Random X-chromosome inactivation dynamics in vivo by single-cell RNA sequencing

Random X-chromosome inactivation (rXCI) is important for the maintenance of normal somatic cell functions in female eutherian mammals. The dynamics of X-chromosome inactivation initiation has been widely studied by assessing embryonic stem cell differentiation in vitro. To investigate the phenomenon in vivo, we applied RNA sequencing to single cells from female embryos obtained from a natural intercrossing of two genetically distant mouse strains. Instead of artificially assigning the parental origin of the inactive X chromosome, the inactive X chromosomes in this study were randomly selected from the natural developmental periods and thus included both paternal and maternal origins. The rXCI stages of single cells from the same developmental stage showed heterogeneity. The high resolution of the rXCI dynamics was exhibited. The inactivation orders of X chromosomal genes were determined by their functions, expression levels, and locations; generally, the inactivation order did not exhibit a parental origin preference. New escape genes were identified. Ohno's hypothesis of dosage compensation was refuted by our post-implantation stage data. We found the inactivation orders of X chromosomal genes were determined by their own properties. Generally, the inactivation order did not exhibit a parental origin preference. It provided insights into the gene silencing dynamics during rXCI in vivo.