Integration of Repulsive Guidance Cues Generates Avascular Zones that Shape Mammalian Blood Vessels

Rationale—Positive signals, such as vascular endothelial factor (VEGF), direct endothelial cells (ECs) to specific locations during blood vessel formation. Less is known about repulsive signal contribution to shaping vessels. Recently, ’neuronal guidance cues ’ (NGCs) have been shown to influence EC behavior, particularly in directing sprouting angiogenesis by repelling ECs. However, their role during de novo blood vessel formation remains unexplored. Objective—To identify signals that guide and pattern the first mammalian blood vessels. Methods and Results—Using genetic mouse models, we show that blood vessels are sculpted via the generation of stereotyped avascular zones by EC-repulsive cues. We demonstrate that Semaphorin3E (Sema3E) is a key factor that shapes the paired DA in mouse, as sema3E-/-embryos develop an abnormally branched aortic plexus with a markedly narrowed avascular midline. In vitro cultures and avian grafting experiments show strong repulsion of ECs by Sema3E-expressing cells. We further identify the mouse notochord as a rich source of multiple redundant NGCs. Mouse embryos that lack notochords fail to form cohesive aortic vessels due to loss of the avascular midline, yet maintain lateral avascular zones. We demonstrate that latera