Fig. 5. Identification of a crucial E box for Gata2 vascular endothelium enhancer activity. (A) Consensus binding motifs for candidate regulatory effectors within the evolutionarily conserved 460 bp AlwNI-ApaI endothelium-specific enhancer sequence are highlighted. The 167 bp minimal vascular endothelium-specific (VE) enhancer in TKVEß (B) was generated using the PCR primer pairs indicated by the two convergent arrows. The italicized sequences correspond to the radiolabeled probe used for EMSA studies (see Fig. 6). (B) TKVEß recapitulates widespread vascular (14/17), but not endocardial (0/17, arrow), endothelial lacZ expression in E10.5 transgenic embryos. (C) Simultaneous mutation of all three ETS1-binding consensus sites (A) in TKVEßmEts1,2,3 resulted in far fewer (3/25) transgenic embryos that displayed vascular endothelium-specific lacZ expression. (D) Disruption of the single SCL-binding site (A) in TKVEßmScl completely abrogated vascular endothelium-specific X-gal accumulation (0/24). (E-G) Transverse sections through the hearts of E10.5 embryos bearing TKSRß (E; Fig. 4B), TKAAß (F; Fig. 4C) or TKVEß (G; Fig. 5B) transgenes. Notice the conspicuous absence of X-gal staining in the endocardium of the ventricular chamber of the TKVEß embryo.