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Blair, S. S. Notch signaling: Fringe really is a glycosyltransferase. Curr. Biol. 10, R608-R612
Abstract
Fringe modifies the ligand-selectivity of Notch in ways that are crucial for a number of Notch's developmental functions. Recent results have confirmed the suspicion that Fringe is a glycosyltransferase that works in the Golgi complex by modifying Notch's glycosylation state.
... Il a été montré que l'expression de Lunatic fringe est oscillatoire (Forsberg et al., 1998, McGrew et al., 1998 ; cette protéine modifie Notch, le rendant plus sensible à l'activation par Delta (Blair, 2000), qui est le ligand exprimé dans le PSM. Il a aussi été montré que l'expression oscillatoire peut continuer aprés une section du PSM et l'incubation séparée des deux morceaux (Palmeirim et al., 1997, McGrew et al., 1998, Forsberg et al., 1998 ; cela a été récemment étudié en plus de détail par Maroto et al. (2005)). ...
... Oscillatory expression has been shown in chick and mouse for Lunatic fringe (Forsberg et al., 1998, McGrew et al., 1998, which modifies Notch to make it more sensitive to activation by Delta (Blair, 2000), which is the Notch ligand expressed in the PSM ; zebrafish Lunatic fringe does not show similar oscillatory expression (Prince et al., 2001), but the Notch ligand DeltaC does (Jiang et al., 2000 ; it has also been very recently proposed that expression of the mouse ligand Dll1 oscillates as well, Maruhashi et al., 2005). ...
... (mammals) cycles in both chick and mouse PSM (Jouve et al., 2000), and Lunatic fringe (L-fng), an important regulator of the Notch pathway (Blair, 2000), involved ...
The acquisition of a spatial structure during embryo development involves the differentiation of cells, often according to positional information. The complexity of the molecular networks regulating differentiation and of the mechanisms generating positional information makes it necessary to study them by means of mathematical modeling. Vertebrate embryos also acquire a segmented structure during somitogenesis; this requires spatial and temporal variations in gene expression, which mathematical modeling can also help understand.
A molecular mechanism for the somitogenesis clock is proposed, which accounts for inter-cellular synchronisation, and is based on positive feedback, even though it is compatible with all experimental data interpreted as showing that the clock is based on negative feedback. Experiments proposed to test this model involve real-time clock reporters, as well as inducible systems to induce spatially-controlled perturbations.
Theoretical and experimental results have led to conflicting ideas as to how useful positional information can be established. In particular, it has been pointed out that some models of extracellular diffusion of morphogen exhibit inadequate traveling waves of receptor saturation. Two alternative (but not mutually exclusive) models are proposed, which are based on recent experimental results highlighting the roles of extracellular glycoproteins and morphogen oligomerization.
The readout of positional information is translated to a discrete set of gene expression patterns. Intriguingly, it has been observed in numerous contexts that genes regulating differentiation are initially co-expressed in progenitors despite their antagonism. We characterise conditions under which three classes of generic "master regulatory networks" can behave as a "multi-switch", directing differentiation in an all-or-none fashion to a specific cell-type chosen among more than two possible outcomes. bHLH dimerisation networks can readily display coexistence of many antagonistic factors when competition is low. Decision-making can be forced by a transient increase in competition, which could correspond to some unexplained experimental observations related to Id proteins.
... that was discovered in Drosophila. Its three homologues have been discovered in mammals namely lunatic fringe (Lfng), manic fringe (Mfng) and radical fringe (Rfng) [5][6][7]. Fringe has O-fucose specific ß 1-3 N-acetylglucosaminyltransferase activity that adds Nacetylglucoseamine to O-linked fucose on epidermal growth factor repeats of Notch [8]. Fringe is a Golgi resident glycosyltransferase that requires a specific DxD active site motif to function. ...
... O-fucose is present between second and third conserved cysteine residue at C2-X-X-G-G-S/T-C3 of EGF like repeat; it exists as mono-, di-or trisaccharide. Fringe increases the incorporation of GLcNAc to fragments containing these EGF like repeats [5,7,8,16]. EGF repeats 27-36 and LNR domain of Notch are found to be specific for binding fringe [7]. ...
... Fringe increases the incorporation of GLcNAc to fragments containing these EGF like repeats [5,7,8,16]. EGF repeats 27-36 and LNR domain of Notch are found to be specific for binding fringe [7]. Characterization of fringe in humans as per chromosomal localization is as follows: Mfng maps to human chromosome 22q13.1, ...
0-Fucosylpeptide-3-beta-N-acetylglucosaminyltransferase (fringe) belongs to the family of glycosyltransferases. The modulator fringe participates in Notch signaling pathway. Various studies have shown that this modulator is involved in many developmental processes. The mutation or overexpression of fringe can lead to various diseases including cancer. Lunatic fringe (ling), manic fringe (Mfng) and radical fringe (Rfng), the paralogues of fringe, have not yet been studied at any level of computational biological research. The work accomplished in this study has been a step forward towards cutting edge research in the field of computational biology. Homology modeling and molecular dynamics simulation have been employed in order to study different structural, theoretical and dynamical properties of the fringe protein family. The 3D structures of fringe protein family which have not been reported previously in any of the structural database have been predicted by utilizing MODELLER tool. Structures were analyzed and validated through different structural and physicochemical characterization tools. Molecular dynamics simulation (MD simulation) has been employed on all three paralogues-Lfng, Mfng and Rfng-to investigate the protein dynamics of the fringe family at the atomic level reaching till the total production run of 15 ns. Time dependent behavior of protein on different time scales has been noticed through root mean square deviation (RMSD) and root mean square fluctuation (RMSF). The average RMSD for Lfng, Mfng and Rfng calculated was 3.07, 2.82 and 335 A, respectively, for the production run of 5 ns each, depicting the stable structural dynamics of protein models. RMSD, RMSF, factor and radius of gyration graphs showed the similar pattern of protein dynamics throughout the production run of 5 ns each for Lfng, Mfng and Rfng. The DxD (aspartate any residue aspartate) motif, a hallmark active site of glycosyltransferases, remained well stabilized throughout the simulation run of fringe glycosyltransferase. This study is significantly important to interpret theoretical and dynamical behavior of the fringe protein family that also infers the conservation of similar dynamic pattern among same family of proteins which further demonstrates the sequence to dynamic similarity of proteins.
... The Serrate ligands have an additional cysteine rich region (CRR) between the EGF-like repeats and the transmembrane domain. Cytoplasmic domains do not show significant sequence similarities or identified motifs (Adapted from Blair, 2000). Unprocessed Notch is cleaved in the golgi by the Furin protease, at the site marked 81. ...
... The function of fringe has been recently elucidated (reviewed in Blair, 2000). In a series of papers. ...
The sensory patches of the inner ear consist of two types of cell: sensory hair cells and supporting cells. The pattern is such that supporting cells surround each hair cell and no two hair cells touch each other. The aim of this study was to uncover the genetic mechanisms that control the differentiation and patterning of these two cell types. The alternating pattern of hair cells and support cells has led to the suggestion that their differentiation is co-ordinately regulated by cell- cell interactions involving the Notch signaling pathway. The key players in this pathway are Delta, a ligand, and Notch, its receptor, mediating a process known as lateral inhibition - a mechanism which forces neighbouring cells of an initially equivalent group to become different. The findings in this study show that two Notch ligands Deltal and Serrate2 are expressed in the nascent hair cells and are thought to deliver lateral inhibition to their neighbours, which become supporting cells. Intriguingly, the supporting cells also express a Notch ligand, Serratel. To functionally test the role of the Notch signaling pathway in the developing chick inner ear, retroviral vectors were used to misexpress components of the Notch signaling pathway. It is shown that a simple lateral inhibition model based on feedback regulation of the Notch ligands is inadequate to explain the generation and patterning of the sensory hair cells. The Notch ligand Serratel is regulated by lateral induction and not lateral inhibition; commitment to become a hair cell is not simply controlled by levels of expression of the Notch ligand Deltal, Serratel, and Serrate2 in the neighbours of the nascent hair cell. At least one factor. Numb, capable of blocking reception of Notch signaling is concentrated in hair cells.
... To date, four Notch receptors have been identified (Notch 1-4) in humans, with five canonical ligands including three members of the Delta family (Dll1, Dll3, Dll4) and two members of the Serrate family (Jag1 and Jag2, homologues of Drosophila Serrate) [6]. At the same time, the family of Fringe-related proteins is a major Notch regulator, which can promote or suppresse Notch signaling, depending on the Notch ligands [7,8]. There is only a single Fringe in Drosophila, while there are three homologues in mammals: Lunatic Fringe (LFng), Manic Fringe (MFng) and Radical Fringe (RFng) [6]. ...
... All these findings prompt us to study the biological relevance of Notch signaling to the glioma cell fate decisions. We explore the effects of Dll1-Jag1-Fringe on glioma cell fate decisions by analyzing the model (5)- (8). The effect of ligand Jag1 and Fringe is shown in Fig. 3. ...
Background
The Notch family of proteins plays a vital role in determining cell fates, such as proliferation, differentiation, and apoptosis. It has been shown that Notch1 and its ligands, Dll1 and Jag1, are overexpressed in many glioma cell lines and primary human gliomas. The roles of Notch1 in some cancers have been firmly established, and recent data implicate that it plays important roles in glioma cell fate decisions. This paper focuses on devising a specific theoretical framework that incorporates Dll1, Jag1, and Fringe in Notch1 signaling pathway to explore their functional roles of these proteins in glioma cells in the tumorigenesis and progression of human gliomas, and to study how glioma cell fate decisions are modulated by both trans-activation and cis-inhibition.
Results
This paper presents a computational model for Notch1 signaling pathway in glioma cells. Based on the bifurcation analysis of the model, we show that how the glioma cell fate decisions are modulated by both trans-activation and cis-inhibition mediated by the Fringe protein, providing insight into the design and control principles of the Notch signaling system and the gliomas.
Conclusions
This paper presents a computational model for Notch1 signaling pathway in glioma cells based on intertwined dynamics with cis-inhibition and trans-activation involving the proteins Notch1, Dll1, Jag1, and Fringe. The results show that how the glioma cell fate transitions are performed by the Notch1 signaling. Transition from grade III ∼ IV with significantly high Notch1 to grade I ∼ II with high Notch1, and then to normal cells by repressing the Fringe levels or decreasing the strength of enhancement induced by Fringe.
... To determine whether expressing spdo during other Notchdependent events also compromised Notch signaling activity, we expressed spdo throughout the wing blade, and assayed for defects in three additional Notch-mediated events: wing blade growth, wing patterning and wing vein formation (reviewed by Blair, 2000;De Celis, 2003). spdo misexpression in the wing resulted in reduced wing size, notched wings and vein thickening (Fig. 2J) -phenotypes indicative of reduced Notch signaling (see Blair, 2000;De Celis, 2003). ...
... To determine whether expressing spdo during other Notchdependent events also compromised Notch signaling activity, we expressed spdo throughout the wing blade, and assayed for defects in three additional Notch-mediated events: wing blade growth, wing patterning and wing vein formation (reviewed by Blair, 2000;De Celis, 2003). spdo misexpression in the wing resulted in reduced wing size, notched wings and vein thickening (Fig. 2J) -phenotypes indicative of reduced Notch signaling (see Blair, 2000;De Celis, 2003). In agreement, when Notch function is reduced by 50% the wing and lateral inhibition phenotypes observed upon spdo 4091 RESEARCH ARTICLE Spdo: a context-dependent regulator of Notch signaling (Tang et al., 2005). ...
Asymmetric cell divisions generate sibling cells of distinct fates ('A', 'B') and constitute a fundamental mechanism that creates cell-type diversity in multicellular organisms. Antagonistic interactions between the Notch pathway and the intrinsic cell-fate determinant Numb appear to regulate asymmetric divisions in flies and vertebrates. During these divisions, productive Notch signaling requires sanpodo, which encodes a novel transmembrane protein. Here, we demonstrate that Drosophila sanpodo plays a dual role to regulate Notch signaling during asymmetric divisions - amplifying Notch signaling in the absence of Numb in the 'A' daughter cell and inhibiting Notch signaling in the presence of Numb in the 'B' daughter cell. In so doing, sanpodo ensures the asymmetry in Notch signaling levels necessary for the acquisition of distinct fates by the two daughter cells. These findings answer long-standing questions about the restricted ability of Numb and Sanpodo to inhibit and to promote, respectively, Notch signaling during asymmetric divisions.
... The development of the wing margin depends on continuous Notch signalling along the dorsoventral (D/V) compartment boundary [37]. The signalling results in the expression of Wg, which is crucial for margin formation and is initiated at the boundary of Fringe (Fng) expressing and non-expressing cells, by mutual signalling via Ser and Dl [38]. We found that the stripe of Wg expression along the D/V boundary was grossly normal in Dl attP -DlK2R-HA/Df wing discs, indicating that DlK2R-HA can provide sufficient activity for expression of Wg and margin formation ( Fig. 4F-G' , arrow). ...
Background
Ubiquitylation (ubi) of the intracellular domain of the Notch ligand Delta (Dl) by the E3 ligases Neuralized (Neur) and Mindbomb1 (Mib1) on lysines (Ks) is thought to be essential for the its signalling activity. Nevertheless, we have previously shown that DlK2R-HA, a Dl variant where all Ks in its intracellular domain (ICD) are replaced by the structurally similar arginine (R), still possess weak activity if over-expressed. This suggests that ubi is not absolutely required for Dl signalling. However, it is not known whether the residual activity of DlK2R-HA is an effect of over-expression and, if not, whether DlK2R can provide sufficient activity for the whole development of Drosophila.
Results
To clarify these issues, we generated and analysed DlattP-DlK2R-HA, a knock-in allele into the Dl locus. Our analysis of this allele reveals that the sole presence of one copy of DlattP-DlK2R-HA can provide sufficient activity for completion of development. It further indicates that while ubi is required for the full activity of Dl in Mib1-dependent processes, it is not essential for Neur-controlled neural development. We identify three modes of Dl signalling that are either dependent or independent of ubi. Importantly, all modes depend on the presence of the endocytic adapter Epsin. During activation of Dl, direct binding of Epsin appears not to be an essential requirement. In addition, our analysis further reveals that the Ks are required to tune down the cis-inhibitory interaction of Dl with Notch.
Conclusions
Our results indicate that Dl can activate the Notch pathway without ubi of its ICD. It signals via three modes. Ubi is specifically required for the Mib1-dependent processes and the adjustment of cis-inhibition. In contrast to Mib1, Neur can efficiently activate Dl without ubi. Neur probably acts as an endocytic co-adapter in addition to its role as E3 ligase. Endocytosis, regulated in a ubi-dependent or ubi-independent manner is required for signalling and also suppression of cis-inhibition. The findings clarify the role of ubi of the ligands during Notch signalling.
... In this current study, we found that the CGRKKSSC (364-371) motif could be contributing to the functional specificity of MCC1. This motif is a known site for fucose residue attachment to serine and is implicated in Notch signaling [17,18]. Notch signaling plays a role in regulating the cell cycle [19]. ...
The MCC family of genes plays a role in colorectal cancer development through various immunological pathways, including the Th17/Treg axis. We have previously shown that MCC1 but not MCC2 plays a role in Treg differentiation. Our understanding of the genetic divergence patterns and evolutionary history of the MCC family in relation to its function, in general, and the Th17/Treg axis, in particular, remains incomplete. In this investigation, we explored 12 species' genomes to study the phylogenetic origin, structure, and functional specificity of this family. In vertebrates, both MCC1 and MCC2 homologs have been discovered, while invertebrates have a single MCC homolog. We found MCC homologs as early as Cnidarians and Trichoplax, suggesting that the MCC family first appeared 741 million years ago (Ma), whereas MCC divergence into the MCC1 and MCC2 families occurred at 540 Ma. In general, we did not detect significant positive selection regulating MCC evolution. Our investigation, based on MCC1 structural similarity, suggests that they may play a role in the evolutionary changes in Tregs' emergence towards complexity, including the ability to utilize calcium for differentiation through the use of the EFH calcium-binding domain. We also found that the motif NPSTGE was highly conserved in MCC1, but not in MCC2. The NPSTGE motif binds KEAP1 with high affinity, suggesting an Nrf2-mediated function for MCC1. In the case of MCC2, we found that the "modifier of rudimentary" motif is highly conserved. This motif contributes to the regulation of alternative splicing. Overall, our study sheds light on how the evolution of the MCC family is connected to its function in regulating the Th17/Treg axis.
... This motif has been implicated in Notch signaling, where it was shown that the O-fucose moieties can act as a substrate for Fringe. Fringe is a known modifier of the Notch function [22] [23]. However, the mechanism of interaction between MCC1 and notch signaling is still unknown. ...
The MCC family of genes plays a role in colorectal cancer development through various immunological pathways, including the Th17/Treg axis. We have previously shown that MCC1 and not MCC2 play a role in Treg differentiation. Our understanding of the genetic divergence patterns and evolutionary history of the MCC family in relation to its function, in general, and the Th17/Treg axis, in particular, remains incomplete. In this investigation, we explored 12 species' genomes to study the phylogenetic origin, structure, and functional specificity of this family. In vertebrates, both MCC1 and MCC2 homologs have been discovered, while invertebrates have a single MCC homolog. We found MCC homologs as early as Cnidarians and Trichoplax, suggesting that the MCC family first appeared 741 million years ago (Ma), whereas MCC divergence into MCC1 and MCC2 families occurred at 540 Ma. In general, we did not detect significant positive selection regulating MCC evolution. Our investigation, based on MCC1 structural similarity, suggests that they may play a role in the evolutionary changes in Tregs' emergence towards complexity, including the ability to utilize calcium for differentiation through the use of the EFH calcium-binding domain. We also found that the motif NPSTGE was highly conserved in MCC1 but not in MCC2. The NPSTGE motif binds KEAP1 with high affinity, suggesting an Nrf2-mediated function for Nrf2. In the case of MCC2, we found that the "Modifier of rudimentary" motif is highly conserved. This motif contributes to the regulation of alternative splicing. Overall, our study sheds light on how the evolution of the MCC family is connected to its function in regulating the Th17/Treg axis.
... While genetic modifiers of Notch phenotypes have been identified (Greenwald and Kovall, 2013;Lehner et al., 2006), C. elegans appears to lack traditional Fringe homologs, but do harbor two divergent Fringe homologs (Yuan et al., 1997), which are not yet linked to Notch activity in C. elegans. In fact, Notch receptors and ligands in C. elegans appear to lack the majority of the required linkage motifs (Blair, 2000). ...
Breaking symmetry in populations of uniform cells, to induce adoption of an alternative cell fate, is an essential developmental mechanism. Similarly, domain and boundary establishment are crucial steps to forming organs during development. Notch signaling is a pathway ideally suited to mediating precise patterning cues, as both receptors and ligands are membrane-bound and can thus act as a precise switch to toggle cell fates on or off. Fine-tuning of signaling by positive or negative feedback mechanisms dictate whether signaling results in lateral induction or lateral inhibition, respectively, allowing Notch to either induce entire regions of cell specification, or dictate binary fate choices. Furthermore, pathway activity is modulated by Fringe modification of receptors or ligands, co-expression of receptors with ligands, mode of ligand presentation, and cell surface area in contact. In this review, we describe how Notch signaling is fine-tuned to mediate lateral induction or lateral inhibition cues, and discuss examples from C.elegans, D. melanogaster and M. musculus. Identifying the cellular machinery dictating the choice between lateral induction and lateral inhibition highlights the versatility of the Notch signaling pathway in development.
... NICD translocates to the nucleus and forms a transcriptional activation complex together with RBPJK and MAML that activates target genes transcription, including Hes and Hey that encode transcriptional repressors are type I transmembrane proteins and their extracellular domains are composed by a repetition of EGF-like domains responsible for the interaction with the receptor and the DSL (Delta, Serrate, Lin12) domain ( Fig. 12.2) [21]. The glycosyltransferase Fringe can modify the EGF-like domain in the NECD of the receptor by adding O-fucose glycans [5] (Fig. 12.2). This modification determines which ligand preferentially binds to the receptor. ...
The heart is the first organ to develop in the embryo, and its formation is an exquisitely regulated process. Inherited mutations in genes required for cardiac development may cause congenital heart disease (CHD), manifested in the newborn or in the adult. Notch is an ancient, highly conserved signaling pathway that communicates adjacent cells to regulate cell fate specification, differentiation, and tissue patterning. Mutations in Notch signaling elements result in cardiac abnormalities in mice and humans, demonstrating an essential role for Notch in heart development. Recent work has shown that endocardial Notch activity orchestrates the early events as well as the patterning and morphogenesis of the ventricular chambers in the mouse and that inactivating mutations in the NOTCH pathway regulator MIND BOMB-1 (MIB1) cause left ventricular non-compaction (LVNC), a cardiomyopathy of poorly understood etiology. Here, we review these data that shed some light on the etiology of LVNC that at least in the case of that caused by MIB1 mutations has a developmental basis.
... Our data implicate mirr as an important Cic target and appendageproducing fate determinant. One candidate regulatory target of Mirr in this process is fringe (fng), which encodes a glycosyltransferase that regulates cell fate decisions through the modulation of Notch signaling (Blair, 2000;Bruckner et al., 2000;Hicks et al., 2000;Moloney et al., 2000). Mirr regulates the expression of fng in a variety of developmental contexts. ...
... In addition to this canonical signaling pathway, non-canonical Notch signaling independent of either CBF1 or γ-secretase cleavage, or both have been identified [20][21][22]25]. Posttranslational modifications including O-fucosylation and O-glycosylation via fringe proteins (lunatic, radical, and manic) regulate the specificity of Notch receptor-ligand binding, and are also critical for its function [27]. ...
The Notch pathway represents a highly conserved signaling network with essential roles in regulation of key cellular processes and functions, many of which are critical for development. Accumulating evidence indicates that it is also essential for fibrosis and thus the pathogenesis of chronic fibroproliferative diseases in diverse organs and tissues. Different effects of Notch activation are observed depending on cellular and tissue context as well as in both physiologic and pathologic states. Close interactions of Notch signaling pathway with other signaling pathways have been identified. In this review, current knowledge on the role of the Notch signaling with special focus on fibrosis and its potential as a therapeutic target is summarized.
... A subset of modulators act on Notch signaling through receptor and ligand posttranslational modifications, particularly ubiquitination and glycosylation (Haines and Irvine, 2003;Kovall and Blacklow, 2010). Such modulators include the well described ubiquitin-ligases mindbomb and neuralized (Yeh et al., 2001;Itoh et al., 2003) and the glycosyltransferase Fringe (Blair, 2000). Although studies have demonstrated the importance of mindbomb-mediated ubiquitination during zebrafish angiogenesis (Lawson et al., 2001;Lawson and Weinstein, 2002), little is known about the roles of other posttranslational modifications, including glycosylation. ...
... Binding of ligand to its receptor induces metalloproteinase-mediated and γ-secretase-mediated cleavage of the Notch receptor (39). Thereby the Notch1 intracellular domain (N1-ICD) was generated and released from the membrane (40). Then N1-ICD is translocated into the nucleus and finally degraded by the ubiquitin-proteasome system with the aid of Fbxw7 (41)(42)(43). ...
Hepatocellular carcinoma (HCC) is one of the most common human malignancies and also the leading cause of cancer-related death in the world. The mechanisms underlying the progression and metastasis of HCC remain unclear. The E3 ubiquitin ligase F-box and WD repeat domain-containing 7 (Fbxw7) is broadly considered as a tumor suppressor gene. However, the role of Fbxw7 in HCC is not clear. To investigate the expression and biological functions of Fbxw7 in HCC, we examined Fbxw7 expression level using HCC tissue microarray and immunohistochemistry. Our data showed that Fbxw7 expression is significantly reduced in HCC compared with non-cancerous tissues (P<0.05). Fbxw7 levels were significantly associated with tumor differentiation (P=0.013), the incidence of portal or hepatic venous invasion (P=0.031), metastasis (P=0.027) and AJCC cancer stage (P=0.047). Then, we observed a strong correlation between low Fbxw7 expression and a worse 5-year survival of HCC patients (P<0.001). Furthermore, multivariate Cox regression analyses demonstrated that the Fbxw7 expression (P<0.001) was an independent factor for the prediction of the overall survival of HCC patients. We also found that both Fbxw7 mRNA and protein levels were significantly reduced in HCC cell lines compared with human liver non-tumor cell line. Moreover, our in vitro experiments showed a remarkable increase of cell migration and invasion in Fbxw7-knockdown cells and a decrease in Fbxw7-overexpress cells. In addition, the present study demonstrated that Fbxw7 is involved in the migration and invasion of HCC cells via regulating Notch1 and the downstream molecules of Notch1. Taken together, our findings indicate that Fbxw7 can be used as a prognostic marker; it has an important role in HCC progression and inhibits HCC cell migration and invasion through the Notch1 signaling pathway.
... Fringe is a glycosyl transferase that glycosylates EGF repeats of Notch resulting in altered ligand binding affinity and increased signalling from Delta/reduced signalling via Serrate/Jagged (Blair, 2000) . Numb, a phosphotyrosine binding domain adaptor protein that binds Notch directly recruits Itch, an E3 ubiquitin ligase, which ubiquitinates Notch1 at the cell surface, increasing proteasomal degradation and reducing its translocation to the nucleus (McGill et al., 2003) . ...
Integrins are heterodimeric transmembrane proteins that provide a bi-directional link
between the cell’s internal biological mechanisms and the extracellular environment.
During inside-out signalling, intracellular messages converge on the integrin
cytoplasmic domain to induce a conformational change. This is transmitted to the
extracellular domain where it results in an alteration in affinity for integrin ligands
such as fibronectin and laminin. In this way the cell has developed the ability to
modulate the critical functions of adhesion and cell movement. In outside-in
signalling, the integrin performs a more complex function than simple adhesion;
upon binding to ligand, the integrin extracellular domain undergoes a conformational
change which is transmitted to the cytoplasmic domain. This alters the integrin’s
cytoplasmic domain affinity for intracellular signalling proteins and results in the
activation of intracellular second messenger pathways. In this way, the extracellular
milieu is able to influence intracellular signalling including those involved in
apoptosis.
This thesis demonstrates data which provide original insights into bi-directional
integrin signalling:
Inside-out signalling: Constitutively active Notch1 increases β3-integrin affinity and
abrogates Hras-mediated integrin suppression without increasing expression of β3-
integrin. Dominant-Negative Rras blocks Notch-mediated integrin activation and
Notch1-mediated reversal of Hras and Raf-mediated integrin suppression and this is
independent of erk phosphorylation. Notch1 induces Rras activation. Functional
adhesion assays confirm that Notch1IC increases K562 adhesion in a β1-integrin
dependent manner and this is abrogated by Dominant-Negative Rras. This data
supports a mechanism in which Notch1 increases integrin affinity via activation of
Rras.
Outside-in signalling: Evidence is presented demonstrating that extracellular matrix
proteins, laminin and fibronectin, activate β1-integrins to protect SCLC cells against
the apoptotic effects of etoposide and ionizing radiation via PI3Kinase activation.
This occurs in two ways: 1) PI3Kinase-dependent β1-integrin signalling resulting in
phosphorylation of Bad and reduced caspase-9 cleavage and 2) a β1-integrinmediated
over-riding of etoposide and radiotherapy-induced cell cycle S phase delay
and G2/M arrest. β1-integrin-mediated outside-in survival signalling was
investigated further in the in vivo setting; MatrigelTM, a basement membrane product
rich in extracellular matrix proteins, promoted SCLC xenograft survival and growth
in a β1-integrin and tyrosine kinase-dependent manner.
This data provides novel insights into the critical functions that integrins play in
adhesion and survival signalling.
... A subset of modulators act on Notch signaling through receptor and ligand post-translational modifications, particularly ubiquitination and glycosylation (Haines and Irvine, 2003;Kovall and Blacklow, 2010). Such modulators include the well described ubiquitin-ligases mindbomb and neuralized (Yeh et al., 2001; and the glycosyltransferase Fringe (Blair, 2000). Although studies have demonstrated the importance of mindbomb-mediated ubiquitination during zebrafish angiogenesis (Lawson et al., 2001;Lawson and Weinstein, 2002), little is known about the roles of other post-translational modifications, including glycosylation. ...
Background:
The interplay between Notch and Vegf signaling regulates angiogenesis in the embryo. Notch signaling limits the responsiveness of endothelial cells to Vegf to control sprouting. Despite the importance of this regulatory relationship, much remains to be understood about extrinsic factors that modulate the pathway.
Results:
During a forward genetic screen for novel regulators of lymphangiogenesis, we isolated a mutant with reduced lymphatic vessel development. This mutant also exhibited hyperbranching arteries, reminiscent of Notch pathway mutants. Positional cloning identified a missense mutation in the carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (cad) gene. Cad is essential for UDP biosynthesis, which is necessary for protein glycosylation and de novo biosynthesis of pyrimidine-based nucleotides. Using a transgenic reporter of Notch activity, we demonstrate that Notch signaling is significantly reduced in cad(hu10125) mutants. In this context, genetic epistasis showed that increased endothelial cell responsiveness to Vegfc/Vegfr3 signaling drives excessive artery branching.
Conclusions:
These findings suggest important posttranslational modifications requiring Cad as an unappreciated mechanism that regulates Notch/Vegf signaling during angiogenesis.
... Thus, Fng potentiates activation of Notch by Dl and prevents activation of Notch by Ser. In the Drosophila wing and eye discs, and in vertebrate limbs the expression of fringe is restricted to dorsal cells, where it prevents Ser from signaling in the dorsal compartment (Irvine and Vogt 1997;Blair 2000). The absence of Fringe in ventral cells diminishes the signaling activity of Dl in this compartment. ...
... In contrast Dl, signals preferentially to DBCs, where Notch is modified. This polarization of signalling results in the symmetrical activation of Notch in DBCs and VBCs [24], [25]. Hence, it is the boundary of Fng expressing and non-expressing cells (Fringe-boundary, FB) that regulates the activation of Notch signalling at the D/V boundary. ...
Wing development in Drosophila requires the activation of Wingless (Wg) in a small stripe along the boundary of Fringe (Fng) expressing and non-expressing cells (FB), which coincides with the dorso-ventral (D/V) boundary of the wing imaginal disc. The expression of Wg is induced by interactions between dorsal and ventral cells mediated by the Notch signalling pathway. It appears that mutual signalling from dorsal to ventral and ventral to dorsal cells by the Notch ligands Serrate (Ser) and Delta (Dl) respectively establishes a symmetric domain of Wg that straddles the D/V boundary. The directional signalling of these ligands requires the modification of Notch in dorsal cells by the glycosyltransferase Fng and is based on the restricted expression of the ligands with Ser expression to the dorsal and that of Dl to the ventral side of the wing anlage. In order to further investigate the mechanism of Notch signalling at the FB, we analysed the function of Fng, Ser and Dl during wing development at an ectopic FB and at the D/V boundary. We find that Notch signalling is initiated in an asymmetric fashion on only one side of the FB. During this initial asymmetric phase, only one ligand is required, with Ser initiating Notch-signalling at the D/V and Dl at the ectopic FB. Furthermore, our analysis suggests that Fng has also a positive effect on Ser signalling. Because of these additional properties, differential expression of the ligands, which has been a prerequisite to restrict Notch activation to the FB in the current model, is not required to restrict Notch signalling to the FB.
... We used wing discs to ask if like Delta, the ligand Serrate requires Epsin's UIMs. Notch is activated in a stripe of cells at the dorsal/ventral (D/V) boundary of the wing disc by Delta from the ventral side and Serrate from the dorsal side ( Fig. 4A) (reviewed by Irvine and Vogt, 1997;Irvine, 1999 andBlair, 2000). In response to Notch activation, the transcription factor Cut is expressed at the D/V boundary. ...
Epsin is an endocytic protein that binds Clathrin, the plasma membrane, Ubiquitin, and also a variety of other endocytic proteins through well-characterized motifs. Although Epsin is a general endocytic factor, genetic analysis in Drosophila and mice revealed that Epsin is essential specifically for internalization of ubiquitinated transmembrane ligands of the Notch receptor, a process required for Notch activation. Epsin's mechanism of function is complex and context-dependent. Consequently, how Epsin promotes ligand endocytosis and thus Notch signaling is unclear, as is why Notch signaling is uniquely dependent on Epsin. Here, by generating Drosophila lines containing transgenes that express a variety of different Epsin deletion and substitution variants, we tested each of the five protein or lipid interaction modules for a role in Notch activation by each of the two ligands, Serrate and Delta. There are five main results of this work that impact present thinking about the role of Epsin in ligand cells. First, we discovered that deletion or mutation of both UIMs destroyed Epsin's function in Notch signaling and had a greater negative impact on Epsin activity than removal of any other module type. Second, only one of Epsin's two UIMs was essential. Third, the lipid-binding function of the ENTH domain was required only for maximal Epsin activity. Fourth, although the C-terminal Epsin modules that interact with Clathrin, the adapter protein complex AP-2, or endocytic accessory proteins were necessary collectively for Epsin activity, their functions were highly redundant; most unexpected was the finding that Epsin's Clathrin binding motifs were dispensable. Finally, we found that signaling from either ligand, Serrate or Delta, required the same Epsin modules. All of these observations are consistent with a model where Epsin's essential function in ligand cells is to link ubiquitinated Notch ligands to Clathrin-coated vesicles through other Clathrin adapter proteins. We propose that Epsin's specificity for Notch signaling simply reflects its unique ability to interact with the plasma membrane, Ubiquitin, and proteins that bind Clathrin.
... In fact, the establishment of developmental boundaries and the segmentation of limbs and appendages is dependent on Notch signaling and coordination of these processes can be regulated by the spatio-temporal distribution of ligand expression (Bishop et al., 1999;de Celis et al., 1998;Klein and Arias, 1998;Panin et al., 1997;Rauskolb and Irvine, 1999). For instance, in the developing wing disc of flies, Serrate is expressed dorsally while higher Delta expression occurs ventrally and Notch signaling directs this ligand expression pattern (Blair, 2000;Doherty et al., 1996). The coexpression of Fringe in the dorsal compartment ensures that Serrate can only signal to adjacent ventral cells that lack Fringe, while ventral Delta signals preferentially to adjacent dorsal cells. ...
Notch signaling induced by canonical Notch ligands is critical for normal embryonic development and tissue homeostasis through the regulation of a variety of cell fate decisions and cellular processes. Activation of Notch signaling is normally tightly controlled by direct interactions with ligand-expressing cells, and dysregulated Notch signaling is associated with developmental abnormalities and cancer. While canonical Notch ligands are responsible for the majority of Notch signaling, a diverse group of structurally unrelated noncanonical ligands has also been identified that activate Notch and likely contribute to the pleiotropic effects of Notch signaling. Soluble forms of both canonical and noncanonical ligands have been isolated, some of which block Notch signaling and could serve as natural inhibitors of this pathway. Ligand activity can also be indirectly regulated by other signaling pathways at the level of ligand expression, serving to spatiotemporally compartmentalize Notch signaling activity and integrate Notch signaling into a molecular network that orchestrates developmental events. Here, we review the molecular mechanisms underlying the dual role of Notch ligands as activators and inhibitors of Notch signaling. Additionally, evidence that Notch ligands function independent of Notch is presented. We also discuss how ligand posttranslational modification, endocytosis, proteolysis, and spatiotemporal expression regulate their signaling activity.
... The current hypothesis is that fringe glycosylates the EGF repeats of the Notch receptors, controlling the specificity of receptorligand binding, and contributing to lateral inhibition. However it was also found that, O- (Blair, 2000) on specific fucose-modified EGF repeats. Fringe is expressed only in a subset of cells; Notch is hyperactivated when Fringe-expressing and -nonexpressing territories abut, forming a boundary (Irvine and Rauskolb, 2001;Wu and Rao, 1999). ...
Freiburg (Breisgau), Univ., Diss., 2005 (Nur beschränkt für den Austausch).
... The glycosyltransferase Fringe is a modifier of the Delta-Notch interaction in imaginal discs (Bruckner et al., 2000;Moloney et al., 2000;Munro and Freeman, 2000;Blair, 2000), and in oogenesis of earlier Notch-dependent processes that allow pinching off to occur in the germarium and define the number of polar cells (Jordan et al., 2000;Zhao et al., 2000;C. A., W.-M. D., K. Jordan and H. R.-B., unpublished). ...
In many developmental processes, polyploid cells are generated by a variation of the normal cell cycle called the endocycle in which cells increase their genomic content without dividing. How the transition from the normal mitotic cycle to endocycle is regulated is poorly understood. We show that the transition from mitotic cycle to endocycle in the Drosophila follicle cell epithelium is regulated by the Notch pathway. Loss of Notch function in follicle cells or its ligand Delta function in the underlying germline disrupts the normal transition of the follicle cells from mitotic cycle to endocycle, mitotic cycling continues, leading to overproliferation of these cells. The regulation is at the transcriptional level, as Su(H), a downstream transcription factor in the pathway, is also required cell autonomously in follicle cells for proper transitioning to the endocycle. One target of Notch and Su(H) is likely to be the G2/M cell cycle regulator String, a phosphatase that activates Cdc2 by dephosphorylation. String is normally repressed in the follicle cells just before the endocycle transition, but is expressed when Notch is inactivated. Analysis of the activity of String enhancer elements in follicle cells reveals the presence of an element that promotes expression of String until just before the onset of polyploidy in wild-type follicle cells but well beyond this stage in Notch mutant follicle cells. This suggests that it may be the target of the endocycle promoting activity of the Notch pathway. A second element that is insensitive to Notch regulation promotes String expression earlier in follicle cell development, which explains why Notch, while active at both stages, represses String only at the mitotic cycle-endocycle transition.
... apterous Serrate, fringe capricious, tartan High Notch activity the ligand Delta. Apterous also drives the dorsal expression of fringe, which encodes a glycosyltransferase that specifically modifies Notch, blocking its response to Serrate, but increasing its response to Delta (reviewed in789). The currently favored hypothesis is therefore that dorsal Serrate signals preferentially to adjacent ventral cells, while ventral Delta signals preferentially to adjacent dorsal cells. ...
Until recently, little was known about the mechanisms that prevent cell migration across compartment boundaries in Drosophila. A new report suggests that the lineage restriction between the dorsal and ventral compartments of the developing wing relies in part on the transmembrane proteins, Capricious and Tartan.
... These examples offer only a few hints of the future directions. Recent commentaries on many of these issues in "high-impact" journals emphasize the importance of glycobiology (Blair, 2000;Alper, 2001a,b;Axford, 2001;Leonard et al., 2001;Lowe, 2001). ...
Defects in nine genes of the N-linked glycosylation pathway cause congenital disorders of glycosylation (CDGs) and serious medical consequences. Although glycobiology is seldom featured in a general medical education, an increasing number of physicians are becoming acquainted with the field because it directly impacts patient diagnosis and care. Medical practice and attitudes will change in the postgenomic era, and glycobiology has an opportunity to be a cornerstone of part of that new perspective. This review of recent developments in the CDG field describes the biochemical and molecular basis of these disorders, describes successful experimental approaches, and points out a few perspectives on current problems. The broad, multisystemic presentations of these patients emphasize that glycobiology is very much a general medical science, cutting across many traditional medical specialties. The glycobiology community is well poised to provide novel perspectives for the dedicated clinicians treating both well-known and emerging human diseases.
... Upon heat shock, this fusion protein, which is membrane spatial and temporal expression pattern of its ligands, Delta and the transmembrane protein Serrate (Ser), and bound, becomes ubiquitously expressed in the embryo. In cells in which the Notch receptor is activated by ligand by the activity of the glycosyltransferase Fringe (Fng)[39][40][41][42]. Fng controls ligand selectivity of Notch and binding, the intracellular Gal4-VP16 transcription factor moiety is cleaved off and is able to subsequently activate plays a major role in the Notch-dependent positioning of sharp compartment boundaries. ...
Gut formation is a key event during animal development. Recent genetic analysis in chick, mice, and Drosophila has identified Hedgehog and TGFbeta signals as essential players for the development of the primitive gut tube along its anterior-posterior (AP) axis. However, the genetic programs that control gut patterning along its dorsoventral (DV) axis have remained largely elusive.
We demonstrate that the activation of the Notch receptor occurs in a single row of boundary cells which separates dorsal from ventral cells in the Drosophila hindgut. rhomboid, which encodes a transmembrane protein, and knirps/knirps-related, which encode nuclear steroid receptors, are Notch target genes required for the expression of crumbs, which encodes a transmembrane protein involved in organizing apical-basal polarity. Notch receptor activation depends on the expression of its ligand Delta in ventral cells, and localizing the Notch receptor to the apical domain of the boundary cells may be required for proper signaling. The analysis of gene expression mediated by a Notch response element suggests that boundary cell-specific expression can be obtained by cooperation of Suppressor of Hairless and the transcription factor Grainyhead or a related factor.
Our results demonstrate that Notch signaling plays a pivotal role in determining cell fates along the DV axis of the Drosophila hindgut. The finding that Notch signaling results in the expression of an apical polarity organizer which may be required, in turn, for apical Notch receptor localization suggests a simple mechanism by which the specification of a single cell row might be controlled.
... The EGF repeats in Delta-like ligands are continuous whereas in the Serrate-like ligands they are interspersed with short linker sequences. Notch can be modified in the Golgi by a glycosyltransferase called Fringe (Blair, 2000) on specific fucose-modified EGF repeats. Fringe is expressed only in a subset of cells; Notch is hyperactivated when Fringe-expressing and -nonexpressing territories abut, forming a boundary (Irvine and Rauskolb, 2001; Wu and Rao, 1999). ...
The fruit fly Drosophila melanogaster is a holometabolous insect that lives in all warm countries. Drosophila serves as an excellent model organism for genetic studies as it has a short life cycle, produces a large number of offspring, has polytene chromosomes and can be maintained at a low cost. As such, Drosophila has become one of the most important model organisms in the fields of classical and molecular genetics as well as developmental biology. Drosophila have also been harnessed in the field of glycobiology to clarify the novel functions of glycans. Indeed, genome-wide screening and functional analyses of glycans have been performed in the context of the whole fly body, as well as the wings, eyes, neuromuscular junctions, and immune organs. Detailed studies using Drosophila mutants of glycosyltransferases, nucleotide sugar transporters, and glycosidases have revealed novel functions of N-linked glycans, glycosaminoglycans, glycolipids, and O-linked glycans including mucin type O-glycan, O-Fuc, O-Man, and O-GlcNAc. Because many of these functions are common between Drosophila and humans, these mutants serve as excellent models for human disease. In this review, We provide a brief introduction to Drosophila melanogaster and then provide an overview of Drosophila glycobiology.
Introduction To date, the roles of individual Notch ligands in liver injury are not well defined. The current study investigated whether and how Notch ligands DLL4 and JAG1 affect liver injury. Methods We examined the expression of Notch ligands and receptors by immunohistochemistry (IHC) in the liver samples of 26 patients with HBV-induced liver cirrhosis. The function of recombinant Dll4 (rDll4) and rJag-1 was investigated in vivo in carbon tetrachloride (CCl4) and bile duct ligation (BDL) animal models and in vitro in hepatocytes, Kupffer cells (KCs), and hepatic stellate cells (HSCs). Results DLL4 and JAG1 were the only Notch ligands expressed in liver sinusoids of examined patients. In the CCl4 animal model, rDll4 and rJag-1 ameliorated liver fibrosis, decreased infiltration of inflammatory cells, and inhibited apoptosis. On the contrary, rDll4 and rJag-1 caused rapid death of all BDL mice within 1 week. rDll4 inhibited the expression of chemokine ligand 2 (CCL2) and infiltration of inflammatory cells in livers of BDL mice, whereas rJag-1 did not have any impact on inflammatory cells infiltration and CCL2 expression. In macrophages and HSCs, rDll4 inhibited LPS-induced CCL2 expression, whereas rJag-1 did not impact CCL2 expression. Inhibition of inflammation by rDll4 caused unrestricted bile infarct and rapid death of BDL animals. Recombinant Ccl2 (rCcl2) restored the infiltration of inflammatory cells, decreased the size of bile infarcts and rescued rDll4-treated BDL animals from death. In ACLF patients, DLL4 expression was negatively associated with expression of CCL2. In contrast to rDll4-treated BDL animals, rCcl2 did not rescue rJag-1-treated BDL animals. Conclusion Etiology determines the effects of DLL4 and JAG1 on liver injury. DLL4 inhibits liver inflammation through inhibiting CCL2, a key chemokine for recruitment of inflammatory cells. How Jag-1 impact liver injury requires further investigation.
Type II lissencephaly, also known as cobblestone lissencephaly, is genetically, embryologically and pathologically distinct from type I lissencephaly. The muscular dystrophy for each of the disorders usually presents in the first year of life but the relative degree of weakness can vary. The examination of the external surfaces of the brain demonstrates numerous abnormalities in type II lissencephaly. Given that the neuropathology has not been reported for patients with mutations, this chapter focuses primarily on the syndromes without direct reference to the genes, except when noted. Unlike type I lissencephaly, the gray-white junction tends to be preserved, although it can have an irregular and nodular appearance, particularly in the most severely affected regions. The chapter also focuses on the pathogenesis on abnormal glycosylation. Finally, it is valuable to note that junctions between normal, or near normal, cortex and malformed cortex may be abrupt and well defined, again in contrast to type I lissencephaly.
Fringe provides a clear example of the role that carbohydrate modifications can play in regulating signal transduction events. Fringe was originally identified for its role in dorsal/ventral boundary formation during Drosophila wing development (Irvine and Wieschaus 1994). It functions by altering the response of the Notch receptor to its ligands, potentiating signaling from Delta and inhibiting that from Serrate (Fleming et al. 1997; Panin et al. 1997). Recent results have shown that fringe modulates Notch activity by altering the structure of the O-fucose glycans on the EGF repeats in the extracellular domain of Notch (Bruckner et al. 2000; Moloney et al. 2000a). O-fucose modifications occur between the second and third conserved cysteines of an EGF repeat at the consensus site C2-X-X-G-G-S/T-C3, where X can be any amino acid and S/T is the modification site (Harris and Spellman 1993). Numerous cell surface and secreted proteins have EGF repeats containing these sites. Fringe catalyzes the addition of a β-linked GlcNAc to the 3′-hydroxyl of O-fucose, which can be further elongated to a tetrasaccharide with the structure NeuAcα2-3/ 6Galβ1-4GlcNAcβ1-3Fuc (Harris and Spellman 1993; Moloney et al. 2000a; Moloney et al. 2000b). The glycosyltransferase activity of fringe is essential for its ability to modulate Notch signaling (Bruckner et al. 2000; Moloney et al. 2000a; Munro and Freeman 2000), demonstrating that signal transduction events can be regulated by alterations in the glycosylation state of receptors.
Notch signaling is a very conservative system of cell-cell communications playing an essential role in vascular development and human vascular diseases. One of such diseases is a hereditary vascular degenerative disorder known as cerebral autosomal dominant arteriopathy with subcortical infarct and leukoencephalopathy (CADASIL). The disorder is caused by mutations in the NOTCH 3 gene encoding a transmembrane receptor of the same name present in vessels only on vascular smooth muscle cells and pericytes. The disease involves mainly small arteries and capillaries in which degeneration and loss of cells expressing Notch 3 receptor is observed. In the affected vessels accumulation of Notch 3 extracellular domain (N3-ECD) and granular osmiophilic material (GOM) containing N3-ECD are also found. Although pathogenesis of CADASIL is still unknown there are two main distinct hypotheses concerning its development. The first of them assumes that the disease is caused by dysfunction of the mutated Notch 3 receptor which acquires a new properties. According to the second hypothesis, CADASIL - as many other neurodegenerative diseases - is a proteinopathy due to accumulation of proteinaceous aggregates in vessel wall. This paper is an overview of recent findings concerning the role of Notch 3 in vascular biology and hypothetical participation of that signaling system in CADASIL pathogenesis.
In a session on the diversity of form, new model systems were introduced that will allow a comparative approach for studying the evolution of morphological diversity. Richard Behringer (University of Texas M.D. Anderson Cancer Center, Houston, TX, USA) is using a small fruit-eating bat (Carollia perspicillata) caught in abandoned cisterns in Trinidad to study limb evolution. Work in arthropods has shown that morphological differences are often the result of changes in gene regulation rather than in changes in protein function. With this in mind, Behringer, with Chris Cretekos and John Rasweiler, has cloned the bat homolog of prx1, a gene shown in mice to be required for normal limb growth and whose regulation in the mouse is well characterized. Behringer plans to determine whether differences in the regulation of prx1 and other genes that function in limb growth and patterning are responsible for the considerable morphological differences between mouse and bat limbs. He proposes to do this by replacing the mouse regulatory sequences with those of the bat to drive expression of the endogenous mouse gene in a bat-specific pattern in knockin mice, and to look for corresponding morphological changes in the resulting limbs.The ideal tool for studying the genetic basis for morphological diversity is a species with morphologically diverse, crossfertile populations. Katie Peichel (Stanford University, CA, USA) spoke about her work in David Kingsley's lab on the three-spine stickleback, a wild fish species that has evolved a dramatic variety of morphologies since populations became geographically isolated in freshwater lakes at the end of the last ice age (10 000 years ago). Peichel and Kingsley have collaborated with Dolph Schluter's lab (University of British Columbia, Canada) to make F2 families from hybrids between fish with different morphologies. Peichel and Kingsley have begun mapping the genes responsible for these morphological differences with the ultimate goal of determining the molecular changes that underlie morphological change. Surprisingly, some traits, such as the number of bony plates on the fish's side and the presence of the pelvic spine, appear to be determined by single major chromosome regions that segregate in a mendelian manner. Peichel also described a trait that evolved independently in two separate populations, and showed that in both populations that trait mapped to the same genomic region. Together, her results show that dramatic adaptive changes in morphology can result from small numbers of genetic changes, and that the same genetic mechanisms could drive morphological change in independent populations. Further work with larger F2 families will allow these loci to be cloned and their function in controlling the development of new morphologies to be studied at the molecular level.Other sessions, not reviewed here, focused on the developmental basis of inherited human diseases, the development of important infectious disease vectors (with the goal of intelligent drug and pesticide design), and on the development of complex systems such as the wiring of the mammalian olfactory system and the acquisition of sensitivity to cocaine in Drosophila. The breadth of this meeting of the SDB – the largest ever – attests to the energy of the developmental biology community and the sophistication of its tools. At sixty, the society is just now entering the most productive years of its life.
Many proteins produced by multicellular organisms contain covalently linked sugar chains and are called glycoproteins. Because of the difficulties associated with the structural study of the sugar chains of glycoproteins, however, the functional aspects of the sugar moieties of glycoproteins were ignored during the long history of protein research.
The human brain is an intricate organ with over a hundred billion cells. Brain activity is precisely coordinated to generate electrical and hormonal signals that control basic body functions, movement, awareness, and behaviors. Intriguingly, the incredible complexity of the brain is formed from a simple cellular sheet called the neural plate. A progressive series of precisely controlled morphogenetic movements, in combination with cell proliferation, differentiation, and specification of regional character, generates functionally distinct brain structures. In this article, I summarize some of the crucial events happening at the cellular and molecular levels during neural development to help understand the complex processes implicated in vertebrate brain organization.
Canonical and noncanonical activities of the Notch receptor combine to organize the morphology of tissues and organs in Drosophila melanogaster.
Since Alfonso Corti’s first microscopic description of the cochlea in the midnineteenth century, biologists have had a deep appreciation for the beautifully ordered arrays of cells within the vertebrate organ of Corti. Sensory hair cells and nonsensory supporting cells are arranged in a regular, alternating pattern that has been compared to the ordered arrays of mosaic tiles (Posakony 1999). Recently, this “hair cell mosaic” has held interest for developmental biologists as well: such highly ordered cellular patterns are often regulated by similar molecular mechanisms and can be manipulated to reveal the specific pathways involved. Based on studies in other systems, it was suggested that a developmental mechanism known as “lateral inhibition” might be involved in the specification of sensory versus nonsensory cell types in the inner ear (Corwin et al. 1991; Lewis 1991). This mechanism, in which one progenitor cell produces a signal that inhibits differentiation in its immediate neighbor, plays an important role in the development of a variety of invertebrate cell types (Fig. 5.1). However, lateral inhibition had not been directly shown to be involved in the development of any vertebrate system. In 1995, Kelley et al. provided the first experimental evidence that supported a role for lateral inhibition in the formation of the hair cell mosaic. Specifically, this study set out to demonstrate that developing hair cells produce an inhibitory signal that prevents neighboring progenitor cells from adopting the hair cell fate. If this hypothesis is true, a decrease in this inhibitory signal should allow progenitor cells that would not normally develop as hair cells to adopt the sensory fate. To test this hypothesis, individual developing hair cells were identified within explant cultures of the organ of Corti and ablated via laser irradiation. The results demonstrated that progenitor cells immediately adjacent to ablated hair cells could change position and phenotype in order to adopt the hair cell fate. This evidence strongly supported a role for lateral inhibition in the generation of the hair cell mosaic but did not examine the specific molecular pathways that might be responsible for mediating the inhibitory signal.
Notch is a single-pass transmembrane receptor that mediates the local cell-cell interactions necessary for many cell-fate decisions. The extra cellular domain of Notch contains a tandem array of epidermal growth factor-like (EGF-like) repeats. Some of these EGF-like repeats are O-fucosylated by protein O-fucosyltransferase 1 (O-fut1), which is essential for Notch signaling in Drosophila and mouse. This O-fucose is further modified by Fringe, a GlcNAc transferase and other glycosyltransferases (O-fut1 in Drosophila and Pofut1 in mouse), to form an O-linked tetrasaccharide, which modulates Notch's selective binding to its ligands.
Vertebrate segmentation is regulated by the "segmentation clock", which drives cyclic expression of several genes in the caudal presomitic mesoderm (PSM). One such gene is Lunatic fringe (Lfng), which encodes a modifier of Notch signalling, and which is also expressed in a stripe at the cranial end of the PSM, adjacent to the newly forming somite border. We have investigated the functional requirements for these modes of Lfng expression during somitogenesis by generating mice in which Lfng is expressed in the cranial stripe but strongly reduced in the caudal PSM, and find that requirements for Lfng activity alter during axial growth. Formation of cervical, thoracic and lumbar somites/vertebrae, but not sacral and adjacent tail somites/vertebrae, depends on caudal, cyclic Lfng expression. Indeed, the sacral region segments normally in the complete absence of Lfng and shows a reduced requirement for another oscillating gene, Hes7, indicating that the architecture of the clock alters as segmentation progresses. We present evidence that Lfng controls dorsal-ventral axis specification in the tail, and also suggest that Lfng controls the expression or activity of a long-range signal that regulates axial extension.
Not available Cellular and Molecular Biology, Institute for
The reiterated structures of the vertebrate axial skeleton, spinal nervous system and body muscle are based on the metameric structure of somites, which are formed in a dynamic morphogenetic process. Somite segmentation requires the activity of a biochemical oscillator known as the somite-segmentation clock. Although the molecular identity of the clock remains unknown, genetic and experimental evidence has accumulated that indicates how the periodicity of somite formation is generated, how the positions of segment borders are determined, and how the rostrocaudal polarity within somite primordia is generated.
O-linked fucose modification is rare and has been shown to occur almost exclusively within epidermal growth factor (EGF)-like modules. We have found that the EGF-CFC family member human Cripto-1 (CR) is modified with fucose and through a combination of peptide mapping, mass spectrometry, and sequence analysis localized the site of attachment to Thr-88. The identification of a fucose modification on human CR within its EGF-like domain and the presence of a consensus fucosylation site within all EGF-CFC family members suggest that this is a biologically important modification in CR, which functionally distinguishes it from the EGF ligands that bind the type 1 erbB growth factor receptors. A single CR point mutation, Thr-88 --> Ala, results in a form of the protein that is not fucosylated and has substantially weaker activity in cell-based CR/Nodal signaling assays, indicating that fucosylation is functionally important for CR to facilitate Nodal signaling.
Activation of the Notch (N) receptor involves an intracellular proteolytic step triggered by shedding of the extracellular N domain (N-EC) upon ligand interaction. The ligand Dl has been proposed to effect this N-EC shedding by transendocytosing the latter into the signal-emitting cell. We find that Dl endocytosis and N signaling are greatly stimulated by expression of neuralized (neur). neur inactivation suppresses Dl endocytosis, while its overexpression enhances Dl endocytosis and Notch-dependent signaling. We show that neur encodes an intracellular peripheral membrane protein. Its C-terminal RING domain is necessary for Dl accumulation in endosomes, but may be dispensable for Dl signaling. The potent modulatory effect of Neur on Dl activity makes Neur a candidate for establishing signaling asymmetries within cellular equivalence groups.
Notch signaling allows cells in contact to adopt different fates. Regulation of the Notch pathway allows for the same signaling mechanism to be used in a wide variety of contexts during development. Intracellular activities of the E3 ubiquitin ligases Sel-10 and Neuralized involve proteasome-dependent degradation in the regulation of Notch pathway activity. Extracellular manipulations of Notch by Fringe and Scabrous regulate the pathway by changing Notch interactions outside the cell. These regulatory mechanisms, along with many others, affect how Notch signaling activity influences cell fate determination.
The regulatory mechanisms governing the parallel alignment of hairs, bristles, and ommatidia in Drosophila have all served as model systems for studying planar signaling and tissue level morphogenesis. Polarity in all three systems is mediated by the serpentine receptor Frizzled and a number of additional gene products. The localized accumulation of these proteins within cells plays a key role in the development of planar polarity. A comparison of the function of these gene products in the different cell types suggests cell-specific modifications of the pathway.
The hematopoietic system is maintained by a rare population of hematopoietic stem cells (HSC) that are thought to undergo self-renewal as well as continuously produce progeny that differentiate into the various hematopoietic lineages. However, the mechanisms regulating cell fate choices by HSC and their progeny have not been understood. Results of most studies support a stochastic model of cell fate determination in which growth factors support only the survival or proliferation of the progeny specified along a particular lineage. In other developmental systems, however, Notch signaling has been shown to play a central role in regulating fate decisions of numerous types of precursors, often inhibiting a particular (default) pathway while permitting self-renewal or differentiation along an alternative pathway. There is also accumulating evidence that the Notch pathway affects survival, proliferation, and cell fate choices at various stages of hematopoietic cell development, including the decisions of HSC to self-renew or differentiate and of common lymphoid precursors to undergo T- or B-cell differentiation. These data suggest that the Notch pathway plays a fundamental role in the development and maintenance of the hematopoietic system.
Notch is a large cell-surface receptor known to be an essential player in a wide variety of developmental cascades. Here we show that Notch1 endogenously expressed in Chinese hamster ovary cells is modified with O-linked fucose andO-linked glucose saccharides, two unusual forms ofO-linked glycosylation found on epidermal growth factor-like (EGF) modules. Interestingly, both modifications occur as monosaccharide and oligosaccharide species. Through exoglycosidase digestions we determined that the O-linked fucose oligosaccharide is a tetrasaccharide with a structure identical to that found on human clotting factor IX: Sia-α2,3-Gal-β1,4-GlcNAc-β1,3-Fuc-α1-O-Ser/Thr. The elongated form of O-linked glucose appears to be a trisaccharide. Notch1 is the first membrane-associated protein identified with either O-linked fucose orO-linked glucose modifications. It also represents the second protein discovered with an elongated form ofO-linked fucose. The sites of glycosylation, which fall within the multiple EGF modules of Notch, are highly conserved across species and within Notch homologs. Since Notch is known to interact with its ligands through subsets of EGF modules, these results suggest that the O-linked carbohydrate modifications of these modules may influence receptor-ligand interactions.
Proteins encoded by the fringe family of genes are required to modulate
Notch signalling in a wide range of developmental contexts. Using a cell co-culture
assay, we find that mammalian Lunatic fringe (Lfng) inhibits Jagged1-mediated
signalling and potentiates Delta1-mediated signalling through Notch1. Lfng
localizes to the Golgi, and Lfng-dependent modulation of Notch signalling
requires both expression of Lfng in the Notch-responsive cell and the Notch
extracellular domain. Lfng does not prevent binding of soluble Jagged1 or
Delta1 to Notch1-expressing cells. Lfng potentiates both Jagged1- and Delta1-mediated
signalling via Notch2, in contrast to its actions with Notch1. Our data suggest
that Fringe-dependent differential modulation of the interaction of Delta/Serrate/Lag2
(DSL) ligands with their Notch receptors is likely to have a significant role
in the combinatorial repertoire of Notch signalling in mammals.
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The LAG-2 membrane protein is a putative signaling ligand for the LIN-12 and GLP-1 receptors of Caenorhabditis elegans. LAG-2, like its Drosophila homologues Delta and Serrate, acts in a conserved signal transduction pathway to regulate cell fates during development. In this article, we investigate the functional domains of LAG-2. For the most part, mutants were constructed in vitro and assayed for activity in transgenic animals. We find a functional role for all major regions except one. Within the extracellular domain, the N-terminal region, which bears no known motif, and the DSL domain are both required. By contrast, the region bearing epidermal growth factor-like repeats can be deleted with no apparent reduction in rescuing activity. The intracellular region is not required for activity but instead plays a role in down-regulating LAG-2 function. Finally, membrane association is critical for mutant rescue.
During the emergence of neural crest cells from the neural tube, the expression of cadherins dynamically changes. In the chicken embryo, the early neural tube expresses two cadherins, N-cadherin and cadherin-6B (cad6B), in the dorsal-most region where neural crest cells are generated. The expression of these two cadherins is, however, downregulated in the neural crest cells migrating from the neural tube; they instead begin expressing cadherin-7 (cad7). As an attempt to investigate the role of these changes in cadherin expression, we overexpressed various cadherin constructs, including N-cadherin, cad7, and a dominant negative N-cadherin (cN390 ), in neural crest-generating cells. This was achieved by injecting adenoviral expression vectors encoding these molecules into the lumen of the closing neural tube of chicken embryos at stage 14. In neural tubes injected with the viruses, efficient infection was observed at the neural crest-forming area, resulting in the ectopic cadherin expression also in migrating neural crest cells. Notably, the distribution of neural crest cells with the ectopic cadherins changed depending on which constructs were expressed. Many crest cells failed to escape from the neural tube when N-cadherin or cad7 was overexpressed. Moreover, none of the cells with these ectopic cadherins migrated along the dorsolateral (melanocyte) pathway. When these samples were stained for Mitf, an early melanocyte marker, positive cells were found accumulated within the neural tube, suggesting that the failure of their migration was not due to differentiation defects. In contrast to these phenomena, cells expressing non-functional cadherins exhibited a normal migration pattern. Thus, the overexpression of a neuroepithelial cadherin (N-cadherin) and a crest cadherin (cad7) resulted in the same blocking effect on neural crest segregation from neuroepithelial cells, especially for melanocyte precursors. These findings suggest that the regulation of cadherin expression or its activity at the neural crest-forming area plays a critical role in neural crest emigration from the neural tube.
The Notch signalling pathway plays an important role during the development of the wing primordium, especially of the wing blade and margin. In these processes, the activity of Notch is controlled by the activity of the dorsal specific nuclear protein Apterous, which regulates the expression of the Notch ligand, Serrate, and the Fringe signalling molecule. The other Notch ligand, Delta, also plays a role in the development and patterning of the wing. It has been proposed that Fringe modulates the ability of Serrate and Delta to signal through Notch and thereby restricts Notch signalling to the dorsoventral boundary of the developing wing blade. Here we report the results of experiments aimed at establishing the relationships between Fringe, Serrate and Delta during wing development. We find that Serrate is not required for the initiation of wing development but rather for the expansion and early patterning of the wing primordium. We provide evidence that, at the onset of wing development, Delta is under the control of apterous and might be the Notch ligand in this process. In addition, we find that Fringe function requires Su(H). Our results suggest that Notch signalling during wing development relies on careful balances between positive and dominant negative interactions between Notch ligands, some of which are mediated by Fringe.
We find that ectopic expression of Delta or Serrate in neurons within developing bristle organs is capable of non-autonomously inducing the transformation of the pre-trichogen cell into a tormogen cell in a wide variety of developmental contexts. The frequencies at which Delta can induce these transformations are dependent on the level of ectopic Delta expression and the levels of endogenous Notch signalling pathway components. The pre-trichogen cell becomes more responsive to Delta- or Serrate-mediated transformation when the level of endogenous Delta is reduced and less responsive when the dosage of endogenous Delta is increased, supporting the hypothesis that Delta interferes autonomously with the ability of a cell to receive either signal. We also find that a dominant-negative form of Notch, ECN, is capable of autonomously interfering with the ability of a cell to generate the Delta signal. When the region of Notch that mediates trans-interactions between Delta and the Notch extracellular domain is removed from ECN, the ability of Delta to signal is restored. Our findings imply that cell-autonomous interactions between Delta and Notch can affect the ability of a cell to generate and to transduce a Delta-mediated signal. Finally, we present evidence that the Fringe protein can interfere with Delta- and Serrate-mediated signalling within developing bristle organs, in contrast to previous reports of the converse effects of Fringe on Delta signalling in the developing wing.
Signaling by the Notch surface receptor controls cell fate determination in a broad spectrum of tissues. This signaling is triggered by the interaction of the Notch protein with what, so far, have been thought to be transmembrane ligands expressed on adjacent cells. Here biochemical and genetic analyses show that the ligand Delta is cleaved on the surface, releasing an extracellular fragment capable of binding to Notch and acting as an agonist of Notch activity. The ADAM disintegrin metalloprotease Kuzbanian is required for this processing event. These observations raise the possibility that Notch signaling in vivo is modulated by soluble forms of the Notch ligands.
The Notch signalling pathway regulates cell fate choices during both vertebrate and invertebrate development. In the Drosophila wing disc, the activation of Notch by its ligands Delta and Serrate is required to make the dorsoventral boundary, where several genes, such as wingless and cut, are expressed in a 2- to 4-cell-wide domain. The interactions between Notch and its ligands are modulated by Fringe via a mechanism that may involve post-transcriptional modifications of Notch. The ligands themselves also help to restrict Notch activity to the dorsoventral boundary cells, because they antagonise the activation of the receptor in the cells where their expression is high. This function of the ligands is critical to establish the polarity of signalling, but very little is known about the mechanisms involved in the interactions between Notch and its ligands that result in suppression of Notch activity. The extracellular domain of Notch contains an array of 36 EGF repeats, two of which, repeats 11 and 12, are necessary for direct interactions between Notch with Delta and Serrate. We investigate here the function of a region of the Notch extracellular domain where several missense mutations, called Abruptex, are localised. These Notch alleles are characterised by phenotypes opposite to the loss of Notch function and also by complex complementation patterns. We find that, in Abruptex mutant discs, only the negative effects of the ligands and Fringe are affected, resulting in the failure to restrict the expression of cut and wingless to the dorsoventral boundary. We suggest that Abruptex alleles identify a domain in the Notch protein that mediates the interactions between Notch, its ligands and Fringe that result in suppression of Notch activity.
Ligands that are capable of activating Notch family receptors are broadly expressed in animal development, but their activity is tightly regulated to allow formation of tissue boundaries. Members of the fringe gene family have been implicated in limiting Notch activation during boundary formation, but the mechanism of Fringe function has not been determined. Here we present evidence that Fringe acts in the Golgi as a glycosyltransferase enzyme that modifies the epidermal growth factor (EGF) modules of Notch and alters the ability of Notch to bind its ligand Delta. Fringe catalyses the addition of N-acetylglucosamine to fucose, which is consistent with a role in the elongation of O-linked fucose O-glycosylation that is associated with EGF repeats. We suggest that cell-type-specific modification of glycosylation may provide a general mechanism to regulate ligand-receptor interactions in vivo.
Signaling by the Notch surface receptor controls cell fate determination in a broad spectrum of tissues. This signaling is
triggered by the interaction of the Notch protein with what, so far, have been thought to be transmembrane ligands expressed
on adjacent cells. Here biochemical and genetic analyses show that the ligand Delta is cleaved on the surface, releasing an
extracellular fragment capable of binding to Notch and acting as an agonist of Notch activity. The ADAM disintegrin metalloprotease
Kuzbanian is required for this processing event. These observations raise the possibility that Notch signaling in vivo is
modulated by soluble forms of the Notch ligands.
Notch locus EGF-like element mutations spl, altering eye development, and AxE2, affecting wing and sensilla development, are modified by mutations at Delta. It is shown that two allele-specific suppressors of spl involve single amino acid substitutions in the 4th (Dlsup5) and 9th (Dlsup4) EGF-like elements of the Delta protein. Cultured cells producing spl or AxE2 aggregate with cells producing wild-type Delta or Dlsup5 protein, and Dlsup5-producing cells adhere to cells producing wild-type Notch protein. However, spl,AxE2, and Dlsup5 are each defective in promoting these cell affinities, as none of the mutant proteins can compete with the corresponding wild-type proteins for formation of cell aggregates. Thus, widely separated EGF-like elements of Notch and Delta appear to participate in functional molecular interactions between the proteins. Dlsup5 does not improve adhesiveness of spl in vitro, so suppression in vivo may involve altered developmental signaling by spl-Dlsup5 complexes, rather than modified cell adhesion.
The neurogenic loci Notch and Delta, which both encode EGF-homologous transmembrane proteins, appear to function together in mediating cell-cell communication and have been shown to interact at the cell surface in vitro. To examine the role of the EGF repeats in this interaction, we performed an extensive deletion mutagenesis of the extracellular domain of Notch. We find that of the 36 EGF repeats of Notch, only two, 11 and 12, are both necessary and sufficient to mediate interactions with Delta. Furthermore, this Delta binding ability is conserved in the corresponding two repeats from the Xenopus Notch homolog. We report a novel molecular interaction between Notch and Serrate, another EGF-homologous transmembrane protein containing a region of striking similarity to Delta, and show that the same two EGF repeats of Notch also constitute a Serrate binding domain. These results suggest that Notch may act as a multifunctional receptor whose 36 EGF repeats form a tandem array of discrete ligand-binding units, each of which may potentially interact with several different proteins during development.
Genetic analyses have raised the possibility of interactions between the gene products of the neurogenic loci Notch and Delta, each of which encodes a transmembrane protein with EGF homology. To examine the possibility of intermolecular association between the products of these two genes, we studied the effects of their expression on aggregation in Drosophila S2 cells. We find that Notch-expressing cells form mixed aggregates specifically with cells that express Delta and that this process is calcium dependent. In addition, we show that Notch and Delta can associate within the membrane of a single cell, and further, that they form detergent-soluble intermolecular complexes. Our analyses suggest that Notch and Delta proteins interact at the cell surface via their extracellular domains.
Three types of unusual post-translational modification have been found within conserved amino acid sequences in epidermal growth factor homology regions (EGF modules) of some multidomain proteins. beta-Hydroxyaspartate and beta-hydroxyasparagine are found within -Cys-Xxx-Asp/Asn-Xxx-Xxx-Xxx-Xxx-Tyr/Phe-Xxx-Cys-Xxx-Cys- sequences. (Xyl alpha 1-->3)Xyl alpha 1-->3Glc beta 1-->O-Ser glycans at conserved sites within -Cys-Xxx-Ser-Xxx-Pro-Cys- sequences have been reported in several proteins. Fuc alpha 1-->O-Thr/Ser modifications have been found at conserved sites within -Cys-Xxx-Xxx-Gly-Gly-Thr/Ser-Cys- sequences. More recently, it has been discovered that the Ser residue corresponding to the potential O-fucosylation site in human factor IX carries the novel tetrasaccharide NeuAc alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->3Fuc alpha 1-->O-Ser; this tetrasaccharide can be considered to be an extension of the Fuc alpha 1-->O moiety. The consensus sequences for these post-translational modifications are in close proximity to each other; e.g. human factor IX has all three unusual modifications within a 12 amino acid linear sequence. In proteins with multiple EGF modules, the O-glycosidic modifications have been found only within the N-terminal EGF module; beta-hydroxyaspartate/asparagine residues are not restricted in the same fashion. Little is known yet about the functions of, or possible relationships between, any of these modifications.
The Notch family of transmembrane receptor proteins mediate developmental cell-fate decisions, and mutations in mammalian Notch genes have been implicated in leukaemia, breast cancer, stroke and dementia. During wing development in Drosophila, the Notch receptor is activated along the border between dorsal and ventral cells, leading to the specification of specialized cells that express Wingless (Wg) and organize wing growth and patterning. Three genes, fringe (fng), Serrate (Ser) and Delta (Dl), are involved in the cellular interactions leading to Notch activation. Ser and Dl encode transmembrane ligands for Notch, whereas fng encodes a pioneer protein. We have investigated the relationship between these genes by a combination of expression and coexpression studies in the Drosophila wing. We found that Ser and Dl maintain each other's expression by a positive feedback loop. fng is expressed specifically by dorsal cells and functions to position and restrict this feedback loop to the developing dorsal-ventral boundary. This is achieved by fng through a cell-autonomous mechanism that inhibits a cell's ability to respond to Serrate protein and potentiates its ability to respond to Delta protein.
In the developing imaginal wing disc of Drosophila, cells at the dorsoventral boundary require localized Notch activity for specification of the wing margin. The Notch ligands Serrate and Delta are required on opposite sides of the presumptive wing margin and, even though activated forms of Notch generate responses on both sides of the dorsoventral boundary, each ligand generates a compartment-specific response. In this report we demonstrate that Serrate, which is expressed in the dorsal compartment, does not signal in the dorsal regions due to the action of the fringe gene product. Using ectopic expression, we show that regulation of Serrate by fringe occurs at the level of protein and not Serrate transcription. Furthermore, replacement of the N-terminal region of Serrate with the corresponding region of Delta abolishes the ability of fringe to regulate Serrate without altering Serrate-specific signaling.
Recent results show that in the developing Drosophila wing, the secreted pioneer protein Fringe regulates the sensitivity of the Notch signaling pathway to different ligands. This provides a likely mechanism by which Fringe-like molecules may control patterning in both Drosophila and vertebrates.
In this review, we have made a simple comparison of functional parallels and molecular similarities between Brn and Frg. Analysis of Brn function in egg chambers suggests that Brn specificity in patterning fields of cells versus lateral specification is achieved by restricting Brn cooperation with N to the apical surface of follicle cells. Misexpression of Brn in the follicular epithelium, where it would have access to the N signaling process on the lateral surface of follicle cells, might be expected to interfere with N signaling processes, but this has not been tested. Furthermore, it will be of interest to test, as we propose for Brn, whether the specificity of Frg in patterning the wing disk relies on restricting Frg activity to a particular epithelial cell surface. We currently have no molecular knowledge of how Brn participates in the N signaling process. The apparent requirement of Frg to inhibit activation of N by Ser, and to potentiate activation of N by D1, suggests avenues for investigation. If conclusions drawn from analysis of Brn function during oogenesis can be applied to Brn function in early embryos, then we might expect to find that Brn is required for maintaining the epithelial integrity of the neurogenic ectoderm during neuroblast segregation but not for lateral specification between cells within the ectoderm (Goode et al. 1996a,b). Interestingly, N expression in mesodermal cells dramatically rescues the epidermal phenotype of N mutant embryos, which may imply that in addition to being required between 'equivalent' ectodermal cells to ensure proper segregation of neural precursor cells, N acts between distinct tissue layers (Baker and Schubiger 1996). We find this hypothesis attractive because it suggests an alternative means by which may imply Brn might be involved in regulating the segregation of neural precursors cells within the neurogenic ectoderm and is consistent with the tenet that Frg and Brn are specifically involved in N processes of induction and/or maintenance between nonequivalent fields of cells. Brn and Frg are the first secreted factors to be implicated in N signaling processes. We would like to know whether Brn and Frg are diffusible factors and if their secretion is crucial for establishing their activity, perhaps by analyzing the functional consequences of expressing forms of the molecules that remain tethered to the membrane. Further understanding of Brn and Frg in N induction and maintenance processes will also depend on determining whether they act as glycosyltransferases or by a distinct mechanism to influence N-ligand interactions. It will also be important to influence N-ligand interactions. It will also be important to understand how Brn and Frg differ in function, and to what degree, if any, they can substitute for each others' function. The proposal that N serves as a multifunctional receptor by using its multitude of EGF repeats to bind distinct ligands (Rebay et al. 1991) suggests the possibility that Brn and Frg might bind N either directly or through a distinct protein. Alternatively, Brn and Frg might modify the glycosylation state of N or another receptor to influence their ability to be activated by distinct ligands. Further analysis of Egh will be essential to demonstrate whether this protein is part of the Brn signaling process and whether Egh or an Egh homolog acts in the Frg signaling pathway.
In addition to the core components of the Notch pathway, a number of proteins have been identified that exert positive or negative influences on Notch signaling. These include extracellular modulators, which may influence binding or activation of Notch by its ligands, cytoplasmic modulators, which presumably influence signal transduction steps after receptor activation, and nuclear modulators, which may influence the transcriptional activity of a Notch-CSL protein complex. Many of the cytoplasmic and nuclear modulators appear to bind directly to discrete domains within the intracellular domain of Notch. Genetic studies indicate that distinct modulators are deployed during distinct modes of Notch signaling.
The Notch signalling pathway is an evolutionarily conserved cell-to-cell communication system utilized multiple times and in many tissues during development. The outcome of an interaction between Notch and its ligands is highly influenced by factors both extrinsic and intrinsic to Notch expressing cells, suggesting that Notch functions either directly or in parallel with other signalling systems to regulate cellular differentiation events. Protein domains common to all ligands and receptors of this system suggest conserved functional properties that likely relate to regulatory mechanisms for Notch signalling. Within this review, the known functional properties of these domains are analyzed with respect to their contributions to ligand/receptor interactions and Notch signalling.
Multiple mechanisms are involved in positioning and restricting specialized dorsal-ventral border cells in the Drosophila wing, including modulation of Notch signaling by Fringe, autonomous inhibition by Notch ligands, and inhibition of Notch target genes by Nubbin. Recent studies have revealed that Fringe also modulates a Notch-mediated signaling process between dorsal and ventral cells in the Drosophila eye, establishing an organizer of eye growth and patterning along the dorsal-ventral midline. Fringe-dependent modulation of Notch signaling also plays a key role in Drosophila leg segmentation and growth. Lunatic Fringe has been shown to be required for vertebrate somitogenesis, where it appears to act as a crucial link between a molecular clock and the regulation of Notch signaling.
Molecular evidence has established that direct heterotypic interactions occur between the Drosophila receptor Notch and the ligands Delta and Serrate, and that homotypic interactions occur between Delta molecules on opposing cell surfaces. Using an aggregation assay developed for Drosophila cultured cells, we have compared the affinities of these interactions. We find that the heterotypic interactions between Notch and the ligands Delta and Serrate have higher affinities than homotypic interactions between Delta molecules. Contrary to previous suggestions, our evidence implies that the interactions between Serrate and Notch are similar in affinity to those between Delta and Notch. We find that Fringe does not detectably affect the ligand-receptor interactions of the Notch pathway in cultured cells. Furthermore, we find that Serrate, like Delta, is a transmembrane ligand that can participate in reciprocal trans-endocytosis of ligand and receptor between expressing cells. Our findings imply that qualitative differences between Delta- and Serrate-mediated Notch signalling depend on characteristics other than intrinsic ligand-receptor affinities or the ability to participate in reciprocal ligand and receptor trans-endocytosis.
The Notch pathway mediates cell-cell interaction in many developmental processes. Multiple proteins regulate the Notch pathway, among these are the products of the fringe genes. The first fringe gene was identified in Drosophila, where it is involved in the formation of the dorsal/ventral border of the wing disc. It has now been found to be crucial for determining the dorsal/ventral border of the Drosophila eye. In vertebrates, fringe genes play roles in the formation of the apical ectodermal ridge, the dorsal/ventral border in the limb bud, and in the development of somitic borders. The roles of fringe in the neural tube or the eyes of vertebrate embryos are not clear, although it is unlikely that these roles are evolutionarily related to those in the same tissues in Drosophila. Genetic evidences suggest that Fringe protein functions by modulating the Notch signaling pathway, perhaps through differential regulation of Notch activation by different ligands; however, the mechanism underlying Fringe function remains to be investigated.
Enzymatic glycosylation of proteins and lipids is an abundant and important biological process. A great diversity of oligosaccharide structures and types of glycoconjugates is found in nature, and these are synthesized by a large number of glycosyltransferases. Glycosyltransferases have high donor and acceptor substrate specificities and are in general limited to catalysis of one unique glycosidic linkage. Emerging evidence indicates that formation of many glycosidic linkages is covered by large homologous glycosyltransferase gene families, and that the existence of multiple enzyme isoforms provides a degree of redundancy as well as a higher level of regulation of the glycoforms synthesized. Here, we discuss recent cloning strategies enabling the identification of these large glycosyltransferase gene families and exemplify the implication this has for our understanding of regulation of glycosylation by discussing two galactosyltransferase gene families.
Notch is a large cell-surface receptor known to be an essential player in a wide variety of developmental cascades. Here we show that Notch1 endogenously expressed in Chinese hamster ovary cells is modified with O-linked fucose and O-linked glucose saccharides, two unusual forms of O-linked glycosylation found on epidermal growth factor-like (EGF) modules. Interestingly, both modifications occur as monosaccharide and oligosaccharide species. Through exoglycosidase digestions we determined that the O-linked fucose oligosaccharide is a tetrasaccharide with a structure identical to that found on human clotting factor IX: Sia-alpha2,3-Gal-beta1, 4-GlcNAc-beta1,3-Fuc-alpha1-O-Ser/Thr. The elongated form of O-linked glucose appears to be a trisaccharide. Notch1 is the first membrane-associated protein identified with either O-linked fucose or O-linked glucose modifications. It also represents the second protein discovered with an elongated form of O-linked fucose. The sites of glycosylation, which fall within the multiple EGF modules of Notch, are highly conserved across species and within Notch homologs. Since Notch is known to interact with its ligands through subsets of EGF modules, these results suggest that the O-linked carbohydrate modifications of these modules may influence receptor-ligand interactions.
While it has been long appreciated that sugar-modified proteins coat the cell surface, their functions are poorly understood. Here, I describe recent genetic studies that demonstrate that one class of sugar-modified proteins, cell-surface proteoglycans, play crucial roles in morphogenesis, growth regulation and tumor suppression. Mutations that affect individual proteoglycans or the enzymes required for glycosaminoglycan synthesis regulate Wingless and Decapentaplegic signaling in Drosophila, and body size in mice and humans. Compromising proteoglycan function is also associated with the development of Wilm's tumors and hereditary multiple exostoses. In this review, these biological findings are placed in the context of proteoglycan biochemistry and molecular function.
Heparan sulphate proteoglycans are abundant cell-surface molecules that
consist of a protein core to which heparan sulphate glycosaminoglycan chains
are attached. The functions of these molecules have remained mostly underappreciated
by developmental biologists; however, the actions of important signalling
molecules, for example Wnt and Hedgehog, depend on them. To understand both
the mechanisms by which ligands involved in development interact with their
receptors and how morphogens pattern tissues, biologists need to consider
the functions of heparan sulphate proteoglycans in signalling and developmental
patterning.
The Fringe protein of Drosophila and its vertebrate homologues function in boundary determination during pattern formation. Fringe has been proposed to inhibit Serrate-Notch signalling but to potentiate Delta-Notch signalling. Here we show that Fringe and Notch form a complex through both the Lin-Notch repeats and the epidermal growth factor repeats 22-36 (EGF22-36) of Notch when they are co-expressed. The Abruptex59b (Ax59b) and AxM1 mutations, which are caused by missense mutations in EGF repeats 24 and 25, respectively, abolish the Fringe-Notch interaction through EGF22-36, whereas the l(1)N(B) mutation in the third Lin-Notch repeat of Notch abolishes the interaction through Lin-Notch repeats. Ax mutations also greatly affect the Notch response to ectopic Fringe in vivo. Results from in vitro protein mixing experiments and subcellular colocalization experiments indicate that the Fringe-Notch complex may form before their secretion. These findings explain how Fringe acts cell-autonomously to modulate the ligand preference of Notch and why the Fringe-Notch relationship is conserved between phyla and in the development of very diverse structures.
Signalling via the Notch receptor is a key regulator of many developmental processes. The differential responsiveness of Notch-expressing cells to the ligands Delta and Serrate is controlled by Fringe, itself essential for normal patterning in Drosophila and vertebrates. The mechanism of Fringe action, however, is not known. The protein has an amino-terminal hydrophobic stretch resembling a cleaved signal peptide, which has led to the widespread assumption that it is a secreted signalling molecule. It also has distant homology to bacterial glycosyltransferases, although it is not clear if this reflects a shared enzymatic activity, or merely a related structure.
We report that a functional epitope-tagged form of Drosophila Fringe was localised in the Golgi apparatus. When the putative signal peptide was replaced by a confirmed one, Fringe no longer accumulated in the Golgi, but was instead efficiently secreted. This change in localisation dramatically reduced its biological activity, implying that the wild-type protein normally acts inside the cell. We show that Fringe specifically binds the nucleoside diphosphate UDP, a feature of many glycosyltransferases. Furthermore, specific mutation of a DxD motif (in the single-letter amino acid code where x is any amino acid), a hallmark of most glycosyltransferases that use nucleoside diphosphate sugars, did not affect the Golgi localisation of the protein but completely eliminated in vivo activity.
These results indicate that Fringe does not exert its effects outside of the cell, but rather acts in the Golgi apparatus, apparently as a glycosyltransferase. They suggest that alteration in receptor glycosylation can regulate the relative efficiency of different ligands.
Notch receptors function in highly conserved intercellular signalling pathways that direct cell-fate decisions, proliferation and apoptosis in metazoans. Fringe proteins can positively and negatively modulate the ability of Notch ligands to activate the Notch receptor. Here we establish the biochemical mechanism of Fringe action. Drosophila and mammalian Fringe proteins possess a fucose-specific beta1,3 N-acetylglucosaminyltransferase activity that initiates elongation of O-linked fucose residues attached to epidermal growth factor-like sequence repeats of Notch. We obtained biological evidence that Fringe-dependent elongation of O-linked fucose on Notch modulates Notch signalling by using co-culture assays in mammalian cells and by expression of an enzymatically inactive Fringe mutant in Drosophila. The post-translational modification of Notch by Fringe represents a striking example of modulation of a signalling event by differential receptor glycosylation and identifies a mechanism that is likely to be relevant to other signalling pathways.
Artavanis-Tsakonas S: Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor
- L Rebay
- R J Fleming
- R G Fehon
- P Chervas
Rebay L, Fleming RJ, Fehon RG, Chervas P, Artavanis-Tsakonas S:
Specific EGF repeats of Notch mediate interactions with Delta
and Serrate: implications for Notch as a multifunctional receptor.
Cell 1991, 67:687-699.
Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor.
- Rebay L.
- Fleming R.J.
- Fehon R.G.
- Chervas P.
- Artavanis-Tsakonas S.
Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor
- Rebay