Fig. 4. Ablation of Frs2 disrupts OFT cushion formation by inhibiting endocardial EMT and NCC contribution. (A,B) Reduced cellularity in Frs2 mutant OFT cushions. (A) Sagittal sections of E10.5 mouse embryos were H&E stained. High-magnification views of boxed areas representing proximal and distal OFT are shown as indicated (a1-c2). (B) Cell numbers in the proximal and distal cushions (Cu) were assessed and statistical data from four individuals are presented (mean ±s.d.). Note that Frs2^cn/Nkx, but not Frs2^cn/Mef, OFT cushions had decreased cellularity. En, endocardium. (C) H&E staining of transverse sections of E11.5 distal OFT showing the fusion defect in Frs2^cn/Nkx (b) OFT cushions. (D) Immunostaining for PECAM (a-c) and NFATC1 (d-f) shows compromised EMT in the Frs2^cn/Nkx proximal OFT cushions. Note that both PECAM and NFATC1 are still expressed in Frs2^cn/Nkx cushion cells (arrows). (E) Ex vivo culture of E9.5 OFTs shows compromised EMT in Frs2^cn (b) OFT myocardium. (F) Increased immunostaining for VEGFA in the Frs2^cn/Nkx OFT myocardium (My). Expression of VEGFA in E9.5 (a-c) and E10.5 (d-f) embryos. Nuclei were stained with To-Pro3. Boxed insets are high-magnification views of the myocardium. Note that VEGF expression in Frs2^cn/Nkx myocardial cells is significantly increased. (G) FRS2 is essential for FGF2 to activate NFAT transcriptional activity. Mouse embryonic fibroblasts carrying homozygous Frs2^flox alleles were transfected with an NFAT-dependent luciferase reporter with or without Cre coexpression. The cells were cultured in the presence or absence of 2 ng/ml FGF2 as indicated. Luciferase activity was then assessed. Data are mean ±s.d. of triplicate samples.