Inferred trajectories reflect two distinct development routes of CMs.

(A) Two distinct trajectories, the MJH trajectory and the PSH trajectory, were inferred during CM differentiation. UMAP layout from Pijuan-Sala et al. (Pijuan-Sala et al. 2019) is highlighted by cells belonging to the WOT predicted developmental trajectories for CM (left), EEM (upper middle) and PM (lower middle), respectively. UMAP layout for the CM trajectory with cells is colored by the difference between FHF- and SHF-gene signature scores. UMAP layout for the MJH or PSH trajectory is colored by cell types. Gene expression levels of Hand1 (upper right) and Isl1 (lower right) of cells along the CM trajectory is overlaid onto the above-mentioned UMAP layout. Epi, epiblast. PS, primitive streak. NM, nascent mesoderm. MM, mixed mesoderm. EEM, early extraembryonic mesoderm. PM, pharyngeal mesoderm. CM, cardiomyocyte.

(B) Independent component (IC) layout showing pseudotemporal trajectories for MJH trajectory (upper) and PSH trajectory (lower) cells, colored by cell type (left), pseudotime (middle) and developmental time (right).

(C) The MJH and PSH trajectories showing distinct contributions to cardiac structures. Spatial plot showing spots in cardiac subregions from E8.5 embryos (left) (Sampath Kumar et al. 2023). White lines denoting the region including outflow tract (OFT), ventricle (V) and Atria (A). ‘Virtual’ in situ hybridization (vISH) confirming spatial specificity of marker genes corresponding to cardiac subregions (middle). Dot plot showing expression difference in subregion specific genes in the MJH and PSH trajectories (right).

(D) Mesodermal lineage segregation at E7.0. UMAP layout for E7.0 CM trajectory cells is colored by cell type (left), trajectory (middle) and pseudotime (right).

(E) UMAP layout (same as d) showing the expression of the E7.0 marker genes of NM (Mesp1), MJH (Hand1) and PSH (Lefty2).

(F) Heatmap showing the expression of pseudotime-dependent genes for the MJH trajectory (right) and the PSH trajectory (left). Rows and columns represent genes and cell bins, respectively. Genes used for the heatmap are listed in Supplementary Table 1.

(G) Smoothened fitting curves showing expression levels of activated (solid line) and inhibited (dotted line) signaling markers in the MJH (red) and PSH (blue) trajectories.

Multi-omics analysis reveals epigenetic signatures of the early MJH and PSH progenitors.

(A-C) Clustering analysis of E7.0 single-cell multi-omics data and comparison between modalities of transcriptome (snRNA-seq) and chromatin accessibility (snATAC-seq). UMAP layout, using only snRNA-seq (A,C) or snATAC-seq (B) data, is colored by cluster identities (left) or cell types (right). For both (A) and (B), cluster identities are determined by snRNA-seq data. For (C), developmental directions, shown as arrows, are indicated by RNA-velocity analysis.

(D) Heatmap showing the activity of 7,206 differentially accessible elements (DAEs) between clusters. Rows and columns represent DAEs and clusters. Colors indicate levels of accessibility averaged among cells from each cluster. The length of the corresponding color bar on the left represents the number of DEAs. Representative DAE-associated genes are shown on the right side.

(E) Dot plot indicating functional enrichment of DAE-associated genes of each cluster.

(F) Epigenetic status of DAEs during mesodermal specification of E6.5-7.5 mice embryo. H3K4me1, H3K27ac and H3K4me3 profiles were averaged among DAEs of cluster C1 (NM), C2 (PSH-direction frontier) and C8 (MJH-direction frontier). Published H3K4me1, H3K27ac and H3K4me3 ChIP-seq data were collected from E6.5 epiblast, E7.0/7.5 posterior epiblast (P) and E7.5 anterior mesoderm (AM) (Yang et al. 2019).

(G) Smoothened heatmap showing dynamic gene expression (right) and enhancer accessibility (left) along E7.0 mesoderm pseudotime trajectories for gene-enhancer pairs. Dashed lines indicating the pseudo-temporal midpoint.

(H) Representative genome browser snapshots of ATAC/RNA-seq (aggregated gene expression and chromatin accessibility for each cluster), H3K4me1 and H3K27ac at the Bmp4 locus. Putative enhancers status at E7.0 are highlighted by colors.

The CRN is identified centering on GATA4, HAND1, FOXF1 and TEAD4 in driving MJH specification.

(A) Identification of top MJH specific DNA-binding motifs and corresponding candidate TFs. The MJH specific DAEs were defined by comparing C8 with C1 snATAC-seq data using SnapATAC (Fang et al. 2021) ‘findDAR’ function. Motif calling was performed by the HOMER (Heinz et al. 2010) ‘findMotifsGenome.pl’ function. Motif activity (colored in red) and TF expression (colored in blue) levels of trajectory specific candidate TFs, are overlaid on the UMAP layout from Figure 2a.

(B) Core regulatory network (CRN) of E7.0 EEM cells. The network is composed of TFs (GATA4, HAND1, FOXF1 and TEAD4), TF binding regions (diamond shapes), target genes (dot shapes), colored lines (TFs to regions) and grey lines (regions to genes). Colors of genes and regions representing the log2 FCs of E7.0 EEM cells over other mesodermal cells, measured by scRNA-seq and snATAC-seq data, respectively. Functional enrichment terms of target genes are shown in boxes with subtitles indicating corresponding TFs. Q-values, using hyper-geometric tests, are shown in parenthesis.

(C) Heatmaps representing the enrichment of MESP1, ZIC2, ZIC3, FLI1, GATA4, HAND1 and FOXF1 across the DAEs of each cluster.

HAND1 and FOXF1 are mutually regulated and required for the expression of the MJH specific genes.

(A) Comparison of the genes affected by Hand1 KO or Foxf1 KO with cluster specific genes of E7.0 mesodermal cells. Dot plot showing that absolute (dot size) and relative (dot color) ratio of the cluster specific genes in WT, Hand1 KO or Foxf1 KO MES cells. >90% cluster specific genes were expressed in MES. Higher enrichment in C0-2 and lower enrichment in other clusters can be observed in Hand1 KO or Foxf1 KO MES cells. Bar plots showing the up- or down-regulated genes in Hand1 KO or Foxf1 KO MES cells. Lengths of bars representing the percentage of cluster specific genes which were up- or down-regulated. Dark colors indicating direct targets of Hand1 (left) and Foxf1 (right).

(B) Heatmap showing the transcriptomic similarity of MES cells and cell types of E7.0 mouse embryo. MES transcriptome data were generated using bulk RNA-seq. Cell-type specific transcriptomes of E7.0 mice were determined as the average of single-cell transcriptomes from each cell type. The gene set for comparison was defined as the collection of top 50 marker genes of each E7.0 cell type. Cosine similarity metric was used. Ant.PS, anterior primitive streak. Haem, haematoendothelial progenitors. Def.end, def.endoderm. Vis.end, visceral endoderm. Par.end, parietal endoderm. ExE.end, ExE endoderm. Surf.ect, surface ectoderm. ExE.ect, ExE ectoderm.

(C) Scatter plot showing gene expression FCs after Hand1 KO or Foxf1 KO. Dots representing genes with FC > 1.5 and adjusted P (Padj)-value < 1e-5 (Wald tests). Red and blue colors indicating genes co-activated/inhibited in Hand1 KO and Foxf1 KO cells. Correlation coefficient of FCs between Hand1 KO and Foxf1 KO is 0.74, P < 2.2e-16, t-test.

(D) Representative genome browser snapshots showing the localization of H3K27ac, H3K4me1, HAND1 and FOXF1 at the Hand1 and Foxf1 loci. The FOXF1-binding Hand1 enhancer (Hand1-eF) and the HAND1-binding Foxf1 enhancer (Foxf1-eH), for enhancer KO experiments, are highlighted.

(E-F) RT-qPCR showing that the reduction in RNA levels of the EEM marker genes after Hand1-eF KO can be rescued by HAND1 OE (e), and after Foxf1-eH KO can be rescued by FOXF1 OE.

(G) Cartoon illustrating target Foxf1-eH of by dCas9-VP64 and the genes in the vicinity of Foxf1-eH.

(H) RT-qPCR showing that the substantial increase in RNA levels of Foxf1 and the FOXF1 target genes, but not the Foxf1-eH neighbouring genes in Foxf1-eH CRISPRa cells. Data are the mean ± standard error of the mean from three independent experiments. Two-tailed unpaired Student’s t-test was performed.

HAND1 and FOXF1 are required for the cardiac lineage specification.

(A) Meta plot analysis showing the occupancies of HAND1 (left) and FOXF1 (right) at the C3/7/8 MJH specific DAEs from Figure 2d.

(B) Bright field microscopic images of WT, Hand1 KO and Foxf1 KO cells at MES, early CP, late CP and CM stages. Scale bar: 100 μm.

(C) Growth curve showing numbers of WT, Hand1 KO and Foxf1 KO cells counted at different time intervals during EB to MES differentiation. Data are the mean ± standard error of the mean from three independent experiments. Two-tailed unpaired Student’s t-test was performed.

(D) Enriched GO terms of the genes up- or down-regulated after Hand1 KO. One-sided Fisher’s Exact test with Benjamini-Hochberg multiple testing correction was performed.

(E) GSEA showing the distribution of the marker genes within the ranking of Foxf1 KO affected genes of in vitro differentiated CP cells (FDR calculated by permutation tests). Foxf1 KO affected genes were ranked from up- (red) to down-regulation (blue).

Hand1 KO in mesodermal cells blocks MJH specification in mouse embryos.

(A) Immunofluorescence staining showing a substantial reduction in protein levels of HAND1 and FOXF1 at the embryonic-extraembryonic boundary transverse sections (indicated by dashed lines and arrows) of E7.0 Hand1 CKO embryos. Bright filed images (lateral view) of corresponding embryos are shown on the left side. The distance for each section to the distal tip of the embryo is labelled at the upper right corner for each image. Scale bars: 100 μm (bright field images, left) and 20 μm (transverse sections, right).

(B) The bright field images of E9.5 Control (Ctrl) and Hand1 CKO mouse embryos. The arrows indicating the embryonic heart (h) and head folds (hf). Scale bar: 500 μm.

(C) UMAP layout of integrated E7.0 mouse embryo scRNA-seq data. Integration was performed using the scRNA-seq data of Ctrl and Hand1 CKO mice. Colors indicating cell types. Dashed lines emphasizing cells with increased (red) or decreased (yellow) numbers in Hand1 CKO mice.

(D) Bar plot showing the percentage of each cell type of Ctrl and Hand1 CKO mice.

(E) Dot plot showing marker gene expression in Ctrl and Hand1 CKO mice. Red highlighting markers of MM and EEM cell type.

(F) URD inferred trajectory tree revealing the developmental hierarchy of E7.0 mesoderm cells (Ctrl and Hand1 CKO snRNA-seq), colored by cell types. Ctrl and Hand1 CKO cells are distributed on both sides of the URD tree trunk, with Ctrl cells on the left and Hand1 CKO cells on the right.

HAND1 is required for exiting from the NM program.

(A) Co-regulated changes in the genes related to negative regulation of cell proliferation (left) and positive regulation of cell migration (right) in vivo and in vitro. Heatmap showing the fold changes (FC) of those co-regulated genes after Hand1 KO and Foxf1 KO in MES cells in vitro (top). Dot plot showing expression levels of those co-regulated genes after Hand1 KO in the MJH and PSH trajectories in vivo (bottom).

(B-C) DAPI staining in the transverse sections of E7.0 Ctrl (b) and Hand1 CKO (c) embryos. The distance for each section to the distal tip of the embryo was labelled at the upper right corner for each image. Dashed white lines denoting locations of the exocoelomic cavity (EC) and NM.

(D) Violin plot showing the distance between neighboring mesoderm cells in the transverse sections of E7.0 Ctrl and Hand1 CKO embryos. P-value was calculated using one-sided Mann-Whitney U test.

(E) H&E staining showing reduced embryo size and accumulation of mesodermal cells of E7.5 Hand1 CKO mouse embryos. Scale bars: 100 μm.

(F) Working model of early cardiac lineage differentiation. Cardiac fate specification initiates around E6.75-7.0 by segregation of the MJH and PSH trajectories. At E7.0, multipotent progenitor cells in MJH lineage present Hand1+ mixed mesodermal (MM) status, in contrast to PSH cells showing Lefty2+ nascent mesodermal (NM) status. MJH and PSH specifically contribute to JCF and SHF, respectively, while they both contribute to the formation of early heart tube (HT). HAND1 and FOXF1 bind to enhancers, regulate the MJH specific genes, and drive mesodermal differentiation toward MJH direction. Hand1 deletion led to blocked MJH specification and accumulation of early mesodermal cells.

Pseudotime-dependent genes for MJH/PSH trajectoyies. Gene-enhancer pairs in E7.0 mesoderm pseudotime trajectories.

Related to Figure 1F Related to Figure 2G

Hand1- and Foxf1-KO affected genes in MES cells.

Attached as an excel file.

Primers/oligos used in the current study

Data integration showing the developmental trajectories of cardiac progenitors

(A) Gene expression levels of the PSH (upper), MJH (middle) and FHF markers (lower) overlaid on the UMAP layout of the CM trajectory. Only cells belonging to the CM trajectory are shown.

(B) Violin plots showing the distribution of mature myocardium markers expression levels in E8.5 CM cells of the MJH and PSH trajectories.

(C) Fraction of labels transferred from Tyser et al. (Tyser et al. 2021) for E7.5 EEM cells and E7.75 PM cells.

Differential gene expression and signaling activaity in MJH and PSH

(A) Smoothened fitting curves showing expression levels of activated (solid line) and inhibited (dotted line) signaling target genes in MJH (red) and PSH (blue).

(B) Dynamic expression of Bmp4/5/7 in MJH and PSH between E7.0-E8.5 stages.

(C) Corn plots showing spatial MJH (upper) and PSH single cell mapping at the E7.0 (left) and E7.5 (right) stages. Columns representing micro-dissected locations in germ layers and rows representing distal (slice 2) to proximal ends (slices 10/11) of the mouse embryos. Colors indicating the number of single cells mapped to each location, or the aggregated expression level of Bmp4. EA, anterior endoderm; EP, posterior endoderm; M, whole mesoderm; MA, anterior mesoderm; MP, posterior mesoderm; A, anterior ectoderm; P, posterior ectoderm; L, left lateral ectoderm; R, right lateral ectoderm.

(D) Dynamic expression of the Bmp signaling target genes Car4 and Arl4c in MJH and PSH between E7.0-E8.5 stages.

Data integration of E7.0 multi-omics snRNA-seq with reference scRNA-seq

(A) UMAP layout of E7.0 snRNA-seq or snATAC-seq data. UMAPs of snRNA-seq was generated by integrating published (Pijuan-Sala et al. 2019) and multi-omics data of this study. Cells are colored by cell types.

(B) Dot plot showing marker gene expression in reference and multi-omics dataset.

(C) Heatmap showing expression levels of marker genes across mesodermal clusters of E7.0 mouse embryos.

Identification of the key lineage specific TFs

(A) UMAP layout of the MJH (C0/3/7/8) and PSH (C1/0/2) developmental trajectories. Color shades indicating pseudotime for developmental stages. Dot plots showing the marker gene expression for MJH specific clusters (C3/7/8) and PSH specific clusters (C1/0/2) in snRNA-seq.

(B) Identification of top PSH (upper)/Haem (lower) specific DNA-binding motifs and corresponding candidate TFs. The PSH/Haem specific DAEs were defined by comparing C2/C6 with C1 snATAC-seq data using SnapATAC (Fang et al. 2021) ‘findDAR’ function. Motif calling was performed by the HOMER (Heinz et al. 2010) ‘findMotifsGenome.pl’ function. Motif activity (colored in red) and TF expression (colored in blue) levels of trajectory specific candidate TFs, are overlaid on the UMAP layout from Figure 2a.

(C) Dynamic expression of Gata4, Tead4, Hand1 and Foxf1 in MJH and PSH between E7.0-E8.5 developmental stages.

Generation of the Hand1 and Foxf1 KO ESC lines

(A,C) Schematic diagram of the positions of sgRNAs targeting the CDS of Hand1 (A) and Foxf1 (C).

(B,D) Genomic PCR analyses using the primer sets flanking the cleavage sites verifying the genomic DNA deletion of Hand1 (B) and Foxf1 (D).

(E) Heatmap showing expression levels of the marker genes across mesodermal clusters of E7.0 mouse embryos (left). Bar plots showing expression FC upon Hand1 and Foxf1 KO (right). Asterisks indicating direct binding by the corresponding TFs.

Mutual regulation between HAND1 and FOXF1

(A-D) RT-qPCR showing the levels of HAND1 and FOXF1 in Hand1 and Foxf1 KO, the EEM marker genes (Foxf1, Bmp4, Pmp22 and Spin2c) after HAND1 and FOXF1 OE.

(E-F) Genomic PCR analyses using the primer sets flanking the cleavage sites verifying the genomic DNA deletion of the Hand1 enhancer (a) and the Foxf1 enhancer (c).

(G) Enriched GO terms of the genes up- or down-regulated after Foxf1 KO. One-sided Fisher’s Exact test with Benjamini-Hochberg multiple testing correction was performed.

(H) RT-qPCR showing that the expression levels of Myh6, Myh7 and Tnnt2 at CM stage are impaired by Foxf1 KO. Data are the mean ± standard error of the mean from three independent experiments. Two-tailed unpaired Student’s t-test was performed.

HAND1 is required for the expression of the EEM specific genes in vivo

(A) Schematic diagram showing strategy for the mesodermal cell specific Hand1 KO embryo generation.

(B) Bright field images of representative E7.0 Ctrl and Hand1 CKO embryos. Scale bar: 0.1 mm.

(C) UMAP layout as in Figure 6C. Colors indicating expression levels of the marker genes of Epi, PS, NM and EEM.

(D-E) Projection of E7.0 scRNA-seq onto the reference UMAP structure from Figure 1d. Reference UMAP layout for E7.0 CM trajectory cells colored by cell type (b) and trajectory (c).

(F) Bar plot showing the percentage of the MJH and PSH trajectories in NM of Ctrl and Hand1 CKO mice.