Abstract
Embryonic Stem Cell (ESC) differentiation requires complex cell signalling network dynamics, although the key molecular events remain poorly understood. Here, we use phosphoproteomics to identify an FGF4-mediated phosphorylation switch centred upon the key Ephrin receptor EPHA2 in differentiating ESCs. We show that EPHA2 maintains pluripotency and restrains commitment by antagonising ERK1/2 signalling. Upon ESC differentiation, FGF4 utilises a bimodal strategy to disable EPHA2, which is accompanied by transcriptional induction of EFN ligands. Mechanistically, FGF4-ERK1/2-RSK signalling inhibits EPHA2 via Ser/Thr phosphorylation, whilst FGF4-ERK1/2 disrupts a core pluripotency transcriptional circuit required for Epha2 gene expression. This system also operates in mouse and human embryos, where EPHA receptors are enriched in pluripotent cells whilst surrounding lineage-specified trophectoderm expresses EFNA ligands. Our data provide insight into function and regulation of EPH-EFN signalling in ESCs, and suggest that segregated EPH-EFN expression coordinates cell fate with compartmentalisation during early embryonic development.
Highlights
Embryonic Stem Cell (ESC) differentiation requires complex cell signalling network dynamics, the key molecular events remain poorly understood
In order to address this in a systematic, unbiased way, we examine the global phosphoproteomic signature associated with mouse ESC (mESC) differentiating in response to fibroblast growth factor 4 (FGF4)
Our findings indicate that FGF4 extensively rewires phosphorylation networks beyond previously known pathways and targets, suggesting that many hitherto unappreciated phosphorylation events may be key regulators of mESC differentiation
Summary
Embryonic Stem Cell (ESC) differentiation requires complex cell signalling network dynamics, the key molecular events remain poorly understood. FGF4-ERK1/2-RSK signalling inhibits EPHA2 via Ser/Thr phosphorylation, whilst FGF4-ERK1/2 disrupts a core pluripotency transcriptional circuit required for Epha[2] gene expression. This system operates in mouse and human embryos, where EPHA receptors are enriched in pluripotent cells whilst surrounding lineagespecified trophectoderm expresses EFNA ligands. We identify the EPHA2 receptor tyrosine kinase as a critical target of the FGF4 signalling network in ESCs. EPH receptors engage transmembrane Ephrin ligands (EFNs)[11] to drive segregation of EPH- and EFN- expressing cell populations[12], and demarcation of distinct cellular compartments[13]. A-type EPH receptors are expressed in the pluripotent compartment of both human and mouse embryos, whilst the first specified lineage, trophectoderm, is enriched for Efna1/EFNA1, suggesting that segregated expression of A-type EPH receptors and EFN ligands plays a role in pluripotency maintenance during early embryonic development
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