Interactive Fly, Drosophila

Nuclear affectors of the Notch pathway

The whole Notch pathway is evolutionarily conserved in vertebrates. Activated forms of mouse Notch (mNotch) associate with the human analogue of Suppressor of Hairless, KBF2/RBP-Jkappa, and act as transcriptional activator of the Hairy enhancer of split locus, acting through the KBF2-binding site (Jarriault, 1996).

In the mouse, targeted mutation of the Notch pathway genes Notch1 and RBP-Jk has demonstrated a role for these genes in somite segmentation. These mutations lead to altered expression of the Notch signalling pathway homologs Hes-5 (Drosophila homologs Hairy and Enhancer of split), Mash-1 (Drosophila homologs: Achaete and Atonal) and Dll1 (Drosophila homolog: Delta), resulting in enhanced neurogenesis. Precocious neuronal differentiation is indicated by the expanded expression domains of Math4A, neuroD and NSCL-1 (a bHLH transcription factor expressed in the nervous system). The RBP-Jk mutation has stronger effects on expression of these genes than does the Notch1 mutation, consistent with functional redundancy of Notch genes in neurogenesis. In the neural tube, Dll1 is expressed in individual, isolated cells in a basal position in the neural epithelium, in cells that are thought to be committed neuronal precursors. Dll1 expression in the neural tube is increased in both RBP-Jk and Notch1 mutants. In Drosophila, a connection between the up-regulation of E(spl) and the downregulation of Delta, after activation of Notch signaling, may be provided by genes of the achaete-scute complex. Mash-1 is upregulated by Notch1 and RBP-Jk mutation in midbrain/hindbrain region and in the anterior neural tube region. Math-4A is upregulated in the midbrain and spinal cord. Neuro-D is upregulated in the midbrain and spinal cord and in the trigeminal and geniculate ganglia. Hes-5 is downregulated by Notch1 and RBP-Jk in the midbrain, hindbrain, spinal cord and presomitic mesoderm. Thus it appears that the murine Notch signaling pathway is involved in the regulation of neural stem cell differentiation (de la Pompa, 1997).

Mash-2 expression begins during preimplantation development, but is restricted to trophoblasts after the blastocyst stage. Within the trophoblast lineage, Mash-2 transcripts are first expressed in the ectoplacental cone and chorion, but not in terminally differentiated trophoblast giant cells. After day 8.5 of gestation, Mash-2 expression becomes further restricted to focal sites within the spongiotrophoblast and labyrinth. Downregulation is probably important for normal development, since overexpression of Mash-2 reduces giant cell formation. The role that the Notch signaling pathway may play in trophoblast development has been investigated. Mash-2 is a homolog of Drosophila achaete/scute complex genes. In the developing mouse placenta, all elements of the Notch pathway are expressed. In particular, the Notch-2, HES-2, and HES-3 genes are coexpressed in trophoblast giant cells and in foci within the spongiotrophoblast at day 10.5 when Mash-2 transcription becomes restricted. Two members of the mammalian Groucho family are expressed in trophoblasts; TLE3 is expressed broadly in the giant cell, spongiotrophoblast, and labyrinthine regions, whereas TLE2 is limited to giant cells and focal regions of the spongiotrophoblast. These data suggest that Notch signaling through activation of HES transcriptional repressors may play a role in murine placental development (Nakayama, 1997).

While the transmembrane protein Notch plays an important role in various aspects of development, and in diseases (including tumors and neurological disorders), the intracellular pathway of mammalian Notch remains very elusive. To understand the intracellular pathway of mammalian Notch, the role of the bHLH genes Hes1 and Hes5 (mammalian hairy and the Enhancer-of-split homolog, respectively) was examined by retrovirally misexpressing the constitutively active form of Notch (caNotch) in neural precursor cells prepared from wild-type, Hes1-null, Hes5-null and Hes1-Hes5 double-null mouse embryos. caNotch, which induces the endogenous Hes1 and Hes5 expression, inhibits neuronal differentiation in the wild-type, Hes1-null and Hes5-null background, but not in the Hes1-Hes5 double-null background. These results demonstrate that Hes1 and Hes5 are essential Notch effectors in the regulation of mammalian neuronal differentiation (Ohtsuka, 1999).

A truncated active form of Notch1 binds CBF1, also known as JkappaRBP, the mammalian homolog of Suppressor of Hairless and activates transcription through a CBF1 response element-containing promoter. One model for the function of mammalian Notch assumes that Notch is cleaved by a membrane protease, and the released membrane domain is translocated to the nucleus. This model is supported by the observation that the untethered intracellular domain of Notch is as active as truncated Notch and is located predominantly in the nucleus, and by the observation that small amounts of CBF1 are associated with Notch1 in the nucleus (Lu, 1996 and references).

Notch is involved in the cell fate determination of many cell lineages. The intracellular region (RAMIC) of Notch1 transactivates genes by interaction with a DNA binding protein RBP-J (Drosophila homolog Suppressor of hairless). The activities of mouse RAMIC and its derivatives were compared in transactivation and differentiation suppression of myogenic precursor cells. RAMIC comprises two separate domains: IC for transactivation (the IC domain includes the whole intracellular domain exclusive of the RAM domain) and RAM (immediately C-terminal to the transmembrane region) for RBP-J binding. Although the physical interaction of ankyrin repeats within IC with RBP-J is much weaker than is RAM interaction with RBP-J, transactivation activity of IC is shown to involve RBP-J by using an RBP-J null mutant cell line. IC shows differentiation suppression activity that is generally comparable to its transactivation activity. The RBP-J-VP16 fusion protein, which has strong transactivation activity, also suppresses myogenesis of C2C12 myogenic precursor cells. The RAM domain, which has no other activity than binding to RBP-J, synergistically stimulates transactivation activity of IC to the level of RAMIC. The RAM domain is proposed to compete with a putative co-repressor for binding to RBP-J because the RAM domain can also stimulate the activity of RBP-J-VP16. Taken together, these results indicate that differentiation suppression of myogenic precursor cells by Notch signalling is due to the transactivation of genes carrying RBP-J binding motifs (Kato, 1997).

Within the human ERBB-2 gene promoter, a 100-base pair region 5' to the TATA box enhances basal transcription 200-fold. Two palindromes present within this 100-base pair region are important for transcription. The palindrome binding protein was purified to homogeneity and found to be identical to RBPJkappa, the mammalian homolog of Drosophila Suppressor of Hairless (Su[H]). Recombinant RBPJkappa binds the ERBB-2 promoter with affinity comparable to that seen with well characterized RBPJkappa binding sites. RBPJkappa activates an ERBB-2 palindrome-containing promoter in 293 cells. Because in Drosophila Su(H) acts downstream of NOTCH and because the NOTCH.Su(H)/RBPJkappa protein complex stimulates transcription from target promoters, NOTCH-IC, a constitutively active form of NOTCH, was tested for effects on the ERBB-2 palindrome. NOTCH-IC further increases RBPJkappa-mediated transcription on wild type but not mutant ERBB-2 palindrome. Thus, RBPJkappa can activate ERBB-2 transcription and serve as an anchor to mediate NOTCH function on the ERBB-2 gene (Y.Chen, 1997).

The ability of Epstein-Barr virus (EBV) latent infection nuclear protein EBNA3C to activate transcription of two EBNA2-responsive genes and to inhibit EBNA2 activation of transcription in transient-transfection assays appears to be due to its ability to interact with RBPJkappa, a cell protein that links EBNA2 to its response elements. EBNA3A and EBNA3B are similar to EBNA3C in binding to RBPJkappa. EBNA3A and EBNA3B can also inhibit the interaction of RBPJkappa with cognate DNA in vitro. Although EBNA3 open reading frames are each close to 1,000 codons long, EBNA3A amino acids 1 to 138, EBNA3B amino acids 1 to 311, and EBNA3C amino acids 1 to 183 are sufficient for RBPJkappa interaction, while EBNA3B amino acids I to 109 have less or no binding. The RBPJkappa interacting domains overlap with the most highly conserved domain (amino acids 90 to 320) among the EBNA3 proteins. Thus, the EBNA3 gene family appears to have evolved to differentially regulate promoters with RBPJkappa binding sites. EBNA2, EBNA3A, and EBNA3C are important in EBV transformation of primary human B lymphocytes. Their interaction with RBPJkappa links EBV transformation to the notch signaling pathway and the effects of activated notch in T-cell leukemogenesis (Robertson, 1966).

Mammalian Notch intracellular domain (NotchIC) interacts with the transcriptional repressor CBF1, which is the human homolog of Drosophila Suppressor of Hairless. The N-terminal 114-amino-acid region of mouse NotchIC contains the CBF1 interactive domain and the cdc10/ankyrin repeats are not essential for this interaction. This result was confirmed in immunoprecipation assays in which the N-terminal 114-amino-acid segment of NotchIC, but not the ankyrin repeat region, coprecipitated with CBF1. Mouse NotchIC itself is targeted to the transcriptional repression domain (aa179 to 361) of CBF1. NotchIC transactivates gene expression via CBF1 tethering to DNA. Transactivation by NotchIC occurs partially through abolition of CBF1-mediated repession. This same mechanism is used by Epstein-Barr virus EBNA2. Thus, mimicry of Notch signal transduction is involved in Epstein-Barr virus-driven immortalization (Hsieh, 1996).

Truncated forms of the NOTCH1 transmembrane receptor engineered to resemble mutant forms of NOTCH1 found in certain cases of human T cell leukemia/lymphoma (T-ALL) efficiently induce T-ALL when expressed in the bone marrow of mice. Unlike full-sized NOTCH1, two such truncated forms of the protein either lacking a major portion of the extracellular domain (DeltaE) or consisting only of the intracellular domain (ICN) are found to activate transcription in cultured cells, presumably through RBP-Jkappa response elements within DNA. Both truncated forms also bind to the transcription factor RBP-Jkappa in extracts prepared from human and murine T-ALL cell lines. Transcriptional activation requires the presence of a weak RBP-Jkappa-binding site within the NOTCH1 ankyrin repeat region of the intracellular domain. Unexpectedly, a second, stronger RBP-Jkappa-binding site, which lies within the intracellular domain close to the transmembrane region and significantly augments association with RBP-Jkappa, is not needed for oncogenesis or for transcriptional activation. While ICN appears primarily in the nucleus, DeltaE localizes to cytoplasmic and nuclear membranes, suggesting that intranuclear localization is not essential for oncogenesis or transcriptional activation. In support of this interpretation, mutation of putative nuclear localization sequences decreases nuclear localization and increases transcriptional activation by membrane-bound DeltaE. Transcriptional activation by this mutant form of membrane-bound DeltaE is approximately equivalent to that produced by intranuclear ICN. These data are most consistent with NOTCH1 oncogenesis and transcriptional activation being independent of association with RBP-Jkappa at promoter sites (Aster, 1997).

Mutations in neurogenic genes result in an expression of nautilus, and a muscle precursor hyperplasia (too many cells) (Corbin, 1991). In the mouse, ectopic expression of the intracellular domain of mNotch, functions as a constitutively activated repressor of myogenesis both in cultured cells and frog embyros. The mNotch intracellular domain contains a nuclear localization signal and localizes to the nucleus. Removal of the nuclear localization signal reduces nuclear localization and diminishes the inhibition of myogenesis caused by Myf-5 or MyoD. The target for Notch inhibition seems to be the bHLH region of MyoD, as MyoD derivatives missing the N terminus, the C terminus and the C/H region (residues 63-98) are all inhibited. However, mNotch does not affect the MyoD activation domain, but may inhibit a co-factor required for MyoD activation (Kopan, 1994).

A thymic tumor of a feline leukemia virus has been isolated that has transduced a fragment of the Notch2 gene. A nuclear form of Notch2 corresponding to the biologically active intracellular domain (N2ICD) is expressed from this recombinant retrovirus through internal ribosome entry. Internal ribosome entry sites (IRESs) are RNA structural motifs that allow 5' cap-independent recruitment of ribosomal subunits to mRNAs. The Notch2 IRES maps exclusively to Notch2 sequences that correspond to the coding region of the cellular gene. Therefore, these studies not only provide insights into aberrant Notch2 expression in tumors, but they may also inform understanding of N2ICD generation in the cellular context (Lauring, 2000).

There is evidence for both ligand-dependent and ligand-independent expression of the NICD in Drosophila. If the internal Notch2 start codon and upstream IRES are functional within the context of the cellular Notch2 mRNA, this single message could give rise to at least two protein species with different activities and subcellular localization. The first and likely most abundant would be a surface transmembrane protein that could give rise to a ligand-dependent N2ICD through proteolysis. The second would be predicted to be an activated nuclear protein that is presumably subject to regulation by proteins other than extracellular ligands. Internal initiation may only occur when 5' cap-dependent translation is inhibited or when specific trans-acting factors that allow for IRES-driven expression are present. Thus, the activity of a Notch2 IRES could potentially be regulated in a cell type- or cell cycle-specific manner as has been shown for certain cellular IRESs. Intriguingly, the recently described PITSRLE IRES also maps to the coding region of its mRNA, and IRES-driven expression of a smaller form of the protein is regulated, occurring specifically during the G2/M phase of the cell cycle (Lauring, 2000 and references therein).

It is noteworthy that a functional translational start site has been identified in the transmembrane domain of Notch1 in the context of artificially truncated alleles. Given the importance of the Notch proteins in directing cell fate decisions in many lineages at multiple stages of development, an alternative means of generating the NICD in a ligand-independent, cell-specific manner would provide another layer of regulation for this critical pathway (Lauring, 2000 and references therein).

Notch receptors are involved in cell-fate determination in organisms as diverse as flies, frogs and humans. In Drosophila, loss-of-function mutations of Notch produce a 'neurogenic' phenotype in which cells destined to become epidermis switch fate and differentiate to neural cells. Upon ligand activation, the intracellular domain of Notch (ICN) translocates to the nucleus, and interacts directly with the DNA-binding protein Suppressor of hairless [Su(H)] in flies, or recombination signal binding protein Jkappa (RBP-Jkappa) in mammals, to activate gene transcription. But the precise mechanisms of Notch-induced gene expression are not completely understood. The gene mastermind has been identified in multiple genetic screens for modifiers of Notch mutations in Drosophila. MAML1, a human homolog of the Drosophila gene Mastermind, has been cloned; it encodes a protein of 130 kD localizing to nuclear bodies. MAML1 binds to the ankyrin repeat domain of all four mammalian NOTCH receptors, forms a DNA-binding complex with ICN and RBP-Jkappa, and amplifies NOTCH-induced transcription of HES1. These studies provide a molecular mechanism to explain the genetic links between mastermind and Notch in Drosophila and indicate that MAML1 functions as a transcriptional co-activator for NOTCH signaling (Wu, 2000).

The Notch receptor that plays an important role in cell fate determination is intracellularly cleaved by interaction with the ligand. The cleaved intracellular region (RAMIC) of Notch is translocated into the nucleus and interacts with a DNA-binding protein RBP-J to activate transcription of genes that regulate cell differentiation. Although RAMIC has been shown to facilitate the RBP-J-mediated transactivation by displacing the histone deacetylase corepressor complex from RBP-J, there is no evidence demonstrating the involvement of histone acetyltransferases (HATs) in the transactivation. Mouse Notch1 RAMIC interacts with two conserved HATs, mouse PCAF and GCN5, and recruits each of the HATs to RBP-J. The ankyrin repeats and the transactivation domain of RAMIC and the N-terminal regions of PCAF and GCN5, respectively, are required for the interaction. Both mouse Notch1 and Drosophila Notch RAMIC interacts with mouse PCAF and GCN5 in mammalian cells. Furthermore, the RBP-J-mediated transactivation activity of RAMIC is repressed by two HAT inhibitor proteins, E1A and Twist. These results suggest that HATs including PCAF and GCN5 play an important role in the RBP-J-mediated transactivation by RAMIC (Kurooka, 2000).

Presenilin is an essential component of the LIN-12/Notch signaling pathway and also plays a critical role in the genesis of Alzheimer's disease. Previously, a screen for suppressors of the egg-laying defective phenotype caused by partial loss of presenilin activity in Caenorhabditis elegans identified a number of new spr genes that are potentially involved in the regulation of LIN-12/Notch signaling or presenilin activity. The molecular identity of two spr genes, spr-1 and spr-5, is reported in this study. Genetic analysis indicates that loss of spr-1 elevates lin-12/Notch gene activity in many different cell fate decisions, suggesting that spr-1 is a negative regulator of LIN-12/Notch signaling. Sequence analysis revealed that spr-1 is an ortholog of human CoREST (Drosophila homolog: CG3878), a known corepressor. SPR-1 is localized to the nucleus and acts in a cell-autonomous manner; furthermore, human CoREST can substitute for SPR-1 in C. elegans. spr-5 encodes a homolog of p110b, another known member of the CoREST corepressor complex. These results suggest that the CoREST corepressor complex might be functionally conserved in worms, and the potential role of SPR-1 and SPR-5 in the repression of transcription of genes involved in, or downstream of, LIN-12/Notch signal transduction is discussed (Jarriault, 2002).

One model for how SPR-1 and SPR-5 might contribute to repression of LIN-12 target genes is that the SPR-1/SPR-5 complex acts as a corepressor of LAG-1 in the absence of LIN-12/Notch activation, and is displaced upon LIN-12/Notch activation. This model is analogous to what has been proposed for CBF1 and SMRT. Although no interaction has been detected between CoREST and CBF1 in mammalian cells, it may be that the presence of CBF1 in association with the CoREST complex is evident only under certain conditions. Another model is that SPR-1/CoREST and SPR-5/p110b are instead part of a corepressor complex that binds to LIN-12/Notch target genes through a sequence that is distinct from the LAG-1/CBF1 binding site. Therefore, there may be a specific DNA binding protein, which recruits the SPR-1/CoREST complex to LIN-12/Notch target genes. This model allows for potential cooperativity between the corepressor complex associated with LAG-1/CBF1 and the SPR-1/CoREST complex associated with factor X. CoREST has been shown to associate with at least two different zinc finger DNA-binding proteins, REST and ZNF217, suggesting that CoREST may have multiple DNA binding protein partners. In this context, it is interesting to note that spr-3 and spr-4 encode zinc finger proteins. Although these do not appear to be orthologous to either REST or ZNF217, they might represent the hypothetical DNA binding factor X that targets SPR-1 and SPR-5 to DNA (Jarriault, 2002).

The Lin12/Notch receptors regulate cell fate during embryogenesis by activating the expression of downstream target genes. These receptors signal via their intracellular domain (ICD), which is released from the plasma membrane by proteolytic processing and associates in the nucleus with the CSL family of DNA-binding proteins to form a transcriptional activator. How the CSL/ICD complex activates transcription and how this complex is regulated during development remains poorly understood. Nrarp is a new intracellular component of the Notch signaling pathway in Xenopus embryos. Nrarp is a member of the Delta-Notch synexpression group and encodes a small protein containing two ankyrin repeats. Nrarp expression is activated in Xenopus embryos by the CSL-dependent Notch pathway. Conversely, overexpression of Nrarp in embryos blocks Notch signaling and inhibits the activation of Notch target genes by ICD. Nrarp forms a ternary complex with the ICD of XNotch1 and the CSL protein XSu(H) and in embryos Nrarp promotes the loss of ICD. By down-regulating ICD levels, Nrarp could function as a negative feedback regulator of Notch signaling that attenuates ICD-mediated transcription (Lamar, 2001).

Both Nrarp and Mastermind form ternary complexes with the CSL proteins and ICD. It was asked, therefore, whether the binding of Nrarp and Mastermind to Su(H) and ICD is mutually exclusive or whether these proteins can exist in a complex together. Binding of human Mastermind (hMM) to XSu(H) and ICD from XNotch1 was examined first by co-IP analysis in which extracts were prepared from embryos injected with RNA encoding Flag-tagged XSu(H), myc-tagged hMM, and myc-tagged ICDDeltaC. Flag-tagged XSu(H) was recovered from total extracts, and associated proteins were analyzed by Western analysis. The results show that hMM is co-IPed detectably with XSu(H), but only in the presence of ICDDeltaC. Moreover, the amounts of ICDDeltaC associated with XSu(H) in a co-IP complex increase markedly in the presence of hMM. Both of these results are consistent with the idea that hMM binds to XSu(H) and ICD in a ternary complex, as reported by others. Myc-tagged Nrarp is also co-IPed with XSu(H) in the presence of hMM and ICDDeltaC, consistent with the formation of multimeric complexes. However, this finding is inconclusive as to whether these proteins can form a quaternary complex. To address this issue, embryos were injected with RNA encoding Flag-tagged Nrarp along with myc-tagged hMM, XSu(H), and ICD. Flag-tagged Nrarp was recovered from extracts by immunoprecipitation, and associated proteins were analyzed by Western analysis using an alpha-myc antibody. The results show that the immunoprecipitation of Nrarp recovers not only XSu(H) and ICD, but hMM as well, indicating that Nrarp and hMM can bind in tandem to XSu(H)/ICD to form a quaternary complex (Lamar, 2001).

Although Nrarp may contribute to the formation of an active Su(H)/ICD transcriptional complex, it is proposed that the primary role of Narp is to regulate Notch signaling by promoting the degradation of ICD. Several lines of evidence indicate that only very small amounts of ICD need to be released from the membrane to produce maximal stimulation of Notch target genes. For example, ICD is very difficult to detect in the nucleus of cells undergoing Notch signaling even though these levels of ICD are apparently sufficient to produce significant changes in gene expression. One would predict, therefore, that under normal physiological conditions Nrarp is likely to have a dramatic effect on ICD-mediated transcription, in which a low level of ICD is already a rate-limiting factor. Moreover, Nrarp binds to ICD only when it is part of a complex with Su(H), suggesting that only after ICD forms an active transcriptional complex does it become a target of degradation via Nrarp. This mechanism is consistent with recent findings showing that activation of some transcription factors is coupled to their degradation. For example, the melanocyte factor Mi is phosphorylated in response to c-Kit signaling, resulting in both recruitment of coactivator proteins and ubiquitination and subsequent degradation of Mi. Similarly, ligand binding converts the estrogen receptor into a transcriptional activator but also promotes its degradation (Lamar, 2001).

The ability of Nrarp to regulate the levels of ICD may constitute a negative feedback loop that attenuates Notch signaling, assuring that activation of Notch target genes is transient. Notch is used ubiquitously to amplify differences between cells, thus allowing certain patterns of cell fate to be established. As a result, Notch signaling is often used in rapid succession to control multiple cell fate decisions in the same lineage. A mechanism for down-regulating the levels of ICD produced after each decision by Nrarp would allow each decision to remain separate from the next. This rapid resetting of the Notch signaling pathway is particularly evident during segmentation of the paraxial mesoderm in vertebrate embryos, in which the transcription of a number of Notch pathway genes has been shown to oscillate in hourly intervals with a pattern suggestive of a segmental clock. The role of Nrarp in the formation of this periodic pattern of gene expression is of particular interest based on its expression in the presomitic mesoderm and its potential role in down-regulating the levels of ICD required for Notch-mediated transcription (Lamar, 2001).

Studies of mammalian homolog of Mastermind prove insight into the molecular interactions of Mastermind as a co-activator in the Notch pathway. Signaling through the Notch pathway activates the proteolytic release of the Notch intracellular domain (ICD), a dedicated transcriptional coactivator of CSL (CBF-1, Suppressor of Hairless, and Lag-1) enhancer-binding proteins. Chromatin-dependent transactivation by the recombinant Notch ICD-CBF1 enhancer complex in vitro requires an additional coactivator, Mastermind (MAM). MAM provides two activation domains necessary for Notch signaling in mammalian cells and in Xenopus embryos. The central MAM activation domain (TAD1) recruits CBP/p300 (Drosophila homolog: Nejire) to promote nucleosome acetylation at Notch enhancers and activate transcription in vitro. MAM expression induces phosphorylation and relocalization of endogenous CBP/p300 proteins to nuclear foci in vivo. Moreover, coexpression with MAM and CBF1 strongly enhances phosphorylation and proteolytic turnover of the Notch ICD in vivo. Enhanced phosphorylation of the ICD and p300 requires a glutamine-rich region of MAM (TAD2) that is essential for Notch transcription in vivo. Thus MAM may function as a timer to couple transcription activation with disassembly of the Notch enhancer complex on chromatin (Fryer, 2002).

Unexpectedly, expression of MAM induces endogenous CBP/p300 proteins to accumulate in multiple nuclear foci in vivo. These structures do not form upon expression of a mutant MAM protein lacking the C-terminal TAD2 region (1-301MM). Thus, binding of MAM to CBP/p300, which is mediated through TAD1, is not sufficient to cause CBP/p300 to accumulate in these structures. Expression of other Notch components (ICD, CBF1) did not affect the subnuclear localization of CBP/p300, indicating that these foci are not a consequence of high levels of Notch signaling in the nucleus. One possibility is that MAM may regulate the expression or modification of CBP/p300 independently of Notch signaling. Indeed, the MAM-induced foci are accompanied by increased phosphorylation of CBP, and this phosphorylation requires the C-terminal TAD2 domain of MAM. Consequently, overexpression of MAM in the nucleus may promote widespread phosphorylation of CBP, which may cause the CBP/p300 proteins to concentrate in these structures. Changes in CBP/p300 phosphorylation have been shown to alter its activity and differentially affect its interactions with other transcription factors. It will therefore be important to assess whether MAM promotes CBP/p300 phosphorylation within the Notch enhancer complex, and whether phosphorylation of CBP/p300 is important for transcriptional activation by Notch (Fryer, 2002).

The timing of Notch signaling is tightly controlled in developmental processes such as somite formation, during which Notch target genes such as cHairy1 and mHES1 undergo periodic cycles of expression at the direction of a molecular oscillator, or vertebrate segmentation clock. This clock may be established through the intrinsic timing of Notch signaling as well as the half-life of Notch-induced transcriptional repressors. The Notch ICD is subject to proteolytic degradation in the nucleus through the action of the ubiquitin ligases such as Sel-10. Rapid turnover of the ICD may be required to allow genes to respond rapidly to subsequent cycles of Notch signaling. Coexpression with MAM and CBF1 promotes the phosphorylation and proteolytic turnover of the ICD in vivo, indicating that MAM couples transcription activation with degradation of the ICD. In this respect, MAM may act as a timer to control the length of time that the Notch complex remains associated with the enhancer. By extension, MAM might contribute to the periodic expression of Notch target genes during somitogenesis through its potential effects on the disassembly of the Notch enhancer complex (Fryer, 2002).

The data indicate that CBF1 acts in concert with MAM to control the proteolytic turnover of the ICD in vivo. Importantly, both MAM and CBF1 appear to be stable upon coexpression with the ICD, and thus it appears that the ICD can be destabilized independently of its interacting partners. The requirement for CBF1 may reflect its ability to enhance binding of MAM to the ICD, or alternatively CBF1 might be needed to target the Notch enhancer complex to DNA. Stability of a mutant ICD protein lacking the PEST domain is unaffected by coexpression with MAM and CBF1, and turnover is accompanied by increased phosphorylation of the ICD. Importantly, the MAM TAD2 domain is necessary for both enhanced phosphorylation and turnover of the ICD. Because p300 has been shown to be critical for the regulated turnover of the p53 transactivator by MDM2, it will be important to assess whether recruitment of p300 by MAM may similarly be required for proteolytic degradation of the ICD. Nevertheless, it is clear that recruitment of CBP/p300 through the MAM TAD1 region is not sufficient to couple activation with turnover of the Notch ICD under the conditions examined in this study (Fryer, 2002).

Thus the TAD2 region is required for MAM to promote the phosphorylation of its two associated factors, CBP/p300 and the Notch ICD. Because MAM does not possess intrinsic ICD protein kinase activity, it is attractive to consider that the Notch ICD and CBP/p300 may instead be targeted for phosphorylation by cyclin-dependent kinases that associate with the transcription complex and are recruited to the promoter by MAM. Phosphorylation events mediated by CDK7 and Srb10 (the CDK8 homolog in yeast) have been implicated in the proteolytic destruction of other enhancer factors. The CDK9 subunit of the positive transcription elongation factor, P-TEFb, also associates with RNAPII, whereas CDK8 interacts with RNAPII as a component of human and yeast mediator complexes that have been variously implicated in activation and repression of transcription. Another possibility is that the ICD is phosphorylated by a protein kinase that associates with MAM directly. It remains to be determined whether the MAM-induced phosphorylation is accompanied by increased ubiquitination of the ICD, and whether the degradation of the ICD observed is caused by ubiquitin-dependent proteolysis such as that described for the nuclear Sel-10 ubiquitin ligase. It will also be important to learn whether modification of the ICD regulates its transcriptional activity, as has been observed for other transcription factors, and whether these steps may ultimately be coupled to disassembly of the Notch enhancer complex and turnover of the Notch ICD (Fryer, 2002).

In summary, MAM is an essential component of the Notch enhancer complex in vitro as well as in vivo. The human MAM protein recruits p300/CBP to the Notch enhancer complex and controls the stability of the Notch ICD through the action of its unique C-terminal activation domain. Further studies will be needed to evaluate whether these properties are shared among the various MAM proteins in different species, and to learn how MAM-induced phosphorylation of the ICD and CBP/p300 proteins is coordinated with the regulation of Notch transcription (Fryer, 2002).

After ligand binding, Notch receptors are cleaved to release their intracellular domains. The intracellular domains, the activated form of Notch receptors, are then translocated into the nucleus where they interact with other transcriptional machinery to regulate the expression of cellular genes. To dissect the molecular mechanisms of Notch signaling, the cellular targets that interact with Notch1 receptor intracellular domain (N1IC) were screened. Endogenous transcription factor Ying Yang 1 (YY1) is associated with exogenous N1IC in human K562 erythroleukemic cells. The ankyrin (ANK) domain of N1IC and zinc finger domains of YY1 are essential for the association of N1IC and YY1 according to the pull-down assay of glutathione S-transferase fusion proteins. Furthermore, both YY1 and N1IC are present in a large complex of the nucleus to suppress the luciferase reporter activity transactivated by Notch signaling. The transcription factor YY1 indirectly regulates the transcriptional activity of the wild-type CBF1-response elements via the direct interaction of N1IC and CBF1. The association between endogenous N1IC and intrinsic YY1 has also been demonstrated in human acute T-cell lymphoblastic leukemia cell lines. Taken together, these results indicate that transcription factor YY1 may modulate Notch signaling via association with the high molecular weight Notch complex (Yeh, 2003).

Targets of Notch signaling

A gene encoding a novel protein has been identified that is transcriptionally regulated by the Notch signaling pathway in mammals. This gene, named Nrarp (for Notch-regulated ankyrin-repeat protein), encodes a 114 amino acid protein that has a unique amino-terminus and a carboxy-terminal domain containing two ankyrin-repeat motifs. A Xenopus homolog of the Nrarp gene was previously identified in a large-scale in situ hybridization screen of randomly isolated cDNA clones. In T-cell and myoblast cell lines, expression of the Nrarp gene is induced by the intracellular domain of the Notch1 protein, and this induction is mediated by a CBF1/Su(H)/Lag-1 (CSL)-dependent pathway. During mouse embryogenesis, the Nrarp gene is expressed in several tissues in which cellular differentiation is regulated by the Notch signaling pathway. Expression of the Nrarp gene is downregulated in Notch1 null mutant mouse embryos, indicating that expression of the Nrarp gene is regulated by the Notch pathway in vivo. Thus, Nrarp transcript levels are regulated by the level of Notch1 signaling in both cultured cell lines and mouse embryos. During somitogenesis, the Nrarp gene is expressed in a pattern that suggests that Nrarp expression may play a role in the formation of somites, and Nrarp expression in the paraxial mesoderm is altered in several Notch pathway mutants that exhibit defects in somite formation. These observations demonstrate that the Nrarp gene is an evolutionarily conserved transcriptional target of the Notch signaling pathway (Krebs, 2001).

Generation of left-right asymmetry is an integral part of the establishment of the vertebrate body plan. The Notch signaling pathway plays a primary role in the establishment of left-right asymmetry in mice by directly regulating expression of the Nodal gene. Embryos mutant for the Notch ligand Dll1 or doubly mutant for the Notch1 and Notch2 receptors exhibit multiple defects in left-right asymmetry. Analysis of the enhancer regulating node-specific Nodal expression reveals the presence of two consensus binding sites (T/CGTGGGAA) for the RBP-J protein. Electrophoretic mobility shift assays confirms that these consensus RBP-Ji sites bind recombinant RBP-J protein. Analysis of RBP-J RNA expression during mouse embryogenesis reveals that RBP-J is expressed ubiquitously, including in node cells that express the Nodal gene. Mutation of the RBP-J-binding sites destroys the ability of this enhancer to direct node-specific gene expression in transgenic mice. These results demonstrate that Dll1-mediated Notch signaling is essential for generation of left-right asymmetry, and that the Notch pathway acts upstream of Nodal expression during left-right asymmetry determination in mice (Luke, 2003).

In neural development, Notch signaling plays a key role in restricting neuronal differentiation, promoting the maintenance of progenitor cells. Classically, Notch signaling causes transactivation of Hairy-enhancer of Split (HES) genes, which leads to transcriptional repression of neural determination and differentiation genes. In addition to its known transcriptional mechanism, Notch signaling also leads to rapid degradation of the basic helix-loop-helix (bHLH) transcription factor human achaete-scute homolog 1 (hASH1). Using recombinant adenoviruses expressing active Notch1 in small-cell lung cancer cells, it has been shown that the initial appearance of Notch1 coincides with the loss of hASH1 protein, preceding the full decay of hASH1 mRNA. Overexpression of HES1 alone is capable of down-regulating hASH1 mRNA but can not replicate the acute reduction of hASH1 protein induced by Notch1. When adenoviral hASH1 is coinfected with Notch1, a dramatic and abrupt loss of the exogenous hASH1 protein is still observed, despite high levels of ongoing hASH1 RNA expression. Notch1 treatment decreases the apparent half-life of the adenoviral hASH1 protein and increases the fraction of hASH1 that is polyubiquitinylated. The proteasome inhibitor MG132 reverses the Notch1-induced degradation. The Notch RAM domain is dispensable but a lack of the OPA and PEST domains inactivates this Notch1 action. Overexpression of the hASH1-dimerizing partner E12 protects hASH1 from degradation. This novel function of activated Notch to rapidly degrade a class II bHLH protein may prove to be important in many contexts in development and in cancer (Sriuranpong, 2002).

Notch signals are important for lymphocyte development but downstream events that follow Notch signaling are not well understood. Signaling through Notch modulates the turnover of E2A proteins including E12 and E47, which are basic helix-loop-helix proteins crucial for B and T lymphocyte development. Notch-induced degradation requires phosphorylation of E47 by p42/p44 MAP kinases. Expression of the intracellular domain of Notch1 (N1-IC) enhances the association of E47 with the SCF(Skp2) E3 ubiquitin ligase and ubiquitination of E47, followed by proteasome-mediated degradation. Furthermore, N1-IC induces E2A degradation in B and T cells in the presence of activated MAP kinases. Activation of endogenous Notch receptors by treatment of splenocytes with anti-IgM or anti-CD3 plus anti-CD28 also leads to E2A degradation, which is blocked by the inhibitors of Notch activation or proteasome function. Notch-induced E2A degradation depends on the function of its downstream effector, RBP-Jkappa, probably to activate target genes involved in the ubiquitination of E2A proteins. Thus it is proposed that Notch regulates lymphocyte differentiation by controlling E2A protein turnover (Nie, 2003).

In addition to controlling a switch to glycolytic metabolism and induction of erythropoiesis and angiogenesis, hypoxia promotes the undifferentiated cell state in various stem and precursor cell populations. The latter process requires Notch signaling. Hypoxia blocks neuronal and myogenic differentiation in a Notch-dependent manner. Hypoxia activates Notch-responsive promoters and increases expression of Notch direct downstream genes. The Notch intracellular domain (ICD) interacts with HIF-1alpha, a global regulator of oxygen homeostasis, and HIF-1alpha is recruited to Notch-responsive promoters upon Notch activation under hypoxic conditions. Taken together, these data provide molecular insights into how reduced oxygen levels control the cellular differentiation status and demonstrate a role for Notch in this process (Gustafsson, 2005).

The data presented here indicate that Notch ICD and HIF-1α are important at the convergence point between the two signaling mechanisms. The importance of Notch ICD is underlined by the ability of γ-secretase inhibitors, which block the S3 cleavage of the Notch receptor and thus liberation of Notch ICD, to strongly reduce the hypoxic response on Notch downstream genes and promoters. Furthermore, the signaling output from an exogenously introduced Notch 1 ICD was modified by hypoxia, leading to increased activation of 12XCSL-luc and Hes-luc in a Notch 1 ICD-dependent manner. The importance of HIF-1α in this process receives support from the observed direct physical interaction between HIF-1α and Notch 1 ICD, the lack of an hypoxia-induced effect on Notch signaling in fibroblasts devoid of HIF-1α, and that both the amount and activity status of HIF-1α correlate with the level of Notch activation. The latter notion is based on the observations that: (1) transfected HIF-1α elevates the Notch downstream response; (2) a transactivation-inactive form of HIF-1α leaves the Notch response unchanged, and (3) the response is augmented in cells lacking VHL. Finally, HIF-1α is recruited to the Hey-2 promoter in C2C12 cells in a Notch- and hypoxia-dependent manner. This suggests a mechanism involving an effect of direct transcriptional activation of a Notch-responsive promoter by HIF-1α, probably as part of a Notch ICD/CSL transcriptional complex. This model is consistent with the observation that a transcriptionally inactive form of HIF-1α, which was capable of interacting with Notch 1 ICD, does not augment the Notch downstream response. It is therefore reasoned that hypoxia-dependent stabilization of Notch 1CD is not sufficient for activation of the Notch response but may require the recruitment of a form of HIF-1α containing the C-terminal transactivation domain to the Notch ICD/CSL regulatory complex, possibly potentiating the interaction with transcriptional coactivators. This hypothesis is based on the finding that HIF-1α is recruited to promoters of Notch downstream genes and the observation that a mutated form of Notch ICD, unable to interact with CBP/p300, is transcriptionally active at hypoxia. In this context, it will be interesting to learn whether HIF-1β also participates in such a regulatory complex (Gustafsson, 2005).

The link between hypoxia and Notch described here may have ramifications for other aspects of hypoxia, such as tumor development, in which deregulation of both HIF-1α- and Notch-mediated signaling events have been implicated. Since many tumors show elevated expression of HIF-1α, caused by hypoxia inherent to growing tumors and/or genetic loss of VHL, it will be interesting to investigate whether the elevated levels of HIF-1α are paralleled by increased Notch signaling, and whether the ensuing Notch induction contributes to tumor development (Gustafsson, 2005).

In conclusion, the data presented here demonstrate a link between hypoxia and Notch signaling and provide insights into how hypoxia maintains the undifferentiated cell state, by using the Notch signaling mechanism. The data also point to an important role for HIF-1α in this process and to the fact that it can interact with the Notch intracellular domain to link hypoxic information to a Notch response. These data advance the understanding of how Notch crosstalks with other signaling mechanisms and may open up possibilities to control various aspects of the hypoxic response by experimentally manipulating Notch signaling (Gustafsson, 2005).

Human acute T-cell lymphoblastic leukemias and lymphomas (T-ALL) are commonly associated with gain-of-function mutations in Notch1 that contribute to T-ALL induction and maintenance. Starting from an expression-profiling screen, c-myc was identified as a direct target of Notch1 in Notch-dependent T-ALL cell lines, in which Notch accounts for the majority of c-myc expression. In functional assays, inhibitors of c-myc interfere with the progrowth effects of activated Notch1, and enforced expression of c-myc rescues multiple Notch1-dependent T-ALL cell lines from Notch withdrawal. The existence of a Notch-c-myc signaling axis was bolstered further by experiments using c-myc-dependent murine T-ALL cells, which are rescued from withdrawal of c-myc by retroviral transduction of activated Notch1. This Notch1-mediated rescue is associated with the up-regulation of endogenous murine c-myc and its downstream transcriptional targets, and the acquisition of sensitivity to Notch pathway inhibitors. Additionally, this study shows that primary murine thymocytes at the DN3 stage of development depend on ligand-induced Notch signaling to maintain c-myc expression. Together, these data implicate c-myc as a developmentally regulated direct downstream target of Notch1 that contributes to the growth of T-ALL cells (Weng, 2006; full text of article).

Notch and epidermal patterning

Hair follicle development and hair formation involve the co-ordinated differentiation of several different cell types in which Notch appears to have a role. Intricate expression patterns for the Notch-1 receptor and three ligands, Delta-1, Jagged-1 and Jagged-2 in the hair follicle are reported. Notch-1 is expressed in ectodermal-derived cells of the follicle, in the inner cells of the embryonic placode and the follicle bulb, and in the suprabasal cells of the mature outer root sheath. Delta-1 is only expressed during embryonic follicle development and is exclusive to the mesenchymal cells of the pre-papilla located beneath the follicle placode. Expression of Jagged-1 or Jagged-2 overlaps Notch-1 expression at all stages. In mature follicles, Jagged-1 and Jagged-2 are expressed in complementary patterns in the follicle bulb and outer root sheath; Jagged-1 in suprabasal cells and Jagged-2 predominantly in basal cells. In the follicle bulb, Jagged-2 is localized to the inner (basal) bulb cells next to the dermal papilla, which do not express Notch-1, whereas Jagged-1 expression in the upper follicle bulb overlaps Notch-1 expression and correlates with bulb cell differentiation into hair shaft cortical and cuticle keratinocytes (Powell, 1998).

Little is known about the mechanisms underlying the generation of various cell types in the hair follicle. To investigate the role of the Notch pathway in this process, transgenic mice were generated in which an active form of Notch1 (Notch^DeltaE) was overexpressed under the control of the mouse hair keratin A1 (MHKA1) promoter. MHKA-Notch^DeltaE is expressed only in one precursor cell type of the hair follicle, the cortex. Transgenic mice could be easily identified by the phenotypes of curly whiskers and wavy, sheen pelage hair. No effects of activated Notch on proliferation were detected in hair follicles of the transgenic mice. Activating Notch signaling in the cortex causes abnormal differentiation of the medulla and the cuticle, two neighboring cell types that do not express activated Notch. These non-autonomous effects, which are likely caused by cell-cell interactions between keratinocytes within the hair follicle and Notch, may function in such interactions either by directing the differentiation of follicular cells or assisting cells in interpreting a gradient emanating from the dermal papilla (Lin, 2000).

Notch and neural diversity

The ventral spinal cord generates multiple inhibitory and excitatory interneuron subtypes from four cardinal progenitor domains (p0, p1, p2, p3). Cell-cell interactions mediated by the Notch receptor play a critical evolutionarily conserved role in the generation of excitatory v2aIN and inhibitory v2bIN interneurons. Lineage-tracing experiments show that the v2aIN and v2bIN develop from genetically identical p2 progenitors. The p2 daughter cell fate is controlled by Delta4 activation of Notch receptors together with MAML factors. Cells receiving Notch signals activate a transcription factor code that specifies the v2bIN fate, whereas cells deprived of Notch signaling express another code for v2aIN formation. Thus, this study provides insight into the cell-extrinsic signaling that controls combinatorial transcription factor profiles involved in regulating the process of interneuron subtype diversification (Peng, 2007).

Notch signaling plays a well-described role in regulating the formation of neurons from proliferative neural precursors in vertebrates but whether, as in flies, it also specifies sibling cells for different neuronal fates is not known. Ventral spinal cord precursors called pMN cells produce mostly motoneurons and oligodendrocytes, but recent lineage-marking experiments reveal that they also make astrocytes, ependymal cells and interneurons. Clonal analysis of pMN cells in zebrafish showed that some produce a primary motoneuron and KA' interneuron at their final division. The possibility was investigated that Notch signaling regulates a motoneuron-interneuron fate decision using a combination of mutant, transgenic and pharmacological manipulations of Notch activity. Continuous absence of Notch activity produces excess primary motoneurons and a deficit of KA' interneurons, whereas transient inactivation preceding neurogenesis results in an excess of both cell types. By contrast, activation of Notch signaling at the neural plate stage produces excess KA' interneurons and a deficit of primary motoneurons. Furthermore, individual pMN cells produce similar kinds of neurons at their final division in mib mutant embryos, which lack Notch signaling. These data provide evidence that, among some postmitotic daughters of pMN cells, Notch promotes KA' interneuron identity and inhibits primary motoneuron fate, raising the possibility that Notch signaling diversifies vertebrate neuron type by mediating similar binary fate decisions (Shin, 2007).

Notch, cell cycle and transformation

Notch genes encode a family of transmembrane proteins that are involved in many cellular processes such as differentiation, proliferation, and apoptosis. Although it is well established that all four Notch genes can act as oncogenes, the mechanism by which Notch proteins transform cells remains unknown. Transformation of RKE cells can be conditionally induced by hormone activation of Notch^ic-estrogen receptor (ER) chimeras. Using this inducible system, it has been shown that Notch^ic activates transcription of the cyclin D1 gene with rapid kinetics. Transcriptional activation of cyclin D1 is independent from serum-derived growth factors and de novo synthesis of secondary transcriptional activators. Moreover, hormone activation of Notch^ic-ER proteins induces CDK2 activity in the absence of serum. Upregulation of cyclin D1 and activation of CDK2 by Notch^ic result in the promotion of S-phase entry. These data demonstrate the first evidence that Notch^ic proteins can directly regulate factors involved in cell cycle control and affect cellular proliferation. Furthermore, nontransforming Notch^ic proteins do not induce cyclin D1 expression, indicating that the mechanism of transformation involves cell cycle deregulation through constitutive expression of cyclin D1. A CSL binding site has been identified within the human and rat cyclin D1 promoters, suggesting that Notch^ic proteins activate cyclin D1 transcription through a CSL-dependent pathway (Ronchini, 2001).

Notch1-deficient epidermal keratinocytes become progressively hyperplastic and eventually produce tumors. By contrast, Notch1-deficient hair matrix keratinocytes have lower mitotic rates, resulting in smaller follicles with fewer cells. In addition, the ratio of melanocytes to keratinocytes is greatly reduced in hair follicles. Investigation into the underlying mechanism for these phenotypes revealed significant changes in the Kit, Tgfβ and insulin-like growth factor (IGF) signaling pathways, which have not been previously shown to be downstream of Notch signaling. The level of Kitl (Scf) mRNA produced by Notch1-deficient follicular keratinocytes was reduced when compared with wild type, resulting in a decline in melanocyte population. Tgfβ ligands were elevated in Notch1-deficient keratinocytes, which correlated with elevated expression of several targets, including the diffusible IGF antagonist Igfbp3 in the dermal papilla. Diffusible stromal targets remained elevated in the absence of epithelial Tgfβ receptors, consistent with paracrine Tgfβ signaling. Overexpression of Igf1 in the keratinocyte reversed the phenotype, as expected if Notch1 loss altered the IGF/insulin-like growth factor binding protein (IGFBP) balance. Conversely, epidermal keratinocytes contained less stromal Igfbp4 and might thus be primed to experience an increase in IGF signaling as animals age. These results suggest that Notch1 participates in a bi-compartmental signaling network that controls homeostasis, follicular proliferation rates and melanocyte population within the skin (Lee, 2007).

In human acute lymphoblastic T-cell leukemia, a chromosomal translocation damages the NOTCH1 gene. The damage apparently gives rise to a constitutively activated version of NOTCH protein. A truncated version of NOTCH1 protein resembling that found in the leukemic cells can transform rat kidney cells in vitro. The transformation requires cooperation with the E1A oncogene of adenovirus. The transforming version of NOTCH protein is located in the nucleus. In contrast, neither wild-type NOTCH protein nor a form of the truncated protein permanently anchored to the plasma membrane produces transformation in vitro. It is concluded that constitutive activation of NOTCH similar to that found in human leukemia can contribute to neoplastic transformation. Transformation may require that the NOTCH protein be translocated to the nucleus. These results sustain a current view of how Notch transduces a signal from the surface of the cell to the nucleus (Capobianco, 1997).

The Notch family of cell surface receptors plays a key role in cell-fate determination and differentiation, functioning in a cell- and context-specific manner. In mammalian cells, Notch activation is generally thought to maintain stem cell potential and inhibit differentiation, thereby promoting carcinogenesis. However, in other contexts such as primary epithelial cells (keratinocytes), increased Notch activity causes exit from the cell cycle and/or commitment to differentiation. Expression of the endogenous Notch1 gene is markedly reduced in a panel of cervical carcinoma cells whereas expression of Notch2 remains elevated, and Notch1 expression is similarly reduced or absent in invasive cervical cancers. Conversely, expression of activated Notch1 causes strong growth inhibition of HPV-positive, but not HPV-negative, cervical carcinoma cells, but exerts no such effects on other epithelial tumor cells. Increased Notch1 signaling, but not Notch2, causes a dramatic down-modulation of HPV-driven transcription of the E6/E7 viral genes, through suppression of AP-1 activity by up-regulation of the Fra-1 family member and decreased c-Fos expression. Thus, Notch1 exerts specific protective effects against HPV-induced transformation through suppression of E6/E7 expression, and down-modulation of Notch1 expression is likely to play an important role in late stages of HPV-induced carcinogenesis (Talora, 2002).

Notch and developmental disorders

Alagille syndrome is a human autosomal dominant developmental disorder characterized by liver, heart, eye, skeletal, craniofacial and kidney abnormalities. Alagille syndrome is caused by mutations in the Jagged 1 (JAG1) gene, which encodes a ligand for Notch family receptors. The majority of JAG1 mutations seen in Alagille syndrome patients are null alleles, suggesting JAG1 haploinsufficiency is a primary cause of this disorder. Mice homozygous for a Jag1 null mutation die during embryogenesis and Jag1/+ heterozygous mice exhibit eye defects but do not exhibit other phenotypes characteristic of Alagille syndrome patients. Mice doubly heterozygous for the Jag1 null allele and a Notch2 hypomorphic allele exhibit developmental abnormalities characteristic of Alagille syndrome. Double heterozygous mice exhibit jaundice, growth retardation, impaired differentiation of intrahepatic bile ducts and defects in heart, eye and kidney development. The defects in bile duct epithelial cell differentiation and morphogenesis in the double heterozygous mice are similar to defects in epithelial morphogenesis of Notch pathway mutants in Drosophila, suggesting that a role for the Notch signaling pathway in regulating epithelial morphogenesis has been conserved between insects and mammals. In the majority of glomeruli of mutant mice, glomerular podocyte precursors differentiate but do not epithelialize, remaining instead as a dysmorphic aggregate of cells). This work also demonstrates that the Notch2 and Jag1 mutations interact to create a more representative mouse model of Alagille syndrome and provides a possible explanation of the variable phenotypic expression observed in Alagille syndrome patients (McCright, 2002).

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