Multiplicity of functions for the otu gene products during Drosophila oogenesis

The ovarian tumor gene behaves as if it encodes a product (OGP), which is required during several early steps in the transformation of oogonia into functional oocytes. Seventeen ethyl methane sulfonate-induced mutations have been studied, and their mutant phenotypes can be explained as graded responses by individual germ cells to different levels of OGP synthesized by the mutant germ cells themselves. The lowest and highest levels of OGP appear to be produced by otu10 and otu14, respectively. The 15 mutants with intermediate OGP levels are temperature sensitive; subnormal temperatures improve ovarian development, while above-normal temperatures suppress it. A subgroup of these mutants are unable to form a system of actin microfilament bundles in the cortical cytoplasm of their nurse cells during stage 10B, and 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, OGP 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.     

Illuminating the role of caspases during Drosophila oogenesis

Cell death is a prominent feature of animal germline development. In Drosophila, the death of 15 nurse cells is linked to the development of each oocyte. In addition, females respond to poor environmental conditions by inducing egg chamber death prior to yolk uptake by the oocyte. To study these two forms of cell death, we analyzed caspase activity in the germline by expressing a transgene encoding a caspase cleavage site flanked by cyan fluorescent protein and yellow fluorescent protein. When expressed in ovaries undergoing starvation-induced apoptosis, this construct was an accurate reporter of caspase activity. However, dying nurse cells at the end of normal oogenesis showed no evidence of cytoplasmic caspase activity. Furthermore, although expression of the caspase inhibitors p35 or Drosophila inhibitor of apoptosis protein 1 blocked starvation-induced death, it did not affect normal nurse cell death or overall oogenesis in well-fed females. Our data suggest that caspases play no role in developmentally programmed nurse cell death.     

The role of the Suppressor of Hairy-wing insulator protein in Drosophila oogenesis

The Drosophila Suppressor of Hairy wing [Su(Hw)] insulator protein has an essential role in the development of the female germline. Here we investigate the function of Su(Hw) in the ovary. We show that Su(Hw) is universally expressed in somatic cells, while germ cell expression is dynamic. Robust levels accumulate in post-mitotic germ cells, where Su(Hw) localization is limited to chromosomes within nurse cells, the specialized cells that support oocyte growth. Although loss of Su(Hw) causes global defects in nurse cell chromosome structure, we demonstrate that these architectural changes are not responsible for the block in oogenesis. Connections between the fertility and insulator functions of Su(Hw) were investigated through studies of the two gypsy insulator proteins, Modifier of (mdg4)67.2 (Mod67.2) and Centrosomal Protein of 190 kDa (CP190). Accumulation of these proteins is distinct from Su(Hw), with Mod67.2 and CP190 showing uniform expression in all cells during early stages of oogenesis that diminishes in later stages. Although Mod67.2 and CP190 extensively co-localize with Su(Hw) on nurse cell chromosomes, neither protein is required for nurse cell chromosome development or oocyte production. These data indicate that while the gypsy insulator function requires both Mod67.2 and CP190, these proteins are not essential for oogenesis. These studies represent the first molecular investigations of Su(Hw) function in the germline, which uncover distinct requirements for Su(Hw) insulator and ovary functions.     

The role of the actomyosin cytoskeleton in coordination of tissue growth during Drosophila oogenesis

The Drosophila egg chamber is an organ composed of a somatic epithelium that covers a germline cyst. After egg-chamber formation, the germline cells grow rapidly without dividing while the surface of the epithelium expands by cell proliferation [1, 2]. The mechanisms that coordinate growth and morphogenesis of the two tissues are not known. Here we identify a role for the actomyosin cytoskeleton in this process. We show that myosin activity is restricted to the epithelium's apical surface, which is facing the growing cyst. We demonstrate that the epithelium collapses in the absence of myosin activity and show that the force that deforms the epithelium originates from the growing cyst. Thus, myosin activity maintains epithelial shape by balancing the force emanating from cyst growth. Further, our data indicate that cyst growth induces cell division in the epithelium. In addition, we show how apical restriction of myosin activity is controlled. Myosin is activated at the apical cortex by localized Rho kinase and inhibited at the basolateral cortex by PP1beta9C. In addition, our data indicate that active myosin is apically anchored by the Baz/Par-6/aPKC complex.     

A translation-independent role of oskar RNA in early Drosophila oogenesis

The Drosophila maternal effect gene oskar encodes the posterior determinant responsible for the formation of the posterior pole plasm in the egg, and thus of the abdomen and germline of the future fly. Previously identified oskar mutants give rise to offspring that lack both abdominal segments and a germline, thus defining the ;posterior group phenotype'. Common to these classical oskar alleles is that they all produce significant amounts of oskar mRNA. By contrast, two new oskar mutants in which oskar RNA levels are strongly reduced or undetectable are sterile, because of an early arrest of oogenesis. This egg-less phenotype is complemented by oskar nonsense mutant alleles, as well as by oskar transgenes, the protein-coding capacities of which have been annulled. Moreover, we show that expression of the oskar 3' untranslated region (3'UTR) is sufficient to rescue the egg-less defect of the RNA null mutant. Our analysis thus reveals an unexpected role for oskar RNA during early oogenesis, independent of Oskar protein. These findings indicate that oskar RNA acts as a scaffold or regulatory RNA essential for development of the oocyte.     

Cell-autonomous roles of the ecdysoneless gene in Drosophila development and oogenesis

Steroid signaling underlies developmental processes in animals. Mutations that impair steroidogenesis in the fruit fly Drosophila melanogaster provide tools to dissect steroid hormone action genetically. The widely used temperature-sensitive mutation ecdysoneless(1) (ecd(1)) disrupts production of the steroid hormone ecdysone, and causes developmental and reproductive defects. These defects cannot be satisfactorily interpreted without analysis of the ecd gene. Here, we show that ecd encodes an as yet functionally undescribed protein that is conserved throughout eukaryotes. The ecd(1) conditional allele contains an amino acid substitution, whereas three non-conditional larval lethal mutations result in truncated Ecd proteins. Consistent with its role in steroid synthesis, Ecd is expressed in the ecdysone-producing larval ring gland. However, development of ecd-null early larval lethal mutants cannot be advanced by Ecd expression targeted to the ring gland or by hormone feeding. Cell-autonomous ecd function, suggested by these experiments, is evidenced by the inability of ecd(-) clones to survive within developing imaginal discs. Ecd is also expressed in the ovary, and is required in both the follicle cells and the germline for oocyte development. These defects, induced by the loss of ecd, provide the first direct evidence for a cell-autonomous function of this evolutionarily conserved protein.     

The role of polyfusomes in generating branched chains of cystocytes during Drosophila oogenesis

Three-dimensional models were constructed utilizing the information gained from electron micrographs of serial sections of two clones of cystocytes undergoing their terminal divisions. In each clone a polyfusome connected all eight cystocytes together. Each of the spindles was oriented so that one pole touched the polyfusomes, while the other pointed away from it. This positioning of spindles ensures that one cell of each dividing pair retains all previously formed canals, while the other receives none. The two cells that eventually come to contain the maximum number of canals and fusomal material are the ones that differentiate as pro-oocytes, while the others become nurse cells. The orientation of each spindle suggests that the polyfusome formed at one division determines the placement of the cytoskeletal fibers that anchor the spindles formed at the next division. There is a centripetal gathering together of new canals following each cycle of cystocyte division, which is thought to result from the subsequent contraction of the polyfusomal system. Females homozygous for the otu1 mutation are characterized by ovarian tumors, which result when germarial cystocytes undergo supernumerary divisions and fail to differentiate into either nurse cells or oocytes. An analysis of electron micrographs taken of serially sectioned, mutant germaria showed that most germ cells were single or belonged to clusters of two or three interconnected cells. Therefore otu1 cystocytes are unable to undergo a sustained series of arrested cleavages. These cystocytes contain fusomal material that shows ultrastructural differences from normal polyfusomes. We conclude: 1) that a normal polyfusomal system is a necessary prerequisite for the production of a branched chain of cystocytes and for their subsequent differentiation into pro-oocytes and nurse cells; and 2) that a product encoded by the otu+ gene is essential for the construction of a functional polyfusome.     

The arrest gene is required for germline cyst formation during Drosophila oogenesis

In Drosophila, oogenesis is initiated when a germline stem cell produces a differentiating daughter cell called the cystoblast. The cystoblast undergoes four rounds of synchronous divisions with incomplete cytokinesis to generate a syncytial cyst of 16 interconnected cystocytes, in which one cystocyte differentiates into an oocyte. Strong mutations of the arrest (aret) gene disrupt cyst formation and cause the production of clusters of ill-differentiated germline cells that retain cellular and molecular characteristics of cystoblasts. These mutant germ cells express high levels of BAM-C and SXL proteins in the cytoplasm but do not accumulate markers for advanced cystocytes or differentiating oocytes, such as the nuclear localization of SXL or the accumulation of osk mRNA, orb mRNA, and cytoplasmic dynein. However, the mutant germ cells do not contain spectrosomes, the cytoplasmic structure that objectifies the divisional asymmetry of the cystoblast. The aret mutant germ cells undergo active mitosis with complete cytokinesis. Their mitosis is accompanied by massive necrosis, so that the number of germ cells in a stem cell-derived cluster ranges from one to greater than 70. These defects of aret mutants reveal a novel function of aret as the first gene with a defined function in the cystoblast to cyst transition during early oogenesis.     

M6 membrane protein plays an essential role in Drosophila oogenesis

We had previously shown that the transmembrane glycoprotein M6a, a member of the proteolipid protein (PLP) family, regulates neurite/filopodium outgrowth, hence, M6a might be involved in neuronal remodeling and differentiation. In this work we focused on M6, the only PLP family member present in Drosophila, and ortholog to M6a. Unexpectedly, we found that decreased expression of M6 leads to female sterility. M6 is expressed in the membrane of the follicular epithelium in ovarioles throughout oogenesis. Phenotypes triggered by M6 downregulation in hypomorphic mutants included egg collapse and egg permeability, thus suggesting M6 involvement in eggshell biosynthesis. In addition, RNAi-mediated M6 knockdown targeted specifically to follicle cells induced an arrest of egg chamber development, revealing that M6 is essential in oogenesis. Interestingly, M6-associated phenotypes evidenced abnormal changes of the follicle cell shape and disrupted follicular epithelium in mid- and late-stage egg chambers. Therefore, we propose that M6 plays a role in follicular epithelium maintenance involving membrane cell remodeling during oogenesis in Drosophila.     

Systems-level questions in Drosophila oogenesis

This paper describes computational and experimental work on pattern formation in Drosophila egg development (oogenesis), an established experimental model for studying cell fate diversification in developing tissues. Epidermal growth factor receptor (EGFR) is a key regulator of pattern formation and morphogenesis in Drosophila oogenesis. EGFR signalling in oogenesis can be genetically manipulated and monitored at many levels, leading to large sets of heterogeneous data that enable the formulation of increasingly quantitative models of pattern formation in these systems.     

The involvement of the Notch locus in Drosophila oogenesis.

The Notch gene in Drosophila encodes a transmembrane protein with homology to EGF that, in a variety of tissues, appears to mediate cell interactions necessary for cell fate choices. Here we demonstrate that oogenesis and spermatogenesis depend on Notch. We examine the phenotypes of the temperature-sensitive Notch allele, Nts1, and, using a monoclonal antibody, determine the cellular and subcellular distribution of Notch protein during oogenesis. We show that Nts1 is associated with a missense mutation in the extracellular, EGF homologous region of Notch and that at non-permissive temperatures oogenesis is blocked and the subcellular distribution of the protein is altered. In wild-type ovaries, Notch protein is found on the apical surface of somatically derived follicle cells, while in the germline-derived cells the protein is not polarized. These findings are discussed in view of the hypothesis that Notch acts as a multifunctional receptor to mediate developmentally important cell interactions.     

DNA repair in Drosophila oogenesis

In experiments with females of lines with an impaired DNA repair systems mei-9 (impaired excision repair) and mei-41 (impaired postreplicative repair), a method of successive irradiation by X-rays (1000 R) and hyperthermia (+37 degrees C) action was used for the purpose of defining a moment when DNA repair takes place in oogenesis. Repair in mature mei-41 oocytes judged of by synergism effect of the both factors acting was ascertained to take place right after X-raying (prior to DNA replication) and being absent at the fertilization period (at the time of or after DNA replication). DNA repair in mei-9 females was not registered in both cases. On the basis of these facts, it is suggested that coordination of various DNA repair systems is necessary for damaged chromosomes to be repaired. It is also concluded that the method used can be regarded as an effective technique in the study of mutation process.     

The ontogeny of germ plasm during oogenesis in Drosophila

Primordial germ cells can be induced at both the anterior and ventral region of the Drosophila egg by transplanted posterior polar plasm. Two questions arise from these results: (1) Is fertilization required for germ plasm to be functional, and (2) at what stage during oogenesis does the posterior polar plasm become established as a germ-cell determinant?Polar plasm from unfertilized eggs and from oocytes at stage 10 to 14 of Drosophila melanogaster was implanted into the anterior region of cleavage embryos. Some injected embryos were analyzed at the ultrastructural level during blastoderm formation. Polar plasm from unfertilized eggs and from oocytes of stages 13 and 14 was found to be integrated into several anterior cells that resembled morphologically normal pole cells. The formation of such cells, however, could not be detected in embryos injected with polar plasm from oogenetic stages 10 to 12. Experimentally induced pole cells proved to be capable of differentiating into functional germ cells when cycled through the germ line of genetically different host embryos. About 5% of the flies developing from these embryos produced progeny that originated from the induced pole cells. Germ-line mosaicism in those flies also could be detected histochemically in their gonads. No germ cells were recovered with polar plasm transplants from oogenetic stages 10 to 12.The results show that posterior polar plasm of the unfertilized egg is functional in germ-cell determination, and that prior to egg maturation this cytoplasm has already acquired its determinative ability. This is the first demonstration that specific developmental information stored in the cytoplasm can be traced back to a particular region of the oocyte.     

Two discrete modes of histone gene expression during oogenesis in Drosophila melanogaster

We have used in situ hybridization to ovarian tissue sections to study the pattern of histone gene expression during oogenesis in Drosophila melanogaster. Our studies suggest that there are two distinct phases of histone gene expression during oogenesis. In the first phase, which occurs during early to middle oogenesis (stages 5-10A), we observe a mosaic pattern of histone mRNA in the 15 nurse cells of the egg chamber: some cells have very high levels of mRNA, while others have little or no mRNA. Our analysis suggests that there is a cyclic accumulation and subsequent degradation of histone mRNA in the egg chamber and that very little histone mRNA is transported into the growing oocyte. Moreover, since the endomitotic replication cycles of the nurse cells are asynchronous during this period, the mosaic distribution of histone message would suggest that the expression of the histone genes in each nurse cell nucleus is probably coupled to DNA replication as in most somatic cells. The second phase begins at stage 10B. During this period, histone gene expression appears to be "induced" in all 15 nurse cells of the egg chamber, and instead of a mosaic pattern, high levels of histone mRNA are found in all cells. Unlike the earlier phase, this expression is apparently uncoupled from the endomitotic replication of the nurse cells (which are completed by the end of stage 10A). Moreover, much of the newly synthesized histone mRNA is transported from the nurse cells into the oocyte where it accumulates and is stored for use during early embryogenesis. Finally, we have also observed tightly clustered grains within nurse cell nuclei in non-denatured tissue sections. As was the case with cytoplasmic histone mRNA, there is a mosaic distribution of nuclear grains from stages 5 to 10A, while at stage 10B, virtually all nurse cell nuclei have grain clusters. These grain clusters appear to be due to the hybridization of nurse cell histone gene DNA to our probe, and are localized in specific regions of the nucleus.     

Drosophila Cappuccino alleles provide insight into formin mechanism and role in oogenesis

During Drosophila development, the formin actin nucleator Cappuccino (Capu) helps build a cytoplasmic actin mesh throughout the oocyte. Loss of Capu leads to female sterility, presumably because polarity determinants fail to localize properly in the absence of the mesh. To gain deeper insight into how Capu builds this actin mesh, we systematically characterized seven capu alleles, which have missense mutations in Capu''s formin homology 2 (FH2) domain. We report that all seven alleles have deleterious effects on fly fertility and the actin mesh in vivo but have strikingly different effects on Capu''s biochemical activity in vitro. Using a combination of bulk and single- filament actin-assembly assays, we find that the alleles differentially affect Capu''s ability to nucleate and processively elongate actin filaments. We also identify a unique “loop” in the lasso region of Capu''s FH2 domain. Removing this loop enhances Capu''s nucleation, elongation, and F-actin–bundling activities in vitro. Together our results on the loop and the seven missense mutations provides mechanistic insight into formin function in general and Capu''s role in the Drosophila oocyte in particular.     

Ovarian pathologies generated by various alleles of the otu locus in Drosophila melanogaster

A comparative cytological study was made of oogenesis in flies carrying various mutant alleles of the female sterile gene otu. It resides at 22.7 on the genetic map and within subdivision 7F of the cytological map of the X-chromosome. Each of the five ethyl methane sulfonate-induced mutations observed falls into one of three classes. In class 1, most mutant ovarioles lack germ cells; in class 2, most mutant ovarioles contain tumorous chambers; and in class 3 mutants, chambers occur that possess defective oocytes. The otu² allele belongs to class 1; otu¹ to class 2; and otu³, otu⁴, and otu⁵ to class 3. The mutations have no effects upon female viability or upon the viability and fertility of hemizygous males. Heterozygous females are fertile and have cytologically normal ovaries. In otu⁵ homozygotes, all ovarioles contain egg chambers, but oogenesis is prematurely terminated to produce a pseudo-stage 12 oocyte. Ovarioles from otu³ and from otu⁴ homozygotes contain both ovarian tumors and oocytes. Pseudonurse cells (PNC), which are cystocytes that have stopped dividing and have entered the nurse cell mode of development, are also abundant. PNCs contain polytene chromosomes. Since the homologs are paired, each nucleus has the haploid number of chromosomes. In chambers lacking an oocyte, the number of PNCs is less than the normal number of nurse cells. In chambers containing an oocyte, the number of accompanying nurse cells may be 15, or above or below normal. In vitellogenic chambers, the chromosomes in the nurse cells connected directly to the oocyte are more expanded than those in more distant nurse cells. The KA14 deficiency lacks the plus allele of otu. KA14 heterozygotes are fertile and have cytologically normal ovaries. When females carry KA14 and otu¹, otu³, otu⁴, or otu⁵, 80% of their ovarioles are agametic. When females carry otu² and one of the other mutant alleles, the ovarioles proceed further in development. So otu² produces a product that has a beneficial effect on the test allele. When two different otu alleles are combined in a single fly, the phenotype of the hybrid ovary usually most resembles that of the ovary homozygous for the “stronger” allele (the otu mutant that allows oogenesis to proceed farthest). The results indicate that the product of the otu⁺ locus functions at least three different times during oogenesis; first to permit oogonia to proliferate, second to control the division and differentiation of germarial cystocytes, and third to facilitate the normal growth of the ooplasm. The gene product appears to be required in higher concentrations at each developmental period. The lesions produced by the mutations are thought to interfere with the stability or functioning of the gene product, and the ovarian phenotype produced by a given genotype depends upon the concentration of functional gene product available to the germ cells.     

The Drosophila ecdysone receptor (EcR) gene is required maternally for normal oogenesis

Oogenesis in Drosophila is regulated by the steroid hormone ecdysone and the sesquiterpenoid juvenile hormone. Response to ecdysone is mediated by a heteromeric receptor composed of the EcR and USP proteins. We have identified a temperature-sensitive EcR mutation, EcR(A483T), from a previously isolated collection of EcR mutations. EcR(A483T) is predicted to affect all EcR protein products (EcR-A, EcR-B1, and EcR-B2) since it maps to a common exon encoding the ligand-binding domain. In wild-type females, we find that both EcR-A and EcR-B1 are expressed in nurse cells and follicle cells throughout oogenesis. EcR mutant females raised at permissive temperature and then shifted to restrictive temperature exhibit severe reductions in fecundity. Oogenesis in EcR mutant females is defective, and the spectrum of oogenic defects includes the presence of abnormal egg chambers and loss of vitellogenic egg stages. Our results demonstrate a requirement for EcR during female reproduction and suggest that EcR is required for normal oogenesis.     

The role of Drosophila hyperplastic discs gene in spermatogenesis

In Drosophila, the ubiquitin ligase Hyd (hyperplastic disc) is required for regulation of cell proliferation during development [ Martin et al. (1977) Dev Biol 55 , 213–232; Mansfield et al. (1994) Dev Biol 165 , 507–526]. Earlier, we demonstrated that the Drosophila tumour suppressor Merlin participates not only in imaginal discs proliferation control, but also performs a separate Nebenkern structural function in Drosophila spermatogenesis [ Dorogova et al. (2008) BMC Cell Biol 9 , 1. Here, we show that the hyd mutants also have spermatogenesis defects: chromosome condensation and attachment to the spindle, centrosome behaviour and cytokinesis in meiosis. The process of spermatid elongation was also greatly affected: nuclei were scattered along the cyst and had an abnormal shape, Nebenkern–axoneme angular relation and attachment was distorted, axonemes themselves lost correct structure. Since Hyd and pAbp protein families share a common PABC [poly(A)‐binding protein C‐terminal] protein domain, we also studied spermatogenesis in pAbp homozygotes and found defects in cytokinesis and spermatid elongation. However, our study of hyd and pAbp genetic interaction revealed only the phenotype of defective nuclei shape at the final stage of spermatid differentiation. So, the PABC domain is unlikely to be responsible for meiotic defects. Thus, our data document that, in addition to the tumour suppressor Merlin, another tumour suppressor, Hyd, also has a function in spermatogenesis.