Conditional Overexpression of Net1 Enhances the Trans-Differentiation of Lgr5⁺ Progenitors into Hair Cells in the Neonatal Mouse Cochlea

Sensorineural hearing loss is mainly caused by damage to hair cells (HC), which cannot be regenerated spontaneously in adult mammals once damaged. Cochlear Lgr5⁺ progenitors are characterised by HC regeneration capacity in neonatal mice, and we previously screened several new genes that might induce HC regeneration from Lgr5⁺ progenitors. Net1, a guanine nucleotide exchange factor, is one of the screened new genes and is particularly active in cancer cells and is involved in cell proliferation and differentiation. Here, to explore in vivo roles of Net1 in HC regeneration, Net1^loxp/loxp mice were constructed and crossed with Lgr5^CreER/+ mice to conditionally overexpress (cOE) Net1 in cochlear Lgr5⁺ progenitors. We observed a large number of ectopic HCs in Lgr5^CreER/+Net1^loxp/loxp mouse cochlea, which showed a dose-dependent effect. Moreover, the EdU assay was unable to detect any EdU⁺/Sox2⁺ supporting cells, while lineage tracing showed significantly more regenerated tdTomato⁺ HCs in Lgr5^CreER/+Net1^loxp/loxptdTomato mice, which indicated that Net1 cOE enhanced HC regeneration by inducing the direct trans-differentiation of Lgr5⁺ progenitors rather than mitotic HC regeneration. Additionally, qPCR results showed that the transcription factors related to HC regeneration, including Atoh1, Gfi1 and Pou4f3, were significantly upregulated and are probably the mechanism behind the HC regeneration induced by Net1. In conclusion, our study provides new evidence for the role of Net1 in enhancing HC regeneration in the neonatal mouse cochlea.