Development Of Japanese Flounder Research Articles

Identification and functional analysis of circpdlim5a generated from pdlim5a gene splicing in the skeletal muscle of Japanese flounder (Paralichthys olivaceus)

Circular RNAs (circRNAs) are non-coding RNAs with endogenous regulatory functions, including regulating skeletal muscle development. However, its role in the development of skeletal muscle in Japanese flounder (Paralichthys olivaceus) is not clear. Therefore we screened a candidate circpdlim5a, which is derived from the gene pdlim5a, from the skeletal muscle transcriptome of Japanese flounder. We characterized circpdlim5a, which was more stable compared to the linear RNA pdlim5a. Distributional characterization of circpdlim5a showed that circpdlim5a was predominantly distributed in the nucleus and was highly expressed in the skeletal muscle of adult Japanese flounder (24 months). When we further studied the circpdlim5a function, we found that it inhibited the expression of proliferation and differentiation genes according to the over-expression experiment of circpdlim5a in myoblasts. We concluded that circpdlim5a may inhibit the proliferation and differentiation of myoblasts and thereby inhibit skeletal muscle development in Japanese flounder. This experiment provides information for the study of circRNAs by identifying circpdlim5a and exploring its function, and offers clues for molecular breeding from an epigenetic perspective.

Migration of Primordial Germ Cells Is Regulated by miR-430 During Embryonic Development of Japanese Flounder

The origin and migration of primordial germ cells (PGCs) were regulated by miRNA during embryogenesis, but the underlying mechanism in marine teleost species was still unclear. Here, we studied the function of miR-430 in the development and migration of PGCs in Japanese flounder. By co-injecting miR-430 with eGFP-vasa 3’UTR mRNA, we found that miR-430 could cause the mis-localization of PGCs in flounder. Besides, the overexpression of miR-430 also inhibited the expression of three PGC-related genes (nanos3, piwil2 and tdrd7a), both target gene prediction and dual-luciferase reporter assay showed that miR-430 were directly binding on the canonical target site of their 3’UTRs. Significantly, the miR-430 target site of piwil2 3’UTR was necessary for PGC specific expression, where the binding seeds “GCACTTT” sequence mutation lead the distribution of eGFP-piwil2 mRNA turned from PGC-specific (wild-type) to ubiquitous (mutant). These findings will provide further evidence on the role of miR-430 in the regulation of the development and migration of PGCs.

The miR-200 Family Targeting amh Affects the Gonadal Development of Japanese Flounder

Four members of the miR-200 family in Japanese flounder (Paralichthys olivaceus) have sex-biased expression patterns, but their target genes and how they work in the development of the gonads are rarely known. Anti-Müllerian hormone (AMH) can inhibit the development of Muller’s duct in female mammals and regulate the formation of gametes after sexual maturity. There is no Muller’s duct in teleosts, but the amh gene still exists. Knockout of amh results in sex reversal from male to female. Therefore, it is essential to explore the relationship between the miR-200 family and amh to clarify what role miR-200 plays in the development of the gonads. In Japanese flounder, the two binding sites for the miR-200 family in the 3′UTR of amh were found through bioinformatic prediction. Double luciferase and green fluorescent protein reporter experiments demonstrated amh to be directly targeted by miR-200a and miR-200b. Moreover, miR-200a and miR-200b reduced the expression of amh through site 1 rather than site 2. To explore the regulatory role of miR-200a in gonadal development, we further overexpressed miR-200a in the primary Sertoli cells of the testis. With the overexpression of miR-200a, the expression of amh decreased, while the expression of the other two male sex-related genes, dmrt1 (doublesex and mab-3 related transcription factor 1) and gsdf (diagonal soma driven factor), increased significantly. This result indicates that the miR-200 family regulates the gonadal differentiation and development by targeting amh in Japanese flounder.

The mechanism of immune related signal pathway Egr2-FasL-Fas in transcription regulation and methylated modification of Paralichthys olivaceus under acute hypoxia stress

Apoptosis genes Egr2, Fas and FasL are related to immune responses. However, the mechanism of these genes inducing apoptosis in fish are still not very clear. An acute hypoxia treatment (1.73 ± 0.06 mg/L) for 24 h was carried out on Japanese flounder (Paralichthys olivaceus). The increasingly dense apoptotic signals at 3 h, 6 h, 12 h by TUNEL in skeletal muscle indicated that hypoxia could quickly affect muscle growth and development. Furthermore, we concluded that the Egr2-FasL-Fas signal pathway, which was located at the upstream of apoptotic executor protein caspases, was related to the apoptosis by quantitative real-time PCR, protein concentration detection in ELISA and double gene in situ hybridization methods. The mechanism of the pathway was researched in transcription regulation and epigenetic modification by dual-luciferase reporter assay and bisulfite modified method, respectively. Egr2, as a transcription factor, could up-regulate the expression of FasL gene. And its binding site was mainly between −479 to −1 of FasL gene promoter. The 5th CpG dinucleotides (−514) methylation levels in FasL gene were significantly affected by hypoxia, and they were negatively correlated with its expressions. These suggested that the −514 site may be a very important site to regulate the FasL gene expression. Above results, we concluded that hypoxia activated the immune related signal pathway Egr2-FasL-Fas to induced skeletal muscle apoptosis to affect growth and development of Japanese flounder. The study revealed the mechanism of hypoxia induced apoptosis, which could provide a reference for fish immunity and aquaculture management.

Hypothalamic-Pituitary-Gonadal (HPG) Axis and Transcriptional Regulatory Elements Regulate piwil2 Gene Expression During Gametogenesis and Gonadal Development in Japanese Flounder (Paralichthys olivaceus)

The P-element induced wimpy testis (Piwi) proteins, which are associated with PIWI-interacting RNAs (piRNAs), play important roles in meiosis, germ cell division, and germline maintenance. In this study, we identified and characterized the Paralichthys olivaceus piwil2 gene, a constituent factor of the piRNA pathways involved in the biogenesis of reproductive development. The biological analysis indicated that piwil2, which contains PAZ and PIWI domains, was highly conserved between teleosts and tetrapods. The piwil2 distribution profile in different tissues confirmed a sexually dimorphic expression pattern, with a higher expression level in testis. In situ hybridization demonstrated that piwil2 was expressed in the oogonia and oocytes of the ovaries as well as in the Sertoli cells and spermatocytes of the testes. Gene piwil2 showed a maternally inherited expression pattern during embryonic development, and was highly expressed during the early embryonic development. Different luciferase reporters were constructed to determine the transcriptional regulatory mechanisms of piwil2. The piwil2 core promoter region was located at −360 bp to −60bp. Furthermore, some representative sex hormones, including human chorionic gonadotropin, 17α-methyltestosterone, and estradiol-17β had distinct regulatory effects on piwil2. In a summery, these results indicate that piwil2, regulated by sex hormones and transcriptional elements, has vital functions in the reproductive cycle and gonadal development.

Roles of Two Sox9 Genes during Gonadal Development in Japanese Flounder: Sex Differentiation, Spermatogenesis and Gonadal Function Maintenance.

The transcription factor sox9 has been implicated in cartilage formation and testis determination in mammals. Here, two duplicates of sox9 were found in Japanese flounder (Paralichthys olivaceus) named Posox9a and Posox9b, respectively. Phylogenetic and gene structure analyses revealed that Posox9a and Posox9b were homologous to that of teleosts and tetrapods. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that both Posox9a and Posox9b expressed higher in testis than in ovary of adult tissues. The in situ hybridization (ISH) analysis of gonads showed that Posox9a and Posox9b mRNA were both detected in oocytes, Sertoli cells and spermatocytes. During sex differentiation, the expression of Posox9a exhibited obvious sexual dimorphic expression from 60 days after hatch (dah) with higher expression in male preferred individuals than female preferred individuals and increased gradually from 30 to 100 dah. A similar pattern was detected in Posox9b expression. After injection of androgen (17α-methyltestosterone) of different concentrations, the expression level of Posox9b increased significantly, whereas Posox9a did not change obviously. These results indicated that the two sox9 genes of Japanese flounder had converse functions in sex differentiation, whereas their differences in 17α-methyltestosterone administration were obvious and worthwhile for exploring evolutionary and adaptive significance. This study provided a foundation for further exploration of the roles of Posox9 genes during the sex determination and differentiation, spermatogenesis and gonadal function maintenance of Japanese flounder.

Roles of GATA6 during Gonadal Development in Japanese Flounder: Gonadogenesis, Regulation of Gender-Related Genes, Estrogen Formation and Gonadal Function Maintenance.

GATA-binding protein 6 (GATA6), a highly-conserved transcription factor of the GATA family plays an important role in gonadal cell proliferation, differentiation and endoderm development. In this study, the full-length cDNA of GATA6 of Paralichthys olivaceus (Japanese flounder) was obtained. Phylogenetic, gene structure and synteny analyses demonstrated that GATA6 of P. olivaceus is homologous to that of teleosts and tetrapods. The P. olivaceus GATA6 transcript showed higher expression in testis than in ovary, demonstrating a sexually dimorphic gene expression. During embryonic development, the expression of P. olivaceus GATA6 increased at the blastula stage, demonstrating that GATA6 is involved in morphogenesis. Results of in situ hybridization showed that GATA6 signals were detected in Sertoli cells, oogonia and oocytes. Moreover, 17α methyl testosterone, a male hormone, could moderately upregulate P. olivaceus GATA6 and downregulate P. olivaceus aromatase CYP19A1 in testis cells. These results suggest that GATA6 may play an important role in gonadal development in P. olivaceus. This study provides valuable information on the function of P. olivaceus GATA6, laying the foundation for further development of breeding techniques in this species.

Expression patterns of IGF-I and its receptor genes during embryonic development of Japanese flounder

Expression patterns of IGF-I and its receptor genes during embryonic development of Japanese flounder

Changes in DNA, RNA, and Protein Content During Early Growth and Development of Japanese Flounder, Paralichthys olivaceus

Changes in DNA, RNA, and Protein Content During Early Growth and Development of Japanese Flounder, Paralichthys olivaceus

Comparison of behavioural development between Japanese flounder (Paralichthys olivaceus) and spotted halibut (Verasper variegatus) during early life stages

Summary Behavioural development was compared between two flatfish species (Japanese flounder and spotted halibut) from hatching to settlement (juvenile stage) in order to speculate on the ecology of their early life stages and to provide fundamental knowledge for improving seedling production techniques for stock enhancement. Fish were cultured under identical rearing conditions (500-L tank maintained at 17.8 ± 0.4°C, 34 ppt, 10L : 14D light regime and an initial stocking density of 20 larvae L−1). Behavioural observations were conducted at about 4-day intervals from hatching to the juvenile stage. Fish were sampled randomly from the rearing tank, and one fish was transferred into a 250-ml observation container. Behaviour was video-recorded for 5 min without food and for an additional 5 min with live feed (rotifer or Artemia). All behavioural data were sorted according to eight developmental stages and compared among developmental stages and between species. The average standard length of the spotted halibut was significantly greater than that of the Japanese flounder in all developmental stages, while the development of Japanese flounder was faster than that of the spotted halibut. For Japanese flounder, feeding, swimming and Ohm-posture (typical shivering behaviour observed during early life stages in flatfishes) frequency were highest before metamorphosis (mean ± SD; 1.0 ± 2.0 attacks min−1, 24.0 ± 9.6 actions min−1, 1.1 ± 1.1 counts min−1, respectively). Spotted halibut expressed feeding behaviour frequently from the beginning of metamorphosis (3.6 ± 5.2 attacks min−1), had relatively low swimming activity during all developmental stages, and showed a peak of Ohm-posture frequency during the flexion stage (2.6 ± 1.0 counts min−1).

The isolation and characterization of myostatin gene in Japanese flounder (Paralichthys olivaceus): Ubiquitous tissue expression and developmental specific regulation

Myostatin (MSTN) is a negative regulator of skeletal muscle growth and development. In the current study, the MSTN gene was cloned from one of flatfish species, Japanese flounder (Paralichthys olivaceus). In the 5520-bp genomic sequence, three exons, two introns, and 5' and 3' flanking sequences were identified. The putative amino acid sequence was 377 residues long, including a signal peptide, nine conserved cysteine residues, and a RXXR proteolytic processing site. The overall amino acid sequence of the P. olivaceus MSTN (pMSTN) was highly conserved with that of other organisms. Phylogenetic analysis suggested the evolutionary relationships of pMSTN with other known MSTN and GDF-11 genes. There were four microsatellite sequences identified in noncoding regions and 19 SNPs identified in coding sequences. Expression analysis revealed that pMSTN gene was expressed not only in the skeletal muscle, but also in other tissues, including cartilage, which has not been reported in fish previously. Although pMSTN was expressed in gill, no transcript could be detected in FG-9307, a continuous gill cell line from P. olivaceus. Quantitative RT-PCR analysis indicated that the expression of pMSTN appeared to be developmentally regulated. The pMSTN mRNA detected in unfertilized eggs indicated the maternal deposit. Considering of the ubiquitous tissue distribution and the continuous expression of pMSTN during the whole embryonic development in Japanese flounder, MSTN gene in fish may play different roles in comparison with that in mammals.

Maternal and genetic effects on individual variation during early development in Japanese flounder Paralichthys olivaceus

To investigate the changes in maternal effects during early development in Japanese flounder Paralichthys olivaceus, four-by-four factorial mating by artificial fertilization was performed to produce 16 families. Larvae and juveniles of each family were reared under common environmental conditions until completion of metamorphosis. The magnitude of maternal effects was estimated as the differences between sire and dam variance. maternal effects were highest at hatching was largely affected by egg size, but this effect decreased as larvae grew. Estimated mean heritability during early development was nearly constant (0.14±0.06 for total length and 0.10±0.05 for body depth). Therefore, it is suggested that early development in Japanese flounder is affected by both maternal effect and genetic factors.