Fig. 4: A migratory wounded epithelium forms within one day post-injury.

a Krt5-CreERT2;R26R-Confetti mice were injected with a single dose of 30 mg of tamoxifen 3 days prior to TM injury to induce minimal labeling of TM KCs. Perforations were made in the left TMs of mice on day 0, and both TMs were harvested at the indicated timepoints. b Control TMs from the right ear (top row) show sparse labeling with the Confetti reporter. Perforated TMs from the left ear (bottom row) show increased labeling over the malleus at early timepoints, with a concentration of labeled cells at the site of injury at later timepoints (n = 5 mice per timepoint). White dotted circles indicate the perforation c Heat-map showing expression of top genes associated with the wounded epithelial cell state in KC clusters from the unwounded TM and day 1 post-injury. Each column represents a single cell. d–f Violin Plots showing expression of BC100530 (d), Gpr15L (e), and Odc1 (f) in KCs from each timepoint. g RNAscope for Gpr15L (green) expression on whole-mount TMs throughout the regenerative time course (top row). Higher magnification regions show expression of Gpr15L (green) over the malleus at day 1 and near the perforation by day 7 (bottom row), suggesting migration of the wounded epithelium. Scale bars: 100 μm (H) Cartoon model of the origination and migration of the wounded epithelium on the TM.