levels of Hmgcr protein would increase dramatically when it is ectopically expressed in the ectoderm during mid-embryogenesis while this would not be true for Gc1 or, for that matter, the other GGPP biosynthetic enzymes. The idea that Hmgcr is a limiting component of signaling pathways that depend upon geranylation Gc1 (or other targets) is also supported by the defects in germ cell migration that are induced by ectopic expression of these proteins. For example, expression of hmgcr in the CNS cause much more severe abnormalities in germ cell migration than those observed when fpps, qm, or gc1 are misexpressed [15]. If these ideas were correct, than inducing or repressing the expression of the hmgcr gene would provide a novel posttranslational mechanism for regulating the potency of signaling molecules like Hh. The effects of gc1, qm and hmgcr on the distribution of Hh in the ectoderm indicates these genes are required for the release of the Hh ligand from hh expressing cells. For Gc1, a role in releasing the Hh ligand from the sending cells would dovetail nicely with a recently discovered function of this G protein and its partners, Gb and Ga in the transport of cargo from the trans-Golgi network (TGN) to the basolateral plasma membrane [19–21]. Since Hh protein appears to be specifically targeted to the basolateral membrane in punctate structures (LPSs) prior to secretion [22–25,38,39], it is not altogether surprising that components of the machinery needed for the transport of cargo from the TGN to the basolateral membrane would play a key a role in transmitting the Hh signal. Moreover, since Gc1 requires geranylation for membrane association and activity, the retention of Hh in qm and hmgcr mutants would also be explained by a disruption in Gc1-dependent TGN-plasma membrane transport. In this context it is interesting to note that while the levels of Wg are reduced in hmgcr [14] and gc1 mutants, there is no obvious over accumulation of the Wg protein inside wg expressing cells like that observed for Hh. That the hmgcrRqmRgc1 pathway would have no apparent effect on the release of Wg would make sense since this is thought to occur preferentially through the targeting of mRNAs to the apical surface of the cell [49]. Though the precise mechanisms for TGN-plasma membrane transport have yet to be elucidated, it is thought that the heterotrimeric G protein complexes mediate the release of cargo from the TGN by promoting membrane fission [25]. In one scenario, interaction of the cargo with an unidentified receptor in the TGN leads to the activation of the trimeric Gc1:Gb: Ga and the release of Ga The Gc1:Gb heterodimer in turn activates several targets including phosphokinase C and a phosphoinostide- specific phospholipase C (PI-PLC) that generates diacylglycerol. PKC participates in cargo release from the TGN by activating Protein Kinase D (PKD) while locally high concentrations of diacylglycerol produced by PI-PLC are thought to change the properties of the TGN membranes and promote membrane fission. After membrane fission, the vesicle containing the cargo is then targeted to the basolateral plasma membrane [24,25]. A requirement in the formation of cargo containing vesicles would fit well with the effects of hmgcr, qm and gc1 on the formation of the puncate Hh particles, or LPSs, that normally assemble along the basolateral membranes of Hh expressing cells. These LPSs are largely absent in hmgcr, qm and gc1 mutant embryos and instead Hh accumulates in much larger aggregates or clumps. While the precise origin of the LPSs is not known, they are thought to be lipid containing vesicles (or micelles) and it would be reasonable to think that they could be generated by the budding of Hh containing vesicles from the TGN. In this case, the large aggregates or clumps of Hh protein seen in hmgcr, qm and gc1 mutants would likely represent Hh trapped either in the TGN or in aberrant vesicles/structures that accumulate in the mutant cells when efficient cargo release from the TGN is disrupted. Gc1 in Hh Signaling and Germ Cell Migration While the idea that Gc1 promotes the transport of Hh from the TGN to plasma membrane would seem to fit best with the known functions of Gc1 and its collaborating G proteins, it is also possible that Gc1 (plus Gb and Ga) functions at earlier steps in the secretion pathway, for example. in the transport of Hh from Endoplasmic Reticulum to the Golgi [50]. Alternatively, it is possible that some novel activity of Gc1 at the plasma membrane rather than in the TGN is needed. For example, it could function to prevent the newly formed LPSs from clumping together into larger aggregates. Further studies will be required to distinguish between these and other possible mechanisms. Gc1 Is Required for Germ Cell Migration Studies by Santos and Lehmann [15] provided convincing evidence that hmgcr is required in the soma for germ cell migration because its biosynthetic product, mevalonate, is the precursor for the synthesis GGPP by Qm. They also found that GGPP is used in turn by geranylgeranyl transferse type 1 (b-GGT1) for the geranylation of some unknown target(s). The experiments presented here indicate that one (if not the only) somatic target in the germ cell migration pathway is Gc1. Thus, the effects of both gain and loss of gc1 function on germ cell migration closely resemble those reported for hmgcr, fpps, qm, and b-GGT1. Also supporting the idea that Gc1 must be a relevant target for the hmgcr-isoprenoid biosynthetic pathway, we find that Gc1 proteins that cannot be geranylated behave as dominant negatives when ectopically expressed in the mesoderm and disrupt germ cell migration. In addition, there are other significant similarities between the two genes that have been studied in most detail, gc1 and hmgcr. First, both genes show synergistic genetic interactions with components of the hh signaling pathway that perturb the process of germ cell migration. Second, germ cell migration can be disrupted when gc1 or hmgcr are ectopically expressed in hh producing cells; however, there are no apparent effects when the genes are ectopically expressed in hh receiving cells. Taken together with the fact that the SGPs are known to be a source of Hh these findings would argue that a critical function of the biosynthetic pathway leading from hmgcr to the geranylation of Gc1 is to upregulate Hh signaling in the SGPs, and it is the Hh ligand produced by these cells that serves to attract the migrating germ cells. Importantly, this model accounts for a number of different observations. Since Hmgcr is expressed at high levels in the SGPs, but is not expressed elsewhere in the mesoderm, it would explain how Hh signaling could be specifically potentiated in a special sub-population of cells. It would also explain why the effects of hmgcr misexpression are much greater than misexpression of the other genes in the hmgcrRgc1 pathway that are more broadly transcribed in the embryo. Finally, it would explain why germ cells can be misdirected by ectopic expression of hh, hmgcr, the downstream genes in the geranylation biosynthetic pathway, and gc1 in a variety of different tissues. By contrast, if the SGPs were to induce germ cell migration by expressing some unique and dedicated hmgcrRgc1 dependent attractant, it is hard to under- stand how misexpression of these different upstream genes would be able orchestrate the production of this special molecule in a variety of cells and tissues that have little resemblance to the SGPs. It should be noted, however, that our results would also be compatible with more complicated models. For example, it is possible that the potentiation of Hh signaling by the hmgcrRgc1 pathway induces the production of a specialized and as yet unknown germ cell attractant. Likewise, we also can not exclude the possibility that there is some other target for geranylation besides Gc1 which is important for the production or activity of a second germ cell attractant and that this unknown molecule PLoS Genetics | www.plosgenetics.org 11 January 2009 | Volume 5 | Issue 1 | e1000333