Chemical Senses Advance Access originally published online on June 8, 2006
Chemical Senses 2006 31(7):627-639; doi:10.1093/chemse/bjl002
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Behavioral Responses to Odorants in Drosophila Require Nervous System Expression of the ß Integrin Gene Myospheroid
1 Department of Human Genetics, Virginia Commonwealth University, Richmond, VA 23298, USA 2 Department of Epidemiology, Michigan State University, East Lansing, MI 48824, USA 3 Cell and Molecular Biology Program, Duke University, Durham, NC 27710, USA 4 Neuroscience Program, Virginia Commonwealth University, Richmond, VA 23298, USA
Correspondence to be sent to: Michael S. Grotewiel, Department of Human Genetics, Virginia Commonwealth University, Richmond, VA 23298, USA. e-mail: msgrotewiel{at}vcu.edu
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Abstract |
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Integrins are cell adhesion molecules that mediate numerous developmental processes in addition to a variety of acute physiological events. Two reports implicate a Drosophila ß integrin, ßPS, in olfactory behavior. To further investigate the role of integrins in Drosophila olfaction, we used Gal4-driven expression of RNA interference (RNAi) transgenes to knock down expression of myospheroid (mys), the gene that encodes ßPS. Expression of mys-RNAi transgenes in the wing reduced ßPS immunostaining and produced morphological defects associated with loss-of-function mutations in mys, demonstrating that this strategy knocked down mys function. Expression of mys-RNAi transgenes in the antennae, antennal lobes, and mushroom bodies via two Gal4 lines, H24 and MT14, disrupted olfactory behavior but did not alter locomotor abilities or central nervous system structure. Olfactory behavior was normal in flies that expressed mys-RNAi transgenes via other Gal4 lines that specifically targeted the antennae, the projection neurons, the mushroom bodies, bitter and sweet gustatory neurons, or Pox neuro neurons. Our studies confirm that mys is important for the development or function of the Drosophila olfactory system. Additionally, our studies demonstrate that mys is required for normal behavioral responses to both aversive and attractive odorants. Our results are consistent with a model in which ßPS mediates events within the antennal lobes that influence odorant sensitivity.
Key words: behavior, odor attraction, odor avoidance, olfaction, RNA interference
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Olfaction is a key sensory modality that animals use for finding food, identifying mates, and avoiding predators (Vosshall, 2000
; Firestein, 2001). A better understanding of olfactory system function is central for a more comprehensive view of how animals extract information from the environment and then respond appropriately via behavioral outputs. A wealth of studies describe the similarities in functional organization of the olfactory system in mammals and the fruit fly, Drosophila melanogaster, as well as other insects (Vosshall, 2000; Firestein, 2001; Eisthen, 2002). These similarities, combined with powerful genetic tools available in Drosophila, make fruit flies an attractive model system for investigating the molecular genetic basis for olfactory system function and development (Warr et al., 2001).
Many of the main neural components of the olfactory system in Drosophila are well described (Stocker, 1994). Most of the primary olfactory sensory neurons are housed within the antennae. These neurons project in a stereotypical pattern to glomeruli within the antennal lobe, a region of the central insect brain that is functionally analogous to the vertebrate olfactory bulb. Olfactory information is processed in antennal lobe glomeruli composed of synapses between olfactory receptor neurons, local interneurons of the antennal lobes, and projection neurons. Projection neurons in turn synapse with neurons in the lateral protocerebrum as well as neurons that form the mushroom bodies. Olfactory information, therefore, proceeds from the olfactory receptor neurons in the antennae through the antennal lobe glomeruli and projection neurons to higher brain centers where it ultimately elicits a variety of behaviors.
The molecular genetic basis for olfactory system development is being vigorously investigated, leading to the identification of numerous genes important for proper formation of this key sensory system. In contrast, the influence of genes on olfactory behavior has not been as extensively studied. Thus, there is a large need to connect individual genes to many aspects of olfactory behavior in numerous species including Drosophila.
In Drosophila, an abrupt presentation of odorant causes flies to jump. Drosophila olfC mutants are defective in this jump response to some but not all odorants (Ayyub et al., 1990). Genetic complementation with standard loss-of-function mutations and other tests indicate that olfC is likely allelic to the myospheroid (mys) gene (Ayyub et al., 2000) that encodes ßPS, a ß integrin (MacKrell et al., 1988). Integrins are a major class of cell adhesion molecules involved in numerous developmental processes as well as a variety of acute physiological events (Hynes, 1992). The studies by Ayyub and coworkers strongly implicate integrins in the development or function of the olfactory system in Drosophila.
Some of the data from studies using standard loss-of-function alleles (Ayyub et al., 2000; Ayyub and Paranjape, 2002), however, are difficult to reconcile with a model in which all the olfactory defects described map to the mys locus. To independently address whether mys plays a role in Drosophila olfactory behavior, we used a strategy based on RNA interference (RNAi) to knock down mys expression. We find that expression of mys-RNAi in the central nervous system causes deficits in avoidance of aversive odorants as well as attraction to appetitive odorants. The deficits in behavioral responses are associated with a reduction in odorant sensitivity but not with altered locomotor behavior or deranged central nervous system structure. Our studies confirm that mys is important for behavioral responses to aversive odorants and establish that mys also has a role in behavioral responses to attractive odorants. Our data are consistent with a model in which mys functions within the local interneurons of the antennal lobes to influence odor sensitivity.
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Materials and methods |
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Fly husbandry and genetics
Flies were reared on standard food medium (10% sucrose, 2% yeast, 3.3% cornmeal, 1% agar) at 25°C/65% relative humidity under a 12-h day/night cycle. Flies were generously supplied by the following sources: decapentapalegic-Gal4 (dpp-Gal4) and engrailed-Gal4, L.S. Shashidhara, CCMB, Hyderabad; H24, Martin Heisenberg, Universitat Wurzburg; MT14, Reinhard Stocker, University of Fribourg; 107, Patrick Callaerts, University of Houston; 72Y, C739, C133, 59Y, 16Y, 125Y, Doug Armstrong, University System of Taiwan; Pox neuro-Gal4 (Poxn-Gal4), Gr66a, Gr5a, Scott Waddell, University of Massachusetts and Kristen Scott, University of California, Berkeley; Or83b, John Carlson, Yale University; mysnj42 flies, Danny Brower, University of Arizona; GH146, Liqun Luo, Stanford University; and tubulin-Gal4, actin-Gal4, and UAS-lacZ, Drosophila Stock Center at Indiana University (Bloomington, Indiana).
The UAS-mys-RNAi (UMR) transgene was constructed by cloning a 1.2-kb XhoI–BglII trigger sequence from expressed sequence tag clone RE55238 (Berkeley Drosophila Genome Project, GeneBank accession no. AY113499) into the SympUAST vector (Giordano et al., 2002) (kindly provided by Ennio Giordano, Universita di Napoli). Trigger sequences in this vector are flanked by two UAS sites that drive both sense and antisense expression (Figure 2A), thereby producing a double-stranded RNA species that elicits RNAi-mediated knockdown of gene expression (Giordano et al., 2002). For UAS-mys+ transgenic animals, a 2.5-kb NotI–SpeI fragment from the mys cDNA (generously provided by Danny Brower, University of Arizona) was cloned into pUAST (Brand and Perrimon, 1993). Transgenic flies carrying both UAS constructs were generated using standard methods (Rubin and Spradling, 1982) in a cantonized-w1118 (w[CS]) genetic background used previously for behavioral analyses (Cook-Wiens and Grotewiel, 2002; Goddeeris et al., 2003; Stoltzfus et al., 2003). Flies harboring independent insertions of the UMR transgene are designated as UMR1, UMR2, and UMR3 (a.k.a. PDM5i, PDM5g, and PDM3, respectively). The integrity of the UMR and UAS-mys+ transgenes was confirmed by polymerase chain reaction on genomic DNA isolated from the respective transgenic flies (data not shown).
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Flies used in these studies were either generated in the w[CS] genetic background (UAS lines) or were moved into this background via backcrossing for at least six generations (Gal4 lines). The exceptions to this strategy were Or83b, Gr5a, Gr66a, and GH146, Gal4 drivers that did not affect olfactory behavior when crossed to UMR flies. In studies with these four Gal4 drivers, all flies tested were in a hybrid genetic background derived from w[CS] and the Gal4 driver. For all studies, control flies containing a single copy of a UAS transgene without a Gal4 driver or a Gal4 driver without a UAS transgene were generated by crossing flies with either transgene construct to w[CS] animals. Flies with Gal4-driven expression of UAS transgenes were generated by crossing flies containing either construct. For determining the viability of adult flies expressing mys-RNAi, balanced UMR flies were crossed to various Gal4 lines. Viability was calculated as the number of nonbalanced adult progeny divided by the number of balanced adult progeny x 100% from these crosses. Viability greater than 100% indicates that more nonbalanced progeny were observed than balanced progeny (Table 1).
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Behavioral assays
Flies for behavioral studies were reared using standard conditions (Cook-Wiens and Grotewiel, 2002; Goddeeris et al., 2003; Stoltzfus et al., 2003; Gargano et al., 2005). All flies for individual experiments were grown, collected, and handled in parallel. Adult flies (4- to 7-days old) were briefly anesthetized with CO2, sorted into fresh food vials in groups of 25, and then allowed to recover for at least 18 h before being used in behavioral tests.
Olfactory behavior was assessed in T-mazes under dim red light at 22–25°C and 55–65% relative humidity as previously described (Cook-Wiens and Grotewiel, 2002; Stoltzfus et al., 2003). Groups of 25 flies were lowered to a choice-point in a T-maze and allowed to choose for 2 min between a maze arm containing an odorant and a maze arm containing no odorant. Odorants were diluted in mineral oil and supplied to the arms of the maze in an air stream at 750 ml/min. At the conclusion of individual tests, the flies in each maze arm were anesthetized, collected, and counted. Avoidance indices were calculated as the percentage of flies that moved into the arm without odorant minus the percentage of flies that moved into the arm with odorant (Connolly and Tully, 1998). The odorants used were 4-methylcyclohexanol (MCH), benzaldehyde (BNZ), linalool (LIN), ethyl acetate (EA), 2-heptanone (HEP), and isoamyl acetate (IAA). Odorants and mineral oil were obtained from Sigma–Aldrich (St Louis, MO).
Negative geotaxis was assessed in rapid iterative negative geotaxis assays as described (Gargano et al., 2005). In brief, groups of 25 flies from multiple genotypes were tested simultaneously for negative geotaxis. Data were captured with digital photography and extracted via computer-aided data analysis using Scion Image (PC version of NIH Image, Scion Corporation, Frederick, MD).
All behavioral data are presented as mean ± SEM. Statistical analyses were performed using Prism (GraphPad Software, San Diego, CA). One-way ANOVAs, Bonferroni's multiple comparisons, and t-tests were used as indicated in the figure legends. P values >0.05 were considered not significant (NS).
Histology
Wing discs were dissected from third instar larvae using standard procedures. For immunostaining, discs were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20–30 min, rinsed in PBS, and then blocked in PBSTB (1x PBS, 0.1% Triton X-100, 2% bovine serum albumin) for 60 min. Fixed and blocked wing discs were incubated overnight at 4°C with a 1:10 dilution of anti-ßPS monoclonal CF.6G11 (generously provided by Danny Brower, University of Arizona) in PBSTB, rinsed, and then incubated with an FITC-conjugated anti-mouse secondary antibody (Sigma, St Louis, MO) for 60 min at 25°C. All incubations were carried out in a rotating tube holder. For ß-galactosidase staining, wing discs were incubated in X-gal at room temperature and monitored until prominent LacZ reaction product was observed.
For analysis of adult brain, fly heads were loaded into a fly collar, embedded in Tissue Tek M (VWR Scientific Products, West Chester, PA), frozen at –20°C, and sectioned at 14 µm on a Microm HM550 cryostat (provided by Paul Ratz, Virginia Commonwealth University). Sections were fixed in 2% or 4% paraformaldehyde in PBS for 5–10 min, rinsed in PBS, and then blocked with PBSTB. Blocked sections were incubated with monoclonal nc82 (provided by Alois Hofbauer, University of Regensburg) or anti-Discs Large (DLG) monoclonal 4F3 (Developmental Studies Hybridoma Bank at the University of Iowa) for 12–16 h at 4°C, rinsed, and then incubated with FITC-conjugated anti-mouse antiserum (Sigma). Coverslips were mounted using Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Digital images were obtained using a Zeiss Axioplan-2 microscope, Axiocam CCD camera, and Axiovision software (Carl Zeiss, Germany).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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ßPS, encoded by the mys locus (MacKrell et al., 1988
; Leptin et al., 1989), is the major ß integrin in flies (Devenport and Brown, 2004). To better define the role of integrins in Drosophila adult behavior, we initially assessed the innate aversion of BNZ in flies harboring the mysnj42 allele, a partial loss-of-function mutation (Bunch et al., 1992). BNZ avoidance was blunted in mysnj42 animals (Figure 1), consistent with previous reports indicating that mys is important for normal behavioral responses to odorants in flies (Ayyub et al., 2000; Ayyub and Paranjape, 2002). Unfortunately, stronger combinations of mys alleles [mysnj42 in trans to a null (mysXG43) or an antimorph (mysXR04)] had very low viability in our laboratory as described by others (Bunch et al., 1992), precluding behavioral testing of animals with greater reductions in mys function. Weaker alleles at the mys locus (mysts1, mysts2) (Bunch et al., 1992) alone or in trans to mysnj42 were viable but had normal avoidance of odors (data not shown), making such mutants uninformative. Finally, neither heat shock induced nor Gal4-driven expression of wild-type ßPS rescued the BNZ avoidance defects in mysnj42 flies. Thus, our studies on loss-of-function mutants added little to our understanding of mys in Drosophila olfactory behavior.
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Given these issues, we adopted a strategy based on targeted expression of mys-RNAi via the Gal4/UAS system (Brand and Perrimon, 1993
) for investigating the role of mys in olfaction. RNAi is a type of gene silencing that relies on the expression of a double-stranded RNA species to elicit degradation of homologous mRNA and consequently reduce expression of the corresponding translation product (Cerutti, 2003). We predicted that such a strategy would circumvent the problems associated with pleiotropic effects of strong loss-of-function mys mutants as well as problems with the lack of phenotype in weak mys mutants. We also predicted that targeted expression of mys-RNAi would allow us to begin to identify regions of the olfactory system that require expression of mys to function properly. Construction and characterization of mys-RNAi flies
To generate mys-RNAi flies, we subcloned a 1.2-kb region of the mys cDNA (corresponding to exons 4 and 5 and a portion of exon 6) into the SympUAST vector (Giordano et al., 2002) (Figure 2A) and then introduced this construct into w[CS], a control strain, via P-element transformation (Rubin and Spradling, 1982). The SympUAST vector contains two opposing UAS sites that drive sense and antisense transcription of the trigger in response to ectopically supplied Gal4 (Giordano et al., 2002). The SympUAST vector eliminates the need to clone inverted repeat sequences for the trigger. The advantages of using this vector are that the trigger is substantially easier to clone and that the transgene is more stable once integrated into the genome (Giordano et al., 2002). Several independent UMR transformants were generated. Two lines with insertions on the second chromosome (UMR1, UMR3) and one on the X-chromosome (UMR2) were chosen for further study.
Strong loss-of-function mutations in mys are lethal (MacKrell et al., 1988; Leptin et al., 1989; Zusman et al., 1990; Brown, 1994). To determine whether widespread expression of mys-RNAi was similarly lethal, we crossed UMR1, UMR2, and UMR3 flies to a series of Gal4 drivers and then assessed the viability of the resulting F1 progeny. Ubiquitous expression of mys-RNAi driven by tubulin-Gal4 or actin-Gal4 resulted in complete lethality (Table 1). Two other Gal4 lines, 72Y and C739, also strongly reduced viability when driving UMR transgenes (Table 1). Like strong loss-of-function mys alleles, ubiquitous expression of mys-RNAi is lethal.
The normal adult wing of Drosophila is a highly ordered structure with tight adhesion between the dorsal and ventral layers in addition to an essentially invariant vein pattern (Figure 2B). Strong loss-of-function mutations in mys disrupt adhesion between the dorsal and ventral layers of the adult wing, resulting in wing blisters (Zusman et al., 1990; Bunch et al., 1992). Additionally, mys loss-of-function mutations disrupt normal vein formation in the adult wing (Zusman et al., 1990). We expressed mys-RNAi in the developing wing to determine whether it would phenocopy the defects in wing structure caused by strong loss-of-function mys alleles. dpp-Gal4 expresses in a central stripe in the developing wing (Figure 2F). Consistent with this expression pattern, adult wings from all flies that expressed mys-RNAi via dpp-Gal4 had a substantial wrinkled area containing vein defects (Figure 2C). Additionally, all flies with mys-RNAi expression driven by dpp-Gal4 had wing blisters within the presumptive wrinkled area (data not shown). Expression of mys-RNAi via engrailed-Gal4 in the wing caused similar wrinkling, blistering, and vein defects (data not shown). These data demonstrate that expression of mys-RNAi phenocopies the wing defects found in severe mys loss-of-function mutants and strongly suggest that mys-RNAi reduces function of the mys locus.
We evaluated expression of ßPS, the mys gene product (MacKrell et al., 1988; Leptin et al., 1989), in third instar larval discs to determine whether mys-RNAi expression knocked down ßPS levels. Consistent with previous studies (Brower et al., 1984), wild-type larvae expressed ßPS in a fairly uniform pattern throughout the wing disc (Figure 2D). Larvae with expression of mys-RNAi driven by dpp-Gal4 had reduced ßPS immunostaining in a central region of the disc (Figure 2E). The reduction in ßPS immunostaining in larvae expressing mys-RNAi corresponded to the expression pattern of dpp-Gal4 (Figure 2F). These data show that expression of mys-RNAi knocks down ßPS expression. Together, our studies on viability, wing structure, and ßPS immunostaining provide strong evidence that expression of mys-RNAi causes loss-of-function at the mys locus by knocking down expression of ßPS.
Expression of mys-RNAi disrupts olfactory behavior
The two main external components of the insect olfactory system are the antenna, a structure that houses most olfactory receptor neurons, and the maxillary palp, a structure that contains most other olfactory receptor neurons. Neurons from these two structures project to the antennal lobe, a region of the central nervous system that is functionally and structurally analogous to the vertebrate olfactory bulb (Stocker, 1994). The antennal lobes are organized into glomeruli composed of synapses between olfactory receptor neurons, local interneurons, and projection neurons. Projection neurons are the output neurons from the antennal lobes and thereby convey olfactory information to a higher brain center, the lateral protocerebrum, either directly or indirectly via the mushroom bodies. The insect mushroom body is intimately involved in associative olfactory memory (Dubnau et al., 2001; McGuire et al., 2001; Pascual and Preat, 2001) and more recently was implicated in the innate responses to odorants in Drosophila (Wang et al., 2003). Additionally, a recent report identified bitter sensing gustatory neurons and Pox neuro (Poxn) chemosensory neurons as important for behavioral responses to BNZ in flies (Keene et al., 2004). Toward characterizing the spatial requirement for ßPS in olfactory behavior, we evaluated BNZ avoidance in flies with Gal4-driven expression of mys-RNAi in the antennae, antennal lobes, mushroom bodies, sweet sensing gustatory neurons, bitter sensing gustatory neurons, and Poxn chemosensory neurons. Flies that expressed mys-RNAi via the Gal4 drivers used in our studies (H24, MT14, 59Y, C133, 16Y, 125Y, 107, C309, Or83b, GH146, Gr66a, Gr5a, and Poxn-Gal4) were viable (Table 1 and data not shown), exhibited no obvious locomotor defects, and seemed generally healthy, making them suitable for behavioral analyses. H24 drives expression in the antennal lobes, ellipsoid body of the central complex, and mushroom bodies (Figure 3A,B) as reported (Martin et al., 1998; Zars et al., 2000). MT14 drives expression in the antennal lobes (Figure 3C) as found previously (Tissot et al., 1997). This line also expresses Gal4 in the mushroom bodies (Figure 3D). As reported by others (Chiang et al., 2004) (Doug Armstrong, http://www.fly-trap.org/), 59Y (Figure 3E), C133 (Figure 3F), 16Y, and 125Y (data not shown) express in the antennal lobes but not in mushroom bodies. Conversely, 107 and C309 drive expression in the mushroom bodies (Figure 3G,H) but not in the antennal lobes, consistent with other data (Connolly et al., 1996). Or83b drives expression in the antenna but not in the central brain (Vosshall et al., 2000; Larsson et al., 2004). GH146, Gr5a, Gr66a, and Poxn-Gal4 express in the projection neurons (Heimbeck et al., 2001), sweet sensing gustatory neurons, bitter sensing gustatory neurons, and Poxn chemosensory neurons, respectively (Keene et al., 2004). H24, MT14, 59Y, C133, and C309 drive expression in the antennae and maxillary palps (data not shown).
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Avoidance of BNZ was substantially reduced in flies with H24- and MT14-driven expression of mys-RNAi (Figure 4A). Avoidance of BNZ was not significantly altered in flies with mys-RNAi expressed via Or83b (Figure 4B), 107 or C309 (Figure 4D), GH146, Gr5a, Gr66a, or Poxn-Gal4 (data not shown). BNZ avoidance was not significantly reduced in flies with mys-RNAi expression driven by C133, 59Y (Figure 4C), 16Y, or 125Y (data not shown), although there was a trend toward decreased BNZ avoidance that was not statistically significant when C133, 59Y, and 16Y were used to drive mys-RNAi. Thus, within our experiments, avoidance of BNZ is significantly disrupted by expression of mys-RNAi only when driven by H24 and MT14. This suggests that these two Gal4 lines define the tissues that require mys expression within the context of BNZ avoidance. Since BNZ avoidance defects or trends toward such defects were associated with mys-RNAi expression in the antennal lobes but not in other regions of the nervous system, our current model is that mys-RNAi expression in local interneurons of the antennal lobes disrupts odor avoidance.
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To determine whether expression of mys-RNAi disrupted behavioral responses to all odorants and whether mys-RNAi expression perturbed avoidance of and attraction to odorants, we assessed the behavioral responses of flies expressing mys-RNAi to a series of olfactory stimuli. Representative odorants from different chemical classes (aldehydes, esters, and alcohols) were tested. Consistent with the data in Figure 4A, expression of mys-RNAi via H24 reduced avoidance of BNZ (Figure 5A). Avoidance of MCH (Figure 5B) and attraction to EA (Figure 5C) were also disrupted in flies with H24-driven expression of mys-RNAi. Expression of mys-RNAi via H24 marginally reduced avoidance of HEP (Figure 5D) but did not significantly affect avoidance of LIN (Figure 5E) or IAA (Figure 5F). Expression of mys-RNAi, therefore, disrupts behavioral responses to particular odorants but not particular classes of compounds. Additionally, mys-RNAi expression causes defects in both odor avoidance and odor attraction.
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0.003, n = 10; Bonferroni's multiple comparison test, P To examine whether odor potency was altered in flies expressing mys-RNAi, we performed dose-response studies with BNZ. This odorant is attractive at relatively low concentrations and becomes aversive at higher concentrations (Devaud et al., 2003
; Wang et al., 2003). As expected, control flies exhibited a strong attraction to BNZ at low concentrations and a robust aversion to this odorant at higher concentrations (Figure 6A). Attraction to low concentrations of BNZ was undetectable in flies expressing mys-RNAi via H24. These flies also had a rightward shift in their dose response to BNZ that is consistent with a decreased aversiveness of this odorant. The maximal avoidance response to BNZ, however, was unaffected by mys-RNAi expression. These studies confirm that odor avoidance as well as attraction are disrupted by expression of mys-RNAi and suggest that expression of mys-RNAi decreases odor potency.
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Confirmation that expression of mys-RNAi disrupts olfactory behavior
We tested BNZ avoidance in two additional mys-RNAi lines, UMR2 and UMR3, to determine whether the defects in olfactory behavior were bona fide outcomes of mys-RNAi expression. As expected, flies harboring either UMR2 (Figure 6B) or UMR3 (Figure 6C) in conjunction with the H24 driver had significant defects in BNZ avoidance as compared to control flies with either the Gal4 driver or the mys-RNAi transgene alone. These data confirm that Gal4-driven expression of mys-RNAi disrupts BNZ avoidance.
The simplest interpretation of the data in Figures 4, 5, and 6A–C is that mys-RNAi expression knocks down ßPS in the brain as it does in the developing wing (Figure 2) and that this reduction in ßPS expression causes defects in olfactory behavior. Unfortunately, despite extensive efforts using multiple anti-ßPS antibodies to immunostain adult head sections, we were unable to unequivocally demonstrate ßPS expression in the central brain and its knockdown by mys-RNAi. Thus, we used an alternative strategy to address the connection between reduced ßPS expression and olfactory defects.
We postulated that if the odor avoidance and attraction defects in flies with H24-driven expression of mys-RNAi were due to reduced expression of ßPS, then expression of wild-type ßPS should rescue the olfactory deficits in these flies. We therefore assessed olfactory behavior in flies expressing mys-RNAi alone, flies overexpressing wild-type mys (i.e., ßPS) alone, and flies expressing mys-RNAi and wild-type mys together using the H24 driver. BNZ avoidance (Figure 6D) and attraction (Figure 6C) were disrupted in flies expressing mys-RNAi alone as expected. Overexpression of wild-type mys via H24 also disrupted BNZ avoidance and attraction. This dominant negative-like activity of overexpressed mys is consistent with other studies showing that overexpressed wild-type integrin can phenocopy integrin loss-of-function (Brabant et al., 1996). More importantly for our studies, flies that expressed mys-RNAi and wild-type mys together had normal BNZ avoidance and attraction. Expression of wild-type mys, therefore, rescued the olfactory defects associated with mys-RNAi expression. These studies indicate that the behavioral changes in flies that express mys-RNAi are due to knockdown of ßPS expression.
Locomotion and central brain morphology are normal in mys-RNAi flies
We assessed negative geotaxis to explore whether decreased locomotion could explain the olfactory defects in flies expressing mys-RNAi. In negative geotaxis assays, flies are tapped to the bottom of a container to elicit an escape response that manifests as walking up the container wall (Gargano et al., 2005). The distance walked is a measure of locomotor ability. Expression of mys-RNAi via H24 had no effect on negative geotaxis (Figure 6F), indicating that the odor avoidance and attraction defects in these flies are not linked to altered locomotor ability. Additionally, since mys-RNAi flies respond normally to some odorants (Figure 5D–F) and have normal maximal avoidance of BNZ (Figure 6A), their locomotor skills are wholly sufficient for good performance within the T-maze.
ßPS is involved in a number of developmental processes, including axon guidance (Hoang and Chiba, 1998) and synapse maturation (Beumer et al., 1999). To investigate whether developmental abnormalities were associated with the olfactory defects in flies expressing mys-RNAi, we evaluated central brain morphology via light microscopy and immunostaining for DLG, the discs large 1 gene product, in adult head cryosections. DLG is expressed in neuropil regions throughout the Drosophila brain (Ruiz-Canada et al., 2002), making it a good marker for central brain anatomy. Careful examination of the morphology as revealed by DLG immunostaining in antennal lobes (Figure 7A,C), mushroom bodies and central complex (Figure 7B,D), and other regions of the central brain revealed no consistent differences between flies expressing mys-RNAi and controls. We also examined central brain anatomy in mys-RNAi flies via immunostaining with another neuropil marker, monoclonal antibody nc82 (Laissue et al., 1999). Consistent with the anti-DLG immunostaining, no reproducible effect of mys-RNAi expression on brain morphology was observed in head sections stained with nc82 (data not shown). The absence of obvious developmental defects as assessed at the light microscope level in mys-RNAi flies raises the possibility that mys plays a role in the acute function of neurons within the Drosophila olfactory system.
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lobes (
lobes ( |
Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Integrins mediate a variety of key biological processes (Hynes, 1992
). In Drosophila, integrins are essential for proper migration of the dorsal epithelium during development (Bunch et al., 1992) and for the normal development of the eye (Zusman et al., 1990), the wing (Brower and Jaffe, 1989), the midgut (Brabant and Brower, 1993), the nervous system (Hoang and Chiba, 1998), and the musculature (Volk et al., 1990). Integrins are also important for development in Caenorhabditis elegans (Gettner et al., 1995) and mice (Kil et al., 1996; Cachaco et al., 2003; Blaess et al., 2004). In addition to their well-established roles in development, integrins also participate in a number of acute physiological processes. Integrins are essential for proper olfactory memory in Drosophila (Grotewiel et al., 1998) and spatial memory in the mouse (Chan et al., 2003). Integrins also play a role in synaptic plasticity in rodents and Drosophila (Staubli et al., 1998; Rohrbough et al., 2000; Chan et al., 2003), functions that might underlie their role in various forms of memory. Additionally, integrins mediate the acute stretch-induced increase in neurotransmitter release at the frog neuromuscular junction (Chen and Grinnell, 1995) and are involved in the increase in neurotransmitter release elicited by hypertonic solutions at the neuromuscular junction in Drosophila (Suzuki et al., 2002). Thus, integrins have important roles in a variety of acute physiological processes.
ßPS is encoded by the mys locus and is the major ß integrin in flies. A series of olfC mutants was previously identified that have defects in their behavioral responses to a subset of odorants (Ayyub et al., 1990). Using a classical genetic approach, Siddiqi and coworkers showed that three independent alleles of mys fail to complement four different olfC mutations for an innate jump response elicited by IAA. This group also found that a wild-type mys transgene rescues the IAA-induced jump defects in two olfC alleles (Ayyub et al., 2000). Additional experiments revealed that flies with two different alleles olfC (presumably mys) in trans to olfE (an allele of "swisscheese") have defects in IAA- and BNZ-induced jumping (Ayyub and Paranjape, 2002). These data support the hypothesis that the olfC and mys mutations are allelic and that mys is involved in the behavioral response to IAA and possibly other odorants.
There are several complexities, however, within the data reported on mys in olfactory behavior. First, although multiple alleles of mys fail to complement two olfC mutations for IAA-induced behavior, they fully complement both olfC alleles for behavioral responses to EA (Ayyub et al., 2000). Similarly, flies with two different mys alleles in trans have defects in IAA behavior but normal responses to EA (Ayyub et al., 2000). These data suggest that mys is important for IAA-induced behavior, while behavioral responses to EA might be independent of the mys locus. Second, the behavioral response to BNZ is normal in the olfC mutants (Ayyub and Paranjape, 2002), making it unclear whether mys is involved in detecting or responding to this or other odorants. Third, some of the chromosomes harboring mys mutations in these studies carried phenotypic markers that could, in principle, account for some of the olfactory behavioral defects observed (Ayyub et al., 2000; Ayyub and Paranjape, 2002). Finally, data from control groups in some experiments were not reported (Ayyub et al., 2000; Ayyub and Paranjape, 2002), making it difficult to directly assess the effects of individual mutations and transgenes on olfactory behavior in all cases. Together, these complexities undermine the strength of the connection between olfactory behavior and mys.
To further investigate the relationship between mys and olfactory behavior in Drosophila, we initially evaluated mys loss-of-function mutants in odor avoidance assays performed in T-mazes. We found that mysnj42 partial loss-of-function mutants have defects in avoidance of BNZ. Additional experiments in our laboratory on these and other mys loss-of-function mutants, however, were inconclusive. Transgenic wild-type mys failed to rescue the olfactory behavior defect in mysnj42 flies [possibly due to a dominant-negative effect of integrin overexpression (Brabant et al., 1996)], stronger combinations of mys alleles were essentially lethal, and a series of weaker alleles or combinations of weaker alleles had no reproducible effect on odor avoidance. Thus, our experiments on mys loss-of-function mutants provided little if any additional insight into the role of integrins in Drosophila olfactory behavior.
To circumvent limitations associated with the mys loss-of-function mutations that we studied, we generated transgenic flies that expressed mys-RNAi. Ubiquitous expression of mys-RNAi is lethal. When expressed in the wing, mys-RNAi produces wing blisters and vein defects. Furthermore, expression of mys-RNAi reduces expression of ßPS, the mys gene product. All these phenotypes are characteristic of strong mys loss-of-function alleles (Zusman et al., 1990; Bunch et al., 1992), demonstrating that expression of mys-RNAi reduces mys expression.
We assessed olfactory behavior in flies that expressed mys-RNAi via a variety of Gal4 drivers that targeted various components of the nervous system associated with olfaction. Expression of mys-RNAi by H24 and MT14, Gal4 lines that express in the antennae, the antennal lobe, and regions of the mushroom bodies, disrupts avoidance of BNZ. Similarly, expression of mys-RNAi by three antennal lobe drivers (C133, 59Y, and 16Y) results in slight decreases in BNZ avoidance that did not reach statistical significance. In contrast, BNZ avoidance is normal in flies that express mys-RNAi via all other Gal4 lines tested (Or83b, 125Y, 107, C309, GH146, Poxn-Gal4, Gr66a, and Gr5a). Of the Gal4 lines we have tested, therefore, only H24 and MT14 significantly disrupt BNZ avoidance when driving mys-RNAi expression. These studies confirm that mys plays a role in odor-induced behavior in Drosophila as suggested previously (Ayyub et al., 2000; Ayyub and Paranjape, 2002).
Our studies are a first step in defining the spatial requirements for mys in olfactory behavior. Since H24, MT14, 59Y, C133, and Or83b express Gal4 in the antennae and maxillary palps in addition to other tissues, yet defects in BNZ avoidance were found only in flies that expressed mys-RNAi via H24 and MT14, our data indicate that expression of mys-RNAi in the antennae and maxillary palps is likely insufficient to disrupt BNZ avoidance. Additionally, since flies that express mys-RNAi via GH146, Poxn-Gal4, Gr66a, and Gr5a do not have defects in BNZ avoidance, the data indicate that projection neurons, Poxn chemosensory neurons, and neurons that detect sweet and bitter gustatory stimuli, respectively, are also not likely to be key sites for mys function within the context of BNZ avoidance. Expression of mys-RNAi in the mushroom bodies via 107 and C309 did not disrupt olfactory behavior, consistent with previous reports indicating that this region of the nervous system is not involved in naive responses to aversive odorants (de Belle and Heisenberg, 1994; McGuire et al., 2001). Based on the overlapping expression patterns of H24, MT14, C133, 59Y, and 16Y, our current model is that expression of mys-RNAi in local interneurons of the antennal lobes disrupts avoidance of BNZ. We note, though, that BNZ avoidance was not statistically reduced in flies with mys-RNAi expression driven in the antennal lobe by C133, 59Y, 125Y, and 16Y. One possible explanation for this apparent discrepancy is that the antennal lobe Gal4 lines we used do not express at high levels in the key interneurons involved in behavioral responses to the odorants we tested. Another possible explanation is that H24- and MT14-driven expression of mys-RNAi disrupts olfactory behavior because these two Gal4 lines drive expression in the antennal lobe interneurons at critical times during development, whereas C133, 59Y, 125Y, and 16Y do not. Our model of mys functioning within local interneurons of the antennal lobes, however, requires additional testing. For example, our studies do not exclude the possibility that H24 and MT14 drive expression in an as yet unidentified component of the olfactory system of Drosophila.
Previous studies suggest that mys is involved in behavioral responses to IAA and possibly BNZ and EA (Ayyub et al., 2000). To further address whether mys is important for detection of all odorants, we assessed behavioral responses to a panel of odorants in flies that express mys-RNAi. H24-driven expression of mys-RNAi disrupted avoidance of BNZ and MCH as well as attraction to EA. In contrast, H24/mys-RNAi flies had only marginal or no defects in avoidance of HEP, LIN, and IAA. Our data indicate that mys is important for behavioral responses to aversive as well as attractive odorants and that behavioral responses to some, but not all, odorants might depend on expression of mys. It is interesting that flies expressing mys-RNAi had no detectable change in IAA, whereas several mys loss-of-function mutants have defects in their behavioral response to this odorant (Ayyub et al., 2000). Additional experiments will be required to determine whether this apparent discrepancy is due to differences between using T-maze and odor jump assays, tissue-limited knockdown of mys versus ubiquitous reduction in mys function, or some other reason. Nevertheless, our data confirm that mys is important for olfactory behavior in Drosophila.
To more rigorously map the defect in olfactory behavior to the mys locus, we performed two additional genetic studies. In the first study, we assessed BNZ avoidance in flies harboring H24 and either of two independently derived mys-RNAi transgenes. As found in all our other experiments, flies expressing mys-RNAi from these additional transgenes have defects in BNZ avoidance. Thus, expression of multiple mys-RNAi transgenes produces the same behavioral phenotype. In the second study, we evaluated the effect of expressing wild-type mys on the olfactory behavior defects in mys-RNAi flies. Overexpression of wild-type mys in an otherwise normal fly disrupts attraction and aversion to BNZ, consistent with a dominant-negative effect of overexpressing integrins found by others (Brabant et al., 1996). Nevertheless, expression of wild-type mys completely rescues the defects in BNZ attraction and aversion. These data are consistent with transgenic wild-type mys compensating for the knockdown of endogenous mys by mys-RNAi. Together, these two studies confirm that olfactory behavior in Drosophila requires expression of mys.
The olfactory behavioral deficits in flies that express mys-RNAi could be caused by developmental abnormalities or a disruption in the acute physiological events involved in the behavior. To begin to address this question, we evaluated brain morphology in mys-RNAi flies using two different neuropil markers, DLG and nc82, on cryosections. At the level of resolution possible with light microscopy, expression of mys-RNAi via H24 had no obvious effect on the morphology of the antennal lobes, mushroom bodies, central complex, or other central brain regions. These data demonstrate that H24/mys-RNAi flies do not have grossly deranged central nervous system development and are consistent with an acute role for mys in olfactory behavior. Additional studies will be required, however, to formally resolve whether mys mediates developmental or acute processes that underlie olfactory behavior in Drosophila.
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The authors thank Danny Brower (University of Arizona), Ennio Giordano (Universita di Napoli), L. S. Shashidhara (CCMB, Hyderabad), Alois Hofbauer (University of Regensburg), Ron Davis (Baylor College of Medicine), Douglas Armstrong (University System of Taiwan), Martin Heisenberg (Universitat Wurzburg), Reinhard Stocker (University of Fribourg), Patrick Callaerts (University of Houston), Liqun Luo (Stanford University), Scott Waddell (University of Massachusetts), Kristen Scott (University of California, Berkeley), John Carlson (Yale University), and Danny Brower (University of Arizona) for providing fly stocks and other reagents. The authors appreciate the technical assistance of Ryan Lewandowski and Jessica Howell. This work was supported by grants to M.S.G. from the National Institutes of Health.
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Accepted 17 May 2006
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