String (Cdc25) regulates stem cell maintenance, proliferation and aging in Drosophila testis - PubMed (original) (raw)

. 2011 Dec;138(23):5079-86.

doi: 10.1242/dev.072579. Epub 2011 Oct 26.

Affiliations

String (Cdc25) regulates stem cell maintenance, proliferation and aging in Drosophila testis

Mayu Inaba et al. Development. 2011 Dec.

Abstract

Tight regulation of stem cell proliferation is fundamental to tissue homeostasis, aging and tumor suppression. Although stem cells are characterized by their high potential to proliferate throughout the life of the organism, the mechanisms that regulate the cell cycle of stem cells remain poorly understood. Here, we show that the Cdc25 homolog String (Stg) is a crucial regulator of germline stem cells (GSCs) and cyst stem cells (CySCs) in Drosophila testis. Through knockdown and overexpression experiments, we show that Stg is required for stem cell maintenance and that a decline in its expression during aging is a critical determinant of age-associated decline in stem cell function. Furthermore, we show that restoration of Stg expression reverses the age-associated decline in stem cell function but leads to late-onset tumors. We propose that Stg/Cdc25 is a crucial regulator of stem cell function during tissue homeostasis and aging.

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Figures

Fig. 1.

Fig. 1.

Stg is highly expressed in Drosophila GSCs and CySCs and is required for their maintenance. (A) Germline stem cell (GSC) and cyst stem cell (CySC) divisions. GSCs and CySCs attach to the hub cells. Each GSC is encapsulated by a pair of CySCs. Each gonialblast (GB), a differentiating daughter of a GSC, is encapsulated by a pair of cyst cells (CCs), which are the progeny of CySCs. (B,B′) FlyTrap Stg-GFP is highly expressed in GSCs and CySCs. A rosette of GSCs (white dots) and CySCs surrounding the hub (asterisk) is indicated by the white line. Arrowheads indicate GBs and spermatogonia that express a moderate level of Stg-GFP. The dotted line indicates a CC with a minimal level of Stg-GFP expression. Green, Stg-GFP; blue, Vasa (germ cells). (C,C′) Stg expression detected by anti-Stg antibody. The arrow indicates a GSC expressing a high level of Stg. Paired arrows indicate GSC-GB pairs that express a high level of Stg in their nuclei. Dotted lines indicate CySCs with cytoplasmic Stg expression. Blue, Vasa; green, Stg; red, Zfh1 (Zfh-1; CySCs). (D) Stg is highly expressed in a GSC/CySC tumor induced by Upd overexpression. Scale bars: 10 μm.

Fig. 2.

Fig. 2.

Stg is required for stem cell maintenance in the Drosophila testis. (A,A′) Stg is specifically downregulated by StgRNAi expressed in the germline [nos>StgRNAi (GD8177)]; compare with Fig. 1B. Blue, Vasa; green, Stg-GFP. The hub is indicated by an asterisk, and GSCs are indicated by dots. (B) Number of GSCs and CySCs (Zfh1-positive cells) following RNAi-mediated knockdown of Stg in the CySC lineage (c587-gal4 driver) or germline (nos-gal4 driver). Mean ± s.d. The _P_-value (two-tailed Student’s _t_-test) is provided for comparison with the control. _n_>25 testes per data point. n.s., not significant. (C-D′) Apical tip of the testis stained for Vasa (blue), Zfh1 (green) and Adducin-like (red) for control (C,C′) and c587>StgRNAi (D,D′) testes, demonstrating reduction in the number of CySCs upon knockdown of Stg in the CySC lineage. Arrows indicate branching fusomes, indicating normal differentiation of spermatogonia. Adducin-like stains the spectrosome and fusome. (E) CySC clones that express StgRNAi are lost over time. Flies (Hs-FLP; act>stop>gal4 UAS-GFP with or without UAS-StgRNAi) were subjected to heat shock (37°C, 30 minutes) and GFP-positive clones were scored 2, 7 and 14 days after heat shock. Error bars indicate mean ± s.d. (F) Mitotic indices (measured as mitotic cells per testis) of GSCs and CySCs upon knockdown of Stg. _n_>120 testes per data point. Error bars indicate mean ± s.d. Scale bars: 10 μm.

Fig. 3.

Fig. 3.

Overexpression of Stg leads to an increase in the number of CySCs/CCs and mitoses. (A) Mitotic indices of GSCs and CySCs (mitotic cells/testis) upon expression of Stg in the germline and/or CySC lineage. Flies carrying the c587-gal4 and nos-gal4 drivers without UAS-Stg are shown as controls. The _P_-value (two-tailed Student’s _t_-test) is provided for comparison with the control. _n_>120 testes for each data point. (B) Number of GSCs and CySCs (Zfh1-positive cells) upon overexpression of Stg in the germline and/or CySC lineage. _n_>30 testes for each data point. (A,B) Error bars indicate mean ± s.d. (C-D′) Examples of testis apical tip in control (C,C′) or in testis expressing Stg in the CySC lineage (c587>Stg) (D,D′). Green, Chd64-GFP (CB03690, a CySC marker); red, Fas III (a marker of hub cells); blue, Vasa (germ cells). Asterisk indicates the hub. (E) Increased number of CySC-like cells upon expression of Stg does not lead to GSC tumor development. Red, Adducin-like (spectrosome/fusome); blue, Vasa (germ cells). Arrowheads indicate branched fusomes (stained with Adducin-like), a hallmark of differentiation. Scale bars: 25 μm.

Fig. 4.

Fig. 4.

GSCs and CySCs show distinct aging kinetics. (A-B′) Expression of Stg-GFP in flies at day 0 (A,A′) and day 10 (B,B′). The first tier of cells (GSCs and CySCs) is indicated by the white line. GSCs are indicated by white dots. Green, Stg-GFP; blue, Vasa (germ cells). Asterisk indicates the hub. (C) Mitotic indices of GSCs and CySCs with age. The _P_-value (two-tailed Student’s _t_-test) is provided for comparison with day 0. _n_>120 testes for each data point. (D) Number of GSCs and CySCs with age. Whereas GSC number did not decrease significantly by day 20, CySC number significantly decreased by day 10. _n_>30 testes for each data point. (C,D) Error bars indicate mean ± s.d. (E-F′) Representative apical tip of the testis at day 0 (E,E′) and day 10 (F,F′) stained for Vasa (blue), Adducin-like (red) and Zfh1 (green), showing a dramatic decrease in Zfh1-positive CySCs with age. Scale bars: 10 μm.

Fig. 5.

Fig. 5.

Expression of Stg in the germline reverses aging phenotypes but leads to late-onset tumors. (A) Mitotic index of GSCs and CySCs with age upon expression of Stg in the germline (nos>Stg). The mitotic index of Stg-expressing GSCs did not decrease with age (for comparison, see Fig. 4C for mitotic indices in wild type). _n_>120 testes for each data point. (B) Change in stem cell number with age. CySC number was well maintained upon expression of Stg in the germline, whereas control flies lost a considerable number of CySCs with age. By contrast, whereas control flies maintained GSC number with age, Stg-expressing testes showed a slight but statistically significant decrease in GSC number. The _P_-value (two-tailed Student’s _t_-test) is provided for comparison with day 0. _n_>30 testes for each data point. (A,B) Mean ± s.d. n.s., not statistically significant when compared with day 0 of each cell type. (C,D) DAPI staining of testes apical tip from control (C) and Stg-expressing (nos>Stg) (D) testis at day 30. Stg-expressing testes frequently developed tumors by day 30. Asterisks indicate the apical end of the testis. (E,F) Testis from control (E) and Stg-expressing (F) flies stained for Vasa (blue), Zfh1 (green) and Adducin-like (red). (G,H) High magnification of Stg-expressing testis. Some germline tumors contained spectrosomes (G, arrows), whereas others contained branched fusomes (H, arrowheads). Scale bars: 25 μm.

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