dominant gain-of-function hh allele that disrupts patterning of the wing as a heterozygote and is lethal as a homozygote. In wild type wing discs, hh expression is confined to the posterior compartment and it orchestrates wing development by signaling to cells in the anterior compartment along the compartment boundary to upregulate the expression target genes such as ptc and decapentaplegic (dpp). In hhMrt/+ animals, in addition to being expressed normally in the posterior compartment, hh is ectopically activated in the anterior compartment of the wing disc. As a result dpp is expressed in a pattern that leads to overgrowth of the anterior tissues and the partial duplication of distal wing structures. The anterior-to- posterior transformations induced by the hhMrt allele can be dominantly suppressed by mutations in hh signaling pathway genes like disp and hmgcr that are required to promote hh signaling in the sending cells. The gain-of-function wing phenotype can also be suppressed by mutations in genes like toutvelu (ttv) that are required to promote hh signaling in the receiving cell (unpublished data). If gc1 functions as the downstream target for hmgcr in the hh signaling pathway, then mutations in gc1 would be expected to dominantly suppress the hhMrt wing defects. To test for suppression we used two different gc1 mutants. The first, gc1N159, is an EMS induced mutation [28]. The gc1 open reading frame encodes a protein of 70 amino acids and this mutation inserts a stop codon at amino acid 59. The second, gc1k0817, has a P-element insertion in Gc1 in Hh Signaling and Germ Cell Migration the splice donor of the first gc1 exon and produces aberrant transcripts. To assess the effects of these gc1 mutations, the Mrt wing blades were assigned to 5 different classes based on the severity of the wing defects, with I being wild type (not shown), and V being the most severely deformed wing (not shown, for details see 27). Under the conditions of this experiment about 70% of the hhMrt/+ flies were abnormal (see the class III wing in panel A of Figure 1). By contrast when the hhMrt/+ flies were heterozygous for gc1N159 quite strong suppression was observed and more than 80% of the wings belonged to class I (panel B). The suppression of the Mrt gain-of-function phenotype does not appear to be due to some non-specific background effect as the wing defects could also be dominantly suppressed by gc1k0817 (data not shown). Thus like hmgcr and other factors that function to promote hh signaling, gc1 shows genetic interactions with the hhMrt. Moreover, the extent of suppression is similar to that observed previously with hmgcr [14]. gc1 Is Required To Maintain wg and en Expression The dominant suppression of the Mrt wing phenotypes suggests that like hmgcr, gc1 functions in hh signaling. To test this possibility further we examined wingless (wg) expression during embryogen- esis. In wild type embryos, wg stripes are activated by the pair-rule genes at the onset of gastrulation. Once the pair-rule gene products decay later in embryogenesis the maintenance of the wg Figure 1. gc1 can dominantly suppress the wing abnormalities including pattern duplication induced by hh MRT. Panel A shows an example of Class III type of wing defects induced by ectopic expression of Hh in the anterior compartment in hh MRT animals. Panel B shows a wing from an animal of the genotype hh MRT/gc1. Almost complete suppression (Classified as Class I or II) of the wing phenotype can be seen. Panel C shows a graphic representation of the suppression of the wing defects. Roughly 250 single wing blades of the indicated genotypes were analyzed and classified into 5 different categories depending on the severity of the phenotype as previously described in Felsenfeld and Kennison, [27]. All the experimental as well as control crosses testing hhMRT suppression were carried out at 18uC. doi:10.1371/journal.pgen.1000333.g001 PLoS Genetics | www.plosgenetics.org 3 January 2009 | Volume 5 | Issue 1 | e1000333