Mutagenesis of zebrafish rrm2b –. (A) Summary schematic depicting the rrm2b sgRNA target site on exon 3 of the zebrafish rrm2b gene. (B) DNA and corresponding peptide sequence of WT zebrafish rrm2b and mutant zebrafish rrm2b^∆26bp. Red = mutation, ^* = premature stop codon. (C) Immunoblotting of Rrm2b and alpha tubulin in rrm2b^+/+ and rrm2b^−/− in 5 dpf tail muscle. N = 10 larvae tails each. D. Quantification of Rrm2b immunoblotting relative to alpha tubulin.

Characterisation of rrm2b^−/− mutants—. (A) Mean velocity (mm/s) after touch stimulus of 2 dpf rrm2b^+/+ (n = 8), rrm2b^+/− (n = 23) and rrm2b^−/− larvae (n = 12). Results are shown as mean ± SEM (statistical significance was determined with a Kruskal-Wallis test, ^* = adj. P < 0.05, ^** = adj. P < 0.01). (B) Total displacement (mm) after touch stimulus of 2 dpf rrm2b^+/+ (n = 8), rrm2b^+/− (n = 23) and rrm2b^−/− larvae (n = 12). Results are shown as mean ± SEM (statistical significance was determined with a Kruskal-Wallis test, ^* = adj. P < 0.05). (C) Peak acceleration (mm/s²) after touch stimulus of 2 dpf rrm2b^+/+ (n = 8), rrm2b^+/− (n = 22) and rrm2b^−/− larvae (n = 12). Results are shown as mean ± SEM. (D) Representative image illustrating the morphological parameters used to assess zebrafish larval development. (E) Standard length (SL) measurements of 5 dpf rrm2b^+/+ (n = 16), rrm2b^+/Δ26bp (n = 48) and rrm2b^−/−(n = 14) larvae. Results are shown as mean ± SEM. (F) Snout-vent length (SVL) of 5 dpf rrm2b^+/+ (n = 16), rrm2b^+/− (n = 48) and rrm2b^−/− (n = 14) larvae. Results are shown as mean ± SEM. (G) Head-trunk angle (HTA) measurements of 5 dpf rrm2b^+/+ (n = 16), rrm2b^+/− (n = 48) and rrm2b^−/− (n = 14) larvae. Results are shown as mean ± SEM. (H) Eye area (EA) measurements of 5 dpf rrm2b^+/+ (n = 16), rrm2b^+/− (n = 48) and rrm2b^−/− (n = 14) larvae. Results are shown as mean ± SEM. (I) Swim bladder area (SBA) measurements of 5 dpf rrm2b^+/+ (n = 16), rrm2b^+/− (n = 48) and rrm2b^−/− (n = 14) larvae. Results are shown as mean ± SEM (statistical significance was determined with a Kruskal-Wallis test, ^**** = adj. P < 0.0001). (J) Representative images of 5 dpf rrm2b^+/+ larvae morphology (top panel) and 5 dpf rrm2b^−/− larvae morphology (bottom panel). White arrow indicates uninflated swim bladder. (K) Relative mtDNA copy number of whole 3 dpf rrm2b^+/+ & rrm2b^+/− (n = 13) and rrm2b^−/− (n = 14) larvae. Results are shown as mean ± SEM. (L) Relative mtDNA copy number in whole 5 dpf rrm2b^+/+ & rrm2b^+/− larvae (n = 50) and rrm2b^−/− larvae (n = 29). Results are shown as mean ± SEM. (M) Relative mtDNA copy number in the head and abdomen of 5 dpf rrm2b^+/+ & rrm2b^+/− larvae (n = 21) and rrm2b^−/− larvae (n = 16). Results are shown as mean ± SEM. (N) Relative mtDNA copy number in tail muscle of 5 dpf rrm2b^+/+ & rrm2b^+/− larvae (n = 15) and rrm2b^−/− larvae (n = 12) results are shown as mean ± SEM (statistical significance was determined with a Student’s t-test, ^* = P < 0.05). (O) Survival of rrm2b larvae beyond 5 dpf by genotype. 5 dpf: rrm2b^+/+: 25% (n = 23), rrm2b^+/−: 52% (n = 48), rrm2b^−/−: 23% (n = 21). 7 dpf: rrm2b^+/+: 18% (n = 6), rrm2b^+/−: 58% (n = 19), rrm2b^−/−: 24% (n = 8). 10 dpf: rrm2b^+/+: 37% (n = 15), rrm2b^+/−: 53% (n = 21), rrm2b^−/−: 10% (n = 4). 12 dpf: rrm2b^+/+: 42% (n = 15), rrm2b^+/−: 50% (n = 18), rrm2b^−/−: 8% (n = 3). 14 dpf: rrm2b^+/+: 16% (n = 5), rrm2b^+/−: 87% (n = 27), rrm2b^−/−: 0% (n = 0). 17 dpf: rrm2b^+/+: 36% (n = 9), rrm2b^+/−: 60% (n = 15), rrm2b^−/−: 4% (n = 1). 19 dpf: rrm2b^+/+: 37% (n = 12), rrm2b^+/−: 63% (n = 20), rrm2b^−/−: 0% (n = 0). 21 dpf: rrm2b^+/+: 60% (n = 21), rrm2b^+/−: 40% (n = 14), rrm2b^−/−: 0% (n = 0).

Deoxynucleoside supplementation in 5 dpf rrm2b and wild type larvae. A. Relative mtDNA copy number in 5 dpf rrm2b larvae treated between 0 dpf and 5 dpf with 50 μM of deoxynucleosides: dAdo (A), dGuo (G), dCtd (C) & dTmd (T) with 5 μM adenosine deaminase inhibitor, EHNA. Untreated 5 dpf rrm2b^+/+ & rrm2b^+/− tail muscle (n = 54) (circles with thin border), treated 5 dpf rrm2b^+/+ & rrm2b^+/− tail muscle (n = 36) (circles with thick border). Untreated 5 dpf rrm2b^−/− tail muscle (n = 21) (squares with thin border), treated 5 dpf rrm2b^−/− tail muscle (n = 20) (squares with thick border). Results are shown as mean ± SEM (statistical significance was determined with a Mann–Whitney U test, ^****P < 0.0001). B–E. Relative mtDNA copy number of pools of 10, 5 dpf WT whole larvae treated with 15 combinations of deoxynucleosides (circles with thick border) or untreated (circles with thin border). 5 μM of ENHA (deaminase inhibitor) was also supplemented with each deoxynucleoside combination. 3 pools of 10 larvae per treatment condition. Results are shown as mean ± SEM (statistical significance was determined with a one-way ANOVA, ^* = adj. P < 0.05).

Assessment of purine deoxynucleoside supplementation in 5 dpf rrm2b larvae—.(A) Relative mtDNA copy number in whole 5 dpf rrm2b larvae, treated with either vehicle (DMSO) (rrm2b^+/+ n = 7 (circles with thin border), rrm2b^−/− n = 9, (squares with thin border)) or purine deoxynucleosides (50 μM dAdo & dGuo + 5 μM ENHA in 0.5%DMSO) (rrm2b^+/+ n = 4 (circles with thick border), rrm2b^−/− n = 10 (squares with thick border)) between 0 dpf and 5 dpf. Results are shown as mean ± SEM. (B) Average movement profile of 5 dpf vehicle treated rrm2b^+/+ (0.5% DMSO vehicle)(n = 22 (circles with thin border)), vehicle treated rrm2b^−/− (0.5% DMSO vehicle)(n = 17 (squares with thin border)) and deoxynucleoside treated rrm2b^−/− (50 μM dAdo & dGuo + 5 μM ENHA in 0.5%DMSO)(n = 21 (squares with thick border)) larvae during a light dark transition test (LDT). (C) Total distance moved in light periods of vehicle treated 5 dpf rrm2b^+/+ larvae (n = 22 (circles with thin border)), vehicle treated rrm2b^−/− (0.5% DMSO vehicle)(n = 17 (squares with thin border)) and deoxynucleoside treated rrm2b^−/− (50 μM dAdo & dGuo + 5 μM ENHA in 0.5%DMSO)(n = 21 (squares with thick border)) larvae during an LDT test. (I) Total distance moved in dark periods of vehicle treated 5 dpf rrm2b^+/+ larvae (n = 22 (circles with thin border), vehicle treated rrm2b^−/− (0.5% DMSO vehicle)(n = 17 (squares with thin border) and deoxynucleoside treated rrm2b^−/− (50 μM dAdo & dGuo + 5 μM ENHA in 0.5%DMSO)(n = 21 (squares with thick border)) larvae during an LDT test. Results are shown as mean ± SEM (statistical significance was determined with a one-way ANOVA, ^**P < 0.01).

Assessment of purine deoxynucleoside supplementation in 7 dpf rrm2b larvae—. (A) Relative mtDNA copy number in vehicle treated (0.5% DMSO) 7dpf rrm2b^+/+ (n = 9)(circles with thin border) & rrm2b^−/− (n = 8)(squares with thin border) larvae and purine deoxynucleoside treated (50 μM dAdo & dGuo +5 μM ENHA in 0.5%DMSO) 7 dpf rrm2b^+/+ (n = 7)(circles with thick border) & rrm2b^−/− (n = 12)(squares with thick border) larvae. All results shown as mean ± SEM (statistical significance was determined with a Kruskal-Wallis test, ^**adj. P < 0.01). (B) Swim bladder area of 7 dpf vehicle treated (0.5% DMSO) rrm2b^+/+ (n = 19) (circles with thin border), rrm2b^+/− (n = 40) (diamonds), rrm2b^−/− (n = 18) (squares with thin border) and purine deoxynucleoside treated (50 μM dAdo & dGuo +5 μM ENHA in 0.5%DMSO) rrm2b^−/− larvae (n = 20) (squares with thick border). All results are shown as mean ± SEM (statistical significance was determined with a Kruskal-Wallis test, ^* = adj. P < 0.05, ^** = adj. P < 0.01, ^*** = adj. p < 0.001). (C) L-lactate in 7 dpf vehicle treated (0.5% DMSO) rrm2b^+/+ & rrm2b^+/− larvae (circles with thin border) and rrm2b^−/− larvae (squares with thin border) and L-lactate in 7 dpf purine deoxynucleoside treated (50 μM dAdo & dGuo +5 μM ENHA in 0.5%DMSO) rrm2b^+/+ & rrm2b^+/− larvae (circles with thick border) and rrm2b^−/− larvae (squares with thick border). For all conditions n = 3 pools of 20, 7 dpf larvae. All results are shown as mean ± SEM (statistical significance was determined with a one-way ANOVA, ^* = adj. P < 0.05,^** = adj. P < 0.01). (D) Deoxyadenosine concentration (nmol/g protein) in pools (n = 80) of 7 dpf vehicle treated (0.5% DMSO) or purine deoxynucleoside treated (50 μM dAdo & dGuo +5 μM ENHA in 0.5%DMSO) rrm2b^+/+ & rrm2b^+/− and rrm2b^−/− larvae. E. Deoxyguanosine concentration (nmol/g protein) in pools (n = 80) of 7 dpf vehicle treated (0.5% DMSO) or purine deoxynucleoside treated (50 μM dAdo & dGuo +5 μM ENHA in 0.5%DMSO) rrm2b^+/+ & rrm2b^+/− and rrm2b^−/− larvae. Deoxycytidine monophosphate, dTMP: Deoxythymidine monophosphate, TK2: Thymidine kinase 2.