Gene name - fringe
Cytological map position - 78A
Function - Secreted signalling protein
Symbol - fng
Genetic map position - 3-47
Classification - novel protein
Cellular location - secreted
|Recent literature||Ling, L., Ge, X., Li, Z., Zeng, B., Xu, J., Chen, X., Shang, P., James, A. A., Huang, Y. and Tan, A. (2015). MiR-2 family targets awd and fng to regulate wing morphogenesis in Bombyx mori. RNA Biol: [Epub ahead of print]. PubMed ID: 26037405
MicroRNAs (miRNAs) are post-transcriptional regulators that target specific mRNAs for repression and thus play key roles in many biological processes, including insect wing morphogenesis. miR-2 is an invertebrate-specific miRNA family that has been predicted in the fruit fly, Drosophila melanogaster, to be involved in regulating the Notch signaling pathway. This study shows that miR-2 plays a critical role in wing morphogenesis in the silkworm, Bombyx mori, a lepidopteran model insect. Transgenic over-expression of a miR-2 cluster using a Gal4/UAS system results in deformed adult wings, supporting the conclusion that miR-2 regulates functions essential for normal wing morphogenesis. Two genes, <abnormal wing disc (awd) and fringe (fng), which are positive regulators in Notch signaling, are identified as miR-2 targets and validated by a dual-luciferase reporter assay. The relative abundance of both awd and fng expression products was reduced significantly in transgenic animals, implicating them in the abnormal wing phenotype. Furthermore, somatic mutagenesis analysis of awd and fng using the CRISPR/Cas9 system and knock-out mutants also resulted in deformed wings similar to those observed in the miR-2 over-expression transgenic animals. The critical role of miR-2 in Bombyx wing morphogenesis may provide a potential target in future lepidopteran pest control.
| Han, H., Fan, J., Xiong, Y., Wu, W., Lu, Y., Zhang, L. and Zhao, Y. (2016). Chi and dLMO function antagonistically on Notch signaling through directly regulation of fng transcription. Sci Rep 6: 18937. PubMed ID: 26738424
Genes apterous (ap), chip (chi) and beadex (bx) play important roles in the dorsal-ventral compartmentalization in Drosophila wing discs. Meanwhile, Notch signaling is essential to the same process. It has been reported that Ap and Chi function as a tetramer to regulate Notch signaling. At the same time, dLMO (the protein product of gene bx) regulates the activity of Ap by competing its binding with Chi. However, the detailed functions of Chi and dLMO on Notch signaling and the relevant mechanisms remain largely unknown. This study reports the detailed functions of Chi and dLMO on Notch signaling. It was found that different Chi protein levels in adjacent cells activate Notch signaling mainly in the cells with higher level of Chi. Also, dLMO induces antagonistical phenotypes on Notch signaling compared to that induced by Chi. These processes depend on their direct regulation of fringe (fng) transcription.
|Harvey, B. M., Rana, N. A., Moss, H., Leonardi, J., Jafar-Nejad, H. and Haltiwanger, R. S. (2016). Mapping sites of O-glycosylation and fringe elongation on Drosophila Notch. J Biol Chem [Epub ahead of print]. PubMed ID: 27268051
Glycosylation of the Notch receptor is essential for its activity and serves as an important modulator of signaling. Three major forms of O-glycosylation are predicted to occur at consensus sites within the epidermal growth factor-like repeats in the extracellular domain of the receptor: O-fucosylation, O-glucosylation and O-GlcNAcylation. Comprehensive mass spectral analyses of these three types of O-glycosylation was performed on Drosophila Notch produced in S2 cells, and peptides containing all twenty-two predicted O-fucose sites were identified, all eighteen predicted O-glucose sites, and all eighteen putative O-GlcNAc sites. Using semi-quantitative mass spectral methods, the occupancy and relative amount of glycans at each site were evaluated. The majority of the O-fucose sites were modified to high stoichiometries. Upon expression of the beta3-N-acetylglucosaminyltransferase Fringe with Notch, varying degrees of elongation were observed beyond O-fucose monosaccharide, indicating that Fringe preferentially modifies certain sites more than others. Rumi modified O-glucose sites to high stoichiometries, although elongation of the O-glucose was site specific. Although the current putative consensus sequence for O-GlcNAcylation predicts eighteen O-GlcNAc sites on Notch, apparent O-GlcNAc modification was observed at only five sites. In addition, mass spectral analysis was performed on endogenous Notch purified from Drosophila embryos, and the glycosylation states were similar to those found on Notch from S2 cells. These data provide foundational information for future studies investigating the mechanisms of how O-glycosylation regulates Notch activity.
|Pandey, A., Harvey, B. M., Lopez, M. F., Ito, A., Haltiwanger, R. S. and Jafar-Nejad, H. (2019). Glycosylation of specific Notch EGF repeats by O-Fut1 and Fringe regulates Notch signaling in Drosophila. Cell Rep 29(7): 2054-2066. PubMed ID: 31722217
Fringe glycosyltransferases differentially modulate the binding of Notch receptors to Delta/DLL versus Serrate/Jagged ligands by adding GlcNAc to O-linked fucose on Notch epidermal growth factor-like (EGF) repeats. Although Notch has 22 O-fucosylation sites, the biologically relevant sites affecting Notch activity during animal development in vivo in the presence or absence of Fringe are not known. Using a variety of assays, this study found important roles in Drosophila Notch signaling for GlcNAc-fucose-O glycans on three sites: EGF8, EGF9, and EGF12. O-Fucose monosaccharide on EGF12 (in the absence of Fringe) is essential for Delta-mediated lateral inhibition in embryos. However, wing vein development depends on the addition of GlcNAc to EGF8 and EGF12 by Fringe, with a minor contribution from EGF9. Fringe modifications of EGF8 and EGF12 together prevent Notch from cis-inhibiting Serrate, thereby promoting normal wing margin formation. This work shows the combinatorial and context-dependent roles of GlcNAc-fucose-O glycans on these sites in Drosophila Notch-ligand interactions.
One might reasonably expect the edge of the Drosophila wing to be analogous to the edge of a piece of paper, a simple cessation of structure. The reality is more complex; a better analogy would be the edge of a continent in which grand and complex events, like plate tectonics, shape and define geophysical boundaries and structure. The edge of the wing serves as a boundary between dorsal and ventral structures, and is shaped by complex boundary phenomenon. Fringe plays a critical role in boundary formation in the developing wing. This critical role bespeaks the complexity of the edge, and exemplifies the importance of boundaries in developmental processes. To begin to understand fringe, is to begin to understand many important principles of developmental biology.
Clones of cells with mutated fringe, on a background of unmutated cells, form ectopic ectopic wing margins along the edge. How do fringe clones function to produce ectopic margins? As with most (if not all) genes, they are best understood as they relate to others: for fringe, one of the genes to examine carefully is apterous. The pattern of apterous expression in the wing disc is established by wingless as an early event in disc development. apterous expression is confined to the dorsal surface of the disc as a result of expression of wingless in the ventral domain (Williams, 1993). As the basis of the early apterous-wingless interaction is unknown, it is not known whether the wingless effects on apterous are direct.
apterous expression in the dorsal domain of the wing disc is sufficient to account for fringe expression, which overlaps that of apterous. In fact apterous has a central role as a "selector" gene, establishing the identity of cells in the dorsal compartment, and regulates such genes as integrins which are responsible for the differential adhesive properties of cells in opposing compartments (Lawrence, 1996). A further discussion of the role of apterous as a selector gene is found in the segment polarity section.
Since fringe expression is not limited to the edge of the wing, how is it that its effects are felt only at the edge? The answer to this question comes from a study of the effects of mutant apterous clones on wing morphology. Clones of cells mutant for apterous in the dorsal region of the wing disc form ectopic wing margins along clone borders, a phenotype resembling clones of cells mutant for fringe. Clone borders (juxtaposing cells expressing apterous and not expressing apterous) simulate the edge of the wing. The ectopic wing margins are composed of both wild type and mutant cells, with the clone boundary always splitting the middle of the ectopic margin. Such clone boundaries should also create a boundary of fringe expression, since Apterous regulates fringe expression. It is concluded that the juxtaposition of cells possessing and lacking fng expression can induce wing margin formation, and implies the existence of a signaling process between such cells (Irvine, 1994).
Signaling at the boundary between fringe-expressing cells and fringe-non-expressing cells induces the expression of Serrate in fringe-expressing cells. Serrate is an alternative ligand of the receptor Notch. Notch expression is confined to ventral cells, but the interaction of Serrate ligand in dorsal cells and Notch in ventral cells induces the expression of wingless at the margin (Kim, 1995). In addition, the activation of vestigial in a narrow band of cells centered on the DV boundary is one of the first signs of wing formation. vestigial has an intronic enhancer, activated by the juxtaposition of fringe-expressing and fringe minus cells at the margin of the dorsal and ventral compartments. It is presumed that vestigial is a target of Notch or of wingless at the dorsoventral boundary (Kim, 1995 and Williams, 1994).
Fringe has been shown to modulate Notch signaling. 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. The relationship between these genes has been investigated by a combination of expression and coexpression studies in the Drosophila wing. Ser is normally expressed in dorsal cells while Dl is initially expressed by all wing cells. However, their expression soon becomes restricted to the dorso-ventral boundary. In order to study Ser and Dl signaling between dorsal and ventral cells, Dl and Ser were expressed ectopically along the anterior side of the anterior-posterior compartment boundary. These experiments confirm that Ser and Dl induce and maintain each other's expression by a positive feedback loop. Importantly, their ability to induce each others expression is dorsal-ventrally asymmetric, because Ser induces Dl strongly in ventral cells, but only very weakly in dorsal cells, whereas Dl induces Ser expression in dorsal cells, but not in ventral cells. 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 (Panin, 1997).
To determine the effects of Fringe protein on Ser and Dl activity, margin gene expression and margin bristle formation were asayed while coexpressing these proteins in ventral cells. Ectopic expression of Ser leads to ectopic wing-margin gene expression and adult wing-margin formation in ventral cells along the edges of the ectopic Ser stripe. Misexpression of Fng in ventral cells inhibits these effects of Ser activity, demonstrating that Fng can inhibit Ser signaling. Misexpression of Dl induces ectopic wing-margin formation and Ser expression in dorsal cells but not in ventral cells. However, when Fng is misexpressed in ventral cells, Dl induces Ser expression and margin formation in both dorsal and ventral cells. Thus Fng potentiates Dl signaling, allowing ventral cells to respond to Dl just as dorsal cells normally do. Experiments show that Fng inhibits Ser activity only when it is expressd in receiving cells, and not when expression is restricted to Ser-signaling cells. Activated Notch can induce both Ser and Dl, and activated Notch has similar effects on dorsal and ventral cells, implying that Fng exerts its effects upstream of Notch activation. Because Fng is extracellular, this implies that activity of cell-associated Fng protein differentially modulates the binding and/or activation of Notch by its two ligands (Panin, 1997).
Fringe has been proposed to execute its boundary determining function by inhibiting the Notch response to Ser and potentiating the Notch response to Dl. Because Fng does not bind Ser or Dl, modulation of Notch signaling by Fng is directly mediated by the complex formation of Fng and Notch during their secretory transits. Notch bound to Fng may have preferential affinity or sensitivity to Delta, whereas free Notch may have a higher affinity or sensitivity to Ser. Upon Dl binding to the Fng-Notch complex, Fng may also antagonize the repressor function of the Lin-Notch repeats and facilitate the activation of Notch signaling (Ju, 2000).
Notch activation at the midline plays an essential role both in promoting the growth of the eye primordia and in regulating eye patterning (see Specification of the eye disc primordium and establishment of dorsal/ventral asymmetry). Specialized cells are established along the dorsal-ventral midline of the developing eye by Notch-mediated signaling between dorsal and ventral cells. D-V signaling in the eye shares many similarites with D-V signaling in the wing. In both cases an initial asymmetry is set up by Wingless expression. Both eye and wing cells then go through a distinct intermediate step: in the wing, Wingless represses the expression of Apterous, a positive regulator of fringe (fng) expression; in the eye, Wingless promotes the expression of mirror (mrr), which encodes a negative regulator of fringe (unpublished observations of McNeill, Chasen, Papayannopoulos, Irvine, and Simon, cited by Papayannopoulos, 1998). Both wing and eye cells share a Fng-Ser-Dl-Notch signaling cassette to effect signaling between dorsal and ventral cells and establish Notch activation along the D-V midline. Local activation of Notch leads to production of diffusible, long-range signals that direct growth and patterning, which in the wing include Wingless, but in the eye remain unknown. At least one downstream target of D-V midline signaling, four jointed (fj), is also conserved. four jointed is also expressed in the wing and its expression there is indirectly influenced by Notch (Papayannopoulos, 1998 and references).
During early eye development, fringe is expressed by ventral cells. This expression appears to be complementary to that of the dorsally expressed gene mrr. During early to mid-third instar, additional expression of fng appears in the posterior of the eye disc. This line of posterior fng expression is just in front of the morphogenetic furrow and moves across the eye ahead of the furrow. In the wing disc, Dl and Ser induce each other's expression, and become up-regulated along the D-V border where they can productively signal. Dl and Ser are also preferentially expressed along the D-V midline during eye development. Ser expression, like fng expression, is complementary to that of mrr, whereas Dl expression partially overlaps that of mrr. The spatial relations among fng, Ser, and Dl expression in the eye are thus similar to those in the wing, although in the wing, their expressions are inverted with respect to the D-V axis (Papayannopoulos, 1998).
The four-jointed gene is expressed in a gradient during early eye development, with a peak of expression along the D-V midline. Together with Ser and Dl, Fj serves as a molecular marker of midline fate. Ubiquitous expression of Fng during early eye development, generated by placing fng under the control of an eyeless enhancer, eliminates detectable expression of Ser and Dl along the midline. Conversely, misexpression of Fng in clones of cells, can result in ectopic expression of Ser and fj that is centered along novel borders of Fng expression in the dorsal eye. Ectopic Ser and fj expression can also be detected along the borders of fng mutant clones in the ventral eye. These observations show that Fng expression borders play an essential and instructive role in establishing a distinct group of cells along the D-V midline of the developing eye. Animals with reduced fng activity have small eyes. Moreover, ubiquitous fng expression also results in a dramatic loss of tissue. Tissue loss is detectable in the developing imaginal disc, before the morphogenetic furrow moves across the eye. Moreover, eye loss is observed when fng is ectopically expressed during early development, but not when fng is ectopically expressed behind the furrow. These observations indictate that a Fng expression border is required for eye growth, specifically during early eye development (Papayannopoulos, 1998).
Fng differentially modulates the action of Notch ligands in the eye just as it does in the wing. Clones of cells ectopically expressing Dl can induce Ser expression in ventral, Fng-expressing cells, but not in dorsal cells. Fng alone can induce Ser expression in dorsal cells, but only near the D-V midline. When Fng and Dl are co-misexpressed, Ser expression can be induced in dorsal cells even when the clones are far from the D-V midline. Clones of cells ectopically expressing Ser are able to induce increased expression of Dl in dorsal cells but not in ventral, Fng expressing cells. However, if Ser is ectopicallly expressed in fng mutant animals, it can induce Dl expression in ventral cells (Papayannopoulos, 1998).
Notch function is also necessary for normal D-V midline cell fate. The ability of Ser and Dl to induce one another's expression indicates that the expression of either one is a marker for Notch activation in the eye. Analysis of loss-of-function mutants of Notch and its ligands, as well as ectopic expression studies, indicate that Notch activation also regulates eye growth. Several observations indicate that the D-V midline is the focus of Notch activation required for growth. Moreover, the midline corresponds to a fng expression border, which is essential for growth and modulates Notch signaling during early eye development. Because local activation of Notch has long-range effects on growth and four-jointed expression, it is inferred that Notch induces the expression of a diffusible growth factor at the midline. Notch activation influences ommatidial chirality. fng mutant clone borders within the ventral eye can be associated with reversals of ommatidial chirality, whereas mutant clones that cross the D-V midline disrupt the normal equator. The equatorial bias in the influence of ectopic Notch activation implies that the equator is the normal source of a Notch-dependent, chirality-determining signal (Papayannopoulos, 1998).
In the mammalian CNS, glial cells expressing excitatory amino acid transporters (EAATs) tightly regulate extracellular glutamate levels to control neurotransmission and protect neurons from excitotoxic damage. Dysregulated EAAT expression is associated with several CNS pathologies in humans, yet mechanisms of EAAT regulation and the importance of glutamate transport for CNS development and function in vivo remain incompletely understood. Drosophila is an advanced genetic model with only a single high-affinity glutamate transporter termed Eaat1. Eaat1 expression in CNS glia was found to be regulated by the glycosyltransferase Fringe, which promotes neuron-to-glia signaling through the Delta-Notch ligand-receptor pair during embryogenesis. Eaat1 loss-of-function mutations were made and it was found that homozygous larvae could not perform the rhythmic peristaltic contractions required for crawling. No evidence was found for excitotoxic cell death or overt defects in the development of neurons and glia, and the crawling defect could be induced by postembryonic inactivation of Eaat1. Eaat1 fully rescued locomotor activity when expressed in only a limited subpopulation of glial cells situated near potential glutamatergic synapses within the CNS neuropil. Eaat1 mutants had deficits in the frequency, amplitude, and kinetics of synaptic currents in motor neurons whose rhythmic patterns of activity may be regulated by glutamatergic neurotransmission among premotor interneurons; similar results were seen with pharmacological manipulations of glutamate transport. These findings indicate that Eaat1 expression is promoted by Fringe-mediated neuron-glial communication during development and suggest that Eaat1 plays an essential role in regulating CNS neural circuits that control locomotion in Drosophila (Stacey, 2010).
Eaat1 expression in embryogenesis is shown in this study to be regulated by the glycosyltransferase Fringe (Fng), which has been shown to promote neuron-to-glia signaling through the Delta-Notch ligand-receptor pair. Eaat1 loss-of-function mutations were generated, and mutant larvae were found have severe defects of locomotion. The electrophysiological and genetic approaches provide evidence that Eaat1 acts in a limited subpopulation of CNS glial cells to influence glutamatergic neurotransmission controlling the rhythmic patterning of motor neuron activity. Thus, this study has identified cellular and molecular interactions during development that affect the emergence of a functionally distinct glial subtype capable of influencing glutamatergic neurotransmission in the CNS, and a discovered an essential role for the Eaat1 glial glutamate transporter in locomotor behavior (Stacey, 2010).
The major nerve tracts of the Drosophila ventral nerve cord (VNC), called commissures and longitudinal connectives, mark a dense neuropil of axon projections, dendrites, and synapses within the segmentally repeated embryonic and larval CNS. A subset of CNS glial cells expresses the gene CG31235, including the nine longitudinal glia (LG) found in each VNC hemi-segment. LG lie just dorsal to the longitudinal connectives and ensheath the neuropil. In building genetic tools to study these glia in vivo, it was found that a 3 kb promoter/enhancer of CG31235 can direct the expression of Gal4 or nuclear GFP (nGFP) transgenes to the nine LG, plus five additional glial cells in each VNC hemi-segment. in situ hybridization was used to examine the expression of Eaat1 transcripts in the VNC of CG31235-nGFP animals and it was noted that Eaat1 was expressed in glial cells, including a subset of LG. Onset of Eaat1 transcript expression occurred rather late in embryogenesis (stages 15-16), and only narrowly precedes the initiation of spontaneous and uncoordinated muscle contractions (Stacey, 2010).
Using Eaat1-Gal4 to mark Eaat1-expressing cells in the VNC, it was found that virtually all of them also expressed CG31235-nGFP. The nine LG in each hemi-segment can be subdivided further because the anterior-most six of these cells express the transcrtiption factor Prospero (Pros). It was found that 84% (173/205) of Eaat1-Gal4 cells are also Pros positive, indicating that a large majority of Eaat1-expressing cells are of the anterior LG subtype. This subtype also expresses Glutamine synthetase 2 (Gs2). Glutamine synthetases convert glutamate to glutamine, which is synaptically inert and can be safely recycled back to neurons. Coexpression of Gs2 and Eaat1 in the anterior LG strongly suggests that this subtype of glial cell is well equipped for the uptake and metabolism of glutamate from CNS synapses in Drosophila, and could potentially modulate glutamatergic neurotransmission. Consistent with this, the presynaptic vesicular glutamate transporter VGlut, and the postsynaptic glutamate receptor KaiRIA (GluR-IID) are both expressed in the dorsal neuropil of the VNC of embryos and larvae, near the cell bodies of LG. To determine whether Eaat1-expressing LG infiltrate the neuropil and express Eaat1 near putative glutamatergic synapses in first instar (L1) larvae, Eaat1-Gal4 was used to drive expression of an Eaat1::GFP fusion protein (UAS-Eaat1::GFP), and colabelling was performed with either the membrane-targeted reporter mCD8-red fluorescent protein (RFP) or anti-VGlut to mark potential sites of glutamatergic presynaptic terminals in first instar larvae. Eaat1::GFP was broadly expressed among the RFP-labeled glial membranes and, relative to RFP, appeared to be enriched in glial membranes that had infiltrated the CNS neuropil. VGlut-positive puncta were located dorsally within the VNC neuropil, similar to the pattern observed previously in third instar larvae. Optical sections through the neuropil revealed extensive Eaat1::GFP labeling in close proximity to VGlut-positive puncta, consistent with the idea that glutamatergic transmission at CNS synapses in Drosophila could be influenced by the Pros-positive anterior LG subtype that express both the glutamate transporter Eaat1 and the glutamine synthetase Gs2 (Stacey, 2010).
This study found that the requirement for Eaat1 in locomotor behavior is limited to a subpopulation of glia marked by the CNS-specific driver CG31235-Gal4. At present, the tools available cannot distinguish the relative importance of glial cells located in the VNC versus the brain lobes. Nonetheless, Eaat1 is expressed in a limited subset of neuropil-associated glia in the VNC, including the anterior LG subtype, where it is coexpressed with the glutamate recycling enzyme Gs2 and its expression is regulated by the glycosyltransferase Fng. Fng sensitizes the Notch receptor on the anterior LG to stimulation from developing axons bearing the Delta ligand and thereby promotes neuron-to-glial signaling during embryogenesis. Anterior and posterior LG are derived from a common glioblast, and so, as a consequence of this interplay between neurons and glia, Fng provides a mechanism for the selective expression of Eaat1 in the anterior LG subtype. Thus, Fng promotes the emergence of a functionally distinct glial cell subtype that can take up glutamate and has the potential to modulate neurotransmission at central synapses (Stacey, 2010).
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, the function of Fng, Ser and Dl was analyzed during wing development at an ectopic FB and at the D/V boundary. Notch signalling was found to be 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, the 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 (Troost, 2012).
During wing development, the activity of the Notch pathway is required to establish a stripe-like domain of expression of several genes along the D/V boundary that control wing growth and patterning, chief among them are wg and vg. The D/V boundary is a FB, which provides an interface that is crucial for the activation of Notch and establishment of this organising centre. The current understanding is that Fng promotes Dl signalling and prevents Ser signalling through the modification of Notch. As a result, Dl signals strongly from ventral to dorsal and Ser from dorsal to ventral cells boundary cells. The simultaneous signalling of the ligands in opposite direction establishes the expression of Notch target genes at both sides of the FB. It is essential for this model to work at the D/V boundary that induction of expression of Ser is restricted to dorsal and that of Dl to ventral cells. If e.g. Dl expression could also be induced in dorsal cells by the Notch pathway, the activity of Notch would immediately spread throughout the dorsal half of the wing anlage. In agreement with this requirement, it has been observed that expression of Ser is restricted to dorsal cells upon expression of activated Notch in dorsal and ventral cells. The combination of spatially restricted expression of the ligands and the Dl/Ser loop restricts the activation of Notch to the D/V boundary during early stages of wing development. At the middle of the third larval instar stage the Dl/Ser/Wg loop takes over to maintain the activity of Notch. Thus, a critical step is the establishment of expression of Wg in boundary cells. Once this is achieved the second feedback-loop assures expression of Wg and Notch signalling throughout wing development (Troost, 2012).
While this model can explain the events at the D/V boundary, it cannot explain the events at an ectopic FB, since differential expression of the ligands is unlikely to occur there. Nevertheless, the expression of Wg is also restricted at the ectopic FB. The presented work provides further evidence for the current model of Fng action, but adds new details that enable it to explain also the events at an ectopic FB. One addition is the initial sequential establishment of the expression domain of Wg through asymmetric Notch signalling during early stages of wing development. Asymmetric expression of Wg was observed only in ventral boundary cells (VBCs) at the D/V boundary, indicating that these cells achieve sufficiently high activation of Notch to initiate expression of Wg. The existence of the early asymmetric phase of Notch activation was surprising given the fact that activation of Notch results in the activation of the expression of Dl and Ser. Consequently, the activation of Notch in ventral cells should immediately lead to up-regulation of expression of Dl in ventral cells and back-signalling to dorsal cells. It was therefore expected that if an asymmetric phase exists, it would be too short in time to be detected. Importantly, the initial asymmetric phase appears to be a general property for Notch signalling at a FB, since it was observed on both analysed FBs. The existence of the asymmetric phase also indicates that a FB can be used to generate activity domains of the Notch pathway where the feedback-loop that regulates the expression of the ligands through Notch activation does not occur. So far the loop has been found only in the wing pouch. In the absence of the loop the asymmetric state would remain and thus, a defined stripe of high Notch activation would be generated in a field of cells that uniformly express Dl even in the absence of Ser (Troost, 2012).
At the ectopic FB, it was found that eliminating the activity of the Notch pathway in PBCs does not result in the loss of expression of Wg in ABCs. It only prevents the late symmetric phase. This indicates that establishment of a Dl/Ser feedback loop in A/P boundary cells is not essential for reaching sufficient high levels of Notch signalling to induce Wg expression during the asymmetric phase. The same was observed for the D/V boundary: Depletion of Su(H) function or over-expression of H causes a restriction of expression of Wg to VBCs, but not its abolishment. Thus, the Dl/Ser loop is probably mainly required for the later occurring patterning of the future wing margin, but not for the establishment of the wing primordium (Troost, 2012).
A further addition is that the basic expression of Dl is independent of Ser signalling. This is indicated by the observation that 1) Dl is expressed throughout the wing anlage in early discs and 2) Dl signals to DBCs at the D/V boundary in absence of Ser function. This holds true also for the ectopic FB: here Dl signals to the anterior boundary cells (ABCs) in the absence of Ser. However, in both cases Dl signalling is not strong enough to initiate Wg expression, which is a crucial event for wing development (Troost, 2012).
The results also reveal an unanticipated requirement of Ser in ABCs for the expression of Wg. This requirement could be explained through Ser signalling from the ABCs to PBCs to up-regulate Dl there, which in turn signals back to ABCs (Ser/Dl loop). This explanation would imply that the levels of Notch activation required for the induction of Dl expression are lower than that for Wg. Otherwise, the expression of Wg would not stay asymmetric as it is observed. However, it was found that Notch signalling is not required in PBCs during the early asymmetric phase. This excludes the mentioned explanation and suggests that Ser must activate Notch in the Fng expressing ABCs. This assumedly weak activation contributes to the total activity of Notch in these cells and guarantees levels of Notch signalling above the threshold required for expression of Wg. In the absence of Ser the activation by Dl from PBCs appears to fail to reach the threshold level in a fraction of discs. In agreement with this notion it has been shown that expression of Ser is broadly induced upon ectopic expression of Fng (Troost, 2012).
It was further observed that concomitant loss of Ser and Dl function in ABCs always abolishes the expression of Wg at the ectopic FB. Hence, Dl must also signal in Fng expressing ABCs and contribute to Notch signalling in these cells. The results suggest that the total amount of Notch activity in ABCs is the sum of signalling by Dl and Ser in the Fng domain and Dl signalling from PBCs to ABCs. Whereby the signal from posteriors to anterior is the more important one, since its loss always abolishes expression of Wg. A similar requirement at on both sides of the boundary for Dl had been described for the D/V boundary. Interestingly, the data indicate that Dl plays a similar role there as Ser at the ectopic FB (Troost, 2012).
Another addition to the current model is that Fng has two antagonistic effects on each ligand. The modification of Notch by Fringe is known to polarise signalling at the D/V-boundary by enhancing Dl- and suppressing Ser-signalling. Loss of function of fng during early stages of wing development abolishes expression of Wg at a time where it is solely dependent on Ser signalling. This observation indicates that Fng has a positive effect on Ser signalling in addition to its known negative one: it enhances Ser-signalling from Fng expressing dorsal to non-expressing ventral cells. This enhancement is required to activate expression of Wg in cells across the boundary. This finding is in good agreement with previous work that reports that Fng can enhance the ability of Ser to induce ectopic wing margins upon their co-overexpression. It is a possibility that this positive influence on Ser is indirect: The modification of Notch mediated by Fng results in a decrease of binding Ser to Notch. As a consequence high levels of free Ser are available in Fng expressing boundary cells at the FB that can bind in trans to the unmodified Notch on the adjacent Fng non-expressing boundary cells. At the analysed ectopic FB, it was found that Dl is required in Fng non-expressing PBCs to raise the activity of Notch signalling to a level that is sufficient for expression of Wg in Fng expressing anterior boundary cells, although Dl is expressed ubiquitously in early discs and thus, present in both cell populations at the same levels. This finding indicates that although Dl can induce activity of Notch in the Fng domain, this activity is insufficient to initiate the expression of Wg. The activity rises beyond the threshold only if the cells receive an additional Fng-enhanced Dl signal from non-expressing. This suggests that Fng has a suppressing effect on Dl signalling within its domain. The opposing effects on the activity of each ligand have important implications for restricted Notch-signalling at the FB. Restricted expression of the ligands on opposite sides of the FB is not required to restrict Notch activation to the FB. Only Dl outside the Fng domain is sufficiently active to induce Wg expression in Fng expressing cells. In turn only Ser in Fng expressing cells is sufficiently active to induce Wg expression in Fng non-expressing cells. These conditions are only met at the FB. These properties assure that expression of Wg is restricted to a FB even in a tissue where Dl or Ser are initially expressed uniformly, as it is the case for the ectopic FB (Troost, 2012).
A difference between the ectopic FB and the D/V boundary is that the roles of the ligands are reversed: at the ectopic FB, Dl signalling is required for the establishment of the initial asymmetric phase and Ser to establish the symmetric phase and to maintain expression of Notch activity at a high level. In contrast, Ser signalling is required for the initial asymmetric phase at the D/V boundary and Dl for the establishment of the later symmetric phase and probably maintenance during later stages. Thus, activation of Notch-signalling at a FB can be initiated by both ligands. Another difference is the location of the asymmetric stripe of wg expression, which is located in Fng expressing cells at the ectopic FB, but in non-expressing cells at the DV boundary. It is believed that the events observed at the ectopic FB represents the more general mode of interactions, since the interactions occur solely in the ventral pouch where the cells differ mainly with respect to the expression of Fng. In contrast, at the D/V boundary dorsal cells differ from ventral cells by expression the selector Ap, which might modify the outcome of the interactions (Troost, 2012).
On the basis of these results, the events at the ectopic boundary can be summarized. During early stages of wing development, Dl is ubiquitously expressed throughout the wing anlage. Ectopic expression of fng with ptcGal4, creates a band-like Fng domain in the ventral pouch. During early stages Dl and Ser (probably induced by Dl) activate Notch signalling throughout the Fng domain at low levels that are not sufficient for activation of Wg. At the sharp posterior FB, Dl signalling from PBCs to ABCs, enhanced by Fng, raises the levels of Notch in ABCs beyond the threshold required for expression of Wg. The asymmetric phase is established. Dl signalling also up-regulates expression of Ser in ABCs. Over time Ser signalling from ABCs to PBCs (enhanced by Fng), induces Wg expression in PBCs. After the solid induction of symmetric expression of Wg, the Dl/Ser/Wg loop takes over and maintains Notch signalling at the D/V boundary. It was observed that the expression of Gbe+Su(H)-lacZ is broader upon ectopic expression of Fng in Ser mutant early third instar discs. Moreover, Wg is ectopically expressed throughout the ventral ptc domain upon ectopic expression of Fng by with ptcGal4 in Ser H, but not in H mutant discs. Thus, Ser probably contributes to keeping the Notch activity in the Fng domain at low level. It is known that the expression of Ser can contribute to the suppression of Notch activity in a cell-autonomous manner through cis-inhibition. Cis-inhibition has been discovered during analysis of wing development and appears to be involved in regulation and directional Notch signalling in several processes. This mechanism causes strong Ser signalling only from Ser expressing to non-expressing cells. Since Ser expression is induced in the Fng domain, strong signalling occurs from ABCs to PBCs. Thus, it is likely that cis-inhibition contributes to the directional signalling of from ABCs to PBCs to induce the later symmetric phase (Troost, 2012).
At the D/V boundary signalling is initiated differently. This study has shown that loss of the activity of the Notch pathway in dorsal cells does not prevent the establishment of expression of Wg along the D/V boundary, but restricts it to VBCs and allows wing development to proceed. Thus, the dorsal to ventral signal, which is mediated by Ser is most important. This notion is also in agreement with the null phenotype of Ser. Ser and Fng are initially expressed in all dorsal cells. Fng enhances Ser signalling to VBCs, but suppresses signalling among dorsal cells. This strong polarised signalling results in the activation of Wg expression and up-regulation of Dl expression in VBCs. It is likely, that the cis-inhibitory effect of Ser contributes to the suppression of the activation of Notch in dorsal cells through the initial phase of wing development, since the expansion of the expression of Gbe+Su(H)–lacZ over the whole dorsal wing anlage in early Ser mutant discs. This is probably induced by the weak ubiquitous expression of Dl that was observed in early wing discs. The analysis of the Ser null and Ser H double mutants indicates that Dl can activate Notch signalling in absence of Ser function, but not strong enough to induce Wg expression, despite the presence of the FB. It appears that at the D/V boundary, Ser has to up-regulate the expression of Dl in VBCs over time beyond the threshold that is required to induce Wg expression in DBCs. Fng contributes to induction of Wg expression by enhancing Dl signalling from Fng non-expressing VBCs to expressing DBCs. The requirement for accumulation of Dl could contribute to the observed delay of the establishment of the symmetric phase of expression. After the initial asymmetric phase, the Ser/Dl/Wg loop is established to maintain Notch signalling and symmetric expression of Wg. These results indicate that the initial Fng enhanced Ser signal is sufficiently strong to induce Wg in VBCs in a manner that enables also the establishment of the Ser/Dl/Wg loop. This is indicated by the observation that suppression of Notch activity in all dorsal cells throughout wing development does not prevent maintenance of expression of Wg in late stages of the third instar and even allows the development of adult wings with only minor patterning problems. Thus, the later signal from VBCs to DBCs is mainly required for positioning and patterning of the wing margin (Troost, 2012).
Asymmetric signalling through the Hh pathway has been shown to establish the organising centre for the A/P axis at the anterior side of the boundary. It has been assumed that one difference in the establishment of the D/V organising centre is its symmetric placement on the D/V boundary. The current results suggest that at least during initial phases, the signalling at the D/V boundary is also asymmetric and that the established organising centre can also work if it is displaced ventrally. Thus, it appears that the signalling events at both boundaries are more similar than previously anticipated (Troost, 2012).
cDNA length - 1.8-1.9 kb
The novel protein includes an N-terminal signal sequence but lacks a transmembrane domain, suggesting that it is secreted. The protein contains seven cysteine residues, three dibasic sites and two potential N-glycosylation sites. The dibasic sites could function as cleavage sites, suggestion that FNG could be proteolytically processed from an inactive into an active form (Irvine, 1994).
Fringe, a secreted protein involved in boundary formation, and Braniac, required for proper contact or adhesion between germline and follicle cells, may both be part of a large family of glycosyltransferases. BRN and FNG share several features: 1) they are developmentally regulated, secreted signaling molecules without known receptors, 2) they are required during epithelial patterning, 3) they interact genetically with the Notch and/or EGF receptor pathways, suggesting that they might modify the signaling mediated by these receptors, and 4) FNG and BRN both have at least two C. elegans homologs and several vertebrate homologs as well, suggesting the presence of multigene families. FNG and BRN show conserved sequence regions homologous with Haemophilus influenzae Lex1, essential for the biosynthesis of parasitic bacterium's extracellular lipooligosaccharides (LOS). Lex1 is a galactosyltransferase, adding galactose to glucose or N-acetylglucosamine residues of LOS. Secreted glycosyltransferases may use their ability to recognize specific carbohydrate moieties on cell surface molecules to trigger particular receptors; they might also play a crucial role in epithelial pattern formation by modifying these carbohydrate moieties at particular locations recognizable by various carbohydrate-binding domains of extracellular proteins. The carbohydrate status of the cell during development might even be a function of neighboring cells and not only of its own expression set of glycosyltransferases (Yuan, 1997).
date revised: 11 December 98
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