Integrated single-cell analysis of enteric glial cells reveals a molecular basis for postnatal neurogenesis and its therapeutic application

Traditional models posit that enteric neurons and glial cells represent distinct terminal lineages derived from a common neural crest precursor. This model, however, does not explain the neurogenic capability of murine postnatal enteric glial cells. To characterize the full diversity of myenteric glial cells and identify a basis for the glial-to-neuronal transition, which we demonstrate using a two-marker system, we applied single-cell RNA sequencing and single-nucleus ATAC sequencing to generate a multiomic atlas of Plp1-expressing glial cells from the small intestine of adolescent mice. We identify nine transcriptionally distinct subpopulations of enteric glial cells, including cells expressing both canonical neural stem cell genes and enteric neuronal transcriptional factors. We refer to these Plp1-positive cells with neural stem cell features as glial neuroblasts. Surprisingly, most glial cells maintain open chromatin at neuronal-associated loci, suggesting enteric glia are primed for neuronal transition. Comparison with the developing embryonic enteric nervous system shows postnatal glial cells maintain a transcriptional program closely matching embryonic neuronal progenitors. Transcription factor motif enrichment analysis and regulon analysis implicate AP-1 transcription factors in maintaining the glial neuroblast gene program. Three-dimensional cultures of postnatal enteric nervous system cells, which are enriched for glial neuroblasts and provide a niche for neuronal development, recapitulate the transcriptional changes seen during embryonic enteric neurogenesis. snATAC analysis shows chromatin closing consistent with terminal differentiation as glial cells become neurons in three-dimensional culture. In conclusion, postnatal myenteric glial cells include a neuroblast population and maintain a chromatin structure primed for neuronal fate acquisition. Significance The enteric nervous system is comprised of neurons and glial cells derived from neural crest precursors that migrate along the gastrointestinal tract during embryonic development. Considerable efforts have been dedicated to exploring enteric neurons, revealing many subtypes based on structure, function, and molecular profile. However, enteric glial cells have yet to be fully characterized. We performed single-cell transcriptional and epigenetic profiling of thousands of enteric glial cells from adolescent mice, providing for the first time a multimodal atlas of these cells. We identify multiple subpopulations of enteric glia and propose a molecular basis for these cells’ ability to generate neurons. Enteric glial cells closely match embryonic neuronal precursors and may be suitable for therapeutic neuronal regeneration. ### Competing Interest Statement Dr. Goldstein is the recipient of research funds from Takeda Pharmaceutical Company for projects unrelated to this work.