Signaling by the Drosophila epidermal growth factor receptor pathway during development

Available online at www.sciencedirect.com R Experimental Cell Research 284 (2003) 140 149 www.elsevier.com/locate/yexcr Signaling by the Drosophila epidermal growth factor receptor pathway during development Ben-Zion Shilo* Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel Received 11 November 2002, revised version received 6 December 2002 Abstract In 1997 we wrote a review entitled A thousand and one roles for the Drosophila epidermal growth factor (EGF) receptor (DER/EGFR). We are not there yet in terms of the number of developmental roles assigned to this receptor in Drosophila. Nevertheless, DER has certainly emerged as one of the key players in development, since it is used repeatedly to direct cell fate choices, cell division, cell survival, and migration. A battery of activating ligands and an inhibitory ligand achieves this versatility. For the ligands that are produced as membrane-bound precursors, trafficking and processing are the key regulatory steps, determining the eventual temporal and spatial pattern of receptor activation. In most cases DER is activated at a short range, in the cells adjacent to the ones producing the active ligand. This activation dictates a binary choice. In some instances DER is also activated over a longer range, and multiple cell fate choices may be induced, according to its level of activation. A battery of negative feedback loops assures the limited range of DER induction. The distinct responses to DER activation in the different tissues depend upon combinatorial interactions with other signaling pathways and tissue-specific factors, at the level of target-gene regulation. 2003 Elsevier Science (USA). All rights reserved. Introduction The Drosophila epidermal growth factor (EGF) receptor (DER/EGFR) is a single member of the EGFR/ErbB family in the fly genome. The DER protein is similar to the mammalian family members in overall structure. At the extracellular region it has the typical four domains, including two cysteine-rich domains, required for ligand binding. Similar to the C. elegans receptor Let-23, the juxta-membrane cysteine-rich domain (domain IV) is duplicated in DER [1]. The signal peptide and extreme N-terminus is represented in alternative splice forms, encoding two different protein isoforms. However, it is not clear whether there is a distinct role to each of the forms [2]. Expression of DER per se is not a critical regulatory step, as the receptor is broadly expressed during development [3]. The ligands activating the receptor and the signals it transduces represent one of the key channels of communication between cells during development. This review will begin with a survey of the diverse roles carried out by DER * Fax: 972-8-9344108. E-mail address: benny.shilo@weizmann.ac.il. during fly development. We will then discuss the versatility of ligand structure and regulation as key factors in providing diverse modes of receptor activation. Emphasis will be placed on the central regulatory events in ligand activation, namely trafficking and processing. The capacity to activate DER in a restricted spatial domain depends largely on a set of negative feedback loops that will be discussed. Finally, interpretation of the signals of receptor activation in a tissue-specific context will be explored. Multiple roles during development The multitude of roles played by DER during development have complicated the identification of its developmental roles by simple analysis of loss of function phenotypes during embryonic or postembryonic stages [4]. Instead, the use of dominant negative receptor constructs, temperaturesensitive or hypomorphic mutations in the receptor, or mutations in distinct ligands identified discrete roles. The list of 30 odd distinct roles for DER is presented in Table 1. These roles encompass the induction of cell fates in a multitude of contexts, triggering cell proliferation, and 0014-4827/03/$ see front matter 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/s0014-4827(02)00094-0

B.-Z. Shilo / Experimental Cell Research 284 (2003) 140 149 141 Table 1 Roles of DER during development DER function Stages Ligands References Embryogenesis Patterning the neuroectoderm during gastrulation 5, 6 Spitz [62 64] Patterning the ventral ectoderm 10, 11 Spitz, Vein, Argos [6,19,46] Specification of muscle precursors 11 Spitz, Vein, Argos [16,65] Specification of tracheal invagination and branching 11, 12 Spitz [66,67] Cell recruitment to chordotonal organs 11 Spitz, Argos [5,68,69] Specification of oenocytes 11 Spitz, Argos [70,71] Segmentation 12, 13 Spitz, Argos [72 74] Patterning the dorsal midline 8 Spitz, Argos [75] Formation and proliferation of malpigian tubules 12 15 Spitz [76,77] Germ band retraction 12, 13? [78] Tip cell invagination in the stomatogastric nervous system 13 Spitz [79] Muscle attachment to tendons 15 Vein [16] Viability of midline cells 14 16 Spitz, Argos [78,80 82] Imaginal discs Determination of wing vs. leg disc E11 Spitz [83] Determination of notum vs. wing pouch field, and wing dorsal compartment 2nd instar Vein [84 87] Determination of eye vs. antennal disc 2nd instar [88] Wing disc Determination of veins 3rd instar Spitz, Keren? [37,89 91] Inter vein identity Pupa [89] Eye disc Spacing R8 photoreceptors 3rd instar [92] Proliferation anterior to the furrow and viability 3rd instar Keren? [92 94] Induction of the second mitotic wave 3rd instar Spitz [95] Neuronal, cone, and pigment cell determination 3rd instar and pupa Spitz, Argos [96,97] Brain Neuronal differentiation in lamina Pupa Spitz, Argos [98] Leg Induction of bract cells Pupa Spitz, Argos [99] Proximal-distal patterning Pupa Vein [17,18] Spermatogenesis Restriction of stem cell renewal capacity in somatic cyst cells Adult Spitz [100] Encapsulation of germ cells by somatic support cells? [44] Oogenesis Encapsulation in germ cells by follicle cells in germarium Adult? [44] Determination of posterior follicle cells Gurken [9,11] Migration of border cells Gurken (in collaboration [58] with PVF1/PVR) Determination of dorsal follicle cells Gurken [10,101] Patterning the dorsal appendages Gurken, Spitz, Argos [20,40,102] Abdomen Dorsoventral patterning of the adult abdomen Adult? [103] DER, Drosophila epidermal growth factor (EGF) receptor. even guidance of cell migration. For the most part, the responses to DER activation are manifested by the induction of sets of target genes. In addition to the diversity in the final outcome, tight and distinct regulation of the duration of activation, as well as the strength and range of activation, is essential to assure the correct developmental response. We will outline below some of the regulatory features providing this diversity. Five DER ligands provide versatile modes of DER activation The presence of four activating ligands and one inhibitory ligand allows versatile combinations of DER activation. Three of the ligands, Spitz, Keren, and Gurken, are produced as transmembrane precursors. The primary activating ligand is Spitz, a transforming growth factor- (TGF- ) homologue that is responsible for DER activation in most tissues [5]. As described below, the active, secreted form of Spitz is produced by tightly regulated cleavage of the membrane-bound precursor [6]. A ligand structurally related to Spitz has recently been identified and termed Keren [7,8]. In general, this ligand is regulated in a similar manner to Spitz, and may complement its activity in certain tissues. In contrast to Spitz, the membrane-bound precursor of Keren can undergo unregulated low-level cleavage that may be utilized in several tissues [7]. Gurken, a third TGF- homologue, is restricted to the activation of DER in the

142 B.-Z. Shilo / Experimental Cell Research 284 (2003) 140 149 follicle cells of the ovary [9 11]. Gurken is tightly regulated at several levels. First, it is transcribed only in the germline cells of the ovary. Second, regulatory sequences on the gurken transcript restrict the localization of the RNA to the vicinity of the oocyte nucleus [10]. Finally, the Gurken protein is concentrated accordingly [12]. Tight localization of gurken transcript and protein play an instructive role in induction of dorsal follicle cells fates [13]. Vein, a secreted ligand, possesses an inherently weaker activation capacity, and is used in tissues where low activation levels are required [14,15]. In some tissues Vein functions as the main ligand. It induces the muscle attachment cell fate, following its accumulation at the receiving cell [16]. In the leg, Vein induces several distal cell fates [17,18]. Vein is also utilized as a positive feedback reinforcement to the initial activation of the receptor by other ligands [19,20]. Finally, Argos functions as a secreted ligand that binds the receptor but inhibits activation by competing with the activating ligands [21 23]. It is induced in response to DER signaling, and plays a major role in restricting the activation range of the activating ligands [24]. The structure of DER ligands in schematized in Fig. 1. Networks of ligands generate the final pattern of DER activation. The most frequently used network involves a primary activation of the pathway by Spitz. Since argos and, in some contexts, also vein are transcriptional targets of DER, a circuitry of ligands is created. In the case of Argos, being an inhibitory ligand, this assures the spatial restriction of DER activation by Spitz. In contrast, the induction of Vein, which is less potent in activating DER, promotes lower levels of DER induction in cells that are more distant from the source of the signal. Ligand processing as a key regulatory step Three of the five DER ligands, Spitz, Keren, and Gurken, are produced as a precursor molecule with a transmembrane domain. Processing of these molecules to produce a secreted ligand was shown to be a key regulatory step in DER activation. This paradigm was first established for Spitz and subsequently applied to the other two ligands. Spitz is produced as an inactive membrane precursor and is ubiquitously expressed. Even when expressed at high levels, the precursor form is inactive [6]. The spatial and temporal pattern of Spitz-induced DER activation is thus dependent upon regulated processing of Spitz. The Spitz precursor is normally retained in the endoplasmic reticulum. In addition, the protein has a high turnover rate. In this state, it is prevented from reaching cellular compartments where cleavage by nonspecific proteases may take place [25,26]. Indeed, Spitz constructs where the retention is compromised exhibit a basal activity [7]. A clue to the mechanisms regulating Spitz processing emerged from the identification of mutations in two genes that give rise to phenotypes similar to spitz, namely Star and rhomboid [27]. The first regulated step in Spitz processing is the trafficking of the protein from the endoplasmic reticulum to the Golgi compartment. This step is carried out by Star, a novel type II transmembrane protein [28], that serves as a cargo receptor and associates with Spitz [25,26]. Once reaching the Golgi, Spitz encounters Rhomboid, a seven-transmembrane domain protein [29]. Rhomboid is essential for Spitz cleavage. Furthermore, different observations suggest that Rhomboid is the protease cleaving Spitz [30]. The catalytic domain of Rhomboid resides within its conserved transmembrane domains, giving rise to regulated intramembrane proteolysis. This mode of cleavage was identified in other systems such as Presenillins and cholesterol metabolism [31]. Furthermore, it is conserved even in bacteria, where Rhomboid homologues are involved in releasing the peptide responsible for quorum sensing [32,33]. The cleavage of Spitz by Rhomboid appears to take place within the Golgi, rather than on the plasma membrane. Trafficking and processing of Spitz is schematized in Fig. 2. While Spitz and Star are broadly expressed, the expression of Rhomboid is extremely dynamic [29,34]. Interestingly, it is synonymous with the pattern of DER-induced MAPK activation (as followed by the double phosphorylated MAPK-dpERK) [35]. It thus appears that the expression of Rhomboid is the limiting step in DER activation. This was indeed demonstrated experimentally, by showing that ectopic expression of Rhomboid in diverse tissues and contexts is sufficient to give rise to high levels of DER activation [36,37]. What regulates the dynamic expression of Rhomboid? Promoter dissection has revealed an extremely complex organization of enhancer elements, which specifically regulate the modular expression of rhomboid gene [38,39]. It is interesting that in some tissues DER activation induces rhomboid expression. In tissues where multiple cycles of DER activation are required, the induction of rhomboid expression in the responding cells leads to additional rounds of ligand processing [20,40]. Elucidation of the mechanisms regulating Spitz processing led to insights regarding the other two transmembrane ligands. It was shown that Gurken can undergo cleavage that is Rhomboid and Star dependent in cell culture [8,41]. In the ovary, Gurken is found predominantly in the cleaved form and is endocytosed by the follicle cells receiving the signal [42]. A member of the Rhomboid family (Brho, Rho2, or Stet) that is expressed in the oocyte may carry out Gurken processing [8,43,44]. The mechanism regulating Gurken trafficking within the oocyte, before and after cleavage, is still not clear. Genetic interactions show that Star may be required [41]. In addition, Cornichon, which is another potential cargo receptor, is also required for Gurken signaling [11]. The capacity of Rho2/Brho to cleave DER ligands within the ER in culture may imply that Gurken is processed in the ER, and its trafficking from the ER is regulated post cleavage.

Fig. 1. Activating and inhibitory Drosophilia epidermal growth factor (EGF) receptor (DER) ligands. Five ligands are interacting with DER. Spitz, Keren, and Gurken are produced as transmembrane precursors and are cleaved (arrows) to generate the active secreted ligand. Vein is produced as a secreted protein and also has an Ig domain. Argos is produced a secreted protein, and its EGF domain (red) mediates binding to DER and inhibits binding of other ligands, as well as receptor dimerization. Fig. 2. Intracellular trafficking and cleavage of Spitz. Processing of transmembrane Drosophilia epidermal growth factor (EGF) receptor (DER) ligands is tightly regulated and has been studied in detail for Spitz. (1) Spitz precursor is normally retained in the endoplasmic reticulum (ER). (2) Star is also localized predominantly to the ER. It can associate with Spitz and facilitate its translocation to the Golgi. (3) Rhomboid is localized to the Golgi. It catalyzes the cleavage of Spitz that has been transported to the Golgi by Star. (4) Following cleavage, the extracellular domain of Spitz is secreted outside the cell.

144 B.-Z. Shilo / Experimental Cell Research 284 (2003) 140 149

B.-Z. Shilo / Experimental Cell Research 284 (2003) 140 149 145 Finally, Keren is a ligand that is most similar in structure to Spitz, and its processing is regulated in an analogous manner by Star and Rho [7,8]. Since the retention of Keren in the ER is less stringent, some cleavage by Rhomboid was observed in culture even in the absence of Star. In flies this feature is reflected by the observation that, in contrast to Spitz, overexpression of the Keren precursor can lead to hyperactivation of DER [7]. Which aspects of this elaborate retention, trafficking, and cleavage mechanism may also be utilized for the ligands of the mammalian EGF receptor family? Homologues of Star protein were found only in Anopheles gambiae and Bombyx mori [26], while other trafficking molecules like Cornichon are highly conserved. Mammalian Rhomboid homologues have been identified [45], and some were even capable of cleaving Spitz [30]. However, it remains to be seen if they also cleave precursors of the vertebrate ligands. Feedback loops The general theme emerging from examination of the diverse array of DER functions, is that this pathway provides a relatively short-range signaling module between cells. However, it is not confined only to the cells immediately contacting the ligand source. DER signaling that takes place several cells away from the source is the exception. The two cases that stand out include the patterning of the embryonic ventral ectoderm by Spitz emanating from the midline [6,36,46], and the induction of leg segments by expression of Vein in the distal tip of the pupal leg disc [17,18]. In both these instances the ligand functions as a morphogen, inducing more than one cell fate depending upon the level of the ligand. The further the cells are from the ligand source, the lower the level of DER activation. Graded activation of DER expressed by the follicle cells is also achieved by the graded distribution of Gurken in the egg. However, in most other cases, activation of DER provides a binary switch, either in the cells adjacent to the ligand source or within the cells producing the processed ligand. The capacity to induce a response that is spatially restricted within a population of identical cells entails several feedback circuits that are inherent to the pathway. Three negative feedback circuits, which play a cardinal role in this restriction, have been identified. Argos expression is induced in the cells receiving high levels of DER activation [24]. The protein is secreted and reaches several cell rows away from the site of production. Argos maintains a steadystate level of signaling such that the cells receiving maximal levels of Spitz maintain DER activation, in spite of the production of Argos, while in the cells further away from the source, Argos attenuates activation by Spitz [35]. The assumption is that the distribution profiles of Spitz and Argos are different, such that Spitz levels are higher closer to the source while Argos levels are higher further away. Visualization of the actual distribution profile of the two ligands and dissection of the factors affecting them awaits further experiments. The other two feedback circuits work in a cell autonomous manner and are less universally used than Argos. Kekkon and Sprouty are induced only in some of the tissues in which DER is activated. In those cases they display a fairly broad expression in cells receiving both high and intermediate levels of DER activation [47 49]. The assumption is that their broad expression leads to a uniform reduction in the response to signaling. Thus, cells in which high levels of DER activation took place retain activation, while cells exposed to lower activation levels shut off signaling. In mutants for sprouty, and in some contexts also for kekkon, broadening of the response to DER activation ensues. Kekkon is a transmembrane protein that binds the DER extracellular domain and attenuates receptor dimerization [50]. Sprouty is an intracellular protein that may interfere with DER signaling at several levels [51]. While Argos and Kekkon associate with DER itself, Sprouty interacts with signaling elements that are shared by other receptor tyrosine kinases including the fibroblast growth factor (FGF) receptors. Indeed, Sprouty was initially shown to be induced by other receptor tyrosine kinases, such as Breathless, and attenuate their activity [52]. The positive and negative DER feedback loops are schematized in Fig. 3. Inhibitors that are present in the tissue where DER is activated but are not transcriptional targets of the pathway were also identified. They include Yan, an ETS-domain protein that competes with Pointed, a transcription factor that is triggered by DER [53]. Expression of DCbl in the Fig. 3. Positive and negative DER feedback loops. (A) High levels of Drosophilia epidermal growth factor (EGF) receptor (DER) activation by Spitz trigger through MAPK a series of positive and negative feedback loops. The positive feedbacks entails induction of Vein expression, which facilitates moderate levels of DER activation in adjacent cells. It also includes, in some cases, the induction of Rhomboid expression, which facilitates processing of Spitz in these cells. Negative feedback responses encompass the induction of expression of Argos, which is secreted to attenuate DER activation in more distant cells. It also entails the induction of Kekkon expression to reduce the levels of free DER, and the expression of Sprouty, which compromises signaling downstream to the activated receptor. While induction of Argos is universal to all DER-responding tissues, induction of the other responses occurs only in the context of some tissues. (B) The feedback responses have different thresholds of induction and consequently distinct domains of expression. Together, they ensure that the final pattern of activated DER (as monitored by activated MAPK-dpERK) will be spatially restricted. The cells expressing Rhomboid provide the source of Spitz. Induction of Vein and Argos takes place only in cells receiving high levels of DER activation, but their biological effect is also exerted on neighboring cells since they are secreted proteins. Kekkon and Sprouty have lower thresholds of induction and are expressed also in cells located further away from the ligand source. They attenuate DER signaling in a cell-autonomous manner.

146 B.-Z. Shilo / Experimental Cell Research 284 (2003) 140 149 follicle cells of the ovary was shown to be critical for attenuating DER activation. In the absence of DCbl, endocytosis and degradation of DER is compromised and elevated signaling ensues [54]. Is the DER pathway linear? In mammalian systems, the activated EGF receptor serves as a docking site for several distinct signaling modules, leading to multiple outputs. This raises the question whether the DER signaling pathway is bifurcating or linear. The issue is especially pertinent when considering the quantitative aspects of DER signaling. Bifurcating signaling may provide a mechanism for generating tighter thresholds in response to small differences in the level of activating ligand. The main intracellular signaling pathway activated by DER is the Ras/MAPK pathway. The obligatory use of this pathway for most aspects of DER signaling is implied by the fact that mutations in the intracellular components of the pathway give rise to phenotypes similar to loss of the receptor or ligand [55]. More specifically, absence of Ras in the wing imaginal disc had no effect of PI3 kinase signaling [56]. Conversely, activated Ras mimics DER gain of function phenotypes [57]. Similarities in phenotypes or genetic interactions were not detected with other intracellular signaling pathways, suggesting that as a rule, activation of DER triggers a linear intracellular cascade. One exception to the activation of the canonical Ras pathway by DER appears to take place in the border cells of the ovary, where a combination of signals from DER and the platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) receptor (PVR) guide the migration of these cells towards the oocyte. In most cases of DER signaling, transcriptional activation is the final output, averaging the cumulative levels of DER activation around the circumference of the cell. In cases of cell migration, the site of receptor activation on the cell surface is critical, and the final response is likely to be local rather than transcriptional. In the migrating border cells, high and uniform activation of DER by ubiquitous expression of an activating ligand stalled migration, while activated Raf had no effect on migration [58]. This suggests that in the context of the border cells, DER triggers a different, yet unknown signaling pathway that is important for migration. Tissue-specific responses to DER activation The wide spectrum of tissues in which DER activation provides a developmental switch raises the question of the basis for tissue specificity. In most of these cases, the actual activation of the DER pathway provides a binary switch, and the output of the switch, in terms of the battery of target genes, depends on the tissue context and on other signaling pathways. The issue has been addressed in detail in three tissues where the regulatory sequence of a target gene have been dissected in detail, shedding light on the interplay of DER signaling with tissue-specific factors and other signaling pathways [59 61]. The emerging theme is that all these inputs are integrated at the level of enhancer sequences. Only in cases where all necessary factors are bound to the DNA will transcription of the target gene ensue. For example, in the case of the D-Pax2 gene, expression in the induced cone cells will take place if three requirements have been simultaneously fulfilled. They include DER pathway activation (as represented by the expression of the transcription factor Pointed), triggering of the Notch pathway (culminating in activation of Su(H)), and the competence of the cells to become photoreceptor cells as indicated by the expression of the Lozenge transcription factor [59]. This combinatorial arrangement allows the integration of tissue-specific factors that will induce a given gene only in the desired context. Furthermore, in terms of interactions between different signaling pathways, it provides a modular and highly versatile system. Integration at the promoter level allows use of a different set of pathways, as well as alteration of their interrelationship (synergistic or antagonistic) for any given tissue. In cases where DER activation is not a binary switch but an instructive signal dictating different cell fates, the mechanisms responsible for converting small changes at the level of activation to tight thresholds of gene expression remain to be identified. Future directions Many of the open issues in DER signaling involve the intersection between cell biology and developmental patterning. Understanding in detail how the ligands are transported within the producing cell to allow regulated cleavage, and how they are distributed in the extracellular milieu, will be critical. One may expect the convergence of players dedicated to the DER pathway with components of the cellular machinery that are shared with other pathways. A detailed understanding of the spatial regulation of DER activation is still missing. Rhomboid expression triggers the processing of Spitz. Following secretion, what is the distribution of Spitz and Argos outside the producing cells, and why is short-range signaling observed in some tissues and long-range signaling in others? In some tissues such as the chordotonal organs, the cells producing secreted Spitz are refractive to DER activation, while in other cases, such as the future wing veins, the cells producing secreted ligand(s) are the ones undergoing DER activation. The mechanistic basis for the refractivity to DER activation in the producing cells remains to be explored. In view of the conservation of the pathway, it will be interesting to examine if some of the mammalian ligands are regulated in a similar manner to Spitz in terms of retention,

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