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. 2017 Oct 30:8:849.
doi: 10.3389/fphys.2017.00849. eCollection 2017.

Members of Bitter Taste Receptor Cluster Tas2r143/Tas2r135/Tas2r126 Are Expressed in the Epithelium of Murine Airways and Other Non-gustatory Tissues

Shuya Liu  1 , Shun Lu  1 , Rui Xu  1 , Ann Atzberger  2 , Stefan Günther  3 , Nina Wettschureck  1   4 , Stefan Offermanns  1   4
Affiliations

Members of Bitter Taste Receptor Cluster Tas2r143/Tas2r135/Tas2r126 Are Expressed in the Epithelium of Murine Airways and Other Non-gustatory Tissues

Shuya Liu et al. Front Physiol. .

Abstract

The mouse bitter taste receptors Tas2r143, Tas2r135, and Tas2r126 are encoded by genes that cluster on chromosome 6 and have been suggested to be expressed under common regulatory elements. Previous studies indicated that the Tas2r143/Tas2r135/Tas2r126 cluster is expressed in the heart, but other organs had not been systematically analyzed. In order to investigate the expression of this bitter taste receptor gene cluster in non-gustatory tissues, we generated a BAC (bacterial artificial chromosome) based transgenic mouse line, expressing CreERT2 under the control of the Tas2r143 promoter. After crossing this line with a mouse line expressing EGFP after Cre-mediated recombination, we were able to validate the Tas2r143-CreERT2 transgenic mouse line and monitor the expression of Tas2r143. EGFP-positive cells, indicating expression of members of the cluster, were found in about 47% of taste buds, and could also be found in several other organs. A population of EGFP-positive cells was identified in thymic epithelial cells, in the lamina propria of the intestine and in vascular smooth muscle cells of cardiac blood vessels. EGFP-positive cells were also identified in the epithelium of organs readily exposed to pathogens including lower airways, the gastrointestinal tract, urethra, vagina, and cervix. With respect to the function of cells expressing this bitter taste receptor cluster, RNA-seq analysis in EGFP-positive cells isolated from the epithelium of trachea and stomach showed expression of genes related to innate immunity. These data further support the concept that bitter taste receptors serve functions outside the gustatory system.

Keywords: Tas2r126; Tas2r135; Tas2r143; chemosensory cells; tuft cells.

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Figures

Figure 1
Figure 1
Generation of Tas2r143-CreERT2 transgenic mice. (A) NanoString analysis of mRNA expression levels for bitter taste receptors in the murine heart. (n = 2, error bars represent mean ± SD). (B) The genomic sequence between the start codon of Tas2r143 and the stop codon of Tas2r126 (35.2 kb) on BAC RP23-316O11 was replaced by a cassette carrying the CreERT2 cDNA followed by a polyadenylation (pA) signal. (C,D) Offspring of transgenic mice was genotyped by genomic PCR with primers P1/P2 and P3/P4 identifying a 614 bp and a 418 bp fragment, respectively, which indicate the correctly inserted CreERT2.
Figure 2
Figure 2
Generation of tamoxifen-inducible Tas2r143-reporter mice. (A) Tas2r143-CreERT2 mice were crossed with Cre-reporter mice (Rosa26flox−mT−stop−flox−mG) to generate Tas2r143-CreERT2; Rosa26flox−mT−stop−flox−mG reporter mice. (B) Cre recombinase activity was induced by tamoxifen injection on 5 consecutive days. Reporter mice were analyzed 5 days after the last injection. (C) After tamoxifen injection, EGFP-positive cells were detected in taste receptor cells, stained by anti-PLCß2 antibody, in taste buds of the tongue. (D) Tas2r143, Tas2r135, and Tas2r126 were detected in the tongue epithelium by real-time qPCR (n = 3, error bars represent mean ± SD). (E) EGFP-positive cells were detected in cryosections from various organs. Nuclei were counterstained with DAPI. Squares indicate enlarged areas. Scale bars: 10 μm (C), 50 μm (E).
Figure 3
Figure 3
Analysis of Tas2r143-reporter mice in the lower respiratory tract. (A) Immunofluorescence staining of cryosections in the trachea, bronchus and bronchiole. EGFP-positive epithelial cells prominently colocalized with the tuft cell marker DCLK1. Nuclei were counterstained with DAPI. Squares indicate enlarged areas. Scale bars: 50 μm. (B–E) RNA-seq analysis of gene expression levels in purified EGFP-positive cells compared with Tomato-positive cells. The relative expression was calculated as log2(EGFP/Tomato). EGFP- and Tomato-positive cells were isolated from the tracheas of the reporter mice (n = 3).
Figure 4
Figure 4
Analysis of Tas2r143-reporter mice in the stomach. (A) Immunofluorescence staining of stomach cryosections. EGFP-positive epithelial cells colocalized with the tuft cell marker DCLK1. Nuclei were counterstained with DAPI. Squares indicate enlarged areas. Scale bars: 50 μm. (B–E) RNA-seq analysis of gene expression levels in purified EGFP-positive cells compared with Tomato-positive cells. The relative expression was calculated as log2(EGFP/Tomato). EGFP- and Tomato-positive cells were isolated from the stomachs of the reporter mice (n = 3).
Figure 5
Figure 5
Expression analysis of Tas2r143-reporter mice in the intestine. Immunofluorescence staining of cryosections from the duodenum, jejunum, and colon. EGFP-positive cells were detected in the epithelium and lamina propria. EGFP-positive epithelial cells were positive for tuft cell marker DCLK1. EGFP-positive cells in the lamina propria were positive for ChAT. Nuclei were counterstained with DAPI. Squares indicate enlarged areas. Scale bars: 50 μm.
Figure 6
Figure 6
Expression analysis of Tas2r143-reporter mice in the urethra, vagina and cervix. Immunofluorescence staining of urethra, vagina, and cervix cryosections. EGFP-positive cells were detected in the epithelium. Mitotically active epithelial cells were stained by anti-K5 antibody. Terminally differentiated epithelial cells were stained by anti-K10 antibody. Nuclei were counterstained with DAPI. Squares indicate enlarged areas. Lu, lumen. Scale bars: 50 μm.
Figure 7
Figure 7
Expression analysis of Tas2r143-reporter mice in the thymus. Immunofluorescence staining of thymic cryosections. T cells were stained by anti-CD3 antibody. Medullar region of the thymus was marked by medullary thymic epithelial cells stained by anti-K5 antibody. Cortical region of the thymus was marked by cortical thymic epithelial cells stained by K18 antibody. EGFP-positive cells could be stained by an anti-ChAT antibody and partially colocalized with K18 and terminally differentiated epithelial cell marker K10. Nuclei were counterstained with DAPI. Arrows indicate enlarged areas. Scale bars: 50 μm.
Figure 8
Figure 8
Expression analysis of Tas2r143-reporter mice in the heart. Immunofluorescence staining of heart cryosections. EGFP-positive cells were detected in a subset of blood vessels in the heart. EGFP-positive cells were adjacent to endothelial cells, stained by anti-CD31 antibody, and were costained with the vascular smooth muscle cell marker αSMA. Nuclei were counterstained with DAPI. Squares indicate enlarged areas. Scale bars: 50 μm.
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