PROJECT SUMMARY A central question in neurobiology is how individual neurons precisely connect with each other to form functional circuits during development. Understanding the mechanisms of neural circuit assembly may provide insights into the etiology of human brain disorders. The fruit fly olfactory circuit has been an excellent model to investigate the mechanisms by which wiring specificity of neural circuits arises during development. In this circuit, axons of 50 types of olfactory receptor neurons (ORNs) match precisely with dendrites of 50 corresponding types of second-order olfactory projection neurons (PNs), forming 50 discrete glomeruli in the antennal lobe. This allows olfactory information to be faithfully delivered from peripheral sensory organs to higher brain centers, enabling innate and learned olfactory behavior. Thanks to the continual support of this grant since 2003, our studies have made the Drosophila antennal lobe one of the best-understood circuits in the molecular, cellular, and developmental underpinnings of wiring specificity. During the last grant cycle, we made three key advances: 1) we determined single-cell transcriptomes of PNs and ORNs across development, which have produced differentially expressed genes as candidate wiring molecules and cell-type-specific drivers for labelling and genetically manipulating individual PN and ORN types; 2) we developed a cell-surface proteomic profiling method that allowed us to identify new wiring molecules; 3) we established an explant culture that recapitulates wiring specificity in vivo, allowing us to examine the dynamic process of circuit assembly in wild-type and mutants using time-lapse imaging. In this proposal, we will take advantage of these advances to further investigate the cellular and molecular mechanisms by which the olfactory circuit is assembled and wiring specificity is achieved. Specifically, we will utilize new genetic tools and time-lapse imaging to determine the cellular events that lead to PN dendrite patterning, segregation, and ORN-PN synaptic partner matching. We will investigate the mechanisms by which cell surface receptor teneurins work with putative ligands and downstream signaling molecules to instruct synaptic partner matching. We will also identify additional instructive wiring molecules and study their mechanisms of actions using data from single-cell transcriptomes and in vivo assays that can distinguish attraction vs. repulsion in ligand-receptor interactions.