(A) The mutant line lmod3^sa13018 harboured a premature stop codon within exon 3 of lmod3, the start codon of which locates at 13’814’978 bp on chromosome 23. Scissors mark single gRNA targets and scale is 1 kbp. (B) The codon GAA encoding for E338 was changed to the nonsense mutation TAA within lmod3^sa13018. (C) Western blot analysis with antibodies against human LMOD3 detected Lmod3 epitope (76 kDa predicted) in siblings (left lane), but not in lmod3^sa13018 homozygotes (right lane). ß-tubulin was used as loading control. (D) In contrast to siblings, the swimming bladder (arrowhead) of 4-dpf-old lmod3^sa13018 homozygotes was not inflated. The bar indicates larval length without the caudal fin. (E) Whereas 4-dpf-old siblings were 3.68 ± 0.03 mm long, the larval length of lmod3^sa13018 homozygotes was with 3.51 ± 0.04 mm significantly shorter (n = 10 larvae). (F) Under polarised light, the muscle of lmod3^sa13018 homozygotes appeared darker compared to their siblings due to a reduction in birefringence. (G) After rescaling to siblings, the birefringence of lmod3^sa13018 homozygotes was significantly reduced to 79 ± 2% compared to their 3-dpf-old siblings. Crosses represent averaged birefringence of clutches with a minimum of 5 larvae per genotype (n = 5 clutches). At 6 dpf, the birefringence of lmod3^sa13018 homozygotes remained significantly reduced to 54 ± 2% of their siblings (n = 8 individual larvae). (H) Signs of fibrosis were absent on cross sections of 6-dpf-old lmod3^sa13018 siblings and homozygotes stained with hematoxylin and eosin (H&E). (I) lmod3^ex3 harboured a deletion of 1385 bp and insertion of 5 bp in exon 3 of lmod3. (J) After rescaling to siblings, the birefringence reduction of lmod3^ex3 homozygotes was 83 ± 2% at 3 dpf and 55 ± 2% at 6 dpf. Similarly, lmod3^ex3/sa13018 compound heterozygotes had a highly significant reduction in birefringence to 83 ± 2% at 3 dpf and to 58 ± 2% at 6 dpf. At 3 dpf, crosses represent averaged birefringence of clutches with a minimum of 5 larvae per genotype (n = 5 clutches) and individual larvae at 6 dpf (n = 8 larvae). Data are presented as mean ± SEM; ** P < 0.01 and *** P < 0.001 were calculated by Student’s t-test. Scale bar sizes are indicated.

(A) Peak active force of single-twitch contractions generated by 6-dpf-old individual lmod3^sa13018 larvae was significantly weaker compared to their siblings. Siblings generated a maximal active force of 0.82 ± 0.01 mN, compared to and 0.19 ± 0.03 mN generated by lmod3^sa13018 homozygotes (n = 4). (B) Z-stack projections of ventral views depict the cephalic musculature marked by GFP driven by the Tg(−503unc:GFP) transgene. In contrast to 4-dpf-old siblings, a gap between the two hyohyoideus (hh) muscles was formed in the lower jaw of lmod3^sa13018 homozygotes (asterix). (C) At 7dpf, focus stacks of Alcian Blue cartilage stains revealed a widened jaw within lmod3^sa13018 mutants. (D) The angle formed by the ceratohyal cartilages (dotted lines) was significantly wider in lmod3^sa13018 homozygotes compared to siblings (87 ± 2° and 65 ± 1°, respectively. n = 6). Data are presented as mean ± SEM; *** P < 0.001 calculated by Student’s t test. Scale bar sizes are 200 μm.

(A) Immunohistochemistry using antibodies against α-Actinin (green) and phalloidin staining (red) marked aggregates located close to vertical myosepta of 4-dpf-old lmod3^sa13018 homozygotes. Detected aggregates (arrowhead) were of various sizes and shapes, including rod-shaped structures (arrow). (B) Labelling of the myofibril with Lifeact-GFP (green) and the sarcolemma with mCherry-CaaX (red) confirmed aggregate (arrowhead) formation in lmod3^sa13018 homozygotes at 4 dpf, again exclusively at vertical myosepta. (C) At 4 dpf, transmission electron micrographs (TEM) of skeletal muscle from siblings showed the typical myofibril striation and well-aligned sarcomeres. Comparable sarcomeres were also present within lmod3^sa13018 homozygotes. However, close to the vertical myosepta, filament deposits reminiscent of fingerprint bodies (star), misaligned sarcomeres (arrows), and electron-dense aggregates of various sizes (arrowheads) were detected. (D) At 5 pdf, Gomori trichrome-stained sagittal sections depicted blue/purple structures close to the vertical myosepta within myofibres of lmod3^sa13018 homozygotes. Scale bar sizes are indicated.

(A) capza1a^ex5 mutants harbour an insertion of 20 bp into exon 5 of capza1a, resulting in a frame shift. (B) The mutant allele capza1b^ex5 was generated by deletion of 23 bp and insertion of 1 bp into exon 5 of capza1b, causing a frame shift. (C) At 3 dpf, in situ hybridization showed that the trunk expression of capza1a found in siblings was strikingly reduced in capza1a^ex5 homozygotes. (D) At 3 dpf, whole-mount in situ hybridization showed that the expression of capza1b was severely reduced in capza1b^ex5 homozygotes compared to siblings. (E) At 3 dpf, the birefringence of single capza1a^ex5/+ and capza1b^ex5/+ heterozygotes as well as single capza1a^ex5/ex5 homozygotes was comparable to WT siblings (capza1a^+/+;capza1b^+/+, capza1a^ex5/+;capza1b^+/+, capza1a^+/+;capza1b^ex5/+ and capza1a^ex5/ex5;capza1b^+/+ all were 100 ± 1%). However, single capza1b^ex5/ex5 homozygotes were significantly reduced in their birefringence and larvae that were additionally heterozygous for capza1a^ex5/ex5 were significantly reduced further (capza1a^+/+;capza1b^ex5/ex5 were 81 ± 1% and capza1a^ex5/+;capza1b^ex5/ex5 were 75 ± 1%). In addition, compound heterozygotes capza1a^ex5/+;capza1b^ex5/ex5 and capza1a^ex5/ex5 homozygotes that were capza1b^ex5/+ heterozygous were also significantly reduced in their birefringence (capza1a^ex5/+;capza1b^ex5/+ were 92 ± 2% and capza1a^ex5/ex5;capza1b^ex5/+ were 86 ± 1%). Data are mean ± SEM; * P < 0.05, ** P < 0.01 and *** P < 0.001 calculated by one-way ANOVA with post hoc Tukey’s test; n = 5 clutches. Scale bar sizes are 300 μm.

(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.

(A) At 2 dpf, Lifeact-GFP (green) and mCherry-CaaX (red) highlighted the sarcomere organisation and myofibril striation within muscle fibres of WT siblings. In two out of four analysed capza1b^ex5 homozygotes, thin filament deposits (arrows) were detected at the peripheral ends of some fibres. Although myofibril striation (arrowheads) was detected in capza1b^ex5 homozygotes that were hetero- or homozygous for capza1a^ex5, thin filament deposits (arrows) were frequently located at the peripheral myofiber ends. (B) At 2 dpf, highly organised sarcomeres were found on transmission electron micrographs of siblings and capza1a^ex5;capza1b^ex5 compound homozygotes. However, electron-dense aggregates with a lattice structure (arrowheads) and filament deposits (star) were present close to the myosepta (arrow) in the compound homozygotes. Scale bar sizes are indicated.

(A) After rescaling to WT siblings (100 ± 1%), the significant reduction of birefringence of single lmod3^sa13018 (78 ± 1%) and capza1b^ex5 (79 ± 1%) homozygotes was further reduced in lmod3^{sa13018/sa13018};capza1b^ex5/ex5 compound homozygotes (61 ± 2%). (B) At 4 dpf, lmod3^sa13018;capza1b^ex5 compound homozygotes featured thin filament deposits at the peripheral ends of myofibres (arrowhead), which were also highlighted in single capza1b^ex5 homozygotes by Lifeact-GFP (green) and mCherry-CaaX (red). The strong myofibril striation seen in WT siblings appeared severely reduced in capza1b^ex5 homozygotes and was rarely detected in lmod3^sa13018;capza1b^ex5 compound homozygotes (arrow). (C) After rescaling to WT siblings (100.0 ± 0.9%), the significant reduction of birefringence of single lmod3^sa13018 (82 ± 1%) and tmod4^trg homozygotes (47 ± 1%) was further reduced in lmod3^sa13018;tmod4^trg compound homozygotes (30 ± 1%). (D) At 3 dpf, the typical myofibril striation was highlighted by antibodies against actinin (green) and actin-labelling with phalloidin (red). In lmod3^sa13018;tmod4^trg compound homozygotes striation was rarely seen (arrowhead). Instead, actin- and actinin-positive aggregates were detected throughout myofibres (arrow) as well as actin-positive deposits close to vertical myosepta (double arrow). Data are presented as mean ± SEM; *** P < 0.001 calculated by one-way ANOVA with post hoc Tukey’s test; n = 5 clutches. Scale bar sizes are 20 μm.