The mechanisms regulating vertebrate heart and endoderm development have recently become the focus of intense study. Here we present evidence from both loss-and gain-of-function experiments that the zinc finger transcription factor Gata5 is an essential regulator of multiple aspects of heart and endoderm development. We demonstrate that zebrafish Gata5 is encoded by the faust locus. Analysis of faust mutants indicates that early in embryogenesis Gata5 is required for the production of normal numbers of developing myocardial precursors and the expression of normal levels of several myocardial genes including nkx2.5. Later, Gata5 is necessary for the elaboration of ventricular tissue. We further demonstrate that Gata5 is required for the migration of the cardiac primordia to the embryonic midline and for endodermal morphogenesis. Significantly, overexpression of gata5 induces the ectopic expression of several myocardial genes including nkx2.5 and can produce ectopic foci of beating myocardial tissue. Together, these results implicate zebrafish Gata5 in controlling the growth, morphogenesis, and differentiation of the heart and endoderm and indicate that Gata5 regulates the expression of the early myocardial gene nkx2.5.

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.

Fli-1 is an ETS-domain transcription factor whose locus is disrupted in Ewing's Sarcoma and F-MuLV induced erythroleukaemia. To gain a better understanding of its normal function, we have isolated the zebrafish homologue. Similarities with other vertebrates, in the amino acid sequence and DNA binding properties of Fli-1 from zebrafish, suggest that its function has been conserved during vertebrate evolution. The initial expression of zebrafish fli-1 in the posterior lateral mesoderm overlaps with that of gata2 in a potential haemangioblast population which likely contains precursors of blood and endothelium. Subsequently, fli-1 and gata2 expression patterns diverge, with separate fli-1 and gata2 expression domains arising in the developing vasculature and in sites of blood formation respectively. Elsewhere in the embryo, fli-1 is expressed in sites of vasculogenesis. The expression of fli-1 was investigated in a number of zebrafish mutants, which affect the circulatory system. In cloche, endothelium is absent and blood is drastically reduced. In contrast to the blood and endothelial markers that have been studied previously, fli-1 expression was initiated normally in cloche embryos, indicating that induction of fli-1 is one of the earliest indicators of haemangioblast formation. Furthermore, although fli-1 expression in the trunk was not maintained, the normal expression pattern in the anterior half of the embryo was retained. These anterior cells did not, however, condense to form blood vessels. These data indicate that cloche has previously unsuspected roles at multiple stages in the formation of the vasculature. Analysis of fli-1 expression in midline patterning mutants floating head and squint, confirms a requirement for the notochord in the formation of the dorsal-aorta. The formation of endothelium in one-eyed pinhead, cyclops and squint embryos indicates a novel role for the endoderm in the formation of the axial vein. The phenotype of sonic-you mutants implies a likely role for Sonic Hedgehog in mediating these processes.

Blood and endothelium arise in close association during development, possibly from a common precursor, the hemangioblast [1-4]. Genes essential for blood and endothelial development contain functional ETS binding sites, and binding and expression data implicate the transcription factor, friend leukaemia integration 1 (Fli1) [5-10]. However, loss-of-function phenotypes in mice, although suffering both blood and endothelial defects, have thus far precluded the conclusion that Fli1 is essential for these two lineages [11, 12]. By using Xenopus and zebrafish embryos, we show that loss of Fli1 function results in a substantial reduction or absence of hemangioblasts, revealing an absolute requirement. TUNEL assays show that the cells are eventually lost by apoptosis, but only after the regulatory circuit has been disrupted by loss of Fli1. In addition, a constitutively active form of Fli1 is sufficient to induce expression of key hemangioblast genes, such as Scl/Tal1, Lmo2, Gata2, Etsrp, and Flk1. Epistasis assays show that Fli1 expression is induced by Bmp signaling or Cloche, depending on the hemangioblast population, and in both cases Fli1 acts upstream of Gata2, Scl, Lmo2, and Etsrp. Taken together, these results place Fli1 at the top of the transcriptional regulatory hierarchy for hemangioblast specification in vertebrate embryos.