The Role of Eya and its Interacting Gene Six in Mammalian Inner Ear Development

描述(申请人提供)：这项提案的长期目标是确定Eya及其辅助因子Six在哺乳动物内耳发育过程中的分子和发育机制。脊椎动物的内耳是通过多种诱导过程从耳盘发育而来的。最近分离出了大量的听觉基因，然而，它们在内耳及其感觉细胞发育中的确切功能在很大程度上是未知的。EYA1已被证明是内耳发育的关键基因：人类EYA1基因的突变导致Branchio-Oto-Renal(Bor)综合征，这是一种先天性出生缺陷，占严重聋儿的2%，而小鼠的Eya1失活会导致内耳在耳囊肿期停止发育。然而，尽管Bor综合征的致病基因已经确定，但Bor综合征中听力缺陷的发育和分子基础以及Eya1在内耳发育中的作用步骤的一致性仍不清楚。在目前的资助期间，我们调查了与眼部缺陷相关的内耳缺陷的发育和分子基础。我们鉴定了BOR患者SIX1或SIX5的突变，并对这些突变进行了功能分析，通过基因打靶鉴定了SIX1在听觉系统中的发育功能，在体内测试了SIX1与Eya1的相互作用，阐明了Pax、Eya与6个基因之间的调控关系。此外，我们还鉴定了在发育中的内耳特定部位直接表达的SIX1调控元件。此外，我们培育了Eya2突变小鼠，纯合子小鼠有听力损失。这一更新应用将继续定义Eya和六个基因在内耳发育过程中发挥作用的分子和发育机制。具体地说，我们建议：(1)确定EYA和6个基因是否在耳源性安慰剂的决定和发育中起关键作用；(2)寻找控制SIX1基因表达的调控元件，并通过遗传和分子方法严格评估EYA1对其调控；(3)确定EYA2在内耳发育中的生物学功能；(4)进一步确定EYA1、SIX1和S/X5-导致Bor综合征的三个基因在内耳发育中的作用。这些研究将为内耳形态发生的发育和分子机制提供重要的新信息，并极大地扩展我们对Eya及其辅助因子6在哺乳动物内耳耳部胎盘诱导和感觉细胞发育中的作用的理解。这一结果将为Bor综合征内耳缺陷的发育和分子发病机制提供重要的见解。

DESCRIPTION (provided by applicant): This proposal's long term goal is to determine the molecular and developmental mechanisms by which Eya and its cofactors Six act during mammalian inner ear development. The vertebrate inner ear develops from the otic placode via multiple inductive processes. A large number of otic genes have been isolated recently; however, their precise functions in inner ear and its sensory cell development are largely unknown. Eya1 has been shown to be a key gene for inner ear development: 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, while inactivation of Eya1 in the mouse causes an arrest of inner ear development at the otocyst stage. 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 inner ear development are unclear. During the current grant period, we investigated the developmental and molecular basis of inner ear defects associated with Eyaf-deficiency. We identified mutations in the human SIX1 or SIX5 from BOR patients and analyzed these mutations functionally, identified the developmental functions of Six1 in the auditory system through gene targeting, tested the interactions between Six1 and Eya1 in vivo, and clarified the regulatory relationship between Pax, Eya and Six genes. In addition, we have identified Six1 regulatory elements that direct expression in specific parts of the developing inner ear. Furthermore, we generated Eya2 mutant mice, and the homozygous mice have hearing loss. This renewal application will continue to define the molecular and developmental mechanisms by which Eya and Six genes act during inner ear development. Specifically, we propose: (1) To determine whether Eya and Six genes play a crucial role in determination and development of the otic placodes, (2) To identify regulatory elements controlling Six1 otic expression and rigorously evaluate its regulation by Eya1 via genetic and molecular approaches, (3) To define the biological function of Eya2 during inner ear development, and (4) To further determine the role of Eya1, Six1 and S/x5-three BOR syndrome causing genes-in inner ear development. These studies should yield important new information about developmental and molecular mechanisms of inner ear morphogenesis, and greatly extend our understanding of the roles of Eya and its cofactors Six in otic placode induction and sensory cell development in the mammalian inner ear. The results will provide important insights into the developmental and molecular pathogenesis of inner ear defects in the BOR syndrome.