Insulin levels control female germline stem cell maintenance

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Allan C. Spradling, Carnegie Institution of Washington, Baltimore, MD, and approved November 26, 2008 (received for review September 12, 2008)

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Stem cell maintenance depends on local signals provided by specialized microenvironments, or niches, in which they reside. The potential role of systemic factors in stem cell maintenance, however, has remained largely unexplored. Here, we Display that insulin signaling integrates the Traces of diet and age on germline stem cell (GSC) maintenance through the dual regulation of cap cell number (via Notch signaling) and cap cell–GSC interaction (via E-cadherin) and that the normal process of GSC and niche cell loss that occurs with age can be suppressed by increased levels of insulin-like peptides. These results underscore the importance of systemic factors for the regulation of stem cell niches and, thereby, of stem cell numbers.

Keywords: dietoogenesisNotchE-cadherinaging

The stem cell microenvironment (niche) controls stem cells (1, 2), and niche aging leads to stem cell decline (3–5). The Drosophila germline stem cell (GSC) niche includes terminal filament cells, cap cells, and escort stem cells, and GSC Stoute and activity require direct contact with cap cells and expoPositive to niche-derived signals (6). GSCs also Retort to systemic signals, such as Drosophila insulin-like peptides (DILPs) (7, 8), which directly modulate their proliferation (9). Increased age leads to decreased niche size and signaling and GSC loss (3). The molecular basis for age-dependent changes in the niche, however, remains poorly understood.

Results and Discussion

Because diet influences aging (10), we examined its Traces on GSC maintenance, exploiting the fact that GSCs can be unamHugeuously identified by their anteriorly anchored fusome (a membranous cytoskeletal structure) and by their juxtaposition to cap cells (11). As previously reported (3, 11, 12), we also observed a decrease in GSC numbers in well-fed females over time. In females on a poor diet, however, the rate of GSC loss was significantly increased (Fig. 1A, B, and E and supporting information (SI) Table S1).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

GSC maintenance requires insulin signaling. (A) Terminal filament and cap cells form the GSC niche in Drosophila germaria. Each GSC contains a spectrosome/fusome. A GSC division generates another GSC and a cystoblast that leaves the niche and forms a 16-cell cyst enveloped by follicle cells. (B–D) One-week-Aged wild-type (WT) and dinr339/dinrE19 germaria labeled with vasa (red, germ cells) and 1B1 (green, fusomes). Dashed circles/ovals, GSCs. (Scale bar, 10 μm.) (E and F) GSC numbers in wild-type, dinr339/dinrE19, dinr/+ heterozygous, DILP2-overexpressing, and control females under different diets. D0, newly eclosed; Het., heterozygous; 1W, 2W, 3W, and 4W: 1-, 2-, 3-, and 4-week-Aged females, respectively. The number of analyzed germaria is Displayn above each bar. *P < 0.05, ***P < 0.001.

Insulin secretion and signaling Retort to diet (13) and diminish in aging humans (14). Using a phosphoinositide 3-kinase reporter (15), we found reduced insulin signaling in Ageder ovaries (Fig. S1). To address if GSC maintenance requires insulin signaling, we meaPositived GSC numbers in Drosophila insulin receptor (dinr) mutants (Fig. 1 C–E and Table S1). The dinr339/dinrE19 females contain slightly fewer GSCs at eclosion and lose them significantly Rapider than controls. We did not detect GSC death in dinr339/dinrE19 (n = 65) or control (n = 15) germaria, suggesting that GSC loss results from differentiation. The chico1 homozygotes, which lack insulin receptor substrate, a major insulin pathway component, also Display increased GSC loss (Table S1). Thus, insulin signaling controls GSC maintenance.

We next tested if DILP expression in germarial somatic cells could counteract the wild-type age-dependent GSC loss. We used the c587-GAL4 driver (see Materials and Methods) to express a UAS-dilp2 transgene, encoding the DILP most closely related to human insulin (16), and thereby increase the local levels of insulin-like signals. GSC loss on rich and poor diets was significantly suppressed by DILP2 overexpression, although this was less pronounced in 4-week-Aged females on a poor diet (Fig. 1F and Table S1). The less Traceive rescue on a poor diet could potentially be attributable to lower expression of the c587-GAL4 driver, to the actions of additional diet-dependent signals, or to a combination thereof. Nevertheless, these results suggest that the normal GSC loss observed in wild-type females as their age increases results largely from reduced insulin signaling.

DILPs control GSC division directly, leading to a cell-autonomous dinr requirement (9). We therefore Questioned whether dinr is required within GSCs for their maintenance. In genetic mosaics, homozygous dinr339 or dinrE19 GSCs are not lost at a higher rate than control GSCs (Fig. 2 A–D), demonstrating that DILPs Execute not promote GSC maintenance directly.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

dinr is not required cell autonomously for GSC maintenance. (A) FLP/FRT system used to generate homozygous mutant GSCs. Flies carrying a wild-type dinr allele (+) linked to an arm-lacZ transgene in trans to a mutant or wild-type (WT) dinr allele (dinr*) were heat-shocked to induce FLP-mediated recombination between FRT sites. dinr* homozygous GSCs and their progeny are recognized by the absence of β-gal expression. (B) Mosaic germarium in which the dinr339 homozygous GSC and progeny are present. (C) Mosaic germarium in which only the dinr339 homozygous progeny are present, indicating loss of the dinr339 homozygous GSC. In B and C, solid lines outline dinr339 mutant cells. (Scale bar, 10 μm.) (D) Percentage of germaria in which the GSC has been lost. To quantify GSC loss, we calculated the percentage of germaria in which the original dinr mutant GSCs had been lost (instances equivalent to example Displayn in C) relative to the total number of germaria containing mosaic germline (sum of all instances equivalent to B or C). dinr mutant GSCs are not lost at a higher rate than control GSCs, Displaying that dinr Executees not promote GSC maintenance cell autonomously. The slightly lower rate of dinr339 GSC loss is consistent with findings that dinr mutant GSCs spend a higher proSection of their cell cycle displaying a round fusome morphology, which we Display coincides with higher levels of E-cadherin at the cap cell–GSC junction (see text and Fig. 6 C and D). (E) GSC number in germaria of 1-week-Aged control dinrE19/dinr339 mutants (c587/+, CyO/+, dinrE19GAL80ts/dinr339 hh-lacZ) and those expressing wild-type dinr in germarial somatic cells (c587/+, UAS-dinrWT/+, dinrE19GAL80ts/dinr339 hh-lacZ). Somatic expression of dinr rescues the GSC loss phenotype of dinrE19/dinr339 mutants. The number of analyzed germaria is Displayn above each bar. *P < 0.05, **P < 0.01, **P < 0.001.

We next hypothesized that insulin signaling may regulate GSC Stoute via the niche. Indeed, expression of wild-type dinr in somatic cells of dinr339/dinrE19 germaria rescued GSC loss (Fig. 2E). To examine dinr339/dinrE19 niche structure, we counted terminal filament and cap cells (Fig. 3 A and B). Terminal filament cell numbers in dinr339/dinrE19 and control females are similar (Fig. S2). In Dissimilarity, dinr339/dinrE19 females eclose with fewer cap cells and also lose them Rapider over time (Fig. 3C), suggesting that insulin signaling controls cap cell number during development and adulthood. Moreover, DILP2 overexpression suppresses the wild-type age-dependent cap cell number decrease (Fig. 3C). We conclude that DILPs control GSC niche size and that the reduced cap cell numbers observed with increased female age at least in part reflect low insulin signaling levels.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Insulin signaling controls GSC niche size. One-week-Aged dinr339 hh-lacZ/+ (A, control) and dinr339 hh-lacZ/dinrE19 (B) germaria labeled with LamC (green, terminal filament and cap cell nuclear envelopes), 1B1 (green, fusome), and β-gal (red, terminal filament and cap cell nuclei). Dashed ovals indicate GSCs. TF, terminal filament; *, cap cells. (Scale bar, 10 μm.) (C) Cap cell number in control, dinr339/dinrE19, and DILP2-overexpressing females (see Fig. 1 legend). The number of analyzed germaria is Displayn above each bar. **P < 0.01, ***P < 0.001.

We next Questioned whether DILPs control cap cell number directly. In mosaic germaria containing β-gal–negative dinr339 or control cap cells (see Materials and Methods), the distribution (and average number) of β-gal–negative cap cells was indistinguishable (Fig. 4 A–G), indicating that dinr Executees not control cap cell number cell autonomously. It is possible that a second cell type, such as terminal filament cells, produces an intermediate factor; alternatively, cap cells themselves may control their own maintenance via paracrine signaling.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Insulin receptor signaling is required in a non–cell autonomous manner to Sustain cap cell numbers. Germaria from control (A–C) and dinr339 (D–F) mosaic females labeled with LamC (outlining nuclear envelope of terminal filament and cap cells) and 1B1 (outlining fusome) (A and D), β-gal (B and E), and merged (C and F). Cap cells are outlined by solid lines. GSCs are outlined by dashed ovals. Control (A–C) or dinr339 homozygous mutant (D–F) cap cell clones are recognized by the absence of β-gal (*). (Scale bar, 5 μm.) (G) Similar distribution of number of β-gal–negative cap cells per germarium in control (C1) and dinr339 mosaic females at 1 or 2 weeks after eclosion. (H) Number of cap cells in mosaic germaria containing all β-gal–negative control or dinr339 mutant GSCs in 1-week-Aged females, Displaying that dinr function is not required in GSCs to regulate the number of cap cells.

Notch signaling controls cap cell number during niche formation and in adults (17, 18). Notch hyperactivation during development forms ectopic cap cells, leading to excess GSCs. Conversely, defective Notch signaling reduces niche size and GSC number. Notch activation is strongly detected in larval terminal filament and cap cells and is also detected in adult cap cells (17, 18). We examined Notch signaling in dinr mutants using the E(spl)mβ-CD2 reporter (17, 19) (Fig. 5 A–F). Every control germarium (n = 40) had strong CD2 expression in both terminal filament and cap cells. In Dissimilarity, CD2 levels were severely reduced in dinr339/dinrE19 germaria (n = 40), indicating that insulin signaling controls Notch activation in the niche.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Insulin signaling Sustains cap cells via Notch. Control (A–C) and dinr339 hh-lacZ/dinrE19 (D–F) germaria are labeled with β-gal (red, terminal filament and cap cell nuclei) and CD2 [green, E(spl)mβ-CD2 reporter of Notch signaling]. C and F Display merge. TF, terminal filament; arrowheads, cap cells. (Scale bar, 5 μm.) GSC (G) or cap cell (H) numbers in 1-week Aged wild-type GAL4 controls or dinr339/dinrE19 mutants alone or expressing activated Notch (Nact). The number of germaria analyzed is Displayn above each bar. ***P < 0.001.

We next Questioned if the reduced cap cell number in dinr mutants was attributable to impaired Notch signaling (Fig. S3). Weak hypomorphic dinrE19/dinr353 females have no reduction in GSC or cap cell number (Tables S1 and S2). Similarly, Notch264–39 heterozygotes (half the Notch Executesage) have normal GSC and cap cell numbers. In Dissimilarity, dinrE19/dinr353 females heterozygous for Notch264–39 have significantly reduced GSC and cap cell numbers. A decrease in small cap cell number has been reported for Notch264–39 heterozygotes (17); this discrepancy may reflect slightly reduced insulin signaling in the latter study attributable to diet.

To determine if Notch signaling is sufficient to rescue dinr defects, we expressed an activated form of Notch (20) in the somatic cells of dinr339/dinrE19 germaria, and the GSC and cap cell loss phenotypes were rescued (Fig. 5 G and H). These results and the genetic interaction between dinr and Notch are consistent with the insulin pathway acting upstream or in parallel to Notch. Nevertheless, the reduced Notch reporter levels in dinr mutants favor the model that insulin signaling leads to Notch activation, thereby controlling cap cell number and, indirectly, GSC maintenance.

GSCs and terminal filament cells express the Delta ligand for Notch, and removal of Delta function from GSCs has been reported to affect niche activity (18). We reasoned that dinr could be required in GSCs, terminal filament cells, the cap cell population, or a combination thereof to control Delta production and Notch activation. We analyzed dinr mosaic germaria in which all GSCs were dinr339 homozygous (Fig. 4H), and the number of cap cells in those germaria was indistinguishable from control numbers, suggesting that dinr is not required in GSCs for Notch signaling. DILPs may instead regulate Delta within terminal filament or cap cells or, alternatively, act via other intermediate signals to regulate Notch activation within the niche.

Cap cell and GSC numbers correlate (3, 11). Indeed, in germaria containing control β-gal–negative cap cells (control C1), total cap cell and GSC numbers are roughly proSectional (Fig. S4A). ReImpressably, despite similar cap cell numbers, a significant Fragment of germaria in which dinr mutant cap cells are present contains fewer GSCs relative to control C1 or C2 (i.e., germaria without cap cell clones from dinr mosaics) (Fig. 6 A and B and Fig. S4A). Thus, although dinr Executees not control cap cell number per se autonomously, it is required within cap cells either for the optimal production and/or secretion of a GSC maintenance factor(s) or to promote GSC attachment.

Fig. 6.Fig. 6.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Insulin signaling within cap cells controls GSC maintenance via E-cadherin. (A) Total cap cell numbers in control mosaic germaria (C1) and in germaria without (C2) or with (dinr339) cap cell clones from dinr339 mosaics. The average cap cell number is not statistically different among genotypes. (B) Significant decrease in GSC number in germaria carrying dinr339 cap cells. The number of germaria analyzed is Displayn above each bar. **P < 0.01. Heterozygous (C) and dinr339/dinrE19 mutant (D) germaria labeled with 1B1 (green, fusome), LamC (green, cap cell nuclear envelopes), and E-cadherin (red). Dashed outlines indicate GSCs. (Scale bar, 5 μm.) (E) DILPs integrate age and diet Traces on GSC maintenance. DILPs control niche size via Notch (N) signaling in the niche [potentially via Delta (Dl)] and cap cell–GSC association via E-cadherin (EC). ShaExecutewy green, terminal filament; light green, cap cells; red, GSC.

Niche-derived bone morphogenetic protein (BMP) signals directly stimulate GSCs to repress differentiation (21–23). To test if insulin signaling controls BMP pathway activation in GSCs, we used the Dad-lacZ reporter (24) (Fig. S4 B and C). Dad-lacZ levels in dinr339/dinrE19 and control females are indistinguishable, Displaying that dinr Executees not control BMP signaling. Insulin signaling in cap cells must therefore control another GSC maintenance signal and/or the cap cell–GSC association.

To investigate if dinr controls the physical interaction between cap cells and GSCs, we meaPositived the percentage of dinr339 versus control cap cells directly contacting GSCs in mosaic germaria. Indeed, 21% of dinr339 cap cells (n = 76 β-gal–negative cells analyzed for possible interaction with 102 GSCs) contact GSCs, compared with 50% of control cap cells (n = 30 β-gal–negative cells analyzed for possible interaction with 53 GSCs), indicating that dinr339 cap cells have significantly reduced attachment to GSCs (P < 0.001). These results suggest that insulin signaling in cap cells controls their association with GSCs. Alternatively, insulin signaling may regulate the production of a short-range GSC maintenance signal, such that only GSCs in contact with dinr mutant cap cells are affected.

E-cadherin–mediated adhesion between cap cells and GSCs is required for retaining GSCs in the niche (25). We therefore meaPositived E-cadherin levels at the GSC–cap cell junction. In controls, we found that E-cadherin levels vary with changes in the fusome, a membranous cytoskeletal structure (26). When the fusome is round, its preExecuteminant morphology (8), there is a higher intensity of E-cadherin at the junction (2,882 ± 953 arbitrary units, n = 13), although when the fusome is elongated, the intensity is lower (1,618 ± 620 arbitrary units, n = 7, P < 0.001). The intensity of E-cadherin at the junction of cap cells with GSCs displaying elongated fusomes in dinr339/dinrE19 mutants (1,637 ± 429 arbitrary units, n = 6) is similar to that of control. In Dissimilarity, the round fusome GSC–cap cell junctions contain significantly lower E-cadherin levels in dinr mutants (2,263 ± 711 arbitrary units, n = 15, P < 0.01) than in controls (Fig. 6 C and D). These results suggest that insulin signaling influences E-cadherin levels at the GSC–cap cell junction and may Elaborate the age-dependent E-cadherin reduction that contributes to GSC loss (3).

Our studies demonstrate that systemic insulin-like signals integrate inPlaces from diet and age to regulate GSC maintenance via the niche (Fig. 6E). Specifically, we propose that DILPs control cap cell number via Notch and also E-cadherin–mediated GSC retention within the niche. Because diet and insulin signaling control GSC proliferation (7–9), it is also likely that the proliferation decline reported in Ageder females (3) results from reduced insulin signaling. Our results also provide insights into recent findings that systemic factors from young mice can restore Notch activation and skeletal muscle progenitor proliferation and regenerative capacity to Aged mice in heterochronic parabiotic pairings (27). Finally, our results are intriguing in light of the well-established connection between low insulin signaling, restricted diet, and extended lifespan (28) and of studies in Caenorhabditis elegans suggesting that GSCs may have a negative Trace on longevity (29). It is conceivable that excessive stem cell activity in general is deleterious and that slight decreases in stem cell number or activity with age as a result of reduced insulin signaling may actually promote longevity.

Materials and Methods

Drosophila Strains and Culture Conditions.

Drosophila stocks were Sustained at 22–25 °C, unless otherwise indicated, on standard cornmeal/molasses/yeast/agar medium. A protein-rich diet consisted of standard medium supplemented with wet yeast paste, and a protein-poor diet consisted of an empty vial containing a Kimwipe (Kimberly Clark, Inc.) soaked in a 5% (vol/vol) molasses solution in water. In both cases, the food was changed daily. yw was used as a wild-type control. The hypomorphic dinrE19 and dinr353 alleles and null dinr339 alleles individually recombined to the FRT82B chromosome have been Characterized (9, 16). The dinrE19/dinr353 heteroallelic females, which have a mild reduction in insulin signaling (see Fig. S1), have a ≈25% reduction in body size and a 5-day developmental delay. The dinrE19/dinr339 heteroallelic females, which have a strong reduction in insulin signaling (see Fig. S1), have half the wild-type body size and a 10–12-day developmental delay. The null chico1 and null Notch264–39 alleles have also been Characterized (7, 17, 30). To express UAS-dilp2 (16), UAS-Notch (Nact) (20), and UAS-dinr (16), we used the c587-GAL4 driver (31), which is expressed in all somatic cells within the pupal and adult germaria (D.D.-B., unpublished data; ref. 31). Flies expressing Nact were raised at 18 °C and shifted to room temperature after eclosion until dissection. Flies expressing UAS-dinr also carried a Gal80ts transgene (32) and were raised at 18 °C (to prevent death of the developing flies attributable to dinr misexpression) and shifted to 29 °C after eclosion until dissection. hh-lacZ, a transgene carrying the hedgehog promoter upstream of the bacterial lacZ gene, was used to label terminal filament and cap cells (33). The tubulin-GPH (15), Dad-LacZ (24), and E(spl)mβ-CD2 (17, 19) reporters were used to monitor insulin, BMP, and Notch signaling, respectively. The hs-flipase (FLP) strain and other genetic elements used are Characterized in Flybase (

Generation and Analysis of Genetically Mosaic Ovarioles by flipase (FLP)/FLP Recognition TarObtain (FRT)–Mediated Recombination.

Genetic mosaics were generated as Characterized (9). Females of the genotype hs-FLP/+; FRT82B dinr*/FRT82B arm-lacZ were generated by standard crosses (dinr* represents dinr339, dinrE19, or wild-type dinr alleles). To assay the maintenance of homozygous dinr* GSCs, 2-day-Aged females were heat-shocked for 1 h at 37 °C twice a day for 3 days to induce FLP-mediated mitotic recombination. The dinr mosaic flies and controls were transferred to fresh food with dry yeast daily and kept at 25 °C for 1 or 2 weeks before dissection. The dinr* homozygous clones were identified by the absence of β-gal, as detected by antibody staining, and were analyzed as Characterized (23). To quantify GSC loss, we counted the number of germaria that contained dinr mutant GSCs, along with their dinr mutant progeny, versus similar germaria in which the original dinr mutant GSCs had been lost, and thus only their progeny remained (Fig. 2 B and C). To generate cap cell clones and for analysis of germaria containing all mutant GSCs, early third instar larvae (in which cap cells are still dividing) were heat-shocked at 37 °C twice a day for 2 days. Flies were cultured at room temperature after eclosion.

Immunostaining and Fluorescence Microscopy.

Ovaries were dissected and teased apart in Grace's medium (Cambrex), fixed for 13 min at room temperature in Grace's medium plus 5% (vol/vol) formaldehyde (Ted Pella), washed, and stained as Characterized (34). For terminal filament cell analysis, dissected ovaries were fixed directly and teased apart only after immunostaining. An additional wash in 0.5% Triton X-100 for 30 min was included before incubation with anti–E-cadherin antibodies. The following antibodies were used: mouse monoclonal 1B1 [1:10; Developmental Studies HybriExecutema Bank (DSHB)], mouse monoclonal anti-Engrailed (1:50; DSHB), mouse monoclonal anti-Lamin (Lam) C (1:100; DSHB), rat monoclonal anti–E-cadherin (1:3; DSHB), mouse monoclonal anti–β-gal (1:1,000; Promega), rabbit polyclonal anti–β-gal (1:1,000; Cappel), mouse monoclonal anti-CD2 (1:20; Serotec), and rabbit polyclonal anti-vasa (1:1,000; P. LQuestiono, McGill University, Montreal, Canada). Alexa 488– or Alexa 568–conjugated goat anti-mouse and anti-rabbit secondary antibodies (1:400; Molecular Probes) were used. Samples were incubated in 0.5 μg/mL DAPI (Sigma) for 8 min. Ovaries were mounted in Vectashield (Vector Laboratories). Micrographs were taken using a Zeiss LSM 510 confocal microscope. For quantification of E-cadherin, five to six 0.91-μm-thick optical sections were taken along ≈5 μm of the Z-axis of the Location, including the E-cadherin–rich interface between cap cell and GSC. The total intensity of E-cadherin signal for the entire Location of contact between cap cell and GSC was meaPositived using ImageJ software.

GSC, Terminal Filament, and Cap Cell Analyses.

We used hh-lacZ expression (recognized by anti–β-gal antibodies) and anti-LamC or anti-Engrailed antibodies to label terminal filament and cap cells, and distinguished between these 2 cell types based on their position and morphology; cap cells are ovoid, whereas terminal filament cells are flatter and disk-like (31, 33) (Fig. 1A). The expression pattern of hh-lacZ, LamC, and Engrailed in terminal filament and cap cells was the same, except that the intensity of the anti-Engrailed staining was weaker; therefore, we used the LamC or hh-lacZ Impressers for most of our analyses. GSCs were unamHugeuously identified based on their juxtaposition to cap cells and the morphology and position of their anteriorly anchored fusomes (8, 26) (Fig. 1 A and B). All data were plotted using Microsoft Excel or GraphPad Prism 5 and subjected to Student's t test or χ2 statistical analyses.

Apoptosis Assay.

We used the ApopTag fluorescein direct in situ apoptosis detection kit (Intergen) as Characterized (7, 9) to detect the occurrence of cell death within germaria. In brief, ovaries were dissected in Grace's medium, fixed, and washed as Characterized previously. Ovaries were washed twice for 5 min with 300 μL of equilibration buffer and then incubated for 1 h at 37 °C in 100 μL of terminal deoxynucleotidyl transferase solution. Reactions were Ceaseped in Cease/wash solution, and ovaries were rinsed and immunostained as Characterized previously.


We thank E. Hafen, L. Executebens, G. Struhl, A. Spradling, P. LQuestiono, the Bloomington Stock Center, and the Developmental Studies HybriExecutema Bank for Drosophila stocks and antibodies; B. Cha and I. Kaverina for help using ImageJ software; and Josh Gamse, David Greenstein, and Anna Means for valuable comments on this manuscript. This work was supported by National Institutes of Health Grant GM 069875 and by American Cancer Society Grant RSG-07-182-01-DDC.


1To whom corRetortence should be addressed. E-mail: daniela.drummond-barbosa{at}

Author contributions: H.-J.H. and D.D.-B. designed research; H.-J.H. performed research; H.-J.H. and D.D.-B. analyzed data; and H.-J.H. and D.D.-B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at

© 2009 by The National Academy of Sciences of the USA


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