Fig. 4

Identification of Runx1-regulated genes within the aortic HE. a Experimental scheme to identify Runx1-regulated genes in the HE (DP-R1^hi). Highlighted cell populations (DP-R1^hi and DN) from un-injected (control) and runx1 MO-injected double transgenic TgBAC(runx1P2:Citrine);Tg(kdrl:mCherry) embryos (28–30 hpf) were used for differential gene expression analysis (DEG) by RNA-seq similar to Fig. 3. b Multidimensional scaling plot (MDS-plot) analysis of the cell populations depicted in (a). cGene list intersection between ARE-specific and Runx1-repressed genes. d Gene list intersection between HE-specific and Runx1-activated genes. e Experimental strategy to analyse potential HE-specific Runx1 target genes by qRT-PCR in runx1^+/+(WT) and runx1^−/− mutant (MUT) embryos. f Timeline highlighting the time-points (red circles) used for subsequent qRT-PCR analysis. g qRT-PCR gene expression analysis of potential Runx1 targets (dnmt3ba, angpt1, irf1b and pik3cd) in the HE and ARE of runx1^+/+ (WT) and the HE of runx1^−/− mutant (MUT) embryos at the time-points depicted in (f). Graphs show the mean of detected expression levels relative to the geometric mean (GM) of the two housekeeping genes eef1a1l and rpl13a. n = 5 independent biological experiments for WT embryos and n = 6 independent biological experiments for MUT embryos. Error bars represent the SEM. 2-way ANOVA; *p < 0.05; **p < 0.01; ***p < 0.001. h ISH analysis for the same genes analysed in (g) performed in runx1^+/+ and runx1^−/− embryos