Modeling epithelial stem cell competition in a dynamic Drosophila ovarian niche

描述(由申请人提供)：这项建议概述了一种战略，以确定果蝇卵巢中一种新型动态干细胞生态位的关键成分，并阐明竞争生态位占据的机制。实现这项提案中的目标将是朝着建立详细的上皮干细胞调控模型的长期目标迈进的一个重要里程碑，以便开发基于再生医学的疗法，并了解在癌症等疾病状态下调控过程是如何失败的。产生卵泡上皮的卵泡干细胞(FSCs)具有许多与哺乳动物上皮祖细胞相同的特征，有望成为上皮干细胞生物学的优秀模型。像果蝇和哺乳动物中的几种其他类型的上皮干细胞一样，FSCs稳定地维持在组织中，但似乎不受固定的、不分裂的利基细胞群的支持。相反，假定的壁龛细胞在成年期分裂，随着周围生殖细胞囊在卵子发生过程中移动，壁龛的位置会发生变化。此外，FSC的子代经常在壁龛之间迁移，争夺壁龛的占有率，这一过程可能与正常的上皮内稳态和癌症的早期阶段都有广泛的相关性。然而，推测的生态位细胞的身份和调节干细胞维持和生态位竞争的机制尚不清楚。基于已发表的研究和重要的初步数据，该提议的中心假设是细胞黏附和细胞极性的差异导致FSC和子细胞的命运分化。具体目标包括：(1)鉴定动态的FSC生态位细胞，确定它们的谱系，并测量它们的翻转率；(2)阐明细胞黏附在FSC维持和FSC生态位竞争中的作用；以及(3)阐明细胞极性在FSC与子代细胞命运的指定中的作用。为了实现第一个目标，将使用细胞类型特异性标记、谱系分析和细胞周期标记来识别可能的利基细胞并测量它们的周转率，结果将通过实时成像得到证实。为了实现第二和第三个目标，将使用免疫荧光来鉴定在FSC生态位中表达的细胞黏附和细胞极性蛋白，并将使用嵌合体分析来确定它们在FSC维持和竞争中的功能。这一项目意义重大，因为它将为上皮干细胞生态位的研究建立一个新的模型。这与以前的研究不同，以前的研究侧重于建立一个全面的FSC生态位模型，并理解动态生态位、生态位竞争和干细胞特有的极性等新概念。最终，它将为了解体内上皮干细胞调节的保守机制提供基础。 公共卫生相关性：拟议的研究与公共卫生相关，因为它将为理解一种新型的上皮干细胞调控提供基础。上皮干细胞对正常的体内平衡和癌症等疾病的进展都是至关重要的，因此了解它们是如何在其天然的、体内的环境中调节的，对于癌症治疗和基于再生医学的治疗的发展非常重要，并将有助于NIH改善健康的使命。拟议的项目还将为NIH促进基本创造性发现的任务做出重大贡献，通过为研究上皮干细胞生态位提供一个新的模型，并提供机会研究干细胞调节的新机制，包括动态生态位组成和竞争生态位占据。

DESCRIPTION (provided by applicant): This proposal outlines a strategy for defining key components of a novel type of dynamic stem cell niche in the Drosophila ovary and for elucidating the mechanism of competition for niche occupancy. Accomplishing the aims in this proposal will be an important milestone toward the long-term goal of building a detailed model of epithelial stem cell regulation in order to develop regenerative medicine-based therapies and understand how the regulatory process fails in disease states such as cancer. The follicle stem cells (FSCs) that produce follicular epithelia have many features in common with mammalian epithelial progenitors and will likely be an excellent model for epithelial stem cell biology. Like several other types of epithelial stem cells in Drosophila and mammals, FSCs are stably maintained in the tissue but do not appear to be supported by a fixed, non- dividing niche cell population. Instead, putative niche cells divide during adulthood, and the position of the niche changes as surrounding germ cell cysts move through oogenesis. In addition, FSC daughters regularly migrate between niches and compete for niche occupancy in a process that may have broad relevance both to normal epithelial homeostasis and the early stages of cancer. However, the identity of the putative niche cells and the mechanisms that mediate stem cell maintenance and niche competition are unknown. Based on published studies and significant preliminary data, the central hypothesis of the proposal is that differences in cell adhesion and cell polarity cause FSC and daughter cell fates to diverge. Specific aims include: (1) Identifying the dynamic FSC niche cells, determining their lineage, and measuring their rate of turn over; (2) Elucidating the role of cell adhesion in FSC maintenance and FSC niche competition; and (3) Elucidating the role of cell polarity in the specification of FSC versus daughter cell fate. To achieve the first aim, cell-type-specific markers, lineage analysis, and cell cycle markers will be used to identify putative niche cells and measure their rate of turnover, and results will be confirmed with live imaging. To achieve the second and third aims, immunofluorescence will be used to identify cell adhesion and cell polarity proteins that are expressed in the FSC niche, and mosaic analysis will be used to determine their function in FSC maintenance and competition. This project is significant because it will establish a new model for the study of epithelial stem cell niches. It is an innovative departure from previous studies that focuses on building a comprehensive model of the FSC niche and on understanding the novel concepts of the dynamic niche, niche competition and stem cell-specific polarity. Ultimately, it will provide a foundation for understanding conserved mechanisms of in vivo epithelial stem cell regulation. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because it will provide a foundation for understanding a novel type of regulation of epithelial stem cells. Epithelial stem cells are central to both normal homeostasis and the progression of diseases such as cancer, so understanding how they are regulated their native, in vivo contexts is important for the development of cancer treatments and regenerative medicine-based therapies and will contribute to the NIH mission to improve health. The proposed project will also contribute significantly to the NIH mission to foster fundamental creative discoveries by providing a new model for the study of epithelial stem cell niches and the opportunity to investigate novel mechanisms for stem cell regulation including a dynamic niche composition and competition for niche occupancy.