The enteric nervous system (ENS) coordinates essential intestinal functions through the concerted action of diverse enteric neurons (ENs). However, integrated molecular knowledge of EN subtypes is lacking. To compare human and mouse ENs, we transcriptionally profiled healthy ENS from adult humans and mice. We aimed to identify transcripts marking discrete neuron subtypes and visualize conserved EN subtypes for humans and mice in multiple bowel regions.

Human myenteric ganglia and adjacent smooth muscle were isolated by laser-capture microdissection for RNA sequencing. Ganglia-specific transcriptional profiles were identified by computationally subtracting muscle gene signatures. Nuclei from mouse myenteric neurons were isolated and subjected to single-nucleus RNA sequencing, totaling more than 4 billion reads and 25,208 neurons. Neuronal subtypes were defined using mouse single-nucleus RNA sequencing data. Comparative informatics between human and mouse data sets identified shared EN subtype markers, which were visualized in situ using hybridization chain reaction.

Several EN subtypes in the duodenum, ileum, and colon are conserved between humans and mice based on orthologous gene expression. However, some EN subtype–specific genes from mice are expressed in completely distinct morphologically defined subtypes in humans. In mice, we identified several neuronal subtypes that stably express gene modules across all intestinal segments, with graded, regional expression of 1 or more marker genes.

Our combined transcriptional profiling of human myenteric ganglia and mouse EN provides a rich foundation for developing novel intestinal therapeutics. There is congruency among some EN subtypes, but we note multiple species differences that should be carefully considered when relating findings from mouse ENS research to human gastrointestinal studies.