Hemogenic and nonhemogenic endothelium can be distinguished by the activity of fetal liver kinase (Flk)–1promoter/enhancer during mouse embryogenesis

Accumulating evidence in various species has suggested that the origin of definitive hematopoiesis is associated with a special subset of endothelial cells (ECs) that maintain the potential to give rise to hematopoietic cells (HPCs). In this study, we demonstrated that a combination of 5′-flanking region and 3′ portion of the first intron of the Flk-1gene (Flk-1p/e) that has been implicated in endothelium-specific gene expression distinguishes prospectively the EC that has lost hemogenic activity. We assessed the activity of this Flk-1p/e by embryonic stem (ES) cell differentiation culture and transgenic mice by using theGFPgene conjugated to this unit. The expression ofGFPdiffered from that of the endogenous Flk-1gene in that it is active in undifferentiated ES cells and inactive in Flk-1+lateral mesoderm. Flk-1p/e becomes active after generation of vascular endothelial (VE)–cadherin+ECs. Emergence of GFP−ECs preceded that of GFP+ECs, and, finally, most ECs expressed GFP both in vitro and in vivo. Cell sorting experiments demonstrated that only GFP−ECs could give rise to HPCs and preferentially expressed Runx1and c-Mybgenes that are required for the definitive hematopoiesis. Integration of both GFP+and GFP−ECs was observed in the dorsal aorta, but cell clusters appeared associated only to GFP−ECs. These results indicate that activation of Flk-1p/e is associated with a process that excludes HPC potential from the EC differentiation pathway and will be useful for investigating molecular mechanisms underlying the divergence of endothelial and hematopoietic lineages.