IgSF protein interactions drive specificity in circuit wiring and synaptic elaboration

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Project Summary In this application, we examine the molecular mechanisms that instruct neural wiring and axon terminal elaboration. We focus on the Drosophila neuromuscular system due to its invariant connectivity, limited synaptic partners, and accessibility. Given that this ‘simple’ circuit has been studied for over four decades, it is somewhat surprising that fundamental questions still exist as to how motor neurons choose their appropriate muscle targets and how each motor neuron develops a unique, yet stereotyped, axon terminal structure that underlies synaptic function. Conceptually, both of these developmental processes rely on specificity cues to guide synaptic partner matching (role 1) and synaptic elaboration at each axon terminal (role 2). In support of the first role, we previously discovered two interacting cell surface proteins (CSPs), DIP-α and Dpr10, that are required for wiring a motor neuron to a subset of muscles. In support of the second role, these CSPs continue to be expressed after connectivity, implying additional functions in synaptic development. Our central hypothesis is that combinatorial Dpr-DIP interactions, in addition to specifying synaptic connections, also participate in determining the structure and function of specified synapses. Insights into circuit development arose in a prior collaboration where we characterized the ‘Dpr-ome’, the set of interactions between two families of the immunoglobulin superfamily, the Dprs and DIPs. These 32 proteins bind to one another in unique combinations, and our preliminary data reveal unique expression patterns in the Drosophila larval neuromuscular circuit. Additionally, our data support a combinatorial Dpr-DIP interaction model that leverages cis/trans interactions to instruct highly specific synaptic partnerships. We also reveal a novel signaling pathway that underlies local synaptic elaboration. Given our findings and genetic reagents, we are in a unique position not only to compare axon branch-specific identification tags but also to ask if synaptic elaboration of neighboring axon terminals can be independently regulated. In the first aim, we capitalize on the Dpr-ome and the expression of 6 DIPs in multi-innervating motor neurons to perform single-cell genetic manipulations and examine how combinatorial Dpr-DIP codes instruct connectivity. In addition, we generate affinity variants to reveal a coordinated cis/trans interaction model that enhances specificity. In the second aim, we utilize functional and genetic approaches to understand how co-innervating inputs develop unique morphological and functional properties. We identify a novel crosstalk signaling pathway between axon arbors that locally sculpts NMJ size. Overall, these studies will uncover fundamental developmental programs required for neural circuit wiring and axon terminal elaboration, with emphasis on how CSP codes modulate each of these processes.