In an effort to uncover genetic components underlying the courtship behavior of Drosophila melanogaster, we have characterized a novel gene, lingerer (lig), mutations of which result in abnormal copulation. Males carrying a hypomorphic mutation in lig fail to withdraw their genitalia upon termination of copulation, but display no overt abnormalities in their genitalia. A severe reduction in the dosage of the lig gene causes repeated attempted copulations but no successful copulations. Complete loss of lig function results in lethality during early pupal stages. lig is localized to polytene segment 44A on the second chromosome and encodes three alternatively spliced transcripts that generate two types of 150-kD proteins, Lig-A and Lig-B, differing only at the C terminus. Lig proteins show no similarity to known proteins. However, a set of homologous proteins in mammals suggest that Drosophila Lig belongs to a family of proteins that share five highly conserved domains. Lig is a cytoplasmic protein expressed in the central nervous system (CNS), imaginal discs, and gonads. Lig-A expression is selectively reduced in lig mutants and the ubiquitous supply of this protein at the beginning of metamorphosis restores the copulatory defects of the lig mutant. We propose that lig may act in the nervous system to mediate the control of copulatory organs during courtship.
THE mating behavior of male Drosophila is characterized by a fixed sequence of actions. The sequence starts with orientation toward a female fly, followed by tracking, tapping of the female’s abdomen, generation of courtship songs by alternate vibration of its wings, and licking of the female’s genitalia (Hall 1985; Greenspan 1995; Yamamotoet al. 1997; Yamamoto and Nakano 1998). As a consequence of such courtship, sexual receptivity in the female increases, inducing a reduction in her locomotion (von Schilcher 1976). This permits the male to mount her back and attempt copulation by curling his abdomen to clasp her genitalia. After ∼15 min of copulation, the male withdraws his genitalia and dismounts the female.
This stereotypic pattern of courtship represents an innate behavior that is primarily under genetic control, although many of the genes controlling it remain to be identified (Yamamotoet al. 1998). To identify these genes, we employed a forward genetic approach in which mutants of a specific phenotype are isolated to track down the genes responsible for the alteration in courtship behavior. Accordingly, we screened ∼2000 P-element insertion lines and isolated eight mutants, each of which showed unique anomalies in mating behavior (Yokokuraet al. 1995; Suzukiet al. 1997; Babaet al. 1999; Nakanoet al. 2001). lingerer (ligP) is one such mutant. The ligP male flies start copulation normally, but often fail to withdraw their genitalia smoothly at the end of copulation. As a result, the male and female tug at each other, pulling in opposite directions for several seconds to tens of minutes.
Another mutant with a phenotype similar to that of ligP is stuck (sk). sk was isolated by Hall et al. (1980), who reported that the mutation was polygenic in nature with a genetic factor on the fourth chromosome contributing significantly to the sk phenotype. Although we also found some influence of the genetic background on the lig mutant phenotype, mutations in the lig locus resulted in an unambiguous sk-like phenotype, suggesting a major role for this gene in genital control. Furthermore, when the lig gene is severely disrupted, copulation is almost completely blocked although the mutant males continue to repeatedly attempt copulation. The relatively clear-cut behavioral phenotypes found in lig mutants enabled us to clone the lig gene.
Here, we report that three forms of mRNA, type 1, type 2, and type 3, are transcribed from the lig locus, and expression of only the type 1 transcript is disrupted in the ligP mutant because of P-element insertion. The lig gene encodes a set of novel cytoplasmic proteins, which are conserved between flies and mammals. Rescue experiments using the type 1 cDNA driven by a heat-shock promoter revealed that the lig function is required only from the late third instar stage to pupation for normal copulation to occur in adults. During the late third instar larval stage, the lig gene is expressed in the central nervous system (CNS) as well as in the imaginal discs. These results suggest a role for this gene in the development of the machinery controlling genital movement.
MATERIALS AND METHODS
Fly stocks: ligP is a homozygously viable P-element insertion line, obtained from a screen for alterations in mating behavior done in our laboratory. Homozygous ligP males are fertile, whereas ligP females are less fertile. Molecular analyses revealed that ligP is a hypomorphic mutation.
The revertant ligR4 and ligR11 were isolated from fly lines in which the P element at 44A was remobilized by introducing the P(ry+Δ2-3) chromosome into the ligP lines. Precise excision of the P element in the revertant lines was confirmed by genomic Southern hybridization and PCR followed by sequencing. ligR4 was used for further analyses.
Deficiency lines lacking the lig locus [Df(2R)ligX4, Df(2R)ligX13, and Df(2R)ligX18] were obtained by X-ray irradiation of ligP/ SM1 males. All three lines were lethal when homozygous and semilethal when placed in trans to ligP. We confirmed that the lig transcription unit was deleted in these lines by genomic Southern blot and PCR analyses. In the text, Df(2R)ligX4, Df(2R)ligX13, and Df(2R)ligX18 are referred to as X4, X13, and X18, respectively.
The lig alleles with double P insertions were generated by the local transposition (Toweret al. 1993; Zhang and Spradling 1993). From the progeny of the males bearing both ligP and P(ry+Δ2-3), the flies with darker red eyes than those of the original ligP males were collected and balanced. Among them, five lines (ligPP1, ligPP2, ligPP3, ligPP4, and ligPP5) were semilethal when placed in trans to ligP. All of the five alleles showed homozygous pupal lethality forming long puparia and failed to complement pupal lethality of each other. The pupal lethality was observed with equal severity for ligPP1-5 homozygotes and ligPP1-5/X4, indicating that ligPP1-5 are strong loss-of-function or null alleles of this gene. We confirmed that the lig product was undetectable in ligPP1 larvae by Western blot analysis (Figure 6B). Plasmid rescue followed by sequencing demonstrated that these five lines retained the ligP original insertion plus a new independent insert arising from local hopping (see Figure 4). The second P element was inserted in the opposite direction at 878, 456, 503, 507, and 885 bp downstream of the original insertion in ligPP1, ligPP2, ligPP3, ligPP4, and ligPP5, respectively. The ligPP1 was used for further analyses.
The lig mutant alleles used in this study had been outcrossed to a w1118 strain, which had a Canton-Special (CS) wild-type genetic background. The mutant phenotypes were maintained in the CS genetic background.
Behavior assays: To observe mating behavior, virgin males were collected at eclosion and placed individually in food vials for 3-5 days. Each male fly was transferred to a circular mating chamber (0.9-cm diameter, 0.5-cm height) with a wild-type virgin female. The behavior of the fly pairs was recorded using a video recorder. If the flies did not start copulation within the first 25 min, they were discarded. The copulating pairs were carefully observed to check whether the males dismounted normally or not upon termination of copulation. When the male released the female genitalia after, and not before, dismounting, it was considered as a stuck phenotype, irrespective of the time spent to become separated. Mating success was estimated as the percentage of pairs that copulated within the 1-hr or 25-min observation period (the observation period in each experiment is described in the relevant figure legend). The sex appeal parameter index (SAPI) was defined as the proportion of time the male exhibited unilateral wing vibration during a 5-min observation period or during the interval before copulation in the case of males that started to copulate within 5 min.
To examine adult locomotor activity, a male fly was transferred to the mating chamber, and its behavior was recorded by a video recorder for 10 min. The locomotor activity was determined as the number of times the fly crossed a straight line on the floor during a 10-min observation period.
Morphological analysis of male genitalia: For scanning electron microscopy (SEM), the flies were prepared for critical point drying and coated with a 2-nm layer of gold. Images were taken on a low-voltage prototype SEM. For genital cuticle preparation, the male adult terminalia were dissected in PBS, boiled in 10% NaOH for 10 min, and washed in water four times.
Isolation of lig genomic DNA and cDNA: Genomic DNA flanking the P-element insertion site was isolated by plasmid rescue and used for screening of the λEMBL3 CS genomic library (CLONTECH, Palo Alto, CA). Drosophila adult and pupal cDNA libraries were constructed with oligo(dT)-primed cDNA and λgt10 vector using the TimeSaver cDNA synthesis kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). Using genomic fragments as probes, the cDNA libraries were screened, and several cDNA clones containing large inserts were isolated. For cloning of mouse homologs, PCR primers were designed according to the expressed sequence tag (EST) sequences, and PCR was performed using phage DNA prepared from the lysates of a mouse embryonic cDNA library (CLONTECH) as a template. The amplified fragments were sequenced and used as probes for screening of the mouse embryonic cDNA library. Nucleotide sequences were determined using a 377 DNA sequencer (Perkin-Elmer, Norwalk, CT).
Northern hybridization and RT-PCR: Total RNA was isolated using TRIzol (Life Technologies, Rockville, MD), and poly(A)+ RNA was prepared by affinity chromatography using oligo(dT)-cellulose Type 7 (Amersham Pharmacia Biotech). Poly(A)+ RNA (0.5 or 2 μg) was separated on a 1% agarose gel containing formaldehyde. Following transfer to Biodyn-Plus (Pall, Port Washington, NY), the filters were hybridized with digoxigenin (DIG)-labeled probes, and signals were detected using a DIG luminescent detection kit (Boehringer Mannheim, Indianapolis). DIG-labeled antisense single-strand DNA probes, L1 and L2, were prepared by PCR (Lehmann and Tautz 1994) using primers as follows:
L1 sense primer, 5′-ATGCTCGTCAAGGAGCAACAG-3′;
L1 antisense primer, 5′-TGCGGATGTTGCTGTATGTTTC-3′;
L2 sense primer, 5′-TAGGCGGAGGGACGGAAAGT-3′;
L2 antisense primer, 5′-AATCACAGTCAACAAGTGTACAA-3′.
As a control for the amount of RNA loaded in each lane, the blots were rehybridized with a Drosophila melanogaster ras2 gene probe (Bishop and Corces 1988), which was provided by V. G. Corces.
For RT-PCR experiments, oligo(dT)-primed first-strand cDNA was synthesized from 0.5 μg of poly(A)+ RNA using a RNA PCR kit (AMV), version 2.1 (Takara, Kyoto, Japan). To prepare Lig-A-specific first-strand cDNA, reverse transcription was performed on 0.6 μg of poly(A)+ RNA with a primer complementary to the sequence in the 3′-untranslated region of type 1 cDNA (Lig-A-3′; 5′-GCGAAGGCTATAAAAGTC GTTC-3′) at 65° for 1 hr using BcaBEST RNA PCR kit, version 1.1 (Takara). PCR was performed with ExTaq DNA polymerase (Takara) and the following primers:
Antibody production and Western blot analysis: A BamHI/ NotI fragment of the lig cDNA (amino acids 82-225; Lig-N) was cloned into the pET-32b(+) vector (Novagen, Madison, WI). Using this vector, the Lig-N fragment was expressed into the periplasmic space of Escherichia coli as a fusion protein with thioredoxin. The fusion protein was extracted by the osmotic shock method according to the manufacturer’s instructions and purified by affinity chromatography using His-Bind Resin (Novagen) immobilizing Ni2+. A synthetic peptide, Lig-A-C, corresponding to the C-terminal 19-amino-acid peptide of Lig-A protein (CQSKSAGKQGYSPSYWAGQN), was conjugated with bovine serum albumin (BSA) using a cross-linking reagent, N-hydroxysuccinimidyl 3-(2-pyridyldithio) propionate (Carlssonet al. 1978), and the BSA-Lig-A-C conjugate was purified by dialysis. Three female BALB/c mice were immunized for each antigen using standard methods (Harlow and Lane 1988). Antibodies in plasma were prepared as follows. Ten to 14 days after injection, the immunized mice were bled from the tail vein. The blood was collected in heparinized glass capillary tubes and centrifuged to remove cells. The titers of antibodies were checked using the dot-immuno-binding assay (Hawkeset al. 1982).
For Western blotting, flies or larvae were homogenized in lysis buffer [50 mm Tris-HCl (pH 6.8), 2% SDS]. After 10 min boiling in the presence of 0.1 m dithiothreitol, the lysate was applied to 7.5% polyacrylamide gels and then transferred to an Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA). The blots were incubated with anti-Lig-A-C (1:10000) and visualized using an alkaline phosphatase-conjugated secondary antibody (Bio-Rad, Hercules, CA).
P-element transformation and rescue experiment: The following transgenic vectors were made and injected into w1118 embryos together with a phsπ helper plasmid (Steller and Pirrotta 1986) to establish transgenic lines (Rubin and Spradling 1982):
pCaSpeR4-Glig: The 12-kb lig genomic fragment (Glig), which included the whole transcribed region of all types of lig transcripts as well as 0.3 and 1 kb of flanking sequences at the 5′ and 3′ ends, respectively (see Figure 4), was isolated using PCR technique and ligated into the EcoRI/XhoI sites of the pCaSpeR4 transformation vector.
pCaSpeR-hs-lig1: The hs-lig1 fusion gene, in which the expression of type 1 lig cDNA was controlled by the hsp70 heat-shock promoter, was constructed by subcloning the full-length type 1 lig cDNA into the EcoRI site of the pCaSpeR-hs vector in the same direction to the hsp70 promoter.
For rescue experiments of stuck phenotype with hs-lig1, two independently generated transgenic lines, hs-lig1(#4) and hs-lig1(#5), were used. The hs-lig1(#4) line showed leaky expression of the Lig-A protein at 25° (Figure 6B). The hs-lig1(#5) line showed little leaky expression under similar conditions (Figure 6B) and therefore was used as a negative control. Fly lines carrying both the ligP mutation and the hs-lig1 transgene were reared at 25° and subjected to behavioral assays.
For rescue experiments of noncopulating phenotype using hs-lig1, fly lines carrying both the ligP mutation and the hs-lig1 transgene were crossed with the X4/SM1 line. The progeny from the third instar larval stage to the pupal stage were exposed to a single heat-shock treatment at 37° for 1 hr. After eclosion, mutant hemizygous male flies carrying the hs-lig1 transgene were collected and subjected to behavioral assays.
In situ hybridization: In situ hybridization in whole-mount tissues and embryos of Drosophila was performed according to the protocol laid out by Lehmann and Tautz (1994) with some modifications (Juniet al. 1996). The larvae at the late third instar larval stage were cut into anterior and posterior halves and turned inside out in Drosophila Ringer’s solution [183 mm KCl, 46 mm NaCl, 3 mm CaCl2, 10 mm Tris-HCl (pH 7.2), 1% BSA] using a fine metal needle. The dissected larvae were fixed with 4% paraformaldehyde in PBS for 15-30 min on ice followed by incubation with 4% paraformaldehyde and 0.5% Triton X-100 in PBS for 15 min at room temperature. All incubations and washes were performed at room temperature. Embryos were dechorionated, fixed, and devitellinized according to the method described by Lehmann and Tautz (1994). After three 5-min washes in PBS/0.1% Tween 20, the fixed larvae and embryos were digested using proteinase K (12.5 μg/ml in PBS/0.1% Tween 20) for 5-15 min. The digestion was stopped by incubation in PBS/0.1% Tween 20 and 2 mg/ml of glycine for 10 min. After three 5-min washes in PBS/0.1% Tween 20, the larvae and embryos were refixed for 20 min with 4% formaldehyde and 0.1% glutaraldehyde in PBS. Thereafter, following five 5-min washes in PBS/0.1% Tween 20, the larvae and embryos were hybridized with DIG-labeled single-strand DNA probes at 48°, and signals were detected using an alkaline-phosphatase-conjugated anti-DIG antibody (Boehringer Mannheim) using the method described by Lehmann and Tautz (1994). Sense or antisense DIG-labeled single-strand DNA probes were prepared by PCR (Lehmann and Tautz 1994) using the following primers that hybridize to the region common to all types of the lig transcripts:
sense primer, 5′-ACAAAGTGCTTACCAGTCAAG-3′;
antisense primer, 5′-CCGCTGTTGCTGCTCACTC-3′.
In situ hybridization to polytene chromosomes was performed using DIG-labeled probes and the DIG DNA detection kit (Boehringer Mannheim), following the method of Engels et al. (1986).
Immunohistochemistry: For immunohistochemistry, the larval brains and discs were dissected in PBS and fixed with 4% formaldehyde in PBS for 1 hr at room temperature. After blocking with 2% normal goat serum in PBS containing 0.3% Triton X-100 for 1 hr at room temperature, samples were incubated with the mouse anti-Lig-N antibody (1:500) overnight at 4°. The signals were visualized using a biotinylated secondary antibody and horseradish peroxidase-conjugated ABC reagent (Vector, Burlingame, CA) with diaminobenzidine as the chromogen according to the manufacturer’s protocol.
Phenotypes of lig mutant: ligP is a hypomophic mutant induced by a P insertion at cytological location 44A on the second chromosome. Homozygous ligP males were completely viable and fertile and did not show any generalized behavioral abnormality, such as inactivity (see Figure 2B), sluggishness, or uncoordinated walking.
To compare mating behavior between mutant and wild-type males, a single male was placed with a single wild-type female in a small mating chamber. In the case of wild-type male and female pairs, 77% of the tested pairs started copulation within 25 min. Just before the termination of copulation, the female fly often kicked the legs of the mounting male with her hindlegs. The male fly then released the female’s genitalia and dismounted from the female (Figure 1A). In contrast, although the ligP homozygous male flies initiated copulation with wild-type females just as often as wild-type males did (mating success within 25 min = 68%), they were frequently unable to release the female’s genitalia while mounted on the female’s back. The mutant males tended to dismount the females without withdrawing their genitalia. As a result, the male and female often tugged at each other, pulling in opposite directions (Figure 1B). In many cases, the ligP male and wild-type female remained stuck for several seconds to tens of minutes after copulation, until finally managing to separate from each other. We call this the “stuck phenotype.” As shown in Figure 1C, 75% of the copulating ligP males exhibited the stuck phenotype, while only a small percentage of the mated wild-type males became stuck. The frequency of the stuck phenotype in the ligR4 line, which was obtained by precise excision of the P element in ligP, was close to that in the wild-type strain (Figure 1C), demonstrating that the stuck phenotype found in ligP resulted from the P-element insertion.
Interestingly, ligP females did not show any abnormality in courtship behavior. ligP females mated normally with wild-type males (mating success within 25 min = 65%, n = 63) although they showed a reduction in fertility (∼25% of the fertility of CS wild-type or ligR4 females).
The ligP mutation was semilethal in both sexes when placed in trans to chromosomes lacking the lig activity, such as null lig alleles (ligPP1, ligPP2, ligPP3, ligPP4, and ligPP5) or deficiency lines (X4, X13, and X18). Male escaper flies in ligP/ligPP1 or ligP/X4 were tested for mating behavior with the expectation that a lower dosage of the lig gene would result in more severe behavioral phenotypes. As shown in Figure 2A, only 26% of ligP/ligPP1 males started copulation, whereas ligR4/ligPP1 or ligP homozygous males mated at normal wild-type rates. Also, mating success in ligP/X4 was significantly decreased in comparison with that in ligR4/X4 flies (Figure 2A). Similarly, ligP/X13 and ligP/X18 males showed lower mating success rates (0%, n = 22 and 6%, n = 35, respectively) than those of ligR4/X13 (77%, n = 36) or ligR4/X18 (87%, n = 31) males. Thus, a further reduction in lig activity resulted in an additional defect in the ability of the male to initiate copulation, hereafter referred to as the “noncopulating phenotype.”
Although in the absence of females, ligP/ligPP1 or ligP/ X4 males showed low locomotor activity (∼35% of the activity of wild-type males; Figure 2B), they courted females as vigorously as wild-type males did; the SAPI (see materials and methods) estimated for the ligP/ X4 males (0.27 ± 0.04, n = 10) was comparable to that for wild-type males (0.30 ± 0.03, n = 10; Mann-Whitney U-test, P = 0.8). Thus, the noncopulating phenotype was unlikely to be a result of a reduced level of courtship activity.
We hypothesized that the lig mutants disrupted one step in the stereotyped sequence of courtship and mating behavior and that this disruption caused the non-copulating phenotype. To address this, we compared the proportion of wild-type and ligP/ligPP1 males who displayed respective steps of mating behavior (Figure 2C). Once they had started chasing (ch) a female, 90% of wild-type males reached the final step, copulation (co), after engaging in all the elementary behavioral steps, including tapping (t), singing (s), licking (l), and attempted copulation (a). The ligP/ligPP1 males were as active as the wild-type males in performing the early steps of mating behavior; practically all the flies performed chasing, tapping, singing, and licking. After these steps, 73% of ligP/ligPP1 males performed attempted copulation, which is defined as a contact of male genitalia with female genitalia. Nevertheless, only 18% of ligP/ ligPP1 males succeeded in copulation. This means that, among mutant males that attempted copulation, only 24% succeeded in copulation, while 100% of males could copulate after attempted copulation in the case of wild type. These results indicate that lig male flies with more severe mutant alleles are impaired in their ability to clasp female genitalia with their own genitalia.
The stuck and noncopulating phenotypes could be due to morphological abnormalities in the mutant male genitalia. To investigate this possibility, we compared the external genitalia of ligP and ligP/X4 males with those of wild-type and revertant (ligR4) males by SEM (Figure 3, A-D). We found that the morphology of the genitalia of mutant males was indistinguishable from that of wild-type or ligR4 males. We also examined the internal genitalia in cuticle preparations and found no obvious abnormalities in mutant males (Figure 3, E-G). These results suggest that both phenotypes do not appear to result from morphological defects in the male genitalia.
The most severe phenotypes were observed in the null ligPP1 allele. ligPP1 homozygotes die during the early pupal stage. Because the mutant larvae form puparia without contraction, they remain long and slender even after pupariation, unlike the barrel-shaped wild-type pupae (Figure 3, H and I). This phenotype is similar to that found in certain mutants of the ecdysone receptor (Benderet al. 1997) and ecdysone-inducible genes such as E74 (Fletcheret al. 1995). Histological analyses of late third instar larvae in the ligPP1 revealed that wing discs are flimsy and morphologically aberrant, as can be seen in Figure 3K (cf. Figure 3J for the wild type). On the other hand, no discernible abnormalities in cellular composition or structure were found in the ligPP1 larval CNS when stained with anti-Elav and anti-Repo antibodies (data not shown).
Cloning and molecular analyses of lig gene: The 34-kb genomic region adjacent to the P-element insertion site was cloned by plasmid rescue and subsequent chromosomal walking. The genomic fragments in this region were used as probes for screening adult and pupal cDNA libraries. A group of cDNA clones, which hybridized to each other, were isolated and analyzed in detail. The longest cDNA clone (4.6 kb) was used in Northern blot analysis. It recognized a 4.6-kb transcript whose abundance was decreased in the ligP mutant compared with wild-type flies (Figure 5A), suggesting that these cDNAs correspond to the lig gene. Sequence analysis of these cDNA clones revealed two types of alternatively spliced lig transcripts of a similar size, designated type 1 and type 2. By comparing the cDNA sequences with the genomic sequence, we determined the exon/intron boundaries and the genomic organization of the lig gene (Figure 4). Type 1 and type 2 mRNAs have different 5′ untranslated regions since they are transcribed from different transcription initiation sites. These transcripts also differ from each other in their 3′ regions including their open reading frames. Type 1 cDNA potentially encodes a novel protein composed of 1332 amino acids (designated Lig-A), and type 2 cDNA encodes a 1375-amino-acid protein (designated Lig-B) similar to the type 1 product, but having a unique C terminus (Figure 8A).
To examine the expression of Lig-A- or Lig-B-encoding transcripts separately, we performed Northern blot analysis using probes hybridized to the Lig-A- or Lig-B-specific exon (Figure 5, B and C). The Lig-A-encoding transcript was expressed abundantly in both sexes and decreased in amount in ligP homozygotes (Figure 5B). To confirm that the amount of the Lig-A protein is reduced in the mutant, a Lig-A-specific antibody (anti-Lig-A-C) that recognizes the C terminus of Lig-A was raised and used for Western blot analysis. As shown in Figure 6A, the amount of the Lig-A protein (150 kD) was significantly reduced in ligP homozygotes. In contrast, the amount of the Lig-B-encoding transcript, which was rare and specific to the male, was unchanged in the mutant compared with the wild type (Figure 5C). These results suggest that the decrease in the amount of the Lig-A, rather than in that of the Lig-B, protein was likely responsible for the ligP phenotypes. We therefore focused our analyses on the Lig-A protein.
Analysis of the genomic structure of the ligP mutant revealed that the P element was inserted into the first intron of the type 1 transcript, 1.5 kb upstream of the transcription initiation site of the type 2 transcript (Figure 4). It is conceivable that type 1 transcription is interrupted by the P-element insertion while type 2 transcription is unaffected. To examine if this was the case, we performed RT-PCR experiments in which the 5′ region of each transcript type was specifically amplified, using the primers shown in Figure 4. In RT-PCR using primers S-U1 and A-U, the product corresponding to the 5′ region of the type 1 transcript was found in the wild type, but not in the ligP mutant (Figure 7B, lanes 1-4). RT-PCR with S-U2 and A-U amplified the DNA fragment corresponding to the 5′ region of the type 2 transcript in both the wild type and the mutant (Figure 7C, lanes 1-4). In addition, when the S-U1 primer was used in combination with the antisense primer specific to the P-element sequence (A-P), a 0.3-kb product was amplified from ligP but not from the wild type (Figure 7A, lanes 1-4). The size of this PCR product coincided with that of the genomic region between the sequences complementary to the S-U1 and A-P primers in the ligP chromosome (Figure 7A, lane 6). It is likely that aberrant transcripts containing the first exon of the type 1 transcript and P-element sequence are produced from the transcription initiation site of the type 1 transcript. This hypothesis is supported by the results of Northern blot analysis using a probe specific to the pUC9 sequence contained in the P element (Bm-Δw); in this experiment, two aberrant transcripts, 7.5 and 4.2 kb in length, were detected in ligP homozygotes but not in the wild type (Figure 5D). These results collectively suggested that the P insertion between the first and second exons led to the generation of the aberrant fusion transcripts in place of the functional type 1 transcript.
In spite of the complete loss of the type 1 transcript, the Lig-A protein was detected in the ligP homozygote (Figure 6A). This suggests the existence of another type of Lig-A-encoding transcript, which should be transcribed from a different transcription initiation site. One candidate for such a transcription initiation site is found in the type 2 transcript. To investigate the 5′ region of Lig-A-encoding transcripts, Lig-A-specific first-strand cDNA was synthesized with a primer, Lig-A-3′, which anneals to the 3′ untranslated region of the Lig-A-encoding transcript (Figure 4), and was used for RT-PCR with the primers shown in Figure 4. Using primers S-D and A-D2, the 3′ region of the male-specific Lig-B-encoding transcript was amplified from oligo(dT)-primed cDNA (Figure 7E, lanes 1-4), but not from Lig-A-3′-primed cDNA (Figure 7E, lanes 7-10). On the other hand, the 3′ region of the Lig-A-encoding transcript was amplified from Lig-A-3′-primed cDNA (Figure 7D, lanes 7-10) as well as from oligo(dT)-primed cDNA (Figure 7D, lane 1-4). These results indicate that the Lig-A-3′-primed first-strand cDNA contains the Lig-A-encoding cDNA but not the Lig-B-encoding cDNA. The fragment corresponding to the 5′ region of the type 1 transcript was amplified from Lig-A-3′-primed cDNA in the wild type but not in the lig mutant (Figure 7B, lanes 7-10). Using primers S-U1 and A-P, the PCR product corresponding to the aberrant transcript in ligP was obtained from oligo(dT)-primed cDNA (Figure 7A, lanes 1-4) but not from Lig-A-3′-primed cDNA (Figure 7A, lanes 7-10). This result suggests that the aberrant transcript in the ligP mutant does not encode the Lig-A protein. Importantly, the 5′ region of the type 2 transcript was amplified with S-U2 and A-U from Lig-A-3′-primed cDNA in both the wild type and the mutant (Figure 7C, lanes 7-10). Therefore, the Lig-A-encoding mRNA must be transcribed from the transcription initiation site of the type 2 transcript. This type of transcript detected in the aforementioned RT-PCR experiment is designated as the type 3 transcript (Figure 4). Thus, the Lig-A protein can be produced by both type 1 and type 3 transcripts in the wild type. The P-element insertion in the ligP mutant inhibits expression of type 1 mRNA, leaving type 3 mRNA intact and reducing the level of the Lig-A protein.
The deduced amino acid sequences of Lig proteins do not contain any known protein motifs, nor any hydrophobic stretches such as a signalpeptide or a transmembrane domain. Our immunohistochemical analysis revealed cytoplasmic localization of the Lig proteins (see Figure 10, A-C). A homology search of the databases indicated that the Lig proteins exhibited no significant homology to any protein with known functions. However, human genes with unknown function, KIAA0144 and KIAA1491 (Nagase et al. 1995, 2000), and several partial sequences from the mouse, human, and rat EST databases were homologous to the lig gene. To determine the entire sequences of the mouse lig homologs (Mlig) found in EST databases, we cloned Mlig cDNAs from the mouse embryonic cDNA library. Sequence analysis revealed two kinds of Mlig genes, Mlig-1 and Mlig-2, and that the Mlig-2 gene produces two types of transcripts, Mlig-2a and Mlig-2b, by alternative splicing (Figure 8B). The proteins encoded by the Mlig-2a and Mlig-2b transcripts, designated MLig-2A and MLig-2B, respectively, differed from each other in the C-terminal portion. The human lig homologs KIAA1491 and KIAA0144 encode proteins homologous to MLig-1 (the protein encoded by Mlig-1) and MLig-2B, respectively. As was the case with Drosophila Lig (DLig), none of these proteins contained known protein motifs or significant hydrophobic stretches.
Comparisons of the amino acid sequences between mouse and Drosophila Lig proteins revealed five segments that are highly conserved (50-80% similarity) among them, i.e., conserved regions 1-5 (CR1-5; Figure 8, B and C). Interestingly, a putative protein kinase C (PKC) phosphorylation site (S/T-X-R/K) exists in CR1 (Figure 8C, arrowhead). Because of alternative splicing, MLig-2B lacked CR5, whereas MLig-2A possessed this region. Furthermore, two conserved sequences, T(G/S)LPYY and DY(S/T) KGGY, were found in the Lig proteins (Figure 8, B and C). The first sequence was flanked by CR4 at the N-terminal side in the Drosophila Lig proteins and at the C-terminal side in the mouse Lig proteins. The second sequence was located between CR4 and CR5 in DLig and Mlig-1, but was absent in MLig-2A and MLig-2B.
Rescue of lig phenotypes: To establish the causal relationship between the mutant phenotypes and the putative lig gene, we performed rescue experiments using a 12-kb Glig, which encompassed the entire putative lig transcription unit but not any other genes (Figure 4). The Glig transgene successfully rescued all the lig phenotypes described above: stuck phenotype in ligP male flies (Figure 1C), noncopulating phenotype in ligP/ligPP1 and ligP/X4 male flies (Figure 2A), low locomotor activity in ligP/ligPP1 and ligP/X4 adults (Figure 2B), and pupal lethality in ligPP1. Hence, we conclude that the transcription unit included in the Glig is the lig gene.
Both the Lig-A and the Lig-B proteins could be generated from the Glig fragment. Molecular analyses suggested that the Lig-A, rather than the Lig-B, was likely to be responsible for the lig phenotypes. To investigate the specific effect of rescue with Lig-A, we constructed the fusion gene (hs-lig1), in which the expression of type 1 lig cDNA that encodes the Lig-A protein was controlled by the hsp70 heat-shock promoter. Among six independent transgenic lines generated, two lines, hs-lig1(#4) and hs-lig1(#5), were characterized in detail and used for rescue experiments. In Western blot analysis with the anti-Lig-A-C antibody (Figure 6B), a significant amount of Lig-A protein was detected in the ligPP1; hs-lig1(#4) larvae raised at 25°. Since ligPP1 larvae expressed no detectable level of the protein, the Lig-A expression observed in ligPP1; hs-lig1(#4) was likely due to the leaky expression from the hs-lig1(#4) transgene at 25°. On the other hand, the Lig-A protein was undetectable in the ligPP1; hs-lig1(#5) larvae raised under similar conditions, suggesting that the hs-lig1(#5) transgene had little leaky expression of Lig-A at 25°. In an attempt at rescuing the stuck phenotype, the ligP males carrying the hs-lig1(#4) or hs-lig1(#5) transgene were reared at 25° and then tested for their performance in mating behavior. As shown in Figure 1C, the stuck phenotype was suppressed in ligP; hs-lig1(#4) males, in which the Lig-A protein was supplied from the hs-lig1(#4) transgene, while the nonleaky transgene, hs-lig1(#5), did not suppress the phenotype. Thus, the stuck phenotype in the ligP males was rescued by the expression of Lig-A.
To confirm that Lig-A could rescue the noncopulating phenotype as well, and to determine when the Lig-A protein is required to rescue this phenotype, expression of type 1 lig cDNA was induced in the ligP/X4 males at different stages of development using the hs-lig1(#5) transgene. As in the case of ligP/X4 males without the hs-lig1(#5), ligP/X4; hs-lig1(#5)/+ males reared at 25° rarely copulated (Figure 2D). When ligP/X4; hs-lig1(#5)/+ males were administered a single heat-shock treatment at 37° for 1 hr during the late third instar larval or 1-day pupal stage, they exhibited a higher mating success score (50 and 61%, respectively) than that of ligP/X4 males, while the same heat-shock treatment at earlier or later stages gave little rescue (Figure 2D). This rescue of the noncopulating phenotype is due to the hs-lig1(#5) transgene, because ligP/X4 males given the same heat-shock treatment exhibited no such increase in mating success. Similar rescue was observed when the hs-lig1(#4) line was used instead of hs-lig1(#5) (data not shown). Thus, the supply of type 1 lig mRNA in the late third instar larvae or early pupae is able to rescue the noncopulating phenotype, suggesting that the Lig-A protein is required during this developmental period for normal copulation at the adult stage.
Expression pattern of lig product: Developmental expression of the lig transcripts in wild-type animals was analyzed by Northern blotting using the full-length type 1 cDNA as a probe, which can detect all types of lig mRNA. As shown in Figure 9, a high level of lig was found throughout development.
To investigate spatial and temporal distribution of lig mRNA during embryonic development, we performed whole-mount in situ hybridization with a probe hybridizing to the sequence common to all types of transcripts. In early cleavage embryos, maternal lig mRNA was distributed uniformly (Figure 9A). Zygotic expression of lig started at the cellular blastoderm stage (Figure 9B). Shortly after gastrulation, lig mRNA was present in the germ band at relatively low levels (Figure 9, C and D); expression increased markedly from stage 9, at which neuroblasts delaminate from the ectoderm (Figure 9E). At stage 11, lig expression became restricted mainly to neuroblasts (Figure 9F). After germ-band shortening, expression was predominantly observed in the CNS, including the brain and the ventral nerve cord (Figure 9G), as well as in the peripheral nervous system (PNS; Figure 9H). During later stages of development, lig expression was detected not only in the nervous system (Figure 9I) but also in the gonads (Figure 9J). In the ligP homozygous embryos, lig expression was uniformly diminished with negligible changes in spatial and temporal patterns (Figure 9, K and L).
The results of the rescue experiment suggest that lig gene function is required in the late third instar larval to early pupal stage for normal copulation behavior of the adult male (Figure 2D). For a survey of tissues expressing the lig gene at this stage, whole-mount in situ hybridization was performed on “inside-out” preparations of late third instar larvae with the same probe as that used for embryos. The lig gene was expressed in the CNS (Figure 9M), gonads (ovaries and testes; data not shown), and imaginal discs, including genital (Figure 9N), eye-antennal, leg, wing, and haltere discs (data not shown). lig mRNA is expressed ubiquitously in these tissues.
The Lig protein localization was visualized by immunostaining of the late third instar larval CNS (Figure 10) with an anti-Lig antibody that recognizes the N terminus common to both Lig-A and Lig-B (anti-Lig-N; Figure 4). Many neuronal and glial cells in brain and ventral ganglion were immunoreactive to the anti-Lig-N antibody. In these cells, the staining was found predominantly in the cytoplasm, but not in the nuclei (Figure 10, A-C). The axons and dendrites appeared unstained. Also in the imaginal discs, the Lig protein was localized in the cytoplasm (data not shown). No staining was observed in the ligPP1 homozygous larvae (Figure 10, D-F), demonstrating that the antibody specifically detected the lig gene products. Similar results were obtained when the Lig-A-specific antibody (anti-Lig-A-C) was used (data not shown).
In the present work, we demonstrated that the reduction in gene activity at a single locus results in defects in adult sexual behavior; mutations in the lig locus interfere with initiation and termination of copulation.
Mutations that affect initiation of copulation have previously been reported. A classic example is fruitless1 (fru1), males of which court females vigorously and attempt to copulate without success. Hall (1994) considered that insufficient bending of the abdomen in fru1 males is a possible cause for the lack of success in copulation (see also Gailey and Hall 1989; Gaileyet al. 1991; Tayloret al. 1994). Since the fru gene encodes a putative transcription factor believed to play a role in the sex determination of certain neurons (Itoet al. 1996; Ryneret al. 1996; Heinrichset al. 1998), the inability of fru1 males to copulate is likely due to incorrect sex determination of these cells (Yamamotoet al. 1996; Villelaet al. 1997; cf. Ferveuret al. 1995). Males with more severe fru mutations do not exhibit attempted copulation with females (Yamamotoet al. 1998).
Another example of mutants with a noncopulating phenotype is dissatisfaction (dsf). The dsf mutant prevents males from bending their abdomens sufficiently to copulate (Finleyet al. 1997). dsf has been postulated to play a role in the neuronal sex determination mechanism, although dsf and fru appear to function in different branches of the cascade (Finleyet al. 1998; Yamamotoet al. 1998). The dsf mutant also affects female-specific behavior, i.e., oviposition. Both the oviposition defect in females and the noncopulating phenotype in males have been shown to result from aberrant innervation to muscles involved in the respective behavior (Finley et al. 1997, 1998).
Unlike fru1 and dsf mutants, the ligP/ligPP1 males bend their abdomen as much as wild-type males do and can make genital-genital contact with a female (Figure 2C). We suggest that the ligP/ligPP1 blocks a behavioral step subsequent to abdominal bending, e.g., clasping of female genitalia. This notion is consistent with the fact that males with a weaker allele (ligP) are able to copulate but show the stuck phenotype failing to release the female’s genitalia.
This latter phenotype has been reported in the sk mutant (Hallet al. 1980). To characterize the sk phenotype, Hall et al. (1980) measured changes in the strength of “genital coupling” over time after the initiation of copulation by counting the number of copulating pairs that released genital connection in response to vortex stimuli applied at various intervals. They found that, in the case of wild-type flies, a larger proportion of pairs tended to separate in response to the stimuli over a period of time after copulation initiation. In other words, the strength of genital coupling declined with time after the start of copulation in wild-type males. In contrast, a larger number of sk pairs tolerate vortex stimulation, maintaining genital connection following the initiation of copulation. Thus, in the case of the sk mutant, the strength of genital coupling increases toward the end of copulation. As a result, the sk males remain stuck at the end of copulation (Hallet al. 1980). This observation, together with the finding that the genital structure is normal, led Hall et al. (1980) to suggest that the sk mutation affects motor control of genital movements rather than the genital structure per se.
In our screening for mating behavior mutants, we isolated a few mutants whose behavioral anomalies seemed to be ascribed to aberrant male genital structures. One such example is the fickle mutant, males of which show shortened copulatory duration (Babaet al. 1999). This behavioral phenotype appeared to be associated with duplication of the apodeme, a structure that supports the penis. Unlike the case of fickle, we were unable to find any obvious deficits in the genital structure of the lig mutant males (Figure 3, A-G). In addition, lig gene products are expressed in neuronal and glial cells in the CNS (Figure 10, A-C), but not found in muscle cells and/or myoblasts, including those associated with genitalia. Taking these observations into account, we consider it likely that the lig mutations affected the neural mechanism controlling genital movements. However, the lig gene is expressed in imaginal discs, including the male genital disc (Figure 9N), and the ligPP1 mutation interferes with development of wing discs (Figure 3K). Therefore, we cannot exclude the possibility that the lack of lig activity generates a very subtle defect in the male genital structure, leading to the observed deficits in mating behavior.
The late third instar larval to the early pupal stage was the critical period for rescue by the expression of lig cDNA of the noncopulating phenotype in the lig hemizygotes (Figure 2D). This observation indicates that the failure of the hypomorphic lig mutants to copulate resulted from developmental defects during the late larval to early pupal period. This developmental stage coincided with the lethal phase of the null allele, ligPP1 (Figure 3I). Thus, the lig gene products are likely essential for normal development at this stage. lig expression at this stage is confined to the imaginal discs, the CNS, and the gonads, which undergo dynamic growth and/or reconstruction during the larval to pupal molt. The lig gene products may be required for processes associated with metamorphosis. In fact, the elongated shape of ligPP1 dead pupae was reminiscent of that reported for mutants in ecdysone-related genes (Fletcheret al. 1995; Benderet al. 1997). The distortion of the wing-disc structure observed in ligPP1 mature larvae also suggests a role for lig in metamorphosis. However, the gross structure of the brain-ventral ganglion complex in the late third instar larvae appeared normal (Figure 10D) although the ligPP1 CNS did not contain Lig proteins at a detectable level. No obvious cellular changes such as degeneration or ectopic proliferation were observed in the ligPP1 CNS. Fine anatomical analysis with single cell resolution is required to determine whether the lig mutations affect structural and/or functional aspects of the nervous system.
Our molecular analyses revealed that the lig locus generates two types of 150-kD proteins, Lig-A and Lig-B, each of which is encoded by type 1 and type 3 or type 2 transcripts, respectively. We further demonstrated that only the type 1 transcript is disrupted by the P-element insertion in ligP. The finding that ubiquitous expression of type 1 cDNA in ligP (Figure 1C) or ligP/X4 males (Figure 2D) rescued the copulatory abnormalities supports the assumption that a reduced level of the Lig-A protein in mutants is responsible for the behavioral phenotypes. However, we have not determined whether overexpression of the type 2 cDNA, which encodes the Lig-B protein, can compensate for the reduction in the amount of Lig-A protein in the lig mutants so as to restore normal copulatory behavior. If this happens to be the case, the Lig-A and Lig-B proteins would be considered to play similar roles.
By comparing the primary structures of the Lig and related proteins, five regions conserved across phyla were identified. None of these corresponded to protein domains with known functions. However, we found an amino acid sequence that matched the consensus sequence for phosphorylation by PKC in one conserved region. It would thus be of interest to examine whether the lig behavioral phenotypes can be modulated by mutations that change the activity of PKC.
We thank T. Matsuo for his help in generating transformant flies, M. B. Sokolowski and M. Suster for helpful comments on the manuscript, and G. V. Corces for the ras2 gene probe. We also thank S. Kondo, Y. Kai, and K. Shukuya for secretarial assistance. D.Y. was supported in part by Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of Japan and Waseda University grant no. 200B-029.
Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AF276963 (type 1 lig), AF276964 (type 2 lig), AF276965 (Mlig-1), AF276966 (Mlig-2a), and AF276967 (Mlig-2b).
Communicating editor: K. V. Anderson
- Received November 13, 2001.
- Accepted September 27, 2002.
- Copyright © 2002 by the Genetics Society of America