DEVELOPMENT 1703 RESEARCH ARTICLE INTRODUCTION In vertebrates, the vascular network is composed of separate blood and lymphatic systems. Although the blood and lymphatic systems are organized in parallel, the blood vasculature develops and is functional prior to lymphangiogenesis. The murine blood vasculature develops from angioblasts that are associated with the blood islands of the yolk sac. This process, known as vasculogenesis, results in the formation of the initial vascular network, which consists of paired dorsal aortae, the cardinal veins, the vitelline artery and vein, and the endocardial tubes. New endothelial cells and vessels are generated later via a process called angiogenesis (Risau, 1997). Further maturation of this new vasculature occurs via pruning of unneeded branches, resulting in the formation of the mature vascular network. In the yolk sac, the blood islands consist of a thin layer of angioblasts surrounding primitive erythrocytes. Similarly, in the aorta-gonads-mesonephros region – the initial embryonic site of definitive hematopoiesis – hematopoietic stem cells can be detected budding from the endothelium of the dorsal aorta (de Bruijn et al., 2002). Given the close physical proximity of the very earliest hematopoietic and endothelial cells, it has been speculated that they originate from a common progenitor cell, which has been termed the hemangioblast. A number of transcription factors have been shown to play a role in the development of both cell lineages: for example, cloche is required for the formation of endothelial and hematopoietic progenitors in zebrafish (Stainier et al., 1995) and Scl (also known as Tal1 – Mouse Genome Informatics), which encodes a basic helix-loop-helix transcription factor, was initially shown to be required for hematopoietic development in mice (Robb et al., 1995; Shivdasani et al., 1995). Subsequent transgenic rescue of the hematopoietic defect in Scl-null embryos revealed a requirement for SCL in the remodeling of the yolk sac vasculature (Visvader et al., 1998), and it has since been shown to play a role in vasculogenesis (Patterson et al., 2005), as well as in the migration and morphogenesis of endothelial cells (Lazrak et al., 2004). Transgenic expression of SCL is able to rescue the phenotypic consequences of cloche mutation in the zebrafish, suggesting that Scl functions downstream of cloche (Liao et al., 1998). LMO2, a member of the LIM domain family, is required for primitive erythropoiesis in the embryo; Lmo2 ablation results in death at embryonic day (E) 9.75 secondary to hematopoietic failure (Warren et al., 1994). Analysis of chimeric mice bearing contributions from Lmo2 –/– embryonic stem (ES) cells revealed that angiogenic remodeling of blood vessels requires Lmo2 (Yamada et al., 2000). Similarly, targeted disruption of the transcription factor Runx1 eliminates definitive hematopoiesis and results in defective angiogenesis and hemorrhaging throughout the CNS (Wang et al., 1996). The most-widely accepted and experimentally supported model for lymphatic development has proposed that the lymphatic vasculature arises from the blood vasculature (Sabin, 1902; Sabin, 1904; Wigle and Oliver, 1999). Expression of the lymphatic endothelial hyaluronan receptor gene (Lyve1; also known as Xlkd1 – Mouse Genome Informatics) at E9-9.5 in endothelial cells lining the anterior cardinal vein is the first sign that these cells are competent to become lymphatic endothelial cells (LECs). The lymphatic regulatory gene Prox1, encoding a homeobox transcription factor, is expressed several hours later in a subset of LYVE1 + cells in the anterior cardinal vein (Oliver, 2004). Expression of the murine vascular endothelial growth factor receptor 3 gene (Vegfr3, also known as Flt4 – Mouse Genome Informatics), which binds VEGFC, is detected in blood and lymphatic vessels during early embryogenesis, but becomes largely restricted to lymphatic vessels after E14.5 (Kaipainen et al., 1995). A Gata2 intronic enhancer confers its pan-endothelia-specific regulation Melin Khandekar 1, *, William Brandt 1, *, Yinghui Zhou 1,† , Susan Dagenais 2 , Thomas W. Glover 2 , Norio Suzuki 3 , Ritsuko Shimizu 1,3 , Masayuki Yamamoto 3 , Kim-Chew Lim 1 and James Douglas Engel 1,‡ 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 the endothelium is known to require SCL (TAL1), and an SCL-E12 (SCL-Tcfe2a) heterodimer can bind the crucial E box in the enhancer in vitro. Thus, GATA-2 is expressed early in lymphatic, cardiac and blood vascular endothelial cells, and the pan-endothelium-specific expression of Gata2 is controlled by a discrete intronic enhancer. KEY WORDS: Gata2, Endothelium, Cardiovascular, Lymphatic, Enhancer, ETS, SCL, Mouse Development 134, 1703-1712 (2007) doi:10.1242/dev.001297 1 Department of Cell and Developmental Biology and 2 Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109-2200, USA. 3 TARA Centre, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8577, Japan. *These authors contributed equally to this work † Present address: Aveo Pharmaceuticals, 75 Sidney Street, Cambridge, MA 02139, USA ‡ Author for correspondence (e-mail: engel@umich.edu) Accepted 26 February 2007