PONE-D-24-42707Loss-of-function of NSD2 causes abnormal placentation accompanied by fetal growth retardation in micePLOS ONE

Dear Dr. Kawai,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Reviewers agreed that more in-depth analysis is warranted. Reviewer 1 was very specific while Reviewer 2’s comments were very general. As a practical matter all requests to clarify definitions, terminology and data presentation (mutations, strains, placental structure (“layers”) and cell types analyzed, observed phenotypes, etc.) must be addressed. This likely requires additional analyses but not necessarily additional experiments (although additional experiments would be considered). In addition, you must address the sentiment that additional clarification, discussion and interpretation are required to understand and evaluate the validity of this work in the context of preexisting knowledge. However, you needn’t address every reviewer comment in the same detail. The most important reviewer comments deal with placentomegaly and fetal growth restriction, a potential “placenta-heart-axis”, potential deviation from Mendelian ratios in survivors and the need for greater clarity and specificity of description of the placental changes observed.

In addition, the title should reflect the fact that the experimental perturbations are limited to genetic loss of and/or altered function (to be clarified). “Loss-of-function mutation of NSD2 is associated with …” rather than “Loss-of-function of NSD2 causes …”. Also, please discuss the possibility that some placental changes are entirely or in part secondary to primary effects in the embryo.

Please submit your revised manuscript by Feb 10 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, the authors describe two separate mouse lines whereby the gene Nsd2 is knocked out (Nsd2-/-) or contains a point mutation (Nsd2^P906L). NSD2 is a histone methyltransferase and so presumably altering its expression/function will have implications for chromatin structure and gene regulation. The authors previously reported that both mutations lead to perinatal lethality at differing levels of penetrance and that at embryonic day (E)18.5, Nsd2-/- and Nsd2^P906L/P906L fetuses were growth restricted. This led the authors to consider how placenta structure is affected by these mutations in the late stages of gestation. They show an exciting placentomegaly phenotype associated with homozygosity for either Nsd2 mutation, which is an exciting and rare phenotype typically associated with models of epigenetic dysregulation. The authors also show a ventricular septal defect (VSD) in at least one mutant fetal heart. While there is substantial emerging data in the literature regarding the placenta-heart axis, this is not properly introduced by the authors and so the rationale for assessing the fetal heart in this context is unclear. The link between placentomegaly and a heart defect is novel and exciting, and this should be better discussed and even represented in the manuscript title. There are some issues in the description and characterisation of the placenta phenotype (listed below). Currently, the discussion is quite superficial without exploring the specific phenotypes observed in this unique context. However, the data in this study are promising and with a little more detail and finesse, this manuscript will contribute well to the literature.

Major concerns:

1. The authors do not indicate the effect of the Nsd2^P906L point mutation: does it knock out the enzyme function partially or completely? More information in the introduction is required so that a proper comparison between the Nsd2-/- and Nsd2^P906/P906L phenotypes can be achieved. Do the authors have any specific information about how histone methylation is affected by the Nsd2 mutations? Is the histone methylation of placenta or heart specific genes particularly affected? This would be a good addition to the manuscript.

Additionally, as the authors discuss the data throughout the manuscript, it would be helpful to the reader if the data from two mouse lines were kept separate. This might be in separate sentences or even separate paragraphs. This will allow the reader to digest the data and better determine the similarities and differences of the models. Also, the notations for the mutations should be consistent throughout the manuscript and clearer. The authors use the word ‘mutation’ and it is unclear which mouse line they are referring to (e.g., Lines 129-132), and, for example, use Nsd2^P906L/P906L and KI-hom terminology interchangeably. It would be particularly helpful to use the actual mutation name when labelling figures.

2. It is interesting that loss-of-function mutations in Nsd2 lead to perinatal lethality. Looking at the genotypic ratios at different stages of development, it is also possible that embryonic lethality of the homozygous conceptuses has occurred. For example, at E15.5, the following non-Mendelian ratios are reported in this manuscript for each genotype.

Nsd2+/+ : Nsd2+/- : Nsd2-/-, 2:4:1

Nsd2+/+ : Nsd2^+/P906L : Nsd2^P906L/P906L, 2:3:1

To fully appreciate this observation, the authors need to confirm that the genotyping procedure is working properly. This data is relevant to the manuscript because a severe placenta phenotype established through poor development and/or function can cause embryonic lethality. The authors should include this interpretation of the data when it is presented.

3a. The rationale for and description of the placenta phenotypes requires some work, and the current analysis is superficial. Is Nsd2 mRNA or protein expressed within the placenta? If so, in what cell types? This can be achieved by immunohistochemistry of Nsd2 on wildtype placenta sections at E15.5, or by assessing publicly available single cell transcriptome datasets obtained in wildtype placenta. The authors state in the discussion that Nsd2 is expressed in the placenta and cite Han et al, 2018 (ref 20) but do not explicitly indicate the cell types that Nsd2 is expressed within. Knowing its location of expression will help to justify why the analysis is focussed on a particular region of the placenta. The placentas from Nsd2-/- and Nsd2^P906L/P906L are clearly larger but the authors do not really explain why this is the case: more cells? bigger cells? which cells?

3b.The majority of the analysis in the manuscript focuses on the labyrinth layer, but it is also possible that the junctional zone of the placenta is also affected by Nsd2 loss-of-function, given its large size. The authors refer to this cell layer as the ‘spongiotrophoblast layer’, which is a slightly outdated term because this layer contains cells that are not spongiotrophoblast cells (e.g., parietal trophoblast giant cells, and glycogen trophoblast cells that play a role in glycogen storage and metabolism, and potentially other secretory roles (see Tunster et al, 2020 Reproduction PMID 32191912)). Defects in glycogen trophoblast cells associate with fetal growth defects and labyrinth defects. Currently, the function of glycogen trophoblast cells is not well understood, but presumably if the stored glycogen cannot be broken down into glucose for placenta and fetal use, fetal growth can be affected.

It is unclear why the authors have chosen to use PAS stain as the main histological stain to assess Nsd2-/- and Nsd2^P906L/P906L placentas. However, PAS stain can be used to identify glycogen trophoblast cells and their location (they are an invasive cell type appearing in the junctional zone and the decidua) and the approximate level of glycogen that is stored (darker pink PAS stain indicates more glycogen content). The authors should assess the glycogen trophoblast population at a higher magnification in their PAS stained histological sections to rule out a potential defect in this cell type. Are there more glycogen trophoblast or spongiotrophoblast cells, and is this why the junctional zone is larger in the mutants?

In the discussion (lines 203-6), the authors describe the placenta phenotype as ‘an erosion of the ST layer’, which is inconsistent with what is observed in Fig 2A and 2B whereby the junctional zone is much larger and not eroded. A better description might be that the boundary between the junctional zone and labyrinth layer is disorganised – however, based on experience, this type of dipping down of the junctional zone into the labyrinth can occur in the wildtype situation (see Simmons et al, 2008 BMC Genomics PMID: 18662396 for a clear example in Fig 4). It would be better for the authors to focus their discussion on the larger size of the junctional zone. What are reasons for the large size? Progenitor cell regulation? Increased cell proliferation? Size or number of cells dysregulated? Etc, etc.

3c. It is possible that there is a labyrinth layer defect, yet how this phenotype is described in the manuscript is concerning. First, the labyrinth structure shown in the control placenta images is not what is typically observed at E15.5 (Fig 2C). This can have a knock-on effect on how the mutant phenotype is described. Are the images from both Nsd2 wildtype placentas (P906L and knockout littermates) taken from the periphery of the placenta rather than in a central region? There is a noticeable lack of fetal vasculature in the labyrinth region particularly in the KO_WT placenta, which is unusual and unexpected for a control. (Are the images reversed for genotype??)

To describe the effect of Nsd2 loss-of-function on labyrinth structure more accurately, the authors should perform simple staining the histological sections with alkaline phosphatase (labels all cells lining the maternal blood sinusoids) and/or isolectin (labels fetal vascular endothelial cells). This will allow the authors to confidently identify and differentiate between maternal and fetal blood spaces. If the maternal blood sinusoids are indeed dilated as indicated in the manuscript and when compared to a proper control, it would suggest that labyrinth villus branching is impeded. In this case, the villi do not extend into the maternal blood sinusoids as they should and might result from a defect in branching morphogenesis. A good article that discusses this principle is Adamson et al, 2002 Dev Biol (PMID: 12376109).

Further characterisation of the effect of Nsd2 loss of function on the labyrinth requires assessment of specific cell subtypes. This can be achieved by immunostaining (e.g., MCT1 or MCT4) to label syncytriotrophoblast cells to determine if they are fusing together or elongating to form normal labyrinth architecture. Measuring nuclear size of sinusoidal trophoblast giant cells in the histological sections might indicate whether endoreduplication of the DNA occurred as normal. The authors should be more specific on lines 205-6 on how disrupting labryinth architecture can affect placenta function. If the labyrinth villi have a stunted structure, it would prevent the establishment of sufficient surface area for nutrient and gas exchange causing fetal growth restriction.

Reports in the literature that show impeded labyrinth villus structure are typically associated with smaller placentas, and so it is important for the authors to consider why their placentas are bigger in the discussion. The current discussion about the placenta is quite superficial and mechanism is not considered.

4. The introduction should contain information about the placenta-heart axis to provide rationale for the experiments and data in this manuscript. This is very interesting and novel dataset (placentomegaly + fetal growth restriction + VSDs), and should be a ‘selling point’ of the manuscript. I would also recommend altering the title to include the heart data. Perhaps something like: “Nsd2 loss-of-function mutations cause placentomegaly and disrupt fetal heart structure in mice”. More rationale or narrative is required at the start of Line 171 to discuss why the fetal heart is assessed here – potentially linking placenta phenotype with heart phenotype.

Were other less severe heart defects present in the mutant hearts that did not display VSD? Was the placenta phenotype more severe when associated with VSD?

5. The discussion section of this manuscript is superficial and does not sufficiently discuss the potential underlying mechanisms for the cause of placentomegaly in this and other models with an epigenetic regulatory link (Placentomegaly has been identified in several models: 1) Plac1 mutant, which is X-linked, 2) miR-127 mutant, which is located in the Rlt1 imprinted locus (see Ito et al, 2015 Development PMID: 26138447), 3) cloned embryos, which presumably is a process that alters the epigenome including at the Sfmbt2 miRNA cluster, 4) Nsd2 mutants, which is a histone methyltransferase). While I understand that the authors attempt to make this link, it is not outrightly stated (starting on line 208 would be a good place). What might be a good experiment to assess common mechanisms among these models including Nsd2 (e.g., assessing Plac1, miR-127, Sfmbt2 expression in the Nsd2 mutant placentas? Are these genes regulated by H3K36me2 in the placenta?).

The discussion of the Dnmt3b knockout manuscript (Andrews et al, 2023 PMID: 36690623) on lines 225-234 is relevant given that DNA hypomethylation occurs alongside labyrinth defects to cause fetal growth restriction. However, there is proportionately too much detail given about this mutant and this should be condensed and better related to the Nsd2 mutants. Why does a histone methyltransferase and DNA methyltransferase have a similar labyrinth phenotype but not the same placental phenotype? Similarly, the discussion of Radford et al, 2023 (PMID: 36859534) is extensive. Instead, the salient points about the placenta-heart axis should be emphasized along with other articles that discuss this principle.

Other issues:

- the y-axis in all graphs should start at zero as is standard practice.

- Lines 117-8: cite the previous results that you are referring to that show fetal growth restriction at E18.5 in these mutant mouse lines.

- lines 150-2: this sentence is confusing and contradictory. It might be more accurate to state that there was a trend towards thickening of the “junctional zone” in the KO-homs but these measurements were not statistically significant.

- Line 197, line 240: E15.5 to E18.5 is late gestation not mid-gestation

- Line 200: While spongiotrophoblast cells have an endocrine function, the junctional zone that contains spongiotrophoblast cells also has glycogen trophoblast cells that have a metabolic function (store and metabolise glycogen). More information about these cells can be found in the following review (Tunster et al, 2020 Reproduction PMID 32191912).

- Figure 2: it would be helpful to the reader to label directly on the placenta images in figure 2 including each placenta layer in panel A, and the region assessed in panel C.

Reviewer #2: This paper presents evidence that loss of function of NSD2, a marker of DNA methylation and transcriptional activity, results in fetal growth retardation and placental enlargement. The paper is a valuable contribution to the field and has potential for publication. Its strength lies in the careful observation of changes in placental morphology. However, the paper does not address the precise mechanism of methylation dysregulation underlying the observed morphological changes. A more comprehensive investigation and/or discussion of the mechanisms behind these changes would enhance the scientific rigor of the paper.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-24-42707R1Loss-of-function mutation of NSD2 is associated with abnormal placentation accompanied by fetal growth retardation in micePLOS ONE

Dear Dr. Kawai,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Statistical analyses require further clarification as detailed below.

Please submit your revised manuscript by Jun 09 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

David S. Milstone

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

The authors have carefully addressed most of the criticisms of the reviewers and the editor. However, statistical analysis requires further clarification. The underlying issue appears to be that pairwise ratios (that reduce the variation in the control “values” to zero) appear to have been used but are not warranted by the experimental design. However, the method used is not clearly described so some ambiguity remains. The individual experimental and individual control observations/values do not appear to be methodologically paired in any empirically meaningful way. Without this built into the experimental design, paired values cannot be used to determine ratios. Instead, ratios must be determined based on the control values as a group and the variability of the control values (the ratios of individual control values to the mean of all control values) must be included in the statistical analyses. One way to do this is to calculate the ratios of the experimental and the control values to the mean of the control values. The experimental and control ratios can then be used to test null hypotheses in a statistically valid manner.

Description of the statistical analysis does not unambiguously state whether paired or non-paired values were used. Doing so will help clarify which statistical analyses are appropriate

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2:  The author has meticulously addressed the reviewer's comments and has submitted a revised paper that is deemed satisfactory. I have no further comments.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #2: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Loss-of-function mutation of NSD2 is associated with abnormal placentation accompanied by fetal growth retardation in mice

PONE-D-24-42707R2

Dear Dr. Kawai,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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David S. Milstone

Academic Editor

PLOS ONE