SummaryLimited clinical benefits derived from anti-VEGF therapy have driven the identification of new targets involved in tumor angiogenesis. Here, we report an integrative meta-analysis to define the transcriptional program underlying angiogenesis in human cancer. This approach identified ELTD1, an orphan G-protein-coupled receptor whose expression is induced by VEGF/bFGF and repressed by DLL4 signaling. Extensive analysis of multiple cancer types demonstrates significant upregulation of ELTD1 in tumor-associated endothelial cells, with a higher expression correlating with favorable prognosis. Importantly, ELTD1 silencing impairs endothelial sprouting and vessel formation in vitro and in vivo, drastically reducing tumor growth and greatly improving survival. Collectively, these results provide insight into the regulation of tumor angiogenesis and highlight ELTD1 as key player in blood vessel formation.

GATA‐6 is expressed in presumptive cardiac mesoderm before gastrulation, but its role in heart development has been unclear. Here we show that Xenopus and zebrafish embryos, injected with antisense morpholino oligonucleotides designed specifically to knock‐down translation of GATA‐6 protein, are severely compromised for heart development. Injected embryos express greatly reduced levels of contractile machinery genes and, at the same stage, of regulatory genes such as bone morphogenetic protein‐4 (BMP‐4) and the Nkx2 family. In contrast, initial BMP and Nkx2 expression is normal, suggesting a maintenance role for GATA‐6. Endoderm is critical for heart formation in several vertebrates including Xenopus, and separate perturbation of GATA‐6 expression in the deep anterior endoderm and in the overlying heart mesoderm shows that GATA‐6 is required in both for cardiogenesis. The GATA‐6 requirement in cardiac mesoderm was confirmed in zebrafish, an organism in which endoderm is thought not to be necessary for heart formation. We therefore conclude that proper maturation of cardiac mesoderm requires GATA‐6, which functions to maintain BMP‐4 and Nkx2 expression.

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The transcription factors, GATA4, 5 and 6, recognize the same DNA sequence and are all expressed in the developing myocardium. However, knockout studies in the mouse have indicated that none of them are absolutely required for the specification of the myocardium. Here we present evidence for redundancy in this family for the first time. Using morpholinos in both Xenopus and zebrafish embryos, we show that GATA4 knockdown, for example, only affects cardiac marker expression in the absence of either GATA5 or GATA6. A similar situation pertains for GATA5 in Xenopus whereas, in zebrafish, GATA5 (faust) plays a major role in driving the myocardial programme. This requirement for GATA5 in zebrafish is for induction of the myocardium, in contrast to the GATA6 requirement in both species, which is for differentiation. This early role for GATA5 in zebrafish correlates with its earlier expression and with an earlier requirement for BMP signalling, suggesting that a mutual maintenance loop for GATA, BMP and Nkx expression is the evolutionarily conserved entity.

SummaryHematopoietic stem cells (HSCs) are self-renewing multipotent stem cells that generate mature blood lineages throughout life. They, together with hematopoietic progenitor cells (collectively known as HSPCs), emerge from hemogenic endothelium in the floor of the embryonic dorsal aorta by an endothelial-to-hematopoietic transition (EHT). Here we demonstrate that transforming growth factor β (TGFβ) is required for HSPC specification and that it regulates the expression of the Notch ligand Jagged1a in endothelial cells prior to EHT, in a striking parallel with the epithelial-to-mesenchymal transition (EMT). The requirement for TGFβ is two fold and sequential: autocrine via Tgfβ1a and Tgfβ1b produced in the endothelial cells themselves, followed by a paracrine input of Tgfβ3 from the notochord, suggesting that the former programs the hemogenic endothelium and the latter drives EHT. Our findings have important implications for the generation of HSPCs from pluripotent cells in vitro.

Over the past few years it has become clear that over half of the mammalian heart derives from outside the heart field as originally defined. Such a second heart field, however, has not been described in zebrafish, which could explain its smaller, two-chambered heart. Instead, zebrafish have a population of haemangioblasts, which is absent in mammalian embryos, raising the possibility that these cells represent the evolutionary ancestor of the second heart field. Here, we show for the first time that the genetic programmes of these anterior haemangioblasts and the adjacent heart field are co-regulated, by transcription factors previously associated with heart but not blood or endothelial development. We demonstrate that gata4, gata5 and gata6 are essential for anterior haemangioblast specification, and for subsequent myelopoiesis, acting as early as cloche and upstream of scl. The requirement for gata4, gata5 and gata6 in myeloid, endothelial and cardiac specification is in the mesoderm, but these factors also control, from within the endoderm and the yolk syncytial layer, the migration of the cardiac precursors as they differentiate. This genetic link between the blood/endothelial and cardiac programmes supports the notion that this haemangioblast population in zebrafish is an evolutionary antecedent of the second heart field, and has implications for the differentiation of haemangioblasts and cardiomyocytes from pluripotent cells, and for the origins of stem cells in the adult heart. KEY WORDS: Myelopoiesis, Cardiogenesis, GATA factors, Second heart field, Haemangioblasts, Transcriptional regulation, Evolution, Adult stem cells, Zebrafish Development 136, 1465Development 136, -1474Development 136, (2009 DEVELOPMENT 1466 the first time that they are crucial for anterior haemangioblast formation and subsequent myelopoiesis. This requirement is within the mesoderm, although we also show that gata5 and gata6 are required in the yolk syncytial layer (YSL) and the endoderm for the correct migration of cardiac precursors. The ablation of both cardiac and haemangioblast programmes within the ALM suggests that these GATA factors lie at the top of a genetic cascade that is initially common to both of these two lineages. This is confirmed by the continued expression of gata4, gata5 and gata6 in scl morphants and cloche mutants, suggesting that these GATA factors lie upstream of, or parallel to, these well-described blood and endothelial regulatory factors. These data genetically link the anterior haemangioblast and cardiac fields, and are consistent with the former being the evolutionary ancestor of the latter. MATERIALS AND METHODS In situ hybridisation of zebrafish embryosWild-type and cloche [Clo m39 ] (Stainier, 2001) zebrafish were bred, maintained, and embryos raised and staged using standard conditions (Westerfield, 1993). In situ hybridisations on zebrafish embryos were carried out as previously described (Jowett, 2001). All RNA probes used were labelled with digoxigenin (DIG) and detection of the antibody-...

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Haematopoietic stem cells are generated from the haemogenic endothelium (HE) located in the floor of the dorsal aorta (DA). Despite being integral to arteries, it is controversial whether HE and arterial endothelium share a common lineage. Here, we present a transgenic zebrafish runx1 reporter line to isolate HE and aortic roof endothelium (ARE)s, excluding non-aortic endothelium. Transcriptomic analysis of these populations identifies Runx1-regulated genes and shows that HE initially expresses arterial markers at similar levels to ARE. Furthermore, runx1 expression depends on prior arterial programming by the Notch ligand dll4 . Runx1 −/− mutants fail to downregulate arterial genes in the HE, which remains integrated within the DA, suggesting that Runx1 represses the pre-existing arterial programme in HE to allow progression towards the haematopoietic fate. These findings strongly suggest that, in zebrafish, aortic endothelium is a precursor to HE, with potential implications for pluripotent stem cell differentiation protocols for the generation of transplantable HSCs.