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Corresponding author: Haifan Lin, Duke University Medical Center, Box 3709, 412 Nanaline Duke Bldg., Durham, NC 27710., h.lin{at}cellbio.duke.edu (E-mail)
Communicating editor: F. S. HAWLEY
 | ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
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The Drosophila piwi gene is the founding member of the only known family of genes whose function in stem cell maintenance is highly conserved in both animal and plant kingdoms. piwi mutants fail to maintain germline stem cells in both male and female gonads. The identification of piwi-interacting genes is essential for understanding how stem cell divisions are regulated by piwi-mediated mechanisms. To search for such genes, we screened the Drosophila third chromosome (
36% of the euchromatic genome) for suppressor mutations of piwi2 and identified six strong and three weak piwi suppressor genes/sequences. These genes/sequences interact negatively with piwi in a dosage-sensitive manner. Two of the strong suppressors represent known genes—serendipity-
and similar, both encoding transcription factors. These findings reveal that the genetic regulation of germline stem cell division involves dosage-sensitive mechanisms and that such mechanisms exist at the transcriptional level. In addition, we identified three other types of piwi interactors. The first type consists of deficiencies that dominantly interact with piwi2 to cause male sterility, implying that dosage-sensitive regulation also exists in the male germline. The other two types are deficiencies that cause lethality and female-specific lethality in a piwi2 mutant background, revealing the zygotic function of piwi in somatic development.
STEM cells possess the unique abilities to self-renew and to produce numerous differentiated daughter cells. Endowed with these properties, stem cells play a central role in the proliferative and regenerative capabilities of tissues. Understanding mechanisms that control stem cell division is of fundamental biological and medical significance.
Germline stem cells in the Drosophila ovary have been an excellent model for studying the mechanism of stem cell division via combined genetic, cell-biological, and molecular approaches. The Drosophila ovary is composed of 16–18 functional units called ovarioles. Each ovariole contains two or, less frequently, three germline stem cells located at its apical tip in a specialized structure called the germarium (Fig 1A, Fig B, and Fig C'). These stem cells have been positively identified by genetic, laser ablation, and cell-biological analyses (
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Research in recent years has revealed both inter- and intracellular mechanisms involved in regulating the division of germline stem cells in the Drosophila ovary (reviewed in
piwi is the founding member of the piwi family (a.k.a. argonaute family, PPD family) of genes whose function in stem cell maintenance is well conserved in both animal and plant kingdoms (reviewed in
The piwi family genes are also involved in other developmental processes. In Drosophila, a gene closely related to piwi, aubergine/sting, is involved in pole cell formation and the translational regulation of such downstream targets as oskar and gurken (
Since piwi family genes are so widely conserved and play essential roles in stem cell division and other developmental processes, identification of piwi-interacting genes is a key step toward understanding the regulation of stem cell division and other piwi-mediated mechanisms. At present, only Yb has been linked to piwi (
We report here the identification of six strong suppressor and three weak suppressor genes of piwi. Two of these strong suppressors represent known genes: serendipity- (sry-) and similar. The sry- and similar genes both encode transcription factors (
| MATERIALS AND METHODS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
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Drosophila strains and culture:
All Drosophila strains were grown at room temperature on yeast-containing cornmeal/molasses medium unless otherwise indicated. Canton-S and Oregon-R strains were used as wild-type flies. The hh-lacZ flies carry an enhancer trap insert in hh as initially characterized by
piwi mutants:
A P-element insertional mutation of piwi, piwi2, was described in
Individual suppressor mutants:
The three sry- alleles tested in this study, sry-12, sry-SF1, and sry-SF2, were kindly provided by Alain Vincent and are described in
Deficiencies:
Laurel Raftery kindly provided the Df(3R)KpnA,ca1,awdK deficiency stock. The following deficiency stocks were provided by the BDSC (with the stock number in parentheses): Df(3R)B81 P{ry+t7.2 = RP49}F2-80A e1 (3546); Df(3R)fafBP st1 (1011); Df(3L)Aprt-1 ru1 h1 (600); Df(3L)R-G5 rhove-1 (2400); Df(3L)Aprt-32 (5411); Df(3L)R (57); Df(3L)R-G7 rhove-1 (2400); Df(3R)L127 (3547); Df(3R)X3F P{ry+t7.2 = RP49}A3-84F e1 (2353); Df(3R)tll-e ca1 (5415); Df(3R)04661 (5415); Df(3L)ri-79C (3127); Df(3L)rdgC-co2 th1 st1 in1 kniri-1 pp (2052); Df(3L)31A (3627); Df(3L)66C-G28 (1541); Df(3L)pbl-X1 (1420); Df(3L)kto2 (3617); Df(3L)VW3 (3000); Df(3L)XS533 (5126); Df(3L)XS572 (5583); Df(3R)ea kniri-1 pp (383); Df(3R)P115 e11 (1467); Df(3R)C4 p* (3071); Df(3R)DG2 (4431); Df(3R)2-2 (3688); Df(3R)p712 red1 e1 (1968); Df(3R)23D1 ry506 (2586); Df(3R)p40 red1 e1 (1945); Df(3R)ry506-85C (1534); Df(3R)ry27 Dfd1 cu1 kar1 (480); and Df(3R)ry615 (3007). Unless otherwise mentioned, most non-piwi2 interacting deficiencies in the Appendix are also from the Bloomington Drosophila Stock Center, included in the deficiency kit. All of the stocks mentioned in this article have been previously mentioned in FlyBase (http://flybase.bio.indiana.edu).
Deficiency screens:
A five-generation scheme was used in pilot screens to produce piwi2/piwi2; Df/+ females, which were then tested for fertility and ovarian germline stem cell phenotypes, as described below. In these screens, neither the piwi nor the deficiency chromosomes were marked with dominant mutations; thus both the second and third chromosomes needed to be balanced or marked in both stocks to identify piwi2/piwi2; Df/+ females. This resulted in very few lines reaching the F4 cross and those crosses yielding very few progeny of the target phenotype. To achieve a more efficient scheme, the piwi2 chromosome was recombined with b cn If to produce a piwi2 chromosome dominantly marked with If. These stocks were tested for retention of the piwi2 mutation by phenotypic and Southern analysis (described below). No change was seen in the piwi2 mutant phenotype, suggesting that If did not interact with the piwi2 mutation. Therefore, the piwi2 If/CyO line designated W was then used for all further crosses (unless otherwise specified), reducing the cross scheme to two generations (Fig 2).
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The screen in Fig 2 started with two vials for each experimental stock, with each containing 5–6 piwi2 If/CyO; Ly1 or TM8/+ virgin females crossed to 5–6 deficiency/balancer males ordered from the deficiency kit at the Bloomington Drosophila Stock Center. In the F1 generation we collected 10–12 piwi2 If/+; deficiency/Ly1 or TM8 males and divided them into two vials each with 5–6 piwi2 If/CyO; +/+ virgin females. The parents in these two vials were transferred many times to optimize the numbers of F2 progeny. We collected 1- to 2-day-old piwi2 If/piwi2 If; deficiency/+ females and crossed them to 5 wild-type males to check fertility. After 2–3 days we dissected the target females and checked for suppression of the piwi2 germline stem cell maintenance defect.
P-element excision:
To create a stronger deletion allele of P{ry+t7.2 = SRS3.9}3, we mobilized the P element. This was accomplished by crossing ry506 P{ry+t7.2 = SRS3.9}3 females to ry506 Sb1 P{ry+ 
2-3}[99B]/TM6 Ubx130 e1 males and collecting ry506 P{ry+t7.2 = SRS3.9}3/ry506 Sb1 P{ry+ 2-3}[99B] males. These single males (150) were then crossed to D1 red1 e1/TM3 ryRK Sb1 Ser1 females. From each cross, two Sb and phenotypically ry- males were collected and made into 300 stocks. The P-excision chromosomes in these stocks were examined for recessive lethality and sterility.
Genomic Southern blot analysis:
Standard molecular biology techniques, such as DNA preparation, cloning, and Southern blotting, were performed as described in
Inverse PCR mapping of the P-element insertion sites:
DNA preparation, cloning, inverse PCR, and other standard molecular biology techniques were performed as described in
We found that parts of the P{ry+t7.2 = SRS3.9}3 flanking genomic DNA sequence are identical to parts of the 5' ends of at least 13 independently isolated expressed sequence tags (ESTs) identified by the Berkeley Drosophila Genome Project (BDGP) and clustered with at least 27 other ESTs to form clot 2858 (
Immunocytochemistry and immunofluorescence microscopy:
Wild-type and mutant ovaries from adult females were dissected, fixed, and stained as described in
| RESULTS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
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Six regions on the third chromosome suppress the piwi2 phenotype:
Using a two-generation cross scheme (Fig 2), we screened deficiencies on the third chromosome for genes that interact with piwi2 and are involved in germline stem cell division in the Drosophila ovary (see MATERIALS AND METHODS). Each of these deficiencies on average deletes <1% of the genome, corresponding to 100 or fewer genes. We limited our search to deficiencies that were suppressors of piwi2 for two reasons. First, the piwi2 mutant itself fails to maintain germline stem cell division, rendering an enhancer screen for a more severe germline stem cell phenotype impractical. Second, the suppressor screen, advantageously, avoids nonspecific enhancers. We did, however, identify a few deficiencies that led to sterility and haplo-lethality in piwi2 mutant backgrounds; we further describe those interactions in three of the following sections.
Pilot screens were used to test the validity of the piwi2 allele as an appropriate starting point for our piwi dominant suppressor screen (see MATERIALS AND METHODS). We chose this allele for three reasons. First, while both piwi1 and piwi2 cause strong defects in germline stem cell division in adult female ovaries, the piwi1 mutant has additional effects on the initial organization of the third instar gonad (
We judged suppression of piwi2 by the ability of the deficiency (or, later, of the mutation) to restore germline stem cell division in the ovary. To examine this restoration, we used Nomarski optics and antibodies that mark germline cells and their subcellular structures. Since germline stem cell division gives rise to a germline cyst in the germarium, which subsequently develops into an egg chamber in the ovariole, the number of germline cysts and egg chambers per ovariole indicates the number of germline stem cell divisions that have been restored (Fig 1A and Fig C). Because the two or three germline stem cells in a piwi mutant germarium frequently differentiate without renewal, the piwi2 mutant ovarioles often contain a rudimentary germarium plus only one egg chamber (Fig 1D and Fig D'). If an ovariole contains an apparently normal-looking germarium and at least three egg chambers, it suggests that the germline stem cell division defect in this ovariole is significantly suppressed. The following markers were used to characterize suppression more precisely. DAPI was used to mark nuclei, anti-Vasa antibodies (
In total, we screened 109 deficiencies, which uncovered 87% of the third chromosome, or 36% of the whole euchromatic genome, for dominant suppression of piwi2 (Fig 3; Appendix). Within this 36% of the genome, we identified six regions defined by deficiencies as suppressing (Fig 3). Further analysis of these deficiencies is reported below (Table 1; Fig 3).
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Df (3R)L127:
This deficiency (99B5–6 to 99E4–F1) suppresses the germline stem cell defect of piwi2 in 43% of the females (Table 1; Fig 3). Because this deficiency overlaps with another suppressing deficiency, Df(3R)B81 (99C8–100F5), it is likely that the region responsible for suppression is 99C8–99E4/F1. We then tested available mutations in 14 genes (Table 1; Fig 3; Appendix) in this region for their suppression of the piwi2 phenotype. Mutations in 5 genes, P{SRS3.9}3, l(3)99Da, l(3)99Ea, sry-, and l(3)j11B7 (similar), showed strong suppression, and mutations in two others, l(3)99De and l(3)99Dg, displayed weak suppression (Table 1; Fig 3). It is interesting to note that, in this case, one suppressor region yielded multiple piwi-suppressing genes involved in the self-renewing division of germline stem cells in the Drosophila ovary.
Df(3R)fafBP:
This deficiency (100D–100F5) suppresses the germline stem cell defect of piwi2 in 47% of the females (Table 1; Fig 3). This deficiency also overlaps with the suppressing deficiency Df(3R)B81 (99C8–100F5) as well as Df(3R)04661 (100D2–100F5; Table 1; Fig 3). However, it is separable from the previous suppressor region of 99C8–99E4/F1, because Df(3R)tll-e (100A2–100C2/3) does not suppress piwi2 (see Appendix). We tested 17 mutations (Table 1; Fig 3; Appendix) that are uncovered by Df(3R)fafBP and found that a P-insertional mutation, P(wA)4-4, strongly suppressed piwi2 (Table 1; Fig 3 and Fig 4). Thus, we narrowed down this suppressor region to a single P-disrupted sequence.
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Df(3L)66C-G28:
This deficiency (66B8/9–66C9/10) suppresses the germline stem cell defect of piwi2 in 42% of the females (Table 1; Fig 3). Because an adjacent deficiency, Df(3L)pbl-X1 (65F3–66B10), did not suppress piwi2, the suppressor interval should be from 66B10 to 66C9/10 (Fig 3). This suppressor interval was not tested further for its suppression activity.
Df(3L)kto2:
This deficiency (76B1/2–76D5) restores germline stem cell division in 27% of piwi2 mutant females (Table 1; Fig 3). Thus, we classified Df(3L)kto2 as a weak suppressor of piwi2. Ten individual mutations and overlapping deficiencies, Df(3L)VW3 (76A3–76B2), Df(3)XS-533 (76B4–77B), and Df(3L)XS572 (76B6–77C1), were tested; none of them suppressed the piwi2 defects in germline stem cell maintenance (Appendix). Despite this, the suppressor in this deficiency may still reside in 76B2–76B4. Alternatively, the suppression could be caused by the effects of the Df(3L)kto2 breakpoints or an unrelated mutation elsewhere on the Df(3L)kto2 chromosome.
Df(3L)Aprt-1:
This deficiency (62A10/B1–62D2/5) suppressed the piwi2 defect in 50% of the females with almost complete penetrance at the ovariolar level (Table 1; Fig 3). To narrow down the region of suppression, we tested four overlapping deficiencies [Df(3L)R-G5 (62A10/B1–62C4/D1), Df(3L)Aprt-32 (62B1–62E3), Df([3L)R (62B7–62B12), and Df(3L)R-G7 (62B8/9–62F2/5)] and six P-element insertional mutations (see Appendix) in the region. None of them suppressed the piwi2 mutation. Since these deficiencies collectively uncover the region defined by Df(3L)Aprt-1, the suppression effect of Df(3L)Aprt-1 may be caused by one of the two breakpoints of Df(3L)Aprt-1 or due to a background mutation outside of the deficiency. Alternatively, it could suggest that the enhancing effect of many haploid genes in these deficiencies cancels the effect of a specific suppressor (see DISCUSSION).
Df(3L)ri-79C:
This deficiency (77B/C–77F/78A) restores germline stem cell division in 44% of the piwi2 mutant females (Table 1; Fig 3). Overlapping deficiencies uncovering 77A1–77D1 (Df[3L]rdgC-co2) and 78A–78E (Df[3L]31A) do not suppress piwi2, narrowing down the potential suppression region to 77D1–78A (Fig 3). None of the five tested mutations (see Appendix) within this region showed suppression. Further mutagenesis in this region may allow the identification of a potential suppressor gene(s), as may also be the case with Df(3L)Aprt-1 and Df(3L)kto2 (see above).
Deficiencies that cause the sterility of heterozygous piwi2 males:
In our screen, we found enhancers of the piwi2 phenotype by identifying deficiencies that cause the sterility of piwi2/+ males. We identified four adjacent deficiencies within cytological regions 88–90 [Df(3R)ea (88E7/13–89A1), Df(3R)P115 (89B7/8–89E7/8), Df(3R)C4 (89E–90A), and Df(3R)DG2 (89E1/F4–91B1/B2)] that cause such trans-heterozygous male sterility (Fig 3; also see Fig 2 for the generation of trans-heterozygous F1 males). These deficiencies may thus contain genes that interact with piwi in a dosage-dependent mechanism and are positively required for germline stem cell maintenance at least in males.
Deficiencies that are haplo-lethal in a piwi2 mutant background:
We also identified three deficiencies that are haplo-lethal in the piwi2 mutant background: Df(3R)2-2 (81F–82F10/11 or 83A), Df(3R)p40 (84E8/9–85B6), and Df(3R)23D1 (93F–94F; Table 2; Fig 3). Of >100 piwi2 mutant progeny from the suppressor cross to each of the deficiencies (Fig 2), none were found to contain the deficiency (Table 2). The haplo-lethality of Df(3R)p40 in the piwi2/piwi2 background was confirmed by its overlapping deficiency, Df(3R)p712 (84D4/6–85B6). Three additional deficiency crosses yielded only very few deficiency-carrying progeny and these deficiencies were thus classified as semi-haplo-lethal in a piwi2 mutant background (Table 2; Fig 3). These two classes of deficiencies may uncover genes that interact with piwi in a dosage-sensitive manner required for somatic development. Although many selected individual mutations in these six regions were checked for lethality and suppression of piwi2 (Appendix), the available overlapping deficiencies and individual mutations did not allow us to identify individual genes responsible for the haplo-lethal interactions. Regardless of the cause of the haplo-lethal interaction, the discovery of these deficiencies reveals the involvement of the zygotic piwi activity in somatic development.
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Female-specific haplo-lethal deficiency in a piwi2 mutant background:
The deficiency Df(3R)ry506-85C (87D1/2–88E5/6) caused female-specific haplo-lethality in the piwi2 mutant background (Table 2; Fig 3). Of 319 piwi2 mutant progeny, 146 contained the deficiency, all of which were males. We then tested seven smaller or overlapping deficiencies, and only Df(3R)ry27 (87D1/2–87F1/2) showed female lethality in a piwi2 mutant background (Table 2; Fig 3). As deficiencies Df(3R)ry506-85C and Df(3R)ry27 were initially recovered separately, it is unlikely that they share common background mutations responsible for the female haplo-lethal effect. Thus, the gene(s) responsible for the female haplo-lethality likely resides between 87D1/2 and 87F1/2. Since an overlapping deficiency, Df(3R)ry615 (87B11/13–87E8/11), shows no lethality or suppression, the gene(s) responsible for female-specific lethality probably resides between 87E8/11 and 87F1/2 (Fig 3; Appendix), even though this gene(s) is not represented by the tested individual mutations in the region (Appendix). The discovery of this haplo-insufficient female-specific lethal interaction with piwi2 further implicates the existence of a female-specific mechanism that interacts with piwi in a dosage-sensitive manner required for somatic development.
Individual suppressor mutations define novel genes or sequences that interact with piwi:
By systematically testing individual mutations uncovered by the deficiency suppressors for their ability to suppress the piwi2 ovarian phenotype, we identified four novel genes/sequences whose mutations strongly suppress the defects in germline stem cell division in the piwi2 mutants. Two such genes are defined by recessive lethal EMS alleles, while two other genes/sequences are defined by P-element insertions that do not display any detectable phenotype as homozygotes. This study reports their high levels of penetrance in suppressing the piwi2 phenotype.
The P{SRS3.9}3 gene:
The P{SRS3.9}3 (99D1) mutation restores germline stem cell division in 58% of piwi2 mutant females (Table 1; Fig 3). This suppression exceeds that of the original suppressing deficiencies (Df[3R]L127 and Df[3R]B81; Table 1; Fig 3). In fact, all the individual genes that have been identified as suppressors of piwi2 share this characteristic of stronger suppression (see below and DISCUSSION). Fig 4A&NDASH;B', shows examples of both the unsuppressed (Fig 4A and A') and the suppressed (Fig 4B and Fig B') ovarioles. The unsuppressed ovariole displays a typical piwi germline stem cell depletion phenotype, which contains only a rudimentary germarium and an egg chamber. In contrast, the suppressed ovariole contains a rescued germarium as well as multiple egg chambers. In the rescued germarium particularly, the presence of multiple spectrosome-containing cells in the germarium suggests the existence of germline stem cells and/or cystoblasts, while the presence of fusomes suggests the existence of developing germline cysts. The presence of multiple germline cysts and egg chambers in the ovariole indicates that the germline stem cells have undergone multiple rounds of divisions. Thus, the germline stem cells and their divisions are significantly restored in these females.
Although P{SRS3.9}3 strongly suppresses the germline stem cell defect of the piwi2 mutant, it has no detectable phenotype of its own. To verify that the P{SRS3.9}3 insertional mutation is responsible for the suppression and to generate potential new alleles with phenotype, we conducted P-element excision experiments (see MATERIALS AND METHODS). Out of 150 independent P-excised lines, a few lines displayed lethal phenotype, but the lethality did not map to the insertion site. The rest were viable and fertile and showed no apparent phenotype, as did the original P{SRS3.9}3 line. We tested two such lines for their ability to suppress the piwi2 phenotype. Neither line showed suppression. Genomic Southern blotting analysis of these two excision lines (see MATERIALS AND METHODS) shows that they are precise excisions (data not shown). These data support the assertion that the original P{SRS3.9}3 P-element insertion was the agent of piwi2 suppression.
To verify that the suppression is not due to the effect of If, either alone or in combination with piwi2, we used a longer crossing scheme to generate piwi2/piwi2; P{SRS3.9}3/+ females and examined their ovarian phenotype (see MATERIALS AND METHODS). The strong suppression was maintained in the new crosses without If (Fig 4B and Fig B'), suggesting that the If mutation does not contribute to the suppression effect.
To further test whether the suppression is due to the effect of the P{ry11} sequence in the piwi2 allele or to the genetic background associated with the piwi2 chromosome, we tested the suppression effect of P{SRS3.9}3 toward piwi12, a P-excision allele derived from a completely different P-insertional mutagenesis screen that retains only a 42-nucleotide remnant of the original P{w, lacZ} insertion (see MATERIALS AND METHODS). The P{SRS3.9}3 mutation effectively suppresses the germline stem cell defect of the piwi12 mutant ovaries (data not shown). Thus, P{SRS3.9}3 is most likely a genuine suppressor of the piwi mutations.
The same verification screens were also conducted for piwi suppressors l(3)99Da, P{wA}4-4, similar, and sry-. These experiments demonstrated that the screen using the piwi2 If chromosome was valid for identifying dosage-sensitive and non-allele-specific suppressors of piwi.
To precisely map the P{SRS3.9}3 insertion site to the genome, we cloned the surrounding genomic DNA via inverse PCR and sequenced it (see MATERIALS AND METHODS; Fig 5). Matching sequence data against the Drosophila genome sequence at the BDGP revealed that P{SRS3.9}3 disrupts the 5' untranslated region of the predicted CG7816 gene defined by mRNA CT7108 (
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It has been shown that piwi and hh represent two parallel somatic signaling pathways regulated by Yb in maintaining the stem cell fate in the germline ( (data not shown).
The l(3)99Da gene:
The EMS-induced homozygous lethal mutation l(3)99Da1 (99D3–E1) suppresses the germline stem cell defect of 50% of the piwi2 mutant females (Table 1; Fig 3; Fig 4, C–D'). To confirm that the l(3)99Da1 mutation was responsible for piwi2 suppression, the l(3)99Da1 chromosome was recombined with a ry506 chromosome to within 1 MU of l(3)99Da1. Five such independently derived "clean" l(3)99Da1 chromosomes were tested for suppression of piwi2. All five lines continued to show strong suppression, confirming that the l(3)99Da1 mutation indeed suppresses piwi2 (data not shown).
The l(3)99Ea gene:
A second EMS-induced homozygous lethal mutation, l(3)99Ea1 (99D9–E3), suppresses the germline stem cell defect of 42% of piwi2 mutant females (Table 1; Fig 3; Fig 4, E–F'). A second allele of l(3)99Ea, designated l(3)99Ea4, also strongly suppressed the piwi2 phenotype, confirming the l(3)99Ea1 suppression of piwi2 (data not shown). The suppression effect of l(3)99Ea and l(3)99Da is unlikely to be due to their common background, since other mutations derived from the same screen (see below and Appendix) show either different or no interaction with piwi2. This conclusion is supported by the retention of the suppression effect of the l(3)99Da1 mutation after "cleaning the chromosome" (see above).
The P{wA}4-4 suppressor:
This P-element insertion, P{wA}4-4 (100F), is the strongest suppressor of piwi2, as judged by penetrance both at the individual female level (72%) and at the ovariolar level (Table 1; Fig 3; Fig 4, G–H'). However, P{wA}4-4 is homozygous viable and fertile. Inverse PCR and sequencing the genomic DNA flanking the precise site of insertion of the P element have yielded a sequence that matches subtelo-meric heterochromatic repeats at 100F on chromosome 3R in the BDGP database, a result consistent with the previous mapping by
Weak suppressors of piwi2:
The recessive lethal mutations l(3)99De1 (99D3–9) and l(3)99Dg3 (D3–9) suppress the germline stem cell defect of piwi2 in 36 and 27% of the mutant females, respectively (Table 1; Fig 3). This likely reflects that these are weak alleles and, possibly, that these two genes do not strongly interact with piwi. Both mutations are EMS-induced mutations derived from the same screen as l(3)99Da1 and l(3)99Ea1, yet have different interactions with piwi2 (see above). In addition, five other lines from the same screen show no suppression of piwi2 [l(3)99Db1, c1, d2, h1, and i4 complementation groups], strengthening the conclusion that piwi2 suppression by l(3)99Da1, l(3)99Ea1, l(3)99Ea4, l(3)99De1, and l(3)99Dg3 is not due to their shared background.
Individual suppressor mutations define known genes that interact with piwi:
The l(3)j11B7j11B7 (similar) and tango genes:
The P-element insertion l(3)j11B7j11B7 (99E1) is a recessive lethal mutation and a strong suppressor of piwi2, with 50% of the mutant females displaying the suppressed phenotype (Table 1; Fig 3; Fig 6A&NDASH;B'). This P element is inserted in an intron of the similar gene (
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Since the Similar protein is a member of the bHLH-PAS family transcription factors that are known to function as heterodimers (
The sry- gene:
Three alleles of sry- (99D5) were tested in a piwi2 mutant background. sry-SF1 showed no interaction with piwi2, sry-SF2 showed a strong suppression (50% penetrance), and sry-12 showed a weak suppression (20% penetrance; Table 1; Fig 3; Fig 7A&NDASH;B'). Thus sry- is an allele-specific suppressor of piwi with the strength of the suppression of a sry- allele correlating well to its phenotype (see DISCUSSION).
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Sry- is a Cys-2/His-2 zinc-finger transcriptional activator expressed in the nuclei of germline and somatic cells of the ovary and the testis, as well as in many other tissues and stages of development ( in germline stem cell division, we examined whether the sterile sry- hemizygous and intra-allelic escapers display germline stem cell defects during oogenesis and spermatogenesis. The ovaries of all allelic or hemizygote combinations appeared to have wild-type levels of germline stem cell division, as indicated by long ovarioles with apparently normal germaria and numerous egg chambers (data not shown). However, the testes are considerably smaller, containing fewer bundles of sperm (data not shown). This testicular phenotype is very similar to the piwi male-sterile phenotype ( escapers may have early, piwi-like, stem-cell-related defects in spermatogenesis. Such a function in oogenesis may not be revealed by the existing sry- alleles or may be somewhat redundant in oogenesis.
To examine whether sry- mutations suppress the piwi2 phenotype by enhancing Piwi expression, we examined the expression of a fully functional transgenic myc-piwi gene ( escapers (see MATERIALS AND METHODS). The expression of Myc-Piwi was unchanged in the sry- escapers (Fig 7, C–D'). This implies that sry- does not suppress piwi2 by affecting Piwi expression.
| DISCUSSION |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
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We have screened 36% of the euchromatic genome for suppressors of piwi2 and identified six strong and three weak piwi2-suppressing mutations. These suppressors reveal the role of specific transcription factors that interact with piwi in a negative and dosage-sensitive manner in regulating germline stem cell division. In addition, we found deficiencies that dominantly interact with piwi2 to cause male sterility, as well as two other types of piwi2 interactors that cause haplo-lethal and haplo-female-specific lethal types in a piwi mutant background. The latter two types of interactors reveal the somatic function of piwi during development.
Germline stem cell division is regulated by dosage-sensitive mechanisms:
It is amazing that germline stem cell defects in the piwi mutants can be rescued by the removal of one copy of another gene (i.e., by 50% reduction in the activity). The identification of six dominant suppressors of piwi at the deficiency level, as well as six strong and three weak suppressors at the individual gene/sequence level, shows that one or more dosage-sensitive mechanisms must negatively interact with piwi in regulating germline stem cell division in the Drosophila ovary. The fact that each of these suppressors can restore the self-renewing ability of germline stem cells in piwi mutants suggests the importance of the dosage-sensitive mechanisms. The existence of multiple suppressors further implies that such negatively interacting and dosage-sensitive mechanisms may be a significant component of the molecular machinery that regulates germline stem cell division. Finally, four deficiencies in the 89E–91B region dominantly interact with piwi2 to cause male sterility. This implies that dosage-sensitive mechanisms may also exist in the male germline.
Such dosage-sensitive mechanisms may not be manifested as the solo act of an individual gene. The P{wA}4-4 suppressor mutation is inserted in the subtelomeric heterochromatic repeats of chromosome 3R, but does not interrupt TART or HeT-A elements in that region (see RESULTS and
Germline stem cell division requires dosage-sensitive transcriptional regulation:
The discovery of sry-, similar, and tango as suppressors of piwi further suggests that the dosage-sensitive mechanism operates at least at the transcriptional level. Sry- is a Cys-2/His-2 zinc-finger DNA-binding protein that is present in both germline and somatic cells in the ovary and the testis, as well as in many other tissues and stages of development ( mutants are homozygous late embryonic lethal ('s function during oogenesis. The three sry- alleles used in this study (sry-12, sry-SF1, and sry-SF2) are all single-amino-acid changes in the third zinc-finger domain of the protein (SF2 hemizygotes show many more gonadal defects than the other alleles do, while sry-SF1 shows a lower escaper rate than sry-SF2 does (SF2 mutation tends to perturb a subset of downstream effectors necessary for gonadal function, while the sry-SF1 mutation tends to disrupt more general downstream factors, leading to higher lethality (SF2 is the strongest suppressor of the piwi2 phenotype. It would be interesting to conduct genomic screens to identify the target genes of sry- whose transcription is selectively affected by sry-SF1 but not by sry-SF2 mutation. Such target genes would likely be involved in germline stem cell division and gonadogenesis.
Like Sry-, Similar and Tango play a key role in the dosage-sensitive regulation of germline stem cell division. Similar is homologous to a large group of heterodimerizing transcriptional activators (
(HIF-1) and has been shown to function in hypoxic response in Drosophila ( binds to HIF-1ß to drive transcription of downstream genes (
piwi as a regulator of gene expression:
Although the biochemical properties of the Piwi family proteins have not been systematically characterized, this family of proteins has been extensively implicated in RNA-related processes. Piwi itself is necessary for both transcriptional and post-transcriptional gene silencing in Drosophila (
The results of this study help us to understand the potential biochemical function of piwi. Since Sry- and Similar/Tango are transcription factors, it is possible that they suppress the piwi2 phenotype by promoting the transcription of a transcriptional repressor that represses piwi expression. In piwi2/piwi2; Sry-/+ or piwi2/piwi2; similar/+ flies, the reduced Sry- or Similar level leads to a decreased repressor level, which in turn leads to increased piwi transcription. This possibility, however, is less likely because piwi2 produces a 1.65-kb truncated transcript (D. COX and H. LIN, unpublished data), which suggests that piwi2 is a strong or even null mutation. Upregulating such a truncated transcript is unlikely to restore piwi function.
Therefore, we favor the following two possibilities. One possibility is that the suppression of piwi mutations by Sry-
