ovarian tumor

DEVELOPMENTAL BIOLOGY

Adult

The 4.1 kb mRNA encoding the 104 kDa isoform is expressed throughout adult oogenesis, but is mainly restricted to nurse cells. The 3.2 kb mRNA encoding the 98 kDa protein isoform is selectively localized in the oocyte up to stage 9. Both mRNAs are expressed abundantly in nurse cells at stages 10-11 (Tirronen, 1995).

Otu protein is present in the cytoplasm of cystocytes, nurse cells and oocytes, but no Otu protein is detected in nuclei or follicle cells. Intense immunostaining is present in germaria of wild type ovaries, and staining intensity remains constant or decreases slightly through stage 4. From stage 5 or 6, staining intensity increases steadily, to reach a plateau at stages 9-10B. Staining is gradually lost in nurse cells and follows the degeneration of these cells. In contrast, Otu protein in oocytes is degraded beyond detectability during stage 11, a stage that lasts only 0.4 hours. Staining is severely reduced in the DIF mutant otu14, while two ONC mutants, otu11 and otu13, display considerable staining. ONC mutants exhibit more intense staining in differentiated chambers than in tumorous chambers (Steinhauer, 1992).

The Drosophila melanogaster ovarian tumor (otu) gene, required for normal proliferation and differentiation of the female germ-line, encodes two cytoplasmic protein isoforms: one 98kDa and the other,104 kDa. Mutants with defects in this gene are typically grouped into one of three phenotypic classes: quiescent (germ cells that do not proliferate), oncogenic or tumorous (germ-line cells proliferate uncontrollably), and differentiated (germ-line cells initiate but do not complete differentiation). Analysis of transformants expressing only one of the otu isoforms shows that the 104-kDa isoform (otu-104) can rescue all classes of otu mutants, whereas only differentiated mutants are rescued to a significant extent by the 98-kDa isoform (otu-98). Western analysis of protein extracts prepared from ovaries of various developmental stages indicates that otu-104 predominates in predifferentiated stages, while otu-98 is prevalent in differentiated egg chambers. Immunolocalization experiments demonstrate that Otu protein is present in the cytoplasm of oogonial stem cells, which populate third instar larvae. Otu is also present in all germ-line-derived cells until late in oogenesis. In stage 10 egg chambers, Otu protein shifts to the subcortical region of nurse cells. This type of analysis also shows that upon formation of a 16-cell syncytium, otu-104 (but not otu-98) preferentially accumulates in the developing oocyte cytoplasm. The Otu mutant protein does not show this pattern of enhanced accumulation, nor does it occur in ovaries of egalitarian and Bicaudal-D mutants, which are defective in oocyte determination. Thus, these studies indicate that the 104-kDa isoform is required for normal proliferation of female germline cells and perhaps for oocyte differentiation. The 98-kDa isoform appears to be dispensable but can provide an otu function needed for the completion of oocyte maturation (Sass, 1995).

The Drosophila ovarian tumor (otu) gene encodes two novel protein isoforms that are required at multiple stages of oogenesis. The activity of a set of C-terminal truncation Otu proteins has been examined as well as a GFP-tagged Otu (Otu-GFP). These experiments have shown that a putative Tudor domain in the central region of the large Otu isoform and a separate domain in the C-terminal region are required for regulation of cyst formation and oocyte maturation, respectively. Evidence is presented that a portion of Otu co-fractionates with mRNA/protein complexes (mRNPs) and Otu-GFP has been shown to associate with cytoplasmic aggregates at the periphery of the nucleus at an intermediate stage of oogenesis. This study substantially clarifies the relationship between Otu structure and function and reveals new clues about interacting components (Glenn, 2001).

The otu gene encodes 98 and 104 kDa protein isoforms (811 and 853 amino acids, respectively) that are generated from alternatively spliced transcripts. The 104 kDa Otu isoform, when expressed under the control of the endogenous otu promoter, can perform all Otu functions, whereas the smaller isoform can perform only the late function. Otu is found in the cytoplasm of oogonial stem cells of third instar larvae and in developing egg chambers until stage 10. During the pre-vitellogenic stages of oogenesis large isoform is enriched in the oocyte. The otu promoter is active at the distal tip of the testis; however, this gene is not required for differentiation of male germ cells (Glenn, 2001 and references therein).

By expressing C-terminally truncated derivatives of Otu at high levels, it has been definitively shown that the early and late Otu functions depend on separable domains of the protein. The cumulative data are consistent with the model that the early Otu function in controlling cyst formation and differentiation requires an Otu polypeptide that includes amino acids extending from the N-terminal region to the central region of the large isoform. However, the late function in controlling nurse cell dumping and oocyte maturation depends on a polypeptide that includes the N-terminal and C-terminal regions, but not the central region (Glenn, 2001).

The following evidence supports this model. (1) It is consistent with functional analysis of various otu transgenes in the background of different otu mutant alleles. Amino acids 1-423 are sufficient for the early function of Otu, and amino acids downstream of this region, i.e. aa 697-853, are required for its late function. Furthermore the 98 kDa Otu isoform, which contains the N-terminal and C-terminal amino acids but is missing 42 amino acids in the central region, can perform the late Otu function, but not the early function. (2) The model is consistent with the developmentally regulated alternative splicing pattern of otu pre-mRNA transcripts. During the early stages of oogenesis, otu transcripts are spliced to generate the large Otu isoform, which includes the central domain; a shift in the splicing pattern occurs during the later stages to predominantly produce the small Otu isoform, which excludes this domain. (3) The model is consistent with genetic analysis of EMS-induced alleles. These studies show that otu mutations that affect production of the large isoform (otu¹¹ and otu¹³) disrupt the early Otu function, whereas mutations that cause premature truncation in the C-terminal region (otu⁵ and otu¹⁴) disrupt the late function. Furthermore, mutations in one of these groups partially complement mutations in the other. Thus, the central domain of the large isoform and the C-terminal domain(s) need not be present on the same polypeptide. However, the large Otu isoform, which contains both regions, is capable of performing both the early and the late Otu functions (Glenn, 2001).

Several differences are observed between the Otu-GFP fluorescence pattern and the Otu distribution visualized by indirect immunofluroscence. Since the Otu-GFP is fully functional, presumably, these differences are biologically relevant. The absence of a fluorescence signal from Otu-GFP in region 2 of the germarium and at the nurse cell/follicle cell border at stage 10 may indicate that previous immunofluorescence results were misleading, i.e. fixation of ovarian tissue for indirect immunofluorescence created artifacts. Alternatively, it is possible that Otu-GFP fluorescence signal is masked during these stages due to unique interactions that occur at those stages. The presence of Otu-GFP in perinuclear aggregates at stages 6-9 could possibly be related to the association of Otu with mRNPs emerging from the nucleus. The perinuclear distribution might also be related to the function of Otu in promoting formation of cytoplasmic actin bundles, which extend from the nuclear to the plasma membranes. Additional work will be needed to sort out these possibilities (Glenn, 2001).

Effects of Mutation or Deletion

The ovarian tumor gene behaves as if it encodes a product that is required during several early steps in the transformation of oogonia into functional oocytes. Seventeen ethyl methane sulfonate-induced mutations have been studied; their mutant phenotypes can be explained as graded responses by individual germ cells to different levels of Otu synthesized by the mutant germ cells themselves. The lowest and highest levels of Otu appear to be produced by otu10 and otu14, respectively. The 15 mutants with intermediate Otu levels are temperature sensitive: subnormal temperatures improve ovarian development, while above-normal temperatures suppress it. A subgroup of these mutants is unable to form a system of actin microfilament bundles in the cortical cytoplasm of the nurse cells during stage 10B; these defective nurse cells are unable to transport their cytoplasm to the oocyte, as normally happens between stages 10B and 12. In addition to its role in the actin-mediated transport of nurse cell cytoplasm, Otu also appears to alter the morphology of giant polytene chromosomes, which form as the nurse cells undergo endocycles of DNA replication. Genetic evidence suggests that otu also encodes a second product (SP) that is utilized late in oogenesis. SP is required for the synthesis in the ooplasm of glycogen-rich, beta yolk spheres. Products of the otu gene also play a vital but unknown role in embryogenesis (Storto, 1988).

The ovarian tumor gene is required during both the early and late stages of oogenesis. Mutations produce a range of phenotypes, including agametic ovarioles, tumorous egg chambers, and late stage oogenic arrest. Each of these phenotypes is associated with specific aberrations in actin distribution. In the earliest case, otu mutations cause actin filaments to accumulate ectopically in the fusome. This correlates with abnormal fusome morphology and arrested germ cell development in the germaria. Similarly, ovarian tumor function is required for the localization of actin, which is essential for the maturation of ring canals. This defect gives rise to tumorous egg chambers in which germ cell numbers and morphology are profoundly aberrant. ovarian tumor is required for the formation of the nurse cell cytoplasmic actin array that is essential for the nonspecific transport of cytoplasmic contents to the oocyte during late oogenesis. These data suggest that at this stage ovarian tumor controls the site where actin filaments initiate. Taken together, these studies suggest that the diverse ovarian tumor mutant phenotypes derive from the mislocalization of actin filaments, indicating a role for this gene in organizing the female germline cytoskeleton, and that the misregulation of actin can have profound effects on germ cell division and differentiation (Rodesch, 1997).

The ovarian tumor gene is required in germ-line cells for cyst formation, nurse cell chromosome structure and egg maturation. A gene has been analyzed, fs(2)cup, that participates in many of the same processes and interacts with otu genetically. Both nurse cell and oocyte chromosomes require cup to attain a normal morphology. The cupgene is needed for the oocyte to grow normally by taking up materials transported from the nurse cells. cup encodes a 1132-amino-acid protein containing a putative membrane-spanning domain. Cup protein (but not CUP mRNA) is transported selectively into the oocyte in germarial cysts, like the p104 Otu protein. It is strongly associated with large structures in the cytoplasm and the perinuclear region of nurse cells; like Otu, Cup moves to the periphery of these cells in stages 9-10. Moreover, cup mutations dominantly disrupt meiotic chromosome segregation. It is proposed that cup, otu and another interacting gene, fs(2)B, take part in a common cytoplasmic pathway with multiple functions during oogenesis (Keyes, 1997).

Severe alleles of the ovarian tumor (otu) and ovo genes result in female sterility in Drosophila melanogaster, producing adult ovaries that completely lack egg chambers. ovo- or otu- XX embryonic germ cells are indistinguishable in number and morphology from those present in wild-type siblings. The effects of the mutations are not consistently manifested in the female germline until pupariation, and there was no evidence that either gene is required for germ cell viability at earlier stages of development. The requirement for otu function in the pupal and adult ovary is supported by temperature-shift experiments using a heat-inducible otu gene construct. otu activity limited to prepupal stages is not sufficient to support oogenesis, while induction during the pupal and adult periods causes suppression of the otu mutant phenotype (Rodesch, 1995).

Flies mutant for the most severe otu alleles and carrying a heat shock inducible 98 kD isoform have relatively large ovaries containing many tumorous egg chambers. After prolonged heat treatment, occasional cysts are found with nurse-like cells that manifest an extreme polytene phentoype. These results demonstrate that the induced expression of the Otu 98 kD function is sufficient to allow XX germ cell proliferation and the formation of egg chambers, but cannot consistently support the differentiation of the germ cells into later oogenic stages (Rodesch, 1995)

Gametogenesis in Drosophila requires sex-specific interactions between the soma and germline to control germ cell viability, proliferation, and differentiation. To determine what genetic components are involved in this interaction, an examination was carried out to see ifchanges in the sexual identity of the soma affect the function of the ovarian tumor and ovo genes. These genes are required cell autonomously in the female germline for germ cell proliferation and differentiation. XY germ cells do not require otu when developing in the testis, but become dependent on otu function for proliferation when placed in an ovary. This soma-induced requirement can be satisfied by the induced expression of the 98 x 10(3) M(r) OTU product, one of two isoforms produced by differential RNA splicing. These results indicate that the female somatic gonad can induce XY germ cells to become 'female-like' because they require an oogenesis-specific gene. In contrast, the requirement for ovo is dependent on a cell autonomous signal derived from the X:A ratio. It is proposed that differential regulation of the otu and ovo genes provides a mechanism for the female germline to incorporate both somatic and cell autonomous inputs required for oogenesis (Nagoshi, 1995).

Mutations in the ovarian tumor gene arrest oogenesis at several stages in development. A series of deletion mutations in the otu region were characterized, each of which causes the absence or reduction of the otu transcript. These alleles range from the most severe class, which results in ovaries lacking egg cysts, to relatively mild mutations that allow the development of late stage oocytes. Heteroallelic combinations of these mutations demonstrate that the phenotypic complexity of otu mutant ovaries is due to a dosage dependent requirement for otu activity. Reciprocal cross and developmental Northern blot studies suggest a maternal requirement for otu in the development of the female germline. The otu zygotic null phenotype is variable, ranging from the absence of cysts in the most extreme cases, to the presence of tumorous egg chambers (Geyer, 1993).

Mutations caused by insertion and deletion of P elements at the otu gene have been examined. The P element insertion sites are upstream of the major otu transcription start sites. In deletion derivatives, the P element, regulatory regions and/or protein coding sequences have been removed. In both insertion and deletion mutants, the level of otu expression correlates directly with the severity of the phenotype: the absence of otu function produces the most severe quiescent (QUI) phenotype while the oncogenic (ONC) mutants express lower levels of otu than those which are classified as differentiating (DIF). The results of this study demonstrate that the diverse mutant phenotypes of otu are the consequence of different levels of otu function (Sas, 1993).

Otu and sex determination

Certain female-sterile mutations in Drosophila result in the uncontrolled proliferation of X/X germ cells. It has been proposed that this ovarian tumor phenotype results from the sexual transformation of X/X germ cells to a male identity. Findings are presented that are inconsistent with this model. The tumorous cells produced by mutations in the ovarian tumor, Sex-lethal (Sxl) and sans fille (snf) genes are capable of female-specific transcription and RNA processing. This indicates that these ovarian tumor cells still retain some female identity. It is proposed that mutations in these genes do not cause a male transformation of the X/X germ line, but instead, cause either an ambiguous sexual identity or block specific stages of oogenesis. These findings indicate that while Sxl is the master sex determination gene in somatic cells, it appears to play a more subsidiary role in the germ line. The germ line function of Sxl depends on the activity of a specific OTU isoform (Bae, 1994).

The possible role of otu in the determination of the sexual identity of germ cells has not been extensively explored. Some otu alleles produce a phenotype known as ovarian tumorss: ovarioles are filled with numerous poorly differentiated germ cells. These mutant germ cells have a morphology similar to primary spermatocytes; they express male germ line-specific reporter genes. This indicates that they are engaged along the male pathway of germ line differentiation. Consistent with this conclusion, it has been found that the splicing of Sex-lethal (Sxl) pre-mRNAs occurs in the male-specific mode in otu-transformed germ cells. The position of the otu locus in the regulatory cascade of germ line sex determination has been studied by using mutations that constitutively express the feminizing activity of the Sxl gene. The sexual transformation of the germ cells observed with several combinations of otu alleles can be reversed by constitutive expression of Sxl. This shows that otu acts upstream of Sxl in the process of germ line sex determination. Other phenotypes of otu mutations are not rescued by constitutive expression of Sxl, suggesting that several functions of otu are likely to be independent of sex determination. The gene dosage of otu modifies the phenotype of ovaries heterozygous for the dominant alleles of ovo, another gene involved in germ line sex determination. One dose of otu+ enhances the ovoD ovarian phenotypes, while three doses partially suppress these phenotypes. Synergistic interaction between ovoD1 and otu alleles leads to the occasional transformation of chromosomally female germ cells into early spermatocytes. These interactions are similar to those observed between ovoD and one allele of the sans fille (snf) locus. Altogether, these results imply that the otu locus acts, along with ovo, snf, and Sxl, in a pathway (or parallel pathways) required for proper sex determination of the female germ line (Pauli, 1993).

A soma-to-germline signaling pathway that requires the activity of the cut gene has been identified. In the ovary, CUT mRNA and protein expression are restricted to the follicle cells; moreover, cut mutant germline clones are phenotypically normal. When cut function is lost in the follicle cells, however, germline-derived cysts are mispackaged into egg chambers with abnormal numbers of cells, and the structural organization of oocyte-nurse cell complexes disintegrates, generating binucleate germline-derived cells. To date, cut is the only gene known to be required in the follicle cells that when mutated results in binucleate cells. The assembly of egg chambers and the maintenance of germline cell morphology therefore requires the activity of the cut gene in the soma, revealing a signaling pathway that influences the morphology and function of the germline-derived cells. In support of this conclusion, cut interacts genetically during oogenesis with two genes that influence intercellular communication, Notch and Pka-C1 (Jackson, 1999).

To understand the mechanism by which cut expression influences germline cell morphology, it had to be determined whether binucleate cells form by defective cytokinesis or by fusion of adjacent cells. Egg chambers produced by cut, cappuccino, and chickadee mutants contain binucleate cells in which ring canal remnants stain with antibodies against Hu li tai shao and Kelch, two proteins that are added to ring canals after cytokinesis is complete. In addition, defects in egg chamber morphology are observed only in middle to late stages of oogenesis, suggesting that germline cell cytokineses are normal in these mutants. The evidence suggests therefore that binucleate cells are generated by cell fusion. cut exhibits dose-sensitive genetic interactions with cappuccino but not with chickadee or other genes that regulate cytoskeletal function, including armadillo, spaghetti squash, quail, spire, Src64B, and Tec29A. Genomic regions containing genes that cooperate with cut were identified by performing a second-site noncomplementing screen, using a collection of chromosomal deficiencies. Sixteen regions that interact with cut during oogenesis and eight regions that interact during the development of other tissues were identified. Genetic interactions between cut and the ovarian tumor gene were identified as a result of the screen. In addition, the gene agnostic(agu) was found to be required during oogenesis, and genetic interactions between cut and agnostic were revealed. These results demonstrate that a signaling pathway regulating the morphology of germline cells is sensitive to genetic doses of cut and the genes cappuccino, ovarian tumor, and agnostic. Since these genes regulate cytoskeletal function and cAMP metabolism, the cut-mediated pathway functionally links these elements to preserve the cytoarchitecture of the germline cells (Jackson, 1999).

Because cut is expressed and required only in the follicle cells, its influence on germline cell morphology must be mediated across the soma-germline boundary. The results demonstrate that capu and otu, which are both required in the germline, interact genetically with cut and may facilitate cut-mediated events originating in the soma. Although cut is a transcription factor, the clear separation of cell types in which these genes are expressed suggests that cut does not regulate the transcription of capu or otu directly by binding to their promoters and/or enhancers. Rather, a multistep model is proposed in which cut activity in the follicle cells first directs expression of a gene or set of genes that regulates adhesion or signaling between the somatic and germline cells. This soma-to-germline interaction then influences cAMP-dependent function in the germline cells. The activity of Capu and Otu is in turn regulated by these cAMP-mediated events, perhaps by post-translational modifications or by alterations in the subcellular localization of one or both of these proteins. Finally, the regulation of Capu and Otu by cAMP results in altered cytoskeletal function. This hypothesis makes several testable predictions that are currently under investigation. Since it is not yet known if agn is required in the germline cells or follicle cells, the possibility that cut influences agn levels directly by regulating agn transcription in the follicle cells cannot be ruled out. A less favorable model is that capu, otu, and/or agn may function genetically upstream of cut, and loss of germline function of these genes influences cut activity in the follicle cells. At some point in this model, however, cut activity in the follicle cells must influence the function of the germline cytoskeleton, since loss of cut function in the follicle cells is sufficient to produce binucleate germline cells (Jackson, 1999).

The observed genetic interactions between cut and otu are consistent with the model that cut-mediated events disrupt the function of the germline cytoskeleton. Actin cytoskeleton function is known to be disrupted in otu mutants; it was hypothesized that this defect was the underlying cause for the various otu phenotypes. Although otu has been cloned and antibodies have been raised against the protein, the gene's sequence and the uniform distribution of Otu protein within the germplasm give no clue as to how Otu affects the function of the cytoskeleton. Nevertheless, the findings suggest that otu regulates cytoskeleton function in response to signaling events that occur after the cystoblast cell divisions are completed and egg chambers leave the germarium. Finally, one of the otu mutant phenotypes is the production of tumorous egg chambers filled with extra germline-derived cells. Egg chambers that were tumorous or that contained extra germline cell nuclei are not observe in cut/otu double heterozygotes, suggesting that cut and otu do not interact in the germarium to regulate germline cell divisions (Jackson, 1999).

There is now clear evidence that agnostic is required during oogenesis. Loss of agnostic function affects the morphology of the follicle cell epithelium and, because follicle cells are missing in late-stage egg chambers, may influence the survival of the follicle cells. In addition, loss of agnostic affects the morphology of the germline-derived cells. It is not known whether agnostic is required in the follicle cells, germline cells, or both. Signaling between these two cell layers occurs throughout oogenesis and models can be hypothesized in which loss of agnostic function in one cell type affects the function and morphology of the other cell type. Nevertheless, agnostic is thought to be involved in cAMP metabolism by coding for a protein that regulates calmodulin activity. Since the requirement for Protein kinase A is restricted to the germline cells, it is tempting to speculate that minimally, agnostic is required in the germline cells (Jackson, 1999).

Irrespective of the cell type in which agnostic is required, both adenylyl cyclase and phosphodiesterase enzymatic activity are altered in agnostic mutants. These results raise the possibility that cut function impinges on the activity of either or both of these enzymes. Interestingly, some dunce alleles are female sterile, revealing a role for dunce during oogenesis. Deficiencies uncovering dunce and rutabaga fail to interact with cut in the genetic screen, however, and no morphological defects are observed in the ovaries of double heterozygotes of cut and specific mutant alleles of dunce or rutabaga. Thus, cut does not appear to interact individually with either dunce or rutabaga in the same dose-sensitive manner as agnostic. There are three other adenylyl cyclase genes identified in Drosophila that may be regulated by agnostic and/or cut. Finally, it is interesting to note that both adenylyl cyclase and phosphodiesterase activities are increased in agnostic mutants, suggesting that the role of this gene in regulating cAMP levels is complex (Jackson, 1999).

Otu and RNA transport

Certain mutant alleles of the ovarian tumor locus give rise to polytene chromosomes in the pseudonurse cells (PNCs). The banding pattern of these germ line-derived chromosomes is similar to that in the larval salivary gland chromosomes. In this study, the gene activity of these chromosomes was examined. General gene expression from these chromosomes was studied by uridine autoradiography. The expression of specific genes was monitored by in situ hybridisation to mRNA and also by combining enhancer trap lines with otu mutants. Most of the genes studied are expressed in the PNCs, just as they are in the wild-type nurse cells. Four out of the 12 mRNAs studied accumulate in the nuclei instead of migrating to the cytoplasm. The intensity of accumulation directly correlates with the extent of polytenization in the PNC nuclei. It is suggested that the OTU mRNA remains partly attached to the polytene chromosome template after transcription. The effects of this phenomenon on polytenization of the PNC chromosomes are discussed (Heino, 1995).

The otu locus encodes two protein isoforms that have been proposed to act during different stages of oogenesis. The corresponding OTU mRNAs display a dynamic pattern of expression during oogenesis. The 4.1 kb mRNA encoding the 104 kDa isoform is expressed throughout adult oogenesis, but is mainly restricted to nurse cells. The 3.2 kb mRNA encoding the 98 kDa protein isoform is selectively localized in the oocyte up to stage 9. Both mRNAs are expressed abundantly in nurse cells at stages 10-11. It is proposed that the oocyte-specific function of otu is realised by the 98 kDa isoform. The export of the 3.2 kb mRNA from the nurse cell nuclei requires a functional Otu protein. The Otu protein is also required for the correct distribution of the Pumilio and Oskar mRNAs, while the Bic-D, K10 and Staufen mRNAs are localized in wild type fashion in otu mutants (Tirronen, 1995).

The roles of three loci, quit, ovarian tumor and shut down during oocyte differentiation in Drosophila were examined using in situ hybridization and double mutant analyses. Mutations in qui and otu disturb the cystocyte divisions and the oocyte determination, while mutations in shu affect cystocyte integrity, nevertheless allowing differentiation of normal-looking egg chambers with an oocyte. In all mutants the transport of molecules towards the posterior end of the egg chamber takes place as revealed by the accumulation of Bic-D or K10 transcripts. The transport is ineffective in the qui and otu mutants apparently due to the lack of a properly differentiated oocyte. In the shu mutant the transport collapses and the oocyte is lost, leading to egg chambers with 15 nurse cells. One function of qui+ is to enhance otu+ mRNA expression, suggesting that these genes control the cystocyte maturation via the same pathway (Tirronen, 1993).

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