Asymmetry in the frequency and position of mitosis in the mouse embryo epiblast at gastrulation
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We are grateful for the reviewers’ appreciation and comments. We have tried to address all concerns, and believe that those changes have greatly ameliorated the precision and presentation of our findings. All of our responses are in green in this document, and so are the changes in the manuscript and figure legends.
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
In "An asymmetry in the frequency and position of mitosis in the epiblast precedes gastrulation and suggests a role for mitotic rounding in cell delamination during primitive streak epithelial-mesenchymal transition", Mathiah, Despin-Guitard and colleagues study divisions during …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
We are grateful for the reviewers’ appreciation and comments. We have tried to address all concerns, and believe that those changes have greatly ameliorated the precision and presentation of our findings. All of our responses are in green in this document, and so are the changes in the manuscript and figure legends.
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
In "An asymmetry in the frequency and position of mitosis in the epiblast precedes gastrulation and suggests a role for mitotic rounding in cell delamination during primitive streak epithelial-mesenchymal transition", Mathiah, Despin-Guitard and colleagues study divisions during mouse gastrulation. They perform ex vivo culture, live imaging and immunostaining to observe the frequency and position of mitosis within the embryo as well as the destiny of daughter cells after their divisions. The find that divisions on the posterior side of the embryo tend to be more basally located and could contribute to cell delamination into the mesodermal layer. Authors also affect antero-posterior signaling by genetically preventing the migration of the anterior visceral endoderm, which leads to mitosis away from the apical side of the epithelium on all lateral parts of the embryo.
*This study tackles a key developmental process which is poorly understood in mammals due to its concomitance with the implantation phase. Therefore, any carefully-made description of this process has the capacity to be eye-opening. This is potentially the case for this report, which provides nice images that most likely required skills and important efforts to obtain. The authors have written a clear manuscript with an interesting narrative. However, the quantifications are very poorly described, which makes it impossible for anyone to reproduce these results. I describe below a number of suggestions to clarify the quantifications, which is in my opinion a prerequisite to consider the conclusions from the authors. *
Fig1*: Authors describe differences in the formation of rosettes between the anterior and posterior sides of the embryo. The microscopy images and movies provided are overlaid with drawings from the authors but without this visual help, I, and I assume other readers, see more rosettes than highlighted and fail to see some of the rosettes that are marked. To avoid this subjectivity, a clear methodology is required. In the methods, the authors state: "For quantification, rosettes were manually annotated and counted on Z sections located 5 μm from the basal side of the epiblast." And that is all. What defines a rosette? How many cells need to share a vertex to be considered as part of a rosette? How long do they need to persist not to be considered as occurring by chance? What about cells part of multiple rosettes? Does the rosette organization need to be apical, basal or all the way? Having those clearly defined criteria would be essential for anyone else to reproduce this quantification and would also offer a much more comprehensive description of the phenomenon and allow for more powerful conclusions. *
A rosette is defined as a multicellular transient structure composed of at least 5 cells converging to a central vertex. Practically, a region of interest where cell contours are in focus is determined on the Z-section located 5 mm from the epiblast basal side, which is easily identified as the epithelial architecture changes radically when one enters the visceral endoderm. Only rosettes that are visible throughout the epiblast layer, from the basal to the apical side, are counted. To ensure this, manual segmentation of all cells (for all Z plan acquired, from the basal plane to the apical side) contributing to a rosette was performed for lightsheet imaging. This is illustrated in Video 2. For confocal imaging, segmentation was annotated only at the basal plane, but visual verification that the rosette structure is persistent throughout the layer was performed. One cell could be part of several rosettes, and rosette events were counted even when visible only on one timeframe, but this was consistent for all embryo sides. Due to the time resolution of confocal imaging, rosettes could not be followed overtime. However, the time resolution of lightsheet imaging allowed observing rosettes lifespan and resolution. The protocol for image analysis has been better detailed in the results (lines 162-163) and the methods section of the revised version of the manuscript (lines 452-467, copied hereunder).
"Rosettes: For lightsheet imaging, embryos were dissected at E5.75. Images were acquired for 10 to 12 hours. Quantification focused on the first 20 to 30 frames (around 3 hours) to capture pregastrulation events and reduce the risk of bias from imaging. The rest of the frames showed that the embryo continued growing for several hours. Z-stacks from 4 sides were fused using Zeiss plugin for lightsheet Imaging. Images were then processed using Arivis Vision4D v2.12.3 (Arivis, Germany). Embryo contours were segmented manually on each Z-slice and time point, in order to adjust for embryo rotation manually if necessary. For each side of the embryo, Z stack was cropped to an average of 30 Z slices, from the basal side (5 microns from VE layer, which can be morphologically distinguished due to cell shape and membrane Tomato distribution) to the cavity, marking the apical side. Rosettes were identified and counted on Z sections located 5 µm from the basal side of the epiblast. Practically, vertices were systematically scanned to find those in which 5 cells or more met. Cells contributing to a rosette were then manually segmented on each Z-slice and time point by highlighting cellular membranes using Wacom’s Cintiq 13HD, to create a 3D reconstruction. For confocal imaging, rosettes were identified using the same method, and counted on Z sections located 5 µm from the basal side of the epiblast after visual verification that it was present throughout the Z-stack. For both techniques, presence of associated apical rounding was assessed for each vertex. Cells could contribute to several rosettes."
*In addition, the data are given as "rosette/frame" and as "rosette/mm2". What is the point of giving both data, which are essentially the same? The frame is irrelevant__. __It would be more interesting to know how many cells there are in this area, as cell packing could be a determinant of rosette formation. "Rosette/mm2/min" is very confusing. It should state "rosette.mm-2.min-1" or "rosette/mm2.min". *
Following this comment, we indeed chose to get rid of the data expressed as “rosette/frame”. Cells were counted in the area of the epiblast in focus to present data as number of rosettes normalized by the number of cells in the region of interest for both lightsheet and confocal microscopy data (described in results section lines 140 and 164). These measurements led to a similar conclusion, confirming that rosettes are more frequent in posterior epiblast. Difference in cell packing was indeed essential to rule out. We estimated cell packing as the ratio of cell number to surface area, and found it to be similar in posterior, anterior, and lateral sides of embryos at a given stage, which indicates that cell packing is not a determinant for difference in rosette frequency in this context. We discussed packing in the Results section (lines 169-173, copied hereunder).
"The cell number per surface area was similar on all sides, which indicates that the higher number of rosettes was not due to increased cell packing. Rosettes have also been identified in the chick PS (Wagstaff, Bellett, Mogensen, & Münsterberg, 2008), where they were proposed to facilitate ingression during gastrulation."
We modified the legend to use "rosette/mm2.min”.
*On a conclusive note, I fail to understand how relevant the formation of rosettes would be. The authors should clarify this point. *
Epithelial rosettes have been observed as common intermediates in numerous morphogenesis events. In particular cases, such as Drosophila germ band extension, or zebrafish lateral line development, the mechanisms of formation (planar cell polarity (PCP) and apical constriction, respectively) and resolution have been very well described. In the mouse embryo, anterior visceral endoderm (AVE) migration has been linked to PCP signaling-dependent rosette formation (Trichas 2012). In primitive streak (PS) formation, rosettes with actin-rich centers were described in the chick PS and found to be Nodal dependent (Wagstaff 2008 and Yaganawa 2011). Their mode of formation or resolution is currently unknown. Our observations confirm the findings in chick and highlight the presence of rosettes at an earlier stage, before PS can be identified. Interestingly, rosettes are enriched on the posterior side at the same time when Nodal signaling becomes asymmetric, leading to posterior restriction of basal membrane perforations (Kyprianou 2020). To progress towards understanding rosettes’ significance in the mouse gastrulation context, it would be interesting to study whether the distribution of rosettes is homogenous before anterior-posterior axis specification. Additionally, it would be important to assess whether random epiblast cells delaminate before PS formation, as observed in chick (Voiculescu 2014). We could not attempt those experiments so far, as we perform most experiments by two-photon microscopy, by which only one embryo side can be recorded at a time, and have no way to distinguish embryo orientation before AVE migration. A better understanding of rosette mode of formation and resolution, including the role of Nodal, would also be necessary to assess the importance of our observations. The technical evolution in mouse embryo imaging will probably permit solving those questions in the near future, through prolonged imaging with tracking of every cell fate (McDole 2018). We have tried to improve the discussion (lines 314-326, copied hereunder), and acknowledge the limitations of our findings to a description of a phenomenon without proven significance at this stage.
"However, since we observed a marked imbalance in rosette frequency as soon as the anterior-posterior axis was specified, it is possible that rosettes reflect increased epithelium fluidity in posterior epiblast, which is exposed to a distinct mechanical context, at the very beginning of PS morphogenesis. Indeed, a posterior shift in the distribution of basement membrane perforations was identified just after AVE migration, due to an asymmetry in Nodal signaling dependent metalloproteinase activity (Kyprianou et al., 2020). To progress towards understanding rosette formation significance in this context, it would be interesting to study whether the distribution of rosettes is homogenous before anterior-posterior axis specification, and to assess whether random epiblast cells delaminate before PS formation, as observed in chick (Voiculescu, Bodenstein, Lau, & Stern, 2014). As Nodal plays a major role in PS initiation, the presence and distribution of rosettes should be studied in models in which Nodal signaling can be tuned (Kumar, Lualdi, Lewandoski, & Kuehn, 2008)."
Fig2:* I have essentially the same issue for bottle cells and delamination counting as for rosettes. In this case, there is nothing in the methods section. *
We have added a paragraph to describe the mosaic analysis in the Methods section (lines 472-488):
"Mosaic: Embryos were recorded in a lateral position. As the proportion of GFP positive cells varied between mosaic embryos, normalisation was performed by dividing by the number of green cells in a given embryo. Anterior and posterior halves were defined by drawing a line perpendicular to the embryonic/extraembryonic boundary and passing through the distal tip. Bottle-shaped cells were identified as having a thin attachment on the apical surface (less than a third of the larger section), and the majority of the cell body located in the basal side. Quantification was performed both on the 3D rendering, and through navigating through the Z-stack. The same criteria where used on all sides of the embryo, and quantification was verified by two independent investigators. Delamination was defined as retraction of the apical process, and displacement of the cell body in the mesoderm layer, which could be identified because of the ubiquitous membrane Tomato labelling. Cell division was characterized by cell rounding followed by the appearance of daughter cells. Cell dispersion after mitosis was defined as absence of basolateral contact between daughter cells, which implies presence of at least one epiblast cell (more often 2 or 3) between daughter cells. Mitosis was considered “non-apical” when happening at least 10 µm away from the apical pole, hence not in the first pseudo-layer of nuclei lining the apical pole."
*What defines a cell as bottle shape and not bottle shape (apical vs basal width for example)? *
Bottle-shaped cells were visually identified as having a thin attachment on the apical surface (less than a third of the larger section), and the majority of the cell body located in the basal side. Quantification was performed both on the 3D rendering, and by navigating through the Z-stack. Due to the large variation in shape, no systematic measurement was performed. However, the same criteria were used on all sides of the embryo, and quantification was verified by two independent investigators. As proposed by Reviewer 2, those criteria would include scutoids with smaller apical surface, which explains why we observe bottle-shaped cells both on the anterior and posterior sides. In addition to Methods, we included a better description of the methodology in the Results (lines 196-200).
"The quantification of bottle-shaped cells was performed in 3D and through Z-stack navigation and included all cells with an apical section smaller than a third of the basal section. Some cells had a round basal cell body and a thin apical extension while others resembled the recently described scutoids performing apico-basal transitions (Gómez-Gálvez et al., 2018)."
*Where does a cell need to be to be counted as delaminated (a distance needs to be stated, absolute (better) or relative)? *
Delamination is defined as retraction of the apical process, and displacement of the cell body in the mesoderm layer. Using the ubiquitous membrane tomato marker we could easily distinguish the epiblast, mesoderm and visceral endoderm layers, notably through cell packing, morphology and arrangement. This was described in Results (lines 200-204).
"Asymmetrical cells were present on both sides, but more frequent on the posterior side, and cell delamination (retraction of the apical process and cell body shift in the mesoderm layer) only took place on the posterior side. Cells maintained an apical attachment until their basally located cell body had begun crossing the PS/mesoderm border, and only fully detached after delamination."
*What defines sister cells as dispersing after division? How far apart do they have to be? After how much time? From the movies provided, the acquisition time seems to short to assess cell dispersal. *
Cell dispersion after mitosis was defined as absence of basolateral contact between daughter cells as they extend towards the basal side, which implies intercalation of at least one epiblast cell (more often 2 or 3) between daughter cells. After cytokinesis was completed, extension and separation of daughter cells was visible in the next time point (after 25 min). The time resolution was thus sufficient to note that daughter cells were not adjacent, which is consistent with other studies (Abe 2018).
We have modified the Methods (copied above) and the Results section of the revised version of the manuscript (lines 213-217).
"Upon elongation of daughter cells to reach the basal pole of the epiblast, the majority displayed no basolateral connection between each other and were instead separated by intercalating epiblast cells, which would be expected to result in daughter cells dispersion over time, as described in (Abe, Kutsuna, Kiyonari, Furuta, & Fujimori, 2018)."
Fig3*: Mitotic index calculation is described in the figure legend but not in the methods section. It should also be in the methods section and made explicit that the number of mitotic cells is normalized to green cells only, not the entire cell population. The mitotic index seems higher in this population than in the entire embryo as seen in Fig4. *
The mitotic index (MI) was indeed calculated differently so numbers cannot be directly compared. MI identified for anterior and posterior epiblast is not statistically different from the ones found in Figure 4 for E7 embryos. In mosaic embryos, we do not have a way to delimitate the PS. In Figure 4, measurements of MI in the PS (delimitated by the area where the basement membrane is degraded) include cells that are destined to delaminate as wells as those that won't. In the mosaic embryos, MI is measured in cells that delaminate only, and is indeed higher. This represents a small population, which likely explains why it does not reach statistical significance and manifests as a trend.
We have fixed the Methods (see above) and Results (lines 222-228) sections.
"For systematic quantification, epiblast regions were defined as anterior or posterior by tracing a line passing by the distal pole and perpendicular to the embryonic/extraembryonic border, and GFP positive cells undergoing rounding were followed overtime (Fig. 3a-c). Although the frequency of cell division (normalized to the total number of GFP positive cells) was similar in anterior and posterior epiblast, there was a trend towards a higher division rate specifically in cells undergoing delamination to become mesoderm (Fig. 3d)."
What defines an exiting cell*? *
An exiting cell is characterized by morphological remodelling, apical retraction, as well as the position of the cell body across the mesoderm/epiblast border visualized by the precise membrane Tomato labelling. It is now described in Methods and in Results (lines 201-204: " cell delamination (retraction of the apical process and cell body shift in the mesoderm layer) only took place on the posterior side. Cells maintained an apical attachment until their basally located cell body had begun crossing the PS/mesoderm border, and only fully detached after delamination".
Regarding the non-apical rounding, why not calling it basal rounding? How far from the apical side does a cell need to be counted as non-apical?
The reason for that denomination is that these so-called “non-apical mitoses” are not strictly basal either. Indeed, mitosis is considered “non-apical” when happening at least 10µm away from the apical pole, meaning that these mitoses do not occur within the first pseudo-layer of nuclei lining the apical pole. This is described in Methods.
*In the panel h, with the posterior division outcome, is that for all divisions or only for non-apical divisions? *
The panel (Fig. 3g, there was an error in figure labelling in the previous version) has been modified to better precise cell outcomes. It represents all posterior divisions, and quantifies the outcome according to the position of mitosis along the apical-basal axis of the cell. See Results, line 230-232: "Non-apical mitosis in the posterior epiblast was preferentially associated with EMT, as it resulted in formation of one or two mesoderm cells (Fig. 3g)."
Do basal divisions give rise to more epi?
No, non-apical divisions mainly give rise to mesoderm cells. Indeed, approximatively 66% of basal divisions give rise to two mesoderm cells, and 33% to an epiblast and a mesenchymal cell (Figure 3g). We never observed a non-apical division resulting in two epiblast cells.
Is epi or meso fate only determined by location in a different layer or are fate markers used?
Epiblast or mesenchymal fate was determined by both morphological and localization criteria. Epiblast cells have an apical and a basal pole. Mesoderm cells have no apical process, and display initiation of front-rear polarity often defined by the presence of nascent migration appendix. As stated before, membrane Tomato labelling allows exact distinction of germ layers.
What happens to the non-apical mitosis on the anterior side?
On the anterior side, the very few anterior non-apical mitoses only give epiblast cells (not shown).
Fig4*: Methods state "For Phospho-histone H3 quantifications, sections were chosen at least 10 μm apart to ensure that each cell was only counted once, and counting was performed using the Icy software" and legend states "The PS region is defined by the area where the basal membrane (yellow) is degraded, and the posterior region quantification excludes counts from the PS region". *
*What about cells at the boundary between PS and non-PS regions? This needs to be extended and brought together in the methods section. **Also, the tissue architecture in the PS is not as well defined as in the rest of the tissue. *
A cell was counted as being part of the PS region if at least 50% of its cell body (visual measuring) was within the area where the basal membrane is non-ambiguously degraded, and if the cell retained its attachment to the apical pole (cell contours were determined by F-actin detection using Phalloidin). The Methods section has been completed in the revised version of the manuscript (lines 490-501).
"Phospho-histone H3: For Phh3 quantifications, sections were chosen at least 10 mm apart to ensure that each cell was only counted once, and counting was performed using the Icy software (http://icy.bioimageanalysis.org). For sagittal sections, anterior and posterior regions were defined by drawing a line perpendicular to the embryonic/extraembryonic boundary and passing through the distal tip. For transverse sections, anterior-posterior boundary was placed at mid-distance between the anterior and posterior poles. The PS region was defined by the area where the basement membrane was degraded, and the posterior region quantification excluded counts from the PS region. A cell was counted as being part of the PS region if at least 50% of its cell body was within the area where the basement membrane was non-ambiguously degraded, and if the cell retained its attachment to the apical pole (cell contours were defined by F-actin detection using Phalloidin)."
Is the epithelial polarity clear enough to be determined without AB marker in the PS?
We considered that a cell retained its AB polarity if the cell extended to both apical and basal pole. Even if the pseudostratified epithelium architecture is complex, most cell contours could be delimited when navigating through the Z-stack.
Finally, the number of cells counted is missing. This has been fixed in the Figure legend.
Supp Fig5: based on available images of the Rac1KO embryo, I am not sure that epithelial architecture is established well enough to assess the location of mitosis along the apico-basal axis.
Indeed, the architecture of the Rac1 KO mutants is vastly altered. As a consequence, only a small number of Rac1 mutants in which we could delimitate the germ layers were analysed, and only the cells we could unambiguously locate were considered. The Rac1 VE-deleted phenotype, on the other hand, was not severe enough as there is only a partial AVE migration defect in most mutants (Migeotte et al., 2010). This is why we confirmed the data on AVE migration defective embryos by using the RhoA VE-deleted mutant, which has a strong AVE migration defect but retains good tissue architecture. We tried to increase figure clarity by annotating the embryo cavity as well as the embryonic/extraembryonic boundary. We also submit a less compressed version of the figures, which we hope will facilitate image analysis.
Reviewer #1 (Significance (Required)):
Although I am not as familiar with mouse gastrulation as I would like to be, I am familiar with gastrulation, live imaging and analysis. At this point, I find it difficult to discuss the conclusions of the study since the methodology is so unclear. Nevertheless, any carefully-made description of mammalian gastrulation has the capacity to be eye-opening. This is potentially the case for this report, which provides nice images that most likely required skills and important efforts to obtain.
We hope the changes we made help better understanding the methodology, and thank Reviewer 1 for positive comments and the help in identifying the points we had failed to properly describe.
*Reviewer #2 (Evidence, reproducibility and clarity (Required)): *
This manuscript, from Mathiah and colleagues, describes an in-depth analysis of differences in cell organization and division within the epiblast of the very early mouse embryo, and in particular, with the onset of gastrulation. Their data indicate a difference in the organization of cells between the anterior/lateral and posterior regions of the epiblast even before gastrulation has commenced, as well as differences in the location of mitoses relative to the apical and basal ends of the cells. The data provide new insight into the early regionalization of the epiblast. However, the authors should include reference to, and discussion of, the paper by Michael Snow on growth and regionalization of the epiblast (Snow MHL (1977) Gastrulation in the mouse: Growth and regionalization of the epiblast. J. Emb. Exp. Morph. 42: 293-303), where he did a much more fine-grained analysis of mitotic index across the entire epiblast, defining a proliferative zone in the anterior part of the primitive streak where the mitotic index was higher between E6.5 and E7.5. He also describes non-apical mitoses specifically in the primitive streak region as compared to all other regions of the epiblast. The results of the present study dovetail nicely with the results presented by Snow.
This was indeed a major oversight, and we apologize for it. The work of Snow identifies very nicely a proliferative zone in the anterior part of the PS. We did not comment on that as our study focuses on posterior PS. We included the reference in the revised version of the manuscript, and pointed the fact that he first described non-apical mitosis in the PS (lines 233-236).
"Remarkably, this concurs with the observation by Snow (Snow, 1977) that in the PS of E6.5 and E7 embryos, mitosis could be found at all levels of the tissue, including adjacent to the endoderm, while it was located at the apical surface of the pseudostratified tissue everywhere else."
Overall, this is a very nice study, but some revisions would help with clarity at certain points. The data on rosette formation are interesting, but it is not clear what an increase in rosettes in the posterior region means. The authors contend (lines 169-170) that this represents a dynamic epithelium primed for EMT, but it is not clear how rosettes facilitate or promote EMT, and especially why that would be seen at E5.75 before EMT has begun. An alternative interpretation might be that the shape of the epithelium may be changing and the packing of the epithelial cells has to change to accommodate this. We do know that the overall shape of the embryo changes from elongate medial-lateral to elongate anterior-posterior just as EMT is initiated (Perea-Gomez et al., (2004) Current Biology 14: 197-207) and it may be that changes in cell packing are required to accommodate this. The authors may want to consider whether the rosettes that they observed represent scutoids (Gomez-Galvez et al., (2018) Scutoids are a geometrical solution to three-dimensional packing of epithelia. Nat. Commun. 9:2960). An analysis of the 3-dimensional organization of the cells within the rosettes (i.e. at all Z levels) may shed some light on this.
Following on the comments by Reviewer 1 and 2, we quantified cell packing, and found it to be identical on all sides at a given stage. We have added a better description of rosette quantification (lines 169-172 and lines 452-470), a video showing 3D reconstruction of cells in a rosette (Video 2), and an extended discussion (lines 314-326) in the revised version of the manuscript. Some cells within the epiblast are indeed likely to be shaped as scutoids, some with an apical-basal asymmetry (lines 196-202). The reference was added to the manuscript (line 200).
Figure 2b,c and Figure 3a, a', b, c -* Addition of dotted lines to indicate the apical and basal ends of the epiblast would be helpful in orienting the reader*.
We have added lines to indicate apical and basal ends of the epiblast.
Figure 2c'* - what these graphs represent exactly is somewhat vague, and the figure legend is also very vague. In particular, the third graph on cell dispersion is not clear. Does this mean that the daughter cells are separated from one another following division? Or that they are in different compartments (epiblast/mesoderm) after division? A better description should be included in the figure legend. *
Following on the comments by Reviewer 1 and 2, we have added a better description of cell dispersion in the Results (lines 213-217), Methods (lines 483-486) and figure legend.
Figure 3g* would appear to show the proportion of the total number of posterior divisions that give rise to particular combinations of daughter cells (epi/epi, epi/meso, meso/meso). However, the discussion of this graph in the text (lines 215-219) suggests that it demonstrates that non-apical mitoses always result in meso/meso and epi/meso daughter cells, which it does not. That analysis would be very interesting to add to Figure 3, with the daughter cell types broken down into those coming from apical mitoses and those coming from basal mitoses.*
The analysis was broken down as suggested, and has been added to the revised version of the manuscript (line 352, Figure 3g).
In Figure 4, it is not clear how anterior and posterior are defined, and what criteria were used to distinguish posterior from primitive streak. This is nicely demonstrated in Supplementary Figure 3 - maybe panels A and B could be included in Figure 4 to improve the clarity of the analysis.
We have better described the quantification methodology in the Methods section (lines 490-501), moved panel a from Supplementary Figure 3 to Figure 4a as suggested, and added an explanatory drawing (Figure 4b) to the revised version of the manuscript.
The data on mitotic index in Figures 2, 4, and 5 do not appear to be consistent. The mitotic index for E7.25 in Figure 2e is similar between anterior and posterior, even though the posterior includes the primitive streak, while the mitotic index presented for the three stages in Figure 4b would imply that the mitotic index for the entire posterior region should be higher than the anterior at all three stages. Similarly, in Figure 5a' and b', the mitotic index in anterior and posterior regions of E5.75 and E6.25 embryos are not significantly different despite the primitive streak being included in the posterior count, while the data presented in Figure 4 would imply that the entire posterior region including primitive streak should be much higher than the anterior. The authors should clarify this in the Results.
In Figure 2 and 3, the mitotic index (MI) is calculated as number of cell division among GFP+ cells divided by the total number of GFP+ cells, while in Figure 4 and 5 it is quantified as Phospho-histone H3+ cells per total number of cells (DAPI). We have clarified this in the revised graphs and legends of the novel version of the manuscript. Those numbers cannot be directly compared. Nonetheless, we found no statistical difference between the MI shown in Figure 3d, and the MI shown in Figure 4c third row (E7). In sagittal view, the PS area cannot be delimited, so we compared anterior and posterior regions, with the PS included in the posterior region, and saw no difference in MI. In transverse section, there was no MI difference when comparing anterior and posterior embryo halves. However, when we refined the analysis by defining the PS as the area where the basal membrane was degraded, a higher MI emerged specifically in the PS compared to anterior and posterior (not including PS) regions. This difference was thus lost by dilution when the PS area was included in the posterior region. We have also stated this distinction more clearly in the revised version of the manuscript (lines 269-271).
The data on non-apical mitoses in the RhoA-VE deleted (Figure 6) and Rac1ko embryos (Supplemental figure5) are not particularly compelling. It is hard to see the basal mitoses in the new AVE-opposed regions in the mutant embryos in the images presented. Perhaps the graphs in these two figures could have the AVE-opposed data broken down into two groups - the region that is posterior and the region that is anterior but not adjacent to AVE. Better images would improve the clarity of these data as well.
As explained in response to Reviewer 1, we have attempted to clarify the anatomy through annotation, and provide less compressed images. We agree that the embryos are altered. Nonetheless, especially in RhoA-VE deleted, the germ layers could be distinguished and non-apical mitosis identified through combining 3D analysis and navigation through the Z-stack. We honestly admit those are the best images we could get, and we believe that they allow to make the point that non-apical mitosis are only found in the area further away from the AVE.
Reviewer #2 (Significance (Required)):
The data on differential proliferation and apical vs. basal mitoses are complementary to data already published, but the present study updates the existing data by the addition of live imaging and 3-dimensional reconstruction of cell shapes, providing a more complete insight into the process. The observation that rosettes are detectable at the basal ends of the epiblast, and more so in the posterior, is novel, but the significance for embryonic development is not well rationalized.
*These data are of interest to those investigating the mechanisms of early morphogenesis, as well as those interested in the cellular correlates of molecular regionalization that results from the well-described signaling pathways regulating axis specification. *
My background is in early mouse embryo morphogenesis, therefore I feel that I have sufficient expertise to evaluate the data presented.
We thank Reviewer 2 for positive comments, and are grateful for the constructive criticism and important references.
**Referees Cross Commenting**
*I agree with Reviewer 1 on the lack of detail about the methods - my comments stemmed from the same confusion about how measurements were made, but Reviewer 1 more articulately addressed the key points. I agree with Reviewer 3 on the quality of the videos. It is very difficult to see how they could follow cells with a 20 minute interval. *
I would like to address the comment by Reviewer 3 on the use of agarose in the imaging experiments. The methods section states that agarose was used to make the culture "chambers" used for light-sheet imaging, which was not the major approach used for imaging in this study. Only the data in Figure 1A came from those experiments, and it was validated by confocal data in 1B,C where the embryos were cultured in Ibidi chambers with culture medium and no agarose present. So I don't think agarose effects on embryo development are a major worry. Also, this same approach was used by Ryan Udan in Mary Dickinson's lab to visualize yolk sac vasculogenesis, and it did not appear to have a deleterious effect on development in that case, although the embryos imaged here were much earlier and are definitely differentially sensitive to culture conditions from those cultured at E8.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
This is an essentially descriptive study, looking at primitive streak formation and cell ingression from the epiblast in the mouse from about E5.75 to E7.5 or so using time-lapse microscopy (light sheet and confocal) of cultured embryos. The study also takes advantage of some genetically encoded reporters, some of them inducible by tamoxifen, which allow following cells, closer examination of their shapes, and in some cases unambiguous orientation of the embryos based on expression of the reporter. Overall the study is well designed.
I have two very major concerns about this paper - first, the culture system used in most experiments uses agarose, which has been found in several labs to affect normal cell movements and other cell behaviours. It is essential to determine that embryos cultured in these conditions develop normally for much longer than the period of imaging to ensure that the findings are relevant to normal development rather than an artefact. This is particularly important because mouse embryos develop rather poorly at peri-implantation stages with any culture method, and this one could make matters even worse.
Embryos were mounted into an agarose cylinder in which a tunnel had been created with a 150 microns wide copper wire. Embryos were mounted vertically, with the cone oriented on the bottom, to avoid restriction of growth at distal tip of the embryo. As embryos had a smaller diameter than the tunnel, they could comfortably grow without being restricted (Methods, lines 413-414). Although embryos could not been recovered after the long imaging period (12h), embryos similarly mounted in the agarose cylinder but not imaged were kept in culture, and showed normal growth compared to a free-floating embryo (Methods, lines 431-433). In addition, we focused on the first hours of imaging to reduce the risk of phototoxicity-induced anomalies (Methods, lines 453-455). Moreover, although we identified the asymmetry in rosette abundance through lightsheet imaging, we confirmed the finding through confocal imaging of free-floating embryos, and found similar results (Results lines 153-167, Figure 1b and c, Video 3).
While it has been reported that agarose can affect the development of chick embryos in culture, agarose has been a widely used culture matrix for live imaging particularly for lightsheet imaging in other organisms including drosophila, zebrafish, and mouse. We thank Reviewer 2 (in cross-comments) for highlighting that in Udan et al., (2014), a report from Mary Dickinson’s lab, embryos are cultured in agarose “chambers” for lightsheet. Although some of the experiments in Udan et al., (2014) are performed at E8, this paper also focuses on pre-gastrulation mouse embryos as they culture E6.5 embryos for 24 hours, image from 5 view angles, analyze 572 z-slices representing half of the embryo (Fig5 and Fig6 Udan et al., 2014) and show no adverse effects.
The second concern is that for a paper that is almost entirely about time-lapse microscopy observations of live embryos, the movies are very poor. Although the images are generally good and the 3-d sequences/images from the light-sheet microscope sequences are quite impressive (and have good spatial resolution), the time resolution is extremely poor and the movies very short. It is largely impossible to follow cell behaviours or movements in these sequences.
Indeed, the time lapse between time points as well as the total duration of the acquisition is limited, especially when embryos are imaged by confocal microscopy. These measures were taken mainly to preserve the integrity of the embryo and thus ensure that growth conditions were the closest to optimal in vivo conditions. For rosette analysis, the 20 minutes interval was too long to follow rosette resolution, as stated in the manuscript. For mosaically labelled embryos, we quantified only the cells for which the fate and/or progeny could be identified without ambiguity, which was made easier as we chose a 4OH-tamoxifen posology that resulted in a low proportion of labelling. As both cell delamination and mitosis are relatively slow processes, this time resolution proved sufficient. Time resolution for lightsheet was 7 minutes, which is similar compared to other works on mouse gastrulation (such as Williams et al., 2012), and actually higher than most two-photon or confocal studies, including that of our previous reports (Migeotte et al., 2010, Saykali et al., 2019, Trichas et al., 2012) in which cell tracking could be efficiently performed. This high time resolution allowed following individual rosettes overtime (Sup. Fig.2c__).__
Reviewer #3 (Significance (Required))
The study focuses on cell shape changes and various processes that accompany ingression and reports that ingression may occur through a variety of different mechanisms that occur at the same time, including rosette formation, individual ingression of bottle-shaped cells, and larger population ingression events. This is very similar to what has been described in chick embryos (eg. Voiculescu et al. eLife 2014 - surprisingly this is not cited), although in rodents primitive streak formation occurs in the absence of large-scale movements of cell sheets. Basically there are no surprises in the findings either for mouse or in comparison with other species, but the study is OK in terms of contributing useful information about streak formation and function in mouse (if the above problems are fixed).
We thank Reviewer 3 for helpful comments and references. We respectfully disagree concerning the risk of bias due to agarose cylinder culture, as exposed above. Concerning the videos, we have provided less compressed videos to retain as much image quality as possible. Although it would evidently be better to have a higher time resolution and longer movies, we believe it is not a limitation for the events we study and describe as they can be reliably followed with the time resolution and observation length we provide. The reference to Voiculescu et al., 2014 is indeed important, we have added it to the revised version of the manuscript (line 324) and apologize for the oversight.
**Referees Cross Commenting**
In response to reviewer 2: One issue with this is that one does not know whether there is a "deleterious" effect of the agarose on movements until one is sure that (a) one understands what the movements would look like without agarose and that there are no differences, and (b) (a serious shortcoming here) that embryos need to be shown to develop completely normally in those culture conditions WAY beyond the period of imaging. There are lots of observations by several labs (some unpublished of course, but some are published) suggesting that agar and agarose do interfere with cell movements. In chick for example the Chapman and Schoenwolf method where embryos are placed on agarose, there are always head defects due to impaired movements and the agarose interfering with tissue tensile forces.
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Referee #3
Evidence, reproducibility and clarity
This is an essentially descriptive study, looking at primitive streak formation and cell ingression from the epiblast in the mouse from about E5.75 to E7.5 or so using time-lapse microscopy (light sheet and confocal) of cultured embryos. The study also takes advantage of some genetically encoded reporters, some of them inducible by tamoxifen, which allow following cells, closer examination of their shapes, and in some cases unambiguous orientation of the embryos based on expression of the reporter. Overall the study is well designed.
I have two very major concerns about this paper - first, the culture system used in most experiments …
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 #3
Evidence, reproducibility and clarity
This is an essentially descriptive study, looking at primitive streak formation and cell ingression from the epiblast in the mouse from about E5.75 to E7.5 or so using time-lapse microscopy (light sheet and confocal) of cultured embryos. The study also takes advantage of some genetically encoded reporters, some of them inducible by tamoxifen, which allow following cells, closer examination of their shapes, and in some cases unambiguous orientation of the embryos based on expression of the reporter. Overall the study is well designed.
I have two very major concerns about this paper - first, the culture system used in most experiments uses agarose, which has been found in several labs to affect normal cell movements and other cell behaviours. It is essential to determine that embryos cultured in these conditions develop normally for much longer than the period of imaging to ensure that the findings are relevant to normal development rather than an artefact. This is particularly important because mouse embryos develop rather poorly at peri-implantation stages with any culture method, and this one could make matters even worse.
The second concern is that for a paper that is almost entirely about time-lapse microscopy observations of live embryos, the movies are very poor. Although the images are generally good and the 3-d sequences/images from the light-sheet microscope sequences are quite impressive (and have good spatial resolution), the time resolution is extremely poor and the movies very short. It is largely impossible to follow cell behaviours or movements in these sequences.
Significance
The study focuses on cell shape changes and various processes that accompany ingression and reports that ingression may occur through a variety of different mechanisms that occur at the same time, including rosette formation, individual ingression of bottle-shaped cells, and larger population ingression events. This is very similar to what has been described in chick embryos (eg. Voiculescu et al. eLife 2014 - surprisingly this is not cited), although in rodents primitive streak formation occurs in the absence of large-scale movements of cell sheets. Basically there are no surprises in the findings either for mouse or in comparison with other species, but the study is OK in terms of contributing useful information about streak formation and function in mouse (if the above problems are fixed).
Referees Cross Commenting
In response to reviewer 2
One issue with this is that one does not know whether there is a "deleterious" effect of the agarose on movements until one is sure that (a) one understands what the movements would look like without agarose and that there are no differences, and (b) (a serious shortcoming here) that embryos need to be shown to develop completely normally in those culture conditions WAY beyond the period of imaging.
There are lots of observations by several labs (some unpublished of course, but some are published) suggesting that agar and agarose do interfere with cell movements. In chick for example the Chapman and Schoenwolf method where embryos are placed on agarose, there are always head defects due to impaired movements and the agarose interfering with tissue tensile forces.
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Referee #2
Evidence, reproducibility and clarity
This manuscript, from Mathiah and colleagues, describes an in-depth analysis of differences in cell organization and division within the epiblast of the very early mouse embryo, and in particular, with the onset of gastrulation. Their data indicate a difference in the organization of cells between the anterior/lateral and posterior regions of the epiblast even before gastrulation has commenced, as well as differences in the location of mitoses relative to the apical and basal ends of the cells. The data provide new insight into the early regionalization of the epiblast. However, the authors should include reference to, and …
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Referee #2
Evidence, reproducibility and clarity
This manuscript, from Mathiah and colleagues, describes an in-depth analysis of differences in cell organization and division within the epiblast of the very early mouse embryo, and in particular, with the onset of gastrulation. Their data indicate a difference in the organization of cells between the anterior/lateral and posterior regions of the epiblast even before gastrulation has commenced, as well as differences in the location of mitoses relative to the apical and basal ends of the cells. The data provide new insight into the early regionalization of the epiblast. However, the authors should include reference to, and discussion of, the paper by Michael Snow on growth and regionalization of the epiblast (Snow MHL (1977) Gastrulation in the mouse: Growth and regionalization of the epiblast. J. Emb. Exp. Morph. 42: 293-303), where he did a much more fine-grained analysis of mitotic index across the entire epiblast, defining a proliferative zone in the anterior part of the primitive streak where the mitotic index was higher between E6.5 and E7.5. He also describes non-apical mitoses specifically in the primitive streak region as compared to all other regions of the epiblast. The results of the present study dovetail nicely with the results presented by Snow.
Overall, this is a very nice study, but some revisions would help with clarity at certain points. The data on rosette formation are interesting, but it is not clear what an increase in rosettes in the posterior region means. The authors contend (lines 169-170) that this represents a dynamic epithelium primed for EMT, but it is not clear how rosettes facilitate or promote EMT, and especially why that would be seen at E5.75 before EMT has begun. An alternative interpretation might be that the shape of the epithelium may be changing and the packing of the epithelial cells has to change to accommodate this. We do know that the overall shape of the embryo changes from elongate medial-lateral to elongate anterior-posterior just as EMT is initiated (Perea-Gomez et al., (2004) Current Biology 14: 197-207) and it may be that changes in cell packing are required to accommodate this. The authors may want to consider whether the rosettes that they observed represent scutoids (Gomez-Galvez et al., (2018) Scutoids are a geometrical solution to three-dimensional packing of epithelia. Nat. Commun. 9:2960). An analysis of the 3-dimensional organization of the cells within the rosettes (i.e. at all Z levels) may shed some light on this.
Figure 2b,c and Figure 3a, a', b,c - Addition of dotted lines to indicate the apical and basal ends of the epiblast would be helpful in orienting the reader.
Figure 2c' - what these graphs represent exactly is somewhat vague, and the figure legend is also very vague. In particular, the third graph on cell dispersion is not clear. Does this mean that the daughter cells are separated from one another following division? Or that they are in different compartments (epiblast/mesoderm) after division? A better description should be included in the figure legend.
Figure 3g would appear to show the proportion of the total number of posterior divisions that give rise to particular combinations of daughter cells (epi/epi, epi/meso, meso/meso). However, the discussion of this graph in the text (lines 215-219) suggests that it demonstrates that non-apical mitoses always result in meso/meso and epi/meso daughter cells, which it does not. That analysis would be very interesting to add to Figure 3, with the daughter cell types broken down into those coming from apical mitoses and those coming from basal mitoses.
In Figure 4, it is not clear how anterior and posterior are defined, and what criteria were used to distinguish posterior from primitive streak. This is nicely demonstrated in Supplementary Figure 3 - maybe panels A and B could be included in Figure 4 to improve the clarity of the analysis.
The data on mitotic index in Figures 2, 4, and 5 do not appear to be consistent. The mitotic index for E7.25 in Figure 2e is similar between anterior and posterior, even though the posterior includes the primitive streak, while the mitotic index presented for the three stages in Figure 4b would imply that the mitotic index for the entire posterior region should be higher than the anterior at all three stages. Similarly, in Figure 5a' and b', the mitotic index in anterior and posterior regions of E5.75 and E6.25 embryos are not significantly different despite the primitive streak being included in the posterior count, while the data presented in Figure 4 would imply that the entire posterior region including primitive streak should be much higher than the anterior. The authors should clarify this in the Results.
The data on non-apical mitoses in the RhoA-VEdeleted (Figure 6) and Rac1ko embryos (Supplemental figure5) are not particularly compelling. It is hard to see the basal mitoses in the new AVE-opposed regions in the mutant embryos in the images presented. Perhaps the graphs in these two figures could have the AVE-opposed data broken down into two groups - the region that is posterior and the region that is anterior but not adjacent to AVE. Better images would improve the clarity of these data as well.
Significance
The data on differential proliferation and apical vs. basal mitoses are complementary to data already published, but the present study updates the existing data by the addition of live imaging and 3-dimensional reconstruction of cell shapes, providing a more complete insight into the process. The observation that rosettes are detectable at the basal ends of the epiblast, and moreso in the posterior, is novel, but the significance for embryonic development is not well rationalized.
These data are of interest to those investigating the mechanisms of early morphogenesis, as well as those interested in the cellular correlates of molecular regionalization that results from the well-described signaling pathways regulating axis specification.
My background is in early mouse embryo morphogenesis, therefore I feel that I have sufficient expertise to evaluate the data presented.
Referees Cross Commenting
I agree with Reviewer 1 on the lack of detail about the methods - my comments stemmed from the same confusion about how measurements were made, but Reviewer 1 more articulately addressed the key points.
I agree with Reviewer 3 on the quality of the videos. It is very difficult to see how they could follow cells with a 20 minute interval.
I would like to address the comment by Reviewer 3 on the use of agarose in the imaging experiments. The methods section states that agarose was used to make the culture "chambers" used for light-sheet imaging, which was not the major approach used for imaging in this study. Only the data in Figure 1A came from those experiments, and it was validated by confocal data in 1B,C where the embryos were cultured in Ibidi chambers with culture medium and no agarose present. So I don't think agarose effects on embryo development are a major worry. Also, this same approach was used by Ryan Udan in Mary Dickinson's lab to visualize yolk sac vasculogenesis, and it did not appear to have a deleterious effect on development in that case, although the embryos imaged here were much earlier and are definitely differentially sensitive to culture conditions from those cultured at E8.
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Referee #1
Evidence, reproducibility and clarity
In "An asymmetry in the frequency and position of mitosis in the epiblast precedes gastrulation and suggests a role for mitotic rounding in cell delamination during primitive streak epithelial-mesenchymal transition", Mathiah, Despin-Guitard and colleagues study divisions during mouse gastrulation. They perform ex vivo culture, live imaging and immunostaining to observe the frequency and position of mitosis within the embryo as well as the destiny of daughter cells after their divisions. The find that divisions on the posterior side of the embryo tend to be more basally located and could contribute to cell delamination into the …
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Referee #1
Evidence, reproducibility and clarity
In "An asymmetry in the frequency and position of mitosis in the epiblast precedes gastrulation and suggests a role for mitotic rounding in cell delamination during primitive streak epithelial-mesenchymal transition", Mathiah, Despin-Guitard and colleagues study divisions during mouse gastrulation. They perform ex vivo culture, live imaging and immunostaining to observe the frequency and position of mitosis within the embryo as well as the destiny of daughter cells after their divisions. The find that divisions on the posterior side of the embryo tend to be more basally located and could contribute to cell delamination into the mesodermal layer. Authors also affect antero-posterior signaling by genetically preventing the migration of the anterior visceral endoderm, which leads to mitosis away from the apical side of the epithelium on all lateral parts of the embryo.
This study tackles a key developmental process which is poorly understood in mammals due to its concomitance with the implantation phase. Therefore, any carefully-made description of this process has the capacity to be eye-opening. This is potentially the case for this report, which provides nice images that most likely required skills and important efforts to obtain. The authors have written a clear manuscript with an interesting narrative. However, the quantifications are very poorly described, which makes it impossible for anyone to reproduce these results. I describe below a number of suggestions to clarify the quantifications, which is in my opinion a prerequisite to consider the conclusions from the authors.
Fig1: Authors describe differences in the formation of rosettes between the anterior and posterior sides of the embryo. The microscopy images and movies provided are overlaid with drawings from the authors but without this visual help, I, and I assume other readers, see more rosettes than highlighted and fail to see some of the rosettes that are marked. To avoid this subjectivity, a clear methodology is required. In the methods, the authors state: "For quantification, rosettes were manually annotated and counted on Z sections located 5 μm from the basal side of the epiblast." And that is all. What defines a rosette? How many cells need to share a vertex to be considered as part of a rosette? How long do they need to persist not to be considered as occurring by chance? What about cells part of multiple rosettes? Does the rosette organization need to be apical, basal or all the way? Having those clearly defined criteria would be essential for anyone else to reproduce this quantification and would also offer a much more comprehensive description of the phenomenon and allow for more powerful conclusions. In addition, the data are given as "rosette/frame" and as "rosette/mm2". What is the point of giving both data, which are essentially the same? The frame is irrelevant. It would be more interesting to know how many cells there are in this area, as cell packing could be a determinant of rosette formation. "Rosette/mm2/min" is very confusing. It should state "rosette.mm-2.min-1" or "rosette/mm2.min". On a conclusive note, I fail to understand how relevant the formation of rosettes would be. The authors should clarify this point.
Fig2: I have essentially the same issue for bottle cells and delamination counting as for rosettes. In this case, there is nothing in the methods section. What defines a cell as bottle shape and not bottle shape (apical vs basal width for example)? Where does a cell need to be to be counted as delaminated (a distance needs to be stated, absolute (better) or relative)? What defines sister cells as dispersing after division? How far apart do they have to be? After how much time? From the movies provided, the acquisition time seems to short to assess cell dispersal.
Fig3: Mitotic index calculation is described in the figure legend but not in the methods section. It should also be in the methods section and made explicit that the number of mitotic cells is normalized to green cells only, not the entire cell population. The mitotic index seems higher in this population than in the entire embryo as seen in Fig4. What defines an exiting cell? Regarding the non-apical rounding, why not calling it basal rounding? How far from the apical side does a cell need to be to be counted as non-apical? In the panel h, with the posterior division outcome, is that for all divisions or only for non-apical divisions? Do basal divisions give rise to more epi? Is epi or meso fate only determined by location in a different layer or are fate markers used? What happens to the non-apical mitosis on the anterior side?
Fig4: Methods state "For Phospho-histone H3 quantifications, sections were chosen at least 10 μm apart to ensure that each cell was only counted once, and counting was performed using the Icy software" and legend states "The PS region is defined by the area where the basal membrane (yellow) is degraded, and the posterior region quantification excludes counts from the PS region". This needs to be extended (what about cells at the boundary between PS and non-PS regions?) and brought together in the methods section. Also the tissue architecture in the PS is not as well defined as in the rest of the tissue. Is the epithelial polarity clear enough to be determined without AB marker in the PS? Finally, the number of cells counted is missing.
Supp Fig5: based on available images of the Rac1KO embryo, I am not sure that epithelial architecture is established well enough to assess the location of mitosis along the apico-basal axis.
Significance
Although I am not as familiar with mouse gastrulation as I would like to be, I am familiar with gastrulation, live imaging and analysis. At this point I find it difficult to discuss the conclusions of the study since the methodology is so unclear. Nevertheless, any carefully-made description of mammalian gastrulation has the capacity to be eye-opening. This is potentially the case for this report, which provides nice images that most likely required skills and important efforts to obtain.
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Excerpt
Regional differences in cell division within the gastrulating mouse embryo
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