RNA sequencing of Drosophila melanogaster optic lobe cell types. Drosophila melanogaster

Transcription factors regulate the molecular, morphological, and physiological characters of neurons and generate their impressive cell type diversity. To gain insight into general principles that govern how transcription factors regulate cell type diversity, we used large-scale single-cell mRNA sequencing to characterize the extensive cellular diversity in the Drosophila optic lobes. We sequenced 57,000 single optic lobe neurons and glia and assigned them to 52 clusters of transcriptionally distinct single cells. We validated the clustering and annotated many of the clusters using RNA sequencing of characterized FACS-sorted single cell types, as well as marker genes specific to given clusters. To identify transcription factors responsible for inducing specific terminal differentiation features, we used machine-learning to generate a ‘random forest’ model. The predictive power of the model was confirmed by showing that two transcription factors expressed specifically in cholinergic (apterous) and glutamatergic (traffic-jam) neurons are necessary for the expression of ChAT and VGlut in many, but not all, cholinergic or glutamatergic neurons, respectively. Therefore, the same terminal characters can be regulated by different transcription factors in different cell types, arguing for extensive phenotypic convergence. Our data provide a deep understanding of the developmental and functional specification of a complex brain structure. Overall design: We sequenced 17 FACS-sorted single cell types from the Drosophila optic lobe, as well as FACS-sorted neurons as a control. We also sequenced a line that had GFP expression in two cell types - Tm1 and T1.