Fundamental molecular mechanisms of stem cell system

为了探究干细胞系统的基本分子机制，本文对两类干细胞进行了分析。一种是全能小鼠胚胎干细胞（ES细胞），另一种是生殖系干细胞，即原始生殖细胞（PGC）。已知Myb家族基因在细胞分化中具有重要作用。我们利用条件显性负Myb （MERT）抑制Myb的所有成员（c-， a -和B-Myb）的功能。胚胎干细胞几乎只在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，抑制B-Myb功能并不能改变胚胎干细胞的分化状态。相反，细胞周期和细胞粘附受到影响。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，细胞粘附的改变是一个新的发现。精确检查发现，钙粘蛋白和整合素的表面表达变化是由B-Myb的抑制引起的。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的抑制导致了一些转录后修饰。结合B-Myb敲小鼠的数据，B-Myb在胚胎发生早期对细胞粘附至关重要。制备了小鼠组织非特异性碱性磷酸酶(TNAP)-EGFP敲蛋白，并将其用于从小鼠发育胚胎中分离PGCs。采用SAGE分析基因在ES细胞和PGCs中的表达。从这两个来源中测序了大约10,000个表达标签。两个家族基因在胚胎干细胞或PGCs中优先表达。克隆了一个在胚胎干细胞和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.