Quantitative multi-scale morphodynamic analysis reveals ratchet-like collective DVE migration and epiblast retrograde cell flow during anterior patterning in the mouse embryo

Reviewer #1 (Public review):

Summary:

The authors study how the migration of distal visceral endoderm (DVE) cells in early mouse embryos becomes channeled towards one direction and the corresponding movement of the epiblast on which the DVE cells migrate. To this end, they develop an analysis pipeline of an in toto live data set previously obtained by the authors, which includes superpixel motion tracking of the visceral endoderm surface and subregions thereof. They find that a morphological asymmetry of the ectoplacental cone is indicative of anterior-posterior axis orientation. Even during the phases prior to and after collective migration, DVE cell speed was larger than in the surrounding tissue. The crossover from the pre-migratory to the migratory phase relies on the alignment of DVE cell motion. During the migration phase, counter-rotating vortices appeared in the emVE as expected when a rigid body moves through an incompressible fluid. Furthermore, DVE migration exhibits what the authors term a ratchet-like behavior, where the cells alternate between bursts of collective migration and periods of essentially no net motion. This behavior could be reproduced in vertex-model simulations, where DVE cells were subjected to a constant external force in an otherwise passive environment of cells. The observed intermittent behavior results from building up stress in the surrounding tissue that is released through cell rearrangements involving T1 transitions. These findings are in line with experimental results, although in embryos, T1 transitions are not as abundant as in the simulations and are largely confined to the region ahead of the DVE. Finally, the authors report a distally directed planar motion in the anterior epiblast underlying the visceral endoderm and thus opposite to the motion of the DVE. Cell migration in the posterior epiblast was slower and more random than in the anterior.

Strengths:

The authors provide a detailed analysis of the cell migration patterns in the embryo and show through vertex-model simulations that some of the observed features are really consequences of the properties of incompressible fluids.

Weaknesses:

Naming the intermittent dynamics of DVE cells as ratchet-like seems inappropriate, as it is rather reminiscent of stick-slip dynamics.. Quantitatively, the simulations do not provide much more insight beyond providing the flow profile of the (complex) fluid behavior of the tissue surrounding the DVE. It would be interesting to identify mechanisms that underlie migration alignment of DVE cells and to study in detail the T1 transitions - why are they confined to certain regions of the tissue? Furthermore, the theoretical analysis should be extended so that it also considers the dynamics of epiblast cells.

Reviewer #2 (Public review):

Summary:

The work provides mechanistic insights into the establishment of the anterior-posterior axis of the mouse embryo by quantifying multiple cellular and tissular parameters from high-quality live imaging data. It shows that the direction of the axis is predetermined by the embryo geometry, that the cells whose migration defines the direction of the axis (the anterior visceral endoderm) have a ratchet-like movement probably depending on transient relaxation events of the epithelial cells lying in their way, and that the adjacent cell layer (the epiblast) moves in the opposite direction.

Strengths:

The dataset is large, with multiple embryos from relevant reporter lines integrally imaged at high resolution for long periods of time, and the analysis tools are novel, original and powerful.

Weaknesses:

Since all data are obtained from wild-type unchallenged embryos, the direct causality between events may not be fully guaranteed.

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors investigate the dynamics of distal visceral endoderm (DVE) migration during early anterior-posterior axis formation in the mouse embryo. Using long-term light-sheet imaging combined with geodesic projections and quantitative motion analysis, they characterize DVE migration at both the cellular and tissue levels. The study identifies three distinct phases of DVE migration, describes the intermittent "stop-and-go" nature of DVE movement, and quantifies coordinated tissue behaviors within the visceral endoderm. The authors further report a previously unrecognized posterior movement of the underlying epiblast that occurs concomitantly with anterior DVE migration. Finally, they develop a two-dimensional vertex model to investigate the mechanical basis of the observed intermittent migration, proposing that cycles of stress accumulation and T1-mediated stress relaxation within the surrounding visceral endoderm account for the observed dynamics

Strengths:

Overall, this is a very interesting study combining state-of-the-art live imaging with an impressive quantitative image analysis framework. The imaging quality is excellent, and the authors provide one of the most detailed quantitative descriptions of visceral endoderm (VE) dynamics to date. In particular, the combination of whole-embryo light-sheet imaging, geodesic projections and quantitative analysis provides a rich dataset that will undoubtedly be valuable for the community. The model is also informative and provides a mechanistic hypothesis for the start and stop motion of the VE.

Weaknesses:

(1) Clarification of the Superpixel-based image analysis

The image analysis pipeline is impressive but could be explained more clearly for readers unfamiliar with the authors' previous work. In particular, the manuscript relies extensively on superpixel tracking, but it remains unclear what advantages this approach offers over more conventional Lagrangian particle image velocimetry (PIV). Since this paper should be self-contained, it would be helpful if the authors briefly explained the rationale for choosing superpixel tracking rather than referring readers to their previous eLife publication.

Related to this point, the manuscript appears to use two different levels of coarse-graining. Motion is initially estimated from thousands of superpixels (1000-5000 according to the Methods), whereas the quantitative analyses are ultimately averaged over only 32 spatial sectors. The relationship between these two levels of representation is not entirely clear and would benefit from clarification. Why use such a dense superpixel seeding, which seems oversampled, if the intent is to eventually bin the result?

Relatedly, how was the number of superpixels chosen? What is their effective size relative to the size of a VE or epiblast cell? This information is important because the analysis appears to be oversampled. This is particularly evident in Movie S14/Figure 7, where numerous superpixels appear to span a single epiblast cell. At this spatial scale, the measured motion is likely to include intracellular or subcellular movements, such as interkinetic nuclear migration or transient cell-shape changes, rather than pure tissue displacement. This may be somewhat misleading, as the visual impression is that the tissue itself is moving, whereas in some instances this reflects cellular/subcellular fluctuations. A discussion of the spatial scale of the superpixel analysis, together with a demonstration that the conclusions are robust to the degree of coarse-graining, would greatly strengthen the manuscript, especially regarding he movement of the epiblast (see point 4).

(2) Use of the term "ratchet-like"

We would recommend avoiding the term ratchet-like and instead using start-stop or stop-and-go migration throughout the manuscript. While these terms describe the same observed behavior, ratchet-like implicitly suggests an irreversible mechanism underlying the motion, whereas the present study primarily documents an intermittent migration pattern. In my opinion, stop-and-go is a more descriptive and mechanistically neutral terminology, leaving the mechanistic interpretation to the modelling section.

(3) Mechanistic interpretation of the stop-and-go behavior

The vertex model constitutes the principal mechanistic component of the study and provides an interesting explanation for intermittent DVE migration through stress accumulation followed by T1-mediated stress relaxation. However, the comparison between the model and the experimental data reveals an important discrepancy. As acknowledged by the authors, the model predicts a broader distribution of T1 transitions than observed experimentally, whereas in vivo T1 events appear largely confined to the embryonic visceral endoderm ahead of the migrating DVE.

This discrepancy suggests that an important aspect of junctional mechanics may be missing from the current formulation. Have the authors considered whether an asymmetric constitutive description, in which junctions remodel more readily under compression than under tension, could better account for the observed spatial restriction of T1 events? Such constitutive asymmetry may provide a more biologically realistic mechanism for intermittent migration while preserving the overall framework proposed here.

Overall, we find the modelling direction promising, but at present the model appears somewhat premature or overly simplified relative to the experimental observations. The simulations convincingly demonstrate that T1-mediated stress relaxation can generate intermittent migration, but they do not yet quantitatively, if not qualitatively, reproduce the spatial distribution of T1 events observed in vivo. Since the authors have segmented some samples, could all the cells then provide a movie with T1 annotated? That would be helpful to get an intuition on the level of performance of the model compared to experimental data.

Related to this point, the stop-and-go behavior shown in Figure S7 is not immediately obvious. It would be helpful to display the instantaneous DVE velocity together with the timing of T1 transitions, allowing the proposed correlation to be appreciated more directly. In addition, in Figure 5E, the lower panel appears to be labelled "DVE position", whereas the text suggests that DVE velocity is intended. This should be clarified.

(4) Motion of the epiblast

The observation of coordinated epiblast motion is intriguing. However, it would be helpful if the authors quantified the magnitude of the net displacement. From the movies, the overall displacement appears relatively modest, perhaps on the order of one cell diameter. Is this indeed the case?

More generally, we have some concerns regarding the quantification and representation of epiblast motion. As discussed above, the superpixel analysis appears to operate at a subcellular scale, with many superpixels spanning the apico-basal extent of individual epiblast cells. Consequently, the measured motion may partly reflect transient cell deformations, for example during mitosis or interkinetic nuclear migration, rather than displacement of the tissue itself. Finally, we wonder whether the flattened representation is the most appropriate way to present the epiblast data. Such projections are clearly helpful for analyzing the whole VE motion over a curved epithelial surface. However, the epiblast motion described here is essentially linear, and it is therefore less obvious how the flattening affects the apparent displacement. It would be helpful if the authors could also present the epiblast movement in the original, non-flattened imaging data (e.g. using an optical transverse section through the embryo). At present, the motion is only shown either as a geodesic projection or as a flattened transverse view, such that the reader never directly observes the movement in its native three-dimensional geometry.