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doi: 10.1242/10.1242/dev.00538

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Wnt signalling regulates myogenic differentiation in the developing avian wing

Kelly Anakwe1,*, Lesley Robson2,*, Julia Hadley1, Paul Buxton1, Vicki Church1, Steve Allen1, Christine Hartmann3,{ddagger}, Brian Harfe3, Tsutomu Nohno4, Anthony M. C. Brown5, Darrell J. R. Evans6 and Philippa Francis-West1,§

1 Department of Craniofacial Development, King's College, London SE1 9RT, UK
2 Department of Neuroscience, Bart's and The London, Queen Mary's School of Medicine and Dentistry, London E1 4NS, UK
3 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
4 Department of Molecular Biology, Kawasaki Medical School, 577 Matsushima, Kurashiki, 701-0192, Japan
5 Department of Cell and Developmental Biology, Weill Medical College of Cornell University and Strang Cancer Prevention Center, New York, NY 10021, USA
6 School of Biosciences, Cardiff University, Cardiff CF10 3US, UK



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  		Fig. 1. Correlation of Wnt5a and Wnt11 expression with myogenic differentiation. The expression of Wnt5a (A-C,J-M), MyoD (D-F,N) and Wnt11 (G-I,N) in stage 22 (A,D,G), 25 (B,E,H) and 27 (C,F,I) wing buds was determined by whole-mount in situ hybridization. (J-N) Transverse vibratome sections of wing buds expressing Wnt5a (J-M) and Wnt11 and MyoD (N) at stages 22 (J,K), 26 (N) and 27 (L,M). In (A-M), expression is shown in purple/red. In (N), MyoD expression is shown in red and Wnt11 expression is shown in purple. In (A-N), anterior is uppermost and, in (A-I), distal is to the right. In the vibratome sections, (J) is more distal than (K), and (L) is more distal than (M). In (M), developing cartilage is circled in red, whereas developing muscle is circled in black. DM, dorsal muscle mass; R, radius; U, ulna; VM, ventral muscle mass. Scale bars: 100 µm in A,D,G; 150 µm in B,C,E,F,H,I; 125 µm in J,K; 150 µm in L,M.

 


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  		Fig. 2. Correlation of Wnt expression with muscle differentiation. (A) A sketch through the dorso-ventral axis of a stage 24 wing bud showing the relationship between Wnt expression and the developing myogenic cells. Initially, Wnt7a is expressed throughout the dorsal ectoderm but, by stage 24, its expression is restricted to the dorsal ectoderm overlying the progress zone. (B) Transverse section through an 8-day limb showing the expression of slow MyHC (orange) versus fast MyHC (green). (C) Diagrammatic sketch of (B). ANC, anconeus; EDC, extensor digitorum communis; EIL, extensor indicis longus; EMR, extensor metacarpi radialis; EMU, extensor metacarpi ulnaris; Ent, entepicondyloulnaris; FCU, flexor carpi ulnaris; PP, pronator profundus; PS, pronator superficialis; SUP, supinator.

 


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  		Fig. 3. Effects of Wnt overexpression on fibre-type differentiation in vitro. (A-P) Fluorescent images of stage 21/22 wing micromass cultures showing terminally differentiated myogenic cells that have been visualized with antibody A4.1025, which recognizes both slow and fast MyHCs (A-H, green), and antibody A4.840, which specifically recognizes slow MyHCs (I-P, red). The micromass cultures have been infected with control RCAS(BP) virus (A,I) or retroviruses expressing Wnt3a (B,J), Wnt4 (C,K), Wnt5a (D,L), Wnt6 (E,M), Wnt7a (F,N) Wnt11 (G,O) or Wnt14 (H,P). Scale bars, 100 µm.

 


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  		Fig. 4. The effects of Wnt overexpression on fibre-type differentiation in vitro. The bar chart shows the total number of differentiated myogenic cells, and the number expressing fast or slow MyHCs in 3-day limb micromass cultures that have been infected with either a control RCAS(BP) virus or retroviruses expressing Wnt3a, Wnt4, Wnt5a, Wnt6, Wnt7a, Wnt11 or Wnt14, as shown in Fig. 2. The slow population of myoblasts (red) might express either exclusively slow MyHC or both slow and fast MyHCs, whereas the fast myogenic population (yellow) only expresses fast MyHC isoforms. *, P<0.05; **, P<0.01; ***, P<0.001.

 


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  		Fig. 5. Effects of overexpression of Wnt5a and Wnt11 on fibre-type differentiation in vivo. Fluorescent images of transverse cryosections of day 8 (A-F) and stage 29 (G-J) chick wings that were infected with retroviruses expressing GFP (B), Wnt5a (D,F) or Wnt11 (H,J) at stage 18-20, and of the contralateral control wings (A,C,E,G,I). Wings have been visualized with antibody A4.1025, which recognizes slow and fast MyHCs (C,D,G,H, green), and antibody A4.840, which recognizes slow MyHC (E,F,I,J, red). (B) The embryo was pathogen free and viral spread has been visualized with an anti-GAG antibody. The arrows in (B) indicate viral spread throughout the limb bud and muscles (compare with A). The arrows in (E) and (F) compare equivalent muscles, showing an increase in slow MyHC expression in the manipulated limb (F). In the Wnt11-transfected limb, there are fewer slow fibres in the dorsal and ventral muscle masses (J) than in the contralateral limb (I). d, dorsal muscle mass; EDC, extensor digitorum communis; EIL, extensor indicis longus; EMR, extensor metacarpi radialis; EMU, extensor metacarpi ulnaris; FCU, flexor carpi ulnaris; FDP, flexor digitorum profundus; R, radius; u, ulna; v, ventral muscle mass. Scale bars: 200 µm in A-D; 100 µm in E-H.

 


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  		Fig. 6. Effects of loss-of-function of Wnt signalling in vivo. Fluorescent images of transverse cryosections of day 8 chick wings that were infected with retroviruses expressing Sfrp2 (B,D) and {Delta}Wnt11 (F,H) at stage 18-20, and the contralateral control wings (A,C,E,G), which have been visualized with antibody A4.1025, which recognizes slow and fast MyHCs (A,B,E,F, green), and antibody A4.840, which recognizes slow MyHC (C,D,G,H, red). (A-D) Sections through the whole wing. (E-H) High power pictures of the EMU and EDC muscles. The arrows in (A,B,G,H) compare equivalent muscles, showing the changes in muscle development. In (B), the muscles are absent (EMU, SUP) or are decreased in size (EMR, FCU). In H, the EMU muscle has more slow fibres. EDC, extensor digitorum communis; EIL, extensor indicis longus; EMR, extensor metacarpi radialis; EMU, extensor metacarpi ulnaris; FDP, flexor digitorum profundus; SUP, supinator. Scale bars: 200 µm in AD; 100 µm in E-L.

 


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  		Fig. 7. The effect of overexpression of components and dominant-negative components of the Wnt signalling pathway on fibre-type differentiation in vitro. (A-J) Fluorescent images showing terminally differentiated myogenic cells that have been visualized using antibody A4.1025, which recognizes slow and fast MyHCs (A-D,I,J, green), and antibody A4.840, which recognizes slow MyHC (E-H, red) in stage 21/22 micromass cultures that have been infected with control RCAS virus (A,E,I) or retroviruses expressing Dsh{Delta}PDZ (B,F), {Delta}Lef1 (C,G,J) or activated CamKII (D,H). Scale bars: 100 µm in A-H; 25 µm in I,J.

 


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  		Fig. 8. The effect of overexpression of activated components and dominant-negative components of the Wnt signalling pathway on fibre-type differentiation in vitro. The bar chart shows the total number of differentiated myogenic cells and the number expressing fast or slow MyHCs in 3-day limb micromass cultures that have been infected with either a control RCAS(BP) virus or retroviruses expressing Dsh{Delta}PDZ, {Delta}Lef, ß-catenin or activated CamKII. The slow population of myoblasts (red) might express either exclusively slow MyHC or both slow and fast MyHCs, whereas the fast myogenic population indicated by the yellow bar only expresses fast MyHC. *, P<0.05; **, P<0.01; ***, P<0.001.

 

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© The Company of Biologists Ltd 2003