Activation of the zygotic genome is a prerequisite for the transition from maternal to zygotic control of development. The onset of zygotic transcription has been well studied in somatic cells, but evidence suggests that it is controlled differently in the germline. In Drosophila, zygotic transcription in the soma has been detected as early as one hour after egg laying (AEL). In the germline, general RNA synthesis is not detected until 3.5 hours AEL (stage 8) and poly(A)-containing transcripts are not observed in early germ cell nuclei. However, rRNA gene expression has been demonstrated at this time. Therefore, either there is a general, low level activation of the genome in early germ cells, or specific classes of genes are repressed, such as those transcribed by RNA polymerase (RNAP) II. This issue was addressed by localizing the potent transcriptional activator Gal4-VP16 to the germline. Gal4-VP16-dependent gene expression is repressed in early germ cells. Localization of germ plasm to the anterior reveals that it is sufficient to repress Bicoid-dependent gene expression. Thus, even in the presence of known transcriptional activators, RNAP II dependent gene expression is actively repressed in early germ cells. Once the germ cell genome is activated, vasa is expressed specifically in germ cells beginning at stage 9 (about 4 hours after egg lay). This expression is very soon after the time that germ cells become competent for expression of RNAP II target genes, as judged by experiments with Gal4-VP16. The expression does not require proper patterning of the soma, indicating that it is most likely under the control of the germ plasm (Van Doren, 1998).

Vasa protein is found in ovaries from the late third-instar larvae in precursors of oogonia. It is also found in abundance in the germarium of adult ovaries: in nurse cells, and in the oocyte precursor. It begins to be transported to the oocyte during stage ten, where it is first found in the perinuclear nuage, the same location as early Gurken protein. Vasa protein soon accumulates at the posterior pole of the oocyte. The Vasa transcript is not localized. After fertilization and the completion of mitotic cycle 9, Vasa protein accumulates in the pole cells. The protein persists in pole cells [Images] and later in gonads (Lasko, 1990).

Much of Maelstrom localizes to highly abundant particles within germline cells. The frequency and distribution of Maelstrom particles are reminiscent of that previously described for nuage, to which Vasa localizes. Double labeling of Maelstrom and Vasa shows overlap in perinuclear germline granules from stem cells through stage 10 nurse cells. Double labeling of Maelstrom and a nuclear lamin shows that virtually all distinct Maelstrom particles are closely apposed to the cytoplasmic face of the nuclear envelope in nurse cells. Because nanos-GAL4-driven GFP-tagged Aubergine (AubGFP) localizes to nuage in late stage egg chambers, Aubergine localization was examined in combination with Vasa and Maelstrom immunostaining. Each discrete particle in the germarium and early egg chamber labels for Vasa, Maelstrom and AubGFP, a concordance that is also maintained in stages 7-10. (Owing to the discontinuous nature of the nanos driver, AubGFP is not highly expressed between approximately stages 3 and 6.) At the ultrastructural level, most nuage is lost from the oocyte by stage 1, prior to the formation of the karyosome. However, occasional particles of Vasa and Maelstrom can be detected in the ooplasm as late as stage 4. Although the most conspicuous localization of Maelstrom and Vasa is to nuage, each protein is also present within the nucleus and cytoplasm of all germline cells. Within the oocyte nucleus, both proteins localize to discrete regions in young egg chambers: in single confocal sections, Vasa often appears in discrete dot or dots, exclusive of, but adjacent to an 'aura' of concentrated Maelstrom. Maelstrom persists in the oocyte nucleus as diffuse staining through at least late stage 10B. After onset of pole plasm assembly (stage 8/9), Vasa accumulates in posterior region of the oocyte. Maelstrom, by contrast, never shows a posterior concentration in the ooplasm. Although Maelstrom is present in the mature egg and early embryo, its distribution is again uniform at these stages. Since neither the Maelstrom nor its RNA show preferential posterior accumulation in the ooplasm, Maelstrom is the first described nuage component that is not also concentrated in pole plasm (Findley, 2003).

Isolation of new polar granule components in Drosophila reveals P body and ER associated proteins

Germ plasm, a specialized cytoplasm present at the posterior of the early Drosophila embryo, is necessary and sufficient for germ cell formation. Germ plasm is rich in mitochondria and contains electron dense structures called polar granules. To identify novel polar granule components, proteins were isolated that associate in early embryos with Vasa (Vas) and Tudor (Tud), two known polar granule associated molecules. Maternal expression at 31B (ME31B), eIF4A, Aubergine (AUB) and Transitional Endoplasmic Reticulum 94 (TER94) were identified as components of both Vas and Tud complexes and their localization to polar granules was confirmed by immuno-electron microscopy. ME31B, eIF4A and AUB are also present in processing (P) bodies, suggesting that polar granules, which are necessary for germ line formation, might be related to P bodies. The recovery of ER associated proteins TER94 and ME31B confirms that polar granules are closely linked to the translational machinery and to mRNP assembly (Thomson, 2008).

Little is understood of the molecular events that link the assembly of germ plasm to the formation of germ cells. There is a strong correlation between polar granule formation and germ cell formation, yet their functional relationship is still unclear. In an attempt to understand polar granule formation and function this study set out to isolate polar granule components with a biochemical approach; proteins common to both Tud and Vas complexes were isolated. These complexes were isolated by cross-linking proteins from early embryonic extracts followed by anti-Tud or anti-Vas immunoprecipitation; proteins found in both complexes were then immunolocalized using EM. Using this method it was confirmed that Aub is a polar granule component and three new polar granule components were identified: ME31B, TER94 and eIF4A. Through genetic interaction analysis in transheterozygous embryos it was shown that decreasing the levels of Vas or Tud along with either Aub, ME31B, Ter94 or eIF4A reduces germ cell number. This approach both identified novel polar granule components and implicated novel processes in germ cell formation (Thomson, 2008).

The presence of Aub, ME31B and eIF4A in polar granules supports the hypothesis that polar granules and P bodies are structurally, and perhaps functionally, related. Recovery of CUP, an ME31B-interacting protein, in the Tud complex further supports this. Similar parallels have recently been found for the mouse chromatoid body, an electron dense structure in the male germ line with similarities to Drosophila polar granules and nurse cell nuage. RNAs in P bodies are stored in a translationally quiescent state and can later be either degraded or translationally activated in response to physiological cues (Decker, 2006). Translational repression in P bodies occurs at the level of mRNA recruitment to the ribosome, and through miRNA silencing pathways. The polar granule components that were identified suggest their involvement in both types of post-transcriptional regulation. Aub has been implicated in processing of germ line specific piRNAs. Findings that Aub associates with polar granules implicates piRNAs in germ cell formation, as has a previous study. In contrast, Vas and eIF4A have been closely linked with translational regulation and are not known to participate in miRNA silencing pathways (Thomson, 2008).

The RNA-rich nature of early polar granules supports the idea that specific germ line-specific mRNAs are stored in polar granules in a translationally repressed state. Subsequently, these RNAs are translated and their function may be required for germ cell formation and further development. How could general translational repression mediated by polar granules be overcome? Conceivably Vas could be a key factor. Vas, a highly conserved polar granule component with homologues in other species involved in germ line formation, binds directly to eIF5B. Disrupting the eIF5B-Vas interaction abrogates germ cell formation, presumably due to the loss of the ability of Vas to initiate translation of yet unidentified mRNAs (Johnstone, 2004). Thus, Vas may act as a germ line specific mRNA translation derepression factor. Other tissue specific factors could adapt a P body to a specific function or cell line. Identification of mRNAs that localize in polar granules and are dependent on Vas for their translation will no doubt provide more insight into this mechanism (Thomson, 2008).

Ultrastructural analysis of proteins found in the Vas and Tud containing complexes revealed that polar granules were often in close proximity or in contact with ER. Supporting such a link, Ter94 and ME31B were present in both Tud and Vas complexes, and are enriched in polar granules. Further work is required to elucidate what proportion of polar granules associate with ER, and whether this association is stage dependent. The presence of Ter94, an ER exit site marker, with Vas, ME31B, Aub and eIF4A in the same structure suggests that ER exit sites directly associate with the translational machinery with both activating and repressing factors. Polar granules may form at ER exit sites, which could provide a mechanism for the localization and assembly of mRNPs required for the translational regulation of their constituent mRNAs. There is evidence that P bodies associate with ER exit sites. In the Drosophila ovary, Trailer Hitch (TRAL) associates with ER exit sites and associates with P body components such as ME31B and CUP. The C. elegans homologue of TRAL, Car-1, associates with DCAP-1, a P-body marker, and car-1 mutations affect ER assembly (Squirrell, 2006). A single TRAL peptide was recovered in one immunoprecipitation with Tud, perhaps lending additional support to an association between polar granules and ER exit sites (Thomson, 2008).

Repeated attempts to biochemically isolate polar granules were made over 30 years ago, before the advent of modern analytical techniques that allow the identification of very small amounts of protein. From this work a major polar granule component of approximately 95 kDa was identified. The nature of this protein was not determined although Ter94 has approximately the same molecular mass, as does PIWI, a 97-kDa likely polar granule component that has eluded the currently used screens. PIWI associates with Vas, a polar granule component, as well as with components of the miRNA machinery. PIWI RNA and protein are enriched in germ plasm and piwi mutants have defects in germ cell formation. The screen also did not identify Osk, which was shown by a yeast two-hybrid screen to bind directly to Vas. This may be because these proteins were not present in high enough abundance for detection. Alternatively, since the reactive ends of the cross-linkers that were used specifically cross-link cysteine residues, they would not stabilize a particular protein-protein interaction unless a pair of cysteine residues is within the range of the cross-linker. The work demonstrates that a molecular approach can be a powerful complement to genetics, and that purification schemes based on two independent reagents can reduce signal-to-noise problems that are inherent in co-immunoprecipitation experiments. Molecular approaches such as this one also have the capacity to identify proteins involved in a developmental process that are encoded by genes with multiple functions, or required for cellular viability, that will therefore elude phenotype-based genetic screens (Thomson, 2008).

Effects of Mutation or Deletion

The posterior group of maternal genes is required for the development of the abdominal region in the Drosophila embryo. Genetic as well as cytoplasmic transfer experiments have been used to order seven of the posterior group genes into a functional pathway: nanos, pumilio, oskar, valois, vasa, staufen and tudor. Nanos protein can restore normal abdominal development in posterior group mutants. The other posterior group genes have distinct accessory functions: pumilio acts downstream of nanos and is required for thedistribution or stability of the nanos-dependent activity in the embryo. 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 ofnanos-dependent activity and of germ line determinants (Lehmann, 1991).

Several vasa alleles exhibit a wide range of early oogenesis phenotypes. A detailed analysis of Vasa function during early oogenesis is reported using novel as well as previously identified hypomorphic vasa alleles. vasa is required for the establishment of both anterior-posterior and dorsal-ventral polarity of the oocyte. The polarity defects of vasa mutants appear to be caused by a reduction in the amount of Gurken protein at stages of oogenesis critical for the establishment of polarity. Vasa is required for translation of Gurken mRNA during early oogenesis and for achieving wild-type levels of Gurken mRNA expression later in oogenesis. A variety of early oogenesis phenotypes observed in vasa ovaries, which cannot be attributed to the defect in gurken expression, suggest that vasa also affects expression of other mRNAs (Tomancak, 1998).

A hallmark of germline cells across the animal kingdom is the presence of perinuclear, electron-dense granules called nuage. In many species examined, Vasa, a DEAD-box RNA helicase, is found in these morphologically distinct particles. Despite its evolutionary conservation, the function of nuage remains obscure. A null allele of maelstrom (mael) has been characterized. Maelstrom protein is localized to nuage in a Vasa-dependent manner. By phenotypic characterization, maelstrom has been defined as a spindle-class gene that affects Vasa modification. In a nuclear transport assay, it has been determined that Maelstrom shuttles between the nucleus and cytoplasm, which may indicate a nuclear origin for nuage components. Interestingly, Maelstrom, but not Vasa, depends on two genes involved in RNAi phenomena for its nuage localization: aubergine and spindle-E (spn-E). Furthermore, maelstrom mutant ovaries show mislocalization of two proteins involved in the microRNA and/or RNAi pathways, Dicer and Argonaute2, suggesting a potential connection between nuage and the microRNA-pathway (Findley, 2003).

How germline status is established and maintained in sexually reproducing organisms is a fundamental question in developmental biology. A conserved feature of germ cells in species across the animal kingdom is the presence of a distinct morphological element called nuage. Ultrastructurally, nuage appears as electron-dense granules that are localized to the cytoplasmic face of the nuclear envelope. Despite the breadth of nuage in the animal kingdom, there is currently a lack of depth in understanding its function. In animals ranging from the nematode to vertebrates, the Vasa protein has been detected in these granules. Both nuage and Vasa thus offer potential clues as to what makes a germ cell unique (Findley, 2003).

One system with high potential for understanding the role of nuage is Drosophila. In females, Vasa-positive germline granules are continuously present throughout the life cycle, taking one of two forms, nuage or pole plasm. Pole plasm, which contains polar granules, is a determinant that is both necessary and sufficient to induce formation of the germ lineage in early embryogenesis. In Drosophila, nuage is first detectable when primordial germ cells are formed; it persists through adulthood, where it is present in all germ cell types of the ovary (Findley, 2003).

In Drosophila, three proteins are known to localize to nuage: Vasa, Aubergine and Tudor. The sequence or mutant phenotype of each gene suggests a role in post-transcriptional RNA function. Vasa is a DEAD-box RNA helicase required for nurse cell-to-oocyte transport of several mRNAs critical to oocyte patterning. Vasa is also required for efficient translation of several key proteins in oogenesis, and itself interacts both physically and genetically with a Drosophila homolog of yeast, Translation Initiation Factor 2 (dIF2). Vasa is thus potentially implicated in translational control. Aubergine is a member of the RDE1 (for RNAi defective)/AGO1 (Argonaute1) protein family, homologs of which are required in both RNAi and developmental processes in diverse organisms. Aubergine is required, during oogenesis, for efficient translation of Oskar, which is pivotal in initiating pole plasm assembly. Aubergine is also required for RNAi in late oogenesis. Tudor, a novel protein, comprises ten copies of an ~120 residue motif (the 'Tudor Domain', pfam00567) present in several proteins involved or implicated in RNA-binding capacities. The domain has been suggested to mediate protein-protein interactions. Drosophila Tudor is required to mediate transfer of mitochondrial ribosomal RNAs from mitochondria to the surface of polar granules during pole cell formation in early embryogenesis (Amikura, 2001). A role for Tudor prior to pole plasm assembly, however, has not been described (Findley, 2003 and references therein).

A null allele of the maelstrom gene, which encodes a novel protein with a human homolog, has been identified and characterized. The mutant displays each of the defects in oocyte development common to the spindle-class. Maelstrom localizes to nuage in a Vasa-dependent manner and maelstrom is required for proper modification of Vasa. Through mutant analysis, this study begins to unravel genetic dependencies of nuage particle assembly (Findley, 2003).

It is unknown whether the known nuage proteins act in a common pathway before their convergence in nuage particles. To begin to answer this question, attempts were made to determine genetic dependencies for nuage particle assembly. AubGFP is known to depend on vasa function for its nuage localization. Maelstrom and Vasa localization were analyzed in wild-type, maelstrom, vasa, aubergine and spn-E backgrounds. Maelstrom protein levels proved to be quite variable among ovarioles of single mutant backgrounds. So, in order to compare localization, individual ovarioles were examined in which Maelstrom levels were not significantly reduced. Virtually no Maelstrom immunoreactive signal is present in maelstrom mutants, whereas Vasa is largely maintained in nuage. By contrast, the perinuclear accumulation of Maelstrom is virtually absent in the vasa null mutant, suggesting that Maelstrom localization in nuage is Vasa dependent. Maelstrom's distribution was examined in several vasa point mutants, in the hope of correlating functional domains in the protein with nuage organizational function. Of particular interest were two vasa EMS alleles, vas011 and vas014, each of which produces a protein devoid of RNA binding and unwinding activities. In both of these mutants, Vasa and Maelstrom colocalization in nuage is largely maintained. Vasa and Maelstrom localization were analyzed in several allelic combinations of aubergine, since a null for this gene has not been described. Both aubHN2 and aubN11 alleles encode truncated proteins. In this and other aubergine mutant combinations, the normal concentration of Maelstrom in nuage is severely depleted in all germline cells. Vasa is largely maintained in perinuclear localization in this mutant background, but the normally discrete particles are less obvious; instead, Vasa appears as a more uniform perinuclear band (Findley, 2003).

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 t1/2=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 vasAS 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).

SOLO: a meiotic protein required for centromere cohesion, coorientation, and SMC1 localization in Drosophila melanogaster

Sister chromatid cohesion is essential to maintain stable connections between homologues and sister chromatids during meiosis and to establish correct centromere orientation patterns on the meiosis I and II spindles. However, the meiotic cohesion apparatus in Drosophila remains largely uncharacterized. Sisters on the loose (SOLO), a novel splice product of Vasa, is essential for meiotic cohesion in Drosophila. In solo mutants, sister centromeres separate before prometaphase I, disrupting meiosis I centromere orientation and causing nondisjunction of both homologous and sister chromatids. Centromeric foci of the cohesin protein SMC1 are absent in solo mutants at all meiotic stages. SOLO and SMC1 colocalize to meiotic centromeres from early prophase I until anaphase II in wild-type males, but both proteins disappear prematurely at anaphase I in mutants for mei-S332, which encodes the Drosophila homologue of the cohesin protector protein Shugoshin. The solo mutant phenotypes and the localization patterns of SOLO and SMC1 indicate that they function together to maintain sister chromatid cohesion in Drosophila meiosis (Yan, 2010).

Meiosis is a specialized cell division that functions in sexual reproduction to generate haploid gametes from diploid precursor cells. It consists of two divisions preceded by a single round of DNA replication. Meiosis I is a reductional division in which homologous chromosomes (homologues) segregate to opposite spindle poles. Meiosis II is an equational division in which sister chromatids separate (Yan, 2010).

Two key differences in chromosome behavior underlie the different segregation patterns in meiosis I and II. One is the manner in which segregating chromosomes are connected. Stable connections between segregating chromosomes are essential to prevent them from separating prematurely and to provide the tension required to enable the chromosomes to achieve bipolar alignment on the spindle. In meiosis II, as in mitosis, the critical connections are cohesion between sister centromeres. Cohesion is established during replication and preserved throughout the cell cycle until its removal at the onset of anaphase (anaphase II of meiosis). In meiosis I, stable connections between homologues must be established. In most organisms, these connections take the form of chiasmata, which derive from crossovers between homologous chromatids and which are stabilized by cohesion between sister chromatid arms distal to the crossover sites. Thus, sister chromatid cohesion underlies the connections between segregating chromosomes in both meiotic divisions. However, in some eukaryotes, such as Drosophila males, homologue exchange and chiasmata are absent. In Drosophila, homologue connections are provided by the male meiosis-specific chromosomal proteins stromalin in meiosis (SNM) and mod(mdg4) in meiosis (MNM; Thomas, 2005) (Yan, 2010).

Cohesion is mediated by a conserved cohesin complex consisting of one member each of the SMC1, SMC3, SCC1/RAD21/REC8, and SCC3/SA families. Proteolytic cleavage of the SCC1 subunit (or its meiotic paralogue REC8) of cohesin at anaphase by separase triggers chromosome segregation during mitosis and at both meiotic divisions. In meiosis, this requires two separate rounds of separase activation: one round at anaphase I to cleave arm cohesins, release chiasmata, and allow homologues to segregate, and a second round at anaphase II to cleave centromere cohesin and allow sister chromatids to segregate. Conserved centromeric proteins called shugoshins function to protect centromeric cohesins from premature cleavage by separase during anaphase I (Yan, 2010).

A second critical difference between meiosis I and II is the orientation adopted by sister centromeres. In meiosis II, as in mitosis, sister centromeres orient back to back and establish separate kinetochores that make independent attachments to spindle poles. In meiosis I, sister centromeres adopt a side by side orientation and collaborate in forming a single functional kinetochore, ensuring that only two functional kinetochores are present per bivalent despite the presence of four chromatids. This enables sister centromeres to coorient (become attached to spindle fibers emanating from the same pole), which in turn enables homologous centromeres to biorient (Hauf, 2004). The mechanism of sister centromere coorientation is not well understood. In Saccharomyces cerevisiae, it depends on a centromeric meiosis I -- specific complex called monopolin. The role of cohesin in centromere orientation is unclear. In Schizosaccharomyces pombe, coorientation requires both meiosis-specific Rec8 cohesin and MoaI, a specialized centromere protein that appears to function primarily by stabilizing occupancy of centromere core sequences by Rec8 cohesin. Recently, it has been shown that provision of an artificial tether between sister centromere core sequences suffices for preferential sister centromere coorientation in the absence of Rec8 or MoaI. The mechanism of coorientation in higher eukaryotes is not known in any detail, but the fact that rec8 mutations disrupt centromere orientation in several model eukaryotes suggests a role for cohesin (Yan, 2010).

Although there is considerable evidence that the aforementioned two-stage, cohesin-based meiotic segregation mechanism is widely conserved, the role of cohesin in meiotic cohesion in Drosophila remains unclear. This is due in large part to the absence of a functional rec8 orthologue and of meiosis-specific cohesin mutations. In addition to the four mitotic cohesins, the fly genome encodes two meiosis-specific cohesin paralogues: C(2)M, an SCC1/RAD21 paralogue required for homologue synapsis and recombination in female meiosis, and SNM, an SCC3/SA paralogue required for stable homologue pairing in male meiosis (Manheim, 2003; Heidmann, 2004; Thomas, 2005). However, despite their homology to cohesin proteins, both C(2)M and SNM are dispensable for sister chromatid cohesion in meiosis. Although the orientation disruptor (ord) gene is required for meiotic sister chromatid cohesion, ORD has no homology to cohesins or to any other known proteins, and its subcellular localization pattern differs from that of cohesin. Thus, the relationship between ORD and cohesin and the precise role of ORD in cohesion are unclear (Yan, 2010).

Two lines of evidence support a role for cohesin in Drosophila meiosis. First, immunocytological studies have localized SMC1 to centromeres in both male and female meiosis I and to synaptonemal complexes in female meiosis (Thomas, 2005; Khetani, 2007). Second, mutations in the Drosophila shugoshin homologue mei-S332 cause precocious sister chromatid separation (PSCS) and high frequencies of meiosis II nondisjunction (NDJ), which is consistent with a possible role of MEI-S332 in protection of centromeric cohesin at anaphase I. However, the molecular function of mei-S332 has not been established, and the inviability of cohesin component mutants has thus far prevented their meiotic roles from being characterized. Thus, the molecular basis for meiotic cohesion in Drosophila remains poorly defined (Yan, 2010).

This study describes a novel Drosophila protein, sisters on the loose (SOLO), which is required for sister centromere cohesion and SMC1 localization to centromeres throughout meiosis and colocalizes with SMC1 on centromeres from the onset of meiosis until both proteins disappear at anaphase II. In addition to randomizing chromatid segregation in meiosis II, solo mutations result in a unique 'random 2::2' segregation pattern at meiosis I that reflects complete loss of sister centromere coorientation but partial maintenance of bivalent structure and function. The data indicate that SOLO plays a direct role in sister chromatid cohesion during Drosophila meiosis and suggest that it does so in close association with cohesin (Yan, 2010).

To characterize the solo transcription unit, a nearly full-length cDNA was sequenced as well as several RT-PCR and 5′ and 3′ rapid amplification of cDNA ends (RACE) fragments that include part or all of the intronic exons. Those analyses revealed that in addition to the two intronic exons, solo transcripts also include the three upstream vas exons, which encode several RGG repeats found in RNA-binding proteins but lack the five downstream vas exons, which encode the RNA helicase domain. The three upstream vas exons and the two intronic exons are spliced together to create a continuous ORF that extends from the translation start site of vas in exon 2 to a stop codon in the downstream intronic exon and that could encode a protein 1,031 amino acids in length (Yan, 2010).

Complementation analysis between solo and vas mutations confirmed the proposed exon structure of solo. solo alleles complemented all vas alleles containing mutations in any of the five C-terminal exons, which encode the VASA helicase domain, indicating that the C terminus of VASA is not shared by SOLO. However, vas mutations that map upstream of the SOLO-specific exons, including one nonsense mutation in exon 3, failed to complement the solo alleles, indicating that the 137 amino acids encoded by the upstream exons are present in both proteins. It is unlikely that the SOLO-specific exons are expressed independently of vas in addition to being expressed as a fusion product with the N terminus of VASA, as vas6356-001 behaves as a null allele of solo, giving X-Y NDJ frequencies of 41%-44% in trans with solo alleles. It is concluded that solo encodes a protein that includes the N-terminal 137 amino acids of VASA fused to 894 amino acids encoded within the third intron of vas (Yan, 2010).

Single homologues of SOLO were identified by BLAST analysis in all 12 sequenced Drosophila genomes. Overall conservation is fairly low; Drosophila SOLO exhibits only ~30% amino acid identity with its homologues in Drosophila virilis and Drosophila pseudoobscura. However, in all of the Drosophila genomes, the solo sequences are nested within a large intron upstream of the exons that encode the helicase domain of VASA, and SOLO appears capable of being expressed by the same alternative splice mechanism used in Drosophila (Yan, 2010).

No homologues of SOLO were identified outside of the genus Drosophila, not even in the genome of the mosquito Anopheles gambiae. Although it is possible that solo exists in A. gambiae but is unrecognizable because of divergence, it would have to be located elsewhere in the genome, as there are no large exons nested within introns of the A. gambiae vas gene. Other than the RGG motifs in the common N terminus, SOLO exhibits no significant homologies with other proteins in the sequence database (Yan, 2010).

solo mutants exhibit premature loss of centromere cohesion and high NDJ at both meiosis I and II. Centromere cohesion is strongly impaired by stage S5 of prophase I long before centromere orientation patterns are established at prometaphase I. Although the premature loss of centromere cohesion is likely the underlying cause of NDJ at both divisions, the mechanisms of meiosis I and meiosis II NDJ nevertheless differ in important ways. During meiosis II, sister chromatids are fully separated at metaphase II, and anaphase II segregation appears to involve random assortment of fully independent chromatids to the two poles. However, during meiosis I, fully separated sister chromatids are rarely observed, and bivalents containing the four chromatids of a homologous pair remain intact throughout the division. Moreover, at least for the X-Y pair, chromatid segregation is not fully random. Although random assortment would lead to numerically unequal segregation (3:1 or 4:0) in 62.5% of meiosis I divisions, in solo males, >95% of anaphase I cells exhibit two chromatids at each pole. This restriction probably applies to autosomes as well because in DAPI-stained preparations, >90% of anaphase I spermatocytes exhibit poles with roughly equal DNA content. Nevertheless, segregation is very abnormal, indeed random in a more limited sense. Unlike WT spermatocytes in which sister chromatids always cosegregate at meiosis I, in solo spermatocytes X and Y chromatids exhibit no preference for or against their sister as a segregation partner. The result is a 2:1 ratio of equational (XY::XY) to reductional (XX::YY) segregations. Thus, bivalents in solo males retain their gross structure and the ability to segregate in an orderly fashion but lose sister-specific connections and with them the ability to distinguish sister from homologous chromatids. The resulting bivalents have four functional kinetochores instead of the normal two, and these orient independently of each other yet are somehow constrained to orient two to each pole (Yan, 2010).

How might SOLO perform its role in sister centromere orientation? One possibility is a role similar to the monopolin complex in S. cerevisiae or MoaI in S. pombe, proteins that function specifically in coorientation. However, mutations in these proteins do not disrupt sister centromere cohesion, whereas solo mutations disrupt both cohesion and coorientation. Therefore, a more parsimonious idea is that the primary role of SOLO is in centromere cohesion and that cohesion is required for coorientation. SOLO would thus be more similar to REC8, a meiotic cohesin component that is also required for both cohesion and coorientation in S. pombe (Watanabe, 1999; Watanabe, 2001; Yokobayashi, 2003; Sakuno, 2009). It remains to be determined whether other proteins analogous to monopolin or MoaI are also required for centromere coorientation in Drosophila (Yan, 2010).

Homologue connections, in the form of recombination-generated chiasmata, have been shown in both S. cerevisiae and S. pombe to promote fidelity of sister centromere coorientation to varying degrees both in WT cells and in cells deficient for other centromere orientation factors. Drosophila males lack chiasmata but use the SNM-MNM complex to maintain homologue pairing until anaphase I. The data indicate that SNM (likely in complex with MNM) serves to coordinate chromatid segregation patterns in the absence of centromere cohesion but has only a minimal effect on sister centromere orientation by itself. The fact that the reductional/equational segregation ratio in solo mutants almost exactly matches the random expectation makes it unlikely that SNM does anything to actively promote reductional segregations. The main effect of the loss of SNM in a SOLO-deficient background is abrogation of the restriction against unequal segregations. More than 40% of anaphase I cells in solo; snm males exhibit numerically unequal segregations compared with <5% in solo males. Although the ratio of equational to reductional 2::2 segregations increases somewhat in solo; snm mutants relative to solo mutants, for reasons that are not clear, reductional segregations are nevertheless preserved and indeed occur at approximately the expected random frequency (12.5%). This stands in sharp contrast to spo13 or Moa1 mutants (in S. cerevisiae and S. pombe, respectively), which exhibit mixed reductional/equational meiosis I segregation patterns similar to solo but which revert to 100% equational segregation when homologue connections are removed by spo11 mutations. The basis for this difference is that spo13 and Moa1 interfere with sister centromere orientation without disrupting cohesion before anaphase I so that loss of homologue connections leaves most chromosomes still connected at sister centromeres. However, solo mutations ablate sister chromatid cohesion so leave no basis for regular equational segregation (Yan, 2010).

How does SNM-MNM promote regular chromatid segregation? A plausible scenario is that SNM-MNM provides nonspecific connections among all four chromatids at homologue-pairing sites such as the rDNA locus of the X-Y pair. Although inadequate to direct centromere orientation, such connections would preserve bivalent stability and could provide the resistance necessary for generation of tension on the meiosis I spindle. The 2::2 segregation bias could reflect a checkpoint mechanism that serves to monitor and balance such tension. Alternatively, it could reflect a rigidity of bivalent structure that tends to discourage unbalanced orientations. Further research will be required to understand the basis for the unique meiosis I segregation pattern in solo (Yan, 2010).

In S. cerevisiae and S. pombe, multiple meiotic cohesion functions are performed by cohesin complexes that include meiosis-specific subunits such as REC8, which replaces the mitotic kleisin subunit RAD21. REC8 is widely conserved among eukaryotes and has been shown in several model plants and animals to be critical for many of the same meiotic functions identified in yeast. However, in Drosophila, no true REC8 homologue has been identified, and the role of cohesin in meiotic cohesion has been unclear (Yan, 2010).

These data strongly suggest that SOLO and SMC1 function as partners in mediating centromere cohesion in Drosophila meiosis. First, anti-SMC1 and Venus::SOLO foci overlap extensively on centromeres throughout meiosis until anaphase II when both proteins disappear. Second, both Venus::SOLO and anti-SMC1 foci disappear prematurely at anaphase I in mei-S332 mutants, which is consistent with a role of MEI-S332 to protect meiotic cohesin from proteolytic cleavage by separase. Third, centromere localization of SMC1 is abolished at all meiotic stages in solo spermatocytes. Finally, evidence has been obtained for a physical interaction between SMC1 and SOLO in ovaries (Yan, 2010).

Another protein with an essential role in Drosophila meiotic cohesion is ORD. The phenotypes of solo and ord mutations are very similar, including missegregation of both homologous and sister chromatids and ablation of centromeric SMC1 foci. Like SOLO, ORD is a centromere protein, but there are significant differences in the localization patterns of the two proteins. SOLO localizes to centromeres from the earliest stages of prophase I and remains on the centromeres until anaphase II. ORD has been reported to localize predominantly to interchromosomal spaces in early prophase I nuclei in male meiosis, then to the chromosome arms in late prophase I, finally accumulating on centromeres at prometaphase I where it remains until anaphase II. Nevertheless, the striking phenotypic similarity of solo and ord mutants strongly suggests that both ORD and SOLO are intimately involved in establishing and maintaining cohesion in Drosophila meiosis (Yan, 2010).

The exact role of SOLO (and ORD) in meiotic cohesion remains to be determined. One possibility is that SOLO is a regulatory protein required for stable localization of cohesin to centromeres. Several known cohesin cofactors are required for specific aspects of cohesin function, such as chromosomal loading, establishment of cohesion, removal of cohesin during prophase, protection of centromeric cohesin, etc.. SOLO appears to play a more general role than most of these cofactors: it is involved both in stable chromosome association of cohesin and in the establishment and maintenance of cohesion throughout meiosis. Moreover, unlike the known cofactors that associate with cohesin during certain stages of the cell cycle, SOLO colocalizes with SMC1 throughout meiosis. Thus, except for the lack of homology to any of the four families of cohesin proteins, the data are consistent with the possibility that SOLO is a novel component of a meiosis-specific cohesin complex. It will be of considerable interest to determine the composition of the meiotic cohesin complexes in Drosophila (Yan, 2010).

Modulation of gurken translation by insulin and TOR signaling in Drosophila

Localized Gurken (Grk) translation specifies the anterior-posterior and dorsal-ventral axes of the developing Drosophila oocyte; spindle-class females lay ventralized eggs resulting from inefficient grk translation. This phenotype is thought to result from inhibition of the Vasa RNA helicase. In a screen for modifiers of the eggshell phenotype in spn-B flies, a mutation was identified in the lnk gene. lnk mutations restore Grk expression but do not suppress the persistence of double-strand breaks nor other spn-B phenotypes. This suppression does not affect Egfr directly, but rather overcomes the translational block of grk messages seen in spindle mutants. Lnk was recently identified as a component of the insulin/insulin-like growth factor signaling (IIS) and TOR pathway. Interestingly, direct inhibition of TOR with rapamycin in spn-B or vas mutant mothers can also suppress the ventralized eggshell phenotype. When dietary protein is inadequate, reduced IIS-TOR activity inhibits cap-dependent translation by promoting the activity of the translation inhibitor eIF4E-binding protein (4EBP). It is hypothesized that reduced TOR activity promotes grk translation independent of the canonical Vasa- and cap-dependent mechanism. This model might explain how flies can maintain the translation of developmentally important transcripts during periods of nutrient limitation when bulk cap-dependent translation is repressed (Ferguson, 2012).

Reproduction represents a substantial energy investment for an organism. Many studies have shown that ovarian physiology is exquisitely sensitive to nutritional status. Limitation of dietary protein intake results in a dramatic slowing of egg chamber maturation via developmental arrest, programmed cell death, or loss of germline stem cells. Several signaling pathways are integrated to bring about this response including 20-hydroxyecdysone, Juvenile Hormone (JH), and insulin/insulin-like signaling (IIS). IIS is stimulated by protein feeding and is required for oogenesis to progress. The IIS pathway integrates nutritional signals at two distinct points during oogenesis. The first is in region 2A of the germarium where developing germline cysts undergo apoptosis in the absence of a source of maternal dietary protein. The second point of nutritional control is at stage 8 of oogenesis during the onset of vitellogenesis. In the absence of food, egg chambers develop to stage 8, where they are arrested until a favorable food source is located. These two checkpoints represent points at which the energetically expensive process of oogenesis can be halted if insufficient resources are available (Ferguson, 2012 and references therein).

The IIS pathway elicits its effect on Drosophila physiology through several effector pathways, namely the dFOXO transcription factor and the Target of Rapamycin kinase (TOR). IIS inhibits dFOXO activity by promoting its phosphorylation by PKB/Akt and subsequent exclusion from the nucleus. Starvation or mutations in the insulin pathway allow dFOXO to translocate to the nucleus where it directs the transcription of genes that promote longevity, stress resistance, fat storage, and growth attenuation. TOR activity is stimulated by both IIS through the dRheb GTPase and by amino acids via Rag GTPases. When nutrients are plentiful, high TOR activity stimulates the translation of mRNA by phosphorylating S6K which in turn phosphorylates eIF4B and promotes its interaction with eIF3. These steps are critical for recruiting the translation preinitiation complex (PIC) to the m7G cap at the 5’ end of the mRNA. Once bound, the PIC recruits the small ribosomal subunit and proceeds to scan the transcript for an initiating AUG codon. This process requires the activity of the eIF4A RNA helicase. TOR also phosphorylates and inactivates the inhibitory eIF4E binding protein, 4EBP. Starvation inhibits cap- dependent translation through reduced TOR activity. When nutrients are limiting and TOR activity is low, eIF4B is not phosphorylated and can no longer participate in PIC assembly, furthermore 4EBP inhibition is lifted and it proceeds to inhibit cap-recognition by eIF4E. Both activities have the effect of strongly blocking cap-dependent translation initiation when nutrients are scarce. A select few transcripts escape this translational block by upregulating the utilization of an alternative mechanism that relies on an Internal Ribosomal Entry Site (IRES) that obviates the requirement for cap recognition and start codon scanning. The list of transcripts that contain IRES sequences is growing and includes numerous growth factors such as VEGF-A , PDGF2, and IGF-II. A prominent example of IRES-mediated nutritional adaptation is the Drosophila insulin receptor dInR, the translation of which is upregulated in response to starvation as a way to sensitize the cell to insulin when nutrients become available (Ferguson, 2012 and references therein).

Control of translation is vitally important to developmental patterning. The transcripts of many morphogens, including nanos, oskar, and gurken, are co-transcriptionally packaged into silencing particles and transported in a translationally quiescent form. Once localized, this repression is alleviated and translation proceeds in the developmentally appropriate locale. Gurken (Grk) is a TGF-α related ligand for the Drosophila Egfr. Localized translation of the spatially restricted grk transcript results in signaling by germline-derived Grk to the Egfr in the overlying follicle cells. This signal is required to specify the posterior fate in early oogenesis and the dorsal fate during mid oogenesis. Mutations that reduce grk translation are female sterile due to an inability to correctly pattern the developing oocyte and result in concomitant patterning defects in the embryo. grk translation requires the eIF4A-related DEAD-box helicase Vasa (Vas). Mutations in vas are female sterile owing to a failure to specify dorsal structures in the egg shell or posterior structures in the embryo (Ferguson, 2012 and references therein).

Spindle class genes are responsible for repairing DNA double strand breaks (DSBs) that are induced during homologous recombination in Drosophila oogenesis. In wild type females, DSBs are induced in germ line cells entering pachytene in region 2A of the germarium. This process is initiated by the Spo11 homologue Mei-W68 and Mei-P22, a protein that aids in break site selection. These breaks are then repaired by homologous recombination, a process that requires the RAD-51 homologue spindle-B (spn-B). Mutations in spn-B result in an accumulation of unrepaired DSBs that lead to activation of a meiotic checkpoint. The checkpoint is comprised of the ATR homologue mei-41 and the downstream kinase chk-2. Persistent DSBs in spn-BBU females activate the checkpoint that requires the Mei-41 and Chk2 kinases and leads to inefficient grk translation and ventralized eggshell phenotypes. Checkpoint activation also results in phosphorylation of Vasa, a modification that is thought to inhibit its function. Early in oogenesis, the oocyte nucleus becomes arrested in pachytene and forms a compact structure called the karyosome. The formation of the karyosome is disrupted in spindle-class mutants where the chromatin appears fractured or ellipsoid. Weak grk translation and an inability to properly form the karyosome are both spindle phenotypes that are consistent with reduced Vasa activity (Ferguson, 2012).

This study has identified the SH2B family adaptor gene lnk in a genetic screen for modifiers of the ventralized eggshell phenotype seen in spn-BBU mutant flies. SH2B proteins are known to regulate intracellular signaling by membrane bound receptor tyrosine kinases (RTKs). SH2Bs can promote signaling by scaffolding downstream effectors to the RTK or mediate proteosomal receptor destruction by recruiting the Cbl ubiquitin ligase. Lnk was recently identified as a positive regulator of the Insulin/Insulin-like Signaling (IIS) pathway that functions at the level of the insulin receptor substrate Chico. This study shows that lnk mutations can promote grk translation and suppress the ventralized eggshell phenotype in a spn-BBU mutant background. This suppression occurs independent of Vasa activity and does not suppress the karyosome phenotype. No genetic interactions were found with a weak grk allele nor downstream targets of Egfr suggesting that lnk-mediated suppression of spindle phenotypes does not occur by directly modulating Egfr activity. The data suggest that lnk mutations promote grk translation by inhibiting TOR activity as Rapamycin feeding experiments can also suppress the eggshell phenotype of spn-B and vas mutant flies. A model is proposed in which reduced IIS/TOR signaling inhibits cap-dependent translation and promotes utilization of an alternative translation initiation mechanism of the grk mRNA. This mechanism enables flies to faithfully pattern their oocytes when nutrients are scarce (Ferguson, 2012).

This study demonstrates a novel interaction between a meiotic checkpoint, the insulin/insulin- like signaling pathway, and translation of gurken mRNA in Drosophila oogenesis. Mutations in meiotic DNA repair enzymes such as spn-B result in persistent DSBs in early oogenesis that activate an ATR- Chk2-dependent meiotic checkpoint. Checkpoint activation results in phosphorylation of the eIF4A-like RNA helicase Vasa, the activity of which is important for grk translation. In these mutants, low levels of Grk protein are synthesized which is insufficient to pattern the eggshell correctly and results in ventralized eggs. Using forward genetics, an allele was isolated of the insulin receptor adapter, lnk. This mutation can suppress the weak grk translation phenotype and restore normal patterning to eggs laid by spn-BBU flies. Clonal analysis has shown that lnk mutations reduce IIS in a cell-autonomous manner in the ovary. As in mammals, Drosophila IIS controls the rate of cap-dependent translation initiation in the cell by regulating the activity of the TOR kinase. Rapamycin inhibits TOR activity, and feeding rapamycin can suppress the ventralized eggshell phenotype not only in spn-BBU females, but also in vasaPH165 / vasaRG53 flies. These data suggest an alternative translation initiation mechanism for the grk mRNA by which flies can maintain D/V axis patterning in times of moderate nutrient limitation (Ferguson, 2012).

The discovery that mutations in lnk, a positive regulator of IIS, can suppress the patterning defects in spn-B flies was initially surprising. The eggshell phenotypes of the different genotypes were assessed after keeping the flies on apple or grape juice agar plates on which abundant amounts of yeast paste had been added thus allowing the females to eat a very protein rich diet. A protein rich diet stimulates the activity of the TOR kinase via two mechanisms. Insulin-like peptides (dilps) are secreted into the hemolymph by neuroendocrine cells in response to nutrient availability. This in turn activates the IIS cascade comprised of Chico/Lnk, PI3K, Akt, Tsc1/2, and Rheb which promotes TOR-C1 activity. The second mechanism acts more directly through the levels of intracellular amino acids that are imported in part by the slimfast and pathetic transporters. Both of these mechanisms stimulate TOR-C1 activity which has been shown to promote cap-dependent translation by inhibiting 4EBP sequestration of eIF4E. Therefore, reducing TOR activity either by a mutation in lnk or by addition of rapamycin, would be expected to interfere with cap-dependent translation and therefore further enhance the mutant phenotype. However, in spn-B mutant flies, cap-dependent translation is already inhibited by the activity of the checkpoint, presumably acting via Vasa modification. The fact that a suppression of the ventralized phenotype was observed in lnk mutants indicates that reduction in TOR signaling must activate a second mode of translation that allows Gurken protein to be produced independently of the block in cap-dependent translation (Ferguson, 2012).

Several ovarian phenotypes are shared between mutations in spindle genes and vas mutants, including failure to form a compact karyosome, very weak grk translation, and ventralized eggs. Combined with the reproducible phosphorylation of Vas protein in spindle-class mutants, these phenotypes are consistent with a defect in Vas activity. While the specific effect of this phosphorylation is unknown, Vas serves several functions in cap-dependent translation initiation of grk mRNA. Vasa has been shown to interact with eIF5B and mutations that interfere with this interaction inhibit grk translation. This interaction is thought to facilitate assembly of the 60S ribosomal subunit at the AUG start codon. Furthermore, as a DEAD-box RNA helicase, Vasa may permit the pre-initiation complex to scan the 5’ UTR of grk and negotiate secondary structures that may impede the progress of this complex. IRES sequences adopt strong secondary structures in the 5’ UTR of RNAs that they regulate. If it can be demonstrated in the future that grk possess an IRES sequence, this may explain the requirement for Vasa helicase activity to unwind this structure when translation is initiated from the 5'cap during conditions of adequate nutrient availability. Whether the checkpoint dependent phosphorylation of Vas affects its stability, RNA helicase activity, or its eIF5B interaction, the expected result is a block in cap-dependent translation initiation of grk mRNA and concomitant D/V patterning defects. The observation that grk translation can be induced to occur in spn-BBU and in vasaPH165 / vasaRG53 flies indicates that an alternative mechanism for supporting translation initiation is taking place. Because reduced IIS and TOR activity both block bulk cap-dependent translation initiation through sequestration of eIF4E by 4EBP, yet stimulate IRES activity, it is proposed that the latter may provide an explanation for the results (Ferguson, 2012).

Grk plays a central role in shaping the development of the egg and subsequent embryo. Mutations that disrupt Grk / Egfr signaling during oogenesis result in female sterility. Blocking the translation of this essential morphogen in spindle class mutants that are unable to repair DNA damage is an effective mechanism to prevent the transmission of mutations to the progeny. This reproductive checkpoint is effective when nutrients are abundant, however as this study has demonstrated, the strategy breaks down when IIS/TOR activity is low. Under these conditions, grk can be translated and result in eggs that are patterned correctly, even though the DNA damage and karyosome malformation phenotypes persist. It is proposed that this difference occurs because the DNA-damage checkpoint can only impinge on one of the two mechanisms by which grk translation can be initiated (Ferguson, 2012).

One mechanism by which suppression of the D/V patterning defects of spn-BBU may occur is through the effects of the additional time that lnkCR642 egg chambers spend completing oogenesis. While Grk production is reduced in spn-BBU flies, it is not completely blocked and some Grk protein is made. If the reduced rate of Grk production is integrated over the extended time spent during mid oogenesis, sufficient Grk levels could accumulate and support normal D/V patterning. However, this model is inconsistent with the inability of lnkCR642 to suppress the ventralized eggs laid by grkED22 females. These flies do retain some Grk activity as is evident by the single appendage that is specified, however if the mechanism of suppression were via accumulation, then grkED22 should be suppressed by lnk mutations. Therefore, the IRES-dependent model proposed in this study is favored (Ferguson, 2012).

The selective pressure that may have driven the evolution of this bi-modal translation mechanism for grk can be best understood by considering that in wild populations of Drosophila, females feed and oviposit at locations where yeast is abundant. This behavior ensures adequate nutrition to support oogenesis in the female as well as for the developing larvae. If however nutrients become scarce, females adjust the rate of oogenesis to match nutrient availability. In response to complete starvation, egg chambers undergo apoptosis and are reabsorbed, however moderate reductions in IIS slow the rate of oogenesis until an abundant protein source is found. The conserved response to dietary restriction is to repress cap- dependent translation of most cellular transcripts while a select population of RNAs that are essential for survival escape this repression by utilizing a cap-independent IRES mechanism. It is posited that grk may be one such transcript. Oocytes that are in mid development when nutrients are scarce must still be patterned appropriately so that the resulting eggs are fertile. IRES activity may facilitate Grk expression to maintain normal D/V patterning in times of lean whereas when nutrients are abundant, cap-dependent translation predominates (Ferguson, 2012).

Ectopic expression of germline genes drives malignant brain tumor growth in Drosophila

Model organisms such as the fruit fly Drosophila melanogaster can help to elucidate the molecular basis of complex diseases such as cancer. Mutations in the Drosophila gene lethal (3) malignant brain tumor cause malignant growth in the larval brain. This study shows that l(3)mbt tumors exhibited a soma-to-germline transformation through the ectopic expression of genes normally required for germline stemness, fitness, or longevity. Orthologs of some of these genes were also expressed in human somatic tumors. In addition, inactivation of any of the germline genes nanos, vasa, piwi, or aubergine suppressed l(3)mbt malignant growth. These results demonstrate that germline traits are necessary for tumor growth in this Drosophila model and suggest that inactivation of germline genes might have tumor-suppressing effects in other species (Janic, 2010).

The Drosophila tumor-suppressor gene l(3)mbt was identified as a temperature-sensitive mutation that caused malignant growth in the larval brain. Other l(3)mbt mutant alleles obtained later show the same temperature-sensitive phenotype. L(3)mbt's closest homologs, Drosophila Scm (Sex comb on midleg) and Sfmbt (Scm-related gene containing four mbt domains), encode Polycomb Group proteins. L3MBTL1, the human homolog of Drosophila L(3)MBT, is a transcriptional repressor that is found in a complex with core histones, heterochromatin protein 1γ (HP1γ), and RB (Retinoblastoma protein) and can compact nucleosomes. Drosophila L(3)MBT is a substoichiometric component of the dREAM-MMB complex, which includes the two Drosophila Retinoblastoma-family proteins and the Myb-MuvB (MMB) complex. Depletion of components of the dREAM/MMB complex in Drosophila Kc cells by RNA interference results in genome-wide changes in gene expression. These data strongly suggest that l(3)mbt function might contribute to establishing and maintaining certain differentiated states through the stable silencing of specific genes (Janic, 2010).

To identify the genes whose misexpression might account for the growth of l(3)mbt tumors (henceforth referred to as mbt tumors), genome-wide gene expression profiling was carried out of l(3)mbtE2 and l(3)mbtts1 homozygous and transheterozygous larval brains raised at restrictive temperature (29°C). l(3)mbtts1 tumors were also analyzed at the 1st, 5th, and 10th rounds of allograft culture in adult flies (T1, T5, and T10, respectively). Brains from homozygous white1118 (w1118), l(3)mbtE2, or l(3)mbtts1 larvae raised at permissive temperature (17°C) were used as controls. For comparison, larval brain malignant neoplasms caused by mutation in brain tumor (brat) as well as allograft cultures at T1,T5, and T10 of tumors caused by mutants in brat, lethal giant larvae (lgl), miranda (mira), prospero (pros), and partner of inscuteable (pins), were also profiled (Janic, 2010).

Hierarchical clustering plots of these data reveal three distinct clusters that include control larval brains, mbt larval brain tumors, and cultured l(3)mbtts1 tumors, respectively. From these data, 151 genes were identified that were either overexpressed or underexpressed in all three larval mbt tumor types compared to all three controls. From this list, those genes were removed that were also up- or down-regulated) in larval brat neoplasms and, hence, likely to encode functions generally required for larval brain tumor growth. The expression levels of the remaining 102 up-regulated genes are referred to as as the mbt signature (MBTS). MBTS is notably enhanced in cultured mbt tumors and can be used unequivocally to distinguish mbt tumors from other cultured malignant brain neoplasms like lgl, mira, pros, pins, or brat. Individual MBTS genes, however, are also up-regulated in some of these tumors (Janic, 2010).

The function of most MBTS genes remains unknown. However, a quarter of them (26 of 102) are genes required in the germ line. For instance, nanos (nos), female sterile(1)Yb (fs(1)Yb), and zero population growth (zpg) function in the establishment of the pole plasm in the egg and cystoblasts differentiation. The gonad-specific thioredoxins ThioredoxinT (TrxT) and deadhead (dhd), giant nuclei (gnu), corona (cona), hold'em (hdm), matotopetli (topi), and the female germline-specific γTUB37C isoform function during oocyte differentiation, meiosis, and syncytial embryo development. Also piwi, aubergine (aub), krimper (krimp), and tejas (tej) are involved in the biogenesis of Piwi-interacting RNAs (piRNAs) that protect germline cells against transposable elements and viruses. Some of these genes also have functions that are not germline related. For instance, some piwi alleles display synthetic lethality), and nos is required during nervous system development (Janic, 2010).

Driven by the high percentage of MBTS genes that have germline functions, other germline-related genes were sought that do not meet the stringent criteria applied to select the 102 MBTS genes, but are overexpressed in mbt tumors. Among these, the genes were found that encode the synaptonemal complex protein Crossover suppressor on 3 of Gowen [C(3)G] and the cell cycle kinase Pan gu (PNG), which interact with the proteins encoded by the MBTS genes cona and gnu, respectively. The same applies to Squash (SQU), Spindle-E (SPN-E), Maelstrom (MAEL), and AGO3, components of the piRNA machinery, which colocalize with other MBTS proteins in nuage (Janic, 2010).

To determine whether the mRNAs found ectopically expressed in mbt tumors are translated, protein expression was examined with a selected number of currently available antibodies. Given the key role of VASA in the assembly of the pole plasm and germline development, it was included in this study, even though vasa mRNA levels are not significantly increased in mbt tumors. By Western blot, it was confirmed that PIWI, AUB, and VASA are ectopically expressed in mbt tumors. Immunofluorescence studies also revealed the ectopic expression in l(3)mbtts1 brains raised at 29°C of C(3)G, SQU, and VASA. These results show that some of the germline genes ectopically expressed in mbt tumors are translated. However, it has not been possible to confirm the expression of other proteins, including MAEL, ORB, BAM, GNU, and TOPI, which suggests that, possible technical problems aside, either the corresponding mRNAs are not translated or these proteins might be unstable in such an ectopic environment. The expression of VASA, by contrast, suggests that other mRNAs whose levels are not appreciably increased in mbt tumors might actually be ectopically translated (Janic, 2010).

Prompted by the expression in l(3)mbtts1 brains of several genes involved in the biogenesis and regulation of piRNAs, 23- to 30-nucleotide RNAs were sequenced from l(3)mbtts1 larval brain tumors and from wild-type brains and ovaries. 117 known piRNAs and microRNAs (miRNAs) were detected in l(3)mbtts1 larval brain tumor samples. Of these, 31 are either not expressed in wild-type brains or are expressed there at less than 10% their level in larval brain tumors. Most of them are highly expressed in wild-type ovaries, thus substantiating further the ectopic acquisition of germline traits that characterizes mbt tumors (Janic, 2010).

It is not known which, if any, of the germline genes that are up-regulated in mbt tumors are direct targets of l(3)mbt or if their ectopic expression is a downstream consequence of intermediate events. The putative direct targets of l(3)mbt are many. The dREAM-MMB complex, of which L(3)MBT is a substoichiometric component, has been found to be promoter-proximal to 32% of Drosophila genes, and MMB factors are known to regulate transcription of a wide range of genes in Drosophila Kc cells. In addition, there is no estimate for the number of proteins like VASA that, despite their low mRNA expression levels, might be up-regulated in mbt tumors. Indeed, many of these genes, as well as the piRNAs and miRNAs expressed in mbt tumors, might themselves regulate the basal transcription and translation machineries, adding a further layer of gene expression modulation (Janic, 2010).

The extent to which ectopic expression of germline genes contributes to mbt tumor growth was determined. To this end, larval brain growth was quantified in individuals that were mutant for l(3)mbtts1 alone, or double mutant for l(3)mbtts1 and one of several of the germline genes that are ectopically expressed in mbt tumors. Measured as the total amount of protein, the average brain size in l(3)mbtts1 is about seven times as large as that in control w1118 larvae, a difference that is not significantly reduced by the additional loss of zpg, Pxt, or AGO3. However, brain overgrowth is reduced to a size similar to that of the control in l(3)mbtts1 larvae that are also mutant for either piwi, vasa, aub, or nos. The loss of piwi does not prevent brain overgrowth in brat k06028 mutant larvae. Then tumor growth was quantified after allograft in adult flies. The frequency with which l(3)mbtts1 homozygous larval brain tissue develops tumors in this assay is not significantly reduced by the additional loss of zpg or AGO3 and is only moderately reduced by the loss of Pxt, but it is markedly reduced by the additional loss of piwi, vasa, aub), or nos. The frequency of brat k06028 tumor formation is not affected by the loss of piwi or nos. These results demonstrate that the ectopic expression of germline genes, particularly piwi, vasa, nos, and aub, significantly contributes to mbt tumor growth (Janic, 2010).

A closely reminiscent soma-to-germline transformation observed in mutants in the Caenorhabditis elegans Rb homolog LIN-35, as well as in long-lived C. elegans strains, has led some to propose that the acquisition of germline characteristics by somatic cells might contribute to increased fitness and survival, a mechanism that could contribute to the transformation of mammalian cells. Also in humans, some genes that are predominantly expressed in germline cells and have little or no expression in somatic adult tissues become aberrantly activated in various malignancies, including melanoma and several types of carcinomas. These are known as cancer-testis (CT) genes or cancer-germline (CG) genes. A subset of these CG genes encode antigens that are immunogenic in cancer patients and are being pursued as biomarkers and as targets for therapeutic cancer vaccines (Janic, 2010 and references therein).

Human CG genes are suspected to contribute to oncogenesis germline traits like immortality, invasiveness, and hypomethylation, but their actual role in cancer remains unknown. The current results demonstrate that ectopic germline traits are necessary for tumor growth in Drosophila mbt tumors, suggesting that their inactivation might have tumor-suppressing effects in other species. Some germline genes up-regulated in mbt tumors are orthologs of human CG genes like PIWIL1/piwi, NANOS1/nanos, and SYCP1 /c(3)G. The list of genes up-regulated in mbt tumors includes many other germline genes that might also be relevant in human cancer (Janic, 2010).


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vasa: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 June 2014

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