Embryo‐uterine interaction coordinates mouse embryogenesis during implantation

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1. Version published to 10.15252/embj.2022113280

   Jul 31, 2023
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   Dec 11, 2022

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   Referee #1

   1. The authors should provide more information when...

   Responses

   • The typical domed appearance of a hydrocephalus-harboring skull is apparent as early as P4, as shown in a new side-by-side comparison of pups at that age (Fig. 1A).
   • Though this is not stated in the MS

   1. Figure 6: Why has only...

   Response: We expanded the comparison

   Minor comments:

   1. The text contains several...

   Response: We added...

   Referee #2

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   Referee #3

   Evidence, reproducibility and clarity

   Summary:

   Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). In this paper the authors develop an engineered uterus-like microenvironment to recapitulate peri-implantation development of the whole mouse embryo ex vivo. This new model (3E-uterus) is used for mechanistic studies of embryo implantation. They hint that integrin-mediated adhesion of the embryo to the uterine wall is required for peri-implantation mouse development. The authors use this model also to study the role of tension for embryo development. They postulate that release of tension from the polar …

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   Referee #3

   Evidence, reproducibility and clarity

   Summary:

   Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). In this paper the authors develop an engineered uterus-like microenvironment to recapitulate peri-implantation development of the whole mouse embryo ex vivo. This new model (3E-uterus) is used for mechanistic studies of embryo implantation. They hint that integrin-mediated adhesion of the embryo to the uterine wall is required for peri-implantation mouse development. The authors use this model also to study the role of tension for embryo development. They postulate that release of tension from the polar side of the embryo upon implantation allows for extra embryonic development. By using mathematical modeling of the implanting embryo as a wetting droplet, the authors link the embryo shape dynamics to the underlying changes in trophoblast adhesion and suggests that the adhesion-mediated tension release facilitates egg cylinder formation. Finally, the authors uncover the role of coordination between trophoblast motility and embryo growth, where trophoblast mobility displaces the Reichart's membrane giving the embryo space to grow. In summary, the authors technically advance the field of developmental biology by providing a model to study peri implantation morphogenesis of the mouse embryo.

   Major comments:

   • Are the key conclusions convincing?

   The key conclusions the authors derive from their experiments are somewhat convincing. Suggested experiments below will strengthen their claims.

   • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

   Claim 1: The 3E-uterus is representative of mouse embryo peri-implantation. To claim this a more extensive validation of the embryos cultured in their 3E uterus both via scRNA seq and IF for pluripotency, visceral and parietal endoderm markers is required. It is also interesting that embryos cultured in the 3E-uterus lose the correct timing of development. Could the authors please comment on this? scRNA seq of the embryos cultured in their system at different timepoint (i.e. Day 1-3) compared to control pre, peri and post implantation embryos could help answer this question.

   Claim 2: The release of tension from the polar side upon implantation allows for extra embryonic development. To quantitatively measure the difference in tension before and after implantation is technically very challenging. However, this paper could benefit of further validations including IF stainings for markers such as E-cadherin, F-actin and Phospho myosin. In addition to this, treatments with Y27 and blebbistatin of the embryo would allow to further study the role of cell tension on embryo implantation. Finally, a laser ablation experiment at the cell junctions of the polar region before and after implantation would help to answer this question but this could be technically challenging due to the curvature nature of embryos.

   Claim 3: Integrin-mediated adhesion between the trophoblast and the uterine matrix is required for in utero-like transition of the blastocyst to the egg cylinder. In Figure 2a the authors show that embryos cultured in 3E-uterus without RGD do not develop and hypothesize this is due to lack of integrin binding. A control experiment using a non-integrin binding peptide is beneficial here.

   Claim 4: The spatial orientation of the embryo plays a key role in mouse peri implantation development. In Figure 5i-j, the authors place embryos in a downward (i) and upward (j) orientation. Could the authors also please comment on whether they believe the orientation, the way the embryo feels the gravity plays a role in implantation? Is the amount of space that the embryo has to grow in the limiting factor on development? Could the authors use 3E-uterus models with different lengths (by using molds with different spacing) to see the role of geometry and space that the embryo has for trophoblast mobility and embryo growth. What would happen if the embryo were very close to the bottom of the hydrogel?

   Claim 5: A mathematical model based on the wetting droplet recapitulates the embryo in their system. Could the authors comment on whether their mathematical model considers proliferation, and would proliferation have an impact on the system's kinetics? What is the role of polar TE proliferation and how does that influence the trophectoderm morphology? If the embryo is geometrically confined, can the authors exclude that this confinement is influencing cell shape?

   • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

   In this technical advance paper, the claims will become more robust with the suggested experiments above. Claims of implantation should be changed to accurately reflect that the 3E-uterus models peri implantation as there is no invasion in the 3D hydrogel matrix. In addition to this, the uterine cells are missing which are required to fully recapitulate the mechanisms of embryo peri-implantation.

   • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

   3- 6 months will allow the authors to address all questions above. Yet, the laser ablation experiment might be difficult to perform due to the curvature of the embryo.

   • Are the data and the methods presented in such a way that they can be reproduced?

   We appreciate the details of the materials and methods section particularly of the imaging.

   • Are the experiments adequately replicated and statistical analysis adequate?

   Yes

   Minor comments:

   • Specific experimental issues that are easily addressable.

   1. Laminin staining in Figure 2A is only done in in vitro embryos but not in in vivo embryos. Could the authors add the missing staining?
   2. It would be beneficial to have both active and normal integrin stainings in E4.5 embryos.
   3. Could the authors provide stainings for mesenchymal markers for E4.5 and D2 3E uterus?
   4. Can the authors comment on Figure Supp. 3D where the timing seems to be flipped?
   5. Why was 600 um chosen for the depth of the 3E-uterus?

   • Are prior studies referenced appropriately?

   How do the findings in this paper relate to the findings in Weberling et al (PMID 33472064), where they show that in vivo, the polar trophectoderm exerts physical force upon the epiblast, causing it to transform from an oval into a cup shape?

   • Are the text and figures clear and accurate?

   Overall the text and figures are clear and accurate. In figure 2E and 2G, the outline covers the staining. Would it be possible to have it without the outline in the supplementary?

   • Do you have suggestions that would help the authors improve the presentation of their data and conclusions? It would be very informative to have for each panel in which a representative image is used for that image to be marked into the quantified data (graphs).

   Overall, the manuscript is very well written and the conclusions are informative and clear.

   Significance

   • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

   The advance presented in this paper is technical. In this methodology paper, the authors use their novel model to investigate the mechanics of embryo peri-implantation and hint at new conceptual findings such as the role of wetting properties of the embryo onto the ECM of the uterus.

   • Place the work in the context of the existing literature (provide references, where appropriate).

   Since embryos become hidden in the womb upon implantation, ex vivo cultures provide an experimental setting to monitor, measure and manipulate embryonic development. Ex vivo culture of peri-implantation (mouse) embryos so far relied on embryonic growth on 2D plastic surfaces (PMID: 4930085, 4562729 and 24529478) or 3D bioreactors (PMID: 33731940). Although important, these assays do not recapitulate the interaction with the uterine cells and ECM (the in vivo scenario). In this study initial steps are taken to recapitulate the interaction of the embryo with the uterine ECM during peri-implantation. Uterine cells are however missing from this new system, which is important for understanding the full mechanism of implantation.

   • State what audience might be interested in and influenced by the reported findings.

   Developmental biologists

   • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

   Bioengineer, bioinformatician and developmental biologists working with embryo models and hydrogels. There is not sufficient expertise to evaluate the mathematical modeling.

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   Dec 11, 2022

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   Referee #2

   Evidence, reproducibility and clarity

   The authors developed a new method of ex vivo culture of mammalian embryos. This engineered uterus recapitulates some features of peri-implantation development of the mouse embryo. The authors show that integrin adhesion to the uterine wall through integrin beta 1 is required for proper peri-implantation. They also demonstrate collective migration of the trophoblast on the synthetic hydrogel surface. The authors interpret their results through the physics of wetting, which allows them to conclude that a release of tension enables shape changes in the embryo. Finally, light sheet imaging allows the authors to visualize the interplay …

   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 authors developed a new method of ex vivo culture of mammalian embryos. This engineered uterus recapitulates some features of peri-implantation development of the mouse embryo. The authors show that integrin adhesion to the uterine wall through integrin beta 1 is required for proper peri-implantation. They also demonstrate collective migration of the trophoblast on the synthetic hydrogel surface. The authors interpret their results through the physics of wetting, which allows them to conclude that a release of tension enables shape changes in the embryo. Finally, light sheet imaging allows the authors to visualize the interplay between growth and collective motion.

   Major

   1. The article will be of potential interest to a broader community than mammalian embryo peri-implantation researchers. This broader community will likely not be familiar with the structure and nomenclature of the embryo and surrounding tissues. The introduction of terms in the second paragraph of the introduction should be paralleled by a comprehensive image in Fig. 1. This image should clarify what is considered apical and basal in this context. Similarly, when the model is introduced a more comprehensive scheme should also be provided.
   2. The failure of 2D hydrogels to support mouse blastocysts through peri-implantation (Supp Fig. 1) is insufficiently described. Some panels in this figure and not mentioned in the main text. This discussion should be expanded, especially considering that 2D approaches has been quite successful. A detailed discussion of the authors' cylinder approach compared to the best 2D systems published should be provided (Govindasamy et al, for example).
   3. Is the hydrogel purely elastic or viscoelastic? The mechanical properties of the hydrogel (viscoelasticity and degradability) should be presented in the main text.
   4. The authors call the finding that integrins are required for peri-implantation "striking", but a role for integrins in this process is are already known (see Sutherland et al, for example). The novelty of the authors findings in this regard should be better presented.
   5. Figures 2ef (in utero) and 2gh (3E-uterus) show rather different results. In uterus, pERM and ZO1 look quite compartmentalized in the outer region. This is not the case in 3E-uterus shown in Figure 2gh. These data do not seem to support an agreement between in utero and 3E-uterus as mentioned in the text.
   6. The authors claim that mTE cells lose cell polarity upon adhesion to the uterine matrix and acquire mesenchymal properties. This claim should be clarified. Cells protrude and become migratory but invasion seems to be collective, suggestive that epithelial features such as cadherin adhesion remain. Are these cells mesenchymal or are they simply epithelial cells with motile capacity (as in wound healing, for example)? How do mesenchymal vs epithelial features compare between in utero and 3E-uterus?
   7. If I understand correctly, the model assumes that tension of the droplet-medium interface and is the same in the upper and lower sides of the embryo. However, the mechanical and geometrical properties of cells in both sides are quite different. Is the assumption of same tension justified? Can these tensions be measured or inferred to test this assumption?
   8. Along similar lines, attributing a surface tension to a system that is thick (ie several cell layers) and that undergoes apical constriction (ie a bending modulus) is an oversimplification that should be justified. Cells in the pTE change (potentially) their apical, basal and lateral tension during apical constriction. How do these three components relate to what the model simply refers as tension? Additionally, how does the presence of a bending moment alter the wetting picture?
   9. The physics of wetting were recently generalized to include additional terms attributed to active components (main associated with polarity, see works by Alert and Casademunt). These active components are not explicitly taken into account in the authors' model. Are they not needed? A brief discussion of this aspect should be provided.
   10. In utero, the cavity in which the embryo is implanted is created during implantation. In this situation, the analogy with wetting seems harder to establish because the embryo spreads as it forms the cavity. How does this alter the authors interpretation?
   11. The first paragraph of the supplementary note refers to Fig. 4D. This reference here does not seem correct.

   Referees cross-commenting

   The three of us coincide in appreciating the novelty and potential impact of the new method.

   There is an agreement between all 3 referees to request additional evidence of how well the 3E-uterus captures the in vivo phenomenon. I believe the suggestions provided by my two colleagues in this regard are on point and seem feasible for the applicants within a 6 month period.

   I also agree that tension measurements with laser ablation (or other inference techniques) would provide stronger support to the model.

   Significance

   This article provides an important technical advance to study peri-implantation of the mammalian embryo beyond current methods based on 2D substrates. This work will be of interest to the community of early mammalian embryogenesis but also to the broader field of engineering multicellular systems.

   As list above, main limitations concern 1) The extent to which their method properly captures peri-implantation, 2) The novelty of some of the authors observations, 3) The soundness of the theoretical model.

   My expertise is in experimental biophysics of multicellular systems.

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   Review Commons

   Dec 11, 2022

   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 #1

   Evidence, reproducibility and clarity

   Summary:

   To recapitulate mouse peri-implantation development ex vivo, the authors engineered a uterus-like microenvironment by fabrication of topographically patterned hydrogels and identified the roles of the physical interaction between embryo and hydrogels for egg cylinder formation. Notably, integrin-mediated adhesion between trophoblast and matrix facilitates egg-cylinder shape. Moreover, Live-imaging with light-sheet microscopy led them to propose the hypothesis that the interaction between embryos and hydrogels appeared to be described by a droplet wetting process.

   Major comments:

   Although the authors claim that the …

   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 #1

   Evidence, reproducibility and clarity

   Summary:

   To recapitulate mouse peri-implantation development ex vivo, the authors engineered a uterus-like microenvironment by fabrication of topographically patterned hydrogels and identified the roles of the physical interaction between embryo and hydrogels for egg cylinder formation. Notably, integrin-mediated adhesion between trophoblast and matrix facilitates egg-cylinder shape. Moreover, Live-imaging with light-sheet microscopy led them to propose the hypothesis that the interaction between embryos and hydrogels appeared to be described by a droplet wetting process.

   Major comments:

   Although the authors claim that the interaction between the uterus and embryo is crucial for egg-cylinder formation, they did not utilize uterus-derived cells nor analyze these. They just observed how blastocysts grow autonomously into the egg-cylinder shape in the hydrogel which has solely physical properties of the uterus but not biochemical features except for the RGD peptide-mediated cell adhesion process. Thus, it is still uncertain if similar mechanisms contribute to egg-cylinder formation in utero. To fulfill the gap between ex vivo morphogenesis and in utero, the authors would be expected to analyze the interaction between trophoblast and uterus in utero if uterine mechanisms can follow the integrin-mediated adhesion and a droplet wetting process. For example, whether integrin can contribute to egg-cylinder formation in utero can be proved by analyzing knock-out phenotypes of integrin-related genes. It will take around six months to conduct such suggested experiments. Otherwise, the authors should modify their statement "the interaction between embryo and uterine" into "the interaction between embryos and uterine-like hydrogels" throughout the manuscript.

   Specific point:

   Supplemental figure 1h, page 4 lines 20-23: The authors claim that 1.5-2 % PEG generated the shear modulus at 100-300 Pa, which is in the stiffness range of the E5.5 mouse decidua (Govindasamy et al., 2021). In Govindasamy's paper, elasticity measurements were performed in Petri dishes using an MFP-3D Classic AFM (Asylum Research, Wiesbaden, Germany) and cantilevers with a force constant of 0.08 with spherical tips (2 mm; NanoWorld, Neuchatel, Switzerland). In the present paper, the shear modulus (G′) of hydrogels was determined by performing small-strain oscillatory shear measurements on a Bohlin CVO 120 rheometer with plate-plate geometry. Therefore, it is not appropriate that Govindasamy's modulus is compared to the authors' modulus directly. For a direct comparison of the two modulus values, the authors can measure the stiffness of PEG with AFM or the shear modulus of the E5.5 mouse decidua by their rheometer.

   Significance

   Strong points:

   As described in summary, the authors have newly identified that integrin-mediated adhesion between trophoblast and matrix facilitates egg-cylinder shape and the interaction between embryos and hydrogels is followed by a droplet-wetting process. These findings are considered to be novel by their excellent ex vivo imaging method.

   Limitations:

   It remains unaddressed that ex vivo mechanisms they have identified contribute to the egg-cylinder formation in utero.

   Audience:

   The results demonstrated here by the authors are fascinating in terms of general interest after the above concerns are appropriately addressed.

   Since I am an experimental biologist, I don't have sufficient expertise to assess if the physical droplet model can recapitulate the interaction of the embryo and pre-patterned hydrogel for egg cylinder formation in detail.

   Read the original source
6. 

   Version published to 10.1101/2022.06.13.495767 on bioRxiv

   Jun 16, 2022