Formation of a fully functional artery proceeds through a multistep process. Here we show that Notch3 is required to generate functional arteries in mice by regulating arterial differentiation and maturation of vascular smooth muscle cells (vSMC). In adult Notch3−/− mice distal arteries exhibit structural defects and arterial myogenic responses are defective. The postnatal maturation stage of vSMC is deficient in Notch3 −/− mice. We further show that Notch3 is required for arterial specification of vSMC but not of endothelial cells. Our data reveal Notch3 to be the first cell-autonomous regulator of arterial differentiation and maturation of vSMC.Supplemental material is available at http://www.genesdev.org.

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.Supplemental material is available at http://www.genesdev.org.

Generation of left-right asymmetry is an integral partGeneration of left-right asymmetry during development is an integral part of the establishment of the vertebrate body plan (Capdevila et al. 2000;Mercola and Levin 2001;Wright 2001;Yost 2001;Hamada et al. 2002). Specification of the left-right axis requires multiple steps: (1) generation of an initial asymmetric signal in or near the embryonic node, (2) transfer of asymmetric signals from the node to the lateral plate mesoderm (LPM), (3) induction of an evolutionarily conserved cascade of gene expression in the left LPM, and (4) transformation of these left-right asymmetric signals into morphological asymmetries of the visceral organs. In mice, generation of the initial asymmetric signal requires directional fluid flow on the ventral surface of the node (Nonaka et al. 2002). This fluid flow is generated by motile monocilia on cells of the node, and the presence of nodal cilia is conserved in other vertebrates (Essner et al. 2002). However, the mechanism by which directional fluid flow at the node specifies orientation of the left-right axis is controversial (Stern and Wolpert 2002;Tabin and Vogan 2003). In addition, the mechanism for transfer of the initial asymmetric signal from the node to the LPM is unknown.The Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism. Mutations in Notch pathway components disrupt embryonic development in diverse multicellular organisms and cause in- Here we demonstrate that the Notch signaling pathway plays a primary role in the establishment of leftright asymmetry in mice by directly regulating expression of the Nodal gene. Embryos mutant for the Notch ligand Dll1 or doubly mutant for the Notch1 and Notch2 receptors exhibit multiple defects in left-right asymmetry. Notably, Dll1 −/− embryos do not express Nodal in the region around the node. Analysis of the enhancer regulating node-specific Nodal expression (termed the NDE) revealed the presence of binding sites for the RBP-J protein. Mutation of these sites destroyed the ability of the NDE to direct node-specific gene expression in transgenic mice. These results demonstrate that Dll1-mediated Notch signaling is essential for generation of leftright asymmetry, and indicate that perinodal expression of the Nodal gene is an essential component of left-right asymmetry determination in mice. Results and Discussion Laterality defects in Dll1 mutant and Notch1/Notch2 double-mutant mouse embryosDuring studies on the role of the Dll1 gene during somitogenesis (Zhang et al. 2002), we observed that some Dll1 −/− embryos (Hrabé de Angelis et al. 1997) exhibited reversed heart looping. We examined this phenotype more closely by performing scanning electron micros-

The Notch signaling pathway is an evolutionarily conserved signaling mechanism and mutations in its components disrupt cell fate specification and embryonic development in many organisms. To analyze the in vivo role of the Notch3 gene in mice, we created a deletion allele by gene targeting. Embryos homozygous for this mutation developed normally and homozygous mutant adults were viable and fertile. We also examined whether we could detect genetic interactions during early embryogenesis between the Notch3 mutation and a targeted mutation of the Notch1 gene. Double homozygous mutant embryos exhibited defects normally observed in Notch1-deficient embryos, but we detected no obvious synergistic effects in the double mutants. These data demonstrate that the Notch3 gene is not essential for embryonic development or fertility in mice, and does not have a redundant function with the Notch1 gene during early embryogenesis.

Background Notch1 regulates binary cell fate determination and is critical for angiogenesis and cardiovascular development. However, the pathophysiological role of Notch1 in the postnatal period is not known. We hypothesize that Notch1 signaling in vascular smooth muscle cells (SMC) may contribute to neointimal formation following vascular injury. Methods and Results We performed carotid artery ligation in wild-type (WT), control (smCre-Tg), general Notch1 heterozygous deficient (N1+/-), SMC-specific Notch1 heterozygous deficient (smN1+/-), and general Notch3 homozygous deficient (N3-/-) mice. Compared to WT or control mice, N1+/- and smN1+/- mice showed a 70% decrease in neointimal formation following carotid artery ligation. However, neointimal formation was similar between WT and N3-/- mice. Indeed, SMC derived from explanted aortas of either N1+/- or smN1+/- mice showed decreased chemotaxis and proliferation, and increased apoptosis compared to control or N3-/- mice. This correlated with decreased staining of PCNA positive cells and increased staining of cleaved Caspase-3 in the intima of N1+/- or smN1+/- mice. In SMC derived from CHF1/Hey2-/- mice, activation of Notch signaling did not lead to increase SMC proliferation or migration. Conclusion These findings indicate that Notch1, rather than Notch3, mediates SMC proliferation and neointimal formation following vascular injury through CHF1/Hey2 and suggest that therapies, which target Notch1/CHF1/Hey2 in SMC, may be beneficial in preventing vascular proliferative diseases.