Hematopoietic Cytokines, Transcription Factors and Lineage Commitment

Hematopoietic cytokines, transcription factors and lineage commitment

Jiang Zhu1,2 and Stephen G Emerson*,1,2

1Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, PA 19104, USA; 2Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, PA 19104, USA

The past two decades have witnessed signi®cant advances achieved by using many fewer stem cell-enriched cells, in our understanding of the cellular physiology and isolated by any one of a variety of methods, either from molecular regulation of hematopoiesis. At the heart of bone marrow, peripheral blood or fetal liver. Most stem cell self-renewal and lineage commitment decisions recently, careful studies suggest that within the subset lies the relative expression levels of lineage-speci®c of cells previously viewed as uniquely hematopoietic transcription factors. The expression of these transcrip- stem cells may lie a subset with truly multi-lineage tion factors in early stem cells may be promiscuous and hematopoietic potential, capable of giving rise to many ¯uctuate, but ultimately comes under the in¯uence of non-hematopoietic lineages as well, including myocytes, extracellular regulatory signals in the form of hemato- epithelial cells, hepatocytes, and neurons. poietic cytokines. In this review, we ®rst summarize our The phenotypic speci®cation has become progres- current understanding of the phenotypic characterization sively more sophisticated over the past two decades. of hematopoietic stem cells. Next, we describe key Although the precise relationship between cell surface known transcription factors which govern stem cell self- phenotype and cellular plasticity within the stem cell renewal and lineage commitment decisions. Finally, we compartment is still not clear, the murine Sca-1+ C- review data concerning the role of speci®c cytokines in kit+ Lin7 (SKL) population clearly contain most, if in¯uencing these decisions. From this review, a picture not all of the cells with HSC potential, and the same is emerges in which stem cell fate decisions are governed by likely true of the equivalent population in humans. the integrated eects of intrinsic transcription factors This SKL population is further characterized by the and external signaling pathways initiated by regulatory dierential expression of CD34 and CD38, with SKL cytokines. cells being functionally divided between CD34+ and Oncogene (2002) 21, 3295 ± 3313. DOI: 10.1038/sj/ CD347/lo sub-populations. The CD34+ cells provided onc/1205318 immediate radioprotection for lethally irradiated recipient mice while CD347/lo cells were responsible Keywords: transcription factor; cytokines; hematopoi- for long-term reconstitution of both myeloid and esis lymphoid lineages (Osawa et al., 1996). The authors postulated that CD34+ sub-population basically consists of multilineage progenitor cells with low self- renewal capacity, while the real stem cells are The cell biology of primitive stem cells contained in CD347/lo fraction. Similarly, it was recently reported that only the descendants from Diverse results from many experimental systems CD38+CD347 SKL cells, but not from CD34+ SKL indicate that the proliferative and self-renewal capacity cells could be easily detected in the re-constituted BM of a given hematopoietic cell, as well as the diversity of cells in lethally irradiated secondary, and tertiary lineages to which it can dierentiate, decrease progres- recipients over a long term (Zhao et al., 2000). sively with dierentiation (Figure 1). In the now Developmentally, there are two waves of hematopoi- classical view, the earliest cell in this hierarchy is the esis, so probably with two corresponding origins for pluripotent hematopoietic stem cell (HSC), whose HSCs (Ciau-Uitz et al., 2000). Primitive hematopoiesis, existence was demonstrated by serial bone marrow distinguished from de®nitive hematopoiesis by large transplantation (BMT) experiments. In these studies, all and nucleated erythrocytes and speci®cally expressed lineages of derived hematopoiesis could be successfully fetal hemoglobin isoforms, occurs as a transient wave reconstituted in lethally irradiated tertiary recipient preceding the advent of de®nitive hematopoiesis. mice by bone marrow cells from secondary recipients. Primitive hematopoiesis in the mouse begins within Furthermore, successful reconstitution could also be the blood islands in yolk sac around 7.5 days post coitum (d.p.c.). Independently, the stem cells for de®nitive hematopoiesis originate within embryonic SP/AGM (splanchnopleur, aorta, gonads, and mesone- *Correspondence: SG Emerson, Division of Hematology/Oncology, phros) during 9.5 to 11.5 d.p.c. in mouse and 30 to 37 Departments of Medicine and Pediatrics, Maloney 510, 3600 Spruce Street, University of Pennsylvania School of Medicine, Philadelphia, days of gestation in the human, where the hemato- PA 19104, USA; E-mail: [email protected] poietic cells are found adhering to the ventral wall of Transcriptional regulation of hematopoiesis J Zhu and SG Emerson 3296

Figure 1 Transcriptional regulation of early hematopoietic stem cell development. HSCs, as one progeny of hemangioblasts or hemogenic endothelium, are faced with the cell fate choice either to self-renew or to dierentiate into committed common lymphoid or common myeloid hematopoietic precursors. The transcription factors involved in each development direction are depicted

dorsal aorta (Medvinsky and Dzierzak, 1996). Only tion factors and surface markers, including SCL, these de®nitive hematopoietic stem/progenitor cells GATA-2, C-kit, AA-4.1, CD34, Flit-3 ligand, Sca-1, originating from AGM, but not primitive HSCs' are VEGFR-1 and -2, only with the exception of CD45. able to repopulate the entire hematopoietic system in Actually endothelial speci®c receptor VEGFR-2 was the lethally irradiated adult recipient mice (Cumano recently found a key marker for isolating hematopoie- and Godin, 2001). De®nitive HSCs expand in number tic reconstituting cell in NOD/SCID mice from human in the AGM, and then migrate to and colonize fetal circulating CD34+ cells (Ziegler et al., 1999). This close liver and spleen where they continue to dierentiate relationship between HSC and endothelial development into recognizable hematopoietic precursors. After birth, can be further visualized in embryonic stem cell de®nitive hematopoiesis is primarily con®ned to bone cultures. During the in vitro embryo body development marrow, and in some pathological conditions also to of mouse, the blast colony-forming cells (BL-CFC) extramedullary sites such as spleen, liver, and occa- develop within 4 days in the presence of vascular sionally lung and brain. endothelial growth factor (VEGF) and are lost quickly. Because both endothelial and hematopoietic cells These BL-CFCs within embryonic bodies can produce seemed to simultaneously derive from clusters of adherent endothelial cells, and primitive or de®nitive phenotypically similar cells within the yolk sac, it has erythroid cells, as well as macrophages and neutrophils been suggested that hematopoietic and endothelial cells recognized by morphology examination. These BL- share an immediate embryonic parental cell, either a CFCs may thus indeed be the in vitro counterpart to hemangioblast that serves as the common precursor for the long-hypothesized hemangioblasts (Choi et al., hematopoietic and endothelial lineages, or a hemogenic 1998). It is also possible that hemangioblasts may endothelium that gives rise to hematopoietic stem cells persist after birth. Shi et al. (1998) showed evidence (see Figure 1). Although de®nitive hematopoietic stem/ indicating that the endothelial cells lining the Dacron progenitor cells do not resemble endothelial cells graft implanted in the aorta were actually derived from morphologically, they do share a number of transcrip- the pre-transplanted BM cells 8 weeks before. They

Oncogene Transcriptional regulation of hematopoiesis J Zhu and SG Emerson 3297 also showed that some of CD34+ BM cells could be hematopoietic lineages were expressed normally in induced to endothelial cells in vitro culture system with SCL-null embryonic stem cell lines. However, hemato- cytokines like VEGF. poietic-restricted genes, including transcription factors One of the most exciting ®ndings in the past 2 ± 3 GATA-1, EKLF (erythroid kruppel-like factor), and years has been that cells with the phenotype of PU.1 as well as globin genes and MPO (myeloperox- hematopoietic stem cells, but derived from non- idase), were only expressed in wild type and SCL- hematopoietic tissues such as brain and muscle, can heterozygous ES cells. SCL protein shares homology eciently repopulate hematopoiesis in lethally-irra- within a restricted region of 56 amino acids with a diated mice (Bjornson et al., 1999; Jackson et al., number of bHLH transcription factors, and it is 1999). Conversely, numerous laboratories have found capable of binding to E-box motif of DNA that transplanted BM cells could contribute to the (CANNTG) in vitro, and forms heterodimers with regeneration of multiple tissue cell types, including other HLH factors, such as E47 and E12. Strikingly, brain, muscle, hepatocytes, lung, GI epithelium, and SCL mutants unable to bind to E-box were found to skin (Brazelton et al., 2000; Gussoni et al., 1999; be able to nearly fully rescue the hematopoietic defect Krause et al., 2001; Lagasse et al., 2000; Mezey et al., of SCL homogeneous null Zebra ®sh (Porcher et al., 2000). These two sorts of ®ndings have given rise to 1999). The studies' authors proposed two models to two related hypotheses: (1) that HSCs are ubiquitous explain this phenomenon: (1) the primary role of SCL in mature mammals, and the restricted appearance of here is to sequester an unidenti®ed repressor, for which hematopoietic dierentiation in bone marrow alone is DNA-binding of SCL is dispensable; (2) this de®ciency due to environmental regulatory controls on HSC in DNA binding ability of SCL could be compensated dierentiation; and (2) that HSCs are only a subset, by the anity binding with its transcriptional partners either phenotypically or transcriptionally de®ned, of a retaining an intact DNA binding domain to target pool of ubiquitous multi-tissue stem cells, perhaps genes' promoter. closely related to embryonic stem cells. GATA-2 Transcriptional regulation of early hematopoietic Both primitive hematopoiesis in yolk sac and de®nitive development hematopoiesis in fetal liver and spleen as well as adult bone marrow are greatly aected by the de®ciency of Since purifying and biochemically interrogating very GATA-2. De®cient primitive hematopoiesis, evident by rare HSC populations is so dicult, the role of speci®c severe anemia, leads most GATA-2-de®cient embryos transcription factors in HSC fate decisions has derived to die before 10.5 p.c. When GATA-2 7/7 ES cells largely from genetic strategies, primarily gene-targeting were injected into wild type blastocysts, the contribu- (knockout) and retroviral infection/overexpression tion of GATA-2 7/7 cells in both fetal liver and adult experiments. From this growing body of literature, hematopoietic compartment of chimeric mice were several transcription factors have been found to play nearly undetectable (Tsai et al., 1994). The introduc- critical roles in HSC physiology, including SCL (stem tion into de®cient ES cells with a 250 kb GATA-2 cell leukemia hematopoietic transcription factor), YAC clone, which was strongly expressed in both GATA-2, and Lmo-2, which are essential for primitive primitive and de®nitive compartments, rescued hema- and de®nitive hematopoiesis, and AML-1 that is topoiesis both in vivo and in vitro (Zhou et al., 1998). required for de®nitive hematopoiesis. In patients with aplastic anemia, the GATA-2 expression was found decreased in CD34+ bone marrow cells, while no signi®cant change in SCL and SCL/Tal AML1 expression was detected (Fujmaki et al., 2001). SCL homozygous null embryos died at around 9.5 Both wild type PML and PML-RARa fusion proteins d.p.c., and histological studies showed complete were able to interact with GATA-2 and potentiate its absence of recognizable hematopoiesis in yolk sac, transcription activity, implicating a role of GATA-2 in with GATA-1 and c-Myb expressions being undetect- the molecular pathogenesis of APL (Tsuzuki et al., able (Robb et al., 1995). SCL null ES cells were not 2000). GATA-2 may also be one major mediator of the able to form any lineages of blood cells in chimeric ability of BMP-4 to specify hematopoietic mesoderm, mice. SCL null ES cells were also unable to form blast since BMP-4 expression in the embryo is polarized to colonies in vitro, but only transitional colonies that the ventral wall of the aorta, immediately underlying contained only low numbers of primitive erythroid the site of initial hematopoiesis (Marshall et al., 2000; precursors and a subset of precursors associated with Maeno et al., 1996) (see Figure 2). early stage of de®nitive hematopoiesis (Robertson et al., 2000). In control and SCL-null EB, the temporal LMO2 expression pattern of genes associated with the formation of ventral mesoderm, such as Brachyury, Lim-®nger protein LMO2, which was found to be BMP-4 (bone morphogenetic protein-4), and Flik-1 activated in T cell leukemia by chromosomal transloca- (VEGF receptor) was identical. The GATA-2, CD34, tion, is likewise indispensable for primitive erythropoi- and C-kit that are co-expressed in endothelial and esis and de®nitive hematopoiesis, as shown in a Lmo2

Oncogene Transcriptional regulation of hematopoiesis J Zhu and SG Emerson 3298

Figure 2 Extrinsic stem cell hematopoietic growth factors direct HSC transcription programs. For example, the Notch1 ligand Jagged-1 is present on BM mesenchymal cells. Upon binding to membrane-bound receptor Notch1, the cytoplasmic domain of Notch1 is cleaved, allowing it to diuse to the nucleus where it acts as a transcriptional activator of several HSC gene programs. As another example, BMP-4, which has been implicated to delivering inductive signals for the origin of HSCs in embryo, might act through its eect on the transcriptional regulation of GATA-2

null/wild type chimeric mice (Yamada et al., 1998). the transcription factors described above are absolutely Embryogenic Lmo2 expression was found localized to essential for the survival and proliferation of HSCs, hematopoietic sites (Manaia et al., 2000). other molecules clearly impact strongly on the cell fate decisions of stem cells, both for symmetric expansion and lineage commitment. While we are still at the AML-1 beginning of uncovering these key molecular regula- Homozygous knockout mice showed absent fetal liver tors, work indicates that transcription factors HoxB4 hematopoiesis, and failed to contribute to de®nitive and Ikaros, activated nuclear form of Notch1, cell hematopoiesis in chimeric mice, but primitive yolk sac cycle inhibitor P21, and TGF/BMP-4 family members erythropoiesis was unaected (Okuda et al., 1996). The as well as TNFa receptor P55 signaling are likely targeted disruption of its partner-CBFb similarly involved in the maintenance or promotion of the results in a complete block of fetal liver hematopoiesis. hematopoietic stem cell renewal (see Figures 1 and 2). The rescue of AML-1 de®cient ES cells by knocking-in of a wild type AML-1b restored their ability to contribute to the formation of every lineage of Regulation by transcription factors hematopoietic cells in chimeric mice (Okuda et al., 2000). In vitro rescue experiment of the de®cient ES Homeobox genes cells by the retroviral vectors carrying a series of AML1b mutants indicated that a 61aa of C-terminal Several members of the homeobox gene family are region containing a VWRPY motif, required for expressed dierentially during hematopoieic dierentia- interacting with transcriptional co-repressors, was not tion. Perhaps because of the close similarity and required for the de®nitive hematopoiesis (Okuda et al., overlapping functional roles of these molecules, the 2000). Taken together, these results suggest that the results of gene knockout studies have been modest. transcriptional activation, rather than repression of Overexpression of Hox genes by stem cell retroviral target genes by AML-1 is required for de®nitive transduction, in contrast, have been very revealing. For hematopoiesis. examples, it was ®rst noticed that in vitro dierentiation of the human Lin7, non-adherent peripheral blood cells to either erythroid or granulocytic direction Regulation of self-renewal and dierentiation of HSCs was accompanied by a remarkable and persistent increase in the HoxB4 mRNA level, and that the The transcriptional machinery governing primitive administration of HoxB4 antisense oligo inhibited the stem cell biology is undoubtedly very complex. While colony formation to these two lineages in vitro

Oncogene Transcriptional regulation of hematopoiesis J Zhu and SG Emerson 3299 (Giampaolo et al., 1994). Then it was shown that long- N-terminal ®nger domain mediating DNA binding and term repopulating ability of murine bone marrow cells C-terminal domain mediating dimerization. Generally, was increased to at least 10-fold when HoxB4 cDNA Ikaros proteins modulate transcription by recruiting was over-expressed by retroviral infection, in compar- co-repressor complex to the promoters of target genes ison to control stem cells infected with empty vector and/or sequestering these genes to the vicinity of (Sauvageau et al., 1995; Thorsteinsdottir et al., 1999). hetero-chromatin (Koipally et al., 1999; Sabbattini et Paradoxically, it seemed the CRU (competitive repo- al., 2001; Trinh et al., 2001). Intriguingly, dierent pulating unit) capacity of bone marrow cells from the isoforms of Ikaros are dierentially expressed at HoxB4 over-expression group never exceeded the CRU dierent stages of hematopoiesis (Klug et al., 1998), limit found in the normal mice. It was also reported by suggesting that regulated expression of Ikaros isoforms the same lab that the over-expression of HoxB4 in ES could provide ®ne regulation of the expression of cells prompted the production of de®nitive GMME- lineage speci®c transcription factors. CFU and CFU-E colony in the in vitro dierentiation model (Helgason et al., 1996). Recently, HoxB4 Notch1 expression was found increased in protein level in the myeloid dierentiation of HL-60 cells induced by In contrast to the transcription factors described VitD3. By delivering Hox B4 antisense oligonucleotides above, which are believed to be directly regulated by into HL-60 cells, authors showed that the monocytic only intracellular events, the Notch1-Jagged pathway dierentiation was inhibited (Pan and Simpson, 2001). may provide a key pathway to integrate extracellular These eects may be mediated through HoxB4 proteins regulatory signals with stem cell cycling control. interactions with the c-Myc gene and the cell cycle Following engagement of the Notch1 receptor by machinery (Antonchuk et al., 2001). HoxB4 protein extracellular ligand, cleavage events release the intra- was identi®ed as the transcription elongation-blocking cellular portion of Notch1, which in turn translocates factor binding to MIE1 site within the intron1 of c- to the nucleus and acts as a transcription factor on its Myc gene (Pan and Simpson, 1999). In the non- target genes. By constitutively over-expressing the hematopoietic polyclonal Rat-1 ®broblast cells, the intracellular domain of Notch1, several immortalized over-expression of Hox B4 resulted in the faster cell lines were established from murine BM SKL cells, growth, reduced requirement to serum, and even which still maintained the potential to reconstitute malignant transformation, in cooperation with PBX1. myeloid and lymphoid cell lines both in vivo and in This phenomenon was accompanied by enhanced AP-1 vitro (Varnum-Finney et al., 2000). This phenomenon activity and increased level of cyclin D1 (Krosl et al., may re¯ect the in vivo physiology of Notch/jagged 1998). It is also interesting to note that the over- interactions, as the addition of Notch ligand jagged-1 expression of the HoxB4 paralogue HoxC4 in primitive into in vitro expansion system of human hematopoietic cells also enhances the proliferation of CD34+CD387 Lin7 cord blood cells prompted their both HSCs and committed progenitors, although the ability to repopulate pluripotently in SCID mice HoxC4's eect may be milder than HoxB4's (Daga et (Karanu et al., 2000). al., 2000). Most importantly, the list of transcription factors If HoxB4 gene products are critical regulators of and cell cycle regulatory molecules studied to date may HSC cycling, then the molecular mechanism governing represent only a small subset of the genes that control HoxB4 expression is clearly critically important to stem cell self-renewal and lineage commitment deci- primitive stem cell fate decisions. Recently, the two sions. Other candidate genes critical for stem cell essential DNA binding sites were identi®ed in the regulation may surface from broader screening of genes promoter region of HoxB4 gene functioning in both dierentially expressed in HSCs versus closely related normal and malignant hematopoietic primitive cells, but non-HSC cells. For example, by comparing a just upstream of its transcriptional starting site. One of cDNA library of murine fetal liver HSCs (SKL them was a classical E box and served as the functional AA4.1+) with that made from stem cell-depleted binding site for USF1 and USF2 (Giannola et al., AA4.17 cells of same tissue, investigators have 2000). identi®ed a large number of genes that are preferentially expressed in stem cells versus more dierentiated cells. The known transcription factors or chromatin Ikaros binding proteins on the list include AML-1, ALL-1, Mice lacking all isoforms of Ikaros display decreased Bnmt-3b, Evi-1 and macroH2A1.2 and they may expressed of Flk-2 and C-kit on their SKL cells, and involve the maintenance of gene expression program show a marked reduction in long-term repopulating unique to HSCs (Philips et al., 2000). units as measured by competitive repopulation, and mice homozygous for Ikaros dominant negative mutation (DNA-binding domain mutated) possess no Regulation by stromal cells and cytokines measurable repopulating activity at all (Nichogianno- poulou et al., 1999). The Ikaros gene products belong In many biological systems it has been shown that to zinc ®nger family of transcription factors. Long milieus where the stem cells reside play critical roles in form of Ikaros mainly consists of two zinc ®ngers, an the determination of their self-renewal or dierentia-

Oncogene Transcriptional regulation of hematopoiesis J Zhu and SG Emerson 3300 tion choices. For example, in Drosophila the germ line mice. These data were interpreted to suggest that stem cell renewal/dierentiation balance is maintained failure to maintain stem cells out of cell cycle might by surrounding somatic cells that direct the asymmetric predispose stem cells to rapid exhaust after transplan- division of stem cells. The maintenance of this precise tation (Cheng et al., 2000b). In the contrast, this same balance involves activation through the epidermal experimental system was exploited to show that growth factor receptor in somatic cells (Kiger et al., knockout of another cyclin-dependent kinase inhibi- 2000). In the case of murine spermatogonial stem cells, tor ± p27 prompted the repopulating eciency of BM glial cell line-derived neutrophic factor, a distant cells, possibly through enhancement of proliferating member of transforming growth factor-1b family ability of progenitor cells, and with no disturbance on secreted by Sertoli cells, was found to maintain the the stem cell quiescence (Cheng et al., 2000a). stem cell reserves and inhibit their dierentiation However, upon the stimulation of leukemia cell line (Meng et al., 2000). Likewise, murine ES cells can be Kasumi-1 by TGF-b1, the leukemia fusion protein successfully maintained in an undierentiated state and AML1-ETO can sequester Smads, which are the propagated in vitro by the presence of leukemia essential downstream eectors of TGF-b1 signaling inhibitory factor (LIF) (Ichikawa, 1970), which pathway, so as to inhibit the expression activation of requires the activation of downstream molecule Stat3 Smad target genes, suggesting a quiescence-indepen- in ES cells (Raz et al., 1999; Niwa et al., 1998). LIF is dent mechanism for maintaining leukemia stem cell normally produced in embryonic ®broblasts adjacent to renewal (Jakubowiak et al., 2000). ES cells in vivo. There are several other interesting candidates for the Adjacent cells and local cytokines may also regulate cytokines that might mediate these stem cell main- self-renewal versus dierentiation decisions faced by tenance, self-renewal and expansion in vivo. Early HSCs. For example, one group of investigators experiments indicated that IL-3 and GM-CSF might established two endothelial cell lines from day 11 be involved, as IL-3 sustained the renewal of FDCP murine CD34+ AGM cells by infecting them with cell line while GM-CSF induced them to macrophage- retroviruses over-expressing polyoma middle T antigen. granulocyte dierentiation (Just et al., 1991). However, One of these immortalized stromal lines supported the these molecules are unlikely to play essential roles in expansion of CD34+ SKL fetal liver cells, while a vivo, at least by themselves. IL-3 is not expressed in second line induced the dierentiation of these cells bone marrow stroma. On the other hand, the into erythroid, myeloid and B lymphoid cells. For the combination of SCF, Flt-3L and Tpo with either IL- self-renewal induction, the direct contact of hemato- 11 or IL-3, was able to support in vitro expansion of poietic cells with these established endothelia was long term multilineage repopulating activity of SKL required (Ohneda et al., 1998). Circumstantial evidence CD347 cells for at least ®ve cell divisions (Bryder and suggests that similar local interactions between speci®c Jacobsen, 2000). More recently, newer candidates for stromal cells and HSCs may exist in vivo. For example, soluble molecules known to be present on stromal cells the ability of bone marrow stromal cells to support have arisen as stem cell proliferation promptors. Tie-2, hematopoiesis was found to increase between day 7 a tyrosine kinase receptor for angiopoietin, on the fetal and 10 after birth, correlating with the emergence of liver KLSA (AA4.1+) cell population was found in hematons in the bone marrow, the compact cellular association with the cells with long term multiple- aggregates consisting of various stromal cells and lineage repopulating activity (Hsu et al., 2000). The hematopoietic cells. When studied functionally, this soluble form of sonic hedgehog, along with human hematon fraction was found 3.7-fold enriched in day homologues to its receptors, was found to be expressed 35 LTC-IC over unfractionated non-adherent buy in primitive and mature blood cells. These ®ndings are coat bone marrow cells (Blazsek et al., 2000). very provocative, since high concentration of sonic Evidence from several systems suggest that the most hedgehog protein were found to expand human primitive repopulating HSCs are largely quiescent, with CD38734+ Lin7 cells in vitro, which was dependent the majority of these cells out of cycle at any one time. on the transcription activation of BMP-4 (Bhardwaj et It is well known that low level of TGF-b1 maintain the al., 2001), and polarized expression of BMP-4 has been multiple dierentiation potentials of hematopoietic found in endothelium immediately underneath de®ni- stem/progenitor cells, which is associated with its tive hematopoietic clusters in the embryo. Further- negative controlling eect on cells cycling (Batard et more, in vitro study shows that low doses of BMP-4 al., 2000). Accordingly, in another study, TGF-b1 was promoted the proliferation and dierentiation of found to slow down the growth of human umbilical human CD387Lin7 cells, while higher concentration cord CD34+CD387Lin7 blood cells in vitro, accom- of BMP-4 extended the time from 4 to 6 days until that panied by the up-modulation of cell cycle inhibitor the repopulating capacity of cultivated cells could be p21, while independent of its downregulating eect on maintained (Bhatia et al., 1999). Bcl-2 expression (Ducos et al., 2000; Francis et al., A possible role for TNFa in the regulation of HSC 2000). It was recently suggested that the G1 checkpoint proliferation seems quite possible, however the data regulator p21 is one key factor required for the concerning its eect on HSCs are con¯icting. On one maintenance of this quiescent status, for BM cells hand, TNFa was found to eliminate long-term culture- homozygous null for p21 failed to long-term repopu- initiating cells from human CD34+CD387 bone late the hematopoietic system of lethally irradiated marrow, via signaling through the ceramide pathway,

Oncogene Transcriptional regulation of hematopoiesis J Zhu and SG Emerson 3301 at a concentration lower than what is required for tively. The CD34+FcgRlo cells, like CLPs, only capable initiating apoptosis (Maguer-Satta et al., 2000). In of short-term (2 weeks) repopulating the lethal another study, TNFa, through its binding to the P55 irradiated mice, are considered as the common myeloid receptor, inhibited the maintenance and expansion of precursors (CMP). It was also found that the short- multipotent repopulating ability of CD34+CD387 cells term cultivation of CMP cells generated CD347FcgRlo cultured in vitro in the presence of SCF/FL/TPO, (megakaryocytic/erythroid precursor, MEP) and independent of apoptosis induction and cell cycle CD34+FcgR+ (granulocytic/monocytic precursor, change. This TNFa eect was postulated to re¯ect a GMP) cells as thought of before (see Figure 3). The biased eect on dierentiation over renewal of HSCs, transcription factors SCL, GATA-2, NF-E2, GATA-1, especially to an accelerated myeloid cell commitment C/EBPa, c-Myb, and PU.1, and the cytokine receptors and turnover (Dybedal et al., 2001). In direct contrast, TpoR and EpoR, were found to be expressed in CMP however, the P55 TNFa receptor knockout mice were (Akashi et al., 2000b). found to have impaired HSC repopulating, despite the Additional experiments suggest that production of overt phenotype of increased BM cell cellularity, monocytes may be more promiscuous, and occurs from suggesting that a dosage-dependent eect of TNFa common lymphoid as well as myeloid precursors (Kee on HSC renewal versus dierentiation (Rebel et al., and Murre, 1998) (see Figure 4). Clonogenic fetal liver 1999). cells with the phenotype of AA4.1+B2207Mac-17Sca- 1+ were ®rst shown to contain the bi-potent precursor for B cells and macrophages (Cumano and Godin, Early stem cell decisions: commitment to myeloid and 2001). In a second study, AA4.1FcgRII/III+ cells from lymphoid lineages the murine fetal liver of 13 dpc were found to be precursors possessing potential to produce B cells, T Once HSCs divide and generate more dierentiated cells and macrophages (Lacaud et al., 1998). And more daughter cells, within 10 ± 15 divisions the genetic recently, a postnatal mouse bone marrow cell popula- programs of the descendent cells become ®xed toward tion with phenotype IL-7R+Sca-I7C-kit7Lin7B2207 a single lineage. The ®rst steps in this process of lineage CD19+ was isolated, constituting about 0.5% BM cells restriction are uncertain, but the dominant hypothesis (Montecino-Rodriguez et al., 2001), which produced today proposes that the ®rst decisions involve restric- only B cells or macrophages in vitro. tion to the ability to generate myeloid versus lymphoid progeny. The ®rst cells generated following such decisions have been termed common lymphoid pre- How are lineage commitment decisions from HSCs cursors (CLPs) and common myeloid precursor initiated and regulated? (CMPs) (Figure 1). Kondo et al. exploited the data that IL-7 receptor is widely expressed on mature This critical but most dicult question has been the lymphoid (Peschon et al., 1994), but not myeloid cells, subject of intense and creative debate, and equally to isolate a potential CLP population with the intense and creative experimentation. Among the most phenotype IL-7R+Lin7Sca-1loC-kitlo from adult mur- intriguing set of relevant data are those suggesting that ine BM. This same population was later proven to HSC, CLP and CMP fate decisions might not be possess short-term (4 ± 6 weeks) repopulating ability instantly made, but rather preceded by a phase of restricted to either B, T or NK cells in vivo (Kondo et promiscuousness or hesitation (Enver and Greaves, al., 1997). These cells, which comprise 0.02% of BM 1998; Rothenberg, 2000). For example, the mature cells would appear to meet the criteria for CLPs erythroid dierentiation marker b-globin and mono- (Kondo et al., 1997). Of note, GATA-3 and Aiolos cytic marker MPO were unexpectedly found to be co- were expressed while the expression of GATA-1, C/ expressed, at low levels, in the single cell of multiple- EBPa and NFE2 that are thought to associate with potent FDCP-Mix4 cells (Hu et al., 1997). In a myeloid development were repressed (Akashi et al., transgenic mouse model, an erythroid speci®c, micro- 2000b). LCRb-hemoglobin promoter-coupled Lac-Z was not Committed myeloid precursors, in contrast, were only found to express in erythroid lineage, but also in isolated from IL-7R7/Lin7C-kit+/Sca-17 populations. megakaryotic, and in monocytic/granulocytic progeni- This subset was further divided into three sub- tors in vivo, though to milder extents (Papayannopou- populations by the dierential expression of CD34 lou et al., 2000). These data indicate that the and FcgR: (1) CD34+ FcgRlo, (2) CD347FcgRlo and alternative dierentiation potential might not be (3) CD34+FcgR+ (Akashi et al., 2000b), and the immediately eliminated, but rather repressed in a potential of each sub-population was examined by in graded or gradual way in the cells that are committing vitro methylcellulose CFU culture and in vivo BMT to a given lineage. assays. Of them, the CD34+ FcgRlo cells constitute Presumably, the dierential expression of transcrip- about 0.2% of BM cells and gave rise to daughter cells tion factors triggers the determination of HSC fate: consisting of monocytes, granulocytes, erythrocytes renewal, or commitment to either CLP or CMP. By and megakaryocytes, while CD347FcgRlo cells and using dierential cDNA library and sensitive RT ± PCR CD34+FcgR+ cells exclusively produced megakaryo- method, a number of candidate transcription factors cytes/erythrocytes and granulocytes/monocytes, respec- that are preferentially expressed in cells destined to one

Oncogene Transcriptional regulation of hematopoiesis J Zhu and SG Emerson 3302

Figure 3 Transcriptional regulation of common myeloid precursor (CMP) commitment. CMPs dierentiate into either common precursors for granulocytic and monocytic lineages (GMPs) or common precursors for both erythroid and megakaryocytic lineages (EMPs). A separate, possible, pathway leading to eosinophils is depicted by dotted line. Dual expression of PU.1 and GATA-1 leads HSCs to CMPs, but then dominant expression of PU.1 is restricted to GMPs, while unopposed GATA-1 expression directs dierentiation to EMPs

Figure 4 Transcription regulation of common lymphoid precursor (CLP) commitment. B lymphocytes and T lymphocytes are derived from a common lymphoid precursor (CLP). The early development of B cell is distinguished into distinct stages by the sequential expression of dierent transcription factors that direct Ig gene recombination and the expression of B cell speci®c cell surface phenotypes. A proposed (alternative) dierentiation pathway of macrophages from pro-B is also indicated by a dotted line

of those fates have been found. However, most of these body of published evidence relates to the transcription transcription factors have not been tested in clearly factor PU.1. Predominance of PU.1 could be among interpretable in vivo models. Moreover, the primary one of earliest events biasing HSCs to lineage events and mechanism leading to the induction of commitment, for the co-upregulation of PU.1 and dierentially expressed genetic programs are obscure. GATA-1 heralds the commitment to CMPs, and PU.1 Of the transcription factors studied to date, the largest expression is maintained in CLPs and also absolutely

Oncogene Transcriptional regulation of hematopoiesis J Zhu and SG Emerson 3303 required for lymphoid development, while knockout of composed of the extracellular domain of IL-3 and the PU.1 spared the development of primitive erythropoi- cytoplasmic domain of GM-CSFR was introduced into esis (Scott et al., 1994). PU.1 involves the transcription murine IL-3-dependent multipotent FDCP-mix cells, regulation of both IL-7 receptor and M-CSF receptor, the administration of hIL-3 induced cells to granulo- which are required for proper developments of cytic/moncytic dierentiation instead of cell renewal, lymphoid and myeloid lineages, respectively (DeKoter suggesting that cytoplasmic part of GM-CSFR a chain and Singh, 2000; DeKoter et al., 1998). PU.1 over- participates in delivering speci®c signal leading to expression alone was shown to be able to commit dierentiation (Evans et al., 1999). But this cytokine- multipotent hematopoietic progenitors to monocytic/ induced cell fate convention was not observed in the granulocytic direction (Nerlov and Graf, 1998). Two case where a foreign Epo receptor was overexpressed. clues have been found to account for how the active Again in another study, when GSF-R transgenic mice function of PU.1 could lead to two mutually exclusive were generated under the control of an ubiquitous outcomes: lymphoid versus myeloid cells. Firstly, the MHC H-2L promoter, G-CSF was found of capable of distinct eects of PU.1 on lineage commitment is stimulating the growth of multiple hematopoietic dosage-related, with a higher concentration favoring lineage cells, including BM blast cells from transgenic HSCs commitment to myeloid cells (DeKoter and mice, not only restricted to the neutrophils (Yang et Singh, 2000). Secondly, the PU.1 activity on its target al., 1998). gene could be negatively regulated by the lymphoid cell IL-7, which is produced by BM stromal cells, thymic transcription factor Pax5 (Maitra and Atchison, 2000; and intestinal epithelial cells, binds to IL-7 receptor a Nutt et al., 1999), which is preferentially expressed in B chain and induces its association with gc chain, lymphocytes and absolutely required for B lymphocyte recruiting Jak1 and Jak2, which in turn activates commitment. Pyk2 to prompt lymphoid cell survival (Benbernou et Data is scarcely available in terms of the molecular al., 2000). But IL-7 seems to be a unlikely primary mechanisms regulating the expression of key transcrip- factor to induce the commitment of CLPs from HSCs, tion factors like PU.1, and of how the expression of for in the fetal liver of IL-77/7 mice, the development dierentiation-inducing transcription factors might of earliest unipotent B cell precursors with phenotype predominate over self-renewal-maintaining factors. In Lin7CD197C-kit+IL-7R+AA4.1+ was found normal, one view, the so-called `intrinsic theory,' a transcription while it was severely aected in SDF-17/7 embryos factor expression pattern is initiated and even reached (Egawa et al., 2001). by the stochastic and autonomous induction of transcription factors. Some evidence in support of this view that has been presented involves the stochastic Regulation of commitment of CMPs to MEPs transcription activation of transcription factor Pax5 and GMPs (Nutt and Busslinger, 1999). Whether or not such intrinsic transcription factor induction may occur, the Whereas PU.1 and GATA-1 are co-expressed in stable expression of transcriptional programs might CMPs, their mutually exclusive expression coincides still be subject to instruction from outside signals, as with further commitment to either granulocytic/mono- proclaimed by the `extrinsic theory'. The latter was cytic or megakaryocytic/erythroid dierentiation (Fig- recently supported by experiments in which the ure 3). The expression of GATA-1 decreases in addition of anti-TGF-b1 antibody into in vitro culture granulocytic/monocytic dierentiation while that of system of human CLRPP (cytokine low-responding PU.1 decreases with dierentiation to either megakar- proliferating progenitors) cells resulted in an increased yocytic or erythroid direction. Moreover, gene target- generation of cells expressing CD15/CD11b/glycophor- ing experiments have shown that PU.1 is absolutely in-A, accompanied by a signi®cant upregulation of required for monocytic and B lymphocytic develop- PU.1 and GATA-1 expression level (Pierelli et al., ment while GATA-1 for erythroid and megakaryocytic 2000). Thus, at least in this in vitro setting, TGF-b1 development. would appear to directly inhibit the commitment of If hematopoietic lineage commitment is preceded by HSCs to CMP. a state of transcriptional promiscuity, one may ask Related evidence is accumulating for the potential of what is the mechanism that reinforces and stabilizes extrinsic cytokines to positively direct lineage commit- commitment to speci®c lineages. Studies suggest that ment decisions. Based on the ®nding that low but the expression of a transcription factor, above a detectable levels of GM-CSF receptor were found to be threshold level, has two reinforcing lineage directing expressed on HSCs but not CLPs, it was suggested that eects: (1) auto-upregulation of its own (Chen et al., down-regulation of the GM-CSF receptor was among 1995; Tsai et al., 1991); and (2) down-regulation of the initial events during the speci®cation or commit- alternative transcription factor pathways. In the ment processes to CLPs (Kondo et al., 2000). clearest system studied to date, PU.1 was found to Consistent with this data, when GM-CSF receptor or inhibit the transcriptional activity of GATA-1 upon its M-CSF receptor was overexpressed in CLPs, the cells target genes, and vice versa. The overexpression of were reprogramed to monocytes or granulocyts by PU.1 inhibited DNA binding of GATA-1 to its GM-CSF or M-CSF in vitro (Kondo et al., 2000). consensus element on target gene promoters, and this When a chimeric human IL-3/GM-CSFR a chain inhibiting eect could be relived by the over-expression

Oncogene Transcriptional regulation of hematopoiesis J Zhu and SG Emerson 3304 of GATA-1. Detailed studies showed that this PU.1- Recent ®ndings suggest that the co-expression of imposed eect was mediated by direct physical contact GATA-1 and FOG (friend of GATA-1), instead of of its N-terminal part to the GATA-1 C-terminal zinc GATA-1 alone, destines the precursor cells for ®nger that involves DNA binding. In a reverse way, erythroid and megakaryocytic development (Tsang et possibly through the interaction of the c-®nger of al., 1997) (Figure 3). The dierentiation-rescuing GATA-1 with ets domain of PU.1, GATA-1 precludes experiment demonstrated that the interaction between the recruitment of co-activator c-Jun to the b3/b4 GATA-1 and FOG was required for either erythroid or region of PU.1 in the context of the promoters of its megakaryocytic dierentiation (Crispino et al., 1999; target genes (Nerlov et al., 2000; Zhang et al., 1999). Tsang et al., 1997). In one series of experiments, C/ Additional support for cross-modulation in CMP EBPb overexpression induced the eosinophilic dier- commitment comes from the ®ndings that ectopic entiation of MEP cell line (HD57, corresponding to expression of PU.1 in erythroid/megakaryocytic leuke- CMP ?), and GATA-1 expression was sustained while mia cell K562 could redirect them to dierentiate into FOG expression was abrogated. When an exogenous granulocytes and monocytes, instead of megakaryo- FOG was constitutively expressed, dierentiation to cytes upon Ras pathway activation, and overexpression eosinophil was inhibited, as indicated by the disap- of GATA-1 in granulocytic progenitor 32D cells pearance of the lineage speci®c antigen ESO47. It was redirects them to megakaryopoiesis (Matsumura et further found that the inhibiting eect of FOG on the al., 2000). Taken together, these data suggest that the ESO47 promoter activation was mediated by the initial ratio of GATA-1/PU.1 protein at an early, speci®c interaction of FOG with the NF motif of critical time point in the CMP determines the GATA-1 in the ESO47 promoter (Querfurth et al., subsequent lineage-restricted fate of the cell (Rekhtman 2000). These data suggest that the dierential expres- et al., 1999; Zhang et al., 1999, 2000). sion of FOG in eosinophils or megakaryocytic/ Intriguingly, disturbances in the proper kinetic erythroid cells directly in¯uences the cell fate choice, orchestration of critical multivalent lineage-directing and that FOG may act as either a co-activator or a co- transcription factors may result in dierentiation arrest repressor to GATA-1 in dierent cellular and promoter along CMPs to MEPs or further procession, thus contexts. providing a ground state for developing myeloproli- In addition to these well-documented roles of PU.1, ferative disorder or leukemia (Moreau-Gachelin et al., GATA-1 and M-SCFR in the commitment of GMPs 1988). PU.1 transgenic mice develop erythroleukemia, and MEPs, other transcription factors and cytokines whose evolution proceeds in two steps: First, a clonal have also been implicated in these processes. Over- population of erythroblasts emerges from a back- expression of c-Myb, like PU.1, is able to reprogram ground of severe anemia; subsequently an Epo- the K562 cells dierentiate to granulocytic/monocytic independent subclone emerges, resulting in frank direction (Matsumura et al., 2000). Egr-1 (early growth leukemia (Barnache et al., 1998). Experiments with response gene-1) was found to promote macrophage MEL cells indicate that maintenance of PU.1-driven production in the expense of erythroid and granulocy- erythroleukemia requires the sustained predominance tic development (Krishnaraju et al., 2001). Conversely, of PU.1 activity over GATA-1 function, as ectopic when SCL was introduced into human hematopoietic GATA-1 cDNA overexpression drives the erythro- CD34+ cells, the production of erythroid and mega- blasts to terminal erythroid dierentiation (Rekhtman karyocytic colonies were enhanced (Elwood et al., et al., 1999). 1998). TGF-b1 might be a potent cytokine that biases The execution of lineage-committing function of the commitment of erythroid at the expense of PU.1 might at least partly be mediated through the granulocytic and monocytic development (Drexler et extrinsic M-CSF/M-CSFR and GM-CSF/GM-CSFR al., 1998; Krystal et al., 1994). The secreted protein, signaling pathways. Expression of the M-CSFRa gene WNT, was shown to inhibit the formation of c-fms was absolutely dependent on PU.1 and PU.17/7 macrophage, while prompting the production of RBC progenitor failed to respond to M-CSF stimulation and monocytes from the enriched avian embryonic (DeKoter et al., 1998) and the proper GM-CSFR bone marrow cells or quail mesodermal stem cells expression also needed PU.1 (Anderson et al., 1998a). QCE6 (Brandon et al., 2000), suggesting a possible role When a murine-M-CSF receptor gene was ectopically of WNT pathway in favor of erythroid/megakaryocytic expressed in murine mutiple-potent EML cells (Tsai et dierentiation and blockage of late phase dierentia- al., 1994), their dierentiation potential to erythroid tion of monocytes to macrophages. lineage was compromised and the dierentiation to granulocyte/monocyte was favored (Pawlak et al., 2000), suggesting an instructive role of M-CSF/M- Commitment to granulocytic dierentiation CSFR. In this relay, instructive signals through cytokine receptors may be delivered, in part, through The dierential expression of C/EBPa appears to homeobox transcription factors. GM-CSF stimulation in¯uence the lineage choice of bi-potent G/M pre- increases HoxA5 expression in BM cells, and HoxA5 cursors to either granulocytic or monocytic direction, antisense oligonucleotide administration inhibits GM- while PU.1 is needed for both (Anderson et al., 1998b) CFU formation while amplifying the generation of (Figure 3). CEBP/a was found preferentially expressed BFU-E (Fuller et al., 1999). in and required for granulocytic lineage, but not for

Oncogene Transcriptional regulation of hematopoiesis J Zhu and SG Emerson 3305 monocytic lineage (Zhang et al., 1997). Over-expression upregulating the transcription activation of CDK of CEBP/a alone was sucient to drive the bipotent inhibitor p16 (Passegue and Wagner, 2000). U937 cells to dierentiate into granulocytes, but to hinder their monocytic dierentiation induced by PMA (Radomska et al., 1998). The commitment to monocyte dierentiation G-CSF itself does not appear to exert instructive role for granulocytic commitment, but rather triggers their Several transcription factors have been implicated in maturation once commitment is complete. GSF the commitment of monocytes from bipotent precursor receptor knock-in mice were produced in which the GMPs. The interferon consensus sequence binding cytoplasmic domain of GSF receptor was replaced by protein (ICSBP/IRF-8), a putative transcriptional that of Tpo receptor. Homozygous mutant mice partner of PU.1 (Brass et al., 1999), was recently developed normal numbers of morphologically normal found to be essential for monocytic development while neutrophils, although some GSF-dependent functions, inhibiting the production of neutrophils from GM- such as chemotaxis and mobilization, were impaired CSF-dependent ICSBP7/7 cell line Tot2 and granulo- (Semerad et al., 1999). Likewise, Epo's role may also cytic progenitor 32D cells (Tamura et al., 2000). The be limited to post-commitment events. When the Epo introduction of a foreign ICSBP into Tot2 cells receptor was forcedly expressed in 32D cells, the resulted in the upregulation of transcription factor addition of Epo eectively induced their granulocytic Egr-1 (Early growth response gene-1) and M-CSFR as dierentiation as G-CSF would do. It was thought of well as the downregulation of C/EBPa and G-CSFR, an evidence for the notion that it is not cytokines, but without obvious eects on the expression of PU.1 and rather the intrinsic properties of cells that determine GM-CSFR. Consistent with this interpretation, the the dierentiation direction (Harris et al., 1998). overexpression of Egr-1 alone in myeloid-enriched cells The retinoid receptor RARs/RXRs might act as a has been shown to promote macrophage dierentiation dierentiation-checkpoint switch at the promyelocytic and to inhibit granulocyte dierentiation (Krishnaraju stage of granulopoiesis. The proper concentration of et al., 2001). By the way, the bZip transcription factor retinoid acid is required for its binding to RAR/RXR Maf B is upregulated successively from the multipotent heterodimer when the latter is docking on RARE progenitor to macrophage. The overexpression of (retinoid acid response element) in regulatory se- MafB in transformed myeloblasts led to exclusive quences of its target genes. The result is to transform production of monocyts/macrophages, and such eect RAR/RXR repressor into an transcriptional activator was independent of whether a foreign PU.1 was by releasing co-repressor and recruiting co-activator to overexpressed (Kelly et al., 2000). itself, which is necessary for the induction of target ICSBP may be very crucial for maintaining normal genes for granulocytic dierentiation (Chen et al., myelopoiesis over and above its role in monocyte 1997; Lin et al., 1999). commitment. ICSBP expression was found to be Once granulocyte commitment is triggered to greatly decreased in both CML patients and murine terminal dierentiation, these same critical transcrip- CML-like models generated by transgenic expression of tion factors continue as key players, by down- Bcr-Abl fusion gene. ICSBP7/7 mice develop granulo- modulation of cell cycle progression and proliferation. cytosis with atypical macrophages, resembling a CML- The C/EBPa-induced terminal granulocytic dierentia- like disease (Holtschke et al., 1996). Conversely, forced tion was preceded by an inhibited G1/S transition in expression of ICSBP inhibited Bcr-Abl-induced myelo- cell cycling, as shown in IL-3-dependent cell line 32D proliferation both in vitro and in vivo (Hao and Ren, cells (Wang et al., 1999), and when 32D cell 2000; Schmidt et al., 1998). proliferation was kept on by the overexpresion of c- The instructive role of M-CSF/MCSFR signaling in Myb, the terminal dierentiation of these cells induced monocytic commitment was further supported by a by G-CSF was blocked (Oh and Reddy, 1998). recent study concerning the identi®cation of a novel Likewise, the overexpression of the cDNA encoding speci®c interacting protein FMIP to cytoplasmic an active form Notch1(mNotch1) in 32D cells domain of M-CSF receptor c-fms, probably as a promoted the terminal granulocyte production through transient inhibitor of c-fms signaling (Tamura et al., the activation of downstream RBP-J transcription 1999). When FMIP was overexpressed in G/M factor, which was also accompanied by an accumula- bipotent FDC-P1Mac11, the M-CSF-induced mono- tion of cells in G0/G1 phase without substantially cytic dierentiation was prevented, instead all cells aecting the apoptosis (Schroeder and Just, 2000b). It dierentiated into granulocytes. is interesting to note that truncated forms of C/EBPa As with neutrophils, cell cycle arrest might be one were detected in ®ve AML patients and in all the cases prerequisite for the terminal dierentiation of mono- acted as dominant negative form to wild type C/EBPa cytic cells. ICSBP-induced macrophage dierentiation (Pabst et al., 2001). It was recently found that loss of coincided with cell growth arrest (Tamura et al., 2000). JunB expression alone in murine myeloid cells led to a It was shown that HoxA10 overexpression, via the disorder recapitulating the development of CML, activation of p21, induced cell cycle arrest and arising from an increased proliferation of granulocytic monocytic dierentiation of U937 cell (Bromleigh and progenitors (Passegue et al., 2001). Jun B has been Freedman, 2000). In the presence of IL-3, the over- shown to inhibit ®broblast proliferation through expression of GATA-2/estrogen chimera in FDCP cell

Oncogene Transcriptional regulation of hematopoiesis J Zhu and SG Emerson 3306 lines induced the cells to monocytic dierentiation, MEK-ERK pathway. A recent study suggested that which was also accompanied by cell cycle arrest PKC-e, but not other PKC isoforms, functionally (Heyworth et al., 1999). cooperated with GATA-1 to activate the expression As mentioned previously, monocytes/macrophages of megakaryocytic speci®c antigen aIIb (Racke et al., could derive from a bipotent precursor for either 2001). monocyte or B lymphocytes, and the graded expression Once the erythroid versus megakaryocytic decision is of PU.1 might govern the choice of cell fate (DeKoter made, numerous transcription factors and cytokine and Singh, 2000). When Lin7 fetal liver cells from signaling pathways combine to support survival, PU.1+/7 embryos were cultivated in the presence of proliferation, and the expression of lineage speci®c IL-7 and stromal cell S17 for 10 ± 14 days, the majority genes. For example, GATA-1 maintains the expression of cells were pro-B, with less than 10% being of Bcl-x and Epo-R, thereby promotes the survival and macrophage. But with constitutive expression of PU.1 proliferation of erythroid precursors (Kapur and cDNA in PU.17/7 cells, most cells generated in the Zhang, 2001). One of target genes for Epo in this same culture system turned into macrophages. pathway was identi®ed as CHOP, a distant C/EBP family member, involving in the erythroid growth and dierentiation (Coutts et al., 1999). At least within the The commitment of MEPs to megakaryocytes and erythroid system, acetylation is one mechanism where- erythrocytes by transcription factors themselves are regulated. CREB-binding protein/p300 binds to GATA-1 and The developments of megakaryocytes and erythrocytes upregulate its DNA binding activity through acetyla- are closely related and share most of transcription tion (Blobel et al., 1998; Boyes et al., 1998). Similarly, factors for lineage development, such as GATA-1, the erythroid inductive eects of SCL, requiring its FOG and NF-E2. PHZ-induced hemolytic anemia in dissociation from the transcriptional co-repressor mice provokes not only an accelerated erythropoiesis, mSin3A, was also associated with its acetylated form. but also enhanced megakaryocyte production. In this In DMSO-induced erythroid dierentiation from model, late bipotent MEP Ter119+/4A5+ cells were leukemia cell MEL, SCL was found to be acetylated isolated from both bone marrow and spleen, with by P/CAF, which in turn increased SCL's binding individual cells capable of dierentiating into either anity with its target. This eect was inhibited by megakaryocytes or erythrocytes within 24 ± 48 h, in the overexpression of mSin3A and cooperated by admin- presence of Tpo or Epo, respectively (Vannucchi et al., istration of TSA (Huang and Brandt, 2000; Huang et 2000). al., 2000). Recent studies concerning GATA-2, protein kinase C-e (PKC-e) and extracellular signal-regulated kinases (ERKs) induced by Tpo provided some clues for The development of T lymphocytes and B lymphocytes understanding the process of unilineage commitment from CLPs from MEPs. Overexpression of GATA-2 in human erythroleukemia K562 cells led to cell growth inhibi- It is still unclear whether or not the CLPs with the tion and phenotype shift from erythroid cells to phenotype of IL-7R+Sca-1lowC-kitLowLin7 are only megakaryocytes (Ikonomi et al., 2000). The expressions immediate precursors for T or B lymphoid cells of TpoR and GP Ib/IX as well as GP IIb/IIIa were (Akashi et al., 2000a), or whether macrophages can induced while production of total hemoglobin de- also be produced from these same cells (Montecino- creased. ERK1 and ERK2 were dierentially induced Rodriguez et al., 2001). As with monocytes, dendritic between megakaryocytic and erythroid dierentiations cells can be derived from either monocyte or lymphoid from bipotent cell UT-7/GM, by Tpo and Epo pathway (Liu et al., 2001). respectively. When an exogenous MEK1 (MAP Environmental signals might play a critical role kinase/ERK kinase 1) inhibitor was administrated, instructing the commitment of CLPs to either T the production of hemoglobin-harboring cells was lymphocytes or B lymphocytes. The development of increased (Uchida et al., 2001). Mutations in the TpoR T lymphocyte needs either CLPs or newly committed gene have been repeatedly found in the patients with early T cells ®rstly to migrate and home to thymus. congenital amegkaryocytic thrombocytopenia and The earliest thymic progenitors are CD4loCD87 TpoR7/7 mice displayed a phenotype of selective CD44+CD257C-kit+ cells, which are capable of thrombocytopenia (Gurney et al., 1994; Tonelli et al., dierentiating into B, NK and thymic dendritic cells 2000), indicating its selective role in the commitment or at a low frequency (Wu et al., 1991), suggesting this maturation of megakaryocyte. It has been shown that population might contain a small number of CLPs. the activation of ERKs by Tpo/TpoR coupling could Recent experiments studying Notch1 suggest one be mediated by multiple pathways, including Jak/Stat such mechanism to account for T or B cell commit- and Ras/Raf/MAPK pathways as well as PI3K and ment. Notch1 signaling is not only required for T PKCzeta (Rojnuckarin et al., 2001). A positive role of lymphocyte development, but also promote commit- PKC in the megakaryocytic commitment has already ment of CLPs to thymocytes while inhibiting B cell been established by numerous studies and this eect is production (Radtke et al., 1999; Pui et al., 1999). at least partly mediated through the activation of Raf- Constitutive expression of active Notch1 led to the

Oncogene Transcriptional regulation of hematopoiesis J Zhu and SG Emerson 3307 production of CD4+CD8+ T cells in bone marrow, IFN-g gene and the induction of IL-12 receptorb suggesting the possibility that activation of Notch 1 is expression. The IL-12/Stat4 signaling seemed to normally induced by thymus but not BM mesenchyme, provide a secondary signal involving the co-activator thereby inducing T cells in thymus and allowing B cell CBP, which was only required for sustaining the development in BM (Deftos and Bevan, 2000). secretion of INF-g by the committed Th1 cells. IL-4, in contrast, was found to be able to inhibit the expression of T-bet, thereby skewing the early T lymphocytes speci®cation away from Th1. Notch1 speci®es T cell fate by inhibiting B cell lineage speci®c gene expression, inducing B cells to B lymphocytes apoptosis and arresting cell cycling of granulocyte progenitors. It was ®rst found that Notch1 or Notch2 The early phases of B cell development have been well signaling pathway inhibited the transcriptional activ- dissected into several distinctive stages by cell surface ity of E2A/E47, an initiating transcription factor for phenotype and sequential transcription factor expres- the alternative B cell fate speci®cation, possibly sion (Busslinger et al., 2000) (see Figure 4). E2A and through their inhibiting eect on Ras pathway that EBF are initial transcription factors that specify the B is required for the full function of E47 (Ordentlich et cell fates from the progenitors (Bain et al., 1994; al., 1998). More recently it was found that Notch1 O'Riordan and Grosschedl, 1999). The pro-B cells signaling not only suppressed B cell IgH gene from EBF+/7 E2A+/7 mice displayed reduced expres- expression (target gene of E2A), but also induced sion of lymphocyte-speci®c genes, including Pax5, apoptosis of chicken B cells via the upregulation of Rag1 and Rag2. Dierently, the E2A de®ciency Hairy 1, and G1 cell cycle arrest through other promotes early T lymphocyte expansion (Engel et al., pathways (Morimura et al., 2000, 2001). In another 2001). E2A null mutant mice spontaneously developed study, it was shown that Notch signaling also monoclonal T lymphoma in thymus between 3 ± 6 suppressed the proliferation of granulocytic progeni- months, preceded by a decreased percentage of tor 32D cells, by arresting cells in the G0/G1 phase CD44lowCD25+ cells and increased percentage of (Schroeder and Just, 2000a), suggesting that Notch1 CD44high CD257 DN cell population (Bain et al., prevents or terminates `promiscuous trans-dierentia- 1997). The ectopic expression of E2A induces apoptosis tion' of committed T to myeloid cells. On the other of T lymphoma (Engel et al., 2001). Taken together, hand, it appears that the level of Notch1 expression these data suggest a role of E2A to limit early T cell must be precisely controlled to allow the normal expansion and promote dierentiation. In another maturation of thymocytes. Constitutive overexpres- system, the spontaneous conversion of 70Z/3 pre-B sion Notch1 blocks the dierentiation of CD4+CD8+ cell lymphocytes to cells with a macrophage-like T cells to CD4+ and CD8+ T cells late in thymocyte phenotype was associated with the loss of expression development (Izon et al., 2001). of EBF and other B cell speci®c genes, which could be Ikaros, which in earlier studies was found to play a reversed by the ectopic expression of E2A gene product critical role in the development of all lineage of E12 (Kee and Murre, 1998). Two alternative-splicing lymphoid cells, especially NK, gd T cells and lymphoid bHLH products of E2A gene, E12 and E47, work in an dendritic cells (Georgopoulos et al., 1994; Wang et al., additive way in supporting the B cell development. 1996), was recently found to set signaling thresholds Surprisingly, the B cell production from E2A de®ciency for pre-TCR- and TCR-dependent T lymphocyte could also be rescued by knocking-in of two- but not dierentiation (Winandy et al., 1999). GATA-3 may one- copy of E2A homologue-HEB gene, while HEB likewise regulate early and late stages of T lymphocyte knockout mice showed a basically normal B cell development. The GATA-37/7 embryonic stem cells development phenotype (Zhuang et al., 1998). failed to contribute to the formation of thymocytes The transcription factor Pax5/BSAP may play an past CD47 CD87 stage (Ting et al., 1996), suggesting essential and consolidating role in the committing a role in early T cell development. The GATA-3 process of lymphoid precursors to B cell lineage. binding site has been found in enhancer/promoter Firstly, it was found that Pax57/7 B lymphocytes regions of multiple T cell speci®c genes, such as TCR- could develop through the pre-pro B and early pro-B a,-b and -d sub-unit gene, CD8a and CD4, as well as stages and were arrested at the pro-B stage before IgH IFN-g and IL-5 (Yamagata et al., 2000). A GATA-3 V-DJ gene rearrangement (Nutt et al., 1997) (see mutant, KRR-GATA3 that creates local hypoacetyla- Figure 4). Then unexpectedly, Pax5 homozygous tion, led to defective homing and elongated survival of de®cient pro-B cells were found of capable of peripheral T cells (Yamagata et al., 2000). reconstituting T lymphocytes in vivo and even of The recent study concerning the commitment of Th1 trans-dierentiating into various myeloid cells in vitro from Th (T helper) cells provided an illuminating (Nutt et al., 1999; Rolink et al., 1999). So it was example for how cytokines control dierentiation proposed that the full commitment to B cell fate is set (Mullen et al., 2001; Szabo et al., 2000). The around V-DJ recombination and the phase before this speci®cation of Th1 cell fate was initiated by checkpoint could be regarded as a priming or transcription factor T-bet, independent of IL-12/Stat4 speci®cation phase (Rothenberg, 2000). However, the signaling pathway, with mono-allelic expression of the B cell commitment might happen before pro-B stage

Oncogene Transcriptional regulation of hematopoiesis J Zhu and SG Emerson 3308 (Allman et al., 1999), for Pax5 is expressed in the within this stromal compartment, primarily from a earliest stage of recognizable B cell population (Nutt small, highly proliferative subset of stromal ®broblasts and Busslinger, 1999) and its function might not be which display cell surface markers of smooth muscle only restricted to VDJ recombination. For example, cells as well. Osteoblasts produce both GM-CSF and Pax5 could consolidate B cell commitment at pro-B by G-CSF, and may account for the stimulation of basal acting as a reverter or terminator to the cells that neutrophil production in vivo. Each of these cytokines accidentally slip into monocyte pathway. Additionally, is produced at relatively low levels, such that in vitro the transcriptional activity of PU.1 on its target genes cultures of these stromal cells only accumulate sub- was compromised by the presence of Pax5 (Maitra and nanogram/ml concentrates of each cytokine, in which Atchison, 2000), while a higher expression level of each provides only minimal proliferative stimulus to PU.1 favors macrophage fate rather than B cell CD34+ cells. Within the context of in vivo bone (DeKoter and Singh, 2000), though both cell types marrow, these cytokines are undoubtedly locally require PU.1 for early development. The EML cell line, concentrated, and their concerted activities clearly created by overexpression of a dominant negative form support HSC survival and proliferation. of RARa in primitive BM cells, expresses some marker SCF, which is expressed constitutively in BM associated with B cell, and it could be induced to stromal ®broblasts and endothelial cells, is clearly terminal granulocytes by high concentration of ATRA. essential to the survival, proliferation, adhesion/migra- But when Pax5 was forcedly over-expressed in this cell tion and dierentiation of HSC (Broudy, 1997; Hart- line, cellular proliferation and myeloid terminal man et al., 2001). Absence of SCF protein or its dierentiation induced by ATRA was inhibited receptor C-kit, as displayed in Sl mutation and W (Chiang and Monroe, 1999). In support of this, the mutation respectively, leads to severe macrocytic expression of M-CSFR and MPO in Pax57/7 cells was anemia. In adult mice, as early as 2 days after eciently repressed by the introduction of an ectopi- administration of a neutralizing antibody against C- cally expressed Pax5 cDNA (Nutt et al., 1999). kit, all progenitor cells were depleted, and eventually Conversely, in the Pax7/7 mice, the B220+ cell mature myeloid and erythroid cells in BM were absent production size was greatly compromised. Pax5 was (Ogawa et al., 1991). Of note, the SCF mRNA is found to be transcribed monoallelically at the onset of alternatively spliced so that either a soluble or a cell B cell development, and then transcribed from both membrane-bound form could be produced. Interest- alleles at stages of pre-B and immature B cells, and ingly, the Sld genotype, in which the cytoplasmic part is ®nally was reversed to monoallelic transcription at deleted and so soluble SCF could be normally mature B cells (Nutt and Busslinger, 1999). The produced, failed to rescue the macrocytic anemia monoallelic transcripts of Pax5 mRNA were found to caused by Sl mutation, suggesting an essential physical be randomly copied from either one of two parental contact between stromal cells and hematopoietic alleles. Individual cell colonies were able to switch progenitors. This observation was further supported expression between two alleles within 2 weeks and both by a recent study showing that altered presentation of Pax5 alleles were synchronously replicated during the S membrane-bound SCF caused by an amino acid phase. This Pax5 expression activation pattern is mutation in its cytoplasmic part is associated with a thought to be compatible with the stochastic model reduced SCF responsiveness by erythroid progenitors regarding hematopoietic lineage commitment mechan- (Kapur et al., 1999). ism. Flt3 L, which is also widely expressed on mesenchymal stromal cells, is the ligand of Flit3R that is restrictedly expressed in CD34+ cells but not CD347 Regulation of HSC dierentiation by the cytokine cells within BM or cord blood (Broudy et al., 1996). network The targeted disruption of Flt3 causes signi®cant reduction in BM hematopoietic progenitor pool size Modulation of HSC transcription factor expression by and subsequently decreased amounts of mature hematopoietic cytokines appears to re¯ect two over- myeloid, B, NK and dendritic cells. Tpo, which was lapping layers of regulation. First, basal hematopoiesis, originally found to be synthesized by liver and kidney, sucient to maintain normal basal blood cell produc- has likewise been recently found to be locally produced tion, occurs in the context of low level secretion of by bone marrow stromal cells (Sakamaki et al., 1999). multiple cytokines. Overlaid upon this basal combina- Ampli®ed hematopoiesis in response to physiologic tion of cytokine production are short-lived ampli®ca- need appears to be regulated in true endocrine fashion, tions of speci®c cytokine secretions, in response to by the stimulated secretion of cytokines disparate from speci®c hematopoietic stresses (Figure 5). the bone marrow, which then signals the marrow by The cytokine matrix supporting basal hematopoiesis combined diusion and bulk plasma transport. For appears to be constructed locally, through the secretion example, anemia or hypoxia triggers increased produc- and cell surface presentation of cytokines from tion of Epo from kidney and liver, resulting an mesenchymal cells within the bone marrow. These so- elevated serum Epo level. Since baseline Epo serum called `stromal' cells include specialized ®broblasts, concentration only achieves minimal Epo dose-re- endothelial cells, osteoblasts, and perhaps adipocytes. sponse at the progenitor level, any increase in Epo SCF, Tpo, Flt3L and GM-CSF are all produced from above this baseline stimulates erythropoiesis. This Epo-

Oncogene Transcriptional regulation of hematopoiesis J Zhu and SG Emerson 3309

Figure 5 Cytokine production in basal and induced (ampli®ed) hematopoiesis. (Top) The survival and slow proliferation of hematopoietic stem cells (HSC) is maintained by the local membrane presentation and secretion of low levels of stem cell factor (SCF), Fl3 ligand (Flt-3l) and thrombopoietin, and likely additional cofactors, produced by mesenchymal cells lining the bone marrow cavity, including ®broblasts (FB), endothelial cells (endo) and osteoblasts (OB). Basal dierentiation of HSC to mature granulocytes and monocytes is maintained by the local matrix presentation and secretion of G-CSF and GM-CSF from osteoblasts, Tpo, and the endocrine delivery of low levels of Epo (produced by peritubular endothelial cells in the kidney, and the liver). (Bottom) Lineage-speci®c ampli®cation of hematopoiesis is stimulated by increased secretion and delivery of lineage-speci®c cytokines. Increased Epo is produced in the kidney in response to hypoxia or anemia (local hyoxemia), Fb and endo secrete increased quantities of G-CSF and GM-CSF in response to IL-1a and TNFa produced at sites of in¯ammation and infection by activated monocyte/macrophages, and increased Tpo may be produced by Fb in response to TGFb, produced by activated T cells or activated Fb themselves

dependent erythroid expansion occurs on the back- apy, the serum levels of Flt3L ¯uctuated inversely to ground milieu of basal stromal cytokines, and is in fact the extents of BM failure. Whether Tpo is induced in dependent on SCF/C-kit coupling (Broudy et al., response to thrombocytopenia is less clear, but it is 1996). clearly induced by in¯ammation, perhaps by TGF-b1 Similar induced hematopoietic networks regulate and T cell cytokine production. Interestingly, in other lineages, and perhaps stem cell self-renewal as idiopathic thrombopenic purpura, it is found that well. Acute bacterial infection stimulates monocytes to the elevated TGF-b1 level increased Tpo production secrete IL-1a and TNFa. These monokines, produced from stromal cells, which in turn triggered the locally at the sites of infection, travel to the bone expression of TGF receptor on megakaryocytic marrow where they profoundly stimulate the produc- progenitors, rending them susceptible to the inhibition tion and secretion of GM-CSF and G-CSF from eect of TGF-b1 on dierentiation (Sakamaki et al., stromal ®broblasts and endothelium. These cytokines, 1999). The CD40-ligand expressed on activated CD4+ in turn, stimulate multiple stages of granulopoiesis T cells enhances the myelopoiesis/megakaryopoiesis, and also mobilize mature neutrophils into the which was found to be mediated by the its peripheral blood, thereby causing the paradigmatic stimulating eect on the production of Flt3 by a clinical manifestation of granulocytosis. In patients variety of cell types and of Tpo by stromal cells with aplastic anemia or cancers receiving chemother- (Solanilla et al., 2000).

Oncogene Transcriptional regulation of hematopoiesis J Zhu and SG Emerson 3310 Concluding remarks suggested by, e.g. sperm stem cells? How are kinetic changes in individual transcription factors coordinated? Intensive investigations in many genetic and cellular For example, how can PU.1 coexist with GATA-1 in systems over the past two decades have generated a CMPs and direct the restricted choice of either GMPs rich view of the process of hematopoietic dierentia- or EMPs? How do changes in speci®c transcription tion. With at least some of the molecular and cellular factor levels, and changes in local chromatin organiza- players now identi®ed, we are now in position to tion at their loci cause the dysregulated dierentiation address truly fundamental questions of stem cell and genetic instability of myeloproliferative disorders biology, including: What is the true cellular potential and leukemias? The answers to these questions will of primitive stem cells found within the bone marrow, establish the foundation for the directed production of and what directs their commitment to hematopoietic multiple hematopoietic and non-hematopoietic cells versus non-hematopoietic tissues? What are the earliest and tissues, and for the directed treatment of events that control HSCs renewal or commitment, and hematopoietic disorders over the decades to come. is there any transcription factor speci®cally maintaining stem cell self-renewal status, or is it maintained simply by cell cycling status? What are environmental signals that critically control the renewal/dierentiation and Acknowledgments survival of HSCs in BM? Are there speci®c environ- We thank Diane Giannola for assistance in graphical mental triggers for stem cell symmetric division, as design.

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