The Interactive Fly

Genes involved in tissue and organ development

Development of Genitalia

Drosophila terminalia as an appendage-like structure
Genetic control and evolution of sexually dimorphic characters in Drosophila
Doublesex controls the development of the Drosophila genital disc, modulating the response to Hedgehog, Wingless and Decapentaplegic signals
The Drosophila sex determination hierarchy modulates wingless and decapentaplegic signaling to deploy dachshund sex-specifically in the genital imaginal disc
The sex determination gene doublesex regulates the A/P organizer to direct sex-specific patterns of growth in the Drosophila genital imaginal disc
Doublesex represses branchless in the in female genital discs: Branchless positive cells recruit mesodermal cell into the male genital imaginal disc
The Hox gene Abdominal-B antagonizes appendage development in the genital disc of Drosophila
Requirement of Abdominal-A and Abdominal-B in the developing genitalia of Drosophila breaks the posterior downregulation rule
Regulation of Abd-B expression by Cyclin G and Corto in the abdominal epithelium of Drosophila
Type ID unconventional myosin controls left-right asymmetry in Drosophila
Coupling of apoptosis and L/R patterning controls stepwise organ looping
Apoptosis controls the speed of looping morphogenesis in Drosophila male terminalia
The female-specific doublesex isoform regulates pleiotropic transcription factors to pattern genital development in Drosophila

Drosophila terminalia as an appendage-like structure

This study reports the expression pattern of Dll in the genital disc, the requirement of Dll activity for the development of the terminalia and the activation of Dll by the combined action of the morphogenetic signals Wingless (Wg) and Decapentaplegic (Dpp). In Drosophila, the terminalia comprise the entire set of internal and external genitalia (with the exception of the gonads), and includes the hindgut and the anal structures. They arise from a single imaginal disc of ventral origin that has a complex organization and shows bilateral symmetry. The genital disc shows extreme sexual dimorphism. Early in development, the anlage of the genital disc of both sexes consists of three primordia: the female genital primordium (FGP); the male genital primordium (MGP), and the anal primordium (AP). In both sexes, only two of the three primordia develop: the corresponding genital primordium and the anal primordium. These in turn develop, according to the genetic sex, into female or male analia. The undeveloped genital primordium is the repressed primordium (either RFP or RMP, for the respective female and male genital primordia) (Gorfinkiel, 1999).

During the development of the two components of the anal primordium -- the hindgut and the analia -- only the latter is dependent on Dll and hedgehog (hh) function. The hindgut is defined by the expression of the homeobox gene even-skipped. The lack of Dll function in the anal primordia transforms the anal tissue into hindgut by the extension of the eve domain. Meanwhile targeted ectopic Dll represses eve expression and hindgut formation. The Dll requirement for the development of both anal plates in males and only for the dorsal anal plate in females, provides further evidence for the previously held idea that the analia arise from two primordia. In addition, evaluation was made of the requirement for the optomotor-blind (omb) gene which, as in the leg and antenna, is located downstream of Dpp. These results suggest that the terminalia show similar behavior as the leg disc or the antennal part of the eye-antennal disc, consistent with both the proposed ventral origin of the genital disc and the evolutive consideration of the terminalia as an ancestral appendage (Gorfinkiel, 1999).

The expression pattern of Dll in the genital disc was analyzed. Dll is neither expressed in the embryonic terminalia nor in the embryonic precursor cells of the genital disc. In the female third larval instar genital disc, Dll shows a localized distribution; it is strongly expressed in a large spot in the central part of the anal primordium and in a faint band of cells in the genital primordium. It is not detected in the RMP. Similarly, in the male genital disc, Dll is expressed in a large spot both in the anal primordium and in the male genital primordium but not in the RFP. Several GAL4 insertions in the Dll locus were used and these permitted the identifcation of the adult regions where Dll is expressed according to the observed X-Gal staining. In females, Dll is expressed in the vaginal plates and in the anal plates. In the dorsal anal plate, Dll is expressed in a generalized manner, while the ventral plate shows fainter Dll expression, which is stronger in the distal part of the plate. In males, Dll is expressed in the claspers and anal plates. The expression, both in male and female external terminalia, is as predicted by the prospective fate map of the genital disc. The internal structures are not well defned in term of Dll expression (Gorfinkiel, 1999).

To investigate whether there is a functional requirement for Dll in the terminalia, the phenotype of different viable Dll mutant combinations was analyzed. These Dll hypomorphic mutant combinations were initially described by their phenotype in the leg and antenna. In the allelic series homozygous Dll3, DllIB/DllMP and Dll3/DllMP, the female dorsal anal plate is reduced whereas the ventral anal plate is normal. The vaginal plates are disorganized. In males, the anal plates are strongly reduced. The external genital structures are, however, unaffected. These phenotypes show that the Dll expression domains do not fully correspond to its requirement. This led to a search for alterations in the internal genital structures. In both males and females, the internal genitalia appear normal. Surprisingly, under the strongest hypomorphic conditions (Dll3/DllMP), the hindgut is enlarged in both females and males. The few anal structures that remain are surrounded by hindgut tissue. This result suggests an expansion of hindgut territory at the expense of the anal plates. To further investigate the requirement for Dll, Dll SAI clones (marked with yellow) were induced during the larval stages. Dll2 clones do not develop anal plates (males) or dorsal anal plates (females). These clones were recognized since some of them still differentiate yellow (y) bristles. Dll2 clones do not show detectable alterations in the male external genitalia. Since only a few structures of the genitalia can be analyzed with the y marker, it is possible that minor phenotypic alterations may go undetected. Dll 2clones do not affect the development of the female ventral anal plates. Thus, although Dll is also expressed in the ventral anal plate in females and the claspers in males, it seems that it is not required for the formation of these structures. These results are in agreement with those observed using the viable Dll mutant combinations (Gorfinkiel, 1999).

Both the female and male anal primordia give rise to two different adult structures: the hindgut and the anal plates. These territories are well defined by the complementary expression of the homeotic genes Dll and even-skipped (eve). Adult regions that express Dll and eve were defined by X-Gal staining of Dll-GAL4/UAS-LacZ and eve-lacZ- flies, respectively. These two genes show a complementary expression pattern. Dll is expressed in the anal plates of both females and males but not in the hindgut. In contrast, eve is expressed in the hindgut of both females and males. Some residual Dll expression is detected in the rectal papillae, but these structures are not derived from the genital disc. Thus, the adult analia and hindgut are defined by Dll and eve expression patterns, respectively. Also in the genital disc, eve labels the prospective hindgut that occupies the central part of the anal primordium while Dll marks the primordia of the anal plates located at both ends of the primordia in both females and males. This eve expression both in discs and adults suggests that eve is required for hindgut development (Gorfinkiel, 1999).

In the Dll hypomorphic combinations the hindgut is enlarged and the anal plates are reduced. This phenotype correlates with gene expression, since in Dll2clones, eve expression extends into the anal territory both in females and males. In some Dll2 clones eve is only activated in the part of the clone nearest to the normal eve-expression domain. This indicates that there is a region capable of activating eve that is then transformed to hindgut. This region could correspond to the prospective dorsal analia in females where Dll is specifically required (Gorfinkiel, 1999).

To test if a mutually repressive interaction between the homeotic genes Dll and eve in the anal primordium can lead to their complementary expression domains, either Dll or eve were ectopically expressed in the presumptive cells of both the hindgut and analia using the cad-GAL4 line. In UAS-Dll/cad-GAL4 discs, eve is not expressed and there is a reduction of the whole primordium. The Dll domain is also reduced in all ventral discs upon Dll ectopic expression because an excess of Dll represses its own expression. The adult flies do not show hindgut structure and the anal plates are also reduced. In UAS-eve/cad-Gal4 discs there is a reduction of the Dll domain along with an enlargement of the hindgut primordium, but there are still cells that co-express Dll and eve. Therefore, it is concluded that the complementary expression domains of Dll and eve in the anal primordium is due to eve repression by Dll (Gorfinkiel, 1999).

Hh signal is required to form the genital and anal structures but not the hindgut. In the leg and antennal discs, the expression of Dll depends on the Hh signaling pathway. Using the hh ts2 allele, it was observed that in the genital disc, Hh is also required for Dll activation: after 4 days at the restrictive temperature, the genital discs are very small and show no Dll expression. In the same hh ts2 larvae, residual Dll expression can be detected in the trochanter region of the leg disc. However, eve expression in the anal primordia is maintained and occupies most of the reduced genital disc. This result indicates that Dll, but not eve expression, depends on Hh and that all the terminalia with the exception of the hindgut require Hh function. To further analyze this hh requirement for Dll activation, the effect of smoothened (smo) lack of function was examined. In smo2 cells, Hh reception is impeded because smo is a component of the Hh receptor complex. In the genital disc, Dll expression only disappears in smo2 clones when the clone is large enough to cover most of the Dll expression domain. Accordingly, eve expression is also ectopically activated in smo2 mutant cells; although in Dll2 cells eve cannot be activated in certain regions of the clones. These results indicate once again that Dll is dependent on Hh function while eve is not (Gorfinkiel, 1999).

Large smo2 clones close to the A/P compartment transform some structures of the external genitalia and analia. In the female genitalia, smo2 clones duplicate the long bristle of the vaginal plates and clones in T8 to produce tissue overgrowth with y2 bristles. Large smo2 clones reduce the female dorsal anal plate, whereas the female ventral anal plate is rarely affected. Some clones produce segregated tissue in the female analia labelled with y bristles in the perianal region. However, small clones or clones located outside the A/P compartment border have no effect. In the male genitalia, smo2 clones duplicate the genital arc, part of the claspers and the hypandrium bristle. All these structures are located close to the A/P compartment border. As in Dll2 clones, large smo2 clones delete the anal plate in males. In both males and females, only when the clone is large enough can Dll expression not be activated in the disc primordia, giving rise to the Dll2 phenotype. This result suggests that only in large smo2 clones both wg and dpp are not activated and therefore are unable to induce Dll expression (Gorfinkiel, 1999).

The hh requirement for the analia but not for the hindgut has also been confirmed by the ectopic expression of Cubitus interruptus (Ci). ci encodes a transcription factor that acts as an activator of the target genes of the Hh pathway. The overexpression of Ci in the anal primordia of cad-GAL4/UAS-ci flies, leads to the enlargement and fusion of the anal plates. Accordingly, the Dll expression domain in the genital disc is expanded to cover most of the primordia and the eve domain is reduced. This again demonstrates the complementary and exclusive nature of the eve and Dll domains in the anal primordia (Gorfinkiel, 1999).

The requirement for the Hh signal in Dll activation might be mediated by Wg and Dpp signals. This occurs in other ventral discs. Dll expression arises at the juxtaposition of Wg and Dpp expressing cells as revealed by double staining for Dll and Dpp, and Dll and Wg. In both genital and anal primordia, Dll expressing cells overlap those that express wg and dpp. It has been previously reported that the ectopic expression of both Wg and Dpp produces several phenotypic alterations in both female and male terminalia. Similar types of transformations are also induced by the lack of function of either patched (ptc) or Protein kinase A (Pka). In these mutants, the Hh pathway is constitutively active giving rise to the derepression of Wg and Dpp. The lack of Pka function in the genital disc induces ectopic Dll. This Dll induction requires both Wg and Dpp signals in the same cells since Dll is not activated in Pka2;dpp2 and in Pka2;wg2 double mutant clones, as occurs in other discs of ventral origin (Gorfinkiel, 1999).

In the male repressed primordium (RMP) of the female genital disc, wg is expressed but not dpp. Consequently, Dll is not expressed because Dll is only activated in cells that express both dpp and wg. Ectopic Dpp expression in the wg expression domain driven by the MS248-GAL4 line induces Dll 'de novo' in the RMP, which shows an increase in size. However, these changes do not allow the development of adult structures from this primordium since there is no activation of the male specifc cyto-differentiation genes because the genetic sex has not changed. Dll is not activated in the repressed female primordium (RFP) of the male genital disc despite the fact that, in this primordium, both wg and dpp are normally expressed. This activation does not occur even if the levels of Dpp are increased. These results suggest that specific genes expressed in the RFP can exert a negative control of Dll expression (Gorfinkiel, 1999).

In order to find other genes involved in the development of the terminal structures, the expression pattern and the functional requirement for optomotor-blind (omb) were examined. This gene encodes a protein with a DNA-binding domain (T domain) and behaves as a downstream gene of the Hh pathway in other imaginal discs. In the genital disc, Omb is detected in the dpp expression domains, abutting the wg expressing cells. This behaviour of omb expression is similar to that found in the leg and antennal discs. In the genital disc, omb is also regulated by the Hh signaling pathway since Pka2 clones also ectopically express omb. The phenotypes produced due to omb lack of function using the allele omb282 were examined; homozygous females for this allele could not be obtained but some male pharates were analyz

ed. In males, the dorsal bristles of the claspers and the hypandrium bristles are absent. Also, the hypandrium is devoid of hairs and the hypandrium fragma is reduced. Surprisingly, the anal plates are mostly somewhat enlarged in the ventral region and reduced in the dorsal areas. The structures affected in omb2 are duplicated when omb is overexpressed in the dpp domain using the dpp-GAL4/UAS-omb combination. In males, the dorsal bristles of the clasper and the hypandrium bristles are duplicated. These phenotypes are similar to the ones obtained as a result of ectopic Dpp (Gorfinkiel, 1999).

The present findings provide further evidence that the terminalia can be considered a ventral appendage. In addition, this is the first evidence for a similar genetic pathway operating in both the ventral and genital/anal appendages. From an evolutionary point of view, this lends support to the idea that the terminalia arose as the result of the modification of a primitive appendage in an ancestor common to all arthropods. The Dll requirement for some of the external genital structures broadens the definition of the evolutionarily conserved Dll function to cover a more fundamental role than that of the proximo/distal selector gene in the appendages (Gorfinkiel, 1999).

The Dll requirement for the formation of only the dorsal anal plate in females and for both anal plates in males lends further support to the idea that the anal plates form from two primordia. It is proposed that the anal primordia develop as follows: at the blastoderm stage, the anal primordium divides into two cell populations, one of which will form the dorsal analia in a female or the complete analia in a male (homologous anal primordium), while the other set of cells will form the ventral analia in a female and give rise to no structure in a male (non-homologous primordium). Previous reports using the transformed2ts (tra2ts) mutation are also consistent with this organization. In the switch from male to female development, using the tra2ts mutant, the ventral anal plate requires more time to reach a normal female phenotype than the dorsal anal plate. In the shift from the female-to-male determining temperature, the homologous anal primordium switches into the male program while the non-homologous primordium is brought into the repressed state. The functional requirement for Dll only in the homologous anal primordium supports the idea that the anal plates originate from two primordia. It also suggests the existence of other gene(s) responsible for the formation of the ventral anal plate in females (Gorfinkiel, 1999).

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).

The Drosophila sex determination hierarchy modulates wingless and decapentaplegic signaling to deploy dachshund sex-specifically in the genital imaginal disc

The integration of multiple developmental cues is crucial to the combinatorial strategies for cell specification that underlie metazoan development. In the Drosophila genital imaginal disc, which gives rise to the sexually dimorphic genitalia and analia, sexual identity must be integrated with positional cues, in order to direct the appropriate sexually dimorphic developmental program. Sex determination in Drosophila is controlled by a hierarchy of regulatory genes. The last known gene in the somatic branch of this hierarchy is the transcription factor doublesex (dsx); however, targets of the hierarchy that play a role in sexually dimorphic development have remained elusive. The gene dachshund (dac) is differentially expressed in the male and female genital discs, and plays sex-specific roles in the development of the genitalia. Furthermore, the sex determination hierarchy mediates this sex-specific deployment of dac by modulating the regulation of dac by the pattern formation genes wingless (wg) and decapentaplegic (dpp). The sex determination pathway acts cell-autonomously to determine whether dac is activated by wg signaling, as in females, or by dpp signaling, as in males (Keisman, 2001a).

A number of obstacles make it difficult to demonstrate that the sex determination pathway is responsible for the sex-specific regulation of a gene in the genital disc. These obstacles stem from the fact that the male and female primordia, which are the primary constituents of their respective discs, differ in their segmental origin. This raises the possibility that 'sex-specific' gene regulation is really just segment-specific gene regulation, made to look sex specific by the fact that only one primordium develops in each sex. Attempts were made to address this concern by creating clones of the opposite genetic sex in chromosomally male and female genital discs. Thus, for example, dac regulation could be examined in the male (A9) primordium, in both male and female cells. By varying the genetic sex of cells in a context where segmental identity is uniform, it was hoped that the contributions of sex and segmental identity to dac regulation could be disentangled (Keisman, 2001a).

In the male primordium of both male and female discs, the regulation of dac varies according to the genetic sex of the cell. Genetically female clones in the male (A9 derived) primordium of the male genital disc are unable to express dac in the lateral male (dpp-dependent) domain, but are able to express dac when they extended medially, towards the source of Wg. Conversely, in the female genital disc, genetically male clones in the repressed male primordium (A9) lose their ability to express dac in the medial, wg-dependent domain, and begin to express dac laterally, presumably in response to Dpp. Finally, dac expression is abnormal in intersexual genital discs from dsx mutant larvae: the male primordium of dsx genital discs expresses dac in both the endogenous, lateral male domains, and in a slightly weaker medial domain that corresponds roughly to the region where tra + clones are able to activate dac. Thus, it is concluded that in the male primordium, the sex determination pathway determines how a cell will regulate dac (Keisman, 2001a).

In the female primordium the results fail to show a role for the sex determination pathway in dac regulation. If such a role exists, it would be expected that genetically male clones in the female primordia of a female genital disc would activate dac laterally, like their counterparts in the male primordia. They do not, even when they take up much of the presumptive dpp-expressing domain. It would also be expected that such clones would repress dac medially. Only a few clones were observed to extend into the medial wg-expressing domain, and as expected these appear to repress dac. Interpretation of these results is complicated by the fact that changing the genetic sex of a cell in the genital disc can cause it to enter the 'repressed' state. Thus, for example, if a genetically male clone represses dac when it intersects the medial dac domain in the female primordia, it can be concluded either that the sex determination pathway regulates dac expression or that the cells, which are now male, have adopted a repressed state and are generally unresponsive. A similar caveat prevents interpreting the failure of tra2IR clones to activate dac ectopically in the female primordium. That tra + clones in the male primordium of male genital discs enter such a generally non-responsive state was not of concern, because these clones both repress and activate dac expression. The expression pattern of dac in the female primordium of a dsx mutant genital disc is also difficult to interpret. dac is not activated ectopically in the lateral domains of the dsx female primordium, which is consistent with the failure of tra2IR clones to cause such activation. However, even the medial, wg-dependent dac domain is frequently absent or severely reduced in the dsx female primordium, and thus the authors are reluctant to draw any conclusions from the absence of ectopic dac laterally (Keisman, 2001a).

A model is proposed for dac regulation in the male primordium, in which the different isoforms of Dsx protein modulate dac regulation by wg and dpp. In the absence of dsx, both wg and dpp can activate dac, producing the two domains of dac expression observed in the male primordium of a dsx disc. In the female, Dsxf modulates dpp activity so that dpp becomes a repressor of dac; Dsxf may also potentiate the activation of dac by wg. In the male, Dsxm modulates wg activity so that it becomes a repressor of dac, leaving dpp alone to activate dac. In support of this model, it is noted that the Dsx proteins act in a similar manner to positively or negatively modulate the effect of tissue-specific regulators on the yp genes (Keisman, 2001a and references therein).

The behavior of tra + and tra2IR clones provides insight into the mechanism of repression in the undeveloped genital primordium. It was anticipated that such clones would be difficult to recover when they occurred in the male and female primordium, respectively, because they should adopt the repressed state. Instead, large tra + (female) clones were recovered in the male primordium of a male disc, and large tra2IR (male) clones were recovered in the female primordium of a female disc. Some of these clones constitute a substantial fraction of the primordium in question. Though tra + or tra2IR clones were not scored in adults, previous studies strongly suggest that such clones would fail to differentiate adult genital structures (Keisman, 2001a).

It has been shown that tra - (male) clones cause large deletions in the female genitalia, indicating that genetically male cells like those in a tra2IR clone divide but cannot differentiate female genital structures. Further, it has also been shown that male structures are deleted when the mosaic border passes through the male genitalia, suggesting that female tissue cannot differentiate male structures. To reconcile these data, it is proposed that repression of the inappropriate genital primordium involves two separable processes: repression of growth and the prevention of differentiation. Thus, clones of cells of the inappropriate genetic sex cannot differentiate, but they can grow and contribute to a morphologically normal genital primordium. This poses yet another question. Cells in a tra + clone in the male primordium of a male genital disc are analogous to the cells in the repressed male primordium of a wild-type female genital disc: both are genetically female, and both have A9 segmental identity. Why do tra + clones in the male primordium grow, while the repressed male primordium in a female disc does not? One possibility is that the decision of the male primordium to grow in a male disc is made before tra + clones were induced and cannot be over-ridden by a later switch of genetic sex. However, temperature-shift experiments with tra-2 ts alleles suggest that the decision of a genital primordium to develop can be reversed later in development. Furthermore, occasional, large tra + clones can cause severe reductions in male genital discs. This observation leads to the suggestion of a model in which growth in the genital disc is regulated from within organizing zones, such as the domains of wg and dpp expression. According to this model, the sex of the cells in the organizing regions would determine how the disc grows, while cells in other regions would respond accordingly, regardless of their sex. The tra + clones that cause reduction could result when such a clone intersects with one of the postulated organizing centers within the disc. The implication is that the sex determination pathway acts in yet undiscovered ways to modulate the function of the genes that establish pattern in the genital disc. One such interaction was found in the regulation of dac; further study is needed to determine if others exist, and what role they play in producing the sexual dimorphism of the genital disc and its derivatives (Keisman, 2001a).

The sex determination gene doublesex regulates the A/P organizer to direct sex-specific patterns of growth in the Drosophila genital imaginal disc

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).

The Hox gene Abdominal-B antagonizes appendage development in the genital disc of Drosophila

Abdominal-B is required to specify the posterior abdomen and the genitalia. Homologs of Abdominal-B in other species are also needed to determine the posterior part of the body. The function of Abdominal-B in the formation of Drosophila genitalia has been studied, and the absence of Abdominal-B in the genital disc of Drosophila has been shown to transform male and female genitalia into leg or, less frequently, into antenna. These transformations are accompanied by the ectopic expression of genes such as Distal-less or dachshund, which are normally required in these appendages. The extent of wild-type and ectopic Distal-less expression depends on the antagonistic activities of the Abdominal-B gene (as a repressor), and of the decapentaplegic and wingless genes (as activators). Absence of Abdominal-B also changes the expression of Homothorax, a Hox gene co-factor. These results suggest that Abdominal-B forms genitalia by modifying an underlying positional information and repressing appendage development. It is proposed that the genital primordia should be subdivided into two regions, one of them competent to be transformed into an appendage in the absence of Abdominal-B (Estrada, 2001).

Abd-B clones were induced, and they transform posterior abdominal segments into more anterior ones but are normal in the analia. Rare clones transform to distal antennae (second and/or third antennal segment and arista). Transformations to legs or antennae are cell autonomous. The formation of legs requires the activity of genes such as homothorax (hth), dac and Dll, which specify the proximal, medial and distal parts of the leg, respectively. Dll expression in wild-type discs is regulated by the combined activities of wingless and dpp in the genital primordia, and is confined to two groups of cells in male and female discs, the female domains being smaller and expressing lower levels of Dll protein. Since Abd-B is transcribed in the entire genital primordia of the two sexes, some cells co-express Abd-B and Dll. In the male disc, hth is not expressed in the Dll-expressing cells and is also excluded from a large group of cells surrounding them. Levels of antibody signal vary within the disc, and are higher in the female repressed primordium. In females, the hth domain of expression occupies the whole primordium. Lower levels of Hth are detected in a region encompassing the Dll-expressing cells, whereas higher levels are observed in the male repressed primordium. In both sexes, hth expression is absent from the anal primordium. dac is expressed differently in male and female genital primordia: in male discs, Dac protein is detected in two broad lateral bands, while in female discs it is found in the central region, almost coincident with the wg-expressing region. Therefore, the expression patterns of hth, dac and Dll differ substantially from those observed in legs (Estrada, 2001).

It is known that expression of Dll is not required to make male genitalia and that it has only a minor role in the formation of the female one. To ascertain the role of hth in the genitalia, hth minus clones were induced during the third larval period and they were examined in the adult structures. In the female genitalia, hth minus clones cause extra growths with additional vaginal teeth. In males, these clones show occasionally some abnormalities in the clasper teeth. hth clones in the analia are wild type. Possible interactions between Dll and hth in the genital disc were sought. In these experiments, unless stated, the results apply both to male and female genital primordia. Dll minus clones in the Dll domain of the male disc have no hth expression. Similarly, in hth minus clones Dll is not ectopically expressed. Dll was also expressed ectopically and the effect on hth expression was examined. Dll-expressing cells close to the wild-type Dll domain repress hth expression, although not all the cells do so. By contrast, clones far from the Dll domain do not affect hth expression (Estrada, 2001).

To characterize the transformation of genitalia into leg or antennal tissues, Abd-B minus clones were examined. Abd-B minus clones in the genital primordia tend to segregate from the rest of the tissue, round up and have smooth borders, suggesting they have acquired different affinities. By contrast, clones in the analia have indented borders and do not segregate. Abd-B minus clones in the genital primordium close to the normal Dll domain show ectopic, cell-autonomous Dll expression, whereas those far apart do not show such expression. dac is also activated cell autonomously in many Abd-B minus clones. As expected, Dll target genes, such as Bar, also become activated in these clones (Estrada, 2001).

Abd-B minus clones exhibit differential effects on hth, depending on their position: those close to the Dll domain show no hth expression, whereas those located away from the Dll domain show a slight increase in hth signal. Clones in intermediate positions do not significantly change hth levels. This distribution, however, is clearer in females, since in males there is a wide region with no hth expression. The repression of hth observed in some Abd-B minus clones may be mediated by the ectopic Dll (Estrada, 2001).

In the genital disc, the transcription of Dll depends, as in the leg disc, on dpp and wg signals. Abd-B represses Dll expression. Moreover, increasing Abd-B levels in the Dll domain suppresses Dll transcription. The antagonistic activities of dpp/wg and Abd-B in determining the Dll distribution was analyzed. Mutations in PKA ectopically activate wg and dpp expression. PKA minus clones in the genital primordia activate Dll, although only in some places. This activation is not mediated by changes in Abd-B levels. Similarly, although Dll is derepressed in many late Abd-B minus clones, derepression of either dpp or wg was not observed. It is concluded that there is an antagonism between the activation of Dll by dpp/wg signaling and its repression by Abd-B. This is not mediated by changes in the expression of either dpp, wg or Abd-B (Estrada, 2001).

To characterize this antagonism further, Abd-B minus clones that were made were also unable to transduce the dpp signal. This signal requires the presence of the type II receptor encoded by the gene punt. In put;Abd-B double mutant clones, Dll is not activated, indicating that, in the absence of Abd-B, Dpp (and possibly Wg) are still required to activate Dll. Abd-B minus clones far from the wild-type Dll domain fail to activate Dll ectopically, suggesting that activation of Dll in the absence of Abd-B depends on the range of diffusion of Dpp and Wg, as in the leg disc and in the anal primordium (Estrada, 2001).

Dll is required for the development of legs and antennae, and induces these structures when expressed ectopically in the wing or eye-antennal discs. However, although Dll is also expressed in the genital primordia this expression does not lead to the formation of any of these appendages. To test if Abd-B prevents Dll function Abd-B was eliminated in Dll-expressing cells; these cells formed leg tissue. However, it is possible that the high levels of Dll observed in these mutant cells account for the leg transformation. To test this, use was made of the GAL4/UAS system to increase Dll expression in the genital disc (dpp-GAL4/ UAS-Dll flies). Male and female genitalia of this genotype are abnormal, but not transformed into leg. To extend these observations, the ability of Dll to promote Bar transcription, a gene expressed in the leg disc and activated by Dll, was examined. Bar is not expressed in the female genital primordium and only in a few cells within the Dll domain in the male genital primordium; however, Abd-B minus clones show Bar expression in both sexes. When Dll is ectopically expressed in the genital disc, Bar expression is activated in some of the cells that express Dll. These results suggest that, in females, Dll levels are insufficient to activate Bar when Abd-B is present, but that increasing Dll expression or removing Abd-B activates Bar transcription. Abd-B, therefore, prevents some Dll activity in females. In males, although there is Bar transcription, leg tissue is not formed, probably because Abd-B modifies or prevents the activation of other Dll target genes. A similar case has been reported in the wing disc: ectopic Dll activates bric a brac, a gene downstream of Dll, both in the wing pouch and the body wall region of the wing disc; however, legs appear in the wing, but not in the notum (Estrada, 2001).

The Hox gene Antennapedia is involved in leg development. Therefore, an examination was performed to see whether Antp is derepressed in Abd-B minus clones. Antp is not transcribed in the wild-type genital disc, but some Abd-B clones show Antp signal. The presence of the Antp product, however, is not required to transform the genitalia into a leg, since Antp:Abd-B double mutant clones still form ectopic legs. This result is consistent with the view that the role of Antp in leg specification is simply to repress hth expression. It seems that Dll alone is able to direct leg development, provided that Hox and hth genes are not transcribed. Under these conditions, leg tissue can be formed in several appendages: leg, wing, antennal and genital primordia (Estrada, 2001).

Ubx and abdominal A expression were examined in Abd-B minus clones. Ubx was not derepressed in these clones, whereas some clones presented weak ectopic abd-A expression, but only in some cells (Estrada, 2001).

Requirement of Abdominal-A and Abdominal-B in the developing genitalia of Drosophila breaks the posterior downregulation rule

The genitalia of Drosophila derive from the genital disc and require the activity of the Abdominal-B (Abd-B) Hox gene. This gene encodes two different proteins, Abd-B M and Abd-B R. The embryonic genital disc, like the larval genital disc, is formed by cells from the eighth (A8), ninth (A9) and tenth (A10) abdominal segments, which most likely express the Abd-B M, Abd-B R and Caudal products, respectively. Abd-B m is needed for the development of A8 derivatives such as the external and internal female genitalia, the latter also requiring abdominal-A (abd-A), whereas Abd-B r shapes male genitalia (A9 in males). Although Abd-B r represses Abd-B m in the embryo, in at least part of the male A9 such regulation does not occur. In the male A9, some Abd-B mr or Abd-B r clones activate Distal-less and transform part of the genitalia into leg or antenna. In the female A8, many Abd-B mr mutant clones produce similar effects, and also downregulate or eliminate abdominal-A expression. By contrast, although Abd-B m is the main or only Abd-B transcript present in the female A8, Abd-B m clones induced in this primordium do not alter Distal-less or abd-A expression, and transform the A8 segment into the A4. The relationship between Abd-B and abd-A in the female genital disc is opposite that of the embryonic epidermis, and contravenes the rule that posteriorly expressed Hox genes downregulate more anterior ones (Foronda, 2006).

Abd-B is a complex gene: the use of four different promoters and the existence of specific exons give rise to several transcripts that encode two different proteins. The A (m) transcript encodes the Abd-B M (or Abd-B I) protein, and the B, C (r) and gamma RNAs encode the Abd-B R (or Abd-B II) protein. The Abd-B M protein has 221 amino acids more than the Abd-B R product does in its N-terminal domain but both proteins share a common C-terminal region, which includes the homeodomain. In the embryonic epidermis, the Abd-B M transcript and protein are expressed in parasegments (PS) 10-13 (A5-A8 segments), whereas the Abd-B R transcript and protein are present in PS14-PS15 (A9-A10) initially, and in PS14 (A9) at late stages. The gamma RNA is transcribed in just a few cells of PS14 or PS15 (Foronda, 2006 and references therein).

The role of Abd-B M and Abd-B R products in genital development remains unclear. Abd-B m mutations transform the A5-A8 segments into the A4 segment, both in males and females; the female genitalia are lost whereas male genitalia remain intact. Significantly, the transformations obtained in either Abd-B m or Abd-B r mutants clearly differ from those observed when all Abd-B functions are eliminated: in some of the clones mutant for Abd-B (m and r), part of the male or female genitalia are transformed into leg or antenna. Therefore, the precise role of abd-A, Abd-B m and Abd-B r in genitalia development is not well defined (Foronda, 2006).

This study has analyzed homeotic expression and requirement in terminalia development. It is proposed that in the embryonic genital disc, as in the larval discs, Abd-B m, Abd-B r and cad are expressed in the A8, A9 and A10, respectively. It is also reported that abd-A, Abd-B m and Abd-B r are needed for development of the internal female genitalia, Abd-B m for the development of female external genitalia and Abd-B r for the development of male genitalia. Strikingly, abd-A and Abd-B bear unexpected relationships in mature genital discs. In the A8 of the female genital disc, Abd-B M maintains abd-A expression. In Abd-B m mutant clones, however, another Abd-B protein maintains abd-A expression but does not prevent abd-A function, since these clones transform the A8 segment into the A4. In the male A9, Abd-B r function does not repress the Abd-B m transcript, at least in part of the primordium, and some Abd-B r mutant clones transform male genitalia into leg or antenna. These relationships between Hox genes are different from those reported in the embryonic epidermis and contravene the rule that posteriorly expressed Hox genes repress those expressed more anteriorly (Foronda, 2006).

In the third instar genital disc of Drosophila, Abd-B is expressed in the A8 and A9 segments, and cad in the A10. To study whether these expression domains are established early in development, Abd-B and cad transcription were examined in the embryonic genital disc. This disc is identified by the expression of genes like snail, escargot or headcase (hdc), and the hdc-lacZ B5 line, which reproduces the pattern of hdc RNA expression, was selected to mark the genital disc. At about stage 15, hdc is expressed in three clusters of cells, two anterior ones placed bilaterally, and a third one located in a more posterior and central position. The three clusters fuse later in development to form the genital disc. At stage 15, six to seven cells were counted at each of the two anterior groups, and two to three cells in the posterior one, making up a total of 14-17 cells. Double staining with anti-Abd-B and anti-ß-galactosidase antibodies (in hdc-lacZ embryos), or with GFP and anti-ß-galactosidase antibody (in cad-Gal4/UAS-GFP; hdc-lacZ/+ embryos), shows that Abd-B is expressed in the two anterior clusters and cad in the posterior one (Foronda, 2006).

To ascertain whether the two Abd-B products (Abd-B M and Abd-B R) are present in the genital disc primordium, the expression driven by an Abd-B m-Gal4 line was compared with the signal detected with an antibody that recognizes both Abd-B M and Abd-B R proteins. In UAS-myc-EGFPF/+; Abd-B-Gal4LDN/hdc-lacZ embryos, a GFP signal was seen in about two cells located laterally in each of the two anterior clusters; these cells most likely express Abd-B m, and, therefore, are also labelled with the anti-Abd-B antibody. There are also 8-10 Abd-B-expressing cells not labelled with GFP, and these, probably, correspond to those expressing the Abd-B R protein. Taken together, these results suggest that the embryonic genital primordium includes three groups of cells that probably express Abd-B m, Abd-B r and cad, respectively (Foronda, 2006).

Study of mutant phenotypes reveals that as in the embryonic cuticle, abd-A and Abd-B m are needed in the A8 whereas Abd-B r is required in the A9. The relationship between these homeotic products in the mature genital discs, however, clearly differs from what is observed in the embryonic epidermis. The embryonic genital disc has three distinct cell populations at stages 15/16: some anterior-lateral cells transcribe Abd-B m, anterior-central and middle cells express Abd-B r and posterior cells transcribe cad, although the expression of these products may overlap. Because the genital disc is formed by the fusion of cells coming from the A8, A9 and A10 segments, and by analogy to the expression of these genes in the mature genital discs, it is concluded that Abd-B m, Abd-B r and cad are probably expressed in the A8, A9 and A10 segments, respectively, of the embryonic genital disc (Foronda, 2006). Abd-B is not only expressed, but also required in the embryonic genital primordium. In the absence of Abd-B m, the number of hdc-expressing cells in the disc is reduced, most likely because these cells adopt now a more anterior fate, as occurs in the cuticle. When Abd-B r is absent, the genital primordium lacks some cells and is disorganized, and when both Abd-B products are absent, the primordium is reduced to a few, dispersed cells, some of which express Dll ectopically, suggesting a transformation into a leg primordium (Foronda, 2006).

The A8, A9 and A10 primordia of the mature genital discs bear anterior and posterior compartments, with expression of en and wg in each of these three primordia. Curiously, although three primordia in the embryonic disc can be defined, based on the expression of Abd-B m, Abd-B r and cad, neither en nor wg is expressed in the three separate domains at this stage. This may suggest, as was also recently proposed, that new bands of en and wg expression may be formed later in development, in precise concordance with the three primordia defined by the Abd-B m, Abd-B r and cad genes. It is noted that late en expression is also characteristic of the antennal primordium of the eye-antennal disc (Foronda, 2006).

abd-A is expressed in the whole internal female genitalia except for the parovaria, and this is consistent with experiments indicating that parovaria derive from the female A9 segment. abd-A has been shown to be required for gonad development, and in the abd-Aiab-3/Df mutant, combinations ovaries are also absent. However, the defects observed in the female internal genitalia are not simply due to an indirect effect of the lack of gonads, since iab-4 mutations prevent the formation of the ovaries but do not alter internal genitalia formation (Foronda, 2006).

The results indicate that Abd-B m is required for the development of female external and internal genitalia, both derived from the female A8. The internal genitalia of Abd-B-Gal4LDN/UAS-lacZ females (driving expression only where Abd-B m levels are high) were stained with X-gal except in two structures, the oviducts and parovaria. The absence of oviduct staining in Abd-B-Gal 4LDN/UAS-lacZ females is probably due to the particular expression driven by this reporter, and does not imply an absence of Abd-B m transcription in these organs, for two reasons: (1) Abd-B m transcripts are present in the whole A8 segment of the female genital disc, and (2) oviduct development is affected in Abd-B m mutant females. Parovaria, by contrast, are not stained in Abd-B-Gal 4LDN/UAS-lacZ or abd-A-lacZ females, and this agrees with their A9 provenance. This is supported by the observation that in some Abd-B m mutant females parovaria are the only structures that remain in the internal female genitalia (Foronda, 2006).

Abd-B M seems to be the main or only Abd-B product present in the female A8, so it was expected that elimination in this segment of just Abd-B M or of all Abd-B proteins would give similar results. This is not so. Some Abd-B clones transform part of the female genitalia into leg or antenna, whereas Abd-B m mutant clones convert the eighth tergite, and probably the female genitalia, into an anterior abdominal segment. The differences between Abd-B m and AbdB clones in the A8 of the female genital disc reveal the existence of unsuspected regulatory interactions between the abd-A and Abd-B genes: whereas Abd-B m clones do not affect abd-A, in AbdB clones abd-A expression is eliminated. This is a surprising result, because it is contrary to what is observed in the embryo, where Abd-B represses abd-A (Foronda, 2006).

Abd-B m clones induced in the female A8 do not alter abd-A expression but do not change Abd-B expression levels either. This is observed with mutations that do not make Abd-B M protein, so the Abd-B protein detected is not the Abd-B M product. Surprisingly, although some Abd-B r expression is detected in the female A8, uniform Abd-B r expression is not seen throughout this primordium and Abd-B r transcripts seem not to be derepressed in Abd-BM5 mutant clones. No explanation is available for this conundrum. Perhaps the probe used, although it includes sequences complementary to all of the Abd-B r cDNA sequences that have been published, does not efficiently detect all of the non-Abd-B m transcripts (Foronda, 2006).

The differences in regulatory and functional interactions among gene products in the embryo and the genital discs are not limited to those of Abd-B and abd-A that have been discussed above. Three other possibilities should be considered. (1) There may be changes in phenotypic suppression: the transformation of the eighth tergite to the fourth one in Abd-B m clones is due to abd-A. Because in these clones Abd-B protein is still present, this suggests that abd-A may phenotypically suppress Abd-B, differently from what is generally observed in the embryo. (2) Abd-B r represses Abd-B m in the embryo, but some Abd-B r clones do not activate Abd-B m in the male disc. (3) abd-A represses Dll in the embryo, but not in the female genital disc, and ectopic Dll can repress abd-A instead. abd-A does not repress Dll in the leg discs either, and this resembles Ubx function, which represses Dll only early in development. By contrast, Abd-B represses Dll in the embryo, in the larval genital disc, and in the leg disc when ectopically expressed (Foronda, 2006).

Abd-B r expression is restricted to the A9 segment in male genital discs, but shows expression in the A9 and in some cells of the A8 in female genital discs. In spite of this, Abd-B r clones in the external female genitalia (A8) are phenotypically wild type. In the male A9, some Abd-B r mutant clones eliminate Abd-B, activate Dll and transform part of the genitalia into distal leg or antenna. This is similar to the result obtained in some Abd-B clones, and it implies that Abd-B m is not derepressed in these mutant clones. However, Abd-B m is perhaps derepressed in those Abd-B r mutant clones where Abd-B signal remains (Foronda, 2006).

Although Abd-B r clones affect, almost exclusively, male genitalia development, Abd-B r hemizygous or trans-heterozygous flies lack genitalia and analia in both sexes. This probably reflects the absence of proper interactions between the different primordia needed for the growth of the genital disc. In Abd-B r mutant females, the internal genitalia are abnormal, and in some of these females, an absence of parovaria and the presence of three or four spermathecae is observed. This phenotype is consistent with a segment-autonomous transformation of A9 derivatives (parovaria) into A8 structures (spermathecae), similar to the embryonic cuticular transformation of A9 into A8 observed in Abd-B r mutations. A transformation of parovaria into spermathecae has been described in Polycomblike mutants, and may also indicate a transformation of A9 to A8 (Foronda, 2006).

These results illustrate that there are quite different Hox cross-regulatory interactions in the embryo and in the genital disc. The effects in the genital discs contradict the general rule that genes transcribed more posteriorly suppress or downregulate the expression of more anterior ones. This rule has, nevertheless, some exceptions in genes of the Antennapedia complex. Further, differences in Hox cross-regulation between the embryo and imaginal discs are not unprecedented: the proboscipedia (pb) Hox gene is positively regulated by Sex combs reduced in the embryo, but pb activates Sex combs reduced in the labial imaginal disc (Foronda, 2006).

It has been proposed that the primordia of female and male genitalia could be subdivided into an 'appendage-like' and a 'trunk-like' region). These two regions of the female A8 can now be defined more precisely. The 'appendage-like' region would be that expressing abd-A and low levels of Abd-B, and corresponds approximately to the presumptive internal female genitalia. This domain is roughly coincident with the region of expression of a reporter insertion in buttonhead, the gene that defines ventral appendage development, and this is also, approximately, the domain where Abd-B clones may activate Dll. If this subdivision is correct, the 'appendage' specification defined by buttonhead would be repressed in the wild type by Abd-B, which both limits Dll expression to a few cells of the A8 primordium and prevents Dll function. Abd-B clones in this region eliminate abd-A expression and promote leg or antenna development. This subdivision may also apply to the male disc, the penis apparatus presumptive region being the main 'appendage' domain. Similar to what is described in this study, the labial disc possesses a large 'appendage' region that is revealed by Dll derepression in pb mutations. This characteristic, and the changes in Hox gene cross-regulation between the embryo and the imaginal disc, are two features shared by pb/labial disc and Abd-B/genital disc (Foronda, 2006).

Regulation of Abd-B expression by Cyclin G and Corto in the abdominal epithelium of Drosophila

Polycomb-group (PcG) and trithorax-group (trxG) genes encode important regulators of homeotic genes, repressors and activators, respectively. They act through epigenetic mechanisms that maintain chromatin structure. The corto gene of Drosophila encodes a co-factor of these regulators belonging to the Enhancer of Trithorax and Polycomb class. Corto maintains the silencing of the homeotic gene Abdominal-B in the embryo and it interacts with a cyclin, Cyclin G, suggesting that it could be a major actor in the connection between Polycomb/Trithorax function and the cell cycle. This study shows that inactivation of Cyclin G by RNA interference leads to rotated genitalia and cuticle defects in the posterior abdomen of pupae and that corto genetically interacts with Cyclin G for generating these phenotypes. Examination of these pupae shows that development of the dorsal histoblast nests that will give rise to the adult epithelium is impaired in the posterior segments which identity is specified by Abdominal-B. Using a line that expresses LacZ in the Abdominal-B domain, it was shown that corto maintains Abdominal-B repression in the pupal epithelium whereas Cyclin G maintains its activation. These results prompt a proposal that the interaction between the Enhancer of Trithorax and Polycomb Corto and Cyclin G is involved in regulating the balance between cell proliferation and cell differentiation during abdominal epithelium development (Salvaing, 2008).

Ubiquitous downregulation of CycG by RNA interference (using da::Gal4 or Act::Gal4 drivers) led to a high percentage of lethality in late third instar larvae or pharates depending on the CycG line and on the sex (Salvaing, 2008a). Lethality was complete in Act::Gal4/+; UAS::dsCycG2/+ males which intriguingly never underwent pupariation and stopped their development as third instar larvae, dying after a few days. In contrast, most females died as late pharates. UAS::dsCycG2/+; da::Gal4/+ as well as Act::Gal4/+; UAS::dsCycG2/+ emerging animals presented defects in the abdominal cuticle restricted to the posterior tergites A4 to A6. Apart from disorientation of abdominal bristles, the tergites of these segments exhibit unsclerotized patches of variable size. Males were more strongly affected than females and also frequently exhibited rotated genitalia (Salvaing, 2008).

Genetic interactions between CycG and the loss-of-function alleles corto420 and corto07128 were examined. Their combination with ubiquitous RNAi inactivation of CycG increased lethality, cuticle defects and rotated genitalia. These data suggest that CycG and corto interact genetically and corroborate the existence of a functional relationship between CycG and corto (Salvaing, 2008).

To understand the underlying defects of the cuticular phenotypes observed in RNAi-inactivated CycG flies, the development of the abdominal epithelium in pupae was addressed. In Drosophila, the abdominal epithelium of adults is derived from a fixed number of diploid histoblast cells, nested within the polyploid larval epithelium. Each abdominal hemisegment contains four histoblast nests, anterior and posterior dorsal, ventral and spiracle nests, that contribute to tergite and sternite of each abdominal segment. Histoblasts start to proliferate at the beginning of metamorphosis, replacing the larval cells, to eventually build up the adult abdominal integument. In wild-type pupae, the anterior and posterior dorsal histoblast nests of each hemisegment begin to fuse between 15 and 18 h APF. Fusion is completed at 24 h APF and the histoblasts have replaced all the polyploid larval cells at 48 h APF. In Act::Gal4/+; UAS::dsCycG2/+ 48 h APF pupae, whereas the dorsal histoblast nests of segment A3 fuse, the histoblast nests of segments A4 to A6 still remained small and unfused. Nevertheless, the development of these flies was not notably delayed, with regard either to puparium formation or to emergence of adult escapers. Therefore, it is concluded that RNAi inactivation of CycG especially impedes abdominal epithelium development of segments A4 to A6 where histoblast proliferation seemed to have stopped completely (Salvaing, 2008).

It has been shown that corto is involved in the regulation of Abd-B and that Corto and CycG bind to the iab-7 PRE and to the promoter of Abd-B in embryos (Salvaing, 2008a). Since the epithelium defects of RNAi-inactivated CycG individuals affect abdominal segments A4 to A6, and are enhanced in corto mutants, it was hypothesized that they might be associated with misregulation of Abd-B, which specifies posterior abdominal identity. To address the role of corto and CycG in Abd-B regulation in the abdominal epithelium, genetic interactions between Abd-B and corto or CycG mutants was studied. The Fab-71 allele was used, in which both the Fab-7 boundary and the iab-7 PRE of the Abd-B cis-regulatory sequences have been deleted. This mutation induces a higher level of Abd-B expression in A6 which leads to a shift of A6 cell identity toward A7. As there is no normal sclerotized A7 segment in wild-type males, Fab7 homeotic A6 to A7 transformation results in loss of cells. As a result, Fab-71/+ males thus present a half-reduced A6 segment. It was observed that corto alleles enhance the expressivity of this phenotype leading to complete disappearance of the A6 segment in 100% of the males. Next, the effect of inactivation of CycG was examined in a Fab-71/+ genetic context. The expressivity of the Fab-71 phenotype was slightly enhanced in most (86%) of the UAS::dsCycG2/+; da::Gal4/Fab-71 males but at least a thin A6 segment always persisted. Curiously, no cuticular defects were observed neither in A5 nor in the remaining A6 tergites of these males suggesting that they might partly result from altered Abd-B expression. Overexpression of CycG also led to enhancement of the Fab-71 phenotype expressivity but in this case complete disappearance of A6 was observed in 56% of UAS::CycG/+; da::Gal4/Fab-71 males and in 100% of Act::Gal4/+; UAS::CycG/+; Fab-71/+ males. Lastly, the Fab-71 phenotype was investigated in corto, RNAi-inactivated CycG males. Crosses of Act::Gal4; Fab-71 females with UAS::dsCycG2/CyO; corto07128/TM6b males gave only few Act::Gal4/+; UAS::dsCycG2/+; Fab-71/corto07128 male escapers that all exhibited complete disappearance of A6 segment. These results suggest that both corto and CycG participate in maintenance of A6 cell identity by regulating Abd-B expression. corto clearly acted as a repressor of Abd-B since it enhanced the gain-of-function phenotype of Fab-71. However, it is not possible to conclude about the precise role of CycG on Abd-B expression since overexpression as well as inactivation led to enhancement of the Fab-71 phenotype, although to a lesser extent in case of inactivation. To understand this issue, the expression of Abd-B was addressed in corto mutants and in RNAi-inactivated CycG or overexpressing CycG individuals (Salvaing, 2008).

Thus, Abd-B expression was examined in the abdominal epithelium of pupae. Since monoclonal anti-Abd-B antibodies show unspecific ubiquitous staining in the pupal epithelium, the HCJ199 strain was used where a P{LacZ} element is inserted in the cis-regulatory sequences of Abd-B. In agreement with published reports, it was observed that LacZ expression mimics Abd-B expression forming a decreasing gradient from A7 (in females) to the posterior part of A4, the expression in this segment being very faint and only detectable at high magnification. This pattern was also observed in Act::Gal4/+; HCJ199/+ control female pupae showing that the Act::Gal4 driver has no effect on Abd-B expression per se. At 24 h APF, LacZ was expressed in polyploid larval cells as well as in proliferating diploid histoblasts. Later on (48 h APF), LacZ was still expressed in the proliferating diploid histoblasts of A7, A6 and A5, and a barely discernible staining could be seen in posterior . In HCJ199/corto420 and HCJ199/corto07128 48 h APF pupae, LacZ was also expressed from A7 to the posterior part of A4 but expression in posterior A4 was much stronger than in control pupae. Moreover, some cells in the posterior region of A3 also expressed LacZ. This suggests that, in the abdominal epithelium of pupae as in embryos, corto maintains repression of Abd-B expression. In RNAi-inactivated CycG female pupae (Act::Gal4/+; UAS::dsCycG2/+; HCJ199/+) at 48 h APF, almost complete loss of LacZ expression was observed in A5 and A6, whereas it was still expressed in A7. In contrast, 48 h APF Act::Gal4/+; UAS::CycG/+; HCJ199/+ female pupae that overexpressed ubiquitously CycG showed ectopic expression of LacZ in the whole abdomen. Taken together, these results suggest that CycG has the ability to activate Abd-B expression in the abdominal epithelium and contributes to Abd-B expression maintenance in A6 and A5. Thus, corto and CycG play opposite roles on the control of Abd-B expression in the abdominal epithelium, corto being a repressor and CycG an activator. The expression of Abd-B was addressed in pupae where corto and CycG expressions were simultaneously reduced. In Act::Gal4/+; UAS::dsCycG2/+; HCJ199/corto420 48 h APF female pupae, although it was not possible to precisely determine segment borders due to impaired histoblast nest development, rescue of LacZ pattern was obseved that extended more anteriorly than in Act::Gal4/+; UAS::dsCycG2/+; HCJ199/+ pupae. Then, Abd-B loss of expression in A5 and A6 induced by CycG inactivation was abrogated when the amount of Corto was simultaneously reduced (Salvaing, 2008).

This study has show that ubiquitous downregulation of CycG in pupae results in failure of epithelium formation in the posterior abdomen. Abdominal epithelium of adults derives from imaginal histoblasts that are recruited during embryogenic stages and form small group of diploid cells nested in the polyploid larval epithelium. The anterior dorsal nest is composed of about 15 to 18 cells whereas the posterior dorsal nest is composed of about 5 to 6 cells. These cells stay quiescent being arrested in G2 during the larval stages. At the onset of metamorphosis, they first undergo a phase of rapid proliferation triggered by ecdysone signalling and consisting of three synchronous and fast divisions. Also set off by ecdysone signalling are the second phase of histoblast proliferation which is slow and asynchronous and the simultaneous death of the polyploid larval cells. At 24 h APF, the anterior and posterior histoblast nests have fused. It is shown here that inactivation of CycG impedes the proliferation of histoblasts in the posterior part of the abdomen, the dorsal anterior and posterior nests being still individualized at 48 h APF. This probably results in cuticle defects in the less severely affected individuals that will become adult. Similar cuticle defects have been described in some mutants (Arrowhead, escargot, cdc2, myb, torpedo, EcR) where they extend more often over the entire abdomen. Arrowhead has been shown to be involved in the establishment of abdominal histoblasts during embryogenesis. In RNAi-inactivated CycG larvae, the number of cells in the dorsal anterior and posterior histoblast nests is identical to that of wild-type larvae, suggesting that CycG inactivation does not affect histoblast recruitment during embryogenesis. esgargot and cdc2 are required to maintain diploidy of histoblast cells. In RNAi-inactivated CycG larvae, the size of histoblast nuclei in dorsal nests appears similar to the size of the corresponding wild-type nuclei thus suggesting that CycG inactivation does not affect ploidy. Lastly, the epithelium defects could be related to defects in cell proliferation. This is the case for the myb mutant, which proliferating histoblasts exhibit mitosis defects, or the torpedo mutant, which shows loss of mitotic figures in the histoblast nests at 25 h APF. In RNAi-inactivated CycG pupae at 48 h APF, approximately 100 and 40 cells were observed in the anterior and posterior nests of the A6 segment, respectively, suggesting that they might have undergone the 3 first rounds of division. Then, it could be that cells slow down during the second phase of proliferation. Intriguingly, like in RNAi-inactivated CycG pupae, slowdown of histoblast proliferation in segments A5 and A6 has been observed in the torpedo mutant; torpedo encodes the EGF receptor. This suggests that the role of CycG in the proliferation of the abdominal epithelium could be related to MAP kinase signalling. Furthermore, the use of a dominant-negative form of the Ecdysone-receptor that blocks death of the larval epidermal polyploid cells also induces cuticle defects. In this case, cell-autonomous inhibition of EcR activity leads to abortive delamination and persistence of larval polyploid cells in the pupal epithelium. A similar phenomenon, linked to disruption of ecdysone signal reception, could arise when CycG is inactivated. Interestingly, that Act::Gal4>UAS::dsCycG males never go through pupariation; this could reflect a defect in EcR signalling reception. Although the abdominal cuticle of corto mutants seems to be unaffected, the cuticle defects were enhanced by combining them with CycG inactivation. It suggests that corto and CycG together regulate the formation of the abdominal epithelium during metamorphosis (Salvaing, 2008).

These data also show that corto and CycG oppositely regulate the expression of the Hox gene Abd-B in the growing pupal epithelium, corto behaving as a repressor whereas CycG behaves as an activator. Since Corto also represses Abd-B in embryos, it can be considered as a global repressor of Abd-B. Nevertheless, neither Corto nor CycG were detected on the BX-C locus in salivary glands suggesting that they regulate Abd-B in a tissue-specific manner (Salvaing, 2008a). In accordance with expression data, reduction of corto or overexpression of CycG leads to enhancement of the gain-of-function phenotype of Fab-71 heterozygotes. Surprisingly, whereas loss of Abd-B expression was observed upon inactivation of CycG, a mild enhancement of the gain-of-function phenotype of Fab-71 was seen in the same genetic background. This enhancement may result from perturbation of proliferation in the remaining tergite rather than from homeotic transformation of A6 cells to A7 cells. However, it may also reflect the intrinsic mechanism of action of CycG. Indeed, it has been shown that CycG binds both the iab-7 PRE and the promoter of Abd-B (Salvaing, 2008). It is well known that PREs have a stronger silencing activity when present in two copies in the genome, a phenomenon called pairing-sensitive repression. Then, if CycG activates Abd-B partly by working at the promoter and partly by limiting pairing-sensitive repression, loss of Abd-B activation at promoter could be overwhelmed by loss of pairing-sensitive repression when a single copy of the iab-7 PRE is present which is the case in the Fab-71/+ flies (Salvaing, 2008).

Finally, in pupae combining RNAi-inactivated CycG and corto mutation, histoblast proliferation is still impeded whereas Abd-B expression seems to be restored. It suggests that the ratio between Corto and CycG activities must be preserved to insure appropriate regulation of Abd-B in the posterior abdomen. Altogether, these results suggest that a tripartite interaction between corto, CycG and Abd-B together regulates the balance between proliferation and differentiation during the formation of the abdominal epithelium at metamorphosis. Further experiments are now required to better understand how these processes are coordinated (Salvaing, 2008).

Type ID unconventional myosin controls left-right asymmetry in Drosophila

Breaking left-right symmetry in Bilateria embryos is a major event in body plan organization that leads to polarized adult morphology, directional organ looping, and heart and brain function. However, the molecular nature of the determinant(s) responsible for the invariant orientation of the left-right axis (situs choice) remains largely unknown. Mutations producing a complete reversal of left-right asymmetry (situs inversus) are instrumental for identifying mechanisms controlling handedness, yet only one such mutation has been found in mice (inversin) and snails. The conserved type ID unconventional myosin 31DF gene (Myo31DF) has been identified as a unique situs inversus locus in Drosophila. Myo31DF mutations reverse the dextral looping of genitalia, a prominent left-right marker in adult flies. Genetic mosaic analysis pinpoints the A8 segment of the genital disc as a left-right organizer and reveals an anterior-posterior compartmentalization of Myo31DF function that directs dextral development and represses a sinistral default state. As expected of a determinant, Myo31DF has a trigger-like function and is expressed symmetrically in the organizer, and its symmetrical overexpression does not impair left-right asymmetry. Thus Myo31DF is a dextral gene with actin-based motor activity controlling situs choice. Like mouse inversin, Myo31DF interacts and colocalizes with β-catenin, suggesting that situs inversus genes can direct left-right development through the adherens junction (Spéder, 2006).

In wild-type males, the genital plate, to which the spermiduct is attached, undergoes a 360° clockwise (dextral) rotation when viewed from the posterior pole. This directional looping is reminiscent of other coiling processes such as mammalian heart tube looping and snail spiral development. As in other species, one direction is dominant among the Drosophilidae, the dextral rotation, with no sinistral species reported to date. This study identified an insertional mutation, KG02246, which shows a striking inverted phenotype when combined with deficiencies covering the 31DF genomic region. In KG02246/Df(2L)Exel7048 or KG02246/Df(2L)J3 males, genitalia rotation is variable, with ~60% of individuals showing sinistral rotation. Imprecise excision of KG02246 generated two genomic deletions, KG022461 and KG022462, both of which presented a stronger, 100% inverted (sinistral) genitalia rotation phenotype. Thus, KG02246 alleles identify the first situs inversus mutations in Drosophila and provide genetic evidence for the existence of a left-right axis in this organism (Spéder, 2006).

Drosophila Myo31DF is a conserved myosin belonging to the Myo1D family. It is known to interact with F-actin and it colocalizes with actin-rich structures in different tissues. After mouse inversin, Drosophila Myo31DF represents the second situs inversus gene to be molecularly identified. The mouse inversin gene (Invs) encodes a protein with ankyrin repeats and two IQ domain that bind calmodulin, a Ca2+ -dependent regulatory protein. Like inversin, Myo31DF contains two IQ domains essential for its function. Indeed, a Myo31DF form lacking IQ domains (Myo31DFDeltaIQ) is unable to rescue Myo31DF mutations (Spéder, 2006).

To determine the function of Myo31DF in genitalia rotation, its expression was examined in the genital disc, the precursor of adult genitalia. The genital disc is composed of segments A8, A9 and A10, each with an anterior and a posterior. Immunostaining of wild-type flies with two polyclonal antibodies directed against overlapping regions of the Myo31DF tail domain, anti-Myo31DF-1P and anti-Myo31DF-3P, or using a Myo31DF-Gal4 enhancer-trap line (NP1548) revealed symmetrical expression of Myo31DF in a double chevron-like pattern restricted to the ventral domain of the male genital disc. This expression, starting in third instar larvae and remaining unchanged during this stage, was absent in Myo31DFK2 mutant discs. Double staining with an A8-specific marker in the genital disc (tsh-Gal4 > myr-RFP) indicated that Myo31DF is expressed in the A8 segment, with one chevron in the posterior compartment and the other in the anterior compartment. Consistently, expression of two copies of an inhibitory RNA gene (2 x Myo31DFRNAi) specifically in the A8 segment led to loss of Myo31DF expression and to inverted phenotypes that mimicked Myo31DF mutations. Together, these data identify the A8 segment as a left-right organizer that is required for situs choice in Drosophila (Spéder, 2006).

To investigate the relationship between the anterior-posterior and left-right axes, the putative domain(s) of Myo31DF function was mapped by selectively silencing the gene in different compartments, using specific Gal4 lines driving 2 x Myo31DFRNAi. The combinatorial removal of Myo31DF function in the anterior and/or posterior domains led to distinct phenotypes, indicating a dual function for the Myo31DF protein in A8. First, blocking Myo31DF function posteriorly in A8 (using hh-Gal4 or en-Gal4) resulted in a striking non-rotated genitalia phenotype. This finding was confirmed in a complementary experiment using dpp-Gal4 to rescue Myo31DFK1 in the anterior compartment. The absence of situs choice observed in these experiments indicates that posterior Myo31DF has an instructive role in dextral looping. Second, blocking Myo31DF function solely in the anterior compartment (in dpp-Gal4 > 2 x Myo31DFRNAi or in Myo31DFK1/Myo31DFK1; hh-Gal4 > Myo31DF rescued males) led to partial dextral rotation, suggesting a permissive role for Myo31DF in this compartment. Comparing this outcome to the effect of concomitant removal of both anterior and posterior functions (the only context that led to complete reversal) indicates that the function of Myo31DF in the anterior compartment is to repress sinistral looping. These experiments demonstrate that Myo31DF is essential both anteriorly and posteriorly. A model illustrating the dual function of Myo31DF in regulating dextral development within the A8 segment is presented. In this model, A8 contains information to specify both sinistral and dextral rotation, with sinistral information being anterior and dextral information posterior. Dextral information is dominant over sinistral information, and Myo31DF function is required both posteriorly, to induce dextral development, and anteriorly, to repress sinistral development. Only in the absence of Myo31DF can sinistral development occur as a default state (Spéder, 2006).

As expected of a left-right determinant with a function that precedes asymmetry, Myo31DF is expressed symmetrically in A8. In addition, as with mouse inversin (Watanabe, 2003), symmetrical overexpression does not lead to left-right defects (AbdB-Gal4, tsh-Gal4 and ptc-Gal4 lines). Another predicted feature of left-right determinants is their temporally restricted, trigger-like function, which is later relayed by mechanisms acting to maintain the initial symmetry-breaking event. To test this, temperature-shift experiments were carried out using a genetically engineered temperature-sensitive Myo31DF allele. Single temperature-shift experiments with 24-h or 6-h resolution indicated that Myo31DF function is required at day 6 of development, between 126 and 132 h. Double temperature-shift experiments allowed to determine that Myo31DF function is required for as little as 3 h within this period. These data provide high temporal resolution, allowing the temporal mapping of a left-right symmetry-breaking event and demonstrating that situs choice depends on a peak of Myo31DF function in the left-right organizer (Spéder, 2006).

Cilia have emerged as important cellular structures for generating left-right asymmetry in vertebrate embryos. To address their possible contribution to invertebrate left-right determination, genital discs were stained with GT335, an antibody that labels glutamylated tubulin in cilia across species, including Drosophila. GT335 did not detect any cilia in genital discs. Additionally, mutations in the conserved Rfx gene, which controls the formation of ciliated neurons in Drosophila, did not affect genitalia rotation. Together, these data suggest that cilia are not involved in dextral development in Drosophila. It is proposed that Drosophila uses primarily the actin cytoskeleton to determine left-right asymmetry. Consistently, the small GTPase Drac1 and the JNK pathway, known regulators of the actin cytoskeleton, showed specific genetic interactions with Myo31DF. Notably, actin is also important for situs choice in snails, suggesting that in invertebrates the actin cytoskeleton has a central role in left-right determination (Spéder, 2006).

To start investigating how Myo31DF might act to determine left-right asymmetry, two-hybrid screening was used to identify Myo31DF interactors or cargo(es). Using the Myo31DF tail domain (amino acids 737-1011) as bait, several positive clones were found encoding a carboxy-terminal fragment containing ARM repeats 6-12 of the Armadillo/β-catenin protein. This interaction was direct, as shown by glutathione S-transferase (GST)-pulldown experiments using purified proteins or S2 cell extracts. Furthermore, endogenous Myo31DF and a functional Myo31DF-GFP (green fluorescent protein) fusion protein colocalized with Armadillo at the adherens junctions in A8, suggesting that the two proteins can interact in vivo. Notably, inversin has been shown to colocalize and interact with β-catenin in vertebrate epithelial cells, indicating that an interaction with β-catenin is a common feature of both known situs inversus genes. These results are consistent with a demonstrated role of N-cadherin in left-right asymmetry (Spéder, 2006).

How could anterior-posterior organization of Myo31DF and interaction with Armadillo account for left-right asymmetry? The actin cable network might serve as a track for Myo31DF to deliver specific cargoes or vesicles at the adherens junction. Indeed, it was found that Myo31DF interacts with dynamin, consistent with the role of rat MyoID in vesicular transport. The anterior-posterior boundary itself creates an asymmetric junction that could serve as a scaffold for Myo31DF to assemble a dextral-specific complex on the anterior side of posterior Myo31DF-expressing cells. Anterior-posterior asymmetry of dextral information could later be translated into left-right asymmetry through the remodelling of cell contacts (cell intercalation or rotation), as seen in other epithelia. For example, 90° rotation of epithelial cells relative to the main body axis is observed in the Drosophila eye imaginal disc. A similar, 90° planar rotation of Myo31DF-expressing cells would result in left-right orientation of the dextral junctional complex in the tissue. In this working model, a short pulse of Myo31DF activity would be essential for spatially restricting the dextral junction, as has been observed (Spéder, 2006).

Coupling of apoptosis and L/R patterning controls stepwise organ looping

Handed asymmetry in organ shape and positioning is a common feature among bilateria, yet little is known about the morphogenetic mechanisms underlying left-right (LR) organogenesis. This study utilized the directional 360° clockwise rotation of genitalia in Drosophila to study LR-dependent organ looping. Using time-lapse imaging, it was shown that rotation of genitalia by 360° results from an additive process involving two ring-shaped domains, each undergoing 180° rotation. The results show that the direction of rotation for each ring is autonomous and strictly depends on the LR determinant myosin ID (MyoID: Myo31DF). Specific inactivation of MyoID in one domain causes rings to rotate in opposite directions and thereby cancels out the overall movement. A specific pattern of apoptosis at the ring boundaries is revealed, and this study also shows that local cell death is required for the movement of each domain, acting as a brake-releaser. These data indicate that organ looping can proceed through an incremental mechanism coupling LR determination and apoptosis. Furthermore, they suggest a model for the stepwise evolution of genitalia posture in Diptera, through the emergence and duplication of a 180° LR module (Suzanne, 2010).

Left-right (LR) asymmetric development is essential to the morphogenesis of many vital organs, such as the heart. Directional looping of LR organs is a complex morphogenetic process relying on proper coordination of early LR patterning events with late cell-tissue behaviors. In vertebrates, several developmental models have been proposed for gut coiling downstream of the Nodal-Pitx2 regulatory pathway, including intrinsic asymmetric elongation of the gut in Xenopus or extrinsic force generation by mesenchymal tissue in Zebrafish and by dorsal mesentery in the chick and mouse embryos. However, the cellular mechanisms underlying LR organ morphogenesis are mostly unknown (Suzanne, 2010).

In Drosophila, directional clockwise (or dextral) rotation of the genital plate and gut has been shown only recently to be controlled by the LR determinant myosin ID (MyoID). In myoID mutant flies, LR morphological markers are inverted, leading to counterclockwise (or sinistral) looping of the genital plate, spermiduct, gut, and testis. This indicates that myoID is a unique situs inversus gene in Drosophila. Intriguingly, the expression of MyoID is restricted to two rows of cells within the A8 segment of the genital disc (the analia and genitalia precursor), with one row of expression in the anterior compartment (A8a) and the other in the posterior compartment (A8p) (Suzanne, 2010).

Removal of myoID function specifically in the A8 segment is sufficient to provoke the complete inversion of rotation (360° counterclockwise) of the genitalia and sinistral looping of the spermiduct to which it is attached. The A8 segment therefore represents a LR organizer controlling the directional rotation of the whole genitalia in Drosophila (Suzanne, 2010).

Because circumrotation (the process of 360° rotation) may result from a number of different morphogenetic processes, not deducible from the simple observation of the final adult phenotype, a new and innocuous imaging method was developed to follow the rotation in living pupae (Suzanne, 2010).

To be able to analyze the movement of distinct domains in live developing genitalia, time-lapse imaging was coupled with genital disc 'painting' by expressing different fluorophores in various regions of the genitalia precursor. Analysis of wildtype live genitalia through this method revealed their spatial and temporal organization during rotation. It was first determined that rotation begins at around 25 hr after puparium formation (APF) and lasts 12-15 hr. At 25 hr APF, the genital disc is organized into concentric rings, which, from anterior to posterior, include an A8a ring, an A8p ring, and a large central disc composed of A9-A10 tissues. The analysis of rotation in live pupae coupled to manual tracking allowed the identification of two distinct moving domains: a large posterior domain comprising A8p-A9-A10 (hereafter referred to as A8p) and a smaller anterior domain made of A8a. The A8p domain moves first and is followed by A8a, which starts moving later on. During the entire process, cells from the abdomen, to which the genital disc is connected, remain immobile. The finding of two rotating domains, A8a and A8p, was unexpected. It reveals a complex rotational activity of the genitalia and rules out a simple model in which the genital plate would rotate by 360° as a whole. To further understand how rotation occurs, timelapse imaging of the full, 15-hr-long rotation was performed. This analysis revealed that each ring had a different rotational activity. When viewed from the posterior pole, the A8p ring undergoes 360° clockwise rotation, while the A8a ring makes a 180° clockwise rotation. Whereas the rotation of the central part (A8p-A10) of the disc was inferred from the looping of the spermiduct around the gut, the 180° rotation of A8a was not predicted and could only be revealed by time-lapse analysis because this compartment solely gives rise to a tiny and colorless part of the cuticle. Altogether, these in vivo analyses show that rotation of genitalia in Drosophila is a composite process involving two compartments of the A8 segment, A8a and A8p, each expressing a row of MyoID at its anterior boundary and having its own rotational behavior (Suzanne, 2010).

These findings raise the questions of the contribution of each of the two rings to the entire rotation and of how they interact during rotation. In order to address this question, the intrinsic or real rotational activity of A8a and A8p was determined. So far, each ring movement was analyzed relative to the same immobile referential: the abdomen. Although this referential allows the real movement of A8a to be determined, it cannot be used to determine that of A8p, because A8p moves relatively to a mobile referential, i.e., A8a, to which it is attached. To determine the real movement of A8p, it is thus essential to analyze its angular movement relative to A8a, in other words A8a contribution to motion must be subtracted from the apparent A8p movement. To do so, movies were analyzed by setting A8a as a referential and by determining the angular movement of A8p. Reassessing A8p movement through this approach revealed that A8p rotates clockwise only by 180° relative to A8a. The new angular velocity curve of A8p fits almost perfectly with that of A8a, indicating that both movements have similar features. Importantly, these data also indicate that the observed 360° clockwise rotation is the result of a composite process involving two additive 180° clockwise components: a 180° rotation of the A8a relative to the abdomen and an 180° rotation of A8p relative to A8a (Suzanne, 2010).

To further determine the autonomy of each ring relatively to the other, the role of the LR determinant MyoID in this process was dissected by specifically inactivating myoID in either A8a or A8p or in both. By convention, the presence or absence of myoID is represented by a + or - sign, respectively. Accordingly, the wild-type context is noted 'A8a+A8p+' and the myoID mutant 'A8a-A8p-.' Upon specific inactivation of myoID in the A8a domain (A8a-A8p+ context), the adults showed an apparent 'nonrotation phenotype' (0°, no spermiduct looping and genitalia correctly oriented). However, time-lapse imaging revealed that both rings were spinning, although in opposite directions: the A8a domain rotated counterclockwise by 180° (-180°), whereas the A8p domain rotated clockwise by 180° (+180°, real movement). Reciprocally, the inactivation of myoID in the A8p domain (A8a+A8p- context) also led to an apparent nonrotation phenotype. In this context, the behavior of each domain was inverted compared to the previous condition, with the A8a domain rotating clockwise by 180° (+180°) whereas the A8p domain rotated anticlockwise by 180° (-180°, relative or real movement). In both cases, the movement of each ring is consistent with its myoID genotype and the 'dextralizing' activity of this gene. The strict dependence on MyoID for the direction of the rotation is further confirmed in flies where both A8a and A8p were mutants for myoID (myoIDk1). The rotation is often incomplete in this genotype because of the hypomorphic nature of the myoIDk1 allele analyzed; however, both domains show an anticlockwise movement. Therefore, in all genetic contexts analyzed, all parameters of the rotation remain unaffected except the direction of rotation, as illustrated by the perfect mirror image of the angular velocity curves (Suzanne, 2010).

These experiments reveal that each ring adopts an independent 180° movement relative to more anterior structures (A8a relative to the abdomen and A8p relative to A8a): clockwise in the presence of MyoID, anticlockwise in its absence. When both movements are unidirectional, the net rotation is circumrotation (± 360°), whereas upon opposite movements of A8a and A8p, the net rotation is zero (0°), leading to an apparent nonrotation phenotype. Therefore, the net rotation (or apparent rotation = R) can be modeled through a simple equation in which R equals the addition of A8a and A8p movements, with MyoID acting as a sign function (Suzanne, 2010).

It was next of interest to characterize potential cellular mechanisms acting downstream of LR determination during genitalia rotation. In particular, the cellular events responsible for uncoupling rings at the onset of their rotation was determined. Initial insights came from blocking apoptosis, which leads to genitalia rotation defects, but the role of apoptosis in the process is not completely understood. To determine the morphogenetic function of the apoptotic pathway during genitalia rotation, the spatial and temporal requirements for apoptosis were first characterized by analyzing the expression pattern of hid and reaper (rpr) in the genital disc, using two reporter lines. Both reporters were strongly expressed in the A9 and A10 segments. However, in the A8 segment, only hid expression is observed. This coincides with the phenotype of misrotated genitalia observed specifically when hid function is altered but not in rpr mutants. Then the pattern and timing of cell death was determined in the genital disc. To do so, nuclear fragmentation was followed, and an in vivo reporter of caspase activation (the apoliner construct) was used. At the onset of rotation, a large number of apoptotic cells was detected on the most ventral part of the genital disc, first within the A8p ring bordering A8a, coinciding with the beginning of A8p movement. These data indicate an overlap between the apoptotic field and the domain of MyoID expression. These results have been further confirmed by the detection of apoptotic cells by TUNEL staining of fixed pupal genital discs. Later on, a new wave of apoptosis was detected in the most anterior part of the A8a ring, at the junction between A8a and the abdomen. In contrast, only marginal if any apoptosis was detected before and at the end of rotation. Therefore, two waves of cell death are taking place in the A8 segment, coinciding spatially and temporally with the rotation of A8a and A8p rings (Suzanne, 2010).

Given that rings are initially part of the same epithelium and move independently later, it was reasoned that local cell death may be a mechanism to provide the degree of liberty necessary for proper movement. To test this hypothesis, cell death was inhibited in each compartment separately by expressing the caspase inhibitor p35. Interestingly, inactivation of apoptosis in either A8p or A8a leads to a similar phenotype, with flies showing a high proportion of half-rotated genitalia (180° rotation), suggesting that rotation was blocked in the ring deficient for apoptosis. This has been further demonstrated by following the rotation process in vivo, when apoptosis is specifically blocked in the A8a. In this genetic context, the A8a ring stayed mostly still during the whole process, whereas A8p rotated normally. The resulting 180° rotation is thus exclusively due to the movement of one ring, i.e., A8p, in which apoptosis is unaffected. Inhibiting apoptosis in both domains strongly aggravates the phenotype, with 40% of the flies showing nonrotated genitalia (0°), suggestive of an additive phenotype. The rest of the population had 90° rotated genitalia, which may be due to incomplete inhibition of apoptosis. Alternatively, it is possible that some rotation occurs without apoptosis thanks to tissue elasticity. In any case, the results indicate that cell death is required in each ring for separating them from the neighboring tissues and allowing their free rotation. Consistently, nuclei fragmentation and cell death occur normally in a myoID mutant background. Because local cell death is not likely to provide a direct force for rotation, it is proposed that it contributes to the release of rings from neighboring tissues (Suzanne, 2010).

This study has revealed that organ looping can proceed through discrete steps, breaking down circumrotation into the simple building blocks of 180° each. The incremental nature of genitalia rotation is indeed based upon two 180° LR modules, sharing identical angular velocity and range as well as requirement for MyoID and apoptosis. Modularity in morphogenesis provides interesting control mechanisms (through addition or substraction) and therefore plasticity to the process, both at the organism level and during evolution. Entomologists have described different patterns of genitalia rotation in Diptera, ranging from 0° to 360°, that evolved together with changes in mating position. Interestingly, in the Brachycera suborder, to which Drosophilidae belong, we notice that most ancestral species have a nonrotated genitalia (Stratiomyomorpha and Tabanomorpha), whereas 180° and 360° rotation have appeared progressively later in evolution (in Muscomorpha and Cyclorrhapha, respectively). Together with this sequential organization of rotation amplitude in the phylogenetic tree, these data strongly support a model by which the 360° rotation observed in Brachycera ('modern Diptera') would result from the emergence (transition from 0 to 180°) and duplication (transition from 180° to 360°) of a 180° L/R module (Figure S3), thus providing a simple additive model for both the origin of circumrotation and the evolution of genitalia rotation and mating position. However, it should be noted that alternative mechanisms maylead to a similar pattern of genitalia rotation among Diptera (Suzanne, 2010).

The incremental model presented here also offers a solution to the apparent paradox of circumrotation and the question of its elusive utility, illustrated by the fact that both 360° rotation and the absence of rotation lead to the same final posture of genitalia. A facultative role of 360° rotation is further supported by the finding that D. melanogaster males with nonrotating genitalia (A8a-A8p+ or A8a+A8p-) are normally fertile (data not shown). An incremental origin of 360° rotation in which a second half-turn would be added to the existing 180° rotation would well explain this paradox. Thus, circumrotation can be viewed as recapitulating the evolutionary history of genitalia rotation in Brachycera, and its logic would reveal a case of 'retrograde evolution,' in which duplication of a functional module is used to revoke a previous evolutionary step (Suzanne, 2010).

Finally, this analysis of genitalia rotation highlights a new mechanism of morphogenesis relying on a combination of LR patterning and apoptosis. In this process, a new role for apoptosis is revealed as a releasing mechanism allowing the sliding of two parts of an organ. It will be interesting to test in the future whether this releasing role of apoptosis is used more generally, in other morphogenetic movements requiring important cellular rearrangement (Suzanne, 2010).

Apoptosis controls the speed of looping morphogenesis in Drosophila male terminalia

In metazoan development, the precise mechanisms that regulate the completion of morphogenesis according to a developmental timetable remain elusive. The Drosophila male terminalia is an asymmetric looping organ; the internal genitalia (spermiduct) loops dextrally around the hindgut. Mutants for apoptotic signaling have an orientation defect of their male terminalia, indicating that apoptosis contributes to the looping morphogenesis. However, the physiological roles of apoptosis in the looping morphogenesis of male terminalia have been unclear. This study shows the role of apoptosis in the organogenesis of male terminalia using time-lapse imaging. In normal flies, genitalia rotation accelerates as development proceeded, and completes a full 360° rotation. This acceleration is impaired when the activity of caspases or JNK or PVF/PVR signaling was reduced. Acceleration was induced by two distinct subcompartments of the A8 segment that form a ring shape and surround the male genitalia: the inner ring rotates with the genitalia and the outer ring rotates later, functioning as a 'moving walkway' to accelerate the inner ring rotation. A quantitative analysis combining the use of a FRET-based indicator for caspase activation with single-cell tracking showed that the timing and degree of apoptosis correlates with the movement of the outer ring, and upregulation of the apoptotic signal increases the speed of genital rotation. Therefore, apoptosis coordinates the outer ring movement that drives the acceleration of genitalia rotation, thereby enabling the complete morphogenesis of male genitalia within a limited developmental time frame (Kuranaga, 2011).

To visualize the genitalia rotation in living animals, the His2Av-mRFP Drosophila line was used whose nuclei are ubiquitously marked by a fluorescent protein. The genital disc is a compound disc comprised of cells from three different embryonic segments: A8 (male eighth tergite), A9 (male primordium) and A10 (anal). During metamorphosis, the genital disc is partially everted, exposing its apical surface, and adopts a circular shape. The results captured the male genitalia undergoing a 360° clockwise rotation. Inhibiting apoptosis by expressing the baculovirus pan-caspase inhibitor p35 driven by engrailed-GAL4 (en-GAL4), which is expressed in the posterior compartment of each segment, results in genital mis-orientation at the adult stage (Kuranaga, 2011).

In flies expressing nuclear fluorescent protein driven by en-GAL4, it was observed that the posterior part of the A8 segment (A8p) formed a ring of cells surrounding the A9-A10 part of the disc. First, the images were recorded at a low resolution (10× objective lens) to measure the rotation speed accurately in control and p35-expressing flies, because long-term time-lapse imaging at a high resolution can cause photodamage, and thus alter pupal development. Most of the cells in the A8p that seem to disappear actually moved out of the plane of focus. The imaging results, the rotation started around 24 hours APF (after puparium formation) and stopped about 12 hours later. To confirm whether the mis-oriented genital phenotype in the caspase-inhibited flies was caused by incomplete rotation, the rotation was observed in flies expressing p35 under the en-GAL4 driver. In the p35-expressing flies, the rotation began, but it stopped before it was complete, after about 12 hours, i.e. with the same timing as in control flies. This suggested that the reduced caspase activation in A8p prevented the genitalia from completing the rotation, resulting in mis-oriented adult genitalia (Kuranaga, 2011).

To compare complete rotation with incomplete rotation, the rotation speed was calculated by measuring the angle (thetacontrol and thetap35) of the A9 genitalia every 30 minutes on time-lapse images. The normal rotation was composed of at least four steps: initiation, acceleration, deceleration and stopping. The velocity of rotation V=dtheta/dt was calculated by measuring theta as a function of time t. At first, the genitalia rotated at an average velocity (Vcontrol) of 7.67±3.72°/hour by 1 hour after initiation, then the rotation accelerated, with Vcontrol gradually increasing to 53.83±7.11°/hour by 7 hours after initiation. Interestingly, in the p35-expressing flies, the rotation normally started at 24 hours APF, and the average velocity (Vp35) from the initial rotation to 1 hour later was 7.45± 2.98°/hour, which was not significantly different from the normal rotation. However, the acceleration of the rotation in the p35-expressing flies was lower than normal, with Vp35 gradually increasing to 21.35±7.45°/hour at 5.5 hours after initiation. The first peak of the acceleration rate, which was defined as the initiation of rotation, was observed in the p35-expressing flies (ap35) and was the same as in the control flies (acontrol). However, the duration of the acceleration period was shorter in the p35-expressing flies. These data suggest a relationship between apoptosis and the acceleration of genitalia rotation (Kuranaga, 2011).

Next, the signaling mechanism(s) involved in the acceleration of genitalia rotation wee examined. The inhibition of JNK (c-Jun N-terminal kinase) and PVF (platelet vascular factor) signaling in male flies has been shown to result in mis-oriented adult male terminalia, and it has been hypothesized that the PVF/PVR (PVF receptor) may affect the genitalia rotation via JNK-mediated apoptosis (see Benitez, 2010). Consistent with previous reports, the acceleration of genitalia rotation was significantly impaired in flies expressing dominant-negative forms of JNK (JNK-DN) and PVR (PVR-DN). These data implied that caspase activation and JNK signaling contribute to driving the acceleration of genitalia rotation (Kuranaga, 2011).

To analyze how the genitalia accelerate their rotation, the movement of A8p was traced at the single-cell level. For this experiment, live imaging was performed at a high resolution (20× objective lens), which enabled the cells in A8p to be tracked at single-cell resolution. Cells that were neighbors of A9 rotated with A9, whereas cells located in the anterior half of A8p rotated later than A9. Based on this imaging, A8p was divided into two sheets, named A8pa (anterior of A8p) and A8pp (posterior of A8p). It was found that a part of the cells in A8p underwent apoptosis (Kuranaga, 2011).

To observe caspase activation in living animals, a FRET (fluorescence resonance energy transfer)-based indicator, SCAT3 (sensor for activated caspases based on FRET) was generated. To precisely evaluate apoptosis, a nuclear localization signal-tagged SCAT3 (nls-SCAT3; UAS-nls-ECFP-venus) was used. The nls-SCAT3 signal was clearly observed in A8p. Cells exhibiting high caspase activity were extruded into the body cavity and disappeared, consistent with their apoptotic death and engulfment by circulating hemocytes. Each cell was tracked in the A8p region during the first half of the rotation, and it was found that at least three types of cellular behavior were observed: (1) cells located in A8pp moved with A9, (2) cells underwent apoptosis and (3) cells located in A8pa rotated later (Kuranaga, 2011).

Thus, to observe the behavior of the cells in A8pa, Abdominal B (AbdB) was used as an A8 marker. AbdB is a homeotic gene that is required for the correct development of the genital disc, and AbdB-GAL4LDN is expressed in the segment A8 (in A8a and A8p) of the genital disc during the 3rd instar larval stage. At 24 hours APF, AbdB was expressed in A8 and formed a ring. Time-lapse images were taken, and unexpectedly it was found that most of the cells in the AbdB-expressing region underwent a 180° clockwise movement, suggesting that AbdB was not expressed in the A8pp region that moved 360° with A9. To determine the speed of the AbdB-expressing cells, three individual cells were traced in each fly, and the value of the turning angle of the cells (thetaAbdB) was calculated. The findings confirmed that the AbdB-expressing region moved halfway around. Although cells in the AbdB-expressing region moved only 180°, the A8pp (inner ring), which was encircled by the AbdB-expressing region (outer ring), still moved 360°. Furthermore, the imaging data indicated that the movement of the outer ring started 1-2 hours later than that of the A9 region, when the acceleration of the genitalia rotation occurred. These observations raise the possibility that the outer ring movement is related to the acceleration of the genitalia rotation (Kuranaga, 2011).

It was therefore considered that the outer ring movement was restricted in the p35-expressing flies, resulting in an incomplete genitalia rotation of about 180°, which mimics the movement of only the inner ring. To verify this possibility, the movement of the outer ring was examined in the p35-expressing flies (en-GAL4+UAS-p35). Although the inner ring rotated normally, the rotation of the outer ring was impaired in the p35-expressing flies. The turning angles were determined by tracing cells in the p35-expressing flies and it was found that thetap35 _inner increased, while the increase of thetap35 _outer was impaired. These data suggest that the A8 segment is composed of two independently regulated rings, and when apoptosis is inhibited, the inner ring can move only 180° with no outer ring movement, resulting in incomplete genitalia rotation (Kuranaga, 2011).

Thus, to determine whether apoptosis correlates with the outer ring movement, the apoptosis was quantified in A8pa every 10 minutes from 0-8 hours after the start of genitalia rotation. The frequency of apoptosis (Rapoptosis) was normalized to the total number of apoptotic cells in each individual. Pulsatile increases in Rapoptosis were observed, with peaks at 1, 2.5 and 4 hours after the start of genitalia rotation. To verify the participation of Rapoptosis in the initiation of outer ring movement, the acceleration rate of thetaAbdB (aAbdB) was calculated by measuring VAbdB as a function of time t, and Rapoptosis was compared with aAbdB. The starting time of outer ring movement was characterized by the early peaks of aAbdB. The analysis suggested that the aAbdB was related to the Rapoptosis, because aAbdB showed its first two peaks at about 1 and 2.5 hours after genitalia rotation started. To quantify these observations, the correlation was calculated between Rapoptosis and aAbdB. This analysis confirmed that there was a strong correlation between these parameters, because the correlation between aAbdB and Rapoptosis is approximately linear during this time. Therefore, these data implied a possible mechanism of apoptosis that facilitates the outer ring movement (Kuranaga, 2011).

To verify this possibility, whether the upregulation of apoptotic signals induces an increase in genitalia rotation speed was meastured. Because the expression of apoptotic genes using an en-GAL4 driver, which is expressed at the embryonic stage, is lethal, the TARGET system was used to control gene expression temporally. Flies were allowed to develop at 18°C until the head of the pupae had just everted, to inhibit gene expression. The pupae were then heat-shocked at 29°C for 12 hours to induce gene expression. Live imaging was performed at 22°C, after the heat shock. At this temperature, the genitalia rotation in the control flies was slower than in control flies bred at 25°C, because a low breeding temperature affects the rate of fly development, including genitalia rotation. Therefore, it was necessary in this experiment to compare the rotation speeds at the same temperature. The expression of reaper (rpr), a pro-apoptotic gene, using the TARGET system, showed that the upregulation of apoptotic signaling significantly increased the timing of acceleration and speed of genitalia rotation. These observations led to the proposal that the outer ring functions like a 'moving walkway' to accelerate the speed of the inner part of the structure, including the A9 genitalia, enabling genitalia to complete rotation within the appropriate developmental time window (Kuranaga, 2011).

According to these observations, it was found that apoptosis drives the movement of cell sheets during the morphogenesis of male terminalia. Further questions remain with regard to how apoptosis contributes to the cell sheet movement. A recent study indicated the possibility that local apoptosis acts as a brake release to regulate genitalia rotation, coupled with left-right determination (Suzanne, 2010). However, it has been reported that the cell shape change by apoptosis enables not only the extrusion of dying cells, but also the reorganization of the actin cytoskeleton in neighboring cells. Therefore, apoptosis could affect the behavior of neighboring cells, to act as a main driving force of the cell-sheet movement. Taken together, apoptosis may generally participate in the morphogenetic process of cell-sheet movement during morphogenesis (Kuranaga, 2011).

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).


Ahmad, S. M. and Baker, B. S. (2002). Sex-specific deployment of FGF signaling in Drosophila recruits mesodermal cells into the male genital imaginal disc. Cell 109: 651-661. PubMed ID: 12062107

Benitez S., et al. (2010). Both JNK and apoptosis pathways regulate growth and terminalia rotation during Drosophila genital disc development. Int. J. Dev. Biol. 54: 643-653. PubMed ID: 20209437

Chatterjee, S. S., et al. (2011). The female-specific doublesex isoform regulates pleiotropic transcription factors to pattern genital development in Drosophila. Development 138(6): 1099-109. PubMed ID: 21343364

Estrada, B. and Sánchez-Herrero, E. (2001). The Hox gene Abdominal-B antagonizes appendage development in the genital disc of Drosophila. Development 128: 331-339. PubMed ID: 11152632

Foronda, D., Estrada, B., de Navas, L. and Sanchez-Herrero, E. (2006). Requirement of Abdominal-A and Abdominal-B in the developing genitalia of Drosophila breaks the posterior downregulation rule. Development 133(1): 117-27. PubMed ID: 16319117

Gorfinkiel, N., Sanchez, L. and Guerrero, I. (1999). Drosophila terminalia as an appendage-like structure. Mech. Dev. 86(1-2): 113-23. PubMed ID: 10446270

Keisman, E. L. and Baker, B. S. (2001a). The Drosophila sex determination hierarchy modulates wingless and decapentaplegic signaling to deploy dachshund sex-specifically in the genital imaginal disc. Development 128: 1643-1656. PubMed ID: 11290302

Keisman, E. L., Christiansen, A. E. and Baker, B. S. (2001b). The sex determination gene doublesex regulates the A/P organizer to direct sex-specific patterns of growth in the Drosophila genital imaginal disc. Dev. Cell 1: 215-225. PubMed ID: 11702781

Kopp, A., Duncan, I. and Carroll, S. B. (2000). Genetic control and evolution of sexually dimorphic characters in Drosophila. Nature 408: 553-559. PubMed ID: 11117736

Kuranaga, E., et al. (2011). Apoptosis controls the speed of looping morphogenesis in Drosophila male terminalia. Development 138(8): 1493-9. PubMed ID: 21389055

Salvaing, J., et al. (2008). Regulation of Abd-B expression by Cyclin G and Corto in the abdominal epithelium of Drosophila. Hereditas 145(3): 138-46. PubMed ID: 18667003

Sanchez, L., Gorfinkiel, N. and Guerrero, I. (2001). Sex determination genes control the development of the Drosophila genital disc, modulating the response to Hedgehog, Wingless and Decapentaplegic signals. Development 128: 1033-1043. PubMed ID: 11245569

Spéder P, Adám G, Noselli S. (2006). Type ID unconventional myosin controls left-right asymmetry in Drosophila. Nature 440(7085): 803-7. PubMed ID: 16598259

Suzanne M., et al. (2010). Coupling of apoptosis and L/R patterning controls stepwise organ looping. Curr. Biol. 20: 1773-1778. PubMed ID: 20832313

date revised: 15 June 2011

Genes involved in organ development

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.