Fig. 4

A bona fide population of cardiac LECs identified in human fetal heart (A) UMAP generated by EC subclusters of the cell dataset. (B) Dot plot showing both the expression level and the percentage of cells expressing selected genes from the EC clusters and lymphatic markers. (C) Heatmap generated from the EC clusters showing gene expression from snRNA-seq and genes defining the bona fide lymphatic cluster. (D) Whole-mount dorsal view z-projection of 10PCW human fetal hearts immunostained for PROX1 (green) and secreted forms of RELN (blue) and ETV2 (red) (n = 2). (D′) A magnified view of the region in the dashed box in (D). (E) Immunohistochemistry for RELN (red) and PROX1 (green) co-staining on 11PCW fetal heart sections showing signal of RELN surrounds LECs with high PROX1+ nuclei (white arrowheads) in the epicardium. (F) Spearman correlation plot for selected TFs’ average RNA expression and binding motif presence in open chromatin of endothelial and lymphatic cells. Color intensity of the circle indicates the strength of the correlation according to the scale provided at the side of the graph. (G) UMAP generated by subclustering the LECs from cluster 14 of the snMultiome in Figure 1G (left). FeaturePlots of snRNA-seq + snATAC-seq from the subclusters of the LECs showing different levels of expression of PROX1 and FLT4 RNA (right). (H) Dot plots illustrating RNA (left) and binding motifs (right). High-level expressions for PROX1, NR2F2, RBPJ, ETV6, and ETS2 are found in all 4 subclusters. However, binding motifs for all ETV family members show trends opposite to PROX1, NR2F2, SOX18, and RBPJ in the LEC subclusters. Scale bars, (D) 100 μm and (D′ and E) 10 μm.