expression depends upon hh signaling by the cells immediately posterior to the wg stripe and in hh mutants wg expression begins disappearing by stage 10/11 of embryogenesis. Maintenance of the wg stripes also requires hmgcr activity and in hmgcr mutant embryos the stripes begin to fade around stage 11. However, unlike hh mutants, residual wg expression can still be detected in older hmgcr mutant embryos. Since maternal and zygotic hmgcr activity cannot be completely eliminated, this difference likely reflects (at least in part) the presence of residual Hmgcr in the hmgcr mutant embryos. If gc1 functions downstream of hmgcr in the hh signaling pathway, then defects in wg expression should also be evident in gc1 mutant embryos. To determine if this is the case we compared wg expression in gc12 embryos with their heterozygous gc12/+ sibs. We found that wg expression in the homozygous mutant embryos is initially like wild type (or gc12/+); however as shown in Figure 2B for gc1N159, the accumulation of Wg protein begins to decrease around stage 11–12 (compare the gc1N159 homozygote in panel B with the gc1N159/+ sib control in panel A). Similar results were obtained for the gc1k0817 (compare panel C and D in Figure 1). The extent of reduction in Wg protein in the two gc1 mutants is not as severe as that seen in embryos compromised hh; however, as noted above this was also observed for hmgcr and likely reflects the perdurance of the maternally derived Gc1. Another gene whose expression in mid-embryogenesis depends upon hh signaling is engrailed (en). en is part of an autoregulatory loop that is established between the neighboring hh and wg expressing cells. en is transcribed in the hh expressing cells in response to the Gc1 in Hh Signaling and Germ Cell Migration Wg ligand. When wg signaling is disrupted because of a reduction in hh signaling, en transcription is in turn downregulated. As would be expected from the effects of gc1 mutations on wg expression, we find that the accumulation of En protein is reduced in embryos homozygous mutant for both of the gc1 alleles compared to their wild type (gc12/+) sibs (see Figure S1). Smo Protein Is Mislocalized in gc1 Embryos While the effects of gc1 mutations on wg and en expression would be consistent with a role in hh signaling, it is also possible that gc1 activity is required at some other point in the hh-wg autoregulatory loop, for example, in the expression of the Wg or En proteins. For this reason we next examined the effects of gc1 on the Smo receptor which is a more direct target for the Hh ligand in the receiving cells. Upon reception of the Hh signal the Smo receptor is relocalized from intracellular membrane vesicles to the cell surface [29,30]. When hh signaling is compromised, this relocalization does not occur, and the Smo protein remains predominantly cytoplasmic in the receiving cells. Since Smo is not properly relocalized in hmgcr mutant embryos, a similar defect would be expected in gc1 mutants if gc1 functions downstream of hmgcr in the hh signaling pathway. Figure 3 shows that this prediction holds. In this experiment we compared the localization of the Smo receptor in homozygous gc1 mutant embryos with their heterozygous sibs. The pattern of Smo accumulation in the heterozygous gc12/+ embryos (panels A and B) resembles wild type. There are a series of stripes that are approximately 5 cells wide in which the Smo protein is largely localized to the plasma membrane. These stripes are separated from each other by an equivalent band of about 5 cells that have a lower level of Smo at the surface of the cell. In homozygous gc1 mutant embryos this Smo distribution pattern is disrupted. Although a weak stripe pattern can still be discerned in the homozygous mutant Figure 2. Reduced wingless expression in embryos compro- mised for gc1. Panels A and B: Embryos from gc1N159/ Cy0, en:LacZ stock were collected and fixed using standard procedures. Embryos were genotyped by probing with b-galactosidase antibodies (imaged in red: not shown), while Wg accumulation was visualized by probing with Wg (imaged in green) antibodies. Embryos carrying en:LacZ express b- galactosidase whereas homozygous mutant embryos do not. The embryo in panel A was positive for b-galactosidase (not shown) and has at least one wild type copy of gc1. Note the high level of Wg accumulation in the stripes. The embryo in panel B b-galactosidase negative, and is homozygous for the gc1N159 mutation. Note the lower level of Wg expression. Panels C and D: Embryos from a gc1k0817/Cyo, en:LacZ stock were collected and fixed using standard procedures. As in panels A and B embryos were genotyped by probing with b- galactosidase antibodies (imaged in red: not shown), while Wg accumulation was visualized by probing with Wg (imaged in green) antibodies. The embryo in C is positive for b-galactosidase, while the embryo in D is not. Note the difference in Wg accumulation in the blow- up of three Wg stripes. doi:10.1371/journal.pgen.1000333.g002 Figure 3. Localization of Smoothened receptor is altered in embryos compromised for gc1. Embryos from gc1N159/ Cy0; en:LacZ stock were collected, fixed using standard protocol and were subsequently identified by staining simultaneously with the b- galactosidase and Smo antibodies. Smo was imaged with the secondary antibodies coupled with Alexa 546. The figure shows stage 10–11 embryos. In the wild type control (gc1N159/ Cy0; en:LacZ embryos) the intercellular distribution of Smo protein has a parasegmentally repeating pattern (see arrows in Panel A). In a ,5 cell wide stripe across each parasegment Hh signaling leads to the relocalization of Smo protein to the membrane. In the remaining cells in each parasegment (,5 cell wide stripe) Smo protein remains largely cytoplasmic. This can be seen in the magnified view in Panel B. In gc12 embryos, this parasegmentally repeating pattern of Smo protein localization is largely lost (see Panel C). In most cells in each parasgement the Smo protein remains diffusely distributed through cytoplasm (see Panel D). doi:10.1371/journal.pgen.1000333.g003 PLoS Genetics | www.plosgenetics.org 4 January 2009 | Volume 5 | Issue 1 | e1000333