College of Marine Life Science, Ocean University of China, Key Laboratory of Marine Genetics and Breeding, Ministry of Education, 5 Yushan Road, Qingdao 266003, China.
Gene. 2013 Dec 1;531(2):411-21. doi: 10.1016/j.gene.2013.08.030. Epub 2013 Sep 6.
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.
同源域转录因子 Nanog 在维持哺乳动物胚胎干细胞的多能性和自我更新中发挥着关键作用。干细胞是生成转基因动物和保存遗传资源的重要且有效的工具。海洋鱼类 Nanog 基因的分子遗传组织和表达尚未报道。在这项研究中，我们分离并鉴定了牙鲆 Nanog 基因，作为了解鱼类干细胞多能性机制的第一步，并开发了一种潜在的分子标记，以鉴定体内和体外的干细胞。系统发育、基因结构和染色体共线性分析提供的证据表明，Po-nanog 与哺乳动物 nanog 基因同源。蛋白质序列比较表明，牙鲆 Nanog 与其他脊椎动物同源物的相似度较低，除了保守的同源域外。定量 RT-PCR 分析表明，牙鲆 nanog 呈母源表达，从单细胞期到神经胚期都有转录本存在，在囊胚期达到峰值。整体原位杂交分析表明，转录本存在于早期胚胎的所有卵裂球中。组织分布分析表明，nanog 仅在性腺中可检测到。此外，在卵巢中的表达显著高于睾丸。原位杂交显示，在卵巢中的卵原细胞和卵母细胞的细胞质中存在转录本，仅在精原细胞中存在，而在精母细胞或精子中不存在。还分析了启动子区域，发现有几个基本核心启动子元件和转录因子结合位点。所有这些结果表明，Po-Nanog 可能在硬骨鱼类和哺乳动物之间具有保守功能。
