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

Abstract

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. Here, we report 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, we propose 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.

Stem cells in adult animal tissues have the ability to self-renew and differentiate into functional cells that replenish lost cells. Their self-renewal and differentiation are controlled by concerted actions of extrinsic factors and intrinsic factors (1, 2). Although a plethora of intrinsic factors has been identified for their roles in stem cell regulation, it remains largely unclear how differentiation factors are functionally repressed in stem cells. In this study, we show that a translation initiation factor eIF4A maintains germline stem cell (GSC) self-renewal in the Drosophila ovary by antagonizing the differentiation factor BAM.

In the Drosophila ovary, 2 or 3 GSCs are located at the tip of the germarium, where they are directly anchored to cap cells through E-cadherin-mediated cell adhesion (3). In addition, GSCs are also laterally wrapped around by escort stem cells (4). After GSC division, the daughter attaching to cap cells/escort stem cells renews as a stem cell, while the other daughter moving away from them differentiates (5). In addition, the differentiating GSC daughter, known as the cystoblast (CB), is enveloped by escort cells, which are produced by escort stem cells (4). Genetic and cellular studies have shown that cap cells, possibly along with escort stem cells, form a functional niche for GSCs. Consistently, both Yb/Piwi and BMP, which are expressed in cap cells, maintain GSC self-renewal by repressing expression of differentiation-promoting genes such as bam (6–12).

bam and bgcn were identified for their specific roles in the regulation of GSC lineage differentiation. Mutations in either bam or bgcn completely blocks GSC differentiation, causing the accumulation of GSC-like cells (13–15). bam is transcriptionally repressed by BMP signaling, leaving low levels of BAM expression in GSCs (16). We have recently shown that low levels of BAM expression work along with BGCN to control GSC competition (16). Its expression is dramatically up-regulated in differentiated germ cells ranging from cystoblasts to 8-cell cysts (13). On the other hand, bgcn is expressed in GSCs as well as in differentiated germ cells (14). Genetic studies have shown that bam and bgcn require each other's function to control germ cell differentiation, suggesting that their protein products either form a protein complex or function as a linear genetic pathway (14). Although bgcn encodes a putative DExH RNA binding protein (14), bam encodes a novel protein (15). However, their biochemical functions have remained a mystery.

Genetic and cellular studies have indicated that translation regulation plays essential roles in maintaining GSC self-renewal in different organisms, including Drosophila (3, 17, 18). Translation initiation is the rate-limiting step and thus a primary target for translational control (19). It is a complex process in which ribosomes are assembled by eukaryotic initiation factors (eIFs) to mRNA. eIF4F is a protein complex composed of eIF4A, eIF4E, and eIF4G, which respectively perform functions of an RNA helicase removing the secondary structure at the 5′ UTR, recognition of the mRNA 5′ cap structure, and bridging the mRNA with the ribosome. In this study, we have provided experimental evidence that eIF4A is a target of translation regulation in Drosophila ovarian GSCs. BAM and BGCN form a protein complex to repress E-cadherin expression at the translational level. Although eIF4A genetically antagonizes bam function in a dosage-dependent manner, it is required for GSC self-renewal by regulating E-cadherin expression. We propose that eIF4A regulates self-renewal by antagonizing BAM-mediated translation repression and that BAM controls GSC differentiation by interfering with translation initiation.

Results

eIF4A Is Identified as a BAM Interacting Protein.

BAM is necessary and sufficient to induce GSC differentiation in the Drosophila ovary (13, 15, 20). To understand how BAM controls GSC differentiation mechanistically, we performed a yeast 2-hybrid screen to identify BAM interacting proteins using his3 as the selection marker by screening a GSC-enriched cDNA 2-hybrid library (Fig. 1A). In the 2-hybrid screen, we found that a C-terminal fragment of eIF4A (305–403 aa) fused with the GAL4 activation domain (GAD) and activated his3 expression with GDB-BAM to support yeast growth on the His⁻ medium, suggesting that eIF4A is a putative BAM interacting protein. As a component of the translation initiation complex, eIF4A directly interacts with eIF4E and eIF4G to control translation initiation (19). To confirm the interaction between eIF4A and BAM, we made 4 different fusion proteins between eIF4A and GAD, named full-length GAD (FL-GAD), N terminus GAD (NT-GAD), middle portion GAD (MP-GAD), and C terminus (also the same as the initially identified fragment) GAD (CT-GAD) (Fig. 1B). Interestingly, all of the 3 regions (NT, MP, and CT), but not GAD alone, interact with GDB-BAM, indicating that BAM contacts multiple regions of eIF4A (Fig. 1C). This finding has been shown to be true for the interactions between the human tumor suppressor PDCD4 and eIF4A (21). However, the full-length eIF4A and its N terminus had stronger interactions with BAM than the C-terminal part identified in our initial 2-hybrid screen (Fig. 1 B and C).

Fig. 1.

eIF4A interacts with BAM in yeast and Drosophila S2 cells. (A) The yeast 2-hybrid system uses the his3 gene as the selection marker, supporting yeast growth on the medium lacking histidine (His⁻). X-GAD and Y-GAD represent GAD fused in frame with cDNA fragments generated from Drosophila ovarian GSC tumors. Y, but not X, interacts with BAM to activate his3 expression. (B) Four different regions of eIF4A fused with GAD: FL, NT, MP, and CT. (C) FL-GAD, NT-GAD, MP-GAD, and CT-GAD interact with GDB-BAM to support yeast growth on the His⁻ medium. (D) Flag-tagged BAM can bring down Myc-tagged eIF4A in the presence of RNase or BGCN in S2 cells.

To further substantiate the yeast 2-hybrid results, we also performed coimmunoprecipitation (co-IP) experiments to confirm the physical interaction between eIF4A and BAM in Drosophila S2 cells. Flag-tagged BAM and Myc-tagged eIF4A could reciprocally bring down each other in the co-IP experiments (Fig. 1D and Fig. S1). Furthermore, the interaction between eIF4A and BAM remained unchanged after the removal of RNAs (Fig. 1D). Therefore, we conclude that BAM can physically interact with eIF4A in an RNA-independent manner, raising the interesting possibility that BAM might control translation initiation.

eIF4A Genetically Interacts with bam.

To establish a functional connection between eIF4A and bam, we used a sensitive bam genetic background (bam^Z3-2884/bam^Δ86, referred to hereafter as bam^Z/bam^Δ86) to test genetic interactions between bam and eIF4A in the regulation of GSC differentiation. The ovaries of different genotypes were immunostained with the monoclonal anti-Hu-li tai shao (Hts) antibody, which labels spectrosomes in GSCs and CBs and fusomes in differentiated cysts (22). As reported previously (14), each bam^Z/bam^Δ86 transheterozygous ovariole had a tumorous germarium with zero or one normal egg chambers, which greatly contrasted with the wild-type ovariole, which had 7–10 egg chambers after the germarium (Fig. 2 A–C). In the bam^Z/bam^Δ86 mutant ovarioles, the tumorous germaria carried single spectrosome-containing GSC-like germ cells, whereas egg chambers also often contained single germ cells, which are indicated by spectrosomes (Fig. 2C). These bam^Z/bam^Δ86 mutant females were sterile. In contrast, most of the bam^Z/bam^Δ86; eIF4A/+ ovarioles contained 4–6 normal egg chambers (Fig. 2D). Additionally, those mutant germaria contained fewer single germ cells than bam^Z/bam^Δ86 mutant ones (Fig. 2D). Four mutant eIF4A mutant alleles gave consistent significant suppression of the mutant bam^Z/bam^Δ86 phenotypes, and those mutant females were fertile (Fig. 2B). However, all of the eIF4A alleles in the heterozygous state failed to suppress the bam null (bam^Δ86) mutant tumorous phenotype (Fig. 2E), suggesting that eIF4A modulates BAM function in the regulation of GSC differentiation. We also tested mutations in vasa (an eIF4A-like gene specifically expressed in germ cells) and eIF4E (encoding a component of the translation initiation complex). Although a mutation in either vasa or eIF4E had significant suppression of the bam^Z/bam^Δ86mutant phenotype, the effect was much weaker than the mutations in eIF4A (Fig. 2 F and G). These results indicate that eIF4A specifically antagonizes BAM function in the regulation of GSC differentiation.

Fig. 2.

eIF4A genetically interacts with bam in a dosage-dependent manner. (A) A wild-type ovariole carries a germarium (g) and 5 egg chambers (ec). (B) Barograph showing that eIF4A, and vasa or eIF4E to much less extent, can make the bam^Z/bam^Δ86 transheterozygous ovaries produce dramatically more normal egg chambers (n, total ovarioles examined). P value for a given phenotype is compared with the bam transheterozygotes. (C) A bam^Z/bam^Δ86 ovariole carries a tumorous germarium (Inset shows individual spectrosomes) and 2 egg chambers, one of which (arrowhead) also carries many single germ cells (broken lines). (D) A bam^Z/bam^Δ86;eIF4A^S02439/+ ovariole carries a tumorous germarium and 5 normal egg chambers. (E) A bam^Δ86/bam^Δ86;eIF4A^S02439/+ ovariole carries a tumorous germarium and no normal egg chambers. (F) A bam^Z/bam^Δ86;vasa^RJ36/+ ovariole carries a tumorous germarium, 1 normal egg chamber, and 3 tumorous chambers (arrowheads). (G) A bam^Z/bam^Δ86;vasa^RJ36/+ ovariole carries a tumorous germarium, 1 normal egg chamber, and 1 egg chamber (arrowhead) with single germ cells. (H) Barograph showing that germ cell-specific eIF4A overexpression can enhance the GSC differentiation defect of the bam^Δ86 heterozygous mutant (n, total germania examined). P value for a given phenotype is compared with wild type. (I) A nos-gal4 UASp-eIF4A/+ germarium carries 2 GSCs (arrows) and 1 CB (arrowhead). (J) A bam^Δ86/+ germarium carries 2 GSCs (arrows) and 2CBs (arrowheads). (K) A nos-gal4 UASp-eIF4A/+; bam^Δ86/+ carries 2 GSCs (arrows) and 5 CBs (arrowheads). (Scale bars: A–G, 50 μm; I–K, 10 μm.)

To further investigate genetic interactions between bam and eIF4A, we also examined the effect of increasing eIF4A dosage in the bam^Δ86 heterozygous background. Normally, a wild-type germarium has 2 GSCs and 1–2 CBs (Fig. 2H). eIF4A overexpression did not significantly change the GSC and cystoblast numbers in comparison with the wild-type (Fig. 2 H and I). This lack of change may be due to the abundance of eIF4A protein. Although the GSC number did not change much in the bam^Δ86 heterozygous germaria, the CB number significantly increased, indicating that bam is haploinsufficient in the regulation of GSC differentiation (Fig. 2 H and J). Interestingly, eIF4A overexpression in the bam^Δ86 heterozygous background caused the accumulation of significantly more GSCs and cystoblasts than in the bam^Δ86 heterozygote alone (Fig. 2 H and K). These results further support that bam and eIF4A exhibit dosage-dependent genetic interactions. Since BAM does not regulate eIF4A protein expression in germ cells (Fig. S2), these results also suggest that eIF4A directly antagonizes BAM function to favor GSC self-renewal.

eIF4A Is Required for GSC Self-Renewal and Proliferation.

Because eIF4A can bind to BAM and antagonize its function, we would expect that it promotes GSC self-renewal. To directly investigate the role of eIF4A in the regulation of GSC self-renewal, we used FLP-mediated FRT recombination to generate marked eIF4A mutant GSCs and then studied their maintenance and proliferation as we reported previously (10). The marked GSCs were identified by absence of armadillo-lacZ expression and presence of a spectrosome close to cap cells, and the unmarked GSCs were identified by presence of armadillo-lacZ expression and a spectrosome close to cap cells (Fig. 3 A–C and E–G). In the control, 76% of the marked GSCs detected 1 week after the clone induction (ACI) could be still detected 3 weeks ACI (Fig. 3 A–D). In contrast, only <9% of the marked GSCs mutant for eIF4A¹⁰¹³ and eIF4A¹⁰⁰⁶ detected 1 week ACI were maintained 3 weeks ACI (Fig. 3D). Consequently, most of the marked eIF4A mutant GSCs were lost from the niche 2 weeks ACI (Fig. 3F), and most of the germaria carrying a marked mutant GSC 1 week ACI had only unmarked GSCs in the niche 3 weeks ACI (Fig. 3G). The eIF4A^k01501 showed an intermediate GSC loss phenotype, which may be due to the nature of a weak mutation (Fig. 3D). Because eIF4A has been shown to control cell proliferation in a dosage-dependent manner in the Drosophila imaginal disc (23), we then examined the requirement of eIF4A for GSC division. The relative division rate is calculated by the number of cysts produced by a marked GSC divided by the number of cysts produced by a marked wild-type GSC. The relative division rate of the marked GSC mutant for eIF4A¹⁰⁰⁶ was 0.45, indicating that eIF4A mutant GSCs divide slower than wild-type ones. These results demonstrate that eIF4A is required intrinsically for controlling GSC maintenance and proliferation.

Fig. 3.

eIF4A maintains GSCs by regulating E-cadherin expression but not BMP signaling. Solid circles indicate unmarked wild-type control GSCs, whereas broken circles denote marked eIF4A mutant GSCs. (A–C) A marked wild-type GSC is detected 1 week (A), 2 weeks (B), and 3 weeks (C) ACI. (D) The marked eIF4A mutant GSCs are lost at faster rates than the marked wild-type control GSCs. The initiation percentages 1 week ACI are normalized to 100% for comparison, and the percentages at the subsequent time points are calculated by the actual percentages of the germaria carrying a marked GSC divided by the actual percentages, 1 week ACI. (E) A marked eIF4A mutant GSC and an unmarked wild-type GSC in the germarium, 1 week ACI. (F) Two unmarked wild-type GSCs in the germarium with 1 marked eIF4A mutant cyst (broken lines) developed from the recently lost marked mutant GSC, 2 weeks ACI. (G) Two unmarked wild-type GSCs in the germarium with 1 lost marked mutant GSC exiting the germarium, 3 weeks ACI. (H) A marked eIF4A mutant GSC which is negative for ApopTag labeling. (I and J) The marked eIF4A mutant GSC and the unmarked wild-type GSC in the germarium have similar levels of Dad-lacZ expression. (K and L) Both the marked eIF4A mutant GSC and the unmarked wild-type GSC in the germarium show no bam-GFP up-regulation. (M–P) Confocal images (M–O) and quantitative data (P) show that the marked eIF4A mutant GSC has less E-cadherin expression than the unmarked wild-type GSC in the stem cell-niche junction (highlighted by red and yellow lines in N). The germaria in panels A–C and E–L are shown at the same scale. (Scale bars: A and M, 10 μm.)

To help maintain the GSC, eIF4A could act by either regulating survival or self-renewal. To investigate whether eIF4A is required for GSC survival, we examined the apoptosis of the marked mutant eIF4A¹⁰⁰⁶ GSCs by using an ApopTag labeling kit (Chemicon). After examining 27 marked mutant eIF4A¹⁰⁰⁶ mutant GSCs, we did not find that any of them were positive for ApopTag labeling (Fig. 3H). Consistently, we often observed the existence of one or a few mutant cysts in the germaria that had recently lost their marked mutant GSCs (Fig. 3F and Fig. S3 A and B). These results show that eIF4A maintains GSCs by preventing their differentiation and is also required for germ cell growth and oocyte development in egg chambers.

eIF4A Regulates E-Cadherin Expression, but Not BMP Signaling, in GSCs.

BMP signaling is required for maintaining GSCs by preventing GSC differentiation, at least partly, through repressing bam expression (11, 12). Recently, eIF4A has been shown to negatively regulate BMP signaling during early Drosophila development (24). To investigate if eIF4A modulates BMP signaling in GSCs, we examined the expression of Dad-lacZ and bam-GFP in the marked mutant GSCs. Dad is a direct BMP target gene, and Dad-LacZ is a reliable Dad transcriptional reporter line (25). bam-GFP has been shown to be a reliable transcriptional reporter and is repressed in the GSC (26). Surprisingly, both Dad-lacZ (total 15 marked GSCs examined) and bam-GFP (total 19 marked GSCs examined) showed no difference in expression between the marked eIF4A¹⁰⁰⁶ mutant GSCs and the unmarked control GSCs in the same germaria (Fig. 3 I–L). This finding strongly argues against the role of eIF4A in the regulation of BMP signaling in the GSC.

Another factor essential for GSC maintenance is E-cadherin. Loss of E-cadherin expression in GSCs results in the detachment of GSCs from the niche and their subsequent loss (27). To investigate whether eIF4A regulates E-cadherin expression in the GSC, we used reconstructed 3-D confocal images to quantitatively measure E-cadherin expression in the stem cell-niche junction as previously reported (16). The marked eIF4A¹⁰⁰⁶ mutant GSCs expressed significantly less E-cadherin in the stem cell-niche junction than the unmarked control ones in the same germaria (Fig. 3 M–P). This finding indicates that eIF4A controls GSC maintenance, at least partly, through regulating E-cadherin expression in the stem cell-niche junction.

BAM and BGCN Repress E-Cadherin Translation Through Its 3′ UTR in S2 Cells.

Genetic studies have also shown that bam and bgcn require each other to regulate GSC differentiation, suggesting that they may physically interact (14). Both BAM and BGCN have been recently shown to repress E-cadherin expression in the GSC (16). To further understand how BAM and BGCN regulate E-cadherin expression at the molecular level, we used yeast 2-hybrid and co-IP experiments to investigate whether BAM and BGCN can physically interact. In the yeast 2-hybrid system, GDB-BAM and BGCN-GAD or GDB-BGCN and BAM-GAD, but not BAM-GAD or BGCN-GAD alone with GDB, could activate his3 expression, suggesting that BAM and BGCN can interact with each other (Fig. 4A). In S2 cells, the Flag-tagged BAM could bring down the Myc-tagged BGCN in the presence or absence of RNase (Fig. 4B), whereas the Myc-tagged BGCN could precipitate the Flag-tagged BAM (Fig. S4). Interestingly, the Flag-tagged BAM could not bring down the Myc-tagged version of other known RNA binding proteins VASA, Rm62, and Me31B, indicating that BAM specifically interacts with BGCN to form a protein complex (Fig. S5). Based on the observation that BAM can interact with eIF4A in the presence of BGCN and with BGCN in the presence of eIF4A (Figs. 1D and 4B), we propose that BAM, BGCN, and eIF4A form a ternary protein complex in a RNA-independent manner.

Fig. 4.

BAM and BGCN form a complex for repressing E-cadherin translation. (A) BAM and BGCN interact with each other in the yeast 2-hybrid assay. (B) Flag-BAM can bring down Myc-BGCN in the presence of RNase or eIF4A in S2 cells. (C) BAM and BGCN together can significantly repress the expression of the firefly luciferase reporter carrying a shg 3′ UTR in comparison with BAM or BGCN alone. (D) BAM, BGCN, or BAM and BGCN together cannot affect the mRNA stability of the firefly luciferase reporter carrying a shg 3′UTR. (E) Tethering BAM to the 3′ UTR through the λN-5B box interaction can repress the firefly luciferase reporter in absence of BGCN. λN-tagged GW182 (λN-GW) and eIF4G (λN-4G) function as positive controls because they are known to repress and activate translation, respectively. (F) A working model explaining how eIF4A controls GSC self-renewal by preventing differentiation.

Because BGCN contains a putative DEXH RNA binding domain (14), we then tested if BAM and BGCN can repress E-cadherin expression at the posttranscriptional level in S2 cells. Because the 3′ UTR is frequently the target sequence for mRNA degradation or translation regulation, the shg (encoding E-cadherin) 3′ UTR was fused with the Firefly luciferase reporter and, as the internal control, the actin5C 3′ UTR was fused with the Renilla luciferase reporter. Both reporter gene constructs were expressed under the control of the actin5C promoter. Without BAM and BGCN expression, the expression ratio of the Firefly luciferase versus Renilla luciferase was about 8, which represents their relative protein expression levels (Fig. 4C). In the presence of either BAM or BGCN alone, the ratios were still similar to the absence of both BAM and BGCN (Fig. 4C). Note that S2 cells express very low levels of bam and bgcn mRNAs (Fig. S6). However, in the presence of both BAM and BGCN, the ratio was reduced approximately 4-fold in comparison to neither BAM nor BGCN or with either BAM or BGCN, indicating that BAM and BGCN function together to negatively regulate mRNA expression through the shg 3′ UTR. Such E-cadherin expression could either be due to mRNA degradation or translational repression. We did not observe any obvious changes in mRNA levels between BAM/BGCM co-expression and BAM or BGCN alone based on the RT-PCR results, suggesting that BAM and BGCN do not regulate reporter mRNA stability through the shg 3′ UTR (Fig. 4D). Based on the physical interaction between BAM and eIF4A, we propose that BAM and BGCN repress E-cadherin at the translational level as a protein complex.

Because BGCN is a putative RNA binding protein (14), it is plausible that the role of BGCN in the BAM/BGCN complex is to bring BAM to its target mRNAs. To test this idea, we generated a fusion protein between BAM and λN, which could bind to the Firefly luciferase reporter with the 3′ UTR containing a λN binding sequence known as the 5B box (28). To show that the λN-5B system works properly in S2 cells, we tested it using eIF4G and GW182. eIF4G is a m⁷G cap-binding component of the translation initiation complex, which activates translation when loaded onto mRNA (29), whereas GW182 protein is a potent translational repressor for whichever mRNA it is tethered to (30). Indeed, λN-GW182 repressed the translation of the mRNA carrying the 5B at the 3′ end, whereas λN-eIF4G dramatically enhanced translation of the same mRNA, indicating that the λN-5B system works properly in S2 cells (Fig. 4E). Interestingly, the λN-BAM fusion protein was sufficient to repress its target mRNA expression in the absence of BGCN (Fig. 4E). This result suggests that the major role of BGCN in the complex is to help load BAM to its target mRNAs.

Discussion

This study has revealed the biochemical function of the BAM/BGCN complex as a translational repressor. We have also shown eIF4A in the regulation of GSC self-renewal to be a direct antagonist of BAM function in the Drosophila ovary. Here, we propose a model explaining how GSC self-renewal is controlled by concerted actions of intrinsic factors and the extrinsic BMP signal (Fig. 4F). BMP signaling directly represses bam expression, yet leaves low levels of BAM protein expression in the GSC (10–12, 16). 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 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 our current understanding of how GSC self-renewal and differentiation are regulated by translation factors.

BAM and BCGN Form a Protein Complex Involved in Translation Repression.

bam and bgcn genetically require each other's function to control CB differentiation (13, 14). Although they are expressed at low levels in GSCs, they have an important role in regulating GSC competition (16). However, their biochemical functions remained unclear until this study. We showed that BAM specifically interacts with BGCN, but not other RNA-binding proteins VASA, Rm62, and Me31B, to form a protein complex (Fig. 4). In addition, we have also shown that BAM and BGCN together, but not BAM or BGCN alone, are capable of suppressing the expression of the reporter containing the shg 3′ UTR (Fig. 4). 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, we 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, we propose 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.

Our previous genetic study showed that BAM and BGCN negatively regulate E-cadherin expression in GSCs to control GSC competition (16), but the underlying molecular mechanism remains defined. In this study, we showed that in Drosophila S2 cells BAM and BGCN could repress E-cadherin expression through its 3′ UTR at the translational level. Along with our previous observation that BAM and BGCN negatively regulate E-cadherin expression in GSCs in vivo, we propose 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.

eIF4A Directly Antagonizes BAM Function to Maintain E-Cadherin Expression in GSCs.

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 (30). 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, we used the yeast 2-hybrid screen to identify eIF4A as a BAM interacting protein (Fig. 1). Then, we have provided 2 pieces of genetic of evidence 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^Δ86 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^Δ86 heterozygote. These genetic results support the antagonizing relationship between bam and eIF4A (Fig. 4F).

The antagonizing genetic relationship between bam and eIF4A suggests that eIF4A favors GSC maintenance over differentiation. Our 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, we have 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 (19). 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 (23). In this study, we show that eIF4A is also required for controlling GSC division. Therefore, we propose that eIF4A controls GSC proliferation by regulating cell cycle progression like in Drosophila imaginal tissues.

Materials and Methods

Drosophila Stocks and Genetic Clonal Analysis.

Information about the Drosophila strains is described in the flybase (http://www.flybase.bio.indiana.edu), and the strains are specified in SI Text.

Yeast 2-Hybrid System.

The Drosophila ovarian GSC cDNA pool was constructed by using SMART technology (Clontech), and the mRNAs were isolated from Dpp overexpression-induced GSC tumors according to the instruction manuals. Additional details are described in SI Text.

Immunohistochemistry.

Multiple antisera were used, and a complete list is given in SI Text. The immunostaining protocol and the TUNEL assay using the ApopTag kit from Chemicon have been described in Results.

For the details about plasmid constructions, immunoprecipitation, and luciferase assay, please see SI Text.