Allele-specific RT-PCR expression analysis of the X-linked genes Xist, Ddx3x, Ube1x, Zfx, Rnf12, Atrx, Pdha1, and Utx in individual F1 hybrid WT & Xp-Xist^1lox (a,c) 8-16-cell embryos (morulas) and (b,d) embryonic day 6.5 (E6.5) extra-embryonic tissues. F1 hybrid embryos were generated by a cross of the Mus molossinus (M. mol.) strain JF1 females to largely Mus domesticus (M. dom.)-derived males from wild-type and Xp-Xist^1lox laboratory mice strains. Expression of the two alleles (maternal, Xm; paternal, Xp) was distinguished by single nucleotide polymorphisms between the strains resulting in a strain-specific pattern of fragments after restriction enzyme digestion (see Supplementary Methods and Supplementary Table VI). Lane 1, M. mol. allele (Xm); lane 2, M. dom. allele (Xp); lane 3, equal amounts of M. mol. and M. dom; lanes 4–6, representative F1 hybrid WT samples; lanes 7–9 representative F1 hybrid Xp-Xist^1lox samples. Xist is expressed exclusively from the Xp in WT morulas and in WT E6.5 extra-embryonic tissues and is absent in Xp-Xist^1lox morulas and E6.5 extra-embryonic tissues. Only those genes denoted by a ‘*’ show differences in the degree of silencing of the paternal alleles in Xp-Xist^1lox morulas compared to WT embryos (see Supplementary Table I); all genes show differences in E6.5 extra-embryonic tissues (see Supplementary Table II).

a, Physical map of the eleven X-linked genes assayed. b, c, Single representative nuclei from WT and Xp-Xist^1lox embryos probed for expression of G6pdx (red punctate signal) and Xist (green; in WT embryos) or Atrx (green; in Xp-Xist^1lox embryos). In WT embryos, Xist is expressed exclusively from and marks the paternal X-chromosome (Xp)19,30. In Xp-Xist^1lox embryos, Atrx expression is used to mark the two Xs. In nuclei from 2- and 4-cell embryos, most genes are expressed predominantly biallelically. DNA is stained blue with 4′,6-diamidino-2-phenylindole (DAPI). d, Comparison of the distribution of nuclei displaying biallelism, monoallelism, and no signal in WT and Xp-Xist^1lox embryos. n, numbers of nuclei. Only those genes/stages denoted by a ‘*’ show significant differences in the distribution of the three classes of nuclei (see Supplementary Figures 1–4).

a–b, Wild-type (WT) and Xp-Xist^1lox blastocyst (embryonic day 3.5 [E3.5]) female embryos expressing Xp-GFP in both the trophectoderm (TE) and inner cell mass (ICM) lineages. c–d, WT and Xp-Xist^1lox E4.5 female embryos silencing Xp-GFP in the TE but showing continued expression of the Xp-GFP in the ICM-derived cells. The hatched line demarcates the boundary between the TE and the ICM-derivatives. e,g,i, WT E5.75, E6.25, and E7.5 female embryos devoid of Xp-GFP expression in the extra-embryonic ectoderm and its derivatives (ExEmb; derived from the TE) due to imprinted XCI. The embryonic ectoderm (Emb), descended from the ICM, fluoresces green because of Xp-GFP expression due to random XCI that results in Xp activity in approximately half of the cells. The hatched line demarcates the boundary between the ExEmb and the Emb. f,h,j, Xp-Xist^1lox E5.75, E6.25, and E7.5 female embryos with a mosaic extra-embryonic compartment that is comprised of both cells that silence and express the Xp-GFP. DNA is stained blue with DAPI.