rvdn e ellradmlclrisgt noterl of role hematopoiesis. the developmental into insights in molecular an ZBP-89, and disrupting and cellular GATA1 new by STAT3, providing erythropoiesis phosphorylated of in complex role inhibitory important an that plays imply ZNF148) findings PTPN9 These as expression. the known erythroid (also prevented regulating ZBP-89 from and and GATA1 factors entrapped transcription STAT3 hyper-phosphorylated the vivo el.Mcaitcly elto of depletion Mechanistically, cells. siRNA and C515S) mutation against (with maturation PTPN9 Consistently, dominant-negative cell apoptosis. both and erythroid proliferation inhibiting erythroid by affecting without depleted be to erythrocytes elms w rmtv eaooei ie uigzebrafish during sites hematopoietic primitive of knockdown two Morpholino-mediated embryogenesis. – intermediate and mass mesoderm plate cell lateral posterior the in expressed eeomn,CieeAaeyo eia cecs ejn,109 China. 100094 Beijing, Sciences, Medical of Academy Chinese Development, n ebaeBoehooy colo eiie colo ieSciences, Life China. of 100084 School Beijing, Medicine, University, of Tsinghua School Biotechnology, Membrane and [email protected]) ([email protected]; correspondence for *Authors Medicine, Molecular of China. Institute 100871 Beijing, Drugs, University, Biomimetic Peking and Natural of Laboratory eBu zebrafish Ye in dephosphorylation cell STAT3 erythroid through regulates development PTPN9 phosphatase tyrosine Protein ARTICLE RESEARCH ß eevd2 coe 03 cetd6Arl2014 April 6 Accepted 2013; October 27 Received 4 that (PLPM) dorsal the mesoderm of whereas wall (ICM), 1 ventral plate the mass in cell begins lateral hematopoiesis intermediate definitive the intraembryonic (ALPM) posterior mesoderm form two plate aorta-gonad- subsequently the lateral in anterior the occurs the and as as wave Mu known known primitive regions, 1996; the region al., zebrafish, et distinct (Cumano a hematopoiesis mesonephros hematopoiesis definitive sites in and primitive island, distinct mammals, blood originates yolk-sac In anatomically the 2008). in in occurs in Zon, waves occurs and that definitive (Orkin process and sequential a primitive is hematopoiesis Vertebrate INTRODUCTION cell K562 STAT3, PTPN9, development, cell Erythroid WORDS: KEY well cell in erythroid not of regulator is embryos. crucial involved zebrafish PTPs a in as development many PTPN9, are human of of ortholog (PTPs) function the characterized phosphatases but hematopoiesis, tyrosine Protein ABSTRACT hjeChang Zhijie ejn hngnBoeia opn t,Biig 005China. 100085 Beijing, Ltd, Company Biomedical Shenogen Beijing Key State and Medicine Molecular Cardiometabolic of Laboratory Key Beijing 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,26–70doi:10.1242/jcs.145367 2761–2770 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. ri 52cells K562 in or 1 PTPN9 ui Su Fuqin , nvivo in 2 niie rtri ifrnito nhmnK562 human in differentiation erythroid inhibited u Meng Kun , nvitro in 2 uWang Xu , ee ehv dniidPp9,an Ptpn9a, identified have we Here, . nrae hshrltdSA3 and STAT3, phosphorylated increased 4, n igWiXiong Jing-Wei and * 2 tt e aoaoyo Biomembrane of Laboratory Key State 1 ptpn9 a Gao Hai , 3 ptpn9a nttt fMdcnlPlant Medicinal of Institute nzbaihembryos zebrafish in le ta. 94.In 1994). al., et ¨ller u not but , 1,3 e Lei Lei , ptpn9a ptpn9b caused 1, ptpn9a was , 1 * annChang Nannan , in -Y Pi n o,21) h motneo hs factors these of importance The 2010). Zon, and and RUNX1 (Paik factors transcription c-MYB the by initiated is hematopoiesis TN a negn ulcto into duplication undergone has PTPN9 depletion whereas of of STAT3, Overexpression of 2012). phosphorylation al., tyrosine et decreases (Su cells dephosphorylation cancer cell its breast PTPN9 mediates in that endothelial cancer and shown STAT3 regulating have breast with we interacts and Recently, directly suppressing 2012). 2010) al., al., 2006), et (Hao et function al., (Yuan signaling et growth insulin cell mediating (Cho 2004), hepatocytes homotypic al., promoting in et as (Huynh such fusion functions, vesicle and different EGFR for ERBB2, hyper- FOXO1, shown VEGFR2 disorder have NSF, is studies marrow dephosphorylates Previous PTPN9 bone that 2003). that al., the et phosphatase in (Xu vera progenitors cytoplasmic polycythemia erythroid a in is activated pathways. signaling PTPN9 of differentiation, regulation Human proliferation, delicate and cell through migration in primitive and roles of important play processes (PTPs) the sophisticated control a to form hematopoiesis. factors order definitive transcription (Mucenski in These hematopoiesis 2002; network definitive 1991). al., in al., maintains et HSCs et c-MYB the of Kalev-Zylinska member of myb-family population 2009; wall is The the 1996). ventral al., factor, al., the et et transcription in Okuda (Chen domain HSCs aorta of runt generation dorsal a (Scott the development RUNX1, for cell essential 1994). myeloid al., al., domain, in ETS et role et an specifically al., indispensable (Pevny contains that an is et proerythroblasts factor plays transcription of GATA1, Woo hematopoietic a maturation PU.1, protein, 2006; 1991). the regulates al., finger for et zinc ZBP-89 required (Li Another protein differentiation 2008). hematopoiesis finger cell 1994). al., embryonic et progenitor in Warren zinc 1995; also al., et SCL The Shivdasani 2003; as acts al., et manner factor, (Gering hematopoietic same transcription of domain the regulator LIM in a central LMO2, basic a a differentiation. is with factor motif, transcription a helix-loop-helix SCL, models. mouse cell-based knockout in demonstrated been has as nw as known ( ( (also gene gene leukemia 2 cell stem lim-only the including zebrafish. hematopoietic factors, in caudal kidney the the at bone to colonize the migrate finally to and then but tissue and mammals, liver and fetal in self-renewing the marrow are to generates migrate which HSCs wave (HSCs), pluripotent. second cells the stem primitive and produces hematopoietic wave macrophages, first The and 2005). erythrocytes Zon, and Jong (de aorta rti yoieknssadpoentrsn phosphatases tyrosine protein and kinases tyrosine Protein rmtv eaooei srgltdb eea transcription several by regulated is hematopoiesis Primitive PTPN9 1 igWu Qing , nrae t hshrlto.Hr,w hwthat show we Here, phosphorylation. its increases ZNF148 LMO2 1 ), eigHu Keping , and ) PU.1 GATA1 as nw as known (also xvivo ex 3 ycnrs,definitive contrast, By . iou Zhu Xiaojun , ptpn9a sas swl sin as well as assays, SCL/TAL1 SPI1 and ), ptpn9b 1 ZBP-89 PTPN9 , ,the ), 2761 in

Journal of Cell Science hoooe1 n os hoooe9(supplementary 9 that human suggesting on mouse S1C), zebrafish homologs Fig. and their material the with 15 conserved not PTPN9 chromosome are mouse but 1, synteny chromosome and 25, Furthermore, human zebrafish chromosome S1A,B). that to revealed Fig. similarity analysis 41% material linker of displays (supplementary stretch domains (PTPc) it long catalytic a PTP – and possesses SEC14 the protein between the sequences and acids, and human amino to identity 67% PTPN9. have mouse which zebrafish acids, amino the 572 encodes In homologs, gene shown). two human not found we (data genome, analysis PCR quantitative and screen, wild-type this in between From embryos. mutant candidate of expression uigzbaihembryogenesis zebrafish during ptpn9a RESULTS that and zebrafish, ARTICLE RESEARCH 2762 embryogenesis. zebrafish 1. Fig. somites and brain the in S1F,G), Fig. expressed material predominantly (supplementary ICM was the or and and 24 PLPM 18, the at However, in brain 1D), 1D–F). developing 15 (Fig. (Fig. the ICM hpf at in the 36 expression in PLPM its and to the 1C) addition (Fig. sites, in (hpf) hematopoietic fertilization post supplementary primitive the hours two 1A,B; through in (Fig. gastrulation, evident After observed stages S1D,E). was Fig. gastrulation material expression and RNA their blastula using and by maternally, development embryonic situ zebrafish during mammalian the RNA 2008), al., an of et Xiong performed 1995; downstream al., we et PTPs (Stainier ablation encoding cell endothelial genes essential zebrafish for search To n-el() -p B,1-p C,1-p D,2-p E n 36-hpf and (E) 24-hpf (D), 18-hpf Note (C), embryos. 15-hpf (F) (B), 6-hpf (A), one-cell hshrltdSA3 AA n B-9i zebrafish in ZBP-89 of cells. complex and K562 human protein GATA1 and inhibitory embryos STAT3, an disrupting phosphorylated by development DF,addra iw r hw ihatro otetp(C). top the to anterior left with the shown to are red anterior views (D,E; with dorsal (ICM) shown and mass are (D–F), cell views intermediate Lateral the regions. and arrowheads) arrowhead) red (C; (PLPM) efrtasse h xrsinpten of patterns expression the assessed first We cloche yrdzto.Both hybridization. ptpn9a sepesdi h rmtv eaooei sites hematopoietic primitive the in expressed is cloche uat,wihwsfrhrvrfe ysemi- by verified further was which mutants, sepesdi h rmtv eaooei ie during sites hematopoietic primitive the in expressed is PTPN9 ptpn9a ptpn9b PTPN9 lsia uatwt eaooei and hematopoietic with mutant classical a , ptpn9a Zebrafish . xrsini h otro aea lt mesoderm plate lateral posterior the in expression locnan 3eosbtecds716 encodes but exons 13 contains also RNA rhlgi zebrafish. in ortholog nsitu in u not but , ptpn9a nsitu in ptpn9b cenadte oprdthe compared then and screen ptpn9a snupn ptpn9b yrdzto with hybridization and ptpn9a ptpn9a ptpn9a xrsinddntappear not did expression , ptpn9b euae rtri cell erythroid regulates , otis1 xn and exons 13 contains ptpn9a ptpn9a u not but , and a downregulated was ptpn9a ptpn9b eeexpressed were xrsinwas expression and ptpn9b ptpn9a and sin3aa ptpn9b ou on locus fthe of , rb in probe ptpn9b cloche ,is on in ofre h iiihdepeso fteeyhodgene further embryos erythroid analysis the wild-type of PCR human of expression diminished real-time gata1 that in the Quantitative hpf with confirmed 22 2G,J). compared at (Fig. the ICM 2H,K), of the (Fig. expression in and the marker However, erythroid progenitors. hematopoietic of expression The in 2008). unaffected al., be to et and appeared Liu genes ALPM 2003; three these al., the et within (Gering progenitors PLPM hematopoietic of generation together, that Taken suggest S1J,K). data Fig. our material (supplementary after hematopoiesis embryos of primitive in knockdown defects see morpholino-oligonucleotide-mediated not did we However, rgntr u scuilfrterdfeetainit erythroids. into differentiation their for crucial is but progenitors hypothesis the confirm that observations These 2I,L). (Fig. morphants S2A,C,E), Fig. material (supplementary hpf that 14 suggesting at embryos The factors. the factors transcription transcription into hematopoietic essential insights known several molecular in gain hypoplasia hematopoietic To hematopoiesis. primitive S2S–T Fig. material (supplementary hpf 48 the in that at reduced found substantially we morphants erythroids, was differentiated Tg[ population label DsRed-positive to transgene 2003) al., of the et role specific Using a development. being the there supporting of S1O–T), notion Fig. material (supplementary phenotype ohteanra perneadlc feyhoye were erythrocytes human hpf. of 48 lack of at and in 2D) appearance rescued (Fig. partially embryos abnormal wild-type the of Both that with in compared of decreased number The by 2B). greatly detected (Fig. as moderate hpf cells, 24 with blood at head circulating region short small anterior a the and showed in tail morpholino but necrosis curved hpf of 12 ventrally to length, ng up body period 4 stage. the toxic. in of two-cell were normally developed to doses injection higher one- the but the oligonucleotide, tolerated at embryos embryos Most zebrafish into injected against oligonucleotides morpholino el ntevnrlwl ftedra ot eeuatrdin unaltered were aorta Cmyb-positive dorsal the and of Runx1-positive wall ventral of the in populations cells the that cmyb Neither S1L–N). ptpn9a Fig. material zebrafish hematopoiesis. that S1G– found Fig. We material I). (supplementary embryogenesis early through oivsiaeterl of role the investigate To erythrocytes of Knockdown fetdin affected t3 p Fg F upeetr aeilFg 1) hs these Thus, S1I). Fig. that material supplementary suggest site) 1F; results hematopoietic (Fig. definitive the hpf (the or 36 aorta vessels at dorsal blood the in of enrichment wall without ventral hpf, 24 after trunk the ete drse hte eiiiehmtpissis hematopoiesis definitive whether addressed then We ptpn9a asstecmlt bec fHC.W found We HSCs. of absence complete the causes splmnaymtra i.SW.C-neto of Co-injection S2W). Fig. material (supplementary ora fCl cec 21)17 7127 doi:10.1242/jcs.145367 2761–2770 127, (2014) Science Cell of Journal PTPN9 RAwsal orsu the rescue to able was mRNA ptpn9a sntrqie o h eeaino hematopoietic of generation the for required not is PTPN9 ptpn9a RAflyrescued fully mRNA ptpn9a ptpn9a ptpn9a gata1 ptpn9a ptpn9a RA(0 geby)(supplementary pg/embryo) (100 mRNA scl opat.Dpeino either of Depletion morphants. RA(0p/mro Fg CF,or 2C,F), (Fig. pg/embryo) (80 mRNA ptpn9a ptpn9a / iiihstenme fprimitive of number the diminishes tal1 sntrqie o h eeainof generation the for required not is a eue ntePP t1 hpf 15 at PLPM the in reduced was and opatebysuo co-injection upon embryos morphant ssrnl soitdwt primitive with associated strongly is , ptpn9a ptpn9a ptpn9b lmo2 u not but , M-netdebys(i.2E) (Fig. embryos -MO-injected ptpn9a ptpn9b and npiiiehematopoiesis, primitive in opat,w investigated we morphants, ptpn9a gata1 eedfueyepesdin expressed diffusely were o Daiiiesann,was staining, -Dianisidine fli1 ptpn9a ptpn9b M-netdembryos -MO-injected gata1 xrsinin expression o SEC14-deleted nor r eurdfrthe for required are ( ptpn9a ptpn9a ptpn9a ptpn9a sesnilfor essential is , (Traver :DsRed] neyhodcell erythroid in -knockdown Ms were -MOs) morphants ptpn9b morphant runx1 ptpn9a ptpn9a 9 or in ).

Journal of Cell Science eeosnral,btta yli elmtrto smildly is maturation cell lineages myeloid Our myeloid in shown). that affected, to not but (data commitment normally, cells 2006) the develops al., myeloid that et label suggest Renshaw to observations 2007; used al., widely et been (Hall have which zebrafish, EERHARTICLE RESEARCH oivsiaeteclua ehnssunderlying mechanisms cellular the investigate hematopoiesis primitive To in apoptosis and proliferation ptpn9a Tg( using in in reduction verified genes mild myeloid of The expression embryos S2M,O,Q). the wild-type Fig. in material expression (supplementary their with compared S2N,P,R), in reduced in levels normal at detected ptpn9a were HSCs Consistently, S2I–L). Fig. ptpn9a opatpeoye,w xmndhmtpitccell hematopoietic examined in we apoptosis and proliferation phenotypes, morphant genes myeloid (data hpf the 22 at of region found ( expression ICM the We The myeloperoxidase in later shown). 2005). as not well al., material as (supplementary S2G,H), myelopoiesis hpf et 15 Fig. at embryos Rhodes into wild-type and injected 2005; of erythropoiesis levels al., of comparable primitive et expression (Galloway of ectopic the conversion in 2007) results al., et embryogenesis. during North HSCs that of 2005; specification indicate results al., our S2U–V together, et Fig. material (Lin (supplementary embryos transgenic Tg( n aeig(UE)asy a locmaal in comparable also was nick- UTP the assay, transferase in (TUNEL) terminal cells a labeling using apoptotic end by of for revealed number region, positive the ICM in Furthermore, were 3A,B). region (Fig. that ICM Tg( cells the wild-type and in of We fluorescence 2009). numbers al., green et comparable (Sugiyama fluorescence green found with cells M and rvossuishv hw htls of loss that shown have studies Previous ef1 a M-netdebysa 6hf(upeetr material (supplementary hpf 36 at embryos -MO-injected mGze)taseehsbe hw olblS G2 S, label to shown been has transgene :mAG-zGem) opat sn h Tg( the using morphants ncdw a oefc neyhodcell erythroid on effect no has knockdown ptpn9a ptpn9a ef1 mpo morphants. mpo a opat splmnaymtra Fig. material (supplementary morphants mGze)tasei mro t2 hpf 24 at embryos transgenic :mAG-zGem) pu.1 n yoyeC( C lysozyme and ) GP n Tg( and :GFP) nteAP einof region ALPM the in ptpn9a cd41 ptpn9a ptpn9a kokonebys The embryos. -knockdown 9 GP n Tg( and :GFP) aantson.Taken shown). not data ; pu.1 lyz sntrqie o the for required not is opat a further was morphants ptpn9a dRd transgenic :dsRed) eutn nthe in resulting , lyz gata1 a mildly was ) -MO-injected ptpn9a cmyb expression l-plastin ptpn9a ptpn9a :GFP) -MO- - , opat()adwl-ye()ebysa 4hf CD h UE assay in TUNEL ICM the The in (C,D) cells hpf. apoptotic of 24 numbers comparable at showed embryos (A) wild-type and (B) morphant C.Tenme fpeoyi mro/oa mro sindicated. is embryos embryos/total phenotypic of number The theICM. indicate arrowheads Red (E). controls wild-type with compared (F), embryos RNA (E,F) the hpf. of expression 24 at (C) controls and (D) difference little is there between that ICM Note the cycle. in green-fluorescence-positive cell cells of the numbers of the M in or G2 S, phases in are cells Tg( rlfrto n apoptosis. and proliferation 3. Fig. opat n otosa 4hf(i.3,) sexpected, As 3C,D). (Fig. as hpf known 24 (also at controls and morphants ptpn9a ptpn9a TN yuigete aayial nciedominant-negative inactive catalytically hematopoietic of either using K562 inhibition by Consistently, induce – PTPN9 erythrocytes. to mature a order into in progenitors in hemin used system we differentiation hematopoiesis, developmental mammalian human whether determine the ef1 gata1 a mGze)tasei mro,i hc green-fluorescence-positive which in embryos, transgenic :mAG-zGem) ptpn9a kokonebys(i.3) oijcino both of Co-injection 3F). and (Fig. -MOs embryos -knockdown ora fCl cec 21)17 7127 doi:10.1242/jcs.145367 2761–2770 127, (2014) Science Cell of Journal xrsin in expression, ncdw assn eeti eaooei cell hematopoietic in defect no causes knockdown p53 tp53 i.2 ncdw of Knockdown 2. Fig. oto A,a (A), control erythrocytes. primitive yrdzto a efre with performed was RNA hybridization (G–L) erythrocytes. indicate arrowheads in Human (D). controls PTPN9 with compared (E), in embryos cells erythroid of (D–F) depletion hpf. showed 24 at (C) human mRNA with co-injected been had that morphant a enc-netdwt human with and co-injected (K), been hpf 22 had and (J), (H) hpf hpf 22 15 and at (G) hpf 15 at controls h LM()adIM() hc eepartially human were of which co-injection (K), by ICM rescued and (H) reduced PLPM Note (L). the hpf 22 and (I) hpf mro ssoni h otmlf-adcorner. left-hand bottom the in shown top embryos/total is the phenotypic embryos to of anterior number D–F, with The (A–C, shown (G–I). left are the views to dorsal anterior J–L); Lateral with (J–L). shown ICM are the views indicate arrowheads Red (I,L). p53 eewsntidcdi h C of ICM the in induced not was gene ptpn9a a o peuae nteIMrgo of region ICM the in upregulated not was ) Msddntrvretemrhlg,or morphology, the reverse not did -MOs RAprilyrsudpiiieerythropoiesis primitive rescued partially mRNA ptpn9a (A,B) mrhn mro F.I n ,black F, and D In (F). embryos -morphant PTPN9 ptpn9a ptpn9a opat dt o hw) To shown). not (data morphants nsitu in Mswr netdinto injected were -MOs opat(O B n a and (B) (MO) morphant lopasasmlrrl in role similar a plays also ptpn9a AC rgtil mgsof images Brightfield (A–C) yrdzto eeldthat revealed hybridization o Daiiiestaining -Dianisidine eltsebysof embryos depletes ptpn9a gata1 PTPN9 PTPN9 gata1 ptpn9a ptpn9a ptpn9a ptpn9a opat that morphants rbsin probes xrsinin expression RAa 15 at mRNA mRNA -morphant morphants -morphant morphants nsitu in PTPN9 ptpn9a ptpn9a 2763 p53 -

Journal of Cell Science PTPN9 apoptosis. and proliferation cell erythroid that human for suggest material and on observations (supplementary These S3A–F) effect S3G). proliferation Fig. Fig. no cell material had K562 – (supplementary hemin-induced (shN9) apoptosis siRNA cell PTPN9 515 K562 residue or cysteine serine, of mutation to a carried which (N9CS), PTPN9 ARTICLE RESEARCH 2764 genes, erythroid human of of Co-injection S2Y,Z). expression Fig. controls further diminished analysis in including the PCR in that real-time hpf confirmed with Quantitative 48 compared to 4B,D,F,H,J). (Fig. 26 4A,C,E,G,I) from (Fig. diminished morphants gradually of expression genes The erythrocytes, both hematopoiesis. of primitive differentiation during respectively), terminal pattern the expression of the b markers investigate to two chose of We maturation. cell that hypothesized then We e1-globin srqie o rtri elmaturation cell erythroid for required is b and e1-globin b e2-globin and ptpn9a b as nw as known (also e2-globin ih lyarl nerythroid in role a play might PTPN9 splmnaymaterial (supplementary ptpn9a RA(0p per pg (80 mRNA hbbe1 sntrequired not is and ptpn9a hbbe2 , mro rzebrafish or embryo) h xrsinof expression the ari osrc emdSN- a h otptn short that potent revealed material PCR most real-time a (supplementary quantitative constructs the Furthermore, to S4B). RNAi was Fig. led three ShN9-3 short among also The termed hairpin 4M). (shN9), construct (Fig. erythroids hairpin of constructs benzidine-positive of interfering percentage reduction RNA the specific reduced of knockdownof Consistently, form significantly 4M). dominant-negative (Fig. erythroids benzidine-positive a (N9CS) by function that PTPN9 found PTPN9 we of controls, the vector inhibition in with Compared erythrocytes hemin. mature of presence into progenitors hematopoietic of K562 accumulation the in resulting cells. blocked, erythroid immature is cells erythroid progenitors hematopoietic into of differentiation conclude the whereas We initiated, S1L–N). in Fig. material that supplementary 4K,L; (Fig. ete sdtedfeetainsse nodrt induce to order in system differentiation the used then We PTPN9 ora fCl cec 21)17 7127 doi:10.1242/jcs.145367 2761–2770 127, (2014) Science Cell of Journal ptpn9a ysal silencing stably by , kokonebyspiiiehmtpissis hematopoiesis primitive embryos -knockdown b e1-globin ptpn9a i.4 ncdw of Knockdown 4. Fig. AFKL or (A–F,K,L) with performed was hybridization maturation. cell erythroid K.Ltrlvesaesonwt neirt the indicate to arrowheads anterior Red with (A–L). shown left controls are with views compared Lateral (L), (K). hpf 48 at embryos rescued partially human in of (J) Overexpression hpf (G,I). 48 and (H) hpf ptpn9a 36 at abolished almost (A,C,E). in (F) controls hpf with 48 compared at and (D) hpf 36 diminished substantially and (B) hpf reduced the Note (B,D,F,H,J,L). and (A,C,E,G,I,K) oiieor positive iePRaaye h xrsinof expression the analyzed real- PCR Quantitative time (N–P) independent shown. three are from experiments data and of deviation, means standard the groups. the shN9 represent and bars N9CS Error both in cells reduced h. benzidine-positive were 48 of for percentages hemin The with (M) induced were transfected cells or K562 infected The (shN9). construct or a (Scr) either construct with scrambled transfected were or mutant (N9CS), dominant-negative PTPN9 a or empty (Vec) an vector either containing infected retroviruses were with cells K562 Human (M–P) embryos/total embryos. phenotypic left-hand the bottom indicate the corners in numbers The (D,H). 52clsatrhmnidcinfr4 .Relative h. 48 c for induction hemin after cells K562 CD235a omlzdto normalized experiments independent the three are from Measurements means groups. shN9 and N9CS goi,CD235a -globin, RA(0 gpreby)rescued embryo) per pg (100 mRNA opat,cmae ihcontrols with compared morphants, and b in e2-globin 6 b CD71 e2-globin .. * s.d., GAPDH ptpn9a PTPN9 b and ptpn9a e1-globin P nifce rtransfected or infected in pstv rtrisa 6hpf 36 at erythroids -positive , eerdcdi both in reduced were , M-netdembryos -MO-injected CD71 0.05. PTPN9 GJ ncontrols in (G–J) nK6 el with cells K562 in morphants RNA in xrsinlevels, expression PTPN9 ptpn9a ptpn9a nefrswith interferes b PTPN9 b nsitu in e1-globin b e2-globin e1-globin b b e1-globin RNAi e1-globin morphants, c morphant -globin, mRNA t26 at was - at

Journal of Cell Science 8hf(i.5) ycontrast, By 5A). (Fig. hpf 18 ptpn9a McPP9 no23 el.I h rsneo L6and IL-6 of presence the In cells. PTPN9 Myc-tagged 293T with FLAG-tagged Y705F) co-transfected into was mutation (Myc–PTPN9) or 2006) the al., (with et STAT3 (Rong STAT3 wild-type mutant direct dominant-negative FLAG-tagged possible a STAT3, embryos zebrafish suggest in cells. data PTPN9 K562 human by and These STAT3 or of S4B). S4C) dephosphorylation Fig. Fig. material material supplementary 5B; with transfection (Fig. PTPN9 dominant-negative N9CS the mutant of also overexpression was phosphorylation after STAT3 increased cells, K562 material human (supplementary In S4A). zebrafish Fig. in Stat3 of phosphorylation the also might Ptpn9a found that We hematopoiesis. that zebrafish hypothesized during Stat3 dephosphorylate we hematopoiesis, ope,wihpeet idn fGT1adZP8 to ZBP-89 and GATA1 of binding prevents STAT3–GATA1–ZBP-89 which the of complex, formation of we and 2009), knockdown phosphorylation al., et that Yao 2002; al., assumed et a (Foley maturation cell with erythroid a Fig. consistent material (supplementary is is 2008) murine al., there which S4G). et in (Woo that ZBP-89 cells, report previous and STAT3 and (MEL) GATA1 active S4E,F) erythroleukemia between with interaction interacted Fig. physical ZBP-89 material phosphorylation. that STAT3 (supplementary showed of that also activation indicating We requires STAT3, Fig. interaction dominant-negative, with material the not interacted supplementary FLAG-tagged but with STAT3 5D; wild-type, interacted (Fig. preferentially endogenous differentiation. Myc–GATA1 ZBP-89 S4H). that erythroid and regulate found GATA1 to form we might complex Indeed, ZBP-89 and multiprotein studies, these GATA1 on a STAT3, Based differentiation 2008). al., that al., et hypothesized et erythroid Ohneda Woo we 2006; 2011; and al., al., et et Woo genes (Li 2009; regulate zebrafish globin and in hematopoiesis GATA1 the during with of interact to expression reported the previously Furthermore, 2009). been the al., et to Yao 1998; al., Kru leads et Kirito complex 2002; al., of STAT3 et STAT3–GATA1 expression the the wild-type of inhibition of shown to have formation studies bound Previous that S4D). preferentially Fig. material and associated (supplementary physically STAT3 Myc–PTPN9 with overexpressed against and PTPN9 antibody endogenous 5C) both an (Fig. that revealed using experiments by These was FLAG. complex immunoprecipitation protein by the transfection, isolated after receptor IL-6 soluble nrae TT hshrlto ykokonof knockdown by phosphorylation STAT3 Increased encoding genes specifically, GYPA erythroid (here, the globin of expression hemin-induced ARTICLE RESEARCH u rvoswr a hw htPP9directly PTPN9 that shown 2012). al., has et (Su cells cancer Because work breast in STAT3 dephosphorylates previous complex STAT3–GATA1–ZBP-89 Our the of formation enhances hs aasgetta TN srqie o rtri cell erythroid for required is PTPN9 together, that Taken maturation. 4N–P). suggest (Fig. data shN9 or these N9CS expressed that cells odmntaeapyia neato ewe TN and PTPN9 between interaction physical a demonstrate To wn oteihbtr feto hshrltdSA3on STAT3 phosphorylated of effect inhibitory the to Owing pe-yezn igrtasrpinfco B-9has ZBP-89 factor transcription finger zinc ¨ppel-type ptpn9a and ) erae,Sa3popoyaini erfs mro at embryos zebrafish in phosphorylation Stat3 decreased, STAT3 CD71 ncdw nrae,weesoeepeso of overexpression whereas increased, knockdown PTPN9 as nw as known (also ly nipratrl nmammalian in role important an plays Nicntut sN)(supplementary (shN9) constructs RNAi HBG2 ptpn9b c PTPN9 goi Eo ta. 05 Foley 2005; al., et (Ezoe -globin TFRC ), ncdw a oefc on effect no had knockdown CD235a ih nraeSTAT3 increase might )wasinhibitedinK562 as nw as known (also PTPN9 c - 52clsdcesdtecmlxfrainbtsihl reduced slightly in but formation knockdown complex the ZBP-89 the on decreased and cells ZBP-89 effect S4K), K562 K562 and Fig. no STAT3 material in had total (supplementary but STAT3, knockdown phosphorylated formation of GATA1 complex Fig. expression the S4I,J,M,N). material reduced supplementary Fig. cells 5A; STAT3 and Fig. not human but 2G,H; S2W–X in GATA1 (Fig. shN9, of ZBP-89 and expression N9CS and the using decreased by cells K562 function, its by of In abrogation knockdown of 5G,H). (Fig. PTPN9 binding STAT3 addition, dominant-negative rescued the of fully co-transfection decreased was genes by which of shN9 assays, target controls inhibition immunoprecipitation or its that chromatin in N9CS showed to than further either GATA1 phosphorylated we complexes by more predicted, PTPN9 ZBP-89 As of 5F). and shN9 (Fig. formation using GATA1 we by the results, STAT3, STAT3, caused these phosphorylated with knockdown, increasing Consistent that 5E). found and (Fig. decreasing GATA1 complex STAT3, contrast, PTPN9 and phosphorylated ZBP-89 the By wild-type GATA1 of of 5E). formation more overexpression (Fig. prevented dominant- of by complex STAT3 recruitment of the phosphorylated increased the that into to overexpression found ZBP-89 led We by N9CS, genes. negative phosphorylation, target their STAT3 of promoters the fa niioypoencmlxcmrsn phosphorylated comprising formation complex ZBP-89. the and for protein GATA1 crucial inhibitory STAT3, is data an PTPN9, which by These of phosphorylation, regulated S4L). STAT3 negatively that Fig. is notion the and material support STAT3 (supplementary strongly phosphorylated on STAT3 effect no total had and levels GATA1 ece h ubro ezdn-oiieeyhodcells erythroid genes benzidine-positive erythroid of of expression number the and the rescued by of identified as overexpression erythroids, Additionally, 6C,I,L). (Fig. zebrafish rescued dominant-negative of partially in overexpression human levels Remarkably, ZBP-89 or the cells. K562 or manipulating of human GATA1 and by embryos STAT3, maturation phosphorylated cell investigated complex we protein ZBP-89, inhibitory erythroid and the GATA1 STAT3, on phosphorylated hypothesis of our test further To maturation to cell contributes PTPN9 erythroid by STAT3 of Dephosphorylation fmtrto pntetetwt ei o 8h 48 cell erythroid for in we role hemin crucial together, a with STAT3. Taken dephosphorylating plays by S3H,J). the PTPN9 treatment maturation that Fig. on upon conclude effect induction material STAT3 the no (supplementary maturation after decreasing had erythroids that of alone benzidine-positive PTPN9 of STAT3 found wild-type formation material either dominant-negative we (supplementary of upon or overexpression h However, 48 by erythroids phosphorylation for S3J). benzidine-positive hemin Fig. the with active material had constitutively treatment (supplementary decreased of alone proliferation overexpression STAT3 STAT3 cell Consistently, K562 S3I). active Fig. on constitutively effect or no STAT3 negative found CD71 consistently we dominant-negative h, 48 of shN9, for or overexpression N9CS hemin expressing with that cells treatment K562 In upon 6F). (Fig. morphants gata1 ora fCl cec 21)17 7127 doi:10.1242/jcs.145367 2761–2770 127, (2014) Science Cell of Journal Fg MP,weesoeepeso fdominant- of overexpression whereas 6M–P), (Fig. 5 gprembryo), per pg (50 b e1-globin o xrsinin expression c Daiiiesann,in staining, -Dianisidine -globin STAT3 zbp-89 ptpn9a and c M nzbaih or zebrafish, in -MO 10p e embryo) per pg (100 5 gprembryo) per pg (50 -globin CD235a ptpn9a STAT3 , gata1 CD235a morphants yusing by partially rescued ptpn9a 2765 and

Journal of Cell Science yGT1poenwt rwtottetetwt ei.Tedt r h en rmtreidpnetexperiments independent three from means the with are transfected data or The (N9CS) hemin. PTPN9 with mutant treatment of with occupancy without promoter infected the or evaluate been to with used had protein was GATA1 that PCR Quantitative control. by cells IgG K562 GATA1 or against antibody in an genes using performed target were assays its ChIP to GATA1 of binding the rescued genes target its to GATA1 in reduced but activity. embryos (MO) binding GATA1 morphant the reduces and ZBP-89 and GATA1 EERHARTICLE RESEARCH 2766 immunoprecipitation for prepared were lysates cell before h 6 for IL-6 using by induced or (N9CS) were PTPN9 dominant-negative mutant cells either K562 of Overexpression cells. (G,H) STAT3. against K562 antibody gr in (Vec) (Scr) control construct vector scrambled the a with with compared complex, protein phosphorylated-STAT3–GATA1–ZBP-89 the the and STAT3 complex, dominant-neg of phosphorylated (F) formation decreased the (N9WT) the of Overexpression promoted PTPN9 wild-type (E) and STAT3 phosphorylated cells. of overexpression increased K562 whereas cells in K562 ZBP-89 STAT3 in against and antibody GATA1 (N9CS) an with PTPN9 interacts using mutant physically physi by immunoprecipitated STAT3 complex, endogenous PTPN9 protein endogenous STAT3 that endogenous suggesting that (Vec) The suggesting ZBP-89, vector (D) PTPN9, IP. and empty GATA1 contained control STAT3, an IgG, against cells. with antibody K562 infected an in cells STAT3 using with or by interacts (WT) (IP) immunoprecipitated cells complex, uninfected protein STAT3 with cel compared The K562 (N9CS), human (C) PTPN9C515S in mutant increased dominant-negative was blotting, the western with by infected shown STAT3, as been phosphorylated Endogenous (B) unchanged. relatively were Stat3 of amounts total by STAT3 phosphorylated Increasing 5. Fig. PTPN9 ncdw sN)as nrae hshrltdSA3adpooe h omto ftephosphorylated-STAT3–GATA1–ZBP-89 the of compared formation complex, the promoted and STAT3 phosphorylated increased also (shN9) knockdown c -globin and ptpn9a CD235a mN-netdebys(RA,cmae ihwl-ye(T mro t1 p,a hw ywsenbotn.The blotting. western by shown as hpf, 18 at embryos (WT) wild-type with compared (mRNA), embryos -mRNA-injected oprdwt etr(e)o cabe Sr otos vrxrsino oiatngtv TT (STAT3DN) STAT3 dominant-negative of Overexpression controls. (Scr) scrambled or (Vec) vector with compared , PTPN9 ncdw tmltstefraino nihbtr ope opiigpopoyae STAT3, phosphorylated comprising complex inhibitory an of formation the stimulates knockdown A h hshrlto ptt)sau fedgnu tt rti a lvtdin elevated was protein Stat3 endogenous of status (pStat3) phosphorylation The (A) ora fCl cec 21)17 7127 doi:10.1242/jcs.145367 2761–2770 127, (2014) Science Cell of Journal PTPN9 ncdw sN)i 52clsrdcdtebnigof binding the reduced cells K562 in (shN9) knockdown 6 .. * s.d.; P , .5 s o significant. not ns, 0.05; PTPN9 c hN (shN9). shRNA -globin and ptpn9a ihan with contained , sta had that ls . CD235a oup. cally ative -

Journal of Cell Science EERHARTICLE RESEARCH erfs mro n 52clsta akdfntoa PTPN9. functional that lacked in note that cells We maturation a K562 to and cell due embryos either mainly zebrafish erythroid in was in but defects protein apoptosis by deficiency or inhibitory caused proliferation not cell an was erythroid ZBP- dephosphorylating hypoplasia and of Erythrocyte GATA1 through 89. STAT3, formation phosphorylated acts comprising the complex PTPN9 preventing that STAT3, and into cells progenitors K562 hematopoietic erythroids of human differentiation and that the embryos regulating demonstrated by zebrafish have both in we erythropoiesis study, this In DISCUSSION ua nohla ooyfrigcls(un ta. 01 Xu human ptpn9a 2011; Because al., 2003). or et al., cells (Huang et cells stem colony-forming embryonic endothelial human human from derived colonies erythroid mrhn hntpsi erfs,w ugs htthe that suggest we zebrafish, in phenotypes -morphant PTPN9 ly nesnilrl ntefrainof formation the in role essential an plays PTPN9 a bet atal rescue partially to able was PTPN9 ptpn9a- ucin in functions knockdown idn tblt.A rsn,i ean ob eemndwhether GATA1 determined be of to alteration remains it the present, to At stability. attributed binding GATA1 been suppress al., to has et STAT3 have Yao transactivation phosphorylated 2005; of studies al., ability gene et The (Ezoe 2009). Previous through erythroid interaction GATA1 protein–protein ZBP-89. inhibits direct of a STAT3 and phosphorylated inhibition that GATA1 elucidated the by erythroid through expression impaired development then and cell myeloid phosphorylation of STAT3 target regulation increased downstream the in other involved that are maturation. cell PTPN9 suggesting of dominant-negative shown), proteins human in not decreased of was (data dose cells moderate myeloid a of number We the ptpn9a maturation. that erythroid found in functions also conserved have genes two ehnsial,w eosrtdta ncdw of knockdown that demonstrated we Mechanistically, ora fCl cec 21)17 7127 doi:10.1242/jcs.145367 2761–2770 127, (2014) Science Cell of Journal opat u ol o ersudb vrxrsinof overexpression by rescued be not could but morphants a enc-netdwith co-injected been had in rescued (A–C,G–L) cells. K562 knockdown and embryos morphant (C), in development erythroid M and (M) erythroids benzidine-positive rescued of Overexpression (M–P) embryos. phenotypic embryos/total represent corner left the bottom in numbers The staining. by Dianisidine revealed as (D), wild-type controls with compared (F), morphants in erythrocytes primitive addition, In of hpf. overexpression 48 at (A,G,J) (WT) controls wild-type with compared (L), negative dominant-negative or i.6 Overexpressing 6. Fig. etr(e)o cabe Sr controls. (Scr) scrambled with or compared (Vec) h, vector 48 for hemin with with PTPN9 transfected or mutant (N9CS) either PTPN9 with infected been had CD71 * three of experiments means independent the represent data The P , 0.05. zbp-89 P xrsini 52clsthat cells K562 in expression (P) hN sN)atrinduction after (shN9) shRNA c STAT3 -globin ptpn9a b RA()o dominant- or (I) mRNA e1-globin ( (N), STAT3DN gata1 opat M)that (MO) morphants CD235a a partially was STAT3 STAT3 ptpn9a RArescued mRNA 6 gata1 PTPN9 STAT3DN gata1 s.d., ptpn9a mRNA ) o - O and (O) , rescues PTPN9 mRNA zbp-89 mRNA - 2767 -

Journal of Cell Science twr ta. 00.C4,seiial xrse in expressed specifically zebrafish, In lymphocytes. B CD45, and receptor 2007;T antigen 2010). al., B-cell and et signaling. T-cell al., (Jopling regulates developing cells, cord hematopoietic et the spinal in and Stewart system expression nervous highest central the with embryogenesis rlfrto falhmtpitclnae.Zebrafish lineages. hematopoietic all of proliferation oksoe hthprpopoyae TT impaired In cells. 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Noonan human cause Shp-2 cd45 ak usto aorta- of subset a marks eei mroi lethal embryonic is gene b Stat3-loxP Shp-1 -eedn manner 3-dependent Gp130 epo h- () shp-1 ipa high display D ieand mice / or D jak2a mice, soito o sesetadAceiaino aoaoyAia Care. Animal Laboratory of Peking Accreditation the and the Assessment by of for accredited zebrafish guidelines Association Committee Use the transgenic and with Care and accordance Animal Institutional AB in University handled fin, and long raised were Tupfel Hong Technology, Wild-type and Science China). of University Kong, Kong (Hong Wen Zilong by Tg( CA). Angeles, Los Tg( Angeles, Los California of (University ua eobnn L6(0 gm,RDSses n human and Systems) R&D ng/ml, (100 IL-6 cells. recombinant K562 in differentiation Human erythroid induce to used was Sigma-Aldrich) was nl kit embryos. 2.3 two-cell-stage mMachine of to volume mMessage one- a experiments, into the synthesized microinjection injected using the were all constructs mRNAs In (Ambion). pXT7 Capped linearized S1. from Table material supplementary Plasmid synthesis. mRNA for zebrafish template of probes mRNA a of and as generation the probe used for clones antisense was for which pXT7 vector, and SEC14-deleted Blunt synthesis. pEASY vector into ifrn usrtsi re orglt rmtv n definitive and primitive with regulate interact to might order hematopoiesis. 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(Sugiyama al., previously described as generated, et were cycle cell the of phases Tg( maintenance Fish METHODS AND MATERIALS 52clsadMLclswr utrdi 60mdu.Hmn(50 Hemin medium. 1640 in cultured were cells MEL and cells K562 and 33342 staining Hoechst benzidine transfection, infection, culture, Cell the using instructions. performed manufacturer’s described was TUNEL as 2011). kit Situ al., instructions, et labeling (Danilova manufacturer’s RNA 2008). previously al., the DIG et (Xiong the to previously using according synthesized (Roche) were probes Antisense Whole-mount water. RNase-free in stocks mM 2 as purchased of suspended sequences were and The oligonucleotides Tools Gene antisense from morpholino following microinjection The and synthesis mRNA Morpholinos, (zebrafish synthesis mRNA zbp-89 for Constructs laboratory. zebrafish our of from probes generating for clones gata1 plasmid Other China). Beijing, lmo2 UE assay TUNEL osrcshv endsrbdpeiul S ta. 2012). shN9) al., mutation et PTPN9; (Su double human previously against described the been (shRNA have with shPTPN9 constructs and STAT3 N663C) active STAT3), A661C dominant-negative human (constitutively (a of 2011). STAT3Y705F STAT3CA mutant al., STAT3, dominant-negative et N9CS), (a (Woo PTPN9; PTPN9C515S previously ZBP-89 PTPN9, described human as Human and designed (shGATA1) al., were GATA1 et (shZBP-89) human (Li MA) against Cambridge, shRNA University, 2006). 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Journal of Cell Science endsrbdpeiul S ta. 2012). al., et has (Su PTPN9 previously against (Santa described antibody been polyclonal antibodies rabbit (Sigma-Aldrich), secondary The Biotechnology). HRP-conjugated Cruz Myc and Signaling (Abcam), Stat5 Sigma-Aldrich), secondary antibodies (Cell light-chain-specific (HRP)-conjugated phosphorylated Stat5 (M2, peroxidase horseradish Technology), Biotechnology), FLAG Biotechnology), Signaling Cruz Cell Cruz Technology), Cell (D52H6, D47E7, Santa Santa (Tyr694, GATA1 (H-7, (S-15, Technology), ZBP-89 Technology), ZBP-89 Signaling Signaling Technology), Cell (Cell Signaling D3A7) (124H6, (Tyr705, Stat3 Santa Stat3 Stat3 (C-20, phosphorylated Biotechnology), phosphorylated STAT3 zebrafish Laboratories), study: Biological this Cruz indicated and in the (Medical used (Tyr708) against also antibodies were for following proteins used The were immunoprecipitation. (Sigma-Aldrich) beads FLAG-M2-antibody-conjugated el.K6 el eeifce ihtertoiue pMSCV-vector, retroviruses the (1 with cells K562 infected pMSCV-PTPN9C515S. were K562 or stimulate pMSCV-PTPN9WT cells to used K562 were Systems) cells. R&D U/ml, (50 EPO recombinant ARTICLE RESEARCH oeesnilraet n efre oezbaiheprmns K.M. experiments; zebrafish contributed some X.Z. performed and the and K.H. of reagents Q.W., some essential N.C., performed some L.L., and H.G., writing reagents X.W., to experiments; provided contributed biochemical Z.C. and and experiments, F.S. the manuscript; of the most performed and designed Y.B. contributions Author interests. competing no declare authors The interests Competing assistance. Jing-Wei technical of and members discussion and helpful for manuscript; for China) the laboratory of Hangzhou, Xiong’s University, Zoology, reading (Zhejiang of and Bruce (Institute comments C. Liu critical Iain Feng and China), China) Beijing, Qing- Zoology, Beijing, OK), City, of Oklahoma (Institute Oklahoma, Sun of Yuan (University Zhao J. Zhizhuang thank We Acknowledgements Student’s effect. A significant times. three least at repeated were experiments All analysis Statistical for used using and by harvested performed were were sodium Pierce 2006). assays al., cells (ChIP) et 0.25% immunoprecipitation PMSF). (Hu 293T Chromatin mM previously and described 7.4, 1 as EDTA, cells reactions, mM pH immunoprecipitation MEL 1 NaCl, cells, Tris-HCl mM K562 150 and mM NP-40, protease (50 1% with deoxycholate, supplemented inhibitors been radio-immunoprecipitation had modified that phosphatase buffer in lysis (RIPA) lysed assay were embryos Zebrafish and immunoprecipitation immunoprecipitation chromatin blot, Western was from cDNA or First-strand stages, (Invitrogen). different 2 Trizol at from embryos using synthesized from cells, isolated harvested was RNA Total RT-PCR real-time and Semi-quantitative eeeetooae t20Vad975 and V 280 at electroporated were tiighsbe eotdpeiul Coe ta. 1974). al., Benzidine et cells. (Cooper previously 293T reported stimulate been to has used staining receptor were Systems) IL-6 R&D and ng/ml, IL-6 (100 recombinant Human instructions. manufacturer’s ytm(i-a)adaSB rmxE a i Tkr) Primer (Takara). kit Taq S1. detection Table Ex material PCR Premix supplementary real-time in SYBR listed iQ5 a are an sequences and Quantitative using (Bio-Rad) performed protocol. system was manufacturer’s PCR the real-time to according Technologies) a efre oeaut h infcneo ifrne between differences of groups. significance control the and evaluate to experimental performed was a efre sn Lipofectamine using performed transfection transient was and 1 medium Eagle’s modified and Dulbecco’s in G418 cultured mg/ml selected 0.6 were cell-lines with Stable (Bio-Rad). II shPTPN9 Gene-Pulser using and constructs) shZBP-89 shGATA1, STAT3CA, (STAT3Y705F, plasmid TM grs hPktacrigt h auatrrsprotocol. manufacturer’s the to according kit ChIP agarose m fttlRAb ueSrp IR (Life RT II SuperScript by RNA total of g b atn(elSgaigTechnology), Signaling (Cell -actin m /lprmcn E 9Tclswere cells 293T HEK puromycin. g/ml TM P 00(nirgn codn othe to according (Invitrogen) 2000 , m ih20 with F .5idctsastatistically a indicates 0.05 m fnonlinearized of g 6 10 7 cells) t -test alwy .L,Wnet .A,Tis,C,Tis,B n o,L I. L. Zon, and B. Thisse, C., Thisse, A., R. Wingert, L., J. Galloway, aioa . aaoo .M n i,S. Lin, and M. K. Sakamoto, N., Danilova, I. Godin, and F. A. Dieterlen-Lievre, R. A., Rifkind, and Cumano, A. P. Marks, N., L. Cantor, J., Levy, Orth, C., A., M. P. Cooper, Mak, S., Hedrick, S., Callaway, Y., Wang, H., S. Koo, Y., C. Cho, A. N. Speck, and E. Dzierzak, M., B. Zeigler, T., Yokomizo, J., M. Chen, al . lrs .V,Som . rse,K n rse,P. Crosier, and K. Crosier, T., Storm, V., M. Flores, C., Hall, K. R. Patient, and H. T. Rabbitts, Y., Yamada, M., Gering, and S. B. Baliga, S., Critz, A., Yoshimura, M., F., S. Mizuki, Ofori-Acquah, A., J., H. Foley, Ishikawa, Y., Satoh, K., Gale, I., Matsumura, S., Ezoe, I. L. Zon, and L. J. Jong, de iio . cia . aaou . aaia . iao . ir,Y and Y. Miura, T., Hirano, K., Nakajima, M., Takatoku, M., Uchida, K., Kirito, J. Hertog, den and D. Geemen, van C., Jopling, etad .Y,Km .D,Tn,S n rvr D. Traver, and S. Teng, D., A. Kim, Y., J. Bertrand, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.145367/-/DC1 online available material Supplementary material Supplementary 31221002]. and 31271549 numbers National [grant the China of and of Program 2012CB944501] Foundation Research and Science Basic 2010CB529503 National numbers the from [grant grants China by supported was work This Funding un,X,Gcwn,E,VnHne,B,Ceg . ikl,H .adWitte, and K. H. Mikkola, D., Cheng, B., Handel, Van E., Gschweng, X., Huang, W. Wang, and H. A. Bird, Tang, X., and Nan, K., J. Hu, Z. Zhao, L., H. Ji, B., Samten, Q., Hao, hswr,dsge xeiet,aaye aaadwoetemanuscript. the wrote and data analyzed conceived experiments, J-W.X. data; designed analyzed work, and this experiments designed work, this conceived ae-yisa .L,Hrfed .A,Foe,M . otehat .H,Vitas, H., J. Postlethwait, V., M. Flores, A., J. Horsfield, L., M. Kalev-Zylinska, M. Ernst, and D. Grail, M., Najdovska, W., A. M. Roberts, Ernst, J., and B. R. Jenkins, A. Dunn, D., Grail, W., A. Roberts, C., Quilici, J., B. K., Jenkins, Saito, L., Musumeci, V., Cherepanov, S., Williams, N., Bottini, H., Huynh, uaini erfs ed oheaooei n eaoi defects. metabolic and haematopoietic to intraembryonic leads Haematol. caudal zebrafish J. to in restricted mutation is mouse, in in circulation splanchnopleura. cells before precursor erythroid probed of growth colonial on erythropoietin vitro. of effect The signaling. insulin hepatic al. of et antagonist Metab. M. an Cell Montminy, as E., PTP-MEG2 Saez, phosphatase C., tyrosine not E. but Peters, P., transition A. cell haematopoietic to endothelial the for thereafter. required is Runx1 noedteilclsi h bec fGata1. of absence the in differentiate cells 6199. which endothelial haemangioblasts zebrafish into into in mesoderm non-axial myelopoiesis convert to Scl/Tal1 erythropoiesis converts gata2 not embryos. but gata1 of cells. S. erythroid B. Pace, activity. 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