Northern analysis has revealed a polyadenylated RNA of ~3.0 kb length in accordance with the 2790 bp of the cDNA clone. In wild-type flies, the sti RNA accumulates exclusively in the gonads. Because of a more abundant expression in the ovaries, the sti transcript can be detected easily in total RNA isolated from whole females, but rarely in total RNA isolated from whole males. In homozygous mutant males and females, the expression is deregulated. As a consequence, the 3.0-kb RNA is also intensely transcribed outside the gonads. A larger 4.5-kb RNA also accumulates exclusively in the testes. The fact that the 3.0-kb RNA species continues to be transcribed is compatible with the theory that the integration site is most likely 5' to the transcriptional start site. The misexpression outside the gonads can already be seen in heterozygous flies. All attempts to clarify the structure of the new 4.5-kb testis-specific transcript were unsuccessful (Schmidt, 1999).
In the remobilization experiments mentioned above, new mutant alleles were also found. Most informative for the function of the sting gene was the mutation sti3a, a putative null allele. Because of a large deletion, not only the sti, but also the neighboring RpL9 expression was affected, resulting in severe impairment of vitality. The few escapers had minute bristles, were male and female sterile, and crystal needles could be found in spermatogenic stages. Heterozygous sti/sti3a flies were only male sterile and produced needles. Northern analysis with RNA isolated from a few homozygous sti3a escapers has indicated that sti was not expressed at all. This result was confirmed by the analysis of flies transgenic for a fragment that restored viability by introducing one copy of the RpL9 gene. These flies were still female sterile, thus demonstrating that the sterility was separable from the neighboring Minute gene. Males of the corresponding genotype were sterile needle producers. Hence, the null phenotype of the sti mutation is most likely sterility in both sexes, accompanied by the production of needles in spermatocytes and spermatids (Schmidt, 1999).
In Northern analysis, the 3.0-kb sti RNA could only be detected in 0- to 6-hr-old embryos, but not in older ones. Taking into account the large accumulation of transcripts during oogenesis, this could indicate that only maternally provided RNA is present in the embryos. It was therefore hypothesized that the observed female sterility instead represents a maternal lethality. To distinguish between these alternatives, homozygous sti3a females transgenic for the RpL9 gene were mated to wild-type flies. Those females laid eggs that started development. Embryogenesis, however, stopped at various stages, starting with late blastoderm (stage 5). Only very few embryos survived gastrulation (stage 8), but they died before germ band retraction (stage 12). This proves that the maternal contribution of sti mRNA is essential for early development; i.e., the observed female sterility is indeed a consequence of a maternal lethal effect (Schmidt, 1999).
Two different sting-lacZ fusion genes were introduced into the germline by P-element-mediated transformation to monitor the expression pattern in the gonads. The large construct should contain all regulatory elements because it starts in the neighboring gene. Furthermore, the 5' upstream regulatory sequences are identical to clone MR-3, a clone that can fully restore the wild type. The ß-galactosidase activity produced by the large construct should, therefore, reflect the normal activity of the sti gene. In the testes, enzymatic staining could be detected only at the very tip, i.e., in spermatogonia and early spermatocytes. This activity can also be demonstrated in the larval testis. This is almost complementary to the expression pattern in the original enhancer trap line, where the staining does not start at such early stages, but is observed only in late spermatocytes. If the enhancer trap expression pattern faithfully mirrors the expression of the endogenous sting gene, the apparent loss-of-function phenotype of the sting mutation can be explained easily. The gene is not transcribed, or at least not at the correct level, in the cells where it should be, i.e., early germ cells. Instead, the 3.0-kb mRNA accumulates primarily in late spermatocytes and at later stages, where the crystal needles can also be observed. The only synthesis in late spermatocytes might also be true for the 4.5-kb RNA, whose structure and function is, however, still unknown. The misexpression shown by the original enhancer trap line indicates that the P{lacW} insertion has separated or destroyed important regulatory elements. In accordance with this interpretation, the short construct with sequences from the 3' end of the P{lacW} to the transcriptional start site does not support any expression in testes (Schmidt, 1999).
In females transgenic for the large construct, expression is restricted to the ovary. There is a short, transient expression in the oocyte around stage 6 of oogenesis. At later stages, from stage 9 on, ß-galactosidase activity is found in the nurse cells. Later on, with the dumping of the nurse cell contents, this activity is detected in the oocyte and egg. Such a maternal contribution has to be postulated from the observed maternal lethal effect of a sti null allele. Although the ovary can not be reproducibly stained in the original male sterile enhancer trap line, the short construct does show ß-galactosidase activity in the ovary. Whether this difference is caused by activating or repressing elements that cannot function in one of the two transgenic lines is still an open question (Schmidt, 1999).
The aub transcript is expressed at relatively high levels in the germarium, at lower levels during mid-oogenesis, and again at high levels in the nurse cells and oocyte from about stage 6 of oogenesis. Of greater interest is the distribution of Aub protein. Antibodies, which detect Aub on Western blots, fail to detect Aub protein in whole-mount ovaries. In both wild-type and aub mutant ovaries, a similar low level of uniform staining is observed. As an alternate approach to determine the distribution of Aub protein, the UAS/GAL4 system was used to express a GFP-tagged version of Aub in vivo. Flies carrying a uas-gfp-aub transgene were crossed to those that express a nos-gal4-vp16 transgene which drives expression of GAL4-VP16 in the female germline (Harris, 2001).
The temporal expression of the nos-gal4-vp16 driver parallels that of aub, and uas-gfp-aub driven by nos-gal4-vp16 is sufficient to fully rescue aub mutant defects, indicating both that the fusion protein is functional and that its distribution includes the pattern required of native Aub protein. GFP-Aub protein can be visualized throughout oogenesis. In the nurse cells, the protein is distributed evenly in the cytoplasm and in concentrated foci surrounding the nuclei. These presumptive perinuclear particles remain until around stage 10. In the oocyte, GFP-Aub is at first dispersed evenly in the cytoplasm. Later, beginning at stage 8/9, it becomes strikingly concentrated in the posterior pole plasm, where it largely overlaps the distribution of Osk protein. This posterior concentration appears to occur by recruitment of the GFP-Aub protein, since the aub mRNA is not itself localized within the oocyte (Harris, 2001).
The localization of Aub to the posterior pole of the oocyte raises the possibility that it is a component of polar granules. Polar granules are large, electron-dense structures found in the posterior pole plasm of the oocyte. In the embryo, these granules remain localized to the posterior pole where they are largely incorporated into the budding pole cells. Several other posteriorly localized proteins, including Tudor (Tud), Vas and Osk, have been shown by immunoelectron microscopy to be components of the polar granules. With fluorescence-based detection methods, polar granule components appear in particles, which have been presumed to be the polar granules. Colocalization of Osk and Vas (fused to GFP) in these particles supports this conclusion (Harris, 2001).
To determine if Aub is a polar granule component, confocal microscopy was used to characterize the subcellular distribution of GFP-Aub in embryos. Before pole cell formation, GFP-Aub is found throughout the cytoplasm but strongly concentrates at the posterior pole in distinct particles, most of which also contain Osk protein. The particles are predominantly incorporated into the pole buds, which form at the posterior pole of the embryo and initiate pole cell formation. At this time, the particles increase in size and decrease in number, suggesting that individual particles are fusing. Simultaneously, the level of dispersed, non-particulate GFP-Aub increases in the pole cell cytoplasm. Before cellularization, between nuclear division cycles 11 and 13, the particles often cluster in zones at opposite sides of the pole cell nuclei. Most of the particles continue to display colocalization of GFP-Aub and Osk. However, some Osk-containing particles now begin to appear in the pole cell nuclei, while GFP-Aub is not detectable in these particles and consistently remains excluded from the nuclei. By the time of cellularization, following nuclear division cycle 14, only a few large cytoplasmic particles persist in each pole cell, with much of the GFP-Aub distributed evenly throughout the cytoplasm. Osk continues to colocalize strongly with GFP-Aub in the particles, but most Osk staining is now detected in the nuclear particles and dispersed in the nucleoplasm. Both cytoplasmic and nuclear particles often appear in striking hollow spherical shapes (donut-like in optical sections), which are characteristic of some polar granule clusters as well as similar particles that are present in nuclei, called nuclear bodies. In comparable colocalization experiments, it was determined that Vas-GFP behaves very similarly to Osk: there is always a high level of colocalization in particles, both nuclear and cytoplasmic (Harris, 2001).
These data demonstrate that GFP-Aub is found in particles that (1) also contain Osk and are inferred to contain Vas, both known polar granule components; (2) are located, like polar granules, at the posterior pole of the oocyte and embryo and are incorporated into pole cells; (3) increase in size within the pole cells with similar timing to that previously described for polar granules, and (4) frequently appear, as do polar granules, in spherical structures that are donut-shaped in sections. From these results, it is concluded that Aub is a polar granule component. However, unlike Osk and Vas, which are largely nuclear after the cellular blastoderm stage, Aub is restricted to the cytoplasmic class of particles, indicating that the nuclear bodies and cytoplasmic polar granules do not have identical compositions (Harris, 2001).
Because GFP-Aub concentrates at the posterior pole of the oocyte and is found in polar granules, it was reasoned that Aub localization should depend on the activity of genes required for polar granule assembly. stau and vas are two such genes with somewhat different roles in this process. Stau acts primarily in the localization and translation of osk mRNA, prerequisites for polar granule formation. Although Stau is concentrated at the posterior pole of the oocyte, it is not incorporated into the polar granules early in embryogenesis. By contrast, Vas is a polar granule component and appears to be more directly involved in the formation and integrity of these structures (Harris, 2001).
Not surprisingly, GFP-Aub posterior localization is almost completely defective in stau mutants and greatly reduced in vas mutants, confirming that Aub is localized by the same general mechanism implicated in the localization of other polar granule components. In addition, a second and equally striking defect occurs in only the vas mutants: perinuclear localization of GFP-Aub in the nurse cells is almost entirely eliminated. Taken in conjunction with the known perinuclear concentration of Vas protein, this result raises the possibility that vas-dependent localization of Aub initiates in the nurse cells and involves a complex already containing both Vas and Aub proteins (Harris, 2001).
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).
Nuage, a germ line specific organelle, is remarkably conserved between species, suggesting that it has an important germline cell function. Very little is known about the specific role of this organelle, but in Drosophila three nuage components have been identified, the Vasa, Tudor and Aubergine proteins. Each of these components is also present in polar granules, structures that are assembled in the oocyte and specify the formation of embryonic germ cells. GFP-tagged versions of Vasa and Aubergine were used to characterize and track nuage particles and polar granules in live preparations of ovaries and embryos. Perinuclear nuage is a stable structure that maintains size, seldom detaches from the nuclear envelope and exchanges protein components with the cytoplasm. Cytoplasmic nuage particles move rapidly in nurse cell cytoplasm and passage into the oocyte where their movements parallel that of the bulk cytoplasm. These particles do not appear to be anchored at the posterior or incorporated into polar granules, which argues for a model where nuage particles do not serve as the precursors of polar granules. Instead, Oskar protein nucleates the formation of polar granules from cytoplasmic pools of the components shared with nuage. Surprisingly, Oskar also appears to stabilize at least one shared component, Aubergine, and this property probably contributes to the Oskar-dependent formation of polar granules. Bruno, a translational control protein, is associated with nuage, which is consistent with a model in which nuage facilitates post transcriptional regulation by promoting the formation or reorganization of RNA-protein complexes (Snee, 2004).
Perinuclear nuage contains, in addition to Vas and Aub, the Maelstrom (Mael), and Gustavus (Gus) proteins. Another component, Bruno (Bru), is a protein that acts in translational repression of osk and gurken (grk) mRNAs. By immunolocalization and expression of a GFP-tagged version of this protein, it was found that Bru is concentrated in perinuclear clusters, similar to the distribution of known nuage components. Double labelling experiments with GFPAub confirmed that Bru colocalizes with nuage. However, Bru is also present at high levels in the cytoplasm, raising the question of whether the colocalization reveals an association with nuage or simply reflects random overlap of an abundant protein with the more narrowly distributed nuage. Evidence that Bru is specifically associated with nuage comes from analysis of Bru distribution in vas mutants: as for other nuage components, the perinuclear clusters of Bru are strongly reduced. Given this identification of Bru as a nuage-associated protein, arrest (aret) mutants (the aret gene encodes Bru) were included in a genetic analysis of nuage. The other genes tested were vas, tud, aub and spindle E (spnE), each of which encodes a nuage component or has been shown to be required for nuage formation, or both (Snee, 2004).
Live imaging was used to better characterize the perinuclear nuage defects seen in static images and to extend the analysis to include cytoplasmic nuage particles. GFPAub was used as the nuage marker to test the role of vas, aret and tud, and VasGFP was used to test the roles of aub and spnE. The live imaging confirmed, for the most part, the basic observations from analysis of fixed samples. In vas mutants perinuclear nuage is almost completely absent, with only a few nuage clusters visible. Loss of spnE activity has a less extreme effect: the perinuclear nuage clusters are largely missing, but a perinuclear zone of VasGFP remains. Consistent with the results by using fixed samples, the persistent perinuclear zone of VasGFP is qualitatively different from wild type, appearing almost completely uniform and lacking any visible discontinuities. Similar results were obtained with the aub mutant, except that the VasGFP perinuclear clusters remain present up to stage 8 of oogenesis, after which they disappear. In aret and tud mutants no significant alteration of perinuclear nuage was detected (Snee, 2004).
In mutants whose perinuclear VasGFP is uniform (spnE- and later stage aub-), the protein undergoes rapid exchange with cytoplasmic pools, just as for VasGFP in perinuclear clusters of wild-type egg chambers. In photobleaching experiments the fluorescence-recovery half-time is 50 seconds in aub- and 48.5 seconds in spnE-, similar to the t1/2=59 seconds for wild type (Snee, 2004).
Cytoplasmic nuage particles are affected differently in the vas, aub and spnE mutants. The vas and spnE mutants have few or no cytoplasmic nuage particles. By contrast, aub mutants have no dramatic reduction in the abundance of cytoplasmic nuage particles, even at times well after the disappearance of perinuclear nuage clusters at stage 8, and the particles have a fairly typical size distribution. These particles do not simply represent the default appearance of VasGFP; they are absent in the spnE mutant. Thus, it seems unlikely that perinuclear nuage clusters are required for the formation of cytoplasmic nuage particles, a conclusion consistent with the observation that cytoplasmic particles are produced only infrequently by detachment of perinuclear nuage clusters (Snee, 2004).
The consequences of loss of vas activity were examined in the male germ line. Just as in nurse cells, Vas appears to be concentrated in nuage in spermatocytes. Given the crucial role for Vas in the nuage of other cell types, either male nuage must differ in this requirement or nuage is not essential in the male germ line for fertility. To distinguish between these possibilities vasAS spermatocytes were tested for the presence of nuage, using GFPAub as a marker. Although GFPAub was present in the cytoplasm, there were no visible perinuclear nuage clusters, indicating that nuage does not form in the vas mutant and is therefore not required for spermatocyte function. An alternate and less probable interpretation is that a rudimentary form of nuage, lacking Aub, is present and is sufficient to provide a minimal requirement for nuage in males (Snee, 2004).
In Drosophila, two types of function, not mutually exclusive, have been proposed for nuage. In one model nuage has been suggested to serve as a precursor to polar granules, a view initially based on ultrastructural similarities of the two organelles and supported by the identification of shared components. Another possible role for nuage is based on its position at the periphery of the nucleus, at or near nuclear pores. Specifically, nuage might act in some aspect of remodelling RNPs when RNAs are exported from the nucleus. Analysis of the movements and genesis of nuage particles provides two arguments against the first model: (1) the rate of release of perinuclear nuage clusters in the nurse cells is very low, much lower than expected if the clusters form polar granules; (2) no nuage particles arriving at the posterior pole of the oocyte and becoming incorporated into polar granules were detected. An additional observation that argues against a model where nuage is a precursor for polar granules, is the presence of cytoplasmic nuage particles in aub mutants, despite the fact that these mutants do not assemble polar granules. However, this evidence does not exclude the first model, because the nuage particles in the mutant might not be fully functional. A third argument is provided by the evidence that Osk cannot interact with nuage, leaving de novo assembly of polar granules as the only reasonable option. Overall, the results strongly suggest that nuage is not the precursor to polar granules, and it is believed that the shared features are simply indicative of similar biochemical activities, rather than a precursor-product relationship (Snee, 2004).
The data do not directly test the model that nuage might function as a transition zone in the movements of mRNAs from the nucleus to the cytoplasm, where RNP components might be exchanged or otherwise modified. However, new properties of nuage, and these relate to possible functions, have been identified. (1) It was found that Bruno, an RNA binding protein that acts as a translational repressor of osk and grk mRNAs, is associated with nuage. This extends the correlation of nuage components with factors that act in some aspect on mRNA localization or translational control. Of the previously identified nuage components, Vas and Gus are involved in the regulation of grk mRNA localization and translation, Aub is required for efficient translation of osk mRNA and has also been implicated in RNAi, and mael mutants display defects in the early stages of mRNA localization. Moreover, spnE, which is necessary for normal nuage formation, is required for the localization of multiple mRNAs and acts in RNAi. Thus, every known nuage component has a role in one or more types of post-transcriptional control of gene expression (Snee, 2004).
(2) The second property of nuage reported here, is the remarkably dynamic composition of perinuclear nuage clusters, despite their relatively fixed positions around the nucleus. This is in contrast to studies showing that general protein exchange is slow in mouse nuage. The rapid exchange of both Vas and Aub, the two proteins tested, suggests that the clusters are staging sites where these, and presumably additional proteins, become associated with other molecules and move off into the cytoplasm. Much like shuttling-proteins that escort RNAs in their travels from the nucleus to the cytoplasm, there might be a class of proteins that interact in nuage with newly exported RNAs and then facilitate post-transcriptional control events that occur in the cytoplasm. By this model nuage could be an organelle that concentrates and thus potentiates the activity factors normally present in all cells, but that must be especially active in germline cells because of their intensive reliance on post-transcriptional controls of gene expression (Snee, 2004).
It has been argued that nuage from the nurse cells is not used for polar granule assembly in the oocyte, yet these two subcellular structures clearly share components and may well have similar activities. One feature that clearly distinguishes polar granules from nuage is the presence of Osk protein. Under normal circumstances Osk is never in contact with nuage, because an elaborate set of post-transcriptional control mechanisms serves to prevent Osk accumulation in the nurse cells and to restrict the distribution of Osk protein within the oocyte to the posterior pole. The presence of Osk at this single location provides the cue for the assembly of polar granules, and misdirection of Osk to other sites in the oocyte leads to ectopic polar granule formation. Thus Osk is generally viewed as an anchor for the recruitment of the factors that form polar granules. Given the finding that polar granules are significantly more stable that perinuclear nuage clusters, it might be that Osk not only recruits other factors, but also strengthens their interactions. A further and unanticipated property of Osk was revealed in studies in which Osk was expressed precociously throughout the oocyte. Under these conditions GFPAub levels are substantially elevated in the oocyte. Two general explanations are possible. (1) Osk might stimulate the rate of transfer of GFPAub from the nurse cells to the oocyte. Such a model is not supported by any known property of Osk, and no increase in the rate at which GFPAub particles move into the oocyte was detected. Furthermore, GFPAub levels in the oocyte are enhanced even before the onset of known nurse cell to oocyte movements in the cytoplasm, and so Osk would have to dramatically alter the properties of the egg chamber under this model. (2) Osk could stabilize a normally labile pool of GFPAub in the oocyte. In the simplest form of this model, stabilization would occur as a consequence of the assembly into complexes, which could include factors other than Osk and GFPAub. This model appears to be most compatible with the data. In addition, such a model provides a possible explanation for the curious association of the Fat facets (Faf) protein, a deubiquitinating enzyme, with pole plasm. The role of Faf could be to stabilize one or more polar granule components, thereby enhancing the growth of polar granules (Snee, 2004).
The original mutation was isolated in a screen for male sterile insertions among 2225 P{lacW} insertion lines that were generated by A. Beermann and C. Schultz in the laboratory of J. A. Campos-Ortega at the University of Cologne. The male sterile phenotype displays variable penetrance, leading sometimes to progeny from homozygous males. The P{lacW} insertion was localized by in situ hybridization to polytene region 32D on the left arm of chromosome 2, giving the mutation the provisional name ms(2)32D. Analysis of the spermatogenic stages in homozygous mutant males revealed the existence of needle-like structures in spermatocytes and spermatids. These needles strongly resemble those crystals found in X/0 males of Drosophila melanogaster, although the mutant males clearly have an X/Y genotype, as visualized by the presence of Y-chromosomal lampbrush loops (Schmidt, 1999).
An antibody against the Ste protein was tested to see whether these needles consist of the Ste protein despite the fact that they are produced in X/Y male germ cells. This is indeed the case. Furthermore, the crystal morphology is dependent on the Ste genotype. In mutant male flies carrying a Ste+-containing X chromosome, the crystals have a needle-like shape, whereas in a Ste background, they are similar to the star-shaped crystals found in Ste/cry- males. Thus, the crystals' phenotype is determined by the Ste locus in both the X/Y, cry+ homozygous sting mutant males and in X/Y, cry-; sting+ males. Because the processed Stellate transcript can also be detected in homozygous mutant males by Northern analysis, the gene was renamed sting (sti), as an acronym for Stellate-interacting gene and for the production of sting-like crystals (Schmidt, 1999).
To test the hypothesis that the phenotypes shown by the sti homozygous mutants are related to the Stellate system, the expression of the Stellate RNA was analyzed in these individuals by using different Ste alleles. Total RNA was extracted from the testes of X/Y males homozygous and heterozygous for the sti mutation carrying different Stellate alleles. The patterns of Stellate expression were compared with those of X/Y or X/0 testes by Northern hybridizations. The same smeared 750-nt Stellate RNA, typically present in X/Y, cry- testes, is also always present in testes of homozygous sti males independently of the Ste allele present. It is important to note that a high-molecular-weight RNA (of ~8 kb) is present in the testes of X/Y regular males and in X/Y, heterozygous sti males. This fragment, however, appears reduced or almost absent, concomitantly with the production of the 750-base fragment, in the testes of X/Y, cry+ homozygous sti males or X/Y, cry-; sti+ males, respectively. This suggests that, like the crystal locus (also known as Suppressor of Stellate), sting seems to control Stellate expression, mainly at post-transcriptional levels. The reduction of the high-molecular-weight RNA seen in the testes of X/0 males is observed in sting homozygous males only in combination with the mutant Stellate alleles, while it is not seen with the Ste+ alleles, unless the crystal deletion is also present. Intriguingly, the same high-molecular-weight Ste RNA fragment and another 1.2-kb fragment also are present in other tissues of males and females, such as the larval brains (where they are very abundant), salivary glands, and ovaries, irrespective of the sti genotypes or the absence of the Y chromosome. To determine the origin of these two types of constitutive transcripts, the Ste transcription pattern was analyzed in the testes of males carrying the W12 X chromosome, which lacks the euchromatic Ste cluster. In cry- male testes, the 8.0-kb transcripts decrease concomitantly with the production of the 750- to 850-base Ste transcripts. This suggests that these transcripts are processed in testes of cry- males, producing the 750-nt RNA. Therefore these data show that the Ste sequences are also normally transcribed in somatic tissues, and that the sti gene negatively controls the Ste expression only in male spermatocytes (Schmidt, 1999).
X/Y, cry- males show meiotic chromosome nondisjunction and meiotic drive. To test if the sti homozygous mutant males show the same spectrum of meiotic abnormalities correlated with the number of Ste repeats, three Ste+ alleles with low numbers and seven Ste alleles with different high numbers of Ste repeats, as estimated by slot blots, were chosen. Fertility and sex chromosome behavior were analyzed in sti homozygous or heterozygous males carrying Ste+ or Ste alleles. The derivation of the disjunctional parameter PXY (the probability of disjunction of the X and Y), the drive parameters RX (the recovery of X-bearing sperm) and RY (the recovery of Y-bearing sperm), and the statistical methods used have been described in detail. The data presented led to the following conclusions: (1) the sting mutation affects male fertility, and this is inversely correlated with the Ste repeat copy number; (2) the sting mutation induces meiotic nondisjunction, as seen in the high number of XY and 0 sperm (this is directly correlated with the Ste repeat copy number); (3) the sting mutation affects meiotic drive, since there is an excess of X over Y and 0 over XY sperm. Moreover, it is interesting to note that the spectrum of these defects is present also in the meiosis of heterozygous sti males carrying the Stellate X chromosomes. Taken together, the results of the statistical analysis described above show that sti affects fertility, disjunction, and drive, and that it is semidominant. As with cry1, however, PXY and fertility are much more sensitive to the Ste state (Ste vs. Ste+ or Ste copy number) than are RX or RY. A clear correlation between fertility and the total number of Stellate copies was also found (Schmidt, 1999 and references therein).
The effects of Ste copy number on autosomal behavior were also assessed. sti homozygous males carrying four different Ste alleles were crossed to females carrying the compound second chromosome C(2)EN. These crosses show the same basic pattern reported in previous work for Ste+ combined with synthetic, translocation-generated cry deficiencies or with the cry1Y chromosome. The main features of this pattern are: (1) the recovery of numerous diplo-2 and nullo-2 sperm; (2) a strong disruption of sex chromosome disjunction among these sperm, and (3) an excess recovery of nullo-2 sperm, probably reflective of an autosomal meiotic drive that is not correlated with Ste copy number. The observed drive of chromosome 2, when sex chromosome disjunction is regular, leads to the expectation of a similar preference for 0 over 22 sperm in those cases where the disjunction is destroyed. The lack of such an excess of 0;0 over 0;22 sperm suggests, therefore, that the disjunction of the sex chromosomes and autosomes is not completely independent in frequency, but that it is biased, to some extent, toward opposite poles (Schmidt, 1999).
The precise restriction of proteins to specific domains within a cell plays an important role in early development and differentiation. An efficient way to localize and concentrate proteins is by localization of mRNA in a translationally repressed state, followed by activation of translation when the mRNA reaches its destination. A central issue is how localized mRNAs are derepressed. Regulatory elements for both RNA localization and translational repression are situated in the 3' UTR of OSK mRNA, as they are in NOS. In the case of OSK, premature translation is prevented by Bruno, a 68-kD protein encoded by the arrest (aret) locus. Bruno recognizes a repeated conserved sequence (BRE, for Bruno response element) in the osk 3' UTR, and colocalizes with OSK mRNA to the posterior pole. In contrast to NOS, however, 3' UTR-mediated localization at the posterior pole is not sufficient for translation, as heterologous transcripts localized under the control of the full-length OSK 3' UTR are not translated. This indicates that the OSK 3' UTR, although it may participate, is not sufficient for translational activation, and that sequences elsewhere in the transcript are required for translation of OSK mRNA (Gunkel, 1998).
When OSK mRNA reaches the posterior pole of the Drosophila oocyte, its translation is derepressed by an active process that requires a specific element in the 5' region of the mRNA. This novel type of element is a translational derepressor element, whose functional interaction with the previously identified repressor region in the OSK 3' UTR is required for activation of Oskar mRNA translation at the posterior pole. The derepressor element only functions at the posterior pole, suggesting that a locally restricted interaction between trans-acting factors and the derepressor element may be the link between mRNA localization and translational activation. Specific interaction of two proteins with the OSK mRNA 5' region is shown; one of these also recognizes the 3' repressor element. p50 is a BRE binding protein that recognizes 3' repressor motifs similar to those recognized by Bruno. p50 functions as a second translational repressor independent of Bruno. The involvement of a second repressor protein in OSK translational control is not unexpected. Indeed, aubergine (aub), a gene required for efficient OSK mRNA translation, is required even when Bruno-mediated repression is alleviated by mutations in the BRE, leading to the suggestion that the aub gene product enhances translation by counteracting the action of a second repressor. It is interesting to note that the requirement for aub function in OSK translation is conferred not only by the OSK 3' UTR but also involves the 5' end of OSK mRNA. Consistent with this possible involvement of the OSK 5' end in translational repression, it is found that in transgenic flies containing an inefficient BRE, premature translation increases when the 5' end is truncated. Understanding the extent to which the 5' end of the OSKtranscript might contribute to overall translational repression will require mutations that selectively disrupt 5' repressor function without simultaneously affecting derepressor function (Gunkel, 1998).
Gene silencing by double-stranded RNA is a widespread phenomenon called RNAi, involving homology-dependent degradation of mRNAs. RNAi is established in the Drosophila female germ line. mRNA transcripts are translationally quiescent at the arrested oocyte stage and are insensitive to RNAi. Upon oocyte maturation, transcripts that are translated become sensitive to degradation while untranslated transcripts remain resistant. Mutations in aubergine and spindleE, members of the PIWI/PAZ and DE-H helicase gene families, respectively, block RNAi activation during egg maturation and perturb translation control during oogenesis, supporting a connection between gene silencing and translation in the oocyte (Kennerdell, 2002).
To analyze the effects of dsRNA on mRNA stability in Drosophila oocytes, dsRNAs corresponding to the maternally expressed genes bicoid and hunchback were used. These genes were chosen because their mRNAs are synthesized, processed, and localized to the cytoplasm of oocytes during mid- to late oogenesis. To test the sensitivity of bicoid and hunchback to RNAi, fertilized eggs were initially injected with dsRNA. bicoid dsRNA reduces the expression of Bicoid protein and induces a bicoid loss-of-function phenotype in which embryos have partial transformation of anterior structures to posterior identities. The effect is robust enough that dsRNA-coated gold particles randomly introduced into fertilized eggs by a gene gun generate mutant phenotypes. hunchback dsRNA induces phenotypes in which embryos are missing thoracic and head segments. These phenotypes resemble mutant embryos generated when maternal and zygotic hunchback gene activity is reduced. To determine if dsRNA injection causes mRNA degradation, endogenous mRNA levels were measured using a semiquantitative RT-PCR assay. The level of bicoid mRNA was reduced about fourfold 40 min after injection of bicoid dsRNA. Likewise, injection of hunchback dsRNA resulted in a reduction of hunchback mRNA levels. Coinjection of a pan-specific ribonuclease inhibitor, vanadyl-ribonucleoside, with bicoid dsRNA results in no reduction of bicoid mRNA, indicating the effect requires a ribonuclease activity (Kennerdell, 2002).
Whether and when transcripts become sensitive to dsRNAs during oogenesis was determined. dsRNA was injected into staged oocytes and their consequent levels of bicoid and hunchback mRNAs were examined. Although oocytes earlier than stage 14 could not be injected, stage 14 oocytes could be examined for RNAi activity. Levels of bicoid and hunchback mRNAs were unchanged in stage 14 oocytes after injection of dsRNA, indicating that oocytes at this stage are unable to carry out RNAi (Kennerdell, 2002).
Oocytes of most animals arrest at species-specific stages of meiosis while differentiation of the oocytes occurs. Drosophila oocytes arrest transiently in prophase I while the oocytes are loaded with RNAs and proteins. Some of these molecules are differentially localized within the oocyte, imparting positional information to be used for embryonic axis formation. When Drosophila oocytes reach stage 14, they undergo meiotic arrest once more, this time at metaphase I. These arrested oocytes remain translationally quiescent in the ovary, potentially for weeks. Arrest is relieved as in most animal eggs by the process of maturation or activation that precedes fertilization. In the case of Drosophila, it appears that ovulation triggers activation of the oocyte to resume meiosis. When oocytes are activated, meiosis is completed and translation of maternal RNAs is dramatically elevated. Shortly thereafter, the oocyte is fertilized as it passes into the uterus (Kennerdell, 2002).
RNAi-like effects are not detected in arrested stage 14 oocytes injected with dsRNA. Was this a general feature of the female germ line? To explore this issue, dsRNA was injected into mature activated oocytes. Injection of dsRNA causes reduction in bicoid and hunchback mRNA levels comparable to those seen in embryos. To confirm that mRNA sensitivity to dsRNA is strictly coincident with oocyte maturation, arrested stage 14 oocytes were isolated from dissected ovaries and the oocytes were activated in vitro. This maturation procedure reactivates meiosis, mRNA translation, and vitelline membrane cross-linking. After maturation, oocytes were injected with bicoid dsRNA and assayed for bicoid mRNA levels. These oocytes showed a decrease in bicoid mRNA. Thus, immature Drosophila oocytes that are coordinately blocked for meiosis and translation are resistant to RNAi, and the block to these processes can be released by maturation or activation of oocytes (Kennerdell, 2002).
There are several possible ways in which RNAi might be blocked in arrested oocytes. One possibility is that an essential component of the RNAi machinery might be missing at this stage. Oocyte maturation would then involve synthesis of the component. To address if synthesis of a missing component is responsible, oocytes were activated in the presence of the protein synthesis inhibitor cycloheximide. Arrested stage 14 oocytes were preincubated with cycloheximide and then activated in vitro in the presence of cycloheximide. This treatment inhibits >95% of the protein synthesis that occurs during maturation. These oocytes were injected with bicoid dsRNA and, strikingly, they showed a decrease in bicoid mRNA levels that was comparable to that of normal mature oocytes. RNAi is established during oocyte maturation even when protein synthesis is blocked. Thus, RNAi establishment during oocyte maturation does not likely occur by synthesis of an essential protein component of the RNAi machinery (Kennerdell, 2002).
The stage 14 oocyte is coordinately blocked in both translation and RNAi. The two processes are released near simultaneously from this block, suggesting perhaps that a shared mechanism links their regulation. To test this possibility, the effectiveness of dsRNA was examined against a transcript that is present but not translated after oocyte maturation. The alphaTubulin67C gene encodes one of three alpha-tubulin proteins synthesized during oogenesis and embryogenesis. Transcript accumulates and is actively translated in early immature oocytes. However, after oocyte maturation, no translation of alphaTubulin67C mRNA occurs, even though transcripts at this stage are associated with ribosomes and are competent to drive translation in vitro. The stable pool of alphaTubulin67C mRNA is comparable to levels of bicoid and hunchback mRNA in mature oocytes. When two nonoverlapping dsRNAs against alphaTubulin67C transcript were independently injected into mature activated oocytes, no destruction of mRNA was detected. This suggests that the ability of dsRNAs to destroy transcripts during oogenesis is coupled to the translation activity of the transcript. Successful translation of transcripts is perhaps necessary to link a transcript to dsRNA-triggered degradation (Kennerdell, 2002).
Several Drosophila genes have been identified that affect translation of maternal mRNAs during oogenesis. One of these genes, aubergine (aub), encodes a protein with a PIWI and PAZ domain. To determine whether Aub has any role for RNAi in oocytes, the effect of aub mutations on RNAi activity was examined. bicoid and hunchback dsRNAs were injected into aub mutant oocytes that were activated in vitro. Degradation of bicoid and hunchback mRNAs was not observed in aub mutants, indicating that Aub is necessary for germ-line RNAi. Two independent aub alleles in heteroallelic combination produced the same result, indicating that the effect was not due to the influence of linked modifiers (Kennerdell, 2002).
The aub gene is a member of a family of genes implicated in RNAi and PTGS. Indeed, aub has been implicated in PTGS regulation of the Stellate repeats and Su(Ste) genes on X and Y chromosomes. Another member of the family, piwi, has been implicated in PTGS within somatic cells. A third family member, Ago2, is a subunit of the mRNA-cleaving complex that mediates RNAi in Drosophila embryonic cells. Thus, several members of this gene family in Drosophila have been implicated in RNAi and PTGS at various steps (Kennerdell, 2002).
It was of interest to determine if other translational regulatory genes are involved in RNAi. To test this possibility, two genes that possibly act through interactions with RNA were examined. vasa and spindle-E (spn-E) encode DexH-box RNA helicases. When activated spn-E mutant oocytes were injected with bicoid or hunchback dsRNAs, no reduction in cognate mRNA levels occurred. In contrast, activated vasa mutant oocytes injected with bicoid dsRNA were found to show transcript degradation comparable to wild type. It is concluded that activation of RNAi in oocytes is dependent on the activity of Spn-E but not Vasa (Kennerdell, 2002).
Arrested Drosophila oocytes are unable to generate RNAi silencing of endogenous maternal mRNAs, but selectively establish this capability upon egg maturation. How is RNAi activated by egg maturation? It is argued that RNAi is linked in some way to translation of maternal mRNAs, which is also specifically activated by egg maturation. Establishment of RNAi is probably not caused by translation of a missing RNAi component. Rather, the complete RNAi apparatus may be present and poised for action but is unable to target homologous substrate mRNAs until egg maturation. Translational masking of mRNAs, a mechanism that operates on maternal Drosophila gene expression, may conceivably be one way in which mRNA is blocked from RNAi attack. Alternatively, targeting of mRNA might require transcripts be assembled onto active polysomes. This may be the case, because siRNA-containing RISC complexes physically fractionate with polysomes, and siRNAs associate with polysomes in Trypanosoma brucei. There is no evidence to indicate that dsRNA-targeting requires ribosome translocation on transcripts, because it is found that cycloheximide inhibition of ribosome translocation does not block RNAi activity in activated mature oocytes (Kennerdell, 2002).
Coupling RNAi to translated mRNA might facilitate base-pairing interactions between siRNAs and an unfolded mRNA target, or it might simply be a means to mark RNAs to be scanned for destruction. The key evidence suggesting that transcript translation is linked to transcript degradation by RNAi comes from experiments in which dsRNA against the alphaTubulin67C message was tested. dsRNA is ineffective against the untranslated alphaTubulin67C transcript in mature activated oocytes, which are nevertheless competent to carry out RNAi against translated bicoid and hunchback transcripts. Thus, there is a correlation between the ability of a transcript to be translated and its ability to be destroyed by dsRNA (Kennerdell, 2002).
Aub and Spn-E are required for RNAi in Drosophila oocytes. They also regulate several features of germ-cell development, including translation of certain maternal mRNAs. Germ-line and stem-cell functions have been reported for orthologs of piwi and ago1 in a variety of species. The ego-1 gene of C. elegans is required for both germ-line development and germ-line PTGS. Finally, mutations in dcr-1, the C. elegans gene encoding Dicer, disrupt oogenesis in an unspecified manner. The developmental defects associated with mutations in these genes, including aub and spn-E, might reflect a loss of gene silencing important for oocyte development. Thus, Aub and Spn-E might play a specific role in gene silencing mechanisms, including RNAi, that nevertheless have a widespread impact on many features of development. Alternatively, Aub and Spn-E could be required for RNAi because they activate translation of germ-line transcripts including those for bicoid and hunchback. Although there is no evidence for translational control of bicoid mRNA in aub mutants, these mutants may perturb steps in the translation of transcripts that are essential for triggering RNAi. Future experiments should define the specific roles for Aub and Spn-E in dsRNA-mediated destruction and its relationship to translation control (Kennerdell, 2002).
To date, few natural cases of RNA-silencing-mediated regulation have been described. Repression has been analyzed of testis-expressed Stellate genes by the homologous Suppressors of Stellate [Su(Ste)] repeats that produce sense and antisense short RNAs. The Stellate promoter is dispensable for suppression, but local disturbance of complementarity between the Stellate transcript and the Su(Ste) repeats impairs silencing. Using in situ RNA hybridization, temporal control was found of the expression and spatial distribution of sense and antisense Stellate and Su(Ste) transcripts in germinal cells. Antisense Su(Ste) transcripts accumulate in the nuclei of early spermatocytes before the appearance of sense transcripts. The sense and antisense transcripts are colocalized in the nuclei of mature spermatocytes, placing the initial step of silencing in the nucleus and suggesting formation of double-stranded RNA. Mutations in the aubergine and spindle-E genes, members of the Argonaute and RNA helicase gene families, respectively, impair silencing by eliminating the short Su(Ste) RNA, but have no effect on microRNA production. Thus, different small RNA-containing complexes operate in the male germ line (Aravin, 2004).
A strong correlation is observed between Stellate silencing and the presence in testes of sense and antisense 25- to 27-nt RNAs homologous to Stellate and Su(Ste) sequences. The short RNAs are absent when Stellate genes are derepressed as a consequence of either a Su(Ste) locus deletion or mutations in the aub and spn-E genes. The cloning of short RNA from D. melanogaster testes also demonstrates the presence of short RNAs that are derived from Su(Ste) and are highly homologous to Stellate. A rigid size restriction of 21 to 23 nt has, however, been observed for siRNA in various in vitro studies of D. melanogaster RNAi. Examination of Dicer activity with different dsRNAs suggests a strong specificity of processing to 21- to 23-nt fragments in both Drosophila embryo extracts and cell culture. Furthermore, investigation of the functional anatomy of chemically synthesized siRNAs in embryo extracts defined the optimal length of siRNAs as 21 to 23 nt, while RNAs longer than 24 nt have practically no cognate-mRNA cleavage activity. It has been proposed that only RNAs that meet this size requirement can be loaded into the RISC. However, examples of the existence of two size classes of short RNAs (21 or 22 nt and 24 to 26 nt) involved in silencing have also been reported. Two different size variants of short RNAs were observed during artificial silencing in plants, with the short variant responsible for posttranscriptional gene silencing and the long one most likely participating in DNA methylation and spreading of the silencing signal. Furthermore, only RNAs from the long class have been detected that correspond to endogenous plant transposable elements. Two size classes of short RNAs are produced from dsRNA in plant extracts, and the activity of different Dicer proteins was shown to be responsible for producing each class. Cloning of endogenous short RNAs from D. melanogaster has also identified two size classes of short RNAs, with the short class (21 to 23 nt) including microRNAs and the long class (24 to 26 nt) comprising sequences derived from transcripts of transposable elements and other repetitive heterochromatic sequences (Aravin, 2004).
The larger size of the short Su(Ste) RNA may be explained by specific sequences affecting dsRNA processing by Dicer or by the presence in testes of specific factors that change the cleavage interval of dsRNA. However, exogenous Su(Ste) dsRNA is cleaved into 21- to 23-nt siRNA in testis extracts, most likely reflecting the activity of the same Dicer protein that acts in somatic tissues. The hypothesis is favored that the 25- to 27-nt Su(Ste) RNAs detected in vivo are produced by a mechanism at least partially different from conventional siRNA production. A clue to the origin of the short Su(Ste) RNAs comes from the finding that Su(Ste) dsRNA formation occurs in the nucleus, unlike that of artificial RNAi, in which dsRNA is believed to be processed in the cytoplasm. Both conventional-size siRNA and a longer short RNA have been observed during viroid replication in the plant nucleus. Two size classes of short RNAs may be produced in D. melanogaster by different Dicer proteins, as has been demonstrated in plants. Alternatively, specific nuclear factors may affect how a single Dicer protein processes dsRNA in the nucleus (Aravin, 2004).
Mutations in the aub and spn-E genes lead to elimination of short Su(Ste) RNA in testes. However, neither mutation affects processing of exogenously provided dsRNA to 21- to 23-nt siRNA in testis extracts. It has been observed that both aub and spn-E mutations block RNAi in oocytes produced by injected dsRNA. It has been proposed that both proteins affect RNAi because of their involvement in translational control, but the results suggest that Aub and Spn-E may be involved in the production and/or stabilization of siRNA. Similarly, the rde-1 and mut-7 genes of Caenorhabditis elegans are required for the production of siRNA in vivo but are dispensable for dsRNA processing in vitro. The corresponding proteins are required for long-term stabilization of siRNA rather than for dsRNA processing (Aravin, 2004).
The aub and spn-E mutations eliminate the short Su(Ste) RNA without affecting the abundance of two different microRNAs in testes. It is proposed that distinct protein complexes mediate production and/or stabilization of short Su(Ste) RNA and microRNAs in testes. Similarly, different members of the Argonaute family participate in artificial RNAi and in microRNA processing in C. elegans and plants, despite the central role of Dicer in both processes (Aravin, 2004).
Telomeres in Drosophila are maintained by transposition of specialized telomeric retroelements HeT-A, TAHRE, and TART instead of the short DNA repeats generated by telomerase in other eukaryotes. This study implicates the RNA interference machinery in the control of Drosophila telomere length in ovaries. The abundance of telomeric retroelement transcripts is up-regulated owing to mutations in the spn-E and aub genes, encoding a putative RNA helicase and protein of the Argonaute family, respectively, which are related to the RNA interference (RNAi) machinery. These mutations cause an increase in the frequency of telomeric element retrotransposition to a broken chromosome end. spn-E mutations eliminate HeT-A and TART short RNAs in ovaries, suggesting an RNAi-based mechanism in the control of telomere maintenance in the Drosophila germline. Enhanced frequency of TART, but not HeT-A, attachments in individuals carrying one dose of mutant spn-E or aub alleles suggests that TART is a primary target of the RNAi machinery. At the same time, enhanced HeT-A attachments to broken chromosome ends were detected in oocytes from homozygous spn-E mutants. Double-stranded RNA (dsRNA)-mediated control of telomeric retroelement transposition may occur at premeiotic stages, resulting in the maintenance of appropriate telomere length in gamete precursors (Savitsky, 2006).
The problems of end-under-replication and stability of linear chromosomes are resolved by telomeres. The lengthening of terminal regions of linear eukaryotic chromosomes is often provided by RNA-templated addition of repeated DNA by reverse transcriptase enzyme, telomerase. In most eukaryotes, telomeric DNA is maintained by the action of telomerase, which is responsible for the synthesis of short 6-8-nucleotide (nt) arrays using an RNA component as a template. In contrast, telomeres of Drosophila are maintained as a result of retrotranspositions of specialized telomeric non-long-terminal repeat (LTR) HeT-A, TAHRE, and TART retrotranspositions (Biessmann, 1992b; Levis, 1993; for review, see Pardue, 2003; Abad, 2004b). Retrotransposons are also found in telomeric regions of such diverse organisms as Bombyx mori, Chlorella and Giardia lamblia. HeT-A, TAHRE, and TART are found at Drosophila telomeres in tandem arrays. HeT-A, the most abundant Drosophila telomeric element, contains a single ORF encoding a Gag-like RNA-binding protein, but lacks reverse transcriptase (RT). It is proposed that the RT necessary for its transposition might be provided in trans, perhaps by TART (Rashkova, 2002). TART ORF2 encodes a reverse transcriptase related to the catalytic subunit of telomerase. The recently discovered TAHRE element shows extensive similarity to HeT-A, but contains a second ORF, which encodes a reverse transcriptase (Abad, 2004b). A HeT-A promoter located in the 3' region of the element directs synthesis of a downstream neighbor (Danilevskaya, 1997). The TART element was shown to be transcribed bidirectionally using a putative internal sense promoter and antisense one that was localized within the 1-kb region of the TART 3' end (Danilevskaya, 1999). Maintenance of Drosophila telomere length is mediated by HeT-A and TART transpositions to chromosome ends as well as by terminal recombination/gene conversion (Mikhailovsky, 1999; Kahn, 2000). Most of the observed spontaneous attachments to telomeres are HeT-A transpositions (Biessmann, 1992a; Kahn, 2000; Golubovsky, 2001), but TART attachments (Sheen, 1994) were also detected (Savitsky, 2006 and references therein).
The spn-E and aub genes, encoding an RNA helicase and a protein of Argonaute family, respectively, are involved in double-stranded RNA (dsRNA)-triggered RNA interference (RNAi) in embryos, in transcriptional silencing of transgenes, and in the control of Drosophila retrotransposon transcript abundance in the germline, especially in ovaries. No effects of RNAi gene mutations on HeT-A and TART expression and telomere structure were observed in somatic tissues (Perrini, 2004). This study shows that increased HeT-A and TART transcript abundance in ovaries, owing to RNAi mutations, is correlated with a high frequency of telomeric element attachments to broken chromosome ends. Addition of HeT-A or TART to a truncated X chromosome, with a break in the upstream regulatory region of yellow, activates yellow expression in aristae, which enables monitoring of the elongation events (Kahn, 2000; Savitsky, 2002). Using this genetic system, the effects of RNAi mutations were studied on the frequency and molecular nature of telomeric attachments. A high frequency of TART but not HeT-A attachments in heterozygous RNAi mutants suggests that TART may be the primary target of the RNAi-based silencing mechanism. These results highlight for the first time the importance of TART, but not the more abundant HeT-A element, in Drosophila telomere maintenance. The disappearance of short TART and HeT-A RNAs was found in spn-E mutant ovaries, strongly suggesting an RNAi-based pathway in the control of telomere maintenance in the Drosophila germline (Savitsky, 2006).
An RNAi-based mechanism has been proposed to evolve in order to immobilize transposable elements and was found to control expression of endogenous transposable elements and their mobility in different species. Drosophila telomeres are maintained by successive transpositions of specialized telomeric retroelements HeT-A and TART. This study shows that transposition of both telomeric elements is under the control of the spn-E and aub genes, known to be related to the RNAi machinery. Hence, an RNAi-based mechanism may be considered not only as a defense against retrotransposon expansion, but also as a regulatory system responsible for proper telomere length maintenance in Drosophila (Savitsky, 2006).
spn-E is required for appropriate localization of mRNA and proteins involved in the establishment of axis formation in the embryo and encodes a member of the DEAD/DE-H protein family possessing RNA-binding and RNA helicase activity. aub encodes a protein of the Argonaute family that was shown to be a component of the RNAi effector complex RISC. aub and spn-E mutations strongly diminished effects of the injected dsRNA into mature oocytes. Both genes are implicated in small interfering RNA (siRNA)-dependent silencing of testis-expressed Stellate genes. Thus, spn-E and aub are components of RNAi-based silencing pathways in Drosophila. Mutations in these genes result in the derepression of a wide spectrum of retrotransposons in the germline, including the HeT-A telomeric element (Aravin, 2001; Stapleton, 2001; Kogan, 2003). This study demonstrates that spn-E and aub mutations increase the frequency of telomeric element retrotranspositions to broken chromosome termini, suggesting that the RNAi machinery controls telomere length in Drosophila (Savitsky, 2006).
Both telomeric elements are shown to be the targets of RNAi. The present results emphasize the differences in the response of HeT-A and TART elements to RNAi mutations. Surprisingly, two different spn-E mutant alleles and an aub mutation in the heterozygous state increase considerably TART mobility, whereas attachments of HeT-A to broken chromosome ends were detected much more rarely in spn-E1/+ ovaries and are not observed in ovaries of spn-Ehls3987/+ and aubQC42/+ flies. One copy of a spn-E mutation is sufficient to increase TART transcript abundance. Strong accumulation of HeT-A transcripts is found only in homozygous mutants, correlating with a high frequency of HeT-A attachments to the broken chromosome ends in the developing oocytes. This observation argues that TART is a primary target of the RNAi machinery in ovaries (Savitsky, 2006).
TART and HeT-A, in spite of sharing the region of integration, are dissimilar in their structure and expression strategy. While both sense and antisense TART transcription has been demonstrated, antisense transcripts are more abundant. In situ RNA analysis detected sense and antisense TART transcripts in the cytoplasm of nurse cells in the late-stage egg chambers, suggesting a possibility of dsRNA formation. However, it was found that the level of antisense TART transcripts is not affected in RNAi mutants. Only sense HeT-A transcription was observed by Northern or by in situ RNA analyses. Nevertheless, HeT-A- and TART-specific siRNAs were revealed among the cloned short RNA species in Drosophila, and short RNAs corresponding to both HeT-A and TART elements are detected by Northern analysis. Antisense HeT-A RNA is probably transcribed at a low level from an unidentified promoter, possibly, from the HeT-A internal region. Actually, a low level of antisense activity of the HeT-A 3' end has been observed . While TART transcripts were observed only in the nurse cells, HeT-A transcripts were detected both in the growing oocyte and nurse cells. It is proposed that TART is a primary target of the RNAi controlling system, since one dose of an RNAi mutation causes preferential TART, but not HeT-A, attachments to broken chromosome ends in ovaries. In contrast, one dose of a mutant Su(var)205 gene (HP1) considerably increasess the frequency of HeT-A rather than TART attachments to the chromosome ends (Savitsky, 2002). Thus, a specific effect of RNAi components on telomeric element expression is observed . Although TART copies are much less abundant in the genome than HeT-A and no TART elements are detected in some telomeres, TART is a conserved component of telomeres in distant Drosophila species. TART was considered as a source of RT production, thus ensuring retrotranspositions of both TART and HeT-A elements. One may propose that TART supplies an RNAi-regulated template for RT production, thus providing telomere-specific transpositions of both elements (Savitsky, 2006).
Drosophila telomeres contain a multisubunit protein complex forming a chromosome cap protecting chromosomes from DNA repair and end-to-end fusions. However, no HeT-A or TART sequences were detected at the stably maintained broken chromosome end, which is protected from telomere fusions. Thus, a sequence-independent system performs telomere capping functions. The capping complex contains HP1, HOAP (HP1/ORC associated protein), as well as ATM-kinase and DNA repair MRN complex and the Ku70/Ku80 heterodimer. HP1 and the Ku heterodimer act also as negative regulators of telomere elongation by retrotransposition of telomeric elements. Deficiencies that remove either the Ku70 or the Ku80 gene increase the transposition rate of HeT-A and TART elements but exert no effect on the HeT-A expression, suggesting that Ku proteins control the accessibility of the telomere to transposition events. At the same time, mutations in the Su(var)205 gene increase both transcript abundance of HeT-A and TART and the frequency of their attachments to chromosome ends. RNAi affects both telomeric retrotransposon expression and the rate of transposition to the telomere. Probably, this effect is mediated through HP1 recruitment and silencing of HeT-A and/or TART chromatin (Savitsky, 2006).
siRNAs produced from telomeric elements TART and HeT-A belong to the long size class (25-29 nt) in contrast to 21-22-nt RNAs guiding post-transcriptional RNAi. In plants, long siRNAs are associated with RNA-directed DNA methylation and play an essential role in the transcriptional retrotransposon silencing. dsRNA and proteins of the RNAi machinery can direct chromatin alteration to homologous DNA sequences and induce transcriptional silencing. RNAi mutations cause delocalization of HP1 in yeast and Drosophila. Actually, the increase in accessibility of HeT-A chromatin and its enrichment in K9-acetylated H3 histone were revealed in ovaries of spn-E mutants. It is also possible that TART and/or HeT-A short RNAs can be targeted to telomeric repeats in a transcriptional silencing complex (Savitsky, 2006).
RNAi disruption affects neither HeT-A and TART expression, nor telomere fusions in somatic cells. No effect was observed of spn-E mutations on HeT-A expression, even in actively dividing cells of imaginal discs, where HeT-A expression was found. The data indicate a crucial role of the RNAi machinery in the regulation of telomere elongation in germinal cells. The appearance of a cluster of individuals with identical retroelement attachments indicates that dsRNA-mediated control of terminal elongation may occur at premeiotic stages of oogenesis (Savitsky, 2006).
This study has demonstrated that expression and retrotransposition of specific telomeric repeats is under control of an RNAi-based system in the Drosophila germline. In this case, the telomerase-dependent mechanism of telomere stability is substituted by retrotranspositions. Interestingly, telomerase-dependent telomere functioning during meiosis in the yeasts Schizosaccharomyces pombe and Tetrahymena is also under the control of RNAi machinery. These observations and the current data indicate that dsRNA-mediated regulation of telomere dynamics in the germline may be a general phenomenon independent of a mode of telomere maintenance (Savitsky, 2006).
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 TM (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-2L811fsX 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 aubHN2/aubQC42 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 aubHN2/+ 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-1Q1147X is homozygous lethal, making it impossible to procure dcr-1 mutant ovaries from dcr-1Q1147X/dcr-1Q1147X adult females. Therefore, clones of dcr-1Q1147X/dcr-1Q1147X cells were generated in the ovary by mitotic recombination in flies heterozygous for the dominant female-sterile mutation ovoD1. 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 ovoD1/TM3 and dcr-1/ovoD1 nonrecombinant ovaries. The ovoD1 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 ovoD1/TM3 and dcr-1/ovoD1 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 (ovoD1/TM3 and dcr-1/ovoD1) ovaries by Northern analysis revealed that roo rasiRNA accumulation was unperturbed by the null dcr-1Q1147X 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).
Small repeat-associated siRNAs (rasiRNAs) mediate silencing of retrotransposons and the Stellate locus. Mutations in the Drosophila rasiRNA pathway genes armitage and aubergine disrupt embryonic axis specification, triggering defects in microtubule polarization as well as asymmetric localization of mRNA and protein determinants in the developing oocyte. Mutations in the ATR/Chk2 DNA damage signal transduction pathway dramatically suppress these axis specification defects, but do not restore retrotransposon or Stellate silencing. Furthermore, rasiRNA pathway mutations lead to germline-specific accumulation of γ-H2Av foci characteristic of DNA damage. It is concluded that rasiRNA-based gene silencing is not required for axis specification, and that the critical developmental function for this pathway is to suppress DNA damage signaling in the germline (Klattenhoff, 2007).
Mutations in the Drosophila armi, aub, and spn-E genes disrupt oocyte microtubule organization and asymmetric localization of mRNAs and proteins that specify the posterior apole and dorsal-ventral axis of the oocyte and embryo. Mutations in these genes block homology-dependent RNA cleavage and RISC assembly in ovary lysates, RNAi-based gene silencing during early embryogenesis, rasiRNA production, and retrotransposon and Stellate silencing. Mutations in dcr-2 and ago-2 genes, by contrast, block siRNA function, but they do not disrupt the rasiRNA pathway or embryonic axis specification. The rasiRNA pathway thus appears to be required for embryonic axis specification. However, the function of rasiRNAs in the axis specification pathway has not been previously established (Klattenhoff, 2007).
Cytoskeletal polarization, morphogen localization, and eggshell patterning defects associated with armi and aub are efficiently suppressed by mnk and mei-41, which respectively encode Chk2 and ATR kinase components of the DNA damage signaling pathway. In addition, armi and aub mutants accumulate γ-H2Av foci characteristic of DNA DSBs and trigger Chk2-dependent phosphorylation of Vas, an RNA helicase required for posterior and dorsal-ventral specification. Mutations in spn-E also disrupt the rasiRNA pathway, trigger axis specification defects, and lead to germline-specific accumulation of γ-H2Av foci. Significantly, the mnk and mei-41 mutations do not suppress Stellate or HeT-A overexpression, indicating that axis specification does not directly require rasiRNA-dependent gene silencing. Based on these findings, it is concluded that the rasiRNA pathway suppresses DNA damage signaling in the female germline, and that mutations in this pathway disrupt axis specification by activating an ATR/Chk2 kinase pathway that blocks microtubule polarization and morphogen localization in the oocyte (Klattenhoff, 2007).
The cause of DNA damage signaling in armi, aub, and spn-E mutants remains to be established. In wild-type ovaries, γ-H2Av foci begin to accumulate in region 2 of the germarium, when the Spo11 nuclease (encoded by the mei-W68 gene) initiates meiotic breaks. The axis specification defects associated with DNA DSB repair mutations are efficiently suppressed by mei-W68 mutations, indicating that meiotic breaks are the source of DNA damage in these mutants. The axis specification defects and γ-H2Av focus formation associated with armi, by contrast, are not suppressed by mei-W68. mei-W68 double mutants with aub or spn-E have not been analyzed, but this observation strongly suggests that meiotic DSBs are not the source of DNA damage in rasiRNA pathway mutations. Retrotransposon silencing is disrupted in armi, aub, and spn-E mutants, and transcription of LINE retrotransposons in mammalian cells leads to DNA damage and DNA damage signaling. Loss of retrotransposon silencing could therefore directly induce the DSBs in rasiRNA pathway mutants. However, DNA damage can also lead to loss of retrotransposon silencing. Mutations in the rasiRNA pathway could therefore disrupt DNA repair and thus induce DNA damage, which, in turn, induces loss of retrotransposon silencing. Finally, the HeT-A retrotransposon is associated with telomeres, and overexpression of this element could reflect a loss of telomere protection and could damage signaling by chromosome ends in the rasiRNA pathway mutants. The available data do not distinguish between these alternatives (Klattenhoff, 2007).
In mouse, the piwi-related Argonauts Miwi and Mili bind piRNAs, 30 nt RNAs derived primarily from a single strand that appear to be related to rasiRNAs. Mutations in these genes disrupt spermatogenesis and lead to germline apoptosis, which can be induced by DNA damage signaling. Mammalian piRNAs and Drosophila rasiRNAs may therefore serve similar functions in suppressing a germline-specific DNA damage response (Klattenhoff, 2007).
Gametogenesis is a highly regulated process in all organisms. In Drosophila, a meiotic checkpoint which monitors double-stranded DNA breaks and involves Drosophila ATR and Chk2 coordinates the meiotic cell cycle with signaling events that establish the axis of the egg and embryo. Checkpoint activity regulates translation of the transforming growth-factor-alpha-like Gurken signaling molecule which induces dorsal cell fates in the follicle cells. Mutations in the Drosophila gene cutoff (cuff) affect germline cyst development and result in ventralized eggs as a result of reduced Grk protein expression. Surprisingly, cuff mutations lead to a marked increase in the transcript levels of two retrotransposable elements, Het-A and Tart. Small interfering RNAs against the roo element are still produced in cuff mutant ovaries. These results indicate that Cuff is involved in the rasiRNA pathway and most likely acts downstream of siRNA biogenesis. The eggshell and egg-laying defects of cuff mutants are suppressed by a mutation in chk2. Mutations in aubergine (aub), another gene implicated in the rasiRNA pathway, are significantly suppressed by chk2 mutation. These results indicate that mutants in rasiRNA pathways lead to elevated transposition incidents in the germline, and that this elevation activates a checkpoint that causes a loss of germ cells and a reduction of Gurken protein in the remaining egg chambers (Chen, 2007).
cutoff (cuff) mutations were isolated in a large-scale female-sterile screen of Drosophila, and one additional allele was identified in a screen for P element insertions. Females transheterozygous for cuff alleles lay eggs with various degrees of ventralization. The dorsoventral polarity of the egg and embryo depends on the levels of the Gurken (Grk) ligand, which is produced and secreted by the germline and activates the EGF receptor (Egfr) in the overlying follicle cells. To determine whether Grk-Egfr signaling was affected, the grk expression pattern was analyzed in a strong cuff mutant background. In wild-type egg chambers at stage 9 of oogenesis, grk RNA becomes restricted to the future dorsal-anterior side of the oocyte and forms a cap around the oocyte nucleus. Grk protein is translated from the tightly localized RNA and is also spatially restricted to the membrane overlying the oocyte nucleus. cuff mutants do not significantly disrupt grk RNA localization. However, in many mid-stage egg chambers, the Grk protein level is greatly reduced, such that between 10% and 40% of the egg chambers contain no detectable Gurken protein at all, consistent with defects in grk translation. In wild-type egg chambers by stage 3 of oogenesis, the oocyte nucleus forms a compact structure termed the karyosome. In cuff mutants, karyosome formation is affected in 10%–20% of the egg chambers, in which the DNA assumes various shapes and is often found in separate clumps (Chen, 2007).
Genomic database searches identified the yeast gene Rai1 as a homolog of cuff. This gene has been shown to interact with a nuclear 5′–3′ exoribonuclease (Rat1) that is involved in rRNA processing and transcriptional termination. A cytoplasmic homolog of Rat1, Xrn1, has also been described in yeast and vertebrates and has been implicated in mRNA regulation that is localized to cytoplasmic processing bodies. An HA-tagged Drosophila Rat1 (CG10354) construct was generated and overexpressed with a fully functional FLAG-tagged Cuff in the ovary. Using immunoprecipitation (IP), no any interaction between the exoribonuclease and Cuff was detected. It is therefore possible that Drosophila Rat1 is not the correct partner for Cuff. This is also supported by the observation that overexpressed Rat1, as expected, localizes to the nucleus, whereas overexpressed Cuff localizes to the cytoplasm. It was not possible to to detect endogenous Cuff protein with an anti-Cuff antibody, presumably because of low levels of protein expression. However, overexpressed HA-tagged Cuff partially colocalizes with perinuclear puncta in the nurse cells in younger egg chambers. A similar localization pattern has been described for the helicase Vasa, and it was found that Cuff partially colocalizes with Vasa in the cytoplasm. The perinuclear localization pattern, also designated as nuage in the germ cells and related to mammalian P bodies, has been described for components of the RNAi machinery and for genes involved in RNA degradation (Chen, 2007).
Given the eggshell ventralization and the karyosome defect, cuff has mutant phenotypes similar to those of a group of mutants known as the spindle-class genes. Several members of this group encode DNA-repair genes, for instance, spindle(spn) B (XRCC3) and okra (DmRad54). In these mutants, the DSBs that are created during recombination persist and thus activate Chk2 through the Drosophila ATR homolog mei-41. The activity of these kinases negatively regulates the translation of Grk, possibly through a posttranslational modification of Vasa; this modification in turn leads to ventralization of the eggs laid by mutant females. Inactivation of the checkpoint, for instance through mutations in chk2 or mei-41, suppresses the eggshell defects of the spindle-class DNA-repair mutants. In addition, in double mutants of the DNA-repair genes and the genes required for initiating the DSBs, such as c(3)g, mei-W68, or mei-P22, DSBs are not generated; therefore, the checkpoint is not activated, and the eggshell morphology is normal, even in the presence of the repair mutants. To check whether Cuff is involved in the repair of DSBs initiated in prophase of meiosis I, mei41;cuff and cuff;c(3)g double mutants were generated. Although both mutations suppress the eggshell defect of spnB or okra to wild-type morphology, neither suppresses the eggshell defect of cuff, indicating that Cuff does not function in the meiotic repair pathway. Surprisingly, however, a mutation in chk2 partially suppresses the eggshell defect of cuff as well as the defects in cyst development. chk2 cuff double mutants lay mostly wild-type-looking eggs, and have cysts with highly branched fusomes in the germaria, and the females lay more eggs than cuff single mutants, although the rescue is not 100%. In certain allelic combinations, it was possible to observe a dominant effect in the chk2 suppression of the cuff eggshell defect (Chen, 2007).
Previous work has suggested the DNA-repair checkpoint, upon activation, regulates Grk translation through a posttranslational modification of Vas, and that this modification results in slower Vas electrophoretic mobility. To address whether the checkpoint acts in the same manner in cuff mutants, Vas mobility was assayed in cuff mutant combinations. In cuff mutants, Vas migrates slightly more slowly than wild-type control, consistent with the modification seen in the DNA-repair mutants. The mobility is not changed in mei41;cuff double-mutant background, which is consistent with the fact that mei41 mutants do not significantly suppress the eggshell phenotype of cuff. However, Vasa mobility is restored to wild-type in the chk2 cuff double mutant. This suggests that although the checkpoint is activated through a different sensing mechanism in cuff mutants, upon activation the checkpoint involves Chk2 and acts through similar pathways to affect Gurken translation in the egg chambers that escape the early arrest (Chen, 2007).
Several of the spindle-class genes, such as spnE and aub, have been shown to be essential components of the RNAi machinery. Because overexpressed Cuff has a perinuclear localization, whether Cuff might also be required in RNAi pathways was tested. Recently, a specific branch of the RNAi pathways, that involving the repeat-associated small interfering RNA (rasiRNA), has been implicated in the control of retrotransposable elements in the Drosophila germline. Using qRT-PCR, the level of Het-A and Tart, two of the retrotransposable elements responsible for maintaining the telomere in Drosophila, was studied. Previously, it has been shown that in spnE and aub mutants, Het-A and Tart transcripts are derepressed and that this derepression results in a marked elevation in the transcripts level. Compared with heterozygous controls, spnE homozygous mutant females have Het-A and Tart transcript levels that are upregulated by approximately 10-fold, whereas in aub mutants only Het-A is significantly upregulated. In cuff mutant females, the elevation for both transcripts is even more pronounced. Compared with Het-A levels in the heterozygous control, those in cuff mutants are elevated more than 800-fold, and Tart transcript levels increase by more than 20-fold. Transposable elements are normally silenced in the Drosophila germline by the rasiRNA pathway; this silencing process appears to be strongly impaired in the cuff mutants. Whether the upregulation of the transposable elements in cuff mutants could be due to a reduction in the level of rasiRNAs was further tested. However, it was found that the levels of the 25-nt-long roo interfering RNA are not reduced in cuff mutant ovaries, in contrast to ovaries mutant for aub. This indicates that Cuff is not involved in the biogenesis of the rasiRNAs and points to a function for Cuff in the actual silencing process. Because high transcript levels of the retrotransposable elements in the germline are correlated with elevated transposition incidents, which in turn lead to decreased chromosomal integrity, it is possible that such chromosomal defects activate the checkpoint involving chk2. In addition, because transposable elements are involved in the regulation of chromatin structure, the existence of a chromatin checkpoint that involves Chk2 activity is also possible. Once Chk2 is activated, either by the mutants in DNA-repair pathways or by RNAi components such as Cuff and Aub, Chk2 activity leads to posttranslational Vas modification and a negative regulation of Grk translation. However, unlike DNA repair mutations, cuff and aub mutations are not suppressed to wild-type morphology and fecundity by mutations in mei41, suggesting that they activate the checkpoint through a different, or additional, sensing mechanism. Furthermore, most of the mutants in DNA-repair pathways do not cause defects in cyst development or germline stem cell maintenance. These additional defects seen in cuff mutants could be due to the timing of checkpoint activation. DNA-repair mutants activate the meiotic checkpoint during meiotic prophase, which initiates after the formation of the 16 cell cyst, whereas cuff and aub mutants appear to act earlier in oogenesis, given that they already have effects during the mitotic cycles preceding the onset of meiosis. The transposon-activated checkpoint leads not only to translational arrest of Grk, but also to mitotic cell-cycle arrest. Many of the arrested germline cells and cysts eventually undergo apoptosis, leading to gradual loss of both germline stem cells and developing cysts in cuff mutants. However, germ cells that escape the early arrest encounter the second checkpoint effect, which leads to a reduction in Gurken translation (Chen, 2007).
It was recently discovered that there are a large number of different small RNAs generated in the germline of both mammals and flies. Many of them are associated with Piwi family proteins, and most have no known functions. Because the germline represents a special cell type that will pass its DNA on to future progeny, it is possible that selfish elements have developed a high propensity to remobilize in the germline. Furthermore, it is very plausible that in most organisms the germline has evolved sophisticated mechanisms to defend itself against such transposable elements. Many of the small RNAs found in the germline may be involved in the defense against transposable elements, as well as in the regulation of transcription and translation. When the machinery to generate these small silencing RNAs or the effector complexes that are responsible for transcript degradation are disrupted, chromosomal integrity might be at risk. This study has found that a checkpoint involving the conserved Chk2 kinase monitors the RNAi-mediated events in the Drosophila germline and ensures the genomic integrity of the progeny. Chk2 therefore acts as a surveillance factor for both transposon-generated problems as well as DNA-repair problems in the germline. Whether Chk2 has a similar role in the mammalian germline will be interesting to investigate in the future (Chen, 2007).
Abad, J. P., de Pablos, B., Osoegawa, K., de Jong, P. J., Martin-Gallardo, A., and Villasante, A. (2004a). Genomic analysis of Drosophila melanogaster telomeres: Full-length copies of HeT-A and TART elements at telomeres. Mol. Biol. Evol. 21: 1613-1619. 15163766
Abad, J. P., de Pablos, B., Osoegawa, K., de Jong, P. J., Martin-Gallardo, A., and Villasante, A. (2004b). TAHRE, a novel telomeric retrotransposon from Drosophila melanogaster, reveals the origin of Drosophila telomeres. Mol. Biol. Evol. 21: 1620-1624. 15175413
Aravin, A. A., et al. (2001). Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster germline. Curr. Biol. 11: 1017-1027. 11470406
Aravin, A. A., Klenov, M. S., Vagin, V. V., Bantignies, F., Cavalli, G. and Gvozdev, V A. (2004). Dissection of a natural RNA silencing process in the Drosophila melanogaster germ line. Mol. Cell. Biol. 24(15): 6742-50. Medline abstract: 15254241
Aravin, A. A., Hannon, G. J. and Brennecke, J. (2007). The Piwi-piRNA pathway provides an adaptive defense in the transposon arms race. Science 318: 761-764. Medline abstract: 17975059
Bagchi, M. K., Chakravarty, I., Ahmad, M. F., Nasrin, N., Banerjee, A. C., Olson, C. and Gupta, N. K. (1985). Protein synthesis in rabbit reticulocytes. A study of the roles of Co- eIF-2, Co-eIF-2A80, and GDP in peptide chain initiation. J. Biol. Chem. 260: 6950-6954. 3997855
Biessmann, H., Champion, L. E., O'Hair, M., Ikenaga, K., Kasravi, B. and Mason, J. M. (1992a). Frequent transpositions of Drosophila melanogaster HeT-A transposable elements to receding chromosome ends. EMBO J. 11: 4459-4469. 1330538
Biessmann, H., Valgeirsdottir, K., Lofsky, A., Chin, C., Ginther, B., Levis, R. W. and Pardue, M. L. (1992b). HeT-A, a transposable element specifically involved in 'healing' broken chromosome ends in Drosophila melanogaster. Mol. Cell. Biol. 12: 3910-3918. 1324409
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
Cerutti, L., Mian, N. and 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
Chakravarty, I., Bagchi, M. K., Roy, R., Banerjee, A. C. and Gupta, N. K. (1985). Protein synthesis in rabbit reticulocytes. Purification and properties of an Mr 80,000 polypeptide (Co-eIF-2A80) with Co-eIF-2A activity. J. Biol. Chem. 260: 6945-6949. 3888988
Chen, Y., Pane, A. and Schupbach, T. (2007). cutoff and aubergine mutations result in retrotransposon upregulation and checkpoint activation in Drosophila. Curr. Biol. 17(7): 637-42. Medline abstract: 17363252
Findley, S. D., Tamanaha, M., Clegg, N. J. and Ruohola-Baker, H. (2003). Maelstrom, a Drosophila spindle-class gene, encodes a protein that colocalizes with Vasa and RDE1/AGO1 homolog, Aubergine, in nuage. Development 130: 859-871 . 12538514
Golubovsky, M. D., Konev, A. Y., Walter, M. F., Biessmann, H. and Mason, J. M. (2001). Terminal retrotransposons activate a subtelomeric white transgene at the 2L telomere in Drosophila. Genetics 158: 1111-1123. 11454760
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
Gunkel, N., Yano, T., Markussen, F.-H., Olsen, L. C. and Ephrussi, A. (1998). Localization-dependent translation requires a functional interaction between the 5' and 3' ends of oskar mRNA. Genes Dev. 12: 1652-1664.
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: 2823-2832. 11526087
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
Kahn, T., Savitsky, M. and Georgiev, P. (2000). Attachment of HeT-A sequences to chromosomal termini in Drosophila melanogaster may occur by different mechanisms. Mol. Cell. Biol. 20: 7634-7642. 11003659
Kennerdell, J. R. Yamaguchi, S. and Carthew, R. W. (2002). RNAi is activated during Drosophila oocyte maturation in a manner dependent on aubergine and spindle-E. Genes Dev. 16: 1884-1889. 12154120
Klattenhoff, C., et al. (2007). Drosophila rasiRNA pathway mutations disrupt embryonic axis specification through activation of an ATR/Chk2 DNA damage response. Dev. Cell 12(1): 45-55. Medline abstract: 17199040
Kogan, G. L., Tulin, A. V., Aravin, A. A., Abramov, Y. A., Kalmykova, A. I., Maisonhaute, C. and Gvozdev, V. A. (2003). The GATE retrotransposon in Drosophila melanogaster: Mobility in heterochromatin and aspects of its expression in germline tissues. Mol. Genet. Genomics 269: 234-242. 12756535
Levis, R. W., Ganesan, R., Houtchens, K., Tolar, L.A. and Sheen, F. M. (1993). Transposons in place of telomeric repeats at a Drosophila telomere. Cell 75: 1083-1093. 8261510
Mikhailovsky, S., Belenkaya, T. and Georgiev, P. (1999). Broken chromosomal ends can be elongated by conversion in Drosophila melanogaster. Chromosoma 108: 114-120. 10382073
Pardue, M. L. and DeBaryshe, P. G. 2003. Retrotransposons provide an evolutionary robust non-telomerase mechanism to maintain telomeres. Annu. Rev. Genet. 37: 485-511. 14616071
Perrini, B., Piacentini, L., Fanti, L., Altieri, F., Chichiarelli, S., Berloco, M., Turano, C., Ferraro, A. and Pimpinelli, S. (2004). HP1 controls telomere capping, telomere elongation, and telomere silencing by two different mechanisms in Drosophila. Mol. Cell 15: 467-476. 15304225
Rashkova, S., Karam, S. E., Kellum, R. and Pardue, M. L. (2002). Gag proteins of the two Drosophila telomeric retrotransposons are targeted to chromosome ends. J. Cell Biol. 159: 397-402. 12417578
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
Roy, A. L., Chakrabarti, D., Datta, B., Hileman, R. E. and Gupta, N. K. (1988). Natural mRNA is required for directing Met-tRNA(f) binding to 40S ribosomal subunits in animal cells: involvement of Co-eIF-2A in natural mRNA-directed initiation complex formation. Biochemistry 27: 8203-8209. 3233204
Roy, R., Ghosh-Dastidar, P., Das, A., Yaghmai, B. and Gupta, N. K. (1981). Protein synthesis in rabbit reticulocytes. Co-eIF-2A reverses mRNA inhibition of ternary complex (Met-tRNAf.eIF-2.GTP) formation by eIF-2. J. Biol. Chem. 256: 4719-4722. 6153053
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
Schmidt, A., Palumbo, G., Bozzetti, M. P., Tritto, P., Pimpinelli, S. and Schafer, U. (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: 749-760. 9927466
Schüpbach, T. and Wieschaus, E. (1991). Female sterile mutations on the second chromosome of Drosophila melanogaster. II. Mutations blocking oogenesis or altering egg morphology. Genetics 129: 1119-1136. 1783295
Savitsky, M., Kravchuk, O., Melnikova, L. and Georgiev, P. (2002). Heterochromatin protein 1 is involved in control of telomere elongation in Drosophila melanogaster. Mol. Cell. Biol. 22: 3204-3218. 11940677
Savitsky, M., Kwon, D., Georgiev, P., Kalmykova, A. and Gvozdev, V. (2006). Telomere elongation is under the control of the RNAi-based mechanism in the Drosophila germline. Genes Dev. 20(3): 345-54. 16452506
Sheen, F. M. and Levis, R. W. (1994). Transposition of the LINE-like retrotransposon TART to Drosophila chromosome termini. Proc. Natl. Acad. Sci. 91: 12510-12514. 7809068
Snee, M. J. and Macdonald. P. M. (2004). Live imaging of nuage and polar granules: evidence against a precursor-product relationship and a novel role for Oskar in stabilization of polar granule components. J. Cell Sci. 117(Pt 10): 2109-20. 15090597
Stapleton, W., Das, S. and McKee, B. D. (2001). A role of the Drosophila homeless gene in repression of Stellate in male meiosis. Chromosoma 110: 228-240. 11513298
Styhler, S., Nakamura, A. and Lasko, P. (2002). VASA localization requires the SPRY-domain and SOCS-box containing protein, GUSTAVUS. Dev. Cell 3: 865-876. 12479811
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
Wilson, J. E., Connell, J. E. and Macdonald, P. M. (1996). aubergine enhances oskar translation in the Drosophila ovary. Development 122: 1631-1639. 8625849
Zou, C., Zhang, Z., Wu, S. and Osterman, J. C. (1998). Molecular cloning and characterization of a rabbit eIF2C protein. Gene 211: 187-194. 9602122
date revised: 16 January 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.