Effects of Mutation or Deletion (part 1/2)

The segmented portion of the Drosophila embryonic central nervous system develops from a bilaterally symmetrical, segmentally reiterated array of 30 unique neural stem cells, called neuroblasts. The first 15 neuroblasts form about 30-60 minutes after gastrulation in two sequential waves of neuroblast segregation and are arranged in three dorsoventral columns and four anteroposterior rows per hemisegment. Each neuroblast acquires a unique identity, based on gene expression and the unique and nearly invariant cell lineage that this expression produces. Little is known as to the control of neuroblast identity along the DV axis. The Drosophila Egfr receptor (Egfr) has been shown to promote the formation, patterning and individual fate specification of early forming neuroblasts along the DV axis. Molecular markers identify particular neuroectodermal domains, composed of neuroblast clusters or single neuroblasts, and show that in Egfr mutant embryos (1) intermediate column neuroblasts do not form; (2) medial column neuroblasts often acquire identities inappropriate for their position, while (3) lateral neuroblasts develop normally. Active Egfr signaling occurs in the regions from which the medial and intermediate neuroblasts will later delaminate. The concomitant loss of rhomboid and vein yields CNS phenotypes indistinguishable from Egfr mutant embryos, even though loss of either gene alone yields minor CNS phenotypes. These results demonstrate that Egfr plays a critical role during neuroblast formation, patterning and specification along the DV axis within the developing Drosophila embryonic CNS (Skeath, 1998).

spitz and vein are expressed in the early embryo and appear to function in independent pathways to activate Egfr during embyrogenesis. Thus, it is possible that either Spitz or Vein or both activate Egfr to promote neuroblast formation and specification. rhomboid and Star encode for transmembrane factors that appear to promote the production of secreted Spitz (s-Spitz) and to act in the same linear pathway as spitz. To investigate the extent to which spitz, rhomboid and Star, as well as vein participate in Egfr-mediated control of early CNS development, neuroblast formation and specification was assayed in embryos singly mutant for each gene. Early CNS development is essentially normal in embryos singly mutant for spitz, Star or vein. ac expression is restricted correctly to the medial and lateral columns and medial neuroblast specification appears normal. Embryos that lack rhomboid function exhibit more severe, yet still relatively mild, CNS defects. To determine the CNS phenotype of removing both spitz group activity and vein, a fly stock doubly mutant for rhomboid and vein was constructed. Removal of rhomboid and vein produces CNS defects virtually indistinguishable from those observed in Egfr mutant embryos: ac expression expands completely into the intermediate column in rows 3 and 7 and only two neuroblast columns form; the RP2 motoneuron almost never forms and roughly half of MP2s are mis-specified. These data suggest that the activity of the spitz group and vein are sufficient to account for all signals that activate Egfr during early CNS development (Skeath, 1998).

Activation of the Drosophila EGF-receptor (Egfr) is spatially and temporally controlled by the release of its various ligands. Egfr and its ligand Spitz mediate the formation of specific somatic muscle precursors. A second Egfr ligand, Vein, complements the activity of Spitz in the development of various somatic muscle precursors. In vn mutant embryos, the Egfr-dependent muscle precursors do not form in some of the segments. Double labeling with anti-Vein and anti-Kruppel antibodies reveal that the Kruppel-positive muscle precursors overlap. In vein mutant embryos at stage 12-13 of embryonic development DA1 muscle precursors are missing in one (or occasionally two) segments of mutant embryos. The Kruppel-positive precursors LL1 and VA2 are also missing in vein mutant embryos at similar frequencies as those observed for DA1. It is concluded that the loss of the various muscle precursor cells observed in vein mutant embryos is in line with the expression of Vein in these cells. This phenotype is significantly enhanced in embryos carrying only one copy of wild type spitz. This analysis suggests that Vein activation of Egfr differs qualitatively from that of Spitz in that it does not lead to the expression of the inhibitory protein Argos, possibly leading to a continuous activation of the Egfr signaling pathway. The results support the idea that the role of Vein in tissues where Spitz is the major ligand is to complement Spitz activity. A model of synergistic activation by the two ligands is not favored. This explains the extremely weak vein phenotype observed in comparison to a significant and measurable phenotype obtained in tissues where Vein functions as a single ligand (Yarnitzky, 1998).

The spitz-group mutants (spitz, rhomboid, and pointed) are embryonic lethal and have similar cuticle phenotypes; they are shorter than wild type and have deletions of ventral cuticle. vein mutant are shorter and the Keilin's organs and ventral black dots are closer together than in wild-type. Ventral cuticle is deleted between Keilin's organs. The deletions occur in a similar region in spitz-group mutants; spitz and rhomboid have a larger portion of ventral cuticle deleted than vein mutants, but pointed embryos have similar deletions. In vein mutants sensory hairs surrounding the pit structure of Keilin's organs are missing. Unlike the spi-group genes, vein is not critical for embryonic survival and head skeleton and sense organs are normal. Most vein mutants die either as embryos or as larvae, but a small number do pupariate. Individuals that survive to pupariate secrete a pupal case with pattern abnormalities (Schnepp, 1996).

vein and spitz show a strong genetic interaction suggesting a molecular interdependence. Reducing vein dose in a spi null genotype dramatically worsens the phenotype to produce a collapse embryo with an extruded head skeleton. However, the double mutants are not as severly affected as are Egf-R null mutants. Genetic interactions are also observed between vn, Egf-R and rolled. The gain of function alleles Egf-R-Ellipse and rolled-Sevenmaker rescue proliferation defects in strong and null vein mutants. These defects include a small wing disc and the size of the pupal case (Schnepp, 1996).

Strong vein mutants have small wing and haltere discs but normal leg discs. In prepupal-lethal null combinations the tiny disc is composed of anterior and posterior cells but these only differentiate basal cuticle lacking in any wing characteristics. In less severe mutant combinations wing discs are round in shape, because they lack the notum and are composed of anterior and posterior domains. The few flies that develop into pharate adults have missing nota and round winglets with anterior and posterior margin tissue. The growth of the dorsal and ventral surface of the wings is not coordinated and additional and excessive ventral tissue is produced (Simcox, 1996).

Use of a temperature-sensitive mutant allows analysis of the temporal requirement for vein in the wing disc. The period of requirement for vein extends from the end of embryogenesis to the second/early-third instar boundary. Loss of vein results in varying phenotypes dependent on the strength of the allele. In the most extreme, the tiny null disc and the notumless disc results from loss of vein function early in the larval period, extending into the first part of day 3. Fan-shaped wing discs with a small notum and a broad wing pouch region result from vein loss during days 2-3 (first and second instar). Wing discs with a distinct duplicated wing pouch result from vein loss during the second instar and very early third instar. Morphologically normal discs are recovered if the ts mutants are cultured at the restrictive temperature for embryogenesis or after the very early part of the third instar (Simcox, 1996).

Signaling through the Drosophila EGF receptor (DER) is important for the growth and differentiation of the wing. These processes may be mediated by different DER ligands including Spitz (Spi) and Vein (Vn). The roles of these ligands and other DER pathway components in wing disc development were investigated using in vivo culture to produce mutant discs from genotypes that are normally embryonic lethal. No role for spi in wing disc growth was found, whereas vn is essential. spi mutant wing discs are morphologically normal as judged by expression of the vein marker rhomboid and analysis of the differentiated wing tissue. spi embryos produce mature-size wing discs patterned with vein and intervein territories like wild-type discs and differentiated wing structures look normal. rho, Star and argos, all known to be involved in Spi/DER signaling are likewise not required for wing growth, whereas pointed, which acts at the end of the intracellular pathway, is required. The results suggest different ligands and molecular mechanisms control EGF-R signaling in wing growth and differentiation (Simcox, 1997). x

Mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase SHP-2, cause Noonan syndrome (NS), an autosomal dominant disorder with pleomorphic developmental abnormalities. Certain germline and somatic PTPN11 mutations cause leukemias. Mutations have gain-of-function (GOF) effects with the commonest NS allele, N308D, being weaker than leukemia-causing mutations. To study the effects of disease-associated PTPN11 alleles, transgenic fruitflies were generated with GAL4-inducible expression of wild type or mutant csw, the Drosophila orthologue of PTPN11. All three transgenic mutant CSWs rescued a hypomorphic csw allele's eye phenotype, documenting activity. Ubiquitous expression of two strong csw mutant alleles was lethal, but did not perturb development from some CSW-dependent receptor tyrosine kinase pathways. Ubiquitous expression of the weaker N308D allele causes ectopic wing veins, identical to the EGFR GOF phenotype. Epistatic analyses have established that the cswN308D ectopic wing vein phenotype requires intact EGF ligand and receptor, and that this transgene interacts genetically with Notch, DPP and JAK/STAT signaling. LOF alleles of positive regulators (downstream of receptor kinase, son of sevenless, Ras85D, Downstream of raf1, rolled, pointed, Hsp83) resulted in statistically significant suppression of ectopic vein formation. Most wings showed no or minimal ectopic vein formation in the anterior part of the wing, which were consistently observed in the UAS-cswN308D/+;tub-GAL4/+ control wings. In contrast, LOF alleles of negative regulators (sprouty, Gap1) enhanced the phenotype with longer ectopic veins in the anterior part of the wings andmultiple and complex ectopic vein formation in the posterior part. LOF alleles of Egfr, its ligand (vein) and positive extracellular regulators (Star, rhomboid) suppressed the wing phenotype while LOF alleles for argos, a negative ligand regulator, enhanced it. Expression of the mutant csw transgenes increases RAS-MAP kinase activation, which is necessary but not sufficient for transducing their phenotypes. The findings from these fly models provided hypotheses testable in mammalian models, in which these signaling cassettes are largely conserved. In addition, these fly models can be used for sensitized screens to identify novel interacting genes as well as for high-throughput screening of therapeutic compounds for NS and PTPN11-related cancers (Oishi, 2006).

Regulation of the Drosophila epidermal growth factor-ligand vein is mediated by multiple domains

Vein (Vn), a ligand for the Drosophila epidermal growth factor receptor (Egfr), has a complex structure including a PEST, Ig, and EGF domain. Structure-function relationships of Vn have been analyzed by assaying deletion mutants. The results show that each conserved domain influences Vn activity. A PEST deletion increases Vn potency and genetic evidence suggests that Vn is regulated by proteasomal degradation. The Ig deletion causes toxic effects not seen following expression of native Vn, but the Ig domain is not required for Vn localization or for the activation of Egfr signaling in wing vein patterning. Remarkably, when the EGF domain is deleted, Vn functions as a dominant negative ligand, implying that Vn normally physically interacts with another factor to promote its activity. Additional highly conserved sequences were identified and several regions were found that affect Vn potency and one that may mediate the effect of dominant negative Vn molecules. Together the results show that the activity of Vn is controlled both positively and negatively, demonstrating the existence of additional levels at which Egfr signaling can be regulated (Donaldson, 2004).

Remarkably, when the EGF domain is deleted, Vn becomes an inhibitor. The activity of this mutant molecule is similar to that of a chimeric ligand, Vn::Aos-EGF, that includes the EGF domain from Aos, the natural Egfr antagonist. The inhibitory function of Vn::Aos-EGF has been ascribed to possession of the Aos sequence but in light of the findings described here, it is likely that both Vn::Aos-EGF and Vn:DeltaEGF function by a dominant negative mechanism. This also allows reconciliation of the difference in the activities of Vn::Aos-EGF and Spi::Aos-EGF chimeras; both share the Aos EGF domain, but only the Vn::Aos-EGF chimera is able to function as an inhibitor of Egfr signaling through a dominant negative mechanism involving a critical domain found only in the Vn 'backbone' (Donaldson, 2004).

Several possible models could explain how these DN-Vn mutants are able to inhibit Egfr signaling. In the simplest model, Vn/Egfr signaling could involve dimerization of Vn. A dimer formed between Vn and DN-Vn would likely be inactive. Expression of DN-Vn would thus reduce the number of active Vn-Vn dimers and result in inhibition of Vn/Egfr signaling. However, the recent structure of Egfr in complex with its ligands excludes the possibility that a Vn dimer is part of the receptor-ligand complex because the two ligands are expected to be ~70-80 Å apart on opposite sides of the complex. However, this does not rule out the possibility that Vn-Vn interactions have a role in subsequent multimerization of receptor dimers to form, for example, tetramers. In an alternative model, Vn/Egfr activation could depend on an interaction between Vn and another factor. In this case, overexpression of DN-Vn would compete for binding with this factor and abrogate Vn-mediated receptor activation (Donaldson, 2004).

Both models predict that there must be a region in Vn that mediates the effect of the inhibitors by competing for binding to this factor. The normal role for this region is therefore to potentiate Vn function and hence deletion of the region should lower Vn activity. Furthermore, the model predicts that a molecule lacking the key region would not be influenced by the DN-Vn ligands. In the analysis of Vn deletion mutants two adjacent regions were found (MR177-395 and MR395-476) that reduce Vn function in an ectopic expression assay. While both deletions remove blocks of conserved sequences (MCR), only Vn:DeltaMR395-476 is able to be suppressed by DN-Vn. This suggests that residues 213-395 are important for mediating the dominant negative effect. It will be important to map the required region in more detail to determine if the MCR performs this role, but the data indicate that the C-terminal portion of the MCR (which is relatively less conserved) is not required (Donaldson, 2004).

One question that arises from this work is whether an inhibitory vertebrate ligand can be created. EGF molecules with extended B loops have been generated in attempts to mimic Argos function. None of these factors have inhibitory properties. While it is believed that the inhibitory nature of Vn::Aos-EGF is primarily mediated through a dominant negative mechanism independent of the Aos sequence, it was noted that the Vn::Aos-EGF chimera is a more potent inhibitor than Vn:DeltaEGF because stronger induction of the transgene (elevating the rearing temperature to increase the activity of Gal4 and hence UAS-transgene expression) is required to produce an equivalent phenotype. This suggests that some intrinsic property of the Aos EGF domain also has an effect. But basing the design of an inhibitor on the Aos EGF region is unlikely to be a successful approach, given the results with vertebrate ligands and the lack of activity of Spi::Aos-EGF. Instead it may be efficacious to investigate vertebrate ligands that rely on binding with other factors to potentiate interaction with the receptor (Donaldson, 2004).

Attenuation of signaling can be dependent on ligand destruction; structure-function analysis suggests that Vn may be regulated by degradation. Deletion of two regions in the N-terminal part of Vn produces mutant proteins with increased ability to activate Egfr as judged by their ability to produce ectopic veins. One of these regions (amino acids 58-96) is strongly predicted to contain a PEST sequence by the PestFind algorithm (Donaldson, 2004).

The observation that the removal of the PEST domain of Vn results in a more potent activator suggests that Vn is subject to regulation by protein degradation. This would be a novel mechanism for regulation of an EGF ligand. In support of this idea a genetic interaction was found between a vn transgene and mutants for proteasome subunits. Analyzing this connection further not only will be important for understanding Vn regulation, but also may have broader implications, as PEST domains have been reported in two other EGF ligands, Gurken and Lin-3, and can also be detected in the neuregulins. Therefore, any such degradation mechanism may be conserved and involve multiple ligands (Donaldson, 2004).

Of the four vertebrate neuregulin (NRG) genes, both NRG-1 and NRG-2 are alternatively spliced to produce isoforms that possess an Ig domain. The Ig domain in NRG-1 binds to heparin sulfate proteoglycans. This maintains a high local concentration of ligand that results in enhanced receptor activation and extends the duration of the response. Although Vn resembles the Ig-containing NRG isoforms, the Ig domain in Vn is unlikely to have a similar role. Deletion of the Ig domain apparently did not diminish the activity of Vn or prevent its association with the ECM. However, small changes in activity and binding may not be detectable in the assays. Instead, the Vn:DeltaIg mutant appears to have additional properties and causes a number of detrimental effects when ectopically expressed that are not observed with native Vn. The evolutionary relationship of the vertebrate and invertebrate ligands is not clear. Certain residues in the EGF domain are characteristic of the neuregulins, but these are not conserved in Vn, suggesting that it may be no more related to the neuregulins than any other Drosophila EGF ligand. Furthermore, the Ig domains of Vn and the neuregulins appear to have at least some distinct functions (Donaldson, 2004).

The analysis of the Drosophila TGF-alpha genes has highlighted the importance that processing of the membrane-bound ligand precursors plays in signaling regulation. There is also a remarkable potential to regulate activity of the only secreted agonist, Vn. Vn activity is altered by deleting each of three known conserved domains (PEST, Ig, and EGF) and also a novel domain was identified that is required for activity. In subsequent analysis it will be important to define the mechanisms that govern these activities and to determine which, if any, are conserved in other animals (Donaldson, 2004).

Dual role for Drosophila epidermal growth factor receptor signaling in early wing disc development

Cell fate decisions in the early Drosophila wing disc assign cells to compartments (anterior or posterior and dorsal or ventral) and distinguish the future wing from the body wall (notum). Egf receptor signaling stimulated by its ligand, Vein, has a fundamental role in regulating two of these cell fate choices: (1) Vn/EGFR signaling directs cells to become notum by antagonizing wing development and by activating notum-specifying genes; (2) Vn/EGFR signaling directs cells to become part of the dorsal compartment by induction of apterous, the dorsal selector gene, and consequently also controls wing development, which depends on an interaction between dorsal and ventral cells (Wang, 2000).

To determine when Vn/EGFR signaling is required for notum development, the temperature-sensitive alleles, Egfrtsla and vntsWB240 were used. Inactivating Vn/Egfr activity during the second instar (a 24 hr period) causes loss of the notum. The wing develops but shows pattern abnormalities characteristic of vn hypomorphs. Later shifts during the third instar does not cause loss of the notum. This demonstrates that Vn/Egfr activity is required for notum development in the second instar when wg is required to specify the wing. Thus, Vn and Wg appear to have complementary roles and this relationship has been examined by following their expression in mutants (Wang, 2000).

In second instar wild-type wing discs, wg is expressed distally in a wedge of anterior ventral cells and vn is expressed proximally. In vn null mutants, the initiation of wg expression is normal as is expression of its target gene optomotor-blind (omb). In wg mutants, however, there is a dramatic and early expansion of vn expression to include distal cells, presaging the development of these cells as an extra notum. Together these results suggest that Vn has an early role in establishing the notum and that Wg signaling is required to define a distal domain that is reduced in Egfr activity to allow wing development (Wang, 2000).

To test the role of Vn/Egfr signaling in specifying notum an examination was carried out to see whether the Iroquois complex (Iro-C) genes, ara and cap are targets of the pathway. The Iro-C genes have been implicated in specifying notum cell fate because loss of function causes a transformation of notum to hinge. Furthermore, misexpression of ara causes loss of the wing and a duplication of notum. Ectopic expression of an activated form of the receptor, Egfrlambdatop4.2 greatly reduces the size of the wing and a small ectopic notum forms. vn is expressed in the presumptive notum in early second instar discs and Caup/Ara are expressed in the presumptive notum at the end of the second instar. In early third instar wing discs, Caup/Ara are expressed in a domain that overlaps with vn. In vn mutants, this expression of Caup/Ara is lost and loss of Egfr signaling, in Egfrts clones, in the medial notum results in a loss of Caup/Ara expression. However, clones in the lateral notum continued to express Caup/Ara, suggesting other factors regulate Iro-C gene expression in these cells at this stage (Wang, 2000).

Activation of Iro-C genes could account for the requirement for Egfr activity to specify the notum at the end of the second instar as this correlates with when these genes are first expressed. However, loss of Egfr signaling at a slightly earlier time (mid-first instar to mid-second instar, see below), prior to activation of the Iro-C genes, also results in loss of the notum. A possible explanation for this comes from the finding that vn expression is lost in vn mutants. This suggests Egfr activity must be sustained, via a positive feedback loop involving transcriptional activation of vn, during the second instar, to activate the Iro-C genes and hence specify notum at the end of this period. Interestingly, the vn gene is also a target of Egfr signaling in the embryo (Wang, 2000 and references therein).

It is suggested that the mechanisms by which wg and vn specify alternate cell fates in the early wing disc, wing, or notum are antagonistic. This is based on the observation that loss of Wg results in the spread of vn expression and the supposition that the resulting ectopic Egfr activity causes loss of the wing and a double notum phenotype. Further evidence that Vn/Egfr signaling represses wing development comes from the results of misexpressing a constitutive receptor, Egfrlambdatop4.2, in the presumptive wing. In these flies, the wing is reduced to a stump covered with sensilla characteristic of the proximal wing (hinge) region and expression of the wing specific gene vestigial (vg) is repressed. Ectopic notal structures also form from the ventral pleura. The ability of ectopic Egfr signaling to suppress wing development is cell autonomous because clones of cells expressing Egfrlambdatop4.2 lack vg expression. In adult wings these clones produced outgrowths lacking wing characteristics but are otherwise difficult to characterize (Wang, 2000).

Although vn expression expands in wg mutants, no reciprocal spread of wg expression was observed in vn mutants that would have been indicative of a double wing phenotype. However, when Vn/Egfr signaling is inhibited in the notum by expressing a ligand antagonist (Vn::Aos-EGF) under the control of ptc-Gal4, ectopic wings are induced in ~10% of the flies. This result demonstrates that presumptive notal tissue can be transformed to wing by reducing Egfr signaling. However, the transformation occurs only when Egfr signaling is reduced in a subset of cells, rather than all cells in the notum (as in a vn mutant). This may reflect the indirect requirement for Egfr activity to also promote wing development (Wang, 2000).

The loss of notum phenotype is characteristic of vn hypomorphs but in null vn alleles and some Egfr alleles both the wing and notum primordia fail to develop and the wing discs remain tiny. Thus, although ectopic activity of Egfr in the distal disc represses wing development, the pathway is nevertheless normally required for wing development. Using the temperature-sensitive Egfrtsla allele it was found that this requirement is restricted to the period from mid-first to mid-second instar. Key genes involved in wing development that are active at this time include wg and apterous (ap). ap is expressed in dorsal cells and acts as a selector gene to divide the disc into dorsal and ventral compartments. Regulation of Notch ligands by Ap leads to Notch signaling at the DV boundary and the formation of an organizer for wing outgrowth and expression of the wing-specific transcription factor vg (Wang, 2000 and references therein).

Of these two candidates, wg and ap, it seemed unlikely that wg was the key gene affected by Egfr signaling from mid-first to mid-second instar because wg expression is normal in vn mutants at mid-second instar. However, later in the second instar, wg expression normally expands to fill the growing wing pouch and it was noted that in vn mutants, wg expression fails to undergo this expansion. A similar defect in wg expression is seen in ap mutants consistent with Ap function being impaired in vn mutants. Remarkably, ap expression is completely absent in second instar vn mutant discs. Thus, loss of Ap can explain why there is no wing in vn mutants. This is supported by the demonstration that ectopic ap is capable of rescuing wing development in vn mutants (Wang, 2000).

Several additional lines of evidence demonstrate that ap is a cell autonomous target of Vn/Egfr signaling and that this relationship exists only transiently in early wing development: (1) ap expression partially overlaps that of vn in the second instar; (2) ap can be induced ectopically in ventral clones misexpressing an activated form of the receptor, Egfrlambdatop4.2; (3) Egfrtsla mutant clones generated in the first instar show autonomous loss of ap expression, whereas clones generated in the second instar express ap normally. Finally, loss of Egfr activity in whole discs from mid-first to mid-second instar results in complete loss of ap expression, whereas ap is still expressed in discs from larvae given a temperature shift slightly later during the second instar (Wang, 2000).

The results described here suggest that division of the early wing disc into presumptive wing and body wall regions is defined by the action of two secreted signaling molecules, Wg and Vn. wg, a pro-wing gene, is required to repress vn expression, which at high levels antagonizes wing development. Antagonism between Wg and Egfr signaling has also been demonstrated in segmental patterning of the embryo and in development of the head and third instar wing pouch, suggesting such a relationship between these pathways may be a common theme in a number of cell fate choices. Finding that one of the main functions of Wg in early wing specification is to repress Vn/Egfr signaling in the distal region of the early disc raises the question as to whether this is the only role of Wg in wing specification and hence if wing-cell fate can be specified in the absence of both signals. This seems unlikely, because nubbin, an early wing cell marker, is not misexpressed proximally in a vn mutant, where cells would lack both signals (Wang, 2000).

Vn/Egfr signaling promotes development of the notum by maintaining its own activity through transcriptional activation of vn itself, and also promotes expression of ap. Thus, both vn and ap appear to be targets of Egfr signaling, but the domain of ap is clearly wider than that of vn, indicating that ap can be activated at a lower signaling threshold than vn. Vn is a secreted molecule and thus could generate a gradient of Egfr activity. This provides an explanation for how Egfr signaling can regulate both wing and notum development: vn autoregulation and notum development requires high Egfr signaling activity while ap expression and subsequent wing development requires lower signaling activity (Wang, 2000).

Interestingly, vertebrate Egfr and its ligands are expressed in the chick limb bud in a pattern that appears to overlap with the vertebrate ap homolog Lhx2, and these factors are required for limb outgrowth in the chick. In light of the present results it will be important to determine whether Egfr signaling controls Lhx2 expression and thus plays a role in regulating outgrowth of the vertebrate limb. These results may also have implications for the evolution of insect wings. If the control of body wall development by Egfr signaling is ancestral, and comparative analysis of other arthropods will be required to assert this, then one of the first steps towards evolution of wings could have occurred when Egfr signaling assumed control of ap (Wang, 2000).

EGF domain swap converts a Drosophila EGF receptor activator into an inhibitor

What are the biological roles of the EGF domains of the related EGF ligands of Drosophila? The EGF domain contains a series of six cysteines, which form three disulfide bonds to generate a looped structure, and a number of other highly conserved residues that are known to be required for binding and activating members of the vertebrate ErbB receptor family. The EGF domains of Vn and Spi are not highly related (38% conserved) but have more sequence conservation with each other than with Argos. Additionally, the length of the predicted B loop that forms from the region between cysteines 3 and 4 is significantly longer in Aos than in the activating ligands (Schnepp, 1998).

Chimeric molecules were created by exchanging the EGF domain of Vn for those of either Spi or Aos. The activity of these chimeras was compared with the native factors in vitro and in vivo. Secreted Spi (sSpi, the active form of Spi) and Aos increase or decrease, respectively, the level of Egfr tyrosine phosphorylation in Drosophila S2-DER tissue-culture cells. The Vn:Vn EGF chimera, which serves as a control for the effect of the additional residues introduced during construction of the chimeras, behaves like native Vn. In contrast, possession of the Spi-EGF domain converts Vn into a stronger Egfr activator. The Vn:Aos EGF chimera behaves as an inhibitor, rather than an activator and caused a reduction in Egfr activation resulting from the ligand-independent activation of Egfr. These results show that the properties of Vn are changed when its EGF domain is swapped with that of Spi or Aos so that the chimeras behave like the factors from which the EGF domain is derived (Schnepp, 1998).

In the embryo, ectopic activation of the DER pathway by sSpi, using the Gal4-UAS system, causes an expansion of ventral cell fates that can be monitored by expression of the ventral cell marker orthodenticle (otd). Ectopic expression of native Vn causes no change in the expression of otd. The Vn:Vn EGF chimera causes a very mild expansion of otd expression. This slight effect could be the result of higher expression of the transgene. In contrast, ectopic expression of the Vn:Spi EGF chimera causes a dramatic expansion of otd expression that is similar to that seen with ectopic expression of sSpi. In the wing, ectopic activation of the DER pathway is characterized by the appearance of extra veins. Ectopic expression of native Vn in pupal interveins produces a mild or moderate extra-vein phenotype, whereas ectopic expression of sSpi causes a strong extra-vein phenotype. A direct role for Vn in normal vein development has been demonstrated; such a role has not been demonstrated for sSpi but is likely to take place. Ectopic expression of the Vn:Vn EGF chimera gives extra-vein phenotypes similar to those seen after ectopic expression of native Vn. In contrast, ectopic expression of the Vn:Spi EGF chimera produces a strong extra-vein phenotype like that seen following ectopic expression of sSpi. In the eye, ectopic activation of the Egfr pathway is characterized by loss of ommatidia, over-recruitment of cell types, and blistering. Ectopic expression of native Vn posterior to the morphogenetic furrow in the eye disc has no effect on the adult eye phenotype; in contrast, ectopic expression of sSpi and the Vn:Spi EGF chimera produces small disorganized eyes with blisters. Surprisingly, ectopic expression of the Vn:Vn EGF chimera also showed a strong eye phenotype. This result suggests that regions outside the EGF domain can affect the activity of a factor because the manipulation used to create the chimeras changed 4 residues flanking the EGF domain. These in vivo data corroborate the biochemical data that Vn is a less potent activator of Egfr than sSpi. The EGF domain is a key feature that differentiates Vn and sSpi because Vn can be converted into a more potent Egfr activator if its EGF domain is swapped with that of Spi. The ability to differentially regulate signaling, depending on whether Vn or sSpi is utilized, may be one mechanism by which DER elicits specific cell responses during development (Schnepp, 1998).

To test whether Vn can be converted into an inhibitor by swapping its EGF domain with that of Aos, the effects of ectopic expression of Vn, Aos, and the Vn:Aos EGF chimera were compared in larval wing and eye discs. Native Vn produces an extra-vein phenotype when expressed ectopically in larval wing discs, as expected for an activator of Egfr signaling. In the wing, ectopic suppression of the Egfr pathway is characterized by vein loss; ectopic expression of native Aos or the Vn:Aos EGF chimera results in vein loss. The vein loss phenotype associated with ectopic expression of Vn:Aos EGF is not as severe as that caused by native Aos. In the eye, reduction in activity of the Egfr pathway is characterized by loss of cell types and fusion of ommatidia. There is no observable effect on adult eye phenotype following ectopic expression of native Vn in eye discs, but ectopic expression of the Vn:Aos EGF chimera produces a rough eye phenotype with fused lenses similar to, but not as severe as, that produced by ectopic expression of native Aos. These results show that the EGF domain is a key determinant responsible for the difference between Vn and Aos and that the EGF swap is sufficient to convert an Egfr activator into an inhibitor. The Vn:Aos EGF chimera is apparently not as potent an inhibitor as native Aos in the eye or the wing, suggesting that other regions of the proteins (Vn and/or Aos) may play modulating roles (Schnepp, 1998).

Dual role of extramacrochaetae in cell proliferation and cell differentiation during wing morphogenesis in Drosophila

The function of extra macrochaetae is required during wing morphogenesis. Mitotic recombination clones of both null and gain-of-function alleles of emc, indicate that during wing morphogenesis, emc participates in cell proliferation within the intervein regions (vein patterning), as well as in vein differentiation (de Celis, 1995). The study of relationships between emc and different genes involved in wing development reveal strong genetic interactions with genes of the Ras signaling pathway (torpedo, vein, veinlet and Gap), and with several other genes (blistered, plexus and net) in both adult wing phenotypes and cell behaviour in genetic mosaics. These interactions are also analyzed as variations of emc expression patterns in mutant backgrounds for these genes. In addition, cell proliferation behaviour of emc mutant cells varies depending on the mutant background. The results show that genes of the Ras signaling pathway are co-operatively involved in the activity of emc during cell proliferation, and later antagonistically during cell differentiation, repressing EMC expression (Baonza, 1999).

Evidence that emc acts co-operatively with genes of the Ras pathway consists of studies of wing size in flies with multiple mutations. top1 homozygous mutant wings, mutant for the gene coding for the Epidermal growth factor receptor, are 14%-20% smaller than wild type, whereas top;emc double mutant wings are 36%-42% smaller. Surprising, the interaction between loss of function (LOF) alleles of emc and hypomorphic alleles of top and vein (vn) produces the same effect on reduction of wing size as the interaction between gain of function (GOF) hypermorphic emc and mutations in these genes. Thus, the vn wings are 15% smaller than the control wings, and in combination with the LOF and GOF alleles of emc the wings appear 27%-35% and 21%-29% smaller than wild type wings, respectively, suggesting that GOF alleles may have a LOF component (Baonza, 1999).

Evidence that emc acts antagonistically to Ras pathway genes during vein differentiation consists of observations of vein differentiation in genetic mosaics. LOF alleles of member genes of the Ras pathway exhibit the absence of veins, and conversely, mutations that cause an increase in the activity of that pathway result in the appearance of ectopic veins. Mutant vein phenotypes of LOF alleles of genes of the Ras pathway are suppressed in interactions with the LOF alleles of emc and enhanced with the GOF allele of emc. Reciprocally, the extra veins mutant phenotype of the GOF alleles for genes of the Ras pathway, is increased in interaction with LOF alleles of emc and reduced with the GOF allele of emc, indicating an antagonistic relationship between emc and genes of the Ras pathway. LOF alleles of emc rescue the lack of vein differention in emc;ve;vn triple mutant clones (ve is veinlet, coding for the protein better known as Rhomboid). Contrarily, double mutant clones of emc and alleles of members of the Ras pathway, which correspond to a hyperactivation of this pathway (Gap1 or heat shock rhomboid), differentiate ectopic veins everywhere in the wing vein (Baonza, 1999).

Genetic interactions have also shown synergistic mutant effects on venation between emc, plexus (px whose molecular nature is unknown) and net, which codes for a bHLH transcription factor. The net gene is required for intervein fate in wings. Furthermore, emc expression, which is absent in normal veins, also disappears in pupal extra veins caused by px and net. Given the molecular nature of net, the co-operative behavior wth emc could reflect direct molecular interactions. Similarly, genetic interactions and changes in expression pattern of emc are found with blistered (bs) mutants. blistered, coding for the Serum response factor of Drosophila, is expressed in the future intervein issue of the wing imaginal disc, in a complementary pattern to Ras pathway genes. In wing differentiation, bs plays a dual role in wing development. Two fully active copies of bs are required to ensure that the formation of wing veins is limited to vein territories. In addition Bs protein is essential for proper terminal differentiation of intervein cells. bs causes strong phenotypic interactions with mutants of the Ras pathway. Thus, it is proposed that emc, bs, px, net and the Ras signaling pathway set of genes are intimately related in vein/intervein patterning and differentiation. The Ras signaling pathway is thought to be involved in maintaining low levels of emc expression during vein pattern differentiation in cells that will differentiate as veins. This is consistent with observations of the expression pattern of emc. Emc protein and mRNA are found at highest levels in intervein regions (Baonza, 1999).

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).

Guidance of cell migration by EGF receptor signaling during Drosophila oogenesis

Directed cell migration is important for many aspects of normal animal development, but little is known about how cell migrations are guided or the mechanisms by which guidance cues are translated into directed cell movement. Evidence is presented that signaling mediated by the Epidermal growth factor receptor (Egfr) guides dorsal migration of border cells during Drosophila oogenesis. The transforming growth factor-alpha-like ligand Gurken appears to serve as the guidance cue. To mediate this guidance function, Egfr signals via a pathway that is receptor-specific and independent of Raf-MAP kinase (Duchek, 2001).

Border cells constitute a cluster of 6 to 10 specialized somatic follicle cells that perform a stereotypic migration during Drosophila oogenesis. At the beginning of stage 9, border cells delaminate from the anterior follicular epithelium and initiate their migration between the germline derived nurse cells, toward the oocyte. About 6 hours later, at stage 10, the border cells reach the oocyte and then migrate dorsally toward the germinal vesicle (GV). The migration of border cells is essential for female fertility; however, it is not known what guides this migration. Spatial information may be provided by the surrounding tissue in the form of cell-associated or secreted guidance cues, for example, as attractive gradients. The posterior and dorsal migration phases might be guided by separate cues, or by a single cue and a fixed migration path (Duchek, 2001).

To identify guidance cues, it was reasoned as follows: the gradient of spatial information would be perturbed if a key attractant or repellant were uniformly overexpressed. This would be expected to cause the cells to migrate inefficiently as there would be no difference between signaling in the front and the back of the cell. To identify genes capable of perturbing border-cell migration when expressed uniformly, a modular misexpression screen with the P element EPg was used. Expression was induced in the germline (nanosGAL4:VP16 ) and in the border cells themselves (slboGAL4). Of 8500 independent insertion lines, three showed defects in border-cell migration but no detectable morphological abnormalities in the egg chamber. In one of these, EPg35521, the single EPg element is inserted in such a way that it drives expression of the gene encoding the neuregulin-like EGFR ligand Vein. Border-cell migration is affected both when Vein is expressed in the germline tissue and when it is expressed in the border cells themselves, as might be expected of a secreted molecule (Duchek, 2001).

To determine whether the effect on migration is specific to Vein or common to Egfr ligands, secreted forms of the TGF-alpha-like ligands Gurken and Spitz were expressed in border cells. Both affect border-cell migration, with the potent ligand secreted Spitz having the strongest effect. Border-cell expression of an activated, ligand-independent, form of Egfr (lambda-top) also severely affects migration. Thus, constitutive stimulation of Egfr signaling in border cells effectively inhibits their migration (Duchek, 2001).

When border cells migrate dorsally, activating ligands for Egfr are produced by the oocyte (Gurken) and, in response to Gurken, by dorsal follicle cells (Vein and Spitz). Dorsal migration still occurs when dorsal follicle cells are mutant for vein, spitz, or rhomboid, which is required for Spitz activation. Thus, although ectopic expression of Vein or activated Spitz proteins can affect border-cell guidance, neither is required for the process. Removing Egfr from patches of dorsal follicle cells, which renders them unable to activate secondary signals, also has no effect. In contrast, dorsal migration is perturbed in gurken mutants. Ovaries from grkDC/grk2b6 mutant females show a range of defects. In mildly affected egg chambers where the GV has moved anterior and dorsal, border cells complete posterior migration but fail to migrate dorsally. In stage-10 oocytes, Gurken protein is detected in a membrane-associated gradient with the highest level at the dorsal anterior over the GV. These results are most consistent with Gurken serving as the dorsal guidance cue, although contributions from other Egfr ligands cannot be excluded (Duchek, 2001).

The RTKs of the EGF receptor family are required for growth, survival, differentiation, and migration of various cell types during animal development. EGF signaling also stimulates growth and metastatic potential of human tumors, as well as proliferation and motility of tissue culture cells. These results demonstrate that Egfr signaling can direct cell migration in vivo. Egfr acts as a guidance receptor for border cells during oogenesis and is specifically required for the second phase of their migration. Another RTK with similar signaling properties may serve this function for the first phase of migration. Guidance effects of Egfr are mediated by a noncanonical signaling pathway. The challenge is now to determine which pathways and molecules downstream of Egfr translate guidance information into directed cell movement in vivo (Duchek, 2001).

Vein and distalization of the leg disc

Arthropods and higher vertebrates both possess appendages, but these are morphologically distinct and the molecular mechanisms regulating patterning along their proximodistal axis (base to tip) are thought to be quite different. In Drosophila, gene expression along this axis is thought to be controlled primarily by a combination of transforming growth factor-ß and Wnt signalling from sources of ligands, Decapentaplegic (Dpp) and Wingless (Wg), in dorsal and ventral stripes, respectively. In vertebrates, however, proximodistal patterning is regulated by receptor tyrosine kinase (RTK) activity from a source of ligands, fibroblast growth factors (FGFs), at the tip of the limb bud. This study revises understanding of limb development in flies and shows that the distal region is actually patterned by a distal-to-proximal gradient of RTK activity, established by a source of epidermal growth factor (EGF)-related ligands at the presumptive tip. This similarity between proximodistal patterning in vertebrates and flies supports previous suggestions of an evolutionary relationship between appendages/body-wall outgrowths in animals (Campbell, 2002).

A distal-to-proximal gradient of EGFR activity predicts a source of ligand(s) at the presumptive tip. Potential ligands are the TGF-alpha family members Spitz and Keren, and the neuregulin, Vn; the former require activation by the membrane protein Rhomboid (Rho), or the homolog Roughoid (Ru). Both vn and rho are expressed in the center of the leg disc in early third instars. Genetic studies show that they are redundant so that loss of either gene alone has no effect on tarsus development, but loss of both together along with ru, which shows partial redundancy with rho even though no expression can be detected, has marked effects on leg patterning and growth. Large ru rho vn triple mutant clones can result in truncations of the tarsus, although these are never as extreme as in Egfrts mutants, possibly because of the difficulty of removing all ligand-expressing cells at the center of early leg discs using this technique, or because the ru mutant used is not null. Wild-type tissue located at the tip of adult legs always correlates with rescue of tarsal development. In addition, misexpression of a secreted form of Spitz results in non-autonomous activation of al. Verification of high levels of EGFR signalling in the distal leg is revealed by expression of sprouty in this location; this is upregulated in many tissues by EGFR signalling (Campbell, 2002).

Drosophila wing development is a useful model to study organogenesis, which requires the input of selector genes that specify the identity of various morphogenetic fields and cell signaling molecules. In order to understand how the integration of multiple signaling pathways and selector proteins can be achieved during wing development, the regulatory network that controls the expression of Serrate (Ser), a ligand for the Notch (N) signaling pathway, which is essential for the development of the Drosophila wing, as well as vertebrate limbs, was examined. A 794 bp cis-regulatory element located in the 3' region of the Ser gene can recapitulate the dynamic patterns of endogenous Ser expression during wing development. Using this enhancer element, Apterous (Ap, a selector protein), and the Notch and Wingless (Wg) signaling pathways, are shown to sequentially control wing development through direct regulation of Ser expression in early, mid and late third instar stages, respectively. In addition, later Ser expression in the presumptive vein cells is controlled by the Egfr pathway. Thus, a cis-regulatory element is sequentially regulated by multiple signaling pathways and a selector protein during Drosophila wing development. Such a mechanism is possibly conserved in the appendage outgrowth of other arthropods and vertebrates (Yan, 2004).

The results reported here demonstrate that a 794 bp cis-acting regulatory module in the Ser locus can be temporally regulated by three distinct mechanisms that are employed for the proper establishment of the DV organizer during wing development. (1) The selector protein Ap directly activates Ser expression in the dorsal compartment during the early third instar, which sets up N activation for the next stage. (2) By the middle of the third instar, the N pathway maintains Ser expression by a positive-feedback loop along the DV boundary. This feedback loop maintains Ser and Dl expression, leading to the activation of N signaling at the DV boundary, which is essential for establishing the DV organizer. (3) At the end of the third instar, as a result of Wg signaling, Ser is expressed in two stripes flanking the DV boundary, which limits N activation to the DV border. In addition, Ser expression in provein cells is dependent on input from the Egfr pathway. These results indicate how tissue-specific selector and signaling molecules can work sequentially to achieve a complex developmental process, such as organogenesis, which involves a complex temporal and spatial regulation of genes. However, the conclusion that the Ser minimal wing enhancer is sequentially regulated by Ap, Notch, Wg and Egfr does not exclude the possibility that these molecules/signaling pathways may cooperate and synergistically stimulate gene expression at certain stages. In this case, mutations that specifically impair response to the intended factor would affect Ser-lacZ expression in other phases of disc development (Yan, 2004).

Ser is expressed in provein cells and its expression is regulated by Egfr signaling at the pupal stage. N signaling also plays an important role in determining vein cell fate. The data on Ser expression in provein cells is consistent with a report on Ser function during vein development. Thus, in addition to its essential role in development of the Drosophila leg and vertebrate limbs, Egfr/Fgf signaling also plays a role in Drosophila wing development, suggesting a conserved role of Egfr signaling in 'appendage' development. Ser expression was examined in both gain-of-function (gof) and loss-of-function (lof) Egfr signaling-mutant backgrounds. First, in a rho gof mutant (UAS-rho*), Ser appeared to be ectopically expressed between L3 and L4, exactly where ectopic rho activity was localized. Ser expression in the proveins was eliminated in a rho and vein (vn, encoding a Egfr ligand) double-mutant (Egfr lof) background, in which vein formation is completely abolished. These results suggest that the Egfr pathway may regulate Ser expression during vein development at the pupal stage. Interestingly, the Ser minimal wing enhancer is expressed in provein cells at both larval and pupal stages. Further investigation of this element may shed light on how Egfr signaling regulates vein differentiation (Yan, 2004).

Vein, functioning as neuregulin, maintains glial survival during axon guidance in the CNS

Continued: Vein Effects of Mutation part 2/2

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

date revised: 10 October 98  

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