Fig. 2

Fig. 2

Long-lived dystrophic lama2^−/− fibres maintain fibre polarity, upregulate Dystrophin and undergo hyper-fusion. a–c Dystrophin expression (yellow) in the line GT(dmd-citrine), is mislocalised in lama2^−/− mutants (b) compared to WT (a) in confocal projections. Single confocal section (c) reveals junctional polarity of Dystrophin is maintained in lama2^−/− fibres (indicated by arrowhead). Muscle is marked in blue by BFP from Tg(actc1b:EBFP2). a, b Maximum projections from Supplementary Movies 2 and 3. dlama2^−/− dystrophic fibres undergo fusion during remodelling. Injection of actc1b:H2az2a-EosFPtd-IRES-EGFP-CaaX into lama2^−/− embryos allows photoconversion of muscle nuclei expressing H2az2a-EosFPtd from green to red at 3 dpf (arrrowheads). Fibres are tracked and addition of new nuclei (green, arrows) determined. This reveals fusion which precedes reattachment of the fibre to the opposite vertical myoseptum. Time is expressed as days post-photocoversion. e–k Hyper-fusion in lama2^−/− dystrophic fibres. e, f Maximum projections of WT sibling (e) and lama2^−/− (f) larvae transgenic for Tg(actc1b:H2az2a-mCherry) and Tg(actc1b:EBFP2), which mark muscle nuclei (red) and muscle (blue), respectively. Insets reveal the regular arrays of nuclei in WT are perturbed and clumped in lama2^−/− fibres. g Confocal section of dystrophic fibres within a lama2^−/− mutant transgenic for Tg(actc1b:H2z2a-mCherry) and Tg(actc1b:EGFP-CaaX). Hyper-fused fibres are evident, with the centrally located fibre exhibiting clumped nuclei, a situation never seen in wildtypes. h–k Activation of the stem cell compartment as a source of muscle nuclei. WT (h, j) or lama2^−/− (i, k) larvae transgenic for Tg(cmet:KalTA4; UAS-nlsGFP) and Tg(actc1b:EBFP2). Green marks the cmet-positive satellite cell compartment (54) and blue differentiated fibres, revealing the GFP-positive satellite cell-derived nuclei, specifically in lama2^−/− dystrophic fibres (arrowheads i, k inset). Arrows mark the lateral line and brackets indicate the cell body nuclei of the dorsal motor neurons, which also express cmet at high level (h, i). All panels of 3 dpf larvae unless otherwise denoted. l–n Quantitation of the hyper-fusion phenotype in lama2^−/− mutant larvae. Phalloidin (red) marks muscle fibres and DAPI (green) marks nuclei in wildtype sibling (l) and mutant larvae (m). Quantitation of the myonuclear domain size, which measures that relative amount muscle cell cytoplasm to muscle nuclei within individual fibres (n). More nuclei per unit area is represented by a smaller myonuclear domain size. Significance is tested in a one-way ANOVA, Tukey’s test; ****p < 0.0001. Scale bars, 20 μm