Dosage-sensitive requirement for mouse Dll4 in artery development

Abstract

Involvement of the Notch signaling pathway in vascular development has been demonstrated by both gain- and loss-of-function mutations in humans, mice, and zebrafish. In zebrafish, Notch signaling is required for arterial identity by suppressing the venous fate in developing artery cells. In mice, the Notch4 receptor and the Delta-like 4 (Dll4) ligand are specifically expressed in arterial endothelial cells, suggesting a similar role. Here we show that the Dll4 ligand alone is required in a dosage-sensitive manner for normal arterial patterning in development. This implicates Dll4 as the specific mammalian endothelial ligand for autocrine endothelial Notch signaling, and suggests that Dll4 may be a suitable target for intervention in arterial angiogenesis.

Figure 1.

Generation of Dll4 mutant mice. (A) Schematic representations of the wild-type Dll4 locus, targeting vector, and mutant allele. The probe for Southern blot analysis (ClaI-EcoRV genomic fragment) and the PCR primers (P1-P3) used for genotyping are indicated. (B) Southern blot analysis of control (lane 1) and targeted (lane 2) ES cells. Genomic DNA was digested with EcoRI and hybridized with the ClaI-EcoRV probe. (C) PCR genotyping of wildtype (+/+), heterozygous (+/-), and Dll4 homozygous null (-/-) mice with primers P1, P2, and P3. (D) RT-PCR analysis of Dll4 gene expression in E9.5 wild-type (+/+), heterozygous (+/-), and double mutant embryos (-/-).

Figure 2.

Dll4 haploinsufficiency. (A-H) Whole-mount X-gal staining. (K-P) PECAM immunostaining: whole-mount (K-M) and sections (N-P). (I,J) Unstained specimens. (A) Dll4 expression at twosomite stage in the cardiac crescent (cc) and dorsal aortae (da) primordia. (B,C) At E8.5, Dll4 expression is observed in all arterial vessels (da, dorsal aortae; va, vitelline arteries) and in the endothelial lining of the heart (h). (D-H) Variable penetrance of the hemizygous phenotype: littermate embryos (E10.5, D-F) and arterial vessels of the placenta (E9.5, G,H). (I,J) Morphology of E10.5 yolk sacs. Note the absence of large vitelline vessels and the poor circulation in the Dll4^+/- yolk sac compared with wild-type. (K-P) At E9.5 two types of Dll4^+/- embryos can be found: with reduced dorsal aorta but without defects in the anterior cardinal vein (L,O) and with more severe defects, with a very reduced dorsal aorta, enlarged heart, and the anterior cardinal vein reduced to a disorganized venous plexus (M,P).

Figure 3.

Defective arterial and venous remodeling in Dll4^-/- embryos. Whole-mount PECAM (A,B,I-L) and X-gal staining (C-H) of control and Dll4^-/- embryos. At E8.75, Dll4^-/- embryos display reduced dorsal aorta (da), anterior cardinal vein (acv), and sinus venosus (sv). (C,D) Presence of vitelline arteries (va) in the yolk sac of a E9.0 Dll4^+/- embryo contrasts with Dll4^-/- yolk sac vessels, which appear stalled at the primary capillary plexus stage (inset); in Dll4^-/- embryos the Dll4-lacZreporter expression is observed throughout the yolk sac vessels. (E-H) By E9.0, Dll4^-/- embryos exhibit growth retardation and arrested heart development with pericardial edema (p); the dorsal aorta appears further reduced, and there is an abnormal accumulation of lacZ-positive cells in the apical portion of the intersomitic vessels (isv) forming an enlarged dorsal vessel (dv). By E9.5, the Dll4^-/- embryos are severely retarded with extremely reduced dorsal aortae, a reduced and almost indistinguishable venous structure, sinus venosus, and anterior cardinal vein. (K,L; higher magnification of I,J, respectively) In the more dorsal region, instead of an intricately branched network between arterial and venous intersomitic vessels, the Dll4^-/- embryos display a single fused vessel that did not undergo angiogenic remodeling.

Figure 4.

Loss of arterial identity in Dll4^-/- endothelial cells. In situ hybridization (A-F) and immunostaining (G-J) of adjacent cryosections from E9.0 embryos. (A,B) Flk-1 mRNA is present in the endothelial cells of both dorsal aorta (da) and anterior cardinal vein (acv). (C,F) Endothelial cells in Dll4^-/- embryos fail to express arterial-specific EphrinB2 (Efnb2) and Connexin37 (Cx37) mRNAs. (G-J) The endothelial cells (shown by PECAM immunostaining in G,H) lining the dorsal aortae in Dll4^-/- embryos express the venous-specific EphB4 receptor; notice also the fusion between the dorsal aorta and the anterior cardinal vein on the right side of the picture (J).