Fig 1.
Activation of dsx-GAL4-expressing neurons in females induces oviposition-type extension of the ovipositor and egg-laying.
(A) Extrusion of the ovipositor in response to male courtship in mated wild-type females. (B) Egg-laying in mated wild-type females. (C, C’) The oviposition posture with ovipositor extension (C) and egg-laying (C’) was artificially induced in mated females that express dTrpA1 under the control of dsxGAL4(G) by a temperature increase to 29°C. (D) The proportion of mated (left-hand graph) and virgin (right-hand graph) flies that engaged in the ovipositor extension or egg-laying upon temperature increases up to 32°C was compared among the 4 genotypes indicated at the bottom. (+) and (–) indicate the presence or absence of dsxGAL4(G) and dTrpA1 in the fly groups examined. (E) Cumulative plots of the number of dsxGAL4(G)>dTrpA1 mated females (n = 40) that exhibited ovipositor extension or egg-laying when the ambient temperature was increased from 22°C to 32°C. (F) The proportion of virgin flies (dsxGAL4(G)>dTrpA1) exhibiting ovipositor extension or egg-laying upon a temperature increase to 32°C was compared at different ages: 0 (within 24 h), 3, 5, and 8 days after eclosion. The numbers shown in parentheses (D, F) indicate the number of flies examined.
Fig 2.
Activation of dsx-GAL4 neurons in the brain induced mating-type extrusion and that outside the brain induced oviposition-type extension/egg-laying.
(A, B) Brain-restricted activation of dsx-GAL4 neurons induced mating-type extrusion of the ovipositor in a female with the genotype of UAS>stop>dTrpA1-myc/Otd-nsl:FLP; dsxGAL4(G) / + by an increase in temperature to 32°C. The ventral view (A) and lateral view (B) of a mated female displaying mating-type extrusion are shown. (C, D) Activation of dsx-GAL4 neurons outside the brain induced oviposition-type extension/egg ejection in a female with the genotype of Tub>stop>GAL80/Otd-nsl:FLP; dsxGAL4(G) UAS- dTrpA1 / + by an increase in temperature to 32°C. The ventral view (C) and lateral view (D) of a mated female displaying oviposition-type extension are shown. (E) The proportion of females that exhibited mating-type extrusion/egg-laying (left) or oviposition-type extension/egg-laying (right) upon a temperature increase to 32°C. The numbers shown in parentheses (E) indicate the number of flies examined. The drawings below the graph are nervous systems and the red colored region represents the activating sites by dTRPA1.
Fig 3.
Sex differences in dsx-GAL4-expressing neurons.
(A, B) Posterior view of a female (A) and male (B) brain in the flies expressing UAS-mCD8::GFP under the control of dsxGAL4(G). Islets in A and B are shown in anterior view. The genotype of flies used is y hs-flp;G13 UAS-mCD8::GFP;dsxGAL4(G). (C) The number of neurons contained in 11 dsx-GAL4-expressing neuron clusters was compared between the female and male brain. Values represent the mean ± s.e. (n = 12). (D) Examples of sex differences in dsx-GAL4-expressing neuron clusters. The somata of neuron clusters and single neurons indicated as MARCM clones are shown using white and yellow arrows, respectively. The brains were stained with anti-GFP (or anti-mCD8) antibodies (green) and nc82Mab (magenta). The scale bars represent 50 μm.
Fig 4.
Behavioral MARCM identifies the neurons that initiate mating-type ovipositor extrusion or oviposition-type extension/egg ejection.
(A-D) Oviposition posture and egg-laying (A) and mating-type extrusion (C) observed in flies carrying MARCM clones that express dTrpA1 in dsx-GAL4-positive cells. (B, D) Labeling pattern of GFP expression in MARCM females. Each vertical column represents scores for a single fly. Neuron classes indicated in the left-side column are described in Fig 3A, S1 Fig and S1 Table. * in B indicates that the images shown in Fig 4A, S3A and S3B Fig, and S8 Movie are for this fly, and # in D indicates that the images shown in Fig 4C, S3C and S3D Fig, and S9 Movie are for this fly. (E) The proportion of flies that carried mCD8::GFP-labeled dTrpA1-expressing cells in the indicated cluster is compared between the non-responder group (Responder-N, n = 84) and responder group (Responder-MO, n = 67). The responder group included flies that exhibited either oviposition-type extension/egg-laying or mating-type extrusion. (F) The proportion of flies that carried mCD8::GFP-labeled dTrpA1-expressing cells in the indicated cluster is compared between the fly group that responded with oviposition-type extension/egg-laying (Responder-O, n = 38) and the fly group that did not show oviposition-type extension/egg laying (Responder-NM, n = 113). (G) The proportion of flies that carried mCD8::GFP-labeled dTrpA1-expressing cells in the indicated cluster is compared between the fly group that responded with the mating-type extrusion (Responder-M, n = 29) and the fly group that did not show mating-type extrusion (Responder-NO, n = 122). * p < 0.05 and **** p < 0.0001, by Fisher’s exact probability test.
Fig 5.
Ectopic formation of female-specific pMN2 in the male brain by artificial expression of the cell death inhibitor p35 in dsx-expressing neurons.
(A, B) A pair of cell bodies of female-specific pMN2 neurons (yellow arrowheads in A) and their neurites (yellow arrows in B) are present in a wild-type female of y hs-flp/+; UAS-mCD8::GFP/+; dsxGAL4(G)/+. (C, D) A pair of cell bodies of male-specific pMN3 neurons (white arrowheads in C) are present in a wild-type male of y hs-flp/Y; UAS-mCD8::GFP/+; dsxGAL4(G)/+. (E-H) A pair of cell bodies of pMN2 (yellow arrowheads in G) and its neurites (yellow arrows in H) are labeled together with pMN3 neurons (white arrowheads in G) in the brain of a male fly in which cell-death has been blocked. The fly genotype is y hs-flp/Y; UAS-mCD8::GFP/UAS-p35; dsxGAL4(G)/+. In the female of y hs-flp/+; UAS-mCD8::GFP/UAS-p35; dsxGAL4(G)/+, the cell bodies of pMN2 neurons and their neurites are observed (yellow arrowheads in E and yellow arrows in F, respectively). Brains were doubly stained with anti-GFP (green) and nc82 mAb (magenta). The scale bar represents 50 μm.
Fig 6.
dsx-expressing neurons in dsx mutants.
(A, B) A pair of cell bodies of female-specific pMN2 neurons (yellow arrowheads in A) and their neurites (yellow arrows in B) are present in a control XX female of UAS-mCD8::GFP /+; dsxGAL4(B)/TM6b. (dsxGAL4(B); the GAL4 knock-in null allele of the dsx gene was generated by the Baker group at Janelia Farm Research Campus (Ashburn, VA)). (C, D) A pair of cell bodies of male-specific pMN3 neurons (white arrowheads in C) are present in a wild-type male (XY) of UAS-mCD8::GFP/+; dsxGAL4(B)/TM6b. (E, F) In the female (XX) of UAS-mCD8::GFP/+; dsxGAL4(B)/dsx15, the cell bodies of pMN2 neurons and their neurites are observed (yellow arrowheads in E and yellow arrows in F, respectively). (G, H) A pair of cell bodies of pMN2 (yellow arrowheads in G) and its neurites (yellow arrows in H) are seen ectopically in addition to male-specific pMN3 (white arrowheads in G) in the brain of a male fly (XY) with the genotype UAS-mCD8::GFP/+; dsxGAL4(B)/dsx15. Brains were doubly stained with anti-GFP (green) and nc82 mAb (magenta). The scale bar represents 50 μm.
Fig 7.
Activation of pC1 and pC2l neurons induces courtship in males.
Male flies that carry dTrpA1-expressing MARCM clones were tested for the occurrence of courtship behavior in response to temperature increases, followed by immunohistochemical identification of neurons that were activated in the behavioral assays. We judged that a male responded to a temperature increase by courtship behavior (“Responder-C”) only when it vibrated its wings, while displaying any additional courtship actions, i.e., licking, tapping, and abdominal bending for copulation, and excluded those flies that exhibited wing displays alone without any additional element of courtship behavior, from the count of males positive for courtship responses. (A) Courtship behavior induced by a temperature increase to 35°C in a male carrying MARCM clones expressing dTrpA1. Note that the male is vibrating a wing while licking by extending his proboscis. (B) Labeling pattern of GFP expression in MARCM males. Each vertical column represents the scores of a single fly. Neuronal classes in the brain indicated in the left-hand column are described in Fig 3B and S1 Fig and S1 Table and those in the VNC are described in Ref. 19. § in B indicates that the images shown in Fig 7A, S3G and S3H Fig, and S10 Movie are of this fly. (C) The incidence of GFP-labeling of the indicated neural cluster (signifying dTrpA1 expression) was compared between the Responder-C (n = 51) and the rest of the tested flies (non-Responder, n = 114). GFP-labeling, and thus dTrpA1-mediated activation of pC1 and pC2l but none of the other clusters, was significantly correlated with the occurrence of courtship behavior in mosaic males. **** p < 0.0001, by Fisher’s exact probability test.