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，其将声音信息从感觉感受器毛细胞中继到中枢听觉目标。由于声创伤引起的耳聋与螺旋神经节神经元和它们支配的毛细胞两者中的病理相关，并且修复受损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伙伴。一般预期毛细胞神经再支配将涉及与引导新生毛细胞神经支配的那些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非常重要的反馈回路，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言语辨别。该回路依赖于支配外毛细胞的2型螺旋神经节神经元（SGN 2）。SGN 2的形态发育是独特的，因为它们的外周轴突在朝向耳蜗基部形成明显的90°转弯之前伸出内毛细胞，以便与8至10个外毛细胞突触。虽然SGN 2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Vangl 2有助于该过程中的至少一个步骤;将SGN 2外周轴突导向耳蜗基部的转向事件。这项探索性/发展性研究资助的目标是建立SGN 2外周轴突转向期间Vangl 2功能的两个基本特性，期望这将形成解决螺旋神经节发育的更大的独立研究线的基础。首先是区分外周轴突生长锥或Corti器官中Vangl 2功能的自主和非细胞自主位点。这将使用先前由实验室产生的vangl 2条件性敲除系与选择用于空间限制vangl 2基因缺失的Cre系组合来实现。第二个是测定两种替代的非经典Wnt受体和信号传导途径的相对贡献，所述信号传导途径已被证明在其他情况下在Vangl 2的上游起作用。这将通过基于以下假设的遗传相互作用测定来建立：如果Vangl 2和上游受体在相同途径中起作用，则去除两者将增强SGN 2转化表型。虽然这些实验的重点是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神经支配和修复过程中重现，因此拟议的研究将推进修复受损耳蜗的疗法。

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.