Control of sexual differentiation and behavior by the doublesex gene in Drosophila melanogaster

Abstract

Doublesex proteins, which are part of the structurally and functionally conserved Dmrt gene family, are important for sex determination throughout the animal kingdom. We inserted Gal4 into the doublesex (dsx) locus of Drosophila melanogaster, allowing us to visualize and manipulate cells expressing dsx in various tissues. In the nervous system, we detected differences between the sexes in dsx-positive neuronal numbers, axonal projections and synaptic density. We found that dsx was required for the development of male-specific neurons that coexpressed fruitless (fru), a regulator of male sexual behavior. We propose that dsx and fru act together to form the neuronal framework necessary for male sexual behavior. We found that disrupting dsx neuronal function had profound effects on male sexual behavior. Furthermore, our results suggest that dsx-positive neurons are involved in pre- to post-copulatory female reproductive behaviors.

Figure 1

Expression of GAL4 from the dsx locus recapitulates endogenous dsx expression. (a) Schematic of dsx (dsx⁺) and GAL4 knock-in allele (dsx^GAL4). Transcriptional start sites, arrows; black boxes, exons. (b) Male and female dsx^GAL4 locus predicted transcripts. (c,d) dsx^GAL4, UAS-nlacZ 2-day-old male pupa. anti-Dsx (green), anti-ßGal (magenta). (c) Brain and (d) VNC. Ventral views; anterior up. Scale bars = 50 μm. (e-l) dsx^GAL4 transformation of secondary sexual characteristics; dorsal abdominal cuticular pigmentation; external genitalia; T1 leg basitarsal detail. (e) XY and (f) XX wild-type. (g) XX dsx-null intersexual. (h) XX tra-null pseudo-male. (i) XY UAS-dsx^F;dsx^GAL4 pseudo-female. (j) XX dsx^GAL4/UAS-dsx^M pseudo-male. (k) XX UAS-tra^IR;dsx^GAL4. (l) XX dsx^GAL4/UAS-tra2^IR.

Figure 2

Sexually dimorphic expression of dsx^GAL4-neurons and associated projections and colocalization with Fru^M in 3-5 day adult CNSs. (a) Male brain, dsx neuronal clusters (arrowheads), male-specific SN neurons (only one cell in plane of focus; arrow). The cell bodies of pC1 ,pC2, and pC3 in dorsal inferomedial, inferolateral, and superomedial protocerebral areas respectively. (b,c) Male brain, SN cells position, boxed. (d-f) Female brain; (d) dsx neuronal clusters (arrowheads). (g) Male VNC, Abg cluster (arrowhead), male-specific TN1 and TN2 neurons (arrow). (h) Female VNC, Abg cluster (arrowhead). (i,j) Male VNC. (k) Female VNC, hindleg contralateral projection (arrow). (l) Female VNC. (a,d,g,h) Neuronal cell bodies expressing UAS-pStingerII (nGFP). anti-GFP (green). (b,e,i,k) Neuronal projections expressing UAS-mCD8::GFP (membrane-bound GFP), anti-mCD8 (green). (c,f,j,l) Synaptic expression; expressing UAS-synaptotagmin (pre-synaptic marker) tagged with HA, anti-HA (green). Neuropil counterstained with anti-nC82 (magenta). Ventral views; anterior top. (m) UAS-RedStinger;dsx^GAL4, UAS-mCD8::GFP male and (n) female brain. Dsx neuronal clusters (arrowheads). anti-RFP(magenta); anti-GFP (green). Horizontal view, ventral top. Scale bars = 50 μm.

Figure 3

Sex apecific dsx^GAL4 expression in the foreleg and effect of basitarsal amputations on axonal projections. (a-d) Sexually dimorphic expression in T1 foreleg. (a) Male T1 leg (medial aspect), sex comb (boxed). dsx^GAL4 expressing cells in metatarsus = 96 ± 14.4 (n=7) and tarsi 2-5 = 72 ± 10.0 (n=7) (b) Medial and (c) lateral aspect male T1 tarsi and metatarsus, sex comb (boxed). (d) Female T1 tarsi and metatarsus (lateral aspect), area consistent with sex comb (boxed). dsx^GAL4 expressing cells in metatarsus = 77 ± 12.4 (n=8) and tarsi 2-5 = 58 ± 9.7 (n=7) (e) Male T1 tarsi and metatarsus (medial aspect), elav-GAL80 repression in subset of dsx^GAL4 cells, metatarsal sex comb (boxed). (f) Wild-type male and (h) wild-type female prothoracic axonal projections (close-up from Fig. 2k). (g) Atrophied male and (i) atrophied female prothoracic axonal projections, post-amputation. Point of amputation, arrowheads (a). Scale bar = 50 μm.

Figure 4

Co-localization of Fru^M neuronal cells and dsx^GAL4 cells (expressing UAS-nGFP) in 3-day-old adult male. anti-nGFP (green), anti-Fru^M (magenta). (a) Brain; Dorsal view; anterior up. dsx and Fru^M neuronal cells and clusters designated. (b) VNC. Ventral view; anterior up. dsx and Fru^M neuronal cells and clusters that colocalize, designated. Fru^M clusters as previously described. Scale bars = 50 μm.

Figure 5

dsx^GAL4 expression in CNSs of Fru^M-null males and females expressing Fru^M or the anti-apoptotic transgene UAS-p35. (a,b) Schematic of dsx^GAL4 nGFP expression in wild-type adult male (a) and female (b) CNSs. Individual neuronal clusters designated. (c) nGFP and (d) Membrane-bound GFP in Fru^M-null adult male brain. (e) nGFP and (f) Membrane-bound GFP in Fru^M-null adult male VNC, reduction in prothoracic contralateral (boxed area) and cervical connection projections (arrowhead). (g) nGFP and (h) membrane-bound GFP in adult female brain expressing Fru^M. (i) nGFP and (j membrane-bound GFP in adult female VNC expressing Fru^M. (k) nGFP and (l) membrane-bound GFP in adult female brain expressing both Dsx^F and Dsx^M. Supernumerary SN cells (boxed area). (m) nGFP adult female VNC expressing both Dsx^F and Dsx^M. Supernumerary male-specific TN1 and TN2 cells (boxed area, arrow). (n) Membrane-bound GFP in adult female brain expressing both Dsx^F and Dsx^M. Prothoracic contralateral projections (boxed area). (o) UAS-p35;dsx^GAL4 adult female brain, supernumerary SN cells (boxed area). (p) UAS-p35;dsx^GAL4 adult female VNC, supernumerary TN1 (arrowheads) and TN2 (boxed area) neurons. (q-s) UAS-nLacZ,-mCD8::GFP,-p35;dsx^GAL4 adult female prothoracic ganglion (Prg) (q) Merged image, co-expression of membrane-bound GFP, green (r) and nuclear ßGal, magenta (s). Supernumerary TN1 (arrowheads) and TN2 cells (boxed area) and ectopic contralateral projections. Ventral views; anterior up. (c, e, g, i, k, m) anti-GFP, green. (d, f, h, j, l, n, o, p) anti-mCD8, green and neuropil counter-stained with anti-nC82, magenta. Genotypes as per Methods. Scale bar = 50 μm

Figure 6

dsx^GAL4 neurons control male sexual behavior. (a) Male fertility. p-values * <0.05, ** <0.0001 (Fisher Exact Test). (b) Courtship initiation. Mean ± SEM. p-values * <0.05 (Tukey-Kramer HSD statistical test). (c) Courtship index. Mean ± SEM. p-values * <0.05 (Tukey-Kramer HSD test). (d) Percentage males mating in 4-h. p-values ** <0.0001 (Fisher Exact Test). (e) Song-recording, 5-7 day old males. Pulse (P) and sine (S) song components indicated above traces, courtship display below. Each trace represents a fraction of a 10 min recording. Scale bar= 200 ms. Genotypes indicate males. Target females, wild-type. Wild-type males, (15); 18.6 ± 2.2 sine bouts per min (SBPM), 19.9 ± 1.4 pulse trains per min (PTPM), 8.1 ± 0.3 mean pulses per train (MPPT), 31.7 ± 3.0 interpulse interval (IPI, ms). UAS-TNT_G; dsx^GAL4 males, (10); no recordable data. UAS-TNT_G; dsx^GAL4, elav-GAL80 males, (10); 18.4 ± 1.5 (SBPM), 26.6 ± 3.8 (PTPM), 10.0 ± 0.4 (MPPT), 34.0 ± 0.4 (IPI, ms). Mean ± SEM. n’s in parentheses.

Figure 7

dsx^GAL4 neurons control female sexual behavior. (a) Female fertility. p-values ** <0.0001 (Fisher Exact Test). (b) Egg-laying. Mean ± SEM. p-values * <0.0001 (Dunnett’s Test). (c) Percent copulation over time (10 minute intervals for 1 h). (d) Line crossings during copulation. Mean ± SEM. p-values * <0.05 (Tukey-Kramer HSD test). (e) Copulation duration. Mean ± SEM. p-values * <0.05 (Tukey-Kramer HSD test). (f) Percentage females re-mating with the same male in 4 h. p-values * <0.05 (Tukey-Kramer HSD test). Genotypes indicate females. Target males, wild-type. n’s in parentheses.