Two modes of fibre detachment occur in sof muscle. (A-C) In vivo observation of intact fibres in the wild type (A) in comparison with a retracted fibre (B) and an EFT (arrowhead, C) in sof embryos at 120 hpf. Condensed myofibrils can be seen in the retracted fibre (arrowhead, B), whereas fibres attached to the EFT maintain a normal striated appearance. The vertical myoseptum is indicated by arrows in C. (D-K) Antibody co-staining with anti-dystrophin (E,I) and anti-laminin (F,J) reveals the presence of sarcolemma-BM linkages at EFTs in sof embryos (arrow in K), whereas single detached fibres retain sarcolemma only (arrowheads in K). BM detachment can be seen at the junction of the vertical myoseptum and an EFT (inset in K). (L-O) EFTs seen in lamb2 morphants appear identical to those in sof embryos (arrow in O). (P-R) Time points from the monitoring of a single EFT (bracketed) on the Tg(9.7kb smhyc1:gfp)i104 background, with panels labelled according to the stages at which images were captured. The last 16 hours of this period are depicted in Movie 2 in the supplementary material. (S) Anti-laminin staining of the same somite, in red, overlaid onto the GFP fluorescence image, confirming lesion as an EFT. (T-W) Labelling of fibres adjacent to an EFT (arrow in T) with Rhodamine Dextran illustrates viability of detached fibres over 4 days under load-bearing conditions. New fibres occasionally elongate over the EFT (arrow in W), extending to the myoseptum (dashed lines). Panels are labelled according to the stages at which images were captured. (A-C,D,H,L) DIC images, (E,F,I,J,M,N,P-R) fluorescence images, (G,K,O,S,U,W) merge of fluorescence images, (T,V) merge of DIC and fluorescence images. All panels show lateral views with anterior to the left. dys, anti-dystrophin; lam, anti-laminin.
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