(A-D) Stage 16 embryos stained with anti-FasII to mark all FasII-positive axons. A, wt embryo showing the three FasII-positive longitudinal connectives. B, Syx1A mutant embryo showing the three longitudinal connectives closer to the midline. C, fra3 mutant embryo showing an increased distance between fascicles. D, robo2 mutant embryo showing midline crosses and fascicles closer to the midline. (E) Quantification of the relative distance between fascicles in control (n = 20), Syx1A (n = 20), fra (n = 37) and robo2 (n = 20) VNCs. Significant differences are labelled by asterisks (***p≤0.001). (F-I) Stage 16 embryos stained with anti-FasII to mark all FasII-positive axons and to better observe axonal pathway defects. F, robo2 mutant embryo showing midline crosses (arrows) and fascicle collapses. G-I, robo2;Syx1A double mutant embryos representative of the three phenotypes encountered; G: Weak, H: Intermediate and I: Strong. Asterisks show fascicle collapses in the VNC in all cases. Anterior is up. (J) Quantification of the total number of axon guidance defects encountered in Syx1A (n = 38), robo2 (n = 39) and robo2;Syx1A (n = 31) mutant embryos. Significant differences are labelled by asterisks (***p≤0.001). (K-N) Stage 16 embryos stained with anti-FasII to mark all FasII-positive axons and to better observe axonal pathway defects. K, fra3 mutant embryo showing fascicle defasciculation and collapse phenotypes (arrows). L-N, fra;Syx1A/+ mutant embryos representative of the phenotypes encountered. Arrows point at defasciculation and collapsed phenotypes. (O) Quantification of the total number of axon guidance defects encountered in fra (n = 37), Syx1A (n = 31), fra/+;Syx1A (n = 15), fra;Syx1A/+ (n = 8) and fra;Syx1A (n = 8) mutant embryos. Significant differences are labelled by asterisks (*p≤0.05,***p≤0.001). (P) Quantification of the relative distance between medial fascicles in control (n = 20), Syx1A (n = 20), robo2 (n = 20) and robo2;Syx1A (n = 17) VNCs. Only accounted the cases like panel G, where fascicles had not collapsed.