(A) Schematic of hair cell ablation. P1 Pou4f3^DTR/+ and Pou4f3^DTR/+ Sox2^CreERT2/+ pups were injected with DT, and cochleae were examined on P3 and P4. (B) Cartoon depicts supporting cells forming transitional cells during regeneration. (C) Confocal images of P3 WT cochlea show no Atoh1 or Gfi1 expression in supporting cells. (D) After hair cell damage in the Pou4f3^DTR/+ cochlea, some Sox2⁺ supporting cells expressed Atoh1 (chevrons) and Gfi1 (arrowheads), both early hair cell markers. All Gfi1⁺Sox2⁺ supporting cells expressed Atoh1, but some Atoh1⁺Sox2⁺ supporting cells did not express Gfi1. (E) Quantification of transitional cells (Atoh1⁺Sox2⁺ and Atoh1⁺Sox2⁺Gfi1⁺) from WT and Pou4f3^DTR/+ cochleae. (F) P4 WT cochlea showed Gfi1 expression limited to hair cells and no Atoh1 or Gfi1 expression in Sox2⁺ supporting cells. Atoh1 was absent in hair cells. (G) After DT-induced hair cell loss in Pou4f3^DTR/+ cochlea, Gfi1 was downregulated in the remaining myosin 7a⁺ hair cells. Many transitional cells (arrows) (Atoh1⁺Sox2⁺myosin 7a⁺Gfi1⁺) were detected in all 3 cochlear turns. Like the P3 Pou4f3^DTR/+ cochlea, all transitional cells expressed Atoh1 and Sox2. In contrast to the P3 Pou4f3^DTR/+ cochlea, most transitional cells expressed myosin 7a by P4. Myosin7a+ cells with no expression of Sox2, Atoh1, or Gfi1 (chevron) likely represent surviving hair cells (G and H). (H) In the P4 Pou4f3^DTR/+ Sox2^CreERT2/+ cochlea, there were noticeably more transitional cells (arrows). (I) Quantification of transitional cells (Atoh1⁺Sox2⁺myosin 7a⁺Gfi1⁺ and Atoh1⁺Sox2⁺Gfi1⁺) in WT, Pou4f3^DTR/+, and Pou4f3^DTR/+ Sox2^CreERT2/+ cochleae. Hair cell ablation led to a significantly greater number of transitional cells in each cochlear turn compared with that seen in controls. There were significantly more transitional cells detected in each turn of damaged, Sox2^haplo cochleae than in the damaged-only cochleae. The SD of transitional cell counts in the basal turn of damaged, Sox2^haplo cochleae is zero. Data represent the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Holm-Sidak multiple comparisons test. n = 3. Scale bars: 20 μm.