Fundamental molecular mechanisms of stem cell system

为了研究干细胞系统的基本分子机制，分析了两种干细胞。一种是全能性的小鼠胚胎干细胞（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。我们利用条件显性负性Myb（MERT）抑制Myb的所有成员（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B-Myb功能的抑制。不幸的是，B-Myb功能的抑制并不改变ES细胞的分化状态。相反，细胞周期和细胞粘附受到影响。从以前的报道和B-Myb的表达模式来看，细胞周期的改变是合理的。然而，细胞粘附的改变是一个新的发现。精确的检测表明，钙粘蛋白和整合素的表面表达的变化是由抑制B-Myb启动的。考虑到这两种粘附分子的转录水平没有改变，可能是由于B-Myb的抑制引起了一些转录后修饰。结合B-Myb基因敲除小鼠的研究结果，我们构建了TNAP（tissue non-specific alkaline phosphatase，组织非特异性碱性磷酸酶）-EGFP基因敲除小鼠PGCs，并将其用于小鼠胚胎PGCs的分离。通过SAGE分析ES细胞和PGCs中的基因表达。从两个来源测序了约10，000个表达标签。一对夫妇的家庭基因优先表达在胚胎干细胞或PGCs。并克隆了一个在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.