Flt1 acts as a negative regulator of tip cell formation and branching morphogenesis in the zebrafish embryo

INTRODUCTION Sprouting angiogenesis plays a crucial role in vascular network expansion during normal development and pathophysiological conditions (Carmeliet et al., 2009). Angiogenic sprouting involves the specification of endothelial cells into tip and stalk cells in the growing sprout (Phng and Gerhardt, 2009), with the tip cell being localized at the leading edge of the sprout. Tip cells extend numerous filopodia and respond to guidance cues including vascular endothelial growth factor (VegfA), directing the movement of the sprout. The endothelial cells that follow the tip cells are called stalk cells. Stalk cells have fewer filopodia, they proliferate in response to VegfA, and they contribute to vessel lumen formation. Within the sprout, endothelial cells dynamically compete with each other for the tip cell position (Jakobsson et al., 2010). The full repertoire of genes controlling endothelial tip cell differentiation is unknown, but VEGF receptors including Flk1 (Kdr, Vegfr2) and Flt4 (Vegfr3) and Notch–delta-like 4 (Dll4) signaling pathways play key roles (Benedito et al., 2009; Gerhardt et al., 2003; Hellstrom et al., 2007; Lobov et al., 2007; Siekmann and Lawson, 2007; Suchting et al., 2007; Tammela et al., 2008). Here, we present genetic evidence that flt1 (vegfr1) acts, in a Notch-dependent manner, as a negative regulator of tip cell formation and sprout guidance in the trunk vasculature of zebrafish embryos. In mammals, thus far three VEGF receptors have been identified, namely Flt1, Flk1 and Flt4. In all other vertebrate classes, four receptors exist (Bussmann et al., 2008). These receptors bind VEGF ligands and regulate angiogenesis and lymphangiogenesis (Alitalo et al., 2005; Ferrara et al., 2003). Zebrafish VEGF receptor proteins are encoded by the flt1, kdr (previously kdrb), kdr-like (kdrl, previously kdra) and the flt4 (Bussmann et al., 2008) orthologs. Whereas the roles of flk1, kdr and kdrl, and of flt4 in mediating vascular development in zebrafish are well described (Covassin et al., 2009; Covassin et al., 2006; Habeck et al., 2002; Hogan et al., 2009b; Lawson et al., 2002; Liao et al., 1997; Siekmann and Lawson, 2007), surprisingly little is known about flt1. Thus far, flt1 has been implicated in the regulation of embryonic heart function and cardiac morphogenesis (Bussmann et al., 2007; Rottbauer et al., 2005), but its specific role in endothelial tip cell formation in zebrafish is unknown. In mammals, Flt1 encodes one membrane-bound form, which has an intracellular tyrosine kinase signaling domain, and soluble forms, which only contain the extracellular VEGF-binding domain (Sela et al., 2008). Genetic studies in mice have shown that Flt1 plays a pivotal role in both developmental and pathological forms of angiogenesis (Ferrara et al., 2003; Fong et al., 1995). Flt1-null mice die at ~E8.5 and show disorganized blood vessels and overgrowth of endothelial cells (Fong et al., 1995). However, Flt1 tyrosine kinase-deficient homozygous mice (Flt1TK–/–) survive and appear to develop normal vessels, suggesting that the tyrosine Development 138, 2111-2120 (2011) doi:10.1242/dev.063933 © 2011. Published by The Company of Biologists Ltd