Introduction
Acetylcholine (Ach) is the main transmitter in the enteric nervous system, which is considered an essential component of the intestinal stem cell (ISC) niche. Ach modulates epithelial homeostasis and secretion predominantly via cholinergic, muscarinic G-protein coupled receptors such as M3R and M1R.
Aims & Methods
In our previous work, genetic ablation of M3R in the murine small intestine induced a significant reduction in Lgr5+ ISC and was sensed by Prox1+ progenitor cells, which orchestrated the selective, compensatory expansion of DCLK1+ tuft cells. Surprisingly, this phenotype did not occur in the colon of the same mice, hence we here aimed to elucidate potential regional differences in the modulation of ISC homeostasis by cholinergic signaling in the murine intestine.
Immunohistochemical analysis and RT-PCR analysis of colonic tissues from transgenic Vil-Cre x M3R fl/fl mice and Vil-Cre x M1R fl/fl mice compared to WT mice were performed. Further experiments included in-situ hybridization for Lgr5, flow cytometry analysis of transgenic Lgr5-EGFP-IRES-CreERT2 and Prox1-CreERT2 mice, bulk RNA sequencing analysis and immunoblot analysis of key signaling pathways.
Results
Immunohistochemical analysis of small intestinal and colonic segments from Vil-Cre x M3R fl/fl mice showed significant differences in differentiation. In contrast to an expansion of DCLK1+ tuft cells in small intestine, these showed significantly reduced in the colon of Vil-Cre x M3R fl/fl mice, while p-EGFR+ tuft cells expanded. This pointed to potential differences in sensing cholinergic signaling interruption by ISC subtypes, which we currently investigate employing Lgr5-EGFP-IRES-CreERT2 x M3R fl/fl and Prox1-CreERT2 x M3R fl/fl mice. Similar to small intestine, however, colonic Vil-Cre x M3R fl/fl tissues revealed a pronounced decrease in Lgr5+ ISC concomitant to increased mucosal Ach levels. Also, Vil-Cre x M3R fl/fl tissues showed a prominent upregulation of PI3K, which was abrogated in Vil-Cre x M3R fl/fl x M1R fl/fl mice. In addition, RT-PCR and bulk RNA sequencing results from WT tissues or organoids showed more prominent expression of Chrm1 (gene encoding M1R) in colonic epithelia than Chrm3 (gene encoding M3R), and analysis of Vil-Cre x M1R fl/fl colonic tissues revealed more prominent changes to differentiation and Lgr5+ ISC compared to Vil-Cre x M3R fl/fl mice.
Conclusion
Our data point to prominent regional differences in the modulation of ISC by cholinergic muscarinic niche signaling and local ISC subtypes sensing cholinergic signaling interruption. Moreover, the receptor subtypes M3R and M1R appear to modulate distinct intracellular pathways, and M1R more prominently modulates colonic ISC. Hence, our data point to potentially distinct roles of cholinergic receptor subtype signaling in intestinal homeostasis and regeneration in small and large intestine.
References
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Barker, N. Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration. Nat Rev Mol Cell Biol 15, 19–33 (2014).
Haber, A. L. et al. A single-cell survey of the small intestinal epithelium. Nature 551, 333–339 (2017).
McKinley, E. T. et al. Optimized multiplex immunofluorescence single-cell analysis reveals tuft cell heterogeneity. JCI Insight 2, 11 (2017).
Middelhoff, M., Nienhüser, H., Valenti, G. et al. Prox1-positive cells monitor and sustain the murine intestinal epithelial cholinergic niche. Nat Commun 11, 111 (2020).
