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Data related to: Intermittent ERK oscillations downstream of FGF in mouse embryonic stem cells

Citation

Schröter, Christian; Raina, Dhruv (2022), Data related to: Intermittent ERK oscillations downstream of FGF in mouse embryonic stem cells, Dryad, Dataset, https://doi.org/10.5061/dryad.66t1g1k3q

Abstract

Signal transduction networks generate characteristic dynamic activities to process extracellular signals and guide cell fate decisions such as to divide or differentiate. The differentiation of pluripotent cells is controlled by FGF/ERK signaling. However, only few studies have addressed the dynamic activity of the FGF/ERK signaling network in pluripotent cells at high time resolution. Here we use live cell sensors in wild-type and Fgf4-mutant mouse embryonic stem cells to measure dynamic ERK activity in single cells, for defined ligand concentrations and differentiation states. These sensors reveal pulses of ERK activity. Pulsing patterns are heterogeneous between individual cells. Consecutive pulse sequences occur more frequently than expected from simple stochastic models. Sequences become more prevalent with higher ligand concentration, but are rarer in more differentiated cells. Our results suggest that FGF/ERK signaling operates in the vicinity of a transition point between oscillatory and non-oscillatory dynamics in embryonic stem cells. The resulting heterogeneous dynamic signaling activities add a new dimension to cellular heterogeneity that may be linked to divergent fate decisions in stem cell cultures.

Methods

Live cell imaging

ERK-KTR expressing cells were cultured on ibidi µ-slides, and imaged on a Leica SP8 confocal microscope equipped with an incubation chamber and CO2 supply to maintain temperature at 37°C, CO2 at 5%, and relative humidity at 80%. 4 h before acquisition, live-cell nuclear dye SiR-Hoechst 652/674 (Spirochrome) was added to facilitate tracking of cells. SiR-Hoechst was added at a final concentration of 500 nM for short-term time-lapse experiments, and 250nM for long-term time-lapse experiments. Fluorophores were excited with a 504nm line from a white-light laser (Leica), and images of the KTR-Clover and the nuclear marker were simultaneously captured through a 63x 1.4 N.A. oil objective. For short-term (<4 h) imaging experiments, single frames were acquired once every 20 s, with an XY resolution of 0.251 µm, with a pixel dwell time of 2.6 μs, and a pinhole of 2.4 airy units. For long term (~19 h) imaging experiments, to minimize overall light exposure single frames were acquired once every 105 s, with an XY resolution of 0.401 µm, with a pixel dwell time of 3.1 μs, and a pinhole of 2.6 airy units.

Funding

Max-Planck-Gesellschaft