A screen for dominant mutations applied to components in the Drosophila EGF-R pathway

Abstract

The Drosophila epidermal growth factor receptor (EGF-R) controls many critical cell fate choices throughout development. Several proteins collaborate to promote localized EGF-R activation, such as Star and Rhomboid (Rho), which act sequentially to ensure the maturation and processing of inactive membrane-bound EGF ligands. To gain insights into the mechanisms underlying Rho and Star function, we developed a mutagenesis scheme to isolate novel overexpression activity (NOVA) alleles. In the case of rho, we isolated a dominant neomorphic allele, which interferes with Notch signaling, as well as a dominant-negative allele, which produces RNA interference-like flip-back transcripts that reduce endogenous rho expression. We also obtained dominant-negative and neomorphic Star mutations, which have phenotypes similar to those of rho NOVA alleles, as well as dominant-negative Egf-r alleles. The isolation of dominant alleles in several different genes suggests that NOVA mutagenesis should be widely applicable and emerge as an effective tool for generating dominant mutations in genes of unknown function.

CLAVATA1 Dominant-Negative Alleles Reveal Functional Overlap between Multiple Receptor Kinases That Regulate Meristem and Organ Development

“…Interestingly, the mutations in clv1-1, clv1-2, and clv1-9 lie within or close to the activation loop in the kinase domain (Torii and Clark, 2000). Some mutations in animals are comparable to these: the EGFR DN3 mutation is located in domain VIb, and the bmpr2 mutation is located between domains VIb and VII (Zou and Niswander, 1996;Guichard et al, 2002;Rudarakanchana et al, 2002). Studies on bmpr2 showed that this dominant-negative mutant is not expressed at the plasma membrane but instead is retained in the endoplasmic reticulum (Rudarakanchana et al, 2002).…”

“…Deletion of all or part of the cytoplasmic domain of the receptor-Tyr kinase epidermal growth factor receptors or other receptor-Tyr kinases results in dominant-negative mutations (Kashles et al, 1991;Chen et al, 1993;Prager et al, 1994;Guichard et al, 2002). The two CLV1 alleles that mimic this kind of mutation, clv1-6 and clv1-7, exhibit a weak phenotype similar to that of null alleles.…”

GAL4 system in drosophila: A fly geneticist's swiss army knife

“…This approach has been used successfully for many Drosophila genes and is also being used to address the roles of genes involved in human disease. In a slight twist on this approach, one can also isolate alleles of the gene of interest (X) by directly mutagenizing the strain carrying the UAS-gene X responder (Guichard et al, 2002;Penton et al, 2002).…”

Signalling Pathways in Development and Human Disease: A Drosophila Wing Perspective

“…The rationale of these experiments is straightforward: mutations affecting the same signalling pathway result in a similar phenotype and in general display genetic interactions. Thus, exhaustive genetic screens aimed to identify www.intechopen.com genes regulating embryonic segmentation in flies were instrumental to identify many components of the Notch, BMP, Hh and Wnt pathways (Nusslein-Volhard and Wieschaus, 1980), and genetic screens carried out in sensitized genetic backgrounds resulted in the identification of additional components of these pathways and also of the EGFR and InR pathways (Greaves et al, 1999;Rebay et al, 2000;Huang and Rubin, 2000;Guichard et al, 2002;Mahoney et al, 2006). More recently, mosaic screens in adult structures of the fly uncovered the SWH pathway, because of its contribution to the regulation of cell proliferation, competition and apoptosis (Cho et al, 2006;Tapon, 2007 Tyler et al, 2007).…”