(A–F) Stage 16 embryos of the indicated genotypes stained with anti-HRP. Arrowheads indicate thin/defective commissures, arrows indicate missing commissures and asterisks indicate rescued commissures. Scale bar represents 15µm (F). (A) Thick anterior and posterior commissures are formed as axons cross the midline in every segment. (B) frazzled (fra3/fra4) mutants show thin (29%) and occasionally missing commissures (10%). (C) sema-1a mutants show no obvious signs of commissural defects. (D) Embryos heterozygous for both sema-1a and fra appear wild-type. (E) fra, sema-1a double mutants show a 68% loss of commissures. (F) Pan-neural expression of Sema-1a partially rescues these defects, and reduces missing commissures to 25%. (G) Quantification of commissural defects as absent (black bar), thin/defective (dark gray) or wild-type (light grey) in the genotypes shown in (A–F). Data are represented as mean+SEM. n, number of embryos scored for each genotype. Significance was assessed by multiple comparisons using ANOVA (****p<0.0001).