BIOLOGICAL OVERVIEW

The early processes of Drosophila gametogenesis in both ovaries and testes are remarkably similar to one another: germ-line stem cells are sequestered at the anterior end of either organ. When a germ-line cell divides, one of the daughters remains attached and continues to function as a stem cell, whereas the other daughter becomes the 'founder' of a 16-cell syncytial cyst. Four successive divisions of this cystoblast or spermatoblast (within the ovary or testis, respectively) produce a clone of 16 sister germ line cells that remain interconnected due to an unusual process of incomplete cytokinesis, which generates interconnecting ring canals. In the ovary, a complement of somatic cells encloses each 16-cell cyst sometime after it is formed, as it passes through a specific region of the germarium. By comparison, in the testis during spermatogenesis the founding spermatoblast is enclosed by just two somatic cells prior to the four successive spermatoblast divisions. These differences aside, both processes generate cysts of 16 germ-line-derived cells surrounded by a layer of somatic cells. Shortly after this developmental juncture has been reached, the pathways diverge: female cysts go on to produce a single egg, whereas male cysts produce 64 spermatids. (See betaTub85D for a more detailed account of spermatogenesis; see also the oocyte and alpha Spectrin sites for information about oogenesis).

Of particular interest is the fate transition of descendents of the self-renewing stem cell population to become more specialized daughter cells (termed the 'cystoblast' in oogenesis, and the 'primary spermatogonial cell' in spermatogenesis). Several genes have been characterized in Drosophila that carry a common defect in oogenesis. These include ovarian tumor (otu), ovo, benign gonial cell neoplasm, sans fille, Sex lethal and the topic of this site, bag of marbles (bam). Mutations at these loci result in the absence of mature germ cells, and in the overproliferation of small cells with morphological characteristics of undifferentiated germ cells. The tumorous cyst phenotype points to the importance of gene action in the transition from stem cell to daughter cells of distinct fate. Only bag of marbles and benign gonial cell neoplasm (bgcn) act both in oogeneis and spermatogenesis, suggesting that these two genes regulate a pathway shared in the two processes (Gonczy, 1997).

In bam mutant ovaries, the overproliferating germ cells appear to behave as stem cells, according to two criteria. (1) Germ cells overproliferating in bam mutant ovaries are either not connected cytoplasmically to a neighbouring cell, or are connected to a single neighbouring cell, with a fusome passing between the two cells (see alpha Spectrin for information about the fusome). This pattern of connections is identical to that of wild-type germline stem cells, but different from that of wild-type amplifying germ cells (the differentiating progeny of stem cells), which are connected to several neighbouring germ cells as a result of incomplete cytokineses. Similarly, branched fusomes are rarely observed in bgcn mutant ovaries. (2) Germ cells overproliferating in bam mutant ovaries undergo S phase asynchronously. Again, this behavior is like that observed among germline stem cells, but not among their dividing differentiating progeny, which progress synchronously through the cell cycle. These observations led to the conclusion that bam mutant germ cells behave as stem cells and to the postulate that bam function is normally required in females to assign the fate of the cystoblast (McKearin, 1995).

Does bag of marbles play a similar role in spermatogenesis, that is, does bam mutation result in the overproliferation of germ-line stem cells in the male? Examination of bam testes suggests that gametogenesis is also disrupted at an early stage in males. These testes contain abnormal cysts populated with an excessive number of small cells the size of primary spermatocytes. Normally, the 16 primary spermatocytes, derived from the primary spermatogonial cell by four cell divisions, increase 25-fold in size prior to the onset of meiosis. However, bam spermatocytes remain about the size of early spermatocytes. They never undergo subsequent morphological changes characteristic of meiosis and spermatogenesis. Consistent with an early developmental arrest, expression of betaTub85D (also known as beta2-tubulin) is not detected in mutant testes. Mutant cysts progress into a highly refractile state (McKearin, 1990).

Evidence points to different consequences for bam mutation, depending on whether it occurs in spermatogenesis or oogenesis. In either case, mutations in bam and its kindred gene bgcn result in the overproliferation of undifferentiated germ cells. In males, bam and bgcn appear to be required to restrict the proliferation of amplifying germ cells (the progeny of primary spermatogonial cells) or to promote their entry into the meiotic cell cycle, in the case of spermatogenesis. Germ cells accumulating in bam and bgcn mutant testes have several characteristics of amplifying germ cells: (1) cytoplasmic Bam protein is expressed in bgcn mutant germ cells; (2) bgcn and bam mutant germ cells undergo incomplete cytokinesis, and (3) they proliferate in synchrony within a cyst. All three aspects are characteristic of amplifying germ cells, but not of germline stem cells. It is concluded that bam and bgcn most likely do not regulate the decision to adopt a primary spermatogonial cell fate; rather, they act to restrict the proliferation of amplifying germ cells or promote their entry into the meiotic cell cycle (Gonczy, 1997).

In females, overexpression of bam has a deleterious effect on oogenic germline stem cells, one that is not evident in spermatogenic germline stem cells. Female germ cells are progressively depleted with bam overexpression, suggesting that ectopic bam expression blocks stem cell function. It is though that ectopic bam causes stem cells to divide as committed cystoblasts: as contractile ring formation and closure takes place, fusomes grow and cell-cycle coordination is evident. It is suggested that a presumptive cystoblast daughter (pre-cystoblast) of the stem cell division undergoes a maturation process during which bam+ activation initiates cystoblast/cystocyte mitoses by modifying the germ-cell division cycle. Surprisingly, ectopic bam+ has no deleterious consequences for male germline cells, suggesting again that Bam may regulate somewhat different steps of germ-cell development in oogenesis as compared to spermatogenesis (Ohlstein, 1997).

What is the basis of the difference between functions of Bam in males and females? The function of bam and bgcn may have been adapted to play an essential role in the cystoblast due to a unique requirement of the female germline stem cell lineage. In females, only one of the sixteen amplifying germ cells adopts the oocyte fate, while its fifteen sisters become nurse cells. Oocyte determination relies on polarized microtubule-dependent intercellular transport from the nurse cells into the oocyte through ring canals. The basis for this polarity appears to be established as early as in the cystoblast. Since the fusome is associated with microtubules, it has been suggested that the fusome helps establish and maintain polarity during cystoblast divisions and the amplifying of germ cell divisions. Since a form of Bam protein is associated with the fusome (McKearin, 1995), it is possible that Bam itself plays a role in establishing polarity in the cystoblast. In bam mutant ovaries, polarity cannot be established, and the cystoblast cell fate cannot be assigned, resulting in an overproliferation of cells with stem cell characteristics. By contrast, in males, all sixteen amplifying germ cells are equivalent; there is no evidence of polarity analogous to that observed in females. This might explain why bam and bgcn are not strictly necessary for assigning primary spermatogonial cell fate (Gonczy, 1997).

What is the biochemical function of Bag of marbles? Little can be gleaned from the protein sequence since Bam is a novel protein. Some information is provided by the phenotype especially with regard to fusome appearance. In the female, each round of cystocyte mitosis is accompanied by the growth of a germ cell-specific organelle, the fusome. The fusome contains four membrane cytoskeletal proteins: alpha-Spectrin, beta-Spectrin, the adducin-like Hu-li tai shao and Ankyrin. Stem cells and cystocytes contain a large sphere of fusomal material, termed the spectrosome. During the four cystocyte mitoses, one pole of each spindle associates with the fusome, and following each mitosis, as the spindles disaggregate, additional fusomal material accumulates. Thus, by the fourth division, the fusome forms one large branched structure that extends though the ring canals into all the cells in a cyst. alpha-Spectrin deficient cells were generated in fly ovaries and the effects on cyst formation and oocyte differentiation were observed. This work shows that the fusome acts as a pole for each mitotic spindle by capturing a centriole and, in this capacity, serves to orient the planes of cell division at each cystocyte mitosis (reviewed in McKearin, 1997).

Bam is a component of the fusome. It has been noted the bam mutant fusomes are deficient in the membranous tubular reticulum that normally fills the fusome core. This observation prompts the suggestion that Bam protein might be required to recruit vesicular material into the 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. 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 (Ohlstein, 1997).

Cyclin B3 deficiency impairs germline stem cell maintenance and its overexpression delays cystoblast differentiation in Drosophila ovary

It is well known that cyclinB3 (cycB3) plays a key role in the control of cell cycle progression. However, whether cycB3 is involved in stem cell fate determination remains unknown. The Drosophila ovary provides an exclusive model for studying the intrinsic and extrinsic factors that modulate the fate of germline stem cells (GSCs). Here, using this model, Drosophila cycB3 was shown to plays a new role in controlling the fate of germline stem cells (GSC). Results from cycB3 genetic analyses demonstrate that cycB3 is intrinsically required for GSC maintenance. Results from green fluorescent protein (GFP)-transgene reporter assays show that cycB3 is not involved in Dad-mediated regulation of Bmp signaling, or required for dpp-induced bam transcriptional silencing. Double mutants of bam and cycB3 phenocopied bam single mutants, suggesting that cycB3 functions in a bam-dependent manner in GSCs. Deficiency of cycB3 fails to cause apoptosis in GSCs or influence cystoblast (CB) differentiation into oocytes. Furthermore, overexpression of cycB3 dramatically increases the CB number in Drosophila ovaries, suggesting that an excess of cycB3 function delays CB differentiation. Given that the cycB3 gene is evolutionarily conserved, from insects to humans, cycB3 may also be involved in controlling the fate of GSCs in humans (Chen, 2018).

The cycB3 gene is evolutionarily-conserved among higher eukaryotic organisms examined, from insects to mammalians. The cycB3 protein is present as Cyclin A and B (two other B-type Cyclins) in mitotically-proliferating cells, and is involved in the regulation of mitosis, where it cooperates with Cyclin A and B. It is reported that Cyclin A and B are involved in the regulation of ovarian GSC maintenance in Drosophila. Earlier observation showed that cycB3² homozygous mutant females partially exhibit thinned ovaries. Given reports on Cyclin activity in stem cells, the thinned ovaries prompted a further exploration of the potential involvement of cycB3 in the maintenance of germline stem cells, in the Drosophila ovary. The phenotypic assays indicate that a cycB3 deficiency leads to GSC loss with ageing. The rescue assays and genetic mosaic analyses convincingly suggest that CycB3 functions as an intrinsic factor for controlling the fate of GSC (Chen, 2018).

Previous studies have discovered that the Dpp/Bam pathway is the essential signaling pathway for maintaining GSCs in the Drosophila ovary. The bam gene is a key switch in regulating the fate of GSC. Combining these results, a model is proposed to explain how CycB3 is involved in regulation of GSC/CB fate determination. In GSCs, the data show that CycB3 is not involved in Dpp-mediated bam transcriptional silencing. The cycB3 deficiency triggers GSC pre-differentiation and eventually causes its loss phenotype. In CBs, the bam gene exhibits a high expression level, due to loss of the inhibition by Dpp signaling, and the Bam protein can promote CB differentiation. Genetic interaction analyses strongly shows that cycB3 function is positioned upstream of Bam action in CBs. The excess cycB3 come from cycB3 overexpression, which specifically suppresses CB differentiation, probably through repressing the activity of Bam. However, what are the factors that functionally position upstream of cycB3 in CBs of Drosophila ovary? This still remains elusive (Chen, 2018).

It is reported that cycB3 promotes metaphase–anaphase transition in Drosophila embryos. The current data show that overexpression of cycB3 fails to increase the number of GSCs, suggesting that the excess CycB3 may fail to influence transition into the GSC system, whereas the excess CycB3 is sufficient to delay CB differentiation. The underlying molecular mechanism might be due to the fact that the increased CycB3 activity is sufficient to enhance CB proliferation, by promoting metaphase-anaphase transition (Chen, 2018).

Autophagy promotes tumor-like stem cell niche occupancy

Adult stem cells usually reside in specialized niche microenvironments. Accumulating evidence indicates that competitive niche occupancy favors stem cells with oncogenic mutations, also known as tumor-like stem cells. However, the mechanisms that regulate tumor-like stem cell niche occupancy are largely unknown. This study used Drosophila ovarian germline stem cells as a model and use bam mutant cells as tumor-like stem cells. Interestingly, it was found that autophagy is low in wild-type stem cells but elevated in bam mutant stem cells. Significantly, autophagy is required for niche occupancy by bam mutant stem cells. Although loss of either atg6 or Fip200 alone in stem cells does not impact their competitiveness, loss of these conserved regulators of autophagy decreases bam mutant stem cell niche occupancy. In addition, starvation enhances the competition of bam mutant stem cells for niche occupancy in an autophagy-dependent manner. Of note, loss of autophagy slows the cell cycle of bam mutant stem cells and does not influence stem cell death. In contrast to canonical epithelial cell competition, loss of regulators of tissue growth, either the insulin receptor or cyclin-dependent kinase 2 function, influences the competition of bam mutant stem cells for niche occupancy. Additionally, autophagy promotes the tumor-like growth of bam mutant ovaries. Autophagy is known to be induced in a wide variety of tumors. Therefore, these results suggest that specifically targeting autophagy in tumor-like stem cells has potential as a therapeutic strategy (Zhao, 2018).

This study used Drosophila ovarian germline stem cells to study stem cell competition for niche occupancy. Significantly, it was found that autophagy promotes niche occupancy by bam mutant stem cells. Autophagy is required for proper cell cycle of bam mutant cells, and regulators of growth influence bam mutant stem cell niche occupancy. Previous reports indicate that autophagy is required for stem cell maintenance, proper differentiation, and homeostasis in different stem cell systems. By contrast, the data indicate that loss of autophagy does not have a negative impact on Drosophila ovarian germline stem cells, consistent with data indicating that autophagy is low in normal wild-type stem cells. Drosophila ovarian germline stem cells are the largest cells in germaria, and they have a high metabolic rate that is associated with the activation of BMP signaling, the expression of Myc that is a key regulator of cell growth and ribosome biogenesis, and a high level of rRNA transcription. Therefore, the catabolic autophagy pathway may be dispensable in Drosophila ovarian germline stem cells under normal conditions (Zhao, 2018).

Stem cell renewal is dependent on cell growth and division that is typically regulated by mTOR. In most cell contexts, autophagy is inhibited when mTOR-dependent cell growth is activated. Therefore, it is logical that autophagy levels are low in normal stem cells that are not stressed. By contrast, transformed cells, such as those with activated Ras, have been reported to possess elevated autophagy. Therefore, it seems reasonable that, like Ras transformed cells, bam mutant stem cells may depend on autophagy for cell division and ovarian growth. Interestingly, autophagy is required for the proliferation of fast-dividing germline progenitor cells in the C. elegans larval gonad. It is not clear why autophagy may be compatible with and required for tumor-like germline stem cell growth and division. One possibility is that autophagy is functioning to reduce cell stress associated with increased metabolic rate, protein, and organelle damage, but if this were the case, this would likely be reflected in increased cell death. Because non increase in cell death was observed, an alternative explanation is that autophagy is promoting bioenergetic homeostasis that is needed in tumor-like cells (Zhao, 2018).

Unlike previous studies of epithelial cell competition, the data indicate that loss of regulators of tissue growth, either the insulin receptor or cyclin-dependent kinase 2 function, influence the competition of tumor-like stem cells for niche occupancy. Stem cells possess properties that distinguish them from epithelial cells in the context of cell competition. First, adult stem cells are usually quiescent, while epithelial cell competition often takes place in fast-growing tissues. Second, stem cells compete for niche occupancy and there are no known specialized niches in epithelial cell competition systems. Third, loser stem cells are displaced from the niche and do not die, while loser epithelial cells die, enabling winners to expand during epithelial cell competition. In addition, tumor-like stem cells appear to divide faster than normal adult stem cells. These differences probably make tumor-like stem cells more sensitive to regulators of growth than epithelial cells during competition (Zhao, 2018).

Autophagy can either promote or suppress tumor growth, depending on cell and tissue context. Similar phenomena were also observed in different Drosophila tumor-like overgrowth models, where autophagy either enhanced or suppressed epithelial overgrowth phenotypes, depending on the oncogenic stimulus. The results indicate that autophagy promotes Drosophila ovarian germline tumor-like growth. Significantly, autophagy promotes niche occupancy by bam mutant tumor-like stem cells. Of note, the data indicate that the super-competition of bam mutant stem cells largely depends on their proliferative potential. In addition, the data contradict the current model that the niche stem cell adhesion factor E-cadherin plays a vital role, as no significant difference was observed in E-cadherin between wild-type and bam mutant stem cells. Furthermore, bam mutant stem cells that are out of the niche do not possess an E-cadherin connection with niche cap cells, but they are still more competitive than wild-type stem cells for niche occupancy, further challenging the current model that emphasizes a critical role of E-cadherin. In addition, although the super-competition of tumor-like stem cells for niche occupancy is proposed to be important for tumor initiation, it still cannot be excluded that autophagy also contributes to tumor progression in the tumor model system. Importantly, these studies indicate that specifically targeting autophagy in tumor-like stem cells could have potential for cancer therapy (Zhao, 2018).

H3K36 trimethylation-mediated epigenetic regulation is activated by Bam and promotes germ cell differentiation during early oogenesis in Drosophila

Epigenetic silencing is critical for maintaining germline stem cells in Drosophila ovaries. However, it remains unclear how the differentiation factor, Bag-of-marbles (Bam), counteracts transcriptional silencing. This study found that the trimethylation of lysine 36 on histone H3 (H3K36me3), a modification that is associated with gene activation, is enhanced in Bam-expressing cells. H3K36me3 levels were reduced in flies deficient in Bam. Inactivation of the Set2 methyltransferase, which confers the H3K36me3 modification, in germline cells markedly reduced H3K36me3 and impaired differentiation. Genetic analyses revealed that Set2 acts downstream of Bam. Furthermore, orb expression, which is required for germ cell differentiation, was activated by Set2, probably through direct H3K36me3 modification of the orb locus. These data indicate that H3K36me3-mediated epigenetic regulation is activated by bam, and that this modification facilitates germ cell differentiation, probably through transcriptional activation. This work provides a novel link between Bam and epigenetic transcriptional control (Mukai, 2015).

To examine histone modifications in differentiating germ cells, wild-type ovaries were stained using monoclonal antibodies specific for histone modifications. The H3K36me3 histone modification, associated with active genes, accumulated in differentiating cystoblasts. H3K36me3 signals were increased in the differentiating cystoblasts that expressed the bam reporter gene (bam-GFP). By contrast, the H3K27me3 modification associated with gene repression accumulated in early germ cells, and its signals decreased as the cells differentiated. These results suggest that the H3K36me3 levels were upregulated in differentiating cystoblasts. Next, H3K36me3 levels were examined in the ovaries of the third instar larvae and bam⁸⁶ mutant adult females, both of which contain undifferentiated germ cells. Although H3K27me3 signals were detected in these undifferentiated germ cells, strong H3K36me3 signals were not detected. Taken together, these data supported the idea that H3K36me3-mediated epigenetic regulation may be involved in germ cell differentiation. (Mukai, 2015).

Set2 methyltransferase is responsible for the H3K36me3 modification. Immunostaining revealed that, in the germarium region, Set2 was expressed in most of the germline cells, and that nuclear Set2 levels increased in differentiating cystoblasts. To determine whether Set2 participates in H3K36me3 accumulation and differentiation, Set2 expression was inhibited by using an UAS-Set2.IR line. Set2 levels in germ cells were reduced by the expression of Set2 RNAi. Specifically, while Set2 signals in differentiating cystoblasts were detected in 100% of control (nanos-Gal4/+) germaria, the Set2 signals in the cystoblasts were significantly reduced in 57% of the germaria, when Set2 RNAi was expressed in germ cells under the control of the nanos-Gal4 driver. Next, H3K36me3 levels were investigated in the ovaries expressing Set2 RNAi. As expected, H3K36me3 levels were reduced as a consequence of Set2 RNAi treatment. In control ovaries, H3K36me3 signals in differentiating cystoblasts were detected in 97% of germaria. By contrast, when Set2 RNAi was expressed in germ cells under the control of the nanos-Gal4 driver, H3K36me3 signals in cystoblasts were severely reduced in 41% of the germaria. Moreover, germ cell differentiation was impaired because of the expression of Set2 RNAi. In 96% of the control germaria, cysts with branched fusomes were observed. However, fragmented fusomes were detected in 34% of the germaria expressing Set2 RNAi. These results indicate that Set2 was required for both H3K36me3 accumulation and cyst formation. Mosaic analysis was performed by using a Set2 null allele Set2¹. Strong H3K36me3 signals were observed in 80% of the control germline clones. By contrast, H3K36me3 levels were considerably reduced in 74% of the Set2^- cystoblasts. Furthermore, a differentiation defect was observed that was similar to that induced by Set2 RNAi treatment in 84% of Set2^- mutant cysts. These results suggest that Set2 is intrinsically required both for H3K36me3 accumulation in cystoblasts and for differentiation (Mukai, 2015).

To investigate the potential regulatory link between Set2 and Bam, their genetic interaction was analyzed. Reduction in Set2 activity by introduction of a single copy of Set2¹ dominantly increased the number of germaria with the differentiation defect in bam⁸⁶/+ flies. Fragmented fusomes were observed in 26% of germaria from the Set2¹/+; bam⁸⁶/+ females , as compared to 5% in bam⁸⁶/+ and 3% in Set2¹/+ females. These results indicated that Set2 cooperates with bam to promote cyst formation. To determine whether bam expression requires Set2 activity, Bam expression in Set2^- germline clones by immunostaining. Indeed, Set2 activity in germ cells was dispensable for bam expression. Conversely, nuclear Set2 expression in the germ cells was significantly reduced by bam mutation, suggesting that bam is involved in the regulation of Set2 in these cells. This result is consistent with the observation that H3K36me3 levels were reduced by bam mutation. Moreover, reducing of bam activity by introducing of a single copy of bam⁸⁶ dominantly increased the number of germaria with weaker H3K36me3 signals in Set2¹/+ flies. Decreased H3K36me3 signals in the cystoblasts were observed in 29% of germaria from the Set2¹/+; bam⁸⁶/+ females, as compared to 3% in Set2¹/+ and 2% in bam⁸⁶/+ females. These data prompted an exploration of the mechanism of regulation of Set2 activity by bam (Mukai, 2015).

To address whether bambam is sufficient for H3K36me3 accumulation, H3K36me3 levels were examined in the ovaries carrying the hs-bam transgene, which is used to ectopically express bam⁺ by heat shock treatment (Ohlstein and McKearin, 1997). No GSCs with a strong H3K36me3 signal were observed in germaria from wild-type females 1 hour post-heat shock (PHS; n = 42). However, H3K36me3 levels in the GSCs were significantly increased in 51% of the germaria from hs-bam females 1 hour PHS (n = 65), indicating that ectopic bam expression is sufficient for H3K36me3 accumulation. Because Set2 is responsible for H3K36me3, it is speculated that bam may regulate Set2 activity to control H3K36me3 accumulation and GSC differentiation. To determine whether Set2 activity is required for these bam-mediated processes, the effect was studied of a reduction in Set2 activity on the GSC differentiation induced by bam. When bam⁺ was ectopically expressed by heat shock, GSC differentiation was induced as previously reported. In 71% of ovaries from hs-bam flies dissected 24 hours PHS, it was found that differentiating cysts, instead of GSCs, occupied the tip of germaria. By contrast, when both bam and Set2 RNAi were ectopically expressed, GSC loss was significantly suppressed. These data suggest that Set2 activity is regulated by Bam, and that Set2 acts downstream of bam and promotes differentiation (Mukai, 2015).

Nuclear Set2 levels were increased in differentiating cystoblasts. Furthermore, nuclear Set2 levels in germ cells were reduced by bam mutation. It is speculated that bam may regulate Set2 nuclear localization. Therefore, whether bam expression is sufficient for Set2 nuclear accumulation was examined. The subcellular localization of Set2 was examined in hs-bam flies cultured at 30°C. First, H3K36me3 levels were examined in the GSCs. H3K36me3 levels in GSCs were increased in 36% of the germaria from the hs-bam females, as compared to 6% in wild-type females. This result suggests that the ectopic expression of bam is sufficient for H3K36me3 accumulation. Next, Set2 subcellular localization was examined in GSCs of hs-bam females cultured at 30°C. Nuclear Set2 levels in GSCs were increased in 54% of the germaria from the hs-bam females, as compared to 12% in wild-type females. These results suggest that bam promotes the nuclear accumulation of Set2 (Mukai, 2015).

To understand the mechanism by which Set2 regulates germ cell differentiation, the genetic interaction between Set2 and the differentiation genes A2BP1 and orb, both of which are required for cyst differentiation, were examined. Reduction of Set2 activity by introduction of a single dose of Set2¹ dominantly increased the number of germaria exhibiting a differentiation defect in orb^dec/+ flies. In 24% of germaria from the Set2¹/+; orb^dec/+ females, fragmented fusomes were observed, as compared with 4% in orb^dec/+ and 7% in Set2¹/+ females. By contrast, the reduction of Set2 activity did not significantly affect cyst formation in A2BP1^KG06463/+ ovaries). These results implied that Set2 function is required to specifically regulate orb expression and promote cyst formation. To confirm this, orb expression was examined in Set2^- cyst clones. Deletion of Set2 led to the delayed activation of orb. Although 74% of the control cyst clones located at the boundary of germarium regions 1 and 2a initiated orb expression, only 31% of Set2^- cyst clones expressed orb. Most (61%) of the Set2^- cyst clones in germarium region 2b recovered orb expression. These observations suggest that Set2 was required for the proper activation of orb in differentiating cysts. Next, the H3K36me3 state of the orb locus was investigated in the ovaries. ChIP assays demonstrated that the H3K36me3 enrichment in the 3'-UTR region of orb was significantly higher than in the 5'-UTR region. It has been reported that the H3K36me3 modification exhibits a 3'-bias, such that H3K36me3 is preferentially enriched at the 3' regions of actively transcribed genes. These results support the idea that orb expression in differentiating cysts is controlled in part by H3K36me3-mediated epigenetic regulation (Mukai, 2015).

Next, the H3K36me3 status was investigated in the orb gene in bam⁸⁶ mutant ovaries. ChIP assays showed that bam mutation reduced the amount of H3K36me3 in the 3'-UTR region of the orb gene. The H3K36me3 modification is linked to transcriptional elongation. Therefore, the results suggested that bam activates orb expression through the epigenetic control. Additionally, H3K4me3 and RNA polymerase II levels in the 5'-UTR region of the orb gene were also reduced by bam mutation, implying a role for bam in transcriptional initiation. To investigate this possibility, further investigation will be needed in order to identify the enzymes responsible for H3K4me3 and exploring the interactions between bam and those enzymes (Mukai, 2015).

These results have shown that H3K36me3 levels are regulated by bam. As a cytoplasmic protein, Bam may indirectly regulate Set2 nuclear localization. Set2 exerts its functions through the interactions with cofactors. Understanding the mechanism by which Bam regulates Set2 will require the identification of the cofactors that mediate the nuclear transport of Set2. These data suggest a link between Bam and epigenetic transcriptional control. Bam may counteract epigenetic silencing in GSCs through H3K36me3-mediated epigenetic regulation. This study showed that orb expression is activated by epigenetic regulation. Because orb encodes a cytoplasmic polyadenylation element-binding protein, Orb may control translation in differentiating cysts in a polyadenylation-associated manner. Bam antagonizes the Nanos/Pumilio complex, which suppresses the translation of target mRNAs that encode differentiation factors . However, the ientity of the target mRNAs and the mechanisms for transcriptional activation have not yet been elucidated. Because Set2 is required for bam-induced GSC differentiation, studies focused on identifying the genes marked by H3K36me3 and on their epigenetic regulation will aid in the identification of the differentiation genes. Because Set2 is linked to transcriptional elongation, differentiation genes in GSCs might be poised for expression, but may be kept awaiting bam expression for full activation. It is anticipated that these results will facilitate a better understanding of the epigenetic mechanisms that regulate gametogenesis (Mukai, 2015).

Germline stem cell number in the ovary is regulated by mechanisms that control Dpp signaling: Bam blocks Dpp signaling downstream of Dpp receptor activation, thus establishing the existence of a negative feedback loop between the action of the two genes

The available experimental data support the hypothesis that the cap cells (CpCs) at the anterior tip of the germarium form an environmental niche for germline stem cells (GSCs) of the Drosophila ovary. Each GSC undergoes an asymmetric self-renewal division that gives rise to both a GSC, which remains associated with the CpCs, and a more posterior located cystoblast (CB). The CB upregulates expression of the novel gene, bag of marbles (bam), which is necessary for germline differentiation. Decapentaplegic (Dpp), a BMP2/4 homolog, has been postulated to act as a highly localized niche signal that maintains a GSC fate solely by repressing bam transcription. The role of Dpp in GSC maintenance has been examined in more detail. In contrast to the above model, it is found that an enhancer trap inserted near the Dpp target gene, Daughters against Dpp (Dad), is expressed in additional somatic cells within the germarium, suggesting that Dpp protein may be distributed throughout the anterior germarium. However, Dad-lacZ expression within the germline is present only in GSCs and to a lower level in CBs, suggesting there are mechanisms that actively restrict Dpp signaling in germ cells. One function of Bam is to block Dpp signaling downstream of Dpp receptor activation, thus establishing the existence of a negative feedback loop between the action of the two genes. Moreover, in females doubly mutant for bam and the ubiquitin protein ligase Smurf, the number of germ cells responsive to Dpp is greatly increased relative to the number observed in either single mutant. These data indicate that there are multiple, genetically redundant mechanisms that act within the germline to downregulate Dpp signaling in the Cb and its descendants, and raise the possibility that a Cb and its descendants must become refractory to Dpp signaling in order for germline differentiation to occur (Casanueva, 2004).

The prevalent model for Dpp action within the ovary is that it is a local niche signal whose activity is permissive for GSC maintenance. In this model, only GSCs within the niche are exposed to Dpp protein and removal of the CB from the niche lessens or eliminates exposure to the ligand. Moreover, the only postulated function of Dpp is to repress the transcription of bam within the GSCs. The data presented in this paper reveal additional aspects of Dpp function in GSC maintenance. The results strongly suggest that Dpp ligand is not restricted to the niche but rather is present throughout the anterior germarium. Data is presented that the observed specificity of Dpp signaling to the GSCs and CBs is due to functionally redundant mechanisms that operate in the germline to actively downregulate Dpp signaling during GSC differentiation. One of these mechanisms is Bam itself, thus establishing a negative feedback loop between the actions of the two genes. These findings indicate GSC differentiation is correlated with downregulation of Dpp signaling, raising the possibility that Dpp signaling plays an active role in GSC maintenance, and that GSC differentiation requires both the presence of Bam and the absence of Dpp signaling (Casanueva, 2004).

If GSCs and CBs are exposed to equivalent amounts of Dpp protein, as is suggested by both the transcription pattern of the Dpp gene and the expression of Dad-lacZ in the CpCs of the niche and the ISCs posterior to the niche, then it is likely that the observed reduction in Dad-lacZ expression between the GSC and the CB results from intracellular modulation of the strength of the Dpp signal. One hallmark of the GSC is its invariant plane of division. It is proposed that the differential Dpp signaling between the GSC and CB sign results from an intracellular modulation of Dpp signal strength between the two daughter cells, either by the asymmetric segregation of one or more cellular components that modulate Dpp signaling, or by loss of a contact-based niche signal that elevates Dpp signaling preferentially within the GSCs. Removal of the CB cell from the niche thus results in partial downregulation of Dpp signaling. A lower level of Dpp signaling in the CB cell results in the transcription of Bam, which plays multiple roles in CB differentiation, one of which is to cause the daughters of the CB cell to become refractory to further Dpp signaling. Thus, sequential regulatory mechanisms cooperate to ensure an irreversible change in the fate of the GSC cell within two generations (Casanueva, 2004).

Loss-of-function mutations in Smurf and gain-of-function mutations in sax increase the number of GSCs, suggesting these genes may perturb the proposed intracellular modulation of Dpp signaling that occurs between the GSC and CB. However, these data are not sufficient to determine whether this proposed modulatory pathway acts through direct regulation of the functions of one or both of these gene products, or whether the proposed pathway acts in parallel to these genes. In the embryo, loss of Smurf activity results in a ligand-dependent elevation of Dpp signaling that has greater, but not indefinite, perdurance (Podos, 2001), suggesting that Dpp signaling in Smurf mutants, and by inference sax mutants, is still responsive both to the amount of ligand and to the presence of other negative regulatory mechanisms. In the ovary, the Dad-lacZ-expressing germ cells in the Smurf and sax mutants fill the region of the anterior germarium that roughly corresponds to the spatial extent of Dad-lacZ expression in the somatic cells of region 1 and 2A of a wild-type germarium, suggesting that potentially all germ cells in region 1 and 2A of the Smurf and sax germaria are equally and fully responsive to the Dpp ligand. It is proposed that GSCs in the Smurf and sax germaria ultimately undergo normal differentiation because in the more posterior regions of the germaria the amount of Dpp ligand may be reduced to a level that allows bam transcription, which further reduces Dpp signaling and causes cyst differentiation (Casanueva, 2004).

The reduction in Dpp signaling between the GSC and the CB releases Bam from Dpp-dependent transcriptional repression, and one, but not the only, function of Bam is to downregulate Dpp signaling downstream of receptor activation prior to overt GSC differentiation. This is the first molecular action ascribed to Bam, and these data could provide an entry point to elucidate the biochemical basis of the function of Bam in CB differentiation. Further work will be necessary to determine whether the action of Bam on the Dpp pathway is direct or indirect, whether Bam action results in the reduction or complete elimination of Dpp signaling in the developing cysts, and which step in the intracellular Dpp signal transduction pathway or expression of Dpp target genes is affected by Bam action. However, it is possible that initial insights into Bam function can be made by comparing the thresholds for Dpp signaling readouts in the developing wing disc of the larva to the data obtained in the germarium. In the wing disc, Dpp diffuses from a limited source to form a gradient throughout the disc that displays different thresholds for multiple signaling readouts. Specifically, Dad-lacZ is transcribed in response to high and intermediate levels of Dpp, but does not respond to the lowest levels of ligand. An antibody exists that recognizes the active phosphorylated form of Mad, pMad. In the wing disc, high level staining with the pMad antibody is present in only a subset of cells that express high levels of Dad-lacZ, suggesting that in this tissue the pMad antibody is less sensitive to Dpp signaling than is Dad-lacZ expression. Intriguingly, in the ovariole pMad staining is visible in the GSCs, CBs and the developing cysts. Because Dad-lacZ expression was never observed in the developing cysts, these results could suggest that the relative sensitivities of these two reagents are reversed within the germline. Alternatively, if the reagents have the same relative sensitivities in the two tissues, the data suggest that Bam could act, probably at a post-transcriptional level, to downregulate Dpp signaling downstream of Mad activation (Casanueva, 2004).

The pattern of Dad-lacZ expression observed in the Smurf; bam and sax; bam double mutant ovarioles is qualitatively different from that observed in any of the single mutant ovarioles. Although Dad-lacZ expression is observed only at the anterior tip of the germarium of each single mutant, many, but not all, of the double mutant ovarioles contain germ cells throughout the ovariole that express high levels of Dad-lacZ. From these data, it is concluded that two redundant pathways downregulate Dpp signaling in the germline, and that in the single mutants, the action of the remaining active pathway is sufficient to constrain Dpp responsiveness to the anterior tip of the germarium. However, not all doubly mutant ovarioles display a spatial expansion of Dpp signaling, and this variability can even be observed in ovarioles from a single female. It is proposed that the observed variability results because the Smurf and sax mutations have modulatory effects on Dpp signaling that are both dependent on the presence of ligand and are sensitive to additional mechanisms that downregulate Dpp signaling. In both the Smurf; bam and sax; bam ovarioles, the germ cells that express Dad-lacZ are observed throughout the ovariole, but are more likely to be near somatic cells. It is possible that the variability in Dad-lacZ expression occurs because of a non-uniform distribution of the Dpp ligand. Nevertheless, there is not a consistent correlation between the domains of Dad-lacZ expression in the somatic and germ cells, suggesting that there may be additional germline intrinsic factors that affect Dpp signaling (Casanueva, 2004).

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 was shown 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 bam^Z/bam^δ86 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).

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).

The germline linker histone dBigH1 and the translational regulator Bam form a repressor loop essential for male germ stem cell differentiation

Drosophila spermatogenesis constitutes a paradigmatic system to study maintenance, proliferation, and differentiation of adult stem cell lineages. Each Drosophila testis contains 6-12 germ stem cells (GSCs) that divide asymmetrically to produce gonialblast cells that undergo four transit-amplifying (TA) spermatogonial divisions before entering spermatocyte differentiation. Mechanisms governing these crucial transitions are not fully understood. This study reports the essential role of the germline linker histone dBigH1 during early spermatogenesis. These results suggest that dBigH1 is a general silencing factor that represses Bam, a key regulator of spermatogonia proliferation that is silenced in spermatocytes. Reciprocally, Bam represses dBigH1 during TA divisions. This double-repressor mechanism switches dBigH1/Bam expression from off/on in spermatogonia to on/off in spermatocytes, regulating progression into spermatocyte differentiation. dBigH1 is also required for GSC maintenance and differentiation. These results show the critical importance of germline H1s for male GSC lineage differentiation, unveiling a regulatory interaction that couples transcriptional and translational repression (Carbonell, 2017).

Studies in Drosophila have provided important insights into the cellular pathways governing maintenance, proliferation, and differentiation of adult stem cell lineages, which is central to understanding normal tissue homeostasis and its alteration in disease. In particular, Drosophila spermatogenesis has become an ideal model system to study these questions. In the Drosophila testis, germ stem cells (GSCs) localize anterior, anchored to a niche of somatic cells (hub), and divide asymmetrically for self-renewal and to produce daughter progenitor gonialblast cells (GBs), which start the complex differentiation program that leads to the production of functional gametes. GBs are surrounded by 2 somatic cyst cells (Cs) and undergo four successive rounds of transit-amplifying (TA) mitoses with incomplete cytokinesis to produce a cyst of 16 sister spermatogonial cells that remain interconnected. Then, cysts differentiate to spermatocytes and undergo two meiotic divisions to produce 64 spermatids that develop to mature sperm cells (Carbonell, 2017).

bag-of-marbles (bam) is an important regulator of the first stages of spermatogenesis. Upon asymmetric division, daughter cells move away from the niche and escape Dpp/BMP-mediated repression. Bam expression increases during the first TA divisions, reaching a maximum at the 8-cell stage. Then, at the 16-cell stage, Bam expression decreases rapidly, TA proliferation stops, and differentiation into spermatocytes proceeds. How these crucial developmental transitions occurring during early male GSC lineage differentiation are regulated is not fully understood. Bam is a translational repressor that interacts with Bgcn (Benign gonial cell neoplasm) and Tut (tumorous testis) to repress Mei-P26 expression, establishing a regulatory feedback loop that governs spermatogonia proliferation. Bam also plays an important function in female oogenesis, in which it is repressed in GSCs by Dpp/BMP signaling and interacts with Bgcn to prevent translation of GSC maintenance factors (Carbonell, 2017).

This study report on the essential contribution of the Drosophila germline-specific linker histone H1 (dBigH1) to male GSC lineage development and differentiation. Linker H1s are intrinsic components of chromatin that interact with the nucleosome and regulate chromatin higher-order organization. In comparison to core histones, H1s are less well conserved, with most species containing several variants that play partially redundant functions. A conserved feature in metazoans is the presence of germline-specific variants that replace somatic H1s in germ cells (GCs). Vertebrates generally contain several male-specific variants (i.e., H1t, HILS1, and H1T2 in mice and humans) and one female-specific H1 (i.e., B4 in Xenopus and H1oo in mice and humans). In contrast, a single germline-specific linker histone dBigH1 exists in Drosophila, which is present in both the female and the male germline. Female-specific H1s are generally retained during early embryogenesis until zygotic genome activation (ZGA) . In this regard, in Drosophila, dBigH1 has been shown to maintain the zygotic genome silenced until ZGA is completed at cellularization, when dBigH1 is replaced by somatic dH1 (Carbonell, 2017 and references therein).

Little is known about the functions that germline-specific H1s play in GSC lineage development and differentiation. In mammals, h1t2 mutant mice show several abnormalities during spermatogenesis and have reduced fertility. Similarly, hils1 expression is reduced in men suffering from reduced sperm motility. However, h1t mutants do not show detectable abnormality or fertility defects. In females, H1oo is required for maturation of germinal-vesicle stage oocytes. Finally, in Caenorhabditis elegans, depletion of H1.1/HIS-24, which is abundant in the germline, affects GCs proliferation and differentiation and reduces fertility. This study shows that in Drosophila, dBigH1 is essential for male GSC lineage differentiation. dBigH1 and Bam form a double-repressor loop that regulates progression into spermatocyte differentiation. It study also shows that dBigH1 acts as a general repressor in spermatocytes and that dBigH1 is required for male GSC maintenance. Altogether, these results unveil the essential contribution of germline-specific linker histone H1 variants to GSC lineage development and differentiation (Carbonell, 2017).

This study shows that dBigH1 is required to silence bam, which is a master regulator of spermatogonia proliferation and differentiation. During the first three TA divisions, Mei-P26 facilitates accumulation of Bam, which reaches a maximum at the 8-cell stage. Then, Bam levels decrease and spermatogonia stop proliferation and differentiate to spermatocytes (see dBigH1 and Bam Form a Double-Repressor Loop that Regulates Entrance into Spermatocyte Differentiation). Several mechanisms are known to contribute to Bam downregulation after the 8-cell stage. High Bam levels downregulate Mei-P26 translation, establishing a regulatory feedback loop. In addition, several microRNAs have been shown to downregulate Bam translation. The current results suggest a model by which, in addition to translational regulation, dBigH1-mediated transcriptional repression is required to silence bam during spermatocyte differentiation. In the absence of dBigH1, bam is not silenced; thus, entrance to the spermatocyte differentiation program is blocked and spermatogonial cells accumulate. This accumulation is not accompanied by increased spermatogonia proliferation; since dBigH1 is absent during the TA divisions and, therefore, its depletion is not affecting Bam accumulation to reach the threshold that dictates proliferation stop. The current results indicate that in addition to bam, dBigH1 represses expression of multiple other genes in spermatocytes, suggesting that like in early embryogenesis, dBigH1 acts as a general silencing factor in spermatocytes. Altogether, these observations support a model by which dBigH1 acts after spermatogonia cease proliferation to set up the specific gene expression program that governs spermatocyte differentiation (Carbonell, 2017).

The results also show that Bam, which is an important translational repressor, downregulates dBigH1 expression during TA divisions. dBigH1 expression in TA cells decreases parallel to the progressive accumulation of Bam, being detectable in all 2-cell cysts and in some 4-cell cysts. It is not known whether Bam directly interacts with dBigH1 mRNAs. However, dBigH1 mRNAs are likely present during TA divisions, as they are detected before spermatocyte differentiation and bam-GAL4-induced dBigH1 depletion in TA cells reduces dBigH1 content, blocking spermatocyte differentiation. Moreover, Bam represses dBigH1 expression specifically in TA spermatogonial cells when it is driven by the ubiquitously active vasa promoter. Altogether, these observations suggest that Bam expression during the TA divisions inhibits dBigH1 mRNA translation. Later, when Bam levels decrease, dBigH1 translation resumes, reinforcing Bam downregulation through transcriptional silencing. The results show that this dBigH1/Bam double-repressor loop is crucial to license spermatogonia into spermatocyte differentiation. The important contribution of mechanisms that regulate mRNA translation during spermatogenesis has been extensively studied. However, the actual role of transcription regulation in these processes is not well understood. From this point of view, this work unveils a functional interaction during the early stages of spermatogenesis that integrates both translational and transcriptional regulation (Carbonell, 2017).

These results also suggest that dBigH1 is required for GSC maintenance, as shown by the strong developmental defects observed in nos > bigH1^RNAi testes in which dBigH1 depletion was induced in GSCs. These defects include the lack of testes in ∼10% of cases and the drastic loss of GCs in the rest of the affected testes. bam overexpression results in GSC loss. However, the contribution of dBigH1 to GSC maintenance is not likely reflecting a role in bam repression since, in knockdown nos > bigH1^RNAi testes, no derepression of a bam^P-GFP reporter was observed in vasa-positive hub-attached cells that showed no detectable dBigH1 expression. In this regard, it is known that bam is actively repressed in GSCs by the DNA binding proteins PMad/Medea that are downstream effectors of Dpp/BMP signals emanating from the somatic cells of the niche. Repression imposed by specific DNA binding proteins likely prevails over transcriptional silencing induced by general repressors such as dBigH1. dBigH1 expression is constrained to the primordial GSCs early in embryogenesis, being present in somatic cells as long as their transcriptional program is not turned on. In this scenario, it is tempting to speculate that dBigH1 is required in GSCs to repress the somatic gene expression program throughout development. In this regard, its replacement by somatic dH1 during TA divisions is particularly intriguing. How this replacement takes place and what the consequences of its misregulation are remain to be determined (Carbonell, 2017).

The presence of germline-specific histone H1 is conserved in metazoans. However, to date, detailed functional analysis of their contribution to germline development and differentiation was largely missing. From this point of view, this study unveils the fundamental functions that germline-specific linker histone H1 variants play in male GSC lineage differentiation, providing further understanding of the factors and mechanisms that regulate the dramatic developmental transitions associated with spermatogenesis (Carbonell, 2017).

GENE STRUCTURE

Bases in 5' UTR - 112

Exons - 3

Bases in 3' UTR - 479

PROTEIN STRUCTURE

Amino Acids - 442

Structural Domains

Bam has some sequence homology to the ovarian tumor gene of Drosophila. Weak similarity is detected within three regions spanning about 150 amino acids in the central region of the Bam protein. About 20% of the residues are identical and 15% correspond to conserved replacements within these regions (McKearin, 1990).

date revised: 28 December 2023

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