Chronic replication stress-mediated genomic instability disrupts placenta development in mice

  1. Mumingjiang Munisha
  2. Rui Huang
  3. Jordan Khan
  4. John C. Schimenti

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Abstract

Abnormal placentation drives many pregnancy-related pathologies and poor fetal outcomes, but the underlying molecular causes are understudied. Here, we show that persistent replication stress due to mutations in the MCM2-7 replicative helicase disrupts placentation and reduces embryo viability in mice. MCM-deficient embryos exhibited normal morphology but their placentae had a drastically diminished junctional zone (JZ). Whereas cell proliferation in the labyrinth zone (LZ) remained unaffected, JZ cell proliferation was reduced during development. MCM2-7 deficient trophoblast stem cells (TSCs) failed to maintain stemness, suggesting that replication stress affects the initial trophoblast progenitor pool in a manner that preferentially impacts the developing JZ. In contrast, pluripotency of mouse embryonic stem cells with MCM2-7 deficiency were not affected. Developing female mice deficient for FANCM, a protein involved in replication-associated DNA repair, also had placentae with a diminished JZ. These findings indicate that replication stress-induced genomic instability compromises embryo outcomes by impairing placentation.

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    Reply to the reviewers

    1. General Statements

    We thank the reviewers for providing thoughtful and constructive feedback, which will help us improve the clarity and rigor of the paper. On balance, the reviews were positive. Reviewer 1 mentioned that “This is a strong manuscript with few problems and all important findings well justified, indeed this is a nicely polished…..high-quality manuscript,” and that “this paper makes a major breakthrough, showing that cell autonomous defects in hTSCs are very likely at the heart of the pathology observed in GIN-prone murine mutants.” Reviewer 3 stated that “The study is well designed, and the manuscript is very well written. The conclusions are supported by the evidence presented.” Reviewer 2 was less enthusiastic, with main concerns being that “The paper is mostly descriptive and often quite confusing leaving one not much closer to understanding the mechanistic basis for the interesting sex-biased semi-lethal phenotype.” and felt that figure titles/section headers overstated the results, and finally recommended to improve some technical aspects and tempering conclusions. The proposed edits we think address most issues raised by the reviewers either with re-writing or adding data as described below.

    In response to reviewer #1 comments:

    Major comments:

    • I am confused as to the basis of the sex-skewing phenomenon? Is the problem that lack of maternally loaded WT Mcm4 worsens the phenotype, or is the issue that Mcm4C3/C3 dams are less able to retain pregnancies, perhaps being a more inflammatory environment? Also, while there quite consistent evidence for reduced viability of Mcm4C3/C3McmGt/+ progeny, especially for female progeny, how confident can we be that the genotype of the dam vs. sire is important? Notably on a Ddx58 background, the progeny of the Mcm4C3/C3 sire included seven live male Mcm4C3/C3McmGt/+ but no female.

    Regarding the first point (sex skewing only when female is C3/C3), we also suspected either: 1) the maternal uterine environment, or 2) reduced oocyte quality. Although not reported in this manuscript, we tested #1 by performing embryo transfer experiments. Transferring 2-cell stage embryos from sex-skewing mating to WT females did not rescue the sex-bias. We then examined oocytes from C3/C3 females. We found evidence for compromised mitochondria and transcriptome disruption. However, we are not sure why this happens (poor follicle support? Oocyte intrinsic phenomenon?). We are reserving these results and additional experiments for another paper, especially since this one mainly deals with GIN and placenta development. If the reviewers feel strongly that the embryo transfer data is crucial, we can include it.

    Regarding how confident we are that the genotype of the dam vs. sire is important, this stems from our previous paper by McNairn et al 2019 (the percentage of female C3/C3 M2/+ from sex-skewing mating is 20% compared to 60% from the reciprocal mating), which was quite dramatic. Consistent with this, MCM levels were significantly reduced in the placentae only when the dam was C3/C3 and the sire C3/+ M2/+, but not in the reciprocal cross. The reviewer makes a good observation about the Ddx58 cross; we can only hypothesize that the mutation somehow sensitizes females in this scenario and will make mention of it in the revision. We also realize that we neglected to write in Methods that the Ddx58 allele was coisogenic in the C3H background.

    • I'm not sure what Supplementary Figure 6 is showing (faster differentiation of C3 but less TGC?). Regardless, it's hard to draw too much conclusion from one not-very-pretty Western blot. This figure requires both additional replicates and a better explanation of how it fits with the other conclusions of the paper..

    We hypothesized that the JZ defect observed in the semi-lethal genotype placentas could arise either from impaired maintenance of the progenitor pool or from reduced capacity of mutant trophoblast progenitors to differentiate into the JZ lineage. The blot in Supplementary Figure 6 was intended as a qualitative demonstration that mutant trophoblast stem cells can differentiate into JZ lineages. We recognize that the figure is not definitive and will revise the text to clarify its purpose. A replicate(s) of the Western will be performed as suggested.

    • Supplementary Figure 7F-G is puzzling. Half of the mESCs have gamma-H2AX at all times, including most in S or G2 phase? In Figure S7E, do the quadrants correspond to being negative or positive for gamma-H2AX? At very least, IF images showing clear gamma-H2AX foci would be much more convincing.

    The gates for γH2AX FACS analysis were established using negative controls lacking primary antibody. As reported previously, embryonic stem cells display high basal levels of γH2AX staining (Chuykin et al., Cell Cycle 2008; Turinetto et al., Stem Cells 2012; Ahuja et al., Nat Comm 2016), which likely explains the broad signal observed across cell cycle phases. Regardless, we will provide immunofluorescence staining of γH2Ax and foci count in our revision.

    • The methods section is well detailed, but it would be ideal to clarify how many replicates each Western Blot or flow cytometry experiment is representative of.

    Thanks for the suggestion. We will update this for Fig4 and Fig5.

    Minor comments:

    • Is it possible that cGAS-STING and RIG pathways act redundantly to cause inflammation and lethality, or that other innate immune components are involved? I don't expect the authors to make compound mutants to test this but at least this possibility should be discussed textually.

    We appreciate the reviewer’s point, and had the same suspicion. Supporting this, we will add new RNA-seq analysis of Tmem173 KO placentas revealed elevated inflammatory gene expression compared to C3/C3 M2/+ controls, consistent with potential redundancy or feedback regulation. We will update in supplementary figures to reflect this.

    In response to reviewer #2 comments:

    Major comments:

    A major concern throughout the paper is that conclusions are often overstating their data. The title of figure 2 is "placentae with replication stress have smaller junctional and labyrinth zones". However, there is no measure of replication stress in this figure, just a histological evaluation of the placentae from the different mutants. The title of figure 3 is "Impact of GIN on LZ is less than JZ," but there is no measure of GIN, but instead measurement of number of cells in cell cycle and some bulk RNA-seq analysis. Title of figure 4 is "TSCs with increased genomic instability exhibit abnormal phenotypes." Again there is no measure of GIN, but instead staining of derived TSCs for proliferation, cell death, and a TSC marker. Title of figure 5 is "DNA damage responses and G2/M checkpoint activation drive premature TSC differentiation." However, there does not appear to be a difference in gH2AX between the two mutant genotypes. Checkpoint proteins might be up, but need quantification and reproduction. > 4C is the only marker of differentiation. Importantly, all the analyses here are associations, not connections, so cannot use the word "drive". Similar issues can be raised with a number of the supplementary figures.

    The Chaos3 (chromosome aberrations occurring spontaneously 3) model is a well-established system of intrinsic chronic replication stress and GIN. It is characterized by ~20 fold elevation of blood micronuclei (Shima et al., Nature 2007), a hallmark of GIN (Soxena et al., Mol Cell 2022); a destabilized MCM2-7 helicase prone to replication fork collapse (Bai et al., PLoS Genet 2016); and increased mitotic chromosome abnormalities and decreased dormant origins (Kawabata et al., Mol Cell 2011; Chuang et al., Nucleic Acid Res 2012) that are known to cause GIN and replication stress (Ibarra et al., PNAS 2008 ). Also, in our previous work (McNairn et al Nature 2019), we showed that placentae from C3/C3 dams exhibit significantly elevated γH2Ax as well as reduced MCM2 and MCM4 protein levels. In our current study, we also observe elevated γH2Ax in mutant TSCs (C3/C3 and C3/C3 M2/+), consistent with genomic instability. Nevertheless, we acknowledge that in TSCs, we did not formally demonstrate replications stress(RS), so where appropriate, we will advise figure titles, for example to say that “cells/placentae with a GIN or RS genotype.”

    We acknowledge the reviewers concern regarding western blots. We will provide quantification and statistics in our revision.

    1. A deeper analysis of the cell lines is likely to be the most fruitful path to reveal interesting mechanisms. It is very surprising that there is no phenotype in ESCs. Authors should check for increased apoptosis. Maybe the phenotypic cells are lost. Or do ESCs use different MCMs/mechanisms of DNA replication or are they better able to handle replication stress and GIN? How many passages were the TSCs and ESCs cultured for? Does GIN (i.e. aneuploidy, CNVs) develop in TSCs and ESCs with passaging? How do the MCM mutations impact the molecular identity of the ESC and TSC cells including their heterogeneity in the population.

    We assessed apoptosis using cleaved caspase 3 flow cytometry in mutant ESCs and observed no difference compared to controls (we will add this data as Supplementary Fig. 7).

    We believe there are intrinsic differences in TSCs and ESCs in their ability to respond to and counteract replication stress and DNA damage. ESCs are known to license more replication origins than somatic cells at a higher rate, which protects them from short G1-induced replication stress (Ahuja et al., Nat Comm 2016; Ge et al., Stem Cell Rep 2015; Matson et al., eLife 2017). Human placental cells physiologically exhibit high levels of mutation rate and chromosomal instability in vivo (Coorens et al., Nature 2021). Supporting this, Wang, D., et al (Nat Comm 2025) reported that several cell cycle and DDR regulators are differentially expressed in human TSCs vs human pluripotent stem cells. Whether such transcriptional differences directly contribute to functional outcomes remains to be determined.

    All experiments in this study were conducted using early-passage ESCs and TSCs (i.e. Finally, we showed that close to 90% mutant ESCs are KLF4+ (a naive pluripotency marker) whereas EOMES+ cells were significantly reduced in TSCs carrying the GIN genotype (Fig. 4E–F and Supplementary Fig. 7), highlighting lineage-specific differences.

    Minor Comments:

    1. There is a lack of quantification and repeats for all Westerns. At minimum there should be three repeats for each experiment, quantification including normalization to a reference protein, and stats confirming any proposed differences between conditions.

    We will update our revision with quantification and statistics for western blots.

    1. I would recommend moving the results in supp table 1 to figure 1. While negative, they are the newer results. The results shown in current figure 1 are essentially a reproduction of their previous work.

    The placental observations presented in Fig.1 are new. In particular, the placental and embryonic weight measurements graphed in Fig1B and C have not been published by our group. Fig1A reproduces our previous observation on embryo viability in GIN mutants (McNairn et al., Nature 2019), while the schematic was provided for better flow and readability given the complex mating schemes. We are agnostic on the Suppl Table 1. It could be changed to a new Table 1 in the main section depending on the journal.

    In response to reviewer #3 comments:

    Major Comments

    While the inclusion of bulk RNAseq data of whole placental tissue is appreciated, the interpretation of the results is somewhat problematic, as it is acknowledged that the cell type composition of the placentas is drastically different between groups. Making conclusions based upon GSEA analysis of two different groups with drastically different cell type composition is somewhat misleading, as based on the results, it is a direct reflection of the cell types present. It would be more helpful to perform cell type deconvolution of the RNAseq data to estimate the proportion of each cell type within the bulk samples and compare that to what is seen histologically and not dive too deeply into the pathways since the results could just be a reflection of the cell types e.g. angiogenesis pathways from more endothelial cells. Additionally, the RNAseq data can be leveraged to look at expression of inflammatory genes by sex, which may show interesting patterns based on the other results.

    We agree that the representation of cell types in the placenta is problematic especially for underrepresented genes. We propose to use the BayesPrism tool (Chu et al., Nat Cancer 2022) to deconvolute bulk RNA-seq for better representation of transcriptional changes in the placenta.

    Section: GIN impairs trophoblast stem cell establishment and maintenance. To support the assertion in the first paragraph, beyond measuring apoptosis, it would be helpful at this stage to look at RNA expression levels indicative of the activation of DNA damage checkpoint genes

    We have performed RNA-seq on mutant ESC and TSCs and are in the process of data analysis. We will update these results in the revision.

    Please include additional methodological details in the methods section on the statistical analysis done for differential expression analysis. Specifically, what type of normalization was used, if lowly expressed genes were filtered out and at what cutoff, what statistical model was used (did you include covariates?), what comparisons were made? Did you stratify by sex? What cutoff was used for statistical significance? Did you perform multiple testing correction?

    We will update RNA-Seq data analysis methods in our full revision.

    2. Description of the revisions that have already been incorporated in the transferred manuscript

    Reviewer #1 comments:

    • Supplementary Table 1. would be enhanced greatly showing comparable tables for Mcm4C3/C3 x Mcm4C3/+McmGt/+ in mice without the Tmem173 or Ddx58 mutations. It is fine to recycle data from McNairn 2019 here, as long as the source is indicated, but a comparison is needed.

    Thanks for pointing this out. We have updated this suggestion in Supp table 1.

    • In Figure S3E-F, is the box above each graph supposed to show the genotype of the dam?

    Yes. Thanks for pointing this out. We have added a description in the figure legend to make it clear.

    • "Indeed, the placenta and embryo weights of E13.5 Mcm4C3/C3 Mcm2Gt/+ Mcm3Gt/+ animals were significantly improved vs. Mcm4C3/C3 Mcm2Gt/+ animals, rendering them similar to Mcm4C3/C3 littermates (Fig. 6A-C). The JZ (but not LZ) area in Mcm4C3/C3 Mcm2Gt/+ Mcm3Gt/+ placentae also increased to the level of Mcm4C3/C3 littermates (Fig. 6D-H)." There are two problems here. First, the figure calls are wrong. Second, the description of the data is not quite right, it looks like the C3/C3 and C3/C3 M2/+ M3/+ LZs are a similar size to each and are statistically indistinguishable.

    Thanks for catching this. We have updated these in the main text.

    *Reviewer #2 comments: *

    Minor comment

    • Need to review citations to figures. For example, no citations are made to figure 4a and 4c.

    Thanks for catching this. We have updated the text.

    Reviewer #3 comments:

    Define the first use of >4C DNA content to help readers understand this potentially unfamiliar term.

    We have edited this part to indicate cells with more than 4C DNA content for better clarity.

    iDEP tool - please include citation to manuscript instead of link

    We have updated this citation.

    Check citations. Some citations to BioRxiv that are now published e.g. 13.

    We have updated this citation.

    3. Description of analyses that authors prefer not to carry out

    Reviewer 2

    1. Along similar lines, most of the in vivo phenotypic analyses are performed at E13.5, long after defects are likely beginning to express themselves especially given that they see phenotypes in the TSCs, which represent the polar TE of a E4.5. To understand the primary defects of the in vivo phenotype, they should be looking much earlier. Supplemental figure 5 is a start but represents a rather superficial analysis.

    The peri-implantation period, namely E4.5, represents a “black box” of embryonic development given that this is a critical stage for implantation. Aside from being an extremely difficult stage to analyze technically, we don’t think it is essential to the conclusions (or doable in a timely manner), especially given the use of TSCs. If we complete EdU studies on E6.5 embryos, we will include them.

    1. Fig. 6 would benefit from evidence that MCM3 mutant is rescuing MCM4 levels in the chromatin fraction of cells and the DNA damage phenotype.

    The genetic evidence presented is strong, and although we didn’t do the suggested experiment, we feel that our previous studies (McNairn et al., Nature 2019 and Chuang et al., PLoS Genet 2010) on the effects of MCM3 as a nuclear export factor (as it is in yeast (Liku et al., Mol Biol Cell 2005)) are a reasonable basis for not repeating such experiments. Furthermore, we are no longer maintaining the Mcm3 line and it would take over a year to reconstitute and rebreed triple mutants.

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    Referee #3

    Evidence, reproducibility and clarity

    This manuscript examines chronic replication stress-mediated genomic instability in placental development and concludes that it disrupts placental development in mice. The study is well designed and the manuscript is very well written. The conclusions are supported by the evidence presented. The manuscript would be improved by addressing the comments below.

    Major Comments:

    • While the inclusion of bulk RNAseq data of whole placental tissue is appreciated, the interpretation of the results is somewhat problematic, as it is acknowledged that the cell type composition of the placentas is drastically different between groups. Making conclusions based upon GSEA analysis of two different groups with drastically different cell type composition is somewhat misleading, as based on the results, it is a direct reflection of the cell types present. It would be more helpful to perform cell type deconvolution of the RNAseq data to estimate the proportion of each cell type within the bulk samples and compare that to what is seen histologically and not dive too deeply into the pathways since the results could just be a reflection of the cell types e.g. angiogenesis pathways from more endothelial cells. Additionally, the RNAseq data can be leveraged to look at expression of inflammatory genes by sex, which may show interesting patterns based on the other results.

    • Section: GIN impairs trophoblast stem cell establishment and maintenance. To support the assertion in the first paragraph, beyond measuring apoptosis, it would be helpful at this stage to look at RNA expression levels indicative of the activation of DNA damage checkpoint genes

    Minor Comments:

    • Define the first use of >4C DNA content to help readers understand this potentially unfamiliar term.

    • Please include additional methodological details in the methods section on the statistical analysis done for differential expression analysis. Specifically, what type of normalization was used, if lowly expressed genes were filtered out and at what cutoff, what statistical model was used (did you include covariates?), what comparisons were made? Did you stratify by sex? What cutoff was used for statistical significance? Did you perform multiple testing correction?

    • iDEP tool - please include citation to manuscript instead of link

    • Check citations. Some citations to BioRxiv that are now published e.g. 13.

    Significance

    The manuscript concludes that replication-stress induced genomic instability impairs placental development in mice. This is a significant advance in the field, as it mechanistically links genomic instability to placental development with further study needed in human trophoblast to establish clinical relevance. Strengths of this manuscript include solid study design, interpretation and presentation (both writing and figures). Weakness of the manuscript reside primarily in the RNAseq analysis results, methods and interpretation. The manuscript is of interest to audiences with interests in genome maintenance, development and placental biology. To contextualize this reviewer's point of view, this review is based on expertise in genomics, computational biology and placental biology.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript, "Chronic replication stress-mediated genomic instability disrupts placenta development in mice" by Munisha et al follows up a 2019 paper in Nature by the same group where they show that mutations to the MCM genes lead to a sex-skewed semi-lethal phenotype starting after embryonic day 9.5 and extending to birth. In the paper, they hypothesized that the semi-lethality is secondary to genomic instability (GIN) driven inflammation due to activation of the innate immune pathways sensing cytoplasmic DNA. In this paper, they start by disproving that hypothesis and then go on to present data arguing lethality is due to a placental development defect rather than inflammation. The paper is mostly descriptive and often quite confusing leaving one not much closer to understanding the mechanistic basis for the interesting sex-biased semi-lethal phenotype that was described in their original paper. The most interesting aspect of the paper is the derivation of TSC and ESCs and initial analysis suggesting that the TSCs are more sensitive to the MCM mutations, but the analysis is rather shallow. Importantly it is unclear how the phenotype explains the sex-skewing of the phenotype. Are the TSC phenotypes sex-skewed and if so why? Also, why is the JZ and especially GlyTCs most effected?

    A major concern throughout the paper is that conclusions are often overstating their data. The title of figure 2 is "placentae with replication stress have smaller junctional and labyrinth zones". However, there is no measure of replication stress in this figure, just a histological evaluation of the placentae from the different mutants. The title of figure 3 is "Impact of GIN on LZ is less than JZ," but there is no measure of GIN, but instead measurement of number of cells in cell cycle and some bulk RNA-seq analysis. Title of figure 4 is "TSCs with increased genomic instability exhibit abnormal phenotypes." Again there is no measure of GIN, but instead staining of derived TSCs for proliferation, cell death, and a TSC marker. Title of figure 5 is "DNA damage responses and G2/M checkpoint activation drive premature TSC differentiation." However, there does not appear to be a difference in gH2AX between the two mutant genotypes. Checkpoint proteins might be up, but need quantification and reproduction. > 4C is the only marker of differentiation. Importantly, all the analyses here are associations, not connections, so cannot use the word "drive". Similar issues can be raised with a number of the supplementary figures.

    Major Comments:

    1. A deeper analysis of the cell lines is likely to be the most fruitful path to reveal interesting mechanisms. It is very surprising that there is no phenotype in ESCs. Authors should check for increased apoptosis. Maybe the phenotypic cells are lost. Or do ESCs use different MCMs/mechanisms of DNA replication or are they better able to handle replication stress and GIN? How many passages were the TSCs and ESCs cultured for? Does GIN (i.e. aneuploidy, CNVs) develop in TSCs and ESCs with passaging? How do the MCM mutations impact the molecular identity of the ESC and TSC cells including their heterogeneity in the population.

    2. Along similar lines, most of the in vivo phenotypic analyses are performed at E13.5, long after defects are likely beginning to express themselves especially given that they see phenotypes in the TSCs, which represent the polar TE of a E4.5. To understand the primary defects of the in vivo phenotype, they should be looking much earlier. Supplemental figure 5 is a start but represents a rather superficial analysis.

    3. Fig. 6 would benefit from evidence that MCM3 mutant is rescuing MCM4 levels in the chromatin fraction of cells and the DNA damage phenotype.

    Minor Comments:

    1. There is a lack of quantification and repeats for all Westerns. At minimum there should be three repeats for each experiment, quantification including normalization to a reference protein, and stats confirming any proposed differences between conditions.

    2. I would recommend moving the results in supp table 1 to figure 1. While negative, they are the newer results. The results shown in current figure 1 are essentially a reproduction of their previous work.

    3. Need to review citations to figures. For example, no citations are made to figure 4a and 4c.

    Significance

    As is, the study does not provide much new insight or understanding of how the MCM mutants are driving the sex-skewed semi-lethal phenotype. It would likely take much effort (months) to reach such a goal. However, without such effort, it is unclear what the significance of the story is. It does make the observation that the placenta appears to be impacted more severely and earlier than then the embryo, and that within the placenta, certain zones and cell types are more vulnerable. The reasons for these differential impacts are unclear though.

    If the authors choose not to dig deeper as suggested in the major comments, then at a minimum it would be important to soften their conclusions as raised in the summary and at least perform experiments/edits proposed in minor comments.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In a previous paper (McNairn et al. 2019 "Female-biased embryonic death from inflammation induced by genomic instability" Science), the Schimenti lab demonstrated that mouse embryos with hypomorphic mutations of the heterohexameric minichromosome maintenance complex, mutations that cause increased genomic instability (GIN), show reduced embryonic viability, with greater loss of female embryos and some parent-of-origin effect. Treatment with immunosuppressants, including ibuprofen and testosterone, partially rescued the observed lethality.

    In this new manuscript, the Schimenti lab demonstrates that these GIN-prone mutants feature smaller placentas with fewer cells. Mutations that interfere with the ability of the innate immune system to respond to micronuclei (a consequence of GIN) have no protective effect. Munisha and colleagues then demonstrate that MCM-mutant TSCs are harder to derive and show elevated apoptosis and a greater propensity for differentiation. The mutant TSCs show CHK1 phosphorylation, P53 phosphorylation and higher P21 levels, all consistent with a response to DNA damage. Downstream of this, they also show loss and inhibition of CDK1, which is already established to cause G2/M arrest (generally) and endoreduplication (specifically in trophoblast). The authors advance a model in which GIN results in loss of the TSC pool by apoptosis, cell cycle arrest and premature differentiation, resulting in smaller placentas and particularly fewer junctional zone cells. How this causes inflammation is less clear, but inflammation appears to be a downstream effect rather than cause of poor placentation.

    Major comments:

    This is a strong manuscript with few problems and all important findings well justified, indeed this is a nicely polished manuscript for something just entering peer review. There are a few unclear points textually and a couple places in the supplementary figures where better data quality would help, but generally it is a high-quality manuscript.

    • I am confused as to the basis of the sex-skewing phenomenon? Is the problem that lack of maternally loaded WT Mcm4 worsens the phenotype, or is the issue that Mcm4C3/C3 dams are less able to retain pregnancies, perhaps being a more inflammatory environment? Also, while there quite consistent evidence for reduced viability of Mcm4C3/C3McmGt/+ progeny, especially for female progeny, how confident can we be that the genotype of the dam vs. sire is important? Notably on a Ddx58 background, the progeny of the Mcm4C3/C3 sire included seven live male Mcm4C3/C3McmGt/+ but no female.

    • I'm not sure what Supplementary Figure 6 is showing (faster differentiation of C3 but less TGC?). Regardless, it's hard to draw too much conclusion from one not-very-pretty Western blot. This figure requires both additional replicates and a better explanation of how it fits with the other conclusions of the paper..

    • Supplementary Figure 7F-G is puzzling. Half of the mESCs have gamma-H2AX at all times, including most in S or G2 phase? In Figure S7E, do the quadrants correspond to being negative or positive for gamma-H2AX? At very least, IF images showing clear gamma-H2AX foci would be much more convincing.

    • The methods section is well detailed, but it would be ideal to clarify how many replicates each Western Blot or flow cytometry experiment is representative of.

    The required additional experiments re: Supplementary Figure 6 and 7 could be conducted in a couple of months.

    Minor comments:

    • Supplementary Table 1. would be enhanced greatly showing comparable tables for Mcm4C3/C3 x Mcm4C3/+McmGt/+ in mice without the Tmem173 or Ddx58 mutations. It is fine to recycle data from McNairn 2019 here, as long as the source is indicated, but a comparison is needed.

    • Is it possible that cGAS-STING and RIG pathways act redundantly to cause inflammation and lethality, or that other innate immune components are involved? I don't expect the authors to make compound mutants to test this but at least this possibility should be discussed textually.

    • In Figure S3E-F, is the box above each graph supposed to show the genotype of the dam?

    • "Indeed, the placenta and embryo weights of E13.5 Mcm4C3/C3 Mcm2Gt/+ Mcm3Gt/+ animals were significantly improved vs. Mcm4C3/C3 Mcm2Gt/+ animals, rendering them similar to Mcm4C3/C3 littermates (Fig. 6A-C). The JZ (but not LZ) area in Mcm4C3/C3 Mcm2Gt/+ Mcm3Gt/+ placentae also increased to the level of Mcm4C3/C3 littermates (Fig. 6D-H)." There are two problems here. First, the figure calls are wrong. Second, the description of the data is not quite right, it looks like the C3/C3 and C3/C3 M2/+ M3/+ LZs are a similar size to each and are statistically indistinguishable.

    Significance

    I partially discussed the above in the summary, but this paper makes a major breakthrough, showing that cell autonomous defects in hTSCs are very likely at the heart of the pathology observed in GIN-prone murine mutants.

    Some questions go unsolved. Why are TSCs more prone to die in response to GIN than mESCs, particularly in light of the general observation that karyotypic abnormality is more common in placental lineage? How does the placental abnormality give rise to inflammation? No manuscript can answer every question, and I think this is a mature manuscript that can be published in a good journal with limited modifications.

    I am an expert on gene regulation in placental development, with somewhat less expertise in the DNA damage field. The placenta field will find this paper interesting, as will the DNA damage field. There are also ramifications for cancer research. The question of why some cells tolerate high levels of DNA damage and others die is very relevant to cancer.

  5. Version published to 10.1101/2025.02.28.640689 on bioRxiv