Genetic divergence in wild populations of Medaka,Oryzias latipes (Pisces: Oryziatidae) from Japan and China

Allozyme studies of the cyprinodont fish Oryzias latipes have revealed that the wild populations of this species have differentiated regionally, and that Japanese wild populations are composed of two distinct groups, the Northern Population and the Southern Population. In order to elucidate the genetic relationship and the origin of the two major populations, allozymic characters of the specimens from 21 localities in Japan and China were compared. Although Chinese populations could be distinguished from Japanese ones, they were more closely related to the Southern Population, and showed few allozymic traits of the Northern Population. It is likely that the latter is a relic population which has survived the glacial period along the western coast of north-eastern Japan.     

Genetic dissection of the formation of the forebrain in Medaka, Oryzias latipes

The forebrain, consisting of the telencephalon and diencephalon, is essential for processing sensory information. To genetically dissect formation of the forebrain in vertebrates, we carried out a systematic screen for mutations affecting morphogenesis of the forebrain in Medaka. Thirty-three mutations defining 25 genes affecting the morphological development of the forebrain were grouped into two classes. Class 1 mutants commonly showing a decrease in forebrain size, were further divided into subclasses 1A to 1D. Class 1A mutation (1 gene) caused an early defect evidenced by the lack of bf1 expression, Class 1B mutations (6 genes) patterning defects revealed by the aberrant expression of regional marker genes, Class 1C mutation (1 gene) a defect in a later stage, and Class 1D (3 genes) a midline defect analogous to the zebrafish one-eyed pinhead mutation. Class 2 mutations caused morphological abnormalities in the forebrain without considerably affecting its size, Class 2A mutations (6 genes) caused abnormalities in the development of the ventricle, Class 2B mutations (2 genes) severely affected the anterior commissure, and Class 2C (6 genes) mutations resulted in a unique forebrain morphology. Many of these mutants showed the compromised sonic hedgehog expression in the zona-limitans-intrathalamica (zli), arguing for the importance of this structure as a secondary signaling center. These mutants should provide important clues to the elucidation of the molecular mechanisms underlying forebrain development, and shed new light on phylogenically conserved and divergent functions in the developmental process.     

The Genomic and Genetic Toolbox of the Teleost Medaka (Oryzias latipes)

The Japanese medaka, Oryzias latipes, is a vertebrate teleost model with a long history of genetic research. A number of unique features and established resources distinguish medaka from other vertebrate model systems. A large number of laboratory strains from different locations are available. Due to a high tolerance to inbreeding, many highly inbred strains have been established, thus providing a rich resource for genetic studies. Furthermore, closely related species native to different habitats in Southeast Asia permit comparative evolutionary studies. The transparency of embryos, larvae, and juveniles allows a detailed in vivo analysis of development. New tools to study diverse aspects of medaka biology are constantly being generated. Thus, medaka has become an important vertebrate model organism to study development, behavior, and physiology. In this review, we provide a comprehensive overview of established genetic and molecular-genetic tools that render medaka fish a full-fledged vertebrate system.     

Estrogen-Independent Ovary Formation in the Medaka Fish, Oryzias latipes

To investigate whether a female sex steroid, estrogen, acts as a natural inducer of female gonadal sex determination (or ovary formation) in the medaka fish, Oryzias latipes, the effects of an aromatase inhibitor and anti-estrogens on sexual differentiation of gonads were examined. We found that both drugs did not show any discernible effects on the genetically determined sex differentiation in both sexes. However, the aromatase inhibitor impaired the paradoxical effects of androgen (a male sex steroid), and the anti-estrogens inhibited the male-to-female sex reversal caused by estrogen. Treatments of the fertilized eggs with androgen disturbed the gonadal sex developments in both sexes, suggesting that sex steroid synthesis is detrimental to the gonadal sex developments in the medaka embryos. These results are consistent with the previous observation that sex steroids are not synthesized before the onset of gonadal sex differentiation, and suggest that ovary formation in the genetic females of the medaka fish is not dependent on estrogen.     

Identification of radiation-sensitive mutants in the Medaka, Oryzias latipes

We screened populations of N-ethyl-N-nitrosourea (ENU)-mutagenized Medaka, (Oryzias latipes) for radiation-sensitive mutants to investigate the mechanism of genome stability induced by ionizing radiation in developing embryos. F3 embryos derived from male founders that were homozygous for induced the mutations were irradiated with gamma-rays at the organogenesis stage (48hpf) at a dose that did not cause malformation in wild-type embryos. We screened 2130 F2 pairs and identified three types of mutants with high incidence of radiation-induced curly tailed (ric) malformations using a low dose of irradiation. The homozygous strain from one of these mutants, ric1, which is highly fertile and easy to breed, was established and characterized related to gamma-irradiation response. The ric1 strain also showed higher incidence of malformation and lower hatchability compared to the wild-type CAB strain after gamma-irradiation at the morula and pre-early gastrula stages. We found that the decrease in hatching success after gamma-irradiation, depends on the maternal genotype at the ric1 locus. Terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling assays showed a high frequency of apoptosis in the ric1 embryos immediately after gamma-irradiation at the pre-early gastrula stage but apoptotic cells were not observed before midblastula transition (MBT). The neutral comet assay revealed that the ric1 mutant has a defect in the rapid repair of DNA double-strand breaks induced by gamma-rays. These results suggest that RIC1 is involved in the DNA double strand break repair in embryos from morula to organogenesis stages, and unrepaired DNA double strand breaks in ric1 trigger apoptosis after MBT. These results support the use of the ric1 strain for investigating various biological consequences of DNA double strand breaks in vivo and for sensitive monitoring of genotoxicity related to low dose radiation.     

Geographic variation and diversity of the cytochrome b gene in Japanese wild populations of medaka, Oryzias latipes

We conducted a polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome b gene to elucidate the detailed genetic population structure of Japanese wild populations of medaka, Oryzias latipes. The analysis of 1,225 specimens collected from 303 sites identified 67 mitotypes. Subsequently we determined the nucleotide sequences of the complete cytochrome b gene (1141-bp) to clarify the phylogenetic relationships among mitotypes. The phylogenetic tree based on nucleotide sequences indicated three major clades (A, B and C) that differed by 11.3-11.8%, corresponding to three clusters previously identified by RFLP analysis of entire mitochondrial DNAs. The geographic distribution of mitotypes in clades A and B was fully concordant with the Northern and Southern Populations defined by allozymes. Clade A could be subdivided into three subclades and clade B into eleven, with sequence divergences among subclades of 1.3-5.8%. Each distribution of mitotypes in subclades roughly corresponded to that of mtDNA haplotypes in subclusters previously identified. Mitotypes in clade C were found only in the Kanto district. The phylogenetic relationships and the estimated divergence times suggest that three Japanese clades originated from a common ancestor and were separated during the Pliocene, and that the regional differentiation of subclades was closely connected with the geological history of the Quaternary. This study has also demonstrated the possibility of artificial disturbance of natural distribution especially in the Kanto district and the superior efficacy of PCR-RFLP analysis as a simple method for detecting genetic variation and artificial gene flow of medaka.     

Wild-derived XY sex-reversal mutants in the Medaka, Oryzias latipes

The medaka, Oryzias latipes, has an XX/XY sex-determination mechanism. A Y-linked DM domain gene, DMY, has been isolated by positional cloning as a sex-determining gene in this species. Previously, we found 23 XY sex-reversed females from 11 localities by examining the genotypic sex of wild-caught medaka. Genetic analyses revealed that all these females had Y-linked gene mutations. Here, we aimed to clarify the cause of this sex reversal. To achieve this, we screened for mutations in the amino acid coding sequence of DMY and examined DMY expression at 0 days after hatching (dah) using densitometric semiquantitative RT-PCR. We found that the mutants could be classified into two groups. One contained mutations in the amino acid coding sequence of DMY, while the other had reduced DMY expression at 0 dah although the DMY coding sequence was normal. For the latter, histological analyses indicated that YwOurYwOur (YwOur, Y chromosome derived from an Oura XY female) individuals with the lowest DMY expression among the tested mutants were expected to develop into females at 0 dah. These results suggest that early testis development requires DMY expression above a threshold level. Mutants with reduced DMY expression may prove valuable for identifying DMY regulatory elements.     

Light-Induced Pigment Aggregation in Xanthophores of the Medaka,Oryzias latipes

The response mechanism of medaka xanthophores to light was examined at the cellular level. Innervated and denervated xanthophores of adult medakas responded to light (9,000 lux) within 30 sec by pigment aggregation, and this aggregation was not mediated through α-adrenoceptors on the cell membrane. Maximum sensitivity to light was at wavelengths of 410–420 nm, and the direct effect of light was reversible. Xanthophore responsiveness to light in summer was higher than that in winter. Ca²⁺ and calmodulin were not involved in the response, but rather, an important role for cAMP and phos-phodiesterase (PDE) was suggested. It seems likely that photoreception by visual pigment which is sensitive to light at wavelengths of 410–420 nm increases PDE activity, probably via a G-protein, such as occurs with visual cells in the retina, which causes a decrease in levels of cytosolic cAMP, in turn leading to pigment aggregation within medaka xanthophores.     

Simplex PCR Assay for Positive Identification of Genetic Sex in the Japanese Medaka, Oryzias latipes

The medaka, Oryzias latipes, is a very popular model in biomedical research, particularly for elucidating sex differentiation and determination mechanisms and effects of endocrine disruptors among others. These studies require a sensitive, accurate, rapid, and reliable technique for genetic sexing of eggs, larvae, and adults. In this study, we report a simplex polymerase chain reaction approach that uses a single pair of primers for simultaneous amplification of sex-specific amplicons. Males and females yield a single diagnostic band of 933 and 1,906 bp, respectively, in three different strains of medaka tested, permitting gender identification accurately of both immature and adult fish. This technique will be useful in both ecological and biomedical researches that employ medaka and rely on genetic sexing.     

Allelic expression changes in Medaka (Oryzias latipes) hybrids between inbred strains derived from genetically distant populations

Variations in allele expressions between genetically distant populations are one of the most important factors which affects their morphological and physiological variations. These variations are caused by natural mutations accumulated in their habitats. It has been reported that allelic expression differences in the hybrids of genetically distant populations are different from parental strains. In that case, there is a possibility that allelic expression changes lead to novel phenotypes in hybrids. Based on genomic information of the genetically distant populations, quantification and comparison of allelic expression changes make importance of regulatory sequences (cis-acting factors) or upstream regulatory factors (trans-acting modulators) for these changes clearer. In this study, we focused on two Medaka inbred strains, Hd-rR and HNI, derived from genetically distant populations and their hybrids. They are highly polymorphic and we can utilize whole-genome information. To analyze allelic expression changes, we established a method to quantify and compare allele-specific expressions of 11 genes between the parental strains and their reciprocal hybrids. In intestines of reciprocal hybrids, allelic expression was either similar or different in comparison with the parental strains. Total expressions in Hd-rR and HNI were tissue-dependent in the case of HPRT1, with high up-regulation of Hd-rR allele expression in liver. The proportion of genes with differential allelic expression in Medaka hybrids seems to be the same as that in other animals, despite the high SNP rate in the genomes of the two inbred strains. It is suggested that each tissue of the strain difference in trans-acting modulators is more important than polymorphisms in cis-regulatory sequences in producing the allelic expression changes in reciprocal hybrids.     

Geographic variation and diversity of the cytochrome b gene in wild populations of medaka (Oryzias latipes) from Korea and China

We analyzed the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) of the mitochondrial cytochrome b (cyt b) gene in wild populations of medaka from Korea and China. We surveyed 258 wild specimens from 75 different sites, and identified 17 mitotypes. Sequencing analysis of the complete cyt b gene (1141-bp) was subsequently carried out to infer the phylogenetic relationships among these mitotypes. Phylogenetic trees indicated two major clades, D and E, which were different from the Japanese clades (A, B and C). These two clades were completely identical to two clusters previously identified by RFLP analysis of entire mitochondrial DNAs. The geographic distribution of the mitotypes in clades D and E was consistent with the China-West Korean Population and the East Korean Population as defined by allozymic and karyological analyses. This agreement among different analyses suggests long-term isolation between the two groups. In the region where the distributions of two major clades overlapped, a limited extent of gene flow was observed. These results suggested the existence of some reproductive isolation mechanisms between the two clades, or the introgression between them followed by a random drift in each local population. Furthermore, clade D was subdivided into three subclades (D-I to D-III). The phylogenetic relationship and distribution pattern of subclade D-II suggested a dispersal event of medaka from China to southwest Korea. Our results also showed that the East Korean Population has recently expanded its distribution area because little diversity was observed in clade E.     

Mutations affecting somite formation in the Medaka (Oryzias latipes)

The metameric structure of the vertebrate trunk is generated by repeated formation of somites from the unsegmented presomitic mesoderm (PSM). We report the initial characterization of nine different mutants affecting segmentation that were isolated in a large-scale mutagenesis screen in Medaka (Oryzias latipes). Four mutants were identified that show a complete or partial absence of somites or somite boundaries. In addition, five mutations were found that cause fused somites or somites with irregular sizes and shapes. In situ hybridization analysis using specific markers involved in the segmentation clock and antero-posterior (A-P) polarity of somites revealed that the nine mutants can be compiled into two groups. In group 1, mutants exhibit defects in tailbud formation and PSM prepatterning, whereas A-P identity in the somites is defective in group 2 mutants. Three mutants (planlos, pll; schnelles ende, sne; samidare, sam) have characteristic phenotypes that are similar to those in zebrafish mutants affected in the Delta/Notch signaling pathway. The majority of mutants, however, exhibit somitic phenotypes distinct from those found in zebrafish, such as individually fused somites and irregular somite sizes. Thus, these Medaka mutants can be expected to provide clues to uncovering novel components essential for somitogenesis.     

Field survey of sex-reversals in the medaka, Oryzias latipes: genotypic sexing of wild populations

The medaka, Oryzias latipes, has an XX/XY sex determination mechanism. A Y-linked DM domain gene, DMY, has been isolated by positional cloning as a prime candidate for the sex-determining gene. Furthermore, the crucial role of DMY during male development was established by studying two wild-derived XY female mutants. In this study, to find new DMY and sex-determination related gene mutations, we conducted a broad survey of the genotypic sex (DMY-negative or DMY-positive) of wild fish. We examined 2274 wild-caught fish from 40 localities throughout Japan, and 730 fish from 69 wild stocks from Japan, Korea, China, and Taiwan. The phenotypic sex type agreed with the genotypic sex of most fish, while 26 DMY-positive (XY) females and 15 DMY-negative (XX) males were found from 13 and 8 localities, respectively. Sixteen XY sex-reversals from 11 localities were mated with XY males of inbred strains, and the genotypic and phenotypic sexes of the F(1) progeny were analyzed. All these XY sex-reversals produced XY females in the F(1) generation, and all F(1) XY females had the maternal Y chromosome. These results show that DMY is a common sex-determining gene in wild populations of O. latipes and that all XY sex-reversals investigated had a DMY or DMY-linked gene mutation.     

Phylogenetic aspects of tooth and jaw structure of the Medaka, Oryzias latipes, and other beloniform fishes

Tooth structure is described for adult male, female, and juvenile Oryzias latipes (Temminck & Schlegel), the Medaka. Adult males have enlarged, unicuspid teeth posteriorly on the premaxilla and dentary. Oral teeth are smaller and more numerous in females, in which no tooth is notably larger than the others. Juveniles have numerous small teeth from about 3 mm SL (standard length) onwards. By about 16 mm SL, males begin to develop the large posterior teeth, as well as other secondary sexual characters. Lower and upper pharyngeal teeth of both males and females are fine, and in numerous even rows.
The large, posterior oral teeth of males are fully-ankylosed to the attachment bone, and, hence, are not depressible. In female Medaka, as in the Halfbeak Dermogenys pusillus van Hasselt, the oral teeth have a ring of unmineralized collagen at the base, and are not depressible. Pharyngeal teeth of Medaka have a ring of unmineralized collagen at the base, and a distinct wedge of collagen absent posteriorly, such that the pharyngeal teeth may be depressed.
Bone in adult Medaka is acellular. Incompletely mineralized teeth, acellular bone, a protrus-ible upper oral jaw, and a mobile branchial apparatus with an interhyal bone, form a complex characteristic of advanced teleosts. The Medaka differs in several ways from the model advanced teleost: absence of an interhyal bone, ascending and articular processes of the premaxilla, and the rostral cartilage, as well as presence of cartilaginous symphyses between the dentaries and premaxillae, all contribute to the fixed or nonprotrusible jaws.
Reduction in the premaxilla is a derived character within beloniform fishes for which an enlarged, beaked outer jaw is considered plesiomorphic.     

Evolution of acetylcholinesterase and butyrylcholinesterase in the vertebrates: an atypical butyrylcholinesterase from the Medaka Oryzias latipes

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are thought to be the result of a gene duplication event early in vertebrate evolution. To learn more about the evolution of these enzymes, we expressed in vitro, characterized, and modeled a recombinant cholinesterase (ChE) from a teleost, the medaka Oryzias latipes. In addition to AChE, O. latipes has a ChE that is different from either vertebrate AChE or BChE, which we are classifying as an atypical BChE, and which may resemble a transitional form between the two. Of the fourteen aromatic amino acids in the catalytic gorge of vertebrate AChE, ten are conserved in the atypical BChE of O. latipes; by contrast, only eight are conserved in vertebrate BChE. Notably, the atypical BChE has one phenylalanine in its acyl pocket, while AChE has two and BChE none. These substitutions could account for the intermediate nature of this atypical BChE. Molecular modeling supports this proposal. The atypical BChE hydrolyzes acetylthiocholine (ATCh) and propionylthiocholine (PTCh) preferentially but butyrylthiocholine (BTCh) to a considerable extent, which is different from the substrate specificity of AChE or BChE. The enzyme shows substrate inhibition with the two smaller substrates but not with the larger substrate BTCh. In comparison, AChE exhibits substrate inhibition, while BChE does not, but may instead show substrate activation. The atypical BChE from O. latipes also shows a mixed pattern of inhibition. It is effectively inhibited by physostigmine, typical of all ChEs. However, although the atypical BChE is efficiently inhibited by the BChE-specific inhibitor ethopropazine, it is not by another BChE inhibitor, iso-OMPA, nor by the AChE-specific inhibitor BW284c51. The atypical BChE is found as a glycophosphatidylinositol-anchored (GPI-anchored) amphiphilic dimer (G(2) (a)), which is unusual for any BChE. We classify the enzyme as an atypical BChE and discuss its implications for the evolution of AChE and BChE and for ecotoxicology.     

Methylmercury: some effects on embryogenesis in the Japanese Medaka, Oryzias latipes

Japanese Medaka (Oryzias latipes) eggs were treated with 40, 60, and 80 parts per billion (ppb) methylmercuric chloride (MMC) for a period of 10 to 25 days in order to observe gross teratogenic and lethal effects caused by methylmercury during embryogenesis. Over the period tested, 40 ppb-treated embryos were less affected than other treated animals. Embryos treated with 60 ppb MMC showed greater effects in that there was a higher death rate and those which survived ranged considerably in size, degree of gross teratogenic defects, and behavior if development was normal enough for hatching to occur. Most did not hatch during the 25-day test period. Embryos treated with 80 ppb MMC were very abnormal in development, being stunted, poorly pigmented, and they showed various eye defects. None hatched in 25 days. Many did not incorporate the heart into the body. Methylmercuric chloride in trace amounts caused various defects and presents a potential hazard to development of embryos of aquatic vertebrates.     

Sex-Linked Inheritance of the lf Locus in the Medaka Fish (Oryzias latipes)

The lf (leucophore free) locus was previously reported autosomal recessive in the medaka fish (Oryzias latipes). However, extensive linkage analyses in this study using various strains revealed that the lf locus was closely sex-linked. The recombination frequency between lf and the male determining factor (y) was 2.2% (10 recombinants out of 464 progeny). Because the lf/lf homozygous fish do not have visible leucophores, they are distinguishable from wild type in early developmental stages. In the Qurt strain with heterozygous sex chromosomes (X(lf)/X(lf) in females and X(lf)/Y(+) in males), we can predict sex of each embryo on second day after fertilization. The strain should be a very useful material for studying sex determination or differentiation mechanisms in the medaka fish.     

Mutations affecting the formation of posterior lateral line system in Medaka, Oryzias latipes

We performed a systematic screen for mutations affecting the trajectory of axons visualized by immunohistochemical staining of Medaka embryos with anti-acetylated tubulin antibody. Among the mutations identified, yanagi (yan) and kazura (kaz) mutations caused specific defects in projection of the posterior lateral line (PLL) nerve. In yan and kaz mutant embryos, the PLL nerve main bundle was misrouted ventrally and dorsally or anteriorly. Medaka semaphorin3A, sdf1, and cxcr4 cDNA fragments were cloned to allow analysis of these mutants. There were no changes in semaphorin3A or sdf1 expression in mutant embryos, suggesting that the tissues expressing semaphorin3A or sdf1 that are involved in PLL nerve guidance are present in these mutant embryos. Double staining revealed that the mislocated PLL primordium and growth cone of the ectopically projected PLL nerve were always colocalized in both yan and kaz mutant embryos, suggesting that migration of PLL primordia and PLL nerve growth cones are not uncoupled in these mutants. Although homozygous yan larvae showed incomplete migration of the PLL primordium along the anteroposterior axis, ventral proneuromast migration was complete, suggesting that ventral migration of the proneuromast does not require the signaling affected in yan mutants. In addition to the PLL system, the distribution of primordial germ cells (PGCs) was also affected in both yan and kaz mutant embryos, indicating that yan and kaz genes are required for the migration of both PLL primordia and PGCs. Genetic linkage analysis indicated that kaz is linked to cxcr4, but yan is not linked to sdf1 or cxcr4. These mutations will provide genetic clues to investigate the molecular mechanism underlying formation of the PLL system.     

Some Properties of the Hardening Process in Chorions Isolated from Unfertilized Eggs of Medaka, Oryzias latipes

Chorions isolated from unfertilized eggs of medaka, Oryzias latipes , harden during incubation with Ca²⁺ ions (Masuda et al. , 1991). In this process, i.e. in vitro Ca²⁺-hardening, the amounts of the major proteins of unfertilized egg chorions (83 K, 78 K and 51 K, corresponding to ZI-1, 2 and 3 of oocyte chorions reported by Hamazaki et al , 1987) decreased and new proteins having molecular weights of 148 K or more appeared. Immunoblotting analysis using anti-ZI-1, 2 antisera and anti-ZI-3 antisera showed that the 148 K protein was an intermediate formed during polymerization of the original proteins.
The mechanism of in vitro Ca²⁺-hardening was studied by examining the decrease in ZI-1, 2, and 3, the formation of 148 K protein, and the change in solubility of chorions in 6% sodium dodecylsulfate-1% 2-mercaptoethanol-15% glycerol-0.2 M Tris-HCl (pH 6.8). In vitro Ca²⁺-hardening was inhibited at temperatures higher than 70°C and its optimum pH was about 5.5. It was inhibited by neither aminotriazole nor cadaverine. The results suggested that in vitro Ca²⁺-hardening was generated by some factor(s) other than ovoperoxidase and transglutaminase.