Fig. 3: RA signaling patterns the cardiogenic mesoderm.

a Schematic representation of the genetic modifications in the TBX5^mCherry/NKX2.5^eGFP hESC reporter cell line (ES03 TN). b Representative live images of cells expressing eGFP (NKX2.5; green) Cherry (TBX5; red) at day 30 of differentiation with (RA+) or without RA (RA−). Scale bar = 50 µm. c Percentage of cells expressing mCherry (TBX5) and eGFP (NKX2.5) at the indicated days of differentiation with (RA+) or without RA (RA−) as determined by live flow cytometry. Data are mean; for day 2 n = 2 independent samples; days 3 and 4 n = 5; day 5 n = 7; days 8 and 30 n = 3 examined over seven independent experiments. d Quantification of flow cytometry time course analysis of cells expressing ISL1 and cTnT within mCherry⁺/eGFP⁺ (TBX5⁺/NKX2.5⁺) population from RA+ differentiation; and eGFP⁺ (NKX2.5⁺) population from RA− differentiation. Data are mean; n = 2 independent experiments. e Top: Graphic representation of the experimental design applied for scRNA-Seq analysis at days 1.5 and 4.5. Bottom: Representative flow cytometry plots showing the gating strategy for sorting cells at day 4.5. Cells differentiated without RA (RA−) were isolated based on the expression of eGFP (NKX2.5); cells differentiated with RA (RA+) were isolated based on expression of eGFP (NKX2.5), mCherry (TBX5), or both. f UMAP clustering of single cells captured at days 1.5 and 4.5; main cell types are annotated. Inset: UMAP plot showing the contribution of the indicated samples. CPCs cardiovascular progenitor cells, E-PCs endothelial/endocardial progenitor cells. g Dot-plot showing expression level of selected differentially expressed genes for clusters identified as 15, 11–endoderm; 12–early CPCs; 1, 0–first heart field (FHF); 3–anterior second heart field (aSHF); 17–smooth muscle cell progenitor cells (SMC-PCs); 19–E-PCs; 9, 18, 14–juxta-cardiac field (JCF). h Contribution of cells from each population to the indicated clusters relative to total number of cells in the cluster normalized for total number of cells in each population. i Dendrogram showing hierarchical clustering of the averaged, corrected, normalized expression values per clusters after integration of indicated scRNA-seq clusters with scRNA-seq datasets of mouse early heart development^5,6. Source data are provided as a Source Data file.