oskar

The role of PIWI and the miRNA machinery in Drosophila germline determination; increasing the maternal piwi dose increases Osk and Vasa levels

The germ plasm has long been demonstrated to be necessary and sufficient for germline determination, with translational regulation playing a key role in the process. Beyond this, little is known about molecular activities underlying germline determination. This study reports the function of Drosophila Piwi, Dicer-1, and dFMRP (Fragile X Mental Retardation Protein) in germline determination. Piwi is a maternal component of the polar granule, a germ-plasm-specific organelle essential for germline specification. Depleting maternal PIWI does not affect Osk or Vasa expression or abdominal patterning but leads to failure in pole-plasm maintenance and primordial-germ-cell (PGC) formation, whereas doubling and tripling the maternal piwi dose increases Osk and Vasa levels correspondingly and doubles and triples the number of PGCs, respectively. Moreover, Piwi forms a complex with dFMRP and Dicer-1, but not with Dicer-2, in polar-granule-enriched fractions. Depleting Dicer-1, but not Dicer-2, also leads to a severe pole-plasm defect and a reduced PGC number. These effects are also seen, albeit to a lesser extent, for dFMRP, another component of the miRISC complex. Because Dicer-1 is required for the miRNA pathway and Dicer-2 is required for the siRNA pathway yet neither is required for the rasiRNA pathway, the data implicate a crucial role of the Piwi-mediated miRNA pathway in regulating the levels of Osk, Vasa, and possibly other genes involved in germline determination in Drosophila (Megosh, 2006).

It has been nearly a century since the discovery of germ plasm and its function in germline fate determination in diverse organisms. In recent decades, the components and assembly of the polar granule in Drosophila and its equivalent in C. elegans have been effectively explored. Translational regulation has also been implicated in pole plasm for abdominal patterning and germline determination. In addition, germ cell-less (gcl) and mitochondrial large-subunit ribosomal RNAs (mtlr RNAs) have been shown to be required for germline determination. However, the biochemical activities of these molecules remain largely unknown. This study identified Piwi and likely the miRNA machinery as a germ-plasm regulatory activity that is involved in germline fate determination (Megosh, 2006).

Germ-plasm assembly occurs in a stepwise fashion. Step 1 involves the transport of polar granule materials to the posterior end of the oocyte during oogenesis, a process that involves a microtubule-based transport system as well as genes such as cappuccino and staufen. Step 2 is the assembly of polar-granule components at the posterior end, a process that is almost concurrent with the transport and that is completed by stage 12 of oogenesis. A critical component for the assembly is Osk, which determines the pole-cell number in a dose-dependent manner and has the ability to recruit Vasa and Tud as well as to induce pole-cell formation at ectopic sites within the embryo. Three lines of data suggest that Piwi is downstream of Osk, Tud, and Vasa in the assembly process: (1) Osk, Tud, and Vasa appear to assemble normally into the pole plasm in Piwi-depleted developing oocytes; (2) Piwi cannot recruit Osk or Vasa ectopically to the anterior pole, yet Osk can recruit Piwi to the anterior pole; (3) Osk, Tud, and Vasa all have both germline determination and posterior-patterning functions, but Piwi does not appear to have a detectable function in patterning (Megosh, 2006).

Although the assembly of polar-granule components occurs in a hierarchical fashion, there is growing evidence for interactions between polar-granule components beyond what is required for assembly. For example, a regulatory relationship between nanos and tudor has been reported. In nanos mutant embryos, both Tudor levels and the number of pole cells increase. Other experiments suggest that the presence of mtlrRNA in the polar granules is required for stabilization of the polar-granule components Vasa, Gcl, nos mRNA, and pgc mRNA. The regulatory function reported in this study for Piwi toward Osk, Vasa, and Nos further supports the interplay and interdependency among pole-plasm components. A previous study implicates osk as a rate-limiting factor for all aspects of pole-plasm function. The results suggest that Piwi, likely working through the miRNA pathway, is also a limiting factor for germ-cell formation. This function of Piwi is likely achieved via regulation of the levels of Osk, Tud, and Vasa, and possibly that of other polar-granule components, in a dose-dependent fashion (Megosh, 2006).

The regulation of Piwi toward the expression of Osk, Tud, Vasa, and Nos appears to be dispensable; Piwi-deficient oocytes and early embryos do not display detectable defects in their expression of Osk, Tud, Vasa, and Nos. This redundancy is likely due to an overlapping function of Piwi with other proteins involved in the RNAi pathway and/or colocalized in nuage during oogenesis; such proteins might include Maelstrom, Armitage, and Aubergine. Among these proteins, Aubergine, a close homolog of Piwi, is a known polar-granule component in early embryos. It regulates the translation of Osk during oogenesis and is required for both pole-cell formation and posterior patterning during embryogenesis (Megosh, 2006).

It is intriguing that Piwi regulates Osk and Vasa expression yet does not display a posterior-patterning phenotype. This function is different from that of Aubergine, so it is possible that Piwi and Aubergine each have their own regulatory targets in addition to Osk and Vasa. The Piwi targets may be specifically involved in maintaining polar-granule localization and may not be subject to Aubergine regulation, whereas Aubergine targets might be involved in both germline determination and posterior patterning. In support of this possibility, it has recently been shown that the generation of certain rasiRNAs shows varying dependencies on Piwi and Aubergine. The regulation of Piwi toward its specific target genes may be activated during oocyte maturation, similar to the oocyte maturation-dependent activation of RNAi as observed for aubergine and spindle-E. Thus, Piwi is not required for Osk and Vasa localization during oogenesis but is required for maintaining their localization during embryogenesis. An alternative hypothesis is that Piwi, like Aubergine, also regulates patterning genes but that this function is redundant. This hypothesis, however, does not explain the fact that neither ectopic expression nor overexpression of Piwi causes a detectable defect in posterior patterning (Megosh, 2006).

Given the association of Piwi with Dcr-1 and dFMRP, the Piwi-mediated regulation is likely via the miRNA but not the siRNA mechanism, which is Dcr-2-dependent, or the rasiRNA mechanism, which does not depend on either Dcr-1 or Dcr-2. This hypothesis is further supported by the similar phenotypes observed in embryos depleted of Piwi, Dcr-1, and dFMRP but not Dcr-2. It is possible that Piwi might bind to novel small RNAs to achieve this function, given recent findings that mammalian Piwi subfamily proteins bind to Piwi-interacting RNAs (piRNAs). If so, these novel RNAs must function in a Dcr-1-dependent pathway in the cytoplasm given Piwi's localization to the cytoplasm in early pole cells. The function of the Piwi/DCR-1-mediated miRNA or novel small-RNA pathway in germline specification is very similar to that of other germ-cell regulators, such as gcl and mtlr RNAs, in that these genes are required for pole-cell formation but not for abdominal segmentation. However, unlike embryos from the gcl-bcd females, embryos from the piwi-bcd females exhibit no cell-cycle delays in the anterior nuclei and no significant changes in the morphology of anterior nuclei. Furthermore, GCL mediates a transcriptional repression mechanism [72]. Thus, the effect of the Piwi-miRNA mechanism on pole-cell formation may be distinct from the gcl-mediated mechanism (Megosh, 2006).

It is important to note that the Piwi-mediated miRNA pathway positively regulates the expression of Osk and Vasa, in contrast to the known translational repression role of the miRNA pathway. In support of this observation, the Piwi ortholog in the mouse, MIWI, also appears to positively regulate gene expression, likely by enhancing mRNA stability and translation. Alternatively, it is possible that Piwi regulates an unidentified intermediate protein whose function is to repress the expression of Osk and Vasa (Megosh, 2006).

piwi is essential for the self-renewal of adult germline stem cells in Drosophila. Recent studies have demonstrated that the miRNA pathway is involved in division and self-renewal of adult germline stem cells in the Drosophila ovary. This study further connects Piwi and the miRNA pathway and reveals their crucial role in germline fate determination during embryogenesis. These observations suggest that the germline and stem cells may share a common miRNA-mediated mechanism in defining their fates. Given the high degree of conservation of the miRNA machinery during evolution, this pathway may function in diverse organisms in determining the germline and stem cell fates (Megosh, 2006).

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. This study reports 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. This study also found that these actin regulators are required for Osk-dependent formation of long F-actin projections and cortical anchoring of pole plasm components. It is proposed 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 (Tanaka, 2011a).

In many cell types, asymmetric localization of specific RNAs and proteins is essential for exhibiting proper structure and function. These macromolecules are transported to their final destinations and anchored there. This latter step is particularly important for the long-term maintenance of cell asymmetry. A genetically tractable model for studying intracellular RNA and protein localization is the assembly of the pole (germ) plasm in Drosophila oocytes and embryos. The pole plasm is a specialized cytoplasm that contains maternal RNAs and proteins essential for germline and abdominal development. It is assembled at the posterior pole of the oocyte during oogenesis. Drosophila oogenesis is subdivided into 14 stages, with pole plasm assembly starting at stage 8. The functional pole plasm is assembled by stage 13, stably anchored at the posterior cortex of the oocyte and later inherited by the germline progenitors (pole cells) during embryogenesis (Tanaka, 2011a).

Pole plasm assembly begins with the transport of oskar (osk) RNA along microtubules to the posterior pole of the oocyte. There, the osk RNA is translated, producing two isoforms, long and short Osk, by the alternate use of two in-frame translation start sites. Although short Osk shares its entire sequence with long Osk, the isoforms have distinct functions in pole plasm assembly. Downstream, short Osk recruits other pole plasm components, such as Vasa (Vas), to the oocyte posterior, presumably through direct interactions. By contrast, long Osk prevents pole plasm components from diffusing back into the cytoplasm. Intriguingly, embryonic patterning defects are caused by either the ectopic assembly of pole plasm [elicited by Osk translation at the oocyte anterior directed by the osk-bicoid (bcd) 3'UTR] or the leakage of pole plasm activity into the bulk cytoplasm (induced by overexpressing osk). Thus, the pole plasm must be anchored at the posterior cortex for proper embryonic development (Tanaka, 2011a).

Short and long Osk also differ in their subcellular distributions. Short Osk is located on polar granules, specialized ribonucleoprotein aggregates in the pole plasm, and long Osk is associated with endosome surfaces. Intriguingly, the oocyte posterior, where endocytosis is increased, is highly enriched with markers of early, late and recycling endosomes (Rab5, Rab7 and Rab11, respectively). osk oocytes, however, do not maintain either the accumulation of endosomal proteins or the increased endocytic activity at the posterior. Furthermore, the ectopic expression of long Osk at the anterior pole of the oocyte results in the anterior accumulation of endosomal proteins along with increased endocytosis. Thus, long Osk regulates endocytic activity spatially within the oocyte (Tanaka, 2011a).

The endocytic pathway has two separate roles in pole plasm assembly (see Tanaka, 2008). First, it is required for the sustained transport of osk RNA by maintaining microtubule alignment. For example, in oocytes lacking Rabenosyn-5 (Rbsn-5), a Rab5 effector protein essential for endocytosis, the polarity of the microtubule array is not maintained, disrupting osk RNA localization. A similar defect occurs in hypomorphic rab11 oocytes. Second, the endocytic pathway acts downstream of Osk to anchor the pole plasm components. In rbsn-5 oocytes aberrantly expressing osk at the anterior, Osk and other pole plasm components diffuse from the anterior cortex into the ooplasm, indicating that endocytic activity is essential for stably anchoring them to the cortex (Tanaka, 2011a).

The endocytic pathway is thought to anchor pole plasm components by remodeling the cortical actin cytoskeleton in response to Osk. Pole plasm anchoring is sensitive to cytochalasin D, which disrupts actin dynamics, and requires several actin-binding proteins, such as Moesin, Bifocal and Homer. Osk induces long F-actin projections emanating from cortical F-actin bundles at the posterior pole of the oocyte. Ectopic F-actin projections are also induced at the anterior pole when long Osk is misexpressed at the oocyte anterior (Tanaka, 2008). However, when the endocytic pathway is disrupted, F-actin forms aggregates and diffuses into the ooplasm, along with pole plasm components (Tanaka, 2008). These observations led to the hypothesis that Osk stimulates endocytosis, which promotes actin remodeling, which in turn anchors the pole plasm components at the posterior oocyte cortex. However, the molecular mechanism linking Osk, the endocytic pathway and actin remodeling is still unknown (Tanaka, 2011a).

This study has identified Mon2, a conserved Golgi/endosomal protein, as an essential factor in anchoring pole plasm components at the oocyte posterior cortex. Oocytes lacking Mon2 did not form F-actin projections in response to Osk, but neither did they exhibit obvious defects in microtubule alignment or endocytosis. It was also shown that two actin nucleators that function in osk RNA localization in the oocyte, Cappuccino (Capu) and Spire (Spir), play an essential role in a second aspect of pole plasm assembly: the Osk-dependent formation of long F-actin projections and cortical anchoring of pole plasm components. Finally, it was found that Mon2 interacts with Capu and Spir, and promotes the accumulation of the small GTPase Rho1 at the oocyte posterior. These data support a model in which Mon2 acts as a scaffold, linking Osk-induced vesicles with these actin regulators to anchor the pole plasm to the oocyte cortex (Tanaka, 2011a).

To learn more about how the pole plasm is assembled and anchored during Drosophila oogenesis, a germline clone (GLC) screen was conducted for ethyl methanesulfonate-induced mutations showing the abnormal localization of GFP-Vas, a fluorescent pole plasm marker (Tanaka, 2008). In a screen targeting chromosomal arm 2L, six mutants were identified that mapped into a single lethal complementation group, which was named no anchor (noan). In wild-type oocytes, GFP-Vas was first detectable at the posterior pole at stage 9, where it remained tightly anchored, with a progressive accumulation of protein until the end of oogenesis. In the noan GLC oocyte, GFP-Vas initially localized to the oocyte posterior during stages 9-10a, but its level gradually decreased, becoming undetectable in the mature oocyte. Similarly, the localization of Staufen (Stau) and Osk at the posterior pole, which occurs prior to that of Vas, was not maintained in the noan oocytes. Although the noan oocytes developed into normal-looking mature oocytes, the eggs were fragile and did not develop. Therefore, it was not possible to analyze the effects of the loss of maternal noan activity on the formation of abdomen or germ cells in embryos. Nevertheless, these results indicated that noan mutations cause defective anchoring of pole plasm components to the posterior pole of the oocyte (Tanaka, 2011a).

The genetic mapping and subsequent DNA sequencing of the noan locus revealed that all the noan alleles had a nonsense mutation in CG8683, which encodes a homolog of a budding yeast protein, Mon2p, also termed Ysl2p. noan is referred to as mon2. All the mon2 alleles showed identical defects in the posterior localization of GFP-Vas with full penetrance. As the mutation in the mon2^K388 allele was the most proximal to the translational initiation site among the six alleles identified, mon2^K388 was primarily used to characterize the mon2 phenotype (Tanaka, 2011a).

em>Drosophila Mon2 consists of 1684 amino acids and represents a highly conserved protein among eukaryotes. It has two Armadillo (ARM) repeat domains, which are likely to mediate protein-protein interactions, and a DUF1981 domain, which is functionally uncharacterized. In budding yeasts, mon2 (ysl2) was identified as a gene whose mutation increases sensitivity to the Na⁺/H⁺ ionophore monensin, and is synthetically lethal with a mutation in ypt51, which encodes a Rab5 homolo. Yeast Mon2p (Ysl2p) forms a large protein complex on the surface of the trans-Golgi network and early endosomes, and it is proposed to act as a scaffold to regulate antero- and retrograde trafficking between the Golgi, endosomes and vacuoles (Tanaka, 2011a and references therein).

This study found that Capu and Spir act together to form long F-actin projections and to anchor pole plasm components at the oocyte cortex, and that Mon2 is essential to these processes. Capu and Spir also regulate the timing for initiating ooplasmic streaming and microtubule array polarization in the oocyte (Qualmann, 2009). However, the polarity of microtubule arrays was not affected in mon2 oocytes. Therefore, Mon2 is not always required for Capu and Spir to function. Rather, it appears to regulate specifically these actin nucleators through the Osk-induced endocytic pathway (Tanaka, 2011a).

Mon2 is required for the formation of Osk-induced long F-actin projections at the oocyte posterior. Interestingly, ectopic overexpression of Osk at the anterior pole in the mon2 oocyte induced granular, albeit faint, F-actin structures, indicating that Osk-induced actin remodeling does not totally cease in the mon2 oocyte. Ectopic Osk at the anterior of capu spir double-mutant oocytes also induced faint F-actin granules in the cytoplasm. Thus, additional, as yet uncharacterized, actin regulators appear to function in response to Osk. Notably, two actin-binding proteins, Bifocal and Homer, play redundant roles in anchoring Osk to the cortex. Although the precise roles of Bifocal and Homer in this process remain elusive, they might function independently of Mon2 (Tanaka, 2011a).

Oocytes lacking Rab5 showed disrupted posterior cortical F-actin bundles, which was suppressed by the simultaneous loss of Osk. These results reconfirm that the endocytic pathway needs intact Osk function for actin remodeling (Tanaka, 2008). This study also found that the F-actin disorganization in rab5 oocytes is Mon2-dependent. Therefore, Mon2 can facilitate actin remodeling even when Rab5 is absent, but endosomal trafficking, in which Rab5 is involved, is crucial for regulating Mon2. Mammalian Rab5 is also involved in actin remodeling. For example, Rac1 GTPase, a regulator of F-actin dynamics, is activated by Rab5-dependent endocytosis, and the local activation of Rac1 on early endosomes and its subsequent recycling to the plasma membrane spatially regulate actin remodeling. Thus, local endocytic cycling provides a specific platform for actin remodeling in a wide range of cell types (Tanaka, 2011a).

There is growing evidence that endosomes act as multifunctional platforms for many types of molecular machinery. Intriguingly, Mon2 is located on the Golgi and endosomes, without entirely accumulating at the oocyte posterior. It is therefore proposed that the Osk-induced stimulation of endocytic cycling at the oocyte posterior leads to the formation of specialized vesicles, which instruct a fraction of Mon2 to regulate the activity of Capu, Spir and Rho1 to form long F-actin projections from the cortex. Although the functional property of Osk-induced endocytic vesicles has yet to be ascertained, long Osk is known to associate with the surface of endosomes. Therefore, long Osk might modify endosome specificity to recruit and/or stabilize the machineries responsible for actin remodeling (Tanaka, 2011a).

Oocytes lacking Mon2 can mature without morphological abnormalities, but their eggs are nonviable. Furthermore, Drosophila mon2 mutations show recessive lethality, indicating that Mon2 has additional functions in somatic cell development. It might function in regulating vesicle trafficking or protein targeting, as reported in yeasts. As vesicle trafficking is often linked with establishing and maintaining cell polarity, it is an attractive idea that Mon2 might regulate the polarity protein localization and/or mediate the signal transduction for cell polarization in somatic cells, as well as in germ cells. Supporting this idea, a Mon2 homolog in C. elegans has been implicated in the asymmetric division of epithelial stem cells (Kanamori, 2008; Tanaka, 2011a and references therein).

It has been proposed that long Osk localizes to the endosomal membrane and generates a positive-feedback loop for cortical anchoring of pole plasm components. Osk is also thought to generate another positive-feedback loop to maintain the polarity of microtubule arrays, and the process appears to be endosomal protein-dependent. Although Rbsn-5 is required for both feedback loops, Mon2 acts specifically in the loop regulating actin remodeling for pole plasm anchoring, indicating that the two feedback loops are regulated by distinct mechanisms. The endocytic pathway consists of multiple vesicle trafficking steps, including endocytosis, endosomal recycling, late-endosomal sorting and endosome-to-Golgi trafficking. Therefore, determining which steps in the endocytic pathway are used by the two Osk-dependent positive-feedback loops is an important aim for future exploration (Tanaka, 2011a).

Community effects in regulation of translation

Certain forms of translational regulation, and translation itself, rely on long-range interactions between proteins bound to the different ends of mRNAs. A widespread assumption is that such interactions occur only in cis, between the two ends of a single transcript. However, certain translational regulatory defects of the Drosophila oskar (osk) mRNA can be rescued in trans. It is proposed that inter-transcript interactions, promoted by assembly of the mRNAs in particles, allow regulatory elements to act in trans. This study confirms predictions of that model and shows that disruption of Polypyrimidine tract binding protein (PTB) dependent particle assembly inhibits rescue in trans. Communication between transcripts is not limited to different osk mRNAs, as regulation imposed by cis-acting elements embedded in the osk mRNA spreads to gurken mRNA. It is concluded that community effects exist in translational regulation (Macdonald, 2016).

Cup regulates oskar mRNA stability during oogenesis

The proper regulation of the localization, translation, and stability of maternally deposited transcripts is essential for embryonic development in many organisms. These different forms of regulation are mediated by the various protein subunits of the ribonucleoprotein (RNP) complexes that assemble on maternal mRNAs. However, while many of the subunits that regulate the localization and translation of maternal transcripts have been identified, relatively little is known about how maternal mRNAs are stockpiled and stored in a stable form to support early development. One of the best characterized regulators of maternal transcripts is Cup - a broadly conserved component of the maternal RNP complex that in Drosophila acts as a translational repressor of the localized message oskar. This study found that loss of cup disrupts the localization of both the oskar mRNA and its associated proteins to the posterior pole of the developing oocyte. This defect is not due to a failure to specify the oocyte or to disruption of RNP transport. Rather, the localization defects are due to a drop in oskar mRNA levels in cup mutant egg chambers. Thus, in addition to its role in regulating oskar mRNA translation, Cup also plays a critical role in controlling the stability of the oskar transcript. This suggests that Cup is ideally positioned to coordinate the translational control function of the maternal RNP complex with its role in storing maternal transcripts in a stable form (Broyer, 2016).

Targets of Activity

An activity present in the posterior pole plasm of wild-type embryos can restore normal abdominal development in posterior group mutants. This activity, most likely encoded by the gene nanos, is synthesized during oogenesis. staufen, oskar, vasa, valois and tudor act upstream of nanos. Embryos from females mutant for these genes lack the specialized posterior pole plasm and consequently fail to form germ-cell precursors. The products of these genes provide the physical structure necessary for the localization of nanos-dependent activity and of germ line determinants (Lehmann, 1991).

Overexpression of oskar leads to an excess of Nanos activity present throughout the embryo and a superabundance of posterior pole cells. In addition, presumptive pole cells appear at a novel anterior position. Strikingly, formation of these ectopic pole cells is enhanced in nanos mutants. This observation may reflect competition between Nanos and Oskar for a shared and limiting precursor (Smith, 1992).

To understand the nature of the regulatory signals impinging on the second promoter of the Antennapedia gene (Antp P2), analysis of its expression in mutants and in inhibitory drug injected embryos has been carried out. The maternally-active gene osk is identified as one of two general repressors of P2 which prevent Antp transcription until division cycle 14 (Riley, 1991).

Oskar functions in pole plasm

Oskar interacts directly with Vasa and Staufen, in a yeast two-hybrid assay. These interactions also occur in vitro and are affected by mutations in oskar that abolish pole plasm formation in vivo. In the pole plasm, Oskar protein, like Vasa and Tudor, is a component of polar granules, the germ-line-specific RNP structures. Thus the Oskar-Vasa interaction constitutes an initial step in polar granule assembly. The biological significance of the direct interaction between OSK and STAU is unclear. A complex containing OSK could provide a scaffold for OSK transcript until structures containing Vasa as a component are established. It appears that OSK is a component of different complexes at different developmental stages (Breitwieser, 1996).

oskar has two functions: assembly of germ plasm (and the consequent direction of germ cell fate) and determination of the abdomen through binding of Nanos mRNA and the subsequent inhibition of Hunchback translation (Ephrussi, 1992).

oskar is one of seven Drosophila maternal-effect genes that are necessary for germline and abdomen formation. oskar has been cloned, and 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. The pole plasm is assembled stepwise. Continued interaction among its components appears to be required for germ cell determination (Ephrussi, 1991).

Vasa localization requires Oskar. Females homozygous for any one of the maternal-effect mutations (tudor, oskar, staufen, vasa, or valois) give rise to embryos that lack localized polar granules, fail to form the germ cell lineage and have abdominal segment deletions. Using antibodies against a polar granule component, the Vasa protein, it has been shown that Vasa synthesis or localization is affected by these mutations. In oskar and staufen mutant females, Vasa synthesis appears normal, but the Vasa protein is not localized. Vasa is an ATP dependent RNA helicase and bears additional sequence similarity to known RNA-binding proteins (Hay, 1990).

pipsqueak is downstream of oskar and acts after the establishment of the Oskar posterior anchor but before the localization of Vasa protein during oogenesis. Characterization of multiple alleles at the pipsqueak locus shows that pipsqueak, like vasa, is required for early stages of oogenesis, including but not limited to formation of the egg chamber and progression through Stage 6 of oogenesis. Genetic interaction studies suggest that pipsqueak acts at least partially through vasa; molecular studies indicate that pipsqueak affects vasa levels in the ovary (Siegel, 1993).

The oskar gene directs germ plasm assembly and controls the number of germ cell precursors formed at the posterior pole of the Drosophila embryo. Mislocalization of oskar RNA to the anterior pole leads to induction of germ cells at the anterior. Of the eight genes necessary for germ cell formation at the posterior, only three, oskar, vasa and tudor, are essential at an ectopic site. Thus, of all the factors involved in oskar localization, only vasa and tudor, in addition to oskar itself, are sufficient to direct germ plasm assembly in the anterior of the fly (Ephrussi, 1992).

fs(1)K10 mRNA transport and anterior localization is mediated by a 44 nucleotide stem-loop structure. A similar putative stem-loop structure is found in the 3' untranslated region of the Drosophila ORB mRNA, suggesting that the same factors mediate the transport and anterior localization of both K10 and ORB mRNAs. Apart from ORB, the K10 TLS (transport/localization sequence) is not found in any other localized mRNA, raising the possibility that the transport and localization of other mRNAs, e.g., Bicoid, Oskar and Gurken, are mediated by novel sets of cis- and trans-acting factors. K10 TLS overrides the activity of Oskar cis-regulatory elements that mediate the late stage movement of the mRNA to the posterior pole (Serano, 1995).

Mitochondrial large ribosomal RNA (mtlrRNA) was identified as a cytoplasmic factor that induces pole cells in uv-irradiated embryos and is localized on polar granules in normal embryos at early stages. MtlrRNA is localized in the anterior pole if oskar products are forced to the same loction. This clearly shows that localization of mtlrRNA depends on oskar function even at the anterior pole (Kobayashi, 1995).

Posterior localization of Fat facets protein depends on oskar (Fischer-Vize, 1992).

Maternally synthesized Hsp83 transcripts are localized to the posterior pole of the early Drosophila embryo by a novel mechanism involving a combination of generalized RNA degradation and local protection at the posterior. Hsp83 RNA is not protected at the posterior pole of embryos produced by females carrying maternal mutations that disrupt the posterior polar plasm and the polar granules--cappuccino, oskar, spire, staufen, tudor, valois, and vasa (Ding, 1993).

Shortly after fertilization in Drosophila embryos, the G-protein alpha subunit, Gi alpha, undergoes a dramatic redistribution. Initially granules containing Gi alpha are present throughout the embryonic cortex but during nuclear cleavage they become concentrated at the posterior pole and are lost by the blastoderm stage. Mutations that eliminate anterior structures (bicoid, swallow, and exuperantia) do not prevent the posterior accumulation of Gi alpha. Likewise, embryos from mothers with dominant gain of function mutations in the Bicaudal D gene show normal polarization of Gi alpha granules. By contrast, a subset of mutations that eliminate posterior structures (cappuccino, spire, staufen, mago nashi, valois, and oskar) prevent the posterior accumulation of Gi alpha. It is important to note that mutations in posterior genes found lower in the putative hierarchy (vasa, tudor, nanos, and pumilio) do not affect Gi alpha redistribution. From these results it is concluded that Gi alpha redistribution to the posterior pole depends on maternal factors involved in the localization of the posterior morphogen Nanos (Wolfgang, 1995).

The mitochondrially encoded 16S large ribosomal RNA (16S RNA) is highly concentrated at the posterior pole of early embryos. This high posterior accumulation decreases sharply during the first hour of embryogenesis and reaches the uniform level found throughout the remainder of the embryo by the time pole cells form 1.5 hr after fertilization. Transcripts produced by the 12S small rRNA gene are also concentrated in the posterior polar plasm and exhibit the same dynamic changes in distribution as the 16S RNA. Ectopic localization of the Oskar RNA to the anterior pole of the oocyte and early embryo results in anterior assembly of polar plasm and anterior budding of functional pole cells. 16S and 12S RNAs are not concentrated at the anterior pole of such embryos (Ding, 1994).

Pole plasm formation depends on the stepwise recruitment of a number of posterior group gene products to the posterior pole. The posterior localization of Stauffen and Oskar mRNA leads to the translational activation of the latter to produce Osk protein, which then anchors the complex and recruits Vasa protein. par-1 mutants show a novel polarity phenotype in which Bicoid mRNA accumulates normally at the anterior, but Oskar mRNA is redirected to the center of the oocyte, resulting in embryonic patterning defects. These phenotypes arise from a disorganization of the oocyte microtubule cytoskeleton. To determine where Par-1 lies in this hierarchy, its localization was examined in various posterior group mutants. Par-1 localization at the oocyte posterior is unaffected in vas^PD egg chambers, and in homozygotes for osk missense mutations, in which OSK mRNA is localized and anchored at the posterior, but fail to recruit Vasa. In contrast, the strong osk nonsense allele, osk⁵⁴, completely abolishes the posterior localization of Par-1 and null mutations in stau have a similar effect. Thus, the recruitment of PAR-1 to the posterior is upstream and independent of vasa, but requires osk and stau (Shulman, 2000).

Since the posterior localization of OSK mRNA, Stau, and Osk are interdependent, these experiments do not distinguish which of these components is responsible for recruiting PAR-1 to the posterior pole. Therefore use was made of an osk-bcd 3'-UTR transgene, in which the bcd localization signal directs the Stau-independent localization of OSK mRNA and protein to the anterior of the oocyte. In egg chambers expressing this construct, Par-1 localizes to the anterior as well as the posterior pole of the oocyte. Thus, Par-1 must interact directly or indirectly with either Osk protein or a region of OSK mRNA other than the 3'-UTR, which is absent from the transgene. Taken together, these results are most consistent with a model in which Par-1 associates with OSK mRNA, since both show a transient localization at the anterior of wild-type stage 9 oocytes, whereas Osk protein is not translated until the mRNA reaches the posterior (Shulman, 2000).

Given the lack of pole plasm in embryos derived from par-1 mothers, the earliest steps of pole plasm assembly during oogenesis were examined. In par-1 egg chambers, OSK mRNA localizes normally through stage 7, but then diverges strikingly from the wild-type pattern. In the strongest viable allelic combination, par-1⁶³²³/par-1^W3, OSK mRNA is never detected at the posterior, and is either mislocalized to an ectopic site in the center of the oocyte (73%) or not localized at all (27%). This unusual OSK mRNA 'dot' forms as early as stage 8, and can persist until stage 11, the latest stage that can be examined. Stau protein shows an identical mislocalization to the middle of the oocyte in these mutants. Weaker allelic combinations show a similar abnormal pattern of Stau and OSK mRNA localization, but with lower penetrance. In addition, these egg chambers often show an intermediate phenotype in which some OSK RNA forms a dot in the middle of the oocyte while the rest localizes normally to the posterior cortex. Occasional dots of mislocalized OSK mRNA are even observed in par-1^W3 heterozygotes, indicating that par-1 has a slight dominant haplo-insufficient phenotype. Overall, the penetrance of the OSK mRNA mislocalization phenotype correlates well with that of abdominal defects for each allelic combination (Shulman, 2000).

The localization of OSK mRNA is microtubule-dependent, and several mutants that disrupt this process do so by altering the organization of the oocyte microtubule network. It was therefore examined whether microtubule organization and polarity are also disrupted in par-1 oocytes. In wild-type oocytes, the microtubules are organized in an A/P gradient at stages 7-9 that can be visualized using a Tau:GFP fusion protein. In addition, the polarity of the microtubules can be assayed by expressing microtubule motor proteins fused to beta-galactosidase (beta-gal). A beta-gal fusion to the plus-end-directed motor, Kinesin (Kin:beta-gal), localizes to the posterior of the oocyte during stages 9-10 like OSK mRNA, whereas a Nod:beta-gal fusion localizes to the anterior. In par-1 mutant oocytes, Tau:GFP labels microtubules uniformly around the cortex, including the posterior pole, where they are never seen in wild type. Moreover, like OSK mRNA and Stau, Kin:beta-gal is mislocalized to the center of the oocyte, although a small amount of residual staining is often seen at the posterior. In contrast, Nod:beta-gal shows a normal localization to the anterior in par-1 mutant oocytes, suggesting that the microtubules are still nucleated from this pole. Like the other aspects of the par-1 phenotype, the disruption of microtubule organization is completely penetrant in par-1⁶³²³/par-1^W3 egg chambers, but less so in the weaker allelic combinations (Shulman, 2000).

The abnormal arrangement of microtubules in par-1 is similar to that seen in cappuccino (capu), spire (spir), and chickadee mutants, which disrupt OSK mRNA localization by causing a premature rearrangement of microtubules into a cortical array. Upon careful comparison, however, the microtubules in par-1 appear more diffuse than the tight, parallel bundles of capu egg chambers. Furthermore, whereas mutations in capu or spir cause the premature initiation of cytoplasmic streaming, the cytoplasmic movements of par-1 oocytes are indistinguishable from wild type. The microtubule organization in par-1 mutants also differs from that seen in grk mutants, which fail to disassemble the posterior MTOC, resulting in a focus of microtubules at the posterior pole that is never seen in par-1 oocytes (Shulman, 2000).

Live imaging of nuage and polar granules: evidence against a precursor-product relationship and a novel role for Oskar in stabilization of polar granule components

Nuage, a germ line specific organelle, is remarkably conserved between species, suggesting that it has an important germline cell function. Very little is known about the specific role of this organelle, but in Drosophila three nuage components have been identified, the Vasa, Tudor and Aubergine proteins. Each of these components is also present in polar granules, structures that are assembled in the oocyte and specify the formation of embryonic germ cells. GFP-tagged versions of Vasa and Aubergine were used to characterize and track nuage particles and polar granules in live preparations of ovaries and embryos. Perinuclear nuage is a stable structure that maintains size, seldom detaches from the nuclear envelope and exchanges protein components with the cytoplasm. Cytoplasmic nuage particles move rapidly in nurse cell cytoplasm and passage into the oocyte where their movements parallel that of the bulk cytoplasm. These particles do not appear to be anchored at the posterior or incorporated into polar granules, which argues for a model where nuage particles do not serve as the precursors of polar granules. Instead, Oskar protein nucleates the formation of polar granules from cytoplasmic pools of the components shared with nuage. Surprisingly, Oskar also appears to stabilize at least one shared component, Aubergine, and this property probably contributes to the Oskar-dependent formation of polar granules. Bruno, a translational control protein, is associated with nuage, which is consistent with a model in which nuage facilitates post transcriptional regulation by promoting the formation or reorganization of RNA-protein complexes (Snee, 2004).

Perinuclear nuage contains, in addition to Vas and Aub, the Maelstrom (Mael), and Gustavus (Gus) proteins. Another component, Bruno (Bru), is a protein that acts in translational repression of osk and gurken (grk) mRNAs. By immunolocalization and expression of a GFP-tagged version of this protein, it was found that Bru is concentrated in perinuclear clusters, similar to the distribution of known nuage components. Double labelling experiments with GFPAub confirmed that Bru colocalizes with nuage. However, Bru is also present at high levels in the cytoplasm, raising the question of whether the colocalization reveals an association with nuage or simply reflects random overlap of an abundant protein with the more narrowly distributed nuage. Evidence that Bru is specifically associated with nuage comes from analysis of Bru distribution in vas mutants: as for other nuage components, the perinuclear clusters of Bru are strongly reduced. Given this identification of Bru as a nuage-associated protein, arrest (aret) mutants (the aret gene encodes Bru) were included in a genetic analysis of nuage. The other genes tested were vas, tud, aub and spindle E (spnE), each of which encodes a nuage component or has been shown to be required for nuage formation, or both (Snee, 2004).

Live imaging was used to better characterize the perinuclear nuage defects seen in static images and to extend the analysis to include cytoplasmic nuage particles. GFPAub was used as the nuage marker to test the role of vas, aret and tud, and VasGFP was used to test the roles of aub and spnE. The live imaging confirmed, for the most part, the basic observations from analysis of fixed samples. In vas mutants perinuclear nuage is almost completely absent, with only a few nuage clusters visible. Loss of spnE activity has a less extreme effect: the perinuclear nuage clusters are largely missing, but a perinuclear zone of VasGFP remains. Consistent with the results by using fixed samples, the persistent perinuclear zone of VasGFP is qualitatively different from wild type, appearing almost completely uniform and lacking any visible discontinuities. Similar results were obtained with the aub mutant, except that the VasGFP perinuclear clusters remain present up to stage 8 of oogenesis, after which they disappear. In aret and tud mutants no significant alteration of perinuclear nuage was detected (Snee, 2004).

In mutants whose perinuclear VasGFP is uniform (spnE^- and later stage aub^-), the protein undergoes rapid exchange with cytoplasmic pools, just as for VasGFP in perinuclear clusters of wild-type egg chambers. In photobleaching experiments the fluorescence-recovery half-time is 50 seconds in aub^- and 48.5 seconds in spnE^-, similar to the t_1/2=59 seconds for wild type (Snee, 2004).

Cytoplasmic nuage particles are affected differently in the vas, aub and spnE mutants. The vas and spnE mutants have few or no cytoplasmic nuage particles. By contrast, aub mutants have no dramatic reduction in the abundance of cytoplasmic nuage particles, even at times well after the disappearance of perinuclear nuage clusters at stage 8, and the particles have a fairly typical size distribution. These particles do not simply represent the default appearance of VasGFP; they are absent in the spnE mutant. Thus, it seems unlikely that perinuclear nuage clusters are required for the formation of cytoplasmic nuage particles, a conclusion consistent with the observation that cytoplasmic particles are produced only infrequently by detachment of perinuclear nuage clusters (Snee, 2004).

The consequences of loss of vas activity were examined in the male germ line. Just as in nurse cells, Vas appears to be concentrated in nuage in spermatocytes. Given the crucial role for Vas in the nuage of other cell types, either male nuage must differ in this requirement or nuage is not essential in the male germ line for fertility. To distinguish between these possibilities vas^AS spermatocytes were tested for the presence of nuage, using GFPAub as a marker. Although GFPAub was present in the cytoplasm, there were no visible perinuclear nuage clusters, indicating that nuage does not form in the vas mutant and is therefore not required for spermatocyte function. An alternate and less probable interpretation is that a rudimentary form of nuage, lacking Aub, is present and is sufficient to provide a minimal requirement for nuage in males (Snee, 2004).

In Drosophila, two types of function, not mutually exclusive, have been proposed for nuage. In one model nuage has been suggested to serve as a precursor to polar granules, a view initially based on ultrastructural similarities of the two organelles and supported by the identification of shared components. Another possible role for nuage is based on its position at the periphery of the nucleus, at or near nuclear pores. Specifically, nuage might act in some aspect of remodelling RNPs when RNAs are exported from the nucleus. Analysis of the movements and genesis of nuage particles provides two arguments against the first model: (1) the rate of release of perinuclear nuage clusters in the nurse cells is very low, much lower than expected if the clusters form polar granules; (2) no nuage particles arriving at the posterior pole of the oocyte and becoming incorporated into polar granules were detected. An additional observation that argues against a model where nuage is a precursor for polar granules, is the presence of cytoplasmic nuage particles in aub mutants, despite the fact that these mutants do not assemble polar granules. However, this evidence does not exclude the first model, because the nuage particles in the mutant might not be fully functional. A third argument is provided by the evidence that Osk cannot interact with nuage, leaving de novo assembly of polar granules as the only reasonable option. Overall, the results strongly suggest that nuage is not the precursor to polar granules, and it is believed that the shared features are simply indicative of similar biochemical activities, rather than a precursor-product relationship (Snee, 2004).

The data do not directly test the model that nuage might function as a transition zone in the movements of mRNAs from the nucleus to the cytoplasm, where RNP components might be exchanged or otherwise modified. However, new properties of nuage, and these relate to possible functions, have been identified. (1) It was found that Bruno, an RNA binding protein that acts as a translational repressor of osk and grk mRNAs, is associated with nuage. This extends the correlation of nuage components with factors that act in some aspect on mRNA localization or translational control. Of the previously identified nuage components, Vas and Gus are involved in the regulation of grk mRNA localization and translation, Aub is required for efficient translation of osk mRNA and has also been implicated in RNAi, and mael mutants display defects in the early stages of mRNA localization. Moreover, spnE, which is necessary for normal nuage formation, is required for the localization of multiple mRNAs and acts in RNAi. Thus, every known nuage component has a role in one or more types of post-transcriptional control of gene expression (Snee, 2004).

(2) The second property of nuage reported here, is the remarkably dynamic composition of perinuclear nuage clusters, despite their relatively fixed positions around the nucleus. This is in contrast to studies showing that general protein exchange is slow in mouse nuage. The rapid exchange of both Vas and Aub, the two proteins tested, suggests that the clusters are staging sites where these, and presumably additional proteins, become associated with other molecules and move off into the cytoplasm. Much like shuttling-proteins that escort RNAs in their travels from the nucleus to the cytoplasm, there might be a class of proteins that interact in nuage with newly exported RNAs and then facilitate post-transcriptional control events that occur in the cytoplasm. By this model nuage could be an organelle that concentrates and thus potentiates the activity factors normally present in all cells, but that must be especially active in germline cells because of their intensive reliance on post-transcriptional controls of gene expression (Snee, 2004).

It has been argued that nuage from the nurse cells is not used for polar granule assembly in the oocyte, yet these two subcellular structures clearly share components and may well have similar activities. One feature that clearly distinguishes polar granules from nuage is the presence of Osk protein. Under normal circumstances Osk is never in contact with nuage, because an elaborate set of post-transcriptional control mechanisms serves to prevent Osk accumulation in the nurse cells and to restrict the distribution of Osk protein within the oocyte to the posterior pole. The presence of Osk at this single location provides the cue for the assembly of polar granules, and misdirection of Osk to other sites in the oocyte leads to ectopic polar granule formation. Thus Osk is generally viewed as an anchor for the recruitment of the factors that form polar granules. Given the finding that polar granules are significantly more stable that perinuclear nuage clusters, it might be that Osk not only recruits other factors, but also strengthens their interactions. A further and unanticipated property of Osk was revealed in studies in which Osk was expressed precociously throughout the oocyte. Under these conditions GFPAub levels are substantially elevated in the oocyte. Two general explanations are possible. (1) Osk might stimulate the rate of transfer of GFPAub from the nurse cells to the oocyte. Such a model is not supported by any known property of Osk, and no increase in the rate at which GFPAub particles move into the oocyte was detected. Furthermore, GFPAub levels in the oocyte are enhanced even before the onset of known nurse cell to oocyte movements in the cytoplasm, and so Osk would have to dramatically alter the properties of the egg chamber under this model. (2) Osk could stabilize a normally labile pool of GFPAub in the oocyte. In the simplest form of this model, stabilization would occur as a consequence of the assembly into complexes, which could include factors other than Osk and GFPAub. This model appears to be most compatible with the data. In addition, such a model provides a possible explanation for the curious association of the Fat facets (Faf) protein, a deubiquitinating enzyme, with pole plasm. The role of Faf could be to stabilize one or more polar granule components, thereby enhancing the growth of polar granules (Snee, 2004).

The restriction of Osk protein to the posterior pole of the oocyte is known to be important for limiting the spatial distribution of posterior body patterning activity. By analogy, this restriction might also be important for allowing normal assembly and function of nuage in nurse cells, if Osk can compete with nuage for their shared components. To evaluate this possibility, ovaries were examined in which Osk was allowed to accumulate in the nurse cells as well as the oocyte. Osk does indeed nucleate the formation of large bodies in the nurse cell cytoplasm, but the presence of these bodies does not appear to limit the amount of perinuclear nuage. Notably, no Osk was observed in association with perinuclear nuage, which appears not to be affected by the ectopic Osk. The Osk protein can interact directly with Vas in the two-hybrid assay in yeast, and so its failure to associate with perinuclear nuage -- the regions of greatest Vas concentration -- in nurse cells is notable. One interpretation is that the site of Vas binding to Osk is blocked when it is in nuage. This fits with the model in which Osk protein nucleates polar granule formation not from nuage particles themselves, but from individual nuage components or subassemblies (Snee, 2004).

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 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. 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 (Yoshida, 2004).

To isolate new factors involved in axis formation during Drosophila oogenesis, an FRT-based genetic screen was performed for maternal-effect mutants defective in anteroposterior patterning of the embryo. Embryos derived from germline clones of one line, 18304, showed anterior patterning defects ranging from deletion of the head to complete mirror-image duplication of posterior structures (abdominal segments and filzkörper material), the bicaudal phenotype. oskar plays a central role in abdomen and germline formation. At the posterior pole, oskar assembles the pole plasm, and recruits and activates translation of nanos RNA, which encodes the abdominal determinant. To address whether 18304 affects oskar or downstream factors, flies doubly mutant for 18304 and oskar were generated, and the phenotype of their progeny analyzed. Such embryos show a phenotype indistinguishable from that of the oskar single mutant. This indicates that the 18304 locus is required in the germline to control oskar activity (Yoshida, 2004).

These results reveal that Pka-R1 is an essential gene in Drosophila and that it is specifically required during oogenesis for post-transcriptional regulation of oskar. PKA-R1 forms a complex with the PKA catalytic subunit and controls its activity in response to cAMP. In Drosophila, the DC0 locus was identified as the catalytic subunit gene with the highest homology to its mammalian counterparts, and it serves as the major source of PKA catalytic activity. DC0 mutant germline clones show defective actin structures in the nurse cells and oocyte, indicating a role of PKA in organization of the actin cytoskeleton. Analysis of DC0 loss-of-function alleles in the germline has also revealed a requirement for PKA catalytic activity in the establishment of microtubule polarity along the anteroposterior axis of the oocyte, a prerequisite for oskar RNA localization. The effect of loss of DCO activity on microtubule polarity has prevented analysis of the role of PKA in regulation of Oskar protein expression, because oskar RNA is translationally repressed before its localization, and translation is not activated if the RNA fails to localize to the posterior pole (Yoshida, 2004).

This analysis demonstrates a requirement for precise modulation of PKA activity in the Drosophila germline for correct spatial distribution of Oskar protein. In the Pka-R1¹⁸³⁰⁴ mutant, where PKA activity is upregulated, Oskar protein is overexpressed and accumulates ectopically thoughout the oocyte, although oskar RNA localization and levels are normal. Dorsoventral patterning is correctly established. Anterior patterning also appears normal in the mutant, as revealed by the fact that Pka-R1¹⁸³⁰⁴, oskar double mutants develop a normal head and thorax. In addition, over-expression of the regulatory subunit causes a modest reduction in PKA activity, without affecting oskar RNA localization in the ovary. In such egg chambers, Oskar is underexpressed, suggesting that PKA activity is indeed required for Oskar expression. Taken together, these results suggest that, in addition to its role in the establishment of microtubule polarity and actin cytoskeleton integrity, PKA has a positive role in the regulation of Oskar protein expression at the posterior pole (Yoshida, 2004).

In addition to defects in oogenesis, Pka-R1 alleles have reduced viability, indicating that the control of PKA activity by PKA-R1 is required in several developmental processes. It has been shown that Pka-R1 is expressed throughout the cell-body layer of the central brain and optic lobes, and strongly accumulates in mushroom bodies. Analysis of hypomorphic alleles has revealed that Pka-R1 is involved in olfactory learning and courtship conditioning. The novel Pka-R1 mutants described in this study appear to be stronger alleles, and their analysis should prove useful for investigating the role of Pka-R1 in brain development and function. In addition, the role of PKA in different process of development has been investigated by making use of mutants in DC0, PKA-R2, and factors that modulate cAMP levels such as dunce and rutabaga. For example, it has been demonstrated that PKA antagonizes hh signaling by phosphorylating and inactivating the downstream transcription factor Cubitus interruputus. In addition, PKA-mediated signaling was shown to be involved in learning and behavior, and drug responses. Regulation of PKA by PKA-R1 is likely to be crucial in these processes as well (Yoshida, 2004).

The increase in PKA activity in Pka-R1¹⁸³⁰⁴ mutant extracts and the suppression of both the semi-lethality and the maternal-effect bicaudal phenotype by reduction of a functional copy of the DC0 catalytic subunit reveals that the phenotype of Pka-R1¹⁸³⁰⁴ is due to its failure to repress PKA activity in the mutant. Release of active PKA catalytic subunits from the inactive PKA holoenzyme is controlled by cAMP levels. It is also known that free catalytic subunits are more susceptible to proteolytic degradation than are catalytic subunits in the holoenzyme complex (Park, 2000). An excess of PKA catalytic activity is observed both in the absence and the presence of exogenous cAMP in the mutant extract, suggesting that upregulation of PKA catalytic subunit activity in Pka-R1¹⁸³⁰⁴ is due to a defect of mutant PKA-R1 in inhibiting catalytic subunit activity. In addition, the mutant extract still shows an increase in PKA activity in response to cAMP, which suggests the existence of a holoenzyme complex in the mutant. This is likely to be the case, as the point mutation in Pka-R1¹⁸³⁰⁴ is in a conserved arginine in the 'inhibitory domain' that acts as a catalytic unit pseudosubstrate. However, it is also possible that an autoregulatory feedback loop controlling expression or stability of catalytic subunits contributes to the increase in total PKA activity (Yoshida, 2004).

The premature and ectopic accumulation of Oskar in Pka-R1¹⁸³⁰⁴ suggests a role for PKA-R1 in oskar localization-dependent translation. An alternative explanation is that PKA-R1 is involved in the control of Oskar protein stability. In the case of C. elegans, some germ plasm components are excluded from the somatic cells by cullin-dependent degradation. A similar process might operate to restrict Oskar to the posterior pole. This assumes the existence of a mechanism whereby precociously and ectopically translated Oskar is degraded, and that this process requires PKA-R1 and its inhibition of PKA activity. However, there is no evidence to date of translation of oskar RNA prior to its posterior localization, or of active degradation of mislocalized Oskar (Yoshida, 2004).

Oskar degradation has been shown to be inhibited by phosphorylation. Both the protection and the degradation machineries operate throughout the oocyte and not just at the posterior pole. Because nucleotide substitutions in the 3' UTR of oskar lead to its ectopic translation and detectable accumulation, under normal circumstances, oskar translational regulation seems fairly tight, and is responsible for the specific accumulation of the protein at the posterior. Therefore it is speculated that the ectopic accumulation of Oskar in PKA-R1 mutants reflects a role of PKA in activation of oskar translation at the posterior pole, and that phosphorylation of translation regulatory proteins by PKA might cause the release of oskar mRNA from translational repression outside of the posterior domain (Yoshida, 2004).

It has been demonstated PKA is involved in the reception of the signal from the posterior follicle cells for the establishment of oocyte polarity at mid-oogenesis, specifically for the destabilization of the posterior microtubule organizing center. The signal from the posterior follicle cells might activate PKA at the posterior pole of the oocyte, and this local activation might in turn be responsible for the localized activation of oskar expression. It has been shown that the PKA holoenzyme can be targeted to specific subcellular domains by association with A-kinase anchoring proteins (AKAPs), a mechanism that has been proposed to regulate the spatial distribution of PKA activity. It is tempting to speculate that, in wild-type egg chambers, PKA holoenzyme complexes are targeted to specific AKAPs through PKA-R1, which blocks phosphorylation of specific targets, thus preventing ectopic expression of Oskar outside of the posterior domain. However, an alternative explanation is possible, whereby it is not PKA, but rather a PKA target involved in oskar activation that is asymmetrically distributed, with an enrichment at the posterior pole -- as is the case for oskar mRNA. Uniformly moderate levels of PKA activity throughout the oocyte would result in phosphorylation/activation of the target exclusively at the posterior pole; over-activation of PKA throughout the oocyte would result in activation of the target protein throughout the oocyte, which would be sufficient for ectopic activation of Oskar expression outside of the posterior pole. To address these possibilities, and to address directly the mechanism by which PKA controls the spatial restriction of oskar, it will be important to visualize the localization of the kinase activity, and to identify and determine the subcellular localization of the targets of PKA in this process (Yoshida, 2004).

Rab6 and the secretory pathway affect osk mRNA localization and the organization of microtubule plus-ends

The Drosophila oocyte is a highly polarized cell. Secretion occurs towards restricted neighboring cells and asymmetric transport controls the localization of several mRNAs to distinct cortical compartments. This study describes a role for the Drosophila ortholog of the Rab6 GTPase, Drab6, in establishing cell polarity during oogenesis. Drab6 localizes to Golgi and Golgi-derived membranes and interacts with BicD. Evidence is provided that Drab6 and BicD function together to ensure the correct delivery of secretory pathway components, such as the TGFα homolog Gurken, to the plasma membrane. Moreover, in the absence of Drab6, osk mRNA localization and the organization of microtubule plus-ends at the posterior of the oocyte were both severely affected. These results point to a possible connection between Rab protein-mediated secretion, organization of the cytoskeleton and mRNA transport (Januschke, 2007).

Oskar allows nanos mRNA translation in Drosophila embryos by preventing its deadenylation by Smaug/CCR4

Anteroposterior patterning of the Drosophila embryo depends on a gradient of Nanos protein arising from the posterior pole. This gradient results from both nanos mRNA translational repression in the bulk of the embryo and translational activation of nanos mRNA localized at the posterior pole. Two mechanisms of nanos translational repression have been described, at the initiation step and after this step. This study identifies a novel level of nanos translational control. The Smaug protein bound to the nanos 3' UTR recruits the deadenylation complex CCR4-NOT, leading to rapid deadenylation and subsequent decay of nanos mRNA. Inhibition of deadenylation causes stabilization of nanos mRNA, ectopic synthesis of Nanos protein and head defects. Therefore, deadenylation is essential for both translational repression and decay of nanos mRNA. A mechanism is proposed for translational activation at the posterior pole. Translation of nanos mRNA at the posterior pole depends on oskar function. Oskar prevents the rapid deadenylation of nanos mRNA by precluding its binding to Smaug, thus leading to its stabilization and translation. This study provides insights into molecular mechanisms of regulated deadenylation by specific proteins and demonstrates its importance in development (Zaessinger, 2006).

Post-transcriptional mechanisms of gene regulation play a prominent role during early development. Because the oocyte and developing embryo go through a phase in which no transcription takes place, gene expression relies on a pool of maternal mRNAs accumulated during oogenesis and is regulated at the level of translation or mRNA stability. It has been shown in several biological systems that poly(A) tail shortening contributes to translational silencing, whereas translational activation requires poly(A) tail extension. Poly(A) tail shortening, or deadenylation, is also the first step in mRNA decay. Subsequent steps occur only after the poly(A) tail has been shortened beyond a critical limit. Rapid deadenylation of unstable RNAs is caused by destabilizing elements, for example AU-rich elements (AREs) found in the 3' UTRs of several mRNAs. A number of proteins have been identified that bind to destabilizing RNA sequences and accelerate deadenylation as well as subsequent steps of decay (Zaessinger, 2006).

In yeast, deadenylation is mostly catalyzed by the multi-subunit CCR4-NOT complex, and this complex is also involved in deadenylation in Drosophila (Temme, 2004) and in mammalian cells. A second conserved deadenylase, the heterodimeric PAN2-PAN3 complex, appears to act before the CCR4-NOT complex. A third enzyme, the poly(A)-specific ribonuclease (PARN) is present in most eukaryotes but has not been found in yeast and Drosophila (Zaessinger, 2006).

Translational regulation of maternal mRNAs in Drosophila is essential to the formation of the anteroposterior body axis of the embryo. During embryogenesis, a gradient of the Nanos (Nos) protein arises from the posterior pole and organizes abdominal segmentation. This gradient results from translational regulation of maternal nos mRNA. The majority of nos transcripts is uniformly distributed throughout the bulk cytoplasm and is translationally repressed and subsequently degraded during the first 2-3 hours of embryonic development. A small proportion of nos transcripts is localized in the pole plasm, the cytoplasm at the posterior pole that contains the germline determinants. This RNA escapes repression and degradation, and its translation product forms a concentration gradient from the posterior pole. Both translation activation at the posterior pole and repression elsewhere in the embryo are essential for abdominal development, and head and thorax segmentation, respectively (Zaessinger, 2006 and references therein).

Translation of nos mRNA is repressed in the embryo by Smaug (Smg), which binds two Smaug response elements (SREs) in the proximal part of the nos 3' UTR. The SREs are also essential for the decay of nos mRNA. Repression of nos translation appears to be a multistep process, involving at least one level of regulation at the initiation step and another after nos mRNA has been engaged on polysomes. Repression at the initiation step is thought to involve an interaction between Smg and the protein Cup. The latter associates with the cap-binding initiation factor eIF4E, displacing the initiation factor eIF4G. Translation of nos mRNA at the posterior pole depends on Oskar (Osk) protein, although its mechanism of action has remained unknown (Zaessinger, 2006).

Bulk nos mRNA has a short poly(A) tail, and it was thought that nos translational control was independent of poly(A) tail length regulation. More recently, Smg and its yeast homologue Vts1 were shown to be involved in the degradation of mRNAs. Smg induces degradation and deadenylation of Hsp83 mRNA during early embryogenesis. This appears to result from recruitment by Smg of the CCR4-NOT deadenylation complex on Hsp83 mRNA, although the Smg-binding sites in this mRNA have not been identified. However, Hsp83 mRNA deadenylation was reported not to repress its translation. This study shows that nos mRNA is subject to regulation by active deadenylation by the CCR4-NOT deadenylation complex. This deadenylation depends on Smg and on the SREs in the 3' UTR of nos mRNA. The model is confirmed of the CCR4-NOT complex recruitment by Smg, in this case, onto nos mRNA, using genetic interactions between mutants affecting smg and the CCR4 deadenylase (properly named Twin and not to be confused with Twins), and showing the presence in a same protein complex of endogenous Smg and CAF1 (properly termed Pop2 and not to be confused with Caf1), a protein of the CCR4-NOT complex. Active deadenylation of nos mRNA contributes to its translational repression in the bulk embryo and is essential for the anteroposterior patterning of the embryo. Moreover, Osk activates translation of nos by preventing the specific binding of Smg protein to nos mRNA, thereby precluding active deadenylation and destabilization of nos mRNA (Zaessinger, 2006).

This paper shows that poly(A) tail length regulation is central to nos translational control. Poly(A) tail length regulation is a major mechanism of translational control, particularly during early development. nos translational control has been reported to be independent of poly(A) tail length. This conclusion came from the absence of nos poly(A) tail elongation between ovaries and early embryos, and the lack of nos poly(A) tail shortening between wild-type and osk mutant embryos in which nos mRNA is not translated at the posterior pole. However, later studies suggested that this lack of poly(A) tail change was not unexpected, as nos mRNA translation starts in ovaries, and the pool of translationally active nos mRNA in embryos is very small (4%) and remains undetected among the amount of translationally repressed nos in whole embryos. It has now been found that nos mRNA deadenylation by the CCR4-NOT complex, recruited to the 3' UTR by Smg, is required for nos translational repression in the bulk embryo. In addition, these data also suggest that nos translation at the posterior pole depends on the prevention of this deadenylation. nos mRNA is regulated at several levels, including localization, degradation, translational repression and translational activation. Localization at the posterior pole depends on two mechanisms: an actin-dependent anchoring at late stages of oogenesis, after nurse cells dumping and localized stabilization. Localization and translational control are coupled in that the localized RNA escapes both translational repression and degradation. A mechanism is proposed for this coupling. Translational repression and RNA degradation both involve Smg-dependent deadenylation. Deletion of the SREs in a nos transgene, as well as mutations in smg or in twin, which encodes the major catalytic subunit of the deadenylating CCR4-NOT complex, abrogate poly(A) tail shortening. Lack of deadenylation prevents the timely degradation of the RNA and also relieves translational repression. Deadenylation could repress nos mRNA translation by two mechanisms. Interaction of the cytoplasmic poly(A) binding protein (PABP) with mRNA poly(A) tails is important for the activation of translation initiation. Therefore, poly(A) shortening of nos mRNA would lead to PABP dissociation and inhibition of translation. In addition, deadenylation leads eventually to nos mRNA decay, which should also contribute to translational repression. Consistent with the Smg-dependent deadenylation of nos mRNA, describe in embryos, a recent study documented SRE-dependent deadenylation of chimeric transcripts containing the 3' UTR of nos mRNA in cell-free extracts from Drosophila embryos (Jeske, 2006). In this system, deadenylation of the chimeric RNAs also strongly contributes to translational repression, along with at least another deadenylation-independent mechanism (Zaessinger, 2006).

In this analysis, twin and smg mutants, although both impaired in nos mRNA poly(A) tail shortening, did not show the same defects. twin mutants fail to show nos poly(A) tail shortening during embryogenesis, whereas in smg mutant embryos or when poly(A) tails are measured from nos(ΔTCE) transgene, a poly(A) tail elongation is visible. This suggests that nos mRNA is also regulated by cytoplasmic polyadenylation which balances the deadenylation reaction, and that Smg binding to the RNA reduces the polyadenylation reaction. Consistent with a dynamic regulation of poly(A) tail length of maternal mRNAs resulting from a tight balance between regulated deadenylation and polyadenylation, it was found that in mutants for the GLD2 poly(A) polymerase, which is involved in cytoplasmic polyadenylation, nos mRNAs are precociously degraded in 0-1 hour embryos (Zaessinger, 2006).

Ectopic expression of osk in the bulk cytoplasm of the embryo is sufficient to impair nos mRNA binding to Smg and its deadenylation and destabilization. Therefore, it is proposed that, in wild-type embryos, Osk at the posterior pole inhibits Smg binding to the anchored nos mRNA, preventing deadenylation, decay and translational repression. This results in localized nos stabilization and translation. Osk might achieve this by a direct binding to Smg; it was shown to interact with Smg in vitro, through a region overlapping the RNA-binding domain in Smg. Alternatively, Osk could prevent Smg function independently of its binding to Smg, through its recruitment by another protein in nos-containing mRNPs. Consistent with a potential presence of Smg and Osk in the same protein complex, it was possible to co-immunoprecipitate Osk with Smg in embryos overexpressing Osk (Zaessinger, 2006).

Two mechanisms of nos translational repression have already been described. A first mode of translation inhibition appears to act during elongation, as suggested by polysome analysis and by the involvement of the Bicaudal protein, which corresponds to a subunit of the nascent polypeptide associated complex. The second mode of repression involves Smg and is thought to affect initiation. It requires the association of Smg with the protein Cup, which also binds eIF4E. The association of Cup with eIF4E competes with the eIF4E/eIF4G interaction, which is essential for translation initiation. This study identifies deadenylation by the CCR4-NOT complex as a novel level of nos translational repression, also involving Smg. Smg protein synthesis is probably induced by egg activation during egg-laying. Smg is absent in ovaries and accumulates during the first hours of embryogenesis, with a peak at 1-3 hours. Its amount is low during the first hour and possibly nonexistent during the first 30 minutes. This correlates with the presence at that time of high levels of nos mRNA in the bulk embryo that are not destabilized. nos translational repression is active, however, as this pool of mRNA is untranslated. Thus a Smg-independent mode of repression must be efficient during the first hour of development. This might correspond to repression at the elongation step and/or involve the Glorund protein, a Drosophila hnRNP F/H homologue newly identified as a nos translational repressor in the oocyte (Kalifa, 2006). Glorund has a role in repression of unlocalized nos mRNA in late oocytes and has been suggested to also act at the beginning of embryogenesis while Smg is accumulating to ensure the maintenance of translational repression at the oogenesis to embryogenesis transition (Kalifa, 2006). Analysis of glorund mutants revealed that the embryonic phenotypes are less severe than expected and led to the proposal that at least an additional level of nos translational repression is active in oocytes (Kalifa, 2006). Overexpression of Osk in the germline with nos-Gal4 results in long poly(A) tails of nos mRNA, even in 0-1 hour embryos in which Smg protein is poorly expressed. This suggests that the short poly(A) tail of nos mRNA in 0-1 hour wild-type embryos could in part result from active deadenylation during oogenesis, which would depend on a regulatory protein different from Smg. Deadenylation could therefore be involved in nos regulation during oogenesis, and would also be prevented by Osk in the pole plasm, as in embryos (Zaessinger, 2006).

Genetic evidences indicate that all three levels of translational repression are additive. Although the importance of the Smg/Cup/eIF4E mode of nos translational repression for the anteroposterior patterning of the embryo has not been addressed, the other two levels of repression are essential, as ectopic Nos protein leads to disruption of the embryo anteroposterior axis in twin or bicaudal mutants. This demonstrates that none of the three levels of repression is sufficient by itself and suggests that all three regulations are required to achieve complete translational repression of nos. As Osk acts by preventing the binding of Smg to the nos 3' UTR, it is likely to inhibit both Smg-dependent mechanisms of translational repression (Zaessinger, 2006).

The presence of Smg in discrete cytoplasmic foci and its partial colocalization in these foci with components of the CCR4-NOT deadenylation complex, and with components of P bodies, suggest that Smg-dependent deadenylation and translational control of nos occur in P bodies. P body dynamics and function have not been addressed in a complete organism during development. Consistent with the apparent complexity of P body function, including mRNA decay and translational repression, embryos different subsets of Smg-containing structures were identified in embryos: these subsets either do or do not contain the CCR4-NOT deadenylation complex and the Xrn1 5'-3' exonuclease. This suggests the existence of different types of P bodies that may have distinct functions (Zaessinger, 2006).

The CCR4-NOT complex is involved in default deadenylation of bulk mRNAs in somatic cells (Temme, 2004). This study finds that the same deadenylation complex has a role in active, sequence-specific deadenylation of a particular mRNA. Activation of deadenylation by CCR4-NOT results from the recruitment of the deadenylation complex by a regulatory RNA-binding protein to its specific mRNA target (this study) (Semotok, 2005). Several RNA-binding proteins are expected to interact with the CCR4-NOT complex to regulate the deadenylation of different pools of mRNAs in different tissues. CCR4 controls poly(A) tail lengths of Cyclin A and B mRNAs during oogenesis (Morris, 2005); the regulatory protein has not been identified, but it cannot be Smg, which is not expressed in ovaries. A similar mode of active deadenylation involving the recruitment of the deadenylation complex by ARE-binding proteins has been proposed in mammalian cells. A study in yeast has identified the PUF (Pumilio/FBF) family of RNA-binding proteins as activators of CCR4-NOT-mediated deadenylation through a direct interaction between PUF and POP2 (the CAF1 homologue). Although default deadenylation by CCR4 is not essential for viability (Temme, 2004), active deadenylation by CCR4 of specific mRNAs is essential for certain developmental processes, in particular during early development (Zaessinger, 2006).

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. This study shows 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 (Zimyanin, 2007).

On the basis of these results, a revised model is suggested for how the polarity of the oocyte is established. The polarization of the oocyte is initiated by the posterior-follicle-cell signal, which induces the localization of the PAR-1N1 short and long isoforms to the posterior cortex. This localized PAR-1 then recruits or stabilizes some microtubule plus ends at the posterior cortex, leading to the kinesin-dependent transport of a small amount of oskar mRNA from the center of the oocyte to the posterior pole. Once oskar mRNA reaches the posterior, its translational repression is relieved, and the resulting Oskar protein recruits another population of PAR-1 to the posterior. This PAR-1 can then recruit further microtubule plus ends, which in turn direct the posterior localization of more oskar mRNA. Thus, Oskar protein initiates a positive feedback loop that amplifies the polarization of the microtubule cytoskeleton, leading to an increase in the localization of its own mRNA. The microtubule polarity is not reinforced in oskar protein-null mutants or in mutants that disrupt the localization of translation of oskar mRNA, and this results in only a partial polarization of the microtubule cytoskeleton. On the other hand, oskar mRNA overexpression and premature translation in the middle of the oocyte initiates the positive feedback loop in the wrong place. As a consequence, PAR-1 recruits some of the microtubule plus ends to the center of the oocyte and away from the posterior pole. PAR-1 has also been shown to stabilize Oskar protein through direct phosphorylation. This therefore generates a second positive feedback loop that further reinforces the posterior localization of PAR-1, Oskar protein, and microtubule plus ends (Zimyanin, 2007).

Amplification through positive feedback loops appears to be an emerging theme in cell polarity. For example, the polarization of migrating neutrophils depends on the local enrichment of phosphatidylinositol 3,4,5 triphosphate (PtdInsP₃) at the leading edge of the cell, and this is amplified by a Rho-dependent positive feedback loop, in which PtdInsP₃ recruits PtdIns-3-OH kinase to the leading edge, which in turn generates more PtdInsP₃. A similar mechanism operates during bud-site selection in S. cerevisiae. The bud site is defined by the localization of activated Cdc42-GTP, and this localization induces the assembly of actin cables that extend into the cytoplasm. This signal is then reinforced by the transport of more Cdc42-GTP along the actin cables to the bud site, where it can induce the assembly of further actin cables. This mechanism is enhanced by a second feedback loop, in which Cdc42-GTP recruits the adaptor protein, Bem1, which binds Cdc24, which activates Cdc42. The current results add a third example of the use of multiple feedback loops to reinforce an initially weak cell polarity, and they provide the first case where this amplification involves the microtubule cytoskeleton rather than actin (Zimyanin, 2007).

Dynein-Dependent Transport of nanos RNA in Drosophila Sensory Neurons Requires Rumpelstiltskin and the Germ Plasm Organizer Oskar

Intracellular mRNA localization is a conserved mechanism for spatially regulating protein production in polarized cells, such as neurons. The mRNA encoding the translational repressor Nanos (Nos) forms ribonucleoprotein (RNP) particles that are dendritically localized in Drosophila larval class IV dendritic arborization (da) neurons. In nos mutants, class IV da neurons exhibit reduced dendritic branching complexity. This study investigated the mechanism of dendritic nos mRNA localization by analyzing requirements for nos RNP particle motility in class IV da neuron dendrites. Dynein motor machinery components were shown to mediate transport of nos mRNA in proximal dendrites. Two factors, the RNA-binding protein Rumpelstiltskin and the germ plasm protein Oskar function in da neurons for formation and transport of nos RNP particles. nos was shown to regulate neuronal function, most likely independently of its dendritic localization and function in morphogenesis. These results reveal adaptability of localization factors for regulation of a target transcript in different cellular contexts (Xu, 2013).

This study has combined a method that allows live imaging of mRNA in intact Drosophila larvae with genetic analysis to investigate the mechanism underlying transport of nos mRNA in class IV da neurons. Live imaging over the short time periods allowed has provided a snapshot into the steady-state behavior of nos*RFP particles in the proximal dendrites of mature da neurons. The results indicate that anterograde transport of nos RNP particles into and within da neuron dendrites is mediated by dynein and is consistent with the minus-end out model for microtubule polarity in the proximal dendrites of da neurons (Zheng, 2008). This model predicts that bidirectional trafficking would be mediated by opposite polarity motors and the predominance of retrograde movement of nos*RFP particles when dynein function is partially compromised is consistent with this. Moreover, Rab-5 endosomes, whose accumulation in class IV da neuron dendrites is dynein-dependent, also exhibit bidirectional movement (Satoh, 2008), suggesting that different cargos may use similar dendritic transport strategies. Unfortunately, the severe defects caused by loss of kinesin have thus far hampered confirmation of a role for kinesin in these events (Xu, 2013).

The observed bidirectional movement of nos RNP particles resembles the constant bidirectional transport observed for dendritic mRNAs near synapses in hippocampal neurons. In contrast to da neurons, proximal dendrites of mammalian neurons have mixed microtubule polarity so that bidirectional trafficking could be mediated by a single motor that switches microtubules or by switching between the activities of plus-end and minus-end motors. The association of kinesin with neuronal RNP granule components and inhibition of CaMKIIα RNA transport by dominant-negative inhibition of kinesin has implicated kinesin as the primary motor for dendritic mRNA transport. However, a recent study showed that dynein mediates unidirectional transport of vesicle cargoes into dendrites of cultured hippocampal neurons as well as bidirectional transport within the dendrites. Whether dynein plays a role in RNP particle transport in mammalian dendrites as it does in Drosophila neurons remains to be determined (Xu, 2013).

Despite its prevalence, the role of bidirectional motility is not yet clear. A recently proposed 'sushi belt' model suggests that neuronal RNP particles traffic back and forth along the dendrite until they are recruited by an active synapse and disassembled for translation (Doyle, 2011). Although da neuron dendrites do not receive synaptic input, this continual motility may provide a reservoir of nos mRNA that can be rapidly mobilized for translation locally in response to external signals that regulate dendrite branching (Xu, 2013).

These studies have shown that nos mRNA can be adapted for different localization mechanisms depending on cellular context: diffusion/entrapment in late oocytes that lack a requisite polarized microtubule cytoskeleton and microtubule-based transport during germ cell formation in the embryo and in class IV da neurons. Surprisingly, Rump and Osk are specifically required for nos localization in both oocytes and da neurons, suggesting that they function in the assembly or recognition of a fundamental nos RNP that can be adapted to both means of localization. However, because it is not possible to distinguish individual particles within the cell body, the possibility cannot be ruled out that Rump and/or Osk mediate coupling of nos RNP particles to dynein motors rather than particle formation. Within the germ plasm, nos associates with Vasa (Vas), a DEAD-box helicase, and is transported together with Vas into germ cells. Although dendritic branching complexity is reduced in vas mutants, no effect on dendritic localization of nos RNP particles was detected, suggesting that only a subset of germ plasm components are shared by neuronal localization machinery. A role for osk in learning and memory was proposed based on the isolation of an enhancer trap insertion upstream of osk in a screen for mutants with defective long-term memory, but osk function in memory formation has not been directly tested. Notably, however, a recent study showed that the osk ortholog in the cricket Gryllus bimaculatus functions in development of the embryonic nervous system rather than in germ cell formation. Thus, the ancestral function of osk appears to be in neural development, whereas its role in germ plasm formation is a later adaptation in higher insects. The results showing that Osk protein function is not limited to Dipteran germ plasm organization but also plays an important role in neuronal development and function supports this idea (Xu, 2013).

The data indicate that the Nos/Pum complex is not only required for da neuron morphogenesis, but also for nociceptive function. However, nociception does not appear to require local function of Nos/Pum in the dendrites and reduced dendritic branching does not necessarily correlate with a deficit in nociception. These results suggest that morphogenesis and function are regulated separately and that Nos/Pum plays a second role in regulating the somatic translation of proteins required for the nociceptive response. Systematic identification of Nos/Pum targets will be essential to further investigate these different roles (Xu, 2013).

PROTEIN INTERACTIONS