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Cell fate is determined by the coordinated activity of different pathways, including the conserved Notch pathway. Activation of Notch results in the transcription of Notch targets that are otherwise silenced by repressor complexes. In Drosophila, the repressor complex comprises the transcription factor Suppressor of Hairless (Su(H)) bound to the Notch antagonist Hairless (H) and the general co-repressors Groucho (Gro) and C-terminal binding protein (CtBP). The latter two are shared by different repressors from numerous pathways, raising the possibility that they are rate-limiting. We noted that the overexpression during wing development of H mutants HdNT and HLD compromised in Su(H)-binding induced ectopic veins. On the basis of the role of H as Notch antagonist, overexpression of Su(H)-binding defective H isoforms should be without consequence, implying different mechanisms but repression of Notch signaling activity. Perhaps excess H protein curbs general co-repressor availability. Supporting this model, nearly normal wings developed upon overexpression of H mutant isoforms that bound neither Su(H) nor co-repressor Gro and CtBP. Excessive H protein appeared to sequester general co-repressors, resulting in specific vein defects, indicating their limited availability during wing vein development. In conclusion, interpretation of overexpression phenotypes requires careful consideration of possible dominant negative effects from interception of limiting factors.

Background The Notch signaling pathway is fundamental to the regulation of many cell fate decisions in eumetazoans. Not surprisingly, members of this pathway are highly conserved even between vertebrates and invertebrates. There is one notable exception, Hairless, which acts as a general Notch antagonist in Drosophila. Hairless silences Notch target genes by assembling a repressor complex together with Suppressor of Hairless [Su(H)] and the co-repressors Groucho (Gro) and C-terminal binding protein (CtBP). Now with the availability of genomic databases, presumptive Hairless homologues are predicted, however only in insect species. To further our understanding of Hairless structure and function, we have cloned the Hairless gene from Apis mellifera (A.m.H) and characterized its functional conservation in Drosophila. Results The Apis Hairless protein is only one third of the size of the Drosophila orthologue. Interestingly, the defined Suppressor of Hairless binding domain is interrupted by a nonconserved spacer sequence and the N-terminal motif is sufficient for binding. In contrast to Apis Hairless, the Drosophila orthologue contains a large acidic domain and we provide experimental evidence that this acidic domain is necessary to silence Hairless activity in vivo. Despite the dramatic size differences, Apis Hairless binds to the Drosophila Hairless interactors Su(H), Gro, CtBP and Pros26.4. Hence, Apis Hairless assembles a repressor complex with Drosophila components that may have a different topology. Nevertheless, Apis Hairless is sufficient to repress the Notch target gene vestigial in Drosophila. Moreover, it is able to rescue Hairless mutant phenotypes, providing in vivo evidence for its function as a bona fide Notch antagonist. Conclusion This is the first interspecies-complementation analysis of the Hairless gene. Guided by evolutionary comparisons, we hope to eventually identify all the relevant structural domains and cofactors of Hairless, thereby opening an avenue for further insights into the repressor-complexes that down-regulate Notch signaling also in other, higher eukaryotes.