Fig. 3. Physical or genetic disruption of midline contact affects SerT activity in wild type, Robo gain-of-function and Unc5 gain-of-function (but not Robo2 gain-of-function) mutants. (A-C) Staining of ventral nerve cords for serotonin uptake (red in left panel and white in right) and for engrailed (green in left panel). A cut (line of stars) was made in isolated stage 16 cords asymmetrically down the midline such that one set of cell bodies was separated from the midline. The tissue preparations were allowed to develop for 2 hours prior to fixation. (A) In 15 out of 20 wild-type preparations, SerT activity was lost from the cell bodies on the cut side (arrow). (B) Similar cuts in embryos overexpressing Robo in serotonergic neurons (Robo gain of function; UAS-robo with eagleGal4) also showed a loss of SerT activity in the midline-minus side (arrow; three out of three). (C) However, when Robo2 was overexpressed (eagleGal4), only one out of 20 preparations showed a loss of SerT activity after a midline cut. (D) Expression of Unc5 with eagleGal4 specifically prevents the crossing of the midline by serotonergic neurons and also causes a partial loss of SerT activity as seen by staining for serotonin uptake (red/white), but does not affect whole CNS axon guidance (BP102, green). Arrows (left panel) indicate hemisegments where SerT expression is lost and yellow stars (right panel) indicate axons that extends towards the midline despite expression of Unc5. Scale bar: 10 µm.