maelstrom
The establishment of body axes in multicellular organisms requires accurate control of microtubule polarization. Mutations in Drosophila PIWI-interacting RNA (piRNA) pathway genes often disrupt the axes of the oocyte. This results from the activation of the DNA damage checkpoint factor Checkpoint kinase 2 (Chk2) due to transposon derepression. A piRNA pathway gene, maelstrom (mael), is critical for the establishment of oocyte polarity in the developing egg chamber during Drosophila oogenesis. Mael forms complexes with microtubule-organizing center (MTOC) components, including Centrosomin, Mini spindles, and γTubulin. Mael colocalizes with αTubulin and γTubulin to centrosomes in dividing cyst cells and follicle cells. MTOC components mislocalize in mael mutant germarium and egg chambers, leading to centrosome migration defects. During oogenesis, the loss of mael affects oocyte determination and induces egg chamber fusion. Finally, this study shows that the axis specification defects in mael mutants are not suppressed by a mutation in mnk, which encodes a Chk2 homolog. These findings suggest a model in which Mael serves as a platform that nucleates other MTOC components to form a functional MTOC in early oocyte development, which is independent of Chk2 activation and DNA damage signaling (Sato, 2011).
In this study, it was shown that Mael is an MTOC component and that dynamic organization of MTs does not occur in developing mael oocytes, which correlates with mislocalization of other MTOC components. It was also observed that loss of mael affects the number and position of the oocytes in egg chambers and induces fusion of egg chambers. These results indicate that Mael specifically regulates MTOC formation, and thereby plays a key role in coordinating dynamic MT organization during Drosophila oogenesis (Sato, 2011).
Initial polarization of the oocyte during the oocyte specification phase in the germarium requires replacement of the fusome by a polarized MT network, which correlates with the formation of the MTOC. Mael is concentrated in the centrosomal region and is colocalized with αTub and γTub during cyst cell divisions. γTub does not migrate to a developing oocyte in mael germariums, suggesting that Mael is required for the migration of centrioles from the cytoplasm of cysts to pro-oocytes in the germarium. Currently, the detailed mechanism by which Mael functions in MT organization is not clear. The simplest hypothesis is that Mael might serve as a platform that nucleates other MTOC components to form a functional MTOC. A previous report has shown that weaker mutant alleles of γTub affect the number of nurse cells and oocytes within the egg chamber. These γTub mutant defects are very similar to those found in mael mutants in this study. γTub is involved in the nucleation of MTs and is present in the centrosomes and MTOCs in many different systems. It was hypothesized that reduced activity of γTub could activate the oocyte determination program in one of the nurse cells by ectopically presenting MTOC material. The findings that γTub does not accumulate at centrosomes in the mael germarium and is ectopically expressed in the mael egg chamber suggest that Mael regulates localization of γTub at centrosomes through its complex formation and is thereby involved in properly organizing or positioning the MTOC (Sato, 2011).
PIWI proteins function in transposon silencing via association with piRNAs and maintain genome integrity during germline development. Recent studies have suggested that PIWI proteins in sea urchin (Seawi) and Xenopus (Xiwi) can interact with the MTs of the meiotic spindle, while fly ovarioles with mutations in any of several piRNA pathway genes, including spn-E, aub, and armi, have disorganized MTs. This raises the possibility for either a functional role of PIWI proteins in the machinery that impacts on MT organization (in addition to transposon silencing), a role of the MT cytoskeleton in piRNA generation, or both. The current findings further corroborate a link between components of the piRNA pathway and proper MT organization. Although it was found that Mael forms a complex with MTOC components, components of the piRNA pathway in this complex could not be identified. This is in contrast to observations of the mouse Mael homolog, which functions in the piRNA pathway (similar to fly Mael) and interacts with mouse PIWI proteins in the testes (Costa, 2006). Mouse Mael in the testes is almost exclusively cytoplasmic with accumulation at nuage (Soper, 2008). In contrast, fly Mael is located in both the nucleus and the cytoplasm in the ovary and is known to shuttle between them. Thus, one possibility is that in fly ovaries, there may exist nuclear Mael complexes involved in both piRNA generation and transposon silencing, which are distinct from the cytoplasmic complex containing MTOC components that were identified in this study (Sato, 2011).
Female flies with mutations in several genes in the piRNA pathway often lay eggs with axis patterning defects because of MT cytoskeletal changes that result in the mislocalization of bic, grk, and osk mRNAs within the egg chamber. These defects have been linked to the Chk2 DNA damage checkpoint that may be activated by increased retrotransposon transcript levels in mutants defective in piRNA biogenesis. However, because a mutation in mnk does not suppress the mislocalization of Osk and Grk in the mael oocyte, the axis specification defect of mael oocytes does not appear to be triggered by the activation of germline-specific DNA breaks and damage signaling through Chk2. In addition, a mutation in the mei-W68 locus, which encodes the Drosophila Spo11 homolog and induces meiotic double-strand breaks in chromosomes, cannot suppress the axis specification defect of mael oocytes. Therefore, these results suggest that the axis specification defects of mael oocytes are not a secondary consequence of DNA damage signaling. However, it has been shown that in mael mutant ovaries, Vas is post-translationally modified. These results together imply that, acting not only through Chk2, the functions of Mael in MT organization are in parallel with its function in piRNA generation and transposon silencing. There are mutants—including zuc and spn-E that are piRNA pathway genes; their axis defects cannot be rescued by mnk mutations. Vas also appears modified in these mutants, although the relationship with activated checkpoint-modified Vas is unclear (Sato, 2011).
Given that Mael is a new component of the MTOC in the Drosophila ovary, identification of a domain within Mael that is responsible for binding to other MTOC components could aid in understanding how Mael nucleates and regulates MTOC formation. Because Mael contains an evolutionarily highly conserved domain of unknown function, termed the Mael domain (Zhang, 2008), determination of its crystal structure should prove valuable in elucidating mechanisms of both MTOC formation and piRNA generation processes (Sato, 2011).
The maintenance of stem cells is central to generating diverse cell populations in many tissues throughout the life of an animal. Elucidating the mechanisms involved in how stem cells are formed and maintained is crucial to understanding both normal developmental processes and the growth of many cancers. Previously, studies have shown that Zfrp8/PDCD2 is essential for the maintenance of Drosophila hematopoietic stem cells. This study shows that Zfrp8/PDCD2 is also required in both germline and follicle stem cells in the Drosophila ovary. Expression of human PDCD2 fully rescues the Zfrp8 phenotype, underlining the functional conservation of Zfrp8/PDCD2. The piRNA pathway is essential in early oogenesis, and this study found that nuclear localization of Zfrp8 in germline stem cells and their offspring is regulated by some piRNA pathway genes. Zfrp8 forms a complex with the piRNA pathway protein Maelstrom and controls the accumulation of Maelstrom in the nuage. Furthermore, Zfrp8 regulates the activity of specific transposable elements also controlled by Maelstrom and Piwi. These results suggest that Zfrp8/PDCD2 is not an integral member of the piRNA pathway, but has an overlapping function, possibly competing with Maelstrom and Piwi (Minakhina, 2014).
These studies on Zfrp8 requirement in the Drosophila ovary show that the gene is essential in stem cells. The results suggest that Zfrp8 is not required in cells with limited developmental potential, as transient wild-type and mutant clones were similar in number and size. No difference was found in Zfrp8 and wild-type escort cell clones, indicating that Zfrp8 is not required in these cells that multiply by self-duplication. Furthermore, Zfrp8 and wild-type MARCM clones induced in third instar larvae were indistinguishable in the adult antenna and legs 20 days after induction (ACI). These results support the conclusion that Zfrp8 function is primarily required in stem cells (Minakhina, 2014).
Despite this functional requirement, Zfrp8 protein was not enriched in Drosophila GSCs and FSCs. This is surprising, because in mice Zfrp8/PDCD2 is enriched in several types of stem cell. Zfrp8/PDCD2 is also highly expressed in human bone marrow and cord blood stem and precursor cells with protein levels decreasing significantly as these cells differentiate (Minakhina, 2014).
Loss of Zfrp8 in the Drosophila germline did not affect signaling from the niche to the stem cells. But the stem cells themselves are highly sensitive to loss of Zfrp8. In both Zfrp8germline stem cell clones and Zfr8 KD germaria abnormal spectrosomes reminiscent of fusomes were observed. These phenotypes suggest that these germline stem cells are losing stem identity and show features of a stem cell and a more advanced cystocyte. Germline and somatic stem cells and their daughter cells ultimately stop dividing when depleted of Zfrp8 but continue to survive for several days, as evident from the phenotype of the persistent stem cell clones. Similarly, in leukemia and in cancer cell lines that initially have high levels of the protein, reduction of Zfrp8/PDCD2 correlates with delay or arrest of the cell cycle rather than cell death (Minakhina, 2014).
The most severe abnormalities were observed 10-20 days ACI in Zfrp8 GSC clones induced in larvae and adults. The phenotype of Zfrp8 KD ovarioles also became more pronounced with age, starting from a relatively normal-looking germarium and a few egg chambers in young flies, to ovarioles made up of disorganized cysts, and finally, to ovarioles in which germ cells were almost entirely absent. The temporal change in phenotype can be explained in two ways. First, it is possible that Zfrp8 levels are initially high enough in mutant and KD stem cells to support a few divisions and the formation of mutant cysts. However, as Zfrp8 is gradually depleted the cells stop dividing and are eventually lost. Alternatively, lack of Zfrp8 may induce changes in parental cells that affect the developmental potential of the daughter cells. For instance, chromatin modifications could be affected in the absence of Zfrp8, but it could take several cell generations for these changes to have a phenotypic effect. In both these scenarios, loss of Zfrp8 would predominantly affect cells undergoing constant or rapid divisions, such as stem cells and cancer cells (Minakhina, 2014).
The loss of asymmetry in the stem cells, the mislocalization of BicD and Orb proteins to and within the oocyte, the mislocalization of Zfrp8 protein in GSCs of several piRNA pathway mutants, and the genetic interaction of Zfrp8 with piRNA pathway genes suggested a connection between Zfrp8 and the piRNA pathway. The de-repression of the subset of transposons in Zfrp8 KD ovaries further links the gene with the piRNA pathway (Minakhina, 2014).
Several LTR and non-LTR retroelements were tested that represent three major TE classes based on their tissue-specific activity in the germline, soma or in both tissues (intermediate). When Zfrp8 is depleted in the germline, two out of seven intermediate and germline elements tested, HeT-A and TART, show significant de-repression. These elements are different from the majority of Drosophila TEs. The HeT-A, TART and the TAHRE elements are integral components of fly telomere. Their activity is tightly regulated and is required to protect chromosome ends. These elements, like other TEs, are controlled by the piRNA machinery, but their primary piRNAs are likely to be derived from the same telomeric loci that are also their targets for repression. By contrast, the majority of primary piRNAs are derived from piRNA clusters and target TEs dispersed throughout the genome. Furthermore, the repression of TART and HeT-A in the germline involves an unusual combination of piRNA factors. It was found that at early stages of oogenesis they appear to be regulated by piwi and mael, but not by the germline-specific Piwi family member Aub. This result is in agreement with recent studies on piwi function in the soma and germline that showed that HeT-A and TART elements are among the TEs most strongly regulated by Piwi in the germline. Thus, Zfrp8 may target the same TEs as Piwi and Mael but not those regulated by Aub (Minakhina, 2014).
De-repression of TEs caused by Zfrp8 KD could be responsible for the enhancement of developmental defects seen in piRNA pathway mutants. For instance, the increase of TE transcripts may enhance dorsoventral patterning defects in armi, AGO3, aub, spnE and vas because of the competition between TE transcripts and oocyte polarity factors for the same RNA transport machinery. However, the interaction of Zfrp8 with the piRNA pathway machinery seems to be more complex. Zfrp8 enhanced the egg phenotype of only three mutants, spnE, AGO3 and vas, and in these mutants the nuclear localization of Zfrp8 protein was also affected. These results suggest that Zfrp8 functions downstream of the three factors (Minakhina, 2014).
Both piwi and mael are dominantly suppressed by Zfrp8. Both these factors have important nuclear functions, regulating chromatin modifications and controlling TEs at the transcriptional level, and both are required to repress HeT-A and TART-A elements. Zfrp8 could suppress piwi or mael by inducing a competing chromatin modification at the genomic loci targeted by Piwi or Mael. Chromatin modifications are generally stable through several cell generations. Such a function would therefore be consistent with the temporal changes of phenotypes in Zfrp8 ovarian clones and KD ovarioles (Minakhina, 2014).
Although Piwi and Mael target the same genomic loci, no interaction between the two proteins have been detected. Co-immunoprecipitation experiments suggest that Zfrp8 complexes with Mael but not with Piwi, indicating that the observed genetic interaction between Zfrp8 and piwi may be mediated by mael. Mael is one of the most enigmatic proteins in the piRNA pathway. It is found in the cytoplasm, nuage and nucleus, and has been implicated in diverse cellular processes including the ping-pong piRNA amplification cycle in the germline, MTOC assembly in the oocyte and Piwi-dependent chromatin modification in somatic cells. Zfrp8/PDCD2 is also required in the soma and germline and may function both in the cytoplasm and in nuclei. However, in contrast to mael, Zfrp8 homozygous mutants are lethal and Zfrp8 ovaries show a stronger phenotype. Based on the observation that Mael and Zfrp8 are found in the same complex and that Zfrp8 dominantly suppresses Mael, it is proposed that they act in opposite fashion on a common target, whether during piRNA biogenesis or chromatin modification (Minakhina, 2014).
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: 15 December 2021
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