Tadao Kitagawa

Large-scale isolation of ESTs from medaka embryos and its application to medaka developmental genetics

The medaka is becoming an attractive model organism for the study of vertebrate early development and organogenesis and large-scale mutagenesis projects that are aimed at creating developmentally defective mutants are now being conducted by several groups in Japan. To strengthen the study of medaka developmental genetics, we have conducted a large-scale isolation of ESTs from medaka embryos and developed tools that facilitate mutant analysis. In this study, we have characterized a total of 132,082 sequences from both ends of cloned insert cDNAs from libraries generated at different stages of medaka embryo development. Clustering analysis with 3-prime sequences finally identified a total of 12,429 clusters. As a pilot analysis, 924 clusters were subjected to in situ hybridization to determine the spatial localization of their transcripts. Using EST sequence data generated in the present study, a 60-mer oligonucleotide microarray with 8,091 unigenes (Medaka Microarray 8K) was constructed and tested for its usefulness in expression profiling. Furthermore, we have developed a rapid and reliable mutant mapping system using a set of mapped EST markers (M-marker 2003) that covers the entire medaka genome. These resources will accelerate medaka mutant analyses and make an important contribution to the medaka genome project.

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, Kupffers 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.

Phylogeography and the maternal origin of the tetraploid form of the Japanese spined loach, Cobitis biwae, revealed by mitochondrial DNA analysis

The Japanese spined loach Cobitis biwae includes the tetraploid form, which has 96 chromosomes, as well as the diploid form, which has 48 chromosomes. In the present study, we analyzed the mitochondrial DNA (mtDNA) to examine the genetic relationships among 82 populations of diploid–tetraploid complexes of C. biwae. Restriction fragment length polymorphism (RFLP) analysis of the ND1 region on 202 individuals revealed that C. biwae contains three genetically divergent major groups corresponding to geographical proximities (the Eastern, Western, and Kochi groups). Phylogenetic analyses (neighbor-joining, NJ and maximum-parsimony, MP) of a part of the cytochrome b gene sequence (748 bp) in 31 individuals supported the three major groups recognized by RFLP, and indicated considerable genetic differentiation between the Western group and the other two groups (average, 15.2%). The genetic relationship and distribution pattern of the three major groups hypothesized two major dispersions of C. biwae during the middle Miocene: first, the ancestor of the Eastern and Kochi groups had spread from the West through wide regions of present-day Honshu and Shikoku Islands, and following that period, the ancestor of the Western group with a different mtDNA composition, probably resulting from the mtDNA introgression from C. takatsuensis, moved into western Japan and pushed the former north eastward. All tetraploid form populations were included in the Western group and treated as a monophyletic cluster with low genetic divergence. It is notable that two diploid populations geographically adjacent to the tetraploid range were genetically closely related to the tetraploid forms. This result suggests that these diploid populations were directly related to the maternal origin of the tetraploid form.

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 γ1 (lamc1), providing a unique framework to study the role of extracelluar 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.

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 Kupffers 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. Developmental Dynamics 236:2298–2310, 2007.

Genetic relationships among the Japanese and Korean striated spined loach complex (Cobitidae : Cobitis) and their phylogenetic positions

The “striata complex,” a group of spined loaches included in the genus Cobitis and characterized by a striped coloration pattern on the lateral midline, is distributed in rivers in northeastern Asia to western Japan. The complex comprises 2 continental species (Cobitis tetralineata and Cobitis lutheri) and 3 Japanese races of species rank (large race, middle race, and small race), the small race further comprising 6 local forms of subspecific rank (Tokai form, Biwa form, Yodo form, Sanyo form, San-in form, and Kyushu form). Previous karyological studies have revealed that the large race is an allotetraploid, the others being diploid. In this study, mitochondrial (mt) DNA analyses were conducted for 30 diploid populations of the Cobitis striata complex from Japan and Korea to examine: (1) their phylogenetic relationships and the position of the complex among the major lineages of Cobitis; and (2) the genetic relationships among the Japanese and Korean populations. The results, based on cytochrome b sequences (724 base pairs) analyzed with those of the main lineage of European and Japanese Cobitis, indicated that the striata complex should be considered as a monophyletic group, which evolved in northeastern Asia. Initially considered as a subspecies of Cobitis taenia, widely distributed from Europe to Asia, the striata complex does not have a sister-relationship with the former. Although the Korean species C. tetralineata was formerly believed to be closely related to the middle race in Japan, and a second continental species, C. lutheri, closely related to the Kyushu or San-in forms of the small race in Japan, the trees resulting from the present study revealed that the two Korean species were clustered with each other and separated from all Japanese races.

Origin of the two major distinct mtDNA clades of the Japanese population of the oriental weather loach Misgurnus anguillicaudatus (Teleostei: Cobitidae)

Abstract. The oriental weather loach Misgurnus anguillicaudatus (Teleostei: Cobitidae) inhabits the waters of East Asia including Japanese Islands. The Japanese population of M. anguillicaudatus includes two major mitochondrial DNA (mtDNA) clades, but their evolutionary origin is unknown. In this study, we conducted phylogeographic analyses of M. anguillicaudatus that were based on mtDNA cytochrome b sequences to clarify the evolutionary origin of the two distinct mtDNA clades. This newly obtained data were integrated with the mtDNA sequence data obtained in previous studies and reanalysed. The results showed that one major clade originated because of mtDNA introgression from a loach of the genus Cobitis. The geographic range of the populations carrying non-introgressed mtDNA tended to be limited to the peripheral areas of the Japanese Islands, whereas the range of the populations carrying introgressed mtDNA was spread over wide regions of the Japanese Islands. These distribution and divergence patterns suggested that M. anguillicaudatus populations carrying introgressed mtDNA have spread and replaced the range of populations carrying non-introgressed mtDNA.

Current status of genetic disturbances in wild medaka populations (Oryzias latipes species complex) in Japan

We revealed the range and current status of genetic disturbances in wild medaka populations (Oryzias latipes species complex) using two DNA markers (cytb gene and b-marker). Genetic disturbances were detected in many wild populations throughout Japan and were primarily caused by artificial introduction of the commercial medaka variety, himedaka. We identified native medaka populations without introgressions, which may be significant conservation targets. To conserve the native genetic diversity of the medaka species complex, further introduction of himedaka should be prevented by educating the public about the current status and risks of introducing non-native medaka varieties into the wild.

Differences in the behavior and ecology of wild type medaka (Oryzias latipes complex) and an orange commercial variety (himedaka)

Genetic disturbance in wild populations of medaka (Oryzias latipes complex) has been mainly caused by the introduction of the orange-red commercial variety medaka (himedaka) in Japan. To examine whether survival, reproduction, and species recognition would be influenced by this difference in body coloration, we conducted three laboratory experiments (predatory pressure, mate choice, schooling behavior) using wild type medaka and himedaka. In the predation experiment using dark chub (Candidia temminckii) as a predator, himedaka were predated upon more often than wild type medaka. However, individuals did not choose mates or select schooling groups based on himedaka or wild type medaka phenotypes. The results indicate that himedaka receive higher predation pressure but are able to easily mate with wild type medaka in a natural environment. To conserve the genetic diversity of wild medaka populations, we need to control the risk of genetic disturbance caused by himedaka.

Natural hybridization between two Japanese medaka species ( Oryzias latipes and Oryzias sakaizumii ) observed in the Yura River basin, Kyoto, Japan

To determine the actual state of hybridization between two Japanese medaka species (Oryzias latipes and Oryzias sakaizumii) in their natural environment, we used nuclear DNA markers at 10 loci to analyze 215 individuals from eight wild populations in the middle reaches of the Yura River basin in Japan, where the two species are sympatric. Despite large genetic differentiation between the two species, reproductive isolation between them could not be confirmed. We also discussed the formation of the current distribution patterns of the two species and their hybridization zone in the Yura River basin.