Elucidating the Role of Epithelial PCP signaling in SGN Axon Guidance in the Cochlea

我们的听觉非常依赖于连接声音的螺旋神经节神经元（SGN） Corti器（OC）中的受体与后脑的耳蜗核相连。在开发过程中，SGN建立 刻板的神经支配模式与特定的毛细胞目标在OC。I型SGN支配内毛细胞 第二类SGN（SGNII）神经支配外毛细胞（OHC）以检测声音信号， 外伤尽管它们在听力中起着重要作用，但我们对这些分子机制的理解， 听觉外围的中间线路仍然不完整。 最近的研究表明，SGNII外周投射的引导是由平面细胞调节的， 极性（PCP）途径。细胞间PCP信号传导介导组织平面内极化细胞行为 in a plenty过多of development发展processes过程.在野生型OC中，SGNII传入神经形成特征性的90度角。 转向耳蜗底部并支配多个OHC。在几种PCP突变体中，SGNII传入神经 随机地朝向耳蜗基部或顶端。尽管已经表明PCP蛋白定位于 与支持细胞（SC）-SC连接不对称，并在耳蜗上皮中起作用以引导SGNII传入， 其潜在机制目前尚不清楚。基于初步数据的坚实基础，我们将 检验PCP信号调节包括粘附分子在内的多种下游效应物的假设 和Rho GTP酶影响细胞粘附和干细胞中的细胞骨架，它们作为中间靶点 的SGNII。具体地说，我们发现PCP信号调节Nectin 3的定位，Nectin 3是一个成员， 免疫球蛋白超家族的粘附分子。此外，我们的初步数据表明， 耳蜗上皮需要Rac GTP酶用于SGNII传入引导;然而， 当PCP信号传导被破坏时，活性不足以指导SGNII传入。为了验证我们的假设， 1试图阐明Nectin 3在SGNII传入翻转中的作用。为此，我们生成了Nectin 3 使用CRISPR-Cas9敲除小鼠。我们将表征Nectin 3突变等位基因并分析SGNII传入 转化为Nectin 3敲除突变体。此外，为了确定Nectin 3介导的嗜异性粘附是否 SC和SGNII传入之间的相互作用在其转向中起作用，我们将测试可溶性Nectin 3 外结构域-Fc融合蛋白对耳蜗外植体和分离的SGN中SGN II轴突生长和转向的影响 cultures.目的2将进一步研究Rac信号通路活性与非对称PCP的相互调控 使用新产生的转基因Rac 1活性报告细胞系和Rac条件性敲除进行蛋白定位 变种人此外，我们还将确定是否E3泛素连接酶POSH/Sh 3rf 1，一个已知的Rac 1 下游效应器，参与SGNII传入指导使用功能丧失扰动。统称 拟议的实验将提供新的见解PCP信号如何指导极化细胞的行为和填补 我们对哺乳动物耳蜗SGNII布线机制的知识缺口。

Our sense of hearing is critically dependent on the spiral ganglion neurons (SGNs), which connect the sound receptors in the organ of Corti (OC) to the cochlear nuclei of the hindbrain. During development, SGNs establish stereotyped innervation patterns with specific hair cell targets in the OC. Type I SGNs innervate inner hair cells (IHCs) to transmit sound signals, while type II SGNs (SGNIIs) innervate outer hair cells (OHCs) to detect acoustic trauma. Despite their essential functions in hearing, our understanding of the molecular mechanisms that mediate wiring of the auditory periphery is still fragmentary. It has been shown recently that guidance of SGNII peripheral projections is regulated by the Planar Cell Polarity (PCP) pathway. Intercellular PCP signaling mediates polarized cell behaviors within the plane of a tissue in a plethora of developmental processes. In the wild-type OC, SGNII afferents make a characteristic 90-degree turn toward the base of the cochlea and innervate multiple OHCs. In several PCP mutants, SGNII afferents turn randomly towards either the cochlear base or the apex. Although it has been shown that PCP proteins localize asymmetrically to supporting cell (SC)-SC junctions and act in the cochlear epithelium to guide SGNII afferents, the underlying mechanisms are currently unknown. Based on a strong foundation of preliminary data, we will test the hypothesis that PCP signaling regulates multiple downstream effectors including adhesion molecules and Rho GTPases to influence cell adhesion and the cytoskeleton in SCs, which serve as intermediate targets of SGNIIs. Specifically, we found that PCP signaling regulates the localization of Nectin3, a member of the immunoglobulin superfamily of adhesion molecules, in SCs. Moreover, our preliminary data have suggested that the Rac GTPases are required in the cochlear epithelium for SGNII afferent guidance; however, their constitutive activity was insufficient to direct SGNII afferents when PCP signaling is disrupted. To test our hypothesis, Aim 1 seeks to elucidate the role of Nectin3 in SGNII afferent turning. To this end, we have generated Nectin3 knockout mice using CRISPR-Cas9. We will characterize Nectin3 mutant alleles and analyze SGNII afferent turning in Nectin3 knockout mutants. Additionally, to determine whether Nectin3-mediated heterophilic adhesion between SCs and SGNII afferents plays a role in their turning direction, we will test the effect of soluble Nectin3 ecto domain-Fc fusion proteins on SGNII axon outgrowth and turning in cochlear explants and dissociated SGN cultures. Aim 2 will further investigate mutual regulation between Rac signaling activity and asymmetric PCP protein localization, using a newly generated transgenic Rac1 activity reporter line and Rac conditional knockout mutants. Furthermore, we will also determine whether the E3 ubiquitin ligase POSH/Sh3rf1, a known Rac1 downstream effector, is involved in SGNII afferent guidance using loss-of-function perturbations. Together, the proposed experiments will provide novel insights into how PCP signaling directs polarized cell behaviors and fill our knowledge gaps about SGNII wiring mechanisms in the mammalian cochlea.