Scratch-induced partial skin wounds re-epithelialize by sheets of independently migrating keratinocytes

(A) Measurements of cell areas (μm²) of basal cells based on E-cad-CFP expression in different distances towards the wound, directly after wounding (0), 8, 16, and 24 h post wounding. (n = 2 individual mice, with four wounds each per time point). (B) Measurements of cell areas of basal (B, magenta) and suprabasal cells (SB, yellow), directly after wounding (0), 8, 16 and 24 h post wounding. (n = 2 individual mice, with four wounds each per time point). (C) Schematic representation of three proposed models of modes of migration during re-epithelization. (D) Representative sequential images of intravital microscopy of the suprabasal layer (top panel) and the basal layer (bottom panel) after scratch wounding. Scale bar, 100 μm. See also Video 1. (E) Representative images of intravital microscopy of a defined area in proximity (290 μm away from the wound edge) to the wound bed starting 16 h after wound induction; suprabasal keratinocytes (top panel) and basal keratinocytes (bottom layer). Individual basal keratinocytes highlighted with a yellow outline. Scale bar 100 μm. See also Video 2. (F) Quantification of mean migration velocity in relation to cell size 16 h after induction of scratch wounding. (n = 5 individual mice). Dashed line indicating 380 μm², separating basal from suprabasal keratinocytes. (G) Quantification of mean migration velocity of migratory (>10 μm/8 h) basal keratinocytes in relation to the distance of the wound 16 h after induction of scratch wounding. Median and interquartile distances are plotted for the distance bins <0.5 mm; 0.5–1 mm; >1 mm. (n = 5 individual mice). ****P > 0.0001, Two-tailed Mann–Whitney U-tests were performed for all statistical analyses. (H) Rose plot showing migration directionality in respect to the wound site of representative basal (left) and suprabasal cells (right). Inner rose plot in suprabasal cells represents a zoom of the initial rose plot. Value 0 indicates the relative starting point of migration 16 h post wounding.

Source data are available for this figure.

(A) Representative images of a migrating pairs at different distances to the scratch of recombined GFP-expressing cells in a R26-CreERT2:R26-mTmG mouse 16 h after scratch wound induction. Scale bar, 50 μm. (B) Reconstruction of migrating cell pairs and their neighboring cells, followed over minimal 2 h 30 min, 16 h after scratch wounding. The reconstructions depict three examples of cell group with different distances to the scratch wound. (C) The fraction of original neighbors that are remained is plotted over time. Basal keratinocytes are grouped based on initial distance to scratch wound (>1 mm; 0.5–1 mm; <0.5 mm). Imaging was performed 16 h post scratch wounding for 8 h. Plotted is the mean ± SEM for 25 imaging positions in three mice. ANOVA multiple comparison with *P > 0.1, **P > 0.01, and ****P > 0.0001.

(A) Representative images of migrating keratinocytes in a Fucci2 mouse 16 h after scratch wound induction bypassing a hair follicle (*). Scale bar, 50 μm. Individual keratinocytes are highlighted by their migration tracks. Also see Video 3. (B) Representative sequential images of labelled cells within mTmG mice 16–24 h post wounding within the infundibulum and upper hair follicle (upper panel) and cells within the basal layer (lower panel). Migration/no migration is indicated by the shifts in yellow circles over time. (C) Quantification of total displacement in different distances towards the wound of hair follicle (grey) and basal cells (blue), respectively. Data points represent single cells from two mice, with five different wounds per mouse. ****P < 0.0001; **P = 0.002 (unpaired t test with Welsh correction).

Source data are available for this figure.

(A) Left panel: fluorescence staining of EdU incorporation (green) immediately after wounding and 8, 16 and 24 h post wounding. LN332 (magenta) immunofluorescence counterstain marks the basement membrane. Scale bar 100 μm. Right panel: quantification of EdU incorporation, each dot represents one wound in n = 2 mice. (B) Representative image of a scratch wound in Fucci2 mice. Cells in G1-phase (magenta), in G2/S-phase (green) in their different distances towards the wound. Scale bar 100 μm. (C) Quantification of percentage of proliferating (G2/S, green) and non-proliferating cells (G1, magenta) grouped based on their distance to the wound (0–200, 200–400, 400–600, 600–800, and 800–1,000 μm). **P = 0.0086 (ANOVA multiple comparison). (D) Percentage of proliferating G2/S cells within a distance of 200–400 μm away from the wound bed is plotted between 16 and 24 h post wounding. Plots depict the min., max., and median, the lower and upper quantile, the plus indicates the mean. (E) Linear regression analysis between scratch size (μm) and percentage of G2/S-phase proliferating cells 200–600 μm away from the wound site. R² = 0.8241. (F) Linear regression analysis between scratch size (μm) and average migration velocity (μm/h) of basal keratinocytes 0–200 μm away from the wound site. R² = 0.6129. (G) Quantification of migration velocity of proliferating S/G2-phase cells (green) and non-proliferating G1-phase cells (magenta) within different distances towards the wound. ns, not significant (unpaired t test). (H) Quantification of persistence of migratory proliferating S/G2-phase cells (green) and non-proliferating G1-phase cells (magenta) within different distances towards the wound. ns, not significant (Kruskal–Wallis test).

Source data are available for this figure.