InteractiveFly: GeneBrief

benign gonial cell neoplasm: Biological Overview | References

Gene name - benign gonial cell neoplasm

Synonyms -

Cytological map position - 60A4-60A4

Function - translational regulator

Keywords - germ-line stem cell maintenance, oogenesis, testis, regulation of translation

Symbol - bgcn

FlyBase ID: FBgn0004581

Genetic map position - chr2R:19740801-19746314

Classification - Helicase-associated domain

Cellular location - cytoplasmic

NCBI link: EntrezGene

bgcn orthologs: Biolitmine
Recent literature
McCarthy, A., Deiulio, A., Martin, E. T., Upadhyay, M. and Rangan, P. (2018). Tip60 complex promotes expression of a differentiation factor to regulate germline differentiation in female Drosophila. Mol Biol Cell: mbcE18060385. PubMed ID: 30230973
Germline stem cells (GSCs) self-renew and differentiate to sustain a continuous production of gametes. In the female Drosophila germ line, two differentiation factors, bag of marbles (bam) and benign gonial cell neoplasm (bgcn), work in concert in the stem cell daughter to promote the generation. In GSCs, bam transcription is repressed by signaling from the niche and is activated in stem cell daughters. In contrast, bgcn is transcribed in both the GSCs and stem cell daughters, but little is known about how bgcn is transcriptionally modulated. This study finds that the conserved protein Nipped-A acts through the Tat interactive protein 60kDa (Tip60) Histone acetyl transferase (HAT) complex in the germ line to promote GSC daughter differentiation. Nipped-A is required for efficient exit from the gap phase 2 (G2) of cell cycle of the GSC daughter and for expression of a differentiation factor, bgcn. Loss of Nipped-A results in accumulation of GSC daughters. Forced expression of bgcn, in Nipped-A germline-depleted ovaries rescues this differentiation defect. Together, these results indicate that Tip60 complex coordinates cell cycle progression and expression of bgcn to help drive GSC daughters toward a differentiation program.

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 (Ohlstein, 2000; Lavoie, 1999; Ohlstein, 1997; McKearin, 1995), 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 (Li, 2009; Shen, 2009). 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).

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. Page (2000) identified mei-P26 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, Neumuller (2008) reported that bam required 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).

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-CGK1 loss-of-function allele into a bamΔ86/+ 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 cooperates with Bam, Bgcn and Sxl to promote early germline development in the Drosophila ovary

In the Drosophila female germline, spatially and temporally specific translation of mRNAs governs both stem cell maintenance and the differentiation of their progeny. However, the mechanisms that control and coordinate different modes of translational repression within this lineage remain incompletely understood. This study presents data showing that Mei-P26 associates with Bam, Bgcn and Sxl and nanos mRNA during early cyst development, suggesting that this protein helps to repress the translation of nanos mRNA. Together with recently published studies, these data suggest that Mei-P26 mediates both GSC self-renewal and germline differentiation through distinct modes of translational repression depending on the presence of Bam (Li, 2013).

This study presents data that Mei-P26 cooperates with Bam, Bgcn and Sxl to control the translation of nanos mRNA in the Drosophila female germline. Co-immunoprecipitation experiments indicate Mei-P26 physically associates with the differentiation factors Bam, Bgcn and Sxl and yeast 2-hybrid assays suggest the interaction between Mei-P26 and Bgcn may be direct. Disruption of mei-P26, or snf, which disrupts sxl expression in the germline, results in the upregulation of Nanos protein expression in early differentiating cysts. Both Mei-P26 and Sxl protein associate with nanos mRNA (Chau, 2012). In light of the recently published study that shows mutating Sxl binding sites within the 3′UTR of nanos mRNA leads to mis-regulation of the gene (Chau, 2012), these results suggest that Mei-P26 may be part of a Sxl, Bgcn and Bam complex that serves to promote cyst development by directly repressing the expression of Nanos. However, despite repeated attempts, direct interactions between Bam and Bgcn with nanos mRNA could not be detected. While various technical issues may prevent the detection of these specific interactions, the inability to observe direct association between Bam/Bgcn and nanos mRNA leaves open the possibility that interactions between the components of the Mei-P26, Sxl, Bam and Bgcn complex and its target mRNAs may be dynamic in nature. For instance, Bam and Bgcn may help to prepare Sxl and Mei-P26 for mRNA binding but do not themselves directly interact or only transiently interact with these targets. Further experiments will be needed to clarify the more specific molecular mechanisms that underlie Bam/Bgcn function with respect to the translational repression of nanos mRNA (Li, 2013).

Two other recent studies investigated the role of mei-P26 during germline development. Liu (2009) showed that the RNA helicase Vasa directly regulates the translation of mei-P26 mRNA through poly (U) elements within its 3' UTR. Mutations in each gene strongly enhance the phenotype of the other, resulting in the formation of cystic germline tumors. Neumuller (2008) focused on the function of Mei-P26, showing that it negatively regulates the activity of the miRNA pathway. It is now proposed that Mei-P26 functions in both GSCs and early differentiating germ cells. Within GSCs, Mei-P26 is in a complex with miRISC proteins and enhances miRNA-mediated silencing. In addition, Mei-P26 associates with Nanos protein and promotes BMP signaling within GSCs by repressing the expression of the negative regulator Brat. GSC daughters displaced away from the cap cell niche experience less BMP signaling, allowing for the expression of Bam (Li, 2013).

It is speculated that upon Bam expression, Mei-P26 switches its activity and/or its mRNA targets. This switch allows Mei-P26 to promote germline differentiation by both negatively regulating the miRNA pathway and cooperating with Bam, Bgcn and Sxl to repress the translation of specific mRNAs such as nanos. However the complex functional relationships between Mei-P26, Sxl, Bam and Bgcn remain incompletely understood. While evidence is provided that these factors can physically associate with each other under certain conditions, disruption of these genes results in two discrete phenotypes. mei-P26 and snf mutants exhibit a cystic tumorous phenotype marked by the accumulation of undifferentiated cysts that do not express A2BP1, a molecular marker present in 4-, 8- and 16-cell cysts in wild-type samples. In contrast, disruption of bam or bgcn results in the formation of single cell germ cell tumors. These phenotypic differences suggest that Bam and Bgcn carry out additional functions independent of Mei-P26 and Sxl. A more complete characterization of the regulatory networks that govern the very early steps of germline cyst differentiation will have to await a better biochemical characterization of Bam and Bgcn function (Li, 2013).

Together these data suggest that Mei-P26 has a variety of molecular functions inside and outside of the germline. It remains unclear whether Mei-P26 exhibits the same biochemical activity when complexed with different proteins or whether its function completely changes depending on context. Based on the presence of a RING domain, Mei-P26 may act as an ubiquitin ligase. However this specific enzymatic activity has not been demonstrated nor have any direct in vivo substrates been identified. In regards to the translational repression of specific mRNAs, a model is favored in which Mei-P26 exhibits the same molecular activity within GSCs and their early differentiating daughters. It is further speculated that association of Mei-P26 with different mRNA binding proteins modulates its targeting of specific mRNAs, and/or the degree to which these different targets are repressed. The expression of Bam correlates with changes in the development role of Mei-P26 but the manner in which Bam alters the composition or activity of the Mei-P26 complex remains unknown. Regardless, the findings that Bam can associate with Mei-P26 and Sxl provide further support for the hypothesis that Bam regulates the translation of specific mRNAs to promote the early steps of differentiation within the Drosophila female germline (Li, 2013).

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).

eIF4A controls germline stem cell self-renewal by directly inhibiting BAM function in the Drosophila ovary

Stem cell self-renewal is controlled by concerted actions of extrinsic niche signals and intrinsic factors in a variety of systems. Drosophila ovarian germline stem cells (GSCs) have been one of the most productive systems for identifying the factors controlling self-renewal. The differentiation factor BAM is necessary and sufficient for GSC differentiation, but it still remains expressed in GSCs at low levels. However, it is unclear how its function is repressed in GSCs to maintain self-renewal. This study reports the identification of the translation initiation factor eIF4A for its essential role in self-renewal by directly inactivating BAM function. eIF4A can physically interact with BAM in Drosophila S2 cells and yeast cells. eIF4A exhibits dosage-specific interactions with bam in the regulation of GSC differentiation. It is required intrinsically for controlling GSC self-renewal and proliferation but not survival. In addition, it is required for maintaining E-cadherin expression but not BMP signaling activity. Furthermore, BAM and BGCN together repress translation of E-cadherin through its 3' UTR in S2 cells. Therefore, it is proposed that BAM functions as a translation repressor by interfering with translation initiation and eIF4A maintains self-renewal by inhibiting BAM function and promoting E-cadherin expression (Shen, 2009).

This study has revealed the biochemical function of the BAM/BGCN complex as a translational repressor. eIF4A in the regulation of GSC self-renewal to be a direct antagonist of BAM function in the Drosophila ovary. A model is proposed explaining how GSC self-renewal is controlled by concerted actions of intrinsic factors and the extrinsic BMP signal. BMP signaling directly represses bam expression, yet leaves low levels of BAM protein expression in the GSC. eIF4A and other unidentified germline factors in the GSC can effectively dismantle BAM/BGCN's repression of GSC maintenance factors, including E-cadherin, through physical interactions, leading to high expression of maintenance factors in the GSC. In the cystoblast (CB), high levels of BAM along with BGCN can keep eIF4A proteins out of the active pool and thus effectively repress GSC maintenance factors, promoting CB differentiation. Therefore, this study has significantly advanced current understanding of how GSC self-renewal and differentiation are regulated by translation factors (Shen, 2009).

bam and bgcn genetically require each other's function to control CB differentiation. Although they are expressed at low levels in GSCs, they have an important role in regulating GSC competition. However, their biochemical functions remained unclear until this study. This study showed that BAM specifically interacts with BGCN, but not other RNA-binding proteins VASA, Rm62, and Me31B, to form a protein complex. In addition, BAM and BGCN are shown to act together; BAM or BGCN alone are not capable of suppressing the expression of the reporter containing the shg 3' UTR. Furthermore, BAM and BGCN do not affect the stability of the reporter mRNA, further supporting that they regulate mRNA translation but not stability. To reveal the role of BGCN in the function of the BAM/BCGN complex, this study showed that direct tethering of BAM to the 3' UTR of the target mRNA can bypass the requirement of BGCN and sufficiently suppress the expression of the reporter. Based on the fact that BGCN contains a putative DEXH RNA binding domain, it is proposed that BGCN helps bring BAM to its target mRNAs to repress their translation. Therefore, this study has revealed the biochemical functions of BAM and BGCN (Shen, 2009).

Previous genetic study showed that BAM and BGCN negatively regulate E-cadherin expression in GSCs to control GSC competition, but the underlying molecular mechanism remains defined. This study showed that in Drosophila S2 cells BAM and BGCN could repress E-cadherin expression through its 3' UTR at the translational level. Along with previous observation that BAM and BGCN negatively regulate E-cadherin expression in GSCs in vivo, it is proposed that BAM and BGCN likely repress E-cadherin expression in GSCs at the translational level. In the future, it will be important to show if BAM and BGCN directly bind to the shg 3' UTR to repress E-cadherin expression in the GSC (Shen, 2009).

eIF4A, an RNA helicase, is one component of the translation initiation complex eIF4F, which is required for loading the small 40S ribosome subunit onto the target mRNA to initiate its translation. The helicase activity of eIF4A itself is weak but is enhanced upon binding to eIF4G, another component of eIF4F. Such helicase activity is important to remove the secondary structure of the 5' UTR, facilitating the ribosome scanning along mRNA to find the initiation codon ATG. To reveal how BAM and BGCN confer translation repression, the yeast 2-hybrid screen was used to identify eIF4A as a BAM interacting protein. Then, two pieces of genetic of evidence were provided supporting the idea that eIF4A and bam function together to control the balance between GSC self-renewal and differentiation. First, one copy of the mutations in eIF4A can dramatically promote germ cell differentiation in the hypomorphic bamZ/bamδ86 transheterozygous ovaries. However, a mutation in eIF4A cannot suppress the tumorous phenotype of the bam?86 homozygous ovaries (no bam function at all), suggesting that the reduction of eIF4A dosage helps enhance the remaining BAM function. Second, overexpression of eIF4A can enhance the differentiation defect in the bam?86 heterozygote. These genetic results support the antagonizing relationship between bam and eIF4A (Shen, 2009).

The antagonizing genetic relationship between bam and eIF4A suggests that eIF4A favors GSC maintenance over differentiation. The genetic analysis of the marked eIF4A mutant GSC clones shows that eIF4A is indeed required in GSCs for their self-renewal and division. To uncover the genetic mechanism underlying the function of eIF4A in maintaining GSCs, it was also shown that the marked eIF4A mutant GSC has normal BMP signaling activities in comparison with its neighboring wild-type GSC based on expression results from 2 BMP responses genes, bam and Dad, but has significantly reduced E-cadherin expression in comparison with its neighboring wild-type GSC. These genetic and cell biological results demonstrate that eIF4A controls GSC maintenance at least partly by maintaining E-cadherin expression. In mammalian cells, overexpression of translation initiation factors, such as eIF4A, 4G, and 4E, is implicated in different kinds of cancer due to their ability to increase cell proliferation. In the Drosophila imaginal disc, the block in cell proliferation caused by mutations in eIF4A can be bypassed by E2F overexpression, indicating that eIF4A regulates cell cycle progression and consequently cell proliferation. In this study, it was shown that eIF4A is also required for controlling GSC division. Therefore, it is proposed that eIF4A controls GSC proliferation by regulating cell cycle progression like in Drosophila imaginal tissues (Shen, 2009).

Recurrent positive selection at bgcn, a key determinant of germ line differentiation, does not appear to be driven by simple coevolution with its partner protein bam

Surveys of nucleotide sequence polymorphism in Drosophila melanogaster and Drosophila simulans were performed at two interacting loci crucial for gametogenesis: bam and bgcn. At the polymorphism level, both loci appear to be evolving under the expectations of the neutral theory. However, ratios of polymorphism and divergence for synonymous and nonsynonymous mutations depart significantly from neutral expectations for both loci consistent with a previous observation of positive selection at bam. The deviations suggest either an excess of synonymous polymorphisms or an excess of nonsynonymous fixations at both loci. Synonymous evolution appears to conform to neutrality at bam. At bgcn, there is evidence of positive selection affecting preferred synonymous mutations along the D. simulans lineage. However, there is also a significantly higher rate of nonsynonymous fixations at bgcn within D. simulans. Thus, the deviation from neutrality detected by the McDonald-Kreitman test at these 2 loci is likely due to the selective acceleration of nonsynonymous fixations. Differences in the pattern of amino acid fixations between these two interacting proteins suggest that the detected positive selection is not due to a simple model of coevolution (Bauer DuMont, 2007).

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).

Localization and function of Bam protein require the benign gonial cell neoplasm gene product

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)

bag-of-marbles and benign gonial cell neoplasm act in the germline to restrict proliferation during Drosophila spermatogenesis

Stem cells divide asymmetrically, regenerating a parental stem cell and giving rise to a daughter cell with a distinct fate. In many stem cell lineages, this daughter cell undergoes several amplificatory mitoses, thus generating more cells that embark on the differentiation program specific for the given lineage. Spermatogenesis in Drosophila is a model system to identify molecules regulating stem cell lineages. Mutations at two previously identified loci, bag-of-marbles and benign gonial cell neoplasm (Gateff, 1982), prevent progression through spermatogenesis and oogenesis, resulting in the overproliferation of undifferentiated germ cells. This study investigates how bam and bgcn regulate the male germline stem cell lineage. By generating FLP-mediated clones, it was demonstrated that both bam and bgcn act autonomously in the germline to restrict proliferation during spermatogenesis. By using enhancer trap lines, it was found that 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, it was found that 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 a cyst, which 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).

Functions of Bgcn orthologs in other species

The conserved RNA helicase YTHDC2 regulates the transition from proliferation to differentiation in the germline

The switch from mitosis to meiosis is the key event marking onset of differentiation in the germline stem cell lineage. In Drosophila, the translational repressor Bgcn is required for spermatogonia to stop mitosis and transition to meiotic prophase and the spermatocyte state. This study shows that the mammalian Bgcn homolog YTHDC2 facilitates a clean switch from mitosis to meiosis in mouse germ cells, revealing a conserved role for YTHDC2 in this critical cell fate transition. YTHDC2-deficient male germ cells enter meiosis but have a mixed identity, maintaining expression of Cyclin A2 and failing to properly express many meiotic markers. Instead of continuing through meiotic prophase, the cells attempt an abnormal mitotic-like division and die. YTHDC2 binds multiple transcripts including Ccna2 and other mitotic transcripts, binds specific piRNA precursors, and interacts with RNA granule components, suggesting that proper progression of germ cells through meiosis is licensed by YTHDC2 through post-transcriptional regulation (Bailey, 2017).


Search PubMed for articles about Drosophila Bgcn

Bailey, A. S., Batista, P. J., Gold, R. S., Chen, Y. G., de Rooij, D. G., Chang, H. Y. and Fuller, M. T. (2017). The conserved RNA helicase YTHDC2 regulates the transition from proliferation to differentiation in the germline. Elife 6. PubMed ID: 29087293

Bauer DuMont, V. L., et al. (2007). Recurrent positive selection at bgcn, a key determinant of germ line differentiation, does not appear to be driven by simple coevolution with its partner protein bam. Mol. Biol. Evol. 24(1): 182-91. PubMed ID: 17056645

Chau, J., Kulnane, L. S. and Salz, H. K. (2012). Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis. Proc Natl Acad Sci U S A 109: 9465-9470. PubMed ID: 22645327

Chen, D., Wu, C., Zhao, S., Geng, Q., Gao, Y., Li, X., Zhang, Y. and Wang, Z. (2014). Three RNA binding proteins form a complex to promote differentiation of germline stem cell lineage in Drosophila. PLoS Genet 10: e1004797. PubMed ID: 25412508

Gateff, E. (1982). Gonial Cell Neoplasm of Genetic Origin Affecting Both Sexes of Drosophila melanogaster. New York: Alan Liss.

Gonczy, P., Matunis, E. and DiNardo, S. (1997). bag-of-marbles and benign gonial cell neoplasm act in the germline to restrict proliferation during Drosophila spermatogenesis. Development 124(21): 4361-4371. PubMed ID: 9334284

Insco, M. L., Bailey, A. S., Kim, J., Olivares, G. H., Wapinski, O. L., Tam, C. H. and Fuller, M. T. (2012). A self-limiting switch based on translational control regulates the transition from proliferation to differentiation in an adult stem cell lineage. Cell Stem Cell 11: 689-700. PubMed ID: 23122292

Kim, J. Y., Lee, Y. C. and Kim, C. (2010). Direct inhibition of Pumilo activity by Bam and Bgcn in Drosophila germ line stem cell differentiation. J. Biol. Chem. 285(7): 4741-6. PubMed ID: 20018853

Lavoie C. A., Ohlstein B. and McKearin D. M. (1999). Localization and function of Bam protein require the benign gonial cell neoplasm gene product. Dev. Biol. 212, 405-413. PubMed ID: 10433830

Li Y., Minor N. T., Park J. K., McKearin D. M. and Maines J. Z. (2009). Bam and Bgcn antagonize Nanos-dependent germ-line stem cell maintenance (2009) Proc. Natl. Acad. Sci. 106, 9304-9309. PubMed ID: 19470484

Li, Y., Zhang, Q., Carreira-Rosario, A., Maines, J. Z., McKearin, D. M. and Buszczak, M. (2013). Mei-p26 cooperates with Bam, Bgcn and Sxl to promote early germline development in the Drosophila ovary. PLoS One 8: e58301. PubMed ID: 23526974

Liu, N., Han, H. and Lasko, P. (2009). Vasa promotes Drosophila germline stem cell differentiation by activating mei-P26 translation by directly interacting with a (U)-rich motif in its 3' UTR. Genes Dev 23: 2742-2752. PubMed ID: 19952109

McKearin, D. and Ohlstein, B. (1995). A role for the Drosophila bag-of-marbles protein in the differentiation of cystoblasts from germline stem cells. Development 121(9): 2937-2947. PubMed ID: 7555720

Neumuller, R. A., et al. (2008). Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage. Nature 454: 241-245. PubMed ID: 18528333

Ohlstein, B. and McKearin, D. (1997). Ectopic expression of the Drosophila Bam protein eliminates oogenic germline stem cells. Development 124: 3651-3662. PubMed ID: 9342057

Ohlstein, B., et al. (2000). The Drosophila cystoblast differentiation factor, benign gonial cell neoplasm, is related to DExH-box proteins and interacts genetically with bag-of-marbles. Genetics 155: 1809-1819. PubMed ID: 10924476

Page, S. L., et al. (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. Genetics 155: 1757-1772. PubMed ID: 10924472

Shen, R., Weng C., Yu J. and Xie T. (2009). eIF4A controls germline stem cell self-renewal by directly inhibiting BAM function in the Drosophila ovary. (2009) Proc. Natl. Acad. Sci. U.S.A. 106: 11623-11628. PubMed ID: 19556547

Biological Overview

date revised: 25 March 2015

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