Human hypoblast formation is not dependent on FGF signalling.
Bottom Line: These differentiation processes are associated with restricted expression of key transcription factors (Cdx2, Oct4, Nanog and Gata6).However, the formation of hypoblast in the human is apparently not dependent upon FGF signalling, in contrast to rodent embryos.Nonetheless, the persistence of Nanog-positive cells in embryos following treatment with FGF inhibitors is suggestive of a transient naïve pluripotent population in the human blastocyst, which may be similar to rodent epiblast and ES cells but is not sustained during conventional human ES cell derivation protocols.
Affiliation: Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge CB2 1QR, UK.Show MeSH
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Mentions: FGF/Mek inhibition in mouse preimplantation embryos has a striking effect on lineage segregation (Chazaud et al., 2006; Nichols et al., 2009; Yamanaka et al., 2010). Inhibition of FGF signalling diverts all cells of the ICM to the epiblast fate, bypassing the hypoblast. We were interested in investigating whether the formation of human hypoblast is also dependent on FGF signalling. If so, this would indicate that the segregation of these lineages may be based on a conserved mechanism. We performed experiments with human embryos, adding inhibitors from the 6–8 cell stage. 1 μM PD0325901 is effective to block the formation of hypoblast in murine embryos if applied before its segregation (Nichols et al., 2009). However, all human embryos treated with 1 μM PD0325901 that developed to late blastocysts showed Gata4 expression in a subset of cells, separate from the Nanog-expressing population (Fig. 2A, D). To test whether an alternative downstream pathway for FGF signalling is adopted during human hypoblast induction, we introduced an inhibitor of the FGF receptor, PD173074, in combination with PD0325901. Using this combination, Gata4-positive cells were still identified in all the human embryos investigated (Fig. 2C, D). We examined whether PD0325901 could work synergistically with Chir99021, since these are the components of 2i that effectively maintain naïve pluripotency in cultures of murine ES cells (Ying et al., 2008). Gata4-positive cells were still observed in embryos developed under these conditions (Fig. 2B, D). In all three conditions that target FGF/Erk signalling, human embryos possess Gata4 and Nanog-positive cells that are exclusive to one another. Thus, the segregation of epiblast and hypoblast within the human embryo appears not to be dependent on FGF signalling.
Affiliation: Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge CB2 1QR, UK.