Ectopic activation of the polar body extrusion pathway triggers cell fragmentation in preimplantation embryos

  1. Diane Pelzer
  2. Ludmilla de Plater
  3. Peta Bradbury
  4. Adrien Eichmuller
  5. Anne Bourdais
  6. Guillaume Halet
  7. Jean-Léon Maître

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Abstract

Cell fragmentation occurs during physiological processes, such as apoptosis, migration, or germ cell development. Fragmentation is also commonly observed during preimplantation development of human embryos and is associated with poor implantation prognosis during Assisted Reproductive Technology (ART) procedures. Despite its biological and clinical relevance, the mechanisms leading to cell fragmentation are unclear. Light sheet microscopy imaging of mouse embryos reveals that compromised spindle anchoring, due to Myo1c knockout or dynein inhibition, leads to fragmentation. We further show that defective spindle anchoring brings DNA in close proximity to the cell cortex, which, in stark contrast to previous reports in mitotic cells, locally triggers actomyosin contractility and pinches off cell fragments. The activation of actomyosin contractility by DNA in preimplantation embryos is reminiscent of the signals mediated by small GTPases throughout polar body extrusion (PBE) during meiosis. By interfering with the signals driving PBE, we find that this meiotic signaling pathway remains active during cleavage stages and is both required and sufficient to trigger fragmentation. Together, we find that fragmentation happens in mitosis after ectopic activation of actomyosin contractility by signals emanating from DNA, similar to those observed during meiosis. Our study uncovers the mechanisms underlying fragmentation in preimplantation embryos and, more generally, offers insight into the regulation of mitosis during the maternal-zygotic transition.

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  1. Review Commons

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    Reply to the reviewers

    RC-2022-01805

    We thank all reviewers for their careful analysis of our manuscript, constructive suggestions and support of our work.

    Reviewer 1

    The authors show that proximity of early mouse embryo blastomere chromosomes to the cell cortex activates the Polar Body Extrusion pathway to generate cell fragments. The authors use live cell imaging in control and Myo1C and dynein knockdown embryos to document accumulation of actin and myosin near chromosomes that come in close proximity to the cell cortex, which correlates with the increased fragmentation of the mutant blastomeres. The live imaging data are nicely presented and the results are well quantified. I have two major comments, and some minor comments on clarity, for the authors consideration in revising the manuscript.

    Major comment:

    The authors imply that Myo1 and dynein knockdowns result in an increase in the number of cells where chromosomes come in close proximity to the cell cortex. Apparently the spindle anchoring defects are meant to indicate that such defects are responsible for the increased frequency of abnormal chromosome proximity to the cortex. But the authors never actually document whether chromosomes in fact do come into proximity to the cortex more often in the mutant than in control embryos. The authors should clarify if they think the spindle anchoring defect does result in abnormal chromosome distributions. Can the authors somehow quantify a defect in overall chromosome positioning in mutant vs control blastomeres? Presumably the movies the authors already have could be used to provide such quantification?

    We thank the reviewer for this opportunity to correct our previous assumptions. Following the reviewer’s suggestion, we tracked the distance between the cell surface and the center of the chromosomes cluster throughout mitosis. We found little difference in this distance between control and Myo1cKO embryos (Fig S3a), unlike what we had initially implied. This distance seemed more variable in Myo1cKo embryos than in control ones, suggesting that chromosome movements may be more erratic but analysis of this variation for individual cells did not show consistent differences between control and Myo1cKO embryos either (Fig S3b). Therefore, we cannot explain the increased signaling with differences in proximity of the chromosomes to the cortex during mitosis.

    Instead, as already hinted in our initial manuscript as an additional factor, we find that signaling from chromosomes to the cortex can occur for an extended time in embryos with impaired spindle anchoring.

    We had already measured that mitotic spindles persisted for a longer time in Myo1cKO embryos than in control ones (Fig 2b), as well as in ciliobrevin treated embryos as compared to DMSO treated ones (Fig S2b). To strengthen this data, we performed additional experiments in which we injected mRNA encoding fluorescent lamin-associated protein 2b (Lap2b-GFP) to track the breakdown and reassembly of the nuclear envelope. Consistent with the mitotic spindle persisting for a longer time in Myo1cKO embryos than in control ones, it generally takes more time for Myo1cKO embryos to reassemble their nuclear envelope than for control embryos (50 min vs 70 min, n = 8 control and 15 Myo1cKO embryos, p = 0.0161, Fig S3c-d, Movie 5). Taken together, the nuclear envelope and spindle data indicate that, although chromosomes are not closer to the cortex in Myo1cKO embryos than in control ones, they spend more time outside of the nucleus. This should give chromosomes extended opportunities to signal to the cortex and explains how difficulties with chromosome separation can lead to the hyper-activation of the polar body extrusion pathway.

    We have revised our manuscript accordingly.

    Near the end of the paper, the authors discuss how cell with bent/un-anchored spindles are more prone to fragmentation, referring to Figure 2. But Figure 2 does not document a correlation between blastomeres with bent spindles and increased fragmentation. Rather it shows an increase in bent spindles and in fragmentation in mutant vs control, but does show that they occur together. The authors should more accurately describe their results or provide such a correlation with additional data.

    We thank the referee for pointing out this missing information.

    To support our conclusions, we now provide additional analyses of mitosis duration in non-fragmenting and fragmenting cells from Myo1cKO embryos. When cells fragment, their mitosis is consistently longer, as measured from the persistence of the mitotic spindle, than when not fragmenting (Fig 2c). This provides a direct correlation between spindle defects and fragmentation.

    We now present these analyses in the revised manuscript.

    Finally, in describing the data in Figure 3, the authors refer to persistence of the spindle and bending of the spindle as indicating problems with anchoring. It is not clear to me how either spindle persistence or bending relate to anchoring. The authors should explain how they are related if they are, and it would be better if the authors could document spindle displacement relative to the cell center or cortex to make their point more directly that anchoring is defective.

    We apologize for not making this clearer in our initial manuscript. As others noted before (Kotak et al, 2012; Mangon et al, 2021), poorly anchored spindles show larger displacements or rotations during mitosis. Spindle persistence and bending may not be directly related to spindle anchoring defects but could reflect broader issues with spindle assembly and function caused by spindle anchoring defects. Since a previous in vitro study had identified that Myo1cKO is important for spindle anchoring (Mangon et al 2021) and that ciliobrevin, known for compromising spindle anchoring, phenocopied these aspects, we had initially focused on anchoring defects in our conclusions. We still stand by our conclusion that our data suggest spindle anchoring defects. Nevertheless, we agree that our observations report more general spindle defects and that anchoring may be only one of the defective aspects. Instead of “spindle anchoring defects”, we now simply mention “spindle defects” unless specifically discussing spindle straightness and rotation.

    Minor comment.

    The authors document in Figure 3 that Myo1C KO blastomeres have an enhanced response, with more myosin accumulating at the cortex in response to chromosomes. Why does knocking out one non-muscle myosin lead to enhanced accumulation of another? The authors note this effect but provide no discussion as to how it occurs. Some clarification might be helpful.

    In our manuscript, we report that chromosome proximity to the cortex is associated with Cdc42 activation, which leads to cortical actin recruitment (Fig 4a-d). We also observe that non-muscle myosin II (Myh9) is recruited to the cortex when chromosomes come near (Fig 3d-f). Importantly, these phenomena occur in control embryos as well and not only in Myo1cKO embryos.

    We propose that this recruitment is further increased in Myo1cKO embryos (Fig 3f) because chromosomes spend more time outside of the nuclear envelope (Fig 2). This leads to fragmentation and is not specific to Myo1cKO since the same occurs after ciliobrevin treatment (Fig S2).

    The authors provide a significant advance in our understanding of why early mammalian embryos, especially early human embryos, are so prone to fragmentation. Their data strongly support their conclusion that increased proximity of chromosomes to the cortex does lead to activation of the PBE response, which is an interesting and well documented finding. However, unless the authors can address my major comments and provide more direct evidence for increased displacement of chromosomes being responsible for increased fragmentation, they should revise their manuscript to acknowledge that they have not directly quantified chromosome positioning and thus do not conclusively document that it is responsible for increased fragmentation in the mutant oocytes.

    We thank the reviewer for their thorough analysis of our data and for giving us the opportunity to correct some of the aspects of our study.

    Reviewer 2

    The manuscript "Ectopic activation of the polar body extrusion pathway triggers cell fragmentation in preimplantation embryos" by Pelzer and colleagues is focused on mechanism of cell fragmentation in early preimplantation embryos. This is an important issue, since fragmentation, with subsequent cell loss, has significant impact on early development of human embryos in vitro.

    To study the cell fragmentation within the embryo, authors used mouse model system. However, since during the mouse preimplantation development blastomere fragmentation is less frequent than in human embryos, they used knockout of unconventional myosin-Ic to induce fragmentation of embryonic blastomeres with higher frequency and a similar morphology, known from human embryos.

    Using their Myo1c KO, authors confirmed previous observation that reduction of myosin-Ic impairs spindle anchoring and they further show that the defects in spindle anchoring are linked to cell fragmentation. And that similar defects could be induced by chemical inhibition of dynein. Importantly, the defects in anchoring, causing aberrant spindle movements, bring spindle and chromosomal DNA to the proximity of the cell cortex. This induces local changes in concentration and organization of actin and myosin IIA and leads into fragmentation. Authors show that this pathway shares similarity with mechanism of polar body extrusion (PBE) during meiosis, namely that it requires active Cdc42-mediated actin polymerization or Ect2 signaling. And also, that important role in cell fragmentation is played by cell surface tension. Based on their results, authors propose that cell fragmentation within the embryo is triggered either by hyperactivation of PBE pathway in cells with normal surface tension, or by PBE pathway activation in cells with higher contractility.

    This manuscript brings important information about mechanism, which might contribute to the high incidence of blastomere fragmentation in human embryos. I have not identified any important issues with experimental work or conclusions and therefore I recommend this paper for publication. The results from the mouse model system however need to be verified by further studies in human or similar embryos, which naturally exhibit higher fragmentation.

    We thank the reviewer for their careful examination of our manuscript and data.

    We agree that it would be important to verify the validity of our findings in other species. We have considered performing experiments with human embryos.

    Ideally, we would need embryos in their early cleavage stages (zygote to 4-cell stages) to be able study fragmentation without perturbing morphogenetic movements, which begin at the 8-cell stage. Such early embryos are particularly rare, which further requires careful experimental design.

    Ideally, such carefully designed experiment would not cause additional fragmentation (as we have mostly done in the present study) but rather reduce this deleterious process. In light of our experiments shown in Fig 4c-d, inhibiting Cdc42 would be a good way to reduce polar body extrusion signaling. Injection of DNCdc42 mRNA would be embryo-consuming to setup. We tried a Cdc42 chemical inhibitor on mouse embryos with unreliable results. Therefore, we do not yet feel confident in using precious human embryos with our currently available options.

    Another complication is administrative since this project was funded by the ERC, which does not allow experimentation with human embryos.

    As for studying the phenomenon in species other than mouse or human, we currently have limited access to other mammalian species. Generally, other mammalian embryos are less well characterized and, in particular, the species-specific fragmentation behavior would need to be characterized before initiating any attempt to reduce it.

    We hope that the reviewers will agree that the current manuscript, describing and dissecting a previously unknown mechanism, makes sufficient advances to be published without the need to assess its evolutionary conservation.

    This study revealed important mechanism, which might be responsible for inducing fragmentation of blastomeres in early preimplantation embryos. Authors use mouse knockout model system and therefore the results should be verified in other species, in which the embryos show higher fragmentation naturally. The manuscript provides evidence that pathway, leading into PBE in oocytes, remains operational also in embryos and might contribute to blastomere fragmentation in case when spindle loses anchoring to the membrane. The results of this manuscript should be of interest not only to the researchers in reproduction, but also to the general audience.

    Reviewer 3

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    The manuscript discussed interesting and relevant topics in which the Authors addressed the effects of mouse Myo1C knock out on cell fragmentation and spindle anchoring defects. The authors found that fragmentation occurs in mitosis after ectopic activation of actomyosin contractility by signals emanating from DNA.

    Reviewer #3 (Significance (Required)):

    This is an excellent report dealing with significant technical methodologies. I find no fault in the methods, data analysis, or conclusions. I only have two comments. First, the authors should expand on the previous findings about the of the role of Myo1c during early preimplantation development. Second, the discussion should be expanded to compare the results of this study with those of previous/related studies (e.g., other factors involve in fragmentation and spindle anchoring). Finally, I was not able to open movie#2 and movie#8 so they may need to be re-uploaded.

    We thank the reviewer for their careful assessment of our study.

    We apologize for not discussing enough the previous research on Myo1c. To our knowledge, there is only one previous study reporting the effect of a point mutation on Myo1c on mouse ear physiology (Stauffer et al 2005). This is the first study on the role of Myo1c during mouse development. At this point, we would like to stress that our study, while partially based on the KO of Myo1c, is about cell fragmentation, which we induce experimentally in three independent ways: Myo1c KO, ciliobrevin treatment or Ect2 overexpression.

    Regarding fragmentation, to our knowledge there is simply no convincing mechanism to explain this phenomenon. One study proposed that membrane threads connecting the cell surface to the zona pellucida could pull on cells and promote fragmentation (Derick et al 2017). However, fragmentation also occurs without zona pellucida, and hence without threads pulling on cells’ surfaces (Yumoto et al 2020). Other than that, fragmentation was associated with mitosis and general cytoskeleton defects, without no clear mechanism (Alikani 1999, Fujimoto et al 2011, Daughtry et al 2019).

    We have now expanded these discussions.

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript discussed interesting and relevant topics in which the Authors addressed the effects of mouse Myo1C knock out on cell fragmentation and spindle anchoring defects. The authors found that fragmentation occurs in mitosis after ectopic activation of actomyosin contractility by signals emanating from DNA.

    Significance

    This is an excellent report dealing with significant technical methodologies. I find no fault in the methods, data analysis, or conclusions. I only have two comments. First, the authors should expand on the previous findings about the of the role of Myo1c during early preimplantation development. Second, the discussion should be expanded to compare the results of this study with those of previous/related studies (e.g., other factors involve in fragmentation and spindle anchoring). Finally, I was not able to open movie#2 and movie#8 so they may need to be re-uploaded.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript "Ectopic activation of the polar body extrusion pathway triggers cell fragmentation in preimplantation embryos" by Pelzer and colleagues is focused on mechanism of cell fragmentation in early preimplantation embryos. This is an important issue, since fragmentation, with subsequent cell loss, has significant impact on early development of human embryos in vitro.

    To study the cell fragmentation within the embryo, authors used mouse model system. However, since during the mouse preimplantation development blastomere fragmentation is less frequent than in human embryos, they used knockout of unconventional myosin-Ic to induce fragmentation of embryonic blastomeres with higher frequency and a similar morphology, known from human embryos.

    Using their Myo1c KO, authors confirmed previous observation that reduction of myosin-Ic impairs spindle anchoring and they further show that the defects in spindle anchoring are linked to cell fragmentation. And that similar defects could be induced by chemical inhibition of dynein. Importantly, the defects in anchoring, causing aberrant spindle movements, bring spindle and chromosomal DNA to the proximity of the cell cortex. This induces local changes in concentration and organization of actin and myosin IIA and leads into fragmentation. Authors show that this pathway shares similarity with mechanism of polar body extrusion (PBE) during meiosis, namely that it requires active Cdc42-mediated actin polymerization or Ect2 signaling. And also, that important role in cell fragmentation is played by cell surface tension. Based on their results, authors propose that cell fragmentation within the embryo is triggered either by hyperactivation of PBE pathway in cells with normal surface tension, or by PBE pathway activation in cells with higher contractility.

    This manuscript brings important information about mechanism, which might contribute to the high incidence of blastomere fragmentation in human embryos. I have not identified any important issues with experimental work or conclusions and therefore I recommend this paper for publication. The results from the mouse model system however need to be verified by further studies in human or similar embryos, which naturally exhibit higher fragmentation.

    Significance

    This study revealed important mechanism, which might be responsible for inducing fragmentation of blastomeres in early preimplantation embryos. Authors use mouse knockout model system and therefore the results should be verified in other species, in which the embryos show higher fragmentation naturally. The manuscript provides evidence that pathway, leading into PBE in oocytes, remains operational also in embryos and might contribute to blastomere fragmentation in case when spindle loses anchoring to the membrane. The results of this manuscript should be of interest not only to the researchers in reproduction, but also to the general audience.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    The authors show that proximity of early mouse embryo blastomere chromosomes to the cell cortex activates the Polar Body Extrusion pathway to generate cell fragments. The authors use live cell imaging in control and Myo1C and dynein knockdown embryos to document accumulation of actin and myosin near chromosomes that come in close proximity to the cell cortex, which correlates with the increased fragmentation of the mutant blastomeres. The live imaging data are nicely presented and the results are well quantified. I have two major comments, and some minor comments on clarity, for the authors consideration in revising the manuscript.

    Major comment:

    1. The authors imply that Myo1 and dynein knockdowns result in an increase in the number of cells where chromosomes come in close proximity to the cell cortex. Apparently the spindle anchoring defects are meant to indicate that such defects are responsible for the increased frequency of abnormal chromosome proximity to the cortex. But the authors never actually document whether chromosomes in fact do come into proximity to the cortex more often in the mutant than in control embryos. The authors should clarify if they think the spindle anchoring defect does result in abnormal chromosome distributions. Can the authors somehow quantify a defect in overall chromosome positioning in mutant vs control blastomeres? Presumably the movies the authors already have could be used to provide such quantification?
    2. Near the end of the paper, the authors discuss how cell with bent/un-anchored spindles are more prone to fragmentation, referring to Figure 2. But Figure 2 does not document a correlation between blastomeres with bent spindles and increased fragmentation. Rather it shows an increase in bent spindles and in fragmentation in mutant vs control, but does show that they occur together. The authors should more accurately describe their results or provide such a correlation with additional data.
    3. Finally, in describing the data in Figure 3, the authors refer to persistence of the spindle and bending of the spindle as indicating problems with anchoring. It is not clear to me how either spindle persistence or bending relate to anchoring. The authors should explain how they are related if they are, and it would be better if the authors could document spindle displacement relative to the cell center or cortex to make their point more directly that anchoring is defective.

    Minor comment.

    The authors document in Figure 3 that Myo1C KO blastomeres have an enhanced response, with more myosin accumulating at the cortex in response to chromosomes. Why does knocking out one non-muscle myosin lead to enhanced accumulation of another? The authors note this effect but provide no discussion as to how it occurs. Some clarification might be helpful.

    Significance

    The authors provide a significant advance in our understanding of why early mammalian embryos, especially early human embryos, are so prone to fragmentation. Their data strongly support their conclusion that increased proximity of chromosomes to the cortex does lead to activation of the PBE response, which is an interesting and well documented finding. However, unless the authors can address my major comments and provide more direct evidence for increased displacement of chromosomes being responsible for increased fragmentation, they should revise their manuscript to acknowledge that they have not directly quantified chromosome positioning and thus do not conclusively document that it is responsible for increased fragmentation in the mutant oocytes.

  5. Version published to 10.1101/2022.12.22.521568v1 on bioRxiv