Fig. 8. Non-cell autonomous regulation of islet cells by Phox2b. Pancreas from Phox2b^+/+ (A) and Phox2b^-/- (B) mouse embryos at E17.5 co-stained by immunofluorescence for insulin (FITC, green) and glucagon (Cy3, red). (C) Insulin- and glucagon-positive cells from Phox2b^+/+ (white bars) and phox2b^-/- (black bars) embryos counted and expressed as the total number of cells per total pancreatic area in mm². Each data point represents the mean of five embryos ±s.e.m. (D) Insulin and glucagon mRNA levels measured by real-time RT-PCR (TaqMan) from RNA from pancreas of Phox2b^+/+ (white bars) and Phox2b^-/- (black bars) embryos at E17.5. Each data point represents the mean of four independent litters ±s.e.m. (E) Pancreas from a Phox2b^-/- mouse embryo at E17.5 co-stained by immunofluorescence for Ki67 (FITC, green) and insulin (Cy3, red). (F) The percentage of beta-cells replicating at E17.5 in embryos with the genotypes shown assessed by counting the number of cells co-staining for insulin and Ki67 and dividing by the total number of cells staining for insulin. Each data point represents the mean of five pancreas ±s.e.m. (G) Nkx2.2 (white bars) and neurogenin 3 (black bars) mRNA levels determined by real-time RT-PCR (TaqMan) with RNA isolated from pancreas from E15.5 embryos with the Phox2b genotypes shown. Data represent the mean values of three independent experiments performed in triplicate ±s.e.m. ^*P<0.01, ^**P<0.001, by Student's t-test. Scale bars: 50 µm.