Fig. 2.

Loss of Nf1 in epicardial cells results in a phenotypic change to mesenchymal cells in vitro. (A,B) Seventy-two hour primary cultured epicardial cells from E12.5 mouse hearts. (A) Brightfield images. (B) Cultures stained for adherens junctions (β-catenin) and actin stress fibers (phalloidin). Nuclei were detected with DAPI. (C) mRNA expression of epithelial (Bves, Krt14) and mesenchymal (Sox9, Col7a1, Mmp10, Opg) genes. qRT-PCR was used to quantify gene expression in primary epicardial cell cultures. Data were compared with control cultures represented by a baseline of 1.0. For each gene, at least five independent experiments were quantified in triplicate. Values are mean ± s.d. ^*P<0.0001. (D) Embryonic hearts of the indicated genotype were cultured on collagen gels to measure invasion. Actin stress fibers were stained with phalloidin and nuclei were detected with DAPI. Arrows indicate the center of the heart explants and white dashed lines delineate the invasion front. Invasion of epicardial cells was detected by fluorescence microscopy after sectioning (bottom). Red dashed lines indicate the collagen gel surface and arrowheads indicate invading cells. (E) Differentiation of epicardial cells into smooth muscle cells was detected by IHC for SM-MHC. Actin and nuclei were visualized with phalloidin and DAPI, respectively. (F) Quantification of invasion of epicardial cells into the collagen gel. Values are mean ± s.d. ^*P<0.0001. (G) Quantification of VSMC differentiation. The SM-MHC fluorescent area was normalized to the nuclear area. Data are mean ± s.d. n values are indicated in parentheses. ^*P<0.001; ^**P<0.005; ^***P<0.05. Scale bars: 50 μm in A,B,D; 100 μm in E.