1. Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Dr Bohr Gasse 3, 1030 Vienna, Austria
2. European Molecular Biology Laboratory (EMBL), Meyerhofstrae 1, 69117 Heidelberg, Germany
3. Research Institute of Molecular Pathology (IMP), Dr Bohr-Gasse 7, 1030 Vienna, Austria
4. Present addresses: Wellcome Trust Centre for Stem Cell Research (CSCR), University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK (J.B.); Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, 117604, Singapore (S.M.C.).

Correspondence to: Juergen A. Knoblich¹ Correspondence and requests for materials should be addressed to J.A.K. (Email: juergen.knoblich@imba.oeaw.ac.at).

When Drosophila germline stem cells divide (Fig. 1a), 1 daughter cell (the cystoblast) looses niche contact and undergoes 4 transit-amplifying divisions to create a cyst of 16 cystocytes, which remain connected by the fusome. In each cyst, 1 cell becomes the oocyte, whereas the other cells undergo endoreplication to form 15 so-called nurse cells.

Figure 1: Differentiation and cell cycle defects in mei-P26 mutant ovaries.

a, Overview of Drosophila oogenesis showing cap cells (dark green), escort cells (light green), stem cells (orange), cystoblasts and cystocytes (yellow), follicle cells (blue) and the oocyte (dark grey). The spectrosome and fusome are red. b–f, Wild type (WT) (b, e) and mei-P26 (c, d, f) ovarioles stained with mAb1B1 (1B1, green) and for DNA (red). fs1 and mfs1 represent two different mei-P26 alleles. g, h, Cyclin E (red; green, Vasa; blue, DNA) is downregulated in WT (g, arrowhead) but not in the mei-P26^fs1 mutant (h, arrowhead) cystocytes. i, j, Phospho-H3 positive (green, pH3; red, DNA) mitotic cells (arrowheads) are restricted to the tip of the ovarioles in WT (i) but are found at all stages in mei-P26^fs1 (j) ovarioles. k, l, GFP under the control of the bam promoter (bamP–GFP) (green; red, mAb1B1; blue, DNA) is not expressed in WT (k) and mei-P26^fs1 mutant (l) niche contacting germline cells (arrowheads). m, n, Anti-Armadillo (green; red, DNA) shows integrity of WT (m) and mei-P26^fs1 (n) mutant cap cells. o, p, Orb expression is initiated in WT (o) and mei-P26^fs1 mutant (p) cystocytes but restricted to the oocyte only in WT (o, arrow) and not in mei-P26^fs1/mfs1 mutant (p, arrowhead) egg chambers.

High resolution image and legend (366K)

Because brat¹⁹² mutant germline clones do not show any obvious defects in oogenesis (data not shown), we analysed the ovarian phenotype of mei-P26. Like Brat, Mei-P26 is a Trim–NHL protein⁹ and carries an NHL domain, a coiled-coil region and several B-boxes. Weak mei-P26 mutants have defects in meiosis⁷ whereas stronger alleles cause tumourous overproliferation⁸. mei-P26 mutant ovaries were stained using the monoclonal antibody mAb1B1 (ref. 10), which labels the fusome and the spectrosome—a cytoplasmic organelle only present in stem cells and cystoblasts¹¹ (Fig. 1b–f). Wild-type ovaries contain two stem cells near the tip of the germarium whereas cystoblasts and cysts are separated from the stem cell niche (Fig. 1b). In mei-P26 mutant ovaries, germaria are filled with individual spectrosome-containing cells (19%) and cysts containing varying numbers of cells (<4 in 39%, >4 in 42%; n > 400 cells) connected by a fusome (Fig. 1c, d and Supplementary Fig. 1a, a'). Nurse cells and oocytes are not formed, and fusomes or spectrosomes are maintained at later stages of oogenesis (Fig. 1e, f). In wild-type ovaries, the S-phase marker Cyclin E oscillates with the cell cycle in stem cells and mitotically active cysts, is downregulated as cystocytes exit mitotic proliferation and is re-expressed as nurse cells enter endoreplication¹² (Fig. 1g). In mei-P26 mutant ovaries, however, Cyclin E is highly expressed at all stages of oogenesis (Fig. 1h). Upregulation of Cyclin E occurs even in small mei-P26 clones (Supplementary Fig. 1b, c), and is therefore not an indirect consequence of tumour formation. Phospho-histone-H3-positive mitotic germline cells are restricted to the anterior tip of the germarium in wild-type ovaries (Fig. 1i) but are detected throughout the ovarioles in mei-P26 mutants (Fig. 1j and Supplementary Fig. 1a, a'). Whereas all cells in a wild-type cyst divide synchronously, mei-P26 mutant cysts frequently contain both mitotic and interphase cells (data not shown). Thus, mei-P26 is required for proliferation control and differentiation in the female germline stem cell lineage.

To test stem cell niche signalling, we used a green fluorescent protein (GFP) fusion to the bag of marbles (bam) promoter¹³, which is suppressed by Decapentaplegic (Dpp) from the niche in stem cells but not in cystocytes (Fig. 1k and Supplementary Fig. 1d). In mei-P26 mutants (Fig. 1l and Supplementary Fig. 1e), bam transcription is repressed in niche-contacting cells but is upregulated in cystocytes. Because staining for the niche marker Armadillo (Fig. 1m, n) reveals no structural abnormalities and because ovarian tumours are also observed when mei-P26 is removed exclusively from the female germ line (Supplementary Fig. 1f, g), mei-P26 is not required for sending or receiving the niche signal.

mei-P26 mutant tumour cells have branched fusomes and express Bam. To confirm further that these cells do not have stem cell identity, we analysed the expression of oo18 RNA-binding protein (Orb). Orb expression starts in all cystocytes between the 8- and 16-cell stage¹⁴, but is restricted to the oocyte during later stages (Fig. 1o). In mei-P26 mutants, Orb expression is initiated normally (Fig. 1p) and is detected throughout the tumour. However, Orb is never restricted to a single cell, suggesting that the oocyte is not specified. Thus, mei-P26 mutants develop a 'cystocytoma' in which tumour cells express markers for the cystocyte fate. Single-spectrosome-containing cells in the tumour might arise from occasional disintegration of fusome-connected cysts, a process that has been described before¹⁵.

Brat can inhibit cell growth and ribosome biogenesis¹⁶. mei-P26 might also regulate cell growth because overexpression from the eyeless promoter reduces eye size (Supplementary Fig. 2a, b), even when cell death is inhibited by co-expressing p35 (data not shown). To test whether cell growth is differentially regulated in the ovarian stem cell lineage, we quantified the volume of stem cells and cysts after three-dimensional reconstruction (see Methods). Shortly after stem cell division (elongated spectrosome morphology), stem cells are 314  13 m³ (mean  s.e.m.; n = 4) whereas cystoblasts are 330  30 m³ (n = 4). Stem cells grow to a maximum of 600 m³ (average 437  21 m³, n = 19) and double their volume between each mitotic division (about once per day). To measure cyst volumes, mitotic clones were marked by the absence of GFP (Fig. 2a). Three- and four-day-old 16-cell cysts are 1,215  61 m³ (n = 7) and 1,163  48 m³ (n = 7), respectively. Thus, cell growth slows down as cells exit mitotic proliferation. Consistent with this, cystocytes become progressively smaller during transit-amplifying divisions (Fig. 2g). diminutive (dMyc), the Drosophila Myc homologue, an important regulator of cell growth, is highly expressed in stem cells and cystoblasts but downregulated in 16-cell cysts (Fig. 2e). Nucleoli (stained by anti-Fibrillarin)—the sites of ribosomal RNA transcription—are large in stem cells but much smaller in 16-cell cysts (Fig. 2c and Supplementary Fig. 2c). Because ribosome number is thought to control cellular growth in Drosophila^{17, 18}, a reduction in ribosome biogenesis might be responsible for the reduced cell growth at the end of mitotic proliferation.

Figure 2: Mei-P26 regulates cell and nucleolar size.

a, b, Three-dimensional reconstruction of WT 16-cell cysts (filled arrowheads; a), a WT 8-cell cyst (open arrowhead; a) and a mei-P26^fs1/mfs1 mutant cyst containing 14 cells (b). c, d, Nucleoli (green, anti-Fibrillarin) in mei-P26^fs1 mutant ovaries (d) are larger than those in the WT ovaries (c). e, f, High levels of dMyc (red; green, mAb1B1) are detected in germline stem cells and early cysts (filled arrowhead). In postmitotic cysts, dMyc levels are decreased (open arrowhead) and levels increase again as nurse cells undergo endoreplication (e). In mei-P26^mfs1/fs1 mutants, high levels of dMyc are detected throughout the tumour (arrowheads in f). g, Diameter of the indicated cell types in WT (n > 24 cells) and mei-P26^fs1/mfs1 mutant (n = 11 cells for Mei-P26 ant., n > 43 for all others) ovaries. Abbreviations: Mei-P26 ss, single-spectrosome-containing cells; Mei-P26 3–7 cc (or Mei-P26 >7 cc), cystocytes in mei-P26^fs1 mutant cysts containing either 3–7 (or >7) cells; Mei-P26 ant., anterior niche contacting cells. Error bars, s.e.m. (green bars, WT; red bars, mei-P26 mutant). h, i, Mei-P26 expression peaks in early 16-cell cysts in WT and is not detected at later stages of oogenesis (h). In bam⁸⁶ mutant ovaries, expression is not upregulated (i, compare to WT in h). Arrowheads point at equivalent stages. Note that Mei-P26 staining appears more intense in later stages owing to sample thickness and out-of-focus fluorescence (i). j, Germarium close up: Mei-P26 (red; green, mAb1b1; blue, DNA) expression is low in stem cells (arrowhead), weakly upregulated in 8-cell cysts (open arrowhead) and peaks in 16-cell cystocytes (arrow) (see Supplementary Fig. 2e, f, h).

High resolution image and legend (222K)

In mei-P26 mutants, cellular and nucleolar size are increased and dMyc is highly expressed throughout the tumour (Fig. 2d, f, g and Supplementary Fig. 2c): the volume of 16-cell cysts is increased to 3,956  424 m³ (n = 4; Fig. 2b) and cell diameters no longer decrease as cells are displaced from the stem cell niche (Fig. 2g). Thus, mei-P26 is responsible for the differential regulation of cell growth in the Drosophila ovarian stem cell lineage. Like brat, it might achieve its function by regulating ribosome biogenesis and controlling the expression of dMyc.

To analyse Mei-P26 expression, we generated a specific antibody (Supplementary Fig. 2d–f). Mei-P26 mRNA and protein levels are low in stem cells but are upregulated in cysts as they decrease growth and exit mitotic proliferation (Fig. 2h, j and Supplementary Fig. 2e, g, h). This regulation is functionally important because mei-P26 overexpression in ovaries results in stem cell loss and complete depletion of the female germ line (Fig. 3a, b). In bam mutant ovaries, Mei-P26 levels remain low suggesting that mei-P26 is regulated in a Bam-dependent manner (Fig. 2i). bam overexpression induces premature differentiation of stem cells in a wild-type (Fig. 3d) but not in a mei-P26 mutant (Fig. 3e) background, demonstrating that Mei-P26 is essential for Bam to induce cystocyte differentiation. Consistently, germline stem cells do not differentiate when pumilio is removed in a mei-P26 mutant background (Supplementary Fig. 2k, l). To test whether Mei-P26 is the only target of Bam, we overexpressed mei-P26 in a bam mutant background (Fig. 3f, g and Supplementary Fig. 2i, j). mei-P26 overexpression reduces the size of bam mutant cells (from 10.1  0.17 m (bam, n = 27) to 7.0  0.19 m (bam, nanos gal4  UASP-mei-P26, n = 27)) and the enlarged nucleolus in bam mutants (Supplementary Fig. 2c) but does not rescue the differentiation defects (Supplementary Fig. 2i, j) observed in these mutants. Thus, mei-P26 is upregulated by Bam activity in cystocytes and inhibits cell growth and mitotic proliferation.

Figure 3: Bam requires the AGO1-binding protein Mei-P26 to induce proper cystocyte differentiation.

a, b, c, mei-P26 (b) but not mei-P26^NHL (c) overexpression (OE) (from nanos-Gal4::VP16) depletes the germ line (green, Vasa; red, DNA). d, e, Transient (heat-shock-induced) bam overexpression induces stem cell differentiation and depletes the germ line (red, Vasa; green, mAb1B1; blue, DNA) in a WT (d) but not mei-P26^fs1/mfs1 mutant (e) background. f, g, mei-P26 overexpression (from nanos-Gal4::VP16) in bam⁸⁶ mutants (g) reduces cell and nucleolar size (see statistics in Supplementary Fig. 2c) but does not induce stem cell differentiation (Supplementary Fig. 2i, j). h, i, The NHL domain proteins Brat, Mei-P26 and Dappled interact with AGO1. Reciprocal immunoprecipitations (IP) of Brat and AGO1 from Drosophila embryo extracts (h). GFP-tagged Mei-P26 but not GFP-tagged Mei-P26^NHL (i) coimmunoprecipitates Myc-tagged AGO1 in S2 cells (see Supplementary Fig. 4b).

High resolution image and legend (288K)

In the brain, mei-P26 is weakly expressed in neuroblasts, is absent from ganglion mother cells but is highly expressed in neurons (Supplementary Fig. 3a–d). Although mei-P26 mutants have no obvious defects in neurogenesis, mei-P26 overexpression can induce premature neuronal differentiation (Supplementary Fig. 3e, f), suggesting that mei-P26 and brat might have some common targets. Using mass spectrometry, we identified the RNase Argonaute-1 (AGO1) in anti-Brat immunoprecipitates (Supplementary Fig. 4a). Brat and AGO1 can be co-immunoprecipitated from Drosophila embryos (Fig. 3h), and co-transfection experiments in S2 cells show that AGO1 also binds Mei-P26 and the Trim–NHL protein Dappled (Supplementary Fig. 4b). This interaction is functionally significant because Mei-P26 lacking the NHL domain no longer binds AGO1 (Fig. 3i) and fails to block self renewal when overexpressed in ovarian stem cells (Fig. 3c).

AGO1 is a core component of the RISC complex¹⁹ and is important for microRNA-mediated translational repression and RNA degradation. MicroRNAs are essential for self renewal in Drosophila ovarian stem cells because mutations in AGO1 (Supplementary Fig. 5), dicer-1 (ref. 20) or its binding partner loquacious²¹ result in premature stem cell differentiation. To test whether Mei-P26 regulates microRNAs, we measured microRNA levels by quantitative reverse transcription polymerase chain reaction (RT–PCR). In mei-P26 mutants, most microRNAs are significantly upregulated (Supplementary Fig. 7a, b), whereas overexpression of mei-P26 in bam mutants broadly reduces microRNA levels (Fig. 4a and Supplementary Fig. 7a). Importantly, the mei-P26 mutant phenotype can be partially rescued by removing one copy of Loquacious and thereby reducing microRNA levels (Fig. 4b, c and Supplementary Fig. 6a–f), indicating that mei-P26 acts by inhibiting the microRNA pathway in cystocytes.

Figure 4: Mei-P26 regulates miRNAs.

a, Quantitative PCR experiment comparing the level of mature microRNAs in bam⁸⁶ and bam⁸⁶, nanos-Gal4::VP16pUASp-mei-P26. The y axis shows log₂ of the expression ratio between the two genotypes. b, c, Loss of one copy of loquacious partially rescues the mei-P26^fs1 mutant phenotype (Supplementary Fig. 6). d–g, Downregulation of the bantam sensor reveals bantam microRNA expression in stem cells but not cystocytes (e), whereas the control sensor is uniformly expressed throughout the germ line (d). The bantam sensor (g) but not the control sensor (f) is silenced in the germ line of mei-P26^fs1/mfs1 mutants (arrowheads indicate stem cells).

High resolution image and legend (202K)

One of the best characterized Drosophila microRNAs is bantam, a regulator of proliferation and apoptosis²². We used a sensor that expresses GFP from the tubulin promotor and carries two bantam binding sites in the 3' untranslated region²². In wild-type ovaries, this sensor is repressed by bantam activity in stem cells but is derepressed in 16-cell cystocytes in which high levels of Mei-P26 are present (Fig. 4e). In mei-P26 mutants, the sensor is off in all germline cells (Fig. 4g). In contrast, a control sensor lacking microRNA-binding sites shows high expression in all wild-type and mutant cells (Fig. 4d, f). Although mei-P26 regulates many microRNAs, bantam seems to be an important target: even animals heterozygous for a bantam null mutation have a reduced number of stem cells (1.15  0.12 per germarium (n = 32 germaria) compared to 2.09  0.05 (n = 32 germaria) in 14-day-old bantam heterozygous and wild-type flies, respectively), suggesting a defect in self renewal (Supplementary Fig. 7e, f). To test whether mei-P26 regulates bantam directly, we expressed a luciferase construct carrying a bantam-binding site in its 3' untranslated region in S2 cells. In control cells, the bantam sensor is repressed, but a construct lacking the binding site is not affected (Supplementary Fig. 7d). On cotransfection of mei-P26 (Supplementary Fig. 7c), luciferase expression is significantly derepressed, but no derepression is seen with cotransfection of mei-P26 lacking the NHL domain; this indicates that Mei-P26 can repress bantam activity even in S2 cells.

Our data suggest that brat and mei-P26 might act in a similar manner to control proliferation in stem cell lineages. In both mutants, cells that normally stop self renewal increase ribosome biogenesis, grow abnormally large and fail to exit the cell cycle leading to the formation of a tumour. The general upregulation of microRNAs in mei-P26 mutants leaves several possibilities for how these proteins might regulate the microRNA pathway. The presence of a RING finger in Mei-P26 suggests a role in protein degradation. The high amounts of AGO1 detected in Mei-P26 immunoprecipitates make it unlikely that AGO1 itself is degraded by Mei-P26. However, another member of the RISC complex might be a degradation target of Mei-P26. Equally likely, Mei-P26 could prevent the incorporation or increase the turnover of microRNAs in the RISC complex.

Many human tumours contain cancer stem cells that drive tumour growth and metastasis^{23, 24}. Although the similarities between Drosophila tumours and human cancer are limited, brat mutant brains and mei-P26 mutant ovaries (as well as the other mutant conditions causing stem cell tumours^{25, 26, 27}) provide an invertebrate model for stem-cell-derived tumour formation. In mei-P26 mutants, tumours originate from cystocytes, the transit-amplifying pool of the ovarian stem cell lineage. In mei-P26 mutants, these cells re-gain the ability to self-renew: after bam overexpression—which leads to premature differentiation of stem cells—the germ line is depleted in a wild-type but not in a mei-P26 mutant background. Thus, mei-P26 tumours arise from growth defects in the transit-amplifying compartment of the ovarian stem cell lineage—a mechanism that could occur in human tumours as well.

Our data establish Trim–NHL proteins as regulators of stem cell proliferation. Vertebrate members of this family exist and are downregulated in human cancer cell lines²⁸ suggesting that their tumour-suppressor function might be conserved in vertebrates as well.

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Supplementary Information

Supplementary information accompanies this paper.

Acknowledgements

We thank V. Siegel, K. Mochizuki, G. B. Cebolla and S. Weitzer for comments on the manuscript, J. Stolte for assistance with quantitative PCRs, C. Richter and the other members of the Knoblich laboratory for discussion, B. Dickson, E. Izaurralde, P. Lasko, L. Luo, S. Hawley, D. McKearin, H. Richardson, F. Schnorrer, J. Skeath, D. Stein, L. Wong, the Bloomington Drosophila Stock Center, the Developmental Studies Hybridoma Bank (DSHB) and the Drosophila Genomics Resource Center (DGRC) for reagents, and M. Insco and M. Fuller for communicating results before publication. Work in the Knoblich laboratory is supported by the Austrian Academy of Sciences, the Wiener Wissenschafts-, Forschungs- und Technologiefonds (WWTF), the Austrian Science Fund (FWF) and the EU network ONCASYM; K.M. is supported by the Austrian Proteomics Platform (APP) of the Austrian Genome Program (GENAU).

Author Contributions J.A.K. and R.A.N. designed the study. R.A.N. performed the oogenesis experiments. J.B. and K.M. contributed biochemical data (Fig. 3h and Supplementary Fig. 4a, b). A.F. contributed the S2 luciferase assay (Supplementary Fig. 7c, d). I.P. assisted in the experiment in Fig. 3f, g and performed experiments in the larval brain (Supplementary Fig. 3). S.C. and N.B. designed the microRNA quantitative PCR experiment, which was performed by N.B. (Fig. 4a and Supplementary Fig. 7a, b). J.A.K. wrote the paper.

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