Fig. 5. Effect of Runx1^4/4 on bulge SC proliferation. (A) Sections from 3- or 4-day-old BrdU-labeled skin (PD20-PD23 or 24) show cells that proliferated during anagen onset. Early anagen wild-type follicle (a,b) shows multiple BrdU+ (red) cells in hair germ and several BrdU+(red) and CD34+ (green) bulge cells (arrows). Telogen Runx1^4/4 follicle shows complete lack of BrdU+ cells in CD34+ bulge cells or germ cells (c,d). Ep, epidermis; Bu, bulge; hg, hair germ; DP, dermal papillae; De, dermis. Asterisk shows hair shaft autofluorescence. (B) Fraction of follicles scored on skin section shown in A that displayed BrdU+ cells in bulge or germ. Sixty-seven percent of follicles have BrdU+ bulge cells for wild-type mice and there is a complete lack of BrdU+ bulge cells for 4 mice. Follicles with BrdU+ germ cells are further subdivided into those with more than two BrdU+ cells/germ and one or two BrdU+ cells/germ. Total number of HFs analyzed from five wild-type (black) and five 4 (gray) littermates is shown (802 wild type & 737 4). Error bars underscore variability of BrdU+ follicle fractions in each category. (C) CD34+/6-integrin+ cells from mice in A,B were sorted on slides, fixed and stained as described (Tumbar, 2006). There is a high frequency of cells that are double positive for keratin 5 (K5, red) and β4-integrin (β4, green, bottom panel). BrdU+ (red) and DAPI (blue) staining (top panel) shows lack of proliferation in 4 but not wild-type bulge cells. (D) Sorted bulge cells from C counted for double expression of epithelial K5 and β4 markers. Un, unsorted live cell control. Number of cells is at the top, ID of mice is at the bottom. (E) Quantification of proliferating (BrdU+) sorted bulge cells from (C). Number of cells is at the top, mouse ID is at the bottom. Negative controls were from BrdU-negative mice.