Single-Cell Multiome Sequencing Clarifies Enteric Glial Cell Diversity and Identifies an Intraganglionic Population Poised for Neurogenesis

The enteric nervous system (ENS) consists of glial cells (EGCs) and neurons derived from neural crest precursors. EGCs retain capacity for large-scale neurogenesis in culture, and in vivo lineage tracing has identified neurons derived from glial cells in response to inflammation. We thus hypothesize that EGCs possess a chromatin structure poised for neurogenesis. We use single-cell multiome sequencing to assess EGCs undergoing spontaneous neurogenesis in culture, as well as freshly isolated small intestine myenteric plexus EGCs. Cultured EGCs maintain open chromatin at genomic loci accessible in neurons, and neurogenesis from EGCs involves dynamic chromatin rearrangements with a net decrease in accessible chromatin. Multiome analysis of freshly isolated EGCs reveals transcriptional diversity, with open chromatin at neuron-associated genomic elements. A subset of EGCs, highly enriched within the myenteric ganglia, has a gene expression program and chromatin state consistent with neurogenic potential. hypothesize that heterogeneous EGCs maintain a permissive chromatin structure for neuronal differentiation. In the present study, we apply scRNA-seq and single-cell multiome sequencing (scMulti-seq) to characterize EGCs and study the dynamics of neuronal differentiation. We show that EGCs maintain open chromatin at promoters and regulatory elements controlling neuronal marker genes. Differentiation of EGCs into neurons involves reduced chromatin accessibility, consistent with more limited fate potential. scMulti-seq of EGCs freshly isolated from postnatal mouse intestine reveals considerable transcriptional diversity, consistent with previous reports By integrating gene expression with chromatin structure, we identify a subpopulation of EGCs primed for neurogenesis. In situ hybridization validates this population and demonstrates it is heavily enriched within myenteric ganglia. Our work provides a multiomic atlas of myenteric EGCs, identifies a chromatin state poised for neurogenesis, and shows intraganglionic EGCs to be biologically distinct from extraganglionic populations.