Author Summary Previous studies have shown that HMGCoA reductase (Hmgcr) is required for the production of a germ cell attractant by the somatic gonadal precursor cells (SGPs) and for the release of the Hedgehog (Hh) ligand by hh expressing cells. However, it was not clear what role mevalonate, the biosynthetic product of Hmgcr, played in either of these processes or whether the hmgcr-dependent germ cell attractant corresponds to the Hh ligand (which is known to be expressed by the SGPs). We show here that the downstream target for Hmgcr both in generating the germ cell attractant and in releasing the Hh ligand is the G protein, Gc1. Gc1 must be geranylated in order to function, and the substrate for this posttranslational modification, geranylgeranyl-pyrophosphate, is one of the biosynthetic products of mevalonate. In addition to demonstrating a critical role for Gc1 (as well as the hmgcr isoprenoid biosynthetic pathway) in releasing Hh from hh expressing cells, our findings provide additional evidence that Hh protein produced by the SGPs is an hmgcr- dependent germ cell attractant. that they can produce the actual attractant. However, a number of findings argue that Hh acts directly as an attractant. For one, the two known receptors of the Hh signal, Patched (Ptc) and Smoothened (Smo) are required in the germ cells for their proper migration. In the absence of the Hh ligand, the transmembrane receptor Ptc inhibits the 7-pass transmembrane protein Smo from mediating signal transduction [11–13]. When Hh binds to the Ptc receptor, the physical association between these two proteins is thought to relieve the negative influence of Ptc resulting in the relocalization of Smo to the cell membranes, and this in turn activates the signal transduction cascade downstream of the Hh signal. Consistent with their reciprocal functions in hh signaling, germ cells compromised for ptc or smo activity behave differently. For ptc the germ cells clump prematurely near the midgut as if they had already received the full Hh signal. For smo the germ cells behave as if they are ‘signal-blind’ and scatter randomly in the posterior of the embryo [10]. A second line of evidence supporting a direct role for Hh as the germ cell attractant comes from the discovery that hgmcr is required for the release of the Hh ligand by hh expressing cells. In embryos compromised for hmgcr activity [14], Hh is inappropriately retained in the hh expressing cells. Conversely, the range and strength of the Hh signal can be substantially enhanced by ectopic expression of hmgcr. Critically, ectopic hmgcr only had an effect on hh signaling when it was expressed in cells that normally produce the Hh ligand, while there was no effect when hmgcr is ectopically expressed in cells that normally receive the Hh ligand. One important issue left unresolved by these studies is how hmgcr promotes the release of the Hh ligand by hh expressing cells. The hmgcr gene encodes HMGCoA reductase which is responsible for the conversion of 3-hydroxy-3-methylglutaryl coenzyme A to mevalonate. In mammals, mevalonate is a precursor for cholesterol which is used in the modification of the Hh protein. However, providing precursors for cholesterol biosynthesis is not a likely function for hmgcr in the hh signaling pathway as the genes encoding the enzymes required to synthesize cholesterol from appear to be absent in flies [15]. Mevalonate is also a precursor for many different compounds including carotenoids, isoprenoids, ubiquitones and vitamins A and E [16]. Recent studies by Santos and Lehmann [15] on the role of hmgcr in germ cell migration have implicated the isoprenoid branch of the mevalonate precursor Gc1 in Hh Signaling and Germ Cell Migration pathway. The isoprenoids farnsyl-pyrophosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP) are used in the posttrans- lational modification of proteins and are covalently attached to the C terminus of target proteins by farnysyl transferase and type I or type II geranylgeranyl transferases respectively. Santos and Lehmann showed that mutations in farnesyl-diphosphate synthe- tase (fpps) (which synthesizes FPP), geranylgeranyl diphosphate synthetase (qm) (which in turn converts FPP to GGPP), and geranylgeranyl transferase type I (b-ggt1) disrupt germ cell migration. They also found that germ cell migration is perturbed when fpps and qm are ectopically expressed. Though the effects were much less dramatic than observed for ectopic hmgcr, this is not altogether unexpected since these two genes differ from hmgcr in that they are widely expressed in mid-to-late embryogenesis. While these findings indicate that the pathway leading from hmgcr to GGPP is important in germ cell migration because some critical target protein requires geranylation, the identity of this protein and the nature of its function in the production of the germ cell attractant remain to be established. Additionally, Santos and Lehmann [15] did not test whether hh signaling also depends upon this same isoprenoid biosynthetic pathway. Thus, the possibility remains open, especially if there is another germ cell attractant besides Hh, that hmgcr has some other function in hh signaling beside the production of isoprenoids. A possible answer to these questions comes from recent studies on cardiac development in flies. Hmgcr and downstream enzymes in the mevalonate pathway are required in cardioblasts to ensure their proper adhesion to the neighboring pericardial cells. Yi et al. [17] found that the endpoint for the isoprenoid branch of the hmgcr mevalonate pathway in heart development is the geranylgeranylation of the heterotrimeric G protein c subunit 1 (Gc1) [17,18]. The C-terminus of the Drosophila Gc1 protein has the isoprenylation CAAX motif sequence, Cys- Thr-Val-Leu. The leucine residue at the terminal position (X) specifies lipid modification by geranylgeranylation. Gc1 requires this modification for membrane association and is inactive when geranylation is blocked. Significantly, gc1 would be a quite plausible downstream target for hmgcr activity in the hh signaling pathway (and thus in the production of the germ cell attractant). Though heterotrimeric G proteins are normally thought to mediate the transduction of extracellular signals by G-protein coupled receptors, recent studies indicate that these G protein complexes have other intercellular functions. In particular, the Gc1:Gb heterodimer together with Ga have been implicated in the transport of cargo from the trans-Golgi network (TGN) to the basolateral plasma membrane [19–21]. The involvement of machinery targeting proteins to the basolateral membrane from the TGN would make sense in the context of hh signaling as autoprocessed and fully modified Hh protein is found to preferentially accumulate in a punctate pattern along the basolateral membranes of Hh expressing cells in the embryonic ectoderm [22–24]. This protein is then released from the cell, through a Dispatched (Disp) dependent mechanism that is thought to involve translocation of the Hh puncta from their docking sites along the basolateral membranes to the apical membrane [25,26]. In the studies reported here we have asked whether the hmgcrRGc1 pathway is important for hh signaling and whether gc1 is required for proper germ cell migration as is the case for hmgcr. Results gc1 Mutations Suppress the Gain-of-Function Wing Phenotypes of hhMrt To test whether gc1 is a component of the hh signaling pathway, we took advantage of the hhMoonrat (hhMrt) mutation [27]. hhMrt is a PLoS Genetics | www.plosgenetics.org 2 January 2009 | Volume 5 | Issue 1 | e1000333