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).
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 edMZ 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).
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 ElpB1. 1X5 was isolated as an EMS induced mutation that strongly enhances the rough eye phenotype associated with ElpB1. 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 ElpB1, 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 ElpB1 mutation, as well as rlSEM, a constitutively active MAPK. In addition, flies heterozygous for both 1X5 and Gap1, or both 1X5 and styS88, 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 edlF20 (de Belle, 1993) fails to complement 1X5 and enhances the ElpB1 rough eye phenotype, as well as the extra wing vein phenotype of rlSEM. Thus 1X5 is allelic to ed. All ed mutations are pupal lethal in homozygotes with the exception of edslH8, which is a weaker allele. Homozygous edslH8, as well as edslH8 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 ed1X5/edslH8 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, edslH8 hemizygote animals have more R7 cells than ed1X5/edslH8 transheterozygote animals, indicating that the ed alleles are loss of function. edslH8 hemizygotes have 1.68 R7 cells in average, compared with 1.34 in ed1X5/edslH8 (Bai, 2001).
To determine the origins of the extra photoreceptor cells, ed1X5/edslH8 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 ed1X5/edslH8 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 ed1X5/edslH8 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. sevd2 is a loss-of-function sevenless (sev) allele, and sevd2 mutant flies lack R7 cells. Although ommatidia within ed1X5/edslH8 mutants contain an average of 1.34 R7 cell, ommatidia within a sevd2; ed1X5/edslH8 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, ed1X5 enhances the rough eye phenotype caused by overexpressing constitutive active forms of either the Egfr, RAS1, or RAF. Conversely, ed1X5 suppresses the rough eye phenotype caused by overexpressing dominant negative RAS1. While 61% of ommatidia in a sev-RasN17/+ mutant lack R7 cells, only 10% of ommatidia in ed1X5/edslH8; sev-RasN17/+ double mutants lack R7 photoreceptors. In addition, at 25o C, ed1X5 also rescues the lethality of RafHM7, 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. pntDelta88/pnt1277 and sev-yanACT/+ ommatidia contain an average of 0.69 and 0.05 R7 cells, respectively. However, ed1X5/edslH8; pntDelta88/pnt1277 and ed1X5/edslH8; sev-yanACT/+ ommatidia contain an average of 1.44 and 1.01 R7 cells, respectively. Strikingly, ed1X5/edslH8; 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 edslA12 mutant cells in a sevd2 background. No R7 cells develop in the sevd2 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-yanACT 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 edslH8/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).
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 (ato109-68). Although such overexpression does not alter the expression pattern of ato beyond boosting and extending it within R8 cells, ato109-68 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, ato109-68 was used as the basis of a screen for genetic modifiers to isolate mutations that affect R8 resolution. E(ato109-68)4.12 was isolated as a second site mutation that dominantly enhances ato109-68 when present in one copy. E(ato109-68)4.12 itself was found to be homozygous viable with a strong rough eye phenotype. A lethal allele of ed (edlH23) fails to complement E(ato109-68)4.12: transheterozygous flies are viable, with rough eyes, suggesting that E(ato109-68)4.12 is an allele of ed. Sequencing the ed gene from E(ato109-68)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 ed4.12 (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).
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 Nicd 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. ed2B8 is an amorphic allele of ed. Flies carrying only one functional copy of ed and one copy of the GMR-GAL4 driver (ed2B8/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 (ed2B8/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 ed2B8/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/+; Gap1B2/+ flies show increased roughness and the remaining 70% show no significant change. Pointed is a downstream effector of Egfr signaling pathway. GMR-GAL4/+ pntDelta 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 ed2B8 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).
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).
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 Nact in ed2B8/edts 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 edm1/edts 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 (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 NICD. 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 NICD-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 NECN-specific antibodies, but Ed is not present in the NECN-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-rafgof) 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-EgfrDN) partially suppressed the extra bristle phenotype caused by ed1x5/edslH8. 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-ras1V12) 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).
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date revised: 20 January 2007
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