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

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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.