The exuperantia (exu) gene is necessary for this localization of Bicoid mRNA. A chimaeric gene encoding a fusion between the Acquorea victoria green fluorescent protein (GFP) and the Exu protein is expressed in female germ cells. The fusion protein rescues an exu null allele, restoring full fertility to females, and is expressed and localized in a temporal and spatial pattern similar to native Exu. The fusion protein is found in particles concentrated at ring canals, where transport occurs between nurse cells and the oocyte. Drugs such as colchicine and taxol that affect microtubule stability alter localization of the particles. The particles may be ribonucleoprotein complexes or vesicles that transport BCD mRNA along microtubules and target it to the anterior oocyte cortex (Wang, 1994).

Localization of maternally provided RNAs during oogenesis is required for formation of the antero-posterior axis of the Drosophila embryo. A subcellular structure in nurse cells and oocytes is described which may function as an intracellular compartment for assembly and transport of maternal products involved in RNA localization. This structure, which has been termed 'sponge body,' consists of ER-like cisternae, embedded in an amorphous electron-dense mass. It lacks a surrounding membrane and is frequently associated with mitochondria. The sponge bodies are not identical to the Golgi complexes. It is suggested that the sponge bodies are homologous to the mitochondrial cloud in Xenopus oocytes, a granulo-fibrillar structure that contains RNAs involved in patterning of the embryo. Exuperantia protein, the earliest factor known to be required for the localization of bicoid mRNA to the anterior pole of the Drosophila oocyte, is highly enriched in the sponge bodies but not an essential structural component of these. RNA staining indicates that sponge bodies contain RNA. However, neither the intensity of this staining nor the accumulation of Exuperantia in the sponge bodies is dependent on the amount of bicoid mRNA present in the ovaries. Sponge bodies surround nuage, a possible polar granule precursor. Microtubules and microfilaments are not present in sponge bodies, although transport of the sponge bodies through the cells is implied by their presence in cytoplasmic bridges. It is proposed that the sponge bodies are structures that, by assembly and transport of included molecules or associated structures, are involved in localization of mRNAs in Drosophila oocytes (Wilsch-Brauninger, 1997).

Anterior patterning of the Drosophila embryo depends on localization of Bicoid mRNA to the anterior pole of the developing oocyte: BCD mRNA localization requires both the exuperantia gene and an intact microtubule cytoskeleton. A GFP-Exu fusion protein supports BCD mRNA localization and complements the embryonic anterior axis defects produced by exu mutations. During mid-oogenesis, the GFP-Exu fusion protein assembles into particles that are concentrated around the nurse cell nuclei; these particles cluster at the ring canals that link the germline cells of the egg chamber, and accumulate at the anterior pole of the oocyte. At these stages, BCD mRNA also shows a perinuclear or apical distribution in nurse cells, and accumulates at the anterior cortex of the oocyte. These observations suggest that the GFP-Exu particles are transport ribonuclear proteins containing BCD mRNA, and that these particles are targeted to the anterior cortex of the developing oocyte. Although direct proof for an association of BCD mRNA with these particles has not yet been obtained, microtubule depolymerization disrupts both BCD mRNA and Exu protein localization, suggesting that these two components utilize a similar, if not identical, anterior localization pathway (Theurkauf, 1998 and references).

To gain insight into the mechanism of anterior patterning, time lapse laser scanning confocal microscopy was used to analyze transport of particles containing a Green Fluorescent Protein-Exu fusion (GFP-Exu), and to directly image microtubule organization in vivo. These observations indicate that microtubules are required for three forms of particle movement within the nurse cells, while transport through the ring canals linking the nurse cells and oocyte appears to be independent of both microtubules and actin filaments. As particles enter the oocyte, a final microtubule-dependent step directs movement to the oocyte cortex. Exu protein and BCD mRNA are synthesized in a cluster of 15 nurse cells that are linked to the oocyte by ring canal bridges. The first cytoplasmic transport steps in anterior patterning therefore take place within the nurse cells. The analysis indicates that transport within the nurse cell cytoplasm is composed of at least three microtubule-dependent steps that produce a net movement of GFP-Exu particles toward the oocyte. The majority of the individual GFP-Exu particles within the nurse cell cytoplasm move rapidly and with no apparent net directionality with respect to the egg chamber axis. These movements are reversibly inhibited by the microtubule-disrupting drug colcemid. Microtubules throughout the nurse cell cytoplasm that lack clear orientation with respect to the egg chamber axis have been directly observed. Based on these observations, it is concluded that microtubules mediate random particle movements within the nurse cells. In the absence of microtubules, no movement or redistribution of GFP-Exu particles was observed. Simple diffusion thus appears to be insufficient to efficiently disperse these large particles. It is therefore speculated that the random microtubule-dependent particle movements are essential to dispersing GFP-Exu particles. It is proposed that this particle dispersal is required for efficient net particle transport through the nurse cells (Theurkauf, 1998).

Vectorial particle transport is observed in the region near the ring canals linking the nurse cells with the oocyte. In this region, particles tend to move directly to the cell-cell junctions. These movements, like the random movements observed in bulk nurse cell cytoplasm, are reversibly inhibited by colcemid. In addition, a dynamic population of microtubules associated with the nurse cell-oocyte ring canals is directly observed. It is therefore proposed that GFP-Exu particles approach the ring canal junctions by a microtubule-dependent random walk. The particles then associate with microtubules that are organized around the ring canals, and are transported to the nurse cell-oocyte junctions. It is this second step that imparts net directionality on particle transport through the nurse cell cytoplasm (Theurkauf, 1998).

Previous ultrastructural analysis of Exu distribution failed to identify microtubules in direct association with Exu-containing structures, termed sponge bodies. However, the current in vivo analysis indicates that at least some of the microtubules in the nurse cells turn over within 10 to 20 seconds. These microtubules are therefore likely to be difficult to preserve by standard fixation procedures. The failure to identify microtubules directly associated with sponge bodies may reflect the dynamic nature of these filaments. Perinuclear particle clustering in the nurse cells also appears to be microtubule-dependent. This process is reversibly disrupted by colcemid, and microtubules are associated with the surface of the nurse cell nuclei. The function of microtubule-dependent perinuclear clustering is not yet clear, although it seems unlikely that this process contributes directly to movement through the nurse cells. It has been proposed that Exu particles are RNPs that contain BCD mRNA, as well as other proteins. If so, these particles could form in the perinuclear region as BCD mRNA exits the nurse cell nuclei, and microtubule-dependent transport could facilitate complex formation by concentrating cytoplasmic components of the particles in this region. Consistent with this suggestion, GFP-Exu particle size is decreased by microtubule depolymerization, and particles appear to increase in size and fluorescence intensity on microtubule repolymerization (Theurkauf, 1998).

Essentially all of the GFP-Exu particle movements in the nurse cell cytoplasm are microtubule-dependent: these movements are presumably mediated by microtubule motor proteins. It is speculated that several different microtubule motors function in Exu particle motility. Alternatively, the variability in the rates and directionality of particle movements in the nurse cells could reflect complexities in the underlying microtubule cytoskeleton or particle-specific differences in the regulation of a single motor. However, the use of multiple motors for this transport process would serve to isolate axis specification from complete disruption by mutations in single motor protein genes. Mutations in known motor proteins have not yet been identified that disrupt these transport steps. Once GFP-Exu particles are localized to the nurse cell-oocyte ring canals, a distinct transport process appears to drive movement through the cell-cell junctions. These movements are uniform in direction and velocity, raising the possibility that they reflect a very local flow of cytoplasm through the ring canal junctions. The apparent absence of bulk movement through the ring canal suggests that this step in the transport pathway is not due to cytoplasmic flow, but reflects the action of a more selective mechanism. The best characterized specific transport processes require microtubules or actin filaments, yet movement through the ring canals is relatively insensitive to microtubule and actin assembly inhibitors. These observations raise the possibility that this transport step is independent of both actin filaments and microtubules. However, cytochalasin D and colcemid only affect dynamic filaments that are in equilibrium with subunits in the cytoplasm. It is therefore possible that stable actin filaments or microtubules mediate transport through the ring canals. The inhibitor data reported here, combined with previously published data, suggest that GFP-Exu transport through the nurse cell-oocyte ring canals is independent of both microtubules and actin filaments (Theurkauf, 1998).

This suggests a multi-step model for transport and anterior localization of Exu during stages 9 and 10 of oogenesis. It is speculated that Exu protein assembles into particles within the nurse cell cytoplasm, perhaps at the nuclear periphery, where these particles are localized by a microtubule-dependent process. Particles dissociate from the perinuclear regions, and random microtubule-dependent movements then distribute these large particles throughout the nurse cell cytoplasm. As particles approach the posterior of the nurse cell, they interact with microtubules originating near the nurse cell-oocyte ring canals, and are transported to the cell-cell junctions along these microtubules. Particles are then transported through the ring canals in a second vectorial process that appears to be independent of both actin filaments and microtubules. In the final transport step, particles entering the oocyte interact with microtubules originating at the anterior cortex of the oocyte, and are localized to the anterior in a microtubule-dependent step. At the cortex, particle may associate with asymmetrically localized binding sites that stabilize the asymmetric distribution. These observations and previous studies suggest that the polarity of the oocyte microtubule network is not in itself sufficient to generate anterior asymmetry, and that additional factors are required to restrict morphogens to the anterior pole. Based on these observations, a multi-step anterior localization pathway is proposed (Theurkauf, 1998).

Several observations indicate that factors in addition to egg chamber geometry and microtubule-dependent transport play a role in anterior axis specification. For example, BCD mRNA that is ectopically localized to the posterior of mago nashi and PKA mutant oocytes is dispersed as ooplasmic streaming begins at stage 10b, while the anteriorly localized transcripts in these oocytes are stable in spite of cytoplasmic streaming. Stable association of BCD mRNA with the cortex thus appears to be restricted to the anterior pole. In addition, several mRNAs are localized with BCD to the oocyte anterior during stages 9 and 10 of wild-type oogenesis. However, unlike BCD mRNA, these transcripts are dispersed upon ooplasmic streaming at stage 10b. These observations indicate that anterior transcript binding sites are specific for BCD mRNA, or a complex containing this mRNA. It has been suggested that localization of BCD mRNA depends on both microtubule-dependent movement to the cortex and transcript stabilization by a microtubule-independent mechanism. In a modified model for BCD mRNA transport, BCD mRNA binding activity is restricted to the anterior cortex, where it mediates pole-specific stabilization of transcript accumulation (Theurkauf, 1998 and references).

Effects of Mutation or Deletion

Nine maternal-effect loci in Drosophila melanogaster germline mosaics were tested to determine whether the wildtype gene activity is required in somatic or germline components of the maternal ovary. Mutations in these loci affect the anterior-posterior or dorso-ventral body pattern. In all nine loci (torso, trunk, exuperantia, vasa, valois, staufen, tudor, dorsal, Toll) a mutant genotype in the germ cells is sufficient to produce all aspects of the mutant embryonic phenotype, even when those germ cells are surrounded by wildtype somatic tissues (Schupbach, 1986).

exuperantia (exu) is a maternal effect gene that is needed for proper localization of the BCD mRNA during the formation of oocytes. The gene functions in the male as well as the female germline. Six of seven exu alleles are male-sterile; mutant defects in spermatogenesis first appear during meiosis. A genetic analysis shows that the exu gene does not encode a zygotic vital function. The isolation of two overlapping deficiencies which delete exu function, localizes the gene cytologically to polytene bands 57A4-B1. The molecular cloning and identification of the gene is described; it encodes overlapping sex-specific transcripts of 2.9 kb in the male and 2.1 kb in the female. These two transcripts are limited in expression to the germline. One allele, exuVL57, is a deletion of about 700 bp that results in a loss of both transcripts (Hazelrigg, 1990).

Miranda interacts with Staufen protein to couples oskar mRNA/Staufen complexes to the bicoid mRNA localization pathway: A requirement for Exu

The double-stranded RNA binding protein Staufen is required for the microtubule-dependent localization of bicoid and oskar mRNAs to opposite poles of the Drosophila oocyte and also mediates the actin-dependent localization of prospero mRNA during the asymmetric neuroblast divisions. The posterior localization of oskar mRNA requires Staufen RNA binding domain 2, whereas prospero mRNA localization mediated the binding of Miranda to RNA binding domain 5, suggesting that different Staufen domains couple mRNAs to distinct localization pathways. This study shows that the expression of Miranda during mid-oogenesis targets Staufen/oskar mRNA complexes to the anterior of the oocyte, resulting in bicaudal embryos that develop an abdomen and pole cells instead of the head and thorax. Anterior Miranda localization requires microtubules, rather than actin, and depends on the function of Exuperantia and Swallow, indicating that Miranda links Staufen/oskar mRNA complexes to the bicoid mRNA localization pathway. Since Miranda is expressed in late oocytes and bicoid mRNA localization requires the Miranda-binding domain of Staufen, Miranda may play a redundant role in the final step of bicoid mRNA localization. These results demonstrate that different Staufen-interacting proteins couple Staufen/mRNA complexes to distinct localization pathways and reveal that Miranda mediates both actin- and microtubule-dependent mRNA localization (Irion, 2006).

Asymmetric localization of mRNAs is a common mechanism for targeting proteins to the regions of the cell where they are required. This process is particularly important in the developing oocytes of many organisms, where localized mRNAs function as cytoplasmic determinants. This has been best characterized in Drosophila, where the localization of bicoid (bcd) and oskar (osk) mRNAs to the anterior and posterior poles of the oocyte defines the primary axis of the embryo. bcd mRNA is translated after fertilization to produce a morphogen that patterns the head and thorax of the embryo, whereas osk mRNA is translated when it reaches the posterior of the oocyte, where Oskar protein nucleates the assembly of the pole plasm, which contains the abdominal determinant nanos mRNA, as well as the germ line determinants. Localized mRNAs can also function as determinants during asymmetric cell divisions. For example, the asymmetric inheritance of mating type switching in budding yeast is controlled by the localization of Ash1 mRNA to the bud tip, which segregates the repressor ASH1p into only the daughter cell at mitosis. Similarly, prospero (pros) mRNA localizes to the basal side of Drosophila embryonic neuroblasts and is inherited by only the smaller daughter cell of this asymmetric cell division, where Prospero protein acts as a determinant of ganglion mother cell fate (Irion, 2006).

To be localized, an mRNA must contain cis-acting localization elements that are recognized by RNA-binding proteins, which couple the mRNA to the localization machinery. This process is only well understood for ASH1 mRNA, which contains four localization elements that are recognized by She3p, which then links the mRNA to the myosin motor complex Myo4p/She2p so that it can be transported along actin cables to the bud tip. Biochemical and genetic approaches have led to the identification of a number of RNA-binding proteins that associate with localized mRNAs in higher eukaryotes, but it is not known how these interactions target the mRNA to the correct region of the cell (Irion, 2006).

One of the best candidates for an RNA-binding protein that plays a direct role in mRNA localization is the dsRNA-binding protein Staufen (Stau). Staufen was first identified because it is required for the localization of osk mRNA to the posterior of the oocyte and co-localizes with it at the posterior pole. This localization depends on the polarized microtubule cytoskeleton and the plus end-directed microtubule motor kinesin, suggesting that Staufen may play a role in coupling osk mRNA to kinesin, which then transports the osk mRNA complex along microtubules. The posterior localization of osk mRNA also requires the exon junction complex components Mago nashi (Mago), Y14, eIF4AIII and Barentsz (Btz), as well as HRP48, which is needed for the formation of Staufen/osk mRNA particles (Irion, 2006).

Staufen homologues seem to play a similar role in the microtubule-dependent localization in vertebrates. GFP-Stau particles have been observed to move along microtubules in cultured neurons, and the protein is a component of large ribonucleo-protein complexes that contain kinesin and dendritically localized mRNAs. In addition, a Xenopus Staufen homologue associates with Vg1 mRNA and is required for its microtubule-dependent localization to the vegetal pole of the oocyte, which is also thought to be mediated by a kinesin (Irion, 2006).

As well as this possible conserved role in kinesin-dependent transport, Drosophila Staufen is also required for the last phase of bcd mRNA localization and co-localizes with the mRNA at the anterior of the oocyte from stage 10B onwards. Furthermore, when the bcd 3′ UTR is injected into the early embryo, it recruits Staufen into particles that move in a microtubule-dependent manner to the poles of the mitotic spindles, consistent with minus end-directed microtubule transport (Irion, 2006).

Staufen also binds to prospero mRNA and is required for its localization to the basal side of the embryonic neuroblasts. In contrast to the other examples of Staufen-dependent mRNA localization, this process depends on the actin cytoskeleton and the adapter protein Miranda (Mira) (Irion, 2006).

The varied functions of Staufen raise the question of how the same protein can function in both actin- and microtubule-dependent mRNA localization, as well as in the targeting of osk and bcd mRNAs to opposite ends of the same cell. Some insight into this comes from the analysis of Staufen protein, which contains five conserved dsRNA-binding domains (dsRBDs). In all Staufen homologues, dsRBD2 is split by a proline-rich insertion in one of the RNA-binding loops, and deletion of this insertion disrupts the localization of osk mRNA, but not that of prospero mRNA, leading to the proposal that this domain couples Staufen/mRNA complexes to a kinesin-dependent posterior localization pathway. In contrast, removal of dsRBD5 prevents the localization of prospero mRNA, whereas osk mRNA localizes normally but is not translated at the posterior of the oocyte. Indeed, dsRBD5 binds directly to Miranda to couple Staufen/prospero mRNA complexes to the actin-based localization pathway. The localization of bcd mRNA also requires dsRBD5, although the loss of the insert in dsRBD2 also affects its localization slightly (Irion, 2006).

The results above suggest that different domains of Staufen couple mRNAs to distinct localization pathways, raising the possibility that the fate of Staufen mRNA complexes may depend on which Staufen-interacting proteins are present in the cell. To test this hypothesis, the effects of expressing Miranda during oogenesis were examined to determine whether it can influence the localization of bcd or osk mRNAs (Irion, 2006).

Although Miranda is not required during oogenesis, its ectopic expression causes a striking defect in anterior–posterior axis formation that reveals several important features of the mechanisms that control the targeting and translation of localized mRNAs. Firstly, these results provide strong support for the idea that the destination of Staufen/mRNA complexes is determined by the Stau-interacting factors that are present in the cell. During wild type oogenesis, Staufen associates with osk mRNA to mediate its kinesin-dependent localization to the posterior of the oocyte at stage 9, and this requires the insertion in Staufen dsRBD2, suggesting that this domain couples Staufen/osk mRNA complexes to the posterior localization pathway. However, the expression of Miranda is sufficient to target a proportion of these complexes to the anterior. This localization is mediated through the binding of Miranda to dsRBD5 of Staufen because deletion of this domain abolishes anterior localization without affecting the transport to the posterior pole. By contrast, deletion of the insert in dsRBD2 in the presence of Miranda results in the localization of all Staufen/osk mRNA complexes to the anterior pole. Thus, these two pathways act through different domains of Staufen to direct localization to opposite ends of the same cell. These pathways compete with each other, resulting in the partitioning of the Miranda/Staufen/osk mRNA complexes to the anterior and posterior poles, but each is capable of localizing all of the complexes when the other pathway is compromised. exu and swa mutants abolish the Miranda-dependent anterior localization, and osk mRNA now localizes exclusively to the posterior, whereas btz, mago and TmII mutants block the posterior localization pathway, resulting in the localization of all osk mRNA at the anterior cortex and the formation of reverse polarity embryos (Irion, 2006).

Since dsRBD5, which is not an RNA-binding domain, is necessary and sufficient for the interaction of Staufen with Miranda, the anterior localization of osk mRNA by Miranda provides a simple in vivo assay for the binding of Staufen to osk mRNA. This reveals that neither the insert of dsRBD2 nor the RNA-binding residues of dsRBD3 are required for the stable association of Staufen with the RNA. The lack of a requirement for the insert in dsRBD2 is consistent with the observation that dsRBD2Δloop binds dsRNA in vitro when expressed on its own, whereas the full-length dsRBD2 does not. It is more surprising, however, that the mutations in dsRBD3 have no effect on Staufen binding to osk mRNA since this domain binds to dsRNA with the highest affinity in vitro, and these mutations in the five key amino acids that contact the RNA abolish the domain's RNA-binding activity in vitro. The two other functional dsRNA-binding domains in Staufen (dsRBD1 and 4) must therefore be sufficient to form a stable complex with osk mRNA (Irion, 2006).

The specific effect of a quintuple mutant in dsRBD3 on posterior localization, but not on RNA binding of full-length Staufen, further suggests that these five amino acids play a role in coupling Staufen/osk mRNA complexes to the posterior localization pathway. Although it is possible that these residues are required for an interaction with a trans-acting factor, it seems more likely that it is the association of dsRBD3 with the RNA that is important because this affects either the folding of the RNA or the conformation of Staufen protein. For example, it has been suggested that the binding of Staufen dsRBDs1, 3 and 4 to osk mRNA presents a double-stranded region of the RNA to dsRBD2, which induces a conformational change in dsRBD2 that brings together the two RNA-binding regions of the domain and loops out the large insertion, which is then exposed to interact with the transport machinery. The effect of the point mutations in dRBD3 is consistent with this model and the idea that dsRBD2 functions as an RNA-binding sensor that couples Staufen/osk mRNA complexes to factors that target it to the posterior (Irion, 2006).

Although all mRNAs that accumulate in the oocyte localize at least transiently to the anterior, several lines of evidence indicate that Miranda links Staufen and osk mRNA specifically to the bcd localization pathway. (1) All other anterior mRNAs, except bcd and hu-li tai shao (hts), localize to the anterior only during stages 9–10A and become delocalized at stage 10B when rapid cytoplasmic streaming begins. In contrast, Miranda maintains osk mRNA at the anterior throughout oogenesis, so that it is still localized in a tight anterior cap in the freshly laid egg. (2) Miranda, Staufen and oskar undergo the same change in their anterior localization at stage 10B as bcd mRNA: they initially localize as a ring around the anterior cortex and then move towards the middle of the anterior when the centripetal follicle cells start to migrate inwards. (3) Like bcd, the anterior localization of osk mRNA by Miranda requires Exu, Swallow and Staufen, whereas hts mRNA localization is independent of Exu and Staufen. Since the anterior localization does not require bcd mRNA itself, Miranda cannot simply hitchhike on the bcd mRNA localization complex, and it therefore presumably links osk mRNA to the same microtubule-dependent anterior transport pathway used by bcd mRNA (Irion, 2006).

In addition to its role in osk mRNA localization, Staufen associates with bcd mRNA during the late stages of oogenesis to mediate the final steps in its localization to the anterior cortex of the oocyte. Since this localization requires the Miranda-binding domain of Staufen and Miranda couples Staufen/mRNA complexes to the bcd localization pathway, it is attractive to propose that Miranda normally mediates the late anterior localization of bcd mRNA. mira mutants have no phenotype during oogenesis, however, although the protein is expressed in late oocytes. Thus, if Miranda does play a role in bcd mRNA localization, it must function redundantly with another unidentified factor. This is perhaps to be expected given the previous evidence for redundancy in the localization of bcd mRNA. For example, none of the small deletions within the bicoid localization signal abolishes its anterior localization, indicating that it contains redundant localization elements, and two distinct bcd mRNA recognition complexes have been purified biochemically from ovarian extracts (Irion, 2006).

The elucidation of the role of Miranda in bicoid mRNA localization will require the identification of other factors that couple Staufen/bicoid mRNA complexes to the anterior localization pathway, which may function redundantly with Miranda. There are no obvious candidates for these factors, however, since Staufen is the only known protein that is specifically required for the final step of bicoid mRNA localization. Indeed, one reason why such factors may have been missed in genetic screens for mutants that disrupt bicoid mRNA localization is because they are redundant with Miranda and have no phenotype on their own. For these reasons, it is hard to address the question of redundancy using a genetic approach, but further analysis of how Miranda targets Staufen/mRNA complexes to the anterior may help resolve this issue. For example, mapping the Miranda domains that direct anterior localization may provide a clue as to the molecular nature of the unidentified factors that also fulfil this function, while screens for proteins that interact with this domain could identify other components of the anterior localization pathway (Irion, 2006).

These results reveal that Miranda, like Staufen, has the capacity to mediate both microtubule- and actin-dependent localization, raising the question whether the former plays any role in its well-characterized function during the asymmetric divisions of the embryonic neuroblasts. The localization of Miranda to the basal side of the neuroblast is actin-dependent. However, the protein also accumulates at the apical centrosome during both embryonic and larval neuroblast divisions, and this localization is even more prominent in l(2)gl or dlg mutants. Furthermore, Miranda was independently identified as a component of the pericentriolar matrix and co-localizes with γ-tubulin on all of the centrosomes at syncytial blastoderm stage. Although the centrosomes disappear in the female germ line, the anterior cortex is the major site for microtubule nucleation and γ-tubulin localization in the oocyte. Thus, Miranda may localize to the anterior of the oocyte by the same mechanism as it localizes to centrosomes (Irion, 2006).

The phenotype of mira-GFP also provides insights into the translational control of osk mRNA. In wild type ovaries, osk mRNA is translationally repressed before it is localized, and this repression is then specifically relieved once the mRNA reaches the posterior pole. In principle, translational activation of osk mRNA could occur by a specific signal at the posterior, but it could also be due to some other consequence of localization, such as the concentration of the RNA in a small region or its association with the oocyte cortex. Evidence in favor of a specific posterior signal comes from an experiment in which a LacZ reporter gene under the control of the oskar 5′ region and the first 370 nt of the 3′ UTR was targeted to the anterior by the bcd localization element. Since this anterior RNA was not translated, concentration at the cortex appeared to be insufficient to relieve BRE mediated repression. However, it has recently emerged that this reporter RNA lacks the two clusters of insulin growth factor II mRNA-binding protein (IMP) binding elements in the distal oskar 3′ UTR that are essential for oskar translational activation at the posterior, making it hard to draw any conclusions from the lack of translation of this reporter RNA at the anterior. Mira-GFP provides an alternative way to test this hypothesis because it directs the anterior localization of wild type osk mRNA, with all of its translational control elements intact. This anterior mRNA is not translated during stages 9–13, despite being efficiently localized to the cortex, whereas the osk mRNA at the posterior of the same oocytes is translated normally. Thus, concentration at the cortex is not sufficient to de-repress translation, strongly supporting the idea that activation depends on a specific posterior signal (Irion, 2006).

Although the anterior osk mRNA is not translated at the normal time, the repression system breaks down at the very end of oogenesis, and the mRNA is very efficiently translated in mature oocytes. This suggests that some key component of the repression system disappears at this stage, and a good candidate is the BRE-binding protein Bruno. Bruno is highly expressed during oogenesis but is not detectable in embryos. Furthermore, the addition of Bruno is sufficient to cause the repression of exogenous osk mRNA in an embryonic translation system. These results indicate that Bruno is degraded at the end of oogenesis, whereas all other components necessary for translational repression of osk mRNA are still present in the embryo. Thus, the translation of anterior osk mRNA in mira-GFP oocytes is most probably triggered by the disappearance of Bruno (Irion, 2006).

Once it is translated at the posterior of the oocyte, Oskar protein nucleates the formation of the pole plasm with its characteristic electron-dense polar granules, which gradually assemble during stages 9–14 of oogenesis. This appears to be a stepwise process, in which Oskar protein recruits some polar granule components as soon as it is translated at stage 9, such as Vasa and Fat facets, while other components are added in sequence during the rest of oogenesis. For example, Tudor, Capsuleen and Valois are recruited during stage 10A, whereas nanos, Pgc and gcl mRNAs only become enriched at the posterior at stages 10B–11. It is therefore surprising that the anterior Oskar protein, which is only synthesized in stage 14 oocytes, can still nucleate fully functional pole plasm that induces the formation of anterior pole cells. Thus, although the pole plasm normally assembles in an ordered fashion over the last 5 stages of oogenesis, this whole process can still occur once oogenesis is complete. This indicates that the assembly of the pole plasm does not depend on the order of addition of its components, all of which must still be present and freely diffusible in mature oocytes (Irion, 2006).


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exuperantia: Biological Overview | Regulation | Developmental Biology | Effects of Mutation

date revised: 15 April 2007

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