We cloned and characterized cDNA sequence of insulin-like growth factor binding protein-4 (IGFBP-4) from Japanese flounder (Paralichthys olivaceus). The 1493 bp full-length cDNA sequence contained an open reading frame (ORF) of 780 bp, which encoded a protein of 259 amino acids. The deduced amino acid sequences included a putative signal peptide of 28 amino acid residues resulting in a mature protein of 231 amino acids. Twenty cysteine residues and two conserved IGFBPs motif (GCGCCXXC and CWCV) were found in the N- and C-terminal domain. In the over 13 kbp genomic sequence, four exons, three introns, and 5'-/3'-flanking sequences were identified. Sequence alignment and phylogenetic analysis showed that Japanese flounder IGFBP-4 was indeed the ortholog of the human IGFBP-4 gene and shared high identities with other teleost IGFBP-4 genes. The promoter region was also analyzed and several potential transcription factor (TF) binding sites were determined which may modulate the IGFBP-4 expression. Quantitative real-time PCR analysis revealed that IGFBP-4 mRNA was observed in various tissues, with intestine showing the highest expression. The maternal transcripts of IGFBP-4 gene existed in the early embryonic stages and then increased in the following stages until hatching, suggesting that IGFBP-4 may be involved in the fish early development. The expression level of IGFBP-4 mRNA was relatively higher at 3 days post hatching (dph) and 15 dph, and gradually decreased during the metamorphosis period. All these results indicated that IGFBP-4 plays a significant role in IGF regulating vertebrate growth and development.
Sox2 plays an essential role in maintaining the pluripotency of embryonic and neural stem cells as well as in the neurogenesis. While it has been well studied in mammals, information from lower invertebrate especially marine fish is still limited. In this study, we cloned and sequenced the full-length cDNA and partial 5'-flanking region of the Japanese flounder Sox2. Phylogenetic, gene structure, and protein comparison analyses revealed that Paralichthys olivaceus Sox2 (Po-Sox2) was homologous to mammalian Sox2. Quantitative real-time RT-PCR results showed that Po-Sox2 was not maternal inherited, and the transcripts were present from high blastula-stage onwards, with the highest level at the mid-gastrula stage. Tissue distribution analysis revealed that Po-Sox2 was present not only in neural tissues, but also in gonadal and gill tissues. In addition, we analyzed the Po-Sox2 promoter region for several species-conserved motifs as well as various transcription factor binding sites. The overall hypomethylation status of the identified CpG sites in the 5'-regulatory region revealed that it was not involved in the transcriptional modulation of Po-Sox2. All these results suggest that Po-Sox2 may have a conserved function in neurogenesis and early embryonic development.
The vasa gene encodes an ATP-dependent RNA helicase of the DEAD box protein family that functions in a broad range of molecular events involving duplex RNA. In most species, the germline specific expression of vasa becomes a molecular marker widely used in the visualization and labeling of primordial germ cells (PGCs) and a tool in surrogate broodstock production through PGC transplantation. The vasa gene from tongue sole (Cynoglossus semilaevis) was characterized to promote the development of genetic breeding techniques in this species. Three C. semilaevis vasa transcripts were isolated, namely vas-l, vas-m, and vas-s. Quantitative real-time PCR results showed that C. semilaevis vasa transcripts were prevalently expressed in gonads, with very weak expression of vas-s in other tissues. Embryonic development expression profiles revealed the onset of zygotic transcription of vasa mRNAs and the maternal deposit of the three transcripts. The genetic ZW female juvenile fish was discriminated from genetic ZZ males by a pair of female specific primers. Only the expression of vas-s can be observed in both sexes during early gonadal differentiation. Before PGCs started mitosis, there was sexually dimorphic expression of vas-s with the ovary showing higher levels and downward trend. The results demonstrated the benefits of vasa as a germline specific marker for PGCs during embryonic development and gonadal differentiation. This study lays the groundwork for further application of C. semilaevis PGCs in fish breeding.
The homeodomain-containing transcription factor nanog plays a key role in maintaining the pluripotency and self-renewal of embryonic stem cells in mammals. Stem cells offered as a significant and effective tool for generation of transgenic animals and preservation of genetic resources. The molecular genetic organization and expression of nanog gene in marine fish have not been reported yet. In this study, we isolated and characterized the flounder nanog gene as a first step towards understanding the mechanism of the plurpotency of fish stem cells and develop a potential molecular marker to identify the stem cells in vivo and in vitro. Phylogenetic, gene structure and chromosome synteny analysis provided the evidence that Po-nanog is homologous to the mammalian nanog gene. Protein sequence comparison showed that flounder Nanog shared low similarity with other vertebrate orthologs except for a conserved homeodomain. Quantitative RT-PCR analysis showed that flounder nanog was maternally expressed, and the transcripts were present from the one-cell stage to the neurula stage with the peaking at blastula stage. Whole mount in situ hybridization analyses demonstrated that the transcripts were present in all blastomeres of the early embryo. Tissue distribution analysis indicated that nanog was detectable only in gonads. Further, the expression was significantly high in ovary than in testis. In situ hybridization revealed that the transcripts were located in the cytoplasm of the oogonia and oocytes in ovary, only in the spermatogonia but no spermatocytes or spermatids in testis. The promoter region was also analyzed to have several basal core promoter elements and transcription factor binding sites. All these results suggest that Po-Nanog may have a conservative function between teleosts and mammals.
Vasa is a DEAD box helicase and has shown essential functions during gametogenesis and embryogenesis. In most species, research revealed a specific expression of vasa gene in the germ cells. Thus, vasa has become the candidate gene in identifying germ cells. In this study, the vasa gene was isolated from gonads of Japanese flounder (Paralichthys olivaceus). In the 11.4kb genomic sequence, 23 exons were identified besides 5 and 3 flanking regions. The promoter region contained several putative TF binding sites which may have the function of regulating vasa expression. Quantitative real-time PCR analysis showed that vasa gene expression was restricted to adult gonads, with a higher level in the ovary. Development expression profiling revealed a maternal deposit and constant embryonic expression at early stages, but the relative mRNA amount decreased after gastrula. Nine other PoVasa transcripts were detected and their expression in gonads and during early development was not all the same, implying potential different functions during gametogenesis or early embryonic development. These results together confirmed the feasibility of using vasa as a marker of germ cells and that vasa gene had an important role in spermatogenesis and oogenesis. Furthermore, our study laid the groundwork for identifying fish primordial germ cells (PGCs) and investigating germ cell biology.
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