GLAST-Cre⁺ cells from the GER mitotically regenerate IPhCs.

Representative images of apical turns are shown. (A, C) Cryosections of P4 and P7 control cochleae with Fabp7⁺ Sox2⁺ IPhCs (arrowheads). (B) Most Fabp7⁺ Sox2⁺ IPhCs have degenerated in P4 DT-treated Lgr5^DTR/+ cochlea. (D) In the P7 DT-treated Lgr5^DTR/+ cochlea, many Fabp7⁺ Sox2⁺ IPhCs were found. Sox2⁺ SCs in the lateral compartment have degenerated at this age. (E, G) Merged z-stack images of whole mount preparation of control cochleae at P4 and P7 showing Fabp7⁺ Sox2⁺ IPhCs. (F) Most Fabp7⁺ Sox2⁺ IPhCs have degenerated in the P4 DT-treated Lgr5^DTR/+ cochlea. (H) Replenished Fabp7⁺ Sox2⁺ IPhCs in P7 DT-treated Lgr5^DTR/+ cochlea. (E’-H’) Single-plane images from the same representative sample shown in (E-H) highlighting Fabp7⁺ IPhCs. (I) Quantification showing significantly fewer Fabp7⁺ IPhCs in DT-treated Lgr5^DTR/+ cochlea relative to controls and significantly more Fabp7⁺ IPhCs in all cochlear turns at P7 than P4. (J) Schematic of GLAST-tdTomato expression in the P3 GLAST^CreERT/+; R26R^tdTomato/+ cochlea (tamoxifen at P1). Experimental paradigm using GLAST^CreERT/+; R26R^tdTomato/+ (control) or Lgr5^DTR/+; GLAST^CreERT/+; R26R^tdTomato/+ mice (damage). DT and tamoxifen were injected at P1, EdU from P3 to P5, and cochleae examined at P3 and P7. (K, M) In the P3 and P7 control cochlea, there were no EdU⁺ tdTomato⁺ Sox2⁺ SCs in the GER. Some Sox2⁺ IPhCs were tdTomato⁺, and none were EdU⁺. (L, N) In the P3 damage cochlea, EdU⁺ tdTomato⁺ Sox2⁺ SCs were detected in the GER but not in the IPhC region. At P3, many Sox2⁺ IPhCs had degenerated, and none were tdTomato⁺. At P7, many EdU⁺ tdTomato⁺ Sox2⁺ SCs occupied both the GER and the IPhC regions. Orthogonal images (L’ and N’) showing EdU⁺ tdTomato⁺ Sox2⁺ cells in the GER at P4 and in the IPhC region at P7. L” and N” are high magnification images from L and N. (O) Quantification of (total and EdU⁺) GLAST-tdTomato⁺ Sox2⁺ IPhCs at P3 and P7. Control cochleae had no EdU⁺ GLAST-tdTomato⁺ Sox2⁺ IPhCs at both ages in all turns examined. In the damaged cochleae, there were significantly more tdTomato⁺ Sox2⁺ IPhCs (both EdU⁺ and EdU-negative) at P7 relative to P3. Though present in all 3 turns, EdU⁺ GLAST-tdTomato⁺ Sox2⁺ IPhCs decreased in an apex-to-base gradient at P7. (P) P7 control cochlea with Fabp7⁺ tdTomato⁺ IPhCs. (Q) P7 damage cochlea with many Fabp7⁺ tdTomato⁺ IPhCs (asterisks) arranged in a disarrayed pattern. Q’ and Q” are single and dual channel images of Q. Q”‘ and Q”“are high magnification images of traced Fabp7⁺ IPhCs from Q’ and Q”. Data represent mean ± SD. **p < 0.01, ***p < 0.001. Two-way ANOVA with Tukey’s multiple comparisons test. n = 4–9. See S1 Data for I, O. DC, Deiters’ cell; DT, diphtheria toxin; GER, greater epithelial ridge; IHC, inner hair cell; IPhC, inner phalangeal cell; OHC, outer hair cell; PC, pillar cell; SC, supporting cell.