Interactive Fly, Drosophila

Notch


EVOLUTIONARY HOMOLOGS


Table of contents

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 IC’s 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).

The hope of developing new transplantation therapies for degenerative diseases is limited by inefficient stem cell growth and immunological incompatibility with the host. This study shows that Notch receptor activation induces the expression of the specific target genes hairy and enhancer of split 3 (Hes3) and Sonic hedgehog (Shh) through rapid activation of cytoplasmic signals, including the serine/threonine kinase Akt, the transcription factor STAT3 and mammalian target of rapamycin, and thereby promotes the survival of neural stem cells. In both murine somatic and human embryonic stem cells, these positive signals are opposed by a control mechanism that involves the p38 mitogen-activated protein kinase. Transient administration of Notch ligands to the brain of adult rats increases the numbers of newly generated precursor cells and improves motor skills after ischaemic injury. These data indicate that stem cell expansion in vitro and in vivo, two central goals of regenerative medicine, may be achieved by Notch ligands through a pathway that is fundamental to development and cancer (Androutsellis-Theotokis, 2006).

Notch and testis development

During testis development, fetal Leydig cells increase their population from a pool of progenitor cells rather than from proliferation of a differentiated cell population. However, the mechanism that regulates Leydig stem cell self-renewal and differentiation is unknown. This study shows that blocking Notch signaling, by inhibiting gamma-secretase activity or deleting the downstream target gene Hairy/Enhancer-of-split 1, results in an increase in Leydig cells in the testis. By contrast, constitutively active Notch signaling in gonadal somatic progenitor cells causes a dramatic Leydig cell loss, associated with an increase in undifferentiated mesenchymal cells. These results indicate that active Notch signaling restricts fetal Leydig cell differentiation by promoting a progenitor cell fate. Germ cell loss and abnormal testis cord formation were observed in both gain- and loss-of-function gonads, suggesting that regulation of the Leydig/interstitial cell population is important for male germ cell survival and testis cord formation (Tang, 2008).

Table of contents


Notch continued: Biological Overview | Regulation | Protein Interactions | Post-transcriptional regulation of Notch mRNA | Developmental Biology | Effects of Mutation | References

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