Summary Deciphering how neuronal diversity is established and maintained requires a detailed knowledge of neuronal gene expression throughout development. In contrast to mammalian brains 1 , 2 , the large neuronal diversity of the Drosophila optic lobes 3 and its connectome 4 – 6 are almost completely characterized. However, a molecular characterization of this diversity, particularly during development, has been lacking. We present novel insights into brain development through a nearly exhaustive description of the transcriptomic diversity of the optic lobes. We acquired the transcriptome of 275,000 single-cells at adult and five pupal stages, and developed a machine learning framework to assign them to almost 200 cell-types at all timepoints. We discovered two large neuronal populations that wrap neuropils during development but die just before adulthood, as well as neuronal subtypes that partition dorsal and ventral visual circuits by differential Wnt signaling throughout development. Moreover, we showed that neurons of the same type but produced days apart synchronize their transcriptomes shortly after being produced. We also resolved during synaptogenesis neuronal subtypes that converge to indistinguishable transcriptomic profiles in adults while greatly differing in morphology and connectivity. Our datasets almost completely account for the known neuronal diversity of the optic lobes and serve as a paradigm to understand brain development across species.

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The brain consists of thousands of different neuronal types that are generated through multiple divisions of neuronal stem cells. These stem cells have the capacity to generate different neuronal types at different stages of their development. In Drosophila, this temporal patterning is driven by the successive expression of temporal transcription factors (tTFs). While a number of tTFs are known in different animals and across various parts of the nervous system, these have been mostly identified by informed guesses and antibody availability. We used single-cell mRNA sequencing to identify the complete series of tTFs that specify most Drosophila medulla neurons in the optic lobe. We tested the genetic interactions among these tTFs. While we verify the general principle that tTFs regulate the progression of the series by activating the next tTFs in the series and repressing the previous ones, we also identify more complex regulations. Two of the tTFs, Eyeless and Dichaete, act as hubs integrating the input of several upstream tTFs before allowing the series to progress and in turn regulating the expression of several downstream tTFs. Moreover, we show that tTFs not only specify neuronal identity by controlling the expression of cell type-specific genes. Finally, we describe the very first steps of neuronal differentiation and find that terminal differentiation genes, such as neurotransmitter-related genes, are present as transcripts, but not as proteins, in immature larval neurons days before they are being used in functioning neurons; we show that these mechanisms are conserved in humans. Our results offer a comprehensive description of a temporal series of tTFs in a neuronal system, offering mechanistic insights into the regulation of the progression of the series and the regulation of neuronal diversity. This represents a proof-of-principle for the use of single-cell mRNA sequencing for the comparison of temporal patterning across phyla that can lead to an understanding of how the human brain develops and how it has evolved.

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