Molecular Mechanisms for Maintaining Stem Cell Immaturity

为了揭示干细胞的分子基础，我们分析了三个基因的功能，发现它们都是生殖系干细胞系统所必需的。1)我们分析了肿瘤抑制基因PTEN（来自10号染色体的磷酸酶和紧张素同源物）的功能。PTEN通过其磷酸酶活性从PIP3催化到PIP2，是p13激酶的负调控因子。我们制备了针对小鼠原始生殖细胞（PGC）特异性的PTEN。PGC特异性PTEN缺失小鼠在所有新生雄性小鼠中均显示畸胎瘤形成。小鼠体外增殖活性和胚胎生殖细胞形成活性显著提高。2)克隆了果蝇生殖细胞少基因的小鼠同源基因，该基因在果蝇体内形成PGC至关重要。为了分析基因（小鼠生殖细胞less-1， mgcl-1）的功能，制作了基因靶向小鼠。小鼠未见PGC形成异常。然而，靶向小鼠的生育能力明显降低。在研究中，有报道称mgcl-1蛋白与核膜的一个组成部分LAP2β结合，这促使我们对精子发生过程中的核形态进行研究。无精母细胞的核形态异常，随后的染色质重塑受损。3) Piwi是果蝇生殖细胞自我更新活动所必需的基因。我们克隆了一个小鼠同源基因mili，并通过基因靶向分析了其功能。由于原代精母细胞粗成期凋亡细胞死亡，mili小鼠不能产生精子。这些结果有助于进一步了解干细胞在生殖系统中的分子机制。

To reveal the molecular basis of stem cells, we analyzed the function of three genes and found that all of them are essential for germ line stem cell systems.1) We analyzed the function of a tumor suppressor gene PTEN (phosphatase and tensin homologue from chromosome 10). PTEN is a negative regulator of P13kinase by its phosphatase activity catalyzing from PIP3 to PIP2. We produced primordial germ cell (PGC) specific PTEN targeting mouse. The PGC specific PTEN null mice showed teratoma formation in all new born male mice. And the in vitro proliferative activity and embryonic germ cell forming activity was strikingly increased in the null mice.2) We cloned a mouse homologue of Drosophila germ cell less gene which is essential for PGC formation in the fly. To analyze the function of the gene (mouse germ cell less-1, mgcl-1), gene targeting mice were produced. The mice did not show any abnormality in PGC formation. However, the fertility of the targeting mice was significantly reduced. During the study, it was reported that mgcl-1 protein binds to a component of nuclear envelope, LAP2β, which prompted us to examine the nuclear morphology in spermatogenesis. Nuclear morphology of the null spermatocyte was abnormal and subsequent chromatin remodeling was impaired.3) Piwi is a gene essential for germ cell self renewing activity in Drosophila. We cloned one mouse homologue, mili, and analyzed its function by gene targeting. The mili null mice could not produce sperm due to apoptotic cell death at pachytene phase of primary spermatocytes.All of the results facilitates the understanding of molecular mechanisms of stem cells in germ system.