Abstract
During peri-implantation development in mice, X chromosome inactivation (XCI) status changes dynamically. Here, we examined the expression of Xist and its antisense partner, Tsix, via whole-mount 3D RNA-FISH using strand-specific probes and evaluated XCI status. The results indicate that Xist expression disappears completely by embryonic day (E) 4.5 without Tsix activation in the ICM and that Xist re-expression occurs at E4.75 in some cells, suggesting that random XCI is already initiated in these cells. Intriguingly, epiblast cells exhibiting biallelic Xist expression were observed frequently (~15%) at E5.25 and E5.5. Immunostaining analysis of epigenetic modifications suggests that global change in epigenomic status occurs concomitantly with the transition from imprinted to random XCI. However, global upregulation of H3K27me3 modifications initiated earlier than other modifications, occurring specifically in ICM during progression of Xist erasure. Although both Xist expression and imprinted XCI are thought to be stable in the primitive endoderm/visceral endoderm and trophectoderm/extraembryonic ectoderm lineages, transient loss of Xist clouds was noted only in a subset of extraembryonic ectodermal cells, suggesting distinct features of Xist regulation among the three different embryonic tissue layers. These results will serve as a basis for future functional studies of XCI regulation in vivo.