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Seminars in Immunology 20 (2008) 228–235 Contents lists available at ScienceDirect Seminars in Immunology journal homepage: www.elsevier.com/locate/ysmim Review Gene regulatory networks directing myeloid and lymphoid cell fates within the immune system Peter Laslo 1 , Jagan M.R. Pongubala 1 , David W. Lancki, Harinder Singh ∗ Howard Hughes Medical Institute, Department of Molecular Genetics and Cell Biology, The University of Chicago, 929 East 57th Street, GCIS W522, Chicago, IL 60637, USA a r t i c l e i n f o Keywords: Cell fate determination Lineage restriction Transcription factor antagonism Myeloid and lymphoid lineages Macrophages Neutrophils B and T lymphocytes a b s t r a c t Considerable progress is being achieved in the analysis of gene regulatory networks that direct cell fate decisions within the hematopoietic system. In addition to transcription factors that are pivotal for cell fate specification and commitment, recent evidence suggests the involvement of microRNAs. In this review we attempt to integrate these two types of regulatory components into circuits that dictate cell fate choices leading to the generation of innate as well as adaptive immune cells. The developmental circuits are placed in the context of a revised scheme for hematopoiesis that suggests that both the innate (myeloid) and adaptive (lymphoid) lineages of the immune system arise from a common progenitor. © 2008 Elsevier Ltd. All rights reserved. 1. Introduction Considerable progress is being achieved in the analysis of gene regulatory networks that direct cell fate decisions within the hematopoietic system. Many cell fate decisions appear to be dictated by the antagonistic interplay of transcription factors [1–6]. In addition to transcription factors that are pivotal for cell fate specification and commitment, recent evidence suggests the involvement of regulatory RNAs (miRNAs) [7–9]. Thus the interplay of transcription factors and miRNAs will need to be integrated in order to develop a more comprehensive understanding of these developmental circuits. Hematopoiesis involves a series of hierarchically organized progenitors that arise from a self-renewing stem cell (HSC) (Fig. 1). Increasing evidence suggests that both innate (myeloid lineages) and adaptive (lymphoid lineages) cells of the immune system can arise from a shared lymphoid-primed multipotent progenitor (LMPP) [10]. Cell fate specification involves the action of primary lineage determinants (transcription factors) that initiate and resolve mixed lineage patterns of gene expression by activating lineage appropriate genes and repressing alternate lineage genes [11]. Cell fate choice is reinforced by the induction of secondary transcription factors that function in concert with primary determinants, thereby enabling lineage commitment. In this review, we will discuss the known regulatory factors that dictate cell fate choices ∗ Corresponding author. E-mail address: [email protected] (H. Singh). 1 These authors contributed equally to this work. 1044-5323/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.smim.2008.08.003 within the innate and adaptive immune system and focus on their organization into coherent networks or circuits. 2. Stem cells: self-renewal versus differentiation Self-renewing HSCs are contained within the lineage-negative c-kithi Sca-1+ population of cells in the bone marrow (LSK) [12]. The decision of an HSC to undergo differentiation is associated with loss of self-renewal capacity and results in the generation of a multipotent progenitor, MPP (a transit amplifying cell) that can undergo limited rounds of cell division before differentiating into a series of progressively lineage-restricted progenitors (Fig. 1). Upregulation of the Flt3 receptor is correlated with loss of self-renewal capacity [13]. The regulatory proteins Gfi-1, and Bmi-1 have been shown to be necessary for HSC self-renewal, whereas C/EBP␣ and c-Myc appear to promote their differentiation [14–17]. These results raise the possibility that the choice of an HSC to undergo differentiation involves the transient induction of C/EBP␣ and c-Myc that may in turn antagonize the expression of Gfi-1 and Bmi-1. However, the molecular networks and mechanisms regulating HSC self-renewal versus differentiation remain to be more fully delineated. 3. Progressive lineage restriction of MPPs—a revised roadmap Initial analyses by the Weissman laboratory led to the widely adopted view that MPPs give rise to two major lineage restricted intermediates; a common myeloid progenitor (CMP that generates megakaryocytic, erythroid, granulocytic and macrophage progeny) and a common lymphoid progenitor (CLP that gives rise P. Laslo et al. / Seminars in Immunology 20 (2008) 228–235 229 Fig. 1. Developmental scheme for hematopoiesis. The scheme emphasizes a revised roadmap for hematopoiesis that involves a lymphoid-primed multipotent progenitor (LMPP) from which all innate (myeloid) and adaptive (lymphoid) lineages of the immune system are generated. Cross-antagonism between key transcription factors that function to regulate binary cell fate choices is noted at the appropriate bifurcation points in the developmental scheme. Transcription factors that are important for the generation of particular intermediates are noted within colored circles representing such cells. HSC (hematopoietic stem cell), MPP (Multipotential progenitor), LMPP (Lymphoid-primed multipotential progenitor), MEP (Megakaryocyte–Erythrocyte progenitor), ETP (Early thymic progenitor), CLP (Common lymphoid progenitor), GMP (Granulocyte–Macrophage progenitor). to B and T lymphoid cells) [18]. Recent studies by the Jacobsen laboratory have suggested a revised roadmap for hematopoiesis [10]. Based on Flt3 receptor expression, it was demonstrated that MPPs that are Flt3− differentiate preferentially along the erythroid/megakaryocyte pathway, whereas Flt3+ MPPs have significantly reduced megakaryocyte and erythrocyte potential and give rise primarily to lymphoid (B and T) and myeloid lineages (macrophages and granulocytes) [10]. These analyses have led to the proposal that MPPs initially undergo a binary decision to differentiate into a megakaryocyte/erythroid (MEP) progenitor and a lymphoid/myeloid multipotential progenitor (LMPP) (Fig. 1). This result has important developmental implications as it suggests that both the innate (myeloid) and adaptive (lymphoid) lineages arise from a common progenitor and likely share one or more regulatory components such as the transcription factor PU.1 [19]. 4. Erythroid versus myelo-lymphoid lineage restriction is based on transcriptional cross-antagonism between GATA-1 and PU.1 Genetic analyses of the transcription factors GATA-1 and PU.1 are consistent with the revised developmental scheme for 230 P. Laslo et al. / Seminars in Immunology 20 (2008) 228–235 hematopoiesis. Gene disruption studies have shown that GATA-1 is necessary for erythroid and megakaryocyte development whereas PU.1 is required for the generation of myeloid (macrophage and granulocyte) and lymphoid lineages [20,21]. Based on the findings that PU.1 and GATA-1 could inhibit each other’s molecular activities [22,23] it was proposed that this cross-antagonism is critical for generation of megakaryocyte/erythroid versus myeloid progenitors [24]. In various cell line models, ectopic expression of GATA-1 blocks myeloid development and similarly forced PU.1 expression inhibits erythroid differentiation [25–27]. This functional antagonism appears to involve a direct physical interaction between the two proteins that results in inhibition of each other’s transactivation potential [22,23]. Since GATA-1 and PU.1 positively regulate expression of their respective genes, their cross-antagonism is predicted to lead to mutual gene repression [28,29]. Recently, the reciprocal activation of the GATA-1 and PU.1 genes in MPPs has been shown to promote the specification of erythroid and myelolymphoid lineages, respectively [30]. 5. Regulation of binary myeloid cell fate choices Unlike the cross-antagonism between GATA-1 and PU.1 in lineage restriction, cell fate specification of certain myeloid lineages depends on shared primary lineage determinants. For example, PU.1 and C/EBP␣ are required for the generation of both macrophages and neutrophils. This is also the case for basophils and eosinophils whose development requires the factors C/EBP␣ and GATA-2. Two key studies have provided substantial insight into the underlying basis of these cell fate choices [11,31]. The regulatory mechanisms invoke an initial increase in the concentration of one or the other determinant. Subsequently, the second determinant is also induced but two different cell fates are specified as a consequence of changing the developmental order of induction of the shared regulatory factors. The order of induction of C/EBP␣ and GATA-2 has been shown to regulate the cell fate choice that results in the generation of eosinophils and basophils. C/EBP␣ and GATA-2 were ectopically expressed in a sequential manner utilizing CLPs that do not express either of the above transcription factors [31]. If C/EBP␣ was expressed first, followed by GATA-2 the CLPs generated eosinophils. However, when the order was reversed and GATA-2 expressed before C/EBP␣ the CLPs were specified into basophils. Macrophage and neutrophil cell fate specification require the transcription factors PU.1 and C/EBP␣. The relative concentration of PU.1 and C/EBP␣ in granulocytic-macrophage progenitors (GMPs) was suggested to regulate macrophage versus neutrophil cell fate choice based on alteration of gene dosage [32]. Using PU.1−/− progenitors, more recent analysis has demonstrated that at sub-threshold levels, this Ets transcription factor regulates a mixed pattern (macrophage/neutrophil) of gene expression within individual myeloid progenitors [11]. The onset of this mixed lineage program recapitulates a fundamental feature displayed by many types of hematopoietic progenitors [33]. Increased PU.1 levels refine the mixed lineage pattern and promote macrophage differentiation by modulating a novel regulatory circuit comprised of counter antagonistic repressors, Egr-1,2/Nab-2 and Gfi-1. Egr-1 and Egr-2 function redundantly to activate macrophage genes and to repress the neutrophil program of gene expression. Conversely, Gfi-1 represses expression of macrophage genes while promoting neutrophil differentiation in conjunction with C/EBP␣. Thus the Egr’s and Gfi-1 represent crucial secondary cell fate determinants that function in concert with the primary determinants, PU.1 and C/EBP␣, respectively, to dictate macrophage versus neutrophil cell fate choice (Fig. 2). Mirroring the gene circuit demonstrated for Fig. 2. A gene regulatory network dictating macrophage versus neutrophil cell fates. The network depicts the regulatory connections between primary (PU.1 and C/EBP␣) and secondary (Egr’s and Gfi-1) transcription factors and their macrophage or neutrophil specific target genes in the context of a binary cell fate choice. A key architectural feature of this regulatory network is the sub-circuit that is comprised of counter-acting (mutual) repressors (Egr/Nab and Gfi-1). The Egr’s are components of a feed forward loop with PU.1 that activates macrophage-specific genes. C/EBP␣ and Gfi-1 are proposed to constitute a similar feed forward loop in context of the neutrophil-specific gene expression program. macrophage and neutrophil cell fate specification, erythroid versus megakaryocytic cell fate choice also appears to involve a pair of counter-acting repressors, EKLF and Fli-1 [34] (Fig. 1). The aforementioned results involving PU.1, C/EBP␣, Egr’s and Gfi-1 have been used to assemble and mathematically model a simple gene regulatory network [11]. A key architectural feature of this regulatory network is the sub-circuit that is comprised of counteracting (mutual) repressors (Fig. 2). Mathematical modeling of the overall network architecture reveals that it exhibits both graded and bi-stable (switch-like) behaviors. The model accounts for both the onset and resolution of mixed lineage patterns during cell fate determination. It explains instructed cell fate choice based on the developmental ordering of PU.1 and C/EBP␣ induction. However it also reveals an alternate stochastic basis for cell fate choice by demonstrating that co-induction of a common pair of primary lineage determinants can contribute to specification and commitment from a cell state in which the potential for two distinct fates co-exist [11]. Molecular characterization of the macrophage specific c-fms gene has revealed a two-step mechanism of gene activation that maybe generalized to other lineage-specific genes in the context of cell fate specification. The mechanism involves the promoter being maintained in a primed state by PU.1 in early progenitor cells. Increased levels of PU.1 induce Egr-2 and both transcription factors then function in concert to activate a c-fms enhancer and thereby robust expression of the c-fms gene in differentiating myeloid cells [35]. These observations lead us to propose that primary (PU.1/C/EBP␣) and secondary (Egr’s/Gfi-1) cell fate determinants function in concert with one another as components of feed forward loops to promote transcription of lineage specific genes, thus reinforcing cell fate decisions. 6. miRNAs as new components of gene regulatory networks miRNA molecules are a recently discovered class of non-coding RNAs that can regulate gene expression at the level of transcription, RNA stability or translation and many have been found to play important roles in hematopoiesis [7,36]. Constitutive expression of miR-181 in HSC results in increased generation of B lymphocytes while miR-223 promotes the differentiation of myeloid progenitors into granulocytes [8,37]. Other studies have demonstrated the need to repress miRNA expression to enable cell fate determination with the down-regulation of miR-221 and -222 being required for erythrocyte development [38] and that of miR-17-5p, 20a and 106a for macrophage development [39]. Some of these studies have not only delineated a role for miRNAs in hematopoiesis, but have P. Laslo et al. / Seminars in Immunology 20 (2008) 228–235 231 Fig. 3. Regulation of neutrophil cell fate specification by the interplay of transcription factors and miRNAs. The scheme depicts regulatory interactions between transcription factors (colored circles) and microRNAs (miR) and their target genes (lines with promoter sequences marked by arrows). These regulatory interactions have been experimentally demonstrated to regulate the differentiation of a GMP (Granulocyte–Macrophage progenitor) into a neutrophil precursor (see text for details). also uncovered their molecular mechanisms of action involving interplay with lineage determining transcription factors. Using both gain and loss of function experiments, Fazi and colleagues have demonstrated the requirement of miR-223 during granulocyte differentiation [37]. Notably, two C/EBP␣ binding sites were identified within the miR-223 promoter revealing how miR- 223 is induced during granulocyte differentiation. Bioinformatics identified numerous putative regulatory targets of miR-223, one of them being the mRNA encoding the transcription factor NFI-A. Molecular genetic experiments demonstrated the miR-223 dependent down-regulation of NFI-A as a requirement for granulocyte differentiation. Collectively, these experiments elucidated a novel Fig. 4. Regulation of macrophage cell fate specification by the interplay of transcription factors and miRNAs. The scheme depicts regulatory interactions between transcription factors (colored circles) and microRNAs (miR) and their target genes (lines with promoter sequences marked by arrows). These regulatory interactions have been experimentally demonstrated to regulate the differentiation of a GMP (Granulocyte–Macrophage progenitor) into a macrophage precursor (see text for details). M-CSFR refers to the receptor for the cytokine M-CSF. 232 P. Laslo et al. / Seminars in Immunology 20 (2008) 228–235 regulatory loop involving C/EBP␣, NFI-A and miR-223. In myeloid progenitors, the NFI-A transcription factor is expressed and bound to the miR-223 promoter. With low levels of C/EBP␣ in these progenitors, NFI-A is able to compete for the overlapping C/EBP␣ binding sites in the miR-223 promoter and maintain expression of miR-223 at low levels. During induction of granulocyte differentiation, C/EBP␣ levels are increased which competitively displaces NFI-A from the miR-223 promoter and induces the expression of miR-223. This molecular switch is further reinforced by the translational repression of NFI-A mRNA by miR-223 resulting in a feed forward regulatory loop that enables exit from the progenitor cell state and initiates granulocytic differentiation (Fig. 3). A remarkably similar circuit architecture to the one noted above appears to regulate the levels of NFI-A during macrophage differentiation (Fig. 4). In this context PU.1 induces the expression of miR-424 that also targets the NFI-A mRNA resulting in the down-regulation of NFI-A expression [40]. As with granulocyte differentiation, the down-regulation of NFI-A within myeloid progenitors is required for macrophage development [40]. These studies suggest NFI-A as a key transcription factor in maintaining myeloid progenitors in an undifferentiated state and propose that its down-regulation is necessary for the specification of both granulocyte and macrophage cell fates. Collectively these studies demonstrate analogous roles for miRNA molecules in myeloid cell fate specification. Both primary determinants PU.1 and C/EBP␣ induce distinct miRNAs each of which can down regulate NFI-A expression resulting in the onset of either macrophage or neutrophil differentiation (Figs. 3 and 4). In a related study on macrophage development the expression of the transcription factor AML1 has also shown to be regulated by a miRNA [39]. In this study, the miRNA 17-5p-20a was demonstrated to target the AML1 3 UTR and thus regulate protein expression. Conversely, AML1 appears to repress the transcription of the miRNA 17-5p-20a gene. M-CSF signaling was shown to induce expression of AML1 and repress the expression of miR-17-5p-20a. This in turn alleviated the repression of AML1 translation mediated by the miRNA, thereby enabling the further accumulation of AML1 protein. AML1 also functioned in a feed forward loop to reinforce M-CSFR expression. Thus two inter-connected feed forward loops appear to reinforce macrophage differentiation promoted by M-CSF signaling (Fig. 4). 7. Extrinsic signaling inputs and myeloid gene regulatory networks The requirement for the myeloid cytokines in cell fate determination has been debated over the years. While these cytokine receptors are developmentally expressed in a lineage restricted manner and function to expand myeloid progenitors they are not essential for myeloid development [41]. Nevertheless, two studies reveal key functions for myeloid cytokines in regulating cell fate choice. The first relates to alterations in the relative concentration of C/EBP␣ and PU.1 in the context of neutrophil versus macrophage development. It was shown that PU.1 heterozygosity could partially suppress the neutropenia caused by the mutation of the G-CSF gene. The molecular pathway underlying this genetic suppression appears to involve induction of C/EBP␣ by G-CSF signaling [32]. In the absence of G-CSF signaling lowering the dose of PU.1 is needed to promote efficient neutrophil development thereby compensating for reduced C/EBP␣ expression. In a separate study, the block to granulopoiesis in C/EBP␣ deficient mice was shown to be rescued by administering cytokines in vivo that induce an alternate C/EBP family member [42]. These observations suggest that the primary role of myeloid cytokines, during steady state hematopoiesis, is to maintain progenitor numbers. However, upon infection or stress, altered cytokine levels can direct the generation of specific innate immune cells by modulating the relevant gene regulatory networks. Toll-like receptors (TLRs) are expressed on myeloid cells and function to recognize bacterial structures and initiate the innate immune response. A recent study invokes a novel function for TLRs in promoting myeloid cell fates [43]. Activation of TLR signaling in HSCs promoted macrophage differentiation. Similarly, TLR signaling in lymphoid progenitors resulted in the generation of dendritic cells. Collectively, these observations suggest that during infection by a bacterial pathogen, TLR signaling can be used to direct the rapid generation of innate immune cells from HSC and lymphoid progenitors. 8. Regulation of B-lymphoid versus myeloid cell fate choice According to the revised developmental scheme for hematopoiesis the regulation of B-lymphoid versus myeloid cell fate choice occurs in the context of an LMPP. Several transcription factors that are essential for B cell development, including PU.1, E2A, Ikaros and EBF may function in the context of LMPPs to regulate this cell fate choice. Graded levels of PU.1 have been shown to regulate B versus macrophage development by complementation of PU.1−/− multipotential hematopoietic progenitors [44]. A low concentration of PU.1 is needed to induce B cell development but is not sufficient for promoting the generation of macrophages. In contrast a 4–5-fold higher concentration of PU.1 drives macrophage differentiation and actively blocks B cell development. The molecular basis of this concentration dependent function of PU.1 in regulating B versus myeloid cell fate choice remains to be elucidated. The E2A gene, which encodes the isoforms E12 and E47, is necessary for the generation of B lineage progenitors in the bone marrow [45–47]. Bone-marrow derived E2A−/− progenitors expanded in vitro under B-lymphoid conditions are transplantable and give rise to multiple lineages of the hematopoietic system. These progenitors despite culturing in B-lymphoid inducing conditions are impaired for the expression of B-lineage genes and continue to express substantial levels of GATA-1 [48]. Thus, E2A may initially function to promote the generation of LMPPs by antagonizing the expression of a critical erythroid and megakaryocytic lineage determinant and by priming the transcription of B-lineage specific genes. Ikaros-deficient mice appear to generate LMPPs but these progenitors undergo excessive myelopoiesis and are completely blocked for B cell development [49]. Interestingly, the B cell developmental block in Ikaros−/− hematopoietic progenitors can be rescued by ectopic expression of EBF [50]. Ikaros is likely to regulate B cell fate choice in an LMPP by repressing the expression of myeloid lineage genes and inducing the expression of EBF [50]. Importantly Ikaros is an obligate regulator of B cell fate specification as it activates Rag gene expression and IgH gene rearrangement [50]. It has been demonstrated that EBF−/− progenitors like their E2A−/− counterparts, when cultured with B-lymphoid inducing signals display multilineage developmental capacity both in vitro and in vivo [51]. However, although EBF−/− progenitors efficiently give rise to Mac-1+ and Gr-1+ myeloid lineage progeny they are unable to generate erythroid cells. These studies provide support for a stepwise lineage restriction model with Ikaros and EBF functioning to restrict myeloid lineage developmental capacity during the developmental transition from an LMPP to a CLP [50,51]. EBF−/− mutant cells fail to initiate the early B lineage program of gene expression that includes the activation of the 5 and Vpre-B, mb-1, B29 genes and are impaired for DNA recombination events P. Laslo et al. / Seminars in Immunology 20 (2008) 228–235 233 fate determinant that can initiate alternate myeloid lineage restriction and dictate B cell fate commitment independently of Pax5. However Pax5 is needed to sustain EBF expression and the two factors likely function in a concerted manner in wild-type proB cells to ensure the repression of myeloid genes and B cell fate commitment. Recently, Ikaros has also been shown to be required for the repression of myeloid lineage genes in pro-B cells and to block macrophage developmental potential [50]. Thus, B cell fate commitment is not simply a consequence of the action of a single transcription factor but instead dependent on an elaborate network of regulatory molecules that include EBF, Pax5 and Ikaros. These factors are likely to function in both a sequential and concerted manner during B cell development from LMPPs thereby restricting alternate myeloid options and ultimately leading to B cell fate commitment. Fig. 5. Proposed regulatory network that dictates B-lymphoid versus myeloid cell fate choice. This circuit is suggested to operate during the transition from an LMPP (lymphoid primed multipotent progenitor) to a CLP (common lymphoid progenitor). EBF represents the primary B cell fate determinant and its initial expression is controlled by four developmental inputs (PU.1lo , Ikaros, E2A and IL-7R signaling). EBF is proposed to antagonize alternate cell fate options by interfering with the expression or function of myeloid regulators (PU.1hi , C/EBP␣ and Id2). Importantly, EBF activates early B cell gene expression including the secondary B cell fate determinant, Pax5. Furthermore, Pax5 functions in a feedback loop to augment EBF expression. Positive and negative regulatory inputs are depicted using arrowheads and T-junctions, respectively. at the IgH locus [52]. EBF has been shown to function synergistically with E2A to promote B cell development [53]. EBF also induces the transcription factor Pax5, a secondary B lineage determinant [51,54,55]. Functional by-pass studies have demonstrated that ectopic expression of EBF rescues the B cell developmental block in PU.1−/− progenitors [55]. Similarly, EBF rescues the B cell developmental potential of E2A−/− and IL-7R␣−/− progenitors as well as of progenitors isolated from IL-7 deficient mice [56–58]. Based on these studies we have assembled a gene regulatory network invoking EBF as a primary B cell fate determinant [3] (Fig. 5). Consistent with the proposal, ectopic expression of EBF in multipotential progenitors directs B cell generation at the expense of myeloid cell fates [51]. Importantly, in the regulatory network, EBF expression is regulated by multiple inputs such as PU.1lo , E2A, Ikaros and IL-7R␣ thereby ensuring stringent control of its developmental induction. The identity and functions of miRNAs that interface with this network remain to be elucidated. Unlike disruption of the E2A or EBF genes, mutation of Pax5 results in a block to B cell development at the pro-B cell stage [59]. However, the Pax5−/− pro-B cells are not committed to the B cell fate as they express lineage inappropriate genes such as c-fms and Notch-1 and differentiate into various hematopoietic lineages both in vitro and in vivo. Thus Pax5 is considered to be a crucial factor required for B lineage commitment [60,61]. Conditional disruption of Pax5 in committed pro-B cells also results in mis-expression of lineage inappropriate genes and the acquisition of the capacity to differentiate into macrophages in vitro and T lineage cells in vivo indicating that Pax5 is also necessary to maintain B cell fate commitment [62,63]. Strikingly, sustained expression of EBF in Pax5−/− hematopoietic progenitors restricts their alternate lineage potentials (myeloid and T) in vivo [51]. Increased expression of EBF, in Pax5−/− pro-B cells represses myeloid and T lineage genes, including subsets activated by PU.1 or repressed by Pax5. Restriction of myeloid developmental options by EBF appears to be due to its antagonism of C/EBP␣ and PU.1 expression (Fig. 5). These results along with analysis of the developmental potential of wild type CLPs expressing an EBF regulated transgene [64] demonstrate that EBF is a primary B cell 9. Extrinsic signaling inputs and B cell fate specification Unlike myeloid cell fate determination, B cell fate specification is critically dependent on cytokine signals. The earliest signaling event that appears to trigger B cell development is the activation of the Flt3 receptor within a subset of LMPPs. Targeted inactivation of the Flt3 gene results in a severe deficiency in the generation of B lineage progenitors [65]. Consistent with the requirement for Flt3 signaling in the development of B lineage progenitors, there is a significant decrease in CLPs observed in mice deficient in the Flt3 ligand (FL) [66]. Taken together, these results strongly suggest that specification of the B lymphoid cell fate initiates within the LMPP population as a consequence of expression of Flt3. The signaling pathway through which Flt3 promotes the generation of B lineage progenitors is unknown, but in vitro data suggest that activation of this receptor promotes expression of the IL-7 receptor (IL-7R) [67]. The development of pro-B cells in the bone marrow requires signaling through IL-7R [68]. Furthermore, in a culture system, IL-7R signaling is sufficient to induce the differentiation of CLPs into proB cells. Importantly, two studies have shown that combined loss of Flt3 and IL-7R signaling results in a complete failure to develop B lineage cells during both fetal and adult hematopoiesis [69,70]. Signaling through the IL-7R induces the expression of the EBF gene thereby representing a critical input into the B lineage gene regulatory network (Fig. 5). Consistent with this possibility, it has been shown that the EBF promoter is responsive to STAT5 [71]. 10. Regulation of T-lymphoid versus B and myeloid cell fate choice Recent analyses indicate that early thymic progenitors (ETPs) lack B cell potential but retain myeloid developmental capacity [72–74]. ETPs are likely generated from LMPPs as a consequence of robust Notch signaling in the thymus. Recently, the transcription factor, LRF has been shown to inhibit basal Notch signaling in the bone marrow [75]. Conditional disruption of LRF in HSCs results in generation of T lineage progeny in the bone marrow suggesting that LRF is necessary to block precocious T lineage development in response to basal Notch signaling in the bone marrow. Both lossof-function and gain-of-function studies have demonstrated that Notch-1 signaling in the thymic cortex instructs the T cell fate and inhibits B cell development [76]. The block to B cell development may be due to the inhibition of EBF function and Pax5 expression by Notch signaling [77,78]. In addition to Notch-1, additional transcription factors such as PU.1, AML1, c-Myb, GATA-3 and E2A are necessary for development of T lineage progeny [79]. However their assembly into a coherent gene regulatory network requires further experimental analysis and is still in progress. 234 P. Laslo et al. / Seminars in Immunology 20 (2008) 228–235 11. Reprogramming of cellular fates Ectopic expression of C/EBP␣ in committed B cells results in their transdifferentiation into macrophages [80]. This lineage conversion appears to be due to rapid and efficient down-regulation of Pax5 indicating that C/EBP␣ initiates B-lineage reprogramming by antagonizing Pax5. Given that Pax5 sustains EBF expression, it is likely that down-regulation of Pax5 expression by C/EBP␣ also leads to loss of EBF expression thereby collapsing the B-lineage specific regulatory network. Intriguingly, the expression of C/EBP␣ along with four transcription factors (Oct4, Sox2, Klf4 and c-Myc), that are sufficient to convert fibroblasts into pluripotent stem cells (iPS), also enables the reprogramming of B cells into iPS cells [81,82]. Such dramatic cellular reprogramming can also be achieved via knocking down Pax5 expression along with the ectopic expression of Oct4, Sox2, Klf4 and c-Myc. These results demonstrate that specification and commitment to a lymphocyte cell fate does not appear to involve any irreversible epigenetic modifications. The only irreversible modifications in lymphocyte development are genetically based and involve DNA rearrangements of antigen receptor loci. 12. 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