The regulatory mechanisms of self-proliferation and differentiation in stem cells

Notch基因家族的成员编码跨膜蛋白，在抑制邻近细胞之间的分化和介导干细胞的细胞命运决定中发挥作用。小鼠Notch4是乳腺肿瘤中MMTV的常见整合位点，其胚胎表达主要局限于血管内皮细胞。这些结果表明Notch信号通路在血管发育中起着至关重要的作用。为了研究Notch4基因在血管发育中的作用，本研究试图通过基因靶向的方法产生Notch4基因敲除小鼠。嵌合小鼠已被杂交产生杂合基因敲除小鼠。此外，我们还利用胚胎干细胞体外分化系统培养造血细胞和内皮细胞，研究了激活Notch4对胚胎干细胞分化的影响。对多个标记基因的表达分析表明，激活的Notch4抑制了胚胎干细胞的极早期分化，也抑制了成血管细胞谱系向造血细胞或内皮细胞的分支点。这些结果表明，Notch4在血管发生中发挥了与侧抑制相似的细胞命运决定机制。我们还尝试使用胚胎干细胞内皮细胞诱导和分化系统分析Notch4信号通路的成分。Notch4在未分化的ES细胞中表达，诱导后消失。内皮细胞分化开始时，Notch4重新表达。我们用差分显示法寻找与Notch4表达模式相同的候选基因。鉴定了几个基因来检测与Notch4的相互作用。

Members of Notch gene family encode transmembrane proteins that play roles in inhibition of differentiation among neighboring cells and mediate cell fate decisions of stem cells. The embryonic expression of mouse Notch4, a common integration site for MMTV in mammary tumors, is largely restricted to vascular endothelial cells. These results suggested that the Notch signaling pathway plays a critical role in vascular development.In this study we attempted to produce the knockout mouse of Notch4 by gene targeting in order to investigate the role of the Notch4 gene in vascular development. Chimeric mice have been crossed to generate heterozygous knockout mice. Moreover, we also use the in vitro differentiation system of embryonic stem (ES) cells for hematopoietic and endothelial cells, and examine the effect of activated Notch4 on the differentiation of ES cells. The analysis of expression in several marker genes show that activated Notch4 inhibits the differentiation of ES cells at the very early stage as well as the branching point of hemangioblast cell lineage toward hematopoietic cells or endothelial cells. These results suggested that Notch4 play cell fate decision in vasculogenesis using the similar mechanism of the lateral inhibition.We also attempted to analyze the components of Notch4 signaling pathway using an induction and differentiation system for endothelial cells from embryonic stem (ES) cells. Expression of Notch4 was observed in undifferentiated ES cells, and disappeared after induction. At the onset of endothelial cells differentiation Notch4 was re-expressed. We searched candidate genes with the same expression pattern as Notch4 by the Differential Display method. Several genes were identified to examine the interaction with Notch4.