Fig. 7.

UTX is required for proper reactivation of late-reactivated genes. a Conditional Utx allele: FD = Flp and Dre-recombined conditional allele. Recombination of the third exon of Utx by Cre expression gives a knockout FDC allele. b Individual Utx control and mutant female ICMs analysed by immunolabelling with H3K27me3 (grey), combined with Xist (green) and Kif4 (red) RNA-FISH in late blastocysts (scale bar 20 µm). Cells below the white line illustrate the cluster of cells that have lost Xist coating and H3K27me3 enrichment on the Xp and are presumably the epiblast. Enlarged nuclei are shown for not reactivated (1, 3) and reactivated cells (2, 4) (scale bar 5 µm). c Mean of ICM cells showing enrichment of H3K27me3 on the Xist RNA-coated X-chromosome from E4.5 control (Utx ^FD/FD), heterozygous (Utx ^FDC/+ ) and mutant (Utx ^FDC/FDC) female blastocysts. Below the graph, the total cell number analysed is indicated, followed by the number of females in brackets. p value <0.0057 (**), <0.0018 (**) and <0.021 (*) between control and heterozygous vs. mutant, respectively, for H3K27me3-Xist-negative cells, H3K27me3-Xist-positive cells and H3K27me3-positive, Xist-negative cells, by two-sided Dunn’s test. d Example of Utx mutant female ICM analysed by immunolabelling with H3K27me3 (grey), combined with Xist RNA (green) at late (E4.5) blastocyst stage. Red and blue arrows pointed cells, respectively, with both Xist and H3K27me3 enrichment and only H3K27me3 enrichment on the Xp (scale bar 10 µm). e Percentage (mean) of cells showing biallelic expression for X-linked genes in ICM of E4.5 control (Utx ^FD/FD ) and Utx mutant (Utx ^FDC/FDC) embryos. Kif4, Rnf12 and Pdha1 are late- or very late-reactivated genes, when Abcb7 and Atrx are early-reactivated genes (Supplementary Data 2). f Control (Utx ^FD/FD ) and mutant (Utx ^FDC/FDC) E4.5 blastocysts analysed by immunofluorescence against NANOG (green) and GATA6 (red). DAPI is in dark blue (scale bar 20 µm). Percentage of positive cells for Nanog, Gata6 or both have been summarized as the mean in the graph and total cell number analysed is indicated below, followed by the number of females in brackets. Non-significant (n.s.) by Kruskal–Wallis test. FDC Flp, Dre and Cre-recombined knockout allele, MW Mann–Whitney non-parametric test