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First published online 19 July 2006
doi: 10.1242/dev.02480

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Distinct roles for Hedgehog and canonical Wnt signaling in specification, differentiation and maintenance of osteoblast progenitors

Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.

View larger version (48K): [in a new window]   Fig. 1. Col21-cre3 removal of ß-catenin function results in a failure to develop terminally differentiated osteoblasts. Histological analysis and in situ hybridization of 35S-labeled riboprobes to sections of E18.5 tibia from (A-I) ß-cateninc/c (wild type) and (J-R) Col21-cre3;ß-cateninc/c (mutant) embryos, with specific anti-sense riboprobes for marker genes that identify states of osteoblast and chondrocyte differentiation. Osteoblast: (D,M) Col11, (E,N) Runx2, (F,O) osterix1 and (G,P) osteocalcin. Chondrocyte: (H,Q) Col21 and (I,R) Col101. Boxed areas in B and K are presented at higher magnification in C and L, respectively; black arrows indicate the absence of ossification in the mutant periosteum. White arrowheads indicate the invading wedge of mesenchymal cells into the marrow cavity; black arrowheads indicate sites of ectopic expression of chondrocyte marker genes.

View larger version (71K): [in a new window]   Fig. 2. Generation of an Osx1-GFP::Cre transgenic mouse line. (A) Schematic outlining the linear configuration of the pTGCK cassette used for homologous recombination to the first exon of the osterix1 locus contained within the BAC: RP23-399N14. The correctly targeted BAC was subsequently used to generate a transgenic mouse line by pro-nuclear injection. (B-K) Founder Osx1-GFP::Cre male transgenic mice were crossed to the female Rosa26lacZ reporter line and activity of the transgenic line was observed by way of whole-mount lacZ assay at (B) E14.5, and by lacZ assay or direct fluorescence microscopy on 15 µm cryosections of tibia from (C,D) E14.5, (F,G) E18.5 and (I,J) postnatal day 10 mice. (E,H,K) Negative control specimens for littermates, at each corresponding age, that do not carry the transgene.

View larger version (30K): [in a new window]   Fig. 3. Osx1-GFP::Cre removal of ß-catenin function affects skeletal development. Skeletal preparations of ß-cateninc/n (wild type) and Osx1-GFP::Cre;ß-cateninc/n (mutant) embryos at E14.5, E16.5 and E18.5. Higher magnifications of the fore- and hindlimbs are presented below each embryo. White arrowheads indicate comparable levels of hypertrophy between wild-type and mutant tibia. Arrows indicate the absence of mineralized bone matrix in mutants when compared with wild-type embryos (black arrowheads).

View larger version (87K): [in a new window]   Fig. 4. Osx1-GFP::Cre removal of ß-catenin function results in a failure to develop terminally differentiated osteoblasts. (A-Q') Histological analysis and in situ hybridization of 35S-labeled riboprobes to sections of E18.5 tibia from (A-Q) ß-cateninc/n (wild type) and (A'-Q') Osx1-GFP::Cre;ß-cateninc/n (mutant) embryos. Specific anti-sense riboprobes were used that identify marker genes for: states of osteoblast and chondrocyte differentiation, (D,D') Col11, (E,E') Runx2, (F,F') osterix1, (G,G') osteocalcin, (H,H') Col21 and (I,I') Col101; components of Hh signaling, (J,J') Ihh and (K,K') Ptch1; components of canonical Wnt signaling, (L,L') Dkk1 and (M,M') Tcf1; the early osteoblast marker, (N,N') Bmp3; matrix remodeling, (O,O') Mmp9 and (P,P') Mmp13; and vascularization, (Q,Q') Vegf. Boxed areas in B and B' are presented at higher magnification in C and C', respectively. Black arrows indicate the absence of ossification in the mutant periosteum. White arrowheads indicate the invading wedge of mesenchymal cells into the marrow cavity, whereas black arrowheads indicate sites of ectopic expression of chondrocyte marker genes.

View larger version (63K): [in a new window]   Fig. 5. Osx1-GFP::Cre removal of Smo function does not affect osteoblast differentiation. Skeletal preparations of (A,B) Smoc/n (wild type) and (F,G) Osx1-GFP::Cre;Smoc/n (mutant) fore- and hindlimbs. Histological analysis (C,D,H,I) and in situ hybridization with specific anti-sense 35S-labeled riboprobes for the terminal osteoblast differentiation marker osteocalcin (E,J) to sections of E18.5 tibia from wild-type (C-E) and mutant (H-J) embryos.

View larger version (35K): [in a new window]   Fig. 6. Conditional stabilization of ß-catenin by Osx1-GFP::Cre affects skeletal development. Skeletal preparations of Catnblox(ex3)/+ (wild type) and Osx1-GFP::Cre;Catnblox(ex3)/+ (mutant) embryos at E14.5, E16.5 and P0. Higher magnifications of the fore- and hindlimbs are presented below each embryo. Black arrows indicate the presence of a poor Alcian blue-stained discontinuous matrix in mutants in comparison with wild-type embryos at E14.5 (black arrowheads). White arrows indicate the expansion in mineralized bone matrix observed in mutants when compared with wild-type embryos (white arrowheads).

View larger version (48K): [in a new window]   Fig. 7. Conditional stabilization of ß-catenin by Osx1-GFP::Cre promotes premature mineralization of skeletal elements. Histological analysis and in situ hybridization of 35S-labeled riboprobes to sections of E14.5 tibia from (A-I) Catnblox(ex3)/+ (wild type) and (J-R) Osx1-GFP::Cre;Catnblox(ex3)/+ (mutant) embryos, with specific anti-sense riboprobes for marker genes that identify states of chondrocyte and osteoblast differentiation. Chondrocyte: (E,N) Col21 and (F,O) Col101. Osteoblast: (G,P) Col11, (H,Q) osterix1 and (I,R) osteoprotegerin. Arrows indicate premature mineralization, identified as silver deposition by (K) von Kossa and (L) Alizarin red staining, present in mutants compared with (B,C) wild types. (S,T) Analysis of BrDU incorporation in wild-type and mutant embryos at E14.5. Graphs represent the relative number of proliferative cells within the (S) growth plates or (T) periosteal region of mutant and wild-type tibia. Error bars represent the s.d. of the mean of the results (n3).

View larger version (72K): [in a new window]   Fig. 8. Osx1-GFP::Cre conditional stabilization of ß-catenin does not promote expression of osteocalcin. Histological analysis and in situ hybridization of an osteocalcin-specific anti-sense 35S-labeled riboprobe to sections of E16.5 tibia from (A-E) Catnblox(ex3)/+ (wild type) and (F-J) Osx1-GFP::Cre;Catnblox(ex3)/+ (mutant) embryos. Arrows indicate sites of high osteocalcin expression in the wild type and low-level osteocalcin expression in the mutant in the periosteum.

View larger version (19K): [in a new window]   Fig. 9. Proposed model for the role of Hh and canonical Wnt signaling in regulating the differentiation of skeletal progenitors. Various lines of evidence implicate the importance of canonical Wnt signaling at multiple stages along the osteoblast differentiation pathway, from the specification of early skeletal progenitor cells to a terminally differentiated osteoblast. Observations from these studies (Hu et al., 2005; Day et al., 2005), together with results presented in this study, have been used to synthesize a working model for the specific functions of Hh and canonical Wnt signaling during osteoblast specification and differentiation. See text for details. Red and green arrows indicate the requirement for Hh and canonical Wnt signaling, respectively; the blue line represents the negative regulation of osteoclasts by OPG.