Anterior-Posterior Axis Specification in Drosophila Oocytes: Identification of Novel Bicoid and Oskar mRNA Localization Factors
Chang, Chin-Wen, Nashchekin, Dmitry, Wheatley, Lucy, Irion, Uwe, Dahlgaard, Katja, Montague, Tessa G., Hall, Jacqueline, St Johnston, Daniel, Genetics
ABSTRACT The Drosophila melanogaster anterior-posterior axis is established during oogenesis by the localization of bicoid and oskar mRNAs to the anterior and posterior poles of the oocyte. Although genetic screens have identified some trans-acting factors required for the localization of these transcripts, other factors may have been missed because they also function at other stages of oogenesis. To circumvent this problem, we performed a screen for revertants and dominant suppressors of the bicaudal phenotype caused by expressing Miranda-GFP in the female germline. Miranda mislocalizes oskar mRNA/Staufen complexes to the oocyte anterior by coupling them to the bicoid localization pathway, resulting in the formation of an anterior abdomen in place of the head. In one class of revertants, Miranda still binds Staufen/oskar mRNA complexes, but does not localize to the anterior, identifying an anterior targeting domain at the N terminus of Miranda. This has an almost identical sequence to the N terminus of vertebrate RHAMM, which is also a large coiled-coil protein, suggesting that it may be a divergent Miranda ortholog. In addition, we recovered 30 dominant suppressors, including multiple alleles of the spectroplakin, short stop, a lethal complementation group that prevents oskar mRNA anchoring, and a female sterile complementation group that disrupts the anterior localization of bicoid mRNA in late oogenesis. One of the single allele suppressors proved to be a mutation in the actin nucleator, Cappuccino, revealing a previously unrecognized function of Cappuccino in pole plasm anchoring and the induction of actin filaments by Long Oskar protein.
THE subcellular localization of mRNAs is an important mechanism for restricting specific proteins to the region of a cell where they are required and plays a key role in axis formation, synaptic plasticity, and cell polarity (St. Johnston 2005; Becalska and Gavis 2009). Indeed, nearly 70% of all tested mRNAs show some pattern of localization in the early Drosophila embryo, indicating this is a widespread means for protein targeting (Lécuyer et al. 2007). mRNAs can be delivered to the correct destination in a variety of ways, such as local protection from degradation, diffusion and anchoring, or active transport along either the actin or microtubule cytoskeletons. One of the best-characterized systems for studying the latter mechanism is the formation of the anterior- posterior axis in Drosophila, which is specified by the microtubule-dependent localization of bicoid mRNA to the anterior of the oocyte and of oskar mRNA to the posterior (Bastock and St. Johnston 2008). bicoid mRNA is translated at the anterior after egg activation to produce a protein gradient that acts as a morphogen to pattern the head and thorax of the embryo (Ephrussi and Johnston 2004). oskar mRNA, on the other hand, is translated once it is localized to the posterior pole of the oocyte, where Oskar protein defines the site of pole plasm assembly, leading to the posterior recruitment of the abdominal determinant, nanos RNA (Ephrussi et al. 1991; Kim-Ha et al. 1991).
Mutants that disrupt the localization of bicoid mRNA produce embryos with defective heads, whereas oskar mRNA localization mutants result in embryos without pole cells or an abdomen, and this has allowed the identification of a number of trans-acting factors in screens for maternal-effect lethal mutations (Nusslein-Volhard et al. 1987; Schupbach and Wieschaus 1989). One limitation of these screens is that they could not identify zygotic lethal mutations in essential genes. This problem can be circumvented, however, by using the FLP/FRT system to perform screens in germ line clones, and additional factors have been identified in such screens for mutants with embryonic patterning phenotypes, as well as more targeted screens for mutations that disrupt the localization of GFP-Staufen, an RNA-binding protein that associates with oskar and bicoid mRNAs (Chou and Perrimon 1996; Martin et al. …