Fig. 7. Analysis of Ihh, BMP and TGFß signaling in E18.5 cartilage. (A) distal tibia; (B-H) proximal tibia. (A) Ptch1 RNA in situ hybridization as output for Ihh signaling reveals expression in columnar chondrocytes in both wild-type (i) and mutant (ii) cartilage. (B-D) Phosphorylated Smad1 distribution. Wild-type (i) and mutant (ii) growth plates were stained using an antibody that recognizes Smad1 phosphorylated by activated BMP receptors. (B) pSmad1 (red) is seen in hypertrophic chondrocytes in wild-type cartilage (i), and is reduced in mutant cartilage (ii). (C) Higher magnification of proliferating region (indicated as `C' in B) shows very little pSmad1 staining in both wild-type (i) and mutant (ii) cartilage. (D) Higher magnification of early hypertrophic regions (indicated as `D' in B) shows nuclear pSmad1 staining in wild-type chondrocytes (i), which is reduced in mutant chondrocytes (ii). (E-H) Phosphorylated Smad2 distribution. Wild-type and mutant growth plates were stained using an antibody that recognizes Smad2 phosphorylated by activated TGFß receptors. (E) Very little nuclear pSmad2 staining was seen in wild-type growth plates (i) in columnar/early hypertrophic layers and in lateral aspects of the growth plate (white arrow). In mutant cartilage (ii), strong pSmad2 staining is seen in all cartilage layers. (F-H) Higher magnification of regions indicated in E. Arrows indicate nuclear staining. (F) Higher magnification of proliferative layer. No nuclear pSmad2 staining was seen in wild-type chondrocytes (i), whereas a high proportion of mutant chondrocytes (ii) shows moderate nuclear pSmad2 staining. (G) Higher magnification of late columnar layer. Very little nuclear pSmad2 staining is visible in wild-type cells (i), whereas strong nuclear pSmad2 staining is apparent in all mutant chondrocytes (ii). (H) Higher magnification of hypertrophic layers. Weak nuclear pSmad2 staining is seen in few wild-type chondrocytes (i), whereas almost all mutant chondrocytes show strong nuclear pSmad2 staining (ii). Scale bar: 300 µm for A; 200 µm for B,E; 20 µm for C,D,F,G,H.