Functional analysis of systemic factors regulating germline stem cells

描述(申请人提供)：生殖系干细胞(GSCs)产生精子和卵子，因此对这些干细胞的适当调控对生殖健康至关重要。GSCs受来自附近“小生”细胞的短距离信号的调控，这些细胞使GSCs保持自我更新状态，同时允许子细胞开始分化。然而，最近的研究表明，GSCs也对整个动物的生理信号高度敏感，包括营养、交配状态和年龄。虽然GSCs和生态位之间交换的短程信号得到了很好的表征，但GSCs长程调控的分子机制还不是很清楚。本研究的目的是系统地识别和表征影响GSC增殖的新的循环因素。拟议的实验将在黑腹果蝇中进行，这是一个成熟的GSC细胞生物学模型系统，与脊椎动物干细胞系统有许多相似之处，而且唯一可能进行全基因组、体内遗传筛选的是特定器官释放的影响成年干细胞行为的因素。在具体目标1中，新的工具将被应用于果蝇GSCs，以直接可视化细胞周期的进程，以及GSCs和生态位之间局部信号通路活动的动态。这些技术将克服以往监测GSC增殖率和信号通路活性的技术障碍，并将用于测试高营养和低营养对局部GSC/生态位信号和细胞周期的影响。《特定目标2》将测试这10条特性良好、高度保守的动物信号通路的已知配基中是否有任何一种参与了GSC的组织外在调节。这些因素中的几个最近被认为与各种器官间的通讯过程有关，但它们在调节成体干细胞(如GSCs)中的作用以前还没有被解决。这些分泌的配体中的每一个都将在脂肪体(类似于果蝇的脂肪组织)、肌肉、肠道和大脑中被特异性地击倒，并将测试其对GSC增殖和局部信号通路活性的影响。特异性目标3是一个系统的、组织特异性的RNAi筛选，以确定影响GSC增殖的新的器官分泌因子。果蝇基因组中的所有分泌因子(n=~800)将在与目标2相同的四个组织中被击倒，并将筛选对GSC增殖的影响。然后将对所有已识别的新基因进行测试，以确定它们是否也影响额外的成人干细胞群体的行为，或者它们的影响是否是GSC特有的。总之，这些研究为成人干细胞群体如何整合和响应来自外源的循环信号提供了洞察力，这一过程对人类健康问题(如精子发生、多囊卵巢综合征、癌症风险和全身对营养的反应)具有潜在的影响。

DESCRIPTION (provided by applicant): Germline stem cells (GSCs) produce sperm and egg, and the proper regulation of these stem cells is therefore essential for reproductive health. GSCs are regulated by short-range signaling from nearby "niche" cells that keep GSCs in a self-renewing state while allowing daughter cells to begin differentiation. However, recent studies have demonstrated that GSCs are also highly sensitive to whole-animal physiological cues, including nutrition, mating status, and age. While the short-range signals exchanged between GSCs and the niche are well characterized, the molecular mechanisms underlying long-range regulation of GSCs are not well understood. The goal of this study is to systematically identify and characterize new circulating factors that influence GSC proliferation. The proposed experiments will be conducted in Drosophila melanogaster, an established model system for GSC cell biology, possessing many parallels to vertebrate stem cell systems, and where it is uniquely possible to conduct genome-wide, in vivo genetic screens for factors released by specific organs that influence adult stem cell behavior. In Specific Aim 1, new tools will be applied to Drosophila GSCs to directly visualize the progression of the cell cycle, as well as the dynamics of local signaling pathway activity between GSCs and the niche. These techniques will overcome previous technical obstacles to monitoring GSC proliferation rate and signaling pathway activity, and will be used to test the effects of high- and low-nutrition on local GSC/niche signaling and cell cycling. Specific Aim 2 will test whether any of the known ligands for the 10 well-characterized, highly conserved animal signaling pathways are involved in tissue-extrinsic regulation of GSCs. Several of these factors have recently been implicated in a variety of inter-organ communication processes, but their roles in regulating adult stem cells such as GSCs have not been previously addressed. Each of these secreted ligands will be knocked down specifically in the fat body (the Drosophila analog of adipose tissue), muscles, gut, and brain, and the effects on GSC proliferation and local signaling pathway activity will be tested. Specific Aim 3 is a systematic, tissue-specific RNAi screen to identify novel organ-secreted factors influence GSC proliferation. All of the secreted factors in the Drosophila genome (n = ~800) will be knocked down in the same four tissues as in Aim 2, and effects on GSC proliferation will be screened. All of the new genes identified will then be tested to see whether they also influence the behavior of additional adult stem cell populations, or whether their effect are GSC-specific. Together, these studies provide insight into how adult stem cell populations integrate and respond to circulating signals from exogenous sources, a process with potential implications for such human health issues as spermatogenesis, polycystic ovarian syndrome, cancer risk, and systemic responses to nutrition.