doublesex

Targets of Activity

A single copy a specific enhancer from yolk protein genes directs female- and fat body-specific transcription. The enhancer consists of four protein-binding sites: dsxA, which binds male (DSXM) and female (DSXF) proteins encoded by the doublesex gene; aef1, which binds the AEF1 repressor; bzip1, which binds the DmC/EBP activator encoded by the slbo gene; and ref1, which binds an unknown activator. DSXF activates from dsxA by sterically excluding AEF1 repressor from the aef1 site and synergistically activating transcription, together with a protein at bzip1. Sex specificity in fat bodies arises from the opposite effect of DSXM: repression of the protein at bzip1. Tissue specificity is regulated by all four DNA sites. Separately, bzip1 and ref1 activate transcription in ovarian somatic cells and all nongonadal tissues, respectively, whereas together they activate only in fat bodies. The aef1 site represses ectopic transcription in ovaries and dsxA antirepresses this activity in fat bodies. Thus, in the organism, ref1 and bzip1 act combinatorially to direct the fundamental tissue specificity; aef1 and dsxA modulate this tissue specificity, and dsxA adds sex specificity. Surprisingly, SLBO does not appear to be the bZIP factor regulating the bzip1 site. SLBO is not found in adult fat bodies, and altered SLBO levels do not alter the fat body expression of Yp genes (An, 1995a).

Transcription of the Drosophila yolk protein (Yp) genes is regulated by the somatic sex determination pathway. A gene at the bottom of this pathway, doublesex, encodes the female-specific DSXF and male-specific DSXM proteins that bind to and regulate transcription from several sites in the Yp genes. Site-directed mutagenesis, protein binding and germline transformation experiments have been carried out that identify and characterize the activity of a single binding site (dsxA) for the Doublesex proteins and two binding sites for other regulatory proteins. The fat body enhancer (FBE), located between -197 and -332 of yp transcriptional start, has three bzip binding sites that bind Slbo non-cooperatively. A single copy of the three sites is sufficient to direct the sex and fat body specificities of Yp transcription. The sites form an enhancer with two strongly synergistic enhancer elements. One element (22 bp) consists of dsxA and an overlapping site, bzip1, that binds the DmC/EBP (Slbo) protein, a member of the bZIP family of transcriptional activators. bzip1 is the strongest of the three FBE binding sites for Slbo and dsxA is the strongest FBE binding site for DSXM and DSXF. Overlapping these two elements is a binding site for Aef1, the only FBE binding site for AEF1, a Drosophila repressor protein. The other element is an 11 bp binding site (ref1) for an unknown protein. Tissue-specific activation requires strong cooperation between the ref1 site and the bzip1 or dsxA sites. Sex specificity is regulated exclusively by the dsxA site which connects the sex determination pathway to the target gene through DSXM repression and DSXF activation. The dsxA site is not necessary for tissue specificity. The bzip1 site activates transcription in both sexes, but does not appear to be necessary for tissue specificity (An, 1995b).

Paradoxically, DSX does not regulate Yp1 and Yp2 expression in follicle cells of the ovary. The responsible regulator is an ovary-specific GATA factor, dGATAb, known as Serpent (Lossky, 1995).

Drosophila yolk protein genes are regulated by Doublesex male protein (DSXM) in males and Doublesex female protein (DSXF) in females. Both proteins bind to the same DNA sites from which DSXM represses and DSXF activates transcription. The proteins are identical through 397 N-terminal amino acids including domains for oligomerization and DNA binding. The remaining C-termini are sex-specific and include an essential part of a second oligomerization domain. Dimers of male or female specific proteins bind to a regulatory site, dsxA, with the same affinity, specificity and dependence on monovalent and divalent cations. The first order dependence on unbound DNA suggests that a direct transfer between DNAs is likely to occur when DSX proteins search for specific sites in the many short open DNA regions of chromatin. Overall, dimer binding to individual DNA sites appears to be determined by the sex-nonspecific part of the two proteins. It is inferred that the sex-specific oligomerization domains play roles in binding cooperativity to multiple DNA sites or in other protein:protein interactions (Cho, 1997).

The only sex-differentiation genes shown to be directly controlled by the sex-determination hierarchy are the yolk protein genes in Drosophila. The yp genes are coordinately regulated, that is, transcription occurs only in the female fat body and in a subset of ovarian follicle cells at specific stages of oogenesis. This highly specific expression pattern is the result of a complex regulatory mechanism involving tissue-specific factors and doublesex in the fat body and tissue specific factors in the ovary. Using the yolk protein genes, the conservation of regulatory elements for sex- and tissue-specific gene expression has been examined in three dipteran species: Drosophila melanogaster, Musca domestica and Calliphora erythrocephala. Yolk proteins of the fruitfly, medfly, housefly and blowfly are very well conserved both in their sequence and their expression in ovarian follicle cells and in fat bodies of adult females. yp regulation by both hormonal and nutritional factors shows similar features in all four species. To study conservation of yp regulation in dipteran insects, 5' flanking regions from one Musca yp gene and one Calliphora yp gene were tested for enhancer functions in D. melanogaster. Two fragments of 823 and 1046 bp isolated from Musca and Calliphora yp genes, respectively, are able to direct correct expression of a reporter gene in the ovarian follicle cells of transformed Drosophila at specific stages during oogenesis. Surprisingly, these enhancers do not confer sex-specific reporter gene expression in the fat body, since expression is found in both sexes of the transformed flies. Nonetheless, by in vitro DNA/protein interaction assays, a 284-bp DNA region from the Musca yp enhancer is able to bind the Drosophila Doublesex protein, which in D. melanogaster confers sex-specific expression of yp. It is speculated that the sex-determining pathway is not directly involved in yp regulation in Musca or Calliphora adult females, but depends instead on hormonal controls to achieve sex-specific expression of yp genes in the adult (Tortiglione, 1997).

Sex determination is regulated by diverse pathways. Although upstream signals vary, a cysteine-rich DNA-binding domain (the DM motif) is conserved within downstream transcription factors of Drosophila melanogaster (Doublesex) and C. elegans (MAB-3). Vertebrate DM motif genes have likewise been identified and, remarkably, are associated with human sex reversal (46, XY gonadal dysgenesis). The structure of the Doublesex domain contains a novel zinc module and disordered tail. The module consists of intertwined CCHC and HCCC Zn2+-binding sites: the tail functions as a nascent recognition alpha-helix. Mutations in either Zn2+-binding site or tail can lead to an intersex phenotype. The motif binds in the DNA minor groove without sharp DNA bending. These molecular features, unusual among zinc fingers and zinc modules, underlie the organization of a Drosophila enhancer that integrates sex- and tissue-specific signals. The structure provides a foundation for analysis of DM mutations affecting sexual dimorphism and courtship behavior (Zhu, 2000).

Whereas the genetic regulation of mammalian DM proteins (and in particular their relationship to SRY) is not understood, dsx and mab-3 have been characterized extensively. Intersex phenotypes demonstrate that only a subset of dimorphic lineages in either D. melanogaster or C. elegans are affected by DM mutations, reflecting the function of dsx and mab-3 in one branch of a ramifying pathway. Downstream pathways are not well defined. A model Dsx responsive enhancer (known as the fbe) has been isolated in yolk-protein promoters that is active in the fat body of the female adult fly and inactive in the male. Although an analogous MAB-3 responsive element has not been characterized, positive regulatory sites have been defined in yolk protein-related vitellogenin promoters. Mutations that block binding of MAB-3 in vitro also attenuate sex-specific transcriptional regulation in vivo. Whether the ubiquity of the DM motif indicates conservation of other downstream genes is unknown (Zhu, 2000 and references therein).

The structure of the DM zinc module, like the C₄-C₄ DBD of the nuclear hormone-receptor superfamily, contains two Zn²⁺ atoms and adjoining alpha-helices in a unified core. Although unrelated in detail, these motifs are substantially but not completely folded in the absence of DNA. Like the glucocorticoid and estrogen receptor (GR and ER) DBDs in particular, the DM domain recognizes pseudopalindromic DNA sites as an induced homodimer. Insertion of a symmetrical central base pair to make a palindromic DNA element in each case leads to a 100-fold reduction in affinity with retention of cooperativity. These similarities belie fundamental differences. Whereas the nuclear hormone receptor DBD consists of two C₄ substructures encoded by separate exons, the DM motif contains intertwined CCHC and HCCC ligands encoded by a single exon. Whereas the C₄-C₄ DBD binds in DNA's major groove, the DM domain binds in the minor groove (Zhu, 2000 and references therein).

DNA recognition by the DM domain requires a carboxy-terminal basic tail. The present studies demonstrate that the tail undergoes an independent and noncooperative helix-coil equilibrium. These observations are reminiscent of DNA-dependent folding transitions among the basic arms of major groove-binding motifs. Whereas the dimensions of the major groove of B-DNA are commensurate with those of an alpha-helix, the minor groove is ordinarily too narrow. DM-DNA recognition thus provides an unusual example of the minor groove acting as a template for protein folding. The R91Q intersex mutation in Dsx demonstrates that a native tail is necessary for DNA recognition and sex-specific gene regulation in vivo. Tail sequences share an overall helical propensity. Conservation of tail sequences is more stringent than within the zinc module (exclusive of the invariant cysteines and histidines. The Dsx tail is similar, for example, to those of MAB-3b and the vertebrate DMRT and TERRA families: the motif L(V/T)X(D/E)RQRVMA(A/L)Q(V/T)-ALRR(Q/A)QA (Dsx residues 75-94 including the underlined Dsx intersex site R91Q) is invariant. It is proposed that this segment functions as a DNA recognition helix and therefore, defines a subgroup of DM proteins of related specificity. Such tail-DNA contacts are presumably extended by interactions between the DNA and the Zn module itself (Zhu, 2000).

The tail of MAB-3a is strikingly divergent. In particular, MAB-3a contains Ile rather than Arg at Dsx intersex site R91Q. Such divergence, retained in C. briggsae, suggests that MAB-3a recognizes a different DNA sequence or is positioned differently relative to a DNA half site. Indeed, although Dsx and MAB-3 exhibit overlapping specificities, random binding-site selection has revealed important differences. In particular, MAB-3's consensus sequence exhibits marked asymmetry [5'-AATGTTGCGA(T/A)NT-3' and complement], which contrasts with near-palindromic dsxA half-sites. Because respective Dsx and MAB-3 target sites are similar in length, MAB-3 is proposed to bind DNA as an "internal dimer" of DM domains. It is imagined that the divergent tails of MAB-3a and MAB-3b contact asymmetric half-sites with different intrinsic specificity. Whereas the genetic function of MAB-3 requires both domains, other known DM proteins contain a single domain. It is speculated that such proteins undergo DNA-dependent homo- or hetero-dimerization as an example of combinatorial gene regulation (Zhu, 2000).

The fbe, a model Dsx response element in a dimorphic tissue, contains three overlapping factor-binding sites. Binding of Dsx to the central site is proposed to regulate binding of tissue-specific factors to flanking sites (aef1 and bzip1). Critical bases in dsxA recognized by Dsx are palindromic about a central base pair. The associated tissue-specific factors (AEF1 and an uncharacterized bZIP transcription factor) contain conserved DNA-binding motifs (classic zinc fingers and basic region-leucine zipper, respectively). In the fat body, coordinate binding of Dsx^F and bZIP1 to the fbe displaces AEF1 and recruits an unknown activator to proximal site ref1, resulting in fat body-specific activation. Overlapping DNA target sites are thus proposed to provide a mechanism by which sex- and tissue-specific signals are integrated to regulate gene expression. Although the biochemical basis of fbe regulation remains speculative, functional integration of its discrete factor-binding sites has been demonstrated in transgenic flies. Such features are likely to reflect general principles of transcriptional activation in eukaryotes (Zhu, 2000).

Genetic dissection of the fbe has novel and previously unrecognized structural consequences. Because bZIP proteins bind within the major groove, the An-Wensink model (An, 1995a and b) predicts that the DM domain (like SRY but unlike classic zinc fingers and other zinc modules) would bind in the minor groove. The model further requires that the DM domain (unlike SRY and other HMG boxes) would not induce sharp DNA bending lest associated compression of the major groove were to displace bZIP1. The present studies have tested and confirmed these implicit predictions. Minor groove binding without bending is unusual and may be analogous to that of the T domain-DNA complex of the Brachyury transcription factor. The putative ternary Dsx^F-bZIP1-DNA complex would be remarkable for adaptive binding of one nascent alpha-helix in the DNA major groove (bzip1) and of another nascent alpha-helix in the overlapping DNA minor groove (dsxA). Methylphosphonate interference experiments suggest intimate juxtaposition of Dsx^F and bZIP1 complexes. bZIP1-phosphate contacts, inferred from crystal structures of the GCN4 bZIP-DNA complex, overlap sites of Dsx-methylphosphonate interference. Such interdigitation of protein-DNA phosphate contacts is consistent with a previous 'missing base' interference assay suggesting corecognized bases. Simultaneous occupancy of major and minor grooves is likely to be a general feature of enhanceosome assembly (Zhu, 2000).

The mechanism of synergistic transcriptional activation between Dsx^F and bZIP1 is not well understood. Studies of mammalian liver-specific gene regulation by C/EBPalpha and NF-Y have demonstrated that synergy can occur in the absence of cooperative promoter binding; binding of C/EBP in fact impairs binding of NF-Y and yet their simultaneous binding leads to synergistic activation of a minimal promoter. Nonetheless, synergy between CCAAT enhancer-binding factor alpha (C/EBPalpha) and nuclear factor Y (NF-Y) results in the formation of a preinitiation complex that is stable through multiple rounds of transcription. Additional biochemical studies will be required to distinguish whether (1) DNA binding by Dsx^F and bZIP1 is cooperative (a thermodynamic mechanism), (2) the ternary Dsx^F-DNA-bZIP1 complex dissociates more slowly than either binary complex (a kinetic mechanism), or (3) binding of Dsx induces a conformational change in bZIP1 enhancing its potency in transcriptional activation (an allosteric mechanism). The mechanisms by which Dsx^M represses bzip1 are also not well understood (Zhu, 2000).

doublesex is representative of a class of genes that not only specify aspects of body plan but also influence behavior. Chromosomal female (XX) flies expressing Dsx^M, although male in external appearance, do not court wild-type females. Chromosomal male (haplo-X) flies bearing a deletion in one dsx allele and various point mutations in the other exhibit specific and quantifiable changes in courtship behavior. The haplo-X dsx phenotype differs from that associated with fruitless (male chaining). Dsx-associated anomalies include an altered courtship song, selective lack of vigor in the pursuit of wild-type females, increased elicitation of courtship from wild-type males, and decreased rejection of subsequent attempted copulation. The mechanics of courtship and individual elements of mating behavior are normal with the exception of the extent of abdominal bending during attempted copulation and altered song. The latter is of particular interest: the humming component is absent, whereas changes in the rhythmicity of acoustic pulses (as associated with mutations in the period gene) are not observed. Unlike fruitless, dsx does not regulate the male-specific induction or female-specific suppression of the abdominal Muscle of Lawrence, which thus represents a dsx-independent branch of the ramified sex-determining pathway (Zhu, 2000 and references therein).

The extent and spectrum of behavioral change associated with the various point mutations in dsx are allele specific. The biochemical bases of these phenotypic differences have not been elucidated. Courtship behavior reflects the complex integration of multiple systems, including pheromone production, elaboration, and sensing; development of the peripheral nervous system and its central ramifications; and function of a putative courtship command center in the brain. Whether and how dsx influences the differentiation of the male brain are not well understood. Although dsx controls the sex-specific pattern of post embryonic proliferation of neuroblasts in an abdominal ganglion, its effect on patterning of the central nervous system (and in particular, on the function of a courtship command center) is not well characterized. In light of the allele-specific effects of mutations on extent of courtship anomalies, the Dsx pathway promises to provide an attractive model for study of the biochemical basis of a complex behavior (Zhu, 2000).

Two proteins function together to regulate sex specific genes: (1) the sex specific transcription factor Doublesex (Dsx), and (2) the non-sex specific transcription factor Hermaphrodite (Her). A study by Li and Baker (1998) analyzes the often complex combinatorial interactions between parallel pathways that intersect in the regulation of even a single gene. The targeted yolk protein (yp) genes are transcriptionally activated by two separate pathways. One is a female-specific pathway, which is positively regulated by the female-specific Doublesex protein (Dsx F). The other is a non-sex-specific pathway, that is positively regulated by Her. The Her pathway is prevented from functioning in males by the action of the male-specific Doublesex protein (Dsx M). The Her and Dsx pathways also function independently to control downstream target genes in the precursor cells that give rise to the vaginal teeth and the dorsal anal plate in females, and the lateral anal plates in males. However, a female-specific pathway that is dependent on both Dsx F and Her controls the female-specific differentiation of the foreleg bristles and tergites 5 and 6, and the male-specific differentiation of these tissues does not require the suppression of Hers function by Dsx M (Li, 1998).

Since the only characterized target genes of dsx are the yp genes, an investigation was undertaken to see if her also regulates the expression of the yp genes and if so, whether her functions in their regulation in a manner similar to dsx . Northern analysis was used to examine the effects of her on expression of the yp genes. Since the complete loss of her function is lethal, the temperature-sensitive allele her 1 was used. At 25°C, her 1 flies are intersexual and have severely reduced viability; in contrast to this, at 18°C, they are morphologically normal, and have wild-type viability and fertility. There is a 10-fold activation of yolk protein 2 (yp2) expression by her +, since mutant her females raised at a non-permissive temperature (25°C) show a 10-fold reduction of yp2 transcript levels, as compared to wild-type females and their her 1/+ sisters. This is comparable to the activation effect of the dsx + gene in females. Surprisingly, yp2 expression is also reduced 10-fold in the her 1 homozygous females raised at 18°C. However, when grown at 16°C, her 1 females have levels of yp2 expression comparable to that seen in wild-type females. These results indicate that yp2 expression is more sensitive to the level of her function than is external sexual morphology. In her males, the yp2 transcript level remains unchanged. This is in striking contrast to dsx males where the yp2 level is increased 20-fold compared to that of wild-type males and dsx/+ brothers, consistent with previous findings that Dsx M (the male Dsx splice variant) functions to repress the transcription of the yp genes. This result reflects a fundamental difference between the her and dsx functions in males. The expression of the yp1 and yp3 genes is regulated the same way by dsx and her as is the yp2 gene. It is concluded that Her is required, like Dsx F, for the activation of the yp genes in female fat body cells. But, in contrast to Dsx M , Her is not required for the inhibition of yp gene expression in males (Li, 1998).

The reduction of yp2 transcripts in her mutant females could be due to the involvement of her in the regulation of yp2 transcription or yp2 RNA stability. To distinguish between the two possibilities, the yp reporter gene pCR1 was used. In the pCR1 construct, the intergenic regulatory region of the divergently transcribed yp1 and yp2 genes remains intact while the coding sequences of yp1 and yp2 are replaced by the Drosophila Adh and the Escherichia coli lacZ genes, respectively. The effects of her and dsx on the expression of the lacZ gene of pCR1 are in all cases comparable to their effects on yp gene expression as monitored by Northern blots, demonstrating that her, like dsx, controls yp gene expression at the level of transcription, rather than RNA stability. Similarly, her + activity is also required in females for the transcriptional activation of the yp genes, rather than the stability of their transcripts. These results demonstrate that her, like dsx, activates the transcription of the yp genes in females through the intergenic region of yp1 and yp2 (Li, 1998).

The perceptions that derive from the above experiments concerning the roles of dsx and her in regulating the transcription of the yp genes suggest that both genes function in the activation of the yp genes in females, but that only dsx functions in males, where it acts to repress the yps expression. However, consideration of the quantitative aspects of the data from these experiments indicates that this interpretation is incorrect. In particular, the data with respect to the roles of Dsx M in males and Dsx F in females indicate that dsx function can account for all of the difference between the sexes in the levels of yp gene expression. These findings with regard to dsx clearly contradict the idea that there is a female-specific role for her in the activation of the yp genes. Two alternative views of the role of her in regulating yp gene expression are presented that are consistent with these results. The argument that dsx is the major, if not the only, sex-specific regulator of the yp genes derives from the analysis of the transcriptional regulation of pCR1. The pCR1 lacZ activity in dsx/+ females is about 2000-fold higher than in dsx/+ males (no expression of the yp genes). However, the difference is only about 2.6-fold between the dsx homozygous female and male sibs. Since flies homozygous for the X-linked pCR1 transgene were female, the 2.6-fold difference in the pCR1 activity between the dsx females and males is largely, if not entirely, due to the 2-fold difference in the gene dosage of pCR1 between females (two copies of the pCR1 transgene) and males (one copy of the pCR1 transgene). Therefore, these results demonstrate that in the absence of dsx, the yp genes are expressed at the same levels in both sexes (Li, 1998).

In considering these results, it is important to note that two factors contribute to making the levels of yp gene expression equivalent in dsx mutant males and females: (1) the expression level of the yp genes is elevated in dsx males (compared to wild-type males), due to the absence of repression by Dsx M, and (2) the expression level of the yp genes in dsx females is reduced, due to the absence of activation by Dsx F. Thus in both dsx mutant males and females, there are significant levels of expression of the yp genes, and these levels are equivalent in the two sexes. There are two ways to reconcile these observations with regard to dsx with the observation that her appears to control the expression of the yp genes female-specifically. One model is that her does function female-specifically, but that its female-specific function is dependent on Dsx F. The second model is that her functions sex-independently to activate the expression of the yp genes, but that its action in males is precluded by Dsx M's repression of any yp gene's expression. These two models make different predictions as to the effects expected of her mutants in dsx mutant backgrounds. If the first model is correct, the presence or absence of her should have no effect on the yp genes when Dsx F is absent. If the second model is correct, her should be able to activate the yp genes in dsx mutant males where Dsx M is absent. To examine the effects of her on yp gene expression in the absence of dsx function, the pCR1 reporter gene was used. To test whether her activates yp gene expression in males in the absence of the inhibition by Dsx M, the responsiveness of pCR1 to her regulation was examined in the absence of Dsx M. In males, when Dsx M is present, pCR1 is not expressed whether or not her is present. However, in males without Dsx M, pCR1 is expressed and the pCR1 activity is 5-fold higher when her is present than when her is absent. This finding suggests that wild-type her function is normally present in males and capable of activating the transcription of the yp genes, but its activity is normally overridden by the inhibitory function of Dsx M. In conclusion, there are two separate pathways for the activation of the yp genes. One is the female-specific activation of yp genes, which is Dsx F-dependent. The other is the non-sex-specific activation of ypgenes, which is Her-dependent, Dsx F-independent and inhibited by Dsx M. These results also suggest that her has the same biological function in both sexes, providing further evidence that the expression of her is independent of the sex determination hierarchy. It is further shown that Dsx and Her can activate the yp genes independently in females (Li, 1998).

Further dissection of the yp promoter reveals that the fat body enhancer (FBE), the site of Dsx action, is not sufficient to confer her responsiveness and the major her responsive element is located outside of the FBE, in the Her responsive region (HRR). Thus, the HRR is necessary for the Her-dependent non-sex-specific activation of yp1 and yp2 (Li, 1998).

The fact that her and dsx mutant females have similar external phenotypes raises the possibility that dsx and her may regulate other downstream target genes in a manner similar to how they regulate the yp genes. This predicts that the loss of her should masculinize dsx mutant XX flies and vice versa, since Her and Dsx F regulate the yp genes independently. In addition, the loss of her should also masculinize dsx mutant XY flies, since Dsx M inhibits her's activation of the yp genes. To examine whether these predictions are true, a comparison was made of the phenotypes of five different external cuticular structures (which are sexually dimorphic in wild-type adult flies) among XX and XY sibs of the following four genotypes: (1) her/+; dsx/+, (2) her/her; dsx/+, (3) her/+; dsx/dsx and (4) her/her; dsx/dsx. The first cuticular structure examined was the number of the vaginal teeth. In the precursor cells that give rise to vaginal teeth, her and dsx are shown to act independently as in the case of the regulation of the yp genes in fat body. These results show that the loss of her masculinizes dsx mutant XX flies and vice versa, indicating that her + and dsx + can act in each other's absence in these cells (Li, 1998).

The second set of cuticular structures examined were the anal plates. The dorsal anal plate of females and the two lateral anal plates of males derive from the same precursor cells. In XX and XY intersex flies, there are a pair of anal plates located dorsolaterally to the anal opening and they are often fused at the dorsoanterior side. This pair of anal plates (referred to as DLAP hereafter) represents the intersexual differentiation of the precursor cells, and they are completely fused to form the dorsal anal plate in wild-type females and are completely separated to form the two lateral anal plates in wild-type males. Loss of her masculinizes dsx mutant XX flies and vice versa. These results indicate that, in the precursor cells of vaginal teeth and DLAP, Her controls downstream female-specific differentiation genes non-sex-specifically, and Her's functioning is independent of Dsx F in females and is inhibited by Dsx M in males, analogous to Her's regulation of the yp genes in fat body cells. However, the results also indicate that this is not the only mechanism by which her and dsx act. In the precursor cells of the last (most distal) transverse row of bristles (LTRB) of the basitarsus of the forelegs (LTRB form sex combs in males), Her functions together with Dsx F. In addition to the cuticular structures already described, the number of the 6th sternite (S6) bristles was also examined on dsx mutant, her mutant, and her; dsx mutant XX and XY flies. The results indicate that (1) in XX flies, S6 differentiation follows a default pathway that is independent of dsx (Dsx F ) and her, and (2) in XY flies, S6 differentiation is dependent on both her and dsx (DSX M) (Li, 1998).

In summary, analysis of sexual phenotypes of various tissues in the her and dsx single mutants and the her; dsx double mutants demonstrates that there are three ways by which sexual dimorphism is generated. The first utilizes DsxM in males and does not require DsxF in females. The second utilizes DsxF in females and does not require DsxM in males. The third utilizes both DsxM in males and DsxF in females. Her is involved in the last two modes of regulation, and likely also in at least some cases of the first mode of regulation. On theoretical grounds, the most parsimonious way to generate differences between homologous tissues in the two sexes during evolution is to have a regulatory gene product present in the tissues of one sex and absent in the other sex, thus affecting the pre-existing non-sex-specific differentiation in one sex, but not in the other. For example, the default pathway for T6 is full pigmentation. The sexual dimorphism of T6 is solely due to the suppression of the T6 pigmentation by Dsx F in females, in collaboration with Her, and is irrespective of the presence or absence of Dsx M in males. Another example is the formation of sixth sternite (S6) bristles. The default pathway is to form 18 bristles on S6. The sexual dimorphism of S6 is caused by the suppression of bristle formation by Dsx M in males, likely in collaboration with Her, and is irrespective of Dsx F in females. However, in the presence of selective pressures on both sexes in evolution, one way to increase sexual dimorphism is to have female- and male-specific products of regulatory genes that each have active roles in modifying the effects of pre-existing non-sex-specific regulatory systems in opposite ways, thus generating dramatic sex-specific features. For instance, in the absence of Dsx F in females and Dsx M in males, the expression levels of the yp genes are equivalent between the two sexes due to non-sex-specific control by Her. When females have Dsx F and males do not have Dsx M, there is a 30-fold difference between females and males in the expression levels of the yp genes, and when females do not have Dsx F and males have Dsx M, there is a 180-fold difference between females and males. However, a maximum difference (2000-fold) is observed only when Dsx F is present in females and Dsx M is present in males. The sexually dimorphic differentiation of the precursor cells of the vaginal teeth and DLAP is similarly controlled by Her and both Dsx proteins. Thus, her may be viewed as part of a non-sex-specific regulatory system in these tissues, which is subject to sex-specific modification by Dsx F and Dsx M (Li, 1998).

The Drosophila somatic sex-determination regulatory pathway has been well studied, but little is known about the target genes that it ultimately controls. In a differential screen for sex-specific transcripts expressed in fly heads, a highly male-enriched transcript was identified encoding Takeout, a protein related to a superfamily of factors that bind small lipophilic molecules. Sex-specific takeout transcripts derive from fat body tissue closely associated with the adult brain and are dependent on the sex determination genes doublesex (dsx) and fruitless (fru). The male-specific Doublesex and Fruitless proteins together activate Takeout expression, whereas the female-specific Doublesex protein represses takeout independently of Fru. When cells that normally express takeout are feminized by expression of the Transformer-F protein, male courtship behavior is dramatically reduced, suggesting that male identity in these cells is necessary for behavior. A loss-of-function mutation in the takeout gene reduces male courtship and synergizes with fruitless mutations, suggesting that takeout plays a redundant role with other fru-dependent factors involved in male mating behavior. Comparison of Takeout sequences to the Drosophila genome reveals a family of 20 related secreted factors. Expression analysis of a subset of these genes suggests that the takeout gene family encodes multiple factors with sex-specific functions (Dauwalder, 2002).

To identify genes under the control of the sex-determination regulatory pathway, a PCR-based subtractive hybridization screen was carried out for sex-specific RNAs expressed in adult fly heads. Head RNA of tra-2/tra-2⁺ phenotypically wild-type XX adult females was subtracted against the head RNA of sibling XX tra-2/tra-2 mutants, and vice versa. The latter flies are transformed into males both somatically and behaviorally. One cDNA clone that hybridized preferentially with sequences from phenotypic males was isolated and studied in more detail. Northern blot hybridizations confirmed that this sequence represents a highly male-specific 1.1-kb mRNA that was expressed primarily in adult heads. Expression of this mRNA was repressed by Tra-2 in females, since XX tra-2 mutants expressed levels similar to wild-type males. The sequence of the clone was later found to be identical to that of takeout, an independently identified gene responsive to circadian rhythms and starvation. The takeout gene encodes a secreted protein related to circulating carrier proteins of lipophilic factors, such as the juvenile hormone-binding proteins of other insects. Analysis of RNA prepared at different times during the day failed to reveal any significant variation in takeout levels (Dauwalder, 2002).

How Dsx and Fru affect takeout expression was examined. Since dsx is known to affect sexual differentiation in males and females, it might either activate takeout in males, repress it in females, or both. takeout expression was compared between dsx homozygous mutant animals and their heterozygous siblings. In blot hybridization experiments, XY dsx individuals were found to have takeout RNA levels reduced by 37% relative to XY dsx/+ flies, indicating that the male-specific Dsx-M product functions to activate takeout expression. In chromosomally XX individuals, dsx mutations have an opposite effect. In comparison with XX dsx/+ siblings, XX dsx/dsx animals have levels of takeout mRNA increased by 13-fold, indicating that Dsx-F normally functions to repress takeout expression. Thus, the differential expression in males and females is achieved (at least in part) by dsx-dependent repression in females and activation in males. Curiously, XX dsx/dsx intersexes have more takeout RNA than do XY dsx/dsx intersexes, suggesting that sex-specific factors other than dsx also affect overall takeout expression (Dauwalder, 2002).

The effect of the dominant dsx^SWE allele on takeout expression was examined. Due to a deletion in the female-specific exon that results in constitutive male-specific splicing of the dsx pre-mRNA, this allele produces only Dsx-M. XY flies carrying this allele are phenotypically normal males, and do not have reduced takeout expression. However, in XX; dsx^SWE/+ animals, the presence of Dsx-M antagonizes Dsx-F function, resulting in intersexual flies that are similar in phenotype to those produced by dsx null mutations. takeout was derepressed to intermediate levels in such intersexes, further supporting the idea that takeout is controlled by dsx (Dauwalder, 2002).

Functional analysis of Dsx and Fru has led to the suggestion that they have distinct and complementary roles, with Fru specifying sexual identity of tissues in the CNS that are responsible for courtship behavior, and Dsx specifying sex in other somatic tissues. However, given the observation that dsx mutants also have minor effects on courtship behavior, it is believed that a clear delineation of the roles played by Dsx and Fru will require more information about the specific genes and cell types whose sexual identity these factors specify (Dauwalder, 2002).

Both the dsx and fru genes encode alternatively spliced transcripts that encode distinct forms of the Dsx and Fru proteins in males and females. Thus, both genes could potentially play a role in either activating takeout in males or repressing it in females. Full activation of takeout is not achieved in either dsx null or fru hypomorphic mutant XY individuals and, instead, takeout RNA is present at levels intermediate between those found in males and females. In chromosomal females, only Dsx is required for repression of takeout. The fact that fru mutants do not affect takeout expression is consistent with experiments suggesting that the female-specific form of fru mRNA is not translated into a functional protein. Moreover, all sex-specific Fru functions so far identified have been found in males. Therefore, although a sex-specific Fru mRNA is produced in females that potentially encodes a protein, there is currently no evidence that it functions to regulate sexual differentiation (Dauwalder, 2002).

The fact that dsx is capable of both activating and repressing takeout expression reflects the dsx gene's unusual ability to perform opposite functions in males and females by producing distinct proteins in the two sexes through alternative pre-mRNA splicing. The male-specific (Dsx-M) and the female-specific (Dsx-F) proteins share a common DM domain, which is required for DNA binding. The two proteins differ at their C termini, a region promoting dimerization in both forms. Three potential Dsx-binding sites are located within 1 kb upstream of the takeout translation initiation codon, but further studies will be required to determine whether Dsx proteins associate directly with the takeout promoter. Taken together, the results presented here suggest that Dsx-F and Dsx-M can each either activate or repress the activity of downstream genes. Presumably, the effect Dsx has on any particular gene is also determined by other regulators interacting with the gene's promoter (Dauwalder, 2002).

Genetic control and evolution of sexually dimorphic characters in Drosophila

Sexually dimorphic abdominal pigmentation and segment morphology evolved recently in the melanogaster species group of the Drosophila. These traits are controlled by the bric à brac gene, which integrates regulatory inputs from the homeotic and sex-determination pathways. bab expression is modulated segment- and sex-specifically in sexually dimorphic species, but is uniform in sexually monomorphic species. It is suggested that bab has an ancestral homeotic function, and that regulatory changes at the bab locus played a key role in the evolution of sexual dimorphism. Pigmentation patterns specified by bab affect mating preferences, suggesting that sexual selection has contributed to the evolution of bab regulation (Kopp, 2000).

An approach to bridging this gap between evolutionary genetics and comparative embryology is to analyze and compare the development of rapidly evolving morphological traits. In many animals, secondary sexual characteristics evolve rapidly, making them good candidates for analysis. One such character in Drosophila is the pigmentation of adult abdominal segments. In D. melanogaster, abdominal pigmentation is sexually dimorphic. Segments 1 to 6 in females and 1 to 4 in males carry only a posterior stripe of dark pigment. However, segments 5 and 6 (A5 and A6) in males are completely pigmented, giving the species its name. This pattern is of recent evolutionary origin; in most Drosophila species, male-specific pigmentation is absent, so that females and males are pigmented identically. To understand how this new pattern originated and evolved, the regulatory circuit that controls its development has been characterized, and its operation has been compared in sexually dimorphic and monomorphic species (Kopp, 2000).

The development of sexually dimorphic external characteristics is controlled by the doublesex (dsx) gene. Alternative splicing of the dsx transcript produces a male-specific product in males (dsx^M), and a female-specific product in females (dsx^F). Loss of dsx function in females results in the development of male-like pigmentation, which can be suppressed by heat-shock dsx^F transgenes. Male-specific pigmentation is therefore expressed by default, and must be actively repressed by dsx^F (Kopp, 2000).

Thus, the development of sexually dimorphic pigmentation requires integration of homeotic and sex determination gene inputs. In investigating how this integration is achieved, a newly evolved genetic circuit has been discovered that appears to be responsible for the origin of male-specific pigmentation (Kopp, 2000).

A gene near the left tip of the third chromosome contributes to the variation in female abdominal pigmentation. In investigating this genetic region, it was found that loss of one copy of the bab locus results in the development of male-specific pigmentation in females, but has no effect on the male abdomen. Ectopic pigmentation in heterozygous bab females is suppressed by reducing the dosage of Abd-B, but is not eliminated by loss of omb. This suggests that bab+ represses the development of male-specific pigmentation in females by opposing the function of Abd-B. The bab locus contains two closely related genes, bab1 and bab2, which encode putative transcription factors with multiple roles in development. Ectopic pigmentation in females increases in the order bab1/+ < bab1/bab1 bab1bab2/+ bab1bab2/bab1, indicating that both genes are involved in repressing male pigmentation. For simplicity, the entire locus has been treated as one gene, bab, unless noted otherwise (Kopp, 2000).

The expression pattern of bab at the pupal stage when the adult epidermis develops reflects its sex- and segment-specific function. In females, bab expression is strongest in segments A2 and A3, and progressively weaker in A4, A5 and A6. In males, bab expression is considerably weaker than in females in all segments. Most strikingly, it is completely absent from A5 and A6. This pattern of bab repression correlates with the presence of sex-specific pigmentation in males, and its absence in females (Kopp, 2000).

To test whether bab+ is sufficient to repress pigmentation, the bab genes were ectopically expressed in the pupal abdomen. Low-level expression of bab+ results in the loss of male-specific pigmentation, but has no other effects on external morphology, indicating that differential regulation of bab plays a central role in establishing sexual dimorphism. bab+ can also repress non-sex-specific pigment stripes when expressed at a higher level. This suggests that bab+ acts as a general repressor of pigmentation, but that its effects are overridden by omb in the posterior part of each segment. Consistent with this, complete loss of both bab genes results in ectopic pigmentation of A2 to A7 in both sexes. This phenotype is not caused by expansion of Abd-B expression, which appears normal in these mutants. In bab homozygotes, the intensity of pigmentation is higher in the more posterior segments than in those more anterior. This suggests that pigmentation does not develop by default in the absence of bab, but is actively promoted by Abd-B and abd-A (Kopp, 2000).

The sexually dimorphic repression of bab in the posterior abdomen suggests that bab integrates the homeotic and sex determination regulatory inputs. To test this, bab expression was examined in Abd-B and dsx mutant backgrounds. Ectopic expression of Abd-B in A3 and A4 eliminates bab expression from these segments in males, and downregulates it in females. Conversely, bab is derepressed in A5-A7 in the mutants that lack Abd-B function in these segments. Together, these results indicate that bab expression in A5 and A6 is normally repressed by Abd-B. The slight downregulation of bab in A4 suggests that it is also weakly repressed by abd-A (Kopp, 2000).

In dsx-intersexes, bab is expressed in a male-like pattern, suggesting that dsx^F upregulates bab transcription in females. Abd-B and abd-A expression is identical in males, females and dsx -intersexes, indicating that bab is regulated independently by homeotic and sex-determination inputs. dsx^Dominant intersexes, which express both male- and female-specific dsx products, also show male-like expression of bab, indicating that dsx^M can interfere with dsx^F function. The two dsx isoforms encode transcription factors that bind the same DNA sequence, but have opposite effects on gene expression. dsx-intersexes differ from males in having a small unpigmented region at the anterior-lateral margin of A5, suggesting that dsx^M may have a slight negative influence on bab expression (Kopp, 2000).

These results suggest that bab+ regulates sexually dimorphic pigmentation by integrating regulatory inputs from the homeotic genes and the sex determination pathway. In this regulatory circuit, bab+ acts as a general repressor of pigmentation, and Abd-B and abd-A promote pigmentation in both sexes. In addition, Abd-B, and to a lesser extent abd-A, repress bab transcription. In males, this results in the absence of bab from A5 and A6, allowing Abd-B and abd-A to promote pigmentation in these segments. However, in females, dsx^F prevents bab transcription from being completely repressed by the homeotic genes. As a result, bab is present in A5 and A6 in females, where it blocks the ability of Abd-B and abd-A to promote pigmentation. In A2-A4, abd-A alone is not sufficient either to repress bab or to overcome its inhibitory effect on pigmentation; thus, only the omb-dependent striped pigmentation is generated. Because Abd-B, abd-A and dsx encode transcription factors, they may regulate bab expression directly (Kopp, 2000).

The central role of bab as an integrator of homeotic and sex-determination gene inputs suggests that changes in bab regulation may have been responsible for the evolution of sexually dimorphic pigmentation. In the subgenus Sophophora, male-specific pigmentation is present only in the melanogaster species group. Within this group, sexual dimorphism is seen in all species of the melanogaster subgroup and the closely related oriental subgroups, whereas the ananassae and montium subgroups contain both sexually dimorphic and sexually monomorphic species (Kopp, 2000).

In species with male-specific pigmentation of A5 and A6, bab expression is absent or strongly downregulated in these segments in males, but not in females. Moreover, in the sexually monomorphic species outside the melanogaster species group, bab expression is identical in both sexes and in all segments from A2 to A7. This correlation suggests that changes in the regulation of bab by Abd-B and dsx played an important role in the origin of sexually dimorphic pigmentation (Kopp, 2000).

bab+ regulates segment shape and bristle and trichome patterns in a manner reciprocal to Abd-B. Loss of bab+ function in females enhances posterior characteristics in A6, A7 and A8. No phenotype is seen in males, consistent with the absence of bab expression in posterior segments. Conversely, ectopic expression of bab transforms A6 and A7 to a more anterior identity in both males and females. These observations suggest that bab+ acts as an antagonist of Abd-B homeotic function, and that posterior abdominal characters are determined by the balance between Abd-B and bab activities (Kopp, 2000).

This model predicts that evolutionary changes in bab regulation should result in morphological transformation of Abd-B-expressing segments. Indeed, the entire suite of characteristics that distinguishes A5 and A6 from the more anterior segments in D. melanogaster is of recent evolutionary origin. In D. willistoni, bab is expressed strongly in A5 and A6 in males, whereas Abd-B is expressed in the same pattern as in D. melanogaster. As predicted, A5 and A6 are almost identical to the more anterior, non-Abd-B-expressing segments in the males of this species. In contrast, the melanogaster species group shows great diversity of bristle and trichome patterns in posterior abdominal segments. The two main lineages within this group show different patterns of evolution. In the clade composed of the melanogaster and oriental subgroups, male-specific pigmentation and bristle and trichome patterns have evolved in a concerted fashion. However, in the ananassae + montium lineage, these characteristics vary independently of each other, and sexually dimorphic bristle and trichome patterns are sometimes observed in species that do not show visible modulation of bab expression. This suggests that evolutionary changes have occurred not only in bab regulation, but also in the target genes of bab and in other genes regulated by Abd-B and dsx . Suppression of A7 development in males has occurred earlier in evolution than visible modulation of bab expression, despite the ability of bab to override this suppression (Kopp, 2000).

These findings indicate that changes in bab regulation have played an important part in the evolution of abdominal segment morphology. The presence of bab expression in all Drosophila species examined suggests that its roles in antagonizing the homeotic function of Abd-B and repressing pigmentation are ancestral. However, in the ancestral condition, bab expression was independent of Abd-B and dsx, resulting in sexually monomorphic pigmentation and segment morphology. In the melanogaster species group, bab evolved to be under the control of Abd-B and dsx. This eliminated bab from Abd-B-expressing segments in the male and resulted in a major transformation of male segment morphology. Subsequent diversification of pigmentation, bristle and trichome patterns was probably driven both by the fine-tuning of bab regulation and by changes in the downstream targets of bab and Abd-B (Kopp, 2000).

Two features of this genetic circuit make it highly plastic and evolvable: (1) the adult phenotype is sensitive to quantitative changes in bab expression; (2) the level of bab expression is determined by the balance between Abd-B and dsx^F inputs. If bab is regulated directly by Abd-B and dsx, then the evolution of sexually dimorphic pigmentation and segment morphology may ultimately be traced to the acquisition and modification of binding sites for the Abd-B and Dsx proteins in the cis-regulatory region of bab. Thus, even a subtle molecular change could be expressed phenotypically and become subject to selection (Kopp, 2000).

Doublesex controls the development of the Drosophila genital disc, modulating the response to Hedgehog, Wingless and Decapentaplegic signals

In both sexes, the Drosophila genital disc contains the female and male genital primordia. The sex determination gene doublesex controls which of these primordia will develop and which will be repressed. In females, the presence of Doublesex^F product results in the development of the female genital primordium and repression of the male primordium. In males, the presence of Doublesex^M product results in the development and repression of the male and female genital primordia, respectively. This report shows that Doublesex^F prevents the induction of decapentaplegic by Hedgehog in the repressed male primordium of female genital discs, whereas Doublesex^M blocks the Wingless pathway in the repressed female primordium of male genital discs. It is also shown that Doublesex^F is continuously required during female larval development to prevent activation of decapentaplegic in the repressed male primordium, and during pupation for female genital cytodifferentiation. In males, however, it seems that Doublesex^M is not continuously required during larval development for blocking the Wingless signaling pathway in the female genital primordium. Furthermore, Doublesex^M does not appear to be needed during pupation for male genital cytodifferentiation. Using dachshund as a gene target for Decapentaplegic and Wingless signals, it was also found that Doublesex^M and Doublesex^F both positively and negatively control the response to these signals in male and female genitalia, respectively. A model is presented for the dimorphic sexual development of the genital primordium in which both Doublesex^M and Doublesex^F products play positive and negative roles (Sanchez, 2001).

dpp is expressed in the growing male genital primordium of male genital discs but not in the repressed male primordium (RMP) of female genital discs. This suggests that the developing or repressed status of the male genital primordium is determined by the regulation of dpp expression. As dsx controls the developmental status of the male genital primordium, and the expression of dpp depends on the Hh signal, the relationship between the Hh signal cascade and dsx in the control of RMP development was examined. To this end, a twin clonal analysis for the loss-of-function tra2 mutation was performed in tra2/+ female genital discs. In this way, the proliferation and the induction of dpp expression was examined in the clones homozygous for tra2 (male genetic constitution) and that of the twin wild-type clones, both in the repressed male and the growing female primordia. Recall that the effects of tra2 in the genital disc are entirely mediated by its role in the splicing of DSX RNA: the presence or absence of functional Tra2 product gives rise to the production of female Dsx^F or male Dsx^M product, respectively. Clones for tra2 (expressing Dsx^M) induced in the RMP of female genital discs show overgrowth and are always associated with dpp expression, indicating that the lower proliferation shown by the RMP is probably caused by the absence of dpp expression. This activation of dpp is restricted to only certain parts of the clone and never overlaps with Wg expression. Since wg is normally expressed in the RMP, the possibility exists that the cells that do not express dpp in the clone are expressing wg, owing to their antagonistic interaction. Double staining of Wg and Dpp in tra2 clones reveals an expansion of the normal domain of wg expression that abuts the dpp-expressing cells (Sanchez, 2001).

In the RMP, the two sister clones are different in size: the tra2 clone (male genetic constitution) is bigger than the wild-type twin clone (female genetic constitution). In contrast, when the clones are induced in the growing female genital primordium, both of them are of a similar size. Moreover, the pattern of dpp expression does not change in the tra2 cells induced in this primordium (Sanchez, 2001).

optomotor-blind, a target of the Dpp pathway, also responds to Dpp in the genital disc. Since dpp is de-repressed in tra2 clones induced in the RMP, the activation of omb was monitored in these clones. The activation of dpp in tra2 clones induces the expression of this target gene, whose function is required for the development of specific male genital structures. It is concluded that Dsx^F product prevents the induction of Dpp by Hh in the repressed male genital primordium of female genital discs (Sanchez, 2001).

In the male genital disc, which has Dsx^M product, the low proliferation rate of the repressed female primordium (RFP) cannot be attributed to a lack of dpp or wg, since both genes are expressed in this primordium. Failure to respond to the Dpp signal may also be ruled out because the RFP expresses the Dpp downstream gene, omb, indicating that the Dpp pathway is active in this primordium. However, Dll, a target gene for both Wg and Dpp, is not expressed in the RFP but is expressed in the developing female primordium of female genital discs. This suggests that the Wg pathway cannot activate some of its targets in the RFP. Thus, the analysis of dsx¹ mutant genital discs, where both male and female genital primordia develop, becomes relevant. These mutant discs show neither Dsx^M nor Dsx^F products. The female genital primordium of these discs now expresses Dll. It is concluded that Dsx^M controls the response to the Wg pathway in the RFP of male genital discs (Sanchez, 2001).

The gene dachsund (dac) is also a target of the Hh pathway in the leg and antenna. In the present study, it was found that dac is differentially expressed in female and male genital discs. In the female genital discs, which have Dsx^F product, dac expression mostly coincides with that of wg in both the growing female primordium and the RMP. In contrast, in male genital discs, which have Dsx^M product, dac is not similarly expressed to wg but its expression partially overlaps that of dpp and no expression is observed in the RFP. In pkA minus clones, which autonomously activate Wg and Dpp signals in a complementary pattern, dac was ectopically expressed only in mutant pkA minus cells at or close to the normal dac expression domains in male and female genital discs. In pkA minus;dpp minus double clones, which express wg, dac is not ectopically induced in the male primordium of the male genital disc, but is still ectopically induced in both the growing female genital primordium and the RMP of female genital disc. Conversely, in pkA minus wg minus double clones, which express dpp, dac is not ectopically induced in the growing female or in the RMP of female genital discs, but is ectopically induced in the growing male primordium of the male genital disc. These results indicate that dac responds differently to Wg and Dpp signals in both sexes (Sanchez, 2001).

In dsx^Mas/+ intersexual genital discs, which have both Dsx^M and Dsx^F products, and in dsx¹ intersexual genital discs, which have neither Dsx^M nor Dsx^F products, dac is expressed in Wg and Dpp domains although at lower levels than in normal male and female genital discs. These results suggest that Dsx^M plays opposing, positive and negative roles in dac expression in male and female genital discs, respectively; and that Dsx^F plays opposing, positive and negative roles in dac expression in female and male genital discs, respectively. To test this hypothesis, tra2 clones (which express only Dsx^M ) were induced in female genital discs. The expression of dac is repressed in tra2 clones located in Wg territory. Therefore, Dsx^F positively regulates dac expression in the Wg domain, and Dsx^M negatively regulates dac expression in this domain, otherwise dac would be expressed in tra2 clones at the low levels found in dsx intersexual genital discs. However, when the tra2 clones are induced in the RMP, in the territory competent to activate dpp, they show ectopic expression of dac (Sanchez, 2001).

Therefore, Dsx^M positively regulates dac expression in the Dpp domain, whereas Dsx^F negatively regulates dac expression in this domain, since in normal female genital discs with Dsx^F dac is not expressed in Dpp territory. This is further supported by the induction of dac in the Wg domain and repression of dac in the Dpp domain by ectopic expression of Dsx^F in the male genital primordium of male genital discs. It is concluded that in male genital discs, Dsx^M positively and negatively regulates dac expression in Dpp and Wg domains, respectively; and in female genital discs, Dsx^F positively and negatively regulates dac expression in Wg and Dpp domains, respectively (Sanchez, 2001).

Homozygous tra2^ts larvae with two X-chromosomes develop into female or male adults if reared at 18°C or 29°C, respectively, because at 18°C they produce Dsx^F and at 29°C they produce Dsx^M. A shift in the temperature of the culture is accompanied by a change in the sexual pathway of tra2_ts larvae. Analysis of the growth of genital primordia and their capacity to differentiate adult structures of tra2_ts flies was performed using pulses between the male- and the female-determining temperatures in both directions during development (Sanchez, 2001).

Regardless of the stage in development at which the female-determining temperature pulse was given (transitory presence of functional Tra2_ts product; i.e. transitory presence of Dsx^F product and absence of Dsx^M product), the male genital disc develops normal male adult genital structures and not female ones. This occurs even if the pulse is applied during pupation. Pulses of 24 hours at the male-determining temperature (temporal absence of functional Tra2 ts product; i.e. transitory absence of Dsx^F product and presence of Dsx^M product) before the end of first larval stage produces female and not male genital structures. However, later pulses always give rise to male genital structures, except when close to pupation. Further, the capacity of the female genital disc to differentiate adult genital structures is also reduced when the temperature pulse is applied during metamorphosis (Sanchez, 2001).

When the effect of the male-determining temperature pulses was analyzed in the genital disc, it was found that overgrowth of the RMP is always associated with the activation of dpp in this primordium. However, this activation and the associated overgrowth only occurs when the temperature pulse is given after the end of first larval instar. This suggests that there is a time requirement for induction of dpp (Sanchez, 2001).

The activation of this gene in the RMP and the cell proliferation resumed by this primordium, as well as its capacity to differentiate adult structures is irreversible, because they are maintained when the larvae are returned to the female-determining temperature, which is when functional Tra2^ts product is again available (i.e. the presence of Dsx^F product and absence of Dsx^M product). This time requirement for induction of dpp is also supported by the fact that dsx¹¹ clones (which lack Dsx^M) induce differentiated normal male adult genital structures in the developing male genital primordium of XY; dsx¹¹/+ male genital discs (which express only Dsx^M ) after 24 hours of development. However, when the dsx¹¹ clones are induced in the time period between 0 and 24 hours of development, they do not differentiate normally and give rise to incomplete adult male genital structures. This different developmental capacity shown by the dsx¹¹ clones depending on their induction time is explained as follows. When the clones are induced after 24 hours of development, dpp is already activated. Indeed, these clones show no change in the expression pattern of dpp or their targets. Accordingly, these clones display normal proliferation and capacity to differentiate male adult genital structures. However, when the clones are induced early in development, dpp is not yet activated, since this gene is not expressed in the male genital primordium of male genital discs early in development. Therefore, when the male genital disc reaches the state in development when dpp is induced, the cells that form the clones activate this gene as in dsx mutant intersexual flies because the clones have neither Dsx^M nor Dsx^F products. Consequently, these clones do not achieve a normal proliferation rate, and then do not differentiate normal adult male genital structures (Sanchez, 2001).

As described above, it has been shown that dsx regulates the expression of gene dac. Recall that in male genital discs, Dsx^M positively and negatively regulates dac expression in Dpp and Wg domains, respectively; and in female genital discs, Dsx^F positively and negatively regulates dac expression in Wg and Dpp domains, respectively. The expression of the gene dac was analyzed in genital discs of tra2^ts flies using pulses between the male- and the female-determining temperatures in both directions. It was found that the dac expression pattern switches from a 'female type' to a 'male type' when male-determining temperature pulses were applied to tra2^ts larvae after first larval instar. Note that dac expression is reduced in the Wg domain of the RMP and is progressively activated in the Dpp domain. It should be remembered that these pulses lead to the transient presence of Dsx^M instead of Dsx^F product. Thus, these results are consistent with the previously proposed suggestion that Dsx^M activates dac in the Dpp domain and represses it in the Wg domain (again the converse is true for Dsx^F). When the pulse is given during first larval instar, dac is not activated in the Dpp domain of RMP, in spite of the fact that there is also a transient presence of Dsx^M instead of Dsx^F. This is explained by the lack of competence of cells to express Dpp, which is acquired after first larval instar. When the tra2^ts larvae reach such a developmental stage, these cells now produce Dsx^F because they have returned to the female-determining temperature (Sanchez, 2001).

Dsx^F prevents activation of dpp in the RMP, and consequently no induction of dac expression occurs. In the female genital primordium, dac expression is strongly reduced in the Wg domain and absent in the Dpp domain. Taken together, these results suggest that the development of male and female genital primordia have different time requirements for Dsx^M and Dsx^F products (Sanchez, 2001).

dsx controls which of the two genital primordia will develop and which will be repressed. Nevertheless, since it is expressed in each cell, another gene(s) is required to distinguish between the female and the male genitalia. The female genitalia develop from eighth abdominal segment and the male genitalia develop from ninth abdominal segment. It is also known that Abdominal-B (Abd-B) is responsible for the specification of these posterior segments. It has been proposed that the development of the male and female genitalia requires the concerted action of Abd-B and dsx, and that these two genes control proliferation of each genital primordium through the expression, either directly or indirectly, of dpp and wg. Abd-B produces two different proteins: Abd-Bm and Abd-Br. Abd-Bm is present only in the female genital primordium, whereas Abd-Br is present only in the male genital primordium. It is proposed that Dsx^M and Dsx^F combine with Abd-Bm and Abd-Br to make up the signals that determine the dimorphic sexual development of the genital disc. In the absence of both Dsx^M and Dsx^F products (dsx intersexes), there is a basal expression of dpp and a basal functional level of the Wg signaling pathway in both male and female genital primordia. In females, the concerted signal made up of Dsx^F and Abd-Br cause repression of the development of the male genital primordium by preventing the expression of dpp, resulting in the RMP of female genital discs. In males, the concerted signal formed by Dsx^M and Abd-Bm represses the female genital primordium by blocking the Wg signaling pathway, giving rise to the RFP of male genital discs. It is further proposed that Dsx^M plus Abd-Br increase dpp expression in the male genital primordium of male genital discs, and that Dsx^F plus Abd-Bm enhance Wg signaling pathway function in the female genital primordium of female genital discs. A similar mechanism of modulation of Dpp and Wg responses has been described for the shaping of haltere development by Ultrabithorax. Therefore, Dsx^M would play a positive and a negative role in male and female genital primordia, respectively, whereas Dsx^F would play a positive and a negative role in female and male genital primordia, respectively. This positive role of both Dsx products serves to explain the expression of dpp and the function of the Wg signaling pathway in growing male and female genital primordia, respectively, in dsx ^Mas/+ intersexual flies, where both genital primordia simultaneously have Dsx^M and Dsx^F. Otherwise, dpp would not be expressed in the male genital primordium and the Wg signaling pathway would not be functional in the female genital primordium, as occurs in normal female and male genital discs. If so, this would mean that the two genital primordia of these intersexual genital discs would be kept in the repressed state and would not develop. Contrary to observations, this would result in a lack of male and female adult genital structures in these intersexes (Sanchez, 2001).

It has been shown that homothorax and extradenticle genes are involved in the control of the response to Dpp and Wg signals in the proximal part of the leg. Since these genes are strongly expressed in the repressed male and female primordia of the genital disc, it is proposed here that these two genes may form part of the integrated mechanism comprised by Dsx and Abd-B products for the regulation of the morphogenetic signaling response. During the evolution of the Diptera there has been a tendency towards the fusion of the posterior segments into a single imaginal disc. In primitive Diptera, such as Tipulidae, males and females still produce an eighth tergite and ninth tergite, respectively. Insects such as Musca and Calliphora, which are considered to represent an intermediate evolutionary step between Tipulidae and Drosophila, have two laterals and one single median genital disc. The anlage of the lateral discs corresponds to segment eight and the anlage of the single median disc to the fusion of segments 9 to 11. In females, the lateral discs form the female genitalia, except the parovaria. The median disc develops the parovaria (ninth segment) and the female analia (segments 10-11). In males, the lateral discs produce a reduced eighth tergite. The median disc develops the male genitalia (ninth segment) and the male analia (segments 10-11). A further level of fusion occurred in the Drosophila lineage, where segments 8 to 11 form a single genital disc. The model proposed here for the development of the genital disc of Drosophila can be applied to the above primitive dipteran species (Sanchez, 2001).

In vertebrates, Dmrt1, the dsx homolog, has been implicated in male gonad development and murine Dmrt1 seems to be required for multiple aspects of testis differentiation. This functional similarity could imply a close evolutionary relationship between Dmrt1 and the Drosophila dsx gene. In the same evolutionary context, it has been reported that, in mammals, the signaling molecule Wnt4, one of the mammalian homologs of the Drosophila Wingless gene family, is crucial for female sexual development. Although the relationship between sex determination genes and morphogenetic signals has not been found in mammals yet, the findings reported here suggest the possibility that similar signals might be used across species for implementation of sex differentiation (Sanchez, 2001 and references therein).

Each Drosophila genital imaginal disc contains primordia for both male and female genitalia and analia. The sexually dimorphic development of this disc is governed by the sex-specific expression of doublesex. Data is presented that substantially revises understanding of how dsx controls growth and differentiation in the genital disc. The classical view of genital disc development is that in each sex, dsx autonomously 'represses' the development of the inappropriate genital primordium while allowing the development of the appropriate primordium. Instead, dsx is shown to regulate the A/P organizer to control growth of each genital primordium, and then dsx directs each genital primordium to differentiate defined adult structures in both sexes (Keisman, 2001b).

Recent findings concerning the growth of clones of genital disc cells whose sex was altered genetically suggest that the growth of each genital primordium is controlled by the sex of a subset of its cells. Such clones were expected to develop according to their genetic sex, because sex determination is cell autonomous. For instance, female clones in the male primordium should adopt the 'repressed' state characteristic of that primordium in females. Consistent with this prediction, female clones cannot contribute normally to adult male genital structures. However, such clones frequently grow substantially and contribute to a morphologically normal male genital primordium in the larval genital disc, suggesting that growth and the capacity to differentiate are under separate control. Yet occasional female clones in the male primordium are associated with severe reductions in the size of the corresponding genital primordium in the disc. That some clones in the male primordium disrupt growth while others do not led to a proposal that growth in the genital primordia is controlled nonautonomously from within an unidentified organizing region. Clones that grow normally would lie outside of this organizing region, while those that cause reductions would intersect it. An obvious candidate for this organizing region is the strip of anterior compartment cells along the A/P border that express wg and dpp, which is referred to as the A/P organizer (Keisman, 2001b).

Therefore, it was hypothesized that the sex of the A/P organizer region nonautonomously controls the sex-specific patterns of proliferation in the genital disc. To test this hypothesis, advantage was taken of the fact that the A/P organizer coincides with high levels of expression of the patched (ptc) gene, while the posterior compartment is defined by engrailed (en) expression. Thus, gene expression can be targeted to these regions using ptc-GAL4 and en-GAL4 drivers, respectively. Chromosomally male cells were feminized by expressing a female tra cDNA, while chromosomally female cells were masculinized by expression of a tra-2 inverted repeat construct (tra2IR) that blocks the function of tra-2 through the mechanism of dsRNA-mediated interference. If the hypothesis is correct, changing the sex of cells in the A/P organizer region would cause each primordium to develop as it does in the corresponding sex. Conversely, changing the sex of the posterior compartment cells should have no effect on genital disc morphology (Keisman, 2001b).

When cells of the A/P organizers in chromosomally male genital discs are feminized, a radical change in the morphology of both the male and female genital primordia is observed. The chromosomally male genital discs resemble female genital discs: the female primordium grows to dominate the disc epithelium, while the male primordium is substantially reduced. Feminization of the posterior compartment of chromosomally male genital discs, in contrast, has no discernable effect on disc morphology. As expected, the morphology of chromosomally female genital discs is unaffected by the expression of tra. The transformation produced by ptc-GAL4-driven tra expression in XY animals is not perfect, as the female primordium overgrows and is thrown into folds. Occasionally, these discs have male primordia with vestiges of male morphology. This pattern of growth is usually only on one side of the disc, and it is attributed to variability in tra expression produced by the ptc-GAL4 driver. To confirm that the intended transformation had been produced, the adult phenotypes of the feminized flies were examined. The expected correlation exists between the domain of tra expression and the affected elements of the male and female adult structures (Keisman, 2001b).

The reciprocal transformation, masculinization of the A/P organizer cells in a chromosomally female disc, also produces a striking transformation of disc morphology. Many of these discs are morphologically indistinguishable from those of their male siblings. The male primordium is wild-type or near wild-type in size, while the female primordium is reduced in size. This transformation is not completely penetrant. While the majority of the chromosomally female discs (11/17) had predominantly or completely male morphology, there were a few discs in which the female primordia grew slightly. Nevertheless, for a significant fraction of the masculinized female discs, it would have been impossible to determine their chromosomal sex without anti-Sxl staining to identify them. When the posterior compartment of female discs is masculinized, there are only minor changes in the morphology of these discs. The female primordium overgrows slightly, deepening a normally shallow groove that runs between its left and right halves and occasionally causing extra folds. The male primordium of these discs is also slightly thickened. Taken together, these experiments demonstrate that the primary determinant of disc growth and morphology is the sex of the cells of the A/P organizer, although the sex of other cells makes a minor contribution to morphology (Keisman, 2001b).

Tracing the fate of the male primordium in the female genital disc has revealed that its cells persist throughout metamorphosis and give rise to the parovaria, the internal female accessory glands. The male primordium of the female disc was tracked during metamorphosis by following the expression of reporter genes that reveal the arrangement of the three primordia in the disc. The parovaria bud forms from the female genital disc in the first 12 hr of metamorphosis, during which there is a radical rearrangement of the epithelium's geometry. The major element of this rearrangement is an elongation of the disc along the A/P axis. This elongation is driven by an apparent convergent extension, most pronounced in the thickened ventral epithelium. This convergent extension drives the primordia of the spermathecae, which originate ventrally in the female primordium, onto the dorsal side of the disc. Cells on the lateral edges of the disc are also driven dorsally and medially. Almost immediately after this rearrangement, the emerging parovaria become evident just posterior to the emerging spermathecae. By 12 hr after puparium formation (hAPF), the protrusion of the parovaria and spermathecae becomes more pronounced and the identification of these structures can be made unequivocally (Keisman, 2001b).

That the parovaria arise from the male genital primordium can be seen by following the expression patterns of wg and en. In the third instar female genital disc, wg is expressed in a thin band of cells in the male primordium just anterior to the en-expressing domain. These two domains of gene expression define the male primordium. During the first 4 hr of metamorphosis, the en and wg bands from the male primordium are joined on the dorsal surface by additional, more anterior bands of en and wg that derive from the ventral female primordium and are driven dorsally by the convergent extension of the disc. At 4, 8, and 12 hAPF, it is evident that the parovaria are emerging from within the domain of en expression that, at third instar, defines the posterior compartment of the male primordium (Keisman, 2001b).

Previous cell lineage analysis and gynandromorph fate mapping studies assigned the parovaria to the anal (A10) primordium. Although the anal primordium is physically distant from where the parovaria originate, the data were corroborated by tracking the anal primordium during metamorphosis. Since the anal primordium (A10) is defined by the expression of caudal (cad), a GAL4 enhancer trap insertion in cad was used to drive expression of GFP in the anal primordium and this expression was followed in the female genital disc during the first 12 hr of metamorphosis. In the third instar female disc, cad expression extends from the posterior edge of the disc anteriorly, approximately two-thirds of the way across the disc. This anterior border correlates with the posterior edge of the male primordium as defined by en expression. It is clear that the parovaria bud from a region of the disc well anterior to the domain of cad expression. Thus, the parovaria do not derive from the anal primordium (Keisman, 2001b).

Tracing the cells of the female primordium in male genital discs shows that these cells persist throughout metamorphosis and produce a miniature eighth tergite at the anterior edge of the male genital arch. The topology of the three primordia in the male genital disc epithelium is similar to that in the female. However, the morphogenesis of the male genitalia is substantially more complex than that of the female, and determining the fate of the female primordium requires following its metamorphosis until 48 hAPF (Keisman, 2001b).

The posterior compartment of the female primordium in males corresponds to the long patch of en expression at the posterior edge of the disc. During metamorphosis, the male genital disc opens at its posterior edge and turns partially inside-out to expose the apical surface of the genital disc. If the disc is viewed from the posterior, the female primordium is at the leading edge of the ventral 'lip' when the disc everts. The en domain is toward the back of this lip, preceded by the anterior compartment of the female primordium. Following this group of cells until 24 hAPF reveals that it persists and proceeds to completely encircle the differentiating male genitalia. Importantly, this band can be distinguished from the thick band of en expression in the male genital arch, which corresponds to segment A9. Intermediate time points (at 8, 30, and 36 hr) were used to confirm that these cells are continually present and not lost and then replaced by other cells. By 48 hr the A8 en band can be seen as a tight collar that rings the male genitalia. This band is easily distinguished from the A6 band of en expression and persists in later pupae. This band is also present in the adult, where it labels the anterior rim of the genital arch. The border of the A8 en band in the adult correlates roughly with a seam in the anterior cuticle of the genital arch; it is concluded that the region of the genital arch anterior to this seam is a vestigial male eighth tergite (T8) (Keisman, 2001b).

The analysis was complicated by the presence of en expression in the larval epidermal cuticle (LEC), which persists until it is replaced by the expanding histoblast nests. The male genital disc integrates into the LEC as it everts, making it necessary to confirm that the en expression, which is inferred to derive from the female primordium, is indeed of imaginal origin. Advantage was taken of a GAL4-expressing enhancer trap insertion in escargot (esg) was used to confirm the identity of these cells, since esg is expressed in imaginal cells but not in the larval cuticle. esg is expressed strongly in a thick epithelial mantle just ventral to the male genitalia. Comparison with the expression of en in a separate 24 hr male genitalia shows that the band of en expression that defines the female primordium is well within this same mantle of cells. The imaginal origin of the A8 en band is also supported by the size of the nuclei in these cells: the LEC has large polyploid nuclei, while the imaginal nuclei of the presumptive female primordium at 24 hAPF are diploid and much smaller. At 24 hr the expanding diploid histoblast nests have only partially completed the replacement of the LEC. As a result, bands of en in the LEC consist of a mix of small diploid nuclei and large polyploid nuclei. In contrast, the entire circumference of the en ring in the presumptive female primordium consists of small diploid nuclei. The simplest interpretation of this observation is that this ring of en-expressing cells derives from the diploid genital disc and identifies the female primordium (Keisman, 2001b).

There appears to be expression of GFP in the polyploid cells of the LEC, casting doubt on the reliability of the esg-GAL4 as an imaginal marker at this stage. However, these animals do not express GFP in the LEC at larval stages. Moreover, many enhancer traps become ubiquitously activated in the LEC after 10-12 hr APF. Even though there is some GFP expression in the LEC, the intensity of GFP expression in the everting genitalia is stronger than in the surrounding cells. In whole mounts of esg-GAL4/UAS-GFP abdomens, the genitalia stand out dramatically and there is a perceptible change in the intensity of GFP expression that correlates with where the thick epithelial mantle meets a much thinner epithelium. It is inferred that this mantle is the female primordium, based on its location, the relative intensity of esg-driven GFP expression, and its contiguity with the male genitalia (Keisman, 2001b).

Because the sex determination pathway acts cell autonomously to determine sex, the reduced growth in the 'repressed' primordium has long been thought to reflect a cell autonomously regulated quiescent state. However, the results show that the major factor controlling the growth of the genital primordia is the sex of the cells at the A/P border, not the sex of individual cells. When the cells of the A/P organizer are feminized in a male disc or masculinized in a female disc, both genital primordia respond by switching to growth patterns that reflect the sex of the cells at the organizer. When the sex of posterior compartment cells is genetically altered, there is no major change in disc morphology. It is inferred that these posterior compartment cells continue to grow normally under the influence of the unaffected A/P organizer (Keisman, 2001b).

It is thought that the primary activity of the sex determination hierarchy in the A/P organizer is to regulate wg and dpp signaling. It has been suggested that cell growth in the genital disc is controlled by dsx acting either directly or indirectly through the expression of dpp and wg. In the genital disc, wg and dpp are expressed along the A/P border in the same cells that express the ptc-GAL4 driver and the activity of wg and dpp is the primary determinant of disc size and shape in the thoracic imaginal discs, and the reduced male primordium of a female genital disc does not express dpp. However, the female primordium expresses wg and dpp in both sexes yet grows to different sizes and shapes in each. Thus, it remained a distinct possibility that this difference in growth was attributable to the response of individual cells to wg and dpp. The current results argue otherwise, suggesting that the sex determination pathway produces different patterns of growth by regulating the absolute levels and/or timing of wg and dpp expression (Keisman, 2001b).

The results also suggest that while the A/P organizer is the primary determinant of growth in the two genital primordia, the sex of other cells is not completely irrelevant. ptc-GAL4 driven feminization of the A/P organizer in chromosomally male discs is not perfect, as the female primordia of these discs overgrow and are thrown into folds. Masculinization of the posterior compartment in chromosomally female discs also cause slight overgrowth and subtle alterations in the morphology of the female primordia. The most important nontrivial possibility raised by these results is that the shape that the female primordium adopts remains partially dependent on the sex of its constituent cells (Keisman, 2001b).

The results add to evidence indicating that dsx plays an active role in directing the differentiation of the genital primordia and that dsx acts instructively at multiple steps during development to direct sex-specific differentiation. Specifically, the control of growth and differentiation by dsx are separable processes: dsx controls growth primarily by regulating the activity of the A/P organizer, while differentiation is controlled by dsx cell autonomously (Keisman, 2001b).

The control of growth and the establishment of pattern in imaginal discs are mediated by the same molecules, the morphogens encoded by wg and dpp. This conservation implies that in directing the correct sex-specific differentiation of a given genital primordium, dsx acts on wg and dpp signaling twice: at the A/P organizer, dsx acts to direct the correct patterns of growth via wg and dpp expression; dsx must then act again in individual cells, probably throughout the disc, to direct the correct sex-specific interpretation of the positional identities specified by wg and dpp. This prediction is borne out by recent findings that the expression of individual genes in the genital primordia is under the cell-autonomous control of dsx. For instance, dsx determines whether cells in the male (A9) primordium will express dachshund in response to wg, as in female discs, or in response to dpp, as in male discs (Keisman, 2001b).

Since the homeotic genes specify the identity of segments A8 and A9, they must provide the context for the differential action of dsx on the two genital primordia, both at the A/P organizer (to regulate growth) and in individual cells (to control differentiation). The segmental identities of A8 and A9 are specified by the homeotic genes abd-A and the two genetically distinct functions of the Abd-B gene, Abd-BI, and Abd-BII. The exact division of labor in this respect is not clear, but most evidence suggests that abd-A and Abd-BI specify different parts of segment A8, while Abd-BII specifies segment A9. Removal of Abd-B from the genital disc causes it to switch to a leg-like mode of differentiation in which, for instance, the expression of dac reverts to a broader domain of expression. Thus, sex-specific dac expression requires not only dsx, but also Abd-B, confirming that differentiation in the genital disc requires the collaboration of these two types of genetic inputs. It is proposed that the sex-specific growth and differentiation of A8 and A9 are specified jointly by the homeotic genes and the sex-specific functions of dsx (Keisman, 2001b).

Doublesex represses branchless in the in female genital discs: Branchless positive cells recruit mesodermal cell into the male genital imaginal disc

A central issue in developmental biology is how the deployment of generic signaling proteins produces diverse specific outcomes. Drosophila FGF is used, only in males, to recruit mesodermal cells expressing the FGF receptor to become part of the genital imaginal disc. Male-specific deployment of FGF signaling is controlled by the sex determination regulatory gene doublesex. The recruited mesodermal cells become epithelial and differentiate into parts of the internal genitalia. These results provide exceptions to two basic tenets of imaginal disc biology -- that imaginal disc cells are derived from the embryonic ectoderm and that they belong to either an anterior or posterior compartment. The recruited mesodermal cells migrate into the disc late in development and are neither anterior nor posterior (Ahmad, 2002).

The extensive sexual dimorphisms of the genitalia and analia suggest that the genital disc is relatively enriched in genes expressed downstream of dsx. To identify such genes, a random collection of enhancer traps was screened for sex-specific expression patterns in late third instar genital discs. Enhancer trap insertions in the bnl and btl genes were both isolated as enhancer traps expressed in male but not female genital discs. The sex specificity and the spatial patterns of expression of these enhancer traps accurately reflect the expression of the bnl and btl genes in the genital disc. Of the three primordia that comprise the genital disc, bnl and btl are both expressed in only one: the A9-derived developing 'male' primordium. bnl and btl are also expressed in adjacent domains: bnl is expressed at the base of two bilateral bowl-like infoldings of the disc epithelium, while btl is expressed in a group of loosely packed cells that fills these bowls and extends over the anterior and ventral surfaces of the disc (Ahmad, 2002).

The juxtaposition of btl- and bnl-expressing cells suggested that their proximity to one another might be the result of FGF-mediated cell-cell signaling. The locations of btl-expressing cells in male genital discs were determined at different stages of larval development. At early third instar (70-75 hr after egg laying), while a few btl-expressing cells are associated with the external surface of the disc, none are detected inside the disc. In mid-third instar (89-99 hr AEL), the btl-expressing cells are lying on the external surface of the disc, as well as adjacent to, and filling shallow invaginations in the disc epithelium. And by late third instar (110-120 hr AEL), these invaginations have become much deeper and are completely filled by btl-expressing cells. Thus, these btl-expressing cells are not originally a part of the disc epithelium but are recruited into invaginations in the epithelium during the third instar. Unlike the disc epithelium, the btl-expressing cells in the third instar disc do not express escargot (esg), a classical marker for ectoderm-derived imaginal cells, indicating that the btl-expressing cells have a different origin than do the other cells of the disc. The btl-expressing cells are, in fact, mesodermal in origin and derived from the adepithelial cells associated with the genital disc (Ahmad, 2002).

A priori, there are two possible explanations for the male-specific expression of FGF. One possibility is that bnl is an A9-specific gene, being expressed only in males where the A9-derived primordium grows significantly. The other possibility is that bnl is a target of the sex determination hierarchy, being either repressed by the female-specific Dsx protein (Dsx^F) in females and/or activated by the male-specific Dsx protein (Dsx^M) in males. To distinguish between these possibilities, feminized (Tra protein-expressing) clones of cells were generated in the A9-derived primordium of wild-type male genital discs and the effects of these clones on bnl expression were examined. Whenever feminized clones overlapped domains of bnl expression, the expression of bnl was repressed, indicating that it is cell-autonomous regulation by the sex determination hierarchy that is responsible for the male-specific expression of bnl in the genital disc (Ahmad, 2002).

When a feminized clone completely eliminated bnl expression from one side of a male disc, the lobe lacking bnl expression looked flattened. This was a consequence of btl-expressing cells not migrating into this lobe in the absence of Bnl protein, showing that bnl expression is not simply sufficient, but also necessary for the recruitment of btl-expressing cells. This observation suggests that btl, unlike bnl, is not a target of the sex determination hierarchy, and that the male-specific presence of btl-expressing cells in the genital disc is solely a consequence of Bnl recruiting the btl-expressing cells (Ahmad, 2002).

To examine how dsx regulates bnl expression, bnl expression was examined in wild-type genital discs and discs lacking dsx function. bnl is expressed in the A9-derived primordium of a wild-type male disc, where Dsx^M is present, but is not expressed in the A8-derived primordium of a wild-type female disc, where Dsx^F is expressed. However, in a disc in which neither Dsx protein is expressed, both the A8 and A9 primordia proliferate and bnl expression is seen in both primordia. That the A8 primordium grows in both wild-type and dsx mutant females but bnl is expressed in the A8 primordium only when the Dsx^F protein is absent, implies that bnl expression is repressed in the female genital disc by the presence of Dsx^F protein (Ahmad, 2002).

The ectopic expression of bnl in the A8-derived 'female' primordia of discs lacking dsx function offers an explanation for a puzzling observation: while wild-type males have only two paragonia (mesodermally derived components of the male disc), dsx mutant flies often have as many as four paragonia-like structures. The finding that the ectopic expression of bnl in flies mutant for dsx results in btl-expressing cells from the ventral surface of the disc being recruited into two ectopic invaginating pockets in the A8-derived female primordium of the disc, in addition to the original bowls in the A9-derived primordium, suggests that these ectopic pockets of btl-expressing cells give rise to the supernumerary paragonia when taken together with the observation that the extra paragonia in dsx mutants arise from the female primordium (Ahmad, 2002).

It is concluded that the sex-specific deployment bnl in the genital disc depends on the sex of the individual bnl-expressing cells. Given that bnl is regulated cell autonomously by Dsx^F, an obvious question is whether the Dsx^F protein directly represses bnl. In this regard, it is noted that 0.7 kb and 1.6 kb upstream of the putative bnl transcriptional start site, there are clusters of 5 and 4 sites respectively with at most a 1 bp mismatch to the 13 bp consensus Dsx binding site sequence. This is reminiscent of the 3 Dsx binding sites in a 76 bp stretch of an enhancer for the Yolk protein (Yp) genes, the only known direct targets of dsx (Ahmad, 2002).

The Drosophila sex determination hierarchy acts at multiple levels to control sexual differentiation. Some terminal differentiation genes like the Yp genes are direct transcriptional targets of the Dsx proteins and are continuously subject to their regulation. In other cases, the direct targets of dsx appear to be genes involved in initiating the differentiation of sex-specific tissues; genes expressed subsequently in these sex-specific tissues are governed by a tissue differentiation program, rather than being directly controlled by the sex hierarchy. It seems likely that the targets through which dsx initiates formation of such sex-specific tissues will be the genes where information from several developmental hierarchies are integrated to direct the differentiation of tissues (Ahmad, 2002).

These results suggest that bnl is one of the genes used by the sex determination hierarchy to direct the construction of sex-specific tissues. Bnl recruits btl-expressing cells into the male genital disc, and the recruited cells eventually form the paragonia and vas deferens (another mesodermally derived gonadal organ), tissues that are present only in males. Moreover, three genes expressed in the paragonia, the male-specific transcripts (msts) 316, 355a, and 355b, have been shown to be regulated in a tissue-specific rather than sex-specific manner: while transcription of these three male-specific RNAs begins in the late pupal period, their expression is governed by the sex hierarchy acting earlier, during the third larval instar -- the period when the expression of bnl recruits the paragonia-forming btl-expressing cells into the male genital disc. Thus, the sex-specific expression of the msts is achieved by dsx acting through bnl to generate the sex-specific tissue, the paragonia, in which the msts are subsequently expressed.

bnl also appears to be a gene where information from other regulatory hierarchies and the sex determination hierarchy are integrated in the male genital disc. The genetic hierarchies that control pattern formation and confer positional identity in the thoracic imaginal discs have previously been shown to function analogously in the genital disc. The fact that the bnl expression domain is limited to two specific subsets of the ectoderm-derived disc epithelia in males implies that bnl is also regulated by these pattern formation hierarchies. One area of future exploration will be examining how this coordinated regulation of bnl by dsx and the genes involved in pattern formation is brought about (Ahmad, 2002).

An intriguing aspect of these findings is the gradual transition of the btl-expressing cells, upon recruitment into the male genital disc, from twi-expressing mesodermal cells to epithelial cells with septate junctions. It is not clear if this transformation is also a consequence of FGF signaling, or if it is brought about by a different process. However, three separate observations suggest a role for bnl and btl in this mesoderm-epithelial transition: (1) FGF signaling mediates this process in mice -- during kidney development, FGF2 and leukemia inhibiting factor (LIF) secreted from the epithelial ureteric bud induce the conversion of the undifferentiated mesoderm-derived metanephric mesenchyme to the epithelial tubular structures of the nephron; (2) the converse process can also be mediated by FGF signaling -- FGFR1 regulates the morphogenetic movement and cell fate specification events during gastrulation in mice; it orchestrates the epithelial to mesenchymal transition during morphogenesis at the primitive streak and specifies the mesodermal cell fate of these mesenchymal cells, and (3) stumps, a gene acting downstream of the FGFR-encoding btl, has its expression elevated in the btl-expressing cells undergoing the transition into epithelial cells in the genital disc (Ahmad, 2002 and references therein).

Finally, it is noted that there are striking parallels between the roles of the FGF in sexual differentiation in the fly and FGF9 in sexual differentiation in mice. FGF9 is required for testicular embryogenesis in mice, and in its absence, XY mice undergo male-to-female sex reversal. FGF9 is expressed in the early embryonic gonads of male mice, not in the gonads of female mice, and not in the mesonephros of either sex, while bnl is expressed in the male genital disc, not in the female genital disc, and not in the btl-expressing mesodermal cells that are recruited into the male disc. The mesonephric cells migrate into only the male gonads, and the btl-expressing cells are recruited only into the male genital disc. Exogenous FGF9 induces mesonephric cell migration into female gonads, while ectopic expression of bnl is sufficient to recruit the btl-expressing cells into the female primordium of a dsx disc. The btl-expressing cells are mesodermal in origin, eventually undergo a transition into epithelial cells, and give rise to the vascular paragonia and vas deferens. The mesonephros, too, is derived from the mesoderm, and mesonephric cell migration into the testis contributes to the vascular endothelial, myoepithelial, and peritubular myoid cell populations. Given that there is considerable variation in the earlier aspects of sex determination across species, these findings suggest a possible conserved role for FGF signaling in later aspects of sexual differentiation (Ahmad, 2002 and references therein).

Genes expressed in the Drosophila head reveal a role for fat cells in sex-specific physiology

The downstream effectors of the Drosophila sex determination cascade are mostly unknown and thought to mediate all aspects of sexual differentiation, physiology and behavior. Serial analysis of gene expression (SAGE) has been used to identify male and female effectors expressed in the head; 46 sex-biased genes (>4-fold/P < 0.01) are reported. 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 (tra) and its downstream target doublesex (dsx). 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).

None of the four genes is expressed at significant levels in the brain. Instead, tsx, sxe1, sxe2 and fit are expressed mainly in the fat cells of the head, which suggests that these genes create different physiological conditions in the adult male and female head important for sex-specific functions. It is proposed that fit, sxe1 and sxe2, which are expressed mainly in the head, may exert their functions on the brain. Similar to the pituitary in the head of mammals, the fat cells could play the role of an endocrine organ. Such a function has been shown for the fat cells of females, where the YPs are synthesized and released into the hemolymph (Fujii, 2002).

Based on the predicted protein sequences, sex-specific roles can be envisaged for sxe1 and tsx. tsx encodes a member of the opbp gene family. Odorant/pheromone binding proteins (OPBPs) are expressed generally in support cells of chemosensory sensilla and secreted into the extracellular lymph space, where they interact with odor and taste ligands to increase their solubility, protect them from degradation or remove them from the lymph space. It is suggested that TSX has been co-opted for a role to interact with and transport small molecules in the head. Upon release from the fat cells, TSX bound to a ligand may reach a target organ, for example the brain, to exert its physiological effects. The reduced mating activity of females ectopically expressing TSX is consistent with such a role. In addition, a precedent for a protein related to Drosophila OPBPs with a putative function unrelated to chemosensation has been reported in rats (Fujii, 2002).

The second gene for which a function is proposed, sxe1, encodes a cytochrome P450 protein (CYP); members of this family have been studied extensively in mammals and insects. One major role of these enzymes is liver detoxification, whereby toxic, water-insoluble metabolites are rendered sufficiently water soluble to be excreted in the urine. A second important function for CYPs is their role in steroid hormone metabolism, in both mammals and insects. Of particular interest in this regard is cytochrome P450arom (CYP19), which has been widely implicated in sex-specific functions in vertebrates. In insects, CYPs are involved in ecdysone metabolism, specifically in hydroxylation of cholesterol precursors. Disembodied (dib), the only studied Cyp gene in Drosophila, is involved in ecdysone metabolism during embryogenesis. The expression of sxe1 in the fat cells of the male head suggests the intriguing possibility that small molecules (e.g. steroid hormones) might be synthesized in a sex-specific fashion. Released into the circulatory system, they could reach any organ in the adult male fly, including the brain, and hence mediate sex-specific physiological states that could affect behaviors. One target of such a male-specific hormone might be the neurons in the brain expressing DSF, an orphan nuclear hormone receptor, which controls different male- and female-specific behaviors in adult flies (Fujii, 2002).

Hephaestus functions specifically in the male germline downstream of Doublesex

The mammalian polypyrimidine-tract binding protein (PTB), which is a heterogeneous ribonucleoprotein, is ubiquitously expressed. Unexpectedly, in Drosophila, the abundant transcript of hephaestus, referred to as dmPTB in this publication, is present only in males (third instar larval, pupal and adult stages) and in adult flies is restricted to the germline. Most importantly, a signal from the somatic sex-determination pathway that is dependent on the male-specific isoform of the doublesex protein (DSXM) regulates PTB, providing evidence for the necessity of soma -- germline communication in the differentiation of the male germline. Analysis of a P-element insertion directly links PTB function with male fertility. Specifically, loss of Drosophila PTB affects spermatid differentiation, resulting in the accumulation of cysts with elongated spermatids without producing fully separated motile sperms. This male-specific expression of PTB is conserved in D. virilis. Thus, PTB appears to be a particularly potent downstream target of the sex-determination pathway in the male germline, since it can regulate multiple mRNAs (Robida, 2003).

To analyze PTB function in vivo and complement studies with the vertebrate PTB, the Drosophila PTB was studied. Unexpectedly, dmPTB is expressed in adult males but not females, as determined by Northern analysis using the full-length cDNA probe. Since prior studies have not suggested that PTB has a sex-specific function or regulation, it remained possible that the abundant band results from cross-hybridization via an RRM, a common highly conserved RNA-binding domain. To exclude this possibility, several probes were prepared corresponding to divergent portions of the gene such as the 5' and 3' untranslated regions (5' and 3' UTRs) and the variable linker region between RRMs (inter-RRM). Each of the probes shows an identical male-specific signal. Consistent with this finding, BLAST results show that there is only one sequence match to the dmPTB cDNA (P-value 6.7e–290) in the Drosophila genome. These results confirm that this abundant mRNA expressed in adult males but not females is a genuine dmPTB transcript (Robida, 2003).

Previously, a large-scale P-element insertion mutagenesis screen for male sterility identified the hephaestus² (heph²) mutation (Castrillon, 1993), which was later mapped to the dmPTB locus by the Drosophila Genome Project. Other P-element insertions into the dmPTB locus are homozygous lethal (Dansereau, 2002). However, the molecular basis for the male sterility of the heph² mutant was not studied. Since homozygosity for the heph² allele causes sterility in male but not female flies (Castrillon, 1993), it was reasoned that this phenotype might be due to the absence of the abundant male-specific dmPTB transcript. To directly test this hypothesis, the expression of dmPTB was analyzed in heph² flies. The dmPTB transcript was present in both wild-type and heph² heterozygous males but absent in heph² homozygous males. Thus, the heph² P-element insertion disrupts the expression of the male-specific dmPTB transcript (Robida, 2003).

This study provides the first evidence that there is a major male-specific transcript of the Drosophila PTB that is regulated by the somatic sex-determination pathway. The sex-specific function of the abundant dmPTB transcript is restricted to the male germline. A direct molecular link is found between male fertility and PTB function, which offers a molecular basis for the male sterility of the heph² mutant (Robida, 2003).

It is postulated that the somatic sex-determination pathway, in a DSX^M-dependent manner, provides a signal for the proliferation and differentiation of male germ cells, leading to the expression of dmPTB in the male germline. Since tra and dsx are dispensable within the germline, their effect from the somatic tissue is inductive in nature. Accordingly, the DSX^F isoform in the female soma or lack of the DSX^M isoform in the male soma would fail to provide an appropriate signal for the development of the male germ cells. Thus, dmPTB expression is indirectly regulated by DSX^M in the male germline (Robida, 2003).

There are several differences in the mechanism of sex determination between somatic cells and the female germline, e.g. the mechanism by which the X:A ratio is sensed is different between the two cell types. Furthermore, sexual differentiation is entirely cell autonomous in somatic cells but also requires a somatic inductive signal(s) in germ cells. It is emphasized that, unlike other male-specific transcripts that are either functional in somatic cells or dispensable for germline sex determination and spermatogenesis, dmPTB function is necessary in the germline for spermatogenesis. Thus, dmPTB provides evidence for the necessity of soma-germline communication in the differentiation of the male germline (Robida, 2003).

Several interesting aspects of dmPTB regulation, however, remain to be addressed. For example, relatively little is known about the molecular nature of somatic- or germline-specific activation signals for dmPTB expression. Also, whether the relevant germline-specific signal is repressed in the female germline or is activated only in the male germline cannot be distinguish. Finally, the promoter elements that confer male germline-specific expression remain unknown (Robida, 2003).

The male-germline-specific function of the abundant dmPTB transcript reported in this study directly links dmPTB function to male fertility. Specifically, dmPTB is expressed in primary spermatocytes and affects spermatid differentiation, resulting in the accumulation of cysts with elongated spermatids, but fully separated motile sperms are not observed. This phenotype is reminiscent of the defect seen in late male-sterile mutants such as the individualization-deficient clathrin heavy chain (Chc) mutant, suggesting that dmPTB may control a component(s) of the cytoskeletal machinery. The expression pattern of dmPTB is consistent with the observation that the majority of transcription in germ cells is limited to the premeiotic stages, although protein synthesis and significant morphological changes occur during postmeiotic spermatid differentiation. Accordingly, the idea is favored that dmPTB is expressed early during spermatogenesis but affects either directly or indirectly the events that occur or manifest late during spermatid differentiation. It is emphasized that many male-sterile mutants are known to show secondary effects even though such mutations affect processes early during spermatogenesis. Thus, dmPTB in the male germline may control multiple targets or steps during spermatogenesis. Consistent with the known RNA-binding functions of the mammalian PTB, it could regulate the splicing, polyadenylation or translation of potential mRNAs that participate in spermatogenesis (Robida, 2003).

The male-germline-specific function of dmPTB is not necessarily inconsistent with the ubiquitous expression and multiple known targets of the vertebrate PTB. To reconcile these differences, the idea is favored that dmPTB performs an additional non-sex-specific function(s) vital for both sexes in Drosophila. (1) The male-sterile heph² mutant also affects viability of both sexes. (2) Other mutations in the dmPTB locus are homozygous lethal. The most likely explanation for the different phenotypes of these mutations is that, whereas the heph² mutation perturbs the male germline function but partially supports the vital function, the ema mutation compromises both functions. (3) Based on in situ hybridizations, dmPTB transcripts are expressed in several cell lineages, and minor transcripts are observed in females only upon longer exposure (Robida, 2003).

Given that the dmPTB locus is large (>135 kb) and that there is an indication of two distinct 5' UTRs (distal and proximal), the simplest interpretation for the two phenotypes is that the abundant male germline-specifc transcript reported in this study corresponds to an mRNA that contains the distal 5' UTR and is likely transcribed from an upstream promoter. Accordingly, the idea is favored that a downstream promoter(s) possibly contributes low abundance transcript(s) that are expressed non-sex-specifically in many cell lineages (Davis, 2002). This situation is reminiscent of two types of Sxl transcripts arising from a sex-specific establishment promoter (Pe) that is transiently active early during development (blastoderm stage) in females and from a non-sex-specific maintenance promoter (Pm) that is active in both sexes later during development. Although PTB transcripts are expressed in both male and female gonads in mice and worms, the possibility that the PTB protein is functional only in the male germline or regulates male-germline- specific mRNA(s) in the gonads of these organisms cannot be excluded (Robida, 2003).

The sex-determination genes fruitless and doublesex specify a neural substrate required for courtship song

Courtship song is a critical component of male courtship behavior in Drosophila, making the female more receptive to copulation and communicating species-specific information. Sex mosaic studies have shown that the sex of certain regions of the central nervous system (CNS) is critical to song production. Examination of one of these regions, the mesothoracic ganglion (Msg), revealed the coexpression of two sex-determination genes, fruitless (fru) and doublesex (dsx). Because both genes are involved in creating a sexually dimorphic CNS and are necessary for song production, the individual contributions of fru and dsx to the specification of a male CNS and song production was investigated. A novel requirement is shown for dsx in specifying a sexually dimorphic population of fru-expressing neurons in the Msg. Moreover, by using females constitutively expressing the male-specific isoforms of fru (Fru^M), a critical requirement is shown for the male isoform of dsx (Dsx^M), alongside Fru^M, in the specification of courtship song. Therefore, although Fru^M expression is sufficient for the performance of many male-specific behaviors, this study has shown that without Dsx^M, the determination of a male-specific CNS and thus a full complement of male behaviors are not realized (Rideout, 2007).

Courtship behavior in Drosophila consists of a sequence of behaviors performed by males to interest females in copulation. The male orients to the female, follows her, taps her abdomen with his foreleg, sings a species-specific courtship song, licks her genitals, attempts copulation, and finally copulates. Sex mosaic studies have shown that the sex of the central nervous system (CNS) is critical to the performance of these behaviors, suggesting that sex determination in the CNS is required for male sexual behavior in flies. In particular, one sex-determination gene, fruitless (fru), is a key regulator of many steps in the courtship ritual (Rideout, 2007 and references therein).

Transcripts derived from the fru P1 promoter are spliced in females by the sex-specific splice factor Transformer (Tra) in conjunction with the non-sex-specific Transformer-2 (Tra-2), introducing a premature stop codon into female P1 transcripts. In males, a default splice occurs, giving rise to a class of male-specific fru isoforms (Fru^M proteins) that are expressed in the CNS and peripheral nervous system (PNS) in regions associated with male-specific behaviors (Rideout, 2007).

The constitutive expression of Fru^M isoforms in females triggers many male-specific courtship behaviors. However, these females perform subnorm