Overexpression of delta-like 4 induces arterialization and attenuates vessel formation in developing mouse embryos

The importance of Notch signaling pathway in the regulation of vascular development and angiogenesis is suggested by the expression of Notch receptors and ligands in vascular endothelial cells (ECs) and the observed vascular phenotypes in mutants of Notch receptors or ligands, especially DII4. DLL4 is specifically expressed in arterial ECs during development, and haploinsufficiency is embryonically lethal in mice. To address the role of DII4 in vascular development, we produced mDII4 conditionally overexpressed transgenic mice that were crossed with constitutive recombinase cre lines. Double transgenic embryos displayed grossly enlarged dorsal aortae (DA) and died before embryonic day 10.5 (E10.5), showing a variable degree of premature arteriovenous fusion. Veins displayed ectopic expression of arterial markers. Other defects included reduced vascular sprouting, EC proliferation, and migration. mDII4 overexpression also inhibited VEGF signaling and increased fibronectin accumulation around the vessels. In vitro and in vivo studies of DLL4-FL (DII4-full-length) in ECs recapitulate many of the mDII4 transgenics findings, including decreased tube formation, reduced vascular branching, fewer vessels, increased pericyte recruitment, and increased fibronectin expression. These results establish the role of DII4 in arterial identity determination, and regulation of angiogenesis subject to dose and location.