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 another class of TFs known as terminal selectors (tsTFs). However, it remains largely unclear how the tsTF combinations are specified in newborn neurons, 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 experiments on the Drosophila optic lobes and identified over 250 distinct cell types at four stages of their development. 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 cooperative actions of pan-neuronal and tsTFs on cell type-specific enhancers, and that same effector genes are often regulated by different TF combinations in different cell types. During neurogenesis, patterning TFs are associated with different accessible enhancers before and after cell cycle exit, allowing them to be re-utilized as tsTFs independently from their earlier roles in progenitors. We show that expression of tsTFs Vsx1/2 in a variety of optic lobe neurons is mediated by lineage-specific enhancers, each patterned by different upstream mechanisms. Therefore, neuronal identity specification is a multi-step regulatory program wherein the same TFs can enact distinct regulatory codes at different steps and across cell types.