Genes In Medaka Research Articles

The Chinese medaka (Oryzias sinensis) dmrt1 gene converts females to males in medaka (Oryzias latipes)

BackgroundChinese medaka (Oryzias sinensis) is widely distributed in freshwater rivers in China. Similar to the medaka (Oryzias latipes), Chinese medaka has the characteristics of small size, rapid reproductive cycle, and strong adaptability, which makes it suitable as a model organism for studies in basic biology and environmental toxicology. Chinese medaka exhibits distinct sexual dimorphism. However, due to the lack of complete genomic information, the regulation of sex determination and differentiation-related genes in Chinese medaka remains unclear. MethodsChinese medaka dmrt1 (Osdmrt1) was cloned by PCR, and transgenic individuals of medaka [Tg(CMV:Osdmrt1)] overexpressing Osdmrt1 were generated to investigate the role of Osdmrt1 in sex determination. Western blot was used to validate the integration of the Osdmrt1 into the medaka genome. Tissue sectioning and HE staining were used to identify Tg(CMV:Osdmrt1) physiological gender and phenotype. qRT-PCR was used to analyze the expression of gonad-specific genes. ResultsOsdmrt1 was cloned and identified, and it shared similar evolutionary relationships with medaka dmrt1. Tg(CMV:Osdmrt1) exhibited partial sex reversal from female to male in the F2 generation, with genetically female individuals developing testes and producing functional sperm. Additionally, the secondary sexual characteristics of the transgenic females also changed to males. ConclusionThe Chinese medaka dmrt1 gene could convert females to males in medaka.General significance: These results not only elucidate the function of Chinese medaka dmrt1, but also accumulate knowledge for studying the function of economically important fish genes in model fish by transgenic technology.

Reproductive toxic effects of chronic exposure to bisphenol A and its analogues in marine medaka (Oryzias melastigma)

As awareness of BPA's health risks has increased, many countries and regions have implemented strict controls on its use. Consequently, bisphenol analogues like BPF and BPAF are being increasingly used as substitutes. However, these compounds are also becoming increasingly prevalent in the environment due to production, use and disposal processes. The oceans act as a repository for various pollutants, and recent studies have revealed the extensive presence of bisphenols (BPs, including BPA, BPF, BPAF, etc.) in the marine environment, posing numerous health hazards to marine wildlife. Nevertheless, the reproductive toxicity of these chemicals on marine fish is not comprehensively comprehended yet. Thus, the histological features of the gonads and the gene expression profiles of HPG (Hypothalamic-Pituitary-Gonadal) axis-related genes in marine medaka (Oryzias melastigma) were studied after exposure to single and combined BPs for 70 days. The effects of each exposure group on spawning, embryo fertilization, and hatching in marine medaka were also assessed. Furthermore, the impacts of each exposure group on the genes related to methylation in the F2 and F3 generations were consistently investigated. BPs exposure was found to cause follicular atresia, irregular oocytes, and empty follicles in the ovary; but no significant lesions in the testis were observed. The expression of several HPG axis genes, including cyp19b, 17βhsd, 3βhsd, and fshr, resulted in significant changes compared to the control group. The quantity of eggs laid and fertilization rate decreased in all groups treated with BPs, with the BPAF-treated group showing a notable reduction in the number of eggs laid. Additionally, the hatching rate showed a more significant decline in the BPF-treated group. The analysis of methylated genes in the offspring of bisphenol-treated groups revealed significant changes in the expression of genes including amh, dnmt1, dnmt3ab, mbd2, and mecp2, indicating a potential transgenerational impact of bisphenols on phenotype through epigenetic modifications. Overall, the potential detrimental impact of bisphenol on the reproduction of marine medaka emphasizes the need for caution in considering the use of BPAF and BPF as substitutes.

Modeling familial and sporadic Parkinson's disease in small fishes.

The establishment of animal models for Parkinson's disease (PD) has been challenging. Nevertheless, once established, they will serve as valuable tools for elucidating the causes and pathogenesis of PD, as well as for developing new strategies for its treatment. Following the recent discovery of a series of PD causative genes in familial cases, teleost fishes, including zebrafish and medaka, have often been used to establish genetic PD models because of their ease of breeding and gene manipulation, as well as the high conservation of gene orthologs. Some of the fish lines can recapitulate PD phenotypes, which are often more pronounced than those in rodent genetic models. In addition, a new experimental teleost fish, turquoise killifish, can be used as a sporadic PD model, because it spontaneously manifests age-dependent PD phenotypes. Several PD fish models have already made significant contributions to the discovery of novel PD pathological features, such as cytosolic leakage of mitochondrial DNA and pathogenic phosphorylation in α-synuclein. Therefore, utilizing various PD fish models with distinct degenerative phenotypes will be an effective strategy for identifying emerging facets of PD pathogenesis and therapeutic modalities.

Expression of secretory calcium-binding phosphoprotein (scpp) genes in medaka during the formation and replacement of pharyngeal teeth

BackgroundAnalyses of tooth families and tooth-forming units in medaka with regard to tooth replacement cycles and the localization of odontogenic stem cell niches in the pharyngeal dentition clearly indicate that continuous tooth replacement is maintained. The secretory calcium-binding phosphoprotein (scpp) gene cluster is involved in the formation of mineralized tissues, such as dental and bone tissues, and the genes encoding multiple SCPPs are conserved in fish, amphibians, reptiles, and mammals. In the present study, we examined the expression patterns of several scpp genes in the pharyngeal teeth of medaka to elucidate their roles during tooth formation and replacement.MethodsHimedaka (Japanese medaka, Oryzias latipes) of both sexes (body length: 28 to 33 mm) were used in this study. Real-time quantitative reverse transcription-polymerase chain reaction (PCR) (qPCR) data were evaluated using one-way analysis of variance for multi-group comparisons, and the significance of differences was determined by Tukey’s comparison test. The expression of scpp genes was examined using in situ hybridization (ISH) with a digoxigenin-labeled, single-stranded antisense probe.ResultsqPCR results showed that several scpp genes were strongly expressed in pharyngeal tissues. ISH analysis revealed specific expression of scpp1, scpp5, and sparc in tooth germ, and scpp5 was continually expressed in the odontoblasts of teeth attached to pedicles, but not in the osteoblasts of pedicles. In addition, many scpp genes were expressed in inner dental epithelium (ide), but not in odontoblasts, and scpp2 consistently showed epithelial-specific expression in the functional teeth. Taken together, these data indicate that specific expression of scpp2 and scpp5 may play a critical role in pharyngeal tooth formation in medaka.ConclusionWe characterized changes in the expression patterns of scpp genes in medaka during the formation and replacement of pharyngeal teeth.

Effects of plasticizer diisobutyl adipate on the Japanese medaka (Oryzias latipes) endocrine system.

Plasticizer pollution of the water environment is one of the world's most serious environmental issues. Phthalate plasticizers can disrupt endocrine function in vertebrates. Therefore, this study analyzed thyroid-related, reproduction-related, and estrogen-responsive genes in Japanese medaka (Oryzias latipes) to determine whether non-phthalate diisobutyl adipate (DIBA) plasticizer could affect endocrine hormone activity or not. Developmental toxicity during fish embryogenesis was also evaluated. At a concentration of 11.57 mg/l, embryonic exposure to DIBA increased the mortality rate. Although abnormal development, including body curvature, edema, and lack of swim bladder inflation, was observed at 3.54 and 11.57 mg/l DIBA, growth inhibition and reduced swimming performance were also observed. In addition, DIBA exposure increased the levels of thyroid-stimulating hormone beta-subunit (tshβ) and deiodinase 1 (dio1) but decreased the levels of thyroid hormone receptor alpha (trα) and beta (trβ). These results suggest that DIBA has thyroid hormone-disrupting activities in fish. However, kisspeptin (kiss1 and kiss2), gonadotropin-releasing hormone (gnrh1), follicle-stimulating hormone beta (fshβ), luteinizing hormone beta (lhβ), choriogenin H (chgH), and vitellogenin (vtg1) expression did not change dose-dependently in response to DIBA exposure, whereas gnrh2 and vtg2 expression was elevated. These results indicate that DIBA has low estrogenic activity and does not disrupt the endocrine reproduction system in fish. Overall, this is the first report indicating that non-phthalate DIBA plasticizer is embryotoxic and disrupt thyroid hormone activity in fish.

Synthetic microfiber exposure negatively affects reproductive parameters in male medaka (Oryzias latipes)

Microplastics not only accumulate in the bodies of fishes and cause damage to the organs, but also cause many other problems, such as reduced reproductive capacity, by acting directly or indirectly on the hypothalamus-pituitary-gonad axis (HPG axis). In this study, we investigated the changes in HPG axis-related genes in male medaka (Oryzias latipes) exposed to fiber-type microplastics. We confirmed the progression of vitellogenesis, a sign of endocrine disruption, in male fish. In the microfiber-exposed group, microfiber accumulation was confirmed in the gills and intestines. One week after exposure to two different concentrations of microfibers (500 and 1,000 fibers/L), the fish showed increased expression of gonadotropin-releasing hormone (GnRH) and luteinizing hormone receptor (LH-R) mRNA. From day 10 of exposure to the microfibers, there was an increase in the expression of the gonadotropin-inhibitory hormone (GnIH) mRNA and a decrease in the expression of GnRH and LH-R mRNA. There was an increase in the cytochrome P450 aromatase (CYP19a) mRNA expression and plasma estradiol (E2) concentration in the 1,000 fibers/L exposure group. High vitellogenin (VTG) mRNA expression was confirmed seven days after exposure in the 1,000 fibers/L group, which was consistent with the VTG mRNA expression signals detected in the liver using in situ hybridization. These results suggest that microfiber ingestion may cause short-term endocrinal disruption of the HPG axis in male medaka, which in turn may interfere with their normal maturation process.

Genome Editing of Medaka.

Medaka (Oryzias latipes), along with zebrafish (Danio rerio), is a useful experimental model fish. Here, we describe a simple method for generating medaka gene knockout strains using an automated microchip electrophoresis system. We also describe a method for targeted gene knockin using a plasmid carrying a sequence that does not cause off-target effects in medaka. Additionally, knockin method without plasmid cloning is described.

Adverse effects of thyroid-hormone-disrupting chemicals 6-propyl-2-thiouracil and tetrabromobisphenol A on Japanese medaka (Oryzias latipes)

Thyroid-hormone-disrupting chemicals are increasingly attracting attention because of their potential harmful effects on animal health, including on fishes. Here, we investigated the effects of exposure to the thyroid-hormone-disrupting chemicals 6-propyl-2-thiouracil (PTU) and tetrabromobisphenol A (TBBPA) on swim bladder inflation, eye development, growth, swimming performance, and the expression of thyroid-related genes in Japanese medaka (Oryzias latipes). PTU exposure resulted in reductions in eye size, growth, and swim bladder inflation, and these effects led to poorer swimming performance. These phenotypic effects were accompanied by increased expression of the thyroid-stimulating hormone subunit beta (tshβ) paralog tshβ-like, but there were no significant changes in expression for tshβ, deiodinase 1 (dio1), deiodinase 2 (dio2), and thyroid hormone receptor alpha (trα) and beta (trβ). For PTU exposure, we identified the key event (swim bladder inflation reduction) and an adverse outcome (swimming performance reduction). No significant effects from TBBPA exposure were seen on swim bladder inflation, eye development, growth, or swimming performance. However, expression of tshβ-like and tshβ (significantly enhanced) and trα and trβ (significantly reduced) were affected by TBBPA exposure albeit not in dose-dependent manners. There were no effects of TBBPA on the expression of dio1 and dio2. We thus show that the two thyroid-hormone-disrupting chemicals PTU and TBBPA differ in their effect profiles with comparable effects on the studied phenotypes and thyroid-related gene expression to those reported in zebrafish.

Oryzias latipes (JAPANESE MEDAKA) AS GENETIC MODEL TO STUDY CAUSATIVE GENES OF EPILEPSY DISEASE: AN IN-SILICO APPROACH

Epilepsy is a chronic neurological disorder that has affected around 50-70 million people worldwide. Various animal models have previously been used in epilepsy research. To expand the knowledge of the disease, a new animal model is suggested to be explored considering the genetic and phenotypic heterogeneity that contributes to the complexity of the disease. This study was undertaken to analyze 14 causative genes of epilepsy disease in Japanese medaka (Oryzias latipes), humans, and the established model of this disease which is zebrafish (Danio rerio) by assessing the variation in the genes by using MEGA X and predicting the functional motif and secondary structure of the proteins by using PROSITE and GORIV respectively. Results from the variation analysis showed the lowest percentage of conserved genes in Japanese medaka was 60%.50% of the genes of Japanese medaka were found to be more conserved than zebrafish in comparison to a human. The functional motifs present in all genes in Japanese medaka showed the same motifs present in humans. All the secondary structures of Japanese medaka genes were predicted to contain the alpha helix, extended strand, and random coil. In conclusion, it can be inferred that Japanese medaka could be a reliable animal model for epilepsy disease.

Conserved and diverged asymmetric gene expression in the brain of teleosts.

Morphological left-right brain asymmetries are universal phenomena in animals. These features have been studied for decades, but the functional relevance is often unclear. Studies from the zebrafish dorsal diencephalon on the genetics underlying the establishment and function of brain asymmetries have uncovered genes associated with the development of functional brain asymmetries. To gain further insights, comparative studies help to investigate the emergence of asymmetries and underlying genetics in connection to functional adaptation. Evolutionarily distant isogenic medaka inbred lines, that show divergence of complex traits such as morphology, physiology and behavior, are a valuable resource to investigate intra-species variations in a given trait of interest. For a detailed study of asymmetry in the medaka diencephalon we generated molecular probes of ten medaka genes that are expressed asymmetrically in the zebrafish habenulae and pineal complex. We find expression of eight genes in the corresponding brain areas of medaka with differences in the extent of left-right asymmetry compared to zebrafish. Our marker gene analysis of the diverged medaka inbred strains revealed marked inter-strain size differences of the respective expression domains in the parapineal and the habenulae, which we hypothesize may result from strain-specific gene loss. Thus, our analysis reveals both inter-species differences but also intra-species plasticity of gene expression in the teleost dorsal diencephalon. These findings are a starting point showing the potential to identify the genetics underlying the emergence and modulations of asymmetries. They are also the prerequisite to examine whether variance in habenular gene expression may cause variation of behavioral traits.

Diel rhythm of the inflammatory cytokine il1b in the Japanese medaka (Oryzias latipes) regulated by core components of the circadian clock

In recent years, studies on circadian control in immunity have been actively conducted in mammals, but little is known about circadian rhythms in the field of fish immunology. In this study, we aimed to analyse the regulation of the diel oscillation of inflammatory cytokine interleukin-1β (il1b) gene expression by core components of the circadian clock in Japanese medaka (Oryzias latipes). The expression of il1b and clock genes (bmal1 and clock1) in medaka acclimated to a 12:12 light (L): dark (D) cycle showed diel rhythm. Additionally, higher expression of il1b was detected in medaka embryo cells (OLHdrR-e3) overexpressing bmal1 and clock1. A significant decrease in il1b expression was observed in OLHdrR-e3 cells after bmal1 knockdown using morpholino oligos. These changes may be mediated by transcriptional regulation via clock proteins, which target the E-box sequence in the cis-element of il1b as identified using luciferase reporter assays. Moreover, LPS stimulation and pathogenic bacterial infection at different zeitgeber time (ZT) under LD12:12 conditions affected the degree of il1b expression, which showed high and low responsiveness to both immuno-stimulations at ZT2 and ZT14, respectively. These results suggested that fish IL-1β exhibited diel oscillation regulated by clock proteins, and its responsiveness to immune-stimulation depends on the time of day.

Knockout of lipoprotein lipase with CRISPR/Cas9 causes severe developmental defects and affects lipid deposition in Japanese medaka (Oryzias latipes)

In mammals, lipoprotein lipase (LPL) has been found to play an important role in lipid mentalism and deposition. LPL deficiency in humans (Homo sapiens) and mice (Mus musculus) tends to cause hypertriglyceridemia. The lpl gene is not expressed in adult mammalian liver, but is in adult fish liver. The functions provided by the lpl gene are diverse in vertebrates. Here, we knocked out the lpl gene in Japanese medaka (Oryzias latipes) with the CRISPR/Cas9 system. The lpl-knockout (KO) homozygous individuals showed severe developmental defects with an extremely emaciated and deformed body and only accounted for about 5% of the F2 fish. This is consistent with the findings in mice but disaccords with the results for zebrafish (Danio rerio). Compared with wild-type (WT) madaka, the mRNA level of lpl in lpl-KO heterozygous mutant was significantly higher in the muscle, showed no significant difference in the liver, and was significantly lower in the heart. Under lpl heterozygous deficiency, the relative area of Oil Red O and triglycerides (TG) level in the liver, heart and muscle tissue covaried with levels of lpl mRNA in medaka. The lpl heterozygous deficiency did not affect the levels of TG, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and total cholesterol (TC) in the plasma of medaka, which is inconsistent with the findings in mammals. In general, the lpl gene plays an important role in the growth and development and is closely related to lipid deposition of medaka.

Establishing an effective gene knockdown system using cultured cells of the model fish medaka (Oryzias latipes).

In spite of the growing attention given to medaka (Oryzias latipes) as an excellent vertebrate model, an effective gene knockdown system has not yet been established using cultured cells of this fish species. In this study, a gene knockdown system using short interfering RNA (siRNA) in medaka cell lines was established through the optimization of transfection conditions. By extensive screening of several medaka cell lines and transfection reagents, OLHNI-2 cells and X-tremeGENE siRNA Transfection Reagent were selected as the best combination to achieve high transfection efficiency of siRNA without cytotoxic effect. Knockdown conditions were then refined using the endogenous heat shock protein 90 (Hsp90) genes as the siRNA targets. Among the parameters tested, cell density, serum concentration in the culture medium, and duration of transfection improved knockdown efficiency, where the target mRNA in cells transfected with each of the siRNAs was reduced from 12.0% to 26.7% of the control level. Our results indicate that the established knockdown system using siRNA is a promising tool for functional analysis of medaka genes in vitro.

Zebrafish and Medaka: Important Animal Models for Human Neurodegenerative Diseases.

Animal models of human neurodegenerative disease have been investigated for several decades. In recent years, zebrafish (Danio rerio) and medaka (Oryzias latipes) have become popular in pathogenic and therapeutic studies about human neurodegenerative diseases due to their small size, the optical clarity of embryos, their fast development, and their suitability to large-scale therapeutic screening. Following the emergence of a new generation of molecular biological technologies such as reverse and forward genetics, morpholino, transgenesis, and gene knockout, many human neurodegenerative disease models, such as Parkinson’s, Huntington’s, and Alzheimer’s, were constructed in zebrafish and medaka. These studies proved that zebrafish and medaka genes are functionally conserved in relation to their human homologues, so they exhibit similar neurodegenerative phenotypes to human beings. Therefore, fish are a suitable model for the investigation of pathologic mechanisms of neurodegenerative diseases and for the large-scale screening of drugs for potential therapy. In this review, we summarize the studies in modelling human neurodegenerative diseases in zebrafish and medaka in recent years.

Oryzias curvinotus in Sanya Does Not Contain the Male Sex-Determining Gene dmy.

Simple Summarydmy is considered to be the male-determining gene in Japanese medaka (Oryzias latipes) and Hainan medaka (Oryzias. curvinotus), both of which have the XX/XY sex-determination system. Here, we found a group of medaka in the Sanya River (named SY-medaka) and confirmed that SY-medaka belongs to O. curvinotus by morphological characteristics and mitochondrial phylogenetic analysis. Through genetic sex identification, genome re-sequencing and gonadal transcriptome analysis, it was preliminary confirmed that SY-medaka did not contain dmy. Our results provide a basis for further studies of the mechanism underlying sex determination in Oryzias and functional genomics and reproduction biology in O. curvinotus.Hainan medaka (Oryzias curvinotus) is distributed in the coastal waters of the South China Sea and is able to adapt to a wide range of salinities. In this study, we characterized O. curvinotus in Sanya River (SY-medaka), which lacks dmy (a male sex-determining gene in O. latipes and O. curvinotus). In a comparison of SY-medaka and Gaoqiao medaka (GQ-medaka), the morphological difference between the two populations does not reach the subspecies level and they can be considered two geographic populations of O. curvinotus. A mitochondrial cytochrome oxidase subunit I (CoI) sequence alignment showed that the sequence identities between SY-medaka and other geographic populations of O. curvinotus are as high as 95%. A phylogenetic analysis of the mitochondrial genome also indicated that SY-medaka belongs to O. curvinotus. Molecular marker-based genetic sex assays and whole genome re-sequencing showed that SY-medaka does not contain dmy. Further, in RNA-Seq analyses of the testis and ovaries of sexually mature SY-medaka, dmy expression was not detected. We speculate that high temperatures resulted in the loss of dmy in SY-medaka during evolution, or the lineage has another sex-determining gene. This study provides a valuable dataset for elucidating the mechanism underlying sex determination in Oryzias genus and advances research on functional genomics or reproduction biology in O. curvinotus.

Characterization, Identification and Phylogeny of the Creatine Kinase (ckma) Gene in Medaka (Oryzias latipes)

Creatine kinase (ckma) has been characterized and described in the medaka (Oryzias latipes), an aquatic model organism and the gene structure has been designed using the exons, introns, produced amino acids of the gene, TATA box, poly A tail and 5’ UTR and 3’ UTR regions of the ckma gene. In another step, firstly, the chromosome region of the ckma gene was determined in medaka and then the other genes which placed in the same region were determined. Then the locations of these genes were determined in zebrafish and human which are the orthologs of medaka. Finally, the conserved gene synteny was designed manually, using these data. However, genetic identity and similarity ratio between medaka and its orthologs were calculated. In this study, characterization and identification, phylogenetic relationship, conserved gene synteny of ckma gene in medaka (O. latipes) which is an important model organism were analyzed by using bioinformatics tools (NCBI database, Ensembl genomic database, Expasy, Reverse Complementary and some programs such as MEGA6 program, BLOSUM62 matrix program and BioEdit software). All these data will be used in future studies on molecular stress response in fish and they were presented to the scientific world with this study.

Circadian oscillation of TNF-α gene expression regulated by clock gene, BMAL1 and CLOCK1, in the Japanese medaka (Oryzias latipes)

To date, little information is available on the effects of circadian oscillation on immune regulation in lower vertebrates, such as teleost fish. In the present study, regulation of circadian oscillation of inflammatory cytokine TNF-α gene expression by clock gene was investigated using model fish medaka (Oryzias latipes). Firstly, structural analysis of clock genes was performed, which revealed that medaka BMAL1 and CLOCK1 conserve functionally important domains, such as basic helix-loop-helix (bHLH) and period-aryl hydrocarbon receptor nuclear translocator-single-minded (PAS), seen in their counterparts in other vertebrates. Expression of medaka Bmal1, Clock1, and Per1 genes was confirmed in central and peripheral tissues. Moreover, the expression of these clock genes and TNF-α genes in medaka acclimated to a 12:12 light (L) - dark (D) cycle showed circadian oscillation. In addition, higher expression of TNF-α gene was detected in medaka embryo cells (OLHdrR-e3) overexpressing Bmal1 and Clock1 genes. It was suggested that this increase was mediated by transcriptional regulation by clock proteins, which target E-box sequence in the cis-element of TNF-α gene as was detected by luciferase reporter gene assay. Moreover, in vitro head kidney stimulation with LPS at different zeitgeber time (ZT) under LD12:12 condition affected the degree of TNF-α gene expression, which shows high and low responsiveness to LPS stimulation at ZT18 and ZT10, respectively. These results suggested that fish TNF-α exhibited circadian oscillation regulated by clock proteins and its responsiveness against immune-stimulation depends on time zone.

Using CRISPR/Cas9-mediated gene editing to further explore growth and trade-off effects in myostatin-mutated F4 medaka (Oryzias latipes)

Myostatin (MSTN) suppresses skeletal muscle development and growth in mammals, but its role in fish is less well understood. Here we used CRISPR/Cas9 to mutate the MSTN gene in medaka (Oryzias latipes) and evaluate subsequent growth performance. We produced mutant F0 fish that carried different frameshifts in the OlMSTN coding sequence and confirmed the heritability of the mutant genotypes to the F1 generation. Two F1 fish with the same heterozygous frame-shifted genomic mutations (a 22 bp insertion in one allele; a 32 bp insertion in the other) were then crossbred to produce subsequent generations (F2~F5). Body length and weight of the MSTN−/− F4 medaka were significantly higher than in the wild type fish, and muscle fiber density in the inner and outer compartments of the epaxial muscles was decreased, suggesting that MSTN null mutation induces muscle hypertrophy. From 3~4 weeks post hatching (wph), the expression of three major myogenic related factors (MRFs), MyoD, Myf5 and Myogenin, was also significantly upregulated. Some medaka had a spinal deformity, and we also observed a trade-off between growth and immunity in MSTN−/− F4 medaka. Reproduction was unimpaired in the fast-growth phenotypes.

Targeted deletion of miRNAs and cis-regulatory modules associated with cone opsin genes in medaka

Targeted deletion of miRNAs and cis-regulatory modules associated with cone opsin genes in medaka

Three urocortins in medaka: identification and spatial expression in the central nervous system.

The urocortin (UCN) group of neuropeptides includes urocortin 1/sauvagine/urotensin 1 (UTS1), urocortin 2 (UCN2) and urocortin 3 (UCN3). In recent years, evidence has accumulated showing that UCNs play pivotal roles in mediating stress response and anxiety in mammals. Evidence has also emerged regarding the evolutionary conservation of UCNs in vertebrates, but very little information is available about UCNs in non-mammalian vertebrates. Indeed, at present, there are no reports of the empirical identification of ucn2 in non-mammalian vertebrates or of the distribution of ucn2 and ucn3 expression in the adult central nervous system (CNS) of these animals. To gain insight into the evolutionary nature of UCNs in vertebrates, we cloned uts1, ucn2 and ucn3 in a teleost fish, medaka and examined the spatial expression of these genes in the adult brain and spinal cord. Although all known UCN2 genes except those in rodents have been reported to likely lack the necessary structural features to produce a functional pre-pro-protein, all three UCN genes in medaka, including ucn2, displayed all of these features, suggesting their functionality. The three UCN genes exhibited distinct spatial expression patterns in the medaka brain: uts1 was primarily expressed in broad regions of the dorsal telencephalon, ucn2 was expressed in restricted regions of the thalamus and brainstem and ucn3 was expressed in discrete nuclei throughout many regions of the brain. We also found that these genes were all expressed throughout the medaka spinal cord, each with a distinct spatial pattern. Given that many of these regions have been implicated in stress responses and anxiety, the three UCNs may serve distinct physiological roles in the medaka CNS, including those involved in stress and anxiety, as shown in the mammalian CNS.