Redistribution of fragmented mitochondria ensure symmetric organelle partitioning and faithful chromosome segregation in mitotic mouse zygotes

Reviewer #1 (Public Review):

Summary:

Gekko, Nomura et al., show that Drp1 elimination in zygotes using the Trim-Away technique leads to mitochondrial clustering and uneven mitochondrial partitioning during the first embryonic cleavage, resulting in embryonic arrest. They monitor organellar localization and partitioning using specific targeted fluorophores. They also describe the effects of mitochondrial clustering in spindle formation and the detrimental effect of uneven mitochondrial partitioning to daughter cells.

Strengths:

The authors have gathered solid evidence for the uneven segregation of mitochondria upon Drp1 depletion through different means: mitochondrial labelling, ATP labelling and mtDNA copy number assessment in each daughter cell. Authors have also characterised the defects in cleavage mitotic spindles upon Drp1 loss

Weaknesses:

While this study convincingly describes the phenotype seen upon Drp1 loss, my major concern is that the mechanism underlying these defects in zygotes remains unclear. The authors refer to mitochondrial fragmentation as the mechanism ensuring organelle positioning and partitioning into functional daughters during the first embryonic cleavage. However, could Drp1 have a role beyond mitochondrial fission in zygotes? I raise these concerns because, as opposed to other Drp1 KO models (including those in oocytes) which lead to hyperfused/tubular mitochondria, Drp1 loss in zygotes appears to generate enlarged yet not tubular mitochondria. Lastly, while the authors discard the role of mitochondrial transport in the clustering observed, more refined experiments should be performed to reach that conclusion.

Reviewer #2 (Public Review):

Gekko et al investigate the impact of perturbing mitochondrial during early embryo development, through modulation of the mitochondrial fission protein Drp1 using Trim-Away technology. They aimed to validate a role for mitochondrial dynamics in modulating chromosomal segregation, mitochondrial inheritance and embryo development and achieve this through the examination of mitochondrial and endoplasmic reticulum distribution, as well as actin filament involvement, using targeted plasmids, molecular probes and TEM in pronuclear stage embryos through the first cleavages divisions. Drp1 deletion perturbed mitochondrial distribution, leading to asymmetric partitioning of mitochondria to the 2-cell stage embryo, prevented appropriate chromosomal segregation and culminated in embryo arrest. Resultant 2-cell embryos displayed altered ATP, mtDNA and calcium levels. Microinjection of Drp1 mRNA partially rescued embryo development. A role for actin filaments in mitochondrial inheritance is described, however the actin-based motor Myo19 does not appear to contribute.

Overall, this study builds upon their previous work and provides further support for the role of mitochondrial dynamics in mediating chromosomal segregation and mitochondrial inheritance. In particular, Drp1 is required for redistribution of mitochondria to support symmetric partitioning and support ongoing development.

Strengths:
The study is well designed, the methods appropriate and the results clearly presented. The findings are nicely summarised in a schematic.

Understanding the role of mitochondria in binucleation and mitochondrial inheritance is of clinical relevance for patients undergoing infertility treatment, particularly those undergoing mitochondrial replacement therapy.

Weaknesses:

The authors first describe the redistribution of mitochondria during normal development, followed by alterations induced by Drp1 depletion. It would be useful to indicate the time post-hCG for imaging of fertilised zygotes (first paragraph of the results/Figure 1) to compare with subsequent Drp1 depletion experiments.

It is noted that Drp1 protein levels were undetectable 5h post-injection, suggesting earlier times were not examined, yet in Figure 3A it would seem that aggregation has occurred within 2 hours (relative to Figure 1).

Mitochondria appear to be slightly more aggregated in Drp1 fl/fl embryos than in control, though comparison with untreated controls does not appear to have been undertaken. There also appears to be some variability in mitochondrial aggregation patterns following Drp1 depletion (Figure 2-suppl 1 B) which are not discussed.

The authors use western blotting to validate the depletion of Drp1, however do not quantify band intensity. It is also unclear whether pooled embryo samples were used for western blot analysis.

Likewise, intracellular ROS levels are examined however quantification is not provided. It is therefore unclear whether 'highly accumulated levels' are of significance or related to Drp1 depletion.

In previous work, Drp1 was found to have a role as a spindle assembly checkpoint (SAC) protein. It is therefore unclear from the experiments performed whether aggregation of mitochondria separating the pronuclei physically (or other aspects of mitochondrial function) prevents appropriate chromosome segregation or whether Drp1 is acting directly on the SAC.

Reviewer #3 (Public Review):

Why mitochondria are finely maintained in the female germ cell (oocyte), zygotes, and preimplantation embryos? Mitochondrial fusion seems beneficial in somatic cells to compensate for mitochondria with mutated mtDNA that potentially defuel the respiratory activity if accumulated above a certain threshold. However, in the germ cells, it may rather increase the risk of transmitting mutated mtDNA to the next generation, as authors discussed. Also, finely maintained mitochondria would also be beneficial for efficient removal when damaged. Due in part to the limited suitable model, the physiological role of mitochondrial fission in embryos were obscure. In this study, authors demonstrated that mitochondrial fission prevents multiple adverse outcomes, even including the aberrant demixing of parental genome in zygotic stage. This is an important study that could contribute by proposing a new mechanism for solving problems that actually arise in the field of reproductive medicine. The conclusion is simple and clear, but the high level of technology has made it possible to overcome the difficulties of proving the results, making this an extremely excellent study.

Seemingly, there are few apparent shortcomings. Following are the specific comments to activate the further open discussion.
- Line 246: Comments on cristae morphology of mitochondria in Drp1-depleted embryos would better be added.
- Regarding Figure 2H: If possible, a representative picture of Ateam would better be included in the figure. As the authors discussed in line 458, Ateam may be able to detect whether any alterations of local energy demand occurred in the Drp1-depleted embryos.
- Line 282: In Figure 3-Video 1, mitochondria were seemingly more aggregated around female pronucleus. Is it OK to understand that there is no gender preference of pronuclei being encircled by more aggregated mitochondria?
- Line 317: A little more explanation of the "variability" would be fine. Does that basically mean that the Ca2+ response in both Drp1-depleted blastomeres were lower than control and blastomere with more highly aggregated mitochondria show severer phenotype compared to the other blastomere with fewer mito?
- Regarding Figure 5B (& Figure 1-figure supplement 1B): Do authors think that there would be less abnormalities in the embryos if Drp1 is trim-awayed after 2-cell or 4-cell, in which mitochondria are less involved in the spindle?

Author Response:

We would like to thank the editors and reviewers for the careful consideration of our manuscript and their many helpful comments. We would like to provide provisional author responses to address the public reviews.

Response to Reviewer 1:

Weaknesses:

While this study convincingly describes the phenotype seen upon Drp1 loss, my major concern is that the mechanism underlying these defects in zygotes remains unclear. The authors refer to mitochondrial fragmentation as the mechanism ensuring organelle positioning and partitioning into functional daughters during the first embryonic cleavage. However, could Drp1 have a role beyond mitochondrial fission in zygotes? I raise these concerns because, as opposed to other Drp1 KO models (including those in oocytes) which lead to hyperfused/tubular mitochondria, Drp1 loss in zygotes appears to generate enlarged yet not tubular mitochondria. Lastly, while the authors discard the role of mitochondrial transport in the clustering observed, more refined experiments should be performed to reach that conclusion.

It would be difficult to answer from this study whether Drp1 has a role beyond mitochondrial fission in zygotes. However, there are several possible reasons why the Drp1 KO zygotes differs from the somatic cell Drp1 KO models.

First, the reviewer mentions that the loss of Drp1 in oocytes leads to hyperfused/tubular mitochondria, but in fact, unlike in somatic cells, the EM images in Drp1 KO oocytes show enlarged mitochondria rather than tubular structures (Udagawa et al. Current Biology 2014, Fig. 2C and Fig. S1B-D), as in the case of zygotes in this study.

These mitochondrial morphologies in Drp1-deficient oocytes/zygotes may be attributed to the unique mitochondrial architecture in these cells. Mitochondria in oocytes have the shape of a small sphere with an irregular cristae located peripherally or transversely. These structural features might be the cause of insensitivity or resistance to inner membrane fusion. In addition, in our previous study (Wakai et al., Molecular Human Reproduction 2014, Fig. 2), overexpression of mitochondrial fusion factors in oocytes resulted in mitochondrial aggregation when outer membrane fusion factor Mfn1/Mfn2 was overexpressed, while overexpression of Opa1 did not cause any morphological changes. Thus, while mitochondria in oocytes/zygotes divide actively, complete fusion, including the inner membrane, as seen in somatic cells, is unlikely to occur.

As for mitochondrial transport, we do not entirely discard its role. Althogh mitochondrial intrinsic dynamics such as fission are of primary importance for the mitochondrial distribution and partitioning in embryos, the regulation of dynamics by the cytoskeletons may be important and thus needs further study, as the reviewer pointed out.

Response to Reviewer 2:

Weaknesses:

The authors first describe the redistribution of mitochondria during normal development, followed by alterations induced by Drp1 depletion. It would be useful to indicate the time post-hCG for imaging of fertilised zygotes (first paragraph of the results/Figure 1) to compare with subsequent Drp1 depletion experiments.

We will indicate the time after hCG as the reviewer pointed out. The only problem is that in this experiment, there may be a slight deviation from the actual mitochondrial distribution change (Fig. S1A) due to the manipulation time for Trim-Away (since it was performed outside of the incubator). Also, no significant delay in pronuclear formation or embryonic development was observed with Drp1 depleted zygotes.

It is noted that Drp1 protein levels were undetectable 5h post-injection, suggesting earlier times were not examined, yet in Figure 3A it would seem that aggregation has occurred within 2 hours (relative to Figure 1).

As the reviewer pointed out, the depletion of Drp1 is likely to have occurred at an earlier stage. In this study, due to the injection of various RNAs to visualize organelles such as mitochondria and chromosomes, observations were started after about 5 hours of incubation for their fluorescent proteins to be sufficiently expressed. Therefore, for the western blotting analysis, samples were taken into account their condition at the start of the observation.

Mitochondria appear to be slightly more aggregated in Drp1 fl/fl embryos than in control, though comparison with untreated controls does not appear to have been undertaken. There also appears to be some variability in mitochondrial aggregation patterns following Drp1 depletion (Figure 2-suppl 1 B) which are not discussed.

We would like to add quantitative data on mitochondrial aggregation in Drp1-depleted embryos.

The authors use western blotting to validate the depletion of Drp1, however do not quantify band intensity. It is also unclear whether pooled embryo samples were used for western blot analysis.

We would like to add the quantitative results of the intensity of the bands for the Western blot analysis. The number of embryos analyzed is described in Fig legends, from 20 (Fig. 4) to 30 (Fig. 2) pooled samples were used.

Likewise, intracellular ROS levels are examined however quantification is not provided. It is therefore unclear whether 'highly accumulated levels' are of significance or related to Drp1 depletion.

We will present to indicate quantitative results on the accumulation of ROS.

In previous work, Drp1 was found to have a role as a spindle assembly checkpoint (SAC) protein. It is therefore unclear from the experiments performed whether aggregation of mitochondria separating the pronuclei physically (or other aspects of mitochondrial function) prevents appropriate chromosome segregation or whether Drp1 is acting directly on the SAC.

It has been reported that Drp1 regulates meiotic spindle through spindle assembly checkpoint (SAC) (Zhou et al., Nature Communications 2022). We would like to mention the possibility pointed out in the discussion part.

Response to Reviewer 3:

Seemingly, there are few apparent shortcomings. Following are the specific comments to activate the further open discussion.

- Line 246: Comments on cristae morphology of mitochondria in Drp1-depleted embryos would better be added.

We would like to add a comment regarding cristae morphology.

- Regarding Figure 2H: If possible, a representative picture of Ateam would better be included in the figure. As the authors discussed in line 458, Ateam may be able to detect whether any alterations of local energy demand occurred in the Drp1-depleted embryos.

ATeam fluorescence is analyzed using a regular fluorescence microscope, not a confocal laser microscope, in order to analyze the intensity in the whole embryo (or the whole blastomere). Therefore, we are currently unable to obtain images of localized areas within the cell (e.g., around the spindle) as expected by the reviewer; as shown in the images in Figure 3-figure supplement 1C, there is a tendency to see high ATP levels at the cell periphery, but further analysis is needed for clear and definitive results.

- Line 282: In Figure 3-Video 1, mitochondria were seemingly more aggregated around female pronucleus. Is it OK to understand that there is no gender preference of pronuclei being encircled by more aggregated mitochondria?

Aggregated mitochondria are localized toward the cell center, but do not behave in such a way that they are preferentially concentrated near the female pronucleus.

- Line 317: A little more explanation of the "variability" would be fine. Does that basically mean that the Ca2+ response in both Drp1-depleted blastomeres were lower than control and blastomere with more highly aggregated mitochondria show severer phenotype compared to the other blastomere with fewer mito?

We assume that what the reviewer have pointed out is right. However, although we were able to show the bias in Ca2+ store levels between blastomeres of Drp1 depleted embryos, we did not stain mitochondria simultaneously, so we were unable to say details such as more Ca2+ stores in blastomere that inherited more mitochondria or less Ca2+ stores in blastomere with more aggregated mitochondria

- Regarding Figure 5B (& Figure 1-figure supplement 1B): Do authors think that there would be less abnormalities in the embryos if Drp1 is trim-awayed after 2-cell or 4-cell, in which mitochondria are less involved in the spindle?

The marked accumulation of mitochondria around the spindle is unique to the first cleavage and seems to be coincident with the migration of the pronuclei toward the center. Since the process of assembly of the male and female pronuclei is also an event unique to the first cleavage, abnormalities such as binucleation due to mitochondrial misplacement are thought to be a phenomenon seen only in the first cleavage. Therefore, if Drp1 is depleted at the 2-cell or 4-cell stage, chromosome segregation errors may be less frequent. However, since unequal partitioning of mitochondria is thought to occur, some abnormalities in embryonic development is likely to be observed.