Molecular profiling of enteric nervous system cell lineages
Authors list
Yuuki Obata Alvaro Castano Medina Todd Fallesen Ana Bon Frauches Oliveira Stefan Boeing Almaz Huseynova Sarah Mccallum Reena Lasrado Tiffany Heanue Vassilis PachnisAbstract
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.
Journal details
Journal Nature Protocols
Volume 17
Issue number 8
Pages 1789-1817
Available online
Publication date
Full text links
Publisher website (DOI) 10.1038/s41596-022-00697-4
Europe PubMed Central 35676375
Pubmed 35676375