DEV02721 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

  Adobe PDF

• Supplemental Figure 1 -

  Fig. S1. Haematoxylin-Eosin staining of fresh-frozen dorsal skin sections. (A,C,E,G,I) Wild-type skin; (B,D,F,H,J) K14-Gata3^−/− skin. Control skin is in catagen phase of HF cycle at P17 (A) and at P19 (C). HFs enter telogen and around P20 they start a new anagen (E). Regression takes at least 6 days in K14-Gata3^−/− HFs. At P17, K14-Gata3^−/− HFs are at late anagen (B) and catagen starts around P19 (F, H) and HFs enter resting phase at P27 (H), while control skin is in the middle of anagen (G). At 5 months of age there are no visible hairs produced in K14-Gata3^−/− skin (J) compared to control (I). Scale bar in A: 200 μm.

• Supplemental Figure 2 -

  Fig. S2. Gata3 expression visualized by X-Gal staining in the Gata3LacZ knock-in skin. (A-C)Arrows indicate double-stained cells with K14. Arrowhead indicates sebaceous gland. Scale bar in A: 150 μm for A; 50 μm for Ai-C.

• Supplemental Figure 3 -

  Fig. S3. Thickened layers in K14-Gata3^−/− skin. Suprabasal, basal and corneal layers are much thicker in the K14-Gata3^−/− skin (B) as compared to control skin (A) at P13. Stratum corneum consists of acellular flaky material (B). Thickness of stratum corneum (C) and epidermis (D) measured in control (white bars) and K14-Gata3^−/− skin (black bars) at P0, P7, P11 and P13. SCL, stratum corneum layer; EL, epidermal layer. Data are given as mean±s.e.m; *, P&λτ;0.05; Student’s t-test. Scale bar in A 50 μm

• Supplemental Figure 4 -

  Fig. S4. In situ mRNA hybridization and protein staining of factors associated with the cell cycle. The expression of cell cycle-related factors in the HF of K14-Gata3^−/− (B,D,F,H,J,L) and control mice (A,C,E,G,I,K). Diminished expression of cyclin D2 (A,B), cyclin E1 (C,D), cyclin A2 (E,F) and Cdk4 (G,H) in most cells located within the hair bulb (region ‘a’ in wt and K14-Gata3^−/− skin). Elevated expression of cyclin D2 (A,B), cyclin A2 (E,F), Cdk4 (G,H) and cyclin D1 (I,J) in the ORS (region b). Arrows indicate expression. Scale bar in A: 150 μm for A,B, E-J, 200 μm for C,D.

• Supplemental Figure 5 -

  Fig. S5. BMP pathway molecules in the K14-Gata3^−/− HFs. (A,B) Higher levels of Bmp6 expression in K14-Gata3^−/− HFs (B) compared to wt controls (A). (C-F) Higher and more expanded phosphorylation of Smad1/5/8 proteins in K14-Gata3^−/− HFs (D) and epidermis (F) compared to wt (C, E, respectively). (G,H) Lack of expression of Gremlin in K14-Gata3^−/− HF (H) compared to control (G). (I-L) Lack of expression of Id2 protein (target for Bmp) in K14-Gata3^−/− IRS (J) and upregulation of Id2 expression in the epidermis and distal part of HF in K14-Gata3^−/− skin (L) compared to control (I, K). Scale bar in A: 150 μm for A,B; 100 μm for C-L.

• Supplemental Figure 6 -

  Fig. S6. Stem cells markers in K14-Gata3^−/−. (A-F) Upregulation of K15 expression in K14-Gata3^−/− HFs and epidermis compared to control. (G-J) Upregulation of K15 expression in K14-Gata3^−/− HFs and epidermis compared to control. Scale bar in A: 50 μm for A,B;150 μm for C-J.

• Supplemental Figure 7 -

  Fig. S7. RT-PCR verification of microarray results. Gli1, Nkd2, Wnt5a, cyclin D2, cyclin D1, Bmp6, S100A4, Notch1, Cutl1 and Crym were used to verify the data reported from microarrays.