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Blood First Edition Paper, prepublished online February 3, 2012; DOI 10.1182/blood-2011-10-355826
Anna Bigas and Lluis Espinosa
Program in Cancer Research.
Institut Mar Investigacions Mèdiques (IMIM), Hospital del Mar,
Parc de Recerca Biomèdica de Barcelona, Barcelona, Spain.
Corresponding author:
Anna Bigas
IMIM-Hospital del Mar
Dr. Aiguader 88
08003 Barcelona
Phone: +34 933160440
Fax: +34 9321600410
e-mail:[email protected]
Copyright © 2012 American Society of Hematology
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Notch is a well-conserved signaling pathway and its function in cell fate determination is
crucial in embryonic development and in the maintenance of tissue homeostasis during
adult life. Notch activation depends on cell-cell interactions that are essential for the
generation of cell diversity from initially equivalent cell populations. In the adult
hematopoiesis, Notch is undoubtedly a very efficient promoter of T-cell differentiation and
this has masked for a long time the effects of Notch on other blood lineages, which are
gradually being identified.
However the adult Hematopoietic Stem Cell (HSC) remains
mostly refractory to Notch intervention in experimental systems.
In contrast, Notch is
essential for the generation of the HSCs, which takes place during embryonic development.
This review summarizes the knowledge accumulated in the recent years regarding the role
of the Notch pathway in the different stages of HSC ontology from embryonic life to fetal
and adult bone marrow stem cells. In addition, we will briefly examine other systems where
Notch regulates specific stem cell capacities, in an attempt to understand how Notch
functions in Stem Cell Biology.
Stem Cell Biology is dependent on a few signaling pathways that crosstalk each other to
generate a network of biochemical interactions and are ultimately translated into precise
instructions at the nuclear level. The Notch pathway has been evolutionary preserved not
only biochemically but also functionally, being involved in establishing cell diversification
from equipotent adjacent cells using a mechanism that requires cell-cell interaction.
Essentially, Notch-mediated communication depends on the differential expression of
specific ligands (Jagged or Delta) and receptors (Notch1-4) in adjacent cells. Basically,
Notch signaling is transduced from the sending cell (the one that expresses higher levels of
the ligand) to the nucleus of the receiving cell (expressing higher levels of Notch receptors)
thus impinging on its biochemical network 1. Cell-cell interactions are essential players in
the regulation of Stem Cell and tissue homeostasis of the adult but also in developing
In general, the tag “stem” refers to cells with unlimited capacity for self renewal and with the
ability to generate all the different lineages of a specific system. However, when studying
stem cells one need to distinguish two main categories of stem cells: 1) embryonic stem
cells which are pluripotent and retain the capacity to generate all the cell lineages of the
adult organism and 2) somatic stem cells which are also generated in the developing
embryo, maintain the self renewal capacity, but show a reduced pluripotency since they can
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only generate a limited number of cell types. The latter include the ones involved in tissue
formation and regeneration both in the embryo and the adult 2
The hematopoietic system has served for decades as a pioneer model for studying and
deciphering the behavior of somatic Stem Cells and the mechanisms that regulate cell
differentiation, and in fact, the knowledge generated on hematopoietic stem cell biology has
guided the understanding of other types of somatic stem cells. From these studies, the
Notch pathway has emerged as a principal player on stem cell regulation and differentiation
even though many questions on how or whether Notch functions in hematopoietic stem
cells remain still unanswered. Interestingly, studies in the adult hematopoietic stem cells
first unexpectedly revealed that the maintenance of this type of stem cells was independent
of Notch. However, further understanding of the ontogeny of HSC has now provided new
insights about the predominant role of Notch in the generation of HSC in the embryo. The
aim of this review is to gain a better understanding of the Notch pathway and integrate the
results obtained in the last decades by analyzing HSC from the adult bone marrow and the
mouse embryo.
Some Notch History
Notch is a well-conserved signaling pathway that was first identified in Drosophila mutants.
Lack of the X-linked Notch locus in the fly resulted in embryonic death, yet heterozygous
females showed the serrate/notched wing margin first observed by T.H. Morgan’s group 3, a
phenotype that named the whole pathway.
The Notch gene was initially cloned in
and soon after in Caenorhabditis elegans
, Xenopus8, mouse9 and
human10. In general, Notch works as a determinant factor during binary cell decisions from
adjacent cells, a function that was elegantly demonstrated in C. elegans11 and Drosophila12.
Later on, the sequence of the first Notch ligands was obtained and the analysis of the
proteins indicated that they all corresponded to cell surface proteins with a large
extracellular domain, which further supported a function for Notch as a regulator of cell-cell
interactions between neighboring cells. Deciphering the elements downstream of the
receptor and ligands involved laborious work from embryologists and biochemists that
studied and interpreted the results from different mutant models. Thus, genetic experiments
in invertebrates first directed the investigations of the Notch pathway towards the
identification of Supressor of Hairless (Su(H)) as a key transducer of neurogenic
signals13,14. Further biochemical studies demonstrated the interaction between Notch and
Su(H), the orthologue of the mammalian gene Rbpj (for Recombination-signal Binding
Protein jk)15. Genetic screenings were also crucial to identify the Notch-target genes as
Hairy and Enhancer of Split (Hes), which are HLH (Helix loop Helix) proteins involved in
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suppressing the neuronal genes Acute-Scute16,17. Once again, this connection is conserved
in the mammalian systems.
Later on, efforts were focused on investigating Notch signal transduction. The
demonstration that Notch together with Su(H)/RBP-J regulated the transcription of Hes
genes14,18 provided a strong indication that Notch should function in the nucleus. In fact, it
was initially shown that ectopically expressed intracellular Notch translocated to the nucleus
and was capable of activating transcription19 but ingenious experiments were required to
demonstrate the nuclear activity of endogenous Notch20. Another breakthrough discovery in
the history of the Notch field was the demonstration that this receptor was processed by a
γ-secretase/presenilin complex in response to ligand binding, and the consequent
development of inhibitors that target this activity
. The usage of these Notch/γ-secretase
inhibitors has facilitated the further confirmation of most Notch functions in different cell
types and tissues and provided the first attempts of using Notch as a therapeutical target
During the last decade, research in the Notch field has been growing exponentially, which
has contributed to a better understanding of the relevance of this pathway in the generation
and maintenance of multicellular organisms.
Elements of the Notch signaling pathway
Notch is a single-pass transmembrane receptor that can be activated by different
transmembrane ligands. In general, Notch binds to one of its ligands located in the adjacent
cell, which results in two sequential proteolytic cleavages of the receptor. The latter involves
the release of the intracellular part of Notch receptor (IC-Notch), its translocation to the
nucleus, and its association with RBP-J (also known as CSL for the different orthologue
proteins: CBF1, Supressor of Hairless and Lag1) and the co-activator Mastermind (Mam) to
activate specific transcription. Notch participates in the transcriptional regulation of multiple
genes, some of them being context-dependent. However, the most important Notch
functions have been associated with the regulation of the Hes or Hes-related (Hrt) family of
genes. All these genes encode for bHLH proteins that, in general, function as inhibitors of
cell differentiation.
Notch receptors
There is one Notch receptor in Drosophila, two in C. elegans and four different Notch
receptors (Notch 1-4) in most vertebrate species, being mammalian Notch1 and Notch2 the
most similar to the Drosophila homologue. Notch is a single transmembrane protein
composed of an extracellular part with variable number of Epidermal Growth Factor (EGF)like repeats and an intracellular part containing 7 ankyrin-like repeats, nuclear localization
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signals and a transactivation domain. Notch is codified by a single mRNA molecule that
translates into a polypeptide, which is cleaved in the Golgi apparatus by a furin-like
convertase enzyme24. This processing generates two different fragments (one containing
the extracellular domain and another that includes de transmembrane and intracellular
domains) that remain associated by disulfide bonds involving a small conserved
extracellular domain, LNR (Lin/Notch repeats)25. Hence, the resulting functional Notch is
commonly considered as a “heterodimeric” receptor. The LNR region is also crucial to
prevent ligand-independent signaling, which is supported by the fact that mutations in this
region results in increased Notch activity that associates with T-ALL leukemia26.
Notch ligands
There are at least 5 functional Notch ligands in vertebrates: three orthologues of the
Drosophila Delta (Delta or Delta-like (Dll) 1, 3 and 4) and two of the Drosophila Serrate
(Jagged1 and Jagged2). All ligands are able to interact with the Notch receptor and induce
the second cleavage at the extracellular level. However, all ligands have different
expression patterns and specific deletion/inhibition of specific ligands results in a very
diverse outcome. Notch ligands are also composed by a variable number of EGF-like
repeats in their extracellular domain but a small intracellular portion. To achieve Notch
activation, Notch ligands need to be internalized in endosomes in the signaling sending cell
(before they are presented at the cellular membrane), a process that is regulated through
ubiquitination by the E3-ubiquitin ligases Mindbomb and Neuralized 27. The consequence of
Mindbomb deficiency is a defective Notch activation, in both invertebrates and
Notch modification by glycosylation: pofuts, fringes and pogluts
One of the particularities of Notch is the absence of a downstream signaling cascade.
Instead, following Notch activation IC-Notch travels from the cell surface directly to the
nucleus, where it binds RBP-J and indirectly to the DNA. Despite the apparent simplicity of
this pathway with no intermediate effectors, Notch signaling involves an extremely accurate
regulation, which is multifactorially achieved. For example, multiple enzymes can modify the
Notch protein post-translationally, thus changing its functional properties. Pofut-1 is an Ofucosyl-transferase that catalyzes the O-fucosylation of specific EGF-like repeats, which is
an essential condition for their subsequent Fringe-dependent modification. Fringe proteins
(including Lunatic, Manic and Radical Fringe) are Golgi-localized glycosyltransferases that
add N-acetylglucosamine to O-fucose moieties on EGF-like repeats of the extracellular
domains of Notch. Different Fringe homologues modify specific EGF-like repeats with
distinct efficiencies
. Fringe-modifications enhance the capacity of Notch to be activated
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by ligands of the Delta-like family (Dll1,3 and 4) whereas reduce Notch activation by the
Serrate/Jagged family of ligands (Jag1-2)31. While some studies suggest that knockout
mutants for all three Fringes (in a specific genetic background) do not display more
developmental malformations than single mutants for Lunatic Fringe 32, it is well established
that different Fringe homologues modulate particular Notch-mediated biological processes
in a specific manner
, such is the case of Lunatic Fringe in the hematopoietic lineage
commitment and differentiation 36-39.
Another modification involving the extracellular domain of the Notch receptor is its Oglucosylation mediated by the O-glucosyl-transferase (Poglut), Rumi 40. However, which are
the biochemical effects of this modification is mainly unknown but it has been suggested
that it might regulate the proper folding of Notch that is required for its efficient activation.
Downstream effectors of Notch: The Hes gene family
Hes genes and specifically Hes1 are among the best-characterized Notch target genes.
They codify for bHLH proteins and, in general, function as DNA-binding transcriptional
repressors. Expression of several members of the family (including Hes1, Hes5, Hes7, Hrt1
and Hrt2) depends mostly on Notch activity and participate in many of the Notch-assigned
functions including proliferation, differentiation, apoptosis, self-renewal and asymmetric cell
division regulation (reviewed in 41). Hes genes are generally responsible for Notch functions
that require the inhibition of one specific cell fate to allow the determination of an alternative
fate (lateral inhibition) whereas other Notch inductive functions such as T-cell specification
may not be dependent on Hes. In this sense, Hes1 is only needed for the first stages of TCell determination42 while other important Notch targets such as pTα43, IL7R44 are
expressed and regulate specific stages of T-Cell differentiation.
Embryonic development and Somatic Stem Cell generation
During embryonic development, specification of the different tissues runs in parallel with the
progressive restriction of the stem cell potential. However, significant pools of stem cells are
found in the adult tissues that are continuously renewed during lifetime, such as blood or
intestine. For many years, hematopoietic stem cells remained the best-characterized
somatic stem cells and, consequently, they have been used as a model to study adult
tissue regeneration. In the recent years, similar types of cells have been identified in many
other tissues such as muscle, neuronal, pancreas, lung and breast, among others and it is
now widely accepted that they are crucial players in tissue homeostasis and regeneration.
In the blood system, there is now definitive evidence that the somatic-, blood-specific stem
cells are originated during embryonic life and maintained thereafter. Although Notch is
generally considered as an essential signaling pathway that regulates multiple stem cell
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functions, its participation diverges among the different organs and tissues. For example, in
the mammalian hematopoietic system Notch1 is required for the generation of
hematopoietic stem cells in the embryo but it is dispensable for the maintenance of the
hematopoietic stem cells in the adult organism (reviewed in 45), whereas the neuronal stem
cells depends on Notch in both embryonic and adult life. Our current understanding on how
Notch contributes to tissue homeostasis includes a plethora of functions in both the stem
and progenitor cell populations that cannot be generalized from one tissue to the others. In
this review, we will summarize what is known about Notch functions during generation and
maintenance of HSC, and compare this with its role in other tissue-specific stem cell
Notch in Hematopoietic Stem Cells: Generation and Maintenance
Multiple types of specialized cells, which are responsible for nutrient transport and immunedefense, constitute the hematopoietic system. During the adult life, all hematopoietic cells in
the organism are renewed every 5 to 120 days depending on the cell type. Continuous
production of limited numbers of blood cells is achieved by the differentiation of
Hematopoietic Stem Cells in the bone marrow microenvironment. However, it is the
exclusive capacity of HSC for self-renewal that guarantees the maintenance of this tissue
throughout life. The question that arises here is, how this rare population acquires the
features of self-renewal and pluripotency that define a stem cell. From our knowledge stem
cell properties are acquired during embryonic life, thus in the following sections we will
review what it is known about the mechanisms that regulate the generation and
maintenance of HSCs and the role of Notch in these different processes.
Embryonic hematopoiesis
Before colonizing the bone marrow, newly formed HSCs as well as all different
hematopoietic lineages are found in the developing vertebrates in a tightly-regulated spatial
and temporal manner. More specifically, embryonic hematopoiesis takes place in two
different waves namely primitive and permanent-definitive hematopoiesis (reviewed in46). In
mammals, primitive hematopoiesis occurs primarily in the blood islands of the yolk sac47,48
around murine embryonic day 7.5 (E7.5) 49, and whether Yolk Sac blood cells originate from
a common endothelial progenitor called hemangioblast50,51 is still unclear48. Grafting
experiments using quail-chick chimeras and Colony Forming Unit (CFU) cultures showed
that the yolk sac mainly produces primitive erythrocytes and myeloid progenitors but does
not generate definitive HSCs
. In fact, HSCs are first detected in the yolk sac after
circulation between the yolk sac and the embryo is established (in the mouse embryo HSC
are found at E9 and circulation starts around E8.5)
. Placenta is also an important source
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of erythroid and myeloid progenitors at E9.0 and interestingly it becomes a reservoir of
HSCs around E1258-60. However, the first definitive HSCs considering those cells with
capacity to reconstitute irradiated adult mice appear in the Dorsal Aorta, in a region
surrounded by Gonad and Mesonephros (also known as AGM region) around day 9 of
murine development61,62, closely associated with the endothelial cells. Cell-tracing
experiments demonstrated that murine adult hematopoietic cells originate from embryonic
cells that express VE-cadherin and Runx1 around E9.0, strongly suggesting the endothelial
embryonic origin of HSCs but not excluding other possibilities
. Importantly, endothelial
cells from fetal liver are no longer capable of generating hematopoietic cells indicating that
a putative common endothelial-hematopoietic progenitor should be very restricted both
temporally and spatially to specific embryonic vessels, such as the dorsal aorta 65.
Notch in embryonic arterial and hematopoietic development
One of the best-characterized Notch functions in vertebrates consists in the determination
of the arterial program, and several Notch mutant embryos in both zebrafish and mouse
lack artery specification as determined by the absence of EphrinB2, CD44 or SMA
. Accordingly, since HSC originates in arterial vessels the predicted
phenotype of Notch mutant embryos was indeed lack of hematopoiesis. This is exactly the
phenotype of the Notch1, Rbpj and Mindbomb deficient mutant mice
. In contrast, coup-
TFII mutants show ectopic Notch activation in veins accompanied by the expression of
arterial markers and ectopic hematopoiesis
. Other Notch mutants that fail to define the
artery-vein boundaries such as Activin A-receptor typeII-like1 (acvrl1, alk1) or endoglin
(CD150) show ectopic hematopoiesis in veins or at least in vein-like vessels that do not
express the arterial marker EphrinB2
. This link between arterial specification and
hematopoiesis has complicated a direct assignment of the hematopoietic defects to Notch
deficiency, since secondary effects of arterial development may result in the same
phenotype. Opportunely, experiments in zebrafish showed that ectopic Notch1 activation in
the veins induced specific hematopoietic gene expression including Runx1 and c-myb in the
absence of arterial differentiation 71. However, a formal proof that HSC can be generated in
the absence of arteries is still missing. Further insight into the question on how Notch
regulates hematopoiesis independently of its role on arterial specification was obtained by
the analysis of the Jagged1 mutant. In this work, we demonstrated that Jagged1, Jagged2
and Delta4 are all expressed in the endothelium of the aorta in the developing AGM
However, analysis of the mutants demonstrated that they do not display redundant
functions. Specifically, Delta4 deficient mice show a strong arterial phenotype
at early developmental time. Similarly,
Jagged1 mutants show
and die
some vascular
but still express arterial markers such as EphrinB2, CD44 and SMA and
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importantly they mostly failed to generate hematopoietic cells
. These results undoubtedly
demonstrated that Notch was required for definitive hematopoiesis in vivo, and uncouple
this Notch function from its role in arterial specification.
Another unexpected result that arose from the analysis of mutant mice was the fact that
only definitive hematopoiesis in the AGM region, but not the embryonic hematopoiesis of
the yolk sac, was Notch-dependent. As discussed above murine yolk sac from E7.5 to E8.5
mainly produces primitive nucleated erythrocytes (an event known as primitive
erythropoiesis) and it is not until E9.0 when circulation is already established that the yolk
sac contain different hematotopoietic progenitors capable of generating myeloid, erythroid
or lineage-mixed colonies in culture, but also definitive HSCs that are transplantable and
able to reconstitute busulphan-treated newborn mice. Importantly, the yolk sac from Notchdeficient mutants contains similar numbers of hematopoietic progenitors at different times of
development compared to their wild type or heterozygous counterparts
HSC potential at E9.0 (15-20 somites)
but lacks the
. Together these results indicate that Notch
signaling is dispensable for primitive hematopoiesis whereas, independently of the site of
origin (provided that there is more than one), Notch signaling is essential for generating
definitive hematopoiesis. In agreement with this idea, analysis of chimeric mice generated
from wildtype and Notch1-deficient ES cells demonstrated that Notch1-deficient cells
contribute to all types of embryonic, fetal progenitors and hematopoietic cells present in
fetal liver, but do not contribute to adult hematopoiesis
. Similarly, mutation of Mindbomb
in zebrafish affected the later wave of hematopoietic cells (characterized by expression of
c-myb or Runx1) but not for the early phase of primitive cells 79.
Notch in Fetal hematopoiesis
The final purpose of developing HSCs is to access their niche in the bone marrow that will
allow HSC self-renewal and differentiation throughout the adult life. However, before
reaching the bone marrow newly formed HSCs migrate to the fetal liver, which is the main
hematopoietic organ before birth. During this period, early lymphoid fetal liver progenitors
need to colonize the thymic epithelial primordium where they experienced high Notch
signaling dose and differentiate into T-cells
, whereas HSC will need to colonize the bone
marrow. However, the most remarkable hematopoietic process in the fetal liver is that
HSCs increase in number. For this reason fetal liver signals have been studied for many
years in an attempt to identify candidate molecules that prone HSC expansion. Whether
Notch participates in this particular fetal process is mainly unknown basically because most
of conventional Notch mutants are either lethal or failed to generate hematopoiesis before
the fetal liver stage. Thus, analysis of conditional Notch mutants that specifically affect the
fetal liver stage may be useful to uncover any possible role of Notch in this process.
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Notch in Adult Hematopoietic Stem Cells
Early studies demonstrated that Notch is expressed in the undifferentiated progenitors from
human bone marrow cells
and suggested a putative role for this factor in leukemia
Later on, experiments using myeloid progenitor cell lines indicated that Notch is an
essential regulator of hematopoietic differentiation
, thus opening the possibility that it
might function in preserving the stem cell phenotype. In addition, Parathyroid Hormone that
is a crucial regulator of the osteoblastic HSC niche in the bone marrow, increases the levels
of Jagged1 in this tissue with in vitro evidences of Notch function in HSC expansion84.
However, the analysis of transgenic mice carrying a dominant negative form of the
coactivator Mastermind (that specifically blocks all canonical Notch signaling) or mice
deficient for Rbpj indicated that Notch activity was dispensable for the maintenance of HSC
in the adult bone marrow under physiological conditions
. Further support for these results
can be found in the conditional deletion of Notch1 or Notch1 plus Notch2 under the control
of the interferon-dependent expression of Mx-Cre that specifically and exclusively affected
lymphoid differentiation, but not other hematopoietic lineages86-88. Consistent with the
notion that Notch activity is dispensable in the adult HSC a recently published database
( showed low levels of different Notch receptors in purified HSCs.
Notch-dose dependency in hematopoietic differentiation and hematopoietic disorders
Experimental systems that mostly rely on complete abrogation or strong persistent
activation of the Notch pathway result in phenotypes that are mainly associated with the
lymphoid lineage consistent with the fact that high Notch activity promotes T-cell
differentiation whereas complete absence of Notch induces early B-cell commitment
In agreement with this, activating Notch mutations are commonly found in T-cell leukemias
(for review see91,92). However, whole genome sequencing of B-chronic lymphocytic
leukemia (B-CLL) cells has revealed that 12% of these samples also contain activating
mutations of Notch associated with a Notch-active signature
, suggestive of a regulatory
role for Notch in B-cell differentiation which is also in agreement with the effects of Rbpj or
Notch2 deletion in committed B-cell progenitors that impaired Marginal Zone B-cells and
potentiates follicular B-cell differentiation in the spleen94,95. Unexpectedly, the identified
activating mutations in B-CLL affect the Notch1 receptor, which is not the main effector for
B-cell terminal differentiation, suggesting that aberrant transforming Notch activity can be
delivered by any receptor. In fact, this emphasizes the importance of large-scale genome
sequencing of patients with different hematopoietic disorders in providing a new perspective
about the involvement of Notch in these malignancies. The knowledge on how much, when
and where Notch is required to maintain the correct specification of the hematopoietic
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lineages (including the HSCs), is an essential requirement for further manipulation of the
Notch pathway for biomedical purposes.
However, rather than just being a Notch ON/OFF issue the extent of Notch activity is also
critical for most of the Notch functions involved in the homeostasis of the hematopoietic
lineages. Alternative approaches that overcome this limitation include the intervention on
positive or negative regulators, which might affect the strength of Notch signal. Indeed,
overexpression of Lunatic fringe that reduces the affinity between Notch and delta ligands
forces lymphoid progenitors to become B-cells in a high delta presenting environment39
whereas modulating the density of Delta1 influences the generation of B or T cells in vitro96.
Recent works describing the phenotype associated with loss-of-function mutations of
positive Notch regulators such as Mindbomb
, Nicastrin
, Presenilin99 or Pofut
suggested that reduction in the levels of Notch activity is sufficient to induce a
myeloproliferative syndrome that phenocopies the effects observed in one of the Mx-Cremediated Notch1 and Notch2 deletion98, despite that the same experimental model was
previously generated showing an exclusive lymphoid phenotype88. Consistent with the
possibility that myeloid differentiation/proliferation is favored by reduced Notch activity (see
model in Figure 2), loss-of-function mutations of the pathway are present in about 12% of
patients with chronic myelomonocitic leukemia (CMML)98. Interestingly, mouse transplanted
with hematopoietic cells in which RBPJ-mediated Notch activity is completely abrogated
including Rbpj-deletion or ectopic DN-Mam expression85 did not show any myeloproliferative phenotype, suggesting that RBPJ-independent or non-cell-autonomous Notch
activities are required to develop the disease. In addition, a different study showed that
expression of DN-Mam precludes Megakaryocyte differentiation in vivo101.
On the other hand, most of the phenotypes associated to Notch pathway manipulation
suggest an almost exclusive cell-autonomous requirement for Notch signaling in the
maintenance of the hematopoietic system. However, mice deficient for Pofut1, which is
required upstream of Fringe glycosyltransferases to permit Notch signaling through Delta,
displayed both cell-autonomous and non-cell-autonomous defects that resulted in
myeloproliferative disease100 suggestive of undetermined Notch functions in the regulation
of the stromal HSC niche. In addition, defective Notch signaling in the murine skin results in
an epidermal-barrier-associated inflammatory phenotype, which leads to massive Thymic
Stroma Lymphopoietin (TSLP) expression and produces lethal B-cell102 or myeloid
proliferative disorders103.
In conclusion, and to help understand how Notch impinges in the hierarchy of
hematopoietic lineages, we propose a Notch-dose-based model that is compatible with
recent data and revised models104-107 in which specific Notch activity levels impose
progressive lineage restrictions (see model in Figure 2).
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Notch in ex-vivo hematopoietic cultures and stress conditions
Nowadays there is no evidence for a physiological role of Notch in the maintenance of HSC
in vivo. Despite this fact, I.D. Bernstein’s laboratory has recently demonstrated that Notch
pathway manipulation is a potential way of expanding HSCs or progenitor cells for clinical
applications. The basis for this finding comes from early experiments in which murine
undifferentiated bone marrow cells (Lin-Sca+Kit+) were transduced with retrovirus encoding
active Notch1. In this pioneer experiment, cells with self-renewal and pluripotent myeloid
and lymphoid differentiation capacity in vitro and in vivo were obtained
. To avoid using
retrovirally-infected cells, they have now optimized the culture conditions to activate
endogenous Notch through immobilized Notch ligands109. This strategy turned out to be
particularly efficient for the expansion of short-term lymphoid and myeloid progenitors from
samples with low numbers of these cell populations such as cord blood. This strategy is
now under clinical investigation in immune-ablated patients, which are being transplanted
with one un-manipulated plus one Notch-expanded cord blood units. The first evaluated
patients have significantly reduced the time of neutrophil engraftment compared with
patients transplanted with two un-manipulated cord blood units110. However, Notchdependent expansion protocols can still be improved to enhance the long-term engraftment
of the manipulated unit after the identification of crucial elements provided by endothelial
cells111 or by using different ligands112. At the mechanistic level, the impact of Notch
activation on ex-vivo expansion of LT- or ST-HSC may be directly correlated with its
capacity to regulate cell cycle in HSC113 or/and hematopoietic recovery under stress
What can we learn from other systems: Notch function in other somatic stem cells
Different Notch functions that take place in particular cell types, tissues or developmental
stages involve specific mechanistic strategies that are under intense investigation. For
example, many efforts have been made to understand how Notch regulates stem cell
maintenance and cell fate determination in both embryonic and regenerating adult tissues.
Despite some of the identified mechanistic strategies can be tissue specific, others maybe
not, or in any case they can serve as inspiration for people working on different systems
(see Figure 3). In the next section we have selected some of these examples, which may
apply to the hematopoietic system.
The oscillation model: Neural Stem Cells
Similar to HSC, neural stem cell generation also occurs during embryonic life. However,
some specific regions of the adult hippocampus (subgranular zone, SGZ), lateral ventricle
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(subventricular zone, SVZ) and the olfactory bulb still contain some functional neural cells
with self-renewal capacity. During embryonic development, different phases of both
symmetric and asymmetric cell divisions are required to generate all main neuron lineages:
First, symmetric divisions of the neural stem cells support the expansion of the
neuroepithelial progenitors and the generation of radial glial cells. Next, asymmetric division
of these progenitors gives rise to all types of cells during the neurogenic and gliogenic
phases. It is well established that Notch pathway is a crucial regulator of this process and it
is required to maintain neuronal progenitors and inhibit neuronal differentiation (for review
). A nice example on how Notch is differentially activated in the developing neurons
is found during asymmetric division of cortical neuronal progenitors. In this process, both
the Notch receptor and its inhibitor Numb are asymmetrically distributed in the two daughter
cells. The one carrying Notch induces hes1 transcription that directly inhibits neuronal
genes. However, the system is not so simple since Hes1 protein inhibits its own
transcription thus generating a negative feedback loop that creates an oscillatory pattern of
hes1 expression
. Oscillations on Hes1 protein levels induce the oscillation of its target
genes including Neurogenin2 (Ngn2), but also Delta1 (Dll1), which in turn induces
oscillation of Notch activity in neighboring cells
. Elegant studies with time-lapse imaging
from Kageyama’s laboratory showed that Ngn2 and Dll1 oscillate in neural stem/progenitors
in an opposite phase as hes1, and their expression is sustained in differentiating neurons
where Hes1 expression is repressed (reviewed in
. This oscillatory system is responsible
for maintaining active proliferation and neuronal determination in the developing midbrain,
whereas sustained Hes1 levels in the cells of the boundary of the forebrain, midbrain and
hindbrain maintain low levels of Ngn2 that result in low proliferation and prevent neuronal
In the adult brain, neurogenesis is much more restricted in the number and differentiation
potential of cells undergoing this process. However, Notch seems to be also involved in the
maintenance of the adult neural stem cell population, whereas inactivation of Notch
signaling induces neuronal differentiation and depletes the neural stem cell pool 118,119
Cell versus non-Cell autonomous or embryonic versus adult Notch effects: Skin Stem Cells
The adult skin epidermis contains two different types of progenitor or stem cells: The
proliferative cell compartment that is located in the basal layer of the interfolicullar skin and
can differentiate into a single lineage and the mostly quiescent multipotent stem cell
compartment restricted to the bulge of the hair follicle. The bulge stem cells (that are the
pluripotent skin stem cells) can generate both epidermal and hair follicle cells dependent on
Notch activity, since Notch-RPP-J signaling promotes the follicular fate 120,121. The activation
of Notch in this system is downstream of β-catenin activity through the transcriptional
From by guest on June 14, 2017. For personal use only.
activation of Jagged1
. However, there is no evidence for Notch function in maintaining
the multipotent bulge stem cell compartment.
In the basal epidermal layer, Notch signaling regulates proliferation and differentiation of the
epidermal cells, however some data is still controversial and mainly depends on the
experimental systems used. Early studies using epidermis-specific Notch1 knock out mice
(using Keratin5-Cre) demonstrated that Notch deletion increased skin proliferation by downregulating p21 in the keratinocytes
increased β-catenin levels
thus creating a tumor-promoting environment with
. These findings, together with data from knock-down and
knockout of different Notch elements in the mouse skin supported the idea that Notch
behaved as a tumor-suppressor in this tissue
. Surprisingly, Rbpj-deletion in the
developing skin (by using the keratin14-CRE mouse line) resulted in a hypo-proliferative
phenotype concomitant with a blockage in skin differentiation, that was non-cell
autonomous since it was recovered when mutant skin was grafted onto nude mice
These results, further than creating controversy, highlighted the great complexity of the
interactions that mediate Notch effects and suggested that non-cell autonomous effects
might also influence the effect of Notch in skin tumorigenesis. In fact, using a mouse model
with chimeric pattern of Notch1-deletion in the skin it was recently demonstrated that it is
not the absence of Notch in the keratinocyte progenitors but the skin barrier defects
induced by Notch deletion, what is responsible for tumor promotion in these mutants
Analysis of mice that are mutant for the Notch-processing enzymes γ-secretase
, have further confirmed that Notch plays both cell autonomous and non-cell
autonomous effects in the adult skin. In the embryo, deletion of Rbpj prevented the
transition between basal to spinous layers
, whereas Adam10 mutants showed a
premature differentiation from spinous to granular layers
, which in both cases generates
and aberrantly thin spinous layer. Of note, embryonic inactivation of γ-secretase did not
impose any phenotype to the developing skin.
The niche-forming Stem Cell model: Intestinal Stem Cells
The intestinal epithelium is one of the most rapidly self-renewing tissues of the organism
(every 4-5 days for most cell types). In the adult, all post-mitotic cell lineages are originated
by terminal differentiation of a progenitor pool derived from the intestinal stem cells (ISC).
Few years ago, it was proposed that a highly proliferative population of cells residing in the
bottom of the crypts, characterized by expression of the Lgr5 marker, were the actual ISC.
Importantly, these cells reside adjacent to terminally differentiated Paneth cells that support
their self-renewal and pluripotent capacities by providing Wnt and Notch-ligands 130.
Recently, it has been shown that Lgr5-positive cells can derive from quiescent Bmi1
expressing cells located in the +4 position (relative to the bottom of the crypt)
, which
From by guest on June 14, 2017. For personal use only.
suggest the co-existence of more than one ISC compartment (for review see
. Notch
plays an essential role in the maintenance of intestinal homeostasis, since inhibiting Notch,
both pharmacologically and genetically, impairs the integrity of the intestinal crypt leading to
the accumulation of post-mitotic goblet (mucus-secreting) cells
. However, this effect is
supposed to be dependent on Hes1, which inhibits Atonal that is required for differentiation
into the secretory lineage
. Notch is also expressed in the stem cell compartment of the
and mice carrying specific intestinal deletion of Delta1-4 ligands failed to
activate Notch, lacking the expression of stem cell markers in the crypt cells (including Lgr5
and Olfm4)
. This fact suggests a role for Notch not only in regulating intestinal
differentiation but also in the maintenance of the ISC. However, the fact that secretory
Paneth cells (that depends on Atonal, which is inhibited downstream of Notch) participate of
the intestinal stem cell niche precludes giving a definitive answer about whether Notch
directly regulates intestinal stemness or it is just an indirect effect due to possible Paneth
cell number alterations. However, in a simplistic point of view Notch inhibition should lead to
increased Paneth cell numbers and favors ISC generation, which is not the case.
The role of Notch in regulating the niche for stem cell generation or maintenance is a novel
concept, with emerging evidences not only in the mammalian intestinal system but also in
the development of the drosophila gut
as well as the hematopoietic development of
zebrafish 138, which should be investigated in the near future.
A regenerative medicine hallmark: myogenesis.
Finally, another important function of Notch is as a regulator of myogenesis both during
embryonic somitogenesis and in post-natal muscle repair. Importantly, inducing Notch
signaling by Delta1 potentiate muscle regeneration in old aged mice. Specifically, the
balance between Notch and TGFb/smad3 signals determine the levels of CDK inhibitors
that are crucial regulators of the proliferative capacity of muscle progenitors139. Similarly,
activation of Notch in satellite cells blocks myogenesis whereas inhibition of the pathway
leads to increased cell differentiation, indicating that Notch signal is essential to regulate the
pool of satellite cells and adult tissue repair (reviewed in 140).
One of the take home messages of this review is the essential role of Notch in the
regulation of most binary cellular decisions that take place both in the embryo and in adultregenerating tissues, which is exemplified in the hematopoietic system. It is thus envisioned
that the possibility to modulate Notch signaling will open an avenue for regenerative
medicine applications, including hematological and non-hematological diseases, but also
for cancer treatment. Before that, much more detailed studies including mechanistic data
From by guest on June 14, 2017. For personal use only.
from hematopoiesis but also other embryonic and adult tissues are required to clearly
determine whether and how Notch can be used as a tool for generating clinically relevant
and safe cell populations for transplantation.
We sincerely apologize to those whose work could not be cited due to space limitation. We
thank the members of the Bigas-Espinosa lab for critical discussions, specially to Teresa
D’Altri, Leonor Norton, Julia Inglés-Esteve and Erika López-Arribillaga for critical reading of
the manuscript. The laboratory is funded by Ministerio Ciencia e Innovación (SAF200760080, PLE2009-0111, SAF2010-15450, PI10/01128), Red Temática de Investigación
CONES2010-0006. LE is an investigator of ISCIII program (02/30279).
Authorship: L.E. and A.B. wrote the review.
Conflict of Interest: No financial conflicts of interest apply.
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Figure 1: The Notch signaling pathway
Signal-Sending Cell: Functional Notch ligands are ubiquitinated by the E3-ubiquitin ligases
mindbomb or neuralized. After ligands interact with the Notch receptor, the ligand and the
extracellular part of Notch are endocytosed and ligands may be degraded or recycled.
Signal-Receiving Cell: The Notch mRNA is translated as a precursor protein, which is
cleaved by a furin-like convertase in the Golgi apparatus to produce a functional
heterodimeric receptor. During ER/Golgi transit, Notch is modified by different
glycosyltransferases (Rumi/poglut, pofut, fringes). In cells that express fringe, specific sugar
moieties (diamonds) are conjugated to confer higher affinity to Delta-type ligands. After
ligand binds to the EGF-like repeats of the Notch extracellular domain, an ADAM
metalloprotease cleaves Notch at the S2 site, removing most of the extracellular domain.
The membrane-tethered intracellular domain is then cleaved by the Presenilin complex at
site S3, either in the plasma membrane or after endocytosis, freeing the Notch Intracellular
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domain (ICN). ICN translocates to the nucleus, displaces the corepressor complex,
associates with RBP-J and recruits Coactivators such as Mastermind.
ICN becomes monoubiquitylated (Ub), targeting the receptor for degradation. Several E3
ubiquitin ligases (Deltex, Nedd4, Su(Dx)/Itch, Cbl) can direct Notch receptor trafficking
toward lysosomal degradation or toward recycling. Numb can also promote Notch
degradation in daughters of an asymmetrically dividing cell.
Figure 2: Pleiotropic Notch functions in the hematopoietic system
A) Notch is required during hematopoietic development to generate HSC in the AGM. This
process takes place in the aorta endothelium and is dependent of the Jagged1 ligand and
the Notch1 receptor. Endothelial-like Pre-HSC or HSC cells are the putative target of Notch
activation (light green).
B-C) Models of Notch signaling in Hematopoietic differentiation. B) Notch is involved at
different steps of differentiation of the hematopoietic lineages. The classical hierarchical
model of the hematopoietic tree is here adapted to the concept that a gradient of Notch
activation or signaling determines the different types of hematopoietic cells. C) A
Hematopoietic potential restriction model107 in which lineage restriction is determined by
Notch-doses. HSC progressively lose their capacity to generate different lineages
concomitantly to different exposure to Notch-doses. Intensity of the blue color represents a
speculative gradient of Notch activity. Dashed lines represent cell potential to become a
specific cell type. Solid lines represent progression through normal hematopoietic
development. Long-term HSC (LT-HSC), Common myeloid Progenitor (CMP), Common
Lymphoid Progenitor (CLP), Lymphoid/Myeloid Potential Progenitor (LMP), Common
Megakaryocyte-Erythroid progenitor (CME), Early T-progenitor (ETP), Erythrocyte (Ery),
Megakaryocyte (Meg), Macrophage/Granulocyte (M/Gr). Specific Notch activity is indicated
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Figure 3: Notch-dependent mechanistic strategies for stem cell maintenance and
A and B) A)Neuronal differentiation in the developing neuroepithelium is dependent on an
asymmetric cell division in which Numb is asymmetrically distributed and inhibits Notch
signaling in one of the cells to permit neuronal differentiation. B) In the developing brain,
Notch controls the oscillation of downstream genes (hes1 and Ngn2) that allows controlled
proliferation and differentiation waves in the midbrain compartment. In the boundary (green
boxes), Hes1 protein levels are sustained, Ngn2 levels are low and proliferation and neuron
formation is inhibited (based on revision141).
C and D) C)Notch promotes cell proliferation in the basal layer of the embryonic epidermis
however in adult epidermis D) Notch is inhibiting proliferation and inducing differentiation of
the interfollicular basal layer. In the bulge, Notch and Jag1 are regulating epidermal stem
cells by promoting the follicular stem cell fate downstream of Wnt/β-catenin.
E) Two types of ISC have been identified: Lgr5+ proliferating cells and quiescent +4 cells
(Bmi+). Lgr5+ cells are located at the bottom of the intestinal crypts, next to an intestinal
secretory-type cell called Paneth cell. These cells are crucial ISC-niche elements, they
expresses Notch-ligands Dll1 and Dll4 and are required to maintain ISC (likely through
Notch activation). However, differentiation to the secretory lineage including Paneth Cells
require Atonal, which is a target of Notch/hes1 repression, suggesting that generation of the
ISC-niche is under the control of ISC through Notch.
F) Notch signaling induced by Delta1 is required for muscle regeneration upon injury. The
balance between Notch and TGFb/smad3 signals determine the levels of CDK inhibitors
and the proliferative capacity of muscle progenitors.
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Figure 1. Notch signaling
Signal‐sending Cell
Ligand recycling
Ligand Activation Neuralized
Fng‐modified Notch receptor
γ‐secretase (S3)
Signal‐receiving Cell
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Figure 2. Notch in the hematopoietic system
Circulating cells
ETP T-cell
Ery Meg M/Gr B-cell M
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Figure 3. Notch-dependent mechanistic strategies to generate or maintain Stem Cells
hes oscillation
Numb Astrocyte
Low Proliferation
High Proliferation
Radial Glial Oligodendrocyte
Paneth cells
Satelite cell
Stem Cell
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Prepublished online February 3, 2012;
Hematopoietic stem cells: to be or Notch to be
Anna Bigas and Lluis Espinosa
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