【新进展】《Current Biology》：干细胞与小生境的反应关系到分化

Duke大学医学中心的细胞生物学专家已经确定了干细胞与容纳和调节它们的特化的“生态区位细胞”（niche cell）之间的一个信号系统。这些发现使人们对刺激干细胞自我扩增或分化成一定细胞类型的信号有了更多的了解。这项研究公布在2005年1月26日的Current Biology杂志上。

Duke研究组发现niche cell的调节性基因指引干细胞的基因决定干细胞的前途——是继续扩增还是分化。Niche和germline干细胞都具有能够编码影响干细胞分裂的开关蛋白。干细胞的过量扩增是癌症发生的一个主要原因，而干细胞生产力的缩减又与不育、贫血和免疫系统缺陷有关。了解干细胞如何接受分化信息非常重要，因为任何有潜力的干细胞临床应用都需要考虑niche cell的作用。

研究人员分析了germline干细胞在自我复制或分化成其它类型细胞时的特定基因的表达情况。研究人员发现germline干细胞的行为受到临近niche cell的调节，而且干细胞分裂是一个不对称的过程。这个研究组确定了三个不同的基因——piwi、pumilio（pum）和bam，它们能调节干细胞与控制干细胞命运的niche cell之间的互作。

研究证明piwi和bam蛋白在germline干细胞和成包囊细胞中以互惠模式独立表达。但是，它们中的任意一个过表达都会干预另外一个的作用。

根据他们最新的niche cell——germline干细胞互作模型，niche cell中piwi基因的活化导致了抑制germline干细胞中bam表达的蛋白质的产生。缺少活性的bam基因会使干细胞中的pum和其他基因活化。然后，pum基因抑制与分化有关的蛋白质的制造。这些分子事件的结果就是抑制分化、维持这些细胞的原来特性。

Current Biology Volume 15, Issue 2 , 26 January 2005, Pages 171-178

Report

Regulatory Relationship among piwi, pumilio, and bag-of-marbles in Drosophila Germline Stem Cell Self-Renewal and Differentiation

Akos Szakmary1, Daniel N. Cox1, 2, Zhong Wang and Haifan Lin,

Department of Cell Biology, Box 3709, Duke University Medical Center, Durham, NC 27710, USA

Received 8 August 2004; Revised 7 October 2004; accepted 29 October 2004 Published: January 26, 2005 Available online 24 January 2005.

Abstract
The transition from a Drosophila ovarian germline stem cell (GSC) to its differentiated daughter cell, the cystoblast, is controlled by both niche signals and intrinsic factors. piwi and pumilio (pum) are essential for GSC self-renewal, whereas bag-of-marbles (bam) is required for cystoblast differentiation [1, 2, 3, 4, 5, 6, 7 and 8]. We demonstrate that Piwi and Bam proteins are expressed independently of each other in reciprocal patterns in GSCs and cystoblasts. However, overexpression of either one antagonizes the other in these cells. Furthermore, piwi;bam double mutants phenocopy the bam mutant. This epistasis reflects the niche signaling function of piwi because depleting piwi from niche cells in bam mutant ovaries also phenocopies bam mutants. Thus, bam is epistatic to niche Piwi, but not germline Piwi function. Despite this, bam− ovaries lacking germline Piwi contain approximately 4-fold fewer germ cells than bam− ovaries, consistent with the role of germline Piwi in promoting GSC mitosis by 4-fold [3]. Finally, pum is epistatic to bam, indicating that niche Piwi does not regulate Bam-C through Pum. We propose that niche Piwi maintains GSCs by repressing bam expression in GSCs, which consequently prevents Bam from downregulating Pum/Nos function in repressing the translation of differentiation genes and germline Piwi function in promoting germ cell division.

Article Outline
• Results and Discussion
• Piwi and Bam Are Expressed Independently of Each Other in Reciprocal Patterns in GSCs and Cystoblasts
• Ectopic Bam Expression Downregulates Piwi Protein in GSCs
• bam Is Epistatic to piwi
• The Epistasis of bam over piwi Reflects Piwi Signaling Function from Niche Cells
• The Expressions of Piwi and Pum Are Independent of Each Other
• The Proliferation of Germ Cells in bam Mutants Requires Pum Function
• Conclusions
• Experimental Procedures
• Drosophila Strains and Culture
• Immunohistochemistry
• Bam and Piwi Overexpression Analysis
• Construction of bam Null or Yb Null Flies Containing a Transgenic myc-piwi Gene
• Double Mutant Analysis
• Germline Clonal Analysis of the piwi1 Mutant
• Construction of bam Mutant Flies Expressing piwi in Niche Cells but Not in the Germline
• Acknowledgements
• References

Results and Discussion
Here, we investigate the regulatory relationships between Piwi, Bam, and Pum, three key regulators of GSC versus cystoblast fates. Among them, Pum and Bam are intrinsic factors [1, 4, 5, 6, 7 and 8], whereas Piwi is expressed both in niche cells as an essential component of niche signaling and in GSCs to promote its division [2 and 3]. Pum was originally identified as a maternal effect protein that heterodimerizes with NANOS (Nos) to bind and suppress the translation of its target hunchback mRNA in the posterior of the Drosophila embryo (reviewed in [9]). In addition, Pum and Nos have important germline development zygotic roles, including their cell-autonomous function for GSC maintenance [4, 5 and 10]. In contrast to this function of Pum and Nos, Bam is necessary and sufficient in promoting GSC differentiation, even though its molecular activity is not known [6, 7 and 8]. bam encodes two protein isoforms: the cytoplasmic (Bam-C) and the fusomal (Bam-F) forms, with Bam-C specifically present in cystoblasts and differentiating cysts but absent in GSCs [6 and 7]. Finally, Piwi is the founding member of the evolutionarily conserved Piwi protein family (a.k.a. ARGONAUTE family) involved in stem cell division, RNA interference, transcriptional gene silencing, and other developmental processes [1, 2, 11, 12, 13, 14 and 15]. In the Drosophila ovarian germline, Piwi is a nuclear protein that is preferentially expressed in GSCs but is only weakly expressed in cystoblasts and mitotic cysts, consistent with its germline function [3].

Piwi and Bam Are Expressed Independently of Each Other in Reciprocal Patterns in GSCs and Cystoblasts
To investigate the regulatory relationship between piwi and bam, we first confirmed the reciprocal expression pattern by double immunofluorescence microscopy of wild-type germaria for Piwi and Bam-C. As previously reported [3], a fully functional myc-tagged Piwi is expressed at high levels in GSCs and is downregulated in cystoblasts and early mitotic cysts ( Figure 1A). In contrast, Bam-C is absent from GSCs but accumulates in most cystoblasts and mitotic cystocytes in germarial region 1 (Figure 1B). The downregulation of Piwi coincides with the zone of Bam-C expression (Figure 1C). In a few cases, we observed germ cells expressing both Piwi and Bam-C in cystoblast positions. These cells might represent the transitional stage from GSCs to cystoblasts. At a very low frequency, cystoblast-like cells express low levels of Piwi, but no detectable Bam-C. On the basis of piwi;bam double mutant analysis (see below), these cystoblast-like cells are likely to be undifferentiated or potentially apoptotic. Overall, the reciprocal expression pattern of Piwi and Bam-C proteins supports the opposing functions of piwi and bam genes.
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Figure 1. Piwi and Bam-C Are Expressed Independently of Each Other in Reciprocal Patterns in GSCs and Cystoblasts(A–C) Reciprocal expression patterns of Piwi and Bam. Wild-type germarium stained for myc-Piwi (A) and Bam-C (B) is shown. Piwi is expressed in GSCs and late mitotic cysts but is downregulated in cystoblasts (CB) and early mitotic cysts. Bam-C is absent in GSCs but is abundantly present in Cb and early mitotic cysts. (C) is the merged image of (A) and (B). The bar in (A) indicates magnification for (A–C).(D–H) Piwi and Bam expression are independent of each other. (D) A germarium containing piwi1 germline clones is stained for the germ cell marker VASA (green) and for Bam-C (red). Bam-C is expressed in piwi1 cystoblasts (CB) and early mitotic cysts (MC). (E) The anterior part of a Yb mutant germarium double labeled for Bam-C (red) and DAPI (green). Bam-C expression is unaffected in Yb mutant ovarioles. (F–H) piwi is expressed in bam mutants. A P[myc-piwi];bamΔ86/bamΔ86 ovariole stained with DAPI (F), anti-Myc antibody (G), and anti-VASA antibody (H) is shown. Note that myc-piwi is expressed in all germ cells and apical somatic cells. The bar in (H) indicates magnification for (F–H).

To determine whether this reciprocal expression pattern is a result of mutually negative regulation toward each other's expression, we analyzed Bam expression in piwi mutants and vice versa. Because piwi mutant ovarioles typically contain germaria that are depleted of germline cells [1 and 2], it is difficult to assay Bam-C expression in them. For this reason, we generated piwi1 GSC clones with the FLP-DFS (flipase-mediated dominant female sterile) technique ([16]; see Experimental Procedures). Bam-C is expressed normally in cystoblasts and early mitotic cysts in germaria that contain only piwi1 germline cells (Figure 1D). Moreover, no ectopic Bam expression was detected in GSCs. Therefore, proper Bam-C expression in the adult germline during oogenesis does not require piwi+ function in the germline. To address whether piwi expression in apical somatic cells affects bam expression in GSCs, we eliminated Piwi in somatic niche cells. This was achieved by using Yb mutations that eliminate Piwi expression specifically in niche cells [17 and 18]. Yb mutants are phenotypically very similar to piwi mutants. However, if examined within the first day of eclosion, Yb mutant germaria still contain germ cells. As shown in Figure 1E, Bam-C expression is unaffected in adult Yb mutant ovaries, suggesting that there is no specific requirement for YB or Piwi in niche cells for proper Bam-C expression or localization in the germline. Taken together, the above analyses indicate that neither niche nor germline piwi is required for Bam-C expression.

We next investigated whether the absence of Bam affects Piwi expression. A P[myc-piwi] transgene was introduced into a bam null mutant background to monitor Piwi expression. Ovaries were dissected from these females and stained with Myc antibody to monitor the Piwi expression and with VASA antibodies to label germ cells. Piwi is present in all bam null germline cells (Figures 1F–1H). In addition, Piwi is also strongly expressed in apical somatic cells that correspond to terminal filament, cap cells, and inner sheath cells in the wild-type germarium (Figures 1F–1H). Therefore, bam+ function is dispensable for Piwi expression in the germline and the apical somatic cells.

Ectopic Bam Expression Downregulates Piwi Protein in GSCs
We then tested whether piwi or bam negatively regulates the expression of the other. Previously, we reported that overexpression of Piwi in apical somatic cells increases the number of GSC-like cells. These ectopic stem cell-like cells fill regions 1 and 2a of the germarium and, thus, displace Bam-C-expressing cells to region 2b [3]. If Piwi and Bam expression are mutually antagonistic, the prediction would be that expanding Bam expression to GSCs would downregulate Piwi expression there during oogenesis. To express Bam-C protein ectopically in GSCs, we used a heat shock-inducible bam transgene that places the bam cDNA under the control of the hsp70 promoter [7]. Flies carrying a P[myc-piwi] and a hs-bam transgene were subjected to heat shock twice daily for 3 days after eclosion (see Experimental Procedures). Ovaries were subsequently dissected and stained for Bam-C and Myc to monitor ectopic Bam-C expression and its effects on Piwi expression. As predicted, ectopic expression of Bam in GSCs diminished Piwi expression specifically in these cells (Figures 2A–2C). Interestingly, ectopic Bam expression in both somatic cells and other germline cells within and beyond the germarium had no effect on Piwi expression in these cells (Figures 2A–2C; data not shown). Particularly, Piwi expression in apical somatic cells (i.e., cap cells and inner sheath cells) of the germarium was unaffected by ectopic Bam expression. In control flies lacking the hs-bam transgene, we observed no defects in Piwi expression after heat shock treatment (data not shown). This indicates that ectopic Bam expression may specifically downregulate the germline Piwi expression.
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Figure 2. Ectopic Bam Expression Downregulates Piwi Level in GSCs(A–C) The effects of long-term ectopic Bam overexpression. (A) Anti-Bam-C staining of the germarium reveals ectopic Bam expression in the GSC position. (B) Anti-myc-Piwi staining reveals that ectopic Bam expression sharply downregulates myc-Piwi level in germ cells in the GSC positions. However, ectopic Bam expression has no effect on Piwi level in somatic cap cells (CpC) and inner sheath cells (ISC). (C) is the merged image of (A) and (B).(D–F) The short-term effects of a single pulse of ectopic Bam overexpression. hs-bam;myc-piwi flies were heat shocked at 37°C for 2 hr, dissected 6 hr later, and stained with Myc antibodies (red) and spectrin antibodies (green). (D) A non-heat shocked control showing the normal nuclear expression of myc-Piwi is shown. (E) and (F) show rapid downregulation of myc-Piwi expression in early germ cells (E) and, less frequently, myc-Piwi relocation to the cytoplasm (F), after ectopic Bam expression.(G–I) A single pulse of ectopic Piwi overexpression shows no short-term effects on Bam-C expression. Piwi was overexpressed either in both soma and germline by a hsp70-myc-piwi transgene (G and H) or only in the soma by hsp70-gal4/piwiEP transgenes (I). Bam-C expression was monitored with a Bam-C::GFP protein fusion transgene [27]. Flies were heat shocked as in panels (D)–(F) and stained with anti-1B1 (red) and anti-GFP (green) antibodies. (G) A non-heat shocked control showing the normal Bam-C:GFP transgene expression is shown. A single pulse of somatic (I) and germline (H) overexpression of Piwi shows no effect on Bam-C:GFP transgene expression within 6 hr. The bar in (A) denotes the image scale for all panels.

To determine whether downregulation of germline Piwi after ectopic bam expression is an indirect consequence of bam converting GSCs to cystoblasts or, alternatively, reflects a more direct interaction between the two genes, we subjected the P[myc-piwi];hs-bam flies to only a single 2 hr heat shock, followed by a 6 hr recovery period before ovaries were dissected. We estimated that this period should be sufficient for Bam protein to be expressed and to antagonize Piwi without allowing GSCs sufficient time to completely switch their fate. The dissected ovaries were stained for -spectrin and Myc to monitor the effects of ectopic Bam-C expression on Piwi expression (Figures 2D–2F). Ectopically expressed Bam-C elicits a rapid response in the downregulation of myc-piwi. Expectedly, not all germline cells responded equally strongly or rapidly. Nevertheless, Piwi in GSCs was almost always downregulated. In contrast, Piwi expression in niche cells was almost not affected, whereas Piwi in other early germ cells displayed mixed responses. Interestingly, some cystoblasts showed a cytoplasmic localization of myc-Piwi (Figure 2F) rather than its normal nucleoplasmic localization. This change in myc-Piwi localization might reflect an intermediate state of Piwi between its nuclear localization and its degradation in the cytoplasm.

Flies carrying a bam:GFP transgene together with a hs-myc-piwi or piwiEP;hs-gal4 transgene were heat shocked and allowed to recover as described above and then stained with anti-1B1 and anti-GFP antibodies (see Experimental Procedures) to test whether overexpression of Piwi affects Bam expression. Expression of bam:GFP was unaffected when Piwi was overexpressed either in niche cells (Figure 2I; piwiEP;hsgal4) or in all cells (Figure 2H; hs-myc-piwi). These results, combined with previous findings that Piwi overexpression displaces Bam expression posteriorly within the germarium [3], suggest that either downregulation of Bam-C by Piwi requires more time or that it is an indirect consequence of GSC expansion upon Piwi overexpression.

bam Is Epistatic to piwi
The mutually independent expression of Piwi and Bam does not, however, rule out their regulatory relationship in GSC cell fate, whereas the suppression of piwi in GSCs by ectopic bam expression suggests that these two genes interact antagonistically. To further define the interaction between piwi and bam, we constructed females lacking both piwi and bam function and analyzed the double mutants' ovaries. In contrast to the piwi mutant phenotype, in which ovarioles typically contain a germlineless germarium and 2–3 egg chambers (Figure 3A), the double mutant ovaries are characterized by "tumorous" germaria filled with hundreds of undifferentiated germ cells (Figure 3C). Moreover, there is no apparent egg chamber development in the double mutant ovary (Figure 3C). This phenotype is qualitatively similar to the tumorous phenotype observed in bam mutant ovaries, which can contain up to thousands of undifferentiated germ cells (Figure 3B). The piwi;bam double mutant phenotype therefore indicates that bam is epistatic to piwi. Given the opposing functions of piwi and bam, these results suggest that piwi acts upstream of bam to repress its function in promoting GSC differentiation.
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Figure 3. Epistasis Analysis between piwi and bam Mutants(A–C) piwi;bam double mutants display a bam-like phenotype. (A) A piwi1 mutant ovariole containing a germline-depleted germarium (Ge) connected to two egg chambers representing the products of the two differentiated GSCs as revealed by DAPI nuclear staining is shown. (B) A tumorous bam mutant ovariole containing 300–1000 undifferentiated germ cells as revealed by staining for VASA (green) and myc-Piwi (red). Note that myc-Piwi is present in the nucleus of all undifferentiated bam mutant germ cells, suggesting that these germ cells are GSC-like (also see Figure 5). (C) piwi;bam double mutant ovariole characterized by mildly tumorous germaria filled with 50–300 undifferentiated germ cells with no apparent egg chamber development, as revealed by double labeling for VASA (green) and 1B1 (red).(D–J) bam is epistatic to piwi function in niche cells but not in germ cells. All panels show 1B1 (red) and VASA (green) staining. (D) High magnification images of a Yb;bam double mutant ovariole are shown. (E and G) piwiEP/piwi1 heteroallelic ovaries display a piwi mutant phenotype. (I) piwiEP/piwi1;bamΔ86/bamΔ86 displays bam phenotype. (F) en-gal4 restores PIWI expression in anterior somatic cells by activating piwiEP in those cells. (H) en-gal4 alone does not alter bam phenotype. (J) en-gal4,piwi1/piwiEP;bamΔ86/bamΔ86 displays a mild bam phenotype.

Although the piwi;bam double mutant shows a bam-like phenotype, there is a difference between the defect of the double mutant and that of bam alone. The bam mutant typically contains 300–1000 undifferentiated germ cells, whereas the piwi;bam double mutants contain only 50–300 germ cells. One possible explanation for this difference is the absence of the mitosis-promoting, germline cell autonomous piwi function in the double mutant. The cell autonomous function of piwi in GSCs is to promote mitosis, resulting in a 4-fold increase of mitotic rates [3]. In bam mutants, "tumorous" germ cells are more mitotic because of the presence of piwi+ function, whereas in piwi;bam double mutants, "tumorous" germ cells are less mitotic because of the absence of piwi+ function. Therefore, these analyses suggest that, whereas bam is epistatic to the niche function of piwi, the cell autonomous function of piwi is epistatic to bam.

The Epistasis of bam over piwi Reflects Piwi Signaling Function from Niche Cells
To verify the complex epistasis between bam and distinct somatic versus germline functions of Piwi, we investigated the effect of specifically removing Piwi protein from either the germline or the somatic niche cells of bam mutants. The piwi (somatic);bam double mutant was achieved by generating Yb;bam double mutants because Yb specifically eliminates piwi expression in niche cells [18]. The piwi (germline);bam double mutant was achieved by driving transgenic piwi expression specifically in the niche cells of a piwi;bam double mutant background.

Yb;bam double mutant ovaries display a clear bam phenotype (Figure 3D). This phenotype, however, is not as attenuated as in piwi;bam double mutants, but rather appears to be as pronounced as in bam single mutants. This result supports the assumption that the epistasis of bam over piwi reflects the somatic piwi function, and the attenuated bam phenotype of the double mutant reflects the germline cell autonomous piwi function.

To further verify this hypothesis, we analyzed the phenotype of piwi (germline);bam double mutants. The piwi (germline) mutant was generated with an en-gal4 transgene to drive the expression of piwiEP to produce specific expression of fully functional Piwi in niche cells. Because piwiEP is inserted into the piwi locus, it is therefore a piwi mutant allele in the absence of gal4 expression (Figures 3E and 3G). We generated the en-gal4 piwi1/piwiEP transheterozygotes in bam mutant and wild-type backgrounds. The piwi/piwiEP;bam+ ovaries display the expected piwi mutant phenotype (Figures 3E and 3G). The en-gal4 piwi1/piwiEP;bamΔ86~/TM3 Sb ovaries appear wild-type, aside from a mild reduction in size, and give rise to females capable of laying eggs (Figure 3F). This finding directly confirms that Piwi expression in niche cells is sufficient for GSC maintenance, whereas the observed reduction in ovary size may reflect the absence of germline piwi function in promoting GSC mitoses. As expected, the en-gal4 piwi1/CyO;bamΔ86/bamΔ86 flies display typical bam mutant ovarioles (Figure 3H). Also as expected, piwi1/piwiEP;bamΔ86/bamΔ86 and en-gal4 piwi1/piwiEP;bamΔ86/bamΔ86 ovaries display the phenotypes of piwi (somatic);bam double mutants and the piwi (germline);bam double mutants, respectively (Figures 3I and 3J). These analyses further verified that bam is epistatic to somatic niche piwi function, yet germline piwi is epistatic to bam function.

The Expressions of Piwi and Pum Are Independent of Each Other
The fact that Piwi expression in somatic cells has a downregulating effect on Bam-C expression in GSCs [3] raises the question of how this signal may be relayed. The reciprocal expression patterns of Piwi and Bam-C in the germline closely resemble those of Pum and Bam-C. Pum maintains GSC self-renewal during oogenesis [1 and 4], whereas Bam promotes GSC differentiation [6]. GSCs are depleted in pum mutants but overproliferate in bam mutants. This raised the possibility that Piwi may exert its functions by acting on Pum. We therefore explored Pum expression in piwi1 mutants and Piwi expression in pum1688 and pum2003 mutants. The expression of one gene was not detectably altered in the mutant background of the other (Figures 4A–4C), suggesting that neither gene regulates the other's expression. However, pum encodes two distinct protein isoforms (156 kDa and 130 kDa). Either isoform is sufficient for maternal function, but both are required for zygotic function, including GSC maintenance [4]. Because pum1688 and pum2003 eliminate the expression of the 156 kDa and 130 kDa Pum isoforms, respectively, these results could suggest that either the 156 kDa or the 130 kDa isoform of Pum alone is sufficient for proper germline Piwi expression. Even if this is the case, the niche expression of piwi is independent of pum because pum is not required somatically to maintain GSCs [4].
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Figure 4. Regulatory Relationship between piwi and pum(A–C) Piwi and Pum expressions are independent of each other. (A) A piwi1 mutant germarium shows normal Pum expression GSCs. (B and C) pum2003 and pum1688 mutant ovarioles show normal myc-Piwi expression in the germarium (Ge) and in nurse cells of mutant egg chambers, as well as in terminal filament (TF) and other somatic cells of the germarium.(D–G) Germ cell proliferation in bam mutants requires Pum. (D) pumET1/pumET9 mutant ovarioles characterized by germline-empty germaria (eG) and an egg chamber. (E–G) Ovaries are double stained with anti-VASA (green) to label germ cells and anti-1B1 (red) antibodies to label spectrosomes and outline somatic cells. (E) bamΔ86 mutant germaria (G) are filled with proliferating GSC-like germ cells containing spectrosomes (Sp). (F) Low magnification image of pum,bam double mutants in which the majority of the germaria are devoid of germline, except that a few contain a small number of germ cells. (G) High magnification image of a pum,bam double mutant germaria containing a few proliferating germline cells. Images in (D)–(F) are at the same magnification.

The Proliferation of Germ Cells in bam Mutants Requires Pum Function
To more definitively determine the regulatory relationship between Pum and the Piwi-Bam-C pathway, we constructed and analyzed pum,bam double mutants (see Experimental Procedures). If somatic Piwi acts through Pum to regulate Bam-C, then pum,bam double mutants should resemble piwi;bam double mutants. This, however, was not the case. In pumET1,bamΔ86/pumET9,bamΔ86 double mutant flies, in which both pumET1 and pumET9 are null alleles, the majority of germaria were devoid of germ cells (>90%; Figure 4F). Only a minority of germaria (<10%) contained a number of undifferentiated germ cells with restricted proliferation (Figure 4G). This range of defects is indistinguishable from that of the phenotype of typical pum mutant ovaries [1 and 4] (Figures 4B–4D). These results suggest that pum is epistatic to bam and that the proliferation of germ cells in bam mutants requires Pum function.

Conclusions
In summary, our results show that somatic niche Piwi function antagonizes Bam-C, which in turn antagonizes Pum and germline Piwi. The niche function of PIWI in downregulating BAM function appears to converge with the Dpp signaling pathway that is also required for GSC maintenance [19 and 20] ( Figure 5). This is based on the following observations: First, the expression of dpp does not require piwi (A.S. and H.L., unpublished data). Therefore, Dpp is not a downstream signal of piwi. Second, dpp overexpression does not rescue piwi mutant defects [21]. Therefore, Dpp and niche Piwi are functionally parallel. Third, the dpp signaling pathway directly represses bam transcription [22 and 23]. Likewise, piwi niche signaling also downregulates bam expression because bam is epistatic over piwi and because overexpression of Piwi in germarial somatic cells causes overproliferation of GSCs and displaces Bam-C expression beyond region 1 and 2a of the germarium [3]. Taken together, these results indicate that these two signaling pathways must converge at some point to regulate Bam function. The convergence point could be in niche cells, where piwi directly affects Dpp signal production by aiding in its modifications, stability, and/or secretion (Figure 5, pathway 1). Alternatively, it could be in GSCs, where Piwi suppresses a Dpp agonist(s) or perhaps even the Bam/Bgcn complex ( Figure 5, pathway 2). This scenario would require that Piwi produce an intercellular signal independent of Dpp. At present, we cannot differentiate between these two possibilities.
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Figure 5. A Model for the Regulation of GSC Self-Renewal versus DifferentiationFor simplicity, only the left half of the anterior germarium is depicted. The following abbreviations were used: GSC, germline stem cell; CB, cystoblast; CpC, cap cell-central component of niche cells; TF, terminal filament; ISC, inner sheath cell; SSC, somatic stem cell; Sp, spectrosome; mRNA, unknown differentiation factor suppressed by Pum/Nos; S, unknown signal produced by Piwi; and Smad, dpp transducers. 1 and 2 designate two possibilities for the dpp and piwi signaling pathways to converge to regulate bam function. For a detailed explanation, see text.

How does Bam function as a converging target in promoting GSC differentiation? It has been suggested that benign gonial cell neoplasm (Bgcn) is an obligatory partner for Bam-C as a differentiation factor [24]. Bgcn is expressed in GSCs, but not in somatic cells. This may explain why ectopic bam expression only downregulates Piwi in GSCs, but not in somatic cells.

How is Pum involved in the Piwi-Bam pathway? Piwi and Pum do not affect each other's expression, yet pum is clearly epistatic to bam. This precludes the possibility that Piwi exerts its effect on Bam-C via Pum. The epistasis of pum to bam is best explained by ascribing a translational repressing function of Pum/Nos in the germline toward mRNAs that promote differentiation. This repression is released by Bam/Bgcn. In GSCs, Bam-C is itself transcriptionally silenced; therefore, Pum and Nos are active in suppressing differentiation. In cystoblasts, Bam/Bgcn are expressed, thereby antagonizing Pum/Nos function. This allows differentiation-promoting mRNAs to be translated. Bgcn is related to the DexH-box family of RNA-dependent helicases [24]. Recently, it has been suggested that the majority of RNA helicases function by displacing proteins from RNA strands rather than by unwinding RNA [25]. It is therefore conceivable that the Bam/Bgcn complex displaces Pum/Nos from their target RNAs.

We propose a model for switching between self-renewal and differentiation of GSCs in the Drosophila germarium (Figure 5). The niche cells signal to GSCs by secreting Dpp/Bmp and possibly other proteins. The Dpp signal is received by GSCs through its receptors Punt and Thick Veins (TKV), and it is transduced by pMad to silence bam transcription in these cells. This is achieved via the direct binding of Smads to a discrete silencing element in the bam gene [22 and 23]. Piwi in niche cells has an essential and cooperative function to this signal. Piwi and Dpp signaling pathways converge at some point upstream of bam, in either niche cells or GSCs. The absence of Bam allows Pum and Nos to be active, which suppresses the translation of differentiation genes, thus maintaining the stem cell fate. In the cystoblast and differentiating germline cysts, the Dpp signal is no longer effective, thereby relieving the transcriptional repression of bam. The Bam/Bgcn complexes then repress Pum/Nos function, allowing these cells to differentiate. Therefore, Pum/Nos can be considered a switch between self-renewal and differentiation, whereas niche signaling through Bam/Bgcn regulates this switch at a single cell level.

Experimental Procedures
Drosophila Strains and Culture
All strains were grown at 25°C on yeast-containing cornmeal molasses/agar medium. The following fly strains were used in this study: The piwi1 mutant chromosome [1] was dominantly marked with Irregular facets (If) [26]. The w;piwi1 FRT40A [2] and y w P[hsFLP]12;P[ovoD1]2L FRT40A [16] were used for clonal analysis. The P[myc-piwi]G38-2B insertion on the second chromosome was used to visualize Piwi protein expression [3], and is hereafter referred to as P[myc-piwi]. bamΔ86 ry e is a null allele of the bam gene [6]; P[w+;hsp70-bam+]11d and P[w+;hsp70-bam+]18d are heat shock inducible transgenes inserted on the wild-type chromosome 3 and X, respectively [7]; the P[w+ Bam:GFP]28mc chromosome bears a full length bam gene fused to GFP [22 and 27]; pumET1 and pumET9 are maternal effect null alleles [28] obtained from the Bloomington Stock center; pum1688 and pum2003 are hypomorphic alleles with defects in germline stem cell maintenance [4]; Yb72 is a truncation mutant considered to be a null Yb allele [17]. The en-gal4 and hs-gal4 transgenes were used to drive niche cell and global overexpression of EP(2)1024, respectively. EP(2)1024 is an EP element insertion, from the original Rorth collection, inserted in the piwi gene [3], which hereafter is referred to as piwiEP. Oregon R served as the wild-type strain in all experiments.

Immunohistochemistry
Ovaries and testes were dissected, fixed, and stained as described in Lin et al. [29]. For immunofluorescence staining, the following antisera were used: rabbit polyclonal anti-VASA antibody (1:2000; [30]); mouse monoclonal anti-1B1 antibody recognizing spectrosomes and fusomes (1:1; [31]); rabbit polyclonal anti--spectrin antibody (1:200; [32]); mouse monoclonal anti-Myc epitope antibody 1-9E10.2 (1:50; [33]); rat polyclonal anti-Bam-C antibody (1:50; [6]); rat polyclonal anti-Pum antibody (1:200; [34]); and rabbit polyclonal anti-GFP antibody (1:200; Molecular Probes). All the fluorescence-conjugated secondary antibodies were from Jackson Immunoresearch Laboratory and were used at 1:200 dilution. Immunofluorescently labeled samples were also counterstained with DAPI, as described previously [29]. Micrographs were taken with either a Zeiss Axioplan microscope or a Zeiss LSM410 confocal microscope, as described in Cox et al. [2].

Bam and Piwi Overexpression Analysis
w1118;+/+;P[hsp70-bam+;w+]11d/P[hsp70-bam+;w+]11d males were mated to w;P[myc-piwi]/P[myc-piwi];+/+ virgin females to analyze the long-term effect of bam overexpression on Piwi protein expression. Newly eclosed w;P[myc-piwi]/+;P[hsp70-bam+;w+]11d/+ females were heat shocked twice daily for 1 hr in a 37°C water bath with a 2 hr recovery period at 25°C between the first and second heat shock treatments. Flies were heat shocked according to this regime for 3 days, and 3 hr after the final heat shock ovaries were dissected and processed for antibody staining.

w1118 P[w+;hsp70-bam+]18d virgin females were crossed to w/Y;P[myc-piwi]/P[myc-piwi] males to generate w/w P[w+;hsp70-bam+]18d;P[myc-piwi]/+ females to analyze the short-term effects of bam overexpression on Piwi protein expression. These females were heat shocked once for 2 hr upon eclosion in a 37°C water bath and then given a 6 hr recovery period at 25°C, at which point ovaries were dissected and processed for antibody staining.

Virgin females expressing a transgene carrying a wild-type bam gene fused with GFP, w1118;P[w+;bam:GFP]28mc/P[w+;bam:GFP]28mc;bamΔ86/bamΔ86 were crossed to either w/Y;P[w+;hsp70-myc-piwi]/P[w+;hsp70-myc-piwi] males or wY;piwiEP/CyO;hs-gal4/hs-gal4 males to generate w1118;P[w+;bam:GFP]28mc/+;bamΔ86/P[w+;hsp70-myc-piwi] or w1118;P[w+;bam:GFP]28mc;piwiEP;bamΔ86/hs-gal4 progeny to analyze the short-term effects of piwi overexpression on Bam-C protein expression. Newly eclosed females were heat shocked and processed as described above in this section.

Construction of bam Null or Yb Null Flies Containing a Transgenic myc-piwi Gene
The w;P[myc-piwi]/CyO;+/+ males were mated to w;+/+;bamΔ86 ry e/TM3 Sb ry e virgin females. Newly eclosed w;P[myc-piwi]/+;bamΔ86 ry e/+ males were mated to w;+/CyO;+/TM3 Sb ry e virgin females to generate w;P[myc-piwi]/CyO;bamΔ86 ry e/TM3 Sb ry e flies. Sib-matings between males and virgin females established a stock from which P[myc-piwi]/CyO;bamΔ86 ry e/bamΔ86 ry e females were isolated for fertility tests and for ovary analysis via whole-mount immunofluorescence. The same stock was used to isolate males of the same genotype for fertility and spermatogenesis analysis. Southern analysis of genomic DNA from these flies was performed to verify the presence of the P[myc-piwi] transgene and the homozygous bam mutant genotype. Similar crosses with P[myc-piwi] insertions on the X chromosome and at other sites on second chromosomes were used to verify the results obtained from the transgenic line G38-2B (data not shown).

Yb;P[myc-piwi] flies were constructed by crossing y Yb72 w/FM7 P[act-GFP] virgins to w;P[myc-piwi] males to generate y Yb72 w/Y;P[myc-piwi]/+ males that were crossed to y Yb72 w/FM7 P[act-GFP];Sco/CyO virgins to generate y Yb72 w//FM7 P[act-GFP];P[myc-piwi]/CyO flies, which were selected to establish a stock. The FM7 P[act-GFP] balancer allowed the discrimination between Yb heterozygote versus homozygote larvae.

Double Mutant Analysis
piwi;bam mutant flies were constructed by crossing piwi1-If/CyO;+/+ males to +/+;bamΔ86/TM3 Sbvirgin females. piwi1-If/+;bamΔ86/+ males were recovered among the F1 progeny and mated to +/CyO;+/TM3 Sb virgin females. Sib-mating the resulting piwi1-If/CyO;bamΔ86/TM3 Sb males and virgin females established a stock from which homozygous piwi1-If;bamΔ86 females could be isolated for ovarian analysis by whole-mount immunofluorescence.

Yb;bam double mutant flies were constructed by crossing y Yb72 w/FM6;TM3 Sb e/TM6 Hu Tb e virgins to w;bamΔ86 e/TM3 Sb e males. The resulting y Yb72 w/Y;bamΔ86 e/TM3 Sb e or TM6 Hu Tb e males were recrossed to y Yb72 w/FM6;TM3 Sb e/TM6 Hu Tb e virgins to generate y Yb72 w/FM6;bamΔ86 e/TM3 Sb e flies to establish a stock.

Each pum allele was crossed to the bam-containing chromosome to generate pum,bamΔ86 double mutants. bam was selected by its homozygous sterility and verified by PCR. pum was selected by its homozygous lethality and verified by the sterility in the pumET1/pumET9 transheterozygous mutants.

Germline Clonal Analysis of the piwi1 Mutant
piwi mutant germline stem cell clones were generated with the FLP-DFS technique [16], as previously described [2]. Because the transgenic ovoD gene on the second chromosome produces highly atrophic germaria ([16]; data not shown), morphologically normal germaria associated with a normal complement of developing egg chambers are derived from homozygous piwi1 mutant GSC clones.

Construction of bam Mutant Flies Expressing piwi in Niche Cells but Not in the Germline
The w;piwiEP/CyO; bamΔ86 e/TM3 Sb e stock was constructed as follows: w;piwiEP/CyO;TM3 Sb e/TM6 Hu Tb e were crossed to w;+/+;bamΔ86 e/TM3 Sb e males to generate F1 w;+/CyO;bamΔ86/TM3 Sb e virgins, which were mated to their w/Y;piwiEP/+;bamΔ86/TM3 Sb e sibling males to generate w;piwiEP/CyO;bamΔ86 e/TM3 Sb e, which was recovered by selecting for yellow eyes (EP), Cy e, and Sb. Similarly, piwi1 and bamΔ86 were combined by crossing the previously recovered F1 w;+/CyO; bamΔ86 e/TM3 Sb e virgins to w/Y;piwi1/CyO;MKRS Sb/TM6 Hu Tb e males. Single lines were established from the F2 progeny, and lines that contained sterile non-Cy flies (piwi1/piwi1) were selected because they contained piwi1/CyO instead of +/CyO. From these lines, a w;piwi1/CyO;bamΔ86 e/TM6 Hu Tb e stock was established. In parallel to the above crosses, flies carrying en-gal4 and piwi1 chromosomes were mated to yield w;en-gal4/piwi1 females. These virgins were mated to w/Y;wgSp/CyO;MKRS Sb/TM2 Ubx males. The progeny with pale yellow eyes (en-gal4) and CyO phenotype were selected (w,en-gal4,piwi?/CyO;+/TM2 Ubx), and single sib matings were tested for fertility. Sterility of non-CyO individuals indicated the presence of an en-gal4,piwi1 recombinant chromosome because of the presence of piwi1 in both parents. Lines that also contained third chromosome balancers w,en-gal4,piwi1/CyO;+/TM2 Ubx or MKRS Sb were used to introduce bamΔ86. w;piwi1/CyO; bamΔ86 e/TM6 Hu Tb e virgins were mated to w,en-gal4,piwi1/CyO;+/TM2 Ubx or MKRS Sb males, and flies with pale yellow eyes (en-gal4) and not TM6 Hu Tb balancer (bamΔ86) were selected to establish a stock. The experimental crosses were w;piwiEP/CyO;bamΔ86 e/TM3 Sb e virgins mated to either w/Y;en-gal4,piwi1/CyO;bamΔ86 e/MKRS Sb or w/Y;piwi1/CyO;bamΔ86 e/MKRS Sb.

Acknowledgements
We thank Dr. Anna Chao for assisting this project and for data on myc-piwi expression in bam mutants, Dr. Dennis McKearin for stimulating discussions, for communicating unpublished results, and for providing anti-Bam-C antibody, bam mutants, bam-GFP flies, and hsp70[bam+] transgenes. We also thank Dr. Mike Parisi for constructing pum,bam double mutants, Dr. Yuh-Nung Jan for anti-VASA antibodies, Dr. Paul McDonald for anti-Pum antisera, and Dr. Dan Kiehart for anti-spectrin antisera. We are grateful to Dr. Brigid Hogan and the Lin lab members for valuable comments on the manuscript. Confocal imaging was conducted at the Duke Developmental, Cell, and Mollecular Biology and Cell Biology confocal facilities. This work was supported by the National Institutes of Health (HD33760).

References
1. H. Lin and A.C. Spradling, A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary. Development 124 (1997), pp. 2463–2476. Abstract-EMBASE | Abstract-MEDLINE

2. D.N. Cox, A. Chao, J. Baker, L. Chang, D. Qiao and H. Lin, A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal. Genes Dev. 12 (1998), pp. 3715–3727. Abstract-EMBASE | Abstract-MEDLINE

3. D.N. Cox, A. Chao and H. Lin, piwi encodes a nucleoplasmic factor whose activity modulates the number and division rate of germline stem cells. Development 127 (2000), pp. 503–514. Abstract-EMBASE | Abstract-Elsevier BIOBASE | Abstract-MEDLINE

4. M. Parisi and H. Lin, The Drosophila pumilio gene encodes two functional protein isoforms that play multiple roles in germline development, gonadogenesis, oogenesis and embryogenesis. Genetics 153 (1999), pp. 235–250. Abstract-EMBASE | Abstract-Elsevier BIOBASE | Abstract-MEDLINE

5. A. Forbes and R. Lehmann, Nanos and Pumilio have critical roles in the development and function of Drosophila germline stem cells. Development 125 (1998), pp. 679–690. Abstract-EMBASE | Abstract-MEDLINE

6. D. McKearin and B. Ohlstein, A role for the Drosophila bag-of-marbles protein in the differentiation of cystoblasts from germline stem cells. Development 121 (1995), pp. 2937–2947. Abstract-EMBASE | Abstract-Elsevier BIOBASE | Abstract-MEDLINE

7. B. Ohlstein and D. McKearin, Ectopic expression of the Drosophila Bam protein eliminates oogenic germline stem cells. Development 124 (1997), pp. 3651–3662. Abstract-EMBASE | Abstract-MEDLINE

8. P. Gonczy, E. Matunis and S. DiNardo, bag-of-marbles and benign gonial cell neoplasm act in the germline to restrict proliferation during Drosophila spermatogenesis. Development 124 (1997), pp. 4361–4371. Abstract-EMBASE | Abstract-MEDLINE

9. M. Parisi and H. Lin, Translational repression: A duet of Nanos and Pumilio. Curr. Biol. 10 (2000), pp. R81–R83. SummaryPlus | Full Text + Links | PDF (81 K)

10. Z. Wang and H. Lin, Nanos maintains germline stem cell self-renewal by preventing differentiation. Science 303 (2004), pp. 2016–2019. Abstract-EMBASE | Abstract-Compendex | Abstract-GEOBASE | Abstract-Elsevier BIOBASE | Abstract-MEDLINE | Full Text via CrossRef

11. L. Cerutti, N. Mian and A. Bateman, Domains in gene silencing and cell differentiation proteins: the novel PAZ domain and redefinition of the Piwi domain. Trends Biochem. Sci. 25 (2000), pp. 481–482. SummaryPlus | Full Text + Links | PDF (192 K)

12. A.N. Harris and P.M. Macdonald, aubergine encodes a Drosophila polar granule component required for pole cell formation and related to eIF2C. Development 128 (2001), pp. 2823–2832. Abstract-EMBASE | Abstract-Elsevier BIOBASE | Abstract-MEDLINE

13. W. Deng and H. Lin, miwi, a murine homolog of piwi, encodes a cytoplasmic protein essential for spermatogenesis. Dev. Cell 2 (2002), pp. 819–830. SummaryPlus | Full Text + Links | PDF (979 K)

14. K. Mochizuki, N.A. Fine, F.T. Fujisawa and M.A. Gorovsky, Analysis of a piwi-related gene implicates small RNAs in genome rearrangements in Tetrahymena. Cell 110 (2002), pp. 689–699. SummaryPlus | Full Text + Links | PDF (1187 K)

15. M. Pal-Bhadra, U. Bhadra and J.A. Birchler, RNAi related mechanisms affect both transcriptional and posttranscriptional transgene silencing in Drosophila. Mol. Cell 9 (2002), pp. 315–327. SummaryPlus | Full Text + Links | PDF (558 K)

16. T. Chou and N. Perrimon, The autosomal FLP-DFS technique for generating germline mosaics in Drosophila melanogaster. Genetics 144 (1996), pp. 1673–1679. Abstract-EMBASE | Abstract-MEDLINE

17. F.J. King and H. Lin, Somatic signaling mediated by fs(1)Yb is essential for germline stem cell maintenance during Drosophila oogenesis. Development 126 (1999), pp. 1833–1844. Abstract-EMBASE | Abstract-Elsevier BIOBASE | Abstract-MEDLINE

18. F.J. King, A. Szakmary, D.N. Cox and H. Lin, Yb modulates the divisions of both germline and somatic stem cells through piwi and hedgehog mediated mechanisms in the Drosophila ovary. Mol. Cell 7 (2001), pp. 497–508. SummaryPlus | Full Text + Links | PDF (1243 K)

19. T. Xie and A.C. Spradling, decapentaplegic is essential for the maintenance and division of germline stem cells in the Drosophila ovary. Cell 94 (1998), pp. 251–260. SummaryPlus | Full Text + Links | PDF (476 K)

20. T. Xie and A.C. Spradling, A niche maintaining germ line stem cells in the Drosophila ovary. Science 290 (2000), pp. 328–330. Abstract-EMBASE | Abstract-Elsevier BIOBASE | Abstract-MEDLINE | Full Text via CrossRef

21. Cox, D.N. (1999). Function of the Drosophila piwi gene in the self-renewing division of germline stem cells and in germline development. PhD thesis, Duke University, Durham, North Carolina.

22. D. Chen and D. McKearin, Dpp signaling silences bam transcription directly to establish asymmetric divisions of germline stem cells. Curr. Biol. 13 (2003), pp. 1786–1791. SummaryPlus | Full Text + Links | PDF (373 K)

23. X. Song, M.D. Wong, E. Kawase, R. Xi, B.C. Ding, J.J. McCarthy and T. Xie, Bmp signals from niche cells directly repress transcription of a differentiation-promoting gene, bag-of-marbles, in germline stem cells in the Drosophila ovary. Development 131 (2004), pp. 1353–1364. Abstract-EMBASE | Abstract-Elsevier BIOBASE | Abstract-MEDLINE | Full Text via CrossRef

24. B. Ohlstein, C.A. Lavoie, O. Vef, E. Gateff and D.M. McKearin, The Drosophila cystoblast differentiation factor, benign gonial cell neoplasm, is related to DexH-box proteins and interacts genetically with bag-of-marbles. Genetics 155 (2000), pp. 1809–1819. Abstract-EMBASE | Abstract-Elsevier BIOBASE | Abstract-MEDLINE

25. M.E. Fairman, P.A. Maroney, W. Wang, H.A. Bowers, P. Gollnick, T.W. Nilsen and E. Jankowsky, Protein displacement by DexH/D "RNA Helicases" without duplex unwinding. Science 304 (2004), pp. 730–734. Abstract-EMBASE | Abstract-Compendex | Abstract-GEOBASE | Abstract-Elsevier BIOBASE | Abstract-MEDLINE | Full Text via CrossRef

26. D.L. Lindsley and G.G. Zimm. The Genome of Drosophila melanogaster, Academic Press, New York (1992).

27. D. Chen and D. McKearin, A discrete transcriptional silencer in the bam gene determines asymmetric division in the Drosophila germline stem cell. Development 130 (2003), pp. 1159–1170. Abstract-EMBASE | Abstract-Elsevier BIOBASE | Abstract-MEDLINE | Full Text via CrossRef

28. D.D. Barker, C. Wang, J. Moore, L.K. Dickinson and R. Lehmann, Pumilio is essential for function but not for the distribution of the Drosophila abdominal determinant nanos. Genes Dev. 6 (1992), pp. 2312–2326. Abstract-EMBASE | Abstract-MEDLINE

29. H. Lin, L. Yue and C.S. Spradling, The Drosophila fusome, a germline-specific organelle, contains membrane skeletal proteins, and functions in cyst formation and oocyte determination. Development 120 (1994), pp. 947–956. Abstract-EMBASE | Abstract-Elsevier BIOBASE | Abstract-MEDLINE

30. B. Hay, L.Y. Jan and Y.N. Jan, Localization of vasa, a component of Drosophila polar granules, in maternal-effect mutants that alter embryonic anteroposterior polarity. Development 109 (1990), pp. 425–433. Abstract-EMBASE | Abstract-MEDLINE

31. M. Zaccai and H.D. Lip***z, Differential distributions of two adducin-like protein isoforms in the Drosophila ovary and early embryo. Zygote 4 (1996), pp. 159–166. Abstract-MEDLINE

32. T.J. Byers, R.R. Dubreuil, D. Branton, D.P. Kiehart and L.S.B. Goldstein, Drosophila spectrin II. Conserved features of the alpha-subunit are revealed by analysis of cDNA clones and fusion proteins. J. Cell Biol. 105 (1987), pp. 2103–2110. Abstract-EMBASE | Abstract-MEDLINE | Full Text via CrossRef

33. G.I. Evan, G.K. Lewis, G. Ramsay and J.M. Bishop, Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol. Cell. Biol. 5 (1985), pp. 3610–3616. Abstract-EMBASE | Abstract-MEDLINE

34. P. MacDonald, The Drosophila pumilio gene: An unusually long transcription unit and an unusual protein. Development 114 (1992), pp. 221–232. Abstract-EMBASE | Abstract-MEDLINE