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Tokiro Ishikawa,
Tetsuya Okada,
Tomoko Ishikawa-Fujiwara,
Takeshi Todo,
Yasuhiro Kamei,
Shuji Shigenobu, Minoru Tanaka,
Taro L Saito,
Jun Yoshimura,
Shinichi Morishita,
Atsushi Toyoda,
Yoshiyuki Sakaki,
Yoshihito Taniguchi,
Shunichi Takeda,
Kazutoshi Mori
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ABSTRACT: ATF6α and ATF6β are membrane-bound transcription factors which are activated by regulated intramembrane proteolysis in response to endoplasmic reticulum (ER) stress to induce various ER quality control proteins. ATF6α- and ATF6β-single knockout mice develop normally but ATF6α/β-double knockout causes embryonic lethality, the reason for which remains unknown. Here, we showed in medaka fish that ATF6α is primarily responsible for transcriptional induction of the major ER chaperone BiP and that ATF6α/β-double knockout but not ATF6α- or ATF6β-single knockout causes embryonic lethality, as in mice. Analyses of ER stress reporters revealed that ER stress occurred physiologically during medaka early embryonic development, particularly in the brain, otic vesicle and notochord, resulting in ATF6α- and ATF6β-mediated induction of BiP, and that knockdown of α1 chain of type VIII collagen reduced such ER stress. The absence of transcriptional induction of several ER chaperones in ATF6α/β-double knockout caused more profound ER stress and impaired notochord development, which was partially rescued by overexpression of BiP. Thus, ATF6α/β-mediated adjustment of chaperone levels to increased demands in the ER is essential for development of the notochord, which synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis prior to formation of the vertebra.
Molecular biology of the cell 02/2013; · 5.98 Impact Factor
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ABSTRACT: We established three lines of transgenic medaka, a heat-shock element (HSE) monitor line (hse-GFP line), heat-inducible driver lines (hse-cre lines), and effector lines (gapdh-loxP[DsRed]-GFP lines). We employed these to comprehensively analyze gene induction at different time points in various tissues. These analyses demonstrate a good response of synthetic HSEs by heat treatment during embryogenesis and the mosaic gene induction by cre/loxP-mediated recombination, thus providing practical informations regarding the feasibility of a heat-inducible cre/loxP-mediated system in medaka. We also activated recombination by local heat-treatment using a metal probe and an infra-red laser. Our results collectively indicate that these lines allow us to perform lineage tracing and mosaic analysis and provide the platform to investigate gene functions at later developmental stage and adult. © 2012 Wiley Periodicals, Inc.
genesis 09/2012; · 2.53 Impact Factor
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ABSTRACT: The function of AMH (Anti-Müllerian hormone), a phylogenetically ancient member of the TGFβ family of proteins, in lower vertebrates is largely unknown. Previously, we have shown that the gene encoding the type II anti-Müllerian hormone receptor, amhrII, is responsible for excessive germ cell proliferation and male-to-female sex reversal in the medaka hotei mutant. In this study, functional analyses in cultured cells and of other amhrII mutant alleles indicate that lack of AMH signaling causes the hotei phenotype. BrdU incorporation experiments identified the existence of both quiescent and mitotically active germ cells among the self-renewing, type I population of germ cells in the developing gonad. AMH signaling acts in supporting cells to promote the proliferation of mitotically active germ cells but does not trigger quiescent germ cells to proliferate in the developing gonad. Furthermore, we show that the male-to-female sex reversal phenotype in hotei mutants is not a direct consequence of AMH signaling in supporting cells, but is instead mediated by germ cells. Our data demonstrate that interfollicular AMH signaling regulates proliferation at a specific stage of germ cell development, and that this regulation is crucial for the proper manifestation of gonadal sex directed by sex determination genes.
Development 05/2012; 139(13):2283-7. · 6.60 Impact Factor
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ABSTRACT: The sex determining gene is divergent among different animal species. However, sox9 is up-regulated in the male gonads in a number of species in which it is the essential regulator of testis determination. It is therefore often discussed that the sex determining gene-sox9 axis functions in several vertebrates. In our current study, we show that sox9b in the medaka (Oryzias latipes) is one of the orthologues of mammalian Sox9 at syntenic and expression levels. Medaka sox9b affects the organization of extracellular matrices, which represents a conserved role of sox9, but does not directly regulate testis determination. We made this determination via gene expression and phenotype analyses of medaka with different copy numbers of sox9b. Sox9b is involved in promoting cellular associations and is indispensible for the proper proliferation and survival of germ cells in both female and male medaka gonads. Medaka mutants that lack sox9b function exhibit a seemingly paradoxical phenotype of sex reversal to male. This is explained by a reduction in the germ cell number associated with aberrant extracellular matrices. Together with its identified roles in other vertebrate gonads, a testis-determining role for Sox9 in mammals is likely to have been neofunctionalized and appended to its conserved role in germ cell maintenance.
PLoS ONE 01/2012; 7(1):e29982. · 4.09 Impact Factor
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ABSTRACT: We evaluated the effects of 17(-ethinylestradiol (EE(2)) on sexual differentiation in transgenic olvas-GFP/STII-YI medaka (Oryzias latipes) in terms of the proliferative activity of germ cells. 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. From 0 days post-hatch (0 dph) onwards, juveniles were exposed to graded concentrations of EE(2) (25.2-1710 ng/L) for 35 days. The gonads of live specimens were monitored by measuring their size and calculating their GFP-fluorescence area. GFP-fluorescent area in control females was about 10 times that in control males at 10 days posthatch (dph) whereas the gonadal size of 10 dph males that had been exposed to 158 ng/L of EE(2) significantly increased up to twice the size of control males, indicating that abnormal sexual differentiation towards female might occur in these individuals. Histological examination and identification of the sex-linked marker SL1 indicated that male to female sex reversal occurred at EE(2) exposure ≥45.1 ng/L at 35 dph. These results suggest that observation of proliferative activity of germ cells in the olvas-GFP/STII-YI strain could be applied to facilitated screening fish model to detect adverse effects on sexual differentiation as early as 10 dph juveniles.
Aquatic toxicology (Amsterdam, Netherlands) 08/2011; 104(3-4):177-84. · 3.12 Impact Factor
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Seikagaku. The Journal of Japanese Biochemical Society 07/2011; 83(7):627-32. · 0.04 Impact Factor
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ABSTRACT: 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.
International journal of biological sciences 01/2011; 7(4):403-9. · 2.70 Impact Factor
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ABSTRACT: Germline stem cells continually produce sperm in vertebrate testes, whereas there is no direct evidence showing that germline stem cells are present in adult vertebrate ovaries. By using transgenic methods and clonal analysis, we identified germline stem cells that supported oogenesis and the production of offspring in the ovaries of adult medaka fish. Early-stage germ cells were localized in clusters along interwoven threadlike cords of sox9b-expressing somatic cells (termed germinal cradles) where the germ cells developed. Germline stem cells gave rise to germ cells that divided to produce cysts, which then underwent cell death or separated to form follicles. Our results provide insight into the germline stem cell biology of medaka and provide a model system for studying vertebrate stem cell niches.
Science 06/2010; 328(5985):1561-3. · 31.20 Impact Factor
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ABSTRACT: Control and coordination of eukaryotic gene expression rely on transcriptional and posttranscriptional regulatory networks. Evolutionary innovations and adaptations often require rapid changes of such networks. It has long been hypothesized that transposable elements (TE) might contribute to the rewiring of regulatory interactions. More recently it emerged that TEs might bring in ready-to-use transcription factor binding sites to create alterations to the promoters by which they were captured. A process where the gene regulatory architecture is of remarkable plasticity is sex determination. While the more downstream components of the sex determination cascades are evolutionary conserved, the master regulators can switch between groups of organisms even on the interspecies level or between populations. In the medaka fish (Oryzias latipes) a duplicated copy of dmrt1, designated dmrt1bY or DMY, on the Y chromosome was shown to be the master regulator of male development, similar to Sry in mammals. We found that the dmrt1bY gene has acquired a new feedback downregulation of its expression. Additionally, the autosomal dmrt1a gene is also able to regulate transcription of its duplicated paralog by binding to a unique target Dmrt1 site nested within the dmrt1bY proximal promoter region. We could trace back this novel regulatory element to a highly conserved sequence within a new type of TE that inserted into the upstream region of dmrt1bY shortly after the duplication event. Our data provide functional evidence for a role of TEs in transcriptional network rewiring for sub- and/or neo-functionalization of duplicated genes. In the particular case of dmrt1bY, this contributed to create new hierarchies of sex-determining genes.
PLoS Genetics 01/2010; 6(2):e1000844. · 8.69 Impact Factor
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ABSTRACT: Medaka (Oryzias latipes) is a small freshwater teleost fish that serves as a model vertebrate organism in various fields of biology including development, genetics, toxicology and evolution. The recent completion of the medaka genome sequencing project has promoted the use of medaka as a comparative and complementary material for research on other vertebrates such as zebrafish, sticklebacks, mice, and humans. The Japanese government has supported the development of Medaka Bioresources since 2002. The second term of the Medaka Bioresource Project started in 2007. The National Institute for Basic Biology and Niigata University were selected as the core organizations for this project. More than 400 strains including more than 300 spontaneous and induced mutants, 8 inbred lines, 21 transgenic lines, 20 medaka-related species and 66 wild stock lines of medaka are now being provided to the scientific community and educational non-profit organizations. In addition to these live fish, NBRP Medaka is also able to provide cDNA/EST clones such as full-length cDNA and BAC/fosmid clones covering 90% of the medaka genome. All these resources can be found on the NBRP Medaka website (http://shigen.lab.nig.ac.jp/medaka/), and users can order any resource using the shopping cart system. We believe these resources will facilitate the further use of medaka and help to promote new findings for this vertebrate species.
Experimental Animals 01/2010; 59(1):13-23. · 0.92 Impact Factor
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Tomoko Ishikawa,
Yasuhiro Kamei,
Shinji Otozai,
Jinhyong Kim,
Ayuko Sato,
Yoshikazu Kuwahara, Minoru Tanaka,
Tomonori Deguchi,
Hidenori Inohara,
Tohru Tsujimura,
Takeshi Todo
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ABSTRACT: During the last two decades, DNA sequencing has led to the identification of numerous genes in key species; however, in most cases, their functions are still unknown. In this situation, reverse genetics is the most suitable method to assign function to a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse-genetic strategy that combines random chemical mutagenesis with high-throughput discovery of the induced mutations in target genes. The method has been applied to a variety of plant and animal species. Screening of the induced mutations is the most important step in TILLING. Currently, direct sequencing or nuclease-mediated screening of heteroduplexes is widely used for detection of mutations in TILLING. Both methods are useful, but the costs are substantial and turnaround times are relatively long. Thus, there is a need for an alternative method that is of higher throughput and more cost effective.
In this study, we developed a high resolution melting (HRM) assay and evaluated its effectiveness for screening ENU-induced mutations in a medaka TILLING library. We had previously screened mutations in the p53 gene by direct sequencing. Therefore, we first tested the efficiency of the HRM assay by screening mutations in p53, which indicated that the HRM assay is as useful as direct sequencing. Next, we screened mutations in the atr and atm genes with the HRM assay. Nonsense mutations were identified in each gene, and the phenotypes of these nonsense mutants confirmed their loss-of-function nature.
These results demonstrate that the HRM assay is useful for screening mutations in TILLING. Furthermore, the phenotype of the obtained mutants indicates that medaka is an excellent animal model for investigating genome stability and gene function, especially when combined with TILLING.
BMC Molecular Biology 01/2010; 11:70. · 2.86 Impact Factor
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ABSTRACT: Changes in the steroidogenic pathway in medaka (Oryzias latipes) ovarian follicles during vitellogenesis and oocyte maturation were investigated in vitro by incubation of follicles with several radiolabeled steroid precursors, followed by thin layer chromatography (TLC) fractionation and recrystallization. When vitellogenic follicles collected at 18 hr before the expected time of spawning were incubated with 3H-labeled pregnenolone, the major metabolites were 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, and androstenedione. Incubations of vitellogenic follicles with androstenedione produced testosterone and estradiol-17β By contrast, when maturing follicles (postvitellogenic follicles undergoing maturation) collected at 10 hr before spawning were incubated with 3H-labeled pregnenolone, the major metabolites were 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, and 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20β-DP, maturation-inducing hormone of medaka); androstenedione was not detected. Neither vitellogenic and maturing follicles produced progesterone when they were incubated with 3H-labeled pregnenolone, suggesting that in medaka ovarian follicles both estradiol-17β and 17α, 20β-DP are synthesized by the 5Δ-steroid pathway. Thus, there is a distinct shift in the steroidogenic pathway from estradiol-17β to 17α, 20β-DP production in medaka ovarian follicles, and it is suggested that the decrease in C17, 20-lyase activity is responsible for this shift. The phosphodiesterase inhibitor IBMX enhanced androstenedione production in incubations of vitellogenic follicles with 14C-labeled progesterone. Calcium ionophore A23187 and the phorbol ester TPA (a protein kinase C activator) blocked the stimulatory actions of IBMX on androstenedione production. These findings suggest that multiple signalling pathways may participate in the regulation of ovarian steroidogenesis, and further emphasize the importance of calcium as a regulator of P-450c17 activity.
ZOOLOGICAL SCIENCE 12/2009; · 0.95 Impact Factor
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ABSTRACT: We performed in ovo nanoinjection of 4-nonylphenol (NP) into embryos of a transgenic see-through medaka (Oryzias latipes), olvas-GFP/STII-YI strain, which has two genotypic sex markers, and examined the effects on development and sexual differentiation. The transgene consisted of a green fluorescent protein (GFP) gene fused to the regulatory region of the medaka vasa gene. Germ cell-specific GFP expression was visualized in the gonad through the transparent body wall of the living fish. The development of each embryo was observed after nanoinjection of 2.0, 10, 50, 125, or 250 ng of NP. NP administration caused significant higher mortality at > or = 50 ng egg(-1) and inhibited embryonic development, including abnormal hatch and swim-up failure in all treatment groups except 10 ng egg(-1) group. However, it did not cause adverse effects on germ cell proliferation by 10d posthatch (dph) or sex differentiation of survivors by 100 dph. We concluded that single-dose in ovo exposure to nonylphenol affected embryonic development in the medaka but not gonadal development by 10 dph or sexual differentiation in adult fish by 100 dph. Although further investigations might be needed to elucidate the usefulness of nanoinjection of embryos of this strain, present study indicated that the nanoinjection model using olvas-GFP/STII-YI strain medaka has potential for use in evaluating the effects of chemicals on early development and sexual differentiation.
Chemosphere 10/2009; 77(11):1594-9. · 3.21 Impact Factor
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ABSTRACT: Aromatase is a steroidogenic enzyme catalyzing the production of estrogens and is important for the proper development and function of the reproductive system. The lineage of cyp19a1 (ovarian-type aromatase)-expressing cells in the developing gonad was analyzed using a transgenic medaka (Oryzias latipes) that recapitulates endogenous cyp19a1 expression with EGFP fluorescence. Our results show that cyp19a1-expressing cells arise in the ventral stromal cells of the developing female gonad, then expand anteriorly as the gonadal region extends anteriorly. These cells become located close to the developing follicles, and are distinguishable from the P450c17-I-expressing theca cells. In the adult ovary, the expression of P450c17-I and cyp19a1 are mutually exclusive in the outer theca-cell layer. Cyp19a1 expression in the granulosa cells is found only in the population of large follicles. These observations demonstrate two types of theca cells in the medaka ovary. We also show that the maintenance of cyp19a1-expressing cells depends on germ cells.
Developmental Dynamics 09/2009; 238(10):2652-7. · 2.54 Impact Factor
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ABSTRACT: The gene nanos is essential for germ cell development. Although its functions and expression have been investigated in the mouse, nanos genes have yet to be well characterized in other vertebrates. Based on similarity and a syntenic analysis of nanos, we have identified four different nanos in the genome of medaka (Oryzias latipes). nanos1 is duplicated in teleost fish genomes and named nanos1a and nanos1b. Of all medaka nanos, nanos3 is well conserved in terms of expression and synteny. In contrast to a previous study on mice, nanos2 expression was not detected in the gonads at early stages of sex differentiation; however, both oogonia and spermatogonia in adult gonads exhibit nanos2 expression. nanos1a and 1b are both expressed in the developing brain, consistent with the expression of nanos1 in mice. In the gonads, nanos1a is expressed in the somatic cells surrounding oocytes and spermatocytes, whereas expression of nanos1b is not detectable in the gonads by in-situ hybridization. These results suggest common and distinct functions of nanos genes among vertebrates.
ZOOLOGICAL SCIENCE 03/2009; 26(2):112-8. · 0.95 Impact Factor
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ABSTRACT: Differential gene expression largely accounts for the coordinated manifestation of the genetic programme underlying embryonic development and cell differentiation. The 3' untranslated region (3'-UTR) of eukaryotic genes can contain motifs involved in regulation of gene expression at the post-transcriptional level. In the 3'-UTR of dmrt1, a key gene that functions in gonad development and differentiation, an 11-bp protein-binding motif was identified that mediates gonad-specific mRNA localization during embryonic and larval development of fish. Mutations that disrupt the 11-bp motif leading to in vitro protein-binding loss and selective transcript stabilization failure indicate a role for this motif in RNA stabilization through protein binding. The sequence motif was found to be conserved in most of the dmrt1 homologous genes from flies to humans suggesting a widespread conservation of this specific mechanism.
Nucleic Acids Research 02/2009; 37(5):1510-20. · 8.03 Impact Factor
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ABSTRACT: The fluorescence lifetime image of HeLa cells expressing an enhanced green fluorescent protein (EGFP)-fusion protein changes under stress, which allows noninvasive determination of the status of individual cells.
Photochemical and Photobiological Sciences 07/2008; 7(6):671-4. · 2.58 Impact Factor
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ABSTRACT: To evaluate the possible role of germ cells on sex differentiation of the gonads in vertebrates, the teleost fish, medaka (Oryzias latipes), was used to generate a gonad without germ cells. The germ cell-deficient medaka reveals multiple effects of germ cells on the process of sex differentiation. The previously isolated mutant medaka, hotei, with the excessive number of germ cells may support the contention that the proliferation of germ cells is related to feminization of the gonad. Futhermore, we show that two modes of proliferation for either maintenance of germ cells or commitment to gametogenesis are important components of the sex differentiation of medaka developing gonads. An intimate cross talk between germ cells and gonadal somatic cells during the sex differentiation will be discussed.
Embryologia 06/2008; 50(4):273-8. · 2.21 Impact Factor
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ABSTRACT: Migratory pathways of PGCs to the gonad vary depending on the vertebrate species, yet the underlying regulatory mechanisms guiding PGCs are believed to be largely common. In teleost medaka embryo, PGC migration follows two major steps before colonizing in gonadal areas: (1) bilateral lineup in the trunk and (2) posterior drift of PGCs. kazura (kaz) and yanagi (yan) mutants of medaka isolated in mutagenesis screening were defective in the first and second steps, respectively. kaz(j2-15D) was identified as a missense mutation in chemokine receptor gene cxcr4b expressed in PGCs. Embryonic injection of cxcr4b mRNA with vasa 3' UTR rescued the PGC phenotype of kaz mutant, indicating a cell-autonomous function of cxcr4b in PGCs. yan(j6-29C) was identified as a nonsense mutation in the cxcr7/rdc1 gene encoding another chemokine receptor. cxcr7 transgene with genomic flanking sequences rescued the yan mutant phenotype efficiently at the G0 generation. cxcr7 was expressed in somites rather than PGCs. cxcr7-expressing somitic domain expanded posteriorly with its margin immediately anterior of posteriorly drifting PGCs, as if PGCs were thrusted toward the gonadal area. kaz and yan mutants are also defective in lateral line positioning, suggesting combined employment of these receptor systems in various cell migratory processes.
Developmental Biology 06/2008; 320(2):328-39. · 4.07 Impact Factor
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ABSTRACT: The availability of bacterial artificial chromosome (BAC) offers a good genomic platform for a targeted integration of an exogenous gene by a homologous recombination system in Escherichia coli. In combination with microinjection technology, this system allows for the analysis of various aspects of biological phenomena occurring in vivo using Japanese medaka fish (Oryzias latipes). Here we describe a streamlined procedure for selecting BAC clones based on the medaka University of Tokyo genome browser (UTGB), followed by rapid modification with enhanced green fluorescent protein (EGFP) or DsRed fragments for transgenic analysis in medaka. Experimental procedures for BAC DNA preparation, microinjection of medaka embryos and screening of resulting transgenic medaka carrying EGFP/DsRed modified BAC clones are also described.
Embryologia 05/2008; 50(6):415-9. · 2.21 Impact Factor
