Understanding how innexins direct stem cell niche morphology

背景：干细胞需要小生境的支持，小生境提供调控和 内务职能。在C.在线虫性腺中，干细胞龛是远端尖端细胞（DTC）。 生殖系干细胞通过DTC的长细胞过程诱导其自身包裹。 当性腺基底膜破裂时，生殖细胞会逃逸并溢出到体内 腔逃逸的生殖细胞诱导体壁肌肉包裹形成异位小生境。 广泛、长期的目标：该系统非常适合研究不适合的利基属性。 很好地理解了从干细胞到利基的相互信号传导。初步结果显示 连接蛋白是DTC过程形成和异位包裹所必需的， 肌肉.这个提议的目的是发现连接蛋白和其他基因如何介导这一过程。 内源性和异位小生境的相互作用。 具体目的：本研究的目的1是确定连接蛋白在正常生殖细胞中的确切作用。 细胞-小生境相互作用目的2将确定它们在异位龛中的作用。目标3是RNAi 筛选分化型甲状腺癌过程所需的基因，这些基因也在异位妊娠中起作用。 包装 方法：Innexin基因将使用CRISPR/ Cas9进行内源性标记和突变 基因组编辑与用于相关细胞类型的传统转基因报告基因相结合， 这些基因组编辑的蠕虫将允许在活体动物中进行高分辨率共聚焦显微镜检查， 看看生殖细胞和生态位之间的相互作用。RNAi敲除基因的精确 发育时间和现有突变体将用于分析基因功能。C.优雅的是 非常适合基因筛选，RNAi筛选将根据 建立的协议。 健康相关性：干细胞被其小生境和干细胞之间的间隙连接包裹 细胞和它们的小生境都是广泛分布的，但以前不知道它们是相关的。拟议 研究将确定干细胞中缝隙连接功能的机制， 包裹的DTC和异位龛，与更好地了解干细胞的影响- 在许多情况下，包括再生疗法和癌症转移的利基相互作用。 已知间隙连接在癌症中被错误调节。异位肌肉龛构成了一个 癌症期间由癌症干细胞接种的继发性肿瘤形成的新模型 转移

Background: Stem cells require support from a niche, which provides regulatory and housekeeping functions. In the C. elegans gonad, the stem cell niche is the distal tip cell (DTC). The germline stem cells induce their own enwrapment by long cellular processes of the DTC. When the gonad basement membrane ruptures, germ cells escape and spill into the body cavity. Escaped germ cells induce enwrapment by body wall muscle to form an ectopic niche. Broad, long-term objective: This system is ideal for studying a property of the niche that is not well understood—reciprocal signaling from stem cells to the niche. Preliminary results show that innexin proteins are necessary for DTC processes to form, and for ectopic enwrapment by muscle. The objective of this proposal is to discover how innexins and other genes mediate this interaction in endogenous and ectopic niches. Specific aims: Aim 1 of the study is to determine the precise role of innexins in normal germ cell-niche interactions. Aim 2 will determine their role in the ectopic niche. Aim 3 is an RNAi screen for genes necessary for the elaboration of DTC processes that also function in ectopic enwrapment. Method: Innexin genes will be endogenously tagged and mutated using CRISPR/ Cas9 genome-editing. Combined with traditional transgenic reporters for the relevant cells types, these genome-edited worms will allow for high resolution confocal microscopy in live animals to see the interactions between germ cells and the niche. RNAi knockdown of genes at precise developmental times and existing mutants will be used to analyze gene function. C. elegans are perfectly suited to genetic screens, and the RNAi screen will be performed according to established protocols. Health-relatedness: Enwrapment of stem cells by their niches and gap junctions between stem cells and their niches are both widespread but not previously known to be related. The proposed research will determine the mechanism by which gap junctions function in stem cells to induce enwrapment by the DTC and ectopic niche, with implications for better understanding stem cell- niche interactions in many contexts, including regenerative therapies and cancer metastasis. Gap junctions are known to be misregulated in cancer. The ectopic muscle niche constitutes a new model for the formation of secondary tumors seeded by cancer stem cells during cancer metastasis.