Fig. 3: Transcriptional dynamics of cardiac NCCs at E10.5.

a Seurat UMAP plots of scRNA-seq data from NCC populations with the expression of marker genes. b Immunostaining on traverse sections of Wnt1-Cre;ROSA-EGFP embryos (n = 3) showing GFP, ISL1, and ACTA2 expression. Nuclei stained with DAPI are in blue. ISL1 is expressed in smooth muscle cells of the OFT cushions (arrowheads). c–e RNAscope in situ hybridization sections of Wnt1-Cre;ROSA-EGFP embryos for Egfp, Isl1 and Gata3 (c) (n = 5), Egfp, Cnn1 (d) (n = 3), Egfp and Rgs5 (e) (n = 3) expression. Nuclei stained with DAPI are in blue. Isl1 and Gata3 are expressed in NCCs within the OFT (white arrowheads in c) and at the level of the dorsal aortic sac wall and aortic sac protrusion (blue arrowheads in c). Gata3 but not Isl1 is expressed in the more proximal part of OFT (red arrowheads in c). Cnn1 and Rgs5 are expressed in smooth muscle cells of the OFT (white arrowheads in d and e). f Immunostaining on sections of Wnt1-Cre;ROSA-EGFP embryos (n = 3) showing GFP, TBX2, and TAGLN expression. Expression of TAGLN is in smooth muscle cells of the OFT (blue arrowheads) and PAAs expressing TBX2 (white arrowheads). g–i RNAscope in situ hybridization on traverse sections of Wnt1-Cre;ROSA-EGFP embryos for Egfp, Tbx2 (n = 5) and Gata3 (n = 4) (g), Egfp and Tbx2 (h) (n = 5), and Egfp and Tbx3 (i) (n = 5) expression. Nuclei stained with DAPI are in blue. Tbx2 and Gata3 expression overlaps in the dorsal wall of the OFT (white arrowheads in g). Tbx2 and Tbx3 are expressed in the mesenchyme dorsal to the aorta surrounding the PAAs at the level of the PA3-6 (white arrowheads in h and i). j Schematic representation of a transverse section at the level of the OFT summarizing Tbx2, Tbx3, Isl1, Gata3, Acta2, and Tagln expression in CNCCs at E10.5. NT neural tube, end endoderm, PAA pharyngeal arch artery. Scale bars: 100 μm.