The medfly Ceratitis capitata contains a gene (Cctra) with structural and functional homology to the Drosophila melanogaster sex-determining gene transformer (tra). The Ceratitis homolog of Sex lethal does not appear to have a switch function: the gene is expressed in both sexes, irrespective of whether the male-determining Y is present or absent: this is inconsistent with a main sex-determining function. However, preliminary data suggest that the bottom-most component of the pathway, doublesex (dsx), is not only present in Ceratitis (Ccdsx), but has conserved a role in sexual differentiation. The pre-mRNA of this gene is also alternatively spliced giving rise to sex-specific products that show a remarkable structural conservation when compared with the corresponding male and female products in Drosophila. Sequence analysis of Ccdsx has revealed the presence of putative TRA/TRA-2-binding sites close to the regulated splice site, suggesting that the underlying mechanism of sex-specific splicing is conserved and under the control of proteins homologous to Transformer and Transformer-2. Similar to tra in Drosophila, Cctra is regulated by alternative splicing such that only females can encode a full-length protein. In contrast to Drosophila, however, where tra is a subordinate target of Sex-lethal (Sxl), Cctra seems to initiate an autoregulatory mechanism in XX embryos that provides continuous tra female-specific function and acts as a cellular memory maintaining the female pathway. Indeed, a transient interference with Cctra expression in XX embryos by RNAi treatment can cause complete sexual transformation of both germline and soma in adult flies, resulting in a fertile male XX phenotype. The male pathway seems to result when Cctra autoregulation is prevented and instead splice variants with truncated open reading frames are produced. It is proposed that this repression is achieved by M, the Y-linked male-determining factor (Pane, 2002 and references therein).

A broad variety of genetic cues that determine the sexual fate of a developing individual are known (see Schematic of the sex determination hierarchy in Control of male sexual behavior in Drosophila by the sex determination pathway, Billeter, 2006). Even within a minor taxonomic group, for example, dipteran insects widely different mechanisms of sex determination are found (Marin, 1998; Schutt, 2000); male heterogamety with a male-determining Y chromosome (Musca, Ceratitis) or with a single autosomal factor (Megaselia, Culex), female heterogamety (Musca, Chironomus), chromosomal balance systems (Drosophila, Sciara), and maternal effects (Chrysomya). This variety raises the issues of how these different mechanisms have evolved and how much they differ at the genetic and molecular level. Comparative analyses of different species can be used to address these problems. The economically important medfly (Mediterranean fruitfly), Ceratitis capitata (Tephritidae) has been studied in detail. In this species a Y-linked factor, M, determines maleness, and absence of M leads to female development (Willhoeft, 1996). However, how this signal is relayed to genes responsible for expressing dimorphic traits is completely unknown (Pane, 2002 and references therein).

The genetic cascade regulating sexual development in Drosophila is well known down to molecular details (Cline, 1993; Cline, 1996). In contrast to Ceratitis, the primary signal in Drosophila is polygenic and is formed by the ratio of X chromosomes to sets of autosomes, the so-called X:A ratio. When this ratio is 1.0 (XX:AA), the gene Sex-lethal (Sxl) is activated; with a ratio of 0.5 (X:AA), Sxl remains inactive. Sxl now acts as the key ON/OFF switch that controls all aspects of somatic sexual dimorphism via a short cascade of subordinate regulatory genes (Nagoshi, 1988). When the gene is active, it dictates female development; when it is inactive, male development follows. Once the gene is activated in females, its products initiate a positive autoregulatory mechanism that guarantees the continuous production of Sxl, thus forming a cell memory of the sex and maintaining the cells on the female pathway throughout development (Bell, 1991). In males, however, where Sxl is not activated, the gene will remain functionally OFF. Sxl produces sex-specific mRNAs by alternative splicing: the female-specific mRNAs encode full-length functional Sxl protein, while the male-specific ones have an additional stop-containing exon and encode a truncated non-functional Sxl peptide. The ON/OFF state of Sxl activity is set early during embryogenesis by a complex combination of transcriptional and post-transcriptional gene regulation (Bell, 1991; Keyes, 1992). The initial activation of Sxl in XX embryos relies on the use of an alternative XX-embryo-specific promoter that responds to the genes signaling the X:A ratio (Parkhurst, 1990). Sxl pre-mRNAs produced from this promoter have such a structure that they are spliced in a female-specific mode by the spliceosome independently of additional trans-acting factors, such as the Sxl protein itself (Horabin, 1996; Zhu, 1997). The RNA-binding Sxl proteins translated from these early mRNAs then initiate the autoregulatory loop by directing the female-specific processing of the pre-mRNAs produced from the late Sxl promoter. The late pre-mRNAs, in contrast to the early Sxl pre-mRNAs, can be spliced in the female-specific mode only in the presence of Sxl protein (Pane, 2002 and references therein).

To execute the correct developmental program, Sxl transmits the determined state to transformer (tra) (Boggs, 1997), the next gene in the cascade. At this level, Sxl regulates the choice between two alternative 3' splice sites in the pre-mRNA of tra (Inoue, 1990; Valcárcel, 1993). In absence of Sxl, the more proximal site is used, resulting in a tra mRNA that encodes a truncated inactive protein. When Sxl is present, it will bind to the tra pre-mRNA and enforce the use of the distal 3' splice site to produce an mRNA with a full-length ORF (Sosnowski, 1989). The state of activity of tra is then transmitted to doublesex (dsx) (Burtis, 1989), the last component of the pathway. In females, TRA, together with the constitutively expressed TRA-2, binds to dsx pre-mRNA directing its female-specific splicing, such that a mature mRNA encoding the DSX^F protein is generated (Hoshijima, 1991; Tian, 1993). In males, absence of TRA causes male-specific splicing and the production of a DSX^M protein. The two proteins, DSX^F and DSX^M, are transcription factors (Burtis, 1989) that regulate the activity of sex-specific differentiation genes (Pane, 2002 and references therein)

Studies have indicated that control of sexual development in the medfly follows a different route. In particular, the Ceratitis homolog of Sxl does not appear to have a switch function: the gene is expressed in both sexes, irrespective of whether the male-determining Y is present or absent (Saccone, 1998), which is inconsistent with a main sex-determining function. However, preliminary data suggest that the bottom-most component of the pathway, dsx, is not only present in Ceratitis (Ccdsx), but has conserved a role in sexual differentiation (Saccone, 2000). The pre-mRNA of this gene is also alternatively spliced giving rise to sex-specific products that show a remarkable structural conservation when compared with the corresponding male and female products in Drosophila. Sequence analysis of Ccdsx reveals the presence of putative TRA/TRA-2-binding sites close to the regulated splice site, suggesting that the underlying mechanism of sex-specific splicing is conserved and under the control of proteins homologous to TRA and TRA-2 (Saccone, 2000; Saccone, 2002; Pane, 2002 and references therein).

A Ceratatis homolog of Drosophila transformer gene (Cctra), is highly diverged in sequence. As in Drosophila, Cctra has a female-determining master function. However, in contrast to the Drosophila tra, Cctra plays an essential role in Ceratitis sex determination by maintaining the female sexual cell state through a positive feedback loop and by forming an epigenetic memory of the sex of the organism (Pane, 2002).

Thus Ceratitis and Drosophila sex-determining cascades share a conserved tra-->dsx genetic module to control sex determination and sexual differentiation, but tra sex-specific splicing regulation differs in the two species. In Drosophila, Tra protein, together with Tra-2, binds to the Tra/Tra-2 recognition sequences on the Drosophila dsx pre-mRNA and promotes the use of a nearby female-specific acceptor site. Cctra is needed to impose the female-specific splicing of Ccdsx, most probably by a similar mechanism as in Drosophila, invoking the existence of a Cctra2 homolog. This hypothesis is also supported by the finding of Tra/Tra-2 recognition sequences located in close vicinity to the female-specific acceptor site in Ccdsx pre-mRNA (Pane, 2002).

How does tra sex-specific splicing regulation differ in the two species? In Drosophila, tra female-specific splicing is promoted by Sxl, which blocks the use of the non-sex-specific splice site present in the tra pre-mRNA. In Ceratitis, the presence of multiple Tra/Tra-2-binding elements within the Cctra male-specific exonic sequences strongly suggests that CcTRA and a hypothetical CcTRA-2 protein could bind to these sequences, thus mediating a direct autoregulation. The unusually strong phenotypic effects of the RNAi against CCtra also support this model of Cctra regulation. The localization of the putative regulatory elements within the Cctra gene indicates a repression mode by which CcTRA in females prevents the recognition of male-specific splice sites. The mechanism by which Cctra seems to promote the female mode of processing of its own pre-mRNA by TRA/TRA-2-binding elements appears to be different also from the female-specific splicing of dsx. Rather than activating a splice site nearby the regulated exon, as in the case of dsx, inclusion of male-specific Cctra sequences is suppressed when CcTRA is present. Although this would be a novelty with respect to known Drosophila Tra/Tra-2 activities, the 'behavior' of these cis elements is context dependent and changing the location of splicing enhancers can transform them into negative regulatory elements (Pane, 2002).

In Drosophila, the presence of the Y chromosome is necessary for male fertility but not for male development. By contrast, RNAi-treated Ceratitis embryos with a female XX karyotype can develop into fertile males; this indicates that transient repression of Cctra by RNAi is sufficient to implement fully normal male development. The cases of complete sexual transformation of genetic Ceratitis females (XX) into fertile males by RNAi demonstrate that the Y chromosome, except for the dominant male determiner M, does not supply any other contribution to both somatic and germline male development, as suggested by previous Y-chromosome deletion analysis. Other dipteran species, such as Musca domestica and Chrysomya rufifacies show a female and male germline sex determination that is completely dependent on the sexual fate of the soma. However, in Drosophila, the XX and XY germ cells seem to respond differently to sex determining somatic cues. Indeed the XY germ cells have also an autonomous stage-specific sex determination mechanism that probably integrates the somatic signal. In Ceratitis, Cctra could be required in XX somatic cells to let them induce the XX germ cells to differentiate as oogenic cells. Alternatively, Cctra could be required in XX germ cells to 'feminize' them. This case would be a novelty with respect to the known Drosophila transformer gene functions (Pane, 2002).

Since zygotes that carry a Y chromosome do not activate Cctra female-specific splicing and autoregulation, it is proposed that the Y-linked male-determining M factor prevents this activation. It is conceivable that Cctra is a direct target of the M factor. Presence of this M factor in the zygote may prevent the production of CcTRA protein. The Cctra positive feedback loop is a probable target for regulation, because of its sensitivity (already shown by RNAi). An important question to be addressed is how autoregulation of Cctra is initiated in XX embryos of C. capitata and how this is prevented in XY embryos. A possible explanation is suggested by the Cctra female-specific mRNAs encoding the full-length protein, that have been detected in unfertilized eggs. Depositing these Cctra transcripts in eggs may provide a source of activity that can be used later for 'female-specific' processing when Cctra is zygotically transcribed. Once zygotically activated in XX embryos, Cctra promotes its own female-specific splicing, maintaining the female sex determination and the female-specific splicing of the downstream Ccdsx gene. Taken together, these events induce the female differentiation. In the current model for sex determination of medfly, the M factor is directly involved in the Cctra sex-specific regulation. Thus, in the presence of M Cctra, autoregulation is blocked and the gene produces male-specific transcripts encoding short and possibly non-functional CcTRA peptides. The absence of CcTRA leads Ccdsx to produce male-specific transcripts by default, promoting male differentiation. The control of the M factor upon Cctra expression could be exerted at different levels. The male determiner M could, for example, act at the pre-translational level blocking the production of CcTRA protein from the maternal transcripts. M could act at the post-translational level antagonizing the formation of protein complexes necessary for the female splicing mode. Or M could act as a transient transcriptional repressor of Cctra to reduce the amount of active CcTRA below a threshold needed to maintain the feedback loop. The proposed autoregulatory model of Cctra may also explain the remarkable efficiency of sex reversal by Cctra RNAi: a transient silencing of Cctra by injecting dsRNA is sufficient to let the loop collapse. Furthermore, the sensitivity of this positive autoregulation could be an evolutionary widely conserved pre-requisite to permit a 'faster' recruitment/replacement of different upstream regulators and to easily evolve different sex determining primary signals, as observed in dipteran species (Pane, 2002).

Sex can even be determined by a maternal effect in dipteran species such as Sciara coprophila and Chrysomya rufifacies. The hypothesis of a Cctra maternal contribution to the activation of the zygotic Cctra gene has similarities to the model of sex determination proposed for Musca domestica. In the common housefly, the maternal product of the key switch gene F is needed to activate the zygotic function of F in females. Musca male development results whenever F cannot become active in the zygote. This happens when the male-determining M is present in the zygotic genome, or when maternal F is not functional because of either the presence of M or the mutational loss of function of F (Fman) in the germline. More interesting, embryonic RNAi against the Musca tra-2 homolog causes sex reversion of Musca XX adults into intersex and fertile males, although this gene is not sex-specifically expressed. These recent data in Musca and the results in Ceratitis support the idea that F of Musca functionally corresponds to the Ceratitis tra gene, which seems to autoregulate and maternally contribute to its own activation, rather than to the Drosophila tra gene (Pane, 2002).

These data show that a basic structure of sex determination is conserved in the two dipteran species, namely the flow of 'instructions' from tra to dsx. This confirms the model of 'bottom-up' evolution, suggesting that during evolution, developmental cascades are built from bottom up and that the genes at the bottom are widely conserved, while further upstream new regulatory elements may be recruited. The results show that Ceratitis and Drosophila sex-determining cascade differ at the level of transformer as well as upstream of it. Indeed the gene has conserved its function during evolution, but it has female-specific positive autoregulation in Ceratitis, while in Drosophila it needs Sxl as upstream regulator to express its female determining function. More likely the sex-determining function of Sxl was co-opted after Drosophila and Ceratitis had separated more than 100 Myr ago. Furthermore, it is conceivable that the autoregulatory mechanism of Sxl could have been selected to overcome a mutation impairing the tra autoregulation. Hence, in both species the female pathway is maintained by a single gene positive-feedback mechanism through sex-specific alternative splicing. Single gene autoregulation by alternative splicing seems not to be infrequent in nature, especially in those genes encoding splicing regulators. Indeed, other genes encoding RNA-binding proteins are thought to autoregulate their expression by controlling the processing of their own pre-mRNAs. Such a single-gene network with positive regulation is capable of bistability. This suggests that the emergence of analogous positive autoregulation in different genes such as Drosophila Sxl and Ceratitis tra genes would have been selected, during evolution, to guarantee a similar ON/OFF-female/male bistable cell state (Pane, 2002).

Since Ceratitis capitata is a major agricultural pest in many areas of the world, the isolation of a key sex-determining gene such as Cctra will substantially aid the development of new strategies to optimize the efficacy of currently used male sterile techniques for pest control. It is expected that tra is also a key sex-determining gene in many other insect species. Hence, the isolation of corresponding tra genes will open new means to control not only agricultural pests but also medically relevant vectors of diseases such as Glossina palpalis and Anopheles gambiae (Pane, 2002).

GENE STRUCTURE

cDNA clone length - 1382

Bases in 5' UTR - 270

Exons - 3

Bases in 3' UTR - 213

PROTEIN STRUCTURE

Amino Acids - 197

Structural Domains

While developmentally regulated genes are generally conserved, transformer, a key locus involved in the regulation of sexual differentiation, is highly diverged between species of Drosophila. With an aim to understand its divergence between sibling species, tra sequence variation was investigated among members of the Drosophila melanogaster species complex, D. melanogaster, D. simulans, D. mauritiana, and D. sechellia. In this species group, tra divergence is rapid yet clocklike and exhibits large differences in protein size. D. melanogaster contains a 13 amino acid tandem duplication, whereas D. sechellia possesses a 72 amino acid tandem duplication representing a 30% increase in total amino acid residues. Evidence of a nonrandom distribution of replacement substitutions and heterogeneity in substitution rates was found using clustering statistics and a codon substitution model. tra's rapid divergence in this species complex is the result of generally lower selective constraints around regions that encode arginine-serine (RS) domains and a significantly higher rate of substitutions around the insertion site of D. sechellia's large duplication. The proximity of rapidly diverged regions to sites of nucleotide insertion suggests that higher local rates of mutation may provide a causal mechanism for Tra's rapid divergence in this subgroup. A comparison of tra orthologs across the genus Drosophila suggest that Tra maintains an assortment of RS domains for proper sex determining function while much of the protein evolves relatively unconstrained (Kulathina, 2003).

date revised: 10 April 2004

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