piwi

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of piwi at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

Eggs produced from homozygous piwi mutant germ line stem cell (GSC) clones are arrested in embryogenesis, not rescuable by the paternal piwi⁺ gene. Approximately 30% of the arrested embryos show severe mitotic defects during cleavage stage. The remaining embryos show various morphological defects during late embryogenesis, including a high frequency of severe deformation of the head region. These defects demonstrate that piwi expression in the germ line provides an essential maternal contribution for embryogenesis (Cox, 1998).

Adult

To investigate in which cells piwi is expressed to regulate germ line stem cell (GSC) division, the expression pattern of piwi was examined during oogenesis by in situ hybridization of whole mount Drosophila ovaries with DIG-labeled DNA probes prepared from the piwi cDNA clone. PIWI mRNA is detected in the somatic terminal filament cells apical to GSCs in the germarium and anterior sheath cells as well as in the germ line. In the germ line, it is first abundantly expressed in region 2 of the germarium in which 16-cell germ-line cysts are formed, persists at a lower level through stages 1-6 of oogenesis, is at its lowest level between stage 7-9, becomes strongly expressed again at stage 10, and eventually accumulates in early embryos. Given that removing sheath cells does not affect oogenesis, and the terminal filament cells play a role in regulating GSC division, it is likely that the somatic expression in the terminal filament is responsible for piwi function in regulating GSC division (Cox, 1988).

To elucidate the function of piwi in regulating GSC division, the expression and subcellular behavior of the Piwi protein in vivo was studied. The Piwi protein was tagged by inserting a sequence encoding a myc epitope into the piwi gene, at the 5' end of the piwi open reading frame. The p[5'-myc-piwi] transgene (denoted as myc-piwi) was introduced into Drosophila via P-element mediated germline transformation. myc-piwi fully restores the fertility and gametogenesis of piwi mutant males and females. Thus, the myc-PIWI protein confers wild-type Piwi function. In contrast, a p[piwi-3'- myc] transgene (denoted as piwi-myc) with the myc sequence inserted in the highly conserved C-terminal region fails to rescue any piwi mutant phenotype (Cox, 2000).

The expression of myc-PIWI was confirmed by immunoblot analysis using a monoclonal anti-myc antibody as a probe. A single 97.5 kDa band was revealed in the ovarian extract of myc-piwi. In the third instar larval ovary, where germ-line stem cells (GSCs) begin their asymmetric divisions, myc-PIWI has been found in the nucleus of all germ cells of the ovary. In addition, myc-PIWI is also present in the nuclei of the forming terminal filament cells. In adult ovaries, myc-PIWI is present in the nuclei of both the somatic and germline, as predicted by Piwi RNA in situ analyses (Cox, 1998). Specifically, in the germarium, myc-PIWI is expressed in all the somatic cells, including the main terminal filament cells, the cap cells and the inner sheath cells, consistent with the previous genetic clonal analyses that suggest that piwi is required in these cells to maintain GSCs. Piwi is also expressed in somatic stem cells and the follicle cells in the germarium. In the germline, myc-PIWI shows a dynamic nuclear expression pattern: it is present abundantly in GSCs, but is down-regulated in cystoblasts and developing cysts. In 16-cell cysts in regions 2 and 3, the myc-PIWI regains its high level expression, and remains so in nurse cells and oocytes in post-germarial egg chambers throughout oogenesis. In all post-germarial egg chambers, myc-PIWI is also specifically expressed in the anterior polar follicle cells. The consistent nuclear localization of myc-PIWI in various types of somatic and germline cells during oogenesis indicates that Piwi is a nuclear protein (Cox, 2000).

Piwi is also expressed as a nuclear protein during spermatogenesis. In the third instar larval testes which contain mostly premeiotic germ cells, myc-PIWI is localized to the nuclei of apical somatic cells, including the hub cells, which are the testicular equivalent of terminal filament cells. myc-PIWI is also present in somatic stem cells and their progeny, the cyst progenitor. At the apical germline, Piwi is present in the nuclei of GSCs and their immediate daughter cells. In newly formed 16-cell cysts of primary spermatocytes somewhat away from the apex, the myc-PIWI staining is sharply reduced in the germline. The staining is only present in the cyst progenitor cells, which are equivalent to follicle cells in the ovary. Once the developing cyst enters the spermatocyte growth phase, myc-PIWI expression is completely undetectable. This pattern of expression is maintained in the adult testis which displays the same apical-distal organization but now contains more differentiated post-meiotic germ cells in the basal region of the testis. Given the essential role of piwi in testicular germline stem cell maintenance (Lin, 1997), the nuclear localization of Piwi in the testis should also be functionally important. In interphase somatic and germline nuclei, myc-PIWI is not associated with the chromatin or the nuclear envelope, but is localized in the nucleoplasm (Cox, 2000).

Previous clonal analyses have indicated that piwi functions in the apical somatic cells of the germarium to regulate GSC maintenance (Cox, 1998). However, because Piwi is also present in the nuclei of GSCs, attempts were made to test the potential function of Piwi in GSCs by removing Piwi from a single stem cell using the FLP/FRT-mediated clonal technique. piwi mutations were used for generating piwi minus deficient clones. Mitotic recombination was induced immediately prior to oogenesis and looked-for piwi minus GSC clones were sought 1 week, 2 weeks, and 3 weeks following oogenesis. piwi minus GSCs are present even three weeks following oogenesis, consistent with the observation that Piwi in GSCs is not required for GSC maintenance. The number of marked piwi minus and piwi plus germline cysts in tester and control germaria was examined to compare the division rate between the piwi minus and piwi plus GSCs within the germarium. The analysis reveals that piwi minus GSCs divided four-fold slower than wild-type GSCs. Thus, in addition to its somatic function, Piwi acts cell-autonomously in the stem cells to facilitate their division. The piwi minus cysts and postgermarial piwi minus egg chambers were examined by DAPI staining and by Nomarski microscopy for potential developmental defects. They usually developed normally, suggesting that piwi does not play an important cell-autonomous role in germline cyst development and subsequent stages of oogenesis (Cox, 2000).

Overexpression of Piwi increases the number of germline stem cells. Wild-type germaria typically have 2-3 spectrosome-containing cells, with one often being a cystoblast that is not associated with the terminal filament. Interestingly, the number of the spectrosome-containing germ cells increases to an average of 7.5 cells in germarium in which Piwi is overexpressed. In the most extreme case, up to 15 spectrosome-containing cells are observed in a single germarium. Thus, overexpression of Piwi in the soma leads to a 3- to 4-fold increase in the number of GSCs and/or cystoblasts. To distinguish whether the spectrosome-containing cells are stem cells or cystoblasts, the germaria were stained with anti-cytoplasmic Bag of marbles (Bam) antibody, since Bam is only expressed in cystoblasts and early mitotic cysts but not in GSCs. In piwi-overexpressed germaria, Ban staining is strongly present in early cysts. However, Bam staining is conspicuously absent from all the spectrosome-containing cells. This observation suggests that the ectopically induced spectrosome-containing cells are GSC-like cells (Cox, 2000).

The ectopic GSC-like cells appear to be functional GSCs. (1) They incorporate BrdU, an indicator of DNA replication, at a level similar to the wild-type GSCs. This suggests that they are not arrested in the cell cycle.(2) These GSC-like cells can all differentiate within 4 days following the withdrawal of heat shock, leaving the germaria with only 2-3 GSCs at their normal locale. No signs of cell death, such as pycnotic nuclei or apoptotic bodies, were detected by DAPI staining and Nomarski optics. This suggests that the GSC-like cells are capable of oogenesis. Thus, the dependence of GSC number on the Piwi level reveals that piwi-mediated somatic signaling controls the number of GSCs via a dosage dependent mechanism. Since over-expression of piwi in somatic cells increases the number of GSCs, yet loss of piwi function from the soma and germline in piwi mutants abolishes stem cell division and thus their maintenance, one might expect that over-expression of piwi in the soma would also increase the rate of GSC division. In addition to an increase in the number of stem cells, piwi overexpression also increases the rate of GSC division. Because the GSCs under examination are located in their normal niche, the increase in their mitotic frequency should be mostly, if not entirely, due to the increased Piwi expression in the terminal filament (Cox, 2000).

Since Piwi is a nucleoplasmic protein, it is not likely to be a somatic signal itself, but rather an essential component of the somatic signaling machinery responsible for producing the signal. Given its nucleoplasmic localization, Piwi may be involved in post-transcriptional mRNA processing in the nucleus. Alternatively, it may be involved in nuclear functions indirectly related to gene expression. In either case, the somatic activity of Piwi appears to act via a dosage-dependent mechanism to control the number of GSCs in the germline. Therefore, these results show that piwi may help to define a stem cell niche in the germarium for GSC maintenance, with the size of the niche corresponding to the level of Piwi activity. How can piwi acts in two distinct cell types and in two different mechanisms? This is probably because the Piwi protein directly or indirectly mediates gene expression in the nucleus either at the transcriptional or post-transcriptional level. The different piwi target genes or their products in different cell types then lead to the distinctly different cellular function of piwi essential for germline stem cell maintenance during Drosophila oogenesis (Cox, 2000).

The coordinated division of distinctive types of stem cells within an organ is crucial for organogenesis and homeostasis. Genetic interactions among fs(1)Yb (Yb), piwi, and hedgehog (hh) regulate the division of both germline stem cells (GSCs) and somatic stem cells (SSCs) -- the two constituent stem cell populations of the Drosophila ovary. Yb, coding for an ATP/GTP-binding site motif A (P-loop) domain protein, is required for both GSC and SSC divisions; loss of Yb function eliminates GSCs and reduces SSC division, while Yb overexpression increases GSC number and causes SSC overproliferation. Yb acts via the piwi- and hh-mediated signaling pathways that emanate from the same signaling cells to control GSC and SSC division, respectively. hh signaling also has a minor effect in GSC division (King, 2001).

Yb is expressed in terminal filament and cap cells to control GSC self-renewing divisions. The loss-of-function and overexpression phenotype of Yb reported suggests that Yb is also involved in regulating SSC divisions. It is possible that Yb achieves this dual function indirectly by regulating GSC division, which in turn affects SSC division via an unknown coordination mechanism, or vice versa. These possibilities seem unlikely, since all other mutations are known to only affect either GSCs or SSCs, but not both, as judged from their reported phenotype. For example, piwi and dpp mutations cause failure of GSC maintenance, while bam and bgcn mutations as well as piwi and dpp overexpression cause an accumulation of germline cells without a corresponding increase in somatic cells. Similarly, hh activity regulates SSC division without significant effect on GSC divisions. It is therefore unlikely that all these mutations, except for Yb, have a dual effect on GSC or SSC division and on the coordination mechanism between GSCs and SSCs. Thus, Yb appears to be the only known gene that plays a major role in regulating both GSC and SSC divisions. This dual role of Yb is further supported by the regulatory relationship between Yb, piwi, and hh (King, 2001).

The somatic function of Yb is very similar to that of hh. Like hh, Yb is specifically expressed in cap and terminal filament cells to regulate follicle cell division. Loss of either hh or Yb function leads to reduced follicle cell proliferation, while overexpression of either gene by heat shock leads to overproliferation of follicle cells that exceeds the need for egg chamber formation. The relationship between Yb and hh is further defined by observations that Yb is required for the expression of hh in cap cells and, to a lesser extent, terminal filament cells, and that Yb overexpression significantly elevates hh expression in cap cells and, also to a lesser extent, terminal filament cells. Yb overexpression causes less follicle cell overproliferation than hh overexpression. This can be explained by the fact that Yb overexpression only elevates HH expression in cap cells and terminal filament cells, while heat shock causes HH to be overexpressed all over the germarium. Since hh signaling is the main, if not the only, signaling pathway that controls SSC division, the similar mutant and overexpression phenotype between hh and Yb suggests that Yb is a positive regulator of hh expression in cap and terminal filament cells. In addition, these data provide strong evidence that cap cells play a central role in controlling SSC and GSC divisions, a hypothesis that has been proposed based on the expression pattern and function of Yb, hh, dpp, and piwi, as well as on the mitotic behavior of GSCs (King, 2001).

A parallel situation exists between Yb and piwi in controlling GSC division: (1) both Yb and piwi are expressed in cap and terminal filament cells, and this expression is essential for GSC maintenance; (2) Yb and piwi mutants share a very similar, if not identical, GSC phenotype; (3) overexpressing either Yb or piwi in somatic cells causes a similar increase in the number of GSC-like cells; (4) Yb is required for piwi expression in cap and terminal filament cells. These observations suggest that Yb is also a positive regulator of piwi expression in these somatic cells that controls GSC division. In addition, it suggests that cap cells may play a central role in GSC division, because these cells express higher levels of Yb and piwi and directly contact GSCs. The Yb-piwi mechanism apparently does not control the production of the DPP signal required for GSC maintenance, since overexpression of dpp does not produce similar effects as does Yb or piwi and does not rescue the piwi phenotype (King, 2001).

The hypothesis that Yb controls GSC and SSC divisions by regulating piwi and hh expression, respectively, in cap cells and terminal filament cells is favored. The Piwi protein, as a nuclear factor, in turn controls GSC division by promoting the production of a somatic signal 'S,' which is received by its receptor 'R' in GSCs. In parallel, the Hh signaling molecule suppresses the Ptc receptor activity in SSCs to promote SSC division. Meanwhile, Hh also participates in promoting GSC divisions through the Ptc receptor on the GSC surface, since either overexpressing Hh in Yb mutants or removing PTC activity from GSCs in Yb mutants has a similar effect in rescuing GSC division and maintenance. The expression of hh may also be controlled by engrailed (en), a known transcription regulator of hh that is also specifically expressed in cap cells and terminal filament cells. dpp appears to act independent of the Yb-mediated pathway in regulating GSC division. This bifurcating model with Yb as a common upstream regulator of both GSC and SSC divisions represents a working hypothesis to address how the coordinated division of two distinct types of stem cells is possibly controlled (King, 2001).

An interesting aspect of the above model is that the HH signaling pathway, in addition to its essential role in SSC division, is involved in regulating GSC division. This GSC function of hh, however, appears to be somewhat redundant, since the loss of hh function only affects the maintenance of ~20% of GSCs, while overexpression of hh only stimulates a slight increase in GSC-like cells. Despite this, hh overexpression is sufficient to restore GSC divisions in both Yb and piwi mutants. These observations suggest that the hh signaling pathway is a dispensable mechanism that safeguards the GSC maintenance. It remains to be determined whether other known regulators of hh, such as engrailed, are involved in regulating hh, piwi, or Yb expression in cap cells and the terminal filament. What is the somatic signal and what is its receptor also remains to be determined in the piwi branch of the bifurcating pathway. Finally, it awaits to be established whether or how the Yb-mediated extrinsic signaling mechanism regulates the asymmetric expression and activity of intracellular stem cell genes, such as pumilio, bam, and nanos, during GSC division. The study of these questions should significantly advance understanding of the stem cell mechanism in general (King, 2001).

Effects of Mutation or Deletion

To identify genes that control asymmetric cell divisions, a search was carried for mutations that affect germline stem cell division by screening a collection of single P element enhancer-trap female sterile mutants using anti-Vasa immunostaining (germ cells stain darkly with anti-Vasa antibody) and electron microscopic analyses. Females bearing mutations in stem cell function would be expected to be weakly fertile or sterile and contain small ovaries in which the 2-3 stem cells in each ovariole had differentiated into egg chambers. Several mutations with such effects were identified and found to fall into two complementation groups. The first locus is represented by eight female sterile mutations and was named ovarette; it maps to the pumillo locus (Lin, 1997).

The second gene identified in the screen was named piwi (for P-element induced wimpy testis). The piwi gene is defined by two independent, non-complementing P-element insertional mutations, piwi¹ and piwi⁶⁸⁴³, and a third allele piwi². piwi appears to be a new gene based on complementation tests with known mutations in the region. Among the three alleles, piwi¹ and piwi² show the strongest oogenic phenotype. Moreover, piwi¹ also causes male sterility: mutant males show severe defects in spermatogenesis. The effects of the piwi¹ mutation on germ line stem cell behavior were examined by analyzing mutant germaria labeled with various antisera and by electron microscopy. The ovarioles of piwi¹ females are most commonly devoid of germline cells, as indicated by Nomarski, DAPI, anti-Vasa and electron microscopic imaging analyses. Although individual ovarioles are present as revealed by electron microscopic analysis, they are often difficult to recognize. At low frequency, some ovarioles contain a few developing egg chambers or mature eggs. A small number of ovarioles contain 2-3 small clusters of germ cells, with each cluster composed of only a few germ cells. Given that the mutant ovaries initially contain normal number of germline stem cells, this oogenic phenotype suggests that most of these stem cells either die or differentiate into an egg chamber, and thus fail to maintain themselves. The few surviving egg chambers in the mutant ovaries often show a variety of defects, including abnormal egg chamber polarity and reduced nurse cell number. The spermatogenic defects of piwi¹ mutants parallel its oogenic defects. Wild-type testes contain germline cells at all stages of spermatogenesis, including 5-8 germline stem cells at the apical tip as well as numerous bundles of mature sperm that occupy most of the lumenal space in the testis. In contrast, the piwi¹ mutant testes only contain 1-6 bundles of mature sperm but no other germline cells. Since each sperm bundle derives from a single product of a stem cell division, this defect suggests that germline stem cells in the mutant testes either die or differentiate to found a spermatogenic cyst (Lin, 1997).

To analyze whether the mutant gonads initially contain a normal complement of germline cells, the germline in the piwi¹ mutant was examined during embryonic and larval development using the anti-Vasa antibody to label germ cells and the anti-spectrin antibody to visualize spectrosomes and fusomes. Germ cells in mutant embryos develop normally during embryogenesis. The number of germline stem cells in the third instar larval ovaries is also found to be normal, and the spectrosomes in these cells appear normal following staining with anti-spectrin antibodies. However, the stem cells often do not reside in the middle of the ovary. These analyses suggest that the piwi¹ mutation does not affect the initial proliferation of germline stem cell population but acts later to disrupt stem cell division or maintenance during gametogenesis in both sexes. To confirm this conclusion, the developing germline cysts were examined in piwi¹ mutant third instar testes. In wild-type third instar larval testes, the oldest cysts have progressed into meiosis. Premeiotic cysts contain an elaborate fusome connecting the 16 primary spermatocytes. These cysts grow in size as they leave the apical stem cell region and eventually enter meiosis. The mutant piwi¹ testes at this stage show four defects: (1) they often contain fewer growing cysts, suggesting a defect in germline stem cell division; (2) the premeiotic cysts frequently contain fewer then 16 spermatocytes, suggesting a defect in spermatocyte division; (3) the germline cysts often appear to develop aberrantly, as indicated by defective fusome morphology, suggesting a defect in spermatocyte differentiation; (4) presumably because of the above defects, the mutant testes often are smaller than the wild-type testes. As with the defects seen in females, these results suggest that piwi function is required both to maintain germline stem cells and subsequently for the division and differentiation of the stem cell progeny in both sexes (Lin, 1997).

Since the pumillio ovarette and piwi mutations were induced by enhancer trap P elements, the pattern of lacZ expression was examined in these lines in order to see if there is any overlap in the expression domains of the two genes. pum 2003, as well as four other ovt alleles, show lacZ expression specifically in the terminal filament, a group of apical somatic cells involved in regulating germline stem cell division. No expression in the germline stem cells or other germ line cells is detected. In contrast, the piwi elements cause lacZ to be expressed in the germline both in the third instar larval ovary and in the germarium in the adult ovary. These observations raise the prospect that pum ovt and piwi mutations may act in different cells (Lin, 1997).

In Drosophila, the endogenous retrovirus gypsy is repressed by the functional alleles (restrictive) of an as-yet-uncloned heterochromatic gene called flamenco. Using gypsy-lacZ transcriptional fusions, this repression is shown to take place not only in the follicle cells of restrictive ovaries, as has been previously observed, but also in restrictive larval female gonads. Analyses of the role of gypsy cis-regulatory sequences in the control of gypsy expression are also presented. They rule out the hypothesis that gypsy would contain a single binding region for a putative Flamenco repressor. Indeed, the ovarian expression of a chimeric yp3-lacZ construct was shown to become sensitive to the Flamenco regulation when any of three different 5'-UTR gypsy sequences (ranging from 59 to 647 nucleotides) was incorporated into the heterologous yp3-lacZ transcript. The piwi mutation, which is known to affect RNA-mediated homology-dependent transgene silencing, was also shown to impede the repression of gypsy in restrictive female gonads. Finally, an RNA-silencing model is also supported by the finding in ovaries of short RNAs (25-27 nucleotides long) homologous to sequences from within the gypsy 5'-UTR (Sarot, 2004).

Screens for piwi suppressors in Drosophila identify dosage-dependent regulators of germline stem cell division

The Drosophila piwi gene is the founding member of the only known family of genes whose function in stem cell maintenance is highly conserved in both animal and plant kingdoms. piwi mutants fail to maintain germline stem cells in both male and female gonads. The identification of piwi-interacting genes is essential for understanding how stem cell divisions are regulated by piwi-mediated mechanisms. To search for such genes, the Drosophila third chromosome (~36% of the euchromatic genome) was screened for suppressor mutations of piwi², and six strong and three weak piwi suppressor genes/sequences were identified. These genes/sequences interact negatively with piwi in a dosage-sensitive manner. Two of the strong suppressors represent known genes -- serendipity-delta and similar, both encoding transcription factors. These findings reveal that the genetic regulation of germline stem cell division involves dosage-sensitive mechanisms and that such mechanisms exist at the transcriptional level. In addition, three other types of piwi interactors were identified. The first type consists of deficiencies that dominantly interact with piwi² to cause male sterility, implying that dosage-sensitive regulation also exists in the male germline. The other two types are deficiencies that cause lethality and female-specific lethality in a piwi² mutant background, revealing the zygotic function of piwi in somatic development (Smulders-Srinivasan, 2003).

It is amazing that germline stem cell defects in the piwi mutants can be rescued by the removal of one copy of another gene ( i.e., by 50% reduction in the activity). The identification of six dominant suppressors of piwi at the deficiency level, as well as six strong and three weak suppressors at the individual gene/sequence level, shows that one or more dosage-sensitive mechanisms must negatively interact with piwi in regulating germline stem cell division in the Drosophila ovary. The fact that each of these suppressors can restore the self-renewing ability of germline stem cells in piwi mutants suggests the importance of the dosage-sensitive mechanisms. The existence of multiple suppressors further implies that such negatively interacting and dosage-sensitive mechanisms may be a significant component of the molecular machinery that regulates germline stem cell division. Finally, four deficiencies in the 89E-91B region dominantly interact with piwi² to cause male sterility. This implies that dosage-sensitive mechanisms may also exist in the male germline (Smulders-Srinivasan, 2003).

Such dosage-sensitive mechanisms may not be manifested as the solo act of an individual gene. The P{wA}4-4 suppressor mutation is inserted in the subtelomeric heterochromatic repeats of chromosome 3R, but does not interrupt TART or HeT-A elements in that region. This suggests that the insertion could affect either the transcriptional or the transpositional activity of the retrotransposons or the epigenetic state of that chromosomal region. The latter possibility in turn would suggest that epigenetic effect in the subtelomeric region is involved in regulating germline stem cell division via the piwi-mediated mechanism (Smulders-Srinivasan, 2003).

The discovery of sry-delta, similar, and tango as suppressors of piwi further suggests that the dosage-sensitive mechanism operates at least at the transcriptional level. Sry-delta is a Cys-2/His-2 zinc-finger DNA-binding protein that is present in both germline and somatic cells in the ovary and the testis, as well as in many other tissues and stages of development. It is known to act as a homodimer to activate the transcription of bicoid during oogenesis. Although sry-delta mutants are homozygous late embryonic lethal, hemizygous and intra-allelic escapers are sterile, indicating sry-delta's function during oogenesis. The three sry-delta alleles used in this study (sry-delta¹², sry-delta^SF1, and sry-delta^SF2) are all single-amino-acid changes in the third zinc-finger domain of the protein. However, they are not equivalent mutations. For example, sry-delta^SF2 hemizygotes show many more gonadal defects than do the other alleles, while sry-delta^SF1 shows a lower escaper rate than does sry-delta^SF2. This phenotypic difference leads to a suspicion that the sry-delta^SF2 mutation tends to perturb a subset of downstream effectors necessary for gonadal function, while the sry-delta^SF1 mutation tends to disrupt more general downstream factors, leading to higher lethality. Consistent with this speculation, sry-delta^SF2 is the strongest suppressor of the piwi² phenotype. It would be interesting to conduct genomic screens to identify the target genes of sry-delta whose transcription is selectively affected by sry-delta^SF1 but not by sry-delta^SF2 mutation. Such target genes would likely be involved in germline stem cell division and gonadogenesis (Smulders-Srinivasan, 2003).

Like Sry-delta, Similar and Tango play a key role in the dosage-sensitive regulation of germline stem cell division. Similar is homologous to a large group of heterodimerizing transcriptional activators. It shows closest homology to the human hypoxia inducible factor-1alpha (HIF-1alpha) and has been shown to function in hypoxic response in Drosophila. HIF-1alpha binds to HIF-1ß to drive transcription of downstream genes. Since Tango is the only Drosophila homolog of HIF-1ß, it is likely to be a partner of Similar. Indeed, Tango interacts with Similar in the yeast two-hybrid system. However, Tango is also known to bind to two other Drosophila bHLH-PAS family proteins, Single-minded and Trachealess, to mediate the transcription of their downstream targets. By showing that tango suppresses the germline stem cell phenotype of piwi², this study suggests that Tango heterodimerizes with Similar in the dosage-sensitive transcriptional activation of genes involved in germline stem cell division (Smulders-Srinivasan, 2003).

Although the biochemical properties of the Piwi family proteins have not been systematically characterized, this family of proteins has been extensively implicated in RNA-related processes. Piwi itself is necessary for both transcriptional and post-transcriptional gene silencing in Drosophila. The aubergine (a.k.a. sting) gene, a Drosophila homolog of piwi, functions in the regulation of the stellate transcript, as well as in the translational regulation of oskar and gurken. ago2 in Drosophila, rde-1 in C. elegans, ago1 in A. thaliana, and qde-2 in Neurospora crassa are necessary for post-transcriptional gene silencing. Most recently, the piwi family genes have also been implicated in epigenetic modification and even in genomic rearrangement via microRNA-mediated mechanisms. Then, what are the biochemical activities of Piwi that would allow it to achieve these regulatory functions? Piwi family genes contain the highly conserved PAZ domain in the central region and the Piwi domain at the C terminus of the proteins. The N-terminal portion of the Piwi domain in Miwi, a murine member, has been shown to bind selectively to poly(G) sequences in vitro. Thus, this region of the protein may represent an RNA-binding domain. Consistent with these data, Miwi complexes with its target mRNAs in vivo. All these data suggest that Piwi family proteins possess RNA-binding ability (Smulders-Srinivasan, 2003).

The results of this study help in the understanding of the potential biochemical function of piwi. Since Sry-delta and Similar/Tango are transcription factors, it is possible that they suppress the piwi² phenotype by promoting the transcription of a transcriptional repressor that represses piwi expression. In piwi²/piwi²; Sry-delta/+ or piwi²/piwi²; similar/+ flies, the reduced Sry-delta or Similar level leads to a decreased repressor level, which in turn leads to increased piwi transcription. This possibility, however, is less likely because piwi² produces a 1.65-kb truncated transcript, which suggests that piwi² is a strong or even null mutation. Upregulating such a truncated transcript is unlikely to restore piwi function (Smulders-Srinivasan, 2003).

Therefore, the following two possibilities are favored. One possibility is that the suppression of piwi mutations by Sry-delta and similar may reflect the fact that Piwi interacts with their target mRNAs . Given that Piwi is localized in the nucleoplasm in the ovary, it is possible that Piwi interacts with the pre-mRNAs or mRNAs of Sry-delta and Similar/Tango target genes in the nucleus to attenuate their processing, life span, or their transport into the cytoplasm. If this is the case, in piwi mutants, the mRNAs of Sry-delta and Similar/Tango target genes become more highly expressed, which compromises the self-renewing ability of germline stem cells. A corresponding decrease in the transcriptional activity of Sry-delta and Similar/Tango may then compensate for this effect. Finally, such regulation probably occurs in the somatic signaling cells, since piwi function is required in these cells for germline stem cell maintenance. Alternatively, it is possible that Piwi-mediated signaling in somatic cells indirectly downregulates Similar/Tango-mediated transcription in germline stem cells. If this is the case, in piwi²/piwi²; similar/+ flies, even though there is no Piwi-mediated downregulation of the Similar/Tango activity, the reduced Similar dosage may mimic the effect of the downregulation to keep the transcription at an appropriate level for the self-renewal of the stem cells. Further molecular experiments are required to distinguish between these possibilities (Smulders-Srinivasan, 2003).

It has been shown that piwi and hh represent two parallel pathways under the control of female sterile (1) Yb (Yb) in maintaining germline stem cells and that the defect of piwi mutants can be rescued by overexpressing hh. However, hh transcription is not increased in flies carrying a piwi suppressor mutation. Hence, the suppressors could either act in parallel with the Hh signaling pathway or affect the activity of hh via post-transcriptional mechanisms. Further experiments are required to distinguish between these two possibilities (Smulders-Srinivasan, 2003).

Although Piwi as a maternal component is essential for embryogenesis, it is not known whether its zygotic activity is involved in somatic development. This is partly because the existing mutations of piwi do not show lethal phenotype. The deficiencies that cause the general and female-specific lethality of the piwi² mutant suggest that piwi is involved in somatic development as a zygotic gene. Such synthetic lethality could reflect that piwi is not an essential gene for somatic development. This would not be surprising, given the existence of four piwi homologs in Drosophila. Alternatively, it remains possible that a null allele of piwi will display lethality. In either case, this study reveals the involvement of zygotic piwi activity in somatic development (Smulders-Srinivasan, 2003).

A distinct small RNA pathway silences selfish genetic elements in the germline: Requirement for Piwi

In the Drosophila germline, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences. Whereas small interfering RNAs (siRNAs) derive from both the sense and antisense strands of their double-stranded RNA precursors, rasiRNAs arise mainly from the antisense strand. rasiRNA production appears not to require Dicer-1, which makes microRNAs (miRNAs), or Dicer-2, which makes siRNAs, and rasiRNAs lack the 2',3' hydroxy termini characteristic of animal siRNA and miRNA. Unlike siRNAs and miRNAs, rasiRNAs function through the Piwi, rather than the Ago, Argonaute protein subfamily. These data suggest that rasiRNAs protect the fly germline through a silencing mechanism distinct from both the miRNA and RNA interference pathways (Vagin, 2006).

In plants and animals, RNA silencing pathways defend against viruses, regulate endogenous gene expression, and protect the genome against selfish genetic elements such as retrotransposons and repetitive sequences. Common to all RNA silencing pathways are RNAs 19 to 30 nucleotides (nt) long that specify the target RNAs to be repressed. In RNA interference (RNAi), siRNAs are produced from long exogenous double-stranded RNA (dsRNA). In contrast, ~22-nt miRNAs are endonucleolytically processed from endogenous RNA polymerase II transcripts. Dicer ribonuclease III (RNase III) enzymes produce both siRNAs and miRNAs. In flies, Dicer-2 (Dcr-2) generates siRNAs, whereas the Dicer-1 (Dcr-1)–Loquacious (Loqs) complex produces miRNAs. After their production, small silencing RNAs bind Argonaute proteins to form the functional RNA silencing effector complexes believed to mediate all RNA silencing processes (Vagin, 2006 and references therein).

In Drosophila, processive dicing of long dsRNA and the accumulation of sense and antisense siRNAs without reference to the orientation of the target mRNA are hallmarks of RNAi in vitro. Total small RNA was prepared from the heads of adult males expressing a dsRNA hairpin that silences the white gene via the RNAi pathway. white silencing requires Dcr-2, R2D2, and Ago2. siRNAs were detected with a microarray containing T_M (melting temperature)–normalized probes, 22 nt long, for all sense and antisense siRNAs that theoretically can be produced by dicing the white exon 3 hairpin. Both sense and antisense white siRNAs were detected in wild-type flies but not in dcr-2^L811fsX homozygous mutant flies. The Dcr-2–dependent siRNAs were produced with a periodicity of ~22 nt, consistent with the phased processing of the dsRNA hairpin from the end formed by the 6-nt loop predicted to remain after splicing of its intron-containing primary transcript (Vagin, 2006).

Drosophila repeat-associated small interfering RNAs (rasiRNAs) can be distinguished from siRNAs by their longer length, 24 to 29 nt. rasiRNAs have been proposed to be diced from long dsRNA triggers, such as the ~50 copies of the bidirectionally transcribed Suppressor of Stellate [Su(Ste)] locus on the Y chromosome that in testes silence the ~200 copies of the protein-coding gene Stellate (Ste) found on the X chromosome (Vagin, 2006).

Microarray analysis of total small RNA isolated from fly testes revealed that Su(Ste) rasiRNAs detectably accumulate only from the antisense strand, with little or no phasing. As expected, Su(Ste) rasiRNAs were not detected in testes from males lacking the Su(Ste) loci (cry¹Y). Su(Ste) rasiRNAs were also absent from armitage (armi) mutant testes, which fail to silence Ste and do not support RNAi in vitro. armi encodes a non–DEAD-box helicase homologous to the Arabidopsis thaliana protein SDE3, which is required for RNA silencing triggered by transgenes and some viruses, and depletion by RNAi of the mammalian Armi homolog Mov10 blocks siRNA-directed RNAi in cultured human cells. Normal accumulation of Su(Ste) rasiRNA and robust Ste silencing also require the putative helicase Spindle-E (Spn-E), a member of the DExH family of adenosine triphosphatases (Vagin, 2006).

The accumulation in vivo of only antisense rasiRNAs from Su(Ste) implies that sense Su(Ste) rasiRNAs either are not produced or are selectively destroyed. Either process would make Ste silencing mechanistically different from RNAi. In support of this view, mutations in the central components of the Drosophila RNAi pathway—dcr-2, r2d2, and ago2—did not diminish Su(Ste) rasiRNA accumulation. Deletion of the Su(Ste) silencing trigger (cry¹Y) caused a factor of ~65 increase in Ste mRNA, but null or strong hypomorphic mutations in the three key RNAi proteins did not (Vagin, 2006).

Fly Argonaute proteins can be subdivided into the Ago (Ago1 and Ago2) and Piwi [Aubergine (Aub), Piwi, and Ago3] subfamilies. Unlike ago1 and ago2, the aub, piwi, and ago3 mRNAs are enriched in the germline. Aub is required for Ste silencing and Su(Ste) rasiRNA accumulation. In aub^HN2/aub^QC42 trans-heterozygous mutants, Su(Ste) rasiRNAs were not detected by microarray or Northern analysis, and Su(Ste)-triggered silencing of Ste mRNA was lost completely. Even aub^HN2/+ heterozygotes accumulated less of the most abundant Su(Ste) rasiRNA than did the wild type. That the Ago subfamily protein Ago2 is not required for Ste silencing, whereas the Piwi subfamily protein Aub is essential for it, supports the view that Ste is silenced by a pathway distinct from RNAi. Intriguingly, Su(Ste) rasiRNAs hyperaccumulated in piwi mutant testes, where Ste is silenced normally (Vagin, 2006).

Mutations in aub also cause an increase in sense, but not antisense, Su(Ste) RNA; these results suggest that antisense Su(Ste) rasiRNAs can silence both Ste mRNA and sense Su(Ste) RNA, but that no Su(Ste) rasiRNAs exist that can target the antisense Su(Ste) transcript. The finding that Su(Ste) rasiRNAs are predominantly or exclusively antisense is essentially in agreement with the results of small RNA cloning experiments, in which four of five Su(Ste) rasiRNAs sequenced were in the antisense orientation, but is at odds with earlier reports detecting both sense and antisense Su(Ste) rasiRNAs by non-quantitative Northern hybridization (Vagin, 2006).

Is germline RNA silencing of selfish genetic elements generally distinct from the RNAi and miRNA pathways? The expression of a panel of germline-expressed selfish genetic elementswas examined in mutants defective for eight RNA silencing proteins: three long terminal repeat (LTR)-containing retrotransposons (roo, mdg1, and gypsy); two non-LTR retrotransposons (I-element and HeT-A, a component of the Drosophila telomere), and a repetitive locus (mst40). All selfish genetic elements tested behaved like Ste: Loss of the RNAi proteins Dcr-2, R2D2, or Ago2 had little or no effect on retrotransposon or repetitive element silencing. Instead, silencing required the putative helicases Spn-E and Armi plus one or both of the Piwi subfamily Argonaute proteins, Aub and Piwi. Silencing did not require Loqs, the dsRNA-binding protein required to produce miRNAs (Vagin, 2006).

The null allele dcr-1^Q1147X is homozygous lethal, making it impossible to procure dcr-1 mutant ovaries from dcr-1^Q1147X/dcr-1^Q1147X adult females. Therefore, clones of dcr-1^Q1147X/dcr-1^Q1147X cells were generated in the ovary by mitotic recombination in flies heterozygous for the dominant female-sterile mutation ovo^D1. RNA levels, relative to rp49 mRNA, were measured for three retrotransposons (roo, HeT-A, and mdg1) and one repetitive sequence (mst40) in dcr-1/dcr-1 recombinant ovary clones and in ovo^D1/TM3 and dcr-1/ovo^D1 nonrecombinant ovaries. The ovo^D1 mutation blocks oogenesis at stage 4, after the onset of HeT-A and roo rasiRNA production. Retrotransposon or repetitive sequence transcript abundance was unaltered or decreased in dcr-1/dcr-1 relative to ovo^D1/TM3 and dcr-1/ovo^D1 controls. It is concluded that Dcr-1 is dispensable for silencing these selfish genetic elements in the Drosophila female germline (Vagin, 2006).

roo is the most abundant LTR retrotransposon in flies. roo silencing was analyzed in the female germline with the use of microarrays containing 30-nt probes, tiled at 5-nt resolution, for all ~18,000 possible roo rasiRNAs; the data was corroborated at 1-nt resolution for those rasiRNAs derived from LTR sequences. As observed for Su(Ste) but not for white RNAi, roo rasiRNAs were nonhomogeneously distributed along the roo sequence and accumulated primarily from the antisense strand. In fact, the most abundant sense rasiRNA peak corresponded to a set of probes containing 16 contiguous uracil residues, which suggests that these probes nonspecifically detected fragments of the mRNA polyadenylate [poly(A)] tail. Most of the remaining sense peaks were unaltered in armi mutant ovaries, in which roo expression is increased; this result implies that they do not contribute to roo silencing. No phasing was detected in the distribution of roo rasiRNAs (Vagin, 2006).

As for Su(Ste), wild-type accumulation of antisense roo rasiRNA required the putative helicases Armi and Spn-E and the Piwi subfamily Argonaute proteins Piwi and Aub, but not the RNAi proteins Dcr-2, R2D2, and Ago2. Moreover, accumulation of roo rasiRNA was not measurably altered in loqs ^f00791, an allele that strongly disrupts miRNA production in the female germline (Vagin, 2006).

Loss of Dcr-2 or Dcr-1 did not increase retrotransposon or repetitive element expression, which suggests that neither enzyme acts in rasiRNA-directed silencing. Moreover, loss of Dcr-2 had no detectable effect on Su(Ste) rasiRNA in testes or roo rasiRNA in ovaries. The amount of roo rasiRNA and miR-311 was measured in dcr-1/dcr-1 ovary clones generated by mitotic recombination. Comparison of recombinant (dcr-1/dcr-1) and nonrecombinant (ovo^D1/TM3 and dcr-1/ovo^D1) ovaries by Northern analysis revealed that roo rasiRNA accumulation was unperturbed by the null dcr-1^Q1147X mutation. Pre–miR-311 increased and miR-311 declined by a factor of ~3 in the dcr-1/dcr-1 clones, consistent with about two-thirds of the tissue corresponding to mitotic dcr-1/dcr-1 recombinant cells. Yet, although most of the tissue lacked dcr-1 function, improved, rather than diminished, silencing was observed for the four selfish genetic elements examined. Moreover, the dsRNA-binding protein Loqs, which acts with Dcr-1 to produce miRNAs, was also dispensable for roo rasiRNA production and selfish genetic element silencing. Although the possibility that dcr-1 and dcr-2 can fully substitute for each other in the production of rasiRNA in the ovary cannot be excluded, biochemical evidence suggests that none of the three RNase III enzymes in flies—Dcr-1, Dcr-2, and Drosha—can cleave long dsRNA into small RNAs 24 to 30 nt long (Vagin, 2006).

Animal siRNA and miRNA contain 5' phosphate and 2',3' hydroxy termini. Enzymatic and chemical probing was used to infer the terminal structure of roo and Su(Ste) rasiRNAs. RNA from ovaries or testes was treated with calf intestinal phosphatase (CIP) or CIP followed by polynucleotide kinase plus ATP. CIP treatment caused roo and Su(Ste) rasiRNA to migrate more slowly in polyacrylamide gel electrophoresis, consistent with the loss of one or more terminal phosphate groups. Subsequent incubation with polynucleotide kinase and ATP restored the original gel mobility of the rasiRNAs, indicating that they contained a single 5' or 3' phosphate before CIP treatment. The roo rasiRNA served as a substrate for ligation of a 23-nt 5' RNA adapter by T4 RNA ligase, a process that requires a 5' phosphate; pretreatment with CIP blocked ligation, thus establishing that the monophosphate lies at the 5' end. The rasiRNA must also contain at least one terminal hydroxyl group, because it could be joined by T4 RNA ligase to a preadenylated 17-nt 3' RNA adapter. Notably, the 3' ligation reaction was less efficient for the roo rasiRNA than for a miRNA in the same reaction (Vagin, 2006).

RNA from ovaries or testes was reacted with NaIO₄, then subjected to ß-elimination, to determine whether the rasiRNA had either a single 2' or 3' terminal hydroxy group or had terminal hydroxy groups at both the 2' and 3' positions, as do animal siRNA and miRNA. Only RNAs containing both 2' and 3' hydroxy groups react with NaIO₄; ß-elimination shortens NaIO₄-reacted RNA by one nucleotide, leaving a 3' monophosphate terminus, which adds one negative charge. Consequently, NaIO₄-reacted, ß-eliminated RNAs migrate faster in polyacrylamide gel electrophoresis than does the original unreacted RNA. Both roo and Su(Ste) rasiRNA lack either a 2' or a 3' hydroxyl group, because they failed to react with NaIO₄; miRNAs in the same samples reacted with NaIO₄. Together, these results show that rasiRNAs contain one modified and one unmodified hydroxyl. Because T4 RNA ligase can make both 3'-5' and 2'-5' bonds, the blocked position cannot currently be determined. Some plant small silencing RNAs contain a 2'-O-methyl modification at their 3' terminus (Vagin, 2006).

Drosophila and mammalian siRNA and miRNA function through members of the Ago subfamily of Argonaute proteins, but Su(Ste) and roo rasiRNAs require at least one member of the Piwi subfamily for their function and accumulation. To determine whether roo rasiRNAs physically associate with Piwi and Aub, ovary lysate were prepared from wildtype flies or transgenic flies expressing either myc-tagged Piwi or green fluorescent protein (GFP)–tagged Aub protein; they were immunoprecipitated with monoclonal antibodies (mAbs) to myc, GFP, or Ago1; and then the supernatant and antibody-bound small RNAs were analyzed by Northern blotting. Six different roo rasiRNAs were analyzed. All were associated with Piwi but not with Ago1, the Drosophila Argonaute protein typically associated with miRNAs; miR-8, miR-311, and bantam immunoprecipitated with Ago1 mAb. No rasiRNAs immunoprecipitated with the myc mAb when lysate was used from flies lacking the myc-Piwi transgene (Vagin, 2006).

Although aub mutant ovaries silenced roo mRNA normally, they showed reduced accumulation of roo rasiRNA relative to aub/+ heterozygotes, which suggests that roo rasiRNAs associate with both Piwi and Aub. The supernatant and antibody-bound small RNAs were analyzed after GFP mAb immunoprecipitation of ovary lysate from GFP-Aub transgenic flies and flies lacking the transgene. roo rasiRNA was recovered only when the immunoprecipitation was performed with the GFP mAb in ovary lysate from GFP-Aub transgenic flies. The simplest interpretation of these data is that roo rasiRNAs physically associate with both Piwi and Aub, although it remains possible that the roo rasiRNAs are loaded only into Piwi and that Aub associates with Piwi in a stable complex. The association of roo rasiRNA with both Piwi and Aub suggests that piwi and aub are partially redundant, as does the modest reduction in roo silencing in piwi but not in aub mutants. Alternatively, roo silencing might proceed through Piwi alone, but the two proteins could function in the same pathway to silence selfish genetic elements (Vagin, 2006).

These data suggest that in flies, rasiRNAs are produced by a mechanism that requires neither Dcr-1 nor Dcr-2, yet the patterns of rasiRNAs that direct roo and Ste silencing are as stereotyped as the distinctive siRNA population generated from the white hairpin by Dcr-2 or the unique miRNA species made from each pre-miRNA by Dcr-1. A key challenge for the future will be to determine what enzyme makes rasiRNAs and what sequence or structural features of the unknown rasiRNA precursor lead to the accumulation of a stereotyped pattern of predominantly antisense rasiRNAs (Vagin, 2006).

The role of PIWI and the miRNA machinery in Drosophila germline determination

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

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

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

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

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

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

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

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

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

Aravin, A. A., Naumova, N. M., Tulin, A. A., Rozovsky, Y. M. and Gvozdev V. A. (2001). Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in Drosophila melanogaster germline. Curr. Biol. 11: 1017-1027. 11470406

Aravin, A. A., Sachidanandam, R., Girard, A., Fejes-Toth, K. and Hannon, G. J. (2007a). Developmentally regulated piRNA clusters implicate MILI in transposon control. Science 316(5825): 744-7. Medline abstract: 17446352

Aravin, A. A., Hannon, G. J. and Brennecke, J. (2007b). The Piwi-piRNA pathway provides an adaptive defense in the transposon arms race. Science 318: 761-764. Medline abstract: 17975059

Bohmert, K., et al. (1998). AGO1 defines a novel locus of Arabidopsis controlling leaf development. EMBO J. 17: 170-180.

Brennecke. J., Aravin, A. A., Stark, A., Dus, M., Kellis, M., Sachidanandam, R. and Hannon, G. J. (2007). Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128(6): 1089-103. Medline abstract: 17346786

Brower-Toland, B., et al. (2007). Drosophila PIWI associates with chromatin and interacts directly with HP1a. Genes Dev. 21(18): 2300-11. PubMed citation; Online text

Carmell, M. A., et al. (2002). The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes Dev. 16: 2733-2742. 12414724

Cerutti, L., Mian, N., Bateman, A., 2000. Domains in gene silencing and cell differentiation proteins: the novel PAZ domain and redefinition of the Piwi domain. Trends Biochem. Sci. 25: 481-482. 11050429

Chen, D. and McKearin, D. (2005). Gene circuitry controlling a stem cell niche. Curr Biol. 15(2): 179-84. 15668176

Cox, D. N., et al. (1998). A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal. Genes Dev.12(23): 3715-3727.

Cox, D. N., Chao, A. and Lin, H. (2000). piwi encodes a nucleoplasmic factor whose activity modulates the number and division rate of germline stem cells. Development 127: 503-514. 10631171

Deng, W. and Lin, H. (2002). miwi, a murine homolog of piwi, encodes a cytoplasmic protein essential for spermatogenesis. Dev. Cell 2: 819-830. 12062093

Fagard, M., Boutet, S., Morel, J. B., Bellini, C. and Vaucheret, H. (2000) AGO1, QDE-2, and RDE-1 are related proteins required for posttranscriptional gene silencing in plants, quelling in fungi and RNA interference in animals. Proc. Natl. Acad. Sci. 97: 11650-11654. 11016954

Fire, A., et al. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806-811.

Grewal, S. I. and Rice, J. C. (2004). Regulation of heterochromatin by histone methylation and small RNAs, Curr. Opin. Cell Biol. 16: 230-238. 15145346

Grimaud, C., Bantignies, F., Pal-Bhadra, M., Ghana, P., Bhadra, U. and Cavalli, G. (2006). RNAi components are required for nuclear clustering of Polycomb group response elements. Cell 124(5): 957-71. 16530043

Grishok, A., et al. (2001). Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106: 23-34. 11461699

Gunawardane, L. S., Saito, K., Nishida, K. M., Miyoshi, K., Kawamura, Y., Nagami, T., Siomi, H. and Siomi, M. C. (2007). A slicer-mediated mechanism for repeat-associated siRNA 5' end formation in Drosophila. Science. 315(5818): 1587-90. Medline abstract: 17322028

Harris, A. N. and Macdonald, P. M. (2001). Aubergine encodes a Drosophila polar granule component required for pole cell formation and related to eIF2C. Development 128(14): 2823-32. 11526087

Houwing, S., et al. (2007). A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in Zebrafish. Cell 129(1): 69-82. PubMed citation: 17418787

Jia, S., Noma, K. and Grewal, S. I. (2004). RNAi-independent heterochromatin nucleation by the stress-activated ATF/CREB family proteins. Science 304: 1971-1976. 15218150

Josse, T., et al. (2007). Telomeric trans-silencing: an epigenetic repression combining RNA silencing and heterochromatin formation. PLoS Genet. 3(9): 1633-43. PubMed citation; Online text

King. F. J., et al. (2001). Yb modulates the divisions of both germline and somatic stem cells through piwi- and hh-mediated mechanisms in the Drosophila ovary. Molec. Cell 7: 497-508

Kuramochi-Miyagawa, S., et al. (2001). Two mouse piwi-related genes: miwi and mili. Mech. Dev. 108: 121-133. 11578866

Kuramochi-Miyagawa, S., et al. (2004). Mili, a mammalian member of piwi family gene, is essential for spermatogenesis. Development 131: 839-849. 14736746

Lin, H. and Spradling, A. C. (1997). A novel group of pumilio mutationsaffects the asymmetric division of germline stem cells in the Drosophila ovary. Development 124: 2463-2476.

Lynn, K., et al. (1999). The PINHEAD/ZWILLE gene acts pleiotropically in Arabidopsis development and has overlapping functions with the ARGONAUTE1 gene. Development 126(3): 469-81. 9876176

Megosh, H. B., Cox, D. N., Campbell, C. and Lin, H. (2006). The role of PIWI and the miRNA machinery in Drosophila germline determination. Curr. Biol. 16(19): 1884-94. Medline abstract: 16949822

Mochizuki, K., et al. (2002). Analysis of a piwi-related gene implicates small RNAs in genome rearrangement in Tetrahymena. Cell 110: 689-699. 12297043

Moussian, B., et al. (1998). Role of the ZWILLE gene in the regulation of central shoot meristem cell fate during Arabidopsis embryogenesis. EMBO J. 17: 1799-1809.

Noma, K., et al. (2004). RITS acts in cis to promote RNA interference-mediated transcriptional and post-transcriptional silencing, Nat. Genet. 36: 1174-1180. 15475954

Pal-Bhadra, M., Bhadra, U. and Birchler, J. A. (1997). Cosuppression in Drosophila: gene silencing of Alcohol dehydrogenase by white-Adh transgenes is Polycomb dependent, Cell 90: 479-490. 9267028

Pal-Bhadra, M., Bhadra, U. and Birchler, J. A. (1999). Cosuppression of nonhomologous transgenes in Drosophila involves mutually related endogenous sequences, Cell 99: 35-46. 10520992

Pal-Bhadra, M., Bhadra, U. and Birchler, J. A. (2002). RNAi related mechanisms affect both transcriptional and posttranscriptional transgene silencing in Drosophila. Molec. Cell 9: 315-327. 11864605

Pal-Bhadra, M., et al. (2004). Heterochromatic silencing and HP1 localization in Drosophila are dependent on the RNAi machinery, Science 303: 669-672. 14752161

Parrish, S. and Fire, A. (2001). Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans. RNA 7(10): 1397-402. 11680844

Parker, J. S., Roe, S. M., and Barford, D. (2004). Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity. EMBO J. 23(24): 4727-37. 15565169

Reddien, P. W., et al. (2006). SMEDWI-2 is a PIWI-like protein that regulates planarian stem cells. Science 310(5752): 1327-30. 16311336

Reiss, D., Josse, T., Anxolabehere, D. and Ronsseray, S. (2004). Aubergine mutations in Drosophila melanogaster impair P cytotype determination by telomeric P elements inserted in heterochromatin. Mol. Genet. Genomics 272: 336-343. PubMed citation: 15372228

Roche, S. E. and Rio, D. C. (1998). Trans-silencing by P elements inserted in subtelomeric heterochromatin involves the Drosophila Polycomb group gene, Enhancer of zeste. Genetics 149: 1839-1855. PubMed citation: 9691041

Ronsseray, S., Lehmann, M., Nouaud, D. and Anxolabehere, D. (1996). The regulatory properties of autonomous subtelomeric P elements are sensitive to a Suppressor of variegation in Drosophila melanogaster. Genetics 143: 1663-1674. PubMed citation: 8844154

Ronsseray, S., Boivin, A. and Anxolabehere, D. (2001). P-Element repression in Drosophila melanogaster by variegating clusters of P-lacZ-white transgenes. Genetics 159: 1631-1642. PubMed citation: 11779802

Rossi, L., et al. (2006). DjPiwi-1, a member of the PAZ-Piwi gene family, defines a subpopulation of planarian stem cells. Dev. Genes Evol. 216(6): 335-46. 16532341

Saito, K., et al. (2006). Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev. 20: 2214-2222. Medline abstract: 16882972

Saito, K., Sakaguchi, Y., Suzuki, T., Suzuki, T., Siomi, H. and Siomi, M. C. (2007). Pimet, the Drosophila homolog of HEN1, mediates 2'-O-methylation of Piwi- interacting RNAs at their 3' ends. Genes Dev. 21(13): 1603-8. Medline abstract: 17606638

Sarot, E., et al. (2004). Evidence for a piwi-dependent RNA silencing of the gypsy endogenous retrovirus by the Drosophila melanogaster flamenco gene. Genetics 166: 1313-1321. 15082550

Schmidt, A., et al. (1999). Genetic and molecular characterization of sting, a gene involved in crystal formation and meiotic drive in the male germ line of Drosophila melanogaster. Genetics 151(2): 749-60. 9927466

Smulders-Srinivasan, T. K. and Lin, H. (2003). Screens for piwi suppressors in Drosophila identify dosage-dependent regulators of germline stem cell division. Genetics 165: 1971-1991. 14704180

Szakmary, A., Cox, D. N., Wang, Z. and Lin, H. (2005). Regulatory relationship among piwi, pumilio, and bag-of-marbles in Drosophila germline stem cell self-renewal and differentiation. Curr. Biol. 15(2): 171-8. 15668175

Tabara, H., et al. (1999). The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99(2): 123-32. 10535731

Tijsterman, M., et al. (2002). PPW-1, a PAZ/PIWI Protein required for efficient germline RNAi, is defective in a natural isolate of C. elegans Curr. Biol. 12: 1535-1540. 12225671

Vagin, V.V., Sigova, A., Li, C., Seitz, H., Gvozdev, V. and Zamore, P. D. (2006). A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313(5785): 320-4. 16809489

Verdel, A. et al. (2004). RNAi-mediated targeting of heterochromatin by the RITS complex, Science 303: 672-676. 14704433

Wilson, J. E., Connell, J. E. and Macdonald, P. M. (1996). aubergine enhances oskar translation in the Drosophila ovary. Development 122(5): 1631-9. 8625849

Zou, C., et al. (1998). Molecular cloning and characterization of a rabbit eIF2C protein. Gene 211(2): 187-94. 9602122

date revised: 10 March 2008

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D

The Interactive Fly resides on the
Society for Developmental Biology's Web server.