Download Seminars in Immunology Gene regulatory networks directing

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. Perspective
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
As a consequence of rapid progress being made in the analysis
of transcription factors and miRNAs that regulate the development
of innate and adaptive cells of the immune system, it should be
possible in the near future to assemble them into complex gene
regulatory networks and analyze these intricate control circuits
using mathematical and computational modeling. Such modeling
may yield counter-intuitive predictions that can be experimentally
tested. Progress in this area will also facilitate the directed and efficient generation of specific immune cells and their manipulation
for cell based therapies.
[25]
[26]
[27]
[28]
[29]
Acknowledgments
[30]
We thank Eric Bertolino, Karen Reddy, Damien Reynaud and
Chauncey Spooner for their critical reading of the manuscript and
suggestions. H. Singh is an Investigator with the Howard Hughes
Medical Institute.
[31]
References
[1] Orkin SH, Zon LI. Hematopoiesis: an evolving paradigm for stem cell biology.
Cell 2008;132(4):631–44.
[2] Orkin SH. Diversification of haematopoietic stem cells to specific lineages. Nat
Rev Genet 2000;1(1):57–64.
[3] Singh H, Medina KL, Pongubala JM. Contingent gene regulatory networks and
B cell fate specification. Proc Natl Acad Sci USA 2005;102(14):4949–53.
[4] Laiosa CV, Stadtfeld M, Graf T. Determinants of lymphoid–myeloid lineage
diversification. Annu Rev Immunol 2006;24:705–38.
[5] Nutt SL, Kee BL. The transcriptional regulation of B cell lineage commitment.
Immunity 2007;26(6):715–25.
[6] Rothenberg EV. Cell lineage regulators in B and T cell development. Nat
Immunol 2007;8(5):441–4.
[7] Bartel DP, Chen CZ. Micromanagers of gene expression: the potentially widespread influence of metazoan microRNAs. Nat Rev Genet
2004;5(5):396–400.
[8] Chen CZ, Li L, Lodish HF, Bartel DP. MicroRNAs modulate hematopoietic lineage
differentiation. Science 2004;303(5654):83–6.
[9] McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs.
Nat Rev Genet 2002;3(10):737–47.
[10] Adolfsson J, Mansson R, Buza-Vidas N, et al. Identification of Flt3+ lymphomyeloid stem cells lacking erythro-megakaryocytic potential a revised road
map for adult blood lineage commitment. Cell 2005;121(2):295–306.
[11] Laslo P, Spooner CJ, Warmflash A, et al. Multilineage transcriptional priming and
determination of alternate hematopoietic cell fates. Cell 2006;126(4):755–66.
[12] Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of
mouse hematopoietic stem cells. Science 1988;241(4861):58–62.
[13] Adolfsson J, Borge OJ, Bryder D, Theilgaard-Monch K, Astrand-Grundstrom I,
Sitnicka E, et al. Upregulation of Flt3 expression within the bone marrow
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
Lin(−)Sca1(+)c-kit(+) stem cell compartment is accompanied by loss of selfrenewal capacity. Immunity 2001;15(4):659–69.
Hock H, Hamblen MJ, Rooke HM, Schindler JW, Saleque S, Fujiwara Y, et al. Gfi1 restricts proliferation and preserves functional integrity of haematopoietic
stem cells. Nature 2004;431(7011):1002–7.
Park IK, Qian D, Kiel M, Becker MW, Pihalja M, Weissman IL, et al. Bmi-1 is
required for maintenance of adult self-renewing haematopoietic stem cells.
Nature 2003;423(6937):302–5.
Wilson A, Murphy MJ, Oskarsson T, et al. c-Myc controls the balance
between hematopoietic stem cell self-renewal and differentiation. Genes Dev
2004;18(22):2747–63.
Zhang P, Iwasaki-Arai J, Iwasaki H, et al. Enhancement of hematopoietic stem
cell repopulating capacity and self-renewal in the absence of the transcription
factor C/EBP alpha. Immunity 2004;21(6):853–63.
Akashi K, Traver D, Miyamoto T, Weissman IL. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature
2000;404(6774):193–7.
Singh H, DeKoter RP, Walsh JC. PU.1, a shared transcriptional regulator of lymphoid and myeloid cell fates. Cold Spring Harb Symp Quant Biol 1999;64:13–20.
Scott EW, Simon MC, Anastasi J, Singh H. Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science
1994;265:1573–7.
Orkin SH, Shivdasani RA, Fujiwara Y, McDevitt MA. Transcription factor GATA-1
in megakaryocyte development. Stem Cells 1998;16(Suppl. 2):79–83.
Rekhtman N, Radparvar F, Evans T, Skoultchi AI. Direct interaction of
hematopoietic transcription factors PU.1 and GATA-1: functional antagonism
in erythroid cells. Genes Dev 1999;13(11):1398–411.
Zhang P, Behre G, Pan J, et al. Negative cross-talk between hematopoietic regulators: GATA proteins repress PU.1. Proc Natl Acad Sci USA 1999;96(15):8705–10.
Cantor AB, Orkin SH. Transcriptional regulation of erythropoiesis: an affair
involving multiple partners. Oncogene 2002;21(21):3368–76.
Kulessa H, Frampton J, Graf T. GATA-1 reprograms avian myelomonocytic
cell lines into eosinophils, thromboblasts, and erythroblasts. Genes Dev
1995;9(10):1250–62.
Rao G, Rekhtman N, Cheng G, Krasikov T, Skoultchi AI. Deregulated expression
of the PU.1 transcription factor blocks murine erythroleukemia cell terminal
differentiation. Oncogene 1997;14(1):123–31.
Zhang P, Zhang X, Iwama A, et al. PU.1 inhibits GATA-1 function and erythroid
differentiation by blocking GATA-1 DNA binding. Blood 2000;96(8):2641–8.
Okuno Y, Huang G, Rosenbauer F, et al. Potential autoregulation of transcription factor PU.1 by an upstream regulatory element. Mol Cell Biol
2005;25(7):2832–45.
Tsai SF, Strauss E, Orkin SH. Functional analysis and in vivo footprinting implicate the erythroid transcription factor GATA-1 as a positive regulator of its own
promoter. Genes Dev 1991;5(6):919–31.
Arinobu Y, Mizuno S-I, Chong Y, et al. Reciprocal activation of GATA-1 and PU.1
marks initial specification of hematopoietic stem cells into myeloerythroid and
myelolymphoid lineages. Cell Stem Cell 2007;1(4):416–27.
Iwasaki H, Mizuno S, Arinobu Y, et al. The order of expression of transcription
factors directs hierarchical specification of hematopoietic lineages. Genes Dev
2006;20(21):3010–21.
Dahl R, Walsh JC, Lancki D, Laslo P, Iyer SR, Singh H, et al. Regulation of
macrophage and neutrophil cell fates by the PU.1:C/EBPalpha ratio and granulocyte colony-stimulating factor. Nat Immunol 2003;4(10):1029–36.
Miyamoto T, Iwasaki H, Reizis B, Ye M, Graf T, Weissman IL, et al. Myeloid or
lymphoid promiscuity as a critical step in hematopoietic lineage commitment.
Dev Cell 2002;3(1):137–47.
Starck J, Cohet N, Gonnet C, et al. Functional cross-antagonism between transcription factors FLI-1 and EKLF. Mol Cell Biol 2003;23(4):1390–402.
Krysinska H, Hoogenkamp M, Ingram R, Wilson N, Tagoh H, Laslo P, et
al. A two-step, PU.1-dependent mechanism for developmentally regulated
chromatin remodeling and transcription of the c-fms gene. Mol Cell Biol
2007;27(3):878–87.
Kloosterman WP, Plasterk RH. The diverse functions of microRNAs in animal
development and disease. Dev Cell 2006;11(4):441–50.
Fazi F, Rosa A, Fatica A, Gelmetti V, De Marchis ML, Nervi C, et al. A minicircuitry
comprised of microRNA-223 and transcription factors NFI-A and C/EBPalpha
regulates human granulopoiesis. Cell 2005;123(5):819–31.
Felli N, Fontana L, Pelosi E, et al. MicroRNAs 221 and 222 inhibit normal erythropoiesis and erythroleukemic cell growth via kit receptor down-modulation.
Proc Natl Acad Sci USA 2005;102(50):18081–6.
Fontana L, Pelosi E, Greco P, et al. MicroRNAs 17-5p-20a-106a control monocytopoiesis through AML1 targeting and M-CSF receptor upregulation. Nat Cell
Biol 2007;9(7):775–87.
Rosa A, Ballarino M, Sorrentino A, et al. The interplay between the master
transcription factor PU.1 and miR-424 regulates human monocyte/macrophage
differentiation. Proc Natl Acad Sci USA 2007;104(50):19849–54.
Hibbs ML, Quilici C, Kountouri N, Seymour JF, Armes JE, Burgess AW, et al.
Mice lacking three myeloid colony-stimulating factors (G-CSF, GM-CSF, and
M-CSF) still produce macrophages and granulocytes and mount an inflammatory response in a sterile model of peritonitis. J Immunol 2007;178(10):6435–
43.
Hirai H, Zhang P, Dayaram T, Hetherington CJ, Mizuno S, Imanishi J, et
al. C/EBPbeta is required for ‘emergency’ granulopoiesis. Nat Immunol
2006;7(7):732–9.
P. Laslo et al. / Seminars in Immunology 20 (2008) 228–235
[43] Nagai Y, Garrett KP, Ohta S, Bahrun U, Kouro T, Akira S, et al. Toll-like receptors
on hematopoietic progenitor cells stimulate innate immune system replenishment. Immunity 2006;24(6):801–12.
[44] DeKoter RP, Singh H. Regulation of B lymphocyte and macrophage development
by graded expression of PU.1. Science 2000;288(5470):1439–41.
[45] Bain G, Maandag EC, Izon DJ, et al. E2A proteins are required for proper B
cell development and initiation of immunoglobulin gene rearrangements. Cell
1994;79(5):885–92.
[46] Zhuang Y, Soriano P, Weintraub H. The helix-loop-helix gene E2A is required
for B cell formation. Cell 1994;79(5):875–84.
[47] Kwon K, Hutter C, Sun Q, Bilic I, Cobaleda C, Malin S, Busslinger M. Instructive
role of the transcription factor E2A in early B lymphopoiesis and germinal center
B cell development. Immunity 2008;28(6):751–62.
[48] Ikawa T, Kawamoto H, Wright LY, Murre C. Long-term cultured E2A-deficient
hematopoietic progenitor cells are pluripotent. Immunity 2004;20(3):349–60.
[49] Yoshida T, Ng SY, Zuniga-Pflucker JC, Georgopoulos K. Early hematopoietic lineage restrictions directed by Ikaros. Nat Immunol 2006;7(4):382–91.
[50] Reynaud D, Demarco IA, Reddy KL, et al. Regulation of B cell fate commitment and immunoglobulin heavy-chain gene rearrangements by Ikaros. Nat
Immunol 2008;9(8):927–36.
[51] Pongubala JM, Northrup DL, Lancki DW, et al. Transcription factor EBF restricts
alternative lineage options and promotes B cell fate commitment independently of Pax5. Nat Immunol 2008;9(2):203–15.
[52] Lin H, Grosschedl R. Failure of B-cell differentiation in mice lacking the transcription factor EBF. Nature 1995;376(6537):263–7.
[53] O’Riordan M, Grosschedl R. Coordinate regulation of B cell differentiation by
the transcription factors EBF and E2A. Immunity 1999;11(1):21–31.
[54] Fuxa M, Skok J, Souabni A, Salvagiotto G, Roldan E, Busslinger M. Pax5 induces
V-to-DJ rearrangements and locus contraction of the immunoglobulin heavychain gene. Genes Dev 2004;18(4):411–22.
[55] Medina KL, Pongubala JM, Reddy KL, Lancki DW, Dekoter R, Kieslinger M, et al.
Assembling a gene regulatory network for specification of the B cell fate. Dev
Cell 2004;7(4):607–17.
[56] Dias S, Silva Jr H, Cumano A, Vieira P. Interleukin-7 is necessary to maintain the B
cell potential in common lymphoid progenitors. J Exp Med 2005;201(6):971–9.
[57] Kikuchi K, Lai AY, Hsu CL, Kondo M. IL-7 receptor signaling is necessary for stage
transition in adult B cell development through up-regulation of EBF. J Exp Med
2005;201(8):1197–203.
[58] Seet CS, Brumbaugh RL, Kee BL. Early B cell factor promotes B lymphopoiesis
with reduced interleukin 7 responsiveness in the absence of E2A. J Exp Med
2004;199(12):1689–700.
[59] Urbanek P, Wang ZQ, Fetka I, Wagner EF, Busslinger M. Complete block of early
B cell differentiation and altered patterning of the posterior midbrain in mice
lacking Pax5/BSAP. Cell 1994;79:901–12.
[60] Nutt SL, Heavey B, Rolink AG, Busslinger M. Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature 1999;401(6753):556–62.
[61] Rolink AG, Nutt SL, Melchers F, Busslinger M. Long-term in vivo reconstitution of T-cell development by Pax5-deficient B-cell progenitors. Nature
1999;401(6753):603–6.
[62] Mikkola I, Heavey B, Horcher M, Busslinger M. Reversion of B cell commitment
upon loss of Pax5 expression. Science 2002;297(5578):110–3.
235
[63] Delogu A, Schebesta A, Sun Q, Aschenbrenner K, Perlot T, Busslinger M. Gene
repression by Pax5 in B cells is essential for blood cell homeostasis and is
reversed in plasma cells. Immunity 2006;24(3):269–81.
[64] Mansson R, Zandi S, Andersson K, Martensson IL, Jacobsen SE, Bryder D, et
al. B-lineage commitment prior to surface expression of B220 and CD19 on
hematopoietic progenitor cells. Blood 2008.
[65] Mackarehtschian K, Hardin JD, Moore KA, Boast S, Goff SP, Lemischka IR.
Targeted disruption of the flk2/flt3 gene leads to deficiencies in primitive
hematopoietic progenitors. Immunity 1995;3(1):147–61.
[66] Sitnicka E, Bryder D, Theilgaard-Monch K, Buza-Vidas N, Adolfsson J, Jacobsen
SE. Key role of flt3 ligand in regulation of the common lymphoid progenitor but not in maintenance of the hematopoietic stem cell pool. Immunity
2002;17(4):463–72.
[67] Borge OJ, Adolfsson J, Jacobsen AM. Lymphoid-restricted development from
multipotent candidate murine stem cells: distinct and complimentary functions of the c-kit and flt3-ligands. Blood 1999;94(11):3781–90.
[68] Miller JP, Izon D, DeMuth W, Gerstein R, Bhandoola A, Allman D. The earliest step
in B lineage differentiation from common lymphoid progenitors is critically
dependent upon interleukin 7. J Exp Med 2002;196(5):705–11.
[69] Sitnicka E, Brakebusch C, Martensson IL, et al. Complementary signaling
through flt3 and interleukin-7 receptor alpha is indispensable for fetal and
adult B cell genesis. J Exp Med 2003;198(10):1495–506.
[70] Vosshenrich CA, Cumano A, Muller W, Di Santo JP, Vieira P. Thymic stromalderived lymphopoietin distinguishes fetal from adult B cell development. Nat
Immunol 2003;4(8):773–9.
[71] Roessler S, Gyory I, Imhof S, Spivakov M, Williams RR, Busslinger M, et
al. Distinct promoters mediate the regulation of Ebf1 gene expression by
interleukin-7 and Pax5. Mol Cell Biol 2007;27(2):579–94.
[72] Graf T. Immunology: blood lines redrawn. Nature 2008;452(7188):702–3.
[73] Bell JJ, Bhandoola A. The earliest thymic progenitors for T cells possess myeloid
lineage potential. Nature 2008;452(7188):764–7.
[74] Wada H, Masuda K, Satoh R, Kakugawa K, Ikawa T, Katsura Y, et al. Adult T-cell
progenitors retain myeloid potential. Nature 2008;452(7188):768–72.
[75] Maeda T, Merghoub T, Hobbs RM, et al. Regulation of B versus T lymphoid lineage fate decision by the proto-oncogene LRF. Science 2007;316(5826):860–6.
[76] Maillard I, Fang T, Pear WS. Regulation of lymphoid development, differentiation, and function by the Notch pathway. Annu Rev Immunol 2005;23:945–74.
[77] Smith EM, Akerblad P, Kadesch T, Axelson H, Sigvardsson M. Inhibition of EBF
function by active Notch signaling reveals a novel regulatory pathway in early
B-cell development. Blood 2005;106(6):1995–2001.
[78] Hoflinger S, Kesavan K, Fuxa M, Hutter C, Heavey B, Radtke F, et al. Analysis
of Notch1 function by in vitro T cell differentiation of Pax5 mutant lymphoid
progenitors. J Immunol 2004;173(6):3935–44.
[79] Rothenberg EV. Negotiation of the T lineage fate decision by transcription-factor
interplay and microenvironmental signals. Immunity 2007;26(6):690–702.
[80] Xie H, Ye M, Feng R, Graf T. Stepwise reprogramming of B cells into macrophages.
Cell 2004;117(5):663–76.
[81] Graf T, Busslinger M. B young again. Immunity 2008;28(5):606–8.
[82] Hanna J, Markoulaki S, Schorderet P, et al. Direct reprogramming of
terminally differentiated mature B lymphocytes to pluripotency. Cell
2008;133(2):250–64.