Figure 5. Distribution of Hh ligand is altered in qm embryos. Embryos from qm2/ Cy0, ftz-LacZ stock were collected, fixed using standard protocol and were subsequently identified by staining simultaneously with the b-galactosidase (not shown) and Hh antibodies (imaged in red). Panel A: Wild type control (qm2/Cy0, ftz-LacZ). Panel B: qm2 embryo. In the control embryos, Hh protein synthesized in two rows of cells per parasegment is released and spreads through the segment. Within the Hh expressing cells, Hh protein is localized around the membrane in a grainy or punctate pattern (see arrowheads in Panel A). In qm the release/transmission of the Hh protein is abnormal. The level of Hh in the interstripe region is considerably diminished suggesting that like gc1, the qm gene is required for the efficient release and/or transport of the Hh protein. Consistent with this suggestion, Hh protein appears to accumulate in the Hh expressing cells. Like gc1, the distribution of Hh in the expressing cells is abnormal. Instead of the characteristic grainy or punctate pattern of Hh protein localized around the cell membranes, Hh accumulates in clumps or aggregates (see arrows). doi:10.1371/journal.pgen.1000333.g005 carrying an en:LacZ marked 2nd chromosome balancer were stained with b-galactosidase antibodies to identify the homozygous mutant embryos and Vasa antibodies to visualize the germ cells. In wild type embryos (or in gc12/Cyo en:LacZ embryos) germ cells associate with the SGPs in parasegments 10–13 at stages 12– 13 (see stage 13 WT embryo in Figure 6A) and the two cell types coalesce into the embryonic gonad at stages 14–15 (see stage 15 WT embryo Figure 6B). Although germ cells that fail to coalesce into the embryonic gonad are sometimes seen in wild type embryos, the number of lost germ cells is generally rather low. The germ cells in gc12 embryos appear to have no difficulty in exiting the midgut pocket at stage 9–10, while movement along the surface of the midgut also appears to be comparatively normal. However, as illustrated in panel C of Figure 6, defects in migration are clearly evident by stage 13. In this embryo, several of the germ cells are not properly aligned with the SGPs in PS10–13 (compare with wild type in panel A). This problem persists and in stage 15 gc12 embryos germ cells that haven’t coalesced into the embryonic gonad can be seen scattered in the posterior (see panel D). Quantitation indicates that in wild type the vast majority (90%) of the stage 15 embryos (n = 20) have few if any (0 to 2) scattered germ cells. In contrast, about 36% of the gc11N159 embryos (n = 20) have 3 to 4 scattered germ cells while nearly 40% have 5 or more scattered germ cells. Similar results were obtained for the second gc1 allele, gc1k0817 (see Figure S2). Though the severity of the germ cell migration defects in the two gc12 mutants is similar to that reported for embryos zygotically compromised for either fpps or qm, it is not as strong as that observed for hmgcr mutant embryos [9], or for embryos that lack both zygotic and maternal (m2 z 2 ) fpps [15]. While compromising both maternal and zygotic gc1 would likely increase the severity of the germ cell migration defects as seen for fpps, the very severe patterning abnormalities observed in m2z2 embryos [28] would make effects on germ cell migration impossible to interpret. These findings indicate that gc1 is involved in germ cell migration just like the three enzymes, fpps, qm and b- Gc1 in Hh Signaling and Germ Cell Migration GGT1 that are downstream of hmgcr in the geranylgeranylation branch of the mevalonate pathway. Ectopic Expression of Gc1 Induces Germ Cell Migration Defects Ectopic expression of hmgcr, qm or fpps can induce the production of the germ cell attractant in inappropriate tissues. This ectopic source of attractant competes with the attractant produced by the SGPs and confuses the germ cells, disrupting their migration towards the SGPs [9,15]. If gc1 is functioning in the same pathway as these three enzymes, then it should also be possible to confuse germ cells by ectopically expressing gc1. To test this hypothesis, females carrying the CNS driver elav-GAL4 were mated to males carrying a UAS transgene that drives a flag-tagged Gc1 protein and the resulting elav-GAL4/UAS-flag gc1 embryos were stained with Vasa antibodies to mark the germ cells. Figure 6E and 6F show that misexpression of Gc1 in the central nervous system leads to a weak but reproducible germ cell migration defect. In wild type 90% of the stage 14–15 embryos have 0–2 scattered germ cells, while about 10% have 3 or more scattered or lost germ cells. In contrast in elav-GAL4/UAS-flag gc1 embryos (n = 51), more than 40% of the embryos have 3 or more lost germ cells. The effects of elav driven Gc1 expression are less than that reported for elav driven Hgmcr expresson (100% have 3 or more scattered germ cells) but equivalent to that observed for elav dependent misexpression of either Fpps or Qm (approximately 40% with 3 or more scattered germ cells: see 15). Ectopic Expression of gc1 in hh Producing Cells Induces Aberrant Germ Cell Migration In previous studies we found that expression of Hmgcr protein in hh producing cells was much more effective in inducing aberrant germ cell migration than when it was expressed in hh receiving cells. If gc1 functions downstream of hmgcr in the production of the germ cell attractant, then ectopic Gc1 should also have a more pronounced effect on germ cell migration when it is expressed in hh producing cells then when it is expressed in hh receiving cells. The experiment in Figure 7A shows that this prediction holds. There is little or no effect on germ cell migration when Gc1 expression is induced in hh receiving cells by a ptc-GAL4 driver. In this case, less than 10% of the embryos have 3 or more lost germ cells, which is comparable to that seen in wild type embryos (see bar graph in Figure 7A). In contrast, nearly one half of the embryos have 3 or more lost germ cells when Gc1 is expressed in hh producing cells under the control of hh-GAL4 driver (Figure 7A). While this result indicates that like Hmgcr, Gc1 must be misexpressed in hh producing cells in order to induce aberrant germ cell migration, it is important to note that the effects of ectopic Gc1 are less severe than that produced when Hmgcr expression is driven by the same hh-GAL4 driver [10]. Consistent with this difference, we do not observe any obvious alteration in the parasegmental distribution of Hh protein in UAS-gc1/hh-GAL4 embryos (not shown). By contrast, substantially more Hh protein is found in the interstripe regions when Hmgcr expression is driven by hh-GAL4 in hh producing cells [10]. Synergistic Genetic Interactions between gc1 and Either hmgcr or hh Disrupt Germ Cell Migration While there are few if any defects in germ cell migration in hmgcr2/+ embryos, synergistic interactions are observed when hmgcr2 is combined with mutations in two components of the hh signaling pathway hh and disp [10]. The perturbations in germ cell migration observed in the trans-heterozygotes taken together with PLoS Genetics | www.plosgenetics.org 6 January 2009 | Volume 5 | Issue 1 | e1000333