REGULATORY FUNCTIONS OF NOTCH SIGNALING IN EARLY EMBRYONIC VASCULAR DIFFERENTIATION AND VESSEL REMODELING

Abstract

The signaling cascades that direct the morphological differentiation of the vascular system during early embryogenesis are not well defined. Several signaling pathways, including Notch and VEGF signaling, are critical for the formation of the vasculature in the mouse. However, the relationship between the molecular signals and transcriptional networks directing this process are still not well defined. To further understand the role of Notch signaling during endothelial differentiation and the genes regulated by this pathway, both loss-of-function and gain-of-function approaches were analyzed in vivo and in vitro. Conditional transgenic models were used to expand and ablate Notch signaling in the early embryonic endothelium. Embryos with activated Notch1 in the vasculature displayed a variety of defects, particularly in the yolk sac, and die soon after E10.5. These phenotypes were distinct from endothelial loss-of-function of Rbpj, a transcriptional regulator of Notch activity. Gene expression analysis of RNA isolated from the yolk sac of transgenic embryos indicated aberrant expression in a variety of genes in these models. In particular, a variety of secreted factors, including the VEGF family member, Pgf, displayed coordinate expression defects in the loss-of-function and gain-of-function models. These data indicate that Notch signaling may have potential nonautonomous roles in the remodeling of the yolk sac capillary plexus. To further understand the role of placental growth factor during endothelial differentiation, an in vivo gain-of-function transgenic model was developed. Embryos with expanded expression of Pgf in the vasculature display two distinct phenotypes, which were classified moderate and severe. Most notably, in both classes, the extraembryonic vasculature of the yolk sac displayed remodeling differentiation defects, with few matured vessels. Gene expression analysis of RNA isolated from the yolk sac of transgenic embryos indicated aberrant expression in a variety of genes. In particular, Notch family members showed increased expression in the gain-of-function model. The data from this model demonstrates regulatory connections between the VEGF and Notch signaling pathways during endothelial differentiation. We propose a role for Notch signaling in elaborating the microenvironment of the nascent arteriole, and suggest that novel regulatory connections exist between Notch signaling and other signaling pathways, particularly the VEGF family, during endothelial differentiation.