In vitro capture and characterization of embryonic rosette-stage pluripotency between naive and primed states

Upon implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked, and whether intermediate pluripotent states exist remain controversial. We identify here a rosette pluripotent state, defined by co-expression of naive factors with transcription factor OTX2. Downregulation of blastocyst WNT signals drives transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells (RSCs), a self-renewing naive-primed intermediate. RSCs erase constitutive heterochromatin marks and display a primed chromatin landscape, with bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate where control over both pluripotency progression and morphogenesis pivots from WNT to MEK signals.