Histology and immunohistochemistry

Samples were harvested, fixed overnight in 2–4% paraformaldehyde and dehydrated through an ethanol series. All samples were paraffin embedded and sectioned. Antibodies used for immunofluorescence were anti-ErbB2 (Cell Signaling, 1:50), anti-phoshpo-ErbB2 Y1248 (Cell Signaling, 1:50), anti-Nrp1 (Santa Cruz, 1:25), anti-GFP (Abcam, 1:250), and anti-eNOS (BD Bioscieznces, 1:250). Hematoxylin and eosin staining was completed using a standard protocol.

For whole-mount X-gal staining, adult and embryonic hearts were isolated and fixed with 2% paraformaldehyde in PBS for 20 min at 4 °C. Following fixation, the samples were washed twice for 10 min in PBS at 4 °C. Embryos were stained in with PBS containing 1 mg ml⁻¹ X-gal, 5 mM K₃Fe(CN)₆, 5 mM K₄Fe(CN)₆, 2 mM MgCl₂, 0.01% NP-40, 0.01% sodium deoxycholate and incubated overnight at 37 °C.