Extrinsic Notch Ligand Delta-Like 1 Regulates Tip Cell Selection and Vascular Branching Morphogenesis

Rationale: In developing blood vessels, single endothelial cells (ECs) specialize into tip cells that sense vascular endothelial growth factor (VEGF) and contribute to vessel sprouting and branch formation. Tip cell differentiation is inhibited through lateral Notch signaling between ECs, which is controlled by Notch ligands expressed in vessel sprouts. The contribution of the Notch ligand Delta-like (Dll) 1 herein is unknown. Objective: To investigate the role of Dll1 in vascular morphogenesis and tip cell formation in the mouse retina. Methods and Results: Mice with heterozygous deletion of Dll1 had fewer tip cells during angiogenic sprouting of the superficial vascular plexus but also showed impaired vessel branching into deeper retinal layers and impaired deep plexus angiogenesis. Interestingly, the formation of vertical branches was also guided by filopodia-extending ECs located at the tip of branches, consistent with tip cells, which emerged from established vessels to form a secondary plexus within the deeper neuronal cell layers. During both phases of vascular patterning, Dll1 was not expressed in ECs but in the superficial neuronal layer in close contact with expanding vessels, where Dll1 expression coincided with tip cell formation in a spatiotemporal manner. In vitro, culture of ECs on DLL1 induced essential tip cell genes, including Dll4, VEGF receptor 3, and ephrin-B2, and stimulated VEGF responsiveness and vascular network formation. Conclusions: Dll1 acts as an extrinsic cue involved in tip cell selection, which directs vessel sprouting and branch formation. (Circ Res. 2012; 110:530-535.)