Fringe, Notch, and making developmental boundaries.

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.     

A re-evaluation of the contributions of Apterous and Notch to the dorsoventral lineage restriction boundary in the Drosophila wing

The Drosophila limb primordia are subdivided into compartments: cell populations that do not mix during development. The wing is subdivided into dorsal (D) and ventral (V) compartments by the activity of the selector gene apterous in D cells. Apterous causes segregation of D and V cell populations by at least two distinct mechanisms. The LRR transmembrane proteins Capricious and Tartan are transiently expressed in D cells and contribute to initial segregation of D and V cells. Signaling between D and V cells mediated by Notch and Fringe contributes to the maintenance of the DV affinity boundary. Given that Notch is activated symmetrically, in D and V cells adjacent to the boundary, its role in boundary formation remains somewhat unclear. We re-examine the roles of Apterous and Fringe activities in DV boundary formation and present evidence that Fringe cannot, by itself, generate an affinity difference between D and V cells. Although not sufficient, Fringe is required via Notch activation for expression of an Apterous-dependent affinity difference. We propose that Apterous controls expression of surface proteins that confer an affinity difference in conjunction with activated Notch. Thus, we view Apterous as instructive and Notch activity as essential, but permissive.     

The Mirror transcription factor links signalling pathways in Drosophila oogenesis

Many genetic cascades are conserved in evolution, yet they trigger different responses and hence determine different cell fates at specific times and positions in development. At stage 10 of oogenesis, mirror is expressed in anterior-dorsal follicle cells, and we show that this is dependent upon the Gurken signal from the oocyte. The fringe gene is expressed in a complementary pattern in posterior-ventral follicle cells at the same stage. Ectopic expression of mirror represses fringe expression, thus linking the epidermal growth factor receptor (EGFR) signalling pathway to the Fringe signalling pathway via Mirror. The EGFR pathway also triggers the cascade that leads to dorsal-ventral axis determination in the embryo. We used twist as an embryonic marker for ventral cells. Ectopic expression of mirror in the follicle cells during oogenesis ultimately represses twist expression in the embryo, and leads to similar phenotypes to the ectopic expression of the activated form of EGFR. Thus, mirror also controls the Toll signalling pathway, leading to Dorsal nuclear transport. In summary, we show that the Mirror homeodomain protein provides a link that coordinates the Gurken/EGFR signalling pathway (initiated in the oocyte) with the Fringe/Notch/Delta pathway (in follicle cells). This coordination is required for epithelial morphogenesis, and for producing the signal in ventral follicle cells that determines the dorsal/ventral axis of the embryo.     

Drosophila wing development in the absence of dorsal identity

The developing wing disc of Drosophila is divided into distinct lineage-restricted compartments along both the anterior/posterior (A/P) and dorsal/ventral (D/V) axes. At compartment boundaries, morphogenic signals pattern the disc epithelium and direct appropriate outgrowth and differentiation of adult wing structures. The mechanisms by which affinity boundaries are established and maintained, however, are not completely understood. Compartment-specific adhesive differences and inter-compartment signaling have both been implicated in this process. The selector gene apterous (ap) is expressed in dorsal cells of the wing disc and is essential for D/V compartmentalization, wing margin formation, wing outgrowth and dorsal-specific wing structures. To better understand the mechanisms of Ap function and compartment formation, we have rescued aspects of the ap mutant phenotype with genes known to be downstream of Ap. We show that Fringe (Fng), a secreted protein involved in modulation of Notch signaling, is sufficient to rescue D/V compartmentalization, margin formation and wing outgrowth when appropriately expressed in an ap mutant background. When Fng and alphaPS1, a dorsally expressed integrin subunit, are co-expressed, a nearly normal-looking wing is generated. However, these wings are entirely of ventral identity. Our results demonstrate that a number of wing development features, including D/V compartmentalization and wing vein formation, can occur independently of dorsal identity and that inter-compartmental signaling, refined by Fng, plays the crucial role in maintaining the D/V affinity boundary. In addition, it is clear that key functions of the ap selector gene are mediated by only a small number of downstream effectors.     

Lunatic fringe, manic fringe, and radical fringe recognize similar specificity determinants in O-fucosylated epidermal growth factor-like repeats

Notch signaling is a component of a wide variety of developmental processes in many organisms. Notch activity can be modulated by O-fucosylation (mediated by protein O-fucosyltransferase-1) and Fringe, a beta1,3-N-acetylglucosaminyltransferase that modifies O-fucose in the context of epidermal growth factor-like (EGF) repeats. Fringe was initially described in Drosophila, and three mammalian homologues have been identified, Manic fringe, Lunatic fringe, and Radical fringe. Here for the first time we have demonstrated that, similar to Manic and Lunatic, Radical fringe is also a fucose-specific beta1,3-N-acetylglucosaminyltransferase. The fact that three Fringe homologues exist in mammals raises the question of whether and how these enzymes differ. Although Notch contains numerous EGF repeats that are predicted to be modified by O-fucose, previous studies in our laboratory have demonstrated that not all O-fucosylated EGF repeats of Notch are further modified by Fringe, suggesting that the Fringe enzymes can differentiate between them. In this work, we have sought to identify specificity determinants for the recognition of an individual O-fucosylated EGF repeat by the Fringe enzymes. We have also sought to determine differences in the biochemical behavior of the Fringes with regard to their in vitro enzymatic activities. Using both in vivo and in vitro experiments, we have found two amino acids that appear to be important for the recognition of an O-fucosylated EGF repeat by all three mammalian Fringes. These amino acids provide an initial step toward defining sequences that will allow us to predict which O-fucosylated EGF repeats are modified by the Fringes.     

Dorsal-ventral signaling in limb development

In both Drosophila wings and vertebrate limbs, signaling between dorsal and ventral cells establishes an organizer that promotes limb formation. Significant progress has been made recently towards characterizing the signaling interactions that occur at the dorsal—ventral limb border. Studies of chicks have indicated that, as in Drosophila, this signaling process requires the participation of Fringe. Studies of Drosophila have indicated that Fringe functions by inhibiting the ability of Notch to be activated by one ligand, Serrate, while potentiating the ability of Notch to be activated by another ligand, Delta. Recent studies of both Drosophila and vertebrates have also shed new light on the signaling activity of the dorsal—ventral boundary limb organizer, and have highlighted how this organizer is maintained by feedback mechanisms with neighboring cells.     

Genomic structure, mapping, and expression analysis of the mammalian Lunatic, Manic, and Radical fringe genes

The three members of the mammalian fringe gene family, Manic fringe (Mfng), Radical fringe (Rfng), and Lunatic fringe (Lfng), were identified on the basis of their similarity to Drosophila fringe (fng) and their participation in the evolutionarily conserved Notch receptor signaling pathway. Fringe genes encode pioneer secretory proteins with weak similarity to glycosyltransferases. Both expression patterns and functional studies support an important role for Fringe genes in patterning during embryonic development and an association with cellular transformation. We have now further characterized the expression and determined the chromosomal localization and genomic structure of the mouse Mfng, Rfng, and Lfng genes; the genomic structure and conceptual open reading frame of the human RFNG gene; and the refined chromosomal localization of the three human fringe genes. The mouse Fringe genes are expressed in the embryo and in adult tissues. The mouse and human Fringe family members map to three different chromosomes in regions of conserved synteny: Mfng maps to mouse Chr 15, and MFNG maps to human Chr 22q13.1 in the region of two cancer-associated loci; Lfng maps to mouse Chr 5, and LFNG maps to human Chr 7p22; Rfng maps to mouse Chr 11, and RFNG maps to human Chr 17q25 in the minimal region for a familial psoriasis susceptibility locus. Characterization of the genomic loci of the Fringe gene family members reveals a conserved genomic organization of 8 exons. Comparative analysis of mammalian Fringe genomic organization suggests that the first exon is evolutionarily labile and that the Fringe genes have a genomic structure distinct from those of previously characterized glycosyltransferases. Received: 19 February 1999 / Accepted: 22 February 1999     

In vitro reconstitution of the modulation of Drosophila Notch-ligand binding by Fringe

Notch signaling plays critical roles in animal development and physiology. The activation of Notch receptors by their ligands is modulated by Fringe-dependent glycosylation. Fringe catalyzes the addition of N-acetylglucosamine in a beta1,3 linkage onto O-fucose on epidermal growth factor-like domains. This modification of Notch by Fringe influences the binding of Notch ligands to Notch receptors. However, prior studies have relied on in vivo glycosylation, leaving unresolved the question of whether addition of N-acetylglucosamine is sufficient to modulate Notch-ligand interactions on its own, or whether instead it serves as a precursor to subsequent post-translational modifications. Here, we describe the results of in vitro assays using purified components of the Drosophila Notch signaling pathway. In vitro glycosylation and ligand binding studies establish that the addition of N-acetylglucosamine onto O-fucose in vitro is sufficient both to enhance Notch binding to the Delta ligand and to inhibit Notch binding to the Serrate ligand. Further elongation by galactose does not detectably influence Notch-ligand binding in vitro. Consistent with these observations, carbohydrate compositional analysis and mass spectrometry on Notch isolated from cells identified only N-acetylglucosamine added onto Notch in the presence of Fringe. These observations argue against models in which Fringe-dependent glycosylation modulates Notch signaling by acting as a precursor to subsequent modifications and instead establish the simple addition of N-acetylglucosamine as a basis for the effects of Fringe on Drosophila Notch-ligand binding.     

Fringe family genes and their modulation of Notch signaling in cancer

Fringes are glycosyltransferases that transfer N-acetylglucosamine to the O-linked fucose of Notch receptors. They regulate the Notch signaling activity that drives tumor formation and progression, resulting in poor prognosis. However, the specific tumor-promoting role of Fringes differs depending on the type of cancer. Although a particular Fringe member could act as a tumor suppressor in one cancer type, it may act as an oncogene in another. This review discusses the tumorigenic role of the Fringe family (lunatic fringe, manic fringe, and radical fringe) in modulating Notch signaling in various cancers. Although the crucial functions of Fringes continue to emerge as more mechanistic studies are being pursued, further translational research is needed to explore their roles and therapeutic benefits in various malignancies.     

Fringe glycosyltransferases differentially modulate Notch1 proteolysis induced by Delta1 and Jagged1

Fringe O-fucose-beta1,3-N-acetylglucosaminyltransferases modulate Notch signaling by potentiating signaling induced by Delta-like ligands, while inhibiting signaling induced by Serrate/Jagged1 ligands. Based on binding studies, the differential effects of Drosophila fringe (DFng) on Notch signaling are thought to result from alterations in Notch glycosylation that enhance binding of Delta to Notch but reduce Serrate binding. Here, we report that expression of mammalian fringe proteins (Lunatic [LFng], Manic [MFng], or Radical [RFng] Fringe) increased Delta1 binding and activation of Notch1 signaling in 293T and NIH 3T3 cells. Although Jagged1-induced signaling was suppressed by LFng and MFng, RFng enhanced signaling induced by either Delta1 or Jagged1, underscoring the diversity of mammalian fringe glycosyltransferases in regulating signaling downstream of different ligand-receptor combinations. Interestingly, suppression of Jagged1-induced Notch1 signaling did not correlate with changes in Jagged1 binding as found for Delta1. Our data support the idea that fringe glycosylation increases Delta1 binding to potentiate signaling, but we propose that although fringe glycosylation does not reduce Jagged1 binding to Notch1, the resultant ligand-receptor interactions do not effectively promote Notch1 proteolysis required for activation of downstream signaling events.     

Sequential Notch Signalling at the Boundary of Fringe Expressing and Non-Expressing Cells

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.     

Modulation of receptor signaling by glycosylation: fringe is an O-fucose-beta1,3-N-acetylglucosaminyltransferase

The Notch family of signaling receptors plays key roles in determining cell fate and growth control. Recently, a number of laboratories have shown that O-fucose glycans on the epidermal growth factor (EGF)-like repeats of the Notch extracellular domain modulate Notch signaling. Fringe, a known modifier of Notch function, is an O-fucose specific beta1,3-N-acetylglucosaminyltransferase. The transfer of GlcNAc to O-fucose on Notch by fringe results in the potentiation of signaling by the Delta class of Notch ligands, but causes inhibition of signaling by the Serrate/Jagged class of Notch ligands. Interestingly, addition of a beta1,4 galactose by beta4GalT-1 to the GlcNAc added by fringe is required for Jagged1-induced Notch signaling to be inhibited in a co-culture assay. Thus, both fringe and beta4GalT-1 are modulators of Notch function. Several models have been proposed to explain how alterations in O-fucose glycans result in changes in Notch signaling, and these models are discussed.     

Cell-fate choice and boundary formation by combined action of Notch and engrailed in the Drosophila hindgut

The Drosophila large intestine is initially subdivided into dorsal and ventral domains with distinct cell types, and a one-cell-wide strand of boundary cells is induced between them. Here we show that cell identity and localization of the boundary cells are determined by the combined action of Delta, Notch, and engrailed genes. The prospective dorsal domain of the hindgut primordium expresses engrailed. Engrailed represses Delta, which is ubiquitously expressed throughout the prospective hindgut region in early blastodermal stages, in the dorsal domain, and thus generates a Delta-positive/negative prepattern. Expression of Engrailed protein determines the dorsal domain, while an Engrailed-negative (Delta-positive) region is differentiated into the ventral domain. Delta-positive ventral cells activate a Notch cascade in abutting dorsal cells, and thus induce their differentiation into boundary cells. Mis-expression of a constitutively active Notch intracellular domain causes the entire large intestine to develop as boundary cells. It was also found that the transducing activity of a transmembrane form of activated Notch, which requires further proteolytic processing to generate intracellular fragments, is suppressed in the Delta-positive domain. Delta acts in two distinct ways: it activates the Notch signaling pathway in adjacent Delta-negative cells, and, at the same time, autonomously blocks Notch signaling in Delta-positive cells by affecting Notch processing.     

Excess lunatic fringe causes cranial neural crest over-proliferation.

Lunatic fringe is a vertebrate homologue of Drosophila fringe, which plays an important role in modulating Notch signaling. This study examines the distribution of chick lunatic fringe at sites of neural crest formation and explores its possible function by ectopic expression. Shortly after neural tube closure, lunatic fringe is expressed in most of the neural tube, with the exception of the dorsal midline containing presumptive neural crest. Thus, there is a fringe/non-fringe border at the site of neural crest production. Expression of excess lunatic fringe in the cranial neural tube and neural crest by retrovirally mediated gene transfer resulted in a significant increase ( approximately 60%) in the percentage of cranial neural crest cells 1 day after infection. This effect was mediated by an increase in cell division as assayed by BrdU incorporation. Infected embryos had an up-regulation of Delta-1 in the dorsal neural tube and redistribution of Notch-1 to the lumen of the neural tube, confirming that excess fringe modulates Notch signaling. These findings point to a novel role for lunatic fringe in regulating cell division and/or production of neural crest cells by the neural tube.     

A Fringe-modified Notch signal affects specification of mesoderm and endoderm in the sea urchin embryo

Fringe proteins are O-fucose-specific beta-1,3 N-acetylglucosaminyltransferases that glycosylate the extracellular EGF repeats of Notch and enable Notch to be activated by the ligand Delta. In the sea urchin, signaling between Delta and Notch is known to be necessary for specification of secondary mesenchyme cells (SMCs). The Lytechinus variegatus Fringe homologue is expressed in both the signaling and receiving cells during this first Delta-Notch signal. Perturbation of Fringe expression through morpholino antisense oligonucleotide (MO) injection results in fewer SMCs but also causes decreased and delayed archenteron invagination. Partial endoderm specification occurs but expression of some endoderm genes is compromised. The data are consistent with a Fringe-requiring Notch signal as one upstream component of archenteron morphogenesis. Finally, Fringe perturbations result in more severe phenotypes than those previously reported for Notch dominant-negative (LvN(neg)) injections or reported here for Notch MO (NMO) injections. Injecting a combination of LvN(neg) and NMO results in a more severe phenotype than either treatment alone, and this combination phenocopies the fringe MO embryos. Taken together, the results show that Fringe is necessary both for maternal and zygotic Notch signals, and these Notch signals affect specification of mesoderm and endoderm.     

Fringe modifies O-fucose on mouse Notch1 at epidermal growth factor-like repeats within the ligand-binding site and the Abruptex region

Fringe plays a key role in the specification of boundaries during development by modulating the ability of Notch ligands to activate Notch receptors. Fringe is a fucose-specific beta1,3-N-acetylglucosaminyltransferase that modifies O-fucose moieties on the epidermal growth factor-like (EGF) repeats of Notch. To investigate how the change in sugar structure caused by Fringe modulates Notch activity, we have analyzed the sites of O-fucose and Fringe modification on mouse Notch1. The extracellular domain of Notch1 has 36 tandem EGF repeats, many of which are predicted to be modified with O-fucose. We recently proposed a broadened consensus sequence for O-fucose, C(2)X(3-5)(S/T)C(3) (where C(2) and C(3) represent the second and third conserved cysteines), significantly expanding the potential number of modification sites on Notch. Here we demonstrate that sites predicted using this broader consensus sequence are modified with O-fucose on mouse Notch1, and we present evidence suggesting that the consensus can be further refined to C(2)X(4-5)(S/T)C(3). In particular, we demonstrate that EGF 12, a portion of the ligand-binding site, is modified with O-fucose and that this site is evolutionarily conserved. We also show that endogenous Fringe proteins in Chinese hamster ovary cells (Lunatic fringe and Radical fringe) as well as exogenous Manic fringe modify O-fucose on many but not all EGF repeats of mouse Notch1. These findings suggest that the Fringes show a preference for O-fucose on some EGF repeats relative to others. This specificity appears to be encoded within the amino acid sequence of the individual EGF repeats. Interestingly, our results reveal that Manic fringe modifies O-fucose both at the ligand-binding site (EGF 12) and in the Abruptex region. These findings provide insight into potential mechanisms by which Fringe action on Notch receptors may influence both the affinity of Notch-ligand binding and cell-autonomous inhibition of Notch signaling by ligand.     

Notch ligands are substrates for protein O-fucosyltransferase-1 and Fringe

O-Fucose has been identified on epidermal growth factor-like (EGF) repeats of Notch, and elongation of O-fucose has been implicated in the modulation of Notch signaling by Fringe. O-Fucose modifications are also predicted to occur on Notch ligands based on the presence of the C(2)XXGG(S/T)C(3) consensus site (where S/T is the modified amino acid) in a number of the EGF repeats of these proteins. Here we establish that both mammalian and Drosophila Notch ligands are modified with O-fucose glycans, demonstrating that the consensus site was useful for making predictions. The presence of O-fucose on Notch ligands raised the question of whether Fringe, an O-fucose specific beta 1,3-N-acetylglucosaminyltransferase, was capable of modifying O-fucose on the ligands. Indeed, O-fucose on mammalian Delta 1 and Jagged1 can be elongated with Manic Fringe in vivo, and Drosophila Delta and Serrate are substrates for Drosophila Fringe in vitro. These results raise the interesting possibility that alteration of O-fucose glycans on Notch ligands could play a role in the mechanism of Fringe action on Notch signaling. As an initial step to begin addressing the role of the O-fucose glycans on Notch ligands in Notch signaling, a number of mutations in predicted O-fucose glycosylation sites on Drosophila Serrate have been generated. Interestingly, analysis of these mutants has revealed that O-fucose modifications occur on some EGF repeats not predicted by the C(2)XXGGS/TC(3) consensus site. A revised, broad consensus site, C(2)X(3-5)S/TC(3) (where X(3-5) are any 3-5 amino acid residues), is proposed.