Hiroshi Mitani

Silver nanospheres are cytotoxic and genotoxic to fish cells

Nanoparticles are being widely investigated for a range of applications due to their unique physical properties. For example, silver nanoparticles are used in commercial products for their antibacterial and antifungal properties. Some of these products are likely to result in silver nanoparticles reaching the aquatic environment. As such, nanoparticles pose a health concern for humans and aquatic species. We used a medaka (Oryzias latipes) cell line to investigate the cytotoxicity and genotoxicity of 30nm diameter silver nanospheres. Treatments of 0.05, 0.3, 0.5, 3 and 5microg/cm(2) induced 80, 45.7, 24.3, 1 and 0.1% survival, respectively, in a colony forming assay. Silver nanoparticles also induced chromosomal aberrations and aneuploidy. Treatments of 0, 0.05, 0.1 and 0.3microg/cm(2) induced damage in 8, 10.8, 16 and 15.8% of metaphases and 10.8, 15.6, 24 and 24 total aberrations in 100 metaphases, respectively. These data show that silver nanoparticles are cytotoxic and genotoxic to fish cells.

Infrared laser-mediated local gene induction in medaka, zebrafish and Arabidopsis thaliana

Heat shock promoters are powerful tools for the precise control of exogenous gene induction in living organisms. In addition to the temporal control of gene expression, the analysis of gene function can also require spatial restriction. Recently, we reported a new method for in vivo, single‐cell gene induction using an infrared laser‐evoked gene operator (IR‐LEGO) system in living nematodes (Caenorhabditis elegans). It was demonstrated that infrared (IR) irradiation could induce gene expression in single cells without incurring cellular damage. Here, we report the application of IR‐LEGO to the small fish, medaka (Japanese killifish; Oryzias latipes) and zebrafish (Danio rerio), and a higher plant (Arabidopsis thaliana). Using easily observable reporter genes, we successfully induced gene expression in various tissues in these living organisms. IR‐LEGO has the potential to be a useful tool in extensive research fields for cell/tissue marking or targeted gene expression in local tissues of small fish and plants.

Retinoic acid signaling positively regulates liver specification by inducing wnt2bb gene expression in medaka

During vertebrate embryogenesis, the liver develops at a precise location along the endodermal primitive gut tube because of signaling delivered by adjacent mesodermal tissues. Although several signaling molecules have been associated with liver formation, the molecular mechanism that regulates liver specification is still unclear. We previously performed a screen in medaka to isolate mutants with impaired liver development. The medaka hio mutants exhibit a profound (but transient) defect in liver specification that resembles the liver formation defect found in zebrafish prometheus (prt) mutants, whose mutation occurs in the wnt2bb gene. In addition to their liver abnormality, hio mutants lack pectoral fins and die after hatching. Positional cloning indicated that the hio mutation affects the raldh2 gene encoding retinaldehyde dehydrogenase type2 (RALDH2), the enzyme principally responsible for retinoic acid (RA) biosynthesis. Mutations of raldh2 in zebrafish preclude the development of pectoral fins. Interestingly, in hio mutants, expression of wnt2bb in the lateral plate mesoderm (LPM) directly adjacent to the liver‐forming endoderm was completely lost. Conclusion: Our data reveal the unexpected finding that RA signaling positively regulates the wnt2bb gene expression required for liver specification in medaka. These results suggest that a common molecular mechanism may underlie liver and pectoral fin specification during piscine embryogenesis. (HEPATOLOGY 2009.)

Unrepairable DNA double-strand breaks that are generated by ionising radiation determine the fate of normal human cells

Summary After an exposure to ionising radiation, cells can quickly repair damage to their genomes; however, a few unrepairable DNA double-strand breaks (DSBs) emerge in the nucleus in a prolonged culture and perpetuate as long as the culture continues. These DSBs may be retained forever in cells such as non-dividing ageing tissues, which are resistant to apoptosis. We show that such unrepairable DSBs, which had been advocated by the classical target theory as the ‘radiation hit’, could account for permanent growth arrest and premature senescence. The unrepairable DSBs build up with repeated irradiation, which accounts for an accumulated dose. Because these DSBs tend to be paired, we propose that the untethered and ‘torn-off’ molecular structures at the broken ends of the DNA result in an alteration of chromatin structure, which protects the ends of the DNA from genomic catastrophe. Such biochemical responses are important for cell survival but may cause gradual tissue malfunction, which could lead to the late effects of radiation exposure. Thus, understanding the biology of unrepairable damage will provide new insights into the long-term effects of radiation.

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.

Effects of dioxin isomers on induction of AhRs and CYP1A1 in early developmental stage embryos of medaka (Oryzias latipes).

The aryl hydrocarbon receptor (AhR) binds to polyaromatic compounds, including dioxins, and enhances the expression of several target genes, including drug-metabolizing cytochrome P450s (CYP1As). Four AhR genes (AhR1b-1, AhR1b-2, AhR2a, and AhR2b) were identified in the medaka genome. The molecular machinery involved in the dioxin response has been clarified chiefly in mammals, although fish models, such as zebrafish (Danio rerio), medaka (Oryzias latipes), and Fundulus, are excellent candidates for examining the mechanisms of developmental dioxin toxicity. Using these fish models, several experimental studies investigating the induced expression of CYP1A1 and AhRs, including functional evaluations by 2378T4CDD exposure, have been performed. However, few studies have examined the exposure to other dioxin isomers and it is not certain whether similar induced expressions patterns and toxicity-mediating functions of CYP1A1, AhRs, and AhR repressor (AhRR) compare with 2378T4CDD exposure. In this study, we investigated the toxicity of 13 dioxin isomers, including 2378T4CDD, and the induced expression of AhRs, AhRR, and CYP1A1 (CYP1A1_ORYLA) in the early life stages of medaka embryos. After exposure to dioxin isomers for 24-48h, the expression of AhR2a and CYP1A1_ORYLA correlated to the dioxin toxicity, and AhRR mRNA was widely expressed indicating it modulates AhR activity during the early stages of medaka embryos.

Gamma-ray irradiation promotes premature meiosis of spontaneously differentiating testis–ova in the testis of p53-deficient medaka (Oryzias latipes)

In this study, the roles of p53 in impaired spermatogenic male germ cells of p53-deficient medaka were investigated by analyzing histological changes, and gene expressions of 42Sp50, Oct 4 and vitellogenin (VTG2) by RT-PCR or in situ hybridization in the testes. We found that a small number of oocyte-like cells (testis–ova) differentiated spontaneously in the cysts of type A and early type B spermatogonia in the p53-deficient testes, in contrast to the wild-type (wt) testes in which testis–ova were never found. Furthermore, ionizing radiation (IR) irradiation increased the number of testis–ova in p53-deficient testes, increased testis–ova size and proceeded up to the zygotene or pachytene stages of premature meiosis within 14 days after irradiation. However, 28 days after irradiation, almost all the testis–ova were eliminated presumably by p53-independent apoptosis, and spermatogenesis was restored completely. In the wt testis, IR never induced testis–ova differentiation. This is the first study to demonstrate the pivotal role of the p53 gene in the elimination of spontaneous testis–ova in testes, and that p53 is not indispensable for the restoration of spermatogenesis in the impaired testes in which cell cycle regulation is disturbed by IR irradiation.

Tooth replacement and putative odontogenic stem cell niches in pharyngeal dentition of medaka (Oryzias latipes)

The small-sized teleost fish medaka, Oryzias latipes, has as many as 1000 pharyngeal teeth undergoing continuous replacement. In this study, we sought to identify the tooth-forming units and determine its replacement cycles, and further localize odontogenic stem cell niches in the pharyngeal dentition of medaka to gain insights into the mechanisms whereby continuous tooth replacement is maintained. Three-dimensional reconstruction of pharyngeal epithelium and sequential fluorochrome labeling of pharyngeal bones and teeth indicated that the individual functional teeth and their successional teeth were organized in families, each comprising up to five generations of teeth and successional tooth germs, and that the replacement cycle of functional teeth was approximately 4 weeks. BrdU label/chase experiments confirmed the existence of clusters of label-retaining epithelial cells at the posterior end of each tooth family where the expression of pluripotency marker Sox2 was confirmed by in situ hybridization. Label-retaining cells were also identified in the mesoderm immediately adjacent to the posterior end of each tooth family. These data suggest the importance of existence of slow-cycling dental epithelial cells and Sox2 expressions at the posterior end of each tooth family to maintain continuous tooth formation and replacement in the pharyngeal dentition of medaka.

Intraspecific variation in the mitochondrial genome among local populations of Medaka Oryzias latipes

The draft genome data of Medaka Oryzias latipes shows that it has distinct intraspecific genetic variation. To survey the genetic variations contributing to environmental adaptation, we focused on the mitochondrial DNA (mtDNA). The complete mtDNA sequences of Medaka were compared among 8 local population stocks and 4 inbred strains established from genetically divergent groups. Inbred strain HSOK, derived from the Eastern Korean group of Medaka, has a mitochondrial gene order that was distinct from other Medaka groups. Phylogenetic trees based on the mitochondrial genome sequences indicated that the mitogenome from the Shanghai stock (China) and HSOK strain were highly diverged from Japanese Medaka, and that the Japanese Medaka mitogenome was diverged into two groups; this result was fully consistent with those of the previous study using mtDNA-encode gene sequences. Among tRNA genes, the most divergent was the tRNA(Thr) gene as reported in humans previously. The number of tandemly repeated 11 nucleotide units in the Medaka mtDNA control region (CR) varied greatly among local populations. The number of repeats was more variable in the Northern Japanese group (10-34) than in the Southern group (7-12), while two other Oryzias species, inhabiting tropical regions, had no repeats. A comprehensive comparison between the number of repeat units and meteorological data indicated that the number of repeats correlated to the index data of a cold environment and seasonal climatic change. In cold (5 degrees C) acclimated fish, the mRNA levels varied among mitochondria coding genes. mRNA of the cytochrome oxidase subunit I gene in some local stocks was induced by cold temperature and seemed to be correlated with the number of repeated sequences in the CR. This study revealed that the repeated sequences in the mtDNA CR might function for mtDNA gene expression and that the number of tandem repeats in Medaka mtDNA is likely related to adaptation to a harsh habitat.

Medaka: a promising model animal for comparative population genomics

BackgroundWithin-species genome diversity has been best studied in humans. The international HapMap project has revealed a tremendous amount of single-nucleotide polymorphisms (SNPs) among humans, many of which show signals of positive selection during human evolution. In most of the cases, however, functional differences between the alleles remain experimentally unverified due to the inherent difficulty of human genetic studies. It would therefore be highly useful to have a vertebrate model with the following characteristics: (1) high within-species genetic diversity, (2) a variety of gene-manipulation protocols already developed, and (3) a completely sequenced genome. Medaka (Oryzias latipes) and its congeneric species, tiny fresh-water teleosts distributed broadly in East and Southeast Asia, meet these criteria.FindingsUsing Oryzias species from 27 local populations, we conducted a simple screening of nonsynonymous SNPs for 11 genes with apparent orthology between medaka and humans. We found medaka SNPs for which the same sites in human orthologs are known to be highly differentiated among the HapMap populations. Importantly, some of these SNPs show signals of positive selection.ConclusionThese results indicate that medaka is a promising model system for comparative population genomics exploring the functional and adaptive significance of allelic differentiations.