ovarian tumor

REGULATION (part 2/2)

Otu in germ-line sex determination

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, 1999a 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, 1999a 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, 1999a 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, 1999a 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, 1999a).

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, 1999a).

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, 1999a).

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, 1999a).

The ovo and ovarian tumor genes are required during early and late stages of Drosophila oogenesis. The ovo product, a zinc-finger transcription factor, can bind to sites and influence the level of expression of the ovarian tumor promoter. An examination of ovo null mutant organelles demonstrates that ovo is required for the differentiation of XX germ cells during larval gonial stages, in addition to its known role in maintaining germ cell numbers. In contrast, ovarian tumor is required during pupal and adult stages for the cystocyte divisions that give rise to the egg chamber. Studies on sexually transformed flies indicate that both the ovo and ovarian tumor null mutant phenotypes are distinctive from and more severe than the germline defects produced when male germ cells develop in female soma. This suggests that ovo and ovarian tumor have oogenic functions other than their putative role in germline sex determination. The regulation of ovarian tumor by ovo is stage-specific, because ovarian tumor promoter activity does not require ovo during larval stages; rather, it becomes ovo-dependent in the adult ovary. This coincides with the time when the ovarian tumor promoter becomes responsive to sex-specific signals from the soma, suggesting a convergence of somatic and germline regulatory pathways on ovarian tumor during oogenesis (Hinson, 1999b).

XX germ cells developing in a male soma produce spermatogenic fusomes. This indicates at least partial sexual transformation of the XX germline can occur due to sex-specific somatic interactions. In this study, the reciprocal condition was investigate; it was asked whether the presence of female soma could alter the sexual differentiation of XY germ cells. XY flies were induced to undergo female somatic differentiation by the use of hs-tra, a transgenic construct in which the somatic sex determination gene transformer (tra) is induced by the hsp-70 promoter. tra has no known function in the germline, so it is unlikely that hs-tra expression directly affects the differentiation of germ cells. XY;hs-tra/1 pseudofemales are somatically indistinguishable from XX females, and can support the maturation of transplanted XX germ cells. In contrast, the endogenous pseudofemale XY germline remains immature, producing tumorous egg chambers that superficially resemble those found in certain otu mutants. Surprisingly little is known about the differentiated state of the pseudofemale germ cells, such as (1) when development is aborted; (2) how sexually dimorphic structures are affected, or (3) their sexual identity (Hinson, 1999b).

To define the extent of gametogenic differentiation, the composition of early germline-specific structures were determined by immunohistochemical analyses. All XY pseudo female egg chambers examined contained germ cell clusters with spectrosomes or fusomes. The most mature germ cells were interconnected by multibranched fusomes (polyfusomes) containing f-actin, a phenotype suggestive of spermatogenic differentiation. More aberrant germ cells were also found in small (2±3 cell) clusters connected by short, poorly branched fusomes. The retention of f-actin in this subset was variable, indicating an early defect in the spectrosome-to-fusome transition that occurs at the first cystocyte division. Aberrations were also found in the composition of the ring canals. During normal oogenesis, Hu li tai shao (HTS-RC) becomes localized in the rings prior to the localization of f-actin and concomitantly with the disappearance of the fusome. In XY pseudofemales, it was found that 40% of the tumorous chambers examined by phalloidin displayed germ cell clusters with ring canals containing f-actin, but in no case was HTS-RC deposition found. Furthermore, f-actin incorporation in the ring canals occurred while the fusome was still present, a situation not seen in wildtype oogenesis. These results define a complex and variable set of germline characteristics. The XY germ cells typically initiate gametogenic differentiation (to produce spectrosomes and fusomes), but are arrested shortly thereafter, before the differentiation of more mature (post-germarial) stages. A subset of these cells appear to have initiated spermatogenic development, containing male-like polyfusomes consistent with their XY genotype. The remainder were more abnormal, with short fusomes and unusual ring canals. It has been concluded that the presence of female soma disrupts XY gametogenesis during the cystocyte divisions, but does not appear capable of inducing oogenic differentiation (Hinson, 1999b).

Beginning in larval gonads, null ovo mutants show reduced numbers of XX germ cells, suggesting either reduced viability or proliferation. Surprisingly little is known about how the absence of ovo affects female germ cell differentiation. This is of particular interest given the proposal that mutations in ovo, a putative germline sex determination gene, cause the male transformation of XX germ cells. In this case, one might expect ovo null mutants to display spermatogenic characteristics similar to those seen in XY pseudofemales. Although ovo null mutant XX germ cells are mostly absent in adult ovaries some can perdure to the adult stage and even form egg chambers, providing the opportunity for their morphological examination (Hinson, 1999b).

Germ cells mutant for a null ovo allele were examined using antibodies specific for spectrin and Vasa, a germline-specific protein. No egg chambers were found, but 20% of the ovary lobes contained one or more clusters of Vasa-positive germ cells. None contained either spectrosomes or fusomes although spectrin was present along the cell periphery. Similar results were obtained in a separate, larger experiment using otu-lacZ as the germline marker. In this construct, the bacterial lacZ gene is driven by the germline-specific otu promoter. Out of 36 ovo null mutant lobes examined, 81% had no germ cells, while the remaining seven gonads contained a total of 30 germ cell clusters. The great majority of the clusters contained germ cells with no spectrosomes or fusomes. However, in three ovaries a total of seven small germ cell clusters with spectrosomes or fusomes were found; this is indicative of gametogenic differentiation. In these exceptional cases, the fusomes observed were small and poorly branched, indicating aberrant and aborted differentiation early in gametogenesis. It has been concluded that in the absence of ovo, the great majority of germ cells abort during larval oogonial stages, affecting both the number of germ cells perduring to the adult stage and their ability to undergo gametogenic differentiation. Therefore, ovo null mutant XX germ cells are arrested at an earlier stage than the XY pseudofemale germline, but show no evidence of spermatogenic differentiation (Hinson, 1999b).

Morphological comparison of ovo and otu null phenotypes indicates that ovo is first required prior to otu in female germline development. This timing is consistent with suggestions that ovo regulates otu promoter activity, as indicated by the finding that dominant, antimorphic ovo alleles can reduce otu expression. However, this observation contradicts earlier findings that the otu promoter is active in the absence of ovo function, since otulacZ is expressed in ovo null mutant germ cells. A series of experiments was carried out to clarify this regulatory interaction between ovo and otu. otu can indeed be expressed in the absence of ovo. The pOtu-HA construct has otu tagged with the HA-epitope and expressed from the otu promoter. One copy of pOtuHA is capable of rescuing otu null alleles to fertility, indicating that it is expressed at all necessary oogenic periods, and displays the expected pattern of otu protein distribution in the wildtype germline. In ovo null mutants, the majority of germ cells expressed substantial levels of the OTU-HA protein, confirming the otu-lacZ studies. A different result was observed with hypomorphic ovo allele combinations that abort oogenesis during later, postgermarial stages. In these cases the otu-lacZ transgene had greatly reduced levels of expression, even in relatively mature germ cells that had differentiated into nurse cells and oocytes. The level of otu-lacZ expression is dependent on the severity of the ovo allele combination. It seems unlikely that the block in otu promoter expression by hypomorphic ovo alleles is an indirect consequence of abnormal oogenic development. Instead, it is thought that upon the development of the adult ovary, there is a significant change in the regulation of otu transcription such that it now becomes substantially dependent on positive regulation by ovo. These findings seem at odds with the observation that in the absence of otu, female germ cells are arrested during germarial stages. If the hypomorphic ovo alleles block the great majority of otu gene activity in adult gonads, how can the germ cells differentiate to vitellogenic stages? Despite the ovo mutation, the Otu-HA fusion protein was detected during all oogenic stages. Therefore, otu promoter activity in this mutant is sufficient to allow the accumulation of otu protein (Hinson, 1999b).

These experiments on XY pseudofemales make several contributions toward understanding how the soma and germline interact to regulate gametogenesis. (1) It was found that the presence of female soma causes XY germ cells to abort development during the cystocyte divisions, the same period at which XX germ cells arrest when developing in male soma. This demonstrates a common need for sex-specific somatic interactions during the period of fusome and ring canal morphogenesis in both sexes. (2) The presence of female soma does not promote oogenic fusome differentiation in XY germ cells. In comparison, male soma is able to induce a subset XX germ cells to produce spermatogenic polyfusomes. This suggests possible differences in how male and female soma can influence the determination of sexual identity in the germline. (3) The presence of female soma has an unexpected effect on the ring canals of XY germ cells, producing a phenotype never observed in other genotypes. A number of pseudofemale ring canals contained f-actin, but not HTS-RC. This is particularly unusual because HTS-RC is normally deposited before f-actin during oogenesis. Therefore, the sexual incompatibility between the germline and soma seems to have a specific effect on the deposition of f-actin in the maturing ring canal. Interestingly, this correlates with observations that otu is itself regulated by somatic interactions, and is required for f-actin deposition in the ring canals. (4) The pseudofemale studies also identified the phenotype to be expected when male germ cells develop in female soma. This provided an important comparison for the examination of the ovo null phenotype and the proposal that ovo is required for germline sex determination. The hypothesis predicts that XX germ cells lacking ovo function will take on a male identity, and therefore should display some spermatogenic characteristics. In contrast, it was found that those ovo null germ cells perduring in adult ovaries typically lacked either spectrosomes, fusomes, and/or ring canals. This indicates arrest during the larval oogonial stages, the same period when ovo null mutants first show reductions in germ cell numbers. Such a developmental block could readily give rise to the adult ovo null agametic phenotype. For example, the oogonia are found in a period of proliferation, so an arrest at this time would markedly reduce germ cell numbers. In addition, the immature mutant germ cells might fail to associate normally with the somatic ovary, since it differentiates during late larval and pupal periods. Therefore, the ovo null phenotype could be explained by ovo being required simply for early oogonial differentiation, without having to invoke a role in germline sex determination. A later arrest is observed in otu null mutants, where oogenesis is aborted during the first cystocyte division. Therefore, otu is initially required after the period when ovo first acts. This temporal ordering is consistent with recent evidence that ovo controls otu transcription. These experiments demonstrate that this regulatory interaction is a complicated one. During larval stages, the otu promoter is expressed in both male and female gonads and does not appear to require ovo, although the possibility of regulation by maternally contributed ovo product cannot be precluded. It is only in the adult gonad that otu promoter activity becomes dependent on zygotic ovo expression. Coincidentally, the adult stage also defines two other changes in otu regulation. It is only in adults that the otu promoter exhibits female-specific expression and dependence on sex-specific somatic interactions. In fact, the inhibitory effects of male soma on oogenesis are due primarily to insufficient otu expression. Therefore, the formation of the adult ovary correlates with a change in otu regulation such that it now becomes the target of both ovo- and soma-dependent inputs. In this way, otu may serve to coordinate the development of germline and somatic components of the egg chamber (Hinson, 1999b).

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