Constitutive knock-out analysis indicates a role for Slit2, Slit3 and Robo1 during cardiac innervation development.

A, Representative images of Robo1+/+ and Robo1-/- sections stained by immunohistochemistry for the myocardium using cardiac Troponin I (cTnI) in purple and total innervation using Peripherin (Prph) in green. Arrowhead points to reduced total innervation staining in green in the Robo1-/-. B, Quantification of total ventricular innervation corrected for ventricle volume (myocardial marker cTnI) in constitutive knock-outs and wild-type controls. T-test or one-way ANOVA with Tukey’s Test per mouse line. C, Quantification of total ventricular volume based on myocardial marker cTnI in constitutive knock-outs and wild-type controls. T-test or one-way ANOVA with Tukey’s Test per mouse line. Scale bar 100μm.

The coronary endothelium acts as source of chemo-attractive Slit2 that guides the Robo1-positive nerves during heart development.

A, Immunohistochemistry for Slit2, Slit3, Robo1, Robo2, endothelial marker Erg1, innervation marker Prph and smooth muscle marker Myh11 at E18.5. Box indicates region of magnification. Arrow points to overlap Slit3 and Prph in the large epicardial nerves. White arrowheads point to region of absence or presence of co-localisation of the indicated genes. B. Measurements of total ventricular innervation corrected for ventricle volume in the indicated conditional knock-outs and wild-type controls. T-test or one-way ANOVA with Tukey’s Test. Scale bar 100μm.

Reduced sympathetic innervation in absence of endothelial Slit2 limits sympathetic stimulation of heart rate.

A, Immunohistochemistry for sympathetic innervation marker TH and myocardial marker cTnI on whole mount E18.5 hearts. B, Quantification of sympathetic innervation corrected for ventricle volume in endothelial Slit2 knock-outs versus controls. T-test. C, Immunohistochemistry for total innervation marker Prph and myocardial marker cTnI on E18.5 sections. Arrowheads point to the normal development of the coronary vessels. D, Quantification of coronary vessel volume corrected for ventricle volume in endothelial Slit2 knock-outs versus controls. T-test. E, Immunohistochemistry for Slit2 on endothelial Slit2 knock-outs versus controls, showing the lack of Slit2 in the endothelium. F, E14.5 ventricles and stellate ganglia (STG) cultured in three-dimensional collagen matrix (Chemicon) for 3 days. Immunohistochemistry for Prph and cTnI. G, Quantification of axon outgrowth. One-way ANOVA with Tukey’s Test. H, Quantification of baseline heart rate using electrocardiogram. G, Quantification of heart rate after stimulations with isoproterenol. Scale bar 100μm.