Fig. 4. Pouches show elevated proliferation and express markers of vascular smooth muscle cells. (A,C,E) A pouch from an E17.5 Cx431 knockout heart was consecutively sectioned and stained with cardiac myosin antibody (A) and PCNA antibody (C), and TUNEL labeled to detect apoptosis (E). The walls of the pouches consist of a myosin-free subendocardial layer of tissue (between arrows in A,C,E) that exhibits increased PCNA staining and TUNEL labeling (see dark nuclei between arrows in C,E, respectively). (B) Ventricular myosin light chain 2 (MLC2V) is also absent in the subendocardial layer of a pouch from an E17.5 Cx431^–/– mouse heart. (D,F) SM22 (D) is expressed subendocardially in the pouches of E16.5 hearts. After birth, vascular smooth muscle myosin (F) is also observed in this subendocardial layer. (G) The Masson-Goldner trichrome stain shows abnormal extracellular matrix deposition in the pouches of the Cx431 knockout mouse heart (pale blue staining denoted by white arrow). (H) A heterozygous E16.5 knockout mouse heart stained with anti-smooth muscle -actin exhibits a large pouch breaching the wall of the right ventricular outflow just beneath the pulmonary valve. AV, aortic valve; LV, left ventricle; Po, pouch; PV, pulmonary valve; RV, right ventricle. Scale bars in A,C,E,H, 100 µm; D,F, 50 µm.