Volume 61, Issue 4, Pages 519-526 (February 2009) Control of the Postmating Behavioral Switch in Drosophila Females by Internal Sensory Neurons  Chung-hui Yang, Sebastian Rumpf, Yang Xiang, Michael D. Gordon, Wei Song, Lily Y. Jan, Yuh-Nung Jan  Neuron  Volume 61, Issue 4, Pages 519-526 (February 2009) DOI: 10.1016/j.neuron.2008.12.021 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 ppk-GAL4 Labels SP-Responsive Neurons (A and B) Behavioral effects of mSP expression in fru neurons or subsets thereof (by means of fru-GAL4, Or67d-GAL4, or ILP7–GAL4) and ppk-GAL4 on mating behavior and egg laying in virgin females. (A) Receptivity of virgin females, with the score for rejection behavior as well as genotype of experimental animals given below the graph. In each assay, one female of the indicated genotype was confronted with two naive males in a small chamber. Females were scored as receptive when they mated within 20 minutes. Numbers in parentheses are the numbers of females tested. For scoring rejection behavior, ++ indicates frequent rejection behaviors (such as ovipositor protrusion, kicking, wing flicking or running away) when the females were courted, +/− indicates some rejection behaviors, − indicates absence of rejection. ∗∗p < 0.005, ∗∗∗p < 0.0005, two-tailed Fisher's exact test. (B) Egg laying. For each assay, five females of the indicated group were allowed to lay eggs in a vial with grape media at 25°C. Eggs were counted after 24 hr and the number of eggs in each vial was divided by five. n = 15 assays for all genotypes, error bars indicate SEM, ∗p < 0.05, ∗∗∗p < 0.0005, Student's t test. (C and D) Knockdown of SPR in ppk neurons reduces postmating behaviors in mated females. (C) Effect of SPR RNAi (UAS-SPR-IR) in ppk neurons on receptivity of virgin and mated females. The receptivity of virgins of the indicated genotypes was assayed as in (A), and receptivity of the same females after mating was assessed 24 hr later. ∗∗p < 0.005, ∗∗∗p < 0.0005, Fisher's exact test. v and m indicate virgin and mated, respectively. (D) Effect of SPR RNAi expression under ppk-GAL4 on egg laying of virgins or mated females. Egg laying was scored as in (B), but with three females per vial, ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005, Student's t test. n = 15 for each genotype. In (C) and (D), wild type (w1118) females that were mated to SP mutant males (SP0/Δ) or control males (SP+/Δ) were included as additional controls. (E and F) SPR expression in ppk neurons rescues the behavioral defects of the SPR-deficient line Df(1)Exel6234 (ΔSPR). (E) Mating behavior of control Df(1)Exel6234 females and Df(1)Exel6234 females expressing SPR in ppk neurons. Mating assays were performed as in (C). (F) Egg-laying behavior of females of the same genotype was determined as in (D). Neuron 2009 61, 519-526DOI: (10.1016/j.neuron.2008.12.021) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 ppk-GAL4 and fru-GAL4 Expression Overlap in Sensory Neurons on the Female Reproductive Tract In (A–F), cell bodies and cellular processes were visualized by expression of nuclear DsRed (UAS-Red stinger) and membrane bound GFP (UAS-mCD8-GFP) under the control of ppk-GAL4, followed by mCD8 staining. (A and B) Projections, but no cell bodies of ppk neurons are present in the brain (A) and the ventral nerve chord (VNC) (B). (C) Schematic diagram of the Drosophila female reproductive tract. Green dots indicate the positions of ppk-positive neurons, yellow dots those of fru/ppk neurons. (D–F) ppk-positive neurons on the female reproductive tract. (D) Overview of a reproductive tract with stained ppk neurons. The inset (D′) is a magnified picture of the common oviduct that shows dendrites emanating from ppk neurons on the uterus. (E) and (F) show the lateral oviducts and the uterus, respectively. In (E), cell bodies are marked by arrows. Scale bars are 300, 75, and 100 μm in (D), (E), and (F), respectively. In (F), the ppk neuron clusters are numbered as described in the text. (G–G″ and H–H″) Overlap between fru and ppk markers on the lateral oviducts (G) or the uterus (H). ppk promoter activity was visualized with ppk-eGFP and immunofluorescence with anti-GFP antibodies. fru neurons were visualized by fru-GAL4 and UAS-Red stinger (nuclear DsRed). fru/ppk neurons are marked with arrows, ppk neurons by asterisks. The inset in (H) and (H″) shows the second ppk cluster on the uterus at higher magnification. Abbreviations: com. ov., common oviduct, lat. ov., lateral oviduct. Neuron 2009 61, 519-526DOI: (10.1016/j.neuron.2008.12.021) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 fru/ppk Double-Positive Neurons Are Important Mediators of Postmating Behaviors (A–F) Characterization of ppk-GAL80. (A and B) ppk-GAL80 suppresses UAS-mCD-8GFP expression driven by ppk-GAL4. VNCs of ppk-GAL4, UAS-mCD8GFP (A) or ppk-GAL4, UAS-mCD8GFP, ppk-GAL80 females (B) were stained with anti-mCD8 antibodies (green) and neurons with anti-HRP (red). (C and D) ppk-GAL80 does not affect general fru-GAL4 activity in the VNC. fru-GAL4 was used to drive UAS-Red stinger in the absence (C) or presence (D) of ppk-GAL80. Neurons were stained with HRP antibodies (green). (E and F) ppk-GAL80 suppresses fru-GAL4 activity on the reproductive tract. Shown are reproductive tracts of fru-GAL4, UAS-Red stinger females (E) or fru-GAL4, UAS-Red stinger, ppk-GAL80 females (F). Positions of Red stinger-positive or -negative neurons are marked by arrows. Neurons were counterstained with anti-HRP antibodies (green). (G–J) ppk-GAL80 suppresses postmating responses induced by fru-GAL4 and UAS-mSP and restores postmating behavior after SPR RNAi under fru-GAL4. (G) Female receptivity was scored as in Figure 1A. ∗∗∗p < 0.0005, Fisher's exact test. (H) Egg laying was scored as in Figure 1B (n = 17). ∗∗∗p < 0.0005, Student's t test. Error bar indicates SEM. (I and J) UAS-SPR-IR (SPR RNAi) driven by fru-GAL4 suppresses postmating behaviors in mated females, and ppk-GAL80 restores them. (I) Receptivity. Two to three mated females were put in a vial with three to four naive males and allowed to mate for 20 min. Vials were inspected for copulating animals after 5, 10, and 20 min. ∗∗∗p < 0.0005, two-tailed Fisher's exact test. (J) Egg laying. Three mated females per vial were allowed to lay eggs in a grape vial for 24 hr. n = 15, ∗∗∗p < 0.0005, Student's t test. Error bar indicates SEM. (K and L) Clonal mSP expression in fru neurons and behavioral analysis. (K) Experimental outline. (L) Pictures of the uterus region of receptive and rejecting flies, respectively. ∗∗∗p < 0.0005, n = 15, Student's t test. Errors indicate SEM. Neuron 2009 61, 519-526DOI: (10.1016/j.neuron.2008.12.021) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Silencing Neuronal Activity in fru/ppk Neurons Induces Postmating Behaviors (A and B) Behavioral effects induced by expression of shibirets in ppk neurons. (A) The receptivity of virgin females at the restrictive (32°C) or permissive (20°C) temperatures was assayed as in Figure 3I, ∗p < 0.05, ∗∗∗p < 0.0005, Fisher's exact test. (B) Virgin egg laying at the restrictive (29°C) or permissive (20°C) temperatures, as in Figure 1B, but with three females per vial, ∗∗∗p < 0.0005, Student's t test (n = 15). Error bar indicates SEM. (C–F) Behavioral effects of silencing fru neurons with shibirets and influence of ppk-GAL80. (C and D) Virgin receptivity was assayed as in Figure 3G. For the experiments at the restrictive temperature, vials were preincubated for one hour in a water bath, and then put back into the water bath after addition of males. ∗∗p < 0.005, ∗∗∗p < 0.0005, Fisher's exact test. (E and F) Virgin egg laying at 20°C (E) or 29°C (F) was determined as above. ∗∗p < 0.005, ∗∗∗p < 0.0005, Student's t test, (n = 18 for 20°C, n = 19 for 29°C). Error bar indicates SEM. Neuron 2009 61, 519-526DOI: (10.1016/j.neuron.2008.12.021) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Manipulation of G Protein or PKA Signaling in ppk or fru/ppk Neurons Modulates Postmating Behaviors (A and B) Suppression of postmating responses in mated females by inhibition of Gαi or activation of PKA. (A) Effects on mating/remating. Mating assays with flies expressing Pertussis toxin (UAS-PTX) or the active subunit of mouse PKA (UAS-PKAmc∗) were carried out as in Figure 1C. ∗∗∗p < 0.0005, two-tailed Fisher's exact test. (B) Effects on egg laying of virgin or mated females. Experiments were performed as in Figure 2D. n = 15, ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005, Student's t test. Error bar indicates SEM. (C and D) Inhibition of PKA in fru/ppk neurons induces partial postmating responses in virgins. (C) Receptivity of virgin females was scored as in Figure 5G. ∗∗p < 0.005, Fisher's exact test. (D) Virgin egg laying, as in Figure 1B, but with three females per vial (n = 15), ∗∗p < 0.005, Student's t test. Error bar indicates SEM. (E) Model. fru/ppk neurons are active in virgins and presumably signal to the CNS to maintain virgin behaviors. After mating, SP stops synaptic outputs of fru/ppk neurons and mated behaviors are induced. Neuron 2009 61, 519-526DOI: (10.1016/j.neuron.2008.12.021) Copyright © 2009 Elsevier Inc. Terms and Conditions