The Notch repressor complex in Drosophila: in vivo analysis of Hairless mutants using overexpression experiments

During development of higher animals, the Notch signalling pathway governs cell type specification by mediating appropriate gene expression responses. In the absence of signalling, Notch target genes are silenced by repressor complexes. In the model organism Drosophila melanogaster, the repressor complex includes the transcription factor Suppressor of Hairless [Su(H)] and Hairless (H) plus general co-repressors. Recent crystal structure analysis of the Drosophila Notch repressor revealed details of the Su(H)-H complex. They were confirmed by mutational analyses of either protein; however, only Su(H) mutants have been further studied in vivo. Here, we analyse three H variants predicted to affect Su(H) binding. To this end, amino acid replacements Phenylalanine 237, Leucines 245 and 247, as well as Tryptophan 258 to Alanine were introduced into the H protein. A cell-based reporter assay indicates substantial loss of Su(H) binding to the respective mutant proteins H^FA, H^LLAA and H^WA. For in vivo analysis, UAS-lines H^FA, H^LLAA and H^WA were generated to allow spatially restricted overexpression. In these assays, all three mutants resembled the H^LD control, shown before to lack Su(H) binding, indicating a strong reduction of H activity. For example, the H variants were impaired in wing margin formation, but unexpectedly induced ectopic wing venation. Concurrent overexpression with Su(H), however, suggests that all mutant H protein isoforms are still able to bind Su(H) in vivo. We conclude that a weakening of the cohesion in the H-Su(H) repressor complex is sufficient for disrupting its in vivo functionality.