Department of Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260.
Int J Biol Sci. 2011 Apr 15;7(4):440-51. doi: 10.7150/ijbs.7.440.
Stem cell cultures can be derived directly from early developing embryos and indirectly from differentiated cells by forced expression of pluripotency transcription factors. Pluripotency genes are routinely used to characterize mammalian stem cell cultures at the molecular level. However, such genes have remained unknown in lower vertebrates. In this regard, the laboratory fish medaka is uniquely suited because it has embryonic stem (ES) cells and genome sequence data. We identified seven medaka pluripotency genes by homology search and expression in vivo and in vitro. By RT-PCR analysis, the seven genes fall into three groups of expression pattern. Group I includes nanog and oct4 showing gonad-specific expression; Group II contains sall4 and zfp281 displaying gonad-preferential expression; Group III has klf4, ronin and tcf3 exhibiting expression also in several somatic tissues apart from the gonads. The transcripts of the seven genes are maternally supplied and persist at a high level during early embryogenesis. We made use of early embryos and adult gonads to examine expression in stem cells and differentiated derivatives by in situ hybridization. Strikingly, nanog and oct4 are highly expressed in pluripotent blastomeres of 16-cell embryos. In the adult testis, nanog expression was specific to spermatogonia, the germ stem cells, whereas tcf3 expression occurred in spermatogonia and differentiated cells. Most importantly, all the seven genes are pluripotency markers in vitro, because they have high expression in undifferentiated ES cells but dramatic down-regulation upon differentiation. Therefore, these genes have conserved their pluripotency-specific expression in vitro from mammals to lower vertebrates.
干细胞培养可以直接从早期胚胎中获得，也可以通过强制表达多能性转录因子间接从分化细胞中获得。多能性基因通常用于在分子水平上表征哺乳动物干细胞培养物。然而，在较低等的脊椎动物中，这些基因仍然未知。在这方面，实验室鱼类斑马鱼是独一无二的，因为它具有胚胎干细胞（ES）和基因组序列数据。我们通过同源搜索和体内、体外表达鉴定了 7 种斑马鱼多能性基因。通过 RT-PCR 分析，这 7 个基因分为 3 组表达模式。第 I 组包括 nanog 和 oct4，表现出性腺特异性表达；第 II 组包含 sall4 和 zfp281，表现出性腺优先表达；第 III 组包括 klf4、ronin 和 tcf3，除了性腺外，还在几种体细胞组织中表达。这 7 个基因的转录本是母源提供的，并在早期胚胎发生过程中保持高水平表达。我们利用早期胚胎和成年性腺，通过原位杂交技术检测干细胞和分化衍生物中的表达。引人注目的是，nanog 和 oct4 在 16 细胞胚胎的多能性胚泡中高度表达。在成年睾丸中，nanog 表达特异性地存在于精原细胞，即生殖干细胞中，而 tcf3 表达存在于精原细胞和分化细胞中。最重要的是，这 7 个基因在体外都是多能性标志物，因为它们在未分化的 ES 细胞中高表达，但在分化后显著下调。因此，这些基因在从哺乳动物到较低等脊椎动物的体外都保持了其多能性特异性表达。
