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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
PdgfraH2B-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.
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