Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Sep:51:101231.
doi: 10.1016/j.molmet.2021.101231. Epub 2021 Apr 5.

Adhesion receptor ADGRG2/GPR64 is in the GI-tract selectively expressed in mature intestinal tuft cells

Kaare V Grunddal  1 , Sarah Tonack  2 , Kristoffer L Egerod  3 , Jonathan James Thompson  3 , Natalia Petersen  3 , Maja S Engelstoft  3 , Constance Vagne  4 , Céline Keime  5 , Gérard Gradwohl  4 , Stefan Offermanns  2 , Thue W Schwartz  6
Affiliations

Adhesion receptor ADGRG2/GPR64 is in the GI-tract selectively expressed in mature intestinal tuft cells

Kaare V Grunddal et al. Mol Metab. 2021 Sep.

Abstract

Objective: GPR64/ADGRG2 is an orphan Adhesion G protein-coupled receptor (ADGR) known to be mainly expressed in the parathyroid gland and epididymis. This investigation aimed to delineate the cellular expression of GPR64 throughout the body with focus on the gastrointestinal (GI) tract.

Methods: Transgenic Gpr64mCherry reporter mice were histologically examined throughout the body and reporter protein expression in intestinal tuft cells was confirmed by specific cell ablation. The GPCR repertoire of intestinal Gpr64mCherry-positive tuft cells was analyzed by quantitative RT-PCR analysis and in situ hybridization. The Gpr64mCherry was crossed into the general tuft cell reporter Trpm5GFP to generate small intestinal organoids for time-lapse imaging. Intestinal tuft cells were isolated from small intestine, FACS-purified and transcriptionally compared using RNA-seq analysis.

Results: Expression of the Gpr64mCherry reporter was identified in multiple organs and specifically in olfactory microvillous cells, enteric nerves, and importantly in respiratory and GI tuft cells. In the small intestine, cell ablation targeting Gpr64-expressing epithelial cells eliminated tuft cells. Transcriptional analysis of small intestinal Gpr64mCherry -positive tuft cells confirmed expression of Gpr64 and the chemo-sensors Sucnr1, Gprc5c, Drd3, and Gpr41/Ffar3. Time-lapse studies of organoids from Trpm5GFP:Gpr64mCherry mice revealed sequential expression of initially Trpm5GFP and subsequently also Gpr64mCherry in maturing intestinal tuft cells. RNA-seq analysis of small intestinal tuft cells based on these two markers demonstrated a dynamic change in expression of transcription factors and GPCRs from young to mature tuft cells.

Conclusions: GPR64 is expressed in chemosensory epithelial cells across a broad range of tissues; however, in the GI tract, GPR64 is remarkably selectively expressed in mature versus young immunoregulatory tuft cells.

Keywords: ADGRG2; Chemosensory cells; GPCRs; GPR64; Tuft cells.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Histological examination of the transgenic Gpr64-mCherry reporter mice Representative fluorescence microscopy images showing Gpr64mCherry fluorescence (Red) and DAPI nuclei staining (Blue) from A) epididymis, B) parathyroid gland, C) adrenal Gland, D) liver central vein, E) Main olfactory epithelium exhibited three morphologically distinct types of Gpr64mCherry-positive cells: Pear-shaped cells (X), flask-shaped cells (Y) and olfactory sensory neurons (Z), F) tracheal epithelium, G) pancreatic duct, H) gastric groove, I) gastric antrum, J) Brunner glands, K) duodenum, L) Jejunum, M) distal ileum, N, proximal colon and O) rectum. Arrows indicate mCherry fluorescence in myenteric plexi throughout the intestine. Insets show cells in higher magnification. Male mice n = 3. Bar = 50 μm.
Figure 2
Figure 2
Gastrointestinal Gpr64-mCherry positive cells are enteric nerves and tuft cells Representative fluorescence microscopy images of Gpr64mCherry tissue sections immunostained for selected markers specific for tuft cells, microvillous cells, and enteric nerves. A) GI tract Gpr64mCherry-positive cells (red) stained with doublecortin like kinase 1 (DCLK1), acetylated alpha Tubulin (AC-TUB), cytokeratin 18 (CK18), hematopoietic prostaglandin D synthase (HPGDS), prostaglandin D2 Synthase 1 (PTGS1) and prostaglandin D2 Synthase 2 (PTGS2) antibodies (green) and counterstained with DAPI (blue). Bar = 5 μm. B) Gpr64mCherry-positive submucosal and myenteric nerves immunostained with anti-PGP9.5 Bar = 50 μm. C) Respiratory tract Gpr64mCherry cells stained with DCLK1 and CK18 antibodies (scale bar, 5 μm). Fourth panel displays olfactory epithelium isolated from the double transgenic Trpm5GFP;Gpr64mCherry reporter mouse with both Gpr64 promoter-driven mCherry and Trpm5 promoter-driven GFP expression. Bar = 20 μm. Male mice n = 3. Ab, Antibody. D) Small intestinal sections from GPR64DTA control mice (upper row) and Vil-CreERT2;GPR64DTA mice (lower row) before and 48 h after tamoxifen induced cell ablation. Tuft cells visualized with anti-DCLK1 (red). Bar = 100 μm. Per group n = 6–8 mice.
Figure 3
Figure 3
Non-odorant GPCR expression profile of mature tuft cells A) Representative FACS diagram showing gate (trapezoid) used for sorting Gpr64mCherry-positive tuft cells based on emission at 614 and 513 nm after excitation at 561 and 488 nm, respectively. B) Quantitative RT-PCR analysis of FACS-purified Gpr64mCherry-positive cells. The relative expression of 379 GPCRs in Gpr64mCherry-positive cells (y-axis) vs Gpr64mCherry-negative cells (x-axis). The enriched GPCRs are displayed with gene name. The remaining GPCR are shown as grey dots. N = 5, each consisting of cells derived from 3 pooled mice (total 15 male mice). C) Dual immunohistochemistry and in situ hybridization probing for selected GPCR's (Red fluorescence) on from Gpr64mCherry duodenum sections. mCherry signal was retrieved using mCherry-specific antibodies (Green fluorescence). Each red dot represents a single stained mRNA transcript. Positive control probe: Mus musculus, Peptidylpropyl isomerase B (Ppib). Negative control probe: Bacillus subtilis, dihydrodipicolinate reductase (DapB). Ab: Antibody. Pb: in situ hybridization probe. Nuclei were visualized with DAPI counterstaining (blue). Male mice n = 3.
Figure 4
Figure 4
Histological and organoid studies indicate sequential expression of Trpm5, and subsequently Gpr64, in maturing intestinal tuft cells A) Representative fluorescence microscopy images of the double transgenic reporter Trpm5GFP;Gpr64mCherry mouse duodenum showing Trpm5 promoter-driven GFP fluorescence (Green), Gpr64 promoter-driven mCherry fluorescence (Red) and DAPI nuclei staining (Blue). Merged picture on the right also contain magnification of Trpm5GFP-positive cryptal cells, but no visible Gpr64mCherry fluorescence. Dashed line separates crypt (C) and villus (V) area. Bar = 50 μm. B) Quantification of Trpm5GFP and Gpr64mCherry co-localization in crypt and villus area. Normalized to total cell count of crypt or villus area. GFP signal was enhanced with antibodies. >300 counted cells per animal. Male mice n = 3. Data tested with 2-way ANOVA. C) Representative timelapse images of intestinal Trpm5GFP;Gpr64mCherry organoid treated with recombinant Il-4 and Il-13. Brightfield, Trpm5GFP fluorescence and Gpr64mCherry fluorescence images were captured at timepoint: 0, 3, 6, 15 h. Initially, three Trpm5GFP-fluorescent tuft cells are observed (tagged: x,y and z) with no evident mCherry fluorescence. After 6–24 h, a gradual increase in Gpr64mCherry fluorescence is observed in the three Trpm5GFP-positive tuft cells. Arrowhead indicate another cell first expressing GFP and then subsequently express mCherry. Insets show cells in higher magnification. Bar = 50 μm. D) Quantification of sequential expression of GFP and mCherry fluorescence in Trpm5GFP;Gpr64mCherry organoids. Forty-five organoids containing 119 fluorescent cells were monitored for up to 60 h. Of the 119 cells, 58 cells were initially Trpm5GFP-positive/Gpr64mCherry-negative and then became Trpm5GFP-positive/Gpr64mCherry-positive. Average time from first detected GFP fluorescence to first detected mCherry fluorescence: 10.8 h ± 1.7 SEM. G + R- = Cell displaying Trpm5GFP-positive and Gpr64mCherry-negative fluorescence. G + R+ = Cell displaying Trpm5GFP-positive and Gpr64mCherry-positive fluorescence. G-R+ = Cell displaying Trpm5GFP-negative and Gpr64mCherry-positive fluorescence.
Figure 5
Figure 5
RNA-seq reveal tuft cell signature genes and a dynamic chemosensory GPCR and TF expression in maturing intestinal tuft cell FACS-gating of proximal small intestine preparations from A) WT, B) Trpm5GFP reporter, C) Trpm5GFP;Gpr64mCherry reporter mice. Young tuft cells (Trpm5GFP-positive, Gpr64mCherry-negative), mature tuft cells (Trpm5GFP-positive, Gpr64mCherry-positive) and non-tuft background cells (Trpm5GFP-negative, Gpr64mCherry-negative) were FACS-purified from the proximal small intestine of transgenic Trpm5GFP;Gpr64mCherry reporter mice for mRNA sequencing. Heatmap displays the log2 (x+1) normalized expression of D) canonical tuft cell marker genes, E) selected non-odorant GPCR, F) selected TFs, G) tuft-1 markers, H) tuft-2 markers genes in the young-, mature tuft cells and background cell populations. Mice n = 5.
Figure 6
Figure 6
Comparative transcriptional analysis of young vs. mature intestinal tuft cells Heatmap depicting the log2 (x+1) normalized expression of 50 genes with the highest log2 fold A) downregulation or B) upregulation between young tuft cells (Trpm5GFP-positive, Gpr64mCherry-negative) and mature tuft cells (Trpm5GFP-positive, Gpr64mCherry-positive) from Trpm5GFP;Gpr64mCherry proximal small intestine (all plotted genes had statistically significant log2 fold changes in mature versus young cells, P < 0.01). C) Volcano plot. The vertical dimension represents the log10-transformed p-values (with inverted sign) from a two-sided Mann–Whitney–Wilcoxon geneset enrichment test (Materials and Methods). The horizontal dimension represents the weighted mean of the log2 fold change in expression of the geneset genes in old versus young tuft cells, where each gene's contribution is weighted by the log-transformed p-value (with the sign reversed) corresponding to the log2 fold change. Thus green bubbles left of zero represent genesets which are downregulated in old versus young tuft cells, and red bubbles represent upregulated genesets. The size of the bubble represents the number of genes in the geneset. Mice n = 5.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Crosnier C., Stamataki D., Lewis J. Organizing cell renewal in the intestine: stem cells, signals and combinatorial control. Nature Reviews Genetics. 2006;7(5):349–359. - PubMed
    1. Engelstoft M.S., Egerod K.L., Holst B., Schwartz T.W. A gut feeling for obesity: 7TM sensors on enteroendocrine cells. Cell Metabolism. 2008;8(6):447–449. - PubMed
    1. Rhodin J., Dalhamn T. Electron microscopy of the tracheal ciliated mucosa in rat. Zeitschrift fur Zellforschung und mikroskopische Anatomie. 1956;44(4):345–412. - PubMed
    1. Krasteva G., Canning B.J., Hartmann P., Veres T.Z., Papadakis T., Muhlfeld C. Cholinergic chemosensory cells in the trachea regulate breathing. Proceedings of the National Academy of Sciences of the U S A. 2011;108(23):9478–9483. - PMC - PubMed
    1. Chang L.Y., Mercer R.R., Crapo J.D. Differential distribution of brush cells in the rat lung. The Anatomical Record. 1986;216(1):49–54. - PubMed

Publication types

Substances