Neuropeptide repertoire and 3D anatomy of the ctenophore nervous system

Ctenophores are gelatinous marine animals famous for locomotion by ciliary combs. Due to the uncertainties of the phylogenetic placement of ctenophores and the absence of some key bilaterian neuronal genes, it has been hypothesized that their neurons evolved independently. Additionally, recent whole-body, single-cell RNA sequencing (scRNA-seq) analysis failed to identify ctenophore neurons using any of the known neuronal molecular markers. To reveal the molecular machinery of ctenophore neurons, we have characterized the neuropeptide repertoire of the ctenophore Mnemiopsis leidyi. Using the machine learning NeuroPID tool, we predicted 129 new putative neuropeptide precursors. Sixteen of them were localized to the subepithelial nerve net (SNN), sensory aboral organ (AO), and epithelial sensory cells (ESCs), providing evidence that they are neuropeptide precursors. Four of these putative neuropeptides had a behavioral effect and increased the animals' swimming speed. Intriguingly, these putative neuropeptides finally allowed us to identify neuronal cell types in single-cell transcriptomic data and reveal the molecular identity of ctenophore neurons. High-resolution electron microscopy and 3D reconstructions of the nerve net underlying the comb plates confirmed a more than 100-year-old hypothesis of anastomoses between neurites of the same cell in ctenophores and revealed that they occur through a continuous membrane. Our work demonstrates the unique ultrastructure of the peptidergic nerve net and a rich neuropeptide repertoire of ctenophores, supporting the hypothesis that the first nervous system(s) evolved as nets of peptidergic cells.