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Stem Cell Self-Renewal Controlled by Chromatin Remodeling Factors

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Science  02 Dec 2005:
Vol. 310, Issue 5753, pp. 1487-1489
DOI: 10.1126/science.1120140

Abstract

The self-renewing ability of a stem cell is controlled by its specialized micro-environment or niche, whereas epigenetic regulation of gene expression by chromatin remodeling factors underlies cell fate determination. Here we report that the adenosine triphosphate–dependent chromatin remodeling factors ISWI and DOM control germline stem cell and somatic stem cell self-renewal in the Drosophila ovary, respectively. The iswi mutant germline stem cells are lost rapidly because of defects in responding to bone morphogenetic protein niche signals and in repressing differentiation, whereas the dom mutant somatic stem cells are lost because of defective self-renewal. This work demonstrates that different stem cell types can use different chromatin remodeling factors to control cell self-renewal.

Extrinsic signals from niches are believed to control stem cell behavior, including self-renewal through interacting with intrinsic factors (1). However, it remains largely unclear how niche signals regulate their target gene expression in stem cells at the chromatin level. In the germarium at the tip of the Drosophila ovary, germ-line stem cells (GSCs) and somatic stem cells (SSCs) are attractive systems in which to study stem cells and their niches at the molecular and cellular level (2) (Fig. 1A). Terminal filament cells and cap cells at the tip of the germarium form a GSC niche, whereas posterior inner sheath cells in the middle of the germarium function as a SSC niche (39). GSCs can be reliably identified by their location (contact with cap cells), size, and spherical spectrosome (Fig. 1A). A GSC or SSC divides to generate two daughters: One daughter remains anchored to niche cells through Asp-Glu (DE)–cadherin–mediated cell adhesion and retains stem cell identity (10), whereas the other daughter moves away from niche cells and differentiates (Fig. 1A). Decapentaplegic (Dpp) and Hedgehog (Hh) are important for GSC self-renewal (4, 7), whereas Hh and Wingless are essential for SSC self-renewal (4, 5, 9, 11). In this study, we showed that adenosine triphosphate (ATP)–dependent chromatin remodeling factors control stem cell self-renewal by regulating responses to niche signals.

Fig. 1.

ISWI and DOM are expressed in Drosophila ovarian GSCs and SSCs. (A) A schematic diagram showing a cross section of a Drosophila germarium. 1, 2A, 2B, and 3 indicate different regions of the germarium. All the images shown in all the figures represent one confocal section. (B) A germarium showing a uniform expression pattern throughout, including GSCs (dashed circles) and presumably SSCs. (C and D) A germarium showing that GSCs (dashed circles) express DOM at the highest level and that a GFP-marked SSC (solid circles) expresses DOM at a lower level. Abbreviations: TF, terminal filament; CPC, cap cells; IGS, inner sheath cells; SS, spectrosome; CB, cystoblast; FS, fusome; DCs, developing cysts; FC, follicle cells. Scale bar in (B), 10 μm.

Chromatin remodeling factors are involved in maintaining chromatin structures and modulating gene expression in organisms ranging from yeast to humans (12). In Drosophila, five ATP-dependent remodeling factors, Brahma (BRM), Imitation SWI (ISWI), Domino (DOM), Kismet (KIS) and dMi-2, sharing a SNF2-related domain and a DEAD/DEAH-box helicase domain, appear in distinct protein complexes to control different gene activity in a variety of developmental processes (1317). For example, ISWI plays a global role in chromatin compaction and transcriptional regulation (1719); DOM, highly related to a chromatin remodeler SWR1 involved in exchanging histone variants, functions as a transcriptional repressor by interfering with the chromatin structure (13). dom and iswi are known to be required for normal oogenesis, but their roles in GSC and SSC regulation have not been determined (13, 19). In this study, we focused on investigating the roles of dom and iswi in GSC and SSC regulation.

To analyze potential ISWI and DOM functions in ovarian stem cells, we characterized their expression in the germarium using available antibodies. The ISWI protein is present at high levels in the nuclei of all cell types in the germarium, including GSCs and SSCs (Fig. 1B). Although two DOM isoforms, DOM-A and DOM-B, are generally expressed, DOM-B is present in GSCs at higher levels than in other cells of the germarium (Fig. 1C). Because SSCs cannot be reliably identified by their location, morphology, or molecular markers, we used the MARCM (mosaic analysis with a repressible cell marker) system to generate green fluorescent protein (GFP)–marked SSCs for examining DOM-B expression (20). The positively marked SSCs can be readily identified on the basis of their ability to remain in the same position and continuously generate marked follicle cells (6, 9, 21). Indeed, DOM-B is present in the nuclei of SSCs at low levels (Fig. 1, C and D). These results suggest that DOM and ISWI could potentially have roles in GSC and SSC regulation.

To test whether dom and iswi are required for maintaining GSCs, we applied the Flipase (FLP)–mediated mitotic recombination technique to generate marked mutant GSCs and examined the loss rates of those marked mutant GSCs according to our published experimental procedures (7, 10). The marked mutant GSCs were identified by their absence of LacZ staining and the presence of an anteriorly anchored spectrosome (Fig. 2A). dom1, dom3, iswi1, and iswi2, representing strong loss-of-function mutations, were used to generate marked mutant GSC clones (13, 19). In contrast to the loss of 35% of the marked wild-type control GSC clones during the 2-week period, ranging from 3 days after clone induction (ACI) to 17 days ACI, about 99 and 96% of the marked mutant iswi1 and iswi2 GSC clones were lost during the same period, respectively, indicating that iswi is required for GSC maintenance (Table 1). Consequently, most of the marked control GSC clones that were detected 3 days ACI remained in their niches 17 days ACI (Fig. 2, A and B), whereas the majority of the marked mutant iswi GSC clones detected 3 days ACI were not present in their niches 17 days ACI (Fig. 2, C and D). Consistent with the idea that iswi mutations are responsible for the loss of the marked iswi mutant GSCs, a transgene ISWI-HA, which can fully rescue the lethality of both iswi mutants (19), also rescued the loss phenotype of those marked iswi mutant GSCs (Table 1). The marked mutant dom1 and dom3 GSC clones were lost similarly to the marked control clones, indicating that dom plays a minor, if any, role in GSC maintenance (Table 1). However, dom and iswi mutant oocytes formed normally but completely arrested at mid-oogenesis, which explains their requirements for normal oogenesis (13, 19). These results demonstrate that iswi is required for maintaining GSCs.

Fig. 2.

ISWI is required for controlling GSC self-renewal by modulating responses to BMPs in the Drosophila ovary. In (A) to (F) and (K), marked GSCs are identified by the loss of armadillo(arm)-lacZ expression, whereas in (H) and (I), marked GSCs are identified by the loss of ubiquitin (ubi)-GFP expression. (A and B) Germarial tips carrying a 3-day-old (3d) (A) and a 17-day-old (B) marked wild-type GSC (dashed circles), in which the spectrosome is indicated by an arrow. (C and D) Germarial tips showing a 3-day-old iswi2 mutant clone [dashed circle in (C)] and a lost iswi2 mutant clone evidenced by the presence of a mutant egg chamber (inset) 10 days ACI. (E) A germarial tip showing an ApopTag-negative marked iswi2 mutant (dashed circle) and unmarked wild-type (solid circle) GSCs and a dying IGS cell (arrow). (F and G) A germarial tip showing that a marked iswi2 mutant GSC (dashed circles) but not an unmarked wild-type GSC (solid circles) up-regulates bam-GFP expression. (H) A germarial tip showing that a marked iswi2 mutant GSC (dashed circle) down-regulates Dad-lacZ expression in comparison with an unmarked wild-type GSC (solid circle). (I and J) A germarial tip showing that a marked iswi1 mutant GSC (dashed circles) up-regulates Dad-lacZ expression in comparison with an unmarked wild-type GSC (solid circles). (K and L) A germarial tip showing that both a marked iswi2 mutant GSC (dashed circles) and an unmarked wild-type GSC (solid circles) have comparable levels of nuclear pMAD. Scale bar in (L), 10 μm.

Table 1.

ISWI and DOM are required for maintaining GSCs and SSCs in the Drosophila ovary, respectively. nd, not determined.

Genotypes Marked GSC clones Marked SSC clones
3 days 10 days 17 days Relative division rate 3 days 10 days 17 days
Wild type 33.6%View inline 26.4% 22.1% 1.00View inline 41.0% 31.3% 25.6%
(244) (182) (164) (37) (244) (182) (164)
iswi1 21.3% 4.2% 0.2% 0.37 41.8% 20.8% 11.7%
(338) (471) (470) (13) (492) (471) (470)
iswi1 and 31.2% 29.5% 27.6% 0.90 nd nd nd
    ISWI-HAView inline (247) (285) (217) (15)
iswi2 34.3% 11.0% 1.4% 0.40 32.5% 30.7% 18.4%
(492) (629) (576) (27) (492) (629) (576)
iswi2 and 41.0% 35.6% 29.0% 0.88 nd nd nd
    ISWI-HA (210) (225) (176) (21)
dom1 38.4% 30.1% 15.5% 1.06 40.6% 11.5% 3.5%
(594) (721) (689) (50) (594) (721) (689)
dom3 31.6% 27.0% 26.6% 0.97 35.4% 12.7% 2.0%
(263) (371) (357) (44) (263) (371) (357)
  • View inline* The percentage of germaria carrying marked GSCs for a given genotype is determined by the number of germaria carrying one or more marked GSCs divided by the number of total germaria shown in parentheses.

  • View inline The relative division rate for a marked GSC is determined by the average number of marked cysts generated by a marked GSC divided by the average number of unmarked cysts generated by an unmarked GSC. The number of total marked GSCs examined is shown in parentheses.

  • View inline SWI-HA is a transgene.

  • To investigate whether dom and iswi are required for normal GSC division, we determined relative division rates for dom and iswi mutant GSCs by quantifying the average number of cysts produced by a marked mutant GSC, divided by the average number of cysts produced by a marked wild-type GSC (10). The marked dom mutant GSCs had a relative division rate close to 1.0, indicating that dom is not required for GSC division (Table 1). In contrast, the marked iswi1 and iswi2 mutant GSCs had much smaller relative division rates, 0.37 and 0.40, respectively. This result demonstrates that iswi is also required for stimulating GSC division.

    iswi has been implicated in the regulation of cell survival in other developmental processes (19). Thus, we used the ApopTag cell death assay to determine whether marked iswi mutant GSCs undergo apoptosis. After 164 iswi2 mutant GSCs were examined, none of them were apoptotic, indicating that the loss of iswi mutant GSCs is likely not due to apoptosis but results from differentiation (Fig. 2E). These results suggest that ISWI regulates GSC self-renewal but not survival.

    Two bone morphogenetic protein (BMP) niche signals, Dpp and Gbb, control GSC self-renewal by producing phosphorylated MAD (pMAD), activating Dad transcription, and repressing the expression of the differentiation-promoting bag of marbles (bam) gene in GSCs (2224). To test the possibility that iswi is required in GSCs for properly responding to BMP signals, we determined pMAD expression, Dad transcription, and bam repression in marked iswi mutant and unmarked wild-type GSCs of the same germaria. The Dad-lacZ line and the bam-GFP line were used to recapitulate Dad and bam gene expression (2224). We found that 35 and 39% of the 1-week-old marked iswi1 (63 marked GSCs examined) and iswi2 (81 marked GSCs examined) mutant GSCs up-regulated bam-GFP expression in comparison with their neighboring unmarked wild-type GSCs (144 GSCs examined), which showed no bam-GFP expression, indicating that iswi is required for BMP signaling–mediated bam transcriptionalrepression in GSCs (Fig. 2, F and G). In addition, 41.0 and 44.6% of the 1-week-old marked iswi1 (139 marked GSCs examined) and iswi2 (130 marked GSCs examined) mutant GSCs showed misregulated Dad-lacZ expression (down-regulation and up-regulation) in comparison with their neighboring wild-type GSCs of the same germaria (Fig. 2, H to J), suggesting that iswi is required for proper BMP signaling–mediated transcriptional activation in GSCs. However, the pMAD levels in all the mutant iswi GSCs examined (41 iswi2 mutant GSCs) remained similar to those in the wild-type GSCs in the same germaria, suggesting that iswi is dispensable for BMP signal transduction in GSCs (Fig. 2, K and L). These results show that ISWI facilitates the proper interpretation of BMP signaling in GSCs. Furthermore, we showed that ISWI protein expression levels in GSCs are not regulated by BMP signaling (25) (fig. S1). Together, these results demonstrate that ISWI is involved in BMP signaling–mediated gene repression and activation in GSCs.

    We then generated marked dom and iswi mutant SSC clones to investigate their role in SSC regulation in the same way as we examined the marked GSCs. A SSC marked by loss of arm-lacZ expression was identified by its location (in the middle of the germarium) and its ability to generate marked follicle cells in and outside of the germarium. As a control, 37.5% of the marked wild-type SSC clones detected 3 days ACI were lost 17 days ACI, whereas most of them remained (Fig. 3, A to C, and Table 1). During the same period, 43.3 and 72.0% of the marked iswi1 and iswi2 mutant SSC clones were lost, respectively, indicating that iswi plays a minor role in maintaining SSCs (Table 1). In contrast, 91.4 and 94.4% of the marked dom1 and dom3 mutant SSC clones detected 3 days ACI were lost 17 days ACI, indicating that dom is essential for maintaining SSCs (Fig. 3, D to F, and Table 1). Furthermore, we used the ApopTag cell death assay and overexpression of a cell death inhibitor, p35, to show that mutations in dom did not increase apoptosis in the marked dom mutant SSCs (a total of 66 mutant dom1 and dom3 SSCs examined) and that p35 overexpression in the marked dom mutant SSC clones failed to prevent their loss (25) (table S1 and fig. S2). Our results demonstrate that dom specifically controls SSC self-renewal but not survival.

    Fig. 3.

    DOM is required for SSC maintenance. Putative marked lacZ-negative SSCs are indicated by dashed lines; their marked progeny are indicated by solid lines. (A to C) Germaria carrying a 3-day-old (A), 10-day-old (B), and 17-day-old (C) marked wild-type SSC clone. (D) A germarium carrying a 3-day old marked dom1 mutant SSC clone. (E and F) Germaria showing loss of marked mutant dom1 SSC clones, evidenced by the presence of mutant follicle cells outside the germarium (E) or inalateeggchamber(F). Scale bar in (A), 10 μm.

    This study reveals that ATP-dependent chromatin remodeling factors are required to control stem cell self-renewal. ISWI controls GSC self-renewal at least in part through regulating responses of stem cells to BMP niche signals. Furthermore, we also show that DOM is essential only for SSC self-renewal, whereas ISWI is essential only for GSC self-renewal. This suggests that different stem cell types depend on different chromatin remodeling factors to control their self-renewal. Recent studies show that Polycomb-like bmi-1 is required for maintaining the self-renewal of blood stem cells and neural stem cells (26, 27). Because extensive knowledge about Polycomb and other chromatin factors already exists regarding Drosophila, further studies on the roles of ISWI and DOM in the regulation of Drosophila ovarian stem cells would provide insight into how chromatin remodeling factors control stem cell self-renewal. Given an evolutionarily conserved function of SWI/SNF chromatin remodeling complexes, their function in stem cell regulation is likely to be conserved evolutionarily in mammals, including humans.

    Supporting Online Material

    www.sciencemag.org/cgi/content/full/310/5753/1487/DC1

    Materials and Methods

    Figs. S1 and S2

    Table S1

    References

    References and Notes

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