Supplemental materials and methods Zebrafish husbandry Wild-type AB and pu.1G242D mutant zebrafish were maintained as described previously. 1, 2 Whole-mount in situ hybridization (WISH) Antisense digoxigenin (DIG)-labeled RNA probes and WISH were performed according to standard protocols.2 Images were captured using a Nikon AZ100 microscope. For WISH, probes of cebpa,3 cebp1,4 mpx,5 lyz,6 mfap4,7 ande1-globin8 were prepared as reported. Sudan Black Staining Fixed embryos incubated in Sudan black (SB; Sigma-Aldrich) solution as described.9 The SB-stained embryos washed by 70% ethanol, and signal were detected under microscope. Tyramide-based detection of endogenous peroxidase activity Tyramide-based detection of endogenous peroxidase activity performed essentially as described previously.9 Cytological analysis Hematopoietic cell were isolated as described previously.10 Zebrafish were euthanized with tricaine. PB of fish was obtained by gill puncture with a 10-μl heparinized glass microhematocrit tube. Blood cells were isolated from the kidney marrow (KM). Both PB and KM were re-suspended in ice-cold PBS with 5% FBS, followed by cytospins at 400 rpm for 3 minutes and subjected to May-Grunwald Giemsa (Merck) staining according to the manufacturer's instructions. Blood cells of KM and PB in WT and pu.1G242D fish were calculated manually based on their morphology.10 For 2-month-old fish, 2 groups were analyzed. Every group of PB and KM from wild-type or pu.1G242D was collected from 4 fishes. For older than 2-month-old fish, more than 9 groups were analyzed. Each group of PB and KM was collected from 1 fish. Chemotherapy treatment of pu.1G242D embryos Embryos (1 dpf) were soaped in egg water containing 103 mg/L cytarabine or 30 mg/L daunorubicin for 4 days. The treated larvae (5 dpf) were collected for Sudan Back B (SB) staining. BrdU and TUNEL labeling Bromodeoxyuridine (BrdU) labeling was performed as described11 with minor modiﬁcations12. pu.1G242D/Tg(lyc:dsred) intercross embryos at 72 hpf were incubated in 10 mM BrdU (Sigma-Aldrich) for 2 h, fixed in 4% paraformaldehyde, stained with mouse-anti-BrdU (Roche) and goat-anti-dsred Abs (Abcam), followed by Alexa Fluor 488 anti–mouse (Abcam) and Alexa 555 anti-goat (Abcam) for ﬂuorescent visualization. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay was carried out as described,13 followed by goat-anti-dsred Abs (Abcam) and Alexa 555 anti-goat (Abcam). Images were captured using a Zeiss LSM 510 confocal microscope. Statistical analysis Fisher exact tests were used to determine the differences between categorical variables. T-tests were applied to compare continuous variables. ALL Statistical data were tested for significance by 2-tailed analysis. Supplemental references 1. 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Zakrzewska A, Cui C, Stockhammer OW, Benard EL, Spaink HP, Meijer AH: Macrophage-specific gene functions in Spi1-directed innate immunity, Blood 2010, 116:e1-11 8. Quinkertz A, Campos-Ortega JA: A new beta-globin gene from the zebrafish, betaE1, and its pattern of transcription during embryogenesis, Development genes and evolution 1999, 209:126-131 9. Le Guyader D, Redd MJ, Colucci-Guyon E, Murayama E, Kissa K, Briolat V, Mordelet E, Zapata A, Shinomiya H, Herbomel P: Origins and unconventional behavior of neutrophils in developing zebrafish, Blood 2008, 111:132-141 10. Traver D, Paw BH, Poss KD, Penberthy WT, Lin S, Zon LI: Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants, Nature immunology 2003, 4:1238-1246 11. Wallace KN, Akhter S, Smith EM, Lorent K, Pack M: Intestinal growth and differentiation in zebrafish, Mechanisms of development 2005, 122:157-173 12. Zhang Y, Jin H, Li L, Qin FX, Wen Z: cMyb regulates hematopoietic stem/progenitor cell mobilization during zebrafish hematopoiesis, Blood 2011, 118:4093-4101 13. Du L, Xu J, Li X, Ma N, Liu Y, Peng J, Osato M, Zhang W, Wen Z: Rumba and Haus3 are essential factors for the maintenance of hematopoietic stem/progenitor cells during zebrafish hematopoiesis, Development 2011, 138:619-629 Supplemental Figure Legends Figure S1 Expansion of immature myeloid cells in larvae, juvenile and adult pu.1G242D fish. (A) WISH ofβe1-globin expression in 5 dpf (Fisher exact tests, n≥17, P=0.497, no statistical significance). (B-I) blood cell counts of KM and PB. KM blood cells (B, F) and peripheral blood (PB) cells (D, H) in 2 month-old (B, D) and 12 month-old (F, H) WT and pu.1G242D fish stained with May-Grunwald/Giemsa staining. Arrowheads, arrows, and asterisks indicate progenitors, myeloid cells, and lymphocytes respectively. Original magnification is 100x. Blood cell counts of KM (C, G) and PB (E, I) in 2 month-old (C, E) and 1 year (G, I) WT (blue bars) and pu.1G242D (red bars) fish were calculated manually based on their morphology (t-test, n≥7, mean±SD, *P≤0.05, **P≤0.01).