Fig. 5

Activity of the Ephb4-2 enhancer requires SMAD4. a Two sets of E10.5 Smad4^+/+;Ephb4-2:LacZand Smad4^EC/EC;Ephb4-2:LacZ littermates demonstrates loss of Ephb4-2 enhancer activity after loss of SMAD4 regardless of extent of growth retardation (n = 4 from 4 litters). Although some embryos exhibited severe growth retardation, similar sized younger embryos had robust Ephb4-2:LacZ expression (see Supplementary Fig. 5). Grey scale bars are 1000 μm. b, c Mutation of two composite SMAD-binding elements (SBE2/3 and SBE6/7) within the Ephb4-2 enhancer resulted in near-total loss of enhancer activity compared to wild-type enhancer in F0 transgenic zebrafish (b, mosaic due to nature of Tol2-mediated F0 transgenesis) and mice (c). Transverse section taken through the strongest lacZ-expressing F0 Ephb4-2mutSBE(2/3,6/7) embryo confirmed lack of vascular expression. Grey numbers in bottom right corner indicate the number of F0 mouse with similar expression patterns to the image shown. White and black scale bars both represent 100 μm, grey scale bars are 1000 μm. da = dorsal aorta, cv = cardinal vein, nt = neural tube; red bracket = dorsal artery; white bracket = axial veins.