Fig. 4. Localisation of Nanog, Gata4 and GFP in Pdgfra^H2B-GFP/+ blastocysts. (A) In a 32-cell blastocyst, Nanog and GFP show broad, overlapping expression; Gata4 is undetectable. (B) Salt-and-pepper distribution of Nanog, Gata4 and GFP in a 64-cell blastocyst. Gata4 is expressed only in a subset of Nanog-negative and GFP-positive cells. (C) Partial segregation of Nanog-positive and Gata4-/GFP-positive cells in a 115-cell blastocyst. Gata4 and GFP are co-expressed in cells mostly localised to the nascent PrE layer. Nanog is expressed in the EPI layer and no longer colocalises with Gata4 or GFP. (D) In a 118-cell blastocyst, PrE markers (Gata4 and GFP) are co-expressed and mutually exclusive from the EPI marker Nanog; cells expressing PrE and EPI markers are fully restricted to their respective layers. Each row represents a single optical section of one embryo. bf, bright field; green, GFP; white, Nanog; red, Gata4 (A-D); blue, Hoechst. Scale bar: 20 µm. (E) Comparison of the number of Gata4- and Nanog-positive cells versus total cell number. An abrupt decrease in the number of Nanog-expressing cells at around the 64-cell stage coincides with the emergence of Gata4-expressing cells. Thereafter, the number of Nanog-expressing cells appears to increase only slightly, whereas the number of Gata4-expressing cells increases approximately linearly. (F) Comparison of the number of Gata4- and GFP-expressing cells with respect to total cell number.