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.     

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.     

Characterization of mutations affecting embryonic hematopoiesis in the medaka, Oryzias latipes

In a genetic screen for mutations affecting organogenesis in the medaka, Oryzias latipes, we identified eight mutants with defects in embryonic hematopoiesis. These mutations were classified into seven complementation groups. In this paper, we characterize the five mutants that were confirmed in the next generation. The beni fuji mutant was defective in the generation of blood cells, exhibiting reduced blood cells at the initiation of circulation. Mutations in two genes, lady finger and ryogyoku, caused abnormal morphology of blood cells, i.e., deformation, along with a progressive decrease in the number of blood cells. The sekirei mutant exhibited photosensitivity with autofluorescent blood cells. Mutations in kyoho resulted in huge blood cells that were approximately three times longer than the wild-type blood cells. The spectrum of phenotypes identified in this study is similar to that of the zebrafish hematopoietic mutants except for the huge blood cells in kyoho. Our results demonstrate that medaka, as well as zebrafish, is a useful model to study hematopoiesis.     

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.     

Transient expression of foreign DNA during embryonic and larval development of the medaka fish (Oryzias latipes)

Summary Species of small fish are becoming useful tools for studies on vertebrate development. We have investigated the developing embryo of the Japanese medaka for its application as a transient expression system for the in vivo analysis of gene regulation and function. The temporal and spatial expression patterns of bacterial chloramphenicol acetyltransferase and galactosidase reporter genes injected in supercoiled plasmid form into the cytoplasm of one cell of the two-cell stage embryo was promoter-specific. The transient expression was found to be mosaic within the tissue and organs reflecting the unequal distribution of extrachromosomal foreign DNA and the intensive cell mixing movements that occur in fish embryogenesis. The expression data are consistent with data on DNA fate. Foreign DNA persisted during embryogenesis and was still detectable in some 3- and 9-month-old adult fish; it was found in high molecular weight form as well as in circular plasmid conformations. The DNA was replicated during early and late embryogenesis. Our data indicate that the developing medaka embryo is a powerful in vivo assay system for studies of gene regulation and function.This work contains part of the PhD thesis of C. Winkler     

Characterization of teleost Mdga1 using a gene‐trap approach in medaka (Oryzias latipes)

MAM domain containing glycosilphosphatidilinositol anchor 1 (MDGA1) is an IgCAM protein present in many vertebrate species including humans. In mammals, MDGA1 is expressed by a subset of neurons in the developing brain and thought to function in neural cell migration. We identified a fish ortholog of mdga1 by a gene‐trap screen utilizing the Frog Prince transposon in medaka (Japanese killifish, Oryzias latipes). The gene‐trap vector was inserted into an intronic region of mdga1 to form a chimeric protein with green fluorescent protein, allowing us to monitor mdga1 expression in vivo. Expression of medaka mdga1 was seen in various types of embryonic brain neurons, and specifically in neurons migrating toward their target sites, supporting the proposed function of MDGA1. We also isolated the closely related mdga2 gene, whose expression partially overlapped with that of mdga1. Despite the fact that the gene‐trap event eliminated most of the functional domains of the Mdga1 protein, homozygous embryos developed normally without any morphological abnormality, suggesting a functional redundancy of Mdga1 with other related proteins. High sequential homology of MDGA proteins between medaka and other vertebrate species suggests an essential role of the MDGA gene family in brain development among the vertebrate phylum. genesis 47:505–513, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

Classification of late embryonic stages of medaka,Oryzias latipes

To indicate more clearly the stages of development in late embryo of the medaka,Oryzias latipes, the quotients obtained from the length of tail (measured from the vent to distal end of caudal fin) in microns divided by 100 μm were adopted to nominate stage numbers. The 25 stages thus defined were checked against the calcification of 8 elements in the chondrocranium, and also 4 other structures which develop numerically. By the observations of development (cartilages and calcification indicated by alizarin red S staining) of the 8 elements—parasphenoid, clavicle, operculum, occipital arch, hyomandibular, ceratohyal, anterior otic process and mandibular, the stages defined here were found to classify the order and degree of development of bony elements more precisely than other stage classification in the past. The numerical structures—branchiostegal rays, pharyngeal teeth, vertebrae and mandibular teeth also demonstrated clearly their development corresponding to tail length. The “critical” stages in embryonic development of the medaka as shown by the length of notochord with vacuolized cells associated with development of bony elements were also noted.     

p53 Mutation suppresses adult neurogenesis in medaka fish (Oryzias latipes)

Acid-base imbalance leads to pathological cognition and behaviors in the clinical practices. In the comparison with acidosis, the cellular mechanisms underlying alkalosis-induced brain dysfunction remain unclear. By using electrophysiological approach, we investigated the influences of high extracellular pH environment on cortical GABAergic neurons in terms of their responsiveness to synaptic inputs and their ability to produce action potentials. Artificial cerebral spinal fluid in high pH impairs excitatory synaptic transmission and spike initiation in cortical GABAergic neurons. The alkalosis-induced dysfunction of GABAergic neurons is associated with the decrease of receptor responsiveness and the increases of spike refractory periods and threshold potentials. Our studies reveal that alkalosis impairs cortical GABAergic neurons and subsequently deteriorate brain functions. The molecular targets for alkalosis action include glutamate receptor-channels and voltage-gated sodium channels on GABAergic neurons.     

Characterization of the estrogenic response to genistein in Japanese medaka (Oryzias latipes)

This study was designed to determine the estrogenic effect of the phytoestrogen genistein on several measures of endocrine function in adult Japanese medaka (Oryzias latipes) relative to 17-beta-estradiol. Adult animals of both sexes were exposed to 75, 750 and 30,000 ng/fish (average fish weight equals 0.26 g) of genistein by i.p. injection, with a positive control group treated with 300 ng/fish of 17-beta-estradiol, while a negative control group received a vehicle-only (corn oil) injection. Content of vitellogenin, the yolk glycoprotein made in the liver in response to estradiol stimulation, was measured using Western blots. Circulating estradiol and testosterone levels were measured using a steroid-enzyme immunosorbant assay. The ability of ovaries and testes to synthesize and release estradiol and testosterone was determined by ex vivo incubation of gonads with 25-hydroxycholesterol. Vitellogenin, while induced by 17-beta-estradiol, was not increased in the liver of individuals treated with genistein. In females, genistein treatment at 750 and 30,000 ng increased the estradiol production of ovaries more than the 17-beta-estradiol treatment. In males, genistein treatment resulted in decreased testosterone production from ex vivo testis and a comparable reduction in circulating testosterone level. The changes in vitellogenin, circulating steroids and ex vivo steroidogenesis in medaka in response to genistein are similar to that of 17-beta-estradiol. However, some endpoints are more sensitive to estradiol treatment (vitellogenin), while others are more sensitive to genistein (male testosterone and ovarian estrogenesis).     

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.     

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.     

Comparative study of embryonic thermoresistance of two inbred strains of the medaka (Oryzias latipes)

Summary By using inbred strains (HO4C and HB32C) of the medaka,Oryzias latipes, the involvement of genetic factor(s) in the determination of thermoresistance of fish was investigated. The thermoresistance of embryos of the medaka was quantitated by the fraction of the embryos surviving 1 day after heat treatment. At early stages of development (st. 13 and st. 20–21), the HO4C strain was more resistant than the HB32C strain. At st. 20–21, the HO4C strain was more resistant than the HB32C strain at all temperatures used (42, 43, and 44°C). At later stages of development (st. 27 and st. 32), however, the HB32C strain was more resistant than the HO4C strain.The results of genetic cross experiments raised the following possibilities; the thermoresistance of embryos at early developmental stages can be lowered by some factor(s) inherited in the HO4C strain and/or increased by those in the HB32C strain. By contrast, the sensitivity of embryos at later stages of development was not affected by factor(s) of their parents, but by their own genetic constitution.     

A genetic screen for mutations affecting embryonic development in medaka fish (Oryzias latipes)

In a pilot screen, we assayed the efficiency of ethylnitrosourea (ENU) as a chemical mutagen to induce mutations that lead to early embryonic and larval lethal phenotypes in the Japanese medaka fish, Oryzias latipes. ENU acts as a very efficient mutagen inducing mutations at high rates in germ cells. Three repeated treatments of male fish in 3 mM ENU for 1 h results in locus specific mutation rates of 1.1-1.95 x10(-3). Mutagenized males were outcrossed to wild type females and the F1 offspring was used to establish F2 families. F2 siblings were intercrossed and the F3 progeny was scored 24, 48 and 72 h after fertilization for morphological alterations affecting eye development. The presented mutant phenotypes were identified using morphological criteria and occur during early developmental stages of medaka. They are stably inherited in a Mendelian fashion. The high efficiency of ENU to induce mutations in this pilot screen indicates that chemical mutagenesis and screening for morphologically visible phenotypes in medaka fish allows the genetic analysis of specific aspects of vertebrate development complementing the screens performed in other vertebrate model systems.     

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.