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Genetics. Oct 2005; 171(2): 615–624.
PMCID: PMC1456775

Evolutionary Conservation of Ceratitis capitata transformer Gene Function

Abstract

Transformer functions as a binary switch gene in the sex determination and sexual differentiation of Drosophila melanogaster and Ceratitis capitata, two insect species that separated nearly 100 million years ago. The TRA protein is required for female differentiation of XX individuals, while XY individuals express smaller, presumably nonfunctional TRA peptides and consequently develop into adult males. In both species, tra confers female sexual identity through a well-conserved double-sex gene. However, unlike Drosophila tra, which is regulated by the upstream Sex-lethal gene, Ceratitis tra itself is likely to control a feedback loop that ensures the maintenance of the female sexual state. The putative CcTRA protein shares a very low degree of sequence identity with the TRA proteins from Drosophila species. However, in this study we show that a female-specific Ceratitis Cctra cDNA encoding the putative full-length CcTRA protein is able to support the female somatic and germline sexual differentiation of D. melanogaster XX; tra mutant adults. Although highly divergent, CcTRA can functionally substitute for DmTRA and induce the female-specific expression of both Dmdsx and Dmfru genes. These data demonstrate the unusual plasticity of the TRA protein that retains a conserved function despite the high evolutionary rate. We suggest that transformer plays an important role in providing a molecular basis for the variety of sex-determining systems seen among insects.

IN the fruit fly Drosophila melanogaster, the X:A ratio serves as the primary signal for somatic sex determination. The primary signal acts via Sex lethal (Sxl), which functions as the binary switch gene that controls sexual differentiation. When the X:A ratio is 1 (2X/2A embryos), Sxl is switched “on” and signals female identity, whereas a value of 0.5 (X/2A embryos) turns Sxl “off,” allowing male development to ensue (Cline and Meyer 1996). During early development, Sxl is transiently transcribed from an early promoter (SxlPe) in only the XX embryos in response to the primary signal, while at later stages the transcription of Sxl is triggered by a maintenance promoter (SxlPm) in both males and females (Keyes et al. 1992). The early SXL protein establishes a positive feedback loop in the XX embryos by directing the female-specific productive splicing of Sxl pre-mRNA transcribed from SxlPe (Cline 1984; Bell et al. 1991). This autoregulatory mechanism guarantees the memory of the female sexual state throughout the life cycle of the fly. By contrast, in males, transcription from SxlPe is not activated and therefore the feedback loop is never initiated. Sxl controls the sexual differentiation program through the downstream transformer (tra) gene. In females, SXL protein directs the splicing of tra RNA so that the full-length TRA protein is produced only in females (Sosnowski et al. 1989; Inoue et al. 1990; Valcárcel et al. 1993). In males, the Sxl gene is “off”; therefore tra splicing is governed by a default mechanism resulting in small nonfunctional TRA peptides (Butler et al. 1986; Boggs et al. 1987; McKeown et al. 1987). TRA subsequently controls the downstream components of the cascade, namely the double-sex (dsx) and fruitless (fru) genes. Indeed, it has been shown that TRA is able to direct the female-specific processing of dsx and fru precursor mRNAs (Baker and Ridge 1980; McKeown et al. 1988; Hoshijima et al. 1991; Heinrichs et al. 1998). This process requires the product of the sex-non-specific transformer2 (tra2) gene and general splicing factors (Amrein et al. 1988; Inoue et al. 1992). Female-specific mRNAs are produced from dsx and fru encoding the DSXF and FRUF proteins in the presence of the TRA-TRA2 complex. While in males, in the absence of TRA protein, dsx and fru give rise to the male-specific DSXM and FRUM isoforms (Burtis and Baker 1989; Ryner et al. 1996). The protein isoforms produced from the dsx and fru genes are responsible for the development of sex-specific somatic tissues and behavioral traits.

We have recently reported on the molecular cloning of the first tra homolog (Cctra) in a nondrosophilid insect species, namely the Mediterranean fruit fly (Medfly) Ceratitis capitata (Pane et al. 2002), which diverged from D. melanogaster ~100 million years ago (Beverly and Wilson 1984). The primary signal in Medfly relies on a Y-linked male-determining factor (M-factor) that signals male identity. Consequently, XY embryos develop into adult males, while XX embryos turn into females. The Sxl homolog in Medfly (CcSxl) does not function as a key switch gene in the sex-determination process (Saccone et al. 1998). Instead, a master sex-determining activity in Ceratitis is exerted by the tra homolog. Interestingly, Cctra shares several key features with both Dmtra and DmSxl. Similar to Dmtra, Cctra generates mRNAs encoding the full-length CcTRA protein only in females. Also, Cctra seems to act via a conserved Ceratitis double-sex (Ccdsx) homolog (Saccone et al. 2000). Therefore, the tra>dsx genetic segment seems to be conserved in these species, while the Sxl>tra one is present in Drosophila, but not in Ceratitis. This genetic architecture is sustained by the fact that Cctra displays an autocatalytic function in Ceratitis, similar to that of DmSxl, which guarantees the cell memory of the female sexual state. In XY embryos, Cctra autoregulation is likely to be inhibited by the Y-linked male-determining factor (M-factor), and male development is launched. In support of this hypothesis, when Cctra activity is transiently abolished by RNA interference (RNAi) in XX early embryos, the emerging XX adults express male-specific mRNA variants of Cctra and develop as males (Pane et al. 2002). The sex choice established by the state of activity of Cctra is then transmitted to a downstream dsx homolog (Ccdsx). This process appears to be very similar to that observed in Drosophila, since Ccdsx displays a high degree of sequence and structural identity with Dmdsx as well as a similar sex-specific expression (Saccone et al. 2002). In agreement with these observations, when Cctra is turned by RNAi from the female to the male mode of splicing in XX individuals, the expression pattern of Ccdsx changes accordingly (Pane et al. 2002).

It has been shown that the tra gene undergoes a rapid evolutionary divergence even though it governs a fundamental aspect of fly development (O'Neil and Belote 1992; McAllister and McVean 1999; Kulathinal et al. 2003). A low degree of amino acid sequence conservation is consistently revealed when the TRA proteins from several Drosophila species are aligned. For instance, the protein products of Drosophila virilis and D. melanogaster, which separated 60 MYA, share 36% amino acid sequence identity (O'Neil and Belote 1992). In agreement with the phylogenetic distance between Ceratitis and Drosophila, we found that Ceratitis TRA is 17.9% identical to DmTRA and 18% identical to DvTRA, and it is also more than twice as long (429 aa vs. 197 and 199 aa, respectively). Although the evolution of the tra sequences has been analyzed in several studies, the functional conservation of this gene has been poorly investigated through in vivo experiments. To date, only one report has shown that the D. virilis tra can partially supply the tra+ function when introduced in D. melanogaster tra− mutant strains (O'Neil and Belote 1992).

To test the activity of Ceratitis tra in D. melanogaster, we expressed a Cctra cDNA encoding the full-length CcTRA protein in Drosophila transgenic lines. We show that the Cctra transgene is able to promote sexual transformation of Drosophila males into pseudofemales and to rescue the phenotype of XX; tra mutant females. Consistent with the phenotypic effect of Cctra, endogenous dsx and fru gene expression are turned from a male to a female mode of splicing in the presence of CcTRA protein. Our results show that despite the minimal conservation, CcTRA is able to recognize DmTRA target sequences and protein partners during development of the fly.

MATERIALS AND METHODS

Molecular biology techniques:

Restriction digestion, plasmid construction, blotting, or hybridization were essentially as in Sambrook et al. (1989).

Plasmid construction:

The CctraF1 cDNA was removed as an XbaI/Xho fragment from Bluescript vector and subcloned into XbaI and SalI-digested pHSS6-hsp-t (Seifert et al. 1986), which resulted in pHSS6-hsp-CctraF1-t intermediate plasmid. The cassette containing the CctraF1 cDNA under D. melanogaster hsp70 promoter (hsp70p) and terminator (hsp70t) regions was isolated as a NotI fragment. This fragment was then cloned into the unique NotI site of the pSLfa1180fa shuttle vector (Horn and Wimmer 2000), yielding the pSL-hsp-CctraF1-taf plasmid. The pSLfa1180fa cloning vector contains FseI and AscI restriction sites flanking the multiple cloning site of pSL1180 (Amersham Pharmacia). An FseI/AscI fragment, carrying the hsp70pr-CctraF1-hsp70t gene cassette, was removed from pSL-hsp-CctraF1-taf and cloned into FseI/AscI-digested piggyBac-[3xP3-EGFP] vector (Handler and Harrell 1999). The final transformation vector was named pBac(3G)-hs-CctraF1 (Figure 1A).

Figure 1.
(A) Structure of the pBac-[3xP3-EGFP]-CctraF1 transformation vector. (B) Phenotypic analysis of hs-CctraF1 transgenic lines at 25°. Wild-type males and females are reported in a and d, respectively. XY individuals from the weak line 9a develop ...

Germline transformation:

Germline transformation experiments were performed as described by Rubin and Spradling (1982). Preblastoderm embryos of D. melanogaster were injected with 500 ng/μl of pBac(3G)-hs-CctraF1 and 150 ng/μl of the helper phsp-pBac vector (Handler and Harrell 1999). The phenotypic marker carried by the pBac(3G)-hs-CctraF1 plasmid is a cDNA encoding the enhanced green fluorescent protein (EGFP) under the control of the 3xP3 artificial promoter (Horn and Wimmer 2000). This promoter drives the expression of the EGFP in the larval central nervous system (CNS) and in the eyes of Drosophila adults (Horn and Wimmer 2000). Transgenic individuals were detected in the G1 progeny for the presence of green fluorescent eyes. All EGFP fluorescence observations were conducted employing the LEICA MZ FLIII stereomicroscope and the GFP2 (GFP Plus) filter set (excitation filter, 480/40 nm; barrier filter, 510 nm).

We collected 32 transgenic individuals showing the expected eye fluorescence, which were generated by four F0 single crosses. Genomic Southern blots on three lines, 9a, 9i, and 33h, revealed that each line is characterized by a single insertion of the pBac(3G)-hs-CctraF1 transposable element and confirmed that the transgenic lines were derived from independent integration events (data not shown). Line 9a had the transposable element inserted on chromosome II and could be established by crossing transgenic individuals with the double-balanced marker strain yw; CyO/Sco; TM3. Other lines described in this article were kept unbalanced. Line 33h had a single insertion of the pBac(3G)-hs-CctraF1 on the X chromosome. Lines 9a and 33h were chosen as the best representatives of the different phenotypic classes, since they show, respectively, the weakest and the strongest penetrance of the transgene at 25°.

To induce the expression of the cDNA CctraF1 from the Hsp70 promoter, flies were exposed to a daily heat-shock regimen at 37° for 1 hr from embryonic stages until adulthood. PCR-based karyotipic analysis and genetic markers (Bs allele on the Y chromosome) were used to distinguish XX females from XY pseudofemales recovered after heat-shock treatment (data not shown).

RT-PCR:

Total RNA was extracted from adult Drosophila individuals as described elsewhere (Andres and Thummel 1994). Oligo(dT)-primed cDNA was made from DNAase-treated total RNA of adult flies using the SuperScript first-strand synthesis system for RT-PCR (Invitrogen, San Diego). RT-PCR experiments to investigate the state of activity of Drosophila sex-determining genes were performed with the primers reported in Table 1.

TABLE 1
Oligonucleotides used in this study

Cycling conditions were denaturation at 94° for 5 min, followed by 35 cycles of 94° for 30 sec, annealing at 58° (for dsx), 62° (for tra), and 64° (for fru) for 30 sec, and extension at 72° for 1 min, with a final 5-min extension at 72°.

RT-PCR products were transferred to Hybond NX nylon membranes (Amersham Pharmacia) and blotted with a 32P-radiolabeled probe corresponding to a sex-non-specific region of fruitless. The clone containing the fru probe was kindly provided by Stephen Goodwin.

Fly strains:

Transgenic strains were produced by micro-injection of DNA constructs into a D. melanogaster white-eye (w1118) strain. tra mutant lines (BsY; tra, e, ca/TM3, sb, ser) and [BsY; Df(3L)E34, st, tra, e/TM3, sb, ser] were kindly provided by Daniel Bopp. These lines accumulated recessive lethal alleles on chromosome III. Hence, hs-CctraF1 activity was analyzed in a trans-heterozygous tra mutant background. tra2 mutant strains DfTRIX/CyO and tra2B/CyO were kindly provided by William Mattox.

RESULTS

hs-CctraF1 directs female development of XY transgenic flies:

The evolutionary conservation of TRA function was investigated by testing the feminizing activity of the C. capitata TRA protein when expressed in the distantly related species D. melanogaster. To this aim, we generated transgenic strains of Drosophila that carry the Ceratitis female CctraF1 cDNA, encoding the full-length putative CcTRA protein (Pane et al. 2002) under the control of the Drosophila hsp70 promoter (Figure 1A). Transgenic lines were grouped into phenotypic classes according to the severity of perturbation in male development at 25°. In the weak class (Figure 1B), the majority of transgenic males and females had no obvious sex transformation when raised at 25°. They had normal external morphology and appeared fully viable and fertile. However, a small percentage (2%) of males showed male intersexual phenotypes at 25°. These males had reduced pigmentation of the abdomen and incomplete development of sex-comb bristles. Also, their external genitalia exhibited a variable extent of feminization. In the intermediate class, transgenic males displayed a variety of phenotypes, ranging from male to female-like external morphology. In this class, a high percentage of animals (30%) were intersexes and revealed a phenotype similar to that observed in the weak lines. Finally, in the strong class (Figure 1B), transgenic males were always strongly feminized. They exhibited female-like reduced abdominal pigmentation and no sex combs on the forelegs. In addition, external genitalia were made up of both male and female tissues.

Males from all three classes of lines examined were strongly transformed toward femaleness when exposed to a daily heat-shock regimen (Figure 2). Indeed, these individuals developed female-like abdominal pigmentation (Figure 2A) and apparently normal female-like external genitalia (Figure 2B). Furthermore, sex-comb bristles on their forelegs were reduced or absent (Figure 2C). However, pseudofemales from all the classes were sterile most likely because their germline failed to undergo a complete sex transformation. Males of line 9a with one copy of the transgene did not undergo a complete somatic sex transformation and developed as intersexes. XX; hs-CctraF1 transgenic females neither were affected by the basal activity of the transgene at 25° nor displayed sexually altered somatic tissues after heat-shock treatment.

Figure 2.
Phenotypic analysis of XY; hs-CctraF1 adults. Heat-shock induction of the hs-CctraF1 transgene drives the development of females from otherwise male XY embryos. (A) Abdominal pigmentation. (a) w males show fully pigmented fifth and sixth tergites; (b) ...

In Drosophila, one of the most evident dimorphic traits is represented by the body size, with males being smaller than females. It has been previously reported that this character relies on the state of activity of Sxl and is independent of Dmtra (Cline 1984; Waterbury et al. 2000). In agreement with these previous reports, we observed that hs-CctraF1 pseudofemales express male mRNA variants of Sxl and develop a body size similar to that of wild-type males (Figure 2D).

dsx and fru splicing is altered after hs-CctraF1 induction:

In D. melanogaster, the presence of a functional TRA protein triggers the expression of female-specific protein isoforms from dsx and fru, which in turn direct female differentiation. Conversely, when TRA is absent, a default mechanism gives rise to male-specific proteins from dsx and fru and male development ensues. We have shown that ectopic expression of hs-CctraF1 in Drosophila XY transgenic flies is sufficient to trigger feminization of somatic tissues. Hence we thought that the CcTRA protein, encoded by the transgene, is able to mimic the function of DmTRA and to promote the expression of dsx and fru female splice variants. To test whether hs-CctraF1 causes a shift in dsx and fru splicing patterns from a male to a female mode, RT-PCR experiments were performed on RNA extracted from hs-CctraF1 adults (Figure 3). In Drosophila, dsx message is spliced in a sex-specific manner. In the case of females, the mature dsx transcript consists of both exons 3 and 4, whereas in males an alternative splicing event leads to elimination of exon 4. Therefore, mature transcripts contain exon 3 fused directly to exon 5. The expression pattern of dsx was determined with a common forward primer located in exon 3, and female- and male-specific reverse primers located, respectively, in exons 4 and 5 (Figure 3A). The set of primers that we designed allowed us to visualize the sex-specific splice variants of dsx in wild type as well as in transgenic males and females (Figure 3A). A slowly migrating band is detected by RT-PCR in the wild-type female sample as compared to the male lane (Figure 3B, lanes 1 and 3). RT-PCR on XY; hs-CctraF1 adults produce a male-specific amplification product in the absence of heat-shock treatment (Figure 3B, lane 5). However, upon heat shock, an additional band is readily detectable in the XY; hs-CctraF1 sample lane. The size of this band corresponds to the female-specific amplification product (Figure 3B, compare lane 1 to lane 7).

Figure 3.
Expression of the Dmdsx and Dmfru genes in adult XY; hs-CctraF1 pseudofemales. (A) Position of the synthetic oligonucleotides within the Dmdsx gene (arrows). (B) RT-PCR reactions with Dmdsx-specific primers on w males (lane 1) and females (lane 3) generate ...

Another known target of tra in Drosophila is the fruitless gene. It was previously shown that fru expression is restricted to a small number of neurons in the CNS to regulate sex-specific behavior (Villella et al. 1997). The precursor mRNAs transcribed from the fru locus undergo alternative splicing and generate differential mature transcripts in females and males. Consistently, in females the Tra-Tra2 complex interacts with the “Tra-Tra2-binding sites” in fru exon 2 and engineers the inclusion of this exon in the mature transcripts (Heinrichs et al. 1998). Conversely, in males, in the absence of TRA protein, the spliceosome recognizes an upstream 5′ splice site, resulting in exclusion of a portion of exon 2 from the mature fru mRNAs.

We determined the expression pattern of fru by RT-PCR using forward primers located in exon 2, sex-non-specific and male-specific regions, and a reverse primer in exon 3 (Figure 3C). The RT-PCR reaction generates a male product with lower molecular weight with respect to the female amplification fragment (Figure 3D, lanes 1–3). In the absence of heat-shock induction, XY; hs-CctraF1 individuals generate only the male-specific amplification product (Figure 3D, lane 5). By contrast, male- and female-specific bands are detectable in the XY; hs-CctraF1 pseudofemales (Figure 3D, lane 7).

Does Cctra regulate endogenous Dmtra expression?

We have shown that hs-CctraF1 can regulate female-specific processing of dsx and fru precursor transcripts and thereby promote female-like differentiation of Drosophila XY individuals. However, these data, a priori, do not rule out the possibility that hs-CctraF1 actually directs the splicing of Dmtra, rather than acting directly on dsx and fru. Indeed, in Ceratitis, Cctra is thought to regulate its own expression through a positive feedback loop (Pane et al. 2002). The model proposes that CcTRA protein autoregulates by binding to the “Tra-Tra2-binding sites” in Cctra intron 1 and directs the exclusion of this intron from the female splice variants. This mechanism in turn sustains the continuous expression of CcTRA protein needed for remembering the female identity. To determine whether CcTRA might direct the female splicing of Dmtra, RT-PCR experiments were carried out using primers that allow detection of both the sex-non-specific and the female-specific transcripts of Dmtra in the transgenic lines (Figure 4A). As reported previously, the wild-type male samples show only the sex-non-specific transcript (Figure 4B, lane 1), while female samples show both the female-specific and the sex-non-specific transcripts (Figure 4B, lane 3). Only the sex-non-specific class of Dmtra transcripts are detectable in XY; hs-CctraF1 individuals grown at 25° (Figure 4B, lane 5). Furthermore, the expression pattern of Dmtra is not altered even after heat-shock induction of the hs-CctraF1 transgene (Figure 4B, lane 7). We therefore conclude that Dmtra is not a target for the hs-CctraF1 transgene and that Cctra must regulate alternative splicing of dsx and fru directly.

Figure 4.
Analysis of the Dmtra splicing pattern in adult transgenic individuals. (A) Schematic of synthetic primers in the Dmtra gene (arrows). (B) RT-PCR on w males (lane 1) detects a 0.7-kb fragment corresponding to the sex-non-specific Dmtra mRNA. In w females ...

Analysis of hs-CctraF1 activity in a Drosophila tra mutant background:

Since XY animals are morphologically feminized under the control of transgene-derived CcTRA, we wished to determine the extent to which gonad-genitalia formation is influenced by the presence of the hs-CctraF1 transgene. The feminizing activity of hs-CctraF1 during germline development was analyzed by crossing the strong transgenic lines with Drosophila Dmtra mutant strains. Similar to XY; hs-CctraF1 flies, XY; hs-CctraF1; tra individuals are also transformed into somatic females at 25° since the hs-CctraF1 transgene does not act through the endogenous Dmtra. However, analysis of internal genitalia revealed that these individuals either have malformed testes (Figure 5c) or lack internal genitalia. After heat-shock treatment, a higher percentage of individuals apparently lack germline tissues, but ovarian-like tissues are never formed. A similar phenotype is observed when XX; hs-CctraF1; tra females grown at 25° are dissected. However, the ovarian morphology of germline tissues in XX; hs-CctraF1; tra females can be rescued after heat induction of the transgene. Indeed, reinforcement of the transgene activity by heat-shock exposure results in the development of rudimentary or even almost-normal ovaries (Figure 5d). Despite the fact that Cctra can support female differentiation of both somatic and germline tissues in XX; tra individuals, these animals fail to lay eggs, thus suggesting that the rescue of the female phenotype is, however, not complete.

Figure 5.
(A) Internal genitalia of hs-CctraF1 adults after heat-shock treatment. (a) Wild-type testes. (b) Wild-type ovaries. (c) Induction of the hs-CctraF1 transgene induces the development of malformed testes in XY; hs-CctraF1 pseudofemales. (d) Ovarian development ...

Activity of the hs-CctraF1 transgene is dependent upon a functional endogenous tra2 gene:

In D. melanogaster, TRA protein along with its protein partner TRA2 controls all aspects of female-specific differentiation. Therefore, we decided to address if the hs-CctraF1 transgene requires the activity of the endogenous tra2 gene to promote female differentiation. Indeed, it was shown that DmTRA must assemble into a complex containing DmTRA2 and serine-arginine (SR) splicing factors to direct female expression of dsx and fru genes. Accordingly, XX flies carrying a nonfunctional tra2 fail to execute the female program and develop into sterile males (Fujihara et al. 1978; Baker and Ridge 1980). To assess whether CcTRA also requires a functional Dmtra2 gene, we tested for genetic interaction between hs-CctraF1 and tra2. To this end we introduced the hs-CctraF1 transgene into a tra2 mutant background. Two different tra2 alleles were used in this experiment, namely tra2B and DfTRIX (Waterbury et al. 2000). The chromosomes carrying these alleles accumulated recessive lethal mutations and cannot be kept in a homozygous state. Therefore, we performed the test by generating tra2B/DfTRIX trans-heterozygous flies, which lack a functional TRA2 protein and develop as adult pseudomales. We found that both strong and intermediate hs-CctraF1 transgenes were unable to direct female differentiation of XY; tra2B/DfTRIX individuals at 25°. Nor could they rescue the tra2 mutant phenotype of the XX mutant flies. In addition, both XX and XY; hs-CctraF1; tra2B/DfTRIX flies showed only male somatic and germline tissues after heat-shock treatment. We conclude that the putative CcTRA protein, produced by hs-CctraF1, requires a functional endogenous TRA2 protein to direct female development of Drosophila transgenic flies.

DISCUSSION

The transformer gene plays a fundamental role in the sex determination of D. melanogaster. It is responsible for the transduction of the initial sex choice, established by the X/A counting mechanism, to downstream effectors that ultimately control the development of sex-specific traits. Despite its key role in the development of the fly, tra reveals an unusual evolutionary rate even when comparison is restricted to Drosophila species. The evolutionary divergence of this gene becomes even more apparent when a non-drosophilid species, namely C. capitata, is considered (Pane et al. 2002). However, we show here that CcTRA can support not only the somatic, but also the female germline development of Drosophila transgenic strains. Our results demonstrate that CcTRA can substitute for DmTRA during Drosophila sex determination, despite the fact that these proteins share only 17.9% sequence identity and are significantly different in size (429 aa vs. 197 aa, respectively).

Cctra can replace Dmtra in the sex determination of Drosophila:

A comparison of Ceratitis TRA with homologous proteins from drosophilid species highlighted only a few short regions of identity (Pane et al. 2002). All of the TRA proteins are characterized by a variable number of interspersed serine/arginine dipeptides and a proline/valine motif in the C-terminal domain. In addition, a unique feature shared by these molecules is an 11-amino-acid stretch with a PYF(A/I)DE core sequence, whose function is still unknown. In Ceratitis, however, TRA does not contain the MDMDSS motif at the very N-terminal region, which is present in all of the drosophilids. In this study, we demonstrate that the function of the TRA protein in Ceratitis and Drosophila is conserved despite the high degree of sequence divergence. Consistently, the expression of an hs-CctraF1 transgene, encoding the full-length CcTRA protein, in Drosophila transgenic lines drives the female somatic sexual differentiation of otherwise male individuals. Sex transformation affects all somatic tissues and occurs in the strong transgenic lines even in the absence of heat-shock treatment. A nearly complete somatic sex transformation can also be induced in weak lines after heat-shock exposure, indicating its dependence upon the activity of the hs-CctraF1 transgene. Given the high degree of sequence divergence between CcTRA and DmTRA, it was surprising to discover that hs-CctraF1 can also support germline female differentiation. Indeed, we observe that the expression of a strong hs-CctraF1 transgene can rescue the ovarian morphology in XX; tra mutant flies. These flies develop apparently normal ovaries, but upon close inspection, they appear to be sterile and fail to lay eggs. We also observed that ovarian tissues cannot be induced in XY and XY; tra individuals. This result is rather unexpected, given the positive effect of heat shock on rescue of XX; tra mutants. However, it was previously shown that expression of an hs-DmtraF transgene is not able to feminize the germline of Drosophila XY individuals (McKeown et al. 1988). The absence of ovarian tissues in XY flies might be due to the fact that XY germ cells appear to be less responsive to feminizing signals than XX germ cells (Waterbury et al. 2000).

In Drosophila, the sexual identity of the developing individual is controlled by tra via the downstream dsx and fru genes. In the XX individuals the TRA protein controls the female-specific splicing of the dsx and fru pre-mRNAs, which leads to the production of DSX and FRU female protein isoforms. By contrast, in males TRA is not expressed and therefore dsx and fru are spliced in the default male mode. This event leads to the production of the male-specific DSX and FRU proteins. The dsx gene is responsible mainly for the sexual dimorphism of the somatic traits, while fru has been shown to control all aspects of male courtship behavior (Villella et al. 1997). In agreement with the sex transformation of Drosophila hs-CctraF1 transgenic flies, the endogenous dsx and fru genes are expressed in the female mode in the resulting XY pseudofemales. Our data strongly suggest that CcTRA directs the female-specific processing of dsx and fru using a mechanism similar to that of DmTRA. In Drosophila, the TRA protein acts in conjunction with TRA2, a sex-non-specific RNA-binding protein, to trigger female development. TRA and TRA2 assemble into a complex with SR-type splicing factors and recognize short repeats of 13 nucleotides known as “TRA-TRA2 binding sites,” located in dsx exon 4 and fru exon 2 (Hedley and Maniatis 1991; Heinrichs et al. 1998). When the TRA-TRA2 complex binds these cis-regulatory elements, it reinforces the usage of suboptimal splicing sites in the dsx and fru primary transcripts and promotes female-specific splicing events (Tian and Maniatis 1993). Accordingly, XX; tra2 mutants that lack a functional TRA2 protein fail to activate the female processing of dsx and fru and develop as adult pseudomales. Similar to DmTRA, CcTRA feminizing activity is also abolished in Drosophila tra2− flies, thus suggesting that CcTRA requires a functional endogenous tra2 gene. We conclude that CcTRA is able to substitute for DmTRA and interact with the endogenous TRA2 protein. The CcTRA-TRA2 complex maintains the specificity for the “TRA-TRA2 binding sites” and the ability to direct the proper female-specific splicing of dsx and fru.

Molecular mechanisms for Cctra and Ccdsx regulation in Ceratitis:

One important finding of this study is that CcTRA is able to “recognize” the TRA-TRA2-binding sites in vivo, although they are located in entirely divergent contexts, namely the Dmfru and Dmdsx genes of Drosophila. It is therefore tempting to speculate that the TRA-TRA2-binding sites are also target sequences for CcTRA activity in Ceratitis. In this species, TRA-TRA2 elements are present in exon 4 of the dsx homolog (Ccdsx) and in intron 1 of the Cctra gene. Ccdsx reveals a significant structural and sequence identity when compared to Dmdsx and shows a sex-specific expression pattern (Saccone et al. 2000, 2002; Pane et al. 2002). The distribution of the cis elements in the Ccdsx gene is also similar to that of Dmdsx, since they are located in exon 4, which is sex-specifically regulated in both Ceratitis and Drosophila (Saccone et al. 2000). Given that CcTRA can promote the proper female splicing of Dmdsx, it is conceivable that it also controls Ccdsx expression using a similar mechanism. In Ceratitis females, CcTRA is likely to bind the cis elements in Ccdsx exon 4 and to promote the fusion of exon 3 to exon 4. The resulting female mature transcripts encode the CcDSXF protein. By contrast, in males, where the CcTRA protein is absent, exon 4 is not included in the mature mRNA, with exon 3 being fused directly to exon 5. The mature mRNAs generated in males encode the CcDSXM isoform. Consistent with this model, when the Cctra gene is turned off by RNAi in XX individuals, Ccdsx expression pattern is switched from the female to the male mode of splicing (Pane et al. 2002). As in Drosophila, the CcDSX isoforms are likely to control the development of sexually dimorphic traits in Ceratitis.

We have shown that putative TRA-TRA2 elements are also surprisingly contained in intron 1 of the Cctra gene (Pane et al. 2002). This observation pointed to a role for the CcTRA protein in the processing of Cctra precursor mRNA. In Ceratitis, Cctra is sex-specifically expressed through post-transcriptional alternative splicing events. In females, intron 1 is removed from the primary transcript and mature mRNAs that encode the full-length CcTRA protein are produced. Differently, mature mRNAs generated in males retain portions of the intron 1 (i.e., male-specific exons), which contain stop codons and thus prematurely interrupt the translation of the CcTRA protein. We have reported that the female splicing of the Cctra primary transcripts is dependent upon a functional Cctra gene. When Cctra is switched off by RNAi in early embryos, the emerging XX adults are males and express male variants from Cctra. These observations lead to the hypothesis that, in Ceratitis females, Cctra controls its own expression by means of a positive feedback loop. The results reported here further support this hypothesis and suggest that, in females, the CcTRA protein might bind the TRA-TRA2-binding sites in the Cctra pre-mRNA and promote female splicing events. The binding of CcTRA to the cis elements might prevent the usage of male splicing sites, thus leading the splicing machinery to use the criptic female sites. Consequently, intron 1 is removed from Cctra precursor transcripts to produce the female mature mRNAs. An alternative possibility is represented by an activation mechanism in which CcTRA would enforce the usage of female splice sites. This model stems from the observation that the TRA-TRA2 elements are located mainly within the male-specific exons and therefore are included in male mature mRNAs (Pane et al. 2002; A. Pane, unpublished results). It is possible that, in females, “male” transcripts are produced by the default mechanism and might behave as splicing intermediates and substrates for CcTRA activity. In this case, the binding of the CcTRA protein to the cis-regulatory elements would favor the use of the female splice sites and promote the removal of the male-specific exons. Both the repression and the activation mechanisms that we propose would involve a new property for the TRA proteins as well as an intronic function for the TRA-TRA2 elements, which has not been described before. In females, Cctra mature mRNAs have a long open reading frame and represent the source of CcTRA protein to keep the feedback loop active and guarantee the memory of the female sexual state. In males, the M-factor is likely to impair the positive feedback loop at early stages, thus promoting the male developmental program.

Interestingly, CcTRA activity in the Drosophila transgenic lines is dependent upon a functional endogenous Dmtra2 gene. CcTRA is not able to direct female splicing of dsx and fru pre-mRNAs in Drosophila when the DmTRA2 protein is absent. We believe that, in Ceratitis also, female development involves the cooperation between CcTRA and a putative TRA2 homolog (CcTRA2), which is yet to be identified. Several observations further support this hypothesis. tra2 appears to be highly conserved in evolution and tra2 homologs were described even in humans (Dauwalder et al. 1996). Recently, a tra2 homolog was identified in the housefly Musca domestica, which diverged from Drosophila ~100 million years ago. Transient depletion of the tra2 function in Musca by RNAi triggers the sexual transformation of XX embryos, which normally become females, toward maleness (Dübendorfer et al. 2002). These observations all point to the existence of a conserved tra2 homolog in Medfly as strongly suggested by the sequence conservation of Tra/Tra-2-binding sites observed in the Ceratitis dsx homolog (Saccone et al. 2000). The CcTRA2 protein might interact with CcTRA to control both the female-specific splicing of Ccdsx and the positive feedback loop established by the Cctra gene.

Evolution of sex-determining networks:

A wide variety of sex-determination systems can be observed in nature, even when the investigation is restricted to small taxonomic groups (reviewed in Marin and Baker 1998; Schutt and Nothiger 2000; Saccone et al. 2002). In the dipteran insects, for instance, some species use a male-determining factor on the Y chromosome (Ceratitis, Musca), while others use a single autosomal factor (Megaselia, Culex). Yet, in other species, the primary signal is given by chromosome balance (Drosophila, Sciara) or maternal effects (Chrysomya). A growing body of evidence suggests that, despite the striking diversity of primary systems, the sex choice is translated to terminal differentiation genes via a short hierarchy of control elements. It has been proposed that these genetic hierarchies have evolved in a “bottom-up” fashion (Wilkins 1995), with new upstream regulators being recruited, in the course of evolution, to control ancient downstream elements. Therefore, the bottom-most components of the cascades are expected to be conserved across species, while upstream players can vary. Genetic dissection of the sex-determination process in a number of model organisms has so far supported this model. Homologs of the dsx gene, which is the last regulatory element in the cascade, appear to be conserved not only in flies (Shearman and Frommer 1998; Kuhn et al. 2000; Ohbayashi et al. 2001; Hediger et al. 2004), but also in worms and mammals (Raymond et al. 1998), while upstream regulators, such as Sxl, are not shared (Meise et al. 1998; Saccone et al. 1998; Serna et al. 2004). In this scenario, the tra gene represents a key element in gaining insight into the molecular mechanisms underlying the evolution of the sex-determining networks. To date, tra homologs have been described only in the drosophilids and in the distantly related species C. capitata. In both Drosophila and Ceratitis, tra determines the sexual fate of the developing individual by regulating the expression of the bifunctional dsx gene. In this study, we show that the CcTRA protein of Medfly can replace DmTRA in Drosophila sex determination and direct female expression of the endogenous dsx and fru, despite the high degree of evolutionary divergence. In agreement with previous reports (O'Neil and Belote 1992; McAllister and McVean 1999; Kulathinal et al. 2003), we demonstrate that wide rearrangements can occur in both the coding and noncoding region of tra, without affecting the sex-determining function of the gene. Our results support the idea that divergent tra homologs might control sex determination in yet other dipteran species, where a dsx gene has been identified (Pane et al. 2002; Saccone et al. 2002). Consistent with this hypothesis, a locus encoding a TRA-like protein, named complementary sex determiner (csd), was discovered to be the primary signal in sex determination of the honeybee Apis mellifera (Beye et al. 2003; Beye 2004). Although the hymenopteran diverged from the dipteran insects ~270 MYA, Apis retains a conserved dsx homolog, thus suggesting that the tra/csd>dsx genetic segment exists in the honeybee, similar to the Medfly and Drosophila (Beye 2004). These species, however, evolved different systems to regulate the sex-specific expression of tra/csd. In Drosophila, tra occupies an intermediate position in the hierarchy and is regulated by the upstream Sxl gene. By contrast, in Ceratitis, tra is the master switch gene that controls its own expression by means of a positive feedback loop. Finally, in Apis, csd encodes the primary signal and has no further upstream regulatory elements. It is likely that the unusual plasticity allowed tra to maintain the sex-determining activity through the dsx gene, while new upstream regulatory mechanisms were developed. An expectation of this hypothesis is that conserved tra>dsx modules might control sex determination in many other insects, which may have explored alternative systems to regulate its state of activity. We believe that the flexibility of tra and the recruitment of different players to control its expression provided a genetic basis for the evolution of different primary sex-determining signals in the dipteran as well as in more distantly related species.

Acknowledgments

We are grateful to Gerish Deshpande, Patricia Graham, Paul Schedl, Trudi Schupbach, and Andrew Swan for their critical reading of the manuscript and for stimulating discussions. We thank Daniel Bopp, Stephen Godwin, and William Mattox for kindly providing fly strains and Ennio Giordano and Rosaria Rendina for helpful suggestions. We also thank Rosaria Terracciano and Giovanni Manno for technical assistance. This work was supported by Ministero della Ricerca Scientifica e Tecnologica (PRIN 2001-03; FIRB 2003-05).

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