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Gene names - echinoid & friend of echinoid
Synonyms - Cytological map positions - 24D4--6 Function - Transmembrane proteins Keywords - cell adhesion, Egf receptor pathway, Notch pathway |
Symbols - ed & fred FlyBase IDs: FBgn0000547 & FBgn0051774 Genetic map positions - 2-11.0 Classification - Ig and fibronectin domains protein Cellular location - surface |
echinoid (ed) encodes a cell-adhesion molecule (CAM) that contains immunoglobulin domains and regulates the Egfr signaling pathway during Drosophila eye development (Bai, 2001). Genetic mosaic and epistatic analysis, has suggested that Ed, via homotypic interactions, activates a novel, as yet unknown pathway that antagonizes Egfr signaling (Bai, 2001). Alternatively, later studies indicate that Ed inhibits Egfr through direct interactions (Rawlins, 2003; Spencer, 2003). Another body of work suggests that Ed functions as a homophilic adhesion molecule, and also engages in a heterophilic trans-interaction with Drosophila Neuroglian (Nrg), an L1-type CAM. Co-expression of ed and nrg in the eye exhibits a strong genetic synergy in inhibiting Egfr signaling. This synergistic effect requires the intracellular domain of Ed, but not that of Nrg (Islam, 2003). A model for this interaction suggest that Nrg acts as a heterophilic ligand and activator of Ed, which in turn antagonizes Egfr signaling (Islam, 2003).
Complicating the picture even further is an analysis of a paralogue of Ed termed friend of echinoid (fred). ed and fred transcription units are adjacent to one another, approximately 100 kilobases apart on chromosome arm 2L, but they are divergently transcribed in opposite directions. Fred acts in close concert with the Notch signaling pathway. Suppression of fred function results in specification of ectopic SOPs in the wing disc and a rough eye phenotype. Overexpression of N, Su(H), and E(spl)m7 suppresses the fred RNAi phenotypes. Accordingly, decreasing Su(H) or overexpression of Hairless enhances the fred RNAi phenotypes. Thus fred, a paralogue of ed, shows close genetic interaction with the Notch signaling pathway. The weak genetic interaction observed between fred and components of the Egfr pathway also links fred to the Egfr pathway; however, analysis of additional components of the Egfr pathway are necessary to determine Fred's role in the Egfr signaling (Chandra, 2003).
This overview of Ed function will first summarize the role of Ed in antagonizing the Egfr pathway through direct interaction with Egfr receptor and will then treat Ed antagonism of the Egfr pathway through the engagement of Neurogenin.
Echinoid is required to downregulate Egfr activity in the developing Drosophila eye, ensuring a normal array of R8 photoreceptor neurons. Echinoid is an L1-type transmembrane molecule that is expressed in all cells of the eye imaginal discs and, unlike many other Egfr inhibitors, does not appear to be regulated transcriptionally. Echinoid co-precipitates with Egfr from cultured cells and eye imaginal discs, and Egfr activity promotes tyrosine phosphorylation of Echinoid. These observations suggest that Echinoid inhibits Egfr through direct interactions (Spencer, 2003; Rawlins, 2003).
Egfr signaling is essential for the correct patterning and specification of all cell types in the Drosophila eye. Loss of echinoid leads to stabilization of Egfr signaling and Rolled ERKA MAP kinase phosphorylation. Activation of ERKa is closely correlated with expression of the R8 specification factor Atonal, and echinoid mutants show commensurate stabilization of Atonal expression, resulting in the formation of multiple R8 cells in many ommatidia. Mutations in echinoid and Egfr show strong mutual genetic interactions, suggesting that they influence R8 differentiation through a common pathway. Consistent with this view, Echinoid and Egfr are found to co-precipitate from cultured cells, and Echinoid is found to be phosphorylated in response to Egfr signaling in vivo. These data suggest that Echinoid is required to downregulate Egfr signaling after a period of activation in order to limit the number of R8 cells, and may do so through direct interactions (Spencer, 2003).
R8 patterning reflects at least two processes: spacing of emerging R8 equivalence groups and selection from these groups of single R8 cells. It has been suggested that expression of Egfr inhibitors is important for setting the spacing between R8 cells, a view supported by mispatterning in loss-of-function Egfr clones. It is found, however, that echinoid plays no role in this process: while loss of echinoid does increase the duration of Egfr signaling, it does not affect the initial pattern of Egfr activity or the position of R8 equivalence groups within the morphogenetic furrow. Rather, Echinoid appears to be essential only for the second step in R8 specification, the selection of a single R8 cell from the 2-3 cell equivalence group. The role of Echinoid is to ensure that Egfr activity is downregulated within the group in a timely fashion; persistent Egfr activation appears to trigger all cells of the equivalence group to differentiate as R8s. Consistent with this, expression of an activated-Ras, activated-Raf or Pointed-P1 (Rawlins, 2003) promotes multiple R8 cells within individual ommatidia (Spencer, 2003).
Interestingly, Echinoid is the second example of a co-factor required for fine-tuning a major signaling pathway during R8 selection. Selection of R8 from the equivalence group also requires scabrous, a modifier of Notch signaling. Egfr and Notch signaling are used in a number of developing tissues. Echinoid and Scabrous appear to fill the need for high precision during resolution of the R8 equivalence group; this precision is almost unique in the developing nervous system. Therefore, Echinoid and Scabrous appear to have evolved to fine-tune these two pathways for the stringent requirements of the retina. It is anticipated that other factors might provide similar fine-tuning to Egfr and Notch signaling in other tissues (Spencer, 2003).
In an echinoid null allele, only 54% of ommatidia contain multiple R8s (fewer by Boss staining), suggesting that another factor may be acting redundantly to downregulate Egfr signaling in some cells. One candidate for a redundant factor is a highly homologous gene distal to echinoid on the second chromosome. Preliminary data indicates that this gene, fred (friend of echinoid), is expressed in the same tissues as echinoid and displays similar interactions with EgfrEllipse. Further examination of the fred phenotype and creation of fred;ed lines will be necessary to determine if fred acts in a manner similar to echinoid (Spencer, 2003).
In its extracellular domain, Echinoid appears similar to other members of the L1 family of proteins: it undergoes homophilic binding and ectodomain shedding, presumably to regulate cell-cell adhesion. Although some L1 cell adhesion proteins have been shown to interact with receptor tyrosine kinases such as Egfr, those that have been described to date lead to activation, not inhibition, of MAP kinase phosphorylation. In addition, Echinoid lacks two intracellular motifs common to many L1 proteins: a clathrin sorting motif (YRSLE), which regulates internalization, and an ankyrin-binding domain (NEDGSFIGQY), which controls association with the cytoskeleton, suggesting that Echinoid acts by a different mechanism from other L1 proteins. Since overexpression of Echinoid in tissue has no effect on the level of phosphorylated MAP kinase, a read-out of Egfr signaling, it appears that Echinoid does not act as a general inhibitor of Egfr. Instead, the prolonged presence of phosphorylated MAP kinase in echinoid mutants suggests that the role of Echinoid is to downregulate Egfr signaling after a period of activation (Spencer, 2003).
The ability of Egfr to signal depends on its localization and its downstream targets. Ligand-induced endocytosis is a well-documented mechanism for downregulating Egfr activity, and the prolonged Egfr signaling observed in echinoid mutants suggests that one possible role for Echinoid is to facilitate Egfr endocytosis after a period of activity. Another notable feature of Echinoid is its unusual intracellular domain, which differs from other members of the L1 superfamily. This domain is likely required for at least some aspects of Echinoid function (Bai, 2001), and suggests that Echinoid may target downstream signaling molecules. Based on the results, this unknown pathway would intersect with Egfr signaling prior to MAPK phosphorylation (Spencer, 2003).
What downstream molecules might be targeted by Echinoid? One potential model for the function of Echinoid is provided by work on the vertebrate SIRPalpha proteins, the only group of Ig-containing proteins shown to inhibit receptor-tyrosine kinase (RTK) signaling. SIRP-alpha proteins are phosphorylated on tyrosine in response to RTK activation; these phosphorylated residues provide binding sites for the SHP2 tyrosine phosphatase. Analysis of the Drosophila genomic sequence uncovered no clear Drosophila orthologs of SIRP-alpha proteins, but the overall structural similarity of Echinoid, its phosphorylation in response to Egfr signaling and its importance in downregulating Egfr signaling suggest that it may function in a manner analogous to the SIRP-alpha proteins. Genetic interactions have been observed between echinoid and corkscrew, the Drosophila homolog of SHP2, and binding between these proteins has been detected in cultured cells. However, the significance of these interactions will require further study in vivo (Spencer, 2003).
Since Egfr activity is required for the differentiation of both photoreceptor (except R8) and cone cells, the numbers of these cell types per ommatidum was used as a readout for Egfr activity in the eye disc. Flies with a mutation in the ed gene produce extra photoreceptor and cone cells. By contrast, overexpression of ed in the eye leads to a reduction of the number of photoreceptor cells per ommatidium. These findings together with additional genetic evidence indicates that Ed uses an independent pathway to antagonize Egfr signaling, and it is postulated that this inhibition might be initiated by a homophilic binding activity of Ed (Bai, 2001). To explore the possibility that Ed could be involved in heterophilic interactions with other Ig domain CAMs, a genetic overexpression screen was constructed. It was reasoned that if ed acts as a heterophilic receptor, overexpression of both the Ed receptor and its potential ligand(s) should have a synergistic effect on the inhibition of Egfr signaling, which results in a reduced number of cone and photoreceptor cells. In addition, both adhesion molecules must normally be co-expressed and colocalized in the developing eye disc in order to engage in a functional heterophilic adhesive interaction (Islam, 2003).
The GMR-GAL4 driver line was used to co-express UAS-ed with several available UAS and EP lines that drive overexpression of various Ig domain-containing adhesion molecules. Ectopic expression of Ed in the eye results in a rough eye phenotype and a loss of photoreceptor and cone cells (Bai, 2001). On average, 10%-15% of ommatidia were missing photoreceptor or cone cells. By contrast, overexpression of either the neuronal nrg180 or the non-neuronal nrg167 isoform alone has no effect on the number of photoreceptor or cone cells. However, co-expression of both ed and nrg180 (or nrg167) results in a more severe rough eye phenotype with a reduction of the number of ommatidia, a varying size of ommatidia and a decrease in the number of bristles. In addition, a significantly higher percentage of ommatidia contained fewer photoreceptor and cone cells. No synergistic effects were detected when ed was overexpressed together with other CAMs, such as Drosophila Fasciclin 2 or human L1CAM (Islam, 2003).
To document the interaction between Ed and Nrg further, the effect of overexpression of ed was examined in female flies, that had only one copy of the nrg gene. nrg1 is a nrg null allele. A reduction in half of the nrg gene dosage significantly suppresses the cone cell loss phenotype, but not the loss of photoreceptor cells; both these effects were caused by GMR-GAL4-driven UAS-ed expression. Together, these results demonstrate a specific genetic interaction between ed and both protein isoforms of nrg (Islam, 2003).
Both a genetic interaction between ed and nrg and their direct heterophilic trans-binding have been demonstrated. The synergistic effect of ed and nrg could be caused by a unidirectional signaling mechanism with either Ed as the receptor (and Nrg as the ligand) or Nrg as the receptor (and Ed as the ligand). Another possibility is that both Ed and Nrg act as receptor molecules (with Nrg and Ed as ligands, respectively) in mediating a bi-directional signaling process. To distinguish between these three possibilities, the UAS-Gal4 system was used to co-express in the developing Drosophila eye disc ed and nrgGPI, an artificial isoform of Nrg that lacks the intracellular Nrg domain. Overexpression of nrgGPI alone causes no phenotype. However, the synergistic effect between Ed and Nrg on the percentage of ommatidia lacking photoreceptor and cone cell was fully retained for this genetic combination. By contrast, co-expression of native nrg180 and a truncated artificial isoform of Ed, which lacks the intracellular Ed domain (ed intra), does not exhibit a genetic synergy in the eye disc. Similar results were obtained when ed intra and either nrg167 or nrgGPI were co-expressed. This indicates that the intracellular domain of Ed is essential for repressing Egfr signaling (Islam, 2003).
In summary, these results suggest that in this context Nrg primarily functions as a heterophilic ligand of Ed and thereby activates Ed in the signal-receiving cell. As a result of its interaction with Ed, Nrg antagonizes Egfr signaling non-autonomously. By contrast, there is no evidence from the experimental assay system for suggesting any signaling from Ed to Nrg. Consistent with this model, it was found that the ectopic expression of edC50, which contains only the transmembrane domain and the last 50 amino acids of the Ed intracellular domain, but lacks the extracellular Ed domain, also causes a reduced number of photoreceptor and cone cells (Islam, 2003).
Taken together, these results support a model whereby Nrg functions as a heterophilic ligand of Ed and activates Ed in the signal-receiving cells to antagonize Egfr signaling. Ed is the first identified heterophilic, extracellular partner of Nrg. In this context, Nrg functions as a ligand to activate Ed in the signal-receiving cells. This unidirectional signaling mechanism from Nrg to Ed is further supported by the observation that overexpression of edC50 alone can reduce Egfr signaling. By contrast, co-expression of nrg180 and ed intra does not exhibit any genetic synergy in influencing Egfr signaling. Thus, the results fail to support a bi-directional signaling mechanism from Ed to Nrg. Because it is not known whether the intracellular domain of Ed may also be required for signaling out and for activating Nrg in neighboring cells, a signaling process from Ed to Nrg still remains a possibility. The overexpression effect of edC50 on the Egfr signaling varies between different lines and tends to be weaker than that observed for ed and nrg co-expression. It is not clear whether this simply reflects differential expression levels for EdC50 or whether it lacks the full activity of a wild-type Ed (Islam, 2003).
The non-neuronal isoform of Nrg (Nrg167) is expressed in the non-neuronal, epithelial cells of eye imaginal discs. It exhibits a similar effect on Ed (and thereby the Egfr signaling pathway) as does the neuronal Nrg isoform (Nrg180), which is expressed by the photoreceptor cells. Therefore, Nrg167 is probably the major Nrg isoform that inhibits the intrinsic Egfr signaling for basally located, undifferentiated cells. Although cell mixing experiments clearly show that Ed and Nrg protein interact with each other in a trans-type modus, the results neither prove nor disprove that they might also interact in a cis-type modus. In fact, some Ig-domain CAMs, such as axonin 1/TAG1, interact with L1-type proteins exclusively in a functional cis-type interaction (Islam, 2003).
Genetic evidence indicates that Nrg is a cell-autonomous, positive regulator of Egfr signaling in neuronal cells that express both Nrg and Egfr. However, in the developing Drosophila eye disc Nrg functions non-autonomously as a ligand of Ed and activates Ed in the neighboring cells to repress downstream Egfr signaling. Thus, depending on the cellular context, Nrg can act both as an autonomous activator, as well as a non-autonomous inhibitor of the Egfr signaling pathway (Islam, 2003).
Genetic mosaic analysis indicates that ed acts in a cell non-autonomous manner (Bai, 2001). Since the intracellular domain of Ed is required for Egfr signal repression, it is proposed that through its homophilic interaction Ed transmits a negative signal in the receiving cell and antagonizes the Egfr pathway. In this study, a homophilic adhesive activity of Ed has been demonstrated, and it is further shown that ed also acts autonomously as a heterophilic receptor of Nrg. Thus, Ed appears to influence Egfr signaling through both homophilic (non-autonomous) and heterophilic (autonomous) interactions, but the relative contribution derived from either interaction is unknown. Flies that are mutant for ed have extra photoreceptor and cone cells. By contrast, when shifting temperature-sensitive nrg3 larvae to the restrictive temperature during the third instar larval stage, a wild-type number of Elav- and Cut-positive cells was observed. Therefore, the Nrg-mediated heterophilic activity of Ed in repressing Egfr signaling appears to be redundant with the homophilic activity of Ed (Islam, 2003).
Further studies are required to reveal the molecular mechanism by which ed inhibits the Egfr signaling pathway. Equally, with both ed and nrg widely expressed in the developing Drosophila eye disc, it remains to be revealed how the two opposing effects of nrg on Egfr activity might contribute to a differential cellular segregation and the development of different ommatidial cell types (Islam, 2003).
ed encodes an open reading frame of 3996 bp, that predicts a protein of 1332 amino acids. The translated protein contains six immunoglobulin (Ig) C2 type domains, a fibronectin type III domain and a transmembrane domain, followed by a 315 amino acid C-terminal tail with no identifiable functional motif. A comparison of the genomic and cDNA sequence indicates that the P-element l(2)k1102 is inserted in the first intron, which is upstream of the coding region (Bai, 2001).
Conceptual translation of the largest open reading frame (ORF) of the fred cDNA (CG3390) predicts a protein of 1198 amino acids. This protein product has a putative signal sequence, seven immunoglobulin (Ig) C2 type domains, followed by two fibronectin type III (Fn type-III) domains, a transmembrane domain, and a 188-amino-acid C-terminal region with no readily identifiable structural or functional motif. Examination of Ed sequence indicates the presence of an additional Ig C2 domain upstream to the first IgC2 domain and another Fn type-III domain downstream to the Fn type III domain. fred appears to be a paralogue of ed since the overall structural arrangement of Fred closely mimicks that of Ed. Both proteins contain seven Ig C2 type domains, two Fn type-III domains followed by a transmembrane domain, and an intracellular region. The Ig C2 domains of Fred exhibit a high sequence similarity to the corresponding Ig C2 domains of Ed, ranging from 62% to 91% identity. In contrast, Ig C2 domain similarity between unrelated Ig C2 proteins are generally in the 40% range. The proteins also exhibit sequence similarities in regions between the Ig C2 domains. The overall identity between the extracellular regions of the two proteins is 69%. In contrast, the putative intracellular regions of the two proteins exhibit only limited sequence similarity (30% identity) (Chandra, 2003).
BLAST searches of the recently completed Mosquito, Anopheles gambiae, genome sequence using Ed and Fred, reveal the presence of two highly similar genes. The similarity is particularly evident in the comparison of individual IgC2 domains. Like ed and fred, these genes are arranged in tandem, but the predicted transcription units are in the same orientation. Thus, while the predicted Mosquito ed/fred genes appear to be transcribed from the same strand, the Drosophila ed and fred transcription units are transcribed in opposite directions. Direct comparison of the predicted amino acid sequences of Drosophila Fred and Ed with the Anopheles orthologs shows a significantly higher overall similarity of both predicted Anopheles proteins to Drosophila Ed. This suggests that Drosophila Ed is likely to be closer in sequence to the ancestral gene than Fred. This possibility is further supported by the observation that both predicted Mosquito Ed/Fred proteins show sequence similarity to the entire Ed intracellular region, but only limited similarity to the Fred intracellular domain (Chandra, 2003).
date revised: 1 July 2003
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