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Comparative Study
. 2004 Oct 15;18(20):2469-73.
doi: 10.1101/gad.1239204. Epub 2004 Oct 1.

Haploinsufficient lethality and formation of arteriovenous malformations in Notch pathway mutants

Luke T Krebs  1 , John R Shutter, Kenji Tanigaki, Tasuku Honjo, Kevin L Stark, Thomas Gridley
Affiliations
Comparative Study

Haploinsufficient lethality and formation of arteriovenous malformations in Notch pathway mutants

Luke T Krebs et al. Genes Dev. .

Abstract

The Notch signaling pathway is essential for embryonic vascular development in vertebrates. Here we show that mouse embryos heterozygous for a targeted mutation in the gene encoding the DLL4 ligand exhibit haploinsufficient lethality because of defects in vascular remodeling. We also describe vascular defects in embryos homozygous for a mutation in the Rbpsuh gene, which encodes the primary transcriptional mediator of Notch signaling. Conditional inactivation of Rpbsuh function demonstrates that Notch activation is essential in the endothelial cell lineage. Notch pathway mutant embryos exhibit defects in arterial specification of nascent blood vessels and develop arteriovenous malformations. These results demonstrate that vascular remodeling in the mouse embryo is sensitive to Dll4 gene dosage and that Notch activation in endothelial cells is essential for embryonic vascular remodeling.

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Figures

Figure 1.
Figure 1.
Vascular defects in Dll4+/- embryos. PECAM-1-stained yolk sacs (a,b) and embryos (c,d) at E9.5. (b) The Dll4+/- mutant yolk sac has failed to remodel the primary vascular plexus to form the large vitelline blood vessels. (d) The vascular network in the Dll4+/- embryo appears less intricate and more primitive than the capillary network of the control littermate. (e,f) Histological sections of PECAM-1-stained E9.5 embryos at the level of the otic vesicle (asterisk). (f) The dorsal aorta of this Dll4+/- embryo is reduced in diameter (arrowhead) on one side and is atretic (i.e., contains no lumen) on the other side (arrow).
Figure 2.
Figure 2.
Vascular defects and loss of arterial marker expression in Rbpsuh-/- embryos. (a-d) PECAM-1-stained E9.5 embryos and yolk sacs. (b) The Rbpsuh-/- embryo exhibits severe growth retardation and a very primitive vascular network. (d) The Rbpsuh-/- yolk sac exhibits no vascular remodeling. (e-h) Efnb2-tau-lacZ expression in E9.5 embryos and yolk sacs. In the wild-type embryo, Efnb2-taulacZ is expressed in the dorsal aorta (arrow) and intersomitic arteries of the embryo (e) and the vitelline arteries of the yolk sac (g). Expression is also observed in somites, nephrogenic mesoderm, branchial arches and hindbrain. Arterial Efnb2-tau-lacZ expression is lost in the Rbpsuh-/- embryo (f) and yolk sac (h). (i-l) Whole mount immunohistochemistry of E9.5 embryos. CD44 (i,j) and αSMA (k,l) expression in the dorsal aorta (arrow) of wild-type embryos is down-regulated in Rbpsuh-/- embryos.
Figure 3.
Figure 3.
Notch signal activation is required in endothelial cells. The Rbpsuh gene was conditionally inactivated in the endothelial cell lineage by crossing mice containing a floxed Rbpsuh allele to mice expressing Cre recombinase under control of the Tek promoter. (a,b) Whole mounts of E9.5 embryos. (b) The Tek-Cre Rbpsuhfl/null embryo exhibits growth retardation and pericardial effusion. (c,d) PECAM-1-stained E9.5 embryos and yolk sacs. (d) The Tek-Cre Rbpsuhfl/null mutant yolk sac has failed to remodel the primary vascular plexus to form large vitelline blood vessels. (e,f) Sections of PECAM-1-stained E9.5 embryos. The dorsal aortae (arrows) of the Tek-Cre Rbpsuhfl/null mutant are reduced in diameter.
Figure 4.
Figure 4.
Arteriovenous malformations in Notch pathway mutant embryos. India ink was injected into the proximal outflow tract of the heart in order to visualize blood flow and arteriovenous malformations. (a,d) In wild-type embryos, ink injected into the heart exited through the branchial arch arteries, entered the paired dorsal aortae, and traversed the entire length of the embryo. In Dll4+/- (b,e) and Tek-Cre Rbpsuhfl/null (c,f) embryos, injected ink exited the distal outflow tract, then entered the venous circulation via small-diameter anastamoses with the anterior cardinal vein. The Dll4+/- and Tek-Cre Rbpsuhfl/null embryos shown in b and c are different embryos than those shown in e and f. (a-c) Low-magnification views. (d-f) High-magnification views.
Figure 5.
Figure 5.
Histological analysis of arteriovenous malformations in Notch pathway mutants. (a-c) Sections of wild-type, Dll4+/- and Tek-Cre Rbpsuhfl/null PECAM-1-stained embryos at E9.5. (d,e) Higher-magnification views of the embryos in b and c. In each mutant, an arteriovenous malformation (arrowhead) consisting of a small-diameter anastomosis connecting the cardinal vein (CV) with the dorsal aorta (DA) can be observed. (f,g) Sections of wild-type and Rbpsuh-/- PECAM-1-stained embryos at E9.5. (g) The mutant embryo exhibits an arteriovenous malformation (arrowhead) caused by the fusion of the dorsal aorta with the common cardinal vein (CCV).
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References

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