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）的分支形态发生产生肾集合管系统是正常尿路和肾脏形成的关键过程。这一过程的异常会导致出生缺陷，如肾发育不全或发育不良、肾单位数量少或先天性梗阻性尿路病。肾单位数量低可能会促进肾脏疾病的进展和高血压的发生。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产生新的策略来预防此类出生缺陷、修复肾损伤或生长人工肾。虽然正常UB分支形态发生所需的许多基因已被鉴定，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。一般来说，人们对这些细胞行为知之甚少。该项目重点研究 GDNF 信号通过 Ret 受体的作用，这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，分支 UB 上皮经历广泛的细胞重排，其中许多是由 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。我们试图利用各种最先进的遗传和成像技术来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。 我们建议使用几种遗传方法用荧光蛋白标记个体野生型输尿管芽细胞，或生成 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培养物中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相关的新型细胞运动的重要性，有丝分裂发生在 UB尖端上皮。我们询问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全局 4D 跟踪，从而以前所未有的细节分析正常和异常 UB 分支期间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 尖端细胞的广泛突出活动，以及 检查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 信号传导以及作用于 Ret 上游或下游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 细胞行为中的作用来推进该领域的发展；通过全面了解 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；并为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提供新的工具、方法和范例。