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

摘要 生殖细胞能够自我更新、分化，并确保将基因组传给下一代。胚茎 细胞依赖于小生境，这是指导干细胞自我更新的专门微环境。利基市场 伸展是一种现象，在这种现象中，壁龛细胞在茎周围延伸出精心设计的细胞突起 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 仍然不为人所知。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、薄薄的细胞外基质，在结构上支持利基，并可以调节 干性和细胞增殖。IV型胶原是基底膜的主要成分。发现 结构域受体(DDR)是受体酪氨酸激酶的一个子集，它被胶原特异性地激活， 并且通常通过丝裂原活化蛋白激酶(MAPK)级联传递信号。IV型胶原调节失调 哺乳动物DDR的突变都与不孕症有关，然而，它们在促进 繁殖力是未知的。秀丽线虫(C.elegans)种系生态位只由一个 细胞，远端尖端细胞(DTC)，它延伸包裹和维持生殖细胞的长过程，通过 激活Notch信号，从而维持生殖细胞和生育能力。遗传学和视觉实验 线虫的易操纵性允许严格识别生态位扩展的介体及其在 生育能力。通过表达和DTC特异的RNAi筛选，我发现线虫的Discoidin结构域 受体DDR-2促进沿着DTC巢突形成IV型胶原聚集体/斑点。在……里面 缺乏DDR-2，IV型胶原在DTC巢和巢突延展内积累 大大减少了。基于强有力的初步数据，我的中心假设是DDR-2和类型 IV型胶原蛋白在正反馈环中发挥作用，直接分泌IV型胶原蛋白锚定 它推动了利基的扩展，扩大了生殖细胞干细胞库。在目标1中，我将确定 DDR-2和IV型胶原如何在DTC中相互作用以驱动生态位延伸和生殖系维持 通过作用部位研究、基因分析、囊泡运输的活细胞成像和生育分析。在……里面 目的2利用遗传上位性分析确定DDR-2在DTC延伸中的下游效应 以及细胞生物学研究。通过大规模RNAi筛查的初步发现，我将重点介绍 MAPK/细胞外信号调节蛋白激酶(ERK)信号和囊泡运输调节因子。最终我 期待我的研究将确立盘状结构域受体功能在生殖系生态位形成中的新作用 和生育，这将有助于我们理解和治疗人类的生育障碍。 好了！

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. !