Sex-specific splicing of the honeybee doublesex gene reveals 300 million years of evolution at the bottom of the insect sex-determination pathway

Sex-determination mechanisms vary greatly among taxa. It has been proposed that genetic sex-determination pathways evolve in reverse order from the final step in the pathway to the first step. Consistent with this hypothesis, doublesex (dsx), the most downstream gene in the Drosophila sex-determination cascade that determines most sexual phenotypes also determines sex in other dipterans and the silk moth, while the upstream genes vary among these species. However, it is unknown when dsx was recruited to the sex-determination pathway during insect evolution. Furthermore, sex-specific splicing of dsx, by which dsx determines sex, is different in pattern and mechanism between the moth and the fly, raising an interesting question of how these insects have kept the executor of sex determination while allowing flexibility in the means of execution. To address these questions, here we study the dsx gene of the honeybee Apis mellifera, a member of the most basal lineage of holometabolous insects. We report that honeybee dsx is sex-specifically spliced and that it produces both the fly-type and moth-type splicing forms, indicating that the use of different splicing forms of Dsx in controlling sexual differentiation was present in the common ancestor of holometabolous insects. Our data suggest that in ancestral holometabolous insects the female Dsx form is the default and the male form is generated by suppressing the splicing of the female form. Thus, it is likely that the dsx splicing activator system in flies, where the male form is the default, arose during early dipteran evolution.     

Sex determination in the Drosophila germline is dictated by the sexual identity of the surrounding soma

It has been suggested that sexual identity in the germline depends upon the combination of a nonautonomous somatic signaling pathway and an autonomous X chromosome counting system. In the studies reported here, we have examined the role of the sexual differentiation genes transformer (tra) and doublesex (dsx) in regulating the activity of the somatic signaling pathway. We asked whether ectopic somatic expression of the female products of the tra and dsx genes could feminize the germline of XY animals. We find that Tra(F) is sufficient to feminize XY germ cells, shutting off the expression of male-specific markers and activating the expression of female-specific markers. Feminization of the germline depends upon the constitutively expressed transformer-2 (tra-2) gene, but does not seem to require a functional dsx gene. However, feminization of XY germ cells by Tra(F) can be blocked by the male form of the Dsx protein (Dsx(M)). Expression of the female form of dsx, Dsx(F), in XY animals also induced germline expression of female markers. Taken together with a previous analysis of the effects of mutations in tra, tra-2, and dsx on the feminization of XX germ cells in XX animals, our findings indicate that the somatic signaling pathway is redundant at the level tra and dsx. Finally, our studies call into question the idea that a cell-autonomous X chromosome counting system plays a central role in germline sex determination.     

The sex determination gene doublesex regulates the A/P organizer to direct sex-specific patterns of growth in the Drosophila genital imaginal disc.

Each Drosophila genital imaginal disc contains primordia for both male and female genitalia and analia. The sexually dimorphic development of this disc is governed by the sex-specific expression of doublesex (dsx). We present data that substantially revises our understanding of how dsx controls growth and differentiation in the genital disc. The classical view of genital disc development is that in each sex, dsx autonomously "represses" the development of the inappropriate genital primordium while allowing the development of the appropriate primordium. Instead, we show that dsx regulates the A/P organizer to control growth of each genital primordium, and then directs each genital primordium to differentiate defined adult structures in both sexes.     

The sex-determining gene doublesex in the fly Megaselia scalaris: conserved structure and sex-specific splicing.

The well-known sex-determining cascade of Drosophila melanogaster serves as a paradigm for the pathway to sexual development in insects. But the primary sex-determining signal and the subsequent step, Sex-lethal (Sxl), have been shown not to be functionally conserved in non-Drosophila flies. We isolated doublesex (dsx), which is a downstream step in the cascade, from the phorid fly Megaselia scalaris, which is a distant relative of D. melanogaster. Conserved properties, e.g., sex-specific splicing, structure of the female-specific 3' splice site, a splicing enhancer region with binding motifs for the TRA2/RBP1/TRA complex that activates female-specific splicing in Drosophila, and conserved domains for DNA-binding and oligomerization in the putative DSX protein, indicate functional conservation of dsx in M. scalaris. Hence, the dsx step of the sex-determining pathway appears to be conserved among flies and probably in an even wider group of insects, as the analysis of a published cDNA from the silkmoth indicates.     

Runaway evolution of the male-specific exon of the doublesex gene in Diptera

In Diptera (Insecta), alternatively spliced male-specific and female-specific products of the doublesex (dsx) gene play a key role in regulating development of the adult genital structures from the genital disc. Analysis of the pattern of nucleotide substitution of different domains of the dsx gene in 29 dipteran species showed that, over short evolutionary times, purifying selection predominated on the domain common to both sexes, the female-specific exons, and the and male-specific exon. However, over longer the evolutionary time frames represented by between-family comparisons, the male-specific exon accumulated nonsynonymous substitutions at a much more rapid rate than either the common domain or the female-specific exon. Overall, the accumulation of nonsynonymous substitutions in the male-specific exon occurred at a significantly greater than linear rate relative to the common domain, whereas the accumulation of nonsynonymous substitutions in the female-specific exon occurred at less than linear rate relative to the common domain. The evolution of the male-specific exon of dsx thus shows a pattern reminiscent of that seen in the "runaway" evolution of male secondary sexual characters at the morphological level, consistent with the hypothesis that female choice is an important factor in the morphological diversification of insect male genitalia.     

The genital disc of Drosophila melanogaster

 The genital disc of Drosophila, which gives rise to the genitalia and analia of adult flies, is formed by cells from different embryonic segments. To study the organization of this disc, the expressions of segment polarity and homeotic genes were investigated. The organization of the embryonic genital primordium and the requirement of the engrailed and invected genes in the adult terminalia were also analysed. The results show that the three primordia, the female and male genitalia plus the analia, are composed of an anterior and a posterior compartment. In some aspects, each of the three primordia resemble other discs: the expression of genes such as wingless and decapentaplegic in each anterior compartment is similar to that seen in leg discs, and the absence of engrailed and invected cause duplications of anterior regions, as occurs in wing discs. The absence of lineage restrictions in some regions of the terminalia and the expression of segment polarity genes in the embryonic genital disc suggest that this model of compartmental organization evolves, at least in part, as the disc grows. The expression of homeotic genes suggests a parasegmental organization of the genital disc, although these genes may also change their expression patterns during larval development. Received: 4 February 1997 / Accepted: 22 May 1997     

The hierarchical relation between X-chromosomes and autosomal sex determining genes in Drosophila

The classical balance concept of sex determination in Drosophila states that the X-chromosome carries dispersed female-determining factors. Besides, a number of autosomal genes are known that, when mutant, transform chromosomal females (XX) into pseudomales (tra), or intersexes (ix, dsx, dsx). To test whether large duplications of the X-chromosome have a feminizing effect on the sexual phenotype of these mutants, we constructed flies that were mutant for ix, dsx, dsx or tra and had two X-chromosomes plus either a distal or a proximal half of an X-chromosome. These or even smaller X-chromosomal fragments had a strong feminizing effect when added to triploid intersexes (XX; AAA). In the mutants, however, no shift towards femaleness was apparent. We conclude that enhancing the female determining signal is ineffective in flies that are mutant for an autosomal sex determining gene, and therefore, that these genes are under hierarchical control of the signal given by the X:A ratio. Parallels between sex-determining and homeotic genes are drawn.     

The development of the Drosophila genital disc

The imaginal discs of Drosophila melanogaster, which form the adult epidermal structures, are a good experimental model for studying morphogenesis. The genital disc forms the terminalia, which are the most sexually dimorphic structures of the fly. Both sexes of Drosophila have a single genital disc formed by three primordia. The female genital primordium is derived from 8(th) abdominal segment and is located anteriorly, the anal primordium (10 and 11(th) abdominal segments) is located posteriorly, and the male genital primordium from the 9(th) abdominal segment lies between them. In both sexes, only two of these three primordia develop to form the adult terminalia. The anal primordium develops in both sexes but, depending on the genetic sex, will form either male or female analia. However, only one of the genital primordia develops in each sex, forming either the male or the female genitalia. This depends on the genetic sex of the fly. Therefore, the genital disc is a very good experimental model of how the sex-determination and homeotic genes - which determine cell identity - interact to direct the development of a population of cells into male or female terminalia. It has been proposed that the sexually dimorphic development of the genital disc is the result of an integrated genetic input, made up by the sex-determination gene doublesex and the homeotic gene Abdominal-B. This input acts by modulating the response to Hedgehog, Wingless, and Decapentaplegic morphogenetic signals.     

The Orthologue of the Fruitfly Sex Behaviour Gene Fruitless in the Mosquito Aedes aegypti: Evolution of Genomic Organisation and Alternative Splicing

In Drosophila melanogaster the doublesex (dsx) and fruitless (fru) regulatory genes act at the bottom of the somatic sex determination pathway. Both are regulated via alternative splicing by an upstream female-specific TRA/TRA-2 complex, recognizing a common cis element. dsx controls somatic sexual differentiation of non-neural as well as of neural tissues. fru, on the other hand, expresses male-specific functions only in neural system where it is required to built the neural circuits underlying proper courtship behaviour. In the mosquito Aedes aegypti sex determination is different from Drosophila. The key male determiner M, which is located on one of a pair of homomorphic sex chromosomes, controls sex-specific splicing of the mosquito dsx orthologue. In this study we report the genomic organization and expression of the fru homologue in Ae. aegypti (Aeafru). We found that it is sex-specifically spliced suggesting that it is also under the control of the sex determination pathway. Comparative analyses between the Aeafru and Anopheles gambiae fru (Angfru) genomic loci revealed partial conservation of exon organization and extensive divergence of intron lengths. We find that Aeadsx and Aeafru share novel cis splicing regulatory elements conserved in the alternatively spliced regions. We propose that in Aedes aegypti sex-specific splicing of dsx and fru is most likely under the control of splicing regulatory factors which are different from TRA and TRA-2 found in other dipteran insects and discuss the potential use of fru and dsx for developing new genetic strategies in vector control.