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Epithelial morphogenesis is guided by mechanical forces and biochemical signals that vary spatiotemporally. As many morphogenetic events are driven by rapid cellular processes, understanding morphogenesis requires monitoring development in real time. Here, we discuss how live-imaging approaches can help identify morphogenetic mechanisms otherwise missed in static snapshots of development. We begin with a summary of live-imaging strategies, including recent advances that push the limits of spatiotemporal resolution and specimen size. We then describe recent efforts that employ live imaging to uncover morphogenetic mechanisms. We conclude by discussing how information collected from live imaging can be enhanced by genetically-encoded biosensors and spatiotemporal perturbation techniques to determine the dynamics of patterning of developmental signals and their importance for guiding morphogenesis.

Receptor tyrosine kinases (RTKs) are major signaling hubs in metazoans, playing crucial roles in cell proliferation, migration, and differentiation. However, few tools are available to measure the activity of a specific RTK in individual living cells. Here, we present pYtags, a modular approach for monitoring the activity of a user-defined RTK by live-cell microscopy. pYtags consist of an RTK modified with a tyrosine activation motif that, when phosphorylated, recruits a fluorescently labeled tandem SH2 domain with high specificity. We show that pYtags enable the monitoring of a specific RTK on seconds-to-minutes time scales and across subcellular and multicellular length scales. Using a pYtag biosensor for epidermal growth factor receptor (EGFR), we quantitively characterize how signaling dynamics vary with the identity and dose of activating ligand. We show that orthogonal pYtags can be used to monitor the dynamics of EGFR and ErbB2 activity in the same cell, revealing distinct phases of activation for each RTK. The specificity and modularity of pYtags opens the door to robust biosensors of multiple tyrosine kinases and may enable engineering of synthetic receptors with orthogonal response programs.HighlightspYtags report on signaling of user-defined RTKs in living cellsEGFR signaling dynamics depend on ligand identity and dimer strengthrthogonal pYtags enable reporter multiplexingpYtags can report on signaling of endogenously expressed RTKs