In situ activation pattern of Drosophila EGF receptor pathway during development

Signaling cascades triggered by receptor tyrosine kinases (RTKs) participate in diverse developmental processes. The active state of these signaling pathways was monitored by examination of the in situ distribution of the active, dual phosphorylated form of mitogen-activated protein kinase (ERK) with a specific monoclonal antibody. Detection of the active state of the Drosophila epidermal growth factor receptor (DER) pathway allowed the visualization of gradients and boundaries of receptor activation, assessment of the distribution of activating ligands, and analysis of interplay with the inhibitory ligand Argos. This in situ approach can be used to monitor other receptor-triggered pathways in a wide range of organisms.     

Regulation of EGF receptor signaling establishes pattern across the developing Drosophila retina

Developing epithelia use a variety of patterning mechanisms to place individual cells into their correct positions. However, the means by which pattern elements are established are poorly understood. Here, we report evidence that regulation of Drosophila EGF receptor (DER) activity plays a central role in propagating the evenly spaced array of ommatidia across the developing Drosophila retina. DER activity is essential for establishing the first ommatidial cell fate, the R8 photoreceptor neuron. R8s in turn appear to signal through Rhomboid and Vein to create a patterned array of 'proneural clusters' which contain high levels of phosphorylated ERKA and the bHLH protein Atonal. Finally, secretion by the proneural clusters of Argos represses DER activity in less mature regions to create a new pattern of R8s. Propagation of this process anteriorly results in a retina with a precise array of maturing ommatidia.     

The Drosophila EGF receptor controls the formation and specification of neuroblasts along the dorsal-ventral axis of the Drosophila embryo

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 it produces. Recent experiments indicate that the segmentation genes specify neuroblast identity along the AP axis. However, little is known as to the control of neuroblast identity along the DV axis. Here, I show that the Drosophila EGF receptor (encoded by the DER gene) promotes the formation, patterning and individual fate specification of early forming neuroblasts along the DV axis. Specifically, I use molecular markers that identify particular neuroectodermal domains, all neuroblasts or individual neuroblasts, to show that in DER 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. Furthermore, I show that active DER signaling occurs in the regions from which the medial and intermediate neuroblasts will later delaminate. In addition, I demonstrate that the concomitant loss of rhomboid and vein yield CNS phenotypes indistinguishable from DER mutant embryos, even though loss of either gene alone yields minor CNS phenotypes. These results demonstrate that DER plays a critical role during neuroblast formation, patterning and specification along the DV axis within the developing Drosophila embryonic CNS.     

The role of selectins in Drosophila eye and bristle development

Mutations in the furrowed (fw) gene of Drosophila result in defects in the development of the eye and mechanosensory bristles. The eyes are reduced in size, have furrows or crevices in the retina, and show a disturbed patterning of ommatidia. In addition, the ommatidia have an altered morphology and often contain abnormal numbers of cells. The bristles show altered structure and polarity, and are occasionally duplicated or missing. These results suggest that the product of thefw gene is involved in cell determination events within sensory organs. Thefw gene has been cloned and shown to encode a protein homologous to vertebrate selectins. Like selectins, Fw contains a lectin-binding domain, ten complement binding repeats, and a transmembrane domain. The Fw protein is expressed in the larval imaginal discs where it might mediate carbohydrate-protein interactions necessary for proper development of a subset of adult sensory organs.     

Interaction between Drosophila EGF receptor and vnd determines three dorsoventral domains of the neuroectoderm

Neurogenesis in Drosophila melanogaster starts by an ordered appearance of neuroblasts arranged in three columns (medial, intermediate and lateral) in each side of the neuroectoderm. Here we show that, in the intermediate column, the receptor tyrosine kinase DER represses expression of proneural genes, achaete and scute, and is required for the formation of neuroblasts. Most of the early function of DER is likely to be mediated by the Ras-MAP kinase signaling pathway, which is activated in the intermediate column, since a loss of a component of this pathway leads to a phenotype identical to that in DER mutants. MAP-kinase activation was also observed in the medial column where esg and proneural gene expression is unaffected by DER. We found that the homeobox gene vnd is required for the expression of esg and scute in the medial column, and show that vnd acts through the negative regulatory region of the esg enhancer that mediates the DER signal, suggesting the role of vnd is to counteract DER-dependent repression. Thus nested expression of vnd and the DER activator rhomboid is crucial to subdivide the neuroectoderm into the three dorsoventral domains.     

Identification of the minimal requirements for binding to the human epidermal growth factor (EGF) receptor using chimeras of human EGF and an EGF repeat of Drosophila Notch

Many proteins contain so-called epidermal growth factor (EGF)-like domains that share the characteristic spacing of cysteines and glycines with members of the EGF family. They are, however, functionally unrelated, despite the fact that the three-dimensional structure of these EGF-like domains, also, is often very similar to that of the EGF receptor agonists. In the present study, we linked an EGF-like repeat from the Drosophila Notch protein to the N- and C-terminal linear tail sequences of human EGF (hEGF), and we showed that this chimera (E1N6E) is unable to bind or activate the hEGF receptor. This recombinant protein was then used as a basic construct for identifying the minimal requirements for high affinity EGF receptor binding and activation. We selectively reintroduced a limited number of important hEGF-derived residues, and by using this unique approach, we were able to make hEGF/Notch chimeras that, compared with wild type hEGF, showed nearly 100% binding affinity and mitogenic activity on HER-14 cells expressing the hEGF receptor.     

Differential effects of EGF receptor signalling on neuroblast lineages along the dorsoventral axis of the Drosophila CNS

The Drosophila ventral nerve cord derives from a stereotype population of about 30 neural stem cells, the neuroblasts, per hemineuromere. Previous experiments provided indications for inductive signals at ventral sites of the neuroectoderm that confer neuroblast identities. Using cell lineage analysis, molecular markers and cell transplantation, we show here that EGF receptor signalling plays an instructive role in CNS patterning and exerts differential effects on dorsoventral subpopulations of neuroblasts. The Drosophila EGF receptor (DER) is capable of cell autonomously specifiying medial and intermediate neuroblast cell fates. DER signalling appears to be most critical for proper development of intermediate neuroblasts and less important for medial neuroblasts. It is not required for lateral neuroblast lineages or for cells to adopt CNS midline cell fate. Thus, dorsoventral patterning of the CNS involves both DER-dependent and -independent regulatory pathways. Furthermore, we discuss the possibility that different phases of DER activation exist during neuroectodermal patterning with an early phase independent of midline-derived signals.     

Requirement for EGF receptor signalling in neural recruitment during formation of Drosophila chordotonal sense organ clusters

The different lesions of the glenoid labrum are described. They may involve the antero-inferior, the posterior or the superior (SLAP lesions) part of the labrum. CT-arthrography is the gold standard imaging modality in this field of shoulder abnormalities.     

A subset of notch functions during Drosophila eye development require Su(H) and the E(spl) gene complex

The Notch signalling pathway is involved in many processes where cell fate is decided. Previous work showed that Notch is required at successive steps during R8 specification in the Drosophila eye. Initially, Notch enhances atonal expression and promotes atonal function. After atonal autoregulation has been established, Notch signalling represses atonal expression during lateral specification. In this paper we investigate which known components of the Notch pathway are involved in each signalling process. Using clonal analysis we show that a ligand of Notch, Delta, is required along with Notch for both proneural enhancement and lateral specification, while the downstream components Suppressor-of-Hairless and Enhancer-of-Split are involved only in lateral specification. Our data point to a distinct signal transduction pathway during proneural enhancement by Notch. Using misexpression experiments we also show that particular Enhancer-of-split bHLH genes can differ greatly in their contribution to lateral specification.     

Argos transcription is induced by the Drosophila EGF receptor pathway to form an inhibitory feedback loop

Argos is a secreted molecule with an atypical EGF motif. It was recently shown to function as an inhibitor of the signaling triggered by the Drosophila EGF receptor (DER). In this work, we determine the contribution of Argos to the establishment of cell fates in the embryonic ventral ectoderm. Graded activation of DER is essential for patterning the ventral ectoderm. argos mutant embryos show expansion of ventral cell fates suggesting hyperactivation of the DER pathway. In the embryonic ventral ectoderm, argos is expressed in the ventralmost row of cells. We show that argos expression in the ventral ectoderm is induced by the DER pathway: argos is not expressed in DER mutant embryos, while it is ectopically expressed in the entire ventral ectoderm following ubiquitous activation of the DER pathway. argos expression appears to be triggered directly by the DER pathway, since induction can also be observed in cell culture, following activation of DER by its ligand, Spitz. Argos therefore functions in a sequential manner, to restrict the duration and level of DER signaling. This type of inhibitory feedback loop may represent a general paradigm for signaling pathways inducing diverse cell fates within a population of non-committed cells.     

mirror encodes a novel PBX-class homeoprotein that functions in the definition of the dorsal-ventral border in the Drosophila eye

The Drosophila eye is composed of dorsal and ventral mirror-image fields of opposite chiral forms of ommatidia. The boundary between these fields is known as the equator. We describe a novel gene, mirror (mrr), which is expressed in the dorsal half of the eye and plays a key role in forming the equator. Ectopic equators can be generated by juxtaposing mrr expressing and nonexpressing cells, and the path of the normal equator can be altered by changing the domain of mrr expression. These observations suggest that mrr is a key component in defining the dorsal-ventral boundary of tissue polarity in the eye. In addition, loss of mrr function leads to embryonic lethality and segmental defects, and its expression pattern suggests that it may also act to define segmental borders. Mirror is a member of the class of homeoproteins defined by the human proto-oncogene PBX1. mrr is similar to the Iroquois genes ara and caup and is located adjacent to them in this recently described homeotic cluster.     

Epidermal growth factor (EGF) and EGF receptor in hypospadias

Environmental oncology is a discipline concerned with studies of multi-aspect relationships between environment and living organisms exposed to the modifying influence of carcinogenic agents. It also deals with general biological regularities involved in neoplasm development as well as their prevention in different species including man, animals and plants. Various investigations conducted at the Laboratory and supported with Russian and foreign grants (1991-1996) are briefly discussed. Among them are biotesting environmental carcinogens (aminoanthraquinons, by-products of drinking water chlorination, development of new testing systems and objects of detection involved in identification of genotoxic substances (criteria for formation of short-term test batteries and evaluation of perspectives, methods and results), investigation of xenobiotic metabolic activation (enzyme imprinting in adult animals), search for anticarcinogens (classification of carcinogenesis inhibitors, development of testing systems for modifiers selection), and establishing environment-related regularities of tumor growth. Vistas in environmental oncology development are discussed.     

Several levels of EGF receptor signaling during photoreceptor specification in wild-type, Ellipse, and null mutant Drosophila

Dominant Ellipse mutant alleles of the Drosophila EGF receptor homologue (DER) dramatically suppress ommatidium development in the eye and induce ectopic vein development in the wing. Their phenotype suggests a possible role for DER in specifying the founder R8 photoreceptor cells for each ommatidium. Here we analyze the basis of Ellipse mutations and use them to probe the role of DER in eye development. We show that Elp mutations result from a single amino acid substitution in the kinase domain which activates tyrosine kinase activity and MAP kinase activation in tissue culture cells. Transformant studies confirmed that the mutation is hypermorphic in vivo, but the DER function was elevated less than by ectopic expression of the ligand spitz. Ectopic spi promoted photoreceptor differentiation, even in the absence of R8 cells. Pathways downstream of DER activation were assessed to explore the basis of these distinct outcomes. Elp mutations caused overexpression of the Notch target gene E(spl) mdelta and required function of Notch to suppress ommatidium formation. The Elp phenotype also depended on the secreted protein argos and was reverted in Elp aos double mutants. Complete loss of DER function in clones of null mutant cells led to delay in R8 specification and subsequently to loss of mutant cells. The DER null phenotype was distinct from that of either spitz or vein mutants, suggesting that a combination of these or other ligands was required for aspects of DER function. In normal development DER protein was expressed in most retinal cells, but at distinct levels. We used an antibody specific for diphospho-ERK as well as expression of the DER target gene argos to assess the pattern of DER activity, finding highest activity in the intermediate groups of cells in the morphogenetic furrow. However, studies of mutant genotypes suggested that this activity may not be required for normal ommatidium development. Since we saw distinct phenotypic effects of four different levels of DER activity associated with wild-type, null mutant, Elp mutant, or fully activated DER function, we propose that multiple thresholds separate several aspects of DER function. These include activation of N signaling to repress R8 specification, turning on argos expression, and recruiting photoreceptors R1-R7. It is possible that during normal eye development these thresholds are attained by different cells, contributing to the pattern of retinal differentiation.     

decapentaplegic is required for arrest in G1 phase during Drosophila eye development

During eye development in Drosophila, cell cycle progression is coordinated with differentiation. Prior to differentiation, cells arrest in G1 phase anterior to and within the morphogenetic furrow. We show that Decapentaplegic (Dpp), a TGF-&bgr; family member, is required to establish this G1 arrest, since Dpp-unresponsive cells located in the anterior half of the morphogenetic furrow show ectopic S phases and ectopic expression of the cell cycle regulators Cyclins A, E and B. Conversely, ubiquitous over-expression of Dpp in the eye imaginal disc transiently inhibits S phase without affecting Cyclin E or Cyclin A abundance. This Dpp-mediated inhibition of S phase occurs independently of the Cyclin A inhibitor Roughex and of the expression of Dacapo, a Cyclin E-Cdk2 inhibitor. Furthermore, Dpp-signaling genes interact genetically with a hypomorphic cyclin E allele. Taken together our results suggest that Dpp acts to induce G1 arrest in the anterior part of the morphogenetic furrow by a novel inhibitory mechanism. In addition, our results provide evidence for a Dpp-independent mechanism that acts in the posterior part of the morphogenetic furrow to maintain G1 arrest.     

Human lens epithelial cells proliferate in response to exogenous EGF and have EGF and EGF receptor

While epidermal growth factor (EGF) has been found to exist in the human aqueous humor, which is necessary for maintaining the physiological condition of the lens, our immunohistochemical study confirmed the presence of EGF receptors and EGF in human lens epithelial cells (LECs), with EGF promoting growth potential and morphological change of the cells. Based on these findings, we feel that the effect of EGF upon human LECs is related to the movement of the cells in vivo.     

Abnormalities of the EGF receptor gene in brain tumors

In this prospective study, 30 patients with motor conversion disorder were assessed for key psychiatric and demographic variables. At reassessment 2 to 5 years later, 19 patients had completely recovered and 8 patients had improved, whereas only 3 were unchanged or worse. Contrary to other follow-up studies, none of the patients received a rediagnosis of neurological disease. The presence of a personality disorder and overall personality pathology, particularly within cluster C, the presence of a concomitant somatic disease, low DSM-IV Axis V score, and high score on the Becks Hopelessness Scale proved to be associated with poor outcome.     

Expression of EGF receptor subfamily in tumor

The members of EGF receptor subfamily are transmembrane glycoproteins with tyrosine kinase activity. Once EGF or EGF-related peptides binds to the receptors, they undergo autophosphorylation and binding to the SH2 protein, which in turn activates the intracellular signaling cascade. In the squamous carcinoma of head and neck, esophagus and lung, the EGF receptor gene amplification and EGF receptor hyperproduction are frequently observed. In the adenocarcinoma of pancreas, breast and stomach, hyperproduction of the EGF receptor subfamily is common, suggesting the involvement of these growth factor receptors in the tumorigenesis. The prognostic value of EGF receptor hyperproduction appears considerable when combined with other factors. EGF receptor subfamily members are useful targets for the immunotoxin therapy and immunogene therapy.     

The Drosophila embryonic midline is the site of Spitz processing, and induces activation of the EGF receptor in the ventral ectoderm

The Drosophila EGF receptor (DER) is activated by secreted Spitz to induce different cell fates in the ventral ectoderm. Processing of the precursor transmembrane Spitz to generate the secreted form was shown to be the limiting event, but the cells in which processing takes place and the mechanism that may generate a gradient of secreted Spitz in the ectoderm were not known. The ectodermal defects in single minded (sim) mutant embryos, in which the midline fails to develop, suggested that the midline cells contribute to patterning of the ventral ectoderm. This work shows that the midline provides the site for Spitz expression and processing. The Rhomboid and Star proteins are also expressed and required in the midline. The ectodermal defects of spitz, rho or Star mutant embryos could be rescued by inducing the expression of the respective normal genes only in the midline cells. Rho and Star thus function non-autonomously, and may be required for the production or processing of the Spitz precursor. Secreted Spitz is the only sim-dependent contribution of the midline to patterning the ectoderm, since the ventral defects observed in sim mutant embryos can be overcome by expression of secreted Spitz in the ectoderm. While ectopic expression of secreted Spitz in the ectoderm or mesoderm gave rise to ventralization of the embryo, increased expression of secreted Spitz in the midline did not lead to alterations in ectoderm patterning. A mechanism for adjustment to variable levels of secreted Spitz emanating from the midline may be provided by Argos, which forms an inhibitory feedback loop for DER activation. The production of secreted Spitz in the midline, may provide a stable source for graded DER activation in the ventral ectoderm.