Intermittent ERK oscillations downstream of FGF in mouse embryonic stem cells

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 a 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.

信号转导网络可产生特征性动态活性，以处理胞外信号并指导细胞命运决定，如细胞增殖或分化。多能细胞的分化受成纤维细胞生长因子/细胞外调节蛋白激酶（FGF/ERK）信号通路调控。然而，目前仅有少数研究以高时间分辨率探究了多能细胞中FGF/ERK信号转导网络的动态活性。本研究借助野生型及Fgf4敲除小鼠胚胎干细胞的活细胞传感器，在确定的配体浓度与分化状态下，检测单细胞内的ERK动态活性。

实验结果显示ERK活性存在脉冲式波动，且不同细胞间的脉冲模式存在异质性。连续脉冲序列的出现频率高于简单随机模型的预测结果。配体浓度越高，连续脉冲序列越常见；而在分化程度更高的细胞中，此类序列则更为罕见。本研究结果表明，胚胎干细胞中的FGF/ERK信号通路处于振荡与非振荡动态行为之间的转变点附近。由此产生的异质性动态信号活性为细胞异质性增添了新维度，这可能与干细胞培养体系中不同的细胞命运决定相关。

实验方法
活细胞成像
将表达ERK激酶转位报告基因（ERK-KTR）的细胞接种于ibidi µ-slide培养皿中，使用配备孵育腔与CO₂供气系统的Leica SP8共聚焦显微镜进行成像，维持培养环境温度37℃、CO₂浓度5%、相对湿度80%。成像前4小时，加入活细胞核染料SiR-Hoechst 652/674（Spirochrome）以辅助细胞追踪：短期延时成像实验中，该染料终浓度为500 nM；长期延时成像实验中终浓度为250 nM。使用白光激光器（Leica）的504 nm波长激发荧光基团，通过63× 1.4数值孔径的油镜同时采集KTR-Clover与核标记物的图像。
对于短期（<4小时）成像实验，每20秒采集单帧图像，XY分辨率为0.251 µm，像素驻留时间为2.6 μs，针孔光阑设为2.4艾里单位。对于长期（约19小时）成像实验，为减少整体光暴露，每105秒采集单帧图像，XY分辨率为0.401 µm，像素驻留时间为3.1 μs，针孔光阑设为2.6艾里单位。