germ cell migration and about 75% of the twist-GAL4/UAS-gc1- DCAAX transgene embryos have 3 or more lost germ cells. (Note: Figure S3 shows that ectopic expression of Gc1-DCAAX in germ cells also disrupts their migration.) With the caveat that there may be differences in expression levels of the UAS transgene, it would appear that the germ cell migration defects induced by the geranylation defective Gc1-DCAAX protein are somewhat more pronounced than those observed with wild type Gc1. Consistent with this possibility, the Gc1-C67S mutant protein also induces more extensive germ cell migration defects than wild type (not shown). Discussion Gc1 Is Required for Hh Signaling Hh functions as an instructive cue in many different biological contexts. The signaling molecule is secreted from hh expressing cells and it induces morphogenesis in a concentration dependent fashion in neighboring cells by regulating the transcription of downstream target genes. Several mechanisms control the range and inductive activity of the Hh protein. These include the autoprocessing and lipidation [33–35]. Hh has two different lipid modifications that are important for the proper functioning of the Hh ligand. One is the palmitoylation of the N terminus which seems to be critical for signaling activity, while the other is the addition of cholesterol to the C terminus. The C-terminal cholesterol moiety is thought to be important for the dimerization of the Hh protein and for its assembly into LPSs (Large Punctate Particles) prior to secretion [36–39]. The LPSs appear to be lipid vesicles or micelles and they are thought to provide a hydrophobic environment for the lipid modified Hh which facilitates its movement through the extracellular matrix after it is secreted. The release and subsequent transport of the Hh ligand also requires specialized proteins that function in either Hh producing cells or in cells/compartments that are destined to receive the Hh ligand. The transporter class protein, Disp, and a secreted protein Shifted (Shf) are required in hh expressing cells for the efficient release and transmission of the Hh ligand [25,40–42]. In shf mutants, the basolateral accumulation of Hh protein in the wing disc is disrupted, while apical accumulation appears to be normal. The subsequent transport of the Hh ligand to the receiving cells depends upon the glypicans Dally-like (Dlp) and Dally, which are components of the extracellular matrix, and enzymes that are needed for glycosaminoglycan biosynthesis namely Sulfateless and Tout-velu [43–45]. The glycosaminoglycan is thought to promote long range signaling by Hh and other signaling molecules such as Wg by passing the ligand from one cell to its neighbor instead of presenting the ligand to the receptor [46]. It is thought to do so by directing the ligands to the lateral membranes where endocytosis is less efficient [47]. There are likely to be an extensive array of accessory factors like disp and dally that are required for the efficient release of the Hh ligand from hh expressing cells and its subsequent transport or transmission from one neighboring receiving cell to the next. We have previously shown that one such gene encodes the mevalonate biosynthetic enzyme Hmgcr [14]. We found that hmgcr is required in Hh expressing cells to facilitate the release or transmission of the Hh ligand; however, it was not clear from our studies why the biosynthesis of mevalonate would be important for the release/ transmission of the Hh ligand in flies. The obvious explanation, that it is required for the synthesis of the cholesterol that is used to modify Hh, was not likely to be correct as flies do not have the downstream enzymes for cholesterol biosynthesis [15]. In the work reported here we have resolved this question. We show that the Gc1 in Hh Signaling and Germ Cell Migration downstream target for hmgcr in the hh signaling pathway is the heterotrimeric G protein, Gc1, which must be geranylated in order to be active [17,31,48]. Like hmgcr and other genes that are required to promote hh signaling, mutations in gc1 dominantly suppress the gain-of-function wing phenotypes of hhMrt in adult flies. In the embryo, the expression of wg which is activated by hh in the receiving cells is downregulated in both hmgcr and gc1 mutants. This is also true for the en gene which is normally activated by wg signaling in hh expressing cells as part of the autoregulatory circuit that sustains hh and wg expression as the embryo develops. These transcriptional defects arise because the Hh signal is not properly conveyed to hh receiving cells. In wild type embryos Smo protein is redistributed to the membranes of the receiving cells when they receive the Hh signal transmitted from the neighboring Hh producing cells. As observed for hmgcr, Smo protein is not correctly relocalized in gc1 mutant embryos, and instead it remains largely cytoplasmic. Finally, in the ectoderm of wild type embryos there is a gradient of Hh protein extending into the parasegment from the two cell wide stripe of hh expressing cells. Like hmgcr, this gradient is not properly formed in gc1 mutant embryos, and instead Hh protein is inappropriately retained in the Hh producing cells. Since isoprenoid modifications, either farnesylation or geranyla- tion, are known to be critical for the functioning of the Gc family of proteins, these observations would argue that hmgcr is required for the release of the Hh ligand because it provides a precursor that is needed for the geranylgeranylation of Gc1. This conclusion is supported by the finding that qm, which synthesizes the activated substrate, GGPP, that is used to geranylate Gc1, is also required for the release of the Hh ligand from hh expressing cells. While these results implicate the biosynthetic pathway leading from mevalonate to the geranylation of Gc1 in the proper release of the Hh ligand, we cannot exclude the possibility that there are important targets for geranylation in addition to Gc1 or that other products of mevalonate might play some role in the hh signaling pathway. These possibilities remain open for a number of reasons. First, the defects in the release of Hh observed in antibody staining experiments seem to be more severe in the qm mutant (and to a lesser extent in hmgcr: see 14) than in the gc1 mutants we examined. One explanation for this difference is that the Qm enzymatic product, GGPP, is used for the geranylation of other proteins that are important for the release of the Hh ligand. However, this could also be due to, for example, differences in the perdurance of the maternal Qm and Gc1 proteins. Second, ectopic expression of Hmgcr in hh expressing cells causes a readily discernible change in Hh protein distribution across the parasegment and relatively high levels of Hh are found even near the middle of the interstripe region. By contrast, we could not detect an equivalent alteration in Hh distribution when Gc1 was ectopically expressed in hh producing (or receiving) cells. This difference could mean that the mevalonate produced by Hmgcr has uses in hh signaling besides the synthesis of GGPP and the geranylation of Gc1. An alternative and perhaps more interesting possibility is that the differences in the effects of misexpression on the release/ transmission of Hh protein reflect the fact that Hmgcr is limiting whereas Gc1 is not. Consistent with this idea, the distribution of hmgcr mRNAs becomes progressively restricted as development proceeds and by mid-embryogenesis (stages 10–15) hmgcr mRNAs are only detected in the SGPs [9]. By contrast, mRNAs encoding Gc1, as well as several of the enzymes that are downstream of Hmgcr in the biosynthesis of GGPP, are much more widely expressed in the embryo at this stage [15,28]. A possible consequence of this difference in mRNA distribution is that the PLoS Genetics | www.plosgenetics.org 10 January 2009 | Volume 5 | Issue 1 | e1000333