Transcription Factors in Early Kidney Development

描述（由申请人提供）：本提案的长期目标是通过检查转录共激活因子 Eya1、其相互作用的同源域蛋白 Six1 及其辅助因子的作用，阐明控制早期肾脏发育过程中功能性后肾间质形成的分子机制。哺乳动物肾脏通过后肾间质和输尿管芽上皮之间的诱导相互作用在后中间中胚层区域发育。最近的遗传和分子研究表明，后肾间充质可能提供促进输尿管芽形成的初始信号。然而，哪些基因和调控层次控制后肾间质的形成仍不清楚。最近，发现小鼠 Eya1 基因在后肾间充质中表达，而人类 EYA1 基因突变会导致鳃-耳-肾 (BOR) 综合征，这是一种以鳃、耳和肾脏异常为特征的先天性出生缺陷。然而，尽管已鉴定出相关基因，但肾脏缺陷的发育和分子基础以及 Eya1 在早期肾脏形态发生中发挥作用的步骤仍不清楚。在Eya1 -/- 小鼠中，后肾间充质从未形成，间充质细胞从E10.5开始发生异常凋亡，表明Eya1是早期肾脏形态发生所需的关键间充质基因。同源盒基因 Six1 还被发现在早期肾脏发育过程中发挥着重要作用，其基因产物与 Eya1 发生物理相互作用。此外，最近通过使用Eya1作为“诱饵”的酵母双杂交筛选分离出转录因子N-myc，并且它在体外与Six1和Eya1发生物理相互作用。有趣的是，N-myc 基因突变也会导致肾脏缺陷。基于这些数据，我们假设这些转录因子在后肾间充质中共同发挥作用，调节早期肾脏发育。这笔赠款将使用强大的遗传系统来检验这一假设，并将几个间充质基因整合到控制早期肾脏发育的遗传和分子调控途径中。具体来说，我建议：（1）测试Eya1和Six1在早期肾脏发育过程中的功能作用，（2）测试Six1、Eya1和Pax2在早期肾脏发育过程中可能的相互作用，（3）测试N-myc在早期肾脏发育过程中是否与Eya1或Six1相互作用。这些研究将阐明 Pax2、Eya1、Six1、N-myc 和其他基因之间的关系，并且很有可能在分子和遗传水平上为肾脏形态发生的早期发育过程提供重要的见解。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to elucidate the molecular mechanisms controlling the formation of a functional metanephric mesenchyme during early kidney development by examining the role of the transcriptional coactivator Eya1, its interacting homeodomain protein Six1 and their cofactors. The mammalian kidney develops in a region of posterior intermediate mesoderm by inductive interactions between the metanephric mesenchyme and the ureteric bud epithelium. Recent genetic and molecular studies have indicated that the metanephric mesenchyme may provide initial signals to promote ureteric bud formation. However, what genes and the regulatory hierarchy controlling the formation of the metanephric mesenchyme still remain unclear. Recently, the murine Eya1 gene was found to be expressed in the metanephric mesenchyme and mutations in the human EYA1 gene cause Branchio-Oto-Renal (BOR) syndrome, a congenital birth defect characterized by combinations of branchial, otic and renal anomalies. However, despite the identification of the responsible gene, the developmental and molecular basis for renal defects and the identity of the steps at which Eya1 functions in early kidney morphogenesis are unclear. In Eya1 -/- mice, the metanephric mesenchyme never forms and the mesenchymal cells undergo abnormal apoptosis from E10.5, indicating that Eya1 is a key mesenchymal gene required for early kidney morphogenesis. The homeobox gene Six1 was also found to play an essential role during early kidney development and its gene product physically interacts with Eya1. Moreover, the transcription factor N-myc has been recently isolated through yeast two-hybrid screen using Eya1 as "bait" and it physically interacts with both Six1 and Eya1 in vitro. Interestingly, mutations in the N-myc gene also cause kidney defects. Based on these data, we hypothesize that these transcription factors function together in the metanephric mesenchyme to regulate early kidney development. This grant will use a powerful genetic system to test this hypothesis and integrate several mesenchymal genes into a genetic and molecular regulatory pathway governing early kidney development. Specifically, I propose to: (1) test the functional role of Eya1 and Six1 during early kidney development, (2) test the possible interactions between Six1, Eya1 and Pax2 in early kidney development, (3) test whether N-myc interacts with Eya1 or Six1 during early kidney development. These studies will clarify the relationship between Pax2, Eya1, Six1, N-myc and other genes, and have a strong likelihood of providing significant insight at the molecular and genetic level into the early developmental process of kidney morphogenesis.