Chemical Senses Advance Access originally published online on January 4, 2006
Chemical Senses 2006 31(3):213-219; doi:10.1093/chemse/bjj021
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Faithful Expression of GFP from the PLCß2 Promoter in a Functional Class of Taste Receptor Cells
1 Department of Physiology and Biophysics and 2 Program in Neurosciences, Department of Physiology and Biophysics, University of Miami Miller School of Medicine (RMSB 4040), 1600 NW 10th Avenue, Miami, FL 33136, USA
Correspondence to be sent to: Nirupa Chaudhari, Department of Physiology and Biophysics, University of Miami Miller School of Medicine (RMSB 4040), 1600 NW 10th Avenue, Miami, FL 33136, USA. e-mail: nchaudhari{at}miami.edu
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Abstract |
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Phospholipase C–type ß2 (PLCß2) is expressed in a subset of cells within mammalian taste buds. This enzyme is involved in the transduction of sweet, bitter, and umami stimuli and thus is believed to be a marker for gustatory sensory receptor cells. We have developed transgenic mice expressing green fluorescent protein (GFP) under the control of the PLCß2 promoter to enable one to identify these cells and record their physiological activity in living preparations. Expression of GFP (especially in lines with more than one copy integrated) is strong enough to be detected in intact tissue preparations using epifluorescence microscopy. By immunohistochemistry, we confirmed that the overwhelming majority of cells expressing GFP are those that endogenously express PLCß2. Expression of the GFP transgene in circumvallate papillae occurs at about the same time during development as endogenous PLCß2 expression. When loaded with a calcium-sensitive dye in situ, GFP-positive taste cells produce typical Ca2+ responses to a taste stimulus, the bitter compound cycloheximide. These PLCß2 promoter–GFP transgenic lines promise to be useful for studying taste transduction, sensory signal processing, and taste bud development.
Key words: GFP, mouse, PLCß2, taste bud, taste-specific promoter, transgenic
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Introduction |
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In mammals, the G-protein–coupled taste receptors for sweet, bitter, and umami tastes are each expressed in limited subsets of cells within taste buds of rats and mice (Chandrashekar et al., 2000
; Chaudhari et al., 2000; Nelson et al., 2001, 2002). Signal transduction downstream of taste receptors results in transient elevation of cytoplasmic Ca2+ concentration by release of stored Ca2+. Phospholipase C (PLC) is a key mediator of Ca2+ release, and many taste cells express the beta-2 isoform [phospholipase C–type ß2 (PLCß2)] of this enzyme (Rossler et al., 1998, 2000). In situ hybridization and immunocytochemical analyses show that taste cells expressing taste receptors also express PLCß2 (Miyoshi et al., 2001; Chaudhari et al., 2003; Zhang et al., 2003). In mice, genetic ablation of PLCß2 results in a reduction or loss of sensitivity to these taste modalities, indicating that this enzyme is important for the transduction sweet, bitter, and umami stimuli (Zhang et al., 2003; Dotson et al., 2005). In sum, our current understanding of gustatory transduction is that stimulating taste receptors leads to activation of PLCß2, generation of inositol 1,4,5-triphosphate (IP3), and release of Ca2+ from intracellular stores (Ming et al., 1998; Huang et al., 1999; Yan et al., 2001). Therefore, identifying taste cells that express PLCß2 in vivo would provide a useful tool for studying taste transduction. To achieve this, we have developed transgenic mice expressing GFP under the control of the PLCß2 promoter. Our data indicate that a 2.9-kb fragment of the PLCß2 promoter is sufficient to confer cell-type–specific expression in taste cells. |
Materials and methods |
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Generation of PLCß2 promoter–GFP transgenic mice
We obtained the PLCß2 gene and its 5' upstream region on mouse chromosome 2 from BAC clone RP23-172B16 (accession number AL772255) from BACPAC Resources Center (Oakland, CA). The PLCß2 promoter was isolated as an 
8-kb XhoI–SmaI fragment that includes the presumed transcription and translation start sites within exon 1. The translated region of this fragment was removed using polymerase chain reaction (PCR)–based mutagenesis, and the remainder was cloned into the EGFP-BasicII vector to yield the 8.0-PLC–GFP promoter–reporter construct (Figure 1A). We previously generated this vector by removing the lacZ gene from the pßGal-Basic vector (Clontech, Mountain View, CA) and replacing it with cDNA for enhanced green fluorescent protein (herein referred to as GFP) from pIRES-EGFP (Clontech). The 2.9-PLC–GFP construct was then produced by removing a KpnI–KpnI 5.0-kb segment from the 5' end of the aforementioned 8.0-PLC–GFP promoter–reporter construct. Both constructs were validated by sequencing their ends. The plasmids were linearized with SmaI and SalI to remove vector sequences, and transgenic fragments were gel purified (Figure 1A) prior to injection. DNA injection into the pronuclei of fertilized C57BL/6J x SJL/J mouse eggs and transfer into foster mothers were performed by the University of Miami Transgenic Facility. Founder mice were bred with wild-type C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) to establish the transgenic lines.
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Genotyping
All experimental procedures followed the National Institutes of Health (NIH) Guidelines for the Care and Use of Animals and were approved by the University of Miami Animal Care and Use Committee. To assess the presence of transgene, we carried out genotyping PCR on tail DNA from offspring using Platinum supermix (Invitrogen, Carlsbad, CA). The number of integrated transgene copies was determined with the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) using IQ SYBR Green Supermix (Bio-Rad Laboratories). Genotyping PCR primers (0.1 µM in reaction) were designed using Beacon Designer (Bio-Rad Laboratories). In conventional and real-time PCRs, primers (see locations in Figure 1A) used were as follows—for endogenous PLCß2: (a) 5'-TGCCATGTGAGCCTAGCCTAAG-3' and (c) 5'-GCAATAGAACAGGGTTGAGCAAAG-3'; for either transgene: (a) as above and (b) 5'-CTCCTGGACGTAGCCTTCGG-3'; to distinguish the 8.0-PLC–GFP transgene: (d) 5'-TGAGAAGGAGTAAGAAAGGGACAG-3' and (f) 5'-TTGAAGAAGTCGTGCTGCTTCA-3'; and to distinguish the 2.9-PLC–GFP transgene: (e) 5'-GCATCAGGTGAGAAAATTATCCAC-3' and (f) as above. Each reaction contained 100 ng of tail DNA (or water as a negative control). Optimal annealing temperatures were identified for each primer pair by employing a temperature gradient PCR in the iCycler (Bio-Rad Laboratories). After 40 cycles of amplification, the homogeneity of the PCR product was confirmed through a melting temperature paradigm. Quantitative standard DNA templates were derived from DNA sequence–validated PCR products for the transgene and endogenous gene versions of PLCß2. Transgene copy number per genome was calculated as the ratio of transgene copies to endogenous PLCß2 gene copies in F1 (heterozygous) mice (Figure 1C).
GFP fluorescence and immunocytochemistry
GFP expression was confirmed within whole-mounted tongue and palate using a Zeiss Axioplan epifluorescent microscope. Mouse circumvallate, foliate, fungiform, and palate tissue were fixed in 4% paraformaldehyde and cryoprotected in 30% sucrose overnight at 4°C. Frozen sections (25 µm) were prepared and blocked in 4% bovine serum albumin and 0.3% TritonX-100 in 1x phosphate-buffered saline for one and a half hours at room temperature. Sections were then incubated overnight with rabbit anti-PLCß2, diluted 1:1000 (#SC-206, Santa Cruz Biotechnology, Santa Cruz, CA), followed by donkey anti-rabbit conjugated to Alexa 594 (1:1000) (Molecular Probes, Eugene, OR). In some cases, GFP was visualized by immunocytochemistry using mouse anti-GFP, diluted 1:1000 (#11814460001, Roche Applied Science, Columbia, NY), and donkey anti-mouse conjugated to Alexa 488 (1:1000) (Santa Cruz Biotechnology). Similar results were obtained whether GFP was viewed by its native fluorescence or by immunocytochemistry. Both these antibodies are extensively characterized and highly specific (e.g., Clapp et al., 2004).
Immunostained slides were photographed on a Zeiss Axioplan epifluorescent microscope using Axiovision version 3.0 software. Approximately, 5–15 images of at least 25 vallate and foliate taste buds were used to quantify the expression patterns. Only cells with visible nuclei (inferred from the absence of PLCß2 staining) were counted.
Calcium imaging
We obtained vallate papillae from adult transgenic mice (5288 line, 
8 weeks old) and loaded taste cells with the fluorescent calcium indicator dye, Calcium Orange (CaO; 1 mM in H2O; Molecular Probes) as previously described (Richter et al., 2003). Briefly, CaO was injected iontophoretically (–3.5 µA square pulses, 10 min) through a glass micropipette (40-µm tip diameter) into the crypts of the vallate papilla. The tissue was then sliced at 100 µm with a Leica VT1000S vibratome (Nussloch, Germany), and slices containing vallate taste buds were mounted on a glass coverslip coated with Cell-Tak (Becton Dickinson, Franklin Lakes, NJ), placed in a recording chamber, and superfused with Tyrode's solution at 30°C at a rate of 2 ml/min. Stimuli in Tyrode's solution (in mM: 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 NaHCO3, 10 4-(2-hydroxyethyl)-1-piperazineethane sulfonate, 10 glucose, and 10 sodium pyruvate, pH 7.3) were perfused for 60 s. GFP and CaO fluorescence were viewed with an Olympus (Melville, NY) Fluoview scanning laser confocal microscope using 488- or 568-nm excitation, respectively. To measure 
Ca2+, CaO fluorescence signals were captured at 5-s intervals and expressed as relative fluorescence change: F/F = (F – F0)/F0, where F0 denotes the resting fluorescence level corrected for bleaching. Using F/F corrects for variations of baseline fluorescence, cell thickness, total dye concentration, and illumination (Helmchen, 2000).
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Results |
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Generation of PLCß2–GFP transgenic mice
Prior to injection for producing transgenic mice, we validated two GFP reporter constructs (including 2.9 or 8.0 kb of the PLCß2 promoter) by lipofection into taste buds (Landin et al., 2005). Injected eggs produced 30 and 20 live births (for the 2.9- and 8.0-kb constructs, respectively), of which 11 and 3 were genotype positive, respectively (Table 1 and Figure 1B,C). Progeny of all the genotype-positive founder mice were evaluated for GFP expression. GFP fluorescence was observed in two lines each of the 2.9-kb and two 8.0-kb transgenics, and all of these mice have multiple copies of the transgene.
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GFP expression in four taste fields
GFP expression was initially observed in freshly dissected, that is, living whole-mounted lingual and palatal tissues by epifluorescent microscopy (data not shown) to identify transgenic lines for further study. Tissues from four GFP-expressing lines were then fixed and processed for immunocytochemical detection of endogenous PLCß2 using a well-characterized antibody. Four major taste fields were analyzed: circumvallate, foliate papillae, the anterior tongue, and the palate. By fluorescence microscopy, we analyzed the extent to which PLCß2 immunoreactivity overlapped with GFP fluorescence (Table 2). In some instances, this analysis was repeated using an antibody against GFP in double immunocytochemistry. Qualitatively similar results were obtained with anti-GFP (not shown) as when directly viewing GFP fluorescence. In taste tissue, PLCß2 immunoreactivity was strictly limited to a subset of cells within taste buds. To confirm specificity, we immunostained sections of vallate or foliate papillae from PLCß2-knockout mice (Jiang et al., 1997). As expected, no immunofluorescence was detected in these taste cells (Figure 2C).
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We quantified overlapping expression of GFP and endogenous PLCß2 and found that three of the transgenic lines showed GFP consistently in cells expressing PLCß2 (Table 2; Figure 2). Mice in which GFP was a faithful reporter for PLCß2 were derived from founders injected with either the 2.9-PLC–GFP or the 8.0-PLC–GFP construct. In all of the animals showing overlap between GFP and PLCß2 expression, there were no differences in the degree of overlap among the different taste fields—circumvallate, foliate, fungiform, or palate. These data indicate that the 2.9-kb fragment of the PLCß2 promoter is sufficient to confer cell-type–specific expression in taste cells. One of the transgenic lines from the 8.0-kb promoter showed poor overlap between GFP and PLCß2. We speculate that this may reflect a position effect of the integration site.
GFP expression in the developing circumvallate papillae
Next, we studied how the expression of the transgene was regulated during the postnatal development. We analyzed circumvallate papillae from 1-, 3-, and 10-week-old transgenic mice from the 5288 line (one of the lines derived from the 2.9-kb promoter). At 1 week, only a few cells per taste bud expressed either GFP or PLCß2. Nevertheless, in those cells in which expression could be detected, there was a high degree of overlap between GFP and PLCß2. We noted that GFP fluorescence was relatively less intense as compared to adults (compare Figure 4A to Figure 3A), but this was not systematically investigated. By 3 weeks, the number of cells positive for GFP and PLCß2 had reached adult levels, and there was excellent overlap of expression (Figure 4G–I). We quantified this pattern by counting the cells and found that at all three stages, 94–99% of cells that expressed either GFP or PLCß2 expressed both (60 of 64 cells at P7; 191 of 197 cells at P21; and 312 of 316 cells at P70). Overall, our results suggest that the expression of GFP was regulated similarly to the endogenous PLCß2 promoter throughout the postnatal development.
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GFP cells respond to tastant stimuli
PLCß2 is believed to act downstream of the T1R and T2R receptors (Zhang et al., 2003), leading to IP3 formation and Ca2+ release inside taste receptor cells upon stimulation with tastants. We asked whether cells expressing GFP and PLCß2 in the transgenic mice responded to taste stimuli, as predicted. We conducted calcium imaging experiments on living tissue preparations in which GFP cells could be identified in situ. Taste buds in lingual slices of vallate papillae were loaded with the indicator dye, CaO, to monitor the intracellular Ca2+ levels with confocal laser scanning microscopy (Richter et al., 2003). We identified cells expressing "both" GFP and CaO and tested whether they responded to cycloheximide, a bitter tastant that effectively stimulates many taste cells in mice (Caicedo et al., 2002). Figure 4 illustrates a typical experiment showing a GFP/CaO cell that responded to stimulation with cycloheximide (Figure 4D). The results demonstrate that GFP is a successful marker of tastant-responsive (i.e., receptor) cells in in vitro preparations from the transgenic mice and that GFP expression does not interfere with Ca2+ imaging when appropriate indicator dyes, such as CaO, are employed.
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Discussion |
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Recent evidence suggests that taste buds are ensembles of molecularly and functionally diverse neuroepithelial cells, including gustatory receptor cells, supporting cells, stem cells, and others (Bigiani, 2001
; Yee et al., 2001; Medler et al., 2003; Chaudhari and Roper, 2005). Yet, most cells in taste buds possess a generally similar fusiform shape and cannot be readily distinguished in living preparations. We have produced transgenic mice that faithfully express GFP in taste cells that have a key signaling element of the taste transduction cascade, PLCß2. The transgene thus specifically identifies gustatory sensory receptor cells. Further, expression of GFP and PLCß2 increase in parallel during postnatal development. GFP fluorescence in each of these lines is robust enough to be detected in living and fixed tissue without the need for immunodetection. GFP thus serves as an effective means to identify tastant-sensitive receptor cells for a variety of experimental approaches. Specifically, our data show that EGFP-expressing taste cells respond to tastant stimulation, as anticipated for gustatory receptor cells. These transgenic mice should prove valuable for studying transduction, development, and signal processing in taste buds and may also be useful for analyzing living interstitial cells in testis and other cell types that express PLCß2. Previously reported transgenic mice expressing GFP in taste cells have used the promoter for gustducin (Wong et al., 1999). These have proved valuable for interpreting functional responses in taste cells (Ogura et al., 2002; Medler et al., 2003).
We noted that of 14 genotype-positive founder mice only four, that had integrated multiple copies of the transgene, expressed GFP at a sufficiently robust level to be useful for functional studies. In summary, 2.9 kb of the PLCß2 promoter is sufficient to confer cell-type–specific expression of the GFP transgene. Our transgenic lines appear to express GFP in a copy number–dependent fashion. Most transgenes that express in a strictly copy number–dependent, integration site–independent manner tend to include large (tens of kb) promoter segments. Instances of short promoters conferring copy number–dependent and cell-type–specific expression are less common (Dale et al., 1992; Talbot et al., 1994). One of our lines (5302) expresses GFP in mature taste cells but with inaccurate cell-type specificity. This suggests that the precise spatial and temporal regulation of the transgene might be sensitive to insertional position (Grieshammer et al., 1995; Umezawa et al., 1997).
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Acknowledgements |
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This work was supported through grants from the National Institutes of Health/National Institute of Deafness and Other Communication Disorders to N.C. (DC006021, DC006308) and S.R. (DC000374). We thank Kristina Trubey, Sukhdeep Rao, and Jonathan Hernandez for helping to characterize the transgenic mice.
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References |
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Accepted November 30, 2005
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