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Directing Gene Expression to Gustducin-Positive Taste Receptor Cells

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Directing Gene Expression to Gustducin-Positive Taste Receptor Cells The Journal of Neuroscience, July 15, 1999, 19(14):5802–5809 Directing Gene Expression to Gustducin-Positive Taste Receptor Cells Gwendolyn T. Wong, Luis Ruiz-Avila, and Robert F. Margolskee Howard Hughes Medical Institute, Department of Physiology and Biophysics, The Mount Sinai School of Medicine, New York, New York 10029 We have demonstrated that an 8.4 kb segment (GUS8.4 )from Expression of the lacZ transgene from the GUS8.4 promoter and the upstream region of the mouse a-gustducin gene acts as a of endogenous gustducin was coordinately lost after nerve fully functional promoter to target lacZ transgene expression to section and simultaneously recovered after reinnervation, con- the gustducin-positive subset of taste receptor cells (TRCs). firming the functionality of this promoter. Transgenic expression a The GUS8.4 promoter drove TRC expression of the of rat -gustducin restored responsiveness of gustducin null b-galactosidase marker at high levels and in a developmentally mice to both bitter and sweet compounds, demonstrating the appropriate pattern. The gustducin minimal 1.4 kb promoter utility of the gustducin promoter. (GUS1.4 ) by itself was insufficient to specify TRC expression. We also identified an upstream enhancer from the distal portion Key words: gustducin; taste receptor cells; guanine nucleo- of the murine gustducin gene that, in combination with the tide binding regulatory proteins; gustation; transgenic mice; minimal promoter, specified TRC expression of transgenes. promoter identification; b-galactosidase Current models of taste perception in humans suggest four or five sensory cells of the stomach, duodenum, and pancreas (Ho¨fer et primary taste submodalities: sweet, bitter, salty, sour, and umami al., 1996; Ho¨fer and Drenckhahn, 1998). Thus, a-gustducin may (glutamate). Transduction of responses to sapid molecules (“tas- mark chemoresponsive cells regardless of their anatomical loca- tants”) occurs in specialized neuroepithelial taste receptor cells tion. The a-subunit of rod transducin is also expressed in TRCs, (TRCs) organized within taste buds (for review, see Kinnamon although at much lower levels than is a-gustducin (Ruiz-Avila et 1 1 and Margolskee, 1996; Lindemann, 1996). Na and H ions al., 1995). The expression of gustducin and transducin in TRCs (salty and sour tastants, respectively) are thought to interact with suggests that these two G-proteins may function in taste trans- ion channels located at the apical surface of TRC membranes duction as transducin does in phototransduction, i.e., to couple (Heck et al., 1984; Gilbertson et al., 1992; Gilbertson, 1993; seven transmembrane–helix taste receptors to TRC-specific Lindemann, 1996; Ugawa et al., 1998). On the other hand, several phosphodiesterases to regulate intracellular cyclic nucleotide lev- lines of evidence suggest that TRC responses to both sweet and els (Hargrave and McDowell, 1992; Khorana, 1992; McLaughlin bitter compounds are transduced via seven transmembrane–helix et al., 1994; Kolesnikov and Margolskee, 1995; Stryer, 1996). receptors coupled to guanine nucleotide-binding regulatory pro- We tested the role of gustducin in taste transduction in vivo by teins (G-proteins) (for review, see Kinnamon and Margolskee, generating a-gustducin null mice and analyzing their taste re- 1996; Lindemann, 1996). sponses (Wong et al., 1996). The null mice were indistinguishable Biochemical and molecular biological studies have identified from wild type (WT) in their behavioral and chorda tympani specific components of the transduction pathways mediating re- nerve responses to NaCl and HCl. However, in contrast to WT, sponses to bitter and sweet compounds (McLaughlin et al., 1992; the null mice showed greatly reduced aversion to denatonium Ruiz-Avila et al., 1995). Gustducin is a transducin-like G-protein benzoate and quinine hydrochloride (compounds that are bitter ; expressed in 30–40% of mammalian TRCs, all papillae of the to humans) and reduced preference for sucrose and SC-45647 lingual epithelium (McLaughlin et al., 1992; Takami et al., 1994; (compounds that are sweet to humans). Furthermore, the null Boughter and Smith, 1998), and in TRC-like apparent chemo- mice had diminished chorda tympani nerve responses to these same compounds. These data indicate that gustducin is a princi- Received Feb. 22, 1999; revised April 19, 1999; accepted April 27, 1999. pal mediator of both bitter and sweet signal transduction. This research was supported by National Institutes of Health Grants RO1DC03055 Identifying the cis-acting elements within the a-gustducin gene and R01DC03155 (R.F.M.) and F32DC00142 (G.T.W.), and Comissio´ Interdeparta- mental de Recerca i Innovacio´ Tecnolo´gica Grant BEAI-300089 (L.R.-A.). R.F.M. is that target expression to TRCs would provide useful information an Associate Investigator of the Howard Hughes Medical Institute. Dr. K. S. pertinent to the control of TRC expression of gustducin and other Gannon performed the glossopharyngeal nerve sectioning. We thank Dr. C. Mis- tretta for helpful discussions. TRC-specific factors. These elements could also be used as Correspondence should be addressed to Robert F. Margolskee, Howard Hughes probes to identify and characterize taste-specific transcriptional Medical Institute, Department of Physiology and Biophysics, The Mount Sinai factors and to selectively target expression to TRCs of cDNA- School of Medicine, Box 1677, One Gustave L. Levy Place, New York, NY 10029. Dr. Wong’s present address: Department of Central Nervous System/Cardiovas- encoded markers and signal transduction components. To iden- cular Discovery Research, Schering-Plough Research Institute, 2015 Galloping Hill tify such elements and test their functionality, we fused portions Road, Kenilworth, NJ 07033. of the upstream region of the mouse a-gustducin gene to a Dr. Ruiz-Avila’s present address: Almirall Prodesfarma, Cardener 64, 08022 Barcelona, Spain. b-galactosidase reporter and assayed expression of the chimeras Copyright © 1999 Society for Neuroscience 0270-6474/99/195802-08$05.00/0 in transgenic mice. Wong et al. • Targeting Expression to Taste Receptor Cells J. Neurosci., July 15, 1999, 19(14):5802–5809 5803 MATERIALS AND METHODS For immunofluorescence, sectioned tissues were blocked in PBS con- a taining 2% BSA, 1% horse serum, and 0.3% Triton X-100 for 30 min at Cloning and mapping the murine -gustducin gene and construction of b transgenes. Genomic fragments from the a-gustducin locus were obtained room temperature. The primary antibodies used were goat anti- - galactosidase (Arnel Laboratories, New York, NY) diluted 1:500 and from a 129/Sv mouse genomic library (Lambda FIX II; Stratagene, La a Jolla, CA) by screening with the rat a-gustducin cDNA (pSPORTgus) rabbit anti- -gustducin (Takami et al., 1994) diluted 1:500. Primary [see Results and as described previously (Wong et al., 1996)]. Two of 29 antibodies were added to sections, which were incubated in a humidified independent clones isolated (l4 and l27) contained sequences that hy- atmosphere for 1 hr at room temperature. Sections were then washed, bridized with the 59 probe [containing the first 485 nucleotides (nt) of rat secondary antibodies were applied, and incubation continued for 30 min gustducin cDNA]. The genomic fragments of a-gustducin clone l4 were at room temperature. Secondary antibodies used were mouse anti-goat subcloned into pBluescript II (Stratagene, La Jolla, CA). The other immunoglobulin conjugated with lissamine rhodamine and mouse anti- genomic clones were only partially characterized but indicate that the rabbit immunoglobulin conjugated with fluorescein isothiocyanate murine gustducin gene spans at least 60 kb. The lacZ expression vectors (FITC) (both from Jackson ImmunoResearch, West Grove, PA). Slides used in this study were pPSDKlacZpA1 (Echelard et al., 1994) and were washed and mounted in fluorescence mounting medium (Vector phspPTlacZpA (Kothary et al., 1989). Laboratories, Burlingame, CA), and photomicrographs were obtained on GUS1.4-lacZ was generated by subcloning into pBluescript a 1.4 kb a Zeiss Axiomat microscope using Kodak Ektachrome P1600 film, with NdeI restriction endonuclease fragment containing sequences homolo- either FITC or rhodamine wavelength filters placed in the light path. gous to the 59-most portion of the rat a-gustducin cDNA and is presumed Denervation. Animals were anesthetized via intraperitoneal injection to contain the transcription initiation site. The 1.4 kb segment was of sodium pentobarbital (60 mg/kg), secured in a nontraumatic head excised as a BamHI fragment and then ligated into the BglII site of holder and placed in a supine position. After shaving and sterilization of pPSDKlacZpA1. GUS8.4-lacZ was generated by insertinga7kbHindIII the throat area,a1cmventral midline incision was made caudal to the fragment (pH7) into the HindIII site of GUS1.4-lacZ. GUS(1.412.5)-lacZ lower lip and extending over the hyoid bone. Bilaterally, a small section and GUS(1.412.5rev)-lacZ were generated by isolating a 2.5 kb HindIII- of each glossopharyngeal nerve was surgically exposed as described EcoRV cleaved fragment from pH7, filling in the HindIII end using the previously (Oakley, 1995) and pinched approximately five to seven times Klenow fragment, and ligating the blunt ends into the SnaBI site of with fine forceps to disrupt neural transmission. After completion of the GUS1.4-lacZ. GUS5.9-lacZ was generated by isolating a 4.5 kb EcoRV- surgery, the incisions were sutured and sterilized. Water and liquefied HindIII fragment from pH7, filling in the HindIII end, and ligating as sterilized mouse chow were provided for 48 hr after surgery. Thereafter, above into the SnaBI site of GUS1.4-lacZ. GUS2.5-hsp68-lacZ was gener- mouse chow and water were available ad libitum. ated by cloning a blunt-ended 2.5 kb HindIII-EcoRV fragment isolated Behavioral analysis. Mice were genotyped by PCR for endogenous from pH7 directly upstream of the 600 bp hsp68 promoter in phspPT- a-gustducin and neo (to determine whether they were GUS/gus or gus/ lacZpA. GUS8.4-gustducin was generated by isolating the GUS8.4 pro- gus) and for the rat a-gustducin cDNA transgene.
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