Fig. 5. Changes in EPI and PrE marker expression correlate with GFP-positive cell behaviour during in vitro culture of Pdgfr^H2B-GFP/+ blastocysts. (A-D) Immunolocalisation of Gata4 and Nanog in the ICM of embryos of 64 cells or more. (A) Salt-and-pepper distribution of Gata4 and Nanog in a 65-cell embryo. Some cells expressing Gata4 are also Nanog positive. (B) Salt-and-pepper distribution of Gata4 and Nanog in a 72-cell embryo. (C) Partial segregation of Gata4- and Nanog-positive cells in a 108-cell embryo. (D) Gata4- and Nanog-positive cells are completely segregated in a 115-cell embryo. (E-H) Changes in the GFP-positive cell distribution within the ICM of a Pdgfra^H2B-GFP/+ embryo dissected 90 hpc, visualized by 3D time-lapse microscopy. From 20 minutes (E) to 235 minutes (F) of culture, GFP-positive cells are randomly distributed in the ICM. Then, after around 370 minutes, partial segregation of the GFP-positive cells to the layer of cells lining the cavity can be observed (G). Complete segregation of GFP-positive cells to the PrE layer is achieved by 575 minutes (H). (E'-H') High-magnification views of ICMs of embryos at successive time-points E-H. (H') PrE layer can be distinguished by different refractive properties on a phase contrast (bright field) image from EPI cells (arrowheads). Each row represents single section time-lapse images of the same embryo (E-H, GFP fluorescence overlaid on a bright-field image; E'-H', bright-field image only). Green, GFP; red, Gata4; white, Nanog; blue, Hoechst. Scale bar: 20 µm. (I) Quantification of embryos exhibiting salt-and-pepper, partially sorted, or sorted distribution of PrE precursors relative to cell number.