Identification and characterization of a nanog homolog in Japanese flounder (Paralichthys olivaceus)

• We first isolated and characterized the full length cDNA and genomic DNA sequences of nanog gene from the flatfish Japanese flounder (Paralichthys olivaceus).
• The entire promoter region of flounder nanog has been cloned, sequenced and analyzed for potential transcription factor binding sites.
• We analyzed the special and temporal expression profile of flounder nanog during early embryo development and among different tissues by qRT-PCR and ISH.
• Flounder nanog may have a conservative function between teleosts and mammals.

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.