During embryogenesis, hematopoiesis occurs in sequential waves, producing specific blood cell types to meet the needs of the growing embryo at different developmental stages. The first transient wave of primitive erythrocyte production occurs in the yolk sac soon after gastrulation, and is rapidly followed by the production of definitive blood cell types (Palis et al., 1999). Shifting to an intra-embryonic site, a second wave of hematopoiesis occurs along the ventral aspect of the dorsal aorta and in all major arteries of the developing embryo. This wave of hematopoiesis has been shown to produce the hematopoietic stem cell population that will subsequently seed and expand in the fetal liver before migrating to the bone marrow, where it maintains the life-long supply of blood cells in the adult organism (Dzierzak and Speck, 2008). Due to the close proximity of the endothelial and hematopoietic lineages within the extra-embryonic yolk sac blood islands, hematopoietic and endothelial development in the early embryo have long been believed to be closely linked (Sabin, 1920). Studies using differentiating embryonic stem (ES) cells and gastrulating embryos have shown this to be the case by identifying the hemangioblast, a multi-potent precursor that gives rise to hematopoietic, endothelial and smooth muscle lineages (Kennedy et al., 1997; Choi et al., 1998; Nishikawa et al., 1998; Huber et al., 2004). The hemangioblast emerges in the primitive streak from Brachyury-expressing mesoderm and expresses the vascular endothelial growth factor receptor fetal liver kinase 1 (FLK1) (Fehling et al., 2003; Huber et al., 2004). Furthermore, there is a large body of evidence demonstrating that definitive blood cells, emerging from intra-embryonic sites, have an endothelial origin (Jaffredo et al., 1998; de Bruijn et al., 2000; Zovein et al., 2008; Bertrand et al., 2010; Boisset et al., 2010; Kissa and Herbomel, 2010), but it was not until recently that yolk sac hematopoiesis was also demonstrated to proceed from hemangioblast to hematopoiesis by means of an endothelial intermediate with hematopoietic potential, known as hemogenic endothelium (Eilken et al., 2009; Lancrin et al., 2009). This yolk sac hemogenic endothelium population expresses TIE2 and cKIT, but is negative for the αIIb integrin CD41 (Lancrin et al., 2009). Acquisition of CD41 expression followed by the down-regulation of endothelial markers is indicative of the progression to fully committed hematopoietic progenitors (Ferkowicz et al., 2003; Mikkola et al., 2003; Sroczynska et al., 2009a).
Studies of the ETS family transcription factor ETV2 within the context of early embryonic development have revealed a vital role for this protein during mesoderm specification. ETV2 has been shown to be critically required for both hematopoiesis and vasculogenesis as ETV2-deficient embryos die by E11.0 with a complete absence of blood progenitors and severe vascular defects (Lee et al., 2008). Interestingly, while the function of ETV2 in vasculogenesis seems to be well conserved across evolution in higher vertebrates, the role of this transcription factor in hematopoiesis is much less conserved with no apparent role in Xenopus blood formation and only a limited function in the formation of myeloid progenitors in zebrafish (Sumanas and Lin, 2006; Pham et al., 2007; Liu and Patient, 2008; Sumanas et al., 2008; Neuhaus et al., 2010; Ren et al., 2010; Salanga et al., 2011). Nevertheless, despite significant advances in understanding the roles of ETV2 in development and more recently its fundamental requirement for hematovascular mesoderm specification (Kataoka et al., 2011), the precise stage and requirement of ETV2 expression in early hematopoietic precursor populations remains to be fully characterized. To address this, we generated ES cells and corresponding mice carrying a transgene expressing green fluorescent protein (GFP) under the control of ETV2 regulatory sequences. Analysis of ETV2::GFP expression both in vitro and in vivo allowed us to accurately define ETV2 expression at the onset of hematopoiesis. Specifically we demonstrate that both in differentiating ES cells and gastrulating embryos ETV2 is detected in the FLK1+ population but that it also marks the next stage of specification, the hemogenic endothelium. Importantly, knocking out ETV2 function reveals that ETV2 is not just expressed in the hemogenic endothelium, it is also essential for its formation.