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First published online 2 December 2004
doi: 10.1242/dev.01564

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Sequential roles of Hedgehog and Wnt signaling in osteoblast development

Hongliang Hu^1,*, Matthew J. Hilton^1,*, Xiaolin Tu¹, Kai Yu², David M. Ornitz² and Fanxin Long^1,2,

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 (95K): [in a new window]   Fig. 1. ß-Catenin is required for bone formation. (A-B') In situ hybridization using digoxigenin-labeled riboprobes against Tcf1 (A,A') or Dkk1 (B,B') on sections of E14.5 humeri of wild-type (A,B) or Dermo1Cre; ß-cateninc/c (CKO) embryos (A',B'). Arrows indicate the perichondrium. (C-E') Von Kossa staining of sections through the humerus (C-D') or the parietal bone (E,E') of E18.5 wild-type (C,C',E) or CKO embryos (D,D',E'). Boxed areas in C,D are shown at higher magnification in C',D', respectively. Arrows indicate bone (stained black, C' and E) or equivalent regions where bone is missing (D',E'). Asterisks indicate mineralization in the cartilage (stained black, C',D'). (F-K') In situ hybridization using digoxigenin-labeled (F-H') or 35S-labeled riboprobes (I-K') against Col1a1 (F,F',I,I'), Bsp (G,G',J,J') or OC (H,H',K,K') on sections of E18.5 humeri (F-H') or heads (I-K') of wild type (F-K) or CKO embryos (F'-K'). Arrows indicate the bone collar (F-H) or the frontal bone (I-K) in the wild-type animal or the equivalent regions in the mutant embryo (F'-K'). Asterisks indicate the primary spongiosa. (LO') Histological analyses of the humerus from wild-type (L-O) versus CKO (L'-O') embryos. The distal end of the humerus is towards the right in each panel. Boxed regions are shown at a higher magnification as insets. H, hypertrophic chondrocytes. Red arrow in M indicates a red blood cell present in a blood vessel invading the cartilage. Magenta arrow in M' indicates a cell undergoing hypertrophy, whereas the orange arrows indicate immature chondrocytes. Green arrows in N and O indicate the primary spongiosa and the bone collar, respectively. Blue arrows in O' point to perichondrial cells. B, brain; M, marrow cavity; P, proliferating chondrocytes; H, hypertrophic chondrocytes.

View larger version (146K): [in a new window]   Fig. 2. Molecular basis of osteoblast defects in Dermo1Cre; ß-cateninc/c (CKO) and Ihh-/- embryos. In situ hybridization using 35S-labeled riboprobes against Col1a1 (A-A''), AP (BB''), Runx2 (C-C'') or Osx (D-D'', E-F') on sections of humeri from wild type (A-F), CKO (A'-F') or Ihh-/- embryos (A''-D'') at E14.5 (A-D''), E15.5 (E,E') or E18.5 (F,F'). H, hypertrophic chondrocytes; L, ligament; M, marrow cavity. Asterisks indicate expression in chondrocytes. Arrowheads and arrows indicate various regions of the perichondrium (see text).

View larger version (84K): [in a new window]   Fig. 3. ß-Catenin promotes chondrocyte proliferation and maturation. (A-C') In situ hybridization using 35S-labeled riboprobes against Col1a1 (A,A'), Ihh (B,B') or Ptc1 (C,C') on sections of E15.5 humeri of wild type (A-C) or Dermo1Cre; ß-cateninc/c (CKO) embryos (A',B',C'). H, hypertrophic chondrocytes. M, marrow cavity. Arrow in A' denotes a cluster of hypertrophic chondrocytes. Arrows in C and C' indicate the perichondrium. (D) BrdU labeling index of chondrocytes in the humeri of E15.5 wild type (WT) or Dermo1Cre; ß-cateninc/c (MT) embryos.

View larger version (100K): [in a new window]   Fig. 4. Distribution of ß-catenin in the developing cartilage. Immunohistochemistry with an antibody against ß-catenin on sections of tibia from E14.5 wild-type (A,A') versus Ihh-/- embryos (B,B'), or from E16.5 wild type (C,C') versus Ihh-/- embryos (D,D'). (E) In situ hybridization using a 35S-labeled riboprobe against Col10a1 on a section immediately adjacent to that shown in D. Boxed regions in A-D are shown at a higher magnification in A'-D'. Red arrows in A' and C' indicates nuclear staining of ß-catenin. Yellow arrow in C' marks a nucleus negative for ß-catenin staining. Arrows in C and D indicate peripheral chondrocytes with nuclear staining. Arrowheads in A', B' and D' indicate staining at the cell junctions. Asterisk in B denotes the cartilage proper. Asterisks in D mark chondrocytes with ß-catenin staining. P, proliferating chondrocytes; PH, prehypertrophic chondrocytes; EH, early hypertrophic chondrocytes; LH, late hypertrophic chondrocytes; C, columnar chondrocytes.

View larger version (156K): [in a new window]   Fig. 5. Canonical Wnt signaling impaired in the Ihh-/- embryo. In situ hybridization using digoxigenin-labeled riboprobes against Tcf1 (A,A'), Dkk1 (B,B'), Lef1 (C,C'), Tcf3 (D,D') or Tcf4 (E,E') on sections of E14.5 tibia of wild type (A-E) versus Ihh-/- embryos (A'-E'). Arrows indicate the perichondrium. P, proliferating chondrocytes; PH, prehypertrophic chondrocytes; H, hypertrophic chondrocytes; C, chondrocytes.

View larger version (62K): [in a new window]   Fig. 6. Hh signaling induces osteogenesis in C3H10T1/2 cells. (A) Induction of AP by N-Shh. Results are shown for triplicate samples from a representative experiment. This applies to all AP assays that follow. (B,B') Cell-autonomous induction of AP by the constitutively active Smo*. (B) A fluorescence picture showing cells expressing GFP in the nucleus. (B') A bright-field picture of the same field showing cells stained for AP activity. Numbers indicate cells expressing both GFP and AP. Asterisk indicates a cell expressing GFP but not AP. (C,C') Bone nodule formation induced by the constitutively active Smo*. Retroviruses encoding Smo* induced bone nodules (arrows) stained positive by Alizarin Red (C'), whereas a control virus did not (C). (D-G) Expression of Col1a1 (D), Bsp (E), Runx2 (F) or Osx (G) in Hh-treated (red bars) versus control cells (grey bars) by real-time PCR. The mRNA levels were normalized to the level of glyceraldehyde-3-phosphate dehydrogenase (GAPD) mRNA. Results are shown for triplicate samples from a representative experiment. This also applies to other real-time PCR experiments that follow. (H,I) Effects of cycloheximide on Hh induction of Runx2 (H) and Osx (I) expression. Runx2 and Osx mRNA levels were determined by real-time PCR and normalized to Gapd for cells stimulated for 24 hours with N-Shh in the presence of either DMSO (C) or 10 µg/ml cyclohexamine (CHX).

View larger version (43K): [in a new window]   Fig. 7. Wnt signaling induces osteogenesis in C3H10T1/2 cells. (A) Induction of AP by Wnt3a-conditioned medium versus the control L medium. (B-F) Expression of Runx2 (B), Osx (C), Col1a1(I) (D), OP (E) and OC (F) in cells treated with either Wnt3a-conditioned medium (red bars) or the control L medium (grey bars), as determined by real-time PCR. (G-I) Partial inhibition of Smo*-induced osteogenesis by either Dkk1 or dnTCF4. Cells were infected with Smo* virus mixed at equal titer with either Dkk1 virus, dnTcf4 virus or a control GFP virus. AP expression was determined by both an activity assay (G) and real-time PCR (H). Bsp expression was determined by real-time PCR (I). NV, no virus.

View larger version (54K): [in a new window]   Fig. 8. Wnt7b as a potential endogenous signal regulating osteogenesis. (A,B) Induction of Wnt genes in C3H10T1/2 cells stimulated with NShh for 24 hours (A) or 48 hours (B), as determined by real-time PCR. Results are presented as fold changes over unstimulated cells. Wnt proteins not detectable in either stimulated or unstimulated cells are not included in the graphs. *P 0.05. (C-E) Effects of cycloheximide on Hh induction of Wnt5a (C), Wnt7b (D) or Wnt9a (E) expression. mRNA levels were determined by real-time PCR and normalized to Gapd for cells stimulated for 24 hours with N-Shh in the presence of either DMSO (C) or 10 µg/ml cyclohexamine (CHX). (F-H') In situ hybridization using 35S-labeled riboprobes against Wnt5a (F,F'), Wnt7b (G,G') or Wnt9a (H,H') on sections of E14.5 humeri (F,F',H,H') or ulna (G,G') from wild type (F-H) or Ihh-/- (F'-H') embryos. Red arrows indicate the perichondrium. Yellow arrows indicate the joint regions. PH, prehypertrophic chondrocytes; H, hypertrophic chondrocytes. (I) Expression of Wnt7b in cartilage isolated from the hindlimbs of E14.5 wild-type or Ihh-/- embryos, assayed by real-time PCR. The average is presented for three pairs of wild type versus Ihh-/- littermates. The levels of mRNA were normalized to Gapd and presented in arbitrary units with the level in the wild-type embryo set at 1. (J) AP assay for C3H10T1/2 cells transfected with either the empty vector pCIG or constructs expressing Wnt5a, Wnt7b or Wnt9a. Average is shown for triplicate samples from a representative experiment.

View larger version (16K): [in a new window]   Fig. 9. A model that integrates Hh and Wnt signaling in osteoblast development. Red arrows represent events supported by genetic evidence. Blue arrows indicate events revealed by studies in C3H10T1/2 cells. See text for details.