(A, B, D, E) Stage 17 embryos stained with anti-FasII and anti-HRP. (A) Sos embryos show mild ectopic crossing defects of 15% in FasII axons (arrowheads) and no phenotype in HRP. (B) Simultaneous removal of sos and cyfip results in a very strong enhancement of the ectopic FasII crossing defects to 82% and a strong HRP phenotype with thickening and fusion of commissures (asterisk). Similarly, (D) robo2 mutants show mild ectopic crossing defects of 17% in FasII axons and a mildly disorganized axon scaffold in HRP while (E) double mutants for robo2 and cyfip show strong ectopic FasII crossing defects of 77% and thickening and fusion of commissures in HRP. (C, F) Quantitation shows the percentage of segments in which FasII axons ectopically cross the midline. Data are presented as mean ± SEM, number of embryos, n = 15 and 16 (for E) and 20 and 9 (for F). Significance was assessed using Student’s t-test. Scale bars in (A) and (D) represent 20 µm.