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.
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).
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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