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First published online 1 September 2005
doi: 10.1242/dev.02025

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Ihh controls cartilage development by antagonizing Gli3, but requires additional effectors to regulate osteoblast and vascular development

¹ Department of Medicine, Washington University Medical School, St Louis, MO 63110, USA
² Department of Molecular Biology and Pharmacology, Washington University Medical School, St Louis, MO 63110, USA

View larger version (74K): [in a new window]   Fig. 1. Partial rescue of the skeletal growth in the DKO embryo at E18.5. (A1-A5) Forelimbs of embryos of various genotypes: wild type (A1), Gli3-/- (A2), DKO (A3), Ihh-/-; Gli3+/- (I-/-; G+/-, A4) and Ihh-/- (A5). (A1) s, scapula; h, humerus; r, radius; u, ulna; mc, metacarpal; p, phalanges; *, deltoid tuberosity. (A2) Numbers 1-7 indicates polydactyly. (A3) Red arrowhead denotes bifurcation of the proximal phalange (P1). Inset shows higher magnification of boxed region. Green arrowhead indicates ectopic bone. (B1-B5) Hindlimbs from embryos of various genotypes: wild type (B1), Gli3-/- (B2), DKO (B3), Ihh-/-; Gli3+/- (I-/-; G+/-, B4) and Ihh-/- (A5). (B1) fe, femur; t, tibia; fi, fibula; mt, metatarsal; p, phalanges. (B2) Numbers 1-6 indicates polydactyly. (B2,B3) Big red arrowheads indicate truncation of the tibia (t*). Small red arrowhead indicates bifurcation of the proximal phalange (P1). Inset shows higher magnification of boxed region. Green arrowhead indicates ectopic bone. (C1-C4) Rib cages from embryos of various genotypes: wild type (C1), Ihh-/- (C2), DKO (C3) and Gli3-/- (C4). Double-headed arrows (C1-C4) indicate the width of the rib cage. Asterisks (C3,C4) indicate the split sternum. (D1-D3) Skulls of embryos of various genotypes: wild type (D1), Ihh-/- (D2) and DKO (D3). Asterisks (D1-D3) denote the chondrocranium. (E) Detection of Gli3 in limb cartilage proteins. Gli3A, the full-length (190 kDa) activator form of Gli3; Gli3R, the truncated (83 kDa) repressor form of Gli3. -cat., -catenin (102 kDa) used as normalizer. Inset shows longer exposure of the Gli3A band. Asterisk indicates a non-specific protein (80 kDa) reacting with the antibody. (F1-H2) In situ hybridization using 35S-labeled riboprobes against Gli1 (F1,F2), Gli2 (G1,G2) and Gli3 (H1,H2) on longitudinal sections through the ulna (F1,F2,H1,H2) or the humerus (G1,G2) from E14.5 wild type (F1-H1) or Ihh-/- (F2-H2) embryos. Shown is the distal region of each element. Green arrows indicate expression in the perichondrium. Green asterisks indicate the periarticular region. P, proliferative chondrocytes; H, hypertrophic chondrocytes. r, h, u (F2,H2) indicate radius, humerus and ulna, respectively.

View larger version (109K): [in a new window]   Fig. 2. Restoration of chondrocyte morphology and perichondrium growth in the humerus of the DKO embryo at E18.5. (A-C) Low-magnification images of Hematoxylin and Eosin stained sections from the wild type (A), Ihh-/- (B) and DKO (C) embryos. Red arrows indicate the entire growth region. Green and black arrows (A,C) mark the non-hypertrophic, and the hypertrophic regions, respectively. Asterisks indicate areas of vascularization. Rectangle boxes of different colors identify various areas shown at a higher magnification below. (D1-D3,E1-E3,F1-F3,G1-G3) Images at a higher magnification of areas in A-C that are boxed in different colors: green, red, blue and black respectively. Red and green arrows (F1,F3,G1-G3) indicate cuboidal and elongated cells, respectively. Double-headed arrows (F1-F3) indicate width of perichondrium. C (F1-F3), chondrocytes; M (F2,G2), muscle. Yellow contours (F1,G1,G3) indicate the bone collar. Green lines (G1-G3) demarcate the outer layers of the perichondrium. Asterisks (G2,G3) indicate areas of vascularization.

View larger version (116K): [in a new window]   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.

View larger version (94K): [in a new window]   Fig. 4. Defects in bone formation and cartilage vascularization in the DKO embryo. (A1-A4,B1-B4,C1-C4) Von kossa staining on longitudinal sections of the ribs (A1-A4), the humeri (B1-B4) and the femurs (C1-C4) from E18.5 embryos of wild type (A1-C1), Ihh-/- (A2-C2) and DKO (A3-C3,A4-C4). (A1-A3,B1-B3,C1-C3) Images at a low magnification. The ribs (A1-A3) were oriented with the ventral side towards the left, and the humeri (B1-B3) and the femurs (C1-C3) the distal end towards the left. Arrows denote either the normal bone collar (A1-C1), or the corresponding perichondrium where the bone collar is missing (A2-C2,A3-C3). Red asterisks, mineralized cartilage; purple asterisk (A3), marrow cavity. (A4-C4) Images at a higher magnification of boxed areas in A3-C3, respectively. Arrows indicate ectopic bone. Double-headed arrows (B4,C4) indicate the perichondrium separating the ectopic bone from mineralized cartilage. (D1-D3) Hematoxylin and Eosin staining of sections through the equivalent ribs from E18.5 embryos of wild type (D1), Ihh-/- (D2) and DKO (D3). Double-headed arrows indicate the length of the hypertrophy zone. Asterisks (D1,D3) indicate areas of vascularization.

View larger version (103K): [in a new window]   Fig. 5. Molecular defects in osteoblast development in the DKO embryo. (A1-E3) In situ hybridization using 35S-labeled riboprobes against Col10a1 (A1-A3), Runx2 (B1-B3), Ap (C1-C3), Osx (D1-D3) and Bsp (E1-E3), on longitudinal sections through the humerus from E15 embryos of wild type (A1-E1), Ihh-/- (A2-E2) or DKO (A3-E3). Shown are the distal regions of the humerus for the wild type (A1-E1) and the DKO (A3-E3) embryo, but the full-length for the Ihh-/- embryo (A2-E2). Hybridization signals are in red. Red and green arrows indicate signals in the perichondrium; red and green asterisks indicate signals in chondrocytes (see text). e, epiphysis; d, diaphysis; t, tendon. (F1,F2,G1,G2) In situ hybridization using 35S-labeled riboprobes against Tcf1 (F1,F2) and Dkk1 (G1,G2) on longitudinal sections through the humerus from E14.5 wild type (F1-G1) and DKO (F2,G2) embryos. Shown are the distal regions of the humerus. Red arrows indicate the perichondrium. (H1,H2) In situ hybridization using 35S-labeled riboprobes against Tcf1 on longitudinal sections through the humerus from E18.5 embryos of wild type (H1) and DKO (H2). Red arrows indicate either signals in the perichondrium (H1) or the corresponding regions missing the signals (H2). Red asterisk (H2) indicates the ectopic bone. H, hypertrophic zone; PS, primary spongiosa.

View larger version (147K): [in a new window]   Fig. 6. Lack of orthotopic bone formation at E18.5. In situ hybridization using 35S-labeled riboprobes against Ap (A1,A2), Bsp (B1,B2), Osx (C1,C2) and Oc (D1,D2), on longitudinal sections through the humerus from E18.5 wild type (A1-D1) and DKO (A2-D2) embryos. Shown are the distal halves of the humerus. Green arrows indicate either signals in the perichondrium (A1-D1) or the corresponding regions missing the signals (A2-D2). Black double-headed arrows (A2-D2) indicate the widened hypertrophy zone. Green asterisks (A2-D2) indicate the ectopic bone. BC, bone collar; PH, prehypertrophic zone; H, hypertrophic zone; PS, primary spongiosa.

View larger version (79K): [in a new window]   Fig. 7. Osteoblast versus chondrocyte differentiation from the perichondrial progenitors. (A-D) Histology of the femur from an E18.5 DKO embryo. Areas in yellow, green and blue boxes (in A) are shown at a higher magnification in B-D, respectively. H, hypertrophy zone; PC, perichondrium; RC, red blood cells. Red asterisks in A,B indicate mesenchymal cells; green asterisk in C marks the death zone. (B1-B3) In situ hybridization using 35S-labeled riboprobes against Ap (B1), Osx (B2) and Col10a1 (B3), on sections adjacent to that in A. Areas similar to that in B are shown at the same magnification. Hybridization signals are in red. PC, perichondrium. (E,F) Histology of the humerus from an E18.5 Dermo1-Cre; ß-cateninc/c embryo. Area in purple box (E) is shown at a higher magnification (F). Asterisks in E,F indicate ectopic chondrocytes. (F1,F2) In situ hybridization using 35S-labeled riboprobes against Col2a1 (F1) and Col10a1 (F2), on sections adjacent to that in E. Areas similar to that in F are shown at the same magnification. Hybridization signals are in red. PC, perichondrium. (G-G1) Histology of the humerus from an E18.5 Ihh-/- embryo. Asterisk in G indicates areas of vascularization. (G1) Area in yellow box (in G) is shown at a higher magnification in G1. Green lines indicate the outer (2) and inner (1) regions of the perichondrium; red and yellow arrows indicate cuboidal and elongated cells, respectively. (H-J1) In situ hybridization using 35S-labeled riboprobes against Runx2 (H,H1), Osx (I,I1) and Ap (J,J1) on sections adjacent to that in G. (H1-J1) Areas similar to that in G1, marked by yellow boxes in H-J, are shown at the same magnification as in G1. Hybridization signals are in red. Green lines demarcate the outer and inner regions of the perichondrium.

View larger version (29K): [in a new window]   Fig. 8. Gli3 mediates multiple but not all aspects of Ihh signaling in the endochondral skeleton. (A) A summary of the roles of Gli3 and other effectors in mediating Ihh activity. GliA, activated form of Gli molecules. (B) A model for Ihh-versus vascularization-dependent osteoblast development. See text for details.

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