Figure 2: Global XCI and Xist expression states of Xm at late preimplantation stages.
(a) Analysis of Xist expression using qPCR of individual embryos in the morula. An asterisk indicates P<0.05 (Student’s t-test) compared with Egfp-PEs. FEs, fertilized embryos. (b) Large-scale qPCR analysis of Xist and eight X-linked genes in individual blastocysts after culturing for 96 h. Coloured bars indicate expression levels. (c,d) IF (H3K27me3, green) combined with RNA FISH (Xist, red) analysis in 96-h blastocyst stage. 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei are shown in blue. (c) Representative confocal projection. Scale bars, 50 μm. (d) The graph shows Xist expression and H3K27me3 modification states in individual embryos. The horizontal axis indicates the average percentage in the group. *P<3.1 × 10−28 (Fisher’s exact test). n, number of embryos analysed. (e,f) Xm-Xist biallelic expression states in PEs at 96 h. The asterisk indicates cells with biallelic expression. Scale bars, 20 μm. (f) Summary of the ratio of biallelic cells to Xist-positive cells in 96-h blastocyst stage in each group. The number of cells is shown in Supplementary Table 3. (g) qPCR analysis of Xist and eight X-linked genes in individual blastocysts after culturing for 120 h. (h,i) IF (H3K27me3, green) combined with RNA FISH (Xist, red) analysis in 120 h blastocysts. *P<5.4 × 10−23 (Fisher’s exact test). Scale bars, 50 μm. (j) The ratio of biallelic cells to Xist-positive cells in 120 h blastocysts. In qPCR analysis, the average expression level of Xm-Xist in Egfp-PEs was set as 1 (also see the Methods section). Gapdh and β-actin were used as internal controls. Data are summarized in Supplementary Table 4.
