EGF receptor

Effects of Mutation or Deletion

Table of contents

Egfr and the peripheral nervous system

The selection of Drosophila sense organ precursors (SOPs) for sensory bristles is a progressive process: each neural equivalence group is transiently defined by the expression of proneural genes (proneural cluster), and neural fate is refined to single cells by Notch-Delta lateral inhibitory signalling between the cells. Unlike sensory bristles, SOPs of chordotonal (stretch receptor) sense organs are tightly clustered. It has been shown that for one large adult chordotonal SOP array (the adult femoral chordotonal sense organ), clustering results from the progressive accumulation of a large number of SOPs from a persistent proneural cluster. This is achieved by a novel interplay of inductive epidermal growth factor- receptor (EGFR) and competitive Notch signals. EGFR acts in opposition to Notch signaling in two ways: it promotes continuous SOP recruitment despite lateral inhibition, and it attenuates the effect of lateral inhibition on the proneural cluster equivalence group, thus maintaining the persistent proneural cluster. SOP recruitment is reiterative because the inductive signal comes from previously recruited SOPs (zur Lage, 1999).

The adult femoral chordotonal sense organ arises from a group of some 70-80 SOPs. A developmental analysis of Ato expression has revealed that these SOPs accumulate over an extended period of time in the dorsal region of each leg imaginal disc during the third larval instar and early pupa. The continued expression of Ato implies a sustained requirement for proneural function throughout the process of SOP accumulation. Unusually, Ato is persistently expressed in a group of ectodermal cells identified as the proneural cluster (PNC). From this PNC, cells are funnelled inward into a cavity formed by the folding of the disc. This invagination later becomes visible as a distinctive 2-cell wide intrusion, which is referred to as the 'stalk'. Cells at the deepest end of the stalk undergo shape changes to form an amorphous inner SOP mass. Invaginating cells are characterised by upregulation of Ato expression, a characteristic of SOP commitment. Surprisingly, SOP markers (Ase protein and the A101 enhancer trap line) are not expressed in all the stalk SOPs. Instead, these markers are only apparent in older cells, particularly at the time when they become part of the inner mass (which is therefore referred to as mature SOPs). Despite this, entry into the stalk seems to mark SOP commitment, since both the stalk and the mature SOPs are absent in discs from ato mutant larvae. This apparent intermediate stage may not have a counterpart in external sense organ precursor formation, although there is some evidence for multiple steps between the uncommitted cell and the SOP (the so-called pre-sensory mother cell state). Initially, Ato remains activated in all invaginated SOPs. This extended period of proneural gene expression is unusual since AS-C proneural expression is typically switched off in SOPs shortly after commitment. Later, at approximately 6 hours before puparium formation (BPF), Ato expression is switched off synchronously in the mature SOPs, although expression remains in the stalk SOPs and the PNC. At this point there is very little overlap between Ato and Ase or A101 (zur Lage, 1999).

The process of chordotonal SOP formation described above is at odds in several respects with the well-known paradigm of SOP selection for sensory bristles. In the latter, the solitary SOP expresses Delta, which triggers expression in the PNC of genes of the E(spl)-C, thereby preventing further SOP commitment and forcing loss of AS-C expression and neural competence. In the case of the femoral chordotonal organ, newly committed cells from the PNC are in contact with previously committed SOPs in the stalk, but are apparently not receiving (or not responding to) lateral inhibition signals from these to prevent their commitment. Likewise, the presence of committed SOPs does not switch off ato expression in the PNC. Nevertheless, components of the N-Dl pathway are expressed in patterns consistent with lateral inhibition. The newly formed SOPs express Dl, suggesting that they send inhibitory signals, while the PNC expresses mgamma, a member of the E(spl)-C, suggesting that these cells are responding to the Notch-Delta signal. Indeed, mgamma is coexpressed with ato in the PNC throughout the development of the SOP cluster. Chordotonal SOP formation is shown to be sensitive to N inhibitory signaling. Strong activation of N signaling or its effectors can inhibit chordotonal SOP formation. Thus, N signaling has an important role to play: it acts to limit the process of SOP selection from the PNC. Some mechanism, however, must prevent N signaling from completely inhibiting multiple SOP formation (zur Lage, 1999).

The progressive accumulation of chordotonal SOPs suggests that a recruitment mechanism could explain the clustering of SOPs. The Drosophila Egfr signaling pathway is involved in a number of recruitment processes in development, and a role for Egfr signaling has been demonstrated in the induction of embryonic chordotonal precursors (zur Lage, 1997). Although there appear to be significant differences in the process of SOP formation in imaginal discs, as compared with the embryo, it was asked whether Egfr signaling is also involved in forming the femoral chordotonal cluster. To address this question, the pathway was conditionally disrupted by expressing a dominant negative form of Egfr protein. Expression of UAS-Egfr DN results in a dramatic loss of chordotonal SOPs in late third instar imaginal leg discs (as judged by Ase protein expression or the A101 enhancer trap line). This demonstrates that Egfr signaling is required for the process of femoral chordotonal SOP formation. In contrast, the appearance of bristle SOPs is unaffected, arguing against the possibility of a nonspecific effect on SOPs in general (zur Lage, 1999).

To determine whether Egfr signaling controls SOP number, expression of components of the Egfr pathway that determine the level of signaling was forced, thus resulting in hyperactivation of the pathway. pointed (pnt) is an effector gene that encodes a transcription factor and is activated in cells responding to Egfr signaling. Both rho and pnt are expressed during chordotonal SOP formation. Indeed, forced expression of rho or pnt increases chordotonal SOP formation. Egfr could promote SOP formation by stimulating the commitment of PNC cells or by stimulating proliferation of SOPs. Both functions would be consistent with known Egfr roles, but the current investigations favour the former. Analysis of Ato expression in leg discs in which rho has been misexpressed reveals a large invagination of cells and a smaller PNC. Shrinking of the PNC was confirmed by the reduced extent of mgamma expression. These observations are consistent with an increased rate of SOP commitment upon Egfr hyperactivation. Moreover, this effect is reminiscent of the effect of N loss of function on Ato expression, suggesting that Egfr signaling supplies the mechanism that interferes with lateral inhibition of SOP commitment (zur Lage, 1999).

Although it seems that cells of the PNC and stalk are held in a state of mitotic quiescence throughout the time that SOP fate decisions are being made, BrdU is incorporated in the older (mature) SOPs. The experiments so far have indicated that Egfr signaling affects SOP commitment from the PNC. To determine more precisely the spatial patterning of Egfr activity required for SOP clustering and N antagonism, the expression patterns of key components of the pathway were characterized. Localized expression of rho appears to play a central role in spatial restriction of Egfr activity in cases where Spi is the ligand; in these cases it appears to mark the cells that are a source of signaling. During development of the femoral chordotonal organ, rho is expressed in a very restricted pattern: RHO mRNA is only detected in the SOPs, becoming confined in the late third instar larva to the youngest SOPs at the top of the stalk. To identify the cells responding to rho-effected signaling, an antibody that detects the dual-phosphorylated (activated) form of the ERK MAP kinase (dp-ERK) was used. In leg imaginal discs, dp-ERK is detected in a confined area corresponding to the uppermost (youngest) stalk SOPs. Thus, like rho, dp-ERK is expressed in the newly formed stalk SOPs. Double labelling for RHO RNA and dp-ERK confirms this, but also suggests that the overlap in expression is not complete: dp-ERK is detected above the uppermost rho-expressing cells of the stalk, probably in one or a few cells of the proneural cluster as they funnel into the stalk. This suggests that Egfr promotes SOP commitment as a consequence of direct signaling from previous SOPs to overlying PNC cells. Since rho expression is itself activated upon SOP commitment, this process occurs cyclically: the newly recruited SOPs are in turn able to signal to further overlying PNC cells. That is, recruitment is reiterative. Egfr signaling via Spitz has been shown to help to maintain neural competence by attenuation of Notch directed lateral inhibition. The opposing forces of Notch and Egfr signaling are thought to be played out through direct Notch and Egfr signaling between the epidermal proneural cells, which bear Notch, and the SOP, which sends inhibitory signals through the Delta ligand, and stimulatory signals through the Spitz ligand (zur Lage, 1999).

Reiterative recruitment alone cannot entirely explain the accumulation of SOPs. Such an accumulation also relies on the persistence of the competent pool of PNC cells from which SOPs can be recruited. For AS-C PNCs, this does not occur, because the mutual inhibition required for continued competence is unstable and resolves quickly to a state of lateral inhibition once the SOP emerges from the PNC. This results in rapid shutdown of AS-C expression and hence competence within the PNC. It is possible that the members of E(spl)-C that are expressed in the PNC (notably mgamma and mdelta) are less aggressive inhibitors of proneural gene expression than the E(spl)-C members expressed in AS-C PNCs (m5 and m8). The results obtained in the femoral SOP suggest, however, that Egfr has a role to play in maintaining the PNC by partially attenuating lateral inhibition on a PNC-wide scale. Thus, the PNC is not completely shut off by inhibition from SOPs, but instead kept in check, allowing continued mutual inhibition and maintenance of competence but not allowing general SOP commitment. Since neither rho nor dp-ERK are detected in the PNC as a whole, this function of Egfr could be indirect and achieved through partial attenuation of Dl signaling from the stalk SOPs themselves. The trans- or auto-activation of EGFR signaling between the stalk SOPs (as suggested by the co-expression of dp-ERK and rho) might be an indicator of this function. It is also possible, however, that Egfr signaling is direct and that the dp-ERK antibody is not sensitive enough to detect expression in the PNC cells (zur Lage, 1999).

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

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

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

Thus far, the results indicate that Ed cooperates with the N pathway to control the determination of notum macrochaetae. Because Egfr and N pathways act antagonistically in macrochaetae development, the genetic interactions between ed and members of the Egfr signaling pathway were examined. Overexpression of wild-type Egfr (UAS-Egfr) alone by sca-Gal4, has a very weak effect on the number of notum bristles. However, the co-expression of both UAS-edDeltaECD and UAS-Egfr results in a severe tufting phenotype. Similar results were obtained when edDeltaECD and a constitutively activated form of Raf (UAS-rafgof) were co-expressed. As expected, increased number of SOPs were observed in proneural clusters, as detected with anti-Sens antibody. The interaction between Ed and Egfr pathways was verified by observing that a decrease of Egfr activity (overexpression of a dominant-negative form of Egfr, UAS-EgfrDN) partially suppressed the extra bristle phenotype caused by ed1x5/edslH8. Together, these results demonstrate an antagonism between Ed and Egfr signaling pathways in bristle development. However, considering the known antagonism between the Egfr and N pathways in macrochaetae development, these results open the possibility that the Egfr pathway might mediate, at least in part, the interaction between ed and the N pathway. If this were the case, one would expect that modifications of the activity of the Egfr pathway would affect the activity of the N pathway. Apparently, this did not occur. The levels of E(spl)m8 mRNA accumulation in proneural clusters were essentially unmodified by overexpressing either a constitutively activated form of Ras (UAS-ras1V12) or the Egfr-negative ligand Argos (UAS-aos). These conditions mimicked a strong stimulation and an inhibition of the pathway, since they respectively led to formation of many ectopic SOPs or to the removal of most macro and microchaetae. It is concluded that it is unlikely that the interaction of Ed and N is mediated by the Egfr pathway (Escudero, 2003).

Egfr and the regulation of axon branching dynamics

Axonal branching allows a neuron to connect to several targets, increasing neuronal circuit complexity. While axonal branching is well described, the mechanisms that control it remain largely unknown. This study found that in the Drosophila CNS branches develop through a process of excessive growth followed by pruning. In vivo high-resolution live imaging of developing brains as well as loss and gain of function experiments show that activation of Epidermal Growth Factor Receptor (EGFR) is necessary for branch dynamics and the final branching pattern. Live imaging also reveals that intrinsic asymmetry in EGFR localization regulates the balance between dynamic and static filopodia. Elimination of signaling asymmetry by either loss or gain of EGFR function results in reduced dynamics leading to excessive branch formation. In summary, it is proposed that the dynamic process of axon branch development is mediated by differential local distribution of signaling receptors (Hiesinger, 2014).

The dorsal cluster neurons (DCNs) establish a complex neurite network in the Drosophila adult optic lobes. A small subset of neurons from this cluster extend their axons in the outer part of the optic lobe, the medulla (Me), where they form a stereotypic pattern of axonal branches. This pattern can be readily visualized using the ato-Gal4 driver in combination with a UAS-driven marker of choice such as CD8-GFP. Flip-out single cell clones reveal the branch pattern of an individual axon derived from a single neuron of the 12 medulla innervating. False color labeling and tracing of single DCN Me axons and their branches reveals that each axon generates 6-8 primary branches, with a mean of 7 branches. This stereotypic pattern is achieved by hot spots of branches extending in dorsal and ventral direction from each main axon shaft. The first main branch point is located at the border between lobula and Me with one or two branches. The next major branch point with often two branches is situated in Me layers M7-M8 and in this location branches from distinct neighboring axons are often in close contact forming a grid-like pattern. The terminal set of up to four branches is distributed over the M1-M3 layers and is more often intermingled with neighboring axon branches. In between the two most distal branch points intermediate branches occur occasionally. DCN branches never extend beyond the Me neuropil (Hiesinger, 2014).

The refinement of exuberant branches is a crucial step during the development of a neuronal network. This work exploited an adult-specific model circuit, the dorsal cluster neurons, to study developmental neurite pruning processes in the CNS of Drosophila. DCN axons form a stereotyped number of branches innervating the medulla through initial excessive axon branch formation followed by a refinement process. The data suggest a model whereby uneven distribution of EGFR to developing DCN axonal branches is required to eliminate exuberant branches and help generate the correct adult connectivity pattern (Hiesinger, 2014).

During mammalian development neurites are generally formed in excessive numbers and subsequently refined to form the mature circuit. This mechanism ensures that all targets are properly innervated, it enables further specification of connections by the target environment like neighboring neurons and glia and permits the removal of exuberant or mistargeted branches. Studying real-time events in the mammalian system involving CNS refinement is challenging. The Drosophila developing brain culture system used in this work combined with live imaging allows examination and manipulation of neuronal growth dynamics. The data suggest that EGFR signaling, in part triggered by the co-innervation of the target neuropil by sensory neurons from the retina, is a crucial determinant of axonal branch refinement by the regulation of filopodial growth and retraction dynamics. Finally, EGFR activity regulates actin polymerization dynamics at the branch tips. Consistent with this notion, it was found that interfering with actin dynamics in vivo by inhibition of the small GTPase RhoA or constitutive activation of the actin filament severing protein Cofilin, is sufficient to cause ectopic axon branch formation in the DCNs. EGFR expression has been observed in neurites of mammalian neurons and knock-out of the EGFR in the mouse results in increased neurite branching in the skin, suggesting that the mechanism this study has identified in the fly CNS may be more generally utilized (Hiesinger, 2014).

In summary, this study reports evidence for the notion that differential branch signaling is a determinant of connection specificity. Intrinsically asymmetric EGFR localization and signaling is required for efficient branch pruning. Several lines of evidence support this conclusion. First, EGFR is asymmetrically localized in branches and filopodia both in vivo and in cultured primary neurons. Second, both inhibition and constitutive activation result in failure of axonal branch refinement. Third, overexpression of the wildtype receptor, which is differentially localized and trafficked, is not sufficient to produce a phenotype. This argues that receptor localization dynamics-possibly mediated by endocytosis-rather than total EGFR levels, is the cue for filopodial collapse and subsequent axonal branch pruning. What explains the link between regulation of dynamic behavior and the generation of a specific number of axonal branches? A hint to this comes from three observations. First, both loss and gain of EGFR function increase proportion of static filopodia from less than 10% to more than 30%, subsequently increasing the number of axonal branches. Second, this filopodial behavior correlates with small, but significant and highly dynamic differences in EGFR localization. Third, loss of EGFR signaling increases the variability in axon branch number. Based on these observations it is proposed that in wildtype neurons most dynamic filopodia collapse over time, resulting in continuous redistribution of EGFR among fewer and fewer remaining filopodia. This process stops usually when only one filopodium remains at a given branching point, and occasionally when EGFR happens to distribute equally between the last two filopodia. This probabilistic process does not require an additional mechanism of branch 'tagging and selection' and can explain both EGFR loss of function phenotypes: increased branch number and increased variability. What remains to be determined is the interaction between EGFR-dependent branch dynamics and the specificity of the spatial pattern of branches (Hiesinger, 2014).

Egfr and bract development

A long-standing mystery in Drosophila has been how certain bristles induce adjacent cells to make bracts (a type of thick hair) on their proximal side. The apparent answer, based on loss- and gain-of-function studies, is that these bristles emit a signal that neighbors then transduce via the epidermal growth factor receptor pathway. Suppressing this pathway removes bracts, while hyperactivating it evokes bracts indiscriminately on distal leg segments. Misexpression of the diffusible ligand Spitz (but not its membrane-bound precursor) elicits extra bracts at normal sites. What remains unclear is how a secreted signal can have effects in one specific direction (Held, 2002).

The recent availability of a temperature-sensitive LOF allele for the Egfr gene (Egfrts1a) makes it possible to define the sensitive period when the Egfr protein is needed for signal transduction. In the upshift series, Egfrts1a/EgfrCO mutants (EgfrCO is a null allele) were raised at the permissive temperature of 18°C and then shifted to the restrictive temperature of 29°C at different times for the duration of development. In the downshift series, flies of the same genotype were raised at 18°C (to bypass earlier lethal periods), transferred to 29°C at pupariation (before the sensitive period for bract induction begins) and shifted back to 18°C at different times. Flies raised continuously at 18°C have a wild-type pattern of bracts (99%Ti and 100%Ba), while those kept at 29°C during the pupal period lack all bracts (0%Ti and 0%Ba). For the Ti, the 50% midpoint for bract removal is 17 h AP for upshifts and 28 h AP for downshifts. The sensitive period for the Ti would thus be defined as 17-28 h AP. For the Ba, this period begins 4 h earlier (13-28 h AP) (Held, 2002).

Basitarsal bristle rows are heterogeneous in their timecourses. Relative to the bracts of the ventral rows, the bracts of the dorsal rows acquire immunity to upshifts (Egfr inactivation) later but lose their ability to be rescued by downshifts (restoration of Egfr function) earlier (Held, 2002).

To confirm the role of the EGFR pathway, attempts were made to activate or repress the pathway by the Gal4-UAS method ('driver>slave' ). Two types of Gal4 drivers were used: scabrous-Gal4 (sca-Gal4) is expressed in bristle SOPs and in the proneural clusters (PNCs) whence they arise, whereas Distal-less-Gal4 (Dll-Gal4) is expressed throughout the tarsus and distal Ti. In the first series of experiments, the UAS slaves encoded ligands: UAS-mSpi, UAS-sSpi, and UAS-argos. In the second series of experiments, the UAS slaves encoded various versions of Egfr itself: UAS-Egfr (wild-type product), UAS-Egfr*top4.2 (constitutively activated form), and UAS-EgfrDN (dominant-negative form). Misexpressing the normal Egfr was expected to cause extra bracts, but in fact it eliminated bracts (0%Ti and 0%Ba for sca>Egfr and 10%Ti and 0%Ba for Dll>Egfr. Also surprising were the findings that (1) misexpressing the activated receptor had no detectable effect on bracts with either driver and (2) misexpressing the DN form via Dll-Gal4 likewise had no effect. These negative results cannot be ascribed to impotence of the transgenes, since other defects were obvious. To wit, tarsal segments 2-4 were shortened and fused in Dll>EgfrDN (and Dll>Egfr) legs, and the entire tarsus was reduced to a bump on the end of a swollen Ti in Dll>Egfr*top4.2 legs. No sca>EgfrDN flies survived to the adult stage (Held, 2002).

Table of contents

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

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.