InteractiveFly: GeneBrief

echinoid & friend of echinoid: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

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.

Ed antigonism of the Egfr pathway through through direct interaction with Egfr receptor

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 Egfr^Ellipse. 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).

Differential adhesion and actomyosin cable collaborate to drive Echinoid-mediated cell sorting

Cell sorting involves the segregation of two cell populations into 'immiscible' adjacent tissues with smooth borders. Echinoid (Ed), a nectin ortholog (see Ogita, 2011), is an adherens junction protein in Drosophila, and cells mutant for ed sort out from the surrounding wild-type cells. However, it remains unknown which factors trigger cell sorting. This study dissected the sequence of this process and found that cell sorting occurs when differential expression of Ed triggers the assembly of actomyosin cable. Conversely, Ed-mediated cell sorting can be rescued by recruitment of Ed, via homophilic or heterophilic interactions, to the wild-type cell side of the clonal interface, even when differential Ed expression persists. It was found, unexpectedly, that when actomyosin cable was largely absent, differential adhesion was sufficient to cause limited cell segregation but with a jagged tissue border (imperfect sorting). It is proposed that Ed-mediated cell sorting is driven both by differential Ed adhesion that induces cell segregation with a jagged border and by actomyosin cable assembly at the interface that smoothens this border (Chang, 2011).

This study has dissected the sequence of events in Ed-mediated cell sorting and concludes that both differential adhesion and the induction of actomyosin cable formation are required and act cooperatively to mediate cell sorting. It was also demonstrated that the relocalization of Ed by Ed, Fred and Ed^δintra, but not Nrg-Ed, to the clonal interface of the wild-type cells is sufficient to dprevent actomyosin cable formation in the wild-type cells. How differential expression of Ed induces actomyosin cable formation only at the Ed⁺ interface cells (but not the Ed^- cells) to generate a polarized response remains unknown. It has been suggested that interfacial tension is the result of cortical tension decreased by adhesion energy at this interface. Moreover, cortical myosin II recruitment is regulated by tension in a positive-feedback loop that could promote actomyosin cable formation (Fernandez-Gonzalez, 2009). Therefore, it is postulated that the reduction of adhesion energy caused by the loss of Ed would increase the interfacial tension so as to induce actomyosin cable formation at that interface. However, although interfacial tension also increases in ed mutant cells no prominent actomyosin cable formation was detected in these cells. Thus, interfacial tension alone is insufficient to explain this polarized effect (Chang, 2011).

Laplante (2011) proposed that, during dorsal closure, asymmetric distribution of Ed is required in the dorsal-most epidermal (DME) cells for the polarized accumulation of actin regulators (such as Enabled, Diaphanous and RhoGEF2) in the actin-nucleating centers (ANCs) of DME cells, and that this in turn promotes actomyosin cable assembly at the leading edge. Ed-mediated cell sorting resembles embryonic dorsal closure, where the DME cell is equivalent to the Ed⁺ interface cell, the leading edge is equivalent to the interface of ed mutant clones, and the ANC is equivalent to the interfacial tricellular junction of Ed⁺ interface cells. A similar polarized accumulation of actin regulators, such as Enabled, was also found at the tricellular junction of Ed⁺ interface cells of ed-RNAi clones. As cells within ed mutant clones cannot have a polarized distribution of Ed and actin regulators to form actomyosin cable, this provides an alternative mechanism for generating a polarized effect. Moreover, as both Nilson's group and the current demonstrated that the intracellular domain of Ed is required for actomyosin cable formation, it is possible that the asymmetric distribution of Ed might, via its intracellular domain, regulate the polarized accumulation of actin regulators at the interfacial tricellular junctions of Ed⁺ cells that in turn promotes actomyosin cable assembly (Chang, 2011).

The induction of actomyosin cable formation only at the Ed⁺ interface cells was observed not only when a large number of wild-type cells surrounded a few ed-depleted cells (in a small ed-RNAi clone) but also when a large number of ed-RNAi cells surrounded a few wild-type cells (in a very large ed-RNAi clone). The actomyosin cable at the interface supplies the tension needed to form a smooth border, tension that can be supplied either by the Ed⁺ interface cells surrounding a small ed-RNAi clone or by the Ed⁺ interface cells within a large ed-RNAi clone. However, apical constriction was present in ed-depleted cells surrounded by a large number of wild-type cells (in small ed-RNAi clones). Similarly, apical constriction was also detected when a few wild-type cells were surrounded by a large number of ed-RNAi cells (in large ed-RNAi clones). Since significant p-MLC accumulation was not detected in the apically constricted ed-RNAi nor wild-type cells, it is suggested that myosin-mediated contraction is not important in the generation of apical constriction (Chang, 2011).

Ed-mediated cell sorting is similar to the process of dorsal closure. However, during dorsal closure, amnioserosa cells actively undergo pulsed contraction that leads to a reduction in their apical surfaces. This, together with the actomyosin cable acting as a ratchet, pulls the surrounding epidermal cells towards the midline. By contrast, the apical surface of Ed-deficient cells gradually increases when the ed-RNAi clones expand. Moreover, the actomyosin cable of the interface cells acts not as a ratchet but instead as a mechanical fence to smoothly separate wild-type and Ed-deficient cells. Finally, Ed-mediated cell sorting involves the polarized assembly of actomyosin cable only in the wild-type interface cells. This is in contrast to the formation of the anteroposterior boundary in the embryo, where the formation of actomyosin cable by cells on both sides of the boundary is postulated to be the primary mechanism of cell sorting. This study suggests that differential adhesion of Ed alone is sufficient to trigger the segregation of cells into separate populations with jagged borders, but it remains unknown whether differential adhesion mediated by differential expression of as yet unidentified compartment-specific CAMs plays a role in establishing the initial anteroposterior boundary, where actomyosin cable ensures that this boundary remains straight (Chang, 2011).

Ed antigonism of the Egfr pathway through engagement of Neurogenin

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 nrg^GPI, an artificial isoform of Nrg that lacks the intracellular Nrg domain. Overexpression of nrg^GPI 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 nrg^GPI 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 ed^C50, 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 ed^C50 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 ed^C50 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 Ed^C50 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).

REGULATION

Protein Interactions

The genetic synergy between Ed and Neuroglian (Nrg) suggests that both proteins might also physically interact with each other. Using antibodies that specifically recognize Ed and both isoforms of Nrg for an immunocytochemical analysis, this possibility was first tested by examining Nrg and Ed expression patterns in the developing Drosophila eye disc. Both colocalize to all cells throughout the third instar larval eye disc, including undifferentiated cells and developing ommatidial clusters (Chandra, 2003).

Similar to several vertebrate Ig-domain CAMs, which interact with members of the L1 family, Ed might exhibit both homophilic and heterophilic adhesive activities. Genetic results support this possibility. To investigate the adhesive function of Ed, Ed protein was expressed in Drosophila Schneider 2 (S2) cells. Owing to their lack of endogenous CAMs, S2 cells have been successfully used for the functional analysis of a range of adhesive proteins. HA epitope-tagged Ed protein is expressed by S2 cells at a high and stable level. This epitope-tagged version of the Ed protein exhibits an apparent molecular weight of about 160 kDa, about the size to be expected from the Ed amino acid sequence. When S2 cells expressing Ed protein are allowed to aggregate, they formed small to medium-sized cell clusters. In order to establish whether this cell aggregation is due to a homophilic activity of Ed or to a heterophilic interaction with an endogenous ligand, which might be expressed on the S2 cell surface, cell-mixing experiments were performed. Aggregation experiments, in which unlabeled, Ed-expressing cells were mixed with DiI-labeled, Ed-expressing cells, resulted in mixed cell clusters, consisting of labeled and unlabeled cells. By contrast, DiI-labeled, native S2 cells that do not express Ed protein are not recruited into Ed cell aggregates. These results demonstrate that Ed acts as a homophilic adhesion protein and exhibits no heterophilic affinity to any endogenous S2 cell protein (Islam, 2003).

Mixing experiments were performed to determine whether Nrg and Ed engage in a heterophilic interaction. In comparison with Ed-expressing S2 cells, S2 cells expressing Nrg exhibit a much stronger homophilic cell adhesion capability, resulting in very large S2 cell aggregates. The co-aggregation of Ed-expressing cells with Nrg-expressing S2 cells yielded mixed cell aggregates, consisting of cells from both cell populations. DiI-labeled, native S2 cells are not incorporated into Nrg-cell clusters and quantitative evaluation demonstrates that the heterophilic interaction of Ed is specific to S2 cells expressing Nrg and that Ed does not interact with other Drosophila adhesion molecules, such as Drosophila Fasciclin 1. Thus, Ed and Nrg engage in a robust and specific heterophilic trans-interaction (Islam, 2003).

The strength and stability of this interaction was demonstrated by co-immunoprecipitation experiments. Since native Nrg interacts with ankyrin and the S2 cell membrane skeleton, it becomes resistant to non-ionic detergent extraction after engaging in cell adhesion. Therefore, an artificial, GPI-anchored form of Nrg was used for these co-immunoprecipitation experiments. S2 cells that expressed Ed or Nrg^GPI were mixed, co-aggregated and subsequently extracted with Triton X-100. Soluble proteins and protein-complexes were immunoprecipitated with an anti-Nrg or a control antibody and analyzed on Western blots for the presence of Ed protein. Ed protein was efficiently co-immunoprecipitated together with Nrg^GPI, suggesting that a stable and tight interaction is formed between these two adhesive molecules (Islam, 2003).

The extracellular structure of immunoglobulin and fibronectin repeats in Echinoid suggests that it may be a member of the L1 class of cell-adhesion proteins, which are important in mediating axon guidance and cell fate decisions. To examine the ability of Echinoid to mediate homophilic adhesive interactions, cultured S2 cells were transfected with epitope-tagged versions of Echinoid. EdFLAG is capable of co-precipitating Edmyc, suggesting that Echinoid can form multimers. In the converse experiment, immunoprecipitation of Edmyc also co-precipitates EdFLAG. A form of EdFLAG lacking extracellular sequences (EdFLAGdeltaN) does not co-precipitate Edmyc, suggesting that the proteins associate through their extracellular domains. Binding of EdFLAG to Edmyc is unaffected by transfection with Egfr. These results are consistent with the potential of Echinoid to act as a cell adhesion molecule; in keeping with this, defects in R8 differentiation have been observed in wild-type cells just outside echinoid mutant patches of tissue (Rawlins, 2003). However, the nature of this potential adhesive interaction on signaling is not clear, since overexpression of Echinoid, which might be expected to affect adhesion, has no effect on Egfr activity (Spencer, 2003).

Another common property of L1 immunoglobulin proteins is cleavage by plasmin or ADAM-class proteases at sites within or near FN3 domains. A similar cleavage of Echinoid was observed in S2 cultured cells. Immunoprecipitations of C-terminally tagged Echinoid produces two bands on SDS-PAGE gels: one, which corresponds to full-length Echinoid, migrates as a 190 kDa protein (larger than the predicted molecular weight of 146 kDa, possibly owing to glycosylation); the other migrates at approximately 55 kDa, corresponding approximately to the C-terminal 500 amino acids of Echinoid. Consistent with this, an antibody to the N-terminal region of Echinoid detects a ~130 kDa form of the protein in media from transfected S2 cells. This suggests that, in addition to full-length Echinoid, cells also contain a truncated form which lacks most of the extracellular motifs, and that the Ig/Fibronectin domains of the protein are shed into the surrounding extracellular space. The cleavage appears to be constitutive in S2 cells, and is not altered by transfection of Egfr (Spencer, 2003).

Echinoid has been proposed to act in a pathway parallel to Egfr, with signaling converging at the nucleus (Bai, 2001). Data from the current study suggests, in contrast, that loss of echinoid acts upstream of the nucleus to stabilize ERKA phosphorylation. Given the localization of Echinoid at the cell surface (Bai, 2001) the potential for direct interactions between Echinoid and Egfr was examined in S2 cultured cells. FLAG-tagged Echinoid was immunoprecipitated and analyzed by Western blots: transfection with increasing amounts of Echinoid co-precipitates increasing amounts of Egfr, suggesting that this is a specific interaction. Similarly, Echinoid containing a Myc tag also co-precipitates Egfr. In the converse experiment, immunoprecipitation of Egfr co-precipitates EdFLAG. The site of binding between Echinoid and Egfr could not be shown conclusively from these experiments: both EdFLAGdeltaC and EdFLAGdeltaN efficiently immunoprecipitate Egfr, suggesting that their interaction occurs near the transmembrane domain or through multiple domains (Spencer, 2003).

L1 cell-adhesion proteins are frequently regulated by phosphorylation, which controls their binding to other cytoplasmic signaling proteins. The physical association of Echinoid with Egfr, a receptor tyrosine kinase, suggested that it might be a substrate for tyrosine phosphorylation. To examine this possibility, S2 cell culture was sued to assess tyrosine phosphorylation in response to Egfr signaling. Echinoid exhibits a low level of tyrosine phosphorylation in the absence of added Egfr. Co-transfection with Egfr, even without added ligand, leads to a dramatic increase in the phosphorylation of Echinoid. This phosphorylation is increased further by addition of media containing a soluble form of the activating Egfr ligand Spitz. Phosphorylation is limited to the tyrosine-rich intracellular region: a form of Echinoid lacking the intracellular domain (EdFLAGdeltaC) is not phosphorylated. Interestingly, EdFLAGdeltaN, which lacks the extracellular immunoglobulin and fibronectin motifs, is phosphorylated in response to Egfr signaling, suggesting that these extracellular motifs are unnecessary for phosphorylation to occur. Bai (2001) suggested that Echinoid acts in a pathway parallel to Egfr, and that their activities converge in the nucleus. This study finds, however, that expression of the activated Ras isoform dRas1^Val12 does not lead to increased Echinoid phosphorylation, indicating that phosphorylation of Echinoid in response to Egfr signaling occurs upstream of Ras activation. These data do not resolve whether Echinoid is phosphorylated directly by Egfr or by cytoplasmic tyrosine kinases immediately downstream of its activity, an issue also unresolved for other cell-adhesion proteins. Phosphorylation of Echinoid in response to Egfr signaling was confirmed in vivo: Echinoid immunoprecipitated from w¹¹¹⁸ eye discs (which are essentially wild type at this stage) shows a low level of phosphorylation. Echinoid immunoprecipitated from discs in which Rhomboid (an activator of Egfr) was transiently expressed shows an approximately fourfold increase in tyrosine phosphorylation. Transient expression of Argos, an inhibitor of Egfr, produces either no change or a slight decrease in Echinoid phosphorylation levels over wild type. The importance of this phosphorylation to the function of Echinoid is not clear from these data. It has been noted, however, that strong overexpression of Echinoid in the developing retina leads to a rough eye in adults (Bai, 2001). This phenotype requires the presence of the Echinoid intracellular domain (Bai, 2001), suggesting that this domain may be essential for the function of Echinoid (Spencer, 2003).

The cytokine-activated Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays an important role in the control of a wide variety of biological processes. When misregulated, JAK/STAT signaling is associated with various human diseases, such as immune disorders and tumorigenesis. To gain insights into the mechanisms by which JAK/STAT signaling participates in these diverse biological responses, a genome-wide RNA interference (RNAi) screen was carried out in cultured Drosophila cells. One hundred and twenty-one genes were identified whose double-stranded RNA (dsRNA)-mediated knockdowns affected STAT92E activity. Of the 29 positive regulators, 13 are required for the tyrosine phosphorylation of STAT92E. Furthermore, it was found that the Drosophila homologs of RanBP3 and RanBP10 are negative regulators of JAK/STAT signaling through their control of nucleocytoplasmic transport of STAT92E. In addition, a key negative regulator of Drosophila JAK/STAT signaling was identified, protein tyrosine phosphatase PTP61F; it is a transcriptional target of JAK/STAT signaling, thus revealing a novel negative feedback loop. This study has uncovered many uncharacterized genes required for different steps of the JAK/STAT signaling pathway (Baeg, 2005).

echinoid (ed) was identified as a positive regulator required for Upd-dependent STAT92E tyrosine phosphorylation. ed encodes a cell adhesion molecule and has been shown to be a negative regulator of the EGFR signaling pathway during Drosophila eye development. Previous experiments have shown both positive and negative interactions between the JAK/STAT pathway and the EGFR pathway. For example, STAT92E mutants phenocopy mutants in the EGFR pathway. Furthermore, studies using mammalian tissue culture systems have demonstrated that EGFR signaling activates both JAK1 and STAT1. In addition, EGFR-induced cell migration is mediated predominantly by the JAK/STAT pathway in primary esophageal keratinocytes. Similarly, ed has been shown to be responsible for defective cell migration in Caenorhabditis elegans. Therefore studying the role of ed in JAK/STAT signaling in different contexts may facilitate understanding of the genetic and biochemical mode of STAT activation by EGFR signaling, and provide insights into the mechanisms governing cancer cell metastasis in humans (Baeg, 2005).

Echinoid is a component of adherens junctions that cooperates with DE-Cadherin to mediate cell adhesion

Echinoid is an immunoglobulin domain-containing transmembrane protein that modulates cell-cell signaling by Notch and the EGF receptors. In the Drosophila wing disc epithelium, Echinoid is a component of adherens junctions that cooperates with DE-Cadherin in cell adhesion. Echinoid and β-catenin (a DE-Cadherin interacting protein) each possess a C-terminal PDZ domain binding motif that binds to Bazooka/PAR-3; these motifs redundantly position Bazooka to adherens junctions. Echinoid also links to actin filaments by binding to Canoe/AF-6/afadin. Moreover, interfaces between Echinoid^- and Echinoid⁺ cells, like those between DE-Cadherin^- and DE-Cadherin⁺ cells, are deficient in adherens junctions and form actin cables. These characteristics probably facilitate the strong sorting behavior of cells that lack either of these cell-adhesion molecules. Finally, cells lacking either Echinoid or DE-Cadherin accumulate a high density of the reciprocal protein, further suggesting that Echinoid and DE-Cadherin play similar and complementary roles in cell adhesion (Wei, 2005).

Several observations prompted the study of Ed as a canonical CAM in the monolayered wing imaginal disc. Thus, mitotic recombination clones of cells mutant for the null allele ed^1x5 exhibit rounded and smooth contours, in contrast to clones of wild-type cells that show wiggly shapes. This indicated that ed^{- /-} cells have distinct adhesive properties and assort with themselves rather than with the surrounding ed^+/- M^+/- cells. (ed^1x5 clones were M⁺, since without a growth advantage they hardly survive). It was also observed that Ed was absent from the membrane of the heterozygous cells that contacted the mutant cells, a finding consistent with the observation that Ed forms homophilic interactions and that these are required to incorporate/stabilize Ed at the cell membrane. Finally, Ed was found to localize basally to the apical marker Crb and apically to the basolateral marker Dlg. In fact, Ed colocalizes with both DE-Cad and Arm, and, therefore, it might be part of AJs. AJs are structures important for cell-cell contact and recognition. So, these results suggested that Ed plays a role in cell-cell adhesion (Wei, 2005).

Whether Ed affects components of AJs was examined by analyzing the localization of Arm within ed mutant clones. Arm strongly accumulates at the apical membranes of ed^{- /-} cells, and these cells have a reduced apical surface. Both effects are clear in small clones, but cells within larger clones (over hundreds of cells) had both the density of Arm and the apical surface more similar to those of the wild-type cells. Similar observations were made with DE-Cad and Actin. It is suggested that the increased concentration of these molecules in small clones most probably results from the apical constriction as supported by the accumulation of nonmuscle myosin II, without a net per cell increment of these proteins. Alternatively, it could result from increased stability of these proteins. The apical constriction continued through the SJs and ended at the planes just below the GJs as revealed by an Innexin antibody. Hence, these ed^{- /-} cells adopt a bottle shape. In contrast, the apposed ed^{- /-} and ed^+/- cells that form the border of the clone enlarge and adopte a rectangular shape. At this interface, the ed^{- /-} cells often contacted the heterozygous cells by their long sides, as if in an attempt to minimize the number of cells that formed the interface (Wei, 2005).

Interestingly, Arm and DE-Cad, but not Actin, are depleted at the interface membrane of both small and large clones. This suggests that ed^{- /-} and ed heterozygous cells discriminate one another and that AJs do not form properly in between them (Wei, 2005).

ed clones are surrounded by an Actin 'cable'. High-magnification images suggest that the cable is contained within the ed heterozygous cells surrounding the clone and that it is therefore generated by these cells. Several observations suggest that this Actin cable exerts a force. The cells surrounding an ed clone elongate toward the clone and accumulate nonmuscle myosin II at the interface membrane, as if attempting to cover the space exposed by the apically constricted ed^{- /-} cells. This effect is reminiscent of the stretching of the leading-edge cells that will cover the underlying amnioserosa during dorsal closure of the embryos. In the wing disc, the boundary that separates the dorsal (D) and ventral (V) regions of the wing pouch has the shape of a smooth arc and contains an actin 'fence'. After the second instar, this boundary corresponds to a compartment border that imposes absolute restrictions to cell lineages. Large ed^{- /-} clones close to or touching this boundary displace it toward the clones. In contrast, ed clones that straddle the boundary do not overtly distorted it, although the boundary could be less smooth within the clone. (Straddling clones might be originated before the compartment border was established or might be formed of D and V clones that fuse together). Moreover, the Actin cable surrounding the clones fuse with the Actin fence at the D/V boundary, suggesting that the distortion of this boundary is effected through this Actin linkage. Control ed⁺ M⁺ clones do not induce such distortions. These observations suggest that the Actin cable may contribute to the roundish shape of the ed clones and help confine their cells (Wei, 2005).

DE-Cad is a classical homophilic cell adhesion molecule of AJs. It interacts with β-catenin/Arm, which in turn binds α-catenin. Through the association between α-catenin and F-Actin, DE-Cad establishes links between cells that connect to the Actin cytoskeleton. This study shows that Ed is another CAM that, at the resolution of confocal microscopy, is also located at the AJs of imaginal disc cells. While cells in clones mutant for ed still seem to form normal AJs, the cells at the border of the clone seem impaired in forming them. It is hypothesized that this may help them segregate from surrounding ed^+/- cells. Ed was identified as a binding partner for PDZ proteins that, similarly to Arm, helps localize Baz to AJs. Moreover, it was found that through the binding of Cno, Ed, like DE-Cad/β-catenin, may link to F-Actin. Hence, Ed has functions in cell-cell adhesion similar to those of DE-Cad (Wei, 2005).

The differential adhesion hypothesis proposes that cell sorting may be driven by differences in the quantity and/or quality of adhesive molecules displayed on the surface of cells. In keeping with this hypothesis, it was found that ed^{- /-} cells sort out from ed^+/- cells, as shown by the remarkably round shapes and smooth contours of the ed clones. Moreover, their differential adhesiveness is also manifest by the fusion of different ed clones to yield composite but still roundish clones. It is suggested that contraction of the apically enriched Actin network and of the actin cable surrounding the clone, possibly by interaction with nonmuscle myosin II also present there, may contribute to the the apical constriction of the ed^{- /-} cells. It was also observed that the interface between ed^+/- and ed^{- /-} cells is depleted of DE-Cad, Arm and Baz, besides completely lacking Ed. This strongly suggests that this interface is deficient in AJs and probably helps to insulate ed^{- /-} cells from the surrounding ed heterozygous cells. It is hypothesized that this deficiency of AJs, which may reduce adhesion between ed^+/- and ed^{- /-} cells, and the inward-pulling force generated by apical constriction and the actin cable may help create the smooth and rounded contour of the clones at the level of AJs. At the plane of SJs, the clonal boundary is not as smooth. This may be due to the presence of normal levels of SJs, since seemingly wild-type amounts of Dlg were detected at the interface membrane. Normal levels of SJs may allow the clones to remain integrated in the epithelium. It is stressed that when ed clones grow large, the apical constriction disappears, suggesting that the forces responsible for this constriction become insufficient or no longer operate. If the force is exerted, at least in part, by the Actin cable surrounding the clone, as in a purse-string mechanism, it would make sense that this force becomes ineffectual as the number of cells within the clone increases. Remarkably, these differences of apical cell constriction observed in small and large ed clones have a correlate on the adult wing blade: small clones display an increased density of trichomes, implying that their cells are small or more tightly packed, whereas large clones have cells of normal size. This indicates that the apical constriction is retained through imaginal disc eversion, when the disc epithelium changes from columnar to planar (Wei, 2005).

In the embryonic epithelium, Baz, localized to both AJs and the marginal zone, is the initial apical regulator. How is Baz recruited to the apical domain? In the follicular epithelium, Baz is localized to this domain through lateral exclusion mediated by PAR-1/14-3-3 and apical anchoring by Crb/Sdt/Patj. The data support an additional mechanism to localize Baz to the apical domain. Both Ed and Arm can bind Baz through their C-terminal PDZ binding motif and therefore they may redundantly localize Baz to AJs. Indeed, the localization of Baz to AJs is relatively normal in the absence of either one. Most Baz is lost only when both Arm and Ed are depleted, as occurs at the interface membrane of ed clones or in large shg clones where Ed gradually breaks down. In the latter case, there is good colocalization between Baz and the sites maintaining residual Ed. It is suggested that in the epithelium of the wing disc, Baz localizes to AJs by the combined effects of its binding to Ed/Arm and the lateral exclusion of PAR-1/14-3-3. Additionally, apical anchoring of Baz may be mediated by direct association between the Baz and Crb apical complexes. During early embyogenesis, Ed is also present at pseudocleavage furrows. This observation, together with the ability of Ed to localize Baz to AJs, may explain the finding that during cellularization, Baz can accumulate apically in the absence of Arm. Ed also binds to the PDZ domain of Cno and mediates its localization to AJs, where Cno interacts with F-Actin either directly or indirectly through the association with Polychaetoid/ZO-1. Interestingly, the evolutionally conserved EIIV domain of Ed binds Baz and Cno in a mutually exclusive manner. Thus, the concentrations of and differential affinities between Ed, Baz, and Cno should determine their dynamic equilibrium at AJs (Wei, 2005).

Although Baz is critical to form AJs in the blastoderm and in the follicular epithelium, removal of Baz (or Par-6) from cells of the wing disc does not affect the localization of DE-Cad or Ed to AJs. This is consistent with the report that in imaginal discs, Baz does not affect the localization of DE-Cad and Dlg but is required for the asymmetric localization of cell fate determinants. Together, these results suggest that in wing discs, the Baz complex is not critical for the formation of AJs, and that the effect of the loss of Ed on AJs formation/maintenance is not due to Baz depletion (Wei, 2005).

Several similarities between the roles of DE-Cad and Ed in the wing disc epithelium are worth noting. Both Ed and DE-Cad are CAMs that establish homophilic interactions and localize to AJs. The absence of either Ed or of DE-Cad in cells of small clones causes their apical constriction and strong segregation from wild-type cells, giving rise to smooth round borders. In both cases, the mutant cells are impaired in forming AJs with neighboring wild-type or heterozygous cells and are surrounded by an Actin cable. Ed interacts with Cno, and DE-Cad with Arm, and both Cno and Arm directly or indirectly associate with F-Actin. Thus, Ed and DE-Cad represent two distinct classes of CAMs, with widely different chemical compositions, that connect to F-Actin, contribute to cell adhesion in the wing disc, and seem to have partially overlapping functions (Wei, 2005).

In contrast, DE-Cad and Ed differ in their ability to regulate the apical/basal cell polarity. Ed affects components of AJs, but not those of the apical Crb and the basolateral Dlg complexes. In contrast, DE-Cadherin is necessary for Crb localization, but similarly to Ed, it is not required for Dlg localization. Furthermore, the maintenance of Ed at AJs requires DE-Cad. In contrast, localization of DE-Cad to AJs is independent of Ed. Interestingly, the DE-Cad/Arm complex is not essential for the formation of the follicular epithelium, but upon removal of this complex, the integrity of the epithelium is lost slowly over the period of several days. This suggests that other molecules may be maintaining the epithelial structure. During stages 1 to 10 of oogenesis Ed is mainly expressed in the follicle cells, and these cells, if mutant for ed, show at low frequency a multilayered structure with disrupted expression of some polarity markers. Thus, it will be of interest to elucidate whether, in this epithelium, Ed and DE-Cad/Arm also play partially redundant roles in cell adhesion and apical/basal polarity. While both Ed and DE-Cad contribute to cell adhesion and recognition, it is unclear whether each molecule imparts specific recognition properties to cells, so that the final cell-cell affinity results from the sum of distinct affinities mediated by these different CAMs. More specifically, can an increased level (density) of DE-Cad replace the absence of Ed? The results showing that ed^{- /-} cells, with either normal levels (in large clones) or high density (in small clones) of DE-Cad, do not intermix with wild-type cells suggests that the binding specificity provided by a given CAM is not overruled by a higher level (density) of a different CAM. Moreover, the cell sorting properties conferred by Ed cannot account for the separation of cells at both sides of the A/P compartment boundary of the wing disc because A and P cells do not intermingle within composite ed, smo double mutant clones. (Similarly, DE-Cad is not responsible for the sorting out of A and P cells. Hence, cell-cell adhesion in the wing disc appears to depend on multiple CAMs (Ed, DE-Cad, etc.), each imparting specific cell recognition properties. Although Ed and its C-terminal EIIV motif are conserved in invertebrates, no clear vertebrate homolog with 7 Ig domains and a PDZ domain binding motif has been found. Nectin1-4 comprises a family of 3 Ig domain-containing CAM that have several differentially spliced forms and localize to AJs. Most spliced forms share a conserved C-terminal E/A-X-Y-V that binds the PDZ domain of Afadin. Moreover, this motif also interacts with Par-3, the vertebrate homolog of Baz. In spite of these similarities, overexpression of either nectin 1-α or 3-α does not rescue the remarkable clonal phenotype of ed (Wei, 2005).

Complex interaction of Drosophila GRIP PDZ domains and Echinoid during muscle morphogenesis

Glutamate receptor interacting protein (GRIP) homologues, initially characterized in synaptic glutamate receptor trafficking, consist of seven PDZ domains (PDZDs), whose conserved arrangement is of unknown significance. The Drosophila GRIP homologue (DGrip) is needed for proper guidance of embryonic somatic muscles towards epidermal attachment sites, with both excessive and reduced DGrip activity producing specific phenotypes in separate muscle groups. These phenotypes were utilized to analyze the molecular architecture underlying DGrip signaling function in vivo. Surprisingly, removing PDZDs 1-3 (DGripΔ1-3) or deleting ligand binding in PDZDs 1 or 2 convert DGrip to excessive in vivo activity mediated by ligand binding to PDZD 7. Yeast two-hybrid screening identifies the cell adhesion protein the Echinoid (Ed) type II PDZD-interaction motif as binding PDZDs 1, 2 and 7 of DGrip. ed loss-of-function alleles exhibit muscle defects, enhance defects caused by reduced DGrip activity and suppress the dominant DGripΔ1-3 effect during embryonic muscle formation. It is proposed that Ed and DGrip form a signaling complex, where competition between N-terminal and the C-terminal PDZDs of DGrip for Ed binding controls signaling function (Swan, 2006).

This study used genetics to develop a mechanistic model concerning a well-defined function mediated by Drosophila Grip-embryonic muscle guidance. Functional requirements were not transmitted by single domains, but were found to be distributed over the whole length of this 7 PDZD protein in an unexpectedly complex manner. Binding ligands via PDZDs 1 and 2 repressed the activity of the protein, binding to PDZD 3 was involved in de-repression, and PDZ-ligand binding via PDZD 7-mediated DGrip function after its de-repression. Despite the fact that there was no critical dependence on PDZDs 4-5 or interdomains for function, it cannot be excluded that interactions over these domains play a subthreshold role. In fact, the DGripΔ1-3x7 construct showed some residual functionality in terms of muscle rescue. Thus, the whole protein might be used as an 'intelligent frame' designed to execute fine controls such as thresholding functions or coincidence detections. In fact, all attempts to provide DGrip activity or to repress DGrip activities with only partial fragments (DGripΔ4-7, DGripΔ1-5) failed, leading to the belief that DGrip is responsible for the organization of a macromolecular complex, of which the transmembrane protein Ed is part (Swan, 2006).

This analysis suggests that a critical number of PDZDs are utilized for DGrip function, with both negative and positive interactions occurring. Such dependence between PDZDs may be due to structural chaperoning. Alternatively, a fixed orientation might be required for high-affinity binding to its targets as found for tandem PDZDs 1 and 2 in PSD-95, with a complex of two PDZDs having higher binding affinity than either PDZD alone. Moreover, allosteric changes upon PDZD-ligand binding could change binding affinities of neighboring domains or via bridging interactions where one molecule contacts multiple sites on a PDZ protein to effect conformational change. Such mechanisms might be the substrate for integrating ligand binding and functional output over a large 'multivalent' PDZD protein (Swan, 2006).

Point mutations of PDZD 1 and PDZD 2 recapitulated the DGripΔ1-3 phenotype in the lateral transverse muscles (LTM) group of muscles, indicating that the repressive function of the PDZDs 1-3 region is not 'structural' (i.e. by covering other PDZDs on the protein). Instead, it is suggested that ligand interactions are communicated over the whole protein to steer equilibrium between two different functional modes of DGrip signaling (Swan, 2006).

Ed was identified as a novel DGrip interactor. Ed is cell adhesion protein with 7 Ig and 2 FNIII domains, described to have both adherence and signaling roles in Drosophila tissues. It is highly conserved among invertebrates and its closest vertebrate homologues are Nectins, which exhibit 3 Ig domains and end in the PDZ-binding motif E/A-X-Y-V. Functionally, both protein families are similar: although not functionally redundant with Ed, Nectins are present at mammalian adherens junctions (AJs) along with l-afadin and, like Ed, regulate Cadherin-based adherence at AJs. Several lines of evidence link Ed to DGrip:Ed interacted with DGrip in a yeast two-hybrid screen, dependent on the C-terminal EIIV motif, mediated via PDZDs 1, 2 or 7. Myc-tagged DGrip specifically interacts with a peptide representing the last 10 amino acids of the Ed protein, including the EIIV PDZ-binding motif. ed zygotic mutants have defects in the morphogenesis of embryonic muscles qualitatively similar to DGripΔ1-3 overexpression. The dgrip^ex36 muscle phenotype in embryos is enhanced by heterozygosity for ed^1x5. Here, LTMs (unaffected in pure dgrip^ex36) are affected as well. dgrip^ex36 mutant muscles (both VLMs and LTMs) are sensitive to Ed overexpression. These synthetic defects suggest that DGrip, while itself not essential for LTM morphogenesis, regulates Ed in this group of muscles. Homozygosity for hypomorphic ed^slH8 chromosome strongly reduced the severity of the phenotype evoked by pan-muscular expression of DGripΔ1-3, indicating that Ed acts downstream of activated DGrip (Swan, 2006).

Notably, the pattern of Ed-PDZD binding correlates with the DGrip-dependent LTM phenotype. Expression of DGrip missing PDZDs 1, 2 and 3 together, or ligand binding in PDZD 1 and PDZD 2 only, showed a strong dominant active phenotype. Mutation of PDZD 2 caused a dominant phenotype in LTMs. In a yeast-two hybrid test, Ed interacted strongly with PDZD 2 with and PDZD 7, and more weakly with PDZD 1 (Swan, 2006).

In imaginal discs, Ed binds two different PDZD proteins via its EIIV motif: Canoe, an F-actin interacting protein and PAR-3/Bazooka. This interaction is mutually exclusive, thereby influencing cell adhesion and the remodeling of subcortical actin at AJs. This study proposes a similar mechanism, in that both functional states of Ed are established via binding to the same protein (DGrip) at different sites. In this model, DGrip may assist in maintaining equilibrium between active and inactive signaling states of Ed, which in its inactive state binds to PDZDs 1 and 2, and in its active form to PDZD 7 of DGrip. This interaction appears tissue specific in nature, since DGrip mutants do not display the full spectrum of defects of ed mutants (such as neurogenic phenotypes) and since there are as yet unknown members of the DGrip-Ed complex, such as that member that binds to the 'de-repressing' PDZD 3 (Swan, 2006).

Both Ed loss of function and overexpression can produce similar phenotypes in muscles, which are strongly enhanced by the absence of DGrip. Ed is described as a homophilic cell adhesion molecule, and is maternally expressed in the epidermis, over which nascent muscles 'crawl' during the muscle guidance process to reach their target apodeme. ed clones in wing discs show cell sorting behavior, causing aggregation and adhesion of only those cells expressing the same complement of cell adhesion molecules. Thus, both reduction and excess of Ed on the 'muscle side' of transient muscle-epidermal adhesions could lead to significant changes in the cell adhesion properties of the developing muscle. The experiments shown in this study for DGripΔ1-3 overexpression in muscle 5 and for VLMs in dgrip mutants imply that a tight balance of DGrip activity might particularly be needed to keep navigating muscle projections motile and to avoid their premature stabilization at ectopic epidermis contacts during the 'steering' process-ultimately instructed by Slit/Robo or other guidance systems. It is likely that Ed and DGrip form complexes enriched at muscle projection membranes to locally control adhesiveness. Ectopic adhesions among muscles cells with aberrant DGrip activity are in fact indicative of changes in muscle adhesiveness (Swan, 2006).

Natural variants of mGRIP missing PDZDs 1-3 have been localized to mammalian synapses, and it has recently been found that the type 5 metalloproteinase MT5-MMP is recruited by GRIP1/2 to growth-cone filopodia and to both mature and developing synapses, where it proteolyses N-cadherins. GRIP2 was also observed to be a member of a Δ-catenin containing complex. Drosophila Echinoid is known to regulate DE-Cadherin in homeotypic cell-cell junctions. Given these promising indications, it will prove interesting to see whether in the context Grip proteins became famous for synapse assembly -- similar molecular strategies are used by the GRIP protein as those described here in the context of muscle morphogenesis (Swan, 2006).

Cell adhesion molecule Echinoid associates with unconventional myosin VI/Jaguar motor to regulate cell morphology during dorsal closure in Drosophila

Echinoid (Ed) is a homophilic immunoglobulin domain-containing cell adhesion molecule (CAM) that localizes to adherens junctions (AJs) and cooperates with Drosophila epithelial (DE)-cadherin to mediate cell adhesion. This study shows that Ed takes part in many processes of dorsal closure, a morphogenetic movement driven by coordinated cell shape changes and migration of epidermal cells to cover the underlying amnioserosa. Ed is differentially expressed, appearing in epidermis but not in amnioserosa cells. Ed functions independently from the JNK signaling pathway and is required to regulate cell morphology, and for assembly of actomyosin cable, filopodial protrusion and coordinated cell migration in dorsal-most epidermal cells. The effect of Ed on cell morphology requires the presence of the intracellular domain (Ed^intra). Interestingly, Ed forms homodimers in vivo and Ed^intra monomer directly associates with unconventional myosin VI/Jaguar (Jar) motor protein. ed genetically interacts with jar to control cell morphology. It has previously been shown that myosin VI is monomeric in vitro and that its dimeric form can associate with and travel processively along actin filaments. Thus, it is proposed that Ed mediates the dimerization of myosin VI/Jar in vivo which in turn regulates the reorganization and/or contraction of actin filaments to control changes in cell shape. Consistent with this, it was found that ectopic ed expression in the amnioserosa induces myosin VI/Jar-dependent apical constriction of this tissue (Lin, 2007).

Dorsal closure involves cell shape changes and migration of dorsal-most epithelial (DME) cells over the apically constricted amnioserosa. At later stages of dorsal closure, myosin II/Zipper is the major motor protein generating force to drive the contraction of both DME and amnioserosa cells. However, its role in establishing/maintaining early DME cell morphology (prior to the assembly of visible actomyosin cable) has not yet been documented. This study showns that Ed is required to regulate cell morphology at an early stage. Ed forms homodimers and monomeric Ed can directly associate with myosin VI/Jar. Moreover, the data suggest that the effect of ed on cell shape changes is mediated through jar. How does Ed cooperate with Jar to cause cell shape changes? Since myosin VI is monomeric in vitro and cannot itself efficiently initiate dimerization unless two molecules are held in close proximity (Park, 2006), it is proposed that Ed, via homodimerization, might promote the assembly of functional myosin VI/Jar dimer in vivo that either tethers Ed to actin filaments (as an anchor) or moves processively along actin filaments (as a transporter). Interestingly, Jar also associates with Arm to stabilize DE-cadherin/Arm during border cell migration. Thus, it might be feasible that Ed recruits dimeric Jar to AJs where Jar associates with the actomyosin network and stabilizes the DE-cadherin/Arm complex that in turn also interacts with the actomyosin network. In this scenario, myosin VI/Jar acts as an anchor molecule to link homophilic CAMs like Ed and DE-cadherin of AJs to actin filaments. Due to the large step size of myosin VI, the resulting dimeric Ed/Jar complex might simultaneously associate with and cross-link neighboring actin filaments. In this aspect, the function of Jar in DME cells would be similar to Canoe, which links Ed to actin filaments in wing disc cells. Interestingly, Ed associates with Canoe and Jar via different domains (C-terminal PDZ domain-binding motif and N-terminal 80 amino acids, respectively). However, unlike Jar, the distribution of Canoe at AJs of DME cells is unaffected in ed^lF20 M/Z embryos, indicating the importance of tissue-specific interactions (Lin, 2007).

Following the engagement of myosin VI/Jar to actin filaments at AJs, myosin II/Zipper, myosin VI/Jar or other myosin motors might be responsible for the force generation to establishing/maintaining early DME cell morphology. For example, the plus end-directed myosin II/Zipper generates pulling force to drive contraction of DME cells during the zippering phase of dorsal closure. Moreover, at stage 12, the apical constriction caused by ectopic ed expression in amnioserosa cells is also associated with the accumulation of myosin II. However, its role in early DME cell morphology remains unknown. In contrast, the minus end-directed myosin VI/Jar might theoretically generate a pushing force to cause cell expansion. This is, however, in contrast with the observation that Ed cooperates with Jar to cause cell contraction. It is likely that myosin VI/Jar functions only as an anchor to link Ed to actin filaments but not as a force-generating motor. However, since the organization and orientation of actin filaments in early DME cells are currently unknown, the possibility cannot be completely excluded that myosin VI plays an additional role in force generation (Lin, 2007).

It has been shown that loss of Ed in the ed mutant clones induces apical constriction in the wing imaginal disc and this study demonstrates that ectopic ed expression in the amnioserosa also induces apical constriction of this tissue. While both loss of ed and ectopic ed expression can induce apical constriction, the mechanisms, however, differ in these two systems. In the former case, apical constriction of ed^−/− cells is caused by the accumulation of a higher density of DE-cadherin, Arm and actin (and by their interaction with myosin II). According to the differential adhesion hypothesis, these ed^−/− cells thus achieve stronger adhesiveness (affinity) and self-sort out from the surrounding wild-type cells. In contrast, overexpression of ed in amnioserosa might promote, via myosin VI/Jar dimer, the assembly of actin filaments that in turn interact with myosin II/Zipper, myosin VI/Jar or other myosin motors to produce apical constriction.

The strong genetic interaction between ed and jar is detectable not only during dorsal closure but also during germband retraction, a process that is associated with dramatic cell shape change of germband cells. Thus, Ed might also cooperate with Jar to regulate germband cell elongation along the D/V axis (Lin, 2007)

There are two types of AJs present in DME cells. The AJs facing LE contain only DE-cadherin, while the AJs connecting adjacent DME cells possess both DE-cadherin and Ed which both associate with actin filaments. The difference in AJ composition might regulate their stability and the strength of cell-cell adhesion. For example, AJs possessing both DE-cadherin and Ed might be more stable and rigid. This scenario, together with the tension exerted by Ed in the DME cells, can prevent each connecting DME cell from moving prematurely even at stage 12. In contrast, the presence of only DE-cadherin at AJs of the LE front might allow faster turnover of AJs that in turn results in more efficient cell migration. Upon removal of Ed, DME cells lose their tension and contain only DE-cadherin at their AJs, which together permit uncoordinated and premature migration of these cells at an earlier stage (Lin, 2007)

This study demonstrated that ed is also required for the assembly of actomyosin cable from stage 13 of dorsal closure onwards. Because the presence of the actomyosin cable can maintain a taut LE front, the irregular migration defect of ed mutant DME cells during early dorsal closure will become even more obvious when actomyosin cable assembly also fails in these cells. Thereafter, all DME cells migrate toward the dorsal midline with different speeds. The faster-moving DME cells of a given segmental stripe may extend filopodia to sense and preferentially zip up with the other faster-moving DME cells derived from either an adjacent segmental stripe or another stripe not directly opposite it (instead of in the opposing stripe) to cause misalignment. Although Ed is critical for actomyosin cable formation, Ed accumulates only at ANC but not at LE. It is possible that Ed might recruit additional factors to promote actomyosin cable assembly. Alternatively, Ed might function as a scaffold protein to regulate ANC formation, as all ANC-associated molecules tested mislocalize in the absence of Ed (Lin, 2007)

During the course of this study, Laplante (2006) proposed that differential Ed expression in lateral epidermis and amnioserosa (some vs. no Ed expression) promotes the generation of actomyosin cable and therefore, dorsal closure. In contrast, this study demonstrated that the elimination of Ed expression border by ectopic expression of Ed in amnioserosa (i.e., Ed expression at both sides of the border) still induces assembly of actomyosin cable in DME cells. It is possible that different levels of Ed expression across the border, but not necessary 'some vs. no Ed expression', are sufficient to trigger the generation of actomyosin cable (Lin, 2007).

Echinoid regulates Flamingo endocytosis to control ommatidial rotation in the Drosophila eye

Planar cell polarity (PCP) refers to a second polarity axis orthogonal to the apicobasal axis in the plane of the epithelium. The molecular link between apicobasal polarity and PCP is largely unknown. During Drosophila eye development, differentiated photoreceptors form clusters that rotate independently of the surrounding interommatidial cells (ICs). This study demonstrates that both Echinoid (Ed), an adherens junction-associated cell adhesion molecule, and Flamingo (Fmi), a PCP determinant, are endocytosed via a clathrin-mediated pathway in ICs. Interestingly, it was found that Ed binds AP-2, an adaptor that acts between cargo receptors and clathrin-coated vesicles during endocytosis, and is required for the internalization of Fmi into ICs. Loss of ed led to increased amounts of Fmi on the cell membrane of non-rotating ICs and also to the misrotation of photoreceptor clusters. Importantly, overexpression of fmi in ICs alone was sufficient to cause misrotation of the adjacent photoreceptor clusters. Together, it is proposed that Ed, when internalized by AP-2, undergoes co-endocytosis with, and thereby decreases, Fmi levels on non-rotating ICs to permit correct rotation of ommatidial clusters. Thus, co-endocytosis of Ed and Fmi provides a link between apicobasal polarity and PCP (Ho, 2010).

This study found that Ed interacts with AP-2 and promotes Fmi internalization via a clathrin-dependent pathway in ICs. Moreover, loss of ed leads to accumulation of Fmi (and of several receptors/CAMs of AJs) on the membrane of these non-rotating ICs. This, together with the observation that overexpression of fmi in the non-rotating ICs is sufficient to cause photoreceptor misrotation, led an Ed-mediated co-endocytosis model to be proposed to explain the rotation defects associated with the ed mutant tissue. Thus, the homophilic CAMs, Ed and Fmi, play crucial roles in ICs to allow coordinated rotation of ommatidial clusters (Ho, 2010).

Ed specifically regulates Fmi endocytosis in ICs. Although Fmi was previously shown to be endocytosed in the photoreceptor cluster, it is argued that Fmi levels in the rotating photoreceptor clusters are regulated by an Ed-independent mechanism. First, Fmi levels in the photoreceptor clusters are not affected in the ed mutant clones. Second, the Fmi distribution pattern in R3/R4 is largely unchanged even when Ed-GFP is overexpressed in photoreceptors by Elav-Gal4 to mimic the ed-expressing ICs. It was also demonstrated that Ed levels in ICs, but not in photoreceptor clusters, are regulated via an AP-2-dependent endocytic pathway. It remains unknown how Ed is downregulated in the photoreceptor cluster. Egfr signaling has been proposed to regulate the morphological and adhesive changes of cells within the photoreceptor cluster, and it is possible that Egfr signaling directly or indirectly downregulates the Ed levels in R8/R2/R5 and later in R3/R4 (Ho, 2010).

Interestingly, ed affects the levels of Fmi, DE-cad and Egfr, but not of Dlg. Therefore, Ed seems to only affect receptors/CAMs at AJs. One intriguing possibility is that Ed, via its interaction with AP-2, triggers the co-endocytosis of most, if not all, of the receptor/CAM at AJs. Although Ed has been shown to associate with Egfr, there is currently no evidence that Ed interacts directly with Fmi. Thus, Ed might undergo co-endocytosis either directly or indirectly. Although Ed contains five putative protein-sorting motifs, it is not the only molecule with a protein-sorting motif: Egfr, for example, also contains the YXXphi signal (phi is a bulky hydrophobic amino acid which could potentially interact with the AP-50 subunit of the AP-2 complex). It is possible that different receptors/CAMs might cooperate, via their interaction with AP-2 or other adaptors, to promote the co-endocytosis of other receptors/CAMs (Ho, 2010).

Because Ed facilitates the endocytosis of many receptors/CAMs of AJs, although to different extents, multiple functions of ed in the eye disc would be expected. Indeed, ed plays crucial roles in PCP (this study) and in Egfr signaling during eye development. It was previously shown that loss of ed leads to sustained MAPK (Rolled - FlyBase) activation only in cells of the proneural clusters and over several rows. This is consistent with the observation that Egfr is upregulated in an enlarged group of arc cells that contains two R8 photoreceptors as well as in cells of developing ommatidia up to two rows posterior to the MF. Thus, it is plausible that Ed was co-endocytosed with Egfr in the proneural clusters to downregulate Egfr activity within these cells and thus ensure that only one R8 is selected from the two to three R8 cell-equivalent group. When ed is absent, Egfr cannot be internalized efficiently and therefore persists on the membrane to cause sustained MAPK activation and multiple R8 selection. Although the level of Egfr is also upregulated in cells more posterior to the MF, these levels might not be high enough to cause sustained MAPK activation. In the wing disc, Ed also facilitates Notch signaling to promote mesothorax bristle patterning. In fact, Ed colocalizes with Notch/Delta in Hrs-containing early endosomes. However, it remains unclear whether the endocytosis of Ed plays any role in facilitating Notch signaling in the wing discs (Ho, 2010).

It has been shown that each photoreceptor cluster, as a group, moves independently of the adjacent ICs. Most rotation-specific genes identified thus far have been proposed to function mainly in the rotating clusters to modulate rotation. This study provides evidence that Ed plays crucial roles in the ICs to modulate ommatidial rotation. It is proposed that Ed, via co-endocytosis, reduces the level of Fmi on the non-rotating ICs to prevent homotypic interactions with the enriched Fmi on the rotating cluster. This allows free and coordinated rotation of photoreceptor clusters, a process regulated by effectors such as Zipper and Nemo. It is reasoned that in the absence of ed, as seen in ed mutant clones, the upregulated Fmi on the non-rotating ICs might affect the free rotation of ommatidial clusters not only within the ed clone, but also in the adjacent wild-type clusters abutting the ed clones. This might contribute, at least in part, to the non-autonomous effect of ed on ommatidial rotation. The dynamic and differential expression of Ed (and of its paralog Friend of Echinoid) in the rotating clusters and non-rotating ICs has also been proposed to modulate ommatidial rotation (Fetting, 2009). Thus, differential expression of Ed, Fmi, DE-cad and Friend of Echinoid in the rotating clusters and non-rotating ICs prevents the homotypic interaction of these four CAMs to allow free rotation of photoreceptor clusters. The largely complementary expression pattern between Ed and DE-cad/Arm in a photoreceptor cluster is similar to that observed during the generation of ed mutant clones in the wing discs, where Ed-non-expressing cells accumulate high levels of DE-cad/Arm and sort out from the surrounding Ed-expressing cells. Thus, cell sorting-like behavior of a photoreceptor cluster, mediated by differential expression of Ed and DE-cad/Arm, might help photoreceptors in the cluster to rotate as a group (Ho, 2010).

Fetting (2009) recently showed that ed genetically interacts with Egfr pathway members, and proposed that ed, via inhibiting Egfr signaling in the photoreceptors, regulates ommatidial rotation. However, this study demonstrated that, after row 2, Ed facilitates the endocytosis of Egfr only in the non-rotating ICs, but not in the photoreceptor clusters. Thus, if Ed indeed inhibits Egfr signaling in the photoreceptors as suggested, it probably employs mechanisms other than to reduce the levels of Egfr on the photoreceptors. It is currently unknown whether the effect of Ed on Egfr levels in ICs plays any role in the modulation of ommatidial rotation. Moreover, this study found that in the absence of ed, not only Fmi, Fz and Dsh (the R3-specific PCP proteins), but also Strabismus (Van Gogh) and its associated Prickle (the R4-specific PCP proteins) were all upregulated in ICs, but their enrichment at R3/R4 borders in the photoreceptor cluster was largely unaffected. As ed affects the levels of all the core PCP proteins tested in ICs, it remains unclear how ed only interacts genetically with the R3-specific PCP genes to modulate the degree of ommatidial rotation (Fetting, 2009). Finally, ed also weakly affects the initial R3/R4 specification, as a small proportion of ed mutant cells also show randomized chirality and symmetrical ommatidia. It is possible that ed might exert this effect through mechanisms other than the promotion of Fmi endocytosis. Alternatively, as overexpression of Fmi in the non-rotating ICs (generated by fmi-overexpressing clones) can affect both ommatidial rotation and reversal of R3/R4 cell fate of the adjacent clusters, it is possible that the Fmi upregulation in the ICs (generated by ed mutant clones) might also affect, to some extent, the asymmetric distribution of Fmi in R3/R4 and, thereby, the R3/R4 specification of the adjacent clusters (Ho, 2010).

The cell adhesion molecule echinoid functions as a tumor suppressor and upstream regulator of the Hippo signaling pathway

The Hippo (Hpo) signaling pathway controls tissue growth and organ size in species ranging from Drosophila to mammals and is deregulated in a wide range of human cancers. The core pathway consists of the Hpo/Warts (Wts) kinase cassette that phosphorylates and inactivates the transcriptional coactivator Yorkie (Yki). This study reports that Echinoid (Ed), an immunoglobulin domain-containing cell adhesion molecule, acts as an upstream regulator of the Hpo pathway (see Ed regulates organ size through the Hpo-Yki pathway). Loss of Ed compromises Yki phosphorylation, resulting in elevated Yki activity that increases Hpo target gene expression and drives tissue overgrowth. Ed physically interacts with and stabilizes the Hpo-binding partner Salvador (Sav) at adherens junctions. Ed/Sav interaction is promoted by cell-cell contact and requires dimerization of Ed cytoplasmic domain. Overexpression of Sav or dimerized Ed cytoplasmic domain suppressed loss-of-Ed phenotypes. It is proposed that Ed may link cell-cell contact to Hpo signaling through binding and stabilizing Sav, thus modulating the Hpo kinase activity (Yue, 2012).

The Hpo signaling pathway has emerged as a conserved regulatory pathway that controls tissue growth and organ size. Although the core pathway components (i.e., the Hpo/Sav/ Wts/Mats kinase cassette and its effector Yki/Yap), have been well defined, the upstream regulators, especially the membrane receptors that link cell-cell communication to Hpo signaling, remain poorly defined. This study provides both genetic and biochemical evidence that the transmembrane cell adhesion molecule Ed functions as a upstream regulator of the Hpo pathway. Evidence is provided that Ed physically interacts with Sav/Hpo and regulates the abundance of Sav at adherens junctions. Loss of Ed compromises the ability of Hpo/Wts kinase cassette to phosphorylate Yki, leading to elevated levels of nuclear Yki that drive tissue overgrowth. Ed/Sav association is facilitated by cell-cell contact, raising an interesting possibility that Ed may serve as a mechanism that links Hpo signaling to cell contact inhibition (Yue, 2012).

The atypical cadherin Ft functions as a receptor for the Hpo pathway; however, Ft mainly acts through Dachs to control the stability of Wts. Genetic study indicated that Ed does not act through Ft-Dachs to regulate Yki activity because inactivation of Dachs did not block Yki activation induced by loss of Ed. Furthermore, loss of Ed synergized with loss of Ds to induce the expression of Hpo-responsive genes, supporting a model in which Ed acts in parallel with Ds/Ft in the Hpo pathway. Several lines of evidence suggest that Ed regulates Hpo signaling, at least in part, through Sav. (1) Using coimmunoprecipitation, colocalization, and FRET assays, it was demonstrated that Ed physically interacts with Sav. (2) Deleting the Sav-interacting domain in Ed compromises its in vivo activity. (3) Ed regulates the abundance and subcellular localization of Sav both in vitro and in vivo. (4) Overexpression of Sav suppresses tissue overgrowth induced by loss of Ed. Sav is a binding partner and activator of Hpo. Hence, Ed could influence the Hpo kinase activity by regulating the abundance and subcellular location of the Sav/Hpo complex. How Ed regulates Sav stability is currently unknown; however, it was found that Sav is degraded in a proteasome-dependent manner. It is possible that binding of Ed to Sav leads to some modifications of Sav and prevents it from ubiquitin/proteasome-mediated degradation (Yue, 2012).

Sav binds Ed and Hpo through its N- and C-terminal regions, respectively, and thus may function as a bridge to bring Hpo to Ed. Indeed, enhanced Ed/Hpo association was observed in the presence of cotransfected Sav. It has been suggested that apical membrane recruitment of Hpo promotes phosphorylation of Wts. Thus, it is conceivable that Ed may regulate the Hpo kinase by recruiting Sav/Hpo complex to the apical membrane. It was found that Ed/Sav interaction requires membrane association and dimerization/oligomerization of Ed intracellular domain. As Sav also forms a dimer/oligomer, dimerization/oligomerization of Ed intracellular domain may enhance binding to Sav through multimeric interactions. It is also possible that membrane association and dimerization/oligomerization could lead to a modification of Ed intracellular domain, resulting in increased binding affinity toward Sav (Yue, 2012).

It has been shown that the Hpo pathway can mediate cell contact inhibition in mammalian cultured cells, although the underlying mechanism has remained poorly defined. Interestingly, this study found that cell-cell contact can facilitate the recruitment of Sav to Ed at the contact site. Cell-cell contact may facilitate homophilic interaction in trans and clustering of Ed intracellular domain or induce posttranslational modification of Ed C-tail at the contact site, leading to enhanced Sav association. It is proposed that regulation of Ed/Sav association may provide a mechanism for cell-cell contact to modulate Hpo signaling and tissue growth (Yue, 2012).

The mechanism by which Ed regulates Hpo signaling is likely to be more complex than simply regulating Sav/Hpo. For example, it was also observed that Ed interacts with Ex/Mer/Kibra as well as Yki. It has been proposed that enrichment of Hpo pathway components to the apical membrane domain may facilitate the activation of the kinase cassette and increase the accessibility of Yki to its kinase (Genevet, 2011). The finding that Ed facilitates the apical localization of Sav lends further support to this notion. Through interacting with multiple components of the Hpo pathway, Ed could function as a molecular scaffold to facilitate Hpo activation and Yki phosphorylation. Loss of Ed did not alter the apical membrane localization of Ex and Mer in wing discs even though overexpression of Ed in S2 cells facilitates membrane recruitment of Ex. The apical localization of Ex and Mer is likely to be mediated by other upstream components such as Ft and Crb in the absence of Ed. Indeed, Crb physically interacts with Ex, and both loss and gain of function of Crb caused mislocalization of a fraction of Ex to the basal region. It has been shown that Ex physically interacts with Yki, which may sequester Yki in the cytoplasm independent of Yki phosphorylation. The finding that Ed interacts with Yki through a domain distinct from those mediating its binding to the upstream Hpo pathway components raises a possibility that Ed may also directly sequester Yki in the cytoplasm in addition to regulating its subcellular localization through phosphorylation (Yue, 2012).

It is interesting to note that Ed is related to TSLC1, a tumor suppressor implicated in human non-small-cell lung cancer and other cancers including liver, pancreatic, and prostate cancers. Like Ed, TSLC1 also mediates cell-cell adhesion through homophilic interactions. TSLC1 interacts with MPP3, a human homolog of Drosophila tumor suppressor Discs large (Dlg) that has been implicated in the Hpo pathway, as well as DAL-1, a FERM-domain containing tumor suppressor related to Ex/Mer. Therefore, it would be interesting to determine whether TSLC1 inhibits tumor formation through the Hpo pathway (Yue, 2012).

DEVELOPMENTAL BIOLOGY

To detect the expression pattern of Ed, embryos were stained with an antibody generated against the N-terminal Ed peptide. The Ed protein is widely expressed in the epidermis and is localized to the plasma membrane. Further, Ed is found uniformly detected in all cells throughout the third instar larval eye and wing disc. The expression of aos and kek1, two other negative regulators of the Egfr pathway, is regulated by the Egfr pathway. To determine whether ed is regulated by the Egfr pathway, the expression of ed was examined in GMR-aos and sev- RasV12 eye discs. In each case, the level of ed mRNA is not affected, as revealed by either the X-gal staining of the P insertion l(2)k1102 or the ed-specific relative RT-PCR. These results indicate that ed, unlike aos and kek1, is not transcriptionally regulated by the activation of Egfr pathway (Bai, 2001).

The fred mRNA expression pattern was determined by in situ hybridization. fred shows a rather general expression pattern. In the embryo, fred is expressed in most tissues, including the central nervous system (CNS) and epidermis. In third instar larval wing and eye discs, fred is also rather uniformly expressed (Chandra, 2003).

Differential expression of the adhesion molecule Echinoid drives epithelial morphogenesis in Drosophila

Epithelial morphogenesis requires cell movements and cell shape changes coordinated by modulation of the actin cytoskeleton. A role has been identified for Echinoid, an immunoglobulin domain-containing cell-adhesion molecule, in the generation of a contractile actomyosin cable required for epithelial morphogenesis in both the Drosophila ovarian follicular epithelium and embryo. Analysis of ed mutant follicle cell clones indicates that the juxtaposition of wild-type and ed mutant cells is sufficient to trigger actomyosin cable formation. Moreover, in wild-type ovaries and embryos, specific epithelial domains lack detectable Ed, thus creating endogenous interfaces between cells with and without Ed; these interfaces display the same contractile characteristics as the ectopic Ed expression borders generated by ed mutant clones. In the ovary, such an interface lies between the two cell types of the dorsal appendage primordia. In the embryo, Ed is absent from the amnioserosa during dorsal closure, generating an Ed expression border with the lateral epidermis that coincides with the actomyosin cable present at this interface. In both cases, ed mutant epithelia exhibit loss of this contractile structure and subsequent defects in morphogenesis. It is proposed that local modulation of the cytoskeleton at Ed expression borders may represent a general mechanism for promoting epithelial morphogenesis (Laplante, 2006).

In a genetic screen for defects associated with follicle cell clones, a mutation, initially designated F72, was discovered with a novel effect on the organization of the imprints on the eggshell surface. The pattern of these imprints reflects the organization of the cells in the follicular epithelium, which secretes the eggshell and degenerates before the egg is laid. Eggs produced by females bearing mitotic follicle cell clones homozygous for this mutation display subsets of eggshell imprints organized into groups with smooth borders. When the F72 mutant clones were marked with the defective chorion 1 (dec-1) marker, which confers a distinct appearance on the eggshell secreted by the mutant cells, the dec-1-marked imprints were contained exclusively within the smooth borders, indicating that these borders occur at the interface of imprints produced by mutant and non-mutant cells (Laplante, 2006).

Consistent with the mosaic eggshell phenotype, clones of homozygous mutant follicle cells exhibit smooth borders with adjacent heterozygous or homozygous wild-type cells. Interfaces between mutant cells within the clone, however, appear normal. Interestingly, the smooth clone border is detectable only at the apical side of the epithelium, while the basal aspect of the clone displays no obvious phenotype. This mosaic phenotype also exhibits a surprising temporal profile. The smooth clone border phenotype is completely penetrant in early stage egg chambers but, during stage 10 of mid-oogenesis, the border of F72 mutant clones becomes indistinguishable from adjacent intercellular interfaces. The disappearance of the phenotype is transient, however, and by stage 11 the marked smoothness of the clone border is again readily detectable and completely penetrant, and persists for the remainder of oogenesis. Sequencing revealed a single nucleotide substitution, which generates a premature termination at codon 205 of the ed open reading frame (Laplante, 2006).

The borders of ed mutant follicle cell clones display a reduced apical circumference and apical enrichment of F-actin and the phosphorylated form of the light chain of non-muscle myosin II (p-MLC), suggesting that the juxtaposition of follicle cells with and without Ed is sufficient to trigger the assembly of an apical actomyosin cable at their interface. Based on these observations, it is proposed that the smooth, constricted border of ed mutant clones is the result of a contractile force generated by this structure. Consistent with this interpretation, ed clone borders do not exhibit this phenotype if the adjacent wild-type cells, owing to their position or developmental stage, also lack Ed. Thus, the generation of this contractile structure is a result of an interface between cells with and without Ed, rather than the loss of Ed per se (Laplante, 2006).

The apical constriction associated with the loss of Ed appears to be restricted to the Ed expression boundary itself; individual ed mutant follicle cells that do not contact the clone border do not display pronounced apical constriction. Although the apical circumference of follicle cells in the interior of ed mutant clones occasionally appears reduced, this effect is not observed in larger clones. The reduction of apical circumference observed in individual ed mutant cells may therefore be a secondary consequence of the contractile force generated at the clone border, rather than a direct effect of the absence of Ed (Laplante, 2006).

Although a smooth border has been reported previously for ed mutant clones in the wing imaginal disc, the data are the first to reveal a developmentally regulated absence of Ed in specific cell types associated with epithelial sheet movements. Ed is absent from the presumptive roof cells of the appendage primordia prior to tube morphogenesis, and from the embryonic amnioserosa prior to dorsal closure. In both cases, the resulting endogenous Ed expression borders are smooth and display features of a contractile actomyosin cable, and loss of Ed results in defects in epithelial closure. Because generation of ectopic Ed expression borders is sufficient to generate a smooth contractile intercellular interface, these defects are interpreted as a result of the elimination of the endogenous Ed expression borders between these tissues. It is proposed that the juxtaposition of cells with and without Ed at these endogenous interfaces induces local contractility of the actin cytoskeleton that in turn drives the convergence of opposing epithelial domains during morphogenesis (Laplante, 2006).

Ed does not appear to play a role, however, in the generation of the actin-rich smooth interface observed at the boundary between dorsal and ventral compartments of the wing imaginal disc. Differential expression of Ed between dorsal and ventral compartments is not detected, and ed mutant clones in either compartment exhibit smooth borders. Therefore, despite a general morphological similarity, differential Ed expression does not appear to play a role at this epithelial boundary (Laplante, 2006).

Although the data demonstrate that differential Ed expression generates a contractile interface that is required for proper appendage tube formation and dorsal closure, other forces also contribute to these processes. The involvement of multiple forces is best understood for dorsal closure where, in addition to the contractile actin cable at the epidermis/amnioserosa interface, apical constriction of the individual amnioserosa cells also drives the movement of the leading edge, particularly in the initial stages of the process. In later stages, interactions between filopodia of opposing leading edge cells also contribute to the completion of closure. Consistent with the involvement of multiple forces, the lateral epidermal edges do ultimately approach the dorsal midline in ed^MZ embryos, suggesting that the elimination of the Ed expression border specifically disrupts the actin cable, while the other forces remain functional (Laplante, 2006).

The cell movements and shape changes associated with the morphogenesis of the appendage primordia appear very similar to those observed in dorsal closure. In addition to the convergence of opposing floor cell domains to form the tube floor, the individual roof cells constrict apically, similar to the amnioserosa cells. This roof cell behavior is probably a consequence of roof cell fate determination rather than the absence of Ed, since ed mutant cells outside of this domain do not exhibit this same pronounced reduction in apical circumference. Presumably the epithelial groove generated by the coordinated apical constriction of the roof cells, together with the elongation of the floor cells, can generate the rudimentary tubes that give rise to the severely shortened and malformed appendages observed in the absence of floor closure in ed mutant primordia (Laplante, 2006).

Given the proposed role of Ed as a homophilic adhesion molecule, selective affinity may also contribute to morphogenesis. For example, as the anterior and medial floor cells elongate towards the midline of the primordium, preferential affinity for the opposing floor cells, which also express Ed, over the roof cells, which lack Ed, may favor floor cell association. In dorsal closure, Ed-mediated interactions between opposing leading edge cells could play a similar role. It is also possible that differential Ed expression may have a dual function, contributing to morphogenesis through generation of both a contractile interface and differential affinity between cell types (Laplante, 2006).

In irradiated cultured epithelia, a smooth contractile interface has been observed between apoptotic epithelial cells and their neighbors, suggesting that active extrusion of dying cells preserves the integrity of the epithelium. This effect resembles the ed mosaic phenotype, but the presence on the eggshell surface of imprints produced by ed mutant cells indicates that these cells do not die before the secretion of eggshell at the end of oogenesis. Moreover, ed mutant clones are not detectably smaller than their associated twin spots and no evidence of DNA fragmentation or the active form of the proapoptotic enzyme caspase 3 was detected in ed mutant follicle cells, confirming that the contractile border of ed mutant clones is not induced by premature cell death (Laplante, 2006).

Reduced levels and altered distribution of DE-cad and Arm are observed at the border between cells with and without Ed. By contrast, the distribution and level of DE-cad and Arm at the interfaces between ed mutant follicle cells within a clone appear normal. This observation demonstrates that, although recent evidence suggests that Ed is a component of adherens junctions, Ed is not generally required for adherens junction stability (Laplante, 2006).

A border effect on adherens junction components has also been reported in ed mutant clones in the wing disc epithelium, where it has been proposed to play a causative role in the generation of a smooth clone border by mediating cell sorting. However, at the border of ed mutant follicle cell clones, this effect is frequently mild and occasionally undetectable, whereas the contractile phenotype is completely penetrant. This difference could suggest that a functionally relevant alteration in adherens junction distribution is only occasionally reflected by diminished immunoreactivity. Alternatively, this effect on adherens junction components could be instead a consequence of contraction of the actin cable assembled at the Ed expression border. Indeed, an actomyosin-based contractile force has been proposed to be capable of disrupting adherens junctions. However, no disruption of adherens junction components at endogenous Ed expression borders were observed, raising the possibility that this effect is not involved in Ed expression border function (Laplante, 2006).

How an Ed expression border induces the local assembly of a contractile actin cable remains unclear. A potential connection between Ed and the actin cytoskeleton is suggested by the reported interaction between Ed and Canoe (Cno), which is homologous to mammalian Afadin and contains a actin filament binding domain, suggesting that Ed may function as a Nectin, the Afadin binding partner. However, in ed mosaic wing imaginal discs, Cno distribution is altered throughout ed mutant clones, not just at the border. This observation does not exclude a role for Cno in Ed function but, because this effect on Cno is not restricted to the clone border, it alone cannot explain the localized effect on the actin cytoskeleton. Interestingly, an interaction with Ed does not appear to be strictly required for proper membrane localization of Cno, as Ed is lost from the amnioserosa during dorsal closure while Cno remains detectable (Laplante, 2006).

An obvious distinguishing feature of Ed expression borders is the absence of Ed from the apposing face of the Ed-expressing population, presumably owing to the absence of trans homophilic interactions. The mechanism that removes or redistributes Ed from this interface, rather than the absence of Ed itself, might therefore mediate the border-specific effect on the actin cytoskeleton. If the machinery that removes Ed, e.g. through endocytosis, is not completely specific, such a model could also account for altered levels of DE-cad and Arm at these interfaces. Alternatively, the absence of homophilic interactions across Ed expression borders could favor the interaction of Ed with other factors, which could in turn mediate border specific effects (Laplante, 2006).

During Drosophila dorsal closure, lateral sheets of embryonic epidermis assemble an actomyosin cable at their leading edge and migrate dorsally over the amnioserosa, converging at the dorsal midline. This study shows that disappearance of the homophilic cell adhesion molecule Echinoid (Ed) from the amnioserosa just before dorsal closure eliminates homophilic interactions with the adjacent dorsal-most epidermal (DME) cells, which comprise the leading edge. The resulting planar polarized distribution of Ed in the DME cells is essential for the localized accumulation of actin regulators and for actomyosin cable formation at the leading edge and for the polarized localization of the scaffolding protein Bazooka/PAR-3. DME cells with uniform Ed fail to assemble a cable and protrude dorsally, suggesting that the cable restricts dorsal migration. The planar polarized distribution of Ed in the DME cells thus provides a spatial cue that polarizes the DME cell actin cytoskeleton, defining the epidermal leading edge and establishing its contractile properties (Laplante, 2011; full text of article).

EFFECTS OF MUTATION

Echinoid antagonizes the Drosophila EGF receptor signaling pathway

Photoreceptor and cone cells in the Drosophila eye are recruited following activation of the epidermal growth factor receptor (Egfr) pathway. echinoid (ed) is a novel putative cell adhesion molecule that negatively regulates Egfr signaling. The ed mutant phenotype is associated with extra photoreceptor and cone cells. Conversely, ectopic expression of ed in the eye leads to a reduction in the number of photoreceptor cells. ed expression is independent of Egfr signaling and Ed is localized to the plasma membrane of every cells throughout the eye disc. Evidence is presented that ed acts nonautonomously to generate extra R7 cells by a mechanism that is sina-independent but upstream of Tramtrack (Ttk88). Together, these results support a model whereby Ed defines an independent pathway that antagonizes Egfr signaling by regulating the activity, but not the level, of the Ttk88 transcriptional repressor (Bai, 2001).

ElpB1 is a gain-of-function allele of the Egfr. A genetic modifier screen was carried out for components of the Egfr pathway that dominantly enhance or suppress the rough eye phenotype caused by Elp^B1. 1X5 was isolated as an EMS induced mutation that strongly enhances the rough eye phenotype associated with Elp^B1. The dominant enhancer activity of 1X5 is similar to the effect of Gap1 or yan mutations, two known negative regulators of the Egfr signaling pathway. Consistent with the genetic interaction with Elp^B1, 1X5 also enhances the eye phenotype caused by sev-tor⁴⁰²¹Egfr, another constitutively active form of the Egfr. To define further the role of 1X5 in the Egfr signaling pathway, the genetic interactions between 1X5 and rho, a specific activator of Egfr pathway, and aos, a specific Egfr inhibitor, were examined. Interestingly, it was found that 1X5 enhances the rough eye phenotype caused by ectopic expression of rho, and suppresses the rough eye phenotype caused by misexpression of aos. Further genetic interactions between the Egfr pathway and 1X5 were also detected in the wing. 1X5 enhances the extra wing-vein phenotype caused by the overactive Elp^B1 mutation, as well as rl^SEM, a constitutively active MAPK. In addition, flies heterozygous for both 1X5 and Gap1, or both 1X5 and sty^S88, exhibit extra vein materials, although heterozygosity for either mutation alone causes no phenotype. Therefore, the genetic interactions observed between 1X5 and several components of the Egfr pathway suggest that 1X5 is a negative regulator of the Egfr signaling pathway during eye and wing vein development (Bai, 2001).

1X5 was mapped to 24D3-4 using three overlapping deficiencies. This region contains the ed gene and it was found that ed^lF20 (de Belle, 1993) fails to complement 1X5 and enhances the Elp^B1 rough eye phenotype, as well as the extra wing vein phenotype of rl^SEM. Thus 1X5 is allelic to ed. All ed mutations are pupal lethal in homozygotes with the exception of ed^slH8, which is a weaker allele. Homozygous ed^slH8, as well as ed^slH8 in combination with all other ed alleles are semi-lethal. Emerging adults have rough eyes and extra wing veins. When sectioned, 33% of ommatidia contain extra R7-like cells with small and centrally positioned rhabdomeres. To exclude the probability that these extra cells with small rhabdomeres are R8, third instar larval imaginal discs of ed^1X5/ed^slH8 transheterozygotes were stained with anti-Boss, an R8-specific antibody. Single R8 cell was seen in each mature ommatidium, confirming that the extra photoreceptor cells are indeed R7. In addition, 26% of ommatidia exhibit extra outerphotoreceptor cells while 6% of the ommatidia show reduced outer-photoreceptor cells. Further, ed^slH8 hemizygote animals have more R7 cells than ed^1X5/ed^slH8 transheterozygote animals, indicating that the ed alleles are loss of function. ed^slH8 hemizygotes have 1.68 R7 cells in average, compared with 1.34 in ed^1X5/ed^slH8 (Bai, 2001).

To determine the origins of the extra photoreceptor cells, ed^1X5/ed^slH8 transheterozygote discs were stained with the anti-Elav neural marker. Extra Elav-positive cells were first detected in rows 2 and 3, where R8/R2/R5 are located. However, these four-cell clusters contain only single R8. In addition, one or two extra Elav-positive mystery cells were detected adjacent to R3 and R4 cells, four rows of cells behind the furrow. Mystery cells will normally leave the five cell precluster and disappear; however, as in sty or yan mutants, they are transformed into neuronal photoreceptor cells in the ed mutant discs. The ed mutant phenotype was also examined during pupariation. At this stage there are four cone cells and two primary pigment cells in wild-type discs. However, 69% of ommatidia in ed^1X5/ed^slH8 transheterozygotes exhibit five or six cone cells and 10% contain three primary pigment cells. Together, the overrecruitment of photoreceptor, cone and pigment cells in ed mutants is consistent with Ed acting as a negative regulator of Egfr because previous analyses have shown that Egfr is required for differentiation of these three cell types (Bai, 2001).

Thus, loss of ed function is required for the formation of photoreceptor, cone and primary pigment cells. To determine the effect of overexpression of ed in the eye, UAS-ed was overexpressed using the GMR-Gal4 driver. GMR-Gal4; UAS-ed flies exhibit a small rough eye and a reduced number of photoreceptors; this effect correlates with the reduced number of Elav-positive cells in the eye disc. There are only four or five Elav-positive cells per cluster. In contrast, no obvious defects in the formation of cone cells were observed in response to ed overexpression, since most ommatidia still contain four Cut-positive cells. Flies carrying two copies of GMR-GAL4-driven UAS-ed exhibit complete absence of the eye. To further document the interaction between Ed and the Egfr pathway, the effect of ectopic expression of ed was examined in flies where other regulators were overexpressed. Overexpression of UAS-sty alone by GMR-GAL4 produces small rough eye. This phenotype can be partially suppressed by halving the dose of ed, and enhanced by GMR-GAL4-driven UAS-ed. Similar genetic interactions can also be observed between ed and kek1. The rough eye phenotype caused by GMR-GAL4-driven UAS-kek1 is enhanced by GMR-GAL4-driven UAS-ed. Therefore ed, like sty and kek1, is a repressor of Egfr signaling during eye development. Similarly, during wing vein development, ed genetically interacts with several components in the Egfr pathway. Flies of ed^1X5/ed^slH8 have increased size of wing and extra wing vein. However, ectopic expression of ed using MS1096 GAL4 results in severe reduction in the size of the wing, ranging from one quarter to one fifth normal wing size. In addition, there is no vein material present (Bai, 2001).

Ed contains six Ig domains and a 315 amino acid intracellular domain. To determine whether the intracellular domain of Ed is required for the repression of the Egfr signaling, UAS-edDeltaintra flies were created. Overexpression ofUAS-edDeltaintra using GMR-GAL4 has no phenotypes in the eye, indicating that the cytoplasmic domain of Ed is required for the repression of the Egfr signaling pathway (Bai, 2001).

To determine where in the RAS/RAF/MAPK signaling pathway ed acts, a number of genetic epistasis experiments were conducted. sev^d2 is a loss-of-function sevenless (sev) allele, and sev^d2 mutant flies lack R7 cells. Although ommatidia within ed^1X5/ed^slH8 mutants contain an average of 1.34 R7 cell, ommatidia within a sev^d2; ed^1X5/ed^slH8 double mutant contain an average of 1.37 R7 cells. This demonstrates that in ed mutants, the formation of supernumary R7 cells is independent of sev function. In addition, ed^1X5 enhances the rough eye phenotype caused by overexpressing constitutive active forms of either the Egfr, RAS1, or RAF. Conversely, ed^1X5 suppresses the rough eye phenotype caused by overexpressing dominant negative RAS1. While 61% of ommatidia in a sev-Ras^N17/+ mutant lack R7 cells, only 10% of ommatidia in ed^1X5/ed^slH8; sev-Ras^N17/+ double mutants lack R7 photoreceptors. In addition, at 25^o C, ed^1X5 also rescues the lethality of Raf^HM7, a temperature-sensitive Raf allele. Therefore, ed acts either downstream of the Ras/Raf pathway or in parallel (Bai, 2001).

To determine whether Ed acts in the nucleus, flies double mutant for ed;pnt, ed;yan or ed;sina were created. pnt^Delta88/pnt¹²⁷⁷ and sev-yan^ACT/+ ommatidia contain an average of 0.69 and 0.05 R7 cells, respectively. However, ed^1X5/ed^slH8; pnt^Delta88/pnt¹²⁷⁷ and ed^1X5/ed^slH8; sev-yan^ACT/+ ommatidia contain an average of 1.44 and 1.01 R7 cells, respectively. Strikingly, ed^1X5/ed^slH8; sina²/sina³ ommatidia contain an average of 1.29 R7 cells, as compared with 0.01 R7 cells in the sina²/sina³ mutant. Therefore, in ed mutants, the formation of supernumary R7 cells is independent of sina function. Finally, loss of ttk activity has been shown to produce ectopic R7 cells in a sina-independent manner. To determine whether ed acts downstream of ttk, ttk was overexpressed in ed mutants. Overexpression of Ttk88 under the control of the GMR enhancer completely inhibits photoreceptor cell development, while overexpression of Ttk88 under the control of the sev enhancer only deletes R3, R4 and R7 photoreceptors. However, this Ttk88-mediated neuronal repression cannot be suppressed by removing ed activity, indicating that ed acts upstream of ttk to specify R7 development. Together, these genetic epistatic analyses suggest that ed acts either parallel or downstream of Ras, Raf, pnt, yan and sina, but upstream of ttk to specify R7 cell fates (Bai, 2001).

Genetic epistatic analyses suggest that ed acts upstream of ttk88 to specify R7. ed might regulate ttk88 mRNA expression or Ttk88 protein levels. Alternatively, ed might regulate the activity of Ttk88 through protein modification, i.e., phosphorylation. To determine whether ed regulates ttk expression, the expression of ttk was examined in ed mutant disc using the X-gal staining of the P-element insertion ttk⁰²¹⁹; no obvious changes were detected. Furthermore, Ttk88 is expressed at high levels in the cone cells but is not expressed in developing photoreceptor cells. To determine whether ed regulates Ttk88 protein levels, Ttk88 levels were examined in ed and GMR-Gal4; UAS-ed eye discs. In each case, the level of Ttk88 is unaffected. Together, these results suggest that Ed does not regulate ttk88 mRNA expression or Ttk88 protein stability (Bai, 2001).

To determine in which cells ed is required, ey-FLP was used to generate clones of homozygous ed^slA12 mutant cells in a sev^d2 background. No R7 cells develop in the sev^d2 background. Fifty-four mosaic ommatidia that contain R7-like cells were scored. Among them, 57% of the R7-like cells were ed minus, while 43% were ed plus. Similar results were obtained when ed mutant clones were generated in sina and sev-yan^ACT mutant backgrounds. The observation that R7 cells can be derived from either wild-type or ed mutant cells, leads to the proposal that the ed mutation acts cell non-autonomously in the generation of supernumerary R7 cells (Bai, 2001).

Ed is uniformly expressed in the follicle cells during stage 1-10 oogenesis. To determine whether ed acts during oogenesis in the establishment of Egfr-dependent dorsal/ventral polarity, the eggs derived from ed^slH8/Df(2L)ed-dp females were examined. These females are fertile and do not exhibit any overt morphological defects (Bai, 2001).

Since loss-of-function mutations in many cell adhesion molecule have subtle mutant phenotypes, UAS-ed was overexpressed in the follicle cells using the GAL4 drivers T155 or CY2. The eggs derived from such females have completely normal dorsal appendages suggesting that Ed does not interfere with Egfr signaling in follicle cells (Bai, 2001).

It is concluded that ed genetically interacts with several components in the Egfr pathway. Flies of ed mutant produce extra photoreceptor and cone cells. Conversely, ectopic overexpression of ed in the eye leads to reduction of photoreceptor number. Ed acts by converging on Ttk88, the most downstream component known in EGF receptor signaling. These results not only demonstrate the active role of an adhesion molecule in the Egfr signal transduction pathway but also identify a previously unknown regulatory mechanism (Bai, 2001).

Ed is expressed in every cell of the eye disc. In addition, genetic analysis demonstrates that ed acts in a cell nonautonomous manner to generate extra R7 cells. If Ed transmits the negative signal from the sending cell via homophilic interaction to the receiving cell, loss of ed in either sending or receiving cells would result in the same phenotype, owing to the failure to receive the inhibitory signal. Therefore the extra R7 cells found in the receiving cells could be either wild type or mutant for ed. However, if Ed transmits the negative signaling via heterophilic interaction, ed is required only in the sending cells but not the receiving cells. Therefore, the extra R7 cells found in the receiving cells could be either wild type or mutant for ed. Alternatively, Ed might act as a ligand that activates an unidentified receptor on receiving cells. All three models are consistent with the results showing that ed functions cell nonautonomously. However, only the homophilic interaction model would require that the cytoplasmic domain of Ed be required in both the sending and receiving cells. Since the cytoplasmic domain of Ed was found to be required for the repression of the Egfr pathway, the homophilic interaction model between Ed molecules to specify photoreceptor cell formation is favored (Bai, 2001).

Echinoid limits R8 photoreceptor specification by inhibiting inappropriate EGF receptor signalling within R8 equivalence groups

The activity of EGF receptor must be carefully regulated in a variety of ways to control the time, pattern, intensity and duration of signalling. The cell surface protein Echinoid is required to moderate Egfr signalling during R8 photoreceptor selection by the proneural gene atonal during Drosophila eye development. In echinoid mutants, Egfr signalling is increased during R8 formation, and this causes isolated R8 cells to be replaced by groups of two or three cells. This mutant phenotype resembles the normal inductive function of Egfr in other developmental contexts, particularly during atonal-controlled neural recruitment of chordotonal sense organ precursors. It is suggested that echinoid acts to prevent a similar inductive outcome of Egfr signalling during R8 selection (Rawlins, 2003a).

atonal (ato) can be overexpressed in the developing R8 precursor using an R8 specific Gal4 driver (109-68Gal4) to drive UAS-ato (ato^109-68). Although such overexpression does not alter the expression pattern of ato beyond boosting and extending it within R8 cells, ato^109-68 exhibits several defects in eye development. One of these defects is R8 twinning, indicating failure of R8 resolution within the equivalence group. This is unexpected because overexpressing ato in R8 should increase Notch-mediated lateral inhibition, not reduce it. This non-autonomous effect therefore suggests that undefined signalling mechanisms that impinge on R8 resolution are being affected by ato misexpression (Rawlins, 2003a).

To investigate the process of R8 selection further, ato^109-68 was used as the basis of a screen for genetic modifiers to isolate mutations that affect R8 resolution. E(ato^109-68)4.12 was isolated as a second site mutation that dominantly enhances ato^109-68 when present in one copy. E(ato^109-68)4.12 itself was found to be homozygous viable with a strong rough eye phenotype. A lethal allele of ed (ed^lH23) fails to complement E(ato^109-68)4.12: transheterozygous flies are viable, with rough eyes, suggesting that E(ato^109-68)4.12 is an allele of ed. Sequencing the ed gene from E(ato^109-68)4.12 homozygotes reveals in the predicted extracellular domain a single amino acid substitution compared with the published Ed protein sequence and with that of the parent line used for the mutagenesis. This mutation was therefore renamed ed^4.12 (Rawlins, 2003a).

ed is shown to be an Egfr antagonist that inhibits Egfr protein itself or a closely associated component of the signalling pathway. The Egfr signalling pathway functions in diverse inductive events during development. Clearly such a commonly deployed pathway must be tightly regulated to prevent inappropriate inductive events occurring at other times and locations. Analysis of ed suggests that it is a mediator of such regulation. Although Egfr signalling is not required directly for wild-type R8 fate specification, derepressed signalling in ed mutants induces multiple R8 cells (the R8 twinning phenotype). ed is notable, therefore, because its mutation exposes a new and unexpected outcome of signalling (R8 specification), rather than expansion of an existing Egfr function (Rawlins, 2003a).

The finding that Egfr signalling can induce R8 specification even though it does not normally do so may resolve the contradictory evidence for Egfr function in R8 specification. Recent studies show that R8 cells can be specified in the absence of Egfr, albeit aberrantly. Yet other results strongly suggest a link between R8 selection and Egfr/Ras signalling; expression of activated Ras has been shown to result in strong ato upregulation and ectopic R8 cells and that argos misexpression inhibits R8 formation. The latter findings may allude not to an Egfr requirement during R8 selection, but to the ability of aberrant Egfr signalling to induce R8s (Rawlins, 2003a and references therein).

Bai (2001) suggested that ed acts downstream of the Egfr target gene pnt-P1 in R7 specification; based on this, a hypothetical parallel signalling pathway that antagonizes Egfr was proposed. The current observations are more consistent with membrane-associated Ed interacting directly with Egfr or with immediate downstream components. Increased activated MAPK and pnt-P1 expression is observed in ed mutants, which suggests that ed acts upstream of MAPK activation in the Egfr signalling pathway. Moreover, forced expression of pnt-P1 or activated Raf can bypass the inhibitory function of ed, whereas spi cannot. This is entirely consistent with the finding that Ed is colocalized with Egfr at the cell surface (unpublished observation cited in Rawlins, 2003a) and that Ed can bind Egfr protein and is phosphorylated in response to Egfr activation (Spencer, 2003). Moreover, these findings are consistent with known features of the L1 family of cell adhesion molecules (CAMs), with which Ed protein shares extensive homology in its extracellular portion (Bai, 2001). L1 CAMs are involved in the control of axon outgrowth, where they are associated with regulation of Fgfr and Egfr activity). In brain extracts, L1 physically associates with the MAPK cascade components Raf1 and Erk2, while in vitro Erk2 can phosphorylate the L1 cytoplasmic domain. Interestingly, clonal analysis suggests both autonomy and nonautonomy, suggesting that Ed might be able to interact with Egfr in trans as well as in cis. If so, this might imply an association between the extracellular domains of the two proteins. The molecular mechanism of L1 function is unclear, although its endocytosis may be important for downstream events. This may have implications for Ed function. However, the intracellular domain of Ed is distinct from that of L1 and there is evidence that tyrosine phosphorylation within this domain is important for function, and that Ed may act on Egfr via an interaction with the phosphatase encoded by corkscrew (Rawlins, 2003a and references therein).

Unlike negative regulators such as argos, mutation of ed does not alter the pattern of Egfr activation, just the intensity, suggesting that the function of ed is to limit the level or duration of activation. In support of this, Spencer (2003) provide biochemical evidence that the inhibitory activity of Ed is dependent post-translationally on Egfr signalling, thereby providing a negative feedback mechanism to damp down Egfr signalling. ed does not completely suppress Egfr signalling around the morphogenetic furrow, presumably because such signalling has some role to play. Indeed this wild-type level of signalling may be important for mediating the proposed inhibitory Egfr/Ras/Raf process in which one row of IGs helps to pattern the next row. Such activity occurs at the same time that R8 fate must be restricted within the IGs by lateral inhibition. Given the inductive nature of Egfr signalling generally, such signalling could therefore interfere with R8 resolution. Therefore, in R8 proneural clusters ed must suppress a potential outcome of Egfr signalling in the morphogenetic furrow (induction of R8 fate) rather than the signalling itself (Rawlins, 2003a).

Ed protein at the cell surface may provide a contact mechanism that preferentially inhibits short range R8 inductive signalling rather than long-range signalling in which the diffusible antagonist Argos may participate. This may explain why simply increasing EGF receptor activity does not normally cause R8 twinning. For example, mutations of other negative regulators of Egfr (argos, sprouty, kekkon I) do not show R8 twinning, despite raising levels of Egfr signalling. Neither does increased expression of Spi ligand. The wild-type function of ed must be sufficient to quash any level of Egfr signalling specifically in the context of R8 selection (Rawlins, 2003a).

Why does Egfr signalling induce R8 fate in ed mutants? It may reflect the general inductive ability of Egfr in the context of cells primed to become R8s. An alternative, however, is suggested by the close relationship between Egfr and ato function. The wild-type level of Egfr signalling in the morphogenetic furrow is dependent on ato. Moreover, increased ato expression in R8 precursors can provoke R8 twinning in a non-autonomous manner, presumably by hyperactivation of Egfr signalling. This relationship between ato and Egfr is reminiscent of the normal function of ato during chordotonal SOP selection. In the femoral chordotonal organ, ato triggers SOP recruitment by activating Egfr signalling. In turn, Egfr signalling activates ato and SOP fate in uncommitted cells in a manner that is suggestive of the aberrant effect of Egfr on R8 specification in ed mutants. It is speculated therefore that R8 twinning might be an aberrant outcome of an ato-Egfr neural recruitment network in the wrong time and place. It is notable that chordotonal recruitment is unaffected in ed mutants. Thus, by modulating Egfr signalling specifically in the eye, ed enables the ato-Egfr network to be customised to the specific needs of R8 precursor patterning, where Egfr signalling must be activated by ato but supernumerary R8 specification must be prevented. A key principle of development is the continual redeployment of a handful of intercellular signalling pathways such as Egfr. As such, much of development must involve similar instances of suppression of potential developmental outcomes that would result from the re-use of signalling networks (Rawlins, 2003a).

Friend-of-Echinoid limits the number of sensory organ precursors in the wing disc and interacts with the Notch signaling pathway

In order to study the function of fred, the heritable and inducible double-stranded RNA-mediated interference (RNAi) method was used. For this study, transcript sequence of fred was cloned as a dyad symmetric molecule in the pUAST vector and transgenic lines established. Expression of the construct was induced by crossing the transgenic lines to tissue- and/or stage-specific GAL4 driver lines. Transcription of a dyad symmetric molecule results in a RNA that snaps back to give rise to a dsRNA with a hairpin loop; this mediates the degradation of the corresponding endogenous mRNA. A 638-bp region of fred was selected for this analysis based on minimal similarity to ed sequence (Chandra, 2003).

The effectiveness of the UAS-fred RNAi construct in mediating the degradation of fred transcripts was tested in third-instar larval wing discs by using the pannier-GAL4 (pnr-GAL4) driver. pnr-GAL4-mediated expression of the UAS-fred RNAi construct in the dorsal-most region of the wing disc results in a strong reduction of fred mRNA. Staining for ed mRNA or protein did not show any decrease in expression, verifying the specificity of the fred RNAi construct. Many of the pnr-GAL4/UAS-fred RNAi larvae develop into adults that display a range of phenotypes, including a loss of epithelium resulting in a smaller notum and scutellum and loss or duplication of sensory bristles. These phenotypes are generally more severe when these flies are raised at 29ºC. Here, loss of epithelium is so extensive that approximately one-third of the eclosed adults have holes in the dorsal cuticle. In addition, a third of the pharate adults fail to eclose and display defects in dorsal cuticle. A similar phenotype is also observed by using the Eq-GAL4 driver, which directs expression in the anterior region of the future notum, with a stronger expression in the anterior midline. Degradation of fred mRNA in this region also results in the loss of epithelial tissue, resulting in a pinched appearance of the nota (Chandra, 2003).

The loss of epithelia and the misspecification of sensory bristles might indicate a role for fred in sensory organ formation and/or in cell survival. To test these possibilities, sensory organ formation was followed by analyzing the expression of the SOP markers neur-A101lacZ and SRV-lacZ. A101 is an early marker for the SOP cell fate; in wild type wing discs, it labels a single nucleus in each proneural cluster. SRV-lacZ is a sc lacZ reporter construct that specifically labels the SOPs. Suppression of fred function in the dorsal-most part of the wing discs results in a dramatic increase in the number of cells expressing the A101 SOP marker. The ectopic A101-positive cells are generally arranged in a single large, continuous patch. Ectopic expression of the SOP marker A101 was also observed when another GAL4 driver, apterous-GAL4 (ap-GAL4), which drives expression in almost the entire dorsal compartment of the wing disc, was used. Ectopic SOPs were also obtained in the UAS-fred RNAi; pnr-GAL4/SRV-lacZ wing discs. Testing, using the Df(1)sc10-1 line. was carried out to determine whether the proneural genes ac and sc are required for the specification of these ectopic SOPs. Males hemizygous for this deficiency lack both ac and sc and have a bald nota as no SOPs are specified. The induction of ectopic SOPs upon fred suppression requires ac and sc as male pharate adults and the occasionally eclosed adults of the genotype Df(1)sc10-1/Y; UAS-fred RNAi/pnr-GAL4 show no bristles and have a near wild type notum morphology. The specification of ectopic SOPs upon fred suppression is accompanied by extensive cell death, as revealed by acridine orange staining (Chandra, 2003).

The effects of fred RNAi was also examined in the developing eye. At 25ºC, flies transheterozygous for UAS-fred RNAi and the eye-antennal disc specific driver line GMR-GAL4 show fused ommatidia and mispositioned and/or missing bristles. Again, this phenotype is enhanced if flies are raised at 29ºC (Chandra, 2003).

The Notch signaling pathway is involved in limiting the SOP fate to a single cell within each proneural cluster. Since degradation of fred mRNA leads to formation of ectopic SOPs, it was of interest to see if the Notch signaling pathway genes functionally interact with fred in this process and, thus, may modulate the fred RNAi phenotype. To this end, four Notch pathway genes, Notch (N), Suppressor of Hairless [Su(H)], Hairless (H), and E (spl) m7 were tested for genetic interactions with fred (Chandra, 2003).

Overexpression of Notch leads to a loss of sensory organs and hair to socket transformation. Expression of a UAS-Notch (UAS-N) construct with pnr-GAL4 results in flies that show loss of most of the bristles from the dorsal-most region of the thorax. In addition, occasionally, bristle to socket transformation is observed. When fred dsRNA and Notch are expressed simultaneously by using the pnr-GAL4 driver, the flies show a phenotype that is intermediate between that of the two individual phenotypes. Although overexpression of Notch could suppress the cuticular holes and ectopic microchaeta formation, the thoraces of these flies still had some of the phenotypes associated with RNAi-mediated suppression of fred, such as a pinched notum and a smaller scutellum (Chandra, 2003).

Following Notch activation, the N^icd translocates to the nucleus, where it forms a complex with the transcription factor Su(H) and switches on the transcription of E (spl) complex. Loss of Su (H) results in the formation of ectopic sensory bristles, while overexpression results in suppression of sensory organ specification. Ectopic expression of Su(H), using the pnr-GAL4 driver, results in the absence of sensory organs in the medial region of the notum. Simultaneous expression of both Su(H) and the fred RNAi construct in the pnr domain produces flies that are similar to the UAS-Su(H); pnr-GAL4 flies. Moreover, the ectopic cell death associated with fred suppression was alleviated by Su(H) overexpression. Thus, ectopic expression of Su(H) effectively suppresses the phenotype associated with the reduction of fred function. The effect was tested of loss of Su(H) function on the fred RNAi phenotype. Reduction of one functional copy of Su (H) in UAS-fredRNAi/pnr-GAL4 flies did not show a consistent modulation of the phenotype, indicating that this assay might not be sensitive enough. However, eye morphogenesis has been proven to be very sensitive to dosage-sensitive interactions. Therefore, the effect of loss of one functional Su (H) copy on the rough eye phenotype generated by expression of UAS-fred RNAi was tested in eye with the GMR-GAL4 driver. A consistent enhancement of the fred RNAi induced rough eye phenotype was observed upon decreasing Su (H) function (Chandra, 2003).

H antagonizes Notch target gene activation by binding to the Notch signal transducer, Su(H). Accordingly, overexpression of H phenocopies reduction of Notch activity. Ectopic expression of H in the pnr domain results in the formation of multiple/split bristles and loss of epidermal tissue. This phenotype is enhanced in animals with suppressed fred activity in the pnr domain. Functional interactions between H and fred are also evident in the eye. UAS-H/GMR-GAL4 flies have eyes that are slightly smaller along the anterior-posterior axis and show ommatidial fusion and interommatidial bristle tufting, as well as bristle loss. When fred activity is suppressed in this genetic background, there is an enhanced disruption of the eye morphology. Ommatidia lack definition, bristle tufting is more severe, and loss of bristles is observed (Chandra, 2003).

Among the best characterized targets of Notch signaling in Drosophila are the seven Enhancer of split [E (spl)] complex genes. Activation of the Notch signaling pathway results in the activation of the expression of various E(spl) complex genes. Overexpression of E (spl)m8, E (spl)m7, E (spl)mß, E (spl)mgamma, E (spl)m 3, and E (spl)mDelta in wing discs results in loss of sensory organs. To determine whether the phenotype associated with suppression of fred could be modulated by expression of an E(spl) complex gene, E (spl) m7 was expressed simultaneously with fred dsRNA using the pnr-GAL4 driver. Flies that overexpress both fred dsRNA and E (spl) m7 are indistinguishable from those expressing only E (spl) m7. Third instar larval wing discs from these crosses were also analyzed for A101-lacZ expression. Ectopic expression of E(spl)m7 by pnr-GAL4 results in the loss of dorsocentral and scutellar SOPs, while suppression of fred activity results in large domains of A101-positive cells. Notably, wing discs of UAS-E(spl)m7; UAS-fred RNAi/pnr-GAL4: A101-lacZ larvae show the same SOP pattern as UAS-E(spl) m7; pnr-GAL4: A101-lacZ larvae. Therefore, ectopic expression of E (spl)m7 suppresses the phenotype associated with the reduction of fred in the wing disc (Chandra, 2003).

Whether this is also the case in the developing eye was also tested. Degradation of fred mRNA in the eye with GMR-GAL4 results in a rough eye phenotype with missing or duplicated bristles and fused ommatidia. Ectopic expression of E (spl) m7 by GMR-GAL4 results in the loss of most of the bristles in the eye. While the ommatidia remain highly organized, bristle sockets are present only infrequently or are entirely missing. If present, sockets are mispositioned and sometimes duplicated. The phenotype of eyes of animals expressing both E (spl) m7 and fred ds RNA under the control of GMR-GAL4 is very similar to the phenotype of UAS-E(spl)m7/GMR-GAL4 flies, with the exception of a few fused ommatidia that can still be observed in the posterior part of the eye (Chandra, 2003).

fred shares high sequence similarity with ed. fred and ed are both uniformly expressed in third-instar larval eye and wing discs. To address the possibility that ed and fred are functioning in close concert, a dosage-sensitive genetic interaction assay was employed. ed^2B8 is an amorphic allele of ed. Flies carrying only one functional copy of ed and one copy of the GMR-GAL4 driver (ed^2B8/GMR-GAL4) show near wild-type morphology. RNAi-mediated suppression of fred in the developing eye results in a mild rough eye phenotype. In contrast, suppression of fred in eye-antennal discs of animals with only one functional allele of ed (ed^2B8/GMR-GAL4; UAS-fred RNAi/+) leads to a severe rough eye phenotype, which is easily distinguishable from that of GMR-GAL4; UAS-fred RNAi flies. The ommatidial fusion seen in GMR-GAL4; UAS-fred RNAi eyes is significantly enhanced, and there is increased bristle loss as well as pitting and scarring of the ommatidia of ed^2B8/GMR-GAL4; UAS-fred RNAi flies (Chandra, 2003).

Since ed has been shown to be a negative regulator of the Egfr pathway, tests were performed for dosage-sensitive interaction with members of this pathway. Gap1 (GTP-activating protein) is a negative regulator of the Egfr pathway. Egfr signaling is transduced by the Ras/Raf/MAP kinase cascade. Gap1 inactivates RAS1 (RAS1 is activated by exchanging GDP for GTP) by stimulating its intrinsic GTPase activity. Reduction of Gap1 in the GMR-GAL4 background does not result in any eye abnormality. Reduction of Gap1 in UAS-fred RNAi/+; GMR-GAL4/+ flies results in a moderate enhancement of the rough eye phenotype seen in UAS-fred RNAi/+; GMR-GAL4/+ flies. This enhancement, however, is not very consistent since only 30% of UAS-fredRNAi/+; GMR-GAL4/+; Gap1^B2/+ flies show increased roughness and the remaining 70% show no significant change. Pointed is a downstream effector of Egfr signaling pathway. GMR-GAL4/+ pnt^{Delta 88}/+ flies have a normal eye morphology. UAS-fred RNAi/+; GMR-GAL4/+; pntDelta 88/+ flies show a suppression of the rough eye phenotype caused by fred RNAi. Again, this suppression was not very consistent since only 40% of the UAS-fred RNAi/+; GMR-GAL4/+; pntDelta 88/+ flies showed this suppression. While genetic interactions are observed between fred and the two members of the Egfr pathway, these interactions are consistently weaker than that observed with the Notch signaling pathway. Egfr activity was monitored by staining for the doubly phosphorylated mitogen-activated protein (MAP) kinase (dp-ERK). While the wild type expression pattern of dp-ERK could be consistently detected in the wing discs, a significant change in the dp-ERK expression is not detected in the wing discs of either UAS-fred RNAi; pnr-GAL4 or UAS-fred RNAi; ap-GAL4 larvae (Chandra, 2003).

Therefore, using inducible RNAi, it has been shown that fred function is required in eye morphogenesis and to restrict SOP cell fate in wing disc. Suppression of fred function in the developing wing disc results in ectopic SOPs, as revealed by the SOP markers, neur-A101-lacZ and SRV-lacZ. In the wing discs of the mid to late third-instar larva, only few SOPs are present. However, ap-GAL4-driven degradation of fred mRNA results in specification of a continuous patch of A101 lacZ-expressing cells in the wing pouch region. In the case of pnr-GAL4-driven fred mRNA degradation, SOPs are induced at positions where, in the wild type wing disc, no SOPs yet exist. Similar results are obtained with SRV-lacZ, a SOP marker. However, normally, SOPs do form in these regions of the wing disc at later stages of development. Thus, suppression of Fred function may result in precocious formation of SOPs. Moreover, the presence of the ectopic SOPs in large, continuous patches, without any intervening epidermal cells, indicates a disruption of the process of lateral inhibition. Adult flies of the UAS-fred RNAi/+; pnr-GAL4: A101neur-LacZ/+ genotype show a moderate increase in the number of microchaeta. These extra microchaeta could originate from the ectopic SOPs. Furthermore, frequent bristle duplications are also observed. These phenotypes suggest that Fred function might be required during SOP specification and bristle development (Chandra, 2003).

In these experiments, specific regions of the wing disc appear to be more sensitive to suppression of Fred function, as indicated by the positions occupied by ectopic SOPs. While pnr-GAL4 drives expression in the dorsal-most region of the wing disc, only some regions in the pnr domain of wing discs of UAS-fred RNAi/+; pnr-GAL4: A101neur-LacZ/+ larvae show ectopic expression of the SOP markers, A101 and SRV-lacZ. The same observation is made by using ap-GAL4. ap-GAL4 drives expression of UAS constructs in almost the entire dorsal domain; however, in ap-GAL4/+; UAS-fred RNAi: A101neur-LacZ/+ third-instar-larvae, ectopic expression of the SOP marker A101-lacZ is only detected in a part of that region. These observations might point to a higher requirement for fred function in certain regions of the wing disc and/or slightly different levels in the expression by the respective GAL4 drivers that would result in different levels of fred mRNA degradation (Chandra, 2003).

RNAi-mediated suppression of fred also results in an increase in cell death. Presently, it is not clear whether this is a direct or indirect consequence of cell fate changes associated with the formation of ectopic SOPs, which subsequently undergo cell death, or if there is a separate requirement for fred function in epidermal cells. However, the strong suppression of cell death upon overexpression of Su(H) and the wild type morphology of the notum of males lacking ac and sc strongly suggest that the ectopic cell death is associated with the change in cell fate (Chandra, 2003).

The observations that changes in the activity of four genes of the Notch signaling pathway can either suppress or enhance the phenotypes associated with the suppression of fred function suggest that fred is functioning in close concert with the Notch signaling pathway. Reduction in the activity of a Notch signaling pathway gene, Su(H) results in an enhancement of the fred RNAi phenotype. In contrast, ectopic expression of Notch signaling pathway genes, Notch, Su(H), and E(spl)m7 suppresses, to different degrees, different aspects of the fred RNAi phenotype in the developing wing, thorax, and eye. In contrast, overexpression of Hairless (a negative regulator of the Notch pathway) enhances the phenotypes induced by Fred suppression. It is presently not clear whether Fred defines a separate pathway for SOP determination or if it shares downstream components of the Notch signaling pathway. The remarkable degree to which ectopic expression of an E(spl) complex bHLH transcription factor results in a nearly complete suppression of phenotypes associated with fred degradation strongly supports the idea of very close functional interactions. These observations, furthermore, raise the possibility that E(spl) complex genes and/or other genes of the Notch signaling pathway act downstream of fred function (Chandra, 2003).

Reduction in ed gene dosage results in a very pronounced, dominant enhancement of the fred-RNAi eye phenotype despite the fact that ed^2B8 has no dominant visible phenotype. This suggests that ed and fred closely interact in processes that require Fred function. The similarity in protein structure and overlapping expression patterns would support such a functional interaction and may also point to the possibility of functional redundancy. Both Ed and Fred contain highly similar Ig C2 domains in their respective extracellular regions. Ig C2 domains are frequently involved in homophilic or heterophilic interactions with other Ig domain containing adhesion molecules. Thus, it is possible that Fred and Ed might communicate via interactions of their extracellular domains. Future research will have to address this possibility (Chandra, 2003).

The weak genetic interaction observed between fred and two members 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).

In summary, 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 H enhances the fred RNAi phenotypes. Thus fred, a paralogue of ed, is a new gene that shows close genetic interactions with the Notch signaling pathway (Chandra, 2003).

Echinoid facilitates Notch pathway signalling during Drosophila neurogenesis through functional interaction with Delta

The Notch intercellular signalling pathway is important throughout development, and its components are modulated by a variety of cellular and molecular mechanisms. Ligand and receptor trafficking are tightly controlled, although context-specific regulation of this is incompletely understood. During sense organ precursor specification in Drosophila, the cell adhesion molecule Echinoid colocalises extensively with the Notch ligand, Delta, at the cell membrane and in early endosomes. Echinoid facilitates efficient Notch pathway signalling. Cultured cell experiments suggest that Echinoid is associated with the cis-endocytosis of Delta, and is therefore linked to the signalling events that have been shown to require such Delta trafficking. Consistent with this, overexpression of Echinoid protein causes a reduction in Delta level at the membrane and in endosomes. In vivo and cell culture studies suggest that homophilic interaction of Echinoid on adjacent cells is necessary for its function (Rawlins, 2003b).

Therefore, both in vivo and in culture Ed protein is strongly associated with Dl at the cell membrane and in the early endosome compartment. Several lines of evidence suggest that Ed self associates in trans. Ed expression promotes the adhesion of cultured cells, while genetic clonal analysis shows that in vivo Ed protein cannot accumulate at the cell membrane if it is absent from the adjacent cell. Moreover, this genetic analysis suggested that such a trans interaction might be important for function (Rawlins, 2003b).

Ed is not essential for Notch signalling but has a modulatory effect. The basis of this effect must be relatively subtle, since no strongly visible difference is found in expression pattern, level, or subcellular localization of Dl, N or E(spl) in ed mutant clones. The idea is favored that Ed influences PNC resolution as part of the specific process that drives the singling out of individual SOPs. In other words, it is a part of a 'symmetry breaking' apparatus. There are two lines of evidence to suggest that Ed functions to inhibit the transition from PNC cell to SOP. (1) No more than four SOPs are selected from each PNC even in null ed alleles. (2) ed interacts particularly strongly with ase, which is expressed on the transition from PNC to SOP. It is suggested that the role of ed is analogous to that proposed for sca. Based on analysis in the eye, it is envisaged that singling out causes several cells to begin to become resistant to Dl ('pre-SOPs'), but a specific genetic mechanism involving sca and gp150, encoding a leucine-rich repeat (LRR) protein that is required for viability, fertility and proper development of the eye, wing and sensory organs, causes all but one of these unwanted SOPs to revert and once again become responsive to Dl from the selected precursor. It is hypothesized is that, like sca, ed functions to promote N receptor activation in these pre-SOPs. Despite these similarities between sca and ed function, genetic evidence suggests that they take part in parallel processes. Moreover, Sca and Gp150 are located in late endosomes, whereas Ed is located at the membrane and in early endosomes (Rawlins, 2003b).

In vivo and in cultured cells, Ed protein colocalizes very strongly with Dl in cis, both at the membrane and in early endosomes. It is possible that there is a direct molecular interaction between the two proteins, but no evidence has yet been found for this. Such an association may require Ed-Ed homophilic binding. Nevertheless, colocalization suggests a close and specific association with Dl-N signalling. One possibility is that Ed promotes Dl function in the 'true' SOP, leading to more efficient suppression of the emergence of unwanted SOPs. Cis-endocytosis of Dl into the signalling cell is apparently required for activation of the Notch receptor, and one could envisage that ed may enhance this process in the SOP. This is supported by the colocalization of Ed with N and Dl during N activation as observed in this study's cell culture analysis (Rawlins, 2003b).

An alternative is that ed may inhibit Dl activity in recipient (non-SOP) cells. There is evidence that such reduction of Dl activity may promote unidirectional signalling in two ways: (1) it would free an SOP from inhibition by surrounding cells; (2) it has been suggested that Dl in recipient cells antagonizes their response to trans signalling, perhaps by cis association of Dl and N. Therefore, Ed inhibition of this antagonistic function of Dl would make non-SOP cells more vulnerable to signalling from the SOP. No difference is seen in Dl distribution and level in ed mutant clones, but it is suspected that this might only be apparent in the pre-SOPs. However, after overexpression of Ed, a striking and specific decrease in Dl is observed both at the membrane and in vesicles. Remarkably, this correlates with SOP loss, which is the opposite phenotype to that normally expected for loss of Dl. Thus, Ed function may be connected to the downregulation of Dl in recipient cells. Proteolysis and endocytosis of Dl have both been implicated as causing its downregulation. It is feasible that Ed promotes one of these processes, for example by helping to present Dl to Kuzbanian for cleavage (Rawlins, 2003b).

ed mutants have twinned R8 photoreceptors in the eye and additional es organ SOPs everywhere. A priori one would imagine these phenotypes to have the same genetic and mechanistic basis. They appear, however, to indicate the interaction of ed with two different signalling pathways. Ed negatively regulates Egfr signalling (through direct interaction with pathway components) during R8 specification. This is in contrast to the role of Ed during es organ specification, where it modulates Notch pathway signalling. There are several other reasons for concluding that the R8 and SOP phenotypes of ed mutants, although superficially similar, have different origins. The latter, but not the former, is sensitive to overexpression of Ed protein. For R8, this is explained because Ed is regulated by EGFR post-translationally and so absolute protein levels are unimportant. Sensitivity of SOP singling out to Ed protein levels suggests a different mechanism is at play. Most strikingly, Ed protein is colocalized extensively with N and Dl in the wing disc cells, but not in the eye disc, where interestingly there appears to be very little N and Dl on the cell . Therefore, all this suggests the conclusion that the two phenotypes do indeed have different origins, and moreover that there are significant differences in SOP singling out compared with R8 precursor selection (Rawlins, 2003b).

echinoid mutants exhibit neurogenic phenotypes and show synergistic interactions with the Notch signaling pathway

During neurogenesis in Drosophila, groups of ectodermal cells are endowed with the capacity to become neuronal precursors. The Notch signaling pathway is required to limit the neuronal potential to a single cell within each group. Loss of genes of the Notch signaling pathway results in a neurogenic phenotype: hyperplasia of the nervous system accompanied by a parallel loss of epidermis. Echinoid (Ed), a cell membrane associated Immunoglobulin C2-type protein, has been shown to be a negative regulator of the EGFR pathway during eye and wing vein development. Using in situ hybridization and antibody staining of whole-mount embryos, Ed has been shown to have a dynamic expression pattern during embryogenesis. Embryonic lethal alleles of ed reveal a role of Ed in restricting neurogenic potential during embryonic neurogenesis, and result in a phenotype similar to that of loss-of-function mutations of Notch signaling pathway genes. In this process Ed interacts closely with the Notch signaling pathway. Loss of ed suppresses the loss of neuronal elements caused by ectopic activation of the Notch signaling pathway. Using a temperature-sensitive allele of ed it has been shown that Ed is required to suppress sensory bristles and for proper wing vein specification during adult development. In these processes also, ed acts in close concert with genes of the Notch signaling pathway. Thus the extra wing vein phenotype of ed is enhanced upon reduction of Delta (Dl) or Enhancer of split [E(spl)] proteins. Overexpression of the membrane-tethered extracellular region of Ed results in a dominant-negative phenotype. This phenotype is suppressed by overexpression of E(spl)m7 and enhanced by overexpression of Dl. This work establishes a role for Ed during embryonic nervous system development, as well as adult sensory bristle specification and shows that Ed interacts synergistically with the Notch signaling pathway (Ahmed, 2003).

A main difference between the mutant phenotypes of neurogenic genes and of ed alleles lies in the severity of the observed hyperplasia of the embryonic CNS. Embryos homozygous for apparently amorphic ed alleles show a less extensive neural hyperplasia than that caused by loss of genes such as N or Dl and resemble embryos homozygous/hemizygous for hypomorphic alleles of other neurogenic genes. Strong maternal effects contribute to weak phenotypes of various amorphic mutant alleles of neurogenic genes such as N, mam or groucho. Indeed, maternal ed transcripts can be readily detected in Northern blot analysis of 0-2 hour-old embryos. However, it remains to be determined if a maternal contribution can account for the relatively weak neural hyperplasia exhibited by ed mutant embryos (Ahmed, 2003).

ed RNA and protein expression during early neurogenesis indicates that ed gene products become restricted to the neuroectodermal cell layer, whereas no ed products are detectable in the delaminated neuroblasts. The dynamics of ed RNA and protein distribution during neuroblast delamination implies that ed function might be required in cells that remain in the ectodermal cell layer. In such a scenario, similar to N, ed function would be required in the cells receiving the lateral inhibitory signal. However, it should be noted that at the time when the differential distribution of ed RNA and protein becomes detectable, the neuroblast segregation has already been initiated (Ahmed, 2003).

Ed expression during embryogenesis is dynamic and seen in many developing organ systems. The widespread expression of ed indicates that ed might be required for the development of multiple organs. Indeed, analysis of the trachea and muscles in ed mutant embryos reveals defects in the proper formation of these organ systems. The requirement of ed for normal development of multiple tissues is not limited to embryogenesis. Adult flies with reduced ed activity show defects in leg, wing and eye development. A similar widespread expression and requirement in multiple organs has also been observed for the Notch signaling pathway during Drosophila development (Ahmed, 2003).

Ed protein missing its intracellular region interferes with the process of lateral inhibition; overexpression of EdExt in the developing wing disc results in an increase in the number of macrochaetae and microchaetae. Additional phenotypes include the irregular thickening of wing vein II and infrequent notching of the wing margin. These phenotypes are similar to those seen upon reduced ed function. Thus, ectopic expression of EdExt interferes with the function of endogenous Ed. A dominant-negative activity of the extracellular portion is not unusual for receptors that bind to ligands and then transduce a signal intracellularly. Thus, it is possible that the EdExt competes with the WT Ed for a limited amount of ligand. Because EdExt is missing its intracellular region, its binding to the ligand may have no functional consequence other than limiting the amount of available ligand. The ability of the extracellular domain to act as a dominant-negative molecule and the observation that the temperature-sensitive allele of ed has a mutation associated with Ig C2 domain V implies that the interaction of the extracellular domain with a putative ligand is an essential component of Ed function. Two isoforms of Neuroglian (Nrg) have been identified as activating ligands for the antagonistic effect of Ed on the EGFR pathway in the eye disc. Both isoforms (Nrg¹⁸⁰ and Nrg¹⁶⁷) are expressed in the wing disc and thus overlap in their expression with Ed. It has yet to be determined whether Nrg also functions as a ligand for Ed during sensory organ development. Ed has been shown to act as a homophilic cell adhesion molecule. In the eye disc, it has been shown that the Nrg-mediated heterophilic activity of Ed in repressing the EGFR signaling pathway is redundant with the homophilic activity of Ed. Thus, it is possible that the dominant-negative construct interferes with Ed activity by competing for homophilic binding (Ahmed, 2003).

Ectopic expression of an activated form of N results in suppression of neuronal specification. In contrast, reduced ed gene activity results in increased specification of neurons. Ectopic expression of N^act in ed^2B8/ed^ts embryos results in a near WT nervous system. The observation of compensating, as opposed to an epistatic phenotype, does not support the formulation of a straightforward epistatic relationship between ed and N gene function. Rather, although both WT N and ed have a similar antineurogenic function during neurogenesis, they might be acting in functionally synergistic, yet possibly parallel regulatory pathways (Ahmed, 2003).

The observation of dosage-sensitive interactions between mutations in two genes can also be indicative of closely related roles. Dosage-sensitive interactions have been observed between ed and Dl and ed and E(spl). The mild wing phenotype exhibited by ed^m1/ed^ts flies raised at 25°C is enhanced by loss of one copy of Dl. Similarly, the wing phenotype of E(spl)^8D06/+ flies is enhanced by reduction of Ed activity. These observations imply that, in the wing disc also, the Notch signaling pathway and ed are acting synergistically (Ahmed, 2003).

Genetic interaction between ed and the Notch signaling pathway is also observed during the development of the adult PNS. Ectopic expression of EdExt results in specification of extra macrochaetae and microchaetae. Overexpression of Dl results in an increase in the number of sensory bristles. Simultaneous ectopic expression of EdExt and Dl has a phenotype much stronger than what would be the result of additive combination of the individual phenotypes. The EdExt protein interferes with the activity of endogenous Ed and the decrease in Ed activity increases the neurogenic phenotype caused by Dl overexpression. These observations imply that Ed acts in concert with Dl. An epistatic interaction was also observed with E(spl)m7. Ectopic expression of E(spl)m7 completely suppressed the extra bristles phenotype obtained upon EdExt expression. The complete suppression of the dominant-negative phenotype would imply that E(spl)m7 functions downstream of ed. However, it is possible that this suppression is a result of the strong antineurogenic activity of E(spl)m7. Although it is presently not clear whether ed and the genes of the Notch signaling pathway function in the same or parallel pathway, these observations establish that ed and the Notch signaling pathway genes act synergistically in both embryonic and postembryonic development (Ahmed, 2003).

The data point to a role for Ed in the cell-cell communication processes that lead to the selection of the future neural precursor from the proneural cluster. In this process, Ed functions synergistically with the Notch signaling pathway. In this role Ed might be part of the cell-cell communication process itself. However, keeping in mind its cell-cell adhesion function, it could be argued that Ed may also be involved in the execution of the developmental decisions that result from cell-cell communication. In this scenario downregulation of ed expression in the future neuroblast may contribute to neuroblast delamination, whereas continued ed expression in the future epidermal precursors maintains cell adhesion and stabilizes their fate. Thus, Ed might be functioning at the level of cell-cell communication and at the level of coordinating cell-cell signaling with morphogenesis (Ahmed, 2003).

Echinoid synergizes with the Notch signaling pathway in Drosophila mesothorax bristle patterning

echinoid (ed) encodes an immunoglobulin domain-containing cell adhesion molecule that negatively regulates the Egfr signaling pathway during Drosophila photoreceptor development. A novel function of Ed is shown, i.e., the restriction of the number of notum bristles that arise from a proneural cluster. Thus, loss-of-function conditions for ed give rise to the development of extra macrochaetae near the extant ones and increase the density of microchaetae. Analysis of ed mosaics indicates that extra sensory organ precursors (SOPs) arise from proneural clusters of achaete-scute expression in a cell-autonomous way. ed embryos also exhibit a neurogenic phenotype. These phenotypes suggest a functional relation between ed and the Notch (N) pathway. Indeed, loss-of-function of ed reduces the expression of the N pathway effector E(spl)m8 in proneural clusters. Moreover, combinations of moderate loss-of-function conditions for ed and for different components of the N pathway show clear synergistic interactions manifested as strong neurogenic bristle phenotypes. It is concluded that Ed is not essential for, but it facilitates, N signaling. It is known that the N and Egfr pathways act antagonistically in bristle development. Consistently, it is found that Ed also antagonizes the bristle-promoting activity of the Egfr pathway, either by the enhancement of N signalling or, similar to the eye, by a more direct action on the Egfr pathway (Escudero, 2003).

Epistatic and clonal analyses are compatible with Ed facilitating N signaling by acting at a step previous to the release of the N^ICD. Accordingly, the possibility that Ed might physically interact with N was tested. First, the subcellular localization of both proteins was examined in the wing imaginal disc. Using antibodies that recognize the C terminus of Ed and the zonula adherens marker Armadillo (Arm), Ed was observed to mainly, if not exclusively, accumulate at the zonula adherens where it colocalizes with Arm. This is in sharp contrast to the eye disc, where Ed resides throughout the cell membrane of all cells. Using N^ICD-specific antibodies, it was further observed that N is mainly colocalized with Ed. Similar colocalization with Ed at zonula adherens can also be detected with N^ECN-specific antibodies, but Ed is not present in the N^ECN-containing internalized vesicles (Escudero, 2003).

The colocalization of Ed and N at zonula adherens and the observation that the intracellular domain of Ed is required for the dominant-negative effect prompted a determination of whether the intracellular domain of both proteins might also physically interact with each other. Both GST pull-down and yeast two-hybrid assays were performed. No detectable binding between the intracellular domain of N and either the entire intracellular domain or the last 50 amino acids of Ed was observed. This suggests that the functional interaction between Ed and N is not mediated by a direct interaction between both proteins, although the possibility still remains that a physical interaction might occur via their extracellular domains (Escudero, 2003).

Thus far, the results indicate that Ed cooperates with the N pathway to control the determination of notum macrochaetae. Because Egfr and N pathways act antagonistically in macrochaetae development, the genetic interactions between ed and members of the Egfr signaling pathway were examined. Overexpression of wild-type Egfr (UAS-Egfr) alone by sca-Gal4, has a very weak effect on the number of notum bristles. However, the co-expression of both UAS-edDeltaECD and UAS-Egfr results in a severe tufting phenotype. Similar results were obtained when edDeltaECD and a constitutively activated form of Raf (UAS-raf^gof) were co-expressed. As expected, increased number of SOPs were observed in proneural clusters, as detected with anti-Sens antibody. The interaction between Ed and Egfr pathways was verified by observing that a decrease of Egfr activity (overexpression of a dominant-negative form of Egfr, UAS-Egfr^DN) partially suppressed the extra bristle phenotype caused by ed^1x5/ed^slH8. Together, these results demonstrate an antagonism between Ed and Egfr signaling pathways in bristle development. However, considering the known antagonism between the Egfr and N pathways in macrochaetae development, these results open the possibility that the Egfr pathway might mediate, at least in part, the interaction between ed and the N pathway. If this were the case, one would expect that modifications of the activity of the Egfr pathway would affect the activity of the N pathway. Apparently, this did not occur. The levels of E(spl)m8 mRNA accumulation in proneural clusters were essentially unmodified by overexpressing either a constitutively activated form of Ras (UAS-ras1^V12) or the Egfr-negative ligand Argos (UAS-aos). These conditions mimicked a strong stimulation and an inhibition of the pathway, since they respectively led to formation of many ectopic SOPs or to the removal of most macro and microchaetae. It is concluded that it is unlikely that the interaction of Ed and N is mediated by the Egfr pathway (Escudero, 2003).

The cell adhesion molecules Echinoid and Friend of Echinoid coordinate cell adhesion and cell signaling to regulate the fidelity of ommatidial rotation in the Drosophila eye

Directed cellular movements are a universal feature of morphogenesis in multicellular organisms. Differential adhesion between the stationary and motile cells promotes these cellular movements to effect spatial patterning of cells. A prominent feature of Drosophila eye development is the 90° rotational movement of the multicellular ommatidial precursors within a matrix of stationary cells. This study shows that the cell adhesion molecules Echinoid (Ed) and Friend of Echinoid (Fred) act throughout ommatidial rotation to modulate the degree of ommatidial precursor movement. It is proposed that differential levels of Ed and Fred between stationary and rotating cells at the initiation of rotation create a permissive environment for cell movement, and that uniform levels in these two populations later contribute to stopping the movement. Based on genetic data, it is proposed that ed and fred impart a second, independent, `brake-like' contribution to this process via Egfr signaling. Ed and Fred are localized in largely distinct and dynamic patterns throughout rotation. However, ed and fred are required in only a subset of cells -- photoreceptors R1, R7 and R6 -- for normal rotation, cells that have only recently been linked to a role in planar cell polarity (PCP). This work also provides the first demonstration of a requirement for cone cells in the ommatidial rotation aspect of PCP (Fetting, 2009).

ed and fred also genetically interact with the PCP genes, but affect only the degree-of-rotation aspect of the PCP phenotype. Significantly, this study demonstrates that at least one PCP protein, Stbm, is required in R7 to control the degree of ommatidial rotation (Fetting, 2009).

This study demonstrates that ed and fred have partially overlapping functions during the two phases of ommatidial rotation. It is proposed that different levels of Ed and Fred in rotating and non-rotating cells modulate the adhesivity of these cells, a prerequisite for rotation to occur. In the second phase, Ed and Fred are required in R1, R6, R7 and the cone cells, where they are likely to regulate the Egf receptor to contribute to the slowing of rotation (Fetting, 2009).

There are two phases of rotation distinguishable by the rate at which the ommatidia rotate. The initial phase (rows 4-7) is fast, with ommatidia rotating 10-15° per row, whereas rotation slows to 5-10° per row in the slow phase (rows 7-15). The data demonstrate that Ed and Fred function during both phases and that they play unique roles in each phase (Fetting, 2009).

In the first phase, it is proposed that the tight regulation of Ed and Fred levels between rotating and stationary cells creates an environment that is permissive to rotation. Immediately before rotation starts, Ed begins to be endocytosed in the ommatidial precluster cells. Concurrently, Ed levels fall dramatically in these cells while remaining high in the stationary interommatidial cells (IOCs), setting up an imbalance in Ed levels between these two populations of cells. It is proposed that the resulting differential adhesion between these two cell populations enables the rotating cells to slide past their stationary neighbors in accordance with Steinberg's differential adhesion hypothesis (DAH) (Steinberg, 2007). The DAH suggests that cell populations maximize the strength of adhesive bonding between them and minimize the adhesive free energy, and use tension generated by adhesion between cells to drive events such as cell rearrangements during morphogenesis. Cells with equivalent levels of Ed (or Fred) adhere more tightly to one another and adhesion is reduced between cells with different levels of Ed (or Fred), thereby enabling the two groups to slide past one another. In support of this hypothesis, artificially equalizing levels of Ed or Fred significantly slows rotation (Fetting, 2009).

The data are consistent with Ed and Fred playing two key roles in the slow phase by both directly and indirectly (through Egfr signaling) affecting the physical component of the process. It is suggested that in both cases the outputs produce adhesive forces that slow/stop rotation. Ed and Fred are required in photoreceptors R1, R6 and R7 and the cone cells for normal ommatidial rotation. These cells do not become fully integrated into the ommatidial cluster until the second half of rotation. Furthermore, R1, R6 and R7 constitute the rotation interface until the cone cells are recruited, at which point the cone cells co-opt this position and role. Consequently, Ed and Fred are required in the right place (the subset of cells that lie at the rotation interface) and at the right time (the slower phase of rotation) to play a role in slowing rotation (Fetting, 2009).

It is proposed that Ed and Fred activity in R1, R6, R7 and the cone cells regulates Egfr signaling in these cells to slow/stop rotation as follows. Egfr signaling promotes rotation via the Ras/Cno and Ras/Mapk/Pnt effectors (Brown, 2003; Gaengel, 2003), so its output must be dampened to slow rotation. Ed binds and inhibits the Egf receptor, whereas Fred binds Ed and interferes with this inhibition. Therefore, cooperation between Ed and Fred precisely titrates Egfr activity in the cells in which Ed and Fred function. As R1, R6 and R7 are recruited into the ommatidial cluster, Ed levels are high in these cells, thereby decreasing Egfr signaling at their side of the rotation interface, thus impeding rotation. This inhibitory role switches to the cone cells when they are recruited, creating a new rotation interface (Fetting, 2009).

Rotation may be slowed through Egfr signaling activity via its effector Cno, the fly homolog of Afadin/AF-6, an actin-binding adherens junction (AJ) protein. Afadin and its binding partners, nectins and α-actinin, build and stabilize dynamic AJs that undergo remodeling (Ooshio, 2007). The majority of cno mutant ommatidia over-rotate, indicating that Cno inhibits ommatidial rotation. Since Egfr signaling promotes and Cno inhibits rotation, Egfr signaling is likely to suppress Cno activity during rotation thereby blocking stable junction formation. In this scenario, high levels of Egfr would be required during the early phase of rotation to prevent Cno from promoting stable junctions between rotating and non-rotating cells. Consistent with this hypothesis, levels of Ed, an Egfr inhibitor, are very low in ommatidial cells both when rotation commences and during the fast phase of rotation (Fetting, 2009).

Early in the second half of rotation, it is proposed that higher levels of Ed activity are necessary to repress Egfr signaling at the rotation interface, possibly increasing the amount of active Cno and consequently increasing the number of stable AJs between the moving and stationary cells. The more tightly the cells adhere to one another, the less permissive the environment is for movement, and the more difficult rotation becomes. Ed levels are high in the cells in which it would need to be high, i.e., R1, R6, R7 and the cone cells. Once rotation is complete, Ed and Fred are at high levels at the cell boundaries between the interommatidial and ommatidial cells, an indication that stable AJs now cement the fully rotated ommatidia in place (Fetting, 2009).

ed and fred interact genetically with the R3 and R4 genes, respectively, modifying only the degree-of-rotation aspect of the PCP phenotype. Genetic and molecular epistasis data suggest that ed and fred act in a pathway either downstream of, or parallel to, the PCP genes. First, localization of Ed and Fred does not require the PCP complex, nor do the PCP proteins require Ed and Fred for their localization. Second, mutations in ed and fred affect only one aspect of the PCP phenotype (Fetting, 2009).

Nectins and afadins have been implicated in numerous human diseases and developmental defects, including breast cancer, metastasis and cleft palate. Defective cell adhesion and cell signaling also underlie these problems. Given the interspecies conservation of AJ genes, similar mechanisms might control ommatidial rotation and contribute to these human diseases (Fetting, 2009).

Echinoid regulates tracheal morphology and fusion cell fate in Drosophila

Morphogenesis of the Drosophila embryonic trachea involves a stereotyped pattern of epithelial tube branching and fusion. This study reports unexpected phenotypes resulting from maternal and zygotic (M/Z) loss of the homophilic cell adhesion molecule Echinoid (Ed), as well as the subcellular localization of Ed in the trachea. ed(M/Z) embryos have convoluted trachea reminiscent of septate junction (SJ) and luminal matrix mutants. However, Ed does not localize to SJs, and ed^M/Z embryos have intact SJs and show normal luminal accumulation of the matrix-modifying protein Vermiform. Surprisingly, tracheal length is not increased in ed^M/Z mutants, but a previously undescribed combination of reduced intersegmental spacing and deep epidermal grooves produces a convoluted tracheal phenotype. In addition, ed^M/Z mutants have unique fusion defects involving supernumerary fusion cells, ectopic fusion events and atypical branch breaks. Tracheal-specific expression of Ed rescues these fusion defects, indicating that Ed acts in trachea to control fusion cell fate (Laplante, 2010; full text of article).

REFERENCES

Search PubMed for articles about Drosophila echinoid & friend of echinoid

Ahmed, A., et al. (2003). echinoid mutants exhibit neurogenic phenotypes and show synergistic interactions with the Notch signaling pathway. Development 130: 6295-6304. 14623819

Baeg, G. H., Zhou, R. and Perrimon, N. (2005). Genome-wide RNAi analysis of JAK/STAT signaling components in Drosophila. Genes Dev. 19: 1861-1870. 16055650

Bai, J., Chiu, W., Wang, J., Tzeng, T., Perrimon, N. and Hsu, J. (2001). The cell adhesion molecule Echinoid defines a new pathway that antagonizes the Drosophila EGF receptor signaling pathway. Development. 128(4): 591-601. 11171342

Brown, K. E. and Freeman, M. (2003). Egfr signalling defines a protective function for ommatidial orientation in the Drosophila eye. Development 130: 5401-5412. PubMed Citation: 14507785

Chandra, S., Ahmed, A. and Vaessin, H. (2003). The Drosophila IgC2 domain protein Friend-of-Echinoid, a paralogue of Echinoid, limits the number of sensory organ precursors in the wing disc and interacts with the Notch signaling pathway. Dev Biol. 256(2): 302-16. 12679104

Chang, L. H., et al. (2011). Differential adhesion and actomyosin cable collaborate to drive Echinoid-mediated cell sorting. Development 138(17): 3803-12. PubMed Citation: 21795280

de Belle, J. S., Sokolowski, M. B. and Hilliker, A. J. (1993). Genetic analysis of foraging microregion of Drosophila melanogaster. Genome 36: 94-101. 8458574

Escudero, L. M., et al. (2003). Echinoid synergizes with the Notch signaling pathway in Drosophila mesothorax bristle patterning. Development 130: 6305-6316. 14623820

Fernandez-Gonzalez R., et al. (2009). Myosin II dynamics are regulated by tension in intercalating cells. Dev. Cell 17: 736-743. PubMed Citation: 19879198

Fetting, J. L., Spencer, S. A. and Wolff, T. (2009). The cell adhesion molecules Echinoid and Friend of Echinoid coordinate cell adhesion and cell signaling to regulate the fidelity of ommatidial rotation in the Drosophila eye. Development 136: 3323-3333. PubMed Citation: 19736327

Gaengel, K. and Mlodzik, M. (2003). Egfr signaling regulates ommatidial rotation and cell motility in the Drosophila eye via MAPK/Pnt signaling and the Ras effector Canoe/AF6. Development 130: 5413-5423. PubMed Citation: 14507782

Genevet, A., Wehr, M.C., Brain, R., Thompson, B.J. and Tapon, N. (2010). Kibra is a regulator of the Salvador/Warts/Hippo signaling network. Dev. Cell 18: 300-308. PubMed Citation: 20159599

Ho, Y. H., et al. (2010). Echinoid regulates Flamingo endocytosis to control ommatidial rotation in the Drosophila eye. Development 137(5): 745-54. PubMed Citation: 20110316

Islam, R., Wei, S. Y., Chiu, W. H., Hortsch, M. and Hsu, J. C. (2003). Neuroglian activates Echinoid to antagonize the Drosophila EGF receptor signaling pathway. Development. 130(10): 2051-9. 12668620

Laplante, C. and Nilson, L. A.. (2006). Differential expression of the adhesion molecule Echinoid drives epithelial morphogenesis in Drosophila. Development 133(16): 3255-64. 16854971

Laplante, C., Paul, S. M., Beitel, G. J. and Nilson, L. A. (2010). Echinoid regulates tracheal morphology and fusion cell fate in Drosophila. Dev. Dyn. 239: 2509-2519. PubMed Citation: 20730906

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date revised: 15 July 2012

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