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Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing

Nature Neuroscience volume 24, pages 34–46 (2021)Cite this article

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Abstract

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.

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Fig. 1: Molecular definition of 12 enteric neuron classes in the mouse small intestine myenteric plexus.
Fig. 2: Gene categories conferring functional characteristics in enteric neuron classes.
Fig. 3: Immunohistochemical validation of marker genes for enteric neuron classes.
Fig. 4: Assessment of IPAN characteristics in ENCs 6, 7 and 12.
Fig. 5: Single-cell transcriptome analysis of the developing ENS reveals transcriptional programs of generic cell states.
Fig. 6: ENC identities emerge through binary branching followed by postmitotic neuronal diversification.
Fig. 7: Loss of PBX3 expression impairs the ENC8/9 to ENC12 transition.
Fig. 8: Schematic of the major principle of neuronal diversification in the developing CNS versus ENS.

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Data availability

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.

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Acknowledgements

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.

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  1. Rakesh Kumar

    Present address: Zoology Department, Ravenshaw University, Cuttack, India

  2. These authors contributed equally: Khomgrit Morarach, Anastassia Mikhailova.

Authors and Affiliations

  1. Division of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

    Khomgrit Morarach, Anastassia Mikhailova, Viktoria Knoflach, Fatima Memic, Rakesh Kumar, Wei Li, Patrik Ernfors & Ulrika Marklund

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Contributions

Study concept and design: A.M., K.M., V.K. and U.M. Acquisition of data: A.M., F.M., K.M., V.K., R.K., W.L. and U.M. Analysis and interpretation of data: A.M., F.M., K.M., V.K., R.K., W.L. and U.M. Drafting of the manuscript: A.M., K.M., V.K., P.E. and U.M. Obtained funding: F.M., P.E. and U.M.

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Correspondence to Ulrika Marklund.

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Extended data

Extended Data Fig. 1 Supportive Data related to Fig. 1d–g.

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.

Extended Data Fig. 2 Supportive data related to Fig. 1h–j.

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.

Extended Data Fig. 5 Validation of Cre mouse lines for the investigation of ENC6 and ENC7.

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.

Extended Data Fig. 6 Morphological characterization of ENC6, 7, 12 and 5-HT+ ENC12.

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.

Extended Data Fig. 7 Supportive data related to Fig. 5a,b.

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.

Extended Data Fig. 8 Feature plots displaying expression of Notch signaling genes at E18.5.

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.

Extended Data Fig. 10 Supportive data related to Fig. 6f.

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 information

Supplementary Information

Supplementary Figs. 1–6 and Supplementary Table 3.

Reporting Summary

Supplementary Table 1

Enriched genes in ENC1–12.

Supplementary Table 2

Table showing average and individual AUROC scores for HGNC gene families in ENCs.

Supplementary Table 4

Enriched genes in generic cell clusters at E15.5 and E18.5.

Supplementary Table 5

Coordinated gene patterns at E15.5.

Supplementary Table 6

Enriched genes in refined clusters at E15.5 and E18.5.

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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

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