In toto live imaging of Erk signaling dynamics in developing zebrafish hepatocytes

Abstract

Regional and tissue-wide regulation of signaling pathways orchestrates cellular proliferation and differentiation during organ development. Among these pathways, mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (Erk) and c-Jun N-terminal kinase (Jnk) play fundamental roles in coordinating organ morphogenesis by modulating cellular behaviors, including proliferation, differentiation, and migration. Dysregulation of these pathways can result in developmental abnormalities or disease, highlighting the importance of understanding their spatial and temporal activity during development. Traditional biochemical assays provide static snapshots of signaling states but advances in live imaging and biosensor technologies now allow single-cell resolution tracking of pathway activity over extended developmental periods.In this study, we established an imaging platform for the longitudinal analysis of liver development in live zebrafish. We generated hepatocyte-specific transgenic lines expressing kinase translocation reporters (KTRs) for Erk and Jnk signaling, enabling real-time visualization of pathway activity at single-cell resolution. These biosensors allow quantitative assessment of pathway dynamics by tracking nuclear-to-cytoplasmic translocation of fluorescent reporters, providing a robust approach for analyzing signaling events in developing tissues. To complement these biosensors, we integrated neural network-based image segmentation tools such as StarDist to enhance cellular resolution in three-dimensional tissues. These computational methods enable precise identification of individual hepatocytes, allowing for high-fidelity quantification of signaling activity across developmental stages. This framework provides a powerful means to investigate how Erk and Jnk signaling contribute to liver morphogenesis. This work introduces a scalable approach for tracking pathway activity in complex tissues by combining live imaging, genetically encoded biosensors, and deep-learning-based segmentation. Future applications of this methodology could extend to investigating interactions between Erk, Jnk, and other regulatory pathways during organogenesis, advancing our understanding of how signaling networks shape liver development in vertebrates.