Notch
Mammalian Notch homologs The Notch receptor, which is involved in numerous cell fate decisions in both invertebrates and vertebrates, is synthesized as a 300-kDa precursor molecule (p300). Proteolytic processing of p300 is an essential step in the formation of the biologically active receptor because only the cleaved fragments are present at the cell surface. These results confirm and extend recent reports indicating that the Notch receptor exists at the plasma membrane as a heterodimeric molecule, but disagree as to the nature of the protease that is responsible for the cleavage that takes place in the extracellular region. Constitutive processing of murine Notch1 involves a furin-like convertase. This enzyme belongs to a family of subtilisin-like, calcium-dependent convertases that process proproteins in the constitutive secretory pathway. The calcium ionophore A23187 and the alpha1-antitrypsin variant, alpha 1-PDX, a known inhibitor of furin-like convertases, inhibit p300 processing. When expressed in the furin-deficient Lovo cell line, p300 is not processed. In vitro digestion of a recombinant Notch-derived substrate with purified furin allowed mapping of the processing site to the carboxyl side of the sequence RQRR (amino acids 1651-1654). Mutation of these four amino acids (and of two secondary dibasic furin sites located nearby) completely abolishes processing of the Notch1 receptor (Logeat, 1998).
Recently it has been suggested that the disintegrin metalloprotease Kuzbanian is required for the constitutive cleavage of Notch. Overexpression of a dominant negative mutant of Drosophila Kuz (KUZ-DN) lacking the pro- and metallo-protease domains results in no detectable p120 Notch, neither in transfected S2 cells nor in Drosophila imaginal discs. No processing of Notch can be observed in kuz null Drosophila embryos. In the current experiments, the expression of KUZ-DN does not affect mouse Notch1 processing, irrespective of the amount of this dominant negative molecule introduced into cells. In any case, further work will be necessary to clarify these apparent discrepancies between the Drosophila and mammalian results and determine whether these discrepancies are because of differences in the experimental systems used, for example, between Drosophila and mammals. Because Kuz is required for the lateral inhibition process during Drosophila neurogenesis and its target seems to be the extracellular region of Notch, it is possible that Kuz is not involved in the constitutive maturation of the receptor but in a subsequent processing step that would follow interaction with the ligand. This might explain the recently published observation that a new band migrating slightly faster than p120 is induced by contact between cells expressing Notch1 and cells expressing Jagged2. Favoring this hypothesis is the fact that membrane metalloproteases of the ADAM family are postulated to act at the cell surface is in favor of this hypothesis. KUZ-DN is shown not to be able to inhibit transactivation of a target gene of the Notch pathway induced by ligand binding to the receptor. In conclusion, mammalian Notch molecules are constitutively processed by a furin-like convertase as part of their normal maturation process, and this processing is required for cell surface expression of a heterodimeric functional receptor (Logeat, 1998).
Notch1 and 2 are active in development in the mouse. Notch2 also serves some postnatal functions of mouse central nervous system. Notch2 is expressed in the embryonic ventricular
zone, the postnatal ependymal cells, and the choroid plexus throughout embryonic and postnatal
development. Notch1 is also expressed in the ventricular zone between embryonic days 10 and
14, but its expression decreases gradually as the embryo develops. The postnatal mouse brain
strongly expresses Notch2, but not Notch1, in the granular cell layer of hippocampal dentate
gyrus, where neurogenesis continues even in adult rodents. The most remarkable finding is the
detection of a strong signal for Notch2 mRNA in two circumventricular organs: the subfornical
organ and the area postrema (Higuchi, 1995).
The
effects of excess murine Notch 3 activity have been analyzed in central nervous system (CNS) progenitor cells. A mutated Notch
gene encoding the intracellular domain of mouse Notch 3 transcribed from the nestin promoter was
expressed in CNS progenitor cells in transgenic mice. This mutation results in a phenotypic series of
neural tube defects in embryonic day 10.5-12.5 embryos and proves lethal to embryos beyond this age.
In the milder phenotype the neural tube displays a zig-zag morphology and the CNS is slightly
enlarged. More severely affected embryos show a lack of closure of the anterior neural pore,
resulting in the externalization of neural tissue and the complete collapse of the third and fourth
ventricles. The expanded ventricular zone of the neuroepithelium, a correspondingly enlarged area of
nestin expression, and an increase in the number of proliferating cells in the neural tube suggest that
these phenotypes result from an expanded CNS progenitor cell population. These data provide
support in vivo for the notion that Notch activity plays a role in mammalian CNS development and may
be required to guide CNS progenitor cells in their choice between continued proliferation or neuronal
differentiation (Lardelli, 1997).
Neurons in the mammalian central nervous system are generated from progenitor cells of the ventricular zone, a columnar epithelium lining the lumen of the cerebral cortex. Horizontally dividing cells produce basal daughters that behave like young migratory neurons, and apical daughters that remain within the proliferative zone. Notch1 is distributed asymmetrically in mitotic cells, with Notch1 inherited selectively by the basal (neuronal) daughter of horizontal divisions. The idea that Notch signaling directs cells into a nonneuronal fate is at odds with this result. It is conceivable that Notch1 activity delays differentiation of the young neurons long enough to escape the ventricular zone (Chenn, 1995).
Elongation and branching of epithelial ducts is a crucial event during the development of the mammary gland. Branching morphogenesis of the mouse mammary epithelial TAC-2 cell line was used as an assay to examine the role of Wnt, HGF, TGF-beta, and the Notch receptors in branching morphogenesis. Wnt-1 induces the elongation and branching of epithelial tubules, like HGF and TGF-beta2, and strongly cooperates with either HGF or TGF-beta2 in this activity. Wnt-1 displays morphogenetic activity in TAC-2 cells as it induces branching even under conditions that normally promote cyst formation. The Notch4(int-3) mammary oncoprotein, an activated form of the Notch4 receptor, inhibits the branching morphogenesis normally induced by HGF and TGF-beta2. The minimal domain within the Notch4(int-3) protein required to inhibit morphogenesis consists of the CBF-1 interaction domain and the cdc10 repeat domain. Coexpression of Wnt-1 and Notch4(int-3) demonstrates that Wnt-1 can overcome the Notch-mediated inhibition of branching morphogenesis. These data suggest that Wnt and Notch signaling may play opposite roles in mammary gland development, a finding consistent with the convergence of the wingless and Notch signaling pathways found in Drosophila (Uyttendaele, 1998).
The Notch signaling pathway is important for cellular
differentiation. The current view is that the Notch receptor
is cleaved intracellularly upon ligand activation. The
intracellular Notch domain then translocates to the
nucleus, binds to Suppressor of Hairless (RBP-Jk in
mammals), and acts as a transactivator of Enhancer of Split
(HES in mammals) gene expression. The Notch 3 intracellular domain (IC), in contrast to
all other analyzed Notch ICs, is a poor activator, and in fact
acts as a repressor by blocking the ability of the Notch 1
IC to activate expression through the HES-1 and HES-5
promoters. A model is presented in which Notch 3 IC
interferes with Notch 1 IC-mediated activation at two
levels. (1) Notch 3 IC competes with Notch 1 IC for
access to RBP-Jk and does not activate transcription when
positioned close to a promoter. (2) Notch 3 IC appears
to compete with Notch 1 IC for a common coactivator
present in limiting amounts.
Further support for the existence of a
coactivator comes from the finding that the Notch 3 ankyrin
repeat construct, which lacks the strong RBP-Jk-binding
RAM23 domain, is still able to repress Notch 1 IC-mediated
activation. The common coactivator is most likely not
required for all transcriptional complexes, since activation via the
GAL4/VP16 fusion protein is not inhibited by Notch 3 IC.
In keeping with this, cotransfection of the general coactivators
TIF2, SRC1 and p300, does not neutralize Notch 3 ICs
repressor activity.
In conclusion, this is the first
example of a Notch IC that functions as a repressor in
Enhancer of Split/HES upregulation, and shows that
mammalian Notch receptors have acquired distinct
functions during evolution (Beatus, 1999).
What is the structural basis for the difference in
transactivating capacity between Notch 1 IC and Notch 3 IC?
All Notch receptors, including Notch 3, are highly structurally
related in the intracellular domains, in particular in the ankyrin
repeat region. The ankyrin repeat region is important for the
transactivating activity in Drosophila Notch, LIN-12 and
Notch1. The high degree of conservation
between Notch 1 IC and Notch 3 IC in this domain may at first
seem paradoxical. It should however be noted that relatively
subtle mutations in the ankyrin repeat region can dramatically
alter its transactivation competence. The RAM23 region is
conserved to a somewhat lesser extent, but apparently the
conservation is sufficient for both Notch 1 IC and Notch 3 IC
to bind to RBP-Jk. The most obvious
differences between Notch 1 IC and Notch 3 IC are found at
the C-terminal end, where Notch 3 IC is shorter and lacks the
OPA repeats found in other Notch homologs. It remains to be tested, however, whether this region
plays a role in transactivation (Beatus, 1999).
What is the role of Notch3 IIC in vivo? A partial reduction of HES-5 expression in the rhombomere
region was observed in a nestinp/Notch 3 IC transgenic mouse
embryo with a distinct CNS phenotype. This suggests that
Notch 3 IC also acts as a repressor of HES expression in vivo.
Downregulation of HES-5 is evident around the rhombic lip
and in the myelencephalic region, but not in more anterior and
posterior CNS regions. Interestingly, this is reminiscent of the
situation in RBP-Jk and Notch 1 -/- mice, in which HES-5
expression is also reduced in this region. This further supports a role of Notch 3 IC as a repressor
of Notch 1-signaling, but also suggests that HES-5 expression
is, at least in part, regulated by other factors in other regions
of the CNS.
A role for Notch 3 as a repressor of HES expression in vivo
receives further support from comparisons of the phenotypes
resulting from targeting of HES-1 and overexpression of Notch
3 IC in transgenic mice. Expression of Notch 3 IC in the
developing CNS of transgenic mouse embryos produces an
embryonically lethal phenotype. The
transgenic embryos have an undulating spinal cord, fail to close
the anterior neural pore and exhibit protrusions of neural tissue
from the anterior neural pore region.
Although initially interpreted differently,
the latter phenotype may be a consequence of the open neural
pore, in particular considering that the transgenic embryos
show a relatively modest increase in proliferative rate in the
CNS. Embryos lacking the HES-1 gene die just after birth, and
show a kinked neural tube, open anterior neural pore and an
everted neuroepithelium. Thus, the
HES-1 -/- phenotype shows clear similarities to those observed
in embryos overexpressing Notch 3 IC in the early CNS (Beatus, 1999 and references).
The finding that Notch 3 IC acts as a negative modulator of
HES expression will be important for understanding of the
CADASIL (Cerebral Autosomal Dominant with Arteriopathy
and Subcortical Infarcts with Leukoencephalopathy).
CADASIL is a familial disease which leads to migraine,
subcortical brain infarcts and dementia and is caused by
missense mutations in the EGF-repeat region of the human
Notch 3 gene. CADASIL is a
dominant disease, but it is not yet known whether the mutations
in Notch 3 lead to haploinsufficiency (a condition whereby normal function
is impaired by loss of one functional allele), or if the CADASIL
mutations result in gain-of-function receptors. Since Notch 1,
2 and 3, and HES genes are expressed in the adult brain, it is conceivable
that the function of Notch 3 as a negative modulator of HES
expression may be affected in the disease. Thus, if CADASIL
mutations produce gain-of-function Notch 3 receptors, this
would result in decreased HES expression. Conversely, in the
haploinsufficiency scenario, repression of HES expression
would be reduced (Beatus, 1999 and references).
Mammalian Notch homologs: Cell surface and cytoplasmic interactions Starting from probes resembling that of Drosophila Serrate, a full-length human cDNA was assembled (termed human Jagged2) from overlapping cDNA clones. The cDNA encodes a polypeptide having extensive sequence homology to Serrate (40.6%
identity and 58.7% similarity) and even greater homology to several putative mammalian Notch ligands
that have subsequently been described. Expression of the
murine Jagged2 homolog is found to be highest in fetal thymus, epidermis, foregut, dorsal root
ganglia, and inner ear. In Northern blot analysis of RNA from tissues of 2-week-old mice, the 5.0-kb
Jagged2 transcript is most abundant in heart, lung, thymus, skeletal muscle, brain, and testis.
Coexpression of Jagged2 and Notch1 occurs within thymus and other fetal murine tissues, consistent with interaction of the two proteins in vivo. Coculture of fibroblasts
expressing human Jagged2 with murine C2C12 myoblasts inhibits myogenic differentiation, accompanied by increased Notch1 and the appearance of a novel 115-kDa Notch1 fragment.
Exposure of C2C12 cells to Jagged2 leads to increased amounts of Notch mRNA as well as mRNAs for
a second Notch receptor, Notch3, and a second Notch ligand, Jagged1. Constitutively active forms of
Notchl in C2C12 cells also induce increased levels of the same set of mRNAs, suggesting positive
feedback control of these genes initiated by the binding of Jagged2 to Notch1. This feedback control may
function in vivo to coordinate differentiation across certain groups of progenitor cells adopting identical
cell fates (Luo, 1997).
Notch controls cell fate by inhibiting cellular differentiation, presumably through activation of
the transcriptional regulator human C promoter Binding Factor (CBF1), which transactivates
the hairy and Enhancer of split (HES-1) gene. However, constitutively active
forms of Notch1 are described, which inhibit muscle cell differentiation but do not interact with CBF1 or
upregulate endogenous HES-1 expression. In addition, Jagged-Notch interactions that
prevent the expression of muscle cell specific genes do not involve the upregulation of
endogenous HES-1. In fact, exogenous expression of HES-1 in C2C12 myoblasts does not
block myogenesis. These data demonstrate the existence of a CBF1-independent pathway by
which Notch inhibits differentiation. It has been proposed that Notch signaling activates at
least two different pathways: one which involves CBF1 as an intermediate and one which
does not. Since the truncated Notch proteins used in this experiment either contain no intracellular domain, or they possess intracellular domains but no extracellular domain, it is difficult to see how active intracellular signaling of altered receptors could be responsible for the inhibition of myogenesis. The inhibition of muscle cell differentiation might be accomplished by sequestering of functional Notch proteins in an inactive complex (Shawber, 1996).
The Notch receptor is involved in many cell fate determination events in vertebrates and invertebrates. It has been shown in Drosophila that Delta-dependent Notch signaling activates the transcription factor Suppressor of Hairless, leading to an increased expression of the Enhancer of Split genes. Genetic evidence has also implicated the Kuzbanian gene, which encodes a disintegrin metalloprotease, in the Notch signaling pathway. A two-cell coculture assay has shown that vertebrate Dl-1 activates the Notch-1 cascade. Consistent with previous data obtained with active forms of Notch-1, a HES-1-derived promoter construct is transactivated in cells expressing Notch-1 in response to Dl-1 stimulation. Impairing the proteolytic maturation of the full-length receptor leads to a decrease in HES-1 transactivation, further supporting the hypothesis that only mature processed Notch is expressed at the cell surface and activated by its ligand. Dl-1-induced HES-1 transactivation is dependent both on Kuzbanian and RBP-J activities, consistent with the involvement of these two proteins in Notch signaling in Drosophila. Exposure of Notch-1-expressing cells to Dl-1 results in an increased level of endogenous HES-1 mRNA. Finally, coculture of Dl-1-expressing cells with myogenic C2 cells suppresses differentiation of C2 cells into myotubes, as previously demonstrated for Jagged-1 and Jagged-2, and also leads to an increased level of endogenous HES-1 mRNA. Thus, Dl-1 behaves as a functional ligand for Notch-1 and has the same ability to suppress cell differentiation as do the Jagged proteins (Jarriault, 1998).
EBNA2 is essential for immortalization of B cells by Epstein-Barr virus. EBNA2 is tethered to
responsive promoters through a cellular factor, CBF1. CBF1 also binds to the activated form of
mammalian Notch1, providing a linkage between EBNA2 function and Notch signaling. However,
Notch2 is the predominant form expressed in spleen. The degree to which these Notch homologs are
functionally convergent is not known. Evidence is presented that Notch2 also signals through CBF1. As
is the case for Notch1, Notch2 interacts with the minimal repression domain of CBF1 and is
targeted to CBF1 through the intracellular, subtransmembrane domain. Additional characterization
suggests that the interaction domain of Notch may be bipartite. The intracellular domain of Notch2
(Notch2IC) locates to the nucleus. This activated form of Notch2 transactivates expression of a target
gene containing upstream CBF1 binding sites. The use of CBF1 mutants carrying amino acid
substitutions in the transcriptional repression domain reveals that activation of gene expression by
Notch2 is also based on masking of CBF1-mediated repression. Targeting of Notch1 and targeting of
Notch2 are found to be identical and distinguishable from targeting by EBNA2. Mutation of CBF1 at
codons 249 to 251 abolishes interaction with both Notch proteins but not with EBNA2. In a biological
examination of Notch2 function in muscle cells, Notch2IC activates endogenous HES-1 gene
expression and blocks muscle cell differentiation. Overall, the data imply that at least a subset of the
intracellular events following signaling in cells expressing Notch2 are common to those in
Notch1-expressing cells. The concept that EBNA2 functions by mimicking Notch signaling is
therefore viable whether cells are expressing Notch1 or Notch2 (Hsieh, 1997).
Mouse numblike is the second mammalian gene to be isolated that shows significant
sequence similarity to Drosophila numb. Numblike protein shows 76% homology to mouse Numb and 63.6% homology to Drosophila Numb. It is known that both m-Numb and Numblike, two murine numb proteins, can physically interact with the intracellular domain of Notch 1. m-Numb and Numblike each have distinct characteristics that suggest that Numblike would be unable to fully substitute for m-Numb. When expressed in dividing neural
precursors in Drosophila, Numblike is symmetrically distributed in the cytoplasm, unlike
endogenous Drosophila Numb or expressed m-Numb, both of which are
asymmetrically localized to one half of the cell membrane. In Drosophila numb loss-of-function mutant
embryos, expression of Numblike allows both daughter cells of a neural precursor to adopt
the fate of the cell that normally inherits Drosophila Numb. In mice, numblike mRNA is preferentially
expressed in the adult and embryonic nervous system. In the developing neocortex, Numblike
is expressed in postmitotic neurons in the cortical plate, but not in progenitors within the
ventricular zone where m-Numb and Notch1 are expressed. Numblike appears to be a cytoplasmic protein while m-Numb is a membrane associated protein, as is Drosophila Numb. In
dividing cortical progenitors, Notch1 is distributed around the entire membrane, unlike
m-Numb, which is asymmetrically localized to the apical membrane. It is concluded that Numblike functions in postmitotic cells, with m-Numb to suppress the residual Notch1 activity in the cell and allow it to fully differentiate into a neuron. In contrast, m-Numb and Numb function to ensure that daughter cells of asymmetric divisions acquire distinct fates. It is proposed that an
interplay between cell-intrinsic mechanisms (executed by m-numb and numblike) and
cell-extrinsic mechanisms (mediated by Notch1) may be involved in both progenitor cell
proliferation and neuronal differentiation during mammalian cortical neurogenesis (Zhong, 1997).
Approximately 10% of cases of Alzheimer's disease are familial and associated with autosomal
dominant inheritance of mutations in genes encoding the amyloid precursor protein, presenilin 1 (PS1)
and presenilin 2 (PS2). Mutations in PS1 are linked to about 25% of cases of early-onset familial
Alzheimer's disease. PS1, which is endoproteolytically processed in vivo, is a multipass transmembrane
protein and is a functional homologue of SEL-12, a C. elegans protein that facilitates
signaling mediated by the Notch/LIN-12 family of receptors. To examine potential roles for PS1 in
facilitating Notch-mediated signaling during mammalian embryogenesis, mice were generated with
targeted disruptions of PS1 alleles (PS1-/- mice). PS1-/- embryos exhibit abnormal patterning of the
axial skeleton and spinal ganglia, phenotypes traced to defects in somite segmentation and
differentiation. Moreover, expression of mRNA encoding Notch1 and Dll1 (delta-like gene 1), a
vertebrate Notch ligand, is markedly reduced in the presomitic mesoderm of PS1-/- embryos, as compared
to controls. Hence, PS1 is required for the spatiotemporal expression of Notch1 and Dll1, which are
essential for somite segmentation and maintenance of somite borders (Wong, 1997).
Notch proteins are ligand-activated transmembrane receptors involved in cell-fate selection throughout development. No
known enzymatic activity is contained within Notch and the molecular mechanism by which it transduces signals across the cell
membrane is poorly understood. In many instances, Notch activation results in transcriptional changes in the nucleus through
an association with members of the CSL family of DNA-binding proteins (where CSL stands for CBF1, Su[H], Lag-1). Since
Notch is located in the plasma membrane and CSL is a nuclear protein, two models have been proposed to explain how they
interact. The first suggests that the two interact transiently at the membrane. The second postulates that Notch is cleaved by a
protease, enabling the cleaved fragment to enter the nucleus. Signaling by a constitutively active
membrane-bound Notch-1 protein is shown to require the proteolytic release of the Notch intracellular domain (NICD), which interacts
preferentially with CSL. Very small amounts of NICD are active, explaining why it is hard to detect in the nucleus in vivo. It is ligand binding that induces release of NICD (Schroeter, 1998).
Two of the positive regulators of the Notch pathway of Drosophila are encoded by the Suppressor of hairless ([Su(H)]) and deltex (dx) genes. Drosophila dx encodes a ubiquitous, novel cytoplasmic protein of unknown biochemical function. A human deltex homolog has been cloned and characterized in parallel with its Drosophila counterpart, in biochemical assays to assess Deltex function. Both human and Drosophila Deltex bind to Notch across species and carry putative SH3-binding domains. Using the yeast interaction trap system, it has been found that Drosophila and human Deltex bind to the human SH3-domain containing protein Grb2. Results from two different reporter assays demonstrate the association of Deltex with Notch-dependent transcriptional events. Evidence is presented linking Deltex to the modulation of basic helix-loop-helix (bHLH) transcription factor activity (Matsuno, 1998).
Notch signal transduction is mediated by proteolysis of the receptor and translocation of the intracellular domain (IC) into the nucleus,
where it functions as a regulator of HES gene expression after binding to the DNA-binding protein RBP-Jk. The mammalian Notch receptors
are structurally very similar, but have distinct functions. Most notably, Notch 1 IC is a potent activator of the HES promoter, while Notch 3 IC
is a much weaker activator and can repress Notch 1 IC-mediated HES activation in certain contexts. This report explores the molecular
basis for this functional difference between Notch 1 and Notch 3 IC. Notch 3 IC, like Notch 1 IC, can bind the SKIP and PCAF
proteins. Furthermore, both Notch 1 and Notch 3 ICs displace the co-repressor SMRT from the DNA-binding protein RBP-Jk on the HES
promoter. The latter observation suggests that both Notch 3 IC and Notch 1 IC can access RBP-Jk in vivo, and that the difference in activation
capacity instead stems from structural differences in the two ICs when positioned on RBP-Jk. Two distinct regions in the Notch
IC are critical for the difference between the Notch 1 and Notch 3 IC; (1) the origin of the ankyrin repeat region is important, i.e. only
chimeric ICs containing a Notch 1-derived ankyrin repeat region are potent activators; (2) a novel important region has been identified in the
Notch IC. This region, named the RE/AC region (for repression/activation), is located immediately C-terminal to the ankyrin repeat region,
and is required for Notch 1 IC's ability to activate and for Notch 3 IC's ability to repress a HES promoter. The interplay between the RE/AC region and the ankyrin repeat region provides a basis to understand the difference in HES activation between structurally similar Notch receptors (Beatus, 2001).
Previous work on Notch ICs has focused on three different domains: the RAM, ankyrin repeat and C-terminal regions. Another region in the Notch IC,
the RE/AC region, is described in this study and has been shown to be important for regulation of HES promoters. Several lines of evidence support the
importance of the RE/AC region, which is 120 amino acid
residues long, located immediately C-terminal to the
ankyrin repeat region. In Notch 1 IC, deletion constructs in
which only the RE/AC region is removed fail to activate
transcription, while more C-terminal deletions result in a
much less dramatic decrease in activation. Similarly,
removal of the RE/AC region from Notch 1 IC fused to
GAL4 DB (G4-1101) largely abolishes activation from a
GAL4 responsive promoter. In Notch 3 IC, the RE/AC
region is also important, but here it plays a critical role in
repression in trans of Notch 1 IC-mediated activation of the
HES-1 promoter. Deletion constructs of Notch 3 IC, where
the RE/AC region is removed, largely fail to repress Notch 1
IC-mediated activation on HES promoters. Moreover, a
small region encompassing the Notch 3 IC RE/AC region,
together with the last four ankyrin repeats, is sufficient to
mediate substantial repression. The importance of this
region has been shown in ESR-1 activation in Xenopus, in activation of the TP-1 promoter and for cellular transformation. Furthermore, it has been
shown that Notch 1 IC and Notch 2 IC inhibit myeloid
differentiation in response to different cytokines and that a
region corresponding to RE/AC is involved in mediating the
cytokine specificity of Notch 1 IC and Notch 2 IC. The finding that the RE/AC region is crucial for HES promoter regulation, oncogenesis and cytokine specificity underlines its importance for Notch function in
various contexts (Beatus, 2001).
It has been proposed that the Notch 3 IC-mediated repression of Notch 1 IC activation is caused
both by competition for access to RBP-Jk and by competition
for a nuclear factor present in limiting amounts. The most parsimonial explanation for the role of the
RE/AC region is to postulate that it constitutes a binding
domain for the putative factor present in limiting amounts.
This line of reasoning receives support from the finding that
the Notch 3 IC RE/AC region can replace the Notch 1 IC RE/
AC region in the full IC context. It is interesting to note that there
are local stretches of very high amino acid sequence similarity when the RE/AC regions of Notch 1 and Notch 3 ICs are
compared. However, these highly conserved domains are not
binding sites for known transcription factors.
The C-terminal region of Notch 1 IC functions as a transcriptional activator on a multimerized RBP-Jk binding site,
and has consequently also been referred to as TAD (transcription activation domain). The C-terminal region, alone or with the RE/AC region, is a potent
activator also on the GAL4 responsive promoter. In
contrast, the C-terminal region of Notch 1
IC is dispensable for potent activation on an endogenous
250 bp HES promoter. This indicates that the C-terminal
region is crucial only on the multimerized RBP-Jk binding
sites and in the GAL4 context, but not on the endogenous
HES promoter (Beatus, 2001).
While the presence of a RE/AC region is required for
activation from Notch 1 IC and repression from Notch 3
IC, the origin of the RE/AC region is not important for
activation, since it can be exchanged between the two ICs
without altering the effect. Therefore, the RE/AC region per se, does not explain why Notch 1 IC is a good activator
and why Notch 3 IC is not. To address whether the origin of other
regions is important, a set of chimeric Notch 1 IC/Notch 3
IC molecules was tested for activation and repression. It is
concluded that the origin of the ankyrin repeat region is the
most critical determinant for activation from HES promoters. This is based on the observation that only chimeric ICs
containing the ankyrin repeat region from Notch 1 are
good activators, while Notch 3 ankyrin repeat-containing
constructs are not. The origin of the ankyrin repeat
region is also most important for repression in trans. Only
chimeric ICs harboring Notch 3 IC-derived ankyrin repeats
are repressors of the same magnitude as Notch 3 itself, while
proteins with ankyrin repeats derived from Notch 1 are
considerably less potent repressors (Beatus, 2001).
How can the data presented here be incorporated into a
model explaining the difference in HES promoter activation
by Notch 1 and Notch 3? There could be two principally
different explanations for Notch 3 IC's poor activation capacity; (1) Notch 3 IC may not get access to RBP-Jk on the
HES promoter in vivo or (2) something in the structure
of Notch 3 IC makes it an inferior activator once positioned
on the HES promoter. The first explanation appears less
likely, based on the data from the SMRT and SKIP experiments. Notch 3 IC, like Notch 1 IC, is capable of displacing VP16-SMRT
from RBP-Jk fused to GAL4 in a two-hybrid assay. Furthermore, addition of SMRT to both Notch 1 IC and Notch 3 IC
results in repression of activation on the HES promoter; in
the case of Notch 1 IC a dramatic decrease, and in the case
of Notch 3 IC a decrease from a very low level of activation.
It is also observed that both Notch 1 IC and Notch 3 IC can
bind to SKIP, which facilitates Notch function and has been
shown to bind Notch 1 IC and SMRT in a mutually exclusive manner. In conclusion, it therefore
appears reasonable to assume that Notch 3 IC can access
RBP-Jk on a HES promoter in a manner similar to Notch 1
IC, i.e. by displacing SMRT from binding to SKIP/RBP-Jk.
Assuming that both Notch 1 IC and Notch 3 IC have
access to RBP-Jk in vivo, three different models
are proposed to explain the differences in activation. These
models take into account that the origin of the ankyrin
repeat regions is important and that a Notch IC requires
the presence of a RE/AC region, which binds a factor,
present in limiting amounts. In the first model, the
ankyrin repeat region would be important for the conformation of the Notch IC/factor complex, and the factor binding
to the RE/AC region would only be optimally presented to
the transcription machinery when the ankyrin repeat region
is of Notch 1 IC origin. In the second model, the
ankyrin repeat region of Notch 1 IC serves as a docking site
for a second co-activator, which can not bind or binds less
well to the ankyrin repeat region of Notch 3 IC. Only the
cooperative binding of the co-activator on the Notch 1 IC
ankyrin repeats and the factor binding to the RE/AC region
would lead to potent activation. PCAF binds less well to
Notch 3 IC than to Notch 1 IC, and could thus be a candidate
factor. PCAF has indeed been shown to bind both
to the ankyrin repeats and the C-terminal region in Notch 1
IC. The less efficient PCAF binding to Notch 3 IC could result in a more compacted chromatin structure at the promoter, as compared to when Notch 1 IC-
PCAF is present. In the third model, the presence
of an additional factor is also postulated, but in this model
the co-factor would be a co-repressor specifically recruited
to the Notch 3 IC ankyrin repeat region. This could quench
the activity of the factor binding to the RE/AC region, thus
rendering Notch 3 IC incapable of activating transcription.
Irrespective of the finer details of how different factors
work together to fine-tune transcriptional regulation, the
discovery of the novel RE/AC region in the Notch IC is
important for a more complete understanding of Notch
signal transduction. The RE/AC region, combined with
the observation that the origin of the ankyrin repeat region
is important for activation, helps to explain why different
Notch ICs are endowed with different activation properties
on downstream HES promoters (Beatus, 2001).
The Drosophila Seven in absentia (Sina) gene product originally was described as a protein that controls cell fate decisions during eye
development. Its mammalian homolog, Siah-1, recently was found to be involved in p53-dependent and -independent pathways of apoptosis and
G1 arrest. Siah-1 is shown to interact directly with and promote the degradation of the cell fate regulator Numb. Siah-1-mediated Numb
degradation leads to redistribution of endogenous cell-surface Notch to the cytoplasm and nucleus and to augmented Notch-regulated
transcriptional activity. These data imply that through its ability to target Numb for degradation, Siah-1 can act as a key regulator of Numb-related
activities, including Notch signaling (Susini, 2001).
Numb physically interacts with and inhibits the signaling of Notch1, a cell-surface receptor that promotes cell fate decisions by activating downstream transcription factors of the CSL family. This is achieved by proteolytic cleavage within an intracellular site of Notch that results in the release and subsequent translocation of its cytosolic fragment (NICD) into the nucleus. The consequences of Siah-1 overexpression on Notch subcellular localization were investigated.
Confocal microscopy analysis of Notch1 immunofluorescence in control U937 cells has revealed a rim-like staining pattern typical of cell-surface receptors. In striking contrast, Siah-1-overexpressing cells exhibited a redistribution of Notch1 immunofluorescence in the cytoplasm and in the nucleus.
Confocal imaging within the Z plane of the nucleus confirms Notch1 nuclear localization. To show that the observed pattern of Notch expression in
Siah-1-overpressing U937 cells resembles that of an activation state, U937 vector control cells were analyzed for Notch translocation after EDTA treatment, which
mimics the effects of ligand-induced nuclear translocation of Notch. By 30 min after EDTA removal, Notch1 immunofluorescence was visualized in and around
the nucleus. Together, these observations suggest further that Siah-1 overexpression promotes Notch1 activation. This conclusion was validated
directly by monitoring endogenous NICD activity in MCF-7 cells stably transfected with vector control or pBK-RSV-Siah-1. A luciferase reporter construct whose
activation is proportional to NICD translocation to the nucleus was transfected into these cells. The presence of endogenous Notch1 was verified by Western blot
analysis. A consistent, >3-fold increase in endogenous NICD activity was observed in MCF-7 cells overexpressing Siah-1, and this
increase was reduced by transient transfection of Numb (Susini, 2001).
Genetic studies have identified human Itch, which is homologous to the E6-associated protein carboxyl terminus (Hect) domain-containing E3 ubiquitin-protein ligase that is disrupted in non-agouti lethal mice or Itchy mice. Itch is a homolog of Drosophila Suppressor of Deltex. Itch-deficiency results in abnormal immune responses and constant itching in the skin. Itch associates with Notch, a protein involved in cell fate decision in many mammalian cell types, including cells in the immune system. Itch binds to the N-terminal portion of the Notch intracellular domain via its WW domains and promotes ubiquitination of Notch through its Hect ubiquitin ligase domain. Thus, Itch may participate in the regulation of immune responses by modifying Notch-mediated signaling (Qiu, 2000).
Pancreatic endocrine cells originate from precursors that express the transcription factor Neurogenin3 (Ngn3). Ngn3 expression is repressed by active Notch signaling. Accordingly, mice with Notch signaling pathway mutations display increased Ngn3 expression and endocrine cell lineage allocation. To determine how the Notch ligand Jagged1 (Jag1) functions during pancreas development, Jag1 was deleted in foregut endoderm and postnatal and embryonic endocrine cells and precursors were examined. Postnatal Jag1 mutants display increased Ngn3 expression, α-cell mass, and endocrine cell percentage, similar to the early embryonic phenotype of Dll1 and Rbpj mutants. However, in sharp contrast to postnatal animals, Jag1-deficient embryos display increased expression of Notch transcriptional targets and decreased Ngn3 expression, resulting in reduced endocrine lineage allocation. Jag1 acts as an inhibitor of Notch signaling during embryonic pancreas development but an activator of Notch signaling postnatally. Expression of the Notch modifier Manic Fringe (Mfng) is limited to endocrine precursors, providing a possible explanation for the inhibition of Notch signaling by Jag1 during mid-gestation embryonic pancreas development (Golson, 2009).
Mammalian Notch homologs: Effects of mutation The Notch genes encode single-pass transmembrane receptors that transduce extracellular signals responsible for cell fate determination during several steps of metazoan development. The mechanism by which extracellular signals affect gene transcription
and ultimately cell fate decisions is beginning to emerge for the Notch signaling pathway. One paradigm is that ligand binding to Notch
triggers a Presenilin1-dependent proteolytic release of the Notch intracellular domain from the membrane, resulting in low amounts of
Notch intracellular domain that form a nuclear complex with CBF1/Su(H)/Lag1 to activate transcription of downstream targets. Not
all observations clearly support this processing model, and the most rigorous test of it is to block processing in vivo and then determine
the ability of unprocessed Notch to signal. The phenotypes associated with a single point mutation at the intramembranous processing site of Notch1, Valine 1,744 to Glycine,
resemble the null Notch1 phenotype. The data show unequivocally that efficient Notch1 intracellular processing is required for viability and proper formation of yolk-sac vasculature in the
mouse embryo. A processing-independent Notch1 signaling event would be predicted to function in a manner indistinguishable from that of wild-type Notch. Analysis of mice expressing the processing-deficient Notch1 allele reveals that all the phenotypes described in the null genotypes are qualitatively reproduced.
However, quantitative differences are described: the null-like phenotype in the anterior occurs at lower penetrance. The quantitative differences that have been observed in the
regions of high Notch1 expression may reflect either residual processing observed in V1744G mutants, or proteolysis-independent Notch1 signaling acting at the
posterior but not the anterior of the embryo. The latter possibility is unlikely given that no fundamental differences between anterior and posterior somitogenesis are
known to exist. Collectively, the data establish the processing-deficient allele as a strong hypomorph of Notch1. It remains to be determined whether late functions of
Notch1 also depend on efficient cleavage at V1744. Thus efficient
intramembranous processing of Notch1 is indispensable for embryonic viability and proper early embryonic development in vivo (Huppert, 2000).
The Notch gene family encodes large transmembrane
receptors that are components of an evolutionarily
conserved intercellular signaling mechanism. To assess
the in vivo role of the Notch2 gene, a
targeted mutation, Notch2del1 was constructed. Unexpectedly, it was found that
alternative splicing of the Notch2del1 mutant allele leads to
the production of two different in-frame transcripts that
delete either one or two EGF repeats of the Notch2 protein,
suggesting that this allele is a hypomorphic Notch2
mutation. Mice homozygous for the Notch2del1 mutation
die perinatally from defects in glomerular development
in the kidney. Notch2del1 /Notch2del1 mutant kidneys are hypoplastic and mutant glomeruli lack a normal
capillary tuft. The Notch ligand encoded by the Jag1 gene
is expressed in developing glomeruli in cells adjacent to
Notch2-expressing cells. Mice heterozygous
for both the Notch2del1 and Jag1dDSL mutations exhibit a
glomerular defect similar to, but less severe than, that of
Notch2del1/Notch2del1 homozygotes. The co-localization and genetic interaction of Jag1 and Notch2 imply that this
ligand and receptor physically interact, forming part of
the signal transduction pathway required for glomerular
differentiation and patterning. Notch2del1/Notch2del1
homozygotes also display myocardial hypoplasia, edema
and hyperplasia of cells associated with the hyaloid
vasculature of the eye. These data identify novel developmental roles for Notch2 in kidney, heart and eye development (McCright, 2001).
The role of Notch signaling in growth/differentiation control of mammalian epithelial cells is still poorly defined. Keratinocyte-specific deletion of the Notch1 gene results in marked epidermal hyperplasia and deregulated expression of multiple differentiation markers. In differentiating primary keratinocytes in vitro endogenous Notch1 is required for induction of p21WAF1/Cip1
expression, and activated Notch1 causes growth suppression by inducing p21WAF1/Cip1 expression. Activated Notch1 also induces
expression of 'early' differentiation markers, while suppressing the late markers. Induction of p21WAF1/Cip1 expression and early
differentiation markers occur through two different mechanisms. The RBP-Jkappa protein binds directly to the endogenous
p21 promoter and p21 expression is induced specifically by activated Notch1 through RBP-Jkappa-dependent transcription. Expression of early differentiation markers is RBP-Jkappa-independent and can be induced by both activated Notch1 and Notch2, as well as the highly conserved ankyrin repeat domain of the Notch1 cytoplasmic region. Thus, Notch signaling triggers two distinct pathways leading to keratinocyte growth arrest and differentiation (Rangarajan, 2001).
Notch is a critical component of evolutionarily conserved signaling mechanisms that regulate development and may contribute to plasticity-related processes, including changes in neurite structure and maintenance of neural stem cells. Deficits in the Notch pathway are responsible for Alagille (Li, 1997) and Cadasil (Harris, 2001) syndromes, which are associated with mental retardation and dementia. Additionally, in postmitotic neurons, Notch proteins interact with presenilins and with ß-amyloid precursor protein and could therefore have a role in the memory deficits associated with familial and sporadic Alzheimer's disease. To test if alterations in Notch signaling can lead to learning and memory deficits, mice with mutations in this pathway were studied. Null heterozygous mutations in Notch1 are shown to result in deficits in spatial learning and memory without affecting other forms of learning, motor control, or exploratory activity. Null heterozygous mutations in the downstream cofactor RBP-J result in similarly specific spatial learning and memory deficits. These data indicate that a constitutive decrease in Notch signaling can result in specific learning and memory deficits and suggest that abnormalities in Notch-dependent transcription may contribute to the cognitive deficits associated with Alzheimer's disease and Alagille and Cadasil syndromes (Costa, 2003).
The exquisite specificity of these learning and memory deficits argues against the possibility that the mutations studied result in gross developmental abnormalities that could account for the learning deficits. Also, long-term spatial memory deficits were observed in experiments in which Notch mutant mice had normal acquisition and short-term spatial memory. This strongly argues against a general deficit in hippocampal information processing and points toward a specific functional deficit due to changes in Notch signaling. It is important to notice that previous studies have shown that different hippocampal-dependent functions are differentially affected by hippocampal manipulations. Results with heterozygotes indicate that Notch mutations affect a specific domain of hippocampal function that is especially important for spatial learning and memory, but it is possible that complete deletions would result in more generalized deficits. Also, it is conceivable that very specific and undetected neuroanatomical changes taking place during development account for the specific spatial learning deficits of the RBP-J+/- and Notch1+/- mice. Even if this would be the case, the findings presented here would still be important for understanding the deficits associated with Alagille and Cadasil syndromes and Alzheimer's disease. However, reducing Notch function specifically in adult Drosophila leads to progressive neurological dysfunction (Costa, 2003).
The results presented here also have important implications for the development of therapeutic strategies for AD. Moreover, they may help in the understanding of the mechanisms associated with the generation of cognitive deficits in AD. It is possible that the synaptic loss and the synaptic dysfunction observed in AD, which correlate with the cognitive deficits, could result from an interaction between the effects of extracellular soluble ß-amyloid and intracellular alterations in Notch and APP signaling (Costa, 2003).
In conclusion, these results are consistent with the hypothesis that Notch signaling is involved in learning and memory processes in the adult brain, and they suggest that abnormalities in Notch-dependent transcription may contribute to the learning and memory deficits associated with Alzheimer's disease and Alagille and Cadasil syndromes (Costa, 2003).
Notch receptors play various roles for cell fate decisions in developing organs, although their functions at the cell level are poorly understood. Notch1 and its ligand are each expressed in juxtaposed cell compartments in the follicles of the bursa of Fabricius, the central organ for chicken B cell development. To examine the function of Notch1 in B cells, a constitutively active form of chicken Notch1 was expressed in a chicken B cell line, DT40, by a Cre/loxP-mediated inducible expression system. Remarkably, the active Notch1 causes growth suppression of the cells, accompanied by a cell cycle inhibition at the G(1) phase and apoptosis. The expression of Hairy1, a gene product up-regulated by the Notch1 signaling, also induces the apoptosis, but no cell cycle inhibition. Thus, Notch1 signaling induces apoptosis of the B cells through Hairy1, and the G(1) cell cycle arrest through other pathways. This novel function of Notch1 may account for the recent observations indicating the selective inhibition of early B cell development in mice by Notch1 (Morimura, 2001).
Formation of a fully functional artery proceeds through a multistep process. Notch3 is required to generate functional arteries in mice by regulating arterial differentiation and maturation of vascular smooth muscle cells (vSMC). In adult Notch3-/- mice distal arteries exhibit structural defects and arterial myogenic responses are defective. The postnatal maturation stage of vSMC is deficient in Notch3-/- mice. Notch3 is shown to be required for arterial specification of vSMC but not of endothelial cells. These data reveal Notch3 to be the first cell-autonomous regulator of arterial differentiation and maturation of vSMC (Domenga, 2004).
Transcriptional regulation of Notch Cyclin D1 belongs to the core cell cycle machinery, and it is frequently overexpressed in human cancers. The full repertoire of cyclin D1 functions in normal development and oncogenesis is unclear at present. This study developed Flag- and haemagglutinin-tagged cyclin D1 knock-in mouse strains that allowed a high-throughput mass spectrometry approach to search for cyclin D1-binding proteins in different mouse organs. In addition to cell cycle partners, several proteins involved in transcription were uncovered. Genome-wide location analyses (chromatin immunoprecipitation coupled to DNA microarray; ChIP-chip) showed that during mouse development cyclin D1 occupies promoters of abundantly expressed genes. In particular, it was found that in developing mouse retinas - an organ that critically requires cyclin D1 function - cyclin D1 binds the upstream regulatory region of the Notch1 gene, where it serves to recruit CREB binding protein (CBP) histone acetyltransferase. Genetic ablation of cyclin D1 resulted in decreased CBP recruitment, decreased histone acetylation of the Notch1 promoter region, and led to decreased levels of the Notch1 transcript and protein in cyclin D1-null (Ccnd1-/-) retinas. Transduction of an activated allele of Notch1 into Ccnd1-/- retinas increased proliferation of retinal progenitor cells, indicating that upregulation of Notch1 signalling alleviates the phenotype of cyclin D1-deficiency. These studies show that in addition to its well-established cell cycle roles, cyclin D1 has an in vivo transcriptional function in mouse development. This approach, which termed 'genetic-proteomic', can be used to study the in vivo function of essentially any protein (Bienvenu, 2010).
Notch and stem cells Neural stem cells, which exhibit self-renewal and multipotentiality,
are generated in early embryonic brains and maintained throughout the
lifespan. The mechanisms of their generation and maintenance are
largely unknown. This study shows, by using RBP-Jkappa-/- embryonic stem cells in an embryonic stem cell-derived neurosphere assay, that neural stem cells are generated independent of RBP-Jkappa, a key molecule in Notch signaling. However, Notch pathway molecules are essential for the maintenance of neural stem cells; stem cells are depleted in the early embryonic brains of RBP-Jkappa-/- or Notch1-/- mice.
Neural stem cells also are depleted in embryonic brains deficient for
the presenilin1 (PS1) gene, a key regulator in Notch signaling, and are reduced in PS1+/- adult brains.
Both neuronal and glial differentiation in vitro are enhanced by
attenuation of Notch signaling and suppressed by expressing an active
form of Notch1. These data are consistent with a role for Notch
signaling in the maintenance of the neural stem cell, and inconsistent
with a role in a neuronal/glial fate switch (Hitoshi, 2002).
Historically, Notch signaling in Drosophila was thought to
maintain cells in an undifferentiated state. More recently, gain-of-function evidence in mammals has suggested that Notch signaling directly and
instructively induces glial differentiation. Some Notch-signaling loss-of-function studies in mammals seem consistent with this neuronal/glial fate switch idea, in that there is a premature appearance and increased number of postmitotic neurons expressing MAP2 or ßIII tubulin between E10.5 and E13.5 in the
PS1-/- brain. Similarly, mice with mutations in other Notch-signaling molecules such as Notch1, RBP-Jkappa, or Hes1/5 have revealed premature
neuronal differentiation. However, it is worth noting that such mice
with null mutations in Notch-signaling genes die in mid-to-late
embryogenesis, when neurogenesis predominates over gliogenesis in vivo. A clonal analysis of E10 cortical cells in vitro shows that neuronal differentiation from single neural stem cells precedes gliogenesis in clonal cell colonies. Thus, the in vivo analyses of Notch mutants may not allow sufficient time to assess whether
gliogenesis is increased or decreased (Hitoshi, 2002).
The present study of the loss-of-function and gain-of-function in Notch
pathway molecules in vitro reveals that the PS1 homozygous mutation drives E14.5 neural stem cells to differentiate both into more
neurons and more astroglia, and that the expression of the active form
of Notch1 suppresses the differentiation of postnatal neural stem-cell
progeny both into neurons and into astroglia. These findings are
therefore inconsistent with the idea that Notch signaling controls a
neuronal/glial fate switch of neural stem cells in the central nervous
system, although it remains possible that the
different times of the introduction of active Notch in neural stem
cells (and thus the different in vivo progenitor cell environments)
result in the apparently contradictory findings. These data are more
consistent with the idea that Notch signaling keeps cells in an
undifferentiated state. PS1-/- neural stem cells
have a greater probability of dividing asymmetrically to produce
neuronal progenitors early in vivo (and neuronal and glial progenitors
in vitro), rather than of dividing symmetrically to produce two
daughter neural stem cells as wild-type neural stem cells often do
during early embryogenic development. Hence, neuronal progenitor cells
in the PS1-/- brain may differentiate prematurely
from early asymmetric neural stem-cell divisions. Note that this
hypothesis of premature neuronal division as a by product of the
failure of symmetric divisions of forebrain neural stem cells with
deficits in Notch signaling can be seen as an alternative to the idea
that Notch signaling is directly and instructively involved in the fate
choice between neuronal and glial differentiation in the mammalian
central nervous system. These findings, therefore, are consistent with the idea of a primary defect in symmetric stem-cell self-renewal within the central nervous system (Hitoshi, 2002).
The gain-of-function study in vivo shows that enhanced Notch signaling
(by transducing an active form of Notch1 via retroviral infection)
increases the number of postnatal neural stem cells in the subependyma
of the forebrain lateral ventricle. The cells expressing the active
form of Notch1 shows self-renewal and multipotentiality, and, thus,
they are neural stem cells. These data suggest that Notch signaling
encourages neural stem cells to divide symmetrically to increase the
size of the neural stem-cell population, rather than to divide
asymmetrically to produce progenitor cells in the embryonic brain,
consistent with other gain-of-function studies showing that
constitutively active Notch signaling inhibits the differentiation of
neural progenitor cells in mammals (Hitoshi, 2002).
The role of Notch signaling on the generation of neurons and glia from neural stem cells was examined by using neurospheres that are clonally derived from neural stem cells. Neurospheres prepared from Dll1lacZ/lacZ mutant embryos deficient for Delta-like gene 1 segregate more neurons at the expense of both oligodendrocytes and astrocytes. This mutant phenotype could be rescued when Dll1lacZ/lacZ spheres were grown and/or differentiated in the presence of conditioned medium from wild-type neurospheres. Temporal modulation of Notch by soluble forms of ligands indicates that Notch signaling acts in two steps. Initially, it inhibits the neuronal fate while promoting the glial cell fate. In a second step, Notch promotes the differentiation of astrocytes, while inhibiting the differentiation of both neurons and oligodendrocytes (Grandbarbe, 2003).
As a result of Notch function, precursors are generated that are fated
either to a neuronal (P1) or a glial fate (P2). However, these precursors do
not necessarily give rise to the more mature cell type that expresses the
appropriate differentiation marker. The experimental temporal modulation of
Notch activity is consistent with the notion that neuron precursors, as well
as glial precursors, could be blocked in a non-differentiating state, and that
their further differentiation depends on secondary Notch signaling.
Neuronal precursors that were normally generated in
Dll1lacZ/lacZ mutant spheres, owing to the absence of
Notch activity during the proliferation phase, do not develop into
MAP2-expressing cells when Notch is activated during the differentiation phase. On the contrary, precursors that were fated to a glial cell type upon
transient activation of Notch will not differentiate into GFAP-expressing
astrocytes unless Notch is re-activated through the presence of soluble
ligand during the differentiation phase. It is assume that these cells, which are
blocked in a non-differentiated state, are likely to undergo cell death by
apoptosis, as usually described for cells that are misdirected and do not
differentiate properly (Grandbarbe, 2003).
In keeping with its role in the specification of cell types, Notch is
positively acting for the differentiation of astrocytes and negatively acting
for the differentiation of neurons. By contrast, Notch signaling has two
contradictory effects on the production of oligodendrocytes. In a first step
it acts positively to promote OPC production, whereas it negatively regulates
their subsequent differentiation into oligodendrocytes; however, only the
latter effect has been previously reported in other systems that were already
committed to the oligodendroglial lineage.
A model postulates that P2 is restricted to a glial fate with the
potential to differentiate into either astrocytes or oligodendrocytes. Owing
to the absence of specific markers, P2 cannot be identified in neurospheres.
The existence of such a precursor with both astrocytic and oligodendroglial
potential is controversial in vivo. The OPCs (formally called 0-2A) have long
been investigated and have been shown to differentiate in vitro (in the
presence of 10% FBS) into both oligodendrocytes and type II astrocytes that
are positive for both GFAP and A2B5. Cells were never observed with
characteristics of type II astrocytes. P2 is therefore different from PDGFR
cells, which, it is assumed, are already committed to an oligodendroglial lineage
and are likely, under the conditions employed, to give rise only to
04-expressing oligodendrocytes. Unfortunately, GFAP is likely to be a marker of astrocyte maturation rather
than of lineage commitment, thereby hindering the direct comparison of OPCs
with astrocyte precursors regarding Notch signaling. However, the observations
show that in no case were oligodendrocytes and astrocytes mutually exclusive
regarding Notch activation. It is therefore concluded that the segregation between
oligodendrocyte and astrocyte lineages is independent of Notch signaling and
might derive from another mechanism, involving for example the transcription
factors OLIG1 and OLIG2 (Grandbarbe, 2003).
Basic fibroblast growth factor (FGF2)-responsive definitive neural stem cells first appear in embryonic day 8.5 (E8.5) mouse embryos, but not in earlier embryos, although neural tissue exists at E7.5. Leukemia inhibitory factor-dependent (but not FGF2-dependent) sphere-forming cells are present in the earlier (E5.5-E7.5) mouse embryo. The resultant clonal sphere cells possess self-renewal capacity and neural multipotentiality, cardinal features of the neural stem cell. However, they also retain some nonneural properties, suggesting that they are the in vivo cells' equivalent of the primitive neural stem cells that form in vitro from embryonic stem cells. The generation of the in vivo primitive neural stem cell was independent of Notch signaling, but the activation of the Notch pathway was important for the transition from the primitive to full definitive neural stem cell properties and for the maintenance of the definitive neural stem cell state (Hitoshi, 2004).
In the mouse embryo deficient for Notch pathway molecules, the size of the definitive neural stem cell pool is reduced. Activation of Notch signaling is indispensable for maintaining the neural stem cell by enhancing its self-renewal
capacity and by repressing differentiation into progenitor cells. The current
results suggest an additional role for Notch signaling in neural stem
cell ontogenesis; activation of Notch pathway is required for the
transition from the primitive neural stem cell to the definitive neural
stem cell, which subsequently acquires EGF responsiveness. The reduction
of definitive neural stem cells observed in E8.0 Notch1-/- embryos in vivo, as well as the reduction seen in the EGF-responsive tertiary spheres from E7.5 Notch1-/- primitive neural stem cell spheres in
vitro, are consistent with this model. Certainly, the appearance of some
definitive neural stem cells in the E7.5 Notch-/-
embryos suggests that other signaling pathways may permit some
transition from primitive to definitive neural stem cells. However,
another model also is possible: definitive neural stem cells are
generated from the primitive neural stem cells independent of Notch
signaling, but definitive neural stem cells may require Notch signaling
for their maintenance, as suggested previously. These two possibilities
are not mutually exclusive and current data it is not yet possible to
discriminate between them. Later in development, Notch signaling may
play additional roles in enhancing the symmetric and self-renewing
divisions of definitive neural stem cells and suppressing asymmetric
division of neural stem cells to produce neuronal and glial progenitor
cells. This later function appears inconsistent with the
recent notion of an instructive role for Notch signaling to produce glia
from neural progenitor cells in the mammalian central nervous system.
However, this inconsistency disappears if adult forebrain neural stem
cells acquire some glial features (but do not differentiate into
unipotential glial cells), as suggested by demonstrations that Notch
signaling enhances glial fibrillary acidic protein (GFAP) transcription in adult neural progenitor cells and that at least some of the GFAP-expressing astrocytes in the adult forebrain subependyma are, indeed, neural stem cells (Hitoshi, 2004).
During development of the mammalian brain, many neural precursor cells (NPCs) undergo apoptosis. The regulation of such cell death, however, is poorly understood. The survival of mouse embryonic NPCs in vitro is increased by culture at a high cell density and this effect is attributable to activation of Notch signaling. Expression of an active form of Notch1 thus markedly promotes NPC survival. Hes proteins, key effectors of Notch signaling in inhibition of neurogenesis, are not sufficient for the survival-promoting effect of Notch1. This effect of Notch1 requires a region of the protein containing the RAM domain and is accompanied by up-regulation of the anti-apoptotic proteins Bcl-2 and Mcl-1. Moreover, knockdown of these proteins by RNA interference results in blockade of the Notch1-induced survival. These results reveal a new function of Notch, the promotion of NPC survival (Oishi, 2004).
Radial glia function during CNS development both as neural progenitors and as a
scaffolding supporting neuronal migration. To elucidate pathways involved in
these functions, the promoter for Blbp, a radial glial gene, was mapped in vivo. A binding site for the Notch effector CBF1 is essential for
all Blbp transcription in radial glia, and BLBP expression is significantly
reduced in the forebrains of mice lacking the Notch1 and Notch3 receptors. These
results identify Blbp as the first predominantly CNS-specific Notch target gene
and suggest that it mediates some aspects of Notch signaling in radial glia (Anthony, 2004).
The identification of Blbp as a Notch target indicates that the role of Notch signaling in neural progenitors varies as development proceeds, and that the
spectrum of downstream target genes change. Blbp
transcription in the neocortex does not begin until the onset of neurogenesis at
E10.5, and significant levels of BLBP protein are not detectable in this
region until E12.5. In contrast, neocortical expression of
Hes5 is detectable as early as E9.5, and high levels of
both Notch1 and Hes5 are present by E10.5. These data
demonstrate that distinct developmental stages are accompanied by distinct
patterns of Notch target gene expression. These shifts in Notch target gene
expression appear to be mediated by additional regulatory factors that interact
with the CBF1 coactivator complex; the existence of these additional factors is
evident from findings that the CBF1-binding site within the Blbp promoter is
necessary but not sufficient for transcription, and that multiple promoter
elements mediate transcription of Blbp at different times and places. POU domain
transcription factors have been implicated in regulating Blbp
transcription in the embryonic forebrain, and thus
represent one possible class of CBF1-interacting proteins that function in
radial glia. Interestingly, Notch and the POU domain protein Nubbin positively
interact to promote gliogenesis in certain Drosophila cell lineages.
The fact that constitutively active Notch could promote glial fate in
many but not all murine radial glia may reflect a
dependence of Notch signaling on other factors such as POU domain proteins.
Additional candidates include factors downstream of Neuregulin and Reelin; these
signaling molecules have been shown to induce radial glial expression
of BLBP (Anthony, 2004).
Finally, it is noted that whereas radial glia serve as neuronal progenitors,
Bergmann glia do not. This suggests that in addition to its well-documented role
as a cell fate regulator, Notch signaling may also function to support neuronal
migration. Previous studies have demonstrated that Notch signaling promotes a
radial glial phenotype in the forebrain,
radializes cerebellar astrocytes, and induces expression of
ErbB2, a receptor implicated in radial glial differentiation. Since
antibody blocking experiments have implicated BLBP
in regulating glial morphology,
the available data suggest that Notch signaling may induce and/or
maintain the radial glial scaffold, and that it does so in part through its
induction of BLBP. Further insight into the mechanisms regulating radial glial
function will likely be gained by the elucidation of BLBP function as well as
the identification of additional radial glial Notch target genes (Anthony, 2004).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Notch
continued:
Biological Overview
| Regulation
| Protein Interactions | Post-transcriptional regulation of Notch mRNA
| Developmental Biology
| Effects of Mutation
| References
Society for Developmental Biology's Web server.