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

Abstract

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

Fig. 1.

Disruption of mei-P26 results in GSC loss. (A-D) Germaria stained for Vasa (green), Hts (red) and DNA (blue). (A) Wild-type germaria contain two to three germline stem cells (GSCs) (arrow) marked by small round fusomes. mei-P26^mfs1 homozygotes (B) and mei-P26^mfs1/mei-P26^mfs2 transheterozygotes (C) often have multicellular germline cysts, marked by branched fusomes (arrowheads), adjacent to the cap cells. (D) mei-P26^mfs1 homozygotes carrying a duplication that contains the mei-P26 gene exhibit the normal number of GSCs (arrow). (E) Quantification of the mei-P26 mutant phenotype. Genotypes are displayed on the x-axis. The y-axis lists the number of GSCs per terminal filament (TF). Each point signifies a TF counted (mei-P26^mfs1, n=41; mei-P26^mfs1 cDNA, n=35; mei-P26^mfs1/mfs2, n=27; mei-P26^mfs1/mfs2 cDNA, n=27). A full-length wild-type mei-P26 cDNA transgene driven by a nanos-gal4::VP16 germline driver rescued the mei-P26 phenotype. Error bars indicate s.e.m. (F) Quantification of clonal analysis using the mei-P26^mfs1 allele. Germline clones of a control chromosome (red line) and four independent recombinant mei-P26^mfs1 mutant chromosomes (green lines) were induced using FRT/FLP-mediated mitotic recombination. The number of GSC clones was assayed over three time points (n≥100 germaria/sample/time point). Although nearly 50% of the germaria counted contained control clones, the number of clones observed for mei-P26 mutant chromosomes rapidly decreased over time, indicating that disruption of mei-P26 results in a stem cell loss phenotype. (G) A mei-P26^mfs1 clonal germarium stained for GFP (green), Hts (red) and DNA (blue). A cystic tumor clone is outlined. Scale bars: 10 μm.

Fig. 2.

Mei-P26 associates with miRISC components in undifferentiated germ cells. (A) Co-immunoprecipitation (co-IP) from bam^Δ86 mutant ovarian extracts using an anti-Ago1 antibody. The resulting pellets were analyzed by western blot using antibodies against Mei-P26 and Ago1. (B) co-IP from c587-gal4;UAS-dpp (dpp overexpressing, OE) ovarian extracts using an anti-Mei-P26 antibody. The resulting pellets were analyzed by western blot using antibodies against Mei-P26, Ago1 and GW182. (C) Western blot comparing levels of Mei-P26 protein in mei-P26^mfs1, mei-P26^mfs1;nanos-gal4::VP16>UAS-full-length mei-P26 (FL) and mei-P26^mfs1;nanos-gal4::VP16>UAS-mei-P26ΔNHL (ΔNHL) ovaries. Actin served as a loading control. (D) Quantification of the mei-P26 mutant phenotype. Genotypes are displayed on the x-axis. The y-axis lists the number of GSCs per TF. Each point signifies a TF counted (mei-P26^mfs1, n=24; mei-P26^mfs1 cDNA, n=25; mei-P26^mfs1 ΔNHL, n=26). A full-length wild-type mei-P26 cDNA transgene driven by a nanos-gal4::VP16 germline driver rescued the mei-P26 GSC loss phenotype but a transgene lacking the NHL domain did not. Error bars indicate s.e.m. (E-G) mei-P26^mfs1 (E), FL (F) and ΔNHL (G) ovaries stained for Vasa (green), Hts (red) and DNA (blue). The arrows point to where GSCs would normally reside. Both mei-P26^mfs1 and ΔNHL ovaries often had multicellular cysts at the anterior of the germarium, whereas FL ovaries appeared to be rescued. Scale bars: 10 μm.

Fig. 3.

Ago1 and Mei-P26 regulate orb translation in GSCs. (A) mei-P26^mfs1 clonal germarium stained for GFP (green), Orb (red) and DNA (blue). The negatively marked mei-P26 mutant GSC (arrow) expresses higher levels of Orb protein than its heterozygous neighbor (arrowhead). (B) Ago1¹⁴ clonal germarium stained for GFP (green), Orb (red) and DNA (blue). The negatively marked Ago1 mutant GSC (arrow) expresses higher levels of Orb protein than its heterozygous neighbor (arrowhead). (C) A germarium expressing an Hsp83-lacZ-orb3′UTR reporter stained for lacZ (green), Orb (red) and DNA (blue). (D) RT-PCR of orb and Actin 5C mRNA from Ago1 and Mei-P26 IPs. orb mRNA was consistently enriched in the Ago1 and Mei-P26 IP pellets but not in the IgG control pellets. (E) The ratio of mRNA pulled down in a Mei-P26 IP versus a control IP and in an Ago1 IP versus a control IP as measured by real-time PCR. Error bars indicate s.d. Scale bars: 10 μm.

Fig. 4.

The 3′UTR of orb mRNA contains miRNA binding sites and responds to changes in Mei-P26 levels. (A) The reporter genes used to evaluate the importance of predicted miRNA binding sites within the 3′UTR of orb mRNA. (B,C) Full-length orb 3′UTR (FL-orb3′UTR) (B) and an orb 3′UTR reporter that contains mutated miRNA binding sites (ΔmiR-orb3′UTR) (C) stained for Venus (green), Hts (red) and DNA (blue). The FL-orb3′UTR reporter exhibits very low levels of expression in GSCs and high levels in 16-cell cysts, similar to the Hsp83-lacZ-orb3′UTR reporter and Orb protein (see Fig. 3C). By contrast, the ΔmiR-orb3′UTR reporter displays elevated levels of expression in GSCs. The arrows point to GSCs. (D) The average signal ratio between Venus expression in GSCs and that in 16-cell cysts for the indicated reporters (n=11; germaria from at least two different transgenic lines were evaluated for each reporter; ^*P<0.005). Error bars indicate s.d. (E) Cells adjacent to the cap cells express the FL-orb 3′UTR reporter in a mei-P26 mutant background. Scale bars: 10 μm.

Fig. 5.

mei-P26 mutants display ectopic Bam expression. (A,B) w¹¹¹⁸ control (A) and mei-P26^mfs1 homozygous (B) germaria stained for Nanos (Nos; green), Bam (red) and DNA (blue). In control samples, Nanos and Bam are expressed in mutually exclusive patterns. By contrast, mei-P26 mutants display overlapping Nanos and Bam expression. (C,D) mei-P26^mfs1 bam (C) and mei-P26^mfs1 bgcn (D) double mutants stained for Hts (green) and DNA (blue). Both double mutants form ovarian tumors that contain single cells with round fusomes. Scale bars: 10 μm.

Fig. 6.

mei-P26 mutants display defects in BMP signal transduction. (A-B′) w¹¹¹⁸ control (A,A′) and mei-P26^mfs1 homozygotes (B,B′) stained for Dad-lacZ (green), Hts (red) and DNA (blue). (A′,B′) Dad-lacZ staining alone. Arrows indicate germ cells adjacent to cap cells. (C-D′) w¹¹¹⁸ control (C,C′) and mei-P26^mfs1 homozygous (D,D′) germaria stained for pMad (green), Hts (red) and DNA (blue). (C′,D′) pMad staining alone. Lack of Dad-lacZ and pMad expression in germline cells immediately adjacent to cap cells in mutant samples suggests that BMP signaling is compromised in the absence of mei-P26. Arrows indicate germ cells adjacent to cap cells. (E-F′) w¹¹¹⁸ control (E) and mei-P26^mfs1 (F) germaria stained for Brat (green), Hts (red) and DNA (blue). (E′,F′) Brat expression shown alone. Germline cells throughout mei-P26 mutant germaria express higher levels of Brat than the control samples. Arrows indicate germ cells adjacent to cap cells. (G-G″) mei-P26^mfs1 clonal germarium stained for Brat (green), GFP (red) and DNA (blue) (G). The homozygous mei-P26 mutant GSC clone (green arrow) expresses more Brat protein than the heterozygous control clone (red arrow). (G′,G″) Brat and GFP staining alone. Scale bars: 10 μm.

Fig. 7.

Mei-P26 binds to Nanos and regulates Brat protein levels. (A) Ethidium bromide-stained agarose gel showing products from RT-PCR reactions with (RT) and without (No RT) reverse transcriptase. Three different primer pairs specific for different regions of brat mRNA were used to amplify total RNA isolated from bam mutant and mei-P26 bam double-mutant samples. RpL32 was amplified as a loading control. In the RT samples, bam and mei-P26 bam mutants exhibited similar levels of brat mRNA. (B) Western blot analysis of ovarian extracts from bam and mei-P26 bam mutants probed with Brat and Actin antibodies. The mei-P26 bam double-mutant extracts exhibited higher levels of Brat expression than the control extract. (C) Western blot of an anti-Myc co-IP from ovary extract expressing a Myc-tagged nanos transgene. The specificity of the anti-Myc resin was tested using extract from ovaries that did not express the Myc-tagged nanos transgene. (D) Western blot of an anti-Myc co-IP from bam^Δ86 mutant extract expressing a Myc-tagged nanos transgene. A rabbit IgG IP of bam^Δ86 mutant extract was used as a negative control. co-IP of Mei-P26 with Myc-Nanos was seen in both control ovary and bam mutant ovary extracts (arrows). (E) Model describing the translational regulatory cascades within wild-type and mei-P26 mutant GSCs. Niche cells (light brown ovals) produce Dpp and Gbb, which activate a signal transduction cascade in wild-type GSCs that results in the transcriptional repression of bam. Within wild-type GSCs, Mei-P26 cooperates with both Nanos and miRISC to repress the translation of specific mRNAs. In the absence of Mei-P26, Brat is inappropriately translated resulting in the repression of Mad. Loss of pMAD results in the expression of bam, which causes these mei-P26 mutant GSCs to partially differentiate.