Studies with embryonic explants and embryonic stem cells have suggested a role for Hedgehog (Hh) signaling in hematopoiesis. However, targeted deletion of Hh pathway components in the mouse has so far failed to provide in vivo evidence. Here we show that zebrafish embryos mutant in the Hh pathway or treated with the Hh signaling inhibitor cyclopamine display defects in adult hematopoietic stem cell (HSC) formation but not in primitive hematopoiesis. Hh is required in the trunk at three consecutive stages during vascular development: for the medial migration of endothelial progenitors of the dorsal aorta (DA), for arterial gene expression, and for the formation of intersomitic vessel sprouts. Interference with Hh signaling during the first two stages also interferes with HSC formation. Furthermore, HSC and DA formation also share Vegf and Notch requirements, which further distinguishes them from primitive hematopoiesis and underlines their close relationship during vertebrate development.

The SCL gene encodes a basic helix-loop-helix (bHLH) transcription factor that is essential for the development of all haematopoietic lineages. SCL is also expressed in endothelial cells, but its function is not essential for specification of endothelial progenitors and the role of SCL in endothelial development is obscure. We isolated the zebrafish SCL homologue and show that it was co-expressed in early mesoderm with markers of haematopoietic, endothelial and pronephric progenitors. Ectopic expression of SCL mRNA in zebrafish embryos resulted in overproduction of common haematopoietic and endothelial precursors, perturbation of vasculogenesis and concomitant loss of pronephric duct and somitic tissue. Notochord and neural tube formation were unaffected. These results provide the first evidence that SCL specifies formation of haemangioblasts, the proposed common precursor of blood and endothelial lineages. Our data also underline the striking similarities between the role of SCL in haematopoiesis/vasculogenesis and the function of other bHLH proteins in muscle and neural development.

Blood and endothelial cells arise in close association in developing embryos, possibly from a shared precursor, the hemangioblast, or as hemogenic endothelium. The transcription factor, Scl/Tal1 (stem cell leukemia protein), is essential for hematopoiesis but thought to be required only for remodeling of endothelium in mouse embryos. By contrast, it has been implicated in hemangioblast formation in embryoid bodies. To resolve the role of scl in endothelial development, we knocked down its synthesis in zebrafish embryos where early precursors and later phenotypes can be more easily monitored. With respect to blood, the zebrafish morphants phenocopied the mouse knockout and positioned scl in the genetic hierarchy. Importantly, endothelial development was also clearly disrupted. IntroductionDuring mammalian and avian embryogenesis, hematopoietic precursors arise in close association with the vasculature in the blood islands of the extra-embryonic yolk sac (YS) and in the ventral wall of the dorsal aorta (DA), in the aorta-gonads-mesonephros (AGM) region. These observations have led to the hypothesis that the 2 cell types arise from a common precursor, the hemangioblast. Coexpression of blood and endothelial genes, and the dependence of both lineages on several of them, are cited as evidence (for a review, see Keller 1 ). In addition, single cells expressing the vascular endothelial growth factor (VEGF) receptor, flk1, isolated from embryoid bodies (EBs), differentiate in vitro into blast colonies, consisting of both hematopoietic and endothelial progenitors. These blast colonyforming cells (BL-CFCs) therefore represent the in vitro equivalent of the hemangioblast. 2 However, the definitive lineage-labeling experiment to prove the in vivo existence of the hemangioblast has yet to be performed.The concept of hemogenic endothelium has also acquired significant experimental support over recent years. Label targeted to endothelial cells (ECs) has been found later in the emerging clusters of cells containing the first hematopoietic stem cells (HSCs) in the DA (Jaffredo et al 3 and references therein). Consistent with these data, purified ECs from both mouse and human embryos have been shown to possess hematopoietic potential. [4][5][6] Taken together, the hemangioblast and hemogenic endothelium data strongly link hematopoiesis with at least a subset of EC development.One gene, expressed in hemangioblast populations, is scl/tal1, hereafter referred to as scl. The gene encodes a basic helix-loophelix (bHLH) transcription factor, which was initially discovered at the sites of chromosomal translocations in leukemic T cells (for a review, see Begley and Green 7 ). Gene ablation studies in the mouse have revealed an essential role for scl in hematopoiesis. Scl Ϫ/Ϫ mouse embryos die at embryonic day 9.5 (E9.5) due to a complete absence of primitive blood cells. 8,9 Subsequent studies in chimeras revealed that scl Ϫ/Ϫ ES cells were also unable to contribute to definitive hematopoiesis. 10,11 Using conditional knockouts, scl req...