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 HFA, HLLAA and HWA. For in vivo analysis, UAS-lines HFA, HLLAA and HWA were generated to allow spatially restricted overexpression. In these assays, all three mutants resembled the HLD 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.
KeywordsDrosophila melanogaster Notch antagonist Hairless Notch signalling Notch repression Repressor complex Suppressor of Hairless Overexpression
We are indebted to Rafael Saup for establishing the NTCTLL245/247AA replacement mutation, to Adriana Schulz, Helena Mastel and Thomas Stößer for technical assistance and to Anja C. Nagel for critical reading of the manuscript, manifold input and discussions.
D.M. and A.P. conceived and designed the experiments; T.K.S., M.M., and D.M. conducted the experiments, T.K.S., M.M., A.P. and D.M. collected and analysed the data and performed the statistical analysis; A.P. wrote the manuscript; and all authors have approved the final manuscript.
This work was supported by grants from the German Science Foundation to DM (MA 1328/10-1 and MA 1328/11-1). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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- Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401–415Google Scholar
- de Celis JF (1998) Positioning and differentiation of veins in the Drosophila wing. Int J Dev Biol 42:335–344Google Scholar
- de Celis JF, Bray SJ (1997) Feed-back mechanisms affecting Notch activation at the dorsoventral boundary in the Drosophila wing. Development 124:3241–3251Google Scholar
- Go MJ, Eastman DS, Artavanis-Tsakonas S (1998) Cell proliferation control by Notch signaling in Drosophila development. Development 125:2031–2040Google Scholar
- Hay BA, Wolff T, Rubin GM (1994) Expression of baculovirus P35 prevents cell death in Drosophila. Development 120:2121–2129Google Scholar
- Lindsley D, Zimm GG (1992) The genome of Drosophila melanogaster. Academic Press, San DiegoGoogle Scholar
- Praxenthaler H, Smylla TK, Nagel AC, Preiss A, Maier D (2015) Generation of new Hairless alleles by genomic engineering at the Hairless locus in Drosophila melanogaster. PLoS ONE 10(10):e0140007Google Scholar