dissatisfaction, a gene involved in sex-specific behavior and neural development of Drosophila melanogaster

Abstract

Few mutations link well defined behaviors with individual neurons and the activity of specific genes. In Drosophila, recent evidence indicates the presence of a doublesex-independent pathway controlling sexual behavior and neuronal differentiation. We have identified a gene, dissatisfaction (dsf), that affects sex-specific courtship behaviors and neural differentiation in both sexes without an associated general behavioral debilitation. Male and female mutant animals exhibit abnormalities in courtship behaviors, suggesting a requirement for dsf in the brain. Virgin dsf females resist males during courtship and copulation and fail to lay mature eggs. dsf males actively court and attempt copulation with both mature males and females but are slow to copulate because of maladroit abdominal curling. Structural abnormalities in specific neurons indicate a role for dsf in the differentiation of sex-specific abdominal neurons. The egg-laying defect in females correlates with the absence of motor neuronal innervation on uterine muscles, and the reduced abdominal curling in males correlates with alteration in motor neuronal innervation of male ventral abdominal muscles. Epistasis experiments show that dsf acts in a tra-dependent and dsx-independent manner, placing dsf in the dsx-independent portion of the sex determination cascade.

Reproductive behavior in Drosophila can be divided into a series of stereotypical sex-specific activities, performed by males or females, which are released and modulated by specific environmental cues (see refs. 1 and 2 for reviews). Male courtship begins with orientation toward the female and continues through tapping, following, “singing” a species-specific song by vibrating an extended wing, licking, and copulation. Virgin female responses to male courtship include mild rejection of a courting male as well as receptive behaviors that facilitate copulation (3). Mated females respond to male courtship with the strong rejection response of ovipositor extrusion (3, 4).

Understanding the neural circuitry underlying sexual behavior has provoked intense scientific study in a wide variety of species (e.g., refs. 5–9). Pioneering studies using XX and XO tissue mosaics in Drosophila have identified regions of the nervous system that are involved in the generation of sex-specific behaviors (see refs. 2 and 10 for reviews). Male orientation, following, and wing extension all require a part of the dorsal posterior region of the brain to be male (XO; refs. 11 and 12), and courtship song requires additional male thoracic centers (13). Similar studies of female behavior indicate that a region of the anterior dorso-medial protocerebrum must be female for receptivity to male courtship (14). More recent studies using direct sexual transformations of small sets of cells also implicate the brain as a site controlling sex partner choice. Specifically, misexpression of the transformer gene (see below) in parts of the mushroom body or antennal lobe of the brain appears to cause males to engage in bisexual courtship behavior (7, 8).

By comparison, relatively little is known about the genes involved with specific control of sexual behavior. The primary determinant of sex in Drosophila is the ratio of X chromosomes to sets of autosomes (X:A ratio). Information about this ratio is passed through Sex-lethal (Sxl), transformer (tra), transformer-2 (tra2), and doublesex (dsx) to the genes involved in sexual differentiation (refs. 15–23; Fig. 1). In females (X:A = 1), this cascade leads to the expression of an active female form of dsx protein. In males (X:A = 0.5), the Sxl and tra proteins are not made, and an active male form of dsx protein is expressed, leading to male differentiation.

Figure 1.

Regulation of the sex determination cascade. The top line shows a schematic diagram of the regulatory relationships of the sex differentiation cascade. Solid lines represent definitively characterized interactions. Dotted lines represent possible additional relationships (2, 24, 25). Genetic data imply the existence of other genes that control behavior and neural development, which are under the control of tra and tra2 but not regulated by dsx (24, 26). One gene proposed as a candidate for such regulation is fruitless (fru; refs. 2, 24, and 25). The work in this paper suggests dissatisfaction (dsf) as a second candidate for a member of a dsx-independent pathway controlling behavior.

Although dsx is involved in the development of one set of neurons (27) and one aspect of the courtship song (28), recent genetic evidence indicates the existence of a regulatory pathway for the control of sex-specific neuronal development and behavior, which is dependent on tra and tra2 but independent of dsx (24, 26). The development of a pair of bilaterally symmetrical muscles of the dorsal abdomen, the Muscles of Lawrence (MOLs), depends on the sex of the neurons that innervate them (29, 30). tra or tra2 mutations that completely transform females into somatic males cause XX animals to have MOLs. However, loss or gain of function dsx mutations do not alter the sex specificity of MOLs in either XY or XX animals (26). Similar results are seen for sex-specific behaviors (24, 31).

Since dsx provides the primary, if not exclusive, genetic control of external sexual differentiation, we expect that mutations in dsx-independent genes controlling sex-specific neural development and behavior should have little or no effect on external differentiation, gametogenesis, or development of the reproductive tract but should affect aspects of courtship or fertility. A synthetic transformer mutation in which a female tra cDNA is expressed from a basal level heat shock promoter (hs-tra) was used as a model for this study (32). XX; tra⁻; hs-tra animals express dsx in its female form, have superficially normal gonads and internal genitalia, as well as normal female cuticular development. Late-stage eggs are produced and appear normal, and motile sperm are stored correctly (32). However, these animals are not fully tra⁺ due to poor nervous system expression from the basal heat shock promoter (J. Thomas, personal communication). As a result, these animals behave abnormally. XX; tra⁻; hs-tra females resist male courtship before and during copulation and exhibit active male-like courtship of both females and males. They have MOLs (unpublished observations). Finally, in spite of making apparently normal eggs and storing motile sperm, they never lay eggs (32). Using this array of phenotypes, we have identified a mutation in a gene that acts in a dsx-independent pathway.

MATERIALS AND METHODS

Drosophila were raised on standard cornmeal/molasses/yeast/agar media. Mutants and balancer chromosomes are listed in Lindsley and Zimm (33). Existing female sterile mutations, as homozygotes or as mutant/deficiency heterozygotes, were tested for male-by-male courtship, female-by-female courtship, and female resistance to male courtship, as judged by delayed copulation. Those mutations showing any of these phenotypes were subjected to further tests.

For the experiments shown in this paper, mating tests involved flies aged 4–10 days and were performed in circular mating chambers (2.4-cm diameter, 0.7-cm height). Wild-type in all assays was the Canton-Strain. P values relative to wild type were calculated using two-tailed t tests. Center crossing and 180° turns, as well as abdominal bending, were determined from video recordings of courting or mating pairs using 1.2-cm diameter × 0.6-cm height chambers. The center crossing assay involved counting the number of times the mating pair crossed a horizontal diameter in the videotaped image of the mating chamber. For tests of male-by-male courtship, males of appropriate genotypes were collected within 4 hr of eclosion and kept in vials of 10 flies for 4–10 days before testing, as in Gailey and Hall (34). Courtship indexes were determined from videotaped records of courtship bouts.

To label synapses and nerve terminals, the abdominal body wall and/or internal genitalia were dissected, fixed in 4% paraformaldehyde, and then processed for immunohistochemistry using appropriate biotinylated secondary antibodies and horseradish peroxidase-conjugated ABC reagents (VectaKit, Vector Laboratories) with diaminobenzidine as the chromagen, according to Taylor and Knittel (35). Four different primary antibodies that recognize synaptic antigens and/or nerve terminals were used, anti-cysteine string protein (at 1:1000, a gift from E. Buchner, Universitat Wuerzberg, Wuerzberg, Germany; ref. 36 and data not shown), anti-synaptotagmin (at 1:1000, a gift from H. Bellen & Baylor College of Medicine, Houston; ref. 37), anti-horseradish peroxidase (at 1:10,000, Cappel), and mab22C10 (at 1:100, a gift from S. Benzer, California Institute of Technology, Pasadena).

RESULTS

A number of female-sterile mutations that make but fail to lay mature eggs were examined for abnormalities in male and female sexual behavior (38–40). One mutation originally isolated by Schüpbach and Wieschaus (40) as RC32 and renamed dissatisfaction (dsf) was notable for its effects on both male and female courtship and mating behaviors, as well as female sterility. dsf maps to salivary chromosome region 26A, between the right hand end of Df(2L)Gpdh78 (also known as Df(2L)50078a) and the right hand end of Df(2L)cl7. All studies in this paper use dsf¹ (RC32). “Df” in the text indicates Df(2L)cl7. Similar phenotypes are observed using other deficiencies that remove dsf. Additional alleles (data not shown) show similar phenotypes. All phenotypes are observed with equal severity for both dsf/dsf and dsf/Df, indicating that dsf¹ is a strong loss-of-function or null allele of this gene. Close observation of dsf males and females in vials or mating chambers, either directly or from video images, failed to identify any generalized behavioral abnormalities, such as inactivity, sluggishness, or uncoordinated walking, flying, or grooming. Information presented in this paper demonstrating the avid resistance to courtship by dsf females and the normal courtship indexes of dsf males is an indication of general good health and freedom of movement.

dsf Females Retain Mature Eggs.

Homozygous dsf females fail to lay eggs voluntarily or under CO₂ anesthesia. Eggs mature normally in the ovary and pass through the oviducts to lodge within the uterus, where they degenerate. No fertilized eggs were detected among the eggs found lodged in the uterus, even though motile sperm are transferred to the female during copulation and are stored normally in sperm storage organs (n = 30). Thus, mature eggs are able to reach the uterus from the ovaries, suggesting that the failure to lay eggs results from defects within or distal to the uterus and not in the upper portions of the reproductive tract.

dsf Females Respond Aberrantly to Male Courtship.

To see if dsf affects other aspects of female reproductive behavior, we examined the response of dsf females to wild-type male courtship partners (Fig. 2A). In these assays, a single wild-type male was paired with a single female in a small mating chamber. The time from the addition of the second fly to the initiation of courtship was used as a measure of “attractiveness” of mutant females relative to wild-type controls. For all genotypes, tested courtship began, on average, within 2 min (in min ± SEM: +/+, 1.68 ± 0.29; dsf/+, 2.09 ± 0.42; Df/+, 1.58 ± 0.86; dsf/dsf, 0.94 ± 0.17; dsf/Df, 1.12 ± 0.16). The rapid initiation of courtship and the avidity with which wild-type males court dsf females indicate that dsf females are as attractive as wild-type females.

Figure 2.

dsf behavioral phenotypes. The genotypes examined are (genotype 1) wild type (+/+), (genotype 2) dsf/+, (genotype 3) Df/+, (genotype 4) dsf/dsf, and (genotype 5) dsf/Df. (A) dsf females resist male courtship. Individual aged virgin females of the genotypes shown were paired with wild-type (Canton-S) males, and the time from the initiation of singing to copulation was measured. +/+, n = 30; dsf/+, n = 14; Df/+, n = 9; dsf/dsf, n = 19; and dsf/Df, n = 18. Mutants are significantly different from wild type (dsf/dsf, P < 0.0001; dsf/Df, P < 0.0001). (B and C) dsf females resist during copulation, as judged by excess movement during copulation. (B) The number of times the mating pair made a 180° turn was determined from the videotapes. +/+, n = 15; dsf/dsf, n = 11, P = 0.0037; and dsf/Df, n = 14, P = 0.0004. (C) The number of times the mating pair crossed a predetermined center line of the chamber was scored from the same videotapes as in B. +/+, n = 15; dsf/dsf, n = 11, P = 0.0009; and dsf/Df, n = 14, P = 0.0072. (D) Three homozygous dsf males forming a short courtship chain, including a copulation attempt by the middle male. Both homozygous dsf and dsf/deletion males demonstrate this phenotype but with incomplete penetrance. dsf males spend a substantial amount of time orienting and following other males, but dsf chains contain few individuals and are not continued over extended periods of time, probably due to the robust rejection response exhibited by dsf males. dsf males court wild-type males, but are not themselves courted by wild-type males. (E) dsf males are delayed in copulation. Single males of the genotypes shown were paired with aged wild-type (Canton-S) virgin females. The time from initiation of courtship to copulation was measured. +/+, n = 30; dsf/+, n = 13; Df/+, n = 10; dsf/dsf, n = 13, P < 0.0001; and dsf/Df, n = 17, P = 0.0003. (F) Abdominal curling is altered in dsf males. Courtship events involving males of the genotypes shown, and wild-type (Canton-S) females were recorded on videotape. The magnitude of abdominal bending for each observable bend was determined from the videotaped images, with ≤45° being the minimal bend observable and 180° being the maximum magnitude bend observable. Bending at 180° is necessary to achieve the genital–genital contact associated with copulation. Intermediate bending categories (data not shown) have generally intermediate percentages of total bends.

The time from the initiation of courtship until copulation was used as a measure of female receptiveness to male courtship. Wild-type pairs mated in under 3 min, with kinetics that were not significantly different from that of dsf/+ or Df/+ and were similar to previously reported latencies for wild-type pairs (41). Both dsf/dsf and dsf/Df mutant females showed substantially longer and significantly different (P < 0.0001) times until copulation relative to wild type. Differences between dsf/dsf and dsf/Df are not significant. Close observation of courting pairs shows that the copulation delay results from active resistance, including running about the mating chamber, wing flicking, and kicking the male. These behaviors are reminiscent of rejection behaviors of wild-type virgin females but not of those of mated females (3, 4) and resemble the extreme rejection behaviors of XX; tra⁻; hs-tra females.

We also determined whether dsf females exhibit resistance during copulation. As a quantitative measure of resistance, we examined female movement during copulation, judged either by the number of times a mating pair crossed a predetermined center line of the mating chamber or the number of 180° turns a pair made. Wild-type females move very little while mutant females are significantly more active as judged by either measure (Fig. 2 B and C). Besides excess activity, dsf females try to dislodge copulating males by flicking their wings, bucking, and kicking at the males.

dsf Males Show Striking Courtship Abnormalities.

We examined courtship and mating behavior of dsf males to determine if components of their behavior are abnormal. Both dsf/dsf and dsf/Df males actively court mature males even with virgin females present. In tests involving male-only groups, courtship included all behaviors up to and including attempted copulation (see Fig. 2D) with the production of short-lived chains of courting males. dsf males court both mutant and wild-type males but are not themselves courted by wild-type males.

dsf males were also assayed for courtship of mature wild-type virgin females. Although dsf males initiate courtship as rapidly as wild type (in min ± SEM: +/+, 1.68 ± 0.29; dsf/+, 0.85 ± 0.16; Df/+, 0.47 ± 0.10; dsf/dsf, 2.25 ± 0.73; dsf/Df, 0.96 ± 0.22), actively court females (courtship index ± SEM: +/+, 0.88 ± 0.04; dsf/dsf, 0.89 ± 0.03; dsf/Df, 0.90 ± 0.03), and are fertile, single-pair tests demonstrate that dsf males are substantially delayed in time to copulation (6- to 8-fold increase; Fig. 2E). From video observation of courting pairs, we noted that target females respond normally to dsf courtship by stopping and positioning themselves for copulation (41–43), while the dsf males are defective in the final step of courtship, abdominal curling.

Males need to bend their abdomen about 180° to make genital–genital contact with females and copulate. From data collected from videotaped records (Fig. 2F), it is clear that dsf males make fewer bends falling into the maximum degree category (180°), which would be sufficient for copulation, and more shallow bends (<45°) than wild-type males during courtship of females. Since only ≈10% of the bends made by dsf males were in the range sufficient for copulation, this inability to produce deep bends likely accounts for the increase in time to copulation noted for dsf males.

dsf Females and Males Show Sex-Specific Neuronal Abnormalities.

The observation of failure to lay eggs in females and defective abdominal curling in males led us to concentrate our initial studies of neuronal changes in dsf on abdominal motor neurons and their targets. To detect a wide variety of axonal and/or nerve terminal structures, we used antibodies directed against four different neuronal antigens as noted in Materials and Methods. All antibodies gave similar results. These studies reveal specific abnormalities in a subset of motor endplates on muscles in males and females.

In dsf female abdomens, the full complement of segmental abdominal muscles and genital muscles is present, and there is no evidence of masculinization of any muscles. No muscles with male-like MOL morphology are found. Innervation of the segmental abdominal muscles and all but one set of genital muscles appeared normal. In the single exception, no synapses are detected on the circumferential muscles of the uterus of mutant females (Fig. 3 C and D), while these muscles in wild-type females are extensively innervated (Fig. 3 A and B). This absence of innervation was found with all four antibodies, indicating that synapses are not present as opposed to the loss of one individual neuronal antigen. The antibodies mab22c10 and anti-horseradish peroxidase also label the axons of both sensory and motor axons. Only sensory axons appear to contact the uterus in dsf females (data not shown), which suggests that the two to four motorneurons that innervate the uterus in wild-type females (B.J.T., unpublished results) do not reach the uterus in dsf females. Nearby visceral muscles associated with the spermathecae, sperm receptacle, and oviduct are innervated and appear normal. The lack of uterine muscle innervation is likely sufficient to account for the egg-laying deficit. Likewise, examination of XX; tra⁻; hs-tra females (Fig. 3 E and F) shows that there is a substantial reduction in the number of boutons present on the circular uterine muscles, consistent with the idea that XX; tra⁻; hs-tra and dsf mutants fail to lay eggs for a similar reason.

Figure 3.

Motor neuronal innervation of the female reproductive tract. Synaptic boutons have been stained with anti-synaptotagmin (37). All views are side views. U, uterus, SR, sperm receptacle. (A) Wild-type female internal genitalia (n = 11). Synaptic boutons are present on the muscles of the oviduct, sperm receptacle, spermathecae, and uterus and on other muscles associated with the external genitalia. (B) Higher power photomicrograph of wild-type uterine muscles and their innervation. (C) Internal genitalia from a dsf female. There are no boutons on the uterine muscles, but boutons are visible on the muscles of the oviduct, sperm receptacle, and spermathecae and on other muscles associated with the external genitalia. This phenotype is seen in both dsf/dsf (n = 16) and dsf/Df (n = 11). (D) Higher power photomicrograph of the uterine musculature of a dsf female (arrows). (E) Internal genitalia (side view) from an XX; tra⁻; hs-tra female (n = 32). There are few boutons on the uterine muscles, but boutons are visible on the the oviduct, sperm receptacle, and spermathecae and on other muscles associated with the external genitalia. (F) Higher power photomicrograph of the uterine musculature of an XX; tra⁻; hs-tra female. Dorsal is to the left. (Bar = 20 μm.)

The poor abdominal curling of dsf males prompted us to examine their abdominal musculature and innervation. A full complement of abdominal muscles, including normal MOLs and genital muscles, is present in dsf/dsf and dsf/Df males. Examination of muscle innervation with all four anti-neuronal antibodies shows that the nerve terminals, including those on the MOL, are apparently morphologically normal, except those on one muscle group. Innervation of the ventral longitudinal muscles of abdominal segment 5 (A5) is abnormal in dsf mutants (Fig. 4D) when compared with other abdominal segments of mutant males (A3; Fig. 4C) and wild-type males (Fig. 4 A and B). The most striking feature of ventral A5 innervation in mutants is the presence of a few large spherical boutons on each fiber rather than strings of small boutons cascading from the point of nerve contact with the muscle (compare Fig. 4 A–C with D). This mutant phenotype does not appear in dsf females (Fig. 4 E and F) and reveals, for the first time, that innervation of the ventral muscles of A5 is sex-specific, even though the normal pattern of neural connections looks similar between males and females. The improper innervation of ventral A5 is consistent with a causal role for the slow abdominal bends made by dsf males, although we cannot rule out additional dysfunction in central nervous system neural connections.

Figure 4.

Motor neuron innervation of the ventral abdominal muscles in wild-type and mutant males. Nerve terminals are stained with anti-synaptotagmin (37). (A) The third abdominal segment in a wild-type male. (B) The fifth abdominal segment in a wild-type male. (C) Morphologically normal third abdominal segment in a dsf male. (D) The fifth abdominal segment of a dsf/Df male. Fewer and enlarged boutons are found on the muscles. This phenotype is seen in both dsf/dsf and dsf/Df. (E) Morphologically normal third abdominal segment in a dsf female. (F) Morphologically normal fifth abdominal segment in a dsf female. Wild-type male, n = 12. XY; dsf/dsf, n = 10. XY; dsf/Df, n = 11. XX; dsf/dsf, n = 7. XX; dsf/Df, n = 12. Anterior is to the top. (Bar = 20 μm.)

dsf Acts Below tra in the Control of Neural Differentiation.

Analysis of sex-specific neural and behavioral phenotypes suggests that genes regulating these phenotypes act downstream of tra (24, 26). If so, XX; dsf⁻ animals masculinized by mutations in tra will have the male-specific ventral neuronal phenotype shown by XY; dsf⁻ males. To test this, we have examined ventral abdominal innervation of XX; tra⁻ and XX; dsf⁻; tra⁻ individuals. As shown in Fig. 5 A and B, XX; dsf⁻; tra⁻ animals show the dsf phenotype while their dsf⁺; tra⁻ siblings do not. Thus, we infer that dsf acts downstream of tra for development of ventral abdominal neuromuscular junctions. At the same time, these data rule out models in which some alternative pathway involving upstream elements such as the X chromosome to autosome ratio or Sxl, which are identical in both tested XX genotypes, independently regulates this process.

Figure 5.

dsf acts downstream of tra but independently of dsx. (A and B) dsf acts downstream of tra. Motor neuron innervation of the ventral muscles of abdominal segment 5 in XX animals transformed to maleness by a transformer mutation. Nerve terminals are stained with anti-synaptotagmin (37). (A) Abnormal innervation in XX; dsf⁻; tra⁻ pseudomales (n = 4). (B) Morphologically normal innervation in XX; dsf⁺; tra⁻ pseudomales (n = 3). (C–E) dsf is not under the control of dsx. Motor neuron innervation of the ventral muscles of abdominal segment 5 in XX and XY dsx^D/Df animals. Such animals differentiate as normal-looking males without regard for chromosomal sex or the action of tra and tra2. Nerve terminals are stained with anti-synaptotagmin (37). (C) Abnormal innervation in an XY; dsf⁻; dsx^D/Df male (n = 11). (D) Normal innervation in an XX; dsf⁻; dsx^D/Df pseudomale (n = 6). (E) Normal innervation in an XX; dsf⁺; dsx^D/Df pseudomale (n = 23). (Bar = 20 μm.)

dsf Acts in the dsx-Independent Pathway Controlling Sex-Specific Neural Differentiation.

To test dsf dependence on dsx function, we took advantage of a gain-of-function dsx mutant, dsx^D, which expresses the male dsx protein regardless of tra and tra2 activity. In the absence of a wild-type dsx allele, both sexes develop external male morphology (44). Even so, XX animals express tra and XY flies do not. If dsf acts independently of dsx, we expect XX; dsf⁻; dsx^D/Df (tra ON) animals to have normal ventral innervation and XY; dsf⁻; dsx^D/Df (tra OFF) animals to have abnormal ventral innervation, the dsf mutant phenotype. If dsf is dependent on the activity of dsx, then XX and XY; dsf⁻; dsx^D/Df animals should have equivalent and mutant phenotypes. We examined the neurons in ventral A5 for different genotypes of dsx^D/Df mutant animals. XX; dsf/Df; dsx^D/Df are wild type in appearance while XY; dsf/Df; dsx^D/Df have a dsf phenotype (Fig. 5 C–E). This result is consistent with the idea that dsf is part of a dsx-independent pathway.

DISCUSSION

Relatively few cases exist in which alterations in behavior, neuronal abnormalities, and specific genes have been linked. This work suggest that a solid link can be forged between complicated sexual behaviors in Drosophila, differentiation of specific neurons implicated in these behaviors, and the function of a single gene, dissatisfaction. Both dsf males and females exhibit behaviors that can be interpreted as defects in the recognition of and/or response to appropriate sexual partners; dsf males will court other mature males, as well as females, while dsf virgin females show strong rejection of courting males. These behavioral abnormalities are likely due to abnormalities in brain tissue based on the work of others mapping the brain as the critical area of the nervous system involved in these behaviors (2, 7, 8, 11, 12, 14, 24, 45, 46). We have not yet examined specific regions of the brain for abnormal sex-specific neuronal patterns, due to a lack of clearly defined sex-specific neuronal patterns within the brain and because of the highly complex nature and structure of the brain.

Clearly, dsf males and females are defective in “downstream” sexual behaviors: dsf males have difficulty in abdominal curling associated with mating and dsf females are unable to lay eggs. Our work has demonstrated that these defects correlate with obvious abnormalities in motor neuronal innervation. Males have abnormal innervation of the ventral muscles of abdominal segment 5, while females lack innervation to the circular muscles of the uterus. These neural defects are extremely specific, being limited to defined sets of sex-specific neurons. These data indicate a role for dsf in the genetic pathway controlling sex-specific behavior and neuronal development.

A body of work underscores the existence of a tra-dependent, dsx-independent mechanism for the control of some aspects of male and female sexual behavior and neural development. We have demonstrated that one dsf function, proper ventral A5 abdominal innervation in males, is downstream of tra but not downstream of dsx. These results lead us to infer that dsf functions in a tra-dependent and dsx-independent process (Fig. 1).

Another gene, fruitless (fru), has also been postulated to be part of a dsx-independent pathway controlling behavior and neuronal development (2, 24, 25). Males with strong fru mutations: (i) fail to differentiate MOLs (47); (ii) show abnormal courtship partner choice, courting both mature males and females (34, 47, 48); (iii) are sterile as a result of an inability to curl their abdomens into a copulatory position (48); (iv) generate an abnormal courtship song (49, 50). Mutant phenotypes have not been reported for fru females.

The identification of dsf as a second dsx-independent gene controlling sexual behavior and neural development raises questions about whether dsf and fru are part of a single regulatory pathway or of two different regulatory pathways (Fig. 1). There are both substantial similarities and substantial differences in phenotype between these two genes. dsf and fru are similar in that mutations in both genes lead to male by male courtship and to abnormality (dsf) or failure (fru) in abdominal curling during copulation (34, 47, 48). On the other hand, there are behavioral and neurological differences between dsf and fru mutants. The most notable among these is the lack of any reported abnormalities for fru females in either courtship or fertility relative to the substantial abnormalities exhibited by dsf females. There are also phenotypic differences between dsf and fru males (41). Males with strong fru alleles do not produce male-like MOLs, while dsf males produce normal MOLs with normal innervation (data not shown). In addition, the failure of abdominal bending is absolute in males carrying strong fru alleles and only partial in dsf mutants, while ventral abdominal muscles are innervated normally in fru males (B.J.T., unpublished observations) and abnormally in dsf males. As diagrammed in Fig. 1, the multiple differences between the fru and dsf phenotypes lead us to conclude that these genes act in separate regulatory pathways, each of which is required for appropriate sexual behavior.

Note added in proof.

Two recent papers (51, 52) describe the cloning of fru and various aspects of its regulation.