A scRNAseq atlas of larval Drosophila ventral cord - a resource for studying motor systems function and development

Drosophila larval ventral nerve cord (VNC) shares many similarities with the spinal cord of vertebrates and has emerged as a major model for understanding the development and function of motor systems. We use high quality single cell RNA sequencing to create a comprehensive atlas of larval VNC cell types. Our atlas provides a high-resolution characterization of larval VNC capturing primary neurons, glia and the functional landscape that coordinates larval behavior. At the same time, this atlas offers unique insights into neurogenesis and into the strategies and signaling networks utilized for generation of the adult VNC. Overall design: We have previously described in detail the single-cell RNA sequencing protocols that we optimized for the analysis of Drosophila larval tissues (Nguyen et al., 2021; Vicidomini et al., 2021). Here we analyzed VNCs from Drosophila third instar larvae of five different genotypes. The VNCs were manually dissected and dissociated using a combination of mechanical and enzymatic treatments. The cell suspensions were processed using droplet microfluidics (10x Genomics Chromium platform) and the resulting libraries were sequenced on an Illumina platform. To characterize larval motor neurons, selective neuronal populations were GFP-marked and FACS-enriched before processing. This strategy permitted separation and identification of all motor neuron bundles, linking individual neuron clusters to morphologically characterized known subtypes.