Integrated pseudotime analysis of human preimplantation embryo single-cell transcriptomes reveals the dynamics of lineage specification Graphical abstract Highlights d Distinct trophectoderm/epiblast signatures arise at the B2-B3 blastocyst stages d IFI16 is broadly expressed in the ICM and then restricted to epiblast after implantation d NR2F2 arises from the polar TE in late blastocysts and then spreads to all TE cells

Activation of the ERK signalling pathway is essential for the differentiation of the inner cell mass (ICM) during mouse preimplantation development. We show here that ERK phosphorylation occurs in ICM precursor cells, in differentiated primitive endoderm (PrE) cells as well as in the mature, formative state epiblast (Epi). We further show that DUSP4 and ETV5, factors often involved in negative-feedback loops of the FGF pathway, are differently regulated. Whereas DUSP4 presence clearly depends on ERK phosphorylation in PrE cells, ETV5 localises mainly to Epi cells. Unexpectedly, ETV5 accumulation does not depend on direct activation by ERK but requires NANOG activity. Indeed ETV5, like Fgf4 expression, is not present in Nanog mutant embryos. Our results lead us to propose that in pluripotent early Epi cells, NANOG induces the expression of both Fgf4 and Etv5 to enable the differentiation of neighbouring cells into the PrE while protecting the Epi identity from autocrine signalling.