In vitro endoderm emergence and self-organisation in the absence of extraembryonic tissues and embryonic architecture

  1. Stefano Vianello
  2. Matthias P. Lutolf

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Abstract

The endoderm is the cell lineage which gives rise in the embryo to the organs of the respiratory and gastrointestinal system. Uniquely, endodermal tissue does not just derive from descendants of the embryo proper (the epiblast) but instead arises from their gradual incorporation into an extraembryonic substrate (the visceral endoderm). Given the configuration of the early embryo, such a paradigm requires epiblast endodermal progenitors to negotiate embryonic compartments with very diverse epithelial character, a developmental contingency reflected by the fact that key early endodermal markers such as Foxa2 and Sox17 have been consistently found to be embedded within gene programmes involved in epithelialisation.

To explore the underlying cell biology of embryonic endoderm precursors, and to explore the relationship between endoderm development, epithelial identity, and extraembryonic mixing, we leveraged Gastruloids, in vitro models of early development. These self-organising three-dimensional aggregates of mouse embryonic stem cells do not possess an extraembryonic component, nor do they appear to display typical tissue architecture. Yet, they generate cells expressing endodermal markers. By tracking these cells throughout in vitro development, we highlight a persistent and uninterrupted pairing between epithelial and endodermal identity, with FoxA2+/Sox17+ endoderm progenitors never transitioning through mesenchymal intermediates and never leaving the epithelial compartment in which they arise. We also document the dramatic morphogenesis of these progenitors into a macroscopic epithelial primordium extending along the entire anterior-posterior axis of the Gastruloid. Finally, we find that this primordium correctly patterns into broad domains of gene expression, and matures cells with anterior foregut, midgut, and hindgut identities within 7 days of culture. We thus postulate that Gastruloids may serve as a potential source of endodermal types difficult to obtain through classical 2D differentiation protocols.

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    ## Comments by Reviewer 1

    For the sake of clarity, it may help to provide some table illustrating the proportion of gastruloids behaving precisely as the best example shown

    We thank the reviewer for raising this point. For the expression patterns of each of the main endodermal markers analyzed, at the timepoints considered, we will provide additional context on the variability among Gastruloids. We envisage to provide such results in a format similar to what used e.g. in Supplementary Figure 4 of https://doi.org/10.1038/s41467-021-23653-4 [Xu, Peng-Fei, et al. "Construction of a mammalian embryo model from stem cells organized by a morphogen signalling centre." (2021)]; i.e. categorization of the phenotypes observed among gastruloids, and quantification of their proportions.

    It would also strengthen the message even more to provide some quantitation of co-expression for the main markers. As the behaviour seems very consistent, it is likely that such quantification would not be very arduous, and it would show the strength of the model.

    We thank again the reviewer for highlighting the value of co-localisation data, and in fact this is a suggestion also put forward by our other reviewer. We are currently developing a pipeline to segment the nuclei on the DAPI channel of each immunostained Gastruloid, and extract marker intensities within each cell. We envisage the results to be presented in a format similar to what shown e.g. in Fig. 2 of https://doi.org/10.1242/dev.159103 [Mulas, Carla, et al. "Oct4 regulates the embryonic axis and coordinates exit from pluripotency and germ layer specification in the mouse embryo." (2018)]. Such data would undoubtedly strengthen the reliability of the claims we here draw mostly from a qualitative inference of colocalisation.

    The introduction could be shortened, and the results a bit more to the point.

    As this is also a point raised by both reviewers, we will make sure to go back over the entirety of the manuscript and do the necessary adjustments, especially for what concerns the Results section. We would however like to point out that the unusual length of our Introduction section is to be understood as a deliberate departure from the limits and conventions prescribed by traditional publishing formats. In line with our intentional choice of a journal-independent publication platform (i.e. a preprint server), and of journal-independent review process, we also presented and contextualized our research in a journal-independent format. We see this as an opportunity to present research in a voice and in a form truer to that of the researchers that carried it out. We thank both reviewers for their feedback on the format and will certainly still make sure to minimize redundancy of information throughout our manuscript.

    ## Comments by Reviewer 2

    The following conclusions were not warranted by the findings:

    __Endoderm emergence can occur in the absence of extraembryonic tissues and embryonic architecture: It is unclear if extraembryonic tissues (e.g., primitive endoderm and visceral endoderm-like cells) are absent in the early phase of gastruloid development. __

    The reviewer raises an important point regarding the presence/absence of cells with extraembryonic endoderm identity within the early developing gastruloid. Our claim of absence of these cell types is grounded on previous gastruloid research (Turner, David A., et al. "Anteroposterior polarity and elongation in the absence of extra-embryonic tissues and of spatially localised signalling in gastruloids: mammalian embryonic organoids." (2017); van den Brink, Susanne C., et al. "Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids." Nature 582.7812 (2020): 405-409.), which has indeed found no evidence of extraembryonic endoderm in Gastruloids. While other datasets do not include early Gastruloid stages where such cells may instead be detected, transcriptional analyses of later timepoint Gastruloids [Rossi, Giuliana, et al. "Capturing cardiogenesis in gastruloids." (2021)] also do not seem able to uniquely define extraembryonic endoderm signatures. Accordingly, we did not see this claim as having to be further substantiated.

    In light of most recent reports (see https://doi.org/10.1038/s41467-021-23653-4 [Xu, Peng-Fei, et al. "Construction of a mammalian embryo model from stem cells organized by a morphogen signalling centre." (2021)]), the necessary absence of extraembryonic endoderm types within self-organising embryonic models may appear to need to be at least revisited. Xu et al indeed report extraembryonic endoderm cells in an embryoid model in many ways analogous to the Gastruloid system. These claims are mainly based on the recovery of DBA (Dolichos Biflorus Agglutinin) signal, a marker traditionally associated with extraembryonic endoderm in vivo. Yet, and based on our own unpublished exploration of the topic, we presently cannot confirm DBA lectins to be an exclusive marker of extraembryonic endoderm in an in vitro setting (i.e. in the cell types obtained from and amongst differentiating stem cells), as not only we detect DBA positivity in wide cellular domains that are incompatible with any realistic estimate of the extraembryonic makeup of Gastruloids, but we also see this marker decorating the membrane of 2D colonies of pluripotent mouse embryonic stem cells maintained under 2iLIF conditions. When counterstaining for Ttr (Transthyretin; aka prealbumin) (a major discriminant of extraembryonic vs embryonic endoderm, as recovered from single-cell datasets; https://endoderm-explorer.com/), we further cannot detect positivity in either DBA+ or DBA- cells.

    To the best of the knowledge and evidence available to us, we thus still consider the absence of extraembryonic endoderm in Gastruloids to be a substantiated claim. We thank however the reviewer for raising this important point and agree that dedicated characterization of extraembryonic endoderm signatures/markers in the developing Gastruloid could certainly help to support the validity of the claims made in previous Gastruloid literature, from which we draw the premise that no extraembryonic endoderm is present. To facilitate this process, and in fact to better contextualize our results in the light of what now published in Xu et al, we will now include detected DBA and Ttr (absent) patterns in early Gastruloids, as well as the expression patterns of extraembryonic endoderm markers for the timepoints for which single cell transcriptomics data is available (i.e. 96h onwards).

    While the gastruloid does not replicate the morphological feature of the post-gastrula embryo, it nevertheless has a certain degree of tissue organization. Perhaps the emergence of DE-like cells in 2-D culture would be a more convincing model for "the absence of extraembryonic tissues and embryonic architecture".

    The observation is correct: gastruloids do not replicate the architecture of the peri gastrulation mouse embryo. Concomitantly, they display a striking degree of tissue (re-/)organization as they proceed throughout differentiation. We were deliberate in the presentation of our data, and as such we always refer to “absence of embryonic architecture” rather than “absence of architecture” (i.e. of any architecture), as the latter assertion would in fact be contrary to a main finding of our investigation.

    Inde ed, the observation that Gastruloids, and specifically endodermal cells within them, can give rise to such developed tissue architectures, by self-organisation and without the need of externally-supplied matrices, is a major focus of our manuscript. Since Gastruloids start as an unstructured, epithelioid, cluster of stem cells, the architectural rearrangements observed in the mature Gastruloid highlight an intrinsic propensity of endodermal cells to forming epithelia. Key aspect to this point, is that these architectural rearrangements are carried out by cells in a landscape that is not architecturally similar to that of the embryo (specifically, where endoderm progenitors do not and cannot arise from a columnar epithelium and do not and cannot have a visceral-endoderm-like destination in which to intercalate).

    Considering what discussed in this and in the previous point, we struggle to frame the Gastruloid as not a “convincing model for the absence of extraembryonic tissues and embryonic architecture", given that it satisfies both criteria. More complete and articulated discussions and appraisals of the value of Gastruloids and other 3D *in vitro *models towards uncovering fundamental features of embryonic development are available elsewhere, including in their comparison to 2D differentiation assays (van den Brink, Susanne C., and Alexander van Oudenaarden. "3D gastruloids: a novel frontier in stem cell-based in vitro modeling of mammalian gastrulation." (2021); Simunovic, Mijo, and Ali H. Brivanlou. "Embryoids, organoids and gastruloids: new approaches to understanding embryogenesis." (2017); Turner, David A., Peter Baillie‐Johnson, and Alfonso Martinez Arias. "Organoids and the genetically encoded self‐assembly of embryonic stem cells." (2016)). We refer readers to these reviews to help inform their own assessment on the matter.

    Of course, this is not to say that 2D culture models are not an equally valuable system to study peri-gastrulation development (if only as exemplified by https://doi.org/10.1186/s12915-014-0063-7 [Turner, David A., et al. "Brachyury cooperates with Wnt/β-catenin signalling to elicit primitive-streak-like behaviour in differentiating mouse embryonic stem cells." (2014)] and by the numerous studies on micropatterned stem cells). To the best of our knowledge, however, no 2D model of spontaneous, undirected endodermal differentiation has been investigated in detail and from a developmental perspective. We share the reviewer’s interest on the insight that this kind of approach could provide. Still, claims of absence of some degrees of architectural organisation or of extraembryonic tissues are not straightforward in self-organising 2D systems either.On the other hand, 2D approaches that consist in directed differentiation of stem cells to specific endodermal fates are clearly not the type of investigation we were interested within the scope of our experimental questions. We also believe that e.g. differentiation of 2D epithelia that then bud to form 3D spheres are generally contexts that are too decoupled from embryonic modes of development to provide the same degree of developmental insight than e.g. Gastruloids. Having both 2D and 3D platforms at our availability, we opted for latter.

    The following conclusions were not warranted by the findings: […]

    The FoxA2+/Sox17+ endoderm progenitors never transitioning through the mesenchymal intermediates and never leaving the epithelial compartment that they arise: In view of that the stereotypic morphogenetic activity was not documented during the development of the gastruloid, it is not possible to exclude the possibility of the progenitors undergo a partial EMT (loss of epithelial feature and cellular polarity and display of morphogenetic movement, as in vivo) in the transition from progenitor to the epithelial endoderm cells. The DE-like cells when first discerned in the gastruloid are apparently epithelialized. In the absence of lineage tracing results, It is not clear whether they are still residing in the "epithelial compartment that they arise".

    We agree with the reviewer’s comment: the ability to trace endodermal cells throughout their journey in the Gastruloid and throughout differentiation, specifically in conjunction with a live monitor of their epithelial status (e.g. overlayed with a Cdh1 reporter) would provide clear and definitive insight on the endodermal and epithelial transitions taking place in this system. Our conclusions are based on timed immunostaining showing that in early Gastruloids all cells are epithelioid. Finding FoxA2+ (and Sox17+) cells consistently within a Cdh1+ context, while also having necessarily emerged within an epithelioid context, suggests that these cells never leave an epithelial compartment. Within the text, we also put forwards alternative hypotheses equally consistent with our observations: namely that these cells would indeed leave the epithelial compartment (still, through incomplete EMT processes not relying on Snai1 programmes), but reintegrate it at short timescales. We do not find FoxA2+ cells within the mesodermal compartment, as one would expect from comparison with the embryo, and we do not see Snai1 expression within Cdh1+ cells. We would like a live tracing system whereas we could track endodermal identity (e.g. a FoxA2 reporter) while being also able to track its epithelial (E-cadherin, Cdh1) status. We do not foresee to be able to perform such experiment in the near future. Live tracking of Cdh1+ cells in Gastruloids has been described in [Hashmi, Ali, et al. "Cell-state transitions and collective cell movement generate an endoderm-like region in gastruloids." BioRxiv (2020).].

    We use the term “mesenchymal” to signify “not-epithelial”, and as such “Cdh1-negative”. When we hypothesise endoderm cells not to go through EMT, we imply a classical, complete, Snai1-mediated EMT. By “leaving the epithelial compartment” we mean “losing Cdh1 expression” (and as such not being associated anymore with the epithelial/epithelioid compartment). As such, we are not excluding the possibility put forwards by the reviewer: i.e. endodermal progenitors going through a partial EMT with loss of epithelial architecture, but not epithelial markers, and movement in this epithelioid state. In fact, this is the interpretation we are favoring in our report. We will clarify our use of each term within the text of the preprint and provide more clarity to these points. Accordingly, we will rephrase each of the terms above (“mesenchymal”, “absence of EMT”, and “epithelial compartment”) in terms of the Cdh1 and Snai1 status of the cells.

    __The mature endoderm cells are patterned segmentally in the gastruloid. The findings that the molecular phenotype (marker expression) of the mature endoderm cells "aligns with (cellular) identities along the entire length of the embryonic gut tube" are not sufficient evidence of spatial A-P patterning of endoderm cells. The expression pattern of Foxa2/Cdh1 (Fig 5d) was not informative of tissue patterning. __

    We share the reviewer’s point that alignment of the molecular phenotype (transcript expression) of Gastruloid cells with that of cells along varying position of the gut tube of the embryo does not in fact necessarily imply that these cells are spatially patterned within the Gastruloid. We were deliberate in our presentation of the data on this point, and in fact explicitly presented to readers the equally probable possibility that “the variety of cell identities uncovered in the single cell dataset are intermingled throughout the core of the Gastruloid” (rather than being spatially patterned; lines 787-789). This possibility is in fact provided as the rationale to highlight the need for alternative investigations able to provide spatial information (provided in the following section).

    Promisingly, the markers we did investigate (Pax__9__ for anterior endoderm; Cdx2 and TBra for posterior identities) were found to not be intermingled throughout the primordium but correctly expressed at anterior and posterior domains (as already known for TBra and Cdx2 from previous Gastruloid literature). We do agree that showing the distribution of AP markers within a same sample would provide a more immediate and compelling visual of the AP patterning of anterior and posterior markers. We also agree that the number of markers we investigated spatially is still restricted, and further characterisation, specifically of middle markers, would provide a more complete picture of the extent to which the Gastruloid primordium is effectively patterned. We would like however to point out that we do not make claims of spatial patterning beyond those supported by the markers we did confirm by immunostaning/HCR; i.e. we only claim spatial patterning of anterior and posterior domains (“Gastruloid endoderm contains patterned anterior and posterior endodermal types”; line 663).

    We agree with the reviewer that the expression pattern of Foxa2/Cdh1 is not informative of tissue patterning. When using these markers on Gastruloids, we indeed use them as “pan-endodermal” markers to identify the general cellular domain at the core of the Gastruloid.

    […] Whether endoderm cells are patterned or not is, however, irrelevant for the understanding of the mode of endoderm formation, unless the timing and the mechanism of allocation of endoderm cells of specific segmental property has been studied in the gastruloid.

    The relevance of the specific set of results indicated by the reviewer (i.e. the topic of patterning of endodermal cells in the Gastruloid) is maybe better appreciated in the context of understanding the modes of later development and maturation of endoderm in vitro, and its self-organising and self-patterning abilities after it has formed, rather than provide direct insights into the formation of the germ layer itself. In the preprint, we indeed present this investigation as a segue to the first set of experiments that instead focus on the formation of the endoderm itself. As the reviewer points out, we have not investigated the specific aspect of timing and mechanisms of allocation of endoderm cells to specific segmental identities as these cells are emerging within the Gastruloid. Our reporting that Gastruloids contain endodermal identities that do end up specified to different segmental identities in the first place sets the basis for the kind of investigation the reviewer suggests.

    __it is also unclear if a structure reminiscent of the embryonic gut (closed or partly open) was formed (or self-organised) in the gastruloid. __

    We thank the reviewer for raising this point. The structures we describe are initially multi-branched and whisk-shaped (120h), and in turn resolve a single rod-like tissue that follows the outer geometry of the Gastruloid (144h), interfacing with an outer envelope of mesenchymal (non-endodermal) cells. We provide depth-coded maximal intensity projection of these structures (under Cdh1 immunostaining) to try to best convey images of their three-dimensional shape (Figure 4A, Figure 5C). While we believe this epithelial primordium to be fully closed and non-hollow, in fact quite different than the folding/folded epithelium of the post-gastrulating mouse gut endoderm, a better idea of the 3D organisation of this primordium could certainly be provided by outputs from light sheet imaging and series of optical sections along the z-axis of our immunostained samples. We plan to include these alternative visualisations and in the meantime refer to already published light-sheet data as found in [Rossi, Giuliana, et al. "Capturing cardiogenesis in gastruloids." (2021)]. Here too, the endodermal primordium appears as a dense, closed mass of cells. To contextualise these structures with respect to most recent literature, we do not see the kind of vacuolated and segmented structures described instead in https://doi.org/10.1038/s41467-021-23653-4 [Xu, Peng-Fei, et al. "Construction of a mammalian embryo model from stem cells organized by a morphogen signalling centre." (2021)]

    The information regarding the spatial localization of specific germ layer markers in the gastruloids at different timepoints would be important to understand how the morphology progresses and how it is comparable to the developing embryo itself. How is the organisation of the mesoderm and endoderm layers in comparison to embryo in the early timepoints and later timepoints of gastruloids?

    We agree with the reviewer about the helpfulness of such characterisations. While a temporal characterisation of the evolution of the endoderm compartment in relationship to the other cell types is provided in Figure 4A, cells of the other two germ layers are not explicitly labelled. We will provide analogous immunostaining series across Gastruloid development (early to late timepoints), choosing markers able to highlight cells of each of the three germ layers. Given the difficulty of finding specific germ layer markers, and the often-unforgiving limitations on the markers that can be chosen for simultaneous staining due to host-species antibody cross-reactivity, we also find useful to piece together relative germ layer localisation from the much wider imaging data now widely available in previous Gastruloid literature. We thus point interested readers to complement the endoderm descriptions from our preprint with dedicated characterisations of the axial organisation and distribution of the other two germ layers in Gastruloids published in https://doi.org/10.1038/s41586-018-0578-0 [Beccari, Leonardo, et al. "Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids." Nature 562.7726 (2018): 272-276.], which also specifically discusses these aspects in relation to the germ layer organisation of the embryo proper.

    Clarify if Foxa2 and Sox17 double positive cells exist in the Cdh1 patches (Fig 3a). In Fig 4, authors have demonstrated the development of epithelial primordium with overlaying mesodermal wings, however it is important to show if Foxa2, Sox17, or other definitive endoderm markers co-express in these cells.

    We thank the reviewer for highlighting the value of co-localisation data, and in fact this is a suggestion also put forward by our other reviewer. We are currently developing a pipeline to segment the nuclei on the DAPI channel of each immunostained Gastruloid, and extract marker intensities within each cell. We envisage the results to be presented in a format similar to what shown e.g. in Fig. 2 of https://doi.org/10.1242/dev.159103 [Mulas, Carla, et al. "Oct4 regulates the embryonic axis and coordinates exit from pluripotency and germ layer specification in the mouse embryo." (2018)]. Such data would undoubtedly strengthen the reliability of the claims we here draw mostly from a qualitative inference of colocalisation. When showing the distribution of markers within the 120h epithelial primordium, the assumption was that since the entire primordium is FoxA2+, any other marker would colocalise with a subset of them, when expressed within the primordium. We do note the importance of colocalisation data for a more exact description of the cell type identities contained within the primordium.

    It was suggested that E-Cadherin is maintained during endoderm differentiation. N-cadherin expression may be examined to determine if N-cad is expressed in the other region of gastruloids.

    We share the interest in describing N-cadherin expression patterns, especially in the context of EMT development and endoderm epitheliality, and thank the reviewer for highlighting the value of this marker. To the time of publication, we had been unfortunately unable to source a N-cadherin antibody giving good signal quality in our hands. We are planning further staining of early and late Gastruloids for this marker. Given what recently reported in https://doi.org/10.1038/s41556-021-00694-x [Scheibner, Katharina, et al. "Epithelial cell plasticity drives endoderm formation during gastrulation." (2021)] we expect endodermal cells to display double cadherin expression (Cdh1+/Cdh2+), and the mesodermal compartment to display N-cadherin instead only.

    In Fig 6, FACS quantification is not proportional to the expression of the TBra:GFP as shown in the microscopic images at 96 hr, 120hr. Fig 6D does not show the TBra:GFP positive cells on the y -axis in the top-left quadrant, even though it is quite visible in microscopy - at 96, 120 hr. Microscopic images suggest TBra signal is almost completely lost at 128hr whereas FACS does not represent that. Infact, at 120 hours, the plot shows opposite of what microscopy shows.

    The reviewer is correct in pointing out these discrepancies and we will make sure to flag them explicitly in the main body of our report. It appears that the trend in reporter expression highlighted by FACS data is delayed with respect to what visible by live imaging (where TBra loss of expression can already be seen starting at t=120h). The decrease in TBra expression does not instead seem to be recovered even by the last FACS timepoint. Concomitantly, the TBra signal detected during time-lapse seems to decrease to abnormally low level (indeed, be lost), especially if compared to equivalent timepoints processed for FACS. We will investigate whether TBra reporter signal is particularly vulnerable to sustained illumination (i.e. timelapse conditions), as it appears to be still be present at later timepoints in both FACS and end-imaged Gastruloids (see clear posterior expression in Fig 6B). Maintained presence of TBra expression is also what expected and detected by immunostaining in both our data and in general Gastruloid literature. In light of the possible effects of sustained imaging on our reporters, we only use timelapse data to describe global cell movement (and of the FoxA2+ cells, rather than changes in reporter expression), and instead rely on the FACS data for claims based on intensity values. While we resolve the effect of timelapse imaging on TBra reporter detection, we will explicitly highlight the discrepancy between the two investigative approaches within the Results section, and thank the reviewer for bringing this point to our attention.

    Gastruloids were sampled at 96-168 hours for single cell transcriptome analysis. However, the specimens documented in this study were those only up to 144 hours. How does the gastruloid morphology look at 168 hours? It is essential to show the morphology and characterise the further development from 144 to 168 hours, to compare the single cell RNA seq data with the morphology of the gastruloid.

    The reviewer is correct in pointing out the absence of imaging data showing the internal endodermal primordium at t=168h. Examples of 168h Gastruloids (but not of the internal primordium) are shown in Figure 8D, indeed when we compare transcriptomics data with spatial patterning and verify the spatial distribution of late gut markers. At this stage, the internal endodermal primordium is mostly similar to its configuration at t=144h. We will incorporate additional morphological data for Gastruloids at 168h to show the organisation of the endodermal primordium at this later stage and to facilitate morphologytranscriptome comparisons.

    In Fig 7, it is surprising to see that the proportion of cells in the two clusters 13, 4 that mark endoderm are a minor portion of the whole dataset collected, whereas the microscopic images suggest that the majority of the gastruloid structure from 120hr onwards is marked by Foxa2 and shows the epithelial primordium morphology as claimed.

    Microscopic images are optical sections through the midplane of each Gastruloid as to capture the full extent of the internal Gastruloid epithelial primordium. We believe that these pictures fail to accurately convey the degree to which this primordium is in fact completely surrounded by a thick and dense layer of mesenchymal cells, not only in the lateral dimension as can be appreciated e.g. in Figure 4B, but also above and below the plane of the microphotographs. A better idea of the volume occupied by such cells, and the proportion of endodermal vs non-endodermal cells, could be provided by lightsheet imaging. At later timepoints, and as hinted by e.g. panel 4B [144h], the volume occupied by non-endodermal cells can be considerable. Linking back to a previous suggestion from the reviewer, we believe documentation of Gastruloid morphology at 168h could help to further clarify the relationship between data coming from the single cell dataset and morphological data as seen from the Gastruloid themselves. This might also be a further opportunity to underscore that the single-cell dataset accessed for our endoderm-targeted analysis was produced in the context of a different study https://doi.org/10.1016/j.stem.2020.10.013 [Rossi, Giuliana, et al. "Capturing cardiogenesis in gastruloids." (2021)], in which Gastruloids made with this same cell line were additionally treated with cardiogenesis-inducing factors. The proportion of cells classified as cardiac mesoderm and associated mesodermal types is thus likely much over-represented compared to what present in non-induced Gastruloids (i.e. those considered in this report), and as in fact illustrated by the imaging data presented in the paper.

    The single-cell RNA-seg data should be analysed for the co-expression of multiple segment-specific cell markers to ensure that the mature endoderm cells align with high-confidence with the known cell types in different segments of the embryonic gut, and that the localization of representative cell types can be validated spatially along an endoderm structure in single gastruloids.

    We will analyze the single-cell RBAseq dataset to show the co-expression of segment specific markers. As the reviewer points out we have only shown the patterns of expression of single markers at a time. As mentioned in a previous point, we also agree that the number of markers we verified to be spatially pattern is still restricted, and further characterisation, specifically of middle markers, would provide a more complete picture of the extent to which the Gastruloid primordium is effectively patterned. We do also agree that showing the distribution of AP markers within a same sample would provide a more immediate and compelling visual of the AP patterning of anterior and posterior markers.

    It is not clearly indicated how many replicates were performed to assure consistency/reproductivity of the gastruloid results. Statistical results were not provided for most of the immunostaining experiments, either in the main text or in the figure legends.

    Both reviewers highlight the qualitative nature of much of the data that is presented. Accordingly, we will more clearly and more consistently indicate the number of samples analysed and the number of replicates considered. For what concerns the “statistical results” of immunostaining experiments, and as indicated in response to a previous comment, we envisage to provide such results in a format similar to what used e.g. in Supplementary Figure 4 of https://doi.org/10.1038/s41467-021-23653-4 [Xu, Peng-Fei, et al. "Construction of a mammalian embryo model from stem cells organized by a morphogen signalling centre." (2021)]; i.e. categorization of the phenotypes observed among gastruloids, and quantification of their proportions. To convey statistics on the variability among Gastruloids, our current pipeline can output scatterplots like the one presented in Figure 4C, where the datapoint spread informs about the variability of the data. We will provide statistics on these plots in a numerical format.

    Majority of the images presented in the manuscript are shown as Maximum Intensity Projections, and it is not clearly stated if the localisation of the cells expressing specific protein markers are present on the surface or in the internal layers of the gastruloids. Optical slices of the gastruloid images may be presented as supplementary information.

    Most of the images presented in the manuscript are presented as optical cross-section through the midplane of the Gastruloid. We can make this clearer in the text. Indeed, and since this report focuses on on the spatial distribution of markers along the AP axis of the Gastruloids (and not DV or LR axes), we found midplane optical sections to best capture the entirety of the axis and thus the full extent of marker pattern (where those patterns are not asymmetric along the DV axis). All cells positive for immunostained markers are thus to be interpreted to be within the midplane of the Gastruloid, and as such internal to the Gastruloid if falling internally to the midplane, and external to the Gastruloid when falling towards the edges. As suggested by the reviewer we will acquire optical sections along the z-stack of our immunostained samples and provide them as supplementary information. Maximum intensity projections were only shown when wanting to better show the 3D structure of the internal epithelial primordium, and were always depth-coded to aid visualisation (Figure 4A, rightmost panel, Figure 5C).

    - Are prior studies referenced appropriately?

    Yes, except for the study on endoderm formation by lineage tracing in vivo, high-resolution single-cell analytics and functional analysis of genetic mutant embryos.

    We thank the reviewer for pointing out inappropriate referencing of studies on these three topics, yet given the breadth of each of these topics and the absence of any more specific information we remain unsure on how to address this comment appropriately. We would thus like to warn readers that this comment might have remained unaddressed.

    Results may be presented with reference to the data figures in the appropriate sequential order, or the figures may be re-organised to match the presentation in the Results.

    We thank the reviewer for bringing this to our attention. The figures are automatically organised in the order they are presented in the Results, yet since most of the figures are page-long their placement may appear odd. We will look into the matter and readjust figure positioning where possible, and/or reduce mentions of data shown in previous figures.

    Reduce the verbosity throughout the manuscript, especially the Results and Discussion.

    As this is also a point raised by both reviewers, we will make sure to go back over the entirety of the manuscript and do the necessary adjustments, especially for what concerns the Results section. We would however like to point out that the unusual length of our Introduction section is to be understood as a deliberate departure from the limits and conventions prescribed by traditional publishing formats. In line with our intentional choice of a journal-independent publication platform (i.e. a preprint server), and of journal-independent review process, we also presented and contextualized our research in a journal-independent format. We see this as an opportunity to present research in a voice truer to that of the researchers that carried it out. We thank both reviewers for their feedback on the format and will certainly still make sure to minimize redundancy of information throughout our manuscript.

    __This study on endoderm development, however, is confounded by the inherent limitation of the experimental model: lack of extraembryonic tissue components, the atypical morphological structure and the deviation from the in vivo schedule of development and morphogenesis. This may raise doubt of the relevance of the findings to the __

    We find difficult to share the view expressed by the reviewer here. The “lack of extraembryonic tissue components, the atypical morphological structure, and the deviation from the in vivo schedule of development and morphogenesis”, which they identify here as the inherent limitations of the model, represent in fact its value for us and for most in the Gastruloid field. For more complete and articulated discussion on how it is precisely the differences with the embryo proper that provides insights when studying in vitro models of embryonic development, we refer interested readers to dedicated discussions on the topic [van den Brink, Susanne C., and Alexander van Oudenaarden. "3D gastruloids: a novel frontier in stem cell-based in vitro modeling of mammalian gastrulation." (2021); Simunovic, Mijo, and Ali H. Brivanlou. "Embryoids, organoids and gastruloids: new approaches to understanding embryogenesis." (2017); Turner, David A., Peter Baillie‐Johnson, and Alfonso Martinez Arias. "Organoids and the genetically encoded self‐assembly of embryonic stem cells." (2016)).]. In fact, we share the reviewer’s concern about the relevance of these findings to “understanding of the morphogenetic activity and molecular control of endoderm formation during gastrulation in the embryo”. The constant questioning of such relevance is in fact a central point in Gastruloid research, where insight comes from a dialectic comparison between what observed in vitro and what known to happen in vivo. The reviewer questions the relevance of the findings “to the understanding of the morphogenetic activity and molecular control of endoderm formation during gastrulation in the embryo”. The relevance of our findings might be better framed in that they provide a better “understanding of the morphogenetic activity and molecular control of endoderm formation” *tout court. *In this case, outside of the embryo (and inside a self-organising developmental system), and reflections on this inform better understanding of how endoderm might be developing in vivo.

    Our findings in the Gastruloid open lines of inquiry to be verified and tested in the embryo. Both similarity and differences being equally informative on the intrinsic and extrinsic elements of endoderm behaviour. In fact, where some of the aspects we describe have been investigated in the embryo proper (see [Scheibner, Katharina, et al. "Epithelial cell plasticity drives endoderm formation during gastrulation." (2021)]) many of the same themes have emerged.

    Knowledge gleaned from the present study on the gastruloid study added little to that of a recent study of the morphogenetic program of endoderm formation in the mouse embryo and the ESC differentiation model (https://doi.org/10.1038/s41556-021-00694-x ) .

    The reviewer is absolutely correct in mentioning [Scheibner, Katharina, et al. "Epithelial cell plasticity drives endoderm formation during gastrulation." (2021)] to the readers of this public review. Given that these results were published posteriorly to our preprint, we could only reference them in our later versions, and we did this extensively throughout the text. We consider the paper mentioned by the reviewer [Scheibner, Katharina, et al. "Epithelial cell plasticity drives endoderm formation during gastrulation." (2021)] to represent a major contribution to the topic of (mouse) endoderm development, specifically in its investigation of endoderm EMT mechanisms as they take place within the mouse embryo itself. In relationship with what we describe here in Gastruloids, we see what reported by Scheibner’s et al extremely validating and as a very strong example of the investigative validity of in vitro models of development. Here is the in vivo exploration of the same topic highlighting many of the endoderm features we had inferred from *in vitro *observations, or that our observations further supported (specifically, incomplete EMT, tight association with epithelioid character, low evidence for mesendodermal intermediates etc..).

    To say that our study added very little to what now available in [Scheibner, Katharina, et al. "Epithelial cell plasticity drives endoderm formation during gastrulation." (2021)] represents however an inaccurate view of our study as being a study of endoderm development in vivo (see also answer to previous point). We would also want to point out that a major portion of this preprint describes the self-organisation of endoderm in vitro, the emergence and development of almost all AP-endoderm identities by self-organisation, the effective spatial patterning of at least some of these (waiting further characterisation), and the description of an accessible, tractable, reproducible in vitro model system to study endoderm development and provide populations of interest for culture. Our study also provides further insight on the necessary inputs to endoderm development and patterning, and whether extracellular matrices and extraembryonic tissues are part of such necessary inputs. Sharing the view expressed by the other reviewer, we see great insight from all of these aspects, and these are certainly not the topic or focus of the study referenced by this reviewer.

    As we do throughout the preprint, we strongly encourage readers interested in the topic to refer to [Scheibner, Katharina, et al. "Epithelial cell plasticity drives endoderm formation during gastrulation." (2021)] for insights coming from the embryo proper. We also take the opportunity to stress the value of all types of science, be it incremental, consolidating, or complementary.

  2. Review Commons

    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

    • Are the key conclusions convincing?

    The following conclusions were not warranted by the findings: Endoderm emergence can occur in the absence of extraembryonic tissues and embryonic architecture: It is unclear if extraembryonic tissues (e.g., primitive endoderm and visceral endoderm-like cells) are absent in the early phase of gastruloid development. While the gastruloid does not replicate the morphological feature of the post-gastrula embryo, it nevertheless has a certain degree of tissue organization. Perhaps the emergence of DE-like cells in 2-D culture would be a more convincing model for "the absence of extraembryonic tissues and embryonic architecture".
    The FoxA2+/Sox17+ endoderm progenitors never transitioning through the mesenchymal intermediates and never leaving the epithelial compartment that they arise: In view of that the stereotypic morphogenetic activity was not documented during the development of the gastruloid, it is not possible to exclude the possibility of the progenitors undergo a partial EMT (loss of epithelial feature and cellular polarity and display of morphogenetic movement, as in vivo) in the transition from progenitor to the epithelial endoderm cells. The DE-like cells when first discerned in the gastruloid are apparently epithelialized. In the absence of lineage tracing results, It is not clear whether they are still residing in the "epithelial compartment that they arise".

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

    The mature endoderm cells are patterned segmentally in the gastruloid. The findings that the molecular phenotype (marker expression) of the mature endoderm cells "aligns with (cellular) identities along the entire length of the embryonic gut tube" are not sufficient evidence of spatial A-P patterning of endoderm cells. Only the spatial regionalization of Pax6-expressing cells (Fig. 8) and Cdx2-expressing cells (Fig 4C) were shown on different gastruloid specimens. The expression pattern of Foxa2/Cdh1 (Fig 5d) was not informative of tissue patterning. It is also unclear if a structure reminiscent of the embryonic gut (closed or partly open) was formed (or self-organised) in the gastruloid. Whether endoderm cells are patterned or not is, however, irrelevant for the understanding of the mode of endoderm formation, unless the timing and the mechanism of allocation of endoderm cells of specific segmental property has been studied in the gastruloid.

    • 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.

    Specific points:

    1. The information regarding the spatial localization of specific germ layer markers in the gastruloids at different timepoints would be important to understand how the morphology progresses and how it is comparable to the developing embryo itself. How is the organisation of the mesoderm and endoderm layers in comparison to embryo in the early timepoints and later timepoints of gastruloids?
    2. Clarify if Foxa2 and Sox17 double positive cells exist in the Cdh1 patches (Fig 3a). In Fig 4, authors have demonstrated the development of epithelial primordium with overlaying mesodermal wings, however it is important to show if Foxa2, Sox17, or other definitive endoderm markers co-express in these cells.
    3. It was suggested that E-Cadherin is maintained during endoderm differentiation. N-cadherin expression may be examined to determine if N-cad is expressed in the other region of gastruloids.
    4. In Fig 6, FACS quantification is not proportional to the expression of the TBra:GFP as shown in the microscopic images at 96 hr, 120hr. Fig 6D does not show the TBra:GFP positive cells on the y -axis in the top-left quadrant, even though it is quite visible in microscopy - at 96, 120 hr. Microscopic images suggest TBra signal is almost completely lost at 128hr whereas FACS does not represent that. Infact, at 120 hours, the plot shows opposite of what microscopy shows.
    5. Gastruloids were sampled at 96-168 hours for single cell transcriptome analysis. However, the specimens documented in this study were those only up to 144 hours. How does the gastruloid morphology look at 168 hours? It is essential to show the morphology and characterise the further development from 144 to 168 hours, to compare the single cell RNA seq data with the morphology of the gastruloid.
    6. In Fig 7, it is surprising to see that the proportion of cells in the two clusters 13, 4 that mark endoderm are a minor portion of the whole dataset collected, whereas the microscopic images suggest that the majority of the gastruloid structure from 120hr onwards is marked by Foxa2 and shows the epithelial primordium morphology as claimed.
    7. The single-cell RNA-seg data should be analysed for the co-expression of multiple segment-specific cell markers to ensure that the mature endoderm cells align with high-confidence with the known cell types in different segments of the embryonic gut, and that the localization of representative cell types can be validated spatially along an endoderm structure in single gastruloids.
    • 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.

    The suggested experiments may be accomplished in a few months.

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

    Yes for the methods. It is not clearly indicated how many replicates were performed to assure consistency/reproductivity of the gastruloid results.

    • Are the experiments adequately replicated and statistical analysis adequate?

    Statistical results were not provided for most of the immunostaining experiments, either in the main text or in the figure legends.

    Minor comments:

    • Specific experimental issues that are easily addressable.

    Majority of the images presented in the manuscript are shown as Maximum Intensity Projections, and it is not clearly stated if the localisation of the cells expressing specific protein markers are present on the surface or in the internal layers of the gastruloids. Optical slices of the gastruloid images may be presented as supplementary information.

    • Are prior studies referenced appropriately?

    Yes, except for the study on endoderm formation by lineage tracing in vivo, high-resolution single-cell analytics and functional analysis of genetic mutant embryos.

    • Are the text and figures clear and accurate?

    Results may be presented with reference to the data figures in the appropriate sequential order, or the figures may be re-organised to match the presentation in the Results.

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    While taking into consideration the limitation of this embryo model for studying morphogenesis, highlight the interesting/unique findings of the gastruloid study, albeit they may have been discovered in the study of embryos in vivo.

    Reduce the verbosity throughout the manuscript, especially the Results and Discussion.

    Significance

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

    This study demonstrated that definitive endoderm (DE)-like cells can be generated in the stem cell-derived embryo-like structure, the gastruloid. This observation is significant for that gastruloids can serve as an amenable experimental model, in comparison to other 2D invitro differentiation models, for elucidating the requisite cellular process in endoderm differentiation and the acquisition of cell identity. It was inferred that, in the gastruloid, epithelial-mesenchyme transition may not be a requisite cellular process for the formation of the DE-like cells and that endoderm formation may not involve progression through an intermediate mesenchymal state. The progenitor cells may have acquired and retained the attribute of epithelization during differentiation and organization the DE-like cells into an endoderm layer.
    This study on endoderm development, however, is confounded by the inherent limitation of the experimental model: lack of extraembryonic tissue components, the atypical morphological structure and the deviation from the in vivo schedule of development and morphogenesis. This may raise doubt of the relevance of the findings to the understanding of the morphogenetic activity and molecular control of endoderm formation during gastrulation in the embryo.

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

    Knowledge gleaned from the present study on the gastruloid study added little to that of a recent study of the morphogenetic program of endoderm formation in the mouse embryo and the ESC differentiation model (https://doi.org/10.1038/s41556-021-00694-x ) . This recent study has advanced the understanding of the functional attributes that segregate the endoderm progenitors and mesoderm progenitors in the primitive streak and the posterior epiblast, and has characterised the role of epithelial plasticity and the modulation of EMT activity under the control of Forkhead box transcription factor A2 and modulation of WNT signalling in the formation of the definitive endoderm.

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

    Developmental biologists, stem cell scientists and embryo modellers

    • 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.

    Mouse embryogenesis, gastrulation, in vitro stem cell differentiation, advanced microscopy, single cell transcriptomics.

  3. Review Commons

    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

    The study adresses a long-standing question in mouse gastrulation: the existence of a mesendodermal progenitor, similar to other species. Recent data in the mouse point towards a direct transition from epiblast to endoderm without going through a bipotent progenitor, and without loss of epithelial characteristics (notably Probst 2021). The authors take advantage of the gastruloid system to follow the emergence of endoderm cells. Through co-staining with markers of various germ layers at different timepoints, live imaging, and reanalysis of single cell RNASeq data, they propose a model in which endoderm cells differentiate from E-cadherin positive cells without going through a bipotent stage. Interestingly, they then organise a rod like structure along the anterior-posterior axis of the gastruloid, that display some polarity illustrated by a marker of anterior gut.

    The data present show very high reproducibility, and authors fully exploit the organoid system by analysing a large amount of samples and showing very similar results. The results are qualitatively very convincing. For the sake of clarity, it may help to provide some table illustrating the proportion of gastruloids behaving precisely as the best example shown. It would also strengthen the message even more to provide some quantitation of co-expression for the main markers. As the behaviour seems very consistent, it is likely that such quantification would not be very arduous, and it would show the strength of the model.

    The manuscript is pleasantly written and all statements are clearly explained. It is a bit long though, in particular the introduction, some of which might read more like a review of the field. It is less striking in the Results part, but there is some level of repetition that is a bit distracting.

    Significance

    The question is important and timely, and the data are clear and convincing. There have been a number of publications addressing it in the last years, either in the embryo or in gastruloids (notably Hashmi 2021). All data appear to point in the same direction, and this study is certainly an important contribution. An original aspect to my knowledge is the self organisation of the central rod. The system is simple, reproducible, and opens novel possibilities to dispose of a large number of cells to explore the emergence of endoderm subpopulations.

    The fact that several studies converge is rather an advantage as they all have specificities, and I believe they are adequately cited here.

    In summary this is an important and well conducted study that may just benefit from some additional quantification to prove robustness. In terms of writing, it is pleasant and quite literary, but perhaps a little bit too much so. The introduction could be shortened, and the results a bit more to the point.

  4. Version published to 10.1101/2020.06.07.138883v3 on bioRxiv
  5. Version published to 10.1101/2020.06.07.138883v2 on bioRxiv
  6. Version published to 10.1101/2020.06.07.138883v1 on bioRxiv