DEV0012799 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Runx1^LacZ/b+ detection during hair development. X-Gal staining of Runx1^LacZ/+ skin documents dynamic Runx1 expression in hair compartments. Note expression in bulge and other hair compartments during morphogenesis and hair cycle. No expression is found in the epidermis (Ep) but some cells of the dermis underlining the dermal-epidermal junction in newborn skin (PD0) were found positive see De* below broken line demarcating epidermis (Ep) in the upper left corner panel. Bu, bulge; ORS, outer root sheath; DP, dermal papillae; hg, hair germ; ES, epithelial strands; De, dermis.

• Supplemental Figure S2 -

  Fig. S2. Runx1 expression in skin and hair follicle. (A) Skin from Runx1^LacZ/+ mice at telogen-anagen transition immunolabeled for β-Gal and proliferation marker Ki67 and counterstained for β4-integrin. Left and right panels are serial sections of the same hair follicles. (B) Skin from wild-type mice in anagen (PD24) shows triple co-immunostaining for Runx1 (red), Ki67 (green) and CD34 (blue). Arrows show Runx1+/Ki67+ cells, arrowheads show Runx1+/Ki67−, and asterisk shows Runx1−/Ki67+ cells. Bu, bulge; Mx, matrix; HS, hair shaft; ORS, outer root sheath; Ep, epidermis; hg, hair germ. Scale bar: 50 µm.

• Supplemental Figure S3 -

  Fig. S3. Remodeling of hair germ structure during prolonged quiescence of Runx1^Δb4/b/Δb4 follicles. (A) H&E staining of PD21 WT and Δ4 skin shows lower ORS or germ morphology consistent with end of catagen (stage VIII) or telogen. Note larger size of germ areas marked by dotted lines in Δ4 follicles. (B) H&E staining of Δ4 skin sections shows progressive decrease in germ size and cell numbers at stages indicated. WT follicles are in anagen (Fig. 3F). Images are representative of germ morphology analyzed in n=11 animals (3 WT mice PD21, 3 Δ4 mice at PD21 and 5 Δ4 mice at PD23-PD29).

• Supplemental Figure S4 -

  Fig. S4. Effect of Runx1 disruption on apoptosis and proliferation. All follicles are from 4-day BrdU-labeled PD24 Δ4 or WT skin as shown on each panel, with quadruple staining. Left and right panels are different stains of the same follicle. Stains are indicated on each image in appropriate color. (A) Arrow points to weakly labeled BrdU+ cell in the hair germ, which was negative for caspase co-staining. (B) Conversely, caspase positive cell shown by arrow does not have BrdU. (C) Caspase-positive cell expresses K5 (arrow). (D) WT follicles show proliferating but no caspase+ cells at this stage. DP, dermal papillae. (E) Stat3 shows ∼2.8 fold increase in bulge cells of 3 Δ4 vs 3 WT animals by qRT-PCR in experiments performed in duplicate (P<0.1).

• Supplemental Figure S5 -

  Fig. S5. Effect of Runx1 disruption on weight and hair follicle differentiation. (A) Weight of Δ4 and WT animals at PD28-PD30. Average weight was 12.57g (±2.9) for Δ4 animals (n=25), 13.7g (±1.2) for small WTs (n=10) and 20.4g (±2g) for big WTs (n=19). (B) Percentage of animals with telogen (black) skin at P28-30 in the 3 weight categories defined in A. Skin color was assessed by visual inspection of shaved animals. Mice with gray/black patches on the back were scored in anagen.

• Supplemental Figure S6 -

  Fig. S6. Lack of all differentiated lineages in Runx1 mutant hair follicles. (A) Runx1^Δ4/Δ4 hair follicles show no differentiated cell lineage marker expression. Skin sections from wild type (WT) and mutant (Δ4) animals at PD21 and PD29 (n=2 in each group to a total of 8 mice analyzed) show expression of markers indicated in corresponding color. Bu, bulge; hg, hair germ; Dp, dermal papillae, Mx, matrix; IRS, inner root sheath; preC, precortex; Me, medulla. Scale bars: 10 µm (note scale difference in anagen and telogen follicles). *Auto-fluorescence from the hair shaft. Θ, non-specific streptavidin staining of the sebaceous gland. (B) Schematic summarizing the Runx1 phenotypes, which suggest its role at the stem cell level.

• Supplemental Figure S7 -

  Fig. S7. Skin color indicates hair growth in uninjured WT and injured Δ4 mice. (A) Skin color of Δ4 and WT shaved mice at PD29 is pink (telogen) or black (anagen), respectively. (B) Hair growth spreading from injured into un-injured Δ4 skin. Skin is pink after hair pluck. Hair growth (black) and its spreading indicated by arrow. Gray color is early anagen. (C) Left: mouse at the second round of wounding shows gray skin patch in the previously wounded area (arrow) and pink skin in the newly plucked area (arrowhead). Right: same mouse 2 weeks later shows black skin indicative of hair growth in both the old (arrow) and newly (arrowhead) injured areas.