Extended Data Fig. 4 |. Cochlear hair cells transdifferentiating postnatally transiently co-express markers of mature IHCs (VGLUT3) and OHCs (Prestin and PMCA2), but not of nascent hair cells (SOX2) nor of differentiating OHCs (Insm1 and Bcl11b).

Fgf8^CreER; Tbx2^F/F; R26^{LSL-tdTomato/+} newborn (P0) pups were treated with tamoxifen at P0 and collected for examination at each subsequent day from P1 to P8. (a-c) Immunohistochemistry at P5 reveals that hair cells in the position of the IHCs express the IHC functional marker VGLUT3 (and not the OHC developmental market BCL11b) (a; confocal optical section), but also the OHC functional markers Prestin (b; confocal optical section) and PMCA2 (c; confocal projection). In (c), former IHCs are identified by the expression of TdTomato. Hence, at this time transdifferentiating hair cells display features of both IHCs and OHCs. (d) Immunohistochemistry at P1, displayed as a confocal radial optical section, reveals that cells transdifferentiating from IHCs to OHCs (at this point ambiguously termed ic-HCs; TdTomato+ in the lower panel) do not express SOX2, a nuclear marker of both supporting cells and nascent hair cells, and are beginning to express PMCA2 in their stereocilia (bottom panel). For a better visualization of SOX2, the top panel displays its immunoreactivity only with the nuclear counterstain DAPI. (e,f) In situ hybridization combined with immunohistochemistry (for Myosin VIIa, to label IHCs and OHCs) on cryostat sections from P1 cochleae reveal that ic-HCs transdifferentiating from IHCs to ic-OHCs do not express Bcl11b (e) or Insm1 (f) mRNAs, which are expressed by OHCs during their early (embryonic to postnatal) differentiation. Images were taken at apical, middle and basal cochlear positions at P1 and subsequent postnatal days. Images shown are examples of results obtained in at least three separate tissue samples (n=3 biologically independent samples). All scale bars are 20 μm. (g) Schematic summary of all the stainings performed on mice at P1 to P8 in which Tbx2 ablation had been induced by tamoxifen administration at P0. Blue shading denotes the period (P1 to P7) during which transdifferentiating ic-HCs co-express VGLUT3 with OHC markers PMCA and/or Prestin. At no point from P1 to P8 do ic-HCs express the nascent HC marker SOX2 or the OHC differentiation markers Bcl11b (mRNA or protein) or Insm1. Expression levels are subjectively categorized as strong (+++) to undetectable above background (−). Expression in ic-HCs (red font) is provided by comparison to expression in the following control cells (blue font) in the same tissues or stages: Wild type (WT) IHCs for VGLUT3; adjacent OHCs for Prestin, PMCA2, Bcl11b and Insm1; and Supporting Cells (SC) for SOX2. Supporting and other cells labeled in (d) as expressing SOX2 are epithelial cells of Kölliker’s Organ (KO), Inner Border (IBC), Inner Phalangeal (IPhC), Inner Pillar (IPC), Outer Pillar (OPC), Deiters’ (DC) and Hensen’s (HeC) cells.