More SR proteins are recruited, and spliceosome assembly occurs on nascent premRNP

function is required there. General cell motility and the movements of germ cells toward the gonad do not depend on Tre1, which suggests that Tre1 specifically regulates the onset of migration. To understand the cellular mechanisms underlying the onset of directed migration, we used two-photon LGX-818 site imaging to visualize the cellular transformations that occur in vivo as germ cells migrate through the midgut epithelium. Comparison of wild-type and tre1 mutant germ cells suggests that regulated activation of the Tre1 GPCR controls three phases of early migration: polarization of germ cells, dispersal into individual cells, and transepithelial migration. Germ cell polarization leads to a redistribution of cellcell adherens proteins, such that D. melanogaster E-cadherin levels are reduced from the leading edge of the migrating cells and accumulate in the tail region. Tre1 likely signals via the G proteins G 1 and G 13f as well as Rho-1, as we detect G and Rho-1 protein localization in the tail region, and deletion of their function specifically in germ cells causes the same phenotype as mutation in tre1. Our results suggest a novel function for GPCR signaling in initiating cell migration by polarizing the migrating cell. This polarization leads to the redistribution of signaling components and adherens proteins and may trigger cell dispersal and directed migration. Results Live imaging of early steps in germ cell migration To visualize germ cell migration in developing embryos, we used two-photon microscopy and a germ cellspecific expression system, which translates the actin-binding domain of Moesin fused to EGFP under the control of nanos regulatory sequences. Germ cells appeared motile soon after their formation at the blastoderm stage, as they produced small protrusions away from their neighbors. Despite this apparent motility, germ cells only rarely separated from their neighbors and migrated directly through the underlying blastoderm cells. Subsequently, during gastrulation, as germ cells were internalized together with the invaginating posterior midgut primordium, they rounded up and showed less protrusive activity. At stage 9, germ cells were found inside the midgut primordium in a tight cluster; they were in close contact with each other and showed little contact with the surrounding somatic midgut cells. During this stage, germ cells started to reorganize, changed their shape, and took on a highly polarized morphology. Using electron microscopy, a radial organization of germ cells within the midgut was clearly visible, with the large germ cell nuclei pointed toward the midgut while fine membranous material, apparently corresponding to the tail region, filled the inside of the cluster. This organization oriented the leading edge of each germ cell toward the surrounding midgut primordium. Next, the germ cells lost adhesion to each other, and extensions reached from the germ cells toward the midgut epithelium. Subsequently, germ cells dispersed as they migrated through the midgut primordium to reach the basal side of the midgut cells by stage 10. Long cytoplasmic extensions connected germ cells with each other immediately after the onset of transepithelial migration. As germ cells transmigrated through the midgut epithelium, they appeared completely individualized, displayed amoeboid behavior, and were type. However, tre1 mutant germ cells are unable to disperse and remain in the midgut. High-magnification confocal images PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19836835 of regions in the