Generation, screening and validation of mcu−/− zebrafish. (A) CRISPR/Cas9-based gene editing was used to generate mcu−/− zebrafish. Schematics showing the targeted genomic sequence for the introduction of the indel mutation in the exon 3 containing the mcu coding gene. The identified 18 base pair deletion (shown in faded font) and 70 bp insertion (shown in highlighted font) is denoted in the cDNA sequence. (B) The indel mutation leads to a frameshift mutation with a premature stop codon. The predicted protein product for mcu mutant allele is shown in the lower panel. (C) RT-PCR data showing wt mcu allele in the first column; the second column shows the wt and mutant allele in heterozygous mutants and the third column shows the mcu mutant allele in mcu homozygous zebrafish. (D) q-PCR data show significant downregulation (***P<0.0001) of mcu gene expression in mcu−/− zebrafish when compared to wt, indicating mutation-induced RNA decay. Statistical analysis with t-test of three different experiments with n=3. (E) Representative images showing in situ hybridization using mcu riboprobe. mcu expression is abolished in mcu−/− 3 dpf zebrafish. (F) Impaired mitochondrial calcium ions influx/efflux in isolated mitochondria from 24 hpf larvae in mcu−/− zebrafish. Control mitochondria (green line) had an uptake in calcium added to solution, which created a drop in the fluorescence. After ∼5 min, we observed calcium leakage (rise in fluorescence). Blank control (black line) was mitochondria without added calcium. Ruthenium Red (pink line) prevented calcium uptake. CCCP (yellow line) caused leakage of calcium, observed as rising fluorescence. Uptake in mcu−/− (blue line) was completely blocked in given circumstances, being like the dynamics of Ruthenium Red treatment.
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