The transformer gene of Drosophila is necessary for all aspects of female somatic sexual differentiation. tra uses a single set of precursor RNAs to produce female- and non-sex-specific RNAs by alternative splicing. Ectopic expression of the female-specific RNA causes chromosomal males to develop as females, indicative of a linear pathway of regulated genes controlling sex. Genetic and molecular tests with this ectopically expressed gene are consistent with the following order of gene action: X chromosome to autosome ratio ---> Sex lethal ---> transformer ---> transformer-2 ---> doublesex ---> intersex ---> terminal differentiation. Expression of the female-specific tra RNA in tra mutants is sufficient to lead to female differentiation. Expression of the non-sex-specific tra RNA in tra mutants is not sufficient to lead to female differentiation. The tra female-specific activity is not required for female-specific splicing of the tra precursor RNAs (McKeown, 1988).
In Drosophila, the sex of germ cells is determined by cell autonomous and inductive signals. XY germ cells autonomously enter spermatogenesis when developing in a female host. In contrast, XX germ cells non-autonomously become spermatogenic when developing in a male host. In first instar larvae with two X chromosomes, XX germ cells enter the female or the male pathway depending on the presence or absence of transformer activity in the surrounding soma. In somatic cells, the product of tra regulates the expression of the gene doublesex which can form a male-specific or a female-specific product. In dsx mutant larvae, XX and XY germ cells develop abnormally, with a seemingly intersexual phenotype. This indicates that female-specific somatic dsx products feminize XX germ cells, and male-specific somatic dsx products masculinize XX and XY germ cells. The results show that tra and dsx control early inductive signals that determine the sex of XX germ cells and that somatic signals also affect the development of XY germ cells. XX germ cells that develop in pseudomales lacking the sex-determining function of Sxl are spermatogenic. If, however, female-specific tra functions are expressed in these animals, XX germ cells become oogenic. Furthermore, transplanted XX germ cells can become oogenic and form eggs in XY animals that express the female-specific function of tra. Therefore, Tra product present in somatic cells of XY animals or in animals lacking the sex-determining function of Sxl, is sufficient to support developing XX germ cells through oogenesis (Steinmann-Zwicky, 1994).
In Drosophila, the sex of germ cells is determined by cell-autonomous and inductive signals. XY germ cells autonomously enter spermatogenesis when developing in a female host. In contrast, XX germ cells non-autonomously become spermatogenic when developing in a male host. In first instar larvae with two X chromosomes, XX germ cells enter either the female or the male pathway depending on the presence or absence of transformer activity in the surrounding soma. In somatic cells, the product of tra regulates the expression of the gene doublesex which can form a male-specific or a female-specific product. In dsx mutant larvae, XX and XY germ cells develop abnormally, with a seemingly intersexual phenotype. This indicates that female-specific somatic dsx products feminize XX germ cells, and male-specific somatic dsx products masculinize XX and XY germ cells. The results show that tra and dsx control early inductive signals that determine the sex of XX germ cells and that somatic signals also affect the development of XY germ cells. XX germ cells that develop in pseudomales lacking the sex-determining function of Sxl are spermatogenic. If, however, female-specific tra functions are expressed in these animals, XX germ cells become oogenic. Furthermore, transplanted XX germ cells can become oogenic and form eggs in XY animals that express the female-specific function of tra. Therefore, TRA product present in somatic cells of XY animals or in animals lacking the sex-determining function of Sxl, is sufficient to support developing XX germ cells through oogenesis (Steinmann-Zwicky, 1994).
Relatively little is known about the neural circuitry underlying sex-specific behaviors. The feminizing gene transformer was expressed in genetically defined subregions of the brain of male Drosophila, and in particular within different domains of the mushroom bodies. Mushroom bodies are phylogenetically conserved insect brain centers implicated in associative learning and various other aspects of behavior. Expression of transformer in lines that mark certain subsets of mushroom body intrinsic neurons, and in a line that marks a component of the antennal lobe, causes males to exhibit nondiscriminatory sexual behavior: they court mature males in addition to females. Expression of transformer in other mushroom body domains, and in control lines, has no such effect. These data support the view that genetically defined subsets of mushroom body intrinsic neurons perform different functional roles (O'Dell, 1995).
Pheromones are intraspecific chemical signals important for mate attraction and discrimination. In Drosophila, hydrocarbons on the cuticular surface of the animal are sexually dimorphic in both their occurrence and their effects: Female-specific molecules stimulate male sexual excitation, whereas the predominant male-specific molecule tends to inhibit male excitation. Female flies produce dienes (two double bonds) with 27 and 29 carbons (cis,cis-7,11-heptacosadiene and cis,cis-7,11-nonacosadiene). A few tens of nanograms of both dienes together can elicit vigorous male precopulatory behavior. Male flies synthesize monoenes (one double bond) with 23 and 25 carbons (cis-7tricosene and cis-7-pentacosene). cis-7tricosene can inhibit dose-dependent male excitation, whereas cis-7pentacosene stimulates males of some strains. According to a previously proposed biosynthetic scheme, an elongase, controlled by ecdysone, perhaps coupled with a desaturase, would be sufficinet to replace 7-monoenes by 7,11-dienes. Complete feminization of the pheromone mixture produced by males is induced by targeted expression of the transformer gene in adult oenocytes (subcuticular abdominal cells) or by ubiquitous expression during early imaginal life. Early adult life is the critical period during which sexually dimorphic hydrocarbons replace immature hydrocarbons on the fly cuticle. The resulting flies, genetically male, generally exhibit male heterosexual orientation but elicit homosexual courtship from other males. Two out of seven transformed lines exhibit some bisexual behavior, possibly because they were feminized in the calyces of their mushroom bodies (strain A) and in a dorso-medial subset of their antennal lobes (strains A and B). However, these two brain structures, which function in mate recognition, are not feminized in the other tranformed strains, showing that these structures are not required for feminization of the pheromonal profile. This analysis shows that two aspects of individual sexual identity in Drosophila -- the perception of others and the presentation of self to others -- are under separate genetic and anatomical control (Ferveur, 1997).
Sexual differentiation in Drosophila is controlled by a short cascade of regulatory genes, the expression pattern of which determines all aspects of maleness and femaleness, including complex behaviors displayed by males and females. One sex-determining gene is transformer (tra): tra activity is needed for female development. Flies with a female karyotype (XX) but which are mutant for tra develop and behave as males (and are termed X2 flies). In such flies, a female phenotype can be restored by a transgene that carries the female-specific cDNA of tra under the control of a heat-shock promoter. This transgene, called hs[trafem], also transforms XY animals into sterile females. When these XX and XY 'females' are raised at 25 degrees C, however, they display vigorous male courtship while at the same time, as a result of their female pheromone pattern, they are attractive to males. Intriguingly, their male courtship behavior is indiscriminately directed toward both females and males. When expression of tra is forced by heat shock, applied during a limited period around puparium formation, male behavior is abolished and replaced by female behavior. The temperature-sensitive period extends from shortly before puparium formation into early metamorphosis. Cuticular hydrocarbons function as pheromonal cues between the sexual partners. Consistent with their attractiveness to males, the normal female pattern of pheromones is found in transformed flies, irrespective of the heat shock. Could the indiscriminate male behavior of these flies result from continuous self-stimulation by their own female pheromones? The female pheromone pattern was replaced with the male pattern without affecting the CNS. This was achieved by introducing the male-determining mutation dsxD, which acts downstream of tra. This mutation causes XX flies to produce a male pheromone pattern. They are no longer attracted to males. Similarly, X2 and XY 'females', now with male body and pheromonal status due to dsxD, are completely unattrative to males; but they still court both sexes. Such flies even manage to copulate with females, showing that X2 and XY 'females' are capable of performing the full repertoire of male courtship. These results rule out self-stimulation and leave a disturbed nervous system as the most probable cause for the indiscriminate mating behavior. It is concluded that sexual behavior is irreversibly programmed during a critical period as a result of the activity or inactivity of a single control gene. Such programming takes place during a period of accelerated growth in the brain and especially in the mushroom bodies, suggesting that sexual behavior is 'hard wired' in the CNS (Arthur, 1998).
The isolation and analysis of Drosophila mutants with altered sexual orientation lead to the identification of novel branches in the sex-determination cascade that govern the sexually dimorphic development of the nervous system. One such example is the fruitless (fru) gene, the mutation of which induces male-to-male courtship and malformation of a male-specific muscle, the muscle of Lawrence (MOL). Since the MOL is formed in wild-type flies when the innervating nerve is male, regardless of the sex of the MOL itself, the primary site of Fru function is likely to be the motoneurons controlling the MOL. The fru gene produces multiple transcripts including sex-specific ones. A female-specific mRNA from the fru locus has a putative Transformer (Tra) binding site in its 5' untranslated region, suggesting that fru is a direct target of Tra. The fru transcripts encode a set of proteins similar to the BTB (Bric a brac, Tramtrack and Broad-complex)-Zn finger family of transcription factors. Mutations in the dissatisfaction (dsf) gene result in male-to-male courtship and reduced sexual receptivity of females. The dsf mutations also give rise to poor curling of the abdomen in males during copulation and in females, a failure of egg-laying. The latter phenotypes are ascribable to aberrant innervation of the relevant muscles. A genetic analysis reveals that expression of the dsf phenotypes depends on Tra but not on Doublesex (Dsx) or Fru, suggesting that dsf represents another target of Tra. The effect of dsf mutation was determined in chromosomal females after sexual transformation with tra- (Finley, 1997). In the dsf+;tra- XX flies, the ventral abdominal muscles are innervated by normal locking boutons, as is the case in wild-type males (XY dsf+). In contrast, the corresponding muscles in dsf-;tra- XX flies are associated with enlarged round boutons, just like those seen in mutant males (XY dsf-). The expression of the dsf mutant phenotype depends on tra due to the fact that dsf is located downstream of tra in the sex-determination cascade. In accordance with the idea that dsf is downstream of tra, a moderate reduction in tra+ activity in females eliminates motor innervation of the uterine muscle, inducing a dsf phenotype. Taken together, these findings suggest that the sex-determination protein Tra has at least three different targets: dsx, fru and dsf, each of which represents the first gene in a branch of the sex-determination hierarchy functioning in a mutually-exclusive set of neuronal cells in the Drosophila central nervous system (Yamamoto, 1998).
The downstream effectors of the Drosophila sex determination cascade are mostly unknown and thought to mediate all aspects of sexual differentiation, physiology and behavior. Here, serial analysis of gene expression (SAGE) was employed to identify male and female effectors expressed in the head, and report 46 sex-biased genes. Four novel, male- or female-specific genes have been characterized; all are expressed mainly in the fat cells in the head. Tsx (turn on sex-specificity), sxe1 and sxe2 (sex-specific enzyme 1/2) are expressed in males, but not females, and are dependent on the known sex determination pathway, specifically transformer and its downstream target doublesex. Female-specific expression of the fourth gene, fit (female-specific independent of transformer), is not controlled by tra and dsx, suggesting an alternative pathway for the regulation of some effector genes. These results indicate that fat cells in the head express sex-specific effectors, thereby generating distinct physiological conditions in the male and female head. It is suggested that these differences have consequences on the male and female brain by modulating sex-specific neuronal processes (Fujii, 2002).
In Drosophila melanogaster, the main cuticular hydrocarbons (HCs) are some of the pheromones involved in mate discrimination. These are sexually dimorphic in both their occurrence and their effects. The production of predominant HCs has been measured in male and female progeny of 220 PGa14 lines mated with the feminising UAS-transformer transgenic strain. In 45 lines, XY flies were substantially or totally feminised for their HCs. Surprisingly, XX flies of 14 strains were partially masculinised. Several of the PGa14 enhancer-trap variants screened in this study seem to interact with sex determination mechanisms involved in the control of sexually dimorphic characters. A good relationship was found between the degree of HC transformation and GAL4 expression in oenocytes. The fat body was also involved in the switch of sexually dimorphic cuticular hydrocarbons but its effect was different between the sexes (Savarit, 2002).
In many animal species, copulation involves the coordinated release of both sperm and seminal fluid, including substances that change female fertility and postmating behavior. In Drosophila, these substances increase female fertility and prevent mating with a second male. By using a PGal4 strain, a dozen cholinergic neurons found only in the male abdominal ganglion (Abg-MAch), together with other cells, were targeted for feminization via conditional transformer expression. Genetic feminization apparently deleted these neurons in males and significantly increased their copulation duration, blocked their fertility in 60% of cases, and only weakly repressed remating in females. Genetic repression of Gal4 activity in all cholinergic neurons completely rescued copulation duration and fertility, and totally prevented remating, indicating that Abg-MAch neurons were functional. The conditional blocking of the synaptic activity of these neurons during copulation induces separate effects on the transfer of the seminal substances involved in fertilization and those involved in remating. These effects were dissociated only when Abg-MAch neurons were feminized, indicating that their presence is required to synchronize the emission of the male substance(s) that changes reproductive behaviors (Acebes, 2004).
The feminization of a dozen male-specific cholinergic neurons in the abdominal ganglion (Abg-MAch), possibly with that of other undetected cells, substantially reduced the probability that Drosophila males produce offspring (from 90% to 35%) and altered the coordination of several evolutionarily important reproductive characters normally induced during copulation by control males. This finding is consistent with a number of studies on both insects and mammals and may reveal a high degree of evolutionary conservation of the neural control of these key male-reproductive characters. In insects, acetylcholine (Ach) is a neurotransmitter commonly used by mechanosensory neurons. Food-deprivation of choline, an essential precursor of Ach, alters D. melanogaster male normal copulatory behavior and reduces sperm motility. Studies in vertebrates have reported a functional relation between Ach and male ejaculation. In rats, cholinergic sensitivity is correlated with the intensity of the response of muscarinic agonists, which in turn facilitate ejaculation. In dogs, seminal emission during long copulatory events also involves cholinergic pathways (Acebes, 2004).
The distinct and simultaneous manipulation by UAS-tra and UAS-shits transgenes of the Abg neurons targeted by Gal4 allowed two distinct cell populations eliciting distinct effects on reproduction to be distinguished. Non-sex-specific neurons mostly permit the transfer of the substances that repress female remating. Abg-MAch neurons also decreased the transfer of these substances together with the substances necessary for fertilization, but not in a coordinated manner. The feminization of Abg-MAch neurons (probably leading to their ablation in 55B-tra and 55B-tra- shits males) induced variation for fertility and for repression of remating, but these effects did not coincide either in individuals or in groups of females. The dissociation of these effects, together with the decrease in fecundity induced by feminized males, indicates that the transfer (and the amount) of these substances stochastically varies within a given male, probably because the control of ejaculation is randomly affected (Acebes, 2004).
Apart from these two populations of Abg neurons, a distinct set of eight serotoninergic Abg male-specific neurons also changed copulation duration, fertility, and female remating. It is not yet known whether these three groups of Abg neurons innervate different or overlapping regions of male internal reproductive organs, and it is possible that the ablation of any of these sets of Abg neurons induces the uncoordinated release of male substances (Acebes, 2004).
Could the loss of the synchronized emission of such substances be reflected by the high interindividual variability for copulation duration shown by feminized 55B-tra and 55B-tra-shits males? Two other fly studies suggest that variation in copulation duration reflects a variation for the transfer of male reproductive substances. In Musca domestica, males temporarily exhausted for sperm and associated substances induced increased copulation duration and remating frequency. In D. melanogaster strains selected for copulation duration, short-copulation males (15 min) fertilized females much faster than long-copulation males (25 min). Since the synchronized release of these substances is crucial for reproduction and is probably involved in the evolution of conflicts between the sexes, male-specific neural networks that control the release of these substances should be subject to strong selective forces. That could imply that some of the neural mechanisms underlying ejaculation may have been conserved during evolution. It is predicted that similar cholinergic pathways will be found to underlie ejaculation and the coordinated emission of seminal fluids in others species including vertebrates (Acebes, 2004).
Stem cells are found in specialized microenvironments, or 'niches', which
regulate stem cell identity and behavior. The adult testis and ovary in
Drosophila contain germline stem cells (GSCs) with well-defined niches, and are
excellent models for studying niche development. This study investigates the
formation of the testis GSC niche, or 'hub', during the late stages of
embryogenesis. By morphological and molecular criteria, the development of an embryonic hub that forms from a subset of anterior somatic gonadal precursors (SGPs) were identified and followed in the male gonad. Embryonic hub cells form a discrete cluster apart from other SGPs, express several molecular markers in common with the adult hub and organize anterior-most germ cells in a rosette pattern characteristic of GSCs in the adult. The sex determination genes transformer and doublesex ensure that hub formation occurs only in males. Interestingly, hub formation occurs in both XX and XY gonads mutant for doublesex, indicating that doublesex is required to repress hub formation in females. This work establishes the Drosophila male GSC niche as a model for understanding the mechanisms controlling niche formation and initial stem cell recruitment, as well as the development of sexual dimorphism in the gonad (Le Bras, 2006).
The evidence indicates that an embryonic hub, which appears to give rise to
the adult hub and create the male GSC niche, forms during the late stages of
embryogenesis. A subset of anterior SGPs initiates expression of several
molecular markers that are also expressed in the adult hub. These SGPs segregate
into a tight cluster in a distinct region of the gonad, and a subset of germ
cells organizes around these SGPs in a manner similar to the organization of
GSCs around the adult hub. Since spermatogenesis begins by early larval stages,
it is possible that
the embryonic hub already forms a functional GSC niche. The formation of the
hub, or indeed any stem cell niche, can be divided into the distinct issues of
niche cell identity, niche morphogenesis, and stem cell recruitment (Le Bras, 2006).
The data indicate that the specification of hub cell identity occurs in two stages. During the first stage, some SGPs acquire an anterior identity that is sexually dimorphic, as indicated by the male-specific expression of esg and upd. Anterior SGP identity is positively regulated by abd-A, and is repressed by Abd-B, while sexual identity is regulated by tra and dsx. During the second stage of hub cell specification, a subset of these anterior SGPs acquires hub cell identity during stage 17 of embryogenesis. Only some anterior SGPs maintain esg expression, and the control of late gene expression in the hub appears to be distinct from early expression in anterior SGPs, since some esg and upd enhancer traps only exhibit gonad expression in the hub at this later stage. Furthermore, cells that maintain esg expression
during stage 17 also express every other marker of adult hub identity tested,
including Fasciclin 3, cdi, DN-cadherin and DE-cadherin. It is concluded that
these cells are specified as hub cells at this time. The fate of the anterior
SGPs that lose esg expression and do not form part of the hub is unknown.
An intriguing possibility is that these cells could form another important
somatic cell type: the cyst progenitor cells (somatic stem cells) that associate
with the hub along with the GSCs (Le Bras, 2006).
Based on its expression pattern, the transcription factor esg would seem to be an excellent candidate for specifying hub cell identity. However, no changes were observed in the expression of other hub markers in esg null mutants; this includes expression of DE-cadherin, which is known to
be regulated by esg in other tissues. It has been reported, however,
that esg is required for hub maintenance, and that the hub is severely
defective at later stages in esg mutants that survive embryogenesis.
Thus, esg is critical for
the male GSC niche, but is either not important for the initial formation of
this structure, or acts redundantly with another factor (Le Bras, 2006).
It has been possible to follow the
morphogenesis of the hub from the time of gonad formation until the embryonic
hub is fully formed. At the time of gonad coalescence, anterior SGPs interact
with other SGPs, and with the germ cells, in a manner that is indistinguishable
from posterior SGPs. However, during stage 17, the hub cells undergo dramatic changes in
their relationship to other SGPs and germ cells. Hub cells segregate away from
other SGPs to one pole of the gonad, and coalesce tightly with one another.
In addition, hub cells do not ensheath the germ cells
at this stage. Instead, a defined
interface between hub cells and germ cells forms which is labeled by DE- and
DN-cadherin, but not Fasciclin 3. Thus, hub cells
appear to maximize their interactions with one another, and minimize their
interactions with other cells in the gonad, although they clearly still contact
a subset of germ cells (Le Bras, 2006).
It is apparent that the changes in cell–cell
contact and morphology that occur during hub formation require changes in cell
adhesion. Indeed, characteristic changes have been found in expression of the
homophilic adhesion molecules Fasciclin 3, DN-cadherin and DE-cadherin occur
during hub formation; all three are significantly upregulated in the embryonic
and adult hub. Increased homophilic adhesion among hub cells could account for
their ability to maximize their contacts with one another, and sort away from
other SGPs. However, no changes were observed in embryonic hub formation
in mutants for these cell adhesion molecules.
Thus, these proteins, and possibly others, may act redundantly in
this process (Le Bras, 2006).
It is clear that a subset of germ
cells organizes specifically with the developing hub as it forms. During the
last stage of hub formation, germ cells become oriented in a rosette
distribution around the developing hub in a manner characteristic of GSCs in the
adult. These may
represent the subset of germ cells that will become GSCs. The presence of DE-
and DN-cadherin at sites of hub–germ cell contact suggests that
cadherin-mediated adhesion may be important for niche–GSC interaction in
the testis, as has been observed in the ovary. Interestingly, germ cells are not required
for hub formation. Analysis of a number of hub identity markers indicates that these
cell form normally from a subset of anterior SGPs in embryos that lack germ
cells. The hub does not appear as well compacted in
these embryos, consistent with observations of the adult hub,
indicating that hub–germ cell contact (or hub–germ cell signaling)
affects the final shape of the hub. Nevertheless, the GSC niche can form in the
absence of one of its stem cell populations (somatic stem cells may still be
present). It will be of great interest in the future to determine if the subset
of germ cells organized around the male embryonic hub are, indeed, developing
GSCs, and to study how their transition to stem cell identity might be regulated
by the niche (Le Bras, 2006).
The formation of the male GSC niche is a sex-specific
characteristic of anterior SGPs. Male-specific expression of esg and hub
formation both require the sex determination genes tra and dsx.
In some tissues, DSXM is required to
promote male development and repress female development, while the opposite is
true for DSXF. Interestingly, it was found
that embryonic hub development is entirely masculinized in dsx null
mutants; XX and XY individuals appear identical when mutant for dsx and
both resemble wild type males. Thus, no role is seen for DSXM in
promoting embryonic hub formation, while DSXF is required in females
to repress hub formation. Since esg is expressed male-specifically, it is
one candidate for being directly regulated by DSX (Le Bras, 2006).
We can compare the development of the anterior SGPs and hub with the development of another sexually dimorphic cell type, the msSGPs that join the posterior of the male gonad. First of all, these two cell types are distinct and do not depend on one another for their proper development. The hub still forms in Abd-B mutants that lack msSGPs, while msSGPs are still found in the gonad in Pc mutants, in which no anterior SGPs or hub cells form. Second, the
mechanism for how sexual dimorphism is created differs between the two cell
types. msSGPs are present only in males because they have undergone sex-specific
apoptosis in females. In contrast, no apoptosis was observed in anterior SGPs.
These cells appear to remain present in both sexes,
but only form a hub in males. Thus, although the sex determination genes
tra and dsx regulate sex-specific development of both cell types,
the cellular mechanisms employed are different. Finally, as was observed for the
hub, development of the msSGPs is completely masculinized in dsx mutant
embryos. Thus,
for both of these cell types, the male pattern of development in the embryonic
gonad is the default state in the absence of dsx function, and it is the
role of DSXF to repress male development in females. However,
DSXM may well play a role in development of one or both of these
gonad cell types at later stages, since proper testis development in males
clearly requires dsx (Le Bras, 2006).
The sex determination pathway must also ensure that GSC niches form
in females and are different from those in males. Recently, it has been shown
that germ cells populating the anterior of the gonad in female embryos are
predisposed to become GSCs in the adult ovary, while germ cells populating the
posterior rarely become GSCs. This suggests that anterior SGPs in the female embryonic gonad
may promote GSC identity, similar to what is proposed to happen in the male during
hub formation. One possibility is that anterior SGPs give rise to GSC niches in
both sexes, while genes such as tra and dsx control whether these
niches will be male or female (Le Bras, 2006).
In conclusion, the development has been followed of the embryonic hub, which may represent the nascent GSC niche for the testis. This work provides a basis for further understanding the mechanisms controlling niche formation and GSC recruitment in Drosophila, and determining if these mechanisms are conserved in other
stem cell systems, including the GSC niche of the mammalian testis (Le Bras, 2006).
The transformer and doublesex genes produce sex-specific transcripts that are generated by differential RNA processing. The effects were examined of mutants in other regulatory genes controlling sexual differentiation on the patterns of processing of the tra and dsx RNA transcripts. The genes suggested by genetic studies to act upstream of tra or dsx in the sex determination hierarchy regulate these two loci at the level of RNA processing. The data suggest that the order of interaction of the factors controlling sex is X:A ---> Sxl ---> tra ---> tra-2 ---> dsx ---> ix ---> terminal differentiation. While these results cannot preclude regulatory interactions at other levels, the regulation of RNA splicing revealed by these experiments is sufficient to account for all of the known functional interactions between the regulatory genes in this hierarchy (Nagoshi, 1988).
The transformer gene is one of a set of regulatory genes that form the hierarchy controlling all aspects of somatic sexual differentiation in Drosophila melanogaster. The gene transformer occupies an intermediate position in this hierarchy. Analysis of this gene has allowed determination of the mechanism by which transformer is regulated in a sex-specific manner and to examine the way in which the regulatory hierarchy is organized. The female-specific expression of the tra gene, previously inferred from genetic observations, is based on sex-specific alternative splicing of tra pre-mRNA and is not the result of sex-specific transcriptional activation. The female-specific RNA produced by this alternative splicing is the functional mediator of tra activity. Multiple genetic, molecular, and transformation experiments show that female-specific activation of genes or gene products occurs in the order Sex lethal --> transformer --> transformer-2 --> doublesex --> intersex --> female differentiation. The results do not distinguish the level at which transformer might regulate the downstream gene transformer-2. Neither transformer nor any of the down-stream genes feedback on, or participate in, alternative splicing of transformer RNA. The mechanism by which Sex lethal regulates transformer splicing appears to be a repression of the use of one of a pair of splice acceptor sites (Belote, 1989).
Few mutations link well defined behaviors with individual neurons and the activity of specific genes. In Drosophila, recent evidence indicates the presence of a doublesex-independent pathway controlling sexual behavior and neuronal differentiation. A gene, dissatisfaction (dsf), has been identified that affects sex-specific courtship behaviors and neural differentiation in both sexes without an associated general behavioral debilitation. Male and female mutant animals exhibit abnormalities in courtship behaviors, suggesting a requirement for dsf in the brain. Virgin dsf females resist males during courtship and copulation and fail to lay mature eggs. dsf males actively court and attempt copulation with both mature males and females but are slow to copulate because of maladroit abdominal curling. Structural abnormalities in specific neurons indicate a role for dsf in the differentiation of sex-specific abdominal neurons. The egg-laying defect in females correlates with the absence of motor neuronal innervation on uterine muscles, and the reduced abdominal curling in males correlates with alteration in motor neuronal innervation of male ventral abdominal muscles. Epistasis experiments show that dsf acts in a tra-dependent and dsx-independent manner, placing dsf in the dsx-independent portion of the sex determination cascade (Finley, 1997).
What are the genetic requirements for high ovo expression in gonads? In autosomes, the X to autosome ratio is sufficient for Sex lethal expression. Does a similar mechanism hold in the gonads? To study this question, expression of ovo was examined in gonads of mutant flies where the chromosomal sex does not match the somatic sexual identity. If a 2X karyotype is necessary and sufficient for high levels of ovo expression, then female somatic sex should have no effect on ovo expression. XY females can be produced by using a strain of flies bearing a transformer gene driver by a heat-shock promoter (trahs). In XY genetic males bearing the trahs transgene, TRA activity is sufficient to direct female somatic development in flies that would otherwise develop as males, but the germ line remains male. If a female somatic sexual identity is sufficient for a high level of ovo expression, then both XX females and XY trahs females should be expected to show similar ovo expression. In fact ovo expression is not activated to a high level by a female somatic identity, suggesting that either an XX karyotype or female germ-line identity is required for high ovo expression (Oliver, 1994).
In Drosophila, compatibility between the sexually differentiated state of the soma and the constitution of the sex chromosome in the germline is required for normal gametogenesis. In this study, important aspects of the soma-germline interactions controlling early oogenesis are defined. In particular, the sex-specific germline activity of the ovarian tumor (otu) promoter has been demonstrated to be dependent on somatic factors controlled by the somatic sex differentiation gene transformer. This regulation defines whether there is sufficient ovarian tumor expression in adult XX germ cells to support oogenesis. In addition, the ovarian tumor function required for female germline differentiation is dependent on the activity of another germline gene, ovo, whose regulation is transformer-independent. These and other data indicate that ovarian tumor plays a central role in coordinating regulatory inputs from the soma (as regulated by transformer) with those from the germline (involving ovo). transformer-dependent interactions influence whether XX germ cells require ovarian tumor or ovo functions to undergo early gametogenic differentiation. These results are incorporated into a model that hypothesizes that the functions of ovarian tumor and ovo are dependent on an early sex determination decision in the XX germline -- a decision that is at least partially controlled by somatic transformer activity (Hinson, 1999 and references).
With respect to interactions with the germline, transformer (tra) is the most extensively studied of the somatic sex regulatory genes. The masculinization of XX soma due to loss-of-function tra mutations causes germ cell aberrations during first instar larval stages and misregulates sex-specific germline gene expression in the embryo. Furthermore, when XY soma is feminized by ectopic tra expression (to form 'pseudofemales', the somatic components of the ovaries are sufficiently 'female' so that they can support the maturation of transplanted XX germ cells. The pseudofemale soma also appears to partially feminize the XY germline, since these cells now require the normally female-specific otu function for optimal proliferation. These observations indicate that tra controls a substantial portion of the somatic-germline interactions affecting early gametogenic differentiation (Hinson, 1999 and references).
In Drosophila, the sexual differentiation of the germline requires a complex interplay between cell autonomous factors controlled by the X:A ratio of the germ cells and sex-specific somatic functions. For example, certain allele combinations of transformer, transformer-2 and doublesex can cause chromosomally female (XX) flies to develop with most of their somatic tissues having a male identity, i.e., ëXX pseudomalesí. In these flies, oogenesis is aborted and there is even occasionally what appears to be early spermatogenic development. Since the germline expressions of these sex regulatory genes are not required for early stages of gametogenesis, the aberrant germline phenotypes must result from the male transformation of the soma (Hinson, 1999 and references).
It is not clear which germline genes are influenced by the proposed somatic interactions. Three possible candidates based on their early and sex-specific roles in female germline differentiation are ovarian tumor, ovo and Sex-lethal (Sxl). During oogenesis, the expression of otu is required in the germline at several stages, if not continually. The null mutant phenotype is characterized by the absence of egg chambers in an otherwise normal ovary, denoted as the quiescent phenotype, although substantial numbers of germ cells are still present in the germarium. Null and severe loss-of-function mutations can also produce 'ovarian tumors', a phenotype characterized by egg chambers containing hundreds of seemingly undifferentiated germ cells. Both the quiescent and tumorous cells are aborted at early oogenic stages, during the cystocyte divisions prior to cyst formation. Mutations in otu have no significant effect on spermatogenesis, although some aberrations in male courtship behavior have been reported. The ovo gene has been implicated in regulating sex determination and dosage compensation in the germline. This is based primarily on observations that ovo null XX germ cells are typically not found in the adult ovary, presumably because of reduced cell viability. In addition, certain ovo allele combinations produce tumorous germ cells that morphologically resemble primary spermatocytes. These phenotypes make ovo a candidate target for a somatic signal regulating early oogenesis, although the expression of ovo in adult germ cells does not appear to be responsive to somatic influences. ovo might directly regulate otu. The Ovo protein can bind to sites in the otu promoter, which displays sensitivity to changes in the dosage of ovo + function. It is not known when this putative regulation of otu occurs nor what role it plays in oogenesis (Hinson, 1999 and references).
The effects of an ovo null mutation on XX germ cells developing in pseudomale testes and female ovaries were examined. In females, ovo mutant XX germ cells typically arrest beginning at larval gonial stages. Occasionally, mutant germ cells survived to the adult stage. However, these cells generally failed to undergo gametogenic differentiation as seen by the absence of spectrosomes, fusomes or ring canals. It was reasoned that, if the requirement for ovo is solely dependent on the X:A ratio, then the phenotype of ovo mutant germ cells in pseudomales should be at least as severe. In this case, the ovo mutant XX pseudomale gonads should be either atrophic or contain a few clusters of mostly undifferentiated germ cells. There is an increase in the frequency of atrophic gonads (82%) compared to normal pseudomales (48%), many of the non-oogenic type. The non-oogenic gonads contained VASA-positive germ cells. This indicates that not only are a substantial fraction of the mutant germ cells viable in adults, but gametogenic differentiation occurs as well. The frequency of the non-oogenic gonads in ovo mutant pseudomales is essentially unchanged from that observed in normal pseudomales. This suggests that the observed increase in the atrophic category is due primarily to the loss of the oogenic class. Mutations in otu gave results similar to those described for ovo. This suggests that otu and ovo mutations specifically disrupt only those germ cells attempting female differentiation, rather than the indiscriminate elimination of the entire XX germline (Hinson, 1999).
Heat shock-otu can alter the XX pseudomale gonadal phenotype; to examine whether and to what degree otu expression could induce oogenic development in pseudomales, immunohistochemical studies were performed. When continually cultured at 20-25ƒC, hs-otu pseudomale gonads are as much as two to three times longer than normal. In addition, 88% of the hs-otu gonads examined show extensive Hu-li tai shao-labeling of ring canals (Hts is an adducin-like protein). These feminized gonads display a developmental progression of gametogenic stages. In section III of the gonad, the pseudomale germ cells have differentiated to postgermarial stages as defined by the expression of kelch. Kelch, an actin binding protein, is localized to female ring canals after the ring canal deposition of Hts and f-actin . Kelch is first detected in female ring canals in stage 1 egg chambers, but is not seen in all ring canals until stage 4. In hs-otu XX pseudomales, the germ cell clusters in section III contain thick ring canals, with virtually all of them showing Kelch deposition along the inner surface of the f-actin layer. In comparison, no Kelch-labeled ring canals are observed in XX pseudomales without hs-otu, indicating that oogenesis is not only less frequent, but also more limited. Taken together, these results indicate the masculinizing effect of male soma (or the absence of female soma) on XX germ cells can be partially, but consistently, overridden by the expression of otu from a heterologous promoter. The resulting fusome and ring canal development follows the same sequence of events as occurs in normal oogenesis. Therefore, pseudomale germ cells are competent to both initiate and undergo substantial oogenesis if provided with adequate levels of otu. Both ovo and Sxl were shown to be required for otu induced oogenic differentiation in XX pseudomales. However, an additional role for otu in some process affecting germline viability and/or proliferation can be identified that is separable from oogenic differentiation and independent of ovo and, possibly, Sxl functions (Hinson, 1999).
The finding that hs-otu can feminize XX pseudomale germ cells suggests oogenesis is blocked because of insufficient otu levels. Therefore, an examination was carried out to see whether tra-induced sexual transformation affects the level of otu gene expression. otu-lacZ is expressed in most, if not all, larval and pupal germ cells in both female and male gonads. Sex-specific regulation only becomes apparent in the adult testis where male germline expression become restricted to a few cells at the apical tip. As with otu, the ovo promoter is initially active in both male and female larval gonads. However, ovo-lacZ becomes sex-specific at an earlier stage, showing restricted expression in male gonads during the third instar larval and pupal periods. These results demonstrate that the otu and ovo promoters are under different regulatory control in the pre-adult germline. However, otu, but not ovo, promoter activity is influenced by tra-induced sexual transformation. These data demonstrate that the tra-induced sexual transformation specifically inhibits otu promoter activity. Also carried out was the reciprocal experiment, in which otu-lacZ activity was examined in XY germ cells developing in a female somatic background. XY pseudofemales produced by the ectopic expression of tra result in ovaries containing tumorous egg chambers. Because XY pseudofemale germ cells become sufficiently 'feminized' so that they acquire a need for otu function for optimal proliferation, it was anticipated they would also be permissive for otu promoter activity. This is in fact the case. Even in the absence of ovo function, XY pseudofemale germ cells consistently express otu-lacZ. This indicates that the feminizing effects of tra, but not ovo, are necessary for otu transcription. In comparison, the ovo promoter is not detectably active in XY pseudofemales, again illustrating differential regulation of ovo and otu (Hinson, 1999).
It is thought that during the
pupal and adult stages, two critical events occur in the female
germarium: (1) ovo activity allows XX germ cells
to become receptive to the otu function controlling oogenic
differentiation, and (2) tra-dependent somatic signals allow
continued expression of otu in the female germline by
maintaining otu promoter activity. The combination of these events constitutes a mechanism by which the otu gene serves to link the somatic sex
differentiation pathway controlled by tra with a female
germline developmental pathway controlled by ovo (Hinson, 1999).
It has been suggested that sexual identity in the germline depends upon the combination of a nonautonomous somatic signaling pathway
and an autonomous X chromosome counting system. The roles of the sexual differentiation genes transformer (tra) and doublesex (dsx) in regulating the activity of the somatic signaling pathway have been examined. It was asked
whether ectopic somatic expression of the female products of the tra and dsx genes could feminize the germline of XY animals. TraF, the female form of transformer, is sufficient to feminize XY germ cells, shutting off the expression of male-specific markers and activating the
expression of female-specific markers. Feminization of the germline depends upon the constitutively expressed transformer-2 (tra-2)
gene, but does not seem to require a functional dsx gene. However, feminization of XY germ cells by TraF can be blocked by the
male form of the Dsx protein (DsxM). Expression of the female form of dsx, DsxF, in XY animals also induces germline expression of female markers. Taken together with a previous analysis of the effects of mutations in tra, tra-2, and dsx on the feminization of XX
germ cells in XX animals, these findings indicate that the somatic signaling pathway is redundant at the level of tra and dsx. Finally, these studies call into question the idea that a cell-autonomous X chromosome counting system plays a central role in germline sex determination (Waterbury, 2000).
Transplantation experiments and clonal analysis have suggested that germline sexual identity in XX animals depends upon a combination of cell-autonomous factors that somehow assess the X/A ratio and nonautonomous factors that signal sexual identity from the soma to the germline. A plausible pathway for linking somatic sexual identity to the mechanism that generates the nonautonomous signal is the well-characterized Sxl -> tra/tra-2 -> dsx cascade. In previous studies, the effects were tested of mutations in tra, tra-2, and dsx on the sexual identity of germ cells in XX animals. Unexpectedly, only in the case of the sex-nonspecific gene, tra-2, does loss-of-function mutation lead to a switch in sexual identity of the XX germ cells from female to male. To account for these findings, it has been proposed that the somatic signal must be generated by a novel tra-2-dependent regulatory cascade. Since dsx is dispensable for this process in XX animals, it has been postulated that an unidentified tra-2 regulatory target, z, directly or indirectly generates the signal. To explain the fact that XX germ cells retain partial female identity in tra mutants, it has been suggested that there must be another gene q whose activity overlaps or is redundant with tra. In this view, both tra and q would be able to function with the cofactor tra-2 to promote the female-specific expression of z (Waterbury, 2000).
This model was tested by introducing transgenes that ectopically express the female forms of tra and dsx into XY animals and by assaying their effects on germline sexual identity. The findings are generally consistent with predictions of the original model: there were some unexpected results that altered an understanding of the nature of the germline sex determination process and the role of dsx. Experiments with the tra transgene are considered below (Waterbury, 2000).
According to the model, ectopically expressed tra is predicted to activate the regulatory cascade that signals female identity from the soma to the germline. Activation of this signaling pathway should require tra-2 and the target gene z, while dsx would be dispensable. The results are generally consistent with these predictions. Ectopically expressed Tra switches the sexual identity of germ cells in XY animals from male to female, turning off male-specific germline markers and inducing female-specific markers. This switch in sexual identity is blocked by mutations in tra-2, but is not prevented by loss-of-function mutations in dsx (Waterbury, 2000).
In XX animals, the available evidence indicates that the tra/q -> tra-2 -> z feminization pathway functions in the soma. Hence, an expectation of the model is that ectopically expressed Tra would also feminize XY germ cells through its action in the soma, not in the germline. However, since the hsp83 promoter is known to be active in both soma and germline, it is possible that Tra protein ectopically expressed in XY germ cells feminizes these cells by a novel mechanism that is independent of the somatic signaling pathway that normally operates in XX animals. Two lines of evidence argue against this: (1) since several of the hsp83-traF lines were recovered from males, it would appear that expression of Tra in XY germ cells is not in itself sufficient to feminize these cells; (2) the available evidence suggests that ectopically expressed Tra feminizes the germline in XY animals by a pathway resembling that used in XX animals; it requires tra-2 but is independent of a functional dsx (Waterbury, 2000).
Although the hsp83-traF transgene does not require dsx to feminize the germline of XY animals, feminization can be prevented if the only source of Dsx protein is provided by an allele that constitutively expresses DsxM. This result was unexpected since DsxM has no effect on the sexual identity of the germline in XX animals. There are several possible explanations for this discrepancy. It has been proposed that there is another gene, q, which occupies the same position in the regulatory cascade as tra. If this gene is downstream of Sxl, as expected, it would be expressed in the male mode in XY; P[hsp83-traF] animals and hence would not contribute to the production of the feminizing signal. Because DsxM alters the development of the soma surrounding the germline and consequently the cell-cell contacts between soma and germline, the signal produced by tra alone might not be sufficient to feminize. A second possibility is that XY germ cells are intrinsically less responsive to the feminizing signal than XX germ cells. For example, given the lack of strong evidence for germline dosage compensation, the signal could be sensitive to a twofold difference in X-linked genes. A third possibility is that DsxM produces a masculinizing signal that is able to counteract the effects of the feminizing signal produced by TraF. At the present, these explanations cannot be distinguished (Waterbury, 2000).
Since the dsx gene can be removed or expressed exclusively in the male mode without affecting germline sexual identity in XX animals, it has been suggested that dsx has no role in germline sex determination. However, contrary to this suggestion, ectopic expression of DsxF in XY; dsx- animals can feminize the germline and this feminizing activity can be blocked if DsxM is also present in the soma (Waterbury, 2000).
Why is DsxF capable of feminizing XY germ cells, yet dispensable in XX animals? One way to reconcile these two observations is to postulate that z regulates the synthesis of the feminizing signal ('fes') instead of encoding the signal itself. If this were the case, both Z and DsxF could independently promote the production of fes. In females, since q and tra would be active, Z would be able to induce sufficient levels of fes to feminize the germline in the absence of DsxF or in the presence of DsxM. Furthermore, since the female and male Dsx proteins recognize the same target sequences, ectopically expressed DsxF would be able to activate fes synthesis in XY animals only when DsxM is absent (Waterbury, 2000).
In the revised model for the somatic signaling pathway, Sxl has been placed at the top of the regulatory cascade where it is responsible for activating the female-specific expression of both tra and q. While Sxl is known to be required for sex-specific regulation of tra, it should be noted that there is no evidence that it is responsible for controlling the activity of q. However, unless q is itself a target for the X/A counting system, there are no other known mechanisms that could promote female expression. If q is downstream of Sxl, results with SxlM1,fm3 and SxlM1,fm7 (revertant alleles of SxlM1)
suggest that q is regulated by a different mechanism than tra. q and/or tra, together with tra-2, would then activate the female-specific expression of z and dsx. The female products of z and dsx would in turn direct the synthesis of the feminizing signal. By this model, the germline would assume male identity whenever Sxl is off in the soma. However, it is not clear whether the male pathway requires production of a male somatic signal by the male form of Dsx (or Z) or occurs in XY germ cells by default in the absence of a female signal. In favor of the former possibility is the finding that constitutively expressed DsxM prevents Tra from feminizing XY germ cells. However, functional dsx is not required in XY animals to select male identity (Waterbury, 2000).
One question raised by these studies is the role of the postulated autonomous X chromosome counting system in germline sex determination. In particular, it has been argued from pole cell transplantation experiments that this autonomous system overrides input from the soma in XY germ cells, forcing them to assume male identity. However, data has been presented indicating that the sexual identity of XY germ cells can be switched from male to female by ectopic expression of TraF and DsxF. If TraF and DsxF activate the signaling pathway(s) that normally functions in XX animals, this result would imply that there may be no cell-autonomous system that selects sexual identity by measuring the germ cell X/A ratio. In this view, the autonomous components of the germline sex determination system would play an entirely different role. They would be subordinate to the somatic signaling pathway, being responsible only for responding correctly to the somatic signal and having no role in making the actual choice. Of course, if the default pathway within the germline is male, then this pathway will be followed 'autonomously' in the absence of a feminizing signal from the soma (Waterbury, 2000).
From a phylogenetic perspective, the simplest solution for germline sex determination is that germ cells strictly follow the same sexual fate as that of the soma in securing the development of a fully functional organism. In fact, this appears to be the mechanism for germline sex determination in other dipteran species such as Musca domestica and Chrysomya rufifacies. In these organisms, somatic sex alone is necessary and sufficient to dictate sexual fate to the germline. Irrespective of their sexual karyotype, when germ cells are surrounded by ovarian tissue, eggs are produced, and when surrounded by testicular tissue, sperm are produced. Studies in the nematode C. elegans and in the mouse further support the idea that somatic sex is widely used to dictate the sexual fate of gametes. In Drosophila, it is believed that somatic sex is the primary determinant. Why then is this soma-to-germline signaling mechanism insufficient to direct complete female or male germline differentiation independent of the chromosome composition of the germ cells in Drosophila? A likely explanation is that XY and XX germ cells in Drosophila have lost the ability to respond equally well to somatic cues. For example, in XY; P[hsp83-traF] pseudofemales, most of the ovarioles have a tumorous ovary phenotype and ovarioles that have normal-looking egg chambers are observed very infrequently. Given that there is no strong evidence for germline dosage compensation, one plausible explanation for the abnormal development of these sex-transformed XY germ cells is that the dose of X-linked gene products is insufficient to properly execute an oogenic developmental program. The Sxl gene would be a good example of a gene that is required for oogenesis and, because of its autoregulatory activity, is highly sensitive not only to its own dose but also to the dose of other X-linked genes such as the splicing factor snf. It is reasonable to suppose that there may be a variety of steps in oogenesis (or spermatogenesis) that are sensitive to the dose of X-linked genes (Waterbury, 2000).
Within the germline, otu, ovo, and Sxl have been identified as candidate genes that respond to the feminizing signal from the soma and determine the sex of the germ cells. Mutations in all three genes have sex-specific effects on germline development. Perhaps the most striking result is the fact that loss-of-function ovo and otu mutations markedly reduce the viability of XX but not XY germ cells. Thus one important question is whether these mutations have similar effects on the viability of XY germ cells feminized by the hsp83-traF transgene. Somewhat surprisingly, it was found that otu and ovo mutations behave differently. Strong loss-of-function otu mutations reduce the viability of XY germ cells feminized by the traF transgene. This finding suggests that the lethal effects of strong otu mutations arise because the germ cells assume a female identity, and not because of their number of X chromosomes. In contrast, ovo mutations have no apparent effect on the viability of feminized XY germ cells. One explanation for this difference is that lethal effects are not observed in ovo mutants because the feminizing signal produced by the traF transgene in XY animals is weaker than the feminizing signal found in wild-type XX animals. Alternatively, it is possible that XX germ cell death in ovo mutants does not depend upon the choice of sexual identity, but rather is a function of the X chromosome dose (Waterbury, 2000).
If otu, ovo, or Sxl functions as a master sex determination switch within the germline, one would expect to find that mutations in any of these genes would completely block the feminization of germ cells much like mutations in Sxl prevent feminization in the soma. While the results indicate that none of these genes fits this criterion for a master regulatory switch, effects are observed in the expression of sex-specific markers. Mutations in all three genes prevent the traF transgene from inducing the expression of female bruno (and Sxl) gene products. However, in all three cases the transgene still induces the expression of female orb gene products. One interpretation of these findings is that the sex determination pathway in the germline is split into at least two branches -- one branch that contains bruno and Sxl and another branch that contains orb. For both bruno and orb, sex-specific expression depends upon the activation of distinct sex-specific promoters. If these two genes are in independent branches of the germline sex determination pathway, this would imply that there must be distinct 'male' and 'female' transcription factors for the four promoters. Moreover, it seems likely that one important function of the somatic signaling system would be to control the expression of these transcription factors. Clearly, it will be important to identify these transcription factors and to learn how they are regulated (Waterbury, 2000).
The integration of multiple developmental cues is crucial
to the combinatorial strategies for cell specification that
underlie metazoan development. In the Drosophila genital
imaginal disc, which gives rise to the sexually dimorphic
genitalia and analia, sexual identity must be integrated
with positional cues, in order to direct the appropriate
sexually dimorphic developmental program. Sex
determination in Drosophila is controlled by a hierarchy of
regulatory genes. The last known gene in the somatic
branch of this hierarchy is the transcription factor
doublesex (dsx); however, targets of the hierarchy that play
a role in sexually dimorphic development have remained
elusive. The gene dachshund (dac) is
differentially expressed in the male and female genital
discs, and plays sex-specific roles in the development of the
genitalia. Furthermore, the sex determination hierarchy
mediates this sex-specific deployment of dac by modulating
the regulation of dac by the pattern formation genes
wingless (wg) and decapentaplegic (dpp). The
sex determination pathway acts cell-autonomously to
determine whether dac is activated by wg signaling, as in
females, or by dpp signaling, as in males (Keisman, 2001).
The behavior of tra + and tra2IR clones provides insight into
the mechanism of repression in the undeveloped genital
primordium. It was anticipated that such clones would be
difficult to recover when they occurred in the male and female
primordium, respectively, because they should adopt the
repressed state. Instead, large tra + (female)
clones were recovered in the male primordium of a male disc, and large tra2IR
(male) clones were recovered in the female primordium of a female disc.
Some of these clones constitute a substantial fraction of the
primordium in question. Though tra + or
tra2IR clones were not scored in adults, previous studies strongly suggest that such clones would fail to differentiate adult genital structures (Keisman, 2001).
It has been shown that tra - (male) clones cause large deletions in the female genitalia, indicating that genetically male cells like those in a tra2IR clone divide but cannot differentiate female genital structures. Further, it has also been shown that male structures
are deleted when the mosaic border passes through the male
genitalia, suggesting that female
tissue cannot differentiate male structures. To reconcile these
data, it is proposed that repression of the inappropriate genital
primordium involves two separable processes: repression of
growth and the prevention of differentiation. Thus, clones of
cells of the inappropriate genetic sex cannot differentiate, but
they can grow and contribute to a morphologically normal
genital primordium. This poses yet another question. Cells in a tra + clone in the male primordium of a male genital disc are analogous to the
cells in the repressed male primordium of a wild-type female
genital disc: both are genetically female, and both have A9
segmental identity. Why do tra + clones in the male primordium
grow, while the repressed male primordium in a female disc
does not? One possibility is that the decision of the male
primordium to grow in a male disc is made before tra + clones
were induced and cannot be over-ridden by a later switch of
genetic sex. However, temperature-shift experiments with tra-2
ts alleles suggest that the decision of a genital primordium to
develop can be reversed later in development. Furthermore,
occasional, large tra + clones can cause severe reductions in
male genital discs. This observation leads to the suggestion of a model in which growth in the genital disc is
regulated from within organizing zones, such as the domains
of wg and dpp expression. According to this model, the sex of
the cells in the organizing regions would determine how the
disc grows, while cells in other regions would respond
accordingly, regardless of their sex. The tra + clones that cause
reduction could result when such a clone intersects with one
of the postulated organizing centers within the disc. The
implication is that the sex determination pathway acts in yet
undiscovered ways to modulate the function of the genes that
establish pattern in the genital disc. One such
interaction was found in the regulation of dac; further study is needed to
determine if others exist, and what role they play in producing
the sexual dimorphism of the genital disc and its derivatives (Keisman, 2001).
Sexually dimorphic development of the gonad is essential for germ cell development and sexual reproduction. The Drosophila embryonic gonad is already sexually dimorphic at the time of initial gonad formation. Male-specific somatic gonadal precursors (msSGPs) contribute only to the testis and express a Drosophila homolog of Sox9 (Sox100B: Loh, 2000), a gene essential for testis formation in humans. The msSGPs are specified in both males and females, but are recruited into only the developing testis. In females, these cells are eliminated via programmed cell death dependent on the sex determination regulatory gene doublesex. This work furthers the hypotheses that a conserved pathway controls gonad sexual dimorphism in diverse species and that sex-specific cell recruitment and programmed cell death are common mechanisms for creating sexual dimorphism (DeFalco, 2003).
To investigate when sexual dimorphism is first manifested in the somatic gonad, expression of SGP markers were examined in embryos whose sex could be unambiguously identified, at a developmental stage (stage 15) soon after gonad coalescence has occurred. Analysis of Eya expression reveals anti-Eya immunoreactivity throughout the female somatic gonad, though Eya expression is somewhat stronger in the posterior. In males, anti-Eya immunoreactivity is also found throughout the somatic gonad. However, the expression at the posterior of the gonad is much more intense than in females, as there appears to be a cluster of Eya-expressing cells at the posterior of the male gonad that is not present in females. In blind experiments, the sex of an embryo could be accurately identified by the Eya expression pattern in the gonad. Thus, sexual dimorphism is already apparent in the somatic gonad soon after initial gonad formation. A sex-specific expression pattern is also observed with Wnt-2 at this stage. As is observed with Eya, Wnt-2 is expressed in the SGPs of the female gonad, but its expression is greatly increased at the posterior of the male gonad. The SGP marker bluetail (see Galloni, 1993) exhibits a similar sex-specific pattern as Eya; however, the SGP marker 68-77 is expressed equally in both sexes (see below). Thus, the somatic gonad is sexually dimorphic by stage 15, but only a subset of SGP markers reveals this sexual dimorphism (DeFalco, 2003).
During Drosophila embryogenesis, Sox100B is expressed in a number of cell types, including the gonad (Loh, 2000). Since Sox100B is closely related to Sox9 (an important sex determination factor in humans and mice), whether Sox100B expression is sexually dimorphic in Drosophila was tested. Interestingly, it was found that after gonad coalescence (stage 15), Sox100B expression in the gonad is male-specific. Sox100B immunoreactivity is not observed in the coalesced female gonad, whereas it is detected in a posterior cluster of SGPs in the male gonad. While this expression pattern is seen in most wild-type backgrounds (including Canton-S and faf-lacZ), in certain 'wild-type' lines, such as w1118, a few Sox100B-positive cells are observed in the posterior of the coalesced female gonad (however, this is still clearly distinguishable from the number of Sox100B-positive cells in the male). Unlike Eya and Wnt-2, Sox100B is not expressed in all SGPs, since it is usually absent from female gonads and from the anterior region of the male gonad and does not colocalize with the SGP marker 68-77. Sox100B expression appears restricted to the posterior cluster of SGPs that is observed only in the male gonad. Thus, like Sox9 expression in vertebrates, Sox100B exhibits a male-specific pattern of expression in the Drosophila embryonic gonad, suggesting that it may indeed be an ortholog of Sox9 (DeFalco, 2003).
After having identified sexually dimorphic markers of the embryonic gonad, these markers were used to investigate how sexual dimorphism is established. It was asked whether proper gonad formation is necessary for the establishment of sexual dimorphism by examining Sox100B expression in fear-of-intimacy (foi) mutant embryos. In foi mutants, germ cells migrate and associate normally with the SGPs, but these two cell types fail to coalesce into a round and compact gonad. Despite the failure of gonad coalescence, a cluster of Sox100B-expressing cells was still observed at the posterior of the male gonad, while no Sox100B-expressing cells are observed in the female at this stage. Whether the presence of germ cells is necessary for the establishment of sexual dimorphism in the embryonic gonad was examined. Embryos that lack germ cells due to a hypomorphic mutation in oskar, a gene required for germ cell formation, were examined. Other aspects of embryonic development occur normally in these embryos, including the formation and coalescence of the SGPs. Agametic gonads show identical sexual dimorphism to wild-type embryos. Sox100B is coexpressed with Eya in the cluster of somatic cells in the posterior of the male gonad, but Sox100B expression is not observed in the female gonad. Thus, sexual dimorphism of the embryonic somatic gonad does not require proper gonad morphogenesis or the presence of germ cells (DeFalco, 2003).
The posterior cluster of Eya and Sox100B coexpressing cells could result from sex-specific differences in gene expression within the cells of the gonad. Alternatively, it could reflect a difference in gonad morphology, in which these cells are only present in males and not in females. To distinguish between these possibilities, the morphology of the male and female coalesced (stage 15) gonad was examined, using approaches that do not depend on cell-type-specific SGP markers. First, a CD8-GFP fusion protein was expressed broadly in the mesoderm. The fusion of the extracellular and transmembrane regions of mouse CD8 with GFP allows for visualization of cell and tissue morphology. A cluster of mesodermal cells is consistantly observed attached to the posterior of the male gonad that is not observed in the female. In blind experiments, the sex of the embryo can be predicted based on the presence of this posterior cluster of cells. Male and female gonads were also examined by transmission electron microscopy (TEM). Male and female embryos were first sorted using an X chromosome-linked GFP expression construct and then processed separately for TEM. In this analysis, a cluster of cells that is not present in the female gonad was consistently at the posterior of the male gonad. Both the size and morphology of these cells indicate that they are somatic cells rather than germ cells. Thus, the observed sexual dimorphism reflects a change in gonad morphology, not just a change in gene expression. Since the additional cells at the posterior of the male gonad express at least some markers in common with SGPs (e.g., Eya), these cells are referred to as male-specific SGPs (msSGPs) (DeFalco, 2003).
Since no sex-specific differences were observed in SGP proliferation in the gonad, it seems unlikely that the SGPs are dividing to produce the msSGPs. Therefore, Sox100B was used as a marker for the msSGPs to determine where and when these cells are first specified. At stages prior to gonad coalescence (stages 12 and 13), a cluster of Eya/Sox100B double-immunopositive cells is observed posterior and ventral to the developing clusters of SGPs, which express Eya alone. Interestingly, this cluster of Eya/Sox100B double-positive cells is initially observed in both males and females and appears identical, although Eya expression may be somewhat lower in the female cluster. During stage 13, as the SGPs and germ cells associate closely along PS 10-12, the Eya/Sox100B double-positive cells move toward the gonad in both sexes. In males, these cells join the posterior of the coalescing gonad. In contrast, these cells do not join the gonad in females, and only Eya-positive, Sox100B-negative cells are found in the coalesced gonad. It is concluded that the Eya/Sox100B double-positive cells are the msSGPs and that they form separately from the SGPs. These cells are initially specified in both males and females and move anteriorly to join the gonad in males. In females, these cells do not form part of the gonad, as judged by the above morphological analysis, and are no longer detected using available markers (DeFalco, 2003).
Since the msSGPs develop separately from the SGPs, it was of interest to address where the msSGPs arise and what controls their specification. By marking the anterior of each parasegment using an antibody against Engrailed, it was determined that the msSGPs are specified in PS13. This observation is consistent with these cells arising posterior to the SGPs, which form in PS 10, 11, and 12. Other Sox100B expression is observed in nongonadal tissues. Whether, like the SGPs, the msSGPs are specified in the dorsolateral domain of the mesoderm was also addressed. Mesodermal cell types that form in this region, such as the SGPs and the fat body, require the homeodomain proteins Tinman and Zfh-1 for their specification. However, in embryos double-mutant for tinman and zfh-1, the msSGPs are still specified, even though the SGPs fail to develop. Thus, msSGPs do not arise from the dorsolateral domain, consistent with the fact that the msSGPs are first observed in a position ventral to the SGPs. The msSGPs also differ from the SGPs in terms of their requirements for the homeotic gene abd-A. SGP specification absolutely requires abd-A, while msSGPs are still present in these mutants. Thus, despite the fact that the msSGPs and the SGPs share expression of some molecular markers such as Eya and Wnt-2, their specification is under independent control (DeFalco, 2003).
Since the msSGPs express both Eya and Sox100B, the requirements for each of these genes in msSGP specification was investigated. In eya mutants, Sox100B-positive cells are still observed posterior to the germ cells at early stages, in a position where the msSGPs normally develop. Since the SGPs are not maintained in these mutants, the germ cells disperse and the gonad does not coalesce. Therefore, it is impossible to tell if the msSGPs would join the posterior of the male gonad in eya mutants. However, initial msSGP specification does not require eya. Similarly, in a deletion that removes the Sox100B locus, a large cluster of Eya-positive cells was still observed at the posterior of the male gonad that does not appear in females. Thus, the initial development of the msSGPs does not require Sox100B. Expression of Eya and Sox100B are mutually independent and are likely to be downstream of factors controlling initial msSGP specification (DeFalco, 2003).
Since the msSGPs are initially specified in both males and females, a determination was made of how these cells receive information about their sexual identity that allows them to behave differently in the two sexes. tra plays a key role in the sex determination pathway in Drosophila and is required to promote female differentiation in somatic tissues. tra mutant gonads were examined to test if tra function is required for gonad sexual dimorphism (XX embryos are masculinized by mutations in tra). Sox100B-immunopositive cells are observed in the posterior somatic gonad of both XX and XY tra mutant embryos in a manner comparable to wild-type males. Analysis of the Sox100B expression pattern in the gonad reveals that there are no differences between XX and XY tra mutants, or between either of these genotypes and wild-type males. Conversely, when Transformer is expressed in XY embryos (UAS-traF, tubulin-GAL4), Sox100B-immunopositive cells are no longer observe in these gonads, and they now appear similar to wild-type females (DeFalco, 2003).
In most somatic tissues, the principle sex determination factor downstream of tra is dsx. Unlike tra, dsx is required for both the male and female differentiation pathway, since both XX and XY dsx mutant adults show an intersexual phenotype. However, in the somatic gonad, dsx mutant XY embryos are indistinguishable from wild-type males and show no change in Sox100B expression. Thus, unlike in most somatic tissues, this early characteristic of male development does not require dsx. In XX embryos that are mutant for dsx, a completely masculinized phenotype is observed, in which Sox100B expression in the gonad is similar to a wild-type male. When a dominant allele of dsx, dsxD, is used to express DsxM (dsxD/dsx) in XX embryos, these gonads are no more masculinized than dsx null XX gonads. Therefore, while DsxF is required for the proper female phenotype in XX gonads, it appears that the male Sox100B expression pattern is the 'default' state in the absence of dsx function (DeFalco, 2003).
Since the msSGPs join the posterior of the male gonad but are no longer detected in the female, the basis for the sexually dimorphic behavior of these cells was investigated. In the female, these cells could turn off Sox100B and Eya and contribute to some other tissue, or they might be eliminated altogether. To test this latter hypothesis, whether msSGPs are eliminated by sex-specific programmed cell death in the female was addressed. Since programmed cell death occurs in a caspase-dependent manner, the gonad phenotype was examined in embryos in which caspase activity was inhibited by expressing the baculovirus p35 protein in the mesoderm. In these embryos, XX gonads now appear masculinized; Sox100B-positive cells (msSGPs) persist and join the posterior of female gonads, and coexpress Eya, as in wild-type male embryos. There are not as many Sox100B-positive cells in females as in males, suggesting that p35 may not be completely suppressing cell death. The presence of such cells in the female gonad does not appear to drastically affect ovary formation or oogenesis, since embryos develop into fertile adult females (DeFalco, 2003).
To investigate how programmed cell death might be controlled in the msSGPs, the genes of the H99 region (head involution defective [hid], reaper [rpr], and grim), which are regulators of apoptosis in Drosophila, were examined. A small deletion (DfH99) removes all three of these genes and blocks most programmed cell death in the Drosophila embryo. In DfH99 mutants, an equivalent cluster of Sox100B-positive cells is observed in both males and females. Again, these posterior cells are also Eya positive. Furthermore, XX embryos mutant for hid alone also contain Sox100B-positive cells in the posterior of the gonad, although the posterior cluster of cells is slightly smaller than in the male. It is concluded that the msSGPs are normally eliminated from females through sex-specific programmed cell death, controlled by hid and possibly also other genes of the H99 region. However, if cell death is blocked in females, these cells can continue to exhibit the normal male behavior of the msSGPs, including proper marker expression and recruitment into the gonad. Therefore, the decision whether or not to undergo apoptosis is likely the crucial event leading to the sexually dimorphic development of these cells at this stage (DeFalco, 2003).
It is concluded that proper information from the sex determination pathway is required to control the sexually dimorphic behavior of the msSGPs. The female phenotype in the embryonic gonad is dependent on both tra and dsx. Interestingly, it seems that the male phenotype is the default state; in the absence of any tra or dsx function, msSGPs in both XX and XY embryos behave as in wild-type males. This is a different situation than in most other tissues, in which dsx is required in both sexes to promote proper sexual differentiation. In particular, while no role is found for DsxM in this process, DsxF is positively required either to establish the female fate in the posterior somatic gonad or to repress the male fate. This role for DsxF in msSGP development is analogous to its role in the genital disc, in which DsxF is required to block recruitment of btl-expressing cells into the disc; in both cases, dsx female function serves to repress incorporation of a male-specific cell type. Since the msSGPs are initially specified in a sex-independent manner, this may account for the fact that the persistence of these cells (the male phenotype) is the default state. It will be of interest in the future to address the role of the msSGPs in testis development, and how genes such as dsx, eya, and Sox100B act in this process (DeFalco, 2003).
Although sex determination schemes vary widely in the animal kingdom, there is evidence that the molecular and cellular pathways used to control sexual dimorphism may be conserved, even between vertebrates and invertebrates. One example is Sox9, which has been implicated as an ancestral sex-determining gene in vertebrates given its male-specific gonad expression in diverse species such as human, mouse, turtle, and chicken. A potential Drosophila ortholog of Sox9, Sox100B, is expressed in a male-specific manner in the embryonic somatic gonad. The manner of Sox100B expression is reminiscent of that in the mouse; Sox9 is initially expressed in both sexes, but is maintained and upregulated in the male gonad. It will be very interesting to compare the role that Sox100B plays in the development of the Drosophila testis to the one played by Sox9 in vertebrates (DeFalco, 2003).
Molecular conservation is also observed amongst the members of the Dsx/Mab-3 Related Transcription Factor (DMRT) family. DMRT family members have been shown to be essential for sex-specific development in Drosophila (Dsx), C. elegans (mab-3), medaka fish (DMY), and mice (DMRT1) and have been implicated in human sex reversal. This study demonstrates that dsx is essential for proper sex-specific development of the msSGPs. Thus, increasing evidence indicates that DMRT family members are also conserved regulators of sexual dimorphism (DeFalco, 2003).
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date revised: 20 November 2006
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