Understanding the role of the collagen receptor DDR-2 in germ stem cell niche formation

了解胶原蛋白受体 DDR-2 在生殖干细胞生态位形成中的作用

• 批准号: 10087948
• 负责人: Sara Grace Payne
• 金额: $ 3.81万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10087948
• 关键词: Basement membrane; Behavior; Biochemical; Biological; Biological Assay; Biophysics; C. elegans genome; Caenorhabditis elegans; Cell Proliferation; Cell membrane; Cell physiology; Cells; Collagen; Collagen Receptors; Collagen Type IV; DDR2 gene; Data; Defect; Distal; Environment; Extracellular Matrix; Extracellular Signal Regulated Kinases; Feedback; Fertility; Fertility Disorders; Gene Family; Genes; Genetic; Genetic Epistasis; Genome; Germ Cells; Germ Lines; Goals; Human; Impairment; Infertility; Link; Maintenance; Mediating; Mediator of activation protein; Membrane Biology; Mitogen-Activated Protein Kinases; Modeling; Molecular; Mus; Mutation; Organism; Pathology; Pathway interactions; Physiology; Process; Production; Property; Proteins; RNA Interference; RNA interference screen; Receptor Protein-Tyrosine Kinases; Regulation; Reproduction; Role; Secure; Signal Transduction; Site; Structure; Thinness; Tissues; Vertebrates; Visual; base; cell behavior; crosslink; discoidin domain receptor 2; discoidin receptor; effective therapy; fluorophore; genetic analysis; genome editing; germline stem cells; infertility treatment; insight; knock-down; live cell imaging; next generation; notch protein; receptor; receptor function; self renewing cell; self-renewal; sex; stem cell niche; stem cell proliferation; stem cell self renewal; stem cells; stemness; trafficking; transmission process

Abstract Germ stem cells self-renew, differentiate, and secure genome transmission to the next generation. Germ stem cells depend on niches, which are specialized microenvironments that instruct stem cell self-renewal. Niche extension is a phenomenon in which the niche cells extend elaborate cellular protrusions that surround stem cells. While niche extension is conserved across organisms, the function and regulators of niche extension remain unknown. An essential component of niches is the basement membrane. Basement membranes are secreted, cell-associated, thin sheets of extracellular matrix that structurally support niches, and can regulate stemness and cell proliferation. Type IV collagen is a major component of basement membranes. Discoidin domain receptors (DDRs), are a subset of receptor tyrosine kinases that are specifically activated by collagen, and often signal through mitogen-activated protein kinase (MAPK) cascades. Misregulation of Type IV collagen and mutations in mammalian DDRs have both been linked to infertility, however, their role in promoting fecundity is unknown. The Caenorhabditis elegans (C. elegans) germline niche is composed of only a single cell, the distal tip cell (DTC), which extends long processes that enwrap and maintain germ stem cells by activating Notch signaling, thus sustaining germ stem cells and fertility. The genetic and visual experimental tractability of C. elegans allows for rigorous identification of mediators of niche extension and their role in fertility. Through an expression and DTC-specific RNAi screen, I found that the C. elegans discoidin domain receptor DDR-2 promotes the formation of type IV collagen aggregates/puncta along DTC niche processes. In the absence of DDR-2, type IV collagen accumulates internally in the DTC niche and niche process extension is dramatically reduced. Based on strong preliminary data, my central hypothesis is that DDR-2 and type IV collagen function in a positive feedback loop to direct the secretion of type IV collagen anchoring puncta, which drives niche extension and expands the germ stem cell pool. In Aim 1, I will determine how DDR-2 and type IV collagen interact in the DTC to drive niche extension and germline maintenance through site of action studies, genetic analysis, live cell imaging of vesicular trafficking, and fertility assays. In Aim 2, I will determine the downstream effectors of DDR-2 in DTC extension using genetic epistasis analysis and cell biological studies. Through preliminary findings of a large scale RNAi screen, I will focus on the role of MAPK/ extracellular signal-regulated kinases (ERK) signaling and vesicular trafficking regulators. Ultimately I expect my studies will establish a new role for discoidin domain receptor function in germ line niche formation and fertility, which will help in our understanding and treatment of fertility disorders in humans. !

摘要 生殖干细胞自我更新，分化，并确保基因组传递到下一代。生殖干 细胞依赖于小生境，小生境是指导干细胞自我更新的专门微环境。利基 延伸是一种现象，其中小生境细胞延伸围绕茎的精细细胞突起， 细胞虽然生态位扩展在生物体中是保守的，但生态位扩展的功能和调节因子 仍然未知。壁龛的一个重要组成部分是基底膜。基底膜是 分泌的，细胞相关的，细胞外基质的薄片，结构上支持小生境，并可以调节 干细胞和细胞增殖。IV型胶原是基底膜的主要成分。盘状 结构域受体（DDRs）是由胶原特异性激活的受体酪氨酸激酶的子集， 并且通常通过丝裂原活化蛋白激酶（MAPK）级联信号传导。IV型胶原的失调 哺乳动物DDRs和突变都与不育有关，然而，它们在促进生育方面的作用， 生殖力未知。秀丽隐杆线虫（C. habditiselegans，C.线虫）生殖系生态位仅由单一的 细胞，远侧尖端细胞（DTC），它延伸长的过程，包裹和维持生殖干细胞， 激活Notch信号，从而维持生殖干细胞和生育能力。遗传和视觉实验 C.易处理性elegans允许严格鉴定生态位扩展的介质及其在 生育通过表达和DTC特异性RNAi筛选，我发现C。秀丽隐杆线虫盘状结构域 受体DDR-2促进沿着DTC龛过程的IV型胶原聚集体/斑点的形成。在 缺乏DDR-2，IV型胶原在DTC龛和龛突延伸部内部积累 会大幅减少基于强有力的初步数据，我的中心假设是DDR-2和类型 IV型胶原在正反馈回路中起作用，以指导IV型胶原锚定的分泌 puncta，它驱动生态位延伸并扩大生殖干细胞库。在目标1中，我将确定 DDR-2和IV型胶原蛋白如何在DTC中相互作用以驱动生态位扩展和种系维持 通过作用部位研究、遗传分析、囊泡运输的活细胞成像和生育力测定。在 目的二，利用遗传上位性分析方法，确定DDR-2在DTC延伸中的下游效应子 和细胞生物学研究。通过大规模RNAi筛选的初步发现，我将重点关注 MAPK/细胞外信号调节激酶（ERK）信号传导和囊泡运输调节剂。最终我 我希望我的研究能为盘状结构域受体在生殖系生态位形成中的作用建立一个新的机制 这将有助于我们理解和治疗人类的生育障碍。 !