Abstract

Sensory hair cells (HCs) are highly susceptible to damage by noise, ototoxic drugs, and aging. Although HCs cannot be spontaneously regenerated in adult mammals, previous studies have shown that signaling pathways are involved in HC regeneration in the damaged mouse cochlea. Here, we used a Notch antagonist (DAPT), a Wnt agonist (QS11), and recombinant Sonic hedgehog (SHH) protein to investigate their concerted actions underlying HC regeneration in the mouse cochlea after neomycin-induced damage both in vivo and in vitro. With DAPT, the numbers of HCs increased, and supporting cell (SC) proliferation was seen in both the intact and damaged cochlear sensory epithelia, while these numbers were unchanged in the presence of QS11. When simultaneously treated with DAPT and QS11, the number of HCs increased dramatically, and much greater SC proliferation was seen in the cochlear epithelium. In transgenic mice with both Notch1 conditional knockout and β-catenin over-expression, cochlear SC proliferation and HC regeneration were more obvious than in either Notch1 knockout or β-catenin over-expressing mice separately. When cochleae were treated with DAPT, QS11, and SHH together, SC proliferation was even greater, and this proliferation was seen in both the HC region and the greater epithelial ridge. High-throughput RNA sequencing was used to identify the differentially expressed genes between all groups, and the results showed that the SHH and Wnt signaling pathways are involved in SC proliferation. Our study suggests that co-regulation of the Notch, Wnt, and SHH signaling pathways promotes extensive cell proliferation and regeneration in the mouse cochlea.

Highlights

  • The mammalian cochlea has very limited spontaneous hair cell (HC) regeneration ability, and such regeneration is only seen at embryonic or very early neonatal ages and the quantity and quality of this limited spontaneous hair cells (HCs) regeneration cannot restore cochlear function (Jung et al 2013)

  • In the DAPT-only cochleae, numerous EdU + /Sox2 + cells and EdU + / Myo7a + cells were observed with a gradient from the apex to the base, with most cells seen in the apex, which was consistent with the literature (Matei et al 2005)

  • The EdU + cells of the co-treated group were spread throughout the HC region, and the number of HCs ­(HCDAPT+QS11 100.6 ± 3.731/100 μm, N = 13) increased more than in the QS11-only ­(HCQS11 42.24 ± 1.390/100 μm, N = 5) (p < 0.001, DAPT + QS11 vs. QS11) and the DAPT-only groups (p < 0.01, DAPT + QS11 vs. DAPT) (Fig. 1(a–c, e–f))

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Summary

Introduction

The mammalian cochlea has very limited spontaneous hair cell (HC) regeneration ability, and such regeneration is only seen at embryonic or very early neonatal ages and the quantity and quality of this limited spontaneous HC regeneration cannot restore cochlear function (Jung et al 2013). Regulating these signaling pathways is likely to be an effective way to stimulate sensory progenitor cell proliferation and differentiation into new HCs. The Notch, Wnt, and Sonic hedgehog (SHH) signaling pathways play critical roles in controlling inner ear sensory cell proliferation and differentiation (Roccio and Edge 2019). The Notch and Wnt signaling pathways crosstalk with each other in regulating and maintaining the balance between cell proliferation and differentiation in many tissues (Fre et al 2009; Munnamalai and Fekete 2020). The crosstalk between the Notch, Wnt, and SHH signaling pathways in regulating the proliferation and regeneration of sensory progenitor cells in the mouse cochlea has not been reported in detail

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