Genetic and neuronal mechanisms governing the sex-specific interaction between sleep and sexual behaviors in Drosophila.

Animals execute one particular behavior among many others in a context-dependent manner, yet the mechanisms underlying such behavioral choice remain poorly understood. Here we studied how two fundamental behaviors, sex and sleep, interact at genetic and neuronal levels in Drosophila. We show that an increased need for sleep inhibits male sexual behavior by decreasing the activity of the male-specific P1 neurons that coexpress the sex determination genes fru M and dsx, but does not affect female sexual behavior. Further, we delineate a sex-specific neuronal circuit wherein the P1 neurons encoding increased courtship drive suppressed male sleep by forming mutually excitatory connections with the fru M -positive sleep-controlling DN1 neurons. In addition, we find that FRUM regulates male courtship and sleep through distinct neural substrates. These studies reveal the genetic and neuronal basis underlying the sex-specific interaction between sleep and sexual behaviors in Drosophila, and provide insights into how competing behaviors are co-regulated.Genes and circuits involved in sleep and sexual arousal have been extensively studied in Drosophila. Here the authors identify the sex determination genes fruitless and doublesex, and a sex-specific P1-DN1 neuronal feedback that governs the interaction between these competing behaviors.

Memory Elicited by Courtship Conditioning Requires Mushroom Body Neuronal Subsets Similar to Those Utilized in Appetitive Memory.

An animal's ability to learn and to form memories is essential for its survival. The fruit fly has proven to be a valuable model system for studies of learning and memory. One learned behavior in fruit flies is courtship conditioning. In Drosophila courtship conditioning, male flies learn not to court females during training with an unreceptive female. He retains a memory of this training and for several hours decreases courtship when subsequently paired with any female. Courtship conditioning is a unique learning paradigm; it uses a positive-valence stimulus, a female fly, to teach a male to decrease an innate behavior, courtship of the female. As such, courtship conditioning is not clearly categorized as either appetitive or aversive conditioning. The mushroom body (MB) region in the fruit fly brain is important for several types of memory; however, the precise subsets of intrinsic and extrinsic MB neurons necessary for courtship conditioning are unknown. Here, we disrupted synaptic signaling by driving a shibirets effector in precise subsets of MB neurons, defined by a collection of split-GAL4 drivers. Out of 75 lines tested, 32 showed defects in courtship conditioning memory. Surprisingly, we did not have any hits in the γ lobe Kenyon cells, a region previously implicated in courtship conditioning memory. We did find that several γ lobe extrinsic neurons were necessary for courtship conditioning memory. Overall, our memory hits in the dopaminergic neurons (DANs) and the mushroom body output neurons were more consistent with results from appetitive memory assays than aversive memory assays. For example, protocerebral anterior medial DANs were necessary for courtship memory, similar to appetitive memory, while protocerebral posterior lateral 1 (PPL1) DANs, important for aversive memory, were not needed. Overall, our results indicate that the MB circuits necessary for courtship conditioning memory coincide with circuits necessary for appetitive memory.

Sex-specific regulation of Lgr3 in Drosophila neurons.

The development of sexually dimorphic morphology and the potential for sexually dimorphic behavior in Drosophila are regulated by the Fruitless (Fru) and Doublesex (Dsx) transcription factors. Several direct targets of Dsx have been identified, but direct Fru targets have not been definitively identified. We show that Drosophila leucine-rich repeat G protein-coupled receptor 3 (Lgr3) is regulated by Fru and Dsx in separate populations of neurons. Lgr3 is a member of the relaxin-receptor family and a receptor for Dilp8, necessary for control of organ growth. Lgr3 expression in the anterior central brain of males is inhibited by the B isoform of Fru, whose DNA binding domain interacts with a short region of an Lgr3 intron. Fru A and C isoform mutants had no observed effect on Lgr3 expression. The female form of Dsx (Dsx(F)) separately up- and down-regulates Lgr3 expression in distinct neurons in the abdominal ganglion through female- and male-specific Lgr3 enhancers. Excitation of neural activity in the Dsx(F)-up-regulated abdominal ganglion neurons inhibits female receptivity, indicating the importance of these neurons for sexual behavior. Coordinated regulation of Lgr3 by Fru and Dsx marks a point of convergence of the two branches of the sex-determination hierarchy.

Central neural circuitry mediating courtship song perception in male Drosophila.

Animals use acoustic signals across a variety of social behaviors, particularly courtship. In Drosophila, song is detected by antennal mechanosensory neurons and further processed by second-order aPN1/aLN(al) neurons. However, little is known about the central pathways mediating courtship hearing. In this study, we identified a male-specific pathway for courtship hearing via third-order ventrolateral protocerebrum Projection Neuron 1 (vPN1) neurons and fourth-order pC1 neurons. Genetic inactivation of vPN1 or pC1 disrupts song-induced male-chaining behavior. Calcium imaging reveals that vPN1 responds preferentially to pulse song with long inter-pulse intervals (IPIs), while pC1 responses to pulse song closely match the behavioral chaining responses at different IPIs. Moreover, genetic activation of either vPN1 or pC1 induced courtship chaining, mimicking the behavioral response to song. These results outline the aPN1-vPN1-pC1 pathway as a labeled line for the processing and transformation of courtship song in males.

Constraints on the evolution of a doublesex target gene arising from doublesex's pleiotropic deployment.

"Regulatory evolution," that is, changes in a gene's expression pattern through changes at its regulatory sequence, rather than changes at the coding sequence of the gene or changes of the upstream transcription factors, has been increasingly recognized as a pervasive evolution mechanism. Many somatic sexually dimorphic features of Drosophila melanogaster are the results of gene expression regulated by the doublesex (dsx) gene, which encodes sex-specific transcription factors (DSX(F) in females and DSX(M) in males). Rapid changes in such sexually dimorphic features are likely a result of changes at the regulatory sequence of the target genes. We focused on the Flavin-containing monooxygenase-2 (Fmo-2) gene, a likely direct dsx target, to elucidate how sexually dimorphic expression and its evolution are brought about. We found that dsx is deployed to regulate the Fmo-2 transcription both in the midgut and in fat body cells of the spermatheca (a female-specific tissue), through a canonical DSX-binding site in the Fmo-2 regulatory sequence. In the melanogaster group, Fmo-2 transcription in the midgut has evolved rapidly, in contrast to the conserved spermathecal transcription. We identified two cis-regulatory modules (CRM-p and CRM-d) that direct sexually monomorphic or dimorphic Fmo-2 transcription, respectively, in the midguts of these species. Changes of Fmo-2 transcription in the midgut from sexually dimorphic to sexually monomorphic in some species are caused by the loss of CRM-d function, but not the loss of the canonical DSX-binding site. Thus, conferring transcriptional regulation on a CRM level allows the regulation to evolve rapidly in one tissue while evading evolutionary constraints posed by other tissues.

Central brain neurons expressing doublesex regulate female receptivity in Drosophila.

Drosophila melanogaster females respond to male courtship by either rejecting the male or allowing copulation. The neural mechanisms underlying these female behaviors likely involve the integration of sensory information in the brain. Because doublesex (dsx) controls other aspects of female differentiation, we asked whether dsx-expressing neurons mediate virgin female receptivity to courting males. Using intersectional techniques to manipulate the activities of defined subsets of dsx-expressing neurons, we found that activation of neurons in either the pCd or pC1 clusters promotes receptivity, while silencing these neurons makes females unreceptive. Furthermore, pCd and pC1 neurons physiologically respond to the male-specific pheromone cis-vaccenyl acetate (cVA), while pC1 neurons also respond to male courtship song. The pCd and pC1 neurons expressing dsx in females do not express transcripts from the fruitless (fru) P1 promoter. Thus, virgin female receptivity is controlled at least in part by neurons that are distinct from those governing male courtship.

Neural pathways for the detection and discrimination of conspecific song in D. melanogaster.

BACKGROUND: During courtship, male Drosophila melanogaster sing a multipart courtship song to female flies. This song is of particular interest because (1) it is species specific and varies widely within the genus, (2) it is a gating stimulus for females, who are sensitive detectors of conspecific song, and (3) it is the only sexual signal that is under both neural and genetic control. This song is perceived via mechanosensory neurons in the antennal Johnston's organ, which innervate the antennal mechanosensory and motor center (AMMC) of the brain. However, AMMC outputs that are responsible for detection and discrimination of conspecific courtship song remain unknown. RESULTS: Using a large-scale anatomical screen of AMMC interneurons, we identify seven projection neurons (aPNs) and five local interneurons (aLNs) that outline a complex architecture for the ascending mechanosensory pathway. Neuronal inactivation and hyperactivation during behavior reveal that only two classes of interneurons are necessary for song responses--the projection neuron aPN1 and GABAergic interneuron aLN(al). These neurons are necessary in both male and female flies. Physiological recordings in aPN1 reveal the integration of courtship song as a function of pulse rate and outline an intracellular transfer function that likely facilitates the response to conspecific song. CONCLUSIONS: These results reveal a critical pathway for courtship hearing in male and female flies, in which both aLN(al) and aPN1 mediate the detection of conspecific song. The pathways arising from these neurons likely serve as a critical neural substrate for behavioral reproductive isolation in D. melanogaster.

A small subset of fruitless subesophageal neurons modulate early courtship in Drosophila.

We show that a small subset of two to six subesophageal neurons, expressing the male products of the male courtship master regulator gene products fruitless Male (fru M), are required in the early stages of the Drosophila melanogaster male courtship behavioral program. Loss of fru M expression or inhibition of synaptic transmission in these fru M(+) neurons results in delayed courtship initiation and a failure to progress to copulation primarily under visually-deficient conditions. We identify a fru M-dependent sexually dimorphic arborization in the tritocerebrum made by two of these neurons. Furthermore, these SOG neurons extend descending projections to the thorax and abdominal ganglia. These anatomical and functional characteristics place these neurons in the position to integrate gustatory and higher-order signals in order to properly initiate and progress through early courtship.

Genetic identification and separation of innate and experience-dependent courtship behaviors in Drosophila.

Wild-type D. melanogaster males innately possess the ability to perform a multistep courtship ritual to conspecific females. The potential for this behavior is specified by the male-specific products of the fruitless (fru(M)) gene; males without fru(M) do not court females when held in isolation. We show that such fru(M) null males acquire the potential for courtship when grouped with other flies; they apparently learn to court flies with which they were grouped, irrespective of sex or species and retain this behavior for at least a week. The male-specific product of the doublesex gene (dsx(M)) is necessary and sufficient for the acquisition of the potential for such experience-dependent courtship. These results reveal a process that builds, via dsx(M) and social experience, the potential for a more flexible sexual behavior, which could be evolutionarily conserved as dsx-related genes that function in sexual development are found throughout the animal kingdom.

Male-specific Fruitless isoforms have different regulatory roles conferred by distinct zinc finger DNA binding domains.

BACKGROUND: Drosophila melanogaster adult males perform an elaborate courtship ritual to entice females to mate. fruitless (fru), a gene that is one of the key regulators of male courtship behavior, encodes multiple male-specific isoforms (Fru(M)). These isoforms vary in their carboxy-terminal zinc finger domains, which are predicted to facilitate DNA binding. RESULTS: By over-expressing individual Fru(M) isoforms in fru-expressing neurons in either males or females and assaying the global transcriptional response by RNA-sequencing, we show that three Fru(M) isoforms have different regulatory activities that depend on the sex of the fly. We identified several sets of genes regulated downstream of Fru(M) isoforms, including many annotated with neuronal functions. By determining the binding sites of individual Fru(M) isoforms using SELEX we demonstrate that the distinct zinc finger domain of each Fru(M) isoforms confers different DNA binding specificities. A genome-wide search for these binding site sequences finds that the gene sets identified as induced by over-expression of Fru(M) isoforms in males are enriched for genes that contain the binding sites. An analysis of the chromosomal distribution of genes downstream of Fru(M) shows that those that are induced and repressed in males are highly enriched and depleted on the X chromosome, respectively. CONCLUSIONS: This study elucidates the different regulatory and DNA binding activities of three Fru(M) isoforms on a genome-wide scale and identifies genes regulated by these isoforms. These results add to our understanding of sex chromosome biology and further support the hypothesis that in some cell-types genes with male-biased expression are enriched on the X chromosome.

Genetic and neural mechanisms that inhibit Drosophila from mating with other species.

Genetically hard-wired neural mechanisms must enforce behavioral reproductive isolation because interspecies courtship is rare even in sexually naïve animals of most species. We find that the chemoreceptor Gr32a inhibits male D. melanogaster from courting diverse fruit fly species. Gr32a recognizes nonvolatile aversive cues present on these reproductively dead-end targets, and activity of Gr32a neurons is necessary and sufficient to inhibit interspecies courtship. Male-specific Fruitless (Fru(M)), a master regulator of courtship, also inhibits interspecies courtship. Gr32a and Fru(M) are not coexpressed, but Fru(M) neurons contact Gr32a neurons, suggesting that these genes influence a shared neural circuit that inhibits interspecies courtship. Gr32a and Fru(M) also suppress within-species intermale courtship, but we show that distinct mechanisms preclude sexual displays toward conspecific males and other species. Although this chemosensory pathway does not inhibit interspecies mating in D. melanogaster females, similar mechanisms appear to inhibit this behavior in many other male drosophilids.

doublesex functions early and late in gustatory sense organ development.

Somatic sexual dimorphisms outside of the nervous system in Drosophila melanogaster are largely controlled by the male- and female-specific Doublesex transcription factors (DSX(M) and DSX(F), respectively). The DSX proteins must act at the right times and places in development to regulate the diverse array of genes that sculpt male and female characteristics across a variety of tissues. To explore how cellular and developmental contexts integrate with doublesex (dsx) gene function, we focused on the sexually dimorphic number of gustatory sense organs (GSOs) in the foreleg. We show that DSX(M) and DSX(F) promote and repress GSO formation, respectively, and that their relative contribution to this dimorphism varies along the proximodistal axis of the foreleg. Our results suggest that the DSX proteins impact specification of the gustatory sensory organ precursors (SOPs). DSX(F) then acts later in the foreleg to regulate gustatory receptor neuron axon guidance. These results suggest that the foreleg provides a unique opportunity for examining the context-dependent functions of DSX.

Joint control of Drosophila male courtship behavior by motion cues and activation of male-specific P1 neurons.

Sexual behaviors in animals are governed by inputs from multiple external sensory modalities. However, how these inputs are integrated to jointly control animal behavior is still poorly understood. Whereas visual information alone is not sufficient to induce courtship behavior in Drosophila melanogaster males, when a subset of male-specific fruitless (fru)- and doublesex (dsx)-expressing neurons that respond to chemosensory cues (P1 neurons) were artificially activated via a temperature-sensitive cation channel (dTRPA1), males followed and extended their wing toward moving objects (even a moving piece of rubber band) intensively. When stationary, these objects were not courted. Our results indicate that motion input and activation of P1 neurons are individually necessary, and under our assay conditions, jointly sufficient to elicit early courtship behaviors, and provide insights into how courtship decisions are made via sensory integration.

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.

Direct targets of the D. melanogaster DSXF protein and the evolution of sexual development.

Uncovering the direct regulatory targets of doublesex (dsx) and fruitless (fru) is crucial for an understanding of how they regulate sexual development, morphogenesis, differentiation and adult functions (including behavior) in Drosophila melanogaster. Using a modified DamID approach, we identified 650 DSX-binding regions in the genome from which we then extracted an optimal palindromic 13 bp DSX-binding sequence. This sequence is functional in vivo, and the base identity at each position is important for DSX binding in vitro. In addition, this sequence is enriched in the genomes of D. melanogaster (58 copies versus approximately the three expected from random) and in the 11 other sequenced Drosophila species, as well as in some other Dipterans. Twenty-three genes are associated with both an in vivo peak in DSX binding and an optimal DSX-binding sequence, and thus are almost certainly direct DSX targets. The association of these 23 genes with optimum DSX binding sites was used to examine the evolutionary changes occurring in DSX and its targets in insects.

Functional dissection of the neural substrates for sexual behaviors in Drosophila melanogaster.

The male-specific Fruitless proteins (FruM) act to establish the potential for male courtship behavior in Drosophila melanogaster and are expressed in small groups of neurons throughout the nervous system. We screened ∼1000 GAL4 lines, using assays for general courtship, male-male interactions, and male fertility to determine the phenotypes resulting from the GAL4-driven inhibition of FruM expression in subsets of these neurons. A battery of secondary assays showed that the phenotypic classes of GAL4 lines could be divided into subgroups on the basis of additional neurobiological and behavioral criteria. For example, in some lines, restoration of FruM expression in cholinergic neurons restores fertility or reduces male-male courtship. Persistent chains of males courting each other in some lines results from males courting both sexes indiscriminately, whereas in other lines this phenotype results from apparent habituation deficits. Inhibition of ectopic FruM expression in females, in populations of neurons where FruM is necessary for male fertility, can rescue female infertility. To identify the neurons responsible for some of the observed behavioral alterations, we determined the overlap between the identified GAL4 lines and endogenous FruM expression in lines with fertility defects. The GAL4 lines causing fertility defects generally had widespread overlap with FruM expression in many regions of the nervous system, suggesting likely redundant FruM-expressing neuronal pathways capable of conferring male fertility. From associations between the screened behaviors, we propose a functional model for courtship initiation.

Sex and the single cell. II. There is a time and place for sex.

The Drosophila melanogaster sex hierarchy controls sexual differentiation of somatic cells via the activities of the terminal genes in the hierarchy, doublesex (dsx) and fruitless (fru). We have targeted an insertion of GAL4 into the dsx gene, allowing us to visualize dsx-expressing cells in both sexes. Developmentally and as adults, we find that both XX and XY individuals are fine mosaics of cells and tissues that express dsx and/or fruitless (fru(M)), and hence have the potential to sexually differentiate, and those that don't. Evolutionary considerations suggest such a mosaic expression of sexuality is likely to be a property of other animal species having two sexes. These results have also led to a major revision of our view of how sex-specific functions are regulated by the sex hierarchy in flies. Rather than there being a single regulatory event that governs the activities of all downstream sex determination regulatory genes-turning on Sex lethal (Sxl) RNA splicing activity in females while leaving it turned off in males-there are, in addition, elaborate temporal and spatial transcriptional controls on the expression of the terminal regulatory genes, dsx and fru. Thus tissue-specific aspects of sexual development are jointly specified by post-transcriptional control by Sxl and by the transcriptional controls of dsx and fru expression.

Midline crossing by gustatory receptor neuron axons is regulated by fruitless, doublesex and the Roundabout receptors.

Although nervous system sexual dimorphisms are known in many species, relatively little is understood about the molecular mechanisms generating these dimorphisms. Recent findings in Drosophila provide the tools for dissecting how neurogenesis and neuronal differentiation are modulated by the Drosophila sex-determination regulatory genes to produce nervous system sexual dimorphisms. Here we report studies aimed at illuminating the basis of the sexual dimorphic axonal projection patterns of foreleg gustatory receptor neurons (GRNs): only in males do GRN axons project across the midline of the ventral nerve cord. We show that the sex determination genes fruitless (fru) and doublesex (dsx) both contribute to establishing this sexual dimorphism. Male-specific Fru (Fru(M)) acts in foreleg GRNs to promote midline crossing by their axons, whereas midline crossing is repressed in females by female-specific Dsx (Dsx(F)). In addition, midline crossing by these neurons might be promoted in males by male-specific Dsx (Dsx(M)). Finally, we (1) demonstrate that the roundabout (robo) paralogs also regulate midline crossing by these neurons, and (2) provide evidence that Fru(M) exerts its effect on midline crossing by directly or indirectly regulating Robo signaling.

Manipulation of an innate escape response in Drosophila: photoexcitation of acj6 neurons induces the escape response.

BACKGROUND: The genetic analysis of behavior in Drosophila melanogaster has linked genes controlling neuronal connectivity and physiology to specific neuronal circuits underlying a variety of innate behaviors. We investigated the circuitry underlying the adult startle response, using photoexcitation of neurons that produce the abnormal chemosensory jump 6 (acj6) transcription factor. This transcription factor has previously been shown to play a role in neuronal pathfinding and neurotransmitter modality, but the role of acj6 neurons in the adult startle response was largely unknown. PRINCIPAL FINDINGS: We show that the activity of these neurons is necessary for a wild-type startle response and that excitation is sufficient to generate a synthetic escape response. Further, we show that this synthetic response is still sensitive to the dose of acj6 suggesting that that acj6 mutation alters neuronal activity as well as connectivity and neurotransmitter production. RESULTS/SIGNIFICANCE: These results extend the understanding of the role of acj6 and of the adult startle response in general. They also demonstrate the usefulness of activity-dependent characterization of neuronal circuits underlying innate behaviors in Drosophila, and the utility of integrating genetic analysis into modern circuit analysis techniques.

Expression of the Drosophila secreted cuticle protein 73 (dsc73) requires Shavenbaby.

Low stringency genomic library screens with genomic fragments from the sex determination gene doublesex identified the Drosophila secreted cuticle protein 73 (dsc73) gene, which encodes an 852-residue protein with an N-terminal signal sequence. In embryos, dsc73 RNA and protein are expressed to high levels in the epidermal cells that secrete the larval cuticle as well as in other cuticle-secreting tissues such as the trachea and salivary duct. Embryonic expression of dsc73 requires Shavenbaby, a transcription factor regulating cuticle formation. Double-labeling experiments with alphaCrb and alphaSAS reveal that, as with chitin and other known cuticle proteins, Dsc73 is secreted apically. Zygotic loss of dsc73 results in larval lethality but loss does not result in overt patterning defects or overt morphological defects in the embryonic tissues in which it is expressed. Thus, dsc73 encodes a novel secreted protein, and it is conserved within the Drosophila group. dsc73 may serve as a useful embryonic marker for cuticular patterning.