Summary Lmo 2 and Scl / Tal 1 convert non-axial mesoderm into haemangioblasts which differentiate into endothelial cells in the absence of Gata 1

In mammalian and avian embryos, primitive erythrocytes form in the extraembryonic yolk sac where clusters of mesodermal cells give rise to both haematopoietic cells (HCs) and angioblasts, early progenitors for endothelial cells (ECs). Inside the embryo in the aorta-gonad-mesonephros (AGM) region, definitive HCs develop in association with the floor of the dorsal aorta. This close relationship of HCs and ECs suggests the existence of a common progenitor, the haemangioblast (Keller, 2001). The concept of the haemangioblast has gained support from several findings. Firstly, angioblasts and haematopoietic progenitors share the expression of several genes (Keller, 2001). Secondly, targeted disruption of the gene for the vascular endothelial growth factor (VEGF) receptor-2, flk1, in the mouse (Shalaby et al., 1995) and mutation of the zebrafish cloche locus affect both the blood and endothelial lineages (Stainier et al., 1995). Thirdly, single Flk1-positive (Flk1 +) cells (so called blast colony forming cells; BL-CFCs) derived from embryonic stem (ES) cells can give rise to blast colonies (BL-C) that contain both blood and endothelial progenitors in vitro (Choi et al., 1998; Faloon et al., 2000; Nishikawa et al., 1998). However, haemangioblasts have not yet been isolated from a vertebrate embryo. The best in vivo evidence for the existence of the haemangioblast comes from lineage labelling studies in the chick (Jaffredo et al., 1998; Jaffredo et al., 2000). These studies also suggest that definitive HC clusters directly differentiate from the ventral endothelial lining of the dorsal aorta. Consistent with this idea, ECs isolated from the AGM region and the vitelline and umbilical arteries of murine embryos possess haematopoietic stem cell activity (North et al., 2002), and ECs selected from the AGM region, the foetal liver and the foetal bone marrow of human embryos develop into myelo-lymphoid cells in culture (Oberlin et al., 2002). In zebrafish, primitive erythrocytes and ECs of the major trunk vessels, the dorsal aorta and the axial vein, form in close association in the intermediate cell mass (ICM) in the posterior midline of the embryo 1 day after fertilisation. These cells originate from the posterior lateral mesoderm (PLM) of the post-gastrula embryo (Zhong et al., 2001) (Fig. 1D). Here, overlapping expression patterns of blood and endothelial genes suggest the existence of haemangioblast-like cells (Brown et The LIM domain protein Lmo2 and the basic helix-loophelix transcription factor Scl/Tal1 are expressed in early haematopoietic and endothelial progenitors and interact with each other in haematopoietic cells. While loss-offunction studies have shown that Lmo2 and Scl/Tal1 are essential for haematopoiesis and angiogenic remodelling of the vasculature, gain-of-function studies have suggested an earlier role for Scl/Tal1 in the specification of haemangioblasts, putative bipotential precursors of blood and endothelium. In zebrafish embryos, Scl/Tal1 can induce these progenitors from early mesoderm mainly at the expense of the somitic paraxial mesoderm. We show that this restriction to the somitic paraxial mesoderm correlates well with the ability of Scl/Tal1 to induce ectopic expression of its interaction partner Lmo2. Co-injection of lmo2 mRNA with scl/tal1 dramatically extends its effect to head, heart, pronephros and pronephric duct mesoderm inducing early blood and endothelial genes all along the anteroposterior axis. Erythroid development, however, is expanded only into pronephric mesoderm, remaining excluded from head, heart and somitic paraxial mesoderm territories. This restriction correlates well with activation of gata1 transcription and co-injection of gata1 mRNA along with scl/tal1 and lmo2 induces erythropoiesis more broadly without ventralising or posteriorising the embryo. While no ectopic myeloid development from the Scl/Tal1Lmo2-induced haemangioblasts was observed, a dramatic increase in the number of endothelial cells was found. These results suggest that, in the absence of inducers of erythroid or myeloid haematopoiesis, Scl/Tal1-Lmo2induced haemangioblasts differentiate into endothelial cells.