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First published online November 9, 2007
doi: 10.1242/10.1242/dev.009159

Development 134, 4265-4272 (2007)
Published by The Company of Biologists 2007
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Argonaute 1 regulates the fate of germline stem cells in Drosophila

Lele Yang1,*, Dongsheng Chen1,2,*, Ranhui Duan3,*, Laixin Xia1,2, Jun Wang1, Abrar Qurashi3, Peng Jin3,{dagger} and Dahua Chen1,{dagger}

1 State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology
2 Graduate School, Chinese Academy of Sciences, Beijing, People's Republic of China.
3 Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA.


Figure 1
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  		Fig. 1. Ectopic expression of Ago1 increases the number of GSCs. (A) A schematic diagram of a Drosophila germarium with different cell types labeled by different colors: GSCs blue, CBs and cysts green, terminal filaments (TFs) gray, cap cells (CPCs) deep green, inner germarium sheath cells (IGSs) pink, follicle cells (FCs) and fusomes red. (B) Ovaries collected from wild-type (w1118), P{hs-Ago1} and piwiEP; P{hs-gal4} flies before heat-shock and after 6 days and 10 days heat-shock treatment, as indicated, were stained with anti-Vasa (green) and anti-Hts (red) antibodies. Vasa-positive germ cells carrying spectrosomes were undifferentiated germ cells (GSCs/Cb and GSC-like cells). (C) A germarium from 10-day heat shock was morphologically tumorous; many GSC-like cells carrying spectrosomes (both panels) and differentiated germ cells carrying branched fusomes (right panel) were observed. Scale bar: 10 µm.

 

Figure 2
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  		Fig. 2. Loss of Ago1 disrupts GSC maintenance in Drosophila. Ovaries collected from {hs-Ago1; Ago1/Ago1} flies undergoing different treatments were stained with anti-Vasa (green) and anti-Hts (red) antibodies. (A) Example of a germarium from an {hs-Ago1; Ago1k08121/Ago1k0812} animal 3 days after withdrawal of heat shock, in which two GSCs were maintained well. (B-D) In Ago1 mutants germ cell development was severely defective. There were no GSCs maintained at day 15 after withdrawal of heat shock. Allele combinations of (B) Ago1k08121/Ago1k08121,(C) Ago1k08121/Ago114 and (D) Ago1k08121/Ago1EMS are shown. (E) Example of the germarium of a fly undergoing constitutive heat shock, in which two GSCs were maintained well. (F) AGO1 is expressed almost equally in ovary, embryo and adult. In Ago1k08121/Ago114 mutant flies rescued by the transgene P{hs-Ago1}, the AGO1 protein was undetectable at day 15 after withdrawal of heat-shock treatment. Scale bar: 10 µm.

 

Figure 3
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  		Fig. 3. AGO1 is intrinsically required for the establishment and maintenance of GSCs in Drosophila. Ovaries dissected from flies {hs-flp; frtG13,/frtG13, ubi-gfp} without heat shock (A), {hs-flp; frtG13,/frtG13, ubi-gfp} at day 2 AHST (B), {hs-flp; frtG13,/frtG13, ubi-gfp} at day 10 AHST (C), {hs-flp; frtG13, Ago1k08121/frtG13, ubi-gfp} at day 2 AHST (D), and {hs-flp; frtG13, Ago1k08121/frtG13, ubi-gfp} at day 10 AHST (E,F) were stained with anti-GFP (green) and anti-Hts (red) antibodies. GFP-negatively marked GSCs and cysts were indicated by arrows and arrowheads, respectively. Gonads were dissected from {hs-flp; frtG13,/frtG13, ubi-gfp} (G,G') and {hs-flp; frtG13, Ago114/frtG13, ubi-gfp} (H,H') with constitutive heat-shock treatment at hour 3 after pupa formation were stained with anti-GFP (green) and anti-Hts (red) antibodies. G' and H' were the amplified images from G and H, as indicated, respectively. A putative GSC with negatively marked GFP (an anterior germ cell in a germarium) is indicated by an arrow. A posterior germ cell with negatively marked GFP is indicated by an arrowhead. Scale bar: 10 µm.

 

Figure 4
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  		Fig. 4. Two components of the miRNA pathway, AGO1 and Loqs, are not required for bam silencing in Drosophila. Ovaries dissected from flies {hs-flp; frtG13, Ago1k08121/frtG13, ubi-gfp} with heat-shock treatment were stained with anti-BamC (red). Two wild-type GSCs that are GFP-positive are negative for BamC (indicated by an arrow) (A), and a negatively marked GFP GSC, indicated by an arrow, was also BamC-negative (B). Ovaries dissected from P{bamP-GFP} (C), loqs; P{bamP-GFP} (D), bam, P{bamP-GFP} (E), loqs; bam, P{bamP-GFP} (F) were stained with anti-GFP (green) and anti-Hts (red) antibodies. GSCs with negative GFP expression are indicated by arrows. Scale bar: 10 µm.

 

Figure 5
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  		Fig. 5. In Drosophila, germ cells could differentiate in both Ago1, bam and loqs, bam double mutants. Ovaries dissected from bam86/bamBG mutants (15 days old) (A), hs-dAgo1; Ago1k08121 bam86/bamBG mutants (15 days old) (B), bam86 mutant (15 days old) (C) and loqsf00971; bam86 double mutants (15 days old) (D) were stained with anti-Vasa (green) and anti-Hts (red) antibodies. Branched fusomes in loqs and bam mutant germaria are indicated by arrows. Scale bar: 10 µm.

 

Figure 6
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  		Fig. 6. Model of Ago1-dependent miRNAs in GSC fate determination. In the tip of the Drosophila germarium, BMP/Dpp, as short-range signals from niche cells perceived directly by GSCs, represses bam transcription and results in Bam/Bgcn complex activity loss, thereby depressing both the Pumilio/Nanos complex (Nos, Pum) and GSC-specific miRNA activities. The Pumilio/Nanos complex and GSC-specific miRNAs could function together to repress the translation of mRNAs for GSC/CB differentiation (A), or they might function separately to repress the translation of different groups of mRNA for GSC/CB differentiation (B).

 

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© The Company of Biologists Ltd 2007