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).
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).
The SR proteins represent a family of splicing factors, several of which have been implicated in the regulation of sex-specific alternative splicing of DSX pre-mRNA. Two RNA target sequence motifs recognized by the SR protein RBP1 have been identified. Several copies of these RBP1 target sequences are found within two regions of the DSX pre-mRNA that are important for the regulation of DSX alternative splicing: these areas are the repeat region and the purine-rich polypyrimidine tract of the regulated female-specific 3' splice site. RBP1 target sequences within the DSX repeat region are required for the efficient splicing of DSX pre-mRNA. RBP1 contributes to the activation of female-specific DSX splicing in vivo by recognizing the RBP1 target sequences within the purine-rich polypyrimidine tract of the female-specific 3' splice site (Heinrichs, 1995).
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).
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).
Transformer: a protein that interacts with Transformer 2:
Part 1) Splicing of Transformer mRNA
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).
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).
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).
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).
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).
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).
Transformer: a protein that interacts with Transformer 2:
Part 2) Downstream action of Transformer protein
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).
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).
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).
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 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).
Transformer: a protein that interacts with Transformer 2:
Part 3) Sex-specific apoptosis, functioning downstream of transformer, regulates sexual dimorphism in the Drosophila embryonic gonad
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).
Transformer: a protein that interacts with Transformer 2:
Part 4) Downstream action of Transformer protein - Effects on germ line cells and Part 5) Population and species variation of Transformer sequence
Continued: Transformer 2 Protein interactions part 2/2
transformer 2: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
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