Dynamic changes in paternal X-chromosome activity during imprinted X-chromosome inactivation in mice

Abstract

In mammals, X-chromosome dosage compensation is achieved by inactivating one of the two X chromosomes in females. In mice, X inactivation is initially imprinted, with inactivation of the paternal X (Xp) chromosome occurring during preimplantation development. One theory is that the Xp is preinactivated in female embryos, because of its previous silence during meiosis in the male germ line. The extent to which the Xp is active after fertilization and the exact time of onset of X-linked gene silencing have been the subject of debate. We performed a systematic, single-cell transcriptional analysis to examine the activity of the Xp chromosome for a panel of X-linked genes throughout early preimplantation development in the mouse. Rather than being preinactivated, we found the Xp to be fully active at the time of zygotic gene activation, with silencing beginning from the 4-cell stage onward. X-inactivation patterns were, however, surprisingly diverse between genes. Some loci showed early onset (4-8-cell stage) of X inactivation, and some showed extremely late onset (postblastocyst stage), whereas others were never fully inactivated. Thus, we show that silencing of some X-chromosomal regions occurs outside of the usual time window and that escape from X inactivation can be highly lineage specific. These results reveal that imprinted X inactivation in mice is far less concerted than previously thought and highlight the epigenetic diversity underlying the dosage compensation process during early mammalian development.

Fig. 1.

Transcriptional activity of the paternal X chromosome assayed by gene RNA FISH in 2-cell embryos. (A) Deconvolved images of 3D stacks of a representative 2-cell female embryo labeled for RNA FISH with an Xic-specific probe (green) and Cot 1 RNA (red). Cot 1 is present at the site of the Xist RNA. (B) Deconvolved images of 3D stacks of a representative 2-cell embryo labeled for DNA FISH. Left: Female embryo with an Xic-chromosome probe (green) and chromosome 8 probe (red). Right: Male embryo with an Xic-chromosome probe (green) and chromosome Y probe (red). Y chromosome is not present in the second polar body. All of the chromosomes territories are dispersed. (C) X-chromosome map with the 18 studied genes. In red, genes that were shown to be fully or partially repressed during spermatogenesis in round spermatids (14); in green, gene that was shown to be reactivated in round spermatids (14); in black, genes that have been not studied in Namekawa and colleagues' report (14). (D) Representative 2-cell female embryos are shown for Xist transcript (green), gene primary transcripts (red), and DAPI (blue). Two gene signals can be seen in each blastomere of 2-cell embryos.

Fig. 2.

Representative genes of a specific profile of transcriptional activity during X-imprinted inactivation assayed by RNA FISH in female embryos. (A) Representative gene silenced from the 8-cell stage. Rnf12 primary transcript signals (red) evidenced by specific RNA FISH at 8-cell (Upper Left), 16-cell (Upper Right), and blastocyst (Bottom Left) stages. One signal, distant from the Xist domain (green), is present in the majority of the cells from 8-cell to blastocyst stages. For each cell stage, a representative blastomere is shown. Bottom Right: Kinetics of expression of the 4 genes Rnf12, Atp7a, Chic1, and Kif4 silenced from the 8-cell stage. (B) Representative gene silenced from the morula stage. G6pdx primary transcripts (red) shown by specific RNA FISH at 8-cell (Upper Left), 16-cell (Upper Right), and blastocyst (Bottom Left) stages. Two G6pdx signals, with 1 within the Xist domain (green), could be seen in the majority of the blastomeres at the 8-cell stage, whereas only 1 signal could be observed in the majority of the blastomeres at the 16-cell and blastocyst stages. For each cell stage, a representative blastomere is shown. Bottom Right: Kinetics of expression of the 3 genes G6pdx, Rps4x, and Fmr1 silenced from the 16-cell stage.

Fig. 3.

Representative genes of a specific profile of transcriptional activity during X-imprinted inactivation assayed by RNA FISH in female embryos. (A) Representative gene silenced at the blastocyst stage. Gla primary transcripts (red) shown by specific RNA FISH at 8-cell (Upper Left), 16-cell (Upper Right), and blastocyst (Bottom Left) stages. Two Gla signals, with 1 within the Xist domain (green), could be seen in the majority of the blastomeres at the 8-cell and 16-cell stages, whereas only 1 signal could be observed in the majority of the blastomeres at the blastocyst stage. For each cell stage, a representative blastomere is shown. Bottom Right: Kinetics of expression of the 6 genes Slc25a5, Lamp2, Atrx, Gla, Fgd1, and Pdha1 silenced from the blastocyst stage. (B) Representative gene escaping to silence. Atp6ap2 primary transcripts (red) shown by specific RNA FISH at 8-cell (Upper Left), 16-cell (Upper Right), and blastocyst (Bottom Left) stages. Two Atp6ap2 signals, with 1 within the Xist domain (green), could be seen in the majority of the blastomeres throughout the preimplantation stages. For each cell stage, a representative blastomere is shown. Bottom Right: Kinetics of expression of the 5 genes Atp6ap2, Utx, Mecp2, Huwe1, and Jarid1c escaping from silencing.

Fig. 4.

Specific profile of Atrx and Huwe1 transcriptional activity during X-imprinted inactivation assayed by RNA FISH in pre- and postimplantation stages. (A) Atrx primary transcript signals (red) using specific RNA FISH at blastocyst stage (Top), in extraembryonic tissues at 5 dpc (Upper Middle), proximal endoderm (Bottom Middle), and extraembryonic ectoderm at 6.5 dpc (Bottom). A loss of Atrx signal from the paternal allele was observed in the trophectoderm at the blastocyst stage. Activity from the paternal allele was observed in extraembryonic tissue as early as 5 dpc, as assessed by the presence of 2 Atrx signals. This pattern was confirmed in extraembryonic tissue at 6.5 dpc. Only 1 signal, distant from the Xist RNA domain, was observed in proximal endodermic cells at 6.5 dpc. (B) Huwe1 primary transcript signals (red) using specific RNA FISH at blastocyst stage (Top), in extraembryonic tissues at 5 dpc (Upper Middle), proximal endoderm (Bottom Middle), and extraembryon or embryon at 6.5 dpc (Bottom). Two signals are present, 1 within the Xist domain (green), at the blastocyst stage. The paternal allele is silenced in the extraembryonic tissues as early as 5 dpc because only 1 signal, near the maternal Xist pinpoint, persists. The same pattern was observed in extraembryonic tissues at 6.5 dpc.