Branching Morphogenesis of Urinary Epithelia: from Genes to Cellular Behaviors

泌尿上皮细胞的分支形态发生：从基因到细胞行为

• 批准号: 9245690
• 负责人: FRANKLIN D COSTANTINI
• 金额: $ 50.92万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9245690
• 关键词: Adhesions; Affect; Algorithms; Artificial Kidney; Artificial Organs; Behavior; Behavior Control; Bladder; Candidate Disease Gene; Cell Differentiation process; Cell Nucleus; Cells; Chemicals; Chimera organism; Complex; Congenital Abnormality; Daughter; Defect; Development; Duct (organ) structure; Epithelial; Epithelial Cells; Epithelium; Event; Four-dimensional; GDNF gene; Genes; Genetic; Genetic study; Growth; Human; Hypertension; Image; Imaging technology; Individual; Investigation; Kidney; Kidney Diseases; Label; Lead; Life; Ligands; Link; LoxP-flanked allele; Maps; Metanephric Diverticulum; Methods; Microscopy; Mitosis; Morphogenesis; Mosaicism; Movement; Mus; Mutation; Nature; Nephrons; Nuclear; Organ; Pathway interactions; Pattern; Play; Population; Positioning Attribute; Process; Property; Proteins; Regulation; Research; Resolution; Role; Shapes; Signal Pathway; Signal Transduction; Sister; Site; System; Technology; Testing; Time; Transgenic Mice; Urinary tract; Urine; Urologic Diseases; Work; blood filter; cell behavior; cell dimension; cell motility; cell type; daughter cell; driving force; fetal; gain of function; genetic analysis; genetic manipulation; genetic technology; high resolution imaging; imaging study; in vivo; inhibitor/antagonist; insight; kidney cell; mutant; nephrogenesis; organ growth; prevent; proto-oncogene protein c-ret; public health relevance; receptor; renal agenesis; repaired; response; tool; urinary; urinary tract obstruction; virtual

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.

描述（由申请人提供）：输尿管芽（UB）生成肾脏收集管系统的分支形态发生是形成正常尿路和肾脏的关键过程。此过程中的异常会导致先天缺陷，例如肾脏性发育不全或肾上腺增生，肾单位较低或先天性阻塞性尿症。肾小管低的人数可能会促进肾脏疾病的进展和高血压的发展。因此，对UB分支的基本遗传控制和细胞事件的更好理解可能会导致新的策略，以防止这种出生缺陷，修复肾脏损害或生长人造肾脏。尽管已经确定了正常UB分支形态发生所需的许多基因，但最终，由这些基因控制的特定细胞行为正确地导致了上皮生长和分支。通常，这些细胞行为是很熟悉的。该项目的重点是通过RET受体的GDNF信号传导的作用，RET受体这一信号事件对于小鼠和人类的肾脏发育至关重要。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。我们试图使用各种最先进的遗传和成像技术来描述这些细胞运动，它们的控制及其对肾脏发育的重要性。 我们建议使用几种遗传学方法将具有荧光蛋白的个体，野生型输尿管芽细胞标记，或者产生 在发育中的肾脏中标记了突变UB细胞的克隆。然后，我们通过高分辨率，四维（4D）延时成像在培养过程中遵循其行为。在AIM 1中，我们研究了一种与有丝分裂相关的新型细胞运动的重要性，这发生在 UB尖端上皮。我们询问这些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，以及它们是否因几个候选基因的突变而破坏。在AIM 2中，我们在分支UB尖端中对每个核进行全局4D跟踪，从而分析了未经表述的细节，细胞在正常情况下以及异常的UB分支中发生的各种细胞运动。我们还研究了在高分辨率成像研究中观察到的UB尖端细胞中广泛的突出活性，并且 检查其在上皮细胞运动中的作用。在AIM 3中，我们使用几种强大的克隆遗传分析方法继续研究RET信号传导以及在RET的上游或下游作用的几种基因的活性影响UB分支过程中的细胞运动。总体而言，拟议的研究应通过更深入地阐明GDNF/RET信号在UB细胞行为中的作用来推进该领域。通过提供UB上皮中细胞运动的透彻图片，其调节及其对正常和异常分支形态发生的贡献；通过提供新的工具，方法和范例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。