Functional diversity of enteric glia in the gastrointestinal tract

Abstract

The enteric nervous system (ENS) regulates intestinal motility, immunity, and nutrient absorption, with glia playing essential roles in these processes. However, it is unclear if different types of glia in the ENS have distinct functions, akin to glia in the central nervous system. To address this gap, we developed imaging and sequencing approaches to characterize the cellular behaviors and transcriptional signatures of glia in different spatial compartments along the radial axis of the small intestine. First, we developed an intestinal ring imaging preparation to observe glial behaviors in real time. Using tissues from dual reporter mice (Vil1Cre Rosa26Ai9/+ Plp1eGFP), we found that glia in the healthy intestine exhibit minimal movement, suggesting largely fixed positions and morphologies in their local niches. We then developed a technique to perform bulk-RNA sequencing of glia from the mucosal and muscularis compartments of the small intestine from Plp1eGFP mice, reasoning glia in these two spatial compartments were likely to be the most distinct. While the populations shared core glial gene programs, mucosal glia resembled microglia while muscularis glia were most similar to satellite glia and astrocytes, supporting that enteric glia have identities that reflect their niche and other glial populations outside of the ENS. We also sought to ask whether there were defining markers that distinguished enteric glial subpopulations from other glial cells, which would provide genetic tractability to manipulate these cells. To date, no single genetic handle exists to target enteric glia without affecting another glial population outside of the iii ENS. We identified that muscularis glia showed selective expression of Tacr3, which encodes a G-protein coupled receptor for neuropeptides. Using Tacr3IRES-Cre/+ Rosa26Ai9/+ mice to genetically label Tacr3-expressing cells, we observed that Tacr3 expression emerged in the postnatal ENS. By 21 days of age, over 95% of tdTomato-labeled cells in the muscularis were glia, primarily the glia surrounding neuron somas (intraganglionic). Additionally, tdTomato labeling was not detected in intramuscular or extra-intestinal glia. The restricted expression in intraganglionic glia allowed us to elucidate the transcriptional signatures of glia in other spatial compartments using single cell transcriptomics. Both early postnatal TACR3 inhibition and lack of the high affinity ligand NKB in mice resulted in significant loss of intramuscular glial cells and altered the molecular phenotype of intraganglionic glia, while mucosal glia remained intact, underscoring the importance of NKB- TACR3 signaling in ENS development. In adult mice, pharmacological modulation of TACR3 affected gastrointestinal transit, establishing Tacr3+ intraganglionic glia as a distinct population essential for normal ENS function. During dextran sodium sulfate-induced colitis, Tacr3 expression significantly decreased, and Tac2 haploinsufficient mice showed slower recovery, suggesting therapeutic potential for enhancing TACR3 signaling in treating intestinal inflammation. Taken together, these insights not only advance our understanding of glial biology in the ENS but also have immediate implications for the clinical use of TACR3 inhibitors that are already on the market for women to manage menopausal hot flashes.