Fig. 4

EtOH-induced fiber detachments result from sarcolemmal instability and failure of muscle end attachments. (A–C1) Anterior left, dorsal top, side-mounted, live, 78 hpf embryos treated continuously with 2% EtOH from 30 to 72 hpf, injected with 1% Evans Blue Dye (EBD) (pseudocolored blue) at 72 hpf. White boxes in A, B, and C correspond to zoomed-in, numbered panels A1, B1, and C1, respectively. (A–A1) Control embryo. EBD is in the blood stream (blue arrowheads). (B–B1) Embryo with mild fiber detachment phenotype that was induced by continuous treatment of 2% EtOH from 30 to 72 hpf. EBD is in the blood stream (blue arrowheads). EBD permeates some long muscle fibers (white arrows in B and B1). (C–C1) Embryo with severe fiber detachment phenotype that was induced by continuous treatment of 2% EtOH from 30 to 72 hpf. EBD is in the blood stream (blue arrowheads). EBD permeates long (white arrows in C and C1) and short muscle fibers. (D) Quantification of the relative length of EBD-penetrated myofibers. EBD penetrated myofibers ranging from short, retracted fibers to fully elongated, diagonally arrayed, fast-twitch myofibers in EtOH-treated embryos. (E–I3) Anterior left, dorsal top, side-mounted, 72 hpf embryos stained with phalloidin (pseudo-colored fuchsia) to visualize actin and anti-beta-DG antibodies (green). Lettered panels show phalloidin and beta-DG channels merged. White boxes correspond to numbered panels on the right. Panels numbered 1 show zoomed-in images of phalloidin staining only. Panels numbered 2 show zoomed-in images of beta-DG staining only. Panels numbered 3 show zoomed-in images of phalloidin and beta-DG channels merged. (E–E3) Control embryo. Fibers remained long and beta-DG is localized to the MTJ. (F–G3) Embryos treated continuously with 2% EtOH from 30 to 72 hpf showing mild fiber detachment. White arrowheads in F1–F3 and G1–G3 point to retracted fibers unaccompanied by beta-DG. (H–I3) Embryos treated continuously with 2% EtOH from 30 to 72 hpf with severe fiber detachment. White arrowheads in H1–H3 and I1–I3 point to retracted fibers that retained beta-DG at their detached ends. Scale bar is 50 micrometers.