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doi: 10.1242/10.1242/jcs.00152

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Wnt regulation of chondrocyte differentiation

¹ Department of Craniofacial Development, Guy's, King's and St Thomas' School of Dentistry, Floor 28 Guy's Tower, Guy's Hospital, London Bridge, London SE1 9RT, UK
² Department of Molecular Biology, Kawasaki Medical School, Kurashiki, Japan
³ Developmental Biology Institute, LGPD, Campus de Luminy, case 107, University of Aix-Marseille II, 13288 Marseille Cedex 09, France

View larger version (83K): [in a new window]   Fig. 1. Light field (A,E,I) and corresponding dark field micrographs comparing the expression of Wnt5b (B,F,J) and Wnt11 (D,H,L) with Ihh (C,G,K) in the skeletal elements of the developing wing at stages 28 (A-D), 34 (E-H) and 37 (I-L). Bars, 500 µm. h, humerus; r, radius; u, ulna. HE, haematoxylin and eosin-stained sections. In H and L, arrowheads point to Wnt11 expression in a subset of the prehypertrophic chondrocytes.

View larger version (143K): [in a new window]   Fig. 2. Light field (A,C,E) and corresponding dark field micrographs showing expression of Wnt4 (B,D) and Wnt5a (F) in the developing joints of the stage 28 wing (A,B), stage 37 wing (C,D) and stage 30 leg (E,F). Bars, 500 µm. h, humerus; j, joint interzone; m, mesenchyme; pc, perichondrium; r, radius; sc, synovial capsule; u, ulna. HE, haematoxylin and eosin-stained sections; TolB, toluidine blue-counterstained section.

View larger version (50K): [in a new window]   Fig. 3. Dark field micrographs comparing the expression of Wnt4 (A), Wnt5b (B) and Wnt11 (C) in the stage 40 tibiotarsus. Bars, 500 µm. pr, perichondrial ring.

View larger version (49K): [in a new window]   Fig. 4. Comparison of the effects of Wnts on the initiation of chondrogenic differentiation of stage 23/24 wing mesenchymal cells in micromass cultures after three days. (A) RT-PCR analysis of retroviral transgene expression. (B) Quantification of alcian blue staining (solid bars) and nodule numbers (open bars). (C) Micromass cultures stained for cartilaginous matrix with alcian blue. In B, values shown are the mean+s.d. of 11 cultures from three independent experiments. *P<0.0001; **P<0.0005; ***P<0.005 (Student's t-test) of Wnt/Ihh-infected cultures compared to control cultures.

View larger version (46K): [in a new window]   Fig. 5. Comparison of the effects of Wnts and Ihh on chondrocyte terminal differentiation of stage 23/24 wing mesenchymal cells in micromass cultures after seven days. (A) Quantification of alkaline phosphatase activity. (B) Micromass cultures stained for alkaline phosphatase activity (red staining). (C) Micromass cultures stained for type X collagen (red fluorescence; magnification x10); examples of the stained nodules are outlined. In A, values shown are the mean+s.d. of nine cultures from three independent experiments. *P<0.005; **P<0.001 (Student's t-test) of Wnt/Ihh-infected cultures compared with control cultures.

View larger version (15K): [in a new window]   Fig. 6. Comparison of the effects of Wnts on the proliferation of stage 23/24 wing mesenchymal cells in micromass cultures after 48 and 72 hours. Values shown are the mean+s.d. percentage of BrdU-labelled cells from six cultures from two independent experiments.

View larger version (107K): [in a new window]   Fig. 7. Effect of misexpression of Ihh in vivo. Chick embryonic fibroblasts (CEFs) infected with retrovirus were grafted into the stage 18/19 wing bud. Limbs were fixed after a further 7 days (i.e. at embryonic day 10) and processed for in situ hybridisation. A,C,E and B,D,F are adjacent sections from two different embryos. Light field (A,B) and dark field (C-F) micrographs showing endogenous and misexpressed Ihh (C,D) and the effect of Ihh on the expression of Wnt5b (E) and Wnt11 (F). The inset in A shows a high power view of the chondrocytes. Bars, 500 µm (A,B); 200 µm (inset).

View larger version (76K): [in a new window]   Fig. 8. Effect of misexpression of Wnt5a (A,C,E,G,I) or Wnt11 (B,D,F,H,J) in vivo. Chick embryonic fibroblasts (CEFs) infected with retroviruses expressing either Wnt5a or Wnt11 were grafted into the stage 18/19 wing bud. Limbs were fixed after a further 7 days (i.e. at embryonic day 10) and processed for in situ hybridisation. Comparison of light field (A,B) and dark field (C-J) micrographs showing the endogenous and misexpressed Wnt5a (C) or Wnt11 (D). The effects on chondrogenic differentiation were assessed by analysis of Wnt5b (E,F), Ihh (G,H), and type X collagen (I,J), which are expressed at different stages of chondrocyte differentiation. The insets in A and B show high power views of the chondrocytes from the diaphyseal region in the infected elements. In the Wnt5a-infected elements, the majority of cells are rounded with few prehypertrophic chondrocytes in contrast to the control where the chondrocytes are hypertrophic. In the Wnt11-infected element, the chondrocytes are undergoing hypertrophic differentiation. Bars, 500 µm (A,B); 200 µm (insets).