Promoter

The Drosophila germline lineage depends on a complex microenvironment of extrinsic and intrinsic factors that regulate the self-renewing and asymsmetric 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).

Transcriptional Regulation

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).

Bmp signals directly repress bag of marbles in germline stem cells

The Drosophila ovary is an attractive system to study how niches control stem cell self-renewal and differentiation. The niche for germline stem cells (GSCs) provides a Dpp/Bmp signal, which is essential for GSC maintenance. bam is both necessary and sufficient for the differentiation of immediate GSC daughters (cystoblasts). Bmp signals directly repress bam transcription in GSCs in the Drosophila ovary. Similar to dpp, gbb encodes another Bmp niche signal that is essential for maintaining GSCs. The expression of phosphorylated Mad (pMad), a Bmp signaling indicator, is restricted to GSCs and some cystoblasts, which have repressed bam expression. Both Dpp and Gbb signals contribute to pMad production. bam transcription is upregulated in GSCs mutant for dpp and gbb. In marked GSCs mutant for two essential Bmp signal transducers (Med and punt) bam transcription is also elevated. Finally, Med and Mad are shown to directly bind to the bam silencer in vitro. This study demonstrates that Bmp signals maintain the undifferentiated or self-renewal state of GSCs, and directly repress bam expression in GSCs by functioning as short-range signals. Thus, niche signals directly repress differentiation-promoting genes in stem cells in order to maintain stem cell self-renewal (Song, 2004).

This study reveals a new function for gbb in the regulation of GSCs in the Drosophila ovary. Loss of gbb function leads to GSC differentiation and stem cell loss, similar to dpp mutants. gbb is expressed in somatic cells but not in germ cells, suggesting that gbb is another niche signal that controls GSC maintenance. Like dpp, gbb contributes to the production of pMad in GSCs and also functions to repress bam expression in GSCs. As in the wing imaginal disc, gbb also probably functions to augment the dpp signal in the regulation of GSCs through common receptors in the Drosophila ovary. In both dpp and gbb mutants, pMad accumulation in GSCs is severely reduced but not completely diminished. Since the dpp or gbb mutants used in this study do not carry complete loss-of-function mutations, it remains possible that complete elimination of either dpp or gbb function is sufficient for eradicating pMad accumulation in GSCs. Alternatively, both dpp and gbb signaling are required independently for full pMad accumulation in GSCs, and thus disrupting either one of them only partially diminishes pMad accumulation in GSCs. The lethality of null dpp and gbb mutants, and the difficulty in completely removing their function in the adult ovary, prevent these possibilities from being tested directly (Song, 2004).

Interestingly, dpp overexpression results in complete suppression of cystoblast differentiation and complete repression of bam transcription in the germ cells, whereas gbb overexpression does not have obvious effects on cystoblast differentiation or bam transcription. Even though the UAS-gbb transgene and the c587 driver for gbb overexpression have been demonstrated to function properly, it is possible that active Gbb proteins are not produced in inner sheath cells and somatic follicle cells because of a lack of proper factors that are required for Gbb translation and processing in those cells, which could explain why the assumed gbb overexpression does not have any effect on cystoblast differentiation. However, since active Dpp proteins can be successfully achieved using the same expression method, and Dpp and Gbb are closely related Bmps, it is unlikely that active Gbb proteins are not produced in inner sheath cells and follicle cells. Alternatively, dpp and gbb signals could have distinct signaling properties, and dpp may play a greater role in regulating GSCs and cystoblasts. Recent studies have indicated that Dpp and Gbb have context-dependent relationships in wing development. In the wing disc, duplications of dpp are able to rescue many but not all of the phenotypes associated with gbb mutants, suggesting that dpp and gbb have not only partly redundant functions but also distinct signaling properties. In the wing and ovary, gbb and dpp function through two Bmp type I receptors, sax and tkv. The puzzling difference between gbb and dpp could be explained by context-dependent modifications of Bmp proteins, which render different signaling properties in different cell types. It will be of great future interest to better understand what causes Bmps to have distinct signaling properties (Song, 2004).

All the defined niches share a commonality, structural asymmetry, which ensures stem cells and their differentiated daughters receive different levels of niche signals. In order for a niche signal to function differently in a stem cell and its immediately differentiating daughter cell that is just one cell away, it has to be short-ranged and localized. This study shows that Bmp signaling mediated by Dpp and Gbb results in preferential expression of pMad and Dad in GSCs. Bmp signaling appears to elicit different levels of responses in GSCs and cystoblasts, suggesting that the cap cells are likely to be a source for active short-ranged Bmp signals. These observations support the idea that Bmp signals are active only around cap cells. Consistently, when GSCs lose contact with the cap cells following the removal of adherens junctions they move away from the niche and then are lost. As gbb and dpp mRNAs are broadly expressed in the other somatic cells of the germarium besides cap cells, localized active Bmp proteins around cap cells could be generated by localized translation and/or activation of Bmp proteins. As they can function as long-range signals, it remains unclear how Dpp and Gbb act as short-range signals in the GSC niche (Song, 2004).

Bmp signaling and bam expression are in direct opposition in Drosophila ovarian GSCs. bam is actively repressed in GSCs through a defined transcriptional silencer. These observations lead to a model in which Bmp signals from the niche maintain adjacent germ cells as GSCs by actively suppressing bam transcription and thus preventing differentiation into cystoblasts. The levels of pMad are correlated with the amount of bam transcriptional repression in GSCs and cystoblasts. In the wild-type germarium, pMad is highly expressed in GSCs and some cystoblasts where bam is repressed. In other cystoblasts and differentiated germline cysts, pMad is reduced to very low levels, and thus bam transcriptional repression is relieved. In the GSCs mutant for dpp, gbb or punt, pMad levels are severely reduced, and bam begins to be expressed. The repression of bam transcription as a result of dpp overexpression seems to be a rapid process; bam mRNA is reduced to below detectable levels two hours after dpp is overexpressed. This suggests that repression of bam transcription by Bmp signaling could be direct. Furthermore, Med and Mad can bind to the defined bam silencer in vitro, which also supports the idea that Bmp signaling acts directly to repress bam transcription. Dpp signaling has also been shown to repress brinker (brk) expression in the wing imaginal disc and in the embryo. The repression of brk expression by Dpp signaling is mediated by the direct binding of Mad and Med to a silencer element in the brk promoter. Since the brk silencer is very similar to the bam silencer, the results suggest that bam repression in GSCs is also mediated directly by Dpp and Gbb in a similar manner (Song, 2004).

It remains unclear how the binding of Med and Mad to the bam silencer results in bam transcriptional repression in GSCs. For the brk silencer, Dpp signaling and Shn are both required to repress brk expression in the Drosophila wing disc and embryo. Mad and Med belong to the Smad protein family, which are known to function as transcriptional activators by recruiting co-activators with histone acetyltransferase activity. In the wing disc, Shn is proposed to function as a switch factor that converts the activating property of Mad and Med proteins into a transcriptional repressor property. Possibly, the Mad-Med complex could also recruit Shn to the bam repressor element. Consistent with the possible role of Shn in repressing bam expression in GSCs is the observation that GSCs that lose shn function differentiate, and thus are lost. Also, it remains possible that Mad and Med could recruit a repressor other than Shn when binding to the bam repressor element. In the future, it will be very important to determine whether Shn itself is a co-repressor for Mad/Med proteins or whether it directly recruits a co-repressor to repress bam transcription in GSCs (Song, 2004).

Gbb/Bmp signaling is essential for maintaining germline stem cells and for repressing bam transcription in the Drosophila testis.

Stem cells are responsible for replacing damaged or dying cells in various adult tissues throughout a lifetime. They possess great potential for future regenerative medicine and gene therapy. However, the mechanisms governing stem cell regulation are poorly understood. Germline stem cells (GSCs) in the Drosophila testis have been shown to reside in niches, and thus these represent an excellent system for studying relationships between niches and stem cells. Bmp signals from somatic cells are essential for maintaining GSCs in the Drosophila testis. Somatic cyst cells and hub cells express two Bmp molecules, Gbb and Dpp. Genetic analysis indicates that gbb functions cooperatively with dpp to maintain male GSCs, although gbb alone is essential for GSC maintenance. Furthermore, mutant clonal analysis shows that Bmp signals directly act on GSCs and control their maintenance. In GSCs defective in Bmp signaling, expression of bam is upregulated, whereas forced bam expression in GSCs causes the GSCs to be lost. This study demonstrates that Bmp signals from the somatic cells maintain GSCs, at least in part, by repressing bam expression in the Drosophila testis. dpp signaling is known to be essential for maintaining GSCs in the Drosophila ovary. This study further suggests that both Drosophila male and female GSCs use Bmp signals to maintain GSCs (Kawase, 2004).

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).

Repression of primordial germ cell differentiation parallels germ line stem cell maintenance

In Drosophila, primordial germ cells (PGCs) are set aside from somatic cells and subsequently migrate through the embryo and associate with somatic gonadal cells to form the embryonic gonad. During larval stages, PGCs proliferate in the female gonad, and a subset of PGCs are selected at late larval stages to become germ line stem cells (GSCs), the source of continuous egg production throughout adulthood. However, the degree of similarity between PGCs and the self-renewing GSCs is unclear. Many of the genes that are required for GSC maintenance in adults are also required to prevent precocious differentiation of PGCs within the larval ovary. Following overexpression of the GSC-differentiation gene bag of marbles (bam), PGCs differentiate to form cysts without becoming GSCs. Furthermore, PGCs that are mutant for nanos (nos), pumilio (pum) or for signaling components of the decapentaplegic (dpp) pathway also differentiate. The similarity in the genes necessary for GSC maintenance and the repression of PGC differentiation suggest that PGCs and GSCs may be functionally equivalent and that the larval gonad functions as a 'PGC niche' (Gilboa, 2004).

The embryonic gonad in Drosophila forms when somatic gonadal cells encapsulate about 12 primordial germ cells on each side of the embryo. PGCs proliferate in the female gonad during the subsequent three larval stages until, at the late-third larval stage, the gonad carries over 100 germ cells. At the larval/pupal transition, PGCs at the posterior of the gonad differentiate. Similar to differentiating germ line stem cells, differentiating PGCs undergo several rounds of incomplete mitotic divisions to form cysts and subsequently egg chambers with one oocyte and 15 nurse cells. Differentiation of posterior PGCs at the larval/pupal transition was attributed to the hormonal changes that control pupa formation. At the anterior part of the gonad, the newly formed somatic niche prevents the differentiation of PGCs and these are maintained as self-renewing GSCs throughout adult life. It has therefore been proposed that the transition from PGCs to GSCs coincides with the larval/pupal transition and the formation of the somatic niche. This study examines the genetic mechanisms that control PGCs during the proliferative larval stages, to better understand the PGC to GSC transition (Gilboa, 2004).

To explore how PGC differentiation is inhibited in the larval ovary, tests were performed to see whether any of the genes that are needed for either GSC maintenance or differentiation in the adult are likewise required in PGCs. Bag of marbles (Bam) has been shown to be a critical differentiation factor, because it is both necessary and sufficient to induce adult GSCs to differentiate. Therefore whether overexpression of Bam in the larval ovary is sufficient to drive PGCs to differentiate and form cysts was tested. As a marker for cyst formation, an antibody against an adducin-like molecule (1B1) was used, that stains the fusome, a sub-cellular organelle that is spherical in PGCs and GSCs but extends and branches as the single germ cell forms a multicellular cyst. Larvae carrying the bam gene under control of a heat-shock promoter (hs-bam) were heat-shocked at various time points during larval development and their gonads were examined one or two days later. Control wild-type gonads at the larval/pupal transition (LL3) exhibited either single PGCs carrying a spherical fusome or 2-cell cysts, which shared a bar-like fusome. The latter could be PGCs undergoing division, prior to full dissociation, or PGCs that initiated differentiation at the larval/pupal transition. In contrast, in hs-bam gonads, PGCs differentiate, as shown by the many cysts with branched fusome. Differentiation in hs-bam ovaries depended on the time of heat-shock. No cysts were observed in ovaries derived from larvae that were heat-shocked at the end of embryogenesis. Heat-shocking at the end of the first-larval instar (LL1) led to a high fraction (75%) of gonads that carried many cysts, whereas heat-shocking at the end of the second-larval instar (LL2) led to differentiation of all PGCs in all gonads tested. Thus, PGCs are able to differentiate prior to the larval/pupal transition. The time-dependent response to hs-bam could indicate either that PGCs are more capable of differentiation as the animal matures or that transcription from the hs promoter may be more active in the later larval stages. In support of the latter hypothesis, PGCs are transcriptionally quiescent during early embryogenesis and acquire transcriptional competence as they start to migrate. Indeed, the quantity of bam transcript seems limiting because a less rigorous heat-shock regime induces fewer cysts. Furthermore, with a different expression system, PGCs could be induced to differentiate as early as the end of embryogenesis. In support of the notion that differentiating PGCs follow the normal differentiation program, it was found that the time course of mitotic divisions in cysts that were precociously induced at LL2 was similar to that observed in cysts during normal development in either the pupal or the adult ovary (Gilboa, 2004).

These results suggest that all PGCs in the larval ovary are capable of differentiating following overexpression of Bam. Therefore whether active repression is required to keep PGCs in a proliferative state was tested. In adult GSCs, the Decapentaplegic (Dpp) pathway plays a major role in GSC maintenance. Dpp is produced by niche cells and is perceived directly by GSCs. Dpp signaling activates the downstream components Mothers against dpp (Mad) and Medea (Med), which directly bind to the bam promoter and repress the transcription of bam. In the larval gonad, overexpression of Dpp induces overproliferation of PGCs, suggesting that PGCs can respond to a Dpp signal. However, PGCs have not been shown to require Dpp in larval gonads. It was found that abolishing Dpp signaling in PGCs by overexpression of the negative regulator Daughters against Dpp (Dad) or mutations in thickveins (tkv), the Dpp type I receptor, induced differentiation of PGCs. 16-cell cysts were observed already at LL2 (48 hr after hatching), suggesting that PGCs begin differentiation shortly after the end of embryogenesis. Oocyte determination was detected in LL3 gonads, as indicated by accumulation of Orb, an oocyte marker, in one cell of the cyst (Gilboa, 2004).

To further explore the requirement for Dpp within GSCs, it was asked whether Dpp signaling could be detected directly in larval PGCs by monitoring the accumulation of its target, phosphorylated Mad (pMad) in PGCs. In larval gonads all PGCs accumulate pMad in the nucleus, suggesting that during larval development all PGCs receive a Dpp signal that actively represses their differentiation. In the adult, only germ line cells close to the niche contain significant levels of nuclear pMAD. Thus, the larval ovary may function in a similar manner to the adult niche in the prevention of PGCs from differentiation (Gilboa, 2004).

GSC differentiation is repressed by extrinsic factors, such as Dpp, and also by intrinsic factors. To further test whether PGCs employ the same mechanisms as GSCs to repress differentiation, larval ovaries were examined that were mutant for the translational repressors Nanos (Nos) and Pumilio (Pum), which function within GSCs to repress their differentiation. Indeed, nos mutant LL3 gonads contained many developed cysts. pumilio (pum) mutant gonads also contained cysts, although less so than nos mutants. Gonads that were mutant for both nos and pum did not contain more cysts than gonads that were mutant for nos alone. Because the alleles that were used were very strong, this suggests that nos and pum function together in the repression of PGC differentiation (Gilboa, 2004).

In adult ovaries, the differentiation of cysts requires Bam, and increasing amounts of Bam are present during each subsequent mitotic division. A reporter construct of GFP under control of the bam promoter was used to follow bam expression in the larval cysts. Cysts found in nos ML3 larval gonads also expressed higher amounts of GFP as compared to single PGCs. As in adults, the intensity of GFP labeling corresponds to the developmental state of the cyst. In addition to precocious differentiation, nos mutant germ cells displayed aberrations in the shape of the branched fusome and increased amount of small fusomal material as compared with wild-type. It is concluded that both Nos and Pum, which are required for GSC maintenance, are also required to repress PGC differentiation (Gilboa, 2004).

To further test for a possible partnership between nos and pum in GSC maintenance, the time at which nos or pum mutant germ line clones, generated by the FLP-FRT method, were eliminated from the adult ovary was examined. In wild-type, clones of unmarked GSCs were induced in about 25% of the ovarioles and that percent decreased only slightly during the course of the experiment, probably due to the natural rate of GSC loss. nos and pum mutant GSCs, in contrast, were lost rapidly. GSC loss was observed as early as 4 days after clone induction, and by the 6^th or 7^th day, most ovarioles did not contain a mutant GSC. The striking similarity in the profiles of nos and pum GSC loss therefore suggests that these genes also function together within GSCs (Gilboa, 2004).

As of the fifth and sixth day after clone induction, it was found that many nos mutant cysts were eliminated from the ovary. These results agree with the death of cysts observed in nos and pum mutants and with the death of nos cysts in pupal ovaries, which may be the cause of the empty ovarioles observed in adult nos females. These results and the previously reported phenotypes of nos and pum suggest that these genes are continually required throughout germ cell life. In the embryo, nos and pum are required for correct migration, transcription, and viability. During larval stages, they are required for the repression of PGC differentiation and, in the adult, for the maintenance and viability of GSCs as well as for the viability of differentiating cysts (Gilboa, 2004).

The targets of Nos and Pum within GSCs remain elusive, and the relationship of these 'intrinsic' GSC maintenance factors to the 'extrinsic' Dpp signal is unclear. To test if Dpp could function partly through Nos, the Nos expression pattern was examined in wild-type and in tkv-mutant GSCs. In wild-type germaria Nos is expressed at intermediate levels in GSCs and their immediate daughters, at very low levels during mitotic divisions of the cyst, and at very high levels in a fraction of the 16-cell cysts. This expression pattern was unchanged in tkv-mutant germ cells. Similar results were obtained for larval PGCs; Nos was expressed at intermediate levels in wild-type and tkv mutant PGCs, at lower levels in cysts undergoing mitosis, and at very high levels in 16-cell cysts. This suggests that Nos expression is independent of Dpp signaling (Gilboa, 2004).

Next, whether nos is required for Dpp function was tested, by analyzing nos mutant PGCs that were overexpressing either Dpp or Tkv^QD, a constitutively activated form of Tkv. In nos mutant control gonads, fragmented fusomal material as well as branched cysts could be observed. The spherical fusome within nos mutant germ cells remained small or fragmented in nos gonads overexpressing Dpp. Most strikingly, single PGC/GSC like germ cells accumulated in these gonads, and no cysts could be found. Thus, although increased Dpp signaling cannot fully counteract the nos phenotype, it does prevent precocious differentiation of nos mutant PGCs. Similar results were obtained with PGCs expressing Tkv^QD. In most gonads no cysts could be observed, although occasionally a small branched fusome could be detected, suggesting that Dpp signaling acts directly on PGCs, rather than via a secondary signal. The genetic data show that PGCs that are mutant for nos, can still respond to a Dpp signal, which keeps them in an undifferentiated state (Gilboa, 2004).

During larval stages, PGCs proliferate rather than differentiate. The translational repressors Nos and Pum are required to repress PGCs differentiation during larval stages. It has also been show that the Dpp pathway functions in a similar manner. Both pathways are also required for GSC maintenance. The fact that the spherical fusome remains abnormal in nos mutant gonads even when Dpp is overexpressed may suggest that some of Nos function is downstream of Dpp. However, the Nos expression data and the fact that Dpp signaling can prevent nos mutant PGCs from differentiation are more compatible with the Nos pathway playing a role upstream or in parallel to the Dpp pathway. It remains unclear how these pathways converge within germ cells (Gilboa, 2004).

Germ cells may perceive a Dpp signal from the moment they form at the posterior pole of the embryo until they differentiate to form cysts. Indeed, pMad is present in embryonic pole cells, larval PGCs and adult GSCs. Dpp signaling is not only necessary for GSC maintenance but also required continually through larval stages to actively repress PGC differentiation. Thus, the larval ovary functions in a similar manner to the adult niche with regard to Dpp-mediated repression of differentiation. During the third-larval instar, the adult somatic niche forms, and repression of PGC differentiation may then become limited to the small area of the adult ovary, allowing PGCs outside the confinement of the niche to differentiate (Gilboa, 2004).

Repression of PGC differentiation is required for about 4 days, from the end of embryogenesis to the beginning of pupa formation, whereas GSCs are maintained in the adult for many days. Differences between the 'short-term' and the 'long-term' repression of differentiation may yet be found. However, all the genes tested, dpp, bam, nos, and pum, function similarly in GSCs and PGCs. This similarity suggests that there may not be a clear transition from a 'dividing' PGC to a 'self-renewing' GSC (Gilboa, 2004).

A misexpression screen reveals effects of bag-of-marbles and TGF ß class signaling on the Drosophila male germ-line stem cell lineage

Male gametes are produced throughout reproductive life by a classic stem cell mechanism. However, little is known about the molecular mechanisms for lineage production that maintain male germ-line stem cell (GSC) populations, regulate mitotic amplification divisions, and ensure germ cell differentiation. The Drosophila system has been used to identify genes that cause defects in the male GSC lineage when forcibly expressed. A gain-of-function screen was conducted using a collection of 2050 EP lines and 55 EP lines were found that causes defects at early stages of spermatogenesis upon forced expression either in germ cells or in surrounding somatic support cells. Most strikingly, analysis of forced expression indicated that repression of bag-of-marbles (bam) expression in male GSC is important for male GSC survival, while activity of the TGFß signal transduction pathway may play a permissive role in maintenance of GSCs in Drosophila testes. In addition, forced activation of the TGFß signal transduction pathway in germ cells inhibits the transition from the spermatogonial mitotic amplification program to spermatocyte differentiation (Schulz, 2004).

Expression of the bam gene is normally tightly controlled. bam mRNA is expressed in mitotically amplifying spermatogonia and accumulation of the protein in the cytoplasm (BamC) was detected only in clusters of spermatogonia and not in male GSCs or gonialblasts. BamC protein normally disappears abruptly as germ cells make the transition to spermatocyte differentiation. Wild-type function of bam is required cell autonomously in the male germ line for spermatogonia to cease mitotic amplification divisions and initiate the spermatocyte differentiation program. In males lacking bam function, spermatogonia fail to cease mitosis, producing cysts of 32, 64, and more spermatogonia before eventually undergoing cell death. For some alleles, even testes from bam/+ heterozygous males often contained many clusters of greater than 16 small germ cells. The bam expression pattern and loss-of-function mutant phenotype suggest that in males bam acts primarily to limit the number of mitotic spermatogonial amplification divisions and that a certain threshold level of bam function may be required to trigger the switch to spermatocyte fate (Schulz, 2004).

The data on the effects of forced bam expression suggest that regulation of the bam expression pattern is important for both maintenance of GSCs and differentiation of gonialblasts and spermatogonia. When expression of bam in early germ cells was forced under control of the nos-gal4 transgene driver, early germ cells initially accumulated as single cells at the apical testes tip. Longer or higher forced expression of bam in early germ cells resulted in early germ cell death, first apparent in spermatogonial clusters but also occurring in single cells by 6 days after the temperature shift. By 10 days after the shift, early germ cells were often completely lost, indicating that high levels of bam expression are lethal to early germ cells and that spermatogonia are more sensitive to this lethal effect, possibly because they also express bam protein intrinsically. Although early male germ cells are sensitive to forced expression of bam, it appears that high levels of forced bam expression are necessary to have an effect. Contrary to the female germ line, where pulses of bam expression under the control of a heat-shock promotor forced differentiation of GSCs, no strong effects on male germ cells were noted in initial experiments where pulses of heat shock were applied to male flies carrying the same hs-bam transgene (Schulz, 2004).

It is proposed that sustained ectopic expression of high levels of bam in early germ cells blocks gonialblasts from initiating or carrying out the spermatogonial differentiation program, perhaps by eliciting prematurely the mechanism through which bam normally causes cessation of the spermatogonial mitotic amplification divisions at the 16-cell stage. Alternatively or in addition, forced expression of bam may prematurely activate aspects of the spermatocyte differentiation program that are incompatible with survival of stem cells, gonialblasts, and spermatogonial cysts (Schulz, 2004).

The effects of forced expression of bam suggest that the mechanisms that keep bam expression turned off in early male germ cells may play a role in maintenance of the stem cell population by shielding stem cells from the effects of inappropriate expression of a gene involved in the differentiation pathway for the lineage. Recent studies in the female germ line have identified a silencer element located just downstream of the bam start of transcription that is required to block bam transcript expression in female GSCs. If this same silencer element blocks expression of bam in stem cells and gonialblasts in the male germ line, then the factors that bind to it are likely to play a role in maintenance of the stem cell population. Transcriptional and post-transcriptional mechanisms that negatively regulate expression of differentiation genes may be a general feature of the mechanisms that maintain stem cell populations in many adult stem cell systems (Schulz, 2004).

The TGFß signal transduction pathway clearly plays a role in regulating the transition from the spermatogonial-amplifying mitotic division program to spermatocyte differentiation. However, exactly how TGFß signaling acts to govern this transition remains a puzzle. Mosaic analysis demonstrated that cysts of wild-type spermatogonia undergo extra rounds of mitotic divisions and fail to become spermatocytes when associated with a somatic cyst cell mutant for either punt, the TGFß type 2 receptor, or schnurri, a transcription factor downstream of TGFß signaling during embyrogenesis. These data suggest that receipt of a TGFß class signal by somatic cyst cells induces the somatic cells to send a signal of unknown nature to the germ cells that they enclose, either inducing or permitting the spermatogonia to initiate differentiation as spermatocytes (Schulz, 2004).

Forced expression of the TGFß class signaling molecule dpp specifically in germ cells has effects similar to loss of function of the signal transduction pathway in somatic cyst cells: failure of spermatogonia to stop mitotic divisions and become spermatocytes. This result is surprising, since one would expect that forced expression of a ligand might cause a phenotype opposite that of a receptor's loss of function. One explanation might be that precise levels of the dpp ligand may be critical, for example, for proper temporal or spatial control of activation of the pathway in somatic cyst cells. Another possibility is that dpp may not be the normal ligand, but that high levels of dpp secreted from germ cells may bind to TGFß receptors on cyst cells and block their ability to respond to the normal ligand. Consistent with this hypothesis, the TGFß type II receptor punt and both TGFß type I receptors sax and tkv have been demonstrated to bind dpp in transfected Cos cells. The TGFß homolog Maverick, rather than dpp, may be the ligand normally expressed in spermatogonia for activation of the TGFß signal transduction pathway in surrounding cyst cells, since Maverick mRNA but not dpp mRNA was detected in early germ cells in wild-type testes by in situ hybridization (Schulz, 2004).

Alternatively, TGFß signaling may be required in germ cells. Indeed, forced expression of the activated tkv receptor in early germ cells also caused spermatogonia to continue mitotic proliferation rather than differentiate as spermatocytes. The apparently cell autonomous effect of forced expression of the activated tkv receptor in germ cells suggests a direct role for the TGFß signaling pathway in germ cells. However, results that marked clones of germ cells mutant for the TGFß receptor sax differentiate as spermatocytes, along with similar findings that marked clones of germ cells mutant for punt, schnurri, or Mothers against dpp differentiate as spermatocytes with the normal number of 16 spermatocytes per cyst, indicate that the TGFß signaling pathway may not normally be required in germ cells for proper execution of the spermatogonia-to-spermatocyte transition. These observations raise the possibility that forced expression of dpp or the activated tkv receptor in early germ cells blocks the transition from the spermatogonial mitotic division program to spermatocyte differentiation by artificial and abnormal interference with the germ cell autonomous mechanisms that regulate this critical cell fate transition. The only Drosophila genes previously known to be required cell autonomously in the germ line for spermatogonia to exit the spermatogonial division program and become spermatocytes are bam and its partner, bgcn. The phenotype of males haplo-insufficient for bam suggests that the level of bam expressed in male germ cells is important for the correct transition from spermatogonia to spermatocytes. One model proposed for the female germ line is that dpp secreted from somatic cap cells at the tip of the germarium blocks expression of bam in GSCs, allowing stem cell maintenance. Strikingly, the Pro-bam-GFP reporter was expressed at reduced levels in spermatogonia from males in which UAS-tkv* or UAS-dpp were forcibly expressed in early male germ cells under control of the nos-gal4 germ-line-specific transgene driver, suggesting that activated Tkv or Dpp may suppress bam expression in males as well. It is tempting to speculate that, in the male, forced expression of dpp in spermatogonia may alter levels of bam expression so that bam protein does not reach a critical threshold required for the transition to spermatocyte differentiation. However, consistent with the production of many cysts of differentiating interconnected spermatogonia in UAS-tkv*; nos-gal4 and UAS-dpp; nos-gal4 males, some expression of the Pro-bam-GFP reporter was detected. The expression of the Pro-bam-GFP reporter even in the presence of the activated tkv receptor suggests that there may be mechanisms at work in spermatogonia that can override silencing of bam expression by the TGFß signaling pathway. Because the Pro-bam-GFP transcriptional reporter was expressed in spermatogonia even in cells expressing the activated tkv receptor, these mechanisms are likely either to interfere with the TGFß signal transduction pathway downstream of receptor activation or to act independently of and/or override the TGFß signaling effect. Forced expression of activated tkv in spermatogonia may also somehow affect expression or stability of Bam protein, since no accumulation of BamC protein was detected in spermatogonial cysts in testes from UAS-tkv*; nos-gal4 animals (Schulz, 2004).

Forced expression of the TGFß class signaling molecule dpp or the activated tkv receptor in early male germ cells leads to a mild increase in the number of male GSCs and gonialblasts around the apical hub and to reduced expression levels of the Pro-bam-GFP transcriptional reporter in spermatogonia. If TGFß signaling normally acts on the silencer element in the bam gene to repress expression of bam in male GSCs, as has been shown for female GSCs, then forced activation of TGFß signaling in male early germ cells might delay the transition from stem cell to spermatogonial differentiation by delaying the accumulation of bam protein. However, the effect of activation of TGFß class signaling on male GSCs was much more subtle than the effects noted in female germ cells. The difference between the sexes in this regard may reflect the fundamental difference in the role of bam in male vs. female early germ cells. Loss of function of TGFß class signaling in male GSCs has a subtle, but opposite, effect. Germ-line clones homozygous mutant for the TGFß class receptor sax appear at lower frequency and tend to produce fewer differentiating cysts compared to control clones. Of course, data from clonal analysis must always be interpreted with caution because of the possibility of effects from secondary recessive mutations on the chromosome arm. However, given the observations on the effects of forced expression of bam, it is tempting to speculate that loss of function of sax from germ cells allows bam to be expressed too early in male GSCs and gonialblasts, slowing or arresting differentiation of spermatogonial cysts and eventually leading to early germ cell loss. It is noted that some sax mutant germ-line clones did persist over time, again suggesting that male GSCs appear less sensitive than female early germ cells to either loss of function of TGFß signaling or overactivation of the receptor (Schulz, 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).

twin, a CCR4 homolog, regulates cyclin poly(A) tail length to permit Drosophila oogenesis: Twin/Ccr4 activity is necessary for wild-type Bam expression

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).

Regulatory relationship among piwi, pumilio, and bag-of-marbles in Drosophila germline stem cell self-renewal and differentiation

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).

Gene circuitry controlling a stem cell niche

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).

Otefin, a nuclear membrane protein, determines the fate of germline stem cells in Drosophila via interaction with Smad complexes

Nuclear envelope proteins play important roles in chromatin organization, gene regulation, and signal transduction; however, the physiological role of these proteins remains elusive. This study found that otefin (ote), which encodes a nuclear lamin, is essential for germline stem cell (GSC) maintenance. Ote, as an intrinsic factor, is both necessary and sufficient to regulate GSC fate. Furthermore, ote is required for the Dpp/BMP signaling pathway to silence bam transcription. By structure-function analysis, it was demonstrated that the nuclear membrane localization of Ote is essential for its role in GSC maintenance. Ote physically interacts with Medea/Smad4 at the bam silencer element to regulate GSC fate. Thus, this study demonstrates that specific nuclear membrane components mediate signal-dependent transcriptional effects to control stem cell behavior (Jiang, 2008).

In adult tissues, stem cells are characterized by their unique capacity to produce daughter stem cells for self-renewal as well as differentiated daughter cells for maintaining homeostasis. Understanding how the self-renewal and differentiation processes of stem cells are controlled will not only reveal the fundamental biological mechanisms that govern the formation and maintenance of tissues, but may also influence future stem cell-based therapies for regenerative medicine (Jiang, 2008).

The Drosophila ovarian germline stem cells (GSCs) within the germarium region provide an attractive system to study the regulatory mechanisms that determine stem cell fate. A typical Drosophila ovary is composed of 16-20 ovarioles, each consisting of an anterior functional unit called a germarium and a linear string of differentiated egg chambers posterior to the germarium. In the tip of the germarium, GSCs normally divide asymmetrically to ensure that one daughter remains attached to the stromal somatic cap cells (or niche cells) for self-renewal. The remaining daughter cell is displaced from the niche and becomes a cystoblast (CB), which initiates differentiation and sustains oogenesis. During this process, one gene, bag-of-marbles (bam), has been shown to act autonomously in the germline to play an instructive role in CB differentiation. In contrast, gene products, such as Piwi and Dpp, a homolog of BMP2/4 in mammals, are produced from niche cells; however, they function as maintenance factors for GSC self-renewal. It has been shown that Dpp signaling from stromal cells activates Smad signaling in GSCs, directly silences bam transcription, and blocks the formation of Bam:Bgcn complexes that would otherwise antagonize translational repression. However, the issue of how Dpp/Smad signaling is transduced in the nucleus and acts especially at the bam silencer element to repress bam transcription remains poorly understood (Jiang, 2008).

The nuclear envelope separates the nucleoplasm from cytoplasm and is composed of outer and inner membranes that are separated by the perinuclear space and joined at nuclear pore complexes. The nuclear lamina is a network of lamin polymers and lamin-associated proteins that are embedded in the inner membrane. Increasing evidence indicates that these nuclear membrane proteins play important roles in chromatin organization, gene regulation, and signal transduction at the cellular level. However, the physiological roles of these proteins remain elusive. Otefin (Ote) is one member of the 'LEM' family, which represents an important group of nuclear membrane-associated proteins that share a conserved LEM domain. Previous studies have shown that Ote physically interacted with lamin B and YA proteins and localized at the nuclear envelope. Although inhibition of lamin activity by anti-lamin antibody prevented nuclear assembly in vitro, RNAi experiments demonstrated that knockdown of Ote exhibited no effect on Drosophila Kc167 cells, which suggests that Ote might not be a limiting component for the maintenance of the nuclear architecture. Thus, the function and physiological role of Ote remain elusive (Jiang, 2008).

This study shows that otefin (ote), which encodes a nuclear lamin, is essential for GSC maintenance. Ote, as an intrinsic factor, is both necessary and sufficient for GSC maintenance by silencing bam transcription via interaction with Dpp signaling. Furthermore, nuclear membrane localization of Ote is critical for its function in the maintenance of GSC. Finally, biochemical evidence is presented to support that Ote physically interacts with Medea, a Drosophila Smad4, at the bam silencer element to regulate GSC fate. Thus, these data indicate that an integral membrane protein, the nuclear lamin Ote, functions at target gene loci to mediate BMP signal-dependent repression (Jiang, 2008).

This study has identified and characterized Otefin (Ote) as a protein the plays an important role in the regulation of GSC fate via BMP/Dpp signaling. The data support the notion that specific nuclear membrane components mediate signal-dependent transcriptional effects to control stem cell behavior (Jiang, 2008).

Observation of the abnormality and loss of germ cells in ote mutant ovaries prompted an exploration of whether ote is involved in the regulation of GSC fate. Using germline clonal analysis and rescue tests, it was demonstrated that ote plays an intrinsic role in GSC self-renewal. In addition, it was also observed that ectopic expression of ote increased the number of GSC-like cells, most likely through repression of GSC/CB differentiation. Thus, the results suggest that, like Dpp signaling, Ote is also both necessary and sufficient to regulate GSC fate. A previous study has demonstrated that knockdown of Ote by RNAi interference exhibited no effect on Drosophila Kc167 cells, suggesting that Ote might not be a limiting factor for the maintenance of the nuclear architecture in cultured cells. Consistently, clonal data showed that ote mutant GSCs could develop into normal cysts and egg chambers rather than undergo apoptosis, suggesting that Ote plays a specific role in maintaining GSC self-renewal but not germ cell viability. As supportive evidence, it was also shown that loss of function of ote did not affect the nuclear architecture and the normal expression of other nuclear lamin components in ovaries. In addition, it was found, except in germ cells, neither overexpression nor loss of function of ote exhibited obvious defects in other developmental processes. Together, these data suggest that Ote may play a role in the maintenance of GSC and germ cell development rather than performing a general cell biological function (Jiang, 2008).

Previous studies have revealed two major signaling mechanisms, dpp-dependent bam transcriptional silencing and bam-independent translational repression, that function cooperatively in the repression of GSC differentiation. In GSCs, Pum/Nos-mediated and microRNA-mediated translational control have been proposed to repress translation of the mRNA pool that promotes GSC/CB differentiation; in contrast, Dpp signaling from the niche cells is responsible for silencing bam transcription in GSCs by activating Smad complexes that physically bind the bam silencer element. Thus, the question becomes how Ote integrates into this signal network. Several lines of genetic evidence strongly suggest that Ote acts through the Dpp signaling pathway rather than through a parallel (Dpp-independent) pathway. (1) The removal of Ote activity not only results in the loss of GSCs, but also replicates the mad or med mutant phenotypes. (2) ote suppressed the TKVca-overexpression phenotype, suggesting that the function of Dpp signaling required Ote activity in order to repress germ cell differentiation. (3) Genetic analysis showed that the ote and dpp pathway are functionally dependent on each other. Thus, these results strongly suggest that Ote serves as a positive component in the Dpp signaling pathway rather than acting through a parallel (Dpp-independent) pathway to regulate GSC fate (Jiang, 2008).

The loss of ote results in a female sterile phenotype but does not affect Dpp signaling in other developmental stages, implying that Ote regulates Dpp signaling only in the ovary. It is possible that ote plays a specific role in regulation of the Dpp pathway in ovary, but is dispensable for dpp pathway regulation in other tissues. A similar example is brinker (brk), which also functions in a tissue-specific manner. It has been shown that brk acts as a negative regulator of the dpp pathway in wing growth control; however, it is dispensable for the dpp pathway in the regulation of GSC fate. Another possibility is that ote could have a redundant function with other nuclear membrane protein(s) in the regulation of the dpp pathway in other tissues (Jiang, 2008).

Structure-function analysis revealed that nuclear membrane localization is essential for Ote function in the regulation GSC fate, the co-IP and FRET assays showed a direct interaction between Ote and Med at the nuclear membrane, and the ChIP assay verified that Ote associated with the bam silencer element in a Med-dependent manner, indicating that Ote/Med interaction might be important for recruiting the bam locus to the nuclear envelope. Combined with the data that Ote is necessary and sufficient for bam silencing in vivo, the results further suggest that Ote/Med-mediated relocalization of the bam locus to the nuclear periphery might be important for bam silencing in the regulation of GSC fate. It has been proposed that subnuclear environments at the nuclear periphery promote gene silencing and activation. Silenced regions of the genome, such as centromeres and telomeres, are statically tethered to the nuclear envelope. Thus, Ote/Med interaction recruiting the bam locus to the nuclear periphery that results in bam silencing may provide an interesting example to support the role of the nuclear periphery in target-gene silencing at the transcriptional level to maintain the identity of the specific type cells (Jiang, 2008).

It has been shown that Schnurri (Shn), a negatively acting Mad cofactor, is genetically required for GSC maintenance. The biochemical evidence showed that the bam silencer element could also form a ShnCT-containing protein-DNA complex with high affinity when Dpp signaling was activated. Thus, studies have proposed that Shn probably serves as a component in the bam silencing complexes/Smad complexes required for bam silencing, and germline stem cells are maintained by Shn recruitment to the bam silencer element. However, so far, the direct experimental evidence that loss of shn results in derepression of bam in GSCs is still lacking. Since Shn, like Ote, has tissue-specific functions mediated by its ability to confer repressive activity on Smad complexes, it will be interesting to test whether Shn acts together with Ote at the bam silencer element in GSCs (Jiang, 2008).

The LEM family represents an important group of nuclear membrane-associated proteins that share a conserved LEM domain. A number of studies have focused on the potential biochemical properties of these proteins and their relationship with nuclear assembly and cell division at the cellular level. Recently, several studies revealed that certain nuclear envelope components are involved in signal transductions, such as MAN1, a nuclear membrane protein that binds Smad2 and Smad3 and antagonizes TGF-β signaling in vertebrates. These findings are in contrast with the current results indicating that Ote functions positively to regulate Dpp signaling transduction in the regulation of GSC. It has been reported that a Drosophila LEM domain protein encoded by the annotated gene CG3167, named dman1, is the putative ortholog to vertebrate MAN1. Similar to Ote, downregulation of dMAN1 by RNAi has no obvious effect on Kc167 cells, suggesting that the dMAN1 protein is also not a limiting component of the nuclear architecture either. Since ote and dman1 possess opposite roles in the regulation of TGF-β/BMP signaling, and dMan1 potentially interacts with Mad in yeast two-hybrid assays and co-IP assays in S2 cells, it would be interesting to determine whether Ote and dMan1 collaborate together to balance the self-renewal and differentiation of GSCs by controlling the proper induction of Dpp pathway activity. There is no known counterpart to Ote in mammals; however, Emerin has a domain arrangement similar to Ote, since it also contains a LEM at its N terminus and a single TM at its C terminus. It has been reported that mutations in emerin cause Emery-Dreifuss muscular dystrophy in humans; however, the molecular mechanism of these mutations and their phenotypes remain poorly understood. This study has characterized a new role in the regulation of stem cells for the nuclear lamin Otefin. It will also be interesting to determine whether nuclear lamina components in mammals, including humans, are also involved in fate determination of stem cells, as well as in mediating signal-dependent gene silencing related to human diseases (Jiang, 2008).

DNA damage-induced CHK2 activation compromises germline stem cell self-renewal and lineage differentiation

This study used germline stem cells (GSCs) in the Drosophila ovary to show that DNA damage retards stem cell self-renewal and lineage differentiation in a CHK2 kinase-dependent manner. Both heatshock-inducible endonuclease I-CreI expression and X-ray irradiation can efficiently introduce double-strand breaks in GSCs and their progeny, resulting in a rapid GSC loss and an accumulation of ill-differentiated GSC progeny. Elimination of CHK2 or its kinase activity can almost fully rescue the GSC loss and the progeny differentiation defect caused by DNA damage induced by I-CreI or X-ray. Surprisingly, checkpoint kinases ATM and ATR have distinct functions from CHK2 in GSCs in response to DNA damage. The reduction in BMP signaling and E-cadherin only makes limited contribution to DNA damage-induced GSC loss. Finally, DNA damage also decreases the expression of the master differentiation factor Bam in a CHK2-dependent manner, which helps explain the GSC progeny differentiation defect. Therefore, this study demonstrates, for the first time in vivo, that CHK2 kinase activation is required for the DNA damage-mediated disruption of adult stem cell self-renewal and lineage differentiation, and might also offer novel insight into how DNA damage causes tissue aging and cancer formation. It also demonstrates that inducible I-CreI is a convenient genetic system for studying DNA damage responses in stem cells (Ma, 2016).

Stem cells in adult tissues are responsible for generating new cells to combat against aging, and could also be cellular targets for tumor formation. Although aged stem cells have been shown to accumulate DNA damage, it remains largely unclear how DNA damage affects stem cell self-renewal and differentiation. A previous study has reported that upon weak irradiation apoptotic differentiated GSC progeny can prevent GSC loss by activating Tie-2 receptor tyrosine kinase signaling (Xing, 2015). This study shows that temporally introduced DNA double-stranded breaks cause premature GSC loss and slow down GSC progeny differentiation. Mechanistically, DNA damage causes GSC loss at least via two independent mechanisms, down-regulation of BMP signaling and E-cadherin-mediated GSC-niche adhesion as well as CHK2 activation- dependent GSC loss. In addition, CHK2 activation also decreases Bam protein expression by affecting its gene transcription and translation, slowing down CB differentiation into mitotic cysts and thus causing the accumulation of CB-like cells. Surprisingly, unlike in many somatic cell types, ATM, ATR, CHK1 and p53 do not work with CHK2 in DNA damage checkpoint control in Drosophila ovarian GSCs. Therefore, this study demonstrates that DNA damage-induced CHK2 activation causes premature GSC loss and also retards GSC progeny differentiation. The findings could also offer insight into how DNA damage affects stem cell-based tissue regeneration. In addition, this study also shows that the inducible I-CreI system is a convenient method for studying stem cell responses to transient DNA damage because it does not require any expensive irradiation equipment as the X-ray radiation does (Ma, 2016).

DNA damage normally leads to cell apoptosis to eliminate potential cancer- forming cells. This study, shows that transient DNA damage causes GSC loss not through apoptosis based on twopieces of experimental evidence: first, DNA-damaged GSCs are not positive for the cleaved Caspase-3, a widely used apoptosis marker; Second, forced expression of a known apoptosis inhibitor p35 does not show any rescue effect on DNA damage-induced GSC loss. Thus, DNA damage-induced GSC loss is likely due to self-renewal defects though the possibility could not be ruled out that other forms of cell death are responsible. p53 is known to be required for DNA damage-induced apoptosis from flies to humans. This study, however, demonstrates that p53 prevents the DNA damage-induced GSC loss. Vacating DNA-damaged GSCs from the niche via differentiation might allow their timely replacement and restoration of normal stem cell function. Therefore, the findings argue strongly that DNA damage primarily compromises self-renewal, thus causing GSC loss. Both niche-activated BMP signaling and E-cadherin-mediated cell adhesion are essential for GSC self-renewal. Consistent with the idea that DNA damage compromises GSC self-renewal, it significantly decreases BMP signaling activity and apical accumulation of E-cadherin in GSCs. Since constitutively active BMP signaling alone or in combination with E-cadherin overexpression can only moderately rescue GSC loss caused by DNA damage, it is concluded that decreased BMP signaling and apical E-cadherin accumulation might partly contribute to the DNA damage-induced GSC loss. Therefore, the findings suggest that DNA damage-mediated down-regulation of BMP signaling and E-cadherin-mediated adhesion only moderately contributes to the GSC loss (Ma, 2016).

DNA damage leads to checkpoint activation and cell cycle slowdown, thus giving more time for repairing DNA damage. In various cell types, ATM-CHK2 and ATR-CHK1 kinase pathways are responsible for DNA damage-induced checkpoint activation. During Drosophila meiosis, ATR, but not ATM, is required for checkpoint activity, indicating that ATM and ATR could have different functions in germ cells. Both ATR and CHK2 have been shown to be required for DNA damage-evoked checkpoint control in Drosophilagerm cells and embryonic cells, while CHK1 can control the entry into the anaphase of cell cycle in response to DNA damage, the G2-M checkpoint activation as well as the Drosophila midblastula transition (Ma, 2016).

This study has shown that these four checkpoint kinases function differently in GSCs. First, CHK2 is required for DNA damage-induced GSC loss, but is dispensable for normal GSC maintenance. Particularly, inactivation of its kinase activity can almost fully rescue DNA damage-induced GSC loss. Interestingly, inactivation of CHK2 function can also rescue the female germ cell defect caused by DNA damage in the mouse ovary, indicating that CHK2 function in DNA damage checkpoint activation is conserved at least in female germ cells. However, it remains unclear if CHK2 behaves similarly in mammalian stem cells in response to DNA damage. Second, ATM promotes GSC maintenance in the absence and presence of DNA damage. This is consistent with the finding that ATM is required for the maintenance of mouse male germline stem cells and hematopoietic stem cells. It will be interesting to investigate if ATM also prevents the oxidative stress in Drosophila GSCs as in mouse hematopoietic stem cells. Third, ATR is dispensable for normal GSC maintenance, but it protects GSCs in the presence of DNA damage. Although CHK2 and ATR behave similarly in DNA damage checkpoint control during meiosis and late germ cell development, they behave in an opposite way in GSCs in response to DNA damage. Finally, CHK1 is dispensable for GSC self-renewal in the absence and presence of DNA damage. Consistent with the current findings, the females homozygous for grp, encoding CHK1 in Drosophila, can still normally lay eggs, but those eggs could not develop normally. It will be of great interest in the future to figure out how CHK2 inactivation prevents DNA damage-induced GSC loss and how ATM and ATR inactivation promotes DNA damage-induced GSC loss at the molecular level. A further understanding of the functions of CHK2, ATM and ATR in stem cell response to DNA damage will help preserve aged stem cells and prevent their transformation into CSCs. DNA damage-evoked CHK2 activation retards GSC progeny differentiation by decreasing Bam expression at least at two levels This study has also revealed a novel mechanism of how DNA damage affects stem cell differentiation. Bam is a master differentiation regulator controlling GSC- CB and CB-cyst switches in the Drosophila ovary: CB-like single germ cells accumulate in bam mutant ovaries, whereas forced Bam expression sufficiently drives GSC differentiation. This study shows that DNA damage causes the accumulation of CB-like cells in a CHK2- dependent manner because CHK2 inactivation can fully rescue the germ cell differentiation defect caused by DNA damage. In addition, a heterozygous bam mutation can drastically enhance, and forced bam expression can completely repress, the DNA damage-induced germ cell differentiation defect, indicating that DNA damage disrupts Bam-dependent differentiation pathways. Consistently, Bam protein expression is significantly decreased in DNA damaged mitotic cysts in comparison with control ones. Interestingly, CHK2 inactivation can also fully restore Bam protein expression levels in the DNA-damaged mitotic cysts. Taken together, CHK2 activation is largely responsible forBam down-regulation in DNA damaged mitotic cysts, which can mechanistically explain the DNA damage-induced germ cell differentiation defect. It was further shown that DNA damage decreases Bam protein expression at least at two different levels. First, the bam transcription reporter bam-gfp was used to show that DNA damage decreases bamtranscription in CBs and mitotic cysts. Second, the posttranscriptional reporter Pnos-GFP-bam 3'UTR was generated to show that DNA damage decreases Bam protein expression via its 3'UTR in CBs and mitotic cysts at the level of translation. Although the detailed molecular mechanisms underlying regulation of Bam protein expression by DNA damage await future investigation, these findings demonstrate that DNA damage causes the GSC progeny differentiation defect by decreasing Bam protein expression at transcriptional and translational levels (Ma, 2016).

Taken together, these findings from Drosophila ovarian GSCs could offer important insight into how DNA damage affects stem cell-based tissue regeneration, and have also established Drosophila ovarian GSCs as a new paradigm for studying how DNA damage affects stem cell behavior at the molecular level. Because many stem cell regulatory strategies are conserved from Drosophilato mammals, what has been learned from this study should help understand how mammalian adult stem cells respond to DNA damage (Ma, 2016).

Post-transcriptional regulation

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 and Bgcn antagonize Nanos-dependent germ-line stem cell maintenance

The balance between germ-line stem cell (GSC) self-renewal and differentiation in Drosophila ovaries is mediated by the antagonistic relationship between the Nanos (Nos)-Pumilio translational repressor complex, which promotes GSC self-renewal, and expression of Bam, a key differentiation factor. This study found that Bam and Nos proteins are expressed in reciprocal patterns in young germ cells. Repression of Nos in Bam-expressing cells depends on sequences in the nos 3'-UTR, suggesting that Nos is regulated by translational repression. Ectopic Bam causes differentiation of GSCs, and this activity depends on the endogenous nos 3'-UTR sequence. Previous evidence showed that Bgcn is an obligate factor for the ability of Bam to drive differentiation, and this study reports that Bam forms a complex with Bgcn, a protein related to the RNA-interacting DExH-box polypeptides. Together, these observations suggest that Bam-Bgcn act together to antagonize Nos expression; thus, derepressing cystoblast-promoting factors. These findings emphasize the importance of translational repression in balancing stem cell self-renewal and differentiation (Li, 2009).

Previous studies show GSC maintenance dually depends on Nos expression to suppress CB differentiation and on transcriptional silencing of bam. Expression of Bam acts as a developmental switch, and is both necessary and sufficient to drive germ cell differentiation. Elucidating the biochemical activity of Bam has been impeded, however, because both the low abundance and lack of recognizable functional domains of the protein. The goals of the experiments presented in this article were to provide new insights into the function of Bam by finding protein partners and the downstream targets of Bam action (Li, 2009).

The translational repressor proteins Pum and Nos are critical GSC maintenance factors and suppress CB differentiation, perhaps by repressing translation of a pool of cystoblast (CB) promoting mRNAs stored in GSCs. The dynamic pattern of Nos accumulation in the germarium suggested the protein disappears as CB differentiation begins. Decreasing levels of Nos expression during CB differentiation are unlikely to reflect changes in the transcription, because a GFP reporter fused to the nos promoter remains active throughout the germarium. Instead, Nos elimination within early cysts is mediated by sequences in the 3′-UTR of the transcripts. Substituting a tubulin 3′-UTR for the endogenous nos 3′-UTR resulted in uniform Nos protein expression throughout the germarium. Further work has narrowed down the region responsible for translational repression of nos in the germarium to the first 100 bases of the 3′-UTR of the transcript (Li, 2009).

Genetic experiments with bam and pum alleles has suggested that the 2 genes exerted opposite actions on CB differentiation. Nos accumulation declines when Bam is expressed ectopically in several genetic backgrounds, suggesting that Nos accumulation can be linked directly to Bam protein levels and not to signals from somatic cells in the germarium. Data showing that diminished bam or bgcn gene dosage could suppress the germ cell loss phenotype of nos alleles provided additional evidence for the inverse relationship of bam and nos expression. A reduction in bam or bgcn dose may decrease the likelihood that a nos⁻ GSC differentiates precociously, because stem cells are more likely to be maintained in these ovaries. The relevant antagonism could take place within the transient cell identified as the 'precystoblast'. It is also possible that nos⁻ primordial germ cells are more likely to be captured as stem cells during gonadogenesis when bam or bgcn levels were reduced (Li, 2009).

The nos 3′-UTR is essential for proper regulation, because the CB differentiation induced by ectopic Bam expression fails when [hs-Bam] flies carry a Nos-tub3′-UTR transgene. Surprisingly, cyst formation proceeds normally in ovaries carrying the Nos-tub3′-UTR transgene even though ectopic expression would be expected to promote GSC self-renewal. One possible explanation is provided by observations that Pum levels also fall as CBs differentiate. Pum levels become limiting even as Nos continues to accumulate from the p[nosP-Nos-tub 3′-UTR] transgene. Likewise, redundant pathways may exist to derepress translation of CB-promoting mRNAs, just as multiple pathways appear to exist to silence those mRNAs. If, like Nos-Pum, the miRNA pathway is down-regulated to initiate differentiation, derepression of CB-promoting mRNAs might occur even if Nos expression is maintained during CB and cyst stages. Because ectopic Ago1 expression, but not Nos, produces extra GSCs, miRNAs might be separate and prominent repressors of CB differentiation to maintain GSCs (Li, 2009).

Together, these genetic and biochemical experiments suggest that Bam and Bgcn form a complex that represses nos translation, either directly or indirectly. Mechanistically, it is considered possible that Bam-Bgcn and perhaps other proteins directly repress nos mRNA by binding sequences in the nos 3′-UTR. However, it has not been possible to demonstrate a direct physical interaction between Bam-Bgcn and nos mRNA, either from ovary extracts or in vitro. Likewise, attempts to reconstitute Bam-Bgcn-dependent translational repression of the nos 3′-UTR in S2 cells failed. These experiments may have failed because S2 cells lack important, but as yet unidentified, cofactors found specifically in germ cells, or because Bam-Bgcn regulate nos translation via an indirect mechanism. For example, it is plausible that Bam-Bgcn promote the expression of the early response target mRNAs, and one or more of these factors could repress nos translation. Alternative mechanisms of action for Bam-Bgcn are unclear as Bam lacks any defined sequence motifs and Bgcn, whereas related throughout the length of the protein to RNA/DNA helicases, lacks the motifs to be a functional helicase (Ohlstein, 2000). Outside the Bam-interacting domains, Bgcn contains a pair of ankyrin repeats that could mediate other protein–protein interactions (Li, 2009).

One potential component of the Bam-Bgcn complex, Mei-P26, was suggested by previous genetic experiments. mei-P26 has been identified as a gene required for early germ cell differentiation and meiosis, and showed that mei-P26 activity depends on the proper dosage of bam. Recently, bam has been reported to require mei-P26 to deplete stem cells, and similarly, mei-P26 required bam to function properly. These observations could imply a close working relationship between bam and mei-P26. However, the interactions and interrelated functions of Bam, Bgcn, and Mei-P26 are likely to be complex. For example, although the phenotypes of bam and bgcn mutations are indistinguishable, the mei-P26 mutant phenotype is distinct. Germ cells lacking mei-P26 apparently form CBs, because they produce Bam-positive cysts with branched fusomes. Given the current results, exploring the functional significance of Bam, Bgcn, and Mei-P26 interactions will be important (Li, 2009).

The view of stem cells that emerges from these studies has several striking elements: (1) that repression mechanisms control many stem cell differentiation circuits, and (2) that translational regulation has an integral role in these decisions. The GSC model highlights an intrinsic capacity to differentiate and the need to apply brakes (Nos-Pum) to retard differentiation. Perhaps this mechanism was advantageous to prevent all gametes from maturing at once in animals that developed with a finite number of germ cells. Of course, differentiation would require a mechanism (Bam-Bgcn) to override the brakes. Within this framework, a stem cell population could arise when a group of stromal cells captured germ cells and produced signals that could repress expression of the factor(s) that would antagonize the brakes. Natural selection would rapidly fix this event, because it would greatly expand the number of gametes produced from individuals by establishing a stem cell as a renewable source of germ cells. This mode of niche evolution might also explain the appearance of stem cell populations in most organs, because it would be expected to enhance fitness by permitting larger body size, lengthening the fecund lifespan and increasing survivability of trauma by providing a mechanism for tissue regeneration. If the mechanisms at work in Drosophila GSCs apply to many stem cells, stem cells should be enriched for many more antidifferentiation genes than true stemness genes (Li, 2009).

Direct inhibition of Pumilo activity by Bam and Bgcn in Drosophila germ line stem cell differentiation

The fate of stem cells is intricately regulated by numerous extrinsic and intrinsic factors that promote maintenance or differentiation. The RNA-binding translational repressor Pumilio (Pum) in conjunction with Nanos (Nos) is required for self-renewal, whereas Bam (bag-of-marbles) and Bgcn (benign gonial cell neoplasm) promote differentiation of germ line stem cells in the Drosophila ovary. Genetic analysis suggests that Bam and Bgcn antagonize Pum/Nos function to promote differentiation; however, the molecular basis of this epistatic relationship is currently unknown. This study shows that Bam and Bgcn inhibit Pum function through direct binding. A ternary complex involving Bam, Bgcn, and Pum has been identified in which Bam, but not Bgcn, directly interacts with Pum, and this interaction is greatly increased by the presence of Bgcn. In a heterologous reporter assay to monitor Pum activity, Bam, but not Bgcn, inhibits Pum activity. Notably, the N-terminal region of Pum, which lacks the C-terminal RNA-binding Puf domain, mediates both the ternary protein interaction and the Bam inhibition of Pum function. These studies suggest that, in cystoblasts, Bam and Bgcn may directly inhibit Pum/Nos activity to promote differentiation of germ line stem cells (Kim, 2010).

Two important intrinsic factors, Bam and Bgcn, play critical roles in stem cell differentiation. Loss-of-function mutations in either Bam or Bgcn cause stem cell differentiation to arrest. Conversely, ectopic expression of Bam in stem cells overrides stem cell self-renewal capabilities and promotes differentiation. Genetic analyses have shown that Bam and Bgcn require each other for function. Bgcn is present in stem cells as well as cystoblasts and early mitotic cysts, whereas Bam is not expressed in stem cells but is expressed in cystoblasts and early mitotic cysts. Bam silencing in stem cells is governed by the BMP2/4 homolog Decapentaplegic signal emanating from the niche cells (Kim, 2010 and references therein).

In addition to the extrinsic factors emanating from niche cells, stem cell maintenance requires intrinsic stem cell factors. Pumilio (Pum) and Nanos (Nos) are such intrinsic factors. Pum is an RNA-binding protein with a C-terminal Puf (Pum and Fem3-binding factor) domain, which binds the Nanos response element (NRE) sequences at the 3'-untranslated region of its target mRNAs. Binding of the Puf domain to NRE recruits Nos to this complex, resulting in the repression of the translation of the target mRNAs. Because Pum and Nos are required for repression of differentiation in germ line stem cells, it is conceivable that this complex targets a suite of genes that are required for differentiation, although the identities of these genes are unknown (Kim, 2010 and references therein).

Genetic epistasis analysis of double mutants of Bam and Pum indicated that Bam antagonizes Pum function to promote differentiation of stem cells. For the differentiating cystoblasts to begin differentiation, the Pum/Nos activity must be inhibited in the cystoblast. This study explored the possibility that the Bam-Bgcn complex may inhibit Pum-Nos activity at the protein level and discovered a direct interaction between Bam and Pum. Notably, the Bam-Pum interaction is greatly increased in the presence of Bgcn, and this interaction allows for the formation of a strong ternary complex involving Bam, Bgcn, and Pum. Consistent with this physical interaction, Bam inhibits Pum activity in a heterologous reporter assay, which monitors the activity of Pum. On the other hand, no ternary interaction between Bam, Bgcn, and Nos was detected, suggesting that Bam and Bgcn specifically target Pum directly to negatively regulate Pum/Nos activity and promote stem cell differentiation (Kim, 2010).

Previous genetic analysis suggested that Bam and Bgcn form a complex because they require each other for function. Therefore this study utilized diverse assays to probe the biochemical relevance of these genetic results. Surprisingly, both the fragment complementation analysi (FCA) and the yeast two-hybrid assay failed to detect any interaction between Bam and Bgcn. However, the two assays detected a strong Bam-Bgcn-Pum complex. In contrast, the co-immunoprecipitation assay detected direct Bam-Bgcn interaction without Pum involvement, which is in accord with other recent reports. The inability to detect direct Bam-Bgcn interaction by the FCA and the yeast two-hybrid assay may indicate that Bam-Bgcn interaction is weak in vivo (Kim, 2010).

Both yeast two-hybrid and FCA showed that there is a weak interaction between Bam and Pum. Particularly, the interaction revealed by FCA appears authentic because the Bam-Pum interaction brought the N- and C-terminal fragments of fluorescent reporter mKG (monomeric Kusabira-Green) into the cytoplasm, reflecting the cytoplasmic localization of Bam and Pum. In contrast, the control interaction of the p65 and p55 subunits of NF-kappaB occurred in the nucleus. Importantly, both the FCA and yeast tri-hybrid assay detected a strong ternary interaction involving Bam, Bgcn, and Pum, suggesting that weak interaction between Bam and Pum is greatly enhanced by the presence of Bgcn, through additional Bam-Bgcn interaction (Kim, 2010).

The ternary interaction involving Bam and Bgcn is mediated by the N terminus of Pum, which lacks the C-terminal Puf region. Consistent with this, the Puf region fails to form a ternary complex formation with Bam and Bgcn. It is known that the Puf domain mediates both Nanos response element (NRE) binding and Nos binding of Pum. The binding of Bam and Bgcn to the N-terminal region of Pum appears not to interfere with the binding of Nos to the Puf region, because Bam immunoprecipitates contained Bgcn, Pum, and Nos. Neither Bam nor Bgcn binds to Nos, and a ternary complex involving Bam, Bgcn, and Nos was not observed. Therefore, these results indicate that Pum can recruit both Bam/Bgcn and Nos in distinct sites and thus can account for the fact that Bam precipitates contain Bgcn, Pum, and Nos (Kim, 2010).

Using a luciferase reporter system involving the NRE sequence at the 3'-untranslated region, the relevance of Bam/Bgcn binding to Pum activity was addressed in heterologous cells. Expression of Pum repressed luciferase expression, which requires an intact NRE sequence. Bam was able to abrogate this repression by Pum, suggesting that a weak interaction between Bam and Pum is sufficient for Bam inhibition of Pum activity in this assay. The Bam inhibition of Pum function appears to require Bam binding to Pum, because Bam does not bind to Puf and failed to abrogate Puf-dependent repression. Bgcn failed to interact with Pum or affect Pum repression of the reporter gene expression. These results yield insight into the role of Bgcn in vivo and suggest that Bgcn may be confined to facilitating Bam binding to Pum under physiological conditions where Bam protein levels may not be sufficient for the binding and inhibition of Pum (Kim, 2010).

In conclusion, following stem cell division, one daughter cell moves away from the niche cells and begins to initiate differentiation as a cystoblast. For the cystoblast to begin differentiation, Pum/Nos activity must be inhibited in the cystoblast and early dividing germ cells. One possible mechanism for this inhibition is the decrease of Pum and Nos at the protein level. In fact, these levels are gradually reduced in the cystoblasts and immediate early dividing cysts; however, not all Pum and Nos protein disappears. Thus, other mechanisms must exist to inhibit Pum/Nos activity in the differentiating cells. These data suggest that Bam and Bgcn present in the cystoblast cells play such a role by binding and inhibiting Pum directly at the protein level (see Model depicting Bam/Bgcn binding and inhibition of Pum/Nos activity). This notion is consistent with findings that ectopic Bam expression in stem cells triggers stem cell differentiation, which might occur because of direct Bam/Bgcn inhibition of Pum/Nos activity (Kim, 2010).

A self-limiting switch based on translational control regulates the transition from proliferation to differentiation in an adult stem cell lineage

In adult stem cell lineages, progenitor cells commonly undergo mitotic transit amplifying (TA) divisions before terminal differentiation, allowing production of many differentiated progeny per stem cell division. Mechanisms that limit TA divisions and trigger the switch to differentiation may protect against cancer by preventing accumulation of oncogenic mutations in the proliferating population. This study shows that the switch from TA proliferation to differentiation in the Drosophila male germline stem cell lineage is mediated by translational control. The TRIM-NHL tumor suppressor homolog Mei-P26 facilitates accumulation of the differentiation regulator Bam in TA cells. In turn, Bam and its partner Bgcn bind the mei-P26 3' untranslated region and repress translation of mei-P26 in late TA cells. Thus, germ cells progress through distinct, sequential regulatory states, from Mei-P26 on/Bam off to Bam on/Mei-P26 off. TRIM-NHL homologs across species facilitate the switch from proliferation to differentiation, suggesting a conserved developmentally programmed tumor suppressor mechanism (Insco, 2012).

Adult stem cells act throughout life to replenish differentiated cells lost to turnover or injury. In many adult stem cell lineages, stem cell daughters destined for differentiation first undergo a limited number of mitotic divisions to amplify cell number prior to terminal differentiation. This transit amplifying (TA) division strategy may protect large long-lived animals from tumorigenesis by minimizing the number of stem cell divisions required for tissue homeostasis and preventing accumulation of oncogenic mutations in progenitor cells due to programmed differentiation. The mechanisms that limit the number of TA divisions and initiate terminal differentiation thus may provide tumor suppressor function, and defects may contribute to progression toward cancer in adult stem cell lineages (Insco, 2012).

This study investigated the mechanisms that force TA cells to stop proliferating and initiate terminal differentiation in the Drosophila male germline adult stem cell lineage. Drosophila male germline stem cells (GSCs) reside in a niche at the tip of the testis, attached to somatic hub cells and flanked by somatic cyst stem cells (CySCs). When a GSC divides, one daughter remains in the niche and self-renews, while the other is displaced away and initiates differentiation. The resulting gonialblast, which is enveloped by a pair of CySCs, proceeds through four synchronous TA divisions with incomplete cytokinesis, producing a clone of 16 interconnected germ cells. These 16 mitotic sisters normally stop proliferating, undergo premeiotic DNA synthesis in synchrony, and switch to the spermatocyte program of cell growth, meiosis, and terminal differentiation. Because TA sister cells are contained within a common somatic cell envelope, are joined by cytoplasmic bridges, and divide in synchrony, mutations that cause overproliferation of TA cells can be easily identified (Insco, 2012).

The bag of marbles (bam) gene is required cell autonomously for TA spermatogonia to stop proliferating and enter the spermatocyte differentiation program. Male germ cells mutant for bam undergo several extra rounds of mitotic TA division, fail to differentiate, and eventually die. The number of TA divisions appears to be set by the time required for Bam protein to accumulate to a critical threshold. Bam protein is normally first detected in 4-cell cysts, increases to a peak in 8-cell cysts, and is degraded in early 16-cell cysts immediately after premeiotic DNA replication. Lowering the bam dosage slowed Bam protein accumulation and delayed the transition to differentiation, whereas early accumulation of Bam protein caused a premature switch to differentiation (Insco, 2012).

Bam, a protein with no recognizable domains, acts with a partner, benign gonial cell neoplasm (Bgcn), discovered in a genetic screen for Drosophila tumor suppressors. bam and bgcn have similar mutant phenotypes, and Bam protein directly interacts with Bgcn in Drosophila ovaries or when coexpressed in cultured cells or yeast. Bgcn is related to the DExH-box family of RNA-dependent helicases, indicating that Bgcn, and with it Bam, may regulate RNA (Insco, 2012).

Consistent with a role in translational repression, Bam protein binds the translation initiation factor eIF4A. Furthermore, expression of Bam and Bgcn in Drosophila cultured cells resulted in a 4-fold reduction in expression of a luciferase reporter coupled to the 3′ untranslated region (UTR) of e-cadherin messenger RNA (mRNA), and tethering Bam to the 3′ UTR induced translational repression of the attached reporter. In female germ cells, Bam and Bgcn allow the onset of differentiation through translational repression of nanos (nos) via the nos 3' UTR. However, direct interaction of Bam or Bgcn protein with e-cadherin or nos mRNAs has not been demonstrated (Insco, 2012).

This study identified the microRNA (miRNA) regulator and TRIM-NHL (tripartite motif and Ncl-1, HT2a, and Lin-41 domain) family member Mei-P26 both as a regulator of Bam protein accumulation and, subsequently, as a direct target of translational repression by Bam and Bgcn in male germ cells. Mei-P26 function facilitates both the switch from mitosis to meiosis and spermatocyte differentiation. In mei-P26 mutant males, Bam protein failed to accumulate to its normal peak levels. The overproliferation of TA cells in mei-P26 mutant testes was suppressed by expression of additional Bam, suggesting that the continued TA cell proliferation in mei-P26 mutant males is due to the failure of Bam protein to reach the threshold required for the switch to the spermatocyte state. In turn, Bam specifically binds the mei-P26 3' UTR, and Bam and Bgcn function are required for translational repression of mei-P26 via its 3' UTR in vivo. Mutating two potential let-7 target sites within the mei-P26 3' UTR derepressed reporter expression in vivo and disrupted Bam binding in vitro. These data suggest that a stepwise progression in regulatory states from [Mei-P26 on/Bam off] to [Mei-P26 on/Bam on] to [Bam on/Mei-P26 off], choreographed by translational regulation, accompanies the switch from TA cell proliferation to terminal differentiation in the Drosophila male GSC lineage (Insco, 2012).

It is proposed that Mei-P26 and Bam act in a regulatory cascade based on translational control to affect the switch from TA cell proliferation to spermatocyte differentiation in the Drosophila male GSC lineage. First, wild-type function of Mei-P26 in TA cells facilitates accumulation of Bam protein. Consistent with this model, a mei-P26 hypomorphic allele enhanced the overproliferation of germ cell cysts in a bam/+ heterozygote. Furthermore, the finding that adding one extra copy of bam is sufficient to rescue the early germ cell overproliferation phenotype of mei-P26 mutant males indicates that allowing normal accumulation of Bam is the major role of Mei-P26 in regulating proliferation of early male germ cells. Second, as Bam protein levels rise, Bam and Bgcn repress translation of mei-P26 via its 3' UTR in late TA cell cysts. As a result, GSCs, gonialblasts, and two-cell cysts begin with Mei-P26 expressed and Bam off and transition to 4-cell and early 8-cell cysts wherein both Mei-P26 and Bam protein are expressed. In late 8-cell and early 16-cell cysts, Bam protein levels are high, causing Mei-P26 protein to drop to very low levels. Finally, in early spermatocytes, Mei-P26 levels rise again after Bam protein disappears to facilitate normal differentiation of spermatocytes and spermatids (Insco, 2012).

Recent data suggest that Mei-P26 and related TRIM family proteins may function in the miRNA pathway. Mei-P26, two of its Drosophila homologs, and several mouse homologs have been shown to interact structurally with RISC effector proteins such as Ago-1. Mei-P26 protein localized to cytoplasmic puncta in early male and female germ cells, similar to the punctate distribution of mouse TRIM71. Many of the Mei-P26 puncta colocalized with the RISC component GW182, which accumulates in processing bodies that consist of enzymes involved in mRNA translational repression and degradation. The action of Mei-P26 in early male germ cells may facilitate accumulation of Bam protein by repressing an intermediate negative regulator of Bam. For example, Mei-P26 may function in TA cells to facilitate the accumulation of Bam protein through decreasing the function of the RNA-binding protein HOW. Previous studies suggest that HOW represses Bam expression in early male germ cells. In wild-type testes, HOW protein was expressed in early cells, including GSCs, gonialblasts, and two-cell cysts. However, in mei-P26 mutant testes, HOW protein perdured throughout the overproliferating cysts. Alternatively, given that Mei-P26 also contains a RING domain, it could facilitate degradation of an intermediate that normally degrades Bam. Bam has a C-terminal PEST sequence, a motif that targets proteins for ubiquitination and turnover by the proteasome, and expression of Bam lacking the PEST sequence resulted in early accumulation of high levels of Bam protein and a premature switch to the spermatocyte state (Insco, 2012).

As Bam protein peaks in late TA cells, it acts with its binding partner Bgcn to repress translation of mei-P26 mRNA via sequences in the mei-P26 3' UTR. Bam protein specifically binds the mei-P26 3' UTR, suggesting that Bam and Bgcn act directly as translational repressors. Translational regulation via 3' UTR sequences frequently blocks formation of the translation initiation complex by inhibiting interactions between the cap binding protein eIF4E and the 5′ cap or the rest of the eIF4F complex. Bam protein physically interacts with eIF4A independent of RNA, and eIF4A/+ partially suppressed the phenotype of bam mutants in both the male and female germline systems, raising the possibility that Bam, recruited to a target 3' UTR as part of a translational repressor complex, may block translation initiation by antagonizing eIF4A. Mutating two potential let-7 binding sites within the mei-P26 3' UTR led to derepression of the in vivo reporter and disrupted binding of Bam to the mei-P26 3' UTR. These data raise the possibility that let-7 may work with Bam and Bgcn to translationally repress Mei-P26 in TA cells. In addition, introducing the let-7-C^GK1 loss-of-function allele into a bam^Δ86/+ mutant background enhanced the bam heterozygous mutant phenotype, suggesting that let-7 and Bam may share additional targets within the testes (Insco, 2012).

Mei-P26 appears to play two distinct roles in the female germline as well: an early function in GSC maintenance and a later function required for cystocytes to switch to nurse cell and oocyte differentiation. However, there are also important differences between the male and female germline. Although Mei-P26 protein levels decreased when Bam was expressed in female germ cells, low levels of Mei-P26 were still detected. Bam and Bgcn may inhibit mei-P26 translation in female germ cells, although probably not to the same degree as in males. Notably, the mei-P26 3' UTR cloned from testes lacked the Vasa binding sites shown to be important for Mei-P26 expression in female germ cells. In addition, Bam is active at an earlier stage in the ovary, wherein the function of Bam is necessary for female GSCs to initiate the TA divisions rather than exit the TA divisions, as in males. Finally, Bam and Bgcn may have different mRNA targets in the female germline. In the female, Bam action directly or indirectly represses translation of the translational repressor Nanos, allowing the expression of proteins that initiate germ cell differentiation from the stem cell state. However, Nanos does not appear to play the same role in male as in female GSCs. Thus, the core machinery of Bam, Bgcn, and Mei-P26 probably acts through similar molecular mechanisms in female and male germ cell differentiation, but at a different point in the differentiation pathway, with different regulators and, most likely, on different targets (Insco, 2012).

Strikingly, as is shown in this study for the Drosophila male germline, the switch from mitosis to meiosis is also controlled by a regulatory network based on translational control in the C. elegans germline. BLAST and ClustalW alignments revealed that Bgcn, a core component of the switch mechanism in the Drosophila germline, is a homolog of C. elegans proteins Mog1, Mog4, and Mog5, which are required for stopping mitosis and repressing target-mRNA translation via the 3' UTR (Insco, 2012).

The requirement for TRIM-NHL proteins to facilitate the switch from proliferation to differentiation may be a widely conserved feature in many adult stem cell lineages. In Drosophila, loss of the Mei-P26 homolog dappled causes large melanotic tumors, suggesting the continued proliferation of blood cells. Likewise, loss of the Drosophila Mei-P26 homolog brat in TA cells in certain neural lineages leads to brain tumors that are highly proliferative, invasive, transplantable, and lethal to the animal. In mammals, the mouse Mei-P26 homolog TRIM32 is necessary and sufficient for differentiation in neural lineages, and progenitor cells lacking TRIM32 retain proliferative status. Thus, elucidating the mechanisms by which Mei-P26 homologs and their interacting structural and regulatory partners control the switch from proliferation to differentiation in adult stem cell lineages may uncover a new class of tumor suppressors that act at the level of the developmental program rather than cell-cycle progression (Insco, 2012).

Mei-P26 regulates the maintenance of ovarian germline stem cells by promoting BMP signaling

In the Drosophila ovary, bone morphogenetic protein (BMP) ligands maintain germline stem cells (GSCs) in an undifferentiated state. The activation of the BMP pathway within GSCs results in the transcriptional repression of the differentiation factor bag of marbles (bam). The Nanos-Pumilio translational repressor complex and the miRNA pathway also help to promote GSC self-renewal. How the activities of different transcriptional and translational regulators are coordinated to keep the GSC in an undifferentiated state remains uncertain. Data presented in this study show that Mei-P26 cell-autonomously regulates GSC maintenance in addition to its previously described role of promoting germline cyst development. Within undifferentiated germ cells, Mei-P26 associates with miRNA pathway components and represses the translation of a shared target mRNA, suggesting that Mei-P26 can enhance miRNA-mediated silencing in specific contexts. In addition, disruption of mei-P26 compromises BMP signaling, resulting in the inappropriate expression of bam in germ cells immediately adjacent to the cap cell niche. Loss of mei-P26 results in premature translation of the BMP antagonist Brat in germline stem cells. These data suggest that Mei-P26 has distinct functions in the ovary and participates in regulating the fates of both GSCs and their differentiating daughters (Li, 2012).

Evidence is provided that Mei-P26 promotes GSC self-renewal in addition to its previously described role in negatively regulating the miRNA pathway during germline cyst development. Disruption of mei-P26 results in a bam-dependent GSC loss phenotype and further characterization reveals that Mei-P26 fosters BMP signal transduction within GSCs by repressing Brat protein expression. In addition, Mei-P26 also appears to participate in the miRNA-mediated silencing of orb mRNA in GSCs. These results indicate that Mei-P26 carries out multiple functions within the Drosophila ovary and might be at the center of a molecular hierarchy that controls the fates of GSCs and their differentiating daughters (Li, 2012).

Three observations suggest that mei-P26 functions within GSCs. First, the average number of GSCs per terminal filament decreases from an average of two to well below one in mei-P26 mutant ovaries. Second, mei-P26 mutant germline clones are rapidly lost from the GSC niche. Third, syncytial cysts and Bam-expressing cells are often observed immediately adjacent to the cap cells in mei-P26 mutant ovaries (Li, 2012).

Research over the last ten years has shown that BMP ligands emanating from cap cells at the anterior of the germarium initiate a signal transduction cascade in GSCs that results in the transcriptional repression of bam. Stem cell daughters one cell diameter away from the cap cell niche express bam, suggesting that a steep gradient of Dpp availability or responsiveness exists between GSCs and cystoblasts. Recent work has shed light on how various mechanisms antagonize BMP signaling in cystoblasts. For example, the ubiquitin ligase Smurf (Lack -- FlyBase) promotes germline differentiation and partners with the serine/threonine kinase Fused to reduce levels of the Dpp receptor Tkv in cystoblasts. The TRIM-NHL domain protein Brat also functions in cystoblasts, serving to translationally repress Mad expression. Notably, inappropriate expression of Brat within GSCs results in a stem cell loss phenotype. Brat itself is translationally repressed in GSCs by the Pumilio-Nanos complex. Mutant phenotypes and co-IP experiments presented in this study support a model in which Mei-P26 partners with Nanos to repress Brat expression in GSCs. This negative regulation of Brat expression protects the BMP signal transduction pathway in GSCs from inappropriate deactivation (Li, 2012).

Mei-P26 appears to enhance miRNA-dependent translational silencing within GSCs based on several lines of experimental evidence. First, co-IP experiments using ovarian extracts from c587-gal4>UAS-dpp and bam mutants suggest that Mei-P26 physically associates with Ago1 and GW182 in undifferentiated germ cells. Second, disruption of mei-P26 results in a GSC loss phenotype, similar to the effects of disrupting components of the miRNA pathway tested to date. Third, Mei-P26 and Ago1 can physically associate with the same target mRNA. Finally, disruption of either Ago1 or mei-P26 results in increased expression of this target in GSCs. The evidence that Mei-P26 promotes miRNA action in certain contexts is consistent with the established activities of its close homologs NHL-2 and TRIM32 (Li, 2012 and references therein).

It is proposed that Mei-P26 regulates GSC self-renewal and early germ cell differentiation through distinct mechanisms. In GSCs, Mei-P26 promotes self-renewal by repressing the expression of Brat and potentially other negative regulators of BMP signal transduction. Within stem cells, Mei-P26 also functions together with miRISC to attenuate the translation of specific mRNAs. miRISC does not appear to target brat mRNA based on clonal data. However, the possiblity cannot be ruled out that the enhancement of miRNA-mediated silencing of some mRNAs by Mei-P26 contributes to stem cell self-renewal. Interestingly, recent findings suggest that Pumilio can function together with the miRNA pathway in certain contexts In BJ primary fibroblasts, Pumilio 1, miR-221 and miR-222 regulate the expression of p27 in a 3' UTR-dependent manner. In response to growth factors, Pumilio 1 becomes phosphorylated, which in turn increases its RNA binding activity. Pumilio 1 binding to p27 mRNA results in a conformational change in the 3' UTR that allows miR-221 and miR-222 to bind more efficiently, resulting in greater repression of p27. Perhaps, together, Drosophila Pumilio, Nanos, Ago1 and Mei-P26 also silence specific messages in specific contexts. Identifying more direct in vivo targets for these proteins within GSCs will be crucial for testing this idea (Li, 2012).

In cystoblasts, Mei-P26 promotes germline cyst development by antagonizing the miRNA pathway. This study shows that Mei-P26 can also promote miRNA translational repression in another cell, the GSC. Evidence is provided that Mei-P26 physically associates with miRISC and co-regulates translation of at least one mRNA, orb, through specific elements within its 3′UTR. In cystoblasts and early developing cysts, the induction of Bam expression might cause Mei-P26 to switch from an miRISC-associated silencer to an miRNA antagonist. How Bam activates this switch is currently under investigation. The finding that Mei-P26 functions in both GSCs and differentiating cysts hints at a mechanism whereby different translational repression programs coordinate changes in cell fate (Li, 2012).

Further work will be needed to determine the specific biochemical function of Mei-P26 when it associates with either the Nanos complex or miRISC. Like other TRIM-NHL domain proteins, Mei-P26 contains a RING domain that may have E3 ubiquitin ligase activity. Based on results presented in this study, it is proposed that Mei-P26 and perhaps other TRIM-NHL domain proteins act as effectors for multiple translational repressor complexes. In this model, Mei-P26 is targeted to specific mRNAs through sequence-directed RNA-binding proteins. Specific protein substrates of Mei-P26 in the germline have not yet been determined but identifying these targets will provide key insights into how Mei-P26 and other related TRIM-NHL domain proteins regulate translational repression. Furthermore, the Mei-P26 complex is likely to target additional mRNAs for silencing in both GSCs and developing cysts. Identifying more of these mRNAs will further elucidate the complex translational regulatory hierarchies that control the balance between stem cell self-renewal and differentiation (Li, 2012).

Three RNA binding proteins form a complex to promote differentiation of germline stem cell lineage in Drosophila

In regenerative tissues, one of the strategies to protect stem cells from genetic aberrations, potentially caused by frequent cell division, is to transiently expand the stem cell daughters before further differentiation. However, failure to exit the transit amplification may lead to overgrowth, and the molecular mechanism governing this regulation remains vague. In a Drosophila mutagenesis screen for factors involved in the regulation of germline stem cell (GSC) lineage, a mutation was isolated in the gene CG32364, which encodes a putative RNA-binding protein (RBP) and is designated as tumorous testis (tut). In tut mutant, spermatogonia fail to differentiate and over-amplify, a phenotype similar to that in mei-P26 mutant. Mei-P26 is a TRIM-NHL tumor suppressor homolog required for the differentiation of GSC lineage. Tut was found to bind preferentially a long isoform of mei-P26 3'UTR, and is essential for the translational repression of mei-P26 reporter. Bam and Bgcn are both RBPs that have also been shown to repress mei-P26 expression. Genetic analyses indicate that tut, bam, or bgcn is required to repress mei-P26 and to promote the differentiation of GSCs. Biochemically, this study demonstrates that Tut, Bam, and Bgcn can form a physical complex in which Bam holds Tut on its N-terminus and Bgcn on its C-terminus. Both in vivo and in vitro evidence illustrate that Tut acts with Bam, Bgcn to accurately coordinate proliferation and differentiation in Drosophila germline stem cell lineage (Chen, 2014).

Aubergine controls germline stem cell self-renewal and progeny differentiation via distinct mechanisms

Piwi family protein Aubergine (Aub) maintains genome integrity in late germ cells of the Drosophila ovary through Piwi-associated RNA-mediated repression of transposon activities. Although it is highly expressed in germline stem cells (GSCs) and early progeny, it remains unclear whether it plays any roles in early GSC lineage development. This study reports that Aub promotes GSC self-renewal and GSC progeny differentiation. RNA-iCLIP results show that Aub binds the mRNAs encoding self-renewal and differentiation factors in cultured GSCs. Aub controls GSC self-renewal by preventing DNA-damage-induced Chk2 activation and by translationally controlling the expression of self-renewal factors. It promotes GSC progeny differentiation by translationally controlling the expression of differentiation factors, including Bam. Therefore, this study reveals a function of Aub in GSCs and their progeny, which promotes translation of self-renewal and differentiation factors by directly binding to its target mRNAs and interacting with translational initiation factors (Ma, 2017).

Aub is an essential piRNA pathway component known to be important for silencing TEs to ensure normal late germ cell development in the Drosophila ovary. This study demonstrates that Aub is required intrinsically to maintain GSC self-renewal and promote their progeny differentiation in the Drosophila ovary. aub is required intrinsically in GSCs to maintain self-renewal by preventing DNA-damage-evoked Chk2 activation. Aub promotes cystoblast (CB) differentiation by maintaining Bam expression. In addition, Aub directly binds over 1,100 mRNAs in GSCs, some of which are known to be important for GSC maintenance and differentiation, suggesting that Aub can control GSC maintenance and progeny differentiation also by directly regulating gene expression. Aub directly binds bam, dnc, and Rm62 mRNAs, and regulates their expression at the translation level via 3' UTR, which can also help mechanistically explain how Aub controls GSC maintenance and progeny differentiation. Finally, Aub was shown to physically associated with the translation initiation eIF4 complex and pAbp, suggesting that it promotes gene expression via regulation of translation initiation. Therefore, this study has revealed new roles of Aub in promoting GSC self-renewal and GSC progeny differentiation through different mechanisms (Ma, 2017).

Although Aub represses TEs by controlling the 'ping-pong' piRNA amplification cycle in late germ cells of the Drosophila ovary, its importance in GSCs, CBs, and mitotic cysts has not been previously reported. THE genetic results show that Aub intrinsically maintains GSC self-renewal partly by preventing DNA-damage-evoked checkpoint activation. First, aub mutant ovaries have two GSCs at eclosion, but gradually lose their GSCs within 25 days. Second, marked aub mutant GSC clones are lost from the niche faster than the marked control GSCs. Third, aub mutant GSCs accumulate DNA damage recognized by γ-H2AvD, indicating that Aub is also important to produce piRNAs for repressing TEs, thereby preventing TE-induced DNA damage. Fourth, the GSC loss caused by aub mutations can also be partially and significantly rescued by Chk2 inactivation, indicating that DNA damage is one of the major causes for the loss of the aub mutant GSCs. This interpretation is supported by a recent study showing that Chk2 inactivation can drastically and significantly rescue the DNA-damage-induced GSC loss. This is also consistent with the previous finding that the dorsal-ventral polarity defect of aub mutant egg chambers can be drastically rescued by Chk2 inactivation. Finally, the loss of aub mutant GSCs could be caused primarily by differentiation, but might also be contributed by apoptosis. Taken together, these results demonstrate that Aub intrinsically controls GSC self-renewal partly by preventing DNA-damage-induced Chk2 activation, and have also confirmed that piRNAs are important in GSCs to repress TEs and thus prevent DNA damage. In addition, Aub regulates nanos (nos) mRNA localization in the oocyte and also controls the decay of maternal mRNAs in the early Drosophila embryo, including nos, in cooperation with piRNAs. This study has adopted the iCLIP experimental procedure to the in vitro cultured GSCs for the identification of potential mRNA targets of Aub in GSCs. These cultured GSCs can self-renew, and, more importantly, can also be induced to differentiate into 16-cell cysts upon Bam induction. iCLIP results show that Aub can bind more than 1,100 mRNAs in GSCs primarily via 5' UTR, 3' UTR, or both, 59 of which encode known GSC self-renewal factors, including BMP pathway components, E-cadherin, and chromatin remodeling factors (Ma, 2017).

This study has revealed another role of Aub in promoting GSC progeny differentiation by regulating the expression of differentiation factors via direct binding. First, Aub is required intrinsically to promote GSC progeny differentiation. The aub mutant ovaries accumulate excess undifferentiated CBs, whereas the marked mutant aub GSCs also produce excess undifferentiated CBs. Second, Aub sustains Bam protein expression in mitotic germ cells. Bam is a master regulator for driving GSC progeny differentiation. Genetically, aub interacts with bam to promote germ cell differentiation. Molecularly, Aub is required in mitotic cysts to sustain Bam protein expression, and its binding to the bam 3' UTR is important for regulating the levels of its protein but not mRNA, suggesting that Aub can regulate Bam expression at the translational level. In addition, bam transcription and mRNA levels are significantly lower in aub mutant ovaries than in the control ones, indicating that Aub can also regulate Bam at the transcriptional level. Thus, Aub can regulate Bam expression in early germ cells at the transcriptional and translational levels. Third, Aub is also required to maintain Rm62 expression in CBs and mitotic cysts via its 3' UTR. The deletion of the Aub-binding site from the Rm62 3' UTR significantly decreases its expression in CBs and mitotic cysts, indicating that Aub binding is critical for Rm62 protein expression in CBs and mitotic cysts. This study has confirmed the previously reported role of Rm62 in promoting GSC progeny differentiation. Taken together, this study demonstrates that Aub promotes GSC progeny differentiation at least in part by controlling the expression of differentiation factors at the translational level (Ma, 2017).

Previous studies have shown that Aub can regulate mRNA stability and localization in a piRNA-dependent manner. By examining the existence of the binding sites for known germline-specific piRNAs over the 3' UTRs of 1,189 mRNAs, no significant enrichment was seen of piRNA-binding sites in the Aub-binding regions of these Aub target mRNAs, indicating that Aub binding to its target mRNAs in GSCs is likely independent of piRNAs. More importantly, Aub promotes the expression of its target mRNAs at least partly at the translation level via direct 3' UTR binding in GSCs and their progeny, thereby promoting GSC maintenance, progeny differentiation, or both. Interestingly, co-immunoprecipitation experimental results show that Aub is physically associated with the translation initiation complex eIF4 and the poly(A)-binding protein pAbp in S2 cells. The eIF4 complex and pAbp are known to be able to interact with each other to bring the 5' UTR and 3' UTR in close proximity, facilitating the loading of eIF3 and the 40S ribosome subunit to initiate translation. However, the current findings do not contradict the previous finding that Aub can promote mRNA degradation in different developmental and cellular contexts. Many independent studies have shown that translation regulators can also control mRNA stability, and mRNA stability factors can also regulate translation. Based on these findings, it is proposed that Aub can regulate mRNA translation at the initiation step by facilitating the interaction between the eIF4 complex and pAbp in GSCs and their early progeny (Ma, 2017).

Protein Interactions

Bam-dependent deubiquitinase complex can disrupt germ-line stem cell maintenance by targeting cyclin A

Drosophila germ-line stem cells (GSCs) provide an excellent model to study the regulatory mechanisms of stem cells in vivo. Bag of marbles (bam) has been demonstrated to be necessary and sufficient to promote GSC and cystoblast differentiation. Despite extensive investigation of its regulation and genetic functions, the biochemical nature of the Bam protein has been unknown. This study reports that Bam is an ubiquitin-associated protein and controls the turnover of cyclin A (CycA). Mechanistically, it was found that Bam associated with Otu to form a deubiquitinase complex that stabilized CycA by deubiquitination, thus providing a mechanism to explain how ectopic expression of Bam in GSCs promotes differentiation. Collectively, these findings not only identify a biochemical function of Bam, which contributes to GSC fate determination, but also emphasizes the critical role of proper expression of cyclin proteins mediated by both ubiquitination and deubiquitination pathways in balancing stem cell self-renewal and differentiation (Ji, 2017).

A conserved CAF40-binding motif in metazoan NOT4 mediates association with the CCR4-NOT complex

The multisubunit CCR4-NOT mRNA deadenylase complex plays important roles in the posttranscriptional regulation of gene expression. The NOT4 E3 ubiquitin ligase is a stable component of the CCR4-NOT complex in yeast but does not copurify with the human or Drosophila melanogaster complex. This study shows that the C-terminal regions of human and D. melanogaster NOT4 contain a conserved sequence motif that directly binds the CAF40 subunit of the CCR4-NOT complex (CAF40-binding motif [CBM]). In addition, nonconserved sequences flanking the CBM also contact other subunits of the complex. Crystal structures of the CBM-CAF40 complex reveal a mutually exclusive binding surface for NOT4 and Roquin or Bag of marbles mRNA regulatory proteins. Furthermore, CAF40 depletion or structure-guided mutagenesis to disrupt the NOT4-CAF40 interaction impairs the ability of NOT4 to elicit decay of tethered reporter mRNAs in cells. Together with additional sequence analyses, these results reveal the molecular basis for the association of metazoan NOT4 with the CCR4-NOT complex and show that it deviates substantially from yeast. They mark the NOT4 ubiquitin ligase as an ancient but nonconstitutive cofactor of the CCR4-NOT deadenylase with potential recruitment and/or effector functions (Keskeny, 2019).

Drosophila Bag-of-marbles directly interacts with the CAF40 subunit of the CCR4-NOT complex to elicit repression of mRNA targets

Drosophila melanogaster Bag-of-marbles (Bam) promotes germline stem cell (GSC) differentiation by repressing the expression of mRNAs encoding stem cell maintenance factors. Bam interacts with Benign gonial cell neoplasm (Bgcn) and the CCR4 deadenylase, a catalytic subunit of the CCR4-NOT complex. Bam has been proposed to bind CCR4 and displace it from the CCR4-NOT complex. This study investigated the interaction of Bam with the CCR4-NOT complex by using purified recombinant proteins. Unexpectedly, it was found that Bam does not interact with CCR4 directly but instead binds to the CAF40 subunit of the complex in a manner mediated by a conserved N-terminal CAF40-binding motif (CBM). The crystal structure of the Bam CBM bound to CAF40 reveals that the CBM peptide adopts an alpha-helical conformation after binding to the concave surface of the crescent-shaped CAF40 protein. It was further shown that Bam-mediated mRNA decay and translational repression depend entirely on Bam's interaction with CAF40. Thus, Bam regulates the expression of its mRNA targets by recruiting the CCR4-NOT complex through interaction with CAF40 (Sgromo, 2017).

Developmental Biology

Adult

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 (Gšnczy, 1997).

Bam controls the functional switch of the COP9 complex: Protein competition switches the function of COP9 from self-renewal to differentiation

The balance between stem cell self-renewal and differentiation is controlled by intrinsic factors and niche signals. In the Drosophila melanogaster ovary, some intrinsic factors promote germline stem cell (GSC) self-renewal, whereas others stimulate differentiation. However, it remains poorly understood how the balance between self-renewal and differentiation is controlled. This study used D. melanogaster ovarian GSCs to demonstrate that the differentiation factor Bam controls the functional switch of the COP9 complex (see CSN5) from self-renewal to differentiation via protein competition. The COP9 complex is composed of eight Csn subunits, Csn1-8, and removes Nedd8 modifications from target proteins. Genetic results indicated that the COP9 complex is required intrinsically for GSC self-renewal, whereas other Csn proteins, with the exception of Csn4, were also required for GSC progeny differentiation. Bam-mediated Csn4 sequestration from the COP9 complex via protein competition inactivated the self-renewing function of COP9 and allowed other Csn proteins to promote GSC differentiation. Therefore, this study reveals a protein-competition-based mechanism for controlling the balance between stem cell self-renewal and differentiation. Because numerous self-renewal factors are ubiquitously expressed throughout the stem cell lineage in various systems, protein competition may function as an important mechanism for controlling the self-renewal-to-differentiation switch (Pan, 2014).

Morphogenesis of the Drosophila fusome and its implications for oocyte specification

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).

A niche maintaining germ line stem cells in the Drosophila ovary

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 the germ line. However, a requirement for intercellular signals does not by itself indicate the presence of a niche. A true niche should function independently of resident stem cells and be able to reprogram newly introduced cells to become stem cells. Consequently, it was investigated whether the microenvironment at the ovariolar tip can specify cells to become GSCs (Xie, 2000).

Ovarioles normally lose GSCs by differentiation, but the low rate of GSC loss and the possibility that rapid replacement quickly restores the original GSC configuration complicate observing such events. To study germaria with recently lost stem cells, individual GSCs were genetically marked and destabilized. FRT-mediated recombination was used to generate mutant clones of schnurri (shn), a gene that is likely to reduce GSC lifetime by disrupting dpp signaling, under conditions where the mutant cells also lose an armadillo-lacZ marker. Because cystoblasts require 4 to 5 days to exit the germarium, the only remaining lacZ^- cells 1 week after transiently activating the hs-FLP transgene by means of a heat shock will be clones consisting of shn mutant stem cells and their progeny of 4 to 5 days. With this marking system, marked shn GSCs that differentiate during the last 4.5 days can be recognized because lacZ^- germ cells will remain in the germarium; moreover, the developmental age of the least mature such cell will indicate the elapsed time since GSC loss (Xie, 2000).

The results demonstrate that shn mutant stem cells are lost at an increased rate and are rapidly replaced by wild-type cells. Seventy-nine germaria were found that retained lacZ^- germ cells, revealing that a shn stem cell had been recently lost. In every case they contained two wild-type stem cells, indicating that the lost lacZ^- stem cell had been replaced by a wild-type stem cell. Even when the stem cell was lost so recently that it remained a cystoblast, two lacZ⁺ stem cells were present at the tip. These stem cells are connected by an elongated fusome, indicating that they are recently divided sister cells in early interphase; the fusome is oriented in an unusual manner, perpendicular to the anterior/posterior (A/P) axis. These observations suggest a specific model for GSC replacement. After one GSC is lost, its neighboring stem cell divides perpendicular to the A/Paxis, causing a daughter cell to occupy the environment recently vacated by the departed GSC. For this mechanism to work, the environment at the site of the lost GSC must be capable of programming the incoming cell to become a GSC rather than a cystoblast. Observations indicate that it is capable of doing so, and hence that GSCs reside in a true stem cell niche (Xie, 2000).

The ability of the ovariole tip to act as a stem cell niche is likely to be biologically important. Females produce eggs for months, despite the 4- to 5-week half-life of an individual stem cell. To investigate whether stem cell replacement occurs normally, the number of stem cells and somatic niche cells was measured in aging females. During the first 5 weeks of adult life the average number of GSCs per germarium declines from about 2.5 to 2.0, significantly less than the 50% reduction expected in the absence of replacement. Replacement stem cells must function efficiently because the rate of stem cell loss does not increase with age. Some of the ovarioles that did lose a stem cell started with three GSCs, because the number of such ovarioles declined over the same period (Xie, 2000).

One of the three somatic cell types, cap cells, interacts with stem cells in a manner that suggests they play a role in niche function. Over the 36-day period, the number of cap cells and GSCs remained closely correlated at about 2.5 cap cells per GSC. Moreover, GSCs were observed to always make special contacts with cap cells that characteristically align with the A/P axis of the ovariole. The GSC's fusome remains adjacent to the GSC/cap cell interface during most of the cell cycle. In contrast, the behavior of inner sheath cells and terminal filament cells does not correlate closely with GSCs. As germaria age, terminal filament cells decrease in number from an average of 9.2 (3 days) to 5.0 (36 days) and change from a linear to a ball-like arrangement. Likewise, the relative number of inner germarium sheath (IGS) cells and GSCs vary. However, the number of IGS cells is closely correlated with the number of differentiating germ cells. A functional connection between IGS cells and germ cell cysts has been previously suggested, because ovariole tips that develop without germ cells lack IGS cells (Xie, 2000).

To investigate the role of IGS cells in adult germaria females carrying a hs-bam transgene, whose stem cells can be induced to differentiate, were studied. Over the course of several days after heat shock, GSCs are lost and all germ line cysts completed development and left the germarium. Such germaria also lose all IGS cells, further indicating that developing germ cells control IGS cell number. In contrast, terminal filament and cap cells do not change in the absence of germ cells. Somatic cell divisions continue in their vicinity as in germaria that form in the absence of germ cells. Despite their presence near the GSC niche, these dividing somatic cells do not become GSCs (Xie, 2000).

Because the number of cap cells correlates closely with the number of GSCs, whether they might function by preferentially sending a dpp signal was investigated. Suitable antibodies to Dpp are unavailable, so whole-mount in situ hybridization was used to determine which cells at the ovariole tip express dpp mRNA. These experiments detected low levels of dpp mRNA in both cap cells and inner sheath cells, as well as higher levels in prefollicle cells farther posterior in the germarium. No dpp mRNA was seen in terminal filament cells or in any germ line cells, including GSCs. These results show that cap cells are one of several cell types located near the GSCs that express dpp. Moreover, it does not appear to be the absence of contact with a dpp-expressing cell that causes the posterior stem cell daughter to differentiate as a cystoblast (Xie, 2000).

These studies suggest a working model for a GSC niche. It is proposed that cap cells are critical to the formation, maintenance, and regulation of the GSC niche. Cap cells and terminal filament cells form a characteristic structure with sufficient internal surface area to contact two or three GSCs. A special cell-cell junction is likely to form between GSCs and cap cells to explain their intimate juxtaposition throughout adult life. Such a junction likely holds a GSC at the anterior and prevents it from moving away from the ovariolar tip where it might receive differentiation cues. An intercellular signal, possibly dpp, would be needed to maintain this junction and control the rate of GSC division, but need not be spatially graded. Additional signals appear to be involved in niche function as well. Terminal filament cells and/or cap cells express hedgehog, wingless, and armadillo, although roles for these signaling molecules in regulating GSCs remain unclear. Yb and piwi function outside the germ line in maintaining GSCs. The combined action of these genes ensures that precisely one of the GSC daughters loses cap cell contacts and differentiates (Xie, 2000).

These experiments show that a small group of stromal cells located at the tip of the Drosophila ovariole acts as a stem cell niche. Stem cells in many different tissues and organisms may be regulated in a similar manner. In the Drosophila testis, five to seven stem cells are anchored on terminally differentiated somatic hub cells, suggesting that both the ovary and testis could use similar strategies to regulate their stem cells. In Caenorhabditis elegans, distal tip cells have been directly implicated in the maintenance of the GSC population. In the Arabidopsis shoot meristem, an organizing center located nearby is required to maintain meristem stem cells. The reported plasticity of some mammalian stem cells may result from the existence of niches that can reprogram stem cell identity. The studies presented here provide a basis for detailed comparisons between the structure and regulatory properties of niches supporting different stem cells and will assist efforts to elucidate the molecular signals that control stem cell division and differentiation (Xie, 2000).

Sex-lethal facilitates the transition from germline stem cell to committed daughter cell in the Drosophila ovary

In Drosophila, the female-specific Sex-lethal (Sxl) protein is required for oogenesis, but how Sxl interfaces with the genetic circuitry controlling oogenesis remains unknown. An allele of sans fille (snf) that specifically eliminates Sxl protein in germ cells was used to carry out a detailed genetic and cell biological analysis of the resulting ovarian tumor phenotype. It was found that tumor growth requires both Cyclin B and zero population growth, demonstrating that these mutant cells retain at least some of the essential growth-control mechanisms used by wild-type germ cells. Using a series of molecular markers, it was established that while the tumor often contains at least one apparently bona fide germline stem cell, the majority of cells exhibit an intermediate fate between a stem cell and its daughter cell fated to differentiate. In addition, snf tumors misexpress a select group of testis-enriched markers, which, remarkably, are also misexpressed in ovarian tumors that arise from the loss of bag of marbles (bam). Results of genetic epistasis experiments further reveal that bam's differentiation-promoting function depends on Sxl. Together these data demonstrate a novel role for Sxl in the lineage progression from stem cell to committed daughter cell and suggest a model in which Sxl partners with bam to facilitate this transition (Chau, 2009).

The observation that female germ cells lacking Sxl are tumorigenic was first published >20 years ago, yet the place of this female-specific RNA binding protein in the genetic circuitry controlling oogenesis has remained elusive. This study investigated Sxl's role in the germline by taking advantage of a snf mutant allele that specifically eliminates Sxl expression in the germline. Genetic and cell biological analysis established that Sxl is required for the transition from stem cell to committed daughter cell by showing that the majority of Sxl-deficient germ cells have acquired an intermediate fate. These findings are in contrast to the commonly held view, based on fusome morphology alone, that Sxl mutant germ cells arrest development later in the differentiation pathway. This study also offers new insight into the function of bam by demonstrating that its differentiation-promoting function depends on Sxl and, importantly, that Sxl and bam control the same sex-specific expression network (Chau, 2009).

In current models, maintenance of GSC identity requires contact with the niche to trigger the signal transduction cascade required for transcriptional repression of bam. This in turn provides a permissive environment that allows PUM, which forms a complex with its partner protein Nanos (NOS), to inhibit translation of a yet unidentified set of mRNAs required for differentiation. Differentiation begins when one of the daughter cells is displaced from the niche and can no longer receive the signals that silence bam transcription. Bam then initiates the differentiation program by antagonizing the translation-inhibitory functions of the PUM/NOS complex. This model predicts a strong negative correlation between the expression of bam and the GSC markers, and, while this is true in general, there have been reports of rare single cells that coexpress bam and one or more GSC-specific markers. These and other studies have suggested that cells fated to differentiate first pass through an intermediate stage that transitions, without dividing, to a mature cystoblast (Chau, 2009).

It was shown that Sxl is required to complete the transition from GSC to a mature cystoblast (CB) by demonstrating that the majority of germ cells lacking Sxl resemble an immature CB-like cell. Furthermore, genetic epistasis experiments suggest that the failure to progress beyond this intermediate stage is attributable to a lack of bam function. This conclusion is supported by studies showing that the tumors resulting from the lack of Sxl and bam are remarkably similar. Specifically, the loss of Sxl and bam results in germ cell tumors with the same unique molecular signature including expression of stem cell markers and with the same set of testis-enriched markers. Both types of germ cell tumors also require CycB and zpg for growth. This comparison reveals that snf and bam tumors both result from a failure to initiate the differentiation pathway in stem cell progeny. It will be interesting to determine what role the misregulated testis-enriched markers play in this process (Chau, 2009).

On the basis of these data, it is proposed that Sxl partners with bam to facilitate the transition between GSCs and the daughter cell that is fated to differentiate. In females, differentiation via control of bam transcription is initiated in response to position-dependent extrinsic cues from the somatic gonad. Extrinsic cues from the somatic gonad also provide essential sex-specific information, via control of Sxl expression. These findings suggest that the intrinsic Sxl/bam partnership serves to integrate these two different extrinsic signaling pathways. This proposal is particularly compelling because it explains how bam function is substantially different in males and females (Chau, 2009).

How might Sxl and bam function converge to promote female germ cell differentiation? Sxl acts post-transcriptionally to repress splicing and translation. The molecular function of Bam, on the other hand, is unknown but is also thought to act post-transcriptionally. At a genetic level, one function of bam is to antagonize the differentiation-inhibiting activity of PUM/NOS. The presence of putative high-affinity Sxl-binding sites in both the 5'-UTR and the 3'-UTR of the nos mRNA leads to the speculation that Sxl functions with Bam to promote differentiation by inhibiting the translation of nos. Although this model is consistent with the finding that Sxl and Bam are coexpressed in the appropriate cell type, biochemical studies to address this point have proved to be technically challenging (Chau, 2009).

In summary, these studies support a model in which the Sxl/bam pathway is required for germ cells to progress from a stem cell fate to a differentiation-competent CB fate. These studies also suggest that if this pathway is blocked, germ cells will continue to proliferate, forming a tumor. It is proposed that the block in the developmental progression from stem cell to fully committed daughter cell is the initial tumorigenic event. This model is consistent with the general view that adult stem cells are the source of some, and perhaps all, tumors. Not only do some human germ cell tumors display many of the same characteristics as the Drosophila tumors described in this study, including expression of stem cell markers, but also they occur frequently in individuals with intersex disorders. While true orthologs of Sxl and bam are not found in vertebrates, the processes that they regulate are likely to be conserved. Future studies aimed at understanding the functional connections between the failure to engage the Sxl/bam genetic programs, misexpression of testis-enriched markers, and tumorigenesis will likely provide mechanistic insight into the pathogenesis of germ cell tumors in humans (Chau, 2009).

TSC1/2 tumour suppressor complex maintains Drosophila germline stem cells by preventing differentiation

Tuberous sclerosis complex human disease gene products TSC1 and TSC2 form a functional complex that negatively regulates target of rapamycin (TOR), an evolutionarily conserved kinase that plays a central role in cell growth and metabolism. This study describes a novel role of TSC1/2 in controlling stem cell maintenance. In the Drosophila ovary, disruption of either the Tsc1 or Tsc2 gene in germline stem cells (GSCs) leads to precocious GSC differentiation and loss. The GSC loss can be rescued by treatment with TORC1 inhibitor rapamycin, or by eliminating S6K, a TORC1 downstream effecter, suggesting that precocious differentiation of Tsc1/2 mutant GSC is due to hyperactivation of TORC1. One well-studied mechanism for GSC maintenance is that BMP signals from the niche directly repress the expression of a differentiation-promoting gene bag of marbles (bam) in GSCs. In Tsc1/2 mutant GSCs, BMP signalling activity is downregulated, but bam expression is still repressed. Moreover, Tsc1 bam double mutant GSCs could differentiate into early cystocytes, suggesting that TSC1/2 controls GSC differentiation via both BMP-Bam-dependent and -independent pathways. Taken together, these results suggest that TSC prevents precocious GSC differentiation by inhibiting TORC1 activity and subsequently differentiation-promoting programs. As TSC1/2-TORC1 signalling is highly conserved from Drosophila to mammals, it could have a similar role in controlling stem cell behaviour in mammals, including humans (Sun 2010).

TSC1/2 is known to regulate cell growth via inhibition on TORC1. This study demonstrates that it also functions by inhibiting the activity of TORC1 to maintain GSCs. Treatment with rapamycin, a TORC1-specific inhibitor, can completely rescue GSC loss in Tsc1 mutants. In addition, eliminating S6K, which functions downstream of TORC1 in regulating protein translation, could also completely rescue GSC loss in Tsc2 mutants. Interestingly, the daughters of Tor mutant GSCs can differentiate into germline cyst properly, indicating that TOR is normally not required for differentiation, but its hyperactivation in Tsc1/2 mutants drives precocious GSC differentiation. The simplest explanation of the delayed cystoblast differentiation in rapamycin-treated females might be a non-specific effect of drug treatment. However, it is also possible that TORC1 inhibition by rapamycin might cause repression of some, but not all, aspects of TOR function, which leads to uncoordinated development and/or differentiation of cystoblasts in response to GSC division. Consistently, accumulated cystoblasts where also observed when overexpressing both Tsc1 and Tsc2 in the germline. Together with the observation that TSC1/2-TORC1 signaling controls cell growth of germline cysts, this study suggests that TSC1/2-TORC1 may serve as a signaling integration point that orchestrates germline division, differentiation and development in order to control egg production in response to the local micro-environment and the system environment of the animals (Sun 2010).

In the Drosophila ovary, BMP signaling from the niche directly suppresses bam expression in GSCs to prevent differentiation. This signaling is crucial for GSC maintenance. As revealed by pMad expression, BMP signaling activity is significantly downregulated in Tsc1 mutant GSCs. This study also demonstrated that downregulation of pMad in Tsc1 mutant GSCs is mediated by TORC1 hyperactivation, as rapamycin treatment is able to restore the downregulated pMad level. However, TOR is not required for proper BMP signaling activity because pMad expression is not altered in rapamycin-treated germaria. Therefore, only TORC1 hyperactivation could inhibit BMP signaling in GSCs through unknown mechanisms, and this inhibitory effect occurs specifically in GSCs, as BMP signaling activity is not altered in Tsc1 mutant imaginal disc cells (Sun 2010).

Logically, bam expression could be derepressed in Tsc1 mutant GSCs as a consequence of BMP pathway downregulation. Surprisingly, no significant upregulation of bam-GFP expression could be detected in mutant GSCs, although in other GSCs that were compromised by BMP signaling, such as tkv mutant and mad mutant GSCs, bam transcription is significantly upregulated. Nevertheless, there might still be residual BMP signaling activities in Tsc1/2 mutant GSCs that are sufficient to suppress bam expression. Consistent with this notion is the observation that bam-GFP is not obviously upregulated in aged GSCs, even if BMP signaling activity has been significantly reduced. Together with the observation that bam mutation could not rescue the differentiation of Tsc1 mutant germ cells, it is suggested that the compromised BMP signaling activity may not be primarily responsible for Tsc1/2 mutant GSC loss. It is not clear why the effect of TSC1/2 on BMP signaling occurs specifically in GSCs. Possibly, Tsc1/2 mutant GSCs, once induced, have already primed for differentiation through a Bam-independent mechanism, which may trigger a positive feedback signal to inhibit BMP signaling activity, in order to facilitate differentiation (Sun 2010).

This study also reveals a BMP-Bam-independent mechanism that probably underlies the major role of TSC1/2-TORC1 signaling in GSC maintenance. The phenotype of Tsc1 bam double mutant germ cells differs from the bam alone mutant germ cells, as the double mutant GSCs can still become lost from the niche over time and undergo further differentiation into early cystocytes. Interestingly, the phenotype of Tsc1/2 mutant GSCs is similar to that of pelota (pelo) mutants. Pelo encodes a translational release factor-like protein and may regulate GSC maintenance at the translational level. In pelo mutant GSCs, there is also a downregulation of BMP signaling but no obvious upregulation of bam expression, and bam pelo double mutant germ cells are able to undergo similar limited differentiation into cystocytes, suggesting that TSC1/2 and Pelo might function in the same or parallel pathway to control GSC differentiation. It is proposed that similar to Pelo, TSC1/2 might function in a parallel pathway with the BMP-Bam pathway to control GSC differentiation, possibly by regulating the translation of differentiation-related mRNAs (Sun 2010).

Pum and Nos, which are known to function together to repress translation of the target mRNAs in embryos, are also essential for GSC maintenance. Recent genetic and biochemical studies suggest that Bam/Bgcn may directly inhibit the function of Pum/Nos to allow cystoblast differentiation. However, BMP signaling activation is able to prevent differentiation of nos mutant primordial germ cells, indicating that Pum/Nos could also function in parallel with the BMP-Bam pathway to control germ cell differentiation. In the future, it would be important to determine the functional relationships between the TSC1/2-TORC1 pathway, Pelo and Pum/Nos in regulating GSCs, and whether these factors, together with the microRNA pathway, target similar mRNAs to control GSC differentiation (Sun 2010).

This study has identified a novel role of TSC1/2 in controlling GSC maintenance and differentiation in the Drosophila ovary. Increasing evidence also suggests similar roles for TSC1/2-TOR signaling in regulating adult stem cell differentiation in mammals. For example, TSC1/2-mTOR signaling is also required for maintaining the quiescence of haematopoietic stem cells (HSCs), as Tsc1 deletion drives HSCs from quiescence to rapid cycling, which compromises HSC self-renewal. Thus, TSC1/2-TOR signaling could have an evolutionarily conserved role in regulating stem cell maintenance and differentiation from Drosophila to mammals (Sun 2010).

Ecdysteroids affect Drosophila ovarian stem cell niche formation and early germline differentiation

Steroid hormones are required in Drosophila for progression of oogenesis during late stages of egg maturation. This study shows that ecdysteroids regulate progression through the early steps of germ cell lineage. Upon ecdysone signalling deficit germline stem cell progeny delay switching on a differentiation programme. This differentiation impediment is associated with reduced TGF-β signalling in the germline and increased levels of cell adhesion complexes and cytoskeletal proteins in somatic escort cells. A co-activator of the ecdysone receptor, Taiman is the spatially restricted regulator of the ecdysone signalling pathway in soma. Additionally, when ecdysone signalling is perturbed during the process of somatic stem cell niche establishment enlarged functional niches able to host additional stem cells are formed (König, 2011).

This study shows that in Drosophila ecdysone signalling regulates differentiation of a GSC daughter and modulates ovarian stem cell niche size. The delay in GSC progeny differentiation correlates with reduced expression levels of TGF-β pathway components. Based on expression patterns it appears that germarial somatic cells, niche and ECs are the critical sites of ecdysteroid action and a co-activator of ecdysone receptor, Taiman is the spatially restricted regulator of ecdysone signalling in soma. During adulthood the ecdysone pathway has a specific role in EC differentiation and soma-germline cell contact establishment. In addition, during development the ecdysone signalling pathway has a role in somatic niche formation (König, 2011).

Ecdysteroids in general control major developmental transformations such as metamorphosis and morphogenesis in Drosophila. Different tissues and even different cell types within the same tissue respond to this broad signalling in a specific fashion and in a timely manner. In the developing Drosophila ovary steroid hormone receptors are expressed in a well-timed mode, high levels coinciding with proliferative and immature stages and low levels preceding reduced DNA replication and differentiation. Mutations in ecdysone pathway components affect ovarian morphogenesis, including heterochronic delay or acceleration in the onset of terminal filament differentiation. During the niche establishment the levels of both ecdysone receptors, EcR and USP are greatly downregulated in anterior somatic cells that will contribute to the niche per se. This study shows that perturbation of ecdysone signalling in pre-adult ovarian soma leads to the formation of enlarged niches. The specific response to systemic hormonal signalling in niche precursors is achieved by a specific function of the ecdysone receptor co-activator Taiman. When timely regulation of ecdysone signalling does not occur, more cells are recruited to become niche cells resulting in enlarged niches that are capable to host more stem cells. These data first show that ecdysone steroid hormonal signalling regulates the formation of the adult stem cell niche and suggest that a developmental tuning of ecdysone signalling controls the number of anterior somatic cells that will differentiate into cap cells (König, 2011).

It is logical that stem cell division and germline differentiation are regulated by some systemic signalling depending on the general state of the organism, which depends on age, nutrition, environmental conditions and so on. Hormones are great candidates for this type of regulation as they act in a paracrine fashion and their levels are changing in response to ever-changing external and internal conditions. Steroid binding to nuclear receptors in vertebrates triggers a conformational switch accompanied by increased histone acetylation that permits transcriptional co-activators binding and the transcription initiation complex assembly. In Drosophila, the trithorax-related protein, a histone H3 methyltransferase that like Taiman belongs to the p160 class of co-activators, and an ISWI-containing ATP-dependent chromatin remodelling complex (NURF), that regulates transcription by catalysing nucleosome sliding, both bind EcR in an ecdysone-dependent manner, showing that chromatin modifications can mediate response to this general signalling. Transcriptional regulation has a key role in GSC maintenance and differentiation, for example, the TGF-β ligand Dpp secreted by niche cells induces phosphorylation of the transcription factor Mad in GSCs that in turn suppresses transcription of the differentiation factor Bam. In addition, it has been shown recently that in Drosophila adult GSC ecdysone modulates the strength of TGF-β signalling through a functional interaction with the chromatin remodelling factors ISWI and Nurf301, a subunit of the ISWI-containing NURF chromatin remodelling complex (Ables, 2010). Therefore, it is plausible that ecdysone regulates Mad expression cell autonomously via chromatin modifications. Since pMad directly suppresses a differentiation factor Bam, it is expected that Bam would be expressed in pMad-negative cells. Interestingly, the findings show that ecdysone deficit decreases amounts of phosphorylated Mad in GSCs and also cell non-autonomously suppresses Bam in SSCs. As SSCs that express neither pMad nor Bam are accumulated when the ecdysone pathway is perturbed it suggests that there should be an alternative mechanism of Bam regulation. Even though eventually this still can be done on the level of chromatin modification, the data suggest that the origin of this soma-generated signal may be associated with cell adhesion protein levels. Further understanding of the nature of this signalling is of a great interest (König, 2011).

The progression of oogenesis within the germarium requires cooperation between two stem cell types, germline and somatic (escort) stem cells. In Drosophila, reciprocal signals between germline and escort (in female) or somatic cyst (in male) cells can inhibit reversion to the stem cell state and restrict germ cell proliferation and cyst growth. Therefore, the non-autonomous ecdysone effect can be explained by the necessity of two stem cell types that share the same niche (GSC and ESC) to coordinate their division and progeny differentiation. This coordination is most likely achieved via adhesive cues, as disruption of ecdysone signalling affects turnover of adhesion complexes and cytoskeletal proteins in somatic ECs: mutant cells exhibited abnormal accumulation of DE-Cadherin, β-catenin/Armadillo and Adducin (König, 2011).

Cell adhesion has a crucial role in Drosophila stem cells; GSCs are recruited to and maintained in their niches via cell adhesion. Two major components of this adhesion process, DE-Cadherin and Armadillo/β-catenin, accumulate at high levels in the junctions between GSCs and niche cells, while in the developing cystoblasts and escort cells levels of these proteins are strongly reduced. Levels of DE-Cadherin in GSCs are regulated by various signals, for example, nutrition activation of insulin signalling or chemokine activation of STAT, and this study shows that in ESCs it is regulated by steroid hormone signalling. Possibly, these two stem cell types respond to different signals but then differentiation of their progeny is synchronised via cell contacts. While hormones, growth factors and cytokines certainly manage stem cell maintenance and differentiation, the evidence also reveals that the responses to hormonal stimuli are strongly modified by adhesive cues (König, 2011).

Specificity to endocrine signalling can be achieved via availability of co-factors in the targeted tissue. Tai is a spatially restricted co-factor that cooperates with the EcR/USP nuclear receptor complex to define appropriate responses to globally available hormonal signals. Tai-positive regulation of ecdysone signalling can be alleviated by Abrupt via direct binding of these two proteins that prevents Tai association with EcR/USP (Jang, 2009). Abrupt has been shown to be downregulated by JAK/STAT signalling (Jang, 2009). Interestingly, JAK/STAT signalling also has a critical role in ovarian niche function and controls the morphology and proliferation of ESCs as well as GSCs. JAK/STAT signalling may interact with ecdysone pathway components in ECs to further modulate cell type-specific responses to global endocrine signalling. A combination of regulated by different signalling pathway factors that are also spatially and timely restricted builds a network that ensures the specificity of systemic signalling (König, 2011).

Knowledge of how steroids regulate stem cells and their niche has a great potential for stem cell and regenerative medicine. The current findings open the way for a detailed analysis of a role for steroid hormones in niche development and regulation of germline differentiation via adjacent soma (König, 2011).

The Fused/Smurf complex controls the fate of Drosophila germline stem cells by generating a gradient BMP response

In the Drosophila ovary, germline stem cells (GSCs) are maintained primarily by bone morphogenetic protein (BMP) ligands produced by the stromal cells of the niche. This signaling represses GSC differentiation by blocking the transcription of the differentiation factor Bam. Remarkably, bam transcription begins only one cell diameter away from the GSC in the daughter cystoblasts (CBs). How this steep gradient of response to BMP signaling is formed has been unclear. This study shows that Fused (Fu), a serine/threonine kinase that regulates Hedgehog, functions in concert with the E3 ligase Smurf to regulate ubiquitination and proteolysis of the BMP receptor Thickveins in CBs. This regulation generates a steep gradient of BMP activity between GSCs and CBs, allowing for bam expression on CBs and concomitant differentiation. Similar roles for Fu were observed during embryonic development in zebrafish and in human cell culture, implying broad conservation of this mechanism (Xia, 2010).

Previous studies have demonstrated that BMP/Dpp signals from the niche play primary roles in the self-renewal of GSCs by silencing bam transcription. However, the mechanism by which the differentiating CBs avoid the control of BMP/Dpp and activate bam remains poorly understood. This study has provided direct evidence that the differentiating daughter cells of GSCs, known as CBs, become resistant to BMP signaling through degradation of Tkv in CBs. Fu functions as an antagonistic factor in BMP/Dpp signaling by regulating Tkv degradation during the differentiation of CBs. Moreover, both genetic and biochemical evidence is provided that Fu acts in concert with Smurf, a HECT domain-containing ubiquitin E3 ligase, to regulate the ubiquitination of Tkv in the CB, thereby generating a steep gradient of response to BMP signaling between GSCs and CBs for their fate determination. Finally, a conserved role is shown for fu in antagonizing BMP/ TGFβ signals in zebrafish embryonic development as well as in human cell cultures. These findings not only reveal a conserved function of fu in controlling BMP/TGFβ signal-mediated developmental processes, but also provide a comprehensive view of mechanisms that produce both self-renewal and asymmetry in the division of stem cells (Xia, 2010).

Observations of the existence of a BMP resistance mechanism that controls the proper division of GSCs through the regulation of Tkv prompted an exploration of how Tkv was regulated. Using immunoprecipitation followed by mass spectrometry analysis, it was identified that Fu associates with the Tkv protein. Given that previous studies demonstrated that a loss of fu leads to early germ cell proliferation and a tumorous germarium phenotype and that biochemical evidence showed that Fu forms a complex with Tkv and affects its stability, it was subsequently identified that Fu as a component negatively regulates BMP/Dpp signaling by interacting with the BMP/Dpp type I receptor, Tkv (Xia, 2010).

BMP/TGFβ signals play pivotal roles in controlling diverse normal developmental and cellular processes. In the canonical BMP/TGFβ pathway, the receptors and Smad proteins are the essential components for BMP/TGFβ signal transduction. However, this pathway is known to be modulated by additional factors to reach physiological levels in a cellular context-dependent manner. Smurfs and HECT domain-containing proteins have been shown to antagonize BMP/TGFβ signals through the regulation of the stability of either receptors or Smads in vertebrates. In Drosophila, Smurf has previously been implicated in regulating proteolysis of phosphorylated Smad proteins in somatic cells. In the ovary, Smurf was also proposed to downregulate the level of BMP to promote CB differentiation. The mechanism underlying the action of Smurf in Drosophila early germline cells remains elusive. This study has shown that Fu, Smurf, and Tkv could form a trimeric complex in S2 cells. Importantly, both Fu and Smurf are required for ubiquitination of Tkv in S2 cells and for turnover of Tkv in germ cells. Combined with genetic evidence, it is proposed that Fu and Smurf likely function in a common biochemical process by controlling Tkv degradation. The present study reveals a mechanism by which Fu serves as an essential component in the Smurf-mediated degradation of the BMP/TGFβ receptor, thereby terminating BMP/TGFβ signaling and negatively regulating the downstream target genes of BMP/TGFβ (Xia, 2010).

Because Fu is a putative serine/threonine protein kinase, the question becomes how Fu acts on Tkv regulation in concert with Smurf. Given that knockdown of fu does not significantly change the pattern of autoubiquitination of Smurf itself, it is therefore likely that Tkv is a strong candidate substrate for Fu kinase. Although there is no assay system for analyzing the kinase activity of Fu presently, in this study, mutagenesis assays were perfomred and it was identified that the S238 in Tkv is important for Tkv^ca to respond to Fu and is critical for Tkv^ca ubiquitination and degradation. Of note, it was found that the ubiquitin- resistant form of Tkv^ca [Tkv^caS238A] blocks CB differentiation. A previous study has shown that the S189 site in TGF-β type-I receptor, the corresponding site of S238 in Tkv, was phosphorylated in the cell culture system. The current results suggest that Fu likely acts on Tkv through targeting and phosphorylating the S238 site and subsequently leads to Tkv ubiquitination and degradation by Smurf. Nevertheless, it would be advantageous to develop a kinase assay system for Fu to determine whether the S238 site in Tkv is an authentic phosphorylation site for Fu kinase in the future (Xia, 2010).

Previous genetic analyses revealed that Fu plays an evolutionarily conserved role in the proper activation of the Hh pathway and functions downstream of the Hh receptor. Increasing evidence has shown that the kinase Fu regulates the Hh-signaling complex by targeting Cos2. However, the function of Fu as a component in the Hh pathway is not consistent with its spatiotemporal expression pattern during development. For example, Hh signaling only plays a role in zebrafish embryonic development at late stages, but Fu is expressed ubiquitously at both the early and the late stages of zebrafish embryonic development. These findings suggest that Fu may have Hh-independent functions in different physiological conditions. In this study, by using several different systems, including Drosophila germline, zebrafish embryo, and human tissue cultures, it was demonstrated that Fu is indeed required for balancing proper BMP/TGFβ signals in different developmental processes. Given that both Fu and Smurf are evolutionarily conserved proteins, it would be interesting to determine whether the Fu/Smurf complex also plays roles in other signaling pathways (Xia, 2010).

The niche-dependent feedback loop generates a BMP activity gradient to determine the germline stem cell fate

Stem cells interact with surrounding stromal cells (or niche) via signaling pathways to precisely balance stem cell self-renewal and differentiation. However, little is known about how niche signals are transduced dynamically and differentially to stem cells and their intermediate progeny and how the fate switch of stem cell to differentiating cell is initiated. The Drosophila ovarian germline stem cells (GSCs) have provided a heuristic model for studying the stem cell and niche interaction. Previous studies demonstrated that the niche-dependent BMP signaling is essential for GSC self-renewal via silencing bam transcription in GSCs. It was recently revealed that the Fused (Fu)/Smurf complex degrades the BMP type I receptor Tkv allowing for bam expression in differentiating cystoblasts (CBs). However, how the Fu is differentially regulated in GSCs and CBs remains unclear. This study reports that a niche-dependent feedback loop involving Tkv and Fu produces a steep gradient of BMP activity and determines GSC fate. Importantly, it was shown that Fu and graded BMP activity dynamically develop within an intermediate cell, the precursor of CBs, during GSC-to-CB transition. Mathematic modeling reveals a bistable behavior of the feedback-loop system in controlling the bam transcriptional on/off switch and determining GSC fate (Xia, 1012).

The present data strongly imply that GSC/CB fate finely controlled by Fu protein regulation is important for generating BMP activity gradient between GSCs and CBs. However, the remaining important question is how to understand the mechanism by which the dynamic reciprocal antagonism between Tkv and Fu controls the GSC-to-CB fate switch during GSC division. To clearly answer this question, a mathematical network model was developed based on the experimental evidences with bistable behavior to elucidate how the feedback loop regulation determines the fate specification of GSCs (Xia, 1012).

On the basis of these data, a feedback loop model is proposed to show how the GSC fate is regulated. In the model, the external BMP signal cues stimulate phosphorylation of Tkv protein, the activated Tkv then promotes the synthesis rate of phosphorylated Mad (pMad), and pMad promotes the degradation of Fu protein and represses the transcription of bam. Meanwhile, degradation of the activated Tkv is also controlled by Fu. To assess the dynamic properties of this feedback loop, it was assumed that the transcriptions of genes tkv, mad, and fu are sufficient and that the degradation rate of pMad and the synthesis rate of Fu protein are constants. The network diagram of the feedback loop clearly points out two characteristics of the model: first, the microenvironment-derived BMP ligands serve as a key external signal, the strengths of which are differentially sensed by GSCs, pre-CBs, and CBs, thereby regulating the dynamic expression of the activated Tkv, pMad, and Fu during the asymmetric division of GSCs. Second, although the transcription of the bam gene is regulated negatively by Tkv/pMad, the expressions (and/or regulations) of the activated Tkv, pMad, and Fu are independently of the status of the Bam protein (Xia, 1012).

The dynamic analysis reveals the bistable behavior (i.e., switch behavior) of the system and how the system dynamics respond to the strength of external BMP ligand activity. Specifically, the strong external BMP ligand activity (in GSCs) will lead to a low expression level of Fu as well as high expression levels of the activated Tkv and pMad. Conversely, the weak external BMP ligand activity (in CBs) will lead to a high level of Fu expression (and low levels of the activated Tkv and pMad expression). However, for the transitional stage with intermediate BMP signaling (in pre- CBs), both high and low levels of Fu and pMad expression exist. These theoretical predictions not only exactly match the experimental data, but they also bring an insightful physical interpretation for why the niche dependence of BMP signaling determines the fate of stem cells by precisely balancing of stem cell renewal and differentiation. The current model permits proposal of a comprehensive description of the action of niche signaling that governs the decision between stem cells and differentiating cells (Xia, 1012).

Effects of mutation or deletion

bag-of-marbles expression is necessary for cystoblast differentiation; bam mutant germ cells fail to differentiate, but instead proliferate like stem cells. Ectopic expression of bam is sufficient to extinguish stem cell divisions. Heat-induced bam+ expression specifically eliminates oogenic stem cells, while somatic stem cell populations are not affected. Together with previous studies of the timing of BAM mRNA and protein expression and the state of arrest in bam mutant cells, these data implicate Bam as a direct regulator of the switch from stem cell to cystoblast. It is proposed that a presumptive cystoblast daughter (pre-cystoblast) derived from stem cell divisions undergoes a maturation process during which bam+ activation initiates cystoblast/cystocyte mitosis by modifying the germ-cell division cycle. Surprisingly, ectopic bam+ has no deleterious consequences for male germline cells, suggesting that Bam may regulate somewhat different steps of germ-cell development in oogenesis and spermatogenesis. A model for how bam+ could direct differentiation is presented based on the information that Bam protein is essential to assemble part of the germ-cell-specific organelle, the fusome (a membranous and skeletal structure which connects cystocytes). It is proposed that fusome biogenesis is an obligate step for cystoblast cell fate and that Bam is the limiting factor for fusome maturation in female germ cells. bam mutant fusomes are deficient in the membranous tubular reticulum that fills the core of fusomes. This observation and finding the Bam protein in the cytoplasm of cystocytes and in the fusome prompted the suggestion that cytoplasmic Bam protein might be required to recruit vesicular material into the fusome reticulum. A role for the fusome reticulum in directing a switch from stem cell to cystoblast-like divisions could explain both the bam loss-of-function and ectopic expression phenotypes (Ohlstein, 1997).

Mutations in bag-of-marbles (bam) and benign gonial cell neoplasm (bgcn) prevent progression through spermatogenesis and oogenesis, resulting in the overproliferation of undifferentiated germ cells. Both bam and bgcn act autonomously in the germline to restrict proliferation during spermatogenesis. The overproliferating germ cells express markers specific to amplifying germ cells, while at the same time retaining the expression of some markers of stem cell and primary spermatogonial cell fate. However, germ cells accumulating in bam or bgcn mutant testes most resemble amplifying germ cells, because they undergo incomplete cytokinesis and progress through the cell cycle in synchrony within the cyst; these are two characteristics of amplifying germ cells, but not of stem cells. Taken together, these results suggest that bam and bgcn regulate progression through the male germline stem cell lineage by cell-intrinsically restricting the proliferation of amplifying germ cells (Gonczy, 1997).

The Drosophila cystoblast differentiation factor, benign gonial cell neoplasm, is related to DExH-box proteins and interacts genetically with bag-of-marbles

The bag-of-marbles (bam) gene is an intrinsic factor for cystoblast fate in Drosophila germline cells and it requires active product from the benign gonial cell neoplasm (bgcn) gene. The predicted Bgcn protein is related to the DExH-box family of RNA-dependent helicases but lacks critical residues for ATPase and helicase functions. Expression of the bgcn gene is extremely limited in ovaries, but significantly, BGCN mRNA is expressed in a very limited number of germline cells, including the stem cells. Also, mutations in bgcn dominantly enhance a bam mutant phenotype, further corroborating the interdependence of these two genes' functions. On the basis of known functions of DExH-box proteins, it is proposed that Bgcn and Bam may be involved in regulating translational events that are necessary for activation of the cystoblast differentiation program (Ohlstein, 2000).

Ovarian in situ hybridization with bgcn reveals that the major site of bgcn accumulation is in a small number of cells at the most anterior tip of the germarium. bgcn may be expressed at a low level elsewhere. The most significant aspect of bgcn expression is that, unlike bam mRNA, GSCs are positive for bgcn transcripts. At first, GSC expression appears counterintuitive since bgcn is required for cystoblast, but not GSC, development. It was, however, considered likely that bgcn would be expressed within GSCs based on the consequences of Bam misexpression. Since expression of the P[HS-Bam] transgene ablates wild-type GSCs and apparently converts them to cystoblasts, it was expected that Bam accumulation would be limiting in wild-type GSCs while other cystoblast factors would be expressed in GSCs. From this perspective GSCs, which have no detectable BAM mRNA, are primed to become cystoblasts and lack only a higher expression level of the bam gene (Ohlstein, 2000).

Northern blot analysis revealed that bgcn is expressed at very low levels in ovaries but at significantly higher abundance in testes. In addition, the female transcript is reproducibly smaller than the male. The very low abundance of BGCN mRNA in female poly-A⁺ samples can be explained by the very restricted pattern of expression in ovaries although it is not yet known what factors account for the sexually dimorphic expression levels. Preliminary in situ hybridization suggests that bgcn is expressed throughout the testis but a more clear understanding of Bgcn expression will emerge when antibodies are available (Ohlstein, 2000).

The predicted Bgcn sequence reveals two specific similarities; one to the superfamily of ATP-dependent RNA helicases and a second to ankyrin domains. RNA helicases are a very large family of proteins that are primarily involved in either pre-mRNA processing or in translational control. Bgcn shows position alignment and sequence conservation with dozens of helicase family members. RNA helicases have been recognized and catalogued on the basis of seven conserved domains; four motifs have been implicated in ATP binding and hydrolysis while two others have been implicated in nucleic acid unwinding. The final helicase motif, GRAGR, is implicated in RNA interaction in eIF4A but is required for ATP hydrolysis and RNA unwinding, but not RNA interaction, in the NPH-II protein. Alignment of helicase family members illustrates that sequence conservation extends well beyond the short canonical motifs but no biochemical functions have been associated with conserved sequences outside of domains I-VII. A reasonable hypothesis is that some of the sequences conserved in DExH proteins are involved with RNA interactions, especially since many DExH-box family members lack recognizable RNA-binding motifs (Ohlstein, 2000).

The degree of conservation predicts that Bgcn and helicases share some biochemical activities. Since Bgcn does not have the motifs required for ATP binding and helicase activity, it is postulated that Bgcn shares the RNA interaction activity that characterizes the DExH proteins. Bgcn might represent an ancestral DExH protein that predates the acquisition of domains involved in ATP hydrolysis and RNA helicase catalysis. An alternative is that Bgcn represents a more modern branch that lost those domains involved in ATPase and helicase activity. Irrespective of the protein evolutionary implications of Bgcn and helicase similarities, it is predicted that Bgcn regulates post-transcriptional events (Ohlstein, 2000).

bgcn⁺ is necessary for proper Bam function and it has been proposed that Bam and Bgcn may act together in a complex to accomplish cystoblast differentiation. A weak bam allele allows limited female fertility and creates flies that are sensitive to even small reductions in bam⁺ activity. These flies became sterile and produced tumorous, 'bam-like' egg chambers when these 'sensitized' bam females are made heterozygous for bgcn. The simplest interpretation for this observation is that decreasing bgcn dosage by half can effectively decrease bam⁺ activity and can alter the phenotype in the sensitized genetic background. This implies that Bam and Bgcn work together closely in the molecular pathway leading to cystoblast differentiation (Ohlstein, 2000).

As a member of the DExH-box family, Bgcn may be an RNA interacting protein. To explain the genetic and molecular aspects of their expression, evidence is presented that bgcn and bam functions are interdependent and it is suggested that Bam and Bgcn proteins may interact. This hypothesis predicts that Bgcn action would be cytoplasmic and its role as DExH-box protein would more likely be involved with translational control rather than splicing regulation. Studies of the key role that Pum plays in GSC maintenance implicate translational regulation in the transition between GSC and cystoblast fate. Perhaps a Bgcn-Bam protein complex acts as a translational regulator of cystoblast-promoting transcripts that would be translationally repressed in GSCs. Data suggest association between Bam and the fusome reticulum, a structure resembling a germ cell modification of ER. A role for Bam in translational control could indicate that cystoblast activation depends on ER-associated translation (Ohlstein, 2000).

Genetic studies of mei-P26 reveal a link between the processes that control germ cell proliferation in both sexes and those that control meiotic exchange in Drosophila

mei-P26, a novel P-element-induced exchange-defective female meiotic mutant in Drosophila has been cloned and characterized. Meiotic exchange in females homozygous for mei-P26¹ is reduced in a polar fashion, such that distal chromosomal regions are the most severely affected. Additional alleles generated by duplication of the P element reveal that mei-P26 is also necessary for germline differentiation in both females and males. Tested were double mutant combinations of mei-P26 and bag-of-marbles (bam), a gene necessary for the control of germline differentiation and proliferation in both sexes, in order to further assess the role of mei-P26 in germline differentiation. A null mutation at the bam locus was found to act as a dominant enhancer of mei-P26 in both males and females. Interestingly, meiotic exchange in mei-P26¹; bam^Delta86/+ females is also severely decreased in comparison to mei-P26¹ homozygotes, indicating that bam affects the meiotic phenotype as well. These data suggest that the pathways controlling germline differentiation and meiotic exchange are related and that factors involved in the mitotic divisions of the germline may regulate meiotic recombination (Page, 2000).

mei-P26-induced defects in female germline differentiation appear to occur during the early mitotic divisions in cyst formation, and hypomorphic alleles of mei-P26, such as mei-P26¹, produce egg chambers with abnormal numbers of nurse cells. Cysts with an increased number of nurse cells may be due to an additional round of mitosis occurring in some or all of the cystocytes. Alternatively, a defect in follicle cell packaging of normal cysts may result in chambers containing an excess of cells. However, egg chambers containing two oocytes, which would be expected if two 16-cell cysts were packaged together, were not found. Similarly, cysts with too few nurse cells could also result from packaging of cysts that have abnormally broken their intercellular connections to form smaller clusters of cells. Cysts containing 7 nurse cells and an oocyte may also result from the cystocytes undergoing only three divisions (Page, 2000).

Allelic combinations of mei-P26 that alter the number of nurse cells also reduce the number of normal eggs produced, but they do not cause complete sterility. In addition to the defects in nurse cell number, a tumorous ovary phenotype is more frequent in severe alleles. In females homo- or hemi-zygous for these mutations, the egg chambers become filled with hundreds of small cells, and no oocyte develops. This phenotype is similar to that exhibited by mutants in the bam and benign gonial cell neoplasm (bgcn) genes (Page, 2000).

The most severe mei-P26 allele affects germline differentiation in both males and females. In mei-P26^mfs1, ovaries consist entirely of tumorous egg chambers and, in males carrying this allele, spermatid differentiation progresses only to the point of producing elongated spermatid bundles, and mature spermatozoa are not produced. The mei-P26^mfs1 allele thus bears some similarities to mutants in the bam and bgcn genes, both of which cause a tumorous phenotype in ovaries and arrest of spermatogenesis. However, spermatid differentiation is arrested at an earlier stage in bam and bgcn mutants. Ovary morphology differs somewhat, in that mei-P26 mutant ovarioles consist of a series of defined chambers, while bam and bgcn ovarioles often appear as distended germaria with few distinct chambers. Nevertheless, the interaction of mei-P26 with bam suggests that the similarities in the phenotypes in these mutants are not coincidental (Page, 2000).

Certain mutations in the Sex-lethal (Sxl), ovarian tumor (otu), and ovo genes also cause sterility in females due to the formation of tumorous egg chambers. Interestingly, mild to severe defects in meiotic recombination frequencies are observed in females bearing heteroallelic combinations of female sterile alleles for these loci and in females in which the effects of Sxl mutations have been partially suppressed using genetic modifiers. These results further suggest that the processes that control exchange position and determine germline cyst formation may be coordinately controlled (Page, 2000).

The structure of the MEI-P26 protein suggests a few possibilities for the role of mei-P26. mei-P26 is predicted to encode a member of the RBCC family of proteins, which contain, in their N-terminal regions, a RING finger motif followed by one or two copies of a second cysteine-rich motif called the B-box and a coiled coil region. The RING finger and B-box motifs are believed to mediate physical interactions with other proteins. RBCC proteins are only a subset of the large number of known RING finger proteins, which have diverse roles in oncogenesis, transcriptional regulation, signal transduction, and development (Page, 2000).

Several RBCC proteins, such as PML and the TIF1 family, are known to regulate transcription by binding to nuclear hormone receptors as coactivators or corepressors. While assembling factors for transcriptional regulation is one role for RBCC proteins, certain other RBCC proteins appear to function in capacities such as signal transduction, or by forming ribonucleoprotein complexes (SS-A/Ro). Although a growing number of RBCC proteins have been identified, only a handful also contain NHL repeats, named after the proteins NCL-1, HT2A, and LIN-41. The NHL repeat has been shown to be involved in protein-protein interactions in the RBCC proteins HT2A and BERP (Page, 2000).

The RBCC-NHL proteins are mostly of unknown function or have not been extensively characterized at a molecular level. RBCC-NHL proteins, which contain both RBCC and NHL domains, include at least two potential protein-protein interaction motifs, so a strong possibility is that these also may participate in the formation of multiprotein complexes. One possible candidate for a MEI-P26 partner protein is Bam (Page, 2000).

A genetic interaction between mei-P26 and bam has been demonstrated. Heterozygosity for a null mutant in bam enhances the phenotype of mei-P26, causing sterility in males, an increase in tumor formation in females, and a decrease in meiotic exchange. On the basis of the characterization of the bam and mei-P26 gene products, the nature of the interaction between these two genes is open to speculation (Page, 2000).

(1) MEI-P26 may act as a transcriptional or translational regulator that controls bam expression. A variety of ovarian defects like those observed in mei-P26, including tumorous chambers and cysts with abnormal numbers of nurse cells, are also seen in mutants for the Drosophila Rbp9 gene. The Rbp9 gene has also been shown to encode an RNA binding protein that binds specifically to the bam transcript and may act to regulate Bam expression in the germarium. Similarly, misregulation of bam expression in the encore (enc) mutant may underlie the effect of enc on nurse cell number. However, since heterozygosity for bam exacerbates the meiotic phenotype of mei-P26, this model predicts that the meiotic defects are due to the misregulation of bam, rather than through other genes possibly regulated by mei-P26 (Page, 2000).

(2) mei-P26 could be required for the proper localization or function of Bam. The product of the bam gene is expressed in the cytoplasm of cystoblasts and early germline cysts in females, where it is required for cystoblast differentiation. Bam protein also associates with the fusome, a large organelle, comprised mostly of cytoskeletal and vacuolar components, which is present in early germline cysts. According to this model, MEI-P26 may physically interact with Bam, possibly through the RBCC, NHL, or other motifs in the MEI-P26 protein. Alternatively, this regulation may be indirect, requiring other proteins. For example, MEI-P26 may regulate the bgcn gene product, which is required for Bam function. Again, this suggests that the effects on meiotic exchange are mediated by bam (Page, 2000).

The evidence presented here does not allow a determination of the relative positions of bam and mei-P26 in a pathway. Therefore, (3) bam may be required for mei-P26 function, which in turn would be required for proper germline cyst development and meiotic recombination. In this third model, MEI-P26 may physically interact with Bam and/or other proteins in the cytoplasm, possibly as a component of the fusome, from which MEI-P26 may facilitate normal germline development and meiotic exchange. Alternatively, MEI-P26 might be indirectly controlled by Bam as a downstream effector. While the relationship between mei-P26 and bam has not been fully elucidated, these models are intriguing, as they all suggest a role for bam in a pathway ensuring proper meiotic exchange (Page, 2000).

mei-P26 appears to behave as expected for a female meiotic precondition mutant. This group of Drosophila mutants presents a phenotype in which the total frequency of meiotic exchange is often reduced, although to differing levels, and residual exchanges are abnormally distributed in a polar fashion, with reduced frequencies in the distal parts of the chromosome arms. In mei-P26, exchange is decreased overall and the distribution is polar. In more severe alleles, recombination is more severely affected, and E₀ is increased. To provide a first step toward explaining the various components of the mei-P26 phenotype and the connection with germline differentiation, the following rather speculative model for precondition mutants in general is proposed (Page, 2000).

In many organisms, telomeres have been proposed as sites responsible for initiating at least part of the pairing interactions between homologous chromosomes. Evidence for the clustering of telomeres during meiotic prophase has been gathered through cytological studies in many species. In these studies, chromosomes in meiotic prophase were observed to form what has been described as a 'bouquet' configuration, where the telomeres are positioned together in a small portion of the nuclear volume. The telomeres are often clustered near a region of the nuclear envelope adjacent to the position of the cytoplasmic centrosome. It is thought that the clustering of telomeres may facilitate homolog pairing in meiosis (Page, 2000).

The existence of a bouquet configuration has been demonstrated recently in Saccharomyces cerevisiae, and telomeres are responsible for a delay in meiotic progression observed in haploid yeast strains that are disomic for a single chromosome pair. Furthermore, the delay associated with the presence of telomeres requires an intact NDJ1 gene. In a wild-type background, deletions of NDJ1 interfere with synapsis and alter the distribution of recombination events. Ndj1p localizes to telomeres and is believed to be necessary for telomeric pairing. Defects in synapsis and a decrease in the frequency of recombination are observed in the presence of mutations in yeast KAR3, which encodes a kinesin-like protein. This observation suggests that Kar3p may be involved in chromosome movement that is necessary for proper homolog alignment during meiotic prophase (Page, 2000).

This clustering of telomeres has not been demonstrated in Drosophila oocytes, but the observations by electron microscopy may not have detected transient telomere clustering at the leptotene or zygotene stage of meiosis. Thus, perhaps the association of telomeres is important for homologous chromosome pairing in Drosophila. The mechanism by which telomere clustering occurs may be similar to that in other organisms. In particular, the movement of the chromosomes may require associations with the cytoskeleton, resulting in the clustering of telomeres near centrosomes. Within germline cysts, the fusome acts to orient the spindles in the cystocyte divisions by interacting with one centrosome at each mitosis. Although the fusome begins to break down after the cessation of the mitotic divisions, remnants of this structure may remain in the oocyte during meiotic prophase (germarium regions 2 and 3). Therefore, the fusome may position a determinant, possibly in the form of a cytoskeletal element, necessary for telomere clustering at a point on or near the nuclear membrane. Perhaps Bam, as a component of the fusome, is indirectly necessary for telomere clustering by marking the site of clustering on the nuclear membrane. The polar recombination defect observed in mei-P26 could therefore be the indirect result of abnormal telomeric clustering due to a disruption of Bam function (Page, 2000).

Since proximal euchromatic regions may rely on the pairing of centric heterochromatin, and perhaps not require telomeric clustering, disruption of telomeric clustering may primarily impact the frequency of exchange in the distal regions of the chromosomes. Thus, the specific disruption of telomeric interactions may result in a distribution of residual exchanges like that seen in recombination precondition mutants. Further work will be necessary to determine whether the meiotic defects in precondition mutants are the result of abnormalities in telomeric clustering (Page, 2000).

Identification of TER94, an AAA ATPase protein, as a Bam-dependent component of the Drosophila fusome

The Drosophila fusome is a germ cell-specific organelle assembled from membrane skeletal proteins and membranous vesicles. Mutational studies that have examined inactivating alleles of fusome proteins indicate that the organelle plays central roles in germ cell differentiation. Although mutations in genes encoding skeletal fusome components prevent proper cyst formation, mutations in the bag-of-marbles gene disrupt the assembly of membranous cisternae within the fusome and block cystoblast differentiation altogether. To understand the relationship between fusome cisternae and cystoblast differentiation, attempts have been made to identify other proteins in this network of fusome tubules. Evidence is presented that the fly homolog of the transitional endoplasmic reticulum ATPase (TER94) is one such protein. The presence of TER94 suggests that the fusome cisternae grow by vesicle fusion and are a germ cell modification of endoplasmic reticulum. Fusome association of TER94 is Bam-dependent, suggesting that cystoblast differentiation may be linked to fusome reticulum biogenesis (Leon, 1999).

Antisera raised against a TER94 internal peptide reacts with bands of 94,000 Da in wild-type ovarian extracts and 57,000 Da in Escherichia coli cells expressing a fragment of TER94 as a GST-fusion protein. Both Cdc48p and vertebrate TERs oligomerize to form homohexameric complexes. When ovarian extracts were analyzed on native sucrose gradients, the peak of TER94 from flies sedimented was Mr ~500,000, which is close to the expected size (Mr ~530,000) for a homohexameric complex (Leon, 1999).

TER94 protein is present in both ovarian germ cells and somatic cells. TER94 is largely cytoplasmic in follicle and germ cells. Significantly, germ cells often contain one or several especially intense fluorescent signals, suggesting that TER94 is distributed unevenly in the cytoplasm. In cystocytes in germarial Region 1, these are usually somewhat diffuse bright regions, whereas in more mature cystocytes the bright spots are more sharply defined (Leon, 1999).

The number and positions of the TER-enriched regions suggest that they might correspond to fusomes. Stem cell fusomes in germ cells nearest the anterior tip appear as a single dot of intense staining, whereas those in a more posterior position (i.e. more mature cysts) contain elongated, branched fusomes. Precise colocalization of TER94 and Hu-li tao shao is strongest in Region 1 germ cells and declines in regions containing mature cysts. Because a fraction of TER is nuclear in yeast and mammals, Drosophila nuclei were examined closely. Most germ cell nuclei are faintly TER94 positive. Many examples of strong nuclear and perinuclear staining have been found in nonovarian somatic cells in larvae and adults (Leon, 1999).

Fusomes are the primary site of ER-like cisternae in young germ cells. If TER94 enrichment in fusomes represents accumulation at the fusome reticulum, TER94 distribution might be altered when the reticulum is not properly assembled. Bam is a fusome-associated protein and bam mutant fusomes are deficient in cisternae. The distribution of TER94 protein was examined in bam germ cells; it is distributed uniformly without signs of enrichment at the site of fusomes as is observed in wild-type germaria. Indeed, when the bam stem cell fusomes are visualized with Hts antibodies, it is clear that TER94 is no more abundant within or near stem cell fusomes than in any other cytoplasmic regions. Consistent with this conclusion, the merged images of TER94 and Hts distributions do not show immunofluorescent overlap, indicating that bam fusomes do not accumulate detectable TER94 (Leon, 1999).

TER94 is also enriched at a few sites that do not correspond to fusomes. It is speculated that these may be sites of Golgi bodies or transport vesicles, although unambiguous identification requires additional reagents as markers. These extrafusome sites of TER94 enrichment are also abolished in bam mutant cells (Leon, 1999).

The observation that TER94 fusome association is linked to Bam function can be explained by either a direct or indirect Bam dependent mechanism. Although loss of bam function might block fusome reticulum assembly before TER94 arrival, it is also possible that Bam recruits TER94 to the reticulum as part of the assembly process. This hypothesis has been difficult to test because Bam is a low-abundance protein in ovaries, and in vitro assays for Bam and TER94 interaction have produced inconsistent results. The interaction of Bam and TER94 as two-hybrid partners supports the hypothesis of in vivo interaction. Finding the Drosophila homolog of the S. cerevisiae protein Ufd3p as a second Bam interacting protein strengthens the significance of the Bam-TER94 interaction. Ufd3p and the yeast TER (i.e., Cdc48p) interact with one another directly. Ufd3p is required for efficient organelle vesicle fusion (Leon, 1999 and references therein).

Germ line stem cell differentiation in Drosophila requires gap junctions and proceeds via an intermediate state.

The zero population growth (zpg) locus of Drosophila encodes a germline-specific gap junction protein, Innexin 4, that is required for survival of differentiating early germ cells during gametogenesis in both sexes. Zpg is required during oogenesis for the survival of the germ line stem cell daughter as it moves away from the niche and begins to differentiate. Germ-line stem cells (GSCs) lacking Zpg can divide, but the daughter cell destined to differentiate dies. These results suggest that zpg may be necessary for the differentiation process itself, as well as for survival of differentiated germ cells, and that zpg probably acts in parallel to bam and bgcn. The differentiation of the GSC to a cystoblast is gradual, and it is suggested many of the germ cells in 'stem cell tumors' caused either by strong mutations in bam or by overexpression of Dpp may be at an intermediate state between GSCs and cystoblasts. These findings suggest that germ line stem cells differentiate upon losing contact with their niche, that gap junction mediated cell-cell interactions are required for germ cell differentiation, and that in Drosophila germ line stem cell differentiation to a cystoblast is gradual. (Gilboa, 2003).

Zpg wild-type function is required for the differentiation of GSCs. Two other genes, bam and bgcn, are required for early germ cell differentiation. However, the phenotype of bam and bgcn mutant ovaries is strikingly different from that of zpg mutants. Ovaries mutant for bam or bgcn are filled with many undifferentiated single germ cells harboring a spherical spectrosome, which have been described as GSC tumors. By contrast, ovaries from zpg flies have only a few germ cells at the tip of the ovariole. To determine the functional relationship between these genes, flies were made doubly mutant for zpg with either bam or bgcn. Ovaries from newly eclosed females were stained to visualize the germ line and the spectrosomes. In the double mutant lacking both zpg and bam, only a few germ cells were detected at the ovariole tip. However, most double-mutant ovarioles had somewhat more germ cells than zpg ovarioles. Similar results were obtained with double mutants of zpg and bgcn (Gilboa, 2003).

Since GSCs can survive in a zpg background, the predicted phenotype of a zpg, bam or bgcn; zpg double mutant would be similar to a bam (or bgcn) phenotype (i.e. a germarium filled with undifferentiated GSCs). By contrast, the double-mutant phenotype more closely resembles the zpg phenotype. To test whether slow division of zpg cells accounts for the lack of tumors in young females, older (1- to 2-week-old) females were analyzed; a similar phenotype to that of young females was found. Thus, wild-type Zpg function is required for the accumulation of bam or bgcn mutant germ cell tumors removed from the niche (Gilboa, 2003).

bam tumor cells and germ cells proliferating after Dpp overexpression (hs-dpp tumor cells) are considered to be GSCs because of their round spectrosomes and lack of BamC staining. Yet, these cells do not accumulate in a zpg background. One possible explanation for this observation is that bam and hs-dpp cells, as they move away from the niche, are at an intermediate state (pre-cystoblast) between a stem cell and a cystoblast, and that cystoblast development and survival requires Zpg. To determine whether an intermediate state between GSCs and cystoblasts exists in wild type, ovarioles were triple-labeled with anti-Vasa, 1B1 monoclonal antibody and anti-BamC, to mark the germ line, spectrosomes and cystoblasts, respectively. BamC antibody stains cysts of 4 or 8 cells strongly. Two-cell cysts had notably weaker staining. Only rarely were cystoblasts, i.e., single cells, stained with anti-BamC. In many ovarioles, single cells with a spherical spectrosome were observed that were removed from the stem cell position yet did not stain for the cystoblast marker BamC. The number of cells were counted that carried a spherical spectrosome and did not stain with anti-BamC. These cells would comprise the GSC population plus the presumptive intermediate population. Of 100 ovarioles scored, most had between 3 and 5 single cells that did not stain with anti-BamC. The average number of these cells was 3.9. This is a greater number than the average number of GSCs that populate an ovariole (between 2 and 3), as determined by cell-lineage analysis and electron microscopy. These data support the hypothesis that an intermediate state between a stem cell and a cystoblast exists in wild type (Gilboa, 2003).

Rapid evolution and gene-specific patterns of selection for three genes of spermatogenesis in Drosophila

Hybrid males resulting from crosses between closely related species of Drosophila are sterile. The F1 hybrid sterility phenotype is mainly due to defects occurring during late stages of development that relate to sperm individualization, and so genes controlling sperm development may have been subjected to selective diversification between species. It is also possible that genes of spermatogenesis experience selective constraints given their role in a developmental pathway. The molecular evolution was examined of three genes playing a role during the sperm developmental pathway in Drosophila at an early (bam), a mid (aly), and a late (don juan: dj) stage. The complete coding region of these genes was sequenced in different strains of Drosophila melanogaster and Drosophila simulans. All three genes showed rapid divergence between species, with larger numbers of nonsynonymous to synonymous differences between species than polymorphisms. Although this could be interpreted as evidence for positive selection at all three genes, formal tests of selection do not support such a conclusion. Departures from neutrality were detected only for dj and bam but not aly. The role played by selection is unique and determined by gene-specific characteristics rather than site of expression. In dj, the departure was due to a high proportion of neutral synonymous polymorphisms in D. simulans, and there was evidence of purifying selection maintaining a high lysine amino acid protein content that is characteristic of other DNA-binding proteins. The earliest spermatogenesis gene surveyed, which plays a role in both male and female gametogenesis, was bam, and its significant departure from neutrality was due to an excess of nonsynonymous substitutions between species. Bam is degraded at the end of mitosis, and rapid evolutionary changes among species might be a characteristic shared with other degradable transient proteins. However, the large number of nonsynonymous changes between D. melanogaster and D. simulans and a phylogenetic comparative analysis among species confirms evidence of positive selection driving the evolution of Bam and suggests an yet unknown germ cell line developmental adaptive change between these two species (Civetta, 2006).

Local BMP receptor activation at adherens junctions in the Drosophila germline stem cell niche

According to the stem cell niche synapse hypothesis postulated for the mammalian haematopoietic system, spatial specificity of niche signals is maximized by subcellularly restricting signalling to cadherin-based adherens junctions between individual niche and stem cells. However, such a synapse has never been observed directly, in part, because tools to detect active growth factor receptors with subcellular resolution were not available. This study describes a novel fluorescence-based reporter that directly visualizes bone morphogenetic protein (BMP) receptor activation and shows that in the Drosophila testis a BMP niche signal is transmitted preferentially at adherens junctions between hub and germline stem cells, resembling the proposed synapse organization. Ligand secretion involves the exocyst complex and the Rap activator Gef26, both of which are also required for Cadherin trafficking towards adherens junctions. It is therefore proposed that local generation of the BMP signal is achieved through shared use of the Cadherin transport machinery (Michel, 2011).

In keeping with the stem cell niche synapse hypothesis, a BMP niche signal in the Drosophila testis is transduced at subcellularly confined sites associated with adherens junctions between hub cells and GSCs. Although BMP ligands are also produced by the somatic CySCs, BMP receptor activation is not detected at the GSC surfaces facing the CySCs. There are several nonexclusive explanations that may contribute to this observation. Either, niche signalling is indeed dominated by the homodimeric Dpp or heterodimeric Dpp/Gbb ligands that are produced preferentially by the hub cells. In support of this idea, Dpp but not Gbb can fully suppress bam transcription upon ectopic expression, and is, at least in the wing, thought to have higher signalling activity. Alternatively, signalling from the CySCs may occur diffusely over the entire GSC surface and thus become diluted below the detection threshold of the reporter. Finally, based on the expression profile of the BMP ligands signalling from the CySCs is presumably dominated by Gbb and may therefore preferentially act through the alternative type I BMP receptor Saxophone, thus avoiding detection by a Tkv-specific reporter (Michel, 2011).

However, without artificial Jak/Stat pathway over-activation in the somatic cells of the testis, the CySC-derived BMP signal is by itself not sufficient to maintain GSC fate. Consequently, GSC detachment form the hub induces Bam derepression6 indicating a loss of BMP pathway activation. The junction-associated BMP signal from the hub to the germline, described in this study, is therefore essential for GSC maintenance (Michel, 2011).

In addition, this study shows that trafficking of both Dpp and DE-Cadherin in the hub cells involves the exocyst complex and the Rab11-positive recycling compartment. It is proposed that the local release of the junctional BMP signal is achieved through this shared use of intracellular machinery. Admittedly, RNAi-mediated inactivation of the exocyst complex is bound to have pleiotropic effects, and it cannot be excluded that the secretion of Upd or other growth factors may not also be affected. Can the observed loss of GSC stemness following exocyst knockdown therefore be directly attributed to a loss of BMP signalling from the hub? This is believed to be the case, because loss of Jak/Stat signalling in the germline would primarily be expected to affect adhesion of the GSCs to the hub. Although this loss of contact secondarily causes Bam derepression, Bam expression was also observed in GSCs still adhering to the hub. As Bam expression indicates a loss of BMP signalling also in the testis, this is attributed directly to the loss of the junction-associated BMP signal that is directly detected using a reporter that detects BMP receptor activation (Michel, 2011).

Future studies are required to address what directs Dpp secretion within the hub cells towards the adherens junctions with the overlying GSCs rather than towards those facing the adjacent hub cells. In addition, how the BMP ligands are confined after secretion to prevent lateral diffusion away from the site of release can now be studied. It is likely that for the latter proteoglycans has an essential role (Michel, 2011).

Finally, it was shown that the exocyst is also required for generation of the Dpp signal in the wing disc, where it forms a long-range morphogen gradient rather than a contact-dependent niche signal. It will be interesting to test whether this reflects a specific requirement of planar transcytosis, with the junctions forming a two dimensional network of signalling synapses. Alternatively, as suggested by zebrafish experiments, subcellularly restricted signal transduction at intercellular junctions may be a more general mechanism operating also in systems where BMP ligands spread through extracellular diffusion (Michel, 2011).

Drosophila germ-line modulation of insulin signaling and lifespan

Ablation of germ-line precursor cells in Caenorhabditis elegans extends lifespan by activating DAF-16, a forkhead transcription factor (FOXO) repressed by insulin/insulin-like growth factor (IGF) signaling (IIS). Signals from the gonad might thus regulate whole-organism aging by modulating IIS. To date, the details of this systemic regulation of aging by the reproductive system are not understood, and it is unknown whether such effects are evolutionarily conserved. This study reports that eliminating germ cells (GCs) in Drosophila increases lifespan and modulates insulin signaling. Long-lived germ-line-less flies show increased production of Drosophila insulin-like peptides (dilps) and hypoglycemia but simultaneously exhibit several characteristics of IIS impedance, as indicated by up-regulation of the Drosophila FOXO (dFOXO) target genes 4E-BP and l (2)efl and the insulin/IGF-binding protein IMP-L2. These results suggest that signals from the gonad regulate lifespan and modulate insulin sensitivity in the fly and that the gonadal regulation of aging is evolutionarily conserved (Flatt, 2008).

Ectopic misexpression of bam ⁺ in the female germ line, by using the binary GAL4>UAS system or heat shock-induction, eliminates GCs. Previous data suggest that the lost GCs are germ-line stem cells (GSCs): heat shock-induced bam ⁺ expression causes GC loss, but GCs that were not GSCs at the time of heat shock develop normally. Although grandchildless-like mutants lack pole cells and cannot form primordial GCs, heat shock-induced bam ⁺ overexpression eliminates female GSCs in the third larval instar (L3) or later but not before the L3 stage. When driving constitutive overexpression of UASp-bam ⁺ with the germ-line-specific nanos (nos)-GAL4::VP16 driver, it was found that GC loss continues in adult females, after the ovary has completed development. Females initially have the capacity to lay a small number of eggs but become fully sterile by day 7. Similarly, in males, bam ⁺ overexpression induced GC depopulation in the L3 stage or later. Moreover, bam ⁺ overexpression caused a dramatic expansion of somatic cells in ovaries and testes, reminiscent of the enlarged somatic gonads of agametic grandchildless-like mutants. Thus, grandchildless-like mutants and flies misexpressing bam ⁺ have expanded somatic gonads but complete GC loss at different times (Flatt, 2008).

GC loss induced by misexpression of bam ⁺ significantly increased lifespan in females and males, in several independent experiments. Lifespan was increased by 31.3% and 50% in females and 21% and 27.8% in males by GC ablation in a y w background by driving y w;UASp-bam ⁺ with nos-GAL4::VP16; effects are relative to a coisogenic control (y w;UASp-bam ⁺; control 1) and a control with a heterozygous background (y w/w¹¹¹⁸; nos-GAL4::VP16; control 2). Longevity was also extended when UASp-bam ⁺ was driven by nos-GAL4::VP16 in an independent background (w¹¹¹⁸) lacking one copy of genomic bam. The capacity for GC ablation to extend lifespan was likewise effective with the germ-line driver nos-GAL4-tubulin (NGT-GAL4) in the y w and w¹¹¹⁸ backgrounds. Thus, bam ⁺ misexpression in the germ line is sufficient to force GC loss and to increase lifespan in multiple genetic backgrounds and with different germ-line drivers. Because the failure of grandchildless-like mutants to develop GCs has no consistent major effects on lifespan, it was hypothesize that GC loss during late development or in the adult might promote longevity because GCs associate and interact with somatic cells before loss (Flatt, 2008).

If the germ line produces a signal that shortens lifespan or represses a somatic signal that extends lifespan, GC overproliferation should decrease lifespan. To test this prediction, a sterile heteroallelic null mutant of bam was examined in which mitotically active, nondifferentiating GSCs overproliferate. Thus, eliminating GC proliferation slows aging, whereas GC overproliferation shortens lifespan in the fly, as in the nematode. However, the possibility cannot be completely excluded that the longevity effects of bam are independent of its effects on GCs (Flatt, 2008).

Germ-line loss might slow aging simply by abolishing the survival costs of producing gametes. To rule out that egg production is required for GCs to shorten lifespan, a female-sterile mutant of egalitarian (egl) was examined. Mutants of egl prevent differentiation of cystoblasts into oocytes. Consequently, flies produce eggs with 16 rather than 15 nurse cells, and egg chambers degenerate before they acquire yolk. Lifespan of sterile egl mutant females (egl^PR29/egl^wu50) was reduced compared with fertile controls, suggesting that oogenesis per se might not be sufficient for reproduction to shorten lifespan. This result adds to a growing number of cases showing that the tradeoff between reproduction and survival can be decoupled (Flatt, 2008).

In C. elegans, lifespan extension by GC loss requires the FOXO transcription factor DAF-16; FOXO activity is normally repressed by IIS. Because reduced IIS slows Drosophila aging [by mutations disrupting IIS, constitutive activation of Drosophila FOXO (dFOXO), or ablation of insulin-producing cells, it was reasoned that GC loss might extend lifespan by down-regulating IIS. Accordingly, message abundance was measured for the three Drosophila insulin-like peptides (dilps) produced by median neurosecretory cells (mNSCs), the major insulin-producing cells (IPCs) in the brain of the adult. Rather than reduced message from the dilp2, dilp3, and dilp5 loci, it was found that these transcripts were induced upon GC loss by 1.8- to 26-fold relative to controls, in two independent genetic backgrounds (Flatt, 2008).

Previous attempts to quantify DILPs by Western blot analysis have failed because of low ligand abundance, and current technology does not permit detection of circulating DILPs in the hemolymph. However, several observations suggest that increased dilp message in GC-ablated flies might be biologically meaningful. Immunostaining of brains with DILP antibody indicated that the IPCs of GC-less flies produced as much and, in some cases, more DILP protein than controls, and DILP⁺ staining of IPC axonal projections was strong, suggesting functional DILP transport. Furthermore, neural DILPs homeostatically regulate sugar levels in the hemolymph, and GC-less flies had reduced amounts of stored and circulating carbohydrates (Flatt, 2008).

The hyperinsulinism of GC-less flies is a paradox because lifespan should not be extended in the face of increased DILPs. Because high DILP levels should activate IIS in peripheral tissues and repress dFOXO, transcripts were measured of two major dFOXO targets from body tissue, the translational regulator thor (encoding 4E-BP), and the small heat shock protein l (2)efl, which are normally induced when IIS is repressed and dFOXO is activated. Message levels of both dFOXO targets were up-regulated in GC knockout flies. Although it cannot be ruled out that these targets have transcriptional inputs other than dFOXO, flies with GC loss, despite elevated DILPs, express markers consistent with active dFOXO and reduced IIS (Flatt, 2008).

Because reduced IIS causes dephosphorylation and nuclear translocation of dFOXO, nuclear accumulation of dFOXO can be used to assess IIS pathway activity. To confirm that dFOXO is active in GC-less flies, its localization was examined with immunostaining in peripheral fat body, a major site of IIS activity, and by Western blotting analysis with cell fractionation in whole-body tissue. As expected, dFOXO was predominantly nuclear in GC flies, indicating that dFOXO is active. Yet, despite differential up-regulation of dFOXO targets, GC-less and control flies did not differ in nuclear dFOXO localization, which suggests that GC loss might affect dFOXO activity independent of its subcellular localization, as recently found in C. elegans (Flatt, 2008).

There are many mechanisms by which IIS can be impeded between the site of insulin production and FOXO-dependent responses of peripheral tissues: at the level of insulin secretion or transport and at many steps within intracellular IIS of target tissues. To initiate an understanding of IIS impedance in GC-less flies, whether GC loss might change transcript abundance of two DILP cofactors, dALS and IMP-L2, was assessed. In mammals, circulating IGFs form a complex consisting of IGF-1, IGF-binding proteins (IGF-BPs), and the liver-secreted scaffold protein acid labile substrate (ALS); by creating a pool of circulating IGFs, this ternary complex limits ligand availability. The Drosophila homolog of ALS (dALS) is expressed in DILP-expressing IPCs and the fat body and is up-regulated in dFoxo null mutants. Consistent with the model that dALS functions as a DILP cofactor, dALS forms a circulating trimeric complex containing DILP2 and IMP-L2, an Ig-like homolog of IGF-BP7. Binding of dALS requires prior formation of a dimeric complex containing DILP2 and IMP-L2. In cell culture experiments, IMP-L2 binds mammalian insulin and IGF-1/-2, and fall army worm (Spodoptera frugiperda) IMP-L2 inhibits IIS through the human insulin receptor. Because overexpression of dALS and IMP-L2 can systemically antagonize DILP function and IIS in Drosophila in vivo, message abundance of dALS and IMP-L2 was measured upon GC loss. Although dALS levels did not change, IMP-L2 message was increased 7-fold in GC-less flies. Although this observation is correlational, it might suggest a potential explanation for why IIS might be impeded in GC-less flies in the face of elevated DILP production. It will be of major interest to determine whether GC loss can modulate DILP availability and IIS by affecting IMP-L2 (Flatt, 2008).

Together, these results show that GCs regulate aging and modulate IIS in the fly. Although future work is required to fully characterize IIS state upon GC loss, it was observed that GC-less flies exhibit characteristics of both increased and decreased IIS. Increased DILPs and hypoglycemia are suggestive of increased IIS, but GC-less flies also have markers of IIS impedance. The induction of dFOXO targets is consistent with the finding that lifespan extension by GC loss in the nematode requires FOXO/DAF-16. In the worm, GC loss induces nuclear translocation of DAF-16 and activates DAF-16 targets, but nuclear accumulation is also observed in worms that lack the entire gonad and have normal lifespan. Similarly, it was found that GC-less and control flies differ in dFOXO target activation, but not dFOXO localization, suggesting that IIS can affect aging by modulating FOXO/DAF-16 activity independent of subcellular localization. Indeed, dietary restriction in C. elegans extends longevity by activating AMP-activated protein kinase (AMPK), which phosphorylates and activates DAF-16 but does not promote DAF-16 nuclear translocation (Flatt, 2008).

Because extended longevity by GC loss is associated with up-regulation of DILPs, GC loss might impede IIS downstream of DILP production. In humans, compensatory hyperinsulinemia is a hallmark of severe insulin resistance, and mutations in the tyrosine kinase domain of the insulin receptor can cause hyperinsulinemic hypoglycemia coupled with insulin resistance. Recent studies with fly and mouse also suggest that lifespan can be extended despite hyperinsulinemia. In Drosophila target-of-rapamycin (dTOR) mutants, longevity extension is associated with elevated DILP2 and hypoglycemia, and brain-specific insulin receptor substrate-2 (Irs-2) knockout mice are hyperinsulinemic but insulin-resistant and long-lived. Clearly, further experiments are needed to unravel the mechanisms by which insulin production can be uncoupled from IIS sensitivity and modulation of lifespan (Flatt, 2008).

The finding that GC loss affects neural DILP production also adds to growing evidence suggesting evolutionary conservation of endocrine feedback between brain and gonad. In Drosophila, neural DILPs bind to the insulin-like receptor (dINR) on GSCs to regulate GC proliferation, and neuronal InR knockout (NIRKO) mice show impaired spermatogenesis and ovarian follicle maturation. Conversely, in rats, ovariectomy decreases IGF-1 receptor density in the brain but increases circulating IGF-1 levels. Together with progress made in the worm and mouse, the Drosophila system will allow dissection of the mechanisms underlying the fundamental and intricate relationship among IIS, reproduction, and aging (Flatt, 2008).

Germ line differentiation factor Bag of Marbles is a regulator of hematopoietic progenitor maintenance during Drosophila hematopoiesis

Bag of Marbles (Bam) is a stem cell differentiation factor in the Drosophila germ line. This study demonstrates that Bam has a crucial function in the lymph gland, the tissue that orchestrates the second phase of Drosophila hematopoiesis. In bam mutant larvae, depletion of hematopoietic progenitors is observed, coupled with prodigious production of differentiated hemocytes. Conversely, forced expression of Bam in the lymph gland results in expansion of prohemocytes and substantial reduction of differentiated blood cells. These findings identify Bam as a regulatory protein that promotes blood cell precursor maintenance and prevents hemocyte differentiation during larval hematopoiesis. Cell-specific knockdown of bam function via RNAi expression revealed that Bam activity is required cell-autonomously in hematopoietic progenitors for their maintenance. microRNA-7 (mir-7) mutant lymph glands present with phenotypes identical to those seen in bam-null animals and mutants double-heterozygous for bam and mir-7 reveal that the two cooperate to maintain the hematopoietic progenitor population. By contrast, analysis of yan mutant lymph glands revealed that this transcriptional regulator promotes blood cell differentiation and the loss of prohemocyte maintenance. Expression of Bam or mir-7 in hematopoietic progenitors leads to a reduction of Yan protein. Together, these results demonstrate that Bam and mir-7 antagonize the differentiation-promoting function of Yan to maintain the stem-like hematopoietic progenitor state during hematopoiesis (Tokusumi, 2011).

The findings on Bam, mir-7 and Yan suggest a mechanism for the interaction of these regulators in the control of blood cell homeostasis. The role of Yan is to direct a quiescent hematopoietic progenitor, through a primed intermediate progenitor state, towards a blood cell differentiation fate as crystal cell, plasmatocyte or lamellocyte. The final differentiation status of these cells would be subject to the function of distinct lineage-determining transcription factors. By contrast, Bam and mir-7 cooperate to negatively modulate yan mRNA translation in the quiescent hematopoietic progenitor, thus maintaining the initial prohemocyte state. Although details of this possible translational repression remain to be parsed out, it is likely to include an as yet unidentified protein partner of Bam that would facilitate mir-7 and yan mRNA packaging within an inhibitory RNA-induced silencing complex in prohemocytes (Tokusumi, 2011).

Bag of Marbles controls the size and organization of the Drosophila hematopoietic niche through interactions with the Insulin-like growth factor pathway and Retinoblastoma-family protein

During Drosophila hematopoiesis, Bag of Marbles (Bam) is known to function as a positive regulator of hematopoietic progenitor maintenance in the lymph gland blood cell-forming organ. This study demonstrates a key function for Bam in cells of the lymph gland posterior signaling center (PSC), a cellular domain proven to function as a hematopoietic niche. Bam is expressed in PSC cells and gene loss-of-function results in PSC overgrowth and disorganization, indicating Bam plays a crucial role in controlling the proper development of the niche. It was previously shown that Insulin receptor (InR) pathway signaling was essential for proper PSC cell proliferation. This study analyzed PSC cell number in lymph glands that were double mutant for bam and InR pathway genes, and observed bam genetically interacts with pathway members in the formation of a normal PSC. The elF4A protein is a translation factor downstream of InR pathway signaling and functional knockdown of this critical regulator rescued the bam PSC overgrowth phenotype, further supporting the cooperative function of Bam with InR pathway members. Additionally, the Retinoblastoma-family protein (Rbf), a proven regulator of cell proliferation, is present in cells of the PSC, with this expression dependent on bam function. In contrast, perturbation of Decapentaplegic or Wingless signaling failed to affect Rbf niche cell expression. Together, these findings indicate InR pathway-Bam-Rbf functional interactions represent a newly identified means to regulate the correct size and organization of the PSC hematopoietic niche (Tokusumi, 2015).

The Drosophila bag of marbles gene interacts genetically with Wolbachia and shows female-specific effects of divergence

Many reproductive proteins from diverse taxa evolve rapidly and adaptively. These proteins are typically involved in late stages of reproduction such as sperm development and fertilization, and are more often functional in males than females. Surprisingly, many germline stem cell (GSC) regulatory genes, which are essential for the earliest stages of reproduction, also evolve adaptively in Drosophila. One example is the bag of marbles (bam) gene, which is required for GSC differentiation and germline cyst development in females and for regulating mitotic divisions and entry to spermatocyte differentiation in males. This study shows that the extensive divergence of bam between Drosophila melanogaster and D. simulans affects bam function in females but has no apparent effect in males. It was further found that infection with Wolbachia pipientis, an endosymbiotic bacterium that can affect host reproduction through various mechanisms, partially suppresses female sterility caused by bam mutations in D. melanogaster and interacts differentially with bam orthologs from D. melanogaster and D. simulans. It is proposed that the adaptive evolution of bam has been driven at least in part by the long-term interactions between Drosophila species and Wolbachia. More generally, it is suggested that microbial infections of the germline may explain the unexpected pattern of evolution of several GSC regulatory genes (Flores, 2015).

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date revised: 22 May 2023

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