Fig. 4.

Midline collapse of longitudinal axons in gcm-robo1 double-mutant embryos. Axons are visualised with anti-FasII antibodies (brown) at stage 16 (A-D). (A) Wild-type embryo. (B) robo1 mutant. Note that the medial fascicle runs along the midline (white arrowhead) whereas the outer two fascicles are well formed and run parallel to the midline (black arrowheads). (C) In gcm mutants, axons sporadically misroute across the midline (black arrowheads) and the lateral fascicles can be fused into one that runs parallel to the midline along the central trajectory (white arrowheads). This embryo has also been stained with anti-Repo (in black, there are a few glia present). (D) In gcm-robo1 double-mutant embryos, axons can fuse (white arrowhead) into one single fascicle running along the midline (black arrowhead) or exit the CNS (asterisk) and grow towards the muscle, but no axons run along the lateral trajectories. (E,F) Axons are stained with anti-Robo3 (brown) and midline glia with anti-Slit (black) at stage 16. (E) Robo3 axons never cross the midline in wild-type embryos, and anti-Slit stains the midline glia in each segment. (F) gcm-robo1 double mutant. Slit (black) is still produced by the midline glia, although the midline glia are disorganised. No longitudinal axons remain, instead axons cross the midline despite expressing robo3. Anterior is up.