Fig. 6. The ICM with pluripotent epiblast cells shows a specific and characteristic histone methylation pattern that is distinct from the pattern seen in TE cells. (A) The ICM with pluripotent epiblast cells has high me₁-, me₂- and me₃-K27 methylation levels (pink, middle). These cells show expression of Oct4-GFP, which is a marker of pluripotent epiblast cells (green, right). This pattern of histone methylation differs from that in trophectoderm cells shown in Fig. 4. Cells that show clustering of di- and tri-K27 methylation on the X_i do not express Oct4-GFP (green), which is most obvious in single optical sections (right). The white arrowheads in the middle and right images indicate an Oct4-GFP-expressing cell in the ICM with high me₃-K27 levels. The orange arrowhead indicates an Oct4-GFP negative cell that is undergoing X-inactivation. (B) The high levels of H3-K27 methylation of the ICM is similar to the staining at earlier stages of development, when all cells are pluripotent. The images show a late morula stage just prior to the blastocyst stage. (C) Deletion of Ezh2 from primary embryonic fibroblasts (PEFs) show no detectable effects on growth. Growth of PEFs was monitored in uninfected, GFP-infected and Cre-infected cells after three (1), five (2), six plus one passages (3) and eight days (4) in culture after infection. (F) Immunostaining of control GFP (upper panel) and experimental Cre (lower panel)-infected PEFS. The Ezh2 protein level was already highly reduced three days after Cre infection (lower panel) compared with GFP-infected cells (upper panel) as shown by immunofluorescence (DNA in blue, Ezh2 in red).

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