Figure 1.
Expression analysis of Tas2rs and taste signaling cascade elements in mouse tissues.
(A) and (B) PCR analyses of cDNA from vallate papillae (VP), stomach (Sto), duodenum (Duo), jejunum (Jej), colon (Col) and liver (Liv). (A) PCR products obtained from cDNA of mouse GI tissues (+) and -RT controls (−) with primers specific for Tas2r108, -r138, -r119 and -r118. GAPDH was amplified as quality control, mouse genomic DNA served as positive control (gD). N, negative control (H2O). (B) PCR analysis of α-gustducin, PLCβ2, TRPM5, Gγ13, Gβ1 and Gβ3 expression in mouse GI tract. M - Molecular weight standard (ΦX174 DNA/HaeIII). (C) Quantitative RT-PCR of gustatory and gastrointestinal tissues. cDNA of vallate papillae (VP), duodenum (Duo), proximal (pJej) and distal (dJej) jejunum, ileum (Ile), colon (Col), stomach (Sto) and liver (Liv) was subjected to quantitative real-time PCR analyses using primers specific for Tas2r108 (black bars), -r118 (dark gray bars), -r119 (light gray bars), -r131 (white bars), and –r138 (hatched bars). Y-axis = logarithm of the expression levels relative to β-actin (mean log(2−ΔCT) ±SE, n≥3). (D) Analysis of Tas2r gene expression in whole blood. PCR products obtained from cDNA of mouse whole blood (+) and -RT controls (−) with primers specific for Tas2r108, -r138, and -r118. GAPDH was amplified as quality control, mouse genomic DNA served as positive control (gD). N, negative control (H2O). M - Molecular weight standard (ΦX174 DNA/HaeIII). (E) In situ hybridization. Antisense riboprobes specific for α-gustducin and a mixture of riboprobes for Tas2r108, -r119, -r138, -r131 and -r118 in VP sections resulted in specific signals in taste buds. In situ hybridization of small intestine sections revealed cells positive for α-gustducin, whereas no signals were detected for Tas2r probes. In situ hybridization in colon sections resulted again in the detection of α-gustducin positive cells, but no signals were evident using Tas2r probes. Hybridization with corresponding sense riboprobes (negative control) showed no staining. Scale bars, 100 µm.
Figure 2.
Cre recombinase expression in gastrointestinal tissues of gene-targeted mice.
(A) cDNA was prepared from vallate papillae (VP), duodenum (Duo), proximal (pJej) and distal (dJej)jejunum, ileum (Ile), colon (Col), stomach (Sto), and liver (Liv) of homozygous and heterozygous Tas2r131BLiC/BLiC as well as Tas2r131+/+ control mice. The cDNA samples were subjected to quantitative real-time PCR analyses using primers specific for Tas2r131 (white bars, amplification of cDNAs from Tas2r131+/+; black bars, amplification of cDNAs from Tas2r131+/BLiC; Tas2r131 expression in Tas2r131BLiC/BLiC was not detectable) and Cre-recombinase (dark gray bars, amplification of cDNAs from Tas2r131BLiC/BLiC; light gray bars, amplification of cDNAs from Tas2r131+/BLiC; Cre expression in Tas2r131+/+ was not detectable). Y-axis = logarithm of the expression levels relative to β-actin (mean log(2−ΔCT) ±SE, n≥3). (B) In situ hybridization of colon cross-sections of homozygous Tas2r131BLiC/BLiC mice (left panel) and Tas2r131+/+ control mice (right panel). Note that cells expressing Cre-mRNA were only detected in colon sections of Tas2r131BLiC/BLiC mice, but not in the corresponding control tissue. (C) Comparison of the numbers of tdRFP expressing cells in sections of the stomach, duodenum, jejunum, ileum and colon with the expression levels determined by qRT-PCR analyses. The absolute numbers tdRFP expressing cells (tdRFP+ cells) and the numbers of sections (sect.) monitored for the various GI-tissues are provided and compared to the values obtained for Cre- and Tas2r131-mRNAs in the corresponding homozygous mouse lines by the qRT-PCR analyses shown in A. (D) Graph showing the correlation between Cre-mRNA expression levels and the number of tdRFP expressing cells per section in distal jejunum (Jej), ileum (Ile) and colon (Col).
Figure 3.
Characterization of tdRFP expressing cells in mouse large intestine.
Red tdRFP expressing cells are labeled by arrowheads (left row). Arrows point to cells expressing one of the various marker proteins (green) used in this experiment (middle row). An overlay of the red and green fluorescence is shown in the right row. (A) Labeling of tissue sections with markers of enteroendocrine cells, GLP-1 (upper panel) and chromogranin A (lower panel). (B) Labeling of tissue sections with markers for brush cells and brush border membranes, cytokeratin 18 (upper panel) and villin (lower panel), respectively. (C) Labeling of sections with goblet-cell marker mucin-2. (D) Labeling of tissue sections with markers of taste-like cells, α-gustducin (upper panel) and PLCβ2 (lower panel). Scale bars, 50 µm; 10 µm (insets).