Nuclear distribution and shape indicate that hydrostatic pressure of the notochord is decreased in spzl mutants. ( A–B) Confocal images of cross sections of 14 dpf WT and spzl-/- larvae section. Vacuolated cells are labeled with SAG:gal4;UAS:GFP and nuclei are stained with DAPI. Scale bar = 50 µm ( C) Plot of vacuolated cell nuclear distribution for WT (red, n = 29) and spzl-/- (black, n = 32). ( D) Nuclear volume in WT and spzl-/- at 14 dpf. ( E–F) Reconstructions of nuclei in WT ( E) and spzl-/- ( F) and two different viewing angles. Scale bar = 20 µm ( G) Sphericity of WT and spzl-/- nuclei at 14 dpf. p-values were determined by an un-paired t-test using Welch’s correction.

Loss of Dstyk function causes the spzl phenotype. ( A) Genome view of SNPtrack linkage analysis of exome sequencing data. The spzl locus maps to chromosome 22 (top panel), with a high probability interval (red line) centered around 410–570 Kbps. Recombination events with a panel of 83 mutants at either side of the spzl locus define a ~ 1.2 Mb critical interval. ( B) Genome organization of the critical genomic interval near the spzl locus. A nonsense mutation generates an early stop codon in dstyk. ( C) Cartoon depicting predicted gene product structure of dstyk-202, spzls739 and the Crispr/Cas9 generated dstykpd1133 allele. ( D) Dstyk is a Ser/Thr and Tyr kinase. Heat map depicting amino acid similarity between the human and zebrafish proteins. The kinase domain (highlighted) is 93.73% similar. ( E) In situ hybridization for dstyk expression at the 1 cell stage, 10 somites, 20 somites, and 24 hpf, respectively. Arrows point to highly expressing areas of the notochord. ( F–H) Bright field images of spzl-/-and WT sibling, spzl;dstykpd1133-/- and WT sibling, dstykpd1133 homozygous mutant and WT sibling, scale bars = 1 mm. DIC images of the notochord of 48 hpf embryos. Arrows point to fragmented vacuoles. Scale bars are 100 µm.

gabra1 expression in the developing zebrafish. WISH was performed at 1, 2 and 3 DPF with an anti-sense gabra1 probe. Arrows indicated the expression of gabra1 at each developmental stage. Minimum of ten larvae per group/biological replicate. Abbreviations: FB, forebrain; OT, optic tectum; C, cerebellum; HB, hindbrain; MO, medulla oblongata. Scale bars: 0.2 mm.

Whole-mount in situ hybridization with prokr1a probe (A–F) did not display any signal in the head of zebrafsh embryos. In contrast, prokr1b (G–L) is expressed in cells near the olfactory region at 36 hpf (G,H), 48 hpf (I,J) and 72 hpf (K,L). Tat is similar to gnrh3 expression at these times (M–R). Panels A,C,E,G,I,K,M,O and Q show lateral views. Panels B,D,F,H,J,L,N,P and R show dorsal views of zebrafsh embryos. Scale bar=50µm. Image taken using Leica Application Suite sofware

Generation of capn3b null mutant allele. a Generation of the capn3bΔ19Δ14 mutant allele. Upper panel: diagram showing the genomic structure of the zebrafish capn3b gene and the two mutated sites in capn3bΔ19Δ14 generated by TALEN and CRISPR-Cas9 approaches, respectively. Vertical bar: exon; line connecting vertical bar: intron. Lower panel: highlighting the 19 bp deletion (red letters) in 1st exon generated by the TALEN approach (on the left) and 14 bps deletion (red letters) in 3rd exon by the CRISPR-Cas9 approach (on the right), respectively. The position of nucleotide in the capn3b open reading frame (ORF) from the translation start codon ATG is provided. C120, Capn3b activity center. b Predicted peptide encoded by the capn3bΔ19 (∆19) and capn3bΔ19Δ14 (Δ19Δ14) mutant mRNA, respectively. The Δ19 mutation leads to an early stop codon in the capn3b ORF and resulted in a 30 amino acids (aa) long polypeptide, however, the Δ19 mutation potentially allows the ATG codon encoding M94 residue of WT Capn3b to be used as an alternative translation start codon to translate an N-terminus truncated peptide which harbors the activity center C120. The Δ19Δ14 mutation creates a new early stop codon in the presumed variant initiated from M94 after C120, therefore, capn3bΔ19Δ14 is likely a null allele. c Western blotting analysis of Capn3b in WT, capn3b capn3bΔ19Δ14, capn3bΔ19 at 5dpf. β-Actin: loading control. d Immunostaining of Capn3b in WT and capn3bΔ19Δ14 mutant embryos at 5dpf. Scale bar: 50 μm

AICAR and Metformin Treatment Improve the Muscle Phenotype in a Zebrafish Model of Dysferlin Deficiency (A) Morpholino oligonucleotides (MOs) targeting zebrafish dysferlin. (B) A single RT-PCR product of 301 bp was seen in 4-dpf embryos injected with 1.5 ng of control MO; two differently sized RT-PCR products of 301 and 195 bp were seen in 4-dpf embryos injected with 1.5 ng of dysferlin. (C) Panels represent the results of the birefringence assay (BR). The abnormal structure of the muscles is more clearly seen in dysferlin MO-injected fish (MO, lower panel) than in controls (CMO, upper panel) under BR. Scale bar, 200 μm. (D) Histogram of the percentage of normal, affected (reduced BR), and dead fish upon MO injection. After injecting 1.5 ng of MO, approximately 30.0% of the embryos exhibited reduced birefringence. The white, black, and gray bars show the percentage of normal, reduced BR, and dead fish, respectively. WT, uninjected fish; CMO, CMO-injected fish; MO, MO-injected fish. (E) Representative western blot image of phospho-AMPK, AMPKα, and β-actin in MO-injected fish treated with AICAR for 4 days at 0, 0.1, 1.0, or 10 mM. Quantification of phospho-AMPK expression was normalized to AMPKα in the lower panels. (F) AICAR treatment reduced the percentage of BR-reduced fish. Results for metformin concentrations of 0, 0.1, 1.0, and 10 mM are the average of three independent experiments. Of the untreated MO morphants, 31.1% were BR reduced. AICAR treatment at concentrations of 0.1, 1.0, and 10 mM reduced the percentage of BR-reduced fish to 22.2%, 17.8%, and 17.8%, respectively. Statistically significant differences were determined using ANOVA followed by Tukey’s multiple comparisons test. Statistical significance was set at *p < 0.05. (G) Representative western blot image of phospho-AMPK, AMPKα, and β-actin in MO-injected fish treated with metformin for 4 days at 0, 1, and 10 mM. Quantification of phospho-AMPK expression was normalized to AMPKα in the lower panels. Results for metformin concentrations of 0, 1, and 10 mM are the average of three independent experiments and were normalized to those without metformin treatment. (H) Metformin treatment reduced the percentage of BR-reduced fish. Results for metformin concentrations of 0, 1, and 10 mM are the average of three independent experiments. Of the untreated MO morphants, 33.3% were BR-reduced fish. Metformin treatment at concentrations of 1 and 10 mM reduced the percentage of BR-reduced fish to 25.0% and 20.8%, respectively. Statistically significant differences were determined using ANOVA followed by Tukey’s multiple comparisons test. Statistical significance was set at *p < 0.05.

Dstyk regulates fusion of pre-vacuolar carriers with the notochord vacuole. ( A–B) Maximum projection of a light sheet microscopy live image of WT and spzl-/- embryos at 48hpf and 65hpf, respectively. ( C–D) Still images from a 17 hr light sheet movie of a WT and spzl-/-embryos zebrafish embryo during vacuole biogenesis depicting vacuole fusion events. Vacuole membranes are labeled by NFATC:gal4;UAS:GFPrab32 expression. Arrows point to GFP-Rab32 positive vesicles. Arrowheads point to large vesicle (vacuolinos) docking events. n = 2 animals per genotype. Asterisks mark fusion events. Scale bar = 100 µm.

Defective development of capn3b mutant zebrafish under environment stress. a, b WISH using the fabp10a probe on 3dpf- and 5dpf-old WT and capn3bΔ19Δ14 embryos grown at the low (60 embryo/dish) (left panel) and high (120cm2/dish) (right panel) rearing density in a 9 cm-diameter dish (a). In (b), the data of liver sizes were compared between 3dpf- and 5dpf-old WT or between 3dpf- and 5dpf-old capn3bΔ19Δ14 mutant embryos under low (L) and high (H) rearing intensity. c WISH using the fabp10a and trypsin probes on WT and capn3bΔ19Δ14 embryos at 2dpf and 3dpf. Embryos were shifted to 34.5 °C at 12 hpf till the time of sample harvesting. Numerator/denominator: number of embryos displayed the shown phenotype over total number of genotyped embryos. d Photo images showing the curved body phenotype displayed by the 3dpf-old capn3bΔ19Δ14 mutant but not WT embryos grown at 34.5 °C. e Body lengths of 6-, 8- and 12-months-old WT and capn3bΔ19Δ14 fish. f LBR of 6- and 12-months-old WT and capn3bΔ19Δ14 fish. Student’s T-test for statistical analyses, *, p < 0.05; ***, p < 0.001; ****, p < 0.0001

Elp3 morphants present a ventrally-curved tail. (A) Schematic representation of the anti-codon loop of a tRNA, indicating position 34 (red arrow) where 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) is located. Anti-codon is depicted in green. (B) In situ hybridization of Elp3 mRNA in 4 cell (upper) and 27 hpf (lower) embryos. Scale bar 0.7 mm. (C) Saccharomyces cerevisiae mutants for ELP3 (ΔELP3) were transformed with an expression plasmid that contained zebrafish Elp3 (zElp3). BY4741 was used as control. Image representative of three independent experiments. (D) Western blot detecting Elp3 in 48 hpf samples from control and morphant embryos (MO-Elp3); β-actin was used as loading control. Samples from embryos injected with translation- (ATG) and splicing-blocking (SP). (E) Liquid chromatography-mass spectrometry (LC-MS) analysis of tRNA modification. Peak area values for mcm5s2U are shown normalized to the of purified tRNA for each sample (n = 3, * p < 0.05). (F) qPCR for UPR targets BiP, Chop and Atf4. Fold change in mRNA levels comparing morphant embryos to controls; t-test was performed for statistical analysis. *** p < 0.005). (G) Representative images of control, morphants and MO + ELP3 mRNA injected embryos by 48 hpf (images representative of at least six independent experiments, in which at least 100 embryos were analyzed).