embryos (see panel C) it is much less distinct than in the heteterozygous sibs (panel A). Panel D shows that Smo remains largely cytoplasmic in most of the cells in each segment and is not tightly localized at the plasma membrane as it is in wild type or gc12/+ heterozygous embryos. Embryos Compromised for gc1 Display an Altered Hh Protein Distribution The finding that Smo protein does not properly relocalize in gc1 mutant embryos would be consistent with the idea that Gc1 acts downstream of hmgcr to promote the efficient release and/or transport of Hh protein. To test this hypothesis further, we compared the pattern of Hh accumulation in gc1 mutant embryos with their heterozygous sibs. The distribution of Hh protein in gc12/+ embryos resembles that seen in wild type [22–26]. Hh is expressed in each parasegment in a two cell wide stripe, and most of the protein in these Hh expressing cells is distributed in the cell membrane in a fine grain or punctate pattern (see arrowheads in panel A of Figure 4). Emanating in both directions from the two cell wide stripe is an Hh protein gradient that appears to extend through much of the parasegment. In this gradient the highest levels of Hh protein are observed associated with cells adjacent to the two Hh expressing cells, while lower levels of protein are found in more distant cells. The distribution of Hh protein in gc1 mutant embryos (panels B and C) resembles that seen in hmgcr mutant embryos [14]. First, in spite of the fact that the overall level of Hh expression is expected to be reduced in these embryos because of the disruption in the wg-hh positive autoregulatory loop (see above), the relative amount of Hh in cells in the hh stripes appears higher than in wild type embryos, while there is a concomitant reduction in the amount of Hh in the gradient that extends through the interstripe region (compare panels A with B & C). Second, the normal grainy or punctate pattern of Hh protein localized around the basolateral membrane of the hh expressing cells that is seen in wild type embryos (see arrows in panel A and in the enlargement in panel D) is largely lost. Instead, Hh accumulates in larger ‘‘clumps’’ or aggregates (see arrows in panels B and C and in the enlargement in panels E and F) that in many instances seem to be displaced from the cell membranes (see top arrows in panel E and F). qm Is Required To Promote the Release/Transmission of the Hh Ligand The results described in the previous sections demonstrate that like hmgcr, gc1 is required for the efficient release/transmission of the Hh ligand by hh producing cells. Since the role of the isoprenoid branch of the hmgcr mevalonate pathway in heart development is the geranylgeranylation of Gc1, a plausible idea is that the function of hmgcr in hh signaling is to provide substrates for the modification of the Gc1 protein. If this model is correct, then gene products that are downstream of hmgcr in the geranylger- anylation pathway should also be required for the release/ transmission of the Hh ligand. To test this prediction we examined the distribution of Hh protein in qm mutant embryos. As described above, qm encodes geranylgeranyl diphosphate synthetase and this enzyme produces the substrate, GGPP, that is used by the geranyl transferase to modify Gc1. Figure 5 shows the distribution of Hh in a homozygous qm mutant embryo (panel B) and its heterozygous qm2/+sibs (panel A). As observed for both hmgcr [14] and gc1 (see above), the Hh ligand is inappropriately retained in the hh producing cells in the qm mutant embryos (compare Hh distribution in panels A and B). Like gc1 the characteristic punctate distribution of Hh protein around the membranes of hh expressing cells (arrowheads in pane A) is reduced or lost and Gc1 in Hh Signaling and Germ Cell Migration Figure 4. Spread of Hh ligand is restricted in gc12 embryos. 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 anti-Hh antibodies. Hh specific signal was imaged with the secondary antibodies coupled to Alexa 546 (Red). The figure shows stage 10–11 wild type (panel A) and gc1N159 (panels B and C) embryos. In wild type embryos, two rows of cells per segment express Hh protein. In these cells Hh protein is distributed around the membrane in a grainy or punctate pattern (see arrowheads). The Hh ligand is released and it spreads in a graded fashion through the parasegment. In the gc1N159 embryos (panels B and C) Hh is not properly released from hh expressing cells and high levels of Hh protein accumulate in these cells. The grainy pattern of Hh protein around the cell membrane is less evident and instead Hh protein accumulates in larger clumps or aggregates that appear to be distributed in the cytoplasm rather than just at the membrane (see arrows). Panels D, E and F are enlargements of the Hh stripe in the embryos shown in panels A, B and C respectively. Arrows in panel D show the grainy punctate pattern of Hh protein in wild type embryos (the LPS), while the arrows in panels E and F show the larger clumps or aggregates of Hh protein that accumulate in cells of mutant embryos. doi:10.1371/journal.pgen.1000333.g004 instead Hh accumulates in clumps or large aggregates (arrows in panel B). It should also be noted that this particular qm mutation appears to cause a more pronounced defect in the release/ transmission of the Hh ligand than is observed for gc1 (compare Figures 4 and 5), while the defects in Hh distribution evident in hmgcr mutant embryos [14] are roughly intermediate between that in qm and gc1. Embryos Compromised for gc1 Activity Display Germ Cell Migration Defects The findings describe above indicate that gc1 represents an endpoint for the isoprenoid branch of the hmgcr mevalonate pathway in the hh signaling pathway, and that like hmgcr, gc1 is required for the efficient release/transmission of the Hh ligand. Since several components of this hmgcr mevalonateRisoprenoid pathway have also been implicated in the production or transmission of the germ cell attractant by the SGPs [15], we tested whether mutations in gc1 have any effects on germ cell migration. Embryos collected from gc1k0817 and gc1N159 stocks PLoS Genetics | www.plosgenetics.org 5 January 2009 | Volume 5 | Issue 1 | e1000333