Knockdown of TFAM in zebrafish embryos. Zebrafish embryos injected with indicated amount of either Control-morpholino (Ctrl-MO) or tfam splice-morpholino tfam splice-MO at 1 hpf and analyzed at 4 dpf. (A)tfam splice-morpholino (MO) at the 3′ splice site of exon 2 causes deletion of exon 2, which predicts a frame-shift and premature stop codon (p.(Cys29Hisfs*36)) (figure is not on scale). (B) The relative copy number assessed by mitochondrial ND1/nuclear B2M ratio. Data are normalized to the NIC embryos (n = 7). Bars indicate mean values with SD, n = 20 per 2 ng injected condition and n = 16 per 4 ng injected condition. P-values are calculated using ANOVA followed by Bonferroni’s Multiple Comparison Test to assess copy number, *P-value < 0.05, **P-value < 0.01. (C) The oxygen consumption rate (OCR) measured by Seahorse XF24 in order to assess basal respiration and maximal ATP production capacity at 4 dpf using 2 ng MO. The statistical analysis was performed by using a student t-test (P < 0.05), n = 10 per tfam splice-MO condition and n = 9 per Ctrl-MO condition.

Microscopy of zebrafish embryos following TFAM knockdown. (A–H) Microscopic analysis following serial HE-staining of zebrafish embryos at 4 dpf, which were injected at 1 hpf with either 2 ng tfam splice-MO (B–E) or 2 ng Ctrl-MO, (A,C,E,G)n = 13 each condition. (B) The cerebellum was missing in the phenotype or a delayed development of this cerebellum in these fish. (D) The eyes were smaller and the eye layers were less developed. (F) A muscle phenotype was clearly visible in the dorsal fin, especially in the myotomic area, which was less compact in these somatic muscles. (H) The fish displayed a marked pericardial oedema (red arrow). They did not develop a normally looped heart as in Ctrl-MO (G), but the hearts were dilated in the phenotypes (H).

Schematic overview of alterations in tfam splice-MO zebrafish at 4dpf. The arrow indicates if a process is upregulated (green) or down-regulated (red). Altered gene expression processes and genes are shown in italic. The mitochondrial DNA figure is adapted from Amorim et al. (2019).