The Drosophila germline lineage depends on a complex microenvironment of extrinsic and intrinsic factors that regulate the self-renewing and asymmetric divisions of dedicated stem cells. Germline stem cells (GSCs) must express components of the Dpp cassette and the translational repressors Nanos and Pumilio, whereas cystoblasts require the bam and benign gonial cell neoplasm (bgcn) genes. Bam is especially attractive as a target of GSC differentiation factors because current evidence indicates that bam is both necessary and sufficient for cystoblast differentiation. This study has sought to distinguish between mutually exclusive transcriptional or post-transcriptional mechanisms as the primary regulators of bam expression in GSCs and cystoblasts. bam transcription is active in young germ cells but is repressed specifically in GSCs. Activation depends on a 50 bp fragment that carries at least one germ cell-specific enhancer element. A non-overlapping 18 bp sequence carries a transcriptional silencer that prevents bam expression in the GSC. Promoters lacking this silencer cause bam expression in the GSC and concomitant GSC loss. Thus, asymmetry of the GSC division can be reduced to identifying the mechanism that selectively activates the silencer element in GSCs (Chen, 2003a).

The cystoblast is a differentiated cell because, unlike the GSC, it divides precisely four times to produce 16 daughter cystocytes, which eventually produce 15 nurse cells and one oocyte. Also, unlike the GSC, each cystoblast or cystocyte cell division executes only partial cytokinesis and thus the cystocytes remain interconnected as a cyst. Low levels of Bam protein are expressed in GSCs, where Bam accumulates in fusomes but bam mRNA is undetectable by in situ hybridization. These observations indicate that bam is transcribed at a very low level in GSCs and that this small amount of transcript produces the protein seen associated with GSC fusomes. In the cystoblast and young cystocytes, bam transcripts become readily detectable, reflecting an upregulation in transcription rate or increased stability of the mRNA. This more abundant pool of mRNA produces higher levels of Bam protein that begin to accumulate in the germ cells' cytoplasm, and that continue to decorate the growing fusome. As cysts mature, the abundance of bam transcripts declines such that mRNA is again undetectable in eight-cell cysts and Bam protein levels fall precipitously once the 16-celled cyst forms such that Bam is again restricted to the fusome (Chen, 2003a).

Although loss of bam or bgcn function produces hyperplasia of undifferentiated germ cells, ectopic Bam expression caused GSC elimination similar to that seen when GSC maintenance factors are inactivated. These data are most readily explained by assigning bam the role of a necessary and sufficient cystoblast differentiation factor. A simple model to account for the experimental observations is that the microenvironment, or niche, created by somatic tip cells maintains the most anterior germ cells as GSCs by suppressing bam+ expression, thereby preventing them from differentiating as cystoblasts. The nature of intrinsic and extrinsic regulatory factors can provide clues about which aspects of gene expression are targeted by the signaling that sustains GSCs. The abrupt appearance of bam transcripts in cystoblasts and their persistence in cystocytes for only a few cell divisions is consistent with control of either bam transcription or mRNA stability or both. The demonstration that GSC maintenance depends on the Dpp signaling cassette suggests that Smad-dependent transcriptional control could regulate the choice between GSC or cystoblast fate. Alternatively, the asymmetric distribution of bam mRNA could arise if bam transcripts were more stable in cystoblasts than in GSCs. A reporter gene driven by a partial bam promoter can be transcribed in bgcn- cells that can not become cystoblasts, implying that bam transcriptional activation might precede full cystoblast differentiation. On this basis, the role of transcriptional control has been determined using promoter mutagenesis studies in transgenic flies. This study confirms that bam+ transcriptional control is necessary for proper GSC and cystoblast fate and determines the regions of the bam promoter that are required and sufficient for the GSC-low/cystoblast-high pattern of expression. One transcriptional control element in the 5'-UTR acts as a silencer in the GSC and bam transcriptional silencing is required for GSC fate (Chen, 2003a).

A genomic fragment of 1008 bp immediately upstream from the +1-site plus the 5'-UTR can fully rescue bam phenotype. A 3' series of deletions has shown that promoters that retain at least to -20 can reproduce the wild-type bam expression profile but promoters that retain only to -46 can not. Because fusion of the (-799 to -46) fragment to the P-element minimal promoter can recapitulate bam-like expression, it is concluded that -46 to -20 contains the 'basal elements' that interact with the core RNA polymerase subunits. The bam promoter does not comply with the rules for a TATA-dependent promoter because none of the sequences within the essential regions match the TATA element consensus. Residues -24 to -17 (TTAACAA) can represent a functional variation on the TATA motif, but the fact that promoters that include only to -20 are active means that the full motif is not required for normal transcriptional activity. 'TATA'-less promoters that lack the 'TATAA' box and bind core RNA polymerase proteins with Initiator (Inr) cis-acting sites have also been characterized. The efficiency and accuracy of Inr elements is increased in the presence of two other sequence motifs, the BRE and DPE. The sequences surrounding the bam transcriptional start site, however, does not match the consensus for Inr, BRE or DPE elements. Thus, other elements between -46 and -20 might function as `TATA-less' RNA polymerase recruitment sites in the bam promoter or 'TTAACAA' at -24 to -17 functions as a 'TATAA' box in vivo and can be adequately replaced in transgenic constructs (Chen, 2003a).

The same transgenes that identify the core element(s) of the bam promoter reveal that a germ cell-specific enhancer(s) is located upstream of -47. Deletions from the 5'-side show that the upstream border of this enhancer region is located at -96. Additional deletions show that essential enhancer sequences lay between -86 and -61. The sequence 'gcgacggc' matches the binding consensus site for the Dpp-activated transcription factor Mad and specific deletion of that eight-base pair element inactivates the bam promoter. Genetic studies, however, have shown that Mad is not required for cystoblast and cyst differentiation and is therefore not essential for bam transcription. It is suspected, therefore, that another activating transcription factor with a Mad-like recognition sequence binds to this site in bam. The significance of other sequences within this enhancer region will be determined only by additional transgenic studies with site-directed mutant constructs (Chen, 2003a).

Deletions that cause the spreading of reporter expression into GSCs and constructs that test the sufficiency of specific bam promoter fragments reveal that an 18 bp silencer element (SE) regulates bam expression. Although the SE is located in the 5'UTR, experiments that distinguished between transcriptional and post-transcriptional effects have demonstrated that the bam SE represses transcription. Chimeric promoters that joined heterologous sequences to bam fragments revealed several other features of the silencer element. The bam SE can not block GSC transcription from a nos enhancer/promoter fragment nor when the SE is placed upstream of the bam enhancer/basal region. Both of these observations can be explained if the SE's range is limited because either construct places the silencer much further from the relevant enhancer elements. Such short-range repressive effects have been described for the CtBP family of transcriptional repressors. Other explanations are equally possible, however, and resolution of the silencer's mechanism of action must await biochemical studies with silencer-binding proteins (Chen, 2003a).

The discovery of the silencer element permits refinement of a model explaining the GSC-to-cystoblast switch. This dissection of the bam promoter shows that bam can be expressed in GSCs because promoters lacking the silencer element are transcribed in all germ cells. For the same reason, the silencer does not work by blocking an overlapping enhancer site because silencer sequences can be deleted without disrupting enhancer-dependent transcription. The current working model is that the silencer is occupied in GSCs and bam is transcriptionally quiescent because the germ cell-specific enhancer(s) cannot be engaged. When the presumptive cystoblast is born, silencer element-dependent repression can be relieved, allowing enhancer(s) in the bam promoter to activate transcription and promote cystoblast differentiation. The clear implication of this model is that bam silencer occupancy is the basis of the asymmetric GSC division and that identification of silencer binding proteins can provide a mechanism for the asymmetry (Chen, 2003a).

Stem cells execute self-renewing and asymmetric cell divisions in close association with stromal cells that form a niche. The mechanisms that link stromal cell signaling to self-renewal and asymmetry are only beginning to be identified, but Drosophila oogenic germline stem cells (GSCs) have emerged as an important model for studying stem cell niches. Decapentaplegic sustains ovarian GSCs by suppressing differentiation in the stem cell niche. Dpp overexpression expands the niche, blocks germ cell differentiation, and causes GSC hyperplasty. The bag-of-marbles (bam) differentiation factor is the principal target of Dpp signaling in GSCs; ectopic bam expression restores differentiation even when Dpp is overexpressed. The transcriptional silencer element in the bam gene integrates Dpp control of bam expression. Finally and most significantly, this study demonstrates that Dpp signaling regulates bam expression directly since the bam silencer element (SE) is a strong binding site for the Drosophila Smads, Mad and Medea. These studies provide a simple mechanistic explanation for how stromal cell signals regulate both the self-renewal and asymmetric fates of the products of stem cell division (Chen, 2003b).

GSCs divide in the anterior/posterior axis, and this division produces daughters with different fates. The anterior cell of a GSC division retains contact with the stromal cap cells, maintains high levels of Dpp signaling, and continues as a stem cell. The posterior stem cell daughter dissociates from the cap cells and becomes a cystoblast (CB). The CB divides precisely four times with incomplete cytokinesis, giving rise to a cyst of 16 interconnected cells that differentiate further into 1 oocyte and 15 nurse cells. The progress of cyst formation can be followed by monitoring the morphogenesis of a dynamic organelle, termed the fusome, that grows and branches with each cyst cell mitosis (Chen, 2003b).

Cystoblasts require the product of the bag-of-marbles (bam) gene, which is both necessary and sufficient for differentiation. Germ cells lacking bam fail to differentiate into cystoblasts and continue to divide with full cytokinesis, producing germ cell hyperplasia. The failure of these proliferating germ cells to differentiate can be recognized by following the fusome since it remains spherical instead of growing into a branched structure. Superficially, therefore, bam mutant cells behave like GSCs, but molecular markers to determine the stage of arrest have been lacking (Chen, 2003b).

Studies characterizing bam transcription demonstrate that bam is tightly regulated such that it is off in GSCs and on in CBs. Thus, it was possible to determine if bam mutant cells are 'stem cell-like' since the activity of a bam reporter transgene would distinguish GSCs from CBs precisely. GFP expression was examined in bam mutant animals carrying a transgene with the bam promoter fused to GFP; most germ cells were observed to be GFP positive. Thus, unlike GSCs, germ cells lacking bam advanced to a state of differentiation sufficient to activate bam transcription (Chen, 2003).

Two features of GSC divisions demand molecular explanation: how is the anterior daughter of the GSC division retained as a stem cell (self-renewal) and what causes the posterior daughter to differentiate into a CB (asymmetric division)? Stromal cells, including the cap and inner sheath cells, at the germarial tip express several signaling molecules and are a likely source of dpp. dpp signaling has been shown to be required to maintain GSCs, and transcriptional control over Bam has been shown to distinguish GSCs from CBs. These two phenomena can now be linked directly (Chen, 2003b).

In GSCs, in which dpp signaling and pMad levels are highest, the Mad:Med complex binds to the bam SE and prevents bam transcription. GSCs self-renew because association of the anterior daughter with stromal cells permits sufficient dpp signaling to block CB differentiation by assembling a repressor complex on the bam SE element. This complex is likely to include other factors required for transcriptional antagonism, such as TGIF, a homeodomain-containing transcriptional corepressor of TGF-beta-dependent gene expression, or Ski/Sno factors, which can recruit histone-modifying enzymes. The complex may also contain Schnurri, a negatively acting Mad cofactor, since shn mutant GSCs also differentiate precociously (Chen, 2003b).

During division, a GSC daughter cell is displaced away from the cap cells and into a region of diminished dpp signaling. This cell, the CB precursor (pre-CB), expresses lower pMad levels that would cause the concentration of Mad:Med complexes to fall. Declining occupancy levels of the bam SE would produce derepression of bam transcription and concomittant activation of the CB differentiation program (Chen, 2003b).

Embryonic stem cells are considered totipotent because they can populate any of the adult niches. Although the degree of adult stem cell plasticity is currently receiving much attention, assembly of stem cells into signaling niches during postembryonic development might impose differentiation limits. What are the specific effects of stromal cell niches on captured stem cells? In the case of the Drosophila ovarian niche, GSCs are maintained as 'CBs-in-waiting' because a stromal cell signal represses the expression of one key factor (i.e., Bam). Perhaps other types of stem cells are similarly differentiated but blocked by stromal cell signals and require the expression of only one or a few key molecules to resume development (Chen, 2003b).

The continuous and steady supply of transient cell types such as skin, blood and gut depends crucially on the controlled proliferation of stem cells and their transit amplifying progeny. Although it is thought that signaling to and from support cells might play a key role in these processes, few signals that might mediate this interaction have been identified. During spermatogenesis in Drosophila, the asymmetric division of each germ line stem cell results in its self-renewal and the production of a committed progenitor that undergoes four mitotic divisions before differentiating while remaining in intimate contact with somatic support cells. TGF-ß signaling pathway components punt and schnurri have been shown to be required in the somatic support cells to restrict germ cell proliferation. This study showns, by contrast, that the maintenance and proliferation of germ line stem cells and their progeny depends upon their ability to transduce the activity of a somatically expressed TGF-ß ligand, the BMP5/8 ortholog Glass Bottom Boat. TGF-ß signaling represses the expression of the Bam protein, which is both necessary and sufficient for germ cell differentiation, thereby maintaining germ line stem cells and spermatogonia in their proliferative state (Shivdasani, 2003).

The mechanisms by which Gbb might regulate germ cell proliferation were explored next. One candidate that might interact with the pathway is the bags of marbles (bam) gene since, as with the activation of high-level TGF-β signaling in the male germ line, the loss of bam function is sufficient to induce the overproliferation of spermatogonia-like cells, but not GSCs (Shivdasani, 2003).

It has been reported that cytoplasmic Bam (Bam-C) is expressed in 2- to 16-cell spermatogonia, but not in GSCs, gonialblasts, or spermatocytes. Bam-C levels appear to be highest in late-stage spermatogonia, those farthest away from the apical hub, which are about to cease mitosis and differentiate into spermatocytes. Overexpression of bam using a heat-shock-bam transgene is sufficient to eliminate germ line stem cells in the ovary, but not in the testis. However, since it is not possible to achieve sustained, high-level, targeted overexpression with a heat-shock transgene, this analysis does not exclude a similar activity for Bam in the germ line of both sexes. By driving sustained, high-level overexpression of bam in GSCs and spermatogonia using nos-GAL4, it was found that testes of such animals resemble UAS-dad testes, being dramatically reduced in size, lacking early germ cells, and containing only mature spermatids. These expression and phenotypic data suggest that bam might be required for the differentiation of spermatogonia into spermatocytes. Loss of bam function might forbid differentiation, thereby maintaining spermatogonia in a proliferative state (Shivdasani, 2003).

The similarity between the TGF-β gain of function and bam loss of function phenotypes suggests that TGF-β signaling might act to repress bam activity. Testes were examined in which clones of cells expressing tkv* had been generated; such clones did not express Bam-C even though they overproliferated. It is therefore possible that TGF-β signaling might promote germ cell proliferation by repressing the activity of Bam, thus preventing premature differentiation of GSCs and amplifying spermatogonia, thereby maintaining them in a proliferative state. This possibility was tested by generating germ line clones that lacked both Bam activity and the ability to transduce the TGF-β signal. Such germ cells, doubly mutant for bam and put, behave as bam mutant clones and overproliferate as small cells resembling spermatogonia (Shivdasani, 2003).

It is proposed that Gbb acts as a short-range signal, emanating from cyst cells, signaling only to the GSCs and spermatogonia they enclose, thereby repressing Bam activity and maintaining germ cells in a proliferative state. Such short-range signaling by Gbb is consistent with the independent proliferation and differentiation of individual cysts. As each cyst ages, diminishing Gbb levels result in less TGF-β signal transduction in the spermatogonia, which in turn results in increasing Bam levels. Bam levels might constitute a counting mechanism such that once Bam levels reach a certain threshold, spermatogonia exit the cell cycle and commence differentiation into spermatocytes. This would be consistent with spermatogonia undergoing exactly four mitotic divisions. Bam activity thus forges an intimate link between proliferation and differentiation such that the former can only proceed if the latter is suppressed (Shivdasani, 2003).

To determine the sources for Gbb and Dpp in the testis, RT-PCR was used to study the presence of gbb and dpp mRNAs in the purified hub cells, somatic cyst cells and germ cells using fluorescent-activated cell sorting (FACS). The hub cells were marked by the upd-gal4 driven UAS-GFP expression. The somatic cyst cells and somatic stem cells were marked by the c587-gal4-driven UAS-GFP. vasa is a germline-specific gene. The germ cells were marked by a vasa-GFP transgene. The tips of the testes were isolated and dissociated, and the GFP-positive cells were purified from the dissociated testicular cells by FACS. As a control, vasa mRNAs were present in the whole testis and isolated germ cells but were absent in the somatic cyst cells and hub cells. Interestingly, gbb and dpp mRNAs were present in the hub cells and the somatic cysts/somatic stem cells but were absent in the germ cells. In addition, dpp mRNAs appeared to be less abundant than gbb mRNAs in the testis. These results indicate that both Dpp and Gbb are probably somatic cell-derived Bmp signals that directly regulate GSC maintenance in the testis (Kawase, 2004).

Although parallels between the male and female GSC systems are beginning to emerge, bam and the TGFß signaling pathway appear to play fundamentally different roles in male vs. female early germ cells. In both cases, male GSCs appear to be less sensitive than female GSCs to perturbations. It is proposed that this difference relates, at least in part, to the difference in the primary role of bam in the two sexes. In the female germ line, expression of bam appears to be the key event that produces a cystoblast and drives it to embark on cystocyte differentiation. Thus the mechanisms that suppress bam expression in GSCs and allow it in cystoblasts are likely to be key instructive determinants in the decision between stem cell self-renewal and the onset of differentiation. In contrast, in the male germ line, wild-type function of bam is primarily required at a later step in the differentiation pathway for cessation of the amplifying mitotic spermatogonial divisions and the transition to spermatocyte differentiation. In this case, the mechanisms that block bam protein expression in the GSCs may play a permissive rather than instructive role in allowing stem cell maintenance (Schulz, 2004).

Cyclins regulate progression through the cell cycle. Control of cyclin levels is essential in Drosophila oogenesis for the four synchronous divisions that generate the 16 cell germ line cyst and for ensuring that one cell in each cyst, the oocyte, is arrested in meiosis, while the remaining fifteen cells become polyploid nurse cells. Changes in cyclin levels could be achieved by regulating transcription, translation or protein stability. The proteasome limits cyclin protein levels in the Drosophila ovary, but the mechanisms regulating RNA turnover or translation remain largely unclear. This study reports the identification of twin, a homolog of the yeast CCR4 deadenylase. twin is important for the number and synchrony of cyst divisions and oocyte fate. Consistent with the deadenylase activity of CCR4 in yeast, these data suggest that Twin controls germ line cyst development by regulating poly(A) tail lengths of several targets including Cyclin A (CycA) RNA. twin mutants exhibit very low expression of Bag-of-marbles (Bam), a regulator of cyst division, indicating that Twin/Ccr4 activity is necessary for wild-type Bam expression. Lowering the levels of CycA or increasing the levels of Bam suppresses the defects observed in twin ovaries, implicating CycA and Bam as downstream effectors of Twin. It is proposed that Twin/Ccr4 functions during early oogenesis to coordinate cyst division, oocyte fate specification and egg chamber maturation (Morris, 2005).

twin encodes the Drosophila homolog of the yeast ccr4 gene. ccr4 (carbon-catabolite-repression) was first identified in S. cerevisiae as a regulator of RNA levels of the alcohol-dehydrogenase II gene. Although CCR4 protein was previously shown to associate with basal transcription machinery, recent data demonstrate that CCR4 catalyzes the degradation of poly(A) tails in yeast and flies (Temme, 2004; Morris, 2005 and references therein).

It has been unclear whether mutations in CCR4 have specific developmental defects and whether these defects might reveal specific targets sensitive to CCR4 function. twin mutant cysts divide asynchronously and less than four times; oocyte specification is defective and many egg chambers die and degrade. The mitotic cyclins, CycA and CycB, are misexpressed in twin, and reducing the gene copy number of cycA partially suppresses the twin egg chamber degradation phenotype. Furthermore, the poly(A) tails of cycA, cycE and, to a lesser extent, cycB, are longer in twin extracts, suggesting that Twin/Ccr4 deadenylase activity directly controls the RNA levels of these cell cycle regulators. By contrast, cytoplasmic Bam staining is reduced in twin. Induction of extra bam expression suppresses the cyst division and oocyte fate specification defects in twin mutants, implicating low Bam levels as one of the causes of these twin phenotypes (Morris, 2005).

The twin alleles are viable and specifically affect the female germline. In S. cerevesiae, ccr4 mutations are not lethal, although CCR4 is thought to be the main cytoplasmic deadenylase. It is possible that angel and Dnocturnin (CG4796), two other genes with extensive homology to the ccr4 catalytic domain but lacking the crucial LRR repeats, can partially compensate for loss of Twin function. Alternatively, since the mutations are probably not complete nulls, oogenesis may be more sensitive than the soma to decreased Twin function. Like the ovary, the early embryo relies on precise post-transcriptional gene regulation. The mature egg contains high levels of maternally loaded twin, consistent with a role for Twin in deadenylation, and probably explaining why twin mutants carry out embryogenesis normally (Morris, 2005).

Mitotic cells regulate cyclin levels in order to progress through the cell cycle. At the protein level, Drosophila regulates CycA, CycB and CycE, via proteasome-mediated degradation. In the Drosophila ovary, the novel protein Encore has been proposed to localize components of the proteasome complex to the fusome to regulate CycE. encore mutant cysts undergo an extra cell division and contain 32 cells, probably as a consequence of misexpressing not only CycE, but also CycA. Other experiments have shown that cyst divisions are sensitive to CycA levels. Adding a brief pulse of CycA by inducing a heat-shock construct can lead to an extra round of cyst division, suggesting that downregulation of CycA is crucial for cell cycle progression. Only a small number of cysts respond to such a CycA pulse, suggesting that in the wild type not all germ cells are in a susceptible phase of the cell cycle (G2) during which they can respond to CycA (Morris, 2005).

Cyclin RNA levels are regulated by control of poly(A) tail length. In Xenopus and mouse oocytes, cycB RNA is not translated in the absence of CPEB-mediated poly(A) tail lengthening. Longer poly(A) tails also enhance cyclin translation in Drosophila embryos. In the Drosophila ovary, Orb, the CPEB homolog, regulates poly(A) tail length and expression of its own RNA and oskar RNA. Consistent with a role for Orb in cyclin regulation and cyst division, orb mutant cysts frequently contain eight germ cells (Morris, 2005).

The data suggest that Twin-mediated deadenylation of cyclin RNA regulates cyst divisions. Cyclin polyadenylation has been well studied, but much less is known about cyclin RNA deadenylation. In Drosophila, Nanos and Pumilio have been shown to control deadenylation of cycB mRNA in primordial germ cells. Furthermore, Xenopus Pumilio interacts with CPEB, and Nanos, Pumilio and Orb/CPEB are all expressed early in Drosophila oogenesis. It is intriguing to speculate that Twin may regulate the poly(A) tail lengths in the dividing cyst in conjunction with Nanos, Pumilio and/or Orb (Morris, 2005).

Cytoplasmic Bam expression is reduced in twin germaria; a phenotype that would not be predicted if Twin directly regulated Bam expression via deadenylation. Indeed, no substantial change was detected in bam poly(A) tail length in twin ovaries. It is therefore proposed that bam is an indirect target of Twin/Ccr4 (Morris, 2005).

Although bam is known to control the differentiation of the cystoblast and to promote cyst division, the biochemical role of Bam is unknown. Removing one copy of bam suppresses the extra division in cysts lacking encore or overexpressing CycA. The results further implicate Bam in the events of early oogenesis. Increased bam expression suppresses not only the cyst division defects observed in twin mutants, but also the twin oocyte specification defects. Because Twin regulates cycA directly and may regulate Bam indirectly, the simplest model would posit that high levels of CycA are sufficient to suppress Bam expression. Two pieces of evidence argue against this model: Bam and CycA are both present at high levels in the dividing cyst; and Bam is required for the fifth cyst division induced by high levels of CycA. In addition, hs-bam induces stem cells to develop into normal cysts, indicating that high Bam levels do not disrupt CycA expression. A model is favored by which Bam and CycA act in parallel to each other, downstream of Twin (Morris, 2005).

Although several models could explain the data, it is proposed that increased mitotic cyclin levels together with low Bam expression cause many of the twin phenotypes. If Bam expression were normal, overexpressing cyclins could lead to extra cyst divisions. The low level of Bam in twin germaria does not permit continued cell division, yet cyclin levels remain high, delaying cell cycle progression and probably causing the egg chamber degradation observed in twin. This model is consistent with the fact that reducing the copy number of bam suppresses the extra cyst division phenotype of encore and of hs-cycA. Corroborating evidence comes from the observation that reducing the gene dose of cycA or increasing the dose of bam can partially suppress the degradation phenotype. However, there are likely to be other, unidentified targets of twin that also contribute to the twin phenotype (Morris, 2005).

twin and Hu Li Tai Shao mutants disrupt the number and synchrony of cyst divisions and oocyte specification. This array of defects is not shared by the cell cycle mutants described above or by other mutants such as orb, the M-phase inhibitor tribbles or the M-phase activator string, which affect the number but not the synchrony of cyst divisions. Comparison of twin and hts may therefore be instructive. hts cysts have no fusome, and are thought consequently not to coordinate the cyst divisions. By contrast, cysts in twin mutants contain branched fusomes that are capable of colocalizing with CycA, suggesting the possibility that Twin/Ccr4 gene regulation may mediate the coordination of the cyst divisions with oocyte specification downstream of the fusome (Morris, 2005).

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. This study demonstrate that Piwi and Bam proteins are expressed independently of one another 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. Finally, pum is epistatic to bam, indicating that niche Piwi does not regulate Bam-C through Pum. It is proposed 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 (Szakmary, 2005).

This study investigates the regulatory relationships between Piwi, Bam, and Pum, three key regulators of GSC versus cystoblast fates. Among them, Pum and Bam are intrinsic factors, whereas Piwi is expressed both in niche cells as an essential component of niche signaling and in GSCs to promote its division. 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. In addition, Pum and Nos have important germline development zygotic roles, including their cell-autonomous function for GSC maintenance. 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. 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. 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. 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 (Szakmary, 2005).

To investigate the regulatory relationship between piwi and bam, the reciprocal expression pattern was confirmed by double immunofluorescence microscopy of wild-type germaria for Piwi and Bam-C. A fully functional myc-tagged Piwi is expressed at high levels in GSCs and is downregulated in cystoblasts and early mitotic cysts. In contrast, Bam-C is absent from GSCs but accumulates in most cystoblasts and mitotic cystocytes in germarial region 1. The downregulation of Piwi coincides with the zone of Bam-C expression. In a few cases, germ cells were observed 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, 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 (Szakmary, 2005).

To determine whether this reciprocal expression pattern is a result of mutually negative regulation toward each other's expression, Bam expression was analyzed in piwi mutants and vice versa. Because piwi mutant ovarioles typically contain germaria that are depleted of germline cells, it is difficult to assay Bam-C expression in them. For this reason, piwi¹ GSC clones were generated with the FLP-DFS (flipase-mediated dominant female sterile) technique. Bam-C is expressed normally in cystoblasts and early mitotic cysts in germaria that contain only piwi¹ germline cells. 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, Piwi was eliminated in somatic niche cells. This was achieved by using Yb mutations that eliminate Piwi expression specifically in niche cells. 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. 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 (Szakmary, 2005).

Whether the absence of Bam affects Piwi expression was examined. 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. 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. Therefore, bam⁺ function is dispensable for Piwi expression in the germline and the apical somatic cells (Szakmary, 2005).

Whether piwi or bam negatively regulates the expression of the other was tested. 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. 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, a heat shock-inducible bam transgene was used that places the bam cDNA under the control of the hsp70 promoter. Flies carrying a P[myc-piwi] and a hs-bam transgene were subjected to heat shock twice daily for 3 days after eclosion. 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 diminishes Piwi expression specifically in these cells. Interestingly, ectopic Bam expression in both somatic cells and other germline cells within and beyond the germarium has no effect on Piwi expression in these cells. Particularly, Piwi expression in apical somatic cells (i.e., cap cells and inner sheath cells) of the germarium is unaffected by ectopic Bam expression. Thus ectopic Bam expression may specifically downregulate the germline Piwi expression (Szakmary, 2005).

The mutually independent expression of Piwi and Bam does not 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, females were constructed lacking both piwi and bam function and the double mutants' ovaries were examined. In contrast to the piwi mutant phenotype, in which ovarioles typically contain a germlineless germarium and 2-3 egg chambers, the double mutant ovaries are characterized by 'tumorous' germaria filled with hundreds of undifferentiated germ cells. Moreover, there is no apparent egg chamber development in the double mutant ovary. This phenotype is qualitatively similar to the tumorous phenotype observed in bam mutant ovaries, which can contain up to thousands of undifferentiated germ cells. 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 (Szakmary, 2005).

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. 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 (Szakmary, 2005).

To verify the complex epistasis between bam and distinct somatic versus germline functions of Piwi, the effect of specifically removing Piwi protein from either the germline or the somatic niche cells of bam mutants was investigated. The piwi (somatic);bam double mutant was achieved by generating Yb;bam double mutants because Yb specifically eliminates piwi expression in niche cells. 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 (Szakmary, 2005).

Yb;bam double mutant ovaries display a clear bam phenotype. 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 (Szakmary, 2005).

To further verify this hypothesis, the phenotype of piwi (germline);bam double mutants was analyzed. The piwi (germline) mutant was generated with an en-gal4 transgene to drive the expression of piwi^EP to produce specific expression of fully functional Piwi in niche cells. Because piwi^EP is inserted into the piwi locus, it is therefore a piwi mutant allele in the absence of gal4 expression. The en-gal4 piwi¹/piwi^EP transheterozygotes were generated in bam mutant and wild-type backgrounds. The piwi/piwi^EP;bam⁺ ovaries display the expected piwi mutant phenotype. The en-gal4 piwi¹/piwi^EP;bam^Δ86~/TM3 Sb ovaries appear wild-type, aside from a mild reduction in size, and give rise to females capable of laying eggs. 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;bam^Δ86/bam^Δ86 flies display typical bam mutant ovarioles. Also as expected, piwi¹/piwi^EP;bam^Δ86/bam^Δ86 and en-gal4 piwi¹/piwi^EP;bam^Δ86/bam^Δ86 ovaries display the phenotypes of piwi (somatic);bam double mutants and the piwi (germline);bam double mutants, respectively. These analyses further verified that bam is epistatic to somatic niche piwi function, yet germline piwi is epistatic to bam function (Szakmary, 2005).

The fact that Piwi expression in somatic cells has a downregulating effect on Bam-C expression in GSCs 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, whereas Bam promotes GSC differentiation. 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. Pum expression was therefore examined 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, 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. 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 (Szakmary, 2005).

To more definitively determine the regulatory relationship between Pum and the Piwi-Bam-C pathway, pum,bam double mutants were constructed and analyzed. 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 pum^ET1,bam^Δ86/pum^ET9,bam^Δ86 double mutant flies, in which both pum^ET1 and pum^ET9 are null alleles, the majority of germaria were devoid of germ cells. Only a minority of germaria (<10%) contained a number of undifferentiated germ cells with restricted proliferation. This range of defects is indistinguishable from that of the phenotype of typical pum mutant ovaries. These results suggest that pum is epistatic to bam and that the proliferation of germ cells in bam mutants requires Pum function (Szakmary, 2005).

In summary, these 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. This is based on three observations. (1) the expression of dpp does not require piwi. Therefore, Dpp is not a downstream signal of piwi. (2) dpp overexpression does not rescue piwi mutant defects. Therefore, Dpp and niche Piwi are functionally parallel. (3) The dpp signaling pathway directly represses bam transcription. 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. 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. Alternatively, it could be in GSCs, where Piwi suppresses a Dpp agonist(s) or perhaps even the Bam/Bgcn complex. This scenario would require that Piwi produce an intercellular signal independent of Dpp. At present, these two possibilities cannot be distinguished (Szakmary, 2005).

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. 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 (Szakmary, 2005).

How is Pum involved in the Piwi-Bam pathway? Piwi and Pum do not affect one another'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. Recently, it has been suggested that the majority of RNA helicases function by displacing proteins from RNA strands rather than by unwinding RNA. It is therefore conceivable that the Bam/Bgcn complex displaces Pum/Nos from their target RNAs (Szakmary, 2005).

A model is proposed for switching between self-renewal and differentiation of GSCs in the Drosophila germarium. 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. Piwi in niche cells has an essential and cooperative functional involvement in 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 (Szakmary, 2005).

Many stem cell populations interact with stromal cells via signaling pathways, and understanding these interactions is key for understanding stem cell biology. In Drosophila, germline stem cell (GSC) maintenance requires regulation of several genes, including dpp, piwi, pumilio, and bam. GSCs also maintain continuous contact with cap cells that probably secrete the signaling ligands necessary for controlling expression of these genes. For example, dpp signaling acts by silencing transcription of the differentiation factor, bam, in GSCs. Despite numerous studies, it is not clear what roles piwi, primarily a cap cell factor, and pumilio, a germ cell factor, play in maintaining GSC function. With molecular and genetic experiments, it is shown that piwi maintains GSCs by silencing bam. In contrast, pumilio is not required for bam silencing, indicating that pumilio maintains GSC fate by a mechanism not dependent on bam transcription. Surprisingly, it was found that germ cells can differentiate without bam if they also lack pumilio. These findings suggest a molecular pathway for GSC maintenance. dpp- and piwi-dependent signaling act synergistically in GSCs to silence bam, whereas pumilio represses translation of differentiation-promoting mRNAs. In cystoblasts, accumulating Bam protein antagonizes pumilio, permitting the translation of cystoblast-promoting transcripts (Chen, 2005).

dpp-dependent silencing of bam transcription defines a key -- probably the primary -- mechanism for maintaining GSCs. By repressing bam transcription in the germ cells attached to cap cells, dpp signaling prevents these cells from forming cystoblasts and assigns them as GSCs. It is speculated that all GSC maintenance genes might act by repressing bam transcription and this prediction was tested for piwi and pumilio (Chen, 2005).

Two genetic observations suggested that piwi might negatively regulate bam expression: (1) bam was epistatic to piwi in double mutants, indicating that the piwi GSC-loss phenotype required an active bam gene; (2) Bam coexpression suppressed the formation of extra GSCs induced when piwi was overexpressed. Thus, piwi-dependent GSC formation depends on maintaining low levels of bam expression (Chen, 2005).

Both overexpression and loss-of-function phenotypes could be explained if piwi, like dpp signaling, were necessary to silence bam transcription in GSCs. This possibility was tested by scoring the expression of bam transcriptional reporters in piwi mutant GSCs. Because piwi inactivation causes GSC loss, P{bamP-GFP} reporters were assayed in piwi bgcn (benign gonial cell neoplasm) double mutant flies that preserve GSCs. GSCs lacking bgcn were GFP negative, but GSCs that lacked both piwi and bgcn were GFP positive. Thus, like dpp signaling, piwi⁺ was necessary to silence bam transcription in GSCs (Chen, 2005).

piwi⁺ action in somatic, but not germline, cells is critical for GSC maintenance, and, therefore, piwi must act indirectly to repress bam transcription. A putative piwi target (or targets) in GSCs must integrate with dpp signaling because previous work has established that the Mad:Medea binding site in bam is a sufficient silencer element. Two recent findings drew attention to the E3-ligase Dsmurf as a candidate for a germ cell piwi target: (1) Dsmurf inactivation produces extra GSCs, just as does ectopic piwi expression and (2) Dsmurf suppresses dpp signaling by targeting phosphorylated Mad for degradation (Chen, 2005).

If piwi silences bam transcription by repressing Dsmurf in GSCs, then GSCs might be restored in piwi mutants if Dsmurf were simultaneously removed. Therefore ovaries of piwi Dsmurf double mutant females were examined and it was found that most germaria contained supernumerary GSCs and a continuous supply of egg chambers. It was verified that piwi Dsmurf GSC-like cells behaved as GSCs by noting that they did not express BamC protein. In 62/80 double mutant germaria, no cells expressing BamC were detected, whereas BamC-positive germ cells were detected in 18/80 germaria. In those cases, the most apical cells, in the GSC position, were BamC negative (Chen, 2005).

Dpp signaling and piwi act as GSC maintenance factors by repressing bam transcription. pumilio (pum) is a component of an evolutionarily conserved mechanism of translational control and is also essential for ovarian GSCs. The expression of P{bamP-GFP} reporter was examined in pum mutant germ cells to determine if bam transcriptional silencing also depends on pum⁺. In contrast to piwi, the reporter was properly silenced in pum bam GSCs. For example, GSCs in 84.6% of pum^MSC bam^BG/pum²⁰⁰³ bam^Δ86 germaria were GFP negative. Because pum mutant germ cells are unstable, it was suspected that the few GFP-positive cells in the GSC position had either differentiated or were dying (Chen, 2005).

GSCs required (1) dpp⁺ and piwi⁺ signaling to repress cystoblast (CB) differentiation by silencing bam transcription and (2) pum⁺ to repress CB differentiation by a mechanism that is independent of bam silencing. Because previous work has shown that Pum forms a translational repressor complex with Nanos, it was reasoned that pum⁺ might maintain GSCs by repressing translation of CB-promoting mRNAs. One candidate target mRNA is bam itself, but, because dpp-dependent transcriptional silencing of bam fully accounts for the absence of bam from GSCs, it is unlikely that Pum sustains GSCs by repressing bam translation (Chen, 2005).

The Pum:Nos repressor complex probably blocks translation of other unidentified target mRNAs that are essential for CB differentiation. Cystoblast formation would then depend on relieving this block, and, because bam is both necessary and sufficient to induce CB differentiation, bam might antagonize or bypass translational repression. The phenotypes of double mutants can distinguish between these possibilities. If bam bypasses translational repression, pum bam germ cells would not form CBs and would resemble bam mutant gonads. If, however, bam antagonizes Pum/Nos-mediated translational repression, pum bam germ cells might differentiate (Chen, 2005).

Ovaries formed in various pum and bam genotypes were compared with several alleles of each gene. Double mutant ovaries produced a complex phenotype that was distinct from either single mutant. Staining nuclei with DNA dyes revealed a mixture of apparently undifferentiated cells and overtly polyploid cells. Indeed, in many cases the polyploid chromosomes were also thick and expanded like nurse cell chromosomes. Most remarkably, these pseudo-nurse cells were occasionally organized within an epithelial layer of follicle cells, like a cyst, although these cysts never contained a full complement of 16 cystocytes. Cells with hallmarks of post-CB differentiation occurred only in the pum bam double mutant ovaries, where they were seen in over half the ovarioles scored (see Table S2) (Chen, 2005).

The appearance of pseudo-nurse cells and even cysts suggested that double mutant germ cells had formed functional cystoblasts, remarkably bypassing the requirement for bam⁺ expression. To verify that pum bam germ cells were undergoing differentiation, the double mutant cells were examined with several additional markers of differentiation (Chen, 2005).

Orb is expressed in all germ cells, but its levels increase dramatically in the cystocytes of developing cysts. Orb protein levels remain at very low levels in bam mutant cells. Double mutant cells, however, expressed Orb at levels seen in differentiating cysts and well above the levels in bam cells. Orb accumulation revealed that many of the pum bam germ cells that did not yet have obvious pseudo-nurse cell chromosomes had, in fact, progressed well beyond the "pre-CB" stage of bam cells. Pseudo-nurse cells also had high levels of Orb expression, similar to accumulation seen in developing nurse cells (Chen, 2005).

Ring canal formation is a distinctive feature of germ cell cysts, and the multiple cell pum bam cysts contained ring canals. The incidence of these pum bam cysts was modest but reproducible in all double-mutant combinations, including those containing null alleles of bam and very strong or null alleles of pum. It is suspected that the infrequent appearance of multi-nurse cell cysts is due to a second requirement for bam⁺ to drive cystocyte divisions during cyst formation. This requirement would not have been recognized previously because bam mutations arrested cells as 'pre-CBs' (Chen, 2005).

Although they are not normal, the appearance of these cysts is a striking manifestation that CBs lacking bam could differentiate as long as they also lacked pum. Combined with previous studies showing that ectopic bam expression is sufficient to direct GSC differentiation, the pum bam phenotype strongly suggests that bam acts as a CB-promoting factor by antagonizing, rather than bypassing, pum action. The data suggests further that dpp signaling, which directly regulates bam expression, does not control pum⁺ expression. A similar conclusion has been reached about the relationship between dpp signaling and nanos expression on the basis of studies of primordial germ cell differentiation (Chen, 2005).

A unifying model is proposed to explain the gene circuitry of GSC and CB fate within the GSC niche. The results suggest that Drosophila ovarian GSCs are retained as stem cells because Pum:Nos complexes repress translation of a pool of mRNAs that induce CB differentiation (Chen, 2005).

In wild-type GSCs that contact cap cells, Pum:Nos translational repression remains active because dpp signaling from stromal cells silences bam transcription and thus blocks the formation of Bam:Bgcn complexes that would antagonize Pum:Nos translational repression. Expression of piwi in stromal cells contributes a key, but unknown, signal that stabilizes or strengthens the Dpp response and bam transcriptional silencing (Chen, 2005).

After the GSC divides, the strength of Dpp signaling falls to levels that can no longer efficiently silence bam transcription in the cell displaced to the posterior and away from cap cells. This could be due to declining Dpp levels or diminished piwi-dependent signals that lead to reduced phospho-Mad levels. As bam transcription increases, Bam:Bgcn complexes antagonize Pum:Nos action and cause derepression of CB-promoting mRNAs, initiating the events of CB differentiation. Because pumilio and nanos are evolutionarily conserved proteins, it will be important to determine if a similar 'multiple-negative' circuitry is at work in mammalian stem cell niches (Chen, 2005).

encore (enc) codes for a novel protein that is involved both in regulating the number of germline mitoses and in the process of oocyte differentation. Mutations in encore result in exactly one extra round of mitosis in the germline. Genetic and molecular studies indicate that this mitotic defect may be mediated through the gene bag-of-marbles. The isolation and characterization of multiple alleles of encore reveal that they are all temperature sensitive for this phenotype. Mutations in encore also affect the process of oocyte differentiation. Egg chambers are produced in which the oocyte nucleus has undergone endoreplication often resulting in the formation of 16 nurse cells. It is argued that these two phenotypes produced by mutations in encore represent two independent requirements for encore during oogenesis (Hawkins, 1996). A third defect, one associated with Gurken (Grk), has been found in encore mutants. Post-transcriptional regulation of Grk protein levels is required for correct oocyte pattern formation. encore is required for the accumulation of Grk protein during oogenesis. Enc regulates Grk post-transcriptionally to ensure adequate levels of signaling for the establishment of the anterior-posterior and dorsal-ventral axes. The extra round of germline mitoses in enc mutants is most likely due to an overproduction of bag-of-marbles mRNA early in oogenesis. In contrast, the ventralization phenotype appears to result from a lack of Gurken protein. Encore could be a protein that regulates RNA function and stability in oogenesis, and thus may be involved in the turn-over of BAM mRNA and the translational control of GRK mRNA (Hawkins, 1996 and 1997).

Division of a female Drosophila stem cell produces a daughter stem cell and a cystoblast. The cystoblast produces a syncytial cluster of 16 cells by precisely four mitotic divisions and incomplete cytokinesis. Mutations in genes required for cystoblast differentiation, such as bag-of-marbles, block syncytial cluster formation and produce a distinctive 'tumorous' or hyperplastic germ cell phenotype. In this paper, the oogenic phenotypes of benign gonial cell neoplasm mutations are compared to those of mutations in bam. The data indicate that, like bam, bgcn is required for cystoblast development and that germ cells lacking bgcn become trapped in a stem cell-like state. One indication that germ cells lacking bgcn cannot form cystoblasts is that bgcn stem cells resist genetic ablation by Bam misexpression. Misexpression of Bam eliminates wild-type stem cells, apparently by inducing them to divide as cystoblasts. bgcn stem cells remain active when Bam is misexpressed, probably because they cannot adopt the cystoblast fate. Bgcn activity is not required for Bam protein expression but is essential for the localization of Bam protein to the fusome. Together, the results suggest that Bam and Bgcn cooperatively regulate cystoblast differentiation by controlling localization of Bam protein to the fusome (Lavoie, 1999)

BAM mRNA is present at high levels in only one or two cells located near the anterior tip of the germarium. Following a stack of about 10 somatic 'terminal filament' cells, germaria contain two or three large germ-line stem cells, and then more posteriorly, one or two cystoblasts and two-cell cysts. The hybridization observed is always located in germ-line cells immediately posterior to the stem cells. Transcription appears to be repressed in mature egg chambers because no labeling is detected in subsequent stages of oogenesis (McKearin, 1990).

Oocytic Bam protein is found in two distinct cellular locations. Spectosomal and fusomal Bam is found in all germ cells in the germarium and additionally in nurse cells of young egg chambers. Bam is found in cytoplasm of cystocytes as a halo of antigen. bam gene activity is essential even when formal fusomes cannot form suggesting that cytoplasmic Bam protein is active independent of association with fusomes (McKearin, 1995). Cytoplasmic Bam appears to accumulate to detectable levels only late in the cystoblast's lifetime, shortly before the cystoblast divides. Bam protein is present in the cytoplasm of the primary spermatogonial cell and its amplifying spermatogonial progeny but not in stem cells. It is also found associated with spermatogonial spectosomes (Gönczy, 1997).

The Drosophila oocyte develops within a cyst of 16 germline cells interconnected by ring canals. One of the 16 germline cells becomes the oocyte, while the other 15 become nurse cells. Polarized, microtubule-based transport of unknown determinants from nurse cells to oocyte is required for oocyte formation. Whether polarity is established during or after cyst formation has not been clear. An analysis was carried out of how polarity develops in stem cells and dividing cysts by following the development of ring canals and the growth of the fusome, a vesiculated cytoplasmic organelle that interconnects nurse cells and the oocyte. Fusomes were marked with antiserum to adducin-like Hu-li tai shao (Hts). The ring canal was identified by labeling ovaries with antibodies that recognize the actin-binding protein, Anillin (Field, 1995). In Drosophila embryos and tissue culture cells, Anillin is expressed in actively dividing cells and is localized in a cell-cycle-dependent manner. During interphase, it accumulates in the nucleus, but in mitosis, following nuclear envelope breakdown, it is released into the cytoplasm and moves out to the cortex. At telophase, Anillin becomes highly enriched in the cleavage furrow, where it remains until the connection between the sister cells is severed. It is usually not detected in cells that have left the cell cycle. In germline stem cells and cystocytes, Anillin follows the same cell-cycle-dependent pattern of localization; however, it persists in the ring canals that link the cystocytes long after the cells have stopped dividing, at least until the cyst has left the germarium. Anillin also localizes to a transient ring canal that arises when a stem cell divides to produce a daughter stem cell and a cystoblast. When it is first formed, this ring canal is similar in size and shape to the ring canals that link dividing cystocytes. It is not a permanent structure, however; before either cell divides again, the ring canal shrinks in diameter and the hole in its center disappears, severing the connection between the two cells. Usually no detectable Anillin remains at the site of the severed connection, though occasionally a small spot can be found on the membrane of a dividing cystoblast. This stem cell ring canal was identified previously in electron micrographs of Drosophila germaria, but Anillin is its first molecular component to be identified (de Cuevas, 1998).

Having a marker for the stem cell ring canal has allowed the identification of stem cells that have recently completed mitosis, whose fusomes are likely to be in the process of segregating between the two daughter cells. To analyze the behavior of the fusome in these cells, ovaries were fixed and triple-stained with anti-Anillin antibodies, anti-Hts antibodies (which label the fusome) and the DNA dye DAPI; the cells were then examined by immunofluorescence and confocal microscopy. Stem cells were identified by their location in the germarium, directly abutting the base of the terminal filament. The cell cycle phase of a stem cell was identified from its nuclear morphology, from the location of Anillin within the cell, and from the presence or absence of a ring canal. In late interphase, when the stem cell has a high level of Anillin in its nucleus and there is no ring canal attaching it to any other cell, the fusome is spherical in shape and is located at the anterior tip of the stem cell, adjacent to the base of the terminal filament. Throughout mitosis, when Anillin is enriched at the cell cortex, the fusome stays in this location and usually remains spherical, although occasionally it flattens out along the membrane of the stem cell, forming a wedge-shaped structure. After mitosis, when the nuclear membrane has reformed and the cleavage furrow is well advanced, the fusome begins to migrate posteriorly toward the cleavage furrow. At the same time, a small amount of fusomal material accumulates in the nascent ring canal, forming a 'plug' in the ring. Although its origin(s) could not be determined from this experiment, it is suggested that the plug is formed at least in part from newly synthesized material, since the original spherical fusome does not become noticeably smaller as it forms. As interphase progresses, the plug enlarges and the original fusome elongates along the anterior-posterior axis of the stem cell, eventually contacting the growing plug. The resulting single fusome then reorganizes and/or accumulates new material until it forms a thick bar-shaped structure that extends through the ring canal. Later, as the ring canal closes, it appears to squeeze the bar-shaped fusome into two pieces. Because of the appearance of the fusome, this has been called the 'exclamation point' stage. At this stage, the fusome is usually distributed unequally between the two cells; thus about one-third of it ends up in the cystoblast, and the remainder in the stem cell (de Cuevas, 1998). The bar-shaped fusome and its unequal distribution between the two cells were previously reported by Deng (1997).

When there is no Anillin at the membrane (and presumably no connection) between the stem cell and cystoblast, the stem cell fusome returns to the anterior tip of the cell and regains its spherical shape. In stem cells that appear to have separated only recently from a cystoblast, the fusome looks smaller than in other stem cells; hence it likely accumulates more material before the stem cell divides again. The behavior of the fusome suggests that there is a specific cortical attachment site for the fusome, from which it does not always detach completely. Because most germaria have at least one stem cell, and sometimes several, with a ring canal and an elongated fusome, it was reasoned that these stages of the fusome cycle must occupy a fairly large portion of the cell cycle following mitosis. The ring canal between stem cell and cystoblast stays open until after S-phase of the following cell cycle is complete in both cells. Thus, it is concluded that the exclamation point stage of the fusome cycle occurs in G2 (de Cuevas, 1998).

The results of these experiments also suggest that the stem cell and cystoblast enter and exit S-phase together. By examining the BrdU-labeled stem cell and cystoblast nuclei more closely, it was determined that the BrdU was distributed over the same portion of the nucleus (over euchromatin, over heterochromatin, or over both) in every labeled pair of cells. Thus, it is concluded that the cell cycles of the stem cell and cystoblast are synchronized through S-phase. It is also concluded that the stem cell and cystoblast remain connected and are synchronized at least through S-phase. It is thought that they become asynchronous soon after the cells are separated, however, since a stem cell and a cystoblast are never seen in mitosis at the same time. In stem cells, the ring canal is transient and closes down after the fusome is partitioned through it (de Cuevas, 1998).

The growth and behavior of the fusome in dividing cystoblasts has been studied in ovaries that were triple-labeled with anti-Anillin antibodies, anti-Hts antibodies and DAPI. A new cystoblast, which has separated from the stem cell but not yet entered mitosis, contains a spherical fusome slightly smaller than a stem cell fusome; it is usually located in the anterior half of the cell, near the plasma membrane. In mitotic cystoblasts, the fusome keeps its spherical shape and size but is often located in the posterior half of the cell. Thus, at cytokinesis, the fusome is often retained by the more posterior of the two daughter cells. It was not determined whether the fusome changes its location by migrating through the cystoblast before mitosis begins, or whether the entire cell rotates; it is suggested, however, that the fusome migrates towards the centrosome, which is likely to be located posteriorly. Following mitosis, as in the stem cell, a small plug of fusomal material accumulates in the arrested cleavage furrow. As interphase progresses, the original fusome and plug move closer together, additional fusomal material accumulates between them, and the two pieces of fusome eventually fuse, forming a snowman-shaped structure with its 'neck' in the ring canal. In the cystoblast, however, the original fusome does not elongate toward the plug as fusome and plug fuse. Thus, it could not be determined if the original fusome moves toward the plug, or if the plug and ring canal move towards the original fusome. Because it is formed from two smaller fusomes, the fused fusome has also been called a 'polyfusome'. After fusion, the cell that lacks a fusome at cytokinesis contains less fusomal material than its sister cell and, apparently, it does not accumulate more during the rest of interphase. By the next mitosis, which forms four cells from two, the fusome is still asymmetrically distributed within the cyst (de Cuevas, 1998).

In 4-, 8- and 16-cell cysts, the fusome grows in a similar manner as in 2-cell cysts: after each round of mitosis, plugs form in each nascent ring canal and gradually fuse with the original fusome. Fusion takes place as the plugs move towards the central fusome and as fusomal material accumulates in the gaps between them. All plugs fuse before the next round of mitosis begins. In 16-cell cysts, fusion is always complete before the cyst becomes lens-shaped and the fusome begins to disaggregate. Thus, 16-cell cysts whose fusomes are in the process of fusing are located in region 2a of the germarium. It is likely that some fusomal material is newly synthesized or assembled as the cyst grows, since fusomes in 16-cell cysts are much bigger than cystoblast fusomes. As in 2-cell cysts, after every round of fusome fusion, one cell appears to contain more fusomal material than its sister cystocytes; this cell is always one of the two with the greatest number of ring canals. Based on this observation, the cell with the most fusomal material in a 16-cell cyst is probably the same cell that retained the fusome at cytokinesis in the cystoblast. Thus, the initial asymmetry of the fusome is maintained throughout cyst formation. In 4-, 8- and newly formed 16-cell cysts (found in region 2a of the germarium), the cell with the most fusomal material is positioned at random in the cyst with respect to the axes of the germarium (de Cuevas, 1998).

Fusome morphogenesis was examined in two mutants in which cyst polarity appears to be disrupted. Mutations in Bicaudal-D (Bic-D PA66/R26 ) and egalitarian (egl WU50/RC12 ) cause the formation of cysts that fail to accumulate oocyte-specific factors in a single cell and thus differentiate into 16 nurse cells. Fusomes appear to form normally and are still asymmetrically distributed in these cysts, however, indicating that the polarizing mechanisms that underlie fusome formation are not disrupted by these mutations. Thus, the fact that the oocyte fails to differentiate in these mutants is apparently not caused by a lack of polarity in dividing cysts. Consistent with this result, one of the two cells with four ring canals still localizes to the posterior end of these mutant cysts in region 2b, indicating that some polarity is retained in older cysts as well (de Cuevas, 1998).

After each round of mitosis, as the fusome plugs move toward the central fusome, their associated ring canals move with them. To quantify this movement, the distances between new and old ring canals were measured both before movement (in cysts with newly arrested cleavage furrows, which did not yet contain a distinct plug) and after movement (in cysts whose plugs had all fused with the central fusome). After plug fusion, the newest ring canal in each cell can still be distinguished by its smaller size. In cells with more than one old ring canal, only the distance from the new ring canal to the closest old ring canal was measured. The results of this analysis show that new ring canals move about 2-3 mm closer to old ring canals after each round of mitosis. It could not be determined from this experiment if new ring canals actually flow through the plasma membrane, or if they are brought closer to old ring canals by removal of plasma membrane from one side of the cell and its replacement elsewhere. Regardless of how it happens, the centripetal ring canal 'movement' results in a change in the cyst's geometry. This change occurs after every round of mitosis but is best illustrated in 4-cell cysts. In newly formed 4-cell cysts, just after cytokinesis, the cells are arranged in a linear or V-shaped structure. In older 4-cell cysts, however, the cells radiate out like flower petals from the central clump of ring canals. This arrangement, called a 'rosette', can be seen most clearly in cysts that are just entering mitosis, when Anillin outlines the cortex of the cells. Rosettes have been found in many other insect species and their formation appears to be a general characteristic of cyst morphogenesis. The effect of rosette formation is to shorten the distances between cells that are not directly connected; thus, it is tempting to speculate that this change in cyst geometry facilitates synchronization or communication between cystocytes. It is also suggested that ring canal movement and rosette formation might be facilitated by the reduced amounts of membrane skeletal proteins found at the plasma membrane of dividing cystocytes (de Cuevas, 1998).

To characterize fusome formation in more detail, ovaries were labelled with antibodies against two other fusome components, alpha-Spectrin and Bag-of-marbles (Bam) protein. Both of these proteins, like Hts protein, localize to fusome plugs in nascent ring canals and are distributed asymmetrically within the cyst throughout its formation. No difference in composition has been found between the plugs and central fusome, but it is possible that other components might associate with only one part of the fusome. The plugs are formed initially by material that appears to approach the ring canal from both sides. Moreover, especially when labeled with anti-Hts antibodies, this material appears to approach the ring canal in lines, as if it is travelling along fibers. Thus, it is suggested that the fusome plugs are assembled from material that is transported to the site of the spindle midbody, around which the ring canal forms, perhaps along microtubules that are remnants of the mitotic spindle (de Cuevas, 1998).

Anillin is also present in the somatic ring canals that link ovarian follicle cells. These ring canals, which are first visible in region 2 of the germarium, increase in number as the follicle cells divide; thus by late oogenesis many follicle cells have multiple ring canals associated with them. Unlike germline ring canals, somatic ring canals are very small - about 0.5 mm in diameter - and do not enlarge as oogenesis progresses. They also continue to stain with anti-Anillin antibodies throughout oogenesis. Ring canals are not seen between the cells that comprise the interfollicular stalks (except when the stalks are forming, in stages 1-2) but they are present in the follicle cells that remain over the nurse cells after the others migrate posteriorly in stage 10. Somatic ring canals in later egg chambers also contain actin. In dividing follicle cells that were double-labeled with anti-Anillin and anti-Hts antibodies, no fusome-like structures were seen spanning the ring canals (de Cuevas, 1998).

The work presented in this paper strongly suggests that ovarian cysts are polarized at the first division, that this polarity is maintained during the subsequent rounds of division, and that the fusome is a sensitive indicator of polarity in dividing cysts. Thus, these observations support the idea that the oocyte is specified early, during the cyst divisions, rather than later, after the completion of the cyst mitoses. Previous studies had shown that the fusome associates with only one pole of the spindle in dividing cystoblasts. Since the two cells that are produced by this division give rise to the two cells with four ring canals (at the 16 cell stage), one of which becomes the oocyte, this result suggested that the fusome might mark the future oocyte. It was not clear, however, how or even if the original asymmetry between these two cells was maintained as the cyst continued to divide. The results presented in this paper provide the first evidence suggesting that the initial polarity of the cyst is maintained throughout the cell division process that produces the final 16 cell cyst (de Cuevas, 1998).

The fusome grows by a regular, polarized process throughout the stem cell and cyst cell cycles. Each polarization cycle begins in mitosis, when the fusome segregates to a single daughter cell of each pair. Following mitosis, a 'plug' of fusomal material forms in each nascent ring canal and gradually fuses with the pre-existing fusome. In dividing cysts, as the fusome plugs move toward the pre-existing fusome, their associated ring canals also move, changing the geometry of the cyst. At the end of each cycle of cyst growth, the fusome remains asymmetrically distributed within the cyst; one of the two cells with four ring canals retains a bigger piece of fusome than any other cell, including the other cell with four ring canals. Based on these observations, it is argued that the oocyte is specified at the first cyst division (de Cuevas, 1998).

Stromal cells are thought to generate specific regulatory microenviroments or 'niches' that control stem cell behavior. Characterizing stem cell niches in vivo remains an important goal that has been difficult to achieve. The individual ovarioles of the Drosophila ovary each contain about two germ line stem cells that maintain oocyte production. Anterior ovariolar somatic cells comprising three cell types act as a germ line stem cell niche. Germ line stem cells lost by normal or induced differentiation are efficiently replaced, and the ability to repopulate the niche increases the functional lifetime of ovarioles in vivo. These studies implicate one of the somatic cell types, the cap cells, as a key niche component (Xie, 2000).

The Drosophila ovary is a tissue where stem cells can be studied at the cellular and molecular level in vivo. Near the beginning of each developing egg string (or ovariole) within the ovary reside about two germ line stem cells (GSCs) whose progeny differentiate into eggs within 8 days as they move at predictable rates along the ovariole. These stem cells are surrounded by three differentiated somatic cell types -- terminal filament, cap, and inner sheath cells -- that help make up an anatomically simple tubular structure known as the germarium. GSCs are easily identified by size, location, and the shape of the fusome, an intracellular structure rich in membrane skeleton proteins. Stem cells usually contain a round fusome, but display a distinctive elongated fusome after division when they remain transiently connected with their daughter cell. Under appropriate conditions, GSCs divide about once per day and are randomly lost by differentiation, with a half-life of 4 to 5 weeks. It has been proposed that the somatic cells at the tip of the ovariole are organized into a niche that maintains and controls GSCs (Xie, 2000).

Ovariolar anatomy is consistent with the existence of a niche at the anterior tip. After stem cell division, the daughter that lies closer to the terminal filament and cap cells remains a stem cell, whereas the daughter that more closely adjoins the inner sheath cells differentiates into a cystoblast. Anatomical asymmetry may ensure that equivalent stem cell daughters receive different fate-determining signals. GSCs require a signal mediated by Dpp, a homolog of human bone morphogenetic proteins 2 and 4, in order to remain as stem cells and to divide at a normal rate. Two other proteins needed to maintain GSCs, Piwi and Fs(1)Yb (Yb), act outside