Fig. 6

Aortic HE maintains arterial features in the absence of runx1. a qRT-PCR gene expression analysis of the arterial marker efnb2aand the venous marker ephb4b in the HE and ARE of runx1^+/+ (WT) and the HE of runx1^−/− mutant (MUT) embryos. 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. Two-way ANOVA; *p < 0.05; **p < 0.01; ***p < 0.001. b Heatmaps of whole-genome gene expression data from RNA-seq for the HE of control and runx1 MO embryos showing arterial and venous genes. cRepresentative fluorescent microscopy image of 6 dpf TgBAC(runx1P2:Citrine), Tg(kdrl:mCherry) double transgenic embryos on a runx1^+/+ or runx1^−/− genetic background. Left: Region depicting the region of definitive haematopoietic niches including the thymus (Th) and the kidney (Ki). Right: region depicting the DA and the beginning of the caudal haematopoietic tissue (CHT). Green arrows point to the ventral wall of the DA. d Quantification of embryos derived from the indicated in-crosses (InX) depicting the runx1^−/− mutant phenotype as depicted in (c). e ISH analysis of runx1 in control and dll4 MO embryos. Experiment was performed with two different MOs. Green arrows point to the HE. f Offspring of dll4^+/− heterozygous fish were analysed by ISH for runx1 at 32 hpf. Runx1 expression was quantified by image analysis and subsequent genotyping⁵⁷. Representative examples of runx1 expression in each of the WT, dll4^+/− and dll4^−/− genotypes are shown in the right-hand panels and the corresponding expression values are shown as orange points in the graph. (μ_wt = 33.3, μ_mut = 15.19, p < 0.0001, F(2,50) = 16,79, one way ANOVA). g Model depicting the lineage relationship between the definitive HE giving rise to HSCs and arterial endothelium in the DA during embryogenesis. Dll4⁺ aortic progenitors get exposed to localised signals patterning the DA and inducing haemogenic gene expression. Haemogenic specification includes upregulation of runx1 expression and a subsequent Runx1 induced repression of dll4, thus giving rise to Dll4⁻ mature HE