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.

In Drosophila melanogaster, external sensory organs develop from a single sensory organ precursor (SOP). The SOP divides asymmetrically to generate daughter cells, whose fates are governed by differential Notch activation. Here we show that the clathrin adaptor AP-1 complex, localized at the trans Golgi network and in recycling endosomes, acts as a negative regulator of Notch signaling. Inactivation of AP-1 causes ligand-dependent activation of Notch, leading to a fate transformation within sensory organs. Loss of AP-1 affects neither cell polarity nor the unequal segregation of the cell fate determinants Numb and Neuralized. Instead, it causes apical accumulation of the Notch activator Sanpodo and stabilization of both Sanpodo and Notch at the interface between SOP daughter cells, where DE-cadherin is localized. Endocytosis-recycling assays reveal that AP-1 acts in recycling endosomes to prevent internalized Spdo from recycling toward adherens junctions. Because AP-1 does not prevent endocytosis and recycling of the Notch ligand Delta, our data indicate that the DE-cadherin junctional domain may act as a launching pad through which endocytosed Notch ligand is trafficked for signaling.

In metazoans, unequal partitioning of the cell-fate determinant Numb underlies the generation of distinct cell fates following asymmetric cell division [1-5]. In Drosophila, during asymmetric division of the sensory organ precursor (SOP) cell, Numb is unequally inherited by the pIIb daughter cell, where it antagonizes Notch [1, 6-8]. Numb inhibits Notch partly through inhibiting the plasma membrane localization of Sanpodo (Spdo), a transmembrane protein required for Notch signaling during asymmetric cell division [9, 10]. Numb, by binding to Spdo and α-Adaptin, was proposed to mediate Spdo endocytosis alone or bound to Notch in the pIIb cell, thereby preventing Notch activation [11-16]. However, in addition to endocytosis, Numb also controls the postendocytic trafficking and degradation of Notch in mammals [17, 18] and negatively regulates basolateral recycling in C. elegans [19, 20]. Thus, whether Numb promotes the endocytosis of Spdo is a question that requires experimental demonstration and is therefore investigated in this article. Based on internalization assays, we show that Spdo endocytosis is restricted to cells in interphase and requires AP-2 activity. Surprisingly, the bulk endocytosis of Spdo occurs properly in numb mutant SOP, indicating that Numb does not regulate the steady-state localization of Spdo via Spdo internalization. We report that Numb genetically and physically interacts with AP-1, a complex regulating the basolateral recycling of Spdo [21]. In numb mutant organs, Spdo is efficiently internalized and recycled back to the plasma membrane. We propose that Numb acts in concert with AP-1 to control the endocytic recycling of Spdo to regulate binary-fate decisions.