The Role of the Eyal Gene in Metanephric Induction

描述（申请人提供）：先天性肾病发病率为千分之一，由于肾脏发育不全、发育不良和收集系统缺陷，发病率高。分子遗传学方法揭示了肾脏形成所需的几个基因。其中之一是Eyes absent 1或Eyal，它编码一种含有不同转录激活和蛋白-蛋白相互作用结构域的蛋白质。人EYA 1单倍体不足可导致支气管-耳-肾（BOR）综合征。与患有BOR综合征的个体相似，Eyal杂合小鼠也表现出肾脏发育不全和外显率降低。更引人注目的是，Eyal纯合子小鼠由于输尿管芽生长缺陷而表现出完全渗透的双侧肾发育不全表型。Eyal在后肾间质中表达，而在输尿管芽中不表达。因此，我们假设Eyal调节直接输尿管芽生长的间充质因子。事实上，GDNF（一种能够指导输尿管芽生长的间质因子）的表达在缺钙后肾间质中是检测不到的。在这个应用中，我们寻求解决控制输尿管芽生长的eyal依赖的遗传调控层次。在Aim 1中，我们将使用Eyal基因剂量实验和标记研究来阐明Eyal杂合子和纯合子表型的基础。胚胎肾外植体培养将用于测试GDNF或其他间充质因子是否可以挽救Eyal纯合子输尿管芽的生长。此外，我们将使用其他缺乏Six1、Wtl、c-ret、Gdnf、ld （Fmnl）和Pax2的小鼠肾脏突变体来检测后肾间质和Wolffian导管中基因表达的依赖性。在Aim 2中，我们将通过测试已知的蛋白质，通过进行酵母杂交筛选，以及通过分析肾脏和间充质来源细胞系中与Eyal共免疫沉淀的蛋白质，来确定Eyal在肾脏发育过程中与之相互作用的蛋白质。最后，在Aim 3中，我们将通过验证Eyal复合物可以激活Gdnf启动子或调节其他Eyal下游靶基因表达的假设，寻求建立Eyal在肾形成中的功能的分子模型。

DESCRIPTION (provided by applicant): Congenital kidney disease occurs at a frequency of 1 in 1000 and causes significant morbidity due to renal agenesis, dysplasia and collecting system defects. Molecular genetic approaches have revealed several genes required for kidney formation. One of these is Eyes absent 1 or Eyal, which encodes a protein containing distinct transcriptional activation and protein-protein interaction domains. Haploinsufficiency for human EYA 1 results in Branchio-Oto-Renal (BOR) syndrome. Similar to individuals with BOR syndrome, Eyal heterozygous mice also exhibit renal hypoplasia and agenesis with reduced penetrance. Even more dramatically, Eyal homozygous mice manifest a fully penetrant bilateral renal agenesis phenotype due to defective ureteric bud outgrowth. Eyal is expressed in metanephric mesenchyme but not in ureteric bud. We therefore hypothesize that Eyal regulates mesenchymal factor(s) that direct ureteric bud outgrowth. Indeed, expression of GDNF, one mesenchymal factor capable of directing ureteric bud outgrowth, is undetectable inEyal-deficient metanephric mesenchyme. In this application, we seek to resolve the Eyal-dependent genetic regulatory hierarchy controlling ureteric bud outgrowth. In Aim 1, we will use Eyal gene dosage experiments and marker studies to clarify the basis for the Eyal hetero- and homozygous phenotypes. Embryonic kidney explant cultures will be used to test whether GDNF or other mesenchymal factors can rescue ureteric bud outgrowth in Eyal homozygotes. In addition, we will use other mouse kidney mutants lacking Six1, Wtl, c-ret, Gdnf, ld (Fmnl) and Pax2 to assay dependencies of gene expression in metanephric mesenchyme and Wolffian duct. In Aim 2, we will identify proteins which Eyal interacts with in kidney development by testing known proteins, by conducting a yeast two hybrid screen, and by analyzing proteins that co-immunoprecipitate with Eyal from renal and mesenchymal-derived cell lines. Lastly, in Aim 3, we will seek to develop a molecular model for Eyal function in nephrogenesis by testing the hypothesis that an Eyal complex can activate the Gdnf promoter, or that it can regulate the expression of other Eyal downstream target genes.