tre1 regulates DE-cadherin localization in the tail of germ cells. (A–F) DE-cadherin localization in wild-type and tre1 germ cells at stage 5. Embryos were stained with anti–DE-cadherin (red) and anti-Vasa antibodies (green). DE-cadherin is distributed uniformly in the germ cell membrane in the wild type (A–C) and tre1 mutants (D–F). (G–L) DE-cadherin localization in wild-type and tre1 germ cells at stage 9. In wild-type germ cells, DE-cadherin is enriched in the tail region (G–I). In tre1 mutant germ cells, DE-cadherin is evenly distributed in the cell membrane (J–L). Vasa channel is shown in B, E, H, and K, and the DE-cadherin channel is shown in C, F, I, and L. (M) Quantification of DE-cadherin levels in the cell body membrane of stage 5 and 9 germ cells. Normalized intensity of the DE-cadherin staining is shown (see Materials and methods). DE-cadherin levels are similar in wild-type and tre1 germ cells at stage 5. At stage 9, DE-cadherin levels in tre1 mutant germ cells are significantly higher than wild-type cells. Error bars indicate standard error. Bar, 20 μm.

Gβ13f and Gγ1 act downstream of tre1 in transepithelial migration. (A and B) Phenotype of maternal Gβ13f and Gγ1 mutants. Loss of maternal Gβ13f and Gγ1 results in gastrulation defects and prevents normal germ cell migration. (C) A strategy to rescue the gastrulation phenotype by overexpression of G proteins in the somatic tissue. In the wild type, the product of the G protein X (green) is provided maternally in germ cells and the soma, and is lost from both tissues in maternal G protein X mutants. G protein X product is restored only in the soma by using a soma-specific Gal4 transgene, nullo-GAL4 (yellow), which binds to UAS to turn on transcription in the soma but not in germ cells. (D–O) Phenotype of germ cell migration in the wild type (D–F) and maternal Gβ13f (G–I) and Gγ1 (J–L) mutants with somatic rescue. In these mutants, germ cells (brown, anti-Vasa antibody) display a transepithelial migration defect similar to tre1 mutants (M–O). Embryos are oriented anterior to the left, lateral views except for stage 13 embryos, which are oriented dorsally. Bar, 50 μm.

Reduction of DE-cadherin function or adhesion between germ cells does not rescue the tre1 phenotype. (A and B) In tre1 mutants, germ cells (brown) form a tight cluster inside the midgut (white dotted lines in A–F, J, and K) during stages 8–11. (C and D) In tre1 and maternal shg^A9-49 double mutants, germ cells disperse within the midgut (C, arrow); however, they are not able to migrate through the midgut (D, arrow). (E and F) Single germ cells in maternal tud^A36-38 mutants cross the midgut normally (E, arrow) to reach the gonad (F, yellow dotted circle). (G–I) Polarity of single germ cells. Maternal tud^A36-38 mutant embryos were stained with anti-Vasa (green) and anti–DE-cadherin antibodies (red). Single germ cells display a polarized morphology with a tail at stage 9. Vasa channel is shown in H, and the DE-cadherin channel is shown in I. (J and K) Single germ cells (arrows) in tre1 and maternal tud^A36-38 double mutants fail to migrate through the midgut. Embryos are oriented anterior to the left, lateral view in all panels, except for embryos in A and C, which are oriented dorsally. Bars: (G) 20 μm; (K) 50 μm.

DE-cadherin is required for germ cell–germ cell adhesion. (A, C, and E) Wild-type germ cells (brown) form a tight cluster inside the posterior midgut primordium (dotted lines) during stage 8–9 (A and C). Germ cells disperse just before the onset of migration at stage 10 (E). (B, D, and F) In maternal shg^A9-49 mutants, germ cells precociously disperse within the midgut, yet they do not initiate premature transmigration (B and D). shg^A9-49 mutant germ cells have a slight delay in crossing the midgut, and 38% of embryos displayed partial or no transmigration of germ cells at stage 10 (n = 39; arrow in F). (G and H) High-magnification confocal images of germ cells in wild-type (G) and shg^A9-49 mutants (H) at stage 9. Note the lack of clear polarization and radial organization in shg^A9-49 (m⁻, z⁺) mutant germ cells (H). Germ cells were labeled with anti-Vasa antibody (green), and nuclei were labeled with DAPI (blue). Embryos in A, B, E, and F are oriented anterior to the left, lateral view. Embryos in C and D are oriented anterior to the left, dorsal view. Bars, (F) 50 μm; (H) 20 μm.

Tre1 regulates germ cell polarization and G protein localization. (A and B) Electron micrograph images of wild-type and tre1 germ cells at stage 9. Germ cells were identified by the presence of a large nucleus and the lack of white lipid droplets. (A) In wild-type embryos at stage 9, germ cells are organized into a group with little interaction with the surrounding midgut. Germ cells display polarized morphology, with their nuclei facing the midgut and their tails toward the center of the cluster. (B) In tre1 mutants at stage 9, germ cells are not well organized into a radial cluster and are not polarized like the wild type. (C–J) Gβ13f protein localization in the wild type and tre1 mutants. At stage 5, Gβ13f protein (red) is uniformly distributed along the cell surface in wild-type (C and D) and tre1 mutant (G and H) germ cells (green). At stage 9, Gβ13f protein is localized to the tail region of wild-type germ cells (E and F) but is uniformly distributed in tre1 mutant (I and J) germ cells (Gβ13 channel shown in D, H, F, and J). (K–R) Rho1 protein localization in the wild type and tre1 mutants. At stage 5, Rho1 protein (red) is uniformly distributed along the cell surface in wild-type (K and L) and tre1 mutant (O and P) germ cells (green). At stage 9, Rho1 protein is localized to the tail region of wild-type germ cells (M and N) but is uniformly distributed in tre1 mutant (Q and R) germ cells (Rho1 channel shown in L, P, N, and R). Germ cells are visualized by anti-Vasa antibody (green). Bars, 20 μm.

Live imaging of germ cell migration through the midgut in wild-type and tre1 mutant embryos. (A–F) Migration of germ cells in the wild type. (A and C) Wild-type embryos at stages 9 and 10. Germ cells (anti-Vasa antibody, brown) form a tight cluster inside the midgut (dotted lines) at stage 9 (A), then disperse and migrate through the midgut to reach the basal side of the midgut cell layer at stage 10 (C). (B and D) High-magnification confocal images of the regions in the boxes in A and C, respectively, showing the midgut regions of stage 9 (B) and stage 10 embryos (D). Germ cells are shown in green, and the midgut cell membrane was detected with anti-neurotactin antibody (red). (E) Time-lapse analysis of germ cell migration during stages 9 and 10 with two-photon microscopy. Shown are still images from a time-lapse video (Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200807049/DC1). Germ cells lose adhesion with other germ cells just before the onset of migration through the midgut (E 3 and 4). (F) Trajectory of germ cells shows radial dispersion. (G–K) Migration of germ cells in tre1 mutants. (G and I) tre1 mutant embryos at stages 9 and 10. tre1 germ cells (brown) form a tight cluster in the midgut at stage 9 (G) similar to the wild type. However, tre1 mutant germ cells are unable to disperse and remain in the midgut (I). High-magnification confocal images of regions in the boxes in G and I, respectively, showing the midgut regions of stage 9 (H) and stage 10 tre1 mutant embryos (J). Germ cells are shown in green and the membranes of the midgut cells are labeled in red. (K) Still images of tre1 mutant embryo during stages 9 and 10 from time-lapse analysis shown in Video 5. Germ cells are motile and change their positions; however, they are not able to disperse and remain in a tight group in the midgut. Embryos are oriented anterior to the left, dorsal view in A–D and G–J. Embryos in E and K are oriented anterior to the top, dorsal view. Bars, 50 μm.