Abstract
Female mammals inactivate one of their two X-chromosomes to compensate for the difference in gene-dosage with males that have just one X-chromosome. X-chromosome inactivation is initiated by the expression of the non-coding RNA Xist, which coats the X-chromosome in cis and triggers gene silencing. In early mouse development the paternal X-chromosome is initially inactivated in all cells of cleavage stage embryos (imprinted X-inactivation) followed by reactivation of the inactivated paternal X-chromosome exclusively in the epiblast precursors of blastocysts, resulting temporarily in the presence of two active X-chromosomes in this specific lineage. Shortly thereafter, epiblast cells randomly inactivate either the maternal or the paternal X-chromosome. XCI is accompanied by the accumulation of histone 3 lysine 27 trimethylation (H3K27me3) marks on the condensed X-chromosome. It is still poorly understood how XCI is regulated during early human development. Here we have investigated lineage development and the distribution of H3K27me3 foci in human embryos derived from an in-vitro model for human implantation. In this system, embryos are co-cultured on decidualized endometrial stromal cells up to day 8, which allows the culture period to be extended for an additional two days. We demonstrate that after the co-culture period, the inner cell masses have relatively high cell numbers and that the GATA4-positive hypoblast lineage and OCT4-positive epiblast cell lineage in these embryos have segregated. H3K27me3 foci were observed in ∼25% of the trophectoderm cells and in ∼7.5% of the hypoblast cells, but not in epiblast cells. In contrast with day 8 embryos derived from the co-cultures, foci of H3K27me3 were not observed in embryos at day 5 of development derived from regular IVF-cultures. These findings indicate that the dynamics of H3K27me3 accumulation on the X-chromosome in human development is regulated in a lineage specific fashion.
Highlights
X-chromosome inactivation (XCI) is a complex process whereby one of the two X-chromosomes in female cells is epigenetically silenced, to obtain similar X-linked gene expression levels as male cells, which have just one X-chromosome
Using immunofluorescence for OCT4 and GATA4 we show that these markers are initially co-expressed in all cells of embryos at day 5 of development, but after 72 h of co-culture expression of OCT4 is restricted to the epiblast, GATA4 expression is restricted to the hypoblast and trophectoderm lineages are OCT4- and GATA4-negative
In embryos cultured on decidualized endometrial cells for 72 h, double-immunofluorescence demonstrated the presence of an OCT4-positive cell population covered by a layer of GATA4-positive hypoblast precursors
Summary
X-chromosome inactivation (XCI) is a complex process whereby one of the two X-chromosomes in female cells is epigenetically silenced, to obtain similar X-linked gene expression levels as male cells, which have just one X-chromosome. Xist recruits the polycomb group complex PRC2 to the X-chromosome that subsequently tri-methylates histone 3 on lysine 27 (H3K27me3), a repressive epigenetic mark that leads to further silencing of the X-chromosome [3]. The activity status of the X-chromosomes in pre- and postimplantation development of female mouse embryos is dynamically regulated. The paternal X-chromosome remains inactive in the trophectoderm and the primitive endoderm precursors of blastocyst stage mouse embryos (imprinted XCI), but is reactivated in the epiblast precursors of the ICM between E3.5–E4.5, resulting temporarily in two active X-chromosomes (XaXa) [5,6,7,8,9,10,11,12]. One of the active X-chromosomes is randomly inactivated and the resulting pattern of X-chromosome activity is epigenetically transmitted to all daughter cells [10,11]
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