Project Summary Visuomotor transformation (VMT), a vital process by which the brain converts vision into action, requires precise synaptic connectivity between sensory and motor neural circuits. Impaired visuomotor processing has been associated with a wide range of neurological disorders. Developmental and molecular origins of a VMT remain elusive due to the lack of experimentally tractable model systems. I address this knowledge gap by interrogating the visuomotor interface of Drosophila, where transcriptomics, connectomics and physiology can be integrated to causally link genes and molecules with circuit structure and function. My recent work uncovered a completely new wiring strategy underlying a VMT: visual space coordinates are transformed into synaptic weights. Such synaptic gradient mechanism found in Visual Projection Neurons (VPN) emerges through within-cell-type synaptic specificity. Individual neurons belonging to the same VPN cell type connect to different postsynaptic partners, which elicits multidirectional motor programs in response to differentially localized visual stimuli. Aim 1 of my research during the K99 phase will build on these findings and identify the transcriptomic signatures of synaptic gradients. I hypothesize that within-cell-type synaptic specificity is achieved through transcriptomic uniqueness of individual neurons of the same type (i.e., molecular gradients of wiring genes). In collaboration with my co-mentor Dr. Y. Kurmangaliyev, I will test this hypothesis and generate a developmental transcriptomic atlas of the fly visuomotor interface featuring 20 VPN cell types. Single-cell RNA-seq profiling will be followed by the validation of candidate gene expression patterns using genetics and spatial transcriptomics. This will generate a molecular model of synaptic gradients. Aim 2, pursued in collaboration with my co-mentor Dr. C. von Reyn, will functionally test this model using genetic perturbation screening. Candidate genes will be misexpressed in VPNs and their postsynaptic partners, and the effects on synaptic gradients will be assessed using electrophysiology. This work will provide causal relationships between molecular gradients of wiring genes and within-cell-type synaptic specificity. The mentorship I will continue to receive from Dr. Zipursky, and the training in single-cell data analysis and electrophysiology I will acquire during the K99 phase will facilitate my transition to an independent research program. Aim 3, to be pursued during the R00 phase, will investigate the gene regulatory networks of synaptic specificity in visuomotor circuits. I will examine the role of global extrinsic regulatory programs (e.g., spatially graded Wnt signaling and neural activity) in establishing synaptic gradients using a combination of Perturb-RNA-seq and chromatin accessibility (ATAC-seq) analysis in VPNs. This approach will link extracellular signals with transcription factor mediated differential gene expression in a...