Expansion of EPDCs upon loss of Nf1 in the epicardium. (A) In situ hybridization at E18.5 for cardiac fibroblast marker genes (Col1a1, Col3a1 and Pdgfra). Mouse embryos were induced with tamoxifen at E12.5 and hearts were isolated at E18.5. The boxed regions are shown at higher magnification in the insets. Scale bars: 500 μm; 100 μm in insets. (B) Whole-mount β-galactosidase staining (blue) of X-LacZ4 hearts for detection of VSMCs. Tamoxifen was administrated maternally at E12.5 before heart isolation at E17.5. (C) Quantification of the area positive for Col1a1, Col3a1 or Pdgfra in A from 20× field-of-view images taken in similar regions of the left ventricle. (D) Quantification of multi-branched (three or more) SMC-coated vessels in B. Data are mean ± s.d. n values are indicated in parentheses. *P<0.001; **P<0.005. Ra, right atrium; La, left atrium; Rv, right ventricle; Lv, left ventricle; FOV, field of view.