Abstract
Ed is a putative cell-adhesion molecule (CAM), which contains 7 immunoglobulin (Ig) domains, 2 fibronectin type III (Fn III) domains, and a transmembrane (TM) domain, followed by a 315 amino acid intracellular domain with no identifiable structural or functional amino acid motif whose last four amino acids (EIIV) are responsible for non-typical PDZ binding. Polarized epithelial cells form a monolayer in which the apical is separated with the lateral and basal membrane by the apical junctional complex. Apical junctional comeplex first initiates cell-cell adhesion which in turn activates protein-sorting mechanism for establishing cell polarity and maintaining cell integrity. Adherens junctions (AJs) are mainly targeted by of DE-cadherin, β-catenin/Armadillo (arm), and α-catenin. DE-cadherin form homophilic binding via its extracellular Ig domains while its cytoplasmic region.bind with Arm and α-catenin linking membrane receptors to actin polymers. DE-cadherin complex therefore regulates cell-cell adhesion. To investigate the canonical role of Ed as a CAM, we performed the mosaic analysis and immunochemistry staining in Drosophila wing imaginal disc. The results showed that Ed is colocalized with DE-cadherin, and Arm at the AJs and ed clones exhibit rounded and smooth countour indicating Ed－ cells are sorted out. Removing of Ed from AJs will cause wing disc cells to have greatly reduced apical surface and highly accumulation of DE-cadherin, Arm, and actin at AJs to hold the Ed－ cells together. Based on above results, it was shown that the affinity between Ed－ and Ed－ is higher than Ed－ and Ed＋. Similarly, removing of DE-cadherin will also produce cells with smaller apical surface but elevated Ed and the PDZ-containing protein Bazooka (Baz) at the position of AJs. Yeast two-hybrid analyses and GST pull down assay illustrate that Baz can both bind with Ed and Arm via its PDZ domain, thereby locating to AJs by Ed- or Arm-dependant mechanism. Together, these results indicate that Ed is an essential component of AJs and cooperates with DE-cadherin to mediate cell-cell adhesion. Ed has multiple functions during Drosophila development. Previous studies found that Ed defines a novel pathway which limits R8 photoreceptor specification by inhibiting inappropriate EGFR signaling within R8 equivalence groups. Consequently, flies possess exact number of the photoreceptor and cone cells. Genetic mosaic analysis shows that Ed exerts its function by homophilic binding. In this study, co-expression of Ed and Neuroglian (Nrg), a L1-type CAM, in eye exhibits a strong genetic synergy in inhibiting EGFR signaling. This synergistic effect requires the intracellular domain of Ed, but not that of Nrg. Cell aggregation test and immunoprecipitation results show that Ed not only form homophilic binding but also engages heterophilic trans-interaction with Nrg. Furthermore, Ed co-localizes with Nrg in eye imaginal disc. Together, our results suggest a model in which Nrg acts as a heterophilic ligand and activator of Ed which in turn antagonizes EGFR signaling. Besides ommatidium specification, Ed also involves in mesothoracic bristle development. Loss-of-function of ed leads to the formation of extra macrochaetae near the extant ones and increases the density of microchaetae. Analysis of ed mosaics indicates that extra sensory organ precursors (SOPs) arise from proneural clusters of achaete-scute expression in a cell-autonomous way. ed embryos also exhibit a neurogenic phenotype. These phenotypes suggest a functional relation between ed and the Notch (N) pathway. Indeed, mutation of ed reduces the expression level of the N pathway effector E(spl)m8 in proneural clusters. Moreover, combinations of moderate loss of function for ed and for different components of N pathway show clear synergistic effects on neurogenic bristles phenotypes. We conclude that Ed facilitates N pathway in mesothoracic bristle development, but not like its inhibiting EGFR signal in eye development. However, EGFR also acts in bristle development. We present that Ed antagonizes the bristle promoting activity of the Egfr pathway, either by enhancement of N pathway or, similar to the eye, by more direct effect on EGFR signal. E-cadherin is also a tumor suppressor gene in human. Disruption of E-cadherin-meditated adhesion is a key step in progression toward invasive phase of carcinoma. Loss-of-function conditions for ed and DE-cadherin cause silimar phenotypes. Ed involves in cell-cell adhesion that means Ed also play a role in tumorigenesis and metastasis? Furthermore, ed embryo exhibit extra C.N.S. and P.N.S. neurons. Ed also serves in Drosophila eye development and mesothoracic bristle development. Photoreceptor cells of the ommatidium and bristles are sensory nerves. We suggest that Ed might meditate the development of the nervous tissues.