Evolution of sex-specific traits through changes in HOX-dependent doublesex expression

Abstract

Almost every animal lineage is characterized by unique sex-specific traits, implying that such traits are gained and lost frequently in evolution. However, the genetic mechanisms responsible for these changes are not understood. In Drosophila, the activity of the sex determination pathway is restricted to sexually dimorphic tissues, suggesting that spatial regulation of this pathway may contribute to the evolution of sex-specific traits. We examine the regulation and function of doublesex (dsx), the main transcriptional effector of the sex determination pathway, in the development and evolution of Drosophila sex combs. Sex combs are a recent evolutionary innovation and show dramatic diversity in the relatively few Drosophila species that have them. We show that dsx expression in the presumptive sex comb region is activated by the HOX gene Sex combs reduced (Scr), and that the male isoform of dsx up-regulates Scr so that both genes become expressed at high levels in this region in males but not in females. Precise spatial regulation of dsx is essential for defining sex comb position and morphology. Comparative analysis of Scr and dsx expression reveals a tight correlation between sex comb morphology and the expression patterns of both genes. In species that primitively lack sex combs, no dsx expression is observed in the homologous region, suggesting that the origin and diversification of this structure were linked to the gain of a new dsx expression domain. Two other, distantly related fly lineages that independently evolved novel male-specific structures show evolutionary gains of dsx expression in the corresponding tissues, where dsx may also be controlled by Scr. These findings suggest that changes in the spatial regulation of sex-determining genes are a key mechanism that enables the evolution of new sex-specific traits, contributing to some of the most dramatic examples of phenotypic diversification in nature.

Figure 1. Dsx and Scr expression during sex comb development in D. melanogaster.

Immunostaining with anti-Scr (red) and anti-DsxC (green) antibodies. Ti, tibia; ta, tarsus; AP, after pupariation. All panels except (E) and (F) are merged images. (A) L3 male T1 leg disc. Anterior is to the left, dorsal is up. Scr is expressed at a high level in the anterior part of distal Ti and ta1 (arrow) and in a more proximal region corresponding to the presumptive body wall (arrowhead). Low expression is present in the rest of the disc. (B) Wandering male T1 leg disc. Dsx is expressed in the distal part of the Scr domain (overlap in yellow) and in the more central region (arrow). Inset shows a magnified view of the boxed area. (C) Wandering female T1 leg disc. (D) Wandering male T2 leg disc. The only detectable Scr expression is subepidermal. (E–H) 5 h AP T1 leg (E–G, male; H, female). The tarsal segments are numbered. Dsx is strongly expressed on the ventral-anterior side of distal ta1 in both sexes (arrow). (F) In the male, Dsx is expressed in the distal ta1 and in small dorsal and ventral patches in ta2–4 (asterisks). (H) In the female, Dsx expression is only in the distal ta1 and is weaker than in the male. (I–K) 16 h AP T1 leg (I, J, male; K, female). Arrows point to the rotating sex comb. Note the absence of Dsx expression in ta2. High background staining is caused by the pupal cuticle, which is still attached to the epidermis at this stage. (L–N) 24 h AP T1 leg. (L, M, male; N, female). (O) 36 h AP male T1 leg. (P, Q) Scanning electron micrographs of the distal ta1 in the adult male (P) and female (Q). Ventral is to the right and anterior is facing out of the page.

Figure 2. Transcriptional regulation of dsx.

(A–C) Localization of dsx transcripts by in situ hybridization. Anterior is to the left, dorsal is up. (A) In the wandering L3 male T1 leg disc, dsxM is expressed in an anterior proximal (arrowhead) and a distal (arrow) crescent domain. (B) No detectable signal is seen in male T2. (C) dsxM expression in the 24 h AP male T1 leg. The only epidermal expression is in the presumptive sex comb region (arrow). Strong staining in the center of the leg is non-specific. (D, E) DsxM immunostaining in rn-Gal4/UAS-dsxM male (D) and female (E) T1 leg discs is seen throughout the rn-expression domain.

Figure 3. dsx and Scr control sex comb development.

(A) Wild-type male adult T1 leg. ta, tarsus; bracket, TBRs; arrow, sex combs. (B) tub-Gal80^ts; neur-Gal4/UAS-dsxM male. The bristles in TBRs are transformed into ectopic sex comb teeth (bracket). Arrow points to the normal sex comb. (C) tub-Gal80^ts; rn-Gal4/UAS-dsxRNAi male. The sex comb is only partially rotated and has fewer and thinner teeth (arrow). (D) Scr expression in tub-Gal80^ts; rn-Gal4/UAS-dsxRNAi male at 24 h AP. Scr is down-regulated except in the cells distal to the sex comb (arrow). (E) Wild-type female adult T1 leg. Bracket, TBRs. (F) tub-Gal80^ts; neur-Gal4/UAS-dsxM female. As in the male of the same genotype (B), TBR bristles assume sex comb-like morphology (bracket). (G) tub-Gal80^ts; rn-Gal4/UAS-dsxM female. The two most distal TBRs develop into partially rotated sex combs (arrows). (H) Scr expression in tub-Gal80^ts; rn-Gal4/UAS-dsxM female T1 leg at 24 h AP. Scr is up-regulated in cells distal to the ectopic sex comb (arrow); compare to (D). (I) tub-Gal80^ts; rn-Gal4/UAS-Scr male. Ectopic sex combs are formed on distal tarsal segments (arrow). (J) tub-Gal80^ts; rn-Gal4/UAS-ScrRNAi male. The sex comb and TBR are lost from the distal part of ta1, where rn is expressed (bracket). (K) tub-Gal80^ts; neur-Gal4/UAS-ScrRNAi male. The number of teeth is reduced, but tooth morphology is normal (arrow). (L) T1 leg disc of tub-Gal80^ts/UAS-Gal4; rn-Gal4/UAS-ScrRNAi male. No Dsx is detectable. (M) T2 leg disc of tub-Gal80^ts; rn-Gal4/UAS-Scr male at the wandering stage. Ectopic Dsx expression is detected throughout the rn expression domain.

Figure 4. Dsx and Scr expression in the melanogaster species group.

ta1–2 of adult male T1 legs are shown on the left. Scr (red) and Dsx (green) immunostaining of the same segments in mid-pupal male T1 legs are shown in the right panels. Developing sex combs are indicated by arrows (longitudinal combs) or arrowheads (small and transverse combs). In all species, Dsx expression is highest in the sex comb teeth, while Scr is low in the sex comb teeth but high in the surrounding cells. (A) D. ficusphila. (B) D. biarmipes. (C) D. takahashii. (D) D. nikananu. (E) D. kikkawai. (F) D. bipectinata. (G) D. malerkotliana. (H) Phylogenetic relationships among the species shown in this figure. The latest common ancestor of D. kikkawai and D. nikananu had a sex comb similar to that of D. kikkawai; the latest common ancestor of D. malerkotliana and D. bipectinata had a sex comb similar to D. malerkotliana (Barmina and Kopp 2007) .

Figure 5. Dsx expression in distantly related lineages.

Dsx immunostaining is in green. (A) Simplified phylogeny of the species shown in this figure and Figure 6. (B) Male T1 leg disc of S. lebanonensis. (C) Male T1 leg disc of D. busckii. (D) Adult male T1 leg of D. pseudoobscura carries sex combs on the ta1 and ta2 segments. (E) Dsx expression in the corresponding segments of the male T1 leg at the early pupal stage. (F–L) D. willistoni. (F) Adult male T1 leg. Note the absence of sex combs and the very small number of long and curved chemosensory bristles (compare to M). (G) Male T1 leg disc stained with the DsxC antibody. Arrowhead, an expression domain unique to the male T1 disc. (H) Male T2 leg disc stained with the DsxC antibody. (I) Female T1 leg disc stained with the DsxC antibody. (J) Adult male brain stained with the DsxC antibody, showing the PC1 (arrow) and PC2 (arrowhead) neuronal clusters. (K) Male T1 prepupal leg at 5 h AP stained with the DsxC antibody. (L) Male T1 pupal leg at 24 h AP. (M–Q) D. hydei. (M) Adult male T1 leg. (N) Male T1 leg disc. Arrow and arrowhead point to the dorsal and ventral expression domains respectively. (O) Male T2 leg disc. (P) Male T1 prepupal leg at 8 h AP. The two domains are still visible (arrow, arrowhead). (Q) Male T1 pupal leg at 40 h AP.

Figure 6. Dsx expression in species that evolved lineage-specific sexually dimorphic structures.

Dsx immunostaining is in green. (A–G) D. immigrans. (A, B) Adult male and female T1 legs, respectively. (C) Male T1 leg disc. (D) Male T2 leg disc. (E) Male T1 prepupal leg. (F, G) Male and female T1 pupal legs, respectively, at 48 h AP. (H–L) Zaprionus tuberculatus. (H, I) Adult male and female T1 legs, respectively. (J) Male-specific brush structure shown at higher magnification. (K) Male T1 leg disc. Arrow, dsx expression domain. (L) Male T1 pupal leg at 48 h AP.

Figure 7. A model for the origin of a new sex-specific developmental pathway.

Ancestral regulatory interactions are indicated in black, and newly evolved interactions in red. In the ancestral condition (left), leg patterning genes lay down the basic bristle pattern and establish a domain of high Scr expression on the ventral-anterior surface of the distal Ti and ta1. High levels of Scr organize the ventral-anterior bristles into TBRs. dsx is not expressed in the TBRs so they develop in a sexually monomorphic manner. In the melanogaster-obscura clade (right), dsx was recruited into the TBR development pathway under the control of both Scr and leg patterning genes. Scr activates dsx in T1 at the late larval stage, while dsx modulates Scr at the pupal stage to make its expression sexually dimorphic in some species. Both genes have acquired new downstream targets involved in bristle patterning and morphogenesis.