Supplementary Figure 8: Time lapse imaging of migration of suprabasal Gata6 GL cells during wound healing in adult ear skin.

(a) A single stack image at 0 h indicating the wound edge (magenta dotted line), HFs (white dotted circles), nuclei and collagen (green) and Gata6 genetically labeled (GL) cells (red). The thickness used to reconstruct the orthogonal images to be able to follow the entire group of Gata6 GL cells over time shown in Fig. 7a is indicated by the yellow rectangle. Representative image of 6 independent experiments. Scale bars, 100 μm. (b) Quantification of the speed of cell migration according to whether cells moved upward, downward or straight. (c,d) Representative examples of upward (c) and straight (d) and migration of Gata6 GL cells (white arrows) are shown. Orthogonal images at the indicated time points from the beginning of image acquisition showing tdTomato labeled cells (red), nuclei and collagen (green). White lines indicate the basal membrane. Cartoons summarising the cell movements are shown in the top left panels. Scale bars, 25 μm. (e,f) Cell shape and size analysis of Gata6 GL cells during wound healing. Snapshots from live imaging during wound healing (Fig. 7) were analyzed to assess size and shape of Gata6 GL cells that attached to the basement membrane compared to Gata6 GL cells that maintained a suprabasal position and unlabelled keratinocytes. Heat-mapped XY -view of the shapes of suprabasal Gata6 GL cells (left panel, n = 18 cells) and Gata6 GL cells that attached to the basement membrane (BM) (right panel, n = 9 cells) (e). Individual cells were aligned to the same XY centroid position and rotated to align wound direction horizontally to the right. Note that newly BM-attached GL cells were elongated toward the axis of wound direction as quantified in (f) (Right panel). Quantification of Gata6 GL cell size with reference to control keratinocytes in mouse ear skin (f). The reference cell size was obtained by in vivo labelling of keratinocytes by injection of Isolectin into WT mouse ear skin. Note that Gata6 GL cell that newly attached to the basement membrane had a similar size to normal basal keratinocytes (left panel). Quantification of cell elongation toward the axis of wound direction through the quantification of the migration shape index (MSI), defined as the ratio (α/β) of cell length projected to the axis of wound direction (α) over cell length perpendicular to the axis (β). Blue dotted line indicates MSI = 1, no elongation (right panel). Data are means ± s.d. from n = 21 Basal cells (reference); n = 20 Suprabasal cells (reference); n = 18 Suprabasal Gata6 GL cells; n = 9 newly BM-attached Gata6 GL cells. ^∗P < 0.05; ^∗∗P < 0.005 by unpaired Student’s t-test.