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

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

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

Conditional reduction of JHAMT in mature males causes courtship defects that can be rescued by application of the JH analog Methoprene.

<p>Graphs show the courtship index CI (fraction of time males spend courting during the observation period) ± SEM of the indicated genotypes (A, C), or the performance of males in a control activity assay (# of line crossings ± SEM (B). Data were analyzed by ANOVA followed by Bonferroni multiple comparisons. (A) Courtship index of induced and un-induced experimental (<i>X/Y; UAS-JHAMT-RNAi/+; hsp70-GAL4/+</i>) and control (<i>X/Y; +/+; hsp70-GAL4/+;</i> and <i>X/Y; UAS-JHAMT-RNAi/+;+/+</i>) genotypes. The experimental genotype shows a significant reduction in courtship index (p = 0.0007). (N = 20). Induced flies were heat-shocked at 37°C for 1 hour and let recover for four hours. (B) Activity assay of the induced and un-induced genotypes; genotypes as in (A) (n = 10). (C) Genotypes as in (A); one hour after induction, flies were treated with Methoprene in acetone, or with acetone alone, and courtship was examined 4 hours later. The experimental genotype shows complete rescue (p< 0.003) (N = 20). (Un-induced (-), Induced (+).</p

JHAMT-Gal4 driver directs expression in the corpora allata (CA).

<p>Frozen sections of adult <i>JHAMT-Gal4/UAS-lacZ</i> males were incubated with an anti-ßGal antibody (red) and with an anti-JHAMT antibody (green). Overlapping expression in the CA (marked by arrow) was observed. H:Head; T:Thorax</p

Conditional adult ablation of the corpora allata (CA) causes courtship defects that can be rescued by application of the JH analog Methoprene.

<p>The conditional Gal80^ts system was used to express UAS-DTI in adult males. The flies were created and maintained at 18°C. For induction, mature males were placed at 30°C for 2 days and then kept at room temperature for one day. The induced and un-induced experimental and control genotypes were subjected to courtship assay. Graphs show the courtship index CI (fraction of time males spend courting during the observation period) ± SEM of the indicated genotypes (A, C), or the performance of males in a control activity assay (# of line crossings ± SEM (B). Data were analyzed by ANOVA followed by Bonferroni multiple comparisons. (A) Courtship assay of induced and un-induced experimental <i>X/Y; Gal80</i>^<i>ts</i><i>/Gal80</i>^<i>ts</i><i>; JHAMT-GAL4/ UAS-DTI</i> and control genotypes <i>X/Y; Gal80</i>^<i>ts</i><i>/+; UAS-DTI/ +;</i> and <i>X/Y; Gal80</i>^<i>ts</i><i>/+; JHAMT-GAL4/+</i>. (N = 15). Experimental flies had significantly lower courtship than the controls (p<0.01). (B) Activity assay of the flies described in (A) (N = 10). (C) Methoprene in acetone or acetone alone was applied to induced males four hours prior to testing for courtship. Methoprene application completely rescued the courtship defect of experimental flies (P<0.001). (N = 20). (Un-induced (-), Induced (+).</p

Expression of JHAMT-RNAi lowers the courtship index and can be rescued by application of the JH analog Methoprene.

<p>Graphs show the courtship index CI (fraction of time males spend courting during the observation period) ± SEM of the indicated genotypes (A, C), or the performance of males in a control activity assay (# of line crossings ± SEM) (B). Data were analyzed by ANOVA followed by Bonferroni multiple comparisons. A) Expression of <i>UAS-JHAMT RNAi</i> using our CA-specific <i>JHAMT-Gal4</i> driver significantly reduces male courtship in comparison to the control males. N = 20. (p < 0.001). Genotypes used were <i>UAS-dicer/Y; UAS-JHAMT-RNAi</i>/+; <i>JHAMT-GAL4/+;</i> and control genotypes <i>UAS-dicer/Y; UAS-JHAMT-RNAi/+; +/+</i> and <i>X/Y; +/+; JHAMT-GAL4/+</i>. B) Activity levels in the mutants and in control flies (genotypes as in A). Mutants are not different from the normally courting X/Y; +/+; <i>JHAMT-Gal4</i> control. (N = 10). C) Mature males of the genotypes described in (A) were treated with Methoprene in acetone or acetone alone, and tested four hours later. The experimental genotype shows complete rescue of the courtship index following Methoprene treatment compared to acetone treatment alone. (N = 20).</p

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

Mutants of the \u3cem\u3ewhite\u3c/em\u3e ABCG transporter in \u3cem\u3eDrosophila melanogaster\u3c/em\u3e 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 impairs olfactory learning for wildtype controls, while w1118 mutants are resistant to this deficit. The w1118 mutants display higher levels of cholesterol and cholesterol esters than wildtype under this low cholesterol diet. Increasing serotonin and/or dopamine levels 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

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

Drosophila&rsquo;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