Abstract
Remarkably little is known about the molecular mechanisms that drive sexual behavior. We have identified a new gene, quick-to-court (qtc), whose mutations cause males to show high levels of male-male courtship. qtc males also show a novel phenotype: when placed in the presence of a virgin female, they begin courtship abnormally quickly. qtc mutations are striking in their specificity, in that many aspects of male sexual behavior are normal. We have cloned the qtc gene and found that it encodes a predicted coiled-coil protein and is expressed in the olfactory organs, central nervous system, and male reproductive tract.
SEXUAL behaviors are critical to the reproduction of most animal species, yet remarkably little is known about the molecular mechanisms that drive them. Neural elements associated with sexual behaviors have been defined in a number of systems, but in few species have the molecular elements underlying these behaviors been explored. One organism that exhibits a rich repertoire of sexual behaviors and offers a powerful means of investigating their genetic and molecular underpinnings is Drosophila melanogaster.
Male Drosophila show stereotyped courtship behaviors toward virgin females. The male orients toward the female, follows her, taps her abdomen with his foreleg, extends and vibrates his wing, licks her genitalia, and then mounts her and initiates copulation (Spieth and Ringo 1983; Hall 1994). A number of mutants showing abnormalities in courtship behavior have been identified, many of which show reduced levels of courtship behavior (Hall 1994; Yamamotoet al. 1997). Many of these mutants exhibit sensory defects, defects in the generation of the courtship song, and/or reduced motor activity.
In this article we describe a new gene whose mutations are unique, in that they cause augmentation of male courtship behavior toward both females and other males. The gene was identified by an enhancer trap insertion, which was isolated in a screen of 6400 enhancer trap lines for lines showing expression of a lacZ reporter gene in the olfactory system (Riesgo-Escovaret al. 1992). This particular insertion was distinguished by its sexually dimorphic expression in the Drosophila antenna, exhibiting stronger staining in the male antenna than in the female antenna. Further investigation showed that the staining was due to the insertion of a P-element enhancer trap in cytogenetic region 25C of the second chromosome. The staining pattern was not specific to the antenna, but was also detected in a small number of cells in the adult brain, where it was expressed at comparable levels in males and females. Expression was also observed in association with the larval olfactory organ, the antenno-maxillary complex.
The sexual dimorphism of expression in the antenna prompted the suggestion that the enhancer trap insertion might identify a gene that mediates a sexual behavior (Riesgo-Escovaret al. 1992). In this article we define a gene into which the P-element enhancer trap has inserted. We show that males expressing mutations in this gene exhibit high levels of courtship behavior toward other males and that they are abnormally quick to initiate courtship with females. We show further that the gene, which we call quick-to-court (qtc), encodes a protein with predicted coiled-coil domains and is expressed in the olfactory organs, the brain, and the male reproductive tract.
MATERIALS AND METHODS
Drosophila stocks: The original quick-to-court (qtc) enhancer trap isolate, designated as line 4567, contained two P-element insertions, both on the left arm of the second chromosome (Riesgo-Escovaret al. 1992). The two insertions were separated by meiotic recombination with the al dp b pr c px sp chromosome. The qtc1 b pr chromosome generated from this cross was shown to have a single insertion at cytogenetic position 25C and a lacZ reporter gene expression pattern indistinguishable from that of the original isolate. The qtc1 b+ pr+ chromosome, henceforth designated qtc1, was subsequently generated from the qtc1 b pr chromosome by meiotic recombination. The qtc1 enhancer trap line does not contain a mini-white gene, which has been shown to cause high levels of male-male courtship when ectopically expressed (Zhang and Odenwald 1995; Hing and Carlson 1996).
Chromosomes from which the 25C insertion had been excised were generated by crossing flies homozygous for the qtc1 b pr chromosome, or the qtc1 b+ pr+ chromosome, to flies containing the Δ2-3 transposase (Robertsonet al. 1988). qtcr1, qtcr2, and qtcr3 are lines in which the transposable element excised precisely, as determined by Southern analyses, PCR, and sequence information obtained from PCR products extending from exon 3 to exon 4 (see below). (qtcr1 and qtcr2 were derived from qtc1 b pr, and qtcr3 was derived from qtc1 b+ pr+). qtcEx1 is a line in which the excision of the transposable element was accompanied by a deletion of ~1 kb of flanking DNA (see Figure 3); it was derived from qtc1 b pr.
Wild-type flies, used as controls in some experiments, were obtained from a Canton-S stock called CS-5 (McKennaet al. 1989). In other experiments, control flies were obtained from 6276 (Riesgo-Escovaret al. 1992), an enhancer trap line that is a product of the same crosses that yielded the original qtc1 isolate.
All stocks were maintained on a standard cornmeal-molasses-agar medium at 25°.
Phenotypic analysis: Virgin males and females were separated by sex under carbon dioxide anesthesia within 6 hr of eclosion. While they were anesthetized, half of the males used in the experiment shown in Figure 1C had the distal tip of one wing amputated. After sexing, males and females were maintained in unyeasted food vials until they were 3–6 days old. Males were stored individually; females were stored in groups of 2–12.
Unless otherwise indicated, behavioral observations were initiated by transferring a pair of flies (a mature male and a virgin female or two mature males) without anesthesia to a 0.2-cm3 cylindrical observation chamber. The chamber was illuminated with white light and viewed through a dissecting microscope. The flies were observed for 10 min or until copulation ensued, whichever occurred first. A courtship index (CI) was then calculated, defined as the percentage of the observation period during which any courtship behaviors (orientation, following, tapping, wing vibration, licking, attempted copulation; Tompkins 1984) were performed. The courtship index for a male tested with a virgin female is the percentage of the observation period during which the male performed courtship. If two males were present in the chamber, the courtship index is the percentage of the observation period during which either or both males performed courtship, unless otherwise indicated. The occurrence of specific courtship behaviors was also noted. In some experiments in which mature males courted females, the courtship latency (the time elapsing between the beginning of the observation period and the onset of the first bout of courtship, which is a measure of how long it takes a male to initiate courtship) was determined.
In one series of experiments, groups of males were observed to determine whether they exhibited chaining behavior (i.e., formation of chains or circles of at least three males, in which males courted the male in front of them). Males were collected as virgins as described above and then stored in groups of 30–40 in unyeasted vials. When they were 3 days old, the males in each vial were transferred en masse without anesthesia to a Petri dish containing a piece of filter paper on the bottom and then were observed for 20 min as described in Hing and Carlson (1996).
Hydrocarbons were extracted from the cuticles of mature males and analyzed by gas chromatography/mass spectroscopy (GC/MS) as described in Tompkins and McRobert (1989). Hydrocarbons were extracted by immersing groups of five males in chromatography-grade hexane for 1 min.
Molecular analysis: To clone genomic DNA flanking the P-element insertion at 25C, a genomic library was constructed from qtc1 b pr. The library was screened with a probe containing P-element sequences. This screen identified a 3.6-kb EcoRI fragment that contains P-element sequences and ~3 kb of flanking genomic DNA. The 3.6-kb fragment was then used to screen a wild-type genomic library in the phage λ vector EMBL3, prepared using CS genomic DNA and kindly provided by I. Dawson (Yale University). A genomic clone isolated from this wild-type library was, in turn, used as a probe to isolate clones encompassing more genomic DNA from the region. Two lines of evidence were obtained to indicate that the cloned region corresponds to the site of insertion. First, in situ hybridization to polytene chromosomes showed that the cloned genomic DNA derived from cytogenetic region 25C, the location of the P-element insertion. Second, when used as a probe in genomic Southern blots, the cloned genomic sequences produced different patterns when hybridized to genomic DNA from qtc1 vs. another enhancer trap line (4715) isolated in the same enhancer trap screen. The cDNA clones H5 and H9 were isolated from head cDNA libraries synthesized by M. McKenna (McKennaet al. 1994) and T. Schwarz (Stanford University).
DNA sequences were determined from subcloned fragments using either vector primers or primers designed from previously sequenced DNA regions. The nucleotide sequences shown in Figure 4 were based on a complete analysis of both DNA strands. Sequence comparisons and GenBank database searches were performed with the BLAST search program (National Center for Biotechnology Information). Structural analyses were performed using the GCG program (University of Wisconsin Genetics Computer Group). The positions of introns were determined by comparing sequences from cDNA clones H5 and H9 with those obtained from genomic clones. The RNase protection experiment was performed with an RPA II kit from Ambion (Austin, TX) according to the recommendations of the manufacturer. The probe was 465 bases in length, extending from nucleotide position 241 (in the first exon) to position 705 (in the second exon) of the qtc gene (Figure 3). The results were quantitated on a phosphorimager.
Total RNA used in Northern blots was isolated either by direct phenol extraction in RNA extraction buffer (0.1 m Tris-Cl, 0.1 m NaCl, 20 mm EDTA, and 1% n-lauryl sarcosine) and TE-saturated phenol or by using TRIzol reagent (GIBCO BRL Gaithersburg, MD). Poly(A)+ RNA was isolated from total RNA using the Oligotex mRNA isolation kit (QIAGEN, Chatsworth, CA). Fractionation of RNA and hybridizations were done using standard methods. In situ hybridizations to polytene chromosomes and to RNA in tissue sections were as described elsewhere (McKennaet al. 1994).
PCR reactions to examine the insertion site in qtc1 and in the derived excision lines used two sets of primers. Amplification of sequences between exons 3 and 5 in qtcEx1, qtcr1, qtcr2, and qtcr3 were performed using primers that corresponded to positions 1555–1573 in exon 3 and 1721–1700 in exon 5. Amplifications of sequences between the P element and exon 5 in qtc1 b pr and qtc1 were performed using the primer 5′-CGGCTATCGACGGGACCACC-3′ [derived from the P(lacZ) element] and the exon 5 primer described above.
RESULTS
qtc males show high levels of male-male courtship: While examining the sexual behavior of a collection of laboratory stocks, we observed striking abnormalities in a P(lacZ) enhancer trap line, subsequently designated as qtc1. Sexually mature qtc1 males performed much more courtship in response to each other than did control Canton-S-5 (CS) males (Figure 1A). Specifically, mature qtc1 males yielded a male-male CI of 42 ± 5, indicating that courtship behaviors (orientation, following, tapping, wing vibration, licking, or attempted copulation) were observed on average during 42% of a 10-min observation period (Tompkins 1984), whereas CS control males had a CI of only 4 ± 1. CS was chosen as a control because the sexual behavior of CS flies has been thoroughly characterized (Tompkinset al. 1980; McRobertet al. 1995); however, since qtc1 is not in a CS genetic background, we performed a series of experiments to determine whether the phenotype is in fact due to the P-element insertion in this line.
The behavior of qtc1 males first was compared to that of males from 6276, another line isolated from the same enhancer trap screen as the original isolate of qtc1. Males from the 6276 line perform very low levels of male-male courtship (CI = 2 ± 0; Figure 1A), like CS. We then compared the responses of males from four stocks derived by excision of the P-element insertion: three stocks in which the P element had excised precisely (qtcr1, qtcr2, and qtcr3) and one in which the P element had excised imprecisely, creating a deletion of flanking DNA (qtcEx1, described in detail below). As shown in Figure 1B, males from the precise excision stocks (qtcr1 and qtcr2) showed low levels of male-male courtship, similar to CS (low levels of male-male courtship also were observed in qtcr3, for which fewer data have been collected). In comparison, males from the imprecise excision stock (qtcEx1) had high levels of male-male courtship. Since these excision lines were generated in parallel and are expected to have similar genetic backgrounds, these results indicate that the elevated levels of male-male courtship in the qtc1 stock result from the insertion of the P element.
qtc1 males' courtship of each other also was qualitatively different from that of CS controls. In all pairs of CS males observed (10/10), one or both males performed at least one of the “early” behaviors (orientation, tapping, and following), but in only half of the pairs observed did one or both males vibrate their wings to produce a courtship song, and none of the males licked or attempted copulation. By contrast, in all pairs of qtc1 males observed (10/10), one or both males exhibited wing vibration. In half of the pairs observed, at least one of the qtc1 males licked the other male's genitalia, and one qtc1 male attempted to copulate with the other male in the observation chamber. Some aspects of qtc1 males' responses to each other were normal. When groups of qtc1 males were observed in a Petri dish, the males did not exhibit the chaining behavior shown by certain other mutants (Hall 1994; Zhang and Odenwald 1995; Hing and Carlson 1996). Moreover, like CS males, all of the qtc1 males that we observed in chambers or in Petri dishes ran away from the males that courted them. Thus, qtc1 males' responses to male courtship appeared normal.
Levels of male-male courtship are elevated by qtc mutations. The CI is the percentage of the observation period during which courtship behaviors were performed. Error bars indicate SEM and in some cases are too small to be visible. In all cases, differences between experimental lines and control lines are significant at the P < 0.001 level. Note that scales are not uniform in the different panels. (A) qtc1 shows a higher level of male-male courtship than a CS wild-type control or a control enhancer trap line, 6276; n = 20 pairs for each genotype. (B) Males homozygous for the imprecise excision qtcEx1 also show a higher level of male-male courtship than do either of two precise qtc excision lines, qtcr1 and qtcr2; n = 20 pairs for each genotype. (C) qtc1 males court CS wild-type males at a higher level than CS males court qtc1 males. One CS male was paired with one qtc1 male in each test, and the CI was calculated for each genotype separately. The CI values represent the duration of the observation period during which courtship was performed by the males of the indicated genotype; n = 5 pairs for each mixed genotype experiment.
It was possible that pairs of qtc1 males performed more male-male courtship, in comparison to pairs of CS controls, because the qtc1 mutation affected males' propensity to court other males. Alternatively, the qtc1 mutation could have affected males' ability to elicit courtship from other males. To distinguish between these two possibilities, we performed a series of behavioral experiments in which we paired one qtc1 male with one CS male and determined each male's courtship index individually. In addition, we noted which of the two males initiated courtship first. As shown in Figure 1C, the qtc1 males performed high levels of male-male courtship in response to the CS males, while the CS males performed little or no courtship in response to the qtc1 males. Moreover, in all nine of the pairs in which both males performed at least some courtship (in one pair the CS male did not perform any courtship), the qtc1 male initiated courtship first. The simplest interpretation of these results is that the qtc1 mutation affects mature males' performance of male-male courtship but has no discernible effect on their ability to elicit male courtship. This conclusion is supported by analysis of cuticular hydrocarbons extracted from qtc1 and CS males (see Jallon 1984; Ferveur 1997; Tompkins 1998). This analysis revealed no differences in levels of 7-tricosene and 7-pentacosene, which are abundant hydrocarbons on mature males' cuticles that have courtship-inhibiting and courtship-stimulating activity in bioassays, respectively (not shown). Nor did qtc1 males synthesize detectable quantities of 7,11-heptacosadiene, a female-specific courtship-stimulating pheromone.
qtc males are quick to court females: A second phenotype was observed when qtc1 males were paired with CS virgin females: qtc1 males initiated courtship of females more quickly than did control CS males (Figure 2). To determine whether this phenotype was due to the P-element insertion, we first tested males of line 6276 with CS females. As shown in Figure 2, 6276 males have significantly longer courtship latencies than qtc1 males. We also observed that males homozygous for the imprecise excision (qtcEx1) initiate courtship more rapidly than those homozygous for precise excisions (qtcr1 and qtcr2; Figure 2). Accordingly, we conclude that the rapid initiation of courtship in response to females most likely arises from the P-element insertion in the qtc1 stock. Because the foregoing analysis indicated that the same mutation is responsible for the elevated levels of malemale courtship displayed by qtc1 males, we have designated the gene that is mutated as quick-to-court (qtc), based on the finding that mutant males are quick to initiate courtship of both virgin females and mature males.
Many aspects of sexual behavior are not affected by qtc: Several aspects of the sexual behavior of qtc males and females are normal, in comparison to CS males and females. Mature qtc1 males performed normal levels of courtship toward CS virgin females during the observation period: the CI of qtc1 males paired with CS females was 75 ± 3 (n = 20), compared with 78 ± 3 (n = 21) for CS males paired with CS females (P > 0.05; t-test on arcsin transformed courtship indices). Moreover, mutant males performed “advanced” courtship behaviors toward CS females: qtc1 males showed wing vibration in all of 20 cases, and in most cases they showed licking of females' genitalia and curling of the abdomen to attempt copulation [see Tompkins (1984) for a description of these behaviors]. Moreover, qtc1 and CS males that began to mate with females during the 10-min observation period had copulation latencies that were not significantly different. qtc1 females elicited as much courtship from CS males [CI = 80 ± 3 (n = 20)] as did CS females [78 ± 3 (n = 21)]. Immature qtc1 males elicited the same level of courtship from CS males [70 ± 8 (n = 10)] as did immature CS males [83 ± 4 (n = 10)]. qtc1 females were as likely to mate with CS males (65%; n = 20 pairs) during the observation period as were CS females (67%; n = 21), and the mutant females' copulation latencies were normal. We note that during courtship tests of qtc1 males with CS females, qtc1 males were observed to perform the full repertoire of wild-type courtship behaviors.
qtc males show a reduced latency of male-female courtship. Males of the indicated genotypes were paired with CS virgin females, and the time elapsing between the beginning of the observation period and the onset of courtship was determined. Error bars indicate SEM. In all cases, differences between experimental lines and control lines are significant at the P < 0.01 level. (A) qtc1 males begin to court females faster than do CS wild-type control males or males of a control enhancer trap line, 6276. The difference in latency between 6276 and CS is likely due to differences in their genetic backgrounds; n = 20 pairs for each genotype. (B) Males homozygous for the imprecise excision qtcEx1 also show a faster onset of male-female courtship than do males homozygous for the precise excisions qtcr1 and qtcr2; n = 10 pairs for each genotype.
In addition, sensory function of qtc1 appeared normal in limited testing of visual and olfactory physiology by electroretinogram and electroantennogram recordings and in tests of both adult and larval olfactory behavior. Detailed examination of qtc mutants did not reveal any general behavioral defects in such activities as walking or grooming.
Identification of a novel gene spanning the site of the P-element insertion: Genomic DNA flanking the insertion site of the P-element enhancer trap was cloned and used to isolate cDNA clones from Drosophila head cDNA libraries. Two cross-hybridizing cDNA clones, H9 and H5, were analyzed in detail, and each hybridized to genomic DNA on both sides of the P-element insertion site. H5 was found to hybridize in situ to a single site on the polytene chromosomes, 25C, the site of the P-element insertion. In low-stringency Southern blots, H5 was observed to hybridize only to genomic fragments of sizes consistent with those from the 25C region. Northern blots using H9 as probe revealed a major transcript of 2.4 kb (shown in Figure 5A). Since H9, the longer cDNA, is 2338 bp in length exclusive of a poly(A) tract, the length of the H9 cDNA sequence is close to that of a full-length cDNA.
The genomic organization of the gene defined by the H9 and H5 cDNA clones is shown in Figure 3. Five exons have been identified, spanning at least 6 kb. The P-element insertion causing the qtc1 mutation lies in the third intron, flanked by exons that each carry coding sequences. Specifically, the insertion maps 214 nucleotides downstream from the 3′ end of the third exon, as determined by direct nucleotide sequencing of genomic DNA from the insertion chromosome, and as confirmed by several lines of PCR evidence. A ribosomal protein gene, L37a, is located ~260 bp beyond the 3′ end of the gene (Gaineset al. 1999). We tentatively designate as qtc the gene defined by the H9 and H5 cDNA clones, on the basis of the following evidence. The qtc insertion mutation (qtc1) and the imprecise excision qtcEx1 both disrupt the H9 transcription unit (Figure 3; direct evidence for an effect on the RNA is shown by Northern analysis in Figure 5C); extensive screening of head and antennal cDNA libraries has revealed no transcripts in the third intron of the gene; nucleotide sequence analysis of the third intron has revealed no long open reading frames (ORFs); sequence analysis of the second and fourth introns and of the region between the H9 transcription unit and the L37a gene has revealed no long open reading frames; Northern analysis has revealed no effect of the qtc1 insertion on expression of the L37a gene (see below). The simplest interpretation of these data is that qtc mutations exert their effects by disrupting the H9-defined gene, henceforth referred to as qtc.
qtc undergoes alternative forms of splicing. Sequence analysis of the cDNA clones revealed that H9, but not H5, contains the fourth exon of qtc. In H5 the third exon of qtc is spliced directly to the fifth exon (Figures 3 and 4). qtc also shows alternative forms of processing at the 3′ end. The H5 cDNA clone contains a poly(A) tract 19 bp downstream from an AATACA sequence, which differs from the consensus polyadenylation signal AATAAA at a single nucleotide (see Figure 4 below). In the H9 cDNA, transcription extends 105 bases further than in H5, terminating at a position 22 nucleotides downstream from an AATAAA hexanucleotide.
The qtc sequence reveals an open reading frame of 566 amino acids (Figure 4). The methionine indicated at position 1 is expected to be the translation start site, on the basis of the predictions of two codon preference programs (Testcode and Codonpreference; Geneworks). Portions of the predicted Qtc protein have limited sequence identity to a large family of myosin and myosinlike proteins. These proteins all contain α-helical coiled-coil domains, which differ in sequence but which contain common structural features (Lupas 1996). Specifically, these domains comprise an ordered series of hydrophilic and hydrophobic residues that are repeated every seven residues. Hydrophobic residues are found in the first (a) and fourth (d) positions within the heptad repeats, whereas hydrophilic residues occupy the other five positions (b, c, e, f, and g). At least four of these heptad repeats are needed to form a stable coiled-coil structure in the protein (Lupaset al. 1991).
In qtc, five or six heptad repeats are found in each of three α-helical domains [underlined in Figure 4, with amino acids at positions (a) and (d) shown in boldface and their composition summarized in Table 1], as predicted by the GCG programs Helicalwheel and Coiled-coil. One domain comprises six heptad repeats; two domains, located close to each other, comprise five heptad repeats each. The distributions of hydrophobic and charged residues in the predicted coiled-coil domains are similar to those observed in a number of coiled-coil proteins, as shown in Table 1 (Lupaset al. 1991).
qtc is expressed in the olfactory organs, brain, and male reproductive tract: The qtc enhancer trap line was originally identified by virtue of its reporter gene expression in the olfactory system, with limited expression also detected in the brain. To determine whether the qtc gene is expressed in the olfactory system and brain, we performed Northern blots and in situ hybridizations to RNA in tissue sections.
Genomic organization of qtc. Above the restriction map are the intron-exon maps of qtc and the adjacent L37a ribosomal gene; the solid blocks represent coding regions. The dotted line connecting exons 3 and 5 represents the alternative splice found in the cDNA clone H5. The solid SalI-BamHI fragment was used to screen a head cDNA library. The position of the 5′ exon within the SpeI fragment at the left has not been determined precisely. The transposable element insertion site is 214 bp from the 3′ end of the third exon. The extent of deleted genomic DNA in qtcEx1 is shown below the restriction map, with uncertainty in the endpoints represented by the hatched boxes. The 5′ to 3′ orientations of both the lacZ reporter gene and the rosy marker gene are from right to left. The transposable element is ~14 kb and is not drawn to scale. B, BamHI; E, EcoRI; K, KpnI; N, NheI; S, SalI; Sp, SpeI.
A 2.4-kb transcript in heads and antennae is observed to hybridize to a qtc probe (Figure 5A). The transcript is present in both males and females, in comparable amounts. Quantitative RNase protection experiments revealed no differences between male and female antennae in qtc transcript levels. In another experiment, the 2.4-kb transcript was also observed in bodies from which heads had been removed and in embryos, although the intensity of hybridization was less than that to head RNA (Figure 5B). Following long exposure, an additional band of 4.4 kb could be observed in the head RNA track.
The qtc mutations were found to affect the 2.4-kb transcript, as shown in Figure 5C. Specifically, the P-element insertion in qtc1, which introduces ~14 kb of DNA into the third intron of the qtc gene, greatly reduces the intensity of labeling and affects the band pattern; the same alterations are observed for the parental insertion line qtc1 b pr. The qtcEx1 deletion mutation, generated by imprecise excision of the P-element insertion, also reduces the intensity of labeling and alters the band pattern. The changes in band pattern presumably reflect changes in the length of the transcription unit, the deletion or addition of splice junctions, and stability of the mRNAs: rapid degradation of other disrupted transcripts by mRNA surveillance pathways has been demonstrated (Beelman and Parker 1995). In contrast, wild-type levels of L37a gene expression were observed in both qtc1 insertion lines and in the deletion mutant line qtcEx1 (data not shown).
Localization of the qtc transcript was confirmed and extended by in situ hybridization to RNA in tissue sections. A qtc antisense probe hybridized to sections of the antenna and the maxillary palp, another olfactory organ of the fly head (Figure 6, A and C). Within the antenna, expression is seen in both the second and third segments and is concentrated near the cuticle, where the cell bodies of sensory neurons are located. Expression in the maxillary palp also appears near the cuticle. These hybridization results show that the gene is expressed in two olfactory organs: the third antennal segment and the maxillary palp. However, they also show that expression is not limited to olfactory cells, since the second antennal segment contains no olfactory sensilla.
Abundant expression is also seen in the CNS within the head (Figure 6D), consistent with the strong hybridization to head RNA in Northern blots. There are high levels of expression in the visual system: expression is seen in the retina and the optic lobe. Uniform expression is also seen in the cortex of the brain, where the cell bodies of brain neuropil are located. The probe also hybridized to sections from the thorax and abdomens of males and females. In both sexes, expression is seen in the ventral ganglion within the thorax (Figure 6F, arrowheads). In males, expression is seen near the tip of the abdomen, closely associated with the ejaculatory bulb and testis (Figure 6G, arrowhead). In females, expression is also observed in the distal end of the abdomen, likely associated with the reproductive system (not shown).
DISCUSSION
We have identified a new gene, qtc, mutations in which cause striking abnormalities in sexual behavior. qtc males show elevated levels of male-male courtship and a rapid onset of male-female courtship. These phenotypes are of particular interest in that they represent heightened or accelerated activity, as opposed to a loss of activity, which can be caused indirectly by a wide variety of general defects. The short latency of male-female courtship is a novel phenotype that, to our knowledge, has not previously been described.
Nucleotide and deduced amino acid sequence of qtc. The nucleotide sequence shown is that of the H9 cDNA. The predicted amino acid sequence of the longest ORF is shown. The amino acids in the heptad repeats that constitute the predicted coiled-coil regions are underlined. The residues occupying the a and d positions in the heptad repeats are in boldface. In the second underlined region, the first three residues (QEL) and the last four residues (KVEA) are included because of the predicted likelihood (i.e., 60–70%) that they are part of the domain. Double arrows above the nucleotide sequence designate the locations of four identified introns. The diamond above nucleotide 748 indicates the 5′ end of cDNA clone H5. Presumed polyadenylation signal sequences are underlined or in boldface. The boldface An indicates a poly(A) addition site in the H5 cDNA. No poly(A) track was found at the 3′ end of the H9 cDNA. The nucleotides at positions 1505 and 1508 are C and G, respectively, in the genomic sequence; these differences do not affect the coding potential and may reflect polymorphism.
The qtc phenotype is highly specific. We have detected no morphological, sensory, motor, or general behavioral defects. Many aspects of sexual behavior are normal in qtc mutants. Mature qtc1 males perform the full repertoire of courtship behaviors, show normal levels of courtship toward virgin CS females, and have normal copulation latencies with them. qtc1 females are normal in all courtship behaviors we have examined. qtc1 thus defines two classes of sexual behaviors: those that are affected by qtc1 and those that are not. These results suggest that some sexual behaviors are controlled by a genetic pathway that depends on qtc, and other behaviors are controlled by one or more distinct pathways.
The male-male and male-female courtship phenotypes of qtc are related, in the sense that both may be considered as an increase in sexual activity. One interpretation of these phenotypes is that qtc males show an increased tendency to court: when paired with a female, they begin courting quickly; when paired with a male, they court despite the presence of inhibitory cues associated with the male partner. If this interpretation is correct, qtc may define a novel molecular component required for the control of male sexual drive. Since males expressing qtc mutations show elevated levels of courtship compared to controls, it is tempting to speculate that the qtc mutations affect neural circuitry that, in normal males, inhibits courtship.
Distribution of hydrophobic and charged residues in Qtc and other coiled-coil-containing proteins
While several Drosophila genes have been associated with high levels of male-male courtship, qtc is unique among them in that its wild-type function appears to be required for maintaining low levels of male-male sexual behavior, but not for the ability to execute the various courtship behaviors. The qtc phenotype shows a high degree of specificity in this sense. When fruitless (fru; Gill 1963) males are placed in pairs, they show elevated levels of courtship behavior; when placed in groups, they form courtship chains, in which individual males court the fly ahead and are courted by the fly behind (reviewed in Hall 1994; Tayloret al. 1994). fru has recently been shown to encode a zinc finger transcription factor that defines a branch of the sex determination hierarchy (Itoet al. 1996; Ryneret al. 1996). qtc is different from fru in that qtc males carry out the full repertoire of courtship behaviors and are fertile; males expressing most fru alleles generate an abnormal courtship song (Wheeleret al. 1989; Bernsteinet al. 1992), do not bend their abdomen into a copulating position (Hall 1978; Gailey and Hall 1989), and are sterile. For most fru alleles, males show courtship toward females as well as males, but their courtship latency has not been measured, to our knowledge. Males mutant for the recently described dissatisfaction (dsf) mutation court and attempt copulation with both males and females, but are slow to copulate, at least in part because of abnormal abdominal curling (Finley et al. 1997, 1998). dsf is different from qtc and fru in that dsf affects female, as well as male, courtship. dsf females resist copulation and fail to lay eggs. The abnormalities in female egg-laying and male abdominal curling are correlated with defects in motor neuron innervation of abdominal muscles. Flies in which the white (w) gene is ectopically expressed have also been shown to exhibit high levels of male-male courtship (Zhang and Odenwald 1995; Hing and Carlson 1996). When pairs of male flies that carry a mini-w gene under the control of the hsp70 heat-shock promoter are heat-shocked, they vigorously court each other; groups of heat-shocked males form courtship chains, lariats, and rings. The w gene encodes a polypeptide that is believed to play a role in transmembrane transport (O'Hareet al. 1984; Pepling and Mount 1990; Ewartet al. 1994). Male-male courtship has not been observed in deletion or other loss-of-function mutants of w, and it is difficult to draw conclusions about the normal role of the w+ gene from these ectopic expression studies. Elevated levels of male-male courtship have also been observed in males exhibiting ectopic expression of the feminizing gene transformer in portions of the antennal lobes or mushroom bodies, structures implicated in olfactory processing (Ferveuret al. 1995; O'Dellet al. 1995).
We have cloned the qtc gene, shown that it encodes a predicted protein with coiled-coil domains, and found that the expression pattern includes the olfactory organs, the CNS, and the male reproductive tract. Many proteins with such coiled-coil domains form dimers as a result of hydrophobic interactions between two α-helices that wrap around each other. Some coiled-coil proteins, such as myosin, contain many heptad repeats; others contain few (Table 1). Among the latter are several proteins containing leucine zippers, such as the yeast protein GCN4 (Hinnebusch 1984; Williamset al. 1988), or zinc fingers, such as the mouse protein Rpt-1 (Patarcaet al. 1988). Some of these proteins contain DNA binding domains and function as transcription factors.
RNA blot analysis of qtc expression. (A) Heads and antennae of males and females contain a 2.4-kb transcript that hybridizes to the qtc cDNA clone H9. Although the band in the male antennal track appears more intense than that in the female antennal track in this experiment, this difference was not reproducible and could not be substantiated by quantitative RNase protection experiments. Total RNA was used, and below each blot is a portion of the same blot hybridized with a Drosophila actin 5C probe as a loading control. (B) Embryos and adult bodies also contain RNA that hybridizes to the qtc clone, although at lower levels than adult heads. “Whole male” and “whole female” contain RNA extracted from entire animals. Embryonic RNA was prepared from 0- to 24-hr embryos. RNA molecular weight markers are shown on the left. Poly(A)+ RNA was used, and below each blot is a portion of the same blot hybridized with a Drosophila actin 5C probe as a loading control. (C) The 2.4-kb qtc transcript is affected in RNA from qtc1, its parental line qtc1 b pr, and qtcEx1 whole animals, but not in the precise excision lines qtcr1, qtcr2, or qtcr3. Below are portions of the same blots hybridized with a Drosophila rp49 probe (O'Connell and Rosbash 1984); the qtc1 and qtcEx1 lanes were run on a separate blot from qtc1 b pr and the other genotypes. Poly(A)+ RNA was used, and RNA molecular weight markers are shown on the left.
The qtc expression pattern is much broader than that of lacZ in the qtc1 enhancer trap line. qtc1 was identified in a screen for lines showing lacZ expression in the olfactory organs of adults and larvae, but little expression elsewhere (Riesgo-Escovaret al. 1992). The qtc1 enhancer trap line shows sexually dimorphic lacZ staining in a subregion of the third antennal segment and very limited staining in the CNS. The qtc transcript is distributed extensively in the antenna and the CNS and in some tissues outside the nervous system, e.g., in the male reproductive tract. Moreover, qtc expression in the antenna shows no evidence of sexual dimorphism in quantitative RNase protection experiments. There is ample precedent for P lacZ insertions whose expression patterns vary from those of the genes in which they are inserted (e.g., Bieret al. 1990). It is unlikely that the lacZ pattern of the qtc enhancer trap line reflects precisely the expression of a gene other than qtc. The P lacZ insertion is located within a small intron of qtc, an intron whose sequence does not indicate the presence of any internal genes. There is a gene that lies ~260 bp distal to the 3′ end of qtc, but it encodes a predicted ribosomal protein and is ubiquitously expressed (data not shown). We have not identified genes lying upstream from the 5′ end of qtc, but the 5′ end of qtc lies far (>18 kb) from the 5′ end of the lacZ reporter gene. One possible interpretation of the discrepancy between the qtc and lacZ patterns is that qtc expression is controlled by multiple regulatory elements, one of which lies adjacent to the lacZ insertion in the third intron and drives sexually dimorphic expression in a group of antennal cells, conceivably in cells that receive pheromonal input.
Expression of qtc in the olfactory organs, CNS, and male reproductive tract. qtc RNA expression was visualized by in situ hybridization with a probe generated from the H9 cDNA. (A) Antenna. Frontal section, with dorsal at top; antisense strand probe. Signal is visible around most of the periphery of the third segment (bottom), where cell bodies are located. Signal is also visible directly under the cuticle in the second segment (top). (B) Antenna; sense strand control probe. No signal is visible. (C) Maxillary palp, hybridized with antisense strand. Signal is visible directly beneath the cuticle. (D) Head; frontal section hybridized with antisense strand. Strong signal is visible in much of the visual system, including the retina and a band of cells at the distal region of the lamina that appear to be the interlaminar neurons. Uniform expression is also visible in the cortex of the brain, and punctate signal is associated with the cell bodies of the maxillary nerve (arrowhead). Dorsal is at the top. (E) Head section hybridized with sense strand probe. (F) Male head and thorax, seen in sagittal section, hybridized with antisense probe. Dorsal is at the top and posterior is to the right. Arrowheads indicate some of the labeled regions in the thorax. (G) Male abdomen; sagittal section, hybridized with antisense probe. Dorsal is at the top and posterior is to the right. Signal is visible near the sperm pump (sp, arrowhead).
In summary, qtc encodes a novel protein that is required for the normal regulation of male sexual behavior. The molecular genetic underpinnings of sexual drive are poorly understood, and the identification of new components affecting this process provides new avenues for investigation. Further genetic analysis of qtc, including mosaic analysis or analysis of GAL4-driven qtc misexpression, may determine whether specific parts of the nervous system or reproductive tract require qtc function for normal sexual behavior. Such analysis may also determine whether the male-male and male-female courtship abnormalities have distinct anatomical foci. Finally, further molecular and genetic analysis of qtc may be useful in determining its molecular function and in identifying the pathway through which it exerts its role in the regulation of sexual behavior.
Acknowledgments
We are very grateful to Kathy Raman for excellent assistance with the project, to Marc Freeman for quantitative analysis of transcripts, to Larry Jackson for analysis of pheromone profiles, and to Marien de Bruyne for comments on the manuscript. This work was supported by National Science Foundation grant IBN-9317054 and National Institutes of Health grant DC-02174 (to J.C.) and by a National Institute of Mental Health National Research Service Award (to P.G.). Some of the experiments were performed by L.T. while on sabbatical leave at Yale.
Footnotes
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Communicating editor: V. G. Finnert
- Received August 13, 1999.
- Accepted November 29, 1999.
- Copyright © 2000 by the Genetics Society of America
