FAK-mediated extracellular signals are essential for interkinetic nuclear migration and planar divisions in the neuroepithelium.

During the development of the vertebrate nervous system, mitosis of neural progenitor cells takes place near the lumen, the apical side of the neural tube, through a characteristic movement of nuclei known as interkinetic nuclear migration (INM). Furthermore, during the proliferative period, neural progenitor cells exhibit planar cell divisions to produce equivalent daughter cells. Here, we examine the potential role of extracellular signals in INM and planar divisions using the medaka mutant tacobo (tab). This tab mutant shows pleiotropic phenotypes, including neurogenesis, and positional cloning identified tab as laminin gamma1 (lamc1), providing a unique framework to study the role of extracellular signals in neurogenesis. In tab mutant neural tubes, a number of nuclei exhibit abnormal patterns of migration leading to basally mislocalized mitosis. Furthermore, the orientation of cell division near the apical surface is randomized. Probably because of these defects, neurogenesis is accelerated in the tab neural tube. Detailed analyses demonstrate that extracellular signals mediated by the FAK pathway regulate INM and planar divisions in the neuroepithelium, possibly through interaction with the intracellular dynein-motor system.

Rescue from oculocutaneous albinism type 4 using medaka slc45a2 cDNA driven by its own promoter.

Patients and vertebrate mutants with oculocutaneous albinism type 4 (OCA4) have mutations in the solute carrier family 45 member 2 (slc45a2) gene. However, there is no empirical evidence for this gene-phenotype relationship. There is a unique OCA4 mutant in medaka (b) that exhibits albinism only in the skin, but the mechanism underlying this phenotype is also unknown. In this study, we rescued medaka OCA4 phenotypes, in both the eyes and the skin, by micro-injection of an slc45a2-containing genomic fragment or slc45a2 cDNA driven by its own 0.9-kb promoter. We also identified a spontaneous nucleotide change of 339 bp in the promoter as the b mutation. There are multiple transcription start sites in medaka slc45a2, as in its human ortholog, and only the shortest and eye-specific mRNA is transcribed with the b mutation. Interestingly, we further revealed a conserved pyrimidine (Py)-rich sequence of approximately 10 bp in the promoter by medaka-pufferfish comparative genomics and verified that it plays an indispensable role for expression of slc45a2 in the skin. Further studies of the 0.9-kb promoter identified in this study should provide insights into the cis/trans-regulatory mechanisms underlying the ocular and cutaneous expression of slc45a2.

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.

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.

The DNA sequence of medaka chromosome LG22.

We report the genomic DNA sequence of a single chromosome (linkage group 22; LG22) of the small teleost fish medaka (Oryzias latipes) as a first whole chromosome sequence from a non-mammalian vertebrate. The order and orientation of 633 protein-coding genes were deduced from 18,803,338 bp of DNA sequence, providing the opportunity to analyze chromosome evolution of vertebrate genomes by direct comparison with the human genome. The average number of genes in the "conserved gene cluster" (CGC), a strict definition of "synteny" at the sequence basis, between medaka and human was 1.6. These and other data suggest that approximately 38.8% of pair-wise gene relationships would have been broken from their common ancestor in the human and medaka lineages and further imply that approx 20,000 (15,520-23,280) breaks would have occurred from the entire genome of the common ancestor. These breaks were generated mainly by intra-chromosomal shufflings at a specific era in the vertebrate lineage. These precise comparative genomics allowed us to identify the pieces of ancient chromosomes of the common vertebrate ancestor and estimate chromosomal evolution in the vertebrate lineage.

Targeted reduction of the DNA methylation level with 5-azacytidine promotes excision of the medaka fish Tol2 transposable element.

The Tol2 element of the medaka fish Oryzias latipes is a member of the hAT (hobo/Activator/Tam3) transposable element family. There is evidence for rapid expansion in the genome and throughout the species in the past but a high spontaneous transposition rate is not observed with current fish materials, suggesting that the Tol2 element and its host species have already acquired an interactive mechanism to control the transposition frequency. DNA methylation is a possible contributing factor, given its involvement with many other transposable elements. We therefore soaked embryos in 5-azacytidine, a reagent that causes reduction in the DNA methylation level, and examined amounts of PCR products reflecting the somatic excision frequency, obtaining direct evidence that exposure promotes Tol2 excision. Our results thus suggest that methylation of the genome DNA is a factor included in the putative mechanisms of control of transposition of the Tol2 element.

Vertebrate DNA transposon as a natural mutator: the medaka fish Tol2 element contributes to genetic variation without recognizable traces.

DNA-based transposable elements, or DNA transposons, transpose in a cut-and-paste fashion, involving excision from the chromosome. If this process affects the function of a host gene and the excision rate is high, any gene associated with such an element would clearly be in a genetically "unstable" state, and there are many examples of unstable genes in various organisms. However, none have hitherto been reported in vertebrates. We here document the finding of an unstable mutant gene in the medaka fish, Oryzias latipes, a useful model animal for vertebrate genetics and evolutionary studies. In an inbred strain, excision of the Tol2 element inserted in a pigmentation gene occurs spontaneously, giving rise to different heritable phenotypes and new mutant genes that carry different excision footprint sequences. The phenotypic mutation rate is as high as 2% per gamete, representing a 1000-fold increase from spontaneous mutation rates so far determined with the same organism. With mutations caused by insertion, and then excision, of transposons, one can no longer recognize participation of transposons in their generation. Thus, the impact of DNA transposons on vertebrate genomes may be, and may have been, larger than commonly supposed.

Comparative genomics of medaka and fugu.

A small freshwater fish medaka (Oryzias latipes) has been one of the most attractive experimental systems for research in genetics and developmental biology. We have formed an international consortium Medaka Genome Initiative (MGI) to collect and share various information and resources on medaka. The MGI has set an ambitious goal aiming at the complete sequencing of the medaka genome and as a feasibility study we have begun sequencing one particular chromosome, linkage group 22 (LG22) of approximately 22 Mb in size. Initial sequence analysis revealed unique features of the medaka genome in comparison to fugu genome.

Reversion mutation of ib oculocutaneous albinism to wild-type pigmentation in medaka fish.

We have previously identified three naturally occurring mutations in the medaka fish tyrosinase gene caused by transposable element insertions. Tyr-i(b) is one of these, containing the Tol2 element in the promoter region. Its homozygous carriers exhibit a weak oculocutaneous albino phenotype. We report here spontaneous reversion of the albino phenotype to the wild-type pigmentation, associated with excision of the Tol2 element. The newly arising mutant gene is inherited in the Mendelian fashion. Thus, oculocutaneous albinism is not strictly irreversible, at least in this organism and the results also indicate that the insertion of the Tol2 element is the main, and possibly the only, cause of the i(b) albinism. Importantly our data also suggest that medaka fish possess an active transposase.

Unprecedented intraspecific diversity of the MHC class I region of a teleost medaka, Oryzias latipes.

The major histocompatibility complex (MHC) is present at a single chromosomal locus of all jawed vertebrate analyzed so far, from sharks to mammals, except for teleosts whose orthologs of the mammalian MHC-encoded genes are dispersed at several chromosomal loci. Even in teleosts, several class IA genes and those genes directly involved in class I antigen presentation preserve their linkage, defining the teleost MHC class I region. We determined the complete nucleotide sequence of the MHC class I region of the inbred HNI strain of medaka, Oryzias latipes (northern Japan population-derived), from four overlapping bacterial artificial chromosome (BAC) clones spanning 540,982 bp, and compared it with the published sequence of the corresponding region of the inbred Hd-rR strain of medaka (425,935 bp, southern Japan population-derived) as the first extensive study of intraspecies polymorphisms of the ectotherm MHC regions. A segment of about 100 kb in the middle of the compared sequences encompassing two class Ia genes and two immunoproteasome subunit genes, PSMB8 and PSMB10, was so divergent between these two inbred strains that a reliable sequence alignment could not be made. The rest of the compared region (about 320 kb) showed a fair correspondence, and an approximately 96% nucleotide identity was observed upon gap-free segmental alignment. These results indicate that the medaka MHC class I region contains an approximately 100-kb polymorphic core, which is most probably evolving adaptively by accumulation of point mutations and extensive genetic rearrangements such as insertions, deletions, and duplications.

Germ cell mutagenesis in medaka fish after exposures to high-energy cosmic ray nuclei: A human model.

Astronauts beyond the Earth's orbit are exposed to high-energy cosmic-ray nuclei with high values of linear energy transfer (LET), resulting in much more biological damage than from x-rays or gamma-rays and may result in mutations and cancer induction. The relative biological effectiveness of these nuclei depends on the LET, rising to as high as approximately 50 at LET values of approximately 100-200 keV/microm. An endpoint of concern is germ cell mutations passed on to offspring, arising from exposure to these nuclei. A vertebrate model for germ cell mutation is Medaka fish (Oryzias latipes). We exposed wild type males to doses of 1 GeV per nucleon Fe nuclei or to 290 MeV per nucleon C nuclei. They were mated to females with recessive mutations at five-color loci. The transparent embryos from >100 days of mating (representing exposed sperm, spermatids, or spermatogonia) were observed so as to detect dominant lethal mutations and total color mutations, even though the embryos might not hatch. The relative number of mutant embryos as a function of dose were compared with those induced by gamma-rays. The relative biological effectiveness values for dominant lethal mutations and total color mutations for exposed sperm and spermatids were 1.3-2.1 for exposure to C nuclei and 1.5-3.0 for exposure to Fe nuclei. (The spermatogonial data were uncertain.) These low values, and the negligible number of viable mutations, compared with those for mutations in somatic cells and for neoplastic transformation, indicate that germ cell mutations arising from exposures to cosmic ray nuclei are not a significant hazard to astronauts.

A novel membrane guanylyl cyclase expressed in medaka (Oryzias latipes) intestine.

A novel membrane guanylyl cyclase (GC), OlGC9, was identified in the intestine of the medaka fish Oryzias latipes by the isolation of a full-length cDNA clone (3783 bp). Phylogenetic analysis indicated that OlGC9 belongs in the enterotoxin/guanylin receptor membrane GC subfamily. The nucleotide and deduced amino acid sequences of OlGC9 were highly homologous to those of OlGC6, another enterotoxin/guanylin receptor membrane GC in medaka fish. Linkage analysis of the medaka fish chromosome demonstrated that the OlGC9 gene was mapped to LG8, which distinguishes it from the OlGC6 gene. Determination of the cGMP concentrations in COS-7 cells expressed with OlGC9 indicated that Escherichia coli heat-stable enterotoxin (STa) stimulated the activity of OlGC9 in a concentration-dependent manner, although it did not activate the OlGC6 expressed in the COS-7 cells. The 5'-flanking region of the OlGC9 gene important for its transcription was partially determined using both CACO-2 cells and COS-1 cells, and was not found to be conserved with respect to either the mammalian GC-C gene or the OlGC6 gene.

Dose-rate effect on transgenerational mutation frequencies in spermatogonial stem cells of the medaka fish.

The estimation of transgenerational genetic risk of radiation exposure to non-human species is crucial for the protection of ecosystems. Here we determined the frequency of specific-locus mutations at the five pigmentation loci in medaka spermatogonial stem cells after gamma irradiation at 0.03 cGy/min and 95 cGy/min. At each total dose, the mutation frequency was significantly lower in the 0.03-cGy/min group than in the 95-cGy/min group, suggesting a dose-rate effect. The ratio of the induced mutation frequency at 0.03 cGy/min to that at 95 cGy/min was approximately 0.42 from 0 to 1.9 Gy and approximately 0.33 from 1.9 to 4.75 cGy. In the mouse, this ratio is estimated to be 0.33 (Russell and Kelly, Proc. Natl. Acad. Sci. USA 79, 542-544, 1982). It is thus possible that the magnitude of the dose-rate effect on transgenerational mutation frequencies is comparable between mouse and medaka spermatogonia, suggesting similar dose-rate effects among vertebrates.

Conserved function of medaka pink-eyed dilution in melanin synthesis and its divergent transcriptional regulation in gonads among vertebrates.

Medaka is emerging as a model organism for the study of vertebrate development and genetics, and its effectiveness in forward genetics should prove equal to that of zebrafish. Here, we identify by positional cloning a gene responsible for the medaka i-3 albino mutant. i-3 larvae have weakly tyrosinase-positive cells but lack strongly positive and dendritic cells, suggesting loss of fully differentiated melanophores. The region surrounding the i-3 locus is syntenic to human 19p13, but a BAC clone covering the i-3 locus contained orthologs located at 15q11-13, including OCA2 (P). Medaka P consists of 842 amino acids and shares approximately 65% identity with mammalian P proteins. The i-3 mutation is a four-base deletion in exon 13, which causes a frameshift and truncation of the protein. We detected medaka P transcripts in melanin-producing eyeballs and (putative) skin melanophores on embryos and an alternatively spliced form in the non-melanin-producing ovary or oocytes. The mouse p is similarly expressed in gonads, but not alternatively spliced. This is the first isolation of nonmammalian P, the functional mechanism of action of which has not yet been elucidated, even in mammals. Further investigation of the functions of P proteins and the regulation of their expression will provide new insight into body color determination and gene evolution.

Transgenerational genomic instability as revealed by a somatic mutation assay using the medaka fish.

We previously established a somatic mutation assay of the medaka wl (white leucophores) locus based on visual inspection, and showed that somatic mutations at paternally derived alleles frequently arise during the development of F1 embryos fertilized by sperm/late spermatids that had been exposed to gamma-rays. To further study such delayed mutations, we determined the frequency of mutant embryos obtained from three different crosses between irradiated males and non-irradiated females. When sperm and late spermatids were irradiated, the mutant frequency within non-irradiated maternally derived alleles was approximately 3 times higher than in the control group. In the F2 generation, however, no increase in mutant frequency was observed. Similarly, there was no significant increase in the F1 mutant frequency when stem spermatogonia were irradiated. These data suggest that irradiation of sperm and late spermatids can induce indirect mutations in F1 somatic cells, supporting the idea that genomic instability arises during F1 embryonic development. Moreover, such instability apparently arises most frequently when eggs are fertilized just after the sperm are irradiated.

Somatolactin selectively regulates proliferation and morphogenesis of neural-crest derived pigment cells in medaka.

Species-specific colors and patterns on animal body surfaces are determined primarily by neural-crest-derived pigment cells in the skin (chromatophores). However, even closely related species display widely differing patterns. These contrasting aspects of chromatophores (i.e., the fixed developmental control within species and extreme diversity among species) seem to be a curious and suitable subject for understanding evolution and diversity of organisms. Here we identify a gene responsible for medaka "color interfere" mutants by positional cloning. These mutants do not show any obvious morphological and physiological defects other than defects in chromatophore proliferation and morphogenesis. The mutation has been identified as an 11-base deletion in somatolactin, which causes truncation 91 aa upstream of the C terminus of the protein's 230 aa. Somatolactin transcription changed dramatically during morphological body color adaptation to different backgrounds. This genetic evidence explains somatolactin function. Studying this mutant will provide further insights into the development and regulation of chromatophores and clues for reassessing other functions of somatolactin suggested in other fish.

Medaka as a research organism: past, present and future.

This introductory review briefly describes the history of medaka as a research organism and the previous accomplishments of the medaka field. The medaka genome project currently underway through the efforts of an international consortium, the Medaka Genome Initiative, and the future prospects for medaka research, particularly for genomic analyses, are also discussed.

Genomic organization and developmental expression of globin genes in the teleost Oryzias latipes.

We isolated globin genes from a genomic DNA library of the drR strain of medaka Oryzias latipes, and walked on chromosome. The present study is the first demonstration of the full-length structure of globin gene locus in the teleosts. Two gene clusters were found. One cluster of 36 kbp consisted of nine globin genes and two pseudogenes. Based on structural and phylogenetic similarity of amino acid sequences, the cluster was named embryonic globin gene cluster (E1). The orientation of the genes was in (5')alpha0(3')-(3')beta1(5')-(5')alpha1(3')-(5')beta2(3')-(5')alpha2(3')-(3')alpha3(5')-(5')beta3(3')-(3')beta4(5')-(5')alpha4(3')-(3')psialpha(5')-(5')psibeta(3'). The other cluster of 20 kbp contained three globin genes ((3')ad.alpha1(5')-(5')ad.beta1(3')-(3')ad.alpha2(5')), and was named adult globin gene cluster (A1). Genetic linkage analysis clarified that E1 and A1 were mapped on linkage groups 8 and 19, respectively. The E1 cluster included other genes homologous to human EST clone KIAA0172, Sushi-1 retrotransposon, and protein 14 gene-like gene, while the A1 cluster linked to aquaporin-8 gene-like gene. The expression patterns of the genes were classified into four types: embryo-specific expression (alpha3, beta3, alpha4 and beta4), expression in embryo to young fish (alpha0, beta1, alpha1 and ad.alpha2), expression in young to adult fish (alpha2 and ad.alpha1) and successive expression in embryo to adult (ad.beta1).

A mutation in the gene for delta-aminolevulinic acid dehydratase (ALAD) causes hypochromic anemia in the medaka, Oryzias latipes.

A genetic screen for mutations affecting embryogenesis in the medaka, Oryzias latipes, identified a mutant, whiteout (who), that exhibited hypochromic anemia. The who mutant initially had the normal number of blood cells, but it then gradually decreased during the embryonic and larval stages. The blood cells in the who mutants show an elongated morphology and little hemoglobin activity. Genetic mapping localized who to the vicinity of a LG12 marker, olgc1. By utilizing the highly conserved synteny between medaka and pufferfish, we identified a gene for delta-aminolevulinic acid dehydratase (ALAD), which is the second enzyme in the heme synthetic pathway, as a candidate for who. We found a missense mutation in the alad gene that was tightly linked to the who phenotype, strongly suggesting that the hypochromic anemia phenotype in the who mutant is caused by a loss of the alad function. Thus, who mutants represent a model for the human disease ALAD-deficiency porphyria.