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The brain consists of thousands of neuronal types that are generated by stem cells that produce different neuronal types as they age. In Drosophila, this temporal patterning is driven by the successive expression of temporal transcription factors (tTFs). We used single-cell mRNA sequencing to identify the complete series of tTFs that specify most Drosophila optic lobe neurons. We verified that tTFs regulate the progression of the series by activating the next tTFs and repressing the previous one(s), but also identified more complex regulations. Moreover, we established the temporal window of origin and birth order of each neuronal type in the medulla and provide evidence that these tTFs are sufficient to explain the generation of all the neuronal diversity in this brain region. Finally, we describe the first steps of neuronal differentiation. We find that terminal differentiation genes, such as neurotransmitter-related genes, are present as transcripts, but not as proteins, in immature larval neurons. This comprehensive analysis of a temporal series of transcription factors offers 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 neurodevelopmental mechanisms evolve to generate diverse neuronal types.

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