Matrix metalloproteinase inhibition alters the angle of daughter cell allocation. (A) Snapshot images and cell division tracking data from developing hair follicles in skin explants from eGFP-Col4a2; mem-tdTomato mouse embryos cultured under control or MMP-inhibited conditions. The BM and cell membrane were visualized with eGFP-COL4A2 (green) and mem-tdTomato (magenta), respectively. Scale bars: 20 μm. (B) Frequency of cell division in epithelial basal progenitors in the experiments in A (n = 6 follicles from five explants, each from an independent experiment for the control, n = 6 follicles, each from a separate explant in an independent experiment for the inhibitor-treated). A two-tailed unpaired t test was used. (C) EdU incorporation assays. Dividing cells were labeled with EdU (magenta). All the cells were counterstained with DAPI (blue). Scale bar: 20 μm. (D) Percentage of EdU-positive basal epithelial cells in the tip, lower stalk, and upper stalk regions (n = 7 follicles from three explants, each from an independent experiment for the control, and n = 5 follicles from three explants, each from an independent experiment for the inhibitor-treated). Values: means ± SD. Two-way ANOVA with Tukey’s post-hoc tests was used. (E) Daughter cell allocation angle of proliferating basal epithelial progenitors relative to the BM zone in the experiments in A. Images of perpendicular and horizontal divisions are shown. Scale bar: 5 μm. (F and G) Pie graphs and a plot illustrating the angle of daughter cell allocation relative to the BM under control and MMP inhibitor treatment conditions (n = 48 cells from 11 follicles from seven explants, each from an independent experiment for the control, and n = 36 cells from eight follicles from eight explants, each from an independent experiment for the inhibitor-treated). All statistical tests in this figure assumed normal data distribution, but this was not formally tested.