Figure 4: Immunohistochemical and ES/EB studies support the dpath results.

(a) The average metagene coefficients of cells from the C1, C2 and C3 clusters were illustrated using the barplot. In contrast to C2 where both MG1 (endothelium) and MG3 (endocardium) had high intensity, C1 and C3 were dominated by MG3 and MG1, respectively. (b) Immunohistochemical analysis of Etv2-EYFP transgenic hearts at E7.75 and E8.5 supports the existence of the C2 cell population and the notion that they are progenitors of the cardiac cushion. Fluorescent images are pseudo-coloured after photographing in black and white. Large arrowheads point to EYFP⁺ Endomucin⁺ endothelial cells. Small arrowheads denote EYFP⁺ Endomucin⁺ angioblasts. Small arrows highlight EYFP⁺ Tbx20⁺ Endomucin⁻ C1 cells (a: common atrium, cc: cardiac crescent, ec: endocardium, ivs: intraventricular septum, la: left atrium, lv: left ventricle, nt: neural tube, oft: outflow tract, ra: right atrium, rv: left ventricle). Scale bars indicate 100 μm. (c,d) The expression patterns of Runx1 and Gata1 were illustrated on the metacell landscape. Green: high expression. Black: low expression. (e,f) The aggregated expression pattern of genes related to primitive erythrocyte differentiation (GO:0060215) and definitive erythrocyte differentiation (GO:0060216) were illustrated on the metacell landscape. Yellow: high expression. Blue: low expression. (g) The barplot shows the top 10 KEGG pathways that were enriched in genes that were significantly upregulated in C2, C5 and C6 cell clusters, compared with the remaining clusters (SCDE P value <0.001). (h) The aggregated expression pattern of genes related to hedgehog signalling pathway were illustrated on the metacell landscape. (i) A schematic diagram represents the ES/EB differentiation model system (using Etv2-EYFP transgenic cell lines) and the exposure to the SHH agonist (SAG) or SHH antagonist cyclopamine from days 2 to 4.5. (j) FACS quantification indicates that sonic hedgehog agonist (SAG) (or cyclopamine) significantly promotes (or suppresses) endothelial and haematopoietic progenitors (EYFP⁺/CD41⁺/Tie2⁺), compared with dimethyl sulfoxide control (*Student’s t-test P value <0.05.