Indian Hedgehog produced by postnatal chondrocytes is essential for maintaining a growth plate and trabecular bone

Abstract

Indian hedgehog (Ihh) is essential for chondrocyte and osteoblast proliferation/differentiation during prenatal endochondral bone formation. The early lethality of various Ihh-ablated mutant mice, however, prevented further analysis of its role in postnatal bone growth and development. In this study, we describe the generation and characterization of a mouse model in which the Ihh gene was successfully ablated from postnatal chondrocytes in a temporal/spatial-specific manner; postnatal deletion of Ihh resulted in loss of columnar structure, premature vascular invasion, and formation of ectopic hypertrophic chondrocytes in the growth plate. Furthermore, destruction of the articular surface in long bones and premature fusion of growth plates of various endochondral bones was evident, resulting in dwarfism in mutant mice. More importantly, these mutant mice exhibited continuous loss of trabecular bone over time, which was accompanied by reduced Wnt signaling in the osteoblastic cells. These results demonstrate, for the first time, that postnatal chondrocyte-derived Ihh is essential for maintaining the growth plate and articular surface and is required for sustaining trabecular bone and skeletal growth.

Fig. 1.

Specificity of Col2α1-Cre ER* using floxed ROSA reporter mice. (A) Col2α1-Cre ER* construct. (B–L) LacZ staining of Col2α1-Cre ER*; Rosa26cre mice. (B–G) Col2α1-Cre ER*; Rosa26cre mice were injected with tamoxifen (Tam) or vehicle (Veh) at P0 followed by LacZ staining at P1, P4, P7, and P14. (B) LacZ staining of hindlimbs. (C and D) Sections of proximal tibiae at P7 (C) and P14 (D). (E–G) Ribs and sternum (E), paws (F), and skull (G) at P4. (H–L) LacZ staining of tibiae at P15, P18, P21, and P28 (H) and of ribs (I), sternum (J), paws (K), and tail (L) at P18 after injection of tamoxifen at P14.

Fig. 2.

Phenotype of Col2α1-Cre ER*; Ihh d/d mice at P14 (A and B) and 8 weeks (A and C). (A) At P14, mutant mice were 24% smaller in size than control littermates (∗, P < 0.01); at 8 weeks, the difference in size increased to 45% (∗∗∗, P < 0.0001; n = 7, 5, 8, 5, 7, and 3 in each group, respectively from left to right). (B) X-ray analysis of tibiae at P14. Arrows depict the translucency of the growth plates in the control (Left) and the fusion of the growth plate in the mutant proximal and distal tibia (Right). (C) Macroscopic image of 8-week-old littermates. Mutant (right) did not increase body size after P14 (see also A).

Fig. 3.

Mineralization of various skeletal elements. (A–D) Mineralization pattern of ribs (A, arrow), paws (B), tibia (C), and tail vertebrae (D) of controls (Co) and mutants (M) by Alizarin red/Alcian blue at P14. Arrows in B–D depict the loss of the growth plates. (E) Mutant vertebrae showed loss of annulus fibrosus (arrow) surrounding the significantly enlarged nucleus pulposus (NP). (F–I) von Kossa staining of tibiae of Ihh fl/fl (Upper) and col2α1-Cre ER*; Ihh d/d (Lower) at P7 (F), P14 (G) (complete loss of growth plate is evident), and 8 weeks (H and I) (note loss of trabecular bone in H). Arrows in I point to the decreased thickness of the endosteal seam along the diaphysis in the mutant tibia.

Fig. 4.

Analyses of endochondral bone formation in tibiae at P7. (A) Proliferation was analyzed by PCNA staining and was significantly decreased in col2α1-Cre ER*; Ihh d/d. Lower represents magnifications of the boxed areas in Upper. (B) Atypical invasion of blood vessels in the middle of growth plate (arrows) was detected by PECAM-1 (CD31) staining. (C) Altered expression pattern of PTHrP protein in the mutant proximal tibia. (D) Protein levels for β-catenin were significantly decreased in bone collar (arrowheads) and primary spongiosa (boxed area) of col2α-Cre ER*; Ihh d/d animals.

Fig. 5.

Analyses of osteoblast maturation at P7. (A–D) In situ hybridization using riboprobes for Ihh (A), Ptch (B), collagen type I (Col I) (C), and osteocalcin (OC) (D). The arrow in B points to the complete loss of Ptch expression in the mutant primary spongiosa. Boxed area in C depicts collagen type I expression in the bone collar, which is absent in the mutant. (E) Bar graph showing mRNA expression of Dkk1 and OPG in the primary spongiosa of Ihh fl/fl and col2α1-Cre ER*; Ihh d/d mice at P7. Data are expressed as fold over controls. The expression of both Dkk1 and OPG was significantly reduced in mutants (P < 0.05; n = 3 measured in triplicates).