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). 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.