PROJECT SUMMARY Signaling and adhesive interactions between neighboring cells play an important role in developmental processes, helping determine cell fates and identities, migration pathways, and ultimately cellular function. Currently the best method for characterizing cell to cell contacts is through ultrastructural studies. These provide a wealth of information, but are technically complex, tedious to perform, and require extensive analysis to identify specific interactions of interest. We are working to develop a method of cell to cell contact tracing that provides useful information at the optical microscopy level. Using synthetic notch receptors and the ligands they recognize, it is now possible to design and genetically encode a touch sensitive detection system that operates in vivo. We have chosen the CD19/synNotch receptor system utilized in CART therapy to detect contacts between cells. In our version, recognition of the the CD19 ligand displayed on one cell surface activates a synNotch receptor on an opposing surface, inducing the cleavage and release of a transcriptional activator that induces the expression of a fluorescent marker protein. We are working to perfect a cell to cell contact tracing methodology in the developing olfactory system of the zebrafish. Zebrafish have a number of advantages for this study. Among them are excellent access to all developmental stages, a rapid generation time, outstanding optical properties for imaging, and easy transgenesis. The olfactory system has simple, well characterized circuitry and cell-specific promoters are available to drive transgenic expression in specific classes of olfactory sensory neurons. The aim of this project is to identify the cells an olfactory sensory axon contacts as it extends from its origin in the olfactory epithelium to its target in the olfactory bulb. This information is crucial for understanding how these sensory axons home in on specific target regions within the bulb.