REGULATION OF GERMLINE STEM CELL DIVISION IN DROSOPHILA

描述：研究人员感兴趣的是如何自我更新 干细胞的不对称分裂是实现和调节的。他在利用 以果蝇雌性生殖系为模型系统进行这方面的研究 有问题。作为艾伦·斯普拉林的博士后研究员，PI开发了 一种定位生殖系干细胞的体内培养系统 在细菌馆的尽头附近。他将这种疗法与激光消融相结合， 显示每个生殖室包含2-3个相互接触的干细胞 与形成终丝的体细胞一起。他发现了一种 小说《细胞器》，他把包含这两种成分的光谱体命名为 血影蛋白和HTS基因的内收蛋白样蛋白产物。这个 光谱体位于干细胞核和终末之间。 灯丝。在有丝分裂过程中，光谱体保持在末端附近。 灯丝。中心粒复制后，一个中心粒停留在 有丝分裂纺锤体的一个紫锥体和一个极成为 与光谱组有关。不对称的关联 有丝分裂纺锤体的光谱组显示了茎的不对称性。 细胞分裂。HTS突变导致光谱体丢失和 卵室发育异常。在其他实验中，PI发现 终丝细胞的消融导致细胞分裂增加 表明终丝细胞调控了 干细胞的细胞增殖。PI进一步确定了一个数字 在前部细胞中表达的增强子陷阱 他获得了一些证据表明 HH、WG、EN等极性基因参与调控 卵室的发育。最后，PI确定了两个基因 由P插入定义，这些插入似乎对不对称 干细胞的分裂。其中之一，ovarette(OVT)，导致 卵巢大多有2-3簇生殖细胞出现 彼此相似。没有证据表明存在差异。 然而，少数卵巢只含有2-3个成熟卵子 但没有其他生殖细胞。公安局将此解读为暗示 突变可以阻止干细胞的不对称分裂。第二 基因被称为piwi。该基因的突变导致卵巢小管有两个 或更少的发育中的卵室。圆周率表明这种突变 导致干细胞在体内维持失败 未分化的状态(即，它们失去了茎的质量)。 提出了5个具体目标。一是有系统地 检测不同体细胞在调节干细胞中的作用 组织。一种光化学活化烧蚀方法将用于 消融位于大脑前端部的不同组体细胞 生殖器病。然后将消融的生殖器在体内培养以进行检测。 干细胞。第二个具体目标是检查干细胞。 在没有光谱体的情况下进行分裂。这将通过以下方式完成 用共聚焦显微镜检查HTS突变的病原体。第三 具体目的是确定OVT和Piwi在体细胞中的作用 诱导机制。PI将使用杆状细胞移植来 产生基因马赛克以确定突变是否作用于 胚系或体细胞。PI还将检查这两个参数的表达 在RNA和蛋白质水平上的基因。特别有趣的是， 细胞表达每一种基因。第四个具体目标是分析 这两个基因的分子结构和性质的确定 他们的蛋白质产品。最终的具体目标是分析 这两种蛋白质的细胞行为。

DESCRIPTION: The investigator is interested in how the self renewing asymmetric division of stem cells is achieved and regulated. He is using the female germ line of Drosophila as a model system to study this problem. As a postdoctoral fellow with Allan Spradling the PI developed an in vivo culture system for the germ line stem cells which are located near the end of the germarium. He combined this with laser ablation to show that each germarium contained 2-3 stem cells that are in contact with somatic cells that form the terminal filament. He identified a novel "organelle" that he named the spectrosome that contained both spectrin and the adducin like protein product of the hts gene. The spectrosome is located between the stem cell nuclei and the terminal filament. During mitosis the spectrosome remains next to the terminal filament. Following centriole duplication one centriole stays near the spectrosome, and one aster and one pole of the mitotic spindle becomes associated with the spectrosome. The asymmetric association of the spectrosome with the mitotic spindle reveals the asymmetry of the stem cell division. The hts mutation results in a loss of the spectrosome and abnormal egg chamber development. In other experiments the PI found that ablation of terminal filament cells resulted in increased cell division of the stem cells suggesting that the terminal filament cells regulated cell proliferation of the stem cells. The PI further identified a number of enhancer traps that are expressed in the cells of the anterior region of the germarium and he obtained evidence that a number of segment polarity genes such as hh, wg and en are involved in regulating development of the egg chamber. Finally the PI identified two genes defined by P insertions that appear to be essential for the asymmetric division of the stem cells. One of these, ovarette (ovt), results in ovarioles mostly with two-three clusters of germ line cells that appear similar to one another. There is no evidence for differentiation. However, a small number of ovariole contain only two-three mature eggs but no other germ cells. The PI interprets this as suggesting that the mutation prevents the asymmetric division of the stem cells. The second gene is called piwi. Mutations in this gene result in ovarioles with two or fewer developing egg chambers. The PI suggests that this mutation results in a failure of the stem cells to be maintained in an undifferentiated state (i.e., they lose the quality of stemness). There are 5 specific aims proposed. The first is to systematically examine the role of the various somatic cells in regulating stem cell division. A photochemical activation ablation method will be used to ablate different sets of somatic cells located at the anterior end of the germaria. The ablated germaria would then be cultured in vivo to assay the stem cells. The second specific aim is to examine the stem cell division in the absence of the spectrosome. This will be done by examining hts mutant germaria via confocal microscopy. The third specific aim is to define the role of ovt and piwi in the somatic induction mechanism. The PI will use pole cell transplantation to generate genetic mosaics to determine whether the mutations act in the germ line or soma. The PI will also examine the expression of these two genes at both the RNA and protein level. Of particular interest is which cells express each of the genes. The fourth specific aim is to analyze the molecular structure of these two genes and to determine the nature of their protein products. The final specific aim is to analyze the cellular behavior of the two proteins.