Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
Function and Expression of the Drosophila Gr Genes in the Perception of Sweet, Bitter and Pheromone Compounds
Department of Molecular Genetics and Microbiology, Duke University Medical Center, 252 CARL Bldg/Research Drive, Durham, NC 27710, USA
Correspondence to be sent to: Hubert Amrein, e-mail: hoa1{at}duke.edu
Key words: chemosensory, courtship, feeding, sex-specific, subesophageal ganglion, taste
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Introduction |
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 Top Introduction ResultsA male-specific Gr, Gr68a,...ConclusionReferences |
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Sensory perception enhances and adds dimension to life, while also serving the more primitive purpose of providing information about the environment that is necessary for survival and propagation of the individual. Two primal and essential behaviors—mating and feeding—are thought to be mediated by contact chemosensation in many animals, including Drosophila.
Drosophila engage in courtship before mating. A highly ritualistic and
complex behavior, courtship consists of a series of stereotyped interactions between the
male and female (Hall, 1994
;
Greenspan and Ferveur, 2000) that must
occur before a successful mating event. Pheromone detection is necessary for efficient
recognition of a suitable mate and therefore essential in initiating courtship. Detection
of pheromones is thought to occur when the male makes contact with the female abdomen
with his labellum and forelegs (‘tapping’). Evidence that the contact
chemosensory system may mediate pheromone detection include the observations that males
have significantly more taste bristles on their forelegs (
50) compared to females
(37) (Nayak and Singh, 1983) and
that female pheromones are non-volatile hydrocarbons highly concentrated on the female
abdomen (Coyne et al., 1994;
Ferveur et al., 1996).
Taste perception allows nutritionally rewarding food to be discriminated from food that is contaminated and toxic. Generally, foods that are sweet are fed upon by fruit flies, whereas bitter substances are avoided. Unlike humans, however, fruit fly taste perception is not restricted to one tissue. Instead, flies have taste bristles not only on the labellum (human tongue equivalent), but also on the wings, legs, genitalia and in the pharynx (Figure 1a).
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Taste bristles are associated with two to four taste neurons, the dendrites of which extend into the bristle shaft. These taste neurons express a class of G-protein-coupled-receptors (GPCRs), called gustatory receptors (Grs), that are thought to activate the neuron upon contact with a soluble ligand. Initially identified based on their structural similarity to Drosophila odorant receptors (Ors) (Clyne et al., 2000
), the Gr gene family contains 70 members with relatively
low sequence similarity (15–25%) (Dunipace et al., 2001;
Scott et al., 2001;
Robertson et al., 2003). Due
to their low levels of cellular expression in tissues not amenable to RNA in
situ hybridization, the Gal4/UAS system has proven extremely valuable in determining
the expression pattern of individual Grs by driving the expression of a reporter
gene, such as GFP or ß-Gal, under the control of a Gr promoter. Using this
system,
Dunipace et al. (2001) and
Scott et al. (2001) provided
the first peripheral Gr expression map for a subset of receptors, demonstrating
that Grs are expressed in taste neurons of the labellum, wings, legs and
pharynx. |
Results |
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TopIntroductionResultsA male-specific Gr, Gr68a,...ConclusionReferences |
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To understand how the fly recognizes and discriminates among different soluble compounds involved in taste and pheromone perception, we first created an expression map of the Gr genes. Of primary interest is the extent of co-expression of different Gr genes within the same taste neurons. For example, one might expect similarities to the mammalian taste system in which taste receptors recognizing related compounds (bitter-tasting) and mediating similar behavioral responses (repulsion) might be expressed in identical or largely overlapping neurons (Zhang et al., 2003
). Similarly, Grs
recognizing distinct compounds and thereby mediating different behaviors, might be
expressed in an entirely different group of taste neurons. Thus, using the Gal4/UAS
system, which generally allows accurate expression analysis of a gene of interest
(Brand and Perrimon, 1993), we first
determined the cellular expression of 13 Gr genes (Table
1).
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To determine the functional role of neurons that express these Grs, we characterized the behaviors of flies in which specific sets of taste neurons were functionally inactivated. To achieve this, we combined the Gr-Gal4 drivers with a UAS-tnt reporter. Tnt codes for the tetanus-toxin light chain protein that inhibits synaptic transmission in the neuron in which it is expressed (DiAntonio et al., 1993
), thereby
allowing us to inactivate neurons expressing specific Grs. The expression
pattern of these 13 Grs, as well as functional analysis of the neurons in which
they are expressed, indicate that these receptors are involved in the taste perception of
bitter or sweet tastants or in pheromone detection. Discrimination between two taste modalities—sweet and bitter—requires specific sets of gustatory receptor neurons that express different Grs
Previous expression studies of Grs (Clyne et al., 2000;
Dunipace et al., 2001;
Scott et al., 2001;
Hiroi et al., 2002) have
shown that most of the receptors analyzed are expressed in taste neurons of the labellum.
Labellar taste bristles are arranged in a stereotyped pattern and are morphologically
identified as short (S), intermediate (I), or long (L).
Hiroi et al. (2002)
associated Gr expression with bristle-type and found that several of the
Grs were expressed in a single neuron associated with the same bristle.
In an effort to understand how taste is coded at the periphery, we carried out
experiments to determine whether these Grs were co-expressed in the same taste
neuron or different neurons associated with the same taste bristle (Thorne et al., 2004). We drove expression of
nuclear-GFP under the control of two Gr promoters (Gal4/UAS system) and
then used immunohistochemistry and confocal microscopy to obtain an accurate count of the
number of cells expressing either receptor. Based on cell counts of double
Gr-promoter driver lines compared to single Gr-promoter driver lines,
we found that most of the receptors analyzed were partially co-expressed in neurons
associated with S-type bristles of the labellum (Gr66a, Gr22e,
Gr28be, Gr32a, Gr22b, Gr22f, Gr59b; Table
1 and Figure
1B), indicating that labellar neurons
can be defined by their Gr gene code.
Interestingly, Gr5a— a trehalose receptor—was not expressed in
the neurons associated with S-type bristles in which the other receptors were expressed.
In fact, neurons that express Gr5a had morphologically smaller cell bodies
compared to the S-type bristle-associated neurons that express the other Grs.
Additionally, Gr5a was expressed in a relatively large number of labellar
neurons (70 per palp) and many neurons expressing Gr5a were clustered, with
their dendrites converging to enter the same bristle shaft. Thus, our results indicated
that neurons sensitive to sweet taste, in particular trehalose, may be distinct from
those associated with a different taste modality, represented by Gr66a neuronal
expression.
To determine what substrates neurons expressing Gr66a could recognize, we conducted behavioral studies—specifically, feeding preference assays—on flies with neurons functionally ablated by TNT. For example, flies lacking functional Gr66a-expressing neurons were given a choice to feed on 6 mM caffeine with 2 mM sucrose versus 2 mM sucrose alone. We found that these flies were significantly less sensitive to caffeine—a bitter substance—compared to control flies not expressing TNT in these neurons. However, these flies did not have reduced sensitivity to either 2 mM sucrose or 25 mM trehalose. These results indicated that neurons expressing Gr66a were sensitive to a bitter compound—caffeine—but did not play a role in sweet taste perception. Alternatively, flies in which TNT was driven by Gr5a could not discriminate 25 mM trehalose from water. These flies did not have an altered sensitivity to any bitter compound tested or to sucrose. Thus neurons expressing Gr5a appear to mediate sensitivity to trehalose and do not confer sensitivity to bitter compounds or sucrose.
Our behavioral data combined with peripheral expression analysis indicate that neurons sensitive to repellants may be discrete and separate from neurons sensitive to attractants and that these neurons express different Grs. Therefore, we postulate that the Grs expressed in a given neuron define its substrate specificity and sensitivity.
Since the majority of neurons sensitive to the repellant caffeine were distinct from the trehalose sensitive neurons expressing Gr5a, we expected that the axonal targets in the CNS for these two sets of neurons would be different. Not surprisingly, analysis of the axonal projection patterns of neurons expressing the Gr66a group of co-expressed receptors are qualitatively different from the pattern seen for Gr5a in the fly’s primary taste center in the brain—the tritocerebrum/SOG (Figure 1). Thus, bitter and sweet taste modalities may have different neural circuits that translate into different behaviors.
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A male-specific Gr, Gr68a, is involved in female pheromone detection |
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TopIntroductionResultsA male-specific Gr, Gr68a,...ConclusionReferences |
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We have recently discovered a Gr whose expression is associated with ten male-specific taste bristles in the foreleg (Bray and Amrein, 2003
). This male-specific expression of Gr68a suggested
a role in female pheromone detection, especially during the tapping step of courtship.
Males expressing TNT exclusively in Gr68a-expressing neurons were significantly
impaired in their ability to mate: 41% of these males failed to mate within 30 min
compared to an 5% failure rate for control males. Although mating success appeared to be significantly affected in males lacking Gr68a-expressing neurons, our hypothesis would predict that Gr68a is directly involved in pheromone detection—a process that plays an important role in courtship. To this end, we have analyzed courtship behavior of males lacking functional Gr68a neurons and found that not only did they spend significantly less time courting virgin females compared to control males, but that these males were less successful in proceeding to the wing extension/vibration step of courtship. Thus it appeared that these males were hindered moving through the tapping step of courtship—the step that is thought to involve the detection of female pheromones. It is worth mentioning that these males were not found to court, or attempt to mate, other males.
Our data therefore indicate a functional role for Gr68a-expressing neurons of the male forelegs in female pheromone detection. A direct role for Gr68a in this process was found by using RNA interference—males were made that expressed double-stranded Gr68a RNA in an effort to degrade endogenous Gr68a RNA through RNA interference. We found that these males had a very similar, albeit slightly weaker, courtship phenotype compared to males lacking functional Gr68a neurons. This weaker mating phenotype could indicate expression of additional putative pheromone receptors in these neurons. Possible candidates include genes closely related to Gr68a, such as Gr32a or members of the Gr39 gene cluster. Regardless, Gr68a appears to mediate the detection of an unknown female-specific pheromone required for efficient courtship in Drosophila melanogaster.
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Conclusion |
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TopIntroductionResultsA male-specific Gr, Gr68a,...ConclusionReferences |
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Members of the Gr gene family appear to play a significant role in Drosophila chemosensory perception, including taste perception, pheromone detection and olfaction. These receptors, therefore, must detect a large number of distinct compounds.
Future experiments will address whether flies are able to discriminate between different bitter compounds, as suggested by the partial co-expression of many labellar Grs. An effort will also be made to assign ligand specificity to the pheromone receptor Gr68a. It will also be interesting to determine if other members of the Gr gene family code for pheromone receptors.
Drosophila thus provides an elegant model for coupling the molecular basis of sensory perception (Grs) to behavioral output (feeding and courtship), perhaps giving us insight into how sensory perception influences our apparently sophisticated interactions with our environment and the people around us.
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References |
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TopIntroductionResultsA male-specific Gr, Gr68a,...ConclusionReferences |
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