Molecular profiling of enteric nervous system cell lineages

Area of Science:

• Neuroscience
• Genomics
• Gastroenterology

Background:

• The enteric nervous system (ENS) controls gut physiology but adult functional pathways are poorly understood.
• Technical challenges in isolating adult ENS cells hinder molecular characterization.
• Existing methods are insufficient for detailed analysis of adult ENS neuron and glia function.

Purpose of the Study:

• To develop and validate novel methods for transcriptomic analysis of adult enteric neurons and glia.
• To overcome limitations in purifying healthy cells from adult intestinal tissues.
• To enable high-resolution RNA expression analysis of the adult ENS.

Main Methods:

• Adeno-associated virus-mediated gene transfer for labeling adult mouse enteric neuronal nuclei.
• Isolation of fluorescently labeled nuclei via fluorimetric analysis.
• Nuclear RNA sequencing and Spatial Integration of Granular Nuclear Signals (SIGNS) for RNA visualization.

Main Results:

• A step-by-step protocol for nuclear RNA sequencing of adult mouse ENS cells was established.
• The protocol was successfully adapted for adult zebrafish myenteric plexus preparations.
• High-resolution RNA expression analysis on ENS tissues can be performed within 7 days.

Conclusions:

• The developed protocol provides a robust method for molecular characterization of the adult ENS.
• This technique facilitates the study of molecular pathways regulating ENS function in adult stages.
• The SIGNS method allows for visualization and quantification of RNA within myenteric ganglia.