Fig. 1.

p19^Arf protein expression in ExEn. (A) WT E4.5 embryos were fixed and stained with the indicated antibodies and visualized for immunofluorescence with a confocal microscope. Pluripotent cells (red) in the inner cell mass and differentiated primitive endoderm cells (green) were visualized with antibodies to Oct4 and Gata4, respectively; p19^Arf protein was not detected. DAPI was used to visualize cell nuclei. (Scale bars, 25 µm.) (B) Section of WT embryo recovered from the decidua of the uterus at E7.5 stained as above. p19^Arf-positive ExEn cells (green, asterisks) surround the Oct4-positive epiblast (red). (Scale bars, 200 µm, Upper; 100 µm, Lower.) (C) Cryosectioned WT EBs were stained and visualized as above. Expression of p19^Arf (green) and Bmi1 (red) colocalize (yellow) in a single ExEn cell layer at the EB periphery. (Scale bar, 50 µm.) (D) Lineage tracing. Knock-in mice with Cre under the regulatory control of the cellular Arf promoter were crossed to an indicator strain that expresses LacZ in response to Cre-mediated excision of a “lox–stop–lox” cassette. ES cells obtained from these blastocysts were induced to differentiate to EBs. β-galactosidase was detected at the periphery of EBs expressing Arf–Cre (Lower; magnification: 50×).