Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage - PubMed (original) (raw)

Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage

Ralph A Neumüller et al. Nature. 2008.

Abstract

Drosophila neuroblasts and ovarian stem cells are well characterized models for stem cell biology. In both cell types, one daughter cell self-renews continuously while the other undergoes a limited number of divisions, stops to proliferate mitotically and differentiates. Whereas neuroblasts segregate the Trim-NHL (tripartite motif and Ncl-1, HT2A and Lin-41 domain)-containing protein Brain tumour (Brat) into one of the two daughter cells, ovarian stem cells are regulated by an extracellular signal from the surrounding stem cell niche. After division, one daughter cell looses niche contact. It undergoes 4 transit-amplifying divisions to form a cyst of 16 interconnected cells that reduce their rate of growth and stop to proliferate mitotically. Here we show that the Trim-NHL protein Mei-P26 (refs 7, 8) restricts growth and proliferation in the ovarian stem cell lineage. Mei-P26 expression is low in stem cells but is strongly induced in 16-cell cysts. In mei-P26 mutants, transit-amplifying cells are larger and proliferate indefinitely leading to the formation of an ovarian tumour. Like brat, mei-P26 regulates nucleolar size and can induce differentiation in Drosophila neuroblasts, suggesting that these genes act through the same pathway. We identify Argonaute-1, a component of the RISC complex, as a common binding partner of Brat and Mei-P26, and show that Mei-P26 acts by inhibiting the microRNA pathway. Mei-P26 and Brat have a similar domain composition that is also found in other tumour suppressors and might be a defining property of a new family of microRNA regulators that act specifically in stem cell lineages.

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Figures

Figure 1

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-P26fs1 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-P26fs1 (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-P26fs1 mutant (l) niche contacting germline cells (arrowheads). m, n, Anti-Armadillo (green; red, DNA) shows integrity of WT (m) and mei-P26fs1 (n) mutant cap cells. o, p, Orb expression is initiated in WT (o) and mei-P26fs1 mutant (p) cystocytes but restricted to the oocyte only in WT (o, arrow) and not in mei-P26fs1/mfs1 mutant (p, arrowhead) egg chambers.

Figure 2

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-P26fs1/mfs1 mutant cyst containing 14 cells (b). c, d, Nucleoli (green, anti-Fibrillarin) in mei-P26fs1 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-P26mfs1/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-P26fs1/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-P26fs1 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 bamΔ86 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-offocus 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).

Figure 3

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

a, b, c, mei-P26 (b) but not mei-P26ΔNHL (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-P26fs1/mfs1 mutant (e) background. f, g, mei-P26 overexpression (from nanos-Gal4::VP16)in bamΔ86 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-P26ΔNHL (i) coimmunoprecipitates Myc-tagged AGO1 in S2 cells (see Supplementary Fig. 4b).

Figure 4

Figure 4. Mei-P26 regulates miRNAs

a, Quantitative PCR experiment comparing the level of mature microRNAs in bamΔ86 and bamΔ86, nanos-Gal4::VP16formula imagepUASp-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-P26fs1 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-P26fs1/mfs1 mutants (arrowheads indicate stem cells).

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