Src64 is involved in fusome development and karyosome formation during Drosophila oogenesis

Src family tyrosine kinases respond to a variety of signals by regulating the organization of the actin cytoskeleton. Here, we show that during early oogenesis Src64 mutations lead to uneven accumulation of cortical actin, defects in fusome formation, mislocalization of septins, defective transport of Orb protein into the oocyte, and possible defects in cell division. Similar mutant phenotypes suggest that Src64, the Tec29 tyrosine kinase, and the actin crosslinking protein Kelch act together to regulate actin crosslinking, much as they do later during ring canal growth. Condensation of the oocyte chromatin into a compact karyosome is also defective in Src64, Tec29, and kelch mutants and in mutants for spire and chickadee (profilin), genes that regulate actin polymerization. These data, along with changes in G-actin accumulation in the oocyte nucleus, suggest that Src64 is involved in a nuclear actin function during karyosome condensation. Our results indicate that Src64 regulates actin dynamics at multiple stages of oogenesis.     

F-actin is a component of the karyosome in neuropteran oocyte nuclei

The principles underlying the assembly of intranuclear compartments are only beginning to be understood. The karyosome is an organelle typical of oocyte nuclei. It represents the tightly packed oocyte chromosomes, arrested at the diplotene of meiotic prophase. It has been known from several insect orders that a prominent capsule of unknown materials is built around the karyosome in the course of previtellogenesis and vitellogenesis. Here we show that F-actin, detected by dye-coupled phalloidin, is a major molecular component of the karyosome capsule in Neuroptera. We investigated capsule formation in six species belonging to the family Chrysopidae. Though F-actin was present in the capsules of all six species there were striking interspecific differences in the morphological array of actin filaments and the developmental dynamics of actin deposition in the capsule. The potential biological significance of the karyosome capsule is discussed with respect to the presence of extrachromosomal rDNA in neuropteran oocytes and the molecular functions known from F-actin. Our results corroborate the still controversial hypothesis of a role for actin as a nuclear protein.     

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.     

NHK-1 phosphorylates BAF to allow karyosome formation in the Drosophila oocyte nucleus

Accurate chromosome segregation in meiosis requires dynamic changes in chromatin organization. In Drosophila melanogaster, upon completion of recombination, meiotic chromosomes form a single, compact cluster called the karyosome in an enlarged oocyte nucleus. This clustering is also found in humans; however, the mechanisms underlying karyosome formation are not understood. In this study, we report that phosphorylation of barrier to autointegration factor (BAF) by the conserved kinase nucleosomal histone kinase-1 (NHK-1; Drosophila Vrk1) has a critical function in karyosome formation. We find that the noncatalytic domain of NHK-1 is crucial for its kinase activity toward BAF, a protein that acts as a linker between chromatin and the nuclear envelope. A reduction of NHK-1 or expression of nonphosphorylatable BAF results in ectopic association of chromosomes with the nuclear envelope in oocytes. We propose that BAF phosphorylation by NHK-1 disrupts anchorage of chromosomes to the nuclear envelope, allowing karyosome formation in oocytes. These data provide the first mechanistic insight into how the karyosome forms.     

Oogenesis in phthirapterans (Insecta: Phthiraptera). I. Morphological and histochemical characterization of the oocyte nucleus and its inclusions

We characterize morphological and histochemical changes occurring within the oocyte nucleus (germinal vesicle) during oogenesis in two phthirapteran species: the pig louse, Haematopinus suis (Anoplura) and the pigeon louse, Columbicola columbae (Mallophaga). In previtellogenic oocytes, within the oocyte nucleus the chromatin condenses and forms the karyosome. In contact with the karyosome numerous dense and highly heterogeneous nuclear bodies occur. We demonstrate that these nuclear inclusions have complex structure and contain RNA and argyrophilic proteins of nucleolar organizer (Ag-NOR proteins). Results of immunogold electron microscopy experiments are also presented. The obtained results suggest that the phthirapteran nuclear bodies are assemblages of ribonucleoproteins that are stored in the oocyte nucleus and might be utilized during early stages of embryonic development. In the investigated species, the nuclear envelope of the germinal vesicle is equipped with characteristic protrusions. Ultrastructural analysis revealed striking similarity of these structures to the initial stages of the formation of accessory nuclei. Based on these results, we speculate on the possible evolutionary origin of the accessory nuclei in phthirapterans.     

oskar RNA plays multiple noncoding roles to support oogenesis and maintain integrity of the germline/soma distinction

The Drosophila oskar (osk) mRNA is unusual in that it has both coding and noncoding functions. As an mRNA, osk encodes a protein required for embryonic patterning and germ cell formation. Independent of that function, the absence of osk mRNA disrupts formation of the karyosome and blocks progression through oogenesis. Here we show that loss of osk mRNA also affects the distribution of regulatory proteins, relaxing their association with large RNPs within the germline, and allowing them to accumulate in the somatic follicle cells. This and other noncoding functions of the osk mRNA are mediated by multiple sequence elements with distinct roles. One role, provided by numerous binding sites in two distinct regions of the osk 3′ UTR, is to sequester the translational regulator Bruno (Bru), which itself controls translation of osk mRNA. This defines a novel regulatory circuit, with Bru restricting the activity of osk, and osk in turn restricting the activity of Bru. Other functional elements, which do not bind Bru and are positioned close to the 3′ end of the RNA, act in the oocyte and are essential. Despite the different roles played by the different types of elements contributing to RNA function, mutation of any leads to accumulation of the germline regulatory factors in the follicle cells.     

The RNA-binding protein Squid is required for the establishment of anteroposterior polarity in the Drosophila oocyte

The heterogeneous nuclear ribonucleoprotein (hnRNP) Squid (Sqd) is a highly abundant protein that is expected to bind most cellular RNAs. Nonetheless, Sqd plays a very specific developmental role in dorsoventral (DV) axis formation during Drosophila oogenesis by localizing gurken (grk) RNA. Here, we report that Sqd is also essential for anteroposterior (AP) axis formation. We identified sqd in a screen for modifiers of the Protein Kinase A (PKA) oogenesis polarity phenotype. The AP defects of sqd mutant oocytes resemble those of PKA mutants in several ways. In both cases, the cytoskeletal reorganization at mid-oogenesis, which depends on a signal from the posterior follicle cells, does not produce a correctly polarized microtubule (MT) network. This causes the posterior determinant, oskar (osk) RNA, to localize to central regions of the oocyte, where it is ectopically translated. Additionally, MT-dependent anterior movement of the oocyte nucleus and the grk-dependent specification of posterior follicle cells are unaffected in both mutants. However, in contrast to PKA mutants, sqd mutants do not retain a discrete posterior MT organizing center (MTOC) capable of supporting ectopic posterior localization of bicoid (bcd) RNA. sqd mutants also display several other phenotypes not seen in PKA mutants; these probably result from the disruption of MT polarity in earlier stages of oogenesis. Loss of Sqd does not affect polarity in follicle cells, wings or eyes, indicating a specific role in the determination of MT polarity within the germline.     

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.     

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 Schizosaccharomyces pombe cdc3+ gene encodes a profilin essential for cytokinesis

The fission yeast Schizosaccharomyces pombe divides by medial fission and, like many higher eukaryotic cells, requires the function of an F- actin contractile ring for cytokinesis. In S. pombe, a class of cdc- mutants defective for cytokinesis, but not for DNA replication, mitosis, or septum synthesis, have been identified. In this paper, we present the characterization of one of these mutants, cdc3-124. Temperature shift experiments reveal that mutants in cdc3 are incapable of forming an F-actin contractile ring. We have molecularly cloned cdc3 and used the cdc3+ genomic DNA to create a strain carrying a cdc3 null mutation by homologous recombination in vivo. Cells bearing a cdc3-null allele are inviable. They arrest the cell cycle at cytokinesis without forming a contractile ring. DNA sequence analysis of the cdc3+ gene reveals that it encodes profilin, an actin-monomer-binding protein. In light of recent studies with profilins, we propose that Cdc3-profilin plays an essential role in cytokinesis by catalyzing the formation of the F-actin contractile ring. Consistent with this proposal are our observations that Cdc3-profilin localizes to the medial region of the cell where the F-actin contractile ring forms, and that it is essential for F-actin ring formation. Cells overproducing Cdc3-profilin become elongated, dumbbell shaped, and arrest at cytokinesis without any detectable F-actin staining. This effect of Cdc3-profilin overproduction is relieved by introduction of a multicopy plasmid carrying the actin encoding gene, act1+. We attribute these effects to potential sequestration of actin monomers by profilin, when present in excess.     

Dominant mutations in the Caenorhabditis elegans Myt1 ortholog wee-1.3 reveal a novel domain that controls M-phase entry during spermatogenesis

Regulatory phosphorylation of the Cdc2p kinase by Wee1p-type kinases prevents eukaryotic cells from entering mitosis or meiosis at an inappropriate time. The canonical Wee1p kinase is a soluble protein that functions in the eukaryotic nucleus. All metazoa also have a membrane-associated Wee1p-like kinase named Myt1, and we describe the first genetic characterization of this less well-studied kinase. The Caenorhabditis elegans Myt1 ortholog is encoded by the wee-1.3 gene, and six dominant missense mutants prevent primary spermatocytes from entering M phase but do not affect either oocyte meiosis or any mitotic division. These six dominant wee-1.3(gf) mutations are located in a four amino acid region near the C terminus and they cause self-sterility of hermaphrodites. Second-site intragenic suppressor mutations in wee-1.3(gf) restore self-fertility to these dominant sterile hermaphrodites, permitting genetic dissection of this kinase. Ten intragenic wee-1.3 suppressor mutations were recovered and they form an allelic series that includes semi-dominant, hypomorphic and null mutations. These mutants reveal that WEE-1.3 protein is required for embryonic development, germline proliferation and initiation of meiosis during spermatogenesis. This suggests that a novel, sperm-specific pathway negatively regulates WEE-1.3 to allow the G2/M transition of male meiosis I, and that dominant wee-1.3 mutants prevent this negative regulation.     

Deadlock, a novel protein of Drosophila, is required for germline maintenance, fusome morphogenesis and axial patterning in oogenesis and associates with centrosomes in the early embryo

The deadlock gene is required for a number of key developmental events in Drosophila oogenesis. Females homozygous for mutations in the deadlock gene lay few eggs and those exhibit severe patterning defects along both the anterior-posterior and dorsal-ventral axis. In this study, we analyzed eggs and ovaries from deadlock mutants and determined that deadlock is required for germline maintenance, stability of mitotic spindles, localization of patterning determinants, oocyte growth and fusome biogenesis in males and females. Deadlock encodes a novel protein which colocalizes with the oocyte nucleus at midstages of oogenesis and with the centrosomes of early embryos. Our genetic and immunohistological experiments point to a role for Deadlock in microtubule function during oogenesis.     

Formation of the karyosome in developing oocytes of weevils (Coleoptera, Curculionidae)

A complex structure termed the karyosome forms in the nuclei of the developing oocytes of Anthonomus pomorum, Hylobius abietis and Phyllobius sp. (Coleoptera: Curculionidae). It is composed of highly condensed chromosomes, fused with an electron-dense granular material. There are two types of nuclear body associated with the karyosome. The smaller bodies are found in the immediate vicinity of the karyosome. The larger, and more electron-dense, bodies originate next to the condensing chromosomes. During vitellogenesis, the latter bodies disperse in the karyoplasm, and at least some of them locate in the characteristic irregular projections of the germinal vesicle. Morphologically, these projections resemble the accessory nuclei described in other insects. In the studied species, a proteinaceous sheath, the so-called karyosome capsule, surrounds the karyosome. The formation of the karyosome and its capsule occurs during previtellogenesis, so that these structures are fully formed at the onset of vitellogenesis. An extraction of the oocyte cytoplasm with Triton X-100 showed that the material constituting the karyosome capsule is filamentous. Staining with rhodamine-conjugated phalloidin reveals large amount of F-actin in the karyosome capsule.     

The identification of novel genes required for Drosophila anteroposterior axis formation in a germline clone screen using GFP-Staufen

The anteroposterior axis of Drosophila is defined during oogenesis, when the polarisation of the oocyte microtubule cytoskeleton directs the localisation of bicoid and oskar mRNAs to the anterior and posterior poles, respectively. Although maternal-effect lethal and female-sterile screens have identified many mutants that disrupt these processes, these screens could not recover mutations in essential genes. Here we describe a genetic screen in germline clones for mutants that disrupt the localisation of GFP-Staufen in living oocytes, which overcomes this limitation. As Staufen localises to the posterior with oskar mRNA and to the anterior with bicoid mRNA, it acts as a marker for both poles of the oocyte, allowing the identification of mutants that affect the localisation of either mRNA, as well as mutants that disrupt oocyte polarity. Using this approach, we have identified 23 novel complementation groups on chromosome 3R that disrupt anteroposterior axis formation. Analyses of new alleles of spn-E and orb show that both SPN-E and ORB proteins are required to organise the microtubule cytoskeleton at stage 9, and to prevent premature cytoplasmic streaming. Furthermore, yps mutants partially suppress the premature cytoplasmic streaming of orb mutants. As orb, yps and spn-E encode RNA-binding proteins, they may regulate the translation of unidentified RNAs necessary for the polarisation of the microtubule cytoskeleton.     

Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD.

While all known natural isolates of C. elegans contain multiple copies of the Tc1 transposon, which are active in the soma, Tc1 transposition is fully silenced in the germline of many strains. We mutagenized one such silenced strain and isolated mutants in which Tc1 had been activated in the germline ("mutators"). Interestingly, many other transposons of unrelated sequence had also become active. Most of these mutants are resistant to RNA interference (RNAi). We found one of the mutated genes, mut-7, to encode a protein with homology to RNaseD. This provides support for the notion that RNAi works by dsRNA-directed, enzymatic RNA degradation. We propose a model in which MUT-7, guided by transposon-derived dsRNA, represses transposition by degrading transposon-specific messengers, thus preventing transposase production and transposition.     

Identification and analysis of mutations in bob,Doa and eight new genes required for oocyte specification and development in Drosophila melanogaster

The Drosophila oocyte develops from a cluster of 16 interconnected cells that derive from a common progenitor. One of these cells, the oocyte, arrests in meiosis. The other cells endoreplicate their DNA and produce mRNAs and proteins that they traffic to the oocyte along a polarized microtubule cytoskeleton shared by the entire cyst. Therefore, Drosophila oogenesis is an attractive system for the study of cell cycle control and cell polarity. We carried out a clonal screen on the right arm of chromosome 3 for female sterile mutations using the FLP-FRT-ovo(D) system to identify new genes required for early oogenesis. We identified alleles of oo18 RNA binding protein (orb) and Darkener of apricot (Doa), which had previously been shown to exhibit oogenesis defects. We also identified several lethal alleles of the male sterile mutant, bobble (bob). In addition, we identified eight new lethal complementation groups that exhibit early oogenesis phenotypes. We analyzed mutant clones to determine the aspects of oogenesis disrupted by each complementation group. We assayed for the production and development of egg chambers, localization of ORB to and within the oocyte, and proper execution of the nurse cell cycle (endoreplication of DNA) and the oocyte cell cycle (karyosome formation). Here we discuss the identification, mapping, and phenotypic characterization of these new genes: omelet, soft boiled, hard boiled, poached, fried, over easy, sunny side up, and benedict.     

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.