Selective propagation of functional mitochondrial DNA during oogenesis restricts the transmission of a deleterious mitochondrial variant

Abstract

Although mitochondrial DNA (mtDNA) is prone to mutation and few mtDNA repair mechanisms exist, crippling mitochondrial mutations are exceedingly rare. Recent studies have demonstrated strong purifying selection in the mouse female germline. However, the mechanisms underlying positive selection of healthy mitochondria remain to be elucidated. We visualized mtDNA replication during Drosophila melanogaster oogenesis, finding that mtDNA replication commenced before oocyte determination during the late germarium stage and was dependent on mitochondrial fitness. We isolated a temperature-sensitive lethal mtDNA allele, mt:CoI(T300I), which resulted in reduced mtDNA replication in the germarium at the restrictive temperature. Additionally, the frequency of the mt:CoI(T300I) allele in heteroplasmic flies was decreased, both during oogenesis and over multiple generations, at the restrictive temperature. Furthermore, we determined that selection against mt:CoI(T300I) overlaps with the timing of selective replication of mtDNA in the germarium. These findings establish a previously uncharacterized developmental mechanism for the selective amplification of wild-type mtDNA, which may be evolutionarily conserved to limit the transmission of deleterious mutations.

Figure 1

mtDNA replication occurs around the fusome in the germarium. (a) Diagram of a Drosophila ovariole, illustrating anterior to posterior, successive stages of development and a magnified view of the germarium. The durations of germarium and post-germarium stages at 25°C are labeled. Mitochondrial morphology and subcellular distribution changes in the germarium are illustrated and described in detail in the main text. Fusome (red), mitochondria (blue) and future oocyte (broken line) are shown. (b) Representative Z-stack projection of an wt ovariole (>50 repetitions), showing 5-ethynyl-2′-deoxyuridine (EdU) (green) incorporation into mtDNA (arrowheads), and nuclei (arrows) in the presence of aphidicolin, and α-Hts RC (red) staining at 25°C. Scale bar, 10 μm. (c) Magnified view of boxed region in (b) showing germarium with developmental regions indicated. Scale bar, 5 μm. (d) Quantification of mtDNA replication indicated by the number (mean±s.d., n=5) of EdU puncta in different regions of the germarium. 2B ant., region 2B anterior; 2B pos., region 2B posterior. P< 0.05 for each data point compared to region 2A.

Figure 2

The fusome and mitochondria activity are essential for mtDNA replication in region 2B cysts. (a) A representative image from > 10 repetitions showing EdU incorporation into nuclear DNA, labeled by DAPI, but not mtDNA, in hts (hts⁰¹¹⁰³/Df (2R)BSC26) mutant germaria. Boxes, region 2B. (b) Loss of Hts protein does not perturb mtDNA replication in post-germarium egg chambers. (c-f) Z-stack projections of wt germaria subjected to DMSO (c), or the mitochondria uncoupler FCCP at concentrations of 2 μM (d), 5 μM (e) and 10 μM (f) during incorporation of EdU (green). Note the lack of EdU puncta in region 2B with 2 μM FCCP and in both region 2B and 3 with 5 μM FCCP (boxes). Arrow, nucleus; Arrowheads, mtDNA; Scale bars, 10 μm.

Figure 3

mtDNA replication is reduced in 16-cell cysts of homoplasmic mt:CoI^T300I germaria at 29°C. (a) Mutated nucleotide and amino acid residue (red) in mt:CoI^T300I, including XhoI site (yellow). (b) Eclosion rate (mean±s.d., from total >50 animals in 5 groups) of wt, homoplasmic and heteroplasmic mt:CoI^T300I animals at 18°C and 29°C. * indicates zero mt:CoI^T300I eclosed at 29°C. (c) COX activities of wt and mt:CoI^T300I flies were examined on consecutive days shifting to 29°C. Data represent 3 biological replicates. Values were normalized with average COX activities of wt at 29°C 0 day and shown as mean±s.d. (d) Quantification of EdU puncta (mean±s.d.) in posterior cyst of region 2B at 18°C (wt n= 7, mt:CoI^T300I n= 7) or 29°C (wt n= 5, mt:CoI^T300I n= 8). The number of EdU puncta in mt:CoI^T300I at 29°C is significantly less than that at 18°C or wild type at 29°C (P< 0.0005). (e) Z-stack projections showing EdU incorporation in region 2B of germaria from wt and mt:CoI^T300I at 18°C and 29°C. Arrowheads, mtDNA. Scale bar, 5 μm.

Figure 4

Germline selection against mt:CoI^T300I at the restrictive temperature. (a) Frequency of mt:CoI^T300I mutation in heteroplasmic flies maintained at 29°C or 18°C over generations. (b) XhoI digestion of PCR fragment spanning mt:CoI, amplified from single larvae produced by the same heteroplasmic mother at 18°C or 29°C. (c) Proportion of mutant mtDNA in 10 single larvae at 18°C or 29°C, calculated by quantifying band intensity in b. Average level of mutant mtDNA, 18°C, 83±5%; 29°C, 60±9%, n=10, P< 0.0005. (d) Proportion of mutant mtDNA in 10 single larvae from the same mother at 18°C (45±9%), produced on the first 2 days (40±9%), day 3 (37±6%) and day 4 (37±8%) after being shifted to 29°C. n=10, *P< 0.005.