Medaka vasa is required for migration but not survival of primordial germ cells

Vasa is essential for germline development. However, the precise processes in which vasa involves vary considerably in diverse animal phyla. Here we show that vasa is required for primordial germ cell (PGC) migration in the medakafish. vasa knockdown by two morpholinos led to the PGC migration defect that was rescued by coinjection of Vasa RNA. Interestingly, Vasa knockdown did not alter the PGC number, identity, proliferation and motility even at ectopic locations. We established a cell culture system for tracing PGCs at the single cell level in vitro. In this culture system, control and morpholino-injected gastrulae produced the same PGC number and the same time course of PGC survival. importantly, vasa-depleted PGCs in culture had similar motility and locomotion to normal PGCs. Expression patterns of wt1a, sdf1b and cxcT4b in migratory tissues remained unchanged by Vasa knockdown. By chimera formation we show that PGCs from vasa-depleted blastulae failed to migrate properly in the normal environment, whereas control PGCs migrated normally in vasa-disrupted embryos. Furthermore, ectopic PGCs in vasa-depleted embryos also retained all the PGC properties examined. Taken together, medaka vasa is cell-autonomously required for PGC migration, but dispensable to PGC proliferation, motility, identity and survival. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Antimicrobial activity-specific to Gram-negative bacteria and immune modulation-mediated NF-kappa B and Sp1 of a medaka beta-defensin

Defensins are a group of cationic antimicrobial peptides which play an important role in the innate immune system by exerting their antimicrobial activity against pathogens. In this study, we cloned a novel beta-defensin cDNA from medaka (Oryzias latipes) by rapid amplification of cDNA ends (RACE) technique. The full-length cDNA consists of 480 bp, and the open reading frame (CRF) of 189 bp encodes a polypeptide of 63 amino acids (aa) with a predicted molecular weight of 7.44 kDa. Its genomic organization was analyzed, and Southern blot detection confirmed that only one copy of beta-defensin exists in the medaka HNI strain. RT-PCR, Western blot and immunohistochemistry detections showed that the beta-defensin transcript and protein could be detected in eyes, liver, kidney, blood, spleen and gill, and obviously prevalent expression was found in eyes. Antimicrobial activity of the medaka beta-defensin was evaluated, and the antibacterial activity-specific to Gram-negative bacteria was revealed. Furthermore, the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, was demonstrated to be able to induce about 13-fol up-regulation of the beta-defensin within first 12 h. In addition, promoter and promoter mutagenesis analysis were performed in the medaka beta-defensin. A proximal 100 base pair(bp) sequence (+26 to -73)and the next 1700 bp sequence (-73 to -1755) were demonstrated to be responsible for the basal promoter activity and for the transcription regulation. Three nuclear factor kappa B (NF-kappa B) cis-elements and a Sp1 cis-element were revealed by mutagenesis analysis to exist in the 5' flanking sequence, and they were confirmed to be responsible for the up-regulation of medaka beta-defensin stimulated by LPS. And, the Sp1 cis-element was further revealed to be related to the basal promoter activity, and transcriptional factor II D (TFIID) was found to be in charge of the gene transcription initiation. All the obtained data suggested that the novel medaka beta-defensin should have antimicrobial activity-specific to Gram-negative bacteria, and the antibacterial immune function should be modulated by NF-kappa B and Sp1. (C) 2008 Elsevier Ltd. All rights reserved.

Cloning and expression of medaka dazl during ernbryogenesis and gametogenesis

The Deleted in azoospermia family consists of RNA-binding proteins Bottle, Daz, and Daz-like (Dazl) that are expressed in the germline. Here, we report the cloning and expression of the medakafish (Oryzias latipes) dazl gene (odazl). Interestingly, although the predicted medaka Dazl protein (oDazl) contains a RRM motif and a DAZ repeat characteristic of its mammalian homologs, it lacks 80 aa at the C-terminus. By RT-PCR, RNA in situ hybridization, Western blotting and fluorescent immunohistochemistry using a rabbit anti-DazI antibody (alpha Oazl), we analyzed the expression patterns of odazl and its protein. The odazl transcript persists throughout embryogenesis and delineates with primordial germ cells. In adults, the expression of odazl RNA and its protein is restricted to germ cells of both the testis and ovary. We observed differential expression of RNA and protein at critical stages of gametogenesis. In the testis, the odazl RNA is low at premeiotic stages, abundant at meiotic stages, but absent in postmeiotic stages; whereas the oDazl protein is rich in premeiotic stages, reduced at meiotic stages, becomes barely detectable or absent in postmeiotic round spermatids or sperm, respectively. This is in sharp mature spermatozoa. In the ovary, the odazl RNA contrast to the human situation where the Dazl transcript and protein are present in and protein persist throughout oogenesis and also show differential expression at premeiotic, meiotic and postmeiotic stages. Thus, the odazl or its protein is a marker for germ cells during embryogenesis and at critical stages of gametogenesis in both sexes of medaka. (c) 2006 Elsevier B.V. All rights reserved.

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

Gene and genomic duplications are very important and frequent events in fish evolution, and the divergence of duplicated genes in sequences and functions is a focus of research on gene evolution. Here, we report the identification and characterization of three duplicated Spindlin (Spin) genes from medaka (Oryzias latipes): OlSpinA, OlSpinB, and OlSpinC. Molecular cloning, genomic DNA Blast analysis and phylogenetic relationship analysis demonstrated that the three duplicated OlSpin genes should belong to gene duplication. Furthermore, Western blot analysis revealed significant expression differences of the three OlSpins among different tissues and during embryogenesis in medaka, and suggested that sequence and functional divergence might have occurred in evolution among them. © 2005 Elsevier B.V. All rights reserved.

Biomarker responses and reproductive toxicity of the effluent from a Chinese large sewage treatment plant in Japanese medaka (Oryzias latipes)

The present study was conducted to assess the potential toxicity of the effluent from a large sewage treatment plant (GBD-STP) in Beijing. Japanese medakas (Oryzias latipes) at reproduction active period were exposed to a serial of graded concentrations of the effluent or 100 ng l(-1) of 17-alpha-ethinylestradiol (EE2, positive control). Growth, gonadosomatic index (GSI), hepatosomatic index (HSI), reproductive success, induction potency of vitellogenin (VTG) in male fish and that of 7-ethoxyresorufin-o-deethylase activity (EROD) in male fish liver were used as test endpoints. The growth suppression of fish was observed in a dose-dependent manner, resulting in significant differences in both body length and body weight of medaka above 5% effluent. This effluent can inhibit the growth of gonad of medakas and are more sensitive to male than to female. At exposure concentration of 40% and higher, there was an unexpected decrease of HSI values, which may be resulted from sub-lethal toxicity of effluent to fish liver. VTG of plasma in males were induced in all exposure concentration levels, but not in a dose-dependent manner. The concentration of 5% effluent would be the lowest observed adverse effect level (LOAEL) affecting reproductive success when examining fertile individuals, fecundity and fertilization rate. The overt CYP1A response and higher reproductive toxicity may be indicative of low process efficiency of this STP. (c) 2004 Elsevier Ltd. All rights reserved.

Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.

Quantifiable biomarkers of normal aging in the Japanese medaka fish (Oryzias latipes).

BACKGROUND: Small laboratory fish share many anatomical and histological characteristics with other vertebrates, yet can be maintained in large numbers at low cost for lifetime studies. Here we characterize biomarkers associated with normal aging in the Japanese medaka (Oryzias latipes), a species that has been widely used in toxicology studies and has potential utility as a model organism for experimental aging research. PRINCIPAL FINDINGS: The median lifespan of medaka was approximately 22 months under laboratory conditions. We performed quantitative histological analysis of tissues from age-grouped individuals representing young adults (6 months old), mature adults (16 months old), and adults that had survived beyond the median lifespan (24 months). Livers of 24-month old individuals showed extensive morphologic changes, including spongiosis hepatis, steatosis, ballooning degeneration, inflammation, and nuclear pyknosis. There were also phagolysosomes, vacuoles, and residual bodies in parenchymal cells and congestion of sinusoidal vessels. Livers of aged individuals were characterized by increases in lipofuscin deposits and in the number of TUNEL-positive apoptotic cells. Some of these degenerative characteristics were seen, to a lesser extent, in the livers of 16-month old individuals, but not in 6-month old individuals. The basal layer of the dermis showed an age-dependent decline in the number of dividing cells and an increase in senescence-associated β-galactosidase. The hearts of aged individuals were characterized by fibrosis and lipofuscin deposition. There was also a loss of pigmented cells from the retinal epithelium. By contrast, age-associated changes were not apparent in skeletal muscle, the ocular lens, or the brain. SIGNIFICANCE: The results provide a set of markers that can be used to trace the process of normal tissue aging in medaka and to evaluate the effect of environmental stressors.

Gender-specific modulation of immune system complement gene expression in marine medaka Oryzias melastigma following dietary exposure of BDE-47

BDE-47 is one of the most widely found congeners of PBDEs in marine environments. The potential immunomodulatory effects of BDE-47 on fish complement system were studied using the marine medaka Oryzias melastigma as a model fish. Three-month-old O. melastigma were subjected to short-term (5 days) and long-term (21 days) exposure to two concentrations of BDE-47 (low dose at 290 +/- 172 ng/day; high dose at 580 +/- 344 ng/day) via dietary uptake of BDE-47 encapsulated in Artemia nauplii. Body burdens of BDE-47 and other metabolic products were analyzed in the exposed and control fish. Only a small amount of debrominated product, BDE-28, was detected, while other metabolic products were all under detection limit. Transcriptional expression of six major complement system genes involved in complement activation: C1r/s (classical pathway), MBL-2 (lectin pathway), CFP (alternative pathway), F2 (coagulation pathway), C3 (the central component of complement system), and C9 (cell lysis) were quantified in the liver of marine medaka. Endogenous expression of all six complement system genes was found to be higher in males than in females (p < 0.05). Upon dietary exposure of marine medaka to BDE-47, expression of all six complement genes were downregulated in males at day 5 (or longer), whereas in females, MBl-2, CFP, and F2 mRNAs expression were upregulated, but C3 and C9 remained stable with exposure time and dose. A significant negative relationship was found between BDE-47 body burden and mRNA expression of C1r/s, CFP, and C3 in male fish (r = -0.8576 to -0.9447). The above findings on changes in complement gene expression patterns indicate the complement system may be compromised in male O. melastigma upon dietary exposure to BDE-47. Distinct gender difference in expression of six major complement system genes was evident in marine medaka under resting condition and dietary BDE-47 challenge. The immunomodulatory effects of BDE-47 on transcriptional expression of these complement components in marine medaka were likely induced by the parent compound instead of biotransformed products. Our results clearly demonstrate that future direction for fish immunotoxicology and risk assessment of immunosuppressive chemicals must include parallel evaluation for both genders.

Effects of CO2-driven ocean acidification on early life stages of marine medaka (Oryzias melastigma)

The potential effects of elevated CO2 level and reduced carbonate saturation state in marine environment on fishes and other non-calcified organisms are still poorly known. In present study, we investigated the effects of ocean acidification on embryogenesis and organogenesis of newly hatched larvae of marine medaka (Oryzias melastigma) after 21 d exposure of eggs to different artificially acidified seawater (pH 7.6 and 7.2, respectively), and compared with those in control group (pH 8.2). Results showed that CO2-driven seawater acidification (pH 7.6 and 7.2) had no detectable effect on hatching time, hatching rate, and heart rate of embryos. However, the deformity rate of larvae in pH 7.2 treatment was significantly higher than that in control treatment. The left and right sagitta areas did not differ significantly from each other in each treatment. However, the mean sagitta area of larvae in pH 7.6 treatment was significantly smaller than that in the control (p = 0.024). These results suggest that although marine medaka might be more tolerant of elevated CO2 than some other fishes, the effect of elevated CO2 level on the calcification of otolith is likely to be the most susceptibly physiological process of pH regulation in early life stage of marine medaka.

Ol-Prx 3, a member of an additional class of homeobox genes, is unimodally expressed in several domains of the developing and adult central nervous system of the medaka (Oryzias latipes)

Large-scale genetic screens for mutations affecting early neurogenesis of vertebrates have recently been performed with an aquarium fish, the zebrafish. Later stages of neural morphogenesis have attracted less attention in small fish species, partly because of the lack of molecular markers of developing structures that may facilitate the detection of discrete structural alterations. In this context, we report the characterization of Ol-Prx 3 (Oryzias latipes-Prx 3). This gene was isolated in the course of a large-scale screen for brain cDNAs containing a highly conserved DNA binding region, the homeobox helix-three. Sequence analysis revealed that this gene belongs to another class of homeobox genes, together with a previously isolated mouse ortholog, called OG-12 [Rovescalli, A. C., Asoh, S. & Nirenberg, M. (1996) Proc. Natl. Acad. Sci. USA 93, 10691–10696] and with the human SHOX gene [Rao, E., Weiss, B., Fukami, M., Rump, A., Niesler, B., et al. (1997) Nat. Genet. 16, 54–62], thought to be involved in the short-stature phenotype of Turner syndrome patients. These three genes exhibit a moderate level of identity in the homeobox with the other genes of the paired-related (PRX) gene family. Ol-Prx 3, as well as the PRX genes, are expressed in various cartilaginous structures of head and limbs. These genes might thus be involved in common regulatory pathways during the morphogenesis of these structures. Moreover, this paper reports a complex and monophasic pattern of Ol-Prx 3 expression in the central nervous system, which differs markedly from the patterns reported for the PRX genes, Prx 3 excluded: this gene begins to be expressed in a variety of central nervous system territories at late neurula stage. Strikingly, it remains turned on in some of the derivatives of each territory during the entire life of the fish. We hope this work will thus help identify common features for the PRX 3 family of homeobox genes.

Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells: A useful model to monitor germ cells in a live vertebrate

We have generated transgenic medaka (teleost, Oryzias latipes), which allow us to monitor germ cells by green fluorescent protein (GFP) fluorescence in live specimens. Two medaka strains, himedaka (orange–red variety) and inbred QurtE, were used. The transgenic lines were achieved by microinjection of a construct containing the putative promoter region and 3′ region of the medaka vasa gene (olvas). The intensity of GFP fluorescence increases dramatically in primordial germ cells (PGCs) located in the ventrolateral region of the posterior intestine around stage 25 (the onset of blood circulation). Whole-mount in situ hybridization and monitoring of ectopically located cells by GFP fluorescence suggested that (i) the increase in zygotic olvas expression occurs after PGC specification and (ii) PGCs can maintain their cell characteristics ectopically after stages 20–25. Around the day of hatching, the QurtE strain clearly exhibits sexual dimorphisms in the number of GFP fluorescent germ cells, a finding consistent with the appearance of leucophores, a sex-specific marker of QurtE. The GFP expression persists throughout the later stages in the mature ovary and testis. Thus, these transgenic medaka represent a live vertebrate model to investigate how germ cells migrate to form sexually dimorphic gonads, as well as a potential assay system for environmental substances that may affect gonad development. The use of a transgenic construct as a selective marker to efficiently isolate germ-line-transmitting founders during embryogenesis is also discussed.

Expression of Bf/C2 Gene(S) in the Japanese Medaka Fish

Complement factor B and C2 are two central serine proteases of the alternative and classical complement pathways, respectively, that serve as the catalytic subunits of the C3 convertase. Research has been completed using a female Japanese medaka fish, (Oryzias latipes), and other teleost and elasmobrach species in order to isolate eDNA clones and perform linkage analysis of the Bf/C2 gene(s). To further analyze the evolution of the complement system in teleosts, different tissues than the ones from previous studies of medaka fish were analyzed for the constitutive gene expression of factor B and C2. Bf/C2 sequences were amplified by reverse transcription-polymerase chain reaction with primers corresponding to the common amino acid sequences shared by mammalian Bf and C2. Agarose gel electrophoresis was used to visualize sample bands and to calculate the concentration of gene expression of the Bf/C2 gene(s) in each tissue. All five tissue types, kidney, liver, muscle, testis, and spleen from a male medaka fish demonstrated Bf/C2 gene(s) expression, confirming that the messages of Bf/C2 gene(s) are distributed throughout the medaka fish. Tissues of the spleen, liver, and kidney contained the highest concentrations of expression of Bf/C2 gene( s ), while tissues of the muscle and testis contained the lowest concentrations. This research also determined that RT-PCR allowed for more sensitive analysis of gene expression than other molecular biology techniques such as Northern blotting analysis.

Silver toxicity across salinity gradients: the role of dissolved silver chloride species (AgCl x ) in Atlantic killifish (Fundulus heteroclitus) and medaka (Oryzias latipes) early life-stage toxicity.

The influence of salinity on Ag toxicity was investigated in Atlantic killifish (Fundulus heteroclitus) early life-stages. Embryo mortality was significantly reduced as salinity increased and Ag(+) was converted to AgCl(solid). However, as salinity continued to rise (>5 ‰), toxicity increased to a level at least as high as observed for Ag(+) in deionized water. Rather than correlating with Ag(+), Fundulus embryo toxicity was better explained (R(2) = 0.96) by total dissolved Ag (Ag(+), AgCl2 (-), AgCl3 (2-), AgCl4 (3-)). Complementary experiments were conducted with medaka (Oryzias latipes) embryos to determine if this pattern was consistent among evolutionarily divergent euryhaline species. Contrary to Fundulus data, medaka toxicity data were best explained by Ag(+) concentrations (R(2) = 0.94), suggesting that differing ionoregulatory physiology may drive observed differences. Fundulus larvae were also tested, and toxicity did increase at higher salinities, but did not track predicted silver speciation. Alternatively, toxicity began to increase only at salinities above the isosmotic point, suggesting that shifts in osmoregulatory strategy at higher salinities might be an important factor. Na(+) dysregulation was confirmed as the mechanism of toxicity in Ag-exposed Fundulus larvae at both low and high salinities. While Ag uptake was highest at low salinities for both Fundulus embryos and larvae, uptake was not predictive of toxicity.