Ripply1 and Gsc collectively suppress anterior endoderm differentiation from prechordal plate progenitors

  1. Tao Cheng
  2. Xiang Liu
  3. Yang Dong
  4. Yi-Meng Tian
  5. Yan-Yi Xing
  6. Chen-Yi Chen
  7. Cong Liu
  8. Yun-Fei Li
  9. Ying Huang
  10. Ding-Hao Zhuo
  11. Xiao Xu
  12. Jing-Yun Luan
  13. Xin-Xin Fu
  14. Zi-Xin Jin
  15. Jing Mo
  16. Xiang Xu
  17. Hong-Qing Liang
  18. Peng-Fei Xu

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    eLife assessment

    This study provides a useful analysis of the changes in chromatin organization and gene expression that occur during the differentiation of two cell types (anterior endoderm and prechordal plate) from a common progenitor in zebrafish. Although the findings are consistent with previous work, the evidence presented in the study appears to be incomplete and would benefit from more rigorous interpretation of single-cell data, more in-depth lineage tracing, overexpression experiments with physiological levels of Ripply, and a clearer justification for using an explant system. With these modifications, this paper will be of interest to zebrafish developmental biologists investigating mechanisms underlying differentiation.

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Abstract

During gastrulation, the mesendoderm is firstly specified by morphogens such as Nodal, and then segregates into endoderm and mesoderm in a Nodal concentration-dependent manner. However, the mechanism underlying the segregation and crosstalk of different sub-groups within the meso- and endoderm lineages remains unclear. Here, taking zebrafish prechordal plate (PP) and anterior endoderm (Endo) as research model, using single-cell multi-omics and live imaging analyses, we show that anterior Endo progenitors originate directly from PP progenitors. A single-cell transcriptomic trajectory analysis of wild-type, ndr1 knockdown and lft1 knockout Nodal explants confirms the diversification of anterior Endo fate from PP progenitors. Gene Ontology (GO) enrichment analysis indentifies that the change of chromatin organization potentiates the segregation of endodermal cell fate from PP progenitors. Single-cell ATAC & RNA sequencing further reveals that two transcriptional regulators, gsc and ripply1 , exhibit varied activation patterns in PP and Endo lineages at both the chromatin and RNA expression levels. We further demonstrate that Ripply1 functions coordinately with Gsc to repress endodermal cell fate by directly binding to the cis -elements of sox32 and sox17 . Modulating the expression levels of these regulators tilts the cell fate decision between the PP and Endo lineages.

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  1. Version published to 10.7554/elife.100200.1 on eLife
  2. Version published to 10.7554/elife.100200 on eLife
  3. eLife

    eLife assessment

    This study provides a useful analysis of the changes in chromatin organization and gene expression that occur during the differentiation of two cell types (anterior endoderm and prechordal plate) from a common progenitor in zebrafish. Although the findings are consistent with previous work, the evidence presented in the study appears to be incomplete and would benefit from more rigorous interpretation of single-cell data, more in-depth lineage tracing, overexpression experiments with physiological levels of Ripply, and a clearer justification for using an explant system. With these modifications, this paper will be of interest to zebrafish developmental biologists investigating mechanisms underlying differentiation.

  4. eLife

    Reviewer #1 (Public Review):

    Summary:

    During vertebrate gastrulation, mesendoderm cells are initially specified by morphogens (e.g. Nodal) and segregate into endoderm and mesoderm in part based on Nodal concentrations. Using zebrafish genetics, live imaging, and single-cell multi-omics, the manuscript by Cheng et al presents evidence to support a claim that anterior endoderm progenitors derive primarily from prechordal plate progenitors, with transcriptional regulators goosecoid (Gsc) and ripply1 playing key roles in this cell fate determination. Such a finding would represent a significant advance in our understanding of how anterior endoderm is specified in vertebrate embryos.

    Strengths:

    Live imaging-based tracking of PP and endo reporters (Figure 2) is well executed and convincing, though a larger number of individual cell tracks will be needed. Currently, only a single cell track (n=1) is provided.

    Weaknesses:

    (1) The central claim of the paper - that the anterior endoderm progenitors arise directly from prechordal plate progenitors - is not adequately supported by the evidence presented. This is a claim about cell lineage, which the authors are attempting to support with data from single-cell profiling and genetic manipulations in embryos and explants. The construction of gene expression (pseudo-time) trajectories, while a modern and powerful approach for hypothesis generation, should not be used as a substitute for bona fide lineage tracing methods. If the authors' central hypothesis is correct, a CRE-based lineage tracing experiment (e.g. driving CRE using a PP marker such as Gsc) should be able to label PP progenitor cells that ultimately contribute to anterior endoderm-derived tissues. Such an experiment would also allow the authors to quantify the relative contribution of PP (vs non-PP) cells to the anterior endoderm, which is not possible to estimate from the indirect data currently provided. Note: while the present version of the manuscript does describe a sox17:CRE lineage tracing experiment, this actually goes in the opposite direction that would be informative (sox:17:CRE-marked descendants will be a mixture of PP-derived and non-PP derived cells, and the Gsc-based reporter does not allow for long-term tracking the fates of these cells).

    (2) The authors' descriptions of gene expression patterns in the single-cell trajectory analyses do not always match the data. For example, it is stated that goosecoid expression marks progenitor cells that exist prior to a PP vs endo fate bifurcation (e.g. lines 124-130). Yet, in Figure 1C it appears that in fact goosecoid expression largely does not precede (but actually follows) the split and is predominantly expressed in cells that have already been specified into the PP branch. Likewise, most of the cells in the endo branch (or prior) appear to never express Gsc. While these trends do indeed appear to be more muddled in the explant data (Figure 1H), it still seems quite far-fetched to claim that Gsc expression is a hallmark of endoderm-PP progenitors.

    (3) The study seems to refer to "endoderm" and "anterior endoderm" somewhat interchangeably, and this is potentially problematic. Most single-cell-based analyses appearing in the study rely on global endoderm markers (sox17, sox32) which are expressed in endodermal precursors along the entire ventrolateral margin. Some of these cells are adjacent to the prechordal plate on the dorsal side of the gastrula, but many (most in fact) are quite some distance away. The microscopy-based evidence presented in Figure 2 and elsewhere, however, focuses on a small number of sox17-expressing cells that are directly adjacent to, or intermingled with, the prechordal plate. It, therefore, seems problematic for the authors to generalize potential overlaps with the PP lineage to the entire endoderm, which includes cells in ventral locations. It would be helpful if the authors could search for additional markers that might stratify and/or mark the anterior endoderm and perform their trajectory analysis specifically on these cells.

    (4) It is not clear that the use of the nodal explant system is allowing for rigorous assessment of endoderm specification. Why are the numbers of endoderm cells so vanishingly few in the nodal explant experiments (Figure 1H, 3H), especially when compared to the embryo itself (e.g. Figures 1C-D)? It seems difficult to perform a rigorous analysis of endoderm specification using this particular model which seems inherently more biased towards PP vs. endoderm than the embryo itself. Why not simply perform nodal pathway manipulations in embryos?

    (5) The authors should not claim that proximity in UMAP space is an indication of transcriptional similarity (lines 207-208), especially for well-separated clusters. This is a serious misrepresentation of the proper usage of the UMAP algorithm. The authors make a similar claim later on (lines 272-274).

  5. eLife

    Reviewer #2 (Public Review):

    Summary:

    During vertebrate gastrulation, the mesoderm and endoderm arise from a common population of precursor cells and are specified by similar signaling events, raising questions as to how these two germ layers are distinguished. Here, Cheng and colleagues use zebrafish gastrulation as a model for mesoderm and endoderm segregation. By reanalyzing published single-cell sequencing data, they identify a common progenitor population for the anterior endoderm and the mesodermal prechordal plate (PP). They find that expression levels of PP genes Gsc and ripply are among the earliest differences between these populations and that their increased expression suppresses the expression of endoderm markers. Further analysis of chromatin accessibility and Ripply cut-and-tag is consistent with direct repression of endoderm by this PP marker. This study demonstrates the roles of Gsc and Ripply in suppressing anterior endoderm fate, but this role for Gsc was already known and the effect of Ripply is limited to a small population of anterior endoderm. The manuscript also focuses extensively on the function of Nodal in specifying and patterning the mesoderm and endoderm, a role that is already well known and to which the current analysis adds little new insight.

    Strengths:

    Integrated single-cell ATAC- and RNA-seq convincingly demonstrate changes in chromatin accessibility that may underlie the segregation of mesoderm and endoderm lineages, including Gsc and ripply. Identification of Ripply-occupied genomic regions augments this analysis. The genetic mutants for both genes provide strong evidence for their function in anterior mesendoderm development, although these phenotypes are subtle.

    Weaknesses:

    The use of zebrafish embryonic explants for cell fate trajectory analysis (rather than intact embryos) is not justified. In both transcriptomic comparisons between the two fate trajectories of interest and Ripply cut-and-tag analysis, the authors rely too heavily on gene ontology which adds little to our functional understanding. Much of the work is focused on the role of Nodal in the mesoderm/endoderm fate decision, but the results largely confirm previous studies and again provide few new insights. Some experiments were designed to test the relationship between the mesoderm and endoderm lineages and the role of epigenetic regulators therein, but these experiments were not properly controlled and therefore difficult to interpret.

  6. eLife

    Reviewer #3 (Public Review):

    Summary:

    Cheng, Liu, Dong, et al. demonstrate that anterior endoderm cells can arise from prechordal plate progenitors, which is suggested by pseudo time reanalysis of published scRNAseq data, pseudo time analysis of new scRNAseq data generated from Nodal-stimulated explants, live imaging from sox17:DsRed and Gsc:eGFP transgenics, fluorescent in situ hybridization, and a Cre/Lox system. Early fate mapping studies already suggested that progenitors at the dorsal margin give rise to both of these cell types (Warga) and live imaging from the Heisenberg lab (Sako 2016, Barone 2017) also pretty convincingly showed this. However, the data presented for this point are very nice, and the additional experiments in this manuscript, however, further cement this result. Though better demonstrated by previous work (Alexander 1999, Gritsman 1999, Gritsman 2000, Sako 2016, Rogers 2017, others), the manuscript suggests that high Nodal signaling is required for both cell types, and shows preliminary data that suggests that FGF signaling may also be important in their segregation. The manuscript also presents new single-cell RNAseq data from Nodal-stimulated explants with increased (lft1 KO) or decreased (ndr1 KD) Nodal signaling and multi-omic ATAC+scRNAseq data from wild-type 6 hpf embryos but draws relatively few conclusions from these data. Lastly, the manuscript presents data that SWI/SNF remodelers and Ripply1 may be involved in the anterior endoderm - prechordal plate decision, but these data are less convincing. The SWI/SNF remodeler experiments are unconvincing because the demonstration that these factors are differentially expressed or active between the two cell types is weak. The Ripply1 gain-of-function experiments are unconvincing because they are based on incredibly high overexpression of ripply1 (500 pg or 1000 pg) that generates a phenotype that is not in line with previously demonstrated overexpression studies (with phenotypes from 10-20x lower expression). Similarly, the cut-and-tag data seems low quality and like it doesn't support direct binding of ripply1 to these loci.

    In the end, this study provides new details that are likely important in the cell fate decision between the prechordal plate and anterior endoderm; however, it is unclear how Nodal signaling, FGF signaling, and elements of the gene regulatory network (including Gsc, possibly ripply1, and other factors) interact to make the decision. I suggest that this manuscript is of most interest to Nodal signaling or zebrafish germ layer patterning afficionados. While it provides new datasets and observations, it does not weave these into a convincing story to provide a major advance in our understanding of the specification of these cell types.

    Major issues:

    (1) UMAPs: There are several instances in the manuscript where UMAPs are used incorrectly as support for statements about how transcriptionally similar two populations are. UMAP is a stochastic, non-linear projection for visualization - distances in UMAP cannot be used to determine how transcriptionally similar or dissimilar two groups are. In order to make conclusions about how transcriptionally similar two populations are requires performing calculations either in the gene expression space, or in a linear dimensional reduction space (e.g. PCA, keeping in mind that this will only consider the subset of genes used as input into the PCA). Please correct or remove these instances, which include (but are not limited to):
    p.4 107-110
    p.4 112
    p.8 207-208
    p.10 273-275

    (2) Nodal and lefty manipulations: The section "Nodal-Lefty regulatory loop is needed for PP and anterior Endo fate specification" and Figure 3 do not draw any significant conclusions. This section presents a LIANA analysis to determine the signals that might be important between prechordal plate and endoderm, but despite the fact that it suggests that BMP, Nodal, FGF, and Wnt signaling might be important, the manuscript just concludes that Nodal signaling is important. Perhaps this is because the conclusion that Nodal signaling is required for the specification of these cell types has been demonstrated in zebrafish in several other studies with more convincing experiments (Alexander 1999, Gritsman 1999, Gritsman 2000, Rogers 2017, Sako 2016). While FGF has recently been demonstrated to be a key player in the stochastic decision to adopt endodermal fate in lateral endoderm (Economou 2022), the idea that FGF signaling may be a key player in the differentiation of these two cell types has strangely been relegated to the discussion and supplement. Lastly, the manuscript does not make clear the advantage of performing experiments to explore the PP-Endo decision in Nodal-stimulated explants compared to data from intact embryos. What would be learned from this and not from an embryo? Since Nodal signaling stimulates the expression of Wnts and FGFs, these data do not test Nodal signaling independent of the other pathways. It is unclear why this artificial system that has some disadvantages is used since the manuscript does not make clear any advantages that it might have had.

    (3) ripply1 mRNA injection phenotype inconsistent with previous literature: The phenotype presented in this manuscript from overexpressing ripply1 mRNA (Fig S11) is inconsistent with previous observations. This study shows a much more dramatic phenotype, suggesting that the overexpression may be to a non-physiological level that makes it difficult to interpret the gain-of-function experiments. For instance, Kawamura et al 2005 perform this experiment but do not trigger loss of head and eye structures or loss of tail structures. Similarly, Kawamura et al 2008 repeat the experiment, triggering a mildly more dramatic shortening of the tail and complete removal of the notochord, but again no disturbance of head structures as displayed here. These previous studies injected 25 - 100 pg of ripply1 mRNA with dramatic phenotypes, whereas this study uses 500 - 1000 pg. The phenotype is so much more dramatic than previously presented that it suggests that the level of ripply1 overexpression is sufficiently high that it may no longer be regulating only its endogenous targets, making the results drawn from ripply1 overexpression difficult to trust.

    (4) Ripply1 binding to sox17 and sox32 regulatory regions not convincing: The Cut and Tag data presented in Fig 6J-K does not seem to be high quality and does not seem to provide strong support that Ripply 1 binds to the regulatory regions of these genes. The signal-to-noise ratio is very poor, and the 'binding' near sox17 that is identified seems to be even coverage over a 14 kb region, which is not consistent with site-specific recruitment of this factor, and the 'peaks' highlighted with yellow boxes do not appear to be peaks at all. To me, it seems this probably represents either: (1) overtagmentation of these samples or (2) an overexpression artifact from injection of too high concentration of ripply1-HA mRNA. In general, Cut and Tag is only recommended for histone modifications, and Cut and Run would be recommended for transcriptional regulators like these (see Epicypher's literature). Given this and the previous point about Ripply1 overexpression, I am not convinced that Ripply1 regulates endodermal genes. The existing data could be made somewhat more convincing by showing the tracks for other genes as positive and negative controls, given that Ripply1 has known muscle targets (how does its binding look at those targets in comparison) and there should be a number of Nodal target genes that Ripply1 does not bind to that could be used as negative controls. Overall this experiment doesn't seem to be of high enough quality to drive the conclusion that Ripply1 directly binds near sox17 and sox32 and from the data presented in the manuscript looks as if it failed technically.

    (5) "Cooperatively Gsc and ripply1 regulate": I suggest avoiding the term "cooperative," when describing the relationship between Ripply1 and Gsc regulation of PP and anterior endoderm - it evokes the concept of cooperative gene regulation, which implies that these factors interact with each biochemically in order to bind to the DNA. This is not supported by the data in this manuscript, and is especially confusing since Ripply1 is thought to require cooperative binding with a T-box family transcription factor to direct its binding to the DNA.

    (6) SWI/SNF: The differential expression of srcap doesn't seem very remarkable. The dot plots in the supplement S7H don't help - they seem to show no expression at all in the endoderm, which is clearly a distortion of the data, since from the violin plots it's obviously expressed and the dot-size scale only ranges from ~30-38%. Please add to the figure information about fold-change and p-value for the differential expression. Publicly available scRNAseq databases show scrap is expressed throughout the entire early embryo, suggesting that it would be surprising for it to have differential activity in these two cell types and thereby contribute to their separate specification during development. It seems equally possible that this just mildly influences the level of Nodal or FGF signaling, which would create this effect.

    The multiome data seems like a valuable data set for researchers interested in this stage of zebrafish development. However, the presentation of the data doesn't make many conclusions, aside from identifying an element adjacent to ripply1 whose chromatin is open in prechordal plate cells and not endodermal cells and showing that there are a number of loci with differential accessibility between these cell types. That seems fairly expected since both cell types have several differentially expressed transcriptional regulators (for instance, ripply1 has previously been demonstrated in multiple studies to be specific to the prechordal plate during blastula stages). The manuscript implies that SWI/SNF remodeling by Srcap is responsible for the chromatin accessibility differences between these cell types, but that has not actually been tested. It seems more likely that the differences in chromatin accessibility observed are a result of transcription factors binding downstream of Nodal signaling.

    Minor issues:

    Figure 2 E-F: It's not clear which cells from E are quantitated in F. For instance, the dorsal forerunner cells are likely to behave very differently from other endodermal progenitors in this assay. It would be helpful to indicate which cells are analyzed in Fig F with an outline or other indicator of some kind. Or - if both DFCs and endodermal cells are included in F, to perhaps use different colors for their points to help indicate if their fluorescence changes differently.

    Fig 3 J: Should the reference be Dubrulle et al 2015, rather than Julien et al?

    References:
    Alexander, J. & Stainier, D. Y. A molecular pathway leading to endoderm formation in zebrafish. Current biology : CB 9, 1147-1157 (1999).
    Barone, V. et al. An Effective Feedback Loop between Cell-Cell Contact Duration and Morphogen Signaling Determines Cell Fate. Dev. Cell 43, 198-211.e12 (2017).
    Economou, A. D., Guglielmi, L., East, P. & Hill, C. S. Nodal signaling establishes a competency window for stochastic cell fate switching. Dev. Cell 57, 2604-2622.e5 (2022).
    Gritsman, K. et al. The EGF-CFC protein one-eyed pinhead is essential for nodal signaling. Cell 97, 121-132 (1999).
    Gritsman, K., Talbot, W. S. & Schier, A. F. Nodal signaling patterns the organizer. Development (Cambridge, England) 127, 921-932 (2000).
    Kawamura, A. et al. Groucho-associated transcriptional repressor ripply1 is required for proper transition from the presomitic mesoderm to somites. Developmental cell 9, 735-744 (2005).
    Kawamura, A., Koshida, S. & Takada, S. Activator-to-repressor conversion of T-box transcription factors by the Ripply family of Groucho/TLE-associated mediators. Molecular and cellular biology 28, 3236-3244 (2008).
    Sako, K. et al. Optogenetic Control of Nodal Signaling Reveals a Temporal Pattern of Nodal Signaling Regulating Cell Fate Specification during Gastrulation. Cell Rep. 16, 866-877 (2016).
    Rogers, K. W. et al. Nodal patterning without Lefty inhibitory feedback is functional but fragile. eLife 6, e28785 (2017).
    Warga, R. M. & Nüsslein-Volhard, C. Origin and development of the zebrafish endoderm. Development 126, 827-838 (1999).

  7. Version published to 10.1101/2023.05.29.542713v2 on bioRxiv
  9. 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 #3

    Evidence, reproducibility and clarity

    Summary

    Cheng et al. use single-cell sequencing to determine how Nodal signaling influences endodermal and prechordal plate fate specification in zebrafish. Much data and data analyses are presented, but the conclusions that can be drawn remain vague and do not go far beyond what previous studies have already established. While the datasets are a potentially useful resource, the conceptual impact is limited.

    Major comments

    1. The major weakness of the paper is that previous studies have already shown that differential Nodal signaling (and additional mechanisms) can induce anterior endoderm versus prechordal plate. In particular, the studies of Barone et al. (2017) and Sako et al. (2016) have provided much more convincing insights, because they combine genetic manipulations with in vivo imaging. In contrast, the current study mostly infers fate specification from scRNA-seq data. This approach is fraught with artifacts, because pseudotime trajectories are only a proxy for developmental processes, and UMAPs can misrepresent relationships between different cell states and types. The potentially more novel findings (roles for ripply; role of chromatin accessibility) are quite preliminary. Therefore, the conceptual advances provided by the study are minor.
    2. The study attempts to distinguish between anterior endoderm and prechordal plate, but there is little evidence that anterior endoderm versus most/all endoderm is studied. Clear markers for anterior endoderm would be needed (or live imaging as in Barone et al.).
    3. The claim that prechordal plate gives rise to prechordal plate and endoderm is confusing. The initial prechordal plate is different from the later prechordal plate. Please use a more precise nomenclature.
    4. Gsc is described to be expressed highly in anterior endoderm progenitors but Figures 1C and 1J do not support this.
    5. I am not sure what to make of the Nodal and Lefty manipulations. There is plenty of data but previous studies by the Heisenberg lab have provided much more definitive insihgts into the role Nodal signaling in this fate decision. Please put your results into the context of these studies.
    6. The chromatin accessibility results and conclusions seem trivial in light on previous observations that Nodal signaling (and many other signaling pathways) activate gene expression via enhancers, a hallmark of which is increased accessibility upon activation.
    7. The ripply1 overexpression result is potentially interesting, but needs to be complemented with a loss of function analysis.

    Referee cross-commenting

    It is gratifying to see that all three reviewers appreciate the potential of the data, but they find the results not as conclusive as one might wish, and they question the conceptual novelty of the claims when compared to previous studies. I share their suggestions and concerns.

    Significance

    The study provides new single-cell data and analyses but does not provide major conceptual advances when compared to previous studies (e.g. Barone et al. (2017); Sako et al. (2016)). In its current form a small group of researchers in the zebrafish Nodal field might be interested in further exploring the data in this paper and combine it with in situ gene expression analyses and fate mapping.

  10. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    In this study, Chen and colleagues examine the molecular basis for the segregation of mesodermal and endodermal fates during zebrafish gastrulation. For this, they focused on cell fate separation within the anterior mesendoderm, which gives rise to prechordal plate (pp) and anterior endoderm (endo) progenitors. Using a combination of "-omics" approaches and live imaging in both embryos and explants, the authors find that endo progenitors derive from the pp and are specified by comparatively lower levels of Nodal signaling. Mechanistically, higher Nodal signaling levels in pp cells correlates with chromatin openness. Furthermore, the authors provided evidence that gsc and ripply1 activity can repress endo specification. Overall, the authors suggest a model whereby different Nodal signaling levels promote cell fate diversification through modulation of epigenetic states.

    The paper is well written, and the data presented in this study nicely supports the author's interpretation that anterior endo cells derive from the pp and that it requires lower Nodal signaling levels. However, it is unclear what are the core differences between this model and previous work (Sako, et al. 2016; Barone, et al. 2017) and how to interpret these findings in light of recent work in the field, implicating FGF signaling as a critical regulator of the segregation between mesoderm and endoderm in zebrafish (Economou et al. 2023). To reinforce their findings, it would thus be important for the authors to use their datasets to investigate further between these distinct models and explore the role of FGF signaling in this process. For instance, can they observe differential activation of FGF targets in early progenitors cells with higher vs. lower Nodal signaling?

    Similarly, the link between Nodal signaling and chromatin openness is interesting, however, it is still unclear how causative these differences are for the cell fate segregation investigated in this study (this is in line with the way the authors describe these findings). However, given my previous point, I think it would be important to dissect this link further to strengthen the novelty of the study.

    Finally, to prove that gsc and ripply1 cooperate in wild-type embryos for the segregation between pp and endo progenitors, it would be important to include further functional data. For instance, describe the respective loss-of-function phenotypes, as well as whether, at endogenous levels, they can partially compensate for each other's loss-of-function. A similar analysis should be included for osr1 to validate the need for cooperation between these distinct transcriptional repressors in anterior endo specification (a hypothesis nicely raised by the authors in the discussion of this study). Finally, is the expression of these transcriptional repressors restricted to the ppl? If so, why? Would they require peak levels of Nodal signaling, only present in the pp, for their induction? What are the expression levels of these regulators in the morphants for ndr1 and lefty1 (which show differences in Nodal signaling levels)?

    Significance

    This study tackles an important question in vertebrate gastrulation, which has been under intense investigation over the last years. By integrating sequencing datasets from previously published studies, as well as newly-generated datasets, the authors provide evidence that anterior endo progenitors derive from the pp, which is nicely confirmed using live imaging. The findings that anterior endo progenitors are specified by comparatively lower levels of Nodal signaling than pp constitutes a major part of this manuscript. However, it is not clear i) what is necessarily new compared to previous studies implicating Nodal signalling in this process and, ii) how to interpret these findings in the light of recent work in the field disputing this more Nodal-based model. Accordingly, it was previously shown that the duration of Nodal signaling, partially through the action of gsc, played a key role in the differentiation between pp and endo progenitors (Sako, et al. 2016). Furthermore, previous work showed that endo cells leaving the ppl showed shorter-lived cell-cell contacts and, thus, on average lower Nodal signaling (Barone, et al. 2017). Since a recent model challenged the idea that differences in Nodal signaling are sufficient to account for the segregation between mesoderm and endoderm progenitors and instead suggested that an interplay between Nodal and FGF is necessary for the stochastic switch between these two cell fates (Economou et al. 2023), it would be important for the authors to use their datasets to investigate further these distinct models. This would synthetize both previous and current findings into a conceptual framework explaining how endoderm progenitors are specified.

    Audience: This study would be relevant for a broad audience of cell and developmental biologists, interested in morphogen signaling, cell fate specification and pattern formation.

    Expertise in zebrafish development, gastrulation, morphogen signaling and morphogenesis.

  11. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In zebrafish embryos, progenitor cells for both the prechordal plate and anterior endoderm reside at the dorsal margin in early gastrulation. Both cell populations are induced via signaling through the Nodal signaling pathway, however the mechanisms that send Nodal-exposed cells to one fate versus the other remain a matter of debate. Cheng et al use single-cell RNA sequencing to investigate the mechanistic origins of this developmental decision. They argue that both populations emerge from a common progenitor pool marked by the prechordal-plate marker gene goosecoid (gsc). By adding single-cell ATACseq analysis, they go on to argue that Nodal signaling encourages open chromatin states at target genes, and that this may underly the distinction between prechordal plate and endodermal fates. Finally, they suggest two potential regulators (gsc and ripply1) that may repress commitment to the endodermal fate.

    Major Comments:

    1. In lines 128-136, the authors describe a live imaging experiment to support the argument that anterior endodermal cells emerge from a gsc+ progenitor pool. The claim is that sox17+ cells (marked by RFP fluorescence) arise in gsc+ cells (marked by GFP fluorescence). From the presented data, I find it very hard to evaluate this claim. The GFP signal appears quite close to background in the highlighted cell. Additionally, the argument- as presented-turns on the behavior of a single highlighted cell. I think that this analysis should be clarified and extended to support the claim.

    I suggest that the authors (1) plot average cell fluorescence over time rather than a 'line scan' across the cell, (2) draw cell borders from the mask used in each frame for clarity of presentation, and (3) plot the trajectories of gsc+/sox17+, gsc-/sox17- and gsc+/sox17-cells for comparison.

    Alternatively, it could be helpful to extract fluorescence intensities for each cell in the field of view and scatter the RFP vs. GFP intensity for each cell. If the claim is true, three distinct subpopulations should be visible (i.e. gsc+/sox17-, gsc-/sox17- and gsc+/sox17+). Statistical analysis supporting the significance of these differences (e.g. comparing the means of each reporter within the populations) would be clarifying.

    OPTIONAL: The live imaging experiment the authors present is quite ambitious, but perhaps overly difficult for the task at hand. I think this point could be more easily and clearly demonstrated by using two-color fluorescent in situs or HCR staining for gsc and sox17. Using an endpoint measurement would allow for deeper sampling across multiple embryos, and would likely yield clearer signals for cell type quantifications.

    1. In the same section, I suggest that the authors address the possibility that the sox17+ cells observed don't go on to become part of the anterior endoderm. I commend the authors experimental work to support their scRNA-Seq data, however observation of the expression of a reporter gene (injected on a plasmid) is not equivalent to demonstrating that those cells adopt a given fate in the end. Is it not possible that the sox17 expression is transient, and these cells revert to prechordal plate fate? This point would be sealed by a formal fate mapping study (e.g. photoconversion of sox17::kaede cells), but I don't think this is a necessary bar for publication.
    2. In Figure 1 M, the explant data does not seem to clearly support the claim that higher Nodal signaling intensities favor prechordal plate over endoderm. It appears that, for the endodermal panel, 2/3 replicates for 6 pg and 10 pg injections resulted in no endodermal cells observed. Could the authors clarify how this reflects the certainty of the conclusion? No statistical analysis is indicated on this panel or the one below.
    3. OPTIONAL: The analysis presented in Fig. 1M strikes me as rather indirect (i.e. deconvolution of bulk RNA-Seq data to infer cell population proportions), and not strongly compelling. I think a stronger support of this point would be to inject Nodal into embryos and measure positive cell counts for gsc and an endodermal marker (e.g. sox32 or sox17) via HCR or in situ hybridization. This would yield a direct measurement of the cell counts in question. I think this would greatly support the claim, but I don't think should be considered a requirement for publication.
    4. In Fig. 2H, the authors analyze responses to ectopic Nodal gradients in order to corroborate the results of their LIANA analysis. This experiment is a welcome addition to the argument, but has weak points that should be addressed.
      • a. The description of image analysis procedures used to construct the quantification plots are inadequate. It seems likely that the nuclei were segmented from the DAPI images, but this was not clear from the methods section. The authors should completely describe the segmentation pipeline and include sample code in the supplementary material.
      • b. The methods section seems to suggest that the analysis was performed exclusively on maximum intensity projections. I think this procedure may make the data hard to interpret and should be modified/support with additional analysis. For example, there is no reason that, at any given position in the image, the brightest DAPI and pSmad2 channel pixels occur in the same plane. Segmentation boundaries may therefore not reliably match between channels in the maximum intensity projection. The segmentation should be performed using the full Z-stack images. This can be done using widely-available software packages (e.g. CellProfiler).
      • c. The fluorescence images in 2H (specifically for the pSmad2 channel) look like they may contain some artifacts that carry through into the quantification. Specifically, there appears to be substantial non-specific background (both hazy and punctate) in the lft1 mutant that may artificially elevate the quantified intensity. This is evident in the quantification as a larger 'offset' to which the gradient decays than in the other presented images. This may be another explanation for the observation that pSmad2 staining is stronger in this background. I suggest that the authors (a) present all fluorescence images from the dataset in the supplement to allow for visual inspection, and (b) estimate the effect of fluorescence background on their quantifications to ensure that this artifact is not the source of the claimed difference.
    5. In lines 267-284 and Fig. 4 L, the authors make the argument that ripply1 acts as a cell-autonomous repressor of endodermal fate. I find the argument for the cell autonomous character of its function hard to follow. Specifically, the authors lean on the experiment in which a plasmid with a sox17 promoter-ripply1 construct is injected, resulting in a decrease in endodermal cell count. Could the authors elaborate on how this proves a cell autonomous effect? Is it not possible that ripply1 expressed from this construct induces a signal that influences neighboring cells?
    6. The suggestion that prechordal plate fate is favored (over endodermal fate) by higher Nodal signaling levels is interesting. This claim is supported by the derivation of a 'Nodal score' from RNAseq data. However, I don't see where the score is defined in the Methods section or in the supplementary materials. If this was accidentally omitted (my apologies if I am just missing it), it should be added. Additionally, I found the description in the main text to be opaque, and the paper would benefit from a more intuitive/friendly explanation of this metric.

    Additionally, could the authors comment on what they believe-in terms of Nodal signaling history for a given cell- this score represents? Does it correlate with integrated Nodal exposure? Nodal exposure duration? Peak Nodal exposure? Given the results of Sako et al-that Nodal exposure duration is a critical determinant of prechordal plate fate- it would be useful to know if the authors believe their Nodal score findings point toward a different mechanism.

    Minor Comments:

    1. Line 84: The authors refer to the prechordal plate cells being 'more mature' than endoderm. It is unclear what the claim is here; some elaboration would be helpful.
    2. The fluorescence images in Fig. S2 are virtually invisible in the PDF. The images should be rescaled to make them visible.
    3. Fig. 2H would be easier to make sense of if the image panels were labeled. Please indicate which color corresponds to which stain.

    Significance

    I believe that this study fills in some details on the process of anterior endoderm specification that will be of interest to specialists in zebrafish Nodal signaling. I believe that the strongest and most novel section is the combined scRNA-Seq/ATAC-Seq analysis. This dataset is likely to be of interest to researchers who want to dig into potential mechanisms for the separation anterior endoderm and prechordal plate. Further, the singling out of ripply1 as a potential regulator of endodermal specification is interesting, and I hope that the authors follow this promising lead in future work.

    While this study does provide a useful single-cell view of the specification of anterior endoderm, I didn't feel that it came to a concrete conclusion about the mechanism of separation of the anterior endoderm and prechordal plate. A few interesting processes/players are suggested by the findings- for example, Nodal/Lefty signaling between the populations or ripply1 expression could tip the balance- but I don't believe these hypotheses were tested clearly. The authors correctly point out that models for Nodal-driven endoderm/mesoderm separation have recently emerged in the literature, however the findings presented here don't rule out either of these models or compellingly support an alternative. I don't believe that this should preclude publication, however I do think it will limit the reach of the paper. Experiments that more concretely test the possible mechanisms hinted at here- for example, studying the separation of the two lineages in ripply1 mutants- would strengthen the paper's reach.

    My enthusiasm for the paper is also somewhat reduced by the fact that some key findings of the paper can be found in earlier work. Acknowledgement of this prior work in the relevant sections could be improved. Specifically:

    1. The finding that anterior endoderm cells emerge from a gsc-expressing population in the dorsal margin was strongly suggested in the classic Warga et al paper on the origin of zebrafish endoderm. There, fate mapping experiments demonstrate that dorsal marginal cells (in the first two cell tiers) in the late blastula can go on to form both endoderm and mesoderm. This strongly implies that anterior endoderm cells emerge from a gsc+ population, given that these cells are firmly within the gsc expression domain. I also note that the scRNAseq data from Fig. 2 in Farrell et al directly demonstrates that some sox17+ endoderm cells express gsc in their developmental trajectory. The findings in this paper are a welcome confirmation of these earlier observations, however this context should be discussed.
    2. The observation that squint and lefty single mutants (either lefty1 or lefty2) can alter the propensity to adopt endodermal or mesodermal fates has also been observed previously. See for example Fig.1 in Norris et al, Figs 3 and 4 in Rogers et al, or Fig.1 in Chen et al. Acknowledging some of these earlier findings would benefit the paper.

    As a reviewer, I feel most qualified to comment on the embryological aspects of the presented work. While I am generally familiar with the single-cell genomics toolkit, I am not in a position to rigorously assess the technical merit of that side of this work. Accordingly, I have tried to restrict my comments to the embryology side.

    References:

    1. Warga, R.M. and Nüsslein-Volhard, C., 1999. Origin and development of the zebrafish endoderm. Development, 126(4), pp.827-838.
    2. Farrell, J.A., Wang, Y., Riesenfeld, S.J., Shekhar, K., Regev, A. and Schier, A.F., 2018. Single-cell reconstruction of developmental trajectories during zebrafish embryogenesis. Science, 360(6392), p.eaar3131.
    3. Norris, M.L., Pauli, A., Gagnon, J.A., Lord, N.D., Rogers, K.W., Mosimann, C., Zon, L.I. and Schier, A.F., 2017. Toddler signaling regulates mesodermal cell migration downstream of Nodal signaling. Elife, 6, p.e22626.
    4. Rogers, K.W., Lord, N.D., Gagnon, J.A., Pauli, A., Zimmerman, S., Aksel, D.C., Reyon, D., Tsai, S.Q., Joung, J.K. and Schier, A.F., 2017. Nodal patterning without Lefty inhibitory feedback is functional but fragile. Elife, 6, p.e28785.
    5. Chen, Y. and Schier, A.F., 2002. Lefty proteins are long-range inhibitors of squint-mediated nodal signaling. Current Biology, 12(24), pp.2124-2128.
    6. Sako, K., Pradhan, S.J., Barone, V., Ingles-Prieto, A., Müller, P., Ruprecht, V., Čapek, D., Galande, S., Janovjak, H. and Heisenberg, C.P., 2016. Optogenetic control of nodal signaling reveals a temporal pattern of nodal signaling regulating cell fate specification during gastrulation. Cell reports, 16(3), pp.866-877.
  12. Version published to 10.1101/2023.05.29.542713v1 on bioRxiv