Fig 5

(A) Whereas 6-dpf-old siblings were able to generate a peak active force of 0.76 ± 0.01 mN, the force generated by capza1b^ex5 homozygotes was significantly reduced to 0.14 ± 0.01 mN (n = 4). (B) GFP fluorescence driven by Tg(-503unc:GFP) showed that the two hyohyoideus (hh) muscles of capza1b^ex5 homozygotes and siblings are comparable at 4 dpf (Z-stack projections of ventral views). (C) Focus stacks of Alcian Blue cartilage stains of capza1b^ex5 homozygotes and siblings were comparable at 7 dpf. (D) The ceratohyal angle (dotted lines) of capza1b^ex5 homozygotes was with 62 ± 2° not significantly different from the angle of 63 ± 1° formed in siblings (n = 6). (E) Phalloidin (red) and antibodies against α-Actinin (green) marked aggregates (arrowhead) close to the vertical myosepta within 3-dpf-old capza1b^ex5 homozygotes but not siblings. (F) Lifeact-GFP (green) highlighted organised myofibril (arrow) in life siblings and capza1b^ex5 homozygotes at 3dpf. In addition to the residual striation, capza1b^ex5 homozygotes featured myofibril ruptures (arrowhead) as well as aggregates and thin filament deposits at the peripheral myofibre ends. Transgenic mCherry-CaaX (red) was used to mark the sarcolemma and t-tubules. (G) On 3 dpf sagittal sections, Gomori trichrome-staining exposed blue/purple structures (arrowhead) close to the vertical myosepta of capza1b^ex5 homozygotes, but not siblings. (H) At 3 dpf, sarcomeres were organised within siblings as revealed by transmission electron micrographs. Organised sarcomeres were also present within capza1b^ex5 homozygotes (star). However, sarcomere organisation was lost in capza1b^ex5 close to myofibres’ ends and filament deposits, where isolated sarcomeric structures (arrow) and electron-dense aggregates (arrowhead), often with a lattice structure, were found instead. (I) Interestingly, myofibril ruptures were detected in 4-dpf-old capza1b^ex5/+ heterozygotes but not WT siblings, demonstrating capza1b haploinsufficiency. Data are presented as mean ± SEM; n.s. not significant and *** P < 0.001 calculated by Student’s t test. Scale bar sizes are indicated.