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

Abstract

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.

Figure 1. Mutations in the CG4395 GPCR reduce male courtship.

Graphs show the courtship index CI (fraction of time males spend courting during the observation period) ± SEM of the indicated genotypes, or the performance of males in a control activity assay (# of line crossings ± SEM). N = 10. Data were analyzed by ANOVA followed by Tukey Kramer multiple comparisons. * p<0.001). CG4395 mutants PBac(f06077) and PBac(f04274) have a reduced CI in comparison to the control (A), but no defects in activity (B). A similar reduction in courtship is also observed when the assays are performed in red light with either a young or aged virgin female (C, D). Mutant males do not court other males, in contrast to desat1¹⁵³⁷ males (E). Expression of 4395-RNAi using the promoter construct 4395-Gal4 reduces the CI to the levels observed in the PBac mutants in a wildtype as well as a 4395 mutant background (F); activity in the mutants is not different from the control (G). The PBac(f06077) mutant phenotpye can be rescued by expressing wildtype 4395 using the 4395-Gal4 driver and two independent UAS-4395 responder lines (lines 1 and 2) (H). (I) Conditional rescue in adult males. UAS-4395 expression is restricted by the presence of tubP-Gal80^ts at 18°C (induction −). Placement of 5 day old males at 32°C for 40 hours (induction +) releases the inhibition and leads to expression of 4395. Adult induction of 4395 leads to the rescue of the mutant phenotype.

Figure 2. CG4395-Gal4 expression in male brains.

(A). Z-stack of anti-β-Gal staining of isolated male brains of 4395-Gal4/UAS-lacZ^nls. (B,C). Brains of 4395-Gal4/UAS-lacZ^nls males were double-stained with anti-β-Gal (red) and anti-Dco (green). Dco is present in the cytoplasm of the cell bodies of the mushroom bodies and projections of the mushroom bodies . Optical sections are shown. Anti-β-Gal staining is seen in a number of mushroom body cell bodies (arrowhead), but absent from others (arrow). A magnification of (B) is shown in (C). (D–I). Brains of 4395-Gal4/UAS-mcD8::GFP males were double-stained with anti-GFP (green) and nc82 to visualize neurons (magenta). Projections can be seen in the mushroom bodies (D, Z-stack), the central complex (E, Z-stack) and a subset of glomeruli in the antennal lobes (F, selected optical section). Staining was observed in glomeruli DA1, VA1v, VA1d, VA6, DM3 and DL3 (F). (G–I) To visualize mb projections, double staining was performed with anti-GFP and anti-Dco (G, merged), the single channels are shown in (H) and (I); Z stacks are shown. Abbreviations: (nls) nuclear localization signal; (mcD8::GFP) membrane bound GFP that visualizes neuronal extensions. (mb) mushroom bodies; (ca) calyx of the mushroom bodies; α, β γ: α, β, γ lobes of the mushroom bodies.

Figure 3. The sexual identity of 4395 Neurons is important for courtship.

(A, B) Feminization of 4395-Gal4 expressing cells by the expression of UAS-traF reduces male courtship (A), indicating that these cells are sexually dimorphic. In contrast, conditional feminization in adult 5-day old males does not affect male courtship (B). (C). Expression of 4395-RNAi in the mushroom bodies using the P247-Gal4 driver does not affect courtship. N = 10.

Figure 4. CG4395 is required in fru neurons for male courtship.

Graphs show the courtship index CI (fraction of time males spend courting during the observation period) ± SEM of the indicated genotypes, or the performance of males in a control activity assay (# of line crossings ± SEM). N = 10. Data were analyzed by ANOVA followed by Tukey Kramer multiple comparisons. * p<0.001). (A) A mutation in CG4395 and fru interact genetically. The CI of PBac^f06077; fru⁴/+ males is significantly lower than the CI of PBac^f06077 alone. fru⁴/+ males have a normal CI. (B) Expression of 4395-RNAi by fru-Gal4 reduces the CI to the level of the PBacf⁰⁶⁰⁷⁷ mutants. The mutants have activity levels that are not different from the controls (C). (D,E) The PBac^f06077 mutant phenotype can be rescued by expression of wildtype UAS-4395 by fru-Gal4 (D). (E) Conditional adult expression of UAS-4395 by fru-Gal4 rescues the mutant phenotype. UAS-4395 expression is restricted by the presence of tubP-Gal80^ts at 18°C (induction −). Placement of 5 day old males at 32°C for 16 hours (induction +) releases the inhibition and leads to expression of 4395.

Figure 5. Localization of cells that express both fru and 4395-Gal4.

(A–F) Brains of adult UAS-lacZ^nls/lexAop::GFP^nls; 4395-Gal4/fruP1LexA males were double-stained with anti-β-Gal (magenta) and anti-GFP (green) to identify cells expressing both fru and hector. Although this GFP carries a nuclear localization signal (nls), diffusion of the protein is also seen into the projections. Co-staining is observed in numerous cell bodies in the mb area. (A, D: merged images, B, C, E, F: single images). A–C show the Z-stack of a whole brain, D–F show a representative magnified optical section in the area of the mb calyx. (G–K) Triple staining of UAS-lacZ^nls/lexAop::GFP^nls; 4395-Gal4/fruP1LexA males with anti-β-Gal (blue), anti-GFP (green) and anti Dco (red) shows that cells that express both fru and hector are mushroom body cells. Co-staining is shown in G, single channels are shown in M, N, O. (L–Q) Brains of adult UAS-mcD8::GFP/4395-Gal4; lexAop-myr-mCherry/fruP1LexA were co-stained with anti-GFP (magenta) and anti-RFP (green). The projections of the neurons can be seen. In contrast to 4395-Gal4, fru, in addition to expression in the α, β and γ lobes, is also expressed in the α′ and β′ lobes. (L′) shows a magnification at the level of the β and γ lobes that visualizes overlap as well as close proximity of the labeled projections. (O–Q) Colocalization of fru and 4395-Gal4 staining in glomeruli of the antennal lobes. 4395-Gal4 projections are visible in all fru positive glomeruli.

Figure 6. Reduction of CG4395 RNA in GH146 neurons reduces courtship.

Graphs show the courtship index CI (fraction of time males spend courting during the observation period) ± SEM of the indicated genotypes, N = 10. Expression of 4395-RNAi in GH146 projection neurons reduces male courtship in both white and red light (A, B). (C) Optical sections showing co-expression of UAS-mcD8::GFP/4395-Gal4 (magenta) and UASQ-tomato::GH146-Q (green) in antennal glomeruli. (D) CG4395^f06077 mutant males missing the third antennal segment court less than control males.