with fin/limb endoskeleton development. Thus, fin ray morphogenesis may involve developmental mechanisms for AP patterning in the fin/limb endoskeleton, and may have a specific pattern along the AP axis. RESULTS: We conducted detailed morphological observations on fin rays and their connection to distal radials by comparing intra- and inter-strain zebrafish specimens. Although the number of fin rays varied, pectoral fin rays could be categorized into three domains along the AP axis, according to the connection between the fin rays and distal radials; additionally, the number of fin rays varied in the posterior part of the three domains. This result was confirmed by observation of the morphogenesis process of fin rays and distal radials, which showed altered localization of distal radials in the middle domain. We also evaluated the expression pattern of lhx genes, which have AP patterning activity in limb development, in fin rays and during distal radial development and found these genes to be expressed during morphogenesis in both fin rays and distal radials. CONCLUSION: The fin ray and its connection to the endoskeleton are patterned along the AP axis, and the pattern along the AP axis in the fin ray and the radial connection is constructed by the developmental mechanism related to AP patterning in the limb/fin bud. Our results indicate the possibility that the developmental mechanisms of fin rays and their connection are comparable to those of the distal element of the limb skeleton.

Abnormal nuclear morphology is independent of longevity in a zmpste24-deficient fish model of Hutchinson-Gilford progeria syndrome (HGPS).

Lamin is an intermediate protein underlying the nuclear envelope and it plays a key role in maintaining the integrity of the nucleus. A defect in the processing of its precursor by a metalloprotease, ZMPSTE24, results in the accumulation of farnesylated prelamin in the nucleus and causes various diseases, including Hutchinson-Gilford progeria syndrome (HGPS). However, the role of lamin processing is unclear in fish species. Here, we generated zmpste24-deficient medaka and evaluated their phenotype. Unlike humans and mice, homozygous mutants did not show growth defects or lifespan shortening, despite lamin precursor accumulation. Gonadosomatic indices, blood glucose levels, and regenerative capacity of fins were similar in 1-year-old mutants and their wild-type (WT) siblings. Histological examination showed that the muscles, subcutaneous fat tissues, and gonads were normal in the mutants at the age of 1 year. However, the mutants showed hypersensitivity to X-ray irradiation, although p53target genes, p21 and mdm2, were induced 6 h after irradiation. Immunostaining of primary cultured cells from caudal fins and visualization of nuclei using H2B-GFP fusion proteins revealed an abnormal nuclear shape in the mutants both in vitro and in vivo. The telomere lengths were significantly shorter in the mutants compared to WT. Taken together, these results suggest that zmpste24-deficient medaka phenocopied HGPS only partially and that abnormal nuclear morphology and lifespan shortening are two independent events in vertebrates.

Construction of the Inbred Strain.

Genetically homogeneous populations such as inbred strains are valuable experimental tools in various fields of biomedical analyses. In many animals, inbred strains are established by consecutive sib-pair mating for a minimum of 20 generations. As the generation proceeds, fitness of the population reduces usually. Therefore, in order to establish inbred strains, the important point is the selection of pairs in good condition at each generation. Here, I describe the procedure and tips for generating inbred strains in zebrafish.

Evolutionary diversification of MCM3 genes in Xenopus laevis and Danio rerio.

Embryonic cell cycles of amphibians are rapid and lack zygotic transcription and checkpoint control. At the mid-blastula transition, zygotic transcription is initiated and cell divisions become asynchronous. Several cell cycle-related amphibian genes retain 2 distinct forms, maternal and zygotic, but little is known about the functional differences between these 2 forms of proteins. The minichromosome maintenance (MCM) 2-7 complex, consisting of 6 MCM proteins, plays a central role in the regulation of eukaryotic DNA replication. Almost all eukaryotes retain just a single MCM gene for each subunit. Here we report that Xenopus and zebrafish have 2 copies of MCM3 genes, one of which shows a maternal and the other a zygotic expression pattern. Phylogenetic analysis shows that the Xenopus and zebrafish zygotic MCM3 genes are more similar to their mammalian MCM3 ortholog, suggesting that maternal MCM3 was lost during evolution in most vertebrate lineages. Maternal MCM3 proteins in these 2 species are functionally different from zygotic MCM3 proteins because zygotic, but not maternal, MCM3 possesses an active nuclear localization signal in its C-terminal region, such as mammalian MCM3 orthologs do. mRNA injection experiments in zebrafish embryos show that overexpression of maternal MCM3 impairs proliferation and causes developmental defects, whereas zygotic MCM3 has a much weaker effect. This difference is brought about by the difference in their C-terminal regions, which contain putative nuclear localization signals; swapping the C-terminal region between maternal and zygotic genes diminishes the developmental defects. This study suggests that evolutionary diversification has occurred in MCM3 genes, leading to distinct functions, possibly as an adaption to the rapid DNA replication required for early development of Xenopus and zebrafish.

Genetic control of startle behavior in medaka fish.

Genetic polymorphisms are thought to generate intraspecific behavioral diversities, both within and among populations. The mechanisms underlying genetic control of behavioral properties, however, remain unclear in wild-type vertebrates, including humans. To explore this issue, we used diverse inbred strains of medaka fish (Oryzias latipes) established from the same and different local populations. Medaka exhibit a startle response to a visual stimulus (extinction of illumination) by rapidly bending their bodies (C-start) 20-ms after the stimulus presentation. We measured the rates of the response to repeated stimuli (1-s interval, 40 times) among four inbred strains, HNI-I, HNI-II, HO5, and Hd-rR-II1, and quantified two properties of the startle response: sensitivity (response rate to the first stimulus) and attenuation of the response probability with repeated stimulus presentation. Among the four strains, the greatest differences in these properties were detected between HNI-II and Hd-rR-II1. HNI-II exhibited high sensitivity (approximately 80%) and no attenuation, while Hd-rR-II1 exhibited low sensitivity (approximately 50%) and almost complete attenuation after only five stimulus presentations. Our findings suggested behavioral diversity of the startle response within a local population as well as among different populations. Linkage analysis with F2 progeny between HNI-II and Hd-rR-II1 detected quantitative trait loci (QTL) highly related to attenuation, but not to sensitivity, with a maximum logarithm of odds score of 11.82 on linkage group 16. The three genotypes (homozygous for HNI-II and Hd-rR-II1 alleles, and heterozygous) at the marker nearest the QTL correlated with attenuation. Our findings are the first to suggest that a single genomic region might be sufficient to generate individual differences in startle behavior between wild-type strains. Further identification of genetic polymorphisms that define the behavioral trait will contribute to our understanding of the neural mechanisms underlying behavioral diversity, allowing us to investigate the adaptive significance of intraspecific behavioral polymorphisms of the startle response.

Properties of gene knockdown system by vector-based siRNA in zebrafish.

RNA interference (RNAi) has emerged as a powerful tool to silence specific genes. Vector-based RNAi systems have been developed to downregulate targeted genes in a spatially and temporally regulated fashion both in vitro and in vivo. The zebrafish (Danio rerio) is a model animal that has been examined based on a wide variety of biological techniques, including embryonic manipulations, forward and reverse genetics, and molecular biology. However, a heritable and tissue-specific knockdown of gene expression has not yet been developed in zebrafish. We examined two types of vector, which produce small interfering RNA (siRNA), the direct effector in RNAi system; microRNA (miRNA) process mimicking vectors with a promoter for RNA polymerase II and short hairpin RNA (shRNA) expressing vector through a promoter for RNA polymerase III. Though gene-silencing phenotypes were not observed in the miRNA process mimicking vectors, the transgenic embryos of the second vector (Tg(zU6-shGFP)), shRNA expressing vector for enhanced green fluorescence protein, revealed knockdown of the targeted gene. Interestingly, only the embryos from Tg(zU6-shGFP) female but not from the male fish showed the downregulation. Comparison of the quantity of siRNA produced by each vector indicates that the vectors tested here induced siRNA, but at low levels barely sufficient to silence the targeted gene.

Genetic analysis of vertebral regionalization and number in medaka (Oryzias latipes) inbred lines.

Vertebral number is the most variable trait among vertebrates. In addition to the vertebral number, the ratio of abdominal to caudal vertebrae is a variable trait. The vertebral number and the ratio of abdominal to caudal vertebrae contribute to vertebrate diversity. It is very interesting to know how to determine the vertebral number and the ratio of abdominal to caudal vertebrae. In this study, we identify differences in the vertebral number and the ratio of abdominal vertebrae to vertebral number between two inbred lines of medaka, namely, Hd-rRII1 and Kaga. To identify the genetic factor of those differences, we performed quantitative trait locus (QTL) analysis for vertebral number and the ratio of abdominal vertebrae to vertebral number using 200 F(2) fish. Our results show a suggestive QTL of the ratio of abdominal vertebrae to vertebral number on chromosome 15, and five QTL of vertebral number on chromosomes 1, 10, 11, 17, and 23. The QTL on chromosome 15 contains hoxDb cluster genes. The QTL of vertebral number include some genes related to the segmentation clock and axial elongation. In addition, we show that the difference in vertebral number between two inbred lines is derived from differences in the anteroposterior length of somites. Our results emphasize that the developmental process should be considered in genetic analyses for vertebral number.

Production of zebrafish offspring from cultured spermatogonial stem cells.

Germ-line stem cells have the potential to be a very powerful tool for modifying the genetic information of individual animals. As a first step to use spermatogonial stem cells (SSCs) to enable genetic modification, we here describe effective long-term culture conditions for propagating zebrafish SSCs and for the production of offspring from these cultured SSCs after their differentiation into sperm in transplanted testicular cell aggregates. Dissociated testicular cells were cultured in specific medium with some modified supplements, including several mammalian growth factors. The spermatogonia actively proliferated and retained the expression of exogenous green fluorescent protein under the control of vas and sox17 promoters and also of promyelocytic leukemia zinc finger (Plzf), a marker of undifferentiated spermatogonia, after 1 month in culture. This is a longer period than the entire natural spermatogenic cycle (from SSCs to sperm). The use of subcutaneously grafted aggregates of these cultured spermatogonia and freshly dissociated testicular cells showed that these SSCs could undergo self-renewal and differentiation into sperm. Artificial insemination of these grafted aggregates successfully produced offspring. This culture method will facilitate the identification of new factors for the maintenance of SSCs and enable the future enrichment of genetically modified SSCs that will produce offspring in zebrafish.

Evaluation of Sycp3, Plzf and Cyclin B3 expression and suitability as spermatogonia and spermatocyte markers in zebrafish.

Recent studies in mammals have revealed the heterogeneity of spermatogonial populations which contain differentiated and undifferentiated cells that further divide into actual stem cells and potential stem cells. In fish however, there are no functional definitions, and very few molecular markers, for germ cells. In our present study, specific antibodies were raised against Sycp3, Plzf and Cyclin B3 in zebrafish and then used to determine the localization of these proteins in the testis. We wished to confirm whether these molecules were potential markers for spermatocytes and spermatogonia. Immunohistochemical observations revealed that Sycp3 is specifically localized in spermatocytes in typical nuclear patterns at each meiotic stage. Plzf was found to be localized in the nucleus of both type A and type B spermatogonia until the 8-cell clone, similar to the pattern in Plzf-positive A(single)-A(aligned) undifferentiated spermatogonia in rodents. In addition to Plzf, the localization of Cyclin B3 was predominantly detected in the nuclei of type A and early type B spermatogonia until the 16-cell clone. Additionally, Cyclin B3 protein signals were detected in germ cells in large cysts, possibly corresponding to spermatocytes at the preleptotene stage. Our present data thus show that these molecules have properties that will enable their use as markers of spermatocytes and early spermatogonia in zebrafish.

Erythropoiesis failure due to RPS19 deficiency is independent of an activated Tp53 response in a zebrafish model of Diamond-Blackfan anaemia.

Diamond-Blackfan anaemia (DBA) is a cancer-prone genetic disorder characterized by pure red-cell aplasia and associated physical deformities. The ribosomal protein S19 gene (RPS19) is the most frequently mutated gene in DBA (~25%). TP53-mediated cell cycle arrest and/or apoptosis in erythroid cells have been suggested to be major factors for DBA development, but it is not clear why mutations in the ubiquitously expressed RPS19 gene specifically affect erythropoiesis. Previously, we showed that RPS19 deficiency in zebrafish recapitulates the erythropoietic and developmental phenotypes of DBA, including defective erythropoiesis with severe anaemia. In this study, we analysed the simultaneous loss-of-function of RPS19 and Tp53 in zebrafish to investigate the role of Tp53 in the erythroid and morphological defects associated with RPS19 deficiency. Co-inhibition of Tp53 activity rescued the morphological abnormalities, but did not alleviate erythroid aplasia in RPS19-deficient zebrafish. In addition, knockdown of two other RP genes, rps3a and rpl36a, which result in severe morphological abnormalities but only mild erythroid defects, also elicited an activated Tp53 response. These results suggest that a Tp53-independent but RPS19-dependent pathway could be responsible for defective erythropoiesis in RPS19-deficient zebrafish.

Generation of highly homogeneous strains of zebrafish through full sib-pair mating.

Genetically homogeneous populations, such as inbred strains, are powerful experimental tools that are ideally suited for studying immunology, cancer, and genetics of complex traits. The zebrafish, Danio rerio, has been underutilized in these research areas because homogeneous strains of experimental fish have not been available in tractable condition. Here, we attempted to inbreed two zebrafish wild-type strains, Tübingen and India, through full sib-pair mating. Although the inbred Tübingen strain failed to thrive and was lost after 13 generations, an inbred India strain (IM) has been maintained successfully. The IM strain has endured 16 generations of inbreeding and has maintained a healthy condition. Two additional strains, IM12m and IM14m, were established as closed colonies from the branches of the IM strain. Genotype analyses using genetic markers revealed a dramatic decrease in polymorphisms (62% dropped to 5%) in both IM (generation 14) and the two closed colonies. This indicates a high level of homogeneity in these strains. Furthermore, scale transplantations between individuals within each strain were successful. These data suggest that extremely homogeneous zebrafish strains have been established, thereby creating a valuable resource for practical application.

Regeneration of spermatogenesis and production of functional sperm by grafting of testicular cell aggregates in Zebrafish (Danio rerio).

The self-renewal and differentiation of spermatogonial stem cells (SSCs) is essential for the continuous production of sperm throughout life in male vertebrates. The development of a functional assay to analyze these properties in isolated SSCs remains necessary. In our current study, we have developed a transplantation method for testicular cell aggregates in zebrafish (Danio rerio) in which allogeneic SSCs can undergo self-renewal and differentiation. The immature testes from juveniles are dissociated, aggregated by cultivation, and then transplanted under the abdominal skin of the recipient fish. The grafted aggregates reconstitute the appropriate testicular structures, including the lobule structure, consisting of basement membrane and interstitial steroid-producing cells on the outside, and the cysts, which comprise germ cell clusters and surrounding Sertoli cells. Bromodeoxyuridine incorporation analysis indicated that continuous spermatogenesis is maintained for at least 6 mo in the reconstituted testis. Moreover, when the sperm generated from the aggregates at 3 mo postgrafting were used for artificial insemination, fertilized eggs were obtained that developed sexually mature fish. These results suggest that self-renewal of SSCs takes place in reconstituted testes under the abdominal skin and that their differentiating progeny can develop into functional sperm. Furthermore, allogeneic spermatogonia were also found to proliferate and differentiate into sperm in these grafts. Our method of grafting testicular cell aggregates should thus prove useful not only analyzing the stem cell ability of an individual SSC but also for the production of progeny from cultured SSCs or SSCs of sterile mutants with somatic cell defects.

Nodal/Bozozok-independent induction of the dorsal organizer by zebrafish cell lines.

Formation of the dorsal organizer (Spemann organizer) is an important process in early vertebrate development. In zebrafish, two molecular cascades--Bozozok/Dharma (Boz) and Nodal signaling--act in parallel to induce the dorsal organizer. However, the complete molecular mechanism regulating this event remains unclear. Here we report that zebrafish cell lines derived from various developmental stages can induce a secondary axis when they are implanted into the mid-blastula but not the early gastrula. The implanted cells themselves did not differentiate, but instead induced ectopic expression of dorsal organizer markers in cells around the implanted cells and induced notochord formation in the secondary axis. These results indicate that cultured cell lines have the ability to induce a secondary axis through the initiation of dorsal organizer activity. However, ectopic expression of boz and sqt were not observed in cultured cells. In addition, implanted cell lines could induce the dorsal organizer even in maternal-zygotic one-eyed pinhead mutants, which are not responsive to Nodal signaling. Finally, the Nodal signaling pathway was not activated following implantation of cultured cells. Collectively, these data suggest that zebrafish cell lines induce the dorsal organizer independent of the boz and Nodal signaling pathways.

Genetic analysis of craniofacial traits in the medaka.

Family and twin studies suggest that a substantial genetic component underlies individual differences in craniofacial morphology. In the current study, we quantified 444 craniofacial traits in 100 individuals from two inbred medaka (Oryzias latipes) strains, HNI and Hd-rR. Relative distances between defined landmarks were measured in digital images of the medaka head region. A total of 379 traits differed significantly between the two strains, indicating that many craniofacial traits are controlled by genetic factors. Of these, 89 traits were analyzed via interval mapping of 184 F(2) progeny from an intercross between HNI and Hd-rR. We identified quantitative trait loci for 66 craniofacial traits. The highest logarithm of the odds score was 6.2 for linkage group (LG) 9 and 11. Trait L33, which corresponds to the ratio of head length to head height at eye level, mapped to LG9; trait V15, which corresponds to the ratio of snout length to head width measured behind the eyes, mapped to LG11. Our initial results confirm the potential of the medaka as a model system for the genetic analysis of complex traits such as craniofacial morphology.

Genetic linkage map of medaka with polymerase chain reaction length polymorphisms.

With recent improvements in genetic and genomic infrastructures, great interest has been taken in genetic dissection of multi-factorial traits. A genetic map consisting of markers that are highly polymorphic and rapidly genotyped is essential for the genetic mapping of such a complex trait. Medaka, Oryzias latipes, is an excellent model system for genetic studies. To promote genetic mapping of complex traits in medaka we developed the first high-throughput and genome-wide marker set in the organism by using its genomic information and the bioinformatic techniques. We tested 545 primer pairs and obtained 265 co-dominant markers between two inbred strains, HNI and Hd-rR. Our map, consisting of 231 uniquely mapped markers, covers 1257.3 centimorgan (cM) of the medaka genome with an average interval distance of 5.4 cM. Furthermore, the newly designed markers were examined for polymorphisms among six medaka inbred strains: HNI, Hd-rR and four additional strains. Most of our markers are simple sequence length polymorphisms (SSLPs) and can be rapidly genotyped by an automated system under a single polymerase chain reaction (PCR) condition. Together with the genotyping data of six medaka inbred strains, our new marker set provides a powerful tool for genome-wide analysis of complex biological phenomena found widely in medaka populations.

Whole genome association study of rheumatoid arthritis using 27 039 microsatellites.

A major goal of current human genome-wide studies is to identify the genetic basis of complex disorders. However, the availability of an unbiased, reliable, cost efficient and comprehensive methodology to analyze the entire genome for complex disease association is still largely lacking or problematic. Therefore, we have developed a practical and efficient strategy for whole genome association studies of complex diseases by charting the human genome at 100 kb intervals using a collection of 27,039 microsatellites and the DNA pooling method in three successive genomic screens of independent case-control populations. The final step in our methodology consists of fine mapping of the candidate susceptible DNA regions by single nucleotide polymorphisms (SNPs) analysis. This approach was validated upon application to rheumatoid arthritis, a destructive joint disease affecting up to 1% of the population. A total of 47 candidate regions were identified. The top seven loci, withstanding the most stringent statistical tests, were dissected down to individual genes and/or SNPs on four chromosomes, including the previously known 6p21.3-encoded Major Histocompatibility Complex gene, HLA-DRB1. Hence, microsatellite-based genome-wide association analysis complemented by end stage SNP typing provides a new tool for genetic dissection of multifactorial pathologies including common diseases.

Novel algorithm for automated genotyping of microsatellites.

Microsatellites or short tandem repeats (STRs) are abundant in the human genome with easily assayed polymorphisms, providing powerful genetic tools for mapping both Mendelian and complex traits. Microsatellite genotyping requires detection of the products of polymerase chain reaction (PCR) amplification by electrophoresis, and analysis of the peak data for discrimination of the true allele. A high-throughput genotyping approach requires computer-based automation at both the detection and analysis phases. In order to achieve this, complicated peak patterns from individual alleles must be interpreted in order to assign alleles. Previous methods consider limited types of noise peaks and cannot provide enough accuracy. By pattern recognition of various types of noise peaks, such as stutter peaks and additional peaks, we have achieved an overall average accuracy of 94% for allele calling in our actual data. Our algorithm is crucial for a high-throughput genotyping system for microsatellite markers by reducing manual editing and human errors.

Inhibition of BMP activity by the FGF signal promotes posterior neural development in zebrafish.

The expression patterns of region-specific neuroectodermal genes and fate-map analyses in zebrafish gastrulae suggest that posterior neural development is initiated by nonaxial signals, distinct from organizer-derived secreted bone morphogenetic protein (BMP) antagonists. This notion is further supported by the misexpression of a constitutively active form of zebrafish BMP type IA receptor (CA-BRIA) in the zebrafish embryos. It effectively suppressed the anterior neural marker, otx2, but not the posterior marker, hoxb1b. Furthermore, we demonstrated that the cells in the presumptive posterior neural region lose their neural fate only when CA-BRIA and Xenopus dominant-negative fibroblast growth factor (FGF) receptors (XFD) are coexpressed. The indications are that FGF signaling is involved in the formation of the posterior neural region, counteracting the BMP signaling pathway within the target cells. We then examined the functions of Fgf3 in posterior neural development. Zebrafish fgf3 is expressed in the correct place (dorsolateral margin) and at the correct time (late blastula to early gastrula stages), the same point that the most precocious posterior neural marker, hoxb1b, is first activated. Unlike other members of the FGF family, Fgf3 had little mesoderm-inducing activity. When ectopically expressed, Fgf3 expands the neural region with suppression of anterior neural fate. However, this effect was mediated by Chordino (zebrafish Chordin), because Fgf3 induces chordino expression in the epiblast and Fgf3-induced neural expansion was substantially suppressed in dino mutants with mutated chordino genes. The results obtained in the present study reveal multiple actions of the FGF signal on neural development: it antagonizes BMP signaling within posterior neural cells, induces the expression of secreted BMP antagonists, and suppresses anterior neural fate.