Dissecting Wnt/PCP Signaling in Developing Cochlear Sensory Epithelium

描述（由申请人提供）：听觉毛细胞顶端的肌动蛋白基立体纤毛束（或毛束）具有将声能转化为电信号的关键功能。它的阶梯结构使束对机械刺激的方向敏感。因此，听觉毛束必须均匀定向才能正确地进行声音传导。毛束结构或方向的异常可引起耳聋和听力障碍。听毛束的均匀取向是上皮平面细胞极性（PCP）或上皮片平面内极性的一个突出例子。它受进化保守的Wnt/PCP信号通路控制。PCP通路的破坏可导致严重的发育缺陷，包括神经管闭合缺陷和毛束定向错误，强调其在调节组织形态发生中的关键功能。尽管它很重要，但PCP通路调节毛束发育过程中复杂的形态发生过程的分子和细胞机制仍然知之甚少。本研究的长期目标是了解PCP信号如何指导毛细胞骨架的协调重塑，形成均匀定向的毛束。该项目的主要重点是剖析蛋白酪氨酸激酶7 （PTK7）的功能，PTK7是哺乳动物PCP途径的一个新组成部分，此前在小鼠基因陷阱筛选中发现。PTK7编码一种催化活性不高的受体酪氨酸激酶。小鼠模型、耳蜗器官培养以及遗传、生化和细胞生物学方法的结合将用于验证PTK7部署保守机制和新机制来调节毛束取向的中心假设。目的1将确定PTK7在何处进入保守的PCP通路。需要验证的假设是，PTK7通过其细胞质激酶结构域与PCP信号分子Dishevelled相互作用，激活小GTPase Rac，导致细胞骨架重塑。目的2将确定PTK7在Corti器官中的细胞自主性，以及蛋白水解在调节PTK7细胞内和细胞间信号传导活性中的作用。在Aim 3中，将首先通过测试PTK7的激酶功能，然后分离新的PTK7相互作用蛋白来探索PTK7的新的信号传导机制。了解发束发育过程中控制细胞骨架重塑的PCP信号机制将有助于设计促进毛细胞修复和修复后有意义的功能恢复的治疗方法。此外，由于PCP通路调节包括神经管在内的多种组织的形态发生，对毛细胞形态发生的研究可能会为组织形态发生过程中PCP信号传导的一般原理提供新的见解，这将有助于诊断和治疗许多毁灭性的人类出生缺陷。

DESCRIPTION (provided by applicant): The actin-based stereocilary bundle (or hair bundle) on the apex of auditory hair cells serves the crucial function of converting sound energy to electric signals. Its staircase structure renders the bundle directionally sensitive to mechanical stimuli. As such, auditory hair bundles must be uniformly oriented for correct sound transduction. Abnormalities in hair bundle structure or orientation cause deafness and hearing impairment. The uniform orientation of auditory hair bundles is a prominent example of epithelial planer cell polarity (PCP), or polarity within the plane of the epithelial sheet. It is controlled by an evolutionarily conserved Wnt/PCP signaling pathway. Disruption of the PCP pathway results in severe developmental defects including neural tube closure defect and hair bundle misorientation, underscoring its critical function in regulating tissue morphogenesis. Despite its importance, the molecular and cellular mechanisms by which the PCP pathway regulates the intricate morphogenetic processes during hair bundle development are still poorly understood. The long-term goal of this research is to understand how PCP signaling directs coordinated remodeling of the hair cell cytoskeleton to form uniformly oriented hair bundles. The primary focus of this project is to dissect the function of protein tyrosine kinase 7 (PTK7), a novel component of the mammalian PCP pathway previously identified in a mouse gene-trap screen. PTK7 encodes a catalytically inactive receptor tyrosine kinase. Mouse models, cochlear organ cultures, and a combination of genetic, biochemical and cell biological approaches will be used to test the central hypothesis that PTK7 deploys both conserved and novel mechanisms to regulate hair bundle orientation. Aim 1 will determine where PTK7 feeds into the conserved PCP pathway. The hypothesis to be tested is that PTK7, via its cytoplasmic kinase domain, interacts with the PCP signaling molecule Dishevelled to activate the small GTPase Rac, leading to cytoskeletal remodeling. Aim 2 will determine the cell-autonomy of PTK7 in the organ of Corti and the role of proteolysis in regulating intra- and inter-cellular signaling activity of PTK7. In Aim 3, novel signaling mechanisms of PTK7 will be explored, first by testing the kinase function of PTK7, followed by isolating novel PTK7 interacting proteins. Understanding PCP signaling mechanisms that control cytoskeletal remodeling during hair bundle development will aid in devising therapies to promote hair cell repair and meaningful functional recovery following repair. Moreover, because the PCP pathway regulates morphogenesis of diverse tissues, including the neural tube, studies of hair cell morphogenesis will likely provide novel insights into the general principles of PCP signaling during tissue morphogenesis, which will facilitate the diagnosis and treatment of many devastating human birth defects.