Application of a RiboTag‐based approach to generate and analyze mRNA from enteric neural cells

Abstract

Background: Transcriptional profiling of specific intestinal cell populations under health and disease is generally based on traditional sorting approaches followed by gene expression analysis. Therein, specific cell populations are identified either by expressing reporter genes under a cell type-specific promotor or by specific surface antigens. This method provides adequate results for blood-derived and tissue-resident immune cells. However, in stromal cell analysis, cellular stress due to digestion often results in degraded RNA. Particularly, ramified cells integrated into the tissue, such as enteric neurons and glial cells, suffer from these procedures.These cell types are involved in various intestinal processes, including a prominent immune-regulatory role, which requires suitable approaches to generate cell-specific transcriptional profiles.Methods: Sox10 iCreERT2 and choline acetyltransferase (ChAT Cre ) mice were crossed with mice labeling the ribosomal Rpl22 protein upon Cre activity with a hemagglutinin tag (Rpl22-HA, termed RiboTag). This approach enabled cellular targeting of enteric glia and neurons and the immediate isolation of cell-specific mRNA from tissue lysates without the need for cell sorting. Key results:We verified the specific expression of Rpl22-HA in enteric glia and neurons and provided gene expression data demonstrating a successful enrichment of either Sox-10 + glial or ChAT + neuronal mRNAs by the RiboTag-mRNA procedure using qPCR and RNA-Seq analysis. Conclusions and inferences:We present a robust and selective protocol that allows the generation of cell type-specific transcriptional in vivo snapshots of distinct enteric cell populations that will be especially useful for various intestinal disease models involving peripheral neural cells.

BQ788 reveals glial ETB receptor modulation of neuronal cholinergic and nitrergic pathways to inhibit intestinal motility: Linked to postoperative ileus

“…Expression of the ET B receptor is much greater than ET A receptor (or glial makers, * P < 0.01). RiboTag (Sox10‐EGCs) (Leven et al, 2022) mice confirmed that ET B receptor ribosomal mRNA expression is much greater than ET A receptor (Figure 1b, ** P < 0.01).…”

“…Expression of the ET B receptor is much greater than ET A receptor (or glial makers, *P < 0.01). RiboTag (Sox10-EGCs)(Leven et al, 2022) mice confirmed that ET B receptor ribosomal mRNA expression is much greater than ET A receptor (Figure1b, **P < 0.01).3.1.2 | Protein analysis of ET B receptorsAn anti-ET B receptor antibody revealed bands at 52 and 42 kDa. Both bands were expressed in colon and jejunum ME (Figure1c).…”

“…The source of ET‐1 in the ME was investigated by ET‐1 immunoreactivity, RiboTag (ChAT)‐Neurons (Leven et al, 2022) and neural networks of human myenteric ganglia.…”

“…3-D imaging of ET-1-ir in a thicker z-stack to image through the entire thickness of LMMP-CM shows ET-1 is mainly expressed in intra-ganglionic varicose fibres (FigureS2g-i). Edn1 (ET-1) mRNA expression was confirmed in RiboTag ChAT-Neurons(Leven et al, 2022) (FigureS2j; n = 8 animals per group). ET-1 protein was detectable in isolated human neural networks of myenteric ganglia (FigureS2k,l; n = 8)(Gulbransen & Sharkey, 2009).…”

“…As shown in Figure S4a-d Neuron-to-glial communication involves purinergic signalling (Leven et al, 2022). We tested if purinergic signalling accounts for the entire EFS response ranging from 0.1 to 25 Hz EFS.…”