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Fig. 3. Molecular analyses of chondrocyte maturation, and chondrocyte proliferation assays. (A1-A3,B1-B3,C1-C3,D1-D3) In situ hybridization using ³⁵S-labeled riboprobes against Pthr (A1-A3), Col10a1 (B1-B3), Vegfa (C1-C3) and Mmp13 (D1-D3), on longitudinal sections through the humerus from E18.5 embryos of wild type (A1-D1), Ihh^-/- (A2-D2) or DKO (A3-D3). Shown are the distal halves of the humerus for the wild type (A1-D1) and the DKO (A3-D3) embryo, but the full length for the Ihh^-/- embryo (A2-D2). Hybridization signals are in red. Double-headed arrows (A1-A3,B1-B3) denote zones of immature chondrocytes. Yellow arrows (A1,A3) indicate the perichondrium. Purple arrows (A2-D2) indicate joint fusion with the radius. Yellow asterisks (A2,B2,D2) indicate ectopic hypertrophy of chondrocytes. Purple asterisk (C1) indicates immature chondrocytes. Green asterisk (A3) indicates ectopic bone. PH (A1), prehypertrophic chondrocytes; PS (A1,C1,D1), primary spongiosa; BC (A1), bone collar. Green straight lines (C1,C3,D1,D3) demarcate sub-domains of the hypertrophic zone. Green contours denote the boundary between the hypertrophic cartilage and either the primary spongiosa (C1,D1) or the ectopic bone (C3,D3). (E1-G3) In situ hybridization using ³⁵S-labeled riboprobes against Col10a1 (E1-E3), Pthlh (F1-F3) and Ptc1 (G1-G3), on longitudinal sections from E14.5 embryos of wild type (E1-G1), Ihh^-/- (E2-G2) or DKO (E3-G3), through either the humerus (E1-E3) or the radius (F1-F3,G1-G3). Hybridization signals are in red. Shown are the distal halves of the humerus or the radius for the wild type (E1-G1) and the DKO (E3-G3) embryo, but the full length of either element for the Ihh^-/- embryo (E2-G2). Double-headed arrows (E1-E3) indicate zones of immature chondrocytes. Purple arrows (F1,F3,G1,G3) and blue arrows (G1) indicate the outer and inner layers of the perichondrium, respectively. Yellow asterisks (F1,F3,G1,G3) indicate periarticular chondrocytes. Green asterisk (G1) denotes immature chondrocytes adjacent to the prehypertrophic zone. r, h, u (F2,G2): radius, humerus and ulna, respectively. (H) BrdU labeling index for chondrocytes in the humerus (distal half) of E14.5 embryos of wild type (WT), Ihh^-/- and DKO. The wild-type value is arbitrarily assigned 1.

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