Drosophila Oogenesis Research Articles

Mago Nashi, Tsunagi/Y14, and Ranshi form a complex that influences oocyte differentiation in Drosophila melanogaster

During Drosophila melanogaster oogenesis, a germline stem cell divides forming a cyst of 16 interconnected cells. One cell enters the oogenic pathway, and the remaining 15 differentiate as nurse cells. Although directed transport and localization of oocyte differentiation factors within the single cell are indispensible for selection, maintenance, and differentiation of the oocyte, the mechanisms regulating these events are poorly understood. Mago Nashi and Tsunagi/Y14, core components of the exon junction complex (a multiprotein complex assembled on spliced RNAs), are essential for restricting oocyte fate to a single cell and for localization of oskar mRNA. Here we provide evidence that Mago Nashi and Tsunagi/Y14 form an oogenic complex with Ranshi, a protein with a zinc finger-associated domain and zinc finger domains. Genetic analyses of ranshi reveal that (1) 16-cell cysts are formed, (2) two cells retain synaptonemal complexes, (3) all cells have endoreplicated DNA (as observed in nurse cells), and (4) oocyte-specific cytoplasmic markers accumulate and persist within a single cell but are not localized within the posterior pole of the presumptive oocyte. Our results indicate that Ranshi interacts with the exon junction complex to localize components essential for oocyte differentiation within the posterior pole of the presumptive oocyte.

Role of Scrib and Dlg in anterior-posterior patterning of the follicular epithelium during Drosophila oogenesis

BackgroundProper patterning of the follicle cell epithelium over the egg chamber is essential for the Drosophila egg development. Differentiation of the epithelium into several distinct cell types along the anterior-posterior axis requires coordinated activities of multiple signaling pathways. Previously, we reported that lethal(2)giant larvae (lgl), a Drosophila tumor suppressor gene, is required in the follicle cells for the posterior follicle cell (PFC) fate induction at mid-oogenesis. Here we explore the role of another two tumor suppressor genes, scribble (scrib) and discs large (dlg), in the epithelial patterning.ResultsWe found that removal of scrib or dlg function from the follicle cells at posterior terminal of the egg chamber causes a complete loss of the PFC fate. Aberrant specification and differentiation of the PFCs in the mosaic clones can be ascribed to defects in coordinated activation of the EGFR, JAK and Notch signaling pathways in the multilayered cells. Meanwhile, the clonal analysis revealed that loss-of-function mutations in scrib/dlg at the anterior domains result in a partially penetrant phenotype of defective induction of the stretched and centripetal cell fate, whereas specification of the border cell fate can still occur in the most anterior region of the mutant clones. Further, we showed that scrib genetically interacts with dlg in regulating posterior patterning of the epithelium.ConclusionIn this study we provide evidence that scrib and dlg function differentially in anterior and posterior patterning of the follicular epithelium at oogenesis. Further genetic analysis indicates that scrib and dlg act in a common pathway to regulate PFC fate induction. This study may open another window for elucidating role of scrib/dlg in controlling epithelial polarity and cell proliferation during development.

Notch Signaling and Developmental Cell-Cycle Arrest in Drosophila Polar Follicle Cells

Temporal and spatial regulation of cell division is critical for proper development of multicellular organisms. An important aspect of this regulation is cell-cycle arrest, which in many cell types is coupled with differentiated status. Here we report that the polar cells--a group of follicle cells differentiated early during Drosophila oogenesis--are arrested at G2 phase and can serve as a model cell type for investigation of developmental regulation of cell-cycle arrest. On examining the effects of String, a mitosis-promoting phosphatase Cdc25 homolog, and Notch signaling in polar cells, we found that misexpression of String can trigger mitosis in existing polar cells to induce extra polar cells. Normally, differentiation of the polar cells requires Notch signaling. We found that the Notch-induced extra polar cells arise through recruitment of the neighboring cells rather than promotion of proliferation, and they are also arrested at G2 phase. Notch signaling is probably involved in down-regulating String in polar cells, thus inducing the G2 cell-cycle arrest.

Germline cyst formation and incomplete cytokinesis during Drosophila melanogaster oogenesis

Germline cyst formation via incomplete cytokinesis (IC) is necessary to generate functional eggs and sperm in various organisms. Drosophila melanogaster oogenesis is an ideal system for studying IC. 29 stages of germline cyst formation can be identified in D. melanogaster oogenesis. We have defined necessary terminology to describe IC and have developed a method to measure the sizes of contractile rings and ring canals. Time course study of germline cyst formation demonstrates that contractile ring constriction proceeds to a defined end point unique for each mitotic division. Contractile rings constrict to a greater degree, resulting in smaller ring diameters, for each subsequent round of mitotic division. Contrary to conventional wisdom, ring canal growth is not initiated until well after the fourth mitotic division. Ring canals grow, in an orderly manner, with ring canals derived from the first mitotic division enlarging first followed by those from the second, then those from the third, and finally those from the fourth mitotic division. This work establishes a foundation for identifying genes specific for IC and for elucidating the molecular mechanism underlying this aspect of germline cyst formation.

Cytogenetic effects of 2,4,5-trichlorophenoxy-acetic acid on oogenesis and early embryogenesis in Drosophila melanogaster.

This investigation has shown that even in very small dosages the herbicide 2,4,5-T affects the early oogenesis and causes chromosome disturbances, which may result in sterility in Drosophila melanogaster. The adult flies themselves can apparently live unaffected on higher dosages than those recommended in practice for application, for example to scrubs. Different Drosophila test-methods are discussed.

Role of Mammalian Ecdysoneless in Cell Cycle Regulation

The Ecdysoneless (Ecd) protein is required for cell-autonomous roles in development and oogenesis in Drosophila, but the function of its evolutionarily conserved mammalian orthologs is not clear. To study the cellular function of Ecd in mammalian cells, we generated Ecd(lox/lox) mouse embryonic fibroblast cells from Ecd floxed mouse embryos. Cre-mediated deletion of Ecd in Ecd(lox/lox) mouse embryonic fibroblasts led to a proliferative block due to a delay in G(1)-S cell cycle progression; this defect was reversed by the introduction of human Ecd. Loss of Ecd led to marked down-regulation of E2F target gene expression. Furthermore, Ecd directly bound to Rb at the pocket domain and competed with E2F for binding to hypophosphorylated Rb. Our results demonstrate that mammalian Ecd plays a role in cell cycle progression via the Rb-E2F pathway.

Identification of a Short Spir Interaction Sequence at the C-terminal End of Formin Subgroup Proteins

The actin nucleation factors Spire and Cappuccino interact with each other and regulate essential cellular events during Drosophila oogenesis in a cooperative fashion. The interaction blocks formin actin nucleation activity and enhances the Spire activity. Analogous to Spire and Cappuccino, the mammalian homologs Spir-1 and formin-2 show a regulatory interaction. To get an understanding of the nature of the Spir-formin cooperation, we have analyzed the interaction biochemically and biophysically. Our data shows that the association of Spir-1 and formin-2 is not significantly mediated by binding of the Spir-1-KIND domain to the formin FH2 core domain. Instead, a short sequence motif C-terminal adjacent to the formin-2-FH2 domain could be characterized that mediates the interaction and is conserved among the members of the Fmn subgroup of formins. In line with this, we found that both mammalian Spir proteins, Spir-1 and Spir-2, interact with mammalian Fmn subgroup proteins formin-1 and formin-2.

Translational Repression of Cyclin E Prevents Precocious Mitosis and Embryonic Gene Activation during C. elegans Meiosis

Germ cells, the cells that give rise to sperm and egg, maintain the potential to recreate all cell types in a new individual. This wide developmental potential, or totipotency, is manifested in unusual tumors called teratomas, in which germ cells undergo somatic differentiation. Although recent studies have implicated RNA regulation, the mechanism that normally prevents the loss of germ cell identity remains unexplained. In C. elegans, a teratoma is induced in the absence of the conserved RNA-binding protein GLD-1. Here, we demonstrate that GLD-1 represses translation of CYE-1/cyclin E during meiotic prophase, which prevents germ cells from re-entering mitosis and inducing embryonic-like transcription. We describe a mechanism that prevents precocious mitosis in germ cells undergoing meiosis, propose that this mechanism maintains germ cell identity by delaying the onset of embryonic gene activation until after fertilization, and provide a paradigm for the possible origin of human teratomas.

Wash functions downstream of Rho and links linear and branched actin nucleation factors

Wiskott-Aldrich Syndrome (WAS) family proteins are Arp2/3 activators that mediate the branched-actin network formation required for cytoskeletal remodeling, intracellular transport and cell locomotion. Wasp and Scar/WAVE, the two founding members of the family, are regulated by the GTPases Cdc42 and Rac, respectively. By contrast, linear actin nucleators, such as Spire and formins, are regulated by the GTPase Rho. We recently identified a third WAS family member, called Wash, with Arp2/3-mediated actin nucleation activity. We show that Drosophila Wash interacts genetically with Arp2/3, and also functions downstream of Rho1 with Spire and the formin Cappuccino to control actin and microtubule dynamics during Drosophila oogenesis. Wash bundles and crosslinks F-actin and microtubules, is regulated by Rho1, Spire and Arp2/3, and is essential for actin cytoskeleton organization in the egg chamber. Our results establish Wash and Rho as regulators of both linear- and branched-actin networks, and suggest an Arp2/3-mediated mechanism for how cells might coordinately regulate these structures.

Enabled and Capping protein play important roles in shaping cell behavior during Drosophila oogenesis

During development, cells craft an impressive array of actin-based structures, mediating events as diverse as cytokinesis, apical constriction, and cell migration. One challenge is to determine how cells regulate actin assembly and disassembly to carry out these cell behaviors. During Drosophila oogenesis diverse cell behaviors are seen in the soma and germline. We used oogenesis to explore developmental roles of two important actin regulators: Enabled/VASP proteins and Capping protein. We found that Enabled plays an important role in cortical integrity of nurse cells, formation of robust bundled actin filaments in late nurse cells that facilitate nurse cell dumping, and migration of somatic border cells. During nurse cell dumping, Enabled localizes to barbed ends of the nurse cell actin filaments, suggesting its mechanism of action. We further pursued this mechanism using mutant Enabled proteins, each affecting one of its protein domains. These data suggest critical roles for the EVH2 domain and its tetramerization subdomain, while the EVH1 domain appears less critical. Enabled appears to be negatively regulated during oogenesis by Abelson kinase. We also explored the function of Capping protein. This revealed important roles in oocyte determination, nurse cell cortical integrity and nurse cell dumping, and support the idea that Capping protein and Enabled act antagonistically during dumping. Together these data reveal places that these actin regulators shape oogenesis.

The spinal muscular atrophy protein SMN affects Drosophila germline nuclear organization through the U body–P body pathway

Survival motor neuron protein (SMN) is the determining factor for the human neurodegenerative disease spinal muscular atrophy (SMA). SMN is critical for small nuclear ribonucleoprotein (snRNP) assembly. Using Drosophila oogenesis as a model system, we show that mutations in smn cause abnormal nuclear organization in nurse cells and oocytes. Germline and mitotic clonal analysis reveals that both nurse cells and oocytes require SMN to maintain normal organization of nuclear compartments including chromosomes, nucleoli, Cajal bodies and histone locus bodies. We previously found that SMN-containing U bodies invariably associate with P bodies (Liu, J. L., and Gall, J. G. (2007). U bodies are cytoplasmic structures that contain uridine-rich small nuclear ribonucleoproteins and associate with P bodies. Proc. Natl. Acad. Sci. U. S. A. 104, 11655–11659.). Multiple lines of evidence implicate SMN in the regulation of germline nuclear organization through the connection of U bodies and P bodies. Firstly, smn germline clones phenocopy mutations for two P body components, Cup and Ovarian tumour (Otu). Secondly, P body mutations disrupt SMN distribution and the organization of U bodies. Finally, mutations in smn disrupt the function and organization of U bodies and P bodies. Taken together, our results suggest that SMN is required for the functional integrity of the U body–P body pathway, which in turn is important for maintaining proper nuclear architecture.

Quantitative analysis of epithelial morphogenesis in Drosophila oogenesis: New insights based on morphometric analysis and mechanical modeling

The process of epithelial morphogenesis is ubiquitous in animal development, but much remains to be learned about the mechanisms that shape epithelial tissues. The follicle cell (FC) epithelium encapsulating the growing germline of Drosophila is an excellent system to study fundamental elements of epithelial development. During stages 8 to 10 of oogenesis, the FC epithelium transitions between simple geometries–cuboidal, columnar and squamous–and redistributes cell populations in processes described as posterior migration, squamous cell flattening and main body cell columnarization. Here we have carried out a quantitative morphometric analysis of these poorly understood events in order to establish the parameters of and delimit the potential processes that regulate the transitions. Our results compel a striking revision of accepted views of these phenomena, by showing that posterior migration does not involve FC movements, that there is no role for columnar cell apical constriction in FC morphogenesis, and that squamous cell flattening may be a compliant response to germline growth. We utilize mechanical modeling involving finite element computational technologies to demonstrate that time-varying viscoelastic properties and growth are sufficient to account for the bulk of the FC morphogenetic changes.

Breaking a temporal barrier: signalling crosstalk regulates the initiation of border cell migration

Correct timing of developmental events is crucial for generating a normal organism. During oogenesis in Drosophila melanogaster, migration of border cells occurs in a defined temporal window and requires Jak/Stat and steroid hormone signalling. The initiation of border-cell migration is now shown to be timed by Jak/Stat-mediated downregulation of the BTB domain transcriptional regulator Abrupt, which acts as a negative regulator of steroid hormone signalling.

Bruno negatively regulates germ cell-less expression in a BRE-independent manner

Mechanisms of post-transcriptional control are essential during Drosophila oogenesis and embryogenesis to sequester gene products in discrete regions and ultimately achieve embryonic asymmetry. Maternal germ cell-less ( gcl) mRNA accumulates in the pole plasm of the embryo before Gcl protein is detectable. gcl mRNA, but not Gcl protein, can also be detected in somatic regions of the embryo, suggesting that gcl RNA is subject to translational control. We find that Gcl is expressed during oogenesis, and that it is regulated by the translational repressor Bruno (Bru). Increased levels of Gcl are observed in the oocyte when Bru level is reduced, and overexpression of Bru reduces Gcl expression. Consistently, reduction of the maternal dosage of Bruno leads to ectopic Gcl expression in the embryo, which, in turn, represses anterior hückebein ( hkb) expression. Bru binds directly to the gcl 3’UTR in vitro, but, surprisingly, this binding is independent of a BRE (Bruno response element)-like motif. This motif is also not required for in vivo repression of Gcl expression during oogenesis or early embryogenesis. Bru binds the gcl 3’UTR via its C-terminal domain, which includes RNA recognition motif 3 (RRM3), with little or no contribution from the remainder of the protein. We conclude that repression by Bruno during oogenesis is required to restrict Gcl expression in the early embryo and that Bru represses gcl expression in a manner that involves RRM3 and a sequence unrelated to the BRE.

Microtubules, the ER and Exu: New associations revealed by analysis of mini spindles mutations

During Drosophila oogenesis, organized microtubule networks coordinate the localization of specific RNAs, the positioning of the oocyte nucleus, and ooplasmic streaming events. We used mutations in mini spindles (msps), a microtubule-associated protein, to disrupt microtubule function during mid- and late-oogenesis, and show that msps is required for these microtubule-based events. Since endoplasmic reticulum (ER) organization is influenced by microtubules in other systems, we hypothesized that using msps to alter microtubule dynamics might affect the structure and organization of the ER in nurse cells and the oocyte. ER organization was monitored using GFP-tagged versions of Reticulon-like1 and protein disulfide isomerase. Analyses of living cells indicate microtubule associations mediate the movement of ER components within the oocyte. Surprisingly, the distribution and behavior of tubular ER in the oocyte differs from general ER, suggesting these two compartments of the ER interact differently with microtubules. We find that the morphology of Exu particles is msps-dependent, and that Exu is specifically associated with tubular ER in msps mutants. Our results extend previous descriptions of sponge bodies and the fusome, suggesting both are manifestations of a dynamic structure that interacts with microtubules and persists throughout oogenesis.

Transcriptional autoregulation in development

The authors would like to thank Peter Harte and Mark Ptashne for helpful and stimulating comments on the manuscript. Our lab's research is supported by grants from the NIH and NSF. The UNC Developmental Biology NIH training grant provided support to J.C.P.

Mutagenic effects of chlorinated phenoxyacetic acids in Drosophila melanogaster

HereditasVolume 87, Issue 1 p. 121-123 Open Access Mutagenic effects of chlorinated phenoxyacetic acids in Drosophila melanogaster Jan Magnusson, Jan MagnussonSearch for more papers by this authorClaes Ramel, Corresponding Author Claes RamelEnvironmental Toxicology Unit Wallenberg Laboratory Lilla Frescati S-104 05 Stockholm 50. SwedenSearch for more papers by this authorAnn Eriksson, Ann ErikssonSearch for more papers by this author Jan Magnusson, Jan MagnussonSearch for more papers by this authorClaes Ramel, Corresponding Author Claes RamelEnvironmental Toxicology Unit Wallenberg Laboratory Lilla Frescati S-104 05 Stockholm 50. SwedenSearch for more papers by this authorAnn Eriksson, Ann ErikssonSearch for more papers by this author First published: November 1977 https://doi.org/10.1111/j.1601-5223.1977.tb01252.xCitations: 12AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Literature cited Dävring, L. 1975. Effects of a 2,4,5-T ester on early oogenesis, fertility and development in Drosophila melanogaster. Hereditas 80: 255– 262. Dävring, L. and Sunner, M. 1971. Cytogenetic effects of 2,4,5-trichlorophenoxyacetic acid on oogenesis and early embryogenesis in Drosophila melanogaster. Hereditas 68: 115– 122. Jenssen, D. and Renberg, L. 1976. Distribution and cytogenetic test of 2,4-D and 2,4,5-T phenoxyacetic acids in mouse blood tissues, Chem.-Biol. Interactions 14: 291– 299. Grant, W. F. Cytogenetic effects of chlorinated hydrocarbon pesticides. — Nat. Inst. Health, Bethesda, Maryland, U.S.A., (in press). Majumdar, S. K. and Golia, J. K. 1974. Mutation test of 2,4,5-trichlorophenoxyacetic acid on Drosophila melanogaster. Can. J. Genet. Cytol. 16: 465– 466. Majumdar, S. K. and Hall, R. C. 1973. Cytogenetic effects of 2,4,5-T on in vivo bone marrow cells of Mongolian gerbils. Hered. 64: 213– 216. Mohandas, T. and Grant, W. F. 1972. Cytogenetic effects of 2,4-D and amitrole in relation to nuclear volume and DNA content in some higher plants. Can. J. Genet. Cytol. 14: 773– 783. Vogel, E. and Chandler, J. L. R. 1974. Mutagenicity testing of cyclamate and some pesticides in Drosophila melanogaster. Experientia 30: 621– 623. Citing Literature Volume87, Issue1November 1977Pages 121-123 ReferencesRelatedInformation

Effects of a 2,4,5-T ester on early oogenesis, fertility and development in Drosophila melanogaster.

Effects of a commercial 2,4,5-T butoxyethyl ester product on oogenesis and fertility of a Drosophila melanogaster (Canton-S) strain were measured, as well as the toxicity to adult flies. A concentration of 250 ppm, 2,4,5-T in the food reduced fertility with about 50 %. Females given only 1 ppm still showed significant disturbances in their egg follicle development. The LD50 value (dose required to produce 50 % mortality) for adult female flies had a higher magnitude of about 4700 ppm. Fully developed oocytes showed chromosomal abnormalities which were ascribed to treatment with the 2, 4, 5-T product, also in low concentrations (down to 1 ppm).

The Cyclin-dependent Kinase Inhibitor Dacapo Promotes Genomic Stability during Premeiotic S Phase

The proper execution of premeiotic S phase is essential to both the maintenance of genomic integrity and accurate chromosome segregation during the meiotic divisions. However, the regulation of premeiotic S phase remains poorly defined in metazoa. Here, we identify the p21(Cip1)/p27(Kip1)/p57(Kip2)-like cyclin-dependent kinase inhibitor (CKI) Dacapo (Dap) as a key regulator of premeiotic S phase and genomic stability during Drosophila oogenesis. In dap(-/-) females, ovarian cysts enter the meiotic cycle with high levels of Cyclin E/cyclin-dependent kinase (Cdk)2 activity and accumulate DNA damage during the premeiotic S phase. High Cyclin E/Cdk2 activity inhibits the accumulation of the replication-licensing factor Doubleparked/Cdt1 (Dup/Cdt1). Accordingly, we find that dap(-/-) ovarian cysts have low levels of Dup/Cdt1. Moreover, mutations in dup/cdt1 dominantly enhance the dap(-/-) DNA damage phenotype. Importantly, the DNA damage observed in dap(-/-) ovarian cysts is independent of the DNA double-strands breaks that initiate meiotic recombination. Together, our data suggest that the CKI Dap promotes the licensing of DNA replication origins for the premeiotic S phase by restricting Cdk activity in the early meiotic cycle. Finally, we report that dap(-/-) ovarian cysts frequently undergo an extramitotic division before meiotic entry, indicating that Dap influences the timing of the mitotic/meiotic transition.

Bicaudal-C associates with a Trailer Hitch/Me31B complex and is required for efficient Gurken secretion

Bicaudal-C ( Bic-C) is a multiple KH-domain RNA-binding protein required for Drosophila oogenesis and, maternally, for embryonic patterning. In early oogenesis, Bic-C negatively regulates target mRNAs, including Bic-C, by recruiting the CCR4 deadenylase through a direct association with its NOT3 subunit. Here, we identify a novel function for Bic-C in secretion of the TGF-α homolog Gurken (Grk). In Bic-C mutant egg chambers, Grk is sequestered within actin-coated structures during mid-oogenesis. As a consequence, Egfr signalling is not efficiently activated in the dorsal-anterior follicle cells. This phenotype is strikingly similar to that of trailer hitch ( tral) mutants. Consistent with the idea that Bic-C and Tral act together in Grk secretion, Bic-C co-localizes with Tral within cytoplasmic granules, and can be co-purified with multiple protein components of a Tral mRNP complex. Taken together, our results implicate translational regulation by Bic-C and Tral in the secretory pathway.