A Genetic Screen in Drosophila for Identifying Novel Components of the Hedgehog Signaling Pathway

Abstract

The Hedgehog signaling pathway plays an essential role in the pattern formation and development of metazoan animals. Misregulation of Hedgehog signaling has also been associated with the formation of multiple types of cancer. For these reasons, the Hedgehog pathway has attracted considerable interest. Many proteins required in the Hedgehog pathway have been identified, and while much has been learned about their function in signal transduction, it is clear that this complement of proteins does not comprise the full set necessary for Hedgehog signal transduction. Because significant gaps remain in our knowledge of the molecules required for Hedgehog signaling, we performed an enhancer/suppressor screen in Drosophila melanogaster to identify novel components of the pathway. In addition to the isolation of new alleles of the known pathway components patched and smoothened, this screen identified 14 novel complementation groups and a larger number of loci represented by single alleles. These groups include mutations in the genes encoding the translation factors eRF1 and eIF1A and the kinesin-like protein Pavarotti. It also identified mutations in a gene whose product is necessary for the movement of Hedgehog protein through tissues.

A Screen for Modifiers of Hedgehog Signaling inDrosophila melanogasterIdentifiesswmandmts

“…RNAi lines were screened using both ptcGAL4 P{WIZ-smo} (in which smo activity is reduced in the Hh-receiving cells) and y 1 w*; C765GAL4 UAS-smo 5A (in which a dominant negative allele of smo is expressed in both anterior and posterior compartment cells, causing a reduction in wing size (Collins and Cohen 2005). Any mutant alleles, RNAi lines, or minimal deficiencies found to enhance or suppress both smo RNAi and smo 5a were then tested for nonspecific effects in a cross to y 1 w*; P{GAL4-vg.M} 2 pUAS-cutRNAi 1 (generously provided by Wesley Gruber and Yuh-Nun Jan), which produces a jagged wing-blade margin.…”

“…Expression of a dominant negative Smo protein, Smo 5A (Collins and Cohen 2005), causes wing vein fusions that are similar to smo RNAi. This mutant protein has alanines substituted for serines and threonines in the C-terminal cytoplasmic tail that are phosphorylated upon Hh activation, and its expression in the wing partially blocks Hh signaling (Collins and Cohen 2005).…”

“…1 standing of how the presence of Hh at the cell surface leads to changes in expression of target genes, we know that the responses of Smo, Cos2, and Ci to Hh are mediated, in part, by phosphorylation. Smo requires several phosphorylation sites in its C-terminal tail for activity (Chen et al 1998;Price and Kalderon 2002;Jia et al 2004;Collins and Cohen 2005), phosphorylation of Cos2 by Fu promotes cell surface accumulation and stabilization of Smo (Nybakken et al 2002;Lu et al 2006), and Ci phosphorylation is required for conversion to either its repressor or activator forms (Chen et al 1998;Price and Kalderon 2002).…”

“…Published genetic screens were based on the wing phenotypes of known pathway mutants (Vegh and Basler 2003), on suppression of a gain of function hh mutant (Haines and van den Heuvel 2000), or on enhancement or suppression of a partial loss-of-function phenotype generated by a dominant negative form of Smo (Collins and Cohen 2005). These screens identified a new class of ptc mutants (Vegh and Basler 2003), mutations that affect Hh stability (Haines and van den Heuvel 2000), and implicated the translation factors eRF1 and elF1A and the kinesin-like protein Pavarotti in the Hh response (Collins and Cohen 2005). Genomewide RNA interference (RNAi) assays in cultured cells identified many functions that contribute to Hh responses (Lum et al 2003;Nybakken et al 2005).…”

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.…”