A heteroplasmic deletion reduces the fitness of C. elegans mitofusin (fzo-1) mutant
The stable heteroplasmic C. elegans strain uaDf5/+ harbors a mixture of intact (+mtDNA) and ~ 60% of a 3.1 kb mtDNA deletion (ΔmtDNA) [27]. Although lacking four essential genes (i.e., mt-ND1, mt-ATP6, mt-ND2, and mt-Cytb) and seven tRNAs (i.e., K, L, S, R, I, Q and F), this strain is viable and displays some mitochondrial dysfunction [16, 27, 28]. High heteroplasmy levels are likely not maintained due to mtDNA duplication but by stably maintaining +mtDNA copy number [16]. We showed that dysfunctional PDR-1, the worm orthologue of the key mitophagy factor Parkin (PARK2), led to elevated levels of the truncated mtDNA, suggesting that mitochondrial quality control can modulate the levels of dysfunctional mitochondria [14]. In conjunction with this finding, RNAi knockdown of fzo-1, the C. elegans orthologue of MFN1/2, led to a slight reduction in the levels of the heteroplasmic ΔmtDNA, although without any phenotypic consequences [15]. We, therefore, asked what would be the impact of the fzo-1(tm1133) deletion (hereafter designated as fzo-1(mut)) on the inheritance of the ΔmtDNA.
To this end, we crossed uaDf5/+ heteroplasmic hermaphrodites (+/ΔmtDNA) with fzo-1(mut) heterozygote males (Fig. 1A). After self-cross of the F1 progeny, the distribution of the genotypes in the F2 heteroplasmic progeny did not deviate from the expected Mendelian ratio, namely 26% homozygous fzo-1(mut), 48.7% fzo-1 heterozygotes (ht), and 25.3% fzo-1 wild type (wt) (chi-square test, P = 0.960; Additional file 1: Table S1). However, we noticed that only 13 ± 5% of the progeny of the self-crossed fzo-1(mut);+/ΔmtDNA worms hatched, as compared to fzo-1(mut) animals (67 ± 5%, ANOVA followed by a Tukey’s post hoc test, P < 0.001; Fig. 1B). Although mitochondrial organization and TMRE uptake of self-crossed fzo-1(mut);+/ΔmtDNA adults were similar to fzo-1(mut) (Additional file 1: Fig. S1A-B), fzo-1(mut);+/ΔmtDNA animals were developmentally delayed, and none of them reached adulthood after six days as compared to fzo-1(mut) (ANOVA followed by a Tukey’s post hoc test, P < 0.001). This was in contrast to self-crossed fzo-1(wt);+/ΔmtDNA animals, all of which reached adulthood after six days (Fig. 1C). These findings demonstrate that the interaction between the heteroplasmic ΔmtDNA and the nuclear DNA-encoded fzo-1 mutant led to a severe reduction in fitness.
The adverse effects of the interaction between ΔmtDNA and fzo-1(mut) are reversed across generations
To better characterize the phenotypic impact of the interactions between ΔmtDNA and fzo-1(mut), we monitored the development of progeny of the self-crossed fzo-1(ht);+/ΔmtDNA worms, followed by genotyping the resultant adult animals (generation 1, G1). We continued to follow the hatching and development of their progeny, i.e., fzo-1(mut);+/ΔmtDNA (G2m) and fzo-1(wt);+/ΔmtDNA (G2wt), across four generations (Fig. 2A). Specifically, we measured the duration of the larva-to-adulthood period during development in the G1m-G4m generations (Fig. 2B). While ~ 75% of the G1m animals reached adulthood after 6 days, the development of G2m animals was 1.9-fold delayed (Cox proportional-hazards regression, P < 0.001), with 75% of the animals reaching adulthood only after 9 days. Surprisingly, the G3m animals showed significant improvement (Cox proportional-hazards regression, P < 0.001), with ~ 60% of this population reaching adulthood after six days. Moreover, G4m animals showed a full reversal of ΔmtDNA-associated adverse effects (Cox proportional-hazards regression, P = 0.750; Fig. 2B and Additional file 1: Table S2). We noted a similar pattern across generations when hatching was considered: In contrast to the 13.5% hatching observed among G2m embryos, 60 ± 8% hatching of the G3m embryos was observed (ANOVA followed by a Tukey’s post hoc test, P < 0.001). The hatching percentage of the G4m generation was similar to that of G1 animals (71 ± 9% and 67 ± 5%, respectively, ANOVA followed by a Tukey’s post hoc test P = 0.993) and remained stable over subsequent generations (Fig. 2C). Finally, no phenotypic changes were observed for G1wt-G4wt animals while tracing their developmental pace (Cox proportional-hazards regression, P > 0.140; Additional file 1: Table S2) and hatching percentage (ANOVA followed by a Tukey’s post hoc test, P > 0.266; Additional file 1: Fig. S2A-B). Taken together, our findings demonstrate a full reversal of the adverse effects of the interaction between the ΔmtDNA and the nuclear DNA-encoded mutant fzo-1 gene.
fzo-1(mut) leads to selection against ΔmtDNA heteroplasmy in C. elegans
We next asked how the deleterious interactions between fzo-1(mut) and ΔmtDNA were abrogated. We hypothesized that if the ΔmtDNA is not tolerated in the background of fzo-1(mut), then selection against the ΔmtDNA should occur. To test this prediction, we assessed the levels of ΔmtDNA by quantitative PCR (qPCR, see Methods) across the G1m-G4m generations (using gravid adults) in both the fzo-1(mut);+/ΔmtDNA and the fzo-1(wt);+/ΔmtDNA strains. We found that ΔmtDNA levels declined by 10-fold in the G2m fzo-1(mut) animals, as compared to +/ΔmtDNA parental strain (Fractional regression, odds ratio = 0.08; P < 0.001). Values of ΔmtDNA reached below detection levels in most G3m (N = 18/21) and G4m (N = 20/21) animals (fractional regression, odds ratio < 0.007, P < 0.001; Fig. 2D and Additional file 1: Table S3), while the relative levels of intact +mtDNA molecules increased (Additional file 1: Fig. S2C and Table S3). In contrast, ΔmtDNA and +mtDNA levels did not significantly change across the G1wt-G4wt generations of fzo-1(wt);+/ΔmtDNA animals (fractional regression, P = 0.852; Fig. 2E and Additional file 1: S2D and Table S3).
These results suggest that the ΔmtDNA was completely lost during the G1m-G4m generations. To test this hypothesis, we crossed G4m hermaphrodites with wild type males to isolate fzo-1(wt) progeny (Gm→Gwt). Since traces of ΔmtDNA were neither detected in Gm->Gwt animals (fractional regression, P < 0.001; Fig. 2D) nor in subsequent generations (ANOVA followed by a Tukey’s post hoc test, P < 0.001; Fig. 2F), we concluded that disrupting fzo-1 function indeed resulted in a complete and specific loss of the deleterious heteroplasmic ΔmtDNA. These results provide a proof of concept that mitochondrial fusion is critical for regulating the transmission of the uaDf5 ΔmtDNA heteroplasmy across generations.
Selection against heteroplasmic truncations depends on deletion size or mtDNA position
We next asked whether the deleterious interactions between fzo-1(mut) and ΔmtDNA depend on the size or genomic location of mtDNA deletions. To achieve this goal, we characterized two additional mtDNA deletions obtained from the Million Mutation Project strain collection [29]. Specifically, these deletions comprise two new stable heteroplasmic C. elegans strains: bguDf1 (derived from strain VC40128), harboring a mixture of intact +mtDNA along with mtDNA molecules lacking ~ 1 kb (1kbΔmtDNA) encompassing two essential mtDNA genes (i.e., mt-ATP6 and mt-ND2) and three tRNAs (i.e., K, L, and S); the second strain, bguDf2 (derived from VC20469), harbors in addition to the +mtDNA, a ~4.2 kb mtDNA deletion (4kbΔmtDNA) encompassing four different essential genes (i.e., mt-CO1, mt-CO2, mt-ND3, and mt-ND5) and five tRNAs (i.e., C, M, D, G, and H). Notably, the levels of the 1kbΔmtDNA and 4kbΔmtDNA were stable over > 100 generations (80% and 55%, respectively) in the presence of functional (wild type) fzo-1. Reanalysis of whole-genome sequencing data for the two mutant strains identified +mtDNA and deletion sequences, as previously described [29, 30]. These analyses did not reveal any evidence for duplicated regions, confirming the mtDNA deletion heteroplasmy in these strains (Additional file 1: Fig. S3A-B). As previously observed for uaDf5/+ [16], truncated mtDNA levels highly varied between animals, while intact +mtDNA levels were more constant (Additional file 1: Fig. S3C-D). The animals displayed neither embryonic nor developmental phenotypes, and no impact on mitochondria fusion was observed (Additional file 1: Fig. S3E-G and Table S2).
Heteroplasmic hermaphrodites of both strains were separately crossed with fzo-1(mut) heterozygote males, followed by self-cross of F1 progeny (cross as in Fig. 1A). Like the approach taken with the uaDf5/+ strain, we examined the distribution of the genotypes in the F2 heteroplasmic progeny. We found that the genotypes distribution for the fzo-1(mut);+/1kbΔmtDNA did not deviate from the expected Mendelian ratio (23.5% homozygous fzo-1(mut), 43.2% fzo-1(ht) and 33.3% fzo-1(wt) (P = 0.14, chi-square test; Additional file 1: Table S1). In contrast, this ratio strongly deviated from the expected Mendelian ratio for fzo-1(mut) animals harboring +/4kbΔmtDNA (6.4% homozygous fzo-1(mut), 59.3% fzo-1(ht) and 34.3% fzo-1(wt) (P < 0.001, chi-square test; Additional file 1: Table S1). Hence, these results indicate that fzo-1(mut) differentially tolerates mtDNA deletions based on size and/or mtDNA position.
We next quantified the levels of ΔmtDNA in mutant versus wild type fzo-1 progeny across four subsequent generations (as in Fig. 2A; Fig. 3 and Additional file 1: Table S3). We found that both truncated mtDNA molecules were undetectable after four generations (Fig. 3A, B), yet the decline rates significantly diverged (Fig. 3C). Specifically, mean ΔmtDNA levels were significantly lower in fzo-1(mut) animals harboring the 4kbΔmtDNA than in animals harboring either 1kbΔmtDNA or 3kbΔmtDNA in both G1m and G2m animals (fractional regression followed by within generation pairwise comparisons, P < 0.001; Fig. 3C and Additional file 1: Table S3). By the G3m generation, mean ΔmtDNA levels of 3kbΔmtDNA were also significantly lower than those observed in animals harboring 1kbΔmtDNA. Indeed, the 1kbΔmtDNA was still detected in most of G3m animals (N = 13/18). It is worth noting that the levels of both types of ΔmtDNA did not significantly change across the G1wt-G4wt generations of the fzo-1(wt);+/ΔmtDNA animals (fractional regression, P > 0.118 in both cases; Additional file 1: Fig. S3H-I and Table S3).
To assess whether the truncated mtDNAs were completely lost, we crossed G4m hermaphrodites with wild type males and isolated fzo-1(wt) progeny (Gm→wt). qPCR analyses revealed no traces of the ΔmtDNA copies in subsequent generations (fractional regression, P < 0.001; Fig. 3A-B and Additional file 1: Table S3). Thus, disrupting fzo-1 function resulted in a complete and specific loss of a variety of heteroplasmic ΔmtDNAs. We interpret these results to mean that fzo-1 function is sensitive to either the size or location of deleterious mtDNA heteroplasmy.
Selection against ΔmtDNA molecules occurs during C. elegans development
In C. elegans, mtDNA copy numbers increase significantly during the fourth larval stage (L4) in association with oocyte production [27, 31]. We, therefore, asked at which point during the C. elegans life cycle selection against ΔmtDNA occurred. Given that the relative levels of ΔmtDNA are maintained during normal development [27], we compared ΔmtDNA levels between embryos and adults in G2m animals. Our results indicate that ΔmtDNA levels were dramatically reduced (~ 5-fold) during the development of G2m animals (ANOVA followed by a Tukey’s post hoc test, P < 0.001; Fig. 4A) but not in G2wt animals (Fig. 4B). This observation suggests that ΔmtDNA is most likely selected against during the fzo-1(mut) worm development, in agreement with the observed adverse effect of heteroplasmy on the hatching and development of G2m animals.
To examine the possible association of ΔmtDNA levels with embryo lethality, we compared ΔmtDNA levels of unhatched embryos (unhatched > 48 h after being laid) to newly hatched larvae (L1). As expected, given that ~ 85% of G2 embryos did not hatch, ΔmtDNA levels of unhatched embryos were similar to the relative ΔmtDNA levels of newly laid embryos. In contrast, ΔmtDNA levels in L1 animals were reduced by 2-fold (ANOVA followed by a Tukey’s post hoc test, P < 0.05; Fig. 4A). This suggests that hatching is enabled only in embryos with reduced ΔmtDNA levels.
We next examined whether ΔmtDNA levels associate with developmental delay. To this end, we compared the levels of ΔmtDNA in mildly delayed G2 animals that reached adulthood on days 7–8 to severely delayed animals that reached adulthood on days 9–10. Our results show that ΔmtDNA levels were 2-fold higher in the severely delayed group (Wilcoxon Mann-Whitney rank sum test, P < 0.005 test; Fig. 4C). These data demonstrate increased embryo lethality and aggravation in developmental delay in animals harboring high levels of ΔmtDNA. This supports our interpretation that disruption of mitochondrial fusion in animals carrying ΔmtDNA molecules leads to reduced fitness and suggests that there is selection against such molecules at the level of the organism.
Selection against ΔmtDNA molecules defers between gonad and soma
Previously Lieber et al. demonstrated germline selection acting against high levels of mutant mtDNA in Drosophila oogenesis [22]. In C. elegans, the germline tends to accumulate higher levels of deleterious mitochondrial molecules than somatic tissues, although unfertilized oocytes contain lower levels of ΔmtDNA compared to that of germline tissue [32]. Consistently, we found that ΔmtDNA molecules became undetectable in the resultant embryos of G2m animals (i.e., in G3m animals; Fig. 4A). Hence, it is possible that selection against ΔmtDNA molecules occurred during C. elegans gametogenesis. In support of this claim, a comparison of ΔmtDNA levels between gonads and somatic tissues in G2m animals revealed a two-fold decrease of ΔmtDNA levels in the gonads (Wilcoxon Mann-Whitney rank sum test, P < 0.05; Fig. 4D), whereas the levels of +mtDNA intact molecules were ~1.4-fold higher in the gonad (Fig. 4E). In contrast, both ΔmtDNA and +mtDNA molecules were ~1.5-fold higher in the gonad when comparing gonads and somatic tissues of fzo-1(wt);+/ΔmtDNA animals (P < 0.05 Wilcoxon Mann-Whitney rank sum test; Fig. 4F, G) [32]. Since both the Drosophila experiments [12, 21, 22] and our observations are consistent, we argue that selection against ΔmtDNA molecules during gametogenesis is evolutionarily conserved [12].
PARKIN mutant aggravates the adverse effects of the ΔmtDNA-fzo-1 interactions
Since Parkin mediates the turnover of mitofusins and hence impacts their activity [33, 34], we asked what would be the impact of the pdr-1;fzo1 double mutant on the inheritance of the ΔmtDNA. To address this question, we first crossed +/ΔmtDNA heteroplasmic hermaphrodites with pdr-1(gk448) (here named pdr-1(mut)) males and established a stable pdr-1(mut);+/ΔmtDNA strain. Consistent with previous findings, the ΔmtDNA levels were elevated in this strain (75%) [14,15,16,17]. To establish a strain which is mutant in pdr-1 and fzo-1 in the context of +/ΔmtDNA heteroplasmy, we then crossed pdr-1(mut);+/ΔmtDNA heteroplasmic hermaphrodites with pdr-1;fzo-1 heterozygous males, let the F1 progeny self-cross, and isolated fzo-1(ht);pdr-1(mut) hermaphrodites that are harboring +/ΔmtDNA (Fig. 5A). This strain was allowed to propagate, and the genotypic distribution of the heteroplasmic progeny was assessed. While the genotype distribution of fzo-1(mut);+/ΔmtDNA did not deviate from the expected Mendelian ratio, the genotype distribution of fzo-1(ht);pdr-1(mut);+/ΔmtDNA was strongly affected, as follows: 9% homozygous fzo-1(mut);pdr-1(mut), 55.8% fzo-1(ht);pdr-1(mut), and 35.2% fzo-1(wt);pdr-1(mut) (P < 0.001, chi-square test; Additional file 1: Table S1). This suggests that the heteroplasmic ΔmtDNA cannot be tolerated in the background of fzo-1(mut);pdr-1(mut) double mutant, as reflected in further reduction in fitness.
We next monitored the development and fecundity of these animals. Phenotypic characterization revealed a developmental delay in the fzo-1(mut);pdr-1(mut);+/ΔmtDNA G1m, similar to the parental strain (11/11 were adults by day 7); and most of the G1m progeny (G2m eggs) hatched (85 ± 8%; Additional file 1: Fig. S4A). However, 66 ± 1% of the G2m were developmentally arrested (Additional file 1: Fig. S4B). Only 33 ± 12% of the remaining animals reached adulthood by day 7 (Cox proportional-hazards regression, P < 0.001; Fig. 5B and Additional file 1: Fig. S4C and Table S2), and their progeny production was severely reduced (laying seven eggs or less over 20 h). Moreover, 22 ± 7.5% of the G3m animals were still developmentally arrested (Additional file 1: Fig. S4B), and G3m development was similarly delayed (Cox proportional-hazards regression, P < 0.001; Fig. 5B and Additional file 1: Fig. S4C and Table S2). However, we noticed a significant recovery of animals’ development during subsequent generations (Fig. 5B and S4C). In contrast, heteroplasmic fzo-1(wt);pdr-1(mut);+/ΔmtDNA hatching and development was unaffected (~ 98% hatched and 100% were adults by day 7; Additional file 1: Fig. S4A and S4C). Thus, the adverse effects of the interaction between fzo-1(mut) and ΔmtDNA on fecundity and developmental timing were aggravated by pdr-1(mut), supporting fzo-1-pdr-1 epistasis.
We next asked whether the selection against ΔmtDNA would strengthen in the background of fzo-1(mut);pdr-1(mut);+/ΔmtDNA. To directly examine this, we compared the levels of ΔmtDNA molecules in fzo-1(mut);pdr-1(mut);+/ΔmtDNA animals (Fig. 5C) to fzo-1(mut);+/ΔmtDNA (Fig. 2D) across G1m-G4m generations. We found that ΔmtDNA levels declined more sharply in fzo-1(mut);pdr-1(mut);+/ΔmtDNA as compared to the single mutant strain fzo-1(mut);+/ΔmtDNA. Specifically, we found lower ΔmtDNA levels in G1m and G2m fzo-1(mut);pdr-1(mut);+/ΔmtDNA animals (fractional regression followed by within generation pairwise comparisons, P < 0.001; Additional file 1: Table S3). In contrast, ΔmtDNA levels did not significantly change across generations of fzo-1(wt);pdr-1(mut);+/ΔmtDNA animals (fractional regression, P > 0.167 in all cases; Additional file 1: Fig. S4D and Table S3). Taken together, the concomitant disruption of fusion and mitophagy strongly selected against ΔmtDNA molecules. In support of this interpretation, we noticed that even in the fzo-1(ht);pdr-1(mut);+/ΔmtDNA animals, where only one genomic copy of fzo-1 remained functional, ΔmtDNA levels declined and in some individuals were lost across ~ 25 generations (Wilcoxon Mann-Whitney rank sum test, P < 0.001; Fig. 5D).
Finally, we asked whether impaired mitophagy affects selection against ΔmtDNA in the worm germline. To this end, we examined the ratio of ΔmtDNA and +mtDNA between the gonad and soma in double mutant fzo-1(mut);pdr-1(mut);+/ΔmtDNA animals (Fig. 5E, F) and pdr-1(mut);+/ΔmtDNA (Fig. 5G, H). While mtDNA levels, including both ΔmtDNA and +mtDNA, were ~1.5-fold higher in the gonad vs. the soma of pdr-1(mut) animals (Wilcoxon Mann-Whitney rank sum test, P < 0.05; Fig. 5G, H), similar to wild type animals (Fig. 4F, G). We observed that fzo-1(mut);pdr-1(mut);+/ΔmtDNA animals displayed similar mtDNA levels in the gonad compared to soma, for both truncated and intact mtDNA molecules (Fig. 5E, F). On top of the selection against ΔmtDNA, fzo-1(mut);pdr-1(mut);+/ΔmtDNA double mutant displayed a reduction in total mtDNA levels, again supporting epistasis. Taken together, these data suggest that disrupting parkin-mediated mitophagy increased the organismal selection in fzo-1(mut);pdr-1(mut);+/ΔmtDNA individuals, which is associated with reduced fitness.