Supplementary Figure 5: Characterization and quantification of Lrig1 and Gata6 genetically labelled (GL) cells prior to wounding and during re-epithelialization.

(a) Quantification of Gata6 and Lrig1 genetically labeled (GL) cells after tamoxifen treatment, 48 h after wounding in tail skin (n = 5 mice, 116 HF for Gata6; 27 HF for Lrig1). Data are means ± s.d. (b) Flow cytometry analysis showing lack of EdU incorporation by GL Gata6 tail keratinocytes in unwounded skin. Representative flow cytometry plots of epidermal Lrig1 (dark grey) and Gata6 (red) GL cells from back and tail skin (top left panels). Gates in upper panels mark EdU positive cells analyzed in bottom left density plots (dashed green lines demarcate tdTomato negative controls). Quantification of EdU incorporation in all epidermal cells (top right panels) and tdTomato Gata6 (red) or Lrig1 (dark grey) GL cells (bottom right panels) is shown. (n = 6 mice for Gata6; n = 3 mice for Lrig1). Data are means ± s.d. (c) Histological analysis confirming the absence of EdU incorporation into GL Gata6 in unwounded tail skin. Tail skin sections showing tdTomato GL Gata6 (left) or Lrig1 cells (right)) and EdU localization. EdU masks after color threshold analysis with ImageJ software together with tdTomato mask (top panel) or single tdTomato channel (bottom panel) are shown. Representative images of 2 independent experiments. Quantification of EdU incorporation by GL (Lrgi1 or Gata6) cells in JZ/SD (n = 3 mice each; 49HF) is shown. Scale bars, 50 μm (d) Suprabasal Gata6 GL cells acquire a basement membrane position during wound re-epithelialisation. Position in the epidermal layers of Gata6 GL cells at the indicated day after wounding tail skin is shown. Measurements are position relative to the basement membrane of the lowest Gata6 GL cell of a column of labelled cells in the wound bed (“0” corresponds to the basal cell layer). From n = 3 mice for day 5, 9, 12 and n = 4 mice for day 7 (average of 70 columnar cell units per mouse). The yellow bars in the violin plots represent median and black lines the 25th and 75th percentiles. (e) Quantification of flow cytometry analysis of High-Itga6 and Mid/Low-Itga6 expressing Gata6 and Lrig1 GL cells in the tail wound bed at the indicated days after wounding. Data are means ± s.d. from n = 4 mice for Gata6 GL 5d, 12d and Lrig1 GL 5d; n = 3 for Lrig1 GL 12d. Representative flow cytometric plots (right panels) are shown. Light blue lines represent gating strategy used for quantification in left hand panel¹³. ^∗P < 0.05; ^∗∗P < 0.005, by unpaired Student’s t-test. (f) Quantification of Ki67 + Lrig1 and Gata6 tdTomato GL cells at day 6 after wounding. Data are means ± s.e.m. from n = 25 cell clusters.