whether signals mediated via growth factor receptors ( gfrs ) might influence lineage fate in normal multipotent progenitors ( mpps ) and stem cells remains unclear and disputed . new insights into this process have recently been gained through studies in  vitro ( rieger et  al . , 2009 ) ; however , whether gfr signaling instructs lineage specification in  vivo remains a key unresolved issue ( enver and jacobsen , 2009 ) . studying the impact of gain - of - function mutations is an alternative approach to determine whether and how gfrs might instruct lineage fate in  vivo . indeed , activating mutations of gfrs and downstream signaling pathways are common events in cancer , particularly in hematopoietic malignancies such as acute myeloid leukemia ( aml ) , and often show striking associations with distinct clinical and cell - lineage phenotypes ( croce , 2008 ) . in some cases , this might simply reflect the fact that such mutations primarily target a specific cell lineage . however , more intriguingly , if a mutation targets a primitive multipotent cell , it might instruct lineage - fate decisions . as recent investigations of human aml have suggested that the propagating cell might frequently represent the counterpart of normal mpps ( goardon et  al . , 2011 ) , it is possible but still unclear to what degree gfr signaling mutations might also influence lineage specification in multipotent cells . a good example of aberrant gfr signaling associated with specific leukemia phenotypes relates to the fms - like tyrosine kinase 3 receptor ( flt3 ) , which is expressed in the majority of cases of aml and acute lymphoblastic leukemia ( all ) ( stirewalt and radich , 2003 ) . constitutively activating internal tandem duplications ( itds ) within the juxtamembrane domain of flt3 occur in 25% of cases of aml , conferring an adverse prognosis ( stirewalt and radich , 2003 ) . however , despite the high frequency of flt3 expression in all and the key role of flt3 in early lymphoid development ( sitnicka et  al . , 2002 ) , including high - level flt3 expression in lymphoid - primed mpps ( lmpps ) with combined lymphoid and myeloid potential ( adolfsson et  al . , 2005 ) , flt3-itds are rare in cases of all , occurring in < 1% in larger series ( leow et  al . , 2011 ) . as is the case for most leukemic mutations , it is unclear how  often flt3-itds are a true initiating event in myeloid malignancies . indeed , a number of lines of evidence support the notion that flt3-itds are often acquired secondarily to an initiating clonogenic event ( gale et  al . , 2008 ; jan et  al . , 2012 ) , although when involved in chronic myelomonocytic leukemia ( cmml ) , they may indeed be the initiating mutation ( lee et  al . , 2007 ) nevertheless , in line with the normal expression pattern of flt3 ( adolfsson et  al . , 2005 ) , results from studies in patients are compatible with the notion that flt3-itds occur within the human mpp compartment ( levis et  al . , 2005 ) and are an essential driver mutation within the founding leukemic clone ( ding et  al . , 2012 ; smith et  al . , thus , even though flt3-itds might occur secondarily to an initiating clonogenic event in many cases , these mutations occur in multipotent cells ( levis et  al . , 2005 ) and flt3-itd gfr signaling appears to be an essential requirement for leukemia propagation ( smith et  al . , 2012 ) . it is possible , therefore , that flt3-itds may act to dictate the lineage fate and phenotype of the resulting leukemia . compatible with a role of flt3-itd signaling in lineage determination , two different knockin mouse models of flt3-itd have been reported to develop a myeloproliferative phenotype exclusively ( lee et  al . . however , although it was recently suggested that flt3-itds deplete hematopoietic stem cells ( hscs ) ( chu et  al . , 2012 ) , the key progenitor population that propagates flt3-itd - induced myeloid disease , as well as the cellular and molecular bases of their myeloid lineage bias , remains unclear . using a mouse knockin model of the flt3-itd mutation , we investigated the cellular and molecular mechanisms by which constitutive gfr signaling might subvert lineage specification in mpps and alter the cell fate of early lymphoid progenitors , in order to explain the myeloid bias of the resulting leukemias . to definitively determine whether physiologically expressed flt3-itd impacts the establishment of myeloid versus lymphoid leukemia development , we crossed flt3 with runx1 ( growney et  al . , 2005 ) and mx1-cre mice to induce runx1 deletion in mpps . importantly , runx1 loss - of - function mutation is associated with both lymphoid and myeloid leukemia ( grossmann et  al . , 2011 ; schnittger et  al . , unexpectedly , even without poly i : c induction , flt3runx1mx1-cre mice developed a high - penetrance , short - latency acute leukemia ( figure  1a ) characterized by marked leukocytosis , anemia , and thrombocytopenia ( figures s1a  s1c ) and hepatosplenomegaly . peripheral blood ( pb ) and bone marrow ( bm ) morphology resembled aml in all diseased mice ( figures 1b1d ) , and myeloid lineage was confirmed by flow cytometry ( figures 1e and s1d ) . leukemias showed deletion of the runx1 dna - binding domain ( figure  s1e ) . the uniform myeloid - lineage leukemias in flt3runx1mx1-cre mice demonstrate a key role for flt3-itd signaling in introducing a myeloid - lineage bias during leukemogenesis . as shown previously ( lee et  al . , 2007 ) , the multipotent linsca1c - kit ( lsk ) compartment was expanded in flt3 mice ( figure  2a ) . we applied cd150 and cd48 to determine the nature of the expanded cells within the lsk compartment ( kiel et  al . , 2005 ) , as flt3-itd is not  detectable at the cell surface ( figure  s2a ) . notably , the expansion of  lsks was wholly attributable to a marked expansion of lskcd15048 mpps in 8- to 10-week - old flt3 mice ( figures 2b and 2c ) , in line with the recent observation that flt3-itd may suppress the hsc compartment ( chu et  al . , 2012 ) . heterozygous ( flt3 ) mice had an intermediate phenotype between flt3 and flt3 mice ( figures s2b and s2c ) . high - level flt3 expression in the normal lsk compartment defines lmpps that lack self - renewal and megakaryocytic ( mk ) and erythroid ( e ) potential but sustain lymphomyeloid capability  ( adolfsson et  al . , 2005 ) , a progenitor that is also implicated  in human aml ( goardon et  al . , 2011 ) . because flt3 lmpps reside almost exclusively in the lskcd15048 compartment ( figure  s2d ) , we explored whether the expanded lskcd15048 cells in flt3-itd mice had lmpp - like characteristics . as expected for lmpps ( mnsson et  al . , 2007 ) , hsc- and mke - affiliated gene expression was downregulated in flt3 lskcd15048 mpps ( figures 2d , 2e , and s2e ) , and in line with the molecular data they possessed little or no mk potential in  vitro ( figure  2f ) . in contrast , myeloid - affiliated gene expression was upregulated in flt3 lskcd15048 cells ( figure  2 g ) , paralleled by high granulocyte - macrophage ( gm ) potential in  vitro ( figure  2h ) . early lymphoid transcriptional programs were downregulated in both flt3 and flt3 lskcd15048 mpps ( figures 2i and 2j ) , paralleled by severely reduced b cell potential in  vitro ( figure  2k ) . importantly , the lymphoid transcriptional program was already suppressed in flt3 lskcd15048 mpps in embryonic day 15 ( e15 ) fetal liver ( fl ) ( figure  s2f ) , at which time the phenotype and number of lskcd15048 mpps were unaffected ( figure  s2 g ) . in keeping with this suppression of early lymphoid programs in fetal mpps , the number of b220cd19 b cells was suppressed in flt3-itd e15 fl ( figure  s2h ) , preceding the emergence of a myeloproliferative phenotype at this early stage of ontogeny ( figure  s2i ) . this supports the notion that lymphoid suppression and myeloid bias in mpps occurs as a cell - intrinsic and direct consequence of flt3-itd signaling . because flt3-itds in humans often occur secondarily to an initiating clonogenic event ( jan et  al . , 2012 ) , it is possible that in such cases it is the other genetic events and not the flt3-itd that influence the leukemic phenotype . in order to address this issue , we used vav - cre mediated recombination to examine the impact of runx1 loss of function on lineage priming in mpps in the absence of flt3-itd . the data obtained demonstrate a significant upregulation of il7r and rag1 expression in mpps with no significant impact on sigh expression ( figure  s2j ) . this finding is in keeping with the high incidence of lymphoid malignancies in mouse models of runx1 mutation ( jacob et  al . , 2010 ; kundu et  al . , 2005 ; putz et  al . , 2006 ) and contrasts markedly with the suppression of lymphoid programs caused by flt3-itds in mpps . this supports the notion that it is gfr signaling through flt3-itd that instructs the uniform myeloid phenotype of leukemias resulting from the collaboration between runx1 deletion and flt3-itd in lmpps . because flt3-itd compromised lymphoid - transcriptional priming in lmpps , which have been implicated as critical thymus - seeding progenitors ( luc et  al . , 2012 ) , we next investigated whether t lymphopoiesis might be suppressed in flt3-itd mice . thymic cellularity was found to be progressively reduced , and the earliest thymic progenitors ( double - negative 1 kit+ [ dn1kit ] ) were almost completely lost in flt3 mice ( figures 3a3c ) . also , subsequent stages of dn2kit and dn3 thymocyte progenitors were severely reduced ( figures s3a and s3b ) . of  relevance , gene and protein expression for the chemokine receptor ccr9 , which is critical for migration of thymus - seeding progenitors from the bm to the thymus ( schwarz et  al . , 2007 ) , was suppressed in flt3 lskcd15048 mpps ( figures 3d , s3c , and s3d ) . moreover , dn1 cells showed large reductions in expression of notch1 and its targets ( hes1 and hes5 ; figure  s3e ) , which are critical for early t  cell development . although expression of some early lymphoid genes ( il2ra , il7r , and gata3 ) was maintained , myeloid genes ( cebpa , sfpi1 , csf1r , csf2r , and csf3r ) were highly upregulated in dn1 thymic progenitors ( figure  s3e ) . in keeping with this myeloid bias , flt3 dn1 progenitors showed low t  cell potential in  vitro ( figure  3e ) . previous studies demonstrated a reduction of mature b cells in  flt3-itd mice ( lee et  al . we investigated whether this might reflect flt3-itd - induced perturbation at the  earliest b cell commitment stages , and in support of this found a notable 11-fold expansion of lincd19cd24aa4.1cd43b220 pre - pro - b cells in flt3 mice ( figures 3f and 3 g ) , whereas pro - b cells , the next stage in b cell development , were severely reduced ( figures 3h and s3f ) . because pre - pro - b cells are known to sustain low - level myeloid potential ( rumfelt et  al . , 2006 ) , we next investigated lymphoid and myeloid gene expression in flt3 pre - pro - b cells . key early lymphoid genes ( cd79a , il7r , sigh , ebf1 , pax5 , rag1 , and rag2 ) were all  suppressed ( figure  3i ) , whereas myeloid - affiliated genes ( cebpa , cebpb , sfpi1 , csf1r , and csf2r ) were upregulated in flt3 pre - pro - b cells ( figure  3j ) . in agreement with this , flt3 pre - pro - b cells had severely reduced b cell potential ( figure  3k ) . these findings demonstrate that flt3-itds markedly impair the earliest stage of both t and b lymphopoiesis and upregulate myeloid gene expression in the earliest b and t lymphoid progenitors , in agreement with the notion that myeloid bias initiates in multipotent lmpps . to further explore the mechanistic basis of flt3-itd - induced myeloid bias of early lymphoid progenitors , we examined pu1 protein expression using a pu1-yfp reporter line . whereas in wild - type ( wt ) mice lin cells showed a bimodal distribution between pu1 and pu1 cells , almost all lin cells in flt3 mice were pu1 ( figure  4a ) . flt3 mice showed a modest increase in pu1 expression in lskcd15048 mpps ( figure  4b ) and markedly enhanced levels of pu1-yfp in pre - pro - b cells ( figure  4c ) . in keeping with the observed increased expression of pu1 , gene set enrichment analysis ( gsea ) demonstrated upregulation of pu1 target genes ( steidl et  al . furthermore , gsea also demonstrated a similar upregulation of pu1 target genes in flt3-itd mutated  lmpp - like  leukemia stem cells in human aml ( goardon et  al . because transition from pre - pro - b cells to subsequent stages of b cell development is associated with downregulation of pu1 expression ( figure  s4b ) , we attempted to rescue the suppressed b cell phenotype in flt3-itd mice through generation of flt3 pu1 mice . notably , whereas pu1 haploinsufficiency did not increase the number of b cells or their progenitors on a wt background , it resulted in a 10.3-fold increase in b cells in flt3 mice ( figure  4e ) . furthermore , pu1 haploinsufficiency led to a 2-fold reduction in the expanded pre - pro - b cell population in flt3-itd mice ( figure  4f ) together with a 13.5-fold rescue of the suppression of pro - b cells ( figure  4 g ) . strikingly , in the presence of pu1 haploinsufficiency , the expansion of mpps was also restored to wt levels ( figure  4h ) together with a significant amelioration of the myeloproliferative phenotype in flt3 mice ( figure  4i ) . stat3 is aberrantly activated by flt3-itd signaling ( schmidt - arras et  al . , 2009 ) and pu1 is a direct target gene of stat3 ( hegde et  al . , 2009 ) . in line with this , flt3 bm showed aberrant activation of stat3 ( figure  s4c ) , and mpps and pre - pro - b cells showed significantly increased expression of stat3 target genes ( figures 4j4l ) . collectively , these data suggest that pu1 upregulation is a key mediator of the flt3-itd - induced myeloid bias and progenitor phenotype . in the studies presented here , we explored whether and how gfr signaling might influence lineage specification in  vivo . to that end , we used a knockin model of flt3-itd to investigate whether oncogenic mutations that result in constitutive gfr signaling influence the lineage fate of mpps in  vivo . in order to first establish the physiological relevance of flt3-itd as an aml - inducing mutation , we developed a mouse model in which flt3-itd was coexpressed with an inducible deletion of the dna - binding domain of runx1 ( growney et  al . , 2005 ) in mpps . importantly , although runx1 loss - of - function mutations are found in patients with both all and aml , only the latter is often found in association with flt3-itds ( grossmann et  al . deletion of the dna - binding domain of runx1 during adult murine hematopoiesis results in mild myeloproliferation ( growney et  al . , 2005 ) or lymphoblastic leukemias / lymphomas ( jacob et  al . , 2010 ; kundu et  al . , 2005 ; putz et  al . , , we demonstrate that the combination of runx1 mutation with flt3-itds results in aggressive leukemia with 100% penetrance and a uniform myeloid phenotype . in addition to demonstrating the functional relevance for flt3-itd - induced aml , this aml model has a number of important features . first , in contrast to many other mutation collaboration models , both mutations are targeted to the physiologically relevant endogenous genetic loci . second , in isolation each mutation results in only a modest phenotype , but in combination a minor clone of deleted cells rapidly expands and becomes clonally dominant , paralleling somatic mutations during leukemogenesis . third , the evolution of leukemia is rapid , supporting the notion that it occurs without the requirement for additional genetic events . finally , the model recapitulates a particularly poor prognostic form of aml with a major unmet need for novel therapeutic approaches and thus provides a powerful model for future studies of the cellular and molecular mechanistic bases for collaboration of these mutations in aml . the most primitive progenitor population that was expanded by flt3-itds consisted of lmpp - like cells , demonstrating upregulation of the myeloid program , whereas the transcriptional expression of lymphoid - affiliated genes was markedly reduced in both heterozygous and homozygous flt3-itd mice . this myeloid transcriptional bias and suppression of lymphoid transcripts was also present during early b and t  cell development and , importantly , was already present in flt3-itd fl mpps , preceding the expansion of mpps and development of a myeloproliferative phenotype , supporting the notion that it is a direct consequence of flt3-itd signaling in lmpps . the severe suppression of the earliest thymic progenitors in flt3-itd mice and concomitant lack of ccr9 upregulation in lmpps , which have been implicated as key thymus - seeding progenitors ( luc et  al . , 2012 ) , further suggests that thymic seeding might also be impaired by flt3-itds . these findings demonstrate that the myeloid propensity of flt3-itds results from flt3-itd introducing a myeloid bias in multipotent lymphomyeloid progenitors as well as in the earliest b and t lymphoid progenitors . pu1 is a dosage - sensitive regulator of myeloid - lymphoid cell - fate decisions that promotes myeloid differentiation when overexpressed ( dekoter and singh , 2000 ) . using a pu1-yfp reporter , we demonstrated that flt3-itds upregulate pu1 expression in mpps and the earliest b lymphoid progenitors , paralleled by increased expression of pu1 target genes in mpps . furthermore , we confirmed the relevance of these findings for human aml by demonstrating upregulation of pu1 target genes in flt3-itd mutated lmpp - like human aml stem cells ( goardon et  al . , 2011 ) . in agreement with a key mechanistic role for pu1 overexpression , flt3-itd - induced suppression of b cell development , as well as the flt3-itd - associated progenitor phenotype , constitutive flt3-itd signaling is distinct from wt flt3 signaling due to abnormal anchoring of flt3-itd in the endoplasmic reticulum , with markedly reduced surface flt3 expression resulting in aberrant stat3 activation , as confirmed in our study ( schmidt - arras et  al . , 2009 ) . because pu1 is a direct target gene of stat3 ( hegde et  al . , 2009 ) and stat3 target genes were upregulated in flt3-itd lskcd15048 mpps and pre - pro - b cells , this provides a putative direct link between aberrant constitutive flt3-itd signaling and pu1 overexpression as a cause of aberrant myeloid bias . several lines of evidence indicate that flt3-itds frequently arise early in the transformation process in mpps ( levis et  al . , 2005 ) and as a driver mutation in the founding leukemic clone ( ding et  al . , 2012 ; smith et  al . , 2012 ) , and thus may influence the lineage of resulting leukemias . together with our findings , this is consistent with a model in which lmpps , which normally express high levels of flt3 ( adolfsson et  al . , 2005 ) , acquire flt3-itd mutations that confer both a strong clonal advantage and a marked myeloid bias . this results in myeloid expansion and suppression of early lymphoid development , strongly supporting a fundamental role for flt3-itds in promoting myeloid lineage leukemia development at the mpp level . our study also further highlights lmpps as a key target population in aml , as also supported by recent findings in human aml ( goardon et  al . , 2011 ) . transformation to an aggressive leukemia exclusively of a myeloid phenotype by introduction of runx1 mutation demonstrates the functional relevance of this flt3-itd - induced myeloid bias and clonal dominance , providing insights into the process by which oncogenic mutations might determine the lineage fate of the resulting leukemias at the precommitment stage . our findings are also of considerable relevance for normal hematopoiesis , as it remains disputed whether key cytokine receptor signaling pathways mediate critical in  vivo functions in blood lineage development through purely permissive rather than instructive actions ( enver and jacobsen , 2009 ) . thus , although flt3-itd elicits aberrant signaling , our findings clearly provide support for the notion that cytokine receptors are also capable of eliciting lineage - instructive signaling in mpps in  vivo . all animals used were bred and maintained in accordance with regulations of the uk home office . bm samples from aml patients were obtained with informed consent and the approval of oxford ethics committee b ( protocols 06/q1606/110 and 05/mre07/74 ) . details of the antibody staining panels and protocols are provided in the extended experimental procedures . antigens and antibodies used for identification of specific cell populations are shown in tables s1 and s2 . cells were lysed in 1% np40 buffer and analyzed by western blot as previously described ( pecquet et  al . , 2010 ) with antibodies against pstat3 ( cell signaling ) and beta - actin ( sigma ) . evaluation of megakaryocytic , gm , and lymphoid potentials was carried out as previously described ( mnsson et  al . , 2007 ) and detailed in the extended experimental procedures . multiplex quantitative pcr was performed as previously described ( kharazi et  al . , 2011 ) and detailed in the extended experimental procedures . analysis by affymetrix mouse genome 430 2.0 arrays was carried out at the stanford protein and nucleic acid facility as described previously ( mnsson et  al . , 2007 ) and in the extended experimental procedures . extended experimental proceduresquantification of hematologic indiceshemoglobin , wbc , and platelet count per milliliter of blood were quantified on a sysmex kx-21n 3-part differential hematology analyzer.animalsflt3-itd knockin mice on c57bl/6 background have been previously described ( kharazi et  al . homozygous ( flt3 ) and heterozygous ( flt3 ) flt3-itd mice were studied as specified . runx1 mice have been previously described ( grundler et  al . , 2005 ) and were on a 129s1 x cba background . vavcre mice have been previously described ( de boer et  al . , 2003 ) and were on a c57bl/6 background . mx1-cre mice were on c57bl/6 background and have been previously described ( khn et  al . mice carrying a germline deletion of pu1 ( pu1 ) were derived by crossing a previously reported pu1 line ( iwasaki et  al . , 2005 ) on a c57bl/6   129s1 x cba background with a constitutive cmv - cre recombinase , on a c57bl/6 background . pu1-yfp mice on a c57bl/6 background have been previously described ( kirstetter et  al . , 2006 ) . mice were analyzed with littermate controls of the appropriate genotype when available.flow cytometry analysis and facs purificationantibodies and viability dyes used for flow cytometry analysis are shown in table s1 . combinations of antibodies used for identification and purification of indicated populations are shown in table s2.all antibodies were used at predetermined ( titrated ) optimized concentrations . cell acquisition and analysis were performed on a 4-laser lsrii ( becton dickinson , san jose , ca ) using flowjo software ( treestar , ashland , or ) . cells used in cell sorting experiments were either un - enriched or enriched for cd117 with macs cell separation ( miltenyi biotec ) followed by fc - block incubation and staining with anti - mouse antibodies . fluorescence minus - one controls as well as negative populations were used as gate - setting controls . gates were set using a combination of fluorescence minus one controls and also populations that are known to be negative for the antigen . for example , lskcd150cd48 cells , which are known to not express flt3 , were used to set the gate for surface flt3 expression on lskcd150cd48 cells.microarray analysisglobal gene expression analysis was performed on lskcd150cd48 cells from flt3 and flt3 ( 4 mice of each genotype ) as previously reported ( luc et  al . , 2012 ) . two thousand cells were sorted directly into trizol ( invitrogen ) and the rna extraction carried out as per manufacturer s instructions and previously as described ( luc et  al . , 2012 ) . using the same total amount of input rna , samples were amplified using the nugen kit wt - ovation pico rna amplifications system followed by the wt ovation cdna biotin module v2 for cdna labeling ( nugen ) and fragmentation and finally hybridized to affymetrix mouse genome 430 2.0 arrays using standard protocols ( affymetrix ) at the stanford protein and nucleic acid facility . these data will be available through geo accession number gse35805 ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=dfcvjocouaoccds&acc=gse35805).microarray data were normalized using the robust multi - array averages ( rma)(irizarry et  al . , 2003 ) method in biocondcutor / r and analyzed using gene set enrichment analysis ( gsea ) ( subramanian et  al . , 2005 ) . erythroid , myeloid ( chambers et  al . , 2007 ) , pu1 ( steidl et  al . , 2006 ) , and stat3 ( alvarez et  al . , 2005 ) target gene lists have been previously described . a signature for early b cell commitment was generated using data from the immunological genome project ( heng et  al . , 2008 ) ( http://www.immgen.org ) by comparing the gene expression profile of long term reconstituting stem cells with that of pre - pro - b cells using the limma package ( smyth , 2004 ) in r ; genes which were greater than 2-fold upregulated ( limma b statistic > 3 ) were used for the b cell commitment signature . gsea analysis of lmpp - like human aml stem cells , stratified according to the presence of flt3-itd mutation , was carried out using previously described data set ( goardon et  al . , 2011 ) , using human equivalents of the above murine pu1 target genes ( steidl et  al . , 2006).multiplex quantitative real - time pcr analysismultiplex quantitative pcr was performed as previously described ( kharazi et  al . , 2011 ) . for each cell population , two to four biological replicates ( different mice ) were prepared , and two to three separately sorted cell samples from each mouse were cell sorted and analyzed . for lskcd150cd48 cells , lskflt3 cells , and pre - pro - b cells , 100 cells per replicate were analyzed . for dn1 cells , 20 cells per replicate were analyzed as cell numbers of this population were highly limited in flt3 mice . details of the taqman assays used are shown in table s3.for cdna synthesis and preamplification of target genes cellsdirect one - step qrt - pcr kit ( invitrogen ) was used . cells were sorted directly into pcr plates or 0.2  ml pcr tubes containing 2.5  l gene specific 0.2x taqman gene expression assays ( applied biosystems ) , 5  l of cellsdirect 2x reaction mix ( invitrogen ) , 1.2  l cellsdirect rt / taq mix , 1.2  l te buffer and 0.1  l superase - in rnase inhibitor ( ambion ) . conditions for reverse transcription and target gene amplification were : 15  min at 50c ; 2  min at 95c ; 22 cycles of 95c for 15  s and 60c for 4  min . preamplified products were diluted 1:5 in te buffer and analyzed on dynamic array ( fluidigm ) using the following pcr cycling condition : 95c for 10  min ; 40 cycles of 95c for 15  s and 60c for 60 s. data was analyzed using the ct method ; results were normalized to hprt expression and expressed as mean expression level relative to hprt.in  vitro assays for megakaryocyte , gm , and lymphoid potentialsevaluation of megakaryocytic and gm potentials was carried out as previously described ( luc et  al . , 2008 ) . for evaluation of megakaryocytic potential , 150 cells were sorted into 3  ml of x - vivo 15 ( lonza ) supplemented with 0.5% detoxified bovine serum albumin ( bsa ; stemcell technologies inc ) , 10% fetal calf serum ( fcs ) , 2-mercaptoethanol ( 2-me ; 10 m ; sigma - aldrich co ) , 1% penicillin / streptomycin ( paa ) and 50  ng / ml murine stem cell factor ( mscf ; peprotech , rocky hill , nj ) , 50  ng / ml human fms - like tyrosine kinase 3 ligand ( hfl ; immunex , seattle , wa ) , 50  ng / ml human thrombopoietin ( hthpo ; peptrotech ) , 5  u / ml human erythropoietin ( hepo ; boehringer mannheim , mannheim , germany ) and 20  ng / ml murine interleukin 3 ( mil-3 ; peprotech ) . for evaluation of gm potential , cells were sorted into media as above but with the following cytokines : mscf , hfl , hthpo , hg - csf ( neopogen , amgen , thousand oaks , ca ) ( all 25  ng / ml ) , mgm - csf ( immunex , seattle , wa ) and mil-3 ( both 20  ng / ml ) . the cell suspension was thoroughly mixed and 20  l was pipetted into each well of two 60-well plates per replicate ( nunc minitrays catalog number : 163118 , nunc a / s , roskilde , denmark ) . wells were scored for clonal growth and/or frequency of wells with megakaryocytes with an inverted light microscope after 8  days of culture . the reliability of this approach to score cultures for megakaryocytic potential has been validated by morphological validation of giemsa stained cytospins of the cultures ( mnsson et  al . , 2007 ) . percentage of cloning efficiency was calculated according to the poisson distribution which predicts that 63% of wells should contain 1 or more cells following manual plating ( 76 of 120 wells).b cell and t  cell potential was evaluated by sorting 10 mpps , dn1 or pre - pro - b cells onto approximately 80% confluent monolayers of op9 ( b cell potential ) and op9dl1 ( t cell potential ) stromal cells , as previously described ( adolfsson et  al . , 2005 ; luc et  al . , 2012 ) . clones were identified and picked at 3 to 4  weeks ( depending on clonal size ) , and subsequently analyzed by facs for the presence of b cell ( defined as b220cd19 ) or t  cell ( cd4/8 double positive ) committed progeny.statistical analysisthe statistical significance of differences between samples was determined using the 2-tailed t test . hemoglobin , wbc , and platelet count per milliliter of blood were quantified on a sysmex kx-21n 3-part differential hematology analyzer . flt3-itd knockin mice on c57bl/6 background have been previously described ( kharazi et  al . , 2011 ; homozygous ( flt3 ) and heterozygous ( flt3 ) flt3-itd mice were studied as specified . runx1 mice have been previously described ( grundler et  al . , 2005 ) and were on a 129s1 x cba background . vavcre mice have been previously described ( de boer et  al . , 2003 ) and were on a c57bl/6 background . mx1-cre mice were on c57bl/6 background and have been previously described ( khn et  al . , 1995 ) . mice carrying a germline deletion of pu1 ( pu1 ) were derived by crossing a previously reported pu1 line ( iwasaki et  al . , 2005 ) on a c57bl/6   129s1 x cba background with a constitutive cmv - cre recombinase , on a c57bl/6 background . pu1-yfp mice on a c57bl/6 background have been previously described ( kirstetter et  al . , 2006 ) . combinations of antibodies used for identification and purification of indicated populations are shown in table s2 . all antibodies were used at predetermined ( titrated ) optimized concentrations . cell acquisition and analysis were performed on a 4-laser lsrii ( becton dickinson , san jose , ca ) using flowjo software ( treestar , ashland , or ) . cells used in cell sorting experiments were either un - enriched or enriched for cd117 with macs cell separation ( miltenyi biotec ) followed by fc - block incubation and staining with anti - mouse antibodies . fluorescence minus - one controls as well as negative populations were used as gate - setting controls . gates were set using a combination of fluorescence minus one controls and also populations that are known to be negative for the antigen . for example , lskcd150cd48 cells , which are known to not express flt3 , were used to set the gate for surface flt3 expression on lskcd150cd48 cells . global gene expression analysis was performed on lskcd150cd48 cells from flt3 and flt3 ( 4 mice of each genotype ) as previously reported ( luc et  al . , 2012 ) . two thousand cells were sorted directly into trizol ( invitrogen ) and the rna extraction carried out as per manufacturer s instructions and previously as described ( luc et  al . , 2012 ) . using the same total amount of input rna , samples were amplified using the nugen kit wt - ovation pico rna amplifications system followed by the wt ovation cdna biotin module v2 for cdna labeling ( nugen ) and fragmentation and finally hybridized to affymetrix mouse genome 430 2.0 arrays using standard protocols ( affymetrix ) at the stanford protein and nucleic acid facility . microarray data were normalized using the robust multi - array averages ( rma)(irizarry et  al . , 2003 ) method in biocondcutor / r and analyzed using gene set enrichment analysis ( gsea ) ( subramanian et  al . , 2005 ) . , pu1 ( steidl et  al . , 2006 ) , and stat3 ( alvarez et  al . , 2005 ) target gene lists have been previously described . a signature for early b cell commitment was generated using data from the immunological genome project ( heng et  al . , 2008 ) ( http://www.immgen.org ) by comparing the gene expression profile of long term reconstituting stem cells with that of pre - pro - b cells using the limma package ( smyth , 2004 ) in r ; genes which were greater than 2-fold upregulated ( limma b statistic > gsea analysis of lmpp - like human aml stem cells , stratified according to the presence of flt3-itd mutation , was carried out using previously described data set ( goardon et  al . , 2011 ) , using human equivalents of the above murine pu1 target genes ( steidl et  al . , 2006 ) . multiplex quantitative pcr was performed as previously described ( kharazi et  al . , 2011 ) . for each cell population , two to four biological replicates ( different mice ) were prepared , and two to three separately sorted cell samples from each mouse were cell sorted and analyzed . for lskcd150cd48 cells , lskflt3 cells , and pre - pro - b cells , 100 cells per replicate were analyzed . for dn1 cells , 20 cells per replicate were analyzed as cell numbers of this population were highly limited in flt3 mice . for cdna synthesis and preamplification of target genes cellsdirect one - step qrt - pcr kit ( invitrogen ) was used . cells were sorted directly into pcr plates or 0.2  ml pcr tubes containing 2.5  l gene specific 0.2x taqman gene expression assays ( applied biosystems ) , 5  l of cellsdirect 2x reaction mix ( invitrogen ) , 1.2  l cellsdirect rt / taq mix , 1.2  l te buffer and 0.1  l superase - in rnase inhibitor ( ambion ) . conditions for reverse transcription and target gene amplification were : 15  min at 50c ; 2  min at 95c ; 22 cycles of 95c for 15  s and 60c for 4  min . preamplified products were diluted 1:5 in te buffer and analyzed on dynamic array ( fluidigm ) using the following pcr cycling condition : 95c for 10  min ; 40 cycles of 95c for 15  s and 60c for 60 s. data was analyzed using the ct method ; results were normalized to hprt expression and expressed as mean expression level relative to hprt . evaluation of megakaryocytic and gm potentials was carried out as previously described ( luc et  al . , 2008 ) . for evaluation of megakaryocytic potential , 150 cells were sorted into 3  ml of x - vivo 15 ( lonza ) supplemented with 0.5% detoxified bovine serum albumin ( bsa ; stemcell technologies inc ) , 10% fetal calf serum ( fcs ) , 2-mercaptoethanol ( 2-me ; 10 m ; sigma - aldrich co ) , 1% penicillin / streptomycin ( paa ) and 50  ng / ml murine stem cell factor ( mscf ; peprotech , rocky hill , nj ) , 50  ng / ml human fms - like tyrosine kinase 3 ligand ( hfl ; immunex , seattle , wa ) , 50  ng / ml human thrombopoietin ( hthpo ; peptrotech ) , 5  u / ml human erythropoietin ( hepo ; boehringer mannheim , mannheim , germany ) and 20  ng / ml murine interleukin 3 ( mil-3 ; peprotech ) . for evaluation of gm potential , cells were sorted into media as above but with the following cytokines : mscf , hfl , hthpo , hg - csf ( neopogen , amgen , thousand oaks , ca ) ( all 25  ng / ml ) , mgm - csf ( immunex , seattle , wa ) and mil-3 ( both 20  ng / ml ) . the cell suspension was thoroughly mixed and 20  l was pipetted into each well of two 60-well plates per replicate ( nunc minitrays catalog number : 163118 , nunc a / s , roskilde , denmark ) . wells were scored for clonal growth and/or frequency of wells with megakaryocytes with an inverted light microscope after 8  days of culture . the reliability of this approach to score cultures for megakaryocytic potential has been validated by morphological validation of giemsa stained cytospins of the cultures ( mnsson et  al . , 2007 ) . percentage of cloning efficiency was calculated according to the poisson distribution which predicts that 63% of wells should contain 1 or more cells following manual plating ( 76 of 120 wells ) . b cell and t  cell potential was evaluated by sorting 10 mpps , dn1 or pre - pro - b cells onto approximately 80% confluent monolayers of op9 ( b cell potential ) and op9dl1 ( t cell potential ) stromal cells , as previously described ( adolfsson et  al . , 2005 ; clones were identified and picked at 3 to 4  weeks ( depending on clonal size ) , and subsequently analyzed by facs for the presence of b cell ( defined as b220cd19 ) or t  cell ( cd4/8 double positive ) committed progeny .
summarywhether signals mediated via growth factor receptors ( gfrs ) might influence lineage fate in multipotent progenitors ( mpps ) is unclear . 
 we explored this issue in a mouse knockin model of gain - of - function flt3-itd mutation because flt3-itds are paradoxically restricted to acute myeloid leukemia even though flt3 primarily promotes lymphoid development during normal hematopoiesis . 
 when expressed in mpps , flt3-itd collaborated with runx1 mutation to induce high - penetrance aggressive leukemias that were exclusively of the myeloid phenotype . 
 flt3-itds preferentially expanded mpps with reduced lymphoid and increased myeloid transcriptional priming while compromising early b and t lymphopoiesis . 
 flt3-itd - induced myeloid lineage bias involved upregulation of the transcription factor pu.1 , which is a direct target gene of stat3 , an aberrantly activated target of flt3-itds , further establishing how lineage bias can be inflicted on mpps through aberrant gfr signaling . collectively , these findings provide new insights into how oncogenic mutations might subvert the normal process of lineage commitment and dictate the phenotype of resulting malignancies .