The Drosophila dopamine 2-like receptor D2R (Dop2R) is required in the blood brain barrier for male courtship.

The blood brain barrier (BBB) has the essential function to protect the brain from potentially hazardous molecules while also enabling controlled selective uptake. How these processes and signaling inside BBB cells control neuronal function is an intense area of interest. Signaling in the adult Drosophila BBB is required for normal male courtship behavior and relies on male-specific molecules in the BBB. Here we show that the dopamine receptor D2R is expressed in the BBB and is required in mature males for normal mating behavior. Conditional adult male knockdown of D2R in BBB cells causes courtship defects. The courtship defects observed in genetic D2R mutants can be rescued by expression of normal D2R specifically in the BBB of adult males. Drosophila BBB cells are glial cells. Our findings thus identify a specific glial function for the DR2 receptor and dopamine signaling in the regulation of a complex behavior.

The nuclear receptor Hr46/Hr3 is required in the blood brain barrier of mature males for courtship.

The blood brain barrier (BBB) forms a stringent barrier that protects the brain from components in the circulation that could interfere with neuronal function. At the same time, the BBB enables selective transport of critical nutrients and other chemicals to the brain. Beyond these functions, another recently recognized function is even less characterized, specifically the role of the BBB in modulating behavior by affecting neuronal function in a sex-dependent manner. Notably, signaling in the adult Drosophila BBB is required for normal male courtship behavior. Courtship regulation also relies on male-specific molecules in the BBB. Our previous studies have demonstrated that adult feminization of these cells in males significantly lowered courtship. Here, we conducted microarray analysis of BBB cells isolated from males and females. Findings revealed that these cells contain male- and female-enriched transcripts, respectively. Among these transcripts, nuclear receptor Hr46/Hr3 was identified as a male-enriched BBB transcript. Hr46/Hr3 is best known for its essential roles in the ecdysone response during development and metamorphosis. In this study, we demonstrate that Hr46/Hr3 is specifically required in the BBB cells for courtship behavior in mature males. The protein is localized in the nuclei of sub-perineurial glial cells (SPG), indicating that it might act as a transcriptional regulator. These data provide a catalogue of sexually dimorphic BBB transcripts and demonstrate a physiological adult role for the nuclear receptor Hr46/Hr3 in the regulation of male courtship, a novel function that is independent of its developmental role.

Mutants of the white ABCG Transporter in Drosophila melanogaster Have Deficient Olfactory Learning and Cholesterol Homeostasis.

Drosophila's white gene encodes an ATP-binding cassette G-subfamily (ABCG) half-transporter. White is closely related to mammalian ABCG family members that function in cholesterol efflux. Mutants of white have several behavioral phenotypes that are independent of visual defects. This study characterizes a novel defect of white mutants in the acquisition of olfactory memory using the aversive olfactory conditioning paradigm. The w1118 mutants learned slower than wildtype controls, yet with additional training, they reached wildtype levels of performance. The w1118 learning phenotype is also found in the wapricot and wcoral alleles, is dominant, and is rescued by genomic white and mini-white transgenes. Reducing dietary cholesterol strongly impaired olfactory learning for wildtype controls, while w1118 mutants were resistant to this deficit. The w1118 mutants displayed higher levels of cholesterol and cholesterol esters than wildtype under this low-cholesterol diet. Increasing levels of serotonin, dopamine, or both in the white mutants significantly improved w1118 learning. However, serotonin levels were not lower in the heads of the w1118 mutants than in wildtype controls. There were also no significant differences found in synapse numbers within the w1118 brain. We propose that the w1118 learning defect may be due to inefficient biogenic amine signaling brought about by altered cholesterol homeostasis.

Mate Choice: Should I Mate or Should I Go?

A mature virgin female fruit fly will initially resist copulation, while she assesses the desirability of her suitor. A new study identifies a neural circuit that controls rejection and shows how it changes from rejection to acceptance and copulation.

High functional conservation of takeout family members in a courtship model system.

takeout (to) is one of the male-specific genes expressed in the fat body that regulate male courtship behavior, and has been shown to act as a secreted protein in conjunction with courtship circuits. There are 23 takeout family members in Drosophila melanogaster, and homologues of this family are distributed across insect species. Sequence conservation among family members is low. Here we test the functional conservation of takeout family members by examining whether they can rescue the takeout courtship defect. We find that despite their sequence divergence takeout members from Aedes aegypti and Epiphas postvittana, as well as family members from D. melanogaster can substitute for takeout in courtship, demonstrating their functional conservation. Making use of the known E. postvittana Takeout structure, we used homology modeling and amphipathic helix analysis and found high overall structural conservation, including high conservation of the structure and amphipathic lining of an internal cavity that has been shown to accommodate hydrophobic ligands. Together these data suggest a high degree of structural conservation that likely underlies functional conservation in courtship. In addition, we have identified a role for a conserved exposed protein motif important for the protein's role in courtship.

Endocrine network essential for reproductive success in Drosophila melanogaster.

Ecdysis-triggering hormone (ETH) was originally discovered and characterized as a molt termination signal in insects through its regulation of the ecdysis sequence. Here we report that ETH persists in adult Drosophila melanogaster, where it functions as an obligatory allatotropin to promote juvenile hormone (JH) production and reproduction. ETH signaling deficits lead to sharply reduced JH levels and consequent reductions of ovary size, egg production, and yolk deposition in mature oocytes. Expression of ETH and ETH receptor genes is in turn dependent on ecdysone (20E). Furthermore, 20E receptor knockdown specifically in Inka cells reduces fecundity. Our findings indicate that the canonical developmental roles of 20E, ETH, and JH during juvenile stages are repurposed to function as an endocrine network essential for reproductive success.

Juvenile Hormone Is Required in Adult Males for Drosophila Courtship.

Juvenile Hormone (JH) has a prominent role in the regulation of insect development. Much less is known about its roles in adults, although functions in reproductive maturation have been described. In adult females, JH has been shown to regulate egg maturation and mating. To examine a role for JH in male reproductive behavior we created males with reduced levels of Juvenile Hormone Acid O-Methyl Transferase (JHAMT) and tested them for courtship. JHAMT regulates the last step of JH biosynthesis in the Corpora Allata (CA), the organ of JH synthesis. Males with reduced levels of JHAMT showed a reduction in courtship that could be rescued by application of Methoprene, a JH analog, shortly before the courtship assays were performed. In agreement with this, reducing JHAMT conditionally in mature flies led to courtship defects that were rescuable by Methoprene. The same result was also observed when the CA were conditionally ablated by the expression of a cellular toxin. Our findings demonstrate that JH plays an important physiological role in the regulation of male mating behavior.

Multiple pathways mediate the sex-peptide-regulated switch in female Drosophila reproductive behaviours.

Male-derived sex-peptide (SP) induces profound changes in the behaviour of Drosophila females, resulting in decreased receptivity to further mating and increased egg laying. SP can mediate the switch in female reproductive behaviours via a G protein-coupled receptor, SPR, in neurons expressing fruitless, doublesex and pickpocket. Whether SPR is the sole receptor and whether SP induces the postmating switch in a single pathway has not, to our knowledge been tested. Here we report that the SP response can be induced in the absence of SPR when SP is ectopically expressed in neurons or when SP, transferred by mating, can access neurons through a leaky blood brain barrier. Membrane-tethered SP can induce oviposition via doublesex, but not fruitless and pickpocket neurons in SPR mutant females. Although pickpocket and doublesex neurons rely on G(o) signalling to reduce receptivity and induce oviposition, G(o) signalling in fruitless neurons is required only to induce oviposition, but not to reduce receptivity. Our results show that SP's action in reducing receptivity and inducing oviposition can be separated in fruitless and doublesex neurons. Hence, the SP-induced postmating switch incorporates shared, but also distinct circuitry of fruitless, doublesex and pickpocket neurons and additional receptors.

Sex-specific signaling in the blood-brain barrier is required for male courtship in Drosophila.

Soluble circulating proteins play an important role in the regulation of mating behavior in Drosophila melanogaster. However, how these factors signal through the blood-brain barrier (bbb) to interact with the sex-specific brain circuits that control courtship is unknown. Here we show that male identity of the blood-brain barrier is necessary and that male-specific factors in the bbb are physiologically required for normal male courtship behavior. Feminization of the bbb of adult males significantly reduces male courtship. We show that the bbb-specific G-protein coupled receptor moody and bbb-specific Go signaling in adult males are necessary for normal courtship. These data identify sex-specific factors and signaling processes in the bbb as important regulators of male mating behavior.

Diversification of takeout, a male-biased gene family in Drosophila.

The display of courtship behavior has evolved in response to sexual selection driven by competition to obtain mates. Sexually dimorphic mate selection rituals are likely controlled at least in part by genes with sex-biased patterns of expression. In Drosophila melanogaster, male courtship behavior has been well described and consists of a series of stereotyped behaviors. The takeout gene is predominantly expressed in males and affects male courtship behavior. In this study, we examine the patterns of expression and evolution in takeout and the family of related proteins. We show that a number of genes in the takeout gene family show male-biased expression in D. melanogaster, largely in non-reproductive tissues. Phylogenetic analysis reveals that this gene family is conserved across insects. As expected for genes with male-biased expression, we also find evidence of positive selection in some lineages. Our results suggest that the genes in this family may have evolutionarily conserved sex specific roles in male mating behavior across insects.

The hector G-protein coupled receptor is required in a subset of fruitless neurons for male courtship behavior.

Male courtship behavior in Drosophila melanogaster is controlled by two main regulators, fruitless (fru) and doublesex (dsx). Their sex-specific expression in brain neurons has been characterized in detail, but little is known about the downstream targets of the sex-specific FRU and DSX proteins and how they specify the function of these neurons. While sexual dimorphism in the number and connections of fru and dsx expressing neurons has been observed, a majority of the neurons that express the two regulators are present in both sexes. This poses the question which molecules define the sex-specific function of these neurons. Signaling molecules are likely to play a significant role. We have identified a predicted G-protein coupled receptor (GPCR), CG4395, that is required for male courtship behavior. The courtship defect in the mutants can be rescued by expression of the wildtype protein in fru neurons of adult males. The GPCR is expressed in a subset of fru-positive antennal glomeruli that have previously been shown to be essential for male courtship. Expression of 4395-RNAi in GH146 projection neurons lowers courtship. This suggests that signaling through the CG4395 GPCR in this subset of fru neurons is critical for male courtship behavior.

The roles of fruitless and doublesex in the control of male courtship.

Male courtship in Drosophila melanogaster is a robust innate behavior that is shaped by sensory input and experience. It is regulated by the general sex-determination pathway through the sex-specific forms of fruitless and doublesex. Recent findings have shown that both fruitless and doublesex are required for courtship. This chapter reviews the role of these proteins and the neurons that express them in the regulation of courtship behavior. In particular it discusses how doublesex and fruitless contribute to the generation of sexually dimorphic neurons, the role of cell death, and the emerging information about circuits that underlie the behavior.

The circadian output gene takeout is regulated by Pdp1epsilon.

The circadian clock controls many circadian outputs. Although a large number of transcripts are affected by the circadian oscillator, very little is known about their regulation and function. We show here that the Drosophila takeout gene, one of the output genes of the circadian oscillator, is regulated similarly to the circadian clock genes Clock (Clk) and cry. takeout RNA levels are at constant high levels in Clk(JRK) mutants. The circadian transcription factor PAR domain protein 1 (Pdp1epsilon) is a transcription factor that had previously been postulated to control clock output genes, particularly genes regulated similarly to Clk. In agreement with this, we show here that Pdp1epsilon is a regulator of takeout. Takeout levels are low in flies with reduced Pdp1epsilon and high in flies with increased amounts of Pdp1epsilon. Furthermore, flies with reduced or elevated Pdp1epsilon levels in the fat body display courtship defects, identifying Pdp1epsilon as an important transcriptional regulator in that tissue.

A functional genomics strategy reveals clockwork orange as a transcriptional regulator in the Drosophila circadian clock.

The Drosophila circadian clock consists of integrated autoregulatory feedback loops, making the clock difficult to elucidate without comprehensively identifying the network components in vivo. Previous studies have adopted genome-wide screening for clock-controlled genes using high-density oligonucleotide arrays that identified hundreds of clock-controlled genes. In an attempt to identify the core clock genes among these candidates, we applied genome-wide functional screening using an RNA interference (RNAi) system in vivo. Here we report the identification of novel clock gene candidates including clockwork orange (cwo), a transcriptional repressor belonging to the basic helix-loop-helix ORANGE family. cwo is rhythmically expressed and directly regulated by CLK-CYC through canonical E-box sequences. A genome-wide search for its target genes using the Drosophila genome tiling array revealed that cwo forms its own negative feedback loop and directly suppresses the expression of other clock genes through the E-box sequence. Furthermore, this negative transcriptional feedback loop contributes to sustaining a high-amplitude circadian oscillation in vivo. Based on these results, we propose that the competition between cyclic CLK-CYC activity and the adjustable threshold imposed by CWO keeps E-box-mediated transcription within the controllable range of its activity, thereby rendering a Drosophila circadian clock capable of generating high-amplitude oscillation.

A role for the adult fat body in Drosophila male courtship behavior.

Mating behavior in Drosophila depends critically on the sexual identity of specific regions in the brain, but several studies have identified courtship genes that express products only outside the nervous system. Although these genes are each active in a variety of non-neuronal cell types, they are all prominently expressed in the adult fat body, suggesting an important role for this tissue in behavior. To test its role in male courtship, fat body was feminized using the highly specific Larval serum protein promoter. We report here that the specific feminization of this tissue strongly reduces the competence of males to perform courtship. This effect is limited to the fat body of sexually mature adults as the feminization of larval fat body that normally persists in young adults does not affect mating. We propose that feminization of fat body affects the synthesis of male-specific secreted circulating proteins that influence the central nervous system. In support of this idea, we demonstrate that Takeout, a protein known to influence mating, is present in the hemolymph of adult males but not females and acts as a secreted protein.

PER-dependent rhythms in CLK phosphorylation and E-box binding regulate circadian transcription.

Transcriptional activation by CLOCK-CYCLE (CLK-CYC) heterodimers and repression by PERIOD-TIMELESS (PER-TIM) heterodimers are essential for circadian oscillator function in Drosophila. PER-TIM was previously found to interact with CLK-CYC to repress transcription, and here we show that this interaction inhibits binding of CLK-CYC to E-box regulatory elements in vivo. Coincident with the interaction between PER-TIM and CLK-CYC is the hyperphosphorylation of CLK. This hyperphosphorylation occurs in parallel with the PER-dependent entry of DOUBLE-TIME (DBT) kinase into a complex with CLK-CYC, where DBT destabilizes both CLK and PER. Once PER and CLK are degraded, a novel hypophosphorylated form of CLK accumulates in parallel with E-box binding and transcriptional activation. These studies suggest that PER-dependent rhythms in CLK phosphorylation control rhythms in E-box-dependent transcription and CLK stability, thus linking PER and CLK function during the circadian cycle and distinguishing the transcriptional feedback mechanism in flies from that in mammals.

The Drosophila takeout gene is regulated by the somatic sex-determination pathway and affects male courtship behavior.

The Drosophila somatic sex-determination regulatory pathway has been well studied, but little is known about the target genes that it ultimately controls. In a differential screen for sex-specific transcripts expressed in fly heads, we identified a highly male-enriched transcript encoding Takeout, a protein related to a superfamily of factors that bind small lipophilic molecules. We show that sex-specific takeout transcripts derive from fat body tissue closely associated with the adult brain and are dependent on the sex determination genes doublesex (dsx) and fruitless (fru). The male-specific Doublesex and Fruitless proteins together activate Takeout expression, whereas the female-specific Doublesex protein represses takeout independently of Fru. When cells that normally express takeout are feminized by expression of the Transformer-F protein, male courtship behavior is dramatically reduced, suggesting that male identity in these cells is necessary for behavior. A loss-of-function mutation in the takeout gene reduces male courtship and synergizes with fruitless mutations, suggesting that takeout plays a redundant role with other fru-dependent factors involved in male mating behavior. Comparison of Takeout sequences to the Drosophila genome reveals a family of 20 related secreted factors. Expression analysis of a subset of these genes suggests that the takeout gene family encodes multiple factors with sex-specific functions.