Robert Arnulfo Carrillo, Ph.D. - US grants

Project Summary The experiments outlined in this application, and our long term goals, seek to fill a large knowledge gap in our understanding of the developmental mechanisms that control neural circuit wiring. Our collaboration with Dr. Christopher Garcia at Stanford revealed two Ig superfamily subfamilies, the 9-member Dprs and 21-member DIPs. These cell surface proteins (CSPs) bind to one other in unique and overlapping patterns and are expressed on small and unique subsets of neurons in the Drosophila nervous system. The primary hypothesis underlying this application is that Dprs and their DIP partners control cell-cell interactions that underlie synaptic specificity in neural circuits. Our innovative approach is fueled by the Dpr-DIP interactome since we are able to systematically analyze synaptic partners that express corresponding Dpr and DIP partners. Relevant to this proposal, I have gathered preliminary data suggesting that a Dpr-DIP pair mediates synaptic targeting. The first aim takes advantage of the accessibility and invariant connections of the larval neuromuscular junction to understand how interactions between this Dpr-DIP pair control the formation and targeting of a motor axon branch. The focus of the second aim is to examine the roles of Dpr-DIP interactions in neurite growth and the wiring of motor circuits in the ventral nerve cord. These studies will uncover basic principles governing neural circuit assembly and specifically, how CSP subfamilies contribute to this specificity. These analyses will utilize my training in Drosophila neural development and expertise in genetics, electrophysiology, microscopy, and morphological analyses. As a recently hired, tenure-track assistant professor at the University of Chicago, I will take full advantage of the interdisciplinary, collaborative environment to build a productive and independent career. An exceptional mentoring team, consisting of my mentor, Dr. Ilaria Rebay (UChicago), and co-mentors, Dr. Hugo Bellen (Baylor College of Medicine) and Dr. Benjamin White (NIMH), will evaluate my progress and provide feedback. Under this K01 award I will gain additional technical training and the skills required to create a rigorous, independent research program and compete for independent funding, including an R01. These experiences will lay the groundwork to obtain tenure and run a successful, well-funded laboratory.

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-? 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.