Regulation of early trophoblast differentiation – Lessons from the mouse

Transcriptional regulation of trophoblast lineage identity. (A) The interaction of key trophoblast transcription factors can be divided into three distinct stages. Tead4 triggers, directly or indirectly, the expression of Cdx2 and other transcription factors . Once specified, a positive feedback loop involving Cdx2, Eomes, Tcfap2c, and Elf5 reinforces trophoblast identity . In addition to supporting this network, Gata3, Elf5 and Ets2 subsequently act to drive further differentiation of the lineage into different placental cell types . The dashed lines indicate presumptive, albeit not yet directly proven, interactions between Eomes, Gata3, Elf5 and Ets2 with Tcfap2c. Eomes can be activated directly by Elf5 and Tcfap2c, and directly or indirectly by Cdx2; the effect of other transcription factors on Eomes is yet to be tested . (B) Timing of expression of key trophoblast transcription factors throughout trophoblast differentiation, and (C) pattern of expression in the early post-implantation (E6.0–E6.5) mouse embryo, as indicated by grey shadings. Tead4 is the earliest gene detected at the 4-cell stage by RT-PCR; Cdx2, Tcfap2c and Gata3 are expressed from around the 8-cell stage onwards , while Eomes is detected only after the 16-cell stage and co-localises with the other transcription factors in the trophectoderm of the blastocyst [12]. Ets2 and Elf5 (mRNA and/or protein) are not detected at the early blastocyst stage, but are activated around E4.5–E5.0 and are expressed in the trophoblast compartment immediately after implantation . Co-expression of these transcription factors in the proximal ExE defines the niche of trophoblast cells with stem cell potential. E = embryo; ExE = extraembryonic ectoderm; EPC = ectoplacental cone.

Signalling pathways in pre-implantation embryos determine cell fate. Differential activity of the Ras and Hippo signalling pathways determines lineage segregation in the pre-implantation embryo. Circumferential cell–cell contacts activate the Hippo pathway in the cells located in the interior of the embryo. The transcriptional co-activator Yap becomes phosphorylated and is sequestered in the cytoplasm. Conversely, in exterior cells the Hippo pathway is not activated. Yap is free to translocate to the nucleus where it transactivates Tead4-mediated transcription of key trophoblast transcription factors directly or indirectly . In presumptive embryonic cells, Nanog and Oct4 maintain repression of the trophoblast transcriptional programme in conjunction with epigenetic modifiers like Eset , and Elf5 is methylated and repressed [21]. In presumptive trophectoderm cells Cdx2 is activated by Tead4/Yap and in turn induces a self-reinforcing trophoblast-defining transcriptional circuit. Ras–Mapk signalling in the outside cells is also crucial for trophoblast specification and/or integrity [43] but the precise mechanism has yet to be elucidated.