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Molecular profiling of enteric nervous system cell lineages

Nature Protocols volume 17, pages 1789–1817 (2022)Cite this article

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Abstract

The enteric nervous system (ENS) is an extensive network of enteric neurons and glial cells that is intrinsic to the gut wall and regulates almost all aspects of intestinal physiology. While considerable advancement has been made in understanding the genetic programs regulating ENS development, there is limited understanding of the molecular pathways that control ENS function in adult stages. One of the limitations in advancing the molecular characterization of the adult ENS relates to technical difficulties in purifying healthy neurons and glia from adult intestinal tissues. To overcome this, we developed novel methods for performing transcriptomic analysis of enteric neurons and glia, which are based on the isolation of fluorescently labeled nuclei. Here we provide a step-by-step protocol for the labeling of adult mouse enteric neuronal nuclei using adeno-associated-virus-mediated gene transfer, isolation of the labeled nuclei by fluorimetric analysis, RNA purification and nuclear RNA sequencing. This protocol has also been adapted for the isolation of enteric neuron and glia nuclei from myenteric plexus preparations from adult zebrafish intestine. Finally, we describe a method for visualization and quantification of RNA in myenteric ganglia: Spatial Integration of Granular Nuclear Signals (SIGNS). By following this protocol, it takes ~3 d to generate RNA and create cDNA libraries for nuclear RNA sequencing and 4 d to carry out high-resolution RNA expression analysis on ENS tissues.

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Fig. 1: Experimental outline for labeling and isolation of ENS-derived nuclei from mouse or zebrafish.
Fig. 2: Experimental outline for visualization and quantification of gene expression in ENS tissues.
Fig. 3: Gating strategy to sort neuronal nuclei from mouse or zebrafish intestinal muscularis externa.
Fig. 4: Gene expression analysis of EGFP-labeled colonic neuronal nuclei.
Fig. 5: Quantification of neuronal gene expression in the myenteric plexus.
Fig. 6: RNAscope analysis of enteric neurons in different tissue conditions.

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Data availability

Mouse nRNA-seq data are available at Gene Expression Omnibus (GEO) under accession numbers GSE140293. Data describing the transcriptome of ENS nuclei isolated from adult zebrafish gut tissue are available at GEO (GSE145885) or online (https://biologic.crick.ac.uk/ENS). Source data are provided with this paper.

Code availability

Code for quantification of imaging data using SIGNS available at https://github.com/FrancisCrickInstitute/Pachnis-lab/tree/master/Neuronal-programming-Nature/Project%20Code and citable as https://doi.org/10.5281/zenodo.5817674 (ref. 53).

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Acknowledgements

We thank the Crick Science Technology Platforms for expert support. We thank J. Brock for scientific illustration. We also thank all members of the Pachnis lab for insightful discussions and experimental support and advice. We also thank A. Murray (Sainsbury Wellcome Centre, University College London) for experimental support and advice for generating AAV vectors. Y.O. was supported by an EMBO long-term fellowship (ALTF 1214-2015), an HFSP postdoctoral fellowship (LT000176/2016), the travel grants from Boehringer Ingelheim Fonds and the Society for Mucosal Immunology (SMI), and the Japanese Society for the promotion of Science (JSPS) Grants-in-Aid for Scientific Research (20K16951). Work in the Pachnis lab is funded by the Francis Crick Institute, which receives core funding from Cancer Research UK (FC001128), the UK Medical Research Council (FC001128) and the Wellcome Trust (FC001128). We also acknowledge additional funding from the BBSRC (BB/L022974) and a Wellcome Trust Investigator Award (212300/Z/18/Z).

Author information

Author notes

  1. Yuuki Obata

    Present address: Department of Immunology, Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA

  2. Álvaro Castaño

    Present address: MRC London Institute of Medical Sciences, Imperial College London, London, UK

  3. Sarah McCallum

    Present address: Center for Neural Science and Medicine and Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA

Authors and Affiliations

  1. Development and Homeostasis of the Nervous System Laboratory, The Francis Crick Institute, London, UK

    Yuuki Obata, Álvaro Castaño, Ana Carina Bon-Frauches, Sarah McCallum, Reena Lasrado, Tiffany A. Heanue & Vassilis Pachnis

  2. Crick Advanced Light Microscopy, The Francis Crick Institute, London, UK

    Todd L. Fallesen

  3. Bionformatics & Biostatistics, The Francis Crick Institute, London, UK

    Stefan Boeing

  4. Software Development & Machine Learning Team, The Francis Crick Institute, London, UK

    Stefan Boeing

  5. Kinases and Brain Development Laboratory, The Francis Crick Institute, London, UK

    Almaz Huseynova

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Contributions

Y.O. and V.P. conceived the study. Y.O. developed the method for the targeting and isolation of enteric neuronal nuclei from mouse gut with help from A.C.B.-F.; Y.O., T.A.H. and S.M. applied the protocol for zebrafish ENS study. Y.O., A.C.B.-F., S.M. and T.A.H. performed the experiments. R.L. performed initial RNAscope optimization. Á.C. performed further RNA optimization, and Á.C. and T.A.H. performed the RNAscope in situ hybridization experiments; T.L.F. developed the SIGNS method and helped with the quantification of RNAscope data; A.H. prepared the cDNA library for the bulk nRNA-seq. S.B. performed bioinformatics analysis. Y.O. and T.A.H. wrote the manuscript with help from A.C., and contributions from all authors.

Corresponding authors

Correspondence to Yuuki Obata, Tiffany A. Heanue or Vassilis Pachnis.

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Nature Protocols thanks Isaac Chiu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Obata, Y. et al. Nature 578, 284–289 (2020): https://doi.org/10.1038/s41586-020-1975-8

McCallum, S. et al. eLife 9, e56086 (2020): https://doi.org/10.7554/eLife.56086

Supplementary information

Supplementary Information

Supplementary Figs. 1–3.

Source data

Source Data Fig. 4

Neuronal gene expression analysis

Source Data Fig. 5

Quantification of RNA scope signals per individual neuron

Source Data Fig. 6

RNA scope data quantification

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Obata, Y., Castaño, Á., Fallesen, T.L. et al. Molecular profiling of enteric nervous system cell lineages. Nat Protoc 17, 1789–1817 (2022). https://doi.org/10.1038/s41596-022-00697-4

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