Functional analysis of systemic factors regulating germline stem cells

调节生殖干细胞的系统因素的功能分析

• 批准号: 8997406
• 负责人: Benjamin Scott Ewen-Campen
• 金额: $ 5.43万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8997406
• 关键词: Address; Adipose tissue; Adult; Affect; Age; Animals; Biological Assay; Biological Models; Brain; Candidate Disease Gene; Cell Cycle; Cell Cycle Progression; Cell Proliferation; Cells; Cellular biology; Communication; Cues; Drosophila genome; Drosophila genus; Drosophila melanogaster; Ecdysone; Endocrine; Erinaceidae; Exercise; Fat Body; Female; Fertility; Genes; Genetic Screening; Germ Cells; Goals; Health; Human; Insulin; Intestines; Laboratories; Life; Ligands; MAPK8 gene; Measures; Mediating; Molecular; Monitor; Muscle; Nuclear Hormone Receptors; Nutritional status; Organ; Ovarian; Ovary; Partner in relationship; Pathway interactions; Physiological; Physiology; Process; Proteins; RNA Interference; RNA interference screen; Reading; Receptor Protein-Tyrosine Kinases; Regulation; Reporter; Reproductive Health; Role; Series; Signal Pathway; Signal Transduction; Source; Spermatogenesis; Stem cells; Syndrome; System; Techniques; Testing; Tissues; Whole Organism; Work; adult stem cell; aging nutrition; analog; behavior influence; cancer risk; cell behavior; daughter cell; egg; genome-wide; genome-wide analysis; germline stem cells; in vivo; insight; knock-down; male; microbiota; notch protein; novel; nutrition; public health relevance; release factor; reproductive; research study; response; self-renewal; sensor; sperm cell; stem cell division; stem cell niche; stem cell population; temporal measurement; tool

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.

描述（由申请人提供）：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节对生殖健康至关重要。GSCs受来自附近“生态位”细胞的短距离信号调控，这些信号使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对整个动物的生理信号也高度敏感，包括营养、交配状态和年龄。虽然GSCs与生态位之间的短程信号交换已被很好地表征，但GSCs的远程调控的分子机制尚不清楚。本研究的目的是系统地识别和表征影响GSC增殖的新循环因子。拟议的实验将在果蝇（Drosophila melanogaster）中进行，这是一种已建立的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，并且可以进行全基因组的体内遗传筛选，以寻找影响成体干细胞行为的特定器官释放的因素。在Specific Aim 1中，新工具将应用于果蝇GSCs，以直接可视化细胞周期的进展，以及GSCs与生态位之间局部信号通路活性的动态。这些技术将克服以前监测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。特异性目标2将测试10种特征明确、高度保守的动物信号通路的已知配体是否参与GSCs的组织外源性调节。这些因子中有几个最近与各种器官间通讯过程有关，但它们在调节成体干细胞（如GSCs）中的作用以前没有得到解决。这些分泌的配体中的每一种都将在脂肪体（类似于脂肪组织的果蝇）、肌肉、肠道和大脑中被特异性地击倒，并对GSC增殖和局部信号通路活性的影响进行测试。特异性Aim 3是一种系统的、组织特异性的RNAi筛选，用于鉴定影响GSC增殖的新器官分泌因子。果蝇基因组中的所有分泌因子（n = ~800）将在与Aim 2相同的四个组织中被敲除，并将筛选对GSC增殖的影响。所有新发现的基因都将被测试，以确定它们是否也会影响其他成体干细胞群体的行为，或者它们的影响是否是gsc特异性的。总之，这些研究提供了成体干细胞群体如何整合和响应来自外源的循环信号的见解，这一过程对精子发生、多囊卵巢综合征、癌症风险和对营养的全身反应等人类健康问题具有潜在影响。