Barentsz is essential for the posterior localization of oskar mRNA and colocalizes with it to the posterior pole

The localization of Oskar at the posterior pole of the Drosophila oocyte induces the assembly of the pole plasm and therefore defines where the abdomen and germ cells form in the embryo. This localization is achieved by the targeting of oskar mRNA to the posterior and the localized activation of its translation. oskar mRNA seems likely to be actively transported along microtubules, since its localization requires both an intact microtubule cytoskeleton and the plus end-directed motor kinesin I, but nothing is known about how the RNA is coupled to the motor. Here, we describe barentsz, a novel gene required for the localization of oskar mRNA. In contrast to all other mutations that disrupt this process, barentsz-null mutants completely block the posterior localization of oskar mRNA without affecting bicoid and gurken mRNA localization, the organization of the microtubules, or subsequent steps in pole plasm assembly. Surprisingly, most mutant embryos still form an abdomen, indicating that oskar mRNA localization is partially redundant with the translational control. Barentsz protein colocalizes to the posterior with oskar mRNA, and this localization is oskar mRNA dependent. Thus, Barentsz is essential for the posterior localization of oskar mRNA and behaves as a specific component of the oskar RNA transport complex.     

The endocytic pathway acts downstream of Oskar in Drosophila germ plasm assembly

Cell fate is often determined by the intracellular localization of RNAs and proteins. In Drosophila oocytes, oskar (osk) RNA localization and the subsequent Osk synthesis at the posterior pole direct the assembly of the pole plasm, where factors for the germline and abdomen formation accumulate. osk RNA produces two isoforms, long and short Osk, which have distinct functions in pole plasm assembly. Short Osk recruits downstream components of the pole plasm, whose anchoring to the posterior cortex requires long Osk. The anchoring of pole plasm components also requires actin cytoskeleton, and Osk promotes long F-actin projections in the oocyte posterior cytoplasm. However, the mechanism by which Osk mediates F-actin reorganization remains elusive. Furthermore, although long Osk is known to associate with endosomes under immuno-electron microscopy, it was not known whether this association is functionally significant. Here we show that Rabenosyn-5 (Rbsn-5), a Rab5 effector protein required for the early endocytic pathway, is crucial for pole plasm assembly. rbsn-5(-) oocytes fail to maintain microtubule polarity, which secondarily disrupts osk RNA localization. Nevertheless, anteriorly misexpressed Osk, particularly long Osk, recruits endosomal proteins, including Rbsn-5, and stimulates endocytosis. In oocytes lacking rbsn-5, the ectopic Osk induces aberrant F-actin aggregates, which diffuse into the cytoplasm along with pole plasm components. We propose that Osk stimulates endosomal cycling, which in turn promotes F-actin reorganization to anchor the pole plasm components to the oocyte cortex.     

Oskar anchoring restricts pole plasm formation to the posterior of the Drosophila oocyte

Localization of the maternal determinant Oskar at the posterior pole of Drosophila melanogaster oocyte provides the positional information for pole plasm formation. Spatial control of Oskar expression is achieved through the tight coupling of mRNA localization to translational control, such that only posterior-localized oskar mRNA is translated, producing the two Oskar isoforms Long Osk and Short Osk. We present evidence that this coupling is not sufficient to restrict Oskar to the posterior pole of the oocyte. We show that Long Osk anchors both oskar mRNA and Short Osk, the isoform active in pole plasm assembly, at the posterior pole. In the absence of anchoring by Long Osk, Short Osk disperses into the bulk cytoplasm during late oogenesis, impairing pole cell formation in the embryo. In addition, the pool of untethered Short Osk causes anteroposterior patterning defects, owing to the dispersion of pole plasm and its abdomen-inducing activity throughout the oocyte. We show that the N-terminal extension of Long Osk is necessary but not sufficient for posterior anchoring, arguing for multiple docking elements in Oskar. This study reveals cortical anchoring of the posterior determinant Oskar as a crucial step in pole plasm assembly and restriction, required for proper development of Drosophila melanogaster.     

Drosophila Mon2 couples Oskar-induced endocytosis with actin remodeling for cortical anchorage of the germ plasm

Drosophila pole (germ) plasm contains germline and abdominal determinants. Its assembly begins with the localization and translation of oskar (osk) RNA at the oocyte posterior, to which the pole plasm must be restricted for proper embryonic development. Osk stimulates endocytosis, which in turn promotes actin remodeling to form long F-actin projections at the oocyte posterior pole. Although the endocytosis-coupled actin remodeling appears to be crucial for the pole plasm anchoring, the mechanism linking Osk-induced endocytic activity and actin remodeling is unknown. Here, we report that a Golgi-endosomal protein, Mon2, acts downstream of Osk to remodel cortical actin and to anchor the pole plasm. Mon2 interacts with two actin nucleators known to be involved in osk RNA localization in the oocyte, Cappuccino (Capu) and Spire (Spir), and promotes the accumulation of the small GTPase Rho1 at the oocyte posterior. We also found that these actin regulators are required for Osk-dependent formation of long F-actin projections and cortical anchoring of pole plasm components. We propose that, in response to the Osk-mediated endocytic activation, vesicle-localized Mon2 acts as a scaffold that instructs the actin-remodeling complex to form long F-actin projections. This Mon2-mediated coupling event is crucial to restrict the pole plasm to the oocyte posterior cortex.     

Arginine methylation of Aubergine mediates Tudor binding and germ plasm localization

Piwi proteins such as Drosophila Aubergine (Aub) and mouse Miwi are essential for germline development and for primordial germ cell (PGC) specification. They bind piRNAs and contain symmetrically dimethylated arginines (sDMAs), catalyzed by dPRMT5. PGC specification in Drosophila requires maternal inheritance of cytoplasmic factors, including Aub, dPRMT5, and Tudor (Tud), that are concentrated in the germ plasm at the posterior end of the oocyte. Here we show that Miwi binds to Tdrd6 and Aub binds to Tudor, in an sDMA-dependent manner, demonstrating that binding of sDMA-modified Piwi proteins with Tudor-domain proteins is an evolutionarily conserved interaction in germ cells. We report that in Drosophila tud¹ mutants, the piRNA pathway is intact and most transposons are not de-repressed. However, the localization of Aub in the germ plasm is severely reduced. These findings indicate that germ plasm assembly requires sDMA modification of Aub by dPRMT5, which, in turn, is required for binding to Tudor. Our study also suggests that the function of the piRNA pathway in PGC specification may be independent of its role in transposon control.     

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.     

Distinct mechanisms for mRNA localization during embryonic axis specification in the wasp Nasonia

mRNA localization is a powerful mechanism for targeting factors to different regions of the cell and is used in Drosophila to pattern the early embryo. During oogenesis of the wasp Nasonia, mRNA localization is used extensively to replace the function of the Drosophila bicoid gene for the initiation of patterning along the antero-posterior axis. Nasonia localizes both caudal and nanos to the posterior pole, whereas giant mRNA is localized to the anterior pole of the oocyte; orthodenticle1 (otd1) is localized to both the anterior and posterior poles. The abundance of differentially localized mRNAs during Nasonia oogenesis provided a unique opportunity to study the different mechanisms involved in mRNA localization. Through pharmacological disruption of the microtubule network, we found that both anterior otd1 and giant, as well as posterior caudal mRNA localization was microtubule-dependent. Conversely, posterior otd1 and nanos mRNA localized correctly to the posterior upon microtubule disruption. However, actin is important in anchoring these two posteriorly localized mRNAs to the oosome, the structure containing the pole plasm. Moreover, we find that knocking down the functions of the genes tudor and Bicaudal-D mimics disruption of microtubules, suggesting that tudor's function in Nasonia is different from flies, where it is involved in formation of the pole plasm.     

Glorund interactions in the regulation of gurken and oskar mRNAs

Precise temporal and spatial regulation of gene expression during Drosophila oogenesis is essential for patterning the anterior-posterior and dorsal-ventral body axes. Establishment of the anterior-posterior axis requires posterior localization and translational control of both oskar and nanos mRNAs. Establishment of the dorsal-ventral axis depends on the precise restriction of gurken mRNA and protein to the dorsal-anterior corner of the oocyte. We have previously shown that Glorund, the Drosophila hnRNP F/H homolog, contributes to anterior-posterior axis patterning by regulating translation of nanos mRNA, through a direct interaction with its 3′ untranslated region. To investigate the pleiotropy of the glorund mutant phenotype, which includes dorsal-ventral and nuclear morphology defects, we searched for proteins that interact with Glorund. Here we show that Glorund is part of a complex containing the hnRNP protein Hrp48 and the splicing factor Half-pint and plays a role both in mRNA localization and nurse cell chromosome organization, probably by regulating alternative splicing of ovarian tumor. We propose that Glorund is a component of multiple protein complexes and functions both as a translational repressor and splicing regulator for anterior-posterior and dorsal-ventral patterning.     

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.     

An oskar-dependent positive feedback loop maintains the polarity of the Drosophila oocyte

The localization of oskar mRNA to the posterior of the Drosophila oocyte defines the site of assembly of the pole plasm, which contains the abdominal and germline determinants. oskar mRNA localization requires the polarization of the microtubule cytoskeleton, which depends on the recruitment of PAR-1 to the posterior cortex in response to a signal from the follicle cells, where it induces an enrichment of microtubule plus ends. Here, we show that overexpressed oskar mRNA localizes to the middle of the oocyte, as well as the posterior. This ectopic localization depends on the premature translation of Oskar protein, which recruits PAR-1 and microtubule-plus-end markers to the oocyte center instead of the posterior pole, indicating that Oskar regulates the polarity of the cytoskeleton. Oskar also plays a role in the normal polarization of the oocyte; mutants that disrupt oskar mRNA localization or translation strongly reduce the posterior recruitment of microtubule plus ends. Thus, oskar mRNA localization is required to stabilize and amplify microtubule polarity, generating a positive feedback loop in which Oskar recruits PAR-1 to the posterior to increase the microtubule cytoskeleton's polarization, which in turn directs the localization of more oskar mRNA.     

Targeting and anchoring Tudor in the pole plasm of the Drosophila oocyte

Background

Germline formation is a highly regulated process in all organisms. In Drosophila embryos germ cells are specified by the pole plasm, a specialized cytoplasmic region containing polar granules. Components of these granules are also present in the perinuclear ring surrounding nurse cells, the nuage. Two such molecules are the Vasa and Tudor proteins. How Tudor localizes and is maintained in the pole plasm is, however, not known.

Methodology/Principal Findings

Here, the process of Tudor localization in nuage and pole plasm was analyzed. The initial positioning of Tudor at the posterior pole of stage 9 oocytes was found to occur in the absence of a structurally detectable nuage. However, in mutants for genes encoding components of the nuage, including vasa, aubergine, maelstrom, and krimper, Tudor was detached from the posterior cortex in stage 10 oocytes, suggesting a prior passage in the nuage for its stability in the pole plasm. Further studies indicated that Valois, which was previously shown to bind in vitro to Tudor, mediates the localization of Tudor in the pole plasm by physically interacting with Oskar, the polar granule organizer. An association between Tudor and Vasa mediated by RNA was also detected in ovarian extracts.

Conclusions/Significance

The present data challenge the view that the assembly of the polar granules occurs in a stepwise and hierarchical manner and, consequently, a revised model of polar granule assembly is proposed. In this model Oskar recruits two downstream components of the polar granules, Vasa and Tudor, independently from each other: Vasa directly interacts with Oskar while Valois mediates the recruitment of Tudor by interacting with Oskar and Tudor.     

C-terminal moiety of Tudor contains its in vivo activity in Drosophila

Background

In early Drosophila embryos, the germ plasm is localized to the posterior pole region and is partitioned into the germline progenitors, known as pole cells. Germ plasm, or pole plasm, contains the polar granules which form during oogenesis and are required for germline development. Components of these granules are also present in the perinuclear region of the nurse cells, the nuage. One such component is Tudor (Tud) which is a large protein containing multiple Tudor domains. It was previously reported that specific Tudor domains are required for germ cell formation and Tud localization.

Methodology/Principal Findings

In order to better understand the function of Tud the distribution and functional activity of fragments of Tud were analyzed. These fragments were fused to GFP and the fusion proteins were synthesized during oogenesis. Non-overlapping fragments of Tud were found to be able to localize to both the nuage and pole plasm. By introducing these fragments into a tud mutant background and testing their ability to rescue the tud phenotype, I determined that the C-terminal moiety contains the functional activity of Tud. Dividing this fragment into two parts reduces its localization in pole plasm and abolishes its activity.

Conclusions/Significance

I conclude that the C-terminal moiety of Tud contains all the information necessary for its localization in the nuage and pole plasm and its pole cell-forming activity. The present results challenge published data and may help refining the functional features of Tud.     

A stem–loop structure directs oskar mRNA to microtubule minus ends

mRNA transport coupled with translational control underlies the intracellular localization of many proteins in eukaryotic cells. This is exemplified in Drosophila, where oskar mRNA transport and translation at the posterior pole of the oocyte direct posterior patterning of the embryo. oskar localization is a multistep process. Within the oocyte, a spliced oskar localization element (SOLE) targets oskar mRNA for plus end-directed transport by kinesin-1 to the posterior pole. However, the signals mediating the initial minus end-directed, dynein-dependent transport of the mRNA from nurse cells into the oocyte have remained unknown. Here, we show that a 67-nt stem–loop in the oskar 3′ UTR promotes oskar mRNA delivery to the developing oocyte and that it shares functional features with the fs(1)K10 oocyte localization signal. Thus, two independent cis-acting signals, the oocyte entry signal (OES) and the SOLE, mediate sequential dynein- and kinesin-dependent phases of oskar mRNA transport during oogenesis. The OES also promotes apical localization of injected RNAs in blastoderm stage embryos, another dynein-mediated process. Similarly, when ectopically expressed in polarized cells of the follicular epithelium or salivary glands, reporter RNAs bearing the oskar OES are apically enriched, demonstrating that this element promotes mRNA localization independently of cell type. Our work sheds new light on how oskar mRNA is trafficked during oogenesis and the RNA features that mediate minus end-directed transport.     

Drosophila pericentrin‐like protein promotes the formation of primordial germ cells

Primordial germ cells (PGCs) are the precursors to the adult germline stem cells that are set aside early during embryogenesis and specified through the inheritance of the germ plasm, which contains the mRNAs and proteins that function as the germline fate determinants. In Drosophila melanogaster, formation of the PGCs requires the microtubule and actin cytoskeletal networks to actively segregate the germ plasm from the soma and physically construct the pole buds (PBs) that protrude from the posterior cortex. Of emerging importance is the central role of centrosomes in the coordination of microtubule dynamics and actin organization to promote PGC development. We previously identified a requirement for the centrosome protein Centrosomin (Cnn) in PGC formation. Cnn interacts directly with Pericentrin‐like protein (PLP) to form a centrosome scaffold structure required for pericentriolar material recruitment and organization. In this study, we identify a role for PLP at several discrete steps during PGC development. We find PLP functions in segregating the germ plasm from the soma by regulating microtubule organization and centrosome separation. These activities further contribute to promoting PB protrusion and facilitating the distribution of germ plasm in proliferating PGCs.     

PKA-R1 spatially restricts Oskar expression for Drosophila embryonic patterning

Targeting proteins to specific domains within the cell is central to the generation of polarity, which underlies many processes including cell fate specification and pattern formation during development. The anteroposterior and dorsoventral axes of the Drosophila melanogaster embryo are determined by the activities of localized maternal gene products. At the posterior pole of the oocyte, Oskar directs the assembly of the pole plasm, and is thus responsible for formation of abdomen and germline in the embryo. Tight restriction of oskar activity is achieved by mRNA localization, localization-dependent translation, anchoring of the RNA and protein, and stabilization of Oskar at the posterior pole. Here we report that the type 1 regulatory subunit of cAMP-dependent protein kinase (Pka-R1) is crucial for the restriction of Oskar protein to the oocyte posterior. Mutations in PKA-R1 cause premature and ectopic accumulation of Oskar protein throughout the oocyte. This phenotype is due to misregulation of PKA catalytic subunit activity and is suppressed by reducing catalytic subunit gene dosage. These data demonstrate that PKA mediates the spatial restriction of Oskar for anteroposterior patterning of the Drosophila embryo and that control of PKA activity by PKA-R1 is crucial in this process.     

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.     

Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila

BACKGROUND: Localization of nanos mRNA to the posterior pole of the Drosophila embryo directs local synthesis of Nanos protein that is essential for patterning of the anterior-posterior body axis and germ cell function. While nanos RNA is synthesized by the ovarian nurse cells and appears at the posterior pole of the ooctye late in oogenesis, the mechanism by which this RNA is translocated to and anchored at the oocyte posterior is unknown. RESULTS: By labeling endogenous nanos RNA with GFP, we have been able to follow the dynamic pathway of nanos localization in living oocytes. We demonstrate that nanos localization initiates immediately upon nurse cell dumping, whereby diffusion, enhanced by microtubule-dependent cytoplasmic movements, translocates nanos RNA from the nurse cells to the ooctye posterior. At the posterior, nanos is trapped by association, in particles, with the posteriorly localized germ plasm. Actin-dependent anchoring of nanos RNA complexed to the germ plasm at the posterior maintains localization in the face of rapid cytoplasmic movements. CONCLUSIONS: These results reveal a diffusion-based, late-acting posterior localization mechanism for long-range transport of nanos mRNA. This mechanism differs from directed transport-based localization mechanisms in its reliance on bulk movement of RNA.     

Drosophila tudor is essential for polar granule assembly and pole cell specification, but not for posterior patterning

Pole cells and posterior segmentation in Drosophila are specified by maternally encoded genes whose products accumulate at the posterior pole of the oocyte. Among these genes is tudor (tud). Progeny of hypomorphic tud mothers lack pole cells and have variable posterior patterning defects. We have isolated a null allele to further investigate tud function. While no pole cells are ever observed in embryos from tud-null mothers, 15% of these embryos have normal posterior patterning. OSKAR (OSK) and VASA (VAS) proteins, and nanos (nos) RNA, all initially localize to the pole plasm of tud-null oocytes and embryos from tud-null mothers, while localization of germ cell-less (gcl) and polar granule component (pgc), is undetectable or severely reduced. In embryos from tud-null mothers, polar granules are greatly reduced in number, size, and electron density. Thus, tud is dispensable for somatic patterning, but essential for pole cell specification and polar granule formation.