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GATA-2, a transcription factor that has been shown to play important roles in multiple organ systems during embryogenesis, has been ascribed the property of regulating the expression of numerous endothelium-specific genes. However,the transcriptional regulatory hierarchy governing Gata2 activation in endothelial cells has not been fully explored. Here, we document GATA-2 endothelial expression during embryogenesis by following GFP expression in Gata2-GFP knock-in embryos. Using founder transgenic analyses, we identified a Gata2 endothelium enhancer in the fourth intron and found that Gata2 regulation by this enhancer is restricted to the endocardial, lymphatic and vascular endothelium. Whereas disruption of three ETS-binding motifs within the enhancer diminished its activity, the ablation of its single E box extinguished endothelial enhancer-directed expression in transgenic mice. Development of …