The development of B-lymphoid cells in bone marrow (BM) involves a highly orchestrated collaboration between transcription factors promoting B-cell differentiation and mediating lineage restriction. The identification of surface markers associated with defined stages of B-cell development has allowed for the prospective isolation of progenitor cells and the establishment of a developmental hierarchy. Recent technological advances in cell sorting, global gene expression analysis and chromatin immunoprecipitation has allowed for an increased molecular understanding of events associated with these defined stages of development. Another contributing factor is the increased understanding of the overall relationships between different blood cell lineages. The identification of a progenitor population with combined lymphoid and granulocyte/macrophage potential but with limited megakaryocyte and erythroid potential provided a modified view of the relationship between lymphoid and myeloid lineages. These lymphoid primed multipotent progenitor (LMPP) cells were identified by high expression of the tyrosine kinase receptor FLT-3 within the Lineage negative/low (Lin−/low), SCA1 high KIT high (LSK) cells, known to harbor the majority of the multipotent progenitors, including the hematopoietic stem cells (HSCs) in the mouse BM. Furthermore, detailed analysis of the common myeloid and the common lymphoid progenitor (CLP) populations identified several lineage restricted subpopulations within these compartments thereby further increasing the resolution of the hematopoietic tree. Hence both the development of new techniques and the increasing level of basic knowledge in the area has opened new pathways of investigation that can be used to explore the molecular regulation of hematopoesis.
It has been reported that even the most immature progenitor cells express low levels of genes normally associated with lineage restricted progenitors. This could reflect that the HSC compartment is heterogeneous with a fraction of cells already committed to a defined lineage fate. However, the fact that the same multipotent progenitor cell can express genes associated with several developmental pathways, suggests that this reflects lineage priming rather than signs of early lineage commitment. Even though the early expression of lineage associated genes may result in the establishment of a regulatory network, a commonly accepted view is that this is a mean to maintain lineage specific genes in an accessible chromatin state as to allow for their activation upon differentiation. The regulation of the chromatin context, commonly referred to as epigenetics, is an area of intense investigation and it has been shown that the chromatin status of lineage restricted genes is changed during blood cell maturation. A large part of the epigenetic regulation involves modifications of DNA associated proteins such as acetylation or methylation of histones, but also direct methylation of cytosine residues in the genomic DNA. Since the chromatin status is highly dependent on transcription it could be argued that the epigenetic changes observed during blood cell development simply reflects the transcriptional stage of the lineage restricted genes. In such a scenario, epigenetic changes would be a consequence of the action of lineage specific transcription factors and not any primary determinant in lineage specification events in hematopoiesis. However, the finding that HSCs lacking the polycomb protein BMI-1, a component of the multisubunit Polycomb repressor complex 1 (PRC-1), display reduced long term reconstitution capacity suggests a direct role of epigenetic regulation of hematopoesis. A critical role for Bmi-1 in self renewal of HSCs is also supported by the finding that ectopically expressed BMI-1 increased the in vivo reconstitution capacity and the number of symmetric cell divisions in transduced HSCs. A similar phenotype was observed upon ectopic expression of the PRC-2 complex associated histone methyl transferase enhancer of zeste 2 (Ezh2) involved in de novo methylation of DNA, further supporting the idea that epigenetic mechanisms directly participate in the regulation of hematopoesis. In addition to BMI-1, the PRC-1 complex contains DNA methyltransferase-1 (DNMT-1) important for the preservation of lymphoid lineage potential in early progenitor cells by repression of myeloid programs in the HSCs. On the contrary, Bmi-1 deficient mice was reported to display increased numbers of lymphoid progenitor cells and increased expression of the transcription factors Ebf-1 and Pax-5 in early progenitors. These findings would suggest opposing roles of BMI-1 and DNMT-1 in the regulation of the lineage potential in early hematopoietic progenitors, even though they are both parts of the PRC-1 complex involved in maintenance of methylation patterns. There are also several reports of epigenetic changes in leukemia and it is becoming increasingly clear that the regulation of chromatin is of key importance in both normal and pathological hematopoiesis but the understanding of these processes is far from complete.
Even though the investigations of the functional roles of general chromatin modulating factors provide evidence for that epigenetic modifications are of key importance for normal hematopoiesis, they provide limited information about how transcription factors act to create lineage specific gene expression profiles. One of the crucial factors in early lymphoid development is the Ets protein PU.1 that through interaction with regulatory elements appears to set up an epigenetic landscape for downstream lineage restricted transcription factors. PU.1 appears to be involved already in erythroid versus myeloid lineage determination where the factor acts in a positive feedback loop with C/EBPα to prevent GATA-1 from driving development towards erythroid cell fates. PU.1 has also been suggested to act as a regulator of lymphoid/myeloid cell fate choices in a dose dependent manner since high expression results in the development of myeloid and lower levels in the development of lymphoid cells. It has been proposed that in order to modulate the functional levels of PU.1 to allow for lymphoid cell fate, the transcriptional repressor GFI-1 can replace PU.1 on autoregulatory elements in the PU.1 gene thereby modulating the levels of functional PU.1. Gfi-1 expression is regulated by the zinc finger transcription factor IKAROS (Ikzf-1), known to be of crucial importance for development of the earliest B-lymphocyte progenitors. Mice with a disrupted Ikzf-1 gene have a reduced LMPP compartment, however, using Ikaros reporter mice on an Ikzf1 deficient background, it was possible to isolate reporter positive LSK cells displaying a dramatically reduced MkE potential. This would indicate that IKAROS either is directly involved in the regulation of FLT-3 expression, or that development is blocked after lineage restriction but prior to the activation of the Flt-3 gene. The lineage restricted cells in IKAROS deficient mice lack expression of Dntt, Rag-genes and sterile Ig-transcripts detected in LMPPs from normal mice. A similar phenotype is observed in mice carrying a disruption of the Tcfe2a (E2A)-gene and a bioinformatic analysis of promoters regulating early lymphoid genes supported the idea that Ikzf1 and Tcfe2a encoded proteins share several target genes. Hence, it is likely that these transcription factors collaborate to initiate the lymphoid transcriptional program already within the LSK compartment.
Subsequent maturation of lymphoid progenitors is reflected in an upregulation of IL-7R surface expression and reduced expression of SCA1 and KIT to generate a common lymphoid progenitor. These cells display a dramatically reduced myeloid potential while they retaining the capacity to form B, T and NK cells. More detailed analysis revealed that this population is composed of a heterogenous mixture of cells with different lineage potentials. Using a surrogate light chain Igll 1 (λ5) reporter mouse model, it was possible to isolate a fraction of B-cell committed progenitors within the B220−CD19− progenitor compartment. The heterogeneity became even more obvious upon the characterization of mice carrying both Rag-1 and Igll 1 reporter transgenes and by the use of the surface marker Ly6D. Using these markers it is possible to divide the CLP compartment into distinct subpopulations. Inlay and colleagues separated Ly6D− and Ly6D+CLPs, denoting them as all-lymphoid progenitors (ALP) and B-cell-biased lymphoid progenitors (BLP) respectively. The Ly6D−CLP population expressed low levels of Rag-1 and displayed a robust B and T-lymphoid potential as well as ability to generate NK cells at the single cell level. Furthermore, the limited myeloid potential detected in CLP could be assigned to the Rag-1−Ly6D− compartment. Since Rag-1−CLPs transiently generate Rag-1+ CLPs in vitro, there exist evidence for a direct relationship between these cells proposing a sequential loss of lineage potentials within the previously defined CLP population. Even though the Rag-1+/Ly6D+ cells display a robust B and T-lineage potential in vitro, their capacity to generate NK cells is lost and the ability to generate T-cells in vivo appear to be reduced even after intra-thymic injections. The Ly6D+ cells express lower levels of CCR9 than their Ly6D− counterparts possibly causing a deficiency in their ability to home to their proper microenvironment even after intrathymic injection. The idea that NK cell potential is lost before Rag-1 is expressed in the developing progenitors is further supported by the finding that only a small portion of the mature NK cells were genetically labeled in mice carrying CRE recombinase under the regulatory elements of the Rag-1 gene. Thus, even though there might exist alternative pathways to B-lineage commitment it appears as if one major pathway involves a gradual loss of myeloid and NK cell potential and then finally T-cell potential and that all these events occur before CD19 can be detected on the cell surface. Previous reports have shown B and T-lineage potential in Lin−B220+CD19− progenitors, presumably residing downstream of the CLPs in the developmental hierarchy. However, even though the absolute majority of the Lin−B220+ cells expressed Rag-1, only half of the cells expressed the Igll-1 (λ5) reporter associated with the committed cells. This would suggest that only a fraction of the B220+CD19− cells represent B-lineage restricted progenitors, an idea well in line with reports of a B220+CLP-2 population with combined B- and T-cell potential. Hence, it is likely that even though B220 expression on CD19− progenitors allows for an enrichment of B-lineage specified cells, it does not define any specific developmental stage since committed cells can also be found among the B220− progenitors. Even though this model is still under development and that it is difficult to exclude alternative pathways into B-lineage, it certainly presents new possibilities to examine lineage commitment in lymphoid development.
The increased understanding of the developmental pathways and the identification of lineage restricted subpopulations opens for more detailed investigations of mice lacking critical regulatory components. The basic Helix-Loop-Helix proteins E12 and E47, both encoded by the E2A gene, was reported as crucial for the development of early B-cells. Advanced understanding of developmental pathways and identification of additional subpopulations revealed that the defect was established already in the CLP compartment with a reduced number of cells as well as a profound block in development of Ly6D+CLPs. These findings has resulted in the proposal that the E2A encoded E47 protein is crucial for B-lineage specification at this stage of development. A defective CLP compartment could also be found in mice lacking the helix-loop-helix transcription factor Ebf-1. Mice deficient of this protein were reported to develop B220+CD43+ cells leading to the initial conclusion that even though crucial for the development of CD19+ B-cells, this factor was not crucial for the development of early B-lineage cells. However, upon analysis of the CLP compartment developed from transplanted fetal livers obtained from Ebf-1 deficient mice, it was obvious that even though cells with a surface phenotype of a CLP could be detected, these cells lacked expression of B-lineage associated genes. Furthermore, even though Ly6D+CLPs can be found in Ebf-1 deficient mice, these cells maintain their NK cell potential, strongly supporting that EBF-1 is crucial both for the activation of the B-lineage program and for the suppression of alternative cell fates. Such a crucial role for EBF-1 is also supported from the findings that ectopic expression of this protein in early hematopoetic progenitor cells cause disruptions in the development of T and myeloid lineage cells. EBF-1 appear capable to mediate lineage restriction even in the absence of PAX-5 or E2A-proteins, indicating that EBF-1 itself targets genes involved in the reduction of alternative lineage potentials. A direct function of Ebf-1 in early restriction events is also supported by the expression pattern since the mRNA levels are dramatically increased in the transition from Ly6D− to Ly6D+ cells. Even though it has not yet been possible to identify key target genes for Ebf-1 that can explain the full biological activities of this factor, gene expression analysis and chromatin immunoprecipitation experiments has identified a set of highly interesting EBF-1 target genes. These include several components of the pre-B cell receptor complex including Cd79a (Mb-1), Cd79b, Blk, VpreB1 and Igll-1, but also a set of transcription factors that may be of crucial importance both to activate additional genes at the CLP stage and to drive development into more mature developmental stages. One of these is the transcription factor FOXO-1, shown to be of critical importance for early B-cell development and for the activation of Rag-1 expression. This transcription factor is expressed in early multipotent hematopoetic progenitors and in Ly6D−/Rag-1− CLPs, where it may be involved in the activation of the Il-7 receptor gene. The expression of FoxO-1 message is, however, upregulated upon differentiation into the Ly6D+ stage where the factor is involved in the activation of the Rag-1 gene. Therefore, by regulating the expression of FoxO-1, EBF-1 may create a cascade of events that results in the coordinated activation of genes priming the cells towards B-lymphoid commitment. Furthermore, chromatin immunoprecipitation experiments suggested that several of the EBF-1 target genes contained binding sites for E47 and FOXO-1, suggesting that these factors collaborate in the activation of a B-lymphoid program. Such collaborative activities is further supported by the findings that mice transheterocygous for mutations in E2A and Ebf-1 or E2A and FoxO-1 genes, display an enhanced phenotype with regard to defective development of B-lineage cells as compared to the single heterocygotes. Furthermore, it has been reported that ectopic expression of EBF-1 can rescue the development of CD19+ cells in E2A deficient mice, suggesting a partially redundant function of these factors in development. It should, however, be kept in mind that even though these mice lack E2A proteins the early progenitors may express other E-protein family members such as E2-2 or HEB providing a rudimentary level of E-protein activity that may be sufficient to allow for B-lymphocyte development in scenarios where factors like EBF-1 or PAX-5 are overexprssed.
EBF-1 has also been shown to regulate the expression of the paired domain transcription factor PAX-5, shown crucial for early B-cell development. In contrast to what is observed in E2A and EBF-1 deficient mice, Pax-5 deficiency results in the development of cells expressing basal levels of B-lineage restricted genes. This would argue against that PAX-5 would be directly involved in lineage specification but rather suggest that the factor is crucial for the progression of B-lineage development. Additionally, the finding that Pax-5 expression is low or undetectable in the major part of the CLP compartment makes it difficult to understand how PAX-5 could act as the primary determinant in early lineage restriction events. Even though the Ly6D− CLPs display some residual myeloid potential, this is minimal as compared to that of LMPPs suggesting that normal lymphoid/myeloid restriction occurs before the expression of Pax-5. Furthermore, even though Pax-5 expression can be detected in the Ly6D+CLP compartment, the expression appears heterogeneous and the major part of the Pax-5 expression appears to reside within the B-lineage committed part of the CLPs. Hence, the expression pattern of Pax-5 would argue against any major function in the reduction of myeloid or NK cell potential. The expression of Gfi-1 λ5 Pax-5, does, however, display a good overlap with loss of in vitro T-cell potential, suggesting that this factor is a crucial determinant for B versus T cell fate in normal lymphocyte development. This is also supported by the finding that while ectopic expression of PAX-5 has limited impact on the development of myeoloid cells, T-cell development is impaired. Even though several lines of investigation suggests that the role of PAX-5 in lineage commitment is limited to reduction of T-cell potential in the progenitors, the finding that early B cells from Pax-5 deficient mice displayed a dramatic lineage plasticity suggested that PAX-5 indeed plays a crucial role in B-lineage commitment. One possible explanation to these apparently contradictory findings came from investigations of mice where the Pax-5 gene was inactivated in B-cells. This resulted in de-differentiation of mature B-cells presumably enabling them to adopt alternative cell fates. Hence, even though Pax-5 may not be crucial for B-lineage specification and all the lineage restriction events, the protein is essential for the stable commitment of the B-lineage progenitors.
Since EBF-1 is a critical component of early differentiation and B-cell commitment events, the regulation of Ebf-1 expression becomes central in understanding B-lineage restriction events. Even though the large size of this gene complicates the search for regulatory elements, two promoters regulating Ebf-1 expression have been identified. The most 5′ promoter is located approximately 2000 bp upstream of the first coding exon of the gene. This promoter contains potential binding sites for E2A-proteins, IKAROS, EBF-1 and STAT-5. The latter creates a link between cytokine signaling and B-lineage differentiation since STAT-5 is a downstream mediator of IL-7 signaling. IL-7 receptor signaling is of critical importance for B-cell development. Mice lacking IL-7 have a reduced CLP compartment mainly due to a dramatic reduction in Ly6D+ cells while the effect on the Ly6D− compartment is less pronounced. This deficiency in B-cell development can be partially rescued by ectopic expression of Ebf-1 or constitutively active (ca)STAT-5. Thus, IL-7 may act via STAT-5 in an instructive manner to induce Ebf-1 and cause lineage restriction in the Ly6D+CLPs. An instructive role is also supported by that caSTAT-5 promotes B-cell development in the thymus and that B-cell development only is marginally restored by ectopic expression of the anti-apoptotic protein BCL-2. However, IL-7 appears to exert different functions at different stages of development. The B-lymphoid defect observed upon conditional deletion of STAT-5 in a mouse strain with (re-expression driven by the Rag-1 regulatory elements could be rescued by ectopic expression of anti-apoptotic factors]. Since Rag-1 is not extensively expressed until the later stages of Ly6D+CLPs, this would indicate that once the cells has reached this stage, the function of IL-7 is permissive. While the distal Ebf-1 promoter appears to be involved at the earliest stages of B-cell development, the proximal Ebf-1 promoter contains a PAX-5 binding site and since EBF-1 is suggested to regulate the Pax-5 gene this could result in a positive feedback loop serving to stabilize and preserve B-cell identity. This is likely to be crucial since EBF-1 as well as E47 function is targeted by T-cell promoting Notch signaling. In contrast, PAX-5 activity is not repressed by ectopic expression of the intracellular part of the Notch-1 receptor. Instead PAX-5 acts as a repressor of Notch-1 transcription indicating that PAX-5 ensures that NOTCH signaling cannot disturb the regulatory network established. This idea is supported by the findings that B220+ B cell progenitors from Pax-5 deficient mice remain sensitive to NOTCH signaling and hence can be converted into T-lineage cells upon exposure to Notch ligand. Notch signaling has also been shown to be modulated by the transcriptional repressor LRF (Zbtb7a) suggesting that PAX-5 and LFR may collaborate in order to establish stably committed B-lineage cells. PAX-5 has also been shown to directly bind and inhibit transcription from the c-Fms gene and to activate transcription of the Ikaros family protein Aiolos in early progenitor cells. Activation of an Ikaros family member may be of importance for stable lineage commitment since it has been shown that Ikaros deficient pro-B cells rescued by ectopic expression of EBF-1, display lineage plasticity even though they express apparently normal levels of both Ebf-1 and Pax-5 message.
In all, the emerging picture would suggest that the point of no return in B-lymphoid development is gradually reached through an initial specification event where E47 and IL-7 induce expression of B-lineage genes including Ebf-1 in the Ly6D+ progenitors. This results in loss of all but B and T lymphoid potential and the expression of Pax-5 finally creates a stable state and promotes the progression of B-lymphoid development. It is also notable that these transcription factors often share target genes and even if this could reflect a mechanism to achieve high levels of gene expression, it could also be a way to pass on epigenetic information in the differentiation process. Even though much of the recent discoveries confirm the crucial roles for the classical regulators of B-lymphocyte development, there are a number of transcription factors with less obvious links to the known regulatory networks. Among these are BCL 11a, C-MYB, MEF-2c, IRF-8, SOX-4 and GON-4-L. Hence, a major challenge is to understand how and where in the regulatory pathways these factors exert their crucial functions. In addition to their importance in normal blood cell development, it is obvious that these regulatory networks are involved in the development of leukemias. Genome wide analysis of 242 pediatric leukemias revealed that as much as 40% carried mutations in genes encoding major regulators of B-lymphocyte development. Hence, the increased understanding of the genetic networks regulating normal lineage specification and the identification of human progenitor cell subsets promises to provide information crucial for the understanding of malignant transformation in the hematopoetic system thereby opening new pathways for intervention and diagnosis.