doi: 10.1038/nature05883 SUPPLEMENTARY INFORMATION Supplemental Figure 1 Prostaglandin agonists and antagonists alter runx1/cmyb expression. a-e, Embryos were exposed to (b) PGE2 and (c) PGI2 (20μM) and specific (d) cox1 (SC560 10μM) or (e) cox2 (NS398 20μM) inhibitors from 3-somites until 36hpf and assessed for runx1/cmyb expression in HSCs. f, g, Embryos stained for cox2 expression at 36hpf: (f) antisense probe, (g) sense probe. h, microarray expression profile of FACS sorted cell populations isolated during primitive (gata1 and lmo2) and definitive (lmo2 and cd41) hematopoiesis. h, Relative expression of cox1 (light purple) and cox-2 (dark purple) in each GFP + sorted fraction compared to the GFP - sorted population is shown. i, Quantitative analysis of HSC numbers in bigenic zebrafish embryos detected by confocal microscopy: DMSO 23.3±5.0 (mean±sd), (10μM) 38.0±2.2, indomethacin (10μM) (ANOVA, p<0.00001, n=10/treatment). j-r, In situ hybridization at 36hpf for runx1 (j,m,p), cmyb (k,n,q) and myeloperoxidase (l,o,r) in embryos exposed to control DMSO, (10μM), or indomethacin (10μM). s-d, runx1/cmyb expression following MO knockdown (40μM) of endogenous cox1, cox-2 and PGE2 synthase activity (s-v) and rescue by exogenous (10μM) (y-b ); MO knock-down of (w) EP2 and (x) EP4 on HSCs, and the inability of (10μM) to rescue the MO phenotype (c,d ). e, qpcr analysis of EP2 and EP4 expression in FACS-sorted CD41 + HSC and CD41 - populations; two primer sets were tested for each receptor. www.nature.com/nature 1
Supplemental Figure 1 runx1/cmyb h Decreased Increased PGE2 PGI2 SC560 NS398 a b c d Fold-change HSCs per tail [#] cox1 cox2 gata1 lmo2 Primitive (18 somites) i lmo2 CD41 Definitive (36hpf) e control Indomethacin cox2 sense antisense f g www.nature.com/nature 2
Supplemental Figure 1 runx1 cmyb myeloperoxidase control j k l m n o indo p q r runx1/cmyb DMSO s t u v w x y z a b c d MO COX1 MO COX2 MO E2 Syn. MO EP2 MO EP4 MO e Primer set 1: Primer set 2: -actin water ep2 ep4 ep2 ep4 water ep2 ep4 ep2 ep4 www.nature.com/nature 3 CD41+ CD41- CD41+ CD41-
Supplemental Figure 2 Modulation of PG pathway alters expression of HSCrelated genes and recovery in sublethally irradiated adult zebrafish. a, Effect of treatment on stem cell and endothelial markers, as measured by qpcr on whole KM isolated on day 3 post-irradiation. An asterisk () indicates a statistically significant difference (two-tailed t-test, n=15, runx1: p=0.0001; lmo2: p=0.014; fli1: p=0.049). b, Effect of cox1 (SC560, 10μM) and cox2 (NS398, 10μM) inhibition on irradiation recovery (n=5/treatment). For fish treated with SC560 or NS398 no analysis could be obtained at day 14 due to excessive death in these treatment groups. www.nature.com/nature 4
Supplemental Figure 2 a WKM Relative expression level runx1 lmo2 scl gata2 fli1 flk1 b Precursor Myeloid Lymphoid % WKM Days Post Irradiation 10 μm Indomethacin 10 μm SC560 (cox1) 10 μm NS398 (cox2) www.nature.com/nature 5
Supplemental Figure 3 PG pathway components are present in ES cells and PGE2 influences colony number a, qpcr analysis of PG pathway components in ES cells over the course of ES cell differentiation and hematopoietic colony formation. a, b, -mediated (10μM) rescue of indomethacin (100μM) inhibition of colony formation in methylcellulose (a) and OP9 (b) assays. www.nature.com/nature 6
Supplemental Figure 3 a ESC EB (day 1-6) EB (d5) ESC EB (day 1-6) EB (d5) Ptgs1 Ptger1 ND Ptgs2 Ptger2 Ptges Ptger3 Actin Ptger4 CCE d0 Ainv d0 water d1 d2 d3 d4 d5 d6 EtOH Indo CCE d0 Ainv d0 d1 d2 d3 d4 d5 d6 EtOH Indo water b c Colonies [#] per 100,000 Colonies [#] per 100,000 de GM GEMM OP9 100 um Indomethacin 100 um Indomethacin + 10 um www.nature.com/nature 7
Supplemental Figure 4 Exposure of murine BM to increases spleen weight and long-term competitive repopulating HSCs. a, b, Effect of ex vivo treatment of WBM and isolated HSCs with EtOH control (red) or (blue) on spleen weight on day (a) 8 and (b) 12 (two-tailed t-test, CFU-S 8 : n=5, p=0.339; CFU-S 12 : n=9, p<0.00001). c, Splenic weight following indomethacin treatment (green) compared to control (two-tailed t-test: n=10, p=0.00026). d, Spleen colony number after treatment of KSL cells (two-tailed t-test, 100 cells: n=4, p=0.0013; 300 cells: n=5, p=0.009). e, Representative FACS plots illustrating the levels of CD45.1 engraftment (upper left quadrant) in recipients of control and exposed BM cells. f-j, Average chimerism (f, h, i) and calculated frequency of engraftment (g, j) in recipients of -treated WBM (blue) versus control (red). k, l, Effect of ex vivo treatment of WBM with cox1 (SC560, 10μM) and cox2 (NS398, 10μM) inhibitors in the CFU-S 12 assay on colony number (paired t-test, n=10, SC560 p=0.00016, NS398 p<0.00001 and splenic weight (paired t-test, n=10, SC560 p=0.025, NS398 p=0.00075). m,n, Peripheral blood (day 14 post treatment) and bone marrow (day 16 post treatment) WBC counts following 5-FU bone marrow injury; exposure to SC560 or NS398 significantly inhibited WBC recovery, while enhanced WBC counts. www.nature.com/nature 8
Supplemental Figure 4 a CFU-S8 b CFU-S12 Spleen weight [mg] Spleen weight [mg] c Spleen weight [mg] CFU-S12 d Spleen weight [mg] CFU-S12 e Indomethacin 100 cells 300 cells 0.075:1 0.25:1 1:1 10:1 E3 B WBC F4 B WBC G2 B WBC H3 B WBC 10 4 10 4 10 4 10 4 3.52 87.6 14.4 50.3 82.4 17.3 96.2 0.99 FL2-H 10 3 10 2 FL2-H 10 3 10 2 FL2-H 10 3 10 2 FL2-H 10 3 10 2 10 1 10 1 10 1 10 1 CD45.1 10 0 0.34 8.53 10 0 1.34 34 10 0 0.12 0.12 10 0 0.2 2.65 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 FL1-H FL1-H FL1-H FL1-H A2 B WBC B5 B WBC C1 B WBC D1 B WBC 10 4 10 4 10 4 10 4 9.36 86.5 36 63.6 78.4 17.6 95.4 4.51 FL2-H 10 3 10 2 FL2-H 10 3 10 2 FL2-H 10 3 10 2 FL2-H 10 3 10 2 10 1 10 1 10 1 10 1 10 0 0.31 3.81 10 0 0.34 0.068 10 0 0.17 3.86 10 0 0.068 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 FL1-H FL1-H FL1-H FL1-H CD45.2 www.nature.com/nature 9
Supplemental Figure 4 f g h Chimerism [%] 6 weeks 0.075:1 0.25:1 1:1 10:1 % Negative Recipients Number of Transplanted Cells 1:71308 1:21286 6 weeks Chimerism [%] 12 weeks 0.075:1 0.25:1 1:1 10:1 i j 24 weeks Number of Transplanted Cells Chimerism [%] 0.075:1 0.25:1 1:1 10:1 % Negative Recipients 1:34627 24 weeks 1:80359 k CFU-S12 l CFU-S12 Spleen colonies [#] Spleen weight [mg] SC560 (cox1) NS398 (cox2) SC560 (cox1) NS398 (cox2) m k 5FU Recovery PB (day 14) 5FU Recovery BM (day 16) WBC [x10 3 μl -1 ] WBC [x10 6 /femur] SC560 (cox1) NS398 (cox2) SC560 (cox1) NS398 (cox2) www.nature.com/nature 10
Supplemental Figure 5 PGE2 does not influence homing to the BM. Bone marrow was labeled with CDFA, then treated ex vivo with as described and injected into irradiated recipients. No significant differences in the percentage of GFP+ cells in the BM between recipients of treated cells and controls were found by FACS analysis at 12 hours post transplant (two-tailed t-test, n=10, p=0.83). www.nature.com/nature 11
Supplemental Figure 5 doi: 10.1038/nature05883 SUPPLEMENTARY INFORMATION Homing Analysis # CFDA + cells www.nature.com/nature 12
Compounds # of Times Identified Effect on HSCs Celecoxib 2 Decrease (26/31) Febufen 1 Decrease (20/26) Prostaglandin J2 1 Decrease (12/22) Suxibuzone 1 Decrease (16/30) Sulindac 1 Decrease (18/31) Mead Acid 2 Increase (24/32) Linoelic Acid 1 Increase (22/30) 13(S)-HODE 1 Increase (15/25) Ly-171883 1 Increase (17/26) Epoxyeicosatrienoic Acid 1 Increase (17/25) Supplemental Table 1 Compounds identified in the HSC screen associated with the prostaglandin pathway PG pathway compounds identified as modulating runx1/cmyb + HSCs are listed in column one. Column two denotes the frequency at which a particular compound was identified. The third column shows the effect of the compound on HSC gene expression (# embryos altered/ # embryos scored). www.nature.com/nature 13
PGE2 Indomethacin gene mean SD n mean SD n mean SD n scl 1 0.290 8 2.027 0.116 7 0.641 0.388 8 lmo2 1 0.388 7 1.808 0.316 7 0.517 0.240 6 gata2 1 0.282 6 2.339 0.121 4 0.691 0.237 6 flk1 1 0.327 7 1.654 0.204 7 0.578 0.275 7 runx1 1 0.372 8 4.371 0.052 6 0.558 0.265 8 cmyb 1 0.552 8 3.458 0.191 10 0.147 0.152 10 Supplemental Table 2 Effect of and Indomethacin on HSCs by qpcr qpcr for HSC and endothelial markers in 36hpf embryos following PGE2 and Indomethacin treatment; listed are the mean, standard deviation and n value for each gene/treatment. www.nature.com/nature 14
[10μM] [50μM] day mean SD n mean SD n mean SD n lymphoid 0 15.1 4.1 5 14.0 4.9 5 13.9 2.8 5 2 3.2 1.5 5 2.5 0.9 5 3.1 1.6 5 4 1.8 0.5 10 1.9 0.4 5 2.5 0.6 10 7 2.9 1.2 10 3.2 0.6 5 4.1 2.2 10 10 2.5 1.0 9 5.3 0.3 5 7.6 2.9 10 14 8.2 3.4 10 12.6 1.3 5 14.0 3.2 10 myeloid 0 14.5 3.3 5 13.1 3.2 5 15.4 3.5 5 2 13.9 2.4 5 12.4 2.8 5 13.4 3.2 5 4 5.3 1.0 10 4.2 1.8 5 6.0 2.5 10 7 2.7 1.2 10 2.5 0.7 5 5.0 1.7 10 10 3.1 0.9 9 5.1 1.2 5 9.3 3.7 10 14 5.2 3.1 10 6.1 1.7 5 12.3 3.3 10 precursor 0 9.5 2.1 5 8.1 1.6 5 9.4 1.9 5 2 5.2 0.9 5 6.4 2.1 5 7.0 2.1 5 4 4.1 0.5 10 6.2 1.4 5 8.8 1.8 10 7 5.9 1.3 10 8.1 1.8 5 12.9 4.7 10 10 6.3 2.9 9 11.9 2.2 5 29.4 6.0 10 14 13.1 3.5 10 12.2 1.9 5 24.2 6.3 10 Supplemental Table 3 Effect of and Indomethacin on KM recovery qpcr for HSC and endothelial markers at day 3 of KM recovery following PGE2 and Indomethacin treatment; listed are the mean, standard deviation and n value for each gene/treatment. www.nature.com/nature 15
Colony PGE2 [10μM] PGE2 [20μM] PGE2 [100μM] Indo [20μM] Indo [100μM] type Mean SD n mean SD n mean SD n mean SD n mean SD n mean SD 6.83 1.66 6 9.9 4.14 6 11.83 4.12 6 3.2 2.09 6 4.5 1.00 4 6.0 0.00 M 8.83 3.92 6 13.7 4.81 6 20.83 10.08 6 8.0 9.81 6 7.8 0.96 4 2.8 1.64 EMM 3.50 1.95 6 8.9 5.67 6 10.33 6.52 6 2.0 3.00 6 1.0 0.82 4 1.0 0.00 P9 35.1 20.0 6 100.7 107.5 6 135.4 113.2 6 23.0 15.4 6 8.8 6.8 3 12.6 6.0 Supplemental Table 4 Effect of and Indomethacin in ES cell assays Influence of and Indomethacin treatment on hematopoietic colony numbers; listed are the mean, standard deviation and n value for each gene/treatment. www.nature.com/nature 16
CFU-S 8 (n =5) Ex vivo Treatment Murine Cell Population # Cells Transplanted Weight mg Ave (SD) Colony Number EtOH Whole Marrow Whole Marrow 6000 32.3 (2.1) 10.4 (2.2) 6000 36.4 (9.1) 15.6 (4.5) Supplemental Table 5 Effect of on CFU-S 8 Spleen weight and CFU-S activity were assessed at day 8 in irradiated recipients injected with BM exposed to EtOH or. www.nature.com/nature 17
CFU-S 12 Ex vivo Treatment Murine Cell Population n # Cells Transplanted Weight mg Ave (SD) Colony Number EtOH Whole Marrow 9 6000 41.9 (15.8) 5.8 (2.7) Whole Marrow 9 6000 85.4 (16.5) 15.2 (2.4) EtOH Whole Marrow 10 100000 71.8 (18.1) 8.8 (2.1) Indomethacin Whole Marrow 10 100000 32.7 (8.7) 2.5 (1.4) EtOH Kit+Sca+Lin- 5 100 25.1 (5.9) 3.0 (1.6) Kit+Sca+Lin- 5 100 47.7 (5.6) 6.2 (1.3) EtOH Kit+Sca+Lin- 5 300 46.1 (6.2) 5.0 (1.2) Kit+Sca+Lin- 5 300 88.2 (14.8) 11.0 (1.9) Supplemental Table 6 Effect of on CFU-S 12 Spleen weight and CFU-S activity was assessed at day 12 in irradiated recipients injected with either WBM or ckit+sca1+lineage- FACS sorted cells treated with EtOH, or indomethacin (1μM/10 6 cells). www.nature.com/nature 18
Ex vivo Treatment CD45.1 Test Cell Dose CD45.1/.2 Competitor Cell Dose 6 wk CD45.1 Engraftment Animals with > 5% CD45.1 Mean % CD45.1 chimerism (± SD) CD45.2 Recipients Analyzed Total EtOH 15000 200000 3 4.0 ± 7.8 10 50000 200000 4 5.8 ± 5.7 8 200000 200000 9 32.5 ± 20.7 10 2000000 200000 9 52.4 ± 34.8 9 15000 200000 7 16.9 ± 19.5 10 50000 200000 8 22.7 ± 24.2 10 200000 200000 10 41.9 ± 17.2 10 2000000 200000 10 85.1 ± 3.1 10 Supplemental Table 7 Effect of on radio-protective competitive BM repopulation. WBM (CD45.1) was treated ex vivo with EtOH vehicle or and transplanted into sublethally irradiated recipients (CD45.2) with a fixed number of (CD45.1/CD45.2) competitor cells at the ratios shown in columns 2 and 3. Column 4 illustrates the number of animals with more than 5% CD45.1 chimerism at 6 weeks, and column 5 demonstrates the mean percentage of chimerism. The last column indicates the number of CD45.2 recipients analyzed. www.nature.com/nature 19
Ex vivo Treatment CD45.1 Test Cell Dose CD45.1/.2 Competitor Cell Dose 12 wk CD45.1 Repopulation Animals with > 5% CD45.1 Mean % CD45.1 chimerism (± SD) CD45.2 Recipients Analyzed Total EtOH 15000 200000 2 1.9 ± 2.9 9 50000 200000 3 5.4 ± 4.4 8 200000 200000 10 46.9 ± 22.9 10 2000000 200000 10 82.8 ± 14.1 9 15000 200000 5 15.1 ± 22.2 10 50000 200000 8 18.1 ± 22.2 10 200000 200000 10 40.8 ± 28.7 10 2000000 200000 10 87.8 ± 4.0 10 Supplemental Table 8 Effect of on short-term competitive BM repopulation. WBM (CD45.1) was treated ex vivo with EtOH vehicle or and transplanted into sublethally irradiated recipients (CD45.2) with a fixed number of (CD45.1/CD45.2) competitor cells at the ratios shown in columns 2 and 3. Column 4 illustrates the number of animals with more than 5% CD45.1 chimerism at 12 weeks, and column 5 demonstrates the mean percentage of chimerism. The last column indicates the number of CD45.2 recipients analyzed. www.nature.com/nature 20
Ex vivo Treatment CD45.1 Test Cell Dose CD45.1/.2 Competitor Cell Dose 24 wk CD45.1 Repopulation Animals with > 5% CD45.1 Mean % CD45.1 chimerism (± SD) CD45.2 Recipients Analyzed Total EtOH 15000 200000 1 1.9 ± 2.5 9 50000 200000 2 4.7 ± 4.2 7 200000 200000 10 51.18 ± 26.3 10 2000000 200000 9 81.7 ± 24.4 9 15000 200000 4 10.2 ± 20.3 9 50000 200000 7 14.3 ± 16.6 10 200000 200000 10 39.1 ± 31.0 10 2000000 200000 10 90.7 ± 4.0 10 Supplemental Table 9 Effect of on long-term competitive BM repopulation. WBM (CD45.1) was treated ex vivo with EtOH vehicle or and transplanted into sublethally irradiated recipients (CD45.2) with a fixed number of (CD45.1/CD45.2) competitor cells at the ratios shown in columns 2 and 3. Column 4 illustrates the number of animals with more than 5% CD45.1 chimerism at 24 weeks, and column 5 demonstrates the mean percentage of chimerism. The last column indicates the number of CD45.2 recipients analyzed. www.nature.com/nature 21
Supplemental Table 10 qpcr primers The top section indicates primer sequences used to detect EP2 and EP4 in zebrafish sorted cell RNA extracts. The lower section lists primer sequences utilized to detect PG pathway components in ES cells during differentiation and colony formation. www.nature.com/nature 22
Supplemental Table 10 qpcr Primer Sequences Zebrafish Primers EP2-F1 EP2-R1 EP2-F2 EP2-R2 EP4-F1 EP4-R1 EP4-F2 EP4-R2 Murine Primers PTGER1-F PTGER1-R PTGER2-F PTGER2-R PTGER3-F PTGER3-R PTGER4-F PTGER4-R PTGES-F PTGES-R PTGS1-F PTGS1-R PTGS2-F PTGS2-R 5 - taa cgg gaa cct gtc tga cc-3 5 - atc cca acc aat gtg gtg tt-3 5 - aga ctc atg cgg tgt gtg ag-3 5 - gtg ata cgg ata ccc gat gg-3 5 - tta ctg cgt aaa gcg gtc ct-3 5 - gga cac acc aga cgg aga gt-3 5 - cca tga tgg tca atg caa aa-3 5 - ctg gac gct gtc gtt agt ga-3 5 - ttt att agc ctt ggg cct cgt gga-3 5 - att gca cac taa tgc cgc aag gag-3 5 - aac gga att ggt gct cac tga cct-3 5 - tga gca tcg tgg cca gac taa aga-3 5 - tgt cgg ttg agc aat gca aga cac-3 5 - tct ggc aga act tcc gaa gaa gga-3 5 - aga cac cac ctc gct gag aac ttt-3 5 - aac ctc atc cac caa cag gac act-3 5 - tgg ctt ctt cag cat ctg tga ggt-3 5 - aca gca tgg gtc tta gac acc gaa-3 5 - ctt tgc aca aca ctt cac cca cca-3 5 - ttg aag agc cgc agg tga tac tgt-3 5 - atg agt ggt agc cag caa agc cta-3 5 - tac tga gta cca ggc cag cac aaa-3 www.nature.com/nature 23