Cross-activation of FGF, NODAL, and WNT pathways constrains BMP-signaling-mediated induction of the totipotent state in mouse embryonic stem cells

Embryonic stem cells (ESCs) are an attractive model to study the relationship between signaling and cell fates. Cultured mouse ESCs can exist in multiple states resembling distinct stages of early embryogenesis, such as totipotent, pluripotent, primed, and primitive endoderm. The signaling mechanisms regulating the totipotent state and coexistence of these states are poorly understood. Here we identify bone morphogenetic protein (BMP) signaling as an inducer of the totipotent state. However, we discover that BMP's role is con-strained by the cross-activation of FGF, NODAL, and WNT pathways. We exploit this finding to enhance the proportion of totipotent cells by rationally inhibiting the cross-activated pathways. Single-cell mRNA sequencing reveals that induction of the totipotent state is accompanied by suppression of primed and prim-itive endoderm states. Furthermore, reprogrammed totipotent cells we generate in culture resemble totipo-tent cells of preimplantation embryo. Our findings reveal a BMP signaling mechanism regulating both the totipotent state and heterogeneity of ESCs.