ERK signalling orchestrates metachronous transition from naïve to formative pluripotency

Abstract

Naive epiblast cells in the embryo and pluripotent stem cells in vitro undergo developmental progression to a formative state competent for lineage specification. During this transition, transcription factors and chromatin are rewired to encode new functional features. Here, we examine the role of mitogen-activated protein kinase (ERK1/2) signalling in pluripotent state transition. We show that a primary consequence of ERK activation in mouse embryonic stem cells is elimination of Nanog, precipitating breakdown of the naive state gene regulatory network. Cell variability in pERK dynamics results in metachronous down-regulation of Nanog and naive state exit. Knockdown of Nanog allows exit without ERK activation. However, transition to formative pluripotency does not proceed and cells collapse to an indeterminate identity. This failure is attributable to loss of expression of the central pluripotency factor Oct4. Thus, during formative transition ERK signalling both dismantles the naive state and preserves pluripotency. These results illustrate that a single signalling pathway can both drive exit from a developmental state and safeguard progression to the successor state.

Control of cell state transitions by post-transcriptional regulation

“…We inhibited protein synthesis and determined the half-life of the fusion reporter to be 3.02 h in mouse embryonic stem cells which is in general agreement with other protein-fusion reporter systems [31]. Next, we used bioluminescence live imaging [32] to measure the dynamics of Nanog downregulation during differentiation [26]. Surprisingly, we saw that once Nanog started to royalsocietypublishing.org/journal/rstb Phil.…”

“…To ground the model within biologically plausible parameters, I have populated the model using rate constants for the transcription factor Nanog ( figure 1 a ). Nanog clearance is the rate limiting factor during exit from the embryonic stem cell state in mouse [ 26 ] and overexpression significantly delays/impairs the transition to a differentiated state [ 27 , 28 ]. Therefore, it is likely that the rate parameters of Nanog are optimized for transitions and are a good starting point.…”

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Figure 1 ( a ) Mathematical model showing the key rate parameters controlling the expression of a cell state determinant. The following parameters were used for the simulations: initial Nanog protein levels = 1000 molecules (general number of transcription factor molecules per cell from Biggin [ 23 ]), initial Nanog mRNA levels = 240 molecules [ 24 ] , Nanog k mRNA = 0.19 molecules min −1 (average transcription rate calculated from: mean number of mRNA produced during an ‘ON’ period (=123 molecules) / mean ‘ON’ duration (144 min) * fraction of time ‘ON’ (0.22)—from Skinner et al [ 25 ]), Nanog δ mRNA = 0.0022 molecules min −1 [ 25 ], k prot = 0.91 molecules min −1 (estimated from the rate of protein translation in mouse embryonic stem cells from Ingolia et al [ 18 ]), Nanog δ prot = 0.0038 molecules min −1 (experimentally determined, [ 26 ]). ( b ) Simulation of the number of protein molecules of a cell state determinant over time when all rates are constant.

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“…We also examined the dynamics of Nanog protein downregulation using a protein fusion reporter [ 26 ]. In this case, the sequence of a bioluminescent reporter is inserted in-frame with the Nanog coding sequence before the stop codon, meaning it should reflect both the transcriptional and post-transcriptional regulation on Nanog ( figure 1 f ).…”

“…We inhibited protein synthesis and determined the half-life of the fusion reporter to be 3.02 h in mouse embryonic stem cells which is in general agreement with other protein-fusion reporter systems [ 31 ]. Next, we used bioluminescence live imaging [ 32 ] to measure the dynamics of Nanog downregulation during differentiation [ 26 ]. Surprisingly, we saw that once Nanog started to be downregulated, it reached 50% of its original fluorescent levels in approximately 3.2 h. This means that Nanog downregulation is occurring much closer to the rate limit (given by the protein half-life), compared to what is observed for a purely transcriptional reporter like RGd2.…”