Molecular Genetic Dissection of the Sex-Specific and Vital Functions of the Drosophila melanogaster Sex Determination Gene fruitless

Corresponding author: Bruce S. Baker, Department of Biological Sciences, Stanford University, Stanford, CA 94305., bbaker{at}cmgm.stanford.edu (E-mail)

Communicating editor: R. S. HAWLEY

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A multibranched hierarchy of regulatory genes controls all aspects of somatic sexual development in Drosophila melanogaster. One branch of this hierarchy is headed by the fruitless (fru) gene and functions in the central nervous system, where it is necessary for male courtship behavior as well as the differentiation of a male-specific abdominal structure, the muscle of Lawrence (MOL). A preliminary investigation of several of the mutations described here showed that the fru gene also has a sex-nonspecific vital function. The fru gene produces a complex set of transcripts through the use of four promoters and alternative splicing. Only the primary transcripts produced from the most distal (P1) promoter are sex-specifically spliced under direction of the sex-determination hierarchy. We have analyzed eight new fru mutations, created by X-ray mutagenesis and P-element excision, to try to gain insight into the relationship of specific transcript classes to specific fru functions. Males that lack the P1-derived fru transcripts show a complete absence of sexual behavior, but no other defects besides the loss of the MOL. Both males and females that have reduced levels of transcripts from the P3 promoter develop into adults but frequently die after failing to eclose. Analysis of the morphology and behavior of adult escapers showed that P3-encoded functions are required for the proper differentiation and eversion of imaginal discs. Furthermore, the reduction in the size of the neuromuscular junctions on abdominal muscles in these animals suggests that one of fru's sex-nonspecific functions involves general aspects of neuronal differentiation. In mutants that lack all fru transcripts as well as a small number of adjacent genes, animals die at an early pupal stage, indicating that fru's function is required only during late development. Thus, fru functions both in the sex-determination regulatory hierarchy to control male sexual behavior through sex-specific transcripts and sex-nonspecifically to control the development of imaginal discs and motorneuronal synapses during adult development through sex-nonspecific transcript classes.

MULTIPLE promoters, alternative splicing, and complex sets of cis-acting sequences governing temporal and spatial patterns of gene expression are all employed to allow single genes to carry out multiple unrelated biological functions. The fruitless (fru) gene of Drosophila melanogaster is a particularly interesting gene that is representative of such multifunctional genes. fru has been shown to function as a member of the Drosophila sex determination regulatory hierarchy (RYNER et al. 1996 ; HEINRICHS et al. 1998 ), where it acts to build the potential for male sexual behaviors into the male central nervous system (ITO et al. 1996 ; RYNER et al. 1996 ; VILLELLA et al. 1997 ; GOODWIN et al. 2000 ; LEE et al. 2000 ; USUI-AOKI et al. 2000 ). In addition, fru function is essential for the viability of both sexes (RYNER et al. 1996 ).

The multiple functions of the fru gene are encoded by a complex set of sex-specific and sex-nonspecific transcripts that are generated by the use of four promoters (P1–4) and alternative splicing at both the 5' and 3' ends of the primary transcripts (ITO et al. 1996 ; RYNER et al. 1996 ; GOODWIN et al. 2000 ; USUI-AOKI et al. 2000 ; L. C. RYNER, S. F. GOODWIN, T. CARLO, M. FOSS, J. C. HALL, B. J. TAYLOR and B. S. BAKER, unpublished results). The transcripts from each of fru's promoters have long open reading frames that encode closely related proteins belonging to the BTB-Znf family (cf. ALBAGLI et al. 1995 ; HU et al. 1995 ). Transcripts generated from the distal (P1) fru promoter are spliced sex-specifically under the control of transformer (TRA) and transformer-2 (TRA-2) proteins in females, whereas in males a default splice occurs (RYNER et al. 1996 ; HEINRICHS et al. 1998 ). These sex-specifically spliced P1 transcripts are expressed in a small subset of neurons in the central nervous system (CNS; RYNER et al. 1996 ; LEE et al. 2000 ; USUI-AOKI et al. 2000 ) and are likely responsible for fru's role in male sexual behavior. In females, the splicing of the P1-derived transcripts gives rise to mRNAs with the potential to encode proteins with a BTB domain at their amino termini and one of three alternative Zn finger pairs at their carboxy termini (GOODWIN et al. 2000 ; L. C. RYNER, S. F. GOODWIN, T. CARLO, M. FOSS, J. C. HALL, B. J. TAYLOR and B. S. BAKER, unpublished results). In males, the default splicing of the P1 transcripts generates male-specific mRNAs, encoding proteins that differ from the proteins predicted to be produced from the female mRNAs by the addition of 101 amino acids N-terminal to the BTB domain. Strikingly, immunohistochemistry with anti-FRU antibodies shows that the mRNAs produced from the P1 fru promoter are not translated in females, whereas they are translated in males (LEE et al. 2000 ; USUI-AOKI et al. 2000 ). The transcripts from the three more proximal promoters (P2, P3, and P4) encode sex-nonspecific proteins that differ from the male-specific proteins by having just short stretches of amino acids preceding the BTB domain (L. C. RYNER, S. F. GOODWIN, T. CARLO, M. FOSS, J. C. HALL, B. J. TAYLOR and B. S. BAKER, unpublished results). The roles of these sex-nonspecific proteins are not known, but it has been suggested that one or more of them are responsible for fru's vital function (RYNER et al. 1996 ). Moreover, expression data show that one or more of these products are expressed widely in the nervous system as well as in some other tissues in both sexes (RYNER et al. 1996 ; LEE et al. 2000 ).

Most of the fru mutations or mutant combinations studied to date have been viable hypomorphs (ITO et al. 1996 ; RYNER et al. 1996 ; VILLELLA et al. 1997 ; GOODWIN et al. 2000 ). Nevertheless, these studies have revealed that wild-type fru function is required for most steps in male courtship behavior but has no detectable role in female courtship behavior. fru males typically display reduced levels of courtship to females, as measured by the amount of time that a male courts a prospective mate (HALL 1978 ; GAILEY and HALL 1989 ; GAILEY et al. 1991 ; RYNER et al. 1996 ; VILLELLA et al. 1997 ; GOODWIN et al. 2000 ). In addition, males of many fru genotypes are defective in specific parts of the courtship sequence (GAILEY et al. 1991 ; RYNER et al. 1996 ; VILLELLA et al. 1997 ; GOODWIN et al. 2000 ). For example, wild-type males choose females rather than males as the appropriate sexual partner, while fru males fail to distinguish between the sexes and as a consequence court females and males roughly equally (RYNER et al. 1996 ; VILLELLA et al. 1997 ; GOODWIN et al. 2000 ). Indeed, when fru males are housed together, male-male courtship leads to the formation of groups of courting males (HALL 1978 ; GAILEY and HALL 1989 ; GAILEY et al. 1991 ; ITO et al. 1996 ; RYNER et al. 1996 ; VILLELLA et al. 1997 ; GOODWIN et al. 2000 ). At a slightly later stage of courtship, when wild-type males extend a wing and vibrate it to produce a courtship song, certain fru mutants—for example, fru³, fru⁴, and fru^sat males—rarely extend a wing toward females and do not produce courtship pulse song during the short bouts of wing extension that they produce (RYNER et al. 1996 ; VILLELLA et al. 1997 ). Copulation is not attempted by males of most fru genotypes, rendering these males sterile (but see GAILEY et al. 1991 ; VILLELLA et al. 1997 ; GOODWIN et al. 2000 ). Finally, it was recently found by examining fru mutant combinations that do copulate that behaviors during mating, such as transfer of seminal fluids and sperm and the duration of mating, are also governed by fru (LEE et al. 2001 ). While the fru mutations characterized in these studies have strong effects on male courtship behaviors, the full phenotypic consequence of removing fru function has not been established.

To better understand fru's functions, we conducted mutageneses of existing fru P-element alleles with the hope of identifying loss-of-function fru alleles and alleles that removed subsets of fru's transcript classes. Eight new lesions were obtained in the fru locus. Our initial analysis of some of these mutants (RYNER et al. 1996 ) showed that fru's role in controlling male-specific behavior was more extensive than had been previously recognized and that fru had a vital function in both sexes (see also ITO et al. 1996 ). From analyzing male courtship behaviors in numerous mutant combinations, we show here that fru is involved in every step of the courtship ritual, thus extending the role of fru to encompass all aspects of male courtship. We analyzed in detail the anatomical, neuronal, and behavioral phenotypes associated with these new lethal fru lesions and combinations of these lesions with preexisting mutations and deficiencies of fru. Among the new anatomical phenotypes seen in lethal fru mutants were defects in the adult derivatives of the imaginal discs and a reduction in the terminal arborization of motor axons on adult abdominal muscles, suggesting that fru plays a role in the differentiation of the adult epidermis and CNS. In addition, we determined the effects of the new mutations on the expression of the different fru transcripts and correlated fru's sex-specific and sex-nonspecific functions with the expression of particular classes of transcripts. Our results establish that fru functions sex-specifically in the sex-determination regulatory hierarchy to control male sexual behavior and sex-nonspecifically to control the development of imaginal discs and motorneuronal synapses during adult development.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Generation of new fru mutations:
Fly strains and crosses: Flies were reared at 25° in 70% relative humidity on a standard cornmeal, yeast, molasses (or dextrose), and agar diet with proprionic acid and/or tegosept added to inhibit mold.

Previously outcrossed stocks of fru^1-4 (for details see GOODWIN et al. 2000 ) were balanced with TM3, Sb ry, MKRS, Sb ry, TM6C, Sb Tb, or TM6B, Hu Tb e balancer chromosomes (for details on these markers, see LINDSLEY and ZIMM 1992 ). The fru² allele was recently reextracted by crossing to a "Cantonized" white stock. The fru^sat and Df(3R)fru^sat15 alleles (ITO et al. 1996 ; kindly provided by D. Yamamoto) were maintained over TM6B and TM6C balancer chromosomes, respectively. Crosses with third chromosomal deficiencies that have break-points in, or close to, the fru locus, Df(3R)Cha^M5, Df(3R)Cha^M7, Df(3R)P14, Df(3R)BX5, Df(3R)gl^BX7, and Df(3R)148.5-1 (Table 1 and Fig 1A; GAILEY and HALL 1989 ; GAILEY et al. 1991 ), were used in conjunction with the newly generated fru mutations to analyze mutant phenotypes.

View larger version (25K): In this window In a new window Download PPT slide   Figure 1. Genetic and molecular map of the fru locus. (A) The cytological map of the 90–91 region (right arm of the third chromosome). For deficiencies, the deleted regions are indicated by thick solid lines (see Table 1 for details of the mutations). The locations of the chromosomal breaks in fruw9, fruw12, and fruw27 alleles are denoted by the arrow. (B) The molecular map of the fru locus. The insertion sites of the fru2, fru3, fru4, and frusat P elements and the breakpoints of the fruw9, fruw12, and fruw27 chromosomal aberration mutations are shown at a higher level of resolution. The ends of relevant deficiencies are shown, with the thick solid lines demarcating missing DNA and the dashed lines representing the restriction fragments to which the breaks were mapped. The positions of the four promoters (P1–P4) are mapped to their relevant restriction fragments of the fru genomic DNA (adapted from RYNER et al. 1996 ). Each alternative 3' end encodes a pair of Zn-f domains and is labeled A, B, or C according to its distal-to-proximal location in the gene. The P1 transcripts are sex-specifically spliced by the action of the TRA and TRA2 proteins interacting with 13-nucleotide (nt) repeats, resulting in sex-specific transcripts that produce proteins differing at their amino termini. By Northern analysis, three P1 transcript classes have been detected in male and female heads that correspond to usage of each of the alternative 3' ends (GOODWIN et al. 2000 ). The full transcript complexity is not known for transcripts from the other promoters and so the 3' ends for P2, P3, and P4 are not included. Note that the orientation of the molecular map is reversed with respect to the cytological map.

Mutageneses: To generate more severely mutant derivatives of the fru² allele, which is due to the insertion of a P element (p^p[wA]) in the fru locus, fru² homozygous males were X rayed (4000 R, 115 V, 5 mA, 1-mm Plexiglas filter, Torrex X-ray machine) and crossed to w; Dr Pr/TM3, Sb ry virgin females. From 62,000 progeny, 31 white-eyed flies that had lost the w⁺ marker of the P element in fru were obtained. A total of 16 lines were established by backcrossing three times to w; Dr Pr/TM3, Sb ry flies; lines were not established from the other 15 animals. These 16 lines were tested for sterility over the fru² parental chromosome. Seven new fru alleles, fru^w9, fru^w10, fru^w12, fru^w13, fru^w24, fru^w25, and fru^w27, were identified on the basis of the fact that these chromosomes were weakly fertile or sterile over fru² (Table 4). The remaining nine chromosomes were fertile over fru², cytologically normal in the fru region, and were not studied further.

Excisions of the fru⁴ P element (P[lacZ; ry⁺]; CASTRILLON et al. 1993 ) were generated using a standard transposase source (ROBERTSON et al. 1988 ). Of the 90 ry^- lines generated from the remobilization of the fru⁴ P element, Southern analysis showed that 37 were precise, or nearly precise, excisions of the P element, 23 were small deletions, and 3 were lethal. One of the lethal mutations, Df(3R)fru^4-40, which is lethal over Df(3R)Cha^M5 but not over Df(3R)P14 (Table 3), was used in this study.

Molecular characterization of the new fru alleles:
Determining the nature of their chromosomal rearrangements: To determine the type of lesion present in the new fru mutants, cytological analysis was done on salivary chromosomes from fru^-/+ third instar larvae.

Southern analyses were carried out on fru^w9/TM3, fru^w12/TM3, and fru^w27/TM3 adults to localize the position of these chromosomal breakpoints. We used labeled P^p[wA] DNA (HAZELRIGG et al. 1984 ; Flybase segment ID no. FBmc0000129) to probe Southern blots of restriction digests of genomic DNAs of the heterozygous fru mutants and control genomic DNAs obtained from flies of the parental stock used in the mutagenesis, fru²/TM3, and flies heterozygous for a complete deficiency of the region, Df(3R)fru^w24/TM3.

Df(3R)fru^4-40 was mapped using quantitative Southern blots of genomic DNA from mutant heterozygotes, wild-type control, homozygous fru⁴ control, and balancer control flies probed with a series of phage probes (f3A, f2A, f1H, f4B, and f5C) spanning the fru locus (see Fig 1A, RYNER et al. 1996 ). These experiments showed that Df(3R)fru^4-40 has one end in the fru⁴ P element and extends distally beyond the P1 promoter.

Df(3R)fru ^sat15 was roughly mapped by a combination of Southern blot analysis and genomic PCR using oligo primers derived from plasmid subclones from across fru. To determine the exact endpoints of the deficiency, oligonucleotides flanking the breakpoints were utilized with PCR to amplify a fragment containing the sequences flanking the breakpoints. Sequencing of this product and comparison to the genomic sequence for this region showed that the deletion begins 20 nucleotides into the inverted repeat of the fru^sat P element and deletes all nucleotides from 96,551 to 13,839 of ACC no. 003722, deleting the entire coding sequence of fru (data not shown). In addition, Df(3R)fru^sat15 completely deletes the four transcription units immediately proximal to fru as well as parts of two overlapping transcription units that are the fifth and sixth transcription units proximal to fru.

Transcript analysis by reverse transcription PCR: To determine which fru transcripts were produced by various fru alleles, the following genotypes were analyzed: Df(3R)fru^w24/T(3;het)fru^w12, Df(3R)fru^w24/In(3R)fru^w27, T(3;het)fru^w12/In(3R)fru^w27, Df(3R)fru^4-40/Df(3R)fru^sat15, and Df(3R)Cha^M5/ T(3;het)fru^w12. Since most of these genotypes die before adulthood, we collected 50 fru mutant animals prior to their lethal phase, which was 1- to 2-day-old pupae for Df(3R)fru^w24/T(3;het)fru^w12, Df(3R)fru^w24/In(3R)fru^w27, T(3;het)fru^w12//In(3R)fru^w27 and adult flies for Df(3R)fru^4-40/Df(3R)fru^sat15 and Df(3R)Cha^M5/T(3;het)fru^w12. Each of these genotypes was generated from crosses using stocks balanced with the TM6B, Hu Tb e balancer. Animals were quick frozen and stored at -80°. Total RNA was extracted using the TRIzol reagent (GIBCO-BRL, Grand Island, NY). Reverse transcription was performed on total RNA from an equivalent of 2.5 animals for a given genotype using a primer complementary to a sequence from a region common to all known fru transcripts [primer (fru-8-rev) 5' gtgagaccacgcacctgtgcag-3']. One-tenth of the reverse transcription reactions were used for the PCRs. Six different primer sets were used for the PCR to generate products diagnostic of the following: (A) transcripts containing the common coding portion of fru [primers 5'-aacactgaccaaggagcgatg-3' (fru-25) and 5'-atgggcagcgaactctggcc-3' (fru-26-rev)]; (B) transcripts from the P1 promoter spliced in the female pattern [first round primers 5'-ccagatcgaaagagaatatcatca-3' (fru-2) and 5'-cagcgcaagcagaattgctgg-3' (fru-7-rev); second round primers 5'-taattctaaccgaaagtaagcatag-3' (fru-32) and fru-7-rev]; (C) fru transcripts from the P1 promoter spliced in the male pattern [primers 5'-cttccgcccgcatcccctag-3' (fru-31) and fru-26-rev]; note that this primer pair also amplifies the female-specific product from this region; (D) transcripts initiated from the P2 promoter [primers: both rounds forward primer 5'-atcataaaatcgctcggttttagtt-3' (fru-29); first round reverse primer fru-26-rev; second round reverse primer 5'-catgaactcgagcagagatcgca-3' (fru-55-rev-Xho)]; (E) transcripts initiated from the P3 promoter [first round forward primer 5'-ctgagaacgtgcgcgagtgtt-3' (fru-11); second round forward primer 5'-caaagtgagtgagatacaatcgc-3' (fru-12); reverse primer used in both rounds fru-7-rev]; (F) transcripts initiated from the proximal-most fru promoter, P4 [primers 5'-ttacactaactattggctgctgg-3' (fru-40) and fru-26-rev; (G) an additional set of primers that amplify the male P1 product was used in the case of the Cha^M5/fru^w12 mutant combination: forward primer 5'-gcattacgcggccttggact-3' (fru-28) and reverse primer fru-26-rev. PCR products were size fractionated on agarose gels and analyzed on Southern blots to confirm that they were bona fide fru products. Note that each of the primer sets used amplifies a product that contains at least one splice junction so that products generated from contaminating genomic DNA, if any, are distinguishable from those generated from fru transcripts. The identity of the PCR products detected in these experiments was confirmed in all cases by probing these Southern blots with end-labeled oligonucleotide probes homologous to fru mRNA sequences expected to be amplified by each primer pair or with probes that span at least part of the expected fru products. These oligo probes used did not overlap the primer sequences used for amplification.

In situ hybridization to fru mutant animals: In situ hybridizations to fru mutant animals were carried out as described in GOODWIN et al. 2000 on 20-µm horizontal cryostat sections. Single-stranded riboprobes were synthesized from fragments of fru cDNAs subcloned into Bluescript pSK(+). The labeling reaction used T3 or T7 polymerase with digoxygenin-coupled nucleotides according to manufacturer's instructions (DIG RNA labeling kit; Boehringer Mannheim, Indianapolis). Yields were estimated by dot blot of serially diluted labeled probes by comparison with prelabeled RNA standards (Boehringer Mannheim). The P1 promoter probe (Probe S; RYNER et al. 1996 ) included nucleotides 160,236–159,918 of ACC no. AE003722, plus nucleotides 1–261 of ACC no. U27492. The common coding region probe (Probe C; RYNER et al. 1996 ) consisted of nucleotides 2785–3612 in ACC no. U72492.

Behavioral analysis of fru mutants:
Adult fru mutant males were tested for a variety of courtship and noncourtship behaviors. To test for sterility, males were collected at eclosion and stored either individually or as a group of 8–10 males per vial. After aging for 3–4 days, single males were placed with two to three wild-type virgin females in food vials and the presence or absence of the progeny was scored 7 days later (GAILEY and HALL 1989 ; GAILEY et al. 1991 ; VILLELLA et al. 1997 ). Vials in which the males had died were not counted.

Males to be tested for their courtship behavior were collected just after eclosion and aged individually for 6–10 days. The test male was placed in a small observation chamber with either another male of the same genotype or a virgin Canton-S female. The pair was then video recorded for 5–8 min (cf. VILLELLA et al. 1997 ). The percentage of time that a male spends courting [courtship index (CI)] another male (CI m m) or female fly (CI m f) during the observation period was determined by viewing the video tapes and logging the behaviors with a digital event recorder, leading to elementary computations of accumulated-time-logged/total-observation-time for a given playback (VILLELLA et al. 1997 ). When the male is paired with another male, the CI is calculated only for the first male to initiate courtship toward the other male. When the male is paired with a female, the CI represents the time that the male courts the female. The wing extension index (WEI; VILLELLA et al. 1997 ), which is the amount of time that the male extended his wing during the observation period, was also calculated. In addition, courtship song was simultaneously recorded onto the videotapes, and then a subset of the m f recording was analyzed for various song parameters using LifeSong (VILLELLA et al. 1997 ).

Observations of male courtship behavior in all-male groups were used to assess the degree that mutant males courted each other. Males were collected at eclosion and aged individually for 5–6 days. After grouping eight males of the same genotype together in a food vial for 3–4 days, an observation of multi-male courtship interactions for each genotype was made in the late afternoon or early evening. Animals were housed and observations were made under standard conditions of 25° and 70% humidity. A courtship chain was defined as an episode during which three or more of the eight males courted one another. The percentage of time that three or more males courted in such group interactions during a 10-min observation period was logged using a timer and computed as the chaining index (ChI; VILLELLA et al. 1997 ).

General activity measurements: To measure short-term locomotor activity, males were collected at eclosion and aged individually for 7–9 days (KULKARNI and HALL 1987 ). Single males were then placed in a small plexiglass chamber (1 cm diameter x 6 mm height) in which a filter paper with a single line dividing the chamber into two equal halves was placed at the bottom. After a 2- to 3-min accommodation period (which started just after introducing males into the chambers) the number of times each male crossed the center line in a 3-min observation period was counted with a hand-held counter. These observations were done in the late afternoon, usually 2–4 hr before the lights go off.

Statistics: CIs and ChIs were subjected to arcsine square root transformations (cf. VILLELLA et al. 1997 ) and then the studentized residuals were tested for normal distribution approximations (SOKAL and ROHLF 1995 ). Short-term activity and courtship song (interpulse intervals) data were not transformed. One-way ANOVAs, followed by subsequent planned pairwise comparisons, were performed on all behavioral and song data (see table legends for further statistical details).

Lethal phase and anatomical analysis of fru lethal mutants:
Viability and lethal phase determination: To determine the lethal phase for various fru mutant genotypes, pupae and larvae were collected from the appropriate cross, staged, and allowed to develop. For genotypes where animals reached pharate adult stages, the operculum was removed from pupae and they were allowed to emerge, or they were dissected out of the pupal case. The adults obtained were kept in small groups on agar plates supplemented with yeast. In some cases, their behavior was videotaped.

General anatomical and neuroanatomical analysis: fru mutant animals were subjected to several types of anatomical analysis to characterize morphological phenotypes. For external cuticle preparations, adult or late stage pharate adult animals were fixed in alcohol, macerated in hot 10% KOH, dehydrated through an alcohol series and xylene, and mounted between two coverslips in Permount (SZABAD 1978 ). To examine the motorneuronal innervation of abdominal and genital muscles, adult abdomens were dissected, fixed in 4% paraformaldehyde in PBS, and prepared for immunohistochemistry. Nerve terminals were labeled using an anti-synaptotagmin antibody (1:1000 in PBS, 0.1% Triton-X and 2–10% normal goat serum; LITTLETON et al. 1993 ) to label synapses by the protocol described in FINLEY et al. 1997 . Since all fru lethal genotypes survived past the third instar larval stage, we examined the division pattern of a set of sex-specific abdominal neuroblasts by 5' bromodeoxyuridine (BrDU; Sigma, St. Louis) incorporation. Dissected larval and early pupal CNSs were incubated in BrDU for 4 hr and then processed for immunohistochemistry according to techniques in TAYLOR and TRUMAN 1992 .

All immunohistochemical analyses used biotinylated secondary antibodies and ABC reagents (Vector kit; Vector Laboratories, Burlingame, CA). The color reaction used diaminobenzidine for visualization according to standard techniques (TAYLOR and TRUMAN 1992 ; TAYLOR and KNITTEL 1995 ).

Muscle of Lawrence analysis of fru mutants: Procedures used for the characterization of the muscle of Lawrence (MOL) are detailed elsewhere (GAILEY et al. 1991 ). In brief, dorsal abdominal cuticles were dissected out, fixed, and cleared in methyl salicylate. Musculature was then visualized by birefringence in polarized light. Individual MOL phenotypes were established by a ranking system outlined in VILLELLA et al. 1997 ; see also Table 8 legend.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genetic and molecular analysis of the new fru mutants
Isolation of new fru mutants: To generate new, more severe fru mutations, we mutagenized the fru² and fru⁴ mutations (GAILEY and HALL 1989 ; CASTRILLON et al. 1993 ). Both of these fru mutants are due to P-element inserts: the fru² P element is located between the 5' exons of the P3 and P4 promoters and fru⁴ is between the 5' exons of the P2 and P3 promoters (Fig 1B; ITO et al. 1996 ; RYNER et al. 1996 ; GOODWIN et al. 2000 ). The fru² P element was mutagenized by X rays and the fru⁴ P element was mobilized using standard techniques to generate imprecise excisions (see MATERIALS AND METHODS). From these mutageneses, we recovered eight fru mutations.

Cytological and molecular characterization of new fru mutants: Cytological examination of salivary chromosomes showed that six of the eight new fru^- mutations had visible aberrations (Table 1). The Df(3R)fru^w10, Df(3R)fru^w24, and Df(3R)fru^w25 alleles are deficiencies uncovering the 91B region of the third chromosome. In(3LR)fru^w12 is an inversion and T(3;Y)fru^w9 is a translocation, each with a chromosomal break at 91B, the location of fru. T(3R;het)fru^w12 is an inversion-cum-translocation also broken in 91B. Putting the findings that three of the fru²-derived mutations are inversions or translocations with one breakpoint at the cytological location of fru together with the fact that these rearrangements arose simultaneously with the inactivation of the wild-type white gene in the fru² P element suggests that these three rearrangements are broken in the fru² P element.

Southern analysis was used to examine whether these three rearrangements were broken in the fru² P element. These experiments showed that In(3LR)fru^w12 and In(3R)fru^w27 had lesions within a 4-kb HindIII fragment at the 5' end of the white⁺ gene in the P element. T(3;het)fru^w9 has a lesion located within a 3-kb BamHI fragment, partially overlapping the proximal end of the 4-kb HindIII fragment. These data are thus consistent with the idea that the fru breakpoints associated with these three rearrangements are within the fru² P element. Thus these three rearrangements should separate the P1-3 fru promoters, but not the P4 promoter, from the common fru coding sequences.

We also molecularly mapped the tiny deletions associated with the fru mutations Df(3R)fru^4-40 and Df(3R)fru^sat15. Df(3R)fru^4-40 was found to extend distally from within the fru⁴ P element for at least 70 kb (see MATERIALS AND METHODS). Df(3R)fru^sat15, which is derived from the excision of a P element inserted into the fru gene (ITO et al. 1996 ), extends centromere-proximal from within the fru^sat insert and removes the entire common fru coding region as well as several adjacent genes (Fig 1; see MATERIALS AND METHODS).

Transcript analysis of fru mutants: Since the new fru mutants described above are either deletions of portions of the fru locus or inversions or translocations that separate some fru promoters from fru coding sequences, these mutants are expected to lack various subsets of fru transcripts. They can thus be used to dissect the functions of various fru transcript classes. Rather than rely on results of the above cytological and molecular characterizations to infer which transcripts are produced by these mutant chromosomes, we used reverse transcription (RT)-PCR to directly examine the arrays of transcripts produced by various genotypes [In(3R)fru^w27/Df(3R)fru^w24, T(3R;het)fru^w12/ Df(3R)fru^w24, T(3R;het)fru^w12/ In(3R)fru^w27, Df(3R)fru^sat15/ Df(3R)fru^4-40], and Df(3R)Cha^M5/ T(3R;het)fru^w12 mutant animals and Df(3R)fru^w24/+ adults; the latter were used as a positive control]. RNA was extracted from these genotypes and reverse transcribed. Following the RT reactions aliquots of each reaction were amplified with various primer pairs (see MATERIALS AND METHODS) to determine which transcript classes were produced in each genotype.

We first determined whether any fru transcripts were produced in these genotypes by using primers that amplified a sequence from the protein-coding region common to all known transcript classes. This experiment detected fru transcripts in all five mutant genotypes (Fig 2A). These findings are as expected, since the region between P4 and the common coding region is normal on one of the chromosomes in each of these genotypes.

View larger version (64K): In this window In a new window Download PPT slide   Figure 2. RT-PCR analysis of fru mutants for the detection of sex-specific and sex-nonspecific transcripts. In A–G, lanes are as follows: lane 1, fruw27/fruw24; lane 2, fruw12/fruw24; lane 3, fru12/fruw27; lane 4, fruw24/+; lane 5, fur4-40/frusat15. Arrows point to the fru-specific bands. F, female-specific product; M, male-specific product. (A) The 317-nt RT-PCR product amplified from the fru common protein-coding region, which amplifies transcripts from all of the fru promoters (primers fru-25 and fru-26-rev). The slightly larger product detected is due to contamination of genomic DNA, which contains a 72-nt intron. Transcripts containing fru-coding sequences were detected in all fruw24/fruw27, fruw12/fruw24, fruw12/fruw27, and fru4-40/frusat15 mutants. Midstage pupae were used for fruw24/fruw27, fruw12/fruw24, and fruw12/fruw27 and adults were collected for the control. (B) The 305-nt RT-PCR product of female-specific P1 transcripts (primers fru-2 and fru-7-rev). Transcripts were detected in control fruw24/+ but no P1 transcripts were detected in any of the mutants. (C) The 1931- and 341-nt RT-PCR products amplified from P1 transcripts using primers (fru-31 and fru-26-rev) to amplify a sequence, which includes the male-specific splice to the common coding region. In the control fruw24/+ lane, both male-specific (lower band) and female-specific transcripts (upper band) are detected with these primers. No wild-type P1 transcripts were detected in any of the mutants. Bands in the fruw27/fruw24, fruw12/fruw27, and fru4-40/frusat15 lanes are not the expected size and are likely to be artifacts of the PCR. (D) The 379-nt RT-PCR product amplified from P2 transcripts (primers fru-29 and fru-55-rev-Xho). P2 transcripts were only detected in the control fruw24/+ lane. A faint band was present in the frusat15/fru4-40 lane but is smaller than the expected size and so is likely an artifact. (E) The 255-nt RT-PCR product amplified from P3 transcripts (primers fru-12 and fru-7-rev). P3 transcripts were not detected in fruw12/fruw24 animals. (F) The 360-nt RT-PCR product amplified from P4 transcripts (primers fru-40 and fru-26-rev). The fruw24/fruw27, fruw12/fruw24, fruw12/fruw27, and fru4-40/frusat15 mutants make P4 fru transcripts. (G) The 438-nt male P1 and 304-nt female P1 RT-PCR products amplified from total RNA of ChaM5/W12 adult flies (male primers fru-28 and fru-26-rev, female primers fru-2 and fru-7-rev). An 230-nt product is also detected with the female primers and may represent use of a 5' splice site 73 nt upstream of the female-specific 5' splice site. Note this product is detected in both males and females.

Whether the sex-specifically spliced P1 transcripts were produced in these mutants was tested with two sets of primers. One pair amplified the female product (Fig 2B) and another pair amplified both the male and female-specific products (Fig 2C), since the sequence of the P1 transcripts in males overlaps that in females. These primers amplified the expected products from control animals (Fig 2B and Fig C). No P1 transcripts were detected from In(3R)fru^w27/ Df(3R)fru^w24, Df(3R)fru^w24/ T(3R;het)fru^w12, T(3R;het)fru^w12/ In(3R)fru^w27, and Df(3R)fru^4-40/ Df(3R)fru^sat15 animals (Fig 2B and Fig C; data not shown). These findings are consistent with expectations since In(3R)fru^w27 and T(3R;het)fru^w12 are broken between P1 and the common coding sequences, Df(3R)fru^sat15 deletes the common coding sequences, Df(3R)fru^4-40 deletes the P1 promoter, and Df(3R)fru^w24 deletes the entire fru locus. The absence of P1 transcripts in these mutant combinations was confirmed by carrying out two rounds of nested PCR with primers that amplified the female product (data not shown).

Surprisingly, P1 transcripts were detected in the Df(3R)Cha^M5/T(3R;het)fru^w12 mutant combination (Fig 2G) with both sets of primers. As shown in other mutant combinations, T(3R;het)fru^w12 does not produce P1 transcripts spliced to the common coding region and thus these transcripts must come from the Df(3R)Cha^M5 chromosome. Df(3R)Cha^M5 is a large deletion beginning between the P1 and P2 promoters and extending distally far beyond the P1 promoter. Thus the RT-PCR product detected cannot be a fru transcript initiated from the P1 promoter (Fig 1B). However, the end of the Df(3R)Cha^M5 deletion that is within fru is located in the same large genomic restriction fragment that contains the 5' primer sequences used for the RT-PCR detection of P1 transcripts (Fig 1B). The results showing that RT-PCR products were detected with both the male and female primers indicate that the sequences corresponding to these primers are not deleted. Note that the authenticity of all PCR products was confirmed by Southern analysis using probes that did not contain the primer sequences (see MATERIALS AND METHODS). Therefore the most likely explanation for the "P1" transcripts detected in Df(3R)Cha^M5/T(3;het)fru^w12 is that these transcripts are being produced by an ectopic promoter that was juxtaposed to fru by the Df(3R)Cha^M5 deletion. If this is the case, these P1 transcripts may not be being expressed in the cells in which the P1 fru promoter is normally active. Indeed, antibodies specific to the fru male-specific proteins expressed from the P1 promoter fail to detect these proteins in whole mounts of central nervous systems of Df(3R)Cha^M5/Df(3R)P14 at the pupal stages when these proteins are expressed in wild type (LEE et al. 2000 ).

In addition to the sex-specific P1 products detected in Df(3R)Cha^M5/T(3R;het)fru^w12, a sex-nonspecific product, slightly smaller than the wild-type product, is detected in both sexes with the female-specific primers. There is a 5' splice site consensus sequence upstream of the female 5' splice site that would give a product of this size if it were used. Sex-nonspecific products using this splice site have not been detected in wild type or in any of the other mutants and thus are specific to the Df(3R)Cha^M5 deficiency. There are two possible explanations for why this site might be used in Df(3R)Cha^M5-derived transcripts. Because this shorter product is produced in both sexes, it may be the result of ectopic promoter-driven expression in tissues where this cryptic splice site can be utilized. Alternatively, the Df(3R)Cha^M5 deletion may remove upstream sequences that affect the use of this splice site. The latter explanation seems less likely because the primary P1 transcript from Df(3R)Cha^M5 contains sequences (the male-specific PCR primer) upstream of the male 5' splice site, which is >1.6 kb away from the regulatory sequences that are known to affect the regulation of the female-specific splice site in wild type (HEINRICHS et al. 1998 ). These results indicate that a novel fru transcript is produced from the Df(3R)Cha^M5 deficiency chromosome; whether this transcript is expressed in the proper cells to supply fru function is unknown, but phenotypically there is no evidence for P1 function (see below).

On the basis of the location and nature of the fru rearrangements in these genotypes, transcripts initiated at the P2 promoter and spliced to the common coding region are not expected to be present except in the case of the Df(3R)Cha^M5/ T(3R;het)fru^w12 genotype. As predicted, no P2 transcripts were detected in any of the fru mutant genotypes tested except for Df(3R)Cha^M5/ T(3R;het)fru^w12 (Fig 2D, data not shown). In the latter genotype the Df(3R)Cha^M5 allele is the likely source of the P2 RT-PCR products since it does not delete the P2 promoter (Fig 1B); and the T(3;het)fru^w12 allele alone, as shown in the Df(3R)fru^w24/T(3R;het)fru^w12 lane (Fig 2D), does not produce a P2 product spliced to the common coding region.

With respect to the P3 promoter, the locations of the breakpoints in these fru alleles predict that only the Df(3R)Cha^M5 and Df(3R)fru^4-40 chromosomes should produce P3 transcripts spliced to the common coding region. These expectations are fulfilled in all but one case (Fig 2E). Unexpectedly, in all genotypes involving the In(3R)fru^w27 allele, RT-PCR revealed P3 transcripts. That these products are real is indicated by the facts that they are of the correct size and that they contain the expected fru mRNA sequences between the PCR primer pairs (see MATERIALS AND METHODS). These findings suggest that In(3R)fru^w27 is more complex than indicated by its cytological and molecular characterization. The In(3R)fru^w27 chromosome has one inversion with a break in fru and a second inversion that does not involve fru. Southern analysis indicated that there was a lesion within the white gene of the fru²-associated P element on which In(3R)fru^w27 was induced, and we had inferred that this was the breakpoint of the In(3R)fru^w27-associated inversion. However, the RT-PCR findings that P3 but not P2 transcripts are produced from the In(3R)fru^w27 chromosome suggest that there is a breakpoint in the region between the P2 and P3 promoters as shown in Fig 1B.

In the case of the P4 promoter, all the fru alleles analyzed, except Df(3R)fru^sat15 and Df(3R)fru^w24 (both of which delete P4), are expected to produce P4-derived transcripts spliced to the common coding region. The results (Fig 2F) are in accord with these expectations.

In summary, the above data show that different fru mutants are missing different subsets of fru transcripts (Table 2). As described above, our results are consistent with the idea that Df(3R)Cha^M5 affects only P1 function. Two mutations, Df(3R)fru^4-40 and In(3R)fru^w27, lack both P1- and P2-derived transcripts. One mutation, T(3;het)fru^w12, lacks P1-, P2-, and P3-derived transcripts, and we infer that T(3;Y)fru^w9 does also on the basis of the location of its breakpoint (see above). Finally, Df(3R)fru^sat15, Df(3R)fru^w24, and the previously characterized deletions Df(3R)P14 and Df(3R)Cha^M7 lack functional products from all fru promoters but also delete neighboring genes. By using different combinations of these fru alleles, we have begun to associate specific fru mutant phenotypes with the loss of different fru transcript classes.

Phenotypic analysis of new fru mutants
Origin of fru's vital function: To determine which fru promoters encode its vital function(s) we examined the viability of various heteroallelic fru mutant combinations that lacked particular subsets of fru transcripts (Table 3).

The finding that Df(3R)Cha^M5/ Df(3R)fru^sat15 individuals, which have little or no P1 function and express the products of the other fru promoters, have normal viability (Table 3B) indicates that the products of the P1 fru promoter are not needed for viability. This conclusion is also supported by the findings that Df(3R)Cha^M5 is also viable over Df(3R)P14 (GAILEY et al. 1991 ). Df(3R)P14 is like Df(3R)fru^sat15 in that it is a deletion that has one end in the middle of fru and extends proximally, deleting all fru coding sequences as well as neighboring genes.

The RT-PCR experiments described above suggest there are two aberrations, Df(3R)fru^4-40 and In(3R)fru^w27, which lack P1 and P2 function, but have P3 and P4 function. However, these two aberrations give completely discordant results when used in complementation tests with other fru alleles that lack fru's vital function. Thus Df(3R)fru^4-40 over Df(3R)fru^sat15 or Df(3R)P14 is fully viable (Table 3C), whereas In(3R)fru^w27 over Df(3R)fru^sat15, Df(3R)Cha^M7, Df(3R)fru^w24, T(3;het)fru^w12, or T(3;Y)fru^w9 is lethal (Table 3D). As explained in the DISCUSSION we believe that this disparity is due to In(3R)fru^w27 being defective in more than just fru's P1 and P2 functions and that the Df(3R)fru^4-40 results are reflective of the phenotype resulting from the loss of both P1 and P2 functions. This would indicate that P2-derived products, like those derived from P1, are not needed for viability.

With respect to P3, our analysis suggests that both T(3;het)fru^w12 and T(3;Y)fru^w9 do not produce P3-, P1-, or P2-derived products. Both T(3;het)fru^w12 and T(3;Y)fru^w9 are lethal over the null fru mutants Df(3R)fru^sat15, Df(3R)fru^w24, and Df(3R)Cha^M7, as well as each other (Table 3D). These results indicate that the products of P3 are essential for viability.

We do not have a genotype with which we can assess the viability effects of losing P4 but not P3 function. While we examined the viability effects of genotypes that are complete nulls for fru (Table 3E), and these are invariably lethal as expected, these deficiency combinations are also deleted for genes flanking fru. Nevertheless the phenotypic characterization of the lethal phenotypes in these deficiency combinations (see below) is useful as it places an upper boundary on the severity of the phenotype that can be expected from a null fru genotype.

Male sterility of the fru mutants: Previous studies established that males carrying many fru mutants and mutant combinations are sterile and that this sterility is behavioral in origin: Such males frequently fail to copulate (GAILEY and HALL 1989 ; GAILEY et al. 1991 ; VILLELLA et al. 1997 ). In addition, it was recently found by examining fru mutant combinations that do sometimes mate that fru function is also required during mating for successful insemination (LEE et al. 2001 ). To gain insight into which fru products are required for male fertility we assayed the effects of the new fru alleles on fertility.

That T(3;Y)fru^w9, T(3;het)fru^w12, Df(3R)fru^w24, In(3R)fru^w27, Df(3R)fru^sat15, and Df(3R)fru^4-40 all abolish male fertility is shown by the complete sterility of all combinations of these mutants tested with each other (Table 4D). Since all the genotypes in Table 4D, lack the functions encoded by the P1 and P2 fru promoters, these results indicate that some product(s) from these promoters are required for male fertility. To examine whether the products of the P1 promoter were required for male fertility we examined the effects of trans-heterozygotes between Df(3R)Cha^M5 and T(3;Y fru^w9, T(3;het)fru^w12, In(3R)fru^w27, Df(3R)fru^4-40, or Df(3R)fru^sat15. With one exception, all combinations were completely sterile (Table 4C). The exception was that in one round of tests 3/65 Df(3R)Cha^M5/In(3R)fru^w27 males were fertile, whereas several subsequent rounds of tests found 0/36 fertile. With the exception of these 3 males all the results in Table 4C and Table 4D, are consistent with the hypothesis that functional products of the P1 fru promoter are essential for male fertility.

We also examined the effects on male fertility of the new fru mutations in combination with the hypomorphic alleles fru¹, fru², fru³, and fru⁴ (Table 4E). The fru², fru³, and fru⁴ alleles are due to P-element insertions in fru and have reduced levels of either P1- and P2-derived transcripts (fru³ and fru⁴) or reduced levels of P1-, P2-, and P3-derived transcripts (fru²). P4 transcripts are not affected by these mutations (GOODWIN et al. 2000 ). fru¹ is an inversion that removes sequences upstream of the P1 promoter and results in an altered pattern of expression of P1 in the CNS (GOODWIN et al. 2000 ). The fru³ and fru⁴ alleles show more severe behavioral defects than fru¹ or fru² (VILLELLA et al. 1997 ). Consistent with the latter observation T(3;Y)fru^w9, T(3;het)fru^w12, Df(3R)fru^w24, In(3R)fru^w27, Df(3R)fru^4-40, and Df(3R)fru^sat15 are all completely sterile over either fru³ or fru⁴, whereas a few combinations of these alleles with either fru¹ or fru² show some male fertility. Two of the latter genotypes show results worthy of note: Most fru¹/Df(3R)fru^sat15 and fru²/Df(3R)fru^sat15 males are fertile. This is a surprising result, since males with either fru¹ or fru² over other null deletions of fru [e.g., Df(3R)fru^w24, Df(3R)Cha^M7, and Df(3R)P14] were invariably sterile (GAILEY and HALL 1989 ; VILLELLA et al. 1997 ). This may reflect genetic background effects.

As controls for the above experiments we examined the fertility of the new fru alleles when heterozygous with a wild-type chromosome and two deficiencies flanking fru (Table 4A). Two flanking deletions, gl^BX7, a glass allele associated with a 20-kb deletion centromere-proximal to fru, and Df(3R)148.5-1, a large deletion centromere-distal to fru, complemented the sterility phenotype of these new fru lesions, showing that there are no additional male-sterile mutations in the vicinity of fru on these chromosomes.

Male-male and male-female courtship performed by fru mutant males: To gain more insight into fru's male-specific function, we examined various components of male courtship behavior in a number of heteroallelic fru mutant combinations. One measure of overall courtship levels is given by the courtship index (CI), which indicates the amount of time a male fly spends courting (see MATERIALS AND METHODS). The wing extension index (WEI) provides a second measure of the courtship (see MATERIALS AND METHODS). Wild-type males court females vigorously, but court males at very low levels, as is seen from the CIs when T(3;Y)fru^w9, T(3;het)fru^w12, Df(3R)fru^w24, In(3R)fru^w27, or Df(3R)fru^4-40 are heterozygous with a wild-type chromosome (Table 5A). During courtship wild-type males perform courtship song for roughly one-half of the interval spent courting (WEI; Table 5A).

Previous analyses (RYNER et al. 1996 ; GOODWIN et al. 2000 ) had led us to suggest that it was the products of the P1 fru transcripts that carried out fru's role in male courtship. To assess the effects on courtship of just-impaired P1 function we examined the effects of T(3;Y)fru^w9, T(3;het)fru^w12, Df(3R)fru^sat15, and In(3R)fru^w27 when heterozygous with Df(3R)Cha^M5 (Table 5B). We also examined the effects of the absence of both P1- and P2-encoded functions on male courtship behavior in T(3;Y)fru^w9, T(3;het)fru^w12, In(3R)fru^w27, Df(3R)fru^sat15, and Df(3R)P14 when heterozygous with Df(3R)fru^4-40 (Table 5C). In all of these genotypes male-female and male-male courtship measured by either the CI or the WEI is almost completely abolished [unlike what is seen in less severe fru genotypes (see below)]. Taken together these results indicate that in the absence of fru's P1-encoded functions all aspects of male-male and male-female courtship are abolished.

We also examined male courtship behavior in flies in which the new fru alleles were heterozygous with one of the hypomorphic alleles, fru¹, fru², fru³, or fru⁴ (Table 5D). The new alleles over either fru³ or fru⁴ were nearly as severe in their effects as the above genotypes in which fru's P1 function was absent: Male-female courtship as measured by the CI or WEI was abolished as was male-male courtship as measured by WEI. However, in some genotypes, most notably Df(3R)fru^4-40/fru³ and Df(3R)fru^4-40/fru⁴, there was significant male-male courtship as measured by the CI. The effects of the new alleles over fru² were also severe: male-male courtship was essentially abolished as measured by either the CI or WEI, as was male-female courtship as measured by the WEI. In most genotypes involving fru², measurable male-female courtship was produced. Courtship behavior of males carrying one of the new fru alleles over fru¹ with either a male or female was substantial as measured by both CI and WEI. These males had CIs similar to those of fru¹ homozygote males (VILLELLA et al. 1997 ). Interestingly, these fru¹ heterozygous combinations are almost all sterile (Table 4B). In all fru¹ heterozygous combinations male-female courtship was less frequent than that seen with wild-type males (Table 5D vs. 5A); this may be partially due to these mutant individuals being in general less active than wild type (see below).

In summary, heteroallelic combinations of all of the new fru mutant alleles with either fru², fru³, fru⁴, Df(3R)fru^4-40, or Df(3R)Cha^M5 were not significantly different from wild-type males in their courtship of other males, showing that they no longer had the strong male-male courtship behavior associated with fru¹ or homozygosity for the P-element fru alleles (Table 5; VILLELLA et al. 1997 ). Thus these fru males are significantly different from wild-type males in that they do not court females and different from fru¹, fru³, and fru⁴ homozygotes since they do not court males and females. These findings also show that the reduction in wing extension first observed in fru³ and fru⁴ homozygotes and trans-heterozygotes (VILLELLA et al. 1997 ) is common to other severely affected fru genotypes as well.

Although most fru males that court respond to males and females roughly equally, several previously studied fru mutant genotypes showed a courtship bias toward either males or females. For example, fru³ males courted males more vigorously than females while fru² males courted females more avidly than males (GAILEY and HALL 1989 ; VILLELLA et al. 1997 ). We therefore asked whether these new fru mutants showed any consistent bias in the courtship of males vs. females. Males from twenty-four fru mutant genotypes courted males and females approximately equally (Table 5). Overall, the CIs of males of a particular fru genotype to male or female targets are highly correlated [CI_m-m compared to CI_m-f: F_{(29, 4586)} = 64.9, correlation coefficient = 0.83, R² = 69.1%; linear regression analysis, Statgraphics 5.0], suggesting that fru males are not discriminating between male and female partners independent of whatever level of courtship they generate.

Short-term activity of fru mutant males: One explanation for the failure of fru males to court is that these males might be generally inactive or sluggish (for review, see HALL 1994 ). To evaluate the general behavioral robustness of the new fru genotypes, a short-term activity assay was used to measure the voluntary activity a single male performs in the same chamber used for the courtship assays (VILLELLA et al. 1997 ). Tests of five genotypes that do not express P1 or P2 fru transcripts [Df(3R)fru^4-40 heterozygous with either T(3;Y)fru^w9, T(3;het)fru^w12, Df(3R)fru^sat15, In(3R)fru^w27, or Df(3R)P14] and four genotypes that have little or no functional P1 products [Df(3R)Cha^M5 heterozygous with either T(3;Y)fru^w9, T(3;het)fru^w12, Df(3R)fru^w27, or Df(3R)fru^sat15] showed that nearly all of these genotypes had reduced short-term activity compared to fru⁺ control males (Table 6B vs. A). However, two of these genotypes [Df(3R)fru^4-40/Df(3R)P14 and Df(3R)Cha^M5/In(3R)fru^w27] had wild-type activity levels, suggesting that the reduced activity seen in the other genotypes may be due to background effects rather than the result of reduced fru expression.

We also examined short-term activity in males heteroallelic for one of the new fru alleles and either fru¹, fru², fru³, or fru⁴. Heteroallelic combinations with fru³ or fru⁴ had, with one exception, wild-type levels of activity (Table 6C). However, all heteroallelic combinations of one of the new alleles with fru² showed greatly reduced short-term activity (Table 6C). Since all of the fru^w- alleles tested were induced on a fru² parental chromosome, the reduced activity seen in these genotypes likely represents the effects of this common genetic background. In this regard it is worth noting that In(3R)fru^w27/(T(3;het)fru^w12 escapers (this is an essentially lethal genotype; Table 3) also show low levels of activity.

Overall, almost one-half of the fru heteroallelic combinations tested were significantly less active than wild-type males (Table 6B and Table 6C). However, there is only a weak correlation between the level of short-term activity for these fru mutant males and the mean CIs they produce for either male-male or male-female courtship (Table 6 legend). These findings suggest that the failure of fru mutant males of these genotypes to court prospective mates in pairwise tests is predominantly a specific courtship defect rather than simply the result of these males being less active.

Male-male group courtship (chaining) by fru mutant males: fru mutant males exhibit a dramatic behavioral phenotype, the formation of male-male courtship chains (HALL 1978 ; GAILEY and HALL 1989 ; GAILEY et al. 1991 ; VILLELLA et al. 1997 ). Males homozygous for fru¹ court each other in groups, from a young-adult age onward (VILLELLA et al. 1997 ). For other genotypes, such as fru³ and fru⁴, significant chaining is seen only after the males have been grouped together for a few days (VILLELLA et al. 1997 ). A ChI, measuring the time that three or more males were courting during the observation period, was calculated to quantify the amount of intermale courtship produced by the different genotypes we examined (Table 5).

Males carrying one of the new fru alleles [T(3;Y)fru^w9, T(3;het)fru^w12, Df(3R)fru^w24, In(3R)fru^w27, and Df(3R)fru^4-40] over one of the hypomorphic fru alleles (fru¹, fru², fru³, and fru⁴) exhibited substantial chaining (Table 5D). Chaining was highest in fru¹ heteroallelic combinations, somewhat less in fru³ and fru⁴ heteroallelic combinations, and lower still in fru² heteroallelic combinations. This parallels the levels of chaining seen in homozygotes for these four hypomorphic alleles (VILLELLA et al. 1997 ). While the levels of chaining in fru¹ heteroallelic combinations are lower than that seen in fru¹ homozygotes [41–58% (Table 5D) vs. 70% (VILLELLA et al. 1997 )], in the cases of the fru², fru³, and fru⁴ heteroallelic combinations the levels of chaining are, in general, comparable to those previously reported for the respective homozygous mutants (VILLELLA et al. 1997 ).

Chaining was also examined in four heteroallelic fru combinations that have little or no functional P1 fru promoter-derived products (Table 5B) and six heteroallelic combinations that have no functional P1 or P2 fru promoter-derived products (Table 5C). The results were quite variable with four genotypes [T(3;het)fru^w12/Df(3R)Cha^M5, T(3;het)fru^w12/Df(3R)fru^4-40, T(3;het)fru^w12/In(3R)fru^w27, and Df(3R)fru^4-40/Df(3R)P14], showing no chaining, while the remaining five genotypes [T(3;Y)fru^w9/Df(3R)Cha^M5, In(3R)fru^w27/Df(3R)Cha^M5, T(3;Y)fru^w9/Df(3R)fru^4-40, In(3R)fru^w27/Df(3R)fru^4-40, and Df(3R)fru^4-40/Df(3R)fru^sat15] showed significant levels of chaining. We do not understand the bases for these differences between ostensibly equivalent fru genotypes. Three of the four genotypes that did not chain are less active than wild type (Table 6), but one of the fru mutants that did not chain, Df(3R)fru^4-40/Df(3R)P14, had high levels of short-term activity. On the other hand, some mutant genotypes with low mean short-term activity [In(3R)fru^w27/Df(3R)fru^4-40 and T(3;Y)fru^w9/fru²] still performed moderate levels of chaining (Table 5 and Table 6).

To determine whether there was a general relationship between the level of short-term activity and chaining behavior by fru mutant males, the correlation between the mean ChI vs. the mean short-term activity measurement was calculated for all genotypes (correlation coeffici