Microtubule-mediated mechanisms underlying hair cell development and deafness

听毛细胞顶端的肌动蛋白基静纤毛束（或毛束）是听觉系统的重要组成部分。 将声能转换为电信号的功能。毛束发育和维持缺陷 由于遗传和环境因素是感音神经性耳聋的主要原因。一个长期目标 这项工作的目的是获得毛束形态发生程序的机械理解，包括分泌 形态发生素及其下游信号传导效应物和细胞骨架调节剂。形成V形 毛束与毛细胞顶端细胞骨架的平面极化紧密相连，这是由 动纤毛（毛细胞初级纤毛）的外周迁移。最新研究表明，毛细胞 内在平面极性，我们称之为iPCP，是由几个平面极化的细胞调节的， 内在信号模块和微管马达。在组织水平上，非典型Wnt/Planar Cell 极性（PCP）途径使毛细胞取向沿着耳蜗的内侧-外侧（或神经-神经外）轴排列。 蜗管虽然中断的组织水平PCP信号传导导致毛束定向错误，但有缺陷的iPCP 信号传导导致具有错位动纤毛的错位和畸形的发束。尽管 毛束形态发生的重要性，iPCP如何受发育信号调节以及iPCP如何 和PCP信号的整合仍然知之甚少。为了填补这些知识空白，该应用程序 利用我们最近令人兴奋的发现，旨在阐明一种新的Wnt/G 在毛束形态发生过程中，蛋白质信号通路协调控制iPCP和PCP。而 Wnt/β-catenin和Wnt/PCP通路已被广泛研究，但其在体内的功能尚不清楚 和Wnt/G蛋白信号转导机制。 我们的目标是建立在初步数据的坚实基础上，这些数据表明，由细胞分泌的Wnt配体， 耳蜗上皮激活异源三聚体G蛋白和PI 3 K信号传导以控制iPCP和PCP。我们 已经确定了耳蜗中G蛋白激活所需的基因，为研究G蛋白的分子机制提供了线索。 机制我们建议在体内、离体耳蜗外植体和细胞中使用获得和丧失功能的等位基因。 体外培养研究，以确定Wnt/G蛋白途径的关键组分及其与 Wnt/PCP途径。在目的1中，我们将描述Wnt/G蛋白通路的信号转导机制 通过确定特定Wnt配体、Frizzled受体和下游信号转导子的作用。在Aim中 2、我们研究了iPCP成分Par 3在Wnt诱导的G蛋白激活中的作用。在目标3中，我们 测试在耳蜗形态发生期间Wnt/G蛋白信号传导是否由多个G蛋白GEF调节。 最终，对这种新的Wnt信号通路的深入了解可能有助于设计合理的治疗方法来刺激 损伤后的毛束修复和通过干细胞技术再生听毛细胞。

The actin-based stereociliary bundle (or hair bundle) on the apex of auditory hair cells serves the critical function of converting sound energy to electric signals. Defects in hair bundle development and maintenance due to genetic and environmental factors are a leading cause for sensorineural deafness. A long-term objective of this work is to gain a mechanistic understanding of hair bundle morphogenesis programs, including secreted morphogens and their downstream signaling effectors and cytoskeletal regulators. Formation of the V-shaped hair bundle is integrally linked to planar polarization of the hair cell apical cytoskeleton, which is initiated by the peripheral migration of the kinocilium, the hair cell primary cilium. Recent advances demonstrate that hair cell intrinsic planar polarity, which we name iPCP to be concise, is regulated by several planar polarized, cell- intrinsic signaling modules and microtubule motors. At the tissue-level, the non-canonical Wnt/Planar Cell Polarity (PCP) pathway aligns hair cell orientation along the medial-lateral (or neural-abneural) axis of the cochlear duct. While disrupted tissue-level PCP signaling causes hair bundle misoriention, defective iPCP signaling results in both misoriented and misshapen hair bundles with a mispositioned kinocilium. Despite its importance for hair bundle morphogenesis, how iPCP is regulated by developmental signals and how iPCP and PCP signaling are integrated remain poorly understood. To fill these knowledge gaps, this application leverages our recent exciting discoveries and aims to elucidate the mechanisms by which a novel Wnt/G protein signaling pathway coordinately controls iPCP and PCP during hair bundle morphogenesis. While Wnt/β-catenin and Wnt/PCP pathways have been extensively studied, little is known about the in vivo function and mechanisms of Wnt/G protein signaling. Our aims are built on a strong foundation of preliminary data, showing that Wnt ligands secreted by the cochlear epithelium activate heterotrimeric G protein and PI3K signaling to control both iPCP and PCP. We have identified genes required for G protein activation in the cochlea, providing clues to the molecular mechanism. We propose to use gain- and loss-of-function alleles in vivo, ex vivo cochlear explants and cell culture studies in vitro to define critical components of the Wnt/G protein pathway and its integration with the Wnt/PCP pathway. In Aim 1, we will delineate the signal transduction machinery of the Wnt/G protein pathway by determining the roles of specific Wnt ligand, Frizzled receptors and downstream signal transducers. In Aim 2, we investigate the role of the iPCP component Par3 in Wnt-induced G protein activation. In Aim 3, we will test whether Wnt/G protein signaling is regulated by multiple G protein GEFs during cochlear morphogenesis. Ultimately, insights into this new Wnt signaling pathway may facilitate devising rational therapies to stimulate hair bundle repair following injury and to regenerate auditory hair cells through stem cell technologies.