Lamellipodial protrusion is regulated by Ena/VASP proteins. We identified Lamellipodin (Lpd) as an Ena/VASP binding protein. Both proteins colocalize at the tips of lamellipodia and filopodia. Lpd is recruited to EPEC and Vaccinia, pathogens that exploit the actin cytoskeleton for their own motility. Lpd contains a PH domain that binds specifically to PI(3,4)P2, an asymmetrically localized signal in chemotactic cells. Lpd's PH domain can localize to ruffles in PDGF-treated fibroblasts. Lpd overexpression increases lamellipodial protrusion velocity, an effect observed when Ena/VASP proteins are overexpressed or artificially targeted to the plasma membrane. Conversely, knockdown of Lpd expression impairs lamellipodia formation, reduces velocity of residual lamellipodial protrusion, and decreases F-actin content. These phenotypes are more severe than loss of Ena/VASP, suggesting that Lpd regulates other effectors of the actin cytoskeleton in addition to Ena/VASP.

Notch signalling by the ligand Delta-like 4 (Dll4) is essential for normal vascular remodelling, yet the precise way in which the pathway influences the behaviour of endothelial cells remains a mystery. Using the embryonic zebrafish, we show that, when Dll4-Notch signalling is defective, endothelial cells continue to migrate and proliferate when they should normally stop these processes. Artificial overactivation of the Notch pathway has opposite consequences. When vascular endothelial growth factor (Vegf) signalling and Dll4-Notch signalling are both blocked, the endothelial cells remain quiescent. Thus, Dll4-Notch signalling acts as an angiogenic `off' switch by making endothelial cells unresponsive to Vegf.

When and where to make or break new blood vessel connections is the key to understanding guided vascular patterning. VEGF-A stimulation and Dll4/Notch signaling cooperatively control the number of new connections by regulating endothelial tip cell formation. Here, we show that the Notch-regulated ankyrin repeat protein (Nrarp) acts as a molecular link between Notch- and Lef1-dependent Wnt signaling in endothelial cells to control stability of new vessel connections in mouse and zebrafish. Dll4/Notch-induced expression of Nrarp limits Notch signaling and promotes Wnt/Ctnnb1 signaling in endothelial stalk cells through interactions with Lef1. BATgal-reporter expression confirms Wnt signaling activity in endothelial stalk cells. Ex vivo, combined Wnt3a and Dll4 stimulation of endothelial cells enhances Wnt-reporter activity, which is abrogated by loss of Nrarp. In vivo, loss of Nrarp, Lef1, or endothelial Ctnnb1 causes vessel regression. We suggest that the balance between Notch and Wnt signaling determines whether to make or break new vessel connections.