34 research outputs found
Expression Analysis of Taste Signal Transduction Molecules in the Fungiform and Circumvallate Papillae of the Rhesus Macaque, <em>Macaca mulatta</em>
<div><p>The molecular mechanisms of the mammalian gustatory system have been examined in many studies using rodents as model organisms. In this study, we examined the mRNA expression of molecules involved in taste signal transduction in the fungiform papillae (FuP) and circumvallate papillae (CvP) of the rhesus macaque, <em>Macaca mulatta</em>, using <em>in situ</em> hybridization. <em>TAS1R1</em>, <em>TAS1R2, TAS2Rs, and PKD1L3</em> were exclusively expressed in different subsets of taste receptor cells (TRCs) in the FuP and CvP. This finding suggests that TRCs sensing different basic taste modalities are mutually segregated in macaque taste buds. Individual <em>TAS2Rs</em> exhibited a variety of expression patterns in terms of the apparent level of expression and the number of TRCs expressing these genes, as in the case of human <em>TAS2Rs</em>. <em>GNAT3</em>, but not <em>GNA14</em>, was expressed in TRCs of FuP, whereas <em>GNA14</em> was expressed in a small population of TRCs of CvP, which were distinct from <em>GNAT3</em>- or <em>TAS1R2</em>-positive TRCs. These results demonstrate similarities and differences between primates and rodents in the expression profiles of genes involved in taste signal transduction.</p> </div
The co-expression relationships among taste receptors and G protein α subunits.
<p>(A) In the CvP, <i>GNA14</i> was expressed in a much smaller population of TRCs than <i>GNAT3</i> and in a mutually exclusive manner. The <i>GNA14</i>–positive TRCs were distinct from those expressing <i>TAS1R2</i> and <i>TAS2R13</i>, but they were subsets of the <i>TAS1R3</i>-positive TRCs and partially overlapped with the <i>TAS1R1</i>-positive TRCs. n ≥1 (numbers of sections ≥2). (B) In the CvP, <i>TAS1R2</i> and <i>TAS2R13</i> were expressed in subsets of the <i>GNAT3</i>–positive TRCs, which partially overlapped with the <i>TAS1R1</i>- and <i>TAS1R3</i>-positive TRCs. n ≥2 (numbers of sections ≥4). (C) In the FuP, <i>TAS1R1</i>, <i>TAS1R2</i>, <i>TAS1R3</i>, and <i>TAS2R13</i> were expressed in subsets of <i>GNAT3</i>-positive TRCs. n ≥1 (numbers of sections ≥10). (D) Venn diagram illustrating the co-expression relationships among taste receptors and signal transduction molecules in the macaque and the mouse. Scale bars: 50 µm.</p
The co-expression relationships among three <i>TAS1Rs</i>.
<p>(A) <i>TAS1R1</i> and <i>TAS1R2</i> were exclusively expressed in different subsets of the <i>TAS1R3</i>-positive TRCs in the CvP. <i>In situ</i> hybridization using a mixed probe for <i>TAS1R1</i> and <i>TAS1R2</i> combined with a probe for <i>TAS1R3</i> revealed the presence of TRCs expressing <i>TAS1R3</i> alone. n = 2 (numbers of sections ≥4). (B) <i>TAS1R1</i> and <i>TAS1R2</i> were exclusively expressed in different subsets of the <i>TAS1R3</i>-positive TRCs in the FuP. The <i>TAS1R3</i>-positive TRCs in the FuP and CvP were classified into three types of cells: cells expressing <i>TAS1R1</i>+<i>TAS1R3</i>, those expressing <i>TAS1R2</i>+<i>TAS1R3</i>, and those expressing <i>TAS1R3</i> alone. n = 1 or 2 (numbers of sections ≥10). Scale bars: 50 µm.</p
The mRNA expression of genes encoding taste receptors and signal transduction molecules in the fungiform and circumvallate papillae of the rhesus macaque.
<p>(A) <i>In situ</i> hybridization revealed that three <i>TAS1Rs</i>, <i>TAS2R13</i>, <i>PKD1L3</i>, <i>GNAT3</i>, <i>GNA14</i>, and <i>PLCB2</i> were robustly expressed in subsets of the TRCs in the CvP. These genes, except for <i>GNA14</i>, were also expressed in subsets of the TRCs in the FuP. n≥2 (numbers of sections ≥4) for <i>TAS1R1</i>, <i>TAS1R2</i>, <i>TAS1R3</i>, <i>PKD1L3</i>, <i>GNAT3</i>, and <i>TAS2R13</i> in CvP, n = 1 (numbers of sections ≥2) for <i>GNA14</i> and <i>PLCB2</i> in CvP, n≥2 (numbers of sections ≥20) for <i>TAS1R1</i>, <i>TAS1R2</i>, <i>TAS1R3</i>, <i>PKD1L3</i>, and <i>GNA14</i> in FuP, n = 1 (numbers of sections ≥10) for <i>GNAT3</i>, <i>PLCB2</i>, and <i>TAS2R13</i> in FuP. (B) The <i>TAS2Rs</i> located on chromosome 11 (<i>TAS2R9</i> and <i>TAS2R12-25</i>) appeared to be robustly expressed in subsets of TRCs, whereas only weak signals were observed for the <i>TAS2Rs</i> located on chromosomes 3 (<i>TAS2R1-8</i> and <i>TAS2R10-11</i>) and 6 (<i>TAS2R26</i>). <i>Tas2Rs</i> are arranged according to the locations on the chromosomes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045426#pone.0045426.s001" target="_blank">Figure S1</a>). n = 2 (numbers of sections ≥4) for <i>TAS2R1-8</i>, <i>10</i>-<i>11</i>, <i>21</i>, <i>23</i>, and <i>26</i>, n = 1 (numbers of sections ≥2) for <i>TAS2R9</i>, <i>12, 14</i>-<i>20</i>, <i>22</i>, and <i>24</i>-<i>25</i>. Scale bars: 50 µm.</p
The co-expression relationships among taste receptors.
<p>(A) In the CvP, the <i>TAS1R3</i>-positive TRCs were negative for <i>TAS2R13</i>. The <i>PLCB2</i>-positive TRCs, which include <i>TAS1R1</i>-, <i>TAS1R2</i>-, <i>TAS1R3</i>-, and <i>TAS2R13</i>-positive TRCs, were negative for <i>PKD1L3</i>. n = 1 (numbers of sections ≥2). (B) In the FuP, the <i>TAS1R3</i>-positive TRCs were negative for <i>TAS2R13</i>. The <i>PLCB2</i>-positive TRCs, which include <i>TAS1R1</i>-, <i>TAS1R2</i>-, <i>TAS1R3</i>-, and <i>TAS2R13</i>-positive TRCs, were negative for <i>PKD1L3</i>. n = 1 or 2 (numbers of sections ≥10). Scale bars: 50 µm.</p
Nucleotide sequences of primers used in RT-PCR experiments (Horio et al.).
<p>Upper: outside primers.</p><p>Lower: inside primers.</p
Generation of PKD2L1 knock-out mice.
<p><b>A.</b> Schematic representation showing the structure of the
PKD2L1 gene and the strategy for generating knock-out mice. The
targeting construct deleted predicted transmembrane (TM) motifs 1 to 6.
Ex: exon; Cre: Cre recombinase gene; Neo: neomycin resistant gene; loxP:
loxP site; DT-A: diphtheria toxin A-chain gene. <b>B.</b> Genomic
Southern blot analysis of PKD2L1<sup>+/+</sup>,
PKD2L1<sup>+/−</sup>, and
PKD2L1<sup>−/−</sup> mice. BamHI-digested genomic DNAs
extracted from wild-type, heterozygote, or homozygote mice were
subjected to Southern blot analysis with the 5′-flanking probe
that distinguishes wild type and deletion alleles for PKD2L1.
<b>C.</b><i>In situ</i> hybridization experiments demonstrating
complete loss of PKD2L1 expression in the taste buds of the
circumvallate papillae of PKD2L1<sup>−/−</sup> mice and
robust expression in wild-type mice. Scale bar, 20 µm.</p
Expression of PKDs in taste tissues.
<p><b>A.</b> mRNA expression of PKD1L3 and PKD2L1 in taste tissues of
WT, PKD1L3<sup>−/−</sup>, PKD2L1<sup>−/−</sup>,
PKD1L3/2L1<sup>dbl−/−</sup>, GAD-GFP,
GAD-GFP+PKD1L3<sup>−/−</sup>, and
GAD-GFP+PKD2L1<sup>−/−</sup> mouse. FP: fungiform
taste buds. CV: circumvallate taste buds. ET: epithelial tissue.
Gustducin is a control for taste tissue. β-actin is an internal
control. 100 bp marker was used. <b>B</b>, <b>C.</b>
Immunostaining for PKD1L3 (<b>B</b>) and PKD2L1 (<b>C</b>)
in taste tissues of GAD-GFP+WT mouse. <b>D.</b>
Immunostaining for PKD1L3 in taste tissues of
GAD-GFP+PKD1L3<sup>−/−</sup> mouse. <b>E.</b>
Immunostaining for PKD2L1 in taste tissues of
GAD-GFP+PKD2L1<sup>−/−</sup> mouse. <b>F.</b>
Immunostaining for GAD67 in taste tissue of GAD-GFP+WT mouse.
<b>G.</b> Immunostaining without primary antibody in
GAD-GFP+WT mouse. Green shows GFP fluorescence. Red shows
immunoreactivity (IR) for PKD1L3, PKD2L1 or GAD67, respectively. FP:
fungiform papillae. CV: circumvallate papillae. Scale bar, 10
µm.</p
Sample recordings of gustatory nerve responses of WT, PKD1L3<sup>−/−</sup>, PKD2L1<sup>−/−</sup>, and PKD1L3/2L1<sup>dbl−/−</sup> mice.
<p><b>A.</b> CT nerve responses. <b>B.</b> GL nerve responses.
Taste stimuli were NH<sub>4</sub>Cl (100 mM), HCl (10 mM), citric acid
(10 mM), acetic acid (30 mM), sucrose (500 mM), NaCl (100 mM), quinine
(10 mM), MSG (100 mM). Bars indicate taste stimulation (30 sec for CT
nerve responses; 60 sec for GL nerve responses).</p
Concentration-response relationships of CT and GL nerve responses.
<p>Concentration-response relationships of CT (<b>A</b>) and GL
(<b>B</b>) nerve responses of WT [black rectangle,
n = 8 (CT) and 8 (GL)],
PKD1L3<sup>−/−</sup> [white circle,
n = 5 (CT), and 6 (GL)],
PKD2L1<sup>−/−</sup> [white triangle,
n = 7 (CT) and 6 (GL)], and
PKD1L3/2L1<sup>dbl−/−</sup> mice [white diamond,
n = 6 (CT) and 6 (GL)] for HCl, citric acid,
acetic acid, NaCl, sucrose, quinine, MSG, and MPG. Gustatory nerve
responses were normalized to the response to 100 mM NH<sub>4</sub>Cl.
Values indicated are means ± S.E.M. Statistical differences were
analyzed by ANOVA tests (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020007#pone-0020007-t003" target="_blank">Table 3</a>) and <i>post
hoc</i> Dunnett's tests (*: P<0.05, **:
P<0.01 for PKD2L1<sup>−/−</sup>; <sup>+</sup>:
P<0.05, <sup>++</sup>: P<0.01 for
PKD1L3/2L1<sup>dbl−/−</sup>).</p