ABSTRACT
The Notch signaling pathway plays an important role in development and physiology. In Drosophila, Notch is activated by its Delta or Serrate ligands, depending in part on the sugar modifications present in its extracellular domain. O-fucosyltransferase-1 (OFUT1) performs the first glycosylation step in this process, O-fucosylating various EGF repeats at the Notch extracellular domain. Besides its O-fucosyltransferase activity, OFUT1 also behaves as a chaperone during Notch synthesis and is able to down regulate Notch by enhancing its endocytosis and degradation. We have reevaluated the roles that O-fucosylation and the synthesis of GDP-fucose play in the regulation of Notch protein stability. Using mutants and the UAS/Gal4 system, we modified in developing tissues the amount of GDP-mannose-deshydratase (GMD), the first enzyme in the synthesis of GDP-fucose. Our results show that GMD activity, and likely the levels of GDP-fucose and O-fucosylation, are essential to stabilize the Notch protein. Notch degradation observed under low GMD expression is absolutely dependent on OFUT1 and this is also observed in Notch Abruptex mutants, which have mutations in some potential O-fucosylated EGF domains. We propose that the GDP-fucose/OFUT1 balance determines the ability of OFUT1 to endocytose and degrade Notch in a manner that is independent of the residues affected by Abruptex mutations in Notch EGF domains.
Subject(s)
Animals , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Fucosyltransferases/metabolism , Guanosine Diphosphate Fucose/metabolism , Guanosine Diphosphate Mannose/metabolism , Receptors, Notch/metabolism , Wings, Animal/metabolism , Alleles , Drosophila Proteins/genetics , Drosophila melanogaster/anatomy & histology , Drosophila melanogaster/metabolism , Endocytosis/genetics , Fucosyltransferases/genetics , Guanosine Diphosphate Fucose/genetics , Guanosine Diphosphate Mannose/genetics , Immunohistochemistry , In Situ Hybridization , Intracellular Signaling Peptides and Proteins/genetics , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation/genetics , Phenotype , Reverse Transcriptase Polymerase Chain Reaction , Receptors, Notch/genetics , Signal Transduction , Wings, Animal/anatomy & histologyABSTRACT
The neural crest is induced at the border between the neural plate and the epidermis. A complex set of signals is required for the specification of the crest cells between the epidermis and the neural plate. Here we discuss evidence supporting a model for neural crest induction in which different signals contribute in a sequential order. First, a gradient of bone morphogenic proteins (BMPs) is established in the ectoderm that results in segregation into neural plate, neural folds and epidermis at increasing levels of BMP activity. Thus, the neural folds are induced at a precise threshold concentration of BMP, but this neural fold has an anterior character. In a second step, these anterior neural folds are transformed into prospective neural crest by posteriorizing signals due to fibroblast growth factor, Wnts and retinoic acid. Finally, the induced cells interact to complete neural crest induction by a process that requires Notch/Delta signaling. Once neural crest formation has been induced by this combination of extracellular and intracellular signals, a cascade of transcription factors is activated in these cells that culminates in the ultimate steps of neural crest differentiation (AU)#SP#