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First published online 31 March 2009
doi: 10.1242/dev.025999

Development 136, 1497-1507 (2009)
Published by The Company of Biologists 2009
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Dicer-1-dependent Dacapo suppression acts downstream of Insulin receptor in regulating cell division of Drosophila germline stem cells

Jenn-Yah Yu1,*,{dagger}, Steven H. Reynolds1,*, Steve D. Hatfield1,{ddagger}, Halyna R. Shcherbata1,§, Karin A. Fischer1, Ellen J. Ward1, Dang Long2, Ye Ding2 and Hannele Ruohola-Baker1,¶

1 Department of Biochemistry, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98195, USA.
2 Wadsworth Center, New York State Department of Health, 150 New Scotland Avenue, Albany, NY 12208, USA.


Figure 1
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  		Fig. 1. The Drosophila dap 3'UTR is repressed by miRNAs in the germline stem cells. (A) The control sensor lacks significant 3'UTR. The dapL and dapS sensors contain 866 bp and 630 bp of dap 3'UTR, respectively. Predicted miRNA response elements (MREs) are shown. (B) GFP expression was unchanged in Dcr-1 germline stem cells (GSCs) (arrowhead) in the control sensor background as compared with the neighboring control GSC, but was upregulated in the dapL and dapS sensor lines. (C) The dapL and dapS sensors were upregulated in Dcr-1 GSCs. Mean±s.e.; n≥23 pairs of GSCs. Student's t-test; **P<0.01.

 

Figure 2
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  		Fig. 2. The dap 3'UTR is targeted directly by miR-7, miR-278 and miR-309. (A) The luciferase-dapF reporter consists of tubulin promoter-driven luciferase and the 3'UTR of dap. Predicted MREs are shown. (B) Mutations in the dap 3'UTR predicted MREs are shown in lowercase. miRNA/MRE complementarity is also shown. (C) Normalized luciferase activity upon co-expression of miRNAs. miR-1 served as a control. miR-7, -8, -278, -309 and bantam repress luciferase activity, and the predicted MREs for miR-7, -278 and -309 are required for repression. Mean±s.e. of at least three repeats.

 

Figure 3
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  		Fig. 3. miR-278-deficient GSCs proliferate more slowly than control GSCs. (A) qPCR analysis of the mature miRNA expression level of miR-8 and miR-278 in the whole ovary. RNA from miR-278KO ovaries served as negative control for the expression of miR-278. The miRNA expression level is calculated from the cycle threshold (CT) (see Table S1 in the supplementary material) by 2CT(FRT42D control)/2CT(miR-278KO). Mean±s.e. for at least two repeats. (B) miR-278-deficient GSCs produce fewer progeny than control GSCs. A mutant GSC (arrowhead) and its progeny are marked by the lack of GFP. Add, Adducin. (C) The division kinetics of miR-278 GSCs was reduced to 75-79% as compared with the neighboring heterozygous control GSCs or the FRT42D control GSC clones. Division kinetics of miR-7 GSCs were unaffected by comparison with control. The division kinetics of GSCs mutant for both miR-7 and miR-278 were reduced to 82% as compared with the control. Mean±s.e. of three repeats. Student's t-test; *P<0.05. The number of homozygous GSCs counted: hsFLP;FRT42D, n=32; hsFLP;FRT42DmiR-278KO, n=42; hsFLP;FRT42DmiR-278Gal4KI, n=35; hsFLP;FRT42DmiR-7{Delta}1, n=55; hsFLP;FRT42DmiR-278Gal4KI,miR-7{Delta}1, n=41.

 

Figure 4
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  		Fig. 4. miR-7 expression in GSCs. (A) The GFP-miR-7 sensor is repressed in the anterior-most germline (bracket), as compared with control GFP sensor lacking significant 3'UTR content. Adducin (Add), red; DAPI, blue; GFP, green. (B) Dcr-1 GSC clones lacking β-gal (arrowhead) exhibit elevated GFP-miR-7 sensor expression relative to neighboring heterozygous cells. (C) miR-7{Delta}1 GSC clones exhibit an elevated frequency of CycE-positive staining compared with heterozygous GSCs. Ectopic expression of miR-7 in miR-7{Delta}1 mutant GSCs returns the frequency of CycE staining to near control levels. Mean±s.e. of three repeats. Student's t-test; *P<0.05, **P<0.01. The number of GSCs counted: FRT42DmiR-7{Delta}1/CyO;nosGal4/TM6, n=523; FRT42DmiR-7{Delta}1;nosGal4/TM6, n=87; FRT42DmiR-7{Delta}1;nosGal4/UAS-miR-7, n=110. (D,E) Ectopic expression of miR-7 in the GFP-positive follicle cell clones (y w hsFLP;EP{954};pUAST-GFP-act>CD2>Gal4) decreases the frequency of Dap staining compared with the neighboring wild-type cells. Mean±s.e. of three repeats. Student's t-test; **P<0.01.

 

Figure 5
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  		Fig. 5. The dap 3'UTR responds to InR but not TGF-β signaling in GSCs. (A) In InR mutant GSCs (first four rows, arrowhead), the GFP expression is upregulated in dapL, dapF and dapS, but not in the control sensor. In punt mutant GSCs (bottom row, arrowhead), the GFP expression in dapL is not upregulated. (B) Quantification of GFP intensity in InR, punt or Mad mutant GSCs compared with the neighboring heterozygous GSCs. The GFP intensity is upregulated in InR mutant GSCs with dapL and dapF by 1.39-fold and 1.23-fold, respectively. Mean±s.e. for all pairs of GSCs (at least 12 pairs for InRex52.1 clones, five pairs for punt135 clones, and 24 pairs for Mad12 clones). Student's t-test; **P<0.01.

 

Figure 6
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  		Fig. 6. InR-deficient GSCs show abnormal frequencies of cell cycle marker expression. (A) The percentage of CycE-positive GSCs in two different InR alleles (hsFLP;;FRT82BInRex52.1/FRT82BGFP and hsFLP;;FRT82BInRex15/FRT82BGFP) increased 1.7-fold as compared with the control neighboring GSCs. The percentage of Dap-positive GSCs increased 2-fold, whereas the percentage of CycB-positive GSCs decreased 1.5-fold in InR mutant GSCs. The percentage of dap-5gm-positive GSCs remained the same in InR mutant GSCs as in the control heterozygous neighboring GSCs. Flies were dissected 8 or 12 days after larval/pupal heat shock. Mean±s.e. of two to three repeats. Student's t-test; *P<0.05, **P<0.01. The number of homozygous GSCs counted was as follows. For CycE staining: FRT82B, n=14; InRex52.1, n=58; InRex15, n=47. For Dap staining: FRT82B, n=58; InRex52.1, n=57. For CycB staining: FRT82B, n=71; InRex52.1, n=71. For dap-5gm: InRex52.1, n=43. (B) Dap expression in an InR mutant GSC (yellow arrowhead, expression 2.4-fold higher than background).

 

Figure 7
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  		Fig. 7. miRNAs and dap act downstream of nutritional/InR signaling to regulate cell division. The average number of progeny in germarial region 1-2A counted under conditions of differing nutrition. (A) The cell division of FRT82B control GSCs was reduced dramatically under poor food as compared with rich food conditions, whereas the cell division of Dcr-1 GSCs was not significantly reduced. (B) The cell division of control GSCs was dramatically reduced under poor food as compared with rich food conditions, whereas cell division of dap GSCs was not significantly reduced. The cell division of dap4 GSCs was not significantly increased compared with FRT42B control GSCs in poor food conditions (P=0.14075, Student's t-test). (C) Reduction of dap4 partially rescues cell cycle defects in InR-deficient GSCs. Other dap alleles gave similar results (CyO/+;InRex52.1, 50% division index; dap2x10/+;InRex52.1, 68.75% division index). Mean±s.e. of three repeats. Student's t-test; *P<0.05, **P<0.01. n=~40 per condition.

 

Figure 8
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  		Fig. 8. A model for activation of miRNAs by InR signaling, which inhibits Dap expression and accelerates Drosophila GSC division. Reduced InR signaling reduces the levels of miRNAs that repress Dap. Therefore, Dap is upregulated and the cell cycle slows in GSCs. It is also possible that InR signaling regulates GSC division by additional mechanisms (dashed arrow).

 

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© The Company of Biologists Ltd 2009