Essential role of endothelial Notch1 in angiogenesis

Abstract

Background: Notch signaling influences binary cell fate decisions in a variety of tissues. The Notch1 receptor is widely expressed during embryogenesis and is essential for embryonic development. Loss of global Notch1 function results in early embryonic lethality, but the cell type responsible for this defect is not known. Here, we identify the endothelium as the primary target tissue affected by Notch1 signaling.

Methods and results: We generated an endothelium-specific deletion of Notch1 using Tie2Cre and conditional Notch1(flox/flox) mice. Mutant embryos lacking endothelial Notch1 died at approximately embryonic day 10.5 with profound vascular defects in placenta, yolk sac, and embryo proper, whereas heterozygous deletion had no effect. In yolk sacs of mutant embryos, endothelial cells formed a primary vascular plexus indicative of intact vasculogenesis but failed to induce the secondary vascular remodeling required to form a mature network of well-organized large and small blood vessels, which demonstrates a defect in angiogenesis. These vascular defects were also evident in the placenta, where blood vessels failed to invade the placental labyrinth, and in the embryo proper, where defective blood vessel maturation led to pericardial and intersomitic hemorrhage. Enhanced activation of caspase-3 was detected in endothelial and neural cells of mutant mice, which resulted in enhanced apoptotic degeneration of somites and the neural tube.

Conclusions: These findings recapitulate the vascular phenotype of global Notch1-/- mutants and indicate an essential cell-autonomous role of Notch1 signaling in the endothelium during vascular development. These results may have important clinical implications with regard to Notch1 signaling in adult angiogenesis.

Figure 1

Growth retardation and vascular defects in NEKO animals at day E9.5. A and B, Whole-mount preparation of control Notch1 (N1^flox/+/Tie2Cre^+/-) mice (A) and mice lacking endothelial Notch1 (NEKO, N1^flox/flox/Tie2Cre^+/-; B). C and D, Whole-mount PECAM-1-stained control (C) or NEKO (D) embryos. E through G, Whole-mount LacZ-stained control N1^flox/+/Tie2Cre^+/-/LacZ^-/- (E) and N1^flox/+/Tie2Cre^+/-/LacZ^+/- (F) or N1^flox/flox/Tie2Cre^+/-/LacZ^+/- (G) mutant embryos. Note failure to complete embryonic turning in NEKO embryos. Original magnification ×40.

Figure 2

Angiogenic remodeling in yolk sac and placenta depends on endothelial Notch1. A through D, Yolk sac whole-mount preparation (A) and histological sections of control animals (C) and of NEKO mutants (B, D). Arrows denote major vitelline vessels; arrowheads represent smaller capillaries. There is proper remodeling of vitelline vessels in control animals, which is absent in NEKO mutants. E and F, PECAM-1-stained vasculature of yolk sacs from controls (E) and NEKO embryos (F). G through J, Hematoxylin-and-eosin-stained placental sections of control animals (G, original magnification ×40; I, original magnification ×400) and of NEKO mice (H, original magnification ×40; J, original magnification ×400). Arrows denote invading fetal vessels filled with nucleated erythrocytes. Arrowheads show maternal blood sinus. sp indicates spongiotrophoblast; la, labyrinth; and cp, chorionic plate.

Figure 3

Impaired angiogenesis and hemorrhage in NEKO mice. A through D, Whole-mount PECAM-1 staining of head (A, B) and tails (C, D) in control and NEKO mutants. There is hypoplastic development of cardinal vein (arrow in B) and decreased sprouting of vessels (arrow in D). White arrowheads in A and B denote cardinal vein. Black arrowheads in C and D denote intersomitic vessels. E through H, Whole-mount view and hematoxylin-and-eosin-stained cross sections of heart and pericardium (E, F) and tails (G, H). Arrows denote hemorrhage. h indicates heart.

Figure 4

Neuronal and somitic defects in NEKO mice. A and B, Hematoxylin-and-eosin-stained cross sections through day E9.5 embryos of control (A) and NEKO (B) mice; original magnification ×40. Inset shows hypoplastic cardinal vein at higher magnification. C, Higher magnification (×400) of NEKO mice showing pyknotic nuclei in neural tube. D and E, Hematoxylin-and-eosinstained sagittal sections through embryo trunk at day E9.5 of control (D) and NEKO (E) embryos. Arrows denote intersomitic vessels. F and G, Activated caspase-3 staining of control (F) and NEKO (G) mice. Insets represent aortas at higher magnification. ao indicates aorta; at, atrium; cv, cardinal vein; nt, neural tube; ph, pharynx; and v, ventricle.