GDNF/Ret Regulation of Ureteric Bud Morphogenesis

描述(由申请人提供)：这项建议的目的是更深入地了解输尿管芽(UB)的形成和分支所涉及的细胞行为。原发UB的放置不当可能导致与膀胱或重复输尿管的连接失败，而UB形成的异常可能导致一系列肾缺陷，包括肾发育不全、发育不良或肾单位数量减少。这些缺陷中的大多数会导致慢性肾脏疾病，人们认为肾单位数量减少可能导致肾脏疾病和高血压的易感性。鉴于UB发育缺陷对人类健康的影响，深入了解控制UB正常发育的细胞机制是至关重要的，因为这将为治疗由肾脏发育异常引起的肾脏疾病提供背景。遗传学研究表明，胶质细胞源性神经营养因子(GDNF)及其受体Ret是肾脏发育所必需的，但对驱动肾脏形态发生的细胞行为却知之甚少。UB最初是通过基于Ret信号水平的细胞重排形成的。Ret水平较高的Wolffian Date(WD)细胞在UB末端出现时占据UB末端，而较低水平的细胞则被排除在末端之外。这些发现提出了我们计划解决的两个重要问题：目的1)什么位置线索引导表达Ret的WD细胞重排形成UB尖端？目的2)，在随后的分支阶段，由Ret信号驱动的细胞重排是否继续在UB中发挥重要作用？目的1试图确定GDNF/Ret信号在规定WD细胞在原发UB形成部位的结合中所起的作用。我们结合了在培养的Wolffian导管的单个细胞中进行的可诱导的功能增益研究，以监测去偶联GDNF和Ret下游信号对细胞行为的影响。为了实现这一点，我们表达了不依赖配体的Ret形式，在没有GDNF的情况下，Ret在WD细胞的一个子集中维持Ret信号。此外，我们共表达了一个红色荧光蛋白(RFP)和Ret，这使我们能够使用时间推移显微镜跟踪这些操纵细胞的行为，并验证GDNF引导WD细胞在其正确位置形成初始UB的假设。目的2验证Ret信号在肾脏发育过程中继续促进细胞运动的假说，在分枝形态发生过程中只允许表达Ret的细胞停留在Ub顶端。我们使用可诱导遗传学在UB细胞的子集中表达高水平的野生型Ret，或一种配体无关的Ret形式，专门针对UB TIP结构域。然后，我们使用延时显微镜跟踪这些RFP阳性的受操纵细胞在器官培养中的行为，并询问这些被迫保持Ret表达的细胞是停留在顶端，还是随机分散在UB顶端和主干区域之间。这些研究应该为Ret信号在UB形态发生中的作用提供新的见解，从而为人类RET相关肾缺陷的病因学提供新的见解。

DESCRIPTION (provided by applicant): The goal of this proposal is to obtain a deeper understanding of the cellular behaviors involved in the formation and branching of the ureteric bud (UB). Improper placement of the primary UB can result in a failed connection to the bladder or duplicated ureters, while abnormalities in UB formation can result in a spectrum of kidney defects including renal agenesis, hypoplasia or reduced nephron numbers. Most of these defects lead to chronic renal disease and it is thought that reduced nephron numbers can underlie the susceptibility to renal diseases and hypertension. In light of the impact that defective UB development has on human health, a deeper understanding of the cellular mechanisms that control normal UB development is essential because it will provide a context for treating the renal diseases that result from abnormal kidney development. Genetic studies show that Glial cell-derived neurotrophic factor (Gdnf) and its receptor, Ret, are required for renal development, but the cellular behaviors that drive renal morphogenesis are poorly understood. The UB initially forms by cell rearrangements based on levels of Ret signaling. Wolffian duct (WD) cells with higher levels of Ret occupy the UB tip as it emerges from the WD, while cells with lower levels are excluded from the tip. These findings raise two important questions that we plan to address: Aim 1) what positional cues guide the rearrangement of Ret-expressing WD cells to form the UB tip? Aim 2), do cell rearrangements driven by Ret signaling continue to play an important role in the UB during the subsequent branching stages? Aim 1 seeks to define the roles that GDNF/Ret signaling play in specifying the coalescence of WD cells at the site of primary UB formation. We combine inducible, gain-of-function studies in single cells of cultured Wolffian ducts, to monitor the effects on cell behavior that arise from uncoupling GDNF and the signaling downstream of Ret. To achieve this, we express ligand-independent forms of Ret, which maintain Ret signaling in a subset of WD cells in the absence of GDNF. In addition, we co-express a red fluorescent protein (RFP), along with Ret, which allows us to follow the behavior of these manipulated cells using time-lapse microscopy, and to test the hypothesis that GDNF guides WD cells to form the initial UB at its proper location. Aim 2 tests the hypothesis that Ret signaling continues to promote cell movements during kidney develop- ment, allowing only Ret-expressing cells to stay at the UB tips during branching morphogenesis. We use inducible genetics to express elevated levels of wild type Ret, or a ligand-independent form of Ret, in a subset of UB cells, specifically targeting the UB tip domain. We then follow the behavior of these RFP-positive, manipulated cells in organ culture using time-lapse microscopy, and ask if these cells forced to maintain Ret expression stay at the tips, or become randomly dispersed between the UB tip and the trunk domains. These studies should provide new insight into the role of Ret signaling in UB morphogenesis, and thus into the etiology of RET-associated renal defects in humans.