Wiring between signaling pathways differs according to context, as exemplified by interactions between Notch and epidermal growth factor receptor (EGFR) pathways, which are cooperative in some contexts but antagonistic in others. To investigate mechanisms that underlie different modes of cross talk, this study has focused on argos, an EGFR pathway regulator in Drosophila melanogaster which is upregulated by Notch in adult muscle progenitors but is repressed in the wing. Results show that the alternate modes of cross talk depend on the engagement of enhancers with opposite regulatory logic, which are selected by context-determining factors. This is likely to be a general mechanism for enabling the wiring between these pathways to switch according to context (Housden, 2014).
Analysis of tissue-dependent responses to Notch demonstrates that, in argos, these are determined at the level of specific enhancers. These respond either to Su(H) or to the bHLH repressors downstream of Notch, giving rise to different consequences on argos expression and explaining how the logic of signaling pathway cross talk can be switched. Indeed, the different modes of argos regulation correlate with the relationship between Notch and EGFR pathways, with cooperative cross talk occurring in the adult muscle progenitors (AMPs), where the enhancer directly regulated by Su(H) is active, and antagonistic cross talk taking place in the wing pouch, where the repressive enhancer regulated by bHLH operates. Similar distinctive enhancers may also operate at different stages in development, where Notch first activates and then represses the expression of a gene via independent regulatory elements. In both cases it is likely that context-determining factors will alter the ability of specific enhancers to respond to distinct Notch inputs. These will then dictate how signaling pathways will act on the cognate gene, depending on which regulatory elements they make available (Housden, 2014).
Several observations, such as the inability of HLHmβ or HLHmβ-VP16 to alter expression of argos(p)-lacZ when expressed in the AMPs, suggest that, like Su(H), HLHmβ can occupy its binding sites only when the enhancer becomes accessible. Consistent with this possibility, another HLH family transcriptional repressor, Hairy, was shown to bind and repress only those enhancers that had been rendered accessible by prior binding of other factors. Alternatively, HLHmβ may still be capable of binding to its site in enhancer fragment argos2 but lacks the ability to mediate long-range repression, restricting its effects to transcription factors bound within the same vicinity, as observed for short-range repressors regulating even-skipped enhancers. Given that Hairy bHLH repressors can mediate long-range as well as short-range repression, this explanation seems unlikely. Furthermore, as studies of other bHLH factors, such as Myc, argue that they can only bind to chromatin in open conformations, the model in which enhancer accessibility is regulated seems the more probable explanation (Housden, 2014).
Thus, the context-dependent response of argos to Notch could be explained by a two-stage model. Key determining factors, such as Twist in the AMPs or Vvl in the wing pouch, would first regulate the accessibility of different enhancers in the argos intron. This would enable the second stage, which integrates the effects of Notch and EGFR. For example, in the wing pouch, multiple binding sites for the repressor Cic keep the gene repressed, except in regions where EGFR is active. Superimposed on this is the additional regulation from the E(spl)bHLH repressors, acting downstream of Notch to fine-tune the expression patterning within this active domain. Such a model is broadly consistent with two general principles proposed previously for gene regulation by signaling pathways. The first is the reliance on cooperation with context-determining transcription factors, fulfilled here by the requirements for Twist or Vvl. The second is the pivotal role played by repressors, which prevent enhancer activity in appropriate places, as seen here for Cic and E(spl)bHLH (Housden, 2014).
The disparate activities of the argos enhancers suggests that correct modes of response will also require functional boundaries to enable the enhancers to function independently. As no insulator elements have been reported within the argos intron, based on the binding of known factors such as Su(Hw) and CTCF, the mechanism that separates the different functions remains to be elucidated. Other examples of independently functioning enhancers that lack clearly defined insulator elements include the even-skipped stripe enhancers. In this context, the activators and repressors bound to each enhancer act only over short distances, and the spacer sequences between the enhancers prevent cross-regulation. As spacers of a few hundred base pairs were sufficient to enable the even-skipped enhancers to function independently, it is possible that a similar mechanism enables the argos enhancers to operate properly. Such independent operation of these context-dependent enhancers is pivotal for their alternate modes of Notch regulation, and it is likely that similar mechanisms operate when genes are required to adopt different response modes to other widely active signaling pathways (Housden, 2014).
argos expression in the ventral ectoderm is induced by the EGF-R pathway: argos is not expressed in EGF-R mutant embryos, while it is ectopically expressed in the entire ventral ectoderm following ubiquitous activation of the EGF-R pathway. argos expression appears to be triggered directly by the EGF-R pathway, since induction can also be observed in cell culture, following activation of EGF-R by its ligand, Spitz (Golembo, 1996).
EGF receptor signaling is required in neural recruitment during formation of Drosophila chordotonal sense organ clusters. A total of five neural precursors express atonal in abdominal segments, and this number is too few to explain the formation of the eight scolopidia in each abdominal segment. The remaining precursors require Egf-R signaling for their selection. Signaling by the founder precursors is initiated by atonal activating (directly or indirectly) rhomboid expression in the founder cells. It should be noted that in some developmental processes, rhomboid appears to function in the signal-receiving cells, such as in the patterning of ovarian follicle cells. It is not believed that this is the case in chordotonal-precursor formation, because rho is expressed in precursors that do not require rhomboid function (C1-C5 are formed even in rhomboid mutants). Signaling by these founder precursors, presumably through the EGF receptor ligand Spitz, then provokes a response in the surrounding ectodermal cells, as shown by the activation of expression of the Egf-R target genes pointed and argos. The signal and response then leads to recruitment of some of the ectodermal cells to the chordotonal precursor cell fate. Egf-R hyperactivation by misexpression of rhomboid results in excessive chordotonal precursor recruitment. Argos functions in a feedback mechanism to prevent the excess recruitment of additional ectodermal cells. The increase in the number of scolopidia caused by Egf-R hyperactivation is confined to an enlargement of existing cluster sizes: no new chordotonal clusters are formed. A two step mechanism is postulated for the formation of clusters of chordotonal precursors. In the first step, precursors C1-C5 are selected as founder precursors by the conventional route of proneural gene expression and lateral inhibition. In a distinct second phase, these precursors then signal to adjacent ectodermal cells via the Egf-r pathway, inducing some of them to become chordotonal precursors (secondary or recruited precursors). This two-step process is strongly reminiscent of the way atonal acts in neurogenesis in compound eyes. Here, atonal expression is initially refined by lateral inhibition, until atonal is expressed in only the founding R8 precursor, which then recruits R1-R7 in a mechanism that does not require the activation of atonal in these cells (zur Lage, 1997).
During oogenesis, Egfr activation is required for the establishment of the dorsal/ventral axis of the egg and the embryo. To examine how ectopic Egfr activation affects cell fate determination, an activated version of the protein was constructed. Expression of this activated form (lambda top) in the follicle cells of the ovary induces dorsal cell fates in both the follicular epithelium and the embryo. Different levels of expression result in different dorsal follicle cell fates. Among the anterior follicle cells, a minimum of three cell fates can be distinguished by their contribution to the final eggshell morphology. The most dorsal cells produce the midline/operculum region; dorsolateral cells secrete the respiratory appendages, and the ventral cells contribute to the main body. The three populations of follicle cells in the anterior of the developing egg chamber express different combinations of downstream genes. The dorsal midline cells express argos, kekkon1, rhomboid and pointed. The dorsolateral cells express rho and kek1. The ventral follicle cells are distinguished by the expression of CF2 (Queenan, 1997).
Even in cases where all the follicle cells covering the oocyte expresse lambda top, dorsal cell fates are expanded in the anterior, but not the posterior, of the egg. The expression of genes known to respond to Egfr activation (aos, kek 1 and rho) are also expanded in the presence of the lambda top construct. When lambda top is expressed in all the follicle cells covering the oocyte, kek 1 and argos expression are induced in follicle cells all along the anterior/posterior axis of the egg chamber. In contrast, rho RNA expression is only activated in the anterior of the egg chamber. These data indicate that the response to Egfr signaling is regulated by an anterior/posterior prepattern in the follicle cells. Expression of lambda top in the entire follicular epithelium results in an embryo dorsalized along the entire anterior/posterior axis. Expression of lambda top in anterior or posterior subpopulations of follicle cells results in regionally autonomous dorsalization of the embryos. This result indicates that subpopulations of follicle cells along the anterior/posterior axis can respond to Top/Egfr activation independently of one another (Queenan, 1997).
In addition to essential myogenic functions, mutant Mef2 adult females are weakly fertile and produce defective eggs. Mef2 is expressed in nurse and follicle cells of the wild-type egg chamber. The Mef2 oogenic phenotype has been analyzed and it has been shown that the gene is required for the normal patterning and differentiation of the centripetally migrating follicle cells (CMFCs) that are crucial for development of the anterior chorionic structures. Mef2 alleles exhibit a genetic interaction with a dominant-negative allele of thick veins (tkv), which encodes a type I receptor of the Decapentaplegic-signaling pathway. TKV mRNA is overexpressed in Mef2 mutant egg chambers, and, conversely, forced expression of Mef2 represses tkv expression. These results indicate roles for Mef2 in the regulation of tkv gene expression and Decapentaplegic signal transduction that are essential for proper determination and/or differentiation of the anterior follicle cells. Mef2 is also expressed in both nurse and follicle cells. No defects have been observed in the germ line, either the number of germ cells or the location of the oocyte within the egg chamber. Therefore, a possible requirement for Mef2 in germ-line cells remains to be elucidated (Mantrova, 1999).
At stage 10B, tkv is expressed in the ventral half of the CMFC in addition to two short stripes in the dorsal region of the oocyte-associated follicular epithelium. This expression pattern appears to be complementary to that of the Egfr blocker argos, which forms a T-shaped pattern along the dorsal CMFCs and dorsal midline. argos expression is induced by the highest level of Egfr signaling; Egfr in turn, reduces the signaling strength by blocking the interaction between the receptor and its ligands. Thus, the initial graded distribution of Egfr signaling, extending laterally from the anterodorsal midline of the O-FCs, is transformed into two ridges of the Egfr-signaling level just lateral to the dorsal midline. These two ridges define the two lines of O-FCs that ultimately produce the two dorsal appendages. Interestingly, argos expression is diminished in the Mef2 mutant, consistent with the observed mutant egg chambers possessing broad and fused appendages. Although the notion is favored that argos expression is modulated by Mef2 through the action of Tkv, it cannot be ruled out that Mef2 may directly control the transcription of argos (Mantrova, 1999).
In addition to regulating the expression pattern of argos, Mef2 may play a more general role in modulating the Egfr-signaling level. This is suggested by the presence of Mef2 mutant egg chambers with reduced and fused dorsal appendages, a phenotype typical of hypomorphic Egfr-signaling pathway mutants. Indeed, reduced expression of Egfr-signaling components such as rhomboid has been observed in Mef2 mutants. More detailed and expansive studies are needed to elucidate the possible interaction between the Dpp- and Egfr-signaling pathways with Mef2 as a potential mediator (Mantrova, 1999).
Terminal development is disrupted in the dead ringer maternal and zygotic mutant embryos. Both head and tail defects are invariably observed in dri maternal and zygotic mutant embryos. One of the most consistent and striking phenotypes is severe disruption of the cephalo-pharyngeal skeleton. Germline and zygotic dri mutant embryos still have a recognizable dorsal bridge, dorsal and ventral arms and mouth hooks, but the H-piece and lateralgraten are missing or severely malformed. In addition, the atypical anterior position of pharyngeal muscles, visualized using anti-muscle myosin immunostaining, indicates that head involution does not proceed properly (Shandala, 1999).
The appearance of these defects prompted an examination of genes that play a role in the formation of terminal structures. Expression of the terminal gene tailless and the genes buttonhead, empty spiracles, orthodenticle and argos was examined. Of these genes, disruption to only argos (aos) and buttonhead (btd) expression was observed. In wild-type embryos, aos is initially expressed at stage 5 in two terminal domains and a domain that flanks the position of the cephalic furrow. In embryos lacking dri maternal and zygotic product, expression of aos in the terminal domains is almost completely eliminated while expression in the region of the cephalic furrow is maintained, both before and after division into two stripes at the time of cephalic furrow formation. Zygotic aos mutant embryos exhibit head defects that are similar to those observed in maternal and zygotic dri mutant embryos, indicating that the dri mutant head defects are likely to be the result of loss of anterior aos expression in the dri mutant embryos. Analysis of btd expression reveals a regulatory relationship that accounts for another consistent dri mutant phenotype, the appearance of ectopic cephalic furrows. btd expression is found to be partially derepressed in the trunk of dri germline and zygotic mutant embryos. The cephalic furrow arises where expression of the head specific gap gene btd overlaps the first stripe of expression of the primary pair rule gene eve. The repetitive appearance of ectopic cephalic furrows is therefore likely to be the result of the coincident ectopic trunk expression of btd with the more posterior eve stripes. The ectopic furrows do not progress, most probably due to the incomplete derepression of btd in this region (Shandala, 1999).
The Drosophila EGF receptor (Egfr) is required for the specification of diverse cell fates throughout development. How the activation of Egfr controls the development of vein and intervein cells in the Drosophila wing has been examined. Two distinct events are involved in the determination and differentiation of wing vein cells: (1) the establishment of a positive feedback amplification loop, which drives Egfr signaling in larval stages (at this time, rhomboid, in combination with vein, initiates and amplifies the activity of Egfr in vein cells); (2) the late downregulation of Egfr activity [at this point, the inactivation of MAPK in vein cells is necessary for the maintenance of the expression of decapentaplegic (dpp) and becomes essential for vein differentiation. Subseqently, Egfr becomes activated in intervein territories. During the time that dpp is expressed in vein territiories, MAPK activity builds up in intervein territories, probably due to the presence of Vn, a weak Egfr activator. As a consequence, aos expression relocates to intervein territories. Together, these temporal and spatial changes in the activity of Egfr constitute an autoregulatory network that controls the definition of vein and intervein cell types (Martin-Blanco, 1999).
The reiterated use of Egfr is a common effector of differentiation. In the Drosophila eye, Egfr is required for the determination of all cell types. In this system, cell fate depends on the developmental stage at which the receptor is activated. By interfering with Egfr signaling activity, the specification of veins respond to the activation of receptor tyrosine kinase (RTK) signaling during larval stages, but continued activation of RTK signaling results in a failure of vein cells to differentiate. One explanation for these opposite effects could be that early activation of RTK signaling would specify vein cells, while late RTK signaling would implement intervein cell fates. Several observations provide support for this model. In pupae, MAPK is repressed in veins and activated in intervein cells. This activation of MAPK (and the expression of downstream genes, such as argos) responds to Ras signaling activity, and appears to be involved in the suppression of vein cell fates. Indeed, after ectopical activation of D-Raf during the pupal period, promoting intervein cell fates, the MAPK activity remains stimulated all over the wing blade (Martin-Blanco, 1999).
It seems that Egfr is the only receptor tyrosine kinase at work in the wing, able to activate Ras and Raf. While Egfr is ubiquitously expressed during larval imaginal disc development, EGFR mRNA levels are downregulated in the pupal period in presumptive vein cells. This downregulation of Egfr could be involved in the supression of MAPK activity in vein territories. Furthermore, when a dominant negative-Egfr (DN-Egfr) molecule is overexpressed, titrating the endogenous Egfr, in pupae, extra vein tissue is induced. MAPK dephosphorylation in veins could also be induced by other mechanisms; for instance, the early expression of the inhibitor ligand Argos in veins up to 24 hours APF could cooperate in the inactivation of MAPK in these territories (Martin-Blanco, 1999).
What is the function of this change of expression? The first effect of this developmental switch is a modification in the expression of downstream targets. As a consequence of the reduction in MAPK activity from vein cells, aos is eliminated from veins between 24 and 30 hours APF. Conversely, it is upregulated in intervein territories. This scenario is reminiscent of the induction of Egfr ligands in the ventral ectoderm. Here, the primary signal, Spitz induces a relay mechanism by triggering the expression of Vn (and Aos) in adjacent cells. Aos reduces the overall level of Egfr signaling, whereas Vn provides a lower level of activation, capable of inducing only the lateral cell fates. In the larval wing, high levels of Egfr signaling are achieved in veins through a positive feedback loop. Here, Egfr activity promotes the expression of Aos. It is suggested that Aos diffusion from veins could prevent adjacent cells from responding to the vein inductive signals and producing high levels of Egfr activity ('remote inhibition'). Consistently, aos mutant flies display small deltas and extra veins clustered around vein territories. On the contrary, Aos overexpression in larval stages induces the suppression of veins. It is also proposed that, in pupae, while Egfr activity (and Aos) in veins are lost, Vn and Aos expression in intervein cells will reach a competitive balance leading to the activation of Egfr and MAPK, and intervein cell specification (Martin-Blanco, 1999 and references therein).
The tissue-specific regulation of Vn signaling was investigated by examining vn transcriptional control and Vn target gene activation in the embryo and the wing. The results show a complex temporal and spatial regulation of vn transcription involving multiple signaling pathways and tissue-specific activation of Vn target genes. In the embryo, vn is a target of Spi/Egfr signaling mediated by the ETS transcription factor PointedP1 (PntP1). This establishes a positive feedback loop in addition to the negative feedback loop involving Aos. The simultaneous production of Vn provides a mechanism for dampening Aos inhibition and thus fine-tunes signaling. In the larval wing pouch, vn is not a target of Spi/Egfr signaling but is expressed along the anterior-posterior boundary in response to Hedgehog (Hh) signaling. Repression by Wingless (Wg) signaling further refines the vn expression pattern by causing a discontinuity at the dorsal-ventral boundary. The potential for vn to activate Egfr target genes correlates with its roles in development: vn has a minor role in embryogenesis and does not induce Egfr target genes such as aos and pntP1 in the embryo. Conversely, vn has a major role in wing development and Vn/Egfr signaling is a potent inducer of Egfr target genes in the wing disk. Spi also has the potential to induce Egfr target genes in the wing disk. However, the ligands appear to evoke specific responses that result in different patterns of target gene expression. Other factors modulate the potential of Vn so that induction of Vn/Egfr target genes in the wing pouch is cell specific (Wessells, 1999).
Differences between Vn and Spi are apparent in the patterns of target gene induction resultant from the ectopic expression of Vn and soluble Spitz (sSpi) in the wing pouch. Effects have been noted for three Egfr target genes: aos, pntP1, and kekkon-1 (kek1). In each case, a different response to the ligands is seen. Both ligands induce ectopic aos expression but Vn does so in a broader domain than sSpi, however, neither induces aos in the L3/L4 intervein region. In the embryo, the Egfr target gene pntP1 mediates aos induction by sSpi. Likewise in the wing, ectopic sSpi induces pntP1 expression in cells that also expressed aos. However, following ectopic Vn no detectable change in pntP1 expression is seen using in situ hybridization and only very weak induction of pntP1-lacZ is seen in a domain that does not correspond fully with ectopic aos expression. This suggests either that another transcription factor mediates the induction of aos in response to Vn or that PntP1 is capable of inducing aos, even when changes in its own expression level are too low to be detected by current methods (Wessells, 1999).
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).
Convergent intercellular signals must be precisely integrated in order to elicit specific biological responses. During specification of muscle and cardiac progenitors from clusters of equivalent cells in the Drosophila embryonic mesoderm, the Ras/MAPK pathway -- activated by both epidermal and fibroblast growth factor receptors -- functions as an inductive cellular determination signal, while lateral inhibition mediated by Notch antagonizes this activity. A critical balance between these signals must be achieved to enable one cell of an equivalence group to segregate as a progenitor while its neighbors assume a nonprogenitor identity. Whether these opposing signals directly interact with each other has been investigated, and how they are integrated by the responding cells to specify their unique fates was been examined. Two distinct modes of lateral inhibition, one Notch based and a second based on the epidermal growth factor receptor antagonist Argos, are described that have complementary and reinforcing functions. Argos/Ras and Notch do not function independently; rather, several modes of cross-talk between these pathways have been uncovered. Ras induces Notch, its ligand Delta, and Argos. Delta and Argos then synergize to nonautonomously block a positive autoregulatory feedback loop that amplifies a fate-inducing Ras signal. This feedback loop is characterized by Ras-mediated upregulation of proximal components of both the epidermal and fibroblast growth factor receptor pathways. In turn, Notch activation in nonprogenitors induces its own expression and simultaneously suppresses both Delta and Argos levels, thereby reinforcing a unidirectional inhibitory response. These reciprocal interactions combine to generate the signal thresholds that are essential for proper specification of progenitors and nonprogenitors from groups of initially equivalent cells (Carmena, 2002).
This study involves the origin of two progenitors from a single cell cluster. The two progenitors are characterized by expression of the segmentation gene eve and are specified in a distinct temporal order in the Drosophila embryonic mesoderm. Progenitor 2 (P2) develops first; it originates from the preC2 cluster which develops into the C2 cluster and subsequently gives rise to a single P2 cell. P2 divides asymmetrically to give rise to two founder cells, one specific for a pair of persistently Eve-positive heart-associated or pericardial cells (EPCs) in every hemisegment and a second of previously undetermined identity. This second founder coexpresses Eve along with the gap gene Runt, with Eve levels rapidly fading but Runt persisting as development proceeds. By the time that Eve is evident in the EPCs, Runt labels a single somatic muscle, dorsal oblique muscle 2 (DO2). Runt is also detected in the muscle DO2 precursor during germband retraction (Carmena, 2002).
The second Eve progenitor, P15, which also has its origin in the preC2 cluster (which gives rise to a C15 cluster) forms later than P2 and divides asymmetrically to yield the founders of dorsal acute muscle 1 (DA1) and another cell whose fate cannot be followed since a specific, stably expressed marker for it is unavailable (Carmena, 2002).
Although the net effect of Ras and N signaling in the present system is the result of their antagonistic relationship, several forms of cooperative cross-talk also occur. For example, Ras activation induces the expression of Dl. Since the Ras signal is amplified by a positive feedback loop, this has the effect of biasing Dl expression to the emerging progenitor, thereby generating a polarized, nonautonomous inhibitory signal that acts on adjacent cells of the cluster. Aos is also a target of Ras activation, and Aos acts synergistically with the neurogenic pathway to block inductive RTK signaling. Thus, through its effects on the two inhibitory ligands, Dl and Aos, Ras cooperates with N to ensure that only one cell segregates as a progenitor from each equivalence group (Carmena, 2002).
Further cooperation is evident in the N-mediated down-regulation of Dl and Aos in prospective nonprogenitors, a combination of negative feedback and cross-talk that effectively prevents neighboring cells from sending an inhibitory signal to the emerging progenitor. Yan is yet another Ras-dependent component that reinforces the effect of N: when MAPK is suppressed in cells in which N is active, Yan is a functional repressor that blocks progenitor fate. Other examples of cooperation between Ras and N signaling include mammalian cell tumorigenesis and photoreceptor specification in the Drosophila eye (Carmena, 2002).
These results also revealed an effect of Aos on Htl- but not Egfr-dependent C2 cluster development. Although this could be interpreted as indicating a role for Aos in the inhibition of the Htl Fgfr, the idea that Aos is actually blocking basal and/or spontaneous levels of Egfr activation in C2 cells is favored. This interpretation is supported by the finding that a dominant negative form of Egfr suppressed the effect of aos loss-of-function not only in Egfr-dependent C15, but also in C2, which does not require Egfr for its specification. In this cluster, the requisite Aos expression is dependent on Htl activity (Carmena, 2002).
The stereotyped pattern of Drosophila wing veins is determined by the action of two morphogens, Hedgehog (Hh) and Decapentaplegic (Dpp), which act sequentially to organize growth and patterning along the anterior-posterior axis of the wing primordium. An important unresolved question is how positional information established by these morphogen gradients is translated into localized development of morphological structures such as wing veins in precise locations. In the current study, the mechanism has been examined by which two broadly expressed Dpp signaling target genes, optomotor-blind (omb) and brinker (brk), collaborate to initiate formation of the fifth longitudinal (L5) wing vein. omb is broadly expressed at the center of the wing disc in a pattern complementary to that of brk, which is expressed in the lateral regions of the disc and represses omb expression. A border between omb and brk expression domains is necessary and sufficient for inducing L5 development in the posterior regions. Mosaic analysis indicates that brk-expressing cells produce a short-range signal that can induce vein formation in adjacent omb-expressing cells. This induction of the L5 primordium is mediated by abrupt, which is expressed in a narrow stripe of cells along the brk/omb border and plays a key role in organizing gene expression in the L5 primordium. Similarly, in the anterior region of the wing, brk helps define the position of the L2 vein in combination with another Dpp target gene, spalt. The similar mechanisms responsible for the induction of L5 and L2 development reveal how boundaries set by dosage-sensitive responses to a long-range morphogen specify distinct vein fates at precise locations (Cook, 2004).
Extension of a previous analysis of ab in initiating L5 development has shown that ab functions early in L5 specification. Activation of all known vein genes, including rho, Dl, the caupolican and araucan genes of the Iroquois Complex (IroC), and argos, and repression of the intervein genes bs (also known as DSRF) and net, is lost in cells corresponding to the L5 primordium in ab1 mutant wing discs. A determination was also made whether it is critical that ab expression is confined to a narrow stripe for regulating expression of vein or intervein genes. ab was ubiquitously misexpressed in the wing disc using the MS1096-GAL4 driver; such global activation of ab suppresses expression of vein genes, such as rho and Dl. This ab misexpression also caused vein-specific downregulation of the intervein gene bs, in the wing disc, but did not repress expression of other genes, including hh, ptc and dpp. This phenotype may result from unregulated production of a lateral inhibitory signal normally produced by vein cells to suppress vein development in adjacent intervein cells (Cook, 2004).
Reducing the activity of the Drosophila Runx protein Lozenge (Lz) during pupal development causes a decrease in cell death in the eye. Lz-binding sites were identified in introns of argos (aos) and klumpfuss (klu); these genes were shown to be directly activated targets of Lz. Loss of either aos or klu reduces cell death, suggesting that Lz promotes apoptosis at least in part by regulating aos and klu. These results provide novel insights into the control of programmed cell death (PCD) by Lz during Drosophila eye development (Wildonger, 2005).
These findings, together with what is known about aos and klu, support the following model: Lz induces aos expression in cone cells, wherefrom Aos diffuses to antagonize EGFR activity in the surrounding 2° and 3° cells. The expression pattern of aos923-lacZ indicates that Lz also regulates aos expression in 2° and 3° cells, suggesting that these cells may also send antisurvival signals. The data further suggest that within the 2° and 3° cells, Lz activates klu, which antagonizes EGFR signaling downstream of the receptor. Lz also activates klu expression in cone and 1° cells, but it is unclear what function klu has in these cells. Although two phases of PCD during retinal development have been proposed, these experiments support a role for Lz in promoting only the EGFR-dependent phase. An alternative possibility is that the decrease in cell death in lz mutant retinas is due to an increase in 2° and 3° cell differentiation stimulated by an increase in EGFR signaling. However, given the large body of evidence demonstrating that lz normally functions to promote differentiation, a model in which lz acts to suppress differentiation is not favored (Wildonger, 2005).
The mammalian homolog of Lz, Runx1 (also known as AML1), is also a transcriptional regulator. In humans, translocations that affect Runx1 are associated with acute myelogenous leukemia (AML), which is characterized by the proliferation of undifferentiated hematopoietic cells. Effects on cell cycle regulators have been implicated in contributing to this overproliferation, but it is likely that PCD also plays a role . Changes in the amount of the apoptotic regulator Wilms Tumor 1 (WT1) are often found in AML patients. lz promotes cell death in the Drosophila eye in part by activating the expression of klu, the Drosophila homolog of WT1. It is suggested that these findings may be relevant to how Runx1 chimeras lead to the development of AML in humans. Furthermore, they suggest that WT1 may be a direct target of Runx1 (Wildonger, 2005).
Overexpression of the Notch antagonist Hairless (H) during imaginal development in Drosophila is correlated with tissue loss and cell death. Together with the co-repressors Groucho (Gro) and C-terminal binding protein (CtBP), H assembles a repression complex on Notch target genes, thereby downregulating Notch signalling activity. This study investigated the mechanisms underlying H-mediated cell death in S2 cell culture and in vivo during imaginal development in Drosophila. First, the domains within the H protein that are required for apoptosis induction in cell culture were mapped. These include the binding sites for the co-repressors, both of which are essential for H-mediated cell death during fly development. Hence, the underlying cause of H-mediated apoptosis seems to be a transcriptional downregulation of Notch target genes involved in cell survival. In a search for potential targets, transcriptional downregulation of rho-lacZ and EGFR signalling output were noted. Moreover, the EGFR antagonists lozenge, klumpfuss and argos were all activated upon H overexpression. This result conforms to the proapoptotic activity of H, as these factors are known to be involved in apoptosis induction. Together, the results indicate that H induces apoptosis by downregulation of EGFR signalling activity. This highlights the importance of a coordinated interplay of Notch and EGFR signalling pathways for cell survival during Drosophila development (Protzer, 2008).
This work allows two important conclusions: that overexpression of H induces cell-autonomous apoptosis, and that H requires the co-repressors Gro and CtBP for its proapoptotic activity. It is known that H assembles a repression complex together with the two co-repressors, resulting in transcriptional downregulation of Notch target genes. Hence, the ability of H to induce cell death is most likely a consequence of the repression of Notch target genes that are involved in cell survival. It is noted, however, that not every cell that receives an overdose of H dies. One simple explanation for this observation is that the only cells that die are those in which the relevant Notch target genes are normally active, as these cells require a Notch signal for survival. As H results in a repression of Notch activity, these cells would be driven into cell death, whereas those cells that do not depend on higher Notch levels for survival would be resistant to an H overdose. How is this effect of H realised at the molecular level? So far, it has not been possible to narrow down the analyses towards one target gene, the repression of which by the H repressor complex induces apoptosis. The most straightforward idea, repression of the anti-apoptotic protein Diap1, is not supported by the data. Instead, it was found that EGFR signalling activity is downregulated as a consequence of the upregulation of several negative regulators of EGFR (Protzer, 2008).
The existence of a densely woven network of genetic interactions between the EGFR and Notch signalling pathways is well established. This intensive cross-talk harmonises many developmental processes, such as proliferation, differentiation, cell fate specification, morphogenesis and programmed cell death. Still, the molecular basis of this genetic interplay remains largely obscure. So far, few molecular intersections between the Notch and EGFR pathways have been revealed. For example, EGFR signalling causes phosphorylation of the co-repressor Gro, thereby negatively modulating the transcriptional outputs of Notch signalling via the Enhancer of split [E(spl)] genes. Conversely, a myc-Gro complex was shown to inhibit EGFR signalling during neural development in the Drosophila embryo. Although mutual antagonism is probably the most prominent relationship in EGFR-Notch interactions, in some developmental situations both pathways cooperate to potentiate each other's signalling activities. One such example with regard to cell survival has been described in the retina of rugose mutant flies, where cell type-specific cell death could be reversed by an increase in Notch or EGFR signalling activity, indicating that both pathways adopt an anti-apoptotic function in this developmental context. Also, R7 photoreceptor cell specification requires the combined input of both Notch and EGFR signals. Moreover, Notch defines the scope of rho expression in the Drosophila embryo, thereby activating the EGFR pathway required for early ectodermal patterning. Also, during the development of mouse embryonic fibroblast, the Notch receptor-processing γ-secretase presenilin acts as a positive regulator of ERK basal level activity (Protzer, 2008).
A significant decrease was observed in the levels of activated MAPK (diP-ERK), which provides a good assessment of EGFR pathway activation, upon induction of H. Activated MAPK directly phosphorylates two transcription factors, Aop (Yan) and Pointed (Pntp2). Phosphorylation inactivates Aop, which in the unmodified state, represses EGFR targets. At the same time, phosphorylation activates Pointed, which then causes EGFR target gene transcription. As H is a well-defined transcriptional repressor of Notch target genes, it is most unlikely that it impedes EGFR activity at the level of phosphorylation. Moreover, it is not thought that H acts at the level of transcriptional regulation of EGFR target genes, even though combinatorial and antagonistic activities of the nuclear effectors of the EGFR and Notch signalling pathways have been described during eye development. Instead, the hypothesis is favored that H represses the transcription of EGFR activators, or might indirectly provoke the activation of EGFR repressors that affect, for example, the production of EGFR ligands or signal transduction (Protzer, 2008).
Rho activity is required for a timely and spatially regulated release of EGFR ligands. Accordingly, the expression of rho is highly dynamic during Drosophila development, and precedes the appearance of EGFR-induced activated MAPK. Hence, downregulation of rho by H would eventually result in lower levels of activated MAPK (diP-Erk). In contrast to other components of the EGFR signalling pathway, ectopic expression of rho results in EGFR activation in a wide range of tissues, indicating that Rho is an essential and limiting factor. So far, transcriptional control is the only known means of rho regulation. The complex array of enhancers regulating rho expression reflects the dynamic pattern of EGFR activation throughout Drosophila development (Protzer, 2008).
Interestingly, a transcriptional repression of rho-lacZ was observed in H gain-of-function clones that was dependent on the co-repressors Gro and CtBP. This effect might very well be direct, because it was shown previously that rho transcription is regulated by Su(H) in the neuroectoderm as well as in the gut of the Drosophila embryo. As mentioned above, Notch signalling has also been shown to regulate rho expression in the embryonic ectoderm. Moreover, during egg development, a band of Notch activity establishes the boundary between the two dorsal appendage tube cell types, whereby Notch levels are high in rho-expressing cells. In accordance with this, potential Su(H)-binding sites are present in the regulatory regions of rho1 and rho3, making a direct regulation of rho during eye development via the Notch-Su(H)-H complex very likely. It is noted, however, that the downregulation of rho-lacZ and of activated MAPK were focussed at the morphogenetic furrow, where primary photoreceptor cells are specified and ommatidia are founded. Regulation of rho by H would then be expected to interfere with photoreceptor formation rather than with cell survival, which is in agreement with the disturbed cellular architecture of H gain-of-function flies (Protzer, 2008).
Most interestingly, upon H overexpression, ectopic induction of lz, klu and aos was observed. All three genes are known to be involved in cell death induction during pupal eye development. There it was shown that the Runx protein Lz binds to the regulatory regions of klu and aos, resulting in the direct transcriptional activation of these target genes. Therefore, one might speculate that H executes its effect on klu and aos activity via the activation of lz. Moreover, as klu and aos are well-known inhibitors of EGFR signalling activity, this in itself suggests that H impedes EGFR signalling activity via these factors. This interpretation helps to explain why aos expression is induced in H gain-of-function clones, although it is well known that aos is triggered by EGFR signalling, thereby forming an inhibitory loop that acts on EGFR activity. The high levels of Lz still activate aos in H gain-of-function clones, keeping activity of the EGFR pathway low. Alternatively, aos and klu levels might be increased as a consequence of the downregulation, by H, of an as yet unknown repressor. Since H behaves as a kind of 'multi-adaptor protein', which not only recruits the transcriptional silencers Gro and CtBP to Notch targets but also binds other proteins such as Pros26.4, it is also possible that H interacts with positive regulators of lz, klu and aos (Protzer, 2008).
However, a model is favored whereby H influences EGFR signalling activity on two levels. On the one hand, through transcriptional repression of rho, H causes a loss of EGFR signalling output that interferes with cell specification. On the other hand, by interfering with their repressor(s), H relieves the restriction on lz, klu and aos expression, causing their accumulation. In consequence, the survival/death balance is tipped towards apoptosis in those cells that are susceptible to the effects of a lowered EGFR signal. Those cells that do not depend on high Notch and EGFR activity levels for survival would be resistant to an H overdose (Protzer, 2008).
Finally, one can envisage that a downregulation of Notch and EGFR signalling activities, resulting from the overexpression of H, might leave a cell in a state of 'uncertainty' that does not allow any further differentiation towards a certain cell type, but leaves the cell vulnerable to the apoptotic programme (Protzer, 2008).
Gene expression is regulated in part by protein complexes containing ATP-dependent chromatin-remodelling factors of the SWI/SNF family. In Drosophila there is only one SWI/SNF protein, named Brahma, which forms the catalytic subunit of two complexes composed of different proteins. The protein Osa defines the Bramha associated protein (BAP) complex, and the proteins Polybromo and Bap170 are only present in the complex named PBAP. This work analysed the functional requirements of Osa during Drosophila wing development, and found that osa is needed for cell growth and survival in the wing imaginal disc, and for the correct patterning of sensory organs, veins and the wing margin. Other members of the BAP complex, such as Snr1, Bap55, Mor (Moira) and Brahma, also share these functions of Osa. Focus was placed on the requirement of Osa during the formation of the wing veins. Genetic interactions between osa alleles and mutations affecting the activity of the EGFR pathway suggest that one aspect of Osa is intimately related to the response to EGFR activity. Thus, loss of osa and EGFR signalling results in similar wing vein phenotypes, and osa alleles enhance the loss of veins caused by reduced EGFR activity. In addition, Osa is required for the expression of several targets of EGFR signalling, such as Delta, rhomboid and argos. It is suggested that one role of Osa and Brm in the wing is to establish a chromatin environment in the regulatory regions of EGFR target genes, making them available for both activators and repressors and facilitating transcription in response to EGFR signalling (Terriente-Félix, 2009).
Chromatin structure is critical to modulate gene expression during development, and is affected by a variety of alterations such as histone modification, DNA methylation and changes in conformation. Proteins related to Drosophila Brm, such as yeast SNF2 modify chromatin in an ATP-dependent manner, causing repositioning of nucleosomes along the DNA and re-distribution of histone proteins between nucleosomes. The SWI/SNF complexes are conserved in all eukaryotes, and display specific interactions with distinct transcription factors to regulate different subsets of genes. There are several examples where sequence-specific transcription factors interact specifically with SWI/SNF complexes. For example, the ATPase BRG1 binds Zn-finger proteins and hBRM interacts specifically with CBF-1/Su(H), which recruits hBRM to Notch target promoters such as those of HES1 and HES5 (Terriente-Félix, 2009).
A key aspect in the analysis of Brm function is the identification of targets accounting for the functions of the complex. A necessary step in this analysis is the description of its functional requirements using genetic approaches; which helps to identify the specific processes affected by loss of BAP function. The current data indicate that Osa is required during wing disc development for cell viability, cell proliferation, and for the formation of wing veins and the wing margin. Interestingly, increased expression of Osa in the wing also causes phenotypes related to wing growth and patterning, such as reduced wing size, ectopic sensory organs and hairs and the formation of extra vein tissue in most interveins. This analysis focused mostly on Osa, and this raises the question of whether its requirement reflects the function of the BAP complex. This is the most likely scenario, because the preliminary analysis of other BAP members, such as Snr1, Bap55, Mor and Brm uncovers similar phenotypes in the wing. Thus, lowering Snr1, Bap55 or Mor levels reduces wing size, disrupts the wing epithelium and causes the differentiation of ectopic sensory organs and hairs. These wings also display loss of veins, and in general the overall phenotypes are similar to those of loss of Osa. The phenotype of iRNA expression directed against brm is much milder, perhaps due to a lower efficiency of this construct, but still these wings show a loss of veins phenotype. The reduction of Bap170, a member of the PBAP complex, causes the formation of ectopic veins, which is the opposite phenotype to loss of function in osa and in other members that are present in both the BAP and PBAP complexes. Thus, although Brm is the catalytic subunit in both BAP and PBAP, these complexes could act in opposite manners on the same target genes at least during wing vein formation (Terriente-Félix, 2009).
Some Osa requirements can be explained by modifications in the transcriptional response to the activity of the Wg signalling pathway and by effects on wg expression. The function of Wg is required for the formation of the wing margin, including the development of sensory organs and veins along the anterior wing margin. In the absence of Wg signalling the wing margin does not form, and when Wg signalling is inappropriately activated ectopic sensory organs and hairs differentiate throughout the wing blade. In addition to affecting the response to Wg signalling, Osa is also required for the expression of wg along the dorso-ventral boundary. This requirement might be related to Notch signalling in these cells, and explains why the remnants of wing tissue formed in osa mutant wings do not form the wing margin or ectopic sensory organs (Terriente-Félix, 2009).
This study focused on the characterisation of Osa during the formation of the longitudinal wing veins. This process is independent of Wg signalling, and requires the activities of the Notch, Dpp and EGFR signalling pathways. Osa is needed for the expression of bs in the interveins, because bs is not expressed in cells mutant for osa. The regulation of bs expression involves the activity of Ash2 and the function of the Hh and Dpp pathways. It is suggested that Osa participates in the activation of bs facilitating the availability of its regulatory regions to these activators. This aspect of Osa function does not explain the phenotype of loss of veins characteristic of osa mutant cells, because the loss of Bs expression is normally associated with the differentiation of ectopic veins. The only context where bs mutant cells differentiate as interveins is when the activity of the EGFR pathway is reduced. Therefore, it is suggested that loss of bs expression is accompanied in osa mutant cells by a failure in the response to EGFR activity, leading to the differentiation of intervein tissue. Interestingly, the expression of bs is also severely reduced when Osa is present at higher than normal levels, and in this case loss of Bs is accompanied, as expected, by the formation of ectopic veins. The effects of increased Osa on bs expression can also be explained if Osa facilitates EGFR activity, because this pathway mediates the repression of bs in the proveins. In both cases, the common aspect mediated by Osa might be to regulate bs expression in collaboration with its transcriptional activators and repressors (Terriente-Félix, 2009).
Because the failure of osa mutant cells to differentiate the veins is not due to changes in bs expression, nor to changes in the expression of provein genes such as kni and caup, the search for Osa candidate targets was narrowed to the EGFR pathway. Several results suggest a close relationship between Osa and EGFR signalling in the wing. First, the phenotypes of changing osa expression in the veins are very similar to those resulting from the same manipulation in EGFR activity. Thus, a reduction in any core component of the EGFR pathway eliminates the veins, whereas the increase in EGFR signalling activity causes the formation of extra veins in intervein territories. Second, genetic interactions were observed between osa and several components of the EGFR pathway compatible with a function of Osa promoting EGFR activity in the veins. Finally, the extra veins caused by excess of Osa are suppressed when the activity of EGFR is reduced, indicating that Osa cannot substitute for EGFR activity. The changes in vein and intervein expression patterns are already detected in the wing disc, before other signalling pathways, such as Dpp, act to promote vein formation. Taken together, these observations suggest that Osa facilitates the response to EGFR activity in the wing disc, but cannot promote the transcription of EGFR targets in the absence of EGFR signalling (Terriente-Félix, 2009).
The changes in the expression of EGFR target genes observed in osa mutant cells or in osa gain-of-function experiments are compatible with a direct function of Osa/BAP is the transcriptional regulation of EGFR targets such as Dl, rho and aos. How Osa and the BAP complex are targeted to specific genomic regions is not entirely clear, although it is likely that sequence-specific transcription factors are involved in this process. Transcription in response to EGFR signalling is mediated by proteins belonging to the ETS family, such as Pointed-P2, Pointed-P1 and Yan in Drosophila. However, these genes are not required during wing vein formation, suggesting that other ETS proteins or uncharacterised transcription factors bring about interactions between the regulatory regions of EGFR target genes and the BAP complex (Terriente-Félix, 2009).
It is unlikely that Osa participates in any step of the EGFR pathway previous to the transcription of its target genes. It was noticed, however, that the expression of dP-ERK, a direct read-out of the pathway activity, is also affected in osa mutant cells. Thus, these cells frequently fail to express normal levels of dP-ERK, a result indicating that EGFR activity is reduced. The most likely explanation for this observation is that, in the wing, the EGFR pathway is engaged in a positive feedback loop mediated by the activation of rho expression, which maintains EGFR activity in cells where it has already been activated. Thus, loss of osa leads to a failure to express rho and subsequently to a reduction in the activity of the pathway detected as a loss of dP-ERK expression. There is one experimental situation in which Osa function appears to be dispensable for the expression of EGFR target genes. Thus, when a constitutive active form of Ras, RasV12, is driven in the wing, the augmented expression of Dl and aos, and the accumulation of dP-ERK are not affected by a reduction in Osa levels. It is possible that in this situation of strong and constitutive activity of the pathway, the possible modifications to chromatin structure brought about by Osa/BAP on EGFR target genes are not necessary, perhaps because at this level of EGFR activation the transcriptional repressors antagonising EGFR target gene transcription, such as Cic and Gro, are inactivated by the pathway, and this might make dispensable the function of Osa (Terriente-Félix, 2009).
It is not entirely clear to what extent the link observed between BAP function and EGFR signalling during wing disc development is conserved in other developmental systems and in other organisms. Some phenotypes of osa and brm alleles described in the eye disc, such as the loss of photoreceptor cells, are also observed upon a reduction in EGFR activity. Similarly, the loss of distal growth in the legs is also characteristic of reduced EGFR activity. These data are indicative of a general requirement for Osa in the expression of EGFR target genes at least in imaginal discs. The genetic approach that was used identifies transcription downstream of EGFR signalling as a relevant in vivo function of BAP complexes. Subsequent biochemical analysis should determine whether the functional interactions that were observed are mediated by direct binding of BAP to the regulatory regions of bs and other EGFR target genes (Terriente-Félix, 2009).
Argos protein can inhibit the activation of EGF-R by Spitz (Schweitzer,1994). Presumably, it does this by binding competitively to the EGF receptor, but it is unclear whether Argos binding sends an active off signal, or whether it just fails to signal through the receptor. Alternatively, argos could send an off signal throught another receptor, or it could form non-functional heterodimers with Spitz (Golembo, 1996).
The activation of the Drosophila EGF receptor (DER) by its natural ligand Spitz is inhibited by Argos. Argos and Spitz both have an EGF-like domain, which in the case of Argos differs from that of Spitz and other EGF receptor agonists in that it has an extended B-loop of 20 amino acids instead of 10 amino acids. This B-loop contains an unusual cluster of charged residues. To investigate whether B-loop sequences are an important determinant for receptor activation and play a causal role in the antagonistic activity of Argos, three human (h)EGF mutants were constructed in which amino acids derived from the Argos B-loop were introduced. In one mutant (E3A4E/B10), the replacement of four amino acids in the B-loop of hEGF (123, E24, D27, and K28) by the corresponding Argos residues neither alters the binding affinity of the growth factor for the hEGF receptor nor does it change its ability to induce a mitogenic response. However, the insertion of 2 additional Argos residues (E3A4E/B12) or extension of the B-loop by 10 amino acids (E3A4E/B20) results in a significant loss of binding affinity. In spite of this, both E3A4E/B12 and E3A4E/B20 appear to be strong agonists for the hEGF receptor with similar dose-response curves for mitogenic activity and MAPK activation as wild-type hEGF. These data show that several nonconservative substitutions in the hEGF B-loop are tolerated without affecting receptor binding or activation. They show that receptor binding and receptor signaling efficiency can be uncoupled, which is a prerequisite for the development of receptor antagonists (van de Poll, 1997).
Members of the epidermal growth factor receptor (EGFR) or ErbB/HER family and their activating ligands are essential regulators of diverse developmental processes. Inappropriate activation of these receptors is a key feature of many human cancers, and its reversal is an important clinical goal. A natural secreted antagonist of EGFR signalling, called Argos, was identified in Drosophila. Argos functions by directly binding (and sequestering) growth factor ligands that activate EGFR. This study describes the 1.6-Å resolution crystal structure of Argos bound to Spitz, an EGFR ligand. Contrary to expectations, Argos contains no EGF-like domain. Instead, a trio of closely related domains (resembling a three-finger toxin fold) form a clamp-like structure around the bound EGF ligand. Although structurally unrelated to the receptor, Argos mimics EGFR by using a bipartite binding surface to entrap EGF. The individual Argos domains share unexpected structural similarities with the extracellular ligand-binding regions of transforming growth factor-β family receptors. The three-domain clamp of Argos also resembles the urokinase-type plasminogen activator (uPA) receptor, which uses a similar mechanism to engulf the EGF-like module of uPA. These results indicate that undiscovered mammalian counterparts of Argos may exist among other poorly characterized structural homologues. In addition, the structures presented in this study define requirements for the design of artificial EGF-sequestering proteins that would be valuable anti-cancer therapeutics (Klein, 2008).
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