Fig 1.
Physiological parameters underlying sexually dimorphic growth.
(A–D) Growth curve of male and female larvae throughout larval development, showing there is no sex difference in the overall growth period (A), body size at larval hatching (B), or during L1 (C), with a difference in body size first apparent during L2. Graph in (A) shows raw body mass measurements at each time point (dots) and a line of best fit (locally weighted scatterplot smoothing [LOWESS] curve). (E) Ratio of female to male (F:M) body mass during larval development calculated from time-course data in (A). Individual points show F:M ratios from raw body mass measurements. Solid line shows F:M ratios calculated from lines of best fit in (A). (F) Absolute growth rate throughout larval development, calculated as the slope of the growth curve in (A). (G) Mass-specific growth rate throughout larval development, calculated from the data in (A) as a ratio corresponding to the fold growth per unit body mass over 8-h time intervals. Panels A, E, and F show a time line of larval development depicting the 3 instars (L1–L3) and critical weight (CW, vertical blue line). Developmental time, in this and subsequent figures, is measured as hours (h) after larval hatching (ALH). In contrast to this study, previous studies on SSD have focused on the period after CW (see Discussion). The underlying data for this figure can be found in S1 Data.
Fig 2.
Sxl controls the sexual size dimorphism (SSD) of the larval body.
(A) Schematic of the sex determination pathway in Drosophila and its control of sexual differentiation and dosage compensation. Genes/proteins active in females are displayed in red; genes/proteins active in males are displayed in blue. (B) Transheterozygous SxlM1, Δ33/Sxlf7, M1 mutant female adults are masculinised in terms of morphology and body size (middle panel). Both morphology and body size can be rescued by a wild-type copy of the Sex-lethal (Sxl) gene, Sxl+tCa (right panel). (C) SxlM1, Δ33/Sxlf7, M1 mutant female larvae are also masculinised in terms of larval body mass, and this can be rescued by Sxl+tCa, showing that Sxl functions to control larval SSD. Individual data points, means, and SD are plotted. Asterisks denote significant changes according to a 1-way ANOVA with multiple comparisons (** p < 0.01, **** p < 0.0001), otherwise P values >0.05 are shown. The underlying data for this figure can be found in S1 Data.
Fig 3.
Sxl functions in the nervous system to control larval sexual size dimorphism (SSD).
(A) RNAi-mediated knockdown of Sex-lethal (Sxl) in all neurons (elavc155-Gal4) specifically reduces female body mass in wandering L3 larvae (wL3). This abolishes sex differences in body mass, leading to a female to male ratio (F:M ratio) near 1. Body mass data shows representative results of 3 independent experiments, F:M ratio data shows mean ratio, individual ratios, and SEM of 3–4 independent experiments. (B) Rescue of wL3 body mass of SxlM1, Δ33/Sxlf7, M1 mutant females by neuronal expression of upstream activation sequence (UAS)-Sxl (Insc>Sxl) but not of the female splice variant of transformer, UAS-TraF (Insc>TraF). (C–D) Pan-neuronal knockdown of transformer (tra) (C) or overexpression of the female-specific tra splice variant (TraF) (D) has minor or no effects on body mass and SSD. In (C), tra RNAi 1 individual points represent means from groups of 7–18 larvae each. For (D), body mass data show representative results of 3 independent experiments, F:M ratio data plots mean ratio, individual ratios, and SEM of 3–4 independent experiments. (E) Male-specific lethal 2 (msl-2) knockdown does not rescue the female body mass of elavc155>Sxl RNAi larvae (genotype is elavc155>Sxl RNAi 1 + msl-2 RNAi). Note that elavc155>msl-2 RNAi has no effect on female body mass (p = 0.994) but leads to a very small decrease in male body mass with or without Sxl RNAi (p = 0.0028 and p = 0.049, respectively). Graph shows results representative of 3 independent experiments. (F) Neuronal expression of Msl-2 (elavc155>Msl-2::HA) does not significantly decrease female body mass. Individual points represent means from groups of 10 larvae. Unless otherwise noted, graphs of body mass plot mean, SD, and individual data points, and F:M ratio graphs plot the mean ratio of female to male body mass and SEM. * p < 0.05, ** p < 0.01, and **** p < 0.0001 according to 1-way ANOVA with multiple comparisons. The underlying data for this figure can be found in S1 Data.
Fig 4.
Male-specific lethal 2 (msl-2) knockdown blocks expression of Msl-2 and acetylated histone H4 lys16 (H4K16) in elavc155>Sxl RNAi females.
(A) Expression of Msl-2 and Elav (a neuronal marker) with DNA stained by DAPI in central nervous systems (CNSs) from early L3 larvae of the indicated sex and genotype. Left panels show single confocal sections close to the neuropil, where functional neurons, not immature postembryonic neurons, predominate. Centre and right panels show higher magnification views of boxed regions in the left panels. Msl-2 in control males localises to nuclear foci, consistent with its X chromosome localisation, and it is weak/absent in control females. In elavc155>Dcr2 + Sxl RNAi 1 + msl-2 RNAi females, Msl-2 is expressed at moderate levels, and this ectopic Msl-2 is suppressed in elavc155>Dcr2 + Sxl RNAi 1 females. Bottom graph shows quantification of Msl-2 expression (mean fluorescence intensity) measured in a region of interest similar to the boxed regions. Note that mean fluorescence intensity levels in control female levels likely reflect background signal. (B) Expression of histone H4 acetylated at lysine 16 (Ac-H4K16) with DNA stained by DAPI in CNSs from early L3 larvae of the indicated sex and genotype. Left and right panels show high magnification views from similar regions of the CNS as shown in A. Ac-H4K16 in control males strongly localises to nuclear foci (the X chromosome) and, in both sexes, lower signal intensity is observed throughout the nucleus (autosomes). Ac-H4K16 nuclear foci are observed in elavc155>Dcr2 + msl-2::HA and elavc155>Dcr2 + Sxl RNAi 1 females but not in elavc155>Dcr2 + Sxl RNAi 1 + msl-2 RNAi females. The underlying data for this figure can be found in S1 Data.
Fig 5.
Sex-lethal (Sxl) is required in insulin-producing cells (IPCs) and Gad1-Gal4 neurons to control sexual size dimorphism (SSD).
(A) Gal4-driver screen identifies 7 pan-neuronal, broad peptidergic (dimmc929, amon386Y), IPC (insulin-like peptide 2 [Ilp2]), and GABAergic* drivers with female-specific effects on body mass when combined with upstream activation sequence (UAS)-Sxl RNAi. For details and full results of screen, see S8 Fig. Left graph shows mean body mass and SD of 2–16 replicates of groups of 3–11 larvae. Hatched bars depict controls lacking the Sxl RNAi transgene, identified by the CyO, Dfd-YFP balancer, except for Gad1-Gal4 controls, which also include CyO, Dfd-YFP>Sxl RNAi larvae. In all cases, * p < 0.001 from respective no-driver controls using 2-way ANOVA with multiple comparisons. Right graph shows mean female to male (F:M) body mass ratios and SEMs for the Gal4 driver hits that decrease larval SSD. Expression analysis of elavc155-Gal4 and the other driver hits in this screen suggests that there is no shared secondary site of larval expression outside the nervous system (S1 Table). *Note that the 2 “GABAergic” drivers (Gad1-Gal4 and VGAT-Gal4) show only partially overlapping expression with GABA+ neurons (see S1 and S2 Images). (B) Ilp2-Gal4 and Gad1-Gal4 act additively to decrease female larval body mass via Sxl knockdown. Left graph shows mean body mass and SD of 3–4 replicates of groups of 6–10 larvae. Right graph shows mean F:M body mass ratios and SEMs. Knockdown of Sxl using both Ilp2-GAL4 and Gad1-Gal4 decreases female body mass and SSD more strongly than with each driver alone. Note that for Ilp2>Sxl RNAi and Ilp2 + Gad1>Sxl RNAi genotypes, results were pooled from 3 independent recombinants of Ilp2-Gal4, UAS-Sxl RNAi 1, each crossed to a no-driver control or to Gad1-Gal4, respectively. **** indicates p < 0.0001 using 1-way ANOVA with multiple comparisons. The underlying data for this figure can be found in S1 Data.
Fig 6.
Neuronal Sex-lethal (Sxl) selectively controls the sexual size dimorphism (SSD) of larval versus imaginal tissues.
Effect of neuron-specific depletion of Sxl (elavc155>Sxl RNAi 1) on SSD of the body and different tissues at larval and adult stages. For panels A–E, the histograms on the left show the male and female body or tissue sizes used to calculate the female to male (F:M) ratios in the graph on the right. (A) SSD of body mass in wandering L3 larvae (wL3) is abolished in elavc155>Sxl RNAi animals. (B) SSD of fat body nuclei diameter (a proxy for cell size) in wL3 larvae is abolished in elavc155>Sxl RNAi animals. (C) SSD of wing disc volume in wL3 larvae is not decreased in elavc155>Sxl RNAi animals. Note that 1 of 2 control genotypes has an abnormally high F:M ratio of approximately 1.6, but this is due to a decrease in male size not to a change in female size. (D) SSD of adult body mass is decreased but not abolished in elavc155>Sxl RNAi animals. (E) SSD of adult wing area is decreased but not abolished in elavc155>Sxl RNAi animals. This contrasts with lack of an SSD effect in wing discs at the end of larval development (C). (F) Images of adult flies, showing that elavc155>Sxl RNAi decreases female body size without altering female-specific pigmentation of the abdominal cuticle. For adult body and tissue measurements (D–E), animals were transferred to 18°C from pupariation to adulthood to improve adult viability and analysed 1–2 d posteclosion. All graphs of body or tissue measurements show mean, SD, and individual data points. * p < 0.05, ** p < 0.01, and **** p < 0.0001 using 1-way ANOVA with multiple comparisons. All graphs of F:M ratios show mean and SEM. The underlying data for this figure can be found in S1 Data.
Fig 7.
A relay model for the neuronal control of sexual size dimorphism (SSD) in Drosophila.
Sex-lethal (Sxl) expression in insulin-producing cells (IPCs) and other neurons of the central nervous system (CNS) acts, largely independently of the female-specific tranformer splice variant (TraF), to relay a signal(s) to peripheral larval tissues. This signal specifies the female-specific growth trajectory of larval tissues and does not appear to involve IPC-derived insulin-like peptides (Ilps) or insulin signalling. Imaginal tissue growth during larval stages is insensitive to the neuronal Sxl signal. Sxl also acts cell-autonomously via TraF in both larval and imaginal tissues to increase female growth. Red arrows indicate female-specific regulation; black arrows indicate non-sex-specific growth regulatory pathways. Additional SSD mechanisms not depicted in this model are proposed in other studies [25,43].