Fig. 7. Selective inhibition of FGFR1 and FGFR4 diminishes induction of NKX2.1 in ventral endoderm co-cultured with cardiac mesoderm. (A-C) Sagittal sections of mouse embryos at five- (B) and seven- (C) somite stages, demonstrating the onset and expansion of FGFR4 protein expression (arrows) in the foregut (fg) relative to the cardiac domain (cm); (A) adjacent section at five-somite stage, stained with FGFR1. FGFR4 antibody staining of foregut endoderm cultured alone (D) or in the presence of FGF2 100 ng/ml (E), or FGFR4 antibody staining of foregut endoderm cultured alone (D) or in the presence of FGF2 100 ng/ml (E) or cardiac mesoderm (G). (F-Q) Ventral endoderm/cardiac mesoderm explants cultured for 2 days after a single treatment of FGFR1-IgG or FGFR4-IgG, compared with rabbit IgG control (each 1 µg/ml). Same or adjacent sections (F-I;J-L;MO) labeled for designated marker by double immunofluorescence. Frequent colocalization of FGFR4 and NKX2.1 is shown in a merged image (I), with boxed inset demonstrating membranous staining for FGFR4 (nuclear staining is for NKX2.1). Explants in K,L,O have limited or no appreciable staining with albumin (green) or NKX2.1 (red). (P) Influence of FGFR1 and FGFR4 receptor inhibition on protein expression of albumin and NKX2.1 expression in ventral endoderm co-cultured with cardiac mesoderm. For graphic representation, four out of 10 explants in the respective groups were cryosectioned, immunostained for albumin and NKX2.1, and the number of positive cells out of total cells present in the section was determined. Results expressed as mean±s.e.m. (%). Explants treated with FGFR4-IgG exhibit a significant difference in inhibiting NKX2.1 expression (t-test: P<0.0001) but not albumin expression (t-test: P<0.4). FGFR1-IgG treatment effectively diminishes expression of both albumin and NKX2.1 (t-test; P<0.0001). (Q) RT-PCR analysis of RNA from ventral endoderm co-cultured with cardiac mesoderm and exposed to a single dose of soluble FGFR1/IgG-Fc (sFR1-IgG), FGFR4/IgG-Fc (sFR4-IgG) or SU5402; control explants for SU5402 assay cultured in the presence of DMSO. Expression for cardiac troponin and FOXA2 reflect integrity of the cardiac mesoderm and endoderm, respectively.