Gc1 in Hh Signaling and Germ Cell Migration Figure 6. Either ‘loss’ or ‘gain’ of gc1 function leads to germ cell migration defects. Embryos from either the gc12/ Cy0, en:LacZ stock or from UAS-gc1 X elav-GAL4 cross were collected and fixed using standard histochemical technique. Wild type embryos derived from Oregon R stock were used as control (Panels A and B). Embryos from the gc12/ Cy0, en:LacZ stock were identified by simultaneously staining them with b- galactosidase antibody (not shown). Germ cell migration was assessed using anti-Vasa antibodies. A: Wild type stage 13 embryo. B: Wild type stage 15 embryo. C: gc1N159 stage 13 embryo. D: gc1N159 stage 15 embryo. E: UAS-gc1/ elav-GAL4 stage 15 embryo. F: UAS-gc1/ elav-GAL4 stage 15 embryo. doi:10.1371/journal.pgen.1000333.g006 the effects of hmgcr on the release/transmission of the Hh ligand from hh producing cells lent support to the hypothesis that the primary function of hmgcr in the production of the germ cell attractant by the SGPs is to potentiate the Hh signal emanating from these cells. Since the results presented above suggest that gc1 also functions in the release/transmission of the Hh ligand, we wondered whether equivalent synergistic genetic interactions would also be observed for gc1. We first tested for interactions between gc1 and hmgcr. Like hmgcr, there are at most only very modest defects in germ cell migration in gc1N159/+ embryos. However, more than 60% of the trans-heterozygous embryos have 7 or more lost germ cells (see Figure 8A). Next we tested for genetic interactions between gc1N159/+ and hh. As shown in Figure 8B, the minor germ cell migration defects observed in hh2/+ embryos are greatly enhanced when the hh mutation is combined with gc1N159. These results support the idea that gc1 could function in the germ cell migration pathway by facilitating the release/transmission of the Hh ligand. Misexpression of the gc1 CAAX Deletion Mutant Induces Germ Cell Migration Defects The results described in the previous sections suggest that the hmgcr mevalonate pathway is required in germ cell migration because gc1 must be geranylgeranylated in order for it to potentiate Hh signaling by the SGPs. To test this idea further, we examined the effects of misexpressing either the wild type Gc1 or Gc1 proteins that have mutations in the C-terminal CTVL geranylgeranylation motif in the mesoderm using a twist-GAL4 driver. We anticipated that misexpressing wild type Gc1 using the twist driver would induce aberrant germ cell migration because it would inappropriately potentiate signaling by hh expressing cells elsewhere in the mesoderm such as the fat body precursor cells (FBP). The hh signal emanating from these cells would compete with the signal from the SGPs, and this would confuse the migrating germ cells. The results shown in Figure 7B indicate that this expectation is met. While there are only a few wild type embryos in this experiment which have more than 2 lost germ cells, more than half of the twist-GAL4:UAS-gc1 embyros have at least 3 lost or mismigrated germ cells. We also anticipated that misexpressing Gc1 proteins that have mutations in the C-terminal geranylgeranylation motif would induce germ cell migration defects as well. Gc1 forms a heterodimer with a second G protein Gb and together these two proteins interact with a third G protein, Ga to form a heterotrimeric complex. In order to form a functional complex with Ga and also interact with other factors and effectors, the Gb:Gc1 heterodimer must be anchored to the membrane and this is thought to be dependent upon geranylgeranylation of the Gc1 protein [31,32]. We reasoned that Gc1 mutant proteins that cannot be geranylated would likely behave as dominant negatives because they would compete with the endogenous Gc1 protein for complex formation with Gb, and thus reduce the effective concentration of functional membrane bound Gc1:Gb heterodi- mers. This idea is support by studies on the Drosophila eye specific Gc protein Gce. The C-terminal sequence Gce is C-V-I-M which corresponds to the signal for farnesylation rather than geranylger- anylation. Mutations in Gce that eliminate farnesylation have no effect on the formation of Gce:Gbe heterodimers; however, these heterodimers do not interact with the membrane and are non- functional [32]. When the mutant Gce protein is overexpressed it competes with the endogenous Gce protein for heterodimer formation with Gbe, reducing the amount of functional membrane associated Gce:Gbe heterodimers and disrupting signal transduc- tion. If gernaylation defective Gc1 proteins also behave like dominant negatives, they would be expected to interfere with the efficient release of the germ cell attractant by the SGPs when they are ectopically expressed in mesodermal cells and this should perturb germ cell migration. We tested two different Gc1 mutant proteins, one in which the C-terminal CTVL motif is deleted (Gc1-DCAAX) and the other in which the geranylated Cys residue is replaced by Ser (Gc1-C67S) [16]. As shown in Figure 7B and Figure S3, ectopic expression of the Gc1-DCAAX protein disrupts PLoS Genetics | www.plosgenetics.org 7 January 2009 | Volume 5 | Issue 1 | e1000333