Abstract
High mitochondrial DNA (mtDNA) copy numbers are essential for oogenesis and embryogenesis and correlate with fertility of oocytes and viability of embryos. To understand the pathology and mechanisms associated with low mtDNA copy numbers, we knocked down mitochondrial transcription factor A (tfam), a regulator of mtDNA replication, during early zebrafish development. Reduction of tfam using a splice-modifying morpholino (MO) resulted in a 42 ± 17% decrease in mtDNA copy number in embryos at 4 days post fertilization. Morphant embryos displayed abnormal development of the eye, brain, heart, and muscle, as well as a 50 ± 22% decrease in ATP production. Transcriptome analysis revealed a decrease in protein-encoding transcripts from the heavy strand of the mtDNA, and down-regulation of genes involved in haem production and the metabolism of metabolites, which appear to trigger increased rRNA and tRNA synthesis in the nucleoli. However, this stress or compensatory response appears to fall short as pathology emerges and expression of genes related to eye development are severely down-regulated. Taken together, this study highlights the importance of sufficient mtDNA copies for early zebrafish development. Zebrafish is an excellent model to manipulate the mtDNA bottleneck and study its effect on embryogenesis rapidly and in large numbers of offspring.
Highlights
Mitochondria are responsible for producing the majority of cellular energy in the form of ATP
To establish decreased mitochondrial DNA (mtDNA) copy number, we microinjected an antisense splice-morpholino antisense oligonucleotides (MOs) targeting tfam-mRNA in zebrafish embryos
Using RT-PCR and Sanger sequencing of cDNA from tfam splice-MO-injected 4 dpf embryos (n = 6/group), we showed that the tfam splice-MO causes a 128 base-pair deletion of exon 2 (c.84_211del), predicted to cause a frameshift and a premature stop codon (p.(Cys29Hisfs∗36)) (Figure 1A and Supplementary Figure S1)
Summary
Mitochondria are responsible for producing the majority of cellular energy in the form of ATP. The oocyte mtDNA copy number should be sufficient for normal development until implantation at day 4, and it has been demonstrated that oocytes with less than 50,000 mtDNA copies fail to resume development after implantation (Ebert et al, 1988; Wai et al, 2010). This negative correlation between mtDNA copy number and developmental competence of embryos has been suggested for human oocytes (Yamamoto et al, 2010)
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