Understanding how innexins direct stem cell niche morphology

了解内联蛋白如何指导干细胞生态位形态

• 批准号: 9470269
• 负责人: Kacy Lynn Gordon
• 金额: $ 0.24万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-01 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9470269
• 关键词: Adhesions; Adhesives; Animals; Basement membrane; Body cavities; Body part; CRISPR/Cas technology; Caenorhabditis elegans; Cell Shape; Cell membrane; Cell physiology; Cells; Cellular Structures; Cellular biology; Communication; Confocal Microscopy; Development; Disease Progression; Distal; Embryo; Fellowship; Gap Junctions; Genes; Genetic Screening; Germ; Germ Cells; Goals; Gonadal structure; Health; Housekeeping; Human; Human Development; Intestines; Life; Malignant Neoplasms; Mediating; Membrane; Methods; Microscopic; Modeling; Morphology; Muscle; Mutate; Neoplasm Metastasis; Organism; Outcome; Pathway interactions; Pharyngeal structure; Play; Process; Property; Proteins; Protocols documentation; RNA Interference; RNA interference screen; Recovery; Reporter; Research; Resolution; Role; Rupture; Seeds; Signal Transduction; Site; Stem cells; Structure; System; Testing; Time; Tissues; Transgenic Organisms; body cavity; cancer seeding; cancer stem cell; cell type; experimental study; follow-up; gene function; genome editing; germline stem cells; knock-down; mutant; regenerative therapy; small molecule; stem cell niche; trafficking; tumor

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.

背景：干细胞需要来自一个利基市场的支持，后者提供监管和 内务管理功能。在线虫性腺中，干细胞生态位是远端顶端细胞(DTC)。 生殖系干细胞通过DTC的长细胞过程来诱导自己的包裹。 当性腺基底膜破裂时，生殖细胞逃逸并溢出到体内。 空洞。逃逸的生殖细胞被体壁肌肉包裹，形成异位生态位。 广泛的、长期的目标：这个系统是研究不是利基的属性的理想选择 很好理解-从干细胞到利基细胞的互惠信号。初步结果显示， 内联蛋白是DTC过程形成和异位包裹所必需的。 肌肉。这项提议的目标是发现内联蛋白和其他基因是如何调节这一过程的。 内生和异位生态位中的相互作用。 具体目标：该研究的目标1是确定内联蛋白在正常胚胎中的确切作用 细胞-生态位相互作用。目标2将确定它们在异位利基中的角色。AIM 3是RNAi 筛选在异位中也起作用的DTC过程的精化所必需的基因 包裹物。 方法：使用CRISPR/Cas9内源性标记和突变Innexin基因 基因组编辑。结合传统的针对相关细胞类型的转基因记者， 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 看看生殖细胞和壁龛之间的相互作用。RNAi精确敲除基因 将利用发育时间和现有的突变体来分析基因功能。线虫是 非常适合基因筛查，RNAi筛查将根据 已制定的协议。 与健康相关：干细胞的壁龛和干细胞间的缝隙连接包裹干细胞 细胞和它们的生态位都很普遍，但之前并不知道它们之间有关联。建议数 研究将确定干细胞中缝隙连接发挥作用的机制 DTC和异位生态位的包裹，对更好地理解干细胞- 在许多情况下的利基相互作用，包括再生治疗和癌症转移。 众所周知，缝隙连接在癌症中受到错误的调控。异位肌龛构成了一个 肿瘤干细胞在癌变过程中形成继发性肿瘤的新模型 转移。