Robust axis elongation by Nodal-dependent restriction of BMP signaling

  1. Alexandra Schauer
  2. Kornelija Pranjic-Ferscha
  3. Robert Hauschild
  4. Carl-Philipp Heisenberg

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Abstract

Embryogenesis results from the coordinated activities of different signaling pathways controlling cell fate specification and morphogenesis. In vertebrate gastrulation, both Nodal and BMP signaling play key roles in germ layer specification and morphogenesis, yet their interplay to coordinate embryo patterning with morphogenesis is still insufficiently understood. Here, we took a reductionist approach using zebrafish embryonic explants to study the coordination of Nodal and BMP signaling for embryo patterning and morphogenesis. We show that Nodal signaling triggers explant elongation by inducing mesendodermal progenitors but also suppressing BMP signaling activity at the site of mesendoderm induction. Consistent with this, ectopic BMP signaling in the mesendoderm blocks cell alignment and oriented mesendoderm intercalations, key processes during explant elongation. Translating these ex vivo observations to the intact embryo showed that, similar to explants, Nodal signaling suppresses the effect of BMP signaling on cell intercalations in the dorsal domain, thus allowing robust embryonic axis elongation. These findings suggest a dual function of Nodal signaling in embryonic axis elongation by both inducing mesendoderm and suppressing BMP effects in the dorsal portion of the mesendoderm.

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  5. Version published to 10.1242/dev.202316
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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    The manuscript by Schauer et al. uses embryonic explants to study the coordination of Nodal and BMP signaling for embryo morphogenesis. They show that Nodal signaling triggers explant elongation by inducing mesendodermal progenitors that undergo cell intercalation. Looking at the role of BMP signaling, they show that BMP overactivation ventralizes the explants, reducing cell intercalation and therefore explant elongation. Looking at pSmad5, they then establish that BMP signaling in the explant is attenuated by Nodal signaling, through activation of chordin expression, and through some unidentified chordin-independent mechanisms. Moving to the entire embryo, using combinations of BMP overexpression and Nodal inhibition, authors show that Nodal signaling limits BMP signaling on the dorsal side of the embryo, which is key to proper embryo elongation.

    Major comments:

    • The authors used the sebox::EGFP line to show that the growing region of the explant consists mostly of mesendodermal cells. Although this transgenic line had not been used to do so, the authors and others, had previously demonstrated that the extending part of the explant is mostly made of mesoderm and even shows some patterning (1,2). This should be stated and not presented as a new finding.
    • Explant elongation is driven by cell intercalation. The authors analyzed the shape of the mesendodermal tissue to conclude that cells intercalate. While I do not question this conclusion, as it is well known in the embryo, direct observation of cell intercalation, as was done in the embryo (3), would be a better demonstration.
    • Explant elongation is driven by mesendodermal cell intercalation. I certainly agree from the movies and images that the extending region is mostly made of mesendoderm. However, it seemed clear to me that in Movie 1, starting at about 140 minutes, most of the convergence movement is taking place in a non-green region of the explant, fueling the extension of the mesendodermal region. Also, to demonstrate that cell intercalation is occurring in the mesendoderm, the authors performed clone dispersal analysis, comparing clones of mesendodermal and ectodermal cells. However, the selected ectodermal clone is very far from the extending region. To show that the cell intercalation is specific to mesendoderm, I think the authors should try to compare the behavior of mesendodermal and non-mesendodermal cells that are located at the same distance from the extending region. For example, from the image in Figure 1E (235 mpe), it appears that the right side of the base of the extended region is not green and could be compared to the left side. Currently, the quantification shown in 1G mostly demonstrates that the extending region is extending, and that the non-extending region is not.
    • Based on their observations in explants, the authors propose that Nodal signaling maintains an area of low BMP signaling on the dorsal side of the gastrula for robust axis elongation. While I acknowledge that the experiments performed by the authors have not been previoulsy reported, I did not understand how this differs from the very well established fact that Nodal inhibits BMP signaling, in particular through chordin expression. Von der Hardt for instance already reported that overexpression of bmp and inhibition of chordin leads to severe elongation defects (4). More insight could probably be gained by analyzing the effect in more detail: Is the elongation defect due to cell intercalation defects? How are cell fates affected? Is this Nodal effect mediated by Chordin?...

    Minor comments:

    • Fig6B. Are the curves significantly different? If so, how were they compared?
    • Fig6D-E, I found the quantification a bit confusing. The reader is left with the impression of an all-or-nothing answer (effect only with BMP overexpression and strong Nodal inhibition), whereas the effect on the pSmad5 gradient is gradual. Plotting and comparing the pSmad5 intensity gradients would be better.
    • Fig6G. 'Axis length/embryo height' should appear on the x-axis, not the y-axis.

    Referees cross-commenting

    I feel that the three reviews are very much in agreement, recognising that the experiments carried out are well done and calling for a reasonable amount of additional data. The three reviews also agree that the results obtained here in explants were already known from intact embryos, limiting the relevance to ex vivo research.

    Significance

    Overall, the experiments appear carefully carried out, and very precisely quantified. The paper is well written and easy to read. The results add to our understanding of the morphogenetic events occurring in embryonic explants. I therefore support their publication.

    My main concern is with the significance of the results. I am convinced that embryonic explants are great tools, to reduce the complexity of the embryo and to address questions that cannot be addressed in the embryo, as the authors and others have done, for instance, to address the role of extraembryonic tissues and patterning by maternal contributions. Here, however, I felt that most, if not all, of the experiments essentially demonstrate in embryonic explants, results that have been known for years in the intact embryo. While gathering detailed information on what happens in embryonic explants will certainly prove useful in further understanding the self-organizing abilities of these explants, and is worth publishing, the significance of the results reported here seems limited. Specifically, that elongation is driven by cell intercalation, that BMP-mediated dorsoventral patterning affects cell intercalation, that BMP signaling is attenuated by Nodal through Chordin, that Chordin is required for elongation, has been well established in the embryo over the last 20 years. Again, showing that it works the same way in embryonic explants is of interest, but at this point, does not add to our understanding of embryonic development.

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    Referee #2

    Evidence, reproducibility and clarity

    This paper from the Heisenberg lab takes a reductionist approach to understanding how BMP and Nodal signaling interact to coordinate morphogenesis. They mostly use blastoderm explants that they culture in vitro. These explants elongate over time, with Nodal signaling that induces mesendoderm driving the cell intercalations that explain the elongation. They show that increased BMP signaling inhibits this process, but reducing BMP signaling has no effect. They see that reducing Nodal signaling results in an upregulation of BMP activity as read out by phosphorylated Smad5 staining and increasing Nodal signaling has the opposite effect. They explain this mostly by the observation that Nodal induces the expression of the BMP antagonist, Chordin, and validate this idea by demonstrating that a reduction in Chordin expression reduces explant elongation. Returnign to the embryo, the authors show that manipulation of Nodal signaling levels influences the size of the BMP activity gradient as expected from the in vitro results. Finally, they show that reduction of Nodal signaling with SB505124 sensitises the embryos the effects of bmp2b overexpression, and that BMP overeactivation at 90% epiboly reduced C&E movements.

    Major comments

    In general I think the work is well done and the data justify the conclusions.

    I have several suggestions for additional experiments and discussion that I think would improve the paper.

    1. In Figure S1 they present data on elongation of explants treated with a Nodal inhibitor. It would be good to show some examples of images of the explants.
    2. In Figure 1G and 3A, the same wildtype images are shown. This is mentioned and I assume therefore that the results were all part of the same experiment. How many times were these experiments performed? It would be much better to use different biological replicates in the two figures.
    3. It is important for the authors to make clear how many biological replicates each of the experiments correspond to.
    4. In Figure 4E, it would be good to show the levels of P-Smad2 in the Oep and MZ lefty1, 2 explants.
    5. On page 11 the authors mention chordin-independent inhibition of BMP signaling. The most likely candidate would be noggin as it too is expressed dorsally and is at least in part activated by Nodal. This should be tested in their model.
    6. The authors focus on Chordin as downstream of Nodal signaling, and discuss the role of Nodal signaling in inducing chordin as being due to peak Nodal signaling. However, Chordin has been shown to also be downstream of Fgf signaling and Bozozok (PMIDs 23499658 and 16873584), which likely explains its dorsal expression domain. Furthermore, Rogers et al, (PMID 33174840) who the authors refer to, also show that to disrupt BMP signaling in embryos, inhibition of Nodal and Fgf is required. These issues need to be discussed in more detail. It is the combinatorial signaling that is thought to be responsible for the dorsal location of the chordin (and noggin) expression domains.

    Minor comments

    I think in general the manuscript is well written and the figures are clear. Previous data is generally well cited. My only comment is that there is a wealth of data from Xenopus and zebrafish that BMP antagonists are induced as a result of combinatorial Nodal signaling and other pathways (dorsal wnt and fgf) that inhibit BMP signaling. I think this could be better referenced.

    Significance

    The paper is well done and provides important information about the interactions between Nodal and BMP signaling to induce axis elongation. I think the work would be improved if the authors revise it along the lines suggested above. In terms of novelty, many of the component parts of the paper are known (Nodal signaling is important for elongation via cell intercalation and Nodal and BMP can antagonize on another by the induction of BMP antagonists by Nodal), but it is novel to put them together to investigate axis extension using explants. The paper will be of interest to those interested in how these signaling pathways operate in early vertebrate development and to those interested in morphogenesis.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    The authors presented intriguing observations on the molecular mechanisms regulating morphogenic cell movement, with a particular focus on convergent-extension (CE) movement associated with cell type specification in the zebrafish blastoderm explant. In this manuscript, Schauer et al. identified the CE movement of the mesendoderm as triggering the elongation of the zebrafish embryonic explant. In this process, the Nodal signal represses the BMP signal, which negatively regulates the movement of the mesendoderm precursors, through the induction of its inhibitor chordin. This suggests that the Nodal signal is the key factor coordinating cell fate specification and morphogenesis in the zebrafish blastoderm explant. Finally, suppression of Nodal signalling increases sensitivity to BMP signalling in the CE movement of intact embryos. This suggests that promotion of mesendoderm cell intercalation via BMP suppression by Nodal may be involved in conferring robustness to morphogenic cell movement in vivo.

    Major comments

    1. While one of the main conclusions of this manuscript is that "Nodal signaling regulates CE movement of mesendodermal cells by promoting their intercalation through inhibition of BMP signaling". However, this was predicted by changes in individual cell morphology and cell dispersal, and the authors didn't directly examine the behavior of individual cells. It would be better to confirm intercalation during the process of explant elongation by cell tracking analysis.
    2. Although the authors discuss that Nodal signaling inhibits BMP signaling in the later gastrulation stage, this has not been experimentally tested. If possible, the time window in which Nodal signaling acts should be investigated by temporal inhibition of Nodal signaling using chemical inhibitors.
    3. Only the signal gradient of pSmad5 and axis elongation were examined in the intact embryo part of the study (Fig. 6 and Fig. S7). The information on the domain of pSmad2 and the expression of chordin would be helpful for the comparison of the blastoderm explant and the intact embryos.

    Minor concerns

    The first letter of a gene name should be in lowercase. ( ex. Fig.S3C; Smad5 MO)

    Significance

    The zebrafish blastoderm explant assay has the potential to elucidate the molecular mechanisms regulating the complex processes of morphogenesis during vertebrate gastrulation, as the authors demonstrate in this paper. In this manuscript, the authors addressed the molecular mechanism coordinating cell fate specification and morphogenic cell movement in the blastoderm explant. All of the experiments are well-designed, the interpretation of the results is convincing and the paper is well-written. Also, the conclusion is very clear and well supported by the presented data. These findings provide fundamental and important insights for studying morphogenic cell movements in early vertebrate embryos using zebrafish blastoderm explants. On the other hand, most of the molecular mechanisms reported in this manuscript are already predicted by previous studies using intact embryos. Therefore, the impact of this work may be limited to ex vivo research.

  10. Version published to 10.1101/2023.06.19.545591v1 on bioRxiv