Molecular profiling of enteric nervous system cell lineages.
Publication Year:
2022
PubMed ID:
35676375
Funding Grants:
Public Summary:
This study focuses on improving how scientists study the enteric nervous system (ENS)—the network of neurons and glial cells that control nearly all gut functions. While researchers know a lot about how the ENS develops, it’s been difficult to study how it works in adult tissue because isolating healthy gut neurons and glia is technically challenging. To solve this, the authors created new methods that allow for detailed gene-expression analysis (transcriptomics) of these cells. Their approach uses fluorescent labeling of cell nuclei in adult mouse and zebrafish intestines, followed by sorting, RNA extraction, and sequencing. They also developed a visualization technique called SIGNS (Spatial Integration of Granular Nuclear Signals) to map and measure RNA inside gut ganglia. Together, these tools enable high-quality molecular profiling of ENS cell types in adults, advancing understanding of gut-brain biology and intestinal function.
Scientific 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.