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).
The X chromosome of Drosophila shows a deficiency of genes with male-biased expression (see Sturgill, 2007), whereas mammalian X chromosomes are enriched for spermatogenesis genes expressed premeiosis and multicopy testis genes. Meiotic X-inactivation and sexual antagonism can only partly account for these patterns. This study shows that dosage compensation (DC) in Drosophila may contribute substantially to the depletion of male genes on the X. To equalize expression between X-linked and autosomal genes in the two sexes, male Drosophila hypertranscribe their single X, whereas female mammals silence one of their two X chromosomes. Fine-scale mapping data of dosage compensated regions was combined with genome-wide expression profiles to show that most male-biased genes on the D. melanogaster X are located outside dosage compensated regions. Additionally, X-linked genes that have newly acquired male-biased expression in D. melanogaster are less likely to be dosage compensated, and parental X-linked genes that gave rise to an autosomal male-biased retrocopy are more likely located within compensated regions. This suggests that DC contributes to the observed demasculinization of X chromosomes in Drosophila, both by limiting the emergence of male-biased expression patterns of existing X genes, and by contributing to gene trafficking of male genes off the X (Bachtrog, 2010).
This study found compelling evidence that dosage compensation influences patterns of sex-biased expression in Drosophila, and contributes to movement of male-biased genes off the X. This analysis suggests that the deficiency of male-biased genes on the Drosophila X does not simply reflect a lack of dosage compensation at some genes but instead can partly be accounted for by dosage compensation directly interfering with further upregulation of MSL-bound, already hypertranscribed X-linked genes in males. The X chromosome in male Drosophila is encumbered by the MSL complex and its chromatin structure is modified globally, which may limit subsequent transcription factor binding or chromatin remodeling, and thus inhibit further transcriptional activation. Indeed, direct interference between chromatin remodeling complexes and the dosage compensation machinery has been reported in Drosophila. Additionally, male-biased gene expression originates mainly by increasing transcription of nonbiased genes in males (rather than downregulation in females, and higher expression levels may be harder to achieve on an already hypertranscribed chromosome. High-expression male-biased genes are located less often on the X than low-expression male-biased genes. This is expected if limits in rates of transcription prevent the accumulation of male-biased genes on the X, given that such limitations are less likely to affect genes that are transcribed only at low levels (Bachtrog, 2010).
Not all organisms show a deficiency of male-biased genes on the X. In particular, mammalian X chromosomes are enriched for single-copy genesis genes that are expressed premeiosis, and multicopy testis genes showing postmeiotic expression. This difference in X-chromosomal gene content between taxa could result from fundamental differences in the mechanisms of dosage compensation. Dosage compensation in mammals is achieved by first doubling global expression levels of the X in both sexes, followed by inactivation of one X in females. The chromatin structure of the active X in mammals and baseline transcription rates of X-linked genes thus appear the same between the sexes (even though they might differ from average autosomal rates of transcription), therefore imposing no male-specific restrictions on the evolution of sex-biased expression patterns. Thus, the difference in X-chromosomal gene content between Drosophila and mammals -- with a deficiency versus an accumulation of male-biased genes -- may be understood in light of their vastly different dosage compensation mechanisms (Bachtrog, 2010).
Germ cells must develop along distinct male or female paths to produce the sperm or eggs required for sexual reproduction. In both mouse and Drosophila, sexual identity of germ cells is influenced by the sex of the surrounding somatic tissue, but little is known about how the soma controls germline sex determination. This study shows that the JAK/STAT pathway provides a sex-specific signal from the soma to the germline in the Drosophila embryonic gonad. The somatic gonad expresses a JAK/STAT ligand, unpaired (upd), in a male-specific manner, and activates the JAK/STAT pathway in male germ cells at the time of gonad formation. Furthermore, the JAK/STAT pathway is necessary for male-specific germ cell behavior during early gonad development, and is sufficient to activate aspects of male germ cell behavior in female germ cells. This work provides direct evidence that the JAK/STAT pathway mediates a key signal from the somatic gonad that regulates male germline sexual development (Wawersik, 2005).
While investigating communication between the somatic gonad and germline, the JAK/STAT pathway was found to be specifically activated in male, but not female, germ cells. In Drosophila, JAK/STAT signaling is initiated when an UPD or UPD-like ligand binds a transmembrane receptor (Domeless), activating the JAK Hopscotch (HOP), which phosphorylates the STAT92E transcription factor. STAT activation has been shown to regulate stat gene expression and can induce upregulation of the STAT92E protein, which can be used as an assay for JAK/STAT pathway activation. STAT92E is upregulated specifically in male, but not female germ cells at the time of gonad formation. This reflects male-specific activation of the JAK/STAT pathway since (1) the activated form of STAT92E (phospho-STAT92E) is also detected in only male germ cells, and (2) JAK activity is necessary and sufficient for STAT92E expression. Expression of a JAK inhibitor, Socs36E, results in loss of STAT92E expression in male germ cells and expression of constitutively active JAK (hopTumL) induces STAT92E in female germ cells. The male-specific activation of STAT92E at this time is distinct from STAT92E activation in germ cells in the early embryo, which is not sex-specific and is regulated by the MAP kinase pathway (Wawersik, 2005).
It was also found that STAT92E expression in male germ cells is dependent on their association with the somatic gonad. STAT92E is not detected in germ cells that are migrating to the gonad, but is detected in male germ cells after they contact the somatic gonad. STAT92E expression is greatly reduced or absent in eya mutants, where somatic gonad identity is initiated, but not maintained. Furthermore, STAT92E is not detected in germ cells found outside the gonad in wild type embryos or in mis-localized germ cells in wunen and HMG-CoA reductase mutants which lack guidance cues that target germ cells to the somatic gonad. However, in these same mutants, STAT92E is detected in the few germ cells that contact the somatic gonad in male embryos (Wawersik, 2005).
STAT92E expression in the germline is dependent on the sex of the surrounding soma. When XX (normally female) germ cells were present in a soma that was masculinized by expression of the male form of the somatic sex determination gene doublesex (dsx), germ cells now expressed STAT92E. dsx does not play an autonomous role in germ cells themselves, indicating that STAT92E induction in these embryos is caused by masculinization of the soma. Conversely, when the somatic gonad of an XY (normally male) embryo is feminized by expression of the sex determination gene transformer (tra) in the mesoderm, but not germ cells, STAT92E expression is no longer observed in XY germ cells. Taken together, these data indicate that the male somatic gonad is necessary and sufficient to activate the JAK/STAT pathway in either XX or XY germ cells (Wawersik, 2005).
Consistent with this, it was found that the JAK/STAT ligand, upd, is expressed specifically in the male, but not female, somatic gonad. Expression of STAT92E in male germ cells was no longer detected in embryos in which upd and two homologs, upd2 and upd3, are deleted [Df(os1a]. Since male germ cells from embryos mutant for upd alone still express STAT92E, JAK/STAT activation in the germline may be regulated redundantly by upd and one or more of its homologs. In addition, expression of upd in either the mesoderm or germ cells is sufficient to induce STAT92E expression in XX germ cells. Expression of upd2 or upd3 is also capable of inducing STAT92E in germ cells (Wawersik, 2005).
upd is also important for embryonic patterning and somatic sex determination. Interestingly, upd promotes female identity in the soma, but promotes male development in the germline. To verify that the effects of upd on the germline are not indirectly caused by other effects of upd, indicators of embryonic segmentation (Engrailed), somatic sex determination (Sex lethal), somatic gonad identity (Eyes absent), and somatic gonad sexual identity (Sox100B) were examined. Df(os1a) hemizygous male embryos exhibit segmentation defects as expected, but form gonads that express normal somatic and sex-specific markers. Embryos ectopically expressing upd are normal in all respects examined (Wawersik, 2005).
Whether activation of the JAK/STAT pathway by the male somatic gonad regulates male-specific development of germ cells was examined. In adult testes, the JAK/STAT pathway is required for maintenance of germline stem cells, making it difficult to assess the role of this pathway on male germ cell identity at this stage. Instead, germ cells were examined during embryogenesis and early larval stages, when germ cell development first becomes sexually dimorphic. In the mouse, the earliest manifestation of sex determination in the germline is differential regulation of the germline cell cycle by the soma. In Drosophila, germ cells undergo 1-2 divisions after their formation, but are arrested in the cell cycle during germ cell migration and only resume division shortly after the gonad has formed. Since larval testes contain more germ cells than larval ovaries, whether proliferation is regulated differently in male and female germ cells was examined. Indeed, sex-specific analysis of a mitotic marker (phosphohistone-H3) in the germline indicates that germ cell proliferation is entirely male-specific during early stages of gonad development. Furthermore, male-specific germ cell division is dependent on the male somatic gonad. Male germ cells do not proliferate in eya mutants that lack the somatic gonad, or in lost germ cells within wunen mutant embryos. XX germ cells in a masculinized soma (dsxD/ dsx1) proliferate, while XY germ cells in a feminized soma (UAS-traF; twist-Gal4) do not. Thus, the pattern of germ cell proliferation correlates exactly with activity of the JAK/STAT pathway in germ cells (Wawersik, 2005).
To assess whether JAK/STAT signaling regulates male-specific germ cell division, embryos lacking zygotic Stat92E activity were examined and a dramatic decrease was observed in male germ cell proliferation. Similar reductions in germ cell proliferation are observed in the upd/upd-like mutant (Df(os1a)) and in embryos where the JAK inhibitor Socs36E is expressed in germ cells. Thus, JAK/STAT activity is required within germ cells for proper male-specific germ cell division in the gonad. Expression of upd in the germline is sufficient to induce proliferation in female germ cells. Thus, the JAK/STAT pathway can induce XX germ cells to exhibit this male-specific germ cell behavior (Wawersik, 2005).
Whether the JAK/STAT pathway regulates other aspects of male germ cell development was examined. male germline marker-1 (mgm-1) is a lacZ enhancer trap line that is expressed in male germ cells, but not female germ cells, and therefore is a marker for male germ cell identity. Inhibiting the JAK/STAT pathway by removing zygotic Stat92E activity does not affect mgm-1 expression in the embryo, which is as expected since initial mgm-1 expression is dependent on germ cell autonomous cues. However, removal of zygotic Stat92E activity completely abolished mgm-1 expression in first instar larvae. In wild-type first instar male larvae, mgm-1 expression is observed in most germ cells, which are likely to be developing male germline stem cells and spermatogonia. No mgm-1 expression is observed in Stat92E-mutant larvae, and β-galactosidase expression is only observed in the soma, not the germline, in the pattern expected from the Stat92E P element allele. In an experiment where 25% of larvae were expected to be both male and contain the mgm-1 enhancer trap, 23.2% (n=262) of wild type larvae exhibited mgm-1 expression in the germ cells, while no Stat92E mutant larvae exhibited germ cell mgm-1 expression; this is significantly different from wild type siblings. Thus, Stat92E mutants exhibit a strong effect on male germline development, and some male germline cell types are either missing, or have an altered identity (Wawersik, 2005).
Finally, the extent to which activation of the JAK/STAT pathway can masculinize female germ cells was assessed. Female germ cells expressing upd are not expected to be fully masculinized because, although a male-specific signal is being activated, these germ cells are otherwise still in a female somatic environment and retain female germ cell autonomous cues. Indeed, such embryos give rise to fertile adult females, indicating that at least some germ cells retain, or revert back to, a female identity. This may be due, in part, to the failure of the upd construct to be expressed in the adult female germline. However, upd is sufficient to induce male-specific gene expression in embryonic XX germ cells. While mgm-1 is normally expressed only in germ cells in males, mgm-1 was expressed in all embryos when upd was ectopically expressed. In addition, two new male germline markers, disc proliferation abnormal (dpa) and minichromosome maintenance 5 (mcm5), were identified, that can also be induced by upd. Whereas these genes are normally expressed in germ cells only in males, female embryos exhibit germ cell expression of these genes when upd is ectopically expressed. In an experiment where only 50% of embryos are expected to express ectopic upd in the germline, 32.5% of female embryos expressed dpa and 21.3% expressed mcm5. Therefore, upd expression is sufficient to activate male-specific gene expression in female germ cells (Wawersik, 2005).
These data indicate that the JAK/STAT pathway mediates a critical signal from the male somatic gonad that is required for male germ cell development. This signal likely acts together with male germ cell autonomous cues to promote male germline identity and spermatogenesis. This signal is also sufficient to activate the male pattern of proliferation and gene expression in female germ cells, even when these germ cells retain female germ cell autonomous cues and are present in an otherwise female soma. It will be very interesting in the future to identify additional (e.g. female) somatic signals, along with germ cell autonomous cues, and to assess the relative contribution of these factors to proper germline sexual development. Since one of the earliest aspects of sex-specific germ cell behavior in both Drosophila and mouse is the regulation of the germline cell cycle by the somatic gonad, it will be of further interest to determine if the somatic signals operating in Drosophila play a similar role in germline sex determination in mammals (Wawersik, 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).
Social interactions depend on individuals recognizing each other, and in this context many organisms use chemical signals to indicate species and sex. Cuticular hydrocarbon signals are used by insects, including Drosophila, to distinguish conspecific individuals from others. These chemicals also contribute to intraspecific courtship and mating interactions. However, the possibility that sex and species identification are linked by common chemical signalling mechanisms has not been formally tested. This study provides direct evidence that a single compound is used to communicate female identity among flies, and to define a reproductive isolation barrier between Drosophila melanogaster and sibling species. A transgenic manipulation eliminated cuticular hydrocarbons by ablating the oenocytes (see Insect oenocytes: a model system for studying cell-fate
specification by Hox genes), specialized cells required for the expression of these chemical signals. The resulting oenocyte-less (oe-) females elicited the normal repertoire of courtship behaviours from males, but were actually preferred over wild-type females by courting males. In addition, wild-type males attempted to copulate with oe- males. Thus, flies lacking hydrocarbons are a sexual hyperstimulus. Treatment of virgin females with the aversive male pheromone cis-vaccenyl acetate (cVA) significantly delayed mating of oe- females compared to wild-type females. This difference was eliminated when oe- females were treated with a blend of cVA and the female aphrodisiac (7Z,11Z)-heptacosadiene (7,11-HD), showing that female aphrodisiac compounds can attenuate the effects of male aversive pheromones. 7,11-HD also was shown to have a crucial role in heterospecific encounters. Specifically, the species barrier was lost because males of other Drosophila species courted oe- D. melanogaster females, and D. simulans males consistently mated with them. Treatment of oe(-) females with 7,11-HD restored the species barrier, showing that a single compound can confer species identity. These results identify a common mechanism for sexual and species recognition regulated by cuticular hydrocarbons (Billeter, 2009).
D. melanogaster produces hydrocarbons of various chain lengths, including unbranched alkanes, methyl-branched alkanes, alkenes and derivatives thereof. The alkenes are expressed sex-specifically, and have been associated with both sex and species discrimination. Compared to females, males express high levels of the monoalkene (Z)-7-tricosene (7-T), which has been reported to increase females' receptivity to mating attempts. Moreover, 7-T is repulsive to other males and may prevent male-male interactions. In contrast, females produce sex-specific dienes such as (7Z,11Z)-heptacosadiene (7,11-HD) and (7Z,11Z)-nonacosadiene (7,11-ND), which act as aphrodisiac pheromones for D. melanogaster males. Hydrocarbons are strongly associated with sexual recognition, because wild-type males court males that have been genetically modified to express female hydrocarbons, indicating that the mutants are perceived as females hydrocarbons (Billeter, 2009).
There are still large gaps in knowledge of the functions of individual hydrocarbons and the tissues where these compounds are synthesized. As in other insects, specialized cells called oenocytes, located on the inner surface of the abdominal cuticle, are thought to be the site of hydrocarbon biosynthesis in D. melanogaster. Consistent with this hypothesis, desaturase 1 (desat1), which encodes an enzyme involved in hydrocarbon synthesis, is expressed in Drosophila oenocytes (Marcillac, 2005). Previous studies have demonstrated that genetic feminization of these cells results in production of female hydrocarbons by male flies; however, these and other manipulations have been confounded by the concurrent feminization of cells in many other sexually dimorphic tissues, including the central nervous system. To test the hypothesis that these cells are required for production of chemical signals used in sexual and species recognition, the Gal4-UAS system was used to target transgene expression specifically to the adult oenocytes. An oenocyte Gal4 driver was generated, derived from the regulatory sequence of one of the desat1 promoters (Marcillac, 2005) that is expressed specifically in oenocytes of adult females. The driver is also expressed in the larval oenocytes and in the reproductive organs of adult males. This driver was used to ablate adult oenocytes by inducing expression of the pro-apoptotic gene head involution defective (hid). This approach initially caused lethality in larvae, probably due to the destruction of the larval oenocytes. To circumvent this problem blocked the driver's action was blocked during development using the Tubulin-Gal80ts transgene. Using this method, flies were generated without oenocytes (oe-). Analysis of whole-body hydrocarbon extracts confirmed that both oe- males and females were essentially devoid of these compounds, showing that the oenocytes are necessary for hydrocarbon display in D. melanogaster. The male pheromone cis-vaccenyl acetate (cVA) was unaffected in oe- males because this compound is synthesized in the ejaculatory bulb. The oe- transgenic strain therefore provided a 'blank slate' for evaluating the role of hydrocarbons in intra- and interspecific communication hydrocarbons (Billeter, 2009).
Sexual behaviour of oe- flies was assayed to test hydrocarbon function during reproduction. Despite the association of hydrocarbon signals and Drosophila courtship, absence of these signals did not alter courtship behaviours per se. The oe- males displayed normal courtship behaviour towards wild-type females, but slightly less intense than control males. However, wild-type females were less receptive to oe- males than control males, with oe- males taking almost four times as long to achieve mating. Thus, hydrocarbons of males do not seem to affect their own courtship behaviour, but rather, influence the receptivity of females to their mating attempts. However, the influence of non-oenocyte cells within the male reproductive organs that may have been affected by the ablation cannot be excluded. Notably, oe- males elicited courtship and copulation attempts from both wild-type males and other oe- males, indicating that oe- males were perceived as females, even though all other male characteristics were present. The vigorous courtship of oe- males by each other resulted in unnatural behaviours such as engaging one another by rotating in a head-to-head orientation, and males attempting copulation with each other's heads. These behaviours were suppressed by treatment of oe- males with synthetic 7-T, confirming the function of 7-T in inhibiting male-male interactions hydrocarbons (Billeter, 2009).
Wild-type males exhibited normal courtship behaviour towards oe- females, apparently undeterred by the lack of female . However, mating latency was significantly shorter, and when given a choice between an oe- and a control female, wild-type males preferred oe- females. Together, these data indicate that females lacking hydrocarbons are more attractive than those with a normal hydrocarbon profile. This suggests that female hydrocarbons normally act to slow down male mating attempts, facilitating assessment of a potential partner's species and fitness. Thus, any oe- fly, irrespective of its development as female or male, seems to sexually hyperstimulate males. It is hypothesized that hydrocarbons normally act to superimpose sexual identity on an otherwise attractive fly substrate hydrocarbons (Billeter, 2009).
The results described above suggested that female attractiveness depends on a balance between attraction/stimulation and repulsion/deterrence. This was investigated by treating females with the aphrodisiac compound 7,11-HD, and with cVA, which males transfer to females via the ejaculate to deter further mating attempts by other males. Whereas cVA decreases the probability that females will re-mate, wild-caught females produce offspring from multiple sires, indicating that polyandry is common and that the effect of cVA is not absolute. O- flies were treated with doses of these compounds approximating wild-type levels. The mating latency of wild-type males with oe- females treated with 7,11-HD was not different from that with untreated oe- females, indicating that 7,11-HD alone does not affect attractiveness of oe- females. As expected, treating wild-type females with increasing doses of cVA delayed mating accordingly, and the effect was even more pronounced with oe- females treated with the same doses of cVA. This effect was not due to differences in the rates of release of cVA from the control and oe- flies, as shown by the profiles of cumulative loss of cVA over time for the two genotypes. Instead, the exaggerated effect of cVA on oe- females is consistent with the hypothesis that the aversive effects of this compound are normally moderated by the presence of other hydrocarbons. Indeed, when cVA and 7,11-HD were applied together, the mating latencies of oe- and wild-type females were indistinguishable. Apparently, 7,11-HD mitigated the deterrent effects of cVA. The data suggest that a male's perception of a female's availability is normally regulated by a mixture of attractive and aversive signals. From an evolutionary perspective, the combined effect of a female attractant with a male deterrent may illustrate an instance of post-copulatory sexual conflict in which the attractant solicits additional mates despite the first male's effort to render a female unattractive by marking her with cVA hydrocarbons (Billeter, 2009).
In addition to mediating conspecific reproductive interactions, the hydrocarbons of female D. melanogaster have an important role in reproductive isolation between species. For example, within the nine species of the melanogaster subgroup, only D. melanogaster, D. sechellia and D. erecta produce female-specific dienes. Females in the rest of the subgroup express the same hydrocarbons as males. Males of species with non-sexually dimorphic hydrocarbons generally do not court females from dimorphic species, indicating that the dienes might act as reproductive isolation barriers between these species groups. Furthermore, males from dimorphic species do not vigorously court females from non-dimorphic species. In contrast, males of all species in the melanogaster subgroup have similar hydrocarbons, including abundant 7-T. Finally, D. melanogaster females lacking hydrocarbons are courted by at least two sibling species, D. simulans and D. mauritiana. The behaviour of males from other species in the melanogaster subgroup towards oe- females was tested, to assess the contribution of oenocytes and hydrocarbons to reproductive isolating mechanisms. D. simulans and D. yakuba were tested as test species because they represent species in which the females lack dienes. D. erecta was included because it differs from D. melanogaster in the pattern of dienes expressed hydrocarbons (Billeter, 2009).
Males of all three species courted oe- D. melanogaster females, but exhibited limited or no courtship towards control D. melanogaster females. This indicates that oenocytes and their hydrocarbon products are major components of the reproductive isolation barrier, ensuring that courtship and mating attempts are only initiated between conspecifics. It has been proposed that 7,11-HD functions to create this barrier in D. melanogaster. To test this directly, oe- D. melanogaster females and wild-type females from the different species were treated with synthetic 7,11-HD. Treatment suppressed courtship by males of all three species, demonstrating that 7,11-HD alone is sufficient to create a species barrier. Interestingly, D. erecta males were blocked by 7,11-HD, despite the fact that hydrocarbons of D. erecta females include other dienes in common with those of D. melanogaster. Furthermore, D. melanogaster males actively courted D. erecta females, possibly because the diene 7,11-ND is also expressed by D. melanogaster females. D. simulans and D. yakuba females treated with 7,11-HD elicited strong courtship from D. melanogaster males. These results demonstrate the multifunctional role of 7,11-HD as an attractant and/or stimulant for some species and as a deterrent for others hydrocarbons (Billeter, 2009).
Despite attempting copulation, D. erecta males never mated with oe- females, suggesting that signals other than hydrocarbons are required to induce receptivity in these females. However, within a 24-h period, nearly all oe- D. melanogaster females mated with D. simulans males, whereas no control D. melanogaster females mated with these males. Treatment of oe- females with 7,11-HD completely blocked interspecific mating with D. simulans males, even at a dose five times lower than the amount found in females of wild-type D. melanogaster strain. Similar treatment of D. simulans females with 7,11-HD only delayed mating by D. simulans males. It is hypothesized that 7-T counters the effect of 7,11-HD in D. simulans females. This is because 7-T functions as an aphrodisiac for D. simulans males and is expressed in higher quantities in D. simulans females than in D. melanogaster females. D. simulans males were assayed with oe- females treated with either 7-T alone, or in combination with 7,11-HD. Synthetic 7-T alone induced a slight decrease in mating latency, indicating that 7-T is an attractant for D. simulans males. However, the striking effect of 7-T was to reduce the effect of 7,11-HD in a dose-dependent manner. These data parallel the balancing effect of 7,11-HD on cVA for D. melanogaster males. Thus, this study has demonstrated that female hydrocarbons orchestrate mating both within and between the species. Whereas a single compound such as 7,11-HD may be enough to establish a species barrier, the effect of this compound is moderated by the relative quantity of other signals. Indeed, the effects of 7,11-HD are particularly noteworthy because it functions as an attractant in an intraspecific context, whereas in an interspecific context, it aids in species recognition, thereby placing social communication and speciation on the same continuum hydrocarbons (Billeter, 2009).
The logic of pheromonal communication in Drosophila seems to be based on a foundation that imparts general attractiveness to a fly. In this study female oenocytes are the primary organ for communicating species and sex identity to males. Others have shown that males use species-specific acoustic tags within their love song for females during courtship. Thus, both acoustic and pheromonal tags establish a context for social interactions by regulating sex and species recognition. Given that individual flies regulate their own hydrocarbon display in accord with their social surroundings, it is plausible that these compounds also function in individual recognition hydrocarbons (Billeter, 2009).
The establishment of sexual identity is a crucial step of germ cell development in sexually reproducing organisms. Sex determination in the germline is controlled differently than in the soma, and often depends on communication from the soma. To investigate how sexual identity is established in the Drosophila germline, a molecular screen was conducted for genes expressed in a sex-specific manner in embryonic germ cells. Sex-specific expression of these genes is initiated at the time of gonad formation (stage 15), indicating that sexual identity in the germline is established by this time. Experiments where the sex of the soma was altered relative to that of the germline (by manipulating transformer) reveal a dominant role for the soma in regulating initial germline sexual identity. Germ cells largely take on the sex of the surrounding soma, although the sex chromosome constitution of the germ cells still plays some role at this time. The male soma signals to the germline through the JAK/STAT pathway, while the nature of the signal from the female soma remains unknown. The genes ovo and ovarian tumor (otu) are expressed in a female-specific manner in embryonic germ cells, consistent with their role in promoting female germline identity. However, removing the function of ovo and otu, or reducing germline function of Sex lethal, had little effect on establishment of germline sexual identity. This is consistent with findings that signals from the soma are dominant over germline autonomous cues at the initial stage of germline sex determination (Casper, 2009).
This analysis demonstrates that a sex-specific program of gene expression is present in the germline soon after the time of gonad formation [stage 15, ~12 hours after egg laying (AEL)]. Examples were identified of both male-specific germline genes and female-specific germline genes, with other genes being expressed equally in the two sexes. Therefore, it is likely that the sex-specific pattern of gene expression in the germline represents the establishment of true sexual identity in the germline, as opposed to other differences that might be observed in the germline between the sexes, such as proliferation status or transcriptional competence. It is concluded that sexual identity in the germline is established at least as early as stage 15. The observation that many genes examined initiate sex-specific germline expression at this time might indicate that this is when germline sexual identity is first established. However, it was also found that the soma signals to the germline in a sex-specific manner just prior to gonad formation (stage 13, ~10 hours AEL), indicating that germ cells could determine their sex even earlier. Because the zygotic genome is only robustly activated in the germline during gastrulation (stage 9, ~4 hours AEL), this sets a narrow time window during which sexual identity is established in the germline (Casper, 2009).
It is significant that germline sexual identity appears to be first manifested as the germ cells contact the somatic gonad. This is also the time that sexual dimorphism is first observed in the somatic gonad and that it exhibits sex-specific patterns of gene expression, Jheh2 and CG5149). The finding that germ cells might establish their sexual identity only as they contact the somatic gonad is consistent with the strong role of the soma in determining germline sexual identity. In addition, signaling from the germline back to the soma also occurs at this time (Kitadate, 2007; Casper, 2009).
The establishment of a sex-specific pattern of gene expression in the germline requires these cells to acquire germ cell identity, in addition to sexual identity. Previous work has established that germ cell-specific transcription is independent of the somatic environment, and is autonomously regulated in the germ cells. Indeed, it was seen that germ cell expression of the genes reported in this study can be independent of the somatic gonad. Germ cell-specific gene expression is likely to be regulated by the germ plasm, and several maternally expressed germline transcription factors have recently been identified (Yatsu, 2008; Casper, 2009).
Previously, it was known that sexual identity in the germline requires autonomous cues, along with non-autonomous cues from the surrounding soma, but little was known about how the signals work together to establish sexual identity. XX germ cells cannot develop normally in a male soma, and XY germ cells cannot develop normally in a female soma. Although evidence was found for both autonomous and non-autonomous regulation of germline sexual identity in the embryo, it appears that non-autonomous signals from the soma are dominant over germline autonomous cues. When XX germ cells are present in a male soma (tra mutants), they exhibit a clear increase in expression of male germ cell genes, and decreased expression of the female-specific gene otu. Similarly, XY germ cells present in a female soma (UAS-tra) exhibit a strong repression of male germ cell genes, while otu expression is increased. Thus, in each case the germ cells largely take on the sexual identity of the surrounding soma, independent of their own sex chromosome constitution. However, subtle differences in gene expression between XX and XY germ cells remain in these situations, indicating some germ cell autonomous control of sexual identity. In addition, when germ cells are present outside of the gonad (srp mutants), some differences are also observed between XX and XY germ cells. When outside of the gonad, XY germ cells are more likely than XX germ cells to express male-specific genes, and XX germ cells maintain some otu expression, while otu is largely off in XY germ cells. Thus, there is at least some autonomous contribution of the germ cell sex chromosome genotype to germline sexual identity at this early stage (Casper, 2009).
The genes of the ovarian tumor loci, ovo, otu, stil and Sxl, are thought to contribute to autonomous germline sexual identity by promoting female identity in XX germ cells. At this early stage, no change was observed in sex-specific germ cell gene expression in mutants for ovo, otu or stil, or in embryos with mutations that reduce the function of Sxl in the germline. Although this could indicate that these genes do not play a role in the initial establishment of germline sexual identity, these observations could also be due to the dominant effects of the soma on sex-specific germ cell gene expression at this time. These mutations would be expected to masculinize XX germ cells, causing them to exhibit a more male-like pattern of gene expression. However, even fully male (XY) germ cells exhibit a female pattern of gene expression when in a female soma. Thus, the dominant effect of the female soma might mask any masculinizing effects from the removal of ovarian tumor locus genes at this time (Casper, 2009).
Evidence is seen for at least two types of non-autonomous regulation of germline sexual identity by the soma, one coming from the male soma and the other from the female soma. Both XY and XX germ cells can activate male-specific gene expression when not in contact with the somatic gonad, but expression of these genes does not appear to be as robust as when germ cells are in contact with a male soma. Furthermore, when XX germ cells are in contact with a male soma, expression of the female germ cell gene otu is partially repressed. Thus, the male soma is required for full levels of male gene expression in the germ cells, and for the repression of female genes (Casper, 2009).
The signal from the male soma to the germline appears to be primarily, and perhaps exclusively, acting through the JAK/STAT pathway. Previously, it was shown that the male soma activates the JAK/STAT pathway in the germ cells, and that this is required for proper male germ cell behavior (Wawersik, 2005). This pathway is necessary for the proper male-specific proliferation of embryonic germ cells, and for the maintenance, but not the initiation, of male-specific mgm-1 expression (male germline marker-1, which appears to be an enhancer trap in the escargot locus). The JAK/STAT ligand UPD is also sufficient for the induction of germ cell proliferation and mgm-1 expression, and for partial induction of mcm5 and dpa expression. This study found that, when the JAK/STAT pathway is inactivated, sex-specific gene expression in the germline resembles that of germ cells that are not in contact with the somatic gonad. Ectopic expression of upd in females is able to induce some aspects of male-specific gene expression in XX germ cells, but not as robustly as when XX germ cells are present in a male soma. This is likely to be due to the fact that, when upd is expressed in an otherwise female soma, it is in competition with female signals that repress male gene expression. It is concluded that the JAK/STAT pathway is important for the regulation of germline sex determination by the male soma, and may be the primary or only signal from the male soma to the embryonic germ cells. It is essential for the male-specific pattern of germ cell proliferation, and for the robust initiation and maintenance of male-specific gene expression (Casper, 2009).
It is clear that the female soma also plays a key role in regulating the sexual identity of the germline. Both XX and XY germ cells exhibit some level of male-specific gene expression when outside of the gonad, but this expression is dramatically repressed when in contact with a female soma. This repression has also been observed with mgm-1. In addition, XY germ cells exhibit some female-specific otu expression when in contact with a female soma. Therefore, the female soma is essential for the proper sex-specific regulation of germline gene expression, although the nature of the signal from the female soma to the germ cells remains unknown (Casper, 2009).
Somatic control over germline sex determination is a common feature of germ cell sexual development. In the mouse, germ cells first manifest sex-specific behaviors as they contact the somatic gonad in the genital ridge. The initial behavior of the germ cells is dependent on the sex of the soma, as male germ cells exhibit female behavior (early meiosis) when contacting a female soma, and female germ cells exhibit male behavior when in contact with a male soma. At least some aspects of this sex-specific behavior are regulated by retinoic acid (RA) levels controlled by the soma; female germ cells receive a high level of RA signal, but the male soma degrades RA so that male germ cells receive less of this signal. Interestingly, this study found that the somatic gonad in Drosophila expresses Jheh2 in a male-specific manner. JHEH2 hydrolyzes Juvenile Hormone (JH), which is structurally related to RA. JH analogs have also been observed to influence sex determination in crustaceans, which further supports this interesting parallel between vertebrates and invertebrates (Casper, 2009).
In mammals, the germline sex chromosome constitution is also important for germ cell development. As in Drosophila, mouse and human Y chromosome genes are crucial for spermatogenesis. However, the number of X chromosomes also appears to play an autonomous role in germline sexual development in mammals. XX germ cells in a male soma [e.g. in Sex reversed (Sxr) mice appear initially male and do not enter meiosis, but eventually die. XO germ cells in a male soma survive and progress further in spermatogenesis. Similarly, having two X chromosomes promotes female germ cell identity. XX germ cells are predisposed to enter meiosis on the female timetable compared with XO germ cells under the same conditions, and are biased towards a female pattern of imprinting. Thus, as in Drosophila, the germ cell genotype contributes to germ cell sex determination in the mouse and depends on the number of X chromosomes. Similarly, humans with altered sex chromosome constitutions, such as those with Turner's Syndrome (XO females) or Klinefelter's Syndrome (XXY males) have relatively normal somatic development but exhibit severe germline defects, indicating that the proper number of X chromosomes in the germline is essential for germline development (Casper, 2009).
However, in other species, the sex chromosome constitution of germ cells does not play an important role in germline sex determination. In the housefly, Musca domestica, transplanted germ cells develop normally according to the somatic sex of their host and produce fertile gametes. Some species also exhibit dramatic sexual plasticity in the germline, with the same individual being able to produce both sperm and egg, such as in hermaphrodites (e.g., C. elegans or in species that exhibit natural sex reversal. i.e., wrasses and gobies). Given the diversity of mechanisms animals use for establishing sex determination in the soma, it is not surprising that there might be considerable diversity in the germline as well. However, it appears that an important role for the soma in controlling sexual identity in the germline is a common theme in germ cell development. Whether or not the sex chromosome constitution of the germline is also important for fertility in an organism places additional constraints on the evolution of sex determination mechanisms, sexual plasticity and the sex chromosomes (Casper, 2009).
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