Transformer: a protein that interacts with Transformer 2:
Part 4) Downstream action of Transformer protein - Effects on germ line cells
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).
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).
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).
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).
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).
Transformer: a protein that interacts with Transformer 2:
Part 5) Population and species variation of Transformer sequence
This paper reports the results from an evolutionary approach to investigate tra regulation and function, by isolating the tra-homologous genes from selected Drosophila species, and then using the interspecific 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).
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 both synonymous and nonsynonymous sites. In this study, the hypothesis that the tra gene will be polymorphic in synonymous and nonsynonymous sites within species is tested 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 fail to reject a neutral model of molecular evolution because of low statistical power associated with low levels of genetic variation (Tajima/Fu and Li). The Hudson, Kreitman, and Aguade test rejects a neutral model when the tra region is 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).
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