Branching Morphogenesis of Urinary Epithelia: from Genes to Cellular Behaviors

描述（由申请方提供）：输尿管芽（UB）分支形态发生以产生肾集合管系统是形成正常尿路和肾脏的关键过程。这一过程的异常会导致出生缺陷，如肾发育不全或发育不全，肾单位数量减少或先天性梗阻性尿路病。肾单位数目减少可促进肾脏疾病的进展和高血压的发生。因此，更好地了解遗传控制和细胞事件的基础UB分支可能导致新的策略，以防止这种出生缺陷，修复肾损伤，或生长人工肾。虽然已经鉴定了正常UB分支形态发生所需的许多基因，但最终是由这些基因控制的特定细胞行为导致正确模式的上皮生长和分支。这些细胞行为，一般来说，知之甚少。该项目的重点是GDNF通过Ret受体信号传导的作用，这是一种对小鼠和人类肾脏发育至关重要的信号传导事件。我们最近的研究表明，分支UB上皮经历了广泛的细胞重排，其中许多是由Ret控制。我们假设这些细胞重排是驱动正常UB分支的主要力量。我们试图描述这些细胞运动的性质，它们的控制，以及它们对肾脏发育的重要性，使用各种最先进的遗传和成像技术。 我们建议使用几种遗传学方法，用荧光蛋白标记单个野生型输尿管芽细胞，或产生 发育中肾脏中突变UB细胞的标记克隆。然后，我们通过高分辨率，4维（4D）延时成像来跟踪它们在肾脏发育过程中的行为。在目标1中，我们研究了一种与有丝分裂有关的新型细胞运动的重要性，这种运动发生在 UB尖端上皮。我们想知道这些与有丝分裂相关的细胞运动是否在方向上是非随机的，它们是否影响子细胞的发育命运，以及它们是否被几个候选基因的突变所破坏。在目标2中，我们对分支UB尖端中的每个细胞核进行了全局4D跟踪，从而以前所未有的细节分析了正常和异常UB分支期间发生的细胞运动的多样性。我们还研究了在高分辨率成像研究中观察到的UB尖端细胞中广泛的增殖活性， 研究其在上皮细胞运动中的作用。在目标3中，我们使用几种强大的克隆遗传分析方法来继续研究Ret信号传导以及Ret上游或下游作用的几个基因的活性如何影响UB分支期间的细胞运动。总体而言，拟议的研究应该通过更深入地阐明GDNF/Ret信号在UB细胞行为中的作用，通过提供UB上皮细胞运动、其调节及其对正常和异常分支形态发生的贡献的全面图片，通过提供新的工具，研究其他基因和信号通路如何影响体内肾上皮细胞行为的方法和范例。

DESCRIPTION (provided by applicant): Branching morphogenesis of the ureteric bud (UB) to generate the renal collecting duct system is a critical process for the formation of a normal urinary tract and kidney. Abnormalities in this process cause birth defects such as renal agenesis or hypodysplasia, low nephron number, or congenital obstructive uropathies. Low nephron number may promote the progression of renal diseases and the development of hypertension. Thus, a better understanding of the genetic controls and cellular events underlying UB branching could lead to new strategies to prevent such birth defects, repair renal damage, or grow artificial kidneys. While many of the genes required for normal UB branching morphogenesis have been identified, ultimately it is the specific cell behaviors controlled by these genes that cause correctly patterned epithelial growth and branching. These cellular behaviors are, in general, poorly understood. This project focuses on the role of GDNF signaling through the Ret receptor, a signaling event that is critical for kidney development in mice and humans. Our recent studies show that the branching UB epithelium undergoes extensive cell rearrangements, many of which are controlled by Ret. We hypothesize that these cell rearrangements are a major force driving normal UB branching. We seek to describe the nature of these cell movements, their control, and their importance for kidney development, using a variety of state-of-the art genetic and imaging technologies. We propose to use several genetic methods to label individual, wild-type ureteric bud cells with fluorescent proteins, or to generate labeled clones of mutant UB cells in developing kidneys. We then follow their behaviors during renal development in culture, via high-resolution, 4-dimensional (4D) time-lapse imaging. In Aim 1, we investigate the importance of a new type of cell motility, linked to mitosis, which occurs in the UB tip epithelium. We ask if these mitosis-associated cell movements are non-random in direction, if they influence the developmental fate of the daughter cells, and if they are disrupte by mutations in several candidate genes. In Aim 2 we perform global 4D tracking of every nucleus in a branching UB tip, thus analyzing in unprece- dented detail the variety of cell movements that occur during normal, as well as abnormal, UB branching. We also investigate the extensive protrusive activity in UB tip cells, observed in high-resolution imaging studies, and examine its role in epithelial cell motility. In Aim 3, we use several powerful methods of clonal genetic analysis to continue our investigation of how Ret signaling, as well as the activity of several genes acting upstream or downstream of Ret, influences cell movements during UB branching. Overall, the proposed research should advance the field by more deeply elucidating the role of GDNF/Ret signaling in UB cell behaviors; by providing a thorough picture of cell motility in the UB epithelium, its regulation, and its contribu- tion to normal and abnormal branching morphogenesis; and by providing new tools, methods and paradigms for studying how other genes and signaling pathways affect the behaviors of renal epithelial cells in vivo.