Development and transdifferentiation into inner hair cells require Tbx2

Atoh1 is essential for the development of both outer hair cells (OHCs) and inner hair cells (IHCs) in the mammalian cochlea. Whereas Ikzf2 is necessary for OHC development, the key gene required for IHC development remains unknown. We found that deletion of Tbx2 in neonatal IHCs led to their transdifferentiation into OHCs by repressing 26.7% of IHC genes and inducing 56.3% of OHC genes, including Ikzf2. More importantly, persistent expression of Tbx2 coupled with transient Atoh1 expression effectively reprogramed non-sensory supporting cells into new IHCs expressing the functional IHC marker vGlut3. The differentiation status of these new IHCs was considerably more advanced than that previously reported. Thus, Tbx2 is essential for IHC development, and co-upregulation of Tbx2 with Atoh1 in supporting cells represents a new approach for treating deafness related to IHC degeneration. Overall design: Adult cochlear inner hair cells (IHCs) at P30 (P30_WT IHCs) were manually picked and subjected to smart-seq HT library construction. The transcriptomic comparision between P30_WT IHCs and P30_WT OHCs (from our previous report) identified that Tbx2 is a transcriptional factor that highly expressed in P30_WT IHCs, but not in P30_WT OHCs. We further confirmed that Tbx2 is persistently expressed in IHC lineage from emrbyonic to adult ages. To determine the roles of Tbx2 in IHCs, we constructed the conditional Tbx2 cko model where Tbx2 was specifically deleted in neonatal IHCs. Our phenotypic analysis demonstrates that IHCs tend to become OHCs when Tbx2 is deleted. Thus, the Tbx2 -/- IHCs were defined as induced OHCs (iOHCs). Next, by the same smart-seq HT method, we compared the gene profiles between wild type IHCs at P14 (P14_WT IHCs) and those iOHCs at P14 (P14_iOHCs). The overal cell cluster and trajectorary analysis support that the iOHCs globally behave adult OHCs.