Notch

EVOLUTIONARY HOMOLOGS

Table of contents

Notch, myeloid cells and the immune response

A comparison has been made of the ability of two mammalian Notch homologs, mouse Notchl and Notch2, to inhibit the granulocytic differentiation of 32D myeloid progenitor cells. 32D cells undergo granulocytic differentiation when stimulated with either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression of the activated intracellular domain of Notch1 inhibits the differentiation induced by G-CSF but not by GM-CSF; conversely, the corresponding domain of Notch2 inhibits differentiation in response to GM-CSF but not to G-CSF. The region immediately C-terminal to the cdc10 domain of Notch confers cytokine specificity on the cdc10 domain. The cytokine response patterns of Notch1 and Notch2 are transferred with this region, which is here termed the Notch cytokine response (NCR) region. The NCR region is also associated with differences in posttranslational modification and subcellular localization of the different Notch molecules. These findings suggest that the multiple forms of Notch found in mammals have structural differences that allow their function to be modulated by specific differentiation signals (Bigas, 1998).

Notch influences the choice between CD4 and CD8 T cell lineages. In the thymus, developing T cells rearrange and express their T cell antigen receptor genes and undergo a testing process based on the ability of their antigen receptors to recognize major histocompatability complex (MHC) proteins expressed on thymic epithelial cells. The interaction between developing thymocytes and thymic epithelial cells promotes the survival of thymocytes and also directs their lineage choice. Thymocytes whose receptors recognize class I MHC proteins develop as CD8 cells, whereas thymocytes whose antigen receptors recognize class II MHC proteins develop as CD4 cells. Expression of an activated form of Notch1 in developing T cells of the mouse leads to both an increase in CD8 lineage T cells and a decrease in CD4 lineage T cells. Expression of activated Notch permits the development of mature CD8 lineage thymocytes even in the absence of class I MHC proteins, ligands that are normally required for the development of these cells. However, activated Notch is not sufficient to promote CD8 cell development when both class I and class II MHC are absent. Thus Notch is a participant in the CD4 versus CD8 lineage decision (Robey, 1996).

The choice between the alphabeta or gammadelta T cell fates is influenced by the production of functional, in-frame rearrangements of the TCR genes, but the mechanism that controls the lineage choice is not known. T cells that are heterozygous for a mutation of the Notch1 gene are more likely to develop as gammadelta T cells than as alphabeta T cells, implying that reduced Notch activity favors the gammadelta T cell fate over the alphabeta T cell fate. A constitutively activated form of Notch produces a reciprocal phenotype and induces thymocytes that have functional gammadeltaTCR gene rearrangements to adopt the alphabeta T cell fate. These data indicate that Notch acts together with the newly formed T cell antigen receptor to direct the alphabeta versus gammadelta T cell lineage decision. Possibly, thymocytes that successfully rearrange their gammadelta TCR genes might induce their neighbors to develop as alphabeta T cells via a Notch signal, thus maintaining a feedback mechanism that directs neighboring thymocytes to adopt distinct fates (Washburn, 1997).

The multiplicity of Notch receptors raises the question of the contribution of specific isoforms to T-cell development. Notch3 is expressed in CD4^-8^- thymocytes and is down-regulated across the CD4^-8^- to CD4⁺8⁺ transition, controlled by pre-T-cell receptor signaling. To determine the effects of Notch3 on thymocyte development, transgenic mice were generated, expressing lck promoter-driven intracellular Notch3. Thymuses of young transgenics show an increased number of thymocytes, particularly late CD4^-8^- cells, a failure to down-regulate CD25 in post-CD4^-8^- subsets and sustained activity of NF-kappaB. Subsequently, aggressive multicentric T-cell lymphomas develop with high penetrance. Tumors sustain characteristics of immature thymocytes, including expression of CD25, pTalpha and activated NF-kappaB via IKKalpha-dependent degradation of IkappaBalpha and enhancement of NF-kappaB-dependent anti-apoptotic and proliferative pathways. Together, these data identify activated Notch3 as a link between signals leading to NF-kappaB activation and T-cell tumorigenesis. The phenotypes of pre-malignant thymocytes and of lymphomas indicate a novel and particular role for Notch3 in co-ordinating growth and differentiation of thymocytes, across the pre-T/T cell transition, consistent with the normal expression pattern of Notch3 (Bellavia, 2000).

The Notch signaling pathway regulates the commitment and early development of T lymphocytes. Notch-mediated induction of the pre-T cell receptor alpha (pTa) gene, a T-cell-specific transcriptional target of Notch, was studied. pTa encodes a transmembrane protein that pairs with the newly rearranged TCRß chain to form the essential pre-TCR signaling complex in the developing T cells. The pTa gene is expressed in immature T cells, and its up-regulation coincides with irreversible T cell lineage commitment in both murine and human thymic precursors. Moreover, pTa is required for alphaß but not gammadelta T cell development, and may facilitate the alphaß lineage commitment by providing an instructive signal from the pre-TCR. The pTa enhancer is activated by Notch signaling and contains binding sites for its nuclear effector, CSL. Mutation of the CSL-binding sites abolishes enhancer induction by Notch and delays the up-regulation of pTa transgene expression during T cell lineage commitment. These results show a direct mechanism of stage- and tissue-specific gene induction by the mammalian Notch/CSL signaling pathway (Reizis, 2002).

Both the Notch and TCR signaling pathways play an important role in T cell development, but the links between these signaling pathways are largely unexplored. The adapter protein Numb is a well-characterized inhibitor of Notch and also contains a phosphotyrosine binding domain, suggesting that Numb could provide a link between these pathways. This possibility was explored by investigating the physical interactions among Notch, Numb, and the TCR signaling apparatus and by examining the consequences of a Numb mutation on T cell development. Notch and Numb cocluster with the TCR at the APC contact during Ag-driven T cell-APC interactions in both immature and mature T cells. Furthermore, Numb coimmunoprecipitates with components of the TCR signaling apparatus. Despite this association, T cell development and T cell activation occur normally in the absence of Numb, perhaps due to the expression of the related protein, Numblike. Together these data suggest that Notch and TCR signals may be integrated at the cell membrane, and that Numb may be an important adapter in this process (Anderson, 2005).

Identifying the molecular pathways regulating hematopoietic stem cell (HSC) specification, self-renewal, and expansion remains a fundamental goal of both basic and clinical biology. This study analyzes the effects of Notch signaling on HSC number during zebrafish development and adulthood, defining a critical pathway for stem cell specification. The Notch signaling mutant mind bomb displays normal embryonic hematopoiesis but fails to specify adult HSCs. Surprisingly, transient Notch activation during embryogenesis via an inducible transgenic system leads to a Runx1-dependent expansion of HSCs in the aorta-gonad-mesonephros (AGM) region. In irradiated adults, Notch activity induces runx1 gene expression and increases multilineage hematopoietic precursor cells approximately threefold in the marrow. This increase is followed by the accelerated recovery of all the mature blood cell lineages. These data define the Notch-Runx pathway as critical for the developmental specification of HSC fate and the subsequent homeostasis of HSC number, thus providing a mechanism for amplifying stem cells in vivo (Burns, 2005).

The adult stem cell niche has been characterized in the mouse bone marrow and consists of an endosteal (quiescent) and vascular (proliferative) compartment. Under steady-state conditions, it is thought that most HSCs reside in the G₀ phase of the cell cycle in close contact with stromal cells, including osteoblasts. The balance between quiescent and cycling stem cells appears to rely on the amount of soluble cytokines, which result in HSCs relocating from the osteoblastic to the vascular niche. This mobilization of stem cells into peripheral circulation may be necessary for reconstituting the HSC pool. Many signaling pathways are thought to contribute to stem cell self-renewal in the marrow niche including Notch, Wnt, Hedgehog, and factors that negatively regulate the cell cycle, such as Tie2/Angiopoietin-1. Cooperation of such pathways is thought to maintain stem cell homeostasis in vivo (Burns, 2005).

Several studies have hypothesized that Notch affects HSCs, although direct proof of the activity and the downstream targets have remained to be elucidated. In murine cell culture, constitutive Notch1 expression in HSC/progenitor cells establishes immortalized cell lines able to generate progeny with either lymphoid or myeloid characteristics. Retroviral Notch1 activation in recombination activating gene-1 (RAG-1)-deficient mouse stem cells results in an increase in HSC self-renewal and favors lymphoid over myeloid differentiation (Burns, 2005).

The studies presented here differ from others in that a brief pulse of Notch activity was administered and the cells were able to terminally differentiate. Other experiments with retroviruses and conditional alleles permanently express NICD and thus alter the normal maturation of cells. For instance, in adult assays an increase in the lymphoid cell fate was not concomitant with a decrease in the myeloid lineage, as previously seen. Based on these results, it is proposed that activated Notch expands the stem and progenitor cell compartment by either influencing undifferentiated cells to adopt a HSC fate or by causing a G₀ HSC population to up-regulate runx1-dependent gene expression (Burns, 2005).

These findings that the stem cell markers runx1, scl, and lmo2 are transcriptionally increased in response to NICD indicates that stem and progenitor cells are expanded in the adult marrow, possibly by increasing stem cell self-renewal. Recently, a conditional allele of runx1 was generated in the mouse to study the loss of Runx1 function during adult hematopoiesis. In transplantation studies, Runx1-excised marrow cells show a reduced competitive repopulating ability in long-term engraftment assays, demonstrating that Runx1 is essential for normal stem cell function. The NICD-induced expansion of HSCs in the AGM is dependent on Runx1. The proximal and distal promoters of the human runx1 gene were examined, no DNA-binding sites for RBPjkappa, the primary Notch pathway mediator that physically interacts with DNA to modulate target gene transcription, were found. It is still possible that Notch directly regulates runx1 transcription through alternative binding sites, although it may indirectly activate runx1 expression. In either case, the Notch-Runx pathway is likely operative in both the AGM and adult marrow and may lead to the activation of downstream targets critical for stem cell homeostasis (Burns, 2005).

Notch signaling has been extensively linked to the process of both normal and aberrant stem cell self-renewal. The human Notch1 receptor, TAN-1, was first identified as a partner gene in a (7;9) chromosomal translocation found in <1% of all T-cell acute lymphoblastic leukemias (T-ALL) . Recently, >50% of all human T-ALLs were shown to have activating mutations in the notch1 gene. These data emphasize how dysregulation of the Notch signaling pathway can result in uncontrolled self-renewal that ultimately produces malignancy (Burns, 2005).

Transplantation of HSCs has been successful in the treatment of malignancies and other diseases, such as aplastic and sickle-cell anemia. After irradiation or chemotherapy is given to patients, restoration of normal hematopoiesis is critical to prevent infection and bleeding. This study has shown that a pulse of Notch activity expands stem cell number in the adult marrow without permanently altering blood lineage homeostasis. This finding has obvious therapeutic implications. Small molecule agonists that induce Notch signaling could be used to pharmacologically expand stem cell numbers and blood progenitors. For instance, embryonic cord blood stem cells are often insufficient for adult stem cell transplants. Notch activators may be used to increase mobilization of HSCs for transplantation, similar to the clinical activity of G-CSF in peripheral stem cell harvests. These data provide rationale for future clinical work to focus on methods that manipulate the Notch signaling pathway to amplify blood stem cells, and thus multilineage hematopoiesis (Burns, 2005).

Notch and vascular morphogenesis

To assess the function of the Notch4 gene, Notch4-deficient mice were generated by gene targeting. Embryos homozygous for this mutation develop normally, and homozygous mutant adults are viable and fertile. However, the Notch4 mutation displays genetic interactions with a targeted mutation of the related Notch1 gene. Embryos homozygous for mutations of both the Notch4 and Notch1 genes often displayed a more severe phenotype than Notch1 homozygous mutant embryos. Both Notch1 mutant and Notch1/Notch4 double mutant embryos display severe defects in angiogenic vascular remodeling. Analysis of the expression patterns of genes encoding ligands for Notch family receptors indicate that only the Dll4 gene is expressed in a pattern consistent with that expected for a gene encoding a ligand for the Notch1 and Notch4 receptors in the early embryonic vasculature. These results reveal an essential role for the Notch signaling pathway in regulating embryonic vascular morphogenesis and remodeling, and indicate that whereas the Notch4 gene is not essential during embryonic development, the Notch4 and Notch1 genes have partially overlapping roles during embryogenesis in mice (Krebs, 2000).

Targeted mutations in components of a variety of signaling pathways (e.g., vascular endothelial growth factors, TGF-beta1, angiopoietins, ephrins) have been shown to regulate vascular morphogenesis in mice. Another major intercellular signaling pathway, the Notch pathway, also regulates vascular morphogenesis and angiogenic vascular remodeling. Vascular defects are observed in the placenta, yolk sac, and embryo proper of both Notch1^-/- mutant and Notch1^-/- Notch4^-/- double mutant embryos. In the yolk sac of the mutant embryos, the primary vascular plexus formed normally, indicating that there are no apparent defects in vasculogenesis in the mutants. However, both Notch1^-/- mutant and Notch1^-/- Notch4^-/- double mutant embryos fail to remodel the primary vascular plexus to form the large vitelline blood vessels, a process that occurs by angiogenesis. Defects in angiogenesis are also apparent in the placenta, where embryonic blood vessels fail to invade the placental labyrinth. In the embryo proper, defects in angiogenesis and vascular remodeling are apparent as malformations of major vessels such as the dorsal aortae, the anterior cardinal veins, and the intersomitic blood vessels (Krebs, 2000).

The appearance of molecular differences between arterial and venous endothelial cells before circulation suggests that genetic factors determine these cell types. vascular endothelial growth factor (vegf), synthesized by somites, acts downstream of the notochordal sonic hedgehog signal and upstream of the Notch pathway to determine arterial cell fate. Loss of Vegf or Shh results in loss of arterial identity, while exogenous expression of these factors causes ectopic expression of arterial markers. Microinjection of vegf mRNA into embryos lacking Shh activity can rescue arterial differentiation. Finally, activation of the Notch pathway in the absence of Vegf signaling can rescue arterial marker gene expression. These studies reveal a complex signaling cascade responsible for establishing arterial cell fate and suggest differential effects of Vegf on developing endothelial cells (Lawson, 2002).

Recent evidence indicates that growing blood-vessel sprouts consist of endothelial cells with distinct cell fates and behaviours; however, it is not clear what signals determine these sprout cell characteristics. This study shows that Notch signalling is necessary to restrict angiogenic cell behaviour to tip cells in developing segmental arteries in the zebrafish embryo. In the absence of the Notch signalling component Rbpsuh (recombining binding protein suppressor of hairless) excessive sprouting of segmental arteries is observed, whereas Notch activation suppresses angiogenesis. Through mosaic analysis it was found that cells lacking Rbpsuh preferentially localize to the terminal position in developing sprouts. In contrast, cells in which Notch signalling has been activated are excluded from the tip-cell position. In vivo time-lapse analysis reveals that endothelial tip cells undergo a stereotypical pattern of proliferation and migration during sprouting. In the absence of Notch, nearly all sprouting endothelial cells exhibit tip-cell behaviour, leading to excessive numbers of cells within segmental arteries. Furthermore, flt4 (fms-related tyrosine kinase 4, also called vegfr3) is expressed in segmental artery tip cells and becomes ectopically expressed throughout the sprout in the absence of Notch. Loss of flt4 can partially restore normal endothelial cell number in Rbpsuh-deficient segmental arteries. Finally, loss of the Notch ligand dll4 (delta-like 4) also leads to an increased number of endothelial cells within segmental arteries. Together, these studies indicate that proper specification of cell identity, position and behaviour in a developing blood-vessel sprout is required for normal angiogenesis, and implicate the Notch signalling pathway in this process (Siekmann, 3007).

Notch pathway and somitogenesis

During development, Hox gene transcription is activated in presomitic mesoderm with a time sequence that follows the order of the genes along the chromosome. Hoxd1 and other Hox genes display dynamic stripes of expression within presomitic mesoderm. The underlying transcriptional bursts may reflect the mechanism that coordinates Hox gene activation with somitogenesis. This mechanism appears to depend upon Notch signaling, because mice deficient for RBPJk, the effector of the Notch pathway, show severely reduced Hoxd gene expression in presomitic mesoderm. These results suggest a molecular link between Hox gene activation and the segmentation clock. Such a linkage would efficiently keep in phase the production of novel segments with their morphological specification (Zakany, 2001).

Transcriptional bursts in Hox gene expression in forming somites suggest how Hox complexes integrate a temporal parameter. Cells reaching the region where epithelial somites form (S-I) may respond to a localized signal by activating all Hox genes transcriptionally available. Consequently, the earliest burst would activate only group 1 genes; the subsequent burst (one somite-time later) would activate both group 1 and group 2 genes, etc., leading to a temporal coordination between somite formation and Hox gene activation. This view, however, doesn't suggest any mechanism whereby this oscillating time signal could be transformed into a linear activation of the clusters, i.e., how successive bursts would progressively activate more genes in a colinear fashion. Cells located at the more posterior level at time t3 would thus activate two more Hox genes than cells which were at the same more posterior level but at t1 (two segmentation cycles earlier). This requires that the accessibility of Hox genes be progressively increased within posterior presomitic mesoderm, rather than in the region of the stripes. This increased accessibility must occur either soon after gastrulation, i.e., within the pool of mesoderm cells that will produce the PSM, or during the time cells stay in PSM before reaching the more posterior level. Because many Hox genes are already expressed throughout the PSM, a view is favored whereby mesoderm cells are acquiring their 'state of opening' early on during gastrulation. This would uncouple the transcriptional activation of Hox genes from the mechanism that would regulate their accessibility and would give two temporal components to colinearity: (1) a progressive opening, which may rely upon the release of a silencing mechanism, followed by (2) time-dependent bursts of activation. In this context, PSM cells would express some background level of Hox gene products, due to the opening of the complex, and this expression would be coordinated in time by strong bursts of activation, whenever cells would approach the PSM to SM transition. These bursts would genetically 'label' the newly formed somite and imprint its morphological fate (Zakany, 2001).

In the absence of RBPJk function, expression of both Hoxd1 and Hoxd3 could hardly be detected in presomitic and somitic mesoderm, whereas expression in lateral plate mesoderm (for Hoxd1) and in the CNS (for Hoxd3) remain unchanged. This result is reminiscent of the loss of Lfng transcription in these same mutants and suggests that Hox gene may be under the control of the Notch pathway. Since the first establishment of the segmental pattern seems to occur at the level of Mesp2 (for mesoderm posterior 2), and because this latter gene is controlled by Notch signaling, the possibility exists that Hoxd1 activation be downstream of bHLH protein Mesp2 in S-I. This is supported by the apparent coordination of both expression patterns. In this view, the recurrent activation of the Hox system at the presomitic to somitic boundary would respond to a cyclic exposure to the outcome of the Notch pathway. Accordingly, the Notch-dependent coordination of the segmental pattern would be linked to the timing of activation, or enhanced transcription, of the Hox gene family (Zakany, 2001).

The recurrent activation of anterior Hox genes in PSM indicates that the segmentation mechanism, or the mechanism involved in somite boundary formation, may trigger or coordinate the activation of the Hox system. Interestingly, the expression of Lfng, a modulator of Notch signaling cycles in a way resembling the transcriptional oscillations of the chicken c-hairy-1 and -2 genes. These latter genes were proposed to be part of, or tightly linked to, the molecular oscillator underlying the segmental clock. Mutations of genes in the Notch pathway, such as the Notch genes themselves, Delta-like genes, RBPJk, Lfng, Mesp-2, and Presenilin-1, induce strong alterations of the segmental pattern, confirming their function in this fundamental process. In addition, cyclic expression of Lfng in RBPJk mutants is drastically reduced, and so is that of Hes1 in Dll1 mutants, suggesting a causal role for Notch signaling in the segmentation clock itself (Zakany, 2001).

Tail bud formation in Xenopus depends on interaction between a dorsal domain (dorsal roof) expressing lunatic fringe and Notch, and a ventral domain (posterior wall) expressing the Notch ligand Delta. Ectopic expression of an activated form of Notch, Notch ICD, by means of an animal cap graft into the posterior neural plate, results in the formation of an ectopic tail-like structure containing a neural tube and fin. However, somites are never formed in these tails. BMP signaling is activated in the posterior wall of the tail bud and is involved in the formation of tail somites from this region. Grafts into the posterior neural plate, in which BMP signaling is activated, will form tail-like outgrowths. Unlike the Notch ICD tails, the BMP tails contain well-organized somites as well as neural tube and fin, with the graft contributing to both somites and neural tube. Through a variety of epistasis-type experiments, it has been shown that the most likely model involves a requirement for BMP signaling upstream of Notch activation, resulting in formation of the secondary neural tube, as well as a Notch-independent pathway leading to the formation of tail somites from the posterior wall (Beck, 2001).

Somite formation is thought to be regulated by an unknown oscillator mechanism that causes the cells of the presomitic mesoderm to activate and then repress the transcription of specific genes in a cyclical fashion. These oscillations create stripes/waves of gene expression that repeatedly pass through the presomitic mesoderm in a posterior-to-anterior direction. In both the mouse and the zebrafish, it has been shown that the notch pathway is required to create the stripes/waves of gene expression. However, it is not clear if the notch pathway comprises part of the oscillator mechanism or if the notch pathway simply coordinates the activity of the oscillator among neighboring cells. In the zebrafish, oscillations in the expression of a hairy-related transcription factor, her1 and the notch ligand deltaC precede somite formation. This study focuses on how the oscillations in the expression of these two genes areaffected in the mutants aei/deltaD and des/notch1, in 'morpholino knockdowns' of deltaC and her1 and in double 'mutant' combinations. This analysis indicates that these oscillations in gene expression are created by a genetic circuit comprised of the notch pathway and the notch target gene her1. A later function of the notch pathway can create a segmental pattern even in the absence of prior oscillations in her1 and deltaC expression (Holley, 2002).

Both aei/deltaD and des/Notch1 are necessary to promote the expression of the oscillating genes her1 and deltaC. Meanwhile, her1 regulates deltaC expression and functions, directly or indirectly, in a negative feedback loop to repress its own transcription. Thus, the notch pathway functions upstream of her1 to promote the transcription of her1 mRNA, and her1 functions upstream of the Notch pathway to create the oscillating pattern of deltaC transcription. This identifies a rudimentary genetic loop (notch pathway > her1 > notch pathway) that functions within the PSM. Further, fused somites (fss) functions downstream of the notch pathway but upstream of her1 in the anterior PSM, and the notch pathway and fss function downstream of her1 slightly later in the anteriormost PSM. Therefore, the regulatory circuit consisting of her1 and the notch pathway exists throughout the PSM. Because this genetic circuit comprises genes that are required to create the oscillations in gene expression, these findings suggest that her1 and the notch pathway have cyclical functions at the center of the somitogenesis oscillator (Holley, 2002).

The genetic analysis of her1 and the notch pathway suggest a model in which these genes somehow generate the oscillations in gene expression. The initiation of the oscillations may be coupled to the commitment to become paraxial mesoderm. The expression of each of these genes (her1, deltaC, aei/deltaD and des/notch1) is initiated at the tip of the tailbud as cells subduct to form the paraxial mesoderm. The subsequent activities of these proteins could then initiate the interactions that create the oscillations in gene expression. deltaC, aei/deltaD and des/notch1 signaling would activate the transcription of her1 and deltaC. The subsequent increase in Her1 protein would then act to block the transcription of her1. Since the hairy proteins typically function as transcriptional repressors, an increase in Her1 should result in an increase in repressive activity, and the gradual degradation of this protein would produce a gradual decrease in this repressive activity. Therefore, the anterior progression/activation of a stripe of gene expression could be driven by the gradual loss of a repressive activity generated during the previous somite cycle. The positive regulation via notch could also display a cyclical variation, but ultimately the re-initiation of her1 and deltaC transcription would not occur until the level of Her1 drops below a specific threshold. In essence, this model suggests that the anterior progression of a stripe of gene expression is, at least in part, driven by the degradation of an existing, repressive activity (Her1), as opposed to the de novo synthesis of an activating component (Holley, 2002).

The analysis of deltaC expression in her1^mo embryos uncovers an additional Notch-dependent patterning activity in the anterior PSM. This activity can create a segmental pattern of gene expression in the absence of any evidence of oscillations in her1 and deltaC expression: a smooth domain of deltaC expression is refined anteriorly to create stripes of expression that persist in the somitic mesoderm. This refinement requires the activity of fss, aei/deltaD, des/notch1, deltaC and beamter (bea), indicating that each of these genes has an additional function in the anterior-most PSM, downstream of her1. This is consistent with the fact that aei/deltaD, deltaC and des/notch1 are each transcribed within the PSM and later in the somitic mesoderm. In fact, this refining pattern is likely to be revealed only within the her1^mo embryos because her1 is the only one of these cloned genes whose expression is restricted to the PSM. Ultimately, this indicates that the phenotypes observed in aei/deltaD and des/notch1 embryos are composites of defects that occur both upstream and downstream of her1 (oscillator) function. It has been shown that notch pathway signaling is involved in establishing the anteroposterior pattern within each somite. The late activity of the notch pathway described here probably represents this same anteroposterior patterning function. What is remarkable is that this late function can create a segmental pattern in the absence of prior oscillations in her1 and deltaC expression (Holley, 2002).

The expression of a Hairy/E(spl)-related (Her) gene, her7, has been studied in the zebrafish; its expression in the presomitic mesoderm cycles similarly to her1 and deltaC. A decrease in her7 function generated by antisense oligonucleotides disrupts somite formation in the posterior trunk and tail, and disrupts the dynamic expression domains of her1 and deltaC, suggesting that her7 plays a role in coordinating the oscillations of neighboring cells in the presomitic mesoderm. This phenotype is reminiscent of zebrafish segmentation mutants with lesions in genes of the Delta/Notch signaling pathway, which also show a disruption of cyclic her7 expression. The interaction of HER genes with the Delta/Notch signaling system was investigated by introducing a loss of her7 function into mutant backgrounds. This leads to segmental defects more anterior than in either condition alone. Combining a decrease of her7 function with reduction of her1 function results in an enhanced phenotype that affects all the anterior segments, indicating that Her functions in the anterior segments are also partially redundant. In these animals, gene expression does not cycle at any time, suggesting that a complete loss of oscillator function had been achieved. Consistent with this, combining a reduction of her7 and her1 function with a Delta/Notch mutant genotype does not worsen the phenotype further. Thus, these results identify members of the Her family of transcription factors that together behave as a central component of the oscillator, and not as an output. This indicates, therefore, that the function of the segmentation oscillator is restricted to the positioning of segmental boundaries. Furthermore, these data suggest that redundancy between Her genes and genes of the Delta/Notch pathway is in part responsible for the robust formation of anterior somites in vertebrates (Oates, 2002).

Boundary formation plays a central role in differentiating the flanking regions that give rise to discrete tissues and organs during early development. Mechanisms by which a morphological boundary and tissue separation are regulated have been studied by examining chicken somite segmentation as a model system. By transplanting a small group of cells taken from a presumptive border into a non-segmentation site, a novel inductive event has been found where posteriorly juxtaposed cells to the next-forming border instruct the anterior cells to become separated and epithelialized. The molecular mechanisms underlying these interactions was further studied by focusing on Lunatic fringe, a modulator of Notch signaling, which is expressed in the region of the presumptive boundary. By combining DNA in ovo electroporation and embryonic transplantation techniques, a sharp boundary of Lunatic fringe activity has been ectopically made in the unsegmented paraxial mesoderm and a fissure formed at the interface has been observed. In addition, a constitutive active form of Notch mimics this instructive phenomenon. These suggest that the boundary-forming signals emanating from the posterior border cells are mediated by Notch, the action of which is confined to the border region by Lunatic fringe within the area where mRNAs of Notch and its ligand are broadly expressed in the presomitic mesoderm (Sato, 2002).

In the anterior end of the unsegmented paraxial mesoderm (presomitic mesoderm: PSM), an expression boundary of genes exists, including MesP2, a novel mouse gene expressed in the presegmented mesoderm and essential for segmentation initiation. This expression border, which coincides with the next border being formed, is established prior to a morphological change. The segmental patterns of these genes are thought to be regulated by a 'segmentation clock', first demonstrated by wavy and cyclic expression of c-hairy1. Thus, the segmentation clock operates in the continuous young PSM to establish the segmental patterns of gene expression in the anterior PSM, which eventually implements a morphological fissure formation. Both clock and segmentation genes are tightly related to Notch signaling, as revealed mainly by recent knockout and mutant studies: an animal where Notch signaling is (at least in part) deficient displays perturbed patterns of cyclic and segmental expression of genes in PSM, and also shows its consequent malformation of segmented structures later in development. In general, studies using mutants or knockout animals unveil the 'first stage' where the gene of concern is essential during development. However, if a given gene plays a role in the fissure formation as well as at earlier steps of segmentation, it would be difficult to distinguish between them. This may be the reason why the molecular mechanisms underlying the fissure formation have been poorly addressed (Sato, 2002).

A novel inductive event is described that takes place when a segmentation fissure forms. In this event posterior border cells located immediately posterior to the next forming boundary instruct the anterior ones. Molecular mechanisms underlying these events are addressed by focusing on Notch signals where Lunatic fringe (Lfng) is involved. Lfng is a modulator of the Notch receptor with glycosyltransferase activity, and is expressed in a region coinciding with the segmentation border in PSM. By combining DNA in ovo electroporation with embryological manipulations to make an ectopic boundary of a transgene activity in PSM, it was found that Notch signals play major roles in the formation of a fissure. A model is presented in which specific localization of Lfng determines the site of Notch action relevant to the morphological segmentation (Sato, 2002).

Alterations of the Delta/Notch signalling pathway cause multiple morphogenetic abnormalities in somitogenesis, including defects in intersomitic boundary formation and failure in maintenance of somite regularity. Notch signalling has been implicated in establishing the anteroposterior polarity within maturing somites and in regulating the activity of a molecular segmentation clock operating in the presomitic mesoderm. The pleiotropy of Notch signalling obscures the roles of this pathway in different steps of somitogenesis. One possibility is that distinct Notch effectors mediate different aspects of Notch signalling. In this study, focus was placed on two zebrafish Notch-dependent hairy/Enhancer-of-split-related transcription factors, Her6 and Her4, which are expressed at the transition zone between presomitic mesoderm and the segmented somites. The results of overexpression/gain-of-function and of morpholino-mediated loss-of-function experiments show that Her6 and Her4 are Notch signalling effectors that feedback on the clock and take part in the maintenance of cyclic gene expression coordination among adjacent cells in the presomitic mesoderm. Her6 and Her4 are necessary for normal paraxial mesoderm segmentation and the activities of their protein products are required to maintain synchronization of the cyclical expression of both deltaC and her1 (Pasini, 2004).

During most of somitogenesis, expression of her6 is confined to two stripes in the anterior PSM and to the posterior compartment of the mature somites. Expression in the tailbud is only observed early during somitogenesis and no expression is detected in the intermediate PSM at any stage. Although the two her6 stripes in the anterior PSM show some variability in their strength, their distances from one another, from the myod stripes in the PSM or from the last formed intersomitic cleft do not vary among embryos with the same number of somites. In addition to this, and in contrast to the cycling zebrafish hairy/E(Spl)-related genes her1 and her7, the formation and maintenance of the her6 stripes do not depend on the activity of Her6 protein. Thus, her6 does not show an oscillatory behavior and, despite its high degree of homology to the cycling genes mouse Hes1 and chicken hairy2, its expression pattern in the PSM resembles that of the non-cycling frog gene x-hairy2 (Pasini, 2004).

Differences in the PSM expression pattern of Notch pathway components between zebrafish in one case and mouse and chicken in the other have already been noted. Zebrafish lfng, in contrast to its mouse and chicken homologs, does not cycle in the PSM, whereas Delta genes cycle in zebrafish but not in mouse or chicken. One possible explanation for these discrepancies is that different vertebrate classes exploit different cycling components of Notch pathway to fulfil the same functions during somitogenesis. Alternatively, it is possible that Hes1 and chick hairy2 exert different functions in the PSM and in the segmented somites and that in zebrafish such functions have been shared among distinct hairy/E(Spl)-related genes, of which some – her1 and her7 – cycle within the PSM, while others, such as her6, are expressed in a static fashion within the anterior PSM and the somites (Pasini, 2004).

The data show that the pattern of expression of her6 is dependent on the integrity of the Notch signalling pathway and on its spatially restricted activation: a block of the Notch signal by dominant-negative Su(H) results in a loss of her6 expression in somites and the anterior PSM, while ubiquitous and sustained activation of Notch signalling by constitutively active Su(H) leads to ectopic expression of her6 throughout the PSM. Four zebrafish Notch genes with spatially restricted expression patterns have been identified to date. Ubiquitous expression of constitutively active Notch1a, NIC, which leads to increased and ectopic expression of her1 and her4, fails to induce an ectopic expression of her6 in the posterior PSM. However, the expression pattern of her6 in the anterior PSM and the segmented somites is remarkably similar to that of notch5. Thus, it is possible that Her6 is a specific effector of the Notch5-mediated signal (Pasini, 2004).

To explore whether the progressive disruption of somitogenesis correlates with a gradual breakdown of the segmentation clock as in Notch mutants or embryos depleted of Her7 or Her7 and Her1, deltaC and her1 expression were analysed at different time points on batches of her6MO- and her6MO+her4MO-injected embryos. Regardless of the dose and type of injected MO, embryos at early stages of somitogenesis show normal sharp stripes of deltaC and her1 expression. However, in embryos analyzed at progressively later time points, this periodicity of expression is gradually lost. The time at which abnormalities of deltaC and her1 expression pattern appear depends on the dose and type of injected MO and correlates with the onset of somitic defects. In embryos injected with her6MO+her4MO, the homogeneous deltaC stripes in the anterior PSM are progressively replaced by a broad and irregular band within which cells expressing deltaC at strong and weak levels are intermixed. A similar mixture of cells expressing different levels of deltaC is observed in the posterior PSM. It is concluded that Her6 and Her4 are necessary for normal paraxial mesoderm segmentation and the activities of their protein products are required to maintain synchronization of the cyclical expression of both deltaC and her1 (Pasini, 2004).

Segmentation in vertebrate embryos is controlled by a biochemical oscillator ('segmentation clock') intrinsic to the cells in the unsegmented presomitic mesoderm, and is manifested in cyclic transcription of genes involved in establishing somite polarity and boundaries. The receptor protein tyrosine phosphatase psi (RPTPpsi) gene is essential for normal functioning of the somitogenesis clock in zebrafish. Reduction of RPTPpsi activity using morpholino antisense oligonucleotides results in severe disruption of the segmental pattern of the embryo, and loss of cyclic gene expression in the presomitic mesoderm. Analysis of cyclic genes in RPTPpsi morphant embryos indicates an important requirement for RPTPpsi in the control of the somitogenesis clock upstream of or in parallel with Delta/Notch signalling. Impairing RPTPpsi activity also interferes with convergent extension during gastrulation (Aerne, 2004).

How might the dual effect of RPTPpsi on the somite oscillator and convergent extension be explained? The multiplicity of kinases in the vertebrate genome implies that PTPs have a relatively broad range of substrate specificities. One possibility, therefore, is that RPTPpsi affects factors from independent pathways (e.g., Wnt and Notch) that regulate convergent extension and somitogenesis. Alternatively, RPTPpsi might affect a single pathway/component that impinges on both convergent extension and somitogenesis. Human and mouse RPTPpsi have been shown to associate with ß-catenin and to dephosphorylate ß-catenin both in vivo and in vitro. Both these processes could be modulated by RPTPpsi, e.g. by acting on tyrosine phosphorylation levels of ß-catenin, which is crucial for both instability of the ß-catenin/cadherin bond and for enhanced binding to TBP and the Tcf complex. Thus, RPTPpsi has the potential to promote adhesion and negatively regulate ß-catenin-dependent transcriptional activity. It is therefore possible that changes in RPTPpsi activity impinges both on adhesion and migration processes during convergent extension movements, and on Wnt-directed transcriptional regulation of the somite oscillator. Clearly, further experiments are needed to pinpoint the targets of RPTPpsi activity in both processes. In any case, this study adds an unexpected and novel component to the somitogenesis clock, which, until recently, exclusively implicated members of the Delta/Notch signalling pathway (Aerne, 2004).

Fibroblast growth factor (FGF) signaling plays a crucial role in vertebrate segmentation. The FGF pathway establishes a posterior-to-anterior signaling gradient in the presomitic mesoderm (PSM), which controls cell maturation and is involved in the positioning of segmental boundaries. In addition, FGF signaling was shown to be rhythmically activated in the PSM in response to the segmentation clock. This study shows that conditional deletion of the FGF receptor gene Fgfr1 abolishes FGF signaling in the mouse PSM, resulting in an arrest of the dynamic cyclic gene expression and ultimately leading to an arrest of segmentation. Pharmacological treatments disrupting FGF signaling in the PSM result in an immediate arrest of periodic WNT activation, whereas Notch-dependent oscillations stop only during the next oscillatory cycle. Together, these experiments provide genetic evidence for the role of FGF signaling in segmentation, and identify a signaling hierarchy controlling clock oscillations downstream of FGF signaling in the mouse (Wahl, 2007).

Notch and myogenesis

During Drosophila myogenesis, Notch signaling acts at multiple steps of the muscle differentiation process. In vertebrates, Notch activation has been shown to block MyoD activation and muscle differentiation in vitro, suggesting that this pathway may act to maintain the cells in an undifferentiated proliferative state. In this paper, the role of Notch signaling has been addressed in vivo during chick myogenesis. The Notch1 receptor is expressed in postmitotic cells of the myotome and the Notch ligands Delta1 and Serrate2 are detected in subsets of differentiating myogenic cells and are thus in position to signal to Notch1 during myogenic differentiation. The expression of MyoD and Myf5 during avian myogenesis was investigated, and Myf5 was shown to be expressed earlier than MyoD. Forced expression of the Notch ligand, Delta1, during early myogenesis, using a retroviral system, has no effect on the expression of the early myogenic markers Pax3 and Myf5, but causes strong down-regulation of MyoD in infected somites. Although Delta1 overexpression results in the complete lack of differentiated muscles, detailed examination of the infected embryos shows that initial formation of a myotome is not prevented, indicating that exit from the cell cycle has not been blocked. These results suggest that Notch signaling acts in postmitotic myogenic cells to control a critical step of muscle differentiation (Hirsinger, 2001).

The effect of Notch activation on the expression of the myogenic factors MyoD and Myf5 was assessed 48 hours after infection. In infected somites, MyoD expression is strongly down-regulated in the myotome, whereas Myf5 is still normally expressed. The infected dermomyotome maintains its epithelial structure after it should have undergone an epithelio-mesenchymal transition, allowing the release of dermal precursors. Myf5 is expressed in proliferative cells of the dermomyotome and the dorsal lip in addition to the myotome, whereas MyoD is essentially found in the postmitotic cells of myotome. The absence of MyoD in the infected embryos could be due to an accumulation of proliferative Myf5-expressing cells that are unable to proceed further in their differentiation. This situation would be reminiscent of that in the nervous system where widespread Delta1 overexpression blocks exit of neural progenitor cells from the cell cycle. To examine whether myogenic progenitors are also prevented from exiting the cell cycle, BrdU incorporation together with the expression of MyoD and Myf5 were examined in infected embryos. Postmitotic myogenic cells were found in both infected and uninfected myotomes, indicating that ectopic Notch signaling does not block exit from the cell cycle in this context. This is consistent with the retention of normal, and not dramatically widespread, Pax3 and Myf5 expression in the dermomyotome and myotome. The loss of MyoD expression but maintenance of Myf5 expression in postmitotic cells in the myotomal layer implies that constitutive Notch activation does not affect the production of postmitotic Myf5-expressing cells, but specifically blocks subsequent MyoD expression by these cells (Hirsinger, 2001).

The myogenic basic helix-loop-helix (bHLH) transcription factors, Myf5, MyoD, myogenin and MRF4, are unique in their ability to direct a program of specific gene transcription leading to skeletal muscle phenotype. The observation that Myf5 and MyoD can force myogenic conversion in non-muscle cells in vitro does not imply that they are equivalent. Myf5 transcripts are detected before those of MyoD during chick limb development. The Myf5 expression domain resembles that of Pax3 and is larger than that of MyoD. Moreover, Myf5 and Pax3 expression is correlated with myoblast proliferation, while MyoD is detected in post-mitotic myoblasts. These data indicate that Myf5 and MyoD are involved in different steps during chick limb bud myogenesis, Myf5 acting upstream of MyoD. The progression of myoblasts through the differentiation steps must be carefully controlled to ensure myogenesis at the right place and time during wing development. Because Notch signaling is known to prevent differentiation in different systems and species, attempts were made to determine whether these molecules regulate the steps occurring during chick limb myogenesis. Notch1 transcripts are associated with immature myoblasts, while cells expressing the ligands Delta1 and Serrate2 are more advanced in myogenesis. Misexpression of Delta1 using a replication-competent retrovirus activates the Notch pathway. After activation of this pathway, myoblasts still express Myf5 and Pax3 but have downregulated MyoD, resulting in inhibition of terminal muscle differentiation. It is concluded that activation of Notch signaling during chick limb myogenesis prevents Myf5-expressing myoblasts from progressing to the MyoD-expressing stage (Delfini, 2000).

The bone morphogenetic protein (BMP) and Notch signaling pathways are crucial for cellular differentiation. In many cases, the two pathways act similarly; for example, to inhibit myogenic differentiation. It is not known whether this inhibition is caused by distinct mechanisms or by an interplay between Notch and BMP signaling. Functional Notch signaling is shown to be required for BMP4-mediated block of differentiation of muscle stem cells, i.e., satellite cells and the myogenic cell line C2C12. Addition of BMP4 during induction of differentiation dramatically reduces the number of differentiated satellite and C2C12 cells. Differentiation is substantially restored in BMP4-treated cultures by blocking Notch signaling using either the gamma-secretase inhibitor L-685,458 or by introduction of a dominant-negative version of the Notch signal mediator CSL. BMP4 addition to C2C12 cells increases transcription of two immediate Notch responsive genes, Hes1 and Hey1 (Drosophila homolog: Hairy/E(spl)-related with YRPW motif), an effect that is abrogated by L-685,458. A 3 kb Hey1-promoter reporter construct is synergistically activated by the Notch 1 intracellular domain (Notch 1 ICD) and BMP4. The BMP4 mediator SMAD1 mimics BMP activation of the Hey1 promoter. A synthetic Notch-responsive promoter containing no SMAD1 binding sites responds to SMAD1, indicating that DNA-binding activity of SMAD1 is not required for activation. Accordingly, Notch 1 ICD and SMAD1 interacts in binding experiments in vitro. Thus, the data presented here provide evidence for a direct interaction between the Notch and BMP signaling pathways, and indicate that Notch has a crucial role in the execution of certain aspects of BMP-mediated differentiation control (Dahlqvist, 2003).

Notch and heart morphogenesis

Activation of Notch by its ligand Serrate apportions myogenic and non-myogenic cell fates within the early Xenopus heart field. The crescent-shaped field of heart mesoderm is specified initially as cardiomyogenic. While the ventral region of the field forms the myocardial tube, the dorsolateral portions lose myogenic potency and form the dorsal mesocardium and pericardial roof. The local interactions that establish or maintain the distinct myocardial and non-myocardial domains have never been described. Xenopus Notch1 (Xotch) and Serrate1 are expressed in overlapping patterns in the early heart field. Conditional activation or inhibition of the Notch pathway with inducible dominant negative or active forms of the RBP-J/Suppressor of Hairless [Su(H)] transcription factor indicates that activation of Notch feeds back on Serrate1 gene expression to localize transcripts more dorsolaterally than those of Notch1, with overlap in the region of the developing mesocardium. Moreover, Notch pathway activation decreases myocardial gene expression and increases expression of a marker of the mesocardium and pericardial roof, whereas inhibition of Notch signaling has the opposite effect. Activation or inhibition of Notch also regulates contribution of individual cells to the myocardium. Importantly, expression of Nkx2.5 and Gata4 remains largely unaffected, indicating that Notch signaling functions downstream of heart field specification. It is concluded that Notch signaling through Su(H) suppresses cardiomyogenesis and that this activity is essential for the correct specification of myocardial and non-myocardial cell fates (Rones, 2000).

Ventricular chamber morphogenesis, first manifested by trabeculae formation, is crucial for cardiac function and embryonic viability and depends on cellular interactions between the endocardium and myocardium. Ventricular Notch1 activity is highest at presumptive trabecular endocardium. RBPJk and Notch1 mutants show impaired trabeculation and marker expression, attenuated EphrinB2, NRG1, and BMP10 expression and signaling, and decreased myocardial proliferation. Functional and molecular analyses show that Notch inhibition prevents EphrinB2 expression, and that EphrinB2 is a direct Notch target acting upstream of NRG1 in the ventricles. However, BMP10 levels are found to be independent of both EphrinB2 and NRG1 during trabeculation. Accordingly, exogenous BMP10 rescues the myocardial proliferative defect of in vitro-cultured RBPJk mutants, while exogenous NRG1 rescues differentiation in parallel. It is suggested that during trabeculation Notch independently regulates cardiomyocyte proliferation and differentiation, two exquisitely balanced processes whose perturbation may result in congenital heart disease (Grego-Bessa, 2007).

Notch and endodermal differentiation

The intestinal epithelium comprises differentiated cells of four lineages maintained by precursor cells. Since the Notch pathway controls the fate of proliferating cells in many systems, the effect of conditional expression of an activated Notch mutant in intestinal epithelium was investigated. An increase in the number of goblet cells occurs within 8 h of induction, due to an effect of Notch on post-mitotic cells, not on precursors. This observation broadens the role of Notch into controlling postmitotic differentiation and indicates that the composition of the epithelium is not solely determined by progenitor cells (Zecchini, 2005 ).

Notch and spinal cord development

The role of Zic1 was investigated by altering its expression status in developing spinal cords. Zic genes encode zinc finger proteins homologous to Drosophila Odd-paired. In vertebrate neural development, they are generally expressed in the dorsal neural tube. Chick Zic1 is initially expressed evenly along the dorsoventral axis and its expression becomes increasingly restricted dorsally during the course of neurulation. The dorsal expression of Zic1 is regulated by Sonic hedgehog, BMP4, and BMP7, as revealed by experimentally induced overexpression of these genes in the spinal cord. When Zic1 is misexpressed on the ventral side of the chick spinal cord, neuronal differentiation is inhibited irrespective of the dorsoventral position. In addition, dorsoventral properties are not grossly affected as revealed by molecular markers. Concordantly, when Zic1 is overexpressed in the dorsal spinal cord in transgenic mice, hypercellularity is observed in the dorsal spinal cord. The transgene-expressing cells are increased in comparison to those of truncated mutant Zic1-bearing mice. Conversely, a significant cell number reduction is observed without loss of dorsal properties in the dorsal spinal cords of Zic1-deficient mice. Taken together, these findings suggest that Zic1 controls the expansion of neuronal precursors by inhibiting the progression of neuronal differentiation. Notch-mediated inhibition of neuronal differention is likely to act downstream of Zic genes since Notch1 is upregulated in Zic1-overexpressing spinal cords in both the mouse and the chick (Aruga, 2002).

Notch and neural crest

Avian trunk neural crest cells give rise to a variety of cell types including neurons and satellite glial cells in peripheral ganglia. It is widely assumed that crest cell fate is regulated by environmental cues from surrounding embryonic tissues. However, it is not clear how such environmental cues could cause both neurons and glial cells to differentiate from crest-derived precursors in the same ganglionic locations. To elucidate this issue, expression and function of components of the NOTCH signaling pathway have been examined in early crest cells and in avian dorsal root ganglia. Delta1, which encodes a NOTCH ligand, is expressed in early crest- derived neuronal cells, and NOTCH1 activation in crest cells prevents neuronal differentiation and permits glial differentiation in vitro. NUMB, a NOTCH antagonist, is asymmetrically segregated when some undifferentiated crest-derived cells in nascent dorsal root ganglia undergo mitosis. It is concluded that neuron-glia fate determination of crest cells is regulated, at least in part, by NOTCH-mediated lateral inhibition among crest-derived cells, and by asymmetric cell division (Wakamatsu, 2000).

Expression of NUMB protein was observed in nascent DRGs of stage 22 chicken embryos. NUMB immunoreactivity is present in many but not all the mitotic cells in the periphery of nascent DRGs, as well as in the processes of non-mitotic cells. Importantly, in stage 22 DRGs, nearly 40% of mitotic cells have asymmetrically localized NUMB, in which chromosome orientation would cause NUMB to be inherited unevenly in daughter cells after cytokinesis. In contrast to the basal localization in neuroepithelial cells, however, asymmetry of NUMB localization could not be oriented with respect to any known anatomical landmark within the nascent DRG. At later stages of development, such as stages 25-27, only a few mitotic figures are observed. In those mitotic cells, NUMB localizes diffusely and symmetrically. When crest cells are cultured free from surrounding tissues, NUMB is also seen to be localized asymmetrically in mitotic cells, suggesting that some cell-intrinsic mechanism effects the intracellular localization of NUMB in crest cells. Thus, NUMB is asymmetrically localized, with respect to the cleavage plane in approximately 20%-30% of mitotic cells. In these cells, NUMB would be inherited in high concentration by only one of the daughter cells. The remaining mitotic cells either lack detectable NUMB expression, or appear to segregate NUMB symmetrically. Under these culture conditions, neurogenesis is almost complete by 5 days, and the number of mitotic cells that possess NUMB asymmetrically declines rapidly. In all stages examined, however, NUMB was symmetrically distributed throughout the cytoplasm of mitotic Hu-positive neuronal cells (Hu is a neuron-specific family of RNA binding proteins related to Drosophila ELAV), suggesting the machinery regulating asymmetrical NUMB segregation no longer functions in fate-restricted neuronal cells. NUMB immunoreactivity is enriched in the processes of non-mitotic Hu-negative cells, and consequently sequestered away from the cell body, as also observed in vivo. In non-mitotic Hu-positive neuronal cells, NUMB was observed throughout the cell body and their processes, so that activation of residual NOTCH molecules might be prevented (Wakamatsu, 2000).

Neural crest is induced at the junction of epidermal ectoderm and neural plate by the mutual interaction of these tissues. BMP4 has been shown to pattern the ectodermal tissues, and BMP4 can induce neural crest cells from the neural plate. Epidermally expressed Delta1, which encodes a Notch ligand, is required for the activation and/or maintenance of Bmp4 expression in this tissue, and is thus indirectly required for neural crest induction by BMP4 at the epidermis-neural plate boundary. Notch activation in the epidermis additionally inhibits neural crest formation in this tissue, so that neural crest generation by BMP4 is restricted to the junction (Endo, 2002).

In zebrafish, cells at the lateral edge of the neural plate become Rohon-Beard primary sensory neurons or neural crest. Delta/Notch signaling is required for neural crest formation. ngn1 is expressed in primary neurons; inhibiting Ngn1 activity prevents Rohon-Beard (RB) cell formation but not formation of other primary neurons. Reducing Ngn1 activity in embryos lacking Delta/Notch signaling restores neural crest formation, indicating Delta/Notch signaling inhibits neurogenesis without actively promoting neural crest. Ngn1 activity is also required for later development of dorsal root ganglion (DRG) sensory neurons; however, RB neurons and DRG neurons are not necessarily derived from the same precursor cell. It is proposed that temporally distinct episodes of Ngn1 activity in the same precursor population specify these two different types of sensory neurons (Cornell, 2002).

Neural stem cells become progressively less neurogenic and more gliogenic with development. Between E10.5 and E14.5, neural crest stem cells (NCSCs) become increasingly sensitive to the Notch ligand Delta-Fc, a progliogenic and anti-neurogenic signal. This transition is correlated with a 20- to 30-fold increase in the relative ratio of expression of Notch and Numb (a putative inhibitor of Notch signaling). Misexpression experiments suggest that these changes contribute causally to increased Delta sensitivity. Moreover, such changes can occur in NCSCs cultured at clonal density in the absence of other cell types. However, they require local cell-cell interactions within developing clones. Delta-Fc mimics the effect of such cell-cell interactions to increase Notch and decrease Numb expression in isolated NCSCs. Thus, Delta-mediated feedback interactions between NCSCs, coupled with positive feedback control of Notch sensitivity within individual cells, may underlie developmental changes in the ligand-sensitivity of these cells (Kubu, 2002).

The roles of Notch signaling in the chondrogenesis of mouse mesencephalic neural crest cells was examined. The activation of Notch signaling or the treatment with fibroblast growth factors (FGFs) promotes the differentiation of proliferative and prehypertrophic chondrocytes expressing collagen type II. Notch activation or FGF2 exposure during the first 24 h in culture is critical for the differentiation of proliferative and prehypertrophic chondrocytes. The expression of SOX9, a transcription activator of collagen type II, is also upregulated by Notch activation or FGF2 treatment. The promotion of proliferative and prehypertrophic chondrocyte differentiation by FGF2 is significantly suppressed by the inhibition of Notch signaling using Notch-1 siRNA. These results suggest that FGFs activate Notch signaling and that this activation promotes the chondrogenic specification of mouse mesencephalic neural crest cells. Furthermore, the expression patterns of Notch-1, SOX9, and p75, which is a marker of undifferentiated neural crest cells, was investigated in the mandibular arch, where mesencephalic neural crest cells colonize and undergo chondrogenesis. These in vivo observations, coupled with the results of the present in vitro study, suggest that Notch signaling as well as FGFs is a component of epithelial-mesenchymal interactions that promote the chondrogenic specification of mouse mesencephalic neural crest cells (Nakanishi, 2007).

Notch and gliogenesis

The genesis of vertebrate peripheral ganglia poses the problem of how multipotent neural crest stem cells (NCSCs) can sequentially generate neurons and then glia in a local environment containing strong instructive neurogenic factors, such as BMP2. Notch ligands, which are normally expressed on differentiating neuroblasts, can inhibit neurogenesis in NCSCs in a manner that is completely dominant to BMP2. Contrary to expectation, Notch activation does not maintain these stem cells in an uncommitted state or promote their self-renewal. Rather, even a transient activation of Notch is sufficient to cause a rapid and irreversible loss of neurogenic capacity accompanied by accelerated glial differentiation. These data suggest that Notch ligands expressed by neuroblasts may act positively to instruct a cell-heritable switch to gliogenesis in neighboring stem cells (Morrison, 2000).

The observation of such an irreversible inhibition of neurogenesis is surprising, because prior studies in Xenopus and Drosophila have suggested that neurogenic capacity can be recovered upon decay or deliberate inactivation of ectopic Notch expression. The results obtained here therefore challenge the prevailing view that Notch signaling functions principally to inhibit the differentiation of progenitor cells in a reversible manner so as to maintain competence for alternative fates, although it may do so in some settings. The molecular mechanism that underlies the apparently irreversible and cell-heritable influence of Notch activation in NCSCs will be an interesting subject for future study. The ability of Notch activation to cause an irreversible loss of neuronal potential in NCSCs may seem inconsistent with the fact that multipotent neural crest progenitors can be isolated from tissues such as sciatic nerve and dorsal root ganglia where Notch ligands are expressed. However, these multipotent cells constitute a relatively small proportion (15%) of the cells in these tissues, and are present only transiently. Nevertheless, the existence of such cells indicates that there must be mechanisms by which some neural crest progenitors can escape the influence of Notch ligands and maintain multipotency, at least temporarily. One possibility is that the cells that maintain multipotency are not in direct contact with neuroblasts that express Notch ligands. Another possibility is that cell-intrinsic inhibitors of Notch signaling, such as Numb, are differentially expressed among neural crest progenitors. Whatever the reason, the extent of glial differentiation in neural crest-derived tissues is likely regulated by factors in addition to Notch in vivo (Morrison, 2000).

Notch1 has been shown to induce glia in the peripheral nervous system. However, it has not been known whether Notch can direct commitment to glia from multipotent progenitors of the central nervous system. Evidence is presented that activated Notch1 and Notch3 promote the differentiation of astroglia from the rat adult hippocampus-derived multipotent progenitors (AHPs). Quantitative clonal analysis indicates that the action of Notch is likely to be instructive. Transient activation of Notch can direct commitment of AHPs irreversibly to astroglia. Astroglial induction by Notch signaling has been shown to be independent of STAT3, which is a key regulatory transcriptional factor when ciliary neurotrophic factor (CNTF) induces astroglia. These data suggest that Notch provides a CNTF-independent instructive signal of astroglia differentiation in CNS multipotent progenitor cells (Tanagaki, 2001).

Oligodendrocytes, the myelinating cell type of the central nervous system, arise from a ventral population of precursors that also produces motoneurons. Although the mechanisms that specify motoneuron development are well described, the mechanisms that generate oligodendrocytes from the same precursor population are largely unknown. By analyzing mutant zebrafish embryos, it has been found that Delta-Notch signaling is required for spinal cord oligodendrocyte specification. Using a transgenic, conditional expression system, it was also learned that constitutive Notch activity promotes formation of excess oligodendrocyte progenitor cells (OPCs). However, excess OPCs are induced only in ventral spinal cord at the time that OPCs normally develop. These data provide evidence that Notch signaling maintains subsets of ventral spinal cord precursors during neuronal birth and, acting with other temporally and spatially restricted factors, specifies them for oligodendrocyte fate (Park, 2003).

Because mouse embryos that are homozygous for null mutations of Delta or Notch genes die at early stages of neural development, there is little information that addresses the requirement of Notch signaling for vertebrate CNS glial specification. This limitation can be circumvented through analysis of mice in which Notch1 is conditionally inactivated in the cerebellum. These mice prematurely express neuronal markers and have reduced number of mutant cerebellar cells that express the glial marker GFAP. In an alternative approach, neurospheres can derived from Delta-like 1 mutant mice. After culturing, mutant neurospheres produce excess neurons and a deficit of oligodendrocytes and astrocytes compared with controls. Additionally, retinas of mice that are homozygous for a mutation of Hes5, which encodes a downstream effector of Notch signaling, have fewer Müller glia than the wild type. These observations are consistent with the idea that Delta-Notch signaling regulates neuronal-glial fate decisions (Park, 2003).

Constitutive activation of the Notch pathway can promote gliogenesis by peripheral (PNS) and central (CNS) nervous system progenitors. This raises the question of whether physiological Notch signaling regulates gliogenesis in vivo. To test this, Rbpsuh (Rbpj) was conditionally deleted from mouse PNS or CNS progenitors using Wnt1-Cre or Nestin-Cre. Rbpsuh encodes a DNA-binding protein (RBP/J) that is required for canonical signaling by all Notch receptors. In most regions of the developing PNS and spinal cord, Rbpsuh deletion causes only mild defects in neurogenesis, but severe defects in gliogenesis. These resulted from defects in glial specification or differentiation, not premature depletion of neural progenitors, because undifferentiated progenitors from the PNS and spinal cord could be cultured despite their failure to form glia in vivo. In spinal cord progenitors, Rbpsuh was required to maintain Sox9 expression during gliogenesis, demonstrating that Notch signaling promotes the expression of a glial-specification gene. These results demonstrate that physiological Notch signaling is required for gliogenesis in vivo, independent of the role of Notch in the maintenance of undifferentiated neural progenitors (Taylor, 2007).

Notch and brain development

In vertebrates, Notch signaling is generally thought to inhibit neural differentiation. However, whether Notch can also promote specific early cell fates in this context is unknown. Activated Notch1 (NIC) was introduced into the mouse forebrain, before the onset of neurogenesis, using a retroviral vector and ultrasound imaging. During embryogenesis, NIC-infected cells become radial glia, the first specialized cell type evident in the forebrain. Thus, rather than simply inhibiting differentiation, Notch1 signaling promotes the acquisition of an early cellular phenotype. Postnatally, many NIC-infected cells become periventricular astrocytes, cells previously shown to be neural stem cells in the adult. These results suggest that Notch1 promotes radial glial identity during embryogenesis, and that radial glia may be lineally related to stem cells in the adult nervous system (Gaiano, 2000).

Regardless of whether the effects of Notch1 signaling on radial glial fate is direct or indirect, in light of the many roles played by Notch signaling throughout the embryo, it is highly unlikely that Notch alone instructs progenitors to become this cell type. It seems more likely that Notch signaling influences the response of neural progenitors to secondary cues. Such a hypothesis is supported by recent work demonstrating that the secreted protein glial growth factor (GGF) causes elongation of radial glia and upregulation of the radial glial markers nestin and BLBP. The current study suggests that GGF might act on cells in which the Notch pathway has been activated. Nevertheless, it is believed that the activation of Notch signaling may be an essential component of the molecular specification of radial glia. The expression of Notch1 protein in endogenous radial glia, together with the observation that activated Notch1 promotes a radial glial phenotype, raises the question as to how Notch1 is normally activated in this cell type. The most likely explanation is that newly generated neurons, expressing high levels of a Notch ligand such as Dll1, activate Notch1 in radial glia during migration along the radial processes. This activation would allow radial glia to respond to environmental cues, such as GGF perhaps, which might then maintain their morphology and gene expression (Gaiano, 2000).

The olfactory bulb, neocortex and archicortex arise from a common pool of progenitors in the dorsal telencephalon. The consequences were examined of supplying excess Notch1 signal in vivo on the cellular and regional destinies of telencephalic precursors using bicistronic replication defective retroviruses. After mid-neurogenesis (E14.5) ventricular injections, activated Notch1 retrovirus markedly inhibits the generation of neurons from telencephalic precursors, delays the emergence of cells from the subventricular zone (SVZ), and produces an augmentation of glial progeny in the neo- and archi-cortex. However, activated Notch1 has a distinct effect on the progenitors of the olfactory bulb, markedly reducing the numbers of cells of any type that migrates there. To elucidate the mechanism of the cell fate changes elicited by Notch1 signals in the cortical regions, short- and long-term cultures of E14.5 telencephalic progenitors were examined. These studies reveal that activated Notch1 elicits a cessation of proliferation that coincides with an inhibition of the generation of neurons. Later, during gliogenesis, activated Notch1 triggers a rapid cellular proliferation with a significant increase in the generation of cells expressing GFAP. To examine the generation of cells destined for the olfactory bulb, stereotaxic injections into the early postnatal anterior subventricular zone (SVZa) were used. Precursors of the olfactory bulb respond to Notch signals by remaining quiescent and failing to give rise to differentiated progeny of any type, unlike cortical precursor cells, which then generate glia instead of neurons. These data show that forebrain precursors vary in their response to Notch signals according to spatial and temporal cues, and that Notch signals influence the composition of forebrain regions by modulating the rate of proliferation of neural precursor cells (Chambers, 2000).

In the developing cerebellar cortex, granule neuron precursors (GNPs) proliferate and commence differentiation in a superficial zone, the external granule layer (EGL). The molecular basis of the transition from proliferating precursors to immature differentiating neurons remains unknown. Notch signaling is an evolutionarily conserved pathway regulating the differentiation of precursor cells of many lineages. Notch2 is specifically expressed in proliferating GNPs in the EGL. Treatment of GNPs with soluble Notch ligand Jagged1, or overexpression of activated Notch2 or its downstream target HES1, maintains precursor proliferation. The addition of GNP mitogens Jagged1 or Sonic Hedgehog (Shh) upregulates the expression of HES1, suggesting a role for HES1 in maintaining precursor proliferation (Solecki, 2001).

Gain-of-function experiments suggest that Notch signaling is involved in the early stages of mammalian neurogenesis. On the basis of the expression of Notch1 by putative progenitor cells of the vertebrate CNS, the role of Notch1 in the development of the mammalian brain has been addressed directly. The Floxed Notch1 allele, used for conditional ablation was generated by introducing LoxP sequences upstream and downstream of the first translated exon encoding the signal peptide. Recombination between the LoxP sites ablates the first coding exon of the Notch1 gene and results in a null allele. Mice homozygous for the Floxed Notch1 allele show no abnormal phenotype and were used to analyze the function of Notch1 by temporal and spatial gene ablation. Loss of Notch1 results in premature onset of neurogenesis by neuroepithelial cells of the midbrain-hindbrain region of the neural tube. Notch1-deficient cells do not complete differentiation but are eliminated by apoptosis, resulting in a reduced number of neurons in the adult cerebellum. The effects of Notch1 ablation on gliogenesis were examined in vivo. Notch1 is required for both neuron and glia formation and modulates the onset of neurogenesis within the cerebellar neuroepithelium (Lütolf, 2002).

During segmentation of the vertebrate hindbrain, a distinct population of boundary cells forms at the interface between each segment. Little is known regarding mechanisms that regulate the formation or functions of these cells. A potential role of Notch signaling has been investigated; in the zebrafish hindbrain, radical fringe is expressed in boundary cells and delta genes are expressed adjacent to boundaries, consistent with a sustained activation of Notch in boundary cells. Mosaic expression experiments reveal that activation of the Notch/Su(H) pathway regulates cell affinity properties that segregate cells to boundaries. In addition, Notch signaling correlates with a delayed neurogenesis at hindbrain boundaries and is required to inhibit premature neuronal differentiation of boundary cells. These findings reveal that Notch activation couples the regulation of location and differentiation in hindbrain boundary cells. Such coupling may be important for these cells to act as a stable signaling center (Cheng, 2003).

Studies of neurogenesis in the zebrafish hindbrain have shown that differentiation first occurs at rhombomere centers, and only at late stages are neurons formed at the boundaries between rhombomeres. The spatial and temporal pattern of neurogenesis is reflected by the expression of delta genes that mark early neuroblasts: expression is excluded from rhombomere boundaries, and by 24 hr occurs in stripes adjacent to the boundaries. These observations are consistent with Delta mediating a lateral inhibition in a manner analogous to its widely utilized role in the neural epithelium, in which Delta expression by early neuroblasts activates Notch and suppresses neurogenesis and delta expression in adjacent cells. Indeed, ectopic expression of dominant-active Su(H) suppresses delta expression throughout the hindbrain. An important role of the lateral inhibition of neurogenesis is to maintain the progenitor pool of neural epithelial cells that is required for the continued generation of neurons. mind bomb (mib) mutant embryos have a strong Notch pathway deficiency due to mutation of a ubiquitin ligase required for Delta ligand activity. Boundary markers are severely depleted in mib mutant embryos -- this suggests that lateral inhibition maintains the neural epithelium not only in nonboundary regions but also at hindbrain boundaries. Consistent with a role for Notch activation in maintaining boundary cells, following mosaic expression of dominant-active Su(H) in mib mutants, the expressing cells sort to boundaries and boundary marker gene expression is rescued (Cheng, 2003).

These findings reveal that two responses to the activation of Notch are coupled at rhombomere boundaries in the zebrafish hindbrain: the regulation of cell affinity properties of boundary cells and the suppression of neurogenesis. This begs the question of why neurogenesis is delayed at rhombomere boundaries. An attractive possibility is suggested by the observation that signaling centers in the neural epithelium such as the floor plate and roof plate do not undergo neurogenesis and have a low rate of cell proliferation. By enabling the maintenance of a relatively stable number of signaling cells, the suppression of differentiation and proliferation is a simple way to maintain a constant amount of signal. By analogy, the suppression of neurogenesis and proliferation at rhombomere boundaries may reflect that the radical fringe-dependent expression of wnt1 by rhombomere boundary cells is involved in patterning of the zebrafish hindbrain. The regulation by Notch of both cell affinity and the suppression of differentiation at rhombomere boundaries would thus provide a coupling between maintenance of the location and number of signaling cells (Cheng, 2003).

The mammalian cerebral cortex comprises six layers of neurons. Cortical progenitors in the ventricular zone generate neurons specific to each layer through successive cell divisions. Neurons of layer VI are generated at an early stage, whereas later-born neurons occupy progressively upper layers. The underlying molecular mechanisms of neurogenesis, however, are relatively unknown. In this study, a system was devised where the Notch pathway was activated spatiotemporally in the cortex by in vivo electroporation and Cre-mediated DNA recombination. Electroporation at E13.5 transferred DNA to early progenitors that gave rise to neurons of both low and upper layers. Forced expression of a constitutively active form of Notch (caNotch) at E13.5 inhibited progenitors from generating neurons and kept progenitors as proliferating radial glial cells. After subsequent transfection at E15.5 of a Cre expression vector to remove caNotch, double-transfected cells, in which caNotch was excised, migrated into the cortical plate and differentiated into neurons specific to upper layers. Bromodeoxyuridine-labeling experiments showed that the neurons were born after Cre transfection. These results indicate that cortical progenitors that had been temporarily subjected to Notch activation at an early stage generated neurons at later stages, but that the generation of low-layer neurons was skipped. Moreover, the double-transfected cells gave rise to upper-layer neurons, even after their transplantation into the E13.5 brain, indicating that the developmental state of progenitors is not halted by caNotch activity (Mizutani, 2005).

The maintenance of cortical progenitors by caNotch is consistent with previous results obtained from in vitro cell cultures using loss-of-function mutants of Hes1 and RBP-J, which are downstream effectors of the Notch receptor. The Notch pathway is activated by extracellular ligands such as Delta-like and Jagged. Considering that a large number of neurons are generated from many progenitors simultaneously in the mammalian cerebral cortex, a flexible way to control the number of generated neurons using the balance between symmetric and asymmetric divisions of progenitors may be a more advantageous mechanism than the fixed sequential generation of neurons in Drosophila. ß-Catenin signals have also been shown to regulate the balance between symmetric and asymmetric divisions of cortical progenitors. However, some neurons still differentiate from progenitors in which ß-catenin signaling is active. By contrast, few neurons were generated from caNotch+ progenitors. This may suggest that the ß-catenin signaling pathway is different from the Notch pathway, even if they have similar effects on progenitors. Mice carrying gain-of-function mutations in the ß-catenin signaling pathway exhibit severe malformation in the cortex. caNotch transfection showed milder effects on the morphology of the cortex, presumably because of its spatiotemporally restricted expression (Mizutani, 2005).

Numerous lines of evidence suggest that Notch signaling plays a pivotal role in controlling the production of neurons from progenitor cells. However, most experiments have relied on gain-of-function approaches because perturbation of Notch signaling results in death prior to the onset of neurogenesis. This study examined the requirement for Notch signaling in the development of the striatum through the analysis of different single and compound Notch1 conditional and Notch3 null mutants. Normal development of the striatum depends on the presence of appropriate Notch signals in progenitors during a critical window of embryonic development. Early removal of Notch1 prior to neurogenesis alters early-born patch neurons but not late-born matrix neurons in the striatum. The late-born striatal neurons in these mutants are spared as a result of functional compensation by Notch3. Notably, however, the removal of Notch signaling subsequent to cells leaving the germinal zone has no obvious effect on striatal organization and patterning. These results indicate that Notch signaling is required in neural progenitor cells to control cell fate in the striatum, but is dispensable during subsequent phases of neuronal migration and differentiation (Mason, 2005).

Like Notch1, Notch3 is expressed by progenitor cells within the forebrain. To test the role of Notch1 and Notch3 receptors in regulating neurogenesis in the striatum, the phenotypes occurring in single and compound Notch1 conditional mutants and Notch3 null mutant animals were investigated. The Cre-LoxP system and two different Cre-driver lines were used to produce two distinct conditional deletions of the Notch1 receptor. In one case, Notch1 is removed throughout the telencephalon from the beginning of neurogenesis onwards. In the second case, Notch1 is deleted only after cells have exited the VZ in the ventral telencephalon. Striatal development was assessed in Notch1 conditional mutants; Notch3 null double mutant mice in the context of both of these Cre-driver lines. Removing Notch1 in the forebrain prior to neurogenesis preferentially affects early-born neurons in the striatum, whereas later born cell types are generated normally. In addition, Notch3 functionally compensates for the loss of Notch1 in the nervous system and mediates the conservation of late-born neurons in Notch1 conditional mutants. Notably, removal of Notch1 and Notch3 in cells after they have left the ventricular zone has no effect on striatal development. These experiments reveal that Notch signaling is not required in postmitotic neurons for their migration or the subsequent patterning of the striatum (Mason, 2005).

A Notch pathway - Ras pathway connection

E47 (Drosophila homolog: Daughterless) is a widely expressed transcription factor that activates B-cell-specific immunoglobulin gene transcription and is required for early B-cell development. In an effort to identify processes that regulate E47, and potentially B-cell development, it was found that activated Notch1 and Notch2 effectively inhibit E47 activity. Only the intact E47 protein is inhibited by Notch. Fusion proteins containing isolated DNA binding and activation domains are unaffected. Although overexpression of the coactivator p300 partially reverses E47 inhibition, results of several assays indicate that p300/CBP is not a general target of Notch. Notch inhibition of E47 does not correlate with its ability to activate CBF1/RBP-Jkappa, the mammalian homolog of Suppressor of Hairless, a protein that associates physically with Notch and defines the only known Notch signaling pathway in Drosophila (Ordentlich, 1998).

E47 is inhibited by Deltex, a second Notch-interacting protein. Evidence is provided that Notch and Deltex may act on E47 by inhibiting signaling through Ras. The EGR-1 promoter (see Huckebein) is known to be stimulated by Ras through the action of mitogen-activated protein kinases (MAPKs) on a ternary complex involving ETS proteins (e.g., ELK1) and Serum response factor. The activity of a CAT reporter under the control of the EGR-1 promoter is inhibited by Deltex, both in the presence and in the absence of Ras stimulation by platelet-derived growth factor. To reduce the complexity of the effects, a series of GAL4 promoter fusions were used and their abilities to activate a minimal promoter containing GAL4 binding sites was assessed. GAL4-Jun includes a portion of the c-Jun protein whose activity is dependent on signaling from Ras to SAPK/JNK. A promoter fragment lacking the CBF1 interaction domain inhibits GAL4-Jun activity but has no effect on GAL4-CREB. Similarly, Deltex inhibits GAL4-Jun activity and has no effect on GAL4-CREB. Although it is likely that N2-IC and Deltex have somewhat different effects on cells, these results clearly show that both Notch and Deltex inhibit signaling by Ras, as measured by the ability to stimulate SAPK/JNK activity. It is proposed that this is the mechanism by which Notch and Deltex inhibit E47 (Ordentlich, 1998).

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