Detailed epigenome map of embryonic development in the fat-tailed dunnart (Sminthopsis crassicaudata)

DNA methylation (5mC) is an epigenetic mark that plays a critical role in defining cell fate. In eutherian mammals such as mouse and human, DNA methylation inherited from parental gametes is rapidly erased in the early embryo. Intriguingly, 5mC erasure is not conserved across all vertebrates, with non-mammalian vertebrates such as zebrafish inheriting paternal DNA methylation patterns. It remains unclear whether genome-wide erasure of 5mC is unique to eutherians or more broadly shared across mammals, including metatherians (marsupials), the vast majority of which are concentrated on the Australian continent. To address this outstanding knowledge gap of whether Australian marsupials undergo eutherian-like 5mC reprogramming, we generated a chromosome-level genome assembly and high-resolution epigenome maps of embryonic development in the fat-tailed dunnart Sminthopsis crassicaudata. Similar to what has previously been reported in the South American opossum Monodelphis domestica, the dunnart retains a hypermethylated genome in the epiblast, indicating a conserved lack of extensive embryonic 5mC erasure. However, we identified striking global loss of 5mC in the trophectoderm, revealing species-specific usage of 5mC in the regulation and development of extraembryonic tissues. Additionally, chromosome-level assembly of the dunnart genome revealed widespread hypomethylation of the inactive, paternal X chromosome in female dunnart and identified the master regulator of X chromosome inactivation, lncRNA Rsx previously unannotated in the dunnart's genome. Taken together, these insights advance our understanding of the evolution of epigenome remodeling and illustrate how genome regulation shapes diverse developmental strategies across vertebrates.