Mid-stage inner cell masses (ICMs) exhibit increasing Xp-linked gene expression. (A) A representative ICM assayed for Atp7a (red, left panel) and Xist and Tsix (green, right panel) by RNA fluorescence in situ hybridization (FISH). The Xist probe detects Xist RNA and the anti-sense transcript detects Tsix. Xist marks the Xp as a cloud at this stage. In ICMs, Tsix is detected in some nuclei as a pinpoint signal from the Xm (Lee et al., 1999). In the merge image, all nuclei exhibit monoallelic Atp7a expression, localized away from the Xp-Xist coat (green domains). A single nucleus is highlighted in the right corner of each image. (B) Distributions of biallelic (black), monoallelic (gray) and nonscorable (white) nuclei are presented as the average percentage of nuclei per early or mid-stage ICM. In mid-stage ICMs there is a statistically significant (P<0.01) increase in biallelism and decrease in monoallelism compared with early stage ICMs. (C) A representative nucleus illustrates biallelic Abcb7 (red, first panel) expression. H3K27me3 accumulates on the Xp above genomic levels (cyan, second panel). The H3K27me3 enrichment domain colocalizes with Xp-Xist coating (green cloud, third panel). The merge image (fourth panel) indicates that one of the two Abcb7 RNA FISH signals (marked by an arrowhead) colocalizes with both the Xp Xist and H3K27me3 domains. Nuclei are stained with DAPI.

Dynamics of X-linked gene expression assayed by allele-specific RT-PCR. F1 hybrid mouse embryos were generated by mating JF1 females to CD1 males. Xm and Xp transcripts were distinguished utilizing single nucleotide polymorphisms between the two strains. (A) Allele-specific expression was analyzed for Xist and the X-linked genes Ube1x, Rnf12, Pdha1 and Ddx3x. Lane 1, Xm allele; Lane 2, Xp allele; Lane 3, equal amounts of Xm and Xp allele; Lanes 4-6, early F1 hybrid inner cell mass (ICM) samples; Lanes 7-9, mid-stage F1 hybrid ICM samples. Analysis of Xist expression illustrates that in both early and mid-stage ICMs, Xist is expressed from the Xp. In early ICMs, X-linked gene expression for Ube1x, Rnf12, Pdha1 and Ddx3x is largely from the Xm allele. In mid-stage ICMs there is increasing Xp expression of Xp-linked genes. (B) Average Xm and Xp contributions were calculated as a percentage of total expression (Xm+Xp). There is a statistically significant increase in average Xp expression in mid-stage ICMs compared with early stage ICMs for each X-linked gene analyzed (P<0.01). The number of ICMs examined is presented in the table.

Mid-stage inner cell masses (ICMs) exhibit transcription within the Xp-Xist domain. Cot-1 DNA (red) and Xist (green) probes were used in RNA FISH experiments to determine general Xp transcriptional status. (A) The average distribution of nuclei with mutually excluded or overlapping Cot-1 and Xist RNA FISH signals are shown as a percentage of total nuclei per mid-stage trophectoderm (TE), early ICM and mid-stage ICM. The bracket represents a statistically significant (P<XXXX) increase in the percentage of cells exhibiting overlapping FISH signals in mid-ICMs compared with early ICMs. (B) Representative mid-stage ICM nuclei indicate mutually excluded or overlapping patterns. Arrowheads indicate the Xist domain. Dashed lines indicate the area of the merged figure represented in the line scans. Line scans indicate the relative intensities of DAPI, Cot-1 and Xist signals.

Nanog expression corresponds to biallelic X-linked gene expression in mid-stage inner cell masses (ICMs). (A) Side-by-side box plots graphically represent the percentage of biallelic nuclei (y-axis) grouped as either positive or negative for Nanog transcripts (x-axis). The box plot on the left represents ICMs assayed for Atp7a expression and the right-hand box plot represents ICMs assayed for Mecp2 expression. The medians of the data are indicated (green triangle). The boxes contain the middle 50% of the data. Whiskers represent maximum and minimum values of the dataset. The box plots are nonoverlapping, illustrating that the population of biallelic nuclei are largely skewed towards being Nanog positive. (B) Representative nuclei of a mid-stage ICM showing detection of Atp7a (red), Xist RNA (blue) and Nanog (green) assayed by RNA FISH. Top row: Biallelic Atp7a (red) expression is marked by arrowheads (first panel) with one allele overlapping Xist transcripts (blue, second panel). Nanog transcripts (green) are marked by arrows (third panel). Bottom row: Monoallelic Atp7a expression is marked by an arrowhead (first panel) and colocalizes with Tsix expression (blue pinpoint signal, second panel) from the Xm. In this nucleus, Nanog is not expressed (third panel). (C) The graph represents the distribution of the percentage of nuclei from mid-stage Nanog positive nuclei that exhibit Xp-Xist coating. The red line represents the average. n, the number of embryos analyzed.

Grb2^−/− inner cell mass (ICM) cells exhibit loss of Xist transcripts and EED on the Xp. (A) Mid-stage blastocysts immunostained for NANOG (red, top row) and EED (green, second row). NANOG marks the epiblast progenitors. In wild-type blastocysts (left panels), EED accumulates on the Xp in NANOG positive nuclei as seen by the green foci (merge, third row). However, in all but one NANOG positive nuclei of Grb2^−/− blastocysts (right-hand panels), Xp EED foci are undetectable (merge, third row). DAPI (blue, fourth row) stains all nuclei. (B) Wild-type (WT, top row) and Grb2^−/− (bottom row) mid-stage ICMs assayed by RNA FISH for transcripts of Xist/Tsix (green, left-hand panels) and Ube1x (red, middle panels). Grb2^−/− ICMs exhibit a decrease in Xp-Xist coating (green, left panel). A representative nucleus is highlighted and an arrowhead marks monoallelic Ube1x expression, which occurs despite the absence of Xp-Xist coating. The graph indicates the average percentage of cells per ICM that exhibit Xist coating in Grb2^−/− compared with wild-type littermate controls. Error bars represent xxx. (C) Nuclei from wild-type (upper rows) and Grb2^−/− (bottom rows) ICMs assayed for transcripts of X-linked genes Rnf12, Atp7a and Abcb7 (red signals marked by arrowheads, left panels) and Xist/Tsix (green, middle panels). Grb2^−/− ICM nuclei (bottom rows) show monoallelic expression in the absence of the Xp-Xist domain (merge, right panels). The graph indicates the distribution of biallelic X-linked gene expression in Grb2^−/− ICMs compared with wild type. The data are presented as a percentage of total cells per ICM. Error bars represent s.d.