Fig. 1

The MEF2-dependent HLX-3 enhancer directs gene expression within the developing coronary vasculature. a Schematic of the HLX-3 enhancer:lacZ transgene. Coloured shapes represent verified MEF2 and ETS binding motifs within the human HLX-3 enhancer sequence, upstream of the hsp68 silent minimal promoter and lacZ reporter gene¹⁹. b Whole-mount images of embryonic E11.5–E15.5 HLX-3:lacZ transgenic hearts show enhancer activity, as detected by blue X-gal staining, in the endothelium of selective coronary vessels. All images are from a single transgenic line and are representative of at least five biologically independent samples, little variation in staining pattern was seen. Supplementary Figure 5 shows comparative expression patterns in three other HLX-3:lacZ stable transgenic mouse lines. c Schematic summarising the current model of coronary vessel origin and differentiation, adapted from ref. ^10,16,34. Light blue represents SV-derived vascular plexus, green represents endocardium-derived intramyocardial vessels, turquoise colouring of the E15.5 dorsal side indicates presence of vessels from both origins. SV-derived arterial and pre-arterial vessels are indicated in red, SV-derived veins are indicated in dark blue. d Transverse section through a E13.5 HLX-3:lacZ transgenic heart. Immunostaining and X-gal staining was conducted and imaged sequentially on a single sample. HLX-3 enhancer activity (blue staining) is compared with pan-endothelial marker CD31 (green) and venous/endocardial marker EMCN (yellow). Grey boxed regions are shown in detail in Supplementary Fig. 3b. Representative of four biologically immunohistological independent experiments, with similar staining patterns seen in all. e Transverse section through a E15.5 HLX-3:lacZ transgenic heart. HLX-3 enhancer activity (blue) is compared with pan-endothelial marker CD31 (green), arterial marker α-SMA(red) and venous/endocardial marker EMCN (yellow). Two sections are shown, representing different levels in the heart from base to apex. Grey boxed regions are shown in Supplementary Fig. 4 alongside additional arterial and venous markers. Representative of seven biologically independent immunohistological experiments, with similar staining patterns seen in all. Black/white arrows indicate endocardium, red arrows indicate α-SMA-positive arteries, blue arrows indicate veins determined by EMCN expression and sub-epicardial location. ra, right atrium; la, left atrium; rv, right ventricle; lv, left ventricle; s, septum; vp, vascular plexus. Black scale bars represent 500 μm, grey scale bars represent 200 μm