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First published online 25 August 2004
doi: 10.1242/dev.01345

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Coordination of chondrocyte differentiation and joint formation by 5ß1 integrin in the developing appendicular skeleton

¹ Departamento de Biología Celular y Fisiología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico
² Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico
³ Genetics and Cell Biology Section, National Cancer Center Research Institute, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan

View larger version (50K): [in a new window]   Fig. 1. Blocking ß1 integrin induces formation of an ectopic joint. Forelimbs of E14.5 mouse embryos cultured for 4 days were injected at the wrist with 10 µg of an irrelevant IgG (A-D) or with 10 µg of an anti-ß1 integrin mAb (E-H). (A,E) Complete limb stained with Alcian Blue/Alizarin Red to show skeletal elements. Inset shows the area in the red square. (B,F) Histological section of zeugopod region stained with Safranin O and Fast Green to show cartilage in red. (C,G) In-situ hybridizations for Wnt14. (D,H) In-situ hybridizations for Gdf5. (I) Histological sections of zeugopod region in limbs injected with 2 µg RGE peptide stained with Safranin O and Fast Green. (J) Histological sections of zeugopod region in limbs injected with 2 µg RGD peptide stained with Safranin O and Fast Green. (K,L) TUNEL assay for apoptosis, forelimbs injected at the wrist with 10 µg of an irrelevant IgG (K) or with 10 µg of an anti-ß1 integrin mAb (L).

View larger version (94K): [in a new window]   Fig. 2. Blocking 5ß1 integrin induces molecular changes characteristic of joint formation. Forelimbs of E14.5 mouse embryos cultured for 4 days were injected at the wrist with 10 µg of an anti-5 integrin mAb (A-G,I,K,M,O). Control injected with 10 µg of an irrelevant IgG (H,J,L,N). (A) Histological sections of zeugopod region stained with Safranin O and Fast Green to show cartilage in red. In-situ hybridizations for Wnt14 (B), Gdf5 (C), chordin (D), autotaxin (E). Immunofluorescence for CD44 (F), type III collagen (G), type II collagen (H-I), type I collagen (J-K), type X collagen (L-M) and Indian hedgehog (N-O). Immunofluorescence staining for IHH and for type I, II, III and X collagen is green; cell nuclei were stained with propidium iodide (red).

View larger version (61K): [in a new window]   Fig. 3. Pattern expression of integrins during joint formation. Forelimbs of E14 mice were fixed and tissue sections prepared as described in Materials and methods. Sections at the level of autopod were stained for immunofluorescence with polyclonal antibodies against the 5 integrin subunit. (A) Section of the forming fingers stained for 5 integrins. (B,C) Consecutive sections from a joint in the developing fingers showing immunofluorescence staining for 5 integrin (B) and in-situ hybridization for Wnt14 (C). Serial sections of the ulna stained for 5 integrin (D), type X collagen (E) and IHH (F).

View larger version (73K): [in a new window]   Fig. 4. BMP enhances formation of the ectopic joint induced by inhibition of 5ß1 integrin. Forelimbs of E14.5 mouse embryos cultured for 4 days in the presence of 1 µg/ml BMP7 were injected at the wrist with 10 µg of an irrelevant IgG (A,B,C,H,J,L,N) or with 10 µg of an anti-5 integrin mAb (D,E,F,I,K,M,O). (A,D) Complete limb stained with Alcian Blue/Alizarin Red to show skeletal elements. (B,E) Histological sections stained with Safranin O and Fast Green to show cartilage in red. (C,F) In-situ hybridization for Wnt14. (G) Histological sections of limbs injected with 2 µg RGD peptide stained with Safranin O and Fast Green to show cartilage in red. (H,I) TUNEL assay for apoptosis. (J,K) Immunofluorescence staining for type II collagen (green). (L,M) Immunofluorescence staining for type I collagen (green). (N,O) Immunofluorescence staining for Indian hedgehog (green). In all cases cell nuclei are stained with propidium iodide (red).

View larger version (72K): [in a new window]   Fig. 5. Misexpression of 5ß1 integrins induces fusion of joints and differentiation of pre-hypertrophic cells. Chick legs of HH stage 27 were electroporated with full-length cDNA 5ß1 integrins in the region of the autopod and incubated for 5 days (B,D,F,H,J), as controls, contralateral legs were used (A,C,E,G,I). Chick legs were fixed in paraformaldehyde and photographed to observe phenotype induced by misexpression of human 5ß1 integrin (A,B). Serial section of the legs shown in A and B were prepared for paraffin inclusion and used for histology (C,D), immunofluorescence for human 5ß1 integrin (E,F), IHH (G,H) and in-situ hybridization for Wnt14 (I,J). (K-N) Forelimbs of E14.5 mouse embryos cultured for 4 days in the presence of 1 µg/ml BMP7 were injected at the wrist with 10 µg of an anti-5 integrin mAb. Cultures were added with nothing (K,L), or sonic hedgehog 400 ng/ml (M,N). Histological sections were stained with Safranin O and Fast Green (K,M), or processed for in-situ hybridization for Wnt14 (L,N).