Molecular profiling of enteric nervous system cell lineages.
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
08 2022
08 2022
Historique:
received:
04
06
2021
accepted:
04
03
2022
pubmed:
9
6
2022
medline:
6
8
2022
entrez:
8
6
2022
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 35676375
doi: 10.1038/s41596-022-00697-4
pii: 10.1038/s41596-022-00697-4
doi:
Substances chimiques
RNA
63231-63-0
Types de publication
Journal Article
Review
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1789-1817Subventions
Organisme : Cancer Research UK
ID : FC001128
Pays : United Kingdom
Organisme : Medical Research Council
ID : FC001128
Pays : United Kingdom
Organisme : Wellcome Trust
ID : FC001128
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/L022974
Pays : United Kingdom
Organisme : Wellcome Trust
ID : 212300/Z/18/Z
Pays : United Kingdom
Informations de copyright
© 2022. Springer Nature Limited.
Références
Rao, M. & Gershon, M. D. The bowel and beyond: the enteric nervous system in neurological disorders. Nat. Rev. Gastroenterol. Hepatol. 13, 517–528 (2016).
pubmed: 27435372
pmcid: 5005185
doi: 10.1038/nrgastro.2016.107
Furness, J. B. The enteric nervous system and neurogastroenterology. Nat. Rev. Gastroenterol. Hepatol. 9, 286–294 (2012).
pubmed: 22392290
doi: 10.1038/nrgastro.2012.32
Yoo, B. B. & Mazmanian, S. K. The enteric network: interactions between the immune and nervous systems of the gut. Immunity 46, 910–926 (2017).
pubmed: 28636959
pmcid: 5551410
doi: 10.1016/j.immuni.2017.05.011
Spencer, N. J. & Hu, H. Enteric nervous system: sensory transduction, neural circuits and gastrointestinal motility. Nat. Rev. Gastroenterol. Hepatol. 17, 338–351 (2020).
pubmed: 32152479
pmcid: 7474470
doi: 10.1038/s41575-020-0271-2
Rao, M. & Gershon, M. D. Enteric nervous system development: what could possibly go wrong? Nat. Rev. Neurosci. 19, 552–565 (2018).
pubmed: 30046054
pmcid: 6261281
doi: 10.1038/s41583-018-0041-0
Obata, Y. et al. Neuronal programming by microbiota regulates intestinal physiology. Nature 578, 284–289 (2020).
pubmed: 32025031
doi: 10.1038/s41586-020-1975-8
McCallum, S. et al. Enteric glia as a source of neural progenitors in adult zebrafish. eLife 9, e56086 (2020).
pubmed: 32851974
pmcid: 7521928
doi: 10.7554/eLife.56086
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).
pubmed: 33444816
pmcid: 8099699
doi: 10.1016/j.jcmgh.2020.12.014
Drokhlyansky, E. et al. The human and mouse enteric nervous system at single-cell resolution. Cell 182, 1606–1622 e1623 (2020).
pubmed: 32888429
pmcid: 8358727
doi: 10.1016/j.cell.2020.08.003
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).
pubmed: 33010250
doi: 10.1053/j.gastro.2020.09.032
Gombash, S. E. et al. Intravenous AAV9 efficiently transduces myenteric neurons in neonate and juvenile mice. Front. Mol. Neurosci. 7, 81 (2014).
pubmed: 25360081
pmcid: 4197761
doi: 10.3389/fnmol.2014.00081
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).
pubmed: 17158909
doi: 10.1242/jcs.03295
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).
pubmed: 19672982
pmcid: 2919849
doi: 10.1002/cne.22131
Lasrado, R. et al. Lineage-dependent spatial and functional organization of the mammalian enteric nervous system. Science 356, 722–726 (2017).
pubmed: 28522527
doi: 10.1126/science.aam7511
Roy-Carson, S. et al. Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment. BMC Genomics 18, 290 (2017).
pubmed: 28403821
pmcid: 5389105
doi: 10.1186/s12864-017-3653-2
Zeisel, A. et al. Molecular architecture of the mouse nervous system. Cell 174, 999–1014 e1022 (2018).
pubmed: 30096314
pmcid: 6086934
doi: 10.1016/j.cell.2018.06.021
Memic, F. et al. Transcription and signaling regulators in developing neuronal subtypes of mouse and human enteric nervous system. Gastroenterology 154, 624–636 (2018).
pubmed: 29031500
doi: 10.1053/j.gastro.2017.10.005
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).
pubmed: 31442438
doi: 10.1053/j.gastro.2019.08.019
Morarach, K. et al. Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing. Nat. Neurosci. 24, 34–46 (2021).
pubmed: 33288908
doi: 10.1038/s41593-020-00736-x
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).
pubmed: 33591267
pmcid: 7886338
doi: 10.7554/eLife.60005
van den Brink, S. C. et al. Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations. Nat. Methods 14, 935–936 (2017).
pubmed: 28960196
doi: 10.1038/nmeth.4437
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).
pubmed: 6185286
doi: 10.1002/cyto.990030407
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).
pubmed: 32405060
pmcid: 7220853
doi: 10.1038/s41591-020-0844-1
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).
pubmed: 27565577
pmcid: 5088718
doi: 10.1002/dvdy.24438
Carney, T. J. et al. A direct role for Sox10 in specification of neural crest-derived sensory neurons. Development 133, 4619–4630 (2006).
pubmed: 17065232
doi: 10.1242/dev.02668
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).
pubmed: 32541008
pmcid: 7375481
doi: 10.1242/dev.186619
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).
pubmed: 22522888
doi: 10.1002/dvg.22033
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).
pubmed: 21208992
pmcid: 3026409
doi: 10.1242/dev.059097
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
pubmed: 22743772
doi: 10.1038/nmeth.2019
McQuin, C. et al. CellProfiler 3.0: next-generation image processing for biology. PLoS Biol. 16, e2005970 (2018).
pubmed: 29969450
pmcid: 6029841
doi: 10.1371/journal.pbio.2005970
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).
pubmed: 27342713
pmcid: 5004914
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).
pubmed: 28581495
doi: 10.1038/nmeth.4309
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).
pubmed: 32383753
pmcid: 7293004
doi: 10.1093/nar/gkaa312
Pharris, M. C. et al. An automated workflow for quantifying RNA transcripts in individual cells in large data-sets. MethodsX 4, 279–288 (2017).
pubmed: 28932696
pmcid: 5596354
doi: 10.1016/j.mex.2017.08.002
Foust, K. D. et al. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat. Biotechnol. 27, 59–65 (2009).
pubmed: 19098898
doi: 10.1038/nbt.1515
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).
pubmed: 28671695
pmcid: 5529245
doi: 10.1038/nn.4593
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).
Gabanyi, I. et al. Neuro-immune interactions drive tissue programming in intestinal macrophages. Cell 164, 378–391 (2016).
pubmed: 26777404
pmcid: 4733406
doi: 10.1016/j.cell.2015.12.023
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).
pubmed: 33691135
pmcid: 8133394
doi: 10.1016/j.immuni.2021.02.002
Jarret, A. et al. Enteric nervous system-derived IL-18 orchestrates mucosal barrier immunity. Cell 180, 50–63 e12 (2020).
pubmed: 31923399
pmcid: 7339937
doi: 10.1016/j.cell.2019.12.016
Muller, P. A. et al. Microbiota-modulated CART
pubmed: 32855216
pmcid: 7886298
doi: 10.1126/science.abd6176
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).
pubmed: 21865647
pmcid: 3163972
doi: 10.1172/JCI58200
Grindberg, R. V. et al. RNA-sequencing from single nuclei. Proc. Natl Acad. Sci. USA 110, 19802–19807 (2013).
pubmed: 24248345
pmcid: 3856806
doi: 10.1073/pnas.1319700110
Lacar, B. et al. Nuclear RNA-seq of single neurons reveals molecular signatures of activation. Nat. Commun. 7, 11022 (2016).
pubmed: 27090946
pmcid: 4838832
doi: 10.1038/ncomms11022
Stark, R., Grzelak, M. & Hadfield, J. RNA sequencing: the teenage years. Nat. Rev. Genet. 20, 631–656 (2019).
pubmed: 31341269
doi: 10.1038/s41576-019-0150-2
Kamentsky, L. et al. Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software. Bioinformatics 27, 1179–1180 (2011).
pubmed: 21349861
pmcid: 3072555
doi: 10.1093/bioinformatics/btr095
Krishnaswami, S. R. et al. Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons. Nat. Protoc. 11, 499–524 (2016).
pubmed: 26890679
pmcid: 4941947
doi: 10.1038/nprot.2016.015
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).
pubmed: 17514199
doi: 10.1038/nrn2137
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).
pubmed: 26290232
pmcid: 4540795
doi: 10.1523/JNEUROSCI.5267-14.2015
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).
pubmed: 23881409
doi: 10.1007/s00441-013-1690-y
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).
pubmed: 17919497
doi: 10.1053/j.gastro.2007.07.009
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).
pubmed: 16199517
pmcid: 1239896
doi: 10.1073/pnas.0506580102
Obata, Y. et al. Molecular profiling of enteric nervous system cell lineages. Zenodo https://doi.org/10.5281/zenodo.5817674 (2021).