Eya1 and its Interacting Genes in Inner Ear Development

描述（由申请人提供）：本提案的主要目标是阐明在早期哺乳动物内耳形态发生中由Eya 1及其相互作用基因控制的分子和遗传途径。脊椎动物的内耳是从耳基板（一个细胞厚的上皮细胞）通过多个诱导过程发育而来的。近年来，大量的耳相关基因被分离出来，但其确切的功能尚不清楚，调控内耳发育的分子和遗传途径也尚未建立。 最近，发现鼠Eya 1基因在听觉系统发育过程中表达，人类EYA 1基因突变导致鳃-耳-肾（BOR）综合征，这是一种先天性出生缺陷，占深度耳聋儿童的2%。然而，尽管鉴定了BOR综合征的相关基因，但BOR综合征中发生的听觉缺陷的发育和分子基础以及Eya 1在早期耳发育中起作用的步骤的身份尚不清楚。在Eya 1-/-小鼠胚胎中，内耳发育停滞在耳泡阶段，表明Eya 1是早期耳形态发生所需的关键基因。分子上，Six 1而不是Pax 2和Pax 8在耳泡中的表达取决于Eya 1功能。此外，最近发现小鼠Six 1也是内耳发育的关键基因，其基因产物在培养细胞中与Eya 1发生物理相互作用。因此，我们推测，在早期耳形态发生，Pax 8或Pax 2调节Eya 1和Six 1各自的基因产物相互作用。 这项资助将使用一个强大的遗传系统来验证这一假设。具体而言，我建议：（1）通过表型和分子分析建立Eya 1在早期耳发育中起作用的发育和分子机制，（2）在体内测试Six 1的功能作用以及Six 1和Eya 1之间在早期耳形态发生中的可能相互作用，（3）检验Pax-Eya-通过检查各突变体中的基因表达模式和共定位研究，（4）检测酵母双杂交筛选得到的候选Eya 1相互作用蛋白是否在耳发育早期的Eya 1-Six 1-调控通路中发挥作用。这些研究将阐明Pax、Eya 1、Six 1和其他基因之间的关系，并极有可能在分子水平上对耳形态发生的早期发育过程提供重要的见解。

DESCRIPTION (provided by applicant): The major goal of this proposal is to elucidate the molecular and genetic pathway(s) controlled by Eya1 and its interacting genes in early mammalian inner ear morphogenesis. The vertebrate inner ear develops from the otic placode, a one-cell-thick epithelium via multiple inductive processes. A large number of otic genes have been isolated recently, however, their precise functions are largely unknown and the molecular and genetic pathway(s) regulating the morphogenetic processes of inner ear development have not been established. Recently, it was found that the murine Eya1 gene is expressed during the development of the auditory system and mutations in the human EYA1 gene cause Branchio-Oto-Renal (BOR) syndrome, a congenital birth defect that accounts for as many as 2% of profoundly deaf children. However, despite the identification of the responsible gene for BOR syndrome, the developmental and molecular basis of auditory defects occurring in BOR syndrome and the identity of the steps at which Eya1 functions in early otic development are unclear. In Eya1-/- mouse embryos, the inner ear development arrests at the otic vesicle stage, indicating that Eya1 is a key gene required for early otic morphogenesis. Molecularly, Six1 but not Pax2 and Pax8 expression in the otic vesicle depends upon Eya1 function. Moreover, it was recently found that the murine Six1 is also a key gene for inner ear development and its gene product physically interacts with Eya1 in cultured cells. Thus, we hypothesize that during early otic morphogenesis, Pax8 or Pax2 regulates Eya1 and Six1 whose respective gene products interact. This grant will use a powerful genetic system to test this hypothesis. Specifically, I propose to: (1) establish the developmental and molecular mechanism(s) by which Eya1 acts in early otic development by phenotypic and molecular analyses, (2) test the functional role of Six1 and the possible interactions between Six1 and Eya1 in early otic morphogenesis in vivo, (3) test the hypothesis that the Pax-Eya-Six regulatory hierarchy is utilized during early otic development by examining the pattern of gene expression in the respective mutants and co-localization studies, (4) test whether the candidate Eya1-interacting proteins isolated from yeast two-hybrid screen function in the Eya1-Six1-regulatory pathway in early otic development. These studies will clarify the relationship between Pax, Eya1, Six1 and other genes, and have a strong likelihood of providing significant insight at the molecular level into the early developmental process of otic morphogenesis.