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Enteric Neuromics: How High-Throughput “Omics” Deepens Our Understanding of Enteric Nervous System Genetic Architecture

  • Christine Dharshika
    Affiliations
    Department of Physiology, Neuroscience Program, Michigan State University, East Lansing, Michigan

    College of Human Medicine, Michigan State University, East Lansing, Michigan
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  • Brian D. Gulbransen
    Correspondence
    Correspondence Address correspondence to: Brian Gulbransen, PhD, Department of Physiology, Michigan State University, 567 Wilson Road, East Lansing, Michigan 48824. fax: (517) 355-5125.
    Affiliations
    Department of Physiology, Neuroscience Program, Michigan State University, East Lansing, Michigan
    Search for articles by this author
Open AccessPublished:November 08, 2022DOI:https://doi.org/10.1016/j.jcmgh.2022.10.019
      Recent accessibility to specialized high-throughput “omics” technologies including single cell RNA sequencing allows researchers to capture cell type- and subtype-specific expression signatures. These omics methods are used in the enteric nervous system (ENS) to identify potential subtypes of enteric neurons and glia. ENS omics data support the known gene and/or protein expression of functional neuronal and glial cell subtypes and suggest expression patterns of novel subtypes. Gene and protein expression patterns can be further used to infer cellular function and implications in human disease. In this review we discuss how high-throughput “omics” data add additional depth to the understanding of established functional subtypes of ENS cells and raise new questions by suggesting novel ENS cell subtypes with unique gene and protein expression patterns. Then we investigate the changes in these expression patterns during pathology observed by omics research. Although current ENS omics studies provide a plethora of novel data and therefore answers, they equally create new questions and routes for future study.

      Keywords

      Abbreviations used in this paper:

      ACh (acetylcholine), AH (afterhyperpolarization), CGRP (calcitonin gene-related peptide), ChAT (choline acetyltransferase), DNBS (dinitrobenzene sulfonic acid), ENS (enteric nervous system), GDNF (glial cell line–derived neutrophilic factor), HSCR (Hirschsprung disease), IBS (irritable bowel syndrome), IBD (inflammatory bowel disease), IFAN (intestinofugal/viscerofugal afferent neuron), IL (interleukin), IPAN (intrinsic primary afferent neuron), NPY (neuropeptide Y), scRNA-seq (single cell RNA sequencing), SST (somatostatin), VIP (vasoactive intestinal peptide)
      Novel high-throughput techniques like single cell RNA sequencing expand our understanding of the enteric nervous system. This review integrates high-throughput findings to further characterize established functional subtypes of enteric neurons and glia and how enteric gene expression patterns change during disease.
      High-throughput “omics” research investigates molecular information on a large and comprehensive scale. The flexibility and resolution of omics technologies continue to increase while cost decreases,
      • Shapiro E.
      • Biezuner T.
      • Linnarsson S.
      Single-cell sequencing-based technologies will revolutionize whole-organism science.
      making omics methods increasingly accessible and attractive to basic and clinical researchers. This has led to a rapid growth in the number of published studies using omics approaches to understand the enteric nervous system (ENS). The ENS is embedded within the gut wall and provides local control of gastrointestinal functions through intrinsic neurocircuitry and integration with multiple cell types in the gastrointestinal tract and other organs.
      • Furness J.B.
      The enteric nervous system and neurogastroenterology.
      The ENS is composed of neurons and glia with generally well-known electrophysiological properties, anatomic features, and protein markers.
      • Furness J.B.
      The enteric nervous system and neurogastroenterology.
      • Furness J.
      Types of neurons in the enteric nervous system.
      • Furness J.B.
      • Gulbransen B.D.
      • Sharkey K.A.
      Novel functional roles for enteric glia in the gastrointestinal tract.
      However, much of the complexity of the ENS remains unknown and would benefit from developing a deeper understanding of cellular heterogeneity, functional attributes of cells and cellular networks, and genes that contribute to disease.
      Omics technologies are helping to disentangle complexity within the ENS on a scale that was previously inaccessible. The advent of single cell RNA sequencing (scRNA-seq) now allows characterizing heterogeneity between individual cells,
      • Shapiro E.
      • Biezuner T.
      • Linnarsson S.
      Single-cell sequencing-based technologies will revolutionize whole-organism science.
      and cellular genomic libraries are available to explore the cellular makeup of the ENS in fine resolution. We begin this review by summarizing the “pre-omics” understanding of the cellular makeup of the ENS and describe omics strategies used to study the ENS. Then we focus on how omics data expand known ENS cell diversity and cellular changes in gastrointestinal disease (Figure 1). We conclude by discussing strengths and challenges of current ENS omics data and future directions for the field.
      Figure thumbnail gr1
      Figure 1Deepening the understanding of enteric nervous system function and disease through high-throughput omics approaches. This review focuses on how data obtained by high-throughput approaches such as single cell sequencing deepen our understanding of cell identity, mechanisms of intercellular communication, and disease processes in the enteric nervous system.

      Pre-omics Understanding of ENS Cellular Makeup

      Classification of Enteric Neurons

      Enteric neurons are traditionally classified by their morphology, electrophysiological properties, and neurotransmitter expression. Whereas initial descriptions were based on guinea pigs,
      • Furness J.B.
      The enteric nervous system and neurogastroenterology.
      • Furness J.
      Types of neurons in the enteric nervous system.
      • Furness J.B.
      additional comparative data in mice provided murine-specific ENS characterization.
      • Qu Z.-D.
      • Thacker M.
      • Castelucci P.
      • Bagyánszki M.
      • Epstein M.L.
      • Furness J.B.
      Immunohistochemical analysis of neuron types in the mouse small intestine.
      Enteric neuron morphology was initially described by A. S. Dogiel
      • Dogiel A.S.
      Zur Frage über den feineren Bau der Herzganglien des Menschen und der Säugethiere.
      and has been characterized by imaging techniques that include intracellular dye filling, silver staining, retrograde tracing, immunohistochemistry, and electron microscopy. Neuronal cell bodies are typified by the shape and number of axons and dendrites in addition to where these processes project. Neuronal morphology is more complex and clearly defined in larger species such as pigs and humans. Thus, although several Dogiel subtypes can be identified in these mammals, only Dogiel type I and II morphologies are observed in mice.
      • Furness J.B.
      ,
      • Qu Z.-D.
      • Thacker M.
      • Castelucci P.
      • Bagyánszki M.
      • Epstein M.L.
      • Furness J.B.
      Immunohistochemical analysis of neuron types in the mouse small intestine.
      Type I neurons have flat, elongate, and irregular cell bodies with a single axon and numerous short dendrites, whereas type II neurons have smoother and larger cell bodies with multiple long axons. Type I neurons project and communicate with adjacent ganglia in the plexus and musculature, whereas type II neurons communicate with neurons throughout the gut wall, within and between ganglia, and the mucosa.
      • Furness J.B.
      Neurons are also classified by electrophysiological properties, which have been mainly characterized in guinea pigs
      • Nishi S.
      • North R.A.
      Intracellular recording from the myenteric plexus of the guinea-pig ileum.
      ,
      • Hirst G.D.S.
      • McKirdy H.C.
      A nervous mechanism for descending inhibition in guinea-pig small intestine.
      and mice.
      • Mao Y.
      • Wang B.
      • Kunze W.
      Characterization of myenteric sensory neurons in the mouse small intestine.
      Two main types of enteric neurons are categorized as having either synaptic- or AH (after hyperpolarization)-type electrophysiological properties that differ on the basis of action potential speed and magnitude, the length of AH potentials, and tetrodotoxin sensitivity. Synaptic-type neurons typically display Dogiel type I morphology and include interneurons and motor neurons, whereas AH-type neurons typically display Dogiel type II morphology and are considered sensory neurons.
      • Furness J.B.
      Defining the neurochemical coding of enteric neurons was a significant advancement in identifying neuronal subtypes and understanding how enteric neurons communicate with one another and target tissues. Enteric neurochemical coding has been defined by multiple approaches including immunohistochemistry in combination with retrograde tracing, electrophysiology, and pharmacology. Integrating these biomolecular data with morphologic and electrophysiological properties is the basis for current definitions of enteric neuron subtypes, which include motor neurons, interneurons, and sensory neurons. Although these definitions are based largely on studies in guinea pigs, many of the core features of enteric neuron subtypes are conserved between mice and humans. Excitatory and inhibitory motor neurons reside in the myenteric plexus and innervate the circular and longitudinal muscle of the intestine. Motor neurons are defined by Dogiel type I morphology in guinea pigs,
      • Furness J.B.
      The enteric nervous system and neurogastroenterology.
      • Furness J.
      Types of neurons in the enteric nervous system.
      • Furness J.B.
      but many have an unclear morphology in mice, characterized by small or medium-sized cell bodies without obvious dendrites.
      • Qu Z.-D.
      • Thacker M.
      • Castelucci P.
      • Bagyánszki M.
      • Epstein M.L.
      • Furness J.B.
      Immunohistochemical analysis of neuron types in the mouse small intestine.
      Excitatory motor neurons are cholinergic and release acetylcholine (ACh) but can also release tachykinins. Inhibitory motor neurons are nitrergic and release nitric oxide in addition to vasoactive intestinal peptide (VIP) and purines.
      • Furness J.B.
      The enteric nervous system and neurogastroenterology.
      • Furness J.
      Types of neurons in the enteric nervous system.
      • Furness J.B.
      ,
      • Qu Z.-D.
      • Thacker M.
      • Castelucci P.
      • Bagyánszki M.
      • Epstein M.L.
      • Furness J.B.
      Immunohistochemical analysis of neuron types in the mouse small intestine.
      Excitatory motor neurons express choline acetyltransferase (ChAT) and/or vesicular acetylcholine transporter in guinea pigs and mice; however, although both circular and longitudinal muscle-projecting excitatory motor neurons also express tachykinins in guinea pigs, tachykinins are not always expressed by the latter in mice. All inhibitory motor neurons in both guinea pigs and mice express nitric oxide synthase and VIP, whereas those innervating circular muscles can also express neuropeptide Y (NPY).
      • Furness J.B.
      ,
      • Qu Z.-D.
      • Thacker M.
      • Castelucci P.
      • Bagyánszki M.
      • Epstein M.L.
      • Furness J.B.
      Immunohistochemical analysis of neuron types in the mouse small intestine.
      Secretomotor/vasodilator neurons in guinea pigs have 3 known subtypes categorized as non-cholinergic VIP+ neurons, ChAT+/calretnin (Calb2)+ neurons, and ChAT+/NPY+ neurons.
      • Furness J.B.
      The enteric nervous system and neurogastroenterology.
      • Furness J.
      Types of neurons in the enteric nervous system.
      • Furness J.B.
      In mice these submuscosal neurons are categorized into 2 non-cholinergic and 1 cholinergic subtype(s). Both non-cholinergic sectretomotor and vasodilator neurons express VIP and NPY, whereas secretomotor neurons also express tyrosine hydroxylase. Cholinergic secretomotor neurons express ChAT, calcitonin gene-related peptide (CGRP), and somatostatin (SST).
      • Mongardi Fantaguzzi C.
      • Thacker M.
      • Chiocchetti R.
      • Furness J.B.
      Identification of neuron types in the submucosal ganglia of the mouse ileum.
      At least 4 types of interneurons are present in the small intestine of guinea pigs and mice. Ascending interneurons are cholinergic and also use tachykinins.
      • Furness J.B.
      The enteric nervous system and neurogastroenterology.
      • Furness J.
      Types of neurons in the enteric nervous system.
      • Furness J.B.
      ,
      • Qu Z.-D.
      • Thacker M.
      • Castelucci P.
      • Bagyánszki M.
      • Epstein M.L.
      • Furness J.B.
      Immunohistochemical analysis of neuron types in the mouse small intestine.
      These neurons are involved in local motility reflexes.
      • Furness J.
      Types of neurons in the enteric nervous system.
      ,
      • Furness J.B.
      Subtypes of descending interneurons involved in local motility reflexes include an ACh+/nitric oxide synthase+ subtype that is VIP+ in guinea pig but not mouse and an ACh+/serotonin+ subtype that is involved in secretomotor reflexes.
      • Furness J.B.
      The enteric nervous system and neurogastroenterology.
      ,
      • Furness J.B.
      ,
      • Qu Z.-D.
      • Thacker M.
      • Castelucci P.
      • Bagyánszki M.
      • Epstein M.L.
      • Furness J.B.
      Immunohistochemical analysis of neuron types in the mouse small intestine.
      A third type of descending interneuron is ACh+/SST+ and is involved in small intestinal migrating myoelectric complexes. Whereas all other interneuron subtypes are characterized by Dogiel type I morphology in guinea pig and mouse, this third subtype is characterized by distinct filamentous dendrites.
      • Furness J.
      Types of neurons in the enteric nervous system.
      ,
      • Furness J.B.
      ,
      • Qu Z.-D.
      • Thacker M.
      • Castelucci P.
      • Bagyánszki M.
      • Epstein M.L.
      • Furness J.B.
      Immunohistochemical analysis of neuron types in the mouse small intestine.
      Intrinsic primary afferent neurons (IPANs) regulate intrinsic reflex pathways of the intestine and are involved in chemosensation and mechanosensation. IPANs have Dogiel type II morphology and AH-type electrophysiology,
      • Furness J.
      Types of neurons in the enteric nervous system.
      ,
      • Furness J.B.
      and most express ChAT and CGRP. In guinea pigs IPANs also express tachykinins and isolectin B4.
      • Furness J.B.
      The enteric nervous system and neurogastroenterology.
      ,
      • Furness J.B.
      IPANs can be identified in mice, humans, and pigs by neurofilament (Nefm) staining
      • Qu Z.-D.
      • Thacker M.
      • Castelucci P.
      • Bagyánszki M.
      • Epstein M.L.
      • Furness J.B.
      Immunohistochemical analysis of neuron types in the mouse small intestine.
      and by advillin expression in mice, albeit the latter is expressed by other neuronal subtypes as well.
      • Melo CG. de S.
      • Nicolai E.N.
      • Alcaino C.
      • et al.
      Identification of intrinsic primary afferent neurons in mouse jejunum.
      Intestinofugal/viscerofugal afferent neurons (IFANs) reside in the myenteric plexus and project to prevertebral ganglia where they synapse with post-ganglionic sympathetic neurons. These cells contribute to intestinal functions that involve integration with other gastrointestinal organs. IFANs are rare (<1%) and typically display a Dogiel type I morphology (occasionally type II) in guinea pigs and mice. IFANs use ACh and VIP signaling but also express cholecystokinin, gastrin releasing peptide, and opioid-related peptides.
      • Furness J.B.
      The enteric nervous system and neurogastroenterology.
      ,
      • Furness J.B.
      ,
      • Qu Z.-D.
      • Thacker M.
      • Castelucci P.
      • Bagyánszki M.
      • Epstein M.L.
      • Furness J.B.
      Immunohistochemical analysis of neuron types in the mouse small intestine.
      ,
      • Miller S.M.
      • Szurszewski J.H.
      Relationship between colonic motility and cholinergic mechanosensory afferent synaptic input to mouse superior mesenteric ganglion.

      Classification of Enteric Glia

      Enteric glial heterogeneity and functions were covered extensively in a recent review
      • Seguella L.
      • Gulbransen B.D.
      Enteric glial biology, intercellular signalling and roles in gastrointestinal disease.
      and will not be reiterated here. Current glial subtypes are defined on the basis of morphology and anatomic location in the gut wall and may include differences in marker expression and response to various transmitters.
      • Gulbransen B.D.
      • Sharkey K.A.
      Novel functional roles for enteric glia in the gastrointestinal tract.
      ,
      • Boesmans W.
      • Lasrado R.
      • Vanden Berghe P.
      • Pachnis V.
      Heterogeneity and phenotypic plasticity of glial cells in the mammalian enteric nervous system.
      Canonical markers used to identify enteric glia include glial fibrillary acidic protein, S100B, Sox10,5,15,16 and Plp1.
      • Rao M.
      • Nelms B.D.
      • Dong L.
      • et al.
      Enteric glia express proteolipid protein 1 and are a transcriptionally unique population of glia in the mammalian nervous system.
      However, expression of glial markers within a single cell varies over time and is reflective of their current state.
      • Boesmans W.
      • Lasrado R.
      • Vanden Berghe P.
      • Pachnis V.
      Heterogeneity and phenotypic plasticity of glial cells in the mammalian enteric nervous system.
      ,
      • Rao M.
      • Nelms B.D.
      • Dong L.
      • et al.
      Enteric glia express proteolipid protein 1 and are a transcriptionally unique population of glia in the mammalian nervous system.
      Therefore, whether expression patterns are indicative of different glial subtypes or ongoing cellular dynamics is unclear.

      Omics in the Enteric Nervous System

      The technical details of current omics techniques and the strengths and challenges of applying these techniques to biomedical research are discussed in detail elsewhere.
      • Shapiro E.
      • Biezuner T.
      • Linnarsson S.
      Single-cell sequencing-based technologies will revolutionize whole-organism science.
      ,
      • Hasin Y.
      • Seldin M.
      • Lusis A.
      Multi-omics approaches to disease.
      ,
      • Misra B.B.
      • Langefeld C.
      • Olivier M.
      • Cox L.A.
      Integrated omics: tools, advances and future approaches.
      Here we briefly introduce omics techniques used in ENS research. Genomics identifies variation in DNA sequence, primarily using genome-wide association studies. Genome-wide association studies genetic code from diseased humans to identify genetic mutations (specifically single nucleotide polymorphisms) that may confer disease risk. Sequencing the entire genome or coding exome can also identify mutations. Transcriptomics identifies and quantifies RNA expression. Transcriptomics initially used microarray platforms but now primarily consists of sequencing (RNA-seq). Typically RNA-seq focuses on which genes are expressed and how their expression level changes. However, this method can also identify noncoding RNAs such as microRNAs or long noncoding RNAs that influence transcription of coding genes. Proteomics quantifies protein abundance, modification, and interaction. Compared with transcriptomics, proteomics captures a related but separate understanding of gene expression.
      • Hasin Y.
      • Seldin M.
      • Lusis A.
      Multi-omics approaches to disease.
      Altered pipelines of these fundamental omics modalities are used to attain omics data from specialized sources. For instance, DNA sequencing can specifically target variation in the bacterial 16s rRNA gene to taxonomically identify organisms within the gut microbiome. Transcriptomic studies can capture gene expression signatures from specified cells of interest by combining RNA-seq with cell-specific isolation strategies. These strategies range from using genetic driver mouse lines and performing cell sorting protocols to post hoc computational analyses focusing on known cell-specific pathways. One of the most recent of these is scRNA-seq, which measures gene expression within individual cells.
      • Shapiro E.
      • Biezuner T.
      • Linnarsson S.
      Single-cell sequencing-based technologies will revolutionize whole-organism science.
      ScRNA-seq is the primary technique used in ENS research to further resolve subtypes of enteric neurons and glia by grouping individual cells into clusters based on overall shared gene expression patterns. Similarly, proteomics technology can target specified subsets of proteins such as host and/or microbial metabolites based on their physical and chemical properties.
      • Hasin Y.
      • Seldin M.
      • Lusis A.
      Multi-omics approaches to disease.
      ,
      • Fischer R.
      • Bowness P.
      • Kessler B.M.
      Two birds with one stone: doing metabolomics with your proteomics kit.
      The details of omics methods used by the ENS studies discussed in this review are summarized in Table 1.
      Table 1Omics Dataset Metadata and Review Criteria: Number of Methods Used and Species/Gastrointestinal Regions Examined in ENS Omics Datasets
      SectionOmics methodSpeciesRegion
      Cell subtype markersscRNA-seq20–24,31,32Mouse20–24Colon20,21,23,31,32
      RNA-seq21Human20,21,23,31,32Small intestine20–24
      Compares regionsscRNA-seq20,21,23Mouse20,21,23,30,34Colon20,21,23,30,34
      RNA-seq21,30,34Human20,21,23Small intestine20,21,23,30,34
      Compares speciesscRNA-seq20–23,39Mouse20,21,23,25,39Colon20,21,23,41
      RNA-seq21,25,41Human20,21,23,39Small intestine20,21,23,25,41
      Zebrafish41Cell culture39
      Compares sexesscRNA-seq20–23Mouse20–23Colon20,21,23
      RNA-seq21Human20,21,23Small intestine20–23
      DysmotilityscRNA-seq23Mouse23,43Colon23,44,45,49
      RNA-seq43–45Human23,42,44,45Small intestine23,43
      WES42Rat49
      MALDI-TOF MS49
      DevelopmentscRNA-seq22,54–57,59Mouse22,25,52–54Colon41,50,52,53,55–58
      RNA-seq25,41,50,58Human42,55–59Small intestine22,25,41,50,52,53,55–58
      WES42Zebrafish41,50Cell culture54,59
      Microarray52,53
      Neuroimmune communicationscRNA-seq20,33Mouse20,33,60,61,70Colon20,70,72
      RNA-seq60,61,70,72Human20,71Small intestine20,33,60,61,72
      Microarray71Rat72Cell culture71
      DysbiosisscRNA-seq65Mouse30,34,81,65,73–76,78–80Colon30,34,65,74,76–81
      RNA-seq30,34,77Human75,82Small intestine30,34,73,77
      GWAS82Rat77
      LCMS73
      16S rRNA-seq74–76,78–81
      Gastrointestinal disease markersscRNA-seq20,23Mouse20,23,53,70Colon20,23,45,53,70,83,89,90
      RNA-seq45,70Human20,23,45,83,86,87,89,90Small intestine20,23,53
      Microarray53,89
      GWAS86,87
      LCMS83,90
      GWAS, genome-wide association study; LCMS, liquid chromatography-mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; RNA-seq, RNA-sequencing; 16S rRNA-seq, 16S rRNA gene sequencing; scRNA-seq, single-cell RNA-sequencing; WES, whole exome sequencing.
      Review search criteria: Full-text primary research articles were selected from the PubMed database using the following search terms:
      ("neurons"[MeSH Terms] OR "neuroglia"[MeSH Terms] OR "Ganglia, Spinal"[MeSH Terms] OR "Enteric Nervous System"[MeSH Terms] OR "Colon/innervation"[MAJR] OR "dorsal root ganglia"[All Fields] OR "neuron"[Title/Abstract] OR "enteric glia"[All Fields] OR "glia"[Title/Abstract]) AND ("computational biology"[MeSH Terms] OR "sequence analysis"[MeSH Terms] OR "high throughput"[Title/Abstract] OR "sequencing"[All Fields] OR "next generation"[All Fields]) AND ("gastrointestinal diseases"[MeSH Terms] OR "gastrointestinal tract"[MeSH Terms] OR "Gastrointestinal Microbiome"[MeSH Terms] OR "gastrointestinal"[Title/Abstract] OR "bowel"[Title/Abstract] OR "gut"[Title/Abstract]) NOT Review[Publication Type].
      From these results articles were screened for using high-throughput ‘omics’ methods in the enteric nervous system or referencing enteric nervous cells. A few newer articles were selected outside this due to backlog in MeSH classification.

      Using Omics to Define Cellular Subtypes in the Enteric Nervous System

      Genetic Markers of ENS Cell Subtypes

      Data available from several prominent scRNA-seq studies of the ENS vastly expand the ability to investigate ENS heterogeneity.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • et al.
      Molecular architecture of the mouse nervous system.
      Here we highlight collective findings across these data that identify potential novel cellular markers. Although further examination is required to truly establish whether these markers identify ENS cellular subtypes, these data support additional complexity in the current neurochemical coding of enteric neurons and glia. Our interpretation of synthesized findings is summarized in Figure 2 and Table 2. Some correlations in this table that are not mentioned in the text were based on Wright et al
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      (Table 4) or simply list all clusters the original authors designate as putative neuronal subtypes. Also of note, the dataset from Morarach et al
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      represents an extended capturing of enteric neurons of the same age and region as the dataset presented in Zeisel et al,
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • et al.
      Molecular architecture of the mouse nervous system.
      and thus Morarach et al is primarily discussed below. However, work from Zeisel et al
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • et al.
      Molecular architecture of the mouse nervous system.
      is retained to discuss glia.
      Figure thumbnail gr2
      Figure 2Putative markers of ENS cell subtypes. Novel genetic markers of enteric neuron and glia subtypes from scRNA-seq research. Future genetic and functional research could validate these molecules as important functional or developmental requirements for ENS cell subtypes. EMN, excitatory motor neuron; IFAN, intestinofugal afferent neuron; IMN, inhibitory motor neuron; INT, interneuron; IPAN, intrinsic primary afferent neuron; SM/VD, secretomotor/vasodilator neuron.
      Table 2Putative Enteric Neuron and Glia Subtypes and Co-Expression Markers
      No. of ENS cell subtypes proposed by scRNA-seq
      Drokhlyansky et al
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      May-Zhang et al
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      Morarach et al
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      Wright et al
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      Zeisel et al24,
      Zeisel et al24 include a dataset collected from the same region and stage as Morarach et al.22
      Speciesmouse | humanmousemousemousemouse
      AgeadultadultPN21E17.5 | adultPN21
      Regionileum, colon | colonduodenum + ileum + colonsmall intestineintestine | colonsmall intestine
      No. of EMN3, 3–5 | 4241 | 1–2
      No. of IMN2, 4–7 | 5220–2 | 0–2
      No. of INT2, 2–3 | 242–52–3 | 4–6
      No. of IPAN3, 3–4 | 1231 | 1–2
      No. of Glia3 | 3–6— | 47
      Shared neurochemical markers of putative ENS cell subtypes
      Cell subtype (pre-omics murine markers)Drokhlyansky et al
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      May-Zhang et al
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      Morarach et al
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      Wright et al
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      Zeisel et al24,
      Zeisel et al24 include a dataset collected from the same region and stage as Morarach et al.22
      Novel putative co-markers
      EMN, circular muscle (Chat, Tac1, ±Calb2)PEMN 2Cluster 3ENC3-4Chat 2, Chat 3ENT6Gfra2, Oprk1, Htr4, Piezo1
      EMN, longitudinal muscle (Chat, Calb2, ± Tac1)PEMN 1,3-5Cluster 0ENC1-2Chat 1ENT4-5
      IMN, circular muscle (Nos1/2, Vip, ± Npy)PIMN 1-7Cluster 2ENC8Nos 1-2ENT2-3Ass1, Gfra1, Etv1
      IMN, longitudinal muscle (Nos1/2, Vip)PIMN 1-7Cluster 1ENC9Nos 1-2ENT2-3
      INT, descending (Nos1/2, Chat)PIN 1-3Cluster 10ENC10Chat 4Gad2
      INT, descending (Sst, Chat, ± Calb2PSN 4Cluster 9ENC5
      INT, descending (Chat, 5-HT related genes)PIN 1-3Cluster 6sENC12 (subset)ENT7 (subset?)
      INT, ascending (Chat, Tac1, ± Calb2)PIN 1-3Cluster 3sENC4Nos1ENT6
      IPAN (Chat, Nefm, Calca/b, Calb1, ± Calb2)PSN 1Cluster 5ENC6CalcbENT9Nmu, Nog, Dlx3
      Secretomotor, non-cholinergic (Vip, Npy, Th, ± Calb)PSVN 1Glp2r
      Vasodilator, non-cholinergic (Vip, Npy, Calb, Th-)PSVN 1
      Secretomotor, cholinergic (Chat, Calca/b, Sst, Calb)PSVN 2, PSN 4
      Catecholaminergic neurons (Th)PSVN 1-2, PIN 1, PIMN 3Cluster 9ENC11Nos1 cluster 2
      IFAN (Cck)PSN3Cluster 7sENC7ENT8
      Glia (Sox10, S100β, Gfap, Plp1)Glia 1-3Glia1-4ENTG1-7Slc18a2
      EMN, excitatory motor neuron; IFAN, intestinofugal afferent neuron; IMN, inhibitory motor neuron; INT, interneuron; IPAN, intrinsic primary afferent neuron.
      a Zeisel et al
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • et al.
      Molecular architecture of the mouse nervous system.
      include a dataset collected from the same region and stage as Morarach et al.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.

      Motor Neurons

      At least 2–5 subtypes of putative excitatory motor neurons were identified in single cell transcriptional studies.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • et al.
      Molecular architecture of the mouse nervous system.
      Pre-omics guinea pig data suggest longitudinal muscle-innervating excitatory motor neurons express calretnin (Calb2), whereas those innervating circular muscle do not.
      • Furness J.B.
      On the basis of this expression pattern, cell clusters ENC 1-2 from Morarach et al
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      innervate longitudinal muscle, whereas ENC 3-4 innervate circular muscle. However, pre-omics data in mouse suggest circular muscle-innervating neurons may also express calretnin
      • Qu Z.-D.
      • Thacker M.
      • Castelucci P.
      • Bagyánszki M.
      • Epstein M.L.
      • Furness J.B.
      Immunohistochemical analysis of neuron types in the mouse small intestine.
      and complicates this alignment. This murine pattern of Calb2 expression aligns with excitatory clusters in May-Zhang et al,
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      where cluster 0 innervates longitudinal muscle and cluster 3 innervates circular muscle. Regardless of functional classification, some novel biomarkers are shared across excitatory motor neuron subtypes and may support further functional subtyping with future investigation. These include combinations of Gfra2, Oprk1, Htr4, and Piezo1. However, Piezo1 is also expressed by inhibitory motor neurons, and Gfra2 is also expressed by IPANs and SST+ interneurons; thus, specific combinations of these markers may be required to identify excitatory neuronal subtypes.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      Although broad functional excitatory subtypes within scRNA-seq clusters are still unclear, these datasets suggest a rarer excitatory motor neuron variant with novel markers. In May-Zhang et al
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      and Morarach et al
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      this cell type exists within Cluster 3 and ENC4, respectively. These cells express high levels of the 5-HT2B receptor gene Htr2b in addition to genes encoding a calcium binding protein (Necab2), an enzyme that catalyzes the last step in the biosynthesis of Lewis X antigen (Fut9), and a transcription factor involved in inducible gene transcription during immune responses (Nfatc1). Fut9 and Nfatc1 were also expressed by the Chat 3 neuron cluster defined by Wright et al.
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      This cluster could correspond to PEMN2 in Drokhlyanksy et al
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      because of higher enkephalin (Penk) expression compared with other putative excitatory motor neurons, as also seen in other datasets.
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      ,
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      ,
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • et al.
      Molecular architecture of the mouse nervous system.
      Functional validation of these expression markers may shed light on the existence of this peculiar variant.
      At least 2–4 inhibitory motor neuron subtypes were identified in single cell transcriptional studies based on Nos1 and Vip expression.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • et al.
      Molecular architecture of the mouse nervous system.
      Interestingly, Drokhlyansky et al
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      identified 7 inhibitory neuron clusters, PIMN 1–7. Although all these clusters express Nos1, PIMN 1–4 express higher levels of Vip, suggesting relative Vip expression as a means of stratifying subtypes. Pre-omics murine data suggest inhibitory motor neurons innervating circular muscle may also express neuropeptide y (Npy), whereas those innervating longitudinal muscle do not.
      • Qu Z.-D.
      • Thacker M.
      • Castelucci P.
      • Bagyánszki M.
      • Epstein M.L.
      • Furness J.B.
      Immunohistochemical analysis of neuron types in the mouse small intestine.
      This supports clustering from May-Zhang et al
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      and Morarach et al
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      where cluster 2 and ENC8 innervate circular muscle, whereas cluster 1 and ENC9 innervate longitudinal muscle, respectively. Potential new co-markers for inhibitory motor neurons include argininosuccinate synthase 1 (Ass1),
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      ,
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      Gfra1, the receptor for glial cell line-derived neurotrophic factor (GDNF),
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      and Etv1.
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      ,
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • et al.
      Molecular architecture of the mouse nervous system.
      However, Ass1 is expressed by other neurons as well,
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      ,
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      and Gfra1 and Etv1 are also expressed in putative interneurons and/or IPANs.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      Perhaps only co-expression of all these markers is specific to inhibitory motor neurons.
      Drokhlyansky et al
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      identified 2 clusters that potentially correspond to secretomotor/vasodilator neurons, PSVN 1 and PSVN 2. These clusters were characterized as secretomotor/vasodilator neurons on the basis of expression of the non-prototypical marker glucagon-like peptide 2 receptor (Glp2r). However, PSVN 1 expresses relatively low levels of Chat and is possibly non-cholinergic, whereas PSVN 2 expresses relatively higher Chat and may be a cholinergic secretomotor neuron. A putative sensory neuron cluster PSN 4 expresses Sst and Calcb, the beta form of CGRP, and therefore may also identify cholinergic secretomotor neurons. Other studies did not sequence submucosal plexus tissue and therefore did not describe secretomotor/vasodilator neurons.
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.

      Catecholaminergic Neurons

      Pre-omics research supports catecholaminergic neurons in gut that signal via dopamine or norepinephrine/noradrenaline and express tyrosine hydroxylase (Th). Noradrenergic signaling within the ENS is thought to be solely from extrinsic neuronal projections, whereas dopaminergic neurons reside within the ENS.
      • Memic F.
      • Knoflach V.
      • Morarach K.
      • et al.
      Transcription and signaling regulators in developing neuronal subtypes of mouse and human enteric nervous system.
      • Li Z.S.
      • Pham T.D.
      • Tamir H.
      • Chen J.J.
      • Gershon M.D.
      Enteric dopaminergic neurons: definition, developmental lineage, and effects of extrinsic denervation.
      • Rao M.
      • Gershon M.D.
      The bowel and beyond: the enteric nervous system in neurological disorders.
      Dopaminergic enteric neurons are rare neurons that develop relatively late (after E18) and express dopamine active transporter (Dat) and the dopamine metabolite DOPAC in addition to Th.
      • Memic F.
      • Knoflach V.
      • Morarach K.
      • et al.
      Transcription and signaling regulators in developing neuronal subtypes of mouse and human enteric nervous system.
      ,
      • Li Z.S.
      • Pham T.D.
      • Tamir H.
      • Chen J.J.
      • Gershon M.D.
      Enteric dopaminergic neurons: definition, developmental lineage, and effects of extrinsic denervation.
      These neurons are important regulators of gastrointestinal motility
      • Rao M.
      • Gershon M.D.
      The bowel and beyond: the enteric nervous system in neurological disorders.
      and are involved in motor circuitry, but what specific types of neurons express dopamine is unclear. Interestingly, scRNA-seq studies identify clusters with Th expression, but these clusters do not express Dat and often also express dopamine beta-hydroxylase (Dbh)
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      ,
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      and therefore may actually be noradrenergic neurons. Whether this truly identifies intrinsic noradrenergic neurons or is contamination from extrinsic fibers is unclear, but we will refer to these as catecholaminergic neurons here. Regardless, these data suggest subtypes of motor neurons, interneurons, and sensory neurons are catecholaminergic. This includes PSVN1-2, PIN1, and PIMN3 from Drokhlyanksy et al,
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      cluster 9 from May-Zhang et al,
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      ENC11 from Morarach et al,
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      and Nos1 cluster 2 in E17.5 embryonic mice samples from Wright et al.
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      Many of the clusters also express Npy and thus could use this as a co-expression marker. On the basis of prior investigations of enteric dopaminergic neurons they may also co-express combinations of Ebf1, Meis2, Etv1, Satb1, Klf7, and Sox6.
      • Memic F.
      • Knoflach V.
      • Morarach K.
      • et al.
      Transcription and signaling regulators in developing neuronal subtypes of mouse and human enteric nervous system.
      However, these expression markers were determined during ENS development from gastric tissue and may not reflect mature neuronal expression patterns.

      Interneurons

      Interneurons are more difficult to define by gene signatures in scRNA-seq studies, likely in part because of the overlap of their established markers with other neuron types. Despite this, between 2 and 5 subtypes of putative interneurons were proposed.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • et al.
      Molecular architecture of the mouse nervous system.
      May-Zhang et al
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      and Morarach et al
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      suggest interneuron subtypes that align with current functional classifications. One subtype (cluster 1021 and ENC10
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      ) corresponds to Nos1+ descending interneurons. These clusters express Nos1 and glutamate decarboxlyase (Gad2) in addition to various levels of Chat. Somatostatin/Sst+ descending interneurons may correspond to cluster 921 and ENC5.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      Sensory neuron cluster PSN 4 from Drokhlyansky et al
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      is the main cluster that expresses Sst (very little is expressed in their defined interneuron clusters) and therefore may also correspond to this interneuron. The serotonergic/5-HT+ descending interneuron subtype includes cluster 6s
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      and a subset of ENC12
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      characterized by co-expression of serotonin transporter Sert (Slc6a4), monoamine vesicle transporter Vgat2, and/or dopa decarboxylase (Ddc). The ascending interneuron subtype identified by May-Zhang et al
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      (cluster 3s) was characterized by high Chat and Tac1 with potential co-expression of Calb2. These are also expressed in possible interneurons ENC4 in Morarach et al.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      One cell type is conserved among datasets but classified as both a potential interneuron
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      ,
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      or potential IPAN.
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      ,
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      These cells co-expressed combinations of markers Nxph2, Cckar, Slc17a6, Sstr5, and Ntng1.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • et al.
      Molecular architecture of the mouse nervous system.
      These clusters are highly heterogeneous and likely represent multiple neuronal subtypes merged in one semi-identified entity. This is supported by their expression of Nxph2, which is part of the neurexophilin family and modulates synaptic plasticity. Functional ablation by these suggested markers may help resolve the identities of these cells.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.

      Intrinsic Primary Afferent Neurons

      Between 1 and 4 cell clusters are proposed to correspond to IPANs on the expression of CGRP gene Calcb, coding for CGRPβ. Although Calcb is the primary CGRP gene expressed by and considered an IPAN marker,
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • et al.
      Molecular architecture of the mouse nervous system.
      its paralog gene Calca (coding for CGRPα) is expressed in a subset of IPAN clusters in murine juvenile intestine and adult colon,
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      ,
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      ,
      • Spencer N.J.
      • Sorensen J.
      • Travis L.
      • Wiklendt L.
      • Costa M.
      • Hibberd T.
      Imaging activation of peptidergic spinal afferent varicosities within visceral organs using novel CGRPα-mCherry reporter mice.
      and thus its expression may be age- or region-dependent. One potential Calca+/Calbc+ IPAN subtype co-expresses combinations of neuromedin U (Nmu), noggin (Nog), and homeobox Dlx3, corresponding to PSN 1,
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      cluster 5,
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      ENC6,
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      and Calcb.
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      Another putative Calca+/Calbc+ IPAN subtype expresses a combination of brain-derived neurotrophic factor (Bdnf), mechanosensitive ion channel Piezo2, and Cck. Drokhlyansky et al
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      suggest this is a single subtype (PSN3), whereas Morarach et al
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      identified a Bdnf+Piezo2+ subtype (ENC12) and a separate cluster (ENC7) as Bdnf+Cck+.
      Although CGRP genes are expressed in IPANs and therefore a putative means of identifying IPAN clusters, these genes are also expressed by secretomotor/vasodilator neurons and therefore may confound IPAN clustering.
      • Mongardi Fantaguzzi C.
      • Thacker M.
      • Chiocchetti R.
      • Furness J.B.
      Identification of neuron types in the submucosal ganglia of the mouse ileum.
      Morarach et al
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      reported 3 putative IPAN types by clustering: ENC6, ENC7, and ENC12. They subsequently investigated the morphologies of these subtypes, delineating true IPANs by Dogiel type II morphologies and neuronal projections that traveled through layers of the gut wall. They determined that ENC6 and a subset of ENC12 appeared morphologically as IPANs, whereas ENC7 was characterized as atypical IPANs or perhaps even intestinofugal neurons. Because the ENC12 subset expresses Piezo2, these may be mechanosensitive IPANs. This work demonstrates further validation of omics subtype classifications is necessary and suggests some of the other proposed IPAN subtypes may not be true IPANs or be atypical IPANs or unique subtypes.
      Only 2 datasets categorize clusters as intestinofugal neurons/IFANs. Identification of these clusters was based on Cck expression as a known marker of guinea pig IFANs.
      • Furness J.B.
      In addition to ENC7 identified by Morarach et al,
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      May-Zhang et al
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      identified cluster 7s as intestinofugal. In addition, PSN 3 from Drokhlyansky et al
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      may also represent IFANs because these cells express Cck. Cluster 7s co-expresses Slc24a3 and Carmn in mice and humans. Carmn is a long noncoding RNA critical for cardiac muscle development and pathologic remodeling.
      • Ounzain S.
      • Micheletti R.
      • Arnan C.
      • et al.
      CARMEN, a human super enhancer-associated long noncoding RNA controlling cardiac specification, differentiation and homeostasis.
      Although Carmn may play similar roles in IFAN development and neuroplasticity, this would require additional testing to determine the regulatory targets of the long noncoding RNA. Although not a scRNA-seq study, Muller et al
      • Muller P.A.
      • Matheis F.
      • Schneeberger M.
      • Kerner Z.
      • Jové V.
      • Mucida D.
      Microbiota-modulated CART + enteric neurons autonomously regulate blood glucose.
      identified cocaine- and amphetamine-regulated transcript (encoded by gene Cartpt) as a novel IFAN marker as well. However, in scRNA-seq studies Cartpt is expressed by several other neuronal clusters
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      ,
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      and therefore would need to be used in combination with other markers for IFAN identification.

      Enteric Glia

      Between 1 and 7 subtypes of enteric glia are classified on the basis of expression patterns. Of note, the 2 studies that found only 1 glial subtype sequenced biopsy tissue from IBD patients and thus only captured mucosal glia.
      • Kinchen J.
      • Chen H.H.
      • Parikh K.
      • et al.
      Structural remodeling of the human colonic mesenchyme in inflammatory bowel disease.
      ,
      • Smillie C.S.
      • Biton M.
      • Ordovas-Montanes J.
      • et al.
      Intra- and inter-cellular rewiring of the human colon during ulcerative colitis.
      In mice the number of glial subtypes may differ throughout development or by gut region, because 7 subtypes were identified in postnatal day (PN) PN21 mouse small intestine,
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • et al.
      Molecular architecture of the mouse nervous system.
      whereas adult mouse colon only has 2–4 subtypes.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      ,
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      ,
      • Progatzky F.
      • Shapiro M.
      • Chng S.H.
      • et al.
      Regulation of intestinal immunity and tissue repair by enteric glia.
      Alternatively, this is due to the differences in resolution because these datasets contain varied numbers of captured glial cells. One glial subtype in PN21 mice is classified as a progenitor cell because of topoisomerase Top2a expression.
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • et al.
      Molecular architecture of the mouse nervous system.
      This is not a defining marker identified in adult enteric glia and suggests this glial subtype plays a larger role in the juvenile development period than adulthood. Expression of the vesicular monoamine transporter Slc18a2 and GDNFα receptors further supports developmental convergence of glial subtypes, because these mark 2–3 subtypes at PN21 and only 1 subtype each in adulthood.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      ,
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • et al.
      Molecular architecture of the mouse nervous system.
      Several other markers of enteric glial subtypes are identified only in a single study. These include differential expression of Foxd3 and Aldh1a3 in PN21 subtypes
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • et al.
      Molecular architecture of the mouse nervous system.
      and neurotensin receptor Ntsr1 in adult subtypes.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      In humans, 1 glial subtype expressed P2Y12R, NRXN1, and XKR4,
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      which could also be potential markers. Although differential expression of these markers could reflect developmental and species differences, this heterogeneity may also be due to the dynamic and reactive expression patterns known of enteric glia in varying environments.
      • Boesmans W.
      • Lasrado R.
      • Vanden Berghe P.
      • Pachnis V.
      Heterogeneity and phenotypic plasticity of glial cells in the mammalian enteric nervous system.
      ,
      • Rao M.
      • Nelms B.D.
      • Dong L.
      • et al.
      Enteric glia express proteolipid protein 1 and are a transcriptionally unique population of glia in the mammalian nervous system.
      Regardless, the paucity of glial discussion in ENS scRNA-seq studies cannot resolve this, and glial heterogeneity warrants further investigation. Some of these studies did in fact create glial expression datasets,
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      ,
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      ,
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • et al.
      Molecular architecture of the mouse nervous system.
      and reanalysis or meta-analysis of these data with a glial focus would likely help resolve these differences and identify additional subtype expression patterns.

      Region-, Species-, and Sex-Dependent Expression

      Region-Dependent Expression

      The number of neuronal subtypes is mostly conserved across gut regions
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      ,
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      ; however, the proportion of neuronal types and subtypes varies. Catecholaminergic neurons are more highly concentrated in the duodenum than ileum, whereas Sst+ and Cck+ neuronal subtypes are more prevalent in the ileum.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      The ileum contains more sensory neurons than the colon, and the colon contains more secretomotor/vasodilator neurons. This difference likely reflects the colon’s need to regulate fluid absorption and secretion.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      Subtype-specific genes also vary between regions of the small and large intestine. Chat+/Nos+ descending interneurons, Gad2+ interneurons,
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      and/or Gad2+ secretomotor/vasodilator neurons
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      are more prevalent in the small intestine. Genes including Unc5d, Col25a1,
      • Obata Y.
      • Á Castaño
      • Boeing S.
      • et al.
      Neuronal programming by microbiota regulates intestinal physiology.
      Htr2b,
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      ,
      • Muller P.A.
      • Matheis F.
      • Schneeberger M.
      • Kerner Z.
      • Jové V.
      • Mucida D.
      Microbiota-modulated CART + enteric neurons autonomously regulate blood glucose.
      and Htr3a
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      are all highly enriched in the colon and are putative markers for subtypes of ascending interneurons, inhibitory motor neurons,
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      excitatory motor neurons,
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      ,
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      and IPANs/inhibitory motor neurons,
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      respectively.
      Regional expression patterns also reflect gut physiology. Genes involved in signaling with enteroendocrine cells such as Cckar,
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      Tacr3, Npy, and the glucagon receptor Gcgr are highest in duodenal neurons.
      • Muller P.A.
      • Matheis F.
      • Schneeberger M.
      • Kerner Z.
      • Jové V.
      • Mucida D.
      Microbiota-modulated CART + enteric neurons autonomously regulate blood glucose.
      Meanwhile, distal gut segments are enriched for the glutamate receptor,
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      Sst, Cartpt, Penk, and Grp.
      • Muller P.A.
      • Matheis F.
      • Schneeberger M.
      • Kerner Z.
      • Jové V.
      • Mucida D.
      Microbiota-modulated CART + enteric neurons autonomously regulate blood glucose.
      Glutamate is mostly absorbed in terminal ileum,
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      whereas these other distally enriched genes are important in colonic motility.
      • Muller P.A.
      • Matheis F.
      • Schneeberger M.
      • Kerner Z.
      • Jové V.
      • Mucida D.
      Microbiota-modulated CART + enteric neurons autonomously regulate blood glucose.
      Transcription factor Pou3f3 (Brn1) is also higher in the colon than small intestine.
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      ,
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      ,
      • Muller P.A.
      • Matheis F.
      • Schneeberger M.
      • Kerner Z.
      • Jové V.
      • Mucida D.
      Microbiota-modulated CART + enteric neurons autonomously regulate blood glucose.
      ,
      • Obata Y.
      • Á Castaño
      • Boeing S.
      • et al.
      Neuronal programming by microbiota regulates intestinal physiology.
      This gene is important in central nervous system development,
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      so perhaps it plays a role in colonic ENS development as well. Finally, Ahr is highly expressed in colonic neurons,
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      and neuronal Ahr integrates microbial cues with colonic motility.
      • Obata Y.
      • Á Castaño
      • Boeing S.
      • et al.
      Neuronal programming by microbiota regulates intestinal physiology.
      For other genes with regional variation their expression may be functionally relevant but is currently unclear. Duodenal neurons enrich for growth factors such as Fst1 and Wif1, whereas distal neurons enrich for Agrp,
      • Muller P.A.
      • Matheis F.
      • Schneeberger M.
      • Kerner Z.
      • Jové V.
      • Mucida D.
      Microbiota-modulated CART + enteric neurons autonomously regulate blood glucose.
      Ano5, Pde1c, Panrt2,
      • Obata Y.
      • Á Castaño
      • Boeing S.
      • et al.
      Neuronal programming by microbiota regulates intestinal physiology.
      Pantr1, and Zfhx3.
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      Neurotransmitter ligand/receptor expression differs across the colon as well and highlights colonic region-dependent signaling priorities. Somatostatin (Sst) signaling may be more prominent in proximal colon. Meanwhile, several pathways are distally enriched, including serotonin (Htr3a and Htr3b), glutamate (Gria3 and Grid1), ACh (Chrna7 and Chrm1), chromogranin B (Chgb), enkephalin (Penk), norepinephrine (NE), secretogranin II (Scg2), and Vip.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      However, Htr3a and Htr3b are higher in the duodenum than ileum,
      • Muller P.A.
      • Matheis F.
      • Schneeberger M.
      • Kerner Z.
      • Jové V.
      • Mucida D.
      Microbiota-modulated CART + enteric neurons autonomously regulate blood glucose.
      suggesting additional roles for these receptors proximally. Particular neuronal subtypes also demonstrate regional colonic distribution. Calca+/Nog+/Nmu+ sensory neurons are more highly prevalent in the proximal colon, whereas Lgr5+ inhibitory motor neurons are more common distally.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      Enteric glial gene expression also varies between gut locations, including between colonic mucosa and muscularis externa in humans. Mucosal glia more highly expressed ferritin genes (FTH1 and FLT), heat shock protein CRYAB, and galectin-1 (LGALS1), whereas myenteric glia expressed genes involved in cell adhesion such as NRXN1 and CADM2.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      These are likely also reflective of known gut physiology. Ferritin helps regulate iron absorption in the mucosa,
      • Charlton R.W.
      • Jacobs P.
      • Torrance J.D.
      • Bothwell T.H.
      The role of the intestinal mucosa in iron absorption.
      whereas CRYAB modulates mucosal inflammation and barrier integrity,
      • Xu W.
      • Guo Y.
      • Huang Z.
      • et al.
      Small heat shock protein CRYAB inhibits intestinal mucosal inflammatory responses and protects barrier integrity through suppressing IKKβ activity.
      suggesting mucosal glia participate in these functions. This is not a surprising role for enteric glia because it is important in related peripheral glia such as Schwann cells.
      • Mirsky R.
      • Jessen K.R.
      • Schachner M.
      • Goridis C.
      Distribution of the adhesion molecules N-CAM and L1 on peripheral neurons and glia in adult rats.

      Species-Dependent Expression

      Historically enteric neurons from smaller mammals are considered smaller, simpler, and easier to classify than those from larger species such as humans. Perhaps this is in part due to different proportions of neuronal cell types, because these types display varying cell body size and complexity.
      • Furness J.B.
      ScRNA-seq research supports this phenomenon because the proportions of neuronal types differ between species. Both excitatory and inhibitory motor neurons are enriched in humans, whereas all the other types (sensory neurons, interneurons, and secretomotor/vasodilator neurons) are less abundant. However, single cell collection methods have variable efficacy in capturing rare cells or cells with differing morphologies,
      • Valihrach L.
      • Androvic P.
      • Kubista M.
      Platforms for single-cell collection and analysis.
      so to what extent these findings are due to technical limitations is unknown. ScRNA-seq research highlights both conserved functions and complex molecular differences. For instance, development of the ENS is highly conserved. Parallel scRNA-seq of mouse and human neural crest cells identified similar progression of gene expression patterns between both species, suggesting conserved mechanisms of neural fate determination.
      • Lau S.T.
      • Li Z.
      • Pui-Ling Lai F.
      • et al.
      Activation of hedgehog signaling promotes development of mouse and human enteric neural crest cells, based on single-cell transcriptome analyses.
      Specifically, ligand-receptor interactions important for neuronal development are highly conserved between mice and humans.
      • Memic F.
      • Knoflach V.
      • Morarach K.
      • et al.
      Transcription and signaling regulators in developing neuronal subtypes of mouse and human enteric nervous system.
      However, hedgehog signaling is subtly different between species, promoting both neuronal and glial differentiation in mice but only neuronal differentiation in humans.
      • Lau S.T.
      • Li Z.
      • Pui-Ling Lai F.
      • et al.
      Activation of hedgehog signaling promotes development of mouse and human enteric neural crest cells, based on single-cell transcriptome analyses.
      The number of neuronal subtypes based on neurochemical coding is also relatively conserved between species,
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      ,
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      and some co-markers are shared. Chat+ neurons in mice and humans express Galntl6, Tshz2, Alk, Bnc2,
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      Rbfox1, Pbx3, and Tbx2.
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      Nos+ neurons in both species express Dgkb
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      and Tbx3.
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      Putative interneurons express Grm7, and sensory neurons express Cbln2.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      Interestingly, secretomotor/vasodilator neurons from both species share markers with other neuron types and therefore may require co-expression patterns to identify. These neurons express Vip, Kcnd2, Etv1, and Scgn.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      However, murine and human enteric neuronal expression patterns are more different than similar and may reflect divergent molecular signaling mechanisms. May-Zhang et al
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      estimate that only 40% of neuron-specific genes are conserved between mice and humans, with variations in subtype- and location-dependent expression. This is interesting considering mouse and human gene expressions are considered more similar than different within brain regions.
      • Strand A.D.
      • Aragaki A.K.
      • Baquet Z.C.
      • et al.
      Conservation of regional gene expression in mouse and human brain.
      These findings may also be influenced by technical differences. Nonetheless, these suggest differential regulation of feeding and energy within the ENS through the melanocortin, leptin, and serotonin pathways. Human neurons highly express the melanocortin receptor MC1R, whereas mouse neurons express its antagonist Agrp. Similarly, human neurons highly express leptin receptor LEPR and serotonin synthesis enzyme TPH2. Murine Lepr expression was not detected in scRNA-seq studies, whereas Tph2 was undetected or detected in only a very small proportion (0.2%) of enteric neurons.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      ,
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      Differences in gene expression between mice and humans further complicate discovery of subtype markers as well. Human neurons sampled and sequenced by Drokhlyansky et al
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      did not express CHAT, and the authors used expression of the choline transporter SLC5A7 to mark these neurons instead. They hypothesize the lack of CHAT expression is due to their specific methods,
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      which is likely the case because other studies do not report this same concern in scRNA-seq human data.
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      It is important to note that the strategy of using Slc5a7 to mark cholinergic neurons would not likely be appropriate in mice because Slc5a7 may also be expressed by nitrergic neurons
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • et al.
      Molecular architecture of the mouse nervous system.
      ; however, Slc18a3 as used by Morarach et al
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      where Chat detection was low could be another alternative. Cell-specific markers for IPANs are unclear and therefore make species comparisons somewhat premature. May-Zhang et al
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      suggest that Klhl1 may be a species-specific marker of murine IPANs, labeling an entirely different subset of neurons in humans classified as CALB1+/NXPH2+ Dogiel type III neurons of the small intestine. However, Klhl1 is expressed by non-IPAN mouse neuronal subtypes as well,
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      ,
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      and KLHL1+ human neurons may reflect unidentified neuronal subtype(s) conserved between mice and humans. Nmu expression likely reflects true IPANs based on its clear and conserved expression by murine and human IPANs,
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      expression in putative murine IPAN clusters across datasets,
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • et al.
      Molecular architecture of the mouse nervous system.
      and also morphologically verified.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      Enteric glial subtypes appear somewhat conserved between humans and mice, where 3 clusters were identified in each species by Drokhlyansky et al.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      These clusters may correspond to one other but are not explicitly compared. However, human glia demonstrates higher complexity because patient-specific subtypes also clustered, likely reflecting the impact of human genetic variability and disease status on gene expression. Enteric glial expression involving ENS development is mostly conserved between mice and zebrafish, but canonical marker expression differs. McCallum et al
      • McCallum S.
      • Obata Y.
      • Fourli E.
      • et al.
      Enteric glia as a source of neural progenitors in adult zebrafish.
      found that although some canonical markers such as Sox10 and Plp1b are expressed in zebrafish enteric glia, Gfap and S100b are not. Additional developmental genes such as Sox2 and Foxd3 are conserved between species, further validating the zebrafish as a reasonable organism to study mammalian ENS development.

      Sex-Dependent Expression

      Many of the established concepts regarding ENS neurochemical coding and physiology relied on data from studies that either did not consider sex as a variable or aimed to remove it as a variable. Current omics studies investigating sex differences also remain relatively limited. However, scRNA-seq studies that did assess sex differences did not observe overt sex-related differences in clustering of enteric neuron subtypes, regardless of age or species.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      ,
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      ,
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      Although ENS cell clustering is similar between sexes, there are still differentially expressed genes within all or specific clusters.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      ,
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      Although most of these genes are X- and Y-chromosome related, some are not. May-Zhang et al
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      observed that SLC6A14 and MUC5B are enriched in female human neurons, whereas Cntnap5a is higher in putative IPANs (cluster 5), and Sst is higher in excitatory motor neurons innervating longitudinal muscle (cluster 0) in female mice. Similarly, robust sex differences have not been observed between glial cell subtypes. It is currently unclear whether these data reflect a true lack of sex differences or are too underpowered to detect subtle sex differences. This would be an important area to address in future studies.

      Omics Contribution to Understanding Enteric Nervous System Dysfunction and Disease

      High-throughput omics data highlight ENS expression patterns and how they are altered in abnormal states. Here we discuss ENS gene expression in the context of dysmotility, development, communication with immune cells, and dysbiosis. Finally, we link known genetic disease markers with ENS expression. Highlights of these findings are summarized in Table 3.
      Table 3Genes and Proteins Involved in ENS Dysfunction and Disease Suggested by Omics
      Dysfunction/disease topicMajor finding(s) by omicsGenes/proteins of interestReferences
      DysmotilityGenes that affect the number of enteric neurons and modulate neuronal subtype populationsRAD21, Runx1, Dlx1, Dlx2, Sox626,42,43
      Genes/proteins that affect neuronal excitability/functionGABRG1, HTR2B, Gfra1, Secretoneurin21–23,44,45,49,50
      DevelopmentGenes important for ENS developmentSox6, Pbx3, Dlx1, Dlx2, Ascl1, Phox2b, Elavl426,43,50,52,53
      Genes involved in neuronal differentiation/subtype fate determinationSox6, Ascl1, SEMA3A, Etv1, Bnc222,26,55,58
      Genes expressed by glial progenitor clustersCOL20A1, TFAP2B, GFRA3, ARTN, RXRG55,56
      Neuroimmune communicationGenes/proteins for ligands or receptors in neuron-immune cell communicationCX3CL1, CX3CR1, β2-AR, Arg1, Oprm1, Cnr2, IL-12, IL-1820,60,61,65
      Identified IPAN as neuronal subtype that communicates with ILC2s to modulate immune responseNmu20–24
      Genes involved in glial-immune communicationCxcl10, S100b33,71,72
      DysbiosisGenes/proteins in ENS-gut microbiome communication that modify ENS functionAhr, NGF (host) chaperonin 60, SCFAs (microbes)34,73–76
      Genes in ENS-gut microbiome communication that affect cell survival/cell deathCartpt, IL1830,65
      Host genes affected by microbiome compositionMCT2, GRID2IP82
      Gastrointestinal disease markersKnown/novel markers for Hirschsprung disease (HSCR) expressed in enteric neuronsRET, PHOX2B, GFRA1, ECE1, ARF4, KIF5B, RAB8A20,83
      Known markers for Parkinson's disease (PD) expressed in enteric neuronsDLG2, SNCA, SCN3, Lrrk220
      Known markers for autism spectrum disorder (ASD) expressed in enteric neurons and/or enteric gliaGABRB3, DSCAM, NLGN3, NRXN1, ANK220,85
      Known/novel markers for inflammatory bowel disease (IBD) expressed in enteric neurons and/or enteric gliaPtger4, LSAMP, BACH2, NONHSAG044354, elastase 3a, cathepsin L, proteasome alpha subunit-420,70,87,89,90
      ENS, enteric nervous system; ILC2s, group 2 innate lymphoid cells; IPAN, intrinsic primary afferent neuron.

      Dysmotility

      Genome-wide association studies and related genetic studies have identified mutations associated with dysmotility in humans. How these mutations contribute to disease risk through gene expression is often unclear. Omics data suggest some of these mutations affect expression of genes involved in cell cycling and differentiation in the ENS. For instance, mutations in DNA repair gene RAD21 are associated with chronic intestinal pseudo-obstruction. This mutation lowers expression of neuronal differentiation factor Runx1, which subsequently reduces enteric neuron numbers and slows intestinal transit in zebrafish.
      • Memic F.
      • Knoflach V.
      • Morarach K.
      • et al.
      Transcription and signaling regulators in developing neuronal subtypes of mouse and human enteric nervous system.
      ,
      • Bonora E.
      • Bianco F.
      • Cordeddu L.
      • et al.
      Mutations in RAD21 disrupt regulation of apob in patients with chronic intestinal pseudo-obstruction.
      Transcription factors Dlx1 and Dlx2 are also important for bowel motility, where Dlx1/2 mutants have decreased Vip and increased Penk and Plp1 expression,
      • Wright C.M.
      • Garifallou J.P.
      • Schneider S.
      • et al.
      Dlx1/2 mice have abnormal enteric nervous system function.
      suggesting Dlx1/2 signaling modulates neuronal subtype populations and peripheral glia. Sox6 also helps drive neuronal subtype differentiation, and absence of Sox6-driven dopaminergic neurons contributes to gastroparesis in mice.
      • Memic F.
      • Knoflach V.
      • Morarach K.
      • et al.
      Transcription and signaling regulators in developing neuronal subtypes of mouse and human enteric nervous system.
      This may relate to symptoms of gastroparesis in Parkinson’s disease patients, but the connection to diagnosable human disease requires further investigation.
      Not surprisingly, omics data also support that altered neurotransmitter and neuromodulator signaling in the ENS contribute to dysmotility. A mutation in subunit gene GABRG1 of the excitatory ion channel GABA-A is associated with irritable bowel syndrome (IBS) and decreased GABRG1 expression in IBS patients.
      • Videlock E.J.
      • Mahurkar-Joshi S.
      • Hoffman J.M.
      • et al.
      Sigmoid colon mucosal gene expression supports alterations of neuronal signaling in irritable bowel syndrome with constipation.
      Expression of serotonin receptor HTR2B decreases in obstructed defecation patients.
      • Kim M.
      • Rosenbaum C.
      • Schlegel N.
      • et al.
      Obstructed defecation: an enteric neuropathy? an exploratory study of patient samples.
      Htr2b is primarily expressed by excitatory motor neuron subtypes
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      ,
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      and in the distal gut.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      ,
      • Muller P.A.
      • Matheis F.
      • Schneeberger M.
      • Kerner Z.
      • Jové V.
      • Mucida D.
      Microbiota-modulated CART + enteric neurons autonomously regulate blood glucose.
      These data suggest decreased prevalence or activity of excitatory motor neurons contributes to dysmotility, whereas others suggest roles for inhibitory neurons. GDNF signaling is typically highlighted in neuronal development
      • Gianino S.
      • Grider J.R.
      • Cresswell J.
      • Enomoto H.
      • Heuckeroth R.O.
      GDNF availability determines enteric neuron number by controlling precursor proliferation.
      ,
      • Soret R.
      • Schneider S.
      • Bernas G.
      • et al.
      Glial cell-derived neurotrophic factor induces enteric neurogenesis and improves colon structure and function in mouse models of Hirschsprung disease.
      because loss of this signaling during development leads to colonic aganglionosis in mice.
      • Shen L.
      • Pichel J.G.
      • Mayeli T.
      • Sariola H.
      • Lu B.
      • Westphal H.
      Gdnf haploinsufficiency causes Hirschsprung-like intestinal obstruction and early-onset lethality in mice.
      However, scRNA-seq highlighted a potential role for GDNF in acute dysmotility during adulthood as well, albeit further testing is warranted to see whether this has functional relevance. Wright et al
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      discovered that in adult mice GDNF receptor α (Gfra1) is preferentially expressed by nitrergic neurons and glia and confirmed that GDNF preferentially exerts its effects through Gfra1. Furthermore, GDNF signaling enhanced colonic contractility. For other neuropeptides involved in dysmotility the neuron populations affected are unclear. Secretoneurin is involved in gastrointestinal motility
      • Lopes L.V.
      • Marvin-Guy L.F.
      • Fuerholz A.
      • et al.
      Maternal deprivation affects the neuromuscular protein profile of the rat colon in response to an acute stressor later in life.
      ,
      • Roy-Carson S.
      • Natukunda K.
      • Chou H.
      • et al.
      Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment.
      and expressed by the majority of interganglionic enteric neurons.
      • Schürmann G.
      • Bishop A.E.
      • Facer P.
      • et al.
      Secretoneurin: a new peptide in the human enteric nervous system.
      Its precursor protein secretogranin II increases in these neurons in response to early life stress
      • Lopes L.V.
      • Marvin-Guy L.F.
      • Fuerholz A.
      • et al.
      Maternal deprivation affects the neuromuscular protein profile of the rat colon in response to an acute stressor later in life.
      and therefore may impact stress-dependent dysmotility.

      Development

      Genetic and omics studies investigating neuronal development are often in the context of Hirschsprung disease (HSCR). These studies could encompass their own review, and here we focus instead on increased resolution of the enteric neuronal development timeline and where differential expression of developmental genes may disrupt this.
      Many important transcription factors and signaling regulators for ENS development were discovered in early omics studies. These include now canonical developmental markers such as Sox6, Pbx3, Dlx1, Dlx2, Ascl1, Phox2b, and Elavl4.
      • Memic F.
      • Knoflach V.
      • Morarach K.
      • et al.
      Transcription and signaling regulators in developing neuronal subtypes of mouse and human enteric nervous system.
      ,
      • Wright C.M.
      • Garifallou J.P.
      • Schneider S.
      • et al.
      Dlx1/2 mice have abnormal enteric nervous system function.
      ,
      • Roy-Carson S.
      • Natukunda K.
      • Chou H.
      • et al.
      Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment.
      ,
      • Vohra B.P.S.
      • Tsuji K.
      • Nagashimada M.
      • et al.
      Differential gene expression and functional analysis implicate novel mechanisms in enteric nervous system precursor migration and neuritogenesis.
      ,
      • Heanue T.A.
      • Pachnis V.
      Expression profiling the developing mammalian enteric nervous system identifies marker and candidate Hirschsprung disease genes.
      Dlx2 is decreased in aganglionic mouse bowel,
      • Vohra B.P.S.
      • Tsuji K.
      • Nagashimada M.
      • et al.
      Differential gene expression and functional analysis implicate novel mechanisms in enteric nervous system precursor migration and neuritogenesis.
      and its expression is enriched in neuronal cells compared with non-neuronal cells in murine embryonic gut,
      • Memic F.
      • Knoflach V.
      • Morarach K.
      • et al.
      Transcription and signaling regulators in developing neuronal subtypes of mouse and human enteric nervous system.
      further validating Dlx2 as an important neuronal-specific regulator in development. Interestingly, Dlx2 is enriched in non-neuronal cells in the zebrafish embryo gut,
      • Roy-Carson S.
      • Natukunda K.
      • Chou H.
      • et al.
      Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment.
      suggesting interspecies differences in the role of Dlx2. However, many other canonical ENS development genes are conserved between mice and zebrafish, including Phox2b, Elavl3, and Elavl4.
      • McCallum S.
      • Obata Y.
      • Fourli E.
      • et al.
      Enteric glia as a source of neural progenitors in adult zebrafish.
      Integrating findings from prior omics and newer scRNA-seq studies expands on prior omics work identifying integral genes in ENS development by further resolving cellular subtypes and time points where gene expression differs. In addition, these studies are performed in both humans and mice and may shed some light on interspecies variability in ENS development. In mice at embryonic day (E) E12.5, the ENS clusters into glial progenitors, neuronal progenitors, and mixed groups.
      • Lasrado R.
      • Boesmans W.
      • Kleinjung J.
      • et al.
      Lineage-dependent spatial and functional organization of the mammalian enteric nervous system.
      Recent scRNA-seq studies in humans suggest that neural crest progenitors are present by embryonic week (EW) EW6.5 and have already created the basic architecture of the submucosal and myenteric plexuses by EW8.
      • Elmentaite R.
      • Kumasaka N.
      • Roberts K.
      • et al.
      Cells of the human intestinal tract mapped across space and time.
      ,
      • Fawkner-Corbett D.
      • Antanaviciute A.
      • Parikh K.
      • et al.
      Spatiotemporal analysis of human intestinal development at single-cell resolution.
      However, correlating the timeline of ENS development between mice and humans is complex and likely contains discrepancies. For instance, Cao et al
      • Cao J.
      • O’Day D.R.
      • Pliner H.A.
      • et al.
      A human cell atlas of fetal gene expression.
      could map scRNA-seq human enteric glial clusters to murine clusters but could not replicate this in enteric neurons, suggesting differences in neuronal cluster development timelines. Regardless of timeline differences, canonical markers of functional neuronal types can be traced across development in both mice and humans.
      • Memic F.
      • Knoflach V.
      • Morarach K.
      • et al.
      Transcription and signaling regulators in developing neuronal subtypes of mouse and human enteric nervous system.
      ,
      • Heanue T.A.
      • Pachnis V.
      Expression profiling the developing mammalian enteric nervous system identifies marker and candidate Hirschsprung disease genes.
      ,
      • McCann C.J.
      • Alves M.M.
      • Brosens E.
      • et al.
      Neuronal development and onset of electrical activity in the human enteric nervous system.
      ,
      • Holloway E.M.
      • Czerwinski M.
      • Tsai Y.-H.
      • et al.
      Mapping development of the human intestinal niche at single-cell resolution.
      In humans excitatory neurons emerge first, followed by inhibitory neurons at EW14. TAC1+ and VIP+ neurons continue to differentiate until EW16. Electrical excitability also begins to form at this point with the expression of voltage-gated sodium channel SCN3A.
      • McCann C.J.
      • Alves M.M.
      • Brosens E.
      • et al.
      Neuronal development and onset of electrical activity in the human enteric nervous system.
      In mice Vip is apparent by E15.5,
      • Heanue T.A.
      • Pachnis V.
      Expression profiling the developing mammalian enteric nervous system identifies marker and candidate Hirschsprung disease genes.
      and dopaminergic neurons appear later at E18.
      • Memic F.
      • Knoflach V.
      • Morarach K.
      • et al.
      Transcription and signaling regulators in developing neuronal subtypes of mouse and human enteric nervous system.
      ,
      • Li Z.S.
      • Pham T.D.
      • Tamir H.
      • Chen J.J.
      • Gershon M.D.
      Enteric dopaminergic neurons: definition, developmental lineage, and effects of extrinsic denervation.
      Specific transcription factors help regulate these fates, where in mice Sox6 and Ascl1 help drive dopaminergic differentiation,
      • Memic F.
      • Knoflach V.
      • Morarach K.
      • et al.
      Transcription and signaling regulators in developing neuronal subtypes of mouse and human enteric nervous system.
      and in humans SEMA3A may regulate TAC1+/VIP+ neuron development.
      • McCann C.J.
      • Alves M.M.
      • Brosens E.
      • et al.
      Neuronal development and onset of electrical activity in the human enteric nervous system.
      ScRNA-seq identified a novel early binary split in neuronal development with putative genetic markers that are conserved between humans and mice. In mice this split mapped to the timeline E15.5 to E18, whereas in humans this corresponded to EW6 to EW11. At the binary split one group expresses Etv1/ETV1 and contains inhibitory motor neurons and select sensory neurons/interneurons, whereas another group expresses Bnc2/BNC2 and contains excitatory motor neurons and additional sensory neurons/interneurons.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      ,
      • Elmentaite R.
      • Kumasaka N.
      • Roberts K.
      • et al.
      Cells of the human intestinal tract mapped across space and time.
      Although corroboration between 2 scRNA-seq studies is promising, the exact roles of these genes require further study. Mutant phenotypes focusing on these genes could confirm their importance. Regardless, these scRNA-seq studies identified a novel archetype in neuronal development that may be conserved between species. The developmental fate of these binary split clusters was further investigated in mice, where the gene expression patterns of these initial 2 clusters remain into adulthood as Nos1+Npy+ inhibitory motor neurons and Ndufa4l2+ excitatory motor neurons, respectively. Meanwhile, other neuronal types and subtypes down-regulate these markers to diversify into the other characterized enteric neuronal subtypes.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      The role of these early clusters in adulthood is currently unclear, but because of their shared expression patterns with early life progenitors, these cells may shed additional light on the debated phenomenon of adult neurogenesis.
      Human scRNA-seq studies also discuss development of enteric glia. Glial progenitors are detectable at early time points, with 5 glial progenitor clusters identified by one study at EW7–8
      • Fawkner-Corbett D.
      • Antanaviciute A.
      • Parikh K.
      • et al.
      Spatiotemporal analysis of human intestinal development at single-cell resolution.
      and 1 progenitor and 1 maintained glial cluster at EW6–11 by another study.
      • Elmentaite R.
      • Kumasaka N.
      • Roberts K.
      • et al.
      Cells of the human intestinal tract mapped across space and time.
      In addition to the maintained cluster, 3 additional glial clusters were detected at EW12–17.
      • Elmentaite R.
      • Kumasaka N.
      • Roberts K.
      • et al.
      Cells of the human intestinal tract mapped across space and time.
      Interestingly, this maintained cluster appears shared between both and co-expressed MAL, FGL2, GFRA3, and RXRG.
      • Fawkner-Corbett D.
      • Antanaviciute A.
      • Parikh K.
      • et al.
      Spatiotemporal analysis of human intestinal development at single-cell resolution.
      However, one study suggests this cluster represents non-enteric glia originating in the sacrum or trunk due to TFAP2B expression,
      • Elmentaite R.
      • Kumasaka N.
      • Roberts K.
      • et al.
      Cells of the human intestinal tract mapped across space and time.
      whereas the other study suggests this represents lymphoid associated glia due to expression of immune markers FGL2, MAL, and TGFBR3.
      • Fawkner-Corbett D.
      • Antanaviciute A.
      • Parikh K.
      • et al.
      Spatiotemporal analysis of human intestinal development at single-cell resolution.
      Whether either or both of these studies are correct requires further investigation.

      Neuroimmune Communication

      Neuroimmune communication within the gut was initially suggested by innervation surrounding Peyer’s patches and immune cells in the lamina propria and immunostaining for neurotransmitter receptors on these immune cells.
      • Furness J.
      Types of neurons in the enteric nervous system.
      ,
      • Furness J.B.
      Omics data suggest molecular mechanisms of interaction between specific neuronal types or subtypes and immune cells. For instance, secretomotor/vasodilator neurons may communicate with monocytes via chemokine CX3CL1 to CX3CR1.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      Adrenergic neurons communicate with muscularis macrophages through β2 adrenergic receptors during bacterial infection and increase expression of protective and wound-healing genes such as Fizz1 (Retnla) and Il10 in these cells.
      • Gabanyi I.
      • Muller P.A.
      • Feighery L.
      • Oliveira T.Y.
      • Costa-Pinto F.A.
      • Mucida D.
      Neuro-immune interactions drive tissue programming in intestinal macrophages.
      Muscularis macrophages in turn communicate with enteric neurons using arginase 1 to protect them from NLRP6-inflammasome activation and cell death.
      • Matheis F.
      • Muller P.A.
      • Graves C.L.
      • et al.
      Adrenergic signaling in muscularis macrophages limits infection-induced neuronal loss.
      Together these highlight protective signaling mechanisms in enteric neuroimmune communication.
      Neurons may also communicate with immune cells through opioid and cannabinoid receptors, but it is unclear whether these signals would ameliorate or exacerbate inflammation because the impact of opioid and cannabinoid signaling on gut inflammation is complex.
      • DiPatrizio N.V.
      Endocannabinoids in the gut.
      • Kienzl M.
      • Storr M.
      • Schicho R.
      Cannabinoids and opioids in the treatment of inflammatory bowel diseases.
      • Sharma U.
      • Olson R.K.
      • Erhart F.N.
      • et al.
      Prescription opioids induce gut dysbiosis and exacerbate colitis in a murine model of inflammatory bowel disease.
      Neurons could use enkephalins to signal opioid receptor mu 1 (Oprm1) on T cells and Dagla to signal cannabinoid receptor 2 (Cnr2) on B cells. Inhibitory motor neurons also produce interleukins (ILs) IL12 and IL18, which may interact with T cells.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      However, these are transcriptional data and require protein-level mechanistic studies to validate and determine the role of these specified communications. In addition, neuronal IL18 regulates antimicrobial activity in goblet cells
      • Jarret A.
      • Jackson R.
      • Duizer C.
      • et al.
      Enteric nervous system-derived IL-18 orchestrates mucosal barrier immunity.
      and therefore may play a similar role in T cells.
      ScRNA-seq data have also added resolution to previously known neuroimmune communications. For instance, non-omics data support neuromedin U produced by intestinal neurons communicates with group 2 innate lymphoid cells through their neuromedin U receptor 1 (NMUR1).
      • Cardoso V.
      • Chesné J.
      • Ribeiro H.
      • et al.
      Neuronal regulation of type 2 innate lymphoid cells via neuromedin U.
      ,
      • Klose C.S.N.
      • Mahlakõiv T.
      • Moeller J.B.
      • et al.
      The neuropeptide neuromedin U stimulates innate lymphoid cells and type 2 inflammation.
      Although these neuromedin U–producing neurons were postulated to be cholinergic with mucosal projections, their functional type was yet unknown. ScRNA-seq clusters confirmed that IPANs express Nmu as a specific marker
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      • May-Zhang A.A.
      • Tycksen E.
      • Southard-Smith A.N.
      • et al.
      Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • et al.
      Molecular architecture of the mouse nervous system.
      and therefore clearly identifies IPANs as the cells communicating with group 2 innate lymphoid cells to modulate immune response. This is a rewarding example of novel scRNA-seq subtypes complementing non-omics studies to understand biomolecular function.
      Specific mechanisms of communication between enteric glia and immune cells were recently reported in non-omics work.
      • Grubišić V.
      • McClain J.L.
      • Fried D.E.
      • et al.
      Enteric glia modulate macrophage phenotype and visceral sensitivity following inflammation.
      ,
      • Chow A.K.
      • Grubišić V.
      • Gulbransen B.D.
      Enteric glia regulate lymphocyte activation via autophagy-mediated MHC-II expression.
      Although one recent omics study did highlight a putative mechanism of glial-immune interaction in mice, other findings mainly highlight general immune response. Progatzky et al
      • Progatzky F.
      • Shapiro M.
      • Chng S.H.
      • et al.
      Regulation of intestinal immunity and tissue repair by enteric glia.
      identified up-regulation of enteric glial Cxcl10 as an important mediator of interferon gamma signaling and ultimately inflammatory and granulomatous response to helminth infection. Other studies support glial-immune communication in chemical models of inflammation. In dinitrobenzene sulfonic acid (DNBS) colitis enteric glia up-regulate genes in immune-related pathways including cytokine activity and antigen processing and presentation.
      • Delvalle N.M.
      • Dharshika C.
      • Morales-Soto W.
      • Fried D.E.
      • Gaudette L.
      • Gulbransen B.D.
      Communication between enteric neurons, glia, and nociceptors underlies the effects of tachykinins on neuroinflammation.
      Glia treated with lipopolysaccharide + interferon gamma also up-regulate several proinflammatory cytokines, chemokines, and interleukins in cell culture
      • Liñán-Rico A.
      • Turco F.
      • Ochoa-Cortes F.
      • et al.
      Molecular signaling and dysfunction of the human reactive enteric glial cell phenotype.
      and rat small intestine.
      • Rosenbaum C.
      • Schick M.A.
      • Wollborn J.
      • et al.
      Activation of myenteric glia during acute inflammation in vitro and in vivo.
      Interestingly, glial S100b decreased in both models. Typically S100b release increases inducible nitric oxide synthase expression and nitric oxide production,
      • Liñán-Rico A.
      • Turco F.
      • Ochoa-Cortes F.
      • et al.
      Molecular signaling and dysfunction of the human reactive enteric glial cell phenotype.
      ,
      • Rosenbaum C.
      • Schick M.A.
      • Wollborn J.
      • et al.
      Activation of myenteric glia during acute inflammation in vitro and in vivo.
      so perhaps this is a compensatory/protective mechanism.

      Dysbiosis

      Omics research primarily uses 16s rRNA sequencing to correlate altered microbiome diversity with gastrointestinal disease, but here we will focus on host ENS changes. Not surprisingly, the microbiome alters enteric neuronal gene expression in the ileum and colon but not proximal intestinal regions.
      • Muller P.A.
      • Matheis F.
      • Schneeberger M.
      • Kerner Z.
      • Jové V.
      • Mucida D.
      Microbiota-modulated CART + enteric neurons autonomously regulate blood glucose.
      ,
      • Obata Y.
      • Á Castaño
      • Boeing S.
      • et al.
      Neuronal programming by microbiota regulates intestinal physiology.
      Many genes are regulated by colonic microbes and affect ENS function. For instance, the microbiome impacts colonic motility by up-regulating Ahr expression on enteric neurons.
      • Obata Y.
      • Á Castaño
      • Boeing S.
      • et al.
      Neuronal programming by microbiota regulates intestinal physiology.
      Commensal bacteria release extracellular vesicles containing heat shock system proteins such as chaperonin 60, which increase both colonic motor complex amplitude and IPAN activity,
      • Al-Nedawi K.
      • Mian M.F.
      • Hossain N.
      • et al.
      Gut commensal microvesicles reproduce parent bacterial signals to host immune and enteric nervous systems.
      suggesting roles for this communication in both motor and afferent intrinsic pathways. Microbial dysbiosis also correlates with afferent signaling in visceral hypersensitivity. Specifically, taxa that produce short-chain fatty acids increase in multiple inflammatory disease models.
      • Esquerre N.
      • Basso L.
      • Defaye M.
      • et al.
      Colitis-induced microbial perturbation promotes postinflammatory visceral hypersensitivity.
      • Zhou X.Y.
      • Li M.
      • Li X.
      • et al.
      Visceral hypersensitive rats share common dysbiosis features with irritable bowel syndrome patients.
      • De Palma G.
      • Blennerhassett P.
      • Lu J.
      • et al.
      Microbiota and host determinants of behavioural phenotype in maternally separated mice.
      Intrinsic enteric neurons are not considered directly involved in pain transduction pathways,
      • Furness J.B.
      but communication between the ENS and extrinsic sensory neurons can modulate pain perception.
      • Delvalle N.M.
      • Dharshika C.
      • Morales-Soto W.
      • Fried D.E.
      • Gaudette L.
      • Gulbransen B.D.
      Communication between enteric neurons, glia, and nociceptors underlies the effects of tachykinins on neuroinflammation.
      Short-chain fatty acids increase expression of enteric glial fibrillary acid protein and nerve growth factor, where nerve growth factor contributes to visceral hypersensitivity.
      • Long X.
      • Li M.
      • Li L.-X.
      • et al.
      Butyrate promotes visceral hypersensitivity in an IBS-like model via enteric glial cell-derived nerve growth factor.
      Taken together these data suggest that the gut microbiome and its biomolecular mediators have effects on many aspects of known ENS function.
      Microbial dysbiosis likely impacts motor neuron development. Mice that receive antibiotics at PN10 had increased colonic motility corresponding with increased cholinergic neurons and decreased nitrergic neurons, whereas antibiotic-treated 6-week-old or adult mice had the opposite results.
      • Hung L.Y.
      • Boonma P.
      • Unterweger P.
      • et al.
      Neonatal antibiotics disrupt motility and enteric neural circuits in mouse colon.
      • Hung L.Y.
      • Parathan P.
      • Boonma P.
      • et al.
      Antibiotic exposure postweaning disrupts the neurochemistry and function of enteric neurons mediating colonic motor activity.
      • Yarandi S.S.
      • Kulkarni S.
      • Saha M.
      • Sylvia K.E.
      • Sears C.L.
      • Pasricha P.J.
      Intestinal bacteria maintain adult enteric nervous system and nitrergic neurons via toll-like receptor 2-induced neurogenesis in mice.
      Although these findings suggest age-dependent relationships between enteric neurons and microbiota, these groups also received different antibiotics, and this may also explain these results. Commensal microbiota also regulate the survival of specific IFANs by preventing inflammasome-dependent cell death.
      • Muller P.A.
      • Matheis F.
      • Schneeberger M.
      • Kerner Z.
      • Jové V.
      • Mucida D.
      Microbiota-modulated CART + enteric neurons autonomously regulate blood glucose.
      These IFANs express the marker cocaine- and amphetamine-regulated transcript (Cartpt) and help regulate blood glucose levels through communication with the liver and pancreas. Conversely, enteric neurons prevent infection by pathogenic bacteria. Neuronal IL18 promotes goblet cell production of antimicrobial peptides and subsequently prevents invasion of the pathogenic species Salmonella typhimurium.
      • Jarret A.
      • Jackson R.
      • Duizer C.
      • et al.
      Enteric nervous system-derived IL-18 orchestrates mucosal barrier immunity.
      Together these data highlight signaling mechanisms between enteric neurons and gut bacteria that help regulate homeostasis and prevent infection.
      Dysbiosis is also associated with disease pathogenesis and/or disease markers, particularly in IBS. Although taxonomic changes of the microbiome in IBS are subtle, these bacteria alter host serum metabolites,
      • De Palma G.
      • Lynch M.D.J.
      • Lu J.
      • et al.
      Transplantation of fecal microbiota from patients with irritable bowel syndrome alters gut function and behavior in recipient mice.
      likely reflecting altered bacterial metabolites as well. This metabolic disturbance contributes to enteric neuron dysfunction and dysmotility in IBS. For instance, mice that receive fecal transplants from IBS with diarrhea patients recapitulate decreased colonic transit times despite little change in microbial composition.
      • De Palma G.
      • Lynch M.D.J.
      • Lu J.
      • et al.
      Transplantation of fecal microbiota from patients with irritable bowel syndrome alters gut function and behavior in recipient mice.
      In addition, short-chain fatty acids can regulate colonic motility through the monocarboxylate transporter 2, where mutations in the gene for monocarboxylate transporter 2 ligand delphilin (GRID2IP) confer IBS disease risk.
      • Ek W.E.
      • Reznichenko A.
      • Ripke S.
      • et al.
      Exploring the genetics of irritable bowel syndrome: a GWA study in the general population and replication in multinational case-control cohorts.
      This similarly suggests disturbances in microbial metabolites may be key in IBS dysmotility, although functional studies are required to validate this connection.

      ENS Expression of Gastrointestinal Disease Markers

      Many previously identified disease markers and risk genes are enriched in enteric neurons compared with other colonic cell types. Note that our previous sections on pathologies may also include genes that could be considered disease markers. However, in those contexts we investigated how omics data highlighted potential functions of these genes in pathogenesis. Here we discuss genes that omics studies specifically identify as putative markers of specific medical diagnoses. Drokhlyansky et al
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      highlighted the genes RET, PHOX2B, GFRA1, and ECE1 as markers of HSCR that are enriched in neurons compared with non-neuronal cells.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      ,
      • Morarach K.
      • Mikhailova A.
      • Knoflach V.
      • et al.
      Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.
      ,
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      The inclusion of both RET and GFRA1 is interesting because both are considered receptors for GDNF, but Gfra1 signaling was discussed earlier as a means of dysmotility in adult mice,
      • Wright C.M.
      • Schneider S.
      • Smith-Edwards K.M.
      • et al.
      scRNA-sequencing reveals new enteric nervous system roles for GDNF, NRTN, and TBX3.
      and RET is the canonical receptor for GDNF implicated in HSCR.
      • Shen L.
      • Pichel J.G.
      • Mayeli T.
      • Sariola H.
      • Lu B.
      • Westphal H.
      Gdnf haploinsufficiency causes Hirschsprung-like intestinal obstruction and early-onset lethality in mice.
      Indeed murine haploinsufficiency of either GDNF or RET mimics intestinal agangliosis/hypogangliosis seen in HSCR. This supports the role for GDNF-RET signaling in HSCR, and thus the role of GFRA1 in this context is still unclear. Perhaps this highlights complex signaling patterns across development through adulthood that require further investigation, where milder or altered perturbations of the same genes lead to different diseases that present at different ages. This complexity is further suggested by findings in obstructed defecation patients where expression of HSCR-related genes (including RET, PHOX2B, and GFRA1) are down-regulated.
      • Kim M.
      • Rosenbaum C.
      • Schlegel N.
      • et al.
      Obstructed defecation: an enteric neuropathy? an exploratory study of patient samples.
      However, the functional relevance of this differential expression is unknown. In addition to these HSCR genes involved in neuronal development,
      • Roy-Carson S.
      • Natukunda K.
      • Chou H.
      • et al.
      Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment.
      ,
      • Vohra B.P.S.
      • Tsuji K.
      • Nagashimada M.
      • et al.
      Differential gene expression and functional analysis implicate novel mechanisms in enteric nervous system precursor migration and neuritogenesis.
      ,
      • Lasrado R.
      • Boesmans W.
      • Kleinjung J.
      • et al.
      Lineage-dependent spatial and functional organization of the mammalian enteric nervous system.
      a single recent proteomics study in HSCR patients suggests new markers of disease ARF4, KIF5B, and RAB8A.
      • Zhang Q.
      • Wu L.
      • Bai B.
      • et al.
      Quantitative proteomics reveals association of neuron projection development genes ARF4, KIF5B, and RAB8A with Hirschsprung disease.
      Decreased expression of these proteins in colons of HSCR patients was also validated with Western blot and immunostaining. These genes are involved in cellular trafficking functions and theorized to be important for neuronal processes development. Validation of these targets could highlight specificities in the pathogenesis of HSCR in addition to serving as novel disease markers.
      Meanwhile, ENS expression of Parkinson’s disease risk genes suggests neurodegenerative processes may preferentially affect certain neuronal subtypes. Parkinson’s disease risk genes DLG2, SNCA, and SCN3 are enriched across most neuron subtypes in humans, but murine Lrrk2 is more highly expressed in inhibitory motor neuron and secretomotor/vasodilator neuron subtypes. Lrrk2 expression in enteric neurons also increases with age.
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      LRRK2 dysfunction in the brain contributes to neuroinflammation and subsequent neuronal death in late-onset Parkinson’s disease.
      • Rui Q.
      • Ni H.
      • Li D.
      • Gao R.
      • Chen G.
      The role of LRRK2 in neurodegeneration of Parkinson disease.
      Similar mechanisms may occur in the gastrointestinal tract and preferentially target certain neuronal subtypes to produce symptoms. However, whether these are species differences or whether LRRK2 functions similarly in the ENS is unclear.
      The effects of autism spectrum disorder risk genes in the ENS may also reflect central nervous system pathology. Enteric neurons enrich for genes expressed in the central nervous system such as GABA receptor GABRB3 and adhesion molecules DSCAM and neuroligin-3 (NLGN3).
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      ,
      • Hosie S.
      • Ellis M.
      • Swaminathan M.
      • et al.
      Gastrointestinal dysfunction in patients and mice expressing the autism-associated R451C mutation in neuroligin-3.
      The effects of this autism spectrum disorder NLGN3 mutant in the brain recapitulate in the ENS, where enteric neurons have increased GABA-A sensitivity and subsequently shortened intestinal transit time.
      • Hosie S.
      • Ellis M.
      • Swaminathan M.
      • et al.
      Gastrointestinal dysfunction in patients and mice expressing the autism-associated R451C mutation in neuroligin-3.
      Autism spectrum disorder may also involve enteric glial pathology because glia enrich for risk genes NRXN1 and ANK2 compared with other intestinal cell types,
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      but the effect of this on gastrointestinal dysfunction in autism spectrum disorder is unknown. Ank2 is also enriched in a specific glial subtype in mice,
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • et al.
      The human and mouse enteric nervous system at single-cell resolution.
      and perhaps this glial subtype contributes to disease.
      Glia are also implicated in inflammatory bowel disease (IBD). Mutations in the prostaglandin receptor EP4 (PTGER4) confer risk in IBD,
      • Barrett J.C.
      • Hansoul S.
      • Nicolae D.L.
      • et al.
      Genome-wide association defines more than 30 distinct susceptibility loci for Crohn’s disease.
      • Brant S.R.
      • Okou D.T.
      • Simpson C.L.
      • et al.
      Genome-wide association study identifies African-specific susceptibility loci in African American