Donor embryonic stem cells impede host epiblast specification in 8-cell stage chimeras by crowding and FGF4 signalling

During mouse embryo compaction, outer cells become trophectoderm, while inner cells form the inner cell mass (ICM), later differentiating into primitive endoderm and epiblast - source of embryonic stem cells (ESCs) - during blastocyst formation. Trophectoderm specification is driven by position-governed polarisation, while primitive endoderm specification is positively regulated by FGF4 signalling from the ICM and epiblast. When injected into an 8-cell stage morula, ESCs can exclude host cells from the epiblast, leading to mice derived entirely from these cells. While evidence suggests roles for ESC-produced FGF4 and physical crowding in host cell displacement from the ICM, the interplay between these possible mechanisms has yet to be dissected, in part due to the lack of studies using Fgf4-/- ESCs. Here, we combine chimaera titration assays with mathematical modelling to study these mechanisms of host cell displacement. Both Fgf4+/+ and Fgf4-/- ESCs displaced host cells from the epiblast, while only Fgf4-/- injected embryos reduced primitive endoderm and increased trophectoderm, indicating sequential exclusion by displacement crowding followed by FGF4 signalling. SUMMARY STATEMENTIn mouse blastocyst chimeras, donor cells displace host cells from the epiblast by a combination of crowding and FGF4 signalling.