Gene name - EGF receptor
Synonyms - DER, Ellipse, torpedo
Cytological map position - 57F1
Function - transmembrane signaling
Keywords - Epidermal growth factor pathway
Symbol - Egfr
Genetic map position - 2-
Classification - receptor tyrosine kinase
Cellular location - surface TM protein
The Drosophila EGF receptor homolog, commonly called Torpedo or DER, has a complex biology. It is found in multiple sites and at varying times during development; in signaling processes that determine egg polarity, during early development to determine the identity of cells in the ventral ectoderm, during neurogenesis, in the development of the Malpighian tubules, and during larval stages in the development of the eye and wing.
Egfr interacts with three different ligands: Gurken, Spitz (the principle ligand), and Argos. Two accessory proteins modulate Egfr signaling: Rhomboid and Star. The signaling molecules downstream of Egfr include Shc ( the Drosophila homolog of a mammalian oncogene), DRK (an adaptor protein that docks onto Shc bound to Egfr and a homolog of mammalian GRB2), a guanine nucleotide exchange factor (SOS) activated by DRK, and downstream targets like Ras, Raf and Rolled (Map kinase)(Lai, 1995).
All these downstream signaling molecules are members of the RAS-RAF-MAPK pathway that amplifies and transmits receptor signals to various parts of the cell. Signals to the cytoskeleton result in changes in cell shape; signals to the nucleus result in gene activation.
The signaling pathway used by Egfr is also used by other receptor tyrosine kinases, including the Torso receptor and Sevenless, another Drosophila EFG receptor homolog. How does a single pathway used by a number of different receptors permit the wide variety of specific cell differentiation effects that are receptor-dependent? This question is currently being investigated in a number of developmental pathways, but as yet, no answer is available. The immune system, that most intensely studied cell interaction system, is not driven by a single ligand receptor interaction, but by numerous signaling events involving a number of receptors and a number of ligands. There is no reason to believe that Drosophila differentiation will be any less complex. Thus signals through the Egfr will be one of a number of signals integrated into differentiation decisions.
The complexity of receptor tyrosine kinase signaling is revealed by studies of eye and wing differentiation. Signaling in the eye involves three ligands and two receptors as well as Rhomboid and Star, the two accessory proteins of Egfr signaling. The Boss-Sevenless receptor-ligand pair is involved, as well as the Spitz-Egfr receptor-ligand pair. An additional ligand, Argos, functions either directly or indirectly to inhibit the Egfr signaling (Freeman, 1994).
Rhomboid's particiption in Egfr signaling in the wing currently presents a fascinating mystery. Rhomboid is expressed in a geometric pattern that establishes the future sites of wing vein differentiation. Rhomboid enhances Egfr signaling at the sites of future veins, but exactly how this enhancement occurs is currently unknown. It appears that Rhomboid sets the stage for Egfr signaling and the resultant induction of wing veins (Sturtevant, 1995).
A more detailed review of Gurken's role in signaling dorsoventral and anterior-posterior polarity in the developing oocyte will be found at the gurken site. Maternally transcribed Egfr is used in this signaling process. Egfr has two promoters and two first exons. The different developmental roles for each of the two transcripts have not yet been documented (Clifford, 1994).
Wing and leg precursors of Drosophila are recruited from a common pool of ectodermal cells expressing the homeobox gene Dll. Induction by Dpp promotes this cell fate decision toward the wing and proximal leg. The receptor tyrosine kinase Egfr antagonizes the wing-promoting function of Dpp and allows recruitment of leg precursor cells from uncommitted ectodermal cells. By monitoring the spatial distribution of cells responding to Dpp and Egfr, it has been shown that nuclear transduction of the two signals peaks at different positions along the dorsoventral axis when the fates of wing and leg discs are specified and that the balance of the two signals assessed within the nucleus determines the number of cells recruited to the wing. Differential activation of the two signals and the cross talk between them critically affect this cell fate choice (Kubota, 2000).
In a screen for genes expressed in the embryonic limb primordia, rhomboid was found to be transiently expressed in the central part of Dll-expressing limb primordia in stage 11 embryos. rho transcription is the rate-limiting step of the activation of an EGFR ligand Spitz. As expected from the role of rho as a stimulator of Egfr, a transient expression of an activated, phosphorylated form of MAPK (dpMAPK) is detected in the nucleus of limb primordial cells surrounding the rho-expressing cells. The dpMAPK expression starts after the initiation of Dll transcription and diminishes before the separation of the wing and leg disc primordium. The dpMAPK expression is undetectable in null mutants of rho or Egfr. The peak of dpMAPK expression is located ventrally to the cells expressing dpp. The results suggest that rho-mediated stimulation of Egfr and MAPK occurs at the time of cell fate specification of wing and leg discs (Kubota, 2000).
The spatial distribution of cells responding to Dpp and its relationship to Egfr signals was studied. To this end, an antibody specific to phosphorylated C-terminal sequence of Mad was produced. The phosphorylated sequence corresponds to the site at which the type I BMP receptor phosphorylates SMad1. The antibody detects an antigen distributed in a pattern similar to, but broader than, that of DPP mRNA. This immunoreactivity is dependent on Dpp signaling, as it is absent in stage 11 mutants of thick veins encoding type I Dpp receptor and in dpp mutants. This indicates that other extant TGFbeta-related signaling molecules present in Drosophila embryos do not substitute for Dpp to induce this immunoreactivity. Conversely, ectopic expression of Dpp results in high accumulation of this immunoreactivity. These results suggest that the antibody detects a Dpp-specific signaling event, most likely the phosphorylation and nuclear transport of Mad. Hereafter, the immunoreactivity detected by this antibody is called pSSVS (Kubota, 2000).
pSSVS is found mainly localized in the nucleus and distributed in regions a few cells wider in diameter than those of dpp-expressing cells. These properties are consistent with the previous findings that Mad transduces the Dpp signal to the nucleus. Double labeling of pSSVS and DLL mRNA shows that pSSVS expression is higher in the dorsal region of Dll-expressing cells. Combined with the double-labeling results of dpMAPK and Dll or dpp, it is concluded that cells responding to Dpp and Egfr overlap, but the peak of the responses are shifted. Such differential distribution of the two signals results in an arrangement of cells responding to a different strength of Dpp and Egfr along the dorsoventral axis (Kubota, 2000).
To study the role of Egfr at the stage of wing and leg cell fate determination, specific marker gene expression was examined in Egfr signaling mutants. DLL mRNA is expressed in the entire limb primordium at stage 11 and becomes restricted to distal leg cells at stage 15. Esg protein expression was used to detect both wing and proximal leg cells. In rho mutants, the size of limb primordia at stage 11 is the same as the control, but the later development of leg discs is abnormal. The number of leg disc cells expressing Dll and/or Esg at stage 15 is reduced, and these cells no longer show the circular arrangement typical of leg disc precursors. Amorphic mutation of Egfr cause a ventral expansion of limb primordia as a result of a loss of the early function of Egfr, but the expression of leg markers is severely reduced at stage 15. A similar phenotype is observed in mutants lacking both maternal and paternal copies of Dsor1, which encodes a MAP kinase kinase. In all cases described above, Esg-expressing cells at the dorsal part of leg discs are most frequently lost, suggesting that the development of dorsoproximal leg cells is most sensitive to the loss of Egfr activity. In contrast, wing and leg disc development is normal in vein mutants, suggesting the putative ligand of Egfr encoded by this gene is dispensable. These results suggest that MAPK activation induced by Rho and Egfr is essential for normal leg development (Kubota, 2000).
The temporal requirement for Egfr was studied by the temperature-sensitive allele Egfrf1. When the temperature is increased to the restrictive temperature at 5 hours after egg laying (AEL) prior to the induction of the limb primordium, the expression of Dll is expanded to the ventral midline, as was also observed with the strong Egfr mutants. When the temperature is increased at 6 hour AEL, the initial Dll expression is not altered, but the leg disc development is severely affected. Only mild defect is found in leg discs when the temperature is increased at 7 hours AEL, suggesting that Egfr must function between 6 and 7 hours AEL to correctly specify the leg cell fate. This is the time when the transient activation of MAPK is observed. Furthermore, whether Egfr is required autonomously in limb primordial cells was examined by expressing a dominant-negative form of Egfr using Dll-Gal4. Expression of this driver starts in the limb primordium at stage 11 and persists in a subset of wing discs and in entire leg discs at stage 15 because of the persistence of Gal4 activity. Imaginal disc-specific inhibition of Egfr interfers with leg disc development, while leaving the wing disc intact. These results demonstrated that a transient activation of Egfr in stage 11 limb primordia is essential for the leg disc development (Kubota, 2000).
In contrast to the severe defects in leg discs, none of the mutations in Egfr signaling interfer with wing disc formation. In these mutants, wing primordia consistently express Esg and another wing disc marker Vestigial, and invaginate to form discs. However, an increase in the number of wing disc cells has been noted in Egfr signaling mutants. This effect was analyzed in rho mutants; unlike Egfr mutants, in rho mutants the number of limb primordial cells at stage 11 is the same as the control. The number of Esg-expressing wing disc cells in rho mutants is increased compared to the control, while the number of the proximal leg disc cells is severely reduced. It is concluded that Egfr signaling is required to limit wing disc cell differentiation in limb primordial cells that are not yet fully committed. It is inferred that a subset of prospective leg cells that do not receive a sufficient amount of Egfr signaling fail to differentiate as proximal leg and instead adopt a wing fate (Kubota, 2000).
The increase in the number of wing disc cells in rho mutants resembles the overexpression phenotype of Dpp and raises a possibility that Egfr might prevent wing disc development by negatively regulating Dpp signaling. Such a cross talk could occur at several levels including the following: (1) regulation of dpp transcription, (2) signal transduction from Dpp receptors to the nucleus, and (3) transcriptional regulation of downstream target genes. The analyses excluded the first two possibilities for two reasons. (1) The expression pattern of DPP mRNA is unaffected by the mutation of rho. A previous report showing an expansion of dpp expression in Egfr mutants probably reflects the global patterning role of Egfr in the earlier stage. (2) pSSVS expression around limb primordia does not change in rho mutants. Conversely, the expression pattern of dpMAPK is not changed by a null mutation of tkv. These results suggest that the differential distribution of cells responding to Dpp and Egfr is set up independently of each other's activity (Kubota, 2000).
Dpp and Egfr were found to antagonize each other after signal transduction into the nucleus. Hyperactivation of Egfr by an ectopic expression of an Egfr ligand Spitz causes a great accumulation of dpMAPK. As expected from the negative effect of Egfr on the wing development, this treatment completely eliminates wing disc formation and, in addition, causes a malformation of the leg disc. Since it was found that cells migrating out of the leg primordium express dpMAPK, it is unlikely that the failure in wing disc formation is due to the prevention of cell migration or to cell death. It has been suggested that hyperactivation of Egfr prevents limb primordial cells from adopting the wing cell fate. It is likely that those cells adopt the epidermal fate instead. Overexpression of Dpp causes an accumulation of pSSVS and an increase in the number of wing disc cells. Coexpression of Dpp with Spi partially restores the development of both wing and leg discs, suggesting that wing disc development overcomes the negative effect of Egfr if provided with a sufficient amount of Dpp. The restored wing primordia migrate with high levels of pSSVS and dpMAPK, further supporting the notion that Dpp and Egfr signals are transduced independently of one another (Kubota, 2000).
dad is an immediate transcriptional target gene of Dpp, the expression of which closely parallels that of pSSVS expression in embryos and is inducible by Dpp. dad expression is not affected in Egfr or rho mutants. Furthermore, elevated dad expression induced by Dpp is not affected by sSpi, suggesting that at least one of the immediate transcriptional responses to Dpp is unaffected by elevated Egfr signaling (Kubota, 2000).
The antagonism between Dpp and Egfr during wing disc development raises a question: what is the default state of the wing and leg primordia in the absence of the two signals? Double mutant phenotypes of Dpp and Egfr signaling were examined. tkv mutants lack wing discs and their leg discs are malformed. This phenotype reflects a disc cell autonomous requirement for Dpp signaling, because the phenotype is reproduced by the disc-specific inhibition of Dpp signaling by dad, which inhibits Mad. The phenotype of either tkv;rho or tkv;Egfr double mutants is a simple addition of each mutation, in which wing discs are lost completely and leg discs are severely reduced. Since Dll-expressing limb primordial cells are present in tkv;Egfr double mutants in stage 11, it has been concluded that these cells fail to differentiate as wing discs and their ability to differentiate as leg discs is also compromised. A few Esg-positive cells remain at the position of the leg, and it is speculated that this reflects the presence of a second leg-inducing signal. These results suggest that Dpp is absolutely required for wing disc development irrespective of the activity of Egfr (Kubota, 2000).
Egfr affects the choice of wing vs. leg developmental options differently; it promotes leg development while it inhibits wing development. These two activities of Egfr are the earliest of known events of leg specification, and occur prior to the establishment of proximodistal axis in the leg. In the absence of late functions of Dpp and Egfr, limb primordia are specified but fail to differentiate into wing disc and most of leg disc. Thus it is proposed that early limb primordium at stage 11 consists of cells not yet fully committed to either wing or leg disc fate, and the cells are exposed to different amounts of Dpp and Egfr signaling according to their dorsoventral location. Dpp recruits the cells to the wing disc fate. Egfr antagonizes the cellular response to the wing-inducing function of Dpp and allows the development of wing discs only in the dorsal region. Thus the dorsoventral difference in Dpp and Egfr signaling in the limb primordium provides key information to the separation and differentiation of the wing and leg discs. In contrast to the opposing roles of Dpp and Egfr on wing disc development, leg discs requires both signals. The effect of the loss of Egfr activity on leg disc development is not compensated for by a simultaneous loss of Dpp signaling, indicating that Egfr has an additional activity to promote leg development separately from its role to antagonize Dpp. Because dorsal and ventral limb primordial cells respond to Egfr differently, it is speculated that at least one additional dorsoventral factor influences leg disc formation at stage 11. This idea is consistent with the fact that residual proximal leg cells can still be induced in the almost complete absence of Egfr and Dpp activity. One candidate for the factor is Wg, which is expressed in the limb primordium (Kubota, 2000).
The nuclear transduction of the Dpp signal, as visualized by the distribution of pSSVS and expression of dad, is unaffected by Egfr. The results suggest that the antagonistic effect of Egfr on Dpp signaling occurs after transduction into the nucleus. Therefore, the mechanism of SMad inhibition by direct phosphorylation by MAP kinase does not play a major role in this case (Kubota, 2000).
The finding that Egfr is activated in the limb primordium and prevents wing disc formation suggests that Egfr is a key factor in the diversification of the wing and leg fate. It is proposed that the differential activation of Dpp and Egfr, and the dorsal cell migration brings a subset of limb primordial cells out of the range of Egfr signaling, and thereby allows Dpp to induce wing development. It follows that dorsally migrating cells acquire the wing cell identity only after the separation from leg-promoting signals. Consistent with this idea, expression of wing-specific markers Vg and Sna, start only after the separation of the two primordia. Mechanisms that promote the dorsal cell migration remain to be identified. Given that the basic genetic components for the induction of the wing and leg have been identified in the model organism Drosophila, it can now be asked how the genetic mechanism of wing and leg specification has evolved by comparing the expression and function of these genes in limb primordial cells of primitive insects (Kubota, 2000).
A good example of the function of Egfr in regulating cell development is found by examining the role of Egfr in midline glia maturation. The midline glial cells are required for correct formation of the axonal pattern in the embryonic ventral nerve cord. Initially, six midline cells form an equivalence group with the capacity to develop as glial cells. By the end of embryonic development three to four cells are singled out as midline glial cells. Midline glia development occurs in two steps, both of which depend on activation of the Egfreceptor and subsequent Ras1/Raf-mediated signal transduction (See Drosophila Ras1). In the first step six midline cells in each segment, originating from the anterior-most three of a total of eight midline progenitor cells, are determined as the midline glia equivalence group. The process of generation of the midline glia equivalence group involves Notch function and segmentation genes. It might also depend on the function of single minded, the master regulatory gene of midline development. The single minded transcript accumulates in the midline glia and, depending on the context, can act either as a transcriptional activator or repressor. By the end of embryogenesis the final number of midline glial cells is about 3 to 4. Thus, the final number of cells has to be selected from the initially defined equivalence group in a second step (Scholz, 1997).
Egfr mutants show a reduced number of midline glial cells and argos mutants, which possibly exhibit an increased activation of Egfr in the midline, show an increased number of midline glial cells. Furthermore, expression of activated ras1 (or activated raf) in the midline results in the appearance of extra midline cells. This model suggests that activation of ras1 signaling in the entire midline glial equivalence group promotes survival of all cells in this cluster. Thus, in wild-type flies, about 2-3 cells in each group down-regulate Egfr signaling and are destined for cell death. Both Rhomboid and Argos control activation of the Egfr during midline glia development. It is thought that a graded activation of Egfr is brought about by the activity of Rhomboid, which is thought to promote EGF receptor signaling, possibly by cell autonomous activation of the Egfr ligand Spitz. Ectopic rhomboid leads to extra midline glial cells. Egfr activates PointedP2 through phosphorylation; Pointed in turn activates the transcription of argos. Argos negatively regulates Egfr signaling non-cell autonomously and competes with Spitz function. pointed mutants form extra glial cells. Yan antagonizes PointedP2A in midline glial cells, just as it does in the developing photoreceptor cells (Scholz, 1997).
Egfr signaling is required in a narrow medial domain of the head ectoderm (here called ‘head midline’) that includes the anlagen of the medial brain (including the dorsomedial and ventral medial domain of the brain, termed DMD and VMD respectively), the visual system (optic lobe, larval eye) and the stomatogastric nervous system (SNS). These head midline cells differ profoundly from their lateral neighbors in the way they develop. Three differences are noteworthy: (1) Like their counterparts in the mesectoderm, the head midline cells do not give rise to typical neuroblasts by delamination, but stay integrated in the surface ectoderm for an extended period of time. (2) The proneural gene l’sc, which transiently (for approximately 30 minutes) comes on in all parts of the procephalic neurectoderm while neuroblasts delaminate, is expressed continuously in the head midline cells for several hours. (3) Head midline cells, similar to ventral midline cells of the trunk, require the Egfr pathway. In embryos carrying loss-of-function mutations in Egfr, spi, rho, S and pnt, most of the optic lobe, larval eye, SNS and dorsomedial brain are absent. This phenotype arises by a failure of many neurectodermal cells to segregate (i.e., invaginate) from the ectoderm; in addition, around the time when segregation should take place, there is an increased amount of apoptotic cell death, accompanied by reaper expression, which removes many head midline cells. In embryos where Egfr signaling is activated ectopically by inducing rho, or by argos (aos) or yan loss-of-function, head midline structures are variably enlarged. A typical phenotype resulting from the overactivity of Egfr signaling is a ‘cyclops’ like malformation of the visual system, in which the primordia of the visual system stay fused in the dorsal midline. The early expression of cell fate markers, such as sine oculis in Spitz-group mutants, is unaltered (Dumstrei, 1998).
About the time of gastrulation, Egfr signaling is activated in both mesectoderm and the head midline. The ventromedial domain of Egfr activation, as monitored by rho, aos and phosphorylated ERK expression, continues from the ventral mesectodermal domain into the head ectoderm. The ventromedial stripe of aos expression bifurcates at the level of the stomodeal plate and continues dorsally. Approaching the dorsal midline, it turns posteriorly and continues all the way to the posterior boundary of the head neurectoderm. There is an anterior-median patch of aos expression corresponding to the anlage of the stomatogastric nervous system. At later stages (stage 10-12) expression of aos in head midline structures becomes more complex; however, a high level of expression persists in large parts of the optic lobe, SNS, VMD and DMD. The domain of rho expression and ERK phosphorylation matches closely the aos expression domain. pnt and yan, two transcription factors executing the signal passed to the nucleus by the Egfr signaling cascade, are also expressed in structures of the head midline The Egfr pathway is required in the head midline for proper cytodifferentiation and epithelial maintenance. Both Egfr and its ligand Spitz are ubiquitously expressed in the embryo (Dumstrei, 1998).
The Egfr loss-of-function phenotype in the head midline and the mesectoderm is quite compatible with the postulated function of Egfr signaling in the Drosophila compound eye and in various vertebrate systems where Egfr signaling also promotes differentiation and prevents cell death. In the head midline, no evidence for a transformation of cell fate is observed similar to the that takes place in the ventral neurectoderm of the Drosophila embryo. A transformation of fate would imply that the structures missing (e.g., optic lobe, dorsomedial brain) are replaced by other structures, which then would have to expand in size in order to occupy the space normally allotted to them, as well as the additional space normally taken up by the midline structures. However, there does not appear to be an expansion of the lateral neurectoderm, such as takes place in ventral neurectoderm: for example, the number and pattern of neuroblasts delaminating from the lateral head neurectoderm in Egfr or Spi mutant embryos is normal. Thus, it appears that the loss of head midline structures observed in Egfr-signaling-deficient embryos is effected mainly by cell death (Dumstrei, 1998).
The gain of function of EGFR signaling causes a complex phenotype. In yan loss-of-function mutants, a hyperplasia of both dorsomedial brain and stomatogastric nervous system is observed. In case of the latter, cells of the DMD clearly overproliferate; ultimately, these cells express neural markers and become incorporated into the brain. The optic lobe phenotype is more difficult to interpret. Thus, cells located medial to the optic lobes express fasII, resulting in a single optic lobe (‘cyclops’ phenotype) overgrown by the enlarged brain. One might interpret this phenotype as a cell fate transformation: dorsomedial cells, which would normally not express fasII (and would not become part of the optic lobe) turn on this gene if the output of EGFR signaling is increased. However, to clearly interpret this phenotype, one would have to know much better what normally happens to the dorsomedial cells located between the optic lobes. At the blastoderm stage, the anlagen of the optic lobe map to a dorsomedial position. Shortly thereafter, dorsomedial cells move laterally, generating a thin membrane of amnioserosa like cells in between them. A great deal of cell death takes place in this region, prompting one to speculate that cell death is important for the lateral shift of the optic lobe primordia. Supporting this idea it has been found that in embryos in which no cell death occurs due to a deficiency that removes the reaper complex of genes, the optic lobe primordia are frequently extended toward the midline, similar to what can be observed in yan or aos loss-of-function. It is therefore possible that increased activity of Egfr signaling, rather than inducing another fate in the dorsomedial cells, rescues dorsomedial cells from cell death; the expression of FasII might reflect the fate that these cells would normally show if they were permitted to live. Clearly, more experimental studies are required to grasp the effect of Egfr signaling in this region (Dumstrei, 1998).
In head midline structures, in particular the optic lobe and SNS, there may be a late phase of EGFR signaling (as assayed by the expression of aos and activated ERK) whose significance is not yet known. EGFR signaling could be involved in modifying the inhibitory feed-back loop between neurogenic and proneural genes that exists in other neurectoderm cells. In the head midline neurectoderm, regulation of proneural and neurogenic genes has to be different. Thus, instead of a short burst of proneural gene expression in proneural clusters that is resolved into expression in individual neuroblasts, proneural genes are expressed for a long period of time; at the same time, the expression is never restricted to single neuroblasts. Since genes of the E(spl) complex are expressed in the same cells that express l’sc, the inhibitory loop between E(spl)-C and proneural genes must be interrupted at some level. It is possible that Egfr signaling is causing the interruption of this inhibitory loop. Based on genetic studies of Notch and Egfr signaling in the compound eye, it has been speculated that one of the consequences of Egfr activation (which ultimately is required for all ommatidial cell types to differentiate) is to inhibit N signaling, since constitutively active N inhibits ommatidial cell differentiation by preventing response to differentiative signals. However, the same effect could be achieved if Egfr signaling, similar to what is proposed here for the midline neurectoderm, interrupts the inhibition of proneural genes by E(spl). Although this would not prevent N signaling, it would cancel the effect of N signaling on downregulating proneural genes and thereby keep cells in a state of competency to respond to signals (Dumstrei, 1998).
The alternative 3' exons, termed Type 1 and Type 2 exons, are separated from the downstream common sequences by 45 kb. Each alternative exon codes for a signal sequence. The Type 1 exon codes for 101 amino acids while the Type 2 exon codes for 52 amino acids (Clifford, 1994).
cDNA clones of Egfr were isolated and sequenced. The deduced amino acid sequence shows a similar degree of homology to the human epidermal growth factor receptor and to the rat and human neu proteins; the most striking difference is the addition of a third cysteine-rich extracellular domain in Egfr. In the extracellular region the homology between Egfr and HER (human EGF receptor) is 37% (Schejter, 1986 and Price, 1989). The intracellular region contains one kinase domain (Clifford, 1994).
date revised: 30 September 2000
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