Fig. 4.
Downstream targets regulated by GNAQ R183Q in endothelial cells. (A) Transcriptome changes in response to GNAQ R183Q expression in endothelial cells at E14.5, revealed by RNA-seq. Lists of the top 10 genes with the highest fold change. Magenta, upregulated expression; blue, downregulated expression. (B) Enriched GO terms for the differentially expressed genes in GNAQ R183Q mutant skin compared to the control, analyzed by PANTHER. (C) Heatmap from E14.5 RNA-seq datasets showing the expression of 16 selected endothelial cell-expressing genes in the control and GNAQ R183Q mutant mice: 12 from the top 30 upregulated genes and four from the top 30 downregulated genes, compared to the control. (D) Validation of the RNA-seq data by qRT-PCR using RNA extracted from E14.5 back skins. All data represent mean±s.e.m. of four biological replicates. Gapdh was used as a control. (E) Sagittal sections of E14.5 back skin stained with HMGA2 (green) and PECAM1 (magenta). DAPI was used to highlight nuclei. In control skin, HMGA2 staining was found only in non-endothelial dermal cells (arrowheads). Strong HMGA2 staining was found in endothelial cells of mutant embryos (arrows). Scale bar: 50 μm. (F) Tubule formation assay in vitro showing GNAQ R183Q expression in HMEC-1 cells significantly impairs the tubule formation capability of endothelial cells. GNAQ R183Q is sufficient to drive the expression of HMGA2 in HMEC-1 cells. (G) HMGA2 knockdown can partially rescue the phenotype caused by GNAQ R183Q. GAPDH was used as a control. Scale bar: 200 μm (F,G). **P<0.01; ns, not significant. Data are mean±s.e.m.