And we’re live! – A real time readout of Erk signaling dynamics in zebrafish
Summary by Will Anderson: Wilcockson, S. G., Guglielmi, L., Rodriguez, P.A., Amoyel, M., & Hill, C.S. (2022). "An improved Erk biosensor reveals oscillatory Erk dynamics driven by mitotic erasure during early development." bioRxiv. 10.1101/2022.11.03.515001
Image credit: Midjourney
To make an adult organism with a diverse range of organs and tissues, developing embryos must coordinate the differentiation of unspecified cells into distinct and functional types. At the heart of this differentiation are signaling pathways and their constituents, such as the FGF/Erk pathway. Experiments manipulating these pathways have shown that perturbations can drastically alter the developmental trajectories of cells, leading to patterning defects and death in extreme cases (Rogers and Schier 2011). Visualizing and decoding pathway activity is a key component of understanding embryonic development.
To transmit “signal” from FGF ligand, the protein Erk is phosphorylated to become “P-Erk”, which subsequently enters the nucleus and affects the transcription of specific genes (Wen et al. 2022). Researchers can capitalize on this phosphorylation-mediated Erk activation to visualize active Erk signaling. Previously, Regot et al. developed a fluorescent visualization tool called “Erk-Kinase Translocation Reporter” (Erk-KTR) that will localize to the nucleus by default but will translocate to the cytoplasm upon Erk phosphorylation (Regot et al. 2014). Thus, as more Erk enters the active P-Erk state, cellular fluorescence will change from nuclear to cytoplasmic, providing a readout of activation: the “cytoplasmic to nuclear ratio” of each cell’s fluorescence, or “C/N”, which increases as P-Erk increases.
Here, Wilcockson et al. observed that while Erk-KTR is responsive to Erk activation, it is also sensitive to the activity of Cdk1, a cell cycle protein unrelated to Erk signaling (Wilcockson et al 2022). This additional sensitivity can obscure the readout of Erk signaling, so the group created a modified version of the tool called modErk-KTR. modErk-KTR contains two important changes from its predecessor: first, the authors changed an amino acid in the part of the tool suspected to be sensitive to Cdk1 in order to prevent Cdk1 from affecting the tool’s activity. Then, because this change might cause a decrease in the tool’s Erk sensitivity, they added a second Erk binding site to the sequence to compensate. Through fluorescence imaging experiments comparing the C/N dynamics of the two tools in zebrafish, the authors demonstrated that modErk-KTR has no Cdk1 responsiveness, without compromising P-Erk sensitivity.
During their evaluation of modErk-KTR in zebrafish embryos, the authors discovered that cells approaching mitosis display a rapid decrease in their C/N fluorescence, indicating a rapid decrease in Erk signaling leading up to cell division. They termed this phenomenon “mitotic erasure”. After division and mitotic erasure, resulting daughter cells took variable lengths of time to return to previous levels of P-Erk activity. The final levels that each daughter cell returned to also varied. These findings not only demonstrate the usefulness of a live readout of Erk signaling, but also highlight an avenue for exploring an interesting hypothesis: to change from a group of undifferentiated cells into a diverse set of tissues, an organism needs sources of heterogeneity, and it is possible that the variability in Erk dynamics during and after mitotic erasure provides one such source.
Despite some limitations, modErk-KTR improves upon its predecessor and serves as a useful tool for obtaining insights about the developmental dynamics of zebrafish.
Rogers, K. W. and A. F. Schier (2011). "Morphogen gradients: from generation to interpretation." Annual Rev Cell Dev Biol 27: 377-407. 10.1146/annurev-cellbio-092910-154148
Wen, X., et al. (2022). "MAPK/ERK Pathway as a Central Regulator in Vertebrate Organ Regeneration." Int J Mol Sci 23(3). 10.3390/ijms23031464
Regot, S., et al. (2014). "High-sensitivity measurements of multiple kinase activities in live single cells." Cell 157(7): 1724-1734. 10.1016/j.cell.2014.04.039
Wilcockson, S. G., et al. (2022). "An improved Erk biosensor reveals oscillatory Erk dynamics driven by mitotic erasure during early development." bioRxiv. 10.1101/2022.11.03.515001