Fig. 2. Initial screen of midline axon guidance mutants did not reveal a loss of SerT expression. (A-F) stage 16 ventral nerve cords are stained for serotonin uptake in red (A-D) or in white (E,F). (A-D) show the axon scaffold stained with mAb BP102 in green. (A) The wild-type CNS is organized into two longitudinal axon tracts connected by two commissures per segment. Serotonergic axons are seen crossing in the posterior commissure. Loss-of-function mutants (B) commissureless^e39, (C) slit²and (D) robo^GA285 were identified based on CNS phenotype. In comm mutant embryos, the commissures do not form, while in slit mutants, axons fail to leave the midline and in robo mutants serotonergic axons are disorganized as a result of ectopic midline crossing throughout the CNS. (E,F) eagleGal4 drives expression of (E) robo (UAS-robo) and (F) robo2 (EP2582) in the serotonergic neurons. A gain-of-function in robo2, but not robo, prevents the crossing of the midline by serotonergic neurons. Despite defects in serotonergic neuronal axon guidance seen in these embryos, SerT activity remains normal. Scale bar: 10 µm.