Table of contents

C. elegans Notch homologs

Notch is homologous to C. elegans lin-12 (Kidd, 1986) and glp-1 (Mello, 1994). There are three mammalian homologs as well (Larsson, 1994, Lardelli, 1994 and Higuchi, 1995).

Interactions mediated by Notch and Delta homologs contribute to the establishment of the dorsal-ventral axis in the early C. elegans embryo. The sister blastomeres ABp and ABa are equipotent at the beginning of the 4-cell stage in C. elegans embryos, but soon become committed to different fates. The glp-1 gene, a homolog of the Notch gene of Drosophila, functions in two distinct cell-cell interactions that specify the ABp and ABa fates. These interactions both require maternal expression of glp-1. A second maternal gene, apx-1, functions with glp-1 only in the specification of the ABp fate. apx-1 can encode a protein homologous to the Delta protein of Drosophila (Mello, 1994).

Cell-cell interactions mediated by LIN-12 and GLP-1, members of the LNG (LIN-12, Notch, GLP-1) family of receptors, are required to specify numerous cell fates during development of the nematode Caenorhabditis elegans. Maternally expressed glp-1 participates in two of at least four sequential inductive interactions that specify the fates of early embryonic descendants of the AB founder cell. GLP-1 and LIN-12, and apparently their ligand, LAG-2, as well as a downstream component, LAG-1, are required in the latter two inductions. lag-2 is expressed in the signaling cells and lin-12 is expressed in cells receiving the inductions, consistent with their proposed respective roles as ligand and receptor. Maternal GLP-1 activity is required (1) to repress early zygotic lag-2 expression and (2) to activate zygotic lin-12 expression in the early embryo. The patterning of both receptor and ligand expression by maternal GLP-1 signaling establishes competence for the zygotic LNG-mediated cellular interactions and localizes these interactions to the appropriate cells. It is proposed that activation of maternal GLP-1 regulates zygotic lin-12 and lag-2 expression by a regulatory mechanism analogous to that described for the post-embryonic gonad (Moskowitz, 1996).

C. elegans germ-line proliferation is controlled by an inductive interaction between the somatic distal tip cell and the germ line. GLP-1, a member of the Notch family of transmembrane receptors, is required continuously in the germ line to transduce the proliferative signal. In the absence of GLP-1, all proliferative germ cells exit the mitotic cell cycle and enter meiotic prophase. oz112gf, an activating mutation in glp-1 has been characterized as having the opposite phenotype. Homozygous mutant hermaphrodites and mutant males have a completely tumorous germ line in which germ cells never leave the mitotic cycle. In heterozygotes, germ-line polarity is established correctly, but as adults age, the distal proliferative population expands leading to a late-onset tumorous phenotype. The mutant receptor is constitutively active, promoting proliferation in the absence of ligand. The normal distal-proximal spatial restriction of GLP-1 expression is lost in tumorous and late-onset tumorous animals; ectopically proliferating germ cells contain membrane-associated GLP-1. The correlation between proliferation and expression suggests that GLP-1 signaling positively regulates GLP-1 expression: this is true for both wild-type, where GLP-1 signaling is limited by localized ligand, and in mutants, where signaling is ligand-independent. In addition to germ-line defects, mutation causes inappropriate vulval cell fate specification. A missense mutation in a conserved extracellular residue, Ser642, adjacent to the transmembrane domain, is sufficient to confer the mutant phenotype. Two mammalian Notch family members, TAN-1 and int-3, are proto-oncogenes. Thus, activating mutations in both invertebrate and vertebrate Notch family members can lead to tumor formation (Berry, 1997).

A LIN-12::GFP fusion protein was used to examine LIN-12 accumulation during cell fate decisions important for vulval development. During the naturally variable anchor cell (AC)/ventral uterine precursor cell (VU) decision of the somatic gonad, a transcription-based feedback mechanism biases two equivalent cells so that one becomes the AC while the other becomes a VU. LIN-12::GFP accumulation reflects lin-12 transcription: LIN-12::GFP is initially present in both cells, but disappears from the presumptive AC and becomes restricted to the presumptive VU. During vulval precursor cell (VPC) fate determination, six equipotential cells uniformly transcribe lin-12 and have invariant fates that are specified by multiple cell-cell interactions. The pattern of LIN-12::GFP accumulation in VPCs and in the VPC lineages is dynamic and does not always reflect lin-12 transcription. In particular, LIN-12::GFP is expressed initially in all six VPCs, but appears to be reduced specifically in P6.p as a consequence of the activation of the Ras pathway by an EGF-like inductive signal from the AC. It is proposed that downregulation of LIN-12 stability or translation in response to inductive signalling helps impose a bias on lateral signalling and contributes to the invariant pattern of VPC fates (Levitan, 1998a).

In C. elegans, the GLP-1 receptor acts with a downstream transcriptional regulator, LAG-1, to mediate intercellular signaling. GLP-1 and LAG-1 are homologs of Drosophila Notch and Su(H), respectively. The functions of two regions of the GLP-1 intracellular domain were investigated: the ANK repeat domain, which includes six cdc10/ankyrin repeats plus flanking amino acids, and the RAM domain, which spans approximately 60 amino acids just inside the transmembrane domain. Both ANK and RAM domains interact with the LAG-1 transcription factor. The interaction between the ANK domain and LAG-1 is only observed in nematodes by a co-localization assay and, therefore, may be either direct or indirect. By contrast, the interaction between the RAM domain and LAG-1 is likely to be direct, since it is observed by co-precipitation of the proteins in vitro as well as by yeast two-hybrid experiments. The RAM domain, when expressed in nematodes without a functional ANK repeat domain, does not mimic the unregulated receptor in directing cell fates nor does it interfere with signaling by endogenous components. In yeast the ANK repeats are strong transcriptional activators. Furthermore, missense mutations that eliminate receptor activity also abolish transcriptional activation by the GLP-1 ANK repeats in yeast. One possible function for the ANK repeat domain is to act as a transcriptional co-activator with LAG-1 (Roehl, 1996).

Mutations that influence lin-12 activity in Caenorhabditis elegans may identify conserved factors that regulate the activity of lin-12/Notch proteins. Genetic evidence is described indicating that sel-10 is a negative regulator of lin-12/Notch-mediated signaling in C. elegans. Sequence analysis shows that SEL-10 is a member of the CDC4 family of proteins and has a potential human ortholog. Coimmunoprecipitation data indicate that C. elegans SEL-10 complexes with LIN-12 and with murine Notch4. It is proposed that SEL-10 promotes the ubiquitin-mediated turnover of LIN-12/Notch proteins (Hubbard, 1997).

The ectodomain of LIN-12/Notch proteins is cleaved and shed upon ligand binding. In Caenorhabditis elegans, genetic evidence has implicated SUP-17, the ortholog of Drosophila Kuzbanian and mammalian ADAM10, as the protease that mediates this event. In mammals, however, biochemical evidence has implicated TACE, a different ADAM protein. This study investigated potential functional redundancy of sup-17 and the C. elegans ortholog of TACE, adm-4, by exploring their roles in cell fate decisions mediated by lin-12/Notch genes. It was found that reduced adm-4 activity, like reduced sup-17 activity, suppresses an allele of glp-1 that encodes a constitutively active receptor. Furthermore, concomitant reduction of adm-4 and sup-17 activity causes the production of two anchor cells in the hermaphrodite gonad, instead of one—a phenotype associated with loss of lin-12 activity. Concomitant reduction of both sup-17 and adm-4 activity in hermaphrodites results in highly penetrant synthetic sterility, which appears to reflect a defect in the spermatheca. Expression of a truncated form of LIN-12 that mimics the product of ectodomain shedding rescues this fertility defect, suggesting that sup-17 and adm-4 may mediate ectodomain shedding of LIN-12 and/or GLP-1. The results are consistent with the possibility that sup-17 and adm-4 are functionally redundant for at least a subset of LIN-12/Notch-mediated decisions in C. elegans (Jarriault, 2005: full text of article).

The vulval precursor cells (VPCs) of C. elegans are polarized epithelial cells that adopt a precise pattern of fates through regulated activity of basolateral LET-23/EGF receptor and apical LIN-12/Notch. During VPC patterning, there is reciprocal modulation of endocytosis and trafficking of both LET-23 and LIN-12. sel-2 was identified as a negative regulator of lin-12/Notch activity in the VPCs; SEL-2 is the homolog of two closely related human proteins, neurobeachin (also known as BCL8B) and LPS-responsive, beige-like anchor protein (LRBA). In Drosophila, mutations in the single neurobeachin/LRBA homolog rugose (also referred to as DAKAP550) cause defects in eye development consistent with abnormalities in Notch and EGFR signaling (Schreiber, 2002; Shamloula, 2002; Wech, 2005), but the basis for these defects is unclear. SEL-2/neurobeachin/LRBA appears to form a subfamily within a larger family of BEACH-WD40 domain-containing proteins. There are three other C. elegans BEACH-containing proteins. The most closely related to SEL-2 is VT23B5.2, the ortholog of human ALFY and Drosophila Blue Cheese (Finley, 2003), consisting of little else but the BEACH domain and five WD40 motifs, plus a C-terminal FYVE domain. Loss of sel-2 activity leads to basolateral mislocalization and increased accumulation of LIN-12 in VPCs in which LET-23 is not active, and to impaired downregulation of basolateral LET-23 in VPCs in which LIN-12 is active. Downregulation of apical LIN-12 in the VPC in which LET-23 is active is not affected. In addition, in sel-2 mutants, the polarized cells of the intestinal epithelium display an aberrant accumulation of the lipophilic dye FM4-64 when the dye is presented to the basolateral surface. These observations indicate that SEL-2/neurobeachin/LRBA is involved in endosomal traffic and may be involved in efficient delivery of cell surface proteins to the lysosome. These results also suggest that sel-2 activity may contribute to the appropriate steady-state level of LIN-12 or to trafficking events that affect receptor activation (Wech, 2005).

The Notch signaling pathway controls growth, differentiation and patterning in divergent animal phyla; in humans, defective Notch signaling has been implicated in cancer, stroke and neurodegenerative disorders. Despite its developmental and medical significance, little is known about the factors that render cells to become competent for Notch signaling. This study shows that during vulval development in the nematode C. elegans the HOX protein LIN-39 and its EXD/PBX-like cofactor CEH-20 are required for LIN-12/Notch-mediated lateral signaling that specifies the 2° vulval cell fate. Inactivation of either lin-39 or ceh-20 resulted in the misspecification of 2° vulval cells and suppresses the multivulva phenotype of lin-12(n137) gain-of-function mutant animals. Furthermore, both LIN-39 and CEH-20 are required for the expression of basal levels of the genes encoding the LIN-12/Notch receptor and one of its ligands in the vulval precursor cells, LAG-2/Delta/Serrate, rendering them competent for the subsequent lin-12/Notch induction events. These results suggest that the transcription factors LIN-39 and CEH-20, which function at the bottom of the RTK/Ras and Wnt pathways in vulval induction, serve as major integration sites in coordinating and transmitting signals to the LIN-12/Notch cascade to regulate vulval cell fates (Takács-Vellai, 2007).

Convergent intercellular signals must be precisely coordinated in order to elicit specific biological responses. The C. elegans vulva provides an excellent experimental microcosm for studying how cell fate is specified according to the combined effects of different signaling pathways. This paper has studied the role of the Hox gene lin-39 and the Exd ortholog ceh-20 in vulval development. Genetic and molecular evidence is presented that the HOX protein LIN-39 and its putative cofactor CEH-20 are required for basal expression levels of lin-12 and lag-2 in the VPCs prior to vulval induction; this regulation may be important to render the VPCs competent for the subsequent lin-12/Notch induction events at the L3 larval stage. Identifying transcriptional regulators of lateral signaling in C. elegans vulval development will be essential for understanding how the Notch signaling pathway specifies cell fate in divergent animal species, and how compromised Notch signaling leads to human diseases (Takács-Vellai, 2007).

LIN-39 and CEH-20 are both required at the first larval stage to prevent fusion of the VPCs to the surrounding hypodermis. The data lead to the attractive possibility that LIN-39 and its putative cofactor CEH-20 regulate the competence of the VPCs to respond to any of the patterning signals during vulval formation. Along this line, it is challenging to speculate that, besides regulating lin-12 and lag-2 expression, they might also promote the expression of components of the inductive pathway (such as let-23) or other Notch pathway genes in the VPCs (Takács-Vellai, 2007).

It has been shown that CEH-20 binds in vitro, together with LIN-39, to the promoter of the twist transcription factor ortholog hlh-8 to regulate its expression in postembryonic mesodermal cells. ChIP experiments demonstrate that LIN-39 associates with the lag-2promoter, suggesting that the regulation of lag-2 expression by LIN-39 may be direct. It is proposed that LIN-39 forms a heterodimer with CEH-20 to promote the basal transcription of lag-2 and lin-12 in the VPCs. Based on their different expression pattern in the Pn.p lineages, ceh-20 is assumed to have some functions that are independent of lin-39. Indeed, mab-5 has been shown to be expressed in the descendants of the posterior VPCs, P7.p and P8.p, and to prevent them from adopting an induced vulval fate. Thus, it is possible that CEH-20 also interacts and functions with MAB-5 in controlling certain aspects of vulval fate specification. Furthermore, it is noted that ceh-20(ay9) mutant animals sometimes displayed a dual AC phenotype, whereas lin-39 mutants never did. RNAi-mediated depletion of mab-5 sometimes resulted in 2 ACs, suggesting that the correct AC specification requires the combined activity of mab-5 and ceh-20 (Takács-Vellai, 2007).

Finally, CEH-20 has been shown to be required as a cofactor for autoregulatory expression of the anterior Hox paralog (labial-like) ceh-13 in embryonic cells. Because ceh-13 is expressed all along the anteroposterior body axis in the ventral mid-line during the L1–L4 larval stages and a few percent of the ceh-13(sw1) mutant animals that are able to develop into fertile adults exhibit various defects in vulval formation, it is possible that CEH-13 acts with CEH-20 to control cell fate in the anterior VPC lineages. The future analysis of a potential role of ceh-13 in vulval development would help to establish the role of all of the major body Hox genes in this important process (Takács-Vellai, 2007).

The Notch pathway is the key signal for many cell fate decisions in the nematode Caenorhabditis elegans including the uterine pi cell fate, crucial for a proper uterine-vulval connection and egg laying. Expression of the egl-13 SOX domain transcription factor is specifically upregulated upon induction of the pi lineage and not in response to other LIN-12/Notch-mediated decisions. Dual regulation by LIN-12 and FOS-1 is required for egl-13 expression at specification and for complete rescue of egl-13 mutants. fos-1 mutants exhibit uterine defects and fail to express pi markers. FOS-1 is expressed at pi cell specification was demonstrated and that FOS-1 can bind in vitro to egl-13 upstream regulatory sequence (URS) as a heterodimer with C. elegans Jun (Oommen, 2007).

The lin-11 LIM domain transcription factor, a target of Notch signaling, is necessary for morphogenesis of C. elegans uterine cells

The C. elegans hermaphrodite egg-laying system comprises several tissues, including the uterus and vulva. lin-11 encodes a LIM domain transcription factor needed for certain vulval precursor cells to divide asymmetrically. Lin-11 is homologous to a known, but unnamed and uncharacterized Drosophila Lim domain protein. Based on lin-11 expression studies and the lin-11 mutant phenotype, it has been found that lin-11 is also required for C. elegans uterine morphogenesis. Specifically, lin-11 is expressed in the ventral uterine intermediate precursor (pi) cells and their progeny (the utse and uv1 cells), which connect the uterus to the vulva. Like pi cell induction, the uterine lin-11 expression responds to the uterine anchor cell and the lin-12-encoded receptor (Notch). In wild type animals, the utse, which forms the planar process at the uterine-vulval interface, fuses with the anchor cell. In lin-11 mutants, utse differentiation is abnormal; the utse fail to fuse with the anchor cell and a functional uterine-vulval connection is not made. These findings indicate that lin-11 is essential for uterine-vulval morphogenesis. Activation of LIN-12 results in a variety of responses depending on the cell, raising the issue of how cell-specific responses to a general signal are programmed. lin-11 is activated by lin-12 in both the vulval secondary cells and the uterine po cells. However, lin-11 does not appear to be a general executor of lin-12-mediated responses. According to studies of the lin-11-lacZ fusion gene, lin-11 is not expressed in the VU cell early, or in the SMs or G2 cell, all of which require lin-12 function. Also, there is no evidence for a role of lin-12 in VC neuron specification. LIN-11 may be a component of a partially specific response to LIN-12. The ability of a cell to activate LIN-11 in response to LIN-12 might be one of the factors that contribute to the cell specifity of such response (Newman, 1999).

aph-2 encodes a novel extracellular protein required for GLP-1-mediated signaling

In animal development, numerous cell-cell interactions are mediated by the GLP-1/LIN-12/NOTCH family of transmembrane receptors. These proteins function in a signaling pathway that appears to be conserved from nematodes to humans. The aph-2 gene is a new component of the GLP-1 signaling pathway in the early Caenorhabditis elegans embryo, and proteins with sequence similarity to the APH-2 protein are found in Drosophila (CG7012) and vertebrates. During the GLP-1-mediated cell interactions in the C. elegans embryo, APH-2 is associated with the cell surfaces of both the signaling, and the responding, blastomeres. Analysis of chimeric embryos that are composed of aph-2 plus and aph-2 minus blastomeres suggests that aph-2 plus function may be provided by either the signaling or responding blastomere (Goutte, 2000).

Translational repression of a C. elegans Notch mRNA by the STAR/KH domain protein GLD-1

In C. elegans, the Notch receptor GLP-1 is localized within the germline and early embryo by translational control of glp-1 mRNA. RNA elements in the glp-1 3'untranslated region (3' UTR) are necessary for repression of glp-1 translation in germ cells, and for localization of translation to anterior cells of the early embryo. The direct regulators of glp-1 mRNA are not known. A 34 nucleotide region of the glp-1 3' UTR is shown to contain two regulatory elements, an element that represses translation in germ cells and posterior cells of the early embryo, and an element that inhibits repressor activity to promote translation in the embryo. Furthermore, the STAR/KH domain protein GLD-1 (Drosophila homolog: Held out wings) binds directly and specifically to the repressor element. Depletion of GLD-1 activity by RNA interference causes loss of endogenous glp-1 mRNA repression in early meiotic germ cells, and in posterior cells of the early embryo. Therefore, GLD-1 is a direct repressor of glp-1 translation at two developmental stages. These results suggest a new function for GLD-1 in regulating early embryonic asymmetry. Furthermore, these observations indicate that precise control of GLD-1 activity by other regulatory factors is important to localize this Notch receptor. Such control contributes to the spatial organization of Notch signaling (Marin, 2003).

The lateral signal for LIN-12/Notch in C. elegans vulval development comprises redundant secreted and transmembrane DSL proteins

The vulval precursor cells (VPCs) are spatially patterned by a LET-23/EGF receptor-mediated inductive signal and a LIN-12/Notch-mediated lateral signal. The lateral signal has eluded identification, so the mechanism by which lateral signaling is activated has not been known. Ten genes have been computationally identified that encode potential ligands for LIN-12; three of these genes, apx-1, dsl-1, and lag-2, are functionally redundant components of the lateral signal. Transcription of all three genes is initiated or upregulated in VPCs in response to inductive signaling, suggesting that direct transcriptional control of the lateral signal by the inductive signal is part of the mechanism by which these cell signaling events are coordinated. In addition, DSL-1, which lacks a predicted transmembrane domain, is a natural secreted ligand and can substitute for the transmembrane ligand LAG-2 in different functional assays (Chen, 2004).

Sequence analysis indicates that three DSL (Delta/Serrate/Lag-2) proteins have highly probable predicted transmembrane domains; these are encoded by the three genes that had been identified previously, lag-2, apx-1, and arg-1. Two of the proteins identified by computational analysis, DSL-2 and DSL-6, may also have transmembrane domains, although the potential transmembrane domains were assessed as lower probability in prediction programs. Surprisingly, the five remaining genes (dsl-1, dsl-3, dsl-4, dsl-5, and dsl-7) encode DSL proteins that are predicted to lack transmembrane domains and hence are likely to be secreted (Chen, 2004).

In terms of VPC specification, the cell biology of lateral signaling offers a rationale for why a secreted or peripheral membrane protein ligand might be a component of the lateral signal. The VPCs are polarized epithelial cells: they have an apical region and a basolateral domain, separated by adherens junctions. Since the apical regions of adjacent VPCs appear to be in contact only in the vicinity of the cell junctions, and LIN-12 is distributed over the whole apical surface, a transmembrane ligand on the surface of one VPC might have access to LIN-12 on the apical surface of its neighbor only in a relatively limited area. A ligand that can diffuse may be available to activate LIN-12 over a greater region of the apical domain, affording one solution to such a topographical problem (Chen, 2004).

The T-box transcription factors TBX-37 and TBX-38 link GLP-1/Notch signaling to mesoderm induction in C. elegans embryos

The four-cell C. elegans embryo contains two sister cells called ABa and ABp that initially have equivalent abilities to produce ectodermal cell types. Multiple Notch-mediated interactions occur during the early cell divisions that diversify the ABa and ABp descendants. The first interaction determines the pattern of ectodermal cell types produced by ABp. The second interaction induces two ABa granddaughters to become mesodermal precursors. T-box transcription factors called TBX-37 and TBX-38 are essential for mesodermal induction, and these factors are expressed in ABa, but not ABp, descendants. Evidence is provided that the first Notch interaction functions largely, if not entirely, to prevent TBX-37, TBX-38 expression in ABp descendants. Neither the second Notch interaction nor TBX-37, TBX-38 alone are sufficient for mesodermal induction, indicating that both must function together. It is concluded that TBX-37, TBX-38 play a key role in distinguishing the outcomes of two sequential Notch-mediated interactions (Good, 2004).

At least four distinct interactions occur during the first few cell divisions of the C. elegans embryo, providing a relatively simple experimental system to analyze a network of Notch-mediated cell fate decisions. The anterior cell in the two-cell stage embryo is called AB, and all of the early descendants of AB express the receptors GLP-1/Notch or LIN-12/Notch. Various AB descendants contact one of several ligand-expressing cells that are born at different times and places during the early divisions, and change their fate accordingly. In genetic studies of Notch-mediated, binary cell fate decisions, one cell fate can often be considered as 'primary' and a second cell fate as 'secondary'; Notch function is required for the secondary, but not primary, fate (Artavanis-Tsakonas, 1999). In the absence of all Notch-mediated interactions in C. elegans embryos, AB descendants adopt highly patterned ectodermal fates that will thus be described here as primary fates (Good, 2004 and references therein).

The first Notch interaction occurs at the four-cell stage when the posterior daughter of AB, called ABp, contacts a cell called P2 that expresses a Notch ligand. The interaction between P2 and ABp causes the ABp descendants to adopt new fates that are described in this study as secondary fates; cells with secondary fates remain ectodermal precursors, but have a pattern of differentiation that is distinct from cells with primary fates. The anterior daughter of AB, called ABa, does not contact P2 and thus produces descendants that initially retain their potential for primary fates. At the 12-cell stage, however, two of the ABa granddaughters contact a new ligand-expressing cell called MS. This second Notch interaction induces those two ABa granddaughters to adopt novel, tertiary fates and become mesodermal precursors. During the next few cell divisions, there are third and fourth Notch interactions that further diversify the fates of some ABp descendants. Coincident with the Notch-mediated specification of cell fates, a separate anteroposterior polarity system generates additional differences between sister cells that are born from anteroposterior cell divisions. Thus, there are two types of primary fates (1a and 1p) depending on whether a cell is an anterior sister (1a) or posterior sister (1p). Similarly, there are two types of secondary fates and two tertiary fates. These anteroposterior differences appear to involve POP-1, a transcription factor that is localized asymmetrically after all anteroposterior divisions of the AB descendants (Good, 2004 and references therein).

The mesodermal precursors that are induced by the second Notch interaction form the anterior half of the pharynx, a large muscular structure used in feeding. The posterior half of the pharynx contains many of the identical mesodermal cell types found in the anterior half, but is derived from non-AB descendants through a Notch-independent pathway. Preventing the second Notch interaction results in embryos that lack the anterior pharynx, but that have a posterior half-pharynx produced by non-AB descendants (the Aph phenotype; anterior pharynx defective). Through genetic screens for Aph mutants, two functionally redundant T-box transcription factors called TBX-37 and TBX-38 have been identified that are essential for the development of the anterior pharynx. Notch signaling occurs at the 12-cell stage in tbx-37 tbx-38 mutant embryos, but does not result in mesodermal specification. Evidence suggests that the primary, if not sole, function of Notch signaling at the four-cell stage is to repress TBX-37, TBX-38 expression in ABp descendants. Thus, the first Notch interaction restricts the expression of T-box proteins that are essential to couple the second Notch interaction to mesoderm development (Good, 2004).

During the 12-cell stage of C. elegans embryogenesis, Notch-mediated signaling from MS induces a subset of ABa descendants to express forkhead-class transcription factor PHA-4 and become mesodermal precursors. TBX-37 and TBX-38 are essential for the ABa descendants to become mesodermal precursors. Several examples of T-box proteins with roles in mesodermal development have been described, including the prototype member Brachyury. The present study provides evidence that TBX-37, TBX-38 must act in conjunction with unidentified targets of the Notch signal transduction pathway for mesodermal induction in C. elegans. (1) It has been shown that TBX-37, TBX-38 expression and Notch signal transduction are regulated independently in ABa descendants. Killing the signaling cell, MS, or removing LAG-1, the transcriptional effector of the Notch pathway, does not prevent TBX-37, TBX-38 expression in ABa descendants. Conversely, removing TBX-37, TBX-38 does not prevent Notch-mediated repression of LAG-2 in the ABara granddaughters. (2) It has been shown that neither Notch signal transduction nor TBX-37, TBX-38 alone are sufficient for mesodermal induction. All of the early ABa descendants express TBX-37, TBX-38 in wild-type embryos, but only those ABa descendants that are signaled by MS become mesodermal precursors. Conversely, activation of Notch represses LAG-2 expression in the ABara granddaughters of tbx-37 tbx-38 embryos, but does not induce PHA-4 expression in those same cells (Good, 2004).

Although the second Notch interaction (MS signaling) induces cells to become mesodermal precursors that form the pharynx, the first Notch interaction (P2 signaling) prevents cells from becoming mesodermal precursors. If the first Notch interaction does not occur, embryos have a hyperinduction of pharyngeal tissue. In normal development, MS signaling induces ABa (but not ABp) descendants to become mesodermal precursors. However, MS and its sister cell, called E, both have the ability to signal, and one or both of these cells contact some ABp descendants in addition to contacting ABa descendants. When P2 signaling is blocked, either by physically removing P2 or by mutations in the P2 ligand encoded by the Delta gene apx-1, MS and E induce these additional ABp descendants to become mesodermal precursors. Mutations in apx-1 cause the inappropriate expression of TBX-37, TBX-38 in ABp descendants. In addition, it has been shown that removing TBX-37, TBX-38 activities from apx-1 mutant embryos prevents the hyperinduction of pharyngeal cells. Thus, the competence of both ABa and ABp descendants to become mesodermal precursors in response to the second Notch interaction is determined by the pattern of expression of TBX-37, TBX-38 (Good, 2004).

In summary, these results provide insight into two of the four Notch-mediated interactions that occur in rapid succession in early embryogenesis, and that modify ABa and ABp descendants in distinct ways. It is proposed that the transcription factors TBX-37, TBX-38 can promote 'primary' cell fates independent of Notch. The first Notch-mediated interaction blocks expression of TBX-37, TBX-38 in ABp descendants, thus allowing those cells to adopt novel, 'secondary' fates. Next, TBX-37, TBX-38 are expressed in ABa descendants independently of Notch, but shortly after the second Notch interaction. ABa descendants that do not undergo the second Notch interaction assume primary fates, in part through the action of TBX-37, TBX-38. In the ABa descendants that undergo the second Notch-interaction, TBX-37, TBX-38 collaborate with unidentified Notch targets to promote tertiary fates and mesoderm development (Good, 2004).

FBF-1 and FBF-2 regulate the size of the mitotic region in the C. elegans germline

In the C. elegans germline, GLP-1/Notch signaling and two nearly identical RNA binding proteins, FBF-1 and FBF-2, promote proliferation. Here, the fbf-1 and fbf-2 genes are largely redundant for promoting mitosis but they have opposite roles in fine-tuning the size of the mitotic region. The mitotic region is smaller than normal in fbf-1 mutants but larger than normal in fbf-2 mutants. Consistent with gene-specific roles, fbf-2 expression is limited to the distal germline, while fbf-1 expression is broader. The fbf-2 gene, but apparently not fbf-1, is controlled by GLP-1/Notch signaling, and the abundance of FBF-1 and FBF-2 proteins is limited by reciprocal 3′UTR repression. It is proposed that the divergent fbf genes and their regulatory subnetwork enable a precise control over size of the mitotic region. Therefore, fbf-1 and fbf-2 provide a paradigm for how recently duplicated genes can diverge to fine-tune patterning during animal development (Lamont, 2004).

RNA binding proteins are key regulators of the germline decision between proliferation and differentiation. Of particular importance to this paper are FBF-1 and FBF-2 (for fem-3 Binding Factor) -- two nearly identical regulators of the PUF (Pumilio and FBF) protein family. The FBF-1 and FBF-2 proteins are collectively called FBF, and similarly, fbf-1 and fbf-2 are collectively called the fbf genes. The nucleotide sequences of fbf-1 and fbf-2 are 93% identical, and the amino acid sequences are 91% identical, suggesting that fbf-1 and fbf-2 are recently duplicated genes. During early larval stages, germline proliferation is normal in fbf-1 fbf-2 double mutants, but in the fourth larval stage, the germline precociously leaves the mitotic cell cycle to enter meiosis and differentiate as sperm. In addition, depletion of both fbf-1 and fbf-2 eliminates the hermaphrodite switch from spermatogenesis to oogenesis. Therefore, FBF is required for continued mitotic divisions in the germline as well as for the hermaphrodite sperm/oocyte switch (Lamont, 2004).

PUF proteins bind specifically to regulatory elements, usually in the 3' untranslated region (UTR) of a target mRNA, and repress that mRNA, either by promoting mRNA degradation or inhibiting translation. Pumilio, for example, inhibits translation of hunchback mRNA in the early Drosophila embryo, whereas PUF-5/Mpt5 destabilizes HO mRNA in yeast. In C. elegans, FBF-1 and FBF-2 promote mitosis by repressing mRNAs that encode regulators critical for entry into the meiotic cell cycle, and they promote the sperm/oocyte switch by repressing the fem-3 sperm-promoting mRNA. Both FBF-1 and FBF-2 bind specifically to the same RNA target sequence, which differs from the Pumilio binding site. The molecular mechanism by which FBF represses mRNAs in the C. elegans germline remains unknown, but by analogy with its homologs in yeast and Drosophila, FBF is likely to control the stability or translation of its target mRNAs (Lamont, 2004).

Previous studies have suggested that FBF-1 and FBF-2 are redundant: fbf-1 single mutants are grossly normal, albeit with smaller mitotic regions and more hermaphrodite sperm than wild-type. This study confirms the fbf-1/fbf-2 redundancy but also identify individual roles for each gene in regulating the size of the mitotic region. Like fbf-1, the fbf-2 single mutants are grossly normal, but in contrast to fbf-1, fbf-2 mutant germlines have a larger mitotic region than normal and can be feminized. Consistent with fbf-1 and fbf-2 having individual roles, their mRNAs and proteins are expressed in distinct patterns. Furthermore, the fbf-2 gene appears to be a direct target of GLP-1/Notch signaling, a finding that forges the first molecular link between GLP-1/Notch signaling and the RNA regulatory circuit. fbf-1 and fbf-2 repress each other's expression and this reciprocal repression is likely to be direct via FBF binding sites in the fbf-1 and fbf-2 3' UTRs. It is suggested that GLP-1/Notch signaling and FBF autoregulation work together to control the distribution and amount of FBF and thereby fine-tune the size of the mitotic region (Lamont, 2004).

Endocytosis-mediated downregulation of LIN-12/Notch upon Ras activation in Caenorhabditis elegans

The coordination of signals from different pathways is important for cell fate specification during animal development. A novel mode of crosstalk between the epidermal growth factor receptor/Ras/mitogen-activated protein kinase cascade and the LIN-12/Notch pathway during Caenorhabditis elegans vulval development has been defined. Six vulval precursor cells (VPCs) are initially equivalent but adopt different fates as a result of an inductive signal mediated by the Ras pathway and a lateral signal mediated by the LIN-12/Notch pathway1. One consequence of activating Ras is a reduction of LIN-12 protein in P6.p, the VPC believed to be the source of the lateral signal. A 'downregulation targeting signal' (DTS) has been identified in the LIN-12 intracellular domain, which encompasses a di-leucine-containing endocytic sorting motif. The DTS seems to be required for internalization of LIN-12, and on Ras activation it might mediate altered endocytic routing of LIN-12, leading to downregulation. If LIN-12 is stabilized in P6.p, lateral signalling is compromised, indicating that LIN-12 downregulation is important in the appropriate specification of cell fates in vivo (Shaye, 2002).

The DTS contains two adjacent leucine residues and several serines that are conserved in all known nematode LIN-12/Notch proteins. 'Di-leucine motifs' are well-characterized sorting signals that usually take the form (-)(2-4)XLL, where (-) is often an acidic residue or phosphoserine, although basic residues have also been seen at this position, and X is usually a polar or bulky residue. Functional motifs that contain M, V or I instead of L have also been found. Di-leucine motifs regulate different aspects of protein trafficking, including the constitutive or ligand-stimulated internalization of transmembrane receptors, and the routing of proteins within the endocytic and/or secretory pathways. Different residues within the same di-leucine motif might modulate different aspects of motif activity (namely internalization versus routing), and the activity of di-leucine motifs that contain serines might be regulated by phosphorylation. In several cases the di-leucine motif leads to routing of the protein to lysosomes for degradation.Mutating the two leucine residues of the DTS to two alanines disrupts downregulation of sTM::LIN-12(intra)::GFP. Because downregulation of GFP-tagged LIN-12 fragments requires both membrane association and a di-leucine motif, it is inferred that the mechanism of downregulation involves increased internalization and/or altered endocytic routing of LIN-12 when Ras is activated (Shaye, 2002).

The gene sur-2 encodes a component of the Mediator complex, which activates transcription in response to Ras/MAPK signalling in mammalian cells. SUR-2 also seems to be activated by the Ras pathway in C. elegans, and hermaphrodites lacking sur-2 activity display a failure in lateral signalling. In sur-2(-) hermaphrodites, LIN-12(+)::GFP is not downregulated. This result suggests that the failure of lateral signalling in sur-2(-) hermaphrodites might result at least in part from the failure to downregulate endogenous LIN-12(+). Furthermore, in sur-2(-) hermaphrodites, as in the wild type, LIN-12(+)::GFP accumulates in puncta, suggesting that SUR-2/Mediator-promoted transcription is not necessary for the initial internalization, but instead might affect the rate of internalization and/or routing of endocytosed LIN-12(+)::GFP (Shaye, 2002).

A model is presented for cross-talk between the Ras and LIN-12 pathways in P6.p. LIN-12, like other transmembrane proteins, seems to be constitutively internalized and might be routed to recycling endosomes or to lysosomes. It is proposed that Ras activation leads to transcription of at least one gene whose product regulates the rate of internalization and/or subcellular routing of LIN-12, leading to its degradation. Internalization (and perhaps, in addition, endocytic routing) of LIN-12 is mediated by the DTS: when the DTS is removed, LIN-12 accumulates in the apical membrane of P6.p. Persistence of LIN-12, achieved by deleting the DTS or by removing sur-2 activity, is correlated with inhibition of lateral signalling, demonstrating that downregulation is functionally important (Shaye, 2002).

The novel mode of crosstalk between the Ras and LIN-12/Notch pathways may be conserved. Vertebrate Notch proteins contain a highly conserved potential di-leucine-like motif (1822KKFRFEEPVVL1832 in human Notch1) that, like the LIN-12 DTS, lies between the transmembrane domain and the first ankyrin motif, and furthermore the large intracellular domain of notch proteins contains additional potential endocytic motifs. Whether these motifs function as such, and whether their function is regulated by Ras or other signals, can only be answered through experiments in other systems. Observations of Wingless endocytosis has led to the speculation that Ras might modulate the endocytic routing of Wingless bound to its receptor Frizzled2 via a di-leucine motif on Frizzled2, dependent on the transcription of an unknown factor in response to epidermal growth factor receptor/Ras/MAPK signalling. If this proves to be so, the future identification of the putative factors that recognize the targeting signals of LIN-12/Notch and Frizzled2 will reveal whether Ras works through a common or distinct mechanism to modulate the activity of these different receptors (Shaye, 2002).

C. elegans EVI1 proto-oncogene, EGL-43, is necessary for Notch-mediated cell fate specification and regulates cell invasion

During C. elegans development, LIN-12 (Notch) signaling specifies the anchor cell (AC) and ventral uterine precursor cell (VU) fates from two equivalent pre-AC/pre-VU cells in the hermaphrodite gonad. Once specified, the AC induces patterned proliferation of vulva via expression of LIN-3 (EGF) and then invades into the vulval epithelium. Although these cellular processes are essential for the proper organogenesis of vulva and appear to be temporally regulated, the mechanisms that coordinate the processes are not well understood. egl-43 was computationally identified as a gene likely to be expressed in the pre-AC/pre-VU cells and the AC, based on the presence of an enhancer element similar to the one that transcribes lin-3 in the same cells. Genetic epistasis analyses reveal that egl-43 acts downstream of or parallel to lin-12 in AC/VU cell fate specification at an early developmental stage, and functions downstream of fos-1 as well as upstream of zmp-1 and him-4 to regulate AC invasion at a later developmental stage. Characterization of the egl-43 regulatory region suggests that EGL-43 is a direct target of LIN-12 and HLH-2 (E12/47), which is required for the specification of the VU fate during AC/VU specification. EGL-43 also regulates basement membrane breakdown during AC invasion through a FOS-1-responsive regulatory element that drives EGL-43 expression in the AC and VU cells at the later stage. Thus, egl-43 integrates temporally distinct upstream regulatory events and helps program cell fate specification and cell invasion (Hwang, 2007).

Upregulation of the let-7 microRNA with precocious development in lin-12/Notch hypermorphic Caenorhabditis elegans mutants

The lin-12/Notch signaling pathway is conserved from worms to humans and is a master regulator of metazoan development. lin-12/Notch gain-of-function (gf) animals display precocious alae at the L4 larval stage with a significant increase in let-7 expression levels. Furthermore, lin-12(gf) animals display a precocious and higher level of let-7 gfp transgene expression in seam cells at L3 stage. Interestingly, lin-12(gf) mutant rescued the lethal phenotype of let-7 mutants similar to other known heterochronic mutants. It is proposed that lin-12/Notch signaling pathway functions in late developmental timing, upstream of or in parallel to the let-7 heterochronic pathway. Importantly, the human microRNA let-7a was also upregulated in various human cell lines in response to Notch1 activation, suggesting an evolutionarily conserved cross-talk between let-7 and the canonical lin-12/Notch signaling pathway (Solomon, 2008).

LIN-14 inhibition of LIN-12 contributes to precision and timing of C. elegans vulval fate patterning

Studies of C. elegans vulval development have illuminated mechanisms underlying cell fate specification and elucidated intercellular signaling pathways. The vulval precursor cells (VPCs) are spatially patterned during the L3 stage by the EGFR-Ras-MAPK-mediated inductive signal and the LIN-12/Notch-mediated lateral signal. The pattern is both precise and robust because of crosstalk between these pathways. Signaling is also regulated temporally, because constitutive activation of the spatial patterning pathways does not alter the timing of VPC fate specification. The heterochronic genes, including the microRNA lin-4 and its target lin-14, constitute a temporal control mechanism used in different contexts. lin-4 specifically controls the activity of LIN-12/Notch through lin-14, but not other known targets, and persistent lin-14 blocks LIN-12 activity without interfering with the key events of LIN-12/Notch signal transduction. In the L2 stage, there is sufficient lin-14 activity to inhibit constitutive lin-12. The results suggest that lin-4 and lin-14 contribute to spatial patterning through temporal gating of LIN-12. It is proposed that in the L2 stage, lin-14 sets a high threshold for LIN-12 activation to help prevent premature activation of LIN-12 by ligands expressed in other cells in the vicinity, thereby contributing to the precision and robustness of VPC fate patterning. Because LIN-14 can function as a transcriptional repressor, a simple model is that LIN-14 acts to antagonize expression of a key target, or targets, of the lateral signaling pathway (Li, 2010).

The Notch - Presenilin connection in C. elegans and mammals

sel-12 (sel means suppressor/enhancer of lin-12) is a transmembrane protein that facilitates Notch signaling in C. elegans. It is related to Presenilin-1 (S182), a mammalian gene that when mutated causes aggressive, early-onset Alzheimer's via an unknown mechanism. In C. elegans, Notch signaling is involved in the anchor cell/ventral uterine precursor cell (AC/VU) decision and vulval precursor cell (VPC) specification during gonadogenesis. The AC/VU decision involves an interaction between two initially equivalent cells of the somatic gonad. When lin-12, which codes for a Notch family member, is eliminated, both precursor cells become ACs. When lin-12 is constitutively activated, both precursor cells become VUs. sel-12 was isolated as a suppressor of a lin-12 gain of function mutation. That is, sel-12 mutation acts to reduce lin-12 signaling. Reducing sel-12 activity reduces lin-12 activity in lateral signaling that specifies the secondary fate of VPCs. Cell ablation experiments show that sel-12 functions within a VPC to lower lin-12 activity. The predicted SEL-12 protein contains multiple potential transmembrane domains, consistent with its function as a receptor, ligand, channel or membrane structural protein. SEL-12 might be directly involved in lin-12-mediated reception, functioning for example as a co-receptor or as a downstream effector that activatesupon LIN-12 activation. Alternatively, sel-12 may be involved in a more general cellular process, such as receptor localization or recycling, and hence influence lin-12 activity indirectly (Levitan, 1995). The data presented in this paper suggest that the effect of SEL-12/presenilin on LIN-12/Notch is analogous to its effect on APP. LIN-12/Notch proteins are transmembrane proteins with hallmark motifs: epidermal growth factor-like; LIN-12/Notch repeat, and cdc10/SWI6 (ankyrin) motifs. Like APP, LIN-12/Notch proteins must be correctly sorted and transported to the cell surface, and undergo proteolytic cleavage events. There appears to be at least one constitutive proteolytic cleavage event that occurs in the extracellular domain during the transport to the plasma membrane; the cleaved form produced by this constitutive cleavage event may be the major species present at the cell surface. In addition, binding of ligand appears to induce a cleavage event in or near the transmembrane domain; this apparent cleavage event enables the intracellular domain to translocate to the nucleus, where it participates directly in regulating downstream gene expression. It is conceivable that SEL-12/presenilin is involved in promoting one or more of these cleavage events, either by activating protease(s) or promoting trafficking of either LIN-12 or proteases to an appropriate compartment. The strong accumulation of SEL-12::GFP in the ER/Golgi is consistent with a role for SEL-12 in a constitutive cleavage event involved in maturation of LIN-12/Notch proteins. The fact that less LIN-12::GFP was observed at the cell surface in a sel-12 mutant background could be explained in the context of this model by proposing that abnormal processing of LIN-12 leads to its failure to be transported to the plasma membrane or to its degradation. The putative ligand-dependent cleavage of activated LIN-12 might occur at the plasma membrane or in internalized vesicles. The failure to observe SEL-12::GFP in the plasma membranes of the VPCs does not preclude a role for SEL-12 in ligand-dependent cleavage. It is possible that SEL-12::GFP is present at low abundance in the plasma membrane; the ligand-induced event appears to affect a very small proportion of receptor molecules, suggesting that the agent that promotes the cleavage may not be very abundant. Although the issue of the biochemical mechanism of presenilin function is not resolved in any system, the parallels between APP and LIN-12/Notch trafficking and processing suggest that a common mechanism is involved. An important challenge for the future will be to identify the primary effect of SEL-12/presenilin on APP and LIN-12/Notch, since proteolytic processing, intracellular trafficking and degradation are intimately linked, and altering one process can affect another (Levitan, 1998b).

Mutant presenilins have been found to cause Alzheimer disease. This paper describes the identification and characterization of HOP-1, a Caenorhabditis elegans presenilin that displays much more lower sequence identity with human presenilins than does the other C. elegans presenilin, SEL-12. Despite considerable divergence, HOP-1 appears to be a bona fide presenilin, because HOP-1 can rescue the egg-laying defect caused by mutations in sel-12 when hop-1 is expressed under the control of sel-12 regulatory sequences. HOP-1 also has the essential topological characteristics of the other presenilins. Reducing hop-1 activity in a sel-12 mutant background causes synthetic lethality and terminal phenotypes associated with reducing the function of the C. elegans lin-12 and glp-1 genes. These observations suggest that hop-1 is functionally redundant with sel-12 and underscore the intimate connection between presenilin activity and LIN-12/Notch activity inferred from genetic studies in C. elegans and mammals (Li, 1997). C. elegans gene sel-1 functions as a negative regulator of Notch homolog lin-12 activity. It has been predicted that SEL-1 is a secreted or membrane associated protein. Cell ablation experiments that suggest sel-1 mutations elevate lin-12 activity cell autonomously. The predicted signal sequence of SEL-1 can direct secretion and is important for function, while a C-terminal hydrophobic region is not required for SEL-1 function. SEL-1 is localized intracellularly, with a punctate staining pattern suggestive of membrane bound vesicles. A related yeast protein, L8167.5, a product of the HRD3 gene, acts as a component of the mechanism for degrading a yeast metabolic enzyme. It is suggested that SEL-1 might be involved in down-regulating the activated LIN-12 receptor (Grant, 1997).

The Notch signaling pathway regulates specification and proliferation in a variety of cell lineages in invertebrates and vertebrates. A murine homolog of SEL-1, a key negative regulator of the Notch pathway in Caenorhabditis elegans, has been cloned. C. elegans SEL-1 functions as a negative regulator of Notch homolog Lin-12 activity. It has been predicted that SEL-1 is a secreted or membrane associated protein. Murine SEL-1L (mSEL-1L) protein exhibits a high degree of similarity to SEL-1, including a signal peptide and the C-terminal region required for SEL-1 function in C. elegans. This mammalian homolog of sel-1 is widely expressed in adult mouse and human tissues, with particularly high levels in the pancreas. RNA in situ analysis of developing mouse embryos indicates that mSEL-1L is moderately expressed throughout the neural tube and dorsal root ganglia, with particularly high levels in the floor plate of the neural tube beginning at E10.5 and increasing at E11.5. Expression is high at E14.5 and E17.5 in the acini of the pancreas, and moderate in the epithelial cells of the gut villi. The SEL-1L protein has been localized to the cytosol, possibly in intracellular vesicles, in a beta-islet-derived tumor cell line (Donoviel, 1998).

Mutations in the human presenilin genes PS1 and PS2 cause early-onset Alzheimer's disease. Studies in Caenorhabditis elegans and in mice indicate that one function of presenilin genes is to facilitate Notch-pathway signaling. Notably, mutations in the C. elegans presenilin gene sel-12 reduce signaling through an activated version of the Notch receptor LIN-12. To investigate the function of a second C. elegans presenilin gene hop-1 and to examine possible genetic interactions between hop-1 and sel-12, a reverse genetic strategy was used to isolate deletion alleles of both loci. Animals bearing both hop-1 and sel-12 deletions display new phenotypes not observed in animals bearing either single deletion. These new phenotypes (germ-line proliferation defects, maternal-effect embryonic lethality, and somatic gonad defects) resemble those resulting from a reduction in signaling through the C. elegans Notch receptors GLP-1 and LIN-12. Thus SEL-12 and HOP-1 appear to function redundantly in promoting Notch-pathway signaling. Phenotypic analyses of hop-1 and sel-12 single and double mutant animals suggest that sel-12 provides more presenilin function than does hop-1. There is as yet no evidence that Notch-pathway signaling is involved in the pathophysiology of Alzheimer's disease (AD). Thus, the relationship between the roles of presenilins in proteolytic processing of APP and in facilitating Notch receptor function remains unclear. Intriguingly, recent evidence suggests that multiple proteolytic processing events are required for intracellular trafficking and signal transduction of the Notch receptor: two cleavage events are proposed to occur in the extracellular domain and a third proposed cleavage occurs within or just carboxyl-terminal to the transmembrane region. The apparent similarities between the processing of APP and Notch, particularly the prospect that both are cleaved within the transmembrane domain, raise the possibility that presenilins affect proteolytic processing of APP and Notch in analogous ways. Presenilins might regulate proteolytic processing directly or might do so indirectly, for example, by promoting normal intracellular trafficking of APP or Notch. In support of a role for presenilins in the processing or trafficking of Notch, LIN-12::GFP levels at the plasma membrane are seen to be reduced in a sel-12 mutant background. An understanding of how presenilins affect Notch-receptor activity may be relevant to an understanding of the way in which presenilins affect APP cleavage and to the identification of targets for preventing the pathophysiological effects of presenilin dysfunction in AD (Westlund, 1999).

Presenilin-1 (PS1), a polytopic membrane protein primarily localized to the endoplasmic reticulum, is required for efficient proteolysis of both Notch and beta-amyloid precursor protein (APP) within their trans-membrane domains. The activity that cleaves APP (called gamma-secretase) has properties of an aspartyl protease; mutation of either of the two aspartate residues located in adjacent transmembrane domains of PS1 inhibits gamma-secretase processing of APP. These aspartates are required for Notch processing, since mutation of these residues prevents PS1 from inducing the gamma-secretase-like proteolysis of a Notch1 derivative. Thus PS1 might function in Notch cleavage as an aspartyl protease or di-aspartyl protease cofactor. However, the ER localization of PS1 is inconsistent with that hypothesis, since Notch cleavage occurs near the cell surface. Using pulse-chase and biotinylation assays, evidence is provided that PS1 binds Notch in the ER/Golgi and is then co-transported to the plasma membrane as a complex. PS1 aspartate mutants are indistinguishable from wild-type PS1 in their ability to bind Notch or traffic with it to the cell surface, and do not alter the secretion of Notch. Thus, PS1 appears to function specifically in Notch proteolysis near the plasma membrane as an aspartyl protease or cofactor (Ray, 1999b).

The C. elegans intestine is a simple tube consisting of a monolayer of epithelial cells. During embryogenesis, cells in the anterior of the intestinal primordium undergo reproducible movements that lead to an invariant, asymmetrical 'twist' in the intestine. The development of this twist has been examined to determine how left-right and anterior-posterior asymmetries are generated within the intestinal primordium. The twist requires the LIN-12/ Notch-like signaling pathway of C. elegans. All cells within the intestinal primordium initially express LIN-12, a receptor related to Notch; however, only cells in the left half of the primordium contact external, nonintestinal cells that express LAG-2, a ligand related to Delta. LIN-12 and LAG-2 mediated interactions result in the left primordial cells expressing lower levels of LIN-12 than the right primordial cells. It is proposed that this asymmetrical pattern of LIN-12 expression is the basis for asymmetry in later cell-cell interactions within the primordium that lead directly to intestinal twist. Like the interactions that initially establish LIN-12 asymmetry, the later interactions are mediated by LIN-12. The later interactions, however, involve a different ligand related to Delta, called APX-1. The anterior-posterior asymmetry in intestinal twist involves the kinase LIT-1, which is part of a signaling pathway in early embryogenesis that generates anterior-posterior differences between sister cells (Hermann, 2000).

aph-2 (Drosophila homolog: CG7012) encodes a novel extracellular protein required for GLP-1-mediated signaling (Goutte, 2000). Aph-2, termed Nicastrin (see Drosophila nicastrin) in this study, is a transmembrane glycoprotein that forms high molecular weight complexes with presenilin 1 and presenilin 2. Suppression of nicastrin expression in C. elegans embryos induces a subset of notch/glp-1 phenotypes similar to those induced by simultaneous null mutations in both presenilin homologs of C. elegans (sel-12 and hop-1). Nicastrin also binds carboxy-terminal derivatives of beta-amyloid precursor protein (betaAPP), and modulates the production of the amyloid beta-peptide (Abeta) from these derivatives. Missense mutations in a conserved hydrophilic domain of nicastrin increase Abeta42 and Abeta40 peptide secretion. Deletions in this domain inhibit Abeta production. Nicastrin and presenilins are therefore likely to be functional components of a multimeric complex necessary for the intramembranous proteolysis of proteins such as Notch/GLP-1 and betaAPP (Yu, 2000).

In the absence of homology to other proteins, sequence databases were screened for orthologous genes in other species. A full-length C. elegans nicastrin ortholog was found in public databases (accession no. Q23316; identity = 22%; similarity = 41%). Full-length murine and Drosophila nicastrin orthologs from appropriate cDNA libraries were cloned and sequenced using partial cDNA sequences from these databases as start points (mouse nicastrin accession no. AF24069, identity = 89%, similarity = 93%; D. melanogaster nicastrin accession no. AF240470, identity = 30%, similarity = 48%). The four animal nicastrins have similar predicted topologies and have three domains with significant sequence conservation near residues 306-360, 419-458, and 625-662 of human nicastrin. Within the first conserved domain, all four proteins contained the motif DYIGS (residues 336-340), which is also partially conserved in an Arabidopsis protein. All four animal nicastrins also contain four cysteines spaced at 16 to 17-residue intervals in the N terminus (Cys 195, Cys 213, Cys 230 and Cys 248) (Yu, 2000).

To explore whether nicastrin, like the presenilins, might have a role in Notch signaling in vivo, RNA interference (RNAi) was used in C. elegans. Wild-type worms injected with C. elegans nicastrin double-stranded (ds) RNA produce dead embryos, many of which lack an anterior pharynx. This phenotype is highly reproducible and specific. Except for embryonic lethality, none of the other phenotypes associated with a lack of C. elegans presenilin (sel-12) activity were observed. However, this phenotype is identical to that induced when the activity of genes in the notch/glp-1 pathway (glp-1, aph-1 or aph-2) are reduced, or when the activities of both C. elegans presenilin homologs (sel-12 and hop-1) are reduced simultaneously. Thus nicastrin contributes to some aspects of notch/glp-1 signaling in C. elegans embryos (Yu, 2000).

The Notch signaling pathway plays essential roles during the specification of the rostral and caudal somite halves and subsequent segmentation of the paraxial mesoderm. The role of presenilin 1 (Ps1; encoded by Psen1) during segmentation has been investigated using newly generated alleles of the Psen1 mutation. In Psen1-deficient mice, proteolytic activation of Notch1 is significantly affected and the expressions of several genes involved in the Notch signaling pathway are altered, including Delta-like3, Hes5, lunatic fringe (Lfng) and Mesp2, which encodes a bHLH transcriptional regulator expressed in the rostral region of the presomitic mesoderm. Thus, Ps1-dependent activation of the Notch pathway is essential for caudal half somite development. Defects were observed in Notch signaling in both the caudal and rostral region of the presomitic mesoderm. In the caudal presomitic mesoderm, Ps1 is involved in maintaining the amplitude of cyclic activation of the Notch pathway, as represented by significant reduction of Lfng expression in Psen1-deficient mice. In the rostral presomitic mesoderm, rapid downregulation of the Mesp2 expression in the presumptive caudal half somite depends on Ps1 and is a prerequisite for caudal somite half specification. Chimaera analysis between Psen1-deficient and wild-type cells reveals that condensation of the wild-type cells in the caudal half somite is concordant with the formation of segment boundaries, while mutant and wild-type cells intermingle in the presomitic mesoderm. This implies that periodic activation of the Notch pathway in the presomitic mesoderm is still latent to segregate the presumptive rostral and caudal somite. A transient episode of Mesp2 expression might be needed for Notch activation by Ps1 to confer rostral or caudal properties. In summary, it is proposed that Ps1 is involved in the functional manifestation of the segmentation clock in the presomitic mesoderm (Koizumi, 2001).

Mouse Notch1, which plays an important role in cell fate determination in development, is proteolytically processed within its transmembrane domain by unidentified gamma-secretase-like activity that depends on presenilin. To study this proteolytic event, a cell-free Notch cleavage assay system was established using the membrane fraction of fibroblast transfectants of various Notch constructs with deletion of the extracellular portion (NotchDeltaE). The cytoplasmic portion of Notch1DeltaE is released from the membrane upon incubation at 37°C; this is inhibited by the specific gamma-secretase inhibitor, MW167, or by overexpression of dominant negative presenilin1. Likewise, other members of mouse Notch family are proteolytically cleaved in a presenilin-dependent, MW167-sensitive manner in vivo as well as in the cell-free Notch DeltaE cleavage assay system. All four members of the mouse Notch family migrate to the nucleus and activate the transcription from the promoter carrying the RBP-J consensus sequences after they are released from the membrane. These results demonstrate the conserved biochemical mechanism of signal transduction among mammalian Notch family members (Mizutani, 2001).

Nicastrin is a multi-pass transmembrane protein that has recently been identified as a member of high-molecular weight complexes containing presenilin. The C. elegans homolog of nicastrin, aph-2, is required for GLP-1/Notch signaling in the early embryo. In addition to the maternal-effect embryonic lethal phenotype, aph-2 mutant animals also display an egg-laying defect. This latter defect is related to the SEL-12/presenilin egg-laying defect. aph-2 and sel-12 genetically interact and cooperate to regulate LIN-12/Notch signaling in the development of the somatic gonad. In addition, aph-2 and lin-12/Notch genetically interact. A new role for aph-2 in facilitating lin-12 signaling in the somatic gonad is illustrated, thus providing evidence that APH-2 is involved in both GLP-1/Notch- and LIN-12/Notch-mediated signaling events. Nicastrin can partially substitute for aph-2, suggesting a conservation of function between these proteins (Levitan, 2001).

Re-programming of C. elegans male epidermal precursor fates by Wnt, Hox, and LIN-12/Notch activities

In Caenorhabditis elegans males, different subsets of ventral epidermal precursor (Pn.p) cells adopt distinct fates in a position-specific manner: three posterior cells, P(9-11).p, comprise the hook sensillum competence group (HCG) with three potential fates (1°, 2°, or 3°), while eight anterior cells, P(1-8).p, fuse with the hyp7 epidermal syncytium. This study shows that activation of the canonical BAR-1 β-catenin pathway of Wnt signaling alters the competence of P(3-8).p and specifies ectopic HCG-like fates. This fate transformation requires the Hox gene mab-5. In addition, misexpression of mab-5 in P(1-8).p is sufficient to establish HCG competence among these cells, as well as to generate ectopic HCG fates in combination with LIN-12 or EGF signaling. While increased Wnt signaling induces predominantly 1° HCG fates, increased LIN-12 or EGF signaling in combination with MAB-5 overexpression promotes 2° HCG fates in anterior Pn.p cells, suggesting distinctive functions of Wnt, LIN-12, and EGF signaling in specification of HCG fates. Lastly, wild-type mab-5 function is necessary for normal P(9-11).p fate specification, indicating that regulation of ectopic HCG fate formation revealed in anterior Pn.p cells reflect mechanisms of pattern formation during normal hook development (Yu, 2010).

Overall, vulval precursor cell (VPC) and HCG patterning are quite similar: the precise cell fate is generated by progressive restriction through competence, induction, and lateral inhibition mediated by multiple signal integration at different steps, representing a general scenario of complex pattern formation (Yu, 2010).

Specifically, both VPC and HCG competence are established by Wnt signaling and one of the two Hox genes, lin-39 and mab-5, respectively. Expression patterns of both Hox genes are the same in both hermaphrodite and male, with lin-39 expression in P(3-8).p and mab-5 expression in P(7-11).p. However, sex-specific utilization of these two Hox genes, lin-39 and mab-5, determines whether a hermaphrodite vulva or a male hook, respectively, is formed. In hermaphrodites, lin-39 function is favored in the central Pn.p cells, and the ability of mab-5 to prevent P(9-11).p fusion with hyp7 is somehow blocked. As a transcription factor, mab-5 regulates target gene expression. One possibility is that a negative regulator in hermaphrodites sequesters mab-5 from its targets. Alternatively, mab-5 may act with a co-regulator that is missing in hermaphrodites. The Hox genes appear to play a permissive role in VPC and HCG induction because neither multi-vulvae nor multi-hooks are observed when lin-39 or mab-5, respectively, are overexpressed (Yu, 2010).

A major difference between VPC and HCG development is the major inductive signal used to specify the 1° fate: the EGF pathway induces the 1° VPC fate while Wnt signaling promotes the 1° HCG fate. However, both EGF and Wnt act to induce HCG as well as VPC fates, and it has been observed that excessive Wnt signaling can at least partially substitute for EGF signaling in VPC induction and vice versa in HCG specification. The local abundance of the signal could explain why different inductive signals are utilized in VPC and HCG patterning. The availability of the Wnt and EGF inductive signals differ spatially in hermaphrodites and males, contributing further to the sex-specific bias of Hox gene expression. Although Wnts are present in the central region of the body and the EGF ligand is produced in the tail, the EGF signal emanates from a concentrated source, the gonadal anchor cell, only in the hermaphrodite, while Wnt signaling is more abundant in the tail region as elucidated by extensive tail defects caused by deficient Wnt signaling. As such, only the required Hox gene is promoted in each region in a sex-specific manner -- for example, lin-39 activity in males is not reinforced due to lack of a strong extrinsic signal in the central region. Therefore, different signaling pathways may not be the direct cause of sexually dimorphic organogenesis. The specificity of signaling relies on Hox genes to direct sex-specific pattern formation among competent precursor cells (Yu, 2010).

Increasing Notch signaling antagonizes PRC2-mediated silencing to promote reprogramming of germ cells into neurons

Cell-fate reprogramming is at the heart of development, yet very little is known about the molecular mechanisms promoting or inhibiting reprogramming in intact organisms. In the C. elegans germline, reprogramming germ cells into somatic cells requires chromatin perturbation. This study shows that such reprogramming is facilitated by GLP-1/Notch signaling pathway. This is surprising, since this pathway is best known for maintaining undifferentiated germline stem cells/progenitors (see Drosophila germline). Through a combination of genetics, tissue-specific transcriptome analysis, and functional studies of candidate genes, a possible explanation for this unexpected role of GLP-1/Notch was uncovered. The study proposes that GLP-1/Notch promotes reprogramming by activating specific genes, silenced by the Polycomb repressive complex 2 (PRC2) (see Drosophila E(z)), and identifies the conserved histone demethylase UTX-1 (see Drosophila Utx) as a crucial GLP-1/Notch target facilitating reprogramming. These findings have wide implications, ranging from development to diseases associated with abnormal Notch signaling (Seelk, 2016).

Enzymatic cleavage of Notch

The Notch receptor on the plasma membrane is cleaved. The cleavage of human Notch2 is an evolutionarily conserved, general property of Notch and occurs in the trans-Golgi network as the receptor traffics toward the plasma membrane. Although full-length Notch2 is detectable in the cell, it does not reach the surface. Cleavage results in a C-terminal fragment, N(TM), that appears to be cleaved N-terminal to the transmembrane domain, and an N-terminal fragment, N(EC), that contains most of the extracellular region. These fragments are tethered together on the plasma membrane by a link that is sensitive to reducing conditions, forming a heterodimeric receptor. It is likely that the cleavage occurs between the EGF and Lin-12/Notch (LN) repeats, producing two fragments with a calculated molecular mass of 112 kDa and 180 kDa (Blaumueller, 1997).

A gamma-secretase-like proteolysis at site 3 (S3), within the transmembrane domain, releases the Notch intracellular domain (NICD) and activates CSL-mediated Notch signaling. S3 processing occurs only in response to ligand binding; however, the molecular basis of this regulation is unknown. Ligand binding facilitates cleavage at a second, novel site (S2), within the extracellular juxtamembrane region, which serves to release ectodomain repression of NICD production. Cleavage at S2 generates a transient intermediate peptide termed NEXT (Notch extracellular truncation). NEXT accumulates when NICD production is blocked by point mutations or gamma-secretase inhibitors or by loss of presenilin 1, and inhibition of NEXT eliminates NICD production. These data demonstrate that S2 cleavage is a ligand-regulated step in the proteolytic cascade leading to Notch activation (Mumm, 2000).

Peptide sequencing shows that S2 cleavage occurs between Ala-1710 and Val-1711 residues, approximately 12 amino acids outside the transmembrane domain. Thus, NEXT is a naturally occurring equivalent of constitutively active, membrane-tethered, NDeltaE proteins. Pulse-chase analysis has demonstrated that NDeltaE proteins undergo S3 processing and are converted to NICD. Consistent with this observation, evidence is presented that production of NEXT and NICD is linked: NEXT is enriched by blocking NICD production via point mutation, gamma-secretase inhibitors, and loss of presenilin 1 (PS1), while inhibition of NEXT production eliminates NICD accumulation. An initial inhibitor screen implicates metalloprotease activity in S2 proteolysis. In support of this, TACE (TNFalpha-converting enzyme) has been identified as a protease capable of S2 proteolysis in vitro. These data suggest that a ligand-induced proteolytic cascade activates Notch1; ligand binding serving to promote S2 cleavage, which is required for S3 cleavage (Mumm, 2000).

For NEXT to be a true signaling intermediary, it should be regulated in a ligand-dependant manner. In order to demonstrate that S2 is relevant to activation of the full-length receptor, NFL6MT constructs were cotransfected with CSLRBP-Jkappa and the DSL family member Jagged. NEXT and NICD both accumulate in the presence of Jagged. This result demonstrates that S2 proteolysis occurs in response to ligand activation of the full-length Notch1 receptor. However, one caveat of this and previous cotransfection experiments stems from the question of whether Notch is activated by a ligand presented by the same cell (in cis) or from a neighboring cell (in trans). Therefore, in order to confirm that trans interactions at the cell surface between Notch and its ligands lead to NEXT and NICD production, HeLa cells expressing Notch and CSLRBP-Jkappa were cocultured with HEK 293T cells or HEK 293T expressing exogenous Jagged. Both S2 and S3 proteolysis are strongly induced when receptor-ligand interactions occur exclusively in trans. This result provides further support for the hypothesis that the S2 cleavage occurs extracellularly at the plasma membrane. Taken together, these results support the hypothesis that ligand binding serves to relieve extracellular inhibition of S3 cleavage by promoting S2 cleavage. This ectodomain shedding-like process creates NEXT, a NDeltaE-like molecule, which undergoes S3 cleavage, producing NICD and leading to Notch activation (Mumm, 2000).

TACE is among a number of enzymes (alternatively termed secretases or sheddases) known to mediate a proteolytic process termed ectodomain shedding, whereby transmembrane proteins are cleaved extracellularly and released into the extracellular milieu. Ectodomain shedding is known to play key roles in cancer invasion, metastasis, activation of soluble ligands, and protein turnover. Sheddases generally exhibit low substrate specificity, and some are believed to be more dependent on structural motifs than primary residues, often cleaving at a fixed distance from the transmembrane domain (e.g., TACE). Disruption of the TACE locus in mice unexpectedly results in embryonic lethality, implicating TACE and ectodomain shedding in essential developmental events and suggesting TACE normally cleaves substrates other than pro-TNF. While no direct evidence is presented here that S2 cleavage results in shedding of Notch per se, recent reports have demonstrated that dissociation of the extracellular domain is sufficient for activation. Further, it has been observed that the extracellular domain of Notch alone is trans-endocytosed (thus 'shed') into ligand-expressing cells when Notch is activated during pupal wing vein and retinal pigment cell development in Drosophila. If correct, this model would predict that truly 'soluble' ligands will fail to activate Notch, and that Notch will be activated by membrane-tethered or extracellular matrix associated ligands (Mumm, 2000).

A final consideration must be taken of the involvement of metalloprotease(s) in Notch activation. Notch pathway activation has been shown to serve as a secondary event contributing to the oncogenic transformation of either Myc- or E1A- expressing cells. Elevated metalloprotease activity, which is often linked to transformation and metastasis, may lead to ectopic, ligand-independent activation of the endogenous Notch receptor. This may explain why nuclear Notch staining is often found in various tumors and suggests that ectopic, metalloprotease-mediated Notch activation may be a common event during oncogenesis (Mumm, 2000).

The Notch1 receptor is presented at the cell membrane as a heterodimer after constitutive processing by a furin-like convertase. Ligand binding induces the proteolytic release of Notch intracellular domain by a gamma-secretase-like activity. This domain translocates to the nucleus and interacts with the DNA-binding protein CSL, resulting in transcriptional activation of target genes. An additional processing event occurs in the extracellular part of the receptor, preceding cleavage by the gamma-secretase-like activity. Purification of the activity accounting for this cleavage in vitro shows that it is due to TACE (TNFalpha-converting enzyme), a member of the ADAM (a disintegrin and metalloprotease domain) family of metalloproteases. Furthermore, experiments carried out on TACE-/- bone marrow-derived monocytic precursor cells suggest that this metalloprotease plays a prominent role in the activation of the Notch pathway (Brou, 2000).

The TACE cleavage site in the Notch1 sequence is located between Ala-1710 and Val-1711, 13 amino acids upstream of the transmembrane domain. This site is perfectly conserved in murine or human Notch1 and in Xenopus Notch. In murine Notch2 and Notch3 and Drosophila Notch, similar sites can be found (SV in murine Notch2 and Drosophila Notch at the same position upstream of the transmembrane domain; AV 24 amino acids upstream of the transmembrane domain in murine Notch3). It is difficult to identify a similar site in Lin12 or Glp1, because of the weak general conservation. However, a database search indicates that a TACE ortholog exists in C. elegans. Thus, it is possible that TACE could recognize a degenerated site in C. elegans Notch homologs; alternatively, other mechanisms may exist to lead to a conformational change after ligand binding. Interestingly, some constitutively active mutants of C. elegans Lin12 carry mutations in the region surrounding the putative S2 site. The AV cleavage site in Notch matches perfectly known TACE preferred recognition sites such as those found in pro-TNF or pro-TGF (Brou, 2000).

Notch is a conserved cell surface receptor that is activated through direct contact with neighboring ligand-expressing cells. The primary 300-kDa translation product of the Notch1 gene (p300) is cleaved by a furin-like convertase to generate a heterodimeric, cell-surface receptor composed of 180- (p180) and 120- (p120) kDa polypeptides. Heterodimeric Notch is thought to be the only form of the receptor that is both present on the cell surface and able to generate an intracellular signal in response to ligand. Disruption of furin processing of Notch1, either by coexpression of a furin inhibitor or by mutation of furin target sequences within Notch1 itself, perturbs ligand-dependent signaling through the well-characterized mediator of Notch signal transduction, CSL [CBF1, Su(H), and LAG-1]. Yet contrary to these reports, the full-length p300 Notch1 product can be detected on the cell surface. Moreover, this uncleaved form of Notch1 can suppress the differentiation of C2C12 myoblasts in response to ligand. Taken together, these data support characterizing a CSL-independent Notch signaling pathway and identify this uncleaved isoform of Notch as a potential mediator of this pathway. These results suggest a novel paradigm in signal transduction, one in which two isoforms of the same cell-surface receptor can mediate two distinct signaling pathways in response to ligand (Bush, 2001).

The biological activity of the soluble form of the Notch ligand (sNL) and requirement of the intracellular domain (ICD) of the Notch ligand have been debated. Soluble Delta1 (sD1) has been shown to activate Notch2 (N2), but much more weakly than full-length Delta1 (fD1). Furthermore, tracing the N2 molecule after sD1 stimulation reveals that sD1 has a defect in the cleavage releasing ICD of N2 (intracellular cleavage), although it triggers cleavage in the extracellular domain of N2. This represents the molecular basis of the lower activity of sD1 and suggests the presence of an unknown mechanism regulating activation of the intracellular cleavage. The fact that Delta1 lacking its ICD (D1ICD) exhibits the phenotype similar to that exhibited by sD1 indicates that the ICD of D1 (D1DeltaICD) is involved in such an as yet unknown mechanism. Furthermore, the findings that D1DeltaICD acts in a dominant-negative fashion against fD1 and that the signal-transducing activity of sD1 is enhanced by antibody-mediated cross-linking suggest that the multimerization of Delta1 mediated by D1ICD may be required for activation of the N2 intracellular cleavage (Shimizu, 2002).

Following ectodomain shedding, Notch-1 undergoes presenilin (PS)-dependent constitutive intramembranous endoproteolysis at site-3. This cleavage is similar to the PS-dependent γ-secretase cleavage of the ß-amyloid precursor protein (ßAPP). However, topological differences in cleavage resulting in amyloid ß-peptide (Aß) or the Notch-1 intracellular domain (NICD) indicate independent mechanisms of proteolytic cleavage. The secretion of an N-terminal Notch-1 Aß-like fragment (Nß) is reported in this study. Analysis of Nß by MALDI-TOF MS revealed that Nß is cleaved at a novel site (site-4, S4) near the middle of the transmembrane domain. Like the corresponding cleavage of ßAPP at position 40 and 42 of the Aß domain, S4 cleavage is PS dependent. The precision of this cleavage is affected by familial Alzheimer’s disease-associated PS1 mutations similar to the pathological endoproteolysis of ßAPP. Considering these similarities between intramembranous processing of Notch and ßAPP, it is concluded that these proteins are cleaved by a common mechanism utilizing the same protease, i.e. PS/γ-secretase (Okochi, 2002).

Notch receptors undergo a cascade of endoproteolytic cleavages required for Notch signaling. Upon binding of membrane-anchored ligands from the DSL (Delta/Serrate/Lag-2) family, Notch receptors undergo consecutive cleavages at site-2 (S2) and site-3 (S3). Cleavage of mouse Notch-1 at S2 occurs in its ectodomain by TACE [tumor necrosis factor-α (TNF-α-converting enzyme], a member of the ADAM (a disintegrin and metalloprotease domain) family ~12 amino acids distant from the TM. This 'ectodomain shedding' event results in the generation of NEXT (Notch extracellular truncation), that is cleaved subsequently at S3 within the TM close to the cytoplasmic border. Cleavage of Notch at S3 liberates NICD (Notch intracellular domain) that translocates to the nucleus, where it is involved in target gene transcription. S3 cleavage strictly depends on the biological activity of the presenilin (PS) proteins, which may contribute the catalytic site of γ-secretase, an unusual intramembrane-cleaving aspartyl protease complex (Okochi, 2002 and references therein).

Beside the Notch-1-4 receptors, several other type I TM proteins have been identified as substrates for PS-dependent endoproteolysis, including the Alzheimer's disease (AD)-associated ß-amyloid protein precursor (ßAPP), ErbB-4, E-cadherin and LRP. These proteins undergo ‘ectodomain shedding’ in their large extracellular domains, prior to the consecutive PS-dependent cleavage within the TM. In the case of ßAPP, these cleavages are mediated by α-secretase and ß-secretase. Cleavage of ßAPP by α- and ß-secretase (BACE) results in the generation of the respective ßAPP C-terminal fragments (CTFs), CTFα and CTFß, which are the direct substrates for γ-secretase cleavage. Cleavage of CTFß and CTFα by γ-secretase occurs in the middle of the TM and leads to the liberation of Aß and p3 peptides), respectively. Aß is deposited in the brain of AD patients in 'senile plaques', an invariant pathological hallmark of AD. Recently, the elusive C-terminal cleavage product of γ-secretase, AICD (ßAPP intracellular domain), has been identified and characterized. Surprisingly, AICD results from PS-dependent γ-secretase cleavage of ßAPP-CTFs predominantly after Leu49 (Aß numbering). This cleavage is almost identical to the S3 cleavage of Notch-1 and does not occur after Val40 and Ala42 (Aß numbering) as predicted. Thus, γ-secretase cleaves the ßAPP TM at several sites: one in the middle after position 40 (γ40) and 42 (γ42) (with major γ40 and minor γ42 cleavage) and one close to the cytoplasmic border after position 49 (γ49) of the Aß domain. Interestingly, AICD may translocate to the nucleus where it could have a role in transcriptional regulation similar to NICD (Okochi, 2002 and references therein).

Because of these striking similarities between Notch and ßAPP endoproteolysis, it was hypothesized that an Aß/p3-like species (called Notch ß-peptide, Nß) derived from NEXT intramembranous proteolysis may be secreted into the extracellular space. This study reports the identification and characterization of secreted Nß peptides derived from endoproteolysis of NEXT derivatives. Sequence analysis revealed that Nß is derived from endoproteolytic cleavage near the middle of the Notch-1 TM at site-4 (S4), which is 12 amino acid residues upstream of S3. Like S3 cleavage, S4 cleavage occurs in a PS- and γ-secretase-dependent manner. Strikingly, familial AD (FAD)-associated PS mutants known to cause the increased production of C-terminally elongated pathogenic Aß42 also affect the generation of C-terminally elongated Nß variants, supporting a direct role for PS in the proteolytic cleavage of Notch-1 and ßAPP (Okochi, 2002).

The role of Notch signaling in general and presenilin in particular during mouse somitogenesis was analyzed. Cyclical production of activated Notch (NICD) was visualized and it was established that somitogenesis requires less NICD than any other tissue in early mouse embryos. Indeed, formation of cervical somites proceeds in Notch1; Notch2-deficient embryos. This is in contrast to mice lacking all presenilin alleles, that have no somites. Since Nicastrin-, Pen-2-, and APH-1a-deficient embryos have anterior somites without γ-secretase, presenilin may have a γ-secretase-independent role in somitogenesis. Embryos triple homozygous for both presenilin null alleles and a Notch allele that is a poor substrate for presenilin (N1V→G) experience fortuitous cleavage of N1V→G by another protease. This restores NICD, anterior segmentation, and bilateral symmetry but does not rescue rostral/caudal identities. These data clarify multiple roles for Notch signaling during segmentation and suggest that the earliest stages of somitogenesis are regulated by both Notch-dependent and Notch-independent functions of presenilin (Hupper, 2005).

Several type I integral membrane proteins, such as the Notch receptor or the amyloid precursor protein, are cleaved in their intramembrane domain by a gamma-secretase enzyme, which is carried within a multiprotein complex. These cleavages generate molecules that are involved in intracellular or extracellular signaling. At least four transmembrane proteins belong to the gamma-secretase complex: presenilin, nicastrin, Aph-1, and Pen-2. It is still unclear whether these proteins are the only components of the complex and whether a unique complex is involved in the different gamma-secretase cleavage events. A genetic screen was set up based on the permanent acquisition or loss of an antibiotic resistance depending on the presence of an active gamma-secretase able to cleave a Notch-derived substrate. Clones were selected deficient in gamma-secretase activity using this screen on mammalian cells after random mutagenesis. Two of these clones were examined and previously undescribed mutations were identified in the nicastrin gene. The first mutation abolishes nicastrin production, and the second mutation, a point mutation in the ectodomain, abolishes nicastrin maturation. In both cases, gamma-secretase activity on Notch and APP is impaired (Olry, 2005).

The four highly conserved Notch receptors receive short-range signals that control many biological processes during development and in adult vertebrate tissues. The involvement of Notch1 signaling in tissue self-renewal is less clear, however. This study developed a novel genetic approach N1IP-CRE (Notch1 Intramembrane Proteolysis) to follow, at high resolution, the descendents of cells experiencing Notch1 activation in the mouse. To generate a genetic sensor of Notch1 proteolysis the mouse Notch1 intracellular domain (NICD1), immediately downstream of the transmembrane domain, was replaced with the site-specific recombinase Cre, such that the Cre activity is now governed by ligand-induced proteolysis of the Notch1 transmembrane domain tether. In Cre-reporter strains, Notch activation is visualized by β-galactosidase expression. Because Notch1 proteolysis releases Cre that leads to a cell-heritable expression of lacZ, Notch1 signaling in actively cycling stem/progenitor cells will mark all their descendents producing a 'clone', whereas Notch1 activation in transit amplifying or differentiating cells will result in small clones (2-4 cells) or in salt-and-pepper patterns of individually labeled cells. By combining N1IP-CRE with loss-of-function analysis, Notch activation patterns were correlated with function during development, self-renewal and malignancy in selected tissues. Identification of many known functions of Notch1 throughout development validated the utility of this approach. Importantly, novel roles for Notch1 signaling were identified in heart, vasculature, retina and in the stem cell compartments of self-renewing epithelia. The probability of Notch1 activation in different tissues does not always indicate a requirement for this receptor, and gradients of Notch1 activation are evident within one organ. These findings highlight an underappreciated layer of complexity of Notch signaling in vivo. Moreover, NIP-CRE represents a general strategy applicable for monitoring proteolysis-dependent signaling in vivo (Vooijs, 2007).

Numb promotes degradation of Notch intracellular domain

The cell fate determinant Numb influences developmental decisions by antagonizing the Notch signaling pathway. However, the underlying molecular mechanism of this inhibition is poorly understood. The mammalian Numb protein promotes the ubiquitination of membrane-bound Notch1 receptor. Furthermore, Numb expression results in the degradation of the Notch intracellular domain following activation -- this correlates with a loss of Notch-dependent transcriptional activation of the Hes1 promoter as measured by a Hes1 luciferase reporter assay. The phosphotyrosine-binding (PTB) domain of Numb is required for both Notch1 ubiquitination and down-regulation of Notch1 nuclear activity. Numb-mediated ubiquitination of Notch1 is not dependent on the PEST region, which was previously shown to mediate Sel10-dependent ubiquitination of Notch in the nucleus, suggesting a distinct E3 ubiquitin ligase is involved. In agreement, Numb is shown to interact with the cytosolic HECT domain-containing E3 ligase Itch; Numb and Itch act cooperatively to promote ubiquitination of membrane-tethered Notch1. These results suggest that Numb recruits components of the ubiquitination machinery to the Notch receptor thereby facilitating Notch1 ubiquitination at the membrane, which in turn promotes degradation of the intracellular domain circumventing its nuclear translocation and downstream activation of Notch1 target genes (McGill, 2003).

The beta-amyloid precursor protein (APP) and the Notch receptor undergo intramembranous proteolysis by the Presenilin-dependent gamma-secretase. The cleavage of APP by gamma-secretase releases amyloid-beta peptides, which have been implicated in the pathogenesis of Alzheimer's disease, and the APP intracellular domain (AID), for which the function is not yet well understood. A similar gamma-secretase-mediated cleavage of the Notch receptor liberates the Notch intracellular domain (NICD). NICD translocates to the nucleus and activates the transcription of genes that regulate the generation, differentiation, and survival of neuronal cells. Hence, some of the effects of APP signaling and Alzheimer's disease pathology may be mediated by the interaction of APP and Notch. Membrane-tethered APP binds to the cytosolic Notch inhibitors Numb and Numb-like in mouse brain lysates. AID also binds Numb and Numb-like, and represses Notch activity when released by APP. Thus, gamma-secretase may have opposing effects on Notch signaling; positive by cleaving Notch and generating NICD, and negative by processing APP and generating AID, which inhibits the function of NICD (Roncarati, 2002).

Cis-interactions between Notch and Delta generate mutually exclusive signalling states

The Notch-Delta signalling pathway allows communication between neighbouring cells during development. It has a critical role in the formation of 'fine-grained' patterns, generating distinct cell fates among groups of initially equivalent neighbouring cells and sharply delineating neighbouring regions in developing tissues. The Delta ligand has been shown to have two activities: it transactivates Notch in neighbouring cells and cis-inhibits Notch in its own cell. However, it remains unclear how Notch integrates these two activities and how the resulting system facilitates pattern formation. This paper reports the development of a quantitative time-lapse microscopy platform for analysing Notch-Delta signalling dynamics in individual mammalian cells, with the aim of addressing these issues. By controlling both cis- and trans-Delta concentrations, and monitoring the dynamics of a Notch reporter, the combined cis-trans input-output relationship was measure in the Notch-Delta system. The data revealed a striking difference between the responses of Notch to trans- and cis-Delta: whereas the response to trans-Delta is graded, the response to cis-Delta is sharp and occurs at a fixed threshold, independent of trans-Delta. A simple mathematical model shows how these behaviours emerge from the mutual inactivation of Notch and Delta proteins in the same cell (see The mutual inactivation model in multicellular patterning). This interaction generates an ultrasensitive switch between mutually exclusive sending (high Delta/low Notch) and receiving (high Notch/low Delta) signalling states. At the multicellular level, this switch can amplify small differences between neighbouring cells even without transcription-mediated feedback. This Notch-Delta signalling switch facilitates the formation of sharp boundaries and lateral-inhibition patterns in models of development, and provides insight into previously unexplained mutant behaviours (Sprinzak, 2010).

Fringe proteins modulate Notch-ligand and interactions to specify signaling states

The Notch signaling pathway consists of multiple types of receptors and ligands, whose interactions can be tuned by Fringe glycosyltransferases (see Drosophyla Fringe). A major challenge is to determine how these components control the specificity and directionality of Notch signaling in developmental contexts. This study analyzed same-cell (cis) Notch-ligand interactions for Notch1, Dll1, and Jag1, and their dependence on Fringe protein expression in mammalian cells. Dll1 and Jag1 were found to cis-inhibit Notch1, and Fringe proteins modulate these interactions in a way that parallels their effects on trans interactions. Fringe similarly modulated Notch-ligand cis interactions during Drosophila development. Based on these and previously identified interactions, it was shown how the design of the Notch signaling pathway leads to a restricted repertoire of signaling states that promote heterotypic signaling between distinct cell types, providing insight into the design principles of the Notch signaling system, and the specific developmental process of Drosophila dorsal-ventral boundary formation (LeBon, 2014: PubMed).

Deciphering the Fringe-Mediated Notch Code: Identification of Activating and Inhibiting Sites Allowing Discrimination between Ligands

Fringe proteins (see Drosophila Fringe) are beta3-N-acetylglucosaminyltransferases that modulate Notch activity by modifying O-fucose residues on epidermal growth factor-like (EGF) repeats of Notch. Mammals have three Fringes: Lunatic, Manic, and Radical. While Lunatic and Manic Fringe inhibit Notch1 activation from Jagged1 and enhance activation from Delta-like 1 (see Drosophila Delta), Radical Fringe enhances signaling from both. A mass spectrometry approach was used to determine whether the variable effects of Fringes on Notch1 result from generation of unique glycosylation patterns on Notch1. Lunatic and Manic Fringe were found to modify similar sites on Notch1, while Radical Fringe modified a subset. Fringe modifications at EGF8 and EGF12 enhanced Notch1 binding to and activation from Delta-like 1, while modifications at EGF6 and EGF36 (added by Manic and Lunatic but not Radical) inhibited Notch1 activation from Jagged1. Combined, these results suggest that Fringe modifications 'mark' different regions in the Notch1 extracellular domain for activation or inhibition (Kakuda, 2017).

Transcytosis of the Notch Extracellular Domain

Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneural gene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. These observations support a model for Notch activation where the Delta-Notch interaction is followed by endocytosis of Delta and transendocytosis of the Notch extracellular domain by the signaling cell. This facilitates intramembranous cleavage of the remaining Notch receptor, release of the Notch intracellular fragment, and activation of target genes in neighboring cells (Itoh, 2003).

There are two models that could explain why an E3 that is responsible for ubiquitylation and internalization of Delta would be required for effective Notch signaling. One possibility is based on the proposition that Mib is required in the cell that delivers signals; the other assumes that it is required in the cell that receives them. In the first model, Mib promotes the transendocytosis of the Notch extracellular domain by promoting endocytosis of Delta and, in doing so, facilitates proteolytic events that generate the transcriptionally active NotchICD fragment. This proposal comes from studies of the neurogenic phenotype of shibire and neur mutants in Drosophila, suggesting that transendocytosis of the Notch extracellular domain by the adjacent Delta-expressing cell is essential for efficient Notch activation. In the zebrafish system, transplantation experiments show that cells with reduced mib function are less likely to become neurons when surrounded by wild-type cells. This supports the idea that loss of mib function primarily reduces a cell's ability to produce an effective inhibitory signal in the competition to become a neuron (Itoh, 2003).

The other model that explains why ubiquitylation and internalization of Delta might be essential for Notch signaling postulates a cell-autonomous role for mib in signal reception, as has also been suggested previously for neur. According to this model, mib-mediated Delta turnover would limit Delta's ability to inhibit Notch function cell autonomously. However, cell transplantation results argue against a significant deficit in reception of the inhibitory signal. Furthermore, the luciferase experiments, in which cells were cotransfected with notch and various delta constructs, show that, while Delta does indeed have a cell-autonomous effect in blocking signal reception, mib does not significantly influence this action of Delta. Moreover, this action of Delta does not seem to be ubiquitin dependent: the recombinant addition of ubiquitin does not significantly reduce DeltaDeltaICD's ability to inhibit Notch function. It is possible that in these assays, Mib is ineffective at reducing cell-autonomous inhibition of Notch by Delta because very high levels of artificially expressed Delta in the transfected cells in vitro may overwhelm the capacity of the Mib-dependent machinery. However, in studies in COS7 cells, at least, Mib is effective in removing artificially expressed Delta from the cell surface, suggesting that the inhibitory effect of Delta may be independent of delivery of Delta to the cell surface: it may result from Delta-Notch interactions within the secretory pathway. In short, observations do not support a significant role for Mib in limiting Delta's ability to cell autonomously inhibit Notch function as has been described for Neur, but such a role cannot be completely ruled out (Itoh, 2003).

The role of Mib in signal delivery is strongly supported and tightly correlated with Delta ubiquitylation. Ectopic expression of XDelta1DeltaICD, which cannot be ubiquitylated, permits too many cells to become neurons, while XDelta1 and XDelta1DeltaICD-Ub, ectopically expressed in the embryo in a similar way, both inhibit cells from becoming neurons. These effects correlate with the ubiquitin-dependent reduction of cell surface Delta. The internalization of XDelta1DeltaICD-Ub is consonant with previous studies that have shown that in-frame addition of ubiquitin to stable plasma membrane proteins can serve to target their entry into the endocytic pathway. The obvious suggestion, therefore, is that Mib-induced ubiquitylation drives internalization of Delta by endocytosis, and that this process is critical for effective signaling by Delta (Itoh, 2003).

An additional possibility that cannot as yet be excluded is that Mib-dependent ubiquitylation of Delta also decreases the amount of Delta that reaches the cell surface by sorting Delta directly from the Golgi complex to late endosomes. Such a dual role has been shown in yeast for the E3 ligase Rsp5p, which ubiquitylates its substrate, Gap1p, to regulate the total amount of Gap1p at the cell surface. Ubiquitylation of Gap1p by Rsp5p promotes endocytosis of Gap1p and favors sorting of Gap1p from the Golgi to the vacuole, where it is degraded. Mib may also have dual roles in endocytosis of Delta and in direct sorting of Delta to late endosomes/lysosomes; however, it is not clear at this time how the later function might contribute to Notch signaling (Itoh, 2003).

Although mib mutants express unusually high levels of cell surface Delta, it is unlikely that this is per se the cause of the neurogenic phenotype, since artificial expression of even higher levels of Delta in mib mutants following injection of delta mRNA suppress the neurogenic phenotype (Itoh, 2003).

From these observations, it seems that while all the forms of Delta that were examined can cell autonomously inhibit Notch function, only the forms of Delta that are ubiquitylated and endocytosed can effectively activate Notch in neighboring cells. It is likely that when Delta is driven to high levels in a group of cells, the effect of Delta in trans, as an activator of the Notch pathway, dominates over its effect in cis, as an inhibitor, accounting for the ability of Xdelta1 and Xdelta1DeltaICD-Ub to inhibit neurogenesis in the embryo (Itoh, 2003).

The opposing cell-autonomous and nonautonomous effects on Notch signaling define two synergistic mechanisms by which a cell expressing more Delta than its neighbors gains an enhanced ability to become a neuron. By activating Notch in neighboring cells, Delta reduces the neighbors' ability to express the Notch ligand Delta at high levels. At the same time, Delta interferes with Notch function in the cell where Notch and Delta are coexpressed, making it harder for this cell to be inhibited from becoming a neuron by Delta in neighboring cells (Itoh, 2003).

The role for mib in promoting endocytosis in the signal-delivering cell, as demonstrated in this study, is similar to one role proposed for neur in Drosophila. In vertebrates, however, neur seems to have a much more limited role than has been demonstrated for it in Drosophila. Mice that are homozygous for a neur loss-of-function mutation have restricted defects: one study demonstrated defects in spermatogenesis and in mammary gland development, while another study has shown ethanol hypersensitivity and an olfactory discrimination defect. In Xenopus, interfering with neur function by overexpressing either wild-type Neur or a mutant form that lacks the RING finger domain increases the density of ciliated cells in the epidermis . But none of these studies revealed the dramatic neural phenotypes or defects in somitogenesis that are seen when there is broad loss of Notch signaling. In contrast, mib mutants do show widespread abnormalities, suggesting a deficit in many more Notch-dependent developmental events. Currently being investigated are whether mib has assumed some roles that were originally played by neur in Drosophila or whether a cooperative role for neur and mib in Notch signaling limits the deficit caused by loss of neur alone in vertebrates (Itoh, 2003).

In summary, the analysis of the zebrafish mib mutant has led to the identification of a gene that is essential for effective Notch signaling in many different tissues during development. The function of Mib as a ubiquitin ligase in the internalization of Delta provides new avenues for clarifying the mystery of how endocytosis may increase the ability of cell surface Delta to deliver lateral inhibition signals (Itoh, 2003).

Phosphorylation of Notch by GSK3ß

Notch receptors modulate transcriptional targets following the proteolytic release of the Notch intracellular domain (NotchIC). Phosphorylated forms of NotchIC have been identified within the nucleus and have been associated with CSL members, as well as correlated with regions of the receptor that are required for activity. Genetic studies have suggested that Shaggy, the Drosophila homolog of glycogen synthase kinase-3ß (GSK3ß) may act as a positive modulator of the Notch signaling. GSK3ß is a serine/threonine kinase and is a component of the Wnt/wingless signaling cascade. GSK3ß is able to bind and phosphorylate Notch1IC in vitro, and attenuation of GSK3ß activity reduces phosphorylation of NotchIC in vivo. Functionally, ligand-activated signaling through the endogenous Notch1 receptor is reduced in GSK3ß null fibroblasts, implying a positive role for GSK3ß in mammalian Notch signaling. As a possible mechanistic explanation of the effect of GSK3ß on Notch signaling, it was observed that inhibition of GSK3ß shortens the half-life of Notch1IC. Conversely, activated GSK3ß reduces the quantity of Notch1IC that was degraded by the proteasome. These studies reveal that GSK3ß modulates Notch1 signaling, possibly through direct phosphorylation of the intracellular domain of Notch, and that the activity of GSK3ß protects the intracellular domain from proteasome degradation (Foltz, 2002).

Attenuation of Notch signalling by the Down-syndrome-associated kinase DYRK1A dependent phosphorylation

Notch signalling is used throughout the animal kingdom to spatially and temporally regulate cell fate, proliferation and differentiation. Its importance is reflected in the dramatic effects produced on both development and health by small variations in the strength of the Notch signal. The Down-syndrome-associated kinase DYRK1A is coexpressed with Notch in various tissues during embryonic development. DYRK1A moves to the nuclear transcription compartment where it interacts with the intracellular domain of Notch promoting its phosphorylation in the ankyrin domain and reducing its capacity to sustain transcription. DYRK1A attenuates Notch signalling in neural cells both in culture and in vivo, constituting a novel mechanism capable of modulating different developmental processes that can also contribute to the alterations observed during brain development in animal models of Down syndrome (Fernandez-Martinez, 2009).

The modulation of other signalling events by DYRK1A has been described previously, with the kinase activity of DYRK1A often being found to be dispensable for the regulation. By contrast, this study shows here that the attenuation of Notch signalling by DYRK1A is dependent on its kinase activity, compatible with previous findings indicating that the intracellular domain of Notch is a substrate of several kinases that can modulate its activity. Interestingly, the domains previously described as targets for phosphorylation TAD and OPA/PEST are dispensable for the regulation of Notch signalling by DYRK1A. Indeed, mapping analysis shows that DYRK1A phosphorylates the RAM-ANK domain (Fernandez-Martinez, 2009).

To identify the phosphorylation sites in Notch, the potential sites were examined based on the described consensus sequence (RPXS/TP), Notch crystal structure and their conservation in different species. Single substitutions in any of the putative 18 conserved serines and threonines that could be targets of DYRK1A did not reduce the attenuation caused by the kinase. These substitutions included the best conserved sites corresponding to described consensus sequence (RPXS/TP motifs) within the TPLH sequence at positions 4-7 of the ANK repeats 1, 3, 4 and 6 of Notch1. These results indicate that DYRK1A phosphorylates Notch at multiple sites. Double substitutions in different combinations of ANK repeats dramatically decreased Notch activity, but DYRK1A could still attenuate the diminished response. These data highlight the importance of the threonines in the maintenance of the ANK structure and preclude further analysis of multiple mutations in the context of DYRK1A activity. To overcome this problem, 2-D electrophoresis analysis was carried out, and it was confirmed that DYRK1A induces multiple phosphorylation events in the NICD as shown by the large shift observed when both proteins were coexpressed. Interestingly, whereas the phosphorylation of the PEST region by CDK8 targets the NICD to a degradation pathway, phosphorylation events mediated by DYRK1A do not affect the stability of the Notch protein. Thus, DYRK1A attenuates Notch activity by phosphorylation in multiple residues without affecting its stability (Fernandez-Martinez, 2009).

Although DYRK1A performs some of its activities in a kinase-independent manner, the data indicate that the relationship between DYRK1A and the NICD needs the kinase activity, suggesting an enzyme-substrate type interaction. Indeed, the data indicate that the interaction is very transient and thus, difficult to detect. The use of the kinase-inactive form of DYRK1A greatly favoured the detection of the complex, consistent with the formation of a stable, albeit unproductive, complex with Notch. Hence, it is concluded that DYRK1A and the NICD transiently associate in the nucleus, an association that is stabilized when the kinase is inactive (Fernandez-Martinez, 2009).

Besides the effect that DYRK1A exerts on the transcriptional activity of Notch signalling reporters, whether the expression of DYRK1A could affect some of the activities that Notch signalling performs in vivo was investigated. Notch signalling can prevent the maturation of neurons. Whether the reduction in neurite development in cells with an activated form of the NICD could be reversed by the presence of DYRK1A was examined. Indeed, it was found that the repression in neuritogenesis was released in the presence of DYRK1A, indicating that the response of the cell to Notch signalling was attenuated (Fernandez-Martinez, 2009).

As Notch signalling is used iteratively to control cell proliferation, determination or differentiation in the developing neural tube, it was used as an in vivo model to study the effect of DYRK1A on Notch signalling. The overexpression of DYRK1A in the developing neural tube repressed the expression of Hes5-1, a well known indicator of Notch signalling, confirming that DYRK1A is sufficient to attenuate endogenous Notch signalling in vivo (Fernandez-Martinez, 2009).

In Drosophila the lack of function mutant minibrain (mnb), the fly DYRK1A homologue, results in a reduced brain size because of a decrease of the generation of cells during postembryonic development and similarly, Dyrk1a+/– mice also have smaller brains. Although an abnormal increase in Notch signalling inhibits neuronal differentiation in mice, deficient Notch signalling also leads to a reduction in the number of neurons in the adult cause by the induction of precocious neuronal differentiation. Thus, these data are compatible with the finding that DIRK1A attenuates Notch signalling both in vitro and in vivo. Interestingly, the data may be also relevant for studies of Down syndrome (DS). Indeed, DYRK1A is one of the genes located in the Down syndrome critical region and its expression is upregulated in DS individuals. Notch signalling is also altered in the DS condition, although contrasting data have been obtained in studies in humans and mouse models. Although Notch signalling seems to be upregulated in the cortex of DS individuals, it is repressed in the cerebellum of Ts1Cje mice, a model for DS. Several factors could account for these apparently contradictory results. First, the human DS condition also produces an increase in the expression of the Notch receptor and changes in the expression of other Notch modifiers such as Dlx. The presence of DYRK1A in these cells, with a clear misbalance in gene expression, might result in an attenuation of an otherwise augmented Notch signalling and be coherent with the results in which Notch output is upregulated or downregulated. Nevertheless, it is believed that the clear effects of DYRK1A in the attenuation of Notch signalling that are describe in this study can have a higher impact on DS during embryonic development, when DYRK1A is prominently expressed in Notch expressing areas (Fernandez-Martinez, 2009).

In summary, DYRK1A is able to attenuate Notch signalling both in neuroblastoma cells and in vivo, providing further insight into the mechanisms by which neurogenesis and other cell decisions mediated by Notch signalling can be modulated both in physiological and pathological conditions. Indeed, its ability to downregulate Notch signalling could contribute to the severe alterations in the formation of certain brain regions observed in animal models and associated with the development of Down syndrome in humans. Finally, the widespread expression of DYRK1A opens up the possibility that it can also modulate Notch signalling in other tissues (Fernandez-Martinez, 2009).

Contactin proteins act as ligands for Notch

Axon-derived molecules are temporally and spatially required as positive or negative signals to coordinate oligodendrocyte differentiation. Increasing evidence suggests that, in addition to the inhibitory Jagged1/Notch1 signaling cascade, other pathways act via Notch to mediate oligodendrocyte differentiation. The GPI-linked neural cell recognition molecule F3/contactin (See Drosophila Contactin) is clustered during development at the paranodal region, a vital site for axoglial interaction. F3/contactin acts as a functional ligand of Notch. This trans-extracellular interaction triggers gamma-secretase-dependent nuclear translocation of the Notch intracellular domain. F3/Notch signaling promotes oligodendrocyte precursor cell differentiation and upregulates the myelin-related protein MAG in OLN-93 cells. This can be blocked by dominant negative Notch1, Notch2, and two Deltex1 mutants lacking the RING-H2 finger motif, but not by dominant-negative RBP-J or Hes1 antisense oligonucleotides. Expression of constitutively active Notch1 or Notch2 does not upregulate MAG. Thus, F3/contactin specifically initiates a Notch/Deltex1 signaling pathway that promotes oligodendrocyte maturation and myelination (Hu 2003).

Neurons and glia in the vertebrate central nervous system arise in temporally distinct, albeit overlapping, phases. Neurons are generated first followed by astrocytes and oligodendrocytes from common progenitor cells. Increasing evidence indicates that axon-derived signals spatiotemporally modulate oligodendrocyte maturation and myelin formation. F3/contactin is a functional ligand of Notch during oligodendrocyte maturation, revealing the existence of another group of Notch ligands. NB-3, a member of the F3/contactin family, acts as a novel Notch ligand to participate in oligodendrocyte generation. NB-3 triggers nuclear translocation of the Notch intracellular domain and promotes oligodendrogliogenesis from progenitor cells and differentiation of oligodendrocyte precursor cells via Deltex1. In primary oligodendrocytes, NB-3 increases myelin-associated glycoprotein transcripts. Thus, the NB-3/Notch signaling pathway may prove to be a molecular handle to treat demyelinating diseases (Cui, 2004).

Ubiquitination of Notch

Activation of mammalian Notch receptor by its ligands induces TNFalpha-converting enzyme-dependent ectodomain shedding, followed by intramembrane proteolysis due to presenilin (PS)-dependent gamma-secretase activity. A modification, monoubiquitination, as well as clathrin-dependent endocytosis, is required for gamma-secretase processing of a constitutively active Notch derivative, DeltaE, which mimics the TNFalpha-converting enzyme-processing product. PS interacts with this modified form of DeltaE, DeltaEu. The lysine residue targeted by the monoubiquitination event has been identified, and its importance for activation of Notch receptor by its ligand, Delta-like 1, has been confirmed. A new model is proposed where monoubiquitination and endocytosis of Notch are a prerequisite for its PS-dependent cleavage, and its relevance for other gamma-secretase substrates is discussed (Gupta-Rossi, 2004).

The ubiquitination pathway involves a multiprotein cascade in which the substrate specificity is determined by the E3 component. Multi-ubiquitin chains at least four subunits long are required for efficient recognition and degradation of ubiquitinated proteins by the proteasome, but ubiquitin has more recently been shown to endorse new functions that do not always involve the proteasome (Gupta-Rossi, 2004).

The results show that a monoubiquitination event takes place on the DeltaE molecule, a constitutively active form of the Notch receptor that mimics the intermediate TACE-processing product generated after ligand binding. This modification is a prerequisite for gamma-secretase cleavage and targets one of the subunits of a dimeric membrane-anchored form of Notch DeltaE. The major site of monoubiquitination has been localized to a juxtamembrane, conserved lysine residue K1749 in mNotch1. Access to the monoubiquitinated form DeltaEu was gained by coimmunoprecipitation with endogenous PS1 when gamma-secretase activity was inhibited by a specific drug. Thus, DeltaEu is a labile intermediate appearing before gamma-secretase cleavage. This form could also be detected after coexpression of PS1 or DeltaC4, a PS2-derived construct. These molecules are probably not included in PS-containing high molecular weight complexes; neither are gamma-secretase components when transiently overexpressed. The existence of DeltaEu was varified by extracting and stabilizing it out of the active complexes. It is proposed that this ubiquitination step is required in the context of the full-length receptor activated by ligand binding. Although the modified intermediate species derived from full-length Notch could not be directly accessed, mutating the crucial lysine residue impaired Dll1-mediated Notch signaling, in accordance with the DeltaE results. It remains to be determined which E3 ubiquitin ligase is involved in this modification. Various proteins carrying such an activity have been associated with the Notch cascade and are candidates to be tested, e.g., Deltex, Suppressor of Deltex, and Cbl. Experiments are in progress to answer this question (Gupta-Rossi, 2004).

The results show that endocytosis of Notch DeltaE and of ligand-activated full-length Notch are necessary for gamma-secretase cleavage. The involvement of a clathrin-dependent endocytosis event for Notch activation complies with the mosaic analysis performed in Drosophila, which revealed that shibire function is required in Notch-expressing cells receiving a lateral inhibition signal. It is proposed that monoubiquitination on a juxtamembrane lysine (K1749) and endocytosis occur after ligand-induced cleavage of the Notch extracellular domain by TACE. The data are in apparent contradiction with a previous model, according to which gamma-secretase cleavage occurs at the plasma membrane. However, the previous data can be reinterpreted in light of the new model. The previous study argued that the TACE-processing product of Notch (similar to the DeltaE construct used in this study) remains associated with the apical membrane in Nicastrin or PS mutant cells, and in WT cells only a small amount of this molecule can be found in endocytic vesicles. This result can be explained by the fact that ubiquitination is one of the limiting steps in Notch signaling, or that active PS is needed to direct the final steps of endocytosis of the ubiquitinated forms. Probably for the same reason, DeltaE is very poorly cleaved by gamma-secretase when overexpressed, and the ubiquitination event can hardly be detected. The current results are also in apparent contradiction with those of another study, which postulates that in Drosophila, PS-mediated proteolysis does not appear to require a particular sequence nor the presence of active dynamin. However, the assay used appears unusually sensitive, as it even detects the cleavage of a Notch molecule carrying a G1743V mutation of the gamma-secretase cleavage site, a mutation that prevents activation in most other assays and, when introduced into mice, gives rise to an almost perfect Notch1 null phenotype. Therefore, a leakage due to overexpression might in some cases be responsible for the activity detected. Experiments are in progress to test the effect of mutating the juxtamembrane lysine of Drosophila Notch (Gupta-Rossi, 2004).

Various papers have described monoubiquitination as a signal for internalization of receptors such as EGFR or glycine receptor. The results do not allow one to discriminate between ubiquitination triggering endocytosis or being concomitant with the first steps of endocytosis. However, the observations show a more internal localization of LLFF (the site of gamma-secretase cleavage) compared with the K1749R mutant, and endocytosis of the K1749R DeltaE mutant seems to be blocked at an earlier stage when compared with the WT or LLFF mutant. These data suggest that ubiquitination is necessary for late events driving Notch to compartments where gamma-secretase cleavage can occur (Gupta-Rossi, 2004).

O-glucosylation of Notch

Protein O-glucosylation is a conserved post-translational modification that occurs on epidermal growth factor-like (EGF) repeats harboring the C1-X-S-X-P-C2 consensus sequence. The Drosophila protein O-glucosyltransferase (Poglut) Rumi regulates Notch signaling, but the contribution of protein O-glucosylation to mammalian Notch signaling and embryonic development is not known. This study shows that mouse Rumi encodes a Poglut, and that Rumi-/- mouse embryos die before embryonic day 9.5 with posterior axis truncation and severe defects in neural tube development, somitogenesis, cardiogenesis and vascular remodeling. Rumi knockdown in mouse cell lines results in cellular and molecular phenotypes of loss of Notch signaling without affecting Notch ligand binding. Biochemical, cell culture and cross-species transgenic experiments indicate that a decrease in Rumi levels results in reduced O-glucosylation of Notch EGF repeats, and that the enzymatic activity of Rumi is key to its regulatory role in the Notch pathway. Genetic interaction studies show that removing one copy of Rumi in a Jag1+/- (jagged 1) background results in severe bile duct morphogenesis defects. Altogether, these data indicate that addition of O-glucose to EGF repeats is essential for mouse embryonic development and Notch signaling, and that Jag1-induced signaling is sensitive to the gene dosage of the protein O-glucosyltransferase Rumi. Given that Rumi-/- embryos show more severe phenotypes compared to those displayed by other global regulators of canonical Notch signaling, Rumi is likely to have additional important targets during mammalian development (Fernandez-Valdivia, 2011).

LIN-12/Notch trafficking and regulation of DSL ligand activity during vulval induction in C. elegans

A novel mode of crosstalk between the EGFR-Ras-MAPK and LIN-12/Notch pathways occurs during the patterning of a row of vulval precursor cells (VPCs) in Caenorhabditis elegans: activation of the EGFR-Ras-MAPK pathway in the central VPC promotes endocytosis and degradation of LIN-12 protein. LIN-12 downregulation in the central VPC is a prerequisite for the activity of the lateral signal, which activates LIN-12 in neighboring VPCs. This study characterizes cis-acting targeting sequences in the LIN-12 intracellular domain; in addition to a di-leucine motif, serine/threonine residues are important for internalization and lysine residues are important for post-internalization trafficking and degradation. Two trans-acting factors are identified that are required for post-internalization trafficking and degradation: ALX-1, a homolog of yeast Bro1p and mammalian Alix and the WWP-1/Su(dx)/Itch ubiquitin ligase. By examining the effects of mutated forms of LIN-12 and reduced wwp-1 or alx-1 activity on subcellular localization and activity of LIN-12, evidence is provided that the lateral signal-inhibiting activity of LIN-12 resides in the extracellular domain and occurs at the apical surface of the VPCs (Shaye, 2005).

LIN-12 appears to be downregulated via multivesicular endosomes (MVEs). Although mutation of the conserved lysines near the extracellular 'downregulation targeting sequence' DTS does not affect internalization of LIN-12, degradation in P6.p is blocked, and in all VPCs this mutant form accumulates in large pleiomorphic internal vesicles. Furthermore, the ubiquitin ligase WWP-1 and the MVE-associated factor ALX-1 are required for LIN-12 degradation after internalization. Since sur-2 mutants (referring to mutations in the the MED23 subunit of the 'Mediator' transcription activation complex) display a similar phenotype, transcriptional targets of the EGFR-Ras-MAPK pathway may be involved in directing LIN-12 to MVEs (Shaye, 2005).

Mutating the conserved lysines near the DTS caused the 'Multivulva' phenotype associated with constitutive LIN-12 activation. This phenotype is consistent with an MVE sorting defect. If a transmembrane protein does not go through the MVE sorting step, then upon delivery to the lysosome its extracellular domain will be degraded whereas its intracellular domain will remain exposed to the cytosol. For LIN-12/Notch, the mechanism of signal transduction involves cleavage and release of the intracellular domain. Thus, if MVE sorting is disrupted, degradation of the extracellular domain in the lysosome could mimic ectodomain shedding, creating a substrate for Presenilin-dependent release of the intracellular domain of LIN-12, or perhaps such disruption would result in release the intracellular domain by an alternative mechanism (Shaye, 2005).

Recent reports have described 'ligand-independent' activation of Drosophila Notch in late endosomes. In these studies, overexpression of the protein Deltex was shown to promote internalization and accumulation of Notch in late endosomes, correlated with activation of Notch signaling. It was suggested that such endosomal activation of Notch might represent a novel and relevant mode of activating this pathway. However, the finding that an apparent block in MVE sorting can lead to LIN-12 activation suggests an alternative explanation for the effect of Deltex overexpression: the enhanced internalization and endosomal accumulation of Notch may saturate the MVE sorting machinery, so that some Notch is not correctly internalized into MVE lumenal vesicles, leading to degradation of the extracellular domain without concomitant degradation of the intracellular domain (Shaye, 2005).

Evidence is provided that internalization of LIN-12 is mediated by the di-leucine motif and basal phosphorylation of flanking serine/threonine residues. By contrast, for Drosophila Notch, recent evidence suggests that ubiquitination by the dNedd4 ubiquitin ligase is required for Notch internalization. Drosophila Notch does not have a di-leucine-based motif similar to the one described for LIN-12. Conversely, LIN-12 does not have a C-terminal PPXY signal, which in Drosophila Notch promotes interaction with dNedd4. It is suggested that C. elegans and Drosophila may utilize different mechanisms for targeting LIN-12/Notch for internalization (Shaye, 2005).

Both of these mechanisms may be utilized in vertebrate Notch proteins. Sequence analysis of vertebrate Notch proteins shows an intriguing inverse correlation between the presence of a di-leucine based motif and a PPXY signal. The corresponding juxtamembrane regions of vertebrate Notch1 and Notch2 proteins have a segment that is strikingly similar to the LIN-12 DTS, including conserved flanking lysines, but these proteins do not have a conserved PPXY signal at their C-termini. By contrast, most vertebrate Notch3 proteins appear more divergent in this region, but possess a PPXY signal at their C-termini; it is curious that zebrafish Notch3 lacks the PPXY motif, but has instead a canonical di-leucine motif. These observations raise the possibility that the two modes of internalizing Notch proteins (di-leucine based versus ubiquitination via a PPXY motif) have been conserved in different vertebrate Notch proteins through evolution. Perhaps other modes exist as well, as vertebrate Notch4 does not seem to have either of these conserved motifs. Mutational analysis of these potential internalization sequences in vertebrate Notch proteins will be necessary to test their roles (Shaye, 2005).

MAPK regulation of maternal and zygotic Notch transcript stability in early development

Spatiotemporal modulation of the evolutionarily conserved, intercellular Notch signaling pathway is important in the development of many animals. Examples include the regulation of neural-epidermal fate decisions in neurogenic ectoderm of Drosophila and somitogenesis in vertebrate presomitic mesoderm. In both these and most other cases, it appears that Notch-class transmembrane receptors are ubiquitously expressed. Modulation of the pathway is achieved primarily by the localized expression of the activating ligand or by alteration of receptor specificity through a glycosyl transferase. In contrast, this report presents an instance where the abundance of the Notch-class mRNA itself is dynamically regulated. Taking advantage of the long cell cycle of the two-cell-stage embryo of the leech Helobdella robusta, it was shown that this regulation is achieved at the levels of both transcript stability and transcription. Moreover, MAPK signaling plays a significant role in regulating accumulation of the transcript by virtue of its effect on Hro-notch mRNA stability. Intracellular injection of heterologous reporter mRNAs shows that the Hro-notch 3' UTR, containing seven AU-rich elements (AREs), is key to regulating transcript stability. Thus, this study shows that regulation of the Notch pathway can occur at a previously underappreciated level, namely that of transcript stability. Given that AU-rich elements occur in the 3' UTR of Notch-class genes in Drosophila, human, and Caenorhabditis elegans, regulation of Notch signaling by modulation of mRNA levels may be operating in other animals as well (Gonsalves, 2007).

In conclusion, this study shows that transcript levels of a Notch-class gene (Hro-notch) oscillate antiphasically in the AB and CD blastomeres of the two-cell embryo in the leech H. robusta, i.e., high transcript levels in AB are associated with low levels in CD and vice versa. Moreover, the Hro-notch levels are controlled by dynamic activation of one or more of the MAPK (p38MAPK and ERK) signaling pathways. Initially, the Hro-notch level in each cell reflects primarily inherited maternal transcripts. Later, the production and turnover of zygotic transcripts becomes important. The 3' UTR of Hro-notch mRNA confers a relatively short half-life to the transcripts, apparently because of the presence of multiple AREs. This instability is counteracted by the p38MAPK pathway. Thus, the Hro-notch transcript levels are controlled by MAPK signaling at the level of transcript stability and possibly also at the level of transcription. This link between the p38MAPK and Notch pathways persists into later development, because coincident p38MAPK activation and Hro-notch mRNA accumulation has been observed in the ectodermal precursor cell DNOPQ (Gonsalves, 2007).

Another instance where p38MAPK plays a role in early development is in the axial patterning of the Drosophila oocyte, where it regulates the availability of the EGF ligand (encoded by gurken) and thus controls the activation of the ERK pathway in the follicle cells. Although the mechanistic intricacies of combinatorial effects between p38MAPK and other signaling pathways remain to be elucidated, the current observations suggest that these effects might involve the regulation of mRNA stability. It has already been demonstrated that oscillations in Notch signaling can be achieved by modulating transcript levels for the presumptive ligand (deltaC, in fish presomitic mesoderm) and for a receptor glycosylating enzyme (lunatic fringe, in chick PSM). The results reveal yet a third mechanism by which oscillatory modulation of Notch signaling may be achieved, namely regulating transcript levels for Notch receptor itself in the two-cell leech embryo. Does this mechanism of Notch regulation operate in other organisms as well? This question can only be answered empirically, but it is noted that Notch-class genes in a variety of organisms bear several pentameric AREs in their 3' UTRs. In particular, the 3' UTR of human Notch-1, like that of Hro-notch, bears nonameric AREs, which makes it a strong candidate for regulation by p38MAPK. The occurrence of AREs within the 3' UTRs of other Notch genes could be a mere coincidence, and the regulation of Notch transcript stability by p38MAPK could be a novelty restricted to leech. This would be a noteworthy result in and of itself, given the frequency with which genes and signaling pathways discovered in one organism prove to have broadly distributed homologs. A more likely alternative is that the current observations provide a relatively prominent, experimentally accessible example of a regulatory interaction that operates throughout the animal kingdom (Gonsalves, 2007).

Table of contents

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

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