Project Abstract: Circuit assembly, the collection of sequential developmental steps that ultimately lead to the formation of synapses, is a conserved process that determines organismal function and behavior. In humans, billions of neurons make trillions of synapses, and proper circuit function depends on correct synaptic partnerships. Perturbations in circuit assembly can lead to devastating neurodevelopmental diseases. While it is generally accepted that synaptic connectivity is determined by cell surface receptor interactions, only a relatively small number of these receptors have been identified, and the developmental signaling pathways downstream of these molecules remain mostly uncharacterized. Given the complexity of nervous systems, we need to expedite the discovery of neural receptor/ligand pairs and learn how they signal in order to understand brain development and the physiology of diseases where neural wiring is fundamental. Our labs leverage biochemical and genetic insights into cell surface protein interactions to identify new connectivity codes and corresponding signaling pathways. Previously, in an unbiased protein interaction biochemical screen, we identified two Drosophila interaction networks within the immunoglobulin superfamily – Dprs/DIPs and Beats/Sides. These “interaction codes” guide cell-cell interactions that underlie circuit assembly. Most members of these families bind each other in a complex yet specific manner; for example, each Dpr can interact with a specific subset of DIPs and vice versa. The expression patterns of these proteins are stereotyped, and combinatorial expression of Dprs, DIPs, Beats and/or Sides has been observed, likely serving as unique identity markers on cell surfaces. Deletion of these proteins lead to neural connectivity phenotypes, and specifically for Dpr11 and DIP-γ, misregulation of BMP signaling and neuronal death. Here, we propose to uncover the molecular pathways that are downstream of Dpr-DIP interactions and discover and study other cell surface receptors and secreted proteins that mediate Dpr/DIP function via direct interactions. Our exciting preliminary results already revealed new co-receptors, and follow-up experiments will include biophysical and structural characterization of these new interactions, followed by signaling assays in culture and genetic perturbations in vivo to establish functional roles for these interactions. Furthermore, we will reveal the structural basis of Beat-Side interactions, examine their complexes, manipulate the hetero and homodimeric binding abilities of these proteins and test them using established phenotypes in the embryonic and larval neuromuscular system.