Figure 3. Lack of defective X-inactivation initiation in Eed^-/- blastocysts.

See also Figure 3—figure supplement 1. (A) Allele-specific X-linked gene expression heat map of female Eed^fl/fl, Eed^fl/-, and Eed^-/- blastocysts. Four embryos each of Eed^fl/fl, Eed^fl/-, and Eed^-/- genotypes were sequenced individually and only genes with informative allelic expression in all samples are plotted (see Materials and methods). Genes are ordered on the basis of allelic expression in Eed^fl/fl embryos. (B) Average allelic expression of the RNA-Seq data shown in (A). The mean allelic expression of X-linked genes lacks significant difference between each combination of the three genotypes (p>0.05, Welch’s two-sample T-test). Pairwise statistical comparisons between all genotypes are included in Supplementary file 3. (C) Pyrosequencing-based quantification of allelic expression of X-linked genes Xist, Rnf12, Atrx and Pgk1 in Eed^fl/fl, Eed^fl/-, and Eed^-/- blastocysts. Error bars represent the standard deviation of data from 3 to 6 independent blastocyst embryos. The mean allelic expression of all four genes lack significant difference between each combination of the three genotypes (p>0.05, Welch’s two-sample T-test). Pairwise statistical comparisons for all genes and between all genotypes are included in Supplementary file 4. (D) RNA FISH detection of Xist RNA (green), Rnf12 RNA (red), and IF detection of H3K27me3 (white) in representative Eed^fl/fl or Eed^-/- female blastocysts. Nuclei are stained blue with DAPI. Scale bars, 20 µm. Individual nuclei displaying representative categories of stains are shown to the right of each embryo. Embryos ranged in size from 39 to 100 nuclei. (E) Bar plot of percentage of nuclei with coincident accumulation of Xist RNA and H3K27me3 in individual Eed^fl/fl and Eed^-/- embryos. Each bar is an individual embryo. Embryo numbers under the bars correspond to the same embryos plotted in F). (F) Bar plots of percentage of nuclei with or without Xist RNA-coating and Rnf12 RNA expression in the embryos stained in D) and plotted in E). The numbers under the bars correspond to the same embryos plotted in E).

Figure 3—figure supplement 1. X-linked gene expression analysis in Eed^-/- embryos.

(A) Validation of genotypes of E3.5 Eed^fl/fl, Eed^fl/-, and Eed^-/- female blastocyst embryos. Eed exon 7 RNA-Seq reads are normalized to total mapped RNA-Seq reads. (B) Table describing the RNA-Seq genotypes, number of sequenced embryos, average % maternal X-linked gene expression, average number of SNPs per X-linked gene, and the SNP overlapping read coverage threshold. A comprehensive list of expression levels of all informative genes is included in Supplementary file 2. (C) Pyrosequencing-based quantification of allelic expression of X-linked genes Xist, Rnf12, Atrx, and Pgk1 in individual Eed^fl/fl, Eed^fl/-, and Eed^-/- female blastocysts. Error bars, standard deviation of data from 3 to 6 independent embryos. The mean allelic expression of all four genes lacks significant difference between each combination of the three genotypes (p>0.05, Welch’s two-sample T-test). Pairwise statistical comparisons for all genes and between all genotypes are included in Supplementary file 4. (D) RNA FISH detection of Xist RNA (green), Rnf12 RNA (red), and IF detection of H3K27me3 (white) in representative Eed^fl/fl and Eed^fl/- or Eed^-/- male blastocysts. Nuclei are stained blue with DAPI. Scale bars, 20 µm. Right of each embryo, individual nuclei displaying representative categories of stains. Embryos ranged in size from 56 to 65 nuclei. Bar plot, percentage of nuclei with or without Xist RNA-coating and Rnf12 RNA expression.