Fig. 1

Human Dystrophin expression in the zebrafish embryo.

(A) Main features of the human Dystrophin expression constructs engineered for this study. (B) Schematic illustrating 2 dpf zebrafish embryo. Slow muscle fibres within the chevron-shaped somite, one magnified and highlighted in blue, are typically aligned anterior-posteriorly with their tips (dark blue) attaching at vertical somite borders. (C) Immunofluorescent detection of exogenous huDys (green, arrows) at fibre tips, co-localizing with endogenous zebrafish Dystrophin (zfDys, red) that accumulates at the tips of every muscle fibre, marking the somite border. (D) In vivo expression of control GFP shows accumulation in muscle fibre cytoplasm without enrichment at the fibre tips. (E) Immunodetection with antibody specifically recognizing human Dystrophin on whole mount 2 dpf embryo shows punctate accumulation of exogenous huDys (arrow) suggestive of localization at the NMJ, in addition to fibre tips (arrowheads). (F) Immunodetection on longitudinal cryostat sections of 2 dpf somitic muscle shows enrichment of endogenous zebrafish Dystrophin (zfDys) at NMJ (arrows). Note concentration of most zfDys at fibre tips (arrowheads). (G) Maximum intensity projection of a confocal stack showing accumulation of huDysGFP in a muscle fibre in vivo. Strong enrichment is noticeable at the tips (arrows), membrane protrusions (yellow arrowheads), and NMJ (red arrowheads). (H) Double immunofluorescent detection of GFP in a huDysGFP-expressing embryo (huDysGFP, green) and α-bungarotoxin (BTX, red) confirms co-localization at the NMJ (insert). (I, J) huDysGFP mRNA detected by in situ hybridization (arrows in I, Nomarski) localises at fibre tips like GFP fluorescence detected while in vivo (arrows in J; confocal maximum projection). Scale bars = 10 µm.