A Developmental Atlas of the Drosophila Nerve Cord Uncovers a Global Temporal Code for Neuronal Identity

The assembly of functional neural circuits depends on the generation of diverse neural types with precise molecular identity and connectivity. Unlocking general principles of neuronal specification and wiring across the nervous system requires a systematic and high-resolution characterisation of its diversity, recently enabled by advances in single-cell transcriptomics and connectomics. However, linking the molecular identity of neurons to circuit architecture remains a key challenge. Here, we present a high-resolution developmental transcriptional atlas for the Drosophila melanogaster nerve cord, the central hub for sensory-motor circuits. With an unprecedented 38x coverage, this atlas enabled robust alignment to the adult connectome. We found that birth time sets a discrete versus continuous organisation of neuronal molecular identity, a difference we also identified in the connectome. We discovered a set of 17 transcription factors expressed in a conserved temporal sequence across all lineages, establishing a global temporal code for neuronal identity based on birth order, linking specification to differentiation across the nerve cord. Lastly, by mapping sex-specific transcriptional profiles to the connectome, we uncovered apoptosis and transcriptional divergence as key drivers for sex- specification. By resolving how molecular identity is temporally organized, this atlas opens new avenues to dissect the molecular mechanisms underpinning the development of neural circuits. Highlights – A developmental transcriptional atlas of the Drosophila melanogaster ventral nerve cord (VNC) with 38x coverage – Birth order, segment and lineage annotation enables the assignment of transcriptional signatures to 122 cell type groups in the connectome – 17 transcription factors (TFs) are expressed in a conserved temporal sequence within lineages across the nervous system defining a persistent molecular correlate of birth order – We match sex differences in the developmental atlas and adult connectome revealing the molecular logic of sexual dimorphism