Roles of Transcription Factors in Kidney Development

描述（由申请人提供）：本项目的目标是阐明EYA1控制哺乳动物肾脏肾元祖细胞发育的机制。在哺乳动物中，肾脏的发育包括后肾间质（MM）的指定，输尿管芽（UB）的生长，以及UB和MM之间的相互作用，以产生大量的肾单位以及成熟肾脏中的肾收集系统。破坏这些过程的基因突变导致肾脏发育不全和发育不全。我们和其他人发现EYA1及其辅助因子SIX家族蛋白是肾脏发育的关键间充质因子。人类这些基因的缺陷会导致支气管-耳-肾（BOR）综合征，这是一种以支气管、耳部和肾脏异常为特征的先天性出生缺陷。为了了解BOR综合征发生肾脏缺陷的基础，我们分析了从患者和产生的敲除小鼠中发现的EYA1和SIX1/5突变。我们的研究结果表明，这些基因相互作用，对MM细胞发育和UB分支至关重要。由于MM在Eya1-/-小鼠中不形成，我们最近产生了Eya1flox和Eya1CreERT2等位基因。我们发现，在UB生长后，Eya1的缺失导致MM祖细胞因过早上皮化而耗尽，并且Eya1直接与SIX2和Myc家族蛋白相互作用，这两个蛋白已知对MM祖细胞的维持很重要。我们的分析表明，EYA1在体外使Myc在Thr58位点去磷酸化，以防止Myc蛋白降解，并且在MM细胞中删除EYA1导致磷酸化- t58 -Myc水平升高，但Myc水平降低。然而，目前我们还不清楚这些关键因素是如何在功能上与调节肾元祖细胞的增殖和维持联系在一起的。在本次更新申请中，我们拟采用分子、生物化学、遗传学和基因组学相结合的方法来验证EYA1、Myc和SIX2相互作用调节肾元祖细胞池扩张的假设，并确定由EYA1及其辅助因子控制的靶基因。这项研究将通过揭示EYA1如何与其辅助因子相互作用以调节肾元祖细胞自我更新的新机制来推进这一领域。

DESCRIPTION (provided by applicant): The goal of this project is to elucidate the mechanisms by which EYA1 controls nephron progenitor cell development in the mammalian kidney. In mammals, kidney development involves the specification of the metanephric mesenchyme (MM), the outgrowth of the ureteric bud (UB), and reciprocal interactions between the UB and the MM to generate large numbers of nephrons as well as the renal collecting system in the mature kidney. Mutations in genes that disrupt these processes cause renal agenesis and hypoplasia. We and others identified EYA1 and its cofactor SIX family proteins as critical mesenchymal factors for kidney development. Defects in these genes in humans cause Branchio-Oto-Renal (BOR) syndrome, a congenital birth defect characterized by a combination of branchial, otic and renal anomalies. To understand the basis of kidney defects that occur in BOR syndrome, we analyzed EYA1 and SIX1/5 mutations identified from patients and generated knockout mice. Our results show that these genes interact and are essential for MM cell development and UB branching. Since the MM is not formed in Eya1-/- mice, we have recently generated Eya1flox and Eya1CreERT2 alleles. We found that deletion of Eya1 after UB outgrowth results in depletion of the MM progenitors due to premature epithelialization and that EYA1 directly interacts with SIX2 and Myc family proteins, which are known to be important for the maintenance of the MM progenitors. Our analyses indicate that EYA1 dephosphorylates Myc at Thr58 to prevent the Myc protein from degradation in vitro and that deletion of Eya1 in the MM cells leads to increased levels of phosphor-T58-Myc but decreased levels of Myc. Currently, however, we do not understand how these key factors are functionally linked to regulate the proliferation and maintenance of the nephron progenitors. In this renewal application, we propose to take a combination of molecular, biochemical, genetic and genomic approaches to test the hypothesis that EYA1, Myc and SIX2 interact to regulate the expansion of the nephron progenitor pool and identify the target genes that are controlled by EYA1 and its cofactors. This study should advance the field by revealing novel mechanisms of how EYA1 interacts with its cofactors to regulate self-renewal of the nephron progenitors.