Fig. 4. Expression pattern of ECE1 and conduction cell markers in embryonic chick hearts (A-F), in monolayer (G,H) and organ culture of myocytes (I-L). (A-C) Whole-mount in situ hybridization of E7 (A) and E13 (B) hearts for ECE1. (C) High power view of the boxed area in B. (D) In situ hybridization on a frozen section of E13 ventricle with antisense ECE1 probe. Purple staining in C,D is positive hybridization signal. Arrows and arrowheads in C,D indicate ECE1-positive arterial and endocardial endothelial cells, respectively. Asterisks in D,E indicate the lumen of ventricular chamber. (E) Immunostaining of a frozen section of E15 ventricle with ALD58 (green signal), a Purkinje fiber marker. (F) Same as E but double immunostaining with ALD58 (green signal) and MF20 (red signal), which detects all sarcomeric myosins. Arrows and arrowheads indicate ALD58-positive periarterial and subendocardial Purkinje fibers, respectively. (G) Monolayer culture of myocytes isolated from E3 heart tube. (H) Same as G but exposed to 10^–7 M ET1 for 5 days just after isolation. Blue staining is ALD58-positive signals (arrows). (I) A ventricular segment isolated from E3 heart tube. (J-L) E3 ventricular segment cultured and (J) stained with ALD58, (K) exposed to 10^–7 M ET-1 just after isolation and (L) exposed to ET 1 day after isolation. Green signals in J-L are ALD58-positive staining.