Multiple pathways prevent bi-parental mitochondria transmission in C. elegans

Reviewer #1 (Public review):

Summary:

Melin et al. developed a quantitative assay to measure the fate of paternal mitochondria after fertilization. They combine this assay with C. elegans genetics to show that multiple genes contribute to paternal mitochondrial elimination. However, despite their claims, they unconvincingly place these genes into distinct pathways and fail to determine whether additional unknown genes are involved in the process.

Strengths:

Melin et al. develop a new assay to quantify the fate of paternal mitochondria during embryonic development in C. elegans. They use complex C. elegans genetics to disrupt 5 different genes and nicely measure their contributions to paternal mitochondrial elimination. In an attempt to place these genes into pathways, the authors interrupt genes in various combinations and measure paternal mitochondrial persistence. The authors discovered that disrupting 4 of the genes known to contribute to paternal mitochondrial elimination still resulted in paternal mitochondrial elimination, suggesting that more genes also contribute to this process. Finally, the authors discovered that pink-1, which had previously been discounted, indeed contributes to paternal mitochondrial elimination when the major pathway involving allo-1 is also disrupted.

Weaknesses:

In the introduction, the authors describe the importance of studying the maternal inheritance of mitochondrial DNA. However, the authors mostly study the inheritance of paternally-derived mitochondrial proteins (HSP6::GFP). While the authors do use a PCR approach to measure paternal mitochondrial DNA, their results are not as quantitative and thorough (applied to multiple mutant combinations) as their microscopy assay. Using their microscopy assay, the authors did not combine mutants for all 5 genes. Therefore, they cannot support or discount the possibility that undiscovered paternal mitochondrial elimination mechanisms exist. The author's genetic epistasis experiments are incomplete and occasionally improperly interpreted (as described below). Finally, the authors were unable to achieve paternal mitochondrial transmission to the F2 generation (which admittedly has not been achieved in any experimental system).

Reviewer #2 (Public review):

Summary:

Mitochondrial DNA (mtDNA) is exclusively maternally transmitted in almost all species. Paternal mitochondria, with their mtDNA, must be rapidly degraded after fertilisation to prevent their transmission to progeny, which could lead to subsequent detrimental mito-nuclear incompatibilities. Multiple layers of mechanisms contribute to blocking the transmission of paternal mitochondria and their mtDNA to progeny. Endonuclease activity and mitophagy form a part of these strategies. However, other key regulatory mechanisms remain to be elucidated, as inactivating endonuclease and mitophagy activity only delays the clearance of paternal mitochondria. In this study, the authors mainly focused on genes involved in endonuclease function (csp-6) and autophagy (allo-1) in C. elegans, demonstrating a synergic genetic interaction that potentialize their activity. They also revealed a contribution by pink-1/pink1, in the absence of allo-1.

Strengths:

The majority of data relies on confocal microscopy images and corresponding image analysis and quantification. Images are clear, and quantifications are supported by several biological replicates of >10 n and standard statistical tests. Mutants used were obtained from the Caenorhabditis Genetics Center (CGC) and were previously validated and confirmed by the C. elegans community. The scientific approach is solid and rigorous and in line with state-of-the-art C. elegans methods. Proper controls have been performed to rule out the effect of animal viability on observed results or to confirm the staining validity of TUBES on subcellular structures surrounding paternal mitochondria. Controls validating uaDf5 PCR specificity were conducted.

Weaknesses:

However, the embryonic expression of paternally contributing genes in feminised animals cannot be completely ruled out, as RNAi was used to alter gene expression levels. An issue inherent to RNAi approaches. Also, the impact of pink-1/pink1 is significant, but there is a lack of evidence demonstrating its mitophagic function.

Goal achievements and data supportive of conclusions:

In the first part of the study, the authors strongly and clearly demonstrate the synergistic interaction between the csp-6 and allo-1 in delaying paternal mitochondria degradation and associated mtDNA in the fertilised egg. In wild-type animals, paternal mitochondria are visible (using a mitochondrial HSP-::GFP marker) until the 4-cell stage embryo. In the csp-6; allo-1 double mutant genetic background, paternal mitochondria very significantly perdures until the 2-fold embryonic stage. The uaDf5 mitochondrial deletion, detectable by PCR, that was introduced by crossing with a male, followed the same trend. In addition, loss of fncd-1/fndc1 and phb-2 did not extend the perdurance of paternal mitochondria. In the second part of the study, the authors demonstrate a contribution of the loss of pink-1/pink1, in the absence of allo-1, in delaying paternal mitochondria degradation until the 100-cell stage. Overall, the conclusions are in accordance with the data shown.

Impact on the field:

Endonuclease activity and mitophagy aren't sufficient to prevent the transmission of paternal mitochondria and associated mtDNA to progeny, but they still contribute significantly to regulating the perdurance of paternal mitochondria in early embryos. Understanding how these two functions work in concert to potentialize their activity is important, as they could potentially be manipulated/enhanced to improve paternal mitochondrial degradation in the future. Here, the authors demonstrate a detailed synergistic genetic interaction between these functions. Also, they pointed out a new potential contribution of pink-1/pink1, which may underlie a potentially more complex mitophagic protective function.

Reviewer #3 (Public review):

Summary:

The present study examines the cooperation among four allophagy/mitophagy factors, ALLO-1, CPS-6, FNDC-1, and PHB-2, implicated in the elimination of the sperm-derived mitochondria in C. elegans embryos. The key finding of the cumulative effect of ALLO-1 and CPS-6 inactivation causing delayed sperm mitophagy is significant for the understanding of mitochondrial inheritance in the nematode model and in general. Below are some specific suggestions on how the impact of the article could be elevated:

Abstract:

The authors should shorten the description of previously identified mitophagy factors and provide more detail on the present study results. An impact statement should be added at the end, with significance for understanding mitochondrial inheritance across taxa, all the way to mammals/humans.

Introduction:

The authors should provide more details on ALLO-1 and its interaction with LC-3. Also, it should be specified which of those previously identified allophagy factors are unique to worms and which ones are conserved. See also my comment below about including a diagram and a table of pathways and determinants involved in allophagy/paternal mitophagy.

Results:

If I understand the mtDNA data correctly, paternal mtDNA is maintained throughout the lifespan of the F1 generation but absent from the F2 generation. This is reminiscent of past studies of interspecific Mus musculus/Mus spretus mouse crosses by Kaneda/Shitara in which the paternal mtDNA was maintained F1 generation, resulting in heteroplasmy, but was lost from the F2 generation after back-crossing. Are CPS-6 and ALLO-1 effectors, but not determinants of maternal mtDNA inheritance in the nematode?

The finding that PINK-1 inactivation stabilizes sperm-derived mitochondria in the embryos is interesting. Are the substrates of PINK1 known in C. elegans? This could provide a clue concerning the aforementioned mitophagy determinants acting independently of ALLO-1.

Discussion:

A summary-diagram compiling the intersecting allophagy pathways would be helpful to accompany discussion, in addition to or expanding on the simple diagram presented as Figure 5; also, a table listing all the factors implicated in nematode allophagy next to those implicated in human/mammalian sperm mitophagy, which would highlight the divergences and overlaps between vertebrates and invertebrates.

Is it known how CPC-6 enters/gets imported into the sperm mitochondria inside the embryo? This pathway could potentially be targeted to decipher the allophagy mechanism.

PINK/PARKIN/PACRG and FUNDC1/2 pathways have been implicated in mammalian neurodegeneration as well as in mitophagy, including but not limited to sperm mitophagy after fertilization. These pathways in mammals should be briefly reviewed as they may provide further clues to how the allophagy pathways intersect in C. elegans.