Insulin fine-tunes self-renewal pathways governing naive pluripotency and extra-embryonic endoderm.

Signalling downstream of Activin/Nodal (ActA) and Wnt can induce endoderm differentiation and also support self-renewal in pluripotent cells. Here we find that these apparently contradictory activities are fine-tuned by insulin. In the absence of insulin, the combination of these cytokines supports endoderm in a context-dependent manner. When applied to naive pluripotent cells that resemble peri-implantation embryos, ActA and Wnt induce extra-embryonic primitive endoderm (PrE), whereas when applied to primed pluripotent epiblast stem cells (EpiSC), these cytokines induce gastrulation-stage embryonic definitive endoderm. In naive embryonic stem cell culture, we find that insulin complements LIF signalling to support self-renewal; however, when it is removed, LIF, ActA and Wnt signalling not only induce PrE differentiation, but also support its expansion. Self-renewal of these PrE cultures is robust and, on the basis of gene expression, these cells resemble early blastocyst-stage PrE, a naive endoderm state able to make both visceral and parietal endoderm.

Polycomb enables primitive endoderm lineage priming in embryonic stem cells.

Mouse embryonic stem cells (ESCs), like the blastocyst from which they are derived, contain precursors of the epiblast (Epi) and primitive endoderm (PrEn) lineages. While transient in vivo, these precursor populations readily interconvert in vitro. We show that altered transcription is the driver of these coordinated changes, known as lineage priming, in a process that exploits novel polycomb activities. We find that intragenic levels of the polycomb mark H3K27me3 anti-correlate with changes in transcription, irrespective of the gene's developmental trajectory or identity as a polycomb target. In contrast, promoter proximal H3K27me3 is markedly higher for PrEn priming genes. Consequently, depletion of this modification stimulates the degree to which ESCs are primed towards PrEn when challenged to differentiate, but has little effect on gene expression in self-renewing ESC culture. These observations link polycomb with dynamic changes in transcription and stalled lineage commitment, allowing cells to explore alternative choices prior to a definitive decision.