The sex-determination genes fruitless and doublesex specify a neural substrate required for courtship song

Courtship song is a critical component of male courtship behavior in Drosophila, making the female more receptive to copulation and communicating species-specific information [1-6]. Sex mosaic studies have shown that the sex of certain regions of the central nervous system (CNS) is critical to song production [7]. Our examination of one of these regions, the mesothoracic ganglion (Msg), revealed the coexpression of two sex-determination genes, fruitless (fru) and doublesex (dsx). Because both genes are involved in creating a sexually dimorphic CNS [8, 9] and are necessary for song production [10-13], we investigated the individual contributions of fru and dsx to the specification of a male CNS and song production. We show a novel requirement for dsx in specifying a sexually dimorphic population of fru-expressing neurons in the Msg. Moreover, by using females constitutively expressing the male-specific isoforms of fru (Fru(M)), we show a critical requirement for the male isoform of dsx (Dsx(M)), alongside Fru(M), in the specification of courtship song. Therefore, although Fru(M) expression is sufficient for the performance of many male-specific behaviors [14], we have shown that without Dsx(M), the determination of a male-specific CNS and thus a full complement of male behaviors are not realized.     

Turning males on: activation of male courtship behavior in Drosophila melanogaster

The innate sexual behaviors of Drosophila melanogaster males are an attractive system for elucidating how complex behavior patterns are generated. The potential for male sexual behavior in D. melanogaster is specified by the fruitless (fru) and doublesex (dsx) sex regulatory genes. We used the temperature-sensitive activator dTRPA1 to probe the roles of fru(M)- and dsx-expressing neurons in male courtship behaviors. Almost all steps of courtship, from courtship song to ejaculation, can be induced at very high levels through activation of either all fru(M) or all dsx neurons in solitary males. Detailed characterizations reveal different roles for fru(M) and dsx in male courtship. Surprisingly, the system for mate discrimination still works well when all dsx neurons are activated, but is impaired when all fru(M) neurons are activated. Most strikingly, we provide evidence for a fru(M)-independent courtship pathway that is primarily vision dependent.     

Neural circuitry that governs Drosophila male courtship behavior

Male-specific fruitless (fru) products (Fru(M)) are both necessary and sufficient to "hardwire" the potential for male courtship behavior into the Drosophila nervous system. Fru(M) is expressed in approximately 2% of neurons in the male nervous system, but not in the female. We have targeted the insertion of GAL4 into the fru locus, allowing us to visualize and manipulate the Fru(M)-expressing neurons in the male as well as their counterparts in the female. We present evidence that these neurons are directly and specifically involved in male courtship behavior and that at least some of them are interconnected in a circuit. This circuit includes olfactory neurons required for the behavioral response to sex pheromones. Anatomical differences in this circuit that might account for the dramatic differences in male and female sexual behavior are not apparent.     

fruitless splicing specifies male courtship behavior in Drosophila

All animals exhibit innate behaviors that are specified during their development. Drosophila melanogaster males (but not females) perform an elaborate and innate courtship ritual directed toward females (but not males). Male courtship requires products of the fruitless (fru) gene, which is spliced differently in males and females. We have generated alleles of fru that are constitutively spliced in either the male or the female mode. We show that male splicing is essential for male courtship behavior and sexual orientation. More importantly, male splicing is also sufficient to generate male behavior in otherwise normal females. These females direct their courtship toward other females (or males engineered to produce female pheromones). The splicing of a single neuronal gene thus specifies essentially all aspects of a complex innate behavior.     

New reproductive anomalies in fruitless-mutant Drosophila males: extreme lengthening of mating durations and infertility correlated with defective serotonergic innervation of reproductive organs

Several features of male reproductive behavior are under the neural control of fruitless (fru) in Drosophila melanogaster. This gene is known to influence courtship steps prior to mating, due to the absence of attempted copulation in the behavioral repertoire of most types of fru-mutant males. However, certain combinations of fru mutations allow for fertility. By analyzing such matings and their consequences, we uncovered two striking defects: mating times up to four times the normal average duration of copulation; and frequent infertility, regardless of the time of mating by a given transheterozygous fru-mutant male. The lengthened copulation times may be connected with fru-induced defects in the formation of a male-specific abdominal muscle. Production of sperm and certain seminal fluid proteins are normal in these fru mutants. However, analysis of postmating qualities of females that copulated with transheterozygous mutants strongly implied defects in the ability of these males to transfer sperm and seminal fluids. Such abnormalities may be associated with certain serotonergic neurons in the abdominal ganglion in which production of 5HT is regulated by fru. These cells send processes to contractile muscles of the male's internal sex organs; such projection patterns are aberrant in the semifertile fru mutants. Therefore, the reproductive functions regulated by fruitless are expanded in their scope, encompassing not only the earliest stages of courtship behavior along with almost all subsequent steps in the behavioral sequence, but also more than one component of the culminating events.     

Elements of the fruitless locus regulate development of the muscle of Lawrence, a male-specific structure in the abdomen of Drosophila melanogaster adults

A genetically defined element of the fruitless (fru) locus in Drosophila melanogaster regulates the development of a male-specific muscle spanning the fifth abdominal segment in adult males, the 'muscle of Lawrence' (MOL). The region is defined by two cytological deletions, each with a breakpoint that co-maps with previously described mutant courtship phenotypes at cytogenetic interval 91B on the third chromosome. Flies that carry both of these deletions are viable, and males express abnormalities of courtship similar to those caused by the fru inversion breakpoint at 91B. In addition, these double-deletion males show the complete absence of the MOL, suggesting that they have little or no gene expression of a postulated MOL determinant; the musculature in the fifth abdominal segment of these mutants to indistinguishable from that of a normal female. Other mutant combinations that produce fruitless courtship phenotypes--including deletion and inversion breakpoints, and a marked transposon inserted at 91B--produce intermediate forms of the MOL. A new genetic variant, induced by imprecise excision of the marked transposon, is homozygous lethal and disrupts fru functions related to courtship and the MOL. The MOL is shown to be dispensable for fertility and is therefore not the causative factor of fru-induced behavioral sterility. These genetic variants and their phenotypic results are discussed with regard to a model for the organization of the fru locus.     

Fruitless and doublesex coordinate to generate male-specific neurons that can initiate courtship

Biologists postulate that sexual dimorphism in the brain underlies gender differences in behavior, yet direct evidence for this has been sparse. We identified a male-specific, fruitless (fru)/doublesex (dsx)-coexpressing neuronal cluster, P1, in Drosophila. The artificial induction of a P1 clone in females effectively provokes male-typical behavior in such females even when the other parts of the brain are not masculinized. P1, located in the dorsal posterior brain near the mushroom body, is composed of 20 interneurons, each of which has a primary transversal neurite with extensive ramifications in the bilateral protocerebrum. P1 is fated to die in females through the action of a feminizing protein, DsxF. A masculinizing protein Fru is required in the male brain for correct positioning of the terminals of P1 neurites. Thus, the coordinated actions of two sex determination genes, dsx and fru, confer the unique ability to initiate male-typical sexual behavior on P1 neurons.     

Drosophila retained/dead ringer is necessary for neuronal pathfinding, female receptivity and repression of fruitless independent male courtship behaviors

Mutations in the Drosophila retained/dead ringer (retn) gene lead to female behavioral defects and alter a limited set of neurons in the CNS. retn is implicated as a major repressor of male courtship behavior in the absence of the fruitless (fru) male protein. retn females show fru-independent male-like courtship of males and females, and are highly resistant to courtship by males. Males mutant for retn court with normal parameters, although feminization of retn cells in males induces bisexuality. Alternatively spliced RNAs appear in the larval and pupal CNS, but none shows sex specificity. Post-embryonically, retn RNAs are expressed in a limited set of neurons in the CNS and eyes. Neural defects of retn mutant cells include mushroom body beta-lobe fusion and pathfinding errors by photoreceptor and subesophageal neurons. We posit that some of these retn-expressing cells function to repress a male behavioral pathway activated by fruM.     

Novel genes expressed in subsets of chemosensory sensilla on the front legs of male Drosophila melanogaster

Detection of courtship-activating female pheromones by contact chemoreceptors on the front legs of male Drosophila melanogaster is thought to play an important role in triggering courtship behavior. However, the chemosensory organs, cells, and molecules responsible are not known. We have isolated two genes, CheA29a and CheB42a, expressed in nonneuronal auxiliary cells within two nested subsets of chemosensory sensilla on the front legs of sexually mature, adult males. The proteins encoded by the CheA29a and CheB42a genes have no sequence similarity to each other or any other known protein, but they belong to two novel families of proteins encoded by the D. melanogaster genome. Members of the two families are predicted to have a single transmembrane domain at their amino terminus, probably to serve as a signal peptide, suggesting that they are soluble and secreted. Finally, in addition to CheA29a and CheB42a, other genes within each family are expressed preferentially in appendages where chemosensory organs are concentrated, in several cases in a male-specific manner. Our data suggest that CheA29a and CheB42a and other members of these two protein families are involved in male-specific chemical senses, perhaps pheromone response.     

Differentiation of a Male-Specific Muscle in Drosophila Melanogaster Does Not Require the Sex-Determining Genes Doublesex or Intersex

A pair of muscles span the fifth abdominal segment of male but not female Drosophila melanogaster adults. To establish whether genes involved in the development of other sexually dimorphic tissues controlled the differentiation of sex-specific muscles, flies mutant for five known sex-determining genes were examined for the occurrence of male-specific abdominal muscles. Female flies mutant for alleles of Sex-lethal, defective in sex determination, or null alleles of transformer or transformer-2 are converted into phenotypic males that formed male-specific abdominal muscles. Both male and female flies, when mutant for null alleles of doublesex, develop as nearly identical intersexes in other somatic characteristics. Male doublesex flies produced the male-specific muscles, whereas female doublesex flies lacked them. Female flies, even when they inappropriately expressed the male-specific form of doublesex mRNA, failed to produce the male-specific muscles. Therefore, the wild-type products of the genes Sex-lethal, transformer and transformer-2 act to prevent the differentiation of male-specific muscles in female flies. However, there is no role for the genes doublesex or intersex in either the generation of the male-specific muscles in males or their suppression in females.