Fig. 5.

Enhanced X-Gal staining of −34−31−21-ANF-lacZ transgenic mice in the left ventricle of transcription repressor hearts lacking Hey2. (A) PCR genotyping of Hey2^+/+ (lane 1) and germ line Hey2^−/− mice (lane 2). (B) Representative images of in situ hybridization of ANF in Hey2^+/+ versus Hey2^−/− litters. ANF expression in the left ventricle of Hey2^−/− is marked with an asterisk. The right ventricle was rounder and larger in Hey2 knockout mice, in which ANF mRNA was slightly reduced (arrow). (C) Increased X-Gal intensity in the left ventricle of hearts lacking Hey2 (asterisk) in comparison to the control litters at E13.5 in Tg1 to Tg3. The right ventricles of Hey2^−/− mice are marked with arrows. Representative images from a total of 9 Hey2^+/+ and 8 Hey2^−/− heterozygous −34−31−21-ANF-lacZ-positive embryos are shown. (D) Enlarged images of tissue sections demonstrate ectopic expression of ANF and lacZ in the compact layer (arrowheads). The compact layer is defined morphologically as the appearance of a compact band of uniform tissue, while the endocardial noncompacted trabecular layer consists of trabecular meshwork with deep endomyocardial spaces (16). (E) An E-box sequence is located at −34835 bp, close to the Nkx2-5-binding sequence at −34776 bp. The mutated E-box sequence used in the −34-ANF luciferase reporter construct is indicated. (F) Western blotting demonstrates HA-tagged Hey2 protein expression in the transfected cells (lanes 1 and 2) in comparison to control cells transfected with pcDNA3. (G) Effects of Hey2 on ANF(−451 to +56), −34-ANF promoter, and the E-box mutant of −34-ANF luciferase constructs. Induction of luciferase reporter activities was normalized to β-galactosidase activity, with the value in ANF(−451 to +56) luciferase reporter cotransfected with empty pcDNA3 plasmid defined as 1 (values are means ± SE). *, P < 0.05 (ANOVA). Bars = 1 mm (B and C) and 50 μm (D). LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle.