functions in concert with Hh to direct germ cell migration towards the SGPs. However, in either of these more complicated scenarios, the unknown germ cell attractant would have to be a molecule that can be induced in many different cell types in the embryo, but apparently only if these cells also express the Hh protein. Materials and Methods Immunohistochemistry The embryo stainings were performed essentially as described in 51. Vasa (from Paul Lasko) and Hh (from Tom Kornberg) antibodies are rabbit polyclonal antibodies. Both were used at a 1:500 dilution. Engrailed and Wingless antibodies are mouse monoclonal antibodies and were used at 1;10 dilution. b-Galactosidase antibody was either a rabbit polyclonal purchased from Kappel (used at 1:1000 dilution) or a mouse monoclonal antibody from Developmental Hybridoma Bank (used at 1:10 dilution). Smoothened antibody (anti-rat) was a kind gift from Steve Cohen and was used at 1:500 dilution. For confocal analysis a magnification of 406 was used in almost all the instances and images were collected using identical settings for the control and experimental samples. Multiple pairs of wild type (sibs) and mutant embryos were imaged in each case and representative examples are presented. Mutant and Misexpression Analysis gc1 mutant stocks, gc1N159 and gc1k0817, were obtained from Fumio Matsuzaki while the various 1UAS- gc1 stocks (gc1, gc1 DCAAX and gc1 C67S) were kindly provided by the Olson lab. The other UAS and GAL4 stocks used for the misexpression studies: UAS- hmgcr, hairy-GAL4, elav-GAL4, nanos-GAL4, patched-GAL4, UAS-b-galactosidase, hh-GAL4/TM6 Ubx-LacZ. In most experi- ments, males carrying two of the copies UAS transgene were mated with virgin females carrying two copies of the GAL4 transgene. The resulting progeny embryos were fixed and stained for subsequent analysis [51]. Supporting Information Figure S1 Engrailed expression is not properly maintained in gc1 mutant embryos. Embryos from the gc1N159/ Cy0, en:LacZ stock were collected and fixed using standard procedure. Embryos were genotyped by simultaneously staining them with b-galacto- sidase (imaged in green: not shown) and En (imaged in red) antibodies. Balancer embryos (Panel A) show strong En specific expression in 14 stripes. By contrast, En specific signal starts to decline by Stage 11 in the homozygous gc1N159 embryos (Panels B and C). As illustrated in these two panels there is some variation in the extent of the reduction in En expression. In some embryos, moderate levels of En protein are detected (B) while in others only low levels are observed (C). Because of the variability in En accumulation in gc1N159 homozygous embryos, we classified the En staining pattern. For the heterozygous gc1N159/+ control, 6/7 embryos had high levels of En accumulation, while 1 embryo had a medium level of accumulation. For the homozygous gc1N159 embryos 4/11 (37%) had little En protein (like the example shown in the figure) while 4/11 had a medium level of En protein (like the example shown in the figure). The 3 remaining embryos (27%) resembled wild type. We also examined En expression in homozygous gc1k0817 embryos. In this experiment all of the heterozygous gc1k0817/+ control embryos had a high level of En protein (9 embryos). For the homozygous gc1k0817 mutant embryos 8/18 (44%) had a low level of En protein, while 5/18 (28%) had a medium level of En protein. Finally, 5/18 (28%) homozygous mutant embryos had a high level of En protein. Gc1 in Hh Signaling and Germ Cell Migration Found at: doi:10.1371/journal.pgen.1000333.s001 (3.2 MB TIF) Figure S2 Germ cell migration defects are also observed in gc1k0817 mutant embryos. Embryos from a gc1k0817/Cy0 en:LacZ stock were probed with Vasa to mark the germ cells and b- galactosidase antibodies to identify heterozygous and homozygous mutant embryos. Panels A and B are gc1k0817/Cy0 en:LacZ embryos (note b-galactosidase expression) while panel C and D are mutant. Panel C shows embryo with 3 scattered cells whereas the embryo in panel D has more than 6 scattered germ cell cells. About 15% (4/22) of the mutant embryos had 3–4 scattered germ cells (example in panel C), while about 40% (8/22) of the mutant embryos had 5 or more scattered germ cells (example in panel D). The remaining embryos (10/22 or 45%) had 2 or fewer scattered germ cells. Found at: doi:10.1371/journal.pgen.1000333.s002 (2.0 MB TIF) Figure S3 Ectopic expression of gc1-DCAAX in the mesoderm and in germ cells disrupts germ cell migration. Panels A–F show stage 13–15 twist-GAL4/UAS-Gc1-DCAAX or nos-GAL4/UAS-Gc1- DCAAX embryos probed with Vasa antibodies to visualize migrating germ cells. Panels A–D: Germ cell migration defects in twist-GAL4/UAS-Gc1-DCAAX embryos. Panels E and F: Germ cell migration defects in nos-GAL4/UAS-Gc1-DCAAX embryos. Recent studies by Kunwar et al. [52] on germ cell migration have suggested that gc1 has a cell autonomous requirement in germ cells. To test the cell autonomous function of gc1 in germ cell migration, these authors rescued the gastrulation defects of progeny from gc1 germline clone mothers using a nullo-GAL4 to drive expression of a UAS-gc1 transgene. They reported that the pole cells in these embryos failed to migrate properly out of the midgut and exhibited phenotypes similar to those found for mutations in the G protein-coupled receptor (GPCR) gene trapped in endoderm 1 (tre1). We wondered whether the geranylated form of Gc1 is also required in the germline. To explore this possibility we ectopically expressed the dominant negative gc1 deletion mutant Gc1-DCAAX in the germline. As shown in Figure S3E and S3F, ectopic expression of the Gc1-DCAAX protein in germ cells using a nos-GAL4 driver disrupts germ cell migration. We found that nearly 60% of the stage 13–15 nos-GAL4/UAS-Gc1-DCAAX embryos had 3 or more lost germ cells, while 33% had 5 or more lost germ cells (n = 100 embryos). This is roughly equivalent to the germ cell migration defects evident when Gc1-DCAAX is expressed in the mesoderm using a twist-GAL4 driver. The effects of the dominant negative protein in germ cells would support the findings of Kunwar et al., and argue that gc1 (specifically geranylated Gc1) has a cell autonomous function in these cells during their migration towards the SGPs. On the other hand, our results differ somewhat from those reported by Kunwar et al. in that we did not observe any obvious defects in the ability of the germ cells to exit the midgut when the Gc1-DCAAX protein was expressed using the nos-GAL4 driver. While there are a number of plausible reasons why a tre1-like phenotype wasn’t observed, the most likely explanation is that not enough of the dominant negative Gc1-DCAAX is generated to disrupt the tre-1 dependent migration through the midgut epithelia. In particular, germ cells are known to be transcriptionally quiescent until just before they exit the midgut, and there might not be sufficient time to generate high enough levels of Gc1-DCAAX to effectively inhibit the maternally derived product. If this explanation is correct, it would suggest that gc1 may also function at a later, tre-1 independent step in the germ cell migration pathway since many of the germ cells in nos-GAL4/UAS-Gc1-DCAAX embryos fail to coalesce with the SGPs. Found at: doi:10.1371/journal.pgen.1000333.s003 (4.2 MB TIF) PLoS Genetics | www.plosgenetics.org 12 January 2009 | Volume 5 | Issue 1 | e1000333