Fig. 7.

Dynamic expression of NGF in VSMCs from the subepicardium to the myocardium is responsible for myocardial innervation. (A) RT-PCR analysis of E13.5 and E16.5 myocardium explants. (B-D) Location of EPHB4⁺ coronary veins (B) and ephrin B2⁺ arteries (C) in the cardiac ventricles. Sections of EphB4^lacZ/+ (B) and ephrinB2^lacZ/+ (C) hearts were stained with antibodies for β-gal (green) and PECAM1 (red). The relative location of coronary veins (CV) and arteries (CA) in heart ventricle is shown in D. (E-L) NGF expression analysis on NGF^lacZ/+ reporter heart sections. E15.5 (E-G) or E17.5 (I-K) heart sections of NGF^lacZ/+ reporter embryos were stained with antibodies for β-gal (green), PECAM1 (red) and α-actinin (blue). Magnified images (F,G,J,K) show the boxed regions in E,I. Schematic models illustrating dynamic expression of NGF in VSMCs in parallel with sympathetic axon extension at E15.5 (H) and E17.5 (L). (M-O) SG and myocardium co-culture with anti-NGF NZAb. E16.5 SG were cultured with E16.5 myocardial explants in the presence of 200 ng/ml isotype IgG (M) or 200 ng/ml anti-NGF NZAb (N). The culture was stained with anti-TUJ1 antibody (green) and To-pro-3 (blue). (O) Directional axon growth was quantified as in Fig. 5G. ^*P<0.02 (Student’s t-test); isotype IgG, n=5; 200 ng/ml anti-NGF NZAb, n=5; error bars indicate s.e.m. CV, coronary vein; CA, coronary artery; Myo, myocardium; Se, subepicardium.