Haploinsufficiency of delta-like 4 ligand results in embryonic lethality due to major defects in arterial and vascular development

Abstract

Vascular development depends on the highly coordinated actions of a variety of angiogenic regulators, most of which apparently act downstream of vascular endothelial growth factor (VEGF). One potential such regulator is delta-like 4 ligand (Dll4), a recently identified partner for the Notch receptors. We generated mice in which the Dll4 gene was replaced with a reporter gene, and found that Dll4 expression is initially restricted to large arteries in the embryo, whereas in adult mice and tumor models, Dll4 is specifically expressed in smaller arteries and microvessels, with a striking break in expression just as capillaries merge into venules. Consistent with these arterial-specific expression patterns, heterozygous deletion of Dll4 resulted in prominent albeit variable defects in arterial development (reminiscent of those in Notch knockouts), including abnormal stenosis and atresia of the aorta, defective arterial branching from the aorta, and even arterial regression, with occasional extension of the defects to the venous circulation; also noted was gross enlargement of the pericardial sac and failure to remodel the yolk sac vasculature. These striking phenotypes resulting from heterozygous deletion of Dll4 indicate that vascular development may be as sensitive to subtle changes in Dll4 dosage as it is to subtle changes in VEGF dosage, because VEGF accounts for the only other example of haploid insufficiency, resulting in obvious vascular abnormalities. In summary, Dll4 appears to be a major trigger of Notch receptor activities previously implicated in arterial and vascular development, and it may represent a new opportunity for pro-and anti-angiogenic therapies.

Functional Arterial and Venous Fate Is Determined by Graded VEGF Signaling and Notch Status During Embryonic Stem Cell Differentiation

“…This is in accordance with previous in vivo data from Notch mutants, where endothelial cells do form but fail to remodel properly. 7,8 As was expected, inhibition of Notch signaling resulted in lower transcript levels of direct downstream genes Hey1 and Hey2 ( Figure 4B). Expression of the Notch ligand and arterial marker Dll4 decreased as did EphrinB2 levels, whereas expression of its receptor EphB4 remained unaltered at mRNA level.…”

“…[7][8][9][10][11] To evaluate this, FACS sorted Flk1 ϩ cells were differentiated with or without ␥-secretase inhibitor (GSI) to block Notch signaling. Compared with control cultures, GSI treatment did neither alter CD31 and ␣SMA expression levels ( Figure 4A) nor the numbers of CD31 ϩ cells analyzed by FACS.…”

“…Loss-and gain-of-function experiments have provided evidence that the receptors Notch1 and Notch4, and their ligand Dll4, regulate vascular development and remodeling in addition to being specifically expressed in the arteries. [7][8][9] Hey1 and Hey2 are two direct downstream genes of Notch signaling and both have been reported to promote arterial fate, possibly by upregulating EphrinB2 and inhibiting EphB4 expression. 10,11 In addition, VEGF upregulates both Dll4 and Notch1 in vitro and promotes arterial specification in vivo.…”

Notch expression patterns in the retina: An eye on receptor–ligand distribution during angiogenesis

“…Of the Notch receptors, the loss of Notch1 proved to be the most deleterious to vascular development (Conlon et al, 1995;Krebs et al, 2000;Swiatek et al, 1994). Furthermore, several studies have demonstrated expression of Notch receptors and ligands in blood vessels and mural cells (Alva and Iruela-Arispe, 2004;Benedito and Duarte, 2005;Claxton and Fruttiger, 2004;Duarte et al, 2004;Gale et al, 2004;Shutter et al, 2000;Villa et al, 2001). These studies have also generally agreed that in addition to capillaries, Notch ligands and receptors are mostly confined to arteries, a finding that was subsequently supported by the requirement of Notch in establishing arterial identity through expression of EphrinB2 (Lawson et al, 2001).…”

“…In terms of the ligands, only the loss of either Jag1 or Dll4 results in vascular defects, indicating that the other three Notch DSL ligands may not be as involved in vessel development. Notably, analysis of the phenotypes exhibited by Jag1 (Xue et al, 1999) and Dll4 (Duarte et al, 2004;Gale et al, 2004;Krebs et al, 2004) knockout mice, suggested that these two ligands are not functionally redundant. Indeed, the onset of lethality would indicate that each one of these molecules might provide a different array of signals through activation of Notch receptors.…”