EGF receptor


Table of contents

Egfr and wing morphogenesis

vein and spitz show a strong genetic interaction suggesting a molecular interdependence. Reducing vein dose in a spi null genotype dramatically worsens the phenotype to produce a collapse embryo with an extruded head skeleton. However, the double mutants are not as severly affected as are Egfr null mutants. Genetic interactions are also observed between vn, Egfr and rolled. The gain of function alleles Egfr-Ellipse and rolled-Sevenmaker rescue proliferation defects in strong and null vein mutants. These defects include a small wing disc and the size of the pupal case (Schnepp, 1996).

The blistered function in wing vein development was examined by studying genetic mosaics of mutant cells, genetic interactions with other genes affecting vein development and blistered expression in several mutant backgrounds. Clones of blistered mutant cells proliferate normally but tend to grow along veins and always differentiate as vein tissue. These observations indicate that vein-determined wing cells show a particular behaviour that is responsible for their allocation to vein regions. Strong genetic interactions are observed between blistered, veinlet and genes of the Ras signaling cascade, in particular Egf receptor, rolled (rl) (MAPK), and a putative ligand of Egf receptor, vein, that codes for a neuregulin secreted protein. Hemizygosity for blistered totally suppresses the lack of vein L4 phenotype in Egfr, rolled and vein homozygous mutants, while greatly enhancing the amount of ectopic vein observed in a gain-of-function rolled allele. blistered hemizygosity also suppresses the lack of veins in veinlet hypomorph conditions. Conversely, it dramatically enhances the amount of ectopic vein tissue obtained after ubiquitous expression of veinlet + product (Roch, 1998).

The observed interaction between Egf receptor and blistered in hypomorphic conditions led to the study of double mutants for strong alleles of both genes in mosaic clones. Double mutant clones were generated at 48-72 hours AEL for the top 4A and bs P1292 alleles. top 4A clones appear with a reduced frequency, are smaller, narrower and more elongated than controls, and are composed of small cells that are unable to differentiate vein histotype, leaving a gap of intervein tissue wherever they touch a vein, except in the anterior wing margin vein (L1). Double mutant top 4A;bs P1292 clones tend to occupy vein territories like bs P1292 clones, a preference never observed in top 4A clones, but one which appears with a frequency and size similar to top 4A controls. Double mutant cells differentiate autonomously, in all cases, into pigmented, corrugated and compacted tissue with smaller cells than those characteristic of torpedo. The observation of these typical vein features leads to the conclusion that this tissue has a vein histotype indicating that the blistered extra vein phenotype is epistatic to torpedo (Egfr) lack of veins. It is concluded that during disc proliferation, blistered expression is under the control of the Ras signal transduction pathway, but its expression is independent of veinlet. During the pupal period, blistered and veinlet expression become interdependent and mutually exclusive. These results link the activity of the Ras pathway to the process of early determination of intervein cells, by the transcriptional upregulation of the blistered nuclear factor (Roch, 1998).

Embryos lacking Jun activity exhibit a dorsal closure phenotype, very similar to that of basket and hemipterous mutants, indicating that Jun is a target of Hep/Bsk signaling. In eye and wing development Jun participates in a separate signaling pathway comprised of Ras, Raf, and the ERK-type kinase Rolled. In contrast to the strict requirement for Jun in dorsal closure, its role in the eye is redundant but can be uncovered by mutations in other signaling components. The removal of Jun function in the eye by mutation shows only minor defects. Occasionally, only one or two photoreceptors are lost in mutant ommatidia. Nevertheless, gain- and loss-of-function forms of Jun interfere specifically with the endogenously expressed wild-type protein, and Jun interacts genetically with the Sev/Ras/Raf/ERK signal transduction pathway. For example, when one copy of DJun is removed from transgenic lines expressing gain of function sevenless, ras and rolled mutations, a clear suppression of the mutant extra photoreceptor phenotype can be observed. The redundant function of Jun in eye development may contribute to the precision of photoreceptor differentiation and ommatidial assembly. Analysis of DJun mutants in the wing does not reveal any phenotypic defect characteristic of the Ras pathway. Nevertheless, removal of one copy of DJun suppresses the wing phenotypic defects of Ellipse gain-of-function alleles of the Epidermal growth factor receptor. It is concluded that DJun plays a role both in wing and eye development. It is suggested that the role of DJun in the wing and eye is not essential since other systems maintain proper morphogenesis in the absence of DJun. It is also concluded that DJun is a target of both JNK and MAP kinase in Drosophila (Kockel, 1997).

Cell fate decisions in the early Drosophila wing disc assign cells to compartments (anterior or posterior and dorsal or ventral) and distinguish the future wing from the body wall (notum). Egf receptor signaling stimulated by its ligand, Vein, has a fundamental role in regulating two of these cell fate choices: (1) Vn/EGFR signaling directs cells to become notum by antagonizing wing development and by activating notum-specifying genes; (2) Vn/EGFR signaling directs cells to become part of the dorsal compartment by induction of apterous, the dorsal selector gene, and consequently also controls wing development, which depends on an interaction between dorsal and ventral cells (Wang, 2000).

To determine when Vn/EGFR signaling is required for notum development, the temperature-sensitive alleles, Egfrtsla and vntsWB240 were used. Inactivating Vn/Egfr activity during the second instar (a 24 hr period) causes loss of the notum. The wing develops but shows pattern abnormalities characteristic of vn hypomorphs. Later shifts during the third instar does not cause loss of the notum. This demonstrates that Vn/Egfr activity is required for notum development in the second instar when wg is required to specify the wing. Thus, Vn and Wg appear to have complementary roles and this relationship has been examined by following their expression in mutants (Wang, 2000).

In second instar wild-type wing discs, wg is expressed distally in a wedge of anterior ventral cells and vn is expressed proximally. In vn null mutants, the initiation of wg expression is normal as is expression of its target gene optomotor-blind (omb). In wg mutants, however, there is a dramatic and early expansion of vn expression to include distal cells, presaging the development of these cells as an extra notum. Together these results suggest that Vn has an early role in establishing the notum and that Wg signaling is required to define a distal domain that is reduced in Egfr activity to allow wing development (Wang, 2000).

To test the role of Vn/Egfr signaling in specifying notum an examination was carried out to see whether the Iroquois complex (Iro-C) genes, ara and cap are targets of the pathway. The Iro-C genes have been implicated in specifying notum cell fate because loss of function causes a transformation of notum to hinge. Furthermore, misexpression of ara causes loss of the wing and a duplication of notum. Ectopic expression of an activated form of the receptor, Egfrlambdatop4.2 greatly reduces the size of the wing and a small ectopic notum forms. vn is expressed in the presumptive notum in early second instar discs and Caup/Ara are expressed in the presumptive notum at the end of the second instar. In early third instar wing discs, Caup/Ara are expressed in a domain that overlaps with vn. In vn mutants, this expression of Caup/Ara is lost and loss of Egfr signaling, in Egfrts clones, in the medial notum results in a loss of Caup/Ara expression. However, clones in the lateral notum continued to express Caup/Ara, suggesting other factors regulate Iro-C gene expression in these cells at this stage (Wang, 2000).

Activation of Iro-C genes could account for the requirement for Egfr activity to specify the notum at the end of the second instar as this correlates with when these genes are first expressed. However, loss of Egfr signaling at a slightly earlier time (mid-first instar to mid-second instar, see below), prior to activation of the Iro-C genes, also results in loss of the notum. A possible explanation for this comes from the finding that vn expression is lost in vn mutants. This suggests Egfr activity must be sustained, via a positive feedback loop involving transcriptional activation of vn, during the second instar, to activate the Iro-C genes and hence specify notum at the end of this period. Interestingly, the vn gene is also a target of Egfr signaling in the embryo (Wang, 2000 and references therein).

It is suggested that the mechanisms by which wg and vn specify alternate cell fates in the early wing disc, wing, or notum are antagonistic. This is based on the observation that loss of Wg results in the spread of vn expression and the supposition that the resulting ectopic Egfr activity causes loss of the wing and a double notum phenotype. Further evidence that Vn/Egfr signaling represses wing development comes from the results of misexpressing a constitutive receptor, Egfrlambdatop4.2, in the presumptive wing. In these flies, the wing is reduced to a stump covered with sensilla characteristic of the proximal wing (hinge) region and expression of the wing specific gene vestigial (vg) is repressed. Ectopic notal structures also form from the ventral pleura. The ability of ectopic Egfr signaling to suppress wing development is cell autonomous because clones of cells expressing Egfrlambdatop4.2 lack vg expression. In adult wings these clones produced outgrowths lacking wing characteristics but are otherwise difficult to characterize (Wang, 2000).

Although vn expression expands in wg mutants, no reciprocal spread of wg expression was observed in vn mutants that would have been indicative of a double wing phenotype. However, when Vn/Egfr signaling is inhibited in the notum by expressing a ligand antagonist (Vn::Aos-EGF) under the control of ptc-Gal4, ectopic wings are induced in ~10% of the flies. This result demonstrates that presumptive notal tissue can be transformed to wing by reducing Egfr signaling. However, the transformation occurs only when Egfr signaling is reduced in a subset of cells, rather than all cells in the notum (as in a vn mutant). This may reflect the indirect requirement for Egfr activity to also promote wing development (Wang, 2000).

The loss of notum phenotype is characteristic of vn hypomorphs but in null vn alleles and some Egfr alleles both the wing and notum primordia fail to develop and the wing discs remain tiny. Thus, although ectopic activity of Egfr in the distal disc represses wing development, the pathway is nevertheless normally required for wing development. Using the temperature-sensitive Egfrtsla allele it was found that this requirement is restricted to the period from mid-first to mid-second instar. Key genes involved in wing development that are active at this time include wg and apterous (ap). ap is expressed in dorsal cells and acts as a selector gene to divide the disc into dorsal and ventral compartments. Regulation of Notch ligands by Ap leads to Notch signaling at the DV boundary and the formation of an organizer for wing outgrowth and expression of the wing-specific transcription factor vg (Wang, 2000 and references therein).

Of these two candidates, wg and ap, it seemed unlikely that wg was the key gene affected by Egfr signaling from mid-first to mid-second instar because wg expression is normal in vn mutants at mid-second instar. However, later in the second instar, wg expression normally expands to fill the growing wing pouch and it was noted that in vn mutants, wg expression fails to undergo this expansion. A similar defect in wg expression is seen in ap mutants consistent with Ap function being impaired in vn mutants. Remarkably, ap expression is completely absent in second instar vn mutant discs. Thus, loss of Ap can explain why there is no wing in vn mutants. This is supported by the demonstration that ectopic ap is capable of rescuing wing development in vn mutants (Wang, 2000).

Several additional lines of evidence demonstrate that ap is a cell autonomous target of Vn/Egfr signaling and that this relationship exists only transiently in early wing development: (1) ap expression partially overlaps that of vn in the second instar; (2) ap can be induced ectopically in ventral clones misexpressing an activated form of the receptor, Egfrlambdatop4.2; (3) Egfrtsla mutant clones generated in the first instar show autonomous loss of ap expression, whereas clones generated in the second instar express ap normally. Finally, loss of Egfr activity in whole discs from mid-first to mid-second instar results in complete loss of ap expression, whereas ap is still expressed in discs from larvae given a temperature shift slightly later during the second instar (Wang, 2000).

The results described here suggest that division of the early wing disc into presumptive wing and body wall regions is defined by the action of two secreted signaling molecules, Wg and Vn. wg, a pro-wing gene, is required to repress vn expression, which at high levels antagonizes wing development. Antagonism between Wg and Egfr signaling has also been demonstrated in segmental patterning of the embryo and in development of the head and third instar wing pouch, suggesting such a relationship between these pathways may be a common theme in a number of cell fate choices. Finding that one of the main functions of Wg in early wing specification is to repress Vn/Egfr signaling in the distal region of the early disc raises the question as to whether this is the only role of Wg in wing specification and hence if wing-cell fate can be specified in the absence of both signals. This seems unlikely, because nubbin, an early wing cell marker, is not misexpressed proximally in a vn mutant, where cells would lack both signals (Wang, 2000).

Vn/Egfr signaling promotes development of the notum by maintaining its own activity through transcriptional activation of vn itself, and also promotes expression of ap. Thus, both vn and ap appear to be targets of Egfr signaling, but the domain of ap is clearly wider than that of vn, indicating that ap can be activated at a lower signaling threshold than vn. Vn is a secreted molecule and thus could generate a gradient of Egfr activity. This provides an explanation for how Egfr signaling can regulate both wing and notum development: vn autoregulation and notum development requires high Egfr signaling activity while ap expression and subsequent wing development requires lower signaling activity (Wang, 2000).

Interestingly, vertebrate Egfr and its ligands are expressed in the chick limb bud in a pattern that appears to overlap with the vertebrate ap homolog Lhx2, and these factors are required for limb outgrowth in the chick. In light of the present results it will be important to determine whether Egfr signaling controls Lhx2 expression and thus plays a role in regulating outgrowth of the vertebrate limb. These results may also have implications for the evolution of insect wings. If the control of body wall development by Egfr signaling is ancestral, and comparative analysis of other arthropods will be required to assert this, then one of the first steps towards evolution of wings could have occurred when Egfr signaling assumed control of ap (Wang, 2000).

During Drosophila development Fos acts downstream from the JNK pathway. It can also mediate ERK signaling in wing vein formation and photoreceptor differentiation. Drosophila JNK and ERK phosphorylate D-Fos with overlapping, but distinct, patterns. Analysis of flies expressing phosphorylation site point mutants of D-Fos reveals that the transcription factor responds differentially to JNK and ERK signals. Mutations in the phosphorylation sites for JNK interfere specifically with the biological effects of JNK activation, whereas mutations in ERK phosphorylation sites affect responses to the EGF receptor-Ras-ERK pathway. These results indicate that the distinction between ERK and JNK signals can be made at the level of D-Fos, and that different pathway-specific phosphorylated forms of the protein can elicit different responses (Ciapponi, 2001).

The loss of wing vein tissue on expression of D-FosbZIP resembles phenotypes resulting from defects in the Drosophila epidermal growth factor receptor (DER) pathway, caused, for example, by loss-of-function alleles of DER itself or of other genes required for DER signaling, such as rhomboid, vein, ras, and ERK/rolled. Therefore, the above results might indicate that D-Fos acts as a mediator of the DER/ERK signaling pathway during wing vein differentiation. The artificial activation of this RTK pathway, by gain-of-function alleles or by overexpression of downstream effectors, gives rise to ectopic veins in the wing. To establish whether D-Fos might act epistatically to DER, and whether D-FosbZIP or a reduced D-fos gene dose (kayak alleles) might suppress such a phenotype was examined (Ciapponi, 2001).

The EllipseB1 (ElpB1) allele of DER represents an activated component of the DER/ERK signaling pathway. In addition to other phenotypes, for example, in the eye, ElpB1 animals consistently develop wings with ectopic wing vein material. Strikingly, both D-FosbZIP expression in the wing imaginal disc (using the 32B Gal4 driver) or the removal of one copy of D-fos in a kay heterozygote, suppresses this phenotype almost completely. To confirm that the observed effect is specific and caused by a reduction of endogenous D-Fos function, add-back experiments were performed in which this reduction was compensated by supplying extra wild-type D-Fos from a transgene, driven by the heat shock promoter (hs D-fos. Significantly, the presence of the D-fos transgene abrogates the suppression of ElpB1 by kay and reinstates the extra vein phenotype caused by elevated DER activity. This result confirms that the suppression of the activated DER allele is due to a loss of D-fos activity. Hence, D-Fos mediates wing vein patterning downstream from or in parallel with DER (Ciapponi, 2001).

As part of an effort to dissect quantitative trait locus effects to the nucleotide level, association was assessed between 238 single-nucleotide and 20 indel polymorphisms spread over 11 kb of the Drosophila melanogaster Egfr locus and nine relative warp measures of wing shape. One SNP in a conserved potential regulatory site for a GAGA factor in the promoter of alternate first exon 2 approaches conservative experimentwise significance (P < 0.00003) in the sample of 207 lines for association with the location of the crossveins in the central region of the wing. Several other sites indicate marginal association with one or more other aspects of shape. No strong effects of sex or population of origin were detected with measures of shape, but two different sites were strongly associated with overall wing size in interaction with these fixed factors. Whole-gene sequencing in very large samples, rather than selective genotyping, would appear to be the only strategy likely to be successful for detecting subtle associations in species with high polymorphism and little haplotype structure. However, these features severely limit the ability of linkage disequilibrium mapping in Drosophila to resolve quantitative effects to single nucleotides (Palsson, 2004).

Mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase SHP-2, cause Noonan syndrome (NS), an autosomal dominant disorder with pleomorphic developmental abnormalities. Certain germline and somatic PTPN11 mutations cause leukemias. Mutations have gain-of-function (GOF) effects with the commonest NS allele, N308D, being weaker than leukemia-causing mutations. To study the effects of disease-associated PTPN11 alleles, transgenic fruitflies were generated with GAL4-inducible expression of wild type or mutant csw, the Drosophila orthologue of PTPN11. All three transgenic mutant CSWs rescued a hypomorphic csw allele's eye phenotype, documenting activity. Ubiquitous expression of two strong csw mutant alleles was lethal, but did not perturb development from some CSW-dependent receptor tyrosine kinase pathways. Ubiquitous expression of the weaker N308D allele causes ectopic wing veins, identical to the EGFR GOF phenotype. Epistatic analyses have established that the cswN308D ectopic wing vein phenotype requires intact EGF ligand and receptor, and that this transgene interacts genetically with Notch, DPP and JAK/STAT signaling. LOF alleles of positive regulators (downstream of receptor kinase, son of sevenless, Ras85D, Downstream of raf1, rolled, pointed, Hsp83) resulted in statistically significant suppression of ectopic vein formation. Most wings showed no or minimal ectopic vein formation in the anterior part of the wing, which were consistently observed in the UAS-cswN308D/+;tub-GAL4/+ control wings. In contrast, LOF alleles of negative regulators (sprouty, Gap1) enhanced the phenotype with longer ectopic veins in the anterior part of the wings andmultiple and complex ectopic vein formation in the posterior part. LOF alleles of Egfr, its ligand (vein) and positive extracellular regulators (Star, rhomboid) suppressed the wing phenotype while LOF alleles for argos, a negative ligand regulator, enhanced it. Expression of the mutant csw transgenes increases RAS-MAP kinase activation, which is necessary but not sufficient for transducing their phenotypes. The findings from these fly models provided hypotheses testable in mammalian models, in which these signaling cassettes are largely conserved. In addition, these fly models can be used for sensitized screens to identify novel interacting genes as well as for high-throughput screening of therapeutic compounds for NS and PTPN11-related cancers (Oishi, 2006).

Egfr and eye morphogenesis

The consequences of removing or activating the Egfr at different stages of eye development have been examined. The earliest stages of photoreceptor recruitment occur normally within Egfr- clones: the morphogenetic furrow is unimpeded and the R8 photoreceptor is specified. All Elav-expressing cells within Egfr- clones also express Boss, a marker for the R8 cell. All subsequent photoreceptor recruitment is blocked. Rough, which is normally expressed in the outer photoreceptors R2, R5, R3 and R4, fails to be expressed in Egfr- clones; unexpectedly, it was found that the whole early domain of Rough expression in the furrow is also abolished. This result demonstrates that Egfr positively regulates Rough expression, although it is not known how direct this control is (Dominguez, 1998).

Egfr- clones have a characteristic shape indicating that they have undergone substantial cell death posterior to the furrow, where the differentiation program is normally activated; consistent with this, excess apoptosis is detected. Thus Egfr is required for cell survival of differentiating cells posterior to the furrow. The receptor also regulates cell proliferation in the disc. Consistent with this, excessive tissue growth is found in discs in which the activated Egfr is expressed. This coincides with the observations that increased signaling by the Ras pathway also causes excess proliferation and that too many cells behind the furrow enter S phase in Egfr gain-of-function mutations (Dominguez, 1998).

Egfr has an early function at the disc margin (where the morphogenetic furrow initiates) and contributes to the regulation of spacing of the R8 precursors. Activation of the receptor is sufficient to trigger non-R8 photoreceptor development, even in cells in front of the furrow or in the absence of the proneural gene atonal. This implies that after the beginning of the third instar, activation of the Egfr is sufficient to trigger the differentiation of non-R8 photoreceptors in the absence of Atonal. Conversely, R8s require Atonal, but not the Egfr. It is thought that cells become competent to differentiate as photoreceptors at about the time the endogenous furrow initiates, but that subsequently they acquire an increased disposition to differentiate in respone to Egfr signaling as the furrow approaches them (Dominguez, 1998).

These results demonstrate that the photoreceptors comprise two classes: R8, which requires Atonal but not the Egfr, and non-R8, in which Egfr signaling is necessary and sufficient to trigger neural development. Thus ubiquitous overactivity of Egfr receptor in Ellipse mutations might cause a failure of R8 determination because of atonal repression in the furrow. It is clear that the prior expression of Atonal and the passage of the morphogenetic furrow is not required to make cells competent to respond to Egfr activation. It is concluded that at least five distinct functions of Egfr signaling need to be integrated during fly eye development. These include roles in the disc margin, in cell proliferation, survival, differentiation and recruitment of photoreceptors other than R8 (Dominguez, 1998).

Dominant Ellipse mutant alleles of the Drosophila EGF receptor homolog (Egfr) dramatically suppress ommatidium development in the eye and induce ectopic vein development in the wing. This phenotype suggests a possible role for Egfr in specifying the founder R8 photoreceptor cells for each ommatidium. Ellipse mutations have been used to probe the role of Egfr in eye development: Elp mutations result from a single amino acid substitution in the kinase domain, which activates tyrosine kinase activity and MAP kinase activation in tissue culture cells. Transformant studies confirm that the mutation is hypermorphic in vivo, but the Egfr function is elevated less than by ectopic expression of the ligand Spitz. Ectopic Spi promotes photoreceptor differentiation, even in the absence of R8 cells. Pathways downstream of Egfr activation were assessed to explore the basis of these distinct outcomes. Elp mutations cause overexpression of the Notch target gene E(spl) mdelta and require the function of Notch to suppress ommatidium formation. E(spl) mdelta is known to be expressed in cell clusters in the morphogenetic furrow of wild type eyes. The Elp phenotype also depends on the secreted protein Argos and therefore, in Elp;aos double mutants, the phenotype is reverted. Complete loss of Egfr function in clones of null mutant cells leads to delay in R8 specification and subsequently to the loss of mutant cells. The Egfr null phenotype is distinct from that of either spitz or vein mutants, suggesting that a combination of these or other ligands is required for aspects of Egfr function. No delay in ato refinement is found for spi mutant eyes, and vn mutants have vestigial eye discs that usually fail to differentiate any type of eye cell. In normal development, Egfr protein is expressed in most retinal cells, but at distinct levels. Antibody specific for diphospho-ERK as well as expression of the Egfr target gene argos was used to assess the pattern of Egfr activity; highest activity was found in the intermediate groups of cells in the morphogenetic furrow. However, studies of mutant genotypes suggest that this activity may not be required for normal ommatidium development. Since distinct phenotypic effects for four different levels of Egfr activity associated with wild-type, null mutant, Elp mutant, or fully activated DER function are seen, it is proposed that multiple thresholds separate several aspects of Egfr function. These include activation of N signaling to repress R8 specification; turning on argos expression, and recruiting photoreceptors R1-R7. It is possible that during normal eye development these thresholds are attained by different cells, contributing to the pattern of retinal differentiation. It is suggested that Egfr activation by Spi is the only signal that the R8 cell needs to provide for most photoreceptor cells and that ubiquitous targeted Spi expression converts every retinal cell to an R1-R7 photoreceptor, at the expense of R8 and other cell types. Argos is required for loss of ommatidia in Elp. Nonautonomy of Elp mutations suggests that aos acts nonautonomously to inhibit ommatidium formation (Lesokhin, 1999).

A new conditional Egfr allele was used to elucidate the roles of the receptor in eye development. Egfrtsla is a tight temperature-sensitive allele which gives rise to structural defects in the adult eye with treatments at the non-permissive temperature as short as 1 hour. Subjecting Egfrtsla flies to the non-permissive temperature for 24 hours (beginning at a time when the furrow is about half way across the eye field) results in structural defects, many of which are observed in external views of the adult eye. The most obvious defect is a physical scar that runs across the eye in a dorsal-to-ventral direction. Since new ommatidia are found anterior to the scar, it is concluded that development can recover following restoration of Egfr function. Young Egfrtsla larvae were temperature shifted when the furrow was not yet initiated and a novel and unexpected Egfr function was found: it is required for the initiation of neural differentiation on the posterior margin. Normally the furrow initiates at the posterior margin and is negatively regulated by Wingless expression on the disc margins, in particular on the dorsal side. In this early EGFR-TS condition, an inhibition of neural differentiation is seen at the posterior margin, but not at the dorsal margin (Kumar, 1998).

To directly visualize RAS/MAPK pathway signaling downstream of Egfr a monoclonal antibody specific to the active, di-phosphorylated form of the MAP kinase (dp-ERK) was used. In the developing eye dp-ERK is found in large clusters of cells in the furrow. The dp-ERK pattern was examined in the eye disc at a high resolution. In addition to the identification of dp-ERK in large clusters of cells in the furrow, followed by smaller clusters in later columns of photoreceptors (note: each column is a dorsal-ventral stack of photoreceptor clusters all born at the same time), dp-ERK is also found in additional positions. There is a low level of cytoplasmic antigen anterior to the furrow, then in the furrow the large clusters develop from the midline. Within one column in the furrow, the dp-ERK staining clusters are initially small, then larger, and then smaller again. As clusters within a column are formed at 15-20 minute intervals, this phase of dp-ERK accumulation corresponds to more than 2 hours in time. The clusters ultimately focus to one or a few cells, in which dp-ERK can then be seen for about two columns (corresponding to 4 hours of development). This development from the eye midline of small clusters, through large clusters and then back to one or a few cells is consistent with the expression of Scabrous and with the proneural focusing of Atonal in the founding R8 cells. Thus the developing dp-ERK pattern appears to correlate with the specification of the ommatidial precluster. The large dp-ERK clusters were positioned relative to the early steps of ommatidial formation by double staining for dp-ERK and cytoplasmic actin. The large clusters dp-ERK correspond to a very early stage, one column anterior to the first clear ommatidial clusters, which appears to be the `rosette' stage (Kumar, 1998).

It is important to note that, contrary to expectations, dp-ERK in the developing eye is primarily cytoplasmic. Wild-type discs were stained to reveal dp-ERK and DNA and then these were colocalized in confocal optical sections. The dp-ERK antigen is clearly mostly cytoplasmic in the large furrow clusters since dp-ERK cannot be detected in cell nuclei in the furrow. At a later stage (more posterior in the same field), dp-ERK can be seen in occasional, apical nuclei. The time series of cluster formation in the furrow shows that detectable nuclear dp-ERK can follow cytoplasmic phosphorylation by 2 hours or more. Later, transient dp-ERK antigen can be detected in cell nuclei, such as the developing R3 and R4 cells, for much shorter times. About nine columns (18 hours) after dp-ERK staining first appears in the furrow, it can be seen in the cytoplasm of the future R7 cell. This dp-ERK persists for about four columns (8 hours), and is genetically dependent on the activity of the sev gene since it is absent in discs derived from sev null mutant larvae. It is interesting to note that dp-ERK is detectable in the cytoplasm of the future R7 for an extended period (about 8 hours). Ectopic activation of the RAS/MAPK pathway increases the level of cytoplasmic but not nuclear dp-ERK (Kumar, 1998).

Historically, the first data to suggest that Egfr functions in Drosophila retinal development came from an analysis of Ellipse mutations. Egfr Ellipse alleles appear to be dominant gain-of-function mutations as they are suppressed in trans to null alleles of Egfr. In Ellipse homozygotes, there are fewer ommatidia than normal and they are separated by increased space]. This led to an attractive model: that the preclusters and/or founder (R8) cells are normally spaced evenly by a short-range diffusible inhibitor (a mechanism known as `lateral inhibition') and that the receptor for this inhibitor is Egfr. Thus in Ellipse, hyperactive Egfr leads to increased space between the clusters. This model led to a testable prediction that loss of Egfr function should reduce the space between ommatidia. This paper shows that in the temperature sensitive Egfr mutants, a complete loss-of-function condition (a normally spaced array of single R8 cells) is formed. This strongly suggests that Egfr has no role in cluster spacing. If there is any role for EGFR in establishing cluster and/or R8 cell spacing, it must be a minimal contribution and/or be highly redundant (Kumar, 1998 and references).

A second model has been proposed for the function of Egfr in the Drosophila retina: that reiterative use of the Egf receptor triggers differentiation of all cell types in the Drosophila eye. This was suggested by the loss-of-function phenotype and overexpression of spitz and from the phenotype of dominant-negative Egfr mutant protein. The fact that a regularly spaced array of R8 cells is formed in the Egfr-TS condition suggests that, at least as far as the R8 cell type is concerned, Egfr cannot be said to trigger the differentiation of all cell types in the eye. It is formally possible that Egfr does normally play a role in R8 cell specification, but that this role is redundant or dispensable (ie other RTKs can specify R8 cells in the absence of Egfr function). It is likely that all subsequent recruitment steps require Egfr however (Freeman, 1996). Egfr may act in combination with other receptors such as Sev to raise the level of RAS/MAPK pathway activity over some critical threshold. The observation that dp-ERK staining initially disappears from the furrow, but later rebounds (without Egfr function) strongly suggests that other receptor tyrosine kinases are present in the furrow and can act there. Thus, Egfr function is necessary for morphogenetic furrow initiation, is not required for establishment of the founder R8 cell in each ommatidium, but is necessary to maintain the differentiated state of founder cells. Egfr is required subsequently for recruitment of all other neuronal cells. The initial Egfr-dependent MAP kinase activation occurs in the furrow, but the active kinase (dp-ERK) is observed only in the cytoplasm for over 2 hours. Similarly, Sevenless-dependent activation results in the cytoplasmic appearance of dp-ERK for 6 hours. These results suggest an additional regulated step in this pathway (Kumar, 1998 and references).

The Drosophila retina represents a particularly accessible tissue to address issues of local cell-cell signaling. Correct pattern is achieved in the Drosophila retina in part through the temporal and spatial control of programmed cell death (PCD). The mature retina is composed of an organized array of some 750 unit eyes (ommatidia), each containing eight photoreceptor neurons, four cone cells, two primary pigment cells (1°s), and a hexagonal lattice composed of secondary/tertiary pigment cells (2°/3°s) and sensory bristle organules. With the possible exception of the cells of the bristle organule, cell fates in the retina are not determined through lineage-based restriction but instead rely on local signals passed between cells. These signals result in progressive recruitment of undifferentiated cells by their previously differentiated neighbors. Creation of the interommatidial lattice of 2°/3°s is the result of the final cell fate decision in the retina: some cells are recruited as 2°/3°s, while any remaining excess cells are removed by PCD. Two different cell types have been proposed to be the major regulators of cell death in the retina: 1°s and cells of the bristle organule. 1°s were implicated as potential regulators of PCD by experiments examining Notch loss-of-function alleles: reduction of Notch function led to loss of both 1°s and PCD, leading to the suggestion that 1°s direct PCD. Alternatively, bristles have been proposed as regulators of PCD in the retina due to clustering of apoptotic cells (detected by acridine orange staining) around bristle organules. More recent experiments indicate that cell death can occur in the absence of bristles, although their presence may still influence PCD. Evidence is provided that the cone cells and 1°s provide a signal that promotes survival of cells in the interommatidial lattice. Further evidence is provided that this signal represents part of a balance between signals of the ras and Notch pathways, which appear to act in opposition to regulate the number of interommatidial cells permitted to remain (Miller, 1998).

The first cell types to emerge in the developing retina are the photoreceptor neurons and (non-neuronal) cone cells, which arise within the retinal neuroepithelium of the mature larva. The larva then undergoes pupation as the retina evaginates (disc eversion) and is repositioned to lie distally against the pupa’s cuticle. Soon after disc eversion, the 1°s emerge to enwrap the cone cells (22-24 hours APF). They establish direct contact with the remaining undifferentiated cells which lie between ommatidia, and which are referred to as interommatidial precursor cells or ‘IPCs’. Finally, a hexagonal lattice is formed between ommatidia as IPCs are directed into one of two fates: 2°/3° or PCD. The result is a precise hexagonal array of ommatidia, each surrounded by nine 2°/3°s and three bristles. Each cell type in the developing retina can be recognized by the position of its nucleus. Typically, nuclei are first found in the basal part of the neuroepithelium and rise apically as a cell begins its differentiation. During early pupal stages, cone cell nuclei are arranged as an apical ‘cloverleaf’ at the center of each ommatidium and several microns above the photoreceptor nuclei; two 1° nuclei form an apical ring around the cone cells; and the IPC nuclei are found basally between ommatidia (these nuclei are slow to rise apically except bristle nuclei, which are found at an intermediate level early in their differentiation). This stereotyped arrangement permits identification and ablation of each cell type. Experimentally induced ablation alters the arrangement and subsequent identity of cells in the retina, in order to understand the underlying mechanism of cell fate determination. Once the ablation is performed, pupae are permitted to develop for an additional 24 hours to allow for establishment of all cell types; retinae are then removed and stained with cobalt sulfide to highlight each cell type at the surface. In each experiment, the non-ablated partner is used as an internal control. The effects of ablation are limited to the target cell, with little apparent collateral damage to neighboring cells as assessed by their normal subsequent development (Miller, 1998).

Disc eversion is complete by 18 hours APF at 25°C; the first indication of 1° differentiation is the apical migration of its nucleus at 22-24 hours APF. In initial studies, laser ablation of a 1° at this stage results in its rapid replacement. 1° nuclei ablated after 24 hours APF are not replaced. With regard to establishment of the 1° fate, these results indicate: (1) several cells have the potential to differentiate as 1°s; (2) this decision remains reversible for several hours; and (3) during this period, established 1°s provide a signal inhibiting the 1° fate in their neighbors. Loss of pupal Notch activity blocks both 1° differentiation and PCD, leading to the suggestion that 1°s promote PCD. Ras signaling promotes the 2°/3° fate at the expense of PCD. One pathway which can act in opposition to Notch is the Ras signal transduction pathway Ras is required for a variety of cell fate decisions in the developing retina. To test the role of Ras signaling during PCD, flies were used in which an inducible heat shock promoter was fused to the activated Ras form Dras1 Val12. A 1-hour pulse of Dras1 Val12 throughout the retina beginning at 26 hours APF rescues IPCs from PCD. Early removal of cone cells and 1°s in four neighboring ommatidia has no effect on this rescue. This result indicates that Ras signaling acts to prevent PCD and/or promote the 2°/3° fate. With regard to PCD, therefore, Ras acts in opposition to Notch signaling (Miller, 1998).

This Ras-mediated rescue of cells is similar to, and epistatic to, the rescue provided by cone cells and 1°s. Are the two signals linked? The Ras pathway has been demonstrated to be activated by a variety of extracellular stimuli, including signaling through receptor tyrosine kinases (RTKs). In the developing retina, the Egf receptor ortholog is an RTK that regulates a variety of cell fate determination steps including 2°/3° determination. Consistent with the results described above for activation of Dras1, loss of Egfr activity leads to a loss of 2°/3°s, presumably due to an excess of PCD. To determine whether Egfr signaling is sufficient to block PCD, flies containing an activated form of Egfr (l-DER) fused to an inducible heat shock promoter received a 1-hour heat shock. Expression of l-DER throughout the young pupal retina results in a block in PCD. The loss of PCD is not complete, perhaps due to the relatively weak activation of Egfr provided by the l-DER protein. Egfr is a receptor that acts autonomously: Egfr expression in IPCs is anticipated in the cells where it is active during the stage of PCD. Consistent with this view, Egfr is found to be expressed primarily in the IPCs. These results suggest the possibility that IPCs receive a signal from their neighbors that activates their own Egfr signaling and represses PCD. The ablation results suggest this signal is derived from the 1°s and perhaps the cone cells. Interestingly, the TGFalpha ortholog, Spitz, is expressed at high levels in the cone cells and bristles and can be detected at lower levels in the 1°s. Spitz is a diffusible ligand of Egfr and may represent the 'life' signal provided by the ommatidium. Together, these observations suggest a model in which patterning requires local Spitz/Egfr signaling by (at least two) 1°s to rescue neighboring IPCs from a Notch-imposed apoptotic fate. One important test of this model will require the removal of spitz function specifically in cones cells and 1°s (Miller, 1998).

Whether D-Fos mediates ERK signaling during eye morphogenesis was investigated. Defects in photoreceptor differentiation can be induced by the RTK gain-of-function alleles ElpB1 and sevS11. The ElpB1 allele dominantly causes an abnormal eye phenotype that manifests itself in roughness and the occasional lack of outer photoreceptors. This phenotype can be suppressed largely by the removal of one copy of D-fos and restored subsequently by simultaneous transgenic expression of wild-type D-Fos. A gain-of-function transgene of the RTK-coding gene sevenless (sevS11) causes the characteristic appearance of ectopic R7 photoreceptor cells in nearly all ommatidia. The sevS11 phenotype can be suppressed by the expression of dominant-negative Fos. In flies carrying sevS11 in a heterozygous kay2 background, the ectopic R7 photoreceptor phenotype is suppressed significantly; the number of normal ommatidia increases from 5% to approximately 20%. Reintroduction of D-fos by a transgene in this double mutant background restores the percentage of ommatidia with extra photoreceptors observed in sevS11 heterozygous animals. Taken together, these results indicate that D-Fos can act as a rate-limiting component downstream from the RTKs Sev and DER during eye development (Ciapponi, 2001).

Genetic and biochemical analysis of the role of Egfr in the morphogenetic furrow of the developing Drosophila eye

A key event in patterning the developing Drosophila compound eye is the progressive restriction of the transcription factor Atonal in the morphogenetic furrow. The Atonal pattern evolves from expression in all cells to an over-dispersed pattern of single founder cells (the future R8 photoreceptors). This restriction involves Notch-mediated lateral inhibition. However, there have been inconsistent data on a similar proposed role for the Egf receptor (Egfr). Experiments using a conditional Egfr mutation (Egfrtsla) have suggested that Egfr does not regulate Atonal restriction, whereas experiments using Egfr-null mosaic Minute+ clones have suggested that it does. This study re-examines both approaches. The lesion in Egfrtsla is a serine to phenylalanine change in a conserved extracellular ligand-binding domain. It is shown by biochemical and genetic approaches that the Egfrtsla protein is rapidly and completely inactivated upon shift to the non-permissive temperature. On temperature shift, the protein moves from the cell surface into the cell. Finally, a flaw is reported in the Egfr-null mosaic Minute+ clone approach. Thus, this study demonstrates that Egfr does not play a role in the initial specification or spacing of ommatidial founder cells (Rodrigues, 2005).

The lesion in Egfrtsla is a missense mutation in the conserved ligand-binding, extracellular L2 domain. Biochemical and localization data suggest that the Egfrtsla protein functions normally at 18°C as a ligand-activated receptor. However, after shift to the non-permissive temperature, Egfrtsla rapidly becomes inactive and is removed from the cell surface, probably via a non-signaling endocytic pathway (with or without ligand). It may be that the Egfrtsla protein is conformationally unstable at 30°C and is degraded. Human EGFR is normally only internalized in response to ligand binding, but it has been reported that inhibition of PKA leads to internalization of unbound EGFR. The mutation in the extracellular L2 domain suggests that this domain may be involved in mediating the stability of Egfr in the membrane (Rodrigues, 2005).

Atonal expression and R8/founder cell spacing are normal in Egfrtsla eye discs incubated at the non-permissive temperature. Three possible artifacts that could have invalidated this observation were examined. (1) Egfrtsla could be temperature sensitive for synthesis but not activity. If so, then protein made before the shift to 30°C might continue to supply sufficient function at the non-permissive temperature to support normal R8/founder cell development. However, Egfrtsla has, in fact, been shown to be temperature sensitive for activity, and that activity is lost within minutes of the temperature shift, while Atonal expression and R8/founder cell formation continues normally for 24 hours. (2) Egfrtsla could be leaky (i.e. not null) at 30°C, and some residual activity might supply sufficient function at the non-permissive temperature to support normal R8/founder cell development. Egfrtsla mutants (at the non-permissive temperature) have been shown to be genetically indistinguishable from a null (Egfrf24) for three phenotypes. This study shows in addition that Egfrtsla mutants at 30°C are phenotypically indistinguishable from nulls (Egfrf2 and Egfrtop-18A), in Minute+ mosaic clones, as well as in their growth deficits in clones made without Minute mutations. Furthermore, quantitative biochemical experiments were undertaken using S2 cells to show that Egfrtsla at 30°C is indistinguishable from a null in its ability to drive both the phosphorylation of MAPK and its own autophosphorylation. (3) It could be that, at 30°C, the furrow just freezes in Egfrtsla mutant eyes, leaving an arrested but apparently normal furrow. This study shows that in adjacent Egfrtsla and Egfr+ territories, the furrow advances at the same rate after the temperature shift to 30°C. Thus, it is concluded that observation of normal Atonal and R8/founder cell patterning in Egfrtsla mutant eyes is not invalidated by any of these three possible artifacts (Rodrigues, 2005).

Next, the possible problems associated with the Minute+ mosaic method used to make the same observations with the Egfr nulls were examined. The Minute+ Egfr null experiments were replicated exactly as previously reported, using the same Drosophila stocks, except to run them at 30°C. In parallel, the same experiments were done with Egfrtsla. If the known Egfr nulls were to have a different phenotype to Egfrtsla, it would be necessary to conclude that Egfrtsla is not behaving as a null in this assay. If, however, the two Egfr nulls have the same phenotype in this assay as Egfrtsla does, then it could be concluded that the temperature-sensitive allele is indistinguishable from the nulls at 30°C, and that the difference is due to the Minute technique and not to Egfr. Indeed, it was found that the Egfrtsla and Egfr null phenotypes are indistinguishable, and thus it is concluded that the Egfrtsla phenotypes assayed without the Minute technique are valid and that the discrepancy stems from some aspect of the use of the Minute mutations. Minute clones made without any Egfr mutation present were stained and the very same Atonal expression defects appeared. Taken together, these data suggest that the spacing defects are genetically dependent on the presence of the Minute clones, and are not an affect of the Egfr mutations (Rodrigues, 2005).

Therefore, it is concluded that Egfr has no primary role in R8/founder cell spacing and also that Egfrtsla is a rapidly acting temperature-sensitive mutation that is functionally null at the non-permissive temperature (Rodrigues, 2005).

Egfr and eye morphogenesis (part 2/2)

Table of contents

EGF receptor : Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | References

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