The Interactive Fly
Zygotically transcribed genes
Sex Determination and Dosage Compensation Genes
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The Interactive Fly Sex Determination and Dosage Compensation Genes |
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| A simplified model for sex determination in the somatic gonad and germline |
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).
Sex determination in Drosophila is commonly thought to be a cell-autonomous process, where each cell decides its own sexual fate based on its sex chromosome constitution (XX versus XY). This is in contrast to sex determination in mammals, which largely acts nonautonomously through cell-cell signaling. This study examined how sexual dimorphism is created in the Drosophila gonad by investigating the formation of the pigment cell precursors, a male-specific cell type in the embryonic gonad surrounding the testis. Surprisingly, sex determination in the pigment cell precursors, as well as the male-specific somatic gonadal precursors, was found to be non-cell autonomous. Male-specific expression of Wnt2 within the somatic gonad triggers pigment cell precursor formation from surrounding cells. These results indicate that nonautonomous sex determination is important for creating sexual dimorphism in the Drosophila gonad, similar to the manner in which sex-specific gonad formation is controlled in mammals (DeFalco, 2008).
This study has shown that two distinct male-specific cell types in the Drosophila gonad exhibit nonautonomous sex determination. For both the
male specific somatic gonadal precursors (msSGPs) and the pigment cell (PC) precursors, the sex determination pathway does not act in these cells themselves, and both are dependent on sex-specific signaling from the SGPs in order to develop properly as male or female. These findings are in contrast to the commonly held view that sex determination in Drosophila is a cell-autonomous process, and demonstrate the similarity in sex-specific gonad development between flies and mammals (DeFalco, 2008).
This study has identified a novel, sex-specific cell type in the Drosophila embryonic gonad, the PC precursors, and studied the mechanism by which the sex determination switch controls the sex-specific development of these cells. The data indicate that male-specific expression of Wnt2 in the SGPs of the gonad signals nonautonomously to the fat body to form PC precursors. dsx ensures that PC formation is male-specific by repressing Wnt2 expression in female gonads in late-stage embryos (stage 17). The sex of the fat body itself does not affect PC precursor formation, since cells with a female identity can form PC precursors when associated with a male gonad or with a female gonad that expresses Wnt2. Furthermore, Wnt2 acts directly on the fat body, since blocking Wnt signaling in male fat body cells prevents them from forming PC precursors. PC precursor identity in the fat body is regulated by ems acting upstream of Sox100B in response to the Wnt2 signal. An interesting question is whether Wnt2 is a direct downstream target of DSX in controlling sexual dimorphism. The DNA binding specificity for DSX has been determined, and there are a number of sites upstream of the Wnt2 start of transcription that either exactly match or closely match the DSX binding consensus sequence. Several of these sites are conserved between different Drosophila species. However, a fragment of the Wnt2 promoter has not yet been identified that allows testing of whether Wnt2 expression in the somatic gonad is directly regulated by DSX, since the upstream region that includes the putative DSX binding sites does not promote expression in the gonad (DeFalco, 2008).
The creation of sexual dimorphism in the PC precursors differs from that of the msSGPs. While the PC precursors are apparently only specified in males and recruited to form part of the testis, msSGPs are initially specified in both sexes, and are only present in the male gonad because they undergo programmed cell death specifically in females. Furthermore, the germline stem cell niche in the testis (the hub) is formed from a population of anterior SGPs that are present in the gonads of both sexes, but only form the hub in males and presumably form part of the ovary in females. These events are all regulated by dsx, and demonstrate the diverse cellular mechanisms that a sex determination gene can utilize to control sexual dimorphism. Interestingly, in dsx null mutant embryos each of these cell types develops as if it were male. Thus, the male mode of development can at least be initiated in these cell types in the absence of dsx function, and dsx primarily acts in females to repress male development. dsx is clearly required in males at some point for proper testis formation, therefore some cell types in the gonad may not be entirely masculinized in dsx mutants (DeFalco, 2008).
The nonautonomous nature of PC precursor specification contrasts with the commonly held view that sex determination in Drosophila is a cell-autonomous process, where 'every cell decides for itself' whether it should develop as male or female based on its own intrinsic sex chromosome constitution. This study has shown that the msSGPs undergo nonautonomous sex determination. The data indicate that a male-specific survival signal coming from the SGPs allows the msSGPs to survive and join the male gonad, while they undergo apoptosis in females. Finally, it has been shown that nonautonomous sex determination in the germ cells requires a male-specific signal from the SGPs that acts through the JAK/STAT pathway. Thus, not only does non-cell autonomous sex determination occur in the Drosophila gonad, it appears to be the predominant mechanism of sex determination. Of the cell types tested so far, only the hub cells, which form from a subset of SGPs, appear to decide their sexual fate in an autonomous manner. The current model is that the SGPs determine their sex in a cell-autonomous manner, and then signal to other cell types in the gonad (PC precursors, msSGPs, and germ cells) to control the sex-specific development of these cells via nonautonomous sex determination (DeFalco, 2008).
Nonautonomous sex determination is not limited to the gonad. Other tissues have been shown to decide their sex through cell-cell signaling. In the genital imaginal disc, the sexual identity of a signaling center, the A/P organizer, largely determines whether the disc will develop in the male or female mode. This is controlled non-cell autonomously through Wingless and Decapentaplegic signaling. In addition, sex-specific migration of mesodermal cells into the male genital disc is regulated by male-specific expression of the Fibroblast Growth Factor Branchless in the genital disc. Finally, in the nervous system, male neurons can non-cell autonomously induce the formation of the male-specific muscle of Lawrence from female muscle precursors. Given the large number of tissues and cell types that undergo nonautonomous sex determination, it seems that the conventional view can be abandoned that sex determination in Drosophila is an obligatorily cell-autonomous process; while some cell types utilize a cell-autonomous mechanism, many cell types clearly do not (DeFalco, 2008).
One reason why sex determination has been traditionally thought of as a cell-autonomous process in Drosophila is due to its relationship with X chromosome dosage compensation. This is the process by which gene expression from the single X chromosome in males is regulated to match that from the two X chromosomes in females. Both sex determination and X chromosome dosage compensation are regulated by the number of X chromosomes, acting through the master control gene Sex lethal (Sxl). It is likely that most or all cells count their X chromosomes and use this information to control X chromosome dosage in a cell-autonomous manner. However, as discussed above, it is now clear that cells do not necessarily use this information to decide their sex. Consistent with this idea, the expression of dsx, a key regulator of sex determination downstream of Sxl, is surprisingly tissue-specific. Within the embryo, dsx is only expressed in the SGPs and msSGPs of the gonad. Thus, not all cells even express the machinery to translate their sex chromosome constitution into sexual identity, and it is therefore necessary that sex-specific development of many cell types be controlled nonautonomously (DeFalco, 2008).
The nonautonomous cell-cell interactions that control gonad sexual dimorphism in Drosophila show great similarity to sex-specific gonad development in other species. In mammals, somatic sex determination is based on the presence or absence of the Y chromosome. The critical Y chromosome gene Sry is mainly expressed in a subset of cells in the somatic gonad in the mouse embryo, similar to dsx expression in the Drosophila embryonic gonad. Sry is only thought to be important for formation of Sertoli cells in males, and the sexually dimorphic development of all other cell types is thought to be regulated by local cell-cell interaction or hormonal cues. An excellent example of nonautonomous sex determination in the mouse is the recruitment of cells from the neighboring mesoderm (mesonephros) to form specific cell types in the mouse testis. Recruitment of these cells is dependent on the sex of the gonad, not the sex of the mesonephros. In addition, sex-specific development of other somatic cell types in the mouse gonad is regulated nonautonomously by cell-cell interaction, as is sexual identity in the germline. Thus, the use of non-cell autonomous sex determination and sex-specific cell recruitment are common mechanisms for creating gonad sexual dimorphism in flies and mice (DeFalco, 2008).
Nonautonomous sex determination in the mouse also utilizes signaling through the Wnt pathway. Wnt4 acts as a 'pro-female' gene that opposes Fibroblast growth factor 9 to regulate sex determination in the gonad. In early stages of gonad development, Wnt4 knockout females form a male-specific coelomic blood vessel and exhibit ectopic migratory steroidogenic cells, suggesting that Wnt4 acts to inhibit endothelial cell and steroid cell migration from the mesonephros into the female gonad. Interestingly, Wnt4 also has been shown to have a role in the male gonad, as male knockout mice show defects in Sertoli cell differentiation, downstream of Sry but upstream of Sox9. Wnt7a also has been implicated in sexual dimorphism in the reproductive tract, as Wnt7a knockout mice fail to express Mullerian-inhibiting substance (MIS) type II receptor in the Mullerian duct mesenchyme, which is required for regression of the duct in male embryos. In addition, a number of Wnt genes have been found to be expressed sex-specifically in the gonad through sex-specific gene profiling, indicating that other Wnt family members play a role in creating sexual dimorphism in the mammalian gonad (DeFalco, 2008).
It is also interesting that several conserved transcription factors act during gonad development in diverse species. Sox100B is the fly homolog of SOX9/Sox9, a critical regulator of sex determination and male gonad development in humans and mice. Similarly, a mouse homolog of ems, Emx2, is expressed in the developing gonad and is required for development of the urogenital system. Lastly, dsx homologs of the DMRT family have been implicated in sex-specific gonad development in diverse species. Thus, not only are the cellular mechanisms, such as non-cell autonomous sex determination and cell-cell recruitment, common between flies and mice, but the specific genes that regulate sexually dimorphic gonad development may also be conserved. Since the formation of testes versus ovaries, and sperm versus egg, are critical features of sexual reproduction, they may represent processes that are highly conserved across the animal kingdom (DeFalco, 2008).
DeFalco, T., Camara, N., Le Bras, S. and Van Doren, M. (2008). Nonautonomous sex determination controls sexually dimorphic development of the Drosophila gonad. Dev. Cell 14(2): 275-86. PubMed Citation: 18267095
Haag, E. S. and Doty, A. V. (2005). Sex determination across
evolution: connecting the dots. PLoS Biol. 3(1): e21. 15660158
Saccone, G., Peluso, I., Artiaco, D., Giordano, E., Bopp, D., et
al. (1998). The Ceratitis capitata homologue of the Drosophila
sex-determining gene sex-lethal is structurally conserved, but not
sex-specifically regulated. Development 125: 1495-1500. Medline abstract: 9502730
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