To learn where cup is transcribed during oogenesis, the 4.1 kb cDNA was hybridized in situ to wild-type ovaries. cup RNA is present in germ-line cells throughout pre-vitellogenic development, but is not detected in the somatic follicle cells. The RNA is present in region 1 of the germarium, where it is detectable in stem cells, cystoblasts and dividing cysts. Steady state RNA levels decrease in region 2a, rise again in region 2b, and peak around stage 3 or 4. Subsequently, RNA levels decline and reach undetectable levels by stage 8. A second round of expression begins during stage 10 and continues through stage 14. The transcript is not differentially localized within the germ line at any stage. None of the strong alleles abolish all cup RNA expression (Keyes, 1997).
Polyclonal antibodies were raised against two independent domains of the Cup protein. Antisera against both domains produce similar results when used to analyze ovarian extracts on Western blots. Both sera detect a single predominant band of approximately 150 kDa that is abundant in wild-type ovaries but greatly reduced in cup mutant females. This is similar in size to the 130 kDa product predicted by the 4.1 kb cDNA. A second smaller band of 75 kDa reacts more weakly and variably with all Cup sera tested, but this protein is unaffected by mutants and is presumed to be encoded by a separate locus. Testes contain very little of the 150 kDa protein; the anti-Cup antibodies instead detect bands of approximately 180, 110 and 45 kDa. This suggests that the testis-specific cup RNAs encode non-identical but antigenically related Cup proteins (Keyes, 1997).
The protein products of the EMS- and P-derived cup alleles were examined by Western analysis. All but one of the cup alleles affect the quantity, rather than size of the 150 kDa protein, indicating that these alleles are not the products of premature termination. None of the strong cup alleles completely eliminates all immunoreactive protein, suggesting that null alleles have not been recovered. The level of Cup protein detected in these experiments correlates well with the genetic strength of the allele in question: weak alleles produce much more of the 150 kDa Cup protein than strong alleles such. One allele produced a smaller Cup protein of about 135 kDa, further confirming that the gene analyzed corresponds to cup (Keyes, 1997).
Immunofluorescent antibody staining was used to determine the expression pattern and subcellular localization of the Cup protein. Beginning with the stem cells, Cup protein is found in the cytoplasm of all germ-line cells, but was never detected inside either nurse cell or oocyte nuclei. Staining of early embryos confirms that Cup protein is maternally deposited in the egg. The protein is abundant and uniformly distributed in the cytoplasm of all cleavage stage embryos through stage 9, after which signal intensity declines. Staining is effectively absent by gastrulation, and remains so throughout the remainder of embryogenesis (Keyes, 1997).
The subcellular location and distribution of Cup protein within 16-cell cysts undergoes marked changes during the course of oogenesis. Cup protein accumulates preferentially in the future oocyte within 16-cell cysts of the germarium. This enrichment begins very early: localization to a single cell is detectable in region 2a, prior to overt differentiation of the oocyte. Unlike most oocyte-enriched proteins such as Bic-D or Oskar, no corresponding enrichment of cup mRNA was observed. Cup protein may be selectively transported from the nurse cells and/or differentially translated or stabilized within the oocyte. The retention of relatively high levels of Cup protein in the nurse cells suggests that the protein may function in both cell types (Keyes, 1997).
Cup protein continues to be selectively enriched in the oocyte until approximately stage 8, a time when egg chamber microtubules are extensively reorganized. During stage 9 the nurse cells and oocyte contain similar amounts of Cup protein, and by stage 10 most of the protein lies in the nurse cells. Prior to stage 8 Cup forms a cap at the posterior of the oocyte; later much smaller amounts of protein are found at the oocyte surface and also persist in a small cap at the posterior (Keyes, 1997).
The behavior of Cup protein in the nurse cells is particularly interesting. Cup accumulates almost exclusively in large aggregates, whose location varies as egg chambers develop. In early chambers, and especially around stage 4, Cup aggregates are found predominantly around the periphery of the nurse cell nuclei. After stage 4, Cup leaves the nuclear membrane and becomes dispersed throughout the nurse cell cytoplasm in large aggregates that eventually move toward the cellular periphery. By stage 10, Cup protein is localized almost exclusively in particulate structures along the subcortical surface of the nurse cells. The movement of Cup protein away from the nucleus corresponded in time to the sharp reduction in cup mRNA levels after stage 4, suggesting that a decrease in the rate of Cup synthesis might play a role in these changes (Keyes, 1997).
The ovarian tumor gene is required in germ-line cells for cyst formation, nurse cell chromosome structure and egg maturation. A gene has been analyzed, fs(2)cup, that participates in many of the same processes and interacts with otu genetically. Both nurse cell and oocyte chromosomes require cup to attain a normal morphology. The cup gene is needed for the oocyte to grow normally by taking up materials transported from the nurse cells. cup encodes a 1132-amino-acid protein containing a putative membrane-spanning domain. Cup protein (but not CUP mRNA) is transported selectively into the oocyte in germarial cysts, like the p104 Otu protein. It is strongly associated with large structures in the cytoplasm and the perinuclear region of nurse cells; like Otu, Cup moves to the periphery of these cells in stages 9-10. Moreover, cup mutations dominantly disrupt meiotic chromosome segregation. It is proposed that cup, otu and another interacting gene, fs(2)B, take part in a common cytoplasmic pathway with multiple functions during oogenesis (Keyes, 1997).
The finding that Cup can bind eIF4E implies that Cup may be involved in the translational control of maternal RNAs. The distribution of the Osk protein was examined in cup mutants, because the Cup-associated protein Me31B has been found to be involved in the repression of osk translation in early oogenesis (Nakamura, 2001). Although the translation of osk RNA is repressed until stage 8 of oogenesis in wild-type ovaries, it is prematurely translated in stage 4-7 egg chambers of several cup mutants, including cup21 and cup32. Premature Osk expression was also observed in cup1/cup1355 ovaries. These results show that Cup is required for the repression of osk translation during early oogenesis. However, since the egg chambers of these cup mutants start to degenerate at mid-oogenesis, it was not possible to analyze the effects in later oogenesis. Furthermore, the molecular nature of all EMS-induced cup alleles (Keyes, 1997) remains uncharacterized (Nakamura, 2004).
To isolate a molecularly definitive cup mutation, a series of derivative lines were generated by mobilizing the P element in cup4506. Imprecise excision of the P element causes a small deletion within the cup locus. This line, cupΔ212, produces a truncated Cup, in which the amino-terminal third of the protein is deleted and the conserved eIF4E binding sequence is disrupted. Immunoprecipitation-Western analyses reveal that CupΔ212 protein fails to interact with eIF4E in vivo. Females homozygous for the cupΔ212 mutation produce eggs, although the eggs are fragile and were not analyzed further. Thus, the cupΔ212 allele is sufficiently weak to investigate, in detail, the role of Cup in later oogenesis (Nakamura, 2004).
Immunostaining cupΔ212 ovaries for Osk reveals that osk is prematurely translated starting at early oogenesis, as in the EMS-induced cup mutants. The protein is concentrated in the posterior of the oocyte in stage 4-6 egg chambers. In the stage 8 egg chamber, Osk protein is ectopically concentrated at the anterior of the oocyte. The signal was frequently highest at the anterior-dorsal corner of the oocyte. As oogenesis proceeds, Osk becomes concentrated at the posterior pole of the oocyte. However, large Osk particles remained in the cytoplasm of the oocyte. This type of signal was never observed in wild-type egg chambers. These results show that osk translation is not repressed in the cupΔ212 oocyte (Nakamura, 2004).
osk RNA distribution was examined by fluorescence in situ hybridization. osk RNA forms cytoplasmic particles, which are especially obvious in early oogenesis. In wild-type ovaries, osk RNA signals are concentrated in the oocyte in early egg chambers, transiently accumulate in the anterior side of the oocyte during stage 7-8, and localize to the posterior pole of the oocyte from stage 8 onward. In cupΔ212 egg chambers, osk RNA signals show larger particles in the cytoplasm, suggesting that the assembly of osk RNA particles is also affected in the cupΔ212 egg chambers. However, in spite of the abnormally large osk RNA particles in the cupΔ212 egg chambers, osk RNA is concentrated in the oocyte in early egg chambers, and at the posterior pole of the oocyte from stage 8 onward. At stage 10, osk RNA is accumulated at the posterior pole of the cupΔ212 oocyte, although some signal remains in the cytoplasm. Microtubule polarity is normal in cupΔ212 ovaries. In addition, no defect was found in grk RNA, Grk protein or Bicaudal-D distribution in cupΔ212 ovaries. Thus, the defects observed in cupΔ212 ovaries were most striking in their effects on osk translational regulation (Nakamura, 2004).
In Drosophila, germ cell formation depends on inherited maternal factors localized in the posterior pole region of oocytes and early embryos, known as germ plasm. This study reports that heterozygous cup mutant ovaries and embryos have reduced levels of Staufen (Stau), Oskar (Osk) and Vasa (Vas) proteins at the posterior pole. Moreover, Cup interacts with Osk and Vas to ensure anchoring and/or maintenance of germ plasm particles at the posterior pole of oocytes and early embryos. Homozygous cup mutant embryos have a reduced number of germ cells, compared to heterozygous cup mutants, which, in turn, have fewer germ cells than wild-type embryos. In addition, cup and osk interact genetically, because reducing cup copy number further decreases the total number of germ cells observed in heterozygous osk mutant embryos. Finally, cup mRNA and protein were detected within both early and late embryonic germ cells, suggesting a novel role of Cup during germ cell development in Drosophila (Ottone, 2012).
Germ plasm assembly is a stepwise process occurring during oogenesis. Accumulation of osk mRNA at the posterior of egg chambers is necessary for correct germ plasm assembly, which requires a polarized microtubule network, the plus-end motor kinesin I, and the activity of several genes (cappuccino, spire, par-1. mago nashi, barentz, stau, tsunagi, rab11, and valois). Localization of osk mRNA is strictly linked to the control of its translation, as unlocalized osk mRNA is silent. Upon localization at the posterior pole, the relieve of osk translational repression involves several factors, including Orb, Stau, and Aubergine. Localized Osk protein, in turn, triggers a cascade of events that result in the recruitment of all factors, such as Vas, Tud, and Stau proteins and nanos, germ less mRNAs, necessary for the establishment of functional germline structures (Ottone, 2012).
Posterior anchoring of Osk requires the functions of Vas, as well as Osk itself, to direct proper germ plasm assembly. Misexpression of Osk at the anterior pole of oocytes causes ectopic pole plasm formation, indicating that Osk is the key organizer of pole plasm assembly. Moreover, it has been demonstrated that endocytic pathways acting downstream of Osk regulate F-actin dynamics, which in turn are necessary to attach pole plasm components to the oocyte cortex. As far as Cup is concerned, it has been demonstrated that Cup is engaged in translational repression of unlocalized mRNAs, such as osk, gurken, and cyclinA, during early oogenesis (Ottone, 2012).
The current results establish that Cup is also a novel germ plasm component. First, Cup colocalizes with Osk, Stau, and Vas at the posterior pole of stage 10B oocytes. Second, biochemical evidence indicates that Cup interacts with Stau, Osk, and Vas. Vas localization occurs not through its association with localized RNAs, but rather through the interaction with the Osk protein, which represents an essential step in polar granule assembly (Ottone, 2012).
As a consequence of these interactions, Cup protein is mislocalized in osk and vas mutant stage 10 oocytes, demonstrating that Osk and Vas are essential to achieve a correct localization of Cup at the posterior cortex of stage 10 oocytes. This study suggests that the presence of Cup, Osk, Stau, and Vas are required for a correct germ plasm assembly. Moreover, several immuno-precipitation experiments, using anti- Tud and anti-Vas antibodies, identified numerous P-body related proteins, including Cup, as novel polar granule components (Ottone, 2012).
All the results suggest that Cup plays at least an additional role at stage 10 of oogenesis. Cup, besides repressing translation of unlocalized osk mRNA, is necessary to anchor and/or maintain Stau, Osk, and Vas at the posterior cortex. This novel function of Cup is supported by the findings that, when cup gene dosage is reduced, Stau, Osk, and Vas are partially anchored and/or maintained at the posterior pole, even if these proteins are not degraded. Consequently, pole plasm assembly is disturbed and cup mutant females lay embryos with a reduced number of germ cells. Since the role of Cup, a known multi-functional protein during the different stages of egg chamber development, cannot be easily studied in homozygous cup ovaries, it is not surprising that the involvement of Cup in pole plasm assembly remained undiscovered until now (Ottone, 2012).
During embryogenesis, Cup exerts similar functions. In particular, Osk, Stau, and Vas proteins and osk mRNA are not properly maintained and/or anchored at the posterior pole of embryos laid by heterozygous cup mutant mothers. Surprisingly, osk mRNA is increased in heterozygous cup mutant embryos. Since osk mRNA requires sufficient Osk protein to remain tightly linked at the posterior cortex, the reduced amount of Osk protein observed in heterozygous cup embryos, should be not sufficient to maintain all osk mRNA at the embryonic pole and could stimulate, by positive feedback, de novo osk mRNA synthesis. Also, a direct/indirect involvement of Cup in osk mRNA degradation and/or deadenylation cannot be excluded. The findings that Cup has been found together with Osk, when Osk is ectopically localized to the anterior pole of the embryos, and that reducing cup copy number further decreases the total number of germ cells, observed in heterozygous osk mutant embryos, strengthen the idea that Cup is involved in germ cell formation and/or in maintenance of their identity (Ottone, 2012).
Unlike Osk protein, both cup mRNA and protein were detected within germ cells until the end of embryogenesis. These observations suggest that zygotic cup functions, during germ cell formation and maintenance, are not limited to those carried out in combination with Osk. The finding that homozygous cup mutant embryos display a further decrease of germ cell number, in comparison with heterozygous embryos, supports this hypothesis. Whether or not cup zygotic function is involved in the translational repression of specific mRNAs, different from osk, remains to be explored (Ottone, 2012).
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date revised: 20 November 2012
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