Dual Mechanisms of Supporting Cell Regeneration in the Neonatal Mouse Cochlea

Cochlear supporting cells are critical for proper hearing function and regeneration of hair cells, which are responsible for perceiving sound in the inner ear. In the neonatal mouse cochlea, when supporting cells are ablated, they can regenerate through progenitor cells in the cochlea. Here, we show through single cell RNA sequencing and in vivo fate mapping, that upon supporting cell ablation in the neonatal cochlea, inner phalangeal cells regenerate through two independent mechanisms; one which requires proliferation and the other by which transdifferentiation (i.e. regeneration without mitotic division) of cochlear progenitor cells occurs. These dual mechanisms are spatially distinct where transdifferentiation occurs in all three turns of the cochlea, while proliferation-induced regeneration occurs exclusively in the apical and middle turns. Inhibiting proliferation using aphidicolin, slightly decreased the number of regenerated IPhCs in the apex middle turns, but not the base, after damage. We also observed a medial to lateral transition in the compartmentalization of the GER after supporting cell damage, suggesting de-differentiation of these domains. Overall, our study reveals GER cells as supporting cell progenitors that undergo dual mechanisms of regeneration (mitotic and non-mitotic). Additionally, we uncover transcriptomic changes after supporting cell damage in the neonatal mouse cochlea that could be used as gene targets in therapies for hearing loss. Overall design: Whole cochlea from postnatal days (P) 4, P5, P6 and P7 Ki67CreERT/+;Lgr5DTR/+;Rosa26RtdTomato/+ (damage) and Ki67CreERT/+;Rosa26RtdTomato/+ (control) mice were dissociated in biological duplicate for single cell RNA-sequencing. Cells were processed using 10X Genomics Chromium Single Cell 3' Reagents v3.1. After sequencing, Cell Ranger was used for read mapping and quantification, followed by further analysis using Seurat.