Dissecting Wnt/PCP Signaling in Developing Cochlear Sensory Epithelium

描述(申请人提供)：听觉毛细胞顶端基于肌动蛋白的立体纤毛束(或毛束)起着将声能转化为电信号的关键功能。它的阶梯结构使束在方向上对机械刺激很敏感。因此，听觉发束必须均匀定向，才能正确地进行声音传导。发束结构或方向的异常会导致耳聋和听力障碍。听觉毛束的均匀取向是上皮平面细胞极性(PCP)的一个突出例子，即上皮层平面内的极性。它是由进化上保守的Wnt/PCP信号通路控制的。PCP途径的中断会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，这突显了它在调节组织形态发生中的关键作用。尽管PCP途径很重要，但它在毛束发育过程中调控复杂的形态发生过程的分子和细胞机制仍然知之甚少。这项研究的长期目标是了解PCP信号如何指导毛细胞细胞骨架的协调重塑，以形成统一定向的发束。这个项目的主要焦点是剖析蛋白酪氨酸激酶7(PTK7)的功能，PTK7是以前在小鼠基因陷阱筛选中发现的哺乳动物PCP途径的一个新组成部分。PTK7编码一种催化失活的受体酪氨酸激酶。将使用小鼠模型、耳蜗器官培养以及遗传、生化和细胞生物学方法的组合来检验中心假设，即PTK7采用保守和新颖的机制来调节毛束方向。目标1将确定PTK7在哪里进入保守的PCP途径。需要检验的假设是，PTK7通过其胞浆激活区与散乱的PCP信号分子相互作用，激活小GTP酶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，以及蛋白降解在调节PTK7细胞内和细胞间信号活性中的作用。在目标3中，将探索PTK7的新的信号机制，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。了解在毛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途径调节包括神经管在内的各种组织的形态发生，因此对毛细胞形态发生的研究将可能为组织形态发生过程中PCP信号的一般原理提供新的见解，这将有助于许多毁灭性人类出生缺陷的诊断和治疗。

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.