Transcriptional Regulation

Drosophila Hox and sex-determination genes control segment elimination through EGFR and extramacrochetae activity

The formation or suppression of particular structures is a major change occurring in development and evolution. One example of such change is the absence of the seventh abdominal segment (A7) in Drosophila males. This study shows that there is a down-regulation of EGFR activity and fewer histoblasts in the male A7 in early pupae. If this activity is elevated, cell number increases and a small segment develops in the adult. At later pupal stages, the remaining precursors of the A7 are extruded under the epithelium. This extrusion requires the up-regulation of the HLH protein Extramacrochetae and correlates with high levels of spaghetti-squash, the gene encoding the regulatory light chain of the non-muscle myosin II. The Hox gene Abdominal-B controls both the down-regulation of spitz, a ligand of the EGFR pathway, and the up-regulation of extramacrochetae, and also regulates the transcription of the sex-determining gene doublesex. The male Doublesex protein, in turn, controls extramacrochetae and spaghetti-squash expression. In females, the EGFR pathway is also down-regulated in the A7 but extramacrochetae and spaghetti-squash are not up-regulated and extrusion of precursor cells is almost absent. These results show the complex orchestration of cellular and genetic events that lead to this important sexually dimorphic character change (Foronda, 2012).

The elimination of a part of an animal body is a major change occurring during morphogenesis and evolution. This study has analyzed the mechanisms required for one such change, the absence of the male seventh abdominal segment. The study shows that the suppression of this segment involves the interplay between Hox and the sex determining genes, which regulate targets implementing the morphological change. The reduction or suppression of this segment is also a sexually dimorphic feature characteristic of higher Diptera, so the mechanisms shown here may be relevant for the evolution of morphology (Foronda, 2012).

In early pupa, during the second phase of cell division, there is a reduction in the number of A7 histoblasts, both in males and females, but stronger in males perhaps because wg is not expressed in the male A7 histoblasts. It has been shown that fewer histoblasts result in a smaller adult segment. Therefore, the reduced number of A7 histoblasts may account in part for the reduced size of the A7 segment in females. The control of the second phase of cell division involves the EGFR pathway, and Abd-B was found to reduce the number of histoblasts in the A7 through down-regulation of EGFR activity. If this activity is eliminated in the male A7, an increase is observed the number of histoblasts, that many of these cells remain at the surface at the time of extrusion and that a small A7 forms in the adult. It was also previously reported that a small A7 is observed in the male adult when expressing vein, an EGFR ligand. It is possible that the high number of histoblasts obtained when over-expressing elements of the EGFR pathway makes many of them unable to be extruded by a 'titration' effect, that is, there may be 'too many' histoblasts for the invagination mechanism to extrude them at the correct time. However, the EGFR pathway may also hinder extrusion since lower levels are seen of emc-GFP and also many histoblasts remain at the surface after high EGFR activation (Foronda, 2012).

At later pupal stages (around 35-40 h APF) there is the extrusion of the male A7 histoblasts. It was observed, however, that a few histoblasts also invaginate in the female A7, suggesting the male intensifies a mechanism present in both sexes. The extrusion requires the activity of emc, and correlates with higher emc expression in the male A7 histoblasts at about the time of extrusion. The invagination of histoblasts superficially resembles that of larval cells, and it also requires myosin activity. This would suggest that, due to the higher levels of Abd-B and DsxM, male A7 histoblasts may have adopted a mechanism similar to that used by cuticular larval epidermal cells (LECs) for their elimination. Recent reports, however, suggest an alternative mechanism. It has been demonstrated that an excess of proliferation in the epithelium leads to cell death-independent cell extrusion. Since this study has observed that prevention of cell death in the male A7 does not cause the development of an A7 (although delamination is delayed), the mechanism driving extrusion may be more similar to that of an overproliferating epithelium than to that taking place in larval cells (Foronda, 2012).

The data are consistent with emc increasing the expression of spaghetti-squash to accomplish apical constriction and extrusion. However, high expression of emc may not be sufficient to effectively induce histoblast extrusion, suggesting other genes are required. Besides, a strong reduction of emc leads to a very small and poor differentiated male A7 segment, reflecting that this gene is required for several cellular functions, among them cell survival. Perhaps significantly, emc is also expressed in embryonic tissues preceding invagination of different structures in the embryo, suggesting a common requirement for invagination at different developmental stages. It is thought that emc forms part of complex networks that have, among other cellular functions, that of contributing to the extrusion of A7 histoblasts (Foronda, 2012).

Although regulation of the EGFR pathway and emc are two key events in controlling male A7 development, previous experiments have also shown the contribution of the wingless gene, absent in male A7 but present in male A6 and female A7, in the development of this segment. These results have been confirmed and it was also shown that a reduction in wg expression can partially suppress the Abd-B mutant phenotype. Absence of wg is probably required to reduce cell proliferation in the male A7 but the data suggest wg may also be needed to maintain high emc levels. Apart from the role of wg, it was also shown that some A7a cells are transformed into A6p cells, thus reducing the number of A7 cells that might contribute to the adult segment. Finally, the expression of bric-a-brac must also be down-regulated in male A7 histoblasts to eliminate this metamere. Thus, this suppression is a complex process using different genes and mechanisms (Foronda, 2012).

The suppression of the male A7 depends ultimately on the levels of Abd-B expression. The role of this Hox gene is probably mediated in part by dsx, since Abd-B regulates dsx transcription and dsx governs, in turn, the expression of genes required for cell proliferation and extrusion. That Hox genes regulate dsx expression has also been demonstrated in the male foreleg, suggesting that Hox genes specify the different parts of the body where sexual dimorphism may evolve. The different dsx isoforms (DsxF and DsxM) determine the outcome of this regulation. A significant difference between the activities of these two proteins in the A7 is the regulation of emc levels. In the female, emc expression is similar in the A7 and the A6 and, accordingly, histoblast extrusion in females is small and confined to the central dorsal region, a domain virtually absent in the adult tergite. By contrast, the DsxM isoform increases Emc expression to drive large extrusion of A7 cells and elimination of the segment (Foronda, 2012).

Only the male A7, but not anterior abdominal segments, is eliminated. Therefore, the increase in emc expression, and subsequent events observed in the A7, depends on the higher Abd-B expression in the A7 in relation to the A6. Several Hox loci, like Sex combs reduced, Ultrabithorax or Abd-B are haplo-insufficient, and relatively small differences in the amount of some of these Hox proteins can drive major phenotypic changes, suggesting some downstream genes can sense these slight differences and implement major changes in morphology (Foronda, 2012).

Previous studies have shown the cooperation of Abd-B and the sex determination pathway in controlling the pigmentation of the posterior abdomen. It is thought that Abd-B plays a dual role in regulating the morphology of the posterior abdomen. First, it regulates dsx expression, thus allowing the possibility to develop sexually dimorphic characters; second, it cooperates with Dsx proteins in establishing pattern. Part of the effect implemented by Abd-B may be mediated by the levels of expression of dsx (distinguishing male A6 from male A7), and from the nature of the Dsx proteins (male and female ones). Although there is no conclusive evidence that the different levels of dsx in the A6 and A7 play a role in development, it is noted that this difference correlates with that of Abd-B (and depends on it), that high levels of DsxM are sufficient to increase emc-GFP in the A7 of females and eliminate this segment, and that these same high levels similarly increase emc-GFP and partially rescue the Abd-B mutant phenotype in males. Hox genes, therefore, may provide a spatial cue along the anteroposterior axis to activate dsx transcription and allow the formation of sexually dimorphic characters, but they may also cooperate with Dsx proteins to determine different morphologies. This double control by Hox genes may apply to all the sexually dimorphic characters and be also a major force in evolution (Foronda, 2012).

Hox-mediated regulation of doublesex sculpts sex-specific abdomen morphology in Drosophila

Hox transcription factors are deeply conserved proteins that guide development through regulation of diverse target genes. Furthermore, alteration in Hox target cis-regulation has been proposed as a major mechanism of animal morphological evolution. Crucial to understanding how homeotic genes sculpt the developing body and contribute to the evolution of form is identification and characterization of regulatory targets. Because target specificity is achieved through physical or genetic interactions with cofactors or co-regulators, characterizing interactions between homeotic genes and regulatory partners is also critical. In Drosophila, sexually dimorphic abdominal morphology results from sex-specific gene regulation mediated by the Hox protein Abdominal-B (Abd-B) and products of the sex-determination gene doublesex (dsx). Together these transcription factors regulate numerous sex-specific characters, including pigmentation, cuticle morphology, and abdominal segment number. This study shows that Dsx expression in the developing D. melanogaster pupal abdomen is spatiotemporally dynamic, correlating with segments that undergo sexually dimorphic morphogenesis. Furthermore, genetic analyses show Dsx expression is Abd-B dependent. It is concluded that Doublesex and Abd-B are not only requisite co-regulators of sexually dimorphic abdominal morphology, but also that dsx is itself a transcriptional target of Abd-B. These data present a testable hypothesis about the evolution of sexually dimorphic segment number in Diptera (Wang, 2012).

That dsx is transcriptionally regulated downstream of Abd-B presents testable hypotheses about the evolution of posterior abdominal morphology in the Cyclorrhapha. One possibility is that dsx was an Abd-B target before the Cyclorrhaphan radiation. Ancestral to Cyclorrhapha, this spatial regulation of dsx may have contributed to dimorphic traits more subtle than segment number. Although evidence is still needed, it is proposed that, by analogy with bab regulation, male-specific wg repression is mediated directly by Abd-B and DsxM acting on a wg abdominal CRE. The gain of Abd-B and Dsx binding sites in a wg abdominal CRE may have been a critical step in the evolution of sexually dimorphic segment number in the Cyclorrhapha (Wang, 2012).

Alternatively, evolution of the Cyclorrhaphan body plan may first have required changes to the abdominal trans-regulatory landscape: a critical step being novel dsx abdominal expression under control of Abd-B. This evolution of overlapping positional and sex-specific regulatory proteins may have co-opted target genes of this pair of transcription factors, necessary for sexually dimorphic development of the genital, into processes that pattern development of the posterior abdomen. Novel abdominal specific targets of this regulatory partnership would likewise have been acquired through independent cis-regulatory evolution (Wang, 2012).

The Jak-STAT target Chinmo prevents sex transformation of adult stem cells in the Drosophila testis niche

Local signals maintain adult stem cells in many tissues. Whether the sexual identity of adult stem cells must also be maintained was not known. In the adult Drosophila testis niche, local Jak-STAT signaling promotes somatic cyst stem cell (CySC) renewal through several effectors, including the putative transcription factor Chronologically inappropriate morphogenesis (Chinmo). This study found that Chinmo also prevents feminization of CySCs. Chinmo promotes expression of the canonical male sex determination factor DoublesexM (DsxM) within CySCs and their progeny, and ectopic expression of DsxM in the CySC lineage partially rescues the chinmo sex transformation phenotype, placing Chinmo upstream of DsxM. The Dsx homolog DMRT1 prevents the male-to-female conversion of differentiated somatic cells in the adult mammalian testis, but its regulation is not well understood. This work indicates that sex maintenance occurs in adult somatic stem cells and that this highly conserved process is governed by effectors of niche signals (Ma, 2014).

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 DsxF 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 DsxF-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 DsxF 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 DsxF 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 DsxF and bZIP1 is cooperative (a thermodynamic mechanism), (2) the ternary DsxF-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 DsxM 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 DsxM, 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 dsxSWE 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; dsxSWE/+ 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 (dsxM), and a female-specific product in females (dsxF). Loss of dsx function in females results in the development of male-like pigmentation, which can be suppressed by heat-shock dsxF transgenes. Male-specific pigmentation is therefore expressed by default, and must be actively repressed by dsxF (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 dsxF 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. dsxDominant intersexes, which express both male- and female-specific dsx products, also show male-like expression of bab, indicating that dsxM can interfere with dsxF 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 dsxM 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, dsxF 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 dsxF 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 DoublesexF product results in the development of the female genital primordium and repression of the male primordium. In males, the presence of DoublesexM product results in the development and repression of the male and female genital primordia, respectively. This report shows that DoublesexF prevents the induction of decapentaplegic by Hedgehog in the repressed male primordium of female genital discs, whereas DoublesexM blocks the Wingless pathway in the repressed female primordium of male genital discs. It is also shown that DoublesexF 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 DoublesexM is not continuously required during larval development for blocking the Wingless signaling pathway in the female genital primordium. Furthermore, DoublesexM 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 DoublesexM and DoublesexF 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 DoublesexM and DoublesexF 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 DsxF or male DsxM product, respectively. Clones for tra2 (expressing DsxM) 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 DsxF 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 DsxM 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 DsxM nor DsxF products. The female genital primordium of these discs now expresses Dll. It is concluded that DsxM 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 DsxF 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 DsxM 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 dsxMas/+ intersexual genital discs, which have both DsxM and DsxF products, and in dsx1 intersexual genital discs, which have neither DsxM nor DsxF 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 DsxM plays opposing, positive and negative roles in dac expression in male and female genital discs, respectively; and that DsxF 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 DsxM ) were induced in female genital discs. The expression of dac is repressed in tra2 clones located in Wg territory. Therefore, DsxF positively regulates dac expression in the Wg domain, and DsxM 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, DsxM positively regulates dac expression in the Dpp domain, whereas DsxF negatively regulates dac expression in this domain, since in normal female genital discs with DsxF 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 DsxF in the male genital primordium of male genital discs. It is concluded that in male genital discs, DsxM positively and negatively regulates dac expression in Dpp and Wg domains, respectively; and in female genital discs, DsxF positively and negatively regulates dac expression in Wg and Dpp domains, respectively (Sanchez, 2001).

Homozygous tra2ts 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 DsxF and at 29°C they produce DsxM. A shift in the temperature of the culture is accompanied by a change in the sexual pathway of tra2ts larvae. Analysis of the growth of genital primordia and their capacity to differentiate adult structures of tra2ts 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 Tra2ts product; i.e. transitory presence of DsxF product and absence of DsxM 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 DsxF product and presence of DsxM 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 Tra2ts product is again available (i.e. the presence of DsxF product and absence of DsxM product). This time requirement for induction of dpp is also supported by the fact that dsx11 clones (which lack DsxM) induce differentiated normal male adult genital structures in the developing male genital primordium of XY; dsx11/+ male genital discs (which express only DsxM ) 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 DsxM nor DsxF 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, DsxM positively and negatively regulates dac expression in Dpp and Wg domains, respectively; and in female genital discs, DsxF positively and negatively regulates dac expression in Wg and Dpp domains, respectively. The expression of the gene dac was analyzed in genital discs of tra2ts 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 tra2ts 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 DsxM instead of DsxF product. Thus, these results are consistent with the previously proposed suggestion that DsxM activates dac in the Dpp domain and represses it in the Wg domain (again the converse is true for DsxF). 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 DsxM instead of DsxF. This is explained by the lack of competence of cells to express Dpp, which is acquired after first larval instar. When the tra2ts larvae reach such a developmental stage, these cells now produce DsxF because they have returned to the female-determining temperature (Sanchez, 2001).

DsxF 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 DsxM and DsxF 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 DsxM and DsxF 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 DsxM and DsxF 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 DsxF 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 DsxM 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 DsxM plus Abd-Br increase dpp expression in the male genital primordium of male genital discs, and that DsxF 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, DsxM would play a positive and a negative role in male and female genital primordia, respectively, whereas DsxF 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 DsxM and DsxF. 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 (DsxF) in females and/or activated by the male-specific Dsx protein (DsxM) 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 DsxM is present, but is not expressed in the A8-derived primordium of a wild-type female disc, where DsxF 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 DsxF protein is absent, implies that bnl expression is repressed in the female genital disc by the presence of DsxF 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 DsxF, an obvious question is whether the DsxF 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 DSXM-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 DSXF isoform in the female soma or lack of the DSXM 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 DSXM 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 (FruM), a critical requirement is shown for the male isoform of dsx (DsxM), alongside FruM, in the specification of courtship song. Therefore, although FruM expression is sufficient for the performance of many male-specific behaviors, this study has shown that without DsxM, 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 (FruM 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 FruM isoforms in females triggers many male-specific courtship behaviors. However, these females perform subnormal amounts of courtship and do not attempt copulation, suggesting that fru alone cannot specify all male courtship behaviors (Rideout, 2007).

The role of doublesex (dsx), another sex-determination gene, was examined in the specification of male sexual behavior. dsx transcripts also undergo sex-specific splicing by Tra, producing male- and female-specific isoforms: DsxM and DsxF, respectively. dsx is responsible for somatic sexual differentiation and aspects of sex-specific development in the CNS. dsx is also expressed in the CNS and is necessary for wild-type courtship song in males. dsx has been shown to act alongside fru in the differentiation of male-specific neurons in the abdominal ganglion; however, few other studies have examined the relative contributions of both fru and dsx in specifying a male-specific CNS and regulating male sexual behavior. Therefore, this study examined the individual contributions of both genes in specifying courtship song (Rideout, 2007).

Courtship song in Drosophila is male-specific and is critical to stimulating the female. It consists of a humming sound called sine song, and a rhythmically patterned pulse song, which together stimulate the female to mate, reducing the time to copulation. Pulse song also communicates species-specific information, allowing females to recognize conspecific males (Rideout, 2007).

FruM mutant males lack pulse song, and constitutive FruM expression in the CNS of fruM and fruΔtra females induces the performance of many steps of the male courtship ritual, suggesting an important role for FruM in specifying courtship song. To determine the contribution of FruM in the specification of courtship song, song production was analyzed in females of genotype fruM and fruΔtra (Rideout, 2007).

Song analysis was based on 29 fruM and fruΔtra females because most fruM and fruΔtra females did not perform sufficient courtship behavior or song for analysis. The wing-extension indices (WEIs) of these 29 fruM and fruΔtra females were not significantly different from wild-type and control fruM and fruΔtra males; however, a significant decrease was foudn in the song index (SI) (the percentage of time spent singing during wing extension). Also, the fruM and fruΔtra females' pulse song was highly aberrant. The number of pulse trains per minute (PTPM), mean pulses per train (MPPT) and interpulse interval (IPI) were all significantly lower compared to wild-type and control males. Most striking, however, was the complete absence of sine song in these females. Although the fruM and fruΔtra females were capable of wild-type wing extension, they spent significantly less time singing during courtship and produced song of poor quality. Thus, FruM expression alone cannot specify wild-type song production (Rideout, 2007).

To dissect the individual contributions of both FruM and Dsx to the specification of courtship song, males were analyzed lacking FruM and Dsx [genotype fru3,In(3R)dsx23/fru3,Df(3R)dsx15]. These double mutants had a courtship index (CI) of 0 toward females and no song. FruM expression in females is not sufficient for courtship song. Likewise, the expression of DsxM in females is also not sufficient for song. Thus neither fru nor dsx alone can specify courtship song. In fact, only the presence of both FruM and DsxM, as in transformer (tra) mutant females, renders females capable of wild-type courtship song. Together, these results demonstrate a previously unrecognized requirement for DsxM, in conjunction with FruM, in specifying courtship song (Rideout, 2007).

Studies with male-female mosaics have shown that in gynandromorphs with a male head, the ventral thoracic ganglia of the adult CNS (including the mesothoracic ganglion [Msg]) must also be male for courtship song. This suggests that the neural foci of courtship song are located in the ventral thoracic ganglia and that the sex of this region is critical to song. fru and dsx are both expressed in neurons located in this region, and mutations in both genes cause song defects. In the abdominal ganglion (Abg) of the CNS, FruM and Dsx were shown to colocalize in a proportion of neurons and play critical roles in the development of male-specific clusters of serotonergic neurons. Therefore, it was asked whether FruM and Dsx were also coexpressed in the thoracic ganglia, and whether they act in parallel (if expressed in different neurons) or in concert (if expressed in the same neurons) to determine the neuronal substrate for courtship song in the CNS (Rideout, 2007).

It was determined that Dsx and FruM colocalize in the Msg of the CNS. Colocalization occurred in a subset of Dsx-expressing neurons (TN1 cluster. The number of neurons coexpressing Dsx and FruM in 2-day-old male pupae was 17.4 ± 1.7 per hemisegment. Colocalization occurred in a further two subsets of Dsx-expressing neurons in the posterior brain, pC1 and pC2, in addition to previously reported colocalization in the Abg. Given the critical importance of the sex of the ventral ganglia (including the Msg) to song production, the colocalization of FruM and Dsx in this region suggests that sexually dimorphic developmental mechanisms might be operating in the Msg, contributing to the sex-specific nature of courtship song production (Rideout, 2007).

Electrophysiological studies show that the activity of seven of the direct flight muscles (DFMs) is directly related to the beating of the wing during song. These seven DFMs are the basalar muscles B1-B4, the anterior muscles of the first and third axillaries AX1a and AX3a, and the sternobasalar muscle SB. The axonal morphology and cell-body location of the motor neurons innervating six of these DFMs (mnDFMs) has also been reported. The cell bodies of these six mnDFMs lie in the ventral thoracic ganglia, five having cell bodies in the Msg. Therefore whether male-specific song production could be attributed to fru- and/or dsx-regulated sexually dimorphic characteristics of these motor neurons was investigated (Rideout, 2007).

First, it was asked whether any of the mnDFMs were fru or dsx expressing. By using fruGAL4, a GAL4 driver expressing in all fru neurons, it was determined that only mnB3/B4 (a single motor neuron innervating both B3 and B4 was fruGAL4 positive, and thus is a fru neuron. This neuron was fruGAL4 positive in both males and females, and the innervation was not obviously sexually dimorphic. However, because some dsx-expressing neurons in the Msg are not fru expressing, the axonal morphology of all mnDFMs was examined to eliminate the possibility of sex-specific DFM innervation (Rideout, 2007).

The axonal morphology and expression of common neurotransmitters at the neuromuscular junction (NMJ) of all seven DFMs were examined, and no obvious differences between the sexes were found. Type I and type II synaptic terminals were present on all mnDFMs, where type I terminals expressed glutamate and type II terminals expressed octopamine, in accordance with previous reports of neurotransmitter expression at the adult NMJ. Moreover, no obvious differences were found in either axonal morphology or common neurotransmitters were observed in either fru or dsx mutant males. Therefore, the sexually dimorphic production of song is not likely to be a result of an obvious dimorphism in the neuronal morphology of the mnDFMs or in the neurotransmitter expression at the NMJs. Where might the critical difference(s) then lie (Rideout, 2007)?

It has been showm that FruM expression prevented reaper-mediated programmed cell death in a cluster of cells, resulting in more neurons in this cluster in males. DsxM, in contrast, prolongs neuroblast divisions in the Abg of males, again resulting in more neurons in males. Thus, sexual dimorphisms might be present in regions in which Dsx and FruM colocalize, as suggested by the ability of FruM and Dsx to generate sexually dimorphic neuronal populations. Given that this investigation found no obvious sex-specific dimorphisms in the mnDFMs, the dimorphism might lie in a population of interneurons. Therefore, the Msg was examined so that it could be determined whether a sexually dimorphic population of neurons was present (Rideout, 2007).

By using fruGAL4, which expresses in both males and females, to drive a GAL4-responsive UAS-LacZ.NZ reporter, the number of β-Gal-positive neurons was quantified in males and females. The number of β-Gal-positive neurons was significantly higher in males, with 136.4 ± 3.3 cells per hemisegment (n = 10) versus 111.6 ± 3.1 cells per hemisegment in females. A sexual dimorphism has been reported in the number of neurons expressing fru P1 transcripts in the Msg. Together, these results suggest that a sexually dimorphic population of neurons is present in the Msg; therefore, the individual contributions of FruM and Dsx in the creation of this difference was examined in fruGAL4-positive neuron number in the Msg (Rideout, 2007).

A sexually dimorphic number of fruGAL4-expressing neurons was found in the Msg, a region of the CNS central to song production and in which FruM and Dsx colocalize. To determine the individual contributions of dsx and fru in the creation of this sexually dimorphic number of neurons, fruM and fruΔtra females were examined to see if FruM expression alone abolishes the observed difference in neuronal number in the Msg between the sexes. It was found that the number of FruM-expressing neurons in the Msg of these females was significantly reduced in comparison to wild-type and control males. Furthermore, this decrease in FruM-expressing neurons was comparable to the difference in neuron number observed in the Msg of fruGAL4 males and females driving the UAS-LacZ.NZ reporter (Rideout, 2007).

These results demonstrate that the difference in neuronal populations of males and females in the Msg lies in a subpopulation of FruM-expressing neurons, and that FruM expression alone cannot eliminate this difference. Thus FruM expression cannot, by itself, dictate the creation of the sexually dimorphic population of neurons in the Msg. It was therefore asked whether Dsx, which colocalizes with FruM in the Msg, plays a role in the specification of this sexually dimorphic population of neurons, helping to determine the full complement of FruM neurons (Rideout, 2007).

dsx affects the sex-specific development of other regions of the CNS. To determine whether dsx contributes to creating the sex-specific population of neurons in the Msg, the number of FruM-expressing neurons was tabulated in the Msg of dsx null and dsx heterozygote control males. It was found that dsx mutant males had significantly fewer FruM-expressing neurons in the Msg than did wild-type and control males, demonstrating that Dsx is indeed required to obtain a full complement of FruM-expressing neurons. Because fruM and fruΔtra females (who express the female-specific isoform of dsx, DsxF) do not have a full complement of FruM-expressing neurons in the Msg, this study has demonstrated a critical role for DsxM in the creation of a sexually dimorphic Msg. In fact, only when both FruM and DsxM are present, as in tra mutant females, can a full complement of FruM-expressing neurons in the Msg be obtained. Thus, this study has demonstrated a previously unrecognized requirement for DsxM in the specification of a population of FruM-expressing neurons in the Msg (Rideout, 2007).

DsxM prolongs the division of neuroblasts in the Abg of males, resulting in more neurons in the male Abg. Also in the Abg, DsxM plays a critical role alongside FruM in the differentiation of a male-specific serotonergic population of neurons. The current findings suggest that DsxM operates in a similar manner in the Msg and the posterior brain to create sexually dimorphic neuronal numbers. These differences in neuronal populations suggest a common developmental theme in colocalization regions, where DsxM generates a sexually dimorphic population of neurons, which is exploited by FruM to fashion a male-specific behavioral neural network (Rideout, 2007).

It is not clear why the absence of a sexually dimorphic population of FruM-expressing neurons in the Msg is associated with striking defects in courtship song because the results suggest that this population of FruM-expressing neurons does not directly innervate the DFMs. It is proposed that the FruM-expressing neurons form at least part of a male-specific neural network responsible for controlling the production of courtship song (Rideout, 2007).

Thus, although FruM expression can specify many male-specific behaviors, this study showd that without DsxM, the determination of a complete male-specific CNS, and therefore a full complement of male behaviors, is not realized. This additional gene function is critical to understanding complex sex-specific phenotypes compared to previous interpretations of function, where fru has been described as the only gene needed for a 'genetic switch' to male sexual behavior in Drosophila. Significantly, it adds to the growing evidence that fru and dsx are both necessary for a complete male courtship repertoire, in both neural and nonneural tissues (Rideout, 2007).

The regulation and evolution of a genetic switch controlling sexually dimorphic traits in Drosophila

Sexually dimorphic traits play key roles in animal evolution and behavior. Little is known, however, about the mechanisms governing their development and evolution. One recently evolved dimorphic trait is the male-specific abdominal pigmentation of Drosophila melanogaster, which is repressed in females by the Bric-à-brac (Bab) proteins. To understand the regulation and origin of this trait, the evolution of the genetic switch controlling dimorphic bab expression has been identified and traced. The HOX protein Abdominal-B (ABD-B) and the sex-specific isoforms of Doublesex (DSX) directly regulate a bab cis-regulatory element (CRE). In females, ABD-B and DSXF activate bab expression whereas in males DSXM directly represses bab, which allows for pigmentation. A new domain of dimorphic bab expression evolved through multiple fine-scale changes within this CRE, whose ancestral role was to regulate other dimorphic features. These findings reveal how new dimorphic characters can emerge from genetic networks regulating pre-existing dimorphic traits (Williams, 2008).

bab expression in the abdominal epidermis is regulated by two separate CREs, one of which directs gene expression in the anterior abdomen of both sexes, and a second, dimorphic element that regulates female-specific gene expression in segments A5-A7. The dimorphic element, when bound by ABD-B and sex-specific isoforms of the DSX protein, acts as a genetic switch that allows pigmentation in males and represses pigmentation in females. Changes in the activities of both CREs have evolved in the course of the origin of the trait from a monomorphic ancestor. Furthermore, dimorphic CRE function evolved by multiple fine-scale changes within the CRE. These results bear on understanding of how sexually dimorphic traits develop, how new sex- and segment-restricted traits arise, and how CRE functions evolve (Williams, 2008).

Sex-restricted traits are the product of differences in gene expression between sexes, therefore, understanding how such traits develop requires the identification of those genes with sex-limited expression and elucidation of the genetic and molecular mechanisms governing their regulation. This study showed that dimorphic bab expression is regulated by a discrete CRE whose activity is combinatorally regulated by the direct inputs of both region- (ABD-B) and sex-specific (DSX) transcription factors. In females, ABD-B acts in concert with the DSXF isoform through binding sites in the dimorphic element to activate bab expression in the posterior segments. Whereas in males, ABD-B activity is overridden by the repressive activity of the DSXM isoform which binds to the same sites as DSXF and hence, permits the formation of the male-specific posterior pigmentation (Williams, 2008).

The genetic pathways that regulate sex-determination and sexual differentiation differ greatly across the animal kingdom, so this mode of male-specific trait regulation in Drosophila may not apply in detail to other animals. However, the integration of region- and sex-specific regulatory inputs must be a requirement for the production of dimorphic traits. It is suggested that the integration of such combinatorial inputs by cis-regulatory elements, as demonstrated for bab, is a general feature of genetic switches within the pathways regulating the production of dimorphic traits (Williams, 2008).

The origins of sexually dimorphic traits have long been of central interest in evolutionary biology. One of the key questions that Darwin grappled with, as have many others subsequently, was whether dimorphic traits are limited to one sex at their origin, or whether these traits first appear in both sexes and then become restricted to one sex. This question has been particularly important and challenging in terms of genetics and evolutionary theory, as it has not been resolved previously how the effects of mutations could be restricted to one sex (Williams, 2008).

In the simplest genetic scenarios of sexual dimorphism, male-limited traits are the products of the male-limited expression of specific genes. The main evolutionary question then, as it has been phrased in classical genetic terms, is whether male-limited gene expression evolves via: (1) 'alleles' that are expressed only in males; or (2) alleles expressed in both sexes which are then suppressed in females or promoted in males. The elucidation of the regulation and evolution of male-specific pigmentation provides a unique opportunity to reconstruct the genetic path of the evolution of a dimorphic trait (Williams, 2008).

Although posterior male-specific pigmentation is a relatively simple, two-dimensional morphological trait, it is clear that it did not originate via just one of the alternative genetic paths above. Rather, the evolution of this trait has involved three paths: the evolution of male-limited gene expression, of female-limited gene expression, and of non-sex-restricted gene expression. Specifically, this study shows that in the course of the evolution from a monomorphically pigmented ancestor, the activity of the female-specific bab dimorphic CRE expanded into segments A6 and A5 and that the activity of the monomorphic bab anterior CRE retreated from segments A6 and A5 of both sexes. These two combined changes produced the sex-specific repression of bab expression in male segments A5 and A6. In addition, in previous work it was shown that the yellow pigmentation gene gained high-level expression in segments A5 and A6 via the acquisition of ABD-B binding sites in a specific yellow gene CRE, whose activity was male-limited due to repression by Bab (which is apparently indirect) (Williams, 2008).

It is important to underscore that none of the genes in this newly-evolved regulatory circuit are globally restricted in their expression to one sex. Rather, the sex-specific features of their expression are controlled by modular CREs that are physically separate from those controlling gene expression in other developing body regions. The properties of these CREs resolve the question of how the effects of mutations can be restricted to one sex. Namely, mutations in a CRE that is under the direct (the female-specific bab dimorphic element) or indirect (the male-specific yellow CRE) control of an effector of sex determination will have sex-limited effects on gene expression. The findings here are a further demonstration of the general principle of how the modular CREs of pleiotropic genes enable the modification of gene expression in and morphology of one body part independent of other body parts, or in this case, the same body part in the opposite sex (Williams, 2008).

It is also notable that none of the CREs analyzed are new to the dimorphically pigmented melanogaster species group. It is clear, then, that the ancestral dimorphic CRE was active in segment A7 and modified to govern sexually dimorphic pigmentation in segments A6 and A5. Thus, in this example, one path is seen to evolving a new dimorphic trait is via the co-option of genetic components that regulate other pre-existing dimorphic traits (Williams, 2008).

One of the major questions concerning the evolution of gene expression is how new gene expression patterns arise. The two most obvious mechanisms would appear to be the gain of new regulatory elements or the gain of new transcription factor-CRE linkages. While the deep ancestry of the dimorphic element ruled out the former, it was expected that the novel sex- and segment-specific regulation of this CRE by DSX and ABD-B in the D. mel. lineage would require the gain of binding sites for these two transcription factors. However, it was found that the both DSX binding sites and most ABD-B sites were present in D. wil. and other monomorphic species and therefore were present in the last common ancestor of both monomorphic and dimorphic species. Thus, the expansion of the dimorphic CRE activity was not due to the wholesale gain of new DSX and ABD-B binding sites (Williams, 2008).

Rather, it was discovered that the expanded, high level activity of the D. mel. dimorphic CRE in segments A6 and A5, relative to the A7-restricted activity of the D. wil. element, was due to an amalgam of changes involving the number, polarity, and topology of transcription factor binding sites. The evolution of dimorphic CRE activity demonstrates how changes beyond the simple gain or loss of binding sites shape CRE evolution. Similarly, changes in the topology and helical phasing of transcription factor binding sites have shaped the evolution of a genetic switch controlling galactose utilization in yeast (Hittinger, 2007). These studies strongly support the view that the relationship between function and sequence variation in CREs is complex. A vast body of work on eukaryotic and prokaryotic transcriptional regulation has shown that binding site polarity and spacing influences the output of regulatory elements. Therefore, it is suggested that one important, but generally unappreciated, class of functionally relevant mutations in CRE and trait evolution involves sequences outside of transcription factor binding sites. CREs thus present a very large target area for potential functionally relevant mutations that quantitatively modulate gene expression and trait development (Williams, 2008).

Finally, these observations concerning the mechanisms underlying the expansion of dimorphic CRE activity help to shed light on another general aspect of the evolution of animal body plans -- the evolution of segmental traits. A large number of studies have demonstrated that some of the major differences among arthropod and vertebrate body plans have involved evolutionary shifts in the spatial boundaries of gene expression along the main body axis. However, the path by which such gene expression patterns are shifted has not been elucidated in any molecular detail. It is submitted here that the expansion of the activity of the dimorphic element from the A7 segment into A6 and A5 is a model of this process. The remodeling of the dimorphic CRE in the course of evolution illustrates that one way such shifts can be accomplished is through numerous small, quantitative incremental changes in the activity of Hox-regulated CREs (Williams, 2008).

Nonautonomous sex determination controls sexually dimorphic development of the Drosophila gonad: dsx ensures that PC formation is male-specific by repressing Wnt2 expression in female gonads in late-stage embryos

Sex determination in Drosophila is commonly thought to be a cell-autonomous process, where each cell decides its own sexual fate based on its sex chromosome constitution (XX versus XY). This is in contrast to sex determination in mammals, which largely acts nonautonomously through cell-cell signaling. This study examined how sexual dimorphism is created in the Drosophila gonad by investigating the formation of the pigment cell precursors, a male-specific cell type in the embryonic gonad surrounding the testis. Surprisingly, sex determination in the pigment cell precursors, as well as the male-specific somatic gonadal precursors, was found to be non-cell autonomous. Male-specific expression of Wnt2 within the somatic gonad triggers pigment cell precursor formation from surrounding cells. These results indicate that nonautonomous sex determination is important for creating sexual dimorphism in the Drosophila gonad, similar to the manner in which sex-specific gonad formation is controlled in mammals (DeFalco, 2008).

This study has shown that two distinct male-specific cell types in the Drosophila gonad exhibit nonautonomous sex determination. For both the male specific somatic gonadal precursors (msSGPs) and the pigment cell (PC) precursors, the sex determination pathway does not act in these cells themselves, and both are dependent on sex-specific signaling from the SGPs in order to develop properly as male or female. These findings are in contrast to the commonly held view that sex determination in Drosophila is a cell-autonomous process, and demonstrate the similarity in sex-specific gonad development between flies and mammals (DeFalco, 2008).

This study has identified a novel, sex-specific cell type in the Drosophila embryonic gonad, the PC precursors, and studied the mechanism by which the sex determination switch controls the sex-specific development of these cells. The data indicate that male-specific expression of Wnt2 in the SGPs of the gonad signals nonautonomously to the fat body to form PC precursors. dsx ensures that PC formation is male-specific by repressing Wnt2 expression in female gonads in late-stage embryos (stage 17). The sex of the fat body itself does not affect PC precursor formation, since cells with a female identity can form PC precursors when associated with a male gonad or with a female gonad that expresses Wnt2. Furthermore, Wnt2 acts directly on the fat body, since blocking Wnt signaling in male fat body cells prevents them from forming PC precursors. PC precursor identity in the fat body is regulated by ems acting upstream of Sox100B in response to the Wnt2 signal. An interesting question is whether Wnt2 is a direct downstream target of DSX in controlling sexual dimorphism. The DNA binding specificity for DSX has been determined, and there are a number of sites upstream of the Wnt2 start of transcription that either exactly match or closely match the DSX binding consensus sequence. Several of these sites are conserved between different Drosophila species. However, a fragment of the Wnt2 promoter has not yet been identified that allows testing of whether Wnt2 expression in the somatic gonad is directly regulated by DSX, since the upstream region that includes the putative DSX binding sites does not promote expression in the gonad (DeFalco, 2008).

The creation of sexual dimorphism in the PC precursors differs from that of the msSGPs. While the PC precursors are apparently only specified in males and recruited to form part of the testis, msSGPs are initially specified in both sexes, and are only present in the male gonad because they undergo programmed cell death specifically in females. Furthermore, the germline stem cell niche in the testis (the hub) is formed from a population of anterior SGPs that are present in the gonads of both sexes, but only form the hub in males and presumably form part of the ovary in females. These events are all regulated by dsx, and demonstrate the diverse cellular mechanisms that a sex determination gene can utilize to control sexual dimorphism. Interestingly, in dsx null mutant embryos each of these cell types develops as if it were male. Thus, the male mode of development can at least be initiated in these cell types in the absence of dsx function, and dsx primarily acts in females to repress male development. dsx is clearly required in males at some point for proper testis formation, therefore some cell types in the gonad may not be entirely masculinized in dsx mutants (DeFalco, 2008).

The nonautonomous nature of PC precursor specification contrasts with the commonly held view that sex determination in Drosophila is a cell-autonomous process, where 'every cell decides for itself' whether it should develop as male or female based on its own intrinsic sex chromosome constitution. This study has shown that the msSGPs undergo nonautonomous sex determination. The data indicate that a male-specific survival signal coming from the SGPs allows the msSGPs to survive and join the male gonad, while they undergo apoptosis in females. Finally, it has been shown that nonautonomous sex determination in the germ cells requires a male-specific signal from the SGPs that acts through the JAK/STAT pathway. Thus, not only does non-cell autonomous sex determination occur in the Drosophila gonad, it appears to be the predominant mechanism of sex determination. Of the cell types tested so far, only the hub cells, which form from a subset of SGPs, appear to decide their sexual fate in an autonomous manner. The current model is that the SGPs determine their sex in a cell-autonomous manner, and then signal to other cell types in the gonad (PC precursors, msSGPs, and germ cells) to control the sex-specific development of these cells via nonautonomous sex determination (DeFalco, 2008).

Nonautonomous sex determination is not limited to the gonad. Other tissues have been shown to decide their sex through cell-cell signaling. In the genital imaginal disc, the sexual identity of a signaling center, the A/P organizer, largely determines whether the disc will develop in the male or female mode. This is controlled non-cell autonomously through Wingless and Decapentaplegic signaling. In addition, sex-specific migration of mesodermal cells into the male genital disc is regulated by male-specific expression of the Fibroblast Growth Factor Branchless in the genital disc. Finally, in the nervous system, male neurons can non-cell autonomously induce the formation of the male-specific muscle of Lawrence from female muscle precursors. Given the large number of tissues and cell types that undergo nonautonomous sex determination, it seems that the conventional view can be abandoned that sex determination in Drosophila is an obligatorily cell-autonomous process; while some cell types utilize a cell-autonomous mechanism, many cell types clearly do not (DeFalco, 2008).

One reason why sex determination has been traditionally thought of as a cell-autonomous process in Drosophila is due to its relationship with X chromosome dosage compensation. This is the process by which gene expression from the single X chromosome in males is regulated to match that from the two X chromosomes in females. Both sex determination and X chromosome dosage compensation are regulated by the number of X chromosomes, acting through the master control gene Sex lethal (Sxl). It is likely that most or all cells count their X chromosomes and use this information to control X chromosome dosage in a cell-autonomous manner. However, as discussed above, it is now clear that cells do not necessarily use this information to decide their sex. Consistent with this idea, the expression of dsx, a key regulator of sex determination downstream of Sxl, is surprisingly tissue-specific. Within the embryo, dsx is only expressed in the SGPs and msSGPs of the gonad. Thus, not all cells even express the machinery to translate their sex chromosome constitution into sexual identity, and it is therefore necessary that sex-specific development of many cell types be controlled nonautonomously (DeFalco, 2008).

The nonautonomous cell-cell interactions that control gonad sexual dimorphism in Drosophila show great similarity to sex-specific gonad development in other species. In mammals, somatic sex determination is based on the presence or absence of the Y chromosome. The critical Y chromosome gene Sry is mainly expressed in a subset of cells in the somatic gonad in the mouse embryo, similar to dsx expression in the Drosophila embryonic gonad. Sry is only thought to be important for formation of Sertoli cells in males, and the sexually dimorphic development of all other cell types is thought to be regulated by local cell-cell interaction or hormonal cues. An excellent example of nonautonomous sex determination in the mouse is the recruitment of cells from the neighboring mesoderm (mesonephros) to form specific cell types in the mouse testis. Recruitment of these cells is dependent on the sex of the gonad, not the sex of the mesonephros. In addition, sex-specific development of other somatic cell types in the mouse gonad is regulated nonautonomously by cell-cell interaction, as is sexual identity in the germline. Thus, the use of non-cell autonomous sex determination and sex-specific cell recruitment are common mechanisms for creating gonad sexual dimorphism in flies and mice (DeFalco, 2008).

Nonautonomous sex determination in the mouse also utilizes signaling through the Wnt pathway. Wnt4 acts as a 'pro-female' gene that opposes Fibroblast growth factor 9 to regulate sex determination in the gonad. In early stages of gonad development, Wnt4 knockout females form a male-specific coelomic blood vessel and exhibit ectopic migratory steroidogenic cells, suggesting that Wnt4 acts to inhibit endothelial cell and steroid cell migration from the mesonephros into the female gonad. Interestingly, Wnt4 also has been shown to have a role in the male gonad, as male knockout mice show defects in Sertoli cell differentiation, downstream of Sry but upstream of Sox9. Wnt7a also has been implicated in sexual dimorphism in the reproductive tract, as Wnt7a knockout mice fail to express Mullerian-inhibiting substance (MIS) type II receptor in the Mullerian duct mesenchyme, which is required for regression of the duct in male embryos. In addition, a number of Wnt genes have been found to be expressed sex-specifically in the gonad through sex-specific gene profiling, indicating that other Wnt family members play a role in creating sexual dimorphism in the mammalian gonad (DeFalco, 2008).

It is also interesting that several conserved transcription factors act during gonad development in diverse species. Sox100B is the fly homolog of SOX9/Sox9, a critical regulator of sex determination and male gonad development in humans and mice. Similarly, a mouse homolog of ems, Emx2, is expressed in the developing gonad and is required for development of the urogenital system. Lastly, dsx homologs of the DMRT family have been implicated in sex-specific gonad development in diverse species. Thus, not only are the cellular mechanisms, such as non-cell autonomous sex determination and cell-cell recruitment, common between flies and mice, but the specific genes that regulate sexually dimorphic gonad development may also be conserved. Since the formation of testes versus ovaries, and sperm versus egg, are critical features of sexual reproduction, they may represent processes that are highly conserved across the animal kingdom (DeFalco, 2008).

The female-specific doublesex isoform regulates pleiotropic transcription factors to pattern genital development in Drosophila.

Regulatory networks driving morphogenesis of animal genitalia must integrate sexual identity and positional information. Although the genetic hierarchy that controls somatic sexual identity in Drosophila is well understood, there are very few cases in which the mechanism by which it controls tissue-specific gene activity is known. In flies, the sex-determination hierarchy terminates in the doublesex (dsx) gene, which produces sex-specific transcription factors via alternative splicing of its transcripts. To identify sex-specifically expressed genes downstream of dsx that drive the sexually dimorphic development of the genitalia, genome-wide transcriptional profiling was performed of dissected genital imaginal discs of each sex at three time points during early morphogenesis. Using a stringent statistical threshold, 23 genes that have sex-differential transcript levels at all three time points were identified, of which 13 encode transcription factors, a significant enrichment. This study focused on three sex-specifically expressed transcription factors encoded by lozenge (lz), Drop (Dr) and AP-2. In female genital discs, Dsx activates lz and represses Dr and AP-2. It was further shown that the regulation of Dr by Dsx mediates the previously identified expression of the fibroblast growth factor Branchless in male genital discs. The phenotypes observed upon loss of lz or Dr function in genital discs explain the presence or absence of particular structures in dsx mutant flies and thereby clarify previously puzzling observations. This time course of expression data also lays the foundation for elucidating the regulatory networks downstream of the sex-specifically deployed transcription factors (Chatterjee, 2011).

A common theme in the evolution of development is that a limited 'toolkit' of regulatory factors is deployed for different purposes during morphogenesis. It is therefore not surprising that the key regulators of genital morphogenesis that this study identified are pleiotropic factors with roles in other developmental processes (Chatterjee, 2011).

Two genes that are expressed sex-differentially in the genital disc, branchless (bnl) and dachshund (dac), provide the best picture of how dsx controls genital morphogenesis. Bnl, which is the fly fibroblast growth factor (FGF), is expressed in two bowl-like sets of cells in the A9 primordium in male discs; there is no expression in female discs because DsxF cell-autonomously represses bnl. Bnl recruits mesodermal cells expressing the FGF receptor Breathless (Btl) to fill the bowls; these Btl-expressing cells develop into the vas deferens and accessory glands (Chatterjee, 2011 and references therein).

Dac, a transcription factor, is expressed in male discs in lateral domains of the A9 primordium and in female discs in a medial domain of the A8 primordium. These lateral and medial domains correspond to regions exposed to high levels of the morphogens Decapentaplegic (Dpp) and Wingless (Wg), respectively. Dsx determines whether these signals activate or repress dac. Male dac mutants have small claspers with fewer bristles and lack the single, long mechanosensory bristle. Female dac mutants have fused spermathecal ducts (Chatterjee, 2011 and references therein).

As with bnl and dac, it remains to be determined whether these downstream genes are direct Dsx targets. Each contains at least one match within an intron to the consensus Dsx binding sequence ACAATGT. Future work will determine whether these matches are indeed contained within Dsx-regulated genital disc enhancers. Moreover, efforts are underway to define Dsx binding locations genome-wide through experiments rather than bioinformatics (B. Baker and D. Luo, personal communication to Chatterjee, 2011); combined with the current expression data, these binding data could speed the discovery of a large number of sex-regulated genital disc enhancers (Chatterjee, 2011).

An important future direction will be to determine how spatial and temporal cues are integrated with dsx to regulate downstream genes. Because lz is expressed in the anterior medial region of the female disc, it is hypothesized that, like dac, it is activated by Wg and repressed by Dpp. Such combinatorial regulation could explain the spatially restricted competence of cells in the male disc to activate lz in response to DsxF. Although Dr, AP-2 and lz are expressed at L3, P6 and P20, many other genes are differentially expressed at only one or two of these time points. How these timing differences are regulated is an important unanswered question, especially for genes such as ac, which shifts from highly female biased at P6 to highly male biased at P20. The finding that Dsx binding sites are most enriched in genes with sex-biased expression at L3 suggests that indirect regulation through a cascade of interactions might contribute to expression timing differences (Chatterjee, 2011).

It has already been shown that DsxF indirectly represses bnl by repressing Dr. To date, Dr has been shown to repress, but not activate, transcription. Therefore, activation of bnl by Dr might itself be indirect, via repression of a repressor. The regulation of bnl by Dr is sufficient to explain the sex-specific expression of bnl. However, upstream of bnl are two sequence clusters that match the consensus binding motif of Dsx. Thus, bnl might be repressed both directly and indirectly by Dsx, in a coherent feed-forward loop (FFL). FFLs attenuate noisy input signals. An FFL emanating from Dsx could provide a mechanism of robustly preventing bnl activation in female discs, despite potential fluctuations in DsxF levels (Chatterjee, 2011).

Understanding how Dr controls the morphogenesis of external structures is also important. The posterior lobe will be of particular interest because it is the most rapidly evolving morphological feature between D. melanogaster and its sibling species. Mutations in Poxn and sal also impair posterior lobe development. Understanding how these two regulators work with Dr to specify and pattern the developing posterior lobe could substantially advance efforts to understand its morphological divergence. Likewise, understanding how lz governs spermathecal development could advance evolutionary studies, as this organ also shows rapid evolution (Chatterjee, 2011).

The extent to which the regulators that were identified play deeply conserved roles in genital development remains to be determined. Although sex-determination mechanisms evolve rapidly, some features are shared by divergent animal lineages. The observation that FGF signaling is crucial to male differentiation in mammals, or that mutations in a human sal homolog cause anogenital defects, could reflect ancient roles in genital development or convergent draws from the toolkit (Chatterjee, 2011).

Whether AP-2, Dr and lz play conserved roles in vertebrate sexual development is similarly uncertain. In mice, an AP-2 homolog is expressed in the urogenital epithelium (albeit in both sexes) and at least one AP-2 homolog shows sexually dimorphic expression (albeit in the brain). The mouse Dr homolog Msx1 is expressed in the genital ridge and Msx2 functions in female reproductive tract development. In chick embryos, Msx1 and Msx2 are expressed male specifically in the Müllerian ducts. The mouse lz homolog Aml1 (Runx1) is expressed in the Müllerian ducts and genital tubercle. As more data accumulate on the genetic mechanisms controlling genital development in other taxa, the question of how deeply these mechanisms are conserved might be resolved (Chatterjee, 2011).

Direct targets of the D. melanogaster DSXF protein and the evolution of sexual development

Uncovering the direct regulatory targets of doublesex (dsx) and fruitless (fru) is crucial for an understanding of how they regulate sexual development, morphogenesis, differentiation and adult functions (including behavior) in Drosophila melanogaster. Using a modified DamID approach, 650 Dsx-binding regions were indentified in the genome, from which an optimal palindromic 13 bp Dsx-binding sequence was then extracted. This sequence is functional in vivo, and the base identity at each position is important for Dsx binding in vitro. In addition, this sequence is enriched in the genomes of D. melanogaster (58 copies versus approximately the three expected from random) and in the 11 other sequenced Drosophila species, as well as in some other Dipterans. Twenty-three genes are associated with both an in vivo peak in Dsx binding and an optimal Dsx-binding sequence, and thus are almost certainly direct Dsx targets. The association of these 23 genes with optimum Dsx binding sites was used to examine the evolutionary changes occurring in Dsx and its targets in insects (Lu, 2011).

The DamID technique was modified to take advantage of next-generation sequencing. Previous whole-genome DamID experiments largely used array-based methods. In these approaches, the methylated fragments are isolated by PCR-based methods, and therefore are subjected to PCR bias, although efforts were made to minimize such bias. Still, the recovery of a fragment depended on the GATC sites at both ends being methylated, and large fragments produced from GATC-poor regions would be less efficiently amplified. In this approach, each GATC site is evaluated independently. Two sequence tags are generated from each methylated GATC site, and this does not depend on the methylation status of nearby GATC sites. In addition, there are only minimal PCR cycles (<13) employed during library construction and the sizes of PCR products are very uniform (125+/-1 bp); hence, there is minimal PCR bias (Lu, 2011).

In previous reports where cDNA arrays were used, only binding regions close to exons could be identified. As transcription factor-binding sites are often located in the intergenic regions or in introns, using a cDNA array would probably miss many binding regions. Using tilling arrays may avoid the problem of missing target regions distant from genes, but tilling arrays are limited by the fraction of the genome that can be put on a chip. Moreover, the information is harder to compare between different versions of arrays. To avoid these pitfalls, ultra high-throughput sequencing was used to generate digitized actual sequences that can be directly matched to the genome and can be easily compared with results from different experiments. Furthermore, the array-based methods are more easily saturated, thus resulting in false-negatives, whereas the ultra high-throughput sequencing in theory has an infinite linear range (Lu, 2011).

For a binding region to be identified, the corresponding target gene does not need to be in a transcriptionally active state when the Dam-fusion protein experiment is carried out. In the case of the Yp1 DSX-binding region, a three- to ninefold higher normalized methylation level was observed for the DSXF-Dam sample versus Dam alone sample in third instar larvae, even though the Yp1 gene is not transcribed until after eclosion, some 4 days later (Lu, 2011).

There are two possibilities for this lack of temporal specificity. One is that it is an intrinsic feature of the DamID approach. It has been suggested that transcription factors are always in a dynamic mode of on and off their binding sites. The Dam-fusion protein might be highly efficient in methylating the GATC sites around its binding sites, so that even transient binding of the protein is sufficient to generate methylation. Alternatively, the DSX-binding sites may be readily recognizable by the DSX protein either because of their sequences per se or because of other factors associated with the sites are already in position, so that when the DSX-Dam protein is expressed at an earlier stage, it binds to these pre-arranged binding sites (Lu, 2011).

The findings contribute at several levels to an understanding of the evolution of sex. Considerations of where in developmental regulatory hierarchies the mutations underlying evolutionary changes are most likely has led to the proposal that such changes are more likely to be in the cis-regulatory elements of the targets of the transcription factors encoded by the terminal regulatory genes in a hierarchy, than in the coding sequences of the terminal regulatory genes themselves (Lu, 2011).

With respect to the sex hierarchy, the limited data previously available are consistent with this proposal. Thus, in the Dsx DNA-binding domain (DM domain), the Zn module, as well as the N-terminal region of the tail (amino acids 78-97) immediately following the Zn module [which has been proposed to be the functionally important part of a recognition helix providing the binding specificity, are highly conserved among these Drosophila species. Of the four previously identified direct Dsx targets, only two (bab and Fad2) exhibit changes in their sex-specific expression patterns within the Drosophila genus and in both cases these changes in the sex specificity of their expression are correlated with changes in adjacent Dsx-binding sites (Lu, 2011).

The 23 genes identified as very probable direct Dsx targets provide a broader data set with which to examine this proposal. Within the four other sequenced members of the melanogaster subgroup species, 21/23 of these genes are also associated with putative Dsx-binding sites (13/13 or 12/13 matches) in all four species, whereas 2/23 genes are not associated with close matches to the optimal Dsx-binding site in at least one species, suggesting that the latter may be cases in which the sex-specificity of expression of a gene has changed via an alteration of its cis-acting Dsx-binding sequence. When the data from these 23 genes were examined across all 12 sequenced Drosophila species, a similar result was seen: in 23%-35% of the cases an association between the gene and a Dsx-binding site is retained in all species, while for 65%-72% of these genes, a recognizable Dsx-binding site is not present in one or more of these species. These findings with respect to the latter group of genes are consistent with a potential change in the sex specificity of expression as a consequence of a change in the adjacent Dsx-binding site (Lu, 2011).

A second theme in modern considerations of the evolution of sex is that sex evolves rapidly when compared with other developmental processes. This view derives in part from the wide diversity in sex determination mechanisms used in animals, and findings that the primary sex determination mechanisms can differ in closely related species. Furthermore, and probably independently, at a developmental level evidence for the rapid evolution of sex comes directly from the myriad of secondary sexual characteristics that frequently distinguish closely related species, which otherwise have nearly identical body plans. The data, cited immediately above, provides a relatively broad look at the molecular genetic level at the frequencies with which there are potential changes between sex-specific and sex-nonspecific patterns of expression of downstream effector genes. Consistent with relatively rapid evolutionary changes in the sex-specificity of expression of some of the downstream genes in the Drosophila sex hierarchy, it was found that ~70% of the 23 D. melanogaster genes associated with Dsx-binding sites are not associated with such a site in one or more of the other 11 sequenced Drosophila species (Lu, 2011).

It is also important to note that these data, in addition to pinpointing a set of genes whose roles in sexual development may have changed during the evolution of this genus, also identify another subset of these genes, comprising ~30% of the putative direct Dsx targets in D. melanogaster, that retain an association with putative Dsx-binding sites in all 12 Drosophila species examined. The latter finding suggests that a significant subset of the genes directly regulated by Dsx have functions in aspects of sexual development that are evolutionarily relatively stable. Such evolutionarily conserved aspect of maleness or femaleness might encompass, for example, somatic functions that are necessary for sex-specific germline functions or gametogenesis (Lu, 2011).

Extending this analysis of the optimal Dsx-binding site and the related 13 bp palindromic sequences beyond the Drosophila genus provided additional evolutionary insights. In two mosquito species, Aedes aegypti and Anopheles gambiae, enrichment in the optimal Dsx-binding site is also observed. However, in the mosquito species Culex pipiens, although the number of optimal Dsx-binding sites is still 10-fold more than expected, the related sequence aCgACAATGTcGt had the highest number of perfect matches. Further analysis found that this latter sequence is within a repetitive element of the C. pipiens genome, so it is infered that GCAACAATGTTGC is most probably the optimal Dsx site in this species. The enriched sequence, cgtACAATGTacg, in the silkworm Bombyx mori is also within a repetitive element. None of the 24 sequences are enriched for the more distantly related insect species (Tribolium castaneum, Apis mellifera, Nasonia vitripennis, Acyrthosiphon pisum, and Pediculus humanus corporis) examined. Thus, in insects outside of the Diptera, the Dsx proteins might have diverged sufficiently that they bind to sequences different than the 24 examined (Lu, 2011).

Extending approaches such as those described in this study may provide significant insights into how a DNA-binding domain and its targets have evolved to maintain control of a fundamental developmental process across a broad evolutionary timescale (Lu, 2011).

Recurrent modification of a conserved cis-regulatory element underlies fruit fly pigmentation diversity

The development of morphological traits occurs through the collective action of networks of genes connected at the level of gene expression. As any node in a network may be a target of evolutionary change, the recurrent targeting of the same node would indicate that the path of evolution is biased for the relevant trait and network. Although examples of parallel evolution have implicated recurrent modification of the same gene and cis-regulatory element (CRE), little is known about the mutational and molecular paths of parallel CRE evolution. In fruit flies, the Bric-a-brac (Bab) transcription factors control the development of a suite of sexually dimorphic traits on the posterior abdomen. Female-specific Bab expression is regulated by the dimorphic element, a CRE that possesses direct inputs from body plan (Abd-B) and sex-determination (Dsx) transcription factors. This study finds that the recurrent evolutionary modification of this CRE underlies both intraspecific and interspecific variation in female pigmentation in the melanogaster species group. By reconstructing the sequence and regulatory activity of the ancestral Drosophila melanogaster dimorphic element, this study demonstrates that a handful of mutations were sufficient to create independent CRE alleles with differing activities. Moreover, intraspecific and interspecific dimorphic element evolution proceeds with little to no alterations to the known body plan and sex-determination regulatory linkages. Collectively, these findings represent an example where the paths of evolution appear biased to a specific CRE, and drastic changes in function are accompanied by deep conservation of key regulatory linkages (Rogers, 2013).

In the D. melanogaster pigmentation network, the bab genes function as an Input-Output node through the dimorphic element's integration of patterning inputs that include body plan (ABD-B) and sex determination (DSX) pathway inputs. These inputs are converted into a female-specific pattern of expression that culminates in the repression of the differentiation genes yellow and tan in females. In principle, changes in the expression or activity of a patterning gene, differentiation gene, or the Input-Output gene (bab) could alter pigmentation phenotypes. In application though, it is logical that bab expression and dimorphic element encodings were modified as those alterations minimize negative pleiotropic effects while being sufficient to alter the female pigmentation phenotype. For example, ectopic yellow expression fails to create additional melanic pigmentation, and changes in either DSX or ABD-B expression result in ectopic abdominal pigmentation in addition to several other trait phenotypes. Thus, sufficiency for pigmentation is counterbalanced by the negative pleiotropic affects for these genes. In contrast, increased Bab expression in the A5 and A6 segments is sufficient to suppress pigmentation, and ectopic abdomen pigmentation develops in bab heterozygous and homozygous null mutant females (Rogers, 2013).

Bab though is not dedicated to pigmentation. In the pupa, Bab expression includes the leg tarsal segments, abdomen epidermis, sensory organ precursor cells, oenocytes, and dorsal abdominal muscles, and each of these expression patterns are governed by a modular CRE (s). Thus, Bab itself is highly pleiotropic, however it's CREs are far less pleiotropic. For this reason, mutations altering female pigmentation would maximize sufficiency and minimize pleiotropy if they occurred in the dimorphic element, an expectation borne out in this study. Pigmentation of the A5 and A6 segments, though, is only one of many traits influenced by the regulatory activity of the dimorphic element. This CRE drives Bab expression in the female A7 and A8 segments, regulating numerous female-specific traits, including the size, shape, trichome density, and bristle morphologies of the resident dorsal tergites and ventral sternites. As expression in these more posterior segments require the ABD-B and DSX regulatory linkages, these regulatory linkages remain highly pleiotropic. For this reason, it seems logical that evolution would disfavor mutations that have deleterious consequences to these linkages and favor mutations that alter other CRE properties. This scenario reflects how dimorphic element function was modified in both the intraspecific and interspecific comparisons presented presented in this study as well as the long term conservation of the ABD-B and DSX linkages previously described (Rogers, 2013).

The current findings provide a unique contrast with previous investigations of the relationship between CRE conservation and CRE evolution. Although Drosophila non-coding DNA, including CRE sequences, evolves slower than synonymous sites, several well studied CREs were found to undergo substantial sequence evolution without matching regulatory activity evolution. During Drosophila embryonic development, the pair-rule gene even-skipped (eve) is expressed in seven stripes along the anteroposterior axis, with the second stripe of eve expression being specified by the stripe 2 element (S2E) CRE. In D. melanogaster, the S2E possesses binding sites for four transcription factors that collectively specify the eve expression output. The orthologous S2E from the species D. pseudoobscura differs in sequence for numerous binding sites, the overall content of binding sites, and spacing between conserved binding sites, yet the orthologous S2Es function equivalently in vivo. Hence, the S2E is an exemplar as to how selection acting at the level of the character (eve stripe expression) can accommodate a surprising amount of CRE evolution. Similarly, CRE sequence evolution without corresponding functional evolution was found between Drosophila species for the sparkling (spa) CRE that directs cone cell expression for the dPax2 gene. The content and spatial proximity of binding sites for neurogenic ectoderm enhancers (NEEs) evolved in order to conserve expression pattern outputs in response to changing regulatory inputs. These case studies, demonstrate how CRE sequence conservation is not a prerequisite for CRE functional conservation (Rogers, 2013).

In contrast, this study found little divergence in the content and sequence of known binding sites for the D. melanogaster dimorphic element alleles and orthologous sequences. At the sequence level, these CRE alleles and orthologs respectively posses identities of ~98% and ~80%. Indeed, the vast majority of binding sites in the dimorphic element have been conserved for over 30 million years, showing conservation to D. willistoni. At the functional level, these CREs exhibited striking differences in their regulatory activities. Thus, in contrast to S2E, spa, and the NEEs, the dimorphic element demonstrates how CREs can derive dramatic changes in function that drive phenotypic divergence, with little-to-no alteration to the characterized pre-existing regulatory linkages (Rogers, 2013).

While the regulatory activity of the Light and Dark dimorphic elements alleles correlated with female A5 and A6 pigmentation, some outcomes suggest that these variant sequences are affected by other features within or perhaps outside of the bab locus. For instance, the Light 2 and Dark 2 alleles exhibit the highest and lowest regulatory activities respectively. Surprisingly, the Light 1 and Dark 1 alleles and their intermediate regulatory activities are associated with the more extreme Light and Dark female pigmentation phenotypes. At the expression level, Bab1 and Bab2 showed similar patterns in females from the Light 1 (prominent expression in segments A5 and A6) and Dark 1 (reduced expression is A5 and A6) strains. In the Dark 2 strain, Bab1 but not Bab2 expression was reduced in females. Several possible explanations might explain the uncoupled expression of the Bab paralogs in Dark 2. For example, it is possible that a separate, as of yet unidentified CRE controls Bab2 expression. However, a screen of the entire ~160 kb locus failed to identify such a CRE. A second possibility is that a mutation(s) in the Dark 2 allele has paralog-specific regulatory effects, perhaps by modifying an interaction with the promoter for bab1 but not that of bab2 (Rogers, 2013).

Another possible explanation would involve the existence of CREs that coordinate communication between bab1 and bab2. In such a scenario, the Dark 2 allele could contain mutations that alter interaction with coordinating elements to result in paralog-specific expression patterns in the female A5 and A6 segments. This possibility is consistent with observations of bab locus evolution in another population where females differ in A6 segment pigmentation. For this population, fine-scale genetic mapping found that three disparate non-coding regions of the bab locus collaborate to compose a major effect QTL. One of these regions spans the dimorphic element, though no mutations reside with this CRE's core element. The other two regions include an intergenic sequence between bab1 and bab2 and a large sequence that includes the bab2 promoter. In the future, it will be important to understand what roles these other regions serve, and how they may interact with polymorphisms in the dimorphic element to produce paralog-specific effects on gene expression (Rogers, 2013).

With the centrality of CREs and their evolution to the diversification of phenotypic traits, a major obstacle to reaching this goal is understanding the processes by which CRE regulatory logics were modified to contemporary forms. Often studies of CRE evolution involve comparisons of two divergent derived regulatory states, where one sequence assumes the role of a surrogate for the ancestral function. This approach has been successful in making inferences about the ancestral states for regulatory linkages and identifying gains and losses of other key derived transcription factor binding sites. However, it is important to acknowledge a key limitation of this comparative approach; a CRE derived from an outgroup species that serves as a surrogate for the ancestor has also evolved along a unique lineage since divergence (Rogers, 2013).

Studies into the evolution of divergent protein activities encountered a similar problem when comparing extant proteins forms. For several cases, key amino acid residues necessary for a derived function were identified. When substituted into the surrogate ancestral protein, these changes were insufficient to impart the derived function and thereby indicating that the paths of evolution were more intricate. As a solution, the reconstruction of ancestral protein sequences, combined with functional testing of inferred ancestral proteins has allowed a more realistic simulation of evolutionary events. As a result, inferences about the paths of protein evolution were made that likely would not have been found from comparisons of extant proteins (Rogers, 2013).

A more ideal research program to study CRE evolution would include reconstruction of ancestral CREs as a starting point to trace the paths of evolutionarily relevant mutations. Few studies have used CRE reconstruction. For one study, a novel optic lobe expression pattern for the D. santomea Nep-1 gene occurred via the modification of a CRE that drove an eye field pattern of expression for an ancestor that existed ~0.5 million years ago. Importantly, by reconstructing and evaluating the ancestral CRE, the wrong conclusion - that this optic lobe activity evolved de novo – was avoided and the correct conclusion was found - a latent optic lobe CRE activity was augmented into a robust derived state. In the current study, had the Concestor element not been reconstructed, the Dark 1 and Dark 2 dimorphic element sequences would have been considered hypomorphic CRE alleles compared to the robust wild type-like activity of the Light 1 and Light 2 alleles. The Light alleles possessed activities more similar to a previously characterized dimorphic element allele and consistent with the narrative of D. melanogaster being a sexually dimorphic species where females lack posterior abdominal pigmentation. Reconstruction of the dimorphic element revealed a more complex reality, where neither alleles were good surrogates for the ancestral state. Using ancestral sequences as a starting point, this study found that the evolutionary paths for these alleles to be short in number of steps (one to two mutations) and in time frame (in the last ~60,000 years). Thus, demonstrating how simple and rapid an existing CRE regulatory logic can evolve (Rogers, 2013).

The cases of Nep1 optic lobe CRE and the bab dimorphic element evolution demonstrate the utility for reconstructing ancestral CRE states; though it must be pointed out that these cases involved comparisons of very closely-related species/populations. As a result of these short time frames for divergence, the extant CRE forms differ at fewer than two percent of the nucleotide sites. This made possible ancestral sequence reconstruction by the principle of parsimony. However, not all compelling instances of functional CRE evolution occur over similarly short time frames. Therefore, studies will need to reconstruct CREs that existed further in the past and for which the method of parsimony will need to be replaced by methods of maximum likelihood-based inference coupled with the testing of multiple alternate reconstructions (Rogers, 2013).

doublesex: Biological Overview | Evolutionary Homologs | Protein Interactions and Regulation of Splicing | Developmental Biology | Effects of Mutation | References

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