Non-Canonical Wnt Signaling Regulates Cochlear Outgrowth and Planar Cell Polarity via Gsk3β Inhibition.

Shaylyn Clancy,Xiaowei Lu,Diane Hwang,Margaret Smith,Maya Hatley,Alice Liu,Connor Smith,Christopher Clemens,Andre Landin Malt

doi:10.3389/fcell.2021.649830

Shaylyn Clancy, Xiaowei Lu + Show 7 more

Open Access

https://doi.org/10.3389/fcell.2021.649830

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Abstract

During development, sensory hair cells (HCs) in the cochlea assemble a stereociliary hair bundle on their apical surface with planar polarized structure and orientation. We have recently identified a non-canonical, Wnt/G-protein/PI3K signaling pathway that promotes cochlear outgrowth and coordinates planar polarization of the HC apical cytoskeleton and alignment of HC orientation across the cochlear epithelium. Here, we determined the involvement of the kinase Gsk3β and the small GTPase Rac1 in non-canonical Wnt signaling and its regulation of the planar cell polarity (PCP) pathway in the cochlea. We provided the first in vivo evidence for Wnt regulation of Gsk3β activity via inhibitory Ser9 phosphorylation. Furthermore, we carried out genetic rescue experiments of cochlear defects caused by blocking Wnt secretion. We showed that cochlear outgrowth was partially rescued by genetic ablation of Gsk3β but not by expression of stabilized β-catenin; while PCP defects, including hair bundle polarity and junctional localization of the core PCP proteins Fzd6 and Dvl2, were partially rescued by either Gsk3β ablation or constitutive activation of Rac1. Our results identify Gsk3β and likely Rac1 as downstream components of non-canonical Wnt signaling and mediators of cochlear outgrowth, HC planar polarity, and localization of a subset of core PCP proteins in the cochlea.

Highlights

Wnt signaling regulates a plethora of developmental processes through the canonical β-catenindependent pathway and the non-canonical β-catenin-independent pathway (Komiya and Habas, 2008; Wiese et al, 2018)
We further show that cochlear growth, hair bundle polarity, and core planar cell polarity (PCP) protein localization defects caused by blocking Wnt secretion are partially rescued by genetic ablation of Gsk3β in the cochlear epithelium, and to a lesser extent, constitutive activation of Rac1
We have identified PI3K as a key effector of non-canonical Wnt signaling in the OC; PI3K activity was decreased in the WlscKO OC, and importantly, PI3K activation rescued most of the WlscKO cochlear phenotypes (Landin Malt et al, 2020)

Summary

Introduction

Wnt signaling regulates a plethora of developmental processes through the canonical β-catenindependent pathway and the non-canonical β-catenin-independent pathway (Komiya and Habas, 2008; Wiese et al, 2018). Upon Wnt ligand binding to the Frizzled receptor, non-canonical Wnt signaling controls cell polarity and morphogenetic movements through the Rho family small GTPases or heterotrimeric G-proteins. Because the noncanonical Wnt and PCP pathways share many components, including the Fzd receptor, as well as the effectors Dvl and Rho GTPases, the PCP pathway is often considered to be a branch of non-canonical Wnt signaling. We and others have recently demonstrated that secreted Wnts are required for asymmetric localizations of a subset of PCP proteins in inner ear sensory epithelia, including Fzd3/6 and Dvl (Landin Malt et al, 2020; Najarro et al, 2020). We leverage the inner ear sensory epithelium and genetic tools available to further illuminate the precise relationship between the mammalian non-canonical Wnt and PCP pathways

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