Cell,Vol.119,431–443,October 29,2004,Copyright 2004by Cell Press
JunB Deficiency Leads to a Myeloproliferative Disorder Arising from Hematopoietic Stem Cells
duce mature macrophages and granulocytes (Akashi et al.,1999).
Within this developmental scheme,leukemias can Emmanuelle Passegue ′,1,*Erwin F.Wagner,2and Irving L.Weissman 11
Institute of Cancer and Stem Cell Biology now be viewed as aberrant hematopoietic processes and Medicine
initiated by rare leukemic stem cells (LSC)that undergo Departments of Pathology and Developmental an aberrant and poorly regulated process of organogen-Biology
esis analogous to that of normal HSC (Reya et al.,2001;Stanford University School of Medicine Passegue ′et al.,2003).Since a hallmark of all cancers Stanford,California 94305is the capacity for unlimited self-renewal,it has been 2
Research Institute of Molecular Pathology (IMP)proposed,although never directly demonstrated,that Dr.Bohr-Gasse 7leukemias may be initiated by transforming events that A-1030Vienna directly take place in the self-renewing LT-HSC.Alterna-Austria
tively,leukemias may also arise from more committed progenitors that have re-acquired the capacity for indefi-nite self-renewing proliferation through accumulated Summary
mutations and/or epigenetic changes,as we have re-cently shown for some human acute myeloid leukemia The AP-1transcription factor JunB is a transcriptional (AML)(Miyamoto et al.,2000)and late-stage human regulator of myelopoiesis.Inactivation of JunB in post-chronic myelogenous leukemia (CML)(Jamieson et al.,natal mice results in a myeloproliferative disorder 2004).Identifying the developmental origin of the LSC (MPD)resembling early human chronic myelogenous for each type of leukemia is therefore critical for under-leukemia (CML).Here,we show that JunB regulates standing their respective biology and providing powerful the numbers of hematopoietic stem cells (HSC).JunB diagnostic,prognostic,and therapeutic tools in the overexpression decreases the frequency of long-term clinic.
HSC (LT-HSC),while JunB inactivation specifically ex-At the molecular level,the mechanisms controlling pands the numbers of LT-HSC and granulocyte/mac-HSC self-renewal activity are still poorly understood.rophage progenitors (GMP)resulting in chronic MPD.However,the signaling pathways that have been shown Further,we demonstrate that jun B inactivation must to date to be involved in the regulation of HSC self-take place in LT-HSC,and not at later stages of myelo-renewal,i.e.,homeobox genes,such as HoxB4(Anton-poiesis,to induce MPD and that only jun B-deficient chuk et al.,2002)and HoxA9(Thorsteinsdottir et al.,LT-HSC are capable of transplanting the MPD to recip-2002),Notch (Varnum-Finney et al.,2000),Sonic hedge-ient mice.These results demonstrate a stem cell-spe-hog (Shh )(Bhardwaj et al.,2001),the Wnt signaling path-cific role for JunB in normal and leukemic hematopoie-way (Reya et al.,2003),and Bmi-1(Park et al.,2003;sis and provide experimental evidence that leukemic Lessard and Sauvageau,2003),have also been associ-stem cells (LSC)can reside at the LT-HSC stage of ated with oncogenesis and/or overt leukemia (Reya et development in a mouse model of MPD.al.,2001;Lessard and Sauvageau,2003),suggesting the intriguing hypothesis that similar molecular mechanisms may control self-renewal activity in normal hematopoi-Introduction
etic stem cells and malignant stem cells.
The AP-1transcription factor JunB is a transcriptional Hematopoiesis takes place through the stepwise differ-regulator of myelopoiesis and a potential tumor sup-entiation of multipotent hematopoietic stem cells (HSC)pressor gene in mice (Passegue ′et al.,2001).Inactivation (Spangrude et al.,1988)to generate a hierarchy of pro-of jun B in knockout mice results in severe vascular de-genitor populations with progressively restricted devel-fects in the placenta leading to early embryonic lethality,opmental potential,ultimately leading to the production while constitutive overexpression of jun B from the hu-of multiple lineages of mature cells (Orkin,2000).HSC man ubiquitin-C promoter (Ubi)has no major conse-can be divided into a long-term subset (LT-HSC),capa-quences in Ubi-jun B transgenic mice (Schorpp-Kistner ble of extensive self-renewal,and a short-term subset et al.,1999).Reintroducing jun B expression in the jun B-(ST-HSC),which self-renews for a limited interval and deficient background by intercrossing with Ubi-jun B then gives rise to non-self-renewing multipotent progen-transgenic mice fully rescues the mice from embryonic itors (MPP)(Morrison et al.,1997;Christensen and lethality (Schorpp-Kistner et al.,1999).However,all re-Weissman,2001).MPP differentiate to produce both sulting jun B ?/?Ubi-jun B mice progressively develop a the lymphoid lineage,giving rise to common lymphoid transplantable myeloproliferative disorder (MPD)with progenitors (CLP)(Kondo et al.,1997),and the myeloid some features of human CML,including eventual pro-lineage,giving rise to common myeloid progenitors gression to blast crisis associated with an age-depen-(CMP)(Akashi et al.,1999).CMP,in turn,differentiate dent and what was originally described to be myeloid-into megakaryocytic/erythroid progenitors (MEP)and specific silencing of the Ubi-jun B transgene (Passegue ′myelomonocytic progenitors (GMP),which finally pro-et al.,2001).Remarkably,inactivation of jun B expression is also observed in some human CML patients (Bru-chova et al.,2002;Yang et al.,2003),and downregulation
*Correspondence:passegue@https://www.doczj.com/doc/a35051446.html,
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of jun B expression is consistently found in AML patients and all lineages of mature cells were dramatically re-expressing high levels of HoxA9(Dorsam et al.,2003).
duced in number.Together,these results indicate that Together,these findings suggest that jun B inactivation raising JunB levels in LT-HSC decreases the frequency could be important for MPD development and perhaps
of stem cells.
overt myeloid leukemia including CML.
Here,we use the jun B?/?Ubi-jun B model,as well as Increased Frequency of LT-HSC and GMP
several novel models of conditional inactivation and in jun B?/?Ubi-jun B Mice
overexpression of jun B in hematopoietic cells,to investi-To determine the stages of hematopoietic development gate the function of JunB during normal and leukemic that are affected in the jun B?/?Ubi-jun B model of MPD, hematopoiesis.We find that loss of jun B expression we next examined the frequency and fate of stem cell and leads to MPD development only when occurring at the myeloid progenitor populations using6-to9-month-old stem cell level and that the LT-HSC is the sole develop-jun B?/?Ubi-jun B mice(Figure2).The jun B?/?Ubi-jun B mental stage containing the LSC for the jun B-deficient mice develop by3to6months of age an MPD character-MPD.Moreover,we identify JunB as a key negative ized by a massive granulocytic expansion in the bone regulator of LT-HSC function,controlling the numbers marrow and spleen(Figures2A and2B),eventually fol-of LT-HSC by regulating several cellular mechanisms lowed by the appearance of undifferentiated blast cells that are critical for maintenance of the stem cell com-leading to a myeloid blast crisis in16%of the mice partment,i.e.,proliferation/senescence and apoptosis,(Passegue′et al.,2001).Initial analysis of jun B?/?Ubi-via changes in the expression levels of crucial effector jun B bone marrow revealed a considerable increase in genes,including the cell cycle regulators p16INK4a and HSC-enriched(Lin?/c-Kit?/Sca-1?)and myeloid pro-the antiapoptotic proteins Bcl2and Bcl xl.genitor-enriched(Lin?/c-Kit?/Sca-1?)populations(Fig-
ure2A).Further investigation indicated that the increase
in multipotent bone marrow cells resulted exclusively Results
from an increase in the numbers of LT-HSC(Lin?/c-Kit?/
Sca-1?/Flk-2?),while the numbers of ST-HSC and MPP Enforced Expression of JunB in LT-HSC Causes
remained similar to those found in control mice.In the Stem Cell Loss
myeloid lineage,only the GMP population(Lin?/c-Kit?/ To test the role for JunB during normal hematopoiesis,
Sca-1?/CD34?/Fc?R?)displayed a massive expansion, we analyzed the consequences of enforced expression
which accounted almost entirely for the increased num-of JunB in stem cells(Figure1).Highly purified popula-
bers of myeloid progenitors found in jun B?/?Ubi-jun B tions of LT-HSC(Lin?/c-Kit?/Sca-1?/Thy1.1int)(Morrison
mice,while the numbers of CMP and MEP remained et al.,1997)were isolated from C57BL/6-Ly5.2mice,
very similar to control animals(Figure1A).Abnormally transduced with jun B-containing[jun B(L)]or control
high numbers of cells with the phenotypic characteris-[Co(L)]lentiviruses,and transplanted into lethally irradi-
tics of LT-HSC and GMP were also observed in the ated C57BL/6-Ly5.1congenic recipients(Figure1A).
enlarged spleens of jun B?/?Ubi-jun B mice(Figure2B). Both lentiviruses infected LT-HSC with similar efficiency
(37%to45%transduction efficiency),and FACS analy-
sis of peripheral blood isolated from recipient mice at Stochastic Inactivation of JunB
3weeks post-transplantation revealed similar engraftment
in jun B?/?Ubi-jun B LT-HSC
and short-term multilineage reconstitution(Figure1B;To correlate the specific expansion of the LT-HSC and data not shown).However,over time,a dramatic decline
GMP populations with jun B expression,we investigated in the numbers of transduced Ly5.2?/GFP?cells was by qRT-PCR the expression profile of the Ubi-jun B trans-observed in the blood of mice transplanted with jun B(L)-
gene within the hematopoietic system of both Ubi-jun B transduced HSC compared to the fairly constant level transgenic mice and jun B?/?Ubi-jun B MPD mice(Figure found in the blood of mice transplanted with Co(L)-trans-
2C).QRT-PCR revealed high expression levels of the duced HSC.At9weeks post-transplantation,we used Ubi-jun B transgene among the pool of multipotent bone quantitative real-time RT-PCR(qRT-PCR)analysis of
marrow cells(LT-HSC,ST-HSC,and MPP)isolated from HSC-derived Ly5.2?/GFP?spleen cells to assay the in-Ubi-jun B transgenic mice,with considerably lower levels crease in jun B levels provided by the lentiviral transduc-
in the myeloid progenitor populations(CMP,GMP,and tion(Figure1C).At20weeks post-transplantation,we MEP)and B cells,and no expression in mature granulo-determined the distribution of transduced Ly5.2?/GFP?
cytes.Strikingly,a similar analysis performed on popula-cells in the stem,progenitor,and mature compartments tions isolated from6-to9-month-old jun B?/?Ubi-jun B of recipient mice(Figures1D–1F).Although transduction
mice showed dramatically reduced expression levels of with the jun B(L)lentivirus resulted in a modest1.8-fold the Ubi-jun B transgene in every cell type,including LT-increase in total jun B mRNA levels,dramatically reduced
HSC,and,consequently,a major reduction in total jun B numbers of HSC-derived Ly5.2?/GFP?cells were found mRNA levels in the entire hematopoietic system of the in the bone marrow,spleen,and thymus of the trans-
jun B?/?Ubi-jun B mice(Figure2C).Inactivation of the planted mice.Furthermore,in the bone marrow,almost Ubi-jun B transgene in the myeloid lineage of jun B?/?Ubi-no jun B(L)-transduced Ly5.2?/GFP?cells with the phe-
jun B mice has been described previously(Passegue′et notypic characteristics of LT-HSC(Lin?/c-Kit?/Sca-1?/al.,2001).However,these results indicate that transgene Flk2?)(Christensen and Weissman,2001),myeloid pro-
inactivation occurs already in the stem cell compart-genitors(Lin?/c-Kit?/Sca-1?/CD34/Fc?R)(Akashi et al.,ment,consistent with the hypothesis that clones of LT-1999),or lymphoid progenitors(Lin?/Il-7R?/Thy1.1?/
HSC that inactivate jun B expression have a competitive c-Kit int/Sca-1int)(Kondo et al.,1997)could be detected,advantage over LT-HSC that still express the Ubi-jun B
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Figure1.Enforced Expression of jun B in LT-HSC Causes Stem Cell Loss
(A)Schematic representation of the lentiviral transduction/transplantation protocol.LT-HSC were purified from C57BL/6-Ly5.2mice,infected overnight with an empty control[Co(L)]or a jun B-expressing[jun B(L)]lentivirus and transplanted into lethally irradiated C57BL/6-Ly5.1congenic recipient mice(2,000transduced-HSC/mouse).hPGK:human PGK promoter;MCS:multiple cloning site;ires:internal ribosomal entry site; eGFP:enhanced green fluorescent protein.
(B)Peripheral blood chimerism in mice transplanted with jun B(L)-or Co(L)-transduced HSC.Data are plotted as percent(mean?SD;n?5) of GFP?cells in peripheral blood mononuclear cells(PBMC).
(C)QRT-PCR analysis of jun B mRNA levels in transduced HSC-derived(Ly5.2?/GFP?)spleenocytes at9weeks post-transplantation.Results have been standardized for?-actin levels and are expressed as fold-induction(mean?SD;n?3)compared to the levels detected in Co(L)-transduced cells(set to1).
(D–F)Flow cytometric analysis of Co(L)-and jun B(L)-transduced HSC-derived(Ly5.2?/GFP?)cells at20weeks post-transplantation.(D)Overall percentage in the bone marrow.(E)Distribution in the multipotent cell(MC:Lin?/c-Kit/Sca-1),myeloid progenitor(MP:Lin?/c-Kit?/Sca-1?/ CD34/Fc?R),and lymphoid progenitor(CLP:Lin?/IL7R?/Thy1.1?/c-Kit/Sca-1)populations.(F)Distribution in the indicated hematopoietic lineages in the bone marrow(BM),spleen(Sp),and thymus(Thy).Bar graphs indicate the total cell number(mean?SD;n?3)per four long bones or per spleen for each of the indicated populations.Gr:Gr-1?/Mac-1?;B:B220?/CD19?;T:CD4?.Mice transplanted with jun B(L)-transduced HSC displayed on average20%–50%decrease in total cellularity in both bone marrow and spleen compared to mice transplanted with Co(L)-transduced HSC(not shown).
transgene,such that jun B-deficient LT-HSC and their ment,we crossed mice carrying a floxed allele of jun B progeny progressively take over hematopoiesis in
(jun B f)(Kenner et al.,2004)with a granulocyte lineage-jun B?/?Ubi-jun B mice.specific MRP8-Cre-ires/GFP transgenic mouse(Figure
3).The human MRP8promoter restricts CRE recombi-Targeted Inactivation of JunB during Myelopoiesis
nase activity,as well as expression of the GFP marker, Does Not Cause MPD to granulocytes and a fraction of GMP(10%–20%)(Fig-
ures3A and3B)(Lagasse and Weissman,1994).Consis-To identify directly the developmental stage where inac-
tivation of jun B expression is required for MPD develop-tent with this expression pattern,specific deletion of the
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Figure2.Specific Expansion of the LT-HSC and GMP Populations in jun B-Deficient jun B?/?Ubi-jun B MPD Mice
Analysis of6-to9-month-old control(jun B?/?or jun B?/?with or without the Ubi-jun B transgene)and jun B?/?Ubi-jun B mice.
(A and B)Detailed FACS analysis of the granulocytic(Gr:Gr-1?/Mac-1?),multipotent cell(MC:Lin?/c-Kit/Sca-1),stem cell(HSC:Lin-/c-Kit?/ Sca-1?/Flk-2),and myeloid progenitor(MP:Lin?/c-Kit?/Sca-1?/CD34/Fc?R)populations in the bone marrow(BM)and spleen(Sp).Bar graphs indicate the total cell number(mean?SD;n?3)per four long bones or per spleen for each of the indicated populations.By6months of age,the jun B?/?Ubi-jun B mice display on average a1.4-fold increase in total cellularity in the bone marrow and a2.7-fold increase in the spleen compared to control mice(not shown).
(C)QRT-PCR analysis of Ubi-jun B transgene(upper panel)and total jun B(bottom panel)mRNA levels in wild-type(wt;n?3),Ubi-jun B transgenic(n?2),and jun B?/?Ubi-jun B(n?3)bone marrow.Results are given as mean(?SD)of independently sorted sets of stem,progenitor, and mature cell populations.Each value has been standardized for?-actin expression level and is expressed as fold induction compared to Ubi-jun B levels in unfractionated transgenic bone marrow(set to1;top panel)or to jun B levels in unfractionated wild-type bone marrow(set to1;bottom panel).B:B220?/CD19?spleenocytes;n.d.:not detected.
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Figure3.Myeloid-Restricted Deletion of jun B Does Not Lead to MPD
(A and B)Characterization of MRP8-Cre-ires/GFP transgenic mice.(A)FACS analysis of GFP expression in myeloid progenitor(MP:Lin?/ c-Kit?/Sca-1?/CD34/Fc?R)and granulocytic(Gr:Gr-1?/Mac-1?)populations in the bone marrow(BM).(B)QRT-PCR analysis of Cre and Gfp mRNA levels.Results(mean?SD;n?3)have been standardized for?-actin expression levels and are expressed as a percentage of the maximal expression levels detected in granulocytes(set to100%).HSC:Lin?/c-Kit?/Sca-1?bone marrow cells;B:B220?/CD19?spleenocytes; T:CD4?spleenocytes.
(C–E)Analysis of jun B f/f MRP8-Cre-ires/GFP(jun B(?/?)Gr)mice.(C)PCR analysis(upper panel)of wild-type(wt or jun B?)and floxed jun B(jun B f) alleles in genomic DNA extracted from each of the indicated populations.Genomic DNA from spleenocytes(Sp)of the indicated genotypes were used as control.Real-time PCR quantification(lower panel)of the efficiency of deletion of the floxed jun B alleles in the same genomic DNA.Results have been standardized for?-actin DNA levels and are expressed as percentages of the levels detected in HSC(set to100%).
(D)QRT-PCR analysis of jun B mRNA levels in the myeloid lineage of wild-type and jun B(?/?)Gr mice.Results(mean?SD;n?3)have been standardized for?-actin levels and are expressed as percentages of the levels detected in control CMP(set to100%).(E)Detailed FACS analysis of the myeloid progenitor(MP)and granulocytic(Gr)populations in jun B(?/?)Gr bone marrow.Bar graphs indicate the total cell number (mean?SD;n?3)per four long bones or per spleen for each of the indicated populations.
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floxed jun B allele was observed in a subset of the GMP highly purified subpopulations isolated from6-to and in most,if not all,granulocytes of the jun B f/f MRP8-
9-month-old jun B?/?Ubi-jun B mice in sublethally irradi-Cre-ires/GFP(jun B(?/?)Gr)mice(Figure3C),correlating ated immunocompromised RAG2-deficient congenic with reduced levels of jun B mRNA in GMP and no detect-
recipient mice(Table1;Supplemental Figure S2online). able expression in granulocytes(Figure3D).Increased While unfractionated bone marrow always transferred
the MPD in secondary recipient mice,c-Kit-depleted numbers of GMP and granulocytes were observed in the
bone marrow of jun B(?/?)Gr mice,but no significant expan-bone marrow did not,suggesting that the LSC resides sion of the granulocytic population was observed in the
in the stem or myeloid progenitor pools.However,nei-spleen(Figure3E).Furthermore,none of the jun B(?/?)Gr ther the CMP nor the GMP populations transplanted the mice(n?18)analyzed over an observation period of
MPD or persisted in the primary recipient mice for longer 12months showed evolution of this indolent expansion than6weeks,similar to transplantation of wild-type of the granulocytic lineage to blast crisis,MPD,or overt
progenitor populations(Akashi et al.,1999;Na Nakorn leukemia.Therefore,targeted inactivation of jun B in non-et al.,2002).Furthermore,CMP and GMP isolated from self-renewing myeloid progenitors is not sufficient for
jun B?/?Ubi-jun B mice displayed normal clonogenic pro-MPD development.genitor activity in vitro,with80%–90%of the cells plated
giving rise to colonies.Transplantation of high numbers
of(Lin?/c-Kit?/Sca-1?)bone marrow cells containing a Targeted Inactivation of JunB in LT-HSC
mixture of CMP,GMP,and MEP also failed to repopulate Results in MPD
or transplant the MPD to recipient mice(Table1), We next crossed the floxed jun B mice with two different
thereby excluding the possibility of a higher cell number transgenic mice that can induce CRE expression in stem
requirement.Within the pool of multipotent bone marrow cells(Figure4):the constitutive MORE-Cre knockin mice
cells,only transplantation of LT-HSC resulted in the (Tallquist and Soriano,2000)and the inducible Mx-Cre
propagation of the MPD,while transplantation of the mice(Ku¨hn et al.,1995).The MORE-Cre allele restricts
immediate next developmental stages,ST-HSC and the CRE recombinase activity to the embryo proper,
MPP,did not(Table1).Strikingly,as few as60LT-thereby allowing the birth of jun B f/f MORE-Cre(jun B?/?)
HSC were sufficient to transfer the disease to secondary mice(Figure4A),which develop several pathologies,
recipients(data not shown).The MPD that developed including a severe osteoporosis by4weeks of age(Ken-
in the LT-HSC-transplanted recipient mice closely mim-ner et al.,2004)and,by3to6months,an MPD with
icked the disease occurring in primary donor mice,with massive granulocytic expansion in bone marrow and
increased LT-HSC and GMP populations and eventual spleen(Figure4D;Supplemental Figure S1A at http://
emergence of undifferentiated blast cells(Supplemental https://www.doczj.com/doc/a35051446.html,/cgi/content/full/119/3/431/DC1/).
Figure S3online).Transplantation of jun B?/?Ubi-jun B Three-month-old jun B?/?mice showed undetectable
spleen cells,which contain high numbers of cells with levels of the floxed jun B allele in LT-HSC,correlating
the phenotypic characteristics of LT-HSC,also trans-with a complete absence of jun B mRNA expression(Fig-
planted the MPD in primary recipient mice(Table1). ures4B and4C),and,similarly to the jun B?/?Ubi-jun B
Similar results were obtained using lethally irradiated mice,displayed a massive and specific expansion of
nonimmunocompromised congenic recipient mice,al-the LT-HSC and GMP populations(Figure4D).To further
though this model resulted in longer intervals for MPD study the correlation between stem cell deletion of jun B
development following transplantation of unfraction-and MPD development in adult mice,we also used in-
ated bone marrow or purified LT-HSC(data not shown), ducible Mx-Cre transgenic mice that,upon injection of
possibly implicating a role for immunosurveillance in the interferon?inducer poly(I/C),induces CRE recombi-
this disease.Finally,transplantation experiments were nase activity in numerous cell types including stem cells
also performed with highly purified LT-HSC and a mix-in jun B f/f Mx-cre[jun B(?/?)i]mice(Figure4E).Two months
ture of CMP,GMP,and MEP(Lin?/c-Kit?/Sca-1?)iso-after poly(I/C)injection,we confirmed complete deletion
lated from3-month-old jun B?/?mice or from jun B(?/?)i of the floxed jun B allele in LT-HSC(Figure4F)and com-
mice4months after poly(I/C)injection(Table1).Similarly plete loss of jun B mRNA expression in LT-HSC(Figure
to the jun B?/?Ubi-jun B mice,using these two genetically 4G).Following sustained inactivation of jun B,all jun B(?/?)i
defined mouse models,we found that only transplanta-mice displayed a progressive and specific expansion of
tion of unfractionated bone marrow or purified LT-HSC the LT-HSC and GMP populations,which led by6-9
was capable of propagating the MPD to recipient mice. months post-poly(I/C)injection to the development of
Together,these results clearly demonstrate that the LSC an MPD with massive granulocytic expansion in bone
are contained within the LT-HSC population,thereby marrow and spleen(Figure4H;Supplemental Figures
establishing that the jun B-deficient MPD is a hematopoi-S1B and S1C on the Cell website).Together,these re-
etic stem cell leukemia.
sults indicate that inactivation of jun B in hematopoietic
stem cells is absolutely required for MPD development.
They also suggest that jun B-deficient HSC and GMP are JunB Regulates LT-HSC Numbers through qualitatively different in their ability to promote leukemo-Changes in Proliferation and Apoptosis
genesis.Finally,we analyzed jun B-deficient LT-HSC and jun B(L)-
expressing LT-HSC to identify cellular and molecular The jun B-Deficient MPD
mechanisms that are affected by the loss or gain of jun B Is an HSC Leukemic Disorder expression and are critical for maintenance of the stem
cell compartment(Figure5).Cell cycle analysis using To identify the developmental stage that contains the
LSC for the jun B-deficient MPD,we first transplanted Hoechst-33342showed similar percentages of G0-G1
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Figure4.Stem Cell Deletion of jun B Leads to MPD
(A)Schematic representation of the MORE-Cre-mediated deletion of the floxed jun B alleles during embryonic development.The MORE promoter restricts CRE expression to the embryo proper and allows the birth of jun B f/f MORE-Cre(jun B?/?)mice,which are jun B deficient in every cell type.
(B–D)Analysis of3-month-old jun B?/?mice.(B)PCR analysis of wild-type(wt)and floxed jun B(jun B f)alleles in genomic DNA extracted from bone marrow(BM)or LT-HSC purified from mice of the indicated genotype.(C)QRT-PCR analysis jun B mRNA expression in bone marrow and LT-HSC purified from wild-type and jun B?/?mice.Results(mean?SD;n?3)have been standardized for?-actin levels and are expressed as percentages of the levels detected in wild-type bone marrow(set to100%).(D)Detailed FACS analysis of the granulocytic(Gr:Gr-1?/Mac-1?),multipotent cell(MC:Lin?/c-Kit/Sca-1),stem cell(HSC:Lin?/c-Kit?/Sca-1?/Flk-2),and myeloid progenitor(MP:Lin?/c-Kit?/Sca-1?/CD34/ Fc?R)populations in jun B?/?bone marrow.Bar graphs indicate the total cell number(mean?SD;n?3)per four long bones or per spleen (Sp)for each of the indicated populations.Jun B?/?mice display on average a1.6-fold increase in total cellularity in the bone marrow and a
1.3-fold increase in the spleen compared to wild-type mice(not shown).
(E)Schematic representation of the Mx-Cre-mediated deletion of the floxed jun B alleles following poly(I/C)injection in jun B f/f Mx-Cre (jun B(?/?)i)mice.
(F–H)Analysis of jun B(?/?)i mice.(F)PCR analysis of floxed jun B allele in genomic DNA extracted from bone marrow or LT-HSC purified from mice of the indicated genotypes at2months post-poly(I/C)injection.(G)QRT-PCR analysis of jun B mRNA expression in bone marrow and LT-HSC purified from jun B f/f and jun B(?/?)i mice at2months post-poly(I/C)injection.Results(mean?SD;n?3)have been standardized for ?-actin levels and are expressed as percentages of the levels detected in jun B f/f bone marrow(set to100%).(H)Detailed FACS analysis of the multipotent cell(MC),myeloid progenitor(MP),and granulocytic(Gr)populations in jun B(?/?)i bone marrow at the indicated time post-poly(I/C)injection.Bar graphs indicate the total cell number(mean?SD;n?3)per four long bones or per spleen for each of the indicated populations in control jun B f/f and poly(I/C)-injected jun B(?/?)i mice.
and S-G2/M cells in control LT-HSC populations and LT-HSC compared to control LT-HSC(Figure5B).How-jun B-deficient LT-HSC populations isolated from6-to
ever,jun B-deficient and control LT-HSC gave rise with 9-month-old jun B?/?Ubi-jun B mice(Figure5A).Since similar efficiency to the entire range of myeloid and
erythroid colonies in methylcellulose CFU assays(Figure the absolute numbers of LT-HSC are increased in these
MPD mice,this result indicates an increase in both rest-5C),indicating that both LT-HSC populations are equiva-ing and cycling LT-HSC numbers.Consistently,prolifer-
lent in their differentiation capabilities in vitro and in vivo. ation analyses of single LT-HSC cultured in Terasaki Interestingly,jun B-deficient LT-HSC showed decreased plates indicated faster proliferation rates and increased
mRNA levels of the cell cycle inhibitor p16Ink4a,a direct cell numbers generated from a single jun B?/?Ubi-jun B jun B-target gene(Passegue′and Wagner,2000)and a
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Table1.The Transplantable Leukemic Stem Cells for the jun B-Deficient MPD Reside in the LT-HSC Compartment
Number Number Number Observation
of Donors of Cells of Recipients Time(Months)MPD Blast Crisis
jun B?/?Ubi-jun B
Bone marrow52?106162–1216/162/16
c-Kit-depleted BM45?1065up to120/5
LT-HSC42,00092–59/91/9
ST-HSC&MPP43,00010up to120/10
CMP35,0007up to50/7
GMP310,0008up to50/8
CMP,GMP,and MEP25?1054up to120/4
Spleen25?1066up to125/6
jun B f/f MORE-Cre(jun B?/?)
Bone marrow32?10656–125/50/5
LT-HSC32,00085–128/81/8
CMP,GMP,and MEP35?1055up to120/5
jun B f/f Mx-Cre(jun B(?/?)i)
Bone marrow22?10666–126/60/6
LT-HSC22,00068–126/60/6
CMP,GMP,and MEP25?1054up to120/4
Sublethally irradiated RAG2?/?-Ly5.2recipient mice were injected with the indicated numbers of donor bone marrow cells,spleen cells,or purified stem and/or progenitor populations isolated from6-to9-month-old jun B?/?Ubi-jun B-Ly5.1mice,3-month-old jun B f/f MORE-Cre (jun B?/?)-Ly5.1mice,or jun B f/f Mx-Cre(jun B(?/?)i)-Ly5.1mice4months after poly(I/C)injection.Recipient mice were monitored monthly by flow cytometric analysis of peripheral blood and scored positive for MPD when neutrophil counts were above50%.Spleen and bone marrow cells from the diseased recipient mice were further analyzed by flow cytometry and histology to confirm the MPD diagnosis and to determine the occurrence of blast crisis.
key regulator of stem cell proliferation/senescence(Park leading to MPD development.MPD was observed in et al.,2003),and increased expression of the antiapo-
every mouse model in which we could establish condi-ptotic proteins bcl2and bcl x(L),two critical regulators tions for LT-HSC deletion of jun B,either in a stochastic
manner(jun B?/?Ubi-jun B model)or in a direct manner of stem cell death(Domen,2000)(Figures5D and5E).
Conversely,cell cycle analysis of Ly5.2?/GFP?-trans-(jun B f/f More-Cre and jun B f/f Mx-Cre models).Both jun B duced jun B(L)-expressing LT-HSC,isolated from recipi-
deletion and jun B overexpression deregulate cellular ent mice9weeks after transplantation,revealed a signif-mechanisms,such as proliferation/senescence and icant decrease in the numbers of cycling cells compared
apoptosis,that are critical for maintenance of the stem to Co(L)-expressing LT-HSC(Figure5F);and prolifera-cell compartment.JunB effects are mediated,at least tion analysis of single Ly5.2?/GFP?-transduced jun B(L)-
in part,via regulation of the expression of key effectors expressing LT-HSC in Terasaki plates also indicated a genes,like p16Ink4a,bcl2,and bcl x(L),which also have been reduction in the numbers of cells generated from a single
shown to be important in mediating JunB effects during stem cell(Figure5G).Most importantly,analysis by qRT-myelopoiesis(Passegue′et al.,2001).Recently,Bmi-1, PCR indicated a massive increase in the levels of p16Ink4a
a negative regulator of p16Ink4a,has been shown to be expressed in Ly5.2?/GFP?junB(L)-transduced HSC-critical for the self-renewal capability of normal hemato-derived cells as well as some decrease in the levels of
poietic stem cells(Park et al.,2003)and myeloid leuke-bcl2and bcl x(L)expressed(Figure5H).Taken together,mia cells(Lessard and Sauvageau,2003),as well as of
central nervous system(CNS)and peripheral nervous these findings underscore the key biological function of
JunB in regulating homeostasis of the stem cell com-system(PNS)stem cells(Molofsky et al.,2003).Deregu-partment(Figure6).The expanded population of LT-
lation of other molecular pathways perhaps involved in HSC found in jun B-deficient MPD mice could arise from controlling stem cell self-renewal(Reya et al.,2001), deregulated self-renewing replication,which occurs
inhibition of programmed cell death(Traver et al.,1998), without impaired differentiation,and/or could be medi-or conservation of telomeres(Allsopp and Weissman, ated through blockade of apoptosis and/or senescence,
2002)are also likely to contribute to the leukemic fate while the stem cell loss caused by increased JunB levels of the jun B-deficient LT-HSC and are currently being in-could be due to decreased proliferation,increased se-
vestigated.
nescence,and/or increased apoptosis.Although targeted inactivation of JunB in stem cells
leads to a massive expansion of the LT-HSC population, Discussion
normal numbers of ST-HSC,MPP,CLP,and CMP were
derived from jun B-deficient LT-HSC,indicating that In the present study,we demonstrate that the transcrip-
JunB does not control the population size of these pro-tion factor JunB plays a key biological role in control-genitor compartments.Other genes and/or environmen-ling homeostasis of the hematopoietic stem cell com-
tal influences,which are still unknown,are therefore partment(Figure6).We found that raising JunB levels likely to independently regulate the numbers of these in LT-HSC through lentiviral transduction leads to stem
hematopoietic subsets.In contrast,JunB expression is cell loss,while inactivating JunB expression in LT-HSC critical for homeostasis of the granulocytic lineage since inevitably causes a leukemogenic stem cell expansion
jun B-deficient LT-HSC produce vastly increased num-
JunB Regulates HSC Numbers
439
Figure5.JunB Alters the Homeostasis of the Stem Cell Compartment due to Changes in Proliferation and Apoptosis
(A–E)Analysis of LT-HSC purified from6-to9-month-old control(jun B?/?or jun B?/?with or without the Ubi-jun B transgene)and jun B?/?Ubi-jun B mice.(A)Hoechst-33342staining for DNA content.The percent of cells in S-G2/M phase of the cell cycle(with?2N DNA content)is indicated.(B)Analysis of the proliferation rate of single LT-HSC.Data are plotted as the total number(mean?SD)of cells per Terasaki well over a5day period in culture.(C)Methylcellulose colony-forming units(CFU)assays.Colony types include mix(Mix),granulocyte/macrophage (GM),granulocyte(G),macrophage(M),erythroid(E),megakaryocyte(Mk),and erythroid/megakaryocyte(EMk).Granulocyte-containing colonies derived from jun B?/?Ubi-jun B LT-HSC are always larger in size than colonies derived from control LT-HSC(not shown).(D)QRT-PCR analysis of p16Ink4a,bcl2,and bcl x(L)mRNA levels.Results(mean?SD;n?3)have been standardized for?-actin levels and are expressed as percentages of the levels detected in wild-type(wt)LT-HSC(set at100%).(E)Intracellular staining for mouse Bcl2.Data are plotted as histograms of fluorescence intensity.
(F–G)Analysis of(Ly5.2?/GFP?)LT-HSC purified from mice transplanted with Co(L)-or jun B(L)-transduced HSC at9months post-transplanta-tion.(F)Hoechst-33342staining for DNA content.(G)Analysis of the proliferation rate of single LT-HSC.
(H)QRT-PCR analysis of p16Ink4a,bcl2,and bcl x(L)mRNA levels in(Ly5.2?/GFP?)spleen cells purified from mice transplanted with Co(L)-or jun B(L)-transduced HSC at9months post-transplantation.Results(mean?SD;n?3)have been standardized for?-actin levels and are expressed as percentages of the levels detected in Co(L)-transduced cells(set at100%).
bers of GMP and of mature granulocytes as already stasis of the granulocytic lineage and never leads to reported(Passegue′et al.,2001).Interestingly,enforced
MPD development,clearly indicating that the target cell expression of JunB in LT-HSC leads to a dramatic de-for JunB inactivation is critical for the leukemogenic cline in the numbers of mature cells belonging to all the
process.In fact,transplantation experiments indicate hematopoietic lineages,while jun B inactivation leads that LT-HSC,but not GMP or other multipotent or oligo-
potent progenitor population,are the only cell popula-to a specific increase in LT-HSC and GMP/granulocyte
populations.Since jun B-overexpressing HSC are lost,tion capable of propagating the jun B-deficient MPD in the effects on mature populations may be secondary
recipient mice.Taken together,these results demon-to that loss,although we cannot yet exclude a more strate that the LT-HSC population contains the LSC for promiscuous effect of jun B overexpression that could
the jun B-deficient MPD and identify this disease as a involve additional regulatory pathways distinct from the hematopoietic stem cell leukemia.
ones affected in jun B-deficient LT-HSC.Most impor-
Perhaps the most common MPD in man is the chronic tantly,targeted inactivation of jun B in mouse-committed phase of CML(Sawyers,1999).Acquisition of the t(9;22) myelomonocytic cells only modestly affects the homeo-
Bcr-Abl translocation is an early and almost constant
Cell
440
Figure6.Proposed Model for the Role of
JunB during Normal and Leukemic Hemato-
poiesis
During normal hematopoiesis,JunB regu-
lates the homeostasis of the stem cell com-
partment by controlling the balance between
self-renewal replication and apoptosis.In
jun B-deficient mice,the absence of JunB
leads to increased numbers of LT-HSC,due,
at least in part,to increased proliferation and/
or blockade of apoptosis,while the numbers
of differentiating ST-HSC,MPP,CLP,and
CMP remain normal.JunB also controls the
homeostasis of the granulocytic lineage lead-
ing to a vast expansion of GMP and mature
granulocytes in mice harboring stem cell in-
activation of jun B.The expanded population
of LT-HSC is the LSC of the jun B-deficient
MPD,e.g.,the only population capable of
transplanting the disease to recipient mice,
while the expanded GMP and granulocyte
populations provide the myeloproliferative
features of this stem cell leukemia.
event in human chronic phase CML.The activity of the hypermethylation(Yang et al.,2003).JunB is also regu-Bcr-Abl fusion protein is central in the development of
lated by the homeoprotein HoxA9and consistent down-CML,but it is now clear that other transformation events regulation of jun B expression is observed in AML pa-
tients with high HoxA9expression(Dorsam et al.,2003). are also required for its leukemic progression.The Bcr-
Abl translocation can be found in normal human hemato-Together,these findings suggest that jun B inactivation poietic cells in the absence of leukemic transformation
could be a general mechanism involved in myeloid leu-(Bose et al.,1998;Matioli,2002),suggesting the exis-kemias and a contributing factor for some of the human tence and likely importance of additional mutations and/
CML pathologies that show granulocytocis and myeloid or epigenetic changes that could be required alone or blast crisis.
Here,we clearly demonstrate that jun B inactivation in conjunction with Bcr-Abl for CML pathogenesis(Fial-
kow et al.,1981;Clarkson et al.,2003;Feldman et al.,2003).must occur in LT-HSC,and not at later stages of myelo-Transgenic mice overexpressing the Bcr-Abl oncogene in
poiesis,to induce MPD development.In contrast,re-hematopoietic lineages also have requirements for leuke-stricted expression of Bcr-Abl in mouse-committed my-mic evolution.In fact,Bcl2-mediated inhibition of apoptosis
elomonocytic cells can lead to CML development without in mice(Jaiswal et al.,2003)and recently?-catenin-driven stem cell involvement(Jaiswal et al.,2003).Although
the hMRP8-Bcr-Abl model may not recreate the full self-renewal in humans(Jamieson et al.,2004),have
been implicated as synergistic events for CML progres-spectrum of human CML diseases,particularly the sion to its final blast crisis stage.Remarkably,jun B ex-
lymphoid crisis component,it suggests that expression pression is inactivated in peripheral blood cells of Bcr-of Bcr-Abl may be required only at the committed myelo-Abl-positive CML patients(Bruchova et al.,2002;Yang
monocytic stage to cause the disease,although the et al.,2003),mainly due to,or sustained by,hypermethy-translocation would have to be present(through not
necessarily expressed)in LT-HSC,as these are the only lation silencing of the jun B promoter(Yang et al.,2003).
Furthermore,the demethylating agent Decitabine,which self-renewing cells in the hematopoietic system.To-shows encouraging anti-CML activity and promising re-
gether,these results suggest a progression in the CML sults for the treatment of Imatinib Mesylate-resistant leukemogenesis process that could eventually apply to (Gleevec,formally STI571)patients(Kantarjian et al.,
some human pathologies.Hence,stem cell inactivation 2003),leads to partial reactivation of human jun B ex-of jun B could precede or follow acquisition of Bcr-Abl pression that had previously been silenced by promoter
expression by the stem cells,or by more committed
JunB Regulates HSC Numbers
441
number of granulocyte in the bone marrow and no extramedullar myeloid progenitor populations,and cooperate with
hematopoiesis.Myeloproliferative disease(MPD)corresponds to an Bcr-Abl for the genesis and/or progression of chronic
increase by?2-fold in the total number of granulocytes in the bone phase CML.This role for jun B is currently under investi-
marrow and to an increase in the relative frequency of granulocytes gation and the jun B-deficient MPD mice may prove to be by?20%in the spleen and by?50%in the blood,extramedullar
valuable mouse models to study Bcr-Abl-independent hematopoiesis,and transplantability of the disease.Overt myeloid
mechanisms that may function alone or in cooperation
leukemia corresponds to all the previous plus anemia and?20%
blast cells.
with Bcr-Abl in the genesis and/or progression of CML.
Studying the relationship between Bcr-Abl signaling and
Flow Cytometry
jun B regulation at both the HSC and the GMP level also
Cell staining and enrichment procedures for cell sorting were per-may provide insight as to why granulocytosis predomi-
formed as described(Kondo et al.,1997;Akashi et al.,1999;Chris-nates in human CML.tensen and Weissman,2001).Cells were finally resuspended in
The results presented here provide the first experi-Hanks Buffered Salt Solution(HBSS)with2%heat-inactivated fetal-
mental demonstration of the hematopoietic stem cell
calf serum(staining medium)and1?g/ml propidium iodide(PI)and
analyzed or sorted on a modified488nm argon and599nm dye dual leukemia nature of an MPD similar to human chronic
laser FACS Vantage(Becton Dickinson Immunocytometry System). phase CML.They nicely corroborate the less direct evi-
Dead cells were gated out by high PI staining and forward light dence that the chronic phase of human CML is a clonal
scatter.For proliferation analysis of a single stem cell,one LT-HSC expansion of multiple hematopoietic lineages and there-was clone-sorted per well of a Terasaki plate in10?l of Iscove’s
fore derives from HSC or other multipotent cells(Fialkow Modified Dulbecco’s Media(IMDM)containing5%FCS,50mM
et al.,1977;Li et al.,1999).The identification of the LSC
2-?mercaptoethanol,SCF(20ng/ml),Flt3L(20ng/ml),and IL-11(10
ng/ml)and incubated at37?C for the indicated duration.The num-for the jun B-deficient MPD lends further support to the
bers of cells per well were visually determined using an inverted notion that leukemias and cancers may be sustained by
microscope.For Hoechst analysis,c-Kit-enriched bone marrow rare LSC or cancer stem cells(Reya et al.,2001;Pas-
cells were preincubated for45min at37?C in staining medium con-segue′et al.,2003)that could either derive from the stem taining1g/ml glucose,20?g/ml Hoechst-33342(Molecular Probes),
cell of the tissue of origin,as shown here,or from more and50?g/ml Verapamil before the final staining step,also per-
restricted progenitors that have reacquired the stem cell
formed in the presence of50?g/ml Verapamil.Intracellular mBcl2
staining was performed as described(Domen et al.,1998). capability for self-renewal,as we have recently shown
for MLL-ENL-induced mouse AML(Cozzio et al.,2003),
Cell Transplantation
in human myeloid blast crisis CML(Jamieson et al.,
Unfractionated bone marrow and spleen cells and purified stem and 2004)and in other human AML(Miyamoto et al.,2000).
progenitor populations were obtained from6-to9-month-old Insofar as the only self-renewing cell in the myeloid jun B?/?Ubi-jun B-Ly5.1mice,3-month-old jun B f/f MORE-Cre(jun B?/?)-lineage is the LT-HSC,we expect that only clones of LT-Ly5.1mice,or jun B f/f Mx-Cre(jun B(?/?)I)-Ly5.1mice4months after HSC can accumulate all the mutations and/or epigenetic
poly(I/C)injection and transplanted via retro-orbital injection into
sublethally irradiated(450rad)RAG2?/?-Ly5.2recipient mice.Pe-changes involved in the progression to leukemia,includ-
ripheral blood was obtained from the tail vein of each mouse and ing several independent events to inhibit programmed
analyzed by flow cytometry.Donor-derived cells were distinguished cell death(Traver et al.,1998),to inhibit differentiation
from host cells by the expression of different Ly5antigens(Ly5.1 (Tenen,2003)and to enforce self-renewal(Reya et al.,versus Ly5.2).
2001;Jamieson et al.,2004).Future investigations of the
jun B-deficient LT-HSC should therefore provide a better HSC Lentiviral Transduction and Transplantation
molecular understanding of LSC pathogenesis and may
Jun B cDNA was cloned upstream of an ires/eGFP sequence into
a self-inactivating lentiviral vector plasmid and viral stocks were lead to new insights into some features of human MPD
produced by triple transfection of293T cells along with lentiviral including CML.
gag-pol and vesicular stomatitis virus G glycoprotein constructs.
Lentivirus-containing supernatant medium was collected for2con-
secutive days and concentrated by ultracentrifugation.HSC were Experimental Procedures
purified from C57BL/6-Ly5.2mice and cultured in200?l in96-well
plates with concentrated lentiviral stocks(1:50to1:100dilution)in Mouse
Iscove’s Modified Dulbecco’s Media(IMDM)containing5%FCS, The congenic mouse strains C57BL/6-Ly5.1or Ly5.2and the
50?M2-mercaptoethanol,SCF(20ng/ml),Flt3L(20ng/ml),and IL-RAG2?/?-Ly5.2mice were used as described(Kondo et al.,1997).
11(10ng/ml)for12–16hr at37?C.Transduced HSC were mixed Jun B?/?Ubi-jun B(Schorpp-Kistner et al.,1999),jun B f/f(Kenner et
with a radioprotective dose of2.5?105congenic Sca-1-depleted al.,2004),MORE-Cre(Tallquist and Soriano,2000),and Mx-Cre
C57BL/6-Ly5.1helper bone marrow cells and transplanted via retro-(Ku¨hn et al.,1995)mice were generated as described.The MRP8-
orbital injection into lethally irradiated(960rad)congenic C57BL/ Cre-ires/GFP construct was generated by inserting a Cre-ires/GFP
6-Ly5.1recipients.
cassette into the unique BglII site of the human MRP8expression
vector(Lagasse and Weissman,1994)and two founder transgenic
Methylcellulose CFU Assays
lines were obtained after DNA microinjection into fertilized F1
To support the formation of myeloid colonies,purified LT-HSC(100 (C57BL/6?C3H)eggs.All the mice were backcrossed between
cells)were cultured in Iscove’s Modified Dulbecco’s Media(IMDM)-two to seven times to the C57BL/6background before being used
based methylcellulose media(Methocult M3100,StemCell Technol-for the experiments.To induce Mx-Cre-mediated deletion of jun B
ogies)supplemented as described(Akashi et al.,1999).
during adult hematopoiesis,4-week-old jun B f/f Mx-Cre mice were
injected four times at2-day intervals with250?g poly(I/C)in200
?l PBS.All mice were maintained in Stanford University’s Research Quantitative RT-PCR Analysis
Animal Facility in accordance with Stanford University guidelines.
Total RNA was isolated using Trizol reagent(Invitrogen)from purified The classification of the nonlymphoid hematopoietic neoplasm de-HSC(2,000to5,000cells),progenitors(10,000to30,000cells),or scribed in this study was performed according to the guidelines
mature cell populations(30,000to100,000cells),digested with edited by the hematopathology subcommittee of the mouse models DNase I to remove DNA contamination and used for reverse-tran-of human cancers consortium(Kogan et al.,2002).Indolent granulo-
scription according to the manufacturer’s instructions(SuperScript cytic expansion corresponds to an increase by?2-fold in the total II?kit,Invitrogen).For qRT-PCR analysis,primer sequences were
Cell
442
designed using Primer Express software(Applied Biosystems)and C.,and Lawrence,H.J.(2003).The transcriptome of the leukemo-
genic homeoprotein HOXA9in human hematopoietic cells.Blood are available upon request.All reactions were performed in an ABI-
7000sequence detection system using SYBR Green PCR Core re-103,1676–1684.
agents according to the manufacturer’s instructions(Applied Bio-Feldman,E.,Najfeld,V.,Schuster,M.,Roboz,G.,Chadburn,A.,and systems).PCR amplification was performed in a12?l final volume Silver,R.T.(2003).The emergence of Ph-,trisomy-8?cells in patients containing1X SYBR Green PCR buffer,2mM magnesium chloride,with chronic myeloid leukemia treated with imatinib mesylate.Exp.
0.5mM dNTP mix with dUTP,8ng of each primer,0.1U AmpErase Hematol.31,702–707.
UNG,0.25U AmpliTaq Gold,and2?l of DNA or cDNA templates
Fialkow,P.J.,Jacobson,R.J.,and Papayannopoulou,T.(1977). using50?C for2min and95?C for10min followed by45cycles at
Chronic myelocytic leukemia:clonal origin in a stem cell common 95?C for15s and60?C for1min.For each sample,expression of
to the granulocyte,erythrocyte,platelet and monocyte/macrophage. the?-actin gene was used to normalize the amount of the investi-
Am.J.Med.63,125–130.
gated transcript.
Fialkow,P.J.,Martin,P.J.,and Najfed,V.(1981).Evidence of a multi-
step pathogenesis of chronic myelogenous leukemia.Blood58, Acknowledgments
158–163.
We are grateful to L.Krenner for providing us with the jun B?/?mice.Jamieson,C.H.,Ailles,L.E.,Dylla,S.J.,Muijtjens,M.,Jones,C., We thank L.Ailles,T.Serwold,and A.Wagers for critical reading of Zehnder,J.L.,Gotlib.J.,Li,K.,Manz,M.G.,Keating,A.,Sawyers, the manuscript,L.Jerabek for lab management,M.Muijtjens and C.L.,and Weissman,I.L.(2004).Granulocyte/Macrophage progeni-S.Smith for DNA microinjection,O.Zilian for the Mx-Cre mice,and tors in chronic myelogenous leukemia are candidate leukemic stem L.Hidalgo and https://www.doczj.com/doc/a35051446.html,varro for animal care.E.P.was supported by cells that activate the beta-catenin pathway.N.Engl.J.Med.351, an EMBO long-term fellowship and is currently a fellow of the Jose657–667.
Carreras International Leukemia Foundation.This work was sup-
Jaiswal,S.,Traver,D.,Miyamoto,T.,Akashi,K.,Lagasse,E.,and ported by the NIH(CA55209;CA86017)and a De Villier Award from
Weissman,I.L.(2003).Development of murine chronic and acute the Leukemia and Lymphoma Society to I.L.W.
myelogenous leukemia with targeted expression of BCR/ABL to
myeloid https://www.doczj.com/doc/a35051446.html,A100,10002–10007. Received:June28,2004
Kantarjian,H.M.,O’Brien,S.,Cortes,J.,Giles,F.J.,Faderl,S.,Issa, Revised:August26,2004
J.P.,Garcia-Manero,G.,Rios,M.B.,Shan,J.,Andreeff,M.,Keating, Accepted:September7,2004
M.,and Talpaz,M.(2003).Results of decitabine(5-aza-2?deoxycyti-Published:October28,2004
dine)therapy in130patients with chronic myelogenous leukemia.
Cancer98,522–528.
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晋灵公不君（宣公二年） 原文： 晋灵公不君。厚敛以雕墙。从台上弹人，而观其辟丸也。宰夫胹熊蹯不熟，杀之，寘诸畚，使妇人载以过朝。赵盾、士季见其手，问其故而患之。将谏，士季曰：“谏而不入，则莫之继也。会请先，不入，则子继之。”三进及溜，而后视之，曰：“吾知所过矣，将改之。”稽首而对曰：“人谁无过？过而能改，善莫大焉。诗曰：‘靡不有初，鲜克有终。’夫如是，则能补过者鲜矣。君能有终，则社稷之固也，岂惟群臣赖之。又曰：‘衮职有阙，惟仲山甫补之。’能补过也。君能补过，衮不废矣。” 犹不改。宣子骤谏，公患之，使鉏麑贼之。晨往，寝门辟矣，盛服将朝。尚早，坐而假寐。麑退，叹而言曰：“不忘恭敬，民之主也。贼民之主，不忠；弃君之命，不信。有一于此，不如死也！”触槐而死。 秋九月，晋侯饮赵盾酒，伏甲将攻之。其右提弥明知之，趋登曰：“臣侍君宴，过三爵，非礼也。”遂扶以下。公嗾夫獒焉。明搏而杀之。盾曰：“弃人用犬，虽猛何为！”斗且出。提弥明死之。 初，宣子田于首山，舍于翳桑。见灵辄饿，问其病。曰：“不食三日矣！”食之，舍其半。问之，曰：“宦三年矣，未知母之存否。今近焉，请以遗之。”使尽之，而为之箪食与肉，寘诸橐以与之。既而与为公介，倒戟以御公徒，而免之。问何故，对曰：“翳桑之饿人也。”问其名居，不告而退。——遂自亡也。 乙丑，赵穿①攻灵公于桃园。宣子未出山而复。大史书曰：“赵盾弑其君。”以示于朝。宣子曰：“不然。”对曰：“子为正卿，亡不越竟，反不讨贼，非子而谁？”宣子曰：“呜呼!‘我之怀矣，自诒伊戚。’其我之谓矣。” 孔子曰：“董狐，古之良史也，书法不隐。赵宣子，古之良大夫也，为法受恶。惜也，越竞乃免。” 译文： 晋灵公不行君王之道。他向人民收取沉重的税赋以雕饰宫墙。他从高台上用弹弓弹人，然后观赏他们躲避弹丸的样子。他的厨子做熊掌，没有炖熟，晋灵公就把他杀了，把他的尸体装在草筐中，让宫女用车载着经过朝廷。赵盾和士季看到露出来的手臂，询问原由后感到很忧虑。他们准备向晋灵公进谏，士季说：“如果您去进谏而君王不听，那就没有人能够再接着进谏了。还请让我先来吧，不行的话，您再接着来。”士季往前走了三回，行了三回礼，一直到屋檐下，晋灵公才抬头看他。晋灵公说：“我知道我的过错了，我会改过的。”士季叩头回答道：“谁能没有过错呢？有过错而能改掉，这就是最大的善事了。《诗经》说：‘没有人向善没有一个开始的，但却很少有坚持到底的。’如果是这样，那么能弥补过失的人是很少的。您如能坚持向善，那么江山就稳固了，不只是大臣们有所依靠啊。
1、 Blindness is a very serious disability. 失明是非常严重的残疾。 2、 Though he is disabled, he is a gifted artist. 尽管身残疾 (的 )，他是一个有天赋的艺术家。 3、 person with a hearing and eyesight disability misses many things. 一位听力和视力残疾的人会错过很多东西。 4、 Beware of the love syndrome.当心恋爱综合症。 5、 Rosalyn had been there frequently in past years for treatment for infantile paralysis. 罗莎琳在过去的几年中频频来这里治疗小儿麻痹。 6、 Harris went on top in the last lap. 哈里斯在最后一圈跑到了最前面。 7、 He had a high ambition to be a headmaster. 他的抱负是想成为一名校长。 8、 I will love the ambitious for they can inspire me!我爱雄心壮志的人，因为他们能激励我！ 9、 We will have dictation today.我们今天要听写了。 10、 I wrote the letter at Sally's dictation . 我照萨利的口述写信。 11、 The campus is noisy today.今天校园里很吵。 12、 Do believe there is no best man, only much suitable one. 要相信没有最好男人，只有更适合的。 13、 Trees hid the entry of the cave. 洞穴的入口被树丛遮掩。 14、 Fresh air is beneficial to people's health. 新鲜空气对人的健康有好处。 15、 In other words, a classic fudge. 总之一句话，一派胡言。 16、 Mother reproached me for being too clumsy. 母亲责备我笨手笨脚。 17、 He used to be quiet, but now he is very outgoing. 他以前性格沉默少言，但现在很外向。 18、 How do you adapt to this new environment? 你要怎样适应这种新环境呢?。 19、 The bench often does duty for a table. 这条长凳经常当桌子用。 20、 The doctor told me to cut out meat for my fat. 由于肥胖，大夫叫我停止吃肉 21、 He applied his eye to the microscope. 他把眼睛贴近显微镜。 22、 She was out of breath from climbing the stairs. 爬楼梯使她上气不接下气的。 23、 A servant is known by his master's absence.主人不在就可以看出仆人的品行来。 24、 You are a milk-livered fellow!你真是一个胆小的家伙！ 25、 Thank you, my fellow colleagues. 谢谢，同事们。 26、 No joy without annoy. 没有无烦恼之快乐。 27、 I am really very annoyed about it. 我对此事真是很生气。 28、 I felt annoyance at being teased.我恼恨“别人”取笑我。 29、 I let go of all annoyance. 我放下所有烦恼。 30、 He has his faults, but all in all , he is a good guy. 他有缺点，但总的来说，他是不错的人。 单词拼读举例:Di sa bi li ty di sa bled syn drome in fan tile pa ra ly sis am bi tion am bi tious
Unit 3 Under the sea 【导语】捕鲸业是世界上古老的行业之一，它的历史发展和现状如何呢？ The whaling industry is one of the oldest industries in existence.It has undergone vast changes since its beginning，primarily due to the creation of the International Whaling https://www.doczj.com/doc/a35051446.html,mercial whaling still continues throughout the world，but the whaling quotas are restricted by the commission，thereby helping conserve the whale populations. Whaling refers to hunting wh ales primarily for their oil，meat and bones.Whaling dates back to prehistoric times，most prevalent in Norway and Japan.The hunt became an industry in the 1600s in the Arctic regions，mostly by the Dutch and British.American colonists started whaling in the 1700s.Nantucket whalers were the first to kill a sperm whale，and the discovery of spermaceti in the whale's head made the sperm whale one of the most highly prized catches.Whaling ships set out from New England into the Pacific for extremely long voyages to hunt down these whales. Whaling industry has been a part of a variety of countries' industries.The following countries were the earliest pioneers in the whaling industry：England，France，Germany，Iceland，Japan，the Netherlands，Norway and the American colonies (although they were not a country at the time)．The following countries still practice whaling in some form or fashion：Canada，Greenland，the Grenadines，Iceland，Indonesia，Japan，the Netherlands，Norway，Russia and the United States. 【词海拾贝】 1．vast adj.广阔的；巨大的 2．primarily adv.首先；最初 3．commission n．委员会 4．commercial adj.商业的 5．quota n．配额 6．prevalent adj.盛行的 7．sperm whale 巨头鲸；抹香鲸 【问题思考】 1．What helps conserve the whale populations? _______________________________________________________ 答案：The whaling quotas.
如何翻译古文 学习古代汉语，需要经常把古文译成现代汉语。因为古文今译的过程是加深理解和全面运用古汉语知识解决实际问题的过程，也是综合考察古代汉语水平的过程。学习古代汉语，应该重视古文翻译的训练。 古文翻译的要求一般归纳为信、达、雅三项。“信”是指译文要准确地反映原作的含义，避免曲解原文内容。“达”是指译文应该通顺、晓畅，符合现代汉语语法规范。“信”和“达”是紧密相关的。脱离了“信”而求“达”，不能称为翻译；只求“信”而不顾“达”，也不是好的译文。因此“信”和“达”是文言文翻译的基本要求。“雅”是指译文不仅准确、通顺，而且生动、优美，能再现原作的风格神韵。这是很高的要求，在目前学习阶段，我们只要能做到“信”和“达”就可以了。 做好古文翻译，重要的问题是准确地理解古文，这是翻译的基础。但翻译方法也很重要。这里主要谈谈翻译方法方面的问题。 一、直译和意译 直译和意译是古文今译的两大类型，也是两种不同的今译方法。 1．关于直译。所谓直译，是指紧扣原文，按原文的字词和句子进行对等翻译的今译方法。它要求忠实于原文，一丝不苟，确切表达原意，保持原文的本来面貌。例如： 原文：樊迟请学稼，子曰：“吾不如老农。”请学为圃。子曰：“吾不如老圃。”（《论语?子路》） 译文：樊迟请求学种庄稼。孔子道：“我不如老农民。”又请求学种菜蔬。孔子道：“我不如老菜农。”（杨伯峻《论语译注》） 原文：齐宣王问曰：“汤放桀，武王伐纣，有诸？”（《孟子?梁惠王下》） 译文：齐宣王问道：“商汤流放夏桀，武王讨伐殷纣，真有这回事吗？（杨伯峻《孟子译注》） 上面两段译文紧扣原文，字词落实，句法结构基本上与原文对等，属于直译。 但对直译又不能作简单化理解。由于古今汉语在文字、词汇、语法等方面的差异，今译时对原文作一些适当的调整，是必要的，并不破坏直译。例如： 原文：逐之，三周华不注。（《齐晋鞌之战》） 译文：〔晋军〕追赶齐军，围着华不注山绕了三圈。
鞌之战选自《左传》又名《鞍之战》原文：楚癸酉，师陈于鞌(1)。邴夏御侯，逢丑父为右②。晋解张御克，郑丘缓为右(3)。侯日：“余姑翦灭此而朝食(4)”。不介马而驰之⑤。克伤于矢，流血及屦2 未尽∧6)，曰：“余病矣(7)！”张侯曰：“自始合(8)，而矢贯余手及肘(9)，余折以御，左轮朱殷(10)，岂敢言病吾子忍之！”缓曰：“自始合，苟有险，余必下推车，子岂＿识之(11)然子病矣！”张侯曰：“师之耳目，在吾旗鼓，进退从之。此车一人殿之(12)，可以集事(13)，若之何其以病败君之大事也擐甲执兵(14)，固即死也(15)；病未及死，吾子勉之(16)！”左并辔(17) ，右援拐鼓(18)。马逸不能止(19)，师从之，师败绩。逐之，三周华不注(20) 韩厥梦子舆谓己曰：“旦辟左右！”故中御而从齐侯。邴夏曰：“射其御者，君子也。”公曰：“谓之君子而射之，非礼也。”射其左，越于车下；射其右，毙于车中。綦毋张丧车，从韩厥，曰：“请寓乘。”从左右，皆肘之，使立于后。韩厥俛，定其右。逢丑父与公易位。将及华泉，骖絓于木而止。丑父寝于轏中，蛇出于其下，以肱击之，伤而匿之，故不能推车而及。韩厥执絷马前，再拜稽首，奉觞加璧以进，曰：“寡君使群臣为鲁、卫请，曰：‘无令舆师陷入君地。’下臣不幸，属当戎行，无所逃隐。且惧奔辟而忝两君，臣辱戎士，敢告不敏，摄官承乏。” 丑父使公下，如华泉取饮。郑周父御佐车，宛茷为右，载齐侯以免。韩厥献丑父，郤献子将戮之。呼曰：“自今无有代其君任患者，有一于此，将为戮乎”郤子曰：“人不难以死免其君，我戮之不祥。赦之，以劝事君者。”乃免之。译文1：在癸酉这天，双方的军队在鞌这个地方摆开了阵势。齐国一方是邴夏为齐侯赶车，逢丑父当车右。晋军一方是解张为主帅郤克赶车，郑丘缓当车右。齐侯说:“我姑且消灭了这些人再吃早饭。”不给马披甲就冲向了晋军。郤克被箭射伤，血流到了鞋上，但是仍不停止擂鼓继续指挥战斗。他说:“我受重伤了。”解张说:“从一开始接战，一只箭就射穿了我的手和肘，左边的车轮都被我的血染成了黑红色，我哪敢说受伤您忍着点吧！”郑丘缓说:“从一开始接战，如果遇到道路不平的地方，我必定（冒着生命危险）下去推车，您难道了解这些吗不过，您真是受重伤了。”daier 解张说:“军队的耳朵和眼睛，都集中在我们的战旗和鼓声，前进后退都要听从它。这辆车上还有一个人镇守住它，战事就可以成功。为什么为了伤痛而败坏国君的大事呢身披盔甲，手执武器，本来就是去走向死亡，伤痛还没到死的地步，您还是尽力而为吧。”一边说，一边用左手把右手的缰绳攥在一起，用空出的右手抓过郤克手中的鼓棰就擂起鼓来。（由于一手控马，）马飞快奔跑而不能停止，晋军队伍跟着指挥车冲上去，把齐军打得打败。晋军随即追赶齐军，三次围绕着华不注山奔跑。韩厥梦见他去世的父亲对他说:“明天早晨作战时要避开战车左边和右边的位置。”因此韩厥就站在中间担任赶车的来追赶齐侯的战车。邴夏说:“射那个赶车的，他是个君子。”齐侯说: “称他为君子却又去射他，这不合于礼。”daier 于是射车左，车左中箭掉下了车。又射右边的，车右也中箭倒在了车里。（晋军的）将军綦毋张损坏了自己的战车，跟在韩厥的车后说: “请允许我搭乗你的战车。”他上车后，无论是站在车的左边，还是站在车的右边，韩厥都用肘推他，让他站在自己身后——战车的中间。韩厥又低下头安定了一下受伤倒在车中的那位自己的车右。于是逢丑父和齐侯（乘韩厥低头之机）互相调换了位置。将要到达华泉时，齐侯战车的骖马被树木绊住而不能继续逃跑而停了下来。（头天晚上）逢丑父睡在栈车里，有一条蛇从他身子底下爬出来，他用小臂去打蛇，小臂受伤，但他（为了能当车右）隐瞒了这件事。由于这样，他不能用臂推车前进，因而被韩厥追上了。韩厥拿着拴马绳走到齐侯的马前，两次下拜并行稽首礼，捧着一杯酒并加上一块玉璧给齐侯送上去，
Unit 3 Underthesea 基础知识默写篇 一、分层单词 写作词汇 1. vt.放弃;遗弃;抛弃 2. vt.当场见到;目击 n.目击者;证人;证据 3. vi.& n.暂停;中止 4. vt.拖;拉;扯 5. vi.逃避;逃跑 vt.逃离 6. n.住所;住宿 7. n.关系;血缘关系;交往 8. adj.好的;整齐的;匀称的 9. adj.生动的;鲜明的;鲜艳的 10. adj.纯的;纯粹的;纯洁的 11. prep.在……对面 adj.相对的;相反的 12. vt.催促;极力主张;驱策 13. vi.思考 vt.映射;反射;思考 14. adj.意识到的;知道的 阅读词汇 1.jog vi.&vt. 2.dive vi.& n. 3.suck vt.& vi. 4.conservation n. 5.target n. 6.boundary n.
7.pension n. 8.annual adj. & n. 9.narrow adj. 10.shallow adj. 11.steep adj. 12.awesome adj. 13.seaside n.&adj. 14.telescope n. 15.teamwork n. 16.dimension n. 拓展词汇 1. n.深(度);深处→adj.深的adv. 深深地 2. adj.好吃的;可口的→ n.味道;品位;鉴赏力v.尝;品尝;有……的味道 3. adj.意识到的;知道的→ n.意识;认识 4. vi.思考vt.反射;思考→ n.反射;思考 5. adj.锐利的;锋利的;敏捷的→v.(使)变得锋利 6. vt.催促;极力主张;驱策→ n.紧急;迫切;催促→adj.紧急的;迫切的;催促的 7. vt.恐吓 vi.受惊吓→adj.害怕的 二、高频短语 1. 帮助(某人)摆脱困境或危难 2. 对……知道、明白;意识到…… 3. 举起;阻碍 4. 整理;收拾 5. 在此期间;与此同时 6. 靠近 7. upside down 8. (be) scared to death 9. aim at
《鞌之战》阅读答案（附翻译）原文及翻 译 鞌之战[1] 选自《左传成公二年（即公元前589年）》 【原文】 癸酉，师陈于鞌[2]。邴夏御齐侯[3]，逢丑父为右[4]。晋解张御郤克，郑丘缓为右[5]。齐侯曰：余姑翦灭此而朝食[6]。不介马而驰之[7]。郤克伤于矢，流血及屦，未绝鼓音[8]，曰：余病[9]矣！张侯[10]曰：自始合，而矢贯余手及肘[11]，余折以御，左轮朱殷[12]，岂敢言病。吾子[13]忍之！缓曰：自始合，苟有险[14]，余必下推车，子岂识之[15]？然子病矣！张侯曰：师之耳目，在吾旗鼓，进退从之[16]。此车一人殿之[17]，可以集事[18]，若之何其以病败君之大事也[19]？擐甲执兵，固即死也[20]。病未及死，吾子勉之[21]！左并辔[22]，右援枹而鼓[23]，马逸不能止[24]，师从之。齐师败绩[25]。逐之，三周华不注[26]。 【注释】 [1]鞌之战：春秋时期的著名战役之一。战争的实质是齐、晋争霸。由于齐侯骄傲轻敌，而晋军同仇敌忾、士气旺盛，战役以齐败晋胜而告终。鞌：通鞍，齐国地名，在今山东济南西北。 [2]癸酉：成公二年的六月十七日。师，指齐晋两国军队。陈，
列阵，摆开阵势。 [3]邴夏：齐国大夫。御，动词，驾车。御齐侯，给齐侯驾车。齐侯，齐国国君，指齐顷公。 [4]逢丑父：齐国大夫。右：车右。 [5]解张、郑丘缓：都是晋臣，郑丘是复姓。郤（x ）克，晋国大夫，是这次战争中晋军的主帅。又称郤献子、郤子等。 [6]姑：副词，姑且。翦灭：消灭，灭掉。朝食：早饭。这里是吃早饭的意思。这句话是成语灭此朝食的出处。 [7]不介马：不给马披甲。介：甲。这里用作动词，披甲。驰之：驱马追击敌人。之：代词，指晋军。 [8] 未绝鼓音：鼓声不断。古代车战，主帅居中，亲掌旗鼓，指挥军队。兵以鼓进，击鼓是进军的号令。 [9] 病：负伤。 [10]张侯，即解张。张是字，侯是名，人名、字连用，先字后名。 [11]合：交战。贯：穿。肘：胳膊。 [12]朱：大红色。殷：深红色、黑红色。 [13]吾子：您，尊敬。比说子更亲切。 [14]苟：连词，表示假设。险：险阻，指难走的路。 [15]识：知道。之，代词，代苟有险，余必下推车这件事，可不译。 [16]师之耳目：军队的耳、目（指注意力）。在吾旗鼓：在我们
Unit 3 under the sea 1.witness (1) v 当场见到，目击 . 在我昨天回家的路上路上我亲眼目睹一场事故。 (2) v表明，说明Her flushed（通红）face she felt. 她通红的脸表明他、她感到非常高兴。 (3) v 为…作证常与to 连用witness to 为某事作证说 He enter the room. 他作证说他看到那个人进入房间。 (4) n (C) “目击者，证人”常与to连用be a witness to 是…的目击者 The police found the witness to the murder case. 警察找到了那个谋杀案的目击者 (5) n (C,U) 证词，证据，证明give witness on behalf of 替…作证 The old man an accused person. 那位老人为被告作证。 2. sort (1) vt 把…分类,拣选 She is . 她正忙于给信件分类。 (2) sort out 整理（好） I 我已把信分好 (3) n 种类，类别 A hammer is .斧头是一种工具。 He’s the sort of person I really dislike. (4)词组sort of 颇为，有几分I feel . 我感觉有点生病了。 all sorts of 相当于all kinds of 3. accommodation (1) 房间，住所The accommodation of this hotel is scare. （不足） (2) 膳宿（常用复数） 我们今晚能找到旅馆住宿吗？Can we ____find accommodations at a hotel__ for tonight? (3) accommodate 向…提供住处，接纳(v) 相关短语：(1) make accommodations for 为…提供膳宿(2) book accommodation at a hotel 在旅馆预订房间 (4) accommodate …to = adapt… to 使…适应 You will have to accommodate yourself to the situation. 4. yell（1）v.叫喊。 （1）yell(out) at sb.朝某人大喊大叫 .她对淘气的孩子大喊大叫。 They yelled at him to stop. （2）n. /叫声，喊声 E.g. a yell of delight / warning 5. throw (1) 投，掷，扔，抛 Throw the ball to your younger brother. He was thrown into prison.被关进… (2) 匆忙或随便穿/脱 He threw a blanket over the injured man. 他匆忙给伤者披上毯子。
《鞌之战》阅读答案（附翻译） 《鞌之战》阅读答案（附翻译） 鞌之战[1] 选自《左传·成公二年（即公元前589年）》 【原文】 癸酉，师陈于鞌[2]。邴夏御齐侯[3]，逢丑父为右[4]。晋解张御郤克，郑丘缓为右[5]。齐侯曰：“余姑 翦灭此而朝食[6]。”不介马而驰之[7]。郤克伤于矢， 流血及屦，未绝鼓音[8]，曰：“余病[9]矣！”张侯[10]曰：“自始合，而矢贯余手及肘[11]，余折以御，左轮 朱殷[12]，岂敢言病。吾子[13]忍之！”缓曰：“自始合，苟有险[14]，余必下推车，子岂识之[15]？——然 子病矣！”张侯曰：“师之耳目，在吾旗鼓，进退从之[16]。此车一人殿之[17]，可以集事[18]，若之何其以 病败君之大事也[19]？擐甲执兵，固即死也[20]。病未 及死，吾子勉之[21]！”左并辔[22]，右援枹而鼓[23]，马逸不能止[24]，师从之。齐师败绩[25]。逐之，三周 华不注[26]。 【注释】 [1]鞌之战：春秋时期的著名战役之一。战争的实质是齐、晋争霸。由于齐侯骄傲轻敌，而晋军同仇敌忾、 士气旺盛，战役以齐败晋胜而告终。鞌：通“鞍”，齐
国地名，在今山东济南西北。 [2]癸酉：成公二年的六月十七日。师，指齐晋两国军队。陈，列阵，摆开阵势。 [3]邴夏：齐国大夫。御，动词，驾车。御齐侯，给齐侯驾车。齐侯，齐国国君，指齐顷公。 [4]逢丑父：齐国大夫。右：车右。 [5]解张、郑丘缓：都是晋臣，“郑丘”是复姓。郤（xì）克，晋国大夫，是这次战争中晋军的主帅。又称郤献子、郤子等。 [6]姑：副词，姑且。翦灭：消灭，灭掉。朝食：早饭。这里是“吃早饭”的意思。这句话是成语“灭此朝食”的出处。 [7]不介马：不给马披甲。介：甲。这里用作动词，披甲。驰之：驱马追击敌人。之：代词，指晋军。 [8]未绝鼓音：鼓声不断。古代车战，主帅居中，亲掌旗鼓，指挥军队。“兵以鼓进”，击鼓是进军的号令。 [9]病：负伤。 [10]张侯，即解张。“张”是字，“侯”是名，人名、字连用，先字后名。 [11]合：交战。贯：穿。肘：胳膊。 [12]朱：大红色。殷：深红色、黑红色。 [13]吾子：您，尊敬。比说“子”更亲切。
Unit 3 Under the sea 语言点 编稿：牛新阁审稿：王春霞 学习目标 重点词汇 annual witness accommodation opposite yell pause drag depth urge abandon target reflect scare awesome 重点短语 in the meantime be aware of ahead of ... to death help （...）out upside down 重点句型 It takes sb. some time/ ... to do sth.的变化 a time when... 知识讲解 重点词汇 annual 【原句回放】It was a time when the killer whales, or “killers” as they were then called, helped the whalers catch the baleen whales that were on their annual migration. 那个时期，虎鲸（当时被称为“杀手”）帮助捕鲸人在每年须鲸迁徙时捕捉须鲸。 【点拨】annual adj. 1. 每年的，一年一次的 the annual output 年产量an annual report 年度报告 Steel output reached an annual figure of one million tons. 钢的年产量达到100万吨。 2. 按年度计算的 the annual income 年收入the annual rainfall 【拓展】annually adv. 每年地，年度地 witness 【原句回放】I thought, at the time, that this was just a story but then I witnessed it with my own eyes many times. 当时我以为这只是一个故事罢了，但是后来我亲眼见过多次。 【点拨】witness vt.& vi.亲眼看见，目睹，作证； n.见证人；目击者（与of连用） We were witnessing the most important scientific development of the century. 我们正目睹着本世纪最重要的科学发展。 We witnessed great changes in the city. 我们见证了这个城市的巨大变化。 The police found the witness of the murder case. 警察找到了那起谋杀案的证人。 There was no witness at the scene of the accident. 在事故现场没有目击证人。 【拓展】常见搭配：witness to (doing...)为……作证 bear / give witness to 为......作证 in witness of 作为......的证据 a living witness to 活生生的证人 witness stand 证人席(美)＝witness box (英) He witnessed to having seen the man enter the building.
模块7各单元词汇和短语复习 Unit 1 Living Well Part One 根据首字母或汉语提示写出完整的单词: 1. An unexpected accident made Sang Lan a girl with a physical d____________, but she never gave up. 2. We often observe the tiny cells with m______________ in our biology classes. 3. The production of iron and steel is the most important i______________ of the city. 4. Taiwan is part of China and not an i_______________ state. 5. I have passed all the exams and I am waiting to get the graduation c_______________. 6. A place is a______________ if it is possible to reach it. 7. I was not alone on the journey. Instead, I had so many c________________. 8. You can refer to old people in general as the _______________. 9. When something can help people or improve their lives, we say it is ______________ to people. 10. When the disabled meet with difficulty, we should do what we can to give them a___________. 11. One of his ________________ (志向) is to become a world-wide famous pop singer. 12. He was _____________(笨拙的) with a tool. 13. The patient’s _______________(呼吸) grew stronger and stronger. 14. Who took his place and controlled the company during his ________________ (不在) ? 15. I’m going to give some suggestions to this famous ________________(建筑师). 16. A man’s _______________（尊严）doesn’t depends on his wealth but on his character. Part Two 一、词形变换（用所给词的适当形式填空） 1. The ______________ is good at ______________ TV plays from the stories he reads about or hears. (adapt) 2. The country used to be _____________ on Britain in many ways, but now it has become absolutely_______________. (depend) 3. We have ___________ a lot from the co-operation and I’m sure it will continue to be ________ to both of us. (benefit) 4. Her _____________ lecture ____________ all of us. We won’t forget her ____________. (encourage) 5. ______________ people should be treated equally in spite of their ______________. (disability) 6. You were _____________ yesterday, Tom. Can you tell me the reason for you _____________? (absent) 7. The ______________ serving the passengers on the train is of good_______________. (conduct) 8. ________________ countries pay much attention to the development of ________________. (industry) 二、翻译句子 1. 应该给残疾人尽可能多的鼓励和援助。 ______________________________________________________________________________ 2. 换句话说，这些措施对我们的工业发展是有益的。 ______________________________________________________________________________ 3. 总之，在学校寄宿(board)能帮助学生学会独立。 ______________________________________________________________________________
左传《齐晋鞌之战》原文+翻译+注释 楚癸酉，师陈于鞌(1)。邴夏御侯，逢丑父为右②。晋解张御克，郑丘缓 为右(3)。侯日：“余姑翦灭此而朝食(4)”。不介马而驰之⑤。克伤于矢， 流血及屦2未尽∧?6)，曰：“余病矣(7)！”张侯曰：“自始合(8)，而矢贯余手 及肘(9)，余折以御，左轮朱殷(10)，岂敢言病？吾子忍之！”缓曰：“自始合，苟有险，余必下推车，子岂＿识之(11)？然子病矣！”张侯曰：“师之耳目，在 吾旗鼓，进退从之。此车一人殿之(12)，可以集事(13)，若之何其以病败君之大事也？擐甲执兵(14)，固即死也(15)；病未及死，吾子勉之(16)！”左并辔(17) ，右援拐?鼓(18)。马逸不能止(19)，师从之，师败绩。逐之，三周华不注(20) 韩厥梦子舆谓己曰：“旦辟左右！”故中御而从齐侯。邴夏曰：“射其御者，君子也。”公曰：“谓之君子而射之，非礼也。”射其左，越于车下；射其右，毙于车中。綦毋张丧车，从韩厥，曰：“请寓乘。”从左右，皆肘之，使立于后。韩厥俛，定其右。逢丑父与公易位。将及华泉，骖絓于木而止。丑父寝于轏中，蛇出于其下，以肱击之，伤而匿之，故不能推车而及。韩厥执絷马前，再拜稽首，奉觞加璧以进，曰：“寡君使群臣为鲁、卫请，曰：‘无令舆师陷入君地。’下臣不幸，属当戎行，无所逃隐。且惧奔辟而忝两君，臣辱戎士，敢告不敏，摄官承乏。”丑父使公下，如华泉取饮。郑周父御佐车，宛茷为右，载齐侯以免。韩厥献丑父，郤献子将戮之。呼曰：“自今无有代其君任患者，有一于此，将为戮乎？”郤子曰：“人不难以死免其君，我戮之不祥。赦之，以劝事君者。”乃免之。 在癸酉这天，双方的军队在鞌这个地方摆开了阵势。齐国一方是邴夏为齐侯赶车，逢丑父当车右。晋军一方是解张为主帅郤克赶车，郑丘缓当车右。齐侯说:“我姑且消灭了这些人再吃早饭。”不给马披甲就冲向了晋军。郤克被箭射伤，血流到了鞋上，但是仍不停止擂鼓继续指挥战斗。他说:“我受重伤了。”解张说:“从一开始接战，一只箭就射穿了我的手和肘，左边的车轮都被我的血染成了黑红色，我哪敢说受伤？您忍着点吧！”郑丘缓说:“从一开始接战，如果遇到道路不平的地方，我必定（冒着生命危险）下去推车，您难道了解这些吗？不过，您真是受重伤了。”daier解张说:“军队的耳朵和眼睛，都集中在我们的战旗和鼓声，前进后退都要听从它。这辆车上还有一个人镇守住它，战事就可以成功。为什么为了伤痛而败坏国君的大事呢？身披盔甲，手执武器，本来就是去走向死亡，伤痛还没到死的地步，您还是尽力而为吧。”一边说，一边用左手把右手的缰绳攥在一起，用空出的右手抓过郤克手中的鼓棰就擂起鼓来。（由于一手控马，）马飞快奔跑而不能停止，晋军队伍跟着指挥车冲上去，把齐军打得打败。晋军随即追赶齐军，三次围绕着华不注山奔跑。
第5讲Under the sea 词汇篇 __________________________________________________________________________________ __________________________________________________________________________________ 1.熟练掌握重点单词及其用法； 2.能够熟练运用重点短语和句型。 一. 重点词汇 1.annual adj. 每年的；按年度计算的n. 年刊；年鉴 [重点用法] annually adv. 年年地，每年地 [典例] 1) an annual income. 年收入 2) an annual report年度报告 3) Premier Wen Jiabao noted that the two most important problems would be previous to anything else in the government annual report.温家宝总理在政府年度报告中指出要优先解决这两大问题。 2.witness n. 目击者；证人；证据vt. 当场见到；目击 [典例] 1) He is the witness to the accident. 事故的目击者 2) This old auditorium has witnessed many ceremonies. 这个古老的礼堂内举行过许多次典礼。 3.accommodation住所 [重点用法] accommodate v.向...提供，容纳，调和；适应 accommodation address临时通讯处 accommodation allowance膳宿津贴 [典例] 1) The high cost of accommodation makes life difficult for students in London. 由于住宿费用昂贵，伦敦的学生感到生活困难。 2) The university offers excellent accommodation for summer visitors. 这所大学为夏季来访者提供了很好的住宿。 4.abandon vt. 放弃；遗弃；抛弃n. 放任，狂热 [重点用法] abandon oneself to sth./doing sth. 沉溺于 abandon …to sb. 把……舍弃给…... with abandon放任地；放纵地；纵情地 [典例]
BOOK7 Unit1单词练习题 一．根据所给的首字母写出单词的正确形式。 1.What we choose and decide forms our life, but a_______ decides our choice and decision. 2.We should design more websites that are both technically a________ and also usable for people with disabilities. 3.As is known to all, Mo Yan was awarded the 2012 Nobel Prize for l_________. 4.The whole class c________ me on getting the first prize in the English competition. 5.Recently I watched a cartoon a________ from the famous drama by Shakespeare. 二．根据句意用括号内所给单词的适当形式填空。 1.It is known to all that fresh air is _____ to our health and the new park ______ us all, so we should keep it clean.( benefit ) 2.The little boy made up a tale for his ______ ( absent ) from school. 3.Praise acts as an _____ to the players, and therefore they will feel _____ and get the ______ to continue and improve their performance. ( encourage ) 4.The ______ girls swims well in spite of her ______. (disable ) 5.My brother ______ from a well—known American university. My parents attended his ______ ceremony yesterday. ( graduate )