Fig 2: Effects of Ptch2 abrogation on skin thickness and wound healing. (A, B) Immunofluorescence staining for Ptch2 (purple) and Lhx2 (green) in wild-type sections of P1 (A), P14 (B). White arrowheads indicate Ptch2 expression. Inset in (B) is a cross-section through P50 hair follicle processed with Lxh2 and Ptch2 immunostaining. (C) Immunofluorescence staining for Lhx2 (green) in adult (P50) wild-type and Ptch2 -/- skin. (D, E) Quantitative analysis of skin thickness in wild-type and Ptch2 -/- at P1 and P50. (F) H&E staining of paraffin sectioned skins obtained from wild-type and Ptch2 -/- mice at indicated time points. (G) Quantitative analysis of dermis thickness in wild-type and Ptch2 -/ . Scale bar red = 50 µm, white = 100 µm, black = 0.5 mm, ****p < 0.0001. (H–M) H&E staining (H,I) and immunofluorescence against Keratin 14 (J,K) and pHH3 (L, M) of sectioned wild-type and Ptch2 -/- skin wounds at day 7 post-wounding. Scale bars (black and white) = 0.5 mm. Arrows indicate wound edges in the Ptch2 -/- wounds.

Fig 3: Loss of Ptch2 affects bone size and SC potential. (A) Body weight of P28-35 wild-type, Ptch2 +/- , and Ptch2 -/- mice. (B–F) Quantification?fµCT analysis of P28-33 femora and tibiae isolated from wild-type, Ptch2 +/- , and Ptch2 -/- littermates. (G,H) Radioactive in situ hybridization in 2-week-old tibiae detecting Ptch2 (G) and Ptch1 (F). (I,J) Immunostaining for Ptch1 (green) and Ptch2 (red) in the growth plate (I) and cortical bone (J) of 4-week-old tibiae. Arrowheads indicate Ptch2+ cells in the resting zone of the growth plate (I) and endosteum (J). Arrow in (J) indicates Ptch2+ cells in the periosteum layer. (K–O) Analysis of the mineralization (K–M) and adipogenic (N,O) differentiation potential of bone marrow stromal stem cells. Alizarin Red Staining (L) of terminated cultures at day 21 of culture and Nile Red imaging (N) of live adipocytes in 14-day-old cultures of bone marrow stromal stem cells isolated from 8-week-old animals. P-values: *p = 0.05, **p = 0.01, ***p = 0.001. Scale bars 100 µm.

Fig 4: Analysis of Ptch receptor-dependent inhibition of Hh signaling. (A,C,E) Immunocytochemistry detection of Gli3 (A), Gli1 (C) and Gli2 (E) in the wild-type, Ptch1 -/- , and Ptch2 -/- MEFs. Scale bar 50 µm. (B,D,F) Quantification of the intensity of the nuclear GLI3 (B), GLI1 (D), and GLI2 (F) in wild-type, Ptch1 -/- , and Ptch2 -/- MEFs. **p = 0.005, ***p = 0.0005, ****p < 0.0001. (G) Western blot showing nuclear (N) and cellular (C) expression of Gli2 (FL = full length and RF = repressor form) in wild-type, Ptch1 -/- and Ptch2 -/- MEFs.