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Next Article 
Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2431-2448
REVIEW ARTICLE
Notch as a Mediator of Cell Fate Determination in Hematopoiesis:
Evidence and Speculation
By
Laurie A. Milner and
Anna Bigas
From The Fred Hutchinson Cancer Research Center, Seattle, WA; the
Department of Pediatrics, University of Washington School of Medicine,
Seattle, WA; and the Institut de Recerca Oncologica, Hospital Duran y
Reynals, Barcelona, Spain.
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INTRODUCTION |
HEMATOPOIESIS IS A continuous
developmental process in which pluripotent stem cells and their progeny
make sequential cell fate decisions, producing mature blood cells of
the various lineages. The constant generation of appropriate numbers
and types of mature cells, as well as the maintenance of multipotent
progenitors, requires a complex regulatory network, many aspects of
which remain incompletely understood.1-7 Members of the
Notch family play critical roles in the determination of cell
fates and maintenance of progenitors in many developmental
systems.8-10 Given the extensive evolutionary and
phylogenetic conservation of Notch function, it is not surprising that
signaling through the Notch pathway has been implicated in the
regulation of hematopoiesis. Since the initial demonstration that
Notch1 is expressed in normal bone marrow (BM) hematopoietic
precursors,11 considerable evidence has emerged to support
a conserved role for Notch in the mediation of cell fate
decisions and self-renewal of progenitors during hematopoiesis. This
review presents an overview of the Notch signaling pathway, evidence
regarding Notch function, interactions of the Notch pathway with other
signal transduction pathways, and a model for Notch function in hematopoiesis.
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NOTCH AS A MEDIATOR OF CELL FATE DECISIONS: GENERAL CONSIDERATIONS |
During development, multipotent progenitors undergo lineage commitment
and maturation in a strict temporal and spatial pattern that reflects
the expression of different genes among originally equipotent
cells.2,3,6,12 Although many factors contribute to
differential gene expression, signaling between cells is one of the key
components of gene regulation and consequent appropriate cell fate
specification.6,13-15 The Notch family comprises a group of
highly conserved proteins that function both as cell surface receptors
and direct regulators of gene transcription.9,16-18 As
such, these molecules represent a unique conduit for signal transduction from the cell surface to the nucleus, permitting cells to
directly influence gene expression in their neighbors. In general,
Notch activation leads to transcriptional suppression of
lineage-specific genes, inhibiting differentiation in response to
inductive signals. Notch signaling limits the number of cells that
adopt a particular fate and leaves some progenitors uncommitted but
competent to adopt alternative fates.
The requirement for Notch activation through appropriate cell-cell
interactions, combined with continuous changes in gene expression
during development, permits Notch to influence cell fate decisions in a
wide variety of tissues and cell types. In Drosophila, Notch is
essential for the appropriate specification of many different cell
fates during oogenesis, neurogenesis, myogenesis, and wing and eye
development.9,10,19 The four known mammalian Notch
genes, Notch1-4, are widely expressed during embryogenesis and
also play crucial developmental roles.20-27 New evidence
extending the role of Notch as a general mediator of cell fate
determination in mammalian systems is constantly
emerging.10,28,29
Conservation of Notch Structure
The evolutionary conservation of Notch function is reflected in the
high degree of structural conservation of Notch proteins and other
molecules that mediate signal transduction through the Notch pathway.
Figure 1 depicts the general structure of
Notch, showing conserved regions of known functional significance.
Molecules that directly interact with different Notch domains are noted in Fig 1 and are discussed in subsequent sections. The Notch
extracellular domain contains a variable number of tandem epidermal
growth factor (EGF)-like repeats and three Lin/Notch repeats (LNR),
which function in ligand binding and Notch activation.30-32
The conserved cysteines between the LNR and the transmembrane domain
(TM) are likely involved in disulfide bonding of the heterodimeric
receptor (Fig 1, inset). The putative cleavage sites involved in
generation of the functional Notch receptor and release of
intracellular Notch upon activation are indicated by dark and light
arrows, respectively (see below, Notch Activation).
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| Fig 1.
General structure of Notch proteins, showing conserved
functional domains and proteins that interact with different regions.
Inset shows the Notch heterodimeric receptor, which is generated by
proteolytic processing and reassociation of the extracellular and
intracellular fragments before reaching the cell surface. The putative
cleavage site is indicated in the main figure by the black arrow. The
extracellular domain consists of 29-36 tandem EGF-like repeats and 3 Lin/Notch repeats (LNR) involved in DSL ligand binding and Notch
activation. The ligand-induced proteolytic cleavage site is indicated
by the white arrow. The intracellular domain includes 6 cdc10 repeats,
which mediate protein interactions essential for Notch function; the
RAM domain, which binds CSL effector molecules; and the NCR region
associated with cytokine-specific effects of Notch1 and 2. See text for
discussion of the various proteins that interact with Notch.
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The Notch intracellular domain includes six cdc10/ankyrin repeats
(hereafter referred to as cdc10 repeats), motifs characteristic of
molecules involved in protein-protein interactions; this is the most
highly conserved region and is essential for Notch signal transduction.31-35 The Notch protein does not have any
known enzymatic activity, but rather transmits signals through direct
molecular interactions. The region of Notch C-terminal to the cdc10
repeats has been associated with distinct protein interactions and
transactivation,36-38 and the PEST domain is thought to
regulate protein turnover. Drosophila Notch and the mammalian Notch1,
2, and 3 molecules also contain conserved nuclear localization signals
(nls) and OPA sequences. More recently described regions include the
RAM domain, which binds CSL (CBF1/Suppressor of
Hairless/Lag-1) effectors of Notch signal
transduction,35,39-41 and the Notch Cytokine Response (NCR) region associated with distinct effects of Notch1 and 2 on myeloid differentiation.42
Conservation of the Notch Signaling Pathway
In addition to Notch receptors, the primary components of the
Notch signal transduction pathway include ligands homologous to
Drosophila Delta and Serrate and
Caenorhabditis elegans Lag-2 (DSL
proteins) and intracellular effector molecules homologous to
Suppressor of Hairless (CSL proteins) and Enhancer of split
(E[spl]). Other effectors, targets, and modulators of Notch have
also been evolutionarily conserved. Table 1
summarizes the corresponding components of Notch signaling in flies,
worms, and mammals. For the sake of clarity, this review focuses on
mammalian systems, using Drosophila Notch as the prototype.
Studies of lin-12 and glp-1 function during worm
development have also contributed major insights into the mechanisms of
Notch signaling. Comprehensive discussions of Notch/Lin-12/Glp-1
signaling in flies and worms can be found in a number of recent
reviews.19,43,44
In the prevailing model for Notch signal transduction
(Fig 2), Notch activation through binding
to DSL ligands on adjacent cells results in proteolytic cleavage, with
release and nuclear translocation of the Notch intracellular domain
(Notch-IC). Notch-IC interacts with a number of cytoplasmic and nuclear
proteins, permitting signal transduction through at least two pathways,
one involving CSL proteins and one independent of CSL. The interaction
of Notch-IC with CSL proteins results in transcriptional activation of
E(spl)/HES genes, which function as negative regulators of
lineage-specific gene expression. CSL-independent signaling also
results in transcriptional regulation, but is mediated by different
effector molecules, such as Deltex, and may regulate distinct target
genes.
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| Fig 2.
Signal transduction through the Notch pathway. In the
presence of a specific differentiation signal, activation of Notch
through ligand binding results in proteolytic cleavage and release of
the intracellular domain. Activated intracellular Notch (Notch-IC)
and/or CSL proteins translocate to the nucleus, where they activate
transcription of E(spl)/HES. The transcription factors encoded
by E(spl)/HES in turn suppress transcription of
lineage-specific genes, thereby inhibiting cellular differentiation. An
equivalent cell, in the absence of Notch activation (right), will
respond to the differentiation signal by activating transcription of
lineage-specific genes, permitting differentiation along the induced
pathway.
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Homotypic and Heterotypic Cell-Cell Interactions
Intercellular signaling through the Notch receptor permits equipotent
cells in the same environment to respond differently to developmental
signals. Although Notch and its ligands are often expressed on the same
cell, Notch is activated primarily through binding to its ligand on
adjacent cells.45,46 Notch signaling can occur either among
a group of equivalent cells (homotypic interactions) or between
nonequivalent cells (heterotypic interactions), both of which are
essential during development.16,19,43,44
Homotypic interactions: lateral inhibition.
Notch activation through homotypic interactions results in what has
been termed lateral inhibition: among a group of equipotent cells
exposed to a specific differentiation signal, a limited number will
adopt the specific cell fate, whereas adjacent cells (that express more
Notch) are inhibited from differentiating
(Fig 3A). These uncommitted
cells remain competent to respond to subsequent signals, but the
adoption of alternative fates may again be regulated by Notch. The
dosage-dependent effects of Notch during lateral signaling have been
demonstrated in chimeric flies and mice, in which cells expressing
different amounts of Notch are juxtaposed. In the prototype of lateral
signaling, the neural/epidermal cell fate decision during fly central
nervous system (CNS) development, cells expressing less
Notch adopt the primary (neuronal) fate and adjacent cells, expressing
more Notch, adopt the alternative (epidermal) fate.45
Similarly, during T-cell development, thymocytes expressing less Notch
adopt the primary   cell fate, whereas those expressing more fail
to adopt the primary fate and thus adopt the alternative   T cell
fate.28,47 Figure 3A shows how sequential lateral signaling
among progenitors facilitates the generation of distinct cell types.
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| Fig 3.
Relative levels of Notch expression determine
sequential cell fates. (A) Lateral signaling among a group of
equipotent progenitors permits the generation of cells of distinct
lineages in response to inductive signals. When exposed to signal A,
cells expressing less Notch and more ligand (L) respond by adopting the
primary cell fate A; adjacent cells expressing more Notch (N) are
inhibited from adopting fate A, but remain competent to respond to
subsequent signals. Among these remaining progenitors, differential
expression of Notch again determines which cells will respond to
inductive signal B: those expressing less Notch adopt fate B, whereas
those expressing more are again inhibited from differentiating.
Differential Notch and DSL ligand expression among the remaining
progenitors at each subsequent step similarly restricts the number of
cells responding to signals C and D. Thus, from a group of originally
equipotent progenitors, cells of multiple distinct lineages are
established, and some uncommitted progenitors are maintained. (B) Notch
functions through successive cell divisions to influence the numbers
and types of cells generated from a multipotent progenitor. Normal
Notch expression (left panel) allows the A/B progenitor to give rise to
cells of four distinct lineages; at each cell division, the daughter
cell expressing less Notch adopts the primary fate, whereas the cell
expressing more adopts the alternative secondary fate. The A/B
progenitor gives rise to A (primary) and B (secondary) cells; progeny
of type A cells expressing less Notch subsequently adopt the primary
fate A1, whereas those expressing more Notch adopt the secondary fate
A2; the same occurs for type B cells. The result is balanced production
of cells of all four lineages. When Notch activity is dysregulated, the
result is overproduction of one cell type at the expense of another.
With loss of Notch function (middle panel), all cells adopt the primary
fates resulting in production of only A1 cells. With increased Notch
activity (right panel), daughter cells adopt the secondary fates,
generating only B2 cells.
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Although the mechanisms responsible for initial differences in Notch
expression among equivalent progenitors have not been clearly
established, recent studies suggest that, rather than being strictly
stochastic, specific intrinsic and extrinsic factors dictate which
cells will express more or less Notch.43,44,48 Subsequent
signaling is then biased by a feedback loop that reinforces either
Notch or DSL ligand expression. Many downstream components of the Notch
pathway participate in this feedback regulation, which permits the
amplification of very minor differences in Notch expression.19,44
Heterotypic interactions: inductive signaling.
Notch signaling also occurs between Notch and DSL ligand expressed on
different cell types. Inductive signaling through these heterotypic
interactions is regulated primarily by ligand expression, limiting
Notch activation to those cells in direct contact with ligand-expressing cells. Interactions between Notch and DSL ligands can
also be modulated by other molecules, such as Fringe and
Wingless,49,50 and further regulated by a feedback
loop.51,52 Inductive signaling thus permits the
establishment of finely demarcated boundaries between cell types,
exemplified by dorsoventral boundary formation during fly wing margin
specification and vertebrate limb development.52-54
In the hematopoietic system, Notch interactions may be either homotypic
or heterotypic, and both lateral and inductive signaling mechanisms are
likely to be important. The different extrinsic factors and cell types
involved may provide distinct regulatory mechanisms and increase the
diversity of Notch function. However, despite important differences, in
some respects the effects of Notch activation through either type of
signaling are similar, ie, the generation of distinct cell lineages
from initially equivalent cells. Figure 3B shows the influence of Notch
on the normal production of different cell types from a single
progenitor (left panel) and the effects of loss of Notch activity
(middle panel) or increased Notch activity (right panel). Normally,
differences in Notch expression permit daughter cells to adopt distinct
cell fates, resulting in a balanced distribution of four cell types
after two generations. Dysregulated Notch activity results in cells
adopting only primary fates (loss of Notch activity) or secondary fates
(increased Notch activity), resulting in overproduction of one cell
type at the expense of the others. In the case of increased Notch
activity, adoption of the secondary fate depends on the capacity of the cell to respond to the secondary signal. If Notch expression is constitutive, cells may fail to differentiate in response to any signal, resulting in the lack of production of any mature cells.
Diversification of Notch Function in Mammals
The critical role of Notch signaling in mammalian development is
apparent from Notch1 and 2, Jagged1 and 2, and
Delta1 knockout mice, which have severe defects resulting in
embryonic or perinatal lethality.55-59 However, these mice
also display distinct phenotypic defects, showing the lack of complete
functional redundancy of the different mammalian Notch molecules and
ligands. It seems likely that the evolution of multiple genes encoding
Notch receptors and ligands in mammals reflects a need for
diversification of Notch function in these more complex developmental systems.
Functional diversity of Notch signaling in vivo most likely results
from a combination of intrinsic and extrinsic mechanisms. In some
cases, distinct functions may be dictated by differential tissue
expression or by expression levels of different Notch molecules and
ligands. For example, Notch4 expression is largely restricted to endothelial cells,27 and although Notch1 and
2 are widely expressed, within the lymphoid system they are
preferentially expressed in thymus or spleen,
respectively.23,24 Similarly, Jagged1 and 2 are preferentially expressed in BM or thymus.60-62 Restricted expression of DSL ligands may serve an important regulatory role by limiting Notch activity to a subset of expressing cells and
further regulating Notch expression through a feedback
loop.62 The timing and pattern of expression of different
Notch molecules and ligands may also permit distinct functions
within the same tissue, as suggested by Notch1, 2, and
3 expression in the developing tooth63 and by
Notch, Jagged, and Delta expression during nervous system development.64
It has been suggested that the mammalian Notch orthologues are
biochemically redundant, ie, that they are capable of participating in
the same molecular interactions and thus, in principle, can functionally compensate for each other and that their specific roles in
vivo reflect differential expression. However, it is also possible that
intrinsic differences in the Notch proteins permit distinct
interactions that define functional differences. Specific interactions
with DSL ligands and modulators may primarily be determined by
ligand,29 but may also reflect differences in the Notch
extracellular domains. Structural differences in the intracellular
domain may also define distinct activities, even within the same cell,
as shown by the correlation of physical properties with specific
effects of Notch1 and 2 on 32D
differentiation.42
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NOTCH IN HEMATOPOIESIS |
Defining the cellular and molecular mechanisms responsible for
differentiation and self-renewal of hematopoietic progenitors is
central to achieving a complete understanding of hematopoiesis. Hematopoietic stem cells and multipotent progenitors must continuously undergo lineage commitment, differentiation, and proliferation, while
also maintaining a pool of uncommitted progenitors to support the
production of new blood cells. Despite considerable progress, the
molecular processes that mediate cell fate specification and self-renewal of progenitors remain incompletely understood and controversial.2-7,12,65 Notch is a general regulator of
cell fate determination and interacts with a host of factors that are of known significance in hematopoiesis. The integration of Notch signaling with other cell-cell interactions, cytokine pathways and
transcriptional regulation may be a key to understanding the regulation
of hematopoiesis.
A considerable amount of indirect evidence supporting a role for Notch
in hematopoiesis has emerged over the past several years. Our initial
observation that human Notch1 is expressed in normal BM
hematopoietic precursors provided the foundation for the hypothesis
that Notch functions in hematopoiesis.11 These studies
demonstrated that Notch1 is expressed in marrow CD34+ progenitors, including the immature subset that lacks
expression of lineage-associated antigens
(CD34+lin ). Interestingly,
Notch1 is also expressed in the
CD34+lin+ subset and, at lower levels, in more
mature CD34 cells. We subsequently found that
Notch1 is also expressed in lymphoid, myeloid, and erythroid
precursor populations, as well as in peripheral blood T and B
lymphocytes, monocytes, and neutrophils (L.A.M., unpublished
data), suggesting that Notch functions in multiple
lineages and at various stages of maturation. The observations that DSL
ligands are expressed in BM, fetal liver, and
thymus60,61,66,67 and that Notch2, 3, and 4 are also
expressed by hematopoietic progenitors11,68 (and L.A.M.,
unpublished data) provide further evidence that Notch
signaling plays a significant role in hematopoiesis.
Myeloid Differentiation
The first evidence for Notch function in myelopoiesis came from studies
in 32D cells, a progenitor cell line frequently used as a model system
for myeloid differentiation. 32D cells proliferate as undifferentiated
blasts in the presence of interleukin-3 (IL-3), but can be induced to
differentiate in response to other cytokines, including granulocyte
colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin.69
In initial studies, we found that expression of an activated
intracellular form of Notch1 inhibits differentiation of 32D cells in
response to G-CSF and permits the expansion of undifferentiated cells, findings consistent with the effects of constitutive Notch activity in
other systems.68 In subsequent studies we confirmed that activation of full-length Notch1 by the ligand, Jagged1, results in
comparable phenotypic effects.60 These studies further
validate the use of intracellular forms of Notch as a model for Notch
activity and provide the important demonstration that a complete Notch signaling pathway is intact in 32D cells.
Cytokine-specific effects of Notch1 and 2: The Notch
Cytokine Response (NCR) region.
Notch1 and 2 are both expressed in myeloid progenitors, raising the
possibility that they have distinct functions in these cells. In
studies to address this question, we found that, whereas either Notch1
or Notch2 is capable of inhibiting myeloid differentiation, they do so
in a cytokine-specific manner: Notch1 in response to G-CSF, and Notch2
in response to GM-CSF.42 Furthermore, this cytokine
specificity is associated with a previously uncharacterized region of
Notch, which we have termed the Notch Cytokine
Response (NCR) region (Fig 1). The Notch1 and 2 NCR regions
also confer differences in subcellular localization and electrophoretic
mobility, suggesting that differences in posttranslational modification of the NCR region may define specific molecular interactions. These
studies provide the first evidence that different Notch orthologues may
have distinct functions in the same cell type and indicate a molecular
basis for those differences.
The finding that Notch1 and 2 are active only in the context of
specific cytokines elucidates a potentially important link between
Notch and cytokine signaling pathways in hematopoietic regulation. The
use of truncated intracellular Notch1 and 2 molecules in these
experiments suggests that the Notch1 and 2 intracellular domains
interact specifically with components of the G-CSF or GM-CSF
intracellular signal transduction pathways. (For a model, see
Fig 4B.) The involvement of
distinct JAK/STAT molecules in G-CSF and GM-CSF signaling raises the
possibility that these factors are involved in the cytokine specificity
of Notch. It is also possible that interactions involving the
extracellular domain of Notch and cytokines or cytokine receptors
influence the effects of Notch on hematopoietic differentiation. When
reagents are available, it will be important to confirm cytokine
specificity and define molecular interactions using full-length Notch1
and 2 molecules activated by DSL ligands.
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| Fig 4.
A model for Notch function in hematopoiesis,
showing its role in mediating cell fate decisions through cell-cell
interactions and transcriptional regulation. (A) Cellular interactions
and effects of Notch signaling in different hematopoietic
microenvironments, showing the influence of Notch on hematopoietic
cells of different lineages and at different stages of maturation. Each
compartment is used to emphasize particular features of Notch signaling
that are also applicable to the other compartments. In the progenitor
compartment (top panel), Notch signaling occurs between stromal cells
and hematopoietic progenitors and between equivalent or nonequivalent
hematopoietic cells. Hematopoietic progenitors express multiple Notch
molecules (depicted as Notch1 and 2) and DSL ligands. Stromal cells
express DSL ligands, including Jagged and Delta. In the context of
various cytokines, progenitors are induced to differentiate. Notch
signaling regulates the response of progenitors to cytokine
stimulation, permitting some to differentiate and others to self-renew.
Cells expressing more Notch are inhibited from differentiating and thus
maintain a pool of uncommitted progenitors. Cells expressing less
escape from the Notch signal and undergo the next step in
differentiation. Commitment to the lymphoid or myeloid lineage depends
on specific cytokines and the relative activities of Notch1 and 2. Increased Notch1 activity inhibits myeloid differentiation and thus
favors the lymphoid pathway; however, for lymphoid commitment,
progenitors must also express less Notch2 than their neighbors
(increased Notch1 and 2 results in self-renewal). Myeloid
differentiation is similarly favored by increased Notch2 expression
(which inhibits lymphoid differentiation) and permitted by relatively
low levels of Notch1. At the next step, lymphoid and myeloid precursors
again either differentiate or self-renew: those expressing less Notch
continue to differentiate, whereas those expressing more self-renew at
this stage of maturation. In the myeloid compartment (lower left),
precursors express both Notch1 and 2 and the effects on differentiation
are cytokine-specific, as shown by granulocytic differentiation in
response to G-CSF and GM-CSF. Either activation of Notch1 in the
presence of G-CSF or activation of Notch2 in the presence of GM-CSF
results in inhibition of differentiation and self-renewal of
progenitors. These progenitors remain competent to adopt alternative
fates in response to subsequent signals. In the absence of Notch1 or 2 activity or in the context of different cytokines (eg, GM-CSF for
Notch1 or G-CSF for Notch2), myeloid progenitors differentiate to
produce mature granulocytes. In the lymphoid compartment (lower right),
Notch signaling involves interactions of thymocytes with each other and
with thymic epithelial cells. When induced to differentiate, immature
CD4CD8 thymocytes expressing more Notch1
self-renew, whereas those expressing less undergo the next step in
T-cell maturation. At this next step, in the context of a productive
TCR rearrangement, CD4CD8 precursors
expressing less Notch adopt the primary T-cell fate; those
expressing more Notch fail to adopt the cell fate, subsequently
express both CD4 and CD8, and adopt the alternative  T-cell fate.
These CD4+CD8+ precursors, in turn,
can either develop either as mature CD4 or CD8 T cells. Cells
expressing less Notch adopt the primary CD4 cell fate, normally in
association with class II MCH molecules. Development of CD8 T cells
generally requires MHC class I ligation, and Notch expression in this
context permits cells to adopt the CD8 cell fate. However, expression
of high levels of Notch in the presence of MHC class II molecules will
also permit CD8 development, while preventing cells from adopting the
usual CD4 fate in this context. (B) Distinct intracellular interactions
result in cytokine-specific effects of Notch1 and 2 on myeloid
differentiation. The activated intracellular Notch molecule includes
the cdc10 repeats, which are necessary for Notch function, and the NCR
region, which confers cytokine specificity on the Notch1 and 2 molecules. In an inactive conformation, the cdc10 domain is masked and
therefore unable to participate in molecular interactions required for
Notch activity. Stimulation by G-CSF induces signal transduction
through a pathway that includes molecule X, which can interact with the
NCR domain of Notch1, but not Notch2. The interaction of X with Notch1
results in unmasking of the cdc10 repeats and facilitates the
interaction of Notch1 with nuclear factors. The result is
transcriptional suppression of genes that would otherwise be activated
in response to G-CSF. Because the Notch2 NCR cannot interact with X,
the cdc10 domain remains masked, Notch2 remains inactive, and
transcriptional activation of G-CSF-induced genes results in cellular
differentiation. GM-CSF signals through a different pathway, inducing
molecule Y, which can interact with the NCR domain of Notch2, but not
Notch1. Thus, in the context of GM-CSF stimulation, Notch2 is active
(the cdc10 domain is unmasked) and inhibits transcription of
GM-CSF-induced genes. In contrast, in the presence of GM-CSF, Notch1
remains inactive, lineage-specific gene transcription is permitted, and
cells differentiate.
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Lymphoid Differentiation: T-Cell Development
A role for Notch in lymphoid development was first proposed
when the human Notch homologue, TAN-1
(hNotch1), was cloned from T-cell leukemias containing
translocations involving the Notch1 gene.23
Convincing evidence that Notch1 plays a role in normal T-cell
development has since been provided by expression analyses and
functional studies.28,70 In the developing mouse thymus, Notch1 is expressed at relatively high levels in the least
mature (CD4CD8) thymocytes and at
very low levels in mature CD4+CD8 and
CD4CD8+ cells, an expression pattern
consistent with a role for Notch in maintaining cells in a less
differentiated state.71,72 The expression of
Jagged2 further indicates that Notch signaling occurs in the
developing thymus.61,62 Elegant studies by Robey et al47,72 have demonstrated that Notch1 can influence
both the CD4/CD8 and / T-cell fate decisions during
T-lymphocyte development. The general conclusions from these studies
are that increased Notch activity favors the CD8 and T-cell fate
decisions over the CD4 and cell fates, respectively. However,
the effect of Notch on developing thymocytes is modulated by other
factors, including the productive rearrangement and expression of
T-cell receptors (TCR) and ligation with major histocompatability
complex (MHC) molecules.
The CD4/CD8 lineage decision.
Transgenic mice generated by Robey et al72
carry an activated intracellular form of Notch1 under control
of the proximal Lck promoter, which permits expression of the transgene
early in thymocyte development. Evaluation of thymic T-cell subsets from these mice revealed that expression of activated Notch1
results in an increase in mature CD8+ T cells and a
corresponding decrease in CD4+ T cells.72
Expression of the Notch1 transgene in MHC class I-deficient mice
demonstrated that Notch1 activity permits the development of
CD8+ cells even in the absence of class I MHC molecules,
which are normally required for differentiation to this lineage.
However, expression of Notch1 was not sufficient to promote the
generation of CD8+ cells in the absence of both MHC class I
and II molecules, suggesting that MHC ligation is required for the
developing thymocyte to receive the Notch signal at this maturational
stage. Alternatively, MHC ligation may initiate a developmental program
that can be modulated, but not initiated, by Notch signaling.
The / lineage decision.
At an earlier stage of thymocyte development, Notch1 cooperates with
the TCR in the specification of and cell fates. At this
developmental branch point, immature
CD4CD8 cells either adopt the
T-cell fate or further differentiate to express both CD4 and CD8
as well as the TCR, eventually becoming mature CD4 or CD8
T cells. By analyzing transgenic and chimeric mice having thymocytes
expressing different amounts of Notch1, Washburn et al47
concluded that decreased Notch1 expression permits cells to
adopt the fate, whereas increased expression favors the
lineage. In chimeric mice having thymocytes containing one or two
copies of a functional Notch1 gene, cells heterozygous for
Notch1 were more likely to develop as T cells, whereas
wild-type cells containing two copies were more likely to become
T cells. Although thymocytes having productive TCR gene
rearrangements normally adopt the fate, increased Notch activity
permitted TCR+ cells to develop along the
lineage. However, Notch activity in the absence of either the or
TCR was not sufficient to drive the development of T
cells. Thus, in the CD4/CD8 and / cell fate decisions,
excess Notch activity can override the MCH II or TCR signals,
but cannot dictate cell fates in the complete absence of MHC ligation
or TCR signaling, respectively.
The observations from Notch1 chimeric mice provide a classic
illustration of lateral signaling and the dosage effects of
Notch among developing thymocytes. (See above Homotypic
Interactions: Lateral Inhibition and Fig 3A.) During thymic T-cell
development, cells expressing less Notch than their neighbors
adopt the primary fate, regardless of whether the absolute
amount of Notch reflects one or two copies of the
Notch1 gene. Thymocytes expressing more Notch are
inhibited from adopting the fate, but remain competent to adopt
the alternative fate. The
(CD4+CD8+) T-cell precursors, in turn, may be
subject to lateral signaling through differential expression of
Notch; those expressing less Notch adopt the primary
CD4 fate, whereas those expressing more Notch adopt the
alternative CD8 fate.
B-Cell Development
A number of observations provide circumstantial evidence that
Notch also influences B-lymphocyte development. NF- B and
CBF1/RBP-J, which regulate expression of B-cell-specific genes,
also physically interact with Notch and participate in Notch
signaling.39-41,73-75 Transcriptional regulation by NF-B
is essential for normal B-cell development and
activation.76-79 CBF1 binds to promoters of several B-cell
genes and acts as a transcriptional regulator in B-cell immortalization
induced by Epstein-Barr virus (EBV).40,80 The recent
demonstration that Notch1 and 2 inhibit the bHLH transcription factor
E47 also supports a role for Notch in the regulation of B-cell-specific genes.81 E47 is essential for early
B-lymphocyte development, activation of the Ig heavy chain locus, and
initiation of Ig gene rearrangement. The conserved role of Notch in the
regulation of bHLH activity makes this finding particularly intriguing.
Lymphoid Malignancies
Given the broad developmental role for Notch and its general
function in regulating differentiation of immature cells, it is not
surprising that both unregulated and ectopic expression of
Notch have been implicated in oncogenesis. Although the
Notch1-4 genes are located on different chromosomes, all have
been mapped to regions of neoplasia-associated translocation or
oncogenic viral insertion, and three have been directly associated with malignant transformation. Notch1 and 2 have been
implicated in the development of T-lymphoid
malignancies23,82,83 and contribute to neoplastic
transformation in vitro.84 The Notch homologue now
known as Notch4 was first identified as the int-3
oncogene associated with primary mouse mammary tumors.27,85
In all of these cases, the aberrations in Notch involve
expression of truncated molecules lacking most or all of the
extracellular domain. Similar truncated molecules have been shown to
behave as constitutively active forms of
Notch,31-34,86-89 suggesting that unregulated
intracellular Notch activity might contribute to malignant
transformation by inhibiting normal differentiation and permitting the
continued proliferation of undifferentiated cells.
T-cell malignancies.
In a subset of T-cell acute lymphoblastic leukemias, breakpoint
translocations involving the Notch1 gene predict expression of
truncated intracellular Notch1 proteins that likely function as
constitutively activated forms of Notch1.23 A direct
association between expression of such truncated Notch1 proteins and
the development of T-cell malignancies has been confirmed by Pear et
al90 using a mouse transplantation model. These
investigators found that mice transplanted with BM cells transduced
with activated forms of Notch developed T-cell malignancies at a high
frequency. Interestingly, equivalent tumorigenesis was observed for
Notch constructs containing only the intracellular domain and those
including the transmembrane domain, suggesting that either
membrane-bound or free intracellular Notch molecules are oncogenic.
This is in contrast to other reports associating malignant
transformation primarily with nuclear forms of Notch.
Truncated Notch2 molecules have also been associated with T-cell
malignancies. Rohen et al82 described transduction of
Notch2 sequences in thymic lymphomas from cats infected with
feline leukemia virus. The transduced region of Notch2 included
the conserved extracellular cysteines, the transmembrane domain, and
portions of the intracellular domain, including the cdc10 repeats. In
contrast to the corresponding Notch1 protein in mice (discussed above), the truncated Notch2 protein localized to the nucleus, indicating it
was not tethered to the membrane. These investigators have proposed
that nuclear Notch2 is generated through internal translation initiation at a site immediately downstream of the transmembrane domain
and thus would not be membrane-bound. If correct, this observation
suggests a nonproteolytic mechanism for generating activated
intracellular forms of Notch.
B-cell malignancies.
Notch has also been linked to B-cell malignancies induced by EBV.
Immortalization of B lymphocytes by EBV requires EBNA2, a virally
encoded transcriptional activator. EBNA2 transactivates cellular genes
through its association with the CSL protein
CBF1/RBP-J,80 a primary component of the Notch pathway.
EBV-induced immortalization through EBNA2 involves a mechanism that
mimics intracellular Notch activity,40,73 implicating
dysregulation of Notch/CBF1 signaling in the development of
EBV-associated malignancies.
The normal role of Notch includes mediating cell fate decisions such
that appropriate numbers of different cell types are produced.
Aberrations in Notch signaling disrupt this regulation, with either
excess or insufficient Notch activity resulting in expansion of one
cell type at the expense of another (Fig 3B). Thus, any process
disrupting Notch signaling could potentially contribute to malignant
transformation by permitting inappropriate expansion of a single cell
type. Evidence supporting this possibility includes the association of
other components of the Notch signaling pathway with various
malignancies.91,92 Dysregulated Notch signaling may prove
to be a frequent occurrence in malignant transformation. The
contribution of Notch, if any, to the development of hematopoietic malignancies other than lymphomas should become apparent as the role of
Notch in hematopoiesis and its integration with other signaling
pathways become more clearly elucidated.
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NOTCH LIGANDS IN HEMATOPOIESIS |
Within the hematopoietic microenvironment a complex signaling network
involving soluble and cell-bound cytokines, as well as interactions
among hematopoietic cells and stromal elements, regulates
differentiation and proliferation of hematopoietic
progenitors.1,2,4,5,7,12,93 Although the importance of
signaling through cytokine production has been established, the
influence of direct cell-cell interactions among equivalent or
different hematopoietic cells remains largely undefined. Notch, a
molecule that mediates intercellular interactions and directly
influences cell fate decisions may provide an important adjunct to
other regulatory mechanisms. Expression of Notch ligands by BM and
fetal liver stromal cells, thymic epithelial cells, and hematopoietic
cells60,61,66,67 (and L.A.M., unpublished data) supports a role for Notch signaling through
homotypic and heterotypic interactions in the hematopoietic and
lymphopoietic microenvironments.
Notch Ligands: DSL Proteins
In Drosophila, the two Notch ligands Delta and Serrate
have both distinct and overlapping functions.10,94
Multiple ligands corresponding to each of these two general classes
have been identified in vertebrates, leading to a somewhat confusing
nomenclature. In mammals, ligands having high homology to Delta
are referred to as Delta or Delta-like (Dll), and those
homologous to Serrate are called Serrate or
Jagged (Table 1). DSL ligands, like Notch, are transmembrane
proteins having an extracellular domain containing a variable number of
EGF-like repeats. The extracellular domain also contains a conserved
region unique to this family of molecules: a DSL
(Delta/Serrate/Lag-2) domain that is
required for Notch binding and activation.46,95 Serrate and
Jagged also contain a conserved cysteine-rich region that is not
present in Delta homologues. The intracellular domains of DSL proteins
consist of short, diverse sequences of unknown function, but may be
involved in multimerization.46,96
Jagged1 and 2
Human Jagged1 was cloned from a normal BM cDNA library and is
expressed by a subset of marrow stromal cells,60 indicating that it functions in the hematopoietic microenvironment. The
coexpression of Jagged2 and Notch1 in the developing
thymus suggests that Jagged2 is a ligand for the Notch1 receptor in
this tissue.61,62 Mutant mice lacking a functional
Jagged2 gene further illustrate the significance of
Jagged-Notch signaling in T lymphopoiesis: these mice, in addition to
other severe defects, have abnormal thymic morphology and impaired
differentiation of T cells.58
Expression of Jagged1 by BM stromal cells and Notch1 by
hematopoietic progenitors suggests that, within the marrow
microenvironment, interactions between stromal and hematopoietic cells
include Jagged1-Notch1 signaling. The expression of Jagged1 by
the HS-27a stromal cell line is intriguing in this regard. HS-27a
supports the maintenance and proliferation of hematopoietic progenitors
and promotes cobblestone area formation in long-term marrow
cultures,97 properties that could be attributed to
Jagged-Notch signaling. The demonstration that both HS-27a and a
purified Jagged1 protein inhibit G-CSF-induced differentiation of
Notch1-expressing 32D cells and permit proliferation of
undifferentiated progenitors supports this hypothesis.60 The requirement for both Notch1 expression by 32D cells and presence of
ligand strongly suggests that Jagged1-Notch1 signaling is responsible for these effects. Although Jagged1 was most effective when
endogenously expressed as a membrane-bound protein by the HS-27a cell
line, two soluble forms of Jagged1 produced similar effects. Of
particular interest was the finding that a small peptide corresponding
to the unique DSL domain could activate Notch in this system, raising the possibility that DSL peptides might be useful for stem cell expansion.
Two recent reports provide further evidence that Jagged-Notch signaling
promotes the maintenance and expansion of normal hematopoietic progenitors. Varnum-Finney et al66 found that the addition
of Jagged1 to primary cultures of mouse
linsca-1+c-kit+ BM
progenitors resulted in a twofold to threefold increase in the
subsequent generation of high proliferative potential (HPP)-mix colonies. Jones et al67 reported a similar effect of
Jagged1 on mouse CD34+c-kit+ AGM and fetal
liver hematopoietic progenitors: primary culture on a stromal cell line
expressing Jagged1 resulted in a fourfold (AGM cells) or greater (fetal
liver cells) increase in HPP-mix colonies generated in subsequent
methylcellulose cultures. Although these studies suggest that Notch
ligands may be useful for in vitro expansion of hematopoietic
stem/progenitor cells, the complex interactions involving various Notch
molecules, ligands, and cytokines may present a considerable challenge.
Delta-Like Molecules
At least five distinct vertebrate orthologues of Drosophila
Delta have been identified, including Delta-like
(Dll) 1 and 3 in mice and humans.98,99
Dll1 and 3 have both overlapping and distinct patterns
of expression, suggesting cooperative and specific signaling functions
in several developmental processes. A complete analysis of Dll
expression in hematopoietic tissues has not yet been reported, but
Dll1 is expressed in BM stroma67 and
spleen,62 suggesting possible roles in hematopoiesis and
B-cell regulation.
The delta-like (dlk) molecule expressed by the fetal
liver stromal cell line AFT024 is another potential Notch ligand.
AFT024 maintains transplantable hematopoietic progenitors in
vitro100 and like HS-27a (which expresses Jagged1) supports
the formation of cobblestone areas characteristic of proliferation of
primitive hematopoietic cells.101 Moore et
al101 have provided convincing evidence that dlk
contributes to these properties by demonstrating that a soluble dlk
protein stimulates cobblestone area formation from fetal liver and
adult BM stem cells and that cell lines transfected with dlk
increase the short-term repopulating ability of cultured hematopoietic
progenitors. Although these effects are consistent with Notch
signaling, the precise relationship of dlk to other Notch ligands and
its role in Notch signaling remain to be elucidated; it is noteworthy
that dlk lacks the DSL domain characteristic of established Notch
ligands, and activation of a Notch receptor by dlk binding has not yet
been demonstrated.
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INTRACELLULAR NOTCH SIGNAL TRANSDUCTION |
Evidence regarding signal transduction after activation of Notch is
derived mostly from nonhematopoietic systems. However, the fact that
Notch, its ligands, and the intracellular factors that transmit Notch
signals are all present in hematopoietic cells strongly implies that
these signaling pathways function in hematopoiesis. The following
section is provided as a guide to the known pathways with the
expectation that this will prove to be the case.
Notch Activation
Signal transduction through the Notch pathway is initiated when the
extracellular domain of Notch binds to its ligand on adjacent cells,
resulting in activation of the intracellular domain (Fig 2). Several
recent studies suggest a model for Notch processing and activation that
involves two distinct proteolytic events: the first to generate a
functional Notch receptor and the second to activate Notch in response
to ligand binding. Blaumueller et al102 have demonstrated
that functional Notch receptors are present on the cell surface as
heterodimers, generated by proteolytic cleavage of the full-length
Notch protein and reassociation of the extracellular and intracellular
cleavage products through disulfide bonds. The
metalloprotease-disintegrin Kuzbanian plays a role in Notch
signaling103,104 and has been implicated in the processing
of Drosophila Notch and mammalian Notch2.104 However, Logeat et al105 have found that a furin-like convertase is
responsible for the processing of Notch1, suggesting that Kuzbanian is
not an invariant part of Notch signaling. Different mechanisms may be
involved in processing the different Notch orthologues, or different
cell types may use distinct mechanisms, variables that could contribute
to specificity of Notch signaling in mammals.
The initial proteolytic processing of Notch generates a functional
receptor, but does not result in Notch activation. Studies by Kopan et
al106,107 indicate that a second, ligand-dependent, cleavage is required for Notch activation. In these studies, ligand binding to membrane-bound Notch1 induced proteolytic cleavage to
release Notch-IC, which could then translocate to the nucleus. Together, these studies provide the basis for an appealing model of
Notch processing and activation. However, it remains possible that
other proposed mechanisms for Notch signaling will also prove to be
important.82,103
Nuclear Fu |
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INTRODUCTION
NOTCH AS A MEDIATOR...