Gc1 in Hh Signaling and Germ Cell Migration Figure 7. Manipulations of gc1 activity disrupt germ cell migration. (A) Ectopic expression of gc1 in hh producing cells not in hh receiving cells can induce germ cell migration defects. Whole mount staining of stage 13–15 embryos with antibodies against Vasa protein. Females carrying two copies of UAS-gc1 were mated independently either with the ptc-GAL4/ptc-GAL4 males (panels on left) or hh-GAL4/TM6 Ubx-LacZ males (panels on right). Embryos (10–14 hr old) were collected, fixed and then probed with b-galactosidase and Vasa antibodies. In the case of hh-GAL4 driver, embryos of the correct genotype were identified by the absence of b-galactosidase. The staining was visualized using standard immunohistochemical techniques. As can be seen by the comparison of the panels, gc1 is able to induce germ cell migration defects only when it is overexpressed using hh-GAL4 whereas gc1 overexpression using ptc-GAL4 leads to essentially wild type germ cell migration. As shown in the bar- diagram in the lower half of A, more than 90% of the ptc-GAL4/UAS-gc1 embryos (represented with red colored bars) display less than 2 lost germ cells whereas close to 30% of the hh-GAL4/UAS-gc1 embryos (represented with blue colored bars) have more than 5 lost germ cells. hh-GAL4/UAS-gc1 (n = 74), ptc-GAL4/UAS-gc1 (n = 53). (B) Ectopic expression of gc1 and gc1-DCAAX in the mesoderm induces germ cell migration defects. In this experiment embryos produced by females carrying the UAS:gc1 or UAS gc1-DCAAX mated to twist GAL4 males were stained with Vasa antibody and the number of lost or scattered germ cells in each embryo was counted. As shown in the bar graph, ectopic expression of either Gc1 or Gc1-DCAAX using the twist driver induced germ cell migration defects. As explained in the text, these defects likely arise for different reasons. Ore R (n = 20), UAS- gc1 (N = 69), UAS- gc1-DCAAX (n = 53). doi:10.1371/journal.pgen.1000333.g007 Figure 8. Germ cell migration defects induced by partial loss of gc1 are enhanced further by reducing either hmgcr or hh activity. Embryos between stages 12–15 of the indicated genotype were stained with anti-Vasa and b-galactosidase antibody and staining was visualized with standard immunohistochemcial techniques. Total number of germ cells that failed to associate with SGPs and remained scattered were counted per embryo. 25 embryos of each genotype were analyzed. Top panel shows that when embryos are heterozygous either for gc1 or hmgcr, more than 80% of the embryos display 0–2 lost germ cells (blue and red bars respectively). But when embryos are simultaneously compromised for both gc1 and hmgcr, more than 60% of the embryos have 7 or more lost germ cells (yellow bars). Bottom panel shows similar synergistic interaction between gc1 or hmgcr. Although the enhancement in germ cell migration defects is less severe compared to that seen with hmgcr (30% of the total number of embryos of the genotype gc1/+; hh/+ show more than 7 lost germ cells), the germ cell migration defects in embryos simultaneously compromised for gc1 and hh are clearly more severe as opposed to either gc1/+ or hh/+ embryos. doi:10.1371/journal.pgen.1000333.g008 PLoS Genetics | www.plosgenetics.org 9 January 2009 | Volume 5 | Issue 1 | e1000333