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

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).

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.

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:
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