Zebrafish Models for Dysferlinopathy
(A) Comparison of dysf morpholino-injected (i36e37, e18i18) embryos with uninjected control (first panel) at 72 hpf. The i36e37 morphants have slightly bent (second panel) or curved body (third panel), whereas e18i18 morphants (fourth panel) have dorsally bent trunks.
(B) Embryos at 24 hpf, stained with slow muscle myosin antibody F59. No significant defects were obvious in dysf morphants.
(C) F59 staining at 72 hpf reveals gaps (arrow) in the muscles of i36e37 morphants, whereas no defects were observed in e18i18 morphants.
(D) Actin (FITC-phalloidin) staining demonstrates curved myofibers and gaps (arrow) in the i36e37 morphant muscles. e18i18 morphants have slightly misaligned myofibers.
(E) β-sarcoglycan staining shows differences in myoseptal (arrowhead) angle in i36e37 morphants.
(F) Accumulation of Evans blue dye in i36e37 morphant muscle tissue (m). bv, blood vessel.
(G) Birefringence is drastically reduced in i36e37 morphants at 72 hpf, whereas no difference was noted in e18i18 morphants in comparison with control larvae.
(H–K) Electron microscopy (sagittal sections) of 72 hpf myofibers. Plasma membrane is electron dense. (H) shows uninjected control larval myofiber with correctly aligned sarcomeres and T-tubules (arrows). (I) shows extensive muscle damage in i36e37 morphant myofibers. In (J), a high-magnification image of i36e37 morphant myofiber shows misplaced T-tubule fragments (arrows). In (K), vesicles (arrowheads) are widespread in damaged i36e37 morphant myofibers.
(L) Zebrafish Dysf protein structure: C2, C- and N-terminal Dysf (DC and DN) as well as the TM domain are indicated. Vertical lines point to morpholino target sites.
(M) Sequence alignment of human and zebrafish Dysf C2E domain (underlined). The arrowhead points to the splice site, targeted by morpholino i36e37. Magenta letters outline the region, which is missing in i36e37 morphants. Additional transcript is present in the morphants, lacking the whole protein region, downstream from the morpholino target site. Asterisks indicate mismatch substitutions that cause myopathy in humans; x denotes mutations, resulting in premature stop codon in human DYSF.
(N) Sequence alignment of the Dysf protein region targeted by e18i18 morpholino. Letters in magenta indicate the deleted part in the morphants. Orientation of embryos A–G: anterior left, dorsal up.Scale bars represent 500 μm (A and G); 80 μm (B, C, E, and F); 20 μm (D); 6 μm (C); 2 μm (H–I); 1 μm (J and K). See also Figure S1.
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