GDNF/Ret Regulation of Ureteric Bud Morphogenesis

描述（由申请人提供）：本提案的目标是更深入地了解参与输尿管芽（UB）形成和分支的细胞行为。原发性肾盂放置不当可导致膀胱连接失败或输尿管复制，而肾盂形成异常可导致一系列肾脏缺陷，包括肾发育不全、发育不全或肾单位数量减少。大多数这些缺陷导致慢性肾脏疾病，并且认为减少的肾单位数量可能是肾脏疾病和高血压易感性的基础。鉴于UB发育缺陷对人类健康的影响，更深入地了解控制UB正常发育的细胞机制是必不可少的，因为它将为治疗由肾脏异常发育引起的肾脏疾病提供背景。遗传学研究表明，神经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，但驱动肾脏形态发生的细胞行为尚不清楚。UB最初是由基于Ret信号水平的细胞重排形成的。具有较高Ret水平的Wolffian导管（WD）细胞在UB尖端出现时占据了UB尖端，而低水平的细胞则被排除在尖端之外。这些发现提出了我们计划解决的两个重要问题：目标1)什么位置线索引导表达ret的WD细胞重排形成UB尖端？在随后的分支阶段，由Ret信号驱动的细胞重排是否在UB中继续发挥重要作用？目的1旨在确定GDNF/Ret信号在指定WD细胞在原发性UB形成部位的合并中所起的作用。我们在培养的Wolffian导管的单个细胞中进行了可诱导的功能获得研究，以监测GDNF解耦和Ret下游信号传导对细胞行为的影响。为了实现这一目标，我们表达了不依赖配体的Ret形式，在缺乏GDNF的情况下，它在WD细胞的一个子集中维持Ret信号传导。此外，我们与Ret共同表达了一种红色荧光蛋白（RFP），这使我们能够使用延时显微镜跟踪这些操作细胞的行为，并验证GDNF引导WD细胞在其适当位置形成初始UB的假设。目的2验证了Ret信号在肾脏发育过程中继续促进细胞运动的假设，在分支形态发生过程中，只有表达Ret的细胞停留在UB尖端。我们使用诱导遗传学在UB细胞的一个亚群中表达高水平的野生型Ret，或一种与配体无关的Ret形式，专门针对UB尖端结构域。然后，我们使用延时显微镜跟踪这些rfp阳性的操纵细胞在器官培养中的行为，并询问这些被迫维持Ret表达的细胞是留在尖端，还是随机分散在UB尖端和主干区域之间。这些研究将为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.