Isolation And Characterization Of The Transcriptional Factors In Maintaining Pluripotency Of Stem Cells In Japanese Flounder Paralichthys Olivaceus

With the development of the breeding technology, a large number of aquaticproduct varieties have been bred in recent years. Establishing stable methods for long-term preservation of aquatic genetic information is of great significance to protectendangered biological resources. It can also facilitate the conservation and utilizationof the genetic resources from good varieties and high quality strains. Fish ESCs possessthe ability of replicate indefinitely and can differentiate into all three layers of theembryo. It offers as a significant and effective tool for generation of transgenic animalsand preservation of genetic resources. Up to now, it is encouraged that several fish EScell lines have been attempted and successfully established under a feeder-freecondition by using the blastula stage embryos. In mammals, a lot of works have beencarried out to investigate the molecular mechanism for maintaining the stem cell self-renewal and pluripotency, which mainly focused on the transcription factors Oct4,Nanog and Sox2. However, the information of the related approach and molecularmechanism is limited in P. olivaceus. The aim of this study is to identify the mammalianstem cell marker gene orthologue in Japanese flounder so as to lay a foundation for thefurther investigation.Oct4is a crucial transcription factor that in conjunction with Nanog and Sox2toorchestrate pluripotency and proliferation characteristics of embryonic stem cells. Po-Oct4was cloned through homology cloning method and RACE strategy from theblastula-stage embryos. The genomic DNA sequence of Po-Oct4is3978bp long andis consisted of five exons. There exits two transcript isoforms with an identical5′UTRof352bp and different lengths of3′UTR, which are874bp and1318bp long, respectively. The deduced Po-Oct4contained an open reading frame of1428bpencoding a protein of475residues. The predicted Po-Oct4possessed a characteristicPOU domain that consists of the POU specific domain, the POU homeodomain and alinker region between them. Phylogenetic, gene structure and chromosome syntenyanalysis provided the evidence that Po-Oct4is homologous to the mammalian Oct4gene. We isolated the entire promoter region of Po-Oct4by genome walking strategy.Sequence analysis with the MatInspector program revealed that there were numeroustranscription factor binding sites on the promoter region of Po-Oct4, including Oct3₄,Nanog, PRDM14, FOXP1-ES, and ONST which may regulate the target geneexpression in maintaining pluripotency. Po-Oct4was observed to be maternallyexpressed, and the transcripts were present in the unfertilized egg and throughout earlydevelopment. Po-Oct4reached its highest expression level at high-blastula stage,decreased but still relatively high until mid-gastrula stage, then greatly diminished atthe end of gastrulation stage. From heart-beating stage onward, Po-Oct4can hardly bedetected anymore. Overall, the long transcript Po-Oct4-L exhibited a similar expressionpattern with the total Po-Oct4transcripts. Notable, the short transcripts Po-Oct4-Sshowed a higher expression level than the long transcript. Whole mount in situhybridization （WISH） analyses demonstrated that the transcripts were present in allblastomeres of the early embryo. Po-Oct4transcripts were exclusively restricted to thegonad tissues among ten examined tissues and with much more abundance in ovarythan in testis. Through in situ hybridization and immunohistochemistry analyses, wedemonstrated that Po-Oct4is abundant and continuous in oogonia and different stageoocytes, but only in the spermatogonia in flounder males.The homeodomain-containing transcription factor Nanog plays a key role inmaintaining the pluripotency and self-renewal of embryonic stem cells. Usingdegenerate primers and RACE strategy, we cloned Po-Nanog from the embryo ofJapanese flounder. Overlapping sequences of all fragments yielded a full-length cDNAwith120bp5′UTR,459bp of3′UTR and1260bp of the entire open reading frame.The genomic DNA sequence of Po-Nanog is2416bp long and is consisted of fourexons. The predicted amino acid sequence of Po-Nanog is419residues long and contains a60amino acids homeodomain within positions226–285. Protein sequencecomparison showed that flounder Nanog shared low similarity with other vertebrateorthologs except for a conserved homeodomain. Phylogenetic, gene structure andchromosome synteny analysis provided the evidence that Po-Nanog is homologous tothe mammalian Nanog gene. There are numerous cis-elements and potentialtranscription factor binding sites of important for pluripotency or embryonic stem cellswithin the promoter region. There is a conserved29bp TSS-containing sequenceamong diverse vertebrate and located near the5′UTR start site, suggesting that Nanogmay have a conserved start site for initiate transcription. It is also suggested that the150bp sequence upstream from the TSS which contains a TATA-box element would bethe core promoter regulating the transcription activity of Po-Nanog. Quantitative real-time RT-PCR analysis showed that flounder Nanog was maternally expressed, and thetranscripts were present from the one-cell stage to the neurula stage with the peaking atblastula stage. WISH analyses demonstrated that the transcripts were present in allblastomeres of the early embryo. Tissue distribution analysis indicated that nanog wasdetectable only in gonads. Further, the expression was significantly higher in ovary thanin testis. In situ hybridization revealed that the Po-Nanog transcripts were located inthe cytoplasm of the oogonia and oocytes in ovary, only in the spermatogonia but nospermatocytes or spermatids in testis.Sox2plays an essential role in maintaining the pluripotency of embryonic andneural stem cells as well as in the neurogenesis. Po-Sox2is an one exon gene and thefull-length cDNA sequence is2262bp long that contains a352bp of5′UTR,941bpof3′UTR and969bp of ORF. The predicted amino acid sequence of Po-Sox2is322residues long and contains the characteristic HMG-box DNA binding domain as wellas the SOXp domain. Phylogenetic, gene structure, and protein comparison analysesrevealed that Po-Sox2was homologous to mammalian Sox2. The regulatory region ofPo-Sox2contains numerous essential transcription factor binding sites which areclosely related to the stem cell pluripotency and neurogenesis. Nine conserved regionswere identified in flounder, medaka, and zebrafish, of which three were tandemly linkedand also conserved in the similar upstream region of human and mouse Sox2. Conserved motifs within the promoter region may possess potential and critical functions inregulating the expression of Po-Sox2. Quantitative real-time RT-PCR showed thatflounder Sox2was not maternal inherited, and the transcripts were present from highblastula-stage onwards, with the highest level at the mid-gastrula stage. Tissuedistribution analysis revealed that Sox2was present not only in neural tissues, but alsoin gonadal and gill tissues. In the brain, the highest expression of Po-Sox2mRNAtranscripts was detected in the telencephalon, whereas decreasing levels were observedin the macromyelon, epencephalon, mesencephalon, and diencephalon. Po-Sox2transcripts exhibit a similar subcellular localization with Po-Oct4and Po-Nanog in theadult gonadal tissues, but the signal intensity is gradually weakened during oogenesis.