Abstract
BACKGROUND & AIMS The enteric nervous system (ENS) coordinates essential intestinal functions through the concerted action of diverse enteric neurons (EN). 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.
METHODS Human myenteric ganglia and adjacent smooth muscle were isolated by laser-capture microdissection for RNA-Seq. 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-Seq (snRNA-Seq), totaling over four billion reads and 25,208 neurons. Neuronal subtypes were defined using mouse snRNA-Seq data. Comparative informatics between human and mouse datasets identified shared EN subtype markers, which were visualized in situ using hybridization chain reaction (HCR).
RESULTS 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 one or more marker genes.
CONCLUSIONS 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 GI studies.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Grant support: The laser-capture microdissection instrument was funded by NIH grants P30-CA068485-14 and U24-DK059637-13 to the Tissue Pathology Shared Resource at VUMC. Flow sorting was performed in the VUMC Flow Core, supported by the Vanderbilt Ingram Cancer Center (P30 CA68485) and the Vanderbilt Digestive Disease Research Center (P30 DK058404). RNA sequencing and computing support was provided by the Genome Technology Access Center at Washington University, supported in part by NCI Award P30 CA91842 to the Siteman Cancer Center and ICTS/CTSA UL1TR002345 from the NCRR. This work was funded by NIH awards OT2-OD23850 to EMS2, RO1-DK103831 to KSL, OT2-OD024909 to JRM, with support for AAM on T32-DK007673.
Disclosures: Authors do not have any relevant conflicts of interest.
Abbreviations
- CALB1
- Calbindin 1
- CALB2
- Calbindin 2
- CCKAR
- CCK Receptor Type A
- CDH9
- Cadherin 2
- CHAT
- Choline acetyltransferase
- EN
- Enteric neuron
- ENS
- Enteric nervous system
- FACS
- fluorescence-activated cell sorting
- FISH
- fluorescence in situ hybridization
- GO
- Gene Ontology
- HCR
- Hybridization chain reaction
- IPAN
- Intrinsic Primary Afferent Neuron
- LCM
- Laser-Capture Microdissection
- NEFL
- Neurofilament
- NMU
- Neuromedin U
- NOS1
- Nitric oxide synthase
- NXPH2
- Neurexophilin 2
- PCA
- Principal Components Analysis
- SNAP25
- Synaptosome Associated Protein 25
- snRNA-Seq
- single-nucleus RNA-Sequencing
- Sox10
- SRY (sex determining region Y)-box transcription factor 10
- SST
- Somatostatin
