It has been reported that Maelstrom protein displays no distinct subcellular localization within the germline (Clegg, 1997). Its localization has been reexamined using a 'lighter' fixation-based protocol. As a result, it has been found that in addition to previously observed diffuse nuclear and cytoplasmic germline staining, 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).
Because Maelstrom and Vasa are each present in the nucleus, nuage and cytoplasm of germline cells, it was of interest to determine whether either protein could transit between these compartments. Nuclear shuttling was assayed utilizing Leptomycin B (LMB), a specific inhibitor of nuclear transport receptor, CRM1 (Exportin). CRM1 mediates nuclear export of substrates containing a leucine-rich nuclear export sequence (NES) in cells as diverse as yeast and human (Findley, 2003). Drosophila CRM1 has been shown to be mechanistically indistinguishable from its homologs in other systems, including its specific inactivation by LMB (Fasken, 2000). LMB treatment of Drosophila ovaries has a marked effect on Maelstrom protein localization within the germline, whereas Vasa protein shows only a slight redistribution. The effect is most pronounced in nurse cells and oocytes, where Maelstrom manifests a nuclear accumulation, with a corresponding depletion in cytoplasm. It is surmised that Maelstrom must transit between cytoplasm and nucleus (Findley, 2003).
A mutant, maelstrom (mael), is described that disrupts a previously unobserved step in mRNA localization within the early oocyte, distinct from nurse-cell-to-oocyte RNA transport. Mutations in maelstrom disturb the localization of mRNAs for Gurken (a ligand for the Drosophila Egf receptor), Oskar and Bicoid at the posterior of the developing (stage 3-6) oocyte. maelstrom mutants display phenotypes detected in gurken loss-of-function mutants: posterior follicle cells with anterior cell fates; Bicoid mRNA localization at both poles of the stage 8 oocyte, and ventralization of the eggshell. These data are consistent with the suggestion that early posterior localization of Gurken mRNA is essential for activation of the Egf receptor pathway in posterior follicle cells. mael mutation affects the distribution and dynamics of oocyte microtubules. grk and mael mutants have a defective microtubule cytoskeleton similar to that previously described for the oocyte polarity mutants PKA and mago nashi; however, the grk and mael cytoskeletons are not identical. Both mutants have a high concentration of microtubules at the posterior of the oocyte in stages 8 and 9 when microtubules are normally concentrated at the oocyte anterior. In stage 7 however, mael microtubules are tightly bundled around the cortex, while grk mutants have a more diffuse network. This bundling is similar to the continous subcortical array of microtubules in wild-type stage 10b oocytes. Time-lapse videomicroscopy indicates that the cytoplasm undergoes premature streaming. Posterior localization of mRNA in stage 3-6 oocytes could be one of the earliest known steps in the establishment of oocyte polarity. The maelstrom gene encodes a novel protein with a punctate distribution in the cytoplasm of the nurse cells and the oocyte until the protein disappears in stage 7 of oogenesis (Clegg, 1997).
Bicaudal-D (Bic-D) is essential for the establishment of oocyte fate and subsequently for polarity formation within the developing Drosophila oocyte. To find out where in the germ cells Bic-D performs its various functions, transgenic flies were made expressing a chimeric Bic-D::GFP fusion protein. Once Bic-D::GFP preferentially accumulates in the oocyte, it shows an initial anterior localization in germarial region 2. In the subsequent egg chamber stages 1-6 Bic-D::GFP preferentially accumulates between the oocyte nucleus and the posterior cortex in a focus that is consistently aligned with a crater-like indentation in the oocyte nucleus. After stage 6 Bic-D::GFP fluorescent signal is predominantly found between the oocyte nucleus and the dorso-anterior cortex. During the different phases several genes have been found to be required for the establishment of the new Bic-D::GFP distribution patterns. Dynein heavy chain (Dhc), spindle (spn) genes and maelstrom (mael) are required for the re-localization of the Bic-D::GFP focus from its anterior to its posterior oocyte position. Genes predicted to encode proteins that interact with RNA (egalitarian and orb) are required for the normal subcellular distribution of Bic-D::GFP in the germarium, and another potential RNA binding protein, spn-E, is required for proper transport of Bic-D::GFP from the nurse cells to the oocyte in later oogenesis stages. The results indicate that Bic-D requires the activity of mRNA binding proteins and a negative-end directed microtubule motor to localize to the appropriate cellular domains. Asymmetric subcellular accumulation of Bic-D and the polarization of the oocyte nucleus may reflect the function of this localization machinery in vectorial mRNA localization and in tethering of the oocyte nucleus. The subcellular polarity defined by the Bic-D focus and the nuclear polarity marks some of the first steps in antero-posterior and subsequently in dorso-ventral polarity formation (Pare, 2000).
Maelstrom allele M391 was isolated by imprecise excision of a P element from line P [w+lacZ] 11A4, that is inserted in the genomic region corresponding to the 5'UTR of the maelstrom gene. The sterility of maelM391/Df(3L)79E-F females can be rescued by a transgene containing the 4.5 kb genomic region. Southern analysis of maelM391 genomic DNA revealed that, in addition to the P element, about 1.2 kb of sequence 3' to the element was lost in the excision event. The deletion junction was cloned by PCR, using primers predicted to flank the breakpoint. Sequencing of the resulting genomic fragment revealed a deletion of 1319 basepairs of genomic DNA, leaving a 17 bp P-element residue. Nucleotides corresponding to 124-1270 of the mRNA were thus deleted, resulting in the loss of 73% (codons 1-335) of the predicted coding sequence of maelstrom. Western analysis and immunocytochemistry show that maelM391/Df (3L)79E-F ovaries contain no detectable Maelstrom protein. It is concluded that maelM391 is a null allele of the maelstrom locus (Findley, 2003).
Since hypomorphic alleles of maelstrom showed AP and DV spindle-class-like defects in the developing oocyte (Clegg, 2001; Clegg, 1997), it was of interest to determine whether the maelstrom null (hereafter referred to as maelstrom) shares the meiotic progression defect common to the spindle-class mutants. Specifically, the spn mutants fail to form a karyosome, despite the apparently normal assembly of synaptonemal complexes within the oocyte nucleus. To this end, meiotic progression was examined in the oocyte nucleus (germinal vesicle) using synaptonemal complex component, C(3)G, to assess progression to synaptonemal complex formation; oocyte DNA morphology was used to assess progression to the karyosome stage. C(3)G is normally acquired by oocyte chromosomes in the germarium and dissociated from DNA upon karyosome formation. In more than 90% of stage 2 or 3 maelstrom egg chambers, C(3)G signal is present and restricted to the oocyte, where it colocalizes with DNA in a morphology comparable with wild type. This suggests that meiosis has proceeded in the mutant to at least zygotene phase of prophase I. As reported for other spn mutants, maelstrom ovaries show some delay in restriction of synaptonemal complexes to a single cell. When the karyosome forms in wild-type oocytes, DNA within the germinal vesicle loses it association with C(3)G. Despite the dispersion of C(3)G within the oocyte nucleoplasm by stage 6, maelstrom oocytes never form karyosomes. Instead, the DNA shows a nuclear morphology distinct from both stage 1 and karyosome, forming variably distended loops and threads, often closely apposed to an invariably 'deflated' nuclear envelope. This DNA morphology, maintained in maelstrom through at least stage 10B, is similar to that described for other spn mutants (Findley, 2003).
The axial patterning defects displayed by maelstrom hypomorphs (Clegg, 2001; Clegg, 1997) are fully penetrant in oocytes of the maelstrom null. AP axis determination in the Drosophila oocyte is a multistep process, the first known step of which is the establishment of microtubule-mediated cytoplasmic polarity in the stage 2 oocyte. This asymmetry, which is defective in spn mutants such as spn-A, spn-B and vasa, is a likely prerequisite for efficient Gurken signaling from the oocyte to the follicle cells overlying the posterior oocyte. A number of RNAs and proteins accumulate in the posterior of the wild-type oocyte during stages 2-6 in a distribution that both requires and reflects the oocyte polarity in this interval. Polarity was assayed in the oocyte indirectly by monitoring the localization of Bicaudal D [BicD and multiple RNAs including grk, osk, bicD and oo18 RNA binding (orb)]. In normal stage 5/6 oocytes, BicD forms a distinct gradient emanating from the posterior oocyte cortex. In maelstrom oocytes, although BicD is present at levels comparable with wild type, a wild-type gradient is not established. Instead, about half of stage 5/6 maelstrom oocytes show BicD in a diffuse or only vaguely polarized distribution. In the remaining oocytes, the marker forms a randomly localized focus within the ooplasm. Similarly, the normally polarized distribution of grk and other RNAs is lost in maelstrom oocytes. Gurken protein distribution in wild-type oocytes is comparable with that of BicD, albeit more punctate in appearance. In maelstrom oocytes, not only is the gradient lost, but Gurken levels are either highly reduced (~50%) or undetectable (~50%). The Gurken defect is probably sufficient to account for the observed polarity defects in mid- to late-stage maelstrom oocytes, in which variety of polarity markers, including multiple mRNAs (e.g. osk) and proteins (including Staufen, Oskar, Vasa), fail to accumulate in the posterior ooplasm. Dorsal appendages are also invariably vestigial or absent in the maelstrom null. The failure in establishing AP polarity in the early oocyte, together with reduction in Gurken accumulation, the DV phenotypes of the null and hypomorph, and failure to proceed to karyosome stage collectively puts maelstrom in the spindle-class of mutants (Findley, 2003).
The phenotypes of the double-strand break (DSB) repair specific spn mutants (e.g. spn-B) can be suppressed by a mutation in mei-W68. This locus encodes the Drosophila homolog of the Spo11 protein, which induces double-strand breaks in chromosomes, the initiating event required for subsequent steps in recombination. If DSBs do not occur, then genes normally required in the ensuing recombinational repair steps are not required. Thus, their absence will not be detected by the elements of the meiotic checkpoint, which responds to persistent unrepaired DSBs. To resolve the sphere of maelstrom function, genetic interaction was assessed between mei-W68 and maelstrom by examining Gurken accumulation in early oocytes of mei-W68-maelstrom double mutant ovaries. If maelstrom were required only in recombinational repair, a suppression of the Gurken translation defect of the maelstrom null oocyte would be expected. The Gurken defect of maelstrom oocytes is not, in fact, suppressed by mei-W68, from which it is concluded that maelstrom cannot only be required in a recombinational repair step (Findley, 2003).
How meiotic progression status in the oocyte nucleus is transmitted to effectors of oocyte patterning is a key, and largely unanswered, question. One candidate effector is Vasa, a target of the pachytene checkpoint, which displays a mobility shift in spn-B ovaries, in which the checkpoint is activated. Interestingly, Vasa mobility is aberrant in maelstrom ovaries: two distinct species of Vasa protein are observed -- a minor band with wild-type mobility and a species larger, curiously, than that reported for spn-B. Although the relationship to activated-checkpoint-Vasa is unclear, the data shows that maelstrom is required for proper Vasa modification (or processing). It is thus conceivable that any phenotype(s) of the maelstrom mutant could arise, indirectly, as a result of this Vasa modification. The apparent mass of Maelstrom, by contrast, is unchanged in vasa null (vasPH165) background, and in alleles of each of the spn genes (A-E) and okr (Findley, 2003).
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date revised: 25 February 2009
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