Mesodermal metamerism in the teleost, Oryzias latipes (the medaka)

Previous studies of the metameric pattern in mesodermal tissues of chick, mouse, turtle, and amphibian embryos have indicated that segmental characteristics exist along the entire length of the embryo. This paper describes this phenomenon in a fish embryo, for some differences in the cranial segmental plan exist between the anamniote and the amniote embryos hitherto studied. Embryos of the cyprinodont, Oryzias latipes, were fixed at various times, the examined by means of stereo scanning electron microscopy. As in other vertebrate embryos, the first indication of mesodermal metamerism in this fish embryo is the occurrence of somitomeres, which are orderly, tandemly arranged units of uncondensed mesenchymal cells in the paraxial mesoderm. As many as ten somitomeres can be observed caudal to the last formed somite to the elongating tail region. In addition, 7 somitomeres are present rostral to the first definitive somite, which is segment number eight. As in other vertebrate embryos examined, somitomeres in Oryzias embryos are circular, bilaminar arrays of paraxial mesoderm that form before any indications of segmentation can be seen with the light microscope. In the trunk region these mesodermal units condense to give rise to definitive somites, but in the head they eventually disperse. Despite a fundamentally different mode of gastrulation and a relatively small number of cells in the newly formed somitomeres, cranial segmentation in Oryzias embryos was found to be more similar in number to the metameric pattern of the embryos of the bird, reptile, and mammal than to the situation found in the two amphibians studied thus far.     

Ovarian germline stem cells in the teleost fish, medaka (Oryzias latipes)

In the mammalian testis germline stem cells keep producing many sperms, while there is no direct evidence for the presence of germline stem cells in the ovary. It is widely accepted in mammals that the mature oocytes are supplied from a pool of primordial follicles in the adult ovary. In other vertebrates, such as fish, however, there has been no investigation on the mechanism underlying the high egg-producing ability. In this review, we introduce the recently identified ovarian germline stem cells and the surrounding unique structure in teleost fish, medaka (Oryzias latipes) [Nakamura S et al. Science. 2010; 328: 1561-1563]. We also discuss about the expression and function of sox9 that characterizes this unique structure.     

R-spondins are involved in the ovarian differentiation in a teleost,medaka (Oryzias latipes)

Background

Among the four major bilaterian clades, Deuterostomia, Acoelomorpha, Ecdysozoa, and Lophotrochozoa, the latter shows an astonishing diversity of bodyplans. While the largest lophotrochozoan assemblage, the Spiralia, which at least comprises Annelida, Mollusca, Entoprocta, Platyhelminthes, and Nemertea, show a spiral cleavage pattern, Ectoprocta, Brachiopoda and Phoronida (the Lophophorata) cleave radially. Despite a vast amount of recent molecular phylogenetic analyses, the interrelationships of lophotrochozoan phyla remain largely unresolved. Thereby, Entoprocta play a key role, because they have frequently been assigned to the Ectoprocta, despite their differently cleaving embryos. However, developmental data on entoprocts employing modern methods are virtually non-existent and the data available rely exclusively on sketch drawings, thus calling for thorough re-investigation.

Results

By applying fluorescence staining in combination with confocal microscopy and 3D-imaging techniques, we analyzed early embryonic development of a basal loxosomatid entoproct. We found that cleavage is asynchronous, equal, and spiral. An apical rosette, typical for most spiralian embryos, is formed. We also identified two cross-like cellular arrangements that bear similarities to both, a "molluscan-like" as well as an "annelid-like" cross, respectively.

Conclusions

A broad comparison of cleavage types and apical cross patterns across Lophotrochozoa shows high plasticity of these character sets and we therefore argue that these developmental traits should be treated and interpreted carefully when used for phylogenetic inferences.     

Sex determination in fish: Lessons from the sex-determining gene of the teleost medaka, Oryzias latipes

Although sex determination systems in animals are diverse, sex-determining genes have been identified only in mammals and some invertebrates. Recently, DMY (DM domain gene on the Y chromosome) has been found in the sex-determining region on the Y chromosome of the teleost medaka fish, Oryzias latipes. Functional and expression analyses of DMY show it to be the leading candidate for the male-determining master gene of the medaka. Although some work is required to define DMY as the master sex-determining gene, medaka is expected to be a good experimental animal for investigating the precise mechanisms involved in primary sex determination in non-mammalian vertebrates. In this article, the process of identification of DMY and is summarized and the origins of DMY and sexual development of the medaka's gonads are reviewed. In addition, putative functions of DMY are discussed.     

Artificial fertilization by intracytoplasmic sperm injection in a teleost fish, the medaka (Oryzias latipes)

Intracytoplasmic sperm injection (ICSI) is a technique that has been successfully used for assisting reproduction in mammals. However, this method is still not reliable in nonmammalian species, including teleosts. We succeeded in producing medaka individuals by ICSI with a rate of 13.4% (28 hatched embryos out of 209 eggs fertilized by ICSI), the best value reported so far in teleosts, including zebrafish and Nile tilapia. Although the technique was based on that developed for mammalian eggs, some critical modifications were made to adjust it to the medaka egg, which has a thick and hard envelope (the chorion) and a single sperm entry site (the micropyle). Medaka ICSI was performed by injecting a demembranated spermatozoon into an egg cytoplasm through the micropyle 10-15 sec after egg activation induced by a piezo-actuated vibration, the site and timing of sperm penetration being consistent with those in normal fertilization in medaka. To increase the efficiency of ICSI in medaka, we found that the fertilization by ICSI should precisely mimic the fertilization by insemination with intact sperm, both spatially and temporally. The success rate of ICSI was highly variable in batches of eggs (ranging from 0% to 56%), suggesting that the conditions of eggs are important factors in stabilizing the production of individuals by ICSI. The success in medaka ICSI provides a basis for future research to understand the basic mechanisms in gamete biology of teleosts as well as for development of new technology that can yield valuable applications in fisheries science.     

Identification of three duplicated Spin genes in medaka (Oryzias latipes)

Gene and genomic duplications are very important and frequent events in fish evolution, and the divergence of duplicated genes in sequences and functions is a focus of research on gene evolution. Here, we report the identification and characterization of three duplicated Spindlin (Spin) genes from medaka (Oryzias latipes): OlSpinA, OlSpinB, and OlSpinC. Molecular cloning, genomic DNA Blast analysis and phylogenetic relationship analysis demonstrated that the three duplicated OlSpin genes should belong to gene duplication. Furthermore, Western blot analysis revealed significant expression differences of the three OlSpins among different tissues and during embryogenesis in medaka, and suggested that sequence and functional divergence might have occurred in evolution among them.     

Characterization and expression of embryonic and adult globins of the teleost Oryzias latipes (medaka)

Using the teleost Oryzias latipes (medaka), we isolated three embryonic globin cDNAs (em.alpha-0, em.alpha-1, and em.beta-1) from the embryos 5 days after fertilization (at 30 degrees C) and two adult globin cDNAs (ad.alpha-1 and ad.beta-1) from the kidney of the fully-grown adult fish, and predicted their amino acid sequences. Molecular phylogenetic analysis showed that the embryonic globins were highly homologous in amino acid sequence to the embryonic globins previously identified in rainbow trout and zebrafish, and that they formed a monophyletic group among the teleostean globin molecules. They were clearly discriminated from the adult globin of the medaka. RT-PCR analysis showed that the embryonic globin mRNAs were intensely expressed in stage 30 and 38 embryos and in young fish 30 days after hatching. The level of expression decreased drastically after the young fish stage, and was low in fully-grown adult fish. The adult alpha globin mRNA ad.alpha-1 was scarcely expressed in the embryos, and the level of expression gradually increased in young to fully-grown adult fish. Unexpectedly, the adult beta globin mRNA ad.beta-1 was expressed throughout life, from the early embryonic stage to the fully-grown adult stage. This expression profile was quite different from that of the rainbow trout previously investigated. Some globins of the medaka were expressed both in primitive hematopoiesis and in definitive hematopoiesis.     

Immunoglobulin light chains in medaka (Oryzias latipes)

The gene segments encoding antibodies have been studied in many capacities and represent some of the best-characterized gene families in traditional animal disease models (mice and humans). To date, multiple immunoglobulin light chain (IgL) isotypes have been found in vertebrates and it is unclear as to which isotypes might be more primordial in nature. Sequence data emerging from an array of fish genome projects is a valuable resource for discerning complex multigene assemblages in this critical branch point of vertebrate phylogeny. Herein, we have analyzed the genomic organization of medaka (Oryzias latipes) IgL gene segments based on recently released genome data. The medaka IgL locus located on chromosome 11 contains at least three clusters of IgL gene segments comprised of multiple gene assemblages of the kappa light chain isotype. These data suggest that medaka IgL gene segments may undergo both intra- and inter-cluster rearrangements as a means to generate additional diversity. Alignments of expressed sequence tags to concordant gene segments which revealed each of the three IgL clusters are expressed. Collectively, these data provide a genomic framework for IgL genes in medaka and indicate that Ig diversity in this species is achieved from at least three distinct chromosomal regions.     

Germ cell-less expression in medaka (Oryzias latipes)

The gene germ cell-less (gcl) plays an important role in the early differentiation of germ cells in Drosophila. We isolated the gcl homolog of the model teleost medaka (Oryzias latipes) using degenerated primers and an ovary cDNA bank. The predicted amino acid sequence of medaka gcl showed 92, 68 and 31% overall identity to mouse, human and Drosophila gcl respectively. RT-PCR revealed stronger expression in the ovary and weaker expression in testis, brain, heart, liver and muscle tissue. Expression in early embryos indicates the presence of maternal mRNA. By in situ hybridisation (ISH), gcl could not be detected in embryos. In contrast to vasa, ISH revealed expression of gcl in the ovary but not in the testis. Mol. Reprod. Dev. 67: 15-18, 2004.     

Atypical piscine mycobacteriosis in Japanese medaka (Oryzias latipes)

Japanese medaka, (Oryzias latipes), small, freshwater, tropical cyprinodonts, are principally used for toxicologic and carcinogenicity assays, but are finding more applications in developmental genetic and biological research. An increase in mortality began in brood stock of adult medaka that had been shipped and housed separately by sex. Initially, mortality averaged one fish daily and began in females two weeks after they were received. Cohabitation began eight weeks after arrival. After four to six weeks of cohabitation in different spawning aquaria, mortality was observed in males. Clinical signs of disease included loss of scale luster and color, with subsequent blanching of dorsal flank musculature, small raised nodules on various external surfaces, emaciation, fraying of fin tips, and equilibrium disturbances. Histologic examination of affected adults revealed multi-organ granulomatous inflammation with intracellular acid-fast bacilli. Specimens from 46 juvenile medaka that were spawned from affected adults, were submitted for culture and histologic evaluation. Of 18 fish, two had lesions similar to those of adults. The organism isolated from the remaining fish was identified as Mycobacterium fortuitum. Due to atypical rapid progression of disease, spread of M. fortuitum to progeny, and poor prognosis, the entire colony was euthanized.     

Identification of robust hypoxia biomarker candidates from fin of medaka (Oryzias latipes)

Aquatic hypoxia caused by organic pollution and eutrophication is a pressing worldwide water pollution problem. Better methods for monitoring oxygen levels are needed to assist efforts to maintain and protect the health of natural aquatic environments. In this project, we used a Japanese ricefish (medaka, Oryzias latipes) 8K oligonucleotide array as a platform to identify potential hypoxic biomarkers in different organs (fin, gill, liver and brain) upon exposure to hypoxia. The microarray results were validated by qRT-PCR employing a subset of candidate biomarkers. Interestingly, the largest number and most significant of hypoxia responding array features were detected in hypoxia exposed fin tissues. We identified 173 array features that exhibited a significant response (over 2 fold change in expression) upon exposure to hypoxic conditions and validated a subset of these by quantitative RT-PCR. These gene targets were subjected to annotation and gene ontology mining. Positively identifiable gene targets that may be useful for development of a rapid and accurate biomarker test using fin clips are discussed in relation to previous reports on hypoxia responsive genes.