(a) Image from western blot show the SAX-3 extracellular fragment by N-terminal tagging FLAG transgenes. Stars (*) label bands that are nonspecifically recognized by the anti-FLAG antibody. A schematic diagram at the top panel shows that the FLAG tag was fused at the N-terminal of SAX-3, and the extracellular cleavage fragment detected by the FLAG antibody is about 55 kDa. (b) SYG-1 specifically binds with the SAX-3 extracellular cleavage fragment. SYG-1::HA expression is driven by the pan-glial promoter Pmir-228, and FLAG::SAX-3 is expressed under the pan-neuronal promoter Punc-33. Stars (*) label bands that are nonspecifically recognized by the anti-FLAG antibody. (c) Confocal images of AMsh glia in control and syg-1(ok3640) animals. TB: pharyngeal terminal bulb. Stars (*) label AMsh cell bodies. Scale bar, 10 µm. (d) Quantification of Migration Index (MI) in control, syg-1, sax-3, and syg-1;sax-3 double mutants show that double mutants of syg-1;sax-3 display similar defects as in sax-3 single mutants. (e) syg-1 cell autonomously regulates AMsh glial migration, and the WAVE regulatory receptor sequence is required for syg-1 function. (f and g) gex-3 cell autonomously regulates AMsh glial migration. Quantification of Migration Index (f) and the percentage of animals with AMsh defects (g) show that gex-3 is required for AMsh migration. (h and i) Confocal images (h) and quantification (i) show that ectopic expression of a fragment mimicking the SAX-3 cleavage fragment in the tail neurons (Pitr-1) or hypodermis (Plin-44) causes syg-1-dependent over-migration of AMsh glia. Red arrowheads point to the center of the pharyngeal terminal bulb. Green arrowheads indicate AMsh cell body position. Scale bar, 10 µm. In d, f, and i, data are represented as mean ± SEM. One-way ANOVA test. **, p<0.01; Each point represents at least 30 worms. In (e and g), data are represented as mean ± SEM. Two-way ANOVA test. **, p<0.01; ns, no significant difference. Each point represents three experiments of at least 50 worms each.