Fig. 8. C4st1^gt/gt metatarsal explants are able to respond to exogenous growth factors. Metatarsals were removed from E18.5 wild-type (+/+) and mutant (gt/gt) embryos and cultures for 4 days in either the absence (control) or presence of 2 µg/ml N-Shh, 500 pM TGFß or 10nM BMP2 as indicated. Metatarsals were photographed (A,B) or processed for RNA in situ hybridization (C-F) or immunofluorescence (G-J). (A,B) Appearance of explants after 4 day treatment with no factor (control), N-Shh, BMP2 or TGFß. (C,D) Effects of growth factor treatment on hypertrophic differentiation as visualized by collagen X RNA in situ hybridization. Collagen X staining is reduced in N-Shh and TGFß-treated wild-type (C) and mutant (D) explants and increased in BMP2-treated wild-type and mutant explants. In addition, treatment of wild-type, but not mutant explants with N-Shh lead to increased thickness of the perichondrium (arrows). (E-J) Signaling pathways in metatarsal explants. (E) Ptch1 staining in untreated wild-type and mutant explants is restricted to proliferating chondrocytes. No Ptch1 expression is seen in the perichondrium. (F) Treatment of explants with N-Shh leads to Ptch1 expression throughout the growth plate in both wild-type and mutant explants. Wild-type explants also showed Ptch1 expression in the perichondrium, which was not observed in mutant explants (arrows). (G) Nuclear pSmad2 staining in untreated wild-type explants was seen in some late columnar/early hypertrophic cells (i; see iv for higher magnification), whereas occasional weak staining in proliferating cells was also present (i; iii). In untreated mutant explants, strong pSmad2 staining was apparent in all cells of the growth plate (ii, v, vi). (H) Treatment of wild-type explants with TGFß lead to an increase in the number of pSmad2-stained cells and staining intensity in hypertrophic cells (i, iv), whereas cells in the proliferative layer were still mostly negative for nuclear pSmad2 (i, iii). Arrow in i indicates increased pSmad2 staining in lateral regions of the growth plate. Treatment of mutant explants with TGFß lead to a small increase in pSmad2 staining intensity (ii, v, vi). (I) Nuclear pSmad1 expression in untreated wild-type explants was apparent in a subset of proliferating chondrocytes (iii) and in hypertrophic chondrocytes (iv). Whereas mutant explants also showed nuclear pSmad1 staining in hypertrophic chondrocytes (ii, vi), staining intensity was lower in proliferating chondrocytes (ii, v). (J) Both wild-type (i, iii, iv) and mutant (ii, v, vi) explants treated with BMP2 showed strong nuclear pSmad1 staining in the expanded region of hypertrophy (iii, iv, v, vi). Scale bar: 5 mm for A,D; 1 mm for C-F; 500 µm for G-J, parts i, ii; 20 µm for G-J, parts iii-vi.