DLL1-mediated Notch activation regulates endothelial identity in mouse fetal arteries

Abstract

Notch signaling has been shown to regulate various aspects of vascular development. However, a specific role of the ligand Delta-like 1 (DLL1) has not been shown thus far. Here, we demonstrate that during fetal development, DLL1 is an essential Notch ligand in the vascular endothelium of large arteries to activate Notch1 and maintain arterial identity. DLL1 was detected in fetal arterial endothelial cells beginning at embryonic day 13.5. While DLL4-mediated activation has been shown to suppress vascular endothelial growth factor (VEGF) pathway components in growing capillary beds, DLL1-Notch signaling was required for VEGF receptor expression in fetal arteries. In the absence of DLL1 function, VEGF receptor 2 (VEGFR2) and its coreceptor, neuropilin-1 (NRP1), were down-regulated in mutant arteries, which was followed by up-regulation of chicken ovalbumin upstream promoter-transcription factor II (COUP- TFII IntroductionA functional cardiovascular system with precisely formed and connected arteries and veins is established early during mammalian embryogenesis and is essential for subsequent development and survival. First, endothelial precursors, the angioblasts, differentiate into endothelial cells and form a primitive vascular network in a process called vasculogenesis. These first vessels are simple tubes composed entirely of endothelial cells. Subsequently, the simple vascular network is remodeled into a hierarchical network of veins, arteries and capillaries in a process called angiogenesis. 1 Angiogenesis is a precisely orchestrated multistep process that involves localized cell proliferation and migration, the extension of endothelial sprouts and their conversion into patent tubules, as well as the selective pruning of some vessel connections. During angiogenesis, the division into morphologically, functionally, and molecularly unique arteries and veins is established and supporting cell types, such as smooth muscle cells and pericytes, are recruited to form mature and fully functional vessels. 2 Vascular endothelial growth factor (VEGF) signaling is essential for establishment of the embryonic vasculature as is indicated by absence or severe malformations of vessels in embryos lacking VEGF or its receptors (reviewed in Rossant and Howard 3 ). Early during angiogenesis, ie, before development of a functional circulation, the distinction between arteries and veins is established in a process termed arteriovenous differentiation. Accordingly, certain markers and signaling molecules, such as the transmembrane protein ephrinB2 (EFNB2), are specifically expressed in the endothelium of developing arteries. Conversely, the expression of the receptor tyrosine kinase EPHB4, an interaction partner of ephrinB2, and other gene products are confined to the venous endothelium. Loss of either EphrinB2 or EPHB4 leads to very similar defects in vascular remodeling, suggesting that they mediate bidirectional signaling essential for angiogenesis and the establishment of boundaries between venous and arterial domains. [4][5][6...

Deletion of Rbpj from postnatal endothelium leads to abnormal arteriovenous shunting in mice

“…S1) using an inducible Cre-loxP system. We used the Cdh5(PAC)-CreER T2 transgene for CreER T2 expression in ECs (Sorensen et al, 2009;Wang et al, 2010). We bred Cdh5(PAC)-CreER T2 ; Rbpj flox/flox mice, administered Tamoxifen (TAM) at postnatal day (P) 1 and P2, and harvested tissue at P14 (hereafter referred to as immature Rbpj iΔEC ).…”

“…Mouse lines: Cdh5(PAC)-CreER T2 (Sorensen et al, 2009), Rbpj flox (Tanigaki et al, 2002), Rosa26 mT/mG (Muzumdar et al, 2007), Coup-TFII flox-nlacZ (Takamoto et al, 2005), Efnb2 tau-lacZ (Wang et al, 1998) and Ephb4 tau-lacZ (Gerety et al, 1999) were kindly provided by Ralf Adams (Max Planck Institute for Molecular Biomedicine, Münster, Germany), Tasuku Honjo (Kyoto University, Japan), Liqun Luo (Stanford University School of Medicine, Stanford, CA, USA), Sophia Tsai (Baylor College of Medicine, Houston, TX, USA), and, the latter two, by David Anderson (California Institute of Technology, Pasadena, CA, USA), respectively. 100 μg TAM (Sigma) in 50 μl peanut oil (Planters) was injected into stomach at P1 and P2.…”

Roles of Cyclic Adenosine Monophosphate Signaling in Endothelial Cell Differentiation and Arterial-Venous Specification During Vascular Development

“…In ECs, Notch (Notch1, 4) activation can be induced by various Notch ligands, including Dll1, Dll4, and Jagged2, expressed in arterial ECs, and Jagged1 expressed in ECs and mural cells. 35, 36 All of this Notch signaling is considered to be mediated by the Notch intracellular domain (NICD) and RBP-J transcription factor (also called CSL, CBF-1 in mammals, Suppressor of Hairless [Su(H)] in Drosophila and LAG-1 in Caenorhabditis elegans). Genetic animal studies of the Notch signal related-genes have shown that Notch1 and 4, Dll4, RBP-J, and Hey1/Hey2 are essential for arterial formation in the developing vasculature.…”

Pathogenesis of Arteriovenous Malformations in the Absence of Endoglin

“…17 With the aim of developing a more reproducible model of AVM formation, and bypassing embryonic lethality of the endoglin null mouse, we have taken a conditional knockout approach combining our recently generated endoglin-floxed mouse model 18 with an endothelial specific Cdh5(PAC)-Cre ERT2 transgenic line. 19 As Cre ERT2 is inactive until exposed to tamoxifen, this combination of alleles allows endoglin depletion in ECs at different stages of development and adult life, permitting a high degree of manipulability with which to investigate the role of endoglin in vivo. 2fl/2fl and included both tamoxifen-treated and tamoxifen-untreated groups.…”