RESUMO
BACKGROUND: Asymmetric cell division (ACD) is a key process that allows different cell types to be generated at precisely defined times and positions. In Drosophila, neural precursor cells rely heavily on ACD to generate the different cell types in the nervous system. A conserved protein machinery that regulates ACD has been identified in Drosophila, but how this machinery acts to allow the establishment of differential cell fates is not entirely understood. RESULTS: To identify additional proteins required for ACD, we have carried out an in vivo live imaging RNAi screen for genes affecting the asymmetric segregation of Numb in Drosophila sensory organ precursor cells. We identify Banderuola (Bnd), an essential regulator of cell polarization, spindle orientation, and asymmetric protein localization in Drosophila neural precursor cells. Genetic and biochemical experiments show that Bnd acts together with the membrane-associated tumor suppressor Discs-large (Dlg) to establish antagonistic cortical domains during ACD. Inhibiting Bnd strongly enhances the dlg phenotype, causing massive brain tumors upon knockdown of both genes. Because the mammalian homologs of Bnd and Dlg are interacting as well, Bnd function might be conserved in vertebrates, and it might also regulate cell polarity in higher organisms. CONCLUSIONS: Bnd is a novel regulator of ACD in different types of cells. Our data place Bnd at the top of the hierarchy of the factors involved in ACD, suggesting that its main function is to mediate the localization and function of the Dlg tumor suppressor. Bnd has an antioncogenic function that is redundant with Dlg, and the physical interaction between the two proteins is conserved in evolution.
Assuntos
Divisão Celular Assimétrica/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Proteínas Supressoras de Tumor/genética , Animais , Proteínas de Drosophila/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Microscopia Confocal , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Reação em Cadeia da Polimerase , Pupa/genética , Pupa/crescimento & desenvolvimento , Proteínas Supressoras de Tumor/metabolismoRESUMO
In Drosophila, planar cell polarity (PCP) molecules such as Dachsous (Ds) may function as global directional cues directing the asymmetrical localization of PCP core proteins such as Frizzled (Fz). However, the relationship between Ds asymmetry and Fz localization in the eye is opposite to that in the wing, thereby causing controversy regarding how these two systems are connected. Here, we show that this relationship is determined by the ratio of two Prickle (Pk) isoforms, Pk and Spiny-legs (Sple). Pk and Sple form different complexes with distinct subcellular localizations. When the amount of Sple is increased in the wing, Sple induces a reversal of PCP using the Ds-Ft system. A mathematical model demonstrates that Sple is the key regulator connecting Ds and the core proteins. Our model explains the previously noted discrepancies in terms of the differing relative amounts of Sple in the eye and wing.
Assuntos
Caderinas/metabolismo , Polaridade Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas com Domínio LIM/metabolismo , Animais , Caderinas/genética , Olho Composto de Artrópodes/citologia , Olho Composto de Artrópodes/metabolismo , Proteínas de Ligação a DNA/genética , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas com Domínio LIM/genética , Modelos Genéticos , Especificidade de Órgãos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Asas de Animais/citologia , Asas de Animais/metabolismoRESUMO
Genome-wide RNA interference (RNAi) screens have identified near-complete sets of genes involved in cellular processes. However, this methodology has not yet been used to study complex developmental processes in a tissue-specific manner. Here we report the use of a library of Drosophila strains expressing inducible hairpin RNAi constructs to study the Notch signalling pathway during external sensory organ development. We assigned putative loss-of-function phenotypes to 21.2% of the protein-coding Drosophila genes. Using secondary assays, we identified 6 new genes involved in asymmetric cell division and 23 novel genes regulating the Notch signalling pathway. By integrating our phenotypic results with protein interaction data, we constructed a genome-wide, functionally validated interaction network governing Notch signalling and asymmetric cell division. We used clustering algorithms to identify nuclear import pathways and the COP9 signallosome as Notch regulators. Our results show that complex developmental processes can be analysed on a genome-wide level and provide a unique resource for functional annotation of the Drosophila genome.