Scribble Is Essential for Olfactory Behavior in Drosophila melanogaster

Corresponding author: Robert R. H. Anholt, Campus Box 7617, North Carolina State University, Raleigh, NC 27695-7617., anholt{at}ncsu.edu (E-mail)

Communicating editor: K. V. ANDERSON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The ability to discriminate and respond to chemical signals from the environment is an almost universal prerequisite for survival. Here, we report that the scaffold protein Scribble is essential for odor-guided behavior in Drosophila. Previously, we identified a P-element insert line with generalized sexually dimorphic smell impairment, smi97B. We found that the transposon in this line is located between the predicted promoter region and the transcription initiation site of scrib. A deficiency in this region, Df(3R)Tl-X, and two scrib null alleles fail to complement the smell-impaired phenotype of smi97B. Wild-type behavior is restored by precise excision of the P element, scrib mRNA levels correspond with mutant and wild-type phenotypes, and introduction of a full-length scrib transgene in the smi97B mutant rescues the olfactory deficit. Expression of Scrib is widespread in olfactory organs and the central nervous system. Finally, alternative splicing of scrib generates transcripts that differ in the number of leucine-rich repeats and PDZ domains.

FOR virtually all animals, behavioral responses to odorants are essential for food localization, avoidance of noxious agents, and selection of reproductive partners. Olfactory behavior is a complex trait, attributable to multiple interacting loci with small and environmentally sensitive effects. While the genetic basis of odorant recognition has been extensively investigated (FOX et al. 2001 ; MOMBAERTS 2001 ; VOSSHALL 2001 ; WARR et al. 2001 ; ZHANG and FIRESTEIN 2002 ), little information is available about the genetic networks that subserve processing of olfactory information and directing appropriate behavioral responses to odorants.

Genetic dissection of complex behavior has traditionally followed one of two approaches: analysis of induced mutations with severe behavioral effects and quantitative genetic studies of naturally occurring behavioral variation (HIRSCH 1967 ; BENZER 1973 ; TULLY 1996 ; GREENSPAN 1997 ; SOKOLOWSKI 1998 ). The major advantage of the mutational approach is the ability to identify genes required for producing behavior. However, screens for severe mutations will not detect loci with pleiotropic effects on behavior at which null mutations are homozygous lethal or loci for which functional redundancy precludes large mutational effects. Combining quantitative genetic with mutational analysis to detect adult viable mutations with subtle behavioral effects in controlled, isogenic genetic backgrounds should facilitate the discovery of such new loci (NADEAU and FRANKEL 2000 ; MACKAY 2001 ).

We applied this strategy to identify 14 novel, autosomal, P-element-tagged smell-impaired (smi) loci in Drosophila melanogaster (ANHOLT et al. 1996 ), all of which had subtle but statistically significant reduced avoidance responses to benzaldehyde, a repellent odorant. Interestingly, the mutational effects for 4 of these loci were sexually dimorphic, with larger effects in females than in males. Genes that affect naturally occurring variation in olfactory avoidance response to benzaldehyde also have different effects in males and females (MACKAY et al. 1996 ). Variation in sexual dimorphism is thus an integral aspect of the genetic architecture of olfactory behavior, as well as other quantitative traits in Drosophila (MACKAY 2001 ). In keeping with this observation, as much as 60% of the Drosophila transcriptome shows sex-biased expression (JIN et al. 2001 ; this is an upper estimate, as it includes germline transcripts).

Here, we show that smi97B, a P-element-induced mutation with sex-specific effects on olfactory behavior, is an allele of scribble (scrib), a pleiotropic gene that encodes 16 leucine-rich repeats (LRR) and four PDZ (PSD-95, Dlg, ZO-1) domains and is essential for establishing polarity in epithelial cells during embryonic development (BILDER and PERRIMON 2000 ; BILDER 2001 ), tumor suppression (BILDER et al. 2000 ; LI et al. 2001 ), synaptogenesis (MATHEW et al. 2002 ; ROCHE et al. 2002 ), and immune responsiveness (WU et al. 2001 ). We have identified at least seven alternatively spliced scrib variants in adults, including one that is more abundant in males and another that is female-specific. These studies contribute to the emerging theme that mutations and naturally occurring alleles affecting behavior are subtle variants of pleiotropic genes, often with sexually dimorphic phenotypic effects (BELLEN and KIGER 1987 ; PRICE et al. 1998 ; SHAVER et al. 1998 ; ANAND et al. 2001 ; TOMA et al. 2002 ).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Fly stocks:
The smi97B mutant was generated by mobilization of a P[lArB] element to cytological location 97B2 in the highly inbred Samarkand (Sam); ry⁵⁰⁶ strain (ANHOLT et al. 1996 ). EMS-generated nulls, scrib¹ and scrib², and P[lacW] insertions scrib^S042405 and scrib^j7B3 were balanced over TM3, Sb. Df(3R)Tl-X and scrib^j7B3 were obtained from the Bloomington Drosophila Stock Center. All other scrib stocks, including a transgenic stock containing a full-length scrib cDNA clone on the X chromosome, were provided by D. Bilder. Stocks used in the derivation of P[lArB]-element excision lines of smi97B and those used to introgress the transgenic stock into the smi97B homozygous background were Sam1; Sam2; Sb 2-3/Ubx, Sam1; Sam2; TM3, Sb ry^RK/H, FM4/Sam1; Sam2; ry⁵⁰⁶, and CyO,TM6/Xa. All fly stocks were maintained on molasses-yeast-agar medium at 25° and 70% humidity, on a 12-hr light-dark cycle, with the exception of the transgenic scrib stock, which was raised at 18°.

Behavior assays:
Three- to 5-day-old adults were divided into single-sex sets of five individuals and deprived of food for 3 hr before testing them for avoidance responses to 0.3% (v/v) benzaldehyde (Sigma Aldrich, St. Louis), as previously described (ANHOLT et al. 1996 ). The number of individuals in the compartment away from the odorant source was recorded at 5-sec intervals. Ten consecutive observations were averaged to generate the avoidance score for a single replicate. The mean avoidance score for a genotype was calculated by averaging avoidance scores obtained over multiple replicates. Statistically significant differences in avoidance scores between genotypes were assessed using Student's t-test.

Larval olfactory behavior was quantified by modifying a previously described assay (HEIMBECK et al. 1999 ). To prevent diffusion of odorants through the medium and eliminate larval gustatory responses, filter paper discs saturated with 2 µl of undiluted odorants (either benzaldehyde or isoamylacetate) or distilled water were placed on the lids of 1.5-ml microcentrifuge tubes. The lids were positioned diametrically opposite each other on an 85-mm petri dish containing 10 ml of 1.2% agarose. Ten third instar feeding larvae were placed at the center of the petri dish and the number of individuals within a 10-mm radius from the odorant source was recorded for seven consecutive 30-sec intervals and averaged over 10 replicates for benzaldehyde and 11 replicates for isoamylacetate. Two-way analysis of variance was used to detect differences in the kinetics of odor-guided behavior between genotypes.

Larval motility was quantified by a locomotion assay (YANG et al. 2000 ). Path lengths of individual larvae were traced over a 60-sec time period, in the absence of odorants. Wild type and mutants were compared using Student's t-test.

Northern blot analyses:
Total RNA (60–100 µg) was isolated from whole flies or larvae using TRIzol reagent (GIBCO BRL, Gaithersburg, MD), size fractionated by 1% formaldehyde gel electrophoresis, and transferred onto nylon membranes (Hybond-XL, Amersham Radiochemicals, Arlington Heights, IL). Blots were hybridized sequentially with [³²P]dCTP-labeled full-length (6 kb) scrib and ß-actin cDNA probes. Hybridizations were performed as described previously (KULKARNI et al. 2002 ). Hybridizing bands were detected in a phosphorimager (Molecular Dynamics, Sunnyvale, CA).

Western blot analyses:
Homogenates from 6–10 fly equivalents were prepared by homogenization in 50 mM Tris-HCl, pH 6.8, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% Nonidet NP-40, 0.02% NaN₃, supplemented with a cocktail of protease inhibitors including phenylmethylsulfonyl fluoride, aprotinin, leupeptin, pepstatin A, and iodoacetamide. Samples were subjected to polyacrylamide gel electrophoresis in SDS on 4–15% gradient gels and electrophoretically transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA). Blots were blocked with 1% (w/v) nonfat milk in 10 mM sodium phosphate buffer, 100 mM NaCl, 0.05% Tween 20, pH 7.4, and incubated with a 1000-fold dilution of rabbit antibodies, raised against the C-terminal Scrib peptide DMRNPLDEIEAVFRS, or preimmune serum. Immunoreactive bands were visualized using a horseradish peroxidase-conjugated goat-anti-rabbit antibody with a chemiluminescence detection kit (Amersham Biosciences, Piscataway, NJ) and exposed to Kodak X-Omat AR film.

cDNA library screening:
cDNA libraries were constructed by cloning reverse-transcribed adult head poly(A)⁺ RNA into the Lambda ZAP Express vector according to the manufacturer's directions (Stratagene, Cedar Creek, TX). A digoxigenin-labeled 6-kb scrib cDNA was used to probe the library. Among 2 x 10⁵ recombinants, 11 hybridizing plaques were identified and their inserts were sequenced using T7 and SP6 primers.

In situ hybridization:
In situ hybridization was performed on 12-µm-thick formalin-fixed and paraffin-embedded sections through adult heads with heat-denatured digoxigenin-labeled riboprobes (Boehringer Mannheim, Indianapolis) corresponding to the sense and antisense strands of a 6-kb scrib cDNA, exactly as described previously (KULKARNI et al. 2002 ). Hybridization products were visualized with an alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche Molecular Biochemicals, Indianapolis), using nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate as substrates. Images were digitally captured and processed with Adobe Photoshop.

Immunohistochemistry:
Randomly oriented heads of 3- to 10-day-old male or female flies were fixed overnight in 10% buffered formalin and embedded in paraffin. Following deparaffinization and rehydration, 12-µm-thick sections were blocked with 1% bovine-serum albumin and incubated with a 500-fold dilution of antiserum for 16 hr at 4°. Immunoreactive products were visualized using a horseradish peroxidase-labeled secondary goat-anti-rabbit antibody with H₂O₂ and 3,3-diaminobenzidine as chromogenic substrates.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Phenotypic characterization of smi97B:
The smi97B mutation is one of the strongest smi mutations among the set of previously identified smi lines (ANHOLT et al. 1996 ). The mutation is recessive: olfactory ability, quantified by avoidance responses to benzaldehyde, was reduced in smi97B homozygotes, compared to the P-element-free coisogenic host strain, Sam; ry⁵⁰⁶ (Sam; P < 0.0001), whereas smi97B/Sam heterozygotes displayed avoidance responses that were indistinguishable from wild type (P = 0.56; Fig 1A). Furthermore, the magnitude of the mutational effect of smi97B was sexually dimorphic (P < 0.05); even at high odorant concentrations, males were hyposmic, while females were anosmic (Fig 1B).

View larger version (38K): In this window In a new window Download PPT slide   Figure 1. Phenotypic characterization of smi97B. (A) Avoidance scores of Sam, smi97B, and Sam/smi97B to benzaldehyde. (B) Avoidance scores of homozygous smi97B males and females showing sexual dimorphism (P < 0.05). (C) Larval olfactory responses of smi97B () and Sam () to isoamylacetate and benzaldehyde. Numbers of larvae at the odorant source were averaged over 10 replicates for benzaldehyde and 11 replicates for isoamylacetate. Error bars indicate SEM. (D) Larval motility in the absence of odorants. Bar graphs in A, B, and D represent mean scores with error bars indicating SEM. Number of replicates for each data set is noted above the error bars. ****P < 0.0001.

To determine whether smi97B flies experience smell impairment throughout their life cycle, we examined whether larvae also display aberrant olfactory responsiveness. Since larvae are attracted to most odorants, even those that elicit avoidance behavior in adults, we compared the kinetics of odor-guided responses of wild-type and smi97B third instar feeding larvae toward benzaldehyde and isoamylacetate (Fig 1C). The effect of genotype in the two-way analysis of variance was significant for both odorants (P < 0.0001), but time and genotype x time interaction terms were not significant for either odorant. Larval motility in the absence of an odor cue was not significantly different between the two genotypes (P = 0.57; Fig 1D). Thus, the smi97B mutation causes olfactory deficits in both larvae and adults.

smi97B is a homozygous viable allele of scrib:
We sequenced the genomic fragment flanking the 3' end of the P[lArB] element and determined the insertion site to be 1084 bp upstream of the open reading frame of scrib (Fig 2A). scrib is a pleiotropic gene essential for localization of polarity determinants in developing epithelia (BILDER and PERRIMON 2000 ), regulation of cellular growth and proliferation (BILDER et al. 2000 ), organization of fat bodies involved in immune responsiveness (WU et al. 2001 ), and synaptic maturation and modulation of short-term plasticity at the larval neuromuscular junction (ROCHE et al. 2002 ). Here, we show that smi97B is an allele of scrib.

View larger version (35K): In this window In a new window Download PPT slide   Figure 2. Complementation tests with scrib alleles. (A) Schematic representation of the scrib gene showing the P-element insertion sites in smi97B, scribS0242405, and scribj7B3. (B) Avoidance responses of F1 progeny generated by crossing Df(3R)Tl-X and null mutations, scrib1 and scrib2, to Sam and smi97B. Df(3R)Tl-X, scrib1, and scrib2 fail to complement the smell-impaired phenotype of smi97B. (C and D) Interallelic complementation between smi97B and P[lacW] scrib alleles. Avoidance responses of F1 progeny generated by crossing scribS042405 and scribj7B3 to Sam and smi97B reveal that the smell-impaired phenotype of smi97B was complemented by scribS042405 only in males and by scribj7B3 in both males and females. Avoidance scores of smi97B heterozygotes are compared to wild-type, Sam heterozygotes for each cross. Bar graphs in B and C represent mean avoidance scores with error bars indicating SEM. Number of replicates for each data set is noted above the error bars. **P < 0.01; ***P < 0.001; ****P < 0.0001.

First, we mapped the smi97B mutation to the region of the third chromosome including scrib. Df(3R)Tl-X (breakpoints 97B2;97D2), which uncovers scrib, failed to complement smi97B; hemizygotes generated by crossing Df(3R)Tl-X to smi97B displayed reduced avoidance responses compared to Df(3R)Tl-X/Sam controls (P < 0.0001; Fig 2B). The smell-impaired phenotype of Df(3R)Tl-X/smi97B hemizygotes was also sex specific, with significantly more smell impairment in females than in males (P < 0.0005).

Next, to demonstrate that olfactory deficits in smi97B arise from the P[lArB] element and not a linked mutation, we showed that precise excision of P[lArB] restores the wild-type phenotype; avoidance responses of precise excision alleles, like smi97B^16A, were not significantly different from Sam (P = 0.07; Fig 3A and Fig B). Further, mutations generated by imprecise excision of P[lArB] provided evidence for sex-specific regulation of scrib. The smi97B^15A mutation contains a 3.6-kb P[lArB] fragment at the original insertion site that resulted in male-specific olfactory deficits (Fig 3, A–C): smi97B^15A males were smell impaired compared to controls (P < 0.005), while olfactory responses in females were not statistically different from those in wild type (P = 0.19). In contrast, a 2.5-kb P[lArB] insertion at the same site in smi97B^2A was correlated with mild hyposmia in females (P < 0.05), but not in males (P = 0.86; Fig 3, A–C). In agreement with the sexually dimorphic phenotype, scrib transcripts were markedly reduced in smi97B^15A males, but not in females, whereas transcriptional differences could not be resolved in smi97B^2A, in line with the subtle female-specific phenotype of this imprecise revertant (Fig 3D).

View larger version (45K): In this window In a new window Download PPT slide   Figure 3. Excision alleles generated by the mobilization of P[lArB] from smi97B. (A) Avoidance responses of Sam, smi97B, excision alleles smi97B15A and smi97B2A, and phenotypic revertant smi97B16A. Bar graphs represent mean avoidance scores with error bars indicating SEM. Number of replicates for each data set is noted above the error bars. *P < 0.05; **P < 0.01; ****P < 0.0001. (B) Molecular analysis of excision lines. Southern blot of HindIII-digested genomic DNA from Sam, smi97B, smi97B15A, smi97B2A, and smi97B16A, probed with a [32P]dCTP-labeled 0.54-kb genomic fragment adjacent to the 3' end of the P[lArB] element. The 18.5-kb P[lArB] element in smi97B generates a 3.5-kb HindIII-digested fragment that hybridizes to the radiolabeled probe, compared to the wild-type 1.4-kb fragment, also observed in the phenotypic revertant, smi97B16A. The 5- and 2.9-kb hybridizing fragments in smi97B15A and smi97B2A indicate imprecise excision of the P[lArB] element. (C) Schematic representation of P[lArB] and its fragments inserted 1084 bp upstream of scrib, in smi97B, smi97B15A, and smi97B2A. Arrows show the transcription start site of scrib. (D) Northern blot showing scrib transcripts in males and females of Sam and excision alleles smi97B15A, smi97B2A, and smi97B16A. Note the reduction in transcript levels in smi97B15A males and differences in transcript sizes between males and females (see also Fig 6). The bottom shows ethidium bromide-stained 28S rRNA bands to control for equal loading.

To further implicate scrib in olfactory behavior, we conducted complementation tests with previously identified scrib alleles. Two null alleles, scrib¹ and scrib², failed to complement the smell-impaired phenotype of smi97B (Fig 2B). Avoidance responses of scrib¹/smi97B and scrib²/smi97B heterozygotes were significantly lower than those of the scrib¹/Sam (P < 0.01) and scrib²/Sam (P < 0.0001) controls. We did not, however, observe sexual dimorphism in smell impairment, possibly due to the disparate genetic backgrounds of the scrib stocks and smi97B. We also tested scrib^S0421405 and scrib^j7B3 alleles, which contain P[lacW] insertions in the 5' untranslated region of scrib and the second intron, respectively (Fig 2A). Avoidance responses of scrib^S0421405/smi97B females were significantly lower than those of scrib^S0421405/Sam females (P < 0.0005), while male responses were not significantly different from those of control males (P = 0.38; Fig 2C). Hence, scrib^S0421405 failed to complement the olfactory deficit caused by smi97B, but only in females. However, scrib^j7B3 fully complemented the smell-impaired phenotype of smi97B; avoidance responses of scrib^j7B3/smi97B flies were not significantly different from those of scrib^j7B3/Sam controls (P = 0.96; Fig 2D). Interallelic complementation is consistent with alternative splicing of scrib, which may involve the generation of sex-specific gene products involved in olfaction.

Finally, we demonstrated rescue of the smi97B phenotype by functional complementation with a wild-type scrib allele. We used a scrib transgene under the Hsp70 promoter on the X chromosome (BILDER and PERRIMON 2000 ), which we introgressed into the smi97B background to avoid confounding background genetic effects on behavior (Fig 4). The transgenic stock, scrib^hs; smi97B, was reared at 18° to prevent activation of the Hsp70 promoter and subsequent expression of scrib. Under these conditions no phenotypic rescue is observed in either sex. When raised at 25°, scrib^hs;smi97B was smell impaired compared to Sam (P < 0.01), but the phenotype was sexually dimorphic (P < 0.05). Although scrib^hs; smi97B females were significantly smell impaired (P < 0.005), male responses were not statistically different from Sam males (P = 0.53; Fig 5A). This indicates that leaky expression of scrib at a semipermissive temperature for Hsp70 is sufficient to rescue olfactory deficits in males, but not in females. When heat-shocked at 37°, avoidance responses of scrib^hs;smi97B females were also restored to those of the parental strain (P = 0.68 ; Fig 5A).

View larger version (18K): In this window In a new window Download PPT slide   Figure 4. Crossing scheme illustrating the introduction of a full-length scrib transgene on the X chromosome from a stock generated by BILDER and PERRIMON 2000 into the smi97B genetic background.

View larger version (38K): In this window In a new window Download PPT slide   Figure 5. Rescue of smi97B using a full-length scrib transgene. (A) Behavioral rescue of smi97B by a full-length scrib transgene. Avoidance scores of Sam, smi97B, and the scribhs stock (containing an Hsp-70-regulated full-length scrib cDNA in a homozygous smi97B background) recorded at 25° (-hs; absence of heat shock) and after heat shock (+hs; 37° for 45 min). Partial (male-specific) rescue of the smi97B phenotype occurs in the absence of heat shock due to the leaky expression of scrib at 25°. The smi97B smell-impaired phenotype is rescued after heat shock. Bar graphs represent mean avoidance scores with error bars indicating SEM. Number of replicates for each data set is noted above the error bars. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (B) Immunochemical analysis of Scrib expression in Sam, smi97B, and scribhs at 18° and following heat shock.

Analysis of scrib transcripts:
The sexually dimorphic olfactory phenotype of smi97B (Fig 1B) and evidence for interallelic complementation (Fig 2D) led us to ask whether males and females express alternative splice variants of scrib. Three major RNA species were detected on Northern blots probed with a full-length scrib cDNA: a universal 5.9-kb transcript present in adults and to a lesser extent in larvae, a 7.1-kb transcript expressed predominantly in males and larvae of both sexes, and a 4.6-kb female-specific transcript (Fig 6; see also Fig 3D). Transcript levels were correlated with sex-specific behavioral phenotypes, as evidenced by reductions in the 7.1-kb transcript in smi97B larvae and the 5.9- and 4.6-kb transcripts in mutant females compared to controls.

View larger version (119K): In this window In a new window Download PPT slide   Figure 6. Sex-specific expression of scrib in Sam (lane 1), smi97B (lane 2), and the phenotypic revertant smi97B16A (lane 3). A Northern blot of whole-fly extracts probed with a full-length scrib cDNA (top) detects three major transcripts (arrows). The same blot was probed with ß-actin to control for equal loading of the lanes (bottom). Note the reduction in message in the smi97B mutants.

To investigate the existence of less prominent variants of scrib, we screened a Drosophila head cDNA library with a full-length scrib probe. Seven unique clones were identified and sequenced. The sizes of three inserts correspond with splice variants detected on Northern blots (Fig 7, clones a, b, and c). A 7.1-kb clone encoding 16 LRRs, four PDZ domains, and a unique 3' untranslated exon corresponds to the transcript expressed in males and larvae; a 5.9-kb clone identical to the 7.1-kb fragment, but without the 3' untranslated region, corresponds to the transcript present in both sexes and larvae; and a 4.6-kb clone encoding 16 LRRs and PDZ domains I and II corresponds to the female-specific transcript. In addition, the 4.6-kb clone also contains a unique coding exon at the 3' end. The first 285 bases of this exon were shared by four additional clones (Fig 7, clones d, e, f, and g), two of which were identical except for the number of LRRs they encode (Fig 7, clones d and e). In contrast to the 4.6-kb female-specific transcript that encodes only PDZ domains I and II, we identified two clones that encode only PDZ domains III and IV (Fig 7, clones f and g; note that clone f is a partial cDNA sequence). Since LRRs and PDZ domains mediate protein-protein interactions, these differences suggest variability in the composition of protein assemblies recruited by the various Scrib isoforms.

View larger version (18K): In this window In a new window Download PPT slide   Figure 7. Schematic representation of scrib variants encoded by cDNA clones from a head library. Inserts a–c correspond to the major mRNA species identified in Fig 6. Isoforms a and c correspond to male- and female-specific transcripts, respectively. Sequences d, e, f, and g were not detected on Northern blots. UTR designates untranslated regions. Sequences e and g carry a deletion in LRRs 3–5. The hatched regions in c–g, the red-shaded region and line in c, and the blue-shaded region and line in f represent alternatively spliced exons, not common to those expressed in sequences a and b. Sequence f represents a truncated transcript.

Monospecific antibodies raised against a carboxyl-terminal peptide of Scrib did not visualize the expected 200-kD polypeptide encoded by the transgene. Instead they detected a 120-kD band in both sexes and an 80-kD female-specific immunoreactive band (Fig 5B), likely due to high sensitivity of the protein in adult flies to proteolysis, which cleaves the expected 200-kD translation product in 120- and 80-kD immunoreactive polypeptides in females, whereas in males the latter fragment is proteolyzed further into smaller fragments (even inclusion of a cocktail of protease inhibitors and performing homogenization in boiling SDS-containing buffer did not preserve the expected 200-kD band). Whereas proteolysis prevents a detailed characterization of sex-specific gene products in adult flies, we could, however, use this antiserum to provide molecular evidence for the heat-shock rescue of smell-impaired smi97B by scrib^hs;smi97B. Little or no immunoreactivity was detected in protein extracts from smi97B and transgenic flies raised at 18°, whereas the intensity of immunoreactive bands was restored to wild-type levels in extracts from scrib^hs;smi97B following heat shock (Fig 5B). Preincubation of antibodies with the peptides against which they were raised abolished staining on Western blots, verifying specificity (data not shown).

Expression of scrib in adult chemosensory organs and central nervous system:
Visualization of scrib expression in adult tissues with a riboprobe complementary to the scrib coding region revealed staining in the third antennal segment and maxillary palps (Fig 8, A–D) and the major olfactory organs of Drosophila as well as in Johnston's organ in the second antennal segment, the primary auditory organ (Fig 8A). Staining was also observed in cortical regions of the brain (Fig 8E). Staining was not observed when hybridizations were performed with sense riboprobes (Fig 8B and Fig D). No differences in scrib expression were observed between males and females under these experimental conditions.

View larger version (78K): In this window In a new window Download PPT slide   Figure 8. Localization of scrib mRNA in antennae (A and B), maxillary palps (C and D), and the central nervous system (E) of Sam flies. In situ hybridizations were performed with a digoxigenin-labeled scrib anti-sense riboprobe (A, C, and E) and with a sense probe (B and D) to control for nonspecific staining. III, third antennal segment, the main olfactory organ; II, second antennal segment, which houses the mechanosensory neurons of JO, Johnston's organ; me, medulla; la, lamina; oc, optic chiasma; mp, maxillary palp; and ret, retina.

To localize Scrib in CNS projection areas, we performed immunohistochemistry. Staining in the brain was particularly intense in the antennal nerves and the ventrolateral and superior medial protocerebrum (Fig 9A). Widespread deposition of Scrib was also detected in the antennae and maxillary palps (data not shown). Incubation with preimmune serum revealed no staining, demonstrating specificity (Fig 9B).

View larger version (118K): In this window In a new window Download PPT slide   Figure 9. Immunohistochemical localization of Scrib in frontal sections of the head. (A) Staining with Scrib antiserum. (B) Staining with preimmune serum. VLP, ventrolateral protocerebrum; SMP, superior medial protocerebrum; AN, antennal nerve; ret, retina; and la, lamina.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

scrib is a pleiotropic gene affecting olfactory behavior:
Evidence that smi97B is an allele of scrib comes from (1) the position of the P-element insert between the predicted promoter region and the transcription initiation site (Fig 2A); (2) failure of Df(3R)Tl-X and two scrib null alleles to complement the smell-impaired phenotype of smi97B (Fig 2B); (3) the restoration of wild-type olfactory phenotype by the precise excision of the P element (Fig 3); (4) correlation of scrib mRNA and protein expression levels with mutant and wild-type scrib phenotypes (Fig 5 and Fig 6); and (5) functional rescue of the olfactory deficit of smi97B by a full-length scrib construct (Fig 5).

It is of interest to note that differences in expression levels at the mRNA level between the smi97B hypomorph and the wild type appear small and are difficult to quantify reliably on Northerns blots (Fig 6). Furthermore, quantitation by quantitative PCR approaches is difficult because of the existence of multiple scrib isoforms. At the protein level, however, differences are dramatic and no quantitation is needed as the gene product is distinct in the wild type and virtually absent in smi97B (Fig 5B). Subtle reductions in message that translate into large phenotypic effects have been documented previously in studies of behavior (GREENSPAN 1997 ; SOKOLOWSKI 1998 ). Minor changes in the activity of a calcium/calmodulin-dependent protein kinase profoundly affect associative learning in Drosophila (GRIFFITH et al. 1993 ). Similarly, in the foraging gene as little as a 10% change in the activity of a cyclic GMP-dependent protein kinase causes a dramatic switch in larval feeding behavior (OSBORNE et al. 1997 ).

Scrib is a LAP (LRR and PDZ) family protein containing 16 LRRs and four Type 1A PDZ domains that bind the consensus sequence S/TXV at carboxyl terminals of proteins (BILDER 2001 ; SHENG and SALA 2001 ). These interactions result in the organization and trafficking of multivalent protein complexes (BILDER and PERRIMON 2000 ; ROCHE et al. 2002 ). In the absence of homozygous viable alleles that enable the characterization of adult phenotypes, descriptions of scrib phenotypes have hitherto been restricted to larval and embryonic stages. Interallelic complementation of the sexually dimorphic smell-impaired phenotype of smi97B demonstrates functional compartmentalization of alternatively spliced variants of scrib. The scrib^j7B3 insertion in the second intron of scrib affects embryonic development, but not olfactory behavior, whereas the scrib^S042405 insertion disrupts embryonic development as well as female-specific olfactory behavior (Fig 2A, Fig C, and Fig D).

Sex-specific differences in the penetrance of the smell-impaired phenotype of smi97B:
Quantitative genetic analyses of olfactory behavior in Drosophila reveal that genes contributing to naturally occurring phenotypic variation show sex-specific effects (MACKAY et al. 1996 ; FANARA et al. 2002 ). Hence, directional selection on olfactory behavior in one sex may not lead to large correlated responses in the other, which facilitates maintenance of genetic variation in a trait under selection pressure. Genotype-by-sex-environment interactions has been documented for a number of complex traits (LONG et al. 1995 ; TOWNE et al. 1999 ; BOWERS and WEHNER 2001 ; CORRY 2001 ; KLOTING et al. 2001 ); indeed, microarray analyses of the Drosophila transcriptome also revealed that global gene expression levels are most strongly affected by the sex environment (JIN et al. 2001 ). Characterization of imprecise excision alleles smi97B^15A and smi97B^2A reveals that the size of the P-element insertion is critical for sex-specific smell impairment (Fig 3). Sex-specific differences in the penetrance of the smell-impaired phenotype of smi97B^15A and smi97B^2A are consistent with the observation that scrib^hs;smi97B transgenic flies raised at 25°, a temperature that is semipermissive for the Hsp70 promoter, show rescue of the olfactory phenotype in males, but not females (Fig 5). Sex-specific splicing of scrib in the adult is evident from the expression of a male-biased 7.1-kb transcript and a 4.6-kb female-specific transcript (Fig 6).

Whereas transposon insertions reveal sexually dimorphic smell impairments, chemically induced mutations do not (Fig 2). This may be due either to genetic background differences in the strains, which affect the penetrance of sexually dimorphic effects, or to the fact that point mutations disrupt protein structure, whereas transposon insertions disrupt transcriptional regulation.

Sex-specific gene expression in Drosophila is generally controlled by doublesex (dsx) and fruitless (fru), two transcription factors whose activities depend on the splicing regulators Sex-lethal (Sxl), transformer (tra), and transformer 2 (tra2; CLINE and MEYER 1996 ; ANAND et al. 2001 ; GRAVELEY 2002 ). However, a recent study, which identified 46 sex-biased genes in fat cells in the head, reported tra- and dsx-dependent transcripts, but also a tra- and dsx-independent female-specific transcript, suggesting alternative pathways for the generation of sex-biased gene products (FUJII and AMREIN 2002 ). Whether sex-specific transcriptional regulation of scribble is directly or indirectly controlled by genes of the classical sex determination pathway remains to be determined.

The sexually dimorphic effects associated with different scrib alleles raises the question whether in situ hybridization with different splice variants would resolve male- or female-specific expression patterns. At present, such experiments are hampered by the notion that the isoforms shown in Fig 7 are likely to represent only a subset of all Scribble isoforms and by the likelihood that any given cell may express multiple isoforms. The complexity of the regulation of the scrib gene is illustrated by the observation that lacZ reporter gene expression in the smi97B mutant is restricted to only a few cell groups in the arista at the base of the third antennal segment (ANHOLT et al. 1996 ), whereas lacZ expression in the p[lac]W insertion mutant scrib^SO42405 extends throughout the antenna and no lacZ staining at all is detected in the p[lac]W] mutant scrib^j7B3 (data not shown; Fig 2A). Sexually dimorphic expression patterns were not observed in these enhancer trap studies. These observations demonstrate the complex regulation of the scrib gene; detailed analysis of transcriptional regulation of scrib, therefore, requires further studies.

	FOOTNOTES

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AY167903– AY167906.

	ACKNOWLEDGMENTS

We thank Nalini Kulkarni, Kellie Robinson, and Akihiko Yamamoto for technical assistance and helpful discussions and David Bilder for generously providing us with fly stocks. This work was supported by grants from the National Institutes of Health (GM-59469, GM-45146, and GM-45344) and the W. M. Keck Foundation.

Manuscript received December 9, 2002; Accepted for publication April 4, 2003.

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