Dll4 inhibits arterial Vegfc/Flt4 signalling in the developing zebrafish trunk. (A-C) Expression analysis at 24 hpf for dll4 (A), flt4 (B) and vegfc (C) indicates the arterial restricted expression of dll4, expression of flt4 in all venous and arterial cells, and expression of vegfc restricted to the dorsal aorta. (D) Schematic overview of the developing vasculature in the zebrafish trunk. DA, dorsal aorta; PCV, posterior cardinal vein; ISA, intersegmental artery; ISV, intersegmental vein; PL, parachordal lymphangioblast. (E) Working model of the interplay between Dll4 and Vegfc/Flt4 signalling. Previous studies have shown that vegfa (vegfaa and vegfab duplicates in zebrafish) is necessary for ISA development [a (Bahary et al., 2007; Nasevicius et al., 2000)], that vegfc influences (dashed line) ISA development [b (Covassin et al., 2006)] and that the receptors kdr and kdr-l mediate intracellular signalling to control ISA development [c (Bahary et al., 2007; Covassin et al., 2009; Covassin et al., 2006; Habeck et al., 2002; Lawson et al., 2003; Meng et al., 2008)]. We have previously shown that vegfd is unlikely to play a role in ISA development owing to its restricted expression [d (Hogan et al., 2009)]. Here we demonstrate that signalling through the Flt4 kinase domain (KD) does not contribute to ISA development as it is suppressed by Dll4 (dotted lines indicate that the mechanism of suppression is unknown). In the absence of Dll4, endogenous Vegfc drives a Flt4 (KD)-dependent ISA hyperbranching phenotype. In the vein (which does not express dll4), Vegfc/Flt4 signalling is indispensable for normal venous angiogenesis and lymphangiogenesis.
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