The Ovhts polyprotein is cleaved to produce fusome and ring canal proteins required for Drosophila oogenesis

An essential component of normal development is controlling the transition from cell proliferation to differentiation. One such transition occurs during Drosophila oogenesis. In early oogenesis, germ cells undergo mitotic proliferation and contain a specialized organelle called a fusome, whereas later post-mitotic cells differentiate and lose the fusome as F-actin-rich ring canals form. The hts gene encodes the only Drosophila Adducin, and is a female-sterile mutant that affects both the fusome and ring canals. We show that one Hts protein, Ovhts, is a polyprotein that is cleaved to produce two products, Ovhts-Fus and Ovhts-RC. Whereas Ovhts-Fus localizes to the fusome in mitotic cells, Ovhts-RC localizes to ring canals throughout later oogenesis. We demonstrate that an uncleavable version of Ovhts delays the transition from fusome-containing cells to those that have ring canals. Ovhts is the first polyprotein shown to produce proteins that function in separate structures.     

Milton controls the early acquisition of mitochondria by Drosophila oocytes

Mitochondria in many species enter the young oocyte en mass from interconnected germ cells to generate the large aggregate known as the Balbiani body. Organelles and germ plasm components frequently associate with this structure. Balbiani body mitochondria are thought to populate the germ line, ensuring that their genomes will be inherited preferentially. We find that milton, a gene whose product was previously shown to associate with Kinesin and to mediate axonal transport of mitochondria, is needed to form a normal Balbiani body. In addition, germ cells mutant for some milton or Kinesin heavy chain (Khc) alleles transport mitochondria to the oocyte prematurely and excessively, without disturbing Balbiani body-associated components. Our observations show that the oocyte acquires the majority of its mitochondria by competitive bidirectional transport along microtubules mediated by the Milton adaptor. These experiments provide a molecular explanation for Balbiani body formation and, surprisingly, show that viable fertile offspring can be obtained from eggs in which the normal program of mitochondrial acquisition has been severely perturbed.     

Drosophila par-1 is required for oocyte differentiation and microtubule organization

BACKGROUND: Drosophila oocyte determination involves a complex process by which a single cell within an interconnected cyst of 16 germline cells differentiates into an oocyte. This process requires the asymmetric accumulation of both specific messenger RNAs and proteins within the future oocyte as well as the proper organization of the microtubule cytoskeleton, which together with the fusome provides polarity within the developing germline cyst. RESULTS: In addition to its previously described late oogenic role in the establishment of anterior-posterior polarity and subsequent embryonic axis formation, the Drosophila par-1 gene is required very early in the germline for establishing cyst polarity and for oocyte specification. Germline clonal analyses, for which we used a protein null mutation, reveal that Drosophila par-1 (par-1) is required for the asymmetric accumulation of oocyte-specific factors as well as the proper organization of the microtubule cytoskeleton. Similarly, somatic clonal analyses indicate that par-1 is required for microtubule stabilization in follicle cells. The PAR-1 protein is localized to the fusome and ring canals within the developing germline cyst in direct contact with microtubules. Likewise, in the follicular epithelium, PAR-1 colocalizes with microtubules along the basolateral membrane. However, in either case PAR-1 localization is independent of microtubules. CONCLUSIONS: The Drosophila par-1 gene plays at least two essential roles during oogenesis; it is required early in the germline for organization of the microtubule cytoskeleton and subsequent oocyte determination, and it has a second, previously described role late in oogenesis in axis formation. In both cases, par-1 appears to exert its effects through the regulation of microtubule dynamics and/or stability, and this finding is consistent with the defined role of the mammalian PAR-1 homologs.     

Fusome asymmetry and oocyte determination in Drosophila

Germline cysts containing 16 interconnected cells (cystocytes) are produced at an early stage of Drosophila oogenesis by progenitor cells known as cystoblasts that undergo four synchronous rounds of incomplete division. During cyst formation, a region of specialized, spectrin-rich cytoplasm called the fusome traverses the intercellular Connections (ring canals), linking individual cystocytes. Subsequently, 15 cystocytes begin to transport specific RNAs and other components into the remaining cell, the future oocyte. We used fusome-specific antibodies to characterize the early stages of cyst formation. During the first cystoblast division, a spherical mass of fusome material (the “spectrosome”) was associated with only one pole of the mitotic spindle, revealing that this division is asymmetric. During the subsequent three divisions, the growing fusome always associated with the pole of each mitotic spindle that remained in the mother cell, and only extended through the newly formed ring canals after each division was completed. These observations suggest that fusomes help establish a system of directional transport between cystocytes that underlies oocyte determination. © 1995 Wiley-Liss, Inc.     

Centrosome migration into the Drosophila oocyte is independent of BicD and egl, and of the organisation of the microtubule cytoskeleton

During early Drosophila oogenesis, one cell from a cyst of 16 germ cells is selected to become the oocyte, and accumulates oocyte-specific proteins and the centrosomes from the other 15 cells. Here we show that the microtubule cytoskeleton and the centrosomes follow the same stepwise restriction to one cell as other oocyte markers. Surprisingly, the centrosomes still localise to one cell after colcemid treatment, and in BicD and egl mutants, which abolish the localisation of all other oocyte markers and the polarisation of the microtubule cytoskeleton. In contrast, the centrosomes fail to migrate in cysts mutant for Dynein heavy chain 64C, which disrupts the fusome. Thus, centrosome migration is independent of the organisation of the microtubule cytoskeleton, and seems to depend instead on the polarity of the fusome.     

Orbit/Mast,the CLASP orthologue of Drosophila,is required for asymmetric stem cell and cystocyte divisions and development of the polarised microtubule network that interconnects oocyte and nurse cells during oogenesis

Drosophila oocyte differentiation is preceded by the formation of a polarised 16-cell cyst from a single progenitor stem cell as a result of four rounds of asymmetric mitosis followed by incomplete cytokinesis. We show that the Orbit/Mast microtubule-associated protein is required at several stages in the formation of such polarised 16-cell cysts. In wild-type cysts, the Orbit/Mast protein not only associates with the mitotic spindle and its poles, but also with the central spindle (spindle remnant), ring canal and fusome, suggesting it participates in interactions between these structures. In orbit mutants, the stem cells and their associated fusomes are eventually lost as Orbit/Mast protein is depleted. The mitotic spindles of those cystocytes that do divide are either diminutive or monopolar, and do not make contact with the fusome. Moreover, the spindle remnants and ring canals fail to differentiate correctly in such cells and the structure of fusome is compromised. The Orbit/Mast protein thus appears to facilitate multiple interactions of the fusome with mitotic spindles and ring canals. This ensures correct growth of the fusome into a branched asymmetrically distributed organelle that is pre-determinative of 16-cell cyst formation and oocyte fate specification. Finally the Orbit/Mast protein is required during mid-oogenesis for the organisation of the polarised microtubule network inside the 16-cell cyst that ensures oocyte differentiation. The localisation of CLIP-190 to such microtubules and to the fusome is dependent upon Orbit/Mast to which it is complexed.     

Balbiani body,nuage and sponge bodies – The germ plasm pathway players

In many animal species, germ cells are specified by maternally provided, often asymmetrically localized germ cell determinant, termed the germ plasm. It has been shown that in model organisms such as Xenopus laevis, Danio rerio and Drosophila melanogaster germ plasm components (various proteins, mRNAs and mitochondria) are delivered to the proper position within the egg cell by germline specific organelles, i.e. Balbiani bodies, nuage accumulations and/or sponge bodies. In the present article, we review the current knowledge on morphology, molecular composition and functioning of these organelles in main lineages of arthropods and different ovary types on the backdrop of data derived from the studies of the model vertebrate species.     

A late phase of germ plasm accumulation during Drosophila oogenesis requires lost and rumpelstiltskin

Asymmetric mRNA localization is an effective mechanism for establishing cellular and developmental polarity. Posterior localization of oskar in the Drosophila oocyte targets the synthesis of Oskar to the posterior, where Oskar initiates the assembly of the germ plasm. In addition to harboring germline determinants, the germ plasm is required for localization and translation of the abdominal determinant nanos. Consequently, failure of oskar localization during oogenesis results in embryos lacking germ cells and abdominal segments. oskar accumulates at the oocyte posterior during mid-oogenesis through a well-studied process involving kinesin-mediated transport. Through live imaging of oskar mRNA, we have uncovered a second, mechanistically distinct phase of oskar localization that occurs during late oogenesis and results in amplification of the germ plasm. Analysis of two newly identified oskar localization factors, Rumpelstiltskin and Lost, that are required specifically for this late phase of oskar localization shows that germ plasm amplification ensures robust abdomen and germ cell formation during embryogenesis. In addition, our results indicate the importance of mechanisms for adapting mRNAs to utilize multiple localization pathways as necessitated by the dramatic changes in ovarian physiology that occur during oogenesis.     

PAR-1 is required for the maintenance of oocyte fate in Drosophila

The PAR-1 kinase is required for the posterior localisation of the germline determinants in C. elegans and Drosophila, and localises to the posterior of the zygote and the oocyte in each case. We show that Drosophila PAR-1 is also required much earlier in oogenesis for the selection of one cell in a germline cyst to become the oocyte. Although the initial steps in oocyte determination are delayed, three markers for oocyte identity, the synaptonemal complex, the centrosomes and Orb protein, still become restricted to one cell in mutant clones. However, the centrosomes and Orb protein fail to translocate from the anterior to the posterior cortex of the presumptive oocyte in region 3 of the germarium, and the cell exits meiosis and becomes a nurse cell. Furthermore, markers for the minus ends of the microtubules also fail to move from the anterior to the posterior of the oocyte in mutant clones. Thus, PAR-1 is required for the maintenance of oocyte identity, and plays a role in microtubule-dependent localisation within the oocyte at two stages of oogenesis. Finally, we show that PAR-1 localises on the fusome, and provides a link between the asymmetry of the fusome and the selection of the oocyte.     

Protein Phosphatase 1? Limits Ring Canal Constriction during Drosophila Germline Cyst Formation

Germline cyst formation is essential for the propagation of many organisms including humans and flies. The cytoplasm of germline cyst cells communicate with each other directly via large intercellular bridges called ring canals. Ring canals are often derived from arrested contractile rings during incomplete cytokinesis. However how ring canal formation, maintenance and growth are regulated remains unclear. To better understand this process, we carried out an unbiased genetic screen in Drosophila melanogaster germ cells and identified multiple alleles of flapwing (flw), a conserved serine/threonine-specific protein phosphatase. Flw had previously been reported to be unnecessary for early D. melanogaster oogenesis using a hypomorphic allele. We found that loss of Flw leads to over-constricted nascent ring canals and subsequently tiny mature ring canals, through which cytoplasmic transfer from nurse cells to the oocyte is impaired, resulting in small, non-functional eggs. Flw is expressed in germ cells undergoing incomplete cytokinesis, completely colocalized with the Drosophila myosin binding subunit of myosin phosphatase (DMYPT). This colocalization, together with genetic interaction studies, suggests that Flw functions together with DMYPT to negatively regulate myosin activity during ring canal formation. The identification of two subunits of the tripartite myosin phosphatase as the first two main players required for ring canal constriction indicates that tight regulation of myosin activity is essential for germline cyst formation and reproduction in D. melanogaster and probably other species as well.     

The Drosophilaovarian tumorGene Is Required for the Organization of Actin Filaments during Multiple Stages in Oogenesis

Theovarian tumorgene is required during both early and late stages of oogenesis. Mutations produce a range of phenotypes, including agametic ovarioles, tumorous egg chambers, and late stage oogenic arrest. We demonstrate that each of these phenotypes is associated with specific aberrations in actin distribution. In the earliest case,ovarian tumormutations 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 tumorfunction is required for the localization of actin that 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. We also confirm thatovarian tumoris 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. Our data suggest that at this stageovarian tumorcontrols the site where actin filaments initiate. Taken together, these studies suggest that the diverseovarian tumormutant 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.     

Formation, architecture and polarity of female germline cyst in Xenopus

Little is known about the formation of germline cyst and the differentiation of oocyte within the cyst in vertebrates. In the majority of invertebrates in the initial stages of gametogenesis, male and female germ cells develop in full synchrony as a syncytia of interconnected cells called germline cysts (clusters, nests). Using electron microscopy, immunostaining and three-dimensional reconstruction, we were able to elucidate the process of cyst formation in the developing ovary of the vertebrate Xenopus laevis. We found that the germline cyst in Xenopus contains 16 cells that are similar in general architecture and molecular composition to the cyst in Drosophila. Nest cells are connected by cytoplasmic bridges that contain ring canal-like structures. The nest cells contain a structure similar to the Drosophila fusome that that is probably involved in anchoring of the centrioles and organization of the primary mitochondrial cloud (PMC) around the centriole. We also find that in contrast to other organisms, in Xenopus, apoptosis is a rare event within the developing ovary. Our studies indicate that the processes responsible for the formation of female germline cysts and the establishment of germ cell polarity are highly conserved between invertebrates and vertebrates. The dissimilarities between Drosophila and Xenopus and the uniqueness of each system probably evolved through modifications of the same fundamental design of the germline cyst.     

The Balbiani body in the oocytes of a common cellar spider, Pholcus phalangioides (Araneae: Pholcidae)

Previtellogenic oocytes of a common cellar spider, Pholcus phalangioides, contain a single aggregation of organelles referred here to as the Balbiani body. It is a well defined ooplasmic structure predominantly composed of fine granular nuage, RNA rich material but comprising also mitochondria, vesicles of endoplasmic reticulum and stacks of Golgi cysternae. The Balbiani body originates early during previtellogenesis in the form of a cap-shaped mass in juxtaposition to one pole of the oocyte nucleus. During later stages of previtellogenic growth the Balbiani body translocates as a single body towards the ooplasm periphery. The results presented indicate that Balbiani body translocation is cytoskeleton independent. Balbiani body repositioning does not result in the localization of its components to any distinct, asymmetrically situated region of the ooplasm but, instead, ends up with their even dispersion in the oocyte cortex. The Balbiani body in Pholcus does not seem to be implicated either in germ cell determination or organelle inheritance. Its homology with similar organelle accumulations in the oocytes of other species is discussed.     

Orbit/CLASP Is Required for Germline Cyst Formation through Its Developmental Control of Fusomes and Ring Canals in Drosophila Males

Orbit, a Drosophila ortholog of microtubule plus-end enriched protein CLASP, plays an important role in many developmental processes involved in microtubule dynamics. Previous studies have shown that Orbit is required for asymmetric stem cell division and cystocyte divisions in germline cysts and for the development of microtubule networks that interconnect oocyte and nurse cells during oogenesis. Here, we examined the cellular localization of Orbit and its role in cyst formation during spermatogenesis. In male germline stem cells, distinct localization of Orbit was first observed on the spectrosome, which is a spherical precursor of the germline-specific cytoskeleton known as the fusome. In dividing stem cells and spermatogonia, Orbit was localized around centrosomes and on kinetochores and spindle microtubules. After cytokinesis, Orbit remained localized on ring canals, which are cytoplasmic bridges between the cells. Thereafter, it was found along fusomes, extending through the ring canal toward all spermatogonia in a cyst. Fusome localization of Orbit was not affected by microtubule depolymerization. Instead, our fluorescence resonance energy transfer experiments suggested that Orbit is closely associated with F-actin, which is abundantly found in fusomes. Surprisingly, F-actin depolymerization influenced neither fusome organization nor Orbit localization on the germline-specific cytoskeleton. We revealed that two conserved regions of Orbit are required for fusome localization. Using orbit hypomorphic mutants, we showed that the protein is required for ring canal formation and for fusome elongation mediated by the interaction of newly generated fusome plugs with the pre-existing fusome. The orbit mutation also disrupted ring canal clustering, which is essential for folding of the spermatogonia after cytokinesis. Orbit accumulates around centrosomes at the onset of spermatogonial mitosis and is required for the capture of one of the duplicated centrosomes onto the fusome. Moreover, Orbit is involved in the proper orientation of spindles towards fusomes during synchronous mitosis of spermatogonial cysts.     

The cytoskeleton organizes germ nuclei with divergent fates and asynchronous cycles in a common cytoplasm during oogenesis in the chordate Oikopleura

Germline cysts are conserved structures in which cells initiating meiosis are interconnected by ring canals. In many species, the cyst phase is of limited duration, but the chordate, Oikopleura, maintains it throughout prophase I as a unique cell, the coenocyst. We show that despite sharing one common cytoplasm with meiotic and nurse nuclei evenly distributed in a 1:1 ratio, both entry into meiosis and subsequent endocycles of nurse nuclei were asynchronous. Coenocyst cytoskeletal elements played central roles as oogenesis progressed from a syncytial state of indistinguishable germ nuclei, to a final arrangement where the common cytoplasm had been equally partitioned into resolved, mature oocytes. During chromosomal bouquet formation in zygotene, nuclear pore complexes clustered and anchored meiotic nuclei to the coenocyst F-actin network opposite ring canals, polarizing oocytes early in prophase I. F-actin synthesis was required for oocyte growth but movement of cytoplasmic organelles into oocytes did not require cargo transport along colchicine-sensitive microtubules. Instead, microtubules maintained nurse nuclei on the F-actin scaffold and prevented their entry into growing oocytes. Finally, it was possible to both decouple meiotic progression from cellular mechanisms governing oocyte growth, and to advance the timing of oocyte growth in response to external cues.     

The fusome organizes the microtubule network during oocyte differentiation in Drosophila

Differentiation of the Drosophila oocyte takes place in a cyst of 16 interconnected germ cells and is dependent on a network of microtubules that becomes polarized as differentiation progresses (polarization). We have investigated how the microtubule network polarizes using a GFP-tubulin construct that allows germ-cell microtubules to be visualized with greater sensitivity than in previous studies. Unexpectedly, microtubules are seen to associate with the fusome, an asymmetric germline-specific organelle, which elaborates as cysts form and undergoes complex changes during cyst polarization. This fusome-microtubule association occurs periodically during late interphases of cyst divisions and then continuously in 16-cell cysts that have entered meiotic prophase. As meiotic cysts move through the germarium, microtubule minus ends progressively focus towards the center of the fusome, as visualized using a NOD-lacZ marker. During this same period, discrete foci rich in gamma tubulin that very probably correspond to migrating cystocyte centrosomes also associate with the fusome, first on the fusome arms and then in its center, subsequently moving into the differentiating oocyte. The fusome is required for this complex process, because microtubule network organization and polarization are disrupted in hts(1) mutant cysts, which lack fusomes. Our results suggest that the fusome, a specialized membrane-skeletal structure, which arises in early germ cells, plays a crucial role in polarizing 16-cell cysts, at least in part by interacting with microtubules and centrosomes.     

Oskar organizes the germ plasm and directs localization of the posterior determinant nanos

Oskar is one of seven Drosophila maternal-effect genes that are necessary for germline and abdomen formation. We have cloned oskar and show that oskar RNA is localized to the posterior pole of the oocyte when germ plasm forms. This polar distribution of oskar RNA is established during oogenesis in three phases: accumulation in the oocyte, transport toward the posterior, and finally maintenance at the posterior pole of the oocyte. The colocalization of oskar and nanos in wild-type and bicaudal embryos suggests that oskar directs localization of the posterior determinant nanos. We propose that the pole plasm is assembled stepwise and that continued interaction among its components is required for germ cell determination.     

Bucky ball functions in Balbiani body assembly and animal-vegetal polarity in the oocyte and follicle cell layer in zebrafish

The Balbiani body is an evolutionarily conserved asymmetric aggregate of organelles that is present in early oocytes of all animals examined, including humans. Although first identified more than 150 years ago, genes acting in the assembly of the Balbiani body have not been identified in a vertebrate. Here we show that the bucky ball gene in the zebrafish is required to assemble this universal aggregate of organelles. In the absence of bucky ball the Balbiani body fails to form, and vegetal mRNAs are not localized in oocytes. In contrast, animal pole localized oocyte markers are expanded into vegetal regions in bucky ball mutants, but patterning within the expanded animal pole remains intact. Interestingly, in bucky ball mutants an excessive number of cells within the somatic follicle cell layer surrounding the oocyte develop as micropylar cells, an animal pole specific cell fate. The single micropyle permits sperm to fertilize the egg in zebrafish. In bucky ball mutants, excess micropyles cause polyspermy. Thus bucky ball provides the first genetic access to Balbiani body formation in a vertebrate. We demonstrate that bucky ball functions during early oogenesis to regulate polarity of the oocyte, future egg and embryo. Finally, the expansion of animal identity in oocytes and somatic follicle cells suggests that somatic cell fate and oocyte polarity are interdependent.     

Mitochondrial development during Drosophila oogenesis: distribution, density and in situ RNA hybridizations.

The changes in distribution and density of mitochondria and the level of mitochondrial RNA during Drosophila oogenesis were studied simultaneously in the 3 cell types ie follicle cells, nurse cells and oocyte, making up the egg chamber. Up to stage 6, mitochondrial density (mitochondrial and cellular areas ratio) was elevated and increased similarly in both follicle and nurse cells. Thereafter the mitochondrial density of follicle cells continued to increase and that of the nurse cells declined markedly while the nurse cell mitochondria assembled in dense groups and decreased in size. This can be related to a transfer of nurse cell cytoplasm, including mitochondria, to the oocyte. In the oocyte from stage 4 to stage 7 we observed a significant decrease of the mitochondrial density due to the absence of mitochondrial biogenesis. Then the cytoplasm transfer caused mitochondrial density to increase up to the level found in the nurse cells at the end of oogenesis. The mature oocyte contains enough mitochondria to supply 15,000 somatic cells. Our results strongly suggest that the variations in size, distribution and density of mitochondria relate to the particular energetic requirements of the different cell types during the first half of oogenesis. Later they relate to the developmental requirements of the nurse cells and the oocyte, in particular the storage of mitochondria in the oocyte. The level of mitochondrial RNA was studied through in situ hybridization. Throughout oogenesis the follicle and nurse cell RNA evolved similarly. Up to stage 9, there was no change in RNA densities in these cells, suggesting a correlation with the cell volume and/or the nuclear DNA content. Thereafter the cellular RNA concentration declined rapidly. In the oocyte the RNA concentration evolved differently especially from stage 10 to the end, the RNA density being stabilized. This can be related to the injection of nurse cell mitochondria, followed by their assignment to reserve status. Our results suggest that the mt RNA density is under extramitochondrial control mechanisms.