Cell-state transitions and collective cell movement generate an endoderm-like region in gastruloids
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Abstract
Shaping the animal body plan is a complex process that involves the spatial organization and patterning of the different germ layers. Recent advances in live imaging have started to unravel the cellular choreography underlying this process in mammals, however, the sequence of events transforming an unpatterned cell ensemble into structured territories is largely unknown. Here, using gastruloids –3D aggregates of mouse embryonic stem cells- we study the formation of one of the three germ layers, the endoderm. We show that the endoderm is generated from an epiblast-like homogeneous state by a three-step mechanism: (i) a loss of E-cadherin mediated contacts in parts of the aggregate leading to the appearance of islands of E-cadherin expressing cells surrounded by cells devoid of E-cadherin, (ii) a separation of these two populations with islands of E-cadherin expressing cells flowing toward the aggregate tip, and (iii) their differentiation into an endoderm population. During the flow, the islands of E-cadherin expressing cells are surrounded by cells expressing T-Brachyury, reminiscent of the process occurring at the primitive streak. Consistent with recent in vivo observations, the endoderm formation in the gastruloids does not require an epithelial-to-mesenchymal transition, but rather a maintenance of an epithelial state for a subset of cells coupled with fragmentation of E-cadherin contacts in the vicinity, and a sorting process. Our data emphasize the role of signaling and tissue flows in the establishment of the body plan.
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###Reviewer #3:
In this manuscript Hashmi et al describe the emergence of an endoderm population in a gastruloid model. They observe that endoderm cells are positive for E-Cad, likely express E-Cad continuously from an epiblast state, initially form small islands, and finally coalesce into a larger endoderm region at the pole of the gastruloid. There are several issues with this manuscript in its current form.
No evidence is provided that there is a relationship between how endoderm forms in this gastruloid model and in vivo. In fact, endoderm is believed to derive from a restricted area in the anterior primitive streak. This is evident from the mouse imaging data of Mcdole et al Cell 2018 as well as from more recent genetic labeling experiments (Probst et al bioRxiv 2020). It is well known that cells of different germ layers may …
###Reviewer #3:
In this manuscript Hashmi et al describe the emergence of an endoderm population in a gastruloid model. They observe that endoderm cells are positive for E-Cad, likely express E-Cad continuously from an epiblast state, initially form small islands, and finally coalesce into a larger endoderm region at the pole of the gastruloid. There are several issues with this manuscript in its current form.
No evidence is provided that there is a relationship between how endoderm forms in this gastruloid model and in vivo. In fact, endoderm is believed to derive from a restricted area in the anterior primitive streak. This is evident from the mouse imaging data of Mcdole et al Cell 2018 as well as from more recent genetic labeling experiments (Probst et al bioRxiv 2020). It is well known that cells of different germ layers may self-segregate and this may drive the behavior observed here downstream of heterogeneous differentiation in the gastruloids, but that is not necessarily the mechanism which occurs in vivo. The authors suggest that their experiments show something about endoderm formation in vivo without addressing this point which substantially diminishes from the interest of the manuscript.
The authors suggest that this view of endoderm differentiation, which doesn't require full EMT is novel, however, much of the observations here are already known. It is known that future endoderm cells do not down regulate E-Cadherin but instead must continue to express it. They also are known to migrate collectively rather than as single cells in a cadherin-dependent way (Montero et al Development 2005; reviewed in Nowotschin et al Development 2019). The authors should discuss this literature and make clear which aspects of the proposed mechanisms are novel.
The authors are assessing the status of EMT based on a single marker, E-Cad. If this is a major point of the manuscript other markers e.g. Snail, N-Cad should be examined.
It is well known that embryoid bodies form an outer layer of visceral endoderm, e.g. Concouvanis & Martin Cell 1995, Doughton et al PLOS ONE 2010. None of the markers here are exclusive to definitive endoderm (including Sox17 which is used throughout, see Artus et al Dev Biol 2011). The authors should address the possibility that their observations may be consistent with a similar mechanism and may not reflect definitive endoderm differentiation.
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###Reviewer #2:
In this manuscript, the authors proposed a new mechanism of endoderm formation in 3D gastruloid models based on cell migration and fragmentation. Specifically, they found that E-cad is first uniformly expressed inside mESC aggregates. After exposure to Wnt agonist Chiron (Chi), a gradual repression of E-cad and an increase of T-Bra were detected. Cells in the core are tightly packed and express E-cad. T-Bra expressing cells are sparsely wrapped around the core. A directed flow of E-cad expressing cell islands surrounded by T-Bra expressing cells help to accumulate E-cad expressing cells to the tip of the aggregate and form endoderm domain. I think the dynamical expression of E-cad and T-Bra and the directed cell flow reported in this manuscript are interesting. The results and videos have shown that the elongation and …
###Reviewer #2:
In this manuscript, the authors proposed a new mechanism of endoderm formation in 3D gastruloid models based on cell migration and fragmentation. Specifically, they found that E-cad is first uniformly expressed inside mESC aggregates. After exposure to Wnt agonist Chiron (Chi), a gradual repression of E-cad and an increase of T-Bra were detected. Cells in the core are tightly packed and express E-cad. T-Bra expressing cells are sparsely wrapped around the core. A directed flow of E-cad expressing cell islands surrounded by T-Bra expressing cells help to accumulate E-cad expressing cells to the tip of the aggregate and form endoderm domain. I think the dynamical expression of E-cad and T-Bra and the directed cell flow reported in this manuscript are interesting. The results and videos have shown that the elongation and formation of endoderm region is a collective cell behavior rather than single cells undergo epithelial-to-mensenchymal transition. But I am not convinced that the process is done based on the three-step mechanism proposed by the authors. Moreover, I am not sure if this phenomenon really happened in mouse embryo development, giving the considerable differences between gastruloid model and embryo. Since there are methods culturing mouse embryo in vitro up to the early organogenesis stage, I would suggest the authors provide more evidence showing that the proposed mechanisms might also happen in vivo.
In addition, the manuscript provides too little information to understand the phenomenon. And they did not clearly introduce experimental and computational methods they used to acquire the results. I listed some of my comments below.
Major comments:
Did all 3D aggregates become elongated shape in the presence of Chi? If not, what do E-cad and T-Bra expressions and cell migration dynamics look like in those spherical aggregates? Without Chi, inside the spherical aggregates, do they also have cell migration since the aggregates keep growing larger?
When did the collective cell migration start? Right after exposing to Chi? Or after some percentage of cells become T-Bra positive cells? Did the gastruloid keep elongating with directed cell flow inside it when cultured for a long time?
Are the collective cell migration driven by the T-Bra cells? Is it a spontaneous property of E-cad cells when the E-cad cell density exceed some critical threshold (e.g. glassy dynamics)?
Does the elongation and migration dynamics depend on the concentration of Chi, size of the aggregates? I noticed the authors used different initial seeding densities.
For the elongated cell aggregates, one side of cells express E-cad. How about the other side of cells? Did they all become mesoderm-like (T-Bra+) cells?
Many results are only based on several (3 or 4) gastruloids. For example, figure 1 (d) (e), figure 2 (b), figure 3(c). And in Figure 4 (b), the authors only quantify 13 junctions, probably in the same gastruloid. Due to the heterogeneity among the gastruloids, I am not sure how repeatable the experiments are. Can those observations really reflect phenomenon happened inside the majority of gastruloids? I think the authors should provide some quantifications of the percentage of observing the reported results among a large number of gastruloids.
Unclear results or experimental descriptions:
Can the authors show a schematic of the experimental process, such as the time of adding Chi and fixation?
'We find that 30/37 ... set to 0.125.' How did the authors define and calculate the elongation ratio and E-cadherin polarization ratio? How did the authors define the elongation threshold?
Figure 1 (a): what is the y axis? 1 (d): how did the author measure the E-cad and T-Bra expression? Fixing at different time points or live imaging? If it is live imaging, is the acquisition process influenced by adding and removing Chi? 1(e) how can the authors get continuous results for polarization?
Figure 2 (b) Are those dots represents the nuclear position? Can the authors provide the 3D view of the whole gastruloid? (c) What information the authors are trying to get from the connectivity graph?
Figure 3 (a) What are those white dots in the images, also in movie 6? Can the authors replace t1, t2, t3, t4 with the real time, such as 24h, 36h? (d) How did the authors calculate the intensity? How did the authors normalize the intensity? The schematic in (b) is hard to understand. What do the light and dark colors represent? How did the authors measure theta_1 and theta_2, especially in 3D situation? More quantitative information should be acquired from (a).
I am not able to identify islands of E-cad expressing cells in Figure 3 (a) and movie 6.
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###Reviewer #1:
General assessment:
The manuscript by Hashmi et al describes the emergence of endoderm-like cells in a stem cells based embryo model. The particularity of the protocol is that it stimulates transition through an epiblast-like state, then differentiation towards mesoderm after a pulse of Chiron, a Wnt agonist. In those conditions, islets of E-cadherin positive cells emerge, surrounded by Ecad+Brachyury+, then Brachyury positive cells. Those islets fuse together at the tip, possibly due to distinct surface tension and directed cell movements, and express endoderm markers such as Sox17 and FoxA2.
It is an original approach and concept, raising new questions and possibilities about the mechanisms of endoderm emergence in the mouse embryo. The manuscript is well written and clear.
Concerns:
1)The data would benefit from …
###Reviewer #1:
General assessment:
The manuscript by Hashmi et al describes the emergence of endoderm-like cells in a stem cells based embryo model. The particularity of the protocol is that it stimulates transition through an epiblast-like state, then differentiation towards mesoderm after a pulse of Chiron, a Wnt agonist. In those conditions, islets of E-cadherin positive cells emerge, surrounded by Ecad+Brachyury+, then Brachyury positive cells. Those islets fuse together at the tip, possibly due to distinct surface tension and directed cell movements, and express endoderm markers such as Sox17 and FoxA2.
It is an original approach and concept, raising new questions and possibilities about the mechanisms of endoderm emergence in the mouse embryo. The manuscript is well written and clear.
Concerns:
1)The data would benefit from increased clarity in stating, for each experiment, the proportion of aggregates in which a given phenomenon was observed, as well as the number of cells counted in each aggregate, in particular in supplementary figures.
For the migration analysis, it could be interesting to distinguish each cell trajectory in order to distinguish behaviours of the subpopulations.
In terms of the surface tension analysis, performing a similar analysis at different timepoints might be helpful to understand how the islets come to fuse at the tip.
I am not sure about the specificity of the gata6 staining, not that it adds a lot to the story.
The authors might want to discuss how those aggregates evolve, and whether the endoderm-like cells have a potential for further differentiation.
Conclusion:
Overall it is an interesting and original observation, well substantiated. More details on the quantification methods would help convince about the solidity of the model: chance of obtaining those cells, amount of cells of each subpopulation including those described in supplementary figures, technical possibility of sorting them for transcriptome analysis etc.
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
The reviewers agree that the manuscript reports an interesting and original observation in gastruloids. However there is currently no evidence to propose that such a mechanism would be present in embryos. Additionally, there is a consensus that the methods are not sufficiently explained, the reproducibility is not clearly quantified, and some claims would require a larger number of aggregates/cells to be solid.
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