Supporting Information - pnas.org · Supporting Information Kim et al. 10.1073/pnas.1214361110 SI...

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Supporting Information Kim et al. 10.1073/pnas.1214361110 SI Materials and Methods ES Cell Culture and Differentiation. ES cells were cultured on irra- diated mouse embryonic broblasts in 15% Inactivated fetal se- rum (IFS)/Dulbeccos Modied Eagles Medium (DMEM), 2 mM penicillin/streptomycin (GIBCO), 0.1 mM β-mercaptoethanol, 1,000 U/mL Leukemia inhibitory factor (LIF) (Peprotech), and 0.1 mM nonessential amino acids as described previously (1). Embryoid body (EB) differentiation was performed with the dif- ferentiation media containing 15% IFS/Iscoves Modied Dul- beccos Medium, 200 μg/mL iron-saturated transferrin (Sigma), 4.5 mM monothiolglycerol (Sigma), 50 μg/mL ascorbic acid (Sigma), and 2 mM glutamine. EB differentiation was performed by the hanging drop method as described previously (2). Briey, EBs were plated in hanging drops at 100 cells per 15-μL drop in an inverted bacterial dish, collected from hanging drops on days 22.25, cultured in a rotating suspension culture, manipulated in various conditions, collected on day 6 of differentiation, and dis- sociated for ow cytometry or CFU assays. Doxycycline (Sigma), recombinant human Indian Hedgehog (IHH; 250 ng/mL; R&D), and cyclopamine (Toronto Research) were used. When com- pounds were removed, all EBs were washed with 10 mL PBS two times. For the labeling of endothelial cells, DiI-acetylated low density lipoprotein (DiI-Ac-LL) (10 μg/mL) was used (Biomedical Technologies) to label cells for 36 h for analysis under a uo- rescence microscope. For WrightGiemsa stains, we xed cells in methanol and used the Three-Step Stain (Richard-Allan Scien- tic) according to the manufacturers instructions. CFU Assay. Dissociated cells from day 6 EBs were plated in du- plicate into methylcellulose media containing IL-3, IL-6, erythro- poietin, and Stem Cell Factor (M3434; StemCell Technologies) as described previously (1). CFUs (denitive erythroid, myeloid, granulocyte-macrophage, and granulocyte-erythrocyte-monocyte- megakaryocyte multilineage) were scored based on gross mor- phology 710 d postplating. Blast Colony-Forming Cell (BL-CFC) Assay. Flk1 + cells were sorted from days 33.5 EBs by magnetic bead sorting using Flk1-Phyco- erythrin (PE) (Avas 12α1; BD) and anti-PE microbeads (130-048- 801; Miltenyl) using LS Columns (130-042-401; Miltenyl) ac- cording to the manufacturers instructions. Sorted cells were checked for purity using ow cytometry and mixed into DMEM containing 1% methylcellulose, 10% FCS, 25% D4T conditioned medium, 5 ng/mL mVEGF, 100 ng/mL mSCF, 200 μg/mL holo- transferrin, 25 μg/mL ascorbic acid, and 450 μM monothioglycerol (3). Colonies were counted about 4 d after plating. Hemato-Endothelial Culture. The sorting schema is outlined in Fig. S4. Vascular-endothelial cadherin (VE-cadherin) + CD41 CD45 cells were sorted from day 6 EBs by magnetic bead sorting (Mil- tenyl). Specically, dissociated cells were stained with CD41- Fluorescein isothiocyanate (FITC) (30-F11; BD), CD45-FITC (MWReg30; BD), and VE-cadherin (11D4.1) at 1:10 dilution for 10 min in 4 °C 2% Bovine Serum Albumin (BSA)/IMDM. Cells were washed with 4 °C 2% BSA/IMDM and stained with anti- FITC Microbeads (130-048-701; BD) and anti-rat IgG2a-PE (RG7/1.30) at 1:10 dilution for 10 min in 4 °C 2% BSA/IMDM. Cells were depleted in LS columns (130-042-401; Miltenyl) two times. Cells were then stained with anti-PE Microbeads (130-048- 801; Miltenyl) at a 1:10 dilution for 10 min in 4 °C 2% BSA/ IMDM. Cells were selected in LS columns (130-042-401; Mil- tenyl) two times. Cells must always be 4 °C, and time in LS col- umns must be minimized to promote yield and prevent cellcell attachment. Isolated cells were spun down at 200 × g for 5 min at a density of at least 5e4 cells per cm 2 in IMDM containing 10% FSC, 10% equine serum, 5 ng/mL mVEGF, 10 ng/mL IGF1, 2 U/ mL erythropoietin, 10 ng/mL bFGF, 50 ng/mL IL-11, 100 ng/mL mSCF, 100 μg/mL endothelial cell growth supplement, 2 mM penicillin/streptomycin/glutamate, and 450 μM monothioglycerol on matrigel-coated plates (3). Cells were washed two times with PBS within 12 h of plating to remove the nonadherent fraction. Mouse Embryo Culture. Embryonic days (E) 910-staged mouse embryos were obtained from pregnant C57/Bl6 females. Yolk sacs or paraaortic splanchnopleura were dissected and cultured for 23 d on DMEM containing 10% FCS, 25% D4T cell line conditioned medium, 5 ng/mL mVEGF, 100 ng/mL mSCF, 200 μg/mL holo-transferrin, 25 μg/mL ascorbic acid, and 450 μM monothioglycerol on matrigel-coated plates. Viral Transduction. For Scl transduction, the FUΔGW-reverse tetracycline-controlled transcriptional activator (rtTA) lentiviral vector (Addgene) was used in addition to the doxycycline-in- ducible Scl lentivirus. For viral collection, 293T cells in 15-cm plates were transfected with 10 μg retroviral or lentiviral vector, 2 μg G glycoprotein of the vesicular stomatitis virus (VSV-G) vector, and 5 μg Gag-Pol vector using FuGENE 6 Transfection Reagent (Roche). The supernatant was collected 2 and 3 d after transfection, ltered through 0.45-μm lter, and concentrated by ultra-centrifugation at 50,000 × g for 90 min. During each in- fection, cells were centrifuged at 1,000 × g for 1.5 h in the presence of 5 μg/mL protamine sulfate. Inducible ES Cell Lines. cDNA containing constitutively active Hh signaling mediator Smoothened (SMO-M2) IRESeGFP was cloned into the Ainv15 mouse ES cell (mESC) line to generate the tetracycline-inducible SMO-M2 mESC line (4). The inducible NOTCH1 intracellular domain (NICD) mESC line was similarly generated using cDNA for NICD for cloning (5). Flow Cytometry and Cell Sorting. Dissociated cells were stained with CD41 (MWReg30), CD45 (30-F11), c-Kit (2B8), or VE-cadherin (11D4.1) with anti-rat IgG2a (RG7/1.30) or anti-rat IgG (G28-5), diluted in 4% FCS/PBS for 20 min on ice during each staining, and washed two times with PBS. 7-Aminoactinomycin D was added to exclude dead cells. Flow cytometry was performed on a FACSCalibur or LSRII. Data were analyzed using FlowJo. Sample values were displayed after subtracting isotype controls. Cells were sorted with FACSAria or anti-PE or anti-FITC MicroBeads (Miltenyi) using a magnetic column. Each staining step for the magnetic sorting was done for 10 min in 10% FCS/IMDM. All antibodies were obtained from BD Biosciences unless indicated otherwise. For the Car- boxyuorescein succinimidyl ester (CFSE) assay, dissociated day 6 EB-derived cells were resuspended in 10 μM CFSE (Molecular Probes) at 37 °C for 10 min, and labeling was quenched by washing 5× with ice-cold culture media. On day 5 of the hemato-endo- thelial culture, cells were analyzed by ow cytometry. Gene Expression Analysis. Total RNA was isolated using RNA-Stat 60 (TelTest) or RNeasy Kit (Qiagen) and treated with DNase I (Ambion). cDNAs were prepared with SuperScript II reverse transcriptase according to the manufacturers instructions (In- vitrogen). Real-time quantitative PCR (QPCR) was performed with Brilliant SYBR Green QPCR Mix (Stratagene) on an MX3000/6P Stratagene PCR machine. Primer sequences are lis- Kim et al. www.pnas.org/cgi/content/short/1214361110 1 of 11

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Page 1: Supporting Information - pnas.org · Supporting Information Kim et al. 10.1073/pnas.1214361110 SI Materials and Methods ES Cell Culture and Differentiation. ... Kyba M, Perlingeiro

Supporting InformationKim et al. 10.1073/pnas.1214361110SI Materials and MethodsES Cell Culture and Differentiation. ES cells were cultured on irra-diated mouse embryonic fibroblasts in 15% Inactivated fetal se-rum (IFS)/Dulbecco’s Modified Eagle’s Medium (DMEM), 2 mMpenicillin/streptomycin (GIBCO), 0.1 mM β-mercaptoethanol,1,000 U/mL Leukemia inhibitory factor (LIF) (Peprotech), and0.1 mM nonessential amino acids as described previously (1).Embryoid body (EB) differentiation was performed with the dif-ferentiation media containing 15% IFS/Iscove’s Modified Dul-becco’s Medium, 200 μg/mL iron-saturated transferrin (Sigma),4.5 mM monothiolglycerol (Sigma), 50 μg/mL ascorbic acid(Sigma), and 2 mM glutamine. EB differentiation was performedby the hanging drop method as described previously (2). Briefly,EBs were plated in hanging drops at 100 cells per 15-μL drop inan inverted bacterial dish, collected from hanging drops on days2–2.25, cultured in a rotating suspension culture, manipulated invarious conditions, collected on day 6 of differentiation, and dis-sociated for flow cytometry or CFU assays. Doxycycline (Sigma),recombinant human Indian Hedgehog (IHH; 250 ng/mL; R&D),and cyclopamine (Toronto Research) were used. When com-pounds were removed, all EBs were washed with 10 mL PBS twotimes. For the labeling of endothelial cells, DiI-acetylated lowdensity lipoprotein (DiI-Ac-LL) (10 μg/mL) was used (BiomedicalTechnologies) to label cells for 3–6 h for analysis under a fluo-rescence microscope. For Wright–Giemsa stains, we fixed cells inmethanol and used the Three-Step Stain (Richard-Allan Scien-tific) according to the manufacturer’s instructions.

CFU Assay. Dissociated cells from day 6 EBs were plated in du-plicate into methylcellulose media containing IL-3, IL-6, erythro-poietin, and Stem Cell Factor (M3434; StemCell Technologies)as described previously (1). CFUs (definitive erythroid, myeloid,granulocyte-macrophage, and granulocyte-erythrocyte-monocyte-megakaryocyte multilineage) were scored based on gross mor-phology 7–10 d postplating.

Blast Colony-Forming Cell (BL-CFC) Assay. Flk1+ cells were sortedfrom days 3–3.5 EBs by magnetic bead sorting using Flk1-Phyco-erythrin (PE) (Avas 12α1; BD) and anti-PE microbeads (130-048-801; Miltenyl) using LS Columns (130-042-401; Miltenyl) ac-cording to the manufacturer’s instructions. Sorted cells werechecked for purity using flow cytometry and mixed into DMEMcontaining 1% methylcellulose, 10% FCS, 25% D4T conditionedmedium, 5 ng/mL mVEGF, 100 ng/mL mSCF, 200 μg/mL holo-transferrin, 25 μg/mL ascorbic acid, and 450 μMmonothioglycerol(3). Colonies were counted about 4 d after plating.

Hemato-Endothelial Culture. The sorting schema is outlined in Fig.S4. Vascular-endothelial cadherin (VE-cadherin) +CD41−CD45−

cells were sorted from day 6 EBs by magnetic bead sorting (Mil-tenyl). Specifically, dissociated cells were stained with CD41-Fluorescein isothiocyanate (FITC) (30-F11; BD), CD45-FITC(MWReg30; BD), and VE-cadherin (11D4.1) at 1:10 dilution for10 min in 4 °C 2% Bovine Serum Albumin (BSA)/IMDM. Cellswere washed with 4 °C 2% BSA/IMDM and stained with anti-FITC Microbeads (130-048-701; BD) and anti-rat IgG2a-PE(RG7/1.30) at 1:10 dilution for 10 min in 4 °C 2% BSA/IMDM.Cells were depleted in LS columns (130-042-401; Miltenyl) twotimes. Cells were then stained with anti-PEMicrobeads (130-048-801; Miltenyl) at a 1:10 dilution for 10 min in 4 °C 2% BSA/IMDM. Cells were selected in LS columns (130-042-401; Mil-tenyl) two times. Cells must always be 4 °C, and time in LS col-

umns must be minimized to promote yield and prevent cell–cellattachment. Isolated cells were spun down at 200 × g for 5 min ata density of at least 5e4 cells per cm2 in IMDM containing 10%FSC, 10% equine serum, 5 ng/mL mVEGF, 10 ng/mL IGF1, 2 U/mL erythropoietin, 10 ng/mL bFGF, 50 ng/mL IL-11, 100 ng/mLmSCF, 100 μg/mL endothelial cell growth supplement, 2 mMpenicillin/streptomycin/glutamate, and 450 μM monothioglycerolon matrigel-coated plates (3). Cells were washed two times withPBS within 12 h of plating to remove the nonadherent fraction.

Mouse Embryo Culture. Embryonic days (E) 9–10-staged mouseembryos were obtained from pregnant C57/Bl6 females. Yolksacs or paraaortic splanchnopleura were dissected and culturedfor 2–3 d on DMEM containing 10% FCS, 25% D4T cell lineconditioned medium, 5 ng/mL mVEGF, 100 ng/mL mSCF, 200μg/mL holo-transferrin, 25 μg/mL ascorbic acid, and 450 μMmonothioglycerol on matrigel-coated plates.

Viral Transduction. For Scl transduction, the FUΔGW-reversetetracycline-controlled transcriptional activator (rtTA) lentiviralvector (Addgene) was used in addition to the doxycycline-in-ducible Scl lentivirus. For viral collection, 293T cells in 15-cmplates were transfected with 10 μg retroviral or lentiviral vector,2 μg G glycoprotein of the vesicular stomatitis virus (VSV-G)vector, and 5 μg Gag-Pol vector using FuGENE 6 TransfectionReagent (Roche). The supernatant was collected 2 and 3 d aftertransfection, filtered through 0.45-μm filter, and concentrated byultra-centrifugation at 50,000 × g for 90 min. During each in-fection, cells were centrifuged at 1,000 × g for 1.5 h in thepresence of 5 μg/mL protamine sulfate.

Inducible ES Cell Lines. cDNA containing constitutively active Hhsignaling mediator Smoothened (SMO-M2) IRES–eGFP wascloned into the Ainv15 mouse ES cell (mESC) line to generate thetetracycline-inducible SMO-M2 mESC line (4). The inducibleNOTCH1 intracellular domain (NICD) mESC line was similarlygenerated using cDNA for NICD for cloning (5).

Flow Cytometry and Cell Sorting.Dissociated cells were stained withCD41 (MWReg30), CD45 (30-F11), c-Kit (2B8), or VE-cadherin(11D4.1) with anti-rat IgG2a (RG7/1.30) or anti-rat IgG (G28-5),diluted in 4% FCS/PBS for 20 min on ice during each staining, andwashed two times with PBS. 7-Aminoactinomycin D was added toexcludedeadcells.FlowcytometrywasperformedonaFACSCaliburor LSRII. Data were analyzed using FlowJo. Sample values weredisplayed after subtracting isotype controls. Cells were sorted withFACSAria or anti-PE or anti-FITC MicroBeads (Miltenyi) usingamagnetic column.Each staining step for themagnetic sortingwasdone for 10min in 10%FCS/IMDM.All antibodies were obtainedfrom BD Biosciences unless indicated otherwise. For the Car-boxyfluorescein succinimidyl ester (CFSE) assay, dissociated day 6EB-derived cells were resuspended in 10 μM CFSE (MolecularProbes) at 37 °C for 10min, and labeling was quenched by washing5× with ice-cold culture media. On day 5 of the hemato-endo-thelial culture, cells were analyzed by flow cytometry.

Gene Expression Analysis. Total RNA was isolated using RNA-Stat60 (TelTest) or RNeasy Kit (Qiagen) and treated with DNase I(Ambion). cDNAs were prepared with SuperScript II reversetranscriptase according to the manufacturer’s instructions (In-vitrogen). Real-time quantitative PCR (QPCR) was performedwith Brilliant SYBR Green QPCR Mix (Stratagene) on anMX3000/6P Stratagene PCR machine. Primer sequences are lis-

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ted in Table S1. All QPCR data were normalized toGAPDH andcalculated using the ΔΔct method by the MXPro 3000 software.Gene expression heat maps were generated using R/Bioconductor.The microarray data were either analyzed with GenespringGX oraffy package using R/Bioconductor.

Immunofluorescence. Cells were fixed with 4% paraformaldehyde/PBS for 15min, washed three times, and blocked in PBS blockingsolution (10% goat serum, 0.04% Triton X-100, 0.01 M glycine)for 30 min at 25 °C. Cells were incubated overnight at 4 °C withanti-Hes1 (sc-25392; Santa Cruz), washed three times, and ex-posed with Alexa Fluor 647 goat anti-rabbit (Invitrogen) for 1 hat 25 °C. After washing, cells were rehydrated in PBS with DAPIfor analysis. For vonWillebrand factor (vWF) staining, anti-vWF(ab6994; Abcam) was used with Alexa Fluor 488 donkey anti-rabbit (Invitrogen).

Zebrafish Maintenance. Zebrafish were maintained and staged asdescribed previously (6). AB or Tü lines were used for all WTmicroinjection experiments. Embryos from hsp70:gal4 and uas:NICD matings were heat-shocked at 37 °C for 20 min to induceNotch signaling (7). Chemicals were used at the following doses:cyclopamine at 100 μM and N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) (Sigma) at 100 μM.

mRNA Microinjections. Full-length scl mRNA was transcribedfrom linearized pCS2+-SCLA2.1 (GenBank accession no.AF045432) using mMessage mMachine according to the man-ufacturer’s protocol (Ambion) (6). For cyclopamine rescueexperiments, 150 pg scl mRNA were injected at the one- toeight-cell stage, and cyclopamine was added at indicated de-velopmental stages.

Whole-Mount RNA in Situ Hybridization. Embryos were fixed in 4%paraformaldehyde/PBS. Digoxigenin (DIG) -labeled antisenseRNAs were transcribed from linearized templates using RNALabeling Kit (Sp6/T7) according to the manufacturer’s protocol(Roche). Antisense riboprobes to c-myb, runx1, flk1, vegfa, eph-rinb2a, and scl were used as described previously (6, 8, 9). Whole-mount RNA in situ hybridizations were performed as describedpreviously (6). DIG-labeled riboprobes were detected using al-kaline phosphatase-conjugated anti-DIG antibodies (Roche)followed by detection of alkaline phosphatase activity in blueusing NBT/BCIP substrate (Sigma).

Statistical Analysis. n represents the number of biological repli-cates. Two-tailed unpaired Student t tests were used, and errorbars show SE unless indicated otherwise.

1. Kyba M, Perlingeiro RC, Daley GQ (2002) HoxB4 confers definitive lymphoid-myeloidengraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors.Cell 109(1):29–37.

2. Lengerke C, et al. (2008) BMP and Wnt specify hematopoietic fate by activation of theCdx-Hox pathway. Cell Stem Cell 2(1):72–82.

3. Choi K, Kennedy M, Kazarov A, Papadimitriou JC, Keller G (1998) A common precursorfor hematopoietic and endothelial cells. Development 125(4):725–732.

4. Yu C, et al. (2006) Direct and indirect effects of hedgehog pathway activation in themammalian retina. Mol Cell Neurosci 32(3):274–282.

5. Carlesso N, Aster JC, Sklar J, Scadden DT (1999) Notch1-induced delay of humanhematopoietic progenitor cell differentiation is associated with altered cell cyclekinetics. Blood 93(3):838–848.

6. Dooley KA, Davidson AJ, Zon LI (2005) Zebrafish scl functions independently inhematopoietic and endothelial development. Dev Biol 277(2):522–536.

7. Burns CE, Traver D, Mayhall E, Shepard JL, Zon LI (2005) Hematopoietic stem cell fate isestablished by the Notch-Runx pathway. Genes Dev 19(19):2331–2342.

8. Kalev-Zylinska ML, et al. (2002) Runx1 is required for zebrafish blood and vesseldevelopment and expression of a human RUNX1-CBF2T1 transgene advances a modelfor studies of leukemogenesis. Development 129(8):2015–2030.

9. Lawson ND, Vogel AM, Weinstein BM (2002) sonic hedgehog and vascular endothelialgrowth factor act upstream of the Notch pathway during arterial endothelialdifferentiation. Dev Cell 3(1):127–136.

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Fig. S1. Induction of Hh pathway using recombinant IHH. (A) A representative flow cytometry plot showing the induction of CD41+c-Kit+ cells in day 6 wholeEBs after IHH treatment from day 4 to 5. (B) RNA expression levels by QPCR of Hh targets, Patched1 (Ptch1) and Gli1, at the end of each 1-d IHH inductionperiod (n = 4).

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Fig. S2. Concentration-dependent induction of hematopoiesis using the inducible SMO-M2–IRES–eGFP cell line. (A) Schema of the inducible mESC line usedwith indicated loci carrying alterations allowing for production of the reverse tetracycline-controlled transactivator (rtTA), which binds to the tetracyclineoperator (TetOp) when doxycycline (dox) is present and allows for the expression of the SMO-M2–IRES–eGFP cDNA. (B) Induction of the transgene at variousconcentrations of dox (1–2,500 ng/mL) on day 3 or 4 of EB differentiation and analyzed 1 d postinduction using flow cytometry (n ≥ 3). (C) Dox induction for 1 dincreases the expression of Gli1 as assessed by QPCR (n = 3). (D) One-day dox pulse on day 3 or 4 of EB differentiation increases the number of CFUs in day 6whole EBs (n = 5). (E) Percentages of induced cells (GFP+) in VE-cadherin+ populations from day 6 whole EBs that were treated with 100 ng/mL or 2 μg/mLduring differentiation (n = 3). (F) Percentages of induced cells (GFP+) in VE-cadherin− populations from day 6 whole EBs that were treated with 100 ng/mL or2 μg/mL during differentiation (n = 3). (G) Relative percentages of GFP in VE-cadherin+ vs. VE-cadherin− populations from data shown in E and F. (H) Rep-resentative plot for VE-cadherin+ and GFP+ after 1-d dox induction of SMO-M2–IRES–eGFP.

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Fig. S3. Endogenous expression of Hh-related genes in different cell types. Microarray gene expression data of Scl, Hh ligands (Sonic Hh and Ihh), anddownstream targets (Gli1 and Ptch1) in sorted CD41+VE-cadherin+, CD41−VE-cadherin−, CD41−VE-cadherin+, and CD41+VE-cadherin− cells from day 6 EBs (GeneExpression Omnibus accession no. GSE19951).

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Fig. S4. Characterization of hemato-endothelial culture. (A) Experimental schema for the sorting of VE-cadherin+CD41−CD45− cells for the hemato-endo-thelial culture. Antibody clone and isotypes are listed in parentheses next to their respective antibodies. (B) Sorting efficiency showing depletion of CD41/CD45-positive cells and selection for VE-cadherin cells. (C) CFU assays on sorted VE-cadherin+CD41−CD45− cells (n = 16). (D) Immunofluorescence for DiI-acetylted-lowdensity lipoprotein (DiI-Ac-LDL) and von Willebrand Factor in sorted VE-cadherin+CD41−CD45− cells on day 1 of hemato-endothelial culture. (E) Progression ofendothelial-to-hematopoietic conversion in hemato-endothelial culture in VE-cadherin+CD41−CD45− cells. (F) Cytospins of hematopoietic cells derived fromendothelial cells. (G) Cell counts of isolated CD41+ cells cultured in hemato-endothelial culture and treated with IHH and dox (for NICD induction).

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e R

NA

/ G

APD

H

Day 3 EB expression

(d)Vehicle15uM d2-545uM d2-5

(e)

(b)

veh 15µM 45µM0

10

20

30

40

% li

ve c

ells

day 3

day 5

vehicle d2-545 µM

(c)

d2-515 µM

veh d2-5 45uM1 00 1 01 1 02 1 03 1 04

1 00

1 01

1 02

1 03

1 04

1 9 .1 2 .4 7

6 .2 57 2 .11 00 1 01 1 02 1 03 1 04

1 00

1 01

1 02

1 03

1 04

1 3 .2 1 .3

8 .7 27 6 .8

(a)

CD

41

c-Kit

12.5%

0 200 400 600 800 1K

100

101

102

103

104

9.2%

0 200 400 600 800 1K

100

101

102

103

104(f)

EB d2-5 veh EB d2-5 cyc

CD

45

FSC

16.3%

0 200 400 600 800 1K

100

101

102

103

104

15.1%

0 200 400 600 800 1K

100

101

102

103

104

HE veh HE cycFSC

CD

45

(g)

(h)

(i)

CD41-45-100 101 102 103 104

41.358.7

Ctrl CD41/45+100 101 102 103 104

28.871.2

Dox CD41/45+100 101 102 103 104

26.573.5

VEC+100 101 102 103 104

0.2699.7

CFSE

Fig. S5. Characteristics of cyclopamine- and Scl-induced ES-derived cells. (A) Representative flow cytometric plot of CD41+c-Kit+ cells from day 6 whole EBs thatwere treated with cyclopamine (cyc) during days 2–5 of EB differentiation. (B) Flow cytometric analysis of live cells (excludes cells with loss of membraneintegrity and debris with forward scatter) in day 6 EBs after cyc treatment during days 2–5 of EB differentiation (n = 5). (C) Representative pictures of days 2–5cyc-treated EBs on day 3 or 5. Images were acquired with a 10× objective lens in a standard microscope (Nikon). (D and E) Gene expression analysis by QPCR ofanterior primitive streak genes (Gsc and Foxa2) and ventral posterior mesoderm genes (Mesp1 and Evx) during days 2–5 of cyc treatment. Values are nor-malized to endogenous gene expression (n = 2). (F) Flow cytometric quantification of CD45 after hemato-endothelial culture. VE-cadherin+CD41−CD45− cellssorted from day 6 EBs that were treated with cyc on days 2–5 of EB differentiation and cultured in hemato-endothelial culture. (G) Flow cytometric quan-tification of CD45 after hemato-endothelial culture. VE-cadherin+CD41−CD45− cells sorted from day 6 EBs and cultured in hemato-endothelial culture with cyc.(H) Death dynamics of CD41+/CD45+ in hemato-endothelial culture with or without Scl overexpression by Trypan Blue staining and cell counting on days in-dicated on the x axis. An inducible Scl mESC line was used. (I) CFSE dilution assay assessing the dilution of CFSE intensity with each cell division. The intensity isnot dramatically different between Scl overexpression and control experiments, which shows that there is no significant difference in the proliferation ofCD41+/CD45+ cells within the hemato-endothelial culture. An inducible Scl mESC line was used.

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(b)(a)

(c)

020406080

100120140160180

veh dox dox+cyc

CF

U / c

ells

fro

m 1

00

disso

ciated

c

ore

co

lon

ies

Fig. S6. Characterization of colonies from BL-CFC assays. (A) Blast colony on day 5 of BL-CFC assay using Ainv15- or NICD-inducible mESC lines. (B) Core colonyon day 4 of BL-CFC assay using Ainv15- or NICD-inducible mESC lines. (C) CFU potential of dissociated core colonies derived from BL-CFC assay by culture inmethylcellulose M3434 using NICD-inducible mESC line (n = 2).

Ctrl0 200 400 600 800 1000

100

101

102

103

104

2.6%

+ NICD induction0 200 400 600 800 1000

100

101

102

103

104

4.4%

0 200 400 600 800 1000

100

101

102

103

104

7.1

positive control

CD

45

Fig. S7. Effect of Notch induction on endothelial cells. NICD was induced in preformed endothelial cells (VE-cadherin+CD41−CD45−) during hemato-endo-thelial culture. CD45 cells were analyzed by flow cytometry 5 d after culture. The positive control was using NICD overexpression during days 3–5 of EB dif-ferentiation and subsequent hemato-endothelial culture.

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(d)

veh IHH cyc

CFU

/ 25

000

cells

/ em

bryo

P = 0.02

02468101214161820

GEMMG/M/GMEry

0

0.5

1

1.5

2

2.5

Ctrl IHHRel

ativ

e G

li1 /

GA

PDH

P = 0.03(f)

100 101 102 103 104100

101

102

103

104

0.13 0.077

9.2490.6100 101 102 103 104

100

101

102

103

104

0.039 0.14

20.679.2

c-Kit

CD

41

(e)

veh IHH

(a)

01020304050607080

Ctrl IHH

CFU

/ 10

000

YS c

ells

P = 0.006

GEMMG/M/GMEry

(c)

0

0.5

1

1.5

2

2.5

Ctrl IHH

Gli1

RN

A /

GA

PDH

P = 0.04(b)

100 101 102 103 104100

101

102

103

104

8.82 12.3

19.759.2100 101 102 103 104

100

101

102

103

104

9.69 14.4

14.761.2

c-Kit

CD

41

veh IHH

(g)

0

1

2

3

4

5

CFU

/ 10

00 V

E+ P

-Sp/

AG

M c

ells

GEMMG/M/GMEry

P = 0.065

dox

cyc

--

+-

-+

++

Fig. S8. Effect of altered Hh signaling on E9–E10 cultured embryos. (A) CFUs from yolk sacs treated with IHH (25 ng/mL) for 2 d (n = 5). (B) Flow cytometricanalysis of CD41 and c-Kit in cultured yolk sac after 2-d IHH treatment. (C) Gli1 expression by QPCR in yolk sacs after 2-d IHH treatment (n = 3). (D) CFUs fromparaaortic splanchnopleura treated with IHH (250 ng/mL) for 2 d (n = 7). (E) Flow cytometric analysis of CD41 and c-Kit in cultured paraaortic splanchnopleuraafter 2-d IHH treatment. (F) Gli1 expression IHH-treated paraaortic splanchnopleura (n = 3). (G) Effect of Scl overexpression and/or cyc treatment on sorted VE-cadherin+ cells from E9 to E10 paraaortic splanchnopleura. A doxycycline-inducible Scl lentivirus was used (n = 6).

(a) vehicle (b) cyc

(c)15 hpf hs (d)15 hpf hs+cyc

(e)17.5 hpf hs (f)17.5 hpf hs+cyc

runx1 @ 36 hpfin hsp70:gal4;uas:NICD embryos

n = 3

n = 3 n = 7

n = 8

n = 17 n = 21

Fig. S9. NICD induction by heat shock (hs) in the presence of cyc rescues hematopoiesis hsp70:gal4;uas:NICD embryos. In these zebrafish embryos, (B) cyc treatmentfrom 10-somite stages [14 h postfertilization (hpf); n = 21] eliminated runx1 expression in the aorta compared with the (A) vehicle control, but NICD inductionpresent in cyc-treated embryos at either the (C and D) 12- (15 hpf; n = 8) or (E and F) 17-somite stage (17.5 hpf; n = 7) rescued the expression of runx1 in the aorta.Embryos were fixed at 36 hpf and analyzed by whole-mount in situ hybridization. Lateral views of the trunk are accompanied by dorsal views on the right.

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cyc from 70% epiboly

vehicle

cyc

scl

cyc

(a) (e)

(b) (e)

(c) (f)

(d) (g)scl

cyc from 4 sm stage

runx1 @ 36hpf

Fig. S10. Runx1 expression in zebrafish embryos treated with (B and E) cyc, (C and F ) injected with scl RNA, or (D and G) both as compared with the (A and E)vehicle control. Cyc treatment occurred from 70% epiboly stage or four-somite stage. Embryos were fixed at 36 hpf and analyzed by whole-mount in situhybridization. These representative images are of the information shown in Table 1.

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vehicle cyc

15 hpf hs 17.5 hpf hs

scl @ 19.5 hpf in hsp70:gal4;uas:NICD embryosA

B

cyc

15 hpf hs+cyc

17.5 hpf hs+cyc

n = 1

n = 3

n = 6/21 n = 15/21

cycn = 4

n = 3

scl @ 36 hpf in hsp70:gal4;uas:NICD embryos

vehicle

15 hpf hs

17.5 hpf hs

Fig. S11. Scl expression in NICD-induced hsp70:gal4;uas:NICD zebrafish embryos. Embryos were hs at 12- (15 hpf) or 17-somite (17.5 hpf) stage. Cyc treatmentfrom 10-somite stage (14 hpf). (A) Embryos were fixed at 19.5 hpf (21-somite stage) and analyzed by whole-mount in situ hybridization for expression of scl.Green arrows indicate the presence of anterior lateral mesoderm. Black arrows indicate the presence of anterior of the posterior lateral mesoderm. Red arrowsindicate intermediate cell mass. Left shows lateral views (dorsal up); Right shows corresponding dorsal views. (B) Embryos were fixed at 36 hpf and analyzed bywhole-mount in situ hybridization for expression of scl. Red arrows indicate cell clusters in the aorta that have high scl expression. Black arrows indicate suchclusters in the vein.

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Table S1. List of primers

Primer name Sense Antisense Source

Gapdh AATGCATCCTGCACCACCAACTGCTT AGTGATGGCATGGACTGTGGTCATSmo CGAGCCTTCTGGTGGAGAAGATCAA GAGCTCGTGCCGCTTAGAGAAGPtch1 TGGTCACACGAACAATGGGTCTGA TTCGTATTGCCTGAGCTCCTCACTGli1 TCTCAAACTGCCCAGCTTAACCCA AGGACCTGCGGCTGACTGTGTAAHh interaction protein TGAAGATGCTCTCGTTTAAGCTG CCACCACACAGGATCTCTCC 2; Primer Bank ID 34328503a1Ihh TGCACCTCAGTCGATGCTGCTAGATT AGGCTGGCTGTGGTCTTTCAGTTCTShh TTGTGAGGCCAAGCAACCTGCTGAAA TTTACACTTGCGGGAAAGCAGGAGCABrachyury CCCTGCACATTACACACCAC GTCCACGAGGCTATGAGGAG 1Myogenin ACAATCTGCACTCCCTTACGTCCA TCTCAGTTGGGCATGGTTTCGTCT 1Odd1 TGAGCGACCTTACACCTGTGACAT TGGCAGAATCCTTTCCCACACTCT 1Mesp1 GTTCCAGTACGCAGAAACAGCATC CAAGGAGGGTTGGAATGGTACAGT 1Evx1 TGAAGAAATCGAGGTGAGCTGC CGATGCTCTGGGTAGCAGTCCTA 1Goosecoid ACAACAGCTACTTCTACGGGCA AGATGCTGCCCTACATGAACGT 1Cerebreus AGAGGTTCTGGCATCGGTTCATGT GTCTTCATGGGCAATGGTCTGGTT 1Foxa2 AAAGTATGCTGGGAGCCGTGAAGA ACTCTTCCGTGAGCAACATGAACG 1Fli1 ATGGACGGGACTATTAAGGAGG GAAGCAGTCATATCTGCCTTGG 2; Primer Bank ID 6679807a1Ets1 ACAGACTACTTTCGGATCAAGCA ACGCTCTCAAAAGAGTCCTGG 2; Primer Bank ID 6753782a1Elf1 TGTCCAACAGAACGACCTAGT CACACAAGCTAGACCAGCATAA 2; Primer Bank ID 6679627a1Scl TCCCCATATGAGATGGAGATTTC ATTGATGTACTTCATGGCAAGG 3Lmo2 ATGTCCTCGGCCATCGAAAGG AGATGATCCCATTGATCTTGG 3TIE2 ATGGACTCTTTAGCCGGCTTA CCTTATAGCCTGTCCTCGAA 3Gata1 CATTGGCCCCTTGTGAGGCCAG CGCTCCAGCCAGATTCGACCC 3Gata2 CTG CAA CAC ACC ACC CGA TA GGA GCG AGC CTT GCT TCT C 4Gata3 ATCCGCCCTATGTGCCCGAGTA ATGTGGCTGGAGTGGCTGAAGG 3bH1 AGTCCCCATGGAGTCAAAGA CTCAAGGAGACCTTTGCTCA 3bMajor CTGACAGATGCTCTCTTGGG CACAACCCCAGAAACAGACA 3c-Myb GAGCTTGTCCAGAAATATGGTCCGAAG GGCTGCCGCAGCCGGCTGAGGGAC 3AML1/Runx1 CCAGCAAGCTGAGGAGCGGCG CCGACAAACCTGAGGTCGTTG 3Ikaros CACTACCTCTGGAGCACAGC TCTGAGGCATAGAGCTCTTA 3PU.1 ATGGAAGGGTTTTCCCTCACCGCC GTCCACGCTCTGCAGCTCTGTGAA 3E47 CGCACTGACCACGAGCTTCAC TCCAGGGACAGCACCTCATCTG 3HoxB4 CCTGGATGCGCAAAGTTCA CTTGGGCTCCCCGCC 3Flk1 CACCTGGCACTCTCCACCTTC GATTTCATCCCACTACCGAAAG 3eGFP TTCAAGGACGACGGCAACTACAAGAC TCCTCGATGTTGTGGCGGATCTT 4Noggin GCCAGCACTATCTACACATCC GCGTCTCGTTCAGATCCTTCTC 2; Primer Bank ID 7110675a1Bmp4 AGCCAACACTGTGAGGAGTTTCCA TGCTGCTGAGGTTGAAGAGGAAACGA 1Bmp4 TTCCTGGTAACCGAATGCTGA CCTGAATCTCGGCGACTTTTT 2; Primer Bank ID 6680796a1Notch ICD 162 ATGCTGGAGGACCTCATCAACTCA TGTCTCCTCCCTGTTGTTCTGCATNotch ICD 278 ATGCCAACATCCAGGACAACATGG AACTGCGGCATCCACATTGTTCAC

1. Lengerke C, et al. (2008) BMP and Wnt specify hematopoietic fate by activation of the Cdx-Hox pathway. Cell Stem Cell 2(1):72–82.2. Spandidos A, Wang X, Wang H, Seed B (2010) PrimerBank: A resource of human and mouse PCR primer pairs for gene expression detection and quantification. Nucleic Acids Res

38(Database issue):D792–D799.3. Wang Y, Yates F, Naveiras O, Ernst P, Daley GQ (2005) Embryonic stem cell-derived hematopoietic stem cells. Proc Natl Acad Sci USA 102(52):19081–19086.4. McKinney-Freeman SL, et al. (2008) Modulation of murine embryonic stem cell-derived CD41+c-kit+ hematopoietic progenitors by ectopic expression of Cdx genes. Blood 111(10):

4944–4953.

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