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

耳蜗由螺旋神经节的双极性感觉神经元支配，这些神经元将声音信息从感觉受体毛细胞传递到中枢听觉目标。耳蜗损伤引起的耳聋与螺旋神经节神经元及其支配的毛细胞的病变有关，修复耳蜗的一个重要方面是诱导螺旋神经节神经元重新支配它们的毛细胞伴侣。一般认为毛细胞再神经支配与新生毛细胞神经支配的细胞和分子机制相似。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基于再生的治疗策略的重要先决条件。螺旋神经节中的一个神经元子集致力于一个基本重要的反馈回路，该回路在极端噪声中提供神经保护，并促进背景噪声中的听力和语言辨别。该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在向耳蜗基部做一个明显的90°转弯之前，超出了内毛细胞，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在这一过程中至少有一步起作用；将SGN2外周轴突导向耳蜗基部的转动事件。这项探索性/发展性研究资助的目标是建立SGN2外周轴突转动过程中Vangl2功能的两个基本特性，并期望这将为解决螺旋神经节发育的更大、独立的研究奠定基础。首先是区分外周轴突生长锥或Corti器官中自主和非细胞自主的Vangl2功能位点。这将使用先前由实验室生成的vangl2条件敲除系与选择用于空间限制vangl2基因缺失的Cre系相结合来完成。其次是分析两种非典型Wnt受体和信号通路的相对贡献，这两种非典型Wnt受体和信号通路已被证明在其他情况下在Vangl2上游发挥作用。这将通过基于假设的遗传相互作用分析来建立，假设如果Vangl2和上游受体在同一途径中起作用，那么去除两者将增强SGN2的转化表型。虽然这些实验主要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再神经支配和修复过程中重演，因此本研究将推动耳聋修复的治疗。

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.