Unraveling the developmental logic of cortical long-range projections using in situ sequencing-based neuroanatomy

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Project Summary The connectivity of neurons allows complex functions to emerge from a circuit composed of diverse neuronal types. In the mammalian nervous system, recent advances in single-cell transcriptomics make it appealing to define neuronal types by their gene expression patterns (i.e. transcriptomic types). At a high level, classes of neurons defined by transcriptomics are also distinct in other neuronal properties, including their long-range projection patterns. Finer-level transcriptomic types, however, do not correspond to projection patterns: neurons of different transcriptomic types may share similar projections, and neurons of the same transcriptomic type can project diversely. This lack of correspondence at a fine level raises the question of how neuronal types are wired into complex circuits and, furthermore, how cell types can be defined by both gene expression and connectivity. Knowing the developmental relationship between gene expression and projections may help understand this complex relationship, because the projection pattern of an adult neuron is the cumulative result of many developmental processes. However, interrogating the developmental relationship between gene expression and projections is challenging, because conventional single-cell anatomical approaches can only map the projection patterns of a small number of neurons and are difficult to associate projections with gene expression measured in the same cells. Here I propose to overcome this challenge by massively improving the resolution and scale of in situ sequencing-based neuroanatomical approaches. In situ sequencing-based neuroanatomy achieves high throughput and cellular resolution in mapping projections by labeling each neuron with a unique RNA barcode. These RNA barcodes and endogenous mRNAs can both be sequenced in situ to associate projection patterns with gene expression for many neurons in parallel. By improving both the resolution and the throughput of in situ barcode sequencing, I will generate an unprecedented view of the relationship between gene expression and the complete brain-wide projection patterns of neurons in the primary and higher visual cortex over the course of post-natal development. I will complement this systematic but correlational approach with perturbation experiments to establish causal relationship between key genes and projections. By unraveling how the complex relationship between gene expression and projections is established step-by-step in development, this combined approach will provide insights into the wiring rules of cortical neuronal types. The dataset generated will provide a reference for future research into long-range connectivity defects in neurodevelopmental disease models. Finally, the improvement in in situ sequencing-based neuroanatomy will achieve broad impact beyond the developmental focus of this proposal by enabling similar systematic approaches in understanding long-range projections during aging, across individual animals, and across species.