Activity of the Eng +7 and Eng +9 enhancers require conserved Ets and/or Gata TF binding sites. (A) Nucleotide sequence alignment of the Eng +7 enhancer with conserved Ets binding sites marked in blue and Gata binding sites in red. The nucleotide numbering is relative to the first ATG. (B) Nucleotide sequence alignment of the Eng +9 enhancer with Ets binding sites conserved in human/mouse/opossum marked in blue and human/mouse but not opossum in yellow. (C) Representative X-Gal stained wholemount E11.5 F₀ transgenic embryos generated with various wild-type or mutant Eng +7 and/or Eng +9 fragments subcloned into a SV40 promoter/lacZ reporter (SV/lacZ). Fully conserved Ets (E) and Gata (G) binding sites are represented as circles. The number of transgenic embryos with endothelial and/or fetal liver staining relative to number generated is also shown. The degree of X-gal staining is indicated as weak (+) to strong (+‖+‖+‖+). (i) SV/lacZ/ + 7 embryos show minimal blood/endothelial staining. (ii) SV/lacZ/+9 embryos show strong endothelial but little blood staining. SV/lacZ/+9 (ΔEts; missing region with fully conserved Ets binding sites in Eng +9) embryos show variable endothelial staining (some embryos show none) but no fetal liver staining. (iii) SV/lacZ/+7/+9 embryos show strong blood and endothelial staining. SV/lacZ7 (ΔG1G2)/+9 (missing region with conserved Gata binding sites in Eng +7) embryos show variable endothelial and no blood staining. SV/lacZ/+7(ΔG1G2)/+9 (ΔEts; missing region with conserved Gata sites in Eng +7 and fully conserved Ets sites in Eng +9) embryos show variable endothelial and no blood staining. Tissue sections magnified ×40; inset ×100.