Fig. 1. shot mutants disrupt lumen formation at anastomosis sites. (A-D) 15 µm stacks of 1 µm confocal sections of stage 16 wild-type and shot³ null mutant embryos stained with mAb 2A12, which recognizes a lumenal antigen (Samakovlis et al., 1996a). Anastomosis sites are indicated at hemisegment boundaries in the dorsal (short arrows) and lateral (concave arrows) trunks and the dorsal midline (long arrows). Anterior, leftwards; dorsal, upwards. (A) Lateral view, wild type. The lumen branches in a stereotyped pattern and is continuous at anastomosis sites. (B) Lateral view, shot³. The lumen is discontinuous at anastomosis sites. (C) Dorsal view, wild type. The lumens of dorsal branches join together at the dorsal midline. (D) Dorsal view, shot³. Dorsal branches have migrated towards the dorsal midline, but have not joined their lumens together. The dorsal trunk lumen is discontinuous at most (short arrow), but not all (arrowhead), anastomosis sites. (E-H) 1 µm confocal sections of dorsal trunk tracheal cells. (E) Stage 14, wild type. The mAb 2A12 lumenal antigen is transiently present in vesicular structures (arrow) within tracheal cells. Dorsal trunk anastomoses are complete. (F) Stage 14, shot³. mAb 2A12 labels the lumen and vesicular structures (arrow) within tracheal cells. Vesicles are still present in cells at anastomosis sites that do not form lumen. (G) Stage 15, wild type. The Fus-5 enhancer trap labels two fusion cell nuclei (brackets) with lacZ at each dorsal trunk anastomosis site. Other tracheal nuclei are also weakly labeled. (H) Stage 15, shot³. The Fus-5 enhancer trap labels two fusion cell nuclei at each anastomosis site. shot mutant embryos also express the fusion cell specific Fus-2 and Fus-3 enhancer trap markers normally (data not shown). Scale bars: in D, 25 µm for A-D; in H, 10 µm for E-H.