rm line clones, in which 
embryonic patterning defects were rescued by a wild-type shg+ copy from the father. This suggests that DE-cadherin is required autonomously in germ cells, as they are transcriptionally quiescent and thus likely depend exclusively on maternally contributed DE-cadherin. These results indicate that DE-cadherin is required for germ cellgerm cell adhesion in the wild-type embryo. To understand how DE-cadherin is regulated in the dispersal step, we analyzed the distribution of DE-cadherin in wildtype germ cells. We found that DE-cadherin as well as and catenins were initially uniformly present along the germ cell membrane but became enriched in the tail region during germ cell polarization. In stark contrast, DE-cadherin remained uniformly distributed along the cell surface in tre1 mutant embryos. To quantitate the levels, we compared the fluorescent intensity of DE-cadherin staining on the cell membrane of wildtype and tre1 mutant germ cells. To test directly if Tre1 acts via DE-cadherin in transepithelial migration, we generated embryos that lacked tre1 and maternal shgA9-49 function. The germ cells in these embryos dispersed early, thus displaying a phenotype similar to shgA9-49 mutants; 80% of tre1, shgA9-49 double mutant embryos showed precocious dispersal as opposed to 0% in the tre1 mutant embryos. However, even these dispersed germ cells were not able to transmigrate through the midgut in tre1, shgA9-49 double mutant embryos, thereby resembling tre1 mutant germ cells. This suggests that loss of germ cellgerm cell contact may not be sufficient to trigger transepithelial migration. To test this idea further, we disrupted germ cellgerm cell contact independent of E-cadherin function by reducing the germ cell number. We used alleles of the maternal effect gene tudor to reduce the number of germ cells in the embryo to a single germ cell. Such single, tud mutant germ cells migrated through the midgut and invariably reached the gonad. These germ cells had normal morphology and appeared polarized. Next, we analyzed mutant embryos lacking both tre1 and maternal tud. In the absence of tre1, single germ cells were left inside the midgut and did not migrate to the gonad. Thus, whereas germ cell individualization requires Tre1-mediated down-regulation of DE-cadherin, Tre1 activity has additional roles in transepithelial migration. Discussion We have used live imaging to explore the mechanisms by which germ cells acquire motility and traverse the midgut epithelium. We found that before transepithelial migration, germ cells polarize toward the midgut and down-regulate E-cadherin from the AEB 071 site leading edge and accumulate E-cadherin in the tail region. 163 TRE1 GPCR, GERM CELL POLARITY, AND MIGRATION Kunwar et al. This polarization requires Tre1 GPCR activity. The first localized response to receptor activation is the enrichment of the activated G protein subunits, which results in the activation of phosphoinositide 3 kinase. As a consequence of chemokine sensing, the PI3 kinase product phosphatidylinositide 3,4,5-tris phosphate becomes localized to the leading edge, and the phosphatase PTEN moves to the lagging edge in a Rho dependent manner. These signaling events organize the cytoskeleton leading to cellular polarization and di- rectional movement. Our studies suggest a new mechanism by which GPCR signaling initiates directed cell migration. We find that activation of Tre1 causes a redistribution of G protein, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19836835 the GTPase Rho1,