CHF1/Hey2 is not expressed in epicardium or EPDCs and does not affect early epicardial development. (a) In situ hybridization with CHF1/Hey2 probe for left ventricle of E15.5 wild-type heart (n = 3). Arrows indicate epicardium. (b) The expression of CHF1/Hey2 RNA in left ventricle and primary isolated epicardial-derived cells (EPDCs) of CHF1/Hey2 knockout mice. Total RNA was obtained from E12.5 left ventricle of wild-type (n = 3) or CHF1/Hey2 knockout mice (n = 3), or EPDC of wild-type (n = 3) and CHF1/Hey2 knockout mice(n = 3) mice and was reverse-transcribed into cDNA and then subjected to real-time PCR using specific primers for CHF1/Hey2. The starting quantities were calculated and expressed as a ratio of each gene to that of Gapd. (c-f) Subepicardial space of left ventricle of wild-type mice (c, E11.5, n = 3; d, E13.5, n = 3) and CHF1/Hey2 knockout mice (e, E11.5, n = 3; f, E13.5, n = 3) were indistinguishable. In situ hybridization with WT1 probe for left ventricles of E12.5 wild-type (n = 3) (g) or CHF1/Hey2 knockout mice (n = 3) (h). Scale bars = 100 μm in a, c-f; 200 μm in g and h.