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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).

References

  1. Rao, M. & Gershon, M. D. The bowel and beyond: the enteric nervous system in neurological disorders. Nat. Rev. Gastroenterol. Hepatol. 13, 517–528 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Furness, J. B. The enteric nervous system and neurogastroenterology. Nat. Rev. Gastroenterol. Hepatol. 9, 286–294 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Yoo, B. B. & Mazmanian, S. K. The enteric network: interactions between the immune and nervous systems of the gut. Immunity 46, 910–926 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Spencer, N. J. & Hu, H. Enteric nervous system: sensory transduction, neural circuits and gastrointestinal motility. Nat. Rev. Gastroenterol. Hepatol. 17, 338–351 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Rao, M. & Gershon, M. D. Enteric nervous system development: what could possibly go wrong? Nat. Rev. Neurosci. 19, 552–565 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Obata, Y. et al. Neuronal programming by microbiota regulates intestinal physiology. Nature 578, 284–289 (2020).

    Article  CAS  PubMed  Google Scholar 

  7. McCallum, S. et al. Enteric glia as a source of neural progenitors in adult zebrafish. eLife 9, e56086 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Wright, C. M. et al. scRNA-seq reveals new enteric nervous system roles for GDNF, NRTN, and TBX3. Cell Mol. Gastroenterol. Hepatol. 11, 1548–1592.e1 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Drokhlyansky, E. et al. The human and mouse enteric nervous system at single-cell resolution. Cell 182, 1606–1622 e1623 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. May-Zhang, A. A. et al. Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ. Gastroenterology 160, 755–770 e726 (2021).

    Article  CAS  PubMed  Google Scholar 

  11. Gombash, S. E. et al. Intravenous AAV9 efficiently transduces myenteric neurons in neonate and juvenile mice. Front. Mol. Neurosci. 7, 81 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Wilhelmsen, K., Ketema, M., Truong, H. & Sonnenberg, A. KASH-domain proteins in nuclear migration, anchorage and other processes. J. Cell Sci. 119, 5021–5029 (2006).

    Article  CAS  PubMed  Google Scholar 

  13. van den Pol, A. N. et al. Viral strategies for studying the brain, including a replication-restricted self-amplifying delta-G vesicular stomatis virus that rapidly expresses transgenes in brain and can generate a multicolor golgi-like expression. J. Comp. Neurol. 516, 456–481 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Lasrado, R. et al. Lineage-dependent spatial and functional organization of the mammalian enteric nervous system. Science 356, 722–726 (2017).

    Article  CAS  PubMed  Google Scholar 

  15. Roy-Carson, S. et al. Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment. BMC Genomics 18, 290 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Zeisel, A. et al. Molecular architecture of the mouse nervous system. Cell 174, 999–1014 e1022 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Memic, F. et al. Transcription and signaling regulators in developing neuronal subtypes of mouse and human enteric nervous system. Gastroenterology 154, 624–636 (2018).

    Article  CAS  PubMed  Google Scholar 

  18. Lau, S. T. et al. Activation of Hedgehog signaling promotes development of mouse and human enteric neural crest cells, based on single-cell transcriptome analyses. Gastroenterology 157, 1556–1571 e1555 (2019).

    Article  CAS  PubMed  Google Scholar 

  19. Morarach, K. et al. Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing. Nat. Neurosci. 24, 34–46 (2021).

    Article  CAS  PubMed  Google Scholar 

  20. Howard, A. G. T. et al. An atlas of neural crest lineages along the posterior developing zebrafish at single-cell resolution. eLife 10, e60005 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. van den Brink, S. C. et al. Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations. Nat. Methods 14, 935–936 (2017).

    Article  PubMed  CAS  Google Scholar 

  22. Piwnicka, M., Darzynkiewicz, Z. & Melamed, M. R. RNA and DNA content of isolated cell nuclei measured by multiparameter flow cytometry. Cytometry 3, 269–275 (1983).

    Article  CAS  PubMed  Google Scholar 

  23. Slyper, M. et al. A single-cell and single-nucleus RNA-seq toolbox for fresh and frozen human tumors. Nat. Med. 26, 792–802 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Taylor, C. R., Montagne, W. A., Eisen, J. S. & Ganz, J. Molecular fingerprinting delineates progenitor populations in the developing zebrafish enteric nervous system. Dev. Dyn. 245, 1081–1096 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Carney, T. J. et al. A direct role for Sox10 in specification of neural crest-derived sensory neurons. Development 133, 4619–4630 (2006).

    Article  CAS  PubMed  Google Scholar 

  26. El-Nachef, W. N. & Bronner, M. E. De novo enteric neurogenesis in post-embryonic zebrafish from Schwann cell precursors rather than resident cell types. Development 147, dev186619 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Rodrigues, F. S., Doughton, G., Yang, B. & Kelsh, R. N. A novel transgenic line using the Cre-lox system to allow permanent lineage-labeling of the zebrafish neural crest. Genesis 50, 750–757 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Wang, Y., Rovira, M., Yusuff, S. & Parsons, M. J. Genetic inducible fate mapping in larval zebrafish reveals origins of adult insulin-producing beta-cells. Development 138, 609–617 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  30. McQuin, C. et al. CellProfiler 3.0: next-generation image processing for biology. PLoS Biol. 16, e2005970 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Shah, S. et al. Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing. Development 143, 2862–2867 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Long, X., Colonell, J., Wong, A. M., Singer, R. H. & Lionnet, T. Quantitative mRNA imaging throughout the entire Drosophila brain. Nat. Methods 14, 703–706 (2017).

    Article  CAS  PubMed  Google Scholar 

  33. Maynard, K. R. et al. dotdotdot: an automated approach to quantify multiplex single molecule fluorescent in situ hybridization (smFISH) images in complex tissues. Nucleic Acids Res. 48, e66 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Pharris, M. C. et al. An automated workflow for quantifying RNA transcripts in individual cells in large data-sets. MethodsX 4, 279–288 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Foust, K. D. et al. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat. Biotechnol. 27, 59–65 (2009).

    Article  CAS  PubMed  Google Scholar 

  36. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. B. B. Yoo et al. Neuronal activation of the gastrointestinal tract shapes the gut environment in mice. Preprint at bioRxiv https://doi.org/10.1101/2021.04.12.439539 (2021).

  38. Gabanyi, I. et al. Neuro-immune interactions drive tissue programming in intestinal macrophages. Cell 164, 378–391 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Yan, Y. et al. Interleukin-6 produced by enteric neurons regulates the number and phenotype of microbe-responsive regulatory T cells in the gut. Immunity 54, 499–513 e495 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Jarret, A. et al. Enteric nervous system-derived IL-18 orchestrates mucosal barrier immunity. Cell 180, 50–63 e12 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Muller, P. A. et al. Microbiota-modulated CART+ enteric neurons autonomously regulate blood glucose. Science 370, 314–321 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Grindberg, R. V. et al. RNA-sequencing from single nuclei. Proc. Natl Acad. Sci. USA 110, 19802–19807 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Lacar, B. et al. Nuclear RNA-seq of single neurons reveals molecular signatures of activation. Nat. Commun. 7, 11022 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Stark, R., Grzelak, M. & Hadfield, J. RNA sequencing: the teenage years. Nat. Rev. Genet. 20, 631–656 (2019).

    Article  CAS  PubMed  Google Scholar 

  46. Kamentsky, L. et al. Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software. Bioinformatics 27, 1179–1180 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Krishnaswami, S. R. et al. Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons. Nat. Protoc. 11, 499–524 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Heanue, T. A. & Pachnis, V. Enteric nervous system development and Hirschsprung’s disease: advances in genetic and stem cell studies. Nat. Rev. Neurosci. 8, 466–479 (2007).

    Article  CAS  PubMed  Google Scholar 

  49. Avetisyan, M. et al. Hepatocyte growth factor and MET support mouse enteric nervous system development, the peristaltic response, and intestinal epithelial proliferation in response to injury. J. Neurosci. 35, 11543–11558 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Barrenschee, M. et al. Site-specific gene expression and localization of growth factor ligand receptors RET, GFRα1 and GFRα2 in human adult colon. Cell Tissue Res. 354, 371–380 (2013).

    Article  CAS  PubMed  Google Scholar 

  51. Hoogerwerf, W. A. et al. Clock gene expression in the murine gastrointestinal tract: endogenous rhythmicity and effects of a feeding regimen. Gastroenterology 133, 1250–1260 (2007).

    Article  CAS  PubMed  Google Scholar 

  52. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Obata, Y. et al. Molecular profiling of enteric nervous system cell lineages. Zenodo https://doi.org/10.5281/zenodo.5817674 (2021).

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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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