Fig. 6

The Ephb4-2 enhancer is bound by SMAD1/5. a Mouse (m) and orthologous human (h) DNA sequence of the Ephb4-2 enhancer. Red nt on human sequence correspond to regions bound by SMAD1/5 as determined from SMAD1/5 human umbilical vein endothelial cell (HUVEC) chromatin immunoprecipitation-sequencing (ChIP-seq) data³² (see also Supplementary Fig. 6c). Green boxes denote GC-SBE motifs associated with SMAD1/5 binding, blue boxes denote SBE motifs associated with all SMAD binding and grey boxes denote other over-represented motifs (MEME1, 3 and 5³²). Black outlined boxes denote composite SBE/GC-SBE elements, italic nt indicate linker sequences. Grey text under sequences indicates motifs previously identified as the EBEs and SBEs in earlier analysis (Supplementary Fig. 4). For sequence logos of motifs, see Supplementary Fig. 6b. b SMAD1/5 HUVEC and PASMC ChIP-seq data from Morikawa et al.³² (red peaks = statistically significant peaks after BMP9 stimulation in HUVEC). Significant binding peaks are not observed in PASMC after BMP4 stimulation (green). See also Supplementary Fig. 6c). c Box and whiskers plot of ChIP-qPCR data shows significant enrichment of SMAD1 binding at the Ephb4-2 enhancer in BMP9-stimulated HUVECS (red) compared to a control intergenic region on the same chromosome (p = 0.00015, paired two-tailed t test). SMAD1 binding at the Ephb4-2 enhancer region is not enriched over control in the absence of BMP stimulation (green) (p = 0.19212). No enrichment is observed between IgG control regions (p = 0.3154 and p = 0.19212, paired two-tailed t test). Horizontal lines = medians, boxes = interquartile range (IQR); vertical lines = minimal/maximal values (up to 1.5× IQR) and black dots = data points outside of 3× IQR. Data represents three biological replicates each with three technical replicates performed in triplicate. All data points were included in statistical analysis. d, e Morpholino (MO)-mediated partial knockdown of smad1/5 on GFP expression in arterial tg(Dll4in3:GFP) (d) and venous tg(Ephb4-2:GFP) (e) transgenic zebrafish lines, using 0.5 ng smad1 MO and 0.25 ng smad5 MO. Graphs depict expression in all embryos, Dll4in3:GFP WT n = 58, MO n = 48; Ephb4-2:GFP WT n = 104, MO n = 84. High expression = solid colour, weak = pattern, absent = solid white. Zebrafish embryos shown are representative of the predominant phenotype, red bracket = dorsal aorta, white bracket = posterior cardinal and ventral vein(s). White scale bars represent 100 μm