Figure 6. Both dsx and fru specify the sexual dimorphism of P1^a neurons.

(A–D) Expression of CsChrimson:tdTomato under the control of P1^a-GAL4 (red in A₁–D₁, black in A_2,3-D_2,3) in brains of a male (A), fruM female (B), fruF male (C), and female (D) is visualized together with a neuropil marker BRP (blue) by immunohistochemistry. Circle: soma (right cluster is enlarged in A₃ and C₃), red asterisk: sex-invariant background labeling (see Materials and methods for details). Scale bar: 100 μm (A₁–D₁), 10 μm (A₃, C₃). (E) Mean number of cell bodies per hemibrain visualized by anti-DsRed antibody in each genotype represented in A–D) and Figure 6—figure supplement 1A–D. (F, G) Z-projection of the registered and averaged images of CsChrimson:tdTomato expression under the control of P1^a-GAL4 in male (F) and fruF male (G). A part of the standard Drosophila brain is shown in gray in F₂, G₂. Number of hemibrains used are indicated in F₂) and G₂). Lateral junction (cyan) and dorsal posterior projection (yellow) are segmented and overlaid in F₂) and G₂. H, I. Boxplot of volumes of lateral junction (H) and dorsal posterior projection (I). Genotypes and number of hemibrains are indicated below the plot. **p<0.01, n.s. p>0.05 (Kruskal-Wallis one-way ANOVA and post-hoc Mann-Whitney U-test).

Figure 6—figure supplement 1. Heterozygosity of the fru^4-40 allele leaves the neuroanatomy and function of P1^a neurons largely unaltered.

(A–D) Expression of CsChrimson:tdTomato under the control of P1^a (red in A₁–D₁), black in A_2,3-D_2,3) in representative brains of a fru wild-type male (A), fru wild-type female (B), fru^4-40 heterozygous male (C), and fru^4-40 heterozygous female (D), is visualized together with a neuropil marker BRP (blue in A₁–D₁) by immunohistochemistry. Scale bar: 100 μm. Circle: soma, red asterisk: sex-invariant background labeling.( E, F) Z-projection of the registered and averaged images of CsChrimson:tdTomato expression under the control of P1^a in fru wild-type male (E) duplication from Figure 5E) and fru^M/fru^4-40 male (F); duplication from Figure 6F). Number of used hemibrains are indicated. (G) Boxplots of time orienting (G₁), lunges (G₂), and wing extensions (G₃) performed by male tester flies that express CsChrimson:tdTomato under the control of P1^a. Only values during the time window four are shown (see Figure 1D). Testers’ fru locus genotypes and pair numbers are indicated below the plots. Target flies are either group-housed wild-type males or mated females, as indicated. Plots with gray shades are replots of the data sets shown in Figure 5—figure supplement 1A,B. **p<0.01, n.s. p>0.05 (Kruskal-Wallis one-way ANOVA and post-hoc Mann-Whitney U-test).

Figure 6—figure supplement 2. P1^a neurons in fruF males are defective in promoting both courtship and aggressive behavior.

(A, B) Boxplots of time orienting (A₁, B₁), lunges (A₂, B₂), and wing extension (A₃, B₃) by the tester flies during the time windows 1–4 (see Figure 1D). Their genotypes and numbers are indicated below the plots. Gray lines represent single testers. Target flies are either group-housed wild-type males (A) or mated females (B). In gray: **p<0.01, *p<0.05, n.s. p>0.05 (Kruskal-Wallis one-way ANOVA and post-hoc Wilcoxon signed rank test). In black: **p<0.01, n.s. p>0.05 (Kruskal-Wallis one-way ANOVA and post-hoc Mann-Whitney U-test). (C) Comparison of wing extension performed by flies that express CsChrimson:tdTomato under the control of P1^a in males (data from Figure 6—figure supplement 1G) or in fruF males (data from A, (B), during the time window 4. Sex of tester and target flies is indicated above the panels. Number of pairs tested is indicated below the panels. **p<0.01, n.s. p>0.05 (Mann-Whitney U-test).