Figure 4

Early formation of a restricted group of slow MyHC-expressing fibres in the chick forewing. A. Schematic of muscle fibre types in the wing DMM of the HH28 chick zeugopod showing the location of sections in C-H. Definitive slow (red) and non-slow (green) fibres are indicated. B. Immunohistochemistry of transverse cryosection of HH25 chick wings showing earliest differentiation of embryonic MyHC-reactive cells. No fibre type differences were detected at this stage (data not shown). C-K. Serial cryosections at proximal (C-G) and distal (H-K) positions (as indicated in panel A) of HH28 transverse cryosections stained for pan-MyHC (C,D,E), and slow MyHC-reactive BA-D5 (E,I), A4.840 (F,J) and Na8 (G,K). Proximally the definitive slow fibres are located in posterior DMM (cf D with F, C with G). Distally, slow fibres are localised to the central region of the DMM (cf H with I-K). L-N. Serial transverse cryosections of HH32 proximal zeugopod with pan-MyHC (L) revealing muscle splitting, N3.36-reactive neonatal/fast MyHC (M) and definitive A4.840-reactive slow MyHC (N). Some muscles, such as the EMU, contained both fast and slow fibres (M,N arrowheads). Other muscles, such as the superficial region of the FCU, had many slow fibres but fewer fast fibres (M,N; right-pointing arrows). Other muscles, such as the PP/PS, had few slow but numerous fast fibres (M,N; left-pointing arrows). EDC extensor digitorum communis; Anc anconeus; EMU extensor metacarpi ulnaris; EIL extensor indicis longus; EMR extensor metacarpi radialis; PS pronator superficialis; PP pronator profundus; FDP flexor digitorum profundus; UMV ulni metacarpalis ventralis; FCU flexor carpi ulnaris. O-V. In situ mRNA hybridisation for ptc1 (O-Q), gli1 (R-T) or gli2 (U,V) and subsequent MyHC staining with MF20 (P,Q,S-V) in control (O,P,R,S,U) and RCAS/Shh (Q,T,V) limbs. Wholemount in situ at HH22-26 shows ptc1 and gli1 mRNA in distal/posterior limb (O,R; dorsal views of right wing bud, anterior is up). By HH28, an additional zone of ptc1 mRNA is present proximally in the posterior DMM (O). Gli1 mRNA is also accumulating in distinct zones away from the ZPA (R). Sections of the zeugopod show ptc1 mRNA in both muscle and non-muscle regions, with restriction of ptc1 signal to regions without MyHC stain. RCAS/Shh implant up-regulates ptc1 throughout the dorsal region around the implant (Q, inset shows shh mRNA in/near DMM). Normal gli1 expression (S) is partially reciprocal to gli2 mRNA (U). Gli1 mRNA is up-regulated and gli2 reduced around a RCAS/Shh implant (T,V; inset shows shh mRNA in/near DMM). Dorsal is up and posterior to the left in panels B-N,P,Q,S-V. (u) ulna, (r) radius. Scale bar = 600 μm (A-K), 250 μm (L-N).