Regulatory logic of neuronal identity specification in Drosophila

During neurogenesis, signaling molecules and transcription factors (TFs) pattern neural progenitors across space and time to generate the numerous cell types that constitute neural circuits. In postmitotic neurons, these identities are established and maintained by a class of TFs known as terminal selectors (tsTFs). However, it remains largely unclear how the tsTF combinations are specified, and how they then coordinate the type-specific differentiation programs of each neuron. To investigate these regulatory mechanisms, we performed simultaneous single-cell RNA and ATAC sequencing on the Drosophila optic lobes at four stages of their development and identified over 250 distinct cell types. We characterized the common cis-regulatory features of neuronal enhancers and performed comprehensive inference of gene regulatory networks across cell types and stages. Our results reveal cell-type and stage-specific enhancers of many neuronal genes and the cooperative actions of pan-neuronal and tsTFs on these enhancers. We show that the same effector genes are often regulated by different tsTF combinations in different neurons. During neurogenesis, most temporal TFs and the spatial TF Vsx1 are regulated by different enhancers before and after neurons become postmitotic, allowing them to be re-utilized as tsTFs independently from their roles in progenitors. As proof of concept, we genetically dissected the regulation of Vsx1 as a tsTF by different spatial and temporal patterning mechanisms through lineage-specific enhancers.