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 lateral avascular zones are directly generated by the lateral plate mesoderm (LPM), a critical source of Sema3E. Conclusions These findings demonstrate that Sema3E-generated avascular zones are critical regulators of mammalian cardiovascular patterning, and are the first to identify a repulsive role for the LPM. Integration of multiple, and in some cases redundant, repulsive cues from various tissues is critical to patterning the first embryonic blood vessels.

It is generally believed that proteins of the troponin complex are not expressed in smooth muscle. We have directly assayed for expression of troponin transcripts in mouse vascular smooth muscle and found that troponin sequences normally associated with fast twitch skeletal muscle (fTnT, fTnI, fTnC) were present at significant levels in the thoracic aorta. In situ hybridization experiments demonstrated that fTnT, fTnI and fTnC transcripts were expressed in the smooth muscle layer of mouse blood vessels of all sizes. Protein blot analysis using rat tissue showed that at least two members of the troponin complex, Troponin T and Troponin I, were translated in vascular smooth muscle of the aorta. Finally, immuno-fluorescence microscopy of rat aortic smooth muscle revealed that TnT and TnI are localized in a unique pattern, coincident with the distribution of tropomyosin. It seems likely therefore, that a complete troponin complex is expressed in vascular smooth muscle and is associated with the contractile machinery of the cell. These observations raise the possibility that troponins play a role in regulation of smooth muscle function.

Signaling by the hedgehog (Hh) family of secreted growth factors is essential for development of embryonic blood vessels. Embryos lacking Hh function have abundant endothelial cells but fail to assemble vascular cords or lumenized endothelial tubes. However, the role of Hh signaling during later aspects of vascular patterning and morphogenesis is largely unexplored. We have used small molecule inhibitors and agonists to alter activity of the Hh signaling pathway in the chick embryo. When cyclopamine is added after cord formation, aortal cells form tubes, but these are small and disorganized and the density of the adjacent vascular plexus is reduced. Activation of the Hh pathway with SAG leads to formation of enlarged aortae and increased density of the plexus. The number of endothelial cell filopodia is found to correlate with Hh signaling levels. These studies show that Hh signaling levels must be tightly regulated for normal vascular patterning to be achieved.

The cellular mechanisms regulating branching and growth of the intersegmental vessels (ISVs) are not well understood. We have carried out studies demonstrating that Hedgehog (Hh) signaling is a major regulator of intersomitic vessel growth. Inhibition of Hh activity by cyclopamine completely blocks formation of intersomitic vessels in the avian embryo. Examination of gene expression patterns in Hh deficient embryos shows that components of the VEGF and Notch signaling pathways are down-regulated. However, we find no evidence that Notch signaling plays a significant role in regulation of intersomitic vessel growth. Indeed it appears that Hh modulation of Vascular Endothelial Growth Factor, VEGF, is the primary regulator of growth of intersomitic vessels in the avian embryo. Inhibition of the VEGF pathway results in absence of ISVs, whereas stimulation of VEGF expression leads to precocious branching of ISVs. These results demonstrate that Hh is an essential modulator of VEGF expression during developmental angiogenesis.