Sex lethal
SXL proteins were found in all of the drosophilids examined, and they display a
sex-specific pattern of expression. Characterization of the Sxl gene in the distant
drosophilan relative, D. virilis, reveals that the structure and sequence organization of the gene has
been well conserved and that, like melanogaster, alternative RNA processing is responsible for its
sex-specific expression. Hence, this posttranscriptional on-off regulatory mechanism probably
existed before the separation of the drosophilan and sophophoran subgenera. It seems likely
that Sxl functions as a sex determination switch gene in most species in the Drosophila genus.
Although alternative splicing appears to be responsible for the on-off regulation of the Sxl gene in
D. virilis, this species is unusual in that Sxl proteins are present not only in females but also in
males. The D. virilis female and male proteins appear to be identical over most of their length
except for the amino-terminal approx. 25 aa which are encoded by the differentially spliced exons.
In transcriptionally active polytene chromosomes, the male and female proteins bind to the same
cytogenetic loci, including the sites corresponding to the D. virilis Sxl and tra genes. Hence, though
the male proteins are able to interact with appropriate target pre-mRNAs, they are apparently
incapable of altering the splicing pattern of these pre-mRNAs (Bopp, 1996).
In Drosophila, Sex lethal functions as a binary switch in sex determination. Under the control of the primary sex-determining signal, it produces functional protein only in XX animals to implement female development. In contrast to Drosophila, the Sxl homolog in the Medfly, Ceratitis capitata, expresses the same mRNAs and protein isoforms in both XX and XY animals irrespective of the primary sex-determining signal. Experiments with two inducible transgenes demonstrate that the corresponding Ceratitis Sxl product has no significant sex-transforming effects when expressed in Drosophila. The Medfly belongs to the Acalyptratae group, as does Drosophila, and hence it is closely related in phylogenesis to Drosophila. These findings suggest that Sxl acquired its master regulatory role in sex determination during evolution of the Acalyptratae group, most probably after phylogenetic divergence of the genus Drosophila from other genera of this group. It is suggested that in Ceratitis, Musca (the housefly) and other nondrosophilids, Sxl is primarily or even exclusively used as a translational repressor in both sexes to moduate gene activity in a broad sense. In Drosophila, it may in addition have acquired a function as a splicing regulator for sex-specific control of a small number of targets. Ceratitis and Musca Sxl proteins most notably differ from Drosophila Sxl in the amino terminal region. In Drosophila, this domain has been proposed to be important for cooperative binding of the protein to target RNA. This region may thus have served as a site of alteration in which a change in the molecular properties of the protein took place (Saccone, 1998).
Sex-lethal (Sxl) is the master switch gene for somatic sex determination in Drosophila. In XX animals, Sxl becomes activated and imposes female development; in X(Y) animals, Sxl remains inactive and male development ensues. A switch gene for sex determination, called F, has also been identified in the housefly, Musca domestica. An active F dictates female development, while male development ensues when F is inactive. To test if the switch functions of Sxl and F are founded on a common molecular basis, the homologous Sxl gene was isolated in the housefly. Though highly conserved in sequence, Musca-Sxl is not sex-specifically regulated: the same transcripts and protein isoforms are expressed in both male and female animals throughout development. Musca-Sxl is apparently not controlled by the primary sex-determining signal and, thus, is unlikely to correspond to the F gene. Ectopic expression of Musca-SXL protein in Drosophila does not exert any noticeable effects on the known target genes of endogenous Sxl. Instead, forced overexpression of the transgene eventually results in lethality of both XY and XX animals and in developmental abnormalities in some escaper XY animals. These results suggest that, in non-drosophilid species, Sxl performs a function different from that in sex determination. In Musca the X and Y chromosomes are interchangeable, i.e., genetically equivalent, except for the male determiner on the Y. Thus, there is no obvious need for a mechanism that compensates for different doses of X-linked genes on XX and XY houseflies. A tra-like gene would therefore suffice to act as the first gene in the sex determination cascade in Musca to govern sexual development. It is interesting to think that Sxl may have been recruited in the genus Drosophila to coordinte the controls of dosage compensation and sexual differentiation. It is intriguiing that most of the transcriptional regulators of Sxl in Drosophila also participate in controlling the neural pathway. Thus Sxl and its regulators may have originally functioned as a regulatory cassette in neurogenesis. A role for Sxl in neurogenesis finds support in the observation that male embryos of D. virilis express a male-specific SXL protein that specifically accumulates in the developing central nervous system (Meise, 1998 and references).
The gene homologous to Sex-lethal (Sxl) of Drosophila
melanogaster has been cloned and characterized from
Sciara coprophila, Rhynchosciara
americana, and Trichosia pubescens. This gene plays the
key role in controlling sex determination and dosage compensation in
D. melanogaster. The Sxl gene of the three species
studied produces a single transcript encoding a single protein in
both males and females. Hence, Sxl does not appear to play the key
discriminating role in controlling sex
determination and dosage compensation in sciarids that it plays in
Drosophila. Comparison of the Sxl proteins of these
Nematocera insects with those of the Brachycera shows their two
RNA-binding domains (RBD) to be highly conserved, whereas significant
variation is observed in both the N- and C-terminal domains. The
great majority of nucleotide changes in the RBDs were synonymous,
indicating that purifying selection is acting on them. In both sexes
of the three Nematocera insects, the Sxl protein colocalizes with
transcription-active regions dependent on RNA polymerase II but not
on RNA polymerase I. Together, these results indicate that Sxl
does not appear to play a discriminatory role in the control of sex
determination and dosage compensation in nematocerans. Thus, in the
phylogenetic lineage that gave rise to the drosophilids, evolution
coopted for the Sxl gene, modified it, and converted it into
the key gene controlling sex determination and dosage
compensation. At the same time, however, certain properties of the
recruited ancestral Sxl gene were beneficial, and these are
maintained in the evolved Sxl gene, allowing it to exert its
sex-determining and dose compensation functions in Drosophila (Serna, 2004).
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, 2005; see 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).
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