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. 2020 Jan 8;11:111. doi: 10.1038/s41467-019-13850-7

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Immunostainings for M3R showed distribution of the receptor in the crypt base cell compartment (white arrowhead) as well as in cells in positions +4 to +5 of the crypt (white arrow); bar graph top = 50 µm; magnification = 25 µm. b Representative pictures of co-stainings of M3R with intestinal tissue from Lgr5-EGFP-IRES-CreERT2 and induced Prox1-CreERT2 × R26-tdTom mice, respectively, and WT tissue stained for DCLK1, Lysozyme 1, or ChgA (the stainings were repeated at least twice per antibody; positive overlap indicated with white arrowheads); bar graphs = 50 µm. c DCLK1-positive tuft cell frequency within the Lgr5 lineage following M3R ablation in Lgr5-EGFP-IRES-CreERT2 × M3R fl/fl mice 5 days after induction (n = 3 Lgr5-EGFP-IRES-CreERT2 × M3R fl/fl +/+ mice, Mean = 0.917, SEM = 0.088; n = 5 Lgr5-EGFP-IRES-CreERT2 × M3R fl/fl −/− mice, Mean = 1.3, SEM = 0.057; unpaired t test, two-tailed, t = 3.848, df = 6); bar graphs = 100 µm. d Lgr5-positive ISC tracing is reduced following nonselective muscarinic receptor antagonism (n = 4 mice sham, Mean = 1.78, SEM = 0.1151; n = 3 mice scopolamine, Mean = 0.9367, SEM = 0.2050; unpaired t test, two-tailed, t = 3.851, df = 5); bar graphs = 100 µm. e In contrast, ablation of M3R in Prox1-positive endocrine cells resulted in robust DCLK1-positive tuft cell expansions 5 days after induction of Prox1-CreERT2 × M3R fl/fl mice (n = 5 Prox1-CreERT2 × M3R fl/fl +/+ and M3R fl/fl −/− mice for DCLK1, n = 5 Prox1-CreERT2 × M3R fl/fl +/+ mice, n = 4 Prox1-CreERT2 × M3R fl/fl−/− for ChgA; Prox1-CreERT2 × M3R fl/fl +/+ DCLK1 Mean = 1.090, SEM = 0.109; ChgA Mean = 0.77, SEM = 0.054; Prox1-CreERT2 × M3R fl/fl −/− DCLK1 Mean = 4.480, SEM = 0.286, ChgA Mean = 0.588, SEM = 0.031; ordinary two-way ANOVA, DCLK1 t = 14.90, df = 15; ChgA t = 0.7565, df = 15); bar graphs = 100 µm. Source data are provided as a Source Data file. *p < 0.05, **p < 0.01, ****p < 0.001, ns not significant.