Endothelial Slit2 guides the Robo1-positive sympathetic innervation during heart development.

Abstract

Axon guidance cues direct nerves in the heart during development, disease, and regeneration. These cues determine cardiac innervation patterning by regulating the balance between chemo-attraction and chemo-repulsion. However, the role of one of the most crucial ligand-receptor combinations among axon guidance molecules, the Slit chemo-active ligands and their Roundabout (Robo) transmembrane receptors, remains unknown during cardiac innervation patterning. To test if Slit-Robo signalling is important for cardiac innervation guidance, we analysed Slit and Robo mouse knock-outs. Constitutive <i>Slit2^-/-</i> ventricles showed significantly reduced innervation, while <i>Slit3^-/-</i> hearts showed temporary increased levels of innervation compared to wild-type littermate controls. Whereas innervation was not affected in <i>Robo2^-/-</i>mice, the phenotype seen in <i>Slit2^-/-</i> ventricles was recapitulated in <i>Robo1^-/-</i> mice. Detailed expression analysis identified expression of Slit2 ligand in the endothelium of the coronary vessels, while Slit3 was highly present in the coronary smooth muscle wall and in the innervation. Both the Robo1 and Robo2 receptors were present in the nerves and at low levels in the vessels. Knocking out <i>Slit2</i> specifically in the endothelium recapitulated the defects seen in the constitutive <i>Slit2^-/-</i> hearts. Ex vivo axon guidance cultures showed that attraction of axons extending from the ganglia was strongly reduced in ventricles with absence of endothelial Slit2 compared to wild-type controls. In the absence of endothelial Slit2, adult mice showed reduced response to challenging the sympathetic innervation. In conclusion, we have identified an important new chemo-active Slit2-Robo1 pathway required for correct cardiac innervation development.