The orthologue of the fruitfly sex behaviour gene fruitless in the mosquito Aedes aegypti: evolution of genomic organisation and alternative splicing.
ABSTRACT: 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.
Project description:Sex determination in tephritid fruit flies involves a signaling cascade of alternatively spliced genes. The Transformer (TRA) and Transformer-2 (TRA-2) complex establishes an autoregulatory loop switching sex-specific splicing of tra pre-mRNA in females. The TRA/TRA-2 complex also regulates the sex-specific splicing of downstream effector genes, doublesex (dsx) and fruitless (fru). In Ceratitis capitata, a Maleness-on the-Y (MoY) gene modulates sex-specifically spliced Cctra pre-mRNA and results in the breakdown of the Cctra autoregulatory loop in males. In this study, the tra-2 and fru genes were characterised in two key pests, Bactrocera dorsalis and B. correcta. The tra-2 genes showed high degrees of conservation among tephritids. The complex gene organisation for each of Bdfru and Bcfru were identified. There are sex-specific and non sex-specific transcripts generated by alternative promoters as found in Drosophila melanogaster and other insects. RNAi knockdown of Bdtra transcripts showed that BdTRA controls the sex-specific splicing of Bddsx and Bdfru pre-mRNAs. Developmental expression analysis shows that multiple splice variants of Bdtra and Bctra RNAs are present before and during cellular blastoderm formation and that the mature sex-specific variants become fixed later in embryogenesis. Furthermore, the BddsxM splice variants are found in early embryos at the beginning of gastulation, but BdfruM does not appear until the larval stage. We proposed that the zygotic tra loop is initiated in both female and male embryos before becoming automatised or abolished by MoY, respectively.
Project description:Tribolium castaneum Transformer (TcTra) is essential for female sex determination and maintenance through the regulation of sex-specific splicing of doublesex (dsx) pre-mRNA. In females, TcTra also regulates the sex-specific splicing of its own pre-mRNA to ensure continuous production of functional Tra protein. Transformer protein is absent in males and hence dsx pre-mRNA is spliced in a default mode. The mechanisms by which males inhibit the production of functional Tra protein are not known. Here, we report on functional characterization of transformer-2 (tra-2) gene (an ortholog of Drosophila transformer-2) in T. castaneum. RNA interference-mediated knockdown in the expression of gene coding for tra-2 in female pupae or adults resulted in the production of male-specific isoform of dsx and both female and male isoforms of tra suggesting that Tra-2 is essential for the female-specific splicing of tra and dsx pre-mRNAs. Interestingly, knockdown of tra-2 in males did not affect the splicing of dsx but resulted in the production of both female and male isoforms of tra suggesting that Tra-2 suppresses female-specific splicing of tra pre-mRNA in males. This dual regulation of sex-specific splicing of tra pre-mRNA ensures a tight regulation of sex determination and maintenance. These data suggest a critical role for Tra-2 in suppression of female sex determination cascade in males. In addition, RNAi studies showed that Tra-2 is also required for successful embryonic and larval development in both sexes.
Project description:In Drosophila melanogaster, genes of the sex-determination hierarchy orchestrate the development and differentiation of sex-specific tissues, establishing sex-specific physiology and neural circuitry. One of these sex-determination genes, fruitless (fru), plays a key role in the formation of neural circuits underlying Drosophila male courtship behavior. Conservation of fru gene structure and sex-specific expression has been found in several insect orders, though it is still to be determined whether a male courtship role for the gene is employed in these species due to the lack of mutants and homologous experimental evidence. We have isolated the fru ortholog (Md-fru) from the common housefly, Musca domestica, and show the gene's conserved genomic structure. We demonstrate that male-specific Md-fru transcripts arise by conserved mechanisms of sex-specific splicing. Here we show that Md-fru, is similarly involved in controlling male courtship behavior. A male courtship behavioral function for Md-fru was revealed by the behavioral and neuroanatomical analyses of a hypomorphic allele, Md-tra(man) , which specifically disrupted the expression of Md-fru in males, leading to severely impaired male courtship behavior. In line with a role in nervous system development, we found that expression of Md-fru was confined to neural tissues in the brain, most prominently in optic neuropil and in peripheral sensory organs. We propose that, like in Drosophila, overt sexual differentiation of the housefly depends on a sex-determining pathway that bifurcates downstream of the Md-tra gene to coordinate dimorphic development of non-neuronal tissues mediated by Md-dsx with that of neuronal tissues largely mediated by Md-fru.
Project description:BACKGROUND: In the model system Drosophila melanogaster, doublesex (dsx) is the double-switch gene at the bottom of the somatic sex determination cascade that determines the differentiation of sexually dimorphic traits. Homologues of dsx are functionally conserved in various dipteran species, including the malaria vector Anopheles gambiae. They show a striking conservation of sex-specific regulation, based on alternative splicing, and of the encoded sex-specific proteins, which are transcriptional regulators of downstream terminal genes that influence sexual differentiation of cells, tissues and organs. RESULTS: In this work, we report on the molecular characterization of the dsx homologue in the dengue and yellow fever vector Aedes aegypti (Aeadsx). Aeadsx produces sex-specific transcripts by alternative splicing, which encode isoforms with a high degree of identity to Anopheles gambiae and Drosophila melanogaster homologues. Interestingly, Aeadsx produces an additional novel female-specific splicing variant. Genomic comparative analyses between the Aedes and Anopheles dsx genes revealed a partial conservation of the exon organization and extensive divergence in the intron lengths. An expression analysis showed that Aeadsx transcripts were present from early stages of development and that sex-specific regulation starts at least from late larval stages. The analysis of the female-specific untranslated region (UTR) led to the identification of putative regulatory cis-elements potentially involved in the sex-specific splicing regulation. The Aedes dsx sex-specific splicing regulation seems to be more complex with the respect of other dipteran species, suggesting slightly novel evolutionary trajectories for its regulation and hence for the recruitment of upstream splicing regulators. CONCLUSIONS: This study led to uncover the molecular evolution of Aedes aegypti dsx splicing regulation with the respect of the more closely related Culicidae Anopheles gambiae orthologue. In Aedes aegypti, the dsx gene is sex-specifically regulated and encodes two female-specific and one male-specific isoforms, all sharing a doublesex/mab-3 (DM) domain-containing N-terminus and different C-termini. The sex-specific regulation is based on a combination of exon skipping, 5' alternative splice site choice and, most likely, alternative polyadenylation. Interestingly, when the Aeadsx gene is compared to the Anopheles dsx ortholog, there are differences in the in silico predicted default and regulated sex-specific splicing events, which suggests that the upstream regulators either are different or act in a slightly different manner. Furthermore, this study is a premise for the future development of transgenic sexing strains in mosquitoes useful for sterile insect technique (SIT) programs.
Project description:Insects are the only known animals in which sexual differentiation is controlled by sex-specific splicing. The doublesex transcription factor produces distinct male and female isoforms, which are both essential for sex-specific development. dsx splicing depends on transformer, which is also alternatively spliced such that functional Tra is only present in females. This pathway has evolved from an ancestral mechanism where dsx was independent of tra and expressed and required only in males. To reconstruct this transition, we examined three basal, hemimetabolous insect orders: Hemiptera, Phthiraptera, and Blattodea. We show that tra and dsx have distinct functions in these insects, reflecting different stages in the changeover from a transcription-based to a splicing-based mode of sexual differentiation. We propose that the canonical insect tra-dsx pathway evolved via merger between expanding dsx function (from males to both sexes) and narrowing tra function (from a general splicing factor to dedicated regulator of dsx).
Project description:The doublesex gene controls somatic sexual differentiation of many metazoan species, including the malaria mosquito Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti (Diptera: Culicidae). As in other studied dipteran dsx homologs, the gene maintains functionality via evolutionarily conserved protein domains and sex-specific alternative splicing. The upstream factors that regulate splicing of dsx and the manner in which they do so however remain variable even among closely related organisms. As the induction of sex ratio biases is a central mode of action in many emerging molecular insecticides, it is imperative to elucidate as much of the sex determination pathway as possible in the mosquito disease vectors.Here we report the full-length gene sequence of the doublesex gene in Culex quinquefasciatus (Cxqdsx) and its male and female-specific isoforms. Cxqdsx maintains characteristics possibly derived in the Culicinae and present in the Aedes aegypti dsx gene (Aeadsx) such as gain of exon 3b and the presence of Rbp1 cis-regulatory binding sites, and also retains presumably ancestral attributes present in Anopheles gambiae such as maintenance of a singular female-specific exon 5. Unlike in Aedes aegypti, we find no evidence for intron gain in the female transcript(s), yet recover a second female isoform generated via selection of an alternate splice donor. Utilizing next-gen sequence (NGS) data, we complete the Aeadsx gene model and identify a putative core promoter region in both Aeadsx and Cxqdsx. Also utilizing NGS data, we construct a full-length gene sequence for the dsx homolog of the northern house mosquito Culex pipiens form pipiens (Cxpipdsx). Analysis of peptide evolutionary rates between Cxqdsx and Cxpipdsx (both members of the Culex pipiens complex) shows the male-specific portion of the transcript to have evolved rapidly with respect to female-specific and common regions.As in other studied insects, doublesex maintains sex-specific splicing and conserved doublesex/mab-3 domains in the mosquitoes Culex quinquefasciatus and Cx. pipiens. The cis-regulated splicing of Cxqdsx does not appear to follow either currently described mosquito model (for An. gambiae and Ae. aegypti); each of the three mosquito genera exhibit evidence of unique cis-regulatory mechanisms. The male-specific dsx terminus exhibits rapid peptide evolutionary rates, even among closely related sibling species.
Project description:Regulation of gene expression through alternative pre-mRNA splicing appears to occur in all metazoans, but most of our knowledge about splicing regulators derives from studies on genetically identified factors from Drosophila. Among the best studied of these is the transformer-2 (TRA-2) protein which, in combination with the transformer (TRA) protein, directs sex-specific splicing of pre-mRNA from the sex determination gene doublesex (dsx). Here we report the identification of htra-2 alpha, a human homologue of tra-2. Two alternative types of htra-2 alpha cDNA clones were identified that encode different protein isoforms with striking organizational similarity to Drosophila tra-2 proteins. When expressed in flies, one hTRA-2 alpha isoform partially replaces the function of Drosophila TRA-2, affecting both female sexual differentiation and alternative splicing of dsx pre-mRNA. Like Drosophila TRA-2, the ability of hTRA-2 alpha to regulate dsx is female-specific and depends on the presence of the dsx splicing enhancer. These results demonstrate that htra-2 alpha has conserved a striking degree of functional specificity during evolution and leads us to suggest that, although they are likely to serve different roles in development, the tra-2 products of flies and humans have similar molecular functions.
Project description:The Drosophila melanogaster sex hierarchy controls sexual differentiation of somatic cells via the activities of the terminal genes in the hierarchy, doublesex (dsx) and fruitless (fru). We have targeted an insertion of GAL4 into the dsx gene, allowing us to visualize dsx-expressing cells in both sexes. Developmentally and as adults, we find that both XX and XY individuals are fine mosaics of cells and tissues that express dsx and/or fruitless (fru(M)), and hence have the potential to sexually differentiate, and those that don't. Evolutionary considerations suggest such a mosaic expression of sexuality is likely to be a property of other animal species having two sexes. These results have also led to a major revision of our view of how sex-specific functions are regulated by the sex hierarchy in flies. Rather than there being a single regulatory event that governs the activities of all downstream sex determination regulatory genes-turning on Sex lethal (Sxl) RNA splicing activity in females while leaving it turned off in males-there are, in addition, elaborate temporal and spatial transcriptional controls on the expression of the terminal regulatory genes, dsx and fru. Thus tissue-specific aspects of sexual development are jointly specified by post-transcriptional control by Sxl and by the transcriptional controls of dsx and fru expression.
Project description:Courtship song is a critical component of male courtship behavior in Drosophila, making the female more receptive to copulation and communicating species-specific information [1-6]. Sex mosaic studies have shown that the sex of certain regions of the central nervous system (CNS) is critical to song production . Our examination of one of these regions, the mesothoracic ganglion (Msg), revealed the coexpression of two sex-determination genes, fruitless (fru) and doublesex (dsx). Because both genes are involved in creating a sexually dimorphic CNS [8, 9] and are necessary for song production [10-13], we investigated the individual contributions of fru and dsx to the specification of a male CNS and song production. We show a novel requirement for dsx in specifying a sexually dimorphic population of fru-expressing neurons in the Msg. Moreover, by using females constitutively expressing the male-specific isoforms of fru (Fru(M)), we show a critical requirement for the male isoform of dsx (Dsx(M)), alongside Fru(M), in the specification of courtship song. Therefore, although Fru(M) expression is sufficient for the performance of many male-specific behaviors , we have shown that without Dsx(M), the determination of a male-specific CNS and thus a full complement of male behaviors are not realized.
Project description: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.