Genetic Analysis of Etv4/Etv5 Transcription Factors and Kidney Development

Etv4/Etv5 转录因子与肾脏发育的遗传分析

• 批准号: 8182553
• 负责人: FRANKLIN D COSTANTINI
• 金额: $ 52.61万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-07 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8182553
• 关键词: ADAMTS; Behavior; Binding; Binding Sites; Biological Assay; Bladder; Blood Pressure; Cell physiology; Cells; Child; Chimera organism; Chromatin; Chronic Kidney Failure; Congenital Abnormality; Coupled; DNA Sequence; Data Set; Defect; Development; Duct (organ) structure; Enhancers; Epithelial; Extracellular Matrix Proteins; Family; GDNF gene; Gene Family; Gene Targeting; Genes; Genetic; Genome; Goals; Growth; Human; Hydronephrosis; Hypertension; In Situ Hybridization; In Vitro; Individual; Kidney; Kidney Diseases; Knowledge; Label; Lead; Life; Link; Measures; Metanephric Diverticulum; Methods; Microscopy; Morphogenesis; Mus; Mutant Strains Mice; Nephrons; Obstruction; Organ; Organ Culture Techniques; Organogenesis; Pattern; Peptide Hydrolases; Process; Rattus; Receptor Protein-Tyrosine Kinases; Regulator Genes; Renal Hypertension; Rodent; Role; Shapes; Signal Transduction; Sister; Site; Sorting - Cell Movement; Stream; Structure of mesonephric duct; System; Testing; Time; Transcription factor genes; Ureteral obstruction; Urine; Urologic Diseases; Variant; Vesico-Ureteral Reflux; Viral Vector; Work; blood filter; cell behavior; cell motility; chromatin immunoprecipitation; fetal; gain of function; genetic analysis; in vivo; loss of function; member; migration; mouse model; mutant; nephrogenesis; overexpression; promoter; response; transcription factor; ureter dilatation; urologic

DESCRIPTION (provided by applicant): Our goal is to elucidate the genetic and cellular mechanisms controlling the formation of the ureteric bud (UB) and its growth and branching to give rise to the renal collecting duct (CD) system. Defects in UB forma- tion, growth and branching lead to abnormalities such as renal agenesis, hypoplasia and reduced nephron number, obstruction, hydroureter/hydronephrosis and vesicoureteral reflux, conditions associated with chronic renal disease. Furthermore, the number of nephrons is extremely variable in "normal" humans, and low nephron number is thought to promote the progression of renal diseases and hypertension. One cause of low nephron number is a branching defect that reduces the number of UB tips. Signaling by GDNF through the Ret receptor tyrosine kinase is critical for UB formation and branching. To understand how GDNF and Ret regulate these processes, we identified genes that are upregulated by GDNF. Among these genes were two closely related ETS transcription factors, Etv4 and Etv5, which are themselves jointly required for kidney development. Our central hypothesis is that in response to Ret, Etv4 and Etv5 regulate a set of genes that carry out diverse cellular functions, together promoting branching morphogenesis. Aim 1 is to investigate the specific effects of Etv4/Etv5 expression on the behaviors of UB cells during branching morphogenesis. Specifically, we test the hypothesis that cell migration underlies the patterned epithelial growth that occurs during UB branching morphogenesis, and that Etv4/5 promote this migration. We use genetic, single-cell, gain-of-function and loss-of function methods to manipulate expression of Etv4 and Etv5, coupled with fluorescent cell labeling and time lapse microscopy of renal organ cultures. Etv4 and Etv5 are transcriptional regulators, so to understand their functions in kidney development we need to identify the "target genes" that they regulate. Aim 2 is to define the targets of Etv4 and Etv5 through microarray screens of mutant kidneys (identifying genes requiring Etv4/5 for normal expression), ChIP-Seq to define Etv4/5 binding sites in the genome, and further studies to validate downstream genes. This will elaborate the gene regulatory network that controls UB branching morphogenesis, and will advance our understanding of the causes of urological/renal birth defects in mouse models and potentially humans. Aim 3 examines the potential role in renal development and hypertension of two secreted proteases, Adamts16 and Adamts18, which are likely targets of Etv4/5. While the substrates and in vivo functions of Adamts16 and 18 are unknown, their similar sequences and renal expression suggest they may serve redundant functions in UB branching. Furthermore, Adamts16 has been linked to hypertension in rodents and humans. We hypothesize that the absence of Adamts16 and Adamts18 contributes to the UB branching defects in Etv4/5 mutants. Lack of Adamts16 and/or 18 may cause reduced nephron number and hyper- tension. We test this by examining kidney development, nephron number and blood pressure in mutant mice. PUBLIC HEALTH RELEVANCE: Normal development of the kidney during fetal life is important for the organ to achieve its normal size, number of nephrons (blood-filtering units) and connections to the bladder. Defects in fetal organ development may promote the progression of renal and urological diseases and hypertension. This proposal investigates the genetic mechanisms that control how the kidney grows to the correct size and shape, and how the proper number of nephrons and urine-collecting ducts is formed.

描述（由申请人提供）：我们的目标是阐明控制输尿管芽形成（UB）及其生长和分支的遗传和细胞机制，以引起肾脏收集导管（CD）系统。 UB形成，生长和分支的缺陷导致异常，例如肾脏发育不全，发育不全和肾单位数量减少，阻塞，水力器/肾结通和囊泡分层反流，与慢性肾脏疾病相关的疾病。此外，肾单位的数量在“正常”人类中是极大的，肾单位的数量较低会促进肾脏疾病和高血压的进展。肾单位数低的原因之一是分支缺陷，可减少UB尖端的数量。 GDNF通过RET受体酪氨酸激酶发出的信号对于UB的形成和分支至关重要。为了了解GDNF和RET如何调节这些过程，我们确定了由GDNF上调的基因。在这些基因中，有两个密切相关的ETS转录因子ETV4和ETV5，它们本身是肾脏发育所必需的。我们的中心假设是，在响应RET，ETV4和ETV5时，调节了一组具有多种细胞功能的基因，共同促进了分支形态发生。 目的1是研究ETV4/ETV5表达对分支形态发生过程中UB细胞行为的特定影响。具体而言，我们检验了一个假设，即细胞迁移是UB分支形态发生过程中发生的图案上皮生长的基础，而ETV4/5促进了这种迁移。我们使用遗传，单细胞，功能障碍和功能丧失方法来操纵ETV4和ETV5的表达，并与荧光细胞标记以及肾脏器官培养物的时间失去显微镜结合。 ETV4和ETV5是转录调节剂，因此，要了解它们在肾脏发育中的功能，我们需要确定它们调节的“靶基因”。 Aim 2 is to define the targets of Etv4 and Etv5 through microarray screens of mutant kidneys (identifying genes requiring Etv4/5 for normal expression), ChIP-Seq to define Etv4/5 binding sites in the genome, and further studies to validate downstream genes.这将详细说明控制UB分支形态发生的基因调节网络，并将促进我们对小鼠模型和潜在人类泌尿外科/肾脏出生缺陷的原因的理解。 AIM 3检查了两个分泌蛋白酶ADAMTS16和ADAMTS18在肾脏发育和高血压中的潜在作用，它们可能是ETV4/5的靶标。尽管ADAMTS16和18的底物和体内功能尚不清楚，但它们的相似序列和肾脏表达表明它们可以在UB分支中起冗余功能。此外，Adamts16与啮齿动物和人类的高血压有关。我们假设ADAMTS16和ADAMTS18的缺乏有助于ETV4/5突变体中的UB分支缺陷。缺乏ADAMTS16和/或18可能会导致肾单位数量和超张力减少。我们通过检查突变小鼠的肾脏发育，肾小管数量和血压来测试这一点。 公共卫生相关性：胎儿寿命期间肾脏的正常发育对于器官实现其正常大小，肾单位的数量（过滤单位）和与膀胱的连接很重要。胎儿器官发育中的缺陷可能会促进肾脏和泌尿科疾病和高血压的进展。该提案研究了控制肾脏如何生长到正确的大小和形状的遗传机制，以及如何形成适当数量的肾单位和尿液收集管道。