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Title: Environmental Sex Determination in the Branchiopod Crustacean Daphnia magna: Deep Conservation of a Doublesex Gene in the Sex-Determining Pathway}
Article:
Abstract:
Sex-determining mechanisms are diverse among animal lineages and can be broadly divided into two major categories: genetic and environmental. In contrast to genetic sex determination( GSD), little is known about the molecular mechanisms underlying environmental sex determination( ESD). The Doublesex( Dsx) genes play an important role in controlling sexual dimorphism in genetic sex-determining organisms such as nematodes, insects, and vertebrates. Here we report the identification of two Dsx genes from Daphnia magna, a freshwater branchiopod crustacean that parthenogenetically produces males in response to environmental cues. One of these genes, designated DapmaDsx1, is responsible for the male trait development when expressed during environmental sex determination. The domain organization of DapmaDsx1 was similar to that of Dsx from insects, which are thought to be the sister group of branchiopod crustaceans. Intriguingly, the molecular basis for sexually dimorphic expression of DapmaDsx1 is different from that of insects. Rather than being regulated sex-specifically at the level of pre–mRNA splicing in the coding region, DapmaDsx1 exhibits sexually dimorphic differences in the abundance of its transcripts. During embryogenesis, expression of DapmaDsx1 was increased only in males and its transcripts were primarily detected in male-specific structures. Knock-down of DapmaDsx1 in male embryos resulted in the production of female traits including ovarian maturation, whereas ectopic expression of DapmaDsx1 in female embryos resulted in the development of male-like phenotypes. Expression patterns of another D. magna Dsx gene, DapmaDsx2, were similar to those of DapmaDsx1, but silencing and overexpression of this gene did not induce any clear phenotypic changes. These results establish DapmaDsx1 as a key regulator of the male phenotype. Our findings reveal how ESD is implemented by selective expression of a fundamental genetic component that is functionally conserved in animals using GSD. We infer that there is an ancient, previously unidentified link between genetic and environmental sex determination.
Introduction:
Sex determination is a fundamental biological process. It affects not only the sexual differentiation of gonads, but also the development of most organs, and leads to sex-specific differences in behavior, physiology and morphology. Organisms have evolved a variety of different sex-determining systems[1],[2] that can be broadly divided phenomenologically into two categories: genetic and environmental[3]. Genetic sex determination( GSD) is attributed to the genetic segregation of genes, often residing on sex chromosomes that initiate alternate sex-determining developmental pathways. Environmental sex determination( ESD) is initiated by environmental cues that presumably trigger alternative genetic signals, which regulate male or female sex-determining genes[4]. Although GSD is a more prevalent system in animals, ESD is also phylogenetically widespread, occurring in such diverse taxa as rotifers, nematodes, crustaceans, insects, fishes, and reptiles[5]. Environmental cues involved in ESD include temperature, photoperiod, nutrition, and population density[5]. Temperature is the most widely studied environmental cue, particularly in the case of reptiles where the temperature at which the egg is incubated determines sex[6]. ESD has arisen repeatedly during evolution[7], which may imply the adaptive significance of this system in environments[8]. It has long been suggested that selection forces drove the transition between GSD and ESD[9],[10]. Previous experiments using a temperature-sensitive mutation created artificially in Drosophila melanogaster and Caenorhabditis elegans also demonstrated how GSD could rapidly evolve into ESD as a consequence of a mutation in a single control gene[11],[12]. In addition, orthologs of some genes involved in GSD have been examined in species that use ESD, especially in temperature-dependent sex-determining reptiles[13]. Some of those are expressed in the gonads during the temperature-sensitive period[13]. These observations have led to the hypothesis that both ESD and GSD should have the same origin and share similar genetic components in their sex-determining pathways[4]. However, a complete functional analysis of temperature dependent ESD has not yet been performed. Therefore, analyzing function of genes involved in ESD and unraveling the sex-determining pathways are crucial to understand the origin and evolution of sex-determining pathway. The water flea Daphnia magna is a branchiopod crustacean, which is a common inhabitant in fresh water ponds in Europe and Asia. D. magna is known to switch between parthenogenetic and sexual reproduction when environmental quality declines[14]. Normal, healthy populations are entirely female. However, shortened photoperiod, a lack of food and/or increased population density, lead to the clonal production of males that are genetically identical to their sisters and mothers. First instar male juveniles are easily distinguished from the females by their elongated first antennae[15]. During maturation, daphnids undergo morphological sexual differentiation of various somatic tissues including the first thoracic leg that is armed with the copulatory hook in males, which becomes larger in the fifth instar[16]. Gonads develop and finally settle at both sides of the gut during embryogenesis in both males and females[17]. It has been reported that the gonads exhibit morphological sex differences in the first instar juveniles[18],[19]. The appearance of males allows sexual reproduction to occur[20],[21] when females begin producing haploid eggs requiring fertilization. Recently, we and others have shown that juvenile hormone analogs( JHA) induce male production in cladoceran crustaceans without environmental cues[22],[23]. Interestingly, exposure of D. magna to JHA at the stage corresponding to the environmentally-sensitive period for the other cladoceran Moina sex determination[24], reliably produces exclusively male broods, suggesting that juvenile hormone could be a key molecule for understanding environmental sex determination[22],[25],[26]. Together with the growing genome and transcriptome resources for Daphnia[27]-[30], this system is ideal to study genes responsible for ESD. Mechanisms underlying genetic sex-determining pathways have been extensively studied in model organisms such as D. melanogaster, C. elegans and mouse and key genes have been identified[31]-[33]. A Doublesex( Dsx) gene was originally identified in D. melanogaster as a critical transcription factor considered to be at the end of sex determination cascades in GSD, that directly targets genes conferring sexually dimorphic traits[34]. Dsx contains two conserved domains: one is the Dsx/Mab-3( DM) domain at the N-terminus that is evolutionarily conserved even in vertebrates[35] and another is the oligomerization domain at the C-terminus[36]. Genes encoding DM-domain( DM-domain genes) were discovered to play a related role not only in C. elegans[37],[38], but even in vertebrates[39]. In contrast, results from numerous studies have shown that other genetic sex-determining genes are widely diverse among species[1],[2],[40]. To understand the molecular and evolutionary relationships between GSD and ESD, we analyzed the function of two Dsx genes from D. magna using gene manipulations that we have developed[41]. We provide evidence that one of the homologs, termed DapmaDsx1, plays an important role in directing the major sexually dimorphic development of D. magna. Intriguingly, the function of Dsx is significantly conserved between Daphnia and genetic sex-determining insect species, which are thought to be the sister group of branchiopod crustaceans[42]; however, the factors that regulate Dsx gene expression are independently co-opted in each lineage. Our functional demonstration that Dsx controls sexual dimorphism in an environmental sex-determining organism supports the hypothesis that genetic and environmental sex determination are similar at their most fundamental level.
Discussion:
DapmaDsx1, one of two Dsx homologs from Daphnia, shares several important characteristic features of Drosophila Dsx protein to function as a major effector of sexual differentiation[40],[57],[58]. First, DapmaDsx1 protein is composed of two domains, the phylogenetically conserved DM-domain[35] and the insect-specific oligomerization domain. In contrast, the DapmaDsx2 protein appears to be unable to regulate sexual differentiation and contains mutations at important amino acids of oligomerization domain[36], suggesting that this domain might be necessary for establishing sexual dimorphic traits. Second, male-specific expression of DapmaDsx1 is regulated temporally and spatially during development. Although it has long been believed that Drosophila Dsx gene was cell-autonomously expressed in all cells[59], Robinette et al.[60] recently reported that Drosophila also exclusively expressed Dsx in sexually dimorphic tissues and cells. Third, and perhaps most importantly, knock-down of DapmaDsx1 in male embryos resulted in the production of female traits including ovarian maturation whereas ectopic expression of DapmaDsx1 in female embryos resulted in partial masculinization of the first antennae. These results suggest that Dsx gene expression in sexually dimorphic tissues is a key process to induce sexual differentiation in crustacean Daphnia and insects. In the fruit fly D. melanogaster, Dsx is spliced in the coding region by the Tra protein in a sex-dependent manner[34]. The female-specific RNA produced by alternative splicing is a functional mediator of Tra activity[61]. The female-specific splice variant of the Tra homologs encodes a functional protein not only in the Mediterranean fruit fly[62] and the house fly[63], but also in the honeybee Apis mellifera[64]. In all insect species studied to date( except the silkworm Bombyx mori)[65], Tra regulates sex-specific splicing of Dsx, which produces different mRNAs and proteins, resulting in sex-specific transcriptional activation and repression[66]( Figure 6). Sex-specific splicing of the Dsx gene by the Tra protein might be ancestral in insects. In contrast, DapmaDsx genes do not encode sex-specific Dsx proteins, but instead exhibit sexually dimorphic differences in the abundance of its transcripts. Interestingly, Daphnia has a homolog of the Tra protein but the D. magna Tra gene does not display any detectable sexually dimorphic differences in expression or splicing patterns[26]. We also performed knock-down of the D. magna Tra gene, but could not find any effect for development of sexually dimorphic traits( data not shown). This is consistent with the apparent lack of a sex-specific splicing in the Dsx1 gene. Although it is not yet clear if juvenile hormone directly activates the transcription of DapmaDsx genes, this remains an interesting possibility for future study. We found several motifs in promoter regions of the Dsx1 and Dsx2 genes, which resemble JH-responsive elements previously reported in D. melanogaster[67] and D. magna[68]( data not shown), suggesting that these motifs possibly function as elements to regulate the JH-dependent gene expression. The detection of unfavorable environmental conditions by Daphnia could be transmitted to the endocrine system, leading to the release of juvenile hormone to convey the environmental signals to sexually dimorphic cells. This would be a simple and elegant type of sex determination cascade. Understanding the molecular nature by which the transcription of the DapmaDsx genes is regulated remains an important future goal that will greatly enhance our understanding of not only sex determination, but also invertebrate hormonal systems. Interestingly, expression of the DapmaDsx1 gene utilizes alternative polyadenylation at tandem poly( A) sites, which can yield transcripts that have identical protein-coding sequences but different 3'UTR sequences. Alternative polyadenylation is often associated with tissue- or developmental stage-dependent gene expression[69],[70]. We found that female DapmaDsx1-β mRNAs exclusively use the most promoter-proximal polyadenylation signals. The presence of alternative polyadenylation sites in Dsx genes has been reported in three insects, D. melanogaster, the phorid fly Megaselia scalaris and the mosquito Anopheles gambiae, indicating that regulation of the 3'UTR length might be a common mode to regulate expression of Dsx genes. Despite having last shared a common ancestor with insects about 400 million years ago[42],[71] and differences of the initial cue to determine sex, the DapmaDsx1 maintained the domain structure essential for establishing sexual dimorphism, while regulation of its expression by other factors became complex and diverse. This is consistent with the prediction that new signals are co-opted upstream of a cascade during the course of evolution[72],[73]. Thus, we have established that there were no boundaries between GSD and ESD in evolution of sex-determining genes at their most fundamental level.
Sex determination is a fundamental biological process that can be broadly divided into two major categories . In genetic sex determination ( GSD ) , sex-specific differentiation results from intrinsic genetic differences between males and females , whereas environmental sex determination ( ESD ) relies on environmental signals to induce male or female sex determination . In contrast to model organisms that utilize GSD system , environmental sex-determining organisms are poor genetic models . Therefore , although candidate genes involved in ESD have been found in vertebrates , their functions have remained largely unknown , impairing our understanding of ESD and making the comparison of sex-determining genes between both systems difficult . Here , we report the identification of a gene responsible for the production of males during environmental sex determination in the crustacean Daphnia . This gene is homologous to the Doublesex gene that is functionally conserved in animals that use GSD . Expression of Doublesex was increased primarily in male-specific structures . Gain- and loss-of-function analyses established that Daphnia Doublesex gene is a major effector that regulates the male phenotype in Daphnia . We infer that there is an ancient , previously unidentified link between genetic and environmental sex determination .