Figure 3. Differential functions of Robo2’s Ig1, Ig2, and Ig3 domains in regulating longitudinal pathway formation and midline crossing.

HA-tagged wild type and chimeric receptors were crossed to elavGAL4 and the embryonic CNS axon scaffold and major longitudinal pathways were detected using anti-HRP (magenta) and anti-FasII (green) antibodies. Pro-midline crossing activity of Robo2: Misexpression of any receptor variant that includes Ig2 of Robo2 (B, Robo2; C, Robo1^R2I1+2) resulted in thickened commissures and ectopic midline crossing of FasII-positive axons (compare to wild type, A). This phenotype resembles a reduction in robo function. Full-length Robo1, or chimeric receptors possessing the cytodomain, Fn domains, or Ig1,3,4, or 5 of Robo2 did not enhance midline crossing (D, Robo1; E, Robo2^R1I1+2; and F, Robo1^R2I1). Graph shows frequency of ectopic midline crossing of FasII-positive axons for two transgenic lines each for the variants shown in B–F. Error bars indicate s.e.m. Disruption of FasII pathway formation: Pan-neuronal misepxression of Robo2 disrupts the formation of the intermediate FasII pathway (B, arrowhead with asterisk; compare to wild type, A); the axons that normally select this pathway apparently instead join the medial FasII tract, which becomes thicker in elavGAL4;UAS-Robo2 embryos. Although Robo1^R2I1+2 recapitulates the pro-midline crossing activity of Robo2, it does not mimic Robo2’s effect on FasII pathway formation (C, arrowhead). In contrast, Robo2^R1I1+2 is unable to promote midline crossing in this context, but does reproduce Robo2’s disruption of the intermediate FasII pathway (E, arrowhead with asterisk). A similar reduction of the intermediate pathway is produced by Robo1^R2I3 but not Robo2^R1I1−3 (not shown), indicating that it is due solely to Robo2’s Ig3 domain. Ig1 of Robo2 neither promotes midline crossing nor affects formation of the FasII longitudinal pathways (F).