Fundamental molecular mechanisms of stem cell system

为了研究干细胞系统的基本分子机制，对两种干细胞进行了分析。一种是全能的小鼠胚胎干细胞(ES细胞)，另一种是生殖系干细胞，即原始生殖细胞(PGC)。Myb家族基因在细胞分化中起重要作用。我们利用了一个条件性显性负性Myb(Mert)，它抑制了Myb所有成员(C-、A-和B-Myb)的功能。ES细胞几乎只在Myb家族成员中表达B-Myb。因此，Mert的激活导致了B-Myb功能的抑制。不幸的是，B-Myb功能的抑制并不能改变ES细胞的分化状态。相反，细胞周期和细胞黏附会受到影响。从以往的报道和B-Myb的表达模式来看，细胞周期的改变是合理的。然而，细胞黏附的改变是一个新的发现。精确检测表明，细胞表面钙粘附素和整合素表达的改变是由B-Myb的抑制引起的。考虑到这两个黏附分子的转录水平没有改变，可能是由于B-Myb的抑制引起了转录后的修饰。结合B-Myb基因敲打我们的小鼠的资料，B-Myb在胚胎发育早期对细胞的黏附是必不可少的。SAGE法分析ES细胞和PGCs的基因表达。对这两个来源的约10,000个表达标签进行了测序。有几个家族基因在ES细胞或PGCs中优先表达。克隆了一个在ES细胞和PGCs中表达的新基因。

In order to investigate the fundamental molecular mechanisms of stem cell systems, two kinds of stem cells were analyzed. One is totipotent mouse embryonic stem cells (ES cells) and the other is germ line stem cells, namely, primordial germ cells (PGC).Myb family genes are known to posess essential roles in cell differentiation. We utilized a conditinal dominant negative Myb (MERT), which represses the function of all members of Myb (c-, A- and B-Myb). ES cells express B-Myb almost exclusively among Myb family members. Thus, the activation of MERT brings about the suppression of B-Myb function. Unfortunately, the suppression of B-Myb function does not alter differentiation status of ES cells. In stead, cell cycle and cell adhesion are affected. The alteration of cell cycle is reasonable from previous reports and expression pattern of B-Myb. However, the alteration of cell adhesion is a new finding. Precise examination revealed that the change of surface expression of cadherin and integrin was initiated by the inhibition of B-Myb. Considering that the transcription level of these two adhesion molecules was not changed, it is probable that some post-transcriptional modification was caused by the inhibition of B-Myb. Together with the data of B-Myb knock our mice, B-Myb is essential for cell adhesion at the early embryogenesis.TNAP (tissue non-specific alkaline phosphatase)-EGFP knock in mouse was produced and used for isolating PGCs from mouse developing embryos. Gene expression in ES cells and PGCs was analyzed by SAGE analysis. About 10,000 expression tags were sequenced from both sources. A couple of the family genes were preferentially expressed in either ES cells or PGCs. And one novel gene expressed in ES cells and PGCs was cloned.