InteractiveFly: GeneBrief

transformer: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - transformer

Synonyms -

Cytological map position - 73A10

Function - RNA splicing

Keywords - female somatic sex determination, pre-mRNA splicing factor

Symbol - tra

FlyBase ID: FBgn0003741

Genetic map position - 3-45

Classification - Arg/Ser-rich (RS domain)

Cellular location - nuclear

NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Rideout, E. J., Narsaiya, M. S. and Grewal, S. S. (2015). The Sex determination gene transformer regulates male-female differences in Drosophila body size. PLoS Genet 11: e1005683. PubMed ID: 26710087
Almost all animals show sex differences in body size. For example, in Drosophila, females are larger than males. Although Drosophila is widely used as a model to study growth, the mechanisms underlying this male-female difference in size remain unclear. This study describes a novel role for the sex determination gene transformer (tra) in promoting female body growth. Normally, Tra is expressed only in females. Loss of Tra in female larvae decreases body size, while ectopic Tra expression in males increases body size. Although Tra was found to exerts autonomous effects on cell size, it was also discovered that Tra expression in the fat body augments female body size in a non cell-autonomous manner. These effects of Tra do not require its only known targets doublesex and fruitless. Instead, Tra expression in the female fat body promotes growth by stimulating the secretion of insulin-like peptides from insulin producing cells in the brain. These data suggest a model of sex-specific growth in which body size is regulated by a previously unrecognized branch of the sex determination pathway, and identify Tra as a novel link between sex and the conserved insulin signaling pathway.

Hudry, B., Khadayate, S. and Miguel-Aliaga, I. (2016). The sexual identity of adult intestinal stem cells controls organ size and plasticity. Nature 530: 344-348. PubMed ID: 26887495
Sex differences in physiology and disease susceptibility are commonly attributed to developmental and/or hormonal factors, but there is increasing realization that cell-intrinsic mechanisms play important and persistent roles. This study uses the Drosophila melanogaster intestine to investigate the nature and importance of cellular sex in an adult somatic organ in vivo. It was found that the adult intestinal epithelium is a cellular mosaic of different sex differentiation pathways, and displays extensive sex differences in expression of genes with roles in growth and metabolism. Cell-specific reversals of the sexual identity of adult intestinal stem cells uncovers the key role this identity has in controlling organ size, reproductive plasticity and response to genetically induced tumours. Unlike previous examples of sexually dimorphic somatic stem cell activity, the sex differences in intestinal stem cell behaviour arise from intrinsic mechanisms that control cell cycle duration and involve a new doublesex- and fruitless-independent branch of the sex differentiation pathway downstream of transformer. Together, these findings indicate that the plasticity of an adult somatic organ is reversibly controlled by its sexual identity, imparted by a new mechanism that may be active in more tissues than previously recognized. 

Khericha, M., Kolenchery, J. B. and Tauber, E. (2016). Neural and non-neural contributions to sexual dimorphism of mid-day sleep in Drosophila melanogaster: a pilot study. Physiol Entomol 41: 327-334. PubMed ID: 27840547
Many of the characteristics associated with mammalian sleep are also observed in Drosophila, making the fruit fly a powerful model organism for studying the genetics of this important process. Included among the similarities is the presence of sexual dimorphic sleep patterns, which, in flies, are manifested as increased mid-day sleep ('siesta') in males compared with females. In the present study, targeted mis-expression of the genes transformer (tra) and tra2 is used to either feminize or masculinize specific neural and non-neural tissues in the fly. Feminization of male flies using three different GAL4 drivers that are expressed in the mushroom bodies induces a female-like reduced siesta, whereas the masculinization of females using these drivers triggers the male-like increased siesta. A similar reversal of sex-specific sleep is also observed by mis-expressing tra in the fat body, which is a key tissue in energy metabolism and hormone secretion. In addition, the daily expression levels of takeout, an important circadian clock output gene, are sexually dimorphic. Taken together, these experiments suggest that sleep sexual dimorphism in D. melanogaster is driven by multiple neural and non-neural circuits, within and outside the brain.
Pomatto, L. C., Carney, C., Shen, B., Wong, S., Halaszynski, K., Salomon, M. P., Davies, K. J. and Tower, J. (2016). The mitochondrial Lon protease is required for age-specific and sex-specific adaptation to oxidative stress. J Curr Biol [Epub ahead of print]. PubMed ID: 27916526
Multiple human diseases involving chronic oxidative stress show a significant sex bias, including neurodegenerative diseases, cancer, immune dysfunction, diabetes, and cardiovascular disease. This study reports that Drosophila females but not males adapt to hydrogen peroxide stress, whereas males but not females adapt to paraquat (superoxide) stress. Stress adaptation in each sex requires the conserved mitochondrial Lon protease and is associated with sex-specific expression of Lon protein isoforms and proteolytic activity. Adaptation to oxidative stress is lost with age in both sexes. Transgenic expression of transformer gene during development transforms chromosomal males into pseudo-females and confers the female-specific pattern of Lon isoform expression, Lon proteolytic activity induction, and H2O2 stress adaptation; these effects were also observed using adult-specific transformation. Conversely, knockdown of transformer in chromosomal females eliminates the female-specific Lon isoform expression, Lon proteolytic activity induction, and H2O2 stress adaptation and produces the male-specific paraquat (superoxide) stress adaptation. Sex-specific expression of alternative Lon isoforms was also observed in mouse tissues. The results develop Drosophila melanogaster as a model for sex-specific stress adaptation regulated by the Lon protease, with potential implications for understanding sexual dimorphism in human disease.
Wehr Mathews, K., Cavegn, M. and Zwicky, M. (2017). Sexual dimorphism of body size is controlled by dosage of the X-chromosomal gene Myc and by the sex-determining gene tra in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 28064166
Drosophila females are larger than males. This paper describes how X chromosome dosage drives sexual dimorphism of body size through two means: first, through unbalanced expression of a key X-linked growth regulating gene and second, through female-specific activation of the sex-determination pathway. X-chromosome dosage determines phenotypic sex by regulating the genes of the sex-determining pathway. In the presence of two sets of X-chromosome signal elements (XSEs), Sex-lethal (Sxl) is activated in female (XX) but not male (XY) animals. Sxl activates transformer (tra), a gene that encodes a splicing factor essential for female-specific development. It has previously been shown that null mutations in the tra gene result in only a partial reduction of body size of XX animals, which shows that other factors must contribute to size determination. Whether X dosage directly affects animal size was tested by analyzing males with duplications of X chromosomal segments. Upon tiling across the X chromosome, four duplications were found that increase male size by over 9%. Only one of these, Myc, was found not to be dosage compensated. Together, these results indicate that both Myc dosage and tra expression play crucial roles in determining sex-specific size in Drosophila larvae and adult tissue. Since Myc also acts as an XSE that contributes to tra activation in early, development, a double dose of Myc in females serves at least twice in development to promote sexual size dimorphism.

The medfly Ceratitis capitata contains a gene (Cctra) with structural and functional homology to the Drosophila melanogaster sex-determining gene transformer (tra). The Ceratitis homolog of Sex lethal does not appear to have a switch function: the gene is expressed in both sexes, irrespective of whether the male-determining Y is present or absent: this is inconsistent with a main sex-determining function. However, preliminary data suggest that the bottom-most component of the pathway, doublesex (dsx), is not only present in Ceratitis (Ccdsx), but has conserved a role in sexual differentiation. The pre-mRNA of this gene is also alternatively spliced giving rise to sex-specific products that show a remarkable structural conservation when compared with the corresponding male and female products in Drosophila. Sequence analysis of Ccdsx has revealed the presence of putative TRA/TRA-2-binding sites close to the regulated splice site, suggesting that the underlying mechanism of sex-specific splicing is conserved and under the control of proteins homologous to Transformer and Transformer-2. Similar to tra in Drosophila, Cctra is regulated by alternative splicing such that only females can encode a full-length protein. In contrast to Drosophila, however, where tra is a subordinate target of Sex-lethal (Sxl), Cctra seems to initiate an autoregulatory mechanism in XX embryos that provides continuous tra female-specific function and acts as a cellular memory maintaining the female pathway. Indeed, a transient interference with Cctra expression in XX embryos by RNAi treatment can cause complete sexual transformation of both germline and soma in adult flies, resulting in a fertile male XX phenotype. The male pathway seems to result when Cctra autoregulation is prevented and instead splice variants with truncated open reading frames are produced. It is proposed that this repression is achieved by M, the Y-linked male-determining factor (Pane, 2002 and references therein).

A broad variety of genetic cues that determine the sexual fate of a developing individual are known (see Schematic of the sex determination hierarchy in Control of male sexual behavior in Drosophila by the sex determination pathway, Billeter, 2006). Even within a minor taxonomic group, for example, dipteran insects widely different mechanisms of sex determination are found (Marin, 1998; Schutt, 2000); male heterogamety with a male-determining Y chromosome (Musca, Ceratitis) or with a single autosomal factor (Megaselia, Culex), female heterogamety (Musca, Chironomus), chromosomal balance systems (Drosophila, Sciara), and maternal effects (Chrysomya). This variety raises the issues of how these different mechanisms have evolved and how much they differ at the genetic and molecular level. Comparative analyses of different species can be used to address these problems. The economically important medfly (Mediterranean fruitfly), Ceratitis capitata (Tephritidae) has been studied in detail. In this species a Y-linked factor, M, determines maleness, and absence of M leads to female development (Willhoeft, 1996). However, how this signal is relayed to genes responsible for expressing dimorphic traits is completely unknown (Pane, 2002 and references therein).

The genetic cascade regulating sexual development in Drosophila is well known down to molecular details (Cline, 1993; Cline, 1996). In contrast to Ceratitis, the primary signal in Drosophila is polygenic and is formed by the ratio of X chromosomes to sets of autosomes, the so-called X:A ratio. When this ratio is 1.0 (XX:AA), the gene Sex-lethal (Sxl) is activated; with a ratio of 0.5 (X:AA), Sxl remains inactive. Sxl now acts as the key ON/OFF switch that controls all aspects of somatic sexual dimorphism via a short cascade of subordinate regulatory genes (Nagoshi, 1988). When the gene is active, it dictates female development; when it is inactive, male development follows. Once the gene is activated in females, its products initiate a positive autoregulatory mechanism that guarantees the continuous production of Sxl, thus forming a cell memory of the sex and maintaining the cells on the female pathway throughout development (Bell, 1991). In males, however, where Sxl is not activated, the gene will remain functionally OFF. Sxl produces sex-specific mRNAs by alternative splicing: the female-specific mRNAs encode full-length functional Sxl protein, while the male-specific ones have an additional stop-containing exon and encode a truncated non-functional Sxl peptide. The ON/OFF state of Sxl activity is set early during embryogenesis by a complex combination of transcriptional and post-transcriptional gene regulation (Bell, 1991; Keyes, 1992). The initial activation of Sxl in XX embryos relies on the use of an alternative XX-embryo-specific promoter that responds to the genes signaling the X:A ratio (Parkhurst, 1990). Sxl pre-mRNAs produced from this promoter have such a structure that they are spliced in a female-specific mode by the spliceosome independently of additional trans-acting factors, such as the Sxl protein itself (Horabin, 1996; Zhu, 1997). The RNA-binding Sxl proteins translated from these early mRNAs then initiate the autoregulatory loop by directing the female-specific processing of the pre-mRNAs produced from the late Sxl promoter. The late pre-mRNAs, in contrast to the early Sxl pre-mRNAs, can be spliced in the female-specific mode only in the presence of Sxl protein (Pane, 2002 and references therein).

To execute the correct developmental program, Sxl transmits the determined state to transformer (tra) (Boggs, 1997), the next gene in the cascade. At this level, Sxl regulates the choice between two alternative 3' splice sites in the pre-mRNA of tra (Inoue, 1990; Valcárcel, 1993). In absence of Sxl, the more proximal site is used, resulting in a tra mRNA that encodes a truncated inactive protein. When Sxl is present, it will bind to the tra pre-mRNA and enforce the use of the distal 3' splice site to produce an mRNA with a full-length ORF (Sosnowski, 1989). The state of activity of tra is then transmitted to doublesex (dsx) (Burtis, 1989), the last component of the pathway. In females, TRA, together with the constitutively expressed TRA-2, binds to dsx pre-mRNA directing its female-specific splicing, such that a mature mRNA encoding the DSXF protein is generated (Hoshijima, 1991; Tian, 1993). In males, absence of TRA causes male-specific splicing and the production of a DSXM protein. The two proteins, DSXF and DSXM, are transcription factors (Burtis, 1989) that regulate the activity of sex-specific differentiation genes (Pane, 2002 and references therein)

Studies have indicated that control of sexual development in the medfly follows a different route. In particular, the Ceratitis homolog of Sxl does not appear to have a switch function: the gene is expressed in both sexes, irrespective of whether the male-determining Y is present or absent (Saccone, 1998), which is inconsistent with a main sex-determining function. However, preliminary data suggest that the bottom-most component of the pathway, dsx, is not only present in Ceratitis (Ccdsx), but has conserved a role in sexual differentiation (Saccone, 2000). The pre-mRNA of this gene is also alternatively spliced giving rise to sex-specific products that show a remarkable structural conservation when compared with the corresponding male and female products in Drosophila. Sequence analysis of Ccdsx reveals the presence of putative TRA/TRA-2-binding sites close to the regulated splice site, suggesting that the underlying mechanism of sex-specific splicing is conserved and under the control of proteins homologous to TRA and TRA-2 (Saccone, 2000; Saccone, 2002; Pane, 2002 and references therein).

A Ceratatis homolog of Drosophila transformer gene (Cctra), is highly diverged in sequence. As in Drosophila, Cctra has a female-determining master function. However, in contrast to the Drosophila tra, Cctra plays an essential role in Ceratitis sex determination by maintaining the female sexual cell state through a positive feedback loop and by forming an epigenetic memory of the sex of the organism (Pane, 2002).

Thus Ceratitis and Drosophila sex-determining cascades share a conserved tra-->dsx genetic module to control sex determination and sexual differentiation, but tra sex-specific splicing regulation differs in the two species. In Drosophila, Tra protein, together with Tra-2, binds to the Tra/Tra-2 recognition sequences on the Drosophila dsx pre-mRNA and promotes the use of a nearby female-specific acceptor site. Cctra is needed to impose the female-specific splicing of Ccdsx, most probably by a similar mechanism as in Drosophila, invoking the existence of a Cctra2 homolog. This hypothesis is also supported by the finding of Tra/Tra-2 recognition sequences located in close vicinity to the female-specific acceptor site in Ccdsx pre-mRNA (Pane, 2002).

How does tra sex-specific splicing regulation differ in the two species? In Drosophila, tra female-specific splicing is promoted by Sxl, which blocks the use of the non-sex-specific splice site present in the tra pre-mRNA. In Ceratitis, the presence of multiple Tra/Tra-2-binding elements within the Cctra male-specific exonic sequences strongly suggests that CcTRA and a hypothetical CcTRA-2 protein could bind to these sequences, thus mediating a direct autoregulation. The unusually strong phenotypic effects of the RNAi against CCtra also support this model of Cctra regulation. The localization of the putative regulatory elements within the Cctra gene indicates a repression mode by which CcTRA in females prevents the recognition of male-specific splice sites. The mechanism by which Cctra seems to promote the female mode of processing of its own pre-mRNA by TRA/TRA-2-binding elements appears to be different also from the female-specific splicing of dsx. Rather than activating a splice site nearby the regulated exon, as in the case of dsx, inclusion of male-specific Cctra sequences is suppressed when CcTRA is present. Although this would be a novelty with respect to known Drosophila Tra/Tra-2 activities, the 'behavior' of these cis elements is context dependent and changing the location of splicing enhancers can transform them into negative regulatory elements (Pane, 2002).

In Drosophila, the presence of the Y chromosome is necessary for male fertility but not for male development. By contrast, RNAi-treated Ceratitis embryos with a female XX karyotype can develop into fertile males; this indicates that transient repression of Cctra by RNAi is sufficient to implement fully normal male development. The cases of complete sexual transformation of genetic Ceratitis females (XX) into fertile males by RNAi demonstrate that the Y chromosome, except for the dominant male determiner M, does not supply any other contribution to both somatic and germline male development, as suggested by previous Y-chromosome deletion analysis. Other dipteran species, such as Musca domestica and Chrysomya rufifacies show a female and male germline sex determination that is completely dependent on the sexual fate of the soma. However, in Drosophila, the XX and XY germ cells seem to respond differently to sex determining somatic cues. Indeed the XY germ cells have also an autonomous stage-specific sex determination mechanism that probably integrates the somatic signal. In Ceratitis, Cctra could be required in XX somatic cells to let them induce the XX germ cells to differentiate as oogenic cells. Alternatively, Cctra could be required in XX germ cells to 'feminize' them. This case would be a novelty with respect to the known Drosophila transformer gene functions (Pane, 2002).

Since zygotes that carry a Y chromosome do not activate Cctra female-specific splicing and autoregulation, it is proposed that the Y-linked male-determining M factor prevents this activation. It is conceivable that Cctra is a direct target of the M factor. Presence of this M factor in the zygote may prevent the production of CcTRA protein. The Cctra positive feedback loop is a probable target for regulation, because of its sensitivity (already shown by RNAi). An important question to be addressed is how autoregulation of Cctra is initiated in XX embryos of C. capitata and how this is prevented in XY embryos. A possible explanation is suggested by the Cctra female-specific mRNAs encoding the full-length protein, that have been detected in unfertilized eggs. Depositing these Cctra transcripts in eggs may provide a source of activity that can be used later for 'female-specific' processing when Cctra is zygotically transcribed. Once zygotically activated in XX embryos, Cctra promotes its own female-specific splicing, maintaining the female sex determination and the female-specific splicing of the downstream Ccdsx gene. Taken together, these events induce the female differentiation. In the current model for sex determination of medfly, the M factor is directly involved in the Cctra sex-specific regulation. Thus, in the presence of M Cctra, autoregulation is blocked and the gene produces male-specific transcripts encoding short and possibly non-functional CcTRA peptides. The absence of CcTRA leads Ccdsx to produce male-specific transcripts by default, promoting male differentiation. The control of the M factor upon Cctra expression could be exerted at different levels. The male determiner M could, for example, act at the pre-translational level blocking the production of CcTRA protein from the maternal transcripts. M could act at the post-translational level antagonizing the formation of protein complexes necessary for the female splicing mode. Or M could act as a transient transcriptional repressor of Cctra to reduce the amount of active CcTRA below a threshold needed to maintain the feedback loop. The proposed autoregulatory model of Cctra may also explain the remarkable efficiency of sex reversal by Cctra RNAi: a transient silencing of Cctra by injecting dsRNA is sufficient to let the loop collapse. Furthermore, the sensitivity of this positive autoregulation could be an evolutionary widely conserved pre-requisite to permit a 'faster' recruitment/replacement of different upstream regulators and to easily evolve different sex determining primary signals, as observed in dipteran species (Pane, 2002).

Sex can even be determined by a maternal effect in dipteran species such as Sciara coprophila and Chrysomya rufifacies. The hypothesis of a Cctra maternal contribution to the activation of the zygotic Cctra gene has similarities to the model of sex determination proposed for Musca domestica. In the common housefly, the maternal product of the key switch gene F is needed to activate the zygotic function of F in females. Musca male development results whenever F cannot become active in the zygote. This happens when the male-determining M is present in the zygotic genome, or when maternal F is not functional because of either the presence of M or the mutational loss of function of F (Fman) in the germline. More interesting, embryonic RNAi against the Musca tra-2 homolog causes sex reversion of Musca XX adults into intersex and fertile males, although this gene is not sex-specifically expressed. These recent data in Musca and the results in Ceratitis support the idea that F of Musca functionally corresponds to the Ceratitis tra gene, which seems to autoregulate and maternally contribute to its own activation, rather than to the Drosophila tra gene (Pane, 2002).

These data show that a basic structure of sex determination is conserved in the two dipteran species, namely the flow of 'instructions' from tra to dsx. This confirms the model of 'bottom-up' evolution, suggesting that during evolution, developmental cascades are built from bottom up and that the genes at the bottom are widely conserved, while further upstream new regulatory elements may be recruited. The results show that Ceratitis and Drosophila sex-determining cascade differ at the level of transformer as well as upstream of it. Indeed the gene has conserved its function during evolution, but it has female-specific positive autoregulation in Ceratitis, while in Drosophila it needs Sxl as upstream regulator to express its female determining function. More likely the sex-determining function of Sxl was co-opted after Drosophila and Ceratitis had separated more than 100 Myr ago. Furthermore, it is conceivable that the autoregulatory mechanism of Sxl could have been selected to overcome a mutation impairing the tra autoregulation. Hence, in both species the female pathway is maintained by a single gene positive-feedback mechanism through sex-specific alternative splicing. Single gene autoregulation by alternative splicing seems not to be infrequent in nature, especially in those genes encoding splicing regulators. Indeed, other genes encoding RNA-binding proteins are thought to autoregulate their expression by controlling the processing of their own pre-mRNAs. Such a single-gene network with positive regulation is capable of bistability. This suggests that the emergence of analogous positive autoregulation in different genes such as Drosophila Sxl and Ceratitis tra genes would have been selected, during evolution, to guarantee a similar ON/OFF-female/male bistable cell state (Pane, 2002).

Since Ceratitis capitata is a major agricultural pest in many areas of the world, the isolation of a key sex-determining gene such as Cctra will substantially aid the development of new strategies to optimize the efficacy of currently used male sterile techniques for pest control. It is expected that tra is also a key sex-determining gene in many other insect species. Hence, the isolation of corresponding tra genes will open new means to control not only agricultural pests but also medically relevant vectors of diseases such as Glossina palpalis and Anopheles gambiae (Pane, 2002).

Drosophila switch gene Sex-lethal can bypass its switch-gene target transformer to regulate aspects of female behavior

The switch gene Sex-lethal (Sxl) was thought to elicit all aspects of Drosophila female somatic differentiation other than size dimorphism by controlling only the switch gene transformer (tra). This study shows instead that Sxl controls an aspect of female sexual behavior by acting on a target other than or in addition to tra. The existence of this unknown Sxl target was inferred from the observation that a constitutively feminizing tra transgene that restores fertility to tra- females failed to restore fertility to Sxl-mutant females that were adult viable but functionally tra-. The sterility of these mutant females was caused by an ovulation failure. Because tra expression is not sufficient to render these Sxl-mutant females fertile, this pathway is referred to as the tra-insufficient feminization (TIF) branch of the sex-determination regulatory pathway. Using a transgene that conditionally expresses two Sxl feminizing isoforms, it was found that the TIF branch is required developmentally for neurons that also sex-specifically express fruitless, a tra gene target controlling sexual behavior. Thus, in a subset of fruitless neurons, targets of the TIF and tra pathways appear to collaborate to control ovulation. In most insects, Sxl has no sex-specific functions, and tra, rather than Sxl, is both the target of the primary sex signal and the gene that maintains the female developmental commitment via positive autoregulation. The TIF pathway may represent an ancestral female-specific function acquired by Sxl in an early evolutionary step toward its becoming the regulator of tra in Drosophila (Evans, 2013).

Developmental regulatory pathways are rarely as simple as they first appear, but as the twist to the Drosophila sex-determination pathway this study reports here suggests, complications can provide clues to evolution. It was shown that Sxl, the rapidly evolved target of the Drosophila primary sex-determination signal, no longer can be regarded as transmitting all its feminizing orders other than size dimorphism to the soma exclusively through its well-known switch-gene target tra. Instead, one must distinguish between a major pathway branch, TSF, in which tra is sufficient to dictate feminization, and a minor branch, TIF, in which it is not (Evans, 2013).

Evidence for the TIF branch derives from female-viable but masculinizing combinations of partial-loss-of-function Sxl alleles that fail to induce either TSF or TIF in diplo-X individuals, so that when TSF-branch activity is restored by constitutively feminizing transgene U2af-traF or, even more definitively, by a constitutively feminizing mutant endogenous tra allele, mutant females remain TIF defective and hence sterile. Although TIF-mutant sterility superficially resembles sterility in TSF-mutant transgenics, in that both phenotypes include a failure to lay eggs, the TIF-mutant block to egg laying occurs at ovulation, whereas that in TSF-defective transgenics occurs later at oviposition. The possibility that the kind of branch in the TIF pathway that is reported in this study might exist was suggested first in a previous paper reporting the behavior of some U2af-traF-feminized gynandromorphs (coarse-grained XX//XO mosaics) in which the failure of Sxl to activate what is now known to be the TIF pathway was a consequence of the absence of a female primary sex-determination signal in TRA-F-feminized Sxl+ XO cells (Evans, 2007) rather than a consequence of Sxl mutations in TRA-F–feminized XX cells. Because 38% of the feminized egg-producing gynandromorphs failed to lay their eggs, it is concluded that there must be some functionally Sxl- XO somatic cells that cannot substitute for the XX somatic cells required for egg laying, even when feminized by TRA-F. Although gynandromorphs are not nearly as convenient as Sxl-mutant females for studying TIF, they do strengthen the argument that TIF-defective sterility is not caused either by a upset in dosage compensation or by some idiosyncrasy of U2af-traF in Sxl-mutant females (Evans, 2013).

Strong evidence is necessary to legitimize the TIF claim because of the surprising finding that SXL-F functioning in the TIF pathway takes place in a subset of neurons that sex-specifically express fru mRNAs. Because fru sex-specific splicing is controlled entirely by TRA-F, the simplest model would suggest that any deficiency in the sex-specific functioning of these neurons reflects a TSF defect. Of course, just because fru is sex-specifically regulated in these neurons does not require that fru be solely or even partially responsible for their feminization in every case (Evans, 2013).

At this point the 'I' in TIF necessarily stands for 'insufficient' rather than 'independent.' Because conditions under which the TIF phenotype was studied were all ones in which TRA-F activity for the TSF pathway was provided at a level sufficient to rescue the sterility of tra− females, no evidence for or against independence could be generated. If, as the fru neuron results might suggest, tra works with one or more unknown Sxl targets to achieve full feminization in some neurons, the name ultimately might have to be changed to something like 'tra-partnered feminization.' Discovering the identity of the Sxl TIF-gene targets and the specific neurons in which they are required would provide the tools necessary to resolve this question about the relationship between TSF and TIF. The recent availability of an enormous panel of well-characterized neuronal GAL4 drivers should be a great help in this connection, particularly in view of the finding that GAL4-driven SXL-F expression can rescue the TIF-mutant phenotype in females while causing little damage to males. The gene female-specific-independent-of-transformer seemed to be a promising candidate TIF-pathway target until it was shown that, contrary to a previous study, it is firmly in the TSF pathway (and hence is in need of renaming) (Evans, 2013).

The ovulation block should be particularly amenable to future genetic and developmental analyses designed to identify targets of the TIF because it is particularly suited to positive genetic selection in a suppression screen. Arguing for the potential of such a suppression screen is the fact mentioned above that fertility could be restored to TIF-defective females by a GAL4 driver/ SXL-F target combination that had relatively little adverse effect on male viability or fertility. Such sex specificity suggests that the set of neurons responsible for the TIF ovulation defect may not be very large and that disruption of their normal controls is unlikely to disrupt non–sex-specific aspects of development (Evans, 2013).

This report introduces several genetic tools, among which the GAL4 target UAS-Sxlalt5-C8 is perhaps the most broadly useful. That this transgene, which conditionally generates both exon-5 alternative SXL-F isoforms, provides relatively strong Sxl+ function while having no adverse effect on females indicates that the adverse effect on females caused by the Sxl GAL4 target previously reported, a transgene that encodes only a single exon-5 isoform, may not reflect a normal activity of SXL-F protein. Another useful tool is Dp(1;1)SxlΔPm, which can expand the utility of various partial-loss-of-function Sxl alleles. This tool is a chromosomal duplication of Sxl truncated at its 5′ end so that it lacks the gene's maintenance promoter but retains an intact establishment promoter and all the activities that transiently active promoter elicits. The response of this truncated Sxl allele to the female X-chromosome dose signal, a response that ends during the early blastoderm stage, can facilitate engagement of the Sxl positive-feedback loop for various Sxl-mutant alleles without otherwise influencing their Sxl-mutant phenotype. For example, Dp(1;1)SxlΔPm is particularly useful in combination with the intriguing double mutant Sxlf18,f32 because together they can generate thoroughly masculinized Sxl-mutant adult females (pseudomales) with far higher viability and longevity than any previously described masculinizing Sxl genotype. Last, two dominant temperature-sensitive lethal balancers that were introduced in this study should be generally useful, because they allow crosses to be designed so that daughters with one combination of a maternal and paternal X chromosome of choice are the only progeny to survive (Evans, 2013).

Sex determination for flies in the family Drosophilidae is unlike that for most other higher insects in many fundamental respects, including having Sxl rather than tra as the target of their primary sex-determination signal and having Sxl rather than tra as the gene whose positive-feedback loop on its own pre-mRNA splicing maintains the female developmental pathway commitment. Although the TIF branch could be a recent addition to the Drosophila sex-determination pathway made well after Sxl had taken over tra's role as the master feminizing gene, a more intriguing possibility is that TIF instead may reflect an ancestral function that Sxl acquired in the earliest step on its evolutionary path toward usurping tra's role as master sex switch. Because both TIF and TSF function in neurons that sex-specifically express fru, perhaps the first female- specific function that Sxl acquired was to modify the developmental functioning of fru in some neurons. Initially this function may have been achieved without the need for a sex-specific Sxl product, with sex-specific products coming only later as fine-tuning of that particular function under the control of tra. The switch from tra as a regulator of Sxl to Sxl as a regulator of tra (a switch that could have been facilitated by the development of redundancy in the positive-feedback circuits for the two genes) would make any female-specific gene target of Sxl that existed before the switch be independent of tra regulation today if its control by Sxl persisted. Of course there are many important questions about the remarkable path taken by Sxl functional evolution and the forces that drove those changes for which an understanding of the TIF pathway might not be relevant. How did Sxl come to respond to an X-chromosome dose signal? How did it come to control X-chromosome dosage compensation? Why is Sxl's control of germ-line sex determination so different from its control of sex determination in the soma? On the other hand, because next to nothing is known about any of these questions, it is hard to predict where clues might lead regarding an early female-specific Sxl function that the TIF pathway might help reveal. Regardless of whether the TIF pathway is ancestral or recent, further analysis leading to the discovery of the SXL-F targets in this regulatory branch undoubtedly will advance understanding how genes control behavior and how SXL-F proteins control RNA functioning (Evans, 2013).


Alternative splicing of transformer RNA

The transformer gene regulates female somatic sexual differentiation and has no known function in males. It gives rise to two sizes of RNA, one non-sex-specific and one female-specific. These two RNAs are shown to be present throughout the life cycle, and related by the use of alternative first intron splice acceptor sites. The non-sex-specific RNA has a 73 base first intron, while that in the female-specific RNA is 248 bases. The non-sex-specific RNA has no long open reading frame, while the female-specific RNA has a single long open reading frame beginning at the first AUG. Substitution of a heat shock promoter for the tra promoter still leads to female-specific differentiation of otherwise tra-females. A mechanism is suggested by which Sex-lethal controls itself and tra (Boggs, 1987).

Sex-specific alternative splicing of RNA from the Drosophila transformer gene involves competition between two 3' splice sites. In the absence of Sex-lethal activity (as in males), only one site functions; in the presence of Sex-lethal activity (as in females), both sites function. Information for sex-specific splice site choice is contained within the intron itself. Deletions of the splice site used in males lead to Sex-lethal-independent use of the otherwise female-specific site. The relative amounts of unspliced and spliced RNA derived from these mutant genes do not change with changes in Sex-lethal activity. Specific nucleotide changes in the non-sex-specific splice site do not affect splicing activity but eliminate Sex-lethal-induced regulation. A deletion removing material between the two splice sites does not eliminate sex-specific regulation, while a deletion of the female splice site leads to a female-specific increase in unspliced RNA. These results are consistent with a model in which female-specific factors block the function of the non-sex-specific 3' splice site (Sosnowski, 1989).

Somatic sexual differentiation in Drosophila melanogaster is accomplished by a hierarchy of genes of which one, Sex-lethal, is required for the functional female-specific splicing of the transcripts of the immediately downstream regulatory gene, transformer (tra). The first exon of the tra primary transcript is spliced to one of two acceptor sites. Splicing to the upstream site yields a messenger RNA which is neither sex-specific nor functional, but that produced after splicing to the downstream acceptor site yields a functional female-specific mRNA. This study addresses the question of how the Sxl gene product determines the alternative splicing of tra primary transcripts. One suggestion is that non-sex-specific splicing to the upstream acceptor is blocked in female flies by sex-specific factors, but neither the identity of the female-specific factors nor the mechanism of the blockage has been specified. Co-transfection experiments were performed in which Sxl complementary DNA and the tra gene are expressed in Drosophila Kc cells. Moreover, it was found that female Sxl-encoded protein binds specifically to the tra transcript at or near the non-sex-specific acceptor site, implying that the female Sxl gene product is the trans-acting factor that regulates the alternative splicing (Inoue, 1990).

The protein Sex-lethal activates a female-specific 3' splice site in the first intron of Transformer pre-mRNA while repressing an alternative non-sex-specific site. Using an in vitro system, the molecular basis of the splice site switch has been determined. SXL inhibits splicing to the non-sex-specific (default) site by specifically binding to its polypyrimidine tract, blocking the binding of the essential splicing factor U2AF. This enables U2AF to activate the lower-affinity female-specific site. A splicing 'effector' domain present in U2AF but absent from SXL accounts for the different activities of these two polypyrimidine-tract-binding proteins: addition of the U2AF effector domain to SXL converts it from a splicing repressor to an activator and renders it unable to mediate splice-site switching (Valcarcel, 1993).

Splicing of the transformer gene of Drosophila is regulated by Sex-lethal-dependent 3' splice site blockage. 40 nucleotides immediately upstream of the regulated splice site are sufficient to direct sex-specific regulated splicing in transgenic animals. This entire region appears to be necessary for regulation and for efficient Sex-lethal binding. Natural splice sites containing partial homology to Transformer do not show regulation. Mutations that replace the 16 nucleotides surrounding the branch point or that alter single nucleotides near the splice site eliminate or reduce regulation without eliminating splicing. Mutations that reduce or eliminate regulation in vivo reduce binding to Sex-lethal in vitro, consistent with the hypothesis that these mutations bring about their effects by altering Sex-lethal binding rather than by altering binding sites for additional non-Sex-lethal factors (Sosnowski, 1994).

Sex determination and X chromosome dosage compensation in Drosophila melanogaster are directed by the Sex-lethal (Sxl) protein. In part, Sxl functions by regulating the splicing of the Transformer pre-mRNA by binding to a 3' splice site polypyrimidine tract. Polypyrimidine tracts are essential for splicing of metazoan pre-mRNAs. To unravel the mechanism of splicing regulation at polypyrimidine tracts the interaction of Sxl with RNA was studied. The RNA binding activity of Sxl maps to the two ribonucleoprotein consensus sequence domains of the protein. Quantitation of binding shows that both RNA binding domains (RBDs) are required in cis for site-specific RNA binding. Individual RBDs interacted with RNA more weakly and had lost the ability to discriminate between wild-type and mutant Transformer polypyrimidine tracts. Structural elements in one of the RBDs that are likely to interact with a polypyrimidine tract have been identified using nuclear magnetic resonance techniques. In addition, these data suggest that multiple imino protons of the Transformer polypyrimidine tract are involved in hydrogen bonding. Interestingly, in vitro Sxl binds with equal affinity to the polypyrimidine tracts of pre-mRNAs that it does not regulate in vivo (Kanaar, 1995).

The gene virilizer is needed for dosage compensation and sex determination in females and for an unknown vital function in both sexes. In genetic mosaics, XX somatic cells mutant for vir differentiate male structures. One allele, vir2f, is lethal for XX, but not for XY animals. This female-specific lethality can be rescued by constitutive expression of Sxl or by mutations in msl (male-specific lethal) genes. Rescued animals develop as strongly masculinized intersexes or pseudomales. They have male-specifically spliced mRNA of tra, and when rescued by msl, also of Sxl. These data indicate that vir is a positive regulator of female-specific splicing of Sxl and of tra pre-mRNA (Hilfiker, 1995).

All the tested alleles that cause lethality to males and females are also deficient for the female-specific functions of dosage compensation and sex determination. These pathways are controlled by Sxl, and vir could thus be a regulator of Sxl. The role of Sxl is well established in the pathway of somatic sex determination. Its protein, besides regulating the splicing of its own pre-mRNA (autoregulation), promotes the female-specific splicing of the tra pre-mRNA (transregulation) by blocking the 3' splice site that is used in males. The most obvious and best understood aspect of vir is its role in sex determination, as demonstrated by XX;vir mutant cells forming male structures in genetic mosaics. The temperature-sensitive intersexual phenotype of sturtevant/transformer;vir1ts/vir1ts animals is rescued by the construct hs-tra-female that constitutively expresses the female mode of tra. This indicates that vir acts upstream of tra. The new findings confirm the high hierarchical position of vir in the cascade of sex-determining genes and suggest that it plays a role in the regulation of Sxl and tra (Hilfiker, 1995).

These genetic arguments receive support from molecular analyses that show that surviving XX; vir2f mle pseudomales produce mRNAs of Sxl and tra that are male-specifically spliced. The male-specific products of tra could be the consequence of the male-specific expression of Sxl. The next experiments, however, indicate that vir is also directly engaged in the splicing of tra transcripts (Hilfiker, 1995).

Since SxlM/+;vir2f animals are efficiently rescued from the female lethal effect of vir2f, but remain sexually transformed pseudomales or strongly masculinized intersexes, it is concluded that the sex-transforming effect of vir mutations is not exerted via Sxl alone. When the female-specific function of Sxl is constitutively provided in XX;vir2f animals by SxlM or SxlcF1#19, the female-specific function of tra is still not guaranteed, but in addition appears to require an active product of vir. To test this conclusion the rescued animals were subjected to Western analysis. Substantial levels of Sxl protein are present in strong intersexes and pseudomales. Even animals with normal levels of Sxl, such as the intersexes, still splice the pre-mRNA of their tra gene largely in the male mode, as indicated by their strongly masculinized appearance. These intersexes are not mosaics of male and female cells, but display an intermediate sexual phenotype at the cellular level characteristic of flies expressing only low levels of female-specific products of tra. The results thus support the conclusion that vir is needed not only for female-specific expression of Sxl, but also of tra. This may also be the case for fl(2)d, although to a lesser extent, since XX animals mutant for fl(2)d are rescued by SxlM1 and are sexually transformed into intersexes at 29°C (Hilfiker, 1995).

Sex determination in Drosophila depends on the post-transcriptional regulatory activities of the gene Sex-lethal (Sxl). Sxl maintains the female determined state and activates female differentiation pathways by directing the female-specific splicing of Sxl and tra pre-mRNAs. While there is compelling evidence that Sxl proteins regulate splicing by directly binding to target RNAs, previous studies indicate that the two Sxl RNA-binding domains are not in themselves sufficient for biological activity and that an intact N-terminal domain is also critical for splicing function. To further investigate the functions of the Sxl N terminus, a chimeric protein consisting of the N-terminal 99 amino acids fused to beta-galactosidase (hsp83:N beta-gal) was ectopically expressed. The Nbeta-gal fusion protein behaves like a dominant negative, interfering with the Sxl autoregulatory feedback loop and killing females. This dominant negative activity can be attributed to the recruitment of the fusion protein into the large Sxl:Snf splicing complexes that are found in vivo and the consequent disruption of these complexes (Deshpande, 1999).

Since the Sxl gene can be completely deleted in males without any detectable effects on viability, morphology or behavior, the N beta-gal transgene would not, in principle, be expected to have any effect on male-specific developmental pathways. However, contrary to this expectation, approximately 10% of the males in all of the lines exhibit morphological alterations characteristic of sex transformations. These transformations include reduced or patchy abdominal pigmentation, extra sternite hairs, rotated genitalia and alterations in the number or shape of the sex combs. In females, the phenotypic effects of the hsp83:N beta-gal transgene can be enhanced by raising the temperature and suppressed by lowering the temperature. To test whether this is also true for male feminization, wild-type females were crossed to males heterozygous for the N-41 transgenic line, and examined the viability and morphology of the resulting transgenic animals at either 25°C or 18°C. The lethal effects of a single copy of the transgene on female progeny from this cross decreases from about 16% at the higher temperature to about 6% at the lower temperature. On the contrary, decreasing the temperature does not reduce the frequency of feminization; the number of partially feminized males increases from 7% to about 21% as the temperature is dropped from 25°C to 18°C. A low but readily detectable level of yp1 mRNA is present in transgenic N-172 and N-21 males, but not in the control wild-type males. Expression of yp1 mRNA in transgenic males does not require the Sxl gene but does require tra (Deshpande, 1999).

The gain-of-function activity of the Nbeta-gal fusion protein is difficult to reconcile with the prevailing blockage model for the regulation of tra splicing. In this model, Sxl protein prevents the generic splicing factor U2AF from binding to the polypyrimidine tract of the default 3' splice site, forcing it instead to interact with the weaker downstream female-specific 3' splice site. A strong prediction of this model is that Sxl must be able to bind to the default polypyrimdine tract in order to block the binding of U2AF. Since specific binding to target RNAs requires the two Sxl RRM domains, the Nbeta-gal fusion protein should have absolutely no tra regulatory activity. This prediction is not fulfilled. A second, weaker, prediction is that the two Sxl RRM RNA-binding domains should be sufficient to regulate tra splicing. Taken together these findings call into question the simple blockage model and suggest that regulation of tra splicing may be different from that previously envisioned. The most likely mechanism is through interactions with generic RNA-binding proteins (or other components of the splicing machinery) that associate with TRA pre-mRNAs (Deshpande, 1999).

The Sex-lethal (Sxl) protein regulates alternative splicing of the Transformer (TRA) messenger RNA precursor by binding to the tra polypyrimidine tract during the process of sex-determination. Sxl binds tightly to a characteristic uirdine-rich polypyrimidine tract at the non-sex-specific 3' splice site in one of the TRA introns, preventing the general splicing factor U2AF from binding to this site and forcing it to bind to the female specific 3' splice site. The crystal structure has now been determined at 2.6 A resolution of the complex formed between two tandemly arranged RNA-binding domains of the Sxl protein and a 12-nucleotide, single-stranded RNA derived from the tra polypyrimidine tract. The Sxl-binding polypyrimidine tract of TRA-mRNA does not form a tertiary base-paired structure. The two RNA-binding domains have their beta-sheet platforms facing each other to form a V-shaped cleft. The RNA is characteristically extended and bound in this cleft, where the UGUUUUUUU sequence is specifically recognized by the protein. This structure offers an insight into how a protein binds specifically to a cognate RNA without that RNA possessing any intramolecular base-pairing (Handa, 1999).

In Drosophila, Sex-lethal (Sxl) controls autoregulation and sexual differentiation by alternative splicing but regulates dosage compensation by translational repression. To elucidate how Sxl functions in splicing and translational regulation, a full-length Sxl protein (Sx.FL) and a protein lacking the N-terminal 40 amino acids (Sx-N) were ectopically expressed. The Sx.FL protein recapitulates the activity of Sxl gain-of-function mutations, since it is both sex transforming and lethal in males. In contrast, the Sx-N protein unlinks the sex-transforming and male-lethal effects of Sxl. The Sx-N proteins are compromised in splicing functions required for sexual differentiation, displaying only partial autoregulatory activity and almost no sex-transforming activity. However, the Sx-N protein does retain substantial dosage compensation function and kills males almost as effectively as the Sx.FL protein. In the course of the analysis of the Sx.FL and Sx-N transgenes, a novel, negative autoregulatory activity, in which Sxl proteins bind to the 3' untranslated region of SXL mRNAs and thereby decrease Sxl protein expression, was also uncovered. This negative autoregulatory activity may be a homeostasis mechanism (Yanowitz, 1999).

While the Sx-N protein retains at least some autoregulatory and dosage compensation activities, the most striking finding is that the protein seems to lack the ability to regulate the tra sexual differentiation pathway. The feminization activities of the Sx.FL and Sx-N transgenes were compared in males that do not contain the endogenous Sxl locus. This background allows alterations in somatic sexual differentiation to the Sxl proteins expressed by the transgenes to be unambiguously ascribed. The extent of sex transformation that is seen in escaper Sx.FL males again appears to resemble that for the strong cF1 lines, in which the Sxl cDNA is expressed under the control of the inducible hsp70 promoter. The males are intersexual; they have lighter abdominal pigmentation, rotated genitalia, and fewer sex combs, and they are sterile. In sharp contrast, Sxl minus; Sx-N males are morphologically indistinguishable from wild-type males and are fertile. The absence of sex transformations in Sx-N males most likely reflects an inability to regulate the tra pathway. To determine if this is the case, a RT-PCR assay was used to examine the splicing pattern of tra mRNAs. Three amplification products can be detected in this assay, and these correspond to unspliced RNA, default (male) spliced RNA, and female spliced RNA. Of these, only unspliced and default spliced tra RNAs are observed in wild-type males, while all three species are found in females. Thus, while the Sx.FL transgene has tra regulatory activity, it is not as effective as the endogenous Sxl gene. These findings confirm the suggestion that the Sx-N protein is defective in its tra regulatory function (Yanowitz, 1999).

The Drosophila gene female-lethal(2)d [fl(2)d] interacts genetically with the master regulatory gene for sex determination, Sex-lethal. Both genes are required for the activation of female-specific patterns of alternative splicing on transformer and Sex-lethal pre-mRNAs. P-element-mediated mutagenesis has been used to identify the fl(2)d gene. The fl(2)d transcription unit generates two alternatively spliced mRNAs that can encode two protein isoforms differing at their amino terminus. The larger isoform contains a domain rich in histidine and glutamine but has no significant homology to proteins in databases. Several lines of evidence indicate that this protein is responsible for fl(2)d function. (1) The P-element insertion that inactivates fl(2)d interrupts this ORF. (2) Amino acid changes within this ORF have been identified in fl(2)d mutants, and the nature of the changes correlates with the severity of the mutations. (3) All of the phenotypes associated with fl(2)d mutations can be rescued by expression of this cDNA in transgenic flies. Fl(2)d protein can be detected in extracts from Drosophila cell lines, embryos, larvae, and adult animals, without apparent differences between sexes, as well as in adult ovaries. Consistent with a possible function in posttranscriptional regulation, Fl(2)d protein has nuclear localization and is enriched in nuclear extracts (Penalva, 2000).

How can Fl(2)d affect the function of Sxl? One possibility is that Fl(2)d plays an important role in post-translational modifications or proper subcellular localization of Sxl. A second possibility is that Fl(2)d has a direct role in the regulation of pre-mRNA splicing. It could act, for example, by facilitating Sxl binding to its target pre-mRNAs or by assisting its repressive activities. These putative functions could be based on direct interactions between Sxl and Fl(2)d. Alternatively, Fl(2)d could be part of a complex in which Sxl functions, which could also include the products of the genes snf and vir. Finally, a third possibility is that Fl(2)d facilitates the use of the distal splice sites in Sxl and tra that become activated when Sxl represses the use of the proximal ones. More indirect effects of Fl(2)d, as is likely to be the case for the recently reported effects on Sxl expression of mutations in an aspartyl-tRNA synthetase gene, cannot be ruled out (Penalva, 2000).

The non-sex-specific function of fl(2)d remains to be identified. Because Sxl activity is not required for male development, mutations that affect both male and female viability cannot be attributed to genetic interactions with Sxl. It is also very unlikely that Fl(2)d is a general splicing factor involved in an obligatory step in the splicing reaction for the following reasons: (1) no aberrant splicing pattern is detected in Sxl and tra RNAs in fl(2)d mutant males or females (the normal, default sites are used); (2) fl(2)d mutations are not cell lethal. Particularly interesting are the results of the clonal analysis of fl(2)d2, a mutation that produces a truncated, presumably nonfunctional protein. Clones homozygous for fl(2)d2 induced in fl(2)d2/+ males and females are viable, except that in females they develop male instead of female structures, due to the female-specific function of fl(2)d. Furthermore, transplanted male germ cells homozygous for fl(2)d2 can develop into functional spermatozoa, whereas transplanted female germ cells homozygous for fl(2)d2 follow an abortive spermatogenetic pathway, which is an indication of a sexual transformation of the mutant germ cells, due to the female-specific function of fl(2)d. If Fl(2)d was a component of the general splicing machinery, neither fl(2)d2 homozygous clones nor mutant germ cells would survive. One possible scenario is that Fl(2)d plays a role in splicing regulation of a gene(s) important for development in addition to those involved in sex determination. Recently, it has been reported that fl(2)d appears to be necessary for inclusion of mI and mII microexons in Ubx mRNAs. Other examples of splicing factors that are essential for viability but that are dispensable for processing of multiple pre-mRNAs have been described in yeast, Drosophila, and mammals. For some of these, mutations have been identified that disrupt the splicing of only particular substrates, similar to the effects of the fl(2)d1 mutation (Penalva, 2000).

Protein Interactions

Transformer interaction with other splice factors

Specific recognition and pairing of the 5' and 3' splice sites are critical steps in pre-mRNA splicing. The splicing factors SC35 (the SR protein SRp30b) and SF2/ASF (the SR protein SRp30a) specifically interact with both the integral U1 small nuclear ribonucleoprotein (snRNP U1-70K) and with the 35 kd subunit of the splicing factor U2AF (U2AF35). Previous studies indicate that the U1 snRNP binds specifically to the 5' splice site, while U2AF35-U2AF65 heterodimer binds to the 3' splice site. Together, these observations suggest that SC35 and other members of the SR family of splicing factors may function in splice site selection by acting as a bridge between components bound to the 5' and 3' splice sites. Interestingly, SC35, SF2/ASF, and U2AF35 also interact with the Drosophila splicing regulators Transformer and Transformer-2, suggesting that protein-protein interactions mediated by SR proteins may also play an important role in regulating alternative splicing (Wu, 1993).

The ribonucleoprotein consensus sequence (RNP-CS) of Tra2 is required for male fertility and positive and negative control of alternative splicing in transgenic flies, as well as for in vitro binding of recombinant Tra2 to Doublesex and Tra2 pre-mRNAs. Thus, all of the known functions of Tra2 require specific protein-RNA interactions. One of the two arginine-serine (RS)-rich domains of Tra2 is dispensable, while the other is essential for all of the in vivo functions. Part of this domain is also required for RNA binding in vitro. Significantly, the essential RS domain is also required for specific protein-protein interactions. TRA2 interacts with itself, with the splicing regulator Transformer, and with the general splicing factor SF2 in vitro and in the yeast two-hybrid system. These results demonstrate that both protein-RNA and protein-protein interactions are involved in tra2-dependent activation and repression of alternative splicing (Amrein, 1994).

Both subunits of U2AF, U2AF65 and U2AF65, associate with 3' splice sites during assembly of the E complex, the earliest functional complex formed at splice sites. When pre-mRNAs containing a strong 3' splice site are incubated in nuclear extracts, a mixture of a nonspecific H complex and a functional E complex is formed. The amount of E complex formed can be enhanced significantly by addition of SR proteins to the nuclear extract, indicating that SR proteins are limitiing in these extracts. The pyrimidine tract of the DSX 3' splice site is interrupted by purines and is therefore not a high-affinity binding site for U2AF65. Splicing enhancers promote the binding of U2AF to this weak 3' splice site during E complex formation. Enhancer complexes formed on the dsxRE contain significant amounts of U2AF35. An artificial DSX pre-mRNA lacking the dsxRE forms only an H complex under these conditions; the addition of SR proteins has no effect. Thus, the weak female-specific splice site is not recognized by the splicing machinery in the absence of an enhancer, and SR proteins alone are unable to drive complex formation (Zuo, 1996).

To examine the effect of a constitutive DSX splicing enhancer activity on E complex formation, an artificial dsxR2-5 mRNA was tested in which repeats were located within 100 nucleotides of the 3' splice site (in this case close by in contrast to the usual distal positioning). E complex formation is observed with this RNA, and the addition of SR proteins leads to a significant increase in the amount of complex formed Both the 65- and 35-kD subunits of U2AF are present in E complexes formed on pre-mRNAs containing such a strong 3' splice sites (Zuo, 1996).

Experiments were carried out determine whether the TRA- and TRA2-dependent splicing enhancer recruits U2AF to the weak dsx pre-mRNA enhancer containing the dsxRE at its normal position. DSX pre-mRNA containing the dsxRE at its normal position, distant from the 3' splice site, is not assembled into the E complex in the absence of TRA and TRA2. In the presence of TRA and TRA2, however, a significant amount of E complex is observed, and the addition of SR proteins substantially increases the amount of E complex formed. Both U2AF35 and U2AF65 are detected in the DSX RNA complexes. This association is TRA- and TRA2-dependent and is stimulated by SR proteins. In addition, U2AF35 is required for enhancer-dependent binding of U2AF65 to the weak female-specific 3' splice site. This work suggests that recognition of the correct 3' splice site is accomplished through the formation of a network of protein-protein interactions extending across the downstream exon (containing the enhancer site). A key element in this model is the ability of U2AF35 to form a bridge between U2AF65 and SR proteins (including TRA and TRA2) bound to the exon. Similarly, U2AF35 may play a crucial role in the interaction between the 5' and 3' splice sites by functioning as a bridge in the U1 70K-SR protein-U2AF network of protein interactions across the intron (Zuo, 1996 and references).

Function of Transformer in splicing Doublesex

Sex specific splicing of Dsx mRNA is determined by the splice site acceptor sequences. The three splice donor sites following the common exons and the site following the first male-specific exon agree with a consensus donor sequence. The consensus acceptor sequence is n(T/C),NCAG, where the length of the pyrimidine-rich stretch is somewhat ill-defined. The two common acceptors of the Dsx introns plus one female-specific, and two male-specific acceptors all match the last four nucleotides of this consensus. However, of the next 12 nucleotides upstream, the common and male-specific acceptors have pyrimidines at from 9 to 11 positions, but the female-specific acceptor has pyrimidines at only six positions. Thus, the female specific acceptor is a very poor acceptor by this criterion. Since use of the female-specific acceptor depends on the activity of the tra and tra-2 loci, the products of these two genes may act to redirect the normal preference of the splicing machinery of the cell from the stronger 'consensus' male acceptor sequence to the weaker female acceptor (Burtis, 1989).

Sex-specific alternative processing of doublesex (dsx) precursor messenger RNA (pre-mRNA) regulates somatic sexual differentiation in Drosophila melanogaster. Cotransfection analyses in which the dsx gene and the female-specific transformer and transformer-2 complementary DNAs were expressed in Drosophila Kc cells revealed that female-specific splicing of the dsx transcript is positively regulated by the products of the tra and tra-2 genes. Furthermore, analyses of mutant constructs of dsx showed that a portion of the female-specific exon sequence is required for regulation of dsx pre-messenger RNA splicing (Hoshijima, 1991).

Sex-specific alternative processing of doublesex precursor messenger RNA (pre-mRNA) is one of the key steps that regulates somatic sexual differentiation in Drosophila melanogaster. By transfection analyses using dsx minigene constructs, six copies of the 13-nucleotide sequences TC(T/A)(T/A)C(A/G)ATCAACA were identified in the female-specific fourth exon that act as the cis elements for the female-specific splicing of dsx pre-mRNA. UV-crosslinking experiments revealed that both female-specific transformer and transformer-2 products bind to the 13-nucleotide sequences of dsx pre-mRNA. These results strongly suggest that the female-specific splicing of dsx pre-mRNA is activated by binding of these proteins to the 13-nucleotide sequences (Inoue, 1992).

Exonic sequences are involved in proper splicing of the female-specific acceptor site. The activation of a female-specific 3' splice site by Transformer and Transformer-2 proteins involves their binding to an essential exon sequence. Nuclear proteins in addition to TRA and TRA-2 have been found to bind specifically to this exon sequence. Therefore, TRA and TRA-2 may act by promoting the assembly of a multiprotein complex on the exon sequence. This complex may facilitate recognition of the adjacent 3' splice site by the splicing machinery (Tian, 1992).

Within the doublesex repeat element (dsxRE) that contains six copies of a 13-nucleotide repeat sequence, there is, in addition, a purine-rich enhancer (PRE) sequence. This PRE element functionally synergizes with the repeat sequences. In vitro binding studies show that the PRE is required for specific binding of TRA2 to the dsxRE, and that TRA and SR proteins bind cooperatively to the dsxRE in the presence or absence of the PRE. Thus positive control of dsx pre-mRNA splicing requires the TRA- and TRA2-dependent assembly of a multiprotein complex composed on a site of at least two distinct elements (Lynch, 1995).

The Doublesex splice enhancer functions by assembling specific SR protein complexes. Transformer and Transformer 2 recruit different members of the SR family of splicing factors to the six 13-nucleotide repeats and the purine-rich element (PRE) of the splice enhancer. In mammalian cells, the complexes that form on the repeats consist of TRA, TRA2 and the SR protein 9G8. In Drosophila Kc cell extracts, TRA and TRA2 recruit the SR protein RBP1 to the repeats. These proteins are arranged in a specific order on the repeats, with the SR protein at the 5' end of each repeat, and the TRA2 at each 3' end. Although TRA does not cross-link strongly to the repeats, its presence is essential for the binding of TRA2 to the 3' end of the repeat. Individual SR proteins are also recruited to the PRE by TRA and TRA2; SF2/ASF in mammalian cells and dSRp30 in Drosophila cell extracts. The binding of TRA2, TRA, and the specific SR proteins to the repeats in the PRE is highly cooperative within each complex. Thus, TRA2, which contains a single RNA binding domain, can recognize distinct sequences in the repeats and the PRE in conjunction with specific SR proteins. The cross-linking results presented here strongly suggest that Drosophila RBP1 and human 9G8 are functional homologs. These observations show that the protein composition of each complex is determined by the RNA recognition sequence and specific interactions between SR proteins and TRA and TRA2 (Lynch, 1996).

Splicing of Fruitless by Transformer

The alternatively spliced transcripts from the distal promoter are generated by use of 5' splice sites 1590 nucleotides apart. Splice site selection is sex-specific. A downstream splice site for the transcript of the distal promoter is present in fruitless mRNA in female, but not male heads, indicating that this specific splice site is used only in females. Analysis of the upstream 5' splice site reveals that the transcript class missing the repeats region is only detected in males. Thus, the upstream 5' splice site is used only in males and the downstream 5' splice site is used only in females. In chromosomally XX females mutant for tra or tra-2, the male form of FRU transcripts is produced, while the FRU splice pattern is unaffected in XY flies mutant for either tra or tra-2. These results demonstrate that sex-specific splicing of FRU transcripts is indeed controlled by tra and tra-2. A genetic test also reveals that FRU mRNA splicing is controlled by tra and tra-2. If FRU splicing is controlled by tra and tra-2, then XX flies double mutant for fru and tra (or tra-2) should exhibit the mutant fruitless Muscle of Lawrence and behavioral phenotypes. Double mutants have an abnormal MOL muscle like that of fruitless mutant males, and demonstrate abnormal behavior as well (Ryner, 1996).

In Drosophila melanogaster, the fruitless (fru) gene controls essentially all aspects of male courtship behavior. It does this through sex-specific alternative splicing of the FRU pre-mRNA, leading to the production of male-specific FRU mRNAs capable of expressing male-specific Fru proteins. Sex-specific FRU splicing involves the choice between alternative 5' splice sites, one used exclusively in males and the other used only in females. The Drosophila sex determination genes transformer (tra) and transformer-2 (tra-2) switch FRU splicing from the male-specific pattern to the female-specific pattern through activation of the female-specific FRU 5' splice site (SS). Activation of female-specific FRU splicing requires cis-acting tra and tra-2 repeat elements that are part of an exonic splicing enhancer located immediately upstream of the female-specific FRU 5' SS and are recognized by the Tra and Tra-2 proteins in vitro. This FRU splicing enhancer is sufficient to promote the activation by Tra and Tra-2 of both a 5' splice site and the female-specific doublesex (dsx) 3' splice site, suggesting that the mechanisms of 5' splice site activation and 3' splice site activation may be similar (Heinrichs, 1998).

To determine whether the tra/tra-2 repeat elements are essential for regulation of FRU splicing by Tra and Tra-2, a construct, fruM+FREmut, was tested in which the sequence of the conserved part of the tra/tra-2 repeat elements was changed from TCAATCAACA to GGCAGCTTAC. In construct fruM+FREmut, switching to female FRU splicing by Tra and Tra-2 is almost completely blocked, as indicated by the presence of significant amounts of male splicing product M in the presence of cotransfected tra and tra-2. In contrast, deletion of a 1-kb fragment between the repeat elements and the male-specific 5' SS, as in construct fruM+FB-M, does not affect regulation of FRU splicing by Tra and Tra-2. These findings show that the tra/tra-2 repeat elements are required for regulation of FRU splicing by Tra and Tra-2, suggesting that Tra and Tra-2 promote female-specific FRU splicing by acting through these elements (Heinrichs, 1998).

Is the tra/tra-2 repeat region in FRU sufficient to promote the activation of a 5' SS by tra/tra-2? Since the male-specific FRU 5'SS is normally unaffected by tra/tra-2, a 300-bp fragment of fru containing the tra/tra-2 repeats was inserted 4 nt upstream of the male-specific FRU 5' SS (construct fruM+REwt). Interestingly, spliced male product is detected upon cotransfection with tra/tra-2, suggesting activation of the male-specific Fru 5' SS by tra/tra-2 in this hybrid construct. Thus, the tra/tra-2 repeat elements are essential to promote the activation of a heterologous 5' SS by tra/tra-2. Lack of usage of the male-specific 5' SS in the constructs fruM+REwt and fruM+REmut in the absence of cotransfected tra/tra-2 was found to be due to the deletion of a stretch of sequence upstream of the male-specific 5' SS in these constructs. The insertion of the FRU repeat region in either orientation does not affect the usage of the male-specific FRU 5' SS in the absence of cotransfected tra/tra-2 (Heinrichs, 1998).

The Drosophila fruitless gene encodes a transcription factor that essentially regulates all aspects of male courtship behavior. The use of alternative 5' splice sites generates fru isoforms that determine gender-appropriate sexual behaviors. Alternative splicing of fruitless is regulated by Tra and Tra2 and depends on an exonic splicing enhancer (fruRE) consisting of three 13 nucleotide repeat elements, nearly identical to those that regulate alternative sex-specific 3' splice site choice in the doublesex gene. Doublesex has provided a useful model system to investigate the mechanisms of enhancer-dependent 3' splice site choice. However, little is known about enhancer dependent regulation of alternative 5 splice sites. The mechanisms of this process were investigated using an in vitro system in which recombinant Tra/Tra2 could activate the female-specific 5' splice site of fruitless. Mutational analysis has demonstrated that at least one 13 nucleotide repeat element within the fruRE is required and sufficient to activate the regulated female-specific splice site. As was established for doublesex, the fruRE can be replaced by a short element encompassing tandem 13 nucleotide repeat elements, by heterologous splicing enhancers, and by artificially tethering a splicing activator to the pre-mRNA. Complementation experiments show that SR proteins facilitate enhancer-dependent 5' splice site activation. It is concluded that splicing enhancers function similarly in activating regulated 5' and 3' splice sites. These results suggest that exonic splicing enhancers recruit multiple spliceosomal components required for the initial recognition of 5' and 3' splice sites (Lam, 2003).

Splicing of Fruitless by Transformer: Functional analysis of fruitless gene expression by transgenic manipulations of Drosophila courtship

A gal4-containing enhancer-trap called C309, which is expressed up broadly in central-brain and VNC regions, has been shown to cause subnormal courtship of Drosophila males toward females and courtship among males when driving a conditional disrupter of synaptic transmission (shiTS). These manipulations have been extended to analyze all features of male-specific behavior, including courtship song, which was almost eliminated by driving shiTS at high temperature. In the context of singing defects and homosexual courtship affected by mutations in the fru gene, a tra-regulated component of the sex-determination hierarchy, C309/traF combination was also found to induce high levels of courtship between pairs of males and 'chaining' behavior in groups; however, these doubly transgenic males sang normally. Because production of male-specific FRUM protein is regulated by Tra, it was hypothesized that a fru-derived transgene encoding the male (M) form of an Inhibitory RNA (fruMIR) would mimic the effects of traF; but C309/fruMIR males exhibited no courtship chaining, although they courted other males in single-pair tests. Double-labeling of neurons in which GFP was driven by C309 revealed that 10 of the 20 CNS clusters containing FRUM in wild-type males included coexpressing neurons. Histological analysis of the developing CNS could not rationalize the absence of traF or fruMIR effects on courtship song, because C309 was found to be coexpressed with FRUM within the same 10 neuronal clusters in pupae. Thus, it is hypothesized that elimination of singing behavior by the C309/shiTS combination involves neurons acting downstream of FRUM cells (Villella, 2005).

Various portions of the CNS in Drosophila are inferred to control separate elements of normal male courtship, in part by analysis of abnormal behavior. Some such studies have involved brain-behavioral analyses of the fruitless (fru) gene and its mutants. Different fru mutants exhibit courtship subnormalities to varying degrees and at separate stages of the courtship sequence, depending on the mutant allele. Most fru mutants court other males substantially above levels normally exhibited by pairs or groups of wild-type males. The original fruitless mutation leads to spatially nonrandom decreases of fru-product presence within particular subsets of the normal CNS expression pattern, which may be causally connected with the breakdown of recognition that is a salient effect of fru1 on male behavior. fru-like courtship can be induced by the effects of a transgene that encodes GAL4 (a transcription factor derived from yeast). When this broadly expressed C309 enhancer trap was combined with a GAL4-drivable factor containing a dominant-negative, conditionally expressed variant of the shibire gene (shiTS), heat treatment of doubly transgenic males caused them to court females subnormally and to court other males vigorously. Although this strain had been termed a mushroom body enhancer trap in terms of the gal4 sequence it contains, being expressed 'predominantly' within that dorsal-brain structure, C309 drives marker expression in a widespread manner. Therefore, attempts were made to correlate various CNS regions in which this transgene is expressed with its effects on male behavior, emphasizing a search for 'C309 neurons' that might overlap with elements of the FRUM pattern (Villella, 2005).

The possibility was also entertained that the C309/shiTS combination causes a mere caricature of fruitless-like behavior. Therefore, what would be the courtship effects of C309 driving a transgene that produces the female form of the transformer gene product? This Tra protein participates in posttranscriptional control of fru's primary 'sex transcript,' so that FRUM protein is not produced in females. If C309 and traF are naturally coexpressed in a subset of the to-be-analyzed neurons, feminization of the overlapping cells should eliminate this protein. These transgenic experiments were extended to target fruitless expression specifically by gal4 driving of an inhibitory RNA (IR) construct, which was generated with fru DNA. Their experiments furnish one object lesson as to how 'enhancer–trap mosaics' can delve into the neural substrates of a complex behavioral process, an approach commonly taken to manipulate brain structures and functions in courtship experiments. Because few genetic loci putatively identified by such transposons have been specified, the tactics applied are in the context of CNS regions in which expression of a 'real gene' is hypothesized to underlie well defined behaviors (Villella, 2005).

Mutations at the fruitless locus and the C309/UAS-shiTS transgene combination each cause similar courtship subnormalities and anomalies. In this context, the C309 enhancer trap is expressed in many CNS neurons that contain male-specific FRUM protein. One element of the courtship effects of C309/UAS-shiTS involves subnormal interactions between males and females. From correlating C309/FRUM coexpression with the fact that fruitless mutations lead to lower-than-normal male–female courtship, it is speculated that FRUM brain regions 2, 8, 13, and 14 are connected with the deleterious effects of shiTS. As to why two additional neuronal groups that coexpress FRUM and C309 are not noted here (groups 5 and 7), see below (Villella, 2005).

With respect to males courting females, special attention might be paid to clusters 13 and 14. These posteriorly located groups of FRUM neurons are likely to include brain regions within which genetic maleness is required if sex mosaics are to exhibit orientation toward and following of females. A problem with this interpretation is that feminizing substantial proportions of the C309/FRUM coexpressing cells led to no decrements in male-female interactions, despite the 7- to 10-fold coexpression reductions caused by XY/C309/UAS-traF within clusters 13 and 14. Perhaps the relevant 'overlap percentages' would have had to drop from 47 and 31 to 0 for both of these clusters if a traF-affected courtship decrement were to be realized. An alternative to viewing this matter in the context of C309/FRUM coexpression is that certain neurons in which this gal4 driver is active could be anatomically downstream of the fru-expressing brain cells that influence a male's ability to initiate and sustain courtship of a female (Villella, 2005).

This conception is relevant to the striking elimination of courtship song in recordings of C309/UAS-shiTS males. Once again, UAS-traF has no such effect. Neurogenetic findings pertinent to this matter are that C309 is expressed in imaginal thoracic ganglia; fru is 'song-involved'; this gene makes its products within several regions of the ventral nerve cord, coexpressing C309 within most of them; and genetic maleness within Drosophila's VNC has been implicated in song control. Thus, turning off synaptic transmission emanating from one or more subsets of the C309/UAS-shiTS neurons in the thoracic ganglia could be the etiology of heat-induced songlessness exhibited by these doubly transgenic males. Regarding the absence of a traF effect, what if C309 was not expressed in any FRUM-containing song-relevant neurons during metamorphosis? In other words, C309 expression in VNC neurons underlying song control could be activated late in the life cycle, allowing for the shiTS effect to take hold after adult males are heated; however, the progenitors of such cells might not express C309 during an earlier 'feminization-relevant' stage, so that post-metamorphic activation of traF would occur too late to affect singing ability. However, substantial coexpression of FRUM and C309 was found within the pupal VNC. In this respect, it is submitted that assessing the C309's expression pattern throughout the life cycle is a valuable object lesson as to what must be done properly to interpret the biological effects of a given enhancer trap (Villella, 2005).

As to the divergent effects C309-driven shiTS vs. traF, recall that the former factor seems broadly to impinge on VNC functioning, in that the fly's general ability to vibrate its wings is shut down by the synaptic disruptor; in contrast, songless fruitless mutants fly normally. Thus, consider a scenario in which C309 neurons would include those that mediate wing vibrations during flight, and that this transgene is expressed in separate VNC cells hypothetically dedicated to such vibrations during courtship. Therefore, it is speculated that the expression domain of C309 includes inter- or motor-neurons functioning within and downstream of a 'command center' for flight as well as neurons located in relatively distal regions of a separate anatomical pathway. The latter would originate where fru-expressing cells exert the gene's crucial regulation of courtship song (Villella, 2005).

Turning to anomalous courtship interactions among males, focus shifts back to the brain: FRUM clusters 5 and 7, where fru1 causes an apparent absence of this protein. This mutation minimally affects the gene's expression in other brain regions. It is notable that the C309/UAS-traF combination knocked down driver/FRUM coexpression to ~10% of normal in cluster 5. Cluster 7 was similarly affected, but special attention should be paid to cluster 5. One reason that this group was thought to be the etiology of frantic courtship among fru1 males is that cluster 5 is located near the antennal lobes; and transgenically mediated feminization of a brain region near these structures induces intermale courtships, although none of the gal4 drivers in that study included C309. Therefore, if proper male-specific structure or function of cluster 5 is involved in normal sex recognition, the mutation's demasculinizing effect on this brain region, or transgene-effected feminization of it, could cause this aspect of courtship to break down (Villella, 2005).

Elements of the current findings suggest that abnormal formation of the brain region in question is not necessary for it to mediate anomalous interactions between males; this is because deactivating synaptic transmission in cluster 5, after CNS development has been completed in a male manner, is sufficient to induce intermale courtship. Perhaps this behavioral effect of driving UAS-shiTS involves removal of inhibitory neurotransmission relevant to the functioning of this brain region, which in normal males would block their wherewithal to sustain courtship between males. Therefore, the fru1 effect on cluster 5 and that of driving Tra production in this region might not involve the formation of a sex recognition center (such that a hypothetical circuit involved in inhibiting intermale courtship is not present or miswired), but instead the intracellular quality and function of neurons in the mature brain (Villella, 2005).

Considering further that certain cluster 5 neurons comprise the subset of FRUM's spatial domain for shiTS- or traF-induced intermale courtship, the relevant cells would be those in which both fruitless and C309 generate their gene products (20% of the 35 neurons within this group). One problem with this supposition is that C309/UAS-traF flies elicit fairly high levels of courtship. Thus, groups of C309/UAS-traF males may form chains for reasons extending beyond a given fly's inappropriate 'motivation' to court another male: The extent to which a C309/UAS-traF fly is feminized could include self-stimulation that might contribute to intermale courting. However, recall the case of C309/UAS-traF/Cha-gal80 males, a transgenic type that is similarly feminine externally and elicits courtship. The diminished extent to which C309's gal4 is effective when combined with Cha-gal80 led to weakened homosexual courtship in single-pair tests and dramatically reduced chaining behavior, although there was essentially no effect of Cha-gal80 on the basic courtship ability of these triply transgenic males. Thus, the effects of this 'neurons-only' manipulation suggest that hypothetical self-stimulation, which did not cause C309/UAS-traF/Cha-gal80 males vigorously to court other ones, is minimally operating to induce the homosexual courtships performed by XY/C309/UAS-traF flies. Males carrying C309 and UAS-fruMIR are also not feminized externally; however, they courted other males robustly in single-pair tests, an effect that was diminished by adding Cha-gal80. Therefore, it is surmised that flies carrying a given fruitless-affecting transgene exhibit intermale courtship because the relevant CNS neurons are demasculinized (Villella, 2005).

However, what about neural structures not analyzed in the current study that could be involved in the behavioral effects of C309 driving either traFor fruMIR? Thus, consider that Tra affects the primary transcript emanating from the doublesex (dsx) gene and that dsx null mutations cause XY flies to exhibit modest levels of intermale courtships. C309 driving of traF could lead to the female (F) form of DSX (thus, no DSXM, as in dsx) within brain cells connected to sex recognition other than those analyzed. Indeed, dsx+ is expressed in the brain; however, the functional significance of these cells is unknown, let alone whether any of them also express fru+. In this regard, it was important to home in on disruption of fruitless's CNS expression alone by combining C309 with the UAS-fruMIR transgenes; this was sufficient to induce courtship between a given pair of doubly transgenic males but led to no chaining. Thus, anomalously high levels of courtship between two males has been disconnected from courtship chaining. [The same disconnect between these different kinds of intermale courtship occurred when Cha-gal80 was added to the C309/UAS-traF combination. It is as if the broad neural effects of a genetic abnormality such as a fruitless mutation, or combining C309 with UAS-traF, is necessary to cause sustained courtship among several variant males; however, if the impingement on fru+ expression is more limited, only courtship between a pair of males can occur (Villella, 2005).

In this regard, the C309/UAS-fruMIR flies were substantially less affected in terms of numbers of brain neurons within which FRUM became undetectable, compared with the effect of the same driver combined with UAS-traF. This brings us to the matter of additional neurons that are potentially relevant to courtship and should be analyzed in context of the C309 effects. Here, the many PNS cells recently discovered to express fruitless in external sensory structures are referred to. It is unknown whether any of these neurons coexpress C309, such that sensory inputs relevant to courtship may have been impinged upon by combining that transgene with UAS-shiTS or with the sex-affecting transgenes. However, fru+ expression in external appendages is not required for a fly to recognize, follow, and perform wing extension at a female: when these structures are genetically female in certain gynandromorphs, maleness within the brain is sufficient to trigger mosaic-with-female courtship (Villella, 2005).

The current study aimed to delve into various regions of the male CNS in which the fruitless gene is expressed: Do certain subsets of the spatial pattern govern a male's ability to perform a discrete feature of the reproductive sequence? Using the gal4-containing C309 enhancer trap was valuable, because it leads to impersonations of certain fru-mutant behaviors when this driver is combined with a shiTS-containing factor that broadly disrupts neural functioning. By limiting C309's efficacy to disrupt by causing it to drive sex-related transgenes succeeded in provisionally partitioning fru-related 'sex recognition' neurons to a subset of the normal brain pattern. By subtraction, the partitioning was further delimited by knocking out the driver's efficacy in a subset C309's spatial domain: adding a neurally driven gal80 transgene substantially attenuated anomalous intermale courtships. A pleasant surprise occurred when the C309/UAS-fruMIR combination was found not to mimic the effects on courtship among males of combining the driver with UAS-traF. Thus, the broader pattern of FRUM expression, unaffected by the IR compared with the substantial decrement caused by traF, takes the analysis a further step. For example, the manner by which fru mutations and related factors influence courtship between two males, as opposed to the much more complicated behavioral dynamics that can occur in a group of such Drosophila, are now being teased out (Villella, 2005).

However, inferences about the potentially relevant subsets of a given brain cluster do not approach specifically identifiable neurons. For this, it will be necessary to do more than quantify the cells in which a transgene driver and fruitless are coexpressed. Further brain-behavioral dissections will require assessing the differential connectivity patterns defining a given class of FRUM neurons, along with variations of cellular content that are likely to discriminate one category of such neurons from another. The relevant object lessons stem from analyses of, so far, only the posterior-most component of fruitless's expression domain in the male CNS: partitioning certain abdominal-ganglion neurons that differentially connect with either a male-specific muscle or with internal reproductive organs, and discovering that the latter type of FRUM cells uniquely contain serotonin. Neurons containing another neurotransmitter, acetylcholine, are on point; but not all of the C309 effects can be ascribed to neurons affected by Cha-gal80, because certain courtship defects were found to remain when analyzing males that carried this transgene along with C309 and UAS-shiTS. This finding reinforces that notion that additional neuronal qualities must be uncovered with regard to cells expressing this enhancer-trap, the fruitless gene, or both (Villella, 2005).


The transformer gene of Drosophila is necessary for all aspects of female somatic sexual differentiation. tra uses a single set of precursor RNAs to produce female- and non-sex-specific RNAs by alternative splicing. Ectopic expression of the female-specific RNA causes chromosomal males to develop as females, indicative of a linear pathway of regulated genes controlling sex. Genetic and molecular tests with this ectopically expressed gene are consistent with the following order of gene action: X chromosome to autosome ratio ---> Sex lethal ---> transformer ---> transformer-2 ---> doublesex ---> intersex ---> terminal differentiation. Expression of the female-specific tra RNA in tra mutants is sufficient to lead to female differentiation. Expression of the non-sex-specific tra RNA in tra mutants is not sufficient to lead to female differentiation. The tra female-specific activity is not required for female-specific splicing of the tra precursor RNAs (McKeown, 1988).

In Drosophila, the sex of germ cells is determined by cell autonomous and inductive signals. XY germ cells autonomously enter spermatogenesis when developing in a female host. In contrast, XX germ cells non-autonomously become spermatogenic when developing in a male host. In first instar larvae with two X chromosomes, XX germ cells enter the female or the male pathway depending on the presence or absence of transformer activity in the surrounding soma. In somatic cells, the product of tra regulates the expression of the gene doublesex which can form a male-specific or a female-specific product. In dsx mutant larvae, XX and XY germ cells develop abnormally, with a seemingly intersexual phenotype. This indicates that female-specific somatic dsx products feminize XX germ cells, and male-specific somatic dsx products masculinize XX and XY germ cells. The results show that tra and dsx control early inductive signals that determine the sex of XX germ cells and that somatic signals also affect the development of XY germ cells. XX germ cells that develop in pseudomales lacking the sex-determining function of Sxl are spermatogenic. If, however, female-specific tra functions are expressed in these animals, XX germ cells become oogenic. Furthermore, transplanted XX germ cells can become oogenic and form eggs in XY animals that express the female-specific function of tra. Therefore, Tra product present in somatic cells of XY animals or in animals lacking the sex-determining function of Sxl, is sufficient to support developing XX germ cells through oogenesis (Steinmann-Zwicky, 1994).

In Drosophila, the sex of germ cells is determined by cell-autonomous and inductive signals. XY germ cells autonomously enter spermatogenesis when developing in a female host. In contrast, XX germ cells non-autonomously become spermatogenic when developing in a male host. In first instar larvae with two X chromosomes, XX germ cells enter either the female or the male pathway depending on the presence or absence of transformer activity in the surrounding soma. In somatic cells, the product of tra regulates the expression of the gene doublesex which can form a male-specific or a female-specific product. In dsx mutant larvae, XX and XY germ cells develop abnormally, with a seemingly intersexual phenotype. This indicates that female-specific somatic dsx products feminize XX germ cells, and male-specific somatic dsx products masculinize XX and XY germ cells. The results show that tra and dsx control early inductive signals that determine the sex of XX germ cells and that somatic signals also affect the development of XY germ cells. XX germ cells that develop in pseudomales lacking the sex-determining function of Sxl are spermatogenic. If, however, female-specific tra functions are expressed in these animals, XX germ cells become oogenic. Furthermore, transplanted XX germ cells can become oogenic and form eggs in XY animals that express the female-specific function of tra. Therefore, TRA product present in somatic cells of XY animals or in animals lacking the sex-determining function of Sxl, is sufficient to support developing XX germ cells through oogenesis (Steinmann-Zwicky, 1994).

Relatively little is known about the neural circuitry underlying sex-specific behaviors. The feminizing gene transformer was expressed in genetically defined subregions of the brain of male Drosophila, and in particular within different domains of the mushroom bodies. Mushroom bodies are phylogenetically conserved insect brain centers implicated in associative learning and various other aspects of behavior. Expression of transformer in lines that mark certain subsets of mushroom body intrinsic neurons, and in a line that marks a component of the antennal lobe, causes males to exhibit nondiscriminatory sexual behavior: they court mature males in addition to females. Expression of transformer in other mushroom body domains, and in control lines, has no such effect. These data support the view that genetically defined subsets of mushroom body intrinsic neurons perform different functional roles (O'Dell, 1995).

Pheromones are intraspecific chemical signals important for mate attraction and discrimination. In Drosophila, hydrocarbons on the cuticular surface of the animal are sexually dimorphic in both their occurrence and their effects: Female-specific molecules stimulate male sexual excitation, whereas the predominant male-specific molecule tends to inhibit male excitation. Female flies produce dienes (two double bonds) with 27 and 29 carbons (cis,cis-7,11-heptacosadiene and cis,cis-7,11-nonacosadiene). A few tens of nanograms of both dienes together can elicit vigorous male precopulatory behavior. Male flies synthesize monoenes (one double bond) with 23 and 25 carbons (cis-7tricosene and cis-7-pentacosene). cis-7tricosene can inhibit dose-dependent male excitation, whereas cis-7pentacosene stimulates males of some strains. According to a previously proposed biosynthetic scheme, an elongase, controlled by ecdysone, perhaps coupled with a desaturase, would be sufficinet to replace 7-monoenes by 7,11-dienes. Complete feminization of the pheromone mixture produced by males is induced by targeted expression of the transformer gene in adult oenocytes (subcuticular abdominal cells) or by ubiquitous expression during early imaginal life. Early adult life is the critical period during which sexually dimorphic hydrocarbons replace immature hydrocarbons on the fly cuticle. The resulting flies, genetically male, generally exhibit male heterosexual orientation but elicit homosexual courtship from other males. Two out of seven transformed lines exhibit some bisexual behavior, possibly because they were feminized in the calyces of their mushroom bodies (strain A) and in a dorso-medial subset of their antennal lobes (strains A and B). However, these two brain structures, which function in mate recognition, are not feminized in the other tranformed strains, showing that these structures are not required for feminization of the pheromonal profile. This analysis shows that two aspects of individual sexual identity in Drosophila -- the perception of others and the presentation of self to others -- are under separate genetic and anatomical control (Ferveur, 1997).

Sexual differentiation in Drosophila is controlled by a short cascade of regulatory genes, the expression pattern of which determines all aspects of maleness and femaleness, including complex behaviors displayed by males and females. One sex-determining gene is transformer (tra): tra activity is needed for female development. Flies with a female karyotype (XX) but which are mutant for tra develop and behave as males (and are termed X2 flies). In such flies, a female phenotype can be restored by a transgene that carries the female-specific cDNA of tra under the control of a heat-shock promoter. This transgene, called hs[trafem], also transforms XY animals into sterile females. When these XX and XY 'females' are raised at 25 degrees C, however, they display vigorous male courtship while at the same time, as a result of their female pheromone pattern, they are attractive to males. Intriguingly, their male courtship behavior is indiscriminately directed toward both females and males. When expression of tra is forced by heat shock, applied during a limited period around puparium formation, male behavior is abolished and replaced by female behavior. The temperature-sensitive period extends from shortly before puparium formation into early metamorphosis. Cuticular hydrocarbons function as pheromonal cues between the sexual partners. Consistent with their attractiveness to males, the normal female pattern of pheromones is found in transformed flies, irrespective of the heat shock. Could the indiscriminate male behavior of these flies result from continuous self-stimulation by their own female pheromones? The female pheromone pattern was replaced with the male pattern without affecting the CNS. This was achieved by introducing the male-determining mutation dsxD, which acts downstream of tra. This mutation causes XX flies to produce a male pheromone pattern. They are no longer attracted to males. Similarly, X2 and XY 'females', now with male body and pheromonal status due to dsxD, are completely unattrative to males; but they still court both sexes. Such flies even manage to copulate with females, showing that X2 and XY 'females' are capable of performing the full repertoire of male courtship. These results rule out self-stimulation and leave a disturbed nervous system as the most probable cause for the indiscriminate mating behavior. It is concluded that sexual behavior is irreversibly programmed during a critical period as a result of the activity or inactivity of a single control gene. Such programming takes place during a period of accelerated growth in the brain and especially in the mushroom bodies, suggesting that sexual behavior is 'hard wired' in the CNS (Arthur, 1998).

The isolation and analysis of Drosophila mutants with altered sexual orientation lead to the identification of novel branches in the sex-determination cascade that govern the sexually dimorphic development of the nervous system. One such example is the fruitless (fru) gene, the mutation of which induces male-to-male courtship and malformation of a male-specific muscle, the muscle of Lawrence (MOL). Since the MOL is formed in wild-type flies when the innervating nerve is male, regardless of the sex of the MOL itself, the primary site of Fru function is likely to be the motoneurons controlling the MOL. The fru gene produces multiple transcripts including sex-specific ones. A female-specific mRNA from the fru locus has a putative Transformer (Tra) binding site in its 5' untranslated region, suggesting that fru is a direct target of Tra. The fru transcripts encode a set of proteins similar to the BTB (Bric a brac, Tramtrack and Broad-complex)-Zn finger family of transcription factors. Mutations in the dissatisfaction (dsf) gene result in male-to-male courtship and reduced sexual receptivity of females. The dsf mutations also give rise to poor curling of the abdomen in males during copulation and in females, a failure of egg-laying. The latter phenotypes are ascribable to aberrant innervation of the relevant muscles. A genetic analysis reveals that expression of the dsf phenotypes depends on Tra but not on Doublesex (Dsx) or Fru, suggesting that dsf represents another target of Tra. The effect of dsf mutation was determined in chromosomal females after sexual transformation with tra- (Finley, 1997). In the dsf+;tra- XX flies, the ventral abdominal muscles are innervated by normal locking boutons, as is the case in wild-type males (XY dsf+). In contrast, the corresponding muscles in dsf-;tra- XX flies are associated with enlarged round boutons, just like those seen in mutant males (XY dsf-). The expression of the dsf mutant phenotype depends on tra due to the fact that dsf is located downstream of tra in the sex-determination cascade. In accordance with the idea that dsf is downstream of tra, a moderate reduction in tra+ activity in females eliminates motor innervation of the uterine muscle, inducing a dsf phenotype. Taken together, these findings suggest that the sex-determination protein Tra has at least three different targets: dsx, fru and dsf, each of which represents the first gene in a branch of the sex-determination hierarchy functioning in a mutually-exclusive set of neuronal cells in the Drosophila central nervous system (Yamamoto, 1998).

The downstream effectors of the Drosophila sex determination cascade are mostly unknown and thought to mediate all aspects of sexual differentiation, physiology and behavior. Here, serial analysis of gene expression (SAGE) was employed to identify male and female effectors expressed in the head, and report 46 sex-biased genes. Four novel, male- or female-specific genes have been characterized; all are expressed mainly in the fat cells in the head. Tsx (turn on sex-specificity), sxe1 and sxe2 (sex-specific enzyme 1/2) are expressed in males, but not females, and are dependent on the known sex determination pathway, specifically transformer and its downstream target doublesex. Female-specific expression of the fourth gene, fit (female-specific independent of transformer), is not controlled by tra and dsx, suggesting an alternative pathway for the regulation of some effector genes. These results indicate that fat cells in the head express sex-specific effectors, thereby generating distinct physiological conditions in the male and female head. It is suggested that these differences have consequences on the male and female brain by modulating sex-specific neuronal processes (Fujii, 2002).

In Drosophila melanogaster, the main cuticular hydrocarbons (HCs) are some of the pheromones involved in mate discrimination. These are sexually dimorphic in both their occurrence and their effects. The production of predominant HCs has been measured in male and female progeny of 220 PGa14 lines mated with the feminising UAS-transformer transgenic strain. In 45 lines, XY flies were substantially or totally feminised for their HCs. Surprisingly, XX flies of 14 strains were partially masculinised. Several of the PGa14 enhancer-trap variants screened in this study seem to interact with sex determination mechanisms involved in the control of sexually dimorphic characters. A good relationship was found between the degree of HC transformation and GAL4 expression in oenocytes. The fat body was also involved in the switch of sexually dimorphic cuticular hydrocarbons but its effect was different between the sexes (Savarit, 2002).

Cholinergic control of male reproductive characters in Drosophila

In many animal species, copulation involves the coordinated release of both sperm and seminal fluid, including substances that change female fertility and postmating behavior. In Drosophila, these substances increase female fertility and prevent mating with a second male. By using a PGal4 strain, a dozen cholinergic neurons found only in the male abdominal ganglion (Abg-MAch), together with other cells, were targeted for feminization via conditional transformer expression. Genetic feminization apparently deleted these neurons in males and significantly increased their copulation duration, blocked their fertility in 60% of cases, and only weakly repressed remating in females. Genetic repression of Gal4 activity in all cholinergic neurons completely rescued copulation duration and fertility, and totally prevented remating, indicating that Abg-MAch neurons were functional. The conditional blocking of the synaptic activity of these neurons during copulation induces separate effects on the transfer of the seminal substances involved in fertilization and those involved in remating. These effects were dissociated only when Abg-MAch neurons were feminized, indicating that their presence is required to synchronize the emission of the male substance(s) that changes reproductive behaviors (Acebes, 2004).

The feminization of a dozen male-specific cholinergic neurons in the abdominal ganglion (Abg-MAch), possibly with that of other undetected cells, substantially reduced the probability that Drosophila males produce offspring (from 90% to 35%) and altered the coordination of several evolutionarily important reproductive characters normally induced during copulation by control males. This finding is consistent with a number of studies on both insects and mammals and may reveal a high degree of evolutionary conservation of the neural control of these key male-reproductive characters. In insects, acetylcholine (Ach) is a neurotransmitter commonly used by mechanosensory neurons. Food-deprivation of choline, an essential precursor of Ach, alters D. melanogaster male normal copulatory behavior and reduces sperm motility. Studies in vertebrates have reported a functional relation between Ach and male ejaculation. In rats, cholinergic sensitivity is correlated with the intensity of the response of muscarinic agonists, which in turn facilitate ejaculation. In dogs, seminal emission during long copulatory events also involves cholinergic pathways (Acebes, 2004).

The distinct and simultaneous manipulation by UAS-tra and UAS-shits transgenes of the Abg neurons targeted by Gal4 allowed two distinct cell populations eliciting distinct effects on reproduction to be distinguished. Non-sex-specific neurons mostly permit the transfer of the substances that repress female remating. Abg-MAch neurons also decreased the transfer of these substances together with the substances necessary for fertilization, but not in a coordinated manner. The feminization of Abg-MAch neurons (probably leading to their ablation in 55B-tra and 55B-tra- shits males) induced variation for fertility and for repression of remating, but these effects did not coincide either in individuals or in groups of females. The dissociation of these effects, together with the decrease in fecundity induced by feminized males, indicates that the transfer (and the amount) of these substances stochastically varies within a given male, probably because the control of ejaculation is randomly affected (Acebes, 2004).

Apart from these two populations of Abg neurons, a distinct set of eight serotoninergic Abg male-specific neurons also changed copulation duration, fertility, and female remating. It is not yet known whether these three groups of Abg neurons innervate different or overlapping regions of male internal reproductive organs, and it is possible that the ablation of any of these sets of Abg neurons induces the uncoordinated release of male substances (Acebes, 2004).

Could the loss of the synchronized emission of such substances be reflected by the high interindividual variability for copulation duration shown by feminized 55B-tra and 55B-tra-shits males? Two other fly studies suggest that variation in copulation duration reflects a variation for the transfer of male reproductive substances. In Musca domestica, males temporarily exhausted for sperm and associated substances induced increased copulation duration and remating frequency. In D. melanogaster strains selected for copulation duration, short-copulation males (15 min) fertilized females much faster than long-copulation males (25 min). Since the synchronized release of these substances is crucial for reproduction and is probably involved in the evolution of conflicts between the sexes, male-specific neural networks that control the release of these substances should be subject to strong selective forces. That could imply that some of the neural mechanisms underlying ejaculation may have been conserved during evolution. It is predicted that similar cholinergic pathways will be found to underlie ejaculation and the coordinated emission of seminal fluids in others species including vertebrates (Acebes, 2004).

Development of the male germline stem cell niche in Drosophila

Stem cells are found in specialized microenvironments, or 'niches', which regulate stem cell identity and behavior. The adult testis and ovary in Drosophila contain germline stem cells (GSCs) with well-defined niches, and are excellent models for studying niche development. This study investigates the formation of the testis GSC niche, or 'hub', during the late stages of embryogenesis. By morphological and molecular criteria, the development of an embryonic hub that forms from a subset of anterior somatic gonadal precursors (SGPs) were identified and followed in the male gonad. Embryonic hub cells form a discrete cluster apart from other SGPs, express several molecular markers in common with the adult hub and organize anterior-most germ cells in a rosette pattern characteristic of GSCs in the adult. The sex determination genes transformer and doublesex ensure that hub formation occurs only in males. Interestingly, hub formation occurs in both XX and XY gonads mutant for doublesex, indicating that doublesex is required to repress hub formation in females. This work establishes the Drosophila male GSC niche as a model for understanding the mechanisms controlling niche formation and initial stem cell recruitment, as well as the development of sexual dimorphism in the gonad (Le Bras, 2006).

The evidence indicates that an embryonic hub, which appears to give rise to the adult hub and create the male GSC niche, forms during the late stages of embryogenesis. A subset of anterior SGPs initiates expression of several molecular markers that are also expressed in the adult hub. These SGPs segregate into a tight cluster in a distinct region of the gonad, and a subset of germ cells organizes around these SGPs in a manner similar to the organization of GSCs around the adult hub. Since spermatogenesis begins by early larval stages, it is possible that the embryonic hub already forms a functional GSC niche. The formation of the hub, or indeed any stem cell niche, can be divided into the distinct issues of niche cell identity, niche morphogenesis, and stem cell recruitment (Le Bras, 2006).

The data indicate that the specification of hub cell identity occurs in two stages. During the first stage, some SGPs acquire an anterior identity that is sexually dimorphic, as indicated by the male-specific expression of esg and upd. Anterior SGP identity is positively regulated by abd-A, and is repressed by Abd-B, while sexual identity is regulated by tra and dsx. During the second stage of hub cell specification, a subset of these anterior SGPs acquires hub cell identity during stage 17 of embryogenesis. Only some anterior SGPs maintain esg expression, and the control of late gene expression in the hub appears to be distinct from early expression in anterior SGPs, since some esg and upd enhancer traps only exhibit gonad expression in the hub at this later stage. Furthermore, cells that maintain esg expression during stage 17 also express every other marker of adult hub identity tested, including Fasciclin 3, cdi, DN-cadherin and DE-cadherin. It is concluded that these cells are specified as hub cells at this time. The fate of the anterior SGPs that lose esg expression and do not form part of the hub is unknown. An intriguing possibility is that these cells could form another important somatic cell type: the cyst progenitor cells (somatic stem cells) that associate with the hub along with the GSCs (Le Bras, 2006).

Based on its expression pattern, the transcription factor esg would seem to be an excellent candidate for specifying hub cell identity. However, no changes were observed in the expression of other hub markers in esg null mutants; this includes expression of DE-cadherin, which is known to be regulated by esg in other tissues. It has been reported, however, that esg is required for hub maintenance, and that the hub is severely defective at later stages in esg mutants that survive embryogenesis. Thus, esg is critical for the male GSC niche, but is either not important for the initial formation of this structure, or acts redundantly with another factor (Le Bras, 2006).

It has been possible to follow the morphogenesis of the hub from the time of gonad formation until the embryonic hub is fully formed. At the time of gonad coalescence, anterior SGPs interact with other SGPs, and with the germ cells, in a manner that is indistinguishable from posterior SGPs. However, during stage 17, the hub cells undergo dramatic changes in their relationship to other SGPs and germ cells. Hub cells segregate away from other SGPs to one pole of the gonad, and coalesce tightly with one another. In addition, hub cells do not ensheath the germ cells at this stage. Instead, a defined interface between hub cells and germ cells forms which is labeled by DE- and DN-cadherin, but not Fasciclin 3. Thus, hub cells appear to maximize their interactions with one another, and minimize their interactions with other cells in the gonad, although they clearly still contact a subset of germ cells (Le Bras, 2006).

It is apparent that the changes in cell–cell contact and morphology that occur during hub formation require changes in cell adhesion. Indeed, characteristic changes have been found in expression of the homophilic adhesion molecules Fasciclin 3, DN-cadherin and DE-cadherin occur during hub formation; all three are significantly upregulated in the embryonic and adult hub. Increased homophilic adhesion among hub cells could account for their ability to maximize their contacts with one another, and sort away from other SGPs. However, no changes were observed in embryonic hub formation in mutants for these cell adhesion molecules. Thus, these proteins, and possibly others, may act redundantly in this process (Le Bras, 2006).

It is clear that a subset of germ cells organizes specifically with the developing hub as it forms. During the last stage of hub formation, germ cells become oriented in a rosette distribution around the developing hub in a manner characteristic of GSCs in the adult. These may represent the subset of germ cells that will become GSCs. The presence of DE- and DN-cadherin at sites of hub–germ cell contact suggests that cadherin-mediated adhesion may be important for niche–GSC interaction in the testis, as has been observed in the ovary. Interestingly, germ cells are not required for hub formation. Analysis of a number of hub identity markers indicates that these cell form normally from a subset of anterior SGPs in embryos that lack germ cells. The hub does not appear as well compacted in these embryos, consistent with observations of the adult hub, indicating that hub–germ cell contact (or hub–germ cell signaling) affects the final shape of the hub. Nevertheless, the GSC niche can form in the absence of one of its stem cell populations (somatic stem cells may still be present). It will be of great interest in the future to determine if the subset of germ cells organized around the male embryonic hub are, indeed, developing GSCs, and to study how their transition to stem cell identity might be regulated by the niche (Le Bras, 2006).

The formation of the male GSC niche is a sex-specific characteristic of anterior SGPs. Male-specific expression of esg and hub formation both require the sex determination genes tra and dsx. In some tissues, DSXM is required to promote male development and repress female development, while the opposite is true for DSXF. Interestingly, it was found that embryonic hub development is entirely masculinized in dsx null mutants; XX and XY individuals appear identical when mutant for dsx and both resemble wild type males. Thus, no role is seen for DSXM in promoting embryonic hub formation, while DSXF is required in females to repress hub formation. Since esg is expressed male-specifically, it is one candidate for being directly regulated by DSX (Le Bras, 2006).

We can compare the development of the anterior SGPs and hub with the development of another sexually dimorphic cell type, the msSGPs that join the posterior of the male gonad. First of all, these two cell types are distinct and do not depend on one another for their proper development. The hub still forms in Abd-B mutants that lack msSGPs, while msSGPs are still found in the gonad in Pc mutants, in which no anterior SGPs or hub cells form. Second, the mechanism for how sexual dimorphism is created differs between the two cell types. msSGPs are present only in males because they have undergone sex-specific apoptosis in females. In contrast, no apoptosis was observed in anterior SGPs. These cells appear to remain present in both sexes, but only form a hub in males. Thus, although the sex determination genes tra and dsx regulate sex-specific development of both cell types, the cellular mechanisms employed are different. Finally, as was observed for the hub, development of the msSGPs is completely masculinized in dsx mutant embryos. Thus, for both of these cell types, the male pattern of development in the embryonic gonad is the default state in the absence of dsx function, and it is the role of DSXF to repress male development in females. However, DSXM may well play a role in development of one or both of these gonad cell types at later stages, since proper testis development in males clearly requires dsx (Le Bras, 2006).

The sex determination pathway must also ensure that GSC niches form in females and are different from those in males. Recently, it has been shown that germ cells populating the anterior of the gonad in female embryos are predisposed to become GSCs in the adult ovary, while germ cells populating the posterior rarely become GSCs. This suggests that anterior SGPs in the female embryonic gonad may promote GSC identity, similar to what is proposed to happen in the male during hub formation. One possibility is that anterior SGPs give rise to GSC niches in both sexes, while genes such as tra and dsx control whether these niches will be male or female (Le Bras, 2006).

In conclusion, the development has been followed of the embryonic hub, which may represent the nascent GSC niche for the testis. This work provides a basis for further understanding the mechanisms controlling niche formation and GSC recruitment in Drosophila, and determining if these mechanisms are conserved in other stem cell systems, including the GSC niche of the mammalian testis (Le Bras, 2006).


Position of transformer in the sex determination heirarchy in Drosophila

The transformer and doublesex genes produce sex-specific transcripts that are generated by differential RNA processing. The effects were examined of mutants in other regulatory genes controlling sexual differentiation on the patterns of processing of the tra and dsx RNA transcripts. The genes suggested by genetic studies to act upstream of tra or dsx in the sex determination hierarchy regulate these two loci at the level of RNA processing. The data suggest that the order of interaction of the factors controlling sex is X:A ---> Sxl ---> tra ---> tra-2 ---> dsx ---> ix ---> terminal differentiation. While these results cannot preclude regulatory interactions at other levels, the regulation of RNA splicing revealed by these experiments is sufficient to account for all of the known functional interactions between the regulatory genes in this hierarchy (Nagoshi, 1988).

The transformer gene is one of a set of regulatory genes that form the hierarchy controlling all aspects of somatic sexual differentiation in Drosophila melanogaster. The gene transformer occupies an intermediate position in this hierarchy. Analysis of this gene has allowed determination of the mechanism by which transformer is regulated in a sex-specific manner and to examine the way in which the regulatory hierarchy is organized. The female-specific expression of the tra gene, previously inferred from genetic observations, is based on sex-specific alternative splicing of tra pre-mRNA and is not the result of sex-specific transcriptional activation. The female-specific RNA produced by this alternative splicing is the functional mediator of tra activity. Multiple genetic, molecular, and transformation experiments show that female-specific activation of genes or gene products occurs in the order Sex lethal --> transformer --> transformer-2 --> doublesex --> intersex --> female differentiation. The results do not distinguish the level at which transformer might regulate the downstream gene transformer-2. Neither transformer nor any of the down-stream genes feedback on, or participate in, alternative splicing of transformer RNA. The mechanism by which Sex lethal regulates transformer splicing appears to be a repression of the use of one of a pair of splice acceptor sites (Belote, 1989).

Few mutations link well defined behaviors with individual neurons and the activity of specific genes. In Drosophila, recent evidence indicates the presence of a doublesex-independent pathway controlling sexual behavior and neuronal differentiation. A gene, dissatisfaction (dsf), has been identified that affects sex-specific courtship behaviors and neural differentiation in both sexes without an associated general behavioral debilitation. Male and female mutant animals exhibit abnormalities in courtship behaviors, suggesting a requirement for dsf in the brain. Virgin dsf females resist males during courtship and copulation and fail to lay mature eggs. dsf males actively court and attempt copulation with both mature males and females but are slow to copulate because of maladroit abdominal curling. Structural abnormalities in specific neurons indicate a role for dsf in the differentiation of sex-specific abdominal neurons. The egg-laying defect in females correlates with the absence of motor neuronal innervation on uterine muscles, and the reduced abdominal curling in males correlates with alteration in motor neuronal innervation of male ventral abdominal muscles. Epistasis experiments show that dsf acts in a tra-dependent and dsx-independent manner, placing dsf in the dsx-independent portion of the sex determination cascade (Finley, 1997).

Interactions between the somatic sex regulatory gene transformer and the germline genes ovo and ovarian tumor

What are the genetic requirements for high ovo expression in gonads? In autosomes, the X to autosome ratio is sufficient for Sex lethal expression. Does a similar mechanism hold in the gonads? To study this question, expression of ovo was examined in gonads of mutant flies where the chromosomal sex does not match the somatic sexual identity. If a 2X karyotype is necessary and sufficient for high levels of ovo expression, then female somatic sex should have no effect on ovo expression. XY females can be produced by using a strain of flies bearing a transformer gene driver by a heat-shock promoter (trahs). In XY genetic males bearing the trahs transgene, TRA activity is sufficient to direct female somatic development in flies that would otherwise develop as males, but the germ line remains male. If a female somatic sexual identity is sufficient for a high level of ovo expression, then both XX females and XY trahs females should be expected to show similar ovo expression. In fact ovo expression is not activated to a high level by a female somatic identity, suggesting that either an XX karyotype or female germ-line identity is required for high ovo expression (Oliver, 1994).

In Drosophila, compatibility between the sexually differentiated state of the soma and the constitution of the sex chromosome in the germline is required for normal gametogenesis. In this study, important aspects of the soma-germline interactions controlling early oogenesis are defined. In particular, the sex-specific germline activity of the ovarian tumor (otu) promoter has been demonstrated to be dependent on somatic factors controlled by the somatic sex differentiation gene transformer. This regulation defines whether there is sufficient ovarian tumor expression in adult XX germ cells to support oogenesis. In addition, the ovarian tumor function required for female germline differentiation is dependent on the activity of another germline gene, ovo, whose regulation is transformer-independent. These and other data indicate that ovarian tumor plays a central role in coordinating regulatory inputs from the soma (as regulated by transformer) with those from the germline (involving ovo). transformer-dependent interactions influence whether XX germ cells require ovarian tumor or ovo functions to undergo early gametogenic differentiation. These results are incorporated into a model that hypothesizes that the functions of ovarian tumor and ovo are dependent on an early sex determination decision in the XX germline -- a decision that is at least partially controlled by somatic transformer activity (Hinson, 1999 and references).

With respect to interactions with the germline, transformer (tra) is the most extensively studied of the somatic sex regulatory genes. The masculinization of XX soma due to loss-of-function tra mutations causes germ cell aberrations during first instar larval stages and misregulates sex-specific germline gene expression in the embryo. Furthermore, when XY soma is feminized by ectopic tra expression (to form 'pseudofemales', the somatic components of the ovaries are sufficiently 'female' so that they can support the maturation of transplanted XX germ cells. The pseudofemale soma also appears to partially feminize the XY germline, since these cells now require the normally female-specific otu function for optimal proliferation. These observations indicate that tra controls a substantial portion of the somatic-germline interactions affecting early gametogenic differentiation (Hinson, 1999 and references).

In Drosophila, the sexual differentiation of the germline requires a complex interplay between cell autonomous factors controlled by the X:A ratio of the germ cells and sex-specific somatic functions. For example, certain allele combinations of transformer, transformer-2 and doublesex can cause chromosomally female (XX) flies to develop with most of their somatic tissues having a male identity, i.e., ‘XX pseudomales’. In these flies, oogenesis is aborted and there is even occasionally what appears to be early spermatogenic development. Since the germline expressions of these sex regulatory genes are not required for early stages of gametogenesis, the aberrant germline phenotypes must result from the male transformation of the soma (Hinson, 1999 and references).

It is not clear which germline genes are influenced by the proposed somatic interactions. Three possible candidates based on their early and sex-specific roles in female germline differentiation are ovarian tumor, ovo and Sex-lethal (Sxl). During oogenesis, the expression of otu is required in the germline at several stages, if not continually. The null mutant phenotype is characterized by the absence of egg chambers in an otherwise normal ovary, denoted as the quiescent phenotype, although substantial numbers of germ cells are still present in the germarium. Null and severe loss-of-function mutations can also produce 'ovarian tumors', a phenotype characterized by egg chambers containing hundreds of seemingly undifferentiated germ cells. Both the quiescent and tumorous cells are aborted at early oogenic stages, during the cystocyte divisions prior to cyst formation. Mutations in otu have no significant effect on spermatogenesis, although some aberrations in male courtship behavior have been reported. The ovo gene has been implicated in regulating sex determination and dosage compensation in the germline. This is based primarily on observations that ovo null XX germ cells are typically not found in the adult ovary, presumably because of reduced cell viability. In addition, certain ovo allele combinations produce tumorous germ cells that morphologically resemble primary spermatocytes. These phenotypes make ovo a candidate target for a somatic signal regulating early oogenesis, although the expression of ovo in adult germ cells does not appear to be responsive to somatic influences. ovo might directly regulate otu. The Ovo protein can bind to sites in the otu promoter, which displays sensitivity to changes in the dosage of ovo + function. It is not known when this putative regulation of otu occurs nor what role it plays in oogenesis (Hinson, 1999 and references).

The effects of an ovo null mutation on XX germ cells developing in pseudomale testes and female ovaries were examined. In females, ovo mutant XX germ cells typically arrest beginning at larval gonial stages. Occasionally, mutant germ cells survived to the adult stage. However, these cells generally failed to undergo gametogenic differentiation as seen by the absence of spectrosomes, fusomes or ring canals. It was reasoned that, if the requirement for ovo is solely dependent on the X:A ratio, then the phenotype of ovo mutant germ cells in pseudomales should be at least as severe. In this case, the ovo mutant XX pseudomale gonads should be either atrophic or contain a few clusters of mostly undifferentiated germ cells. There is an increase in the frequency of atrophic gonads (82%) compared to normal pseudomales (48%), many of the non-oogenic type. The non-oogenic gonads contained VASA-positive germ cells. This indicates that not only are a substantial fraction of the mutant germ cells viable in adults, but gametogenic differentiation occurs as well. The frequency of the non-oogenic gonads in ovo mutant pseudomales is essentially unchanged from that observed in normal pseudomales. This suggests that the observed increase in the atrophic category is due primarily to the loss of the oogenic class. Mutations in otu gave results similar to those described for ovo. This suggests that otu and ovo mutations specifically disrupt only those germ cells attempting female differentiation, rather than the indiscriminate elimination of the entire XX germline (Hinson, 1999).

Heat shock-otu can alter the XX pseudomale gonadal phenotype; to examine whether and to what degree otu expression could induce oogenic development in pseudomales, immunohistochemical studies were performed. When continually cultured at 20-25°C, hs-otu pseudomale gonads are as much as two to three times longer than normal. In addition, 88% of the hs-otu gonads examined show extensive Hu-li tai shao-labeling of ring canals (Hts is an adducin-like protein). These feminized gonads display a developmental progression of gametogenic stages. In section III of the gonad, the pseudomale germ cells have differentiated to postgermarial stages as defined by the expression of kelch. Kelch, an actin binding protein, is localized to female ring canals after the ring canal deposition of Hts and f-actin . Kelch is first detected in female ring canals in stage 1 egg chambers, but is not seen in all ring canals until stage 4. In hs-otu XX pseudomales, the germ cell clusters in section III contain thick ring canals, with virtually all of them showing Kelch deposition along the inner surface of the f-actin layer. In comparison, no Kelch-labeled ring canals are observed in XX pseudomales without hs-otu, indicating that oogenesis is not only less frequent, but also more limited. Taken together, these results indicate the masculinizing effect of male soma (or the absence of female soma) on XX germ cells can be partially, but consistently, overridden by the expression of otu from a heterologous promoter. The resulting fusome and ring canal development follows the same sequence of events as occurs in normal oogenesis. Therefore, pseudomale germ cells are competent to both initiate and undergo substantial oogenesis if provided with adequate levels of otu. Both ovo and Sxl were shown to be required for otu induced oogenic differentiation in XX pseudomales. However, an additional role for otu in some process affecting germline viability and/or proliferation can be identified that is separable from oogenic differentiation and independent of ovo and, possibly, Sxl functions (Hinson, 1999).

The finding that hs-otu can feminize XX pseudomale germ cells suggests oogenesis is blocked because of insufficient otu levels. Therefore, an examination was carried out to see whether tra-induced sexual transformation affects the level of otu gene expression. otu-lacZ is expressed in most, if not all, larval and pupal germ cells in both female and male gonads. Sex-specific regulation only becomes apparent in the adult testis where male germline expression become restricted to a few cells at the apical tip. As with otu, the ovo promoter is initially active in both male and female larval gonads. However, ovo-lacZ becomes sex-specific at an earlier stage, showing restricted expression in male gonads during the third instar larval and pupal periods. These results demonstrate that the otu and ovo promoters are under different regulatory control in the pre-adult germline. However, otu, but not ovo, promoter activity is influenced by tra-induced sexual transformation. These data demonstrate that the tra-induced sexual transformation specifically inhibits otu promoter activity. Also carried out was the reciprocal experiment, in which otu-lacZ activity was examined in XY germ cells developing in a female somatic background. XY pseudofemales produced by the ectopic expression of tra result in ovaries containing tumorous egg chambers. Because XY pseudofemale germ cells become sufficiently 'feminized' so that they acquire a need for otu function for optimal proliferation, it was anticipated they would also be permissive for otu promoter activity. This is in fact the case. Even in the absence of ovo function, XY pseudofemale germ cells consistently express otu-lacZ. This indicates that the feminizing effects of tra, but not ovo, are necessary for otu transcription. In comparison, the ovo promoter is not detectably active in XY pseudofemales, again illustrating differential regulation of ovo and otu (Hinson, 1999).

It is thought that during the pupal and adult stages, two critical events occur in the female germarium: (1) ovo activity allows XX germ cells to become receptive to the otu function controlling oogenic differentiation, and (2) tra-dependent somatic signals allow continued expression of otu in the female germline by maintaining otu promoter activity. The combination of these events constitutes a mechanism by which the otu gene serves to link the somatic sex differentiation pathway controlled by tra with a female germline developmental pathway controlled by ovo (Hinson, 1999).

Sex determination in the Drosophila germline is dictated by the sexual identity of the surrounding soma

It has been suggested that sexual identity in the germline depends upon the combination of a nonautonomous somatic signaling pathway and an autonomous X chromosome counting system. The roles of the sexual differentiation genes transformer (tra) and doublesex (dsx) in regulating the activity of the somatic signaling pathway have been examined. It was asked whether ectopic somatic expression of the female products of the tra and dsx genes could feminize the germline of XY animals. TraF, the female form of transformer, is sufficient to feminize XY germ cells, shutting off the expression of male-specific markers and activating the expression of female-specific markers. Feminization of the germline depends upon the constitutively expressed transformer-2 (tra-2) gene, but does not seem to require a functional dsx gene. However, feminization of XY germ cells by TraF can be blocked by the male form of the Dsx protein (DsxM). Expression of the female form of dsx, DsxF, in XY animals also induces germline expression of female markers. Taken together with a previous analysis of the effects of mutations in tra, tra-2, and dsx on the feminization of XX germ cells in XX animals, these findings indicate that the somatic signaling pathway is redundant at the level of tra and dsx. Finally, these studies call into question the idea that a cell-autonomous X chromosome counting system plays a central role in germline sex determination (Waterbury, 2000).

Transplantation experiments and clonal analysis have suggested that germline sexual identity in XX animals depends upon a combination of cell-autonomous factors that somehow assess the X/A ratio and nonautonomous factors that signal sexual identity from the soma to the germline. A plausible pathway for linking somatic sexual identity to the mechanism that generates the nonautonomous signal is the well-characterized Sxl -> tra/tra-2 -> dsx cascade. In previous studies, the effects were tested of mutations in tra, tra-2, and dsx on the sexual identity of germ cells in XX animals. Unexpectedly, only in the case of the sex-nonspecific gene, tra-2, does loss-of-function mutation lead to a switch in sexual identity of the XX germ cells from female to male. To account for these findings, it has been proposed that the somatic signal must be generated by a novel tra-2-dependent regulatory cascade. Since dsx is dispensable for this process in XX animals, it has been postulated that an unidentified tra-2 regulatory target, z, directly or indirectly generates the signal. To explain the fact that XX germ cells retain partial female identity in tra mutants, it has been suggested that there must be another gene q whose activity overlaps or is redundant with tra. In this view, both tra and q would be able to function with the cofactor tra-2 to promote the female-specific expression of z (Waterbury, 2000).

This model was tested by introducing transgenes that ectopically express the female forms of tra and dsx into XY animals and by assaying their effects on germline sexual identity. The findings are generally consistent with predictions of the original model: there were some unexpected results that altered an understanding of the nature of the germline sex determination process and the role of dsx. Experiments with the tra transgene are considered below (Waterbury, 2000).

According to the model, ectopically expressed tra is predicted to activate the regulatory cascade that signals female identity from the soma to the germline. Activation of this signaling pathway should require tra-2 and the target gene z, while dsx would be dispensable. The results are generally consistent with these predictions. Ectopically expressed Tra switches the sexual identity of germ cells in XY animals from male to female, turning off male-specific germline markers and inducing female-specific markers. This switch in sexual identity is blocked by mutations in tra-2, but is not prevented by loss-of-function mutations in dsx (Waterbury, 2000).

In XX animals, the available evidence indicates that the tra/q -> tra-2 -> z feminization pathway functions in the soma. Hence, an expectation of the model is that ectopically expressed Tra would also feminize XY germ cells through its action in the soma, not in the germline. However, since the hsp83 promoter is known to be active in both soma and germline, it is possible that Tra protein ectopically expressed in XY germ cells feminizes these cells by a novel mechanism that is independent of the somatic signaling pathway that normally operates in XX animals. Two lines of evidence argue against this: (1) since several of the hsp83-traF lines were recovered from males, it would appear that expression of Tra in XY germ cells is not in itself sufficient to feminize these cells; (2) the available evidence suggests that ectopically expressed Tra feminizes the germline in XY animals by a pathway resembling that used in XX animals; it requires tra-2 but is independent of a functional dsx (Waterbury, 2000).

Although the hsp83-traF transgene does not require dsx to feminize the germline of XY animals, feminization can be prevented if the only source of Dsx protein is provided by an allele that constitutively expresses DsxM. This result was unexpected since DsxM has no effect on the sexual identity of the germline in XX animals. There are several possible explanations for this discrepancy. It has been proposed that there is another gene, q, which occupies the same position in the regulatory cascade as tra. If this gene is downstream of Sxl, as expected, it would be expressed in the male mode in XY; P[hsp83-traF] animals and hence would not contribute to the production of the feminizing signal. Because DsxM alters the development of the soma surrounding the germline and consequently the cell-cell contacts between soma and germline, the signal produced by tra alone might not be sufficient to feminize. A second possibility is that XY germ cells are intrinsically less responsive to the feminizing signal than XX germ cells. For example, given the lack of strong evidence for germline dosage compensation, the signal could be sensitive to a twofold difference in X-linked genes. A third possibility is that DsxM produces a masculinizing signal that is able to counteract the effects of the feminizing signal produced by TraF. At the present, these explanations cannot be distinguished (Waterbury, 2000).

Since the dsx gene can be removed or expressed exclusively in the male mode without affecting germline sexual identity in XX animals, it has been suggested that dsx has no role in germline sex determination. However, contrary to this suggestion, ectopic expression of DsxF in XY; dsx- animals can feminize the germline and this feminizing activity can be blocked if DsxM is also present in the soma (Waterbury, 2000).

Why is DsxF capable of feminizing XY germ cells, yet dispensable in XX animals? One way to reconcile these two observations is to postulate that z regulates the synthesis of the feminizing signal ('fes') instead of encoding the signal itself. If this were the case, both Z and DsxF could independently promote the production of fes. In females, since q and tra would be active, Z would be able to induce sufficient levels of fes to feminize the germline in the absence of DsxF or in the presence of DsxM. Furthermore, since the female and male Dsx proteins recognize the same target sequences, ectopically expressed DsxF would be able to activate fes synthesis in XY animals only when DsxM is absent (Waterbury, 2000).

In the revised model for the somatic signaling pathway, Sxl has been placed at the top of the regulatory cascade where it is responsible for activating the female-specific expression of both tra and q. While Sxl is known to be required for sex-specific regulation of tra, it should be noted that there is no evidence that it is responsible for controlling the activity of q. However, unless q is itself a target for the X/A counting system, there are no other known mechanisms that could promote female expression. If q is downstream of Sxl, results with SxlM1,fm3 and SxlM1,fm7 (revertant alleles of SxlM1) suggest that q is regulated by a different mechanism than tra. q and/or tra, together with tra-2, would then activate the female-specific expression of z and dsx. The female products of z and dsx would in turn direct the synthesis of the feminizing signal. By this model, the germline would assume male identity whenever Sxl is off in the soma. However, it is not clear whether the male pathway requires production of a male somatic signal by the male form of Dsx (or Z) or occurs in XY germ cells by default in the absence of a female signal. In favor of the former possibility is the finding that constitutively expressed DsxM prevents Tra from feminizing XY germ cells. However, functional dsx is not required in XY animals to select male identity (Waterbury, 2000).

One question raised by these studies is the role of the postulated autonomous X chromosome counting system in germline sex determination. In particular, it has been argued from pole cell transplantation experiments that this autonomous system overrides input from the soma in XY germ cells, forcing them to assume male identity. However, data has been presented indicating that the sexual identity of XY germ cells can be switched from male to female by ectopic expression of TraF and DsxF. If TraF and DsxF activate the signaling pathway(s) that normally functions in XX animals, this result would imply that there may be no cell-autonomous system that selects sexual identity by measuring the germ cell X/A ratio. In this view, the autonomous components of the germline sex determination system would play an entirely different role. They would be subordinate to the somatic signaling pathway, being responsible only for responding correctly to the somatic signal and having no role in making the actual choice. Of course, if the default pathway within the germline is male, then this pathway will be followed 'autonomously' in the absence of a feminizing signal from the soma (Waterbury, 2000).

From a phylogenetic perspective, the simplest solution for germline sex determination is that germ cells strictly follow the same sexual fate as that of the soma in securing the development of a fully functional organism. In fact, this appears to be the mechanism for germline sex determination in other dipteran species such as Musca domestica and Chrysomya rufifacies. In these organisms, somatic sex alone is necessary and sufficient to dictate sexual fate to the germline. Irrespective of their sexual karyotype, when germ cells are surrounded by ovarian tissue, eggs are produced, and when surrounded by testicular tissue, sperm are produced. Studies in the nematode C. elegans and in the mouse further support the idea that somatic sex is widely used to dictate the sexual fate of gametes. In Drosophila, it is believed that somatic sex is the primary determinant. Why then is this soma-to-germline signaling mechanism insufficient to direct complete female or male germline differentiation independent of the chromosome composition of the germ cells in Drosophila? A likely explanation is that XY and XX germ cells in Drosophila have lost the ability to respond equally well to somatic cues. For example, in XY; P[hsp83-traF] pseudofemales, most of the ovarioles have a tumorous ovary phenotype and ovarioles that have normal-looking egg chambers are observed very infrequently. Given that there is no strong evidence for germline dosage compensation, one plausible explanation for the abnormal development of these sex-transformed XY germ cells is that the dose of X-linked gene products is insufficient to properly execute an oogenic developmental program. The Sxl gene would be a good example of a gene that is required for oogenesis and, because of its autoregulatory activity, is highly sensitive not only to its own dose but also to the dose of other X-linked genes such as the splicing factor snf. It is reasonable to suppose that there may be a variety of steps in oogenesis (or spermatogenesis) that are sensitive to the dose of X-linked genes (Waterbury, 2000).

Within the germline, otu, ovo, and Sxl have been identified as candidate genes that respond to the feminizing signal from the soma and determine the sex of the germ cells. Mutations in all three genes have sex-specific effects on germline development. Perhaps the most striking result is the fact that loss-of-function ovo and otu mutations markedly reduce the viability of XX but not XY germ cells. Thus one important question is whether these mutations have similar effects on the viability of XY germ cells feminized by the hsp83-traF transgene. Somewhat surprisingly, it was found that otu and ovo mutations behave differently. Strong loss-of-function otu mutations reduce the viability of XY germ cells feminized by the traF transgene. This finding suggests that the lethal effects of strong otu mutations arise because the germ cells assume a female identity, and not because of their number of X chromosomes. In contrast, ovo mutations have no apparent effect on the viability of feminized XY germ cells. One explanation for this difference is that lethal effects are not observed in ovo mutants because the feminizing signal produced by the traF transgene in XY animals is weaker than the feminizing signal found in wild-type XX animals. Alternatively, it is possible that XX germ cell death in ovo mutants does not depend upon the choice of sexual identity, but rather is a function of the X chromosome dose (Waterbury, 2000).

If otu, ovo, or Sxl functions as a master sex determination switch within the germline, one would expect to find that mutations in any of these genes would completely block the feminization of germ cells much like mutations in Sxl prevent feminization in the soma. While the results indicate that none of these genes fits this criterion for a master regulatory switch, effects are observed in the expression of sex-specific markers. Mutations in all three genes prevent the traF transgene from inducing the expression of female bruno (and Sxl) gene products. However, in all three cases the transgene still induces the expression of female orb gene products. One interpretation of these findings is that the sex determination pathway in the germline is split into at least two branches -- one branch that contains bruno and Sxl and another branch that contains orb. For both bruno and orb, sex-specific expression depends upon the activation of distinct sex-specific promoters. If these two genes are in independent branches of the germline sex determination pathway, this would imply that there must be distinct 'male' and 'female' transcription factors for the four promoters. Moreover, it seems likely that one important function of the somatic signaling system would be to control the expression of these transcription factors. Clearly, it will be important to identify these transcription factors and to learn how they are regulated (Waterbury, 2000).

The 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, 2001).

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

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

Function of transformer in the development of gonadal sexual dimorphism

Sexually dimorphic development of the gonad is essential for germ cell development and sexual reproduction. The Drosophila embryonic gonad is already sexually dimorphic at the time of initial gonad formation. Male-specific somatic gonadal precursors (msSGPs) contribute only to the testis and express a Drosophila homolog of Sox9 (Sox100B: Loh, 2000), a gene essential for testis formation in humans. The msSGPs are specified in both males and females, but are recruited into only the developing testis. In females, these cells are eliminated via programmed cell death dependent on the sex determination regulatory gene doublesex. This work furthers the hypotheses that a conserved pathway controls gonad sexual dimorphism in diverse species and that sex-specific cell recruitment and programmed cell death are common mechanisms for creating sexual dimorphism (DeFalco, 2003).

To investigate when sexual dimorphism is first manifested in the somatic gonad, expression of SGP markers were examined in embryos whose sex could be unambiguously identified, at a developmental stage (stage 15) soon after gonad coalescence has occurred. Analysis of Eya expression reveals anti-Eya immunoreactivity throughout the female somatic gonad, though Eya expression is somewhat stronger in the posterior. In males, anti-Eya immunoreactivity is also found throughout the somatic gonad. However, the expression at the posterior of the gonad is much more intense than in females, as there appears to be a cluster of Eya-expressing cells at the posterior of the male gonad that is not present in females. In blind experiments, the sex of an embryo could be accurately identified by the Eya expression pattern in the gonad. Thus, sexual dimorphism is already apparent in the somatic gonad soon after initial gonad formation. A sex-specific expression pattern is also observed with Wnt-2 at this stage. As is observed with Eya, Wnt-2 is expressed in the SGPs of the female gonad, but its expression is greatly increased at the posterior of the male gonad. The SGP marker bluetail (see Galloni, 1993) exhibits a similar sex-specific pattern as Eya; however, the SGP marker 68-77 is expressed equally in both sexes (see below). Thus, the somatic gonad is sexually dimorphic by stage 15, but only a subset of SGP markers reveals this sexual dimorphism (DeFalco, 2003).

During Drosophila embryogenesis, Sox100B is expressed in a number of cell types, including the gonad (Loh, 2000). Since Sox100B is closely related to Sox9 (an important sex determination factor in humans and mice), whether Sox100B expression is sexually dimorphic in Drosophila was tested. Interestingly, it was found that after gonad coalescence (stage 15), Sox100B expression in the gonad is male-specific. Sox100B immunoreactivity is not observed in the coalesced female gonad, whereas it is detected in a posterior cluster of SGPs in the male gonad. While this expression pattern is seen in most wild-type backgrounds (including Canton-S and faf-lacZ), in certain 'wild-type' lines, such as w1118, a few Sox100B-positive cells are observed in the posterior of the coalesced female gonad (however, this is still clearly distinguishable from the number of Sox100B-positive cells in the male). Unlike Eya and Wnt-2, Sox100B is not expressed in all SGPs, since it is usually absent from female gonads and from the anterior region of the male gonad and does not colocalize with the SGP marker 68-77. Sox100B expression appears restricted to the posterior cluster of SGPs that is observed only in the male gonad. Thus, like Sox9 expression in vertebrates, Sox100B exhibits a male-specific pattern of expression in the Drosophila embryonic gonad, suggesting that it may indeed be an ortholog of Sox9 (DeFalco, 2003).

After having identified sexually dimorphic markers of the embryonic gonad, these markers were used to investigate how sexual dimorphism is established. It was asked whether proper gonad formation is necessary for the establishment of sexual dimorphism by examining Sox100B expression in fear-of-intimacy (foi) mutant embryos. In foi mutants, germ cells migrate and associate normally with the SGPs, but these two cell types fail to coalesce into a round and compact gonad. Despite the failure of gonad coalescence, a cluster of Sox100B-expressing cells was still observed at the posterior of the male gonad, while no Sox100B-expressing cells are observed in the female at this stage. Whether the presence of germ cells is necessary for the establishment of sexual dimorphism in the embryonic gonad was examined. Embryos that lack germ cells due to a hypomorphic mutation in oskar, a gene required for germ cell formation, were examined. Other aspects of embryonic development occur normally in these embryos, including the formation and coalescence of the SGPs. Agametic gonads show identical sexual dimorphism to wild-type embryos. Sox100B is coexpressed with Eya in the cluster of somatic cells in the posterior of the male gonad, but Sox100B expression is not observed in the female gonad. Thus, sexual dimorphism of the embryonic somatic gonad does not require proper gonad morphogenesis or the presence of germ cells (DeFalco, 2003).

The posterior cluster of Eya and Sox100B coexpressing cells could result from sex-specific differences in gene expression within the cells of the gonad. Alternatively, it could reflect a difference in gonad morphology, in which these cells are only present in males and not in females. To distinguish between these possibilities, the morphology of the male and female coalesced (stage 15) gonad was examined, using approaches that do not depend on cell-type-specific SGP markers. First, a CD8-GFP fusion protein was expressed broadly in the mesoderm. The fusion of the extracellular and transmembrane regions of mouse CD8 with GFP allows for visualization of cell and tissue morphology. A cluster of mesodermal cells is consistantly observed attached to the posterior of the male gonad that is not observed in the female. In blind experiments, the sex of the embryo can be predicted based on the presence of this posterior cluster of cells. Male and female gonads were also examined by transmission electron microscopy (TEM). Male and female embryos were first sorted using an X chromosome-linked GFP expression construct and then processed separately for TEM. In this analysis, a cluster of cells that is not present in the female gonad was consistently at the posterior of the male gonad. Both the size and morphology of these cells indicate that they are somatic cells rather than germ cells. Thus, the observed sexual dimorphism reflects a change in gonad morphology, not just a change in gene expression. Since the additional cells at the posterior of the male gonad express at least some markers in common with SGPs (e.g., Eya), these cells are referred to as male-specific SGPs (msSGPs) (DeFalco, 2003).

Since no sex-specific differences were observed in SGP proliferation in the gonad, it seems unlikely that the SGPs are dividing to produce the msSGPs. Therefore, Sox100B was used as a marker for the msSGPs to determine where and when these cells are first specified. At stages prior to gonad coalescence (stages 12 and 13), a cluster of Eya/Sox100B double-immunopositive cells is observed posterior and ventral to the developing clusters of SGPs, which express Eya alone. Interestingly, this cluster of Eya/Sox100B double-positive cells is initially observed in both males and females and appears identical, although Eya expression may be somewhat lower in the female cluster. During stage 13, as the SGPs and germ cells associate closely along PS 10-12, the Eya/Sox100B double-positive cells move toward the gonad in both sexes. In males, these cells join the posterior of the coalescing gonad. In contrast, these cells do not join the gonad in females, and only Eya-positive, Sox100B-negative cells are found in the coalesced gonad. It is concluded that the Eya/Sox100B double-positive cells are the msSGPs and that they form separately from the SGPs. These cells are initially specified in both males and females and move anteriorly to join the gonad in males. In females, these cells do not form part of the gonad, as judged by the above morphological analysis, and are no longer detected using available markers (DeFalco, 2003).

Since the msSGPs develop separately from the SGPs, it was of interest to address where the msSGPs arise and what controls their specification. By marking the anterior of each parasegment using an antibody against Engrailed, it was determined that the msSGPs are specified in PS13. This observation is consistent with these cells arising posterior to the SGPs, which form in PS 10, 11, and 12. Other Sox100B expression is observed in nongonadal tissues. Whether, like the SGPs, the msSGPs are specified in the dorsolateral domain of the mesoderm was also addressed. Mesodermal cell types that form in this region, such as the SGPs and the fat body, require the homeodomain proteins Tinman and Zfh-1 for their specification. However, in embryos double-mutant for tinman and zfh-1, the msSGPs are still specified, even though the SGPs fail to develop. Thus, msSGPs do not arise from the dorsolateral domain, consistent with the fact that the msSGPs are first observed in a position ventral to the SGPs. The msSGPs also differ from the SGPs in terms of their requirements for the homeotic gene abd-A. SGP specification absolutely requires abd-A, while msSGPs are still present in these mutants. Thus, despite the fact that the msSGPs and the SGPs share expression of some molecular markers such as Eya and Wnt-2, their specification is under independent control (DeFalco, 2003).

Since the msSGPs express both Eya and Sox100B, the requirements for each of these genes in msSGP specification was investigated. In eya mutants, Sox100B-positive cells are still observed posterior to the germ cells at early stages, in a position where the msSGPs normally develop. Since the SGPs are not maintained in these mutants, the germ cells disperse and the gonad does not coalesce. Therefore, it is impossible to tell if the msSGPs would join the posterior of the male gonad in eya mutants. However, initial msSGP specification does not require eya. Similarly, in a deletion that removes the Sox100B locus, a large cluster of Eya-positive cells was still observed at the posterior of the male gonad that does not appear in females. Thus, the initial development of the msSGPs does not require Sox100B. Expression of Eya and Sox100B are mutually independent and are likely to be downstream of factors controlling initial msSGP specification (DeFalco, 2003).

Since the msSGPs are initially specified in both males and females, a determination was made of how these cells receive information about their sexual identity that allows them to behave differently in the two sexes. tra plays a key role in the sex determination pathway in Drosophila and is required to promote female differentiation in somatic tissues. tra mutant gonads were examined to test if tra function is required for gonad sexual dimorphism (XX embryos are masculinized by mutations in tra). Sox100B-immunopositive cells are observed in the posterior somatic gonad of both XX and XY tra mutant embryos in a manner comparable to wild-type males. Analysis of the Sox100B expression pattern in the gonad reveals that there are no differences between XX and XY tra mutants, or between either of these genotypes and wild-type males. Conversely, when Transformer is expressed in XY embryos (UAS-traF, tubulin-GAL4), Sox100B-immunopositive cells are no longer observe in these gonads, and they now appear similar to wild-type females (DeFalco, 2003).

In most somatic tissues, the principle sex determination factor downstream of tra is dsx. Unlike tra, dsx is required for both the male and female differentiation pathway, since both XX and XY dsx mutant adults show an intersexual phenotype. However, in the somatic gonad, dsx mutant XY embryos are indistinguishable from wild-type males and show no change in Sox100B expression. Thus, unlike in most somatic tissues, this early characteristic of male development does not require dsx. In XX embryos that are mutant for dsx, a completely masculinized phenotype is observed, in which Sox100B expression in the gonad is similar to a wild-type male. When a dominant allele of dsx, dsxD, is used to express DsxM (dsxD/dsx) in XX embryos, these gonads are no more masculinized than dsx null XX gonads. Therefore, while DsxF is required for the proper female phenotype in XX gonads, it appears that the male Sox100B expression pattern is the 'default' state in the absence of dsx function (DeFalco, 2003).

Since the msSGPs join the posterior of the male gonad but are no longer detected in the female, the basis for the sexually dimorphic behavior of these cells was investigated. In the female, these cells could turn off Sox100B and Eya and contribute to some other tissue, or they might be eliminated altogether. To test this latter hypothesis, whether msSGPs are eliminated by sex-specific programmed cell death in the female was addressed. Since programmed cell death occurs in a caspase-dependent manner, the gonad phenotype was examined in embryos in which caspase activity was inhibited by expressing the baculovirus p35 protein in the mesoderm. In these embryos, XX gonads now appear masculinized; Sox100B-positive cells (msSGPs) persist and join the posterior of female gonads, and coexpress Eya, as in wild-type male embryos. There are not as many Sox100B-positive cells in females as in males, suggesting that p35 may not be completely suppressing cell death. The presence of such cells in the female gonad does not appear to drastically affect ovary formation or oogenesis, since embryos develop into fertile adult females (DeFalco, 2003).

To investigate how programmed cell death might be controlled in the msSGPs, the genes of the H99 region (head involution defective [hid], reaper [rpr], and grim), which are regulators of apoptosis in Drosophila, were examined. A small deletion (DfH99) removes all three of these genes and blocks most programmed cell death in the Drosophila embryo. In DfH99 mutants, an equivalent cluster of Sox100B-positive cells is observed in both males and females. Again, these posterior cells are also Eya positive. Furthermore, XX embryos mutant for hid alone also contain Sox100B-positive cells in the posterior of the gonad, although the posterior cluster of cells is slightly smaller than in the male. It is concluded that the msSGPs are normally eliminated from females through sex-specific programmed cell death, controlled by hid and possibly also other genes of the H99 region. However, if cell death is blocked in females, these cells can continue to exhibit the normal male behavior of the msSGPs, including proper marker expression and recruitment into the gonad. Therefore, the decision whether or not to undergo apoptosis is likely the crucial event leading to the sexually dimorphic development of these cells at this stage (DeFalco, 2003).

It is concluded that proper information from the sex determination pathway is required to control the sexually dimorphic behavior of the msSGPs. The female phenotype in the embryonic gonad is dependent on both tra and dsx. Interestingly, it seems that the male phenotype is the default state; in the absence of any tra or dsx function, msSGPs in both XX and XY embryos behave as in wild-type males. This is a different situation than in most other tissues, in which dsx is required in both sexes to promote proper sexual differentiation. In particular, while no role is found for DsxM in this process, DsxF is positively required either to establish the female fate in the posterior somatic gonad or to repress the male fate. This role for DsxF in msSGP development is analogous to its role in the genital disc, in which DsxF is required to block recruitment of btl-expressing cells into the disc; in both cases, dsx female function serves to repress incorporation of a male-specific cell type. Since the msSGPs are initially specified in a sex-independent manner, this may account for the fact that the persistence of these cells (the male phenotype) is the default state. It will be of interest in the future to address the role of the msSGPs in testis development, and how genes such as dsx, eya, and Sox100B act in this process (DeFalco, 2003).

Although sex determination schemes vary widely in the animal kingdom, there is evidence that the molecular and cellular pathways used to control sexual dimorphism may be conserved, even between vertebrates and invertebrates. One example is Sox9, which has been implicated as an ancestral sex-determining gene in vertebrates given its male-specific gonad expression in diverse species such as human, mouse, turtle, and chicken. A potential Drosophila ortholog of Sox9, Sox100B, is expressed in a male-specific manner in the embryonic somatic gonad. The manner of Sox100B expression is reminiscent of that in the mouse; Sox9 is initially expressed in both sexes, but is maintained and upregulated in the male gonad. It will be very interesting to compare the role that Sox100B plays in the development of the Drosophila testis to the one played by Sox9 in vertebrates (DeFalco, 2003).

Molecular conservation is also observed amongst the members of the Dsx/Mab-3 Related Transcription Factor (DMRT) family. DMRT family members have been shown to be essential for sex-specific development in Drosophila (Dsx), C. elegans (mab-3), medaka fish (DMY), and mice (DMRT1) and have been implicated in human sex reversal. This study demonstrates that dsx is essential for proper sex-specific development of the msSGPs. Thus, increasing evidence indicates that DMRT family members are also conserved regulators of sexual dimorphism (DeFalco, 2003).


Evolution of transformer

An evolutionary approach has been taken to investigate tra regulation and function, by isolating the tra-homologous genes from selected Drosophila species, and then using the interpecific DNA sequence comparisons to help identify regions of functional significance. The tra-homologous genes from two Sophophoran subgenus species, Drosophila simulans and Drosophila erecta, and two Drosophila subgenus species, Drosophila hydei and Drosophila virilis, were cloned, sequenced and compared to the D. melanogaster tra gene. This comparison reveals an unusually high degree of evolutionary divergence among the tra coding sequences. These studies also highlight a highly conserved sequence within intron one that probably defines a cis-acting regulator of the sex-specific alternative splicing event (O’Neil, 1992).

As expected from the known phylogenetic relationships, the degree of similarity is higher when melanogaster and simulans are compared (93.6% identity) than when either of these species is compared to erecta (86.0% and 85.6% identity). The higher degree of conservation of sequences in intron 1 than in intron 2 is not unexpected, since intron 1 exhibits sex-specific alternative splicing while the splicing of intron 2 is presumably not regulated. In D. melanogaster it has been shown that the sex-specific splicing of intron 1 is controlled by sequences contained within the intron itself, and these regulatory sequences are likely conserved. The close relationship between these members of the melanogaster subgroup, however, precludes using these comparisons to accurately delimit any well defined region of the intron that might be implicated in this regulation, since not enough time has passed for the nonimportant sequences to have diverged (O’Neil, 1992).

One obvious difference between the coding region of tra in D. melanogaster and that of the other two species is the presence of a 39-base pair sequence in the central exon of D. melanogaster that is missing in D. simulans and D. erecta. Given the phylogenetic relationships of these three species, the most parsimonious explanation is that there has been a duplication of this sequence in the lineage leading to D. melanogaster rather than two independent deletions occurring in the D. simulans and D. erecta lineages. This is supported by the fact that in D. melanogaster, these extra 39 bases exist as an almost perfect tandem repeat (37/39 identity) of the adjacent sequence. This duplication occurs in the very arginine-serine-rich region of the tra gene, and its presence in either one or two copies is presumably not critical for the functioning of the Tra protein. Other smaller deletions/insertions also are seen when the coding regions of these three species are aligned. For example, D. erecta has an extra three nucleotides, encoding a cysteine, in exon 1 that are not seen in D. melanogaster and D. simulans. In exon 2 there are two sites where an extra six nucleotides, encoding R S, and an extra 15 nucleotides, encoding R E/G S R H, are present in D. melanogaster and D. simulans, but not in D. erecta (O’Neil, 1992).

These differences among the coding regions yield different size Tra proteins in the three species: 197, 184 and 178 amino acids, for D. melanogaster, D. simulans and D. erecta, respectively. Because of the relatively close relationship among these three species, information about what regions of the tra gene are conserved and what regions are not conserved is limited (O’Neil, 1992).

The transformer locus produces an RNA processing protein that alternatively splices the doublesex pre-mRNA in the sex determination hierarchy of Drosophila melanogaster. Comparisons of the tra coding region among Drosophila species have revealed an unusually high degree of divergence in synonymous and nonsynonymous sites. In this study, the hypothesis that the tra gene will be polymorphic in synonymous and nonsynonymous sites within species was examined by investigating nucleotide sequence variation in eleven tra alleles within D. melanogaster. Of the 1063 nucleotides examined, two synonymous sites were polymorphic and no amino acid variation was detected. Three statistical tests were used to detect departures from an equilibrium neutral model. Two tests failed to reject a neutral model of molecular evolution because of low statisitical power associated with low levels of genetic variation. The Hudson, Kreitman, and Aguade test rejected a neutral model when the tra region was compared to the 5'-flanking region of alcohol dehydrogenase (Adh). The lack of variability in the tra gene is consistent with a recent selective sweep of a beneficial allele in or near the tra locus (Walthour, 1994).

The amino acid sequence of the transformer gene exhibits an extremely rapid rate of evolution among Drosophila species, although the gene performs a critical step in sex determination. These changes in amino acid sequence are the result of either natural selection or neutral evolution. To differentiate between selective and neutral causes of this evolutionary change, analyses of both intraspecific and interspecific patterns of molecular evolution of tra gene sequences are presented. Sequences of 31 tra alleles were obtained from Drosophila americana. Many replacement and silent nucleotide variants are present among the alleles; however, the distribution of this sequence variation is consistent with neutral evolution. Sequence evolution was also examined among six species representative of the genus Drosophila. For most lineages and most regions of the gene, both silent and replacement substitutions have accumulated in a constant, clock-like manner. In exon 3 of D. virilis and D. americana evidence is found for an elevated rate of nonsynonymous substitution, but no statistical support for a greater rate of nonsynonymous relative to synonymous substitutions. Both levels of analysis of the tra sequence suggest that, although the gene is evolving at a rapid pace, these changes are neutral in function (McAllister, 2000).

The transformer gene in the medfly Ceratitis capitata

Given the unusually high degree of sequence divergence among tra homologs in Drosophila (O’Neil, 1992), the tra gene in the medfly was cloned by exploiting its close linkage in Drosophila to a well-conserved gene, l(3)73Ah. Hence, as a first step towards the isolation of tra, cDNA and genomic Ceratitis sequences were isolated that cross-hybridized to a 500 bp Drosophila cDNA fragment of l(3)73Ah at reduced stringency. These isolates indeed contained a structurally well conserved homolog of l(3)73Ah as confirmed by sequencing and comparison [Ccl(3)73Ah]. A 4 kb long genomic region downstream of the l(3)73Ah homolog was sequenced and a putative ORF was identified that showed by Blast search significant sequence similarity at the amino acid level to tra in Drosophila (ranging from 32% to 40% identity scattered over 120 amino acids) and contained an arginine-serine-rich domain (SR-rich region) commonly found in splicing regulators. As in Drosophila, the two genes are transcribed in opposite orientation and sequence analysis of corresponding cDNA clones reveals that they overlap by about 200 bp. It is concluded that this gene arrangement must have already existed in the common ancestor of these fly species. Though the significance of this synteny is unknown, it provides an ideal entry point to the molecular identification of the tra homolog in Ceratitis (Cctra) (Pane, 2002).

An alignment of CctraF1, CctraM1 and CctraM2 cDNA sequences with the genomic sequence exposes the organization of tra in Ceratitis. The gene is composed of five exons. The first, fourth and fifth exons are included in the mature transcripts of both sexes, while the second and the third exons are male specific. The most important finding is that the female-specific transcript has a long open reading frame, while the male-specific mRNAs contain stop codons that abort prematurely the protein translation. Indeed partially different intronic sequences are retained in the M1 and M2 cDNA clones, adding stop codons in different positions. This finding suggests that a functional full-length TRA is encoded only by the female-specific transcripts. This mode of sex-specific regulation at the level of splicing is well documented for the tra gene in Drosophila (Boggs, 1987). Different from Drosophila, however, where sex-specific regulation is based on the alternative use of two 3' splice acceptor sites, sex-specific regulation in Ceratitis appears more complex and is achieved by a combination of exon skipping and differential use of 5' donor and 3' acceptor sites (Pane, 2002).

The long ORF in the female-specific CctraF1 encodes a putative protein of 429 amino acids. The CcTRA protein exhibits a low degree of similarity to TRA proteins in Drosophila species and it is significantly larger in size in both N and C termini. Sequence processing tools of MACAW led to the identification of five small blocks of sequence similarity dispersed throughout the longest ORF of the female-specific transcripts. The regions with highest similarity (identified also by FastA analysis) are located between CcTRA positions 150-230, 286-292 and 332-342. The SR-rich region in Ceratitis TRA and possibly the other conserved domains may confer specific RNA binding and protein-protein interactions consistent with a proposed role in splicing regulation (Manley, 1996). The male-specific truncated protein isoforms lack the conserved boxes, the SR-rich region and do not show significant similarity with other known proteins (Pane, 2002).

The confinement of transcripts with a long ORF to females suggests that this gene had an essential role in female development of the medfly. To test its function, RNAi technique, which permits functional studies of genes in genetically less amenable organisms, was used. A 900 bp fragment of CctraF1 was used as a template to produce dsRNA that was then injected as a 15 µM solution into either the anterior or the posterior poles of embryos of two different laboratory strains (Benakeion and white-eye). From a total of 900 injected embryos, 272 adult flies were recovered and grouped by their sexual phenotype. A strong sex ratio bias was observed in favor of males. Out of 272, 231 flies (84.9%) showed a normal male morphology, 37 flies (13.6%) exhibited various degrees of intersexuality and the remaining four (1.4%) were the only flies recovered with a normal female phenotype. All of the 37 intersexes exhibited an anteroposterior pattern of intersexuality. More tellingly, the position of male tissues correlated exactly with the initial injection site in the embryo: injection into the anterior pole resulted in the formation of male-specific spatulated bristles on the head of intersexes, male-specific blue eye reflections, male-like bristles mixed with female-like bristles on the femur toward the coxa of the foreleg, but the genitalia at the posterior remained female-like. Conversely, injection into the posterior pole gave rise to mosaic adults with male genitalia but with female bristles on the head and female-specific green eye reflections. The intersexes showed also various degrees of abnormal gonadal development, with abnormally bent or deformed ovopositor and with mixed male-like and female-like tissues. A few intersexes apparently lacked genitalia (Pane, 2002).

To assess the sexual karyotype of affected flies, a PCR amplification of genomic DNA using Ceratitis Y-specific primers was performed. No products were detected in single preparations of 10 randomly chosen intersexes and six out of 10 phenotypic males did not reveal the presence of a Y chromosome by this test, indicating that all these animals have a female XX karyotype. These results are in agreement with the expected loss of female-promoting activity when tra function is impaired by RNAi. On the contrary, male development of XY flies seems not to be affected by RNAi of tra, suggesting that the gene, as in Drosophila, is dispensable in this sex. The occurrence of intersexes and of few females is most likely due to incomplete penetrance of the RNAi effect. Indeed, when a lower concentration of dsRNA (5 µM versus 15 µM) was injected into the anterior embryonic region, 64 intersexes, 76 males and four females were obtained out of 144 adult flies. Therefore the percentage of intersexes increased from 14% to 44%, while the percentage of males decreased from 84% to 52%, suggesting that XX individuals were only partially masculinized. From these results, it is concluded that tra is required for female development in Ceratitis. Moreover, it is conceivable that absence of tra activity constitutes a signal that triggers the male fate. Thus, as in Drosophila, Ceratitis tra may act as a genetic switch between female (when functionally ON) and male (when functionally OFF) development. The male-specific short peptides encoded by the alternatively spliced male-specific transcripts seem to be non-functional, at least at early embryonic stages, because the RNAi has no evident effects on the development of XY males. Whether they play a function at later stages, when the RNAi starts to lose its efficiency, cannot be evaluated (Pane, 2002).

To investigate the fertility of the RNAi-treated adults, 27 males obtained from embryos injected with 15 µM dsRNA solution were individually crossed with wild-type females. It is predicted that if XX males are fertile than they should give a female-only progeny when crossed with wild-type virgin females. Indeed out of 27, seven crosses gave a unisexual female-only progeny. The karyotype of these seven males was then analyzed by PCR, confirming that they were XX fertile males. As expected, PCR karyotypic analyses of those males giving a bisexual progeny revealed that they were XY males. These data demonstrate that the Y-chromosome does not carry genes necessary for male fertility (Pane, 2002).

Next, the mechanisms which control the activity of tra in Ceratitis were investigated. In Drosophila, regulation of tra activity is achieved at the post-transcriptional level based on 3' splice site selection (Boggs, 1997). When Sxl protein is present, Sxl prevents the use of a distal acceptor site, thereby promoting the use of the next downstream available 3' splice site, and it shifts about 50% of the pre-mRNA molecules from a non-sex-specific splicing to a productive female-specific mRNA. This regulation requires the direct binding of SXL to a poly (U)8 stretch upstream of the regulated splice site (Kanaar, 1995). Several findings argue against a similar mechanism for conferring sex-specific splicing of tra in Ceratitis (Saccone, 1998). (1) Cctra splicing is based on a combination of exon skipping and 5' and 3' splice site regulation, rather than on 3' splice site selection. (2) CcSXL protein is present in both sexes of Ceratitis. However, upon close inspection of the Cctra sequence, an important discovery was made: within the two male-specific exons and the male-specifically retained intron, eight repeats were found by DNA sequence comparison that are structurally related to the TRA/TRA-2 binding sites (13 nucleotides long) in the dsx gene of Drosophila. Similar repeats are also detected in the female-specific exon of the dsx homolog in Ceratitis (Saccone, 2000). Their high sequence similarity to Drosophila Tra/Tra-2 binding sites and peculiar localization within the Cctra gene led to the idea that these sequences are involved in the sex-specific splicing regulation of Cctra itself. In Drosophila dsx and fru genes these cis-elements act as, respectively, 3' and 5' splice enhancers by recruiting the Tra/Tra-2 complex to promote the use of the regulated splice site (Tian, 1993: Heinrichs, 1998). The presence of potential TRA/TRA-2-binding sites in and around the male-specific exons suggests that the female-specific CcTRA could inhibit their usage and led to an investigation of whether an autoregulatory function of Cctra is involved in the process of sex-specific splicing (Pane, 2002).

If female-specific splicing of tra pre-mRNA indeed depends on tra activity, it was reasoned that a transient depletion of tra activity should no longer be able to sustain the female mode of splicing. To test this supposition, sex-reversed XX males recovered from Cctra dsRNA injections were analyzed. By RT-PCR analysis, only male-specific tra products were detected in adult tissues of injected XX and XY individuals, but no female-specific products. In addition, the same males contained predominantly male-specific splice variants of dsx, a probable downstream target of tra also in Ceratitis. It was inferred from these results that early application of RNAi transiently eliminates Cctra mRNAs and, thus, prevents continued production of TRA protein. Once tra pre-mRNA production is resumed at a later stage in development, the unproductive male mode of tra splicing is launched because of the absence of functional TRA. Likewise, absence of TRA causes its direct target dsx to be spliced in the male mode. These results are compatible with the postulate that Cctra sustains the productive mode of its splicing by an autoregulatory feedback loop and mediates female differentiation, at least in part, by the control of its target gene dsx. The initiation of the autoregulatory loop in XX embryos could be based on maternal Cctra mRNAs that have been detected in unfertilized eggs by RT-PCR experiments. These mRNAs are spliced in the female mode and hence could provide a source of CcTRA activity that allows female-specific splicing of zygotic Cctra pre-mRNA (Pane, 2002).

Transformer functions as a binary switch gene in the sex determination and sexual differentiation of Drosophila melanogaster and Ceratitis capitata, two insect species that separated nearly 100 million years ago. The TRA protein is required for female differentiation of XX individuals, while XY individuals express smaller, presumably non-functional TRA peptides and consequently develop into adult males. In both species, tra confers female sexual identity through a well conserved double-sex gene. However, unlike Drosophila tra, which is regulated by the upstream Sex-lethal gene, Ceratitis tra itself is likely to control a feedback loop that ensures the maintenance of the female sexual state. The putative CcTRA protein shares a very low degree of sequence identity with the TRA proteins from Drosophila species. However, a female-specific Ceratitis Cctra cDNA encoding the putative full-length CcTRA protein is able to support the female somatic and germline sexual differentiation of D.melanogaster XX; tra mutant adults. Though highly divergent, CcTRA can functionally substitute for DmTRA and induce the female-specific expression of both Dmdsx and Dmfru genes. These data demonstrate the unusual plasticity of the TRA protein that retains a conserved function despite the high evolutionary rate. It is suggested that transformer plays an important role in providing a molecular basis for the variety of sex-determining systems seen among insects (Pane, 2005).

One important finding that comes out of this study is that CcTRA is able to 'recognize' the TRA-TRA2 binding sites in vivo, though they are located in entirely divergent contexts, namely the Dmfru and Dmdsx genes of Drosophila. It is therefore tempting to speculate that the TRA-TRA2 binding sites are also target sequences for CcTRA activity in Ceratitis. In this species, TRA-TRA2 elements are present in exon 4 of the dsx homolog (Ccdsx) and in intron 1 of the Cctra gene. Ccdsx reveals a significant structural and sequence identity when compared to Dmdsx and shows a sex-specific expression pattern. The distribution of the cis elements in the Ccdsx gene is also similar to that of Dmdsx, since they are located in exon 4, which is sex-specifically regulated in both Ceratitis and Drosophila. Given that CcTRA can promote the proper female splicing of Dmdsx, it is conceivable that it also controls Ccdsx expression using a similar mechanism. In Ceratitis females, CcTRA is likely to bind the cis-elements in Ccdsx exon 4 and promote the fusion of exon 3 to exon 4. The resulting female mature transcripts encode the CcdsxF protein. By contrast, in males, where the CcTRA protein is absent, exon 4 is not included in the mature mRNA, with exon 3 being fused directly to exon 5. The mature mRNAs generated in males encode the CcdsxM isoform. Consistent with this model, when the Cctra gene is turned off by RNAi in XX individuals, Ccdsx expression pattern is switched from the female to the male mode of splicing. As in Drosophila, the Ccdsx isoforms are likely to control the development of sexually dimorphic traits in Ceratitis. Putative TRA-TRA2 elements are also surprisingly contained in intron 1 of the Cctra gene. This observation points to a role for the CcTRA protein in the processing of Cctra precursor mRNA. In Ceratitis, Cctra is sex-specifically expressed through post-transcriptional alternative splicing events. In females, the intron 1 is removed from the primary transcript and mature mRNAs that encode the full-length CcTRA protein are produced (Pane, 2005).

Differently, mature mRNAs generated in males retain portions of the intron 1 (i.e. male-specific exons), which contain stop codons and thus prematurely interrupt the translation of the CcTRA protein. The female splicing of the Cctra primary transcripts is dependent upon a functional Cctra gene. When Cctra is switched off by RNAi in early embryos, the emerging XX adults are males and express male variants from Cctra. These observations lead to the hypothesis that, in Ceratitis females, Cctra controls its own expression by means of a positive feedback loop. The results reported in this study further support this hypothesis and suggest that, in females, the CcTRA protein might bind the TRA-TRA2 binding sites in the Cctra pre-mRNA and promote female splicing events (Pane, 2005).

The binding of CcTRA to the cis-elements might prevent the usage of male splicing sites, thus leading the splicing machinery to use the criptic female sites. Consequently, the intron 1 is removed from Cctra precursor transcripts to produce the female mature mRNAs. An alternative possibility is represented by an activation mechanism in which CcTRA would enforce the usage of female splice sites. This model stems from the observation that the TRA-TRA2 elements are mainly located within the male-specific exons and therefore are included in male mature mRNAs. It is possible that, in females, 'male' transcripts are produced by the default mechanism and might behave as splicing intermediates and substrates for CcTRA activity. In this case, the binding of the CcTRA protein to the cis-regulatory elements would favor the use of the female splice sites and promote the removal of the male-specific exons. Both the repression and the activation mechanisms proposed would involve a new property for the TRA proteins as well as an intronic function for the TRA-TRA2 elements, which has not been described before. In females, Cctra mature mRNAs have a long open reading frame and represent the source of CcTRA protein to keep the feedback loop active and guarantee the memory of the female sexual state. In males, the M-factor is likely to impair the positive feedback loop at early stages, thus promoting the male developmental program. Interestingly, CcTRA activity in the Drosophila transgenic lines is dependent upon a functional endogenous Dmtra2 gene. CcTRA is not able to direct female splicing of dsx and fru pre-mRNAs in Drosophila when the DmTRA2 protein is absent. It is believed that, also in Ceratitis, female development involves the cooperation between CcTRA and a putative TRA2 homolog (CcTRA2), which is yet to be identified. Several observations further support this hypothesis. tra2 appears to be highly conserved in evolution and tra2 homologs have been described even in human. Recently a tra2 homolog was identified in the housefly Musca domestica, which diverged from Drosophila some 100 million years ago. Transient depletion of the tra2 function in Musca by RNAi triggers the sexual transformation of XX embryos, which normally become females, toward maleness. These observations all point to the existence of a conserved tra2 homolog in Medfly as strongly suggested by the sequence conservation of Tra/Tra-2 binding sites observed in the Ceratitis dsx homologue. The CcTRA2 protein might interact with CcTRA to control both the female-specific splicing of Ccdsx and the positive feedback loop established by the Cctra gene (Pane, 2005).

Male sex in houseflies is determined by Mdmd, a paralog of the generic splice factor gene CWC22

Across species, animals have diverse sex determination pathways, each consisting of a hierarchical cascade of genes and its associated regulatory mechanism. Houseflies have a distinctive polymorphic sex determination system in which a dominant male determiner, the M-factor, can reside on any of the chromosomes. This study identified a gene, Musca domestica male determiner (Mdmd), as the M-factor. Mdmd originated from a duplication of the spliceosomal factor gene CWC22 (nucampholin). Targeted Mdmd disruption results in complete sex reversal to fertile females because of a shift from male to female expression of the downstream genes transformer and doublesex The presence of Mdmd on different chromosomes indicates that Mdmd translocated to different genomic sites. Thus, an instructive signal in sex determination can arise by duplication and neofunctionalization of an essential splicing regulator (Sharma, 2017).

Sex determination across evolution - connecting the dots: Evolution of sex determination mechanisms

The variety of primary sex determination cues was appreciated long before the advent of molecular genetics. The two broadest categories are genetic sex determination (GSD), in which the sex of offspring is set by a sex chromosome or an autosomal gene, and environmental sex determination (ESD), in which sex is determined by temperature (as with turtles), local sex ratio (as with some tropical fish), or population density (as with mermithid nematodes). Though little is known about the molecular mechanisms of ESD, within the GSD systems many different mechanisms have been uncovered. Dual sex chromosome systems, in which either the female (ZW/ZZ) or the male (XX/XY) is heterogametic, are common, as are systems set by the ratio of the number of X chromosomes to sets of autosomes (X:A). There are also systems in which heterozygosity at a single locus is required for female development (known as complementary sex determination), as well as systems involving sex determination via multiple genes with additive effects (Haag, 2005see full text of article).

Molecular genetic investigations of GSD in model systems such as Drosophila, Caenorhabditis, and mice have revealed a clear lack of conservation, underscoring the diversity. For example, although the primary sex determination signal in both D. melanogaster and C. elegans is the X:A ratio, the fruit fly pathway consists of a cell-autonomous cascade of regulated mRNA splicing, while that of the nematode follows a Hedgehog-like intercellular signaling pathway. GSD in mammals depends (with some interesting exceptions upon a Y-specific dominant gene (Sry) encoding a transcription factor. In the face of such impressive differences, perhaps the assumption of homology should be questioned: could it be that sex determination in different taxa has arisen independently over and over again in evolution? Until 1998, this seemed like a good bet (Haag, 2005).

The discovery of the homology of the key sex-determining genes doublesex in Drosophila and mab-3 in C. elegans provided the first evidence for a common evolutionary basis of sex determination in animals. Soon, related doublesex-mab-3 (DM)-family genes with roles in male sexual development were discovered in vertebrates and even cnidarians. Here at last was a smoking gun that could link the diverse metazoan sex determination systems. But as satisfying as the result was, it immediately gave birth to another mystery: if the enormous diversity of sex determination systems are all derived from a common ancestor, how could they possibly have been modified so radically? After all, sexual differentiation and reproduction are hardly unimportant developmental processes (Haag, 2005).

To understand how such diversity came to be, differences between closely related species must be examined. This approach allows the discovery and interpretation of small-scale sex determination changes before they are obscured by subsequent changes. The processes discovered in this way might then be reasonably extrapolated to explain the seemingly unrelated systems of more deeply diverged taxa. Work in dipterans has revealed three evolutionary phenomena that characterize shorter-term sex determination evolution (Haag, 2005).

The first of these is the often astounding rate of molecular evolution at the level of nucleotide and aminoacid sequences. Although some sex-determining genes are well conserved, many show unprecedented substitution rates. An extreme example is the central integrator of the X:A ratio in Caenorhabditis, xol-1. The xol-1 orthologues of the closely related nematodes C. elegans and C. briggsae are a mere 22% identical, even though genes surrounding xol-1 are much better conserved. Remarkably, the 3′ neighbor of xol-1, the immunoglobulin dim-1, is only 5 kb away and is essentially identical between species (Haag, 2005).

A second phenomenon, best exemplified by dipteran insects, is the modification of genetic control pathways through the gain or loss of key pathway components. In Drosophila, the first gene to respond to the X:A ratio is Sxl, whose transcription is regulated by both autosomal and X-linked factors very early in development. When X: A = 1 (i.e., in female embryos), Sxl transcription occurs and produces Sxl protein. Later in development, transcription from a second promoter occurs in both sexes, but these transcripts cannot be productively spliced without the earlier burst of Sxl expression. As a result, only females sustain Sxl expression, and in turn only females can productively splice the mRNA of tra, its downstream target. Productive splicing of tra is required to produce the female-specific form of dsx, a founding member of the DM family mentioned above (Haag, 2005).

In a series of groundbreaking papers, Saccone and colleagues investigated the pathway in the more distantly related heterogametic Mediterranean fruit fly Ceratitis capitata. The first surprise was that although a highly conserved Sxl homologue exists in Ceratitis, it does not undergo sex-specific regulation similar to that of Drosophila, which suggests that it does not play a key switch role (Saccone, 1998). Similar results have also been found for the housefly, Musca domestica, indicating that the role of Sxl in sex determination may be restricted to Drosophila and its closest relatives. In contrast, tra and dsx are key sex regulators in all dipterans examined thus far (Haag, 2005).

A further surprise came when the Ceratitis tra homologue was characterized. In the case of this gene, clear evidence for sex-specific regulation was found, and as with Drosophila, only females productively splice tra mRNA. However, this splicing difference can be explained nicely by a positive feedback, similar to that seen in Drosophila Sxl, in which Tra protein regulates its own splicing. It has been proposed that the dominant, male-specifying M factor on the Y chromosome inhibits this autoregulation. As a result, males cannot make functional Tra protein, and the male form of Dsx is produced. These experiments show not only how a pathway can evolve, but also, importantly, how X:A and heterogametic GSD systems can be interconverted by modifying the cue that regulates a conserved molecular switch gene (the splicing of tra mRNA) (Haag, 2005).

Finally, recent studies of Caenorhabditis nematodes have shed light on the genetic basis of the convergent evolution of sex determination related to mating system adaptations. An important factor in this area are new phylogenies of the genus, which consistently suggest the surprising possibility that the closely related hermaphroditic species C. elegans and C. briggsae acquired self-fertilization independently, from distinct gonochoristic (male/female) ancestors. Although this scenario is somewhat uncertain purely on parsimony grounds, recent work on the genetic control of the germline bisexuality that defines hermaphroditism has tipped the balance toward parallel evolution (Haag, 2005).

C. elegans fog-2, a gene required for spermatogenesis in hermaphrodites but not in males, has been cloned. It became clear that fog-2 is part of a large family of F-box genes and was produced by several recent rounds of gene duplication. The C. briggsae genome sequence suggested that while C. briggsae possesses a similarly large family of F-box proteins, the duplication event giving rise to fog-2 was specific to the C. elegans lineage. This work has been extended by the rigorous demonstration that fog-2 is indeed absent in C. briggsae. A short, C-terminal domain has been identified that makes FOG-2 uniquely able to perform its germline sex-determining function. This domain is probably derived from a frame-shifting mutation in an ancestral gene. Working with C. briggsae, evidence has been found of important species-specific regulation of germline sex determination. RNA interference and gene knockout approaches have shown that while C. elegans requires the male-promoting genes fem-2 and fem-3 to produce sperm in hermaphrodites, C. briggsae requires neither. Given that both genes have conserved roles in male somatic sex determination, this suggests that C. briggsae evolved hermaphroditism in a way that bypasses these genes (Haag, 2005).

The long-standing mystery of sex determination and its diversity began by comparisons between distantly related species. Recent work on closer relatives has uncovered processes that through a reasonable extrapolation enable the connection of these disparate dots into a fascinating picture of developmental evolution. Though the divergence is extreme, it is likely that a better understanding of the evolution of sex determination genes and pathways holds lessons about the evolution of development in general. The next major challenge will be to integrate the comparative developmental data with the ecological and population processes that are driving the evolution of sex determination. Only then will it be possible to say that the picture is complete (Haag, 2005).


Search PubMed for articles about Drosophila transformer

Acebes, A., et al. (2004). Cholinergic control of synchronized seminal emissions in Drosophila. Curr. Biol. 14: 704-710. 15084286

Amrein, H., Hedley, M. L. and Maniatis, T. (1994). The role of specific protein-RNA and protein-protein interactions in positive and negative control of pre-mRNA splicing by Transformer 2. Cell 76: 735-46.

Arthur, B. I., et al. (1998). Sexual behaviour in Drosophila is irreversibly programmed during a critical period. Curr. Biol. 8(21): 1187-90.

Bell, L. R., Horabin, J. I., Schedl, P. and Cline, T. W. (1991). Positive autoregulation of Sex-lethal by alternative splicing maintains the female determined state in Drosophila. Cell 65: 229-239.

Belote, J. M., McKeown, M., Boggs, R. T., Ohkawa, R. and Sosnowski, B. A. (1989). Molecular genetics of transformer, a genetic switch controlling sexual differentiation in Drosophila. Dev. Genet. 10(3): 143-54. 2472240

Boggs, R. T., Gregor, P., Idriss, S., Belote, J. M. and McKeown, M. (1987). Regulation of sexual differentiation in D. melanogaster via alternative splicing of RNA from the transformer gene. Cell 50: 739-747. 2441872

Burtis, K. C. and Baker, B. S. (1989). Drosophila doublesex gene controls somatic sexual differentiation by producing alternatively spliced mRNAs encoding related sex-specific polypeptides. Cell 56: 997-1010. 2493994

Cline, T. W. (1993). The Drosophila sex determination signal: how do flies count to two? Trends Genet. 9: 385-390. 8310535

Cline, T. W. and Meyer, B. J. (1996). Vive la difference: males vs females in flies vs worms. Annu. Rev. Genet. 30: 637-702. 8982468

DeFalco, T. J., et al. (2003). Sex-specific apoptosis regulates sexual dimorphism in the Drosophila embryonic gonad. Dev. Cell 5: 205-216. 12919673

Deshpande, G., Calhoun, G. and Schedl, P. D. (1999). The N-terminal domain of Sxl protein disrupts Sxl autoregulation in females and promotes female-specific splicing of tra in males. Development 126: 2841-2853. 10357929

Evans, D. S. and Cline, T. W. (2007). Drosophila melanogaster male somatic cells feminized solely by TraF can collaborate with female germ cells to make functional eggs. Genetics 175: 631-642. PubMed ID: 17110478

Evans, D. S. and Cline, T. W. (2013). Drosophila switch gene Sex-lethal can bypass its switch-gene target transformer to regulate aspects of female behavior. Proc Natl Acad Sci U S A. PubMed ID: 24191002

Ferveur, J.-F., et al. (1997). Genetic feminization of pheromones and its behavioral consequences in Drosophila males. Science 276 (5318): 1555-1558.

Finley, K. D., et al. (1997). dissatisfaction, a gene involved in sex-specific behavior and neural development of Drosophila melanogaster. Proc. Natl. Acad. Sci. 94: 913-918.

Fujii, S. and Amrein, H. (2002). Genes expressed in the Drosophila head reveal a role for fat cells in sex-specific physiology. EMBO J. 21(20): 5353-63. 12374736

Haag, E. S. and Doty, A. V. (2005). Sex determination across evolution: connecting the dots. PLoS Biol. 3(1): e21. 15660158

Handa, N., et al. (1999). Structural basis for recognition of the tra mRNA precursor by the Sex-lethal protein. Nature 398(6728): 579-85.

Heinrichs, V., Ryner, L. C. and Baker, B. S. (1998). Regulation of sex-specific selection of fruitless 5' splice sites by transformer and transformer-2. Mol. Cell. Biol. 18(1): 450-458.

Hilfiker, A., et al. (1995). The gene virilizer is required for female-specific splicing controlled by Sxl, the master gene for sexual development in Drosophila. Development 121: 4017-4026. 8575302

Hinson, S. and Nagoshi, R. N. (1999). Regulatory and functional interactions between the somatic sex regulatory gene transformer and the germline genes ovo and ovarian tumor. Development 126: 861-871. 9927588

Horabin, J. I. and Schedl, P. (1996). Splicing of the Drosophila Sex-lethal early transcripts involves exon skipping that is independent of Sex-lethal protein. RNA 2: 1-10. 8846292

Hoshijima, K., Inoue, K., Higuchi, I., Sakamoto, H. and Shimura, Y. (1991). Control of doublesex alternative splicing by transformer and transformer-2 in Drosophila. Science 252: 833-836. 1902987

Inoue, K., Hoshijima, K., Sakamoto, H. and Shimura, Y. (1990). Binding of the Drosophila Sex-lethal gene product to the alternative splice site of transformer primary transcript. Nature 344: 461-463. 1690860

Inoue, K., Hoshijima, K., Higuchi, I., Sakamoto, H. and Shimura, Y. (1992). Binding of the Drosophila Transformer and Transformer-2 proteins to the regulatory elements of doublesex primary transcript for sex-specific RNA processing. Proc. Natl. Acad. Sci. 89(17): 8092-6. 1518835

Kanaar, R., et al. (1995). Interaction of the sex-lethal RNA binding domains with RNA. EMBO J 14: 4530-4539.

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

Keyes, L. N., Cline, T. W. and Schedl, P. (1992). The primary sex determination signal of Drosophila acts at the level of transcription. Cell 68: 933-943. 1547493

Kulathina, R. J., Skwarek, L., Morton, R. A. and Singh, R. S. (2003). Rapid evolution of the sex-determining gene, transformer: Structural diversity and rate heterogeneity among sibling species of Drosophila Mol. Biol. Evol. 20(3): 441-452. 12644565

Lam, B. J., et al. (2003). Enhancer dependent 5' splice site control of fruitless Pre-mRNA splicing. J Biol Chem. 278(25): 22740-7. 12646561

Le Bras, S and Van Doren, X. (2006). Development of the male germline stem cell niche in Drosophila. Dev. Biol. 294(1): 92-103. 16566915

Loh, H. Y. and Russell, S. (2000). A Drosophila group E Sox gene is dynamically expressed in the embryonic alimentary canal. Mech. Dev. 93(1-2): 185-188. 10781954

Lynch, K. W. and Maniatis, T. (1995). Synergistic interactions between two distinct elements of a regulated splicing enhancer. Genes Dev. 9: 284-293. 7867927

Lynch, K. W. and Maniatis, T. (1996). Assembly of specific SR protein complexes on distinct regulatory element of the Drosophila doublesex splicing enhancer. Genes Dev. 10: 2089-2101. 8769651

Manley, J. L. and Tacke, R. (1996). SR proteins and splicing control. Genes Dev. 10: 1569-1579. 8682289

Marin, I. and Baker, S. B. (1998). The evolutionary dynamics of sex determination. Science 281: 1990-1994. 9748152

McAllister, B. F. and McVean, G. A. T. (2000). Neutral evolution of the sex-determining gene transformer in Drosophila. Genetics 154: 1711-1720. 10747064

McKeown, M., Belote, J. M. and Boggs, R. T. (1988). Ectopic expression of the female transformer gene product leads to female differentiation of chromosomally male Drosophila. Cell 53(6): 887-95. 2454747

Nagoshi, R. N., McKeown, M., Burtis, K. C., Belote, J. M. and Baker, B. (1988). The control of alternative splicing at genes regulating sexual differentiation in D. melanogaster. Cell 53: 229-236. 3129196

O'Dell, K. M., et al. (1995). Functional dissection of the Drosophila mushroom bodies by selective feminization of genetically defined subcompartments. Neuron 15: 55-61.

Oliver, B., et al. (1994). Function of Drosophila ovo+ in germ-line sex determination depends on X-chromosome number. Development 120: 3185-3195.

O’Neil, M. T. and Belote, J. M. (1992). Interspecific comparison of the transformer gene of Drosophila reveals an unsually high degree of evolutionary divergence. Genetics 131: 113-128. 1592233

Pane, A., Salvemini, M., Delli Bovi, P., Polito, C. and Saccone, G. (2002). The transformer gene in Ceratitis capitata provides a genetic basis for selecting and remembering the sexual fate. Development 129(15): 3715-25. 12117820

Pane, A., et al. (2005). Evolutionary conservation of Ceratitis capitata transformer gene function. Genetics 171(2): 615-24. 15998727

Parkhurst, S. M., Bopp, D. and Ish-Horowicz, D. (1990). X:A ratio, the primary sex-determining signal in Drosophila, is transduced by helix-loop-helix proteins. Cell 63: 1179-1191. 2124516

Penalva, L. O. F., et al. (2000). The Drosophila fl(2)d gene, required for female-specific splicing of Sxl and tra pre-mRNAs, encodes a novel nuclear protein with a HQ-rich romain. Genetics 155: 129-139.

Ryner, L. C., et al. (1996). Control of male sexual behavior and sexual orientation in Drosophila by the fruitless gene. Cell 87: 1079-1089. 8978612

Saccone, G., et al. (1998). The Ceratitis capitata homologue of the Drosophila sex-determining gene Sex-lethal is structurally conserved, but not sex-specifically regulated. Development125(8): 1495-500. 9502730

Saccone, G., Pane, A., Testa, G., Santoro, M., de Martino, G., di Paola, F., Louis, C. and Polito, L. C. (2000). Sex determination in medfly: a molecular approach. Area-wide control of fruitflies and other pest insects (ed. K.-H. Tan), pp. 491-496. Penang: Penerbit USM.

Saccone, G., Pane, A. and Polito, L. C. (2002). Sex determination in flies, fruitflies and butterflies. Genetica 116(1): 15-23. 12484523

Savarit, F. and Ferveur, J. F. (2002). Genetic study of the production of sexually dimorphic cuticular hydrocarbons in relation with the sex-determination gene transformer in Drosophila melanogaster. Genet Res. 79(1): 23-40. 11974601

Schutt, C. and Nothiger, R. (2000). Structure, function and evolution of sex determining systems in Dipteran insects. Development 127: 667-677. 10648226

Sharma, A., Heinze, S. D., Wu, Y., Kohlbrenner, T., Morilla, I., Brunner, C., Wimmer, E. A., van de Zande, L., Robinson, M. D., Beukeboom, L. W. and Bopp, D. (2017). Male sex in houseflies is determined by Mdmd, a paralog of the generic splice factor gene CWC22. Science 356(6338): 642-645. PubMed ID: 28495751

Sosnowski, B. A., Belote, J. M. and McKeown, M. (1989). Sex-specific alternative splicing of RNA from the transformer gene results from sequence-dependent splice site blockage. Cell 58: 449-459. 2503251

Sosnowski, B. A., et al. (1994). Multiple portions of a small region of the Drosophila transformer gene are required for efficient in vivo sex-specific regulated RNA splicing and in vitro Sex-lethal binding. Dev. Biol. 161: 302-12.

Steinmann-Zwicky, M., et al. (1994). Sex determination of the Drosophila germ line: tra and dsx control somatic inductive signals. Development 120: 707-16.

Tian, M. and Maniatis, T. (1993). A splicing enhancer complex controls alternative splicing of doublesex pre-mRNA. Cell 16: 105-114. 8334698

Valcárcel, J., Singh, R., Zamore, P. D. and Green, M. R. (1993). The protein Sex-lethal antagonizes the splicing factor U2AF to regulate alternative splicing of transformer pre-mRNA. Nature 362, 171-175. 7680770

Villella, A., Ferri, S. L., Krystal, J. D. and Hall, J. C. (2005). Functional analysis of fruitless gene expression by transgenic manipulations of Drosophila courtship. Proc. Natl. Acad. Sci. 102(46): 16550-16557. 16179386

Waterbury, J. A., et al. (2000). Sex determination in the Drosophila germline is dictated by the sexual identity of the surrounding soma. Genetics 155(4): 1741-56.

Walthour, C. S. and Schaeffer, S. W. (1994). Molecular population genetics of sex determination genes: The transformer Gene of Drosophila melanogaster. Genetics 136: 1367-1372. 8013913

Willhoeft, U. and Franz, G. (1996). Identification of the sex-determining region of the Ceratitis capitata Y chromosome by deletion mapping. Genetics 144: 737-745. 8889534

Wu, J. Y. and Maniatis, T. (1993). Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell 75: 1061-70.

Yamamoto, D., Fujitani, K., Usui, K., Ito, H. and Nakano, Y. (1998). From behavior to development: genes for sexual behavior define the neuronal sexual switch in Drosophila. Mech. Dev. 73(2): 135-146.

Yanowitz, J. L., et al. (1999). An N-terminal truncation uncouples the sex-transforming and dosage compensation functions of Sex-lethal. Mol. Cell. Biol. 19: 3018-3028.

Zhu, C., Urano, J. and Bell, L. R. (1997). The Sex-lethal early splicing pattern uses a default mechanism dependent on the alternative 5' splice sites. Mol. Cell. Biol. 17: 1674-1681. 9032294

Zuo, P. and Maniatis, T. (1996). The splicing factor U2AF35 mediates critical protein-protein interactions in constitutive and enhancer-dependent splicing. Genes Dev. 10: 1356-1368. 8647433

Biological Overview

date revised: 12 January 2018

Home page: The Interactive Fly © 2017 Thomas Brody, Ph.D.