A Screen for Genes Regulating the Wingless Gradient in Drosophila Embryos

Abstract

During the development of the Drosophila embryonic epidermis, the secreted Wingless protein initially spreads symmetrically from its source. At later stages, Wingless becomes asymmetrically distributed in a Hedgehog-dependent manner, to control the patterning of the embryonic epidermis. When Wingless is misexpressed in engrailed cells in hedgehog heterozygous mutant embryos, larvae show a dominant phenotype consisting of patches of naked cuticle in denticle belts. This dose-sensitive phenotype is a direct consequence of a change in Wg protein distribution. We used this phenotype to carry out a screen for identifying genes regulating Wingless distribution or transport in the embryonic epidermis. Using a third chromosome deficiency collection, we found several genomic regions that showed a dominant interaction. After using a secondary screen to test for mutants and smaller deficiencies, we identified three interacting genes: dally, notum, and brahma. We confirmed that dally, as well as its homolog dally-like, and notum affect Wingless distribution in the embryonic epidermis, directly or indirectly. Thus, our assay can be used effectively to screen for genes regulating Wingless distribution or transport. S ECRETED signaling molecules play an essential role distribution (Bellaiche et al. 1998;Takei et al. 2003; Han et al. 2004a,b). in patterning developing metazoans. Work from reGradient formation also depends upon the mechacent years shows that the signaling activity of these molenism by which signaling ligands move in the plane of cules is tightly regulated (Freeman 2000). Among variepithelia. It is not yet clear what these mechanisms are, ous levels of regulation, controlling the distribution of but two main modes of transport have been proposed ligands in a field of cells is necessary to establish stable (reviewed by Vincent and Dubois 2002; Gonzalezgradients that robustly pattern developing tissues (VinGaitan 2003): (1) facilitated diffusion, where ligands cent and Dubois 2002). The distribution of ligands diffuse within the extracellular space, and this diffusion could conceivably be influenced by many factors, includis modulated by cell-surface receptors, and (2) transing the concentration of receptors, the composition of cytosis, where ligands are transported along the plane the extracellular matrix, or the rate of recycling and of the epithelium by repeated cycles of endocytosis and degradation of the ligand following internalization. The recycling to the cell surface. best-documented case is the regulation of ligand distriIn this study, we searched for genes that influence bution by receptors. For example, in Drosophila, Hedgethe distribution or transport of the signaling ligand hog (Hh) range of action is regulated by its receptor Wingless (Wg), the homolog of vertebrate Wnt-1 in DroPatched: if the Hh-binding domain of Patched is musophila. In the embryonic epidermis, Wg acts at a short tated, the spread of Hh is extended in the wing imaginal range to regulate the activity of target genes, wherea...

Wnt/Wingless Signaling in Drosophila

“…In deep orange (dor) mutants that have impaired trafficking to the lysosome, Wg accumulates in multivesicular bodies (Piddini et al 2005). Lastly, in clathrin heavy chain (chc) mutants that cannot initiate clathrin-mediated endocytosis, the Wg gradient extends in the posterior direction, suggesting that clathrin is normally required for establishing and maintaining the asymmetric distribution of Wg (Desbordes et al 2005). However, there is also contradictory evidence that suggests a role for endocytosis in Wg dispersal in the embryo.…”

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

“…In these cases, the screen aims to identify genes belonging to a pre-determined set of interacting genes. Some examples of successful screens aiming to identify members of known signalling pathways are those targeting the Sevenless and EGFR (Karim et al, 1996;Huang and Rubin, 2000;Taguchi et al, 2000;Rebay et al, 2000), Notch (Verheyen et al, 1996;Go and Artavanis-Tsakonas, 1998;Muller et al, 2005a), Dpp (Raftery et al, 1995;Chen et al, 1998;Su et al, 2001), JAK/STAT (Bach et al, 2003;Mukherjee et al, 2006), Hh (Haines and van den Heuvel, 2000;Collins and Cohen, 2005), TNF (Geuking et al, 2005) and Wnt (Greaves et al, 1999;Cox et al, 2000;Desbordes et al, 2005) pathways. Although the use of genetic screens in vivo has many advantages, they are time-consuming and difficult to escalate genome-wide.…”

A cautionary tale on genetic screens based on a gain-of-expression approach: The case of LanB1

“…These screens, aimed to identify enhancers and suppressors of a particular phenotype, have been successfully used, for example, for the identification of additional components of signaling pathways. [14][15][16][17][18][19] A second and general limitation of the loss-of-function approach is the labor involved in the mapping of mutations, which generally involves the use of large collections of deficiencies, and complementation analysis that are not yet suited to the mapping of large numbers of newly generated mutants.…”