Epigenetic delineation of the earliest cardiac lineage segregation by single-cell multi-omics

Reviewer #1 (Public Review):

Summary:

In this study, the authors identified and described the transcriptional trajectories leading to CMs during early mouse development, and characterized the epigenetic landscapes that underlie early mesodermal lineage specification.

The authors identified two transcriptomic trajectories from a mesodermal population to cardiomyocytes, the MJH and PSH trajectories. These trajectories are relevant to the current model for the First Heart Field (FHF) and the Second Heart Field (SHF) differentiation. Then, the authors characterized both gene expression and enhancer activity of the MJH and PSH trajectories, using a multiomics analysis. They highlighted the role of Gata4, Hand1, Foxf1, and Tead4 in the specification of the MJH trajectory. Finally, they performed a focused analysis of the role of Hand1 and Foxf1 in the MJH trajectory, showing their mutual regulation and their requirement for cardiac lineage specification.

Strengths:

The authors performed an extensive transcriptional and epigenetic analysis of early cardiac lineage specification and differentiation which will be of interest to investigators in the field of cardiac development and congenital heart disease. The authors considered the impact of the loss of Hand1 and Foxf1 in-vitro and Hand1 in-vivo.

Weaknesses:

The authors used previously published scRNA-seq data to generate two described transcriptomic trajectories.

(1) Details of the re-analysis step should be added, including a careful characterization of the different clusters and maker genes, more details on the WOT analysis, and details on the time stamp distribution along the different pseudotimes. These details would be important to allow readers to gain confidence that the two major trajectories identified are realistic interpretations of the input data.

The authors have also renamed the cardiac trajectories/lineages, departing from the convention applied in hundreds of papers, making the interpretation of their results challenging.

(2) The concept of "reverse reasoning" applied to the Waddington-OT package for directional mass transfer is not adequately explained. While the authors correctly acknowledged Waddington-OT's ability to model cell transitions from ancestors to descendants (using optimal transport theory), the justification for using a "reverse reasoning" approach is missing. Clarifying the rationale behind this strategy would be beneficial.

(3) As the authors used the EEM cell cluster as a starting point to build the MJH trajectory, it's unclear whether this trajectory truly represents the cardiac differentiation trajectory of the FHF progenitors:
- This strategy infers that the FHF progenitors are mixed in the same cluster as the extra-embryonic mesoderm, but no specific characterization of potential different cell populations included in this cluster was performed to confirm this.

- The authors identified the EEM cluster as a Juxta-cardiac field, without showing the expression of the principal marker Mab21l2 per cluster and/or on UMAPs.

- As the FHF progenitors arise earlier than the Juxta-cardiac field cells, it must be possible to identify an early FHF progenitor population (Nkx2-5+; Mab21l2-) using the time stamp. It would be more accurate to use this FHF cluster as a starting point than the EEM cluster to infer the FHF cardiac differentiation trajectory.

These concerns call into question the overall veracity of the trajectory analysis, and in fact, the discrepancies with prior published heart field trajectories are noted but the authors fail to validate their new interpretation. Because their trajectories are followed for the remainder of the paper, many of the interpretations and claims in the paper may be misleading. For example, these trajectories are used subsequently for annotation of the multiomic data, but any errors in the initial trajectories could result in errors in multiomic annotation, etc, etc.

(4) As mentioned in the discussion, the authors identified the MJH and PSH trajectories as non-overlapping. But, the authors did not discuss major previously published data showing that both FHF and SHF arise from a common transcriptomic progenitor state in the primitive streak (DOI: 10.1126/science.aao4174; DOI: 10.1007/s11886-022-01681-w). The authors should consider and discuss the specifics of why they obtained two completely separate trajectories from the beginning, how these observations conflict with prior published work, and what efforts they have made at validation.

(5) Figures 1D and E are confusing, as it's unclear why the authors selected only cells at E7.0. Also, panels 1D 'Trajectory' and 'Pseudotime' suggest that the CM trajectory moves from the PSH cells to the MJH. This result is confusing, and the authors should explain this observation.

(6) Regarding the PSH trajectory, it's unclear how the authors can obtain a full cardiac differentiation trajectory from the SHF progenitors as the SHF-derived cardiomyocytes are just starting to invade the heart tube at E8.5 (DOI: 10.7554/eLife.30668).

The above notes some of the discrepancies between the author's trajectory analysis and the historical cardiac development literature. Overall, the discrepancies between the author's trajectory analysis and the historical cardiac development literature are glossed over and not adequately validated.

(7) The authors mention analyzing "activated/inhibited genes" from Peng et al. 2019 but didn't specify when Peng's data was collected. Is it temporally relevant to the current study? How can "later stage" pathway enrichment be interpreted in the context of early-stage gene expression?

(8) Motif enrichment: cluster-specific DAEs were analyzed for motifs, but the authors list specific TFs rather than TF families, which is all that motif enrichment can provide. The authors should either list TF families or state clearly that the specific TFs they list were not validated beyond motifs.

(9) The core regulatory network is purely predictive. The authors again should refrain from language implying that the TFs in the CRN have any validated role.

Regarding the in vivo analysis of Hand1 CKO embryos, Figures 6 and 7:

(10) How can the authors explain the presence of a heart tube in the E9.5 Hand1 CKO embryos (Figure 6B) if, following the authors' model, the FHF/Juxta-cardiac field trajectory is disrupted by Hand1 CKO? A more detailed analysis of the cardiac phenotype of Hand1 CKO embryos would help to assess this question.

(11) The cell proportion differences observed between Ctrl and Hand1 CKO in Figure 6D need to be replicated and an appropriate statistical analysis must be performed to definitely conclude the impact of Hand1 CKO on cell proportions.

(12) The in-vitro cell differentiations are unlikely to recapitulate the complexity of the heart fields in-vivo, but they are analyzed and interpreted as if they do.

(13) The schematic summary of Figure 7F is confusing and should be adjusted based on the following considerations:
(a) the 'Wild-type' side presents 3 main trajectories (SHF, Early HT and JCF), but uses a 2-color code and the authors described only two trajectories everywhere else in the article (aka MJH and PSH). It's unclear how the SHF trajectory (blue line) can contribute to the Early HT, when the Early HT is supposed to be FHF-associated only (DOI: 10.7554/eLife.30668). As mentioned previously in Major comment 3., this model suggests a distinction between FHF and JCF trajectories, which is not investigated in the article.
(b) the color code suggests that the MJH (FHF-related) trajectory will give rise to the right ventricle and outflow tract (green line), which is contrary to current knowledge.

Minor comments:

(1) How genes were selected to generate Figure 1F? Is this a list of top differentially expressed genes over each pseudotime and/or between pseudotimes?

(2) Regarding Figure 1G, it's unclear how inhibited signaling can have an increased expression of underlying genes over pseudotimes. Can the authors give more details about this analysis and results?

(3) How do the authors explain the visible Hand1 expression in Hand1 CKO in Figure S7C 'EEM markers'? Is this an expected expression in terms of RNA which is not converted into proteins?

(4) The authors do not address the potential presence of doublets (merged cells) within their newly generated dataset. While they mention using "SCTransform" for normalization and artifact removal, it's unclear if doublet removal was explicitly performed.

Reviewer #2 (Public Review):

Summary of goals:

The aims of the study were to identify new lineage trajectories for the cardiac lineages of the heart, and to use computational and cell and animal studies to identify and validate new gene regulatory mechanisms involved in these trajectories.

Strengths:

The study addresses the long-standing yet still not fully answered questions of what drives the earliest specification mechanisms of the heart lineages. The introduction demonstrates a good understanding of the relevant lineage trajectories that have been previously established, and the significance of the work is well described. The study takes advantage of several recently published data sets and attempts t use these in combination to uncover any new mechanisms underlying early mesoderm/cardiac specification mechanisms. A strength of the study is the use of an in vitro model system (mESCs) to assess the functional relevance of the key players identified in the computational analysis, including innovative technology such as CRISPR-guided enhancer modulations. Lastly, the study generates mesoderm-specific Hand1 LOF embryos and assesses the differentiation trajectories in these animals, which represents a strong complementary approach to the in vitro and computational analysis earlier in the paper. The manuscript is clearly written and the methods section is detailed and comprehensive.

Comments and Weaknesses:

Overall: The computational analysis presented here integrates a large number of published data sets with one new data point (E7.0 single cell ATAC and RNA sequencing). This represents an elegant approach to identifying new information using available data. However, the data presentation at times becomes rather confusing, and relatively strong statements and conclusions are made based on trajectory analysis or other inferred mechanisms while jumping from one data set to another. The cell and in vivo work on Hand1 and Foxf1 is an important part of the study. Some additional experiments in both of these model systems could strongly support the novel aspects that were identified by the computational studies leading into the work.

(1) Definition of MJH and PSH trajectory:
The study uses previously published data sets to identify two main new differentiation trajectories: the MJH and the PSH trajectory (Figure 1). A large majority of subsequent conclusions are based on in-depth analysis of these two trajectories. For this reason, the method used to identify these trajectories (WTO, which seems a highly biased analysis with many manually chosen set points) should be supported by other commonly used methods such as for example RNA velocity analysis. This would inspire some additional confidence that the MJH and PSH trajectories were chosen as unbiased and rigorous as possible and that any follow-up analysis is biologically relevant.

(2) Identification of MJH and PSH trajectory progenitors:
The study defines various mesoderm populations from the published data set (Figure 1A-E), including nascent mesoderm, mixed mesoderm, and extraembryonic mesoderm. It further assigns these mesoderm populations to the newly identified MJH/PSH trajectories. Based on the trajectory definition in Figure 1A it appears that both trajectories include all 3 mesoderm populations, albeit at different proportions and it seems thus challenging to assign these as unique progenitor populations for a distinct trajectory, as is done in the epigenetic study by comparing clusters 8 (MJH) and s (PSH)(Figure 2). Along similar lines, the epigenetic analysis of clusters 2 and 8 did not reveal any distinct differences in H3K4m1, H3K27ac, or H3K4me3 at any of the time points analyzed (Figure 2F). While conceptually very interesting, the data presented do not seem to identify any distinct temporal patterns or differences in clones 2 and 8 (Figure 2H), and thus don't support the conclusion as stated: "the combined transcriptome and chromatin accessibility analysis further supported the early lineage segregation of MJH and the epigenetic priming at gastrulation stage for early cardiac genes".

(3) Function of Hand1 and Foxf1 during early cardiac differentiation:
The study incorporated some functional studies by generating Hand1 and Foxf1 KO mESCs and differentiated them into mesoderm cells for RNA sequencing. These lines would present relevant tools to assess the role of Hand1 and Foxf1 in mesoderm formation, and a number of experiments would further support the conclusions, which are made for the most part on transcriptional analysis. For example, the study would benefit from quantification of mesoderm cells and subsequent cardiomyocytes during differentiation (via IF, or more quantitatively, via flow cytometry analysis). These data would help interpret any of the findings in the bulk RNAseq data, and help to assess the function of Hand1 and Foxf1 in generating the cardiac lineages. Conclusions such as "the analysis indicated that HAND1 and FOXF1 could dually regulate MJH specification through directly activating the MJH specific genes and inhibiting PSH specific genes" seem rather strong given the data currently provided.

(4) Analysis of Hand1 cKO embryos:
Adding a mouse model to support the computational analysis is a strong way to conclude the study. Given the availability of these early embryos, some of the findings could be strengthened by performing a similar analysis to Figure 7B&C and by including some of the specific EEM markers found to be differentially regulated to complement the structural analysis of the embryos.

(5) Current findings in the context of previous findings:
The introduction carefully introduces the concept of lineage specification and different progenitor pools. Given the enormous amount of knowledge already available on Hand1 and Foxf1, and their role in specific lineages of the early heart, some of this information should be added, ideally to the discussion where it can be put into context of what the present findings add to the existing understanding of these transcription factors and their role in early cardiac specification.

Reviewer #3 (Public Review):

(1) In Figure 1A, could the authors justify using E8.5 CMs as the endpoint for the second lineage and better clarify the chamber identities of the E8.5 CMs analysed? Why are the atrial genes in Figure 1C of the PSH trajectory not present in Table S1.1, which lists pseudotime-dependent genes for the MJH/PSH trajectories from Figure 1F?

(2) Could the authors increase the resolution of their trajectory and genomic analyses to distinguish between the FHF (Tbx5+ HCN4+) and the JCF (Mab21l2+/ Hand1+) within the MJH lineage? Also, clarify if the early extraembryonic mesoderm contributes to the FHF.

(3) The authors strongly assume that the juxta-cardiac field (JCF), defined by Mab21l2 expression at E7.5 in the extraembryonic mesoderm, contributes to CMs. Could the authors explain the evidence for this? Could the authors identify Mab21l2 expression in the left ventricle (LV) myocardium and septum transversum at E8.5 (see Saito et al., 2013, Biol Open, 2(8): 779-788)? If such a JCF contribution to CMs exists, the extent to which it influences heart development should be clarified or discussed.

(4) Could the authors distinguish the Hand1+ pericardium from JCF progenitors in their single-cell data and explain why they excluded other cell types, such as the endocardium/endothelium and pericardium, or even the endoderm, as endpoints of their trajectory analysis? At the NM and MM mesoderm stages, how did the authors distinguish the earliest cardiac cells from the surrounding developing mesoderm?

(5) Could the authors contrast their trajectory analysis with those of Lescroart et al. (2018), Zhang et al., Tyser et al., and Krup et al.?

(6) Previous studies suggest that Mesp2 expression starts at E8 in the presomitic mesoderm (Saga et al., 1997). Could the authors provide in situ hybridization or HCR staining to confirm the early E7 Mesp2 expression suggested by the pseudo-time analysis of the second lineage.

(7) Could the authors also confirm the complementary Hand1 and Lefty2 expression patterns at E7 using HCR or in situ hybridization? Hand1 expression in the first lineage is plausible, considering lineage tracing results from Zhang et al.

(8) Could the authors explain why Hand1 and Lefty2+ cells are more likely to be multipotent progenitors, as mentioned in the text?

(9) Could the authors comment on the low Mesp1 expression in the mesodermal cells (MM) of the MJH trajectory at E7 (Figure 1D)? Is Mesp1 transiently expressed early in MJH progenitors and then turned off by E7? Have all FHF/JCF/SHF cells expressed Mesp1?

(10) Could the authors clarify if their analysis at E7 comprises a mixture of embryonic stages or a precisely defined embryonic stage for both the trajectory and epigenetic analyses? How do the authors know that cells of the second lineage are readily present in the E7 mesoderm they analysed (clusters 0, 1, and 2 for the multiomic analysis)?

(11) Could the authors further comment on the active Notch signaling observed in the first and second lineages, considering that Notch's role in the early steps of endocardial lineage commitment, but not of CMs, during gastrulation has been previously described by Lescroart et al. (2018)?

(12) In cluster 8, Figure 2D, it seems that levels of accessibility in cluster 8 are relatively high for genes associated with endothelium/endocardium development in addition to MJH genes. Could the authors comment and/or provide further analysis?

(13) Can the authors clarify why they state that cluster 8 DAEs are primed before the full activation of their target genes, considering that Bmp4 and Hand1 peak activities seem to coincide with their gene expression in Figure 2G?

(14) Did the authors extend the multiomic analysis to Nanog+ epiblast cells at E7 and investigate if cardiac/mesodermal priming exists before mesodermal induction (defined by T/Mesp1 onset of expression)?

(15) In the absence of duplicates, it is impossible to statistically compare the proportions of mesodermal cell populations in Hand1 wild-type and knockout (KO) embryos or to assess for abnormal accumulation of PS, NM, and MM cells. Could the authors analyse the proportions of cells by careful imaging of Hand1 wild-type and KO embryos instead?

(16) Could the authors provide high-resolution images for Figure 7 B-C-D as they are currently hard to interpret?