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6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个诱导过程从耳位发育而来的。近年来已分离出大量的克隆基因；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尚不清楚。Eya1已被证明是内耳发育的关键基因：人类Eya1基因的突变导致支气管-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Eya1的失活导致内耳发育在耳囊肿阶段停滞。然而，尽管已经确定了BOR综合征的相关基因，但对于BOR综合征中发生的听觉缺陷的发育和分子基础，以及Eya1在内耳发育中的作用机制尚不清楚。在目前的资助期内，我们研究了与eyaf -缺乏症相关的内耳缺陷的发育和分子基础。我们从BOR患者中鉴定出人类SIX1或SIX5突变，并对这些突变进行功能分析，通过基因靶向鉴定SIX1在听觉系统中的发育功能，在体内检测SIX1与Eya1的相互作用，明确Pax、Eya和Six基因之间的调控关系。此外，我们已经确定了Six1调控元件，直接在发育中的内耳的特定部位表达。此外，我们产生了Eya2突变小鼠，纯合子小鼠有听力损失。这项更新应用将继续确定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发挥关键作用；(2)通过遗传学和分子生物学方法鉴定控制Six1基因表达的调控元件，并严格评估Eya1对其的调控作用；(3)明确Eya2在内耳发育中的生物学功能；(4)进一步确定Eya1、Six1和S/x5 3种引起BOR综合征的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在哺乳动物内耳听觉基板诱导和感觉细胞发育中的作用的理解。该结果将为了解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.