Fig. 2.

Delayed formation of ExEn cells in Arf-null EBs. (A and B) WT (A) and Arf-null (B) EBs were processed for immunofluorescence. Dab2 expression was greatly reduced when Arf was inactivated. DAPI was used to stain cell nuclei. The intense halo of blue cells in B Upper Left is an artifact dependent upon the confocal plane; these same cells expressed Oct4 (Lower Left). (Scale bars, 50 µm.) (C) Immunoblotting of lysates from WT and three pooled Arf-null ES cell-derived EBs generated after 4 d of suspension culture exhibited quantitative differences in the expression of the indicated proteins. (D) Immunoblotting with the indicated antibodies showed that Arf-null EBs express very low levels of the ExEn marker Dab2 after 4 d of culture, but EBs cultured for 10 d recovered Dab2 expression. (E) EBs derived from functionally Arf-null Arf^Gfp/Gfp ES cells after 10 d of culture express Gata4-positive ExEn cells on their surface. (Magnification: 50×.) Activation of the cellular Arf promoter in “GFP knock-in” cells that do not express the p19^Arf protein was accompanied by GFP signals, which colocalized with Gata4-marked ExEn cells at the periphery.