Supporting Online Material for

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1 Supporting Online Material for Stem Cell Self-Renewal Controlled by Chromatin Remodeling Factors Rongwen Xi and Ting Xie* *To whom correspondence should be addressed. This PDF file includes: Materials and Methods Figs. S1 and S2 Table S1 References Published 2 December 2005, Science 310, 1487 (2005) DOI: /science

2 SUPPLEMENTARY DATA Materials and Methods Fly Stocks and Markers Unless otherwise stated, flies were reared at 25 o C. Information on alleles used in this study can be found in Flybase ( or as otherwise noted. dom 1 and dom 3 are strong loss-of-function alleles (6). iswi 1 and iswi 2 are strong or null alleles (7). DadlacZ is a lacz enhancer trap of Dad gene (8). bam-gfp is a transgenic construct which contains a bam promoter driving expression of GFP (9). Generating Marked Clones Mitotic GSC or SSC clones of loss-of-function mutants for the time-course analysis were generated using the FLP-FRT technique (10, 11). The genotypes of adult females in which the clones were induced are: hs-flp/+; FRT 42D / FRT42D arm-lacz, hs-flp/+; FRT 42D dom 1 / FRT42D arm-lacz, hs-flp/+; FRT 42D dom 3 / FRT42D arm-lacz, hs-flp/+; FRT 42B iswi 1 / FRT 42B arm-lacz, hs-flp/+; FRT 42B iswi 2 / FRT 42B arm-lacz, Flies were heat-shocked at 37 o C in a running water bath for one hour, twice a day for 3 consecutive days. The flies were dissected for ovary staining three days, 10 days and 17days after clone induction (ACI). 1

3 For detecting Dad-lacZ and bam-gfp expression in the mutant GSC clones, the following genotypes of animals were used: hs-flp/+; FRT 42B iswi 1 / FRT 42B ubi-gfp; Dad-lacZ/ +, hs-flp/+; FRT 42B iswi 2 / FRT 42B ubi-gfp; Dad-lacZ/ +, hs-flp/+; FRT 42B iswi 1 / FRT 42B arm-lacz; bam-gfp/ +, hs-flp/+; FRT 42B iswi 2 / FRT 42B arm-lacz; bam-gfp / +, Ovaries were dissected 1 week ACI for immunostaining. The MARCM system was used to generate GFP-positive marked wild-type or mutant dom SSC clones that express p35 (3). The genotypes of adult females in which the clones were induced at 37 o C are: hs-flp/+; FRT 42D / FRT42D tub-gal80; act-gal4 UAS-GFP/+, hs-flp/+; FRT 42D / FRT42D tub-gal80; act-gal4 UAS-GFP/UAS-p35, hs-flp/+; FRT 42D dom 1 / FRT42D tub-gal80; act-gal4 UAS-GFP/+, hs-flp/+; FRT 42D dom 1 / FRT42D tub-gal80; act-gal4 UAS-GFP/UAS-p35, Flies were heat-shocked at 37 o C in a running water bath for one hour, twice a day for 3 consecutive days, and their overies were dissected out for staining 4 days, 11 days and 18 days ACI. Antibody Staining Antibody staining of ovaries was performed using our published protocols (12). The following antisera and dilutions were used: rabbit anti-iswi (a gift from J. Tomkun) at 1:400, rat anti-dom-b (a gift from M. Meister) at 1:400, rabbit anti-β-galactosidase 2

4 (Molecular Probes) at 1:100, mouse anti-β-galactosidase (Promega) at 1:50, rabbit anti- GFP (Molecular Probes) at 1:100, mouse monoclonal anti-hts (1B1, Developmental Studies Hybridoma Bank) at 1:4, rabbit anti-pmad (13) at 1:100. Secondary antibodies including goat anti-rabbit, anti-mouse or anti-rat IgGs conjugated to Alexa 488 or Alexa 568 (Molecular Probes) at 1:200. ApopTag Staining The ApopTag in situ apoptosis detection kit (Serologicals, Clarkston, GA) was used to detect dying cells in ovaries. Ovaries were dissected in Grace s media, fixed in 4% paraformaldehyde for 15 minutes and washed with 1XPBS. The fixed ovaries were further processed as described in the manufacturer s manual. Briefly, the ovaries were put in equilibration buffer for 20 seconds, and then the working solution for 1 hour at 37 o C. The reaction was stopped by adding the stop solution, and the ovaries were washed twice with 1XPBS. Subsequent antibody staining was done as described above. Results The expression of ISWI and DOM in GSCs is not regulated by BMP signaling To first test if the antibodies against ISWI and DOM are specific, we used the FLP-mediated FRT recombination technique to generate marked iswi or dom mutant GSCs for examining ISWI or DOM protein expression. As expected, the nuclear expression of ISWI was severely reduced in iswi 2 mutant GSCs (Fig. SA and A ), indicating that the antibody against ISWI is very specific and the iswi 2 mutation is a 3

5 strong loss-of-function allele. Similarly, the nuclear expression of DOM-B was also dramatically reduced in dom 1 mutant GSCs (Fig. SB and B ), indicating that the antibody against DOM-B is very specific and the dom 1 is a strong loss-of-function allele. Similar results were obtained in the marked mutant somatic follicle cell clones (data not shown). To further test if their expression can be potentially regulated by BMP signaling, we generated marked Medea 26 mutant clones for examining ISWI and DOM expression. Medea is essential for BMP signaling in Drosophila (1, 2). In the marked Medea 26 mutant GSCs, the protein expression levels for both ISWI (Fig. SC and C ) and DOM (Fig. SD and D ) were similar to those in the neighboring wild-type GSCs, suggesting BMP signaling does not regulate ISWI and DOM expression. dom mutant SSC clones are lost not due to apoptosis To further investigate if dom mutant SSCs are lost due to apoptosis, we used the MARCM (mosaic analysis with a repressible cell marker) technique to generate GFP positively marked dom mutant SSCs clones that also express p35, a cell death inhibitor (3). p35 overexpression has been shown repress apoptosis induced by either developmental or environmental insults (4, 5). As expected, the GFP-positively marked wild-type or dom mutant SSCs behaved just like the LacZ-negatively marked wild-type or dom mutant SSCs described in the text (Table 1s, Fig. 2S and Table 1). Also, p35 overexpression in the marked wild-type SSCs did not change the loss rate, suggesting that the natural SSC turnover is likely caused by differentiation not by cell death. Interestingly, p35 overepxression could not prevent loss of the marked dom 1 SSC clones, indicating that the dom mutant SSCs are lost likely not due to cell death. Along with our 4

6 ApopTag labeling result, these results indicate that dom is required for controlling SSC self-renewal but not survival. Figure 1S. ISWI and Dom expression in the GSCs is not regulated by BMP pathway activity. In all panels, marked GSC clones are identified by the lack of LacZ staining (green). In the marked iswi 2 mutant GSC (outlined by dashed lines in A and A ), ISWI protein level (red) is severely reduced compared to the neighboring unmarked wild-type GSC (outlined by solid lines in A and A ). In the marked dom 1 mutant GSC (outlined by dashed lines in B and B ), DOM protein level (red) is dramatically reduced comparing to the neighboring unmarked wild-type GSC (outlined by solid lines in B and B ). In the marked Medea 26 mutant GSCs (outlined by dashed lines in C-D ), ISWI (C and C ) and DOM (D and D ) expression does not 5

7 change in comparison with the unmarked wild-type neighboring GSC (outlined by solid lines in D and D ). Figure 2S. p35 overexpression cannot prevent loss of the marked mutant dom SSC clones. All the germaria are labeled for GFP (green), Hts (red) and DNA (blue). The putative marked (arrows) SSCs are identified as the most anterior GFP-positive cells, while their marked GFP-positive progeny lie posteriorly to the marked SSCs. (A-B ) Germaria carrying a 4-day (A, B), 11-day (A, B ) or 18-day (A, B ) old marked wildtype (A-A ) or p35-overexpressing (B-B ) SSC clone. (C, D) Germaria carrying a 4-day old marked dom mutant SSC clone (C) or dom mutant SSC clone that also overexpresses p35 (D). (C, D ) Germaria showing loss of a marked dom mutant SSC clone (C ) or dom mutant SSC clone that also overexpresses p35 (D ) 11days ACI, which is evidenced by 6

8 the presence of a patch of mutant follicle cells just outside the germarial region. (C, D ) Germaria showing loss of a marked dom mutant SSC clone (C ) or dom mutant SSC clone that also overexpresses p35 (D ) 18 days ACI. The panels A-D are shown at the same scale and the bar in A represents 10 µm. Table 1S. dom is required for controlling SSC self-renewal Genotypes 4 days 11 days 18 days WT 35.6%* (174) 32.5% (280) 26.3% (339) UAS-P % (238) 31.6% (285) 24.4% (361) dom % (281) 11.1% (288) 0.5% (202) dom 1 ; UAS-P % (298) 12.8% (218) 0.8% (353) * Percentage of germaria carrying marked SSCs for a given genotype is determined by the number of germaria carrying one or more marked SSCs divided by the number of total germaria shown in parentheses. References 1. P. Das et al., Development 125, 1519 (1998). 2. R. G. Wisotzkey et al., Development 125, 1433 (1998). 3. T. Lee, L. Luo, Neuron 22, 451 (1999). 4. B. A. Hay, T. Wolff, G. M. Rubin, Development 120, 2121 (1994). 5. N. K. Sah et al., Proc Natl Acad Sci U S A 96, 4838 (1999). 6. M. L. Ruhf et al., Development 128, 1429 (2001). 7. R. Deuring et al., Mol Cell 5, 355 (2000). 8. K. Tsuneizumi et al., Nature 389, 627 (1997). 9. D. Chen, D. M. McKearin, Development 130, 1159 (2003). 10. K. G. Golic, S. Lindquist, Cell 59, 499 (1989). 11. T. Xu, G. M. Rubin, Development 117, 1223 (1993). 12. X. Song, C. H. Zhu, C. Doan, T. Xie, Science 296, 1855 (2002). 7

9 13. H. Tanimoto, S. Itoh, P. ten Dijke, T. Tabata, Mol Cell 5, 59 (2000). 8