Mutations of the mitochondrial DNA (mtDNA) are often the cause behind primary mitochondrial disorders affecting 1:5000 individuals. However, the full extent of the impact that mtDNA mutations have is yet to be comprehensively understood. One of the main reasons behind our slow progress in the field is the multi-copied nature of mtDNA, which suggests that even healthy individuals will carry a small percentage of mutated mtDNA molecules alongside healthy ones, in a state termed heteroplasmy. In cases where the proportion of mutant to healthy mtDNA molecules reaches a critical threshold, diverse and multisystem pathological phenotypes begin to appear. While an individual’s mtDNA heteroplasmy level is largely dependent on that of his maternal germline, studies have shown that there are diverse forces, both intra and extracellular in nature that drive segregation. Further complicating this phenomenon, the observed driving forces appear to be mutation- and cell type-specific in their effect.

In this dissertation I first describe my work on optimising and validating a protocol that allows us to measure single cell heteroplasmy. Developing this in-house technique, enabled us to perform high-throughput analyses of cell populations of interest while revealing for the first time the intricacies governing single mtDNA heteroplasmy variability at the single cell level. With this protocol in place, I set out to study the heteroplasmy of mouse brain- and spleen-derived populations. In this endeavour, I made use of two novel mouse models that carry a mutation on mitochondrial-tRNA Alanine (mt-Ta), m.5019A>G and m.5024C>T. Recording single cell heteroplasmy values at different timepoints throughout development, we observed that both mutations followed the principles of random genetic drift. The rate of drift exhibited mutation-specific patterns.

Moreover, I present a collaborative project geared towards uncovering the impact the two mt-Ta mutations have at the level of the transcriptome on difference cell lineages belonging to E8.5 mouse embryos. I describe the identification of 17 distinct cell lineages and their inherent variability in mtDNA transcript abundance. While no developmental disparities were observed in mutant embryos compared to controls, we did detect an upregulation of mtDNA transcripts in response to the mutation. At the same time, genes that were previously defined as epistatic suppressors/buffers were found to be downregulated. Pseudobulk analysis revealed differential expression of genes both at the level of the organism and that of the cell-lineage. Overall, mice carrying the m.5024C>T mutation seem to mount a greater compensatory transcriptional response compared to their m.5019A>G counterparts.

Finally, I explore the relationship between mtDNA heteroplasmy, copy number and the cell cycle. More specifically, making use of a fluorescent cell cycle reporter, I examine mtDNA changes along the cell cycle. Having established a consistent pattern, I assess the impact of genetic manipulation of mtDNA copy number and restriction of glycolysis on cell cycle progression. Finally, I delve into the consequences of large scale mtDNA deletions on the cell’s respiratory capacity and examine whether that defect impacts their ability to complete the cell cycle.