Fig. 8

huDysGFP rescuing and binding dynamics in dmd^{ta222a/ta222a} embryos.

(A) 3 dpf dmd^{ta222a/ta222a} zebrafish embryo with typical dystrophic muscles as shown by actc1b:mCherry reporter (red) in vivo, with several healthy fibres expressing huDysGFP (green). (B) Control GFP mosaically expressed in dmd^{ta222a/ta222a} embryos is found in both healthy (77%) and dystrophic (23%) fibres (N = 56). Expression of huDysGFP fully rescues the dystrophic phenotype in dmd^{ta222a/ta222a} muscle fibres, as no cells expressing huDysGFP were found detached or unhealthy in any visible aspect (N = 56). p = 0.000126 in Chi-square test for significance between GFP and huDysGFP. GFP and huDysGFP-positive cells in regions of very dystrophic muscles in dmd^{ta222a/ta222a} zebrafish embryos are shown. The actc1b:mCherry reporter filling up the cytoplasm and huDysGFP expression at the tip suggest that the fibre structure is kept intact even without support from neighbouring cells, unlike the GFP-positive cell. (C) huDysGFP ratio tip intensity/cytoplasm intensity in wild-type (mean = 3.7 ± 1.1 s.e.m.; n = 33) and dmd^{ta222a/ta222a} (mean = 9.6 ± 3.2 s.e.m.; n = 13) zebrafish embryos. In the wild-type background, where huDysGFP competes with endogenous Dystrophin for available binding sites, the average ratio is 2.5 times lower than in the mutant background (p = 0.03), where huDysGFP can occupy all available sites. (D) Scatter plot of fractional recovery in bleached tip pixels as a function of the cytoplasmic intensity. (E) Comparative scatter plots, with mean and SD, of huDysGFP fractional recovery in bleached tip pixels in wild-type (wt), dmd^{ta222a/ta222a} (dmd) and their siblings (sibs). There were no statistically significant differences between groups as determined by one-way ANOVA [F(2,67) = 0.8628, ns]. (F) Comparative scatter plots, with mean and SD, of huDysGFP final unbleached tip minus bleached tip intensities in wild-type (wt), dmd^{ta222a/ta222a} (dmd) and their siblings (sibs). There were no statistically significant differences between groups as determined by one-way ANOVA [F(2,67) = 2.845, ns]. (G) huDysGFP fraction of cases showing no recovery, or 50% recovery at the first (<10 s), second (<20 s), or later (>20 s) time points, calculated from unbleached tip minus bleached tip intensities, in wild-type (wt), dmd^{ta222a/ta222a} (dmd) and their siblings (sibs). There were no statistically significant differences between groups as determined by one-way ANOVA [F(2,67) = 0.1521, ns]. Scale bars = 10 µm.