Branching Morphogenesis of Urinary Epithelia: from Genes to Cellular Behaviors

描述(申请人提供)：输尿管芽(UB)的分支形态发生以产生肾集合管系统是形成正常的尿路和肾脏的关键过程。这一过程中的异常会导致出生缺陷，如肾发育不全或发育不良、肾单位数量减少或先天性梗阻性尿路疾病。低肾单位数可能促进肾脏疾病的进展和高血压的发展。因此，更好地了解UB分支背后的遗传控制和细胞事件可能会导致新的策略，以防止此类出生缺陷，修复肾脏损伤，或培育人工肾脏。虽然许多正常的UB分支形态发生所需的基因已经被鉴定，但最终是这些基因控制的特定细胞行为导致了正确的模式上皮生长和分支。一般说来，人们对这些细胞行为知之甚少。该项目的重点是通过Ret受体传递GDNF信号的作用，Ret受体是一种对小鼠和人类肾脏发育至关重要的信号事件。我们最近的研究表明，分枝的UB上皮经历了广泛的细胞重排，其中许多重排是由Ret控制的。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，使用各种最先进的基因和成像技术。我们建议使用几种遗传方法来用荧光蛋白标记单个的、野生型输尿管芽细胞，或者产生 发育中肾脏中突变的UB细胞的标记克隆。然后，我们通过高分辨率的四维(4D)时间推移成像，在培养的肾脏发育过程中跟踪它们的行为。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性的重要性，这种细胞运动性发生在 UB尖端上皮。我们问这些与有丝分裂相关的细胞运动在方向上是否是非随机的，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干扰。在目标2中，我们对UB分支尖端的每个细胞核进行全局4D跟踪，从而以前所未有的细节分析在UB分支正常和异常期间发生的细胞运动的变化。我们还研究了在高分辨率成像研究中观察到的UB尖端细胞的广泛突起活动，以及 检测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我们的研究，即Ret信号以及作用于Ret上游或下游的几个基因的活性如何影响UB分支过程中的细胞运动。总体而言，这项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上皮中细胞运动的全景图，它的调节，以及它对正常和异常分支形态发生的作用，并通过提供新的工具、方法和范式来研究其他基因和信号通路如何影响体内肾上皮细胞的行为，从而推动这一领域的发展。

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.