Figure 1.
Generation of PKD2L1 knock-out mice.
A. 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. Genomic Southern blot analysis of PKD2L1+/+, PKD2L1+/−, and PKD2L1−/− 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. C. In situ hybridization experiments demonstrating complete loss of PKD2L1 expression in the taste buds of the circumvallate papillae of PKD2L1−/− mice and robust expression in wild-type mice. Scale bar, 20 µm.
Table 1.
Nucleotide sequences of primers used in RT-PCR experiments (Horio et al.).
Figure 2.
Expression of PKDs in taste tissues.
A. mRNA expression of PKD1L3 and PKD2L1 in taste tissues of WT, PKD1L3−/−, PKD2L1−/−, PKD1L3/2L1dbl−/−, GAD-GFP, GAD-GFP+PKD1L3−/−, and GAD-GFP+PKD2L1−/− 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, C. Immunostaining for PKD1L3 (B) and PKD2L1 (C) in taste tissues of GAD-GFP+WT mouse. D. Immunostaining for PKD1L3 in taste tissues of GAD-GFP+PKD1L3−/− mouse. E. Immunostaining for PKD2L1 in taste tissues of GAD-GFP+PKD2L1−/− mouse. F. Immunostaining for GAD67 in taste tissue of GAD-GFP+WT mouse. G. 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.
Table 2.
Summary of immunohistochemical data on PKD2L1, PKD1L3, and GAD67 (Horio et al.).
Figure 3.
Sample recordings of gustatory nerve responses of WT, PKD1L3−/−, PKD2L1−/−, and PKD1L3/2L1dbl−/− mice.
A. CT nerve responses. B. GL nerve responses. Taste stimuli were NH4Cl (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).
Figure 4.
Concentration-response relationships of CT and GL nerve responses.
Concentration-response relationships of CT (A) and GL (B) nerve responses of WT [black rectangle, n = 8 (CT) and 8 (GL)], PKD1L3−/− [white circle, n = 5 (CT), and 6 (GL)], PKD2L1−/− [white triangle, n = 7 (CT) and 6 (GL)], and PKD1L3/2L1dbl−/− 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 NH4Cl. Values indicated are means ± S.E.M. Statistical differences were analyzed by ANOVA tests (see Table 3) and post hoc Dunnett's tests (*: P<0.05, **: P<0.01 for PKD2L1−/−; +: P<0.05, ++: P<0.01 for PKD1L3/2L1dbl−/−).
Table 3.
ANOVA results for CT and GL nerve responses to taste compounds (vs. WT mice) (Horio et al.).
Figure 5.
Taste responses of GAD67-GFP taste cells in FP taste buds of WT and PKD2L1−/− mice.
A, B. Sample recording from a GAD67-GFP taste cell (see picture) of WT (A) and PKD2L1−/− (B) mice. Taste responses to 10 mM HCl, 10 mM citric acid (CA), and 30 mM acetic acid (AA) were shown. Red bars indicate duration of taste stimulation. C–E. Concentration-response relationships of taste responses of GAD67-GFP taste cells for HCl (C), citric acid (D) and acetic acid (E). Taste responses of PKD2L1−/− mice (white triangle, HCl: n = 18∼27, citric acid: n = 17∼24, acetic acid: n = 17∼24) were significantly smaller than those of WT mice (black rectangle, HCl: n = 17∼31, citric acid: n = 16∼30, acetic acid: n = 17∼25) in ANOVA tests (see Result) and post hoc t-tests (*: P<0.05, **: P<0.01). Values indicated are means ± S.E.M.