X-chromosome dosage compensation dynamics in human early embryos

In mammals, female cells are obliged to inactivate one of two X chromosomes to achieve dosage parity with the single X chromosome in male cells, and it is also thought that the single active X chromosome is increased 2-fold to achieve dosage balance with two sets of autosomes (X:A ratio = 1, or Ohnos hypothesis). However, the ontogeny of X-chromosome inactivation and augmentation of the single active X remains unclear during human embryogenesis. Here, we perform single-cell RNA-seq analysis to examine the timing of X:A balancing and X-inactivation (XCI) in pre- and peri-implantation human embryos up to fourteen days in culture. We find that X-chromosome gene expression in both male and female preimplantation embryos is approximately balanced with autosomes (X:A ratio = 1) after embryonic genome activation (EGA) and persists through fourteen days in vitro. Cross-species analysis of preimplantation embryo also show balanced X:A ratio within the first few days of development. By single-cell mRNA SNP profiling, we find XCI beginning in day 6-7 blastocyst embryos, but does not affect X:A dosage balance. XCI is most evident in trophoectoderm (TE) cells, but can also be observed in a small number of inner cell mass (ICM)-derived cells including primitive endoderm (PE) and epiblast (EPI) cells. Analysis between individual XaXa and XaXi sister cells from the same embryo reveals random XCI and persistently balanced X:A ratio, including sister cells transitioning between XaXa and XaXi states. We therefore conclude that the male X-chromosome undergoes X chromosome augmentation prior to the simultaneous X-chromosome inactivation and augmentation in females. Together, our data demonstrate an evolutionally conserved model of X chromosome dosage compensation in humans and other mammalian species.