Notch1 activation in mice causes arteriovenous malformations phenocopied by ephrinB2 and EphB4 mutants

Abstract

Notch signaling is essential for embryonic vascular development in mammals and other vertebrates. Here we show that mouse embryos with conditional activation of the Notch1 gene in endothelial cells (Notch1 gain of function embryos) exhibit defects in vascular remodeling increased diameter of the dorsal aortae, and form arteriovenous malformations. Conversely, embryos with either constitutive or endothelial cell-specific Notch1 gene deletion also have vascular defects, but exhibit decreased diameter of the dorsal aortae and form arteriovenous malformations distinctly different from the Notch1 gain of function mutants. Surprisingly, embryos homozygous for mutations of the ephrinB/EphB pathway genes Efnb2 and Ephb4 exhibit vascular defects and arteriovenous malformations that phenocopy the Notch1 gain of function mutants. These results suggest that formation of arteriovenous malformations in Notch1 gain of function mutants and ephrinB/EphB pathway loss of function mutant embryos occurs by different mechanisms.

Figure 1. Vascular defects in Notch1^-/-, Tek-Cre; Notch1^flox/- and Notch1^GOF mutant embryos

(a-d) PECAM-1 stained embryos. In the Notch pathway loss of function mutant embryos Notch1^-/- (b), Tek-Cre; Notch1^flox/- (c), and the Notch1^GOF (Tek-Cre; Notch1-IC) embryo (d), the capillary network appears less intricate and more primitive than that of the wild type control (a). (e-h) PECAM-1 stained yolk sacs. Both the Notch pathway loss of function mutant embryos (f, g) and the Notch1^GOF embryo (h) have failed to remodel the primary vascular plexus to form the large vitelline blood vessels observed in the wild type embryo (e). (i-l) Histological sections of PECAM-1-stained embryos at the level of the otic vesicle. In the wild type embryo (i), both dorsal aortae (green arrows) have open lumens and normal morphology. In the two Notch pathway loss of function mutant embryos Notch1^-/- (j) and Tek-Cre; Notch1^flox/- (k), the dorsal aortae are either reduced in diameter or atretic (i.e., contains no lumen). In contrast, in the Notch1^GOF embryo (l) the lumens of the dorsal aortae are increased in diameter. All embryos shown are at E9.5.

Figure 2. Arteriovenous malformations in Notch1^GOF mutant embryos are distinct from those of Notch pathway loss of function mutant embryos

(a-d) India ink injected embryos. India ink was injected into the proximal outflow tract of the heart in order to visualize blood flow and arteriovenous malformations. (a) In the wild type embryo, ink injected into the heart exited through the branchial arch arteries, entered the paired dorsal aortae and traversed caudally the entire length of the embryo. (b, c) In the Notch pathway loss of function mutant embryos Notch1^-/- (b) and Tek-Cre; Notch1^flox/- (c), injected ink exited the distal outflow tract, then entered the venous circulation in the anterior of the embryo via small diameter anastamoses with the anterior cardinal vein. Caudally, injected ink was shunted back into the heart via fusion of the descending dorsal aorta with the common cardinal vein. (d) In the Notch1^GOF (Tek-Cre; Notch1-IC) embryo, injected ink exited via an enlarged branchial arch artery, entered the descending dorsal aorta, and was shunted back into the heart via fusion of the dorsal aorta with the common cardinal vein. (e-f) Sections of PECAM-1 stained embryos just caudal to the heart. (e) In the wild type embryo, the dorsal aorta and common cardinal vein (black arrowhead) are distinct. (f-h) In both Notch loss of function and gain of function mutant embryos, the dorsal aorta has fused with the common cardinal vein (green arrow), shunting blood back into the heart through the sinus venosus. All embryos shown are at E9.5.

Figure 3. Vascular defects and arteriovenous malformations in Efnb2^-/- and Ephb4^-/- mutant embryos phenocopy those of Notch1^GOF embryos

Efnb2^-/- (a, c, e, g) and Ephb4^-/- (b, d, f, h) mutant embryos at E9.5. (a, b) PECAM-1 stained embryos. (c, d) India ink injected embryos revealing arteriovenous malformations. (e, f) PECAM-1 stained yolk sacs exhibiting the absence of vascular remodeling. (g, h) Histological sections of PECAM-1-stained embryos. Vascular defects in both the Efnb2^-/- and Ephb4^-/- mutant embryos phenocopy those of Notch1^GOF embryos.