EGF receptor


Transcriptional regulation

Pointed, an ETS domain transcription factor, negatively regulates the EGF receptor pathway in Drosophila oogenesis

During oogenesis, dorsal follicle cells differentiate into either appendage-producing or midline cells, resulting in patterning in the dorsal follicle cell layer. Pointed, an ETS transcription factor, is required in dorsal follicle cells for this patterning. Loss of pointed results in the loss of midline cells and an excess of appendage-forming cells, a phenotype associated with overactivation of the EGF receptor pathway in the dorsal region. Overexpression of pointed leads to a phenotype similar to that generated by loss of the EGF receptor pathway. This suggests that Pointed normally down-regulates EGF receptor signaling in the midline to generate patterning in the dorsal region. pointed P1 transcript expression, beginning at stage 6-7, is induced by the EGF receptor pathway. By stage 8, expression is restricted to posterior follicle cells. During stage 9-10, P1 is detected in dorsal-anterior and posterior follicle cells. During later stages, both these expression patterns refine into two areas: two dorsal patches and two posterior half-circles. P2 transcript expression is also detected during oogenesis, both in the germ line and follicle cells. Follicle cell expression is observed in stage 9-10 egg chambers in a pattern resembling the early P1 expression pattern in dorsal-anterior follicle cells. During later stages, P2 expression is restricted to two groups of anterior dorsolateral follicle cells that flank the oocyte nucleus. Whereas two dorsolateral dorsal appendages are detected in control eggs, pointed mutant appendages are four times wider than a single wild type appendage, suggesting that the phenotype does not result from two appendages fusing together, but rather fromcells in the middle region taking on an appendage-producing cell fate. These data indicate a novel antagonistic function for Pointed in oogenesis; in response to activation of the EGF receptor, pointed is expressed and negatively regulates the EGF receptor pathway (Morimoto, 1996).

An autoregulatory function of Dfos during Drosophila endoderm induction

The endoderm of Drosophila is patterned during embryogenesis by an inductive cascade emanating from the adhering mesoderm. An immediate-early endodermal target gene of this induction is Dfos whose expression is upregulated in the middle midgut by Dpp signaling. Previous evidence based on a dominant-negative Dfos construct has indicated that Dfos may cooperate with Dpp signaling to induce the HOX gene labial, the ultimate target gene of the inductive cascade. Here, kayak mutants that lack Dfos were examined to establish that Dfos is indeed required for labial induction. Evidence is provided that Dfos acts on labial through a CRE (cyclic AMP response element)-like sequence, previously identified to be a target for signaling by Dpp and by the Epidermal growth factor receptor (Egfr) in the embryonic midgut. Dfos expression is stimulated by Egfr signaling. Dominant negative Egfr-expressing embryos were stained with antibody against Dfos. These embryos never show high levels of Dfos protein in the endodermal cells of the parasegment (ps) 6/7 region in the midgut as normally seen in the wild type. Finally, Dfos function is found to be required for its own upregulation. Thus, endoderm induction is based on at least four tiers of positive autoregulatory feedback loops (Szuts, 2000).

The CRE-like sequence appears to be a target for Egfr signaling. Furthermore, the embryonic gut phenotype of kay1 mutants in the endoderm is similar to that due to loss of Egfr signaling. In particular, both mutant conditions seem to cause some degree of cell death in the midgut epithelium. Although this cell death may contribute to, it does not account completely for, the mutant phenotypes observed. Finally, it has been shown that Dfos upregulation in the ps6/7 region of the endoderm depends on Egfr function. Taken together, these observations indicate that Dfos, or its DNA-binding partner(s), may be a critical target of Egfr signaling during endoderm induction, and that the effects of Egfr signaling in the endoderm may be partly if not largely mediated by Dfos (Szuts, 2000).

Dfos is a context-dependent transcriptional activator whose function in the embryo requires a dimerization partner, such as Djun, as well as combined Jun N-terminal kinase (JNK) and Dpp signaling. This dimerization partner rather than Dfos itself is likely to be the target factor that is directly modified and activated by JNK signaling. In some embryonic tissues, for example the dorsal epidermal cells, this dimerization partner is probably Djun, a transcription factor known to be targeted directly by JNK and Rolled MAP kinase signaling. However, Djun is not a good candidate for a MAP kinase-activated dimerization partner of Dfos in the midgut since Djun neither detectably affects labial nor CRE-mediated expression in the midgut. This putative signal-activated dimerization partner of Dfos in the midgut thus remains elusive (Szuts, 2000).

Activation of knot(kn) specifies the 3-4 intervein region in the Drosophila wing

Hedgehog (Hh) plays an important role in Drosophila wing patterning by inducing expression of Dpp, which serves to organize the wing globally across the A-P axis. Hh signaling also plays a direct role in patterning the medial wing through the activation of the Hh-target gene, knot (kn). kn is expressed in Hh-responsive cells near the A-P compartment boundary, where its expression is dependent on fu, a component of Hh signaling. kn is required for the proper positioning of veins 3 and 4 and to prevent ectopic venation between them. Furthermore, the anterior expansion of the normal kn expression domain causes an associated anterior shift in the position of vein 3 in the resultant wing. Ectopic expression of kn elsewhere in the wing imaginal disc results in the failure to properly activate the vein initiation genes, rho and Dl. Expression of the gene encoding the EGF-receptor (Egfr), which is required for vein initiation and subsequent differentiation, is normally depressed in the 3-4 intervein region. This downregulation of Egfr in the medial portion of the imaginal disc is dependent on kn activity and ectopic expression of kn inactivates Egfr elsewhere in the wing primordium. It is proposed that kn expression in Hh-responsive cells of the wing blade anlagen during the late third instar creates a zone of cells in the medial wing in which vein primordia cannot be induced. The primordia for veins 3 and 4 are laid down adjacent to the kn-imposed vein-free zone, presumably by a signaling factor (such as Vn) also synthesized in the medial region of the wing (Mohler, 2000).

The original viable kn allele causes veins 3 and 4 to form closer together and produces a corresponding shift in their primordia (as detected by rho expression) in the late third instar disc (Sturtevant, 1995). Mosaic analysis of strong, embryonic kn alleles by Nestoras (1997) indicates that kn is important in suppressing vein formation between veins 3 and 4, but not in other regions of the wing. Because vein 4 runs just posterior to the A-P compartment boundary, the region affected by kn mutant clones corresponds approximately to the Hh-responsive cells along the A-P compartment boundary. kn is also required for suppressing vein formation in ptc minus clones in the anterior compartment; these ptc minus clones mimic Hh-responsive cells in which Hh has bound to the Ptc receptor. Nestoras (1997) proposed that kn functions to separate veins 3 and 4 by imposing a vein-free region in response to Hh signaling. Mosaic analysis of strong, pupal lethal alleles of fu, a serine/thronine kinase required in Hh-responsive cells for a normal response, shows a similar requirement for fu in preventing ectopic vein induction between veins 3 and 4, suggesting a direct role for Hh signaling in controlling the 3-4 intervein space (Mohler, 2000 and references therein).

No alteration of vein expression is found in knot mutant discs or in discs in which kn has been ectopically expressed, indicating that the expression of vn in this region is not controlled by kn. Egfr expression, however, is regulated by knot. Although transcription of Egfr is initially uniform throughout the wing blade, by the end of the third instar Egfr transcription has been repressed along the wing margin and in a medial stripe across the wing blade region. Double in-situ hybridization reveals that the medial region of Egfr downregulation coincides with the region of kn gene expression. To determine whether downregulation of Egfr is necessary for the formation of the 3-4 intervein region, Egfr was ectopically expressed in the region in which it is usually downregulated. Ecoptic expression of EGFR in the medial wing driven by the ptc-GAL4 driver generates wings with fusion of veins 3 and 4 in the proximal portion of the wing. Ectopic expression of Egfr driven throughout the wing blade dorsally does not cause formation of ectopic veins between veins 3 and 4, although a significant amount of ectopic vein material is induced anterior to vein 3 and posterior to vein 4. This suggests that the creation of a vein-free zone between veins 3 and 4 is not likely to be explained solely by downregulation of Egfr by kn (Mohler, 2000).

Connecting Hh, Dpp and EGF signalling in patterning of the Drosophila wing; the pivotal role of collier/knot in the AP organiser

Hedgehog (Hh) signaling from posterior (P) to anterior (A) cells is the primary determinant of AP polarity in the limb field in insects and vertebrates. Hh acts in part by inducing expression of Decapentaplegic (Dpp), but how Hh and Dpp together pattern the central region of the Drosophila wing remains largely unknown. The role played by Collier (Col), a dose-dependent Hh target activated in cells along the AP boundary (the AP organizer in the imaginal wing disc) has been examined. col mutant wings are smaller than wild type and lack L4 vein, in addition to missing the L3-L4 intervein and mis-positioning of the anterior L3 vein. These phenotypes are linked to col requirement for the local upregulation of both emc and N, two genes involved in the control of cell proliferation, the EGFR ligand Vein and the intervein determination gene blistered. Attenuation of Dpp signaling in the AP organizer is also col dependent and, in conjunction with Vein upregulation, required for formation of L4 vein. A model recapitulating the molecular interplay between the Hh, Dpp and EGF signaling pathways in the wing AP organizer is presented (Crozatier, 2002).

It has been been proposed that Hh does directly control the position of L3 vein, although the molecular mechanisms of this control have not been firmly established. In both col and mtv mutant clones, the position of L3 vein is shifted posteriorwards. That both col and mtv control the position of L3 vein suggests that this position is defined by Hh signaling through the modulation of Dpp signaling. iro is required for rho activation in the L3 primordium and formation of L3 vein. iro is activated by both Dpp and Hh signaling and its anterior border of expression is under control of sal/salr, a target of Dpp. The patterns of col, iro and rho expression are intimately connected. Both an increased number of cells expressing rho and a posterior shift of the anterior border of iro expression are observed in col1 mutant discs. This posterior shift is interpreted as reflecting a modified range of Dpp signaling relayed, at least in part, by sal/salr activity. The increased number of rho-expressing cells, for its part, indicates that Col is able to antagonize rho activation by iro in cells, which express both iro and Col. This correlates well with the wing phenotype – anteriorwards shift of the L3 vein, together with gaps in its distal region – which results from anterior extension of Col expression, in UAS-Col/dpp-Gal4 wing discs. The distal gaps could reflect the complete absence of rho expression close to the DV border, because of the complete overlap between col and iro expression where iro expression is narrower. From col loss- and gain-of function experiments, it is therefore concluded that the primordium of L3 vein corresponds to cells that express iro but not col. Col thus appears to play a dual role in defining the position and width of L3 vein: activating Blistered and repressing EGFR in the wing AP organizer cells, endows these cells with an intervein fate, while attenuating Dpp signaling indirectly positions the anterior limit of iro expression domain, and L3 vein competence anterior to the AP organizer (Crozatier, 2002).

Negative regulation of Egfr/Ras pathway by Ultrabithorax during haltere development in Drosophila

In Drosophila, wings and halteres are the dorsal appendages of the second and third thoracic segments, respectively. In the third thoracic segment, homeotic selector gene Ultrabithorax (Ubx) suppresses wing development to mediate haltere development. Halteres lack stout sensory bristles of the wing margin and veins that reticulate the wing blade. Furthermore, wing and haltere epithelia differ in the size, shape, spacing and number of cuticular hairs. The differential development of wing and haltere, thus, constitutes a good genetic system to study cell fate determination. Down-regulation of Egfr/Ras pathway is critical for haltere fate specification: over-expression of positive components of this pathway causes significant haltere-to-wing transformations. RNA in situ, immunohistochemistry, and epistasis genetic experiments suggest that Ubx negatively regulates the expression of the ligand vein as well as the receptor Egf-r to down-regulate the signaling pathway. Electromobility shift assays further suggest that Egf-r is a potential direct target of Ubx. These results and other recent findings suggest that homeotic genes may regulate cell fate determination by directly regulating few steps at the top of the hierarchy of selected signal transduction pathways (Pallavi, 2006).

To identify potential targets of Ubx and thereby mechanism of its function, a gain-of-function genetics strategy was employed. Ubx-GAL4 driver, is expressed in the entire anterior compartment of the haltere imaginal disc. Ubx-GAL4 is also a null allele of Ubx and exhibits characteristic dominant phenotype; the presence of wing-type sensory bristles in the capitellum of the haltere. This GAL4 driver provides a fortuitous sensitive background to carry out large-scale screens for identifying the suppressors and enhancers of Ubx function, which otherwise may be less efficient in a wild type background. Indeed, over-expression of Vestigial (Vg), a pro-wing gene and a target of Ubx function, in the developing haltere results in very high degree of haltere-to-wing homeotic transformations, A candidate gene screen was employed to identify downstream targets of Ubx, in which various genes known to be involved in wing development were ectopically expressed in the developing haltere using the Ubx-GAL4 driver. Criterion for defining haltere-to-wing transformation in this study was the presence of wing-type sensory bristles, although increase in haltere size and enhanced pigmentation was frequently observed. For comparison between different genotypes, the degree of transformation was estimated by counting the number of sensory bristles on the haltere capitellum. UAS stocks for a large number of such wing patterning genes were crossed to Ubx-GAL4 driver and were scored for enhancement and suppression of the dominant phenotype of heterozygous Ubx. Progeny for many of the crosses resulted in early embryonic or early larval lethality, reflecting the fact that the GAL4 driver expresses at early stages during embryonic development. Nevertheless, strong haltere-to-wing transformations were observed upon over-expression/mis-expression of most of the positive components of the Egfr/Ras pathway. For example, the bristle number in the capitellum of the Ubx-GAL4 haltere increased when positive components such as Vn or Egf-r were over-expressed and over-expression of negative components such as Aos completely suppressed the heterozygous Ubx phenotype (Pallavi, 2006).

A significant finding of this study is the down-regulation of Egfr/Ras pathway in haltere discs by Ubx. Earlier reports suggest that a short-range signal originating from the D/V boundary activates Egfr/Ras pathway in a zone of cells on the edges of the D/V boundary and that this activation is essential for vg transcription (Nagaraj, 1999). Egfr/Ras pathway has also been implicated in the developmental events along the A/P axis: in wing vein specification. Consistent with the down-regulation of both A/P and D/V signaling events in haltere discs, expression of most of the Egfr/Ras pathway components is repressed in the entire haltere pouch. Observations on the strengths of haltere-to-wing transformations (at the margin bristle level) in different genetic backgrounds establish the specificity of genetic interactions between Egfr/Ras pathway and Ubx during haltere development (Pallavi, 2006).

The abovementioned results on the down-regulation of Egfr/Ras pathway in haltere discs by Ubx are consistent with the previously reported genetic screen for the modifiers of homeotic genes, which indicates that Ras1 activity modulates functions of the homeotic loci Sex combs reduced (Scr) and Ubx. For example, haploinsufficient (haltere-to-wing) phenotype of Ubx109/+ is significantly enhanced in Gap1Ubx109/Gap1 adults (Gap1 is a negative regulator of the Egfr/Ras pathway). This effect of Gap1 is reversed in Gap1Ubx109/Gap1Rase1b individuals due to the antagonistic roles of Gap1 and Ras1 in the Egfr/Ras pathway (Pallavi, 2006).

All the components of the Egfr/Ras pathway tested so far are differentially expressed between wing and haltere discs. This suggests the utility of developing wings and halteres as assay systems to identify novel components of Egfr/Ras pathway. Indeed, enhancer-trap screens and microarray analyses to identify genes that are differentially expressed between wing and haltere discs have resulted in the identification of CG32062 (Drosophila homologue of human ataxin-2 binding protein) and Mapmodulin (Drosophila homologue of human Inhibitor-1 of protein phosphatase-2A) as potential modulators of Egfr/Ras pathway (Pallavi, 2006).

The activation of dpERK1/ERK2 and aos in the haltere pouch by the ectopic expression of vn or Egf-r suggests that Ubx regulates Egfr/Ras pathway at ligand as well as receptor levels. Clonal analysis of Ubx function (loss of Ubx in haltere discs and gain in wing discs) demonstrates that Ubx controls vn expression in a cell-autonomous manner. Inability of ectopic Hh to activate vn expression in haltere discs suggests that Ubx functions downstream of Hh to repress vn expression. Putative Ubx-binding sites are present in the cis-regulatory regions of both vn and Egf-r, further suggesting their direct regulation by Ubx. Indeed, electromobility shift experiments suggest that, at least, Egf-r is probably a direct target of Ubx function. Thus, it is likely that Ubx independently down-regulates both vn and Egf-r. Ubx-mediated down-regulation of vn and Egf-r appears to be critical; over-expression of normal or the activated form of Egf-r induced stronger phenotypes when expressed in aos heterozygous background than in Ubx heterozygous background. However, Ubx may exert some influence on the pathway downstream of the receptor, since the strength of the phenotypes induced by the over-expression of Vn or Egf-r was stronger in Ubx heterozygous background. At dpERK1/ERK2 level too, the activation was stronger when Egf-r was over-expressed in Ubx heterozygous genetic background than in the wild type background (Pallavi, 2006).

It is interesting to note that Ubx regulates Egfr/Ras pathway at the level of the receptor itself. Although Egfr/Ras pathway is auto-regulated at several levels including the transcription of Egf-r itself, external factors regulating Egfr/Ras pathway during various developmental events mostly act at the level of the ligand/s or downstream effectors such as MAPK or transcription factors Yan, Pointed, etc. This study has found that the receptor itself is a direct target of Ubx indicating a novel mode of regulation of this pathway (Pallavi, 2006).

Specification of the larval oenocyte has been shown to be dependent on the regulation of just one principal target Rho by the homeotic gene abdominal A. Similarly, Hox proteins AbdA and AbdB specify the lineage of the embryonic NB6-4 neuroblast in abdominal segments by down-regulating CycE. Differential expression of CycE is both required and sufficient to generate segmental differences in NB6-4 lineage. This study reports that down-regulation of Vn and Egf-r is critical for Ubx-mediated suppression of wing margin bristles in the haltere. These results suggest that one common mechanism by which homeotic genes may regulate cell fate determination is by directly regulating few steps at the top of the hierarchy of selected signal transduction pathways. In contrast, Wingless and Decapentaplegic signaling pathways, which regulate more complex traits such as wing growth and shape, are regulated by Ubx at multiple levels in the hierarchy of those pathways (Pallavi, 2006 and references therein).

Although absence of veins in the haltere could be attributed to down-regulation of Egfr/Ras pathway, activation of sensory bristle development in Ubx+/Ubx+ halteres over-expressing positive components of Egfr/Ras pathway suggests a role for this pathway in cell fate specification in the wing margin. So far, no direct role for Egfr/Ras pathway has been assigned in the specification of sensory bristles of the wing margin, although it is known to specify macrochaete of the notum. Indeed, preliminary investigations suggest that Wg pathway induces EGFR/Ras pathway expression in cells immediately adjacent to the D/V boundary, and the latter pathway is required and sufficient to specify sensory organs of the wing margin (Pallavi, 2006).

The bristle development in the transformed halteres appears to be organized in two parallel rows when various components of Egfr/Ras pathway are over-expressed in Ubx heterozygous background, while the bristles are positioned in a disorganized way when phenotypes are induced in wild type background. This could be due to partial de-repression of D/V signaling in Ubx heterozygous background, which may allow appropriate positioning of the zone of margin bristle development (Pallavi, 2006).

Targets of Activity

Graded activation of Egfr is essential for patterning the ventral midline ectoderm. argos mutant embryos show expansion of ventral cell fates suggesting hyperactivation of the Egfr pathway. In the embryonic ventral ectoderm, argos is expressed in the ventralmost row of cells. argos expression in the ventral ectoderm is induced by the Egfr pathway: argos is not expressed in Egfr mutant embryos, while it is ectopically expressed in the entire ventral ectoderm following ubiquitous activation of the Egfr pathway. Induction can also be observed in cell culture, following activation of Egfr by its ligand, Spitz. Argos also functions in a sequential manner, to restrict the duration and level of Egfr signaling. This type of inhibitory feedback loop may represent a general paradigm for signaling pathways inducing diverse cell fates within a population of non-committed cells (Golembo, 1996).

Epidermal growth factor receptor induces pointed P1 and inactivates Yan protein in the embryonic ventral ectoderm. Two other candidate genes for Egfr regulation are ventral nervous system defective and Fasciclin III. Ectopic expression of secreted Spitz results in expression of orthodenticle within the entire ventral ectoderm, suggesting that ventral expression of otd is normally induced by higher levels of Egfr activity. Of the two pointed transcripts, only pntP1 is expressed in the ventral ectoderm. It first appears prior to gastrulation in the entire neuroectoderm region. In pointed null mutants, the expression of orthodenticle, argos and tartan in these cells is abolished or significantly reduced. Since Pointed P1 is thought to be a constitutively active transcription factor, with no requirement for modulation of its activity by the Egfr/MAP kinase signaling pathway, a direct induction of pntP1 transcription by Egfr appears possible. Early pntP1 expression is Egfr independent, but at stage 9/10, expression of pnt is not observed in the ventral ectoderm of Egfr mutants. yan, which encodes a negative regulator of ETS transcriptional activators, is first detected at stage 5/6, where it is found in the dorsal ectoderm. yan expression declines in a dorsal-ventral gradient and is not found in mesoderm. Expression of yan does not depend on Egfr, as it is unaltered in Egfr mutants. In yan mutants, the ventralmost markers orthodenticle, argos and tartan show a clear expansion. Absence of the Yan protein may thus allow the Pointed P1 protein, which is expressed earlier in a broader domain, to efficiently induce ventralization. In the absence of Egfr and yan, the early Egfr-independent expression of pntP1 is capable of triggering otd expression. An activated form of Yan, which is unable to undergo phosphorylation by MAP kinase, was expressed in wild-type embryos. Indeed, the expression of orthodenticle and argos is significantly reduced or abolished, in the region where activated Yan is expressed (Gabay, 1996).

Spitz, and spitz group genes are at the top of the regulatory hierarchy in the development of salivary ducts. The salivary primordium consists of two regions, a more dorsal pregland anlage and a ventral preduct anlage. Spitz signaling to ventral cells, through the Egfreceptor acts to block forkhead expression in preduct cells, thereby restricting gland identity to more dorsal cells. Forkhead acts in dorsal pregland cells to block duct fate, specifically acting to repress Serrate, a duct specific gene as well as breathless and trachealess, also required for duct formation. The spitz group genes rhomboid and pointed are required for duct fate (Kuo, 1996).

An important question in neurobiology is how different cell fates are established along the dorsoventral (DV) axis of the central nervous system (CNS). The origins of DV patterning within the Drosophila CNS have been investigated. The earliest sign of neural DV patterning is the expression of three homeobox genes in the neuroectoderm -- ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh) -- which are expressed in ventral, intermediate, and dorsal columns of neuroectoderm, respectively. Previous studies have shown that the Dorsal, Decapentaplegic (Dpp), and EGF receptor (Egfr) signaling pathways regulate embryonic DV patterning, as well as aspects of CNS patterning. This study describes the earliest expression of each DV column gene (vnd, ind, and msh), the regulatory relationships between all three DV column genes, and the role of the Dorsal, Dpp, and Egfr signaling pathways in defining vnd, ind, and msh expression domains. The vnd domain is established by Dorsal and maintained by Egfr, but unlike a previous report vnd is found not to be regulated by Dpp signaling. ind expression requires both Dorsal and Egfr signaling for activation and positioning of its dorsal border, and abnormally high Dpp can repress ind expression. The msh domain is defined by repression: it occurs only where Dpp, Vnd, and Ind activity is low. It is concluded that the initial diversification of cell fates along the DV axis of the CNS is coordinately established by Dorsal, Dpp, and Egfr signaling pathways. Understanding the mechanisms involved in patterning vnd, ind, and msh expression is important, because DV columnar homeobox gene expression in the neuroectoderm is an early, essential, and evolutionarily conserved step in generating neuronal diversity along the DV axis of the CNS (Von Ohlen, 2000).

Early stage 5 embryos express vnd in a narrow domain similar to its final width; ind and msh are not detected. By the end of stage 5, both vnd and ind are expressed with a one to two cell wide gap; again, this expression is seen in domains similar to their final widths. The gap fills in during development resulting in the precise juxtaposition of the vnd and ind domains. Expression of msh in the trunk is not detected until stage 7. Thus, the timing of gene expression progresses from ventral to dorsal: vnd is detected first, ind appears soon after, and msh is observed last (Von Ohlen, 2000).

Initiation and maintenance of ind expression require both Dorsal and Egfr signaling pathways, but not Dpp activity. The ventral border of ind expression is established by the dorsal limit of vnd expression. The dorsal border of ind expression has more complex regulation. Dpp repression does not establish the dorsal border of ind, since the ind domain is normal in dpp embryos. In contrast, both Dorsal and Egfr are required to activate ind and set its dorsal border. In wild-type embryos, the domains of ind and activated Egfr have identical dorsal borders. When Egfr activity is increased throughout the embryo, ind expression shows a partial dorsal expansion, showing that the dorsal border of Egfr activity sets the precise dorsal border of ind expression. Ectopic Dorsal activity can also expand the ind domain (without affecting the Egfr activation domain), showing that sufficiently high levels of nuclear Dorsal protein can independently activate ind expression. As expected, when Egfr activity and nuclear Dorsal levels are simultaneously increased there is a complete dorsal expansion of the ind domain. The data presented here suggest that ind expression is activated by both Dorsal and Egfr pathways, limited ventrally by vnd, and limited dorsally by lack of Dorsal and Egfr activity. The data do not distinguish between a linear pathway in which Egfr signaling activates or potentiates Dorsal to allow ind transcription and a parallel pathway in which Dorsal and Egfr signaling act independently to activate ind expression (Von Ohlen, 2000).

Two thoracic limbs of Drosophila, the leg and the wing, originate from a common cluster of cells that include the source of two secreted signaling molecules, Decapentaplegic and Wingless. Wingless, but not Decapentaplegic, is responsible for the initial distal identity specification of the limb primordia. Limb formation is restricted to the lateral position of the embryo through exertion of negative control by Decapentaplegic and the EGF receptor, both of which determine the global dorsoventral pattern. dpp specifies proximal cell identities. Since Distal-less expression persists and expands dorsally in the absence of Dpp, it is clear that Dpp plays no role in inducing initial Dll expression but that the dorsoventral limit of Dll expression is defined by repression as a result of Dpp expression. Similarly, Egfr is required to repress Dll expression in the ventral ectoderm (Goto, 1997).

Mutations in Egf receptor result in the expansion of muscle segment homeobox domains ventrally, and their ventral margins become graded rather than forming a sharp border. In decapentaplegic mutants, msh expression expands dorsally and extends all the way to the dorsal midline, showing that dpp normally represses msh in the dorsal 30% of the circumference. In short gastrulation mutants, with four copies of dpp, there is a complete repression of msh. Thus the early msh domains in the lateral neuroectoderm are delimited through dorsal repression by DPP and ventral repression by the active Egfreceptor (D'Alessio, 1996).

DRacGAP, a novel Drosophila gene, inhibits Egfr/Ras signaling in the developing imaginal wing disc

GTPase-activating proteins (GAPs) are negative regulators that stimulate the intrinsically low GTPase activity of the Rho proteins, thus reducing the signaling potential of the active GTP bound form of Rho proteins. DRacGAP (CG13345 or acGAP) behaves as a negative regulator of Rho-family GTPases Rac1 and Cdc42. Reduced function of RacGAP or increased expression of Rac1 in the wing imaginal disc causes similar effects on vein and sensory organ development and cell proliferation. These effects result from enhanced activity of the Egfr/Ras signaling pathway. In the wing disc, Rac1 enhances Egfr/Ras-dependent activation of MAP Kinase in the prospective veins. Interestingly, DRacGAP expression is negatively regulated by the Egfr/Ras pathway in these regions. During vein formation, local DRacGAP repression ensures maximal activity of Rac and, in turn, of Ras pathways in vein territories. Additionally, maximal expression of DRacGAP at the vein/intervein boundaries helps to refine the width of the veins. Hence, control of DRacGAP expression by the Egfr/Ras pathway is a previously undescribed feedback mechanism modulating the intensity and/or duration of its signaling during Drosophila development (Sotillos, 2000).

Evidence is presented for the cooperation of Rac and Ras signaling pathways in the context of a whole organism. A Drosophila gene, DRacGAP, is described that encodes a putative GAP for Rac and Cdc42 GTPases. Both DRacGAP and DRac1 are involved in the control of cell proliferation. Moreover, reduced activity of DRacGAP or overexpression of DRac1 in the wing imaginal disc cause similar defects: widening of veins, development of extra sensory organs (SOs), apoptosis and the appearance of enlarged cells that differentiate multiple hairs with abnormal polarity. These phenotypes result from DRac1 enhancement of epidermal growth factor receptor (Egfr)/Ras signaling. The Egfr/Ras pathway pathway, which operates through activation of the Ras/Raf/MEK/MAP kinase cascade controls multiple developmental processes and is accurately regulated. Indeed, this pathway controls the expression of its own negative and positive regulators. Interestingly, expression of DRacGAP is repressed by Egfr/Ras signaling in the prospective veins and accumulates at the vein/intervein boundaries. These results suggest that control of DRacGAP expression by the Egfr/Ras pathway provides a new mechanism to modulate the intensity of this pathway during Drosophila development (Sotillos, 2000).

Overexpression of DN DRacGAP or Rac1 causes vein enlargement and the appearance of extra SOs, two structures requiring Egfr/Ras/Raf/MAPK activity. Since Rac and Ras pathways cooperate in mammalian cells in the control of cell proliferation, an investigation was carried out to see whether the phenotypes associated with increased Rac signaling could be due to overactivity of the Ras pathway. This appears to be the case, since a reduction in Egfr signaling (by expression of DN Raf) reduces vein, wing notching and large cell phenotypes. Similarly, when levels of the Egfr activators rhomboid and vein (vn) are decreased, the wing notching associated with DN DRacGAP expresson is substantially corrected. In contrast, activation of Egfr signaling enhances the mutant phenotype of DN DRacGAP flies. Thus, although flies heterozygous for mutant argos, a repressor ligand of Egfr, are phenotypically wild type, its combination with DN DRacGAP significatively increases the number of campaniform sensilla of DN DRacGAP flies. These results further suggest that the efficacy of the Egfr pathway is enhanced by increased Rac activity. Rac signaling ultimately activates MAPK since expression of DN DRacGAP widens the domains of accumulation of dp-ERK in the presumptive veins and spreads them into the interveins. This effect is enhanced by coexpression of Rac1 (Sotillos, 2000).

Expression of DRacGAP appears to be decreased in the domains of Egfr activation. This is most apparent in the notum region of second instar wing disc, where the Egfr activator vein is expressed, and in the presumptive veins of third instar wing disc, territories of maximal Egfr signaling. This observation suggests that the Egfr pathway may repress the expression of DRacGAP. In agreement with this notion, expression of DRacGAP is either decreased or enhanced in cells ectopically expressing Ras V12 or argos in which the EFGR/Ras pathway is activated or repressed, respectively (Sotillos, 2000).

The enlarged cell size of wing cells overexpressing Rac and the small size of wings with reduced Rac function indicate a role for Rac in cell proliferation control. In mammalian cells, Ras and Rac pathways cooperate in stimulating cell proliferation. Similarly, Drosophila Rac1 and Ras also appear to cooperate in this process since the reduced size of the wings of DN Raf-expressing flies is largely normalized by reduction of DRacGAP function or by overactivity of Rac1. The induction of cell death by DN DRacGAP, where Rac activity is upregulated, is in apparent contradiction with these results. However, note that in mammalian cells, quantitative variations in the level of Ras signaling can cause very different effects. Thus, instead of cell proliferation, high levels of Ras signaling may induce cell cycle arrest at G1 and apoptosis as part of a cell self protective mechanism. In that situation, G1 arrest is caused by Raf-dependent induction of p21 WAF1/CIP1 expression, which inhibits Cdk activity and indirectly represses CycE transcription. The situation appears to be very similar in Drosophila. Cell death of DN DRacGAP flies could be attributed to their arrest at the G1 stage, since the phenotype is rescued by expression of CycE. Moreover, the accumulation of p21 causes apoptosis, enlargement of cell size and polarity defects, phenotypes that are corrected by expression of CycE. Accordingly, it is hypothesised that induction of cell cycle arrest and apoptosis by overactivity of the Rac pathway could be a consequence of increased Ras signaling. Rac would potentiate the reduced Raf signaling occurring in DN Raf flies, allowing wing disc cells to proliferate, but in the presence of wild-type Raf, overactivity of Rac would enhance Raf signal to such a high level as to induce p21 expression and ultimately, apoptosis. This interpretation is supported by the partial rescue of the wing notching phenotype of DN DRacGAP flies in a ve;vn heterozygous background and in DN Raf flies where Egfr/Ras signaling is reduced (Sotillos, 2000).

Interestingly, in the Drosophila wing disc, DRacGAP transcription is repressed by the Egfr/Ras pathway. Activity of this pathway is finely tuned by its control of the expression of its own inhibitors and activators. The results indicate that Ras signaling can self-stimulate through activation of the Rac pathway by repression of DRacGAP. During vein formation, the Egfr/Ras pathway, once it has attained a certain threshold, should repress expression of DRacGAP in the prospective vein regions, thus ensuring maximal activity of Rac and, in turn, of Ras pathways in these territories of the imaginal wing disc, which should trigger vein differentiation. In contrast, maximal expression of DRacGAP at the vein/intervein boundaries should locally decrease Rac and Ras signaling, and in collaboration with Notch and Dpp pathways help to refine the final width of the veins. Hence, this regulatory loop is another feedback mechanism modulating the activity of the Egfr/Ras pathway during Drosophila development (Sotillos, 2000).

Hedgehog activates the EGF receptor pathway during Drosophila head development

The Hedgehog (Hh) and Epidermal growth factor receptor (Egfr) signaling pathways play critical roles in pattern formation and cell proliferation in invertebrates and vertebrates. In this study, a direct link between these two pathways is demonstrated in Drosophila. Hh and Egfr signaling are each required for the formation of a specific region of the head of the adult fruitfly. hh and vein (vn), which encodes a ligand of the Drosophila Egfr, are expressed in adjacent domains within the imaginal primordium of this region. Using loss- and gain-of-function approaches, it has been demonstrated that Hh activates vn expression. Hh activation of vn is mediated through the gene cubitus interruptus (ci) and this activation requires the C-terminal region of the Ci protein. wingless (wg) represses vn expression, thereby limiting the domain of EGFR signaling (Amin, 1999).

In addition to a post-translational regulation of Head involution defective (Hid), the Ras/MAPK pathway promotes cell survival in Drosophila by downregulating the expression of hid. Conversely, downregulation of the Ras/MAPK pathway induces cell death by upregulating hid expression. hid transcript levels are downregulated in dominantly active Dras1- (Dras1Q13) expressing embryos when assayed 3 hr after heat shock. In wild-type embryos, total HID mRNA levels do not change dramatically between stage 11, when Ras expression was ectopically induced, and stage 14, when HID mRNA levels were assayed. This eliminates the concern that developmental arrest might account for the observed difference in HID mRNA levels. It was observed that hid levels return to normal in Dras1Q13 embryos by 5 hr after heat shock. Cell death also resumes in these embryos several hours later. This indicates that a transient increase in Ras activity leads to a transient suppression of hid expression, accompanied by a transient protection from naturally occurring cell death. HID mRNA levels were also assayed through an alternative procedure: whole mount in situ analysis. These results confirm that hid transcript levels decline in dominantly active Dras1- (Dras1Q13) expressing embryos. This is particularly apparent in the midline glia, which strongly express hid. The survival of midline glia is known to depend on the activity of the Epidermal growth factor receptor pathway. To confirm that Ras regulation of hid utilizes the Raf/MAPK pathway, the effect of a constitutively active form of Draf (phlF22) on hid expression has been investigated. In situ analyses were performed on embryos expressing activated Draf under the control of the heat shock promoter. Heat-induced expression of phlF22 results in downregulation of hid transcript levels, suggesting that Ras functions through the Raf/MAPK pathway to downregulate hid expression (Kurada, 1998).

Reduction in pointed (pnt) activity has been observed to enhance ectopic Hid induced cell death in the eye. The pointed transcription factor is a target of MAPK function and acts as a positive regulator in the R7 pathway. The pnt gene encodes two related proteins, pnt1 and pnt2. pnt2 operates downstream of the MAPK rolled in the Ras pathway. Therefore, the consequences of ectopic expression of pnt2 were examined. Embryos were generated that carry UAS-Pnt2 and a midline glia-specific Gal4 driver (52A-Gal4), resulting in the expression of pnt2 in the midline glia cells. Such embryos were tested for hid levels by whole-mount in situ analysis. Like embryos expressing activated Dras1 and activated Draf, pnt2-expressing embryos show decreased hid transcript levels, indicating that the Ras/MAPK pathway, acting through pnt, downregulates hid transcription (Kurada, 1998).

Since upregulation of the Ras/MAPK pathway promotes cell survival and downregulates hid expression, it was predicted that increased hid expression is the cause of the increased apoptosis observed when Ras activity is decreased. Ubiquitous expression of the negative regulator yan is able to induce massive embryonic apoptosis. In these same embryos HID mRNA levels are increased within 2 hr of yanAct induction and continue to rise for many more hours. Thus, downregulation of Ras activity in the embryo results in increased hid transcription and apoptosis, and this transcription is regulated either directly or indirectly by yan. These results imply that Ras activation of MAPK and inactivation of yan is an important cell survival pathway in embryos (Kurada, 1998).

Blocking Epidermal growth factor receptor activity in the developing eye also enhances apoptosis. If hid is a target of Egfr/Ras/MAPK activity in this tissue, then hid levels should increase when Egfr activity is blocked. Expression of a dominant negative Egfr in the developing eye results in a band of increased hid transcription in the eye disc. This band lies several rows posterior to the furrow and corresponds well with the first developmental defects seen in these eye discs. In sum, these data implicate the downregulation of hid transcription as an important component of Egfr antiapoptotic activity. The post-transcriptional modification of Hid appears to be equally important (Kurada, 1998).

Sense organ identity in the Drosophila antenna is specified by the expression of the proneural gene atonal

The bHLH transcription factor Atonal is sufficient for specification of one of the three subsets of olfactory sense organs on the Drosophila antenna. Misexpression of Atonal in all sensory precursors in the antennal disc results in their conversion to coeloconic sensilla. The mechanism by which specific sense organ fate is triggered remains unclear. The homeodomain transcription factor Cut, which acts in the choice of chordotonal-external sense organ does not play a role in olfactory sense organ development. The expression of atonal in specific domains of the antennal disc is regulated by an interplay of the patterning genes, Hedgehog and Wingless, and Drosophila epidermal growth factor receptor pathway (Jhaveri, 2000).

Pattern formation in the epidermis is regulated by a hierarchy of genes; the patterning genes -- engrailed, hh, dpp and wg -- specify co-ordinates of the disc and are expected to influence expression of prepatterning genes. Lz is a putative prepatterning gene in the antennal disc and has been shown to regulate expression of amos; genes regulating ato in the antenna are as yet unclear. The olfactory sense organs are located in a distinct pattern across the antenna, thus requiring co-ordinated control of the different proneural genes. Expression of a dominant negative Egfr results in a large number of ectopic atonal+ cells. Phosphorylated MAP kinase expression does not co-localize in ato+ cells and in the third instar antennal disc phosphorylated MAP kinase and Ato immunoreactivity occupy mutually exclusive domains. Hence the expression pattern of these two molecules is consistent with the model that signaling through the Ras/MAP kinase pathway acts to suppress ato expression. It is therefore proposed that signaling through Egfr and Ras/MAPK cascades plays a key role in linking positional information to the expression of proneural genes (Jhaveri, 2000).

During Drosophila eye development, Hh and Dpp are required to initiate photoreceptors at the furrow while Wg inhibits differentiation at the lateral margins. Wg appears to act by antagonizing signaling through the Egfr pathway. In contrast, Hh may directly regulate ato expression, its diffusion ahead of the morphogenetic furrow turns on Ato, while higher levels behind the furrow lead to its downregulation. There is however evidence that Hh can also influence Egfr signaling since Ci has been shown to activate Mapk through the Egfr ligand Vein (Jhaveri, 2000 and reference therein).

During antennal development, suppression of Egfr activity by dominant negative strategies leads to ectopic ato expression. A possible candidate to link Egfr signaling and Ato is the homeodomain molecule Rough which plays such a role during photoreceptor development. Results from DN-Egfr misexpression, and the observation that Mapk levels are high in domains where Ato-expressing cells are absent leads the authors to suggest that signaling through Ras/MAPK determines the pattern of progenitor cells for coeloconic sensilla. This poses the question of how Egfr activation is regulated across the antennal disc (Jhaveri, 2000).

A genetic hierarchy establishes mitogenic signaling and mitotic competence in the renal tubules of Drosophila

Cell proliferation in the developing renal tubules of Drosophila is strikingly patterned, occurring in two phases to generate a consistent number of tubule cells. The later phase of cell division is promoted by EGF receptor signaling from a specialized subset of tubule cells, the tip cells, which express the protease Rhomboid and are thus able to secrete the EGF ligand, Spitz. The response to EGF signaling, and in consequence cell division, is patterned by the specification of a second cell type in the tubules. These cells are primed to respond to EGF signaling by the transcription of two pathway effectors, PointedP2, which is phosphorylated on pathway activation, and Seven up. While expression of pointedP2 is induced by Wingless signaling, seven up is initiated in a subset of the PointedP2 cells through the activity of the proneural genes. Both signaling and responsive cells are set aside in each tubule primordium from a proneural gene-expressing cluster of cells, in a two-step process: (1) a proneural cluster develops within the domain of Wingless-activated, pointedP2-expressing cells to initiate the co-expression of seven up; (2) lateral inhibition, mediated by the neurogenic genes, acts within this cluster of cells to segregate the tip cell precursor, in which proneural gene expression strengthens to initiate rhomboid expression. As a consequence, when the precursor cell divides, both daughters secrete Spitz and become signaling cells. Establishing domains of cells competent to transduce the EGF signal and divide ensures a rapid and reliable response to mitogenic signaling in the tubules and also imposes a limit on the extent of cell division, thus preventing tubule hyperplasia (Sudarsan, 2002).

To understand how the proneural and neurogenic genes pattern the response to EGFR activation, the expression of genes involved in transduction of the pathway was analyzed. The orphan nuclear-receptor svp functions downstream of the EGF receptor to promote cell divisions in the tubules. In the absence of Svp function, cycE and stg transcription is abolished, with a consequent reduction in EGFR-driven cell divisions. These late divisions in the tubules of stage 12 wild-type embryos were followed and it was found that BrdU incorporation (and hence, cell division) is confined within the svp-lacZ domain. These results define the svp domain of expression as including those cells which will divide in response to Egfr activation. However, the expression of svp-lacZ is initiated in a group of cells surrounding the tip mother cell, before the birth of the TC. This early onset of svp expression occurs before the late divisions start (cycle 17 onwards), when neither Svp function nor Egfr activation is required for cell proliferation. The pattern of gene expression observed suggests that the Svp-positive cells surrounding the tip mother cell derive from the proneural cluster (Sudarsan, 2002).

To test this hypothesis, the expression of svp-lacZ in embryos lacking proneural gene function was examined. Indeed, in AS-C-/- embryos, the expression of svp is not initiated in the tubules. Conversely, in N mutants, where all cells in the cluster adopt the primary tip cell (TC) fate, the expression of svp is confined to the transformed cells. After the initiation of Spi signaling from the TC/SC, svp expression depends on Egf receptor activation. However that the early expression of svp is not dependent on Egfr function is shown in topCO mutants, where svp-lacZ expression is still initiated normally, but is not maintained. In AS-C–/– embryos expressing lambdatop in the tubules, svp expression is not detected. Together these data suggest that the initiation of svp depends on the proneural genes but is independent of Egf receptor signaling, which acts only from cycle 17 to maintain svp expression (Sudarsan, 2002).

These results show that the expression of proneural genes in the tubules not only confers tip cell potential but also initiates the expression of an effector of the Egf pathway, svp. It is suggested that this primes cells to divide in response to EGF receptor activation. Proneural genes are therefore required to specify two cell fates in the tubule proneural clusters (PNCs); the tip mother cell and cells competent to respond to Egfr activation (Sudarsan, 2002).

Svp is not the only effector of the EGF pathway. The ETS domain protein PointedP2 (PntP2) functions downstream of Egfr/Ras signaling. This protein contains a single MAPK phosphorylation site and upon phosphorylation, competes with the ETS domain transcriptional repressor, Yan, to activate the expression of target genes. In the absence of pnt function, cell proliferation in the tubules is reduced in a manner similar to svp mutants (Sudarsan, 2002).

It was therefore asked whether early expression of pnt as well as svp is required to prime the mitogenic response in tubule cells. pntP2 is initiated in the posterior side of each tubule during stage 10. This domain is characterized by high levels of wg expression, which are required for the normal development of AS-C expression in the PNC, during the time it develops within this domain. The domain of wg and pntP2 expression is slightly wider than the PNC and pntP2 expression is initiated well before Egfr activity is required for tubule cell divisions. The expression of pntP2 persists in this posterior domain when the tip mother cell is specified. In wgCX4 mutant embryos, tubule expression of pntP2 is completely abolished, showing that Wg signaling is required to initiate its expression. Conversely, the overexpression of wg, using a hs-wg construct, results in expansion of pntP2 expression to the anterior side of the tubule primordium and elevation of expression to high levels. Thus, Wg is necessary and sufficient to activate the expression of pntP2 in the tubules (Sudarsan, 2002).

These results suggest that, while the segregation of single cells from an equivalence domain is a unifying theme in the generation of tissues from a wide range of organisms, PNCs in specific tissues have developed an additional function: to establish a second cell fate that cooperates with the first to implement the subsequent program of tissue differentiation (Sudarsan, 2002).

Targets of Activity: Egf receptor function in the wing disc

The EGF receptor and N signalling pathways act antagonistically in Drosophila mesothorax bristle patterning

An early step in the development of the large mesothoracic bristles (macrochaetae) of Drosophila is the expression of the proneural genes of the achaete-scute complex (AS-C) in small groups of cells (proneural clusters) of the wing imaginal disc. This is followed by a much increased accumulation of AS-C proneural proteins in the cell that will give rise to the sensory organ, the SMC (sensory organ mother cell). This accumulation is driven by cis-regulatory sequences, SMC-specific enhancers, that permit self-stimulation of the achaete, scute and asense proneural genes. Negative interactions among the cells of the cluster, triggered by the proneural proteins and mediated by the Notch receptor (lateral inhibition), block this accumulation in most cluster cells, thereby limiting the number of SMCs. In addition, proneural proteins trigger positive interactions among cells of the cluster that are mediated by the Epidermal growth factor receptor (Egfr) and the Ras/Raf pathway. These interactions, which are termed 'lateral co-operation', are essential for macrochaetae SMC emergence. Activation of the Efgr/Ras pathway appears to promote proneural gene self-stimulation mediated by the SMC-specific enhancers. Excess Egfr signaling can overrule lateral inhibition and allow adjacent cells to become SMCs and sensory organs. Thus, the Egfr and Notch pathways act antagonistically in notum macrochaetae determination (Culí, 2001).

The earliest stage in macrochaetae development is the formation of the proneural clusters of ac-sc expression. Accumulation of Sc in cells of proneural clusters located at the more central positions of the wing disc decreases upon reduction of the level of Egfr signaling. The effect is cell-autonomous, which indicates that reception of the signal is important for cells to express sc properly. In contrast, more marginally located clusters, like the notopleural or scutellar, are unmodified or slightly enhanced under conditions of insufficient Egfr signaling. It is known that expression of ac-sc in different proneural clusters depends on separate, functionally independent enhancers which are thought to respond to local, specific combinations of transcription factors (prepattern). The different, spatially restricted effects of the insufficiency of Egfr function may thus be due to interference in the deployment or function of particular factors expressed in the affected area. Interestingly, the expression of the homeobox genes of the iroquois complex, necessary for the expression of ac-sc in many notum proneural clusters, is especially sensitive to the expression of the Vein Egfr ligand in the central region of the notum. Alternatively, since Egfr function is a well known requisite for growth and patterning of imaginal discs, the reduced expression of sc may be due to a more general impairment of the patterning of the central area of the disc (Culí, 2001).

Weak hypomorphic Egfr alleles cause the partial removal of several notum macrochaetae. The effect of stronger loss-of-function Egfr mutations has not been determined since these mutations drastically reduce the size of the imaginal wing discs and cause lethality. Moreover, clones of cells homozygous for amorphic or nearly amorphic Egfr mutations do not survive in the prospective notum. Consequently, these findings were reexamined using the temperature sensitive Egfr allelic combination. At a permissive temperature (18°C), three bristles (ASA, PSA and PPA) are often missing and the anterior postalar (APA) and anterior dorsocentral (ADC) are frequently duplicated. When late third instar larvae are placed at a non-permissive temperature (30°C) for 15 hours (pupation takes place during this interval) and complete development at 18°C, the presence of all notum macrochaetae is affected to different extents, excepting the scutellars and the APA, a bristle that is sometimes duplicated. Stronger phenotypes have been obtained by overexpressing a dominant negative form of Egfr (UAS-Egfr DN) with either the drivers sca-Gal4 (expressed in proneural clusters) or ap-Gal4 (expressed in the dorsal compartment of the disc). With ap-Gal4 at 29°C most notum macrochaetae are removed, although microchaetae are unaffected. UAS-aos, which encodes the Argos protein (an Egfr inhibitory ligand), driven in proneural clusters by C253-Gal4 suppresses both macro and microchaetae and only a few bristle sockets remain. These results suggest that Egfr signaling is essential for bristle development (Culí, 2001).

The data support a key role for Egfr signaling in the emergence of the notum macrochaetae SMCs from proneural clusters. Indeed, expression of the Egfr inhibitory ligand Aos exclusively in proneural clusters, a condition that permits essentially wild-type Sc accumulation in these clusters, almost completely suppresses the appearance of SMCs and SOs. SMC emergence is also impaired in discs from heat-treated temperature sensitive Egfr larvae and in clones of cells expressing UAS-rafDN2.1. Moreover, when the cells that accumulate RafDN2.1 occupy positions where SMCs normally appear, wild-type neighboring cells give rise to displaced SMCs. This phenomenon is reminiscent of and in accordance with the observation, made with mosaic individuals, that when the position of a dorsocentral bristle is in ac minus territory, this bristle does not develop, but a nearby ac plus cell can give rise to a dorsocentral bristle displaced from its wild-type position. The cell-autonomous effect of RafDN2.1 indicates that reception of the Egfr signal, mediated by the Ras/Raf/MAP kinase cassette, is essential for notum macrochaetae SMC determination. This was further substantiated by the cell autonomous induction of SMCs and bristles in clones of cells overexpressing a constitutively activated form of Ras. Taken together, these results indicate that reception of the Egfr signal promotes sc expression and SMC determination (Culí, 2001).

In the notum anlagen the expression of rho/ve occurs mainly in proneural clusters and this expression is dependent on ac-sc. Rho/ve facilitates the processing of Spitz, an activating ligand of Egfr. The soluble, active form of Spitz promotes ectopic sc expression and SMC emergence. Hence, these data suggest that, in proneural clusters, Ac-Sc promote expression of rho/ve, which by activating Spitz, would stimulate Egfr signaling in the cells of the cluster. (The Vein Egfr ligand probably does not specifically act in proneural clusters, because many of these lie outside of its expression domain). It is thus proposed that Egfr mediates a mutual positive signaling among cells of the proneural cluster, which promotes SMC emergence by probably reinforcing ac-sc expression. This positive signaling is called lateral cooperation. Evidently, this does not exclude an autocrine activation of the Egfr pathway in the cells that express AS-C proteins, but the lateral cooperation hypothesis is favored since it is well established in other systems that the Egfr pathway is used mainly for intercellular communication. This signaling should facilitate the acquisition of the SMC state by one or a few cells of a proneural cluster (Culí, 2001).

The SMC state is associated with greatly increased levels of proneural protein. These are accomplished by the self-stimulation of ac, sc and ase mediated by AS-C enhancers that activate these genes specifically in the cells that become SMCs. Since Ras1V12 elicits the expression of both sc and SRV-lacZ, it is proposed that, in the extant proneural clusters, the SMC-specific enhancers are targets of Egfr signaling. Unidentified effector(s) of the Egfr/Ras pathway should facilitate the self-stimulation of the proneural genes mediated by the SMC-specific enhancers by, possibly, binding to these enhancers. Conclusive evidence in support of this role requires the identification of the signaling effector(s) and of their interaction with the enhancer. Interestingly, overexpression of the effector Pointed P1 promotes development of many extra macrochaetae on the notum and putative Ets-domain binding sites have been identified in the sc and ase SMC enhancers (GTGGAAAT and ACGGAAAC, respectively) (Culí, 2001).

Egfr-mediated lateral cooperation should tend to activate the SMC-specific enhancers in many cells of the proneural clusters. This, however, is prevented by N signaling, which is activated by Ac and Sc in the cells of the cluster. This signaling, by means of the bHLH proteins of the E(spl)-C, blocks the ac-sc-ase self-stimulatory loop promoted by the SMC-specific enhancers. However, within a proneural cluster the cells of the proneural field accumulate greater amounts of Ac-Sc proteins. As it has been hypothesized that cells that signal the most are the least inhibited by their neighbors, eventually, a cell of the proneural field will be released from the inhibitory loop and its levels of E(spl)-C bHLH protein will become minimal. This cell will turn on the ac-sc-ase self-stimulation and become an SMC. The SMC signals maximally to its neighbors and prevents them from following the same fate (lateral inhibition). These results add to this scenario the requirement for Egfr-mediated signaling for one cell of the proneural field to turn on the ac-sc-ase self-stimulatory loops and become an SMC. According to this model, Ac-Sc activate both the N-and Egfr-mediated signaling pathways, with their SMC-suppressing and SMC-promoting abilities, respectively, and both signaling systems appear to act on the same SMC-specific enhancers. Since an excess signaling by the N or the Egfr pathway will either prevent SMC determination or promote emergence of ectopic SMCs, the respective levels of signaling should balance each other so that only one SMC is determined at a time from each proneural cluster. How is this balance accomplished? This is at present unclear. The large enhancement of rho/ve mRNA in proneural clusters under conditions of insufficient N signaling suggests that this pathway may prevent the Rho/Ve-promoted activation of Egfr from rising to excessively high levels. In contrast, the insensitivity of the levels of E(spl)-m8 protein to the overexpression of UAS-aos in proneural clusters suggests that the Egfr pathway does not affect N signaling. Antagonistic interactions between the N and the Egfr pathways are found in other developing systems, as in the wing preveins and in the reiterative recruitment, from a long-lived atonal proneural cluster, of the precursors of the 70-80 scolopidia of the femoral chordotonal organs. In this later case, Egfr signaling promotes commitment of neural precursors and the Dl-N interaction prevents too many cells from being committed (Culí, 2001).

In the presumptive notum, the inability of available antibodies to reliably detect dp-ERK and, in proneural clusters, the low levels of rho/ve mRNA (compared to those in the wing preveins) suggest that low levels of Egfr activity are sufficient to ensure the emergence of the macrochaetae precursor cells. This may explain the failure of the Egfr hypomorphic alleles -- compatible with cell or adult viability -- to completely eliminate notum macrochaetae. The notum microchaetae appear to be even more resistant to the lowering of Egfr signaling. Perhaps, they do not directly require it for development, similarly to the terguite bristles that can arise within Egfr amorphic clones. An essential difference between notum macrochaetae, in contrast to notum microchaetae and terguite bristles, is that the first appear in fixed positions while the others do not do so, being instead organized in density patterns. It is speculated that Egfr signaling among the cells of the proneural field may make the selection of the SMC less ambiguous and, therefore, spatially more precise. A cell centrally located within this subset would receive the strongest signaling from its neighbors and would become a SMC in preference to more marginally located neighbors. The observation that slight reduction in the level of Egfr signaling causes duplications of some notum macrochaetae, that is, it makes the decision of which cell becomes an SMC less precise and it allows two SMCs to arise from presumably the same proneural cluster, may be consistent with this interpretation (Culí, 2001).

The overexpression of UAS-aos in proneural clusters removes essentially all bristles, including those of the tergites. This may indicate that all SOs require some level of Egfr signaling to develop. However, the fact that tergite clones homozygous for amorphic Egfr minus alleles still develop bristles suggests that the Aos overexpression may be interferring with additional tyrosine kinase receptors that would be redundant with Egfr in the development of these bristles (Culí, 2001).

Role of EGF receptor pathway in the growth and patterning of the Drosophila wing through the regulation of vestigial

Growth and patterning of the Drosophila wing disc depends on the coordinated expression of the key regulatory gene vestigial both in the dorsal-ventral (DV) boundary cells and in the wing pouch. It is proposed that a short-range signal originating from the core of the DV boundary cells is responsible for activating Egfr in a zone of organizing cells on the edges of the DV boundary. Using loss-of-function mutations and ectopic expression studies, it has been shown that Egfr signaling is essential for vestigial transcription in these cells and for making them competent to undergo subsequent vestigial-mediated proliferation within the wing pouch (Nagaraj, 1999).

Third instar wing discs stained with an antibody directed against the N-terminal portion of the Spitz protein show a strong expression of Spi along the DV boundary and weaker expression throughout the disc. This elevated protein level at the DV boundary is likely to reflect post-transcriptional control, since work from several laboratories has shown that SPI mRNA is expressed ubiquitously at low levels throughout the wing disc. To determine if the growth promoting activity of the Notch pathway is mediated by Egfr, the dpp-Gal4 driver system was used to ectopically express an activated, secreted form of Spi (sSpi) that would activate Egfr along this boundary. This results in an extensive outgrowth of the wing pouch. When these discs are stained with an a-Vg antibody, the overproliferating cells are found to express Vg. These results can either imply that Vg expression is activated by the Egfr pathway leading to cell proliferation or that Egfr activation results in random proliferation of cells within the pouch, which then secondarily express the Vg protein. To distinguish between these possibilities, the vg 1 mutant allele in which Vg expression is reduced but not eliminated, was used. In dpp-Gal4/UAS-sspi;vg 1/vg 1 flies there is no expansion of the wing pouch. It is concluded that activation of EGFR leads to expression of Vg, which functions downstream of or parallel to the Egfr pathway for the proper proliferation of cells in the pouch (Nagaraj, 1999).

To address the early role of Egfr in wing patterning, four alternative strategies involving loss-of-function mutations in components of the Egfr pathway were used. First, a temperature-sensitive allele of the Egfr gene (EGFR ts) was used to inactivate the pathway. The heteroallelic combination EGFR ts /EGFR top1 gives rise to a null phenotype at the non-permissive temperature and shows decreased Vg expression in the wing pouch. The expression of Vg in the folds outside the pouch region is unaffected and is therefore not responsive to Egfr signaling. In these discs, the notum is significantly reduced in size, consistent with the fact that the alternative Egfr ligand, Vein, is expressed in the third instar notum. As a second approach, a dominant negative version of Egfr (EGFR DN) was expressed using the UAS/Gal4 system. Beginning in early third instar stages, the A9-Gal4 element causes expression of a reporter gene mostly in the dorsal compartment of the wing pouch and at lower levels in the ventral compartment. A9-Gal4; UAS-EGFR DN wing discs show a dramatic reduction in the dorsal compartment of the wing pouch. This is not a secondary consequence of a perturbation in DV boundary specification since the expression pattern of Wg along the DV boundary is maintained. Rather this effect is mediated through the control of vg, since the expression of boundary enhancers and quadrant enhancers are dramatically reduced. As a third approach to attenuate Egfr signaling during development, a hypomorphic allele of pointed (pnt) was used. This allele encodes an ETS domain transcription factor that functions as a downstream member of the Egfr pathway. pnt mutant wing discs show reduced pouch size when compared with wild type, again supporting the conclusion from previous experiments that the Egfr pathway is necessary for the growth of the wing pouch. Finally, a loss of function in genes belonging to the Egfr pathway interact genetically with vg. Egfr signaling is shown to activate the boundary enhancer and represses the quadrant enhancer of vg (Nagaraj, 1999).

Unlike activation of Notch and Egfr, activation of the Wg pathway using similar experimental paradigms does not induce non-cell-autonomous proliferation in the wing pouch cells. A possible synergistic interaction between Wg and Egfr pathway in the control of Vg expression has not been ruled out, but unlike Egfr activation, Wg on its own is not able to induce high enough levels of Vg expression to cause cell proliferation. Wg does, however, have several important functions in the patterning of the wing. These include distinguishing the identity of the pouch cells from those of the notum; specifying the bristles along the anterior margin, and refining the DV boundary (Nagaraj, 1999 and references).

Activation of the Egfr pathway in cells adjacent to the DV boundary leads to the localized activation of MAPK in thin strips of cells flanking the DV boundary. These regions of MAPK activation are termed the competence zone (CZ). The activation of MAPK in this region is also dependent on a functional Notch signal at the DV boundary. The fact that Egfr signaling is operative in this zone is also supported by the earlier finding that argos and rhomboid are also expressed in this region. Also consistent with the hypothesis that Notch signal is essential for the activation of the Egfr in the CZ region, it has been reported that loss of Notch results in the loss of rho expression along the DV boundary even as the expression of rho in the vein regions is greatly expanded upon loss of the Notch signal. Localized Rhomboid expression has been implicated in Egfr signaling and could therefore account for the localized induction of Egfr activation at the DV boundary. Most importantly, these results show that a localized inactivation of the Egfr signal exclusively at the DV boundary results in dramatic loss of Vg in the remainder of the pouch. Thus, localized activation of the Ras pathway in cells flanking the DV boundary is important for the patterning of the entire pouch. Previous work has suggested that loss of Notch function at the DV boundary has a non-cell-autonomous effect on the expression of Vg in the pouch and the proliferation of cells in the rest of the pouch region. These results suggest that this effect is mediated through the Egfr pathway. It is hypothesized that high levels of Egfr signaling are required in these cells in order to provide them with competence to express Vg and therefore to proliferate (Nagaraj, 1999).

EGFR signalling inhibits Capicua-dependent repression during specification of Drosophila wing veins

Localized activation of the Ras/Raf pathway by epidermal growth factor receptor (Egfr) signalling specifies the formation of veins in the Drosophila wing. However, little is known about how the Egfr signal regulates transcriptional responses during the vein/intervein cell fate decision. Evidence is provided that Egfr signaling induces expression of vein-specific genes by inhibiting the Capicua (Cic) HMG-box repressor, a known regulator of embryonic body patterning. Lack of Cic function causes ectopic expression of Egfr targets such as argos, ventral veinless and decapentaplegic and leads to formation of extra vein tissue. In vein cells, Egfr signaling downregulates Cic protein levels in the nucleus and relieves repression of vein-specific genes, whereas intervein cells maintain high levels of Cic throughout larval and pupal development, repressing the expression of vein-specific genes and allowing intervein differentiation. However, regulation of some Egfr targets such as rhomboid appears not to be under direct control of Cic, suggesting that Egfr signaling branches out in the nucleus and controls different targets via distinct mediator factors. These results support the idea that localized inactivation of transcriptional repressors such as Cic is a rather general mechanism for regulation of target gene expression by the Ras/Raf pathway (Roch, 2002).

There are two key aspects of Cic function as a developmental regulator: its ability to repress specific target genes in defined territories, and its inhibition by the Ras/Raf pathway to allow expression of those targets in complementary positions. In the blastoderm embryo, Cic is required for development of trunk body regions and represses genes mediating differentiation of terminal structures. Torso RTK activation at each pole of the embryo alleviates Cic-dependent repression and initiates the terminal gene expression program. A similar model is proposed for cic function during specification of vein versus intervein fate in the wing. Loss of cic function in the wing causes formation of ectopic vein tissue, implying that Cic mediates intervein specification by restricting vein formation to appropriate regions. In intervein territories, Cic behaves as a repressor of vein-specific genes such as argos and vvl but does not seem to affect directly the expression of blistered, which is required for the specification of intervein fates. Finally, Egfr signaling leads to downregulation of Cic protein levels in vein nuclei, thus relieving Cic-mediated repression and promoting vein development (Roch, 2002).

Nevertheless, several data suggest a more complex regulation of vein specification compared to terminal patterning: (1) it has been shown that expression of rho, a positive target of Egfr signaling in the wing and other tissues, is not affected by cic during third larval instar and early pupariation. This suggests that Egfr signaling can mediate activation of some targets in the wing disc by mechanisms other than Cic inhibition. (2) Similarly, the Egfr pathway has been shown to repress bistered expression in presumptive vein cells, a process that is independent of Cic. These results imply that different transcription factors act downstream of the Egfr cascade to direct changes in gene expression during patterning of wing veins. Indeed, recent results indicate that Egfr signaling activates certain target genes via direct phosphorylation of Fos protein (Roch, 2002).

Moreover, vein differentiation is not a mere result of Egfr activation but depends on other signals such as Dpp and Notch, and on the distribution of additional transcription factors that contribute to wing patterning. For example, the Collier/Knot nuclear factor has been shown to induce high levels of Bs expression between veins L3 and L4, promoting intervein development in this region. All these inputs are linked in a complex circuit of intercellular signaling and gene regulation that progressively refines vein determination during late larval and pupal development. This signaling network could provide an explanation for the observed non-autonomy of cic phenotypes during vein specification. Thus, although cic represses aos expression in a cell-autonomous manner, this and other cic targets are likely to participate in signaling mechanisms that affect adjacent cells. Consistent with this idea, it has been found that cic mutant cells express ectopic Dpp product, a diffusible factor that promotes vein differentiation (Roch, 2002).

Subdivision of the Drosophila wing imaginal disc by EGFR-mediated signaling

Growth and patterning of the Drosophila wing imaginal disc depends on its subdivision into dorsoventral (DV) compartments and limb (wing) and body wall (notum) primordia. Evidence is presented that both the DV and wing-notum subdivisions are specified by activation of the Drosophila Epidermal growth factor receptor (Egfr). Egfr signaling is necessary and sufficient to activate apterous (ap) expression, thereby segregating the wing disc into D (ap-ON) and V (ap-OFF) compartments. Similarly, Egfr signaling directs the expression of Iroquois Complex (Iro-C) genes in prospective notum cells, rendering them distinct from, and immiscible with, neighboring wing cells. However, Egfr signaling acts only early in development to heritably activate ap, whereas it is required persistently during subsequent development to maintain Iro-C gene expression. Hence, as the disc grows, the DV compartment boundary can shift ventrally, beyond the range of the instructive Egfr signal(s), in contrast to the notum-wing boundary, which continues to be defined by Egfr input (Zecca, 2002a).

The subdivision of the wing imaginal disc into AP and DV compartments, as well as prospective body wall (notum) and limb (wing) territories is marked by the expression of particular regulatory genes, such as the selector gene engrailed (en) in the P compartment, the selector gene apterous (ap) in the D compartment, and the genes of the Iroquois Complex (Iro-C) [mirror (mirr), auracan (ara) and caupolican (caup)] in the lateral notum. In mature third instar wing discs, the Iro-C genes are expressed not only within the prospective lateral notum, but in additional locations, including a thin stripe of cells that extends ventrally along the edge of the disc, as well as in specific subpopulations of cells in the prospective wing blade. This study addresses the role of Egfr signaling in controlling notum development and Iro-C gene expression therein, and then focuses on the role of Egfr signaling in inducing ap expression and establishing the DV compartments (Zecca, 2002a).

To assess the requirement for signals transduced by the Egfr during normal wing disc development, the behavior was examined of clones of cells that are homozygous for null or temperature-sensitive mutations of the Egfr gene (referred to subsequently as Egfr- or Egfrts), or for a loss of function mutation of the ras gene (ras-), which encodes the Ras GTPase, a conserved downstream effector of the Egfr signal transduction pathway. Clones of mutant cells were generated during different stages of larval development and their size, shape and distribution assayed in each of the four distinct primordia that make up the mature wing disc: the prospective wing blade, wing hinge, lateral notum and medial notum. In general, loss of Egfr activity caused more penetrant and severe effects than the loss of Ras activity, possibly reflecting a shorter perdurance of Egfr function relative to that of Ras following loss of the wild-type gene, or a restricted requirement for Ras in mediating some, but not all, downstream outputs of Egfr activation. ras- clones, in particular, were more viable than Egfr mutant clones, allowing use of the twin spot method of clonal analysis and allowing the generation of mutant clones of large size using the Minute technique. However, aside from this difference, the effects of Egfr and ras mutant clones on Iro-C gene expression were the same. In these experiments mutant clones were marked either by the presence or absence of the reporter proteins GFP or CD2 (Zecca, 2002a). Egfr- clones induced in the wing disc during the first and second instars do not survive to the late third instar, apparently because of defects in cell proliferation and/or viability. To increase the likelihood that mutant clones might survive, the Minute technique was used to give Egfr- cells a growth advantage relative to surrounding Egfr+ cells. Under these circumstances, Egfr- clones induced during the first or early second instar contribute only to the prospective wing blade, whereas clones induced during the late second or early third instar could also populate the prospective wing hinge and medial notum domains. However, Egfr- clones were invariably excluded from the prospective lateral notum. Similar results were obtained for clones of cells homozygous for the Egfrts mutation, which reduces but does not eliminate Egfr activity at the non-permissive temperature (30-31°C), except that the clones tended to be larger than their Egfr- counterparts. Egfrts clones induced after the mid-second instar could also contribute to the prospective lateral notum, albeit rarely. However, these clones were abnormally round in shape, suggesting they developed abnormally (Zecca, 2002a).

Unlike Egfr- clones, ras- clones induced during the first or second larval instar can survive without the benefit of the Minute technique. Under these conditions, mitotic recombination generates 'twin spots' composed of genetically marked ras- and ras+ sister clones, which descend from the same mother cell. Twin spots could be recovered in the prospective wing blade domain, wing hinge and medial notum domains. However, only single ras+ spots were generally observed in the prospective lateral notum domain, indicating that their ras- sister spots failed to survive in this domain; the few ras- sister spots obtained in this domain appeared abnormal. Similar results were obtained when ras- cells were generated during the first larval instar using the Minute technique. Such ras- clones could form large, and apparently normal, regions of the prospective wing blade and wing hinge. Nevertheless, they appeared to be excluded from the presumptive notum territory. Strikingly, some of the discs obtained under these conditions appeared to lack most or all prospective notal tissue and to consist predominantly of prospective wing blade and hinge tissue (Zecca, 2002a).

In summary, Egfr-, Egfrts and ras- clones can contribute to the prospective wing blade, wing hinge and medial notum. However, all three classes of mutant clones generally failed to populate the prospective lateral notum, indicating that Egfr signaling is essential for the normal development of this region of the wing disc (Zecca, 2002a).

Prospective notum cells are distinguished from wing cells by the activity of the Iroquois Complex (Iro-C) genes. These results demonstrate (1) that activation of Egfr/Ras pathway is both necessary and sufficient to drive Iro-C gene expression in wing disc cells, and (2) that wing disc cells persistently monitor their level of Egfr/Ras input and are allocated to the wing or notum primordium on an ongoing basis, depending on the level of Egfr/Ras input they receive. This means that the wing-notum subdivision is not a stable compartmental partition between differently committed cell types, but rather a labile demarcation that reflects the current distribution of an instructive Egfr ligand (Zecca, 2002a).

Despite the provisional nature of the wing-notum segregation, the boundary between the two primordia is relatively straight and sharp. By manipulating Egfr/Ras signaling, it was shown that presumptive notum cells that lose the capacity to maintain Iro-C gene expression sort out of the notum primordium. Conversely, presumptive wing cells that ectopically activate the Iro-C genes sort out of the wing primordium. Similar results have been obtained by altering Iro-C gene function directly, rather than through the manipulation of Egfr/Ras signaling. Taken together, these results suggest that Iro-C gene activity, under Egfr control, programs prospective notum cells to have a different affinity from prospective wing cells, thereby straightening and sharpening the boundary between the two primordia. Further support for such a mechanism comes from experiments in which clones of cells were generated that ectopically express an activated form of Spi, an Egfr ligand, in the prospective wing hinge. All of the cells within these clones express the Iro-C genes and interdigitate freely with neighboring wild-type cells that are also induced to express the Iro-C genes. However, cells located further away do not receive sufficient Spi to activate Iro-C gene expression and these form a smooth boundary encircling the ectopic Iro-C-expressing cells (Zecca, 2002a).

The subdivision of the wing disc into wing and notum primordia resembles that of several other non-compartmental partitioning events that are correlated with the activation of other 'selector-like' genes such as pnr, tsh, hth, vg, Dll, dac and ey. In most cases, the selector-like gene is expressed, or upregulated, in a relatively well-defined domain in response to known extracellular signals, such as Wingless (Wg) and Decapentaplegic (Dpp), and in some cases (e.g., Dll in the leg disc and pnr in the notum), the activity of the selector-like gene is known to regulate cell affinity. Thus, the wing-notum segregation may reflect a general mechanism for maintaining discrete regional primordia based on cell position rather than on cell ancestry (Zecca, 2002a).

The notum primordium, once established by the activation of Iro-C gene expression, is itself subdivided into distinct lateral and medial primordia by the localized activity of the pnr gene. pnr encodes a transcription factor that represses Iro-C gene expression and specifies medial as opposed to lateral notum differentiation. pnr activity also causes medial cells to adopt a distinct affinity that prevents them from mixing with lateral cells. It is tempting to speculate that pnr expression, like that of the Iro-C genes, is governed by Egfr signaling, e.g., being activated at a higher threshold concentration than the Iro-C genes, and hence in a smaller, more dorsally restricted domain. However, it was found that cells do not require peak levels of Egfr/Ras activity to remain and develop normally within the medial primordium. Conversely, enhanced activation of the Egfr/Ras pathway does not appear to cause lateral cells to sort into the medial primordium or adopt medial characteristics (e.g., the loss of Iro-C gene expression). Instead, it seems that pnr expression and subdivision of the notum into medial and lateral domains may depend on other signals, such as Dpp (Zecca, 2002a).

The relationship between pnr and the Iro-C gene expression in the notum is conserved in corresponding dorsolateral and dorsomedial regions of most of the adult segments, as well as in the embryonic and larval ectoderm. Hence, it has been proposed that the deployment of these genes reflects a fundamental partitioning process reiterated in most or all body segments. However, there are significant differences in the way that the Iro-C genes are deployed in the wing disc compared with the eye-antenna disc, the only other context in which an equivalent analysis has been performed. (1) During eye development, Iro-C gene expression is not governed by persistent signaling, in contrast to the wing disc. Instead, these genes are heritably activated early in eye development and behave as classical selector genes, performing a role that corresponds in most respects to that of ap in the wing. (2) Iro-C gene expression is activated in the eye disc by Hedgehog and Wingless signaling, rather than by Egfr signaling. Thus, it appears that the Iro-C genes are activated by different signals and govern different types of partitioning events in these two contexts, raising the possibility that their deployment in other segments, and at other stages, may reflect similarly diverse inputs and developmental roles (Zecca, 2002a).

As in the case of the Iro-C genes, Egfr/Ras signaling is both necessary and sufficient to activate ap expression in early wing disc cells. Furthermore, evidence is provided that each wing disc cell chooses to express, or not to express, ap at this time, depending on its level of Egfr/Ras activation. However, in contrast to the Iro-C genes, the descendents of each cell then inherit this initial choice without further reference to Egfr/Ras signaling. The results of eliminating Egfr/Ras activity before the establishment of the DV compartments are particularly striking. Early loss of Egfr activity causes dorsally positioned cells within the disc to choose, incorrectly, to become V compartment founders. These cells and their descendents generally sort into the existing V compartment or out of the disc epithelium. In rare cases, they can form an ectopic V compartment within the D compartment. By contrast, later loss of Egfr activity has no effect on the DV compartmental segregation. These findings establish that Egfr signaling is responsible for establishing the D and V compartments through the heritable activation of ap (Zecca, 2002a).

Although the Iro-C and ap genes are activated in overlapping dorsoproximal sectors of the early wing disc, the domain of ap expression expands relative to that of Iro-C gene expression during subsequent development, causing the DV boundary to be positioned up to 30 cell diameters ventral to the notum-wing boundary. It is suggested that this shift occurs because ap-expressing cells no longer depend on Egfr/Ras input to continue to express ap. Hence, as ap-expressing cells within the notum primordium proliferate, some will move out of range of the instructive Egfr ligand, cease to express Iro-C genes and enter the wing primordium. In the accompanying paper (Zecca and Struhl, 2002b), evidence is provided that this shift must occur in order for D and V compartment cells to interact to induce Wg and stimulate wing growth and differentiation (Zecca, 2002a).

These results raise intriguing questions about the mechanism of ap activation. For example, Egfr signaling induces ap expression only during a discrete window of opportunity during the second larval instar, even though Egfr signaling both precedes the initial activation of ap and continues thereafter. What makes the ap gene responsive to Egfr signaling only during this early window of opportunity? In addition, the state of ap gene expression during this period, whether 'on' or 'off', is inherited for the remainder of development. How are both states of expression rendered heritable? It is possible that a temporal signal, such as a flux of a unique combination of hormones (for example, ecdysone and juvenile hormone) or the unique prior history of signaling events in the early wing disc, might prime the ap locus for activation by Egfr signaling during this period. The state of expression chosen during this period might then be maintained subsequently by mechanisms involving positive autoregulation (for the 'on' state) or heritable silencing mediated by the Polycomb Group proteins (for the 'off' state). However, there is little evidence at present to support these speculations and the actual mechanisms remain unknown (Zecca, 2002a).

Control of growth and patterning of the Drosophila wing imaginal disc by EGFR-mediated signaling

The subdivision of the Drosophila wing imaginal disc into dorsoventral (DV) compartments and limb-body wall (wing-notum) primordia depends on Epidermal growth factor receptor (Egfr) signaling, which heritably activates apterous (ap) in D compartment cells and maintains Iroquois Complex (Iro-C) gene expression in prospective notum cells. The source, identity and mode of action of the Egfr ligand(s) that specify these subdivisions has been examined. Of the three known ligands for the Drosophila Egfr, only Vein (Vn), but not Spitz or Gurken, is required for wing disc development. Vn activity is required specifically in the dorsoproximal region of the wing disc for ap and Iro-C gene expression. However, ectopic expression of Vn in other locations does not reorganize ap or Iro-C gene expression. Hence, Vn appears to play a permissive rather than an instructive role in organizing the DV and wing-notum segregations, implying the existance of other localized factors that control where Vn-Egfr signaling is effective. After ap is heritably activated, the level of Egfr activity declines in D compartment cells as they proliferate and move ventrally, away from the source of the instructive ligand. Evidence is presented that this reduction is necessary for D and V compartment cells to interact along the compartment boundary to induce signals, like Wingless (Wg), which organize the subsequent growth and differentiation of the wing primordium (Zecca, 2002b).

Early induced clones that express Egfrlambda, the constitutively active form of the Egfr, can induce the formation of ectopic D compartments that retain organizer activity. However, the level of constitutive Egfr/Ras activity in such Egfrlambda-expressing clones appears to be significantly lower than in clones of RasV12-expressing cells. Consistent with this, it is found that ectopic expression of Egfrlambda considerably reduces but does not completely eliminate vg expression. Hence, it is inferred that the levels of Ras activation in Egfrlambda-expressing cells are not sufficiently high to prevent productive interactions between D and V compartment cells, thus allowing the ectopic DV compartment boundary to acquire organizer activity (Zecca, 2002b).

How might Egfr signaling regulate the capacity of the DV compartment boundary to function as an organizer? One possibility is that high levels of Egfr/Ras activity block the ability of cells to transduce Notch signals. During normal development, D and V cells engage in a positive auto-feedback loop of Delta/Notch and Serrate/Notch signaling that drives the reciprocal induction of Wg and Vg expression on both sides of the DV compartment boundary. Hence, if high levels of Egfr/Ras activity block Notch signal transduction, then persistent high levels of Ras activity on even one side of the DV boundary would suffice to disrupt the feedback loop and block the reciprocal induction of Wg and other 'boundary' genes. Accordingly, the DV boundary might have to be located in a region of low Egfr activity in order to allow reciprocal Notch signaling to induce the expression of these, and perhaps other, organizer genes (Zecca, 2002b).

Another possibility is that the apON-apOFF interface may only be able to function as an organizer when cells on both sides are of prospective wing type. Prior to the initial activation of ap and the Iro-C genes, the nascent wing disc appears to be subdivided into mutually antagonistic domains of Egfr and Wg signaling that at least transiently define the incipient notum and wing primordia. Because ap and the Iro-C genes are initially activated in response to a common source of Egfr signaling, most or all D cells at this stage may be notum type. It is only later, when ventrally situated D cells move out of range of Vn-dependent Egfr signaling and switch to being wing type, that inductive interactions occur across the DV boundary to create a new and stable source of Wg signaling. It is suggested that cells on both sides of the DV boundary may have to be of wing type for the boundary to have organizer activity. One possible reason for why this might be the case is that vg, the selector-like gene that defines the wing state, is itself an integral component of the reciprocal signaling mechanism that allows D and V cells to induce the expression of DV boundary genes. High levels of Egfr/Ras signaling actively maintain Iro-C gene expression (and hence the notum state) and block vg expression. Hence, the DV boundary may normally have to shift ventrally, into a domain of low Egfr/Ras signaling and high Wg signaling that defines the incipient wing state, to allow the positive feedback loop of inductive signaling to initiate across the DV compartment boundary. Once this loop is established, it would provide a stable source of Wg and other signals generated along the DV boundary that govern the subsequent growth and differentiation of the wing blade (Zecca, 2002b).

EGFR targets al and C15 in the leg disc

The distal region of the Drosophila leg, the tarsus, is divided into five segments (ta I-V) and terminates in the pretarsus, which is characterized by a pair of claws. Several homeobox genes are expressed in distinct regions of the tarsus, including aristaless (al) and lim1 in the pretarsus, Bar (B) in ta IV and V, and apterous (ap) in ta IV. This pattern is governed by regulatory interactions between these genes; for example, Al and Bar are mutually antagonistic, resulting in exclusion of Bar expression from the pretarsus. Although Al is necessary, it is not sufficient to repress Bar, indicating another factor is required. This factor has been identified as the product of the C15 gene, also termed clawless, a homeodomain protein that is a homolog of the human Hox11 oncogene. C15 is expressed in the same cells as al -- together, C15 and Al appear to directly repress Bar and possibly to activate Lim1. C15/Al also act indirectly to repress ap in ta V, i.e., in surrounding cells. To do this, C15/Al autonomously repress expression of the gene encoding the Notch ligand Delta (Dl) in the pretarsus, restricting Dl to ta V and creating a Dl+/Dl− border at the interface between ta V and the pretarsus. This results in upregulation of Notch signaling, which induces expression of the bowl gene, the product of which represses ap. Similar to aristaless, the maximal expression of C15 requires Lim1 and its co-factor, Chip. Bar attenuates aristaless and C15 expression through Lim1 repression. Aristaless and C15 proteins form a complex capable of binding to specific DNA targets, which cannot be well recognized solely by Aristaless or Clawless (Campbell, 2005; Kojima, 2005).

To determine if C15 lies downstream of Al or vice versa, their expression was examined in discs from the reciprocal mutant. Each was still expressed, but its expression domain was significantly reduced. In contrast, Lim1 expression is lost completely in both C15 and al mutant discs. In addition, although there is some variation, the expression domains of C15 and Al are only mildly reduced in lim1 mutants (Campbell, 2005).

If C15 is not downstream of the other homeobox genes expressed in the center of the disc, it must be activated by another mechanism. al expression is induced by EGFR signaling, raising the possibility that C15 may also be under EGFR control. This was confirmed by loss and gain of function experiments, as follows: (1) C15 expression was lost in discs from an Egfrts mutant grown at the restrictive temperature (29.1°C) at which al expression is lost; (2) misexpression of a constitutively active form of the EGFR (UAS-Egfr.lambdatop) results in ectopic expression of C15; similar to other EGFR targets, this ectopic expression is restricted to the ventral region (Campbell, 2005).

Drosophila pico and its mammalian ortholog lamellipodin activate serum response factor and promote cell proliferation

MIG-10/RIAM/lamellipodin (MRL) proteins link activated Ras-GTPases with actin regulatory Ena/VASP proteins to induce local changes in cytoskeletal dynamics and cell motility. MRL proteins alter monomeric (G):filamentous (F) actin ratios, but the impact of these changes had not been fully appreciated. This study reports here that the Drosophila MRL ortholog, pico, is required for tissue and organismal growth. Reduction in pico levels resulted in reduced cell division rates, growth retardation, increased G:F actin ratios and lethality. Conversely, pico overexpression reduced G:F actin ratios and promoted tissue overgrowth in an epidermal growth factor (EGF) receptor (EGFR)-dependent manner. Consistently, in HeLa cells, lamellipodin was required for EGF-induced proliferation. pico and lamellipodin share the ability to activate serum response factor (SRF), a transcription factor that responds to reduced G:F-actin ratios via its co-factor Mal. Genetics data indicate that mal/SRF levels are important for pico-mediated tissue growth. It is proposed that MRL proteins link EGFR activation to mitogenic SRF signaling via changes in actin dynamics (Lyulcheva, 2008).

The construction of properly sized and functional tissues and organs during animal development requires tight control of cell growth, proliferation, differentiation, and death. Networks of intracellular signal transduction pathways that respond to various secreted ligands and cell surface proteins coordinate these processes. Elucidating the nature of the intracellular signaling networks that connect extracellular stimuli to basic cellular machinery controlling proliferation, growth, and morphology is not only critical for the understanding of tissue size regulation during normal development, but is also important for the identification of aberrant events underlying numerous disease processes, including cancer (Lyulcheva, 2008).

A number of pathways regulating cellular development are initiated by ligation of transmembrane receptor tyrosine kinases (RTKs), such as the epidermal growth factor (EGF) receptor (EGFR). One of the key mediators of RTK signaling is the Ras GTPase, capable of activating proteins harboring Ras association (RA) domains to initiate downstream signaling pathways, such as the mitogen-activated protein kinase (MAPK) cascade, and ultimately resulting in changes in gene transcription. The Ras/MAPK and other canonical RTK signaling pathways have been well characterized, yet they cannot account for all of the observed effects of their respective extracellular signals (Lyulcheva, 2008).

The MIG-10/Rap1-GTP-interacting adaptor molecule (RIAM)/lamellipodin (Lpd) (MRL) proteins are a family of recently identified molecular adaptors, harboring an RA, pleckstrin homology (PH), and several proline-rich domains (Krause, 2004; Lafuente, 2004). Several lines of evidence indicate that MRL proteins act downstream of Ras-like GTPases and transduce extracellular signals to changes in the actin cytoskeleton, cell motility, and adhesion. In particular, Lpd interacts with active Ras and RIAM with active Rap1. Consistent with this, only RIAM is required for Rap1-induced cell adhesion. Lpd also binds to PI(3,4)P2 via its PH domain, which is sufficient for membrane targeting after platelet-derived growth factor stimulation. Both Lpd and RIAM utilize their proline-rich motifs to directly interact with the Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) actin regulators, known to regulate lamellipodia formation and cell migration. In addition, Lpd knockdown impairs lamellipodia formation, whereas Lpd overexpression increases speed of lamellipodia protrusion in an Ena/VASP-dependent manner. Finally, both Lpd and RIAM have been shown to alter the cellular ratio between monomeric (G) and filamentous (F) actin, suggesting a wider role in regulating cell metabolism. Indeed, control of the G:F actin ratio is an essential way for cells to regulate gene transcription via the transcription factor serum response factor (SRF), and has been linked to changes in proliferation, migration, and differentiation (Lyulcheva, 2008).

This study reports the characterization of the Drosophila MRL ortholog, which has been named pico on the basis of the retarded growth phenotype resulting from pico knockdown or loss-of-function mutant. Reduction in pico levels results in reduced rates of cell growth and proliferation, whereas ectopic expression of pico promotes coordinated cell growth and proliferation, leading to tissue overgrowth. pico's effect on cell proliferation is conserved in its mammalian ortholog, Lpd. Evidence is presented that pico and Lpd link extracellular signaling to tissue growth via changes in actin dynamics and SRF activation. This is the first time that MRL proteins have been implicated in controlling cell proliferation and tissue growth (Lyulcheva, 2008).

Phylogenetic analysis has shown that pico (CG11940) encodes the only member of the MRL family of proteins in Drosophila. Two transcripts that were identified are generated from alternative transcription start sites of the pico transcription unit: pico and pico-L. pico-L encodes a 1159 amino acid protein that is identical to the protein encoded by pico, except for the presence of an additional 128 N-terminal residues. Both pico proteins contain RA and PH domains and proline-rich Ena/VASP binding sites characteristic of the MRL proteins (Lyulcheva, 2008).

This study shows that pico, which encodes the only Drosophila member of the MRL family of proteins, and its mammalian ortholog, Lpd, have a conserved role in the regulation of cellular proliferation. Reduced pico or Lpd levels result in reduced rates of cellular proliferation, but do not impair cell survival. Too much pico promotes coordinated growth and proliferation, leading to larger tissues with more normal-sized cells. In this respect, the effect of pico is distinct from that of many known Drosophila growth drivers. Growth regulators, such as Drosophila S6K, cause cells to accumulate mass faster than they can divide, primarily due to effects on translation, leading to cellular hypertrophy. Other regulators, such as E2F, can drive cell division without stimulating cell growth, leading to hyperplastic cellular hypotrophy and/or apoptosis (Lyulcheva, 2008).

Attenuating EGFR signaling abrogates the effect of ectopic pico on both F-actin accumulation and tissue growth. pico acts cell autonomously and is therefore unlikely to act upstream of Egfr by affecting the level of EGFR ligands. To rule out that pico regulates levels of EGFR, receptor levels and distribution was examined in wing imaginal discs overexpressing pico or picoIR. EGFR levels and distribution in these genetic backgrounds resembled wild-type. Another possibility is that pico regulates EGFR activity. Although suitable reagents were not available to directly monitor EGFR activity levels in wing discs, effects on extracellular signal-regulated kinase (ERK) activation, which provides a molecular readout for EGFR/Ras/Raf signaling, were measured. Diphosphorylated (dp) ERK levels were not affected by ectopic pico. These data suggest that, rather than being upstream of EGFR, Pico needs to be activated by EGFR or a downstream component of EGFR signaling, such as activated Ras. Consistently, both Lpd and Pico bind to activated, but not wild-type, Ras. Furthermore, pico knockdown partially suppresses the effects of ectopic Egfr and activated Ras; in addition, Lpd knockdown impairs the EGF-induced increase in proliferation. Taken together, these data suggest that pico and Lpd are downstream effectors of EGFR (Lyulcheva, 2008).

Ena/VASP has been reported to act downstream of MRL proteins. Correspondingly, it was found that pico-mediated wing overgrowth and F-actin accumulation are sensitive to the levels of ena. Importantly, ena is also sufficient to cause overgrowth and F-actin accumulation when overexpressed. Changes in actin dynamics induced by Ena/VASP proteins can activate SRF-dependent gene expression in mammalian cells. Similarly, it was found that Pico and Lpd can activate SRF activity. Like pico, ectopic mal or bs/SRF in flies are sufficient to cause wing overgrowth. Pico-mediated overgrowth is sensitive to the levels of bs/SRF, but mal-induced overgrowth could not be suppressed by pico knockdown, suggesting that Mal/SRF may act downstream of pico in flies. Collectively, these data suggest that MRL proteins may exert their mitogenic effects by specifically interacting with Ena/VASP proteins and inducing SRF-responsive transcription. Interactions between EGFR, MRL proteins, Ena/VASP, and Mal may provide a mechanism linking growth factor signaling and Mal-mediated SRF activation (Lyulcheva, 2008).

Are MRL proteins uniquely able to stimulate Mal/SRF-mediated tissue growth? Although other actin regulators are known to activate Mal/SRF (Posern, 2006), there is currently little data to indicate that they play a role in proliferation control. This might be explained if different transcriptional responses occur at different Mal-dependent SRF activation thresholds, leading to diverse cellular outcomes. Alternatively, other actin regulators might influence processes that limit net tissue growth. For instance, Rho activates Mal/SRF in mammalian cells, but increased Rho activity in flies is associated with loss of epithelial integrity and cell extrusion, which may negate any potential mal-mediated growth-promoting effects. These issues warrant further study in both flies and mammals. Future studies are also needed to characterize transcriptional targets of Drosophila SRF and resolve the contribution of SRF targets to MRL-mediated growth and proliferation (Lyulcheva, 2008).

Lpd expression appears to be differentially regulated in cancer compared to normal tissues. The data, showing a conserved role for MRL proteins in proliferation control, may provide a potential mechanistic explanation for these observations. In this regard, it is interesting that loss of pico or Lpd can abrogate the effects of EGFR/Erb signaling, deregulation of which has also been implicated in cancer progression. Collectively, these data suggest that MRL proteins might play a role in the pathogenesis of certain cancers and may therefore represent novel molecular targets for therapeutic intervention (Lyulcheva, 2008).

Happyhour, a Ste20 family kinase, implicates EGFR signaling in ethanol-induced behaviors
The consequences of alcohol use disorders (AUDs) are devastating to individuals and society, yet few treatments are currently available. To identify genes regulating the behavioral effects of ethanol, a genetic screen was conducted in Drosophila and a mutant, happyhour (hppy), was identified due to its increased resistance to the sedative effects of ethanol. Hppy protein shows strong homology to mammalian Ste20 family kinases of the GCK-1 subfamily. Genetic and biochemical experiments revealed that the epidermal growth factor (EGF)-signaling pathway regulates ethanol sensitivity in Drosophila and that Hppy functions as an inhibitor of the pathway. Acute pharmacological inhibition of the EGF receptor (EGFR) in adult animals altered acute ethanol sensitivity in both flies and mice and reduced ethanol consumption in a preclinical rat model of alcoholism. Inhibitors of the EGFR or components of its signaling pathway are thus potential pharmacotherapies for AUDs (Corl, 2009).

Two P element mutants in the CG7097/happyhour (hppy) gene region were identified and characterized and it was found that reduced hppy expression resulted in decreased sensitivity to ethanol-induced sedation, whereas neuronal overexpression of hppy caused the opposite effect. By in situ hybridization and QPCR, evidence was found for hppy expression in adult brain, and behavioral rescue experiments demonstrated that neuronal expression of hppy was sufficient to rescue the hppy sedation resistance phenotype (Corl, 2009).

Like its mammalian homologs, the GCK-1 subfamily of Ste20 family kinases, the predicted hppy products contain N-terminal serine/threonine kinase domains and C-terminal regulatory domains known as citron homology domains. In vitro studies of these homologs of Hppy, including GCK, GCK-like kinase, kinase homologous to SPS1/STE20, and hematopoietic progenitor kinase (HPK), have revealed that they activate JNK signaling, but not ERK or p38 signaling. HPK1 and GLK have both been shown to phosphorylate MAP3Ks in the JNK pathway, implying that GCK-1 kinases are MAP4Ks acting upstream of JNK signaling (Corl, 2009 and references therein).

This study provides evidence that Hppy, a presumed MAP4K in the GCK-1 subfamily of Ste20 kinases, can modulate EGFR/ERK signaling in a manner that is consistent with it acting as an inhibitor of the pathway. First, retinal hppy overexpression respectively enhanced and suppressed the rough-eye phenotypes brought about by EGFR/ERK pathway inhibition and activation. Second, increased hppy expression enhanced the semilethality caused by ectopic expression of transgene EGFR pathway inhibitors. Third, decreasing levels of hppy completely suppressed the enhanced ethanol sensitivity brought about by neuronal EGFR downregulation. Finally, ethanol induced robust phosphorylation of ERK/Rolled in a hppy mutant, but not in control flies. What, then, is the biochemical mechanism through which Hppy inhibits EGFR/ERK signaling? The answer to this question is still unknown. However, an in vitro study of another GCK-1 subfamily kinase, HPK1, offers an intriguing possibility. HPK1 has been shown to physically associate with the EGFR adaptor protein Grb2 and that EGF stimulation recruits the Grb2/HPK1 complex to the autophosphorylated EGFR. This recruitment leads to the tyrosine phosphorylation of HPK1. It will be interesting to determine whether such a physical association exists between Hppy and components of the EGFR/ERK signaling cascade and, if so, what the consequences may be on signaling (Corl, 2009).

The experiments cannot completely rule out a role for hppy in regulating JNK signaling, although JNK signaling perturbation did not affect ethanol-induced sedation. In addition, hppy mutant flies responded normally to a variety of stress stimuli known to activate the JNK and p38 pathways, including oxidative stress, heat stress, and starvation. Indeed, studies in HeLa cells show a lack of involvement of hppy in JNK activation in response to osmotic stress and the protein synthesis inhibitor anisomycin (Corl, 2009).

In recent years, studies in vitro and in vivo have revealed an intriguing link between ethanol and the mammalian EGFR/ERK pathway, demonstrating that EGFR autophosphorylation and ERK phosphorylation are both inhibited by pharmacologically relevant concentrations of ethanol. In addition, elevated expression of several MAPKs and their regulators has been reported in the brains of mice and rats selected for high ethanol preference. This study uncover a role for the EGFR/ERK pathway in mediating the behavioral responses to ethanol in Drosophila. Neuronal manipulations that activate the EGFR/ERK pathway resulted in enhanced resistance to the sedative effects of ethanol, whereas neuronal inhibition of the pathway caused increased sensitivity. These effects were seen upon manipulations of several different components of the EGFR/ERK pathway. In contrast, no evidence was found for the other two major MAPK pathways, JNK and p38, in mediating the sedative response to ethanol. The finding that EGFR activation specifically in either insulin-producing cells (IPCs) or dopaminergic cells affects ethanol sensitivity is consistent with previous studies implicating both the IPCs and dopaminergic systems in the behavioral response to ethanol in Drosophila and suggests that the EGFR/ERK pathway may interact with the insulin- and dopamine-signaling pathways to control drug responses. Equally interesting is the observation that EGFR activation in many other brain regions, including those previously shown to play a role in ethanol-related behaviors, such as the ellipsoid body and the cells defined by the 201Y GAL4 line, had no effect on ethanol-induced sedation. Thus, the EGFR pathway appears to play a role in only a subset of brain regions that regulate flies' response to ethanol (Corl, 2009).

The mechanisms through which the EGFR/ERK cascade detects ethanol and how it might transduce those signals into a behavioral response remain unknown. It was found that acute ethanol exposure, at concentrations that are behaviorally relevant, led to a rapid and transient increase in ERK/Rolled phosphorylation in the heads of hppy mutants, an effect that was not observed in wild-type flies. Although this finding supports a role for Hppy as an inhibitor of the pathway, it is unclear whether it explains the increased resistance to ethanol-induced sedation observed in hppy flies or in flies in which the EGFR/ERK pathway was chronically upregulated. It is curious that the data do not reveal an inhibitory effect of ethanol on the EGFR/ERK pathway, as has been reported in rodents. While this may reflect a fundamental dissimilarity in the way that the pathway operates in flies and mammals, this discrepancy is more likely due to the fact that mammalian experiments used chronic ethanol exposure paradigms, whereas the current experiments relied on acute exposure (Corl, 2009).

This study shows that the EGFR has a role in regulating ethanol behaviors in adult flies and mammals. Administration of two well-studied EGFR inhibitors, erlotinib (Tarceva) and gefitinib (Iressa), to adult flies resulted in enhanced sensitivity in the LOR assay. Though the current results do not rule out a developmental role for the EGFR/ERK pathway, they do show that this pathway can function in the adult fly to regulate the sedative effects of ethanol. Similarly, acute administration of erlotinib enhanced sensitivity of mice to the sedating effects of ethanol, implying that the role of the EGFR in this behavior is conserved among flies and rodents. Most importantly, it was found that treatment of adult rats with erlotinib significantly decreased ethanol preference in a two-bottle-choice drinking paradigm. This effect appears to be ethanol specific, as preference for a second rewarding substrate, sucrose, was not altered. Together, these data reveal a potentially conserved role for the EGFR pathway in regulating ethanol behaviors in both flies and rodents. Because both erlotinib (Tarceva) and gefitinib (Iressa) (as well as many other small molecule EGFR inhibitors) are FDA-approved drugs, are known to cross the blood-brain barrier, and are well-tolerated, they offer a possible therapeutic avenue for the treatment of AUDs in humans (Corl, 2009).

New negative feedback regulators of Egfr signaling in Drosophila

The highly conserved epidermal growth factor receptor (Egfr) pathway is required in all animals for normal development and homeostasis; consequently, aberrant Egfr signaling is implicated in a number of diseases. Genetic analysis of Drosophila melanogaster Egfr has contributed significantly to understanding this conserved pathway and led to the discovery of new components and targets. This study used microarray analysis of third instar wing discs, in which Egfr signaling was perturbed, to identify new Egfr-responsive genes. Upregulated transcripts included five known targets, suggesting the approach was valid. The function of 29 previously uncharacterized genes, which had pronounced responses, was investigated. The Egfr pathway is important for wing-vein patterning and using reverse genetic analysis five genes were identified that showed venation defects. Three of these genes are expressed in vein primordia and all showed transcriptional changes in response to altered Egfr activity consistent with being targets of the pathway. Genetic interactions with Egfr further linked two of the genes, Sulfated (Sulf1), an endosulfatase gene, and CG4096, an A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS) gene, to the pathway. Sulf1 showed a strong genetic interaction with the neuregulin-like ligand vein (vn) and may influence binding of Vn to heparan-sulfated proteoglycans (HSPGs). How Drosophila Egfr activity is modulated by CG4096 is unknown, but interestingly vertebrate EGF ligands are regulated by a related ADAMTS protein. It is suggested that Sulf1 and CG4096 are negative feedback regulators of Egfr signaling that function in the extracellular space to influence ligand activity (Butchar, 2012).

Negative Regulation of EGFR/MAPK Pathway by Pumilio in Drosophila melanogaster

In Drosophila melanogaster, specification of wing vein cells and sensory organ precursor (SOP) cells, which later give rise to a bristle, requires EGFR signaling. This study shows that Pumilio (Pum), an RNA-binding translational repressor, negatively regulates EGFR signaling in wing vein and bristle development. Loss of Pum function yielded extra wing veins and additional bristles. Conversely, overexpression of Pum eliminated wing veins and bristles. Heterozygotes for Pum produced no phenotype on their own, but greatly enhanced phenotypes caused by the enhancement of EGFR signaling. Conversely, over-expression of Pum suppressed the effects of ectopic EGFR signaling. Components of the EGFR signaling pathway are encoded by mRNAs that have Nanos Response Element (NRE)-like sequences in their 3'UTRs; NREs are known to bind Pum to confer regulation in other mRNAs. This study shows that these NRE-like sequences bind Pum and confer repression on a luciferase reporter in heterologous cells. Taken together, the evidence suggests that Pum functions as a negative regulator of EGFR signaling by directly targeting components of the pathway in Drosophila (Kim, 2012).

In the absence of Pum, extra bristles and wing veins develop, while over-expression of Pum eliminates bristles and wing veins. Several lines of evidence show that the role of Pum is to negatively regulate development of wing veins and bristles. First, loss- and gain-of Pum function produced aberrant wing vein and bristle phenotypes that are inverse to those produced by altered EGFR signaling. Second, reduction of Pum activity greatly enhanced phenotypes associated with reduced EGFR signaling. Third, concomitant expression of Pum suppressed phenotypes associated with ectopic EGFR signaling. In support of the genetic conclusion, it was shown that Pum binds the NRE-like sequence of EGFR, Rl, Sos, and Drk mRNAs and represses translation of a reporter containing these sequences in heterologous cells, suggesting that Pum is a negative regulator of EGFR signaling (Kim, 2012).

To define Pum's role in the development of wing veins and bristles precisely, attempts were made to locate Pum protein and measure Pum activity through a GFP-NRE construct in the 3rd-instar larval and pupal wing imaginal discs where wing vein and SOP cells are specified. A low- level ubiquitous expression of Pum and broad Pum activity was obtained, suggesting that Pum might function as general attenuator of EGFR signaling (Kim, 2012).

This discovery of negative regulation of EGFR signaling by Pum is not confined to Drosophila somatic cells, since it has also been reported in germline cells of C. elegans, cultured human stem cells, and yeast cells. Thus, it is likely that Pum regulation of EGFR signaling is universal and involves diverse developmental contexts, ranging from C. elegans to Drosophila and humans (Kim, 2012).

RTK signaling modulates the Dorsal gradient

The dorsoventral (DV) axis of the Drosophila embryo is patterned by a nuclear gradient of the Rel family transcription factor, Dorsal (Dl), that activates or represses numerous target genes in a region-specific manner. This study demonstrates that signaling by receptor tyrosine kinases (RTK) reduces nuclear levels and transcriptional activity of Dl, both at the poles and in the mid-body of the embryo. These effects depend on wntD, which encodes a Dl antagonist belonging to the Wingless/Wnt family of secreted factors. Specifically, it was shown that, via relief of Groucho- and Capicua-mediated repression, the Torso and EGFR RTK pathways induce expression of WntD, which in turn limits Dl nuclear localization at the poles and along the DV axis. Furthermore, this RTK-dependent control of Dl is important for restricting expression of its targets in both contexts. Thus, the results reveal a new mechanism of crosstalk, whereby RTK signals modulate the spatial distribution and activity of a developmental morphogen in vivo (Helman, 2012).

Specification of body axes in all metazoans is initiated by a small number of inductive signals that must be integrated in time and space to control complex and unique patterns of gene expression. It is therefore of utmost importance to unravel the mechanisms underlying crosstalk between different signaling cues that concur during early development. This study has elucidated a novel signal integration mechanism that coordinates RTK signaling pathways with the Dl nuclear gradient, and thus with terminal and DV patterning of the Drosophila embryo (Helman, 2012).

Previous work had identified an input by Torso signaling into specific transcriptional effects of Dl. The current results establish a general mechanism, which involves RTK-dependent control of the nuclear Dl gradient itself, and thus affects a large group of Dl targets. This regulatory input is based on RTK-dependent derepression of wntD, a Dl target that encodes a feedback inhibitor of the Dl gradient. Thus, Dl activates wntD effectively only when accompanied by RTK signaling, enabling region-specific negative-feedback control of the nuclear Dl gradient. In the absence of RTK signaling, wntD is not expressed and the levels of nuclear Dl are elevated. Consequently, Dl target genes are ectopically expressed, both at the poles and along the DV axis (Helman, 2012).

Torso RTK signaling depends on maternal cues and is independent of the Dl gradient. Thus, it can be viewed as a gating signal that operates only at the embryonic poles, where it controls Dl-dependent gene regulation. However, the activity of the EGFR RTK pathway later on in development crucially depends on Dl, which induces the neuroectodermal expression of rhomboid, a gene encoding a serine protease required for processing of the EGFR ligand Spitz. In this case, EGFR-dependent induction of WntD represents a negative feedback loop that reduces nuclear levels of Dl laterally and, consequently, limits the expression of multiple Dl targets along the DV axis (Helman, 2012).

It should be noted that the regulatory interactions that have been characterized do not preclude the existence of other mechanisms modulating nuclear Dl concentration or activity. For example, the progressive dilution or degradation of maternal components involved in Toll receptor activation upstream of Dl should cause reduced Dl nuclear accumulation and retraction of its targets as development proceeds. It is also possible that Torso- or EGFR-induced repressors block transcription of Dl target genes directly. Accordingly, the ectopic sna expression observed in embryos mutant for components of the Torso pathway such as DSor and trunk probably reflects both loss of WntD activity on Dl and loss of Hkb-mediated repression of sna. In this context, it is interesting to note that sna expression expands and colocalizes with Hkb at the poles of wntD mutants; perhaps repression of sna by Hkb is not sufficient to override increased Dl activation in this genetic background. Thus, the Torso pathway probably employs more than one mechanism to exclude Dl target expression from the termini. Furthermore, the existence of such additional regulatory mechanisms could explain why wntD mutants do not have a clear developmental phenotype, despite the broad effects on Dl-dependent gene expression patterns caused by the genetic removal of wntD. It is proposec that corrective mechanisms are present, which make the terminal and DV systems robust with respect to removal of the WntD-based feedback, such as RTK-induced repressors. Understanding the basis of this robustness will require additional studies (Helman, 2012).

This work shows that RTK-dependent relief of Gro- and Cic-mediated repression is essential for transcriptional activation of wntD by Dl. Correspondingly, in the absence of cic or gro, the early expression of wntD expands ventrally throughout the domain of nuclear Dl. The early onset of this derepression, and the presence of at least one conserved Cic-binding site in the proximal upstream region of wntD, indicate that repression of wntD may be direct. Interestingly, it is thought that Gro and Cic are also involved in assisting Dl-mediated repression of other targets such as dpp and zen, as gro and cic mutant embryos show derepression of those targets in ventral regions. However, as ectopic wntD expression in these mutants leads to reduced nuclear localization of Dl along the ventral region, it is conceivable that decreased Dl activity also contributes to the derepression of dpp and zen (Helman, 2012).

In conclusion, the data presented in this study demonstrate RTK-dependent control of nuclear Dl via wntD, based on multiple regulatory inputs, including negative gating, feed-forward loops and negative feedback control. Together, these mechanisms provide additional combinatorial tiers of spatiotemporal regulation to Dl target gene expression. Future studies will show whether other signal transduction cascades and/or additional developmental cues also impinge on the Dl morphogen gradient (Helman, 2012).

Intrinsic dorsoventral patterning and extrinsic EGFR signaling genes control glial cell development in the Drosophila nervous system

Dorsoventral patterning and EGFR signaling genes are essential for determining neural identity and differentiation of the Drosophila nervous system. Their role in glial cell development in the Drosophila nervous system is not clearly established. This study demonstrates that the dorsoventral patterning genes, vnd, ind, and msh, are intrinsically essential for the proper expression of a master glial cell regulator, gcm, and a differentiation gene, repo, in the lateral glia. In addition, it was shown that esg is particularly required for their expression in the peripheral glia. These results indicate that the dorsoventral patterning and EGFR signaling genes are essential for identity determination and differentiation of the lateral glia by regulating proper expression of gcm and repo in the lateral glia from the early glial development. In contrast, overexpression of vnd, msh, spi, and Egfr genes repress the expression of Repo in the ventral neuroectoderm, indicating that maintenance of correct columnar identity along the dorsoventral axis by proper expression of these genes is essential for restrictive formation of glial precursor cells in the lateral neuroectoderm. Therefore, the dorsoventral patterning and EGFR signaling genes play essential roles in correct identity determination and differentiation of lateral glia in the Drosophila nervous system (Kim, 2015).

This study demonstrates that the DV patterning genes, ind, msh, and esg, are required for expression of the glial cell identity marker, gcm, and of the glial cell differentiation marker, Repo, in the proper region of the LTG in the Drosophila VNE. msh and esg acts locally in the formation and differentiation of the LG from the lateral column of the VNE, and esg strongly influences the formation and differentiation of the PG. ind is also locally involved in the initial formation and differentiation of the SG from the VNE. Considering that DV patterning genes, such as ind and msh, are required for the identity determination and formation of NBs in the intermediate and lateral columns along the DV axis, it is plausible that these two genes play essential roles in the proper development of the LTG in the corresponding columns. Interestingly, the zinc finger transcription factor, Esg, plays an important role in the formation and differentiation of the PG that originate from the lateral column, where esg is expressed. Although esg, together with snail and worniu, is required for the asymmetric division of NBs, the precise role of esg in embryonic CNS development has not been clearly determined. Thus, experimental results obtained in this study on esg's role in glial cell formation and differentiation is the first of its kind to analyze the role of esg in gliogenesis during embryonic CNS development (Kim, 2015).

Unexpectedly, vnd, which is essential for identity determination of the medial column NBs, showed the strongest influence on the proper formation and differentiation of all glia, including the LG, SG, and even PG in the VNE. Since the region of msh expression is ventrally expanded in the vnd mutant, disruption of the expression of gcm and Repo in the lateral column may have caused a decrease in the number of LG, LTG, and PG that originate from this region. In addition, the overexpression of vnd also repressed the expressions of Repo and MAPK in the Kr domain, presumably by promoting identity determination of the medial column in the intermediate and lateral columns. Original reports on the role of the vnd in formation and identity determination of the medial column NBs using the vnd target gene, NK6, showed that intermediate and lateral column identity markers are repressed by overexpression of vnd in the Kr-expression domain. One of the reasons for the wider influence of vnd in DV patterning than other DV patterning genes may be that vnd is expressed earliest among these genes, repressing expression of other DV patterning genes such as ind and msh in the medial column, in a process termed 'ventral dominance' (Kim, 2015).

The data revealed that the EGFR signaling receptor and ligand, Egfr and spi, play more global roles in glial cell development than do the DV patterning genes. Egfr and spi are required for initial glial cell formation as shown by reduced expression of gcm and Repo in the LGBs of the VNE. In addition, Repo expression in the differentiated glia was markedly reduced, especially in Egfr embryos, and in spi embryos, to a lesser degree. Interestingly, Repo expression is almost absent in the SG and remains only in the LGs of spi as well as of ind embryos. Since ind expression is activated by the EGFR signaling ligand, Spi, in the VNE to establish the identity of the intermediate column, it is plausible that glial phenotypes in spi and ind mutants are similar to each other. This result indicated that once the intermediate column identity is determined by ind-mediated repression of msh expression in the lateral column, EGFR signaling provides a consolidating extrinsic cue to make ind a repressor of some of the target genes in the intermediate column via MAPK-mediated phosphorylation. This interpretation is compatible with the results obtained by overexpression of Spi and Vn through Kr- and sca-Gal4 drivers, which show repressed Repo expression in the VNE due to the repressor activity of Ind, which in turn is activated by EGFR signaling. Thus, the results indicated that EGFR signaling globally activates many types of glial cell lineages in the VNE and delimits the area where glial cells originate by repressor activity that is chemically modified by EGFR signal transduction (Kim, 2015).

Establishment of proper identity along the DV axis by expression of the DV patterning and EGFR signaling genes is essential for correct formation and differentiation of glia from the VNE This study revealed that the DV patterning and EGFR signaling genes play important roles in the initial formation and differentiation of various types of glia in the Drosophila CNS. The DV patterning genes and EGFR signaling genes are locally and globally required, respectively, for glial cell formation and differentiation using loss-of function mutants of the genes. Unexpectedly, overexpression of the DV patterning and EGFR signaling genes also repressed the initial formation and differentiation of glia. Overexpression of vnd showed stronger repressor activity than msh on the Repo expression in most types of glial cells including the LG, whereas msh showed mild reduction in the Repo expression mainly in the SG, but not in the LG. The repressor activity of vnd started from the initial formation of the LGBs and continued until the glial cells differentiated into mature glia (Kim, 2015).

There are several possible explanations for the repressive effect in both loss-of-function and gain-of function mutants. First, vnd and EGFR signaling genes together play important roles in establishing identities of the medial and intermediate columns in DV patterning of the VNE. Therefore, overexpression of these genes also promote identities of the medial and intermediate in the lateral columns, where many glial cells, including the LG, PG, and some of the SG originate after neurons are formed. This identity change may block glial cell formation and differentiation from the lateral neuroectoderm. Second, overexpression of these genes may also promote neurogenesis over gliogenesis during developmental stages when overexpression was driven by Kr- and sca-Gal4. In addition, repressor activity appears to play a more dominant role than activator activity upon overexpression of vnd, considering that the DV patterning genes, vnd, int, and msh, act as successive repressors to establish and maintain their identity in the VNE. The results obtained using the loss-of-function and overexpression mutants demonstrate that the expression of a proper level of the DV patterning genes promote identity determination of neurons, while their overexpression represses formation of the glia in the VNE by default. In addition, repressor activity of the DV patterning genes appears to play a dominant role in the establishment of the three columnar divisions along the DV axis (Kim, 2015).

Similarly, overexpression of the EGFR signaling ligands, Spi and Vn, and the activated form of EGFR signaling receptor, EgfrAC, repressed Repo expression in all types of glial cells in the VNE. This may be due to the repressor activity of int, since activation of EGFR signaling induces phosphorylation of int and vnd to consolidate their repressor activity. In addition, since Egfr overexpression can cause expansion of vnd expression from the medial column to the lateral area, the intermediate and lateral columns may have acquired the medial identity, such that the LG and various types of other glia originating from the VNE are not generated after overexpression of Spi in the VNE (Kim, 2015).

These studies on the glial cell development in the Drosophila VNE revealed that the DV patterning and EGFR signaling genes play prominent roles in promoting neural identity, rather than glial identity during the early stages of CNS development, since their overexpression did not activate glial identity, but rather repressed it. Later, expression of the glial master gene, gcm, is required to promote glial cell identity in the VNE. It appears that the two-step mode of CNS development first ensures generation of a neural circuit and then provides supporting glial cells in the CNS. The results indicated that the DV patterning genes act locally to promote glial cell formation in their expression domains, but EGFR signaling genes act broadly throughout the VNE. Among the DV patterning genes, vnd, appears to influence glial cell formation and differentiation globally, since it represses int and msh to establish and maintain medial identity from the earliest developmental stage. It remains to be investigated how the DV patterning and EGFR signaling genes control the spatial and temporal regulation of glial cell formation and how they interact to promote glial identity in the CNS (Kim, 2015).

Targets of Activity: Egf receptor function in oogenesis

Continued: Targets of Activity part 2/2

EGF receptor : Biological Overview | Evolutionary Homologs | Protein Interactions | Developmental Biology | Effects of Mutation | References

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