Sox5 is involved in germ-cell regulation and sex determination in medaka following co-option of nested transposable elements.

BACKGROUND: Sex determination relies on a hierarchically structured network of genes, and is one of the most plastic processes in evolution. The evolution of sex-determining genes within a network, by neo- or sub-functionalization, also requires the regulatory landscape to be rewired to accommodate these novel gene functions. We previously showed that in medaka fish, the regulatory landscape of the master male-determining gene dmrt1bY underwent a profound rearrangement, concomitantly with acquiring a dominant position within the sex-determining network. This rewiring was brought about by the exaptation of a transposable element (TE) called Izanagi, which is co-opted to act as a silencer to turn off the dmrt1bY gene after it performed its function in sex determination. RESULTS: We now show that a second TE, Rex1, has been incorporated into Izanagi. The insertion of Rex1 brought in a preformed regulatory element for the transcription factor Sox5, which here functions in establishing the temporal and cell-type-specific expression pattern of dmrt1bY. Mutant analysis demonstrates the importance of Sox5 in the gonadal development of medaka, and possibly in mice, in a dmrt1bY-independent manner. Moreover, Sox5 medaka mutants have complete female-to-male sex reversal. CONCLUSIONS: Our work reveals an unexpected complexity in TE-mediated transcriptional rewiring, with the exaptation of a second TE into a network already rewired by a TE. We also show a dual role for Sox5 during sex determination: first, as an evolutionarily conserved regulator of germ-cell number in medaka, and second, by de novo regulation of dmrt1 transcriptional activity during primary sex determination due to exaptation of the Rex1 transposable element.

Sox5 functions as a fate switch in medaka pigment cell development.

Mechanisms generating diverse cell types from multipotent progenitors are crucial for normal development. Neural crest cells (NCCs) are multipotent stem cells that give rise to numerous cell-types, including pigment cells. Medaka has four types of NCC-derived pigment cells (xanthophores, leucophores, melanophores and iridophores), making medaka pigment cell development an excellent model for studying the mechanisms controlling specification of distinct cell types from a multipotent progenitor. Medaka many leucophores-3 (ml-3) mutant embryos exhibit a unique phenotype characterized by excessive formation of leucophores and absence of xanthophores. We show that ml-3 encodes sox5, which is expressed in premigratory NCCs and differentiating xanthophores. Cell transplantation studies reveal a cell-autonomous role of sox5 in the xanthophore lineage. pax7a is expressed in NCCs and required for both xanthophore and leucophore lineages; we demonstrate that Sox5 functions downstream of Pax7a. We propose a model in which multipotent NCCs first give rise to pax7a-positive partially fate-restricted intermediate progenitors for xanthophores and leucophores; some of these progenitors then express sox5, and as a result of Sox5 action develop into xanthophores. Our results provide the first demonstration that Sox5 can function as a molecular switch driving specification of a specific cell-fate (xanthophore) from a partially-restricted, but still multipotent, progenitor (the shared xanthophore-leucophore progenitor).

Dynein axonemal intermediate chain 2 is required for formation of the left-right body axis and kidney in medaka.

Ciliary defects lead to various diseases, such as primary ciliary dyskinesia (PCD) and polycystic kidney disease (PKD). We isolated a medaka mutant mii, which exhibits defects in the left-right (LR) polarity of organs, and found that mii encodes dynein axonemal intermediate chain 2a (dnai2a). Ortholog mutations were recently reported to cause PCD in humans. mii mutant embryos exhibited loss of nodal flow in Kupffer's Vesicle (KV), which is equivalent to the mammalian node, and abnormal expression of the left-specific gene. KV cilia in the mii mutant were defective in their outer dynein arms (ODAs), indicating that Dnai2a is required for ODA formation in KV cilia. While the mii mutant retained motility of the renal cilia and failed to show PKD, the loss of dnai2a and another dnai2 ortholog dnai2b led to PKD. These findings demonstrate that Dnai2 proteins control LR polarity and kidney formation through regulation of ciliary motility.

Overexpression of the dominant-negative form of myostatin results in doubling of muscle-fiber number in transgenic medaka (Oryzias latipes).

In addition to altering the phenotypes of gene-modified animals, transgenesis also has the potential to facilitate access to the various mechanisms underlying the development and functioning of specific phenotypes and genes, respectively. Myostatin (MSTN) is implicated in double-muscling when mutated in mammals, indicating that MSTN is a negative regulator of skeletal muscle formation. In order to elucidate the role of an MSTN equivalent in fish muscle formation, we created a transgenic medaka strain that expresses dominant-negative MSTN exclusively in skeletal muscle, d-rR-Tg(OlMA1-C315Y-MSTN-hrGFPII-FLAG). The transgenic fish exhibited increased production of skeletal muscle fibers at the adult stage (hyperplasia), although gross muscle mass was not altered. During embryogenesis, ectopic accumulation and misalignment of muscle fibers, possibly due to muscle-fiber hypertrophy, were observed in the transgenic medaka. Our findings suggest that MSTN function is required for regulating the appropriate growth of skeletal muscle in medaka. Unlike in mammals, MSTN loss-of-function failed to induce double-muscling in medaka, despite the highly conserved nature of MSTN function among taxa.

Kidney regeneration through nephron neogenesis in medaka.

Although renal regeneration is limited to repair of the proximal tubule in mammals, some bony fish are capable of renal regeneration through nephron neogenesis in the event of renal injury. We previously reported that nephron development in the medaka mesonephros is characterized by four histologically distinct stages, generally referred to as condensed mesenchyme, nephrogenic body, relatively small nephron, and the mature nephron. Developing nephrons are positive for wt1 expression during the first three of these stages. In the present study, we examined the regenerative response to renal injury, artificially induced by the administration of sublethal amounts of gentamicin in adult medaka. Similar to previous reports in other animals, the renal tubular epithelium and the glomerulus of the medaka kidney exhibited severe damage after exposure to this agent. However, kidneys showed substantial recovery after gentamicin administration, and a significant number of developing nephrons appeared 14 days after gentamicin administration (P < 0.01). Similarly, the expression of wt1 in developing nephrons also indicated the early stages of nephrogenesis. These findings show that medaka has the ability to regenerate kidney through nephron neogenesis during adulthood and that wt1 is a suitable marker for detecting nephrogenesis.

Distribution of complete and defective copies of the Tol1 transposable element in natural populations of the medaka fish Oryzias latipes.

DNA-based transposable elements are present in the genomes of various organisms, and generally occur in autonomous and nonautonomous forms, with a good correspondence to complete and defective copies, respectively. In vertebrates, however, the vast majority of DNA-based elements occur only in the nonautonomous form. Until now, the only clear exception known has been the Tol2 element of the medaka fish, which still causes mutations in genes of the host species. Here, we report another exception: the Tol1 element of the same species. This element was thought likely to be a "dead" element like the vast majority of vertebrate elements, but recent identification of an autonomous Tol1 copy in a laboratory medaka strain gave rise to the possibility that the element is still "alive" in medaka natural populations. We examined variation in the structure of Tol1 copies through genomic Southern blot analysis, and revealed that 10 of the 32 fish samples examined contained full-length Tol1 copies in their genomes. The frequency at which these copies occur among Tol1 copies is at most 0.5%, yet some of them still have the ability to produce a functional transposase. The medaka fish thus harbors two active DNA-based elements in its genome, and is in this respect unique among vertebrates.

Cell growth-promoting activity of fluid from eye sacs of the bubble-eye goldfish (Carassius auratus).

The growth-promoting effects of fish body fluids, such as serum and embryonic extract, on fish cell cultures have been widely demonstrated. The bubble-eye variety of aquarium goldfish is characterized as having a large sac filled with fluid (sac fluid) under each eye. These sacs are believed to contain lymph, which is similar in composition to serum or blood plasma. In order to test whether the sac fluid can be used as an additive for fetal bovine serum (FBS) in growth factor supplements, we compared cell growth in media containing FBS together with different concentrations of sac fluid. A dose-dependent growth-promotion effect was observed in early passage caudal fin cells from both medaka and zebrafish. Cell-growth promotion was also confirmed in early passage medaka blastula cells and in a zebrafish embryonic cell line (ZF4). Replacement of the fluid in the eye sacs of bubble-eyes occurs within a couple of months after the sac fluid has been harvested, and the cell-growth promoting activity of the new fluid is similar to that of the fluid that was tapped initially. These findings suggest that sac fluid can be used as a growth-promoting supplement for fish cell culture. Importantly, the ability of the goldfish to replace the fluid combined with the fact that equipotent fluid can be repeatedly harvested from the eye sacs means that a sustainable source of the fluid can be obtained without needing to sacrifice the fish.

Mullerian inhibiting substance is required for germ cell proliferation during early gonadal differentiation in medaka (Oryzias latipes).

Müllerian inhibiting substance (MIS) is a glycoprotein belonging to the TGF-beta superfamily. In mammals, MIS is responsible for the regression of Müllerian ducts in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fish, which have no Müllerian ducts, has yet to be clarified. In the present study, we examined the expression pattern of mis and mis type 2 receptor (misr2) mRNAs and the function of MIS signaling in early gonadal differentiation in medaka (teleost, Oryzias latipes). In situ hybridization showed that both mis and misr2 mRNAs were expressed in the somatic cells surrounding the germ cells of both sexes during early sex differentiation. Loss-of-function of either MIS or MIS type II receptor (MISRII) in medaka resulted in suppression of germ cell proliferation during sex differentiation. These results were supported by cell proliferation assay using 5-bromo-2'-deoxyuridine labeling analysis. Treatment of tissue fragments containing germ cells with recombinant eel MIS significantly induced germ cell proliferation in both sexes compared with the untreated control. On the other hand, culture of tissue fragments from the MIS- or MISRII-defective embryos inhibited proliferation of germ cells in both sexes. Moreover, treatment with recombinant eel MIS in the MIS-defective embryos dose-dependently increased germ cell number in both sexes, whereas in the MISRII-defective embryos, it did not permit proliferation of germ cells. These results suggest that in medaka, MIS indirectly stimulates germ cell proliferation through MISRII, expressed in the somatic cells immediately after they reach the gonadal primordium.

Novel method for the nuclear transfer of adult somatic cells in medaka fish (Oryzias latipes): use of diploidized eggs as recipients.

Until recently, the nuclear transfer of adult somatic cell nuclei in fish has been unsuccessful. This is primarily because of chromosomal aberrations in nuclear transplants, which are thought to arise due to asynchrony between the cell cycles of the recipient egg and donor nucleus. We recently succeeded in circumventing this difficulty by using a new nuclear transfer method in medaka fish (Oryzias latipes). Instead of enucleated eggs, the method uses non-enucleated and diploidized eggs, obtained by retention of the second polar body release, as recipients in the nuclear transfer of primary culture cells from the caudal fin of an adult green fluorescent protein gene (GFP)-transgenic strain. We found that 2.7% of the reconstructed embryos grew into diploid and fertile adults exhibiting donor expression characteristics and transmission of the GFP marker gene to progeny. The mechanism underlying the generation of nuclear transplants using this method is unknown at present; however, analyses of donor and recipient nuclei behavior and the cytoskeletal mechanisms involved in the early developmental stages, as well as the special ability of diploidized eggs to facilitate reprogramming of the donor nuclei will result in elucidation of the mechanism.

Conserved function of medaka pink-eyed dilution in melanin synthesis and its divergent transcriptional regulation in gonads among vertebrates.

Medaka is emerging as a model organism for the study of vertebrate development and genetics, and its effectiveness in forward genetics should prove equal to that of zebrafish. Here, we identify by positional cloning a gene responsible for the medaka i-3 albino mutant. i-3 larvae have weakly tyrosinase-positive cells but lack strongly positive and dendritic cells, suggesting loss of fully differentiated melanophores. The region surrounding the i-3 locus is syntenic to human 19p13, but a BAC clone covering the i-3 locus contained orthologs located at 15q11-13, including OCA2 (P). Medaka P consists of 842 amino acids and shares approximately 65% identity with mammalian P proteins. The i-3 mutation is a four-base deletion in exon 13, which causes a frameshift and truncation of the protein. We detected medaka P transcripts in melanin-producing eyeballs and (putative) skin melanophores on embryos and an alternatively spliced form in the non-melanin-producing ovary or oocytes. The mouse p is similarly expressed in gonads, but not alternatively spliced. This is the first isolation of nonmammalian P, the functional mechanism of action of which has not yet been elucidated, even in mammals. Further investigation of the functions of P proteins and the regulation of their expression will provide new insight into body color determination and gene evolution.

The Tomita collection of medaka pigmentation mutants as a resource for understanding neural crest cell development.

All body pigment cells in vertebrates are derived from the neural crest. In fish the neural crest can generate up to six different types of pigment cells, as well as various non-pigmented derivatives. In mouse and zebrafish, extensive collections of pigmentation mutants have enabled dissection of many aspects of pigment cell development, including fate specification, survival, proliferation and differentiation. A collection of spontaneous mutations collected from wild medaka (Oryzias latipes) populations and maintained at Nagoya University includes more than 40 pigmentation mutations. The descriptions of their adult phenotypes have been previously published by Tomita and colleagues (summarised in Medaka (Killifish) Biology and Strains, 1975), but the embryonic phenotypes have not been systematically described. Here we examine these embryonic phenotypes, paying particular attention to the likely defect in pigment cell development in each, and comparing the spectrum of defects to those in the zebrafish and mouse collections. Many phenotypes parallel those of identified zebrafish mutants, although pigment cell death phenotypes are largely absent, presumably due to the different selective pressures under which the mutants were isolated. We have identified mutant phenotypes that may represent the Mitf/Kit pathway of melanophore specification and survival. We use in situ hybridisation with available markers to confirm a key prediction of this hypothesis. We also highlight a set of novel phenotypes not seen in the zebrafish collection. These mutants will be a valuable resource for pigment cell and neural crest studies and will strongly complement the mutant collections in other vertebrates.

Possible roles of zic1 and zic4, identified within the medaka Double anal fin (Da) locus, in dorsoventral patterning of the trunk-tail region (related to phenotypes of the Da mutant).

Double anal fin (Da) is a spontaneous medaka mutant that exhibits an unique ventralizing phenotype, a mirror-image duplication across the lateral midline in the dorsal trunk-tail region. In the mutant, early D-V specification appears normal but the altered phenotype becomes evident during late embryogenesis. In this study, we genetically specified the mutation to a 174-kb region harboring two zinc-finger type transcription factors, zic1 and zic4, and compared the genomic structures of this region between wild-type and Da mutant fish. No mutation was found in the coding regions of either gene of the mutant, while two fragments, 324 bp and 3-4 kb long, were found inserted downstream of zic1 and zic4, respectively. Probably as a result of this, the expression of both genes is lost in the derivatives of the dorsal (epaxial) somite and the region dorsal to the terminal axis bending. All these tissues are morphologically affected or become ventralized in the mutants. In contrast, the expression in the head region and dorsal spinal cord remained unchanged. Detailed characterization of Da phenotypes revealed a novel defect in the axial skeleton (spina bifida occulta) that was also found in zic1-deficient mice. Finally, zic1-morpholino injection partially phenocopied early Da phenotypes. These findings strongly suggest that zic1 and/or zic4 are required for dorsal identity in the trunk-tail region and that loss of their expression in the epaxial somite derivatives and tail region causes the Da phenotypes.

Generation of fertile and diploid fish, medaka (Oryzias latipes), from nuclear transplantation of blastula and four-somite-stage embryonic cells into nonenucleated unfertilized eggs.

In two experimental series of transplantation of embryonic cell nuclei into nonenucleated unfertilized eggs in medaka (Oryzias latipes), fertile and diploid nuclear transplants were successfully generated. In the first experiment, nuclei from blastula cells of a medaka stock with the wild-type body color were transplanted into 1722 eggs from the orange-red variety. Of 26 adult nuclear transplants with the wild-type body color, 22 were, as expected, triploid and sterile, but the other four were fertile. Three of the four were diploid, and the last one was tetraploid. They transmitted the wild-type body color to the F1 and F2 progenies in a Mendelian fashion. In the second experiment, cell nuclei from four-somite-stage embryos of the orangered variety carrying the green fluorescent protein (GFP) transgene were transplanted into 1688 recipients of the same strain. Three adult nuclear transplants expressing GFP were obtained. Two of them were triploid and sterile, but the remaining one was fertile and diploid. The transgene of the donor nuclei was transmitted to the F(1) and F(2) offspring in a Mendelian fashion. These observations that diploid and fertile nuclear transplants could be obtained without enucleation of the recipient eggs may have important implications for future nuclear transplantation in medaka.

Quantitative bio-imaging analysis for evaluation of sexual differentiation in germ cells of olvas-GFP/ST-II YI medaka (Oryzias latipes) nanoinjected in ovo with ethinylestradiol.

We examined the effects on sexual differentiation of nanoinjecting ethinylestradiol (EE2) into embryos of olvas-GFP/ST-II YI medaka (Oryzias latipes). This strain contains the green fluorescent protein (GFP) gene fused to the regulatory region of the medaka vasa gene, and germ-cell-specific expression of GFP can be visualized in living (transparent) individuals. The number of germ cells in untreated genotypic females (XX) was approximately 10-fold that in untreated genotypic males (XY) at 10 d posthatch (dph). Germ cell proliferation was prevented in XX females that developed from embryos nanoinjected with 0.5, 2.5, or 5.0 ng of EE2. Some 10-dph XY males from embryos injected with 0.5 ng or more of EE2 showed a larger fluorescent area and more germ cells than those of pooled control groups. Males and females from embryos injected with 5.0 ng of EE2 had no significant difference in germ cell number or fluorescent area. Thus, EE2 injection into embryos caused abnormal gonadal development in both sexes. Observations of external secondary sex characteristics and histological examination of adult gonads showed complete sex reversal in some males after 0.5-, 2.5-, and 5.0-ng treatments but no changes in XX females after any treatment. Thus, quantitative bio-imaging can aid in evaluating the effects of endocrine-disrupting chemicals on fish within 10 dph.

Analysis of estrogenic effects by quantification of green fluorescent protein in juvenile fish of a transgenic medaka.

TheChgH-GFP strain of the teleost medaka contains a regulatory region of the estrogen-responsive choriogenin H (chgH) gene fussed to the green fluorescent protein (GFP) gene. The strain was developed for the identification of environmental estrogens by noninvasive analysis of GFP fluorescence. In the present study, a quantification method for GFP by image analysis was establishedand applied to the analysis of time- and concentration-dependent GFP fluorescence in juvenile fish. Concentration-response analyses were performed with fish exposed for 14 d to 17beta-estradiol (0.37-367 pM), genistein (0.37-367 nM), or p-nonylphenol (0.367-1,835 nM). By means of image analysis, it was shown that ChgH-GFP was induced at 183.5 pM or greater 17beta-estradiol. Time-course and recovery experiments indicated a strong accumulation of GFP in the liver. Results of reverse transcriptase-polymerase chain reaction analysis of choriogenin H and vitellogenin demonstrated induction of gene expression for the same rangeof concentrations as that for GFP analysis. Neither expression of these genes nor GFP fluorescence was induced by genistein and p-nonylphenol. Although the ChgH-GFP strain failed to detect these weakly estrogenic compounds, the simplicity of the GFP quantification during early life stages of fish offers promising possibilities for further developments of transgenic strains using different target regulatory sequences.

TWO TYPES OF MELANOMAS IN A NEW EXPERIMENTAL SYSTEM OF PLATYFISHSWORDTAIL HYBRIDS.

A new experimental system for investigating platyfish-swordtail hybrid melanomas was developed using ornamental platyfish (Xiphophorus maculatus) and swordtails (Xiphophorus helleri). The incidence of melanomas in the hybrids was studied in relation to individual age factors. Based on the age of the onset of the melanomas, two types of the melanomas were discerned and were designated as the fry and the adult melanomas, respectively. The fry melanomas were characterized by early onset, wide-ranged lesions, an invasive nature owing to densely pigmented cells, and a tendency toward regression. The adult melanomas were characterized by occurrence in the later stage of life, a small number of primary sites, and rapid, expanding, and invasive growth due to sparsely pigmented cells.

Cellular Heterogeneity in the Late-onset Form of Hereditary Melanomas in the Xiphophorus Fish Hybrids.

The late-onset form of melanomas occurring in the Xiphophorus, fish hybrids carrying a macro-melanophore gene Sp was investigated for its cellular heterogeneity. The melanoma tissues were dissociated enzymatically and cultured for a short term. The cultured melanoma cells were characterized according to cell size, cell shape, pigmentation, and response to epinephrine. The melanoma cells were considerably heterogeneous in these phenotypic traits. Various combinations of these heterogeneous cells gave a great heterogeneity to individual melanomas. The stability of the phenotypic traits was followed during the course of tumor growth. Cell size and cell shape were stable, but pigmentation and response to epinephrine varied. The results are discussed in relation to cell differentiation and tumor progression.

The Degree of Differentiation in Early- and Late-onset Forms of Platyfish-swordtail Hybrid Melanomas: A Morphological and Physiological Study in Primary Culture.

The early- and late-onset forms of platyfish-swordtail hybrid melanomas (fry and adult melanomas) and the macromelanophores of platyfish and melanotic hybrids were cultured and characterized according to their cellular morphology and physiology. The fry melanomas contained many large and broad cells. The pigmentation of these cells was somewhat less than that of the macromelanophores of platyfish. Most of the fry melanoma cells responded rapidly to 10-6 M epinephrine, exhibiting reversible melanosome aggregation. The adult melanomas consisted of small, dendritic, and sparsely pigmented cells. The physiological response of these adult melanoma cells varied widely from tumor to tumor. These findings are discussed in relation to the differentiation of fish melanophores.

Fish Hereditary Melanoma Cell Lines of Different Degrees of Cell Differentiation: (fish/hereditary melanoma/melanoma cell line/pigment cell differentiation).

Four cell lines including two sublines were established from hereditary melanomas in interspecific hybrids between platyfish (Xiphophorus maculatus) carrying the Sp gene and swordtails (X. helleri) and maintained in vitro for more than 34 months. Cells in each cell line grew randomly across each other with an apparent lack of contact inhibition of growth and at a population doubling time of 50 to 72 hr. They retained the characteristics of young pigment cells in regard to ultrastructure, tyrosinase activity, the DOPA and combined DOPA-premelanin reactions. In the degree of differentiation, the cells of the three cell lines seemed comparable to early melanocytes close to melanoblasts, and those of the remaining one cell line seemed comparable to young melanocytes but were in a more differentiated state than the early melanocytes. Colony forming ability on plastic plates was at a level of 10% in the three cell lines but only 1% in the one cell line. All four cell lines failed to form colonies in soft agar. Chromosome analysis revealed that these four cell lines were heteroploid with many abnormal figures of chromosomes and double minute chromosomes. None of the cell lines showed transplantability to fish.

Normal growth of the "see-through" medaka.

The see-through stock in the medaka Oryzias latipes, causes pigments to be absent from the whole body and has a transparent body in the adult stage as well as during embryonic stages. To establish a standard table of growth stages for this model fish, morphological features were examined during the growing period from hatching to adulthood. The main observations were performed on morphological changes in external and internal organs that could be seen through the body wall of the living fish during growth. Finally, five growth stages from just after hatching to the adult stage were defined on the basis of synchronized or definite changes in morphology as follows: (1) stage 40 in which the nodes (joints) in bony rays of the caudal and pectoral fins first appear, (2) the stage 41 in which the ribs and the anal, dorsal and ventral fins are formed by degeneration of the membrane fin folds, as recognized by the first appearance of nodes in the fin rays of the anal, pectoral and dorsal fins, and the parallel distribution of the dorsal artery and ventral vein of the tail, (3) stage 42 in which the 2-spiral pattern of the gut, the ray nodes in the ventral fins, and the scales first appear, (4) stage 43 in which early secondary sexual characters such as urinogenital protruberances (female) and papillar processes (male) appear, (5) stage 44 in which the 3-spiral pattern of the gut and the papillar process on the 2nd ray of pectral fins (male) appear.