X-linked gene expression upon conversion of XEN cells into visceral endoderm. (A) Representative photographs illustrating the epithelium-like cell morphology observed after 5 days of bone morphogenetic protein 4 (BMP4) induction of extraendoderm stem (XEN) female cells (GHP7/9 cell line) are shown. (B) Heatmap of single-cell, steady-state RNA levels for the 18 most stringent discriminants of cells converted into visceral endoderm and undifferentiated XEN cells is shown (P < 10−3 by F-test). Hierarchical clustering has been applied. The complete data set is available in Additional file 2E. n = 72 female XEN cells (red) and n = 82 XEN cells treated with BMP4 (25 μM) for 5 days (yellow). The expected tissue specificity of each gene is indicated: EPI, Epiblast; ICM, Inner cell mass; PE, Parietal endoderm; Pl. Lab, Placental labyrinth; PrE, Primitive endoderm; TE, Trophectoderm; TGC, Trophoblast giant cell; VE, Visceral endoderm. The colour scale is the same as the one shown in Figure 1A. (C) Three-dimensional projections of principal components (PCs) 1, 2 and 3 of single-cell expression profiles of genes shown in (B). (D) Two-colour fluorescence in situ hybridisation on RNA analysis for the indicated genes in XEN cells treated with BMP4 for 5 days. Arrowheads point to transcription signals associated with the Xist-coated inactive X chromosome. Scale bar = 5 μm. (E) Cumulative histogram showing the percentage of nuclei with the depicted expression pattern in XEN cells treated with BMP4 for 5 days (D5). Results obtained in untreated XEN cells (D0) are also shown to facilitate the comparison. No significant differences between X-linked gene expression profiles in BMP4 treated compared to untreated XEN cells could be detected (Χ2 test). n > 100. [K27-high], Enriched in H3K27me3 in female compared to male XEN cells; [K27-low], Low levels of H3K27me3 in female and in male XEN cells; Not exp., genes not significantly expressed; Xa, Active X; Xi, Inactive X.