FIGURE 2

FIGURE 2

Generation and functional validation of naxd^−/− and naxe^−/− knockout lines. (A, B) Schematic representation of the zebrafish naxd and naxe genes, with boxes representing exons. (C, D) Deletion mutations at the CRISPR target sites of naxd and naxe, identified in the mutant lines by sequencing at both genomic DNA and cDNA levels. Agarose gels show PCR amplicons obtained from whole larvae genomic DNA for genotyping purposes. For naxe^−/− identification, amplicons were treated with RsaI enzyme (whose restriction site is lost in the mutant line), prior to gel electrophoresis. (E, F) Schematic representation of WT and predicted truncated Naxd and Naxe proteins. (G, H) Naxd and naxe transcript levels in homozygous mutant larvae relative to WT siblings. qPCR was performed with RNA extracted from 5 dpf whole larvae, and actb1 was used as a reference gene. Data are means ± SDs from four biological replicates, each replicate consisting of a batch of 30 larvae. Percentages of WT, heterozygous, and homozygous mutant fish that reached adulthood are visualized in a vertical slice chart. (I) S‐NADHX (red) and R‐NADHX (orange) concentrations in extracts of 10 dpf larvae. Data are means ± SDs from three to six biological replicates, each replicate consisting of a batch of 10 larvae. (J) Schematic representation of the NAD(P)HX repair system. NAD(P)H can be hydrated, either spontaneously or by enzymatic side reactions, to form two epimers, S‐NAD(P)HX or R‐NAD(P)HX. The latter can further react to irreversibly form cyclic NAD(P)HX. NAXE catalyzes the interconversion between the S and R forms of NAD(P)HX. NAXD stereospecifically dehydrates S‐NAD(P)HX back to NAD(P)H. (K, L) NAD(H) and NADP(H) concentrations in extracts of 10 dpf larvae (raw data are provided in Table S3, experiment FERZF). Data shown are means ± SDs from three biological replicates, each replicate consisting of a batch of 10 larvae. Statistical significance was determined with unpaired t‐tests (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001).