Amniogenesis occurs in two independent waves in primate embryos

In primates the amnion emerges through cavitation of the epiblast during implantation, whereas in other species by folding of the ectoderm later around gastrulation. How the mechanisms of amniogenesis diversified in evolution remains unknown. Here we employed the naïve-to-primed transition of human pluripotent stem cells (hPSC) to model peri-implantation epiblast development. We discovered that during this transition hPSC transiently gain the ability to differentiate into cavitating epithelium that transcriptionally matches the early amnion of human embryos. In contrast, primed hPSC produce cells resembling late amnion, uncovering an independent differentiation route. Unexpectedly, single-cell analysis of primate embryos revealed two transcriptionally distinct amniogenesis waves. The early wave occurs through a trophectoderm-like route and encompasses cavitation, whereas the late wave follows an ectoderm-like pathway during gastrulation. The discovery of two independent waves of amniogenesis both of which can be modelled in hPSC explains how amniogenesis through cavitation emerged during the evolution of primates. (1) Partially primed and primed hPSC were treated with MAPK and NODAL inhibitors for 5 days, then the cells were collected for 10X single-cell RNAseq. (2) Partially primed hPSC were treated with MAPK and NODAL inhibitors for 5 days, the cells were collected on day 0, 1, 3, 5 for bulk RNAseq. Experiment was done in biological duplicates.