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Author(s): Shantha K. Mahadevaiah [sup.1] , Mahesh N. Sangrithi [sup.2] [sup.3] , Takayuki Hirota [sup.1] , James M. A. Turner [sup.1] Author Affiliations: (1) Sex Chromosome Biology Laboratory, The Francis [...], Single-cell RNA sequencing of embryos can resolve the transcriptional landscape of development at unprecedented resolution. To date, single-cell RNA-sequencing studies of mammalian embryos have focused exclusively on eutherian species. Analysis of mammalian outgroups has the potential to identify deeply conserved lineage specification and pluripotency factors, and can extend our understanding of X dosage compensation. Metatherian (marsupial) mammals diverged from eutherians around 160 million years ago. They exhibit distinctive developmental features, including late implantation.sup.1 and imprinted X chromosome inactivation.sup.2, which is associated with expression of the XIST-like noncoding RNA RSX.sup.3. Here we perform a single-cell RNA-sequencing analysis of embryogenesis and X chromosome inactivation in a marsupial, the grey short-tailed opossum (Monodelphis domestica). We resolve the developmental trajectory and transcriptional signatures of the epiblast, primitive endoderm and trophectoderm, and identify deeply conserved lineage-specific markers that pre-date the eutherian-marsupial divergence. RSX coating and inactivation of the X chromosome occurs early and rapidly. This observation supports the hypothesis that--in organisms with early X chromosome inactivation--imprinted X chromosome inactivation prevents biallelic X silencing. We identify XSR, an RSX antisense transcript expressed from the active X chromosome, as a candidate for the regulator of imprinted X chromosome inactivation. Our datasets provide insights into the evolution of mammalian embryogenesis and X dosage compensation. Single-cell RNA-sequencing analysis of embryogenesis and X chromosome inactivation in the opossum (Monodelphis domestica) resolves the developmental trajectory of a marsupial, and sheds light on the evolution of embryogenesis in mammals.