Fringe Gene Research Articles

Functional Characterization of Novel Lunatic Fringe Variants in Spondylocostal Dysostosis Type-III with Scoliosis

Scoliosis affects over four million Americans, with most cases having an idiopathic cause. Pathogenic variants in the LUNATIC FRINGE (LFNG) gene can cause spondylocostal dysostosis type-III (SCD3), which is a rare skeletal dysplasia characterized by the absence, fusion, or partial development of vertebrae and ribs. Acute restrictive lung disease and scoliosis may also be present in some cases. The variability in symptoms suggests that there may be other underlying pathological mechanisms that are yet to be discovered. We conducted an analysis of two novel LFNG variants, c.766G>A (p.G256S) and c.521G>A (p.R174H), that were observed in a patient with SCD3 phenotype and scoliosis. Characterizing these variants can help us better understand the relationship between genotype and phenotype. We assessed both variants for impaired glycosyltransferase activity, subcellular mislocalization, and aberrant pre-proprotein processing. Our results indicate that the p.G256S variant is enzymatically nonfunctional, while the p.R174H variant is functionally less effective. Both variants were correctly localized and processed. Our findings suggest that the hypomorphic variant (p.R174H) may have partially improved the patient’s stature, as evidenced by a lower arm span-to-height ratio, increased height, and more vertebrae. However, this variant did not appear to have any effect on the severity of vertebral malformations, including scoliosis. Further research is necessary to determine the extent to which variations in LFNG activity affect the presentation of SCD3.

Breast cancer regulated by Fringe.

Breast cancers are heterogeneous at clinical and molecular levels. In recent years gene expression profiling has been used to classify breast cancers into at least five molecular subtypes, including basal-like, claudin-low, luminal A/B, HER2-enriched and normal-like. Among them, basal-like and claudin-low subtypes account for the majority of triple negative breast cancer (TNBC), a group of diseases that are defined on the basis of being negative for estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 [1]. TNBC preferentially affects younger patients, is more prevalent in women with African ancestry, and is often more aggressive than other types of breast cancer. Basal-like breast cancer (BLBC) expresses markers of myoepithelium/basal cells and shares features with bipotent progenitor cells, and is thought to have originated from mammary bipotent/luminal progenitor cells. Claudin-low breast cancer (CLBC) shares more features with mammary stem cells and cells that have undergone epithelial-to-mesenchymal transition. Fringe proteins are β3N-acteylglucosaminyl-transferases that are known to modify EGF repeats in the extracellular domains of Notch receptor thereby modulating ligand-mediated Notch activation [2]. There are three Fringe genes in mammals, namely, Lunatic Fringe (Lfng), Manic Fringe (Mfng), and Radical Fringe (Rfng). In the mouse mammary gland, Lfng expression is restricted to the stem/progenitor cell-enriched cap cell layer of the terminal end bud [3]. Deletion of Lfng in mouse mammary epithelium (Lfngflox/flox;MMTV-Cre) resulted in ectopic proliferation, expansion of the basal compartment, and ultimately, development of mammary tumors, in which two-thirds resemble BLBC and one-third are similar to CLBC. In agreement with the mouse experiment, the vast majority of BLBC and a subset of CLBC in humans exhibit significantly decreased LFNG expression [3]. While Lfng deficiency is a hallmark of BLBC, we recently reported that Mfng is highly expressed in CLBC and functions as an oncogene in this context [4]. We showed that Mfng regulates Notch activation in human and mouse CLBC cell lines, as well as in mouse mammary gland. Knockdown of Mfng in CLBC cell lines reduced cell migration and tumorsphere formation associated with a decrease in the stem-like cell population, as well as diminished in vivo tumorigenicity. Deletion of Mfng in Lfngflox/flox;MMTV-Cre mice caused a tumor subtype shift away from CLBC. We identified the phosphoinositide kinase Pik3cg as a direct transcriptional target of Mfng-facilitated RBPJk-dependent Notch signaling in CLBC cells. PIK3CG is aberrantly expressed in many invasive human breast tumors and its expression level correlates with metastatic potential of breast cancer cell lines [5]. We showed that pharmacologic inhibition of PI3Kγ in CLBC cell lines blocked migration and tumorsphere formation, suggesting that Mfng-induced Pik3cg contributes to breast cancer aggressiveness by promoting cell migration and invasion, as well as maintaining cancer cell stemness. Identification of Pik3cg as a Notch target prompts a PI3Kγ-targeting strategy for the treatment of CLBC and perhaps other poor prognosis breast cancers. Interestingly, Met proto-oncogene was amplified in both basal-like and claudin-low subtypes of the Lfngflox/flox;MMTV-Cre mammary tumors [3], and Met synergized with p53 loss to induce mouse mammary tumors resembling CLBC [6]. Therefore combination targeting of both Met and PI3Kγ may prove beneficial for the treatment of this disease. Indeed, we observed synergistic effects of Met and PI3Kγ inhibitors on growth of multiple human CLBC cell lines (Chung and Xu, unpublished data). MFNG expression in human breast cancer is highly correlated with the expression of NOTCH4, but not other Notch receptors. In addition, Mfng silencing in CLBC cell lines consistently decreased Notch4 activation, while Mfng deletion in the mouse mammary gland resulted in decreased Notch4 activation [4]. Thus, Mfng appears to control primarily Notch4-mediated signaling in the mammary epithelium. Given that Notch4 is enriched in mammary stem cells, a putative cell-of-origin of CLBC, the Mfng-Notch4-Pik3cg signaling cascade may be a driving force for CLBC pathogenesis. Next question is what regulates Mfng in this context. To this end, we have found that overexpression of Myc upregulates Mfng in mouse mammary epithelium as well as in human breast cancer cell lines (Zhang and Xu, unpublished data). Intriguingly, Myc was found amplified in almost half of human CLBC cases [7]. A link between Myc and Mfng during CLBC development warrants further investigation. Different subtypes of breast cancer are thought to have different cellular origins. Notch pathway may utilize different Fringes to precisely control signaling in distinct cell types of mammary epithelial hierarchy. Altered Fringe expression/activities may cause dysregulation of Notch signaling in different cells-of-origin, and ultimately, leading to breast cancer of different subtypes. Lfng inhibits Notch activation in luminal progenitor cells to prevent BLBC, whereas Mfng may enhance Notch4 signaling in mammary stem cells to promote CLBC initiation and progression. Of note, loss of both Lfng and Mfng in the mammary epithelium dramatically decreased activation of multiple Notch receptors and induced adenosquamous carcinoma [4], suggesting a redundant role of two genes in certain mammary cell types.

Evidence for non-canonical roles of glucose metabolism in the embryo

In our lab we study the effects of glycolytic flux on development and specifically gene activity. Currently it is known that changes in the embryonic environment such as high glucose in diabetic pregnancies increases the risk of congenital malformations including heart, neural tube and segmentation defects. To investigate the link between glucose metabolism and development we use mouse segmentation as a model. We culture mesodermal explants in a chemically defined medium and add glycolytic intermediates in order to bypass or increase the load on specific enzymes. As a real-time readout for gene activity we monitor the oscillatory expression of a venus reporter driven by the promoter of the lunatic fringe gene in the presomitic mesoderm (PSM) during somitogenesis. To date we have identified conditions in which explants cultured with specific metabolites display altered states of gene activity. We have evidence to suggest altered gene activity is not due to a canonical function of carbohydrate metabolism. Additionally we have found minimal flux through the glycolytic enzyme PFK (phosphofructokinase) a flux regulator, to be required for normal PSM patterning. We propose glucose metabolism has a non-canonical role in regulating embryogenesis and the mechanism may be mediated via the activity of PFK.

Mastermind-like 1 (MamL1) and mastermind-like 3 (MamL3) are essential for Notch signaling in vivo

Mastermind (Mam) is one of the elements of Notch signaling, a system that plays a pivotal role in metazoan development. Mam proteins form transcriptionally activating complexes with the intracellular domains of Notch, which are generated in response to the ligand-receptor interaction, and CSL DNA-binding proteins. In mammals, three structurally divergent Mam isoforms (MamL1, MamL2 and MamL3) have been identified. There have also been indications that Mam interacts functionally with various other transcription factors, including the p53 tumor suppressor, β-catenin and NF-κB. We have demonstrated previously that disruption of MamL1 causes partial deficiency of Notch signaling in vivo. However, MamL1-deficient mice did not recapitulate total loss of Notch signaling, suggesting that other members could compensate for the loss or that Notch signaling could proceed in the absence of Mam in certain contexts. Here, we report the generation of lines of mice null for MamL3. Although MamL3-null mice showed no apparent abnormalities, mice null for both MamL1 and MamL3 died during the early organogenic period with classic pan-Notch defects. Furthermore, expression of the lunatic fringe gene, which is strictly controlled by Notch signaling in the posterior presomitic mesoderm, was undetectable in this tissue of the double-null embryos. Neither of the single-null embryos exhibited any of these phenotypes. These various roles of the three Mam proteins could be due to their differential physical characteristics and/or their spatiotemporal distributions. These results indicate that engagement of Mam is essential for Notch signaling, and that the three Mam isoforms have distinct roles in vivo.

Manic fringe is not required for embryonic development, and fringe family members do not exhibit redundant functions in the axial skeleton, limb, or hindbrain

Tight regulation of Notch pathway signaling is important in many aspects of embryonic development. Notch signaling can be modulated by expression of fringe genes, encoding glycosyltransferases that modify EGF repeats in the Notch receptor. Although Lunatic fringe (Lfng) has been shown to play important roles in vertebrate segmentation, comparatively little is known regarding the developmental functions of the other vertebrate fringe genes, Radical fringe (Rfng) and Manic fringe (Mfng). Here we report that Mfng expression is not required for embryonic development. Further, we find that despite significant overlap in expression patterns, we detect no obvious synergistic defects in mice in the absence of two, or all three, fringe genes during development of the axial skeleton, limbs, hindbrain, and cranial nerves.

Mutation of the fucose-specific β1,3 N-acetylglucosaminyltransferase LFNG results in abnormal formation of the spine

Notch signaling is an evolutionarily conserved mechanism that determines cell fate in a variety of contexts during development. This is achieved through different modes of action that are context dependent. One mode involves boundary formation between two groups of cells. With this mode of action, Notch signaling is central to vertebrate evolution as it drives the segmentation of paraxial mesoderm in the formation of somites, which are the precursors of the vertebra. In this case, boundary formation facilitates a mesenchymal to epithelial transition, leading to the creation of a somite. In addition, the boundary establishes a signaling center that patterns the somite, a feature that directly impacts on vertebral column formation. Studies in Xenopus, zebrafish, chicken and mouse have established the importance of Notch signaling in somitogenesis, and indeed in mouse how perturbations in somitogenesis affect vertebral column formation. Spondylocostal dysostosis is a congenital disorder characterized by formation of abnormal vertebrae. Here, mutation in Notch pathway genes demonstrates that Notch signaling is also required for normal somite formation and vertebral column development in humans; of particular interest here is mutation of the LUNATIC FRINGE (LFNG) gene, which causes SCD type 3. LUNATIC FRINGE encodes for a fucose-specific β1,3-N-acetylglucosaminyltransferase, which modifies Notch receptors and alters Notch signaling activity. This review will focus on Notch glycolsylation, and the role of LUNATIC FRINGE in somite formation and vertebral column development in mice and humans.

Bile duct proliferation in Jag1 /fringe heterozygous mice identifies candidate modifiers of the alagille syndrome hepatic phenotype

Alagille syndrome (AGS) is a heterogeneous developmental disorder associated with bile duct paucity and various organ anomalies. The syndrome is caused by mutations in JAG1, which encodes a ligand in the Notch signaling pathway, in the majority of cases and mutations in the NOTCH2 receptor gene in less than 1% of patients. Although a wide array of JAG1 mutations have been identified in the AGS population, these mutational variants have not accounted for the wide phenotypic variability observed in patients with this syndrome. The Fringe genes encode glycosyltransferases, which modify Notch and alter ligand-receptor affinity. In this study, we analyzed double heterozygous mouse models to examine the Fringe genes as potential modifiers of the Notch-mediated hepatic phenotype observed in AGS. We generated mice that were haploinsufficient for both Jag1 and one of three paralogous Fringe genes: Lunatic (Lfng), Radical (Rfng), and Manic (Mfng). Adult Jag1(+/-)Lfng(+/-) and Jag1(+/-)Rfng(+/-) mouse livers exhibited widespread bile duct proliferation beginning at 5 weeks of age and persisting up to 1 year. The Jag1(+/-)Mfng(+/-) livers showed a subtle, yet significant increase in bile duct numbers and bile duct to portal tract ratios. These abnormalities were not observed in the newborn period. Despite the portal tract expansion by bile ducts, fibrosis was not increased and epithelial to mesenchymal transition was not shown in the affected portal tracts. Mice heterozygous for mutations in Jag1 and the Fringe genes display striking bile duct proliferation, which is not apparent at birth. These findings suggest that the Fringe genes may regulate postnatal bile duct growth and remodeling, and serve as candidate modifiers of the hepatic phenotype in AGS.

Engineering of a mammalian O-glycosylation pathway in the yeast Saccharomyces cerevisiae: production of O-fucosylated epidermal growth factor domains

Development of a heterologous system for the production of homogeneous sugar structures has the potential to elucidate structure-function relationships of glycoproteins. In the current study, we used an artificial O-glycosylation pathway to produce an O-fucosylated epidermal growth factor (EGF) domain in Saccharomyces cerevisiae. The in vivo O-fucosylation system was constructed via expression of genes that encode protein O-fucosyltransferase 1 and the EGF domain, along with genes whose protein products convert cytoplasmic GDP-mannose to GDP-fucose. This system allowed identification of an endogenous ability of S. cerevisiae to transport GDP-fucose. Moreover, expression of EGF domain mutants in this system revealed the different contribution of three disulfide bonds to in vivo O-fucosylation. In addition, lectin blotting revealed differences in the ability of fucose-specific lectin to bind the O-fucosylated structure of EGF domains from human factors VII and IX. Further introduction of the human fringe gene into yeast equipped with the in vivo O-fucosylation system facilitated the addition of N-acetylglucosamine to the EGF domain from factor IX but not from factor VII. The results suggest that engineering of an O-fucosylation system in yeast provides a powerful tool for producing proteins with homogenous carbohydrate chains. Such proteins can be used for the analysis of substrate specificity and the production of antibodies that recognize O-glycosylated EGF domains.

Not all lunatic fringe null female mice are infertile

A recent paper in Development by Hahn and colleagues reports that female mice homozygous for a targeted null mutation of the lunatic fringe ( Lfng ) gene are infertile ([Hahn et al., 2005][1]). In 1998, our laboratory and Randy Johnson's laboratory published independent papers on the construction

Mutation of the LUNATIC FRINGE Gene in Humans Causes Spondylocostal Dysostosis with a Severe Vertebral Phenotype

The spondylocostal dysostoses (SCDs) are a heterogeneous group of vertebral malsegmentation disorders that arise during embryonic development by a disruption of somitogenesis. Previously, we had identified two genes that cause a subset of autosomal recessive forms of this disease: DLL3 (SCD1) and MESP2 (SCD2). These genes are important components of the Notch signaling pathway, which has multiple roles in development and disease. Here, we have used a candidate-gene approach to identify a mutation in a third Notch pathway gene, LUNATIC FRINGE (LFNG), in a family with autosomal recessive SCD. LFNG encodes a glycosyltransferase that modifies the Notch family of cell-surface receptors, a key step in the regulation of this signaling pathway. A missense mutation was identified in a highly conserved phenylalanine close to the active site of the enzyme. Functional analysis revealed that the mutant LFNG was not localized to the correct compartment of the cell, was unable to modulate Notch signaling in a cell-based assay, and was enzymatically inactive. This represents the first known mutation in the human LFNG gene and reinforces the hypothesis that proper regulation of the Notch signaling pathway is an absolute requirement for the correct patterning of the axial skeleton.

MARKED BILE DUCT PROLIFERATION IN MICE HETEROZYGOUS FOR LUNATIC FRINGE AND JAG1 MUTATIONS

Background: The Notch signaling pathway is involved in cell fate determination in many organs. JAG1, encoding a ligand in the Notch pathway, has been identified as the disease gene for Alagille Syndrome (AGS), characterized by bile duct paucity and other manifestations. Mice heterozygous for mutations in both the Notch2 receptor and Jag1 ligand have a phenotype similar to AGS. Notch signaling is regulated by three mammalian Fringe genes. These genes encode glycosyltransferases that modify the activation of the Notch receptor by the ligand. To examine the role of the Lunatic Fringe (Lfng) gene in modifying Jag1 ligand function, we carried out genetic analysis of double heterozygous mice (Jag1tm1Grid/+; Lfngtm1Rjo/+, abbreviated as J1LF). Methods: Liver sections from both J1LF and control mice were examined by H&E staining. Bile ducts were identified by cytokeratin staining, and blood vessels were stained with Factor VIII antibody. Mice were evaluated as newborns and 5-week old adults. Quantitative real time RT-PCR for Hes1, a downstream effector in the Notch pathway, was performed to measure Notch signaling. Results: Adult J1LF mice had a significant increase in the number of bile ducts in the portal tracts, when compared to their single heterozygous and wild type littermates. Numbers of blood vessels in portal tracts were not significantly different between J1LF and control mice. Gross examination revealed that the extrahepatic biliary tree was intact. In adult J1LF livers with bile duct proliferation, real time PCR demonstrated a 4 to 8 fold increase in Hes1 expression levels over wild type and single heterozygous controls. However, in the newborn period, J1LF mice did not display a significant difference in the numbers of bile ducts. Conclusions: Jag1/Lfng double heterozygous mice have a striking proliferation of bile ducts in the adult liver. These differences arise postnatally, pointing to a role for Lfng in regulating Jag1-Notch signaling in bile duct growth and maturation and LFNG as a potential modifier gene for AGS.

Regions of Drosophila Notch That Contribute to Ligand Binding and the Modulatory Influence of Fringe

Two glycosyltransferases that transfer sugars to epidermal growth factor (EGF) domains, OFUT1 and Fringe, regulate Notch signaling. To characterize the impact of glycosylation at the 23 consensus O-fucose sites in Drosophila Notch, we conducted deletion mapping and site-specific mutagenesis and then assayed the binding of soluble forms of Notch to cell-surface ligands. Our results support the conclusion that EGF11 and EGF12 are essential for ligand binding, but indicate that other EGF domains also make substantial contributions to ligand binding. Characterization of Notch deletion constructs and O-fucose site mutants further revealed that no single site or region can account for the influence of Fringe on Notch-ligand binding. Additionally, we observed an influence of Fringe on a Notch fragment including only 4 of its 36 EGF domains (EGF10-13). Together, our observations imply that glycosylation influences Notch-ligand interactions through a distributive mechanism that involves local interactions with multiple EGF domains and led us to suggest a structural model for how Notch interacts with its ligands.

The Mesp2 transcription factor establishes segmental borders by suppressing Notch activity

The serially segmented (metameric) structures of vertebrates are based on somites that are periodically formed during embryogenesis. A 'clock and wavefront' model has been proposed to explain the underlying mechanism of somite formation, in which the periodicity is generated by oscillation of Notch components (the clock) in the posterior pre-somitic mesoderm (PSM). This temporal periodicity is then translated into the segmental units in the 'wavefront'. The wavefront is thought to exist in the anterior PSM and progress backwards at a constant rate; however, there has been no direct evidence as to whether the levels of Notch activity really oscillate and how such oscillation is translated into a segmental pattern in the anterior PSM. Here, we have visualized endogenous levels of Notch1 activity in mice, showing that it oscillates in the posterior PSM but is arrested in the anterior PSM. Somite boundaries formed at the interface between Notch1-activated and -repressed domains. Genetic and biochemical studies indicate that this interface is generated by suppression of Notch activity by mesoderm posterior 2 (Mesp2) through induction of the lunatic fringe gene (Lfng). We propose that the oscillation of Notch activity is arrested and translated in the wavefront by Mesp2.

Lunatic Fringe, FGF, and BMP Regulate the Notch Pathway during Epithelial Morphogenesis of Teeth

Teeth develop as epithelial appendages, and their morphogenesis is regulated by epithelial–mesenchymal interactions and conserved signaling pathways common to many developmental processes. A key event during tooth morphogenesis is the transition from bud to cap stage when the epithelial bud is divided into specific compartments distinguished by morphology as well as gene expression patterns. The enamel knot, a signaling center, forms and regulates the shape and size of the tooth. Mesenchymal signals are necessary for epithelial patterning and for the formation and maintenance of the epithelial compartments. We studied the expression of Notch pathway molecules during the bud-to-cap stage transition of the developing mouse tooth. Lunatic fringe expression was restricted to the epithelium, where it formed a boundary flanking the enamel knot. The Lunatic fringe expression domains overlapped only partly with the expression of Notch1 and Notch2, which were coexpressed with Hes1. We examined the regulation of Lunatic fringe and Hes1 in cultured explants of dental epithelium. The expression of Lunatic fringe and Hes1 depended on mesenchymal signals and both were positively regulated by FGF-10. BMP-4 antagonized the stimulatory effect of FGF-10 on Lunatic fringe expression but had a synergistic effect with FGF-10 on Hes1 expression. Recombinant Lunatic fringe protein induced Hes1 expression in the dental epithelium, suggesting that Lunatic fringe can act also extracellularly. Lunatic fringe mutant mice did not reveal tooth abnormalities, and no changes were observed in the expression patterns of other Fringe genes. We conclude that Lunatic fringe may play a role in boundary formation of the enamel knot and that Notch-signaling in the dental epithelium is regulated by mesenchymal FGFs and BMP.

Zebrafish lunatic fringe demarcates segmental boundaries.

Cell interactions involving Notch signaling are required for the demarcation of tissue boundaries in both invertebrate and vertebrate development. Members of the Fringe gene family encode beta-1,3 N-acetyl-glucosaminyltransferases that function to refine the spatial localization of Notch-receptor signaling to tissue boundaries. In this paper we describe the isolation and characterization of the zebrafish (Danio rerio) homologue of the lunatic fringe gene (lfng). Zebrafish lfng is generally expressed in equivalent structures to those reported for the homologous chick and mouse genes. These sites include expression along the A-P axis of the neural tube, within the lateral plate mesoderm, in the presomitic mesoderm and the somites and in specific rhombomeres of the hindbrain; however, within these general expression domains species-specific differences in lfng expression exist. In mouse, Lfng is expressed in odd-numbered rhombomeres, whereas in zebrafish, expression occurs in even-numbered rhombomeres. In contrast to reports in both mouse and chicken embryos showing a kinematic cyclical expression of Lfng mRNA in the presomitic paraxial mesoderm, we find no evidence for a cyclic pattern of expression for the zebrafish lfng gene; instead, the zebrafish lfng is expressed in two static stripes within the presomitic mesoderm. Nevertheless, in zebrafish mutants affecting the correct formation of segment boundaries in the hindbrain and somites, lfng expression is aberrant or lost.

Gene expression profile in streptozotocin-induced diabetic mice kidneys undergoing glomerulosclerosis

To elucidate the molecular mechanism of diabetic nephropathy, a high-density DNA filter array was employed to survey the gene expression profile of streptozotocin-induced diabetic CD-1 (ICR) mouse kidneys. Ten-week-old CD-1 male mice were divided into four groups: (1) control, (2) unilaterally nephrectomized (UX) mice, (3) streptozotocin (STZ)-induced diabetic (STZ) mice, and (4) STZ mice with unilateral renal ablation (STZ-UX). Pathological changes were examined at 24 weeks after the induction. The gene expression profile was compared between the control and STZ mice by a Gene Discovery Array (GDA). The glomeruli in UX mouse kidney showed prominent glomerular hypertrophy, while the accumulation of mesangial matrix was minimal. Both STZ and STZ + UX mice had significant glomerular hypertrophy and glomerulosclerosis, and the lesions were not enhanced by renal ablation. By comparison between control and STZ mice, 16 clones that increased in expression with the induction of diabetes and 65 clones that decreased in diabetic kidneys were identified. The 37 known genes were related to glucose and lipid metabolism, ion transport, transcription factors, signaling molecules, and extracellular matrix-related molecules. The genes known to be involved in cell differentiation and organogenesis in various tissues (that is, Unc-18 homolog, POU domain transcription factor 2, lunatic fringe gene homolog, fibrous sheath component 1, Sox-17, fibulin 2, and MRJ) were found to be differentially expressed in the early phase of diabetic kidneys. Hyperglycemia is a major determinant of glomerulosclerosis in STZ-induced diabetic CD-1 mice, and the altered gene expression in the early phase of diabetic kidney may be critical for the development of diabetic nephropathy.

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.

Glycosyltransferase activity of Fringe modulates Notch–Delta interactions

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.

A role for Fringe in segment morphogenesis but not segment formation in the grasshopper, Schistocerca gregaria.

Studies of somitogenesis in vertebrates have identified a number of genes that are regulated by a periodic oscillator that patterns the pre-somitic mesoderm. One of these genes, hairy, is homologous to a Drosophila segmentation gene that also shows periodic spatial expression. This, and the periodic expression of a zebrafish homologue of hairy during somitogenesis, has suggested that insect segmentation and vertebrate somitogenesis may use similar molecular mechanisms and possibly share a common origin. In chicks and mice expression of the lunatic fringe gene also oscillates in the presomitic mesoderm. Fringe encodes an extracellular protein that regulates Notch signalling. This, and the finding that mutations in Notch or its ligands disrupt somite patterning, suggests that Notch signalling plays an important role in vertebrate somitogenesis. Although Notch signalling is not known to play a role in the formation of segments in Drosophila, we reasoned that it might do so in other insects such as the grasshopper, where segment boundaries form between cells, not between syncytial nuclei as they do in Drosophila. Here we report the cloning of a single fringe gene from the grasshopper Schistocerca. We show that it is not detectably expressed in the forming trunk segments of the embryo until after segment boundaries have formed. We conclude that fringe is not part of the mechanism that makes segments in Schistocerca. Thereafter it is expressed in a pattern which shows that it is a downstream target of the segmentation machinery and suggests that it may play a role in segment morphogenesis. Like its Drosophila counterpart, Schistocerca fringe is also expressed in the eye, in rings in the legs, and during oogenesis, in follicle cells.

A mutation in the Lunatic fringe gene suppresses the effects of a Jagged2 mutation on inner hair cell development in the cochlea.

Recent studies have demonstrated that the Notch signaling pathway regulates the differentiation of sensory hair cells in the vertebrate inner ear [1] [2] [3] [4] [5] [6] [7] [8] [9]. We have shown previously that in mice homozygous for a targeted null mutation of the Jagged2 (Jag2) gene, which encodes a Notch ligand, supernumerary hair cells differentiate in the cochlea of the inner ear [7]. Other components of the Notch pathway, including the Lunatic fringe (Lfng) gene, are also expressed during differentiation of the inner ear in mice [6] [7] [8] [9] [10]. In contrast to the Jag2 gene, which is expressed in hair cells, the Lfng gene is expressed in non-sensory supporting cells in the mouse cochlea [10]. Here we demonstrate that a mutation in the Lfng gene partially suppresses the effects of the Jag2 mutation on hair cell development. In mice homozygous for targeted mutations of both Jag2 and Lfng, the generation of supernumerary hair cells in the inner hair cell row is suppressed, while supernumerary hair cells in the outer hair cell rows are unaffected. We also demonstrate that supernumerary hair cells are generated in mice heterozygous for a Notch1 mutation. We suggest a model for the action of the Notch signaling pathway in regulating hair cell differentiation in the cochlear sensory epithelium.