Autonomous regulation of the intestine requires the combined activity of functionally distinct neurons of the enteric nervous system (ENS). However, the variety of enteric neuron types and how they emerge during development remain largely unknown. Here, we define a molecular taxonomy of 12 enteric neuron classes within the myenteric plexus of the mouse small intestine using single-cell RNA sequencing. We present cell–cell communication features and histochemical markers for motor neurons, sensory neurons and interneurons, together with transgenic tools for class-specific targeting. Transcriptome analysis of the embryonic ENS uncovers a novel principle of neuronal diversification, where two neuron classes arise through a binary neurogenic branching and all other identities emerge through subsequent postmitotic differentiation. We identify generic and class-specific transcriptional regulators and functionally connect Pbx3 to a postmitotic fate transition. Our results offer a conceptual and molecular resource for dissecting ENS circuits and predicting key regulators for directed differentiation of distinct enteric neuron classes.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Velusetrag rescues GI dysfunction, gut inflammation and dysbiosis in a mouse model of Parkinson’s disease
npj Parkinson's Disease Open Access 02 October 2023
Nature Communications Open Access 22 September 2023
Journal of Molecular Neuroscience Open Access 27 June 2023
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Raw sequence and processed data for Wnt1Cre;R26R-Tomato (E15.5 and E18.5) and for Baf53bCre;R26R-Tomato (P21) are available on the Gene Expression Omnibus database under the identifier GSE149524 and accession number SRP258962 at http://ncbi.nlm.nih.gov/sra/SRP258962. Raw sequencing data from Wnt1Cre;R26R-Tomato P21 and P23 animals is available under accession SRP135960 at https://www.ncbi.nlm.nih.gov/sra/SRP135960.
Furness, J. B. The enteric nervous system and neurogastroenterology. Nat. Rev. Gastroenterol. Hepatol. 9, 286–294 (2012).
Veiga-Fernandes, H. & Pachnis, V. Neuroimmune regulation during intestinal development and homeostasis. Nat. Immunol. 18, 116–122 (2017).
Furness, J. B. Types of neurons in the enteric nervous system. J. Auton. Nerv. Syst. 81, 87–96 (2000).
Sang, Q. & Young, H. M. Chemical coding of neurons in the myenteric plexus and external muscle of the small and large intestine of the mouse. Cell Tissue Res. 284, 39–53 (1996).
Sang, Q., Williamson, S. & Young, H. M. Projections of chemically identified myenteric neurons of the small and large intestine of the mouse. J. Anat. 190, 209–222 (1997).
Qu, Z. D. et al. Immunohistochemical analysis of neuron types in the mouse small intestine. Cell Tissue Res. 334, 147–161 (2008).
Furness, J. B., Jones, C., Nurgali, K. & Clerc, N. Intrinsic primary afferent neurons and nerve circuits within the intestine. Prog. Neurobiol. 72, 143–164 (2004).
Knowles, C. H., Lindberg, G., Panza, E. & De Giorgio, R. New perspectives in the diagnosis and management of enteric neuropathies. Nat. Rev. Gastroenterol. Hepatol. 10, 206–218 (2013).
Rivera, L. R., Poole, D. P., Thacker, M. & Furness, J. B. The involvement of nitric oxide synthase neurons in enteric neuropathies. Neurogastroenterol. Motil. 23, 980–988 (2011).
Fattahi, F. et al. Deriving human ENS lineages for cell therapy and drug discovery in Hirschsprung disease. Nature 531, 105–109 (2016).
Burns, A. J. et al. White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies. Dev. Biol. 417, 229–251 (2016).
Obermayr, F., Hotta, R., Enomoto, H. & Young, H. M. Development and developmental disorders of the enteric nervous system. Nat. Rev. Gastroenterol. Hepatol. 10, 43–57 (2013).
Uesaka, T., Nagashimada, M. & Enomoto, H. Neuronal differentiation in schwann cell lineage underlies postnatal neurogenesis in the enteric nervous system. J. Neurosci. 35, 9879–9888 (2015).
Le Dréau, G. & Martí, E. Dorsal-ventral patterning of the neural tube: a tale of three signals. Dev. Neurobiol. 72, 1471–1481 (2012).
Pham, T. D., Gershon, M. D. & Rothman, T. P. Time of origin of neurons in the murine enteric nervous system: sequence in relation to phenotype. J. Comp. Neurol. 314, 789–798 (1991).
Bergner, A. J. et al. Birthdating of myenteric neuron subtypes in the small intestine of the mouse. J. Comp. Neurol. 522, 514–527 (2014).
Memic, F. et al. Transcription and signaling regulators in developing neuronal subtypes of mouse and human enteric nervous system. Gastroenterology 154, 624–636 (2018).
Zeisel, A. et al. Molecular architecture of the mouse nervous system. Cell 174, 999–1014 (2018).
Zhan, X. et al. Generation of BAF53b-Cre transgenic mice with pan-neuronal Cre activities. Genesis 53, 440–448 (2015).
Paul, A. et al. Transcriptional architecture of synaptic communication delineates GABAergic neuron identity. Cell 171, 522–539 (2017).
Cardoso, V. et al. Neuronal regulation of type 2 innate lymphoid cells via neuromedin U. Nature 549, 277–281 (2017).
Klose, C. S. N. et al. The neuropeptide neuromedin U stimulates innate lymphoid cells and type 2 inflammation. Nature 549, 282–286 (2017).
Muller, P. A. et al. Cross-talk between muscularis macrophages and enteric neurons regulates gastrointestinal motility. Cell 158, 300–313 (2014).
Neureither, F., Stowasser, N., Frings, S. & Möhrlen, F. Tracking of unfamiliar odors is facilitated by signal amplification through anoctamin 2 chloride channels in mouse olfactory receptor neurons. Physiol. Rep. 5, e13373 (2017).
Alcaino, C. et al. A population of gut epithelial enterochromaffin cells is mechanosensitive and requires Piezo2 to convert force into serotonin release. Proc. Natl Acad. Sci. USA 115, E7632–E7641 (2018).
Born, G. et al. Modulation of synaptic function through the α-neurexin-specific ligand neurexophilin-1. Proc. Natl Acad. Sci. USA 111, E1274–E1283 (2014).
Creutz, C. E. et al. The copines, a novel class of C2 domain-containing, calcium-dependent, phospholipid-binding proteins conserved from Paramecium to humans. J. Biol. Chem. 273, 1393–1402 (1998).
Gerke, V., Creutz, C. E. & Moss, S. E. Annexins: linking Ca2+ signalling to membrane dynamics. Nat. Rev. Mol. Cell Biol. 6, 449–461 (2005).
Chan, K. Y. et al. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat. Neurosci. 20, 1172–1179 (2017).
Nurgali, K., Stebbing, M. J. & Furness, J. B. Correlation of electrophysiological and morphological characteristics of enteric neurons in the mouse colon. J. Comp. Neurol. 468, 112–124 (2004).
Furness, J. B., Robbins, H. L., Xiao, J., Stebbing, M. J. & Nurgali, K. Projections and chemistry of Dogiel type II neurons in the mouse colon. Cell Tissue Res. 317, 1–12 (2004).
Spencer, N. J. & Smith, T. K. Mechanosensory S-neurons rather than AH-neurons appear to generate a rhythmic motor pattern in guinea pig distal colon. J. Physiol. 558, 577–596 (2004).
Lasrado, R. et al. Lineage-dependent spatial and functional organization of the mammalian enteric nervous system. Science 356, 722–726 (2017).
Rhee, J. W. et al. Pbx3 deficiency results in central hypoventilation. Am. J. Pathol. 165, 1343–1350 (2004).
Okamoto, T. et al. Extensive projections of myenteric serotonergic neurons suggest they comprise the central processing unit in the colon. Neurogastroenterol. Motil. 26, 556–570 (2014).
Hao, M. M. & Young, H. M. Development of enteric neuron diversity. J. Cell. Mol. Med. 13, 1193–1210 (2009).
Arenas, E., Denham, M. & Villaescusa, J. C. How to make a midbrain dopaminergic neuron. Development 142, 1918–1936 (2015).
Young, H. M. et al. Colonizing while migrating: how do individual enteric neural crest cells behave? BMC Biol. 12, 23 (2014).
Young, H. M., Jones, B. R. & McKeown, S. J. The projections of early enteric neurons are influenced by the direction of neural crest cell migration. J. Neurosci. 22, 6005–6018 (2002).
Laranjeira, C. et al. Glial cells in the mouse enteric nervous system can undergo neurogenesis in response to injury. J. Clin. Invest. 121, 3412–3424 (2011).
Panman, L. et al. Transcription factor-induced lineage selection of stem-cell-derived neural progenitor cells. Cell Stem Cell 8, 663–675 (2011).
Rivetti Di Val Cervo, P. et al. Induction of functional dopamine neurons from human astrocytes in vitro and mouse astrocytes in a Parkinson’s disease model. Nat. Biotechnol. 35, 444–452 (2017).
Taniguchi, H. et al. A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex. Neuron 71, 995–1013 (2011).
Danielian, P. S., Muccino, D., Rowitch, D. H., Michael, S. K. & McMahon, A. P. Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr. Biol. 8, 1323–1326 (1998).
Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).
Bialkowska, A. B., Ghaleb, A. M., Nandan, M. O. & Yang, V. W. Improved swiss-rolling technique for intestinal tissue preparation for immunohistochemical and immunofluorescent analyses. J. Vis. Exp. 113, e54161 (2016). (2016).
Memic, F. et al. Ascl1 is required for the development of specific neuronal subtypes in the enteric nervous system. J. Neurosci. 36, 4339–4350 (2016).
Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies and species. Nat. Biotechnol. 36, 411–420 (2018).
Hafemeister, C. & Satija, R. Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. Genome Biol. 20, 296 (2019).
Zhang, A. W. et al. Probabilistic cell-type assignment of single-cell RNA-seq for tumor microenvironment profiling. Nat. Methods 16, 1007–1015 (2019).
Tirosh, I. et al. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science 352, 189–196 (2016).
Stuart, T. et al. Comprehensive integration of single-cell data. Cell 177, 1888–1902 (2019).
Crow, M., Paul, A., Ballouz, S., Huang, Z. J. & Gillis, J. Characterizing the replicability of cell types defined by single cell RNA-sequencing data using MetaNeighbor. Nat. Commun. 9, 884 (2018).
Braschi, B. et al. Genenames.org: the HGNC and VGNC resources in 2019. Nucleic Acids Res. 47, D786–D792 (2019).
Wolf, F. A. et al. PAGA: graph abstraction reconciles clustering with trajectory inference through a topology preserving map of single cells. Genome Biol. 20, 1–9 (2019).
Wolf, F. A., Angerer, P. & Theis, F. J. SCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 19, 15 (2018).
Haghverdi, L., Büttner, M., Wolf, F. A., Buettner, F. & Theis, F. J. Diffusion pseudotime robustly reconstructs lineage branching. Nat. Methods 13, 845–848 (2016).
Stein-O’Brien, G. L. et al. Decomposing cell identity for transfer learning across cellular measurements, platforms, tissues and species. Cell Syst. 8, 395–411 (2019).
Stein-O’Brien, G. L. et al. PatternMarkers & GWCoGAPS for novel data-driven biomarkers via whole transcriptome NMF. Bioinformatics 33, 1892–1894 (2017).
La Manno, G. et al. RNA velocity of single cells. Nature 560, 494–498 (2018).
Cell sampling was performed at the Eukaryotic Single-cell Genomics core facility at Science for Life Laboratory, Sweden, funded by the Swedish Research Council. The authors acknowledge support from Science for Life Laboratory, the Knut and Alice Wallenberg Foundation, the National Genomics Infrastructure funded by the Swedish Research Council and Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure. U.M. was supported by The Swedish Research Council (Vetenskapsrådet; 2016-03130), Swedish Medical Society, Ruth and Richard Julin Foundation, Ollie and Elof Ericssons Foundation, Magnus Bergvall Foundation, Brain Foundation (Hjärnfonden) and Åke Wiberg Foundation. A.M. was supported by Wenner-Gren Foundations. F.M. was supported by the Brain Foundation (Hjärnfonden). P.E. acknowledges the European Research Council (PainCells; 740491), the Swedish Research Council, a KAW Scholar and project grant, and the Wellcome Trust (200183). The imaging was performed in the Biomedicum Imaging Core with support from the Karolinska Institutet. We thank the Viral Vector Facility of the Neuroscience Center Zurich for excellent service and construction of AAV-virus vectors. We thank G. Tabacaru for laboratory assistance. We thank C. Villascusa (Karolinska Institutet) for sharing the Pbx3 mutant mice and J. Kaltschmidt (Stanford University) for antibodies. We acknowledge J. Frisén and S. Giatrellis for access and assistance with flow cytometry.
The authors declare no competing interests.
Peer review information Nature Neuroscience thanks Laren Becker, Michael Gershon, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Frequency distribution of the number of UMIs, detected genes and percent of mitochondrial genes per cell. Orange bars indicate proportion of cells passing the thresholding for each parameter. b, Box plots showing number of UMIs, detected genes and percent of mitochondrial genes per cell for each of the ENCs. Box-and-whisker plots indicate max-min (whiskers), 25-75 percentile (boxes) with median as a centre line. Points indicate outliers. c, UMAP depicting inferred female (Xist) and male (Eif2s3y, Ddx3y, Kdm5) cells. Pie-chart showing proportions of male and female cells (2:3 ratio). d, Bar-graph showing fraction of male and female cells in each ENC after normalized by total number of cells from each sex and scaled to 1. e, Label transfer relationship between previously proposed enteric neuron types (ENT)1-9 in Zeisel et al., 201818 and ENC1-12 presented in this study. Notably, ENC5 (Sst) and ENC11 (Npy/Th/Dbh) represent new clusters. ENT-clusters representing plausible excitatory (ENT4-6) and inhibitory (ENT2,3) motor neurons were not retained, but distributed into ENC1-4 and ENC8-9, respectively. UMI: Unique Molecular Identifier; ENT: Enteric Neuron Type; ENC: Enteric Neuron Class; UMAP: Uniform Manifold Approximation and Projection.
a, Feature plots related to Tac1+ clusters (ENC1-4). A gene set including Calb2 and stronger Ndufa4l2 demarcated ENC1-2 from ENC3-4, while Gda and Penk expression discriminated ENC2-4 from ENC1. ENC4 resembled ENC3, but displayed a unique expression of Fucosyltransferase 9 (Fut9) and the transcription factor Nfatc1. b, Feature plots related to Rprml+ clusters (ENC8-11). c, Heatmap representing CellAssign score for each cell (columns) to each functional type (rows). d, Rare cells assigned with maximum likelihood to interneuron 3 (IN_3, serotonin-producing), presented on UMAP. e, Feature plots displaying expression of genes correlated to serotonin production (Ddc) and re-uptake (Slc6a4) in ENC12.
Extended Data Fig. 3 Negative markers for ENC1-12, summary table of validated ENC markers and ENC proportions across the small intestine.
a–o, Immunohistochemical validation of negative marker proteins. Pictures show either myenteric peel preparations or transverse sections at P21-P90. white arrow: positive marker; yellow arrow: negative marker. Scale bars indicate 20 µm. p, Table summarizing ENC markers verified by immunohistochemistry (unique markers in bold) q) Graphs indicating proportions of ENCs at week 9-12 (n=3-4 mice) using HUC/D or PGP9.5 for total neuron counts. ENC1-2 was calculated by subtracting CALR+ ENC6, ENC5 and ENC11 percentages from the average total CALR+ neurons. Note the much higher proportion of ENC6 in tissue, than in scRNA-seq data, reflecting the difficulties in isolating big size neurons from tissue. Graph with all ENCs in ileum was normalized to 100% (absolute value 102,6%). Data are presented as mean values±SD.
Extended Data Fig. 4 Schematic tables summarizing marker and ligand/receptor gene expression in each ENC.
We combined information gained from RNA-sequencing (Fig. 1 and Supplementary Fig. 2), immunohistochemical analysis (Fig. 3) and transgenic mice (Fig. 4) to make a reasonable representation of gene expression patterns of (a) marker genes (b) ligand/receptors in the ENCs. (ligand): refers to genes required for the production of a ligand, including enzymes. ENC: Enteric Neuron Class.
a, Myenteric plexus peel from Nmu-Cre;R26R-Tom mouse showing TOM in HUC/D+ neurons (stars) and its exclusion from enteric glia (SOX2+). b, Myenteric plexus peel from Cck-IRES-Cre;R26-Tom mouse showing TOM in both neurons (stars) and enteric glia (stars). c, Myenteric plexus peel of Cck-IRES-Cre mouse injected with AAV-DIO-Ruby3 showing RUBY3 only in neurons and not in glia. d, Transverse sections showing that Nmu and Cck RNA expression correlate with reporter+ neurons in Nmu-Cre;R26R-Tom and Cck-IRES-Cre; AAV-DIO-EYFP/Ruby3 animals. e, Graph showing the percentage of reporter+ neurons expressing the reciprocal RNA. Data are presented as mean values±SD. A total of 638 reporter+ neurons were investigated in three Nmu-Cre;R26R-Tom mice, and 71 reporter+ neurons were investigated in three Cck-IRES-Cre;AAV-DIO-EYFP/Ruby3 mice. TOM: dtTomato. Scale bars indicate 50μm.
Related to Fig. 4e–g. a, Representative examples of each morphological type found within ENC6, 7 and 12 and their size. b, Representative examples of each morphological type and their relative proportion (n=90 neurons from 5 animals) within jejunum and ileum of 5-HT+ ENC12. Scale bars indicates 20μm. Data are presented as mean values±SD.
a, b, Frequency distribution of the number of UMIs, detected genes and percent of mitochondrial genes per cell in E15.5 (a) and E18.5 (b) datasets. Orange bars represent cells that pass the thresholding for each parameter. c-d) Boxplots showing normalized expression (log scale) of known cell state genes: Sox10 (progenitor), Ascl1 (neuroblast), Elavl4 (enteric neuron), Plp1 (Enteric glia) and Dhh (SCP), grouped by Louvian clusters for E15.5 (c) and E18.5 (d). Box-and-whisker plots indicate max-min (whiskers), 25-75 percentile (boxes) with median as a centre line. Points indicate outliers. e, f, Refined clusters on UMAP for E15.5 (e) and E18.5 (f). Clusters in the same state were merged to obtain the generic ENS state clusters shown in Fig. 5a,b. UMI: Unique Molecular Identifier; SCP: Schwann cell precursor; E: Embryonic day; UMAP: Uniform Manifold Approximation and Projection; MT: Mitochondrial.
Related to Fig. 5. a, Ligands; note the predominant expression of Dll1 and Dll3 in neuroblasts. b, Receptors; note the predominant expression of Notch1,2 in progenitors. c, Downstream transcription factor; note the enriched expression of Hes6 in neuroblasts, Hes1 in progenitors and Hes5, Heyl and Hey2 in enteric glia. d, Regulator of activity; note the enriched expression of Mfng in neuroblasts and Lfng in progenitors. Color bar indicate expression level with maximum cut off at the 90th percentile.
Extended Data Fig. 9 Feature plots displaying transcription factors associated with generic cell states of the ENS at E15.5.
Related to Fig. 5f–h. a, progenitors, b) neuroblasts and c) neurons. Color bar indicate expression level with maximum cut-off at the 90th percentile.
a, TFs expressed in one or few ENCs that correlated well with juvenile expression (exception in a’). b, TFs with broad ENC-specific expression that correlated well with juvenile expression (exception in b’). c, TFs only expressed at embryonic stages, and not maintained in juvenile ENCs. d, TFs with wide expression, including bona fide ENS markers Hand2 and Phox2b. See Supplementary Fig. 2c to compare with the gene expressions in juvenile ENCs. Color bar indicate expression level with maximum cut off at the 90th percentile.
Supplementary Figs. 1–6 and Supplementary Table 3.
Enriched genes in ENC1–12.
Table showing average and individual AUROC scores for HGNC gene families in ENCs.
Enriched genes in generic cell clusters at E15.5 and E18.5.
Coordinated gene patterns at E15.5.
Enriched genes in refined clusters at E15.5 and E18.5.
About this article
Cite this article
Morarach, K., Mikhailova, A., Knoflach, V. et al. Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing. Nat Neurosci 24, 34–46 (2021). https://doi.org/10.1038/s41593-020-00736-x
This article is cited by
Nature Communications (2023)
Velusetrag rescues GI dysfunction, gut inflammation and dysbiosis in a mouse model of Parkinson’s disease
npj Parkinson's Disease (2023)
Systemic sclerosis gastrointestinal dysmotility: risk factors, pathophysiology, diagnosis and management
Nature Reviews Rheumatology (2023)
Transcriptomics of Hirschsprung disease patient-derived enteric neural crest cells reveals a role for oxidative phosphorylation
Nature Communications (2023)
Nature Communications (2023)