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

描述（由申请人提供）：本提案的长期目标是确定Eya及其辅因子Six在哺乳动物内耳发育过程中发挥作用的分子和发育机制。脊椎动物的内耳是从耳基板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，但它们在内耳及其感觉细胞发育中的确切功能还不清楚。Eya 1已被证明是内耳发育的关键基因：人类EYA 1基因的突变会导致鳃-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%，而小鼠Eya 1的失活会导致内耳发育在耳囊阶段停止。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在内耳发育中发挥作用的步骤的身份尚不清楚。在目前的资助期间，我们调查了与Eyaf缺陷相关的内耳缺陷的发育和分子基础。我们从BOR患者中鉴定了SIX 1或SIX 5基因的突变，并对这些突变进行了功能分析，通过基因打靶鉴定了Six 1在听觉系统中的发育功能，检测了Six 1和Eya 1在体内的相互作用，阐明了Pax、Eya和Six基因之间的调控关系。此外，我们已经确定了Six 1调控元件，直接在特定部位的发展内耳的表达。此外，我们产生了Eya 2突变小鼠，而纯合子小鼠具有听力损失。该更新申请将继续定义Eya和Six基因在内耳发育过程中起作用的分子和发育机制。具体而言，我们建议：（1）确定Eya和Six基因是否在耳基板的决定和发育中起关键作用，（2）鉴定控制Six 1耳表达的调控元件，并通过遗传和分子方法严格评估Eya 1对其的调控，（3）确定Eya 2在内耳发育中的生物学功能，（4）进一步确定Eya 1的作用，Six 1和S/x5-三个BOR综合征引起内耳发育的基因。这些研究应产生重要的新信息的发展和内耳形态发生的分子机制，并大大扩展了我们的理解Eya及其辅助因子6在哺乳动物内耳耳基板诱导和感觉细胞发育的作用。这些结果将为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.