Abstract The medulla is the largest neuropil of the Drosophila optic lobe. It contains about 100 neuronal types that have been comprehensively characterized morphologically and molecularly. These neuronal types are specified from a larval neuroepithelium called the Outer Proliferation Center (OPC) via the integration of temporal, spatial, and Notch-driven mechanisms. Although we recently characterized the temporal windows of origin of all medulla neurons, as well as their Notch status, their spatial origins remained unknown. Here, we isolated cells from different OPC spatial domains and performed single-cell mRNA-sequencing to identify the neuronal types produced in these domains. This allowed us to characterize in a high-throughput manner the spatial origins of all medulla neurons and to identify two new spatial subdivisions of the OPC. Moreover, our work shows that the most abundant neuronal types are produced from epithelial domains of different sizes despite being present in a similar number of copies. Combined with our previously published scRNA-seq developmental atlas of the optic lobe, our work opens the door for further studies on how specification factor expression in progenitors impacts gene expression in developing and adult neurons.

High-throughput identification of the spatial origins ofDrosophilaoptic lobe neurons using single-cell mRNA-sequencing