Rapid adaptation of molecular resources from zebrafish and medaka to develop an estuarine/marine model

Many estuary and coastal waters are highly threatened by heavy anthropogenic pollutants. Oryzias melastigma, also called O. dancena, a marine medaka that showed sensitive response to hypoxia and estrogenic endocrine disruptors in previous studies, is becoming a sentinel species for marine ecotoxicology studies. However, the lack of strong molecular foundation and knowledge of early developmental stages hampers its practical applications. Combining our research strength on zebrafish embryos, this study revealed both morphological and molecular (at mRNA and protein levels) development of embryos of this emergent model. Whole mount immunostaining technique specific for O. melastigma was successfully developed based on zebrafish standard protocols. We demonstrated that 17 out of 61 primary antibodies, which were previously tested in zebrafish, showed specific immunoreactivity with O. melastigma. These antibodies clearly illustrated the embryonic development of target tissues (principally neurons) in this medaka. Additionally, partial cDNA fragments of 11 organ-specific marker genes were isolated according to genomic resources of zebrafish, Japanese medaka and other fishes. Of the 11 genes, 8 are widely used as organ markers and their expression patterns were remarkably similar to their homologues in zebrafish and Japanese medaka. The expression profiles of the remaining 3 genes in fish are reported for the first time. These molecular markers (17 antibodies and 11 mRNA probes) can be used as responsive indicators in environmental toxicity evaluation. Moreover, this study brought forward and demonstrated the advantage of transferring techniques and resources from one model to another to hasten the research of interest.     

Regulation of vertebrate eye development by Rx genes

The paired-like homeobox-containing gene Rx has a critical role in the eye development of several vertebrate species including Xenopus, mouse, chicken, medaka, zebrafish and human. Rx is initially expressed in the anterior neural region of developing embryos, and later in the retina and ventral hypothalamus. Abnormal regulation or function of Rx results in severe abnormalities of eye formation. Overexpression of Rx in Xenopus and zebrafish embryos leads to overproliferation of retinal cells. A targeted elimination of Rx in mice results in a lack of eye formation. Mutations in Rx genes are the cause of the mouse mutation eyeless (ey1), the medaka temperature sensitive mutation eyeless (el) and the zebrafish mutation chokh. In humans, mutations in Rx lead to anophthalmia. All of these studies indicate that Rx genes are key factors in vertebrate eye formation. Because these results cannot be easily reconciled with the most popular dogmas of the field, we offer our interpretation of eye development and evolution.     

Asian medaka fishes offer new models for studying mechanisms of seawater adaptation

Japanese medaka (Oryzias latipes) is a freshwater (FW) teleost that is popular throughout the world for laboratory use. In this paper, we discuss the utility of Japanese medaka and related species for studying mechanisms of seawater (SW) adaptation. In addition to general advantages as an experimental animal such as their daily spawning activity, transparency of embryos, short generation time and established transgenic techniques, Japanese medaka have some adaptability to SW unlike the strictly stenohaline zebrafish (Danio rerio). Since other species in the genus Oryzias exhibit different degrees of adaptability to SW, comparative studies between Japanese medaka, where molecular-biological and genetic information is abundant, and other Oryzias species are expected to present varying approaches to solving the problems of SW adaptation. We introduce some examples of interspecies comparison for SW adaptabilities both in adult fish and in embryos. Oryzias species are good models for evolutionary, ecological and zoogeographical studies and a relationship between SW adaptability and geographic distribution has been suggested. Medaka fishes may thus deliver new insights into our understanding of how fish have expanded their distribution to a wide variety of osmotic environments.     

High incidence of mosaic mutations induced by irradiating paternal germ cells of the medaka fish, Oryzias latipes.

Delayed-type mutations induced by radiation have recently been demonstrated in various somatic-cell systems. Such mutations are thought to result from the transmission of genetic instability through many cell divisions subsequent to a single exposure to ionizing radiation. Here, we have examined whether 'transgenerational' delayed-type mutations can arise during embryonic development of the medaka fish as a result of exposing the sperm and spermatids of live fish to 137Cs gamma-radiation. To do this, we made use of a sensitive specific-locus test (SLT) for the medaka that we have recently developed. Because the medaka has a transparent egg membrane and embryo body, both visible mosaics and whole-body mutations can be detected during development at an early-expressed pigmentation locus. When wild-type +/+ males were gamma-irradiated and then mated with wl/wl females, the frequency of F1 embryos with both wild-type orange leucophores (wl/+) and mutant-type white leucophores (wl/wl*) (mosaic mutants) was about 5.7x10(-3)/Gy. The frequency of embryos with only white leucophores (whole-body mutants) was about 1.3x10(-3)/Gy. These results suggest that delayed mutations frequently arise in medaka fish embryos that have been fertilized with irradiated sperm. Some possible mechanisms involved in the generation of these delayed mutational events (including genomic instability in the early embryos) are discussed.     

Fish stem cell cultures

Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on "Fish Stem Cells and Nuclear Transfer", we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.     

Genetic and environmental melanoma models in fish

Experimental animal models are extremely valuable for the study of human diseases, especially those with underlying genetic components. The exploitation of various animal models, from fruitflies to mice, has led to major advances in our understanding of the etiologies of many diseases, including cancer. Cutaneous malignant melanoma is a form of cancer for which both environmental insult (i.e., UV) and hereditary predisposition are major causative factors. Fish melanoma models have been used in studies of both spontaneous and induced melanoma formation. Genetic hybrids between platyfish and swordtails, different species of the genus Xiphophorus, have been studied since the 1920s to identify genetic determinants of pigmentation and melanoma formation. Recently, transgenesis has been used to develop zebrafish and medaka models for melanoma research. This review will provide a historical perspective on the use of fish models in melanoma research, and an updated summary of current and prospective studies using these unique experimental systems.     

A Comparative Analysis of Glomerulus Development in the Pronephros of Medaka and Zebrafish

The glomerulus of the vertebrate kidney links the vasculature to the excretory system and produces the primary urine. It is a component of every single nephron in the complex mammalian metanephros and also in the primitive pronephros of fish and amphibian larvae. This systematic work highlights the benefits of using teleost models to understand the pronephric glomerulus development. The morphological processes forming the pronephric glomerulus are astoundingly different between medaka and zebrafish. (1) The glomerular primordium of medaka - unlike the one of zebrafish - exhibits a C-shaped epithelial layer. (2) The C-shaped primordium contains a characteristic balloon-like capillary, which is subsequently divided into several smaller capillaries. (3) In zebrafish, the bilateral pair of pronephric glomeruli is fused at the midline to form a glomerulus, while in medaka the two parts remain unmerged due to the interposition of the interglomerular mesangium. (4) Throughout pronephric development the interglomerular mesangial cells exhibit numerous cytoplasmic granules, which are reminiscent of renin-producing (juxtaglomerular) cells in the mammalian afferent arterioles. Our systematic analysis of medaka and zebrafish demonstrates that in fish, the morphogenesis of the pronephric glomerulus is not stereotypical. These differences need be taken into account in future analyses of medaka mutants with glomerulus defects.     

Medaka as a model of transgenic fish.

The medaka (Oryzias latipes) is an egg-laying fresh-water fish. We describe the medaka as a model system of transgenic fish in germs of biological characteristics, manipulation of embryos, gene expression in development, and basic research in aquaculture. The fish are small (approximately 3 cm in length) and have a short generation time (approximately 3 months). The eggs are easy to manipulate. A foreign gene (e.g., the chicken delta crystallin gene) is transferred and expressed stage-dependently in development of medaka embryos. Growth hormone genes of vertebrates are transferred and expressed and, in some cases, accelerate growth of the fish. Thus, the medaka is one of the most promising models of transgenic fish for basic research of gene expression and aquaculture.     

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out ¹. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species ², thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps ³ so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.Download video file.^{(55M, mov)}     

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.     

Novel evolutionary relationship among four fish model systems

Knowledge of the correct phylogenetic relationships among animals is crucial for the valid interpretation of evolutionary trends in biology. Zebrafish, medaka, pufferfish and cichilds are fish models for development, genomics and comparative genetics studies, although their phylogenetic relationships have not been tested rigorously. The results of phylogenomic analysis based on 20 nuclear protein-coding genes confirmed the basal placement of zebrafish in the fish phylogeny but revealed an unexpected relationship among the other three species, contrary to traditionally held systematic views based on morphology. Our analyses show that medaka (Beloniformes) and cichlids (Perciformes) appear to be more closely related to each other than either of them is to pufferfish (Tetraodontiformes), suggesting that a re-interpretation of some findings in comparative biology might be required. In addition, phylogenomic analyses show that fish typically have more copies of nuclear genes than land vertebrates, supporting the fish-specific genome duplication hypothesis.     

Genetics of salmonid skin pigmentation: clues and prospects for improving the external appearance of farmed salmonids

Skin color is an important commercial trait in fish farming, given that this phenotype influences consumer acceptance, thereby determining the commercial value that fish can reach. This character is genetically determined, either by monogenetic or polygenetic control. Over the past few years, progress has been made in studies of quantitative genetic parameters for commercially important traits related to skin pigmentation and, in the molecular field, the mapping and cloning of some genes involved in fish color determination. This study reviews information regarding the genetic determination of salmonid skin color, along with different strategies to improve this character. Data collected in model fish (medaka and zebrafish) are also considered since this information contributes considerably towards improving understanding of the genes that may participate, and of the mechanisms involved in establishing skin coloration in salmonids.     

Zebrafish xenografts as a tool for in vivo studies on human cancer

The zebrafish has become a powerful vertebrate model for genetic studies of embryonic development and organogenesis and increasingly for studies in cancer biology. Zebrafish facilitate the performance of reverse and forward genetic approaches, including mutagenesis and small molecule screens. Moreover, several studies report the feasibility of xenotransplanting human cells into zebrafish embryos and adult fish. This model provides a unique opportunity to monitor tumor-induced angiogenesis, invasiveness, and response to a range of treatments in vivo and in real time. Despite the high conservation of gene function between fish and humans, concern remains that potential differences in zebrafish tissue niches and/or missing microenvironmental cues could limit the relevance and translational utility of data obtained from zebrafish human cancer cell xenograft models. Here, we summarize current data on xenotransplantation of human cells into zebrafish, highlighting the advantages and limitations of this model in comparison to classical murine models of xenotransplantation.     

Genetics and evolution of pigment patterns in fish

Vertebrate pigment patterns are both beautiful and fascinating. In mammals and birds, pigment patterns are likely to reflect the spatial regulation of melanocyte physiology, via alteration of the colour-type of the melanin synthesized. In fish, however, pigment patterns predominantly result from positioning of differently coloured chromatophores. Theoretically, pigment cell patterning might result from long-range patterning mechanisms, from local environmental cues, or from interactions between neighbouring chromatophores. Recent studies in two fish genetic model systems have made progress in understanding pigment pattern formation. In embryos, the limited evidence to date implicates local cues and chromatophore interactions in pigment patterning. In adults, de novo generation of chromatophores and cell-cell interactions between chromatophore types play critical roles in generating striped patterns; orientation of the stripes may well depend upon environmental cues mediated by underlying tissues. Further genetic screens, coupled with the routine characterization of critical gene products, promises a quantitative understanding of how striped patterns are generated in the zebrafish system. Initial 'evo-devo' studies indicate how fish pigment patterns may evolve and will become more complete as the developmental genetics is integrated with theoretical modelling.     

Diverse forms of guanylyl cyclases in medaka fish -- their genomic structure and phylogenetic relationships to those in vertebrates and invertebrates

Fish species such as medaka fish, fugu, and zebrafish contain more guanylyl cyclases (GCs) than do mammals. These GCs can be divided into two types: soluble GCs and membrane GCs. The latter are further divided into four subfamilies: (i) natriuretic peptide receptors, (ii) STa/guanylin receptors, (iii) sensory-organ-specific membrane GCs, and (iv) orphan receptors. Phylogenetic analyses of medaka fish GCs, along with those of fugu and zebrafish, suggest that medaka fish is a much closer relative to fugu than to zebrafish. Analyses of nucleotide data available on a web site (http://www.ncbi. nlm.nih.gov/) of GCs from a range of organisms from bacteria to vertebrates suggest that gene duplication, and possibly chromosomal duplication, play important roles in the divergence of GCs. In particular, the membrane GC genes were generated by chromosomal duplication before the divergence of tetrapods and teleosts.     

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, Kupffer's 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.     

Differential maturation of rhythmic clock gene expression during early development in medaka (Oryzias latipes)

One key challenge for the field of chronobiology is to identify how circadian clock function emerges during early embryonic development. Teleosts such as the zebrafish are ideal models for studying circadian clock ontogeny since the entire process of development occurs ex utero in an optically transparent chorion. Medaka (Oryzias latipes) represents another powerful fish model for exploring early clock function with, like the zebrafish, many tools available for detailed genetic analysis. However, to date there have been no reports documenting circadian clock gene expression during medaka development. Here we have characterized the expression of key clock genes in various developmental stages and in adult tissues of medaka. As previously reported for other fish, light dark cycles are required for the emergence of clock gene expression rhythms in this species. While rhythmic expression of per and cry genes is detected very early during development and seems to be light driven, rhythmic clock and bmal expression appears much later around hatching time. Furthermore, the maturation of clock function seems to correlate with the appearance of rhythmic expression of these positive elements of the clock feedback loop. By accelerating development through elevated temperatures or by artificially removing the chorion, we show an earlier onset of rhythmicity in clock and bmal expression. Thus, differential maturation of key elements of the medaka clock mechanism depends on the developmental stage and the presence of the chorion.     

鱼类的胚胎干细胞

胚胎干细胞(ES)是未分化的细胞培养物，来自动物的早期胚胎。它们能成为稳定的细胞系和长期冻存。在适当的条件下，ES细胞能分化成各种细胞类型，包括生殖细胞。这样，ES细胞就提供了一个有效的纽带，将动物基因组的体外和体内遗传操作连系起来。ES细胞的魅力就由其在产生和分析基因敲除老鼠中显现出来。目前，ES细胞技术仅见之老鼠，因其它脊椎动物的ES细胞的培养和建系难获成功。在鱼类，人们已做了大量的尝试。我们以青鳉(Oryzias latipes)作为建立鱼类ES细胞技术的模式，通过建立并应用无滋养层细胞的培养条件，获得了来自中期囊胚的ES细胞系。青鳉的ES细胞和老鼠的ES细胞有很多共同特征，如二倍体核型、分化潜力和形成嵌合体。因此，在鱼类建立和应用ES细胞技术是可能的。青鳉ES细胞的培养条件已成功地应用到其它鱼类如斑马鱼甚至海水鱼。本文旨在以青鳉为模式，综述获得和应用模式鱼和经济鱼ES细胞的主要进展和前景。