Genetic Dissection of Vangl2-Dependent Axon Guidance in the Developing Cochlea

耳蜗由螺旋神经节的双极感觉神经元支配，螺旋神经节将声音信息从感觉感受器毛细胞传递到中央听觉目标。听觉损伤所致耳聋与螺旋神经节神经元及其支配的毛细胞的病理改变有关，修复耳聋的一个重要方面是诱使螺旋神经节神经元重新支配其毛细胞伙伴。一般认为，毛细胞的再神经支配将涉及到与指导新生毛细胞神经支配的细胞和分子机制类似的细胞和分子机制。因此，了解螺旋神经节发育和毛细胞神经支配的各个方面是以再生为基础的治疗策略的重要前提。螺旋神经节中的神经元子集专门用于一个基本重要的反馈电路，该电路在极端噪声中提供神经保护，并在背景噪声中促进听力和言语识别。这一回路依赖于支配外毛细胞的2型螺旋神经节神经元(SGN2)。SGN2s的形态发育是独特的，因为它们的外周轴突投射到内毛细胞之外，然后向耳蜗基底部明显地转向90°，以便与8到10个外毛细胞突触。虽然SGN2发育和外毛细胞神经支配的许多方面尚不清楚，但我们的实验室已经发现证据表明，平面细胞极性蛋白Vangl2至少在这一过程中起到了一个作用：将SGN2外周轴突导向耳蜗基的转向事件。这项探索性/开发性研究基金的目标是建立SGN2外周轴突转动过程中Vangl2功能的两个基本特性，期望这将形成一个更大的、独立的研究路线，解决螺旋神经节的发育问题。第一个是区分Vangl2在外周轴突、生长锥或Corti器官中的自主和非细胞自主功能部位。这将通过使用实验室先前产生的Vangl2条件性基因敲除系与选择用于空间限制Vangl2基因缺失的CRE系相结合来实现。第二个是分析两种可供选择的非典型Wnt受体和信号通路的相对贡献，这些受体和信号通路已被证明在Vangl2上游在其他环境中发挥作用。这将通过基于以下假设的遗传相互作用分析来建立：如果Vangl2和上游受体在同一途径中发挥作用，那么去除两者将增强SGN2的转向表型。虽然这些实验集中在指导轴突寻路和靶细胞神经支配的发育过程，但我们预计这些事件必须在毛细胞重新神经支配和修复过程中重现，因此拟议的研究将促进耳聋修复的治疗。

The cochlea is innervated by the bipolar sensory neurons of the spiral ganglia that relay sound information from sensory receptor hair cells to central auditory targets. Deafness due to acoustic trauma is associated with pathologies in both spiral ganglion neurons and the hair cells which they innervate and an important aspect of repairing the deafened cochlea is coaxing spiral ganglion neurons to re-innervate their hair cell partners. It is generally anticipated that hair cell re-innervation will involve similar cellular and molecular mechanisms to those guiding nascent hair cell innervation. Therefore, understanding all aspects of spiral ganglion development and hair cell innervation are important prerequisites of regeneration-based therapeutic strategies. A subset of neurons in the spiral ganglion is dedicated to a fundamentally important feedback circuit that provides neuroprotection in extreme noise and facilitates hearing and speech discrimination in background noise. This circuit is dependent on the Type2 spiral ganglion neurons (SGN2) that innervate the outer hair cells. The morphological development of SGN2s is unique because their peripheral axon projects beyond the inner hair cells before making a distinct 90° turn towards the base of the cochlea in order to synapse with 8 to 10 outer hair cells. While many aspects of SGN2 development and outer hair cell innervation are not known, our laboratory has found evidence that the planar cell polarity protein Vangl2 contributes to at least one step in this process; the turning event that directs the SGN2 peripheral axon to the base of the cochlea. The goal of this Exploratory/Developmental Research grant is to establish two basic properties of Vangl2 function during SGN2 peripheral axon turning with the expectation that this will form the foundation of a larger, independent line of research addressing spiral ganglion development. The first is to distinguish between autonomous and non-cell autonomous sites of Vangl2 function in the peripheral axon growth cone or organ of Corti. This will be accomplished using a vangl2 conditional knockout line previously generated by the lab in combination with Cre lines selected to spatially restrict vangl2 gene deletion. The second is to assay the relative contribution of two alternative non-canonical Wnt receptors and signaling pathways that have been demonstrated to function upstream of Vangl2 in other contexts. This will be established through genetic interaction assays based upon the hypothesis that if Vangl2 and an upstream receptor function in the same pathway, then removing both will enhance SGN2 turning phenotypes. While these experiments are focused on developmental processes guiding axon pathfinding and target cell innervation we anticipate that these events must be recapitulated during hair cell re-innervation and repair, and therefore the proposed research will advance therapies for repairing the deafened cochlea.