Microtubule-mediated mechanisms underlying hair cell development and deafness

位于听觉毛细胞顶端的以肌动蛋白为基础的立体纤毛束(或毛束)是关键的 将声能转换为电信号的功能。发束发育和维护方面的缺陷 由于遗传和环境因素是感觉神经性耳聋的主要原因。长期目标 这项工作的目的是从机制上理解发束形态发生程序，包括分泌的 形态因子及其下游的信号效应器和细胞骨架调节因子。V字形的形成 毛束与毛细胞顶端细胞骨架的平面极化紧密相连，这是由 动纤毛细胞初级纤毛周围性移行。最近的进展表明毛细胞 固有的平面极性，我们称之为iPCP，简明地说，是由几个平面极化的细胞- 固有的信号模块和微管马达。在组织水平上，非规范的Wnt/Planar细胞 极性(PCP)通路将毛细胞的方向沿着内侧-外侧(或神经-非神经)轴排列 耳蜗管。虽然组织水平的PCP信号转导中断会导致发束方向错误，但iPCP缺陷 信号会导致方向错误和畸形的发束和错位的动纤毛。尽管它的 发束形态发生的重要性，iPCP如何受发育信号调控以及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，此应用程序 利用我们最新的令人兴奋的发现，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。而当 Wnt/β-catenin和Wnt/pcp通路已被广泛研究，但对其在体内的作用知之甚少 以及Wnt/G蛋白的信号转导机制。 我们的目标建立在坚实的初步数据基础上，表明WNT配体由 耳蜗上皮细胞激活异源三聚体G蛋白和PI3K信号来控制iPCP和PCP。我们 已经确定了耳蜗中G蛋白激活所需的基因，为分子研究提供了线索 机制。我们建议在体内、体外耳蜗外植体和细胞中使用功能获得和功能丧失的等位基因 体外培养研究确定Wnt/G蛋白通路的关键成分及其与细胞的整合 WNT/PCP途径。在目标1中，我们将描述Wnt/G蛋白通路的信号转导机制 通过确定特定的Wnt配体、Frizzled型受体和下游信号转导的作用。在AIM 2、我们研究了iPCP组分Par3在Wnt诱导的G蛋白激活中的作用。在《目标3》中，我们将 检测Wnt/G蛋白信号转导通路是否受多个G蛋白GEF调控。 最终，对这一新的Wnt信号通路的洞察可能有助于设计合理的疗法来刺激 损伤后的毛束修复和通过干细胞技术再生听觉毛细胞。

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.