polg2^ia304 line generation, survival and developmental delay.

A Schematic representation of zebrafish polg2 and its editing by CRISPR/Cas9. Bpolg2^ia304/ia304 individuals fail to survive to 4 weeks post fertilization. Data were analysed by χ2 test; p = 0.4165; p = 0.9802; p = 0.5617; p = 0.0438; p = 0.0226; p < 0.0001; 3 dpf (n = 120), 6 dpf (n = 115), 12 dpf (n = 89), 20 dpf (n = 70), 25 dpf (n = 50), 30 dpf (n = 46). Gene expression analysis performed to assess polg2 (C) and polg (D) mRNA levels in polg2^ia304 mutant line. Values are reported as mean ± SEM of three independent experiments and analysed by Games-Howell’s multiple comparison test (C) and ordinary one-way ANOVA followed by Tukey’s test (D); *p < 0.05; **p < 0.005; ***p < 0.001; polg2^+/+ (n = 6 × 10-larva pools), polg2^+/ia304 (n = 6 × 10-larva pools), polg2^ia304/ia304 (n = 4 × 10-larva pools). E Representative images of zebrafish polg2^+/+ control and polg2^ia304/ia304 mutant at 20 dpf, and polg2^+/+ control and a polg2^+/ia304 heterozygote at 90 dpf. Scale bar: 2 mm (top); scale bar: 1 cm (bottom). F Body length analysis in wt (polg2^+/+), heterozygous (polg2^+/ia304) and homozygous (polg2^ia304/ia304) individuals. Values are expressed as mean ± SEM. Statistics were assessed by Mann-Whitney test; ****p < 0.0001; polg2^+/+ 6 dpf (n = 25), polg2^+/ia304 6 dpf (n = 41), polg2^ia304/ia304 6 dpf (n = 22); polg2^+/+ 20 dpf (n = 39), polg2^+/ia304 20 dpf (n = 74), polg2^ia304/ia304 20 dpf (n = 45); polg2^+/+ 90 dpf (n = 8), polg2^+/ia304 90 dpf (n = 7).

polg2^ia304 behavioural defects assessed by locomotor analysis.

A, C, E Effect of light stimuli on locomotion in polg2^ia304/ia304 mutants at 6, 15 and 20 dpf, respectively. White fields indicate light exposition time, while grey fields represent dark periods. The analysis assessed the average total distance swum at 6 dpf (B), 15 dpf (D) and 20 dpf (F). Values derived from 3 independent biological replicates and are reported as mean ± SEM. Data were analysed using ordinary one-way ANOVA and Tukey’s test (B) or Kruskal-Wallis together with Dunn’s multiple comparison test (D, F); *p < 0.05; **p < 0.005; ***p < 0.001; for 6 dpf polg2^+/+ (n = 19), polg2^+/ia304 (n = 33), polg2^ia304/ia304 (n = 13); for 15 dpf polg2^+/+ (n = 32), polg2^+/ia304 (n = 58), polg2^ia304/ia304 (n = 45); for 20 dpf polg2^+/+ (n = 57), polg2^+/ia304 (n = 131), polg2^ia304/ia304 (n = 49). G Effect of tapping stimulus on locomotion in wt, heterozygous and polg2^ia304/ia304 individuals at 8 dpf. Tapping stimuli were delivered in a 20-s time window with an interstimulus interval of 1 s. H Average response to maximum tapping stimuli given as the total distance covered by polg2^ia304/ia304 homozygotes during tap stress assay, compared to wt and heterozygous sibs. Results are expressed as mean ± SEM. For statistical analysis, the Kruskal-Wallis test corrected with Dunn’s multiple comparison test was applied; *p < 0.05; polg2^+/+ (n = 38), polg2^+/ia304 (n = 72), polg2^ia304/ia304 (n = 26), from 3 independent experiments.

Effects of polg2 mutation on high energy demanding tissues.

A, C Comparison of cardiac tissues at 20 dpf, reporting differences in atrial (a) and ventricular (v) dimensions and an altered trabecular network (t) in mutants. Scale bar: 5 mm. B Histological sections of polg2^+/+ and polg2^ia304/ia304 skeletal muscle tissue at 20 dpf, displaying disorganized fibres and altered somite boundaries in mutants. Arrows: detachment of skeletal muscle fibres. Scale bar: 5 mm. D TEM analysis of 20 dpf skeletal muscle of wt (polg2^+/+) and homozygous mutants (polg2^ia304/ia304), the latter displaying altered organization of myofibrils. Scale bar: 1 µm. E TEM analysis of mitochondria in 20 dpf heart of wt (polg2^+/+) and homozygous mutants (polg2^ia304/ia304). Scale bar: 500 nm. TEM analysis of 20 dpf skeletal muscle from wt (polg2^+/+) (F) and homozygous mutants (polg2^ia304/ia304) (G) confirms altered morphology of myofibrils and mitochondrial cristae (black arrowheads) in mutants. Scale bar: 500 nm. H Quantification of inter-cristae distance (in nm) in the cardiac region. Data are shown as mean ± SEM and statistics were done using unpaired Student’s t-test. I Quantification of heart mitochondrial (mt) aberrant morphologies. For statistical analysis, the χ² test was applied; N = 8 independent TEM images per condition, in triplicate. J Quantification of inter-cristae distance (in nm) in the skeletal muscle. Values are reported as mean ± SEM. Statistics were assessed using unpaired Student’s t-test; ****p < 0.0001. K Quantification of skeletal muscle mt aberrant morphologies. χ² test was used for statistical analysis, N = 8 independent TEM images per condition, in triplicate. ****p < 0.0001. L Number of wide inter-cristae areas, considered when at least equal to 100 × 100 nm, counted for each organelle in the skeletal muscle. Data are shown as mean ± SEM. Mann-Whitney test was used for statistics; ****p < 0.0001.

Defects in mitochondrial content, morphology and metabolism under polg2 KO.

A Confocal images of the Tg(Hsa.Cox8a:MLS-EGFP)^ia301 mitochondrial marker (mito:EGFP) in polg2^+/+, polg2^+/ia304 and polg2^ia304/ia304 zebrafish at 6 dpf. Scale bar: 10 µm. B Relative quantification of the mito:EGFP transgene at 6 dpf. Data are reported as mean ± SEM. Statistics were assessed using the Kruskal-Wallis test followed by Dunn’s multiple comparison test; *p < 0.05; ****p < 0.0001; n = 10 independent larvae per condition. Comparison of relative abundances of the mitochondrial nd1 (C) and nd2 (D) genes in wt (polg2^+/+), heterozygous (polg2^+/ia304) and homozygous (polg2^ia304/ia304) zebrafish at 6 dpf. Values are shown as mean ± SEM. Ordinary one-way ANOVA (C) or Games-Howell’s multiple comparison test (D) were applied for statistics; **p < 0.005; ***p < 0.001; Cpolg2^+/+ (n = 6 × 10-larva pools), polg2^+/ia304 (n = 6 × 10-larva pools), polg2^ia304/ia304 (n = 6 × 10-larva pools); (D) polg2^+/+ (n = 5 × 10-larva pools), polg2^+/ia304 (n = 8 × 10-larva pools), polg2^ia304/ia304 (n = 5 × 10-larva pools) from 3 independent biological replicates. E Analysis of the nd1 gene at 20 dpf. Values are reported as mean ± SEM. For statistical analysis, the Kruskal-Wallis test corrected with Dunn’s multiple comparison test was used; **p < 0.005; polg2^+/+ (n = 6), polg2^+/ia304 (n = 7), polg2^ia304/ia304 (n = 7) from 3 independent experiments. F Diagram depicting the oxygen consumption rate (OCR) profile by Seahorse assay in 4 dpf wt (polg2^+/+), heterozygous (polg2^+/ia304) and homozygous (polg2^ia304/ia304) larvae under basal conditions, FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone)-induced maximal respiratory capacity stimulation and ROT/AA (Rotenone/Antimycin A)-mediated inhibition. Two-way ANOVA followed by Tukey’s multiple comparison test was applied for statistical significance; *p < 0.05 between polg2^+/+ and polg2^ia304/ia304 individuals. G Quantification of basal OCR in 4 dpf zebrafish. Data are shown as mean ± SEM and were analysed by ordinary one-way ANOVA; *p < 0.05; polg2^+/+ (n = 9), polg2^+/ia304 (n = 24), polg2^ia304/ia304 (n = 8) from 3 independent replicates. H Imaging of HRE:EGFP (Hif-hypoxia) reporter at 6 dpf: an example of wt (above, left), heterozygous (above, right) and homozygous (below) mutant larva. Scale bar: 1 mm. I Integrated density analysis of Hif-hypoxia signalling activation in 6-dpf wt (polg2^+/+), heterozygous (polg2^+/ia304) and homozygous (polg2^ia304/ia304) larvae. Values are expressed as mean ± SEM and analysed by ordinary one-way ANOVA; **p < 0.005; polg2^+/+ (n = 6), polg2^+/ia304 (n = 26), polg2^+/ia304 (n = 52), polg2^ia304/ia304 (n = 21), coming from 3 independent experiments.

Analysis of CLO rescuing effects under polg2 KO.

A mtDNA content in treated (+) and non-treated (-) polg2^+/+, polg2^+/ia304 and polg2^ia304/ia304 individuals, exposed to 3 μM CLO from 2 dpf to 6 dpf; Data are shown as mean ± SEM, each dot corresponds to a 10-fish pool. Statistics were assessed using ordinary one-way ANOVA, followed by Tukey’s multiple comparison test; *p < 0.05; **p < 0.005; ****p < 0.0001; polg2^+/+ (n = 6 ×10-larva pools), polg2^+/+ + CLO (n = 5 x 10-larva pools), polg2^+/ia304 (n = 10 x 10-larva pools), polg2^+/ia304 + CLO (n = 10 x 10-larva pools), polg2^ia304/ia304 (n = 7 x 10-larva pools), polg2^ia304/ia304 + CLO (n = 6 ×10-larva pools), from 3 independent biological replicates. B Effect of light stimuli on locomotion in treated (+) and non-treated (-) polg2^+/+, polg2^+/ia304 and polg2^ia304/ia304 individuals, exposed to 2 μM CLO from 10 dpf to 15 dpf. White fields indicate light exposition time, while grey fields represent dark periods. Two-way ANOVA followed by Dunn’s multiple comparison test was applied for statistical significance; polg2^+/+ (n = 34), polg2^+/+ + CLO (n = 32), polg2^+/ia304 (n = 70), polg2^+/ia304 + CLO (n = 50), polg2^ia304/ia304 (n = 35), polg2^ia304/ia304 + CLO (n = 26). C Maximum response to light stimuli, expressed in mm (distance swum) among the three genotypes (polg2^+/+, polg2^+/ia304 and polg2^ia304/ia304) in treated (+) and non-treated (-) fish. Values are reported as mean ± SEM. Statistical analysis was done by Kruskal-Wallis test followed by Dunn’s multiple comparison test; **p < 0.005; *p < 0.05; polg2^+/+ (n = 34), polg2^+/+ + CLO (n = 32), polg2^+/ia304 (n = 70), polg2^+/ia304 + CLO (n = 50), polg2^ia304/ia304 (n = 35), polg2^ia304/ia304 + CLO (n = 26), from 3 independent replicates and in triplicate.