Live ERK-activity sensors revealed that FGF4 signaling in mouse embryonic stem cells (ESCs) produces intermittent ERK oscillations: single cells alternate between silent epochs and trains of pulses with a modal inter-pulse interval of roughly 7 minutes, the fraction of time spent pulsing rises with FGF4 dose, pulsing decays across the cell cycle, and oscillations are less prevalent in more differentiated epiblast-like stem cells. Raina and colleagues interpreted these statistics as evidence that the FGF/ERK system operates near a transition between non-oscillatory and oscillatory regimes, and left four open questions arising from these results: which feedback loops and molecular components position the system near this transition and set the dose-dependence of oscillation prevalence and precision; what biophysical mechanism underlies the cell-cycle dependence of pulsing; which statistic of the ERK dynamics encodes extracellular FGF4 concentration; and how oscillatory versus silent signaling epochs correlate with transcriptional states and relate to lineage choice. Later work has supplied partial handles - Raf dependence of pulse generation, a NANOG-pERK feedback circuit, optogenetic (light-controlled stimulation) evidence that integrated ERK activity rather than dynamics type controls exit from pluripotency, and mitotic erasure of ERK activity in embryos - but none of the four questions is resolved for ESCs, and the problem remains open.

Background

The extracellular signal-regulated kinase (ERK) pathway is a mitogen-activated protein kinase (MAPK) cascade in which Ras activates the kinase Raf, which activates MEK, which activates ERK; ERK then phosphorylates nuclear and cytoplasmic targets to control proliferation and differentiation. In mouse embryonic stem cells (ESCs), which are pluripotent cells derived from the early embryo, signaling through fibroblast growth factor 4 (FGF4) to ERK is required for cells to exit naive pluripotency and start differentiating. FGF4 is secreted by the ESCs themselves, so the cells signal to each other in a paracrine loop, and the FGF4 concentration a cell experiences emerges from this cell-community behavior rather than from a fixed external stimulus.

Much of what is known about ERK activity in individual cells comes from genetically encoded live reporters. The ERK kinase translocation reporter (ERK-KTR) is a fluorescent ERK substrate that shuttles between nucleus and cytoplasm in proportion to ERK activity, so the ratio of cytoplasmic to nuclear fluorescence gives a readout of ERK activity every few minutes in single living cells. Earlier live-imaging work in differentiating ESCs using FRET (Förster resonance energy transfer, a technique in which one fluorescent protein transfers energy to another in a distance-dependent manner, so that the ratio of the two emission channels reports the activity of the labeled sensor) had found ERK activity to be highly heterogeneous between cells, with dynamic features surprisingly weakly coupled to the loss of pluripotency markers (Deathridge et al. 2019), and ERK dynamics in the preimplantation embryo were shown to be cell-cycle dependent (Pokrass et al. 2020). Separately, Raina and colleagues had shown that FGF4-mediated cell-cell communication generates and maintains robust proportions of differentiated cell types in ESC colonies (Raina et al. 2021).

Raina et al. (2022) combined the ERK-KTR sensor with high time-resolution recording in wild-type and Fgf4-mutant ESCs. They found that ERK activity occurs in short pulses with a modal inter-pulse interval (IPI, the time between consecutive pulse peaks) of roughly 7 minutes. Cells were heterogeneous: in serum plus LIF (leukemia inhibitory factor, a cytokine that supports ESC self-renewal) cells pulsed on average about 32% of the time, some continuously, some alternating between pulsing and silent epochs, some not at all. Simple stochastic pulsing models, in which pulses occur with exponential waiting times, failed to reproduce the data - real cells showed too many long trains of consecutive pulses - so the authors interpreted the dynamics as intermittent oscillations, with single cells transitioning between silent and oscillatory states. Fgf4-mutant cells showed no pulsing, and recombinant FGF4 restored it; individual pulse duration was dose-independent, but the fraction of time pulsing and the pulse rate (the number of pulses per unit total recording time) increased with FGF4 concentration over the 0-5 ng/ml range - reflecting that cells spend more of each recording pulsing, since the modal IPI remained about 7 minutes in all conditions - while the regularity of the IPI distribution and the fraction of consecutive pulses increased across the full dose range tested (consecutive-pulse pairs rose from 49.2% at 2.5 ng/ml to 57.6% at 5 ng/ml and 63.9% at 20 ng/ml). Pulsing decayed across the cell cycle after cell birth, and oscillations were less prevalent in epiblast stem cells (EpiSCs), a more differentiated pluripotent state. Because ESC populations contain a mixture of oscillating and non-oscillating cells at all ligand levels, the authors proposed that the FGF/ERK system is organized in the vicinity of a transition point between a non-oscillatory and an oscillatory state, with higher FGF4 pushing the system closer to that point.

In other mammalian cell types, ERK pulsing is often stochastic and its frequency increases with ligand level, which has been interpreted as frequency-modulated encoding of ligand concentration. Raina et al. (2022) argued that this encoding model is unlikely to apply in ESCs, because their pulse statistics deviate from stochastic pulsing and because the within-train oscillation frequency (set by the IPI, whose modal value stays near 7 minutes at all tested FGF4 concentrations) is comparatively insensitive to dose while the prevalence of oscillation is dose-sensitive. This observation frames the unresolved question of how, in ESCs, extracellular FGF4 concentration is actually represented in the ERK dynamics - for instance by the fraction of time a cell spends pulsing, by the population-level mixture of oscillatory and non-oscillatory cells, or by some other statistic.

Raina et al. (2022) also set out the mechanistic questions left open by these measurements. The feedback loops and molecular components that position the FGF/ERK system near the oscillatory transition remain to be identified, with receptor-level delayed feedbacks, negative feedbacks within the RAF-MEK-ERK cascade, and modulators of feedback strength among the mechanisms considered. The biophysical basis of the cell-cycle decay of pulsing is likewise undetermined, with changes in surface-to-volume ratio, receptor abundance, and cell-cycle-dependent expression of pathway regulators among the possibilities raised. The causal link between pulsatile signaling and lineage outcomes is also unresolved; the source framed the test of whether oscillatory versus silent epochs bias lineage choice as relating single-cell signaling time series to contemporaneous transcriptional state trajectories and eventual differentiation fates.

Problem Statement

Explain the mechanism and function of intermittent FGF4/ERK oscillations in mouse embryonic stem cells, following the open questions arising from the results of Raina et al. (2022). The problem has four linked parts that jointly support one objective - a causal account of how these oscillations arise, how they depend on ligand and cell state, and what they do:

  1. Feedback circuitry and the oscillatory transition. Identify the molecular feedback loops and components that position the FGF/ERK signal-transduction system in ESCs in the vicinity of a transition between non-oscillatory and oscillatory dynamics; determine the nature of the non-oscillatory state and the type of transition (for example whether isolated pulses are short excursions of an oscillatory system or the output of a distinct excitable regime); and establish which components set the observed dose-dependence of oscillation prevalence and precision - that is, of the dose-dependent oscillation statistics described in the Background, such as the fraction of a cell's time spent pulsing and the regularity of the inter-pulse-interval distribution.

  2. Cell-cycle basis. Determine the biophysical mechanism by which ERK pulsing decays across the cell cycle.

  3. Ligand encoding. Determine how extracellular FGF4 concentration is represented in the ERK dynamics in ESCs - whether by the fraction of time a cell spends pulsing, by population-level mixture statistics of oscillatory and non-oscillatory cells, or by other features - given that the frequency-modulation encoding described for stochastic ERK pulsing in other cell types is unlikely to apply.

  4. Functional consequence. Discern how oscillatory versus silent signaling epochs are interlinked with transcriptional network states and lineage outcomes.

References

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