Letter | Published:

Mitochondrial fragmentation drives selective removal of deleterious mtDNA in the germline

Nature (2019) | Download Citation

Abstract

Mitochondria contain their own genomes that, unlike nuclear genomes, are inherited only in the maternal line. Owing to a high mutation rate and low levels of recombination of mitrochondrial DNA (mtDNA), special selection mechanisms exist in the female germline to prevent the accumulation of deleterious mutations1,2,3,4,5. However, the molecular mechanisms that underpin selection are poorly understood6. Here we visualize germline selection in Drosophila using an allele-specific fluorescent in situ-hybridization approach to distinguish wild-type from mutant mtDNA. Selection first manifests in the early stages of Drosophila oogenesis, triggered by reduction of the pro-fusion protein Mitofusin. This leads to the physical separation of mitochondrial genomes into different mitochondrial fragments, which prevents the mixing of genomes and their products and thereby reduces complementation. Once fragmented, mitochondria that contain mutant genomes are less able to produce ATP, which marks them for selection through a process that requires the mitophagy proteins Atg1 and BNIP3. A reduction in Atg1 or BNIP3 decreases the amount of wild-type mtDNA, which suggests a link between mitochondrial turnover and mtDNA replication. Fragmentation is not only necessary for selection in germline tissues, but is also sufficient to induce selection in somatic tissues in which selection is normally absent. We postulate that there is a generalizable mechanism for selection against deleterious mtDNA mutations, which may enable the development of strategies for the treatment of mtDNA disorders.

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Source Data for all graphs are provided with the paper. The Cp values associated with each primer pair and DNA, and confocal image data are available upon request.

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Acknowledgements

We thank J. Chung, M. Guo, P. O’Farrell, H. Jacobs, H. Ma, the Drosophila Species Stock Center, the Bloomington Drosophila Stock Center and the Vienna Drosophila Stock Center for fly stocks; members of the Lehmann laboratory and K. Lau for discussions; Y. Abdu, L. Barton, A. Blum, S. Burden, S. Kidd, M. Murphy, A. McQuibban and D. Siekhaus for comments on the manuscript; and A. Sfeir for experimental suggestions to address the reviewers’ comments. This work was supported by Canadian Institutes of Health Research grant FRN 159510 to T.R.H. and by National Institutes of Health grant R37HD41900 to R.L. T.R.H. is part of the University of Toronto Medicine by Design initiative, which receives funding from the Canada First Research Excellence Fund. R.L. is a Howard Hughes Medical Institute investigator.

Reviewer information

Nature thanks Rachel Cox, Yukiko Yamashita and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Toby Lieber, Thomas R. Hurd

Affiliations

  1. HHMI and Kimmel Center for Biology and Medicine of the Skirball Institute, Department of Cell Biology, New York University School of Medicine, New York, NY, USA

    • Toby Lieber
    •  & Ruth Lehmann
  2. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    • Swathi P. Jeedigunta
    • , Jonathan M. Palozzi
    •  & Thomas R. Hurd

Authors

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Contributions

S.P.J. and J.M.P. contributed equally to this work. T.R.H., T.L. and R.L. designed the experiments; T.R.H., T.L., S.P.J. and J.M.P. performed the experiments; and T.R.H., T.L. and R.L. wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Ruth Lehmann or Thomas R. Hurd.

Extended data figures and tables

  1. Extended Data Fig. 1 FISH probes are specific for either D. yakuba or D. melanogaster mitochondrial DNA.

    a, Schematics of the mitochondrial genome and the D-loops of D. yakuba and D. melanogaster. In the schematic of the D-loop of D. melanogaster, the boxed regions denote two classes of repeated sequences. The open boxes are unique to D. melanogaster. The hatched boxes contain a 300-bp sequence, that is conserved in other Drosophilids46 and is depicted by solid bars above the repeats in D. melanogaster and by a single solid bar above the D. yakuba D-loop. The FISH probes are directed against unique regions of the D-loops; the D.-yakuba-specific probe is depicted as a green bar and the D.-melanogaster-specific probe is depicted as a magenta bar beneath the respective D-loops. be, Confocal images of D. yakuba (b, e) and D. melanogaster (c, d) stage 7 egg chambers hybridized with D.-yakuba-specific probes (green; b, c) and D.-melanogaster-specific probes (magenta; d, e). All egg chambers were also hybridized with probes recognizing mtDNA of both species (common; middle panels, blue). The merged images are in the right panels. The D. yakuba probe hybridizes to D. yakuba mtDNA (b) but not D. melanogaster mtDNA (c). The D. melanogaster probe hybridizes to D. melanogaster mtDNA (d) but not D. yakuba mtDNA (e). f, Schematic illustrating the generation of heteroplasmic flies by the transfer of germ plasm that contains wild-type mitochondria (green) from D. yakuba (D. yak) into D. melanogaster (D. mel) embryos that are homoplasmic for mt:CoIts + mt:ND2del1 mutant mitochondria (magenta). g, Bar plots showing the percentage of mutant and wild-type mtDNA, as assayed by qPCR, in adult female carcasses without ovaries from the original mutant D. melanogaster strain and the heteroplasmic line generated by pole plasm transplantation. The data are an average of four biological replicates. hh′, h″, ii′, i″, Ovarioles of flies heteroplasmic (Het) for D. melanogaster mt:Co1ts (mut) and D. yakuba (wt) genomes that were shifted to 18 °C (permissive temperature) for 10 days or maintained at 29 °C (restrictive temperature), hybridized with fluorescent probes that detect either wild-type D. yakuba (green) or mutant D. melanogaster (magenta) genomes. Selection against the mutant genome is observed in the germline when flies were raised at 29 °C. For mtDNA FISH, at least eight control and experimental ovarioles, germaria or testes were imaged for each experimental condition. Imaging parameters are presented in Supplementary Table 3. Here and in all subsequent Extended Data Figs. the greyscale images are non-background-subtracted and unnormalized. Source data

  2. Extended Data Fig. 2 Selection against mutant mitochondrial DNA does not occur in the male germline.

    ad, Testes of heteroplasmic (Het) flies that were shifted to 18 °C for 7 days (a, c) or maintained at 29 °C (b, d) hybridized with fluorescent probes that detect either wild-type D. yakuba (green) or mutant D. melanogaster (magenta) genomes. The higher magnification images in c and d include the stem cells and spermatogonial cysts. Selection against mutant mtDNA is not observed in testes of flies raised at the restrictive temperature (29 °C). e, Scatter plots showing the percentage of mutant mtDNA, as assayed by qPCR, of adult ovaries (n = 5) and testes of heteroplasmic flies raised at 29 °C (n = 5), and of adult testes of heteroplasmic flies shifted to 18 °C for 7 days (n = 5). The mtDNA qPCR data throughout are presented as medians with interquartile range and compared by two-tailed unpaired t-tests. In Supplementary Table 2, we also present 95% confidence intervals of the difference between the control and experimental means for all datasets and the number of biologically independent samples used to derive the statistics. The dashed line denotes the percentage mutant mtDNA in whole adult-female carcasses lacking ovaries. All testes are oriented with the stem-cell niche towards the left. Source data

  3. Extended Data Fig. 3 Selection manifests in germline cyst cells and does not occur when cyst formation is blocked.

    a, b, Germaria of heteroplasmic females (Het), raised at 29 °C, were hybridized with fluorescent probes that detect either wild-type D. yakuba or mutant D. melanogaster mtDNA, and reacted with anti-Vasa antisera to mark the germline (aa‴) or anti-Hts (1B1) antisera to mark the fusome and somatic cells (bb‴). The dashed outlines delineate the germline in the germarium (a), and egg chambers surrounded by somatic follicle cells (a, b). Wild-type mtDNA (arrows) can first be strongly detected in cysts. cc‴, A germarium of a heteroplasmic fly (Het), raised at 29 °C, in which cyst formation was blocked by expression of an RNAi against bag-of-marbles (Bam; UAS-bam shRNA TRiP.HMJ22155) in the germline under the control of nos-GAL4. The germarium was hybridized with fluorescent probes directed against wild-type (c, c‴) and mutant mtDNAs (c′, c‴) and reacted with anti-Vasa antisera to mark the germline (c″). No increase in wild-type mtDNA is observed. dd‴, A germarium of a heteroplasmic fly, raised at 29 °C, hybridized with fluorescent probes that detect either wild-type D. yakuba mtDNA (d, d‴) or mutant D. melanogaster mtDNA (d′, d‴), and reacted with anti-Orb antisera (d″, blue in d‴) to demarcate all cells of the developing cysts and the oocyte in later egg chambers. Arrows in d and d‴ point to wild-type mtDNA, and dashed outlines delineate the germline in the egg chambers. e, Scatter plots showing the relative amounts of wild-type D. yakuba and mutant D. melanogaster mtDNA, as assayed by FISH, in cysts (n = 7) and egg chambers (EC, n = 6) compared to the amount in stem cells (SC, n = 7). f, Scatter plot showing percentage of mutant mtDNA, as assayed by qPCR, of control (Ctrl; nos-GAL4 driving UAS-mCherry RNAi) heteroplasmic ovaries (n = 5) and of heteroplasmic ovaries in which cyst formation was blocked by the knockdown of bam (Bam KD; n = 5). Here and in all subsequent images, ovarioles are oriented with the stem-cell niche towards the left. Source data

  4. Extended Data Fig. 4 Expression of the C. intestinalis alternative oxidase (AOX) rescues mutant mitochondria.

    a, In wild-type mitochondria the electron transport chain complexes (I–IV) that reside in the inner mitochondrial membrane couple the transfer of electrons to the transfer of protons across the membrane. The resulting proton motive force drives the synthesis of ATP by complex V. b, At the restrictive temperature the CoIts mutation blocks the transfer of electrons through complex IV (cytochrome oxidase, purple) resulting in the absence of both the generation of a proton motive force and ATP production. c, AOX (yellow) catalyses the transfer of electrons from ubiquinone to molecular oxygen, bypassing complexes III and IV. This restores the transfer of protons at complex I and the generation of ATP. d, Scatter plot of the amount of mutant D. melanogaster (purple) and wild-type D. yakuba (green) mtDNA, as assayed by qPCR, in ovaries expressing AOX under the control of nos-GAL4, normalized to the amount of mutant and wild-type mtDNA in control ovaries (Ctrl) expressing mCherry RNAi (Ctrl, n = 20; AOX, n = 23). Expression of AOX rescues the mutant genomes. Source data

  5. Extended Data Fig. 5 Mitochondrial fragmentation is necessary for germline mitochondrial DNA selection.

    ac, Stills of live images illustrating the effect that overexpressing Mitofusin (b) or reducing the expression of Drp1 (c) in the germline has on the morphology of mitochondria compared to controls (a, nos-GAL4 driving UAS-mCherry RNAi; the stills in Fig. 2c, d are higher magnifications of this image). When Mitofusin is overexpressed (nos-GAL4 driving UAS-marf47), or when the expression of Drp1 is reduced (nos-GAL4 driving UAS-Drp1.miRNA.CDS48), the mitochondria in the cysts are no longer discrete as they are in control cysts. The mitochondria (white) were labelled with a mitochondrially targeted eYFP and cell membranes (blue) were labelled with CellMask Deep Red Plasma membrane Stain. Stem cells and cysts are outlined in red. dd′, d″, Germarium of a control heteroplasmic female (nos-GAL4 driving UAS-mCherry RNAi), raised at 29 °C, hybridized with fluorescent probes that detect either wild-type D. yakuba mtDNA (greyscale in d; green in d″) or mutant D. melanogaster mtDNA (greyscale in d′; magenta in d″). Selection for wild-type mtDNA is observed as indicated by the arrows in d and d″. ee′, e″, ff′, f″, Selection for wild-type mtDNA is no longer observed when Mitofusin (Mfn) is overexpressed (nos-GAL4 driving UAS-marf) or when the expression of Drp1 is reduced (nos-GAL4 driving UAS-Drp1.miRNA.CDS). Wild-type D. yakuba mtDNA, greyscale in e, f, green in e″, f″; mutant D. melanogaster mtDNA, greyscale in e′, f′, magenta in e″, f″. The dashed outlines delineate the germline. g, Scatter plot showing the percentage of mutant D. melanogaster mtDNA, as assayed by qPCR, in carcasses (carc.) and ovaries of heteroplasmic flies in which the wild-type mtDNA was either from D. yakuba or D. melanogaster. mCherry RNAi was expressed in the ovaries under control of nos-GAL4. h, Scatter plot of the amount of mutant D. melanogaster (purple) and wild-type D. yakuba (green) mtDNA, as assayed by qPCR, of young embryos laid by heteroplasmic females in which Mitofusin was overexpressed in the germline (Mfn OE, n = 4) normalized to the amount of mutant and wild-type mtDNA in young embryos laid by control heteroplasmic females (Ctrl; nos-GAL4 driving UAS-mCherry RNAi, n = 4). i, Same as g, except the analysis was performed on ovaries in which both wild-type and mutant mtDNAs were from D. melanogaster (Ctrl, n = 24; Mfn OE, n = 21). Mitofusin overexpression increases the levels of mutant mtDNA. Source data

  6. Extended Data Fig. 6 Mitochondrial fragmentation is sufficient for germline mitochondrial DNA selection.

    a, b, Stills of live images illustrating the effect that reducing the expression of Mitofusin in the germline (b) has on the morphology of mitochondria compared to controls (a). When mitofusin is knocked down (nos-GAL4 driving UAS-mfn shRNA2 TRiP.HMC0388332) the mitochondria in the stem cells are fragmented. The mitochondria (white) were labelled with a mitochondrially targeted eYFP and cell membranes (blue) were labelled with CellMask Deep Red Plasma membrane Stain. Stem cells and cysts are outlined in red. c, c′, c″, c‴, The knockdown of mitofusin in the germline by expressing mitofusin RNAi (nos-GAL4 driving UAS-mfn shRNA2 TRiP.HMC03883) results in selection for wild-type mtDNA (green) occurring in stem cells. The germarium was also reacted with anti-Vasa antiserum (c″) to mark the germline and delineate the stem cells and cysts. Wild-type D. yakuba mtDNA, greyscale in c, green in c‴; mutant D. melanogaster mtDNA, greyscale in c′, magenta in c‴. Mutant mtDNA is readily detected in the soma but not in the germline. d, Scatter plot comparing the percentage of mutant mtDNA, as assayed by qPCR, of ovaries in which mitofusin was weakly knocked down in the germline (Mfn KD; nos-GAL4 driving UAS-mfn long hairpin RNA1 TRiP.JF0165049; Ctrl, n = 23; Mfn KD, n = 24) and of ovaries in which Drp1 was overexpressed in the germline (Drp1 OE; nos-GAL4 driving UAS-Drp1.E; Ctrl, n = 11; Drp1 OE, n = 11). The percentage of mutant mtDNA in each case was normalized to the percentage of mutant mtDNA in control ovaries to illustrate that the overexpression of Drp1 enhances selection to a similar extent as does a weak reduction in the expression of Mitofusin. e, Scatter plot of the amount of mutant D. melanogaster (purple) and wild-type D. yakuba (green) mtDNA, as assayed by qPCR, in ovaries in which the expression of Mitofusin was weakly reduced (Ctrl, n = 20; Mfn OE, n = 21) or in which Drp1 was overexpressed in the germline (Ctrl, n = 10; Drp1 OE, n = 10), normalized to the amount of mutant and wild-type mtDNA in control ovaries (Ctrl) expressing mCherry RNAi in the germline. Reducing the expression of Mitofusin or overexpressing Drp1 results in a decrease in mutant mtDNA. fh, The effect of germline overexpression of Mitofusin (Mfn) and Drp1 on copy number (f), ATP levels (g), and mitochondrial motility (h) in homoplasmic wild-type D. melanogaster ovaries (see Supplementary Note 1). f, Scatter plot of the amount of mtDNA, as assayed by qPCR, in homoplasmic ovaries in which Mfn (n = 24) or Drp1 (n = 24) was overexpressed in the germline, normalized to the amount of mtDNA in control ovaries (Ctrl; nos-GAL4 driving UAS-mCherry RNAi, n = 23). g, Scatter plot of the amount of ATP in homoplasmic ovaries overexpressing Mfn (n = 5) or Drp1 (n = 5) in the germline under control of Maternal α-Tubulin Gal4, normalized to the amount of ATP in control ovaries (Ctrl; Maternal α-Tubulin Gal4 driving UAS-mCherry RNAi, n = 5). h, Scatter plot of mitochondrial motility in homoplasmic ovaries overexpressing Mfn (n = 5) or Drp1 (n = 5) in the germline. Motility was assessed by measuring mean mitochondrial displacement using live confocal microscopy and Imaris analysis software. Source data

  7. Extended Data Fig. 7 Mitofusin is downregulated in germline cysts.

    aa′, a″, a‴, A germarium of a female fly expressing haemagglutinin-tagged Mitofusin (Mfn), under control of the Mitofusin promoter (Marf-gHA48), and mitochondrially targeted eYFP (mito-eYFP50), was reacted with anti-haemagglutinin antisera to detect Mitofusin (a), anti-GFP antisera to detect mitochondria (a′) and anti-Vasa antisera to delineate the germline (a″). In a‴, the ratio of the levels of Mitofusin to mito-eYFP is presented in pseudocolour. The colours correspond to the ratios indicated on the pseudocolour bar. The dashed red circles outline the cysts and the dashed white circles demarcate the germline in the egg chambers. b, Scheme for quantifying the levels of mitofusin RNA at different time points during early oogenesis. Females mutant for the differentiation factor Bam—which is required for cyst formation—and carrying a rescuing transgene expressing Bam under the control of a heat-shock promoter were heat-shocked at 37 °C for 2 h, and then allowed to recover for the indicated times. This enables the isolation of ovaries that contain staged cysts, predominantly at the 2-, 4- or 8-cell cyst stage. The morphology of the spectrosome and fusome, as revealed by staining with anti-Hts (1B1) antisera, was used to confirm the staging. RNA for RT-qPCR was isolated from ovaries from flies before heat shock and at the indicated times following heat shock.

  8. Extended Data Fig. 8 The downregulation of Mitofusin in cysts is not mediated by known regulators of Mitofusin protein.

    ah″, Germaria of females expressing haemagglutinin-tagged Mitofusin, under control of the Mitofusin promoter (Marf-gHA48) reacted with anti-haemagglutinin antisera to detect Mitofusin (greyscale in ah, magenta in a″–h″) and anti-Vasa antibody to delineate the germline (greyscale in a′–h′, blue in a″h″). The indicated known regulators of Mitofusin protein levels were knocked down in the germline using RNAi under the control of nos-GAL4. The numbers in parentheses are Bloomington Drosophila Stock Center stock numbers. All ovarioles are oriented with the stem-cell niche towards the left.

  9. Extended Data Fig. 9 Inhibiting mitochondrial fragmentation blocks the decrease in proton motive force and ATP levels in cysts of heteroplasmic flies.

    ad, Germaria of heteroplasmic control flies (a, c; w1118) and heteroplasmic flies in which Mitofusin was overexpressed in the germline (b, d; nos-GAL4 driving UAS-marf47), reacted with TMRM to visualize mitochondrial membrane potential (pseudocoloured in a, b) or with antibodies to phosphorylated pyruvate dehydrogenase (PDH P, purple) and pyruvate dehydrogenase (PDH, green) to measure ATP levels (c, d). e, Diagram showing the essential glutamate at position 121 in c-ring subunit that acts as the proton donor and acceptor in the proton translocation pathway. In the dominant negative c-ring (CV-DN) this glutamate was mutated to a glutamine, which can no longer bind the protons. f, Scatter plot illustrating the reduction in ATP/ADP ratio in embryos laid by mothers expressing CV-DN in the germline under the control of Maternal α-Tubulin Gal4. The ratios were measured using an ADP/ATP Ratio Assay Kit (Abcam ab65313). Data presented are median and interquartile range and were analysed using paired t-tests (ATP, n = 5; ADP, n = 4; ATP/ADP, n = 4). g, Blue native polyacrylamide gel illustrating that the expression of the dominant negative inhibitor of complex V (CV-DN) does not disrupt the Complex V dimer. For gel source data, see Supplementary Fig. 1. Source data

  10. Extended Data Fig. 10 The mitophagy proteins Atg1 and BNIP3 are necessary for germline mitochondrial DNA selection.

    ad″, Germaria of a control heteroplasmic female (a, a″; nos-GAL4 driving UAS-mCherry RNAi) and of heteroplasmic females in which the expression of Atg1 (b, b″), Atg8a (c, c″) or BNIP3 (d, d″) was reduced in the germline, raised at 29 °C, hybridized with fluorescent probes that detect either wild-type D. yakuba mtDNA (greyscale in ad, green in a″–d″) or mutant D. melanogaster mtDNA (greyscale in a′d′, magenta in a″d″). The dashed circles demarcate the germline in the early egg chambers. The arrows point to wild-type mtDNA. e, f, Scatter plots showing the percentage of mutant mtDNA and the amount of mutant (magenta) and wild-type (green) mtDNA, as assayed by qPCR, of control heteroplasmic ovaries (Ctrl, n = 24 in e, f) and of ovaries in which the expression of Atg1was reduced in the germline (Atg1 KD, n = 24 in e; n = 23 in f). In the left panel in f, the amount of mutant and wild-type mtDNA of heteroplasmic ovaries overexpressing Mitofusin (Mfn OE) is plotted to illustrate that overexpressing Mitofusin primarily inhibits selection by increasing the amount of mutant mtDNA, whereas reduced expression of Atg1 primarily inhibits selection by decreasing the amount of wild-type mtDNA. The control and Mitofusin-overexpression data are the same as that presented in Extended Data Fig. 5h. All the dissections and analyses were carried out at the same time. The right panel of f is a magnified view to illustrate the effect of reducing the expression of Atg1 on the level of wild-type mtDNA. In e and f, both wild-type and mutant mtDNAs were from D. melanogaster. Source data

Supplementary information

  1. Supplementary Information

    This file contains Supplementary Note 1 and Supplementary Note 2: References for Supplementary Note 1 and Supplementary Table 1.

  2. Reporting Summary

  3. Supplementary Figures

    This file contains Supplementary Figure 1: gel source data for Extended Data Figure 9g.

  4. Supplementary Table

    This file contains Supplementary Table 1: Drosophila stocks.

  5. Supplementary Table

    This file contains Supplementary Table 2: Statistical Analysis of qPCR data.

  6. Supplementary Table

    This file contains Supplementary Table 3: Microscopy settings and imaging parameters.

  7. Supplementary Table

    This file contains Supplementary Table 4: qPCR primers.

  8. Video 1: Mitochondria undergo fragmentation as stem cells differentiate into cysts.

    Live imaging of mitochondrial morphology in stem cells and cysts. Mitochondria (white) were labeled with a mitochondrially targeted eYFP and cell membranes (blue) with CellMask™ Deep Red Plasma Membrane Stain. Cysts are outlined in green and stem cells in magenta.

Source data

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DOI

https://doi.org/10.1038/s41586-019-1213-4

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