Retinoic acid-dependent establishment of positional information in the hindbrain was conserved during vertebrate evolution.

Zebrafish hoxb1b is expressed during epiboly in the posterior neural plate, with its anterior boundary at the prospective r4 region providing a positional cue for hindbrain formation. A similar function and expression is known for Hoxa1 in mice, suggesting a shared regulatory mechanism for hindbrain patterning in vertebrate embryos. To understand the evolution of the regulatory mechanisms of key genes in patterning of the central nervous system, we examined how hoxb1b transcription is regulated in zebrafish embryos and compared the regulatory mechanisms between mammals and teleosts that have undergone an additional genome duplication. By promoter analysis, we found that the expression of the reporter gene recapitulated hoxb1b expression when driven in transgenic embryos by a combination of the upstream 8.0-kb DNA and downstream 4.6-kb DNA. Furthermore, reporter expression expanded anteriorly when transgenic embryos were exposed to retinoic acid (RA) or LiCl, or injected with fgf3/8 mRNA, implicating the flanking DNA examined here in the responsiveness of hoxb1b to posteriorizing signals. We further identified at least two functional RA responsive elements in the downstream DNA that were shown to be major regulators of early hoxb1b expression during gastrulation, while the upstream DNA, which harbors repetitive sequences with apparent similarity to the autoregulatory sequence of mouse Hoxb1, contributed only to later hoxb1b expression, during somitogenesis. Possible implications in vertebrate evolution are discussed based on these findings.

Polycystic kidney disease in the medaka (Oryzias latipes) pc mutant caused by a mutation in the Gli-Similar3 (glis3) gene.

Polycystic kidney disease (PKD) is a common hereditary disease in humans. Recent studies have shown an increasing number of ciliary genes that are involved in the pathogenesis of PKD. In this study, the Gli-similar3 (glis3) gene was identified as the causal gene of the medaka pc mutant, a model of PKD. In the pc mutant, a transposon was found to be inserted into the fourth intron of the pc/glis3 gene, causing aberrant splicing of the pc/glis3 mRNA and thus a putatively truncated protein with a defective zinc finger domain. pc/glis3 mRNA is expressed in the epithelial cells of the renal tubules and ducts of the pronephros and mesonephros, and also in the pancreas. Antisense oligonucleotide-mediated knockdown of pc/glis3 resulted in cyst formation in the pronephric tubules of medaka fry. Although three other glis family members, glis1a, glis1b and glis2, were found in the medaka genome, none were expressed in the embryonic or larval kidney. In the pc mutant, the urine flow rate in the pronephros was significantly reduced, which was considered to be a direct cause of renal cyst formation. The cilia on the surface of the renal tubular epithelium were significantly shorter in the pc mutant than in wild-type, suggesting that shortened cilia resulted in a decrease in driving force and, in turn, a reduction in urine flow rate. Most importantly, EGFP-tagged pc/glis3 protein localized in primary cilia as well as in the nucleus when expressed in mouse renal epithelial cells, indicating a strong connection between pc/glis3 and ciliary function. Unlike human patients with GLIS3 mutations, the medaka pc mutant shows none of the symptoms of a pancreatic phenotype, such as impaired insulin expression and/or diabetes, suggesting that the pc mutant may be suitable for use as a kidney-specific model for human GLIS3 patients.

Mutation in the abcb7 gene causes abnormal iron and fatty acid metabolism in developing medaka fish.

The medaka fish (Oryzias latipes) is an emerging model organism for which a variety of unique developmental mutants have now been generated. Our recent mutagenesis screening of the medaka isolated a unique mutant that develops a fatty liver at larval stages. Positional cloning identified the responsible gene as medaka abcb7. Abcb7, a mitochondrial ABC (ATP binding cassette) half-transporter, has been implicated in iron metabolism. Recently, human Abcb7 was found to be mutated in X-linked sideroblastic anemia with cerebellar ataxia (XLSA/A). The homozygous medaka mutant exhibits abnormal iron metabolism in erythrocytes and accumulation of lipid in the liver. Microarray and in situ hybridization analyses demonstrated that the expression of genes involved in iron and lipid metabolisms are both affected in the mutant liver, suggesting novel roles of Abcb7 in the development of physiologically functional liver. The medaka abcb7 mutant thus could provide insights into the pathogenesis of XLSA/A as well as the normal function of the gene.

UTGB/medaka: genomic resource database for medaka biology.

Medaka (Oryzias latipes) is a small egg-laying freshwater teleost native to East Asia that has become an excellent model system for developmental genetics and evolutionary biology. The draft medaka genome sequence (700 Mb) was reported in June 2007, and its substantial genomic resources have been opened to the public through the University of Tokyo Genome Browser Medaka (UTGB/medaka) database. This database provides basic genomic information, such as predicted genes, expressed sequence tags (ESTs), guanine/cytosine (GC) content, repeats and comparative genomics, as well as unique data resources including (i) 2473 genetic markers and experimentally confirmed PCR primers that amplify these markers, (ii) 142,414 bacterial artificial chromosome (BAC) and 217,344 fosmid end sequences that amount to 15.0- and 11.1-fold clone coverage of the entire genome, respectively, and were used for draft genome assembly, (iii) 16,519,460 single nucleotide polymorphisms (SNPs), and 2 859 905 insertions/deletions detected between two medaka inbred strain genomes and (iv) 841 235 5'-end serial analyses of gene-expression (SAGE) tags that identified 344 266 transcription start sites on the genome. UTGB/medaka is available at: http://medaka.utgenome.org/.

Phenotypic analysis of a novel chordin mutant in medaka.

We have isolated and characterized a ventralized mutant in medaka (the Japanese killifish; Oryzias latipes), which turned out to have a mutation in the chordin gene. The mutant exhibits ventralization of the body axis, malformation of axial bones, over-bifurcation of yolk sac blood vessels, and laterality defects in internal organs. The mutant exhibits variability of phenotypes, depending on the culture temperature, from embryos with a slightly ventralized phenotype to those without any head and trunk structures. Taking advantages of these variable and severe phenotypes, we analyzed the role of Chordin-dependent tissues such as the notochord and Kupffer's vesicle (KV) in the establishment of left-right axis in fish. The results demonstrate that, in the absence of the notochord and KV, the medaka lateral plate mesoderm autonomously and bilaterally expresses spaw gene in a default state.

Right-elevated expression of charon is regulated by fluid flow in medaka Kupffer's vesicle.

Recent studies have revealed that a cilium-generated liquid flow in the node has a crucial role in the establishment of the left-right (LR) axis in the mouse. In fish, Kupffer's vesicle (KV), a teleost-specific spherical organ attached to the tail region, is known to have an equivalent role to the mouse node during LR axis formation. However, at present, there has been no report of an asymmetric gene expressed in KV under the control of fluid flow. Here we report the earliest asymmetric gene in teleost KV, medaka charon, and its regulation. Charon is a member of the Cerberus/DAN family of proteins, first identified in zebrafish. Although zebrafish charon was reported to be symmetrically expressed in KV, medaka charon displays asymmetric expression with more intense expression on the right side. This asymmetric expression was found to be regulated by KV flow because symmetric and up-regulated charon expression was observed in flow-defective embryos with immotile cilia or disrupted KV. Taken together, medaka charon is a reliable gene marker for LR asymmetry in KV and thus, will be useful for the analysis of the early steps downstream of the fluid flow.

The medaka draft genome and insights into vertebrate genome evolution.

Teleosts comprise more than half of all vertebrate species and have adapted to a variety of marine and freshwater habitats. Their genome evolution and diversification are important subjects for the understanding of vertebrate evolution. Although draft genome sequences of two pufferfishes have been published, analysis of more fish genomes is desirable. Here we report a high-quality draft genome sequence of a small egg-laying freshwater teleost, medaka (Oryzias latipes). Medaka is native to East Asia and an excellent model system for a wide range of biology, including ecotoxicology, carcinogenesis, sex determination and developmental genetics. In the assembled medaka genome (700 megabases), which is less than half of the zebrafish genome, we predicted 20,141 genes, including approximately 2,900 new genes, using 5'-end serial analysis of gene expression tag information. We found single nucleotide polymorphisms (SNPs) at an average rate of 3.42% between the two inbred strains derived from two regional populations; this is the highest SNP rate seen in any vertebrate species. Analyses based on the dense SNP information show a strict genetic separation of 4 million years (Myr) between the two populations, and suggest that differential selective pressures acted on specific gene categories. Four-way comparisons with the human, pufferfish (Tetraodon), zebrafish and medaka genomes revealed that eight major interchromosomal rearrangements took place in a remarkably short period of approximately 50 Myr after the whole-genome duplication event in the teleost ancestor and afterwards, intriguingly, the medaka genome preserved its ancestral karyotype for more than 300 Myr.

Mutant analyses reveal different functions of fgfr1 in medaka and zebrafish despite conserved ligand-receptor relationships.

Medaka (Oryzias latipes) is a small freshwater teleost that provides an excellent developmental genetic model complementary to zebrafish. Our recent mutagenesis screening using medaka identified headfish (hdf) which is characterized by the absence of trunk and tail structures with nearly normal head including the midbrain-hindbrain boundary (MHB). Positional-candidate cloning revealed that the hdf mutation causes a functionally null form of Fgfr1. The fgfr1hdf is thus the first fgf receptor mutant in fish. Although FGF signaling has been implicated in mesoderm induction, mesoderm is induced normally in the fgfr1hdf mutant, but subsequently, mutant embryos fail to maintain the mesoderm, leading to defects in mesoderm derivatives, especially in trunk and tail. Furthermore, we found that morpholino knockdown of medaka fgf8 resulted in a phenotype identical to the fgfr1hdf mutant, suggesting that like its mouse counterpart, Fgf8 is a major ligand for Fgfr1 in medaka early embryogenesis. Intriguingly, Fgf8 and Fgfr1 in zebrafish are also suggested to form a major ligand-receptor pair, but their function is much diverged, as the zebrafish fgfr1 morphant and zebrafish fgf8 mutant acerebellar (ace) only fail to develop the MHB, but develop nearly unaffected trunk and tail. These results provide evidence that teleost fish have evolved divergent functions of Fgf8-Fgfr1 while maintaining the ligand-receptor relationships. Comparative analysis using different fish is thus invaluable for shedding light on evolutionary diversification of gene function.

Development of the endoderm and gut in medaka, Oryzias latipes.

We performed an extensive analysis of endodermal development and gut tube morphogenesis in the medaka embryo by histology and in situ hybridization. The markers used in these analyses included sox17, sox32, foxA2, gata-4, -5, -6 and shh. sox17, sox32, foxA2, and gata-5 and -6 are expressed in the early endoderm to the onset of gut tube formation. Sections of medaka embryos hybridized with foxA2, a pan-endodermal marker during gut morphogenesis, demonstrated that gut tube formation is initiated in the anterior portion and that the anterior and mid/posterior gut undergo distinct morphogenetic processes. Tube formation in the anterior endoderm that is fated to the pharynx and esophagus is much delayed and appears to be independent of gut morphogenesis. The overall aspects of medaka gut development are similar to those of zebrafish, except that zebrafish tube formation initiates at both the anterior and posterior portions. Our results therefore describe both molecular and morphological aspects of medaka digestive system development that will be necessary for the characterization of medaka mutants.

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.