MOLECULAR, MORPHOLOGICAL AND ANATOMICAL CHARACTERIZATION OF ENTERIC GLIAL HETEROGENEITY ACROSS THE MOUSE GASTROINTESTINAL TRACT

Enteric glial cells (EGCs) are a numerically dominant and functionally diverse component of the enteric nervous system (ENS), as they contribute to gastrointestinal motility, epithelial barrier integrity and immune regulation. Increasing evidence indicates that EGCs comprise transcriptionally and morphologically distinct subtypes. However, the relationship between molecular identity, cellular morphology, and anatomical localization across the gastrointestinal tract remains poorly defined. Here, we aim to integrate transcriptional, morphological, and spatial analyses to characterize EGC heterogeneity in the murine small intestine and colon. Using previously published and newly generated single-cell RNA sequencing datasets, we defined molecularly distinct EGC populations within the myenteric and submucosal plexuses and the mucosal region. These included intra-ganglionic, extra-ganglionic, and glial populations with intermediate profiles. To validate cluster-specific markers in situ, we combined RNAscope with immunohistochemistry on whole-mount tissue preparations, allowing for the spatial mapping of transcriptionally defined glial subtypes. To further link molecular identity with cellular morphology, we performed single-cell tracing and reconstruction of individual EGCs using Sox10-CreERT2; tdTomato reporter mice. In parallel, we leveraged a novel mouse line that selectively labels extra-ganglionic enteric glia, allowing detailed analysis of this understudied population. These complementary approaches reveal that molecularly distinct EGC populations correspond to reproducible morphological subtypes occupying defined anatomical niches within the ENS across intestinal regions. To sum up, our work presents an integrated framework that links gene expression, morphology, and anatomical distribution of enteric glial subtypes, advancing our understanding of enteric glial diversity, opening the way for future studies of their roles in gut physiology and disease.