An evolutionary approach has been taken to investigate tra regulation and function, by isolating the tra-homologous genes from selected Drosophila species, and then using the interpecific DNA sequence comparisons to help identify regions of functional significance. The tra-homologous genes from two Sophophoran subgenus species, Drosophila simulans and Drosophila erecta, and two Drosophila subgenus species, Drosophila hydei and Drosophila virilis, were cloned, sequenced and compared to the D. melanogaster tra gene. This comparison reveals an unusually high degree of evolutionary divergence among the tra coding sequences. These studies also highlight a highly conserved sequence within intron one that probably defines a cis-acting regulator of the sex-specific alternative splicing event (O’Neil, 1992).
As expected from the known phylogenetic relationships, the degree of similarity is higher when melanogaster and simulans are compared (93.6% identity) than when either of these species is compared to erecta (86.0% and 85.6% identity). The higher degree of conservation of sequences in intron 1 than in intron 2 is not unexpected, since intron 1 exhibits sex-specific alternative splicing while the splicing of intron 2 is presumably not regulated. In D. melanogaster it has been shown that the sex-specific splicing of intron 1 is controlled by sequences contained within the intron itself, and these regulatory sequences are likely conserved. The close relationship between these members of the melanogaster subgroup, however, precludes using these comparisons to accurately delimit any well defined region of the intron that might be implicated in this regulation, since not enough time has passed for the nonimportant sequences to have diverged (O’Neil, 1992).
One obvious difference between the coding region of tra in D. melanogaster and that of the other two species is the presence of a 39-base pair sequence in the central exon of D. melanogaster that is missing in D. simulans and D. erecta. Given the phylogenetic relationships of these three species, the most parsimonious explanation is that there has been a duplication of this sequence in the lineage leading to D. melanogaster rather than two independent deletions occurring in the D. simulans and D. erecta lineages. This is supported by the fact that in D. melanogaster, these extra 39 bases exist as an almost perfect tandem repeat (37/39 identity) of the adjacent sequence. This duplication occurs in the very arginine-serine-rich region of the tra gene, and its presence in either one or two copies is presumably not critical for the functioning of the Tra protein. Other smaller deletions/insertions also are seen when the coding regions of these three species are aligned. For example, D. erecta has an extra three nucleotides, encoding a cysteine, in exon 1 that are not seen in D. melanogaster and D. simulans. In exon 2 there are two sites where an extra six nucleotides, encoding R S, and an extra 15 nucleotides, encoding R E/G S R H, are present in D. melanogaster and D. simulans, but not in D. erecta (O’Neil, 1992).
These differences among the coding regions yield different size Tra proteins in the three species: 197, 184 and 178 amino acids, for D. melanogaster, D. simulans and D. erecta, respectively. Because of the relatively close relationship among these three species, information about what regions of the tra gene are conserved and what regions are not conserved is limited (O’Neil, 1992).
The transformer locus produces an RNA processing protein that alternatively splices the doublesex pre-mRNA in the sex determination hierarchy of Drosophila melanogaster. Comparisons of the tra coding region among Drosophila species have revealed an unusually high degree of divergence in synonymous and nonsynonymous sites. In this study, the hypothesis that the tra gene will be polymorphic in synonymous and nonsynonymous sites within species was examined by investigating nucleotide sequence variation in eleven tra alleles within D. melanogaster. Of the 1063 nucleotides examined, two synonymous sites were polymorphic and no amino acid variation was detected. Three statistical tests were used to detect departures from an equilibrium neutral model. Two tests failed to reject a neutral model of molecular evolution because of low statisitical power associated with low levels of genetic variation. The Hudson, Kreitman, and Aguade test rejected a neutral model when the tra region was compared to the 5'-flanking region of alcohol dehydrogenase (Adh). The lack of variability in the tra gene is consistent with a recent selective sweep of a beneficial allele in or near the tra locus (Walthour, 1994).
The amino acid sequence of the transformer gene exhibits an extremely rapid rate of evolution among Drosophila species, although the gene performs a critical step in sex determination. These changes in amino acid sequence are the result of either natural selection or neutral evolution. To differentiate between selective and neutral causes of this evolutionary change, analyses of both intraspecific and interspecific patterns of molecular evolution of tra gene sequences are presented. Sequences of 31 tra alleles were obtained from Drosophila americana. Many replacement and silent nucleotide variants are present among the alleles; however, the distribution of this sequence variation is consistent with neutral evolution. Sequence evolution was also examined among six species representative of the genus Drosophila. For most lineages and most regions of the gene, both silent and replacement substitutions have accumulated in a constant, clock-like manner. In exon 3 of D. virilis and D. americana evidence is found for an elevated rate of nonsynonymous substitution, but no statistical support for a greater rate of nonsynonymous relative to synonymous substitutions. Both levels of analysis of the tra sequence suggest that, although the gene is evolving at a rapid pace, these changes are neutral in function (McAllister, 2000).
Given the unusually high degree of sequence divergence among tra homologs in Drosophila (O’Neil, 1992), the tra gene in the medfly was cloned by exploiting its close linkage in Drosophila to a well-conserved gene, l(3)73Ah. Hence, as a first step towards the isolation of tra, cDNA and genomic Ceratitis sequences were isolated that cross-hybridized to a 500 bp Drosophila cDNA fragment of l(3)73Ah at reduced stringency. These isolates indeed contained a structurally well conserved homolog of l(3)73Ah as confirmed by sequencing and comparison [Ccl(3)73Ah]. A 4 kb long genomic region downstream of the l(3)73Ah homolog was sequenced and a putative ORF was identified that showed by Blast search significant sequence similarity at the amino acid level to tra in Drosophila (ranging from 32% to 40% identity scattered over 120 amino acids) and contained an arginine-serine-rich domain (SR-rich region) commonly found in splicing regulators. As in Drosophila, the two genes are transcribed in opposite orientation and sequence analysis of corresponding cDNA clones reveals that they overlap by about 200 bp. It is concluded that this gene arrangement must have already existed in the common ancestor of these fly species. Though the significance of this synteny is unknown, it provides an ideal entry point to the molecular identification of the tra homolog in Ceratitis (Cctra) (Pane, 2002).
An alignment of CctraF1, CctraM1 and CctraM2 cDNA sequences with the genomic sequence exposes the organization of tra in Ceratitis. The gene is composed of five exons. The first, fourth and fifth exons are included in the mature transcripts of both sexes, while the second and the third exons are male specific. The most important finding is that the female-specific transcript has a long open reading frame, while the male-specific mRNAs contain stop codons that abort prematurely the protein translation. Indeed partially different intronic sequences are retained in the M1 and M2 cDNA clones, adding stop codons in different positions. This finding suggests that a functional full-length TRA is encoded only by the female-specific transcripts. This mode of sex-specific regulation at the level of splicing is well documented for the tra gene in Drosophila (Boggs, 1987). Different from Drosophila, however, where sex-specific regulation is based on the alternative use of two 3' splice acceptor sites, sex-specific regulation in Ceratitis appears more complex and is achieved by a combination of exon skipping and differential use of 5' donor and 3' acceptor sites (Pane, 2002).
The long ORF in the female-specific CctraF1 encodes a putative protein of 429 amino acids. The CcTRA protein exhibits a low degree of similarity to TRA proteins in Drosophila species and it is significantly larger in size in both N and C termini. Sequence processing tools of MACAW led to the identification of five small blocks of sequence similarity dispersed throughout the longest ORF of the female-specific transcripts. The regions with highest similarity (identified also by FastA analysis) are located between CcTRA positions 150-230, 286-292 and 332-342. The SR-rich region in Ceratitis TRA and possibly the other conserved domains may confer specific RNA binding and protein-protein interactions consistent with a proposed role in splicing regulation (Manley, 1996). The male-specific truncated protein isoforms lack the conserved boxes, the SR-rich region and do not show significant similarity with other known proteins (Pane, 2002).
The confinement of transcripts with a long ORF to females suggests that this gene had an essential role in female development of the medfly. To test its function, RNAi technique, which permits functional studies of genes in genetically less amenable organisms, was used. A 900 bp fragment of CctraF1 was used as a template to produce dsRNA that was then injected as a 15 µM solution into either the anterior or the posterior poles of embryos of two different laboratory strains (Benakeion and white-eye). From a total of 900 injected embryos, 272 adult flies were recovered and grouped by their sexual phenotype. A strong sex ratio bias was observed in favor of males. Out of 272, 231 flies (84.9%) showed a normal male morphology, 37 flies (13.6%) exhibited various degrees of intersexuality and the remaining four (1.4%) were the only flies recovered with a normal female phenotype. All of the 37 intersexes exhibited an anteroposterior pattern of intersexuality. More tellingly, the position of male tissues correlated exactly with the initial injection site in the embryo: injection into the anterior pole resulted in the formation of male-specific spatulated bristles on the head of intersexes, male-specific blue eye reflections, male-like bristles mixed with female-like bristles on the femur toward the coxa of the foreleg, but the genitalia at the posterior remained female-like. Conversely, injection into the posterior pole gave rise to mosaic adults with male genitalia but with female bristles on the head and female-specific green eye reflections. The intersexes showed also various degrees of abnormal gonadal development, with abnormally bent or deformed ovopositor and with mixed male-like and female-like tissues. A few intersexes apparently lacked genitalia (Pane, 2002).
To assess the sexual karyotype of affected flies, a PCR amplification of genomic DNA using Ceratitis Y-specific primers was performed. No products were detected in single preparations of 10 randomly chosen intersexes and six out of 10 phenotypic males did not reveal the presence of a Y chromosome by this test, indicating that all these animals have a female XX karyotype. These results are in agreement with the expected loss of female-promoting activity when tra function is impaired by RNAi. On the contrary, male development of XY flies seems not to be affected by RNAi of tra, suggesting that the gene, as in Drosophila, is dispensable in this sex. The occurrence of intersexes and of few females is most likely due to incomplete penetrance of the RNAi effect. Indeed, when a lower concentration of dsRNA (5 µM versus 15 µM) was injected into the anterior embryonic region, 64 intersexes, 76 males and four females were obtained out of 144 adult flies. Therefore the percentage of intersexes increased from 14% to 44%, while the percentage of males decreased from 84% to 52%, suggesting that XX individuals were only partially masculinized. From these results, it is concluded that tra is required for female development in Ceratitis. Moreover, it is conceivable that absence of tra activity constitutes a signal that triggers the male fate. Thus, as in Drosophila, Ceratitis tra may act as a genetic switch between female (when functionally ON) and male (when functionally OFF) development. The male-specific short peptides encoded by the alternatively spliced male-specific transcripts seem to be non-functional, at least at early embryonic stages, because the RNAi has no evident effects on the development of XY males. Whether they play a function at later stages, when the RNAi starts to lose its efficiency, cannot be evaluated (Pane, 2002).
To investigate the fertility of the RNAi-treated adults, 27 males obtained from embryos injected with 15 µM dsRNA solution were individually crossed with wild-type females. It is predicted that if XX males are fertile than they should give a female-only progeny when crossed with wild-type virgin females. Indeed out of 27, seven crosses gave a unisexual female-only progeny. The karyotype of these seven males was then analyzed by PCR, confirming that they were XX fertile males. As expected, PCR karyotypic analyses of those males giving a bisexual progeny revealed that they were XY males. These data demonstrate that the Y-chromosome does not carry genes necessary for male fertility (Pane, 2002).
Next, the mechanisms which control the activity of tra in Ceratitis were investigated. In Drosophila, regulation of tra activity is achieved at the post-transcriptional level based on 3' splice site selection (Boggs, 1997). When Sxl protein is present, Sxl prevents the use of a distal acceptor site, thereby promoting the use of the next downstream available 3' splice site, and it shifts about 50% of the pre-mRNA molecules from a non-sex-specific splicing to a productive female-specific mRNA. This regulation requires the direct binding of SXL to a poly (U)8 stretch upstream of the regulated splice site (Kanaar, 1995). Several findings argue against a similar mechanism for conferring sex-specific splicing of tra in Ceratitis (Saccone, 1998). (1) Cctra splicing is based on a combination of exon skipping and 5' and 3' splice site regulation, rather than on 3' splice site selection. (2) CcSXL protein is present in both sexes of Ceratitis. However, upon close inspection of the Cctra sequence, an important discovery was made: within the two male-specific exons and the male-specifically retained intron, eight repeats were found by DNA sequence comparison that are structurally related to the TRA/TRA-2 binding sites (13 nucleotides long) in the dsx gene of Drosophila. Similar repeats are also detected in the female-specific exon of the dsx homolog in Ceratitis (Saccone, 2000). Their high sequence similarity to Drosophila Tra/Tra-2 binding sites and peculiar localization within the Cctra gene led to the idea that these sequences are involved in the sex-specific splicing regulation of Cctra itself. In Drosophila dsx and fru genes these cis-elements act as, respectively, 3' and 5' splice enhancers by recruiting the Tra/Tra-2 complex to promote the use of the regulated splice site (Tian, 1993: Heinrichs, 1998). The presence of potential TRA/TRA-2-binding sites in and around the male-specific exons suggests that the female-specific CcTRA could inhibit their usage and led to an investigation of whether an autoregulatory function of Cctra is involved in the process of sex-specific splicing (Pane, 2002).
If female-specific splicing of tra pre-mRNA indeed depends on tra activity, it was reasoned that a transient depletion of tra activity should no longer be able to sustain the female mode of splicing. To test this supposition, sex-reversed XX males recovered from Cctra dsRNA injections were analyzed. By RT-PCR analysis, only male-specific tra products were detected in adult tissues of injected XX and XY individuals, but no female-specific products. In addition, the same males contained predominantly male-specific splice variants of dsx, a probable downstream target of tra also in Ceratitis. It was inferred from these results that early application of RNAi transiently eliminates Cctra mRNAs and, thus, prevents continued production of TRA protein. Once tra pre-mRNA production is resumed at a later stage in development, the unproductive male mode of tra splicing is launched because of the absence of functional TRA. Likewise, absence of TRA causes its direct target dsx to be spliced in the male mode. These results are compatible with the postulate that Cctra sustains the productive mode of its splicing by an autoregulatory feedback loop and mediates female differentiation, at least in part, by the control of its target gene dsx. The initiation of the autoregulatory loop in XX embryos could be based on maternal Cctra mRNAs that have been detected in unfertilized eggs by RT-PCR experiments. These mRNAs are spliced in the female mode and hence could provide a source of CcTRA activity that allows female-specific splicing of zygotic Cctra pre-mRNA (Pane, 2002).
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 non-functional 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, 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. Though 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. It is suggested that transformer plays an important role in providing a molecular basis for the variety of sex-determining systems seen among insects (Pane, 2005).
One important finding that comes out of this study is that CcTRA is able to 'recognize' the TRA-TRA2 binding sites in vivo, though 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. 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. 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 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. As in Drosophila, the Ccdsx isoforms are likely to control the development of sexually dimorphic traits in Ceratitis. Putative TRA-TRA2 elements are also surprisingly contained in intron 1 of the Cctra gene. This observation points 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, the intron 1 is removed from the primary transcript and mature mRNAs that encode the full-length CcTRA protein are produced (Pane, 2005).
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. 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 in this study 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 (Pane, 2005).
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, the 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 mainly located within the male-specific exons and therefore are included in male mature mRNAs. 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 proposed 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. It is believed that, also in Ceratitis, 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 have been described even in human. Recently a tra2 homolog was identified in the housefly Musca domestica, which diverged from Drosophila some 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. 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 homologue. 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 (Pane, 2005).
The variety of primary sex determination cues was appreciated long before the advent of molecular genetics. The two broadest categories are genetic sex determination (GSD), in which the sex of offspring is set by a sex chromosome or an autosomal gene, and environmental sex determination (ESD), in which sex is determined by temperature (as with turtles), local sex ratio (as with some tropical fish), or population density (as with mermithid nematodes). Though little is known about the molecular mechanisms of ESD, within the GSD systems many different mechanisms have been uncovered. Dual sex chromosome systems, in which either the female (ZW/ZZ) or the male (XX/XY) is heterogametic, are common, as are systems set by the ratio of the number of X chromosomes to sets of autosomes (X:A). There are also systems in which heterozygosity at a single locus is required for female development (known as complementary sex determination), as well as systems involving sex determination via multiple genes with additive effects (Haag, 2005see full text of article).
Molecular genetic investigations of GSD in model systems such as Drosophila, Caenorhabditis, and mice have revealed a clear lack of conservation, underscoring the diversity. For example, although the primary sex determination signal in both D. melanogaster and C. elegans is the X:A ratio, the fruit fly pathway consists of a cell-autonomous cascade of regulated mRNA splicing, while that of the nematode follows a Hedgehog-like intercellular signaling pathway. GSD in mammals depends (with some interesting exceptions upon a Y-specific dominant gene (Sry) encoding a transcription factor. In the face of such impressive differences, perhaps the assumption of homology should be questioned: could it be that sex determination in different taxa has arisen independently over and over again in evolution? Until 1998, this seemed like a good bet (Haag, 2005).
The discovery of the homology of the key sex-determining genes doublesex in Drosophila and mab-3 in C. elegans provided the first evidence for a common evolutionary basis of sex determination in animals. Soon, related doublesex-mab-3 (DM)-family genes with roles in male sexual development were discovered in vertebrates and even cnidarians. Here at last was a smoking gun that could link the diverse metazoan sex determination systems. But as satisfying as the result was, it immediately gave birth to another mystery: if the enormous diversity of sex determination systems are all derived from a common ancestor, how could they possibly have been modified so radically? After all, sexual differentiation and reproduction are hardly unimportant developmental processes (Haag, 2005).
To understand how such diversity came to be, differences between closely related species must be examined. This approach allows the discovery and interpretation of small-scale sex determination changes before they are obscured by subsequent changes. The processes discovered in this way might then be reasonably extrapolated to explain the seemingly unrelated systems of more deeply diverged taxa. Work in dipterans has revealed three evolutionary phenomena that characterize shorter-term sex determination evolution (Haag, 2005).
The first of these is the often astounding rate of molecular evolution at the level of nucleotide and aminoacid sequences. Although some sex-determining genes are well conserved, many show unprecedented substitution rates. An extreme example is the central integrator of the X:A ratio in Caenorhabditis, xol-1. The xol-1 orthologues of the closely related nematodes C. elegans and C. briggsae are a mere 22% identical, even though genes surrounding xol-1 are much better conserved. Remarkably, the 3′ neighbor of xol-1, the immunoglobulin dim-1, is only 5 kb away and is essentially identical between species (Haag, 2005).
A second phenomenon, best exemplified by dipteran insects, is the modification of genetic control pathways through the gain or loss of key pathway components. In Drosophila, the first gene to respond to the X:A ratio is Sxl, whose transcription is regulated by both autosomal and X-linked factors very early in development. When X: A = 1 (i.e., in female embryos), Sxl transcription occurs and produces Sxl protein. Later in development, transcription from a second promoter occurs in both sexes, but these transcripts cannot be productively spliced without the earlier burst of Sxl expression. As a result, only females sustain Sxl expression, and in turn only females can productively splice the mRNA of tra, its downstream target. Productive splicing of tra is required to produce the female-specific form of dsx, a founding member of the DM family mentioned above (Haag, 2005).
In a series of groundbreaking papers, Saccone and colleagues investigated the pathway in the more distantly related heterogametic Mediterranean fruit fly Ceratitis capitata. The first surprise was that although a highly conserved Sxl homologue exists in Ceratitis, it does not undergo sex-specific regulation similar to that of Drosophila, which suggests that it does not play a key switch role (Saccone, 1998). Similar results have also been found for the housefly, Musca domestica, indicating that the role of Sxl in sex determination may be restricted to Drosophila and its closest relatives. In contrast, tra and dsx are key sex regulators in all dipterans examined thus far (Haag, 2005).
A further surprise came when the Ceratitis tra homologue was characterized. In the case of this gene, clear evidence for sex-specific regulation was found, and as with Drosophila, only females productively splice tra mRNA. However, this splicing difference can be explained nicely by a positive feedback, similar to that seen in Drosophila Sxl, in which Tra protein regulates its own splicing. It has been proposed that the dominant, male-specifying M factor on the Y chromosome inhibits this autoregulation. As a result, males cannot make functional Tra protein, and the male form of Dsx is produced. These experiments show not only how a pathway can evolve, but also, importantly, how X:A and heterogametic GSD systems can be interconverted by modifying the cue that regulates a conserved molecular switch gene (the splicing of tra mRNA) (Haag, 2005).
Finally, recent studies of Caenorhabditis nematodes have shed light on the genetic basis of the convergent evolution of sex determination related to mating system adaptations. An important factor in this area are new phylogenies of the genus, which consistently suggest the surprising possibility that the closely related hermaphroditic species C. elegans and C. briggsae acquired self-fertilization independently, from distinct gonochoristic (male/female) ancestors. Although this scenario is somewhat uncertain purely on parsimony grounds, recent work on the genetic control of the germline bisexuality that defines hermaphroditism has tipped the balance toward parallel evolution (Haag, 2005).
C. elegans fog-2, a gene required for spermatogenesis in hermaphrodites but not in males, has been cloned. It became clear that fog-2 is part of a large family of F-box genes and was produced by several recent rounds of gene duplication. The C. briggsae genome sequence suggested that while C. briggsae possesses a similarly large family of F-box proteins, the duplication event giving rise to fog-2 was specific to the C. elegans lineage. This work has been extended by the rigorous demonstration that fog-2 is indeed absent in C. briggsae. A short, C-terminal domain has been identified that makes FOG-2 uniquely able to perform its germline sex-determining function. This domain is probably derived from a frame-shifting mutation in an ancestral gene. Working with C. briggsae, evidence has been found of important species-specific regulation of germline sex determination. RNA interference and gene knockout approaches have shown that while C. elegans requires the male-promoting genes fem-2 and fem-3 to produce sperm in hermaphrodites, C. briggsae requires neither. Given that both genes have conserved roles in male somatic sex determination, this suggests that C. briggsae evolved hermaphroditism in a way that bypasses these genes (Haag, 2005).
The long-standing mystery of sex determination and its diversity began by comparisons between distantly related species. Recent work on closer relatives has uncovered processes that through a reasonable extrapolation enable the connection of these disparate dots into a fascinating picture of developmental evolution. Though the divergence is extreme, it is likely that a better understanding of the evolution of sex determination genes and pathways holds lessons about the evolution of development in general. The next major challenge will be to integrate the comparative developmental data with the ecological and population processes that are driving the evolution of sex determination. Only then will it be possible to say that the picture is complete (Haag, 2005).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.