EGF receptor

Effects of Mutation or Deletion

Table of contents

General effects of Egfr mutation

Mutations in the Egfr gene disrupt a variety of developmental processes in Drosophila. These include the survival of certain embryonic ectodermal tissues, the proliferation of the imaginal discs, and the morphogenesis of several adult ectodermal structures and oogenesis. Egfr is genetically complex: a number of alleles of the gene differentially affect the development of specific tissues, such as the eye, wing, bristles and ovary. In addition, Egfr mutations exhibit interallelic complementation. Alleles that differentially affect specific developmental processes encode receptors with altered extracellular domains. Alleles that fully or partially complement a wide range of embryonic and postembryonic Egfr mutations encode receptors with altered intracellular domains (Clifford, 1994).

The Drosophila epidermal growth factor receptor (Egfr) is a key component of a complex signaling pathway that participates in multiple developmental processes. An F1 screen was performed for mutations that cause dominant enhancement of wing vein phenotypes associated with mutations in Egfr. With this screen, mutations were recovered in Hairless (H), vein, groucho (gro), and three apparently novel loci. All of the enhancers of Egfr mutations [E(Egfr)] identified show dominant interactions in transheterozygous combinations with one another and with alleles of Notch or Su(H), suggesting that they are involved in cross-talk between the N and Egfr signaling pathways. Further examination of the phenotypic interactions between Egfr, H, and gro reveals that reductions in Egfr activity enhances both the bristle loss associated with H mutations, and the bristle hyperplasia and ocellar hypertrophy associated with gro mutations. Double mutant combinations of Egfr and gro hypomorphic alleles leads to the formation of ectopic compound eyes in a dosage sensitive manner. These findings suggest that these E(Egfr)s represent links between the Egfr and Notch signaling pathways, and that Egfr activity can either promote or suppress Notch signaling, depending on its developmental context (Price, 1997).

Genetic interactions between the N and Egfr signaling pathways have been reported previously. There is a mutual enhancement between N gain-of-functions (gof) and Egfr loss-of-functon (lof) mutations and mutual suppression between lof alleles of Delta and Egfr. Egfr gof alleles enhance Notchspl in the eyes and Delta loss-of-function alleles in the wings of double mutant flies. Mutations in Egfr and two other Egfr pathway components (Son of sevenless and pointed) act as enhancers of N signaling in the eye. Mutations in both Hairless and groucho enhance L4 wing vein defects associated with mutations in both Egfr and vein. Egfr. In turn, Egfr mutations enhance the Hairless mutant-associated loss of macrochaetae and microchaetae from the head and thorax; therefore, Egfr and Hairless appear to cooperate in at least two developmental processes. Groucho appears to be required in contexts that appear to be distinct from its function in Notch signaling. Ectopic wing hairs are observed on the wings of Egfr, groucho and rolled;groucho double mutants that are similar to defects seen in hairy mutant flies or groucho/hairy transheterozygotes, and may indicate that Egfr mutations reduce Groucho's activity as a corepressor with Hairy. The spectrum of defects enhanced in Egfr;gro or rolled;groucho double mutants appears to reflect a reduction in most or all aspects of groucho activity. The simplest interpretation of these observations is that both Egfr and Rolled promote the activity of Groucho. Gro and its mammalian homolog, TLE1, are phosphorylated on serine residues and thus may be downstream targets of an Egfr-regulated phosphorylation cascade (Price, 1997 and references).

Eight alleles of Dsor1 encoding a Drosophila homolog of mitogen-activated protein (MAP) kinase kinase were obtained as dominant suppressors of the MAP kinase kinase kinase D-raf. These Dsor1 alleles themselves showed no obvious phenotypic consequences nor any effect on the viability of the flies, although they were highly sensitive to upstream signals and strongly interacted with gain-of-function mutations of upstream factors. They suppress mutations for receptor tyrosine kinases (RTKs) torso, sevenless, and to a lesser extent, Drosophila EGF receptor. Furthermore, the Dsor1 alleles show no significant interaction with gain-of-function mutations of Egfr. The observed difference in activity of the Dsor1 alleles among the RTK pathways suggests Dsor1 is one of the components of the pathway that regulates signal specificity. Expression of Dsor1 in budding yeast demonstrates that Dsor1 can activate yeast MAP kinase homologs if a proper activator of Dsor1 is coexpressed. Nucleotide sequencing of the Dsor1 mutant genes reveal that most of the mutations are associated with amino acid changes at highly conserved residues in the kinase domain. The results suggest that they function as suppressors due to increased reactivity to upstream factors rather than constitutive activity (Lim, 1997).

To investigate a Ras-independent means of activating the Mapk cascade, mutations have been isolated that suppress the lethality of a Drosophila Raf mutation [also referred to as l(1) pole hole]. Six extragenic Su(Raf) loci have also been identified. These mutations not only suppress Raf^C110 but also other partial loss-of-function Raf alleles that do not impair Ras-Raf binding. This suggests that the suppression of Raf^C110 by the extragenic Su(Raf) mutations does not necessarily involve the restoration of Ras-Raf binding. Developmental analyses have shown that all six extragenic Su(Raf) mutations promote signaling in the Sevenless (Sev) and Egfr RTK pathways. Su(Raf)34B is a gain-of-function mutation in the Dsor1 locus that encodes the fly Mek. Recently, Su(Raf)1 has been shown to encode Src42A. The isolation of mutations that suppress the suppressor activity of Su(Raf)1 is reported in this paper. These mutations define two known genes, Egfr and rolled (rl; also referred to as Mapk) and two previously uncharacterized loci. In addition, two alleles of Src42A were also isolated in the screen, although these mutations are not true suppressors of Su(Raf)1 (Zhang, 1999).

One of the novel suppressor loci was named semang (sag). sag is required during both embryonic and imaginal disc development. Mutations in sag cause zygotic lethality. To identify developmental pathways where sag functions, the phenotypes associated with sag mutations were examined with particular attention to those processes controlled by known Drosophila RTKs. The results of these analyses show that sag participates in the Torso (Tor) and Drosophila DFGF-R1 RTK (Breathless) pathways during embryonic development. sag also disrupts the embryonic peripheral nervous system. During imaginal disc development, sag mutations affect two processes known to require Egfr signaling: the recruitment of photoreceptor cells and wing vein formation. Thus sag functions broadly in several RTK-mediated processes. This role of sag in RTK signaling is further supported by the genetic interaction between sag and other known RTK signaling genes. sag dominantly enhances the phenotypes caused by reductions of RTK signaling in loss-of-function Raf or rl mutants. Consistent with this, sag dominantly suppresses the formation of supernumerary R7 cells caused by the activated sev-Ras1^V12 mutation. The sag mutations analyzed are likely to be loss-of-function mutations. These results suggest that sag may have a positive role in RTK signaling (Zhang, 1999).

Drosophila has two other Src family members, Src64 and Tec29, both of which are involved in ring canal development during oogenesis. Src64 does not affect viability when mutated. The isolation of Su(Raf)1 as a mutation in Src42A that restores the viability of Raf mutants and the isolation of Egfr, rl, and sag as extragenic suppressors of Su(Raf)1 provides the first in vivo evidence that both Src42A and sag are modulators of RTK signaling. At this moment, it is not known where Src42A and sag fit into the known RTK signaling cascade. An Src42A cDNA driven by a ubiquitously expressing promoter rescues the lethality of both Su(Raf)1 homozygotes and Su(Raf)1/Df hemizygotes. Based on this, Su(Raf)1 has loss-of-function characteristics, suggesting that Src42A is, unexpectedly, a negative modulator of RTK signaling. However, the genetics of Su(Raf)1 suggest that the suppression of Raf^C110 may be attributed to a dominant-interfering effect because the Raf^C110 lethality is not suppressed in Src42A hemizygotes of genotype Df(2R)nap⁹/+. Because of this, the role of Src42A in RTK signaling is still being investigated. However, the genetic interaction as revealed by the modifying screen suggests that Egfr and other RTKs may possibly regulate Src42A and sag, which in turn modulate the Mapk cascade (Zhang, 1999).

Egfr mutation and components of the Egfr pathway

ebi (the Japanese term for 'shrimp') regulates the epidermal growth factor receptor (Egfr) signaling pathway at multiple steps in Drosophila development. Mutations in ebi and Egfr lead to similar phenotypes and show genetic interactions. However, ebi does not show genetic interactions with other RTKs (e.g., torso) or with components of the canonical Ras/MAP kinase pathway. ebi encodes an evolutionarily conserved protein with a unique amino terminus, distantly related to F-box sequences, and six tandemly arranged carboxy-terminal WD40 repeats. The existence of closely related proteins in yeast, plants, and humans suggests that ebi functions in a highly conserved biochemical pathway. Proteins with related structures regulate protein degradation. Similarly, in the developing eye, ebi promotes Egfr-dependent down-regulation of Tramtrack88, an antagonist of neuronal development (Dong, 1999).

ebi mutations have been identified in a screen for enhancers of an eye mutant called roughex, which plays a key role in regulating cell cycle progression in the developing eye. As a consequence of cell cycle defects, photoreceptor differentiation and pattern formation in the eye are disrupted. Whereas cell cycle regulators enhance and suppress the primary cell cycle phenotype, mutations in other loci, such as Star and Epidermal growth factor receptor, only modify the differentiation phenotype, and not the earlier cell cycle defects. Like Star and Egfr, ebi enhances the differentiation phenotype. These observations led to a consideration of the relationship between the Egfr signaling pathway and ebi. Evidence shows that ebi participates in Egfr signaling pathways. ebi^E4, ebi^E90, and ebi^P7 are null, strong, and weak alleles, respectively (Dong, 1999).

That ebi functions in the Egfr pathway was initially suggested by phenotypes of a viable heteroallelic combination of ebi (i.e., ebi^P7/ebi^E90). These flies exhibit phenotypes similar to weak loss-of-function Egfr alleles (i.e., Egfr^top1/Egfr^f2) including partial female sterility resulting from partially ventralized eggs, wing vein defects, short bristles, and abnormal eyes (i.e., rough eyes). Further evidence that ebi participates in the Egfr pathway was provided by genetic interactions between ebi and Egfr components. For instance, flies carrying two different alleles of Egfr (Egfr^top1/Egfr^f2) have a weak rough-eye phenotype, which is enhanced in flies that are heterozygous for ebi. ebi and Egfr mutant embryos are also similar. Homozygous ebi null mutant embryos (ebi^E4) exhibit a tail-up or U-shaped embryo with head defects. Embryos lacking both the zygotic and maternal contributions of ebi were created using ovo^D and FRT/FLP-induced recombination. This results in a more severe phenotype, including the loss of ventral denticle belt structures and a tightly curled morphology indicating a marked failure in germ-band retraction. Severe head defects are also observed. In contrast to Egfr mutants, some residual ventral cuticular structures remain in embryos lacking both the zygotic and maternal contributions of ebi (Dong, 1999).

Loss of ebi also affects Egfr-dependent expression of genes in the embryo. The Egfr ligand Spitz is expressed along the ventral midline and induces expression of different target genes, including fasciclin III (fasIII) and orthodenticle (otd), in cells located in more lateral positions. In zygotic null Egfr mutants both otd and FasIII expression are lost. In wild-type stage 11/12 embryos, FasIII protein is broadly distributed in the visceral mesoderm and in a bilaterally symmetric cluster of cells flanking the midline of the ventral ectoderm. In ebi mutant embryos lacking both maternal and zygotic contribution, FasIII expression is largely abolished, although some residual patches of staining remain. Egfr-independent expression of FasIII in the anterior-most region of the embryo is unaffected in ebi mutants. In wild-type stage 10/11 embryos, otd mRNA is expressed in the preantennal head region and in the ventral-most ectoderm. In ebi mutant embryos, otd expression is markedly reduced. These data suggested that ebi may be a component in the Egfr signal transduction pathway. To assess whether ebi encoded a hitherto unidentified regulator in the Ras/MAP kinase pathway, its role in the Torso RTK pathway was assessed. Torso controls the development of the anterior and posterior termini of the embryo. Ras, Raf, MEK, and MAPK participate in both the Egfr and Torso RTK pathways. The expression of Torso target genes huckebein (hkb) and tailless (tll) in embryos entirely deficient in ebi (i.e., lacking both maternal and zygotic ebi) are indistinguishable from wild type. In summary, ebi mutant phenotypes assessed using both molecular and morphological criteria are similar to Egfr mutations. Furthermore, ebi does not function in all RTK pathways, since Torso-induced terminal development is ebi independent. These data indicate that ebi, either directly or indirectly, regulates Egfr signaling. As a step toward understanding the role of ebi in the context of a specific developmental process, the role of ebi in R7 development in the compound eye was assessed through both genetic and molecular studies (Dong, 1999).

The R7 equivalence group comprises five cells competent to become R7 neurons. They are the R7 precursor cell and the precursors to the four cone cells. Cone cell precursor cells can be induced to become R7 cells by ectopic activation of the R7 inductive pathway in these cells. Transformation of cone cells into R7 cells leads to a disorganized adult eye or a so-called rough-eye phenotype. The ability of loss-of-function ebi mutations to suppress this transformation was assessed in various genetic backgrounds. Whereas ebi dominantly suppresses R7 development induced by the activated Egfr expressed in the R7 equivalence group under the control of the sev enhancer (sev-Tor^DEgfr), it does not suppress R7 development induced by the activated Sev receptor (sev-Tor^DSev, SevS11, or activated forms of Ras, Raf, and MAPK. Hence, ebi is required for the transformation of cone cell precursors into R7 neurons by the activated Egfr (Dong, 1999).

To assess whether ebi participates in the induction of the R7 precursor cell into an R7 neuron, a genetically sensitized background in which only some 15%-20% of the R7 precursors become R7 neurons was used. The R7 inductive signal is attenuated by using a strong hypomorphic allele of sev (sev^E4) and a weak gain-of-function mutation in the Ras activator, encoded by the son-of-sevenless gene, Sos^JC2. Aside from the loss of the majority of the R7 cells, development of the eye in this genetic background is otherwise indistinguishable from wild type. ebi is a dominant enhancer of this phenotype, as are Egfr loss-of-function mutations. These data are consistent with studies demonstrating a requirement for both the Egfr and Sev receptor in R7 induction. Hence, ebi is required for induction of the R7 precursor cell into an R7 neuron and for transformation of cone cell precursors into R7 in response to ectopic activation of Egfr. Ttk88 down-regulation is required for R7 induction of the R7 precursor cell. This is supported by the finding that Ttk88 mutations are dominant suppressors of the Sev^E4;Sos^JC2/+ phenotype (Dong, 1999).

To assess the role of ebi on R7 development in an otherwise wild-type background, attempts were made to generate homozygous null mutant clones. Such clones could not be generated using X-ray and heat shock Flp-induced mitotic recombination. Hence, like Egfr, ebi is required for cell proliferation and/or survival during the proliferative phase of disc development. To increase the efficiency of Flp-induced mitotic recombination, a Flp source driven by the eyeless (ey) promoter was used. The ey promoter drives expression from the earliest cell divisions in the eye primordium until the last cell division of precursor cells in the third instar. This results in the production of multiple mutant clones throughout development. Mutant clones in the eye disc have been recognized by the loss of Ebi immunoreactivity. Rather small clones have been observed: clusters within these clones contain differentiating R cells. Each cluster contains a single R8 cell (i.e., stained with antibody to the Boss protein), and early clusters appear normal. Although clusters containing eight neurons form, disorganized clusters containing fewer differentiated neurons are also observed (Dong, 1999).

Adult ommatidia containing homozygous mutant cells are frequently highly disorganized and show a marked reduction in R cells. Mutant R cells, including R7 cells, are seen in adult mosaic ommatidia; some 80% of these cells show an altered cellular morphology. Hence, although ebi is required for R7 development in a genetically sensitized background, R7 neurons can develop in an ebi mutant. Although the formal possibility that these R7 neurons develop because of perdurance of Ebi protein in the R7 precursor cell cannot be ruled out, these data strongly suggest that R7 cells can form in an ebi-independent fashion, though less efficiently than in wild type. These data are consistent with ebi subserving a redundant function in R7 development. To gain clues to the molecular pathways regulated by ebi, the gene was cloned and sequenced (Dong, 1999). This transcription unit encodes a protein of 700 amino acids with a carboxy-terminal segment containing six WD40 repeats. The ebi^E4 and ebi^E90 alleles result in missense mutations. In ebi^E4 the methionine encoded by codon 1 is changed to an isoleucine, and in ebi^E90 a highly conserved cysteine, located at amino acid 510 between WD40 repeats 3 and 4, is changed to a tyrosine (Dong, 1999).

ebi-related human cDNA sequences and genomic sequences from S. cerevisiae and Arabidopsis thaliana, have been identified in the database. Because the initial human expressed sequence tag was not complete, additional cDNAs were isolated from adult human spleen cDNA library and sequenced. Both an amino-terminal 89-amino-acid segment and the carboxy-terminal WD40 repeats of fly ebi correspond remarkably well to these regions in the mammalian, plant, and yeast genes; the amino-terminal 89 amino acids and the WD40 repeat region share 81%, 34%, and 51% identity with the human, yeast, and plant sequences, respectively. In addition to these conserved regions the fly protein is predicted to contain an insertion of 160 amino acids between the amino terminus and the WD40 repeats (Dong, 1999).

The bipartite structure of Ebi is reminiscent of three proteins involved in protein degradation: Cdc4 from S. cerevisiae; supernumerary limbs (Slimb) from Drosophila melanogaster, and Sel-10 from C. elegans. All three proteins contain an amino-terminal F-box and carboxy-terminal WD40 repeats; these proteins have been shown (Cdc4) or proposed (Slimb and Sel-10) to target proteins for degradation by linking them to a ubiquitin-conjugase complex. Although the amino-terminal domain of Ebi is divergent from the Cdc4 F box (as is Slimb), it shares weak sequence and structural homology. The amino-terminal half of the F box is more highly related to ebi than the carboxy-terminal region. The periodic spacing of hydrophobic residues in both Ebi and F-box sequences is consistent with these regions being able to assume an alpha-helical amphipathic conformation. Three residues in the amino-terminal region of the Cdc4 F box have been shown to be required for binding to Skp1 (a component in the E3 complex). These amino acids are conserved in Ebi, and correspond to residues P45, I52, and L57 in the Ebi sequence (Dong, 1999).

Ebi is widely expressed in nuclei of the embryo and larvae. Immune staining is largely, if not exclusively, nuclear. Double staining of salivary gland nuclei with anti-Myc antibodies to detect Myc-tagged Ebi and the DNA stain DAPI demonstrates that Ebi was not associated with chromatin but, rather, is distributed in a reticular pattern throughout the nucleoplasm. The similarity of Ebi to F-box/WD40 repeat-containing proteins and its nuclear localization suggests that Ebi may regulate Egfr signaling through degradation of nuclear proteins. Recent studies have revealed an important role for both Egfr and degradation of a specific transcription factor Tramtrack88, for R7 development. The structural similarity between Ebi and F-box/WD40-repeat proteins involved in protein degradation prompted an exploration of the relationship between ebi and Ttk88 protein levels in the developing eye. Ttk88 is expressed at very low levels in undifferentiated cells in the developing eye disc and at high levels in developing cone cell nuclei; it is not expressed in developing photoreceptor cells. Transformation of cone cells into R7 by misexpression of phyl under the sev promoter leads to Ttk88 degradation. Ectopic R7 induction by Tor^DEgfr driven by the sev promoter also leads to marked degradation of Ttk88. sev-Tor^DEgfr-induced Ttk88 degradation is dominantly suppressed by ebi. Similarly, ebi dominantly suppresses the pGMR-phyl-induced decrease in Ttk88, as well as the pGMR-phyl-induced eye phenotype (Dong, 1999).

The role of ebi in regulating Ttk88 levels in an otherwise wild-type eye disc was examined. Analysis of Ttk88 levels on the small mutant clones generated with ey-Flp reveals no obvious differences. To explore this issue further, reduction in ebi was achieved by expressing the dominant-negative form of ebi in all cells posterior to the morphogenetic furrow in an ebi heterozygous background. Dominant-negative ebi contains the amino-terminal half of the protein from amino acids 1-334 expressed under the control of the pGMR promoter. In wild-type eye discs, Ttk88 staining is not observed in a focal plane in which photoreceptor cell nuclei are located. In contrast, in mutant discs, an average of 36 ± 6 Ttk88-positive nuclei are observed in this region. Most Ttk88-positive nuclei are found 8-10 rows posterior to the morphogenetic furrow. This increase in Ttk88-positive cells also parallels a concomitant decrease in the number of cells stained with the pan-neuronal stain, anti-Elav. In wild-type eye discs, all ommatidia 8-10 rows posterior to the morphogenetic furrow have at least seven Elav-positive cells (R1-R6 and R8). However, in mutant discs, many ommatidia in this region contained less than seven stained cells. Interestingly, a considerably smaller fraction of ommatidia in rows 11-13 contain less than eight Elav-positive R cells; in wild-type discs, all clusters contain eight Elav-positive cells in this region. Hence, a reduction in ebi activity delays neuronal development and this is correlated with persistent nuclear expression of the Ttk88 protein. In summary, both ebi and Egfr promote Ttk88 down-regulation, thereby promoting neuronal development. Further work is required to assess the relationship between ebi and ttk in Egfr signaling in other developmental contexts (Dong, 1999).

Calcineurin is a Ca²⁺-calmodulin-activated, Ser-Thr protein phosphatase that is essential for the translation of Ca²⁺ signals into changes in cell function and development. A dominant modifier screen was carried out in the Drosophila eye using an activated form of Calcineurin A1 (FlyBase name: Protein phosphatase 2B at 14D), the catalytic subunit, to identify new targets, regulators, and functions of calcineurin. An examination of 70,000 mutagenized flies yielded nine specific complementation groups, four that enhanced and five that suppressed the activated calcineurin phenotype. The gene canB2, which encodes the essential regulatory subunit of calcineurin, was identified as a suppressor group, demonstrating that the screen was capable of identifying genes relevant to calcineurin function. A second suppressor group was sprouty, a negative regulator of receptor tyrosine kinase signaling. Wing and eye phenotypes of ectopic activated calcineurin and genetic interactions with components of signaling pathways have suggested a role for calcineurin in repressing Egf receptor/Ras signal transduction. On the basis of these results, it is proposed that calcineurin, upon activation by Ca²⁺-calmodulin, cooperates with other factors to negatively regulate Egf receptor signaling at the level of Sprouty and the GTPase-activating protein Gap1 (Sullivan, 2002).

Calcineurin is activated by a sustained increase in intracellular Ca²⁺ levels that can result from the opening of intracellular Ca²⁺ channels in response to phosphoinositide (PI) signaling. PI signaling is initiated by the activation of a phosphatidylinositol-specific phospholipase C, either PLCß by G-protein-coupled receptors (GPCR) or PLCgamma by receptor tyrosine kinases (RTK). PI-PLCs cleave phosphatidylinositol 4,5-bisphosphate (PIP₂) to yield inositol 1,4,5-trisphosphate (InsP₃), which then activates the InsP₃ receptor Ca²⁺ channel (Sullivan, 2002).

GPCRs and RTKs activate an integrated signaling network that includes the Ras/mitogen-activated protein (MAP) kinase cascade, PI3-kinase, and the small GTPase Rho. Depending upon the cellular context, these pathways can either antagonize or cooperate with each other and with PI signaling. For example, T-cell activation requires the activation of both NFAT, which is transduced to the nucleus upon dephosphorylation by calcineurin, and AP1, which acts downstream of Ras and MAP kinase. Conversely, PI signaling has been found to antagonize the Ras pathway in Drosophila. The Egf receptor and Ras/MAP kinase cascade are essential for formation of wing veins and photoreceptor (R) cells in the eye. Mutations in the single phospholipase Cgamma gene, small wing (sl), cause the formation of extra R7 cells and wing vein material and also genetically interact with Egf-receptor-signaling components. A recently proposed model for sl-mediated repression of Egf receptor signaling was based on the identification of the GTPase-activating protein Gap1 as an InsP₄ receptor. PLCgamma-generated InsP₃ is converted to InsP₄, which then activates Gap. Gap converts the active form of Ras, Ras-GTP, to the inactive form, Ras-GDP (Sullivan, 2002 and references therein).

An activated form of Pp2B-14D, canA^act, was made by deleting the autoinhibitory and calmodulin-binding domains. The canA^act construct was expressed in Drosophila under the control of glass response elements, which induce transcription uniformly in cells posterior to the morphogenetic furrow in the eye imaginal disc (Sullivan, 2002).

Flies carrying one copy of the canA^act.gl transgene have mild rough eyes compared to wild type, and the eyes of flies carrying two copies exhibit a stronger phenotype. Consistent with observations in other systems, neither full-length CanA nor activated canA without a functional CanB-binding domain causes any detectable phenotypes when expressed throughout development (Sullivan, 2002).

The canA^act.gl screen yielded 11 complementation groups, 9 of which failed to modify rough eyes caused by other glass-induced transgenes. This demonstrates that the majority of the modifier groups do not act through the glass enhancer. The nine specific modifiers were then divided into class I genes, which act downstream of calcineurin, and class II genes, which act at the level of CanB (Sullivan, 2002).

Two lines of evidence suggest that calcineurin is a negative regulator of Egf receptor/Ras signaling. First, a negative regulator of RTK signaling, sprouty, was isolated as a suppressor of the canA^act.gl rough eye phenotype in the dominant modifier screen. Both sprouty and canA^act suppress wing vein formation and reduce the number of photoreceptor cells per ommatidium. Egf receptor/Ras signaling is essential for both wing vein and R-cell formation (Sullivan, 2002).

A thorough examination of genetic interactions between canA^act and components of RTK and other signaling pathways has confirmed that canA^act specifically represses the Egf receptor/Ras pathway and that it acts upstream in the pathway. The lack of convincing genetic interactions with other signaling pathways in the imaginal eye disc does not rule out a role for calcineurin in these pathways in other developmental contexts. With the exception of pnt, activated calcineurin was not modified by components downstream of Ras and was modified only by a subset of genes that act between the Egf receptor and Ras. While Gap1 and sty alleles modify the effects of activated calcineurin, drk and cbl do not. Thus calcineurin may act downstream of, or parallel to, drk and cbl. The more downstream components of the Ras/MAP kinase pathway may not interact with activated calcineurin because they are too far removed from the point(s) of intersection between calcineurin and the pathway. Alternatively, these components may not be limiting, so that reduction of gene dose, which is the basis of a dominant modifier screen, would have no appreciable effect (Sullivan, 2002).

The hypermorphic allele Egfr^E1 inhibits Ras signaling; thus it might be expected to enhance the effects of activated calcineurin. However, low levels of inappropriate Egf receptor activity in eye development are thought to increase secretion of the Egf receptor antagonist Argos. The Argos protein inhibits subsequent Egf receptor signaling that is required for photoreceptor determination. Thus, suppression of the Egfr^E1 rough eye by canA^act.gl may be the result of activated calcineurin inhibiting inappropriate Egf receptor signaling (Sullivan, 2002).

Consistent with these findings, PLCgamma is a negative regulator of Egf receptor/Ras signaling in eye and wing development. However, PLCgamma was identified in this study as a strong suppressor of activated calcineurin, although biochemically PLCgamma has been placed upstream of calcineurin in the PI signaling pathway. One explanation is that PLCgamma acts on one of the other canA genes. Another possibility is that the signaling pathways activated by PLCgamma parallel to calcineurin are required for calcineurin function (Sullivan, 2002).

A simplified schematic is presented that illustrates upstream Egf receptor signaling components in an eye disc cell. PLCgamma is activated by the Egf receptor and cleaves PIP₂ to yield InsP₃. PLCgamma is proposed to negatively regulate Egf receptor signaling through InsP₄, which is generated from InsP₃ by an InsP₃-3 kinase. Gap1 is then activated by InsP₄, which results in the inhibition of Ras. Sprouty, which may be linked to the Egf receptor by the adaptor protein Drk, may facilitate the inactivation of Ras by Gap. In this model, it is proposed that PLCgamma also acts via Ca²⁺ and calcineurin. Genetic evidence suggests that calcineurin acts at the level of sty and Gap1, although it should be noted that calcineurin may act further upstream, e.g., at the level of InsP₄. In addition, it is possible that calcineurin is activated by other Ca²⁺ signaling pathways (Sullivan, 2002).

In conclusion, it has been demonstrated that a dominant modifier screen can be used successfully to isolate mutations in genes involved in calcineurin function. The mutations in the calcineurin B gene that was isolated in the screen will help determine the roles of calcineurin in Drosophila development. In addition, compelling genetic evidence was presented that calcineurin negatively regulates the Egf receptor/Ras signaling pathway at the level of Gap1 and sprouty. Calcineurin may act directly by dephosphorylating one or more signaling components, or it may target a transcription factor and act indirectly through changes in gene expression. More work will be needed to elucidate the molecular mechanism, and the modifiers isolated in the canA^act.gl screen should prove valuable in this endeavor. Furthermore, given the conservation of signal transduction between fruit flies and vertebrates, it is likely that the signaling network that was identified is employed in other organisms (Sullivan, 2002).

To examine the interaction between calcineurin and individual components of the Egfr pathway, the ability of mutations in these components to modify the activated calcineurin phenotype was tested. Hypomorphic mutations in Egfr, Ras, pnt, sty, Gap1, and small wing modified activated calcineurin, although this was not the case for most downstream components of the Egfr pathway. TCAGB (ectopically expressed activated calcineurin) is enhanced by removing one copy of Egfr, Ras, or pnt and was suppressed by Gap1 and small wing. Both TCAGB and TCAG (another form of ectopically expressed activated calcineurin) suppress the rough eye caused by hypermorphic Egfr alleles: flies that have one copy of Egfr^E1 and TCAGB have a rough eye that closely resembles that of TCAGB alone. TCAG is not detectably modified by hypomorphic Egfr, Ras, or pnt alleles. Aside from CS3-3, none of the modifier groups corresponded to Egf receptor/Ras signaling components that genetically interact with TCAG. However, it is possible that these genes are present among the 61 single hits, which have not been characterized (Sullivan, 2002).

The Cbl family proteins function as both E3 ubiquitin ligases and adaptor proteins to regulate various cellular signaling events, including the insulin/insulin-like growth factor 1 (IGF1) and epidermal growth factor (EGF) pathways. These pathways play essential roles in growth, development, metabolism, and survival. This study shows that in Drosophila Cbl (dCbl) regulates longevity and carbohydrate metabolism through downregulating the production of Drosophila insulin-like peptides (dILPs) in the brain. dCbl is highly expressed in the brain and knockdown of the expression of dCbl specifically in neurons by RNA interference increases sensitivity to oxidative stress or starvation, decreased carbohydrate levels, and shortened life span. Insulin-producing neuron-specific knockdown of dCbl results in similar phenotypes. dCbl deficiency in either the brain or insulin-producing cells upregulates the expression of dilp genes, resulting in elevated activation of the dILP pathway, including phosphorylation of Drosophila Akt and Drosophila extracellular signal-regulated kinase (dERK). Genetic interaction analyses revealed that blocking Drosophila epidermal growth factor receptor (dEGFR)-dERK signaling in pan-neurons or insulin-producing cells by overexpressing a dominant-negative form of dEGFR abolishes the effect of dCbl deficiency on the upregulation of dilp genes. Furthermore, knockdown of c-Cbl in INS-1 cells, a rat β-cell line, also increases insulin biosynthesis and glucose-stimulated secretion in an ERK-dependent manner. Collectively, these results suggest that neuronal dCbl regulates life span, stress responses, and metabolism by suppressing dILP production and the EGFR-ERK pathway mediates the dCbl action. Cbl suppression of insulin biosynthesis is evolutionarily conserved, raising the possibility that Cbl may similarly exert its physiological actions through regulating insulin production in β cells (Yu, 2012).

Fasciclin 2 inhibits EGF receptor signalling

Adhesion proteins not only control the degree to which cells adhere to each other but are increasingly recognised as regulators of intercellular signalling. Using genetic screening in Drosophila, Fasciclin 2 (Fas2), the Drosophila orthologue of neural cell adhesion molecule (NCAM), has been identified as a physiologically significant and specific inhibitor of epidermal growth factor receptor (EGFR) signalling in development. Loss of fas2 genetically interacts with multiple genetic conditions that perturb EGFR signalling. Fas2 is expressed in dynamic patterns during imaginal disc development, and in the eye it was shown that this depends on EGFR activity, implying participation in a negative-feedback loop. Loss of fas2 causes characteristic EGFR hyperactivity phenotypes in the eye, notum and wing, and also leads to downregulation of Yan, a transcriptional repressor targeted for degradation by EGFR activity. No significant genetic interactions were detected with the Notch, Wingless, Hedgehog or Dpp pathways, nor did Fas2 inhibit the FGF receptor or Torso, indicating specificity in the inhibitory role of Fas2 in EGFR signalling. These results introduce a new regulatory interaction between an adhesion protein and a Drosophila signalling pathway and highlight the extent to which the EGFR pathway must be regulated at multiple levels (Mao, 2009).

These results demonstrate that the NCAM orthologue Fasciclin 2 specifically inhibits EGFR signalling activity during the normal development of the Drosophila eye, notum and wing. Interestingly, like other Drosophila EGFR inhibitors, Fas2 participates in a potential negative-feedback loop to regulate signalling, although the developmental significance of this remains to be established. The evidence for the interaction between Fas2 and EGFR relies on genetic interactions, diagnostic phenotypes of loss of function fas2 mutants, and a direct readout in fas2 clones of reduction of Yan, a transcriptional repressor targeted for degradation by EGFR activity. Furthermore, the results in the eye are supported by similar genetic logic in the developing notum and wing. Despite this, fas2 phenotypes are not identical to those of other known EGFR inhibitors. This is less surprising than it first appears, as the phenotypes of none of the known EGFR inhibitors in Drosophila (which currently include Argos, Kekkon-1, Echinoid, Sprouty, as well as some less specific proteins such as Gap-1) are as strong as constitutive activation of the receptor, and all are distinct. The explanation for the variation in strength and detail of phenotype is that each of the inhibitors has a different molecular mechanism and site of action in the pathway, as well as different sites of expression. For example, Argos is specific to the EGFR and is a diffusible molecule that sequesters ligand. By contrast, Sprouty, a cytoplasmic protein, inhibits a range of receptor tyrosine kinases, whereas Echinoid and Kekkon-1 are cell surface proteins that bind directly to the EGFR. It is evident that EGFR regulation depends on a patchwork of overlapping effects of multiple different types of modulators, each of which has greater or less importance in different developmental contexts. Presumably, this network of regulators underlies the observed precision and robustness of signalling (Mao, 2009)

Loss of Fas2 in the eye triggers at least two distinct types of extra photoreceptor recruitment. The ectopic mini-clusters appear at the same time that the normal outer photoreceptors are recruited and, by analogy with argos mutations, it is believed that they are caused by transformation of the 'mystery cells'. In normal development these form part of the precluster, but are ejected prior to the onset of photoreceptor differentiation. It is also possible that some of the mini-clusters are derived from de novo photoreceptor determination occurring in undifferentiated interommatidial cells, which is known to be triggered by excess EGFR activity. The second recognisable type of extra photoreceptors are the R7-like, Prospero-positive cells. These are presumably the product of abnormal recruitment of cone cell precursors as R7s, a switch of fates within the R7 equivalence group, which is sensitive to altered levels of receptor tyrosine kinase signalling (Mao, 2009).

The genetic data do not reveal a molecular mechanism for the inhibition of EGFR by Fas2 - that will require future biochemical analysis - but its location at the plasma membrane and the non-autonomy that was detected at the border of mutant clones point to three classes of models. (1) Fas2 reduces EGFR ligand production, presumably the TGFα homologues Spitz or Keren, for example by direct sequestration of the mature ligand. (2) Fas2 inhibits EGFR signalling, either by direct interaction with the receptor, or by indirectly downregulating its level or activity; in this case the observed non-autonomy would be indirect and caused by the well established positive feedback loop, whereby EGFR signalling activates expression of Rhomboid 1, which itself generates processed ligands. (3) Perhaps slightly less plausibly, the extracellular domain of Fas2 might be able to span the intercellular gap, thereby interacting with and inhibiting EGFR molecules on adjacent cells (Mao, 2009).

Precedence leads to a favouring of the second model. Two other adhesion proteins, Kekkon-1 and Echinoid, interact directly with the EGFR. Similarly, mammalian E-cadherin can inhibit the EGFR by direct binding. Of particular relevance to this work, it has recently been reported that mammalian EGFR can be inhibited by NCAM, the Fas2 orthologue (Povlsen, 2008). In these experiments using explanted mouse neurons combined with transfected mammalian cell lines, NCAM stimulates neurite outgrowth by blocking EGFR function. Preliminary results lead the authors to favour a mechanism of NCAM-induced downregulation of EGFR levels, although direct parallels with the current work are difficult to draw because the cytoplasmic domains of NCAM and Fas2 are not similar (Mao, 2009)

Beyond the evidence for inhibition of the EGFR described in this study and in the recent paper discussed above, Fas2/NCAM has now been implicated in several other signalling systems. The best characterised of these is an interaction with FGFR signalling, where, both in Drosophila and mammals, FGFR activity is required for Fas2/NCAM induced neurite outgrowth and direct binding of NCAM activates FGFR (Kiselyov, 2003; Christensen, 2006). By contrast, and an illustration of the context dependence of such interactions, it has also recently been reported that NCAM can inhibit FGFR activation by its ligand FGF (Francavilla, 2007). Less well studied links between NCAM and growth factors include the observation that NCAM can act as a signalling receptor for GDNF, and that it participates in the response of oligodendrocyte precursors to PDGF. The work reported in this study is the first genetic evidence to imply a role for Fas2 in the physiological inhibition of EGFR activity. It is important to set this discussion in the context of the well established role of Fas2/NCAM as a neural cell-adhesion molecule, with roles in axonal growth and pathfinding, as well as in synaptic maturation (Mao, 2009)

Overall, it is becoming clear that the EGFR pathway is regulated by multiple partially overlapping mechanisms, presumably because of the importance of regulatory precision and robustness of such a central and pleiotropic pathway. Notably, negative-feedback control is a recurring theme. Much less is known about physiologically significant regulators of EGFR signalling in mammals, and it will be interesting to determine whether feedback control is a conserved strategy. As there are many other signalling pathways and adhesion proteins that contribute to normal development, the total potential number of regulatory interactions between these key cell surface proteins is enormous and, indeed, many have been observed in vivo and in vitro. Of course, some of these might not occur in normal biological contexts, emphasising the value of a genetic approach to revealing which relationships between adhesion proteins and signalling pathways are physiologically relevant (Mao, 2009)

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