Genome-wide identification of nonvisual opsin family reveals amplification of RPE-retinal G protein receptor gene (RGR) and offers novel insights into functions of RGR(s) in Paralichthys olivaceus (Paralichthyidae, Teleostei).

Opsins play important roles in the image-forming visual pathways and numerous biological systems such as the biological clock and circadian rhythm. However, the nonvisual opsins involved in nonimage forming process are not clear to date. The aim of this study was to characterize nonvisual opsins in Paralichthys olivaceus. A total of 24 nonvisual opsin genes were identified. Expressions of these genes in eye, brain, heart, testis, and fin were investigated by quantitative real-time polymerase chain reaction (qRT-PCR). Testis contained a surprisingly large number of nonvisual opsins including Opn4m2, Tmt2a, Tmt3b, Opn3, RRH, Opn7a, and Opn7b. Syntenic and phylogenetic analyses confirmed that the RGRa and RGRb originated from the teleost-specific genome duplication (TSGD). qRT-PCR results demonstrated high RGRa and RGRb expression in the eye, while the expression levels in the brain, heart, testis, and fin were relatively weak. In situ hybridization results presented here revealed the presence of both RGRa and RGRb mRNA-positive signals in the ganglion cell layer but absence in the intracellular compartment of retinal pigment epithelium (RPE) and Müller glial cells. Therefore, we hypothesized that RGRa and RGRb had undergone subfunctionalization in P. olivaceus after TSGD. In conclusion, this study provides novel insights into the evolutionary fates of the RGR genes, still, further studies need to be done to explore the mechanism about the lack of RGR genes' expression in RPE.

Edwardsiella tarda-induced miR-7a functions as a suppressor in PI3K/AKT/GSK3ß signaling pathway by targeting insulin receptor substrate-2 (IRS2a and IRS2b) in Paralichthys olivaceus.

To study the effect of Edwardsiella tarda infection on miRNAs expression profile in Japanese flounder, fish were injected intraperitoneally with E. tarda. The miRNAs involved in regulating immune responses were analyzed by high-throughput sequencing. A total of 164 mature miRNAs were identified, of which 17 miRNAs were differentially expressed (DE miRNAs) after E. tarda infection, indicating that they were immune-related miRNAs. To further examine the relationship between the miRNAs and their predicted target mRNAs, a total of 22 predicted target mRNAs, mainly related to endocytic signaling pathway, NF-κB signaling pathway, and p53 signaling pathway, were detected with miRNA mimics in HEK-293T cells by dual-luciferase reporter experiments. Finally, we confirmed that insulin receptor substrate-2 (IRS2a and IRS2b) were regulated by miR-7a. And the target sites of the 3' untranslated region (UTR) of IRS2a and IRS2b were verified by dual-luciferase reporter experiments. Furthermore, we found that the E. tarda and LPS significantly increased host miR-7a expression. In vivo and in vitro studies revealed that IRS2-mediated PI3K/AKT/GSK3ß signaling pathway was suppressed. Taken together, these results implied that miR-7a might be a key regulator of PI3K/AKT/GSK3ß signaling pathway via suppressing the IRS2a and IRS2b genes.

Expression and functional analysis of receptor-interacting serine/threonine kinase 2 (RIP2) in Japanese flounder (Paralichthys olivaceus).

Being a key adaptor protein in NOD1/2 and NF-κB signaling pathways, receptor-interacting serine/threonine kinase 2 (RIP2) plays an important role in innate immune response in vertebrates. In this study, we identified and characterized the Paralichthys olivaceus RIP2 gene (PoRIP2). Phylogenetic, alignment, and genomic analysis of PoRIP2 were conducted to determine its conservation and evolutionary relationship with other RIP2 in vertebrates. qRT-PCR results showed that the expression of PoRIP2 was high in the spleen and head kidney. Meanwhile, embryonic development expression profile revealed that it was high in the early developmental stages and hatching stage. In vivo, we examined the expression pattern in different tissues after being challenged with Edwardsiella tarda. PoRIP2 was up-regulated in tissues at different time points. In vitro, the expression of PoRIP2 was also increased after treatment with Poly I:C, PGN, and E. tarda. Transfection and overexpression experiments indicated that PoRIP2 was located in the cytoplasm of the FG-9307 cell line. The pro-inflammatory cytokines, IL-1ß, IL-6, and IL-8, could be activated and up-regulated by PGN stimulation in PoRIP2 overexpressed cells. The inhibitory action was obvious in PoRIP2 overexpressed cells, and the quantity of E. tarda decreased. These findings highlight the important role of PoRIP2 in regulating innate immune in P. olivaceus. Our results indicated that PoRIP2 might be involved in immune response and the activation of the NF-κB signaling pathways. Our study can improve the knowledge on the immune system of fish and provide a theoretical basis for the study of prevention and treatment of fish diseases.

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.

Functional characterization of the Japanese flounder (Paralichthys olivaceus) Sox2 gene promoter.

Sox2 has essential roles in early embryogenesis and the development of the central nervous system. Sox2 is also necessary in maintaining the identity of progenitor cells. In our study, a 1.8-kb fragment of the 5' flanking region of Paralichthys olivaceus Sox2 (Po-Sox2) gene was cloned and functionally characterized. The activity and specificity of Po-Sox2 promoter were analyzed by comparing various deletion mutants for their ability to direct luciferase and GFP expression in flounder brain cell line. Results indicated that the basal promoter is located between -978 and -442 bp, and the region from -1370 to -978 bp enhances the promoter activity. The regulatory elements in the -1370 to -442 bp fragment were further investigated. Many binding sites of transcription factors closely related to neurogenesis and stem cell properties were found in this region. Mutational analysis indicated that Nanog, Pax6, p53, and POU binding sites play functional roles in the transcription of Po-Sox2 gene, whereas NF-Y binding sites did not affect this gene. In vivo studies using transient transgenic zebrafish embryos showed that the Po-Sox2 promoter region can drive GFP expression in brain, yolk syncytial layer, and notochord. Our results provide valuable information in understanding the molecular regulatory mechanisms of Po-Sox2 gene during neurogenesis and embryonic development.

Transcriptome analysis of the gonads of olive flounder (Paralichthys olivaceus).

Olive flounder (Paralichthys olivaceus) is an economically important cultured marine fish in China, Korea, and Japan. Controlling and managing the breeding of olive flounder in captivity is an imperative step toward obtaining a sustainable supply of this fish in aquaculture production systems. Therefore, investigation on the molecular regulatory mechanism of gonadal development and gametogenesis in this species is of great significance in aquaculture. Furthermore, identification of the expression profile of numerous sex-related genes is the first step to primarily understand such molecular regulatory mechanism. Six female and six male gonads obtained from 2-year-old olive flounders were sequenced using Illumina, which produced 6.68 and 6.24 GB data for ovary and testis, respectively. The reads were mapped to the draft genome of olive flounder, and then the reads per kilobase per million (FPKM) for each gene were calculated. The female-/male-biased expressed genes were investigated based on the FPKM values. Overall, 3086 female-biased and 5048 male-biased genes were screened out. GO enrichment analysis showed that the GO terms "male meiosis," "gamete generation," "fertilization," "spermatogenesis," and "germ plasma" were enriched in male-biased genes. In addition, the GO terms "cell morphogenesis involved in differentiation," "embryonic morphogenesis," "plasma membrane," "steroid hormone receptor activity," and "aromatase activity" were enriched in female-biased genes. Moreover, 373,369 single nucleotide polymorphisms and 32,993 indels were identified in the transcriptome. This work is the largest collection of gonad transcriptome data for olive flounder and provides an extensive resource for future gonadal development and gametogenesis molecular biology studies in this species.

Molecular characterization and expression profiles provide new insights into GATA5 functions in tongue sole (Cynoglossus semilaevis).

GATA5 is a member of the GATA transcription factor family, which serves essential roles in varieties of cellular functions and biological processes. In this study, we have accomplished the molecular cloning, bioinformatic analysis and preliminary function study of C. semilaevis GATA5. The full-length cDNA nucleotide sequence is 1955 bp, with a coding sequence of 1167 bp, which encodes a polypeptide of 388 amino acids. Homology, phylogenetic, gene structure and synteny analysis showed that C. semilaevis GATA5 was highly conserved among vertebrates. Tissue distribution pattern exhibited that C. semilaevis GATA5 was significantly expressed in heart, intestine, liver, kidney and gonad, with a sexual dimorphic feature observed in testis and ovary. Embryonic development expression profiles showed that C. semilaevis GATA5 transcripts increased at the blastula stage, and peaked at the heat-beating period. Strong signals were detected at spermatids of male testis and stage III oocytes of female ovary by ISH. The expression of C. semilaevis GATA5 was regulated by 17α-MT and E2 after hormone stimulation to the ovary. Together, all the results pointed out that GATA5 might play a vital role during gonadal maturation and the reproductive cycle of C. semilaevis. This study lays the foundation for further researches on the sex control breeding in tongue sole.

Roles of piwil1 gene in gonad development and gametogenesis in Japanese flounder, Paralichthys olivaceus.

PIWI family member piwil1, which associates with Piwi-interacting RNA (piRNA), is responsible in regulation of germ cell differentiation and maintenance of reproductive stem cells. In this study, we analyzed the piwil1 gene in Paralichthys olivaceus. Bioinformatics analysis and structure prediction showed that piwil1 had the conserved domains: PAZ domain and PIWI domain. Expression analysis during embryonic development implied that piwil1 gene was maternally inherited. The tissue distribution showed a sexually dimorphic gene expression pattern, with higher expression level in testis than ovary. In situ hybridization results demonstrated that piwil1 was predominantly distributed in oogonia, oocytes, sertoli cells and spermatocytes. A CpG island was predicted in the 5'-flanking region of piwil1 gene, and its methylation levels showed significant disparity between males and females, indicating that the sexually dimorphic expression of piwil1 gene might be regulated by methylation. Furthermore, we explored the distinct roles of human chorionic gonadotropin and 17α-methyltestosterone in regulating the expression of piwil1, and found that piwil1 was interacting with the HPG axis hormones. These results indicated that piwil1 might play a crucial role in gonadal development and gametogenesis in Paralichthys olivaceus.

GATA4 is a transcriptional regulator of R-spondin1 in Japanese flounder (Paralichthys olivaceus).

GATA4 is a well-known transcription factor of the GATA family implicated in regulation of sex determination and gonadal development in mammals. In this study, we cloned the full-length cDNA of Paralichthys olivaceus gata4 (Po-gata4). Phylogenetic, gene structure, and synteny analysis showed that Po-GATA4 is homologous to GATA4 of teleost and tetrapod. Po-gata4 transcripts were detected in Sertoli cells, spermatogonia, oogonia and oocytes, with higher transcript levels overall in the testis than the ovary. The promoter region of P. olivaceus R-spondin1was found to contain a GATA4-binding motif. Results of CBA (cleaved amplified polymorphic sequence-based binding assay) indicated that GATA4 could indeed bind to the promoter sequence of R-spondin1. Moreover, human GATA4 recombinant protein could upregulate R-spondin1 in P. olivaceus ovary cells and FBCs (flounder brain cell line). In FBCs, overexpression of Po-gata4 resulted in elevated transcript levels of R-spondin1. Taken together, our results indicate that Po-GATA4 is involved in gonadal development by regulating R-spondin1 expression.

Characterization and functional analysis of the Paralichthys olivaceus prdm1 gene promoter.

PR domain containing protein 1 (Prdm1) is a transcriptional repressor identified in various species and plays multiple important roles in immune response and embryonic development. However, little is known about the transcriptional regulation of the prdm1 gene. This study aims to characterize the promoter of Paralichthys olivaceus prdm1 (Po-prdm1) gene and determine the regulatory mechanism of Po-prdm1 expression. A 2000bp-long 5'-flanking region (translation initiation site designated as +1) of the Po-prdm1 gene was isolated and characterized. The regulatory elements in this fragment were then investigated and many putative transcription factor (TF) binding sites involved in immunity and multiple tissue development were identified. A 5'-deletion analysis was then conducted, and the ability of the deletion mutants to promote luciferase and green fluorescent protein (GFP) expression in a flounder gill cell line was examined. The results revealed that the minimal promoter is located in the region between -446 and -13bp, and the region between -1415 and -13bp enhanced the promoter activity. Site-directed mutation analysis was subsequently performed on the putative regulatory elements sites, and the results indicated that FOXP1, MSX and BCL6 binding sites play negative functional roles in the regulation of the Po-prdm1 expression in FG cells. In vivo analysis demonstrated that a GFP reporter gene containing 1.4kb-long promoter fragment (-1415/-13) was expressed in the head and trunk muscle fibres of transient transgenic zebrafish embryos. Our study provided the basic information for the exploration of Po-prdm1 regulation and expression.

Identification and expression of piwil2 in turbot Scophthalmus maximus, with implications of the involvement in embryonic and gonadal development.

Piwil2, a member of the Argonaute family, is involved in the biogenesis of PIWI-interacting RNAs (piRNAs) and plays an important role in regulating gametogenesis. In the present study, we identified turbot Scophthalmus maximus piwil2 gene, named Smpiwil2, which contained a PAZ domain and a PIWI domain. Sequence comparison, genomic structure and phylogenetic analyses showed that Smpiwil2 is homologous to that of teleosts and tetrapods. The Smpiwil2 transcript showed higher expression in the ovary than in the testis, demonstrating a sexually dimorphic gene expression pattern. In situ hybridization (ISH) showed that Smpiwil2 was expressed in the oogonia and all the stages of oocytes in the ovary as well as in spermatogonia and spermatocytes in the testis. Embryonic expression profile revealed that Smpiwil2 was maternally inherited, and its level was higher from the zygote to the blastula stage and subsequently decreased until hatching. Moreover, a CpG island was predicted to locate in the 5'-flanking region of Smpiwil2 gene, and its methylation levels detected by sodium bisulfite sequencing showed significant disparity between females and males, implying that the sexually dimorphic expression of Smpiwil2 might be regulated by methylation. These results indicated that Smpiwil2 had potentially vital functions in embryonic and gonadal development in this species. In addition, the temporal and sex differences in Smpiwil2 expression indicated that this gene may play different roles in gonadal development of different sexes.

Sexually dimorphic expression in developing and adult gonads shows an important role of gonadal soma-derived factor during sex differentiation in olive flounder (Paralichthys olivaceus).

Gonadal soma-derived factor (gsdf) is a new member of transforming growth factor beta (TGF-ß) superfamily. As a teleost- and gonad-specific growth factor, gsdf has been indicated to play an important role in early germ cell development. However, little is known about its significance in germ cell development of olive flounder (Paralichthys olivaceus). In the present study, a 1338 bp gsdf gene was isolated from P. olivaceus for the first time. Bioinformatic analysis revealed that the genomic structure and synteny relationship of gsdf in teleosts were conserved. Quantitative real-time PCR (qRT-PCR) showed that gsdf expressed before sex gonadal differentiation, and the expression level increased rapidly after initiation of sex differentiation in males. In adult individuals, the expression of gsdf was higher in testis than that in ovary (P<0.01). In situ hybridization (ISH) indicated that gsdf mRNA was detected in the somatic cells of both males and females, and also in the cytoplasm of oocytes. These results suggested that gsdf might play an important role as initial switches to promote testis differentiation and participate in early germ cell development, such as proliferation and differentiation of spermatogonia and oogonia in P. olivaceus.

Evolution history of duplicated smad3 genes in teleost: insights from Japanese flounder, Paralichthys olivaceus.

Following the two rounds of whole-genome duplication (WGD) during deuterosome evolution, a third genome duplication occurred in the ray-fined fish lineage and is considered to be responsible for the teleost-specific lineage diversification and regulation mechanisms. As a receptor-regulated SMAD (R-SMAD), the function of SMAD3 was widely studied in mammals. However, limited information of its role or putative paralogs is available in ray-finned fishes. In this study, two SMAD3 paralogs were first identified in the transcriptome and genome of Japanese flounder (Paralichthys olivaceus). We also explored SMAD3 duplication in other selected species. Following identification, genomic structure, phylogenetic reconstruction, and synteny analyses performed by MrBayes and online bioinformatic tools confirmed that smad3a/3b most likely originated from the teleost-specific WGD. Additionally, selection pressure analysis and expression pattern of the two genes performed by PAML and quantitative real-time PCR (qRT-PCR) revealed evidence of subfunctionalization of the two SMAD3 paralogs in teleost. Our results indicate that two SMAD3 genes originate from teleost-specific WGD, remain transcriptionally active, and may have likely undergone subfunctionalization. This study provides novel insights to the evolution fates of smad3a/3b and draws attentions to future function analysis of SMAD3 gene family.